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Solitary plasmacytoma SP of the skull is an uncommon clinical entity that is characterized by alocalized proliferation of neoplastic monoclonal plasma cells This case report describes a50yearold male that presented with a headache and an exophytic soft mass on the occiputThe diagnosis of SP was based on the pathological results and imaging examinations The patientunderwent occipital craniotomy skull reconstruction and lower trapezius myocutaneous flapLTMF transplantation under general anaesthesia The tumour was capsulized and extended tothe subcutaneous and the subdural space through the dura mater with skull defects The neoplasm of the occipital bone involved large areas of scalp and subcutaneous tissue which resultedin a large postoperative scalp defect that was repaired using LTMF transplantation All of thetumour was removed and the transplanted flap grew well Followup at months identified anaggressive mass lesion on the right frontallobe The patient received six cycles of the PADchemotherapy regimen bortezomib doxorubicin and dexamethasone and the lesion was significantly reduced This case demonstrates that LTMF is an alternative approach for the repair ofscalp and subcutaneous soft tissue defects caused by the excision of a large malignant tumourof the occipital region Chemotherapy is the choice of treatment for neoplastic recurrenceKeywordsSolitary plasmacytoma lower trapezius myocutaneous flap scalp reconstruction plasma cellsDate received July accepted March 1Department of Neurosurgery Hunan Cancer Hospitaland the Affiliated Cancer Hospital of Xiangya School ofMedicine Central South University Changsha HunanProvince China2Department of Head and Neck Surgery Hunan CancerHospital and the Affiliated Cancer Hospital of XiangyaSchool of Medicine Central South University ChangshaHunan Province ChinaCorresponding authorsLei Wang and ZhengWen He Department ofNeurosurgery Hunan Cancer Hospital and the AffiliatedCancer Hospital of Xiangya School of Medicine CentralSouth University Tongzipo Road Yuelu DistrictChangsha Hunan Province ChinaEmails wangsengyi163com hezhw2001163comCreative Commons Non Commercial CC BYNC This is distributed under the terms of the CreativeCommons AttributionNonCommercial License creativecommonslicensesbync40 which permitsnoncommercial use reproduction and distribution of the work without further permission provided the original work is attributedas specified on the SAGE and Access pages ussagepubcomenusnam accessatsage 0cIntroductionSolitary plasmacytoma SP is the pathological manifestation of the proliferationof monoclonal plasma cells and an SPthat originates in bone tissue is called a solitary plasmacytoma of bone SPB1 Bonedestruction may occur in any osseous location but the most common sites are thepelvis spine femur humerus and ribs2“An SPB of the skull is rare and a hugegrowth in the occipital bone is rarely mentioned in the literature34 Complete tumourremoval is the first and best approach forpatients with no lesions in other parts of thebody3 This current case report describes arare case of SPB of the occipital bone withscalp involvement in which the patient underwent radical resection and reconstruction ofJournal of International Medical Researchthe large scalp defects through lower trapeziusmyocutaneous flap LTMF transplantationCase reportA 50yearold male patient presented inOctober to the Department ofNeurosurgery Hunan Cancer Hospitaland the Affiliated Cancer Hospital ofXiangya School of Medicine CentralSouth University Changsha HunanProvince China with a headache and an exophytic mass on the occiputaTheexaminationphysicalshowed no findings Computed tomographyCT showed a large mass with homogeneousenhancement on the occiput compressing thebilateral occipital lobe b and theneurologicalFigure Preoperative imaging examinations a preoperative appearance of the tumour b preoperativeenhanced computed tomography scan c preoperative enhanced magnetic resonance imaging MRId preoperative enhanced MRI scan “ axial view e preoperative digital subtraction angiographyThe colour version of this figure is available at httpimrsagepubcom 0cWang et alresonanceandbone window revealed a solitary osteolyticlesion involving the whole entire of the occipital bone MagneticimagingMRI showed an intra and extracerebralexpansile osseous lesion 02 mm themass was mostly isointense with the brainparenchyma on both T1 and T2weightedimagesenhancedFigures 1c and 1d Digital subtraction angiography DSA demonstrated that all of thetumour had hypervascularity that was supplied from the occipital artery In order todecrease bleeding volume and shorten thesurgery time the feeding blood vessel wasembolized during DSA ehomogeneouslyThe patient underwent occipital craniotomy skull reconstruction and LTMF transplantation under general anaesthesia Thetumour was capsulized and extended tothe subcutaneous and the subdural spacethrough the dura mater with skull defectsGrossly the tumour had a fishmeat likeappearance mixed with hard cartilage andbroken bone a The tumour had arich blood supply and despite embolizationof the main blood supply artery duringDSA before surgery there was a lot ofblood loss during the operation Thetumour mass underwent extended resectionincluding the marginal bone and involvedscalp forming an 02 cm bone windowand a 02 cm scalp defect The skulldefect was reconstructed using titaniummesh and the scalp defect was transplantedusing LTMF The trapezius and the skinisland 02 cm and the supplying vesselsof the transverse cervical artery and thedorsal scapular artery were marked on theskin b The island flap was excisedand its muscle pedicle dissected up to theFigure Perioperative procedures and imaging examinations a the tumour was fishmeat soft tan inappearance b the trapezius and the skin island and the supplying vessels of the transverse cervicalartery and the dorsal scapular artery marked out on the skin c the island flap and its muscle pedicle wereexcised d the flap was set into the defect with a wellperfused distal end e the stiches were removedafter surgery f postoperative enhanced magnetic resonance imaging MRI scan “ sagittal viewg postoperative enhanced MRI scan “ axial view The colour version of this figure is available at httpimrsagepubcom 0crotation point at the medialsuperior edgeof the scapula c The LTMF wasrotated vertically into the occipital scalpdefect through the neck posterior subcutaneous tunnel d Two weeks afterthe operation the transplanted skin islandwas vital and wound healing undisturbedFigurethattheremovedtumour wasFigures 2f and 2g2e MRIcompletelyindicatedHaematoxylin and eosin staining of thetumour showed the presence of atypicalplasma cells with typical eccentric roundnucleistainingshowed the following staining patterncytokeratinP “ epithelial membrane antigen “ melanA “ CD38 ¾ CD138ImmunohistochemicalJournal of International Medical Research¾ CD20 “ Kappa ¾ Lambda ¾glial fibrillary acidic protein “ S100“CD68 ¾ thyroid transcription factor1“ Vim “ CD3 “ and Ki67 Figure The patient refused further radiotherapyfor financial reasons After a followupperiod of around months he was symptom free and had no clinical evidence ofdisease At the 5month followup visitMRI revealed no eld recurrence butan aggressive mass lesion with enhancementwaslobeFigures 4a and 4b Chemotherapy PADregimen bortezomib pegylated liposomaldoxorubicindexamethasone wasadministered from April in thefound on thefrontalrightandFigure Representative photomicrographs of the tumour a haematoxylin and eosin stained sectionshowing diffuse sheets of plasma cells b immunohistochemical staining for CD138 showing strong positivity in the tumour cells c immunohistochemical staining for CD38 showing strong positivity in thetumour cells d the positive expression of Ki67 was The colour version of this figure is available athttpimrsagepubcom Scale bar mm 0cWang et alFigure Magnetic resonance imaging scans of the patient during followup a b at the 5month followup visit showing no recurrence in situ but an aggressive mass lesion with enhancement on the right frontallobe c d after six consecutive cycles of chemotherapy showing no recurrence in situ and the lesion onthe right frontal lobe was significantly reducedDepartmentof Haematology HunanCancer Hospital and the Affiliated CancerHospital of Xiangya School of MedicineCentralSouth University ChangshaHunan Province China After six consecutive cycles of chemotherapy the lesion on hisright frontal lobe was significantly reducedFigures 4c and 4d Postoperative reviewafter months showed no tumour recurrence in situ of the original SPBAs this was a case report the InstitutionalReview Board of Hunan Cancer Hospitalwaived the need for ethical approval Thepatient provided written informed consentfor publication that was approved by theInstitutional Review Board and the detailsof the patient have been anonymizedDiscussionHuge intra and extracranial SPs of theoccipital bone are very rare and few caseshave been reported34 SPB is characterizedby the presence of a solitary lytic lesion dueto monoclonal plasma cell ltration withsofttissue extension5 SPBsor withoutaccount for of all SP cases and theyoccur primarily in red marrowcontainingbones6 Plasma cells are highly sensitive toradiation78 Radiation therapy remains the 0cJournal of International Medical Researchtreatment of choice for patients after surgery According to recommendations froma European expert panel a total fractionated dose of “ Gy should be given anda margin of atleast cm should beemployed6 In this current caseit wasunfortunate that the patient refused radiationreasonsA review was performed months afterthe operation and a new mass was foundon the right frontal lobe After six cyclesof chemotherapy the tumour had reducedin size significantly which suggests that chemotherapy has a positive impact on thegrowth of recurrent tumourseconomictherapyforThe imaging characteristics of SPB in theskull are complex and can easily lead tomisdiagnosis Enhanced CT scanning combined with observation of the bone windoware credible means by which to diagnoseSPB and they could provide more information about osteolytic lesions In the currentcase the MRI examination allowed for theidentification of the location size and shapeof the tumour as well as its relationship tothe surrounding structures In our opinionpreoperative DSA is necessary for the identification of the blood supply vessels and tofacilitate vessel embolizationDuringthetumourrecurrencecurrent operationthetumour was found to involve the scalpand subcutaneous tissue To reduce theprobability oftheinvolved scalp underwent an extendedresection LTMF was used to facilitateoccipital scalp reconstruction LTMF provides available muscle compartments transferred on a reliable vascular pedicle to thedorsal suprascapular and neck regions9The benefits of LTMF include well vascularized tissue ease of harvest and the provision of a large flap located far enoughaway from the damaged area10 The mainblood supply to the LTMF originates fromthe transverse cervical artery and the dorsalscapular artery1112 This method couldsolve the problem ofinsufficient bloodsupply caused by titanium plate implantation In additionthe musculocutaneouspedicle could fill the subcutaneous cavitycreated by the huge tumourresectionpreventing occipitalia scalp hydrops andsecondary infection LTMF is an alternative approach for the repair of scalp andsubcutaneous soft tissue defects caused byexcision of a malignanttheoccipital regiontumour ofAuthors™ contributionsLW studied the case collected the referencesand wrote the paper ZH designed the reportand wrote the paper HC and HZ wrote thepaper XP analysed the data NR served as thefirst chief during surgery and wrote the paperAll authorsread and approved the finalmanuscriptDeclaration of conflicting interestThe authors declare that there are no conflicts ofinterestFundingThis project was supported by grants from theScientific Research Project of Hunan ProvincialHealth Commission No20200709 the HunanProvincial Natural Science Foundation of ChinaNo2019JJ40182 and the Sailing Programme ofHunan Provincial Cancer Hospitalorcid000000021069ORCID iDLei WangReferences Sabattini E Bacci F Sagramoso C et alWHO classification of tumours of haematopoietic and lymphoid tissues in anoverview Pathologica “ Gee ED and Sadovsky R Multiple myeloma recognition and management AmFam Physician “ Rizea RE Popescu M Ghehit¸ 15a KLet al Neurosurgical rare disease solitary 0cWang et alplasmacytoma of the skull “ case report andliterature review Rom J Morphol Embryol “ Chang MY Shih LY Dunn P et al Solitaryplasmacytoma of bone J Formos Med Assoc “ Weber DM Solitary bone and extramedullary plasmacytoma Hematology Am SocHematol Educ Program “ Caers J Paiva B Zamagni E et alDiagnosis treatment and response assessment in solitary plasmacytoma updated recommendations from a European ExpertPanel J Hematol Oncol Knobel D Zouhair A Tsang RW et alPrognostic factors in solitary plasmacytomaof the bone a multicenter Rare CancerNetwork study BMC Cancer Liebross RH Ha CS Cox JD et alSolitary bone plasmacytoma outcome andprognostic factors following radiotherapyInt J Radiat Oncol Biol Phys “ Mohos G Vass G Kemeny L et alExtended lowertrapezius myocutaneousflap to cover a deep lateral neck defect onirradiated skin a new application J PlastSurg Hand Surg “ U 15gurlu K Ozc¸ elik D Hu¨ thu¨ t I et alExtended vertical trapezius myocutaneousflap in head and neck reconstruction as asalvage procedure Plast Reconstr Surg “ Baek SM Biller HF Krespi YP et al Thelower trapezius island myocutaneous flapAnn Plast Surg “ Netterville JL and Wood DE The lower trapezius flap Vascular anatomy and surgicaltechnique Arch Otolaryngol Head NeckSurg “ 0c'

Thyroid_Cancer

"development of cancer is a problem that has accompanied mankind for years The growing number of cases emerging drug resistance and the need to reduce the serious side effects of pharmacotherapy are forcing scientists to better understand the complex mechanisms responsible for the initiation promotion and progression of the disease This paper discusses the modulation of the particular stages of carcinogenesis by selected physiological factors including acetylcholine ACh peroxisome proliferatoractivated receptors PPAR fatty acidbinding proteins FABPs Bruton™s tyrosine kinase Btk aquaporins AQPs insulinlike growth factor2 IGF2 and exosomes Understanding their role may contribute to the development of more effective and safer therapies based on new binding sitesINTRODUCTIONThe increasing prevalence of various types of cancers is a global phenomenon that has been occurring widely and affecting increasing numbers of people In over million new cases were reported and statistics show that every fifth man and every sixth woman is at risk for developing cancer [] Carcinogenesis is a multistage process that leads to cancer development It includes three main stages initiation promotion and progression and each is characterized by different processes During the promotion stage the genetic material of cells is damaged and if not detected by repair systems before the next division the change is transferred to newly formed cells Under the control of growth factors initiated cells may move on to the second stage of cancer formation“promotion During that stage mutated cells undergo multiple divisions their growth is uninhibited as they become insensitive to apoptotic signals As a result the tumor increases its mass but the cells remain within one an The formation of metastases occurs in the next phase“progression Metalloproteinases secreted by tumor cells destroy the extracellular matrix This allows them to enter the bloodstream and reach a new an at a considerable distance from the primary lesion with the stream of flowing blood The key step for the survival of migrating cells is adhesion to the attacked an and the formation of new blood vessels thanks to which they will receive the components necessary for development Angiogenesis the formation of new blood vessels is the last element of carcinogenesis and is enhanced by tumor cell derived factors metabolic changes and a decrease in available oxygen Each of these processes is controlled by different signal pathways via proteins that naturally occur in the body Figure Dysregulation of their expression and thus activity both to an excessive and insufficient degree ensures the continuity of carcinogenesis and increases the chances cancer cells survive in a host anism Understanding the role of individual substances and components of the body in the subsequent stages of neoplastic transformation provides a more complete picture of cancer pathogenesis This allows to develop new recommendations to improve the quality and safety of pharmacotherapy which has a direct effect on an improvement in the cancer patients™ quality of life It also provides the basis for research aimed at developing new compounds that are effective weapons in the fight against cancer Therefore there is no doubt that any actions that bring scientists closer to solving the problem of insufficient cancer therapy are sensible and much needed The purpose of this paper was to discuss the role of selected physiological factors in the various stages of neoplastic transformationOncotargetwwwoncotargetcomwwwoncotargetcom Oncotarget Vol No pp 0cCrucial mechanisms involved in carcinogenesisThe carcinogenic process is regulated by many molecular and cellular mechanisms The diversity of signaling pathways exploited during cancer initiation promotion and progression is immense Many of them are common in different cancer types but there are also clearly unique molecular and cellular signaling signatures specific for particular cancers [] Pathways such as AktPI3K RasMAPKERK Wntcatenin JAKSTAT occupy crucial roles in the regulation of cell proliferation apoptosis differentiation angiogenesis cell migration and invasion immunological activities and inflammationPI3K phosphatidylinositol 3kinase is a family of enzymes that after activation by growth factors cytokines and hormones are involved in the conversion of PIP2 phosphatidylinositol45bisphosphate into PIP3 phosphatidylinositol 345trisphosphate [] The latter in turn takes part in recruiting Akt into the cell membrane where it is phosphorylated and activates other agents such as mTOR NFkB Wnt or inhibits factors like Bad p27 to enhance carcinogenesis Dysregulation of this pathway occurs in endometrial [] bladder [] or gastric cancers [] The aforementioned PIP2 can be also transformed by phospholipase C into DAG and IP3 which then activate protein kinase C PKC [] One of the PKC substrates is Raf kinase which is an element of the RasMAPKERK pathway which is involved in the pathogenesis of ovary cancer [] or nonsmall cell lung cancer [] Ras proteins activate Raf which then phosphorylates kinases MEK12 Its final substrate is kinase ERK12 which after transportation into the nuclei regulates transcription factors essential for DNA synthesis and cell cycle progression They condition cell growth proliferation and viability [] Because of that targeting those two cascades as an anticancer therapy seems to be beneficial However it is important to notice that the existing crosstalk between both of the pathways can impede treatment and inhibition of one can strengthen the activity of the other Another component that is also connected with the MAPK family Figure Modulators of individual stages of carcinogenesis Inducers red frames inhibitors green frames M1rec muscarinic receptor M3rec muscarinic receptor Nrec nicotinic acetylcholine receptor COX2 cyclooxygenase2 PPARÎ peroxisome proliferatoractivated receptors gamma Btk Bruton™s tyrosine kinase FABP fatty acidbinding protein LFABP Ltype fatty acidbinding protein AQPs aquaporins IGF2 insulinlike growth factor IGFBP3 insulinlike growth factor binding protein IGFBP5 Insulin Like Growth Factor Binding Protein IGF2BP2 Insulin Like Growth Factor2 mRNA Binding Protein2 IGF2BP3 Insulin Like Growth Factor2 mRNA Binding Protein Oncotargetwwwoncotargetcom 0cis a signaling pathway conducted by p38MAPKs This subfamily contains four kinases which are activated mainly by inflammatory cytokines and stress factors p38MAPKs control many aspects of cell physiology such as cell cycle regulation differentiation or skeleton remodelling Additional p38MPAKs can act as tumor promotors by enhancing metalloproteinase and VEGF expression []Wntcatenin signaling plays essential roles in caricinogenesis Activation of canonical Wnt signaling results in the inhibition of GSK3 kinase dissociation of catenin proteins and its transfer to the nucleus where it regulates multiple downstream genes like cMyc and cyclin D [] This axis was found to be associated with several oncogenic events including tumor cell proliferation migration epithelial“mesenchymal transition and invasion An abnormally active WntBcatenin pathway is observed among others in gastric [] colon [] breast [] or adrenocortical cancer [] Another signaling pathway involved in the pathogenesis of many cancer types is that induced by the Jak kinases family It includes four kinases which after activation impact of the STAT molecules causing their translocation into the nuclei [] STAT a family that gathers seven forms of proteins is associated with cancer cell development progression metastasis survival and resistance to treatment [] STAT3 and STAT5 factors seem to be the most important agents in light of cancer progression They have an impact on p53 protein which leads to a disruption in cycle control and apoptosis and induces cell proliferation by the increased cMyc and cyclin D expression By the induction of VEGF gene expression they take part in enhanced angiogenesis and induction of genes such as survivin Bcl2 BclXL conditioning overall survival of the tumor cells Moreover pSTAT3 acts to negatively regulate neutrophils NK cells effector T cells and dendritic cells while positively regulating populations of MDSCs myeloidderived suppressor cells and regulatory T cell leads to a highly immunosuppressive TME tumor microenvironment []Multiple endogenous factors are involved in carcinogenesis which affects particular stages of carcinogenesis in various mechanisms Some of them interact directly with the DNA intracellular signalling molecules others by specific cellular receptors Among them are well known growth factors EGF TGFα and TGF FGF or reactive endogenously generated oxygen molecules as well as less often described agents like ACh PPAR FABPs Btk AQPs IGF2 or exosomes The latter have only recently gained in importance and are more widely studied and discussed therefore are considered in this articleAacetylcholine Ach and its receptorsOne of the factors involved in the carcinogenesis process is acetylcholine and its receptors both muscarinic inhibitors and nicotinic Their expression has been demonstrated in numerous types of cancer cells [“] It has also been proven that these cells contain acetylcholinesterase AChE an enzyme that enables cells to produce ACh in the absence of agonists from the external environment Cancer cells also have the ability to produce autoantibodies that stimulate one of the muscarinic receptor subtypes M3 These facts prompted more extensive research to explain the exact role of ACh and its receptors in the carcinogenesis process Figure Muscarinic receptors can be divided into five subtypes each of which is involved in tumor development through a different mechanism The role of muscarinic M3 receptor is the most widely described subtype It is involved in the pathogenesis of lung colon gastric and breast cancer In the course of colon cancer it has been observed that stimulation of the M3 receptor causes phosphorylation of the Akt and ERK12 kinases which are factors that through the ability to regulate the activity of pro and antiapoptotic proteins affect the intensification of cell proliferation survival and motility In addition coexpression of the M3 receptor and endothelial growth factor receptor EGFR as well as EGFR transactivation after M3 receptor stimulation has been demonstrated [ ] Moreover the administration of EGFR inhibits acetylcholineinduced ERK12 kinase phosphorylation which is reflected in a significant decrease in colon cancer cell proliferation This indicates that acetylcholine is involved in the formation of colon cancer but for its full action it is necessary to activate the EGFR receptor Thus indicating that enriching colon cancer treatment with EGFR inhibitors may improve patient outcomes Acetylcholine is also involved in the formation of vascularlike structures in the vicinity of a developing tumor guaranteeing the supply of essential nutrients to the proliferating cancer cells This is called vascular mimicry The basis of this process is the acetylcholinedependent regulation of metastasisassociated in colon cancer1 MACC1 oncogene expression via the M3RAMPKMACC1 signaling pathway [] High activity of this oncogene is associated with an increase in cell invasiveness intensified epithelial“mesenchymal transition more frequent metastases and hence worse treatment prognosis [“] The first subtype of the M receptor M1 is also involved in the tumor development process It activates the hedgehog pathway [ ] whose physiological role is to regulate the transcription factors affecting the oncogenic activity of Gli zinc finger transcription factors [ ] Excessive stimulation causes acceleration of carcinogenesis intensification of proliferation growth and survival of cancer cells as well as angiogenesis by affecting the expression of genes such as cyclin D antiapoptotic Bcl2 or VEGF Furthermore stimulation of this pathway promotes the creation of a microenvironment conducive to tumor growth through increased expression of extracellular matrix components [] Nicotinic Oncotargetwwwoncotargetcom 0cacetylcholine receptors are part of the ligandgated ion channels and are created from two types of subunits α and Receptors that contain an alpha7 or alpha9 subunit in their structure are characterized by a high degree of permeability to calcium ions thanks to which they are involved in the course of numerous processes based on signaling involving secondary messengers Thus after activating N receptors Ca2 inflows into the cell causing the secretion of growth factors exerting paracrine action on neighboring cells Simultaneously apoptosis is inhibited enabling further proliferation Studies conducted on a lung cancer cell line after exposure to cigarette smoke have shown that nicotine and nitrosamines which have a higher affinity for nicotinic acetylcholine receptors nAChRs than nicotine bind to the alpha7 receptors activating the RasERKMAPK and the JAK2STAT3PI3K signaling pathways [ ] The effect of this interaction is increased proliferation and migration of cancer cells and thus facilitated metastasis creation Moreover by affecting Badrenergic receptors nicotine intensifies COX2 expression which through the p38 MAPK and the JNK pathways leads to an increase in VEGF production a highly proangiogenic factor In addition the high activity of cyclooxygenase affects the inactivation of the PTEN protein [ ] whose biological role is silencing PI3KAkt pathway signaling This leads to cellular signal transition and induces effects that contribute to the enhanced growth and proliferation of cancer cells as well as inhibits the process of their death Under the influence of the Akt kinase proapoptotic transcription factors are inactivated including kinase9 mTOR kinase intensifying cell proliferation is activated and VEGF is activated This indicates the involvement of this pathway in processes that facilitate cancer cell survival Another mechanism contributing to cancer development in which cyclooxygenase is involved is the complex process involving the COX2PGE2EP4 axis as a result of which metalloproteinase expression is stimulated [“] MMP9 causes proteolytic activation of TGF which begins the induction of epithelialmesenchymal transformation EMT During this transition the cells acquire features that increase their invasiveness This leads to epithelial cell phenotype changes the connections between adjacent cells and between cells and the basement Figure Carcinogenic effect of acetylcholine Ach Intracellular signaling pathways activated by ACh via M1 and M3 receptors Induction of process is illustrated by arrows inhibition by horizontal lines M1 muscarinic receptor M3 muscarinic receptor N receptor nicotinic acetylcholine receptor Akt protein kinase B ERK extracellular signalregulated kinase JAK2 Janus kinase PI3K phosphoinositide 3kinase STAT3 signal transducer and activator of transcription proteins MAPK mitogenactivated protein kinaseOncotargetwwwoncotargetcom 0cmembrane are loosened the cytoskeleton is rearranged cells lose contact inhibition thus causing excessive proliferation Furthermore the microenvironment of the growing tumor also changes promoting features that facilitate the movement of the cancer cells from the primary focus to other regions of the body Through the EMT process cells acquire greater migration capacity and thus their invasiveness increases In this cellular process MMP9 also modulates the activity of integrins and other molecules that ensure cell adhesion [] and secrete soluble factors into the bloodstream that facilitate the implantation of migrating cells in ans distal to the primary tumor focus [] Thanks to these processes COX2 enables the formation of cancer metastasesPPAR peroxisome proliferatoractivated receptorsThe family of peroxisome proliferatoractivated receptors PPARs includes isoforms of these proteins that function as transcription factors Each type of steroid receptor differs in its location and affinity for ligands [] The best known is the function of the PPARÎ form Agonists of this receptor are a therapeutic option in the treatment of patients with diabetes Current research provides evidence that stimulation of PPARÎ receptors inhibits cell proliferation vessel formation and induces apoptosis Figure [ ] These properties make PPARÎ agonists attractive candidates for anticancer drugs To achieve full transcriptional activity heterodimerization of PPARÎ with the retinoid X receptor is necessary [ ] This interaction enables the promoter to bind with the target gene and regulate its expression An example of a gene against which PPARÎ acts as a repressor is the vascular endothelial growth factor VEGF gene [] Thus angiogenesis is inhibited and this mechanism has been demonstrated in studies on human hepatocellular carcinoma HCC cells [] For this cancer PPARÎ is also involved in inhibiting cell growth and attenuating cell migration and invasiveness By upregulating KLF4 Krüppellike factor expression and inhibiting cyclin D1 expression the cell cycle is inhibited Furthermore PPARÎ negatively affects the expression of transcription factor STAT3 thereby inhibiting proliferation survival and metastasis Lowering STAT3 levels also negatively affects the angiogenesis process by inhibiting VEGF depriving cancer cells of nutrients PPARÎ activation in HHC cells also impairs their metastatic ability [] This is associated with the inhibition of the expression of metalloproteinases MMP9 and MMP13 the increased expression of their inhibitor and Ecadherin a protein conditioning cell adhesion In addition PPARÎ increases plasminogen activator inhibitor expression thus inhibiting the breakdown of the extracellular matrix and basement membrane proteins which also reduces cancer cell invasiveness PPARÎ has also been shown to inhibit esophageal cancer [] The mechanism responsible for suppressing this tumor is MAPK signaling pathway inhibition This process requires TLR4 Tolllike receptor inhibition which allows PPARÎ agonists to be administered TLR4 is a procarcinogenic protein and affects among others changes in the tumor microenvironment and the severity of angiogenesis Therefore in addition to the indirect effect on the MAPK cascade its inhibition is an important element of tumor suppression Studies on esophageal cancer cells indicate that PPARÎ stimulation leads to a decrease in PCNA proliferating cell nuclear antigen factor expression which indicates inhibition of DNA replication [] In addition PPARÎ activation intensifies the process of apoptosis as evidenced by an increase in the expression of the active form of caspase and the Bax gene and a decrease in Bcl2 expression In nonsmallcell lung cancer PPARÎ stimulation inhibits the neoplastic process by regulating PTEN protein expression [] This protein is a tumor suppressor and its activation causes the dephosphorylation and inactivation of the PIP3 second messenger As a result Akt kinase activity is inhibited leading to a decrease in NFkB expression Due to PPARÎ stimulation by eicosapentaenoic acid EPA a relationship is sought between PPARÎ and COX2 activity that uses EPA as a substrate for prostanoid production The combination of PPARÎ agonists with COX2 inhibitors has demonstrated a synergistic tumorsuppressing effect in studies on nonsmallcell lung cancer Researchers indicate significantly reduced thromboxane TXA2 synthesis using combination therapy compared with monotherapy as the main mechanism of this phenomenon [] A decrease in its concentration limits signaling in four pathways ERK p38 MAPK JAK and catenin limiting tumor growth [] Furthermore it affects the arrest of the cell cycle in the G2M phase preventing further cell division and contributing to the reduction of the expression of survivin an antiapoptotic protein [] The described mechanisms affect the induction of the apoptosis process and inhibit cell cycle progression which limits cancer development The above examples indicate that peroxisome proliferatoractivated receptors play a significant role in inhibiting the development of various types of cancer which is why agonist compounds can be considered potential anticancer drugs However further comprehensive and extensive research is needed to identify potential applications and treatment regimensBtk Bruton™s tyrosine kinaseBruton™s tyrosine kinase Btk also plays an important role in carcinogenesis Figure It is a key point in signal transduction from the BCR receptor leading to the activation of the NFkB signaling pathway [] Btk affects phospholipase CÎ2 [] which after phosphorylation causes PIP2 hydrolysis The result Oncotargetwwwoncotargetcom 0cis IP3 and DAG DAG activates protein kinase C which stimulates NFkB pathway factors Finally genes conditioning cell survival are expressed Btk also contributes to inhibiting the apoptosis process through interacting with the Akt kinase [] The signaling cascade includes active PI3K which recruits Akt to the plasma membrane Under the influence of Btk its phosphorylation occurs and thus its activation Then the active Akt returns to the cytoplasm and induces antiapoptotic pathways dependent on NFkB among others In addition Btk as a kinase belonging to the Tec family affects intercellular signaling causing changes in the tumor microenvironment In most cancer cells the developing microenvironment leads to the inhibition of immune processes which results in protection and protects defective cells from natural defense mechanisms One of the most common mechanisms of this type is the effect of cancer cells on the increased differentiation of T lymphocytes towards Th2 cells The biological role of these cells is the secretion of interleukins that start a humoral response dependent on B lymphocytes Due to the increased differentiation of T lymphocytes in this direction the number of formed Th1 lymphocytes directly destroying cells containing the abnormal genome decreases The tumor microenvironment also interferes with normal dendritic cell activity It inhibits their ability to migrate and to present the antigen to immune response cells Some types of cancer show increased CXCL12 expression [] which attracts immature dendritic cells and prevents their differentiation In addition this cytokine via the CXCR4 receptor causes a reanization of the cytoskeleton [] and increases cell motility [] Tec family tyrosine kinases have also been shown to have the ability to activate CXCR4 which determines the growth survival and migration of tumor cells []FABPs fatty acidbinding proteinsThe fatty acids supplied to the cell are involved in its metabolism Due to their lipid nature they are Figure Carcinogenic effect of peroxisome proliferatoractivated receptors PPARÎ The induction of the process is illustrated by arrows the inhibition by the horizontal line PTEN phosphatase and tensin homolog deleted on chromosome ten PIP3 phosphatidylinositol 345trisphosphate Akt protein kinase B PCNA proliferating cell nuclear antigen TXA2 thromboxane A2 ERK extracellular signalregulated kinases p38MAPK P38 mitogenactivated protein kinases JAK Janusactivated kinases KLF4 Kruppellike factor STAT3 Signal transducer and activator of transcription MMP314 metalloproteinase or PAI Plasminogen activator inhibitor TRL4 tolllike receptor MAPK mitogenactivated protein kinaseOncotargetwwwoncotargetcom 0cunable to enter the aqueous environment of the cellular cytoplasm Solubilizing molecules ie fatty acidbinding proteins FABPs enable their transport to various cellular structures and thus become involved in the processes taking place in the cells including their growth reproduction and inflammatory processes In cells characterized by intensive lipid metabolism such as the liver intestine heart brain etc high transporter expression is observed which is associated with their physiological role in the cell Depending on the type of cancer both the overexpression and a decrease in FABPs can be seen FABP isoforms are not specific to one type of cancer and each plays a different role in the carcinogenesis process Figure In liver cancer LFABP was overexpressed and its high concentration was shown to correlate positively with VEGFA [“] The causes of this phenomenon are sought in the direct interaction between these two proteins which in effect provokes the activation of the SrcFAKcdc42 and the AktmTORP70S6K4EBP1 signaling pathways Activation of the SrcFAKcdc42 pathway increases tumor cell migration and LFABP plays a key role in this process [ ] Moreover by stimulating the second mentioned signaling cascade LFABP enhances VEGFA expression facilitating angiogenesis This process is regulated by HIF1α which additionally induces blood vessel formation [] Due to the high homology in their construction FABP3 and FABP4 isoforms were assigned to the same protein subfamily In addition to their physical features it has been observed that the same factors ie HIF1α VEGF increase their coexpression The physiological role of both proteins is to inhibit excessive cell proliferation and increase apoptosis Excessive FABP3 expression leads to an increase in reactive oxygen species and a decrease in the mitochondrial membrane potential [] resulting in the ing of mitochondrial megachannels which play a key role in initiating apoptosis FABP4 activates the apoptosis process by mediating the response to lipidinduced endoplasmic reticulum stress Due to the biological functions of both proteins their deficiency may be the cause of cancer progression When examining the level of FABP3 expression in embryonic tumor cells breast cancer cells [] and FABP4 in breast ovarian prostate bladder or liver cancer cells low levels of both proteins were observed which was associated with a worse prognosis in terms of overall survival Furthermore high FABP4 levels slowed the development of hepatocellular carcinoma and thus contributed to its reduction in size [] This effect is explained by the inhibition of STAT3 phosphorylation via the RaspSTAT3 signaling pathway and the inhibition of the Snail protein an accelerator of the epithelialmesenchymal transition [] However the increased FABP3 expression in tumors of the stomach brain small and nonsmallcell lung cancer seems paradoxical it has been shown to increase tumor aggressiveness and poorer patient prognosis [ ] Thus far the molecular mechanism responsible for the oncogenic potential of FABP3 and FABP4 is unknown Further research is needed to clarify the reasons these proteins promote the neoplastic process Another subtype of fatty acid binding proteins is FABP5 It is involved in the development of breast prostate liver stomach and colon cancers FABP5 has been shown to transport saturated and unsaturated longchain fatty acids that act as PPARδ ligands [ ] The nature of the supplied fatty acids determines the oncogenic activity if unsaturated fatty acid or a suppressor action is supplied to PPARδ in the case of saturated ligands [] When the Figure Carcinogenic effect of Bruton™s tyrosine kinase Btk The induction of the process is illustrated by arrows the inhibiton by the horizontal line Btk Bruton™s tyrosine kinase CXCR2 CXC chemokine receptor type CÎ2 phospholipase CÎ2Oncotargetwwwoncotargetcom 0cFABP5PPARδ pathway is stimulated the transcription of the genes responsible for cell growth and survival increases Moreover one of the target genes stimulated by PPARδ is the FABP5 gene which increases the expression of the transport protein in question [] In prostate cancer cells by supplying long chain fatty acids FABP5 leads to PPARÎ activation [“] Stimulation of these receptors results in increased expression of vascular endothelial growth factor VEGF thereby accelerating the angiogenesis process This mechanism plays a key role in the development of castrationresistant prostate cancer [“] In the case of colon cancer FABP5 contributes to its development by reducing p21 activity Overexpression of FABP5 increases the expression of cMYC which inhibits the action of a cell cycle inhibitor After silencing FABP5 activity a significant reduction in the invasive capacity of colon cancer cells CRC is observed [] The molecular basis of this interaction is not yet known at the moment High expression of monoacylglycerol lipase MAGL responsible for the production of free fatty acids is observed in highly malignant colon cancer cells [ ] This fact combined with the physiological role of FABP5 consisting of transporting free fatty acids to the appropriate cell compartments prompts to seek the answer to the question whether FABP5 and MAGL come into functional interaction with each other and if so how does this affect CRC progression Another FABP isoform that may be involved in colon cancer development is FABP6 [“] It transports bile acids between the epithelial cells of the large intestine which are transformed into secondary metabolites such as deoxycholic acid DCA and lithocholic acid via cellular metabolism A correlation was demonstrated between increased exposure of intestinal epithelial cells to DCA and an increase in reactive oxygen species inside them which is associated with oxidative stress development and DNA damage [“] This mechanism contributes to the formation of mutations that in the event of a replication of defective cells initiate their malignancy Due to their detergent properties bile acids can cause damage to the cell membrane Compensatory mechanisms lead to an intensified inflammatory response and increased proliferation which is qualified as early tumor development Furthermore bile acids activate the muscarinic M3 receptor [“] Its stimulation induces Figure Carcinogenic effect of fatty acid binding proteins FABPs The induction of the process is illustrated by arrows the inhibition by the horizontal lineSTAT3 signal transducer and activator of transcription Src protooncogene tyrosineprotein kinase Src FAK focal adhesion kinase cdc42 cell division control protein Homolog Akt protein kinase B mTOR mammalian target of rapamycin P70S6K p70 ribosomal protein S6 kinase 4EBP1 eukaryotic translation initiation factor 4E eIF4Ebinding protein VEGFA vascular endothelial growth factor1 M3 muscarinic receptor MMPs metalloproteinases PPARÎ peroxisome proliferatoractivated receptors Î PPARδ peroxisome proliferatoractivated receptors or δOncotargetwwwoncotargetcom 0cmetalloproteinase activity and the WntBcatenin signaling pathway which in turn increases the metastatic potential as well as the proliferation and survival of colon cancer cells Desp

Thyroid_Cancer

emergence of promising targeted therapies for the treatment of hepatocellular carcinoma HCCSorafenib has been the mainstay of treatment for a decade and newer modalities were ineffective and did not confer any increasedtherapeutic benefit until the introduction of lenvatinib which was approved based on its noninferiority to sorafenib Thesubsequent success of regorafenib in HCC patients who progress on sorafenib treatment heralded a new era of secondlinetreatment and was quickly followed by ramucirumab cabozantinib and the most ‚uential immune checkpoint inhibitors ICIsOver the same period combination therapies including antiangiogenesis agents with ICIs dual ICIs and targeted agents inconjunction with surgery or other locoregional therapies have been extensively investigated and have shown promise andprovided the basis for exciting clinical trials Work continues to develop additional novel therapeutic agents which could potentiallyaugment the presently available options and understand the underlying mechanisms responsible for drug resistance with the goalof improving the survival of patients with HCCSignal Transduction and Targeted Therapy 101038s4139202000264xINTRODUCTIONPrimary liver cancer remains a major problem for all health caresystems worldwide and is associated with a significant clinicaleconomic and psychological burden Hepatocellular carcinomaHCC accounts for of cases of nonmetastatic tumors of theliver1 During the past decades research has shed light on theepidemiology risk factors and molecular and genetic profiles ofHCCš contributing to the evolution of strategies for preventionsurveillance early diagnosis and treatment23 Liver resectionablation and liver transplantation are potentially curative butrequire diagnosis at a sufficiently early stage Unfortunately asignificant proportion of HCC patients present with intermediateand advanced stage disease often despite diligent surveillanceand curative treatments are frequently not possible4 In thesepatients systemic therapy remains essential and its pivotal roleand potential have stimulated considerable research over the pastdecade In this review we examine recent advances in targetedtherapy and discuss the impact this has had on the managementof HCC We also provide an overview of the most important areasof HCC research including novel clinical trials and technicalplatforms which promise to facilitate substantial progress withinthe next decadeAPPROVED FIRSTLINE AGENTS FOR HCCSorafenibThe success of SHARP and AsiaPacific trial promoted the approvalof sorafenib as firstline targeted therapy for advanced HCC5“ushering in the era of systemic treatment Subsequently virtuallyall trials were centered around sorafenib and it was used as acontrol with which novel firstline agents were compared andevaluated in an attempt to improve the prognosis of patients withHCC Unfortunately despite a number of trials which comparedthese novel agents including sunitinib10 brivanib11 cediranib12linifanib13 dovotinib14 and immunecheckpoint inhibitors ICIs tosorafenib none achieved the predefined primary end points Fig In addition during the decade when these agents were investigated the median overall survival OS of sorafenib monotherapy asfirstline treatment for advanced HCC increased from monthsSHARP to months CheckMate459 further consolidating itsposition Meanwhile the antitumor activity and safety of sorafenibhave been validated in realworld setting Subanalyses of the SHARPand AsiaPacific trials found sorafenib was effective and safeirrespective of disease etiology disease burden ECOG EasternCooperative Oncology Group performance status status etc15“The safety of sorafenib was consistent across ChildPugh A and Bpatients in clinical practice18 and the occurrence of side effects suchas handfoot syndrome and diarrhea were associated with animproved OS19 Baseline hepatic function clinicopathologicalfactors and etiology also affect the prognosis in HCC patientstreated with sorafenib20 In addition sorafenib exerts antitumoreffects with recurrenttransplantationconferring a survival advantage when compared with bestsupportive care BSC21“ Noticeably the application of sorafenibin clinical practice displays significantregional variations andincompliance with guidelines besides its usage as firstline therapyIt is common that initially unresectable HCCs got downstaged aftersorafenib treatment and underwent curativeintent surgery24“ andlocoregional therapies before sorafenib were commonly encountered in realworld settings2930tumors following liver1Department of Liver Surgery and Transplantation Liver Cancer Institute Zhongshan Hospital Fudan University Shanghai China 2Key Laboratory of Carcinogenesis and CancerInvasion Fudan University Ministry of Education Shanghai China 3Shanghai Key Laboratory of an Transplantation Zhongshan Hospital Fudan University Shanghai China4Department of Hepatobiliary and Pancreatic Surgery University Hospitals of Leicester NHS Trust Leicester UK 5Institute of Biomedical Sciences Fudan University ShanghaiChina and 6State Key Laboratory of Genetic Engineering Fudan University Shanghai ChinaCorrespondence Jian Zhou zhoujianzshospitalshcnThese authors contributed equally Ao Huang XinRong YangReceived April Revised July Accepted July The Authors 0cTargeted therapy for hepatocellular carcinomaHuang et alFirstlineSorafenibSharpAsiaPacificCediranibNCT00427973SunitinibNCT0069937BrivanibBRISKFLLinifanibNCT01009593NintedanibNCT01004003DovitinibNCT01232296ATEZOBEVGO30140IMbrave150LenvatinibREFLECTPEMLENKEYNOTE524NivolumabCheckMate459DonofenibChina SecondlineBrivanibBRISKPSEverolimusEVOLVE1AxitinibNCT01210495RamucirumabREACHRegorafenibRESORCENivolumabCheckMate040PEMKEYNOTE224TivantinibMETIVHCCS1SCUBEPEMKEYNOTE240CabozantinibCELESTIALRamucirumabREACH2NIVIPICheckMate040CAMChinaApatinibChinaFig Overview of the targeted agents approved for HCC ATEZO atezolizumab BEV bevacizumab CAM camrelizumab LEN lenvatinib PEMpembrolizumab NIV nivolumab IPI ipilimumabThe clinical benefit of sorafenib however remains modest andthe complex molecular pathogenesis of HCC stimulated theinvestigation of combinations of sorafenib with other moleculartargeting drugs Sorafenib has been combined with antiangiogenic agents MEKERK pathway inhibitors mTOR pathwayinhibitors histone deacetylase inhibitors EGFEGFR pathwayinhibitors and HGFcMet pathway inhibitors31 Other agentssuch as interferon32 selumetinib33 capecitabine34 tegafururacil35 gemcitabine and oxaliplatin GEMOX3637 and gemcitabinealone38 have also been evaluated but to date no treatmentsinvolving combinations containing sorafenib have succeeded inphase III trialsSince sorafenib and TACE are both recommended therapies foradvanced HCC it is reasonable to expect that their combined usewould confer benefits when compared with monotherapy Resultshowever highlighted regional differences and the heterogeneityof the trial protocolsIn TACE the multicenter randomizedplacebocontrolled phase European trial when compared withTACE alone the addition of concurrent sorafenib unlike the SPACEtrial39 did not improve progression free survival PFS40 It was alsoshown that the addition of sorafenib did not confer any survivalbenefit in patients with unresectable HCCs who had alreadyresponded to TACE41 Contrasting with these findings retrospective studies from China have shown that combination therapywith sorafenib and TACE increased OS by more than compared with TACE alone42“ which was supported by thefindings from a number of other groups5455 RecentlytheTACTICS trial a randomized multicenter prospective trial fromJapan reported an improved PFS for TACE plus sorafenibcompared with TACE alone vs months p although this trial used a redefined PFS not conventional PFS buttime until œunTACEable progression The TACTICS trial also usedtime to any cause of death plus OS as primary endpoints resultsnot reported and compared with sorafenib monotherapy TACEplus sorafenib was only superior in controlling tumor progressionand did not prolong OS5758The acceptance of sorafenib as the standard to which othernewer agents and nonsurgical interventions are compared hasresulted in studies comparing its use as monotherapy with TACEplus external beam radiotherapy59 and TACE plus intensitymodulated radiotherapy combined with sorafenib60 In the SARAHstudy selective internal radiotherapy with yttrium90 resin microspheres did not produce any survival benefit compared withsorafenib in locally advanced and inoperable HCC median OS vs p and did not meet the primary endpoint of OS61Similarly the addition of selective internal radiation therapy tosorafenib did not result in a significant improvement in OScompared with sorafenib alone62 Bettinger et al63 however diddemonstrate that stereotactic body external beam radiotherapyemployed as monotherapy SBRT was able to improve OScompared with sorafenib and SBRT with TACE also providedimproved OS and PFS when compared with sorafenib and TACE incombination64 In a recentinfusionchemotherapy HAIC NCT02774187 He et al65 reported thatsorafenib plus HAIC with FOLFOX improved OS compared withsorafenib alone in advanced HCC when portal vein invasion waspresent which was supported by other studies6667 Although theSCOOP2 trial found sequential HAIC with cisplatin and sorafenibdid not improve the survival benefit compared with sorafenibalone this is likely to have resulted from the study beingunderpowered for the primary and secondary endpoints68trial of hepatic arterialDue to the high recurrence rates following hepatectomy fortherapy has been extensivelyHCC approaches to adjuvantinvestigated although previous attemptsincluding the use ofantiviral agents have been largely unsuccessful Based on thepalliative use and success of sorafenib its potential in the adjuvantsetting was investigated and improved survivals following surgeryanticipated Unfortunately this has not been demonstrated and itfailed to reduce postoperative tumor recurrence in the STORMtrial69 and other western studies70 Explanations for the negativeoutcome in the STORM trial include highdose modification ratesshort treatment durations and the enrollment of patients whowere not at high risk of tumor recurrence with no evidenceof tumor satellites with one lesion and with nomicroscopic vascular invasion71 Consistent with this viewpointWang et al72 reported no case of recurrence during the sorafenibdosing period whereas patients suffered recurrence of theirtumor within months of discontinuation of sorafenib72 andpersistent sorafenib intake following postoperative recurrenceimproved OS73 Considering that patients who respond tosorafenib may belong to limited clinical or biological subsetsthe effectiveness of sorafenib in an unselected population cohortsupports its use in the adjuvant setting A number of studies fromthe Far East including China Japan and Korea include patientswith HCCs who are treated with hepatectomy despite their tumorsbeing outside Barcelona Clinic Liver Cancer Classification BCLCguidelines and although the results are difficult to compare dueto heterogeneity ofthe protocols the results are positiveSorafenib significantly reduces tumor recurrence in BCLC stage Cpatients7475 and increases diseasefree survivalDFS76 andSignal Transduction and Targeted Therapy 0cZhuang et al77 demonstrated that adjuvant therapy increaseddisease free survival DFS and OS Sorafenib treatment followinghepatectomy significantly prolonged the OS of advanced HCCthan intermediate HCC78 In addition to BCLC stageratherpatients who underwent hepatectomy and were pathologicallydiagnosed with microvascular invasion MVI also benefited fromadjuvant sorafenib treatment79 In line with these results a largerecent study with propensity score matching analysis alsodemonstrated that sorafenib significantly improved overall andrecurrencefree survival following resection80 The results fromthese studies which include all eligible patients suggest that moreprecise stratification would enable the identification of thosepatients who will benefit most from the use of adjuvant sorafeniband those in where additional treatment is not appropriateOngoing trials are attempting to evaluate the role of sorafenib inpatients with MVI following radical resection NCT02867280 andNCT02537158LenvatinibFollowing the approval of sorafenib for use in the treatment ofHCC it takes more than a decade before the second firstlinetargeted agent for HCC emerged Lenvatinib was approved for thefirstline therapy in advanced HCC following the results of theREFLECT trial a randomized phase III noninferiority trial publishedby Kudo et al81 Although not approved for long further multicenter data from œrealworld conditions confirmed the efficacy oflenvatinib regardless of previous tyrosine kinase inhibitor TKItherapies8283 and lenvatinib monotherapy demonstrated antitumor activity for more than years in unresectable HCC whenportal vein invasion was present84 In intermediatestage HCCpatients with tumors exceeding the uptoseven criteria for whomTACE is not helpful lenvatinib could provide significant longer OS vs months and PFS vs months85 Lenvatinibpharmacokinetics in HCC is affected by body weight8687 and asufficient dose relative dose intensity RDI is required to achieve agood therapeutic effect and consequently improved outcomesand prognosis are associated with the preservation ofliverfunction which reduces the number of patients who need todiscontinue their treatment88“ With lenvatinib unlike other TKIsthere are issues with thyroid toxicity and surveillance for thyroidabnormalities during treatment is important92 Hypothyroidism isnot unusual and there are also fewer common reports ofthyrotoxicosis and destructive thyroiditis93 From a healtheconomics standpoint however lenvatinib is more cost effectivethan sorafenib9495Secondline targeted agents for HCCStill sorafenib displays limited antitumor activity and someinitially sorafenibsensitive would eventually succumb to thedisease indicating the acquired resistance to sorafenib reducesits beneficial effects and an urgent need for secondline therapyRegorafenibInitial attempts to discover effective secondline agents wereunsuccessful and mirrored attempts to develop firstline agentswhich were superior to sorafenib96 The RESORCE trial was arandomized double blind placebocontrolled and phase III trialdemonstrating the effectiveness of regorafenib in patients whohad progressed on sorafenib treatment Thisstudy finallyconfirmed the potential of secondline agents and ushered inthe era of secondline and sequential therapy97 Regorafenibprovided survival benefitthe rate of diseaseprogression during prior sorafenib treatment or since the lastsorafenib dose98 This was confirmed by Yoo et al99 in aretrospective study of safety and efficacy in Korean patientswhere data were consistent with those from the RESORCE trialRegorafenib was even shown to be effective in patients with HCCrecurrence following liver transplantation with a median OS ofregardless ofSignal Transduction and Targeted Therapy Targeted therapy for hepatocellular carcinomaHuang et al months following regorafenib initiation and monthsfollowing sorafenib initiation CI “ for the sorafenibfollowed by regorafenib sequential therapy100However not all patients who progress on sorafenib arecandidates for secondline therapy101 In clinical practice only of patients are eligible forsecondline regorafenibtreatment102 Good liver functional reserve and ECOG performance status during sorafenib treatment contributed to theefficacy and better outcomes of subsequent treatment103104including lenvatinib105 This may in part be due to the RDIrequired to achieve a clinically significantinprognosis106 This is supported by the demonstration that thenew liver reserve function biomarker albuminbilirubin gradeALBI107 successfully identified regorafenib candidates and thatin the selected cohort a median OS of months was achievedcompared with months for noncandidates108 Even in patientsnot eligible for regorafenib the ones with an ECOGPS score of the absence of MVI and TTP time to progression ‰¥ monthscould still have acceptable postprogression survival109 Longtermtreatment with regorafenib has also been shown to reduceangiogenesis and improve portal hypertension PHT and acuteadministration ameliorates portal haemodynamics suggestingthat it may be especially suitable for patients with PHT andpreserved liver function110improvementCabozantinibCabozantinib is another small molecule inhibitor of the tyrosinekinases which are implicated in the progression of HCC and theacquired resistance to sorafenib Cabozantinib blocks the receptors involved in oncogenesis and angiogenesis including VEGFR hepatocyte growth factor receptor MET AXL and theangiopoietin receptors TIE2 RET cKit and FLT3 in vitro andin vivoIn CELESTIAL trial cabozantinib achieved the primaryendpoint with median OS of months compared with months for the placebo group111 and was consequentlyapproved in the EU and USA There remains a paucity of datahowever from realworld clinical practice examining the sequentialtreatment utilizing cabozantinib as the secondline agent it is acostly option associated with frequent highgrade adverse eventsConsequently several studies have addressed the costeffectivenessof cabozantinib using the cost and utility data extracted from theCELESTIAL trial The conclusion from these studies is consistent andconfirms that at its current cost point the gain of qualityadjustedlifeyears for cabozantinib QALYs “ and the incrementalcosteffectiveness ratio ICER “ mean that it isnot a costeffective treatment option for patients with sorafenibrefractory HCC112“ compared with regorafenib QALY “and ICER “RamucirumabRamucirumab is a fully human recombinant IgG1 monoclonalantibody targeting the VEGF2 receptor Although ramucirumabfailed to meet its primary endpoint as secondline treatment in theREACH trial117 subgroup analysis found survival benefit in patientswith AFP of ngml or higher118“ This was later confirmed inthe REACH2 trial122 which led to the approval of ramucirumab assecondline treatment for advanced HCC REACH2 is the firstpositive phase trialin patients with HCC performed in abiomarkerselected patient cohort and more recent findingsdemonstrated that AFPenriched HCCs displayed significantactivation of VEGF which suggests the underlying mechanism ofaction and confirms the potential value of biomarkerdrivenclinical trials123Immune checkpoint therapy and TKI inhibitorsICIs stand as the mainstream of immunotherapy The CheckMate and KEYNOTE224125 studies evaluated the safety andefficacy of nivolumab and pembrolizumab in patients with 0cTargeted therapy for hepatocellular carcinomaHuang et alphaseIIrandomizedparallelgrouptislelizumab sintilimabrealworld cohort studyadvanced HCC refractory to previous sorafenib treatment whichestablished the basis for accelerated approval by the FDA assecondline treatment Subanalysis of CheckMate040 data validated the safety and efficacy of nivolumab in Asian cohort126 Inan internationalICIs have showedpromising efficacy and safety in advanced HCCs as systemicfirstsecondthirdfourthline treatment with median OS andPFS of and months respectively127 and an excellentresponse to antiPD1 therapy has also been described in casereport128 Although the subsequent phase III KEYNOTE240 trialdid not meet its prespecified statistical significance in respect ofimproved PFS and OS the results were consistent with previousKEYNOTE224129 The KEYNOTE394 presently underway in Asianpatients may clarify the role of pembrolizumab in cases ofadvanced HCC with a viral background NCT03062358 RecentlyCheckMate459 the multicenter phase III randomized sorafenibcontrolled trial evaluating nivolumab as firstline treatment foradvanced HCC failed to achieve its endpoints ESMO butnivolumab did prolong OS vs months and achievelongtime disease control less adverse events AEs and survivalbenefit regardless of the level of PDL1 expression Furthermorenivolumab improved the survival of HCC patients whose etiologywas HBVHCV and did not reactivate hepatitis CamrelizumabSHR1210 Hengrui Pharmaceutical is an antiPD1 inhibitor fromChina investigated for the treatment of Hodgkin lymphoma andHCC It has been shown to have antitumor activity in previouslytreated Chinese patients with advanced HCC in a multicenteropenlabeltrialNCT02989922130 providing evidence for the effectiveness ofPD1 therapy for HBV related HCC in Chinese patients The resultsICIs including durvalumabfrom other trials investigating novelavelumabtremelimumabipilimumabspartalizumab and toripalimab will hopefully yield positive resultsand provide further options for the treatment of patients withHCC particularly those who have relapsed on firstline treatmentsFurther efforts to enhance the treatment effect of ICIs includedual ICIs treatment and combination therapy of ICIs with otherkinds of targeted agents For dual ICIs treatment the initial resultsfrom CheckMate 9DW were astonishing the objective responserate was higher than monotherapy of any ICIs alone FDA hasapproved nivolumab in combination with ipilimumab for patientswith HCC previously treated with sorafenib Treatment modalitiessuch as radiotherapy and antiangiogenesis agents which affectantigen release or modulate the tumor microenvironments havethe potential to increase the efficacy of immunotherapy and thecombination oftargeted agents with ICIs are attracting theattention of a number of research groups and in vitro studies andearlyphase clinical trials assessing combination treatments haveshown promising antitumor effects in patients with advancedIn vitro evidence by Qui et al131 demonstrated thatHCClenvatinib and regorafenib could affect the expression of PDL1and realtime PCR results suggested that the mRNA expression ofPDL1 in the lenvatinib group was significantly higher than that inthe control group while its expression in the regorafenib groupwas significantly lower When combined with antiPD1 lenvatinibcan modulate cancer immunity in the tumor microenvironmentand enhance antitumor activity132133 In July the FDAannounced its approval of the first combination therapy employing the TKI lenvatinib with the ICI pembrolizumab based on theresults from the KEYNOTE524Study NCT03006926 for thetreatment of HCC Recently results from Study Phase IbNCT03418922 showed marginally better results for lenvatinibwith nivolumab than lenvatinib with pembrolizumab METmediated phosphorylation leads to a decreased expression ofPDL1 using the combination of MET inhibitors tivantinib andcapmatinib antiPD1 and antiPDL1 produced an additive effectwhich slows the growth of HCCs in mice134 Clinically based onthe results from the experimental arm A of the GO studyNCT02715531 the FDA approved atezolizumab plus bevacizumab as breakthrough therapy for untreated advanced ormetastatic HCC135 Individual case studies also reported promisingresults for the use of combined TKI and antiPD1PDL1 agents foradvanced HCC136“ Such results were confirmed in the phase IIItrialIMbrave study NCT03434379 which reported thatatezolizumab combined with bevacizumab resulted in better OSand PFS than sorafenib in patients with unresectable HCC139Other combination therapies include Galunisertib with nivolumabNCT02423343 spartalizumab with and without capmatinibNCT02795429 FGF401 with spartalizumab NCT02325739regorafenib with pembrolizumab NCT03347292 cabozantinibwithaxitinibNCT03289533 ramucirumab with durvalumab NCT02572687and XL888 with pembrolizumab NCT03095781 Table avelumab withNCT03299946nivolumabImmunerelated adverse events IRAEs occur frequently duringtreatment with ICIs and the clinical consequences can besignificant140 Activation of the immune system leads to damageof normal healthy tissues and IRAEs can have myriad effects andinvolve a number of different ans and have been reported toproduce colitis hepatitis pneumonitis dermatitis myocarditisendocrine glands ‚ammation and rheumatic and musculoskeletal phenotypesincluding ‚ammatory arthritis arthralgiamyositis and sicca syndrome141 Although the precise pathophysiology underlying the IRAEs side effects during treatment withICIs remains unknown discontinuing administration and the useof steroids is generally effectiveIn severe cases howeveradditional immunosuppressants may be required but based oncurrent available evidence immunosuppression for IRAEs does notappearresponse to the ICItreatment142143to compromise the antitumorPromising agents and treatment regimensDespite abovementioned targeted drugs novel agents have beencontinuously under development Table Of note apatinib anovel inhibitor of VEGFR2 tyrosine kinase has attracted considerable attention and there is now a significant body of workdescribing clinical experience with its use Although less effectivethan sorafenib as a firstline treatment in a retrospective study144apatinib still displayed promising antitumor effects in sorafenibresistant HCC145“ where portal vein invasion was present148when metastases have occured149150 and for unresectable andrelapsed HCCs151152 Combination therapy in studies utilisingapatinib with TACE have achieved better clinical effectivenessthan TACE alone with tolerable AEs153“ Recentlythecombination of apatinib with the antiPD1 monoclonal antibodycamrelizumab achieved partial response rates of Theresults of other ongoing trials including the phase IIItrialcomparing TACE and apatinib with sorafenib as firstline treatmentfor locally advanced or metastatic and unresectable HCC NCT and the adjuvant apatinib after hepatectomy for theprevention of tumor recurrence NCT03722875 and NCT03261791will hopefully prove effective and add to the presently availabletherapeutic optionsThese promising results have stimulated the investigation ofother new agents the combinations of agents and regimenswhich have been thoroughly discussed in a recent review fromZhu and Sun154 The combination of bevacizumab and erlotinibhas been extensively evaluated as first155 or secondline inadvanced HCCs156“ but unfortunately the heterogeneousnature of the results precludes firm conclusions and recommendations Recently a singlearm metaanalysis of prospectivestudies found that combination therapy with bevacizumab anderlotinib used as secondline treatment was associated with afavorable PFS weeks P and OS months P suggesting that future welldesigned and sufficiently poweredlargescale RCTsshould be able to identify the potentialcontribution of these agents163Signal Transduction and Targeted Therapy 0cTable Trials investigating the combination therapy of ICIs and TKIs in HCCTrial nameidentifierPatient No Study type LineInterventionsPrimaryendpointStudy statusTargeted therapy for hepatocellular carcinomaHuang et alLEAP002NCT03713593Phase IIIFirstLenvatinib pembrolizumab vs lenvatinib PFSOSPhase IIIFirstPhase IIIPhase IIIPhase IIIFirstFirstFirstPhase IIIFirstFirstPhase IIFirstPhase IIPhase Ib II FirstFirstPhase IIPhase IbFirstNivolumab ipilimumab vs lenvatinib orsorafenibCabozantinib atezolizumab vs sorafenib PFSOSSintilimab IBI305 vs sorafenibOS ORRCamrelizumab apatinib vs sorafenibOSPFSOSCamrelizumab apatinib vs FOLFOX orsorafenibNivolumab lenvatinibNivolumab sorafenibSorafenib pembrolizumabAnlotinib sintilimabAvelumab axitinibOSORR AEMTD ORRORRORR AEAEActive notrecruitingOngoingOngoingOngoingOngoingOngoingOngoingOngoingOngoingOngoingCompletedCheckMate 9DWNCT04039607COSMIC312NCT03755791ORIENT32NCT03794440SHR1210III310NCT03764293SHR1210III305NCT03605706IMMUNIBNCT03841201NCT03439891NCT03211416KEEPG 04NCT04052152VEGF Liver NCT03289533KN743NCT03347292GOINGNCT04170556REGOMUNENCT03475953NCT02423343aaRegorafenib pembrolizumabFirstPhase ISecond Regorafenib nivolumabPhase IISecond Regorafenib avelumabPhase IIIPhase Ib II Second Galunisertib nivolumabAEAECR PRPhase Ib MTDOngoingOngoingOngoingOngoingPFS progressionfree survival OS overall survival ORR objective response rate AE adverse events MTD maximum tolerated dose CR complete response PRpartial responseaTrials enroll not only HCC patientsTable Trials investigating targeted therapy in advanced HCCTrial nameidentifierPatient noStudy typeLineInterventionsPrimary endpointStudy statusIMbrave150 NCT03434379ZGDH3NCT02645981HIMALYYA NCT03298451RATIONALE301 NCT03412773PHOCUSNCT04344158ALTER0802 NCT02809534AHELPNCT02329860KEYNOTE394 NCT03062358NCT04080154Phase IIIPhase IIIIIPhase IIIPhase IIIPhase IIIPhase IIIPhase IIPhase IIIPhase IIIPhase IIFirstFirstFirstFirstFirstFirstFirstSecond Apatinib vs placeboSecond Pembrolizumab BSC vs placebo BSCSecond AnlotinibAtezolizumab bevacizumab vs Sorafenib OS PFSDonafenib vs sorafenibOSDurvalumab tremelimumab vs sorafenib OSOSTislelizumab vs sorafenibPexaVec sorafenib vs sorafenibOSAK105 anlotinib vs sorafenibOSPFS 12WAnlotinibOSOSPFSOS overall survival PFS progressionfree survival BSC best supportive careCompletedCompletedOngoingOngoingOngoingOngoingOngoingCompletedOngoingOngoingundergoingevaluationandPreclinical evidence for cyclindependent kinase CDK targetingtherapies in HCC has showed promise and supports theirinvestigation164“ especially with the potential ability toabrogate the emergence of sorafenib resistance167 and sensitizeHCC to regorafenib treatment168 A number of CKD inhibitors arepalbociclibpresentlyNCT01356628 milciclibribociclibNCT02524119 The antiMET monoclonal antibody emibetuzumab exhibited the greatest antitumor activity in HCC whencombined with ramucirumab and had an excellentsafetyprofile169 and for HCC with high MET expression there was analmost 3fold increase in PFS vs months relative to thosewith low MET expression suggesting the potential for furtherbiomarkerdriven clinical trials Rigosertib is a synthetic benzylstyryl sulfone small molecule inhibitor which has been used in theNCT03109886includingtreatment of monomyelocytic leukemia and due to its activity as aRAS and PLK1signaling inhibitorit was investigated in HCCpatients who demonstrate upregulation of PLK1 during tumordevelopment and HRAS expression in advanced HCC Highexpression levels of PLK1 are also significantly correlated withpoor patient survival and the multiple effects of rigosertib couldbe beneficially employed to produce a therapeutic œdualhitapproach in selected patients170 Donafenib is a novel multikinaseinhibitor which is similar to sorafenib displaying comparable orbetter safety and efficacy when treating advanced HCC in phase1b trial and phase studies using sorafenib as the controlNCT02645981171 There are ongoing trials evaluating novelagents such as anlotinib another multikinase inhibitor which isorally administered and targets VEGFR fibroblast growth factorreceptor FGFR plateletderived growth factor receptors PDGFRSignal Transduction and Targeted Therapy 0cTargeted therapy for hepatocellular carcinomaHuang et alTable Molecular classification of HCCResearcherBoyault et alHoshida et alSchulze et alSia et alKurebayashi et alShinata et alJiang et alYearClassificationG1“G6S1“S3Msig “iC1“iC3 HCCs with adaptive or exhausted immune responsesImmunehigh mid and “lowMS1 ˆ’ ˆ’SI SII and SIIITypeTranscriptomeTranscriptomeExome sequencingMultiomocisImmune cell profilingImmunomicroenvironmentTranscriptome and gonomeProteomicsCase no and ckit NCT02809534 Tivozanib is another oralinhibitor ofVEGFR123 with promising activity against HCC in vivoNCT01835223 and TRC105 which despite demonstrating clinicaltolerated in HCC patients followingactivity and being wellsorafenib has notto date met prespecified criteria and itsdevelopment in HCC continues as combination therapy withsorafenib NCT02560779Biomarkerdriven targeted therapyDespite extensive research investigating potential biomarkers to aidthe development of protocols for the treatment of HCC none haveso far been identified to be able to predict the effect of or responseto treatment with sorafenib172“ Although the molecularclassification of HCC has been widely reported Table to date itremains unclear whether this basic genomic and proteomic datawill prove valuable in guiding targeted therapies183“The continued belief that the future lies with personalizedtreatment which will be made possible through the rapiddevelopments in next generation sequencing and the precisionmedicine that it underpins have encouraged the development ofnovel trial designs191 These novel trials designs offer new hopethat biomarkerdriven targeted therapies can be modul

Thyroid_Cancer

Pathway‘specific model estimation for improved pathway annotation by network crosstalkMiguel Castresana‘Aguirre Erik L L SonnhammerPathway enrichment analysis is the most common approach for understanding which biological processes are affected by altered gene activities under specific conditions However it has been challenging to find a method that efficiently avoids false positives while keeping a high sensitivity We here present a new network‘based method ANUBIX based on sampling random gene sets against intact pathway Benchmarking shows that ANUBIX is considerably more accurate than previous network crosstalk based methods which have the drawback of modelling pathways as random gene sets We demonstrate that ANUBIX does not have a bias for finding certain pathways which previous methods do and show that ANUBIX finds biologically relevant pathways that are missed by other methodsImprovements in molecular biology have led to an increase in highthroughput data which typically produces lists of differentially expressed genes or proteins These lists are useful for identifying genes with important roles in certain conditions However more insight about the biological mechanisms is often needed eg which functional gene sets are related to genes in the result list The study of the relation between a query gene set differentially expressed gene list and functional gene sets pathways is called pathway enrichment analysisImprovements in molecular biology have led to an increase in highthroughput data which typically produces lists of differentially expressed genes or proteins These lists are useful for identifying genes with important roles in certain conditions However more insight about the biological mechanisms is often needed eg which functional gene sets are related to genes in the result list The study of the relation between a query gene set differentially expressed gene list and functional gene sets pathways is called pathway enrichment analysisThere are four generations of pathway enrichment analysis approaches Overrepresentation analysis ORA calculates how many genes from a list of genes extracted based on a threshold or criteria eg differentially expressed genes are in a certain pathway1 Statistical significance is assessed repeating this process with a background list of genes eg all the genes in the microarray This is known as Gene Enrichment Analysis GEA and famous tools like DAVID2 use it Similar but taking into account all the genes in the experiment and the gene expression values is the Functional Class Scoring algorithms FCS3 for which known algorithms include Gene Set AnalysisGSA4 and Gene Set Enrichment Analysis GSEA5 However both FCS and ORA have limitations They both consider genes as independent which is often not true only taking into account their overlap and not their associations or interactions6 Another issue with overlapbased methods is their low coverage since they are heavily dependent on pathway knowledge which is still incomplete leading to a high rate of false negatives7 Pathway topologybased methods use the same steps as FCS with additional pathway topology information However the reliance on gene overlap leads to similar limitations as ORA and FCSWe could consider the network crosstalk enrichment tools as the fourth generation They rely on a network such as a functional association network like Funcoup8 or STRING9 These networks integrate different experiments from different data types into a single network providing information about gene to gene functional associations which is translated into links in the network With this limitations such as gene independency and low coverage of overlapbased methods are overcome Association between two sets is measured in terms of links between them in the network known as crosstalk In the past few years different ways to assess enrichment between two gene sets have been published like NEA10 EnrichNet11 CrosstalkZ12 NEAT13 NEArender14 BinoX7 and GeneSetDPGeneSetMC15 EnrichNet defines a network enrichment score based on network distances between two gene sets using random walks with restart but is not able to calculate statistical significance Department of Biochemistry and Biophysics Science for Life Laboratory Stockholm University Box Solna Sweden email eriksonnhammerdbbsuseScientific RepoRtS 101038s4159802070239zVol0123456789wwwnaturecomscientificreports 0cof the enrichment The tools NEA and CrosstalkZ assess significance using statistical tests assuming that crosstalk between nonenriched gene sets is normally distributed but this is often not the case Moreover they rely on network randomizations to obtain null model parameters which makes them computationally very slow Computational time is reduced in BinoX which also applies network randomization but uses the binomial distribution to calculate statistical significanceThe methods NEAT NEArender and GeneSetDPGeneSetMC do not use network randomization NEAT calculates the expected number of links between two gene sets based on their degrees and then uses the hypergeometric distribution to assess statistical significance NEArender computes the expected number of links in the same way as NEAT but uses a chisquare test to assess statistical significance GeneSetDP uses dynamic programming to calculate an exact distribution of the expected number of links to a pathway for a certain gene set size GeneSetMC does this approximately using MonteCarlo sampling which is faster These two algorithms are however not implemented to allow large scale pathway enrichment analysisThe null model assumption of NEAT NEArender and BinoX is that compared gene sets are expected to behave like random gene sets For real pathways that are very nonrandom eg highly intraconnected this can lead to underestimating the expected level of crosstalk and produce a high false positive rate FPR To avoid this it is important that the method can cope with the nonrandomness of pathways To this end we have developed a novel networkbased pathway enrichment analysis algorithm called ANUBIX Adaptive NUll distriButIon of Xtalk which is based on scoring random gene sets against real pathways to build its null model We show that ANUBIX clearly outperforms recent network crosstalk methods like BinoX NEArender and NEAT in terms of avoiding False Positives FP showing that it can model expected network crosstalk to pathways more preciselyMaterial and methodsOur networkbased pathway enrichment analysis tool ANUBIX depends on a global functional association network We used the network Funcoup version with a link confidence cutoff of containing genes and links With those genes cid31g1 g2 gnˆ’ gncid30 ˆˆ S and all the pairwise links between them form a symmetric matrix A with dimensions SxS such thataij 1if gi is connected to gj and i � j otherwise aij A gene set Q and a pathway P are a subset of the total number of genes for a certain proteome such that Q P Š† S Notice that S Š† Q we can have some genes from the proteome that are not in the network The crosstalk between Q and P is measured with the degree k cid31iˆˆQcid31jˆˆPaijThe null model is built based on the expected crosstalk between a random gene set of the same size as the original gene set Q and pathway P Since the network connections are binary each link is considered as a Bernoulli trial Y ˆ¼ Bcid31pcid30 where p is the probability of observing a link We also calculate n QP ˆ’ Q ˆ P all the possible links between Q and P We count the links each gene from Q has to the pathway P meaning that if two linked genes are in Q and also in the P we count that link twice boosting the cases where we find overlap Each of these Bernoulli trials are assumed to be independent and the sum of them follows a binomial distributionIn the binomial distribution the mean and variance are defined as µ np and Var npcid311 ˆ’ pcid30 respectively This means that µ ‰¥ Var which may not be true when the random variable is overdispersed leading to an underestimation of its variance16The betabinomial distribution has been extensively used as an alternative to handle overdispersed binomiallike random variables1718 Here the probability of success p is not fixed as it is in the binomial distribution but follows a beta distribution Betaα β with parameters α and β The marginal distribution of the betabinomial is described in Eq a0fcid31kn α βcid30 cid29 nk cid28 Bk α n ˆ’ k βBα βTo estimate the optimal parameters of the betabinomial we use maximum likelihood estimation MLE19 where the loglikelihood is Eq a0lcid31kn α βcid30 logLcid31kn α βcid30 logcid29 n logcid29 nk cid28 logBα k β n ˆ’ k ˆ’ logBα βk cid28 logŴα k logŴβ n ˆ’ kˆ’logŴα β n ˆ’ logŴα ˆ’ logŴβ logŴα βThe negative loglikelihood is optimized with the Nelder and Mead method20 The factorial term in the loglikelihood is removed since it does not depend on the parameters to be optimized Once we have the betabinomial parameters α β of our null distribution we calculate if the crosstalk between Q and P is enriched The null and alternative hypotheses areH0 No more links between Q and P than expected by chanceH1 More links between Q and P than expected by chanceBecause of the discrete nature of the null distributions ordinary pvalues are conservative and therefore mid pvalues were used2122 Mid pvalue is defined as half the probability of the observed statistic plus the probability of observing more extreme values22 The workflow of the ANUBIX algorithm is depicted in Fig a0Scientific RepoRtS 101038s4159802070239zVol1234567890wwwnaturecomscientificreports 0cFigure a0 Workflow of ANUBIX The algorithm assesses the significance of the network crosstalk between a query gene set and a pathway A null distribution is generated for each pathway to model the expected crosstalk of random gene sets of the same size as the original gene set This distribution is then fit to a betabinomial distribution to calculate the probability of reaching at least the number of observed links or more between the query gene set and the pathway Software Inkscape version inksc apeIt is important to point out that the networkbased approaches ANUBIX NEAT NEArender and BinoX test three different types of null hypothesis ANUBIX which takes only enrichment into account computes a one tailed test NEAT computes two onetailed tests for enrichment and depletion and takes the minimum pvalue of them multiplied by to emulate a twotailed test BinoX and NEArender compute both enrichment and depletion but only perform one onetailed test since the hypothesis test changes depending on whether the observed number of links is above or below the expected crosstalkPathways To generate the false positive and true positive benchmarks we used KEGG v70123 pathways and REACTOME v6224 pathways for Homo sapiens REACTOME pathways have a deep hierarchical structure including many small pathways on the lower levels that are very specific To reduce Reactome™s specificity we resolved its hierarchy by collapsing lower level pathways below a certain pathway size to their parents until obtaining an average pathway size similar to KEGG pathways genes per pathwayPerformance measures In the FP benchmark we generated random gene sets and tested them against KEGG and REACTOME pathways To make these gene sets representative of real experiments we took the average size of MSigDB25 gene sets which is genesIn the True Positive TP benchmark we bisected the KEGG pathways and REACTOME pathways into two parts Each part gets a similar number of genes and links7 To be able to benchmark GEA we emulated some overlap between the two bisected parts This overlap corresponded to the average overlap between the MSigDB gene sets and the pathway measured individually for each of the pathways in KEGG and REACTOMECorrection for multiple hypothesis testing was done using the Benjamini“Hochberg procedure26Stability Our null distributions are based on random sampling We take random samples of genes from the genome This stochastic procedure makes the null distributions different every time they are generated Since the pvalues are computed from the null distribution their values may change To analyze stability we generated the null distribution times for the crosstalk between the same gene set to the same pathways for increasing numbers of random samples For each sample size we computed the coefficient of variation CV which is the ratio between the standard deviation SD and the mean We required a CV lower than to limit the dispersion of the mean of the null distribution and this was reached at random samples Once the number of random samples were chosen we measured how much the pvalues were varying in each run For that we ran a randomly selected MSigDB gene set times To compute the confidence interval of the pvalues we used the central limit theorem and applied normal distribution statistics to compute themUsed programs ANUBIX bitbu cketsonnh ammer group anubi xBinoX bitbu cketsonnh ammer group binox NEAT cranrproje ctwebpacka gesneatneatpdfNEArender cranrproje ctwebpacka gesNEAre nderNEAre nderpdfGeneSetDP githu bcomstati stica lbiot echno logygenes etdpScientific RepoRtS 101038s4159802070239zVol0123456789wwwnaturecomscientificreports 0cFigure a0 Overdispersion of KEGG and REACTOME pathways null distributions when sampling random gene sets of size from the proteome The dispersion for each pathway is calculated as the ratio between the variance and the mean of the crosstalk null distribution For each pathway database we illustrate the dispersion values through a boxplot and also by showing the dispersion distribution Software R version wwwrproje ctFigure a0 Observed crosstalk distribution fit with binomial and betabinomial distributions random gene sets of size were used to generate a null distribution of crosstalk to the A œBetaalanine metabolism B œProstate cancer and C œAlzheimer™s disease pathways Betabinomial shows a much better fit to the observed link distribution than the binomial Software R version wwwrproje ctResultsTo correctly assess the statistical significance of an observed network crosstalk between two gene sets eg one experimental gene set and one known pathway it is paramount that the null distribution appropriately models the crosstalk of random query gene sets Note that it is not necessarily appropriate to assume that the pathway gene set behaves like a random gene set ie the null distributions need to model crosstalk between random query gene sets versus real pathway gene sets It is also paramount to model the expected crosstalk distribution with an appropriate distribution Previous methods such as BinoX or NEAT use binomial and hypergeometric distributions respectively which are not appropriate for overdispersed distributions since they do not allow the variance of the distribution to be greater than the mean To showcase this we generated null distributions for KEGG and REACTOME pathways by sampling gene sets of size from the proteome In Fig a0 we show the dispersion for each pathway as the ratio between the variance and the mean of the crosstalk null distribution We observe that almost all of these distributions suffer from overdispersion meaning that the variance of the distribution is greater than the mean Therefore statistical models that cannot cope with overdispersion are not appropriate to model the null distribution of most pathwaysTo visualize the overdispersion in detail we chose pathways that are in different quartiles of the dispersion distribution We show their null distributions in Fig a0 Figure a03A shows the œBetaalanine metabolism Scientific RepoRtS 101038s4159802070239zVol1234567890wwwnaturecomscientificreports 0cFigure a0 Pvalue uniformity test of ANUBIX Binox GEA GeneSetDP NEArender and NEAT random gene sets of genes were tested for crosstalk enrichment against the KEGG pathway œProstate cancer A Reported pvalues are plotted against theoretical quantile rank A perfect method should adhere to the diagonal B Distributions of the FPR for all KEGG and REACTOME pathways tested with ANUBIX BinoX NEArender NEAT and GEA Green distribution for enriched tests and red distribution for depleted The dashed line at FPR denotes the expected FPR level The black triangle and circle represent the mean FPR for enrichment or depletion respectively Software R version wwwrproje ctpathway whose dispersion value is in the first quartile Figure a03B shows the œProstate cancer pathway with a dispersion in the second quartile and Fig a03C shows the œAlzheimer™s disease pathway with a dispersion in the fourth quartile The high variance relative to the mean gives a very poor fit with the binomial distribution yet the betabinomial distribution gives a very good fit This underestimation of variance by the binomial distribution would lead to many false positives With a few pathways there is no overdispersion in the data but these can fit a betabinomial equally well as a binomialBenchmark for false positives Since the null model in ANUBIX is based on random gene sets we expect the pvalue distributions when tested with random query gene sets to behave uniformly for any pathway For almost all pathways we observed a virtually perfectly uniform distribution when plotting ANUBIX pvalues of random gene sets against each KEGG pathway full results at Supplementary Fig a0 A few pathways deviated somewhat from uniform which is the result of the betabinomial fit not being able to model the null distribution with enough precision A second type of deviation from perfect uniform distribution is caused by staggering of observed pvalues This is relatively frequent and arises because the support of the test statistics is limited to a few values and therefore unavoidable We also generated the pvalue distributions for gene sets of size and size against each KEGG pathway Supplementary Fig a0 and respectively which gave similar results However some pathways seem to be affected by the size of the gene set ANUBIX was compared to the top networkbased methods BinoX NEAT and NEArender and a recently published method GeneSetDP For comparison we also tested a popular overlapbased pathway enrichment method GEA Because GeneSetDP and GenesetMC are too computationally heavy for large scale analysis we first tested all the gene sets against one individual pathway We only used GeneSetDP because GeneSetMC produces similar pvalues Pvalues were plotted versus quantiles of a uniform distribution For an unbiased method the pvalues would lie on the diagonal y x Figure a04A shows that for the œProstate cancer pathway Pvalues of ANUBIX adhere to the diagonal much better than for BinoX NEAT NEArender and GEA while performing equally well as GenesetDPFor crosstalk to random gene sets we expect of the pvalues to be lower than However for the œProstate cancer pathway BinoX had of its pvalues lower than NEAT and NEArender GEA whose coverage is small7 had of its pvalues below and highly discrete taking on only four possible values for œProstate cancer due to few overlapping genes ANUBIX and GeneSetDP find a correct fraction of the pvalues with and GeneSetDP under respectivelyWe also ran ANUBIX BinoX NEAT NEArender and GEA for the random gene sets against all pathways in the KEGG database and REACTOME database Full results in Supplementary Data and Data respectively GeneSetDP was not included as it is not implemented to run at a large scale NEAT NEArender and BinoX can also give statistical significance when gene sets have fewer links to a pathway than expected by chance known as depletion To make a more consistent benchmark where all methods can be compared equally we only considered enrichment and depleted pathways were treated as nonsignificant The average FPR for all KEGG pathways was with ANUBIX with BinoX with NEAT with NEArender and with GEA For REACTOME almost the same FPR values were obtained ANUBIX BinoX NEAT NEArender and GEA Roughly the same FPR levels came from significant depletions for BinoX NEAT and NEArender However the averaging of the FPR levels for all pathways does not show the real problem of these methods Some pathways could give very nonconservative pvalues while other pathways could give very conservative pvalues To show how each method performs for each of the pathways we plot the distribution of the FPR fraction of pvalues below for each pathway as violin plots in Fig a04B Since GEA Scientific RepoRtS 101038s4159802070239zVol0123456789wwwnaturecomscientificreports 0cand ANUBIX cannot test for depletion they only have the enriched case A perfect method would have all points close to the dashed line at FPR ANUBIX produces FPR values close to this line meaning that the model is robust GEA greatly underestimates FDR and produces almost no false positives but this leads to very poor sensitivity as shown below NEArender NEAT and BinoX produce similar FPR distributions that are very spread out ie the FPR tends to be very different for different pathways For the tests performed per pathway some pathways reach an FPR of for enriched cases and similar for depleted Summing these two can lead to a total FPR above if we take both enriched and depleted cases into account which is very nonconservative The plot also shows that for some pathways these methods are overly conservative giving considerably lower FPR than they should In other words methods like BinoX NEAT and NEArender have a huge variation in the quality of their pvalues depending on the pathway under studyBinoX is implemented in a web server called PathwAX27 where users can submit a query gene set to test for network crosstalk enrichment By analogy we studied false positive rates assuming independence between gene sets where each user submits a single gene set ie multiple testing correction is only performed for number of pathways each query is compared to random gene sets were used against the KEGG database A FDR threshold of was used and enrichment and depletion were grouped separately as shown in Fig a05A The top pathways with highest FPR for BinoX were plotted full results in Supplementary Data all having a highly nonconservative behaviour for BinoX NEAT and NEArender Every time a user submits a random gene set the chance of getting one of these pathways is very high on average if we take both enriched and depleted cases into account In contrast ANUBIX and GEA have less than FPR We observed a very high correlation between perpathway FPR values for BinoX NEAT and NEArender above for each pairwise comparison This indicates that the pathway enrichment analysis results obtained with these methods are highly similar They all had low Pearson correlation to ANUBIX with for BinoX for NEAT and for NEArender The corresponding Spearman correlations were and As for the pathways we noticed that there is a high overlap between some of them For instance the œAlzheimer™s disease and œParkinson™s disease pathways share of their genes The œAlzheimer™s disease the œParkinson™s disease and the œHuntington™s disease pathways have of the genes in common from the union between them Further the œOxidative phosphorylation the œNonalcoholic fatty liver disease the œAlzheimer™s disease the œParkinson™s disease and the œHuntington™s disease have of the genes in common from the union between them Therefore if there is significant crosstalk to one of them crosstalk to the other pathways is very likely The high dependency between some pathways points to opportunities for further improvement of pathway definitions Further exploration was performed in these pathways™ topologies to understand their tendency to generate many FPsWe computed the fraction of intralinks for each pathway as the ratio between the number of internal links and the total number of links We plotted this ratio against the FPR Fig a05B A higher fraction of intralinks means that more links are within the pathway than to the outside suggesting a more isolated pathway The Spearman correlation coefficient between the fraction of intralinks and FPR for BinoX was indicating that the fraction of internal links plays a major role in causing false positives This dependence is also observed with NEAT with a correlation of and with NEArender at However ANUBIX had a correlation of only and GEA This indicates that methods like NEAT NEArender and BinoX cannot deal properly with pathways that are clearly not random and behave more as isolated communitiesAdditionally we calculated the number of maximal cliques each of the KEGG pathways has and we observed a correlation with the FPR for BinoX with a spearman correlation of These maximal cliques were computed using the igraph package in R We considered cliques as all complete subgraphs and a clique is considered maximal if we cannot add more nodes to it This indicates that the higher the number of maximal cliques in a pathway meaning a less random pathway in terms of topology the higher the FPR isBenchmark of true positives Besides a correct FPR it is also important to verify that the power of the method is sufficient for a high true positive rate TPR To this end we devised a benchmark by splitting each KEGG and REACTOME pathway into two parts and then measured each method™s ability to reconnect these parts The splitting into parts included giving an amount of gene overlap between the two parts emulated based on the average overlap between MSigDB gene sets and KEGG and REACTOME pathways We compared the methods by their Receiver Operating Characteristic ROC curves Figure a06A shows only the tests that are statistically significant FDR and only considering enrichment ANUBIX has a TPR of of the enrichment tests as significant without having any FP BinoX has a TPR of with FPR NEArender a TPR of with FPR and NEAT a TPR of with FPR GEA whose coverage is low gives only TPR and no FPs Figure a06B shows the ROC curve for all the enriched tests performed also including insignificant results This shows the coverage of each method ANUBIX recovers of the TP tests without suffering any FPs BinoX NEArender and NEAT have similar curves recovering and of the enriched TP tests respectively GEA can here maximally find of the TP tests since only those tests have some gene overlap This benchmark shows that GEA has very low coverage of what it can potentially find We note that the maximal TPR obtained by GEA corresponds to the amount of significantly enriched crosstalks obtained when running all of MSigDB against KEGG pathways see Pathway annotation of MSigDB gene setsStability and robustness Considering that the null distributions are based on random sampling we studied the number of iterations required to reach a coefficient of variation CV of Figure a07A shows how many pathways pass that threshold depending on different amounts of random samples of the pathways had a CV lower than when using random samples to model the null distribution To verify that this number of random samples is sufficient for every pathway we computed the enrichment of one randomly selected MSigDB Scientific RepoRtS 101038s4159802070239zVol1234567890wwwnaturecomscientificreports 0cFigure a0 Analysis of why certain pathways are very prone to produce false positives random gene sets of genes were tested independently for crosstalk enrichment against the KEGG pathways A The top ten pathways that produce the highest false positive rate FPR with BinoX and the FPR obtained with other methods B Fraction of intralinks for each of the KEGG pathways against FPR The size of the point reflects the total number of links in each pathway Software R version wwwrproje ctgene set to all KEGG pathways times The null distributions are thus generated times for each pathway and we would expect some changes in the pvalues between runs Figure a07B shows the standard deviation of the pvalues We observe that the pvalues almost did not vary showing that random samples are enough Moreover because of sampling the pvalue is not an exact pvalue but a point estimate of it we also provide with the confidence interval of each of the pvalues Supplementary Data Compute time Our method relies on random sampling to model the null distribution which makes ANUBIX computationally intensive To benchmark its speed we did runs each time with a randomly chosen biological gene set extracted from MSigDB against KEGG REACTOME and KEGG plus REACTOME We measured the compute time for each of the networkbased methods see Fig a0 With this benchmark we can show that ANUBIX is fast when running single gene sets One should take into account that ANUBIX and BinoX need a precomputation step before running the actual analysis However the ANUBIX precomputation step takes around a0s whereas in BinoX it takes around a0min To compute the randomized network for BinoX Scientific RepoRtS 101038s4159802070239zVol0123456789wwwnaturecomscientificreports 0cFigure a0 Receiver Operating Characteristic ROC curve For the TP tests each KEGG and REACTOME pathway is divided into two and a TP is interpreted as the crosstalk between two parts from the same pathway For the FP tests random gene sets of size are tested for enrichment against KEGG and REACTOME pathways A ROC curve for only the significantly enriched tests FDR B ROC curve for all enriched tests Software R version wwwrproje ctFigure a0 Stability analysis of ANUBIX A Fraction of KEGG pathways with Coefficient of variation CV below for different number of iterations B ANUBIX pvalues are stable”their variance is low and proportional to the magnitude of the pvalue A randomly chosen MSigDB gene set DAIRKEE_CANCER_PRONE_RESPONSE_BPA was run times against KEGG pathways Standard deviation of the logpvalue is plotted against the meanlogpvalues for each pathway Software R version wwwrproje ctwe used iterations A drawback for ANUBIX compared to methods like BinoX or NEAT is that the computation time for large scale analyses take more time For instance the time required to compute the large scale pathway annotation study for the MSigDB gene sets against KEGG pathways took a0min for ANUBIX using cores a0min for NEArender a0min for BinoX and a0min for NEAT Compute times were measured on an i77700 CPU a0GHz with a0Gb RAMPathway annotation of MSigDB gene sets We carried out a largescale pathway analysis study by running MSigDB gene sets against KEGG pathways using ANUBIX BinoX NEAT NEArender and GEA Full results are in Supplementary Data In total crosstalk tests were done per method and to get a more fair comparison between different methods we only considered enriched crosstalk considering that ANUBIX and GEA can only test for enrichmentNEArender BinoX and NEAT found the highest number of significantly FDR enriched crosstalks with and of all pairs respectively followed by ANUBIX with and GEA with Many MSigDB gene sets thus appear to have a high occurrence of pathway enrichments Even if we do not know whether those enrichments are TPs or FPs we show above Figs a0 and 5A that BinoX NEArender and NEAT are prone to produce FPsThe Venn diagram in Fig a0 shows that the overlap between BinoX NEAT and NEArender is very high having of their significant pathway annotations in common This was expected since all these methods consider pathways as random The overlap is even higher between NEAT and NEArender because they compute Scientific RepoRtS 101038s4159802070239zVol1234567890wwwnaturecomscientificreports 0cFigure a0 Compute time when running a random experimental gene set from MSigDB different gene sets were tested against KEGG REACTOME and KEGG plus REACTOME pathways for each of the networkbased methods Since ANUBIX allows parallelization we also added another run with cores The error bars show the variability in compute time for each of the methods in each of the databases The BinoX precomputation step is not included since it takes a0min Software R version wwwrproje ctFigure a0 KEGG pathway annotation for MSigDB gene sets with five methods The Venn diagram shows the number of shared pathway annotations at FDR Note that ANU

Thyroid_Cancer

Neurosteroids Biosynthesisand Physiological FunctionsShogo Haraguchi and Kazuyoshi Tsutsui Department of Biochemistry Showa University School of Medicine Tokyo Japan Graduate School of Integrated Sciencesfor Life Hiroshima University Hiroshima JapanSimilar to the adrenal glands gonads and placenta vertebrate brains also producevarious steroids which are known as œneurosteroids Neurosteroids are mainlysynthesized in the hippocampus hypothalamus and cerebellum however it has recentlybeen discovered that in birds the pineal gland a photosensitive region in the brainproduces more neurosteroids than other brain regions A series of experiments usingmolecular and biochemical techniques have found that the pineal gland producesvarious neurosteroids including sex steroids de novo from cholesterol For instanceallopregnanolone and 7αhydroxypregnenolone are actively produced in the pinealgland unlike in other brain regions Pineal 7αhydroxypregnenolone an upregulator oflocomotion enhances locomotor activity in response to light stimuli in birds Additionallypineal allopregnanolone acts on Purkinje cells in the cerebellum and prevents neuronalapoptosis within the developing cerebellum in juvenile birds Furthermore exposure tolight during nighttime hours can cause loss of diurnal variations of pineal allopregnanolonesynthesis during early posthatch life eventually leading to cerebellar Purkinje cell deathin juvenile birds In light of these new findings this review summarizes the biosynthesisand physiological functions of pineal neurosteroids Given that the circadian rhythms ofindividuals in modern societies are constantly interrupted by artificial light exposure thesefindings in birds which are excellent model diurnal animals may have direct implicationsfor addressing problems regarding the mental health and brain development of humansKeywords allopregnanolone 7αhydroxypregnenolone neurosteroid pineal gland cerebellum lightINTRODUCTIONSimilar to the gonads and placenta vertebrate brains actively also produce various steroidhormones These steroid hormones produced in the brain are named œneurosteroids Theproduction of neurosteroids was demonstrated firstly in mammals and then in other vertebrates“ Thus neurosteroid production appears to be a universal feature of the brain in vertebratesIt is known that neurosteroids are produced in glial cells and neurons of the centraland peripheral nervous systems However we have demonstrated thatthe pinealgland produces neurosteroids from cholesterol in birds during early posthatch period “Notably allopregnanolone also known as 3α5αtetrahydroprogesterone 3α5αTHP and 7αhydroxypregnenolone are the two major neurosteroids produced in the pineal gland Of thesetwo pineal allopregnanolone prevents the death of developing Purkinje cells and pineal 7αhydroxypregnenolone functions as an upregulator of locomotion regulating locomotor activity inresponse to light stimuli in birds Edited byVance L TrudeauUniversity of Ottawa CanadaReviewed byVincent M CassoneUniversity of Kentucky United StatesMaria Claudia Gonzalez DeniselleCONICET Instituto de Biolog­a yMedicina ExperimentalIBYME ArgentinaCorrespondenceShogo HaraguchishogoharaguchigmailcomSpecialty sectionThis was submitted toNeuroendocrine Sciencea section of the journalFrontiers in EndocrinologyReceived April Accepted July Published August CitationHaraguchi S and Tsutsui K Pineal Neurosteroids Biosynthesisand Physiological FunctionsFront Endocrinol 103389fendo202000549Frontiers in Endocrinology wwwfrontiersinAugust Volume 0cHaraguchi and TsutsuiPineal NeurosteroidsBIOSYNTHESIS OF PINEALNEUROSTEROIDSThe pineal glands of vertebrates respond to light stimuli andfulfillimportant functions in the anization of circadianrhythms The secretion of melatonin a major hormone producedby the pineal gland shows a clear daily rhythm with its peakconcentration occurring at night However it was notknown whether the pineal gland produces neurosteroids untilrecently We have recently demonstrated that the pineal gland isa newly found neurosteroidogenic an producing a variety ofneurosteroids from cholesterol αhydroxypregnenolone and allopregnanolone are activelyreleased Taken together these findings indicate that the pineal gland injuvenile birds produces various neurosteroids from cholesterolAccordingly this is the first demonstration of neurosteroidsynthesis in the pineal gland in a vertebratePHYSIOLOGICAL FUNCTION OF PINEAL7αHYDROXYPREGNENOLONE INLIGHTDEPENDENT LOCOMOTIONthatgene3hydroxysteroidPregnenolone is an anabolic intermediate of most endogenoussteroid hormones and is produced from cholesterol throughthe mitochondrial cholesterol side chain cleavage enzymecytochrome P450scc P450scc encoded by the Cyp11a geneWe have demonstrated by transcriptionpolymerase chainreaction RTPCR thatthe pineal gland in juvenile birdsexpresses P450scc mRNA Figure The proteinproduct of this mRNA is localized in the cells that form thefollicular structures in the pineal glands of birds We havedemonstrated by highperformance liquid chromatography3HHPLC with radioactive flow detector analysischolesterolis converted to radioactive pregnenolone whenincubated with pineal gland extract from juvenile birds This observation has confirmed the presence offunctionalP450scc in the pineal gland Figure which has also beendetected by gas chromatographymass spectrometry GCMS Subsequent RTPCR“based assessment has revealed thatkey steroidogenic enzymes cytochrome P450 7αhydroxylaseP4507α encoded by the Cyp7b gene 3αhydroxysteroiddehydrogenase 01 014isomerase 3αHSD encoded by thedehydrogenase 01 01Hsd3aisomerase 3HSD encoded by the Hsd3b gene 5αreductaseencoded by the Srd5a gene 5reductase encoded by theSrd5b gene cytochrome P450 17αhydroxylasec1720lyaseP45017αlyase encoded by the Cyp17 gene 17hydroxysteroiddehydrogenase 17HSD encoded by the Hsd17b gene andcytochrome P450 aromatase P450arom encoded by the Cyp19gene are expressed in the pineal gland of birds Figure We further demonstrated that steroid hormones are indeedpresent in the pineal gland Incubation of 3Hpregnenolonewith pineal glands from posthatch birds generates 7α andor7hydroxypregnenolone by the action of P4507α foundin the pineal glands Figure In addition to theseneurosteroid isomers progesterone allopregnanolone 3α 5αTHP andor epipregnanolone 3 5THP androstenedionetestosteroneandestradiol17 are also produced Figure These exvivo observations have confirmed that the pineal glands injuvenile birds have the biosynthetic machinery for majorsteroid hormones which have also been verified to be producedas neurosteroidsFigure Although HPLCanalysis has failed to resolve the isomers of these hormonesalloepipregnanolonesuch as 7αhydroxypregnenoloneand 5αdihydrotestosterone several sets ofisomers havebeen successfully isolated by GCMS analysis Especially5dihydrotestosteronein vivo andor5αanalysis hasactivation oftranscriptomicslightinduced transcriptionalfor studiesThe chick pineal gland is used as a modeltheon the lightdependent phaseshifting mechanism ofcircadian clock To search for genes involved in thisa diï¬erential GeneChip analysis has beenmechanismidentifiedperformed Thisthethefullset of genes in the pineal gland involved in cholesterolbiosynthesis When the pineal gland was exposed tolightit produced cholesterol and 7αhydroxypregnenoloneex vivo Interestingly this lightinduced production of 7αhydroxypregnenolone occurred only when the gland wasexposed to light at early night but not atlate night orduring the daytime During early night time the circadianclock is sensitive to light which causes phasedelay of theclock Thusthe lightsensitive pineal production of7αhydroxypregnenolone appearsto be regulated by thecircadian clockactivateslocomotorIn vertebratesan intracerebroventricularinjection of7αhydroxypregnenoloneactivities“ Thusthe intracerebroventricular injection of 7αhydroxypregnenolone was administered in a dosedependentmanner at early night in chicks After the injection chickswere placed individually for locomotor activity measurementin an open field apparatus for min Spontaneous locomotoractivities of chicks were stimulated by the intracerebroventricularinjection of 7αhydroxypregnenolone in a dosedependentmanner Furthermore when chicks are exposed to lightduring early nightreachthe daytime level These results suggest that pineal 7αhydroxypregnenolone reaches the target sites within the brain byvolume transmission upon light exposure at early nightlocomotor activitiestimetheirPHYSIOLOGICAL FUNCTION OF PINEALALLOPREGNANOLONE IN PURKINJECELL SURVIVAL DURING DEVELOPMENT7αHydroxypregnenolone and allopregnanolone are activelyreleased during early posthatch period compared with adulthood Therefore 7αhydroxypregnenolone and allopregnanolonemay play key roles in birds during early posthatch periodIn vertebrates pinealectomy decreases cell number in thedeveloping brain These findings suggest that these majorneurosteroids secreted from the pineal gland are involved in thedevelopment of brain cellsFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cHaraguchi and TsutsuiPineal NeurosteroidsFIGURE Biosynthetic pathways of pineal neurosteroids Allopregnanolone and 7αhydroxypregnenolone are the major neurosteroids produced in the pineal glandof birds P450scc cytochrome P450 sidechain cleavage enzyme P4507α cytochrome P450 7αhydroxylase 3HSD 3hydroxysteroiddehydrogenase 01 014isomerase 3αHSD 3αhydroxysteroid dehydrogenase 01 014isomerase 5αreductase 5reductase P45017αlyase cytochrome P45017αhydroxylasec1720lyase 17HSD 17hydroxysteroid dehydrogenase and P450arom cytochrome P450 aromataseFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cHaraguchi and TsutsuiPineal Neurosteroidstheincreasessupplementation oftheIn chicks pinealectomy decreases the concentration ofallopregnanolone and the number of cerebellar Purkinjeallopregnanolonecells whereasto pinealectomized birdsconcentration ofallopregnanolone and recovers the number of Purkinje cells Thus pineal allopregnanolone is considered to be an essentialfactor for the normal development of cerebellar Purkinje cellsIt thus appears that pineal allopregnanolone functions as anessential factor for Purkinje cells during posthatch periodIn addition pinealectomy in juvenile birds increases theexpression of active caspase3 in Purkinje cells whereasallopregnanolone supplementation decreases the expressionof active caspase3 during posthatch period Thus theneuroprotective action of pineal allopregnanolone on cerebellarPurkinje cells is exerted by suppressing the activation of caspase3Figure Allopregnanolone acts mainly as a ligand ofthe γaminobutyric acid type A GABAA receptor and may alsoact as an agonist of the membrane progesterone receptors αmPRα as well as the mPR and mPRγ “ Thereforeeither mPR siRNA orisoallopregnanolone an antagonistof allopregnanolone was delivered into the cerebellum ofposthatched chicks It was found that the silencing of mPRαincreases the number of Purkinje cells that express active caspase in the cerebellum of chicks Furthermore to uncoverthe mechanism of neuroprotective action of allopregnanoloneFIGURE A schematic model of the effect of pineal allopregnanolone on Purkinje cell survival immediately after hatching under a h lightdark cycle or with hlight exposure during the dark period lightatnight condition Left panel The normal cerebellar development under a h lightdark cycle during the first weekafter hatching Pineal allopregnanolone induces the expression of pituitary adenylate cyclaseactivating polypeptide PACAP a neuroprotective factor through themembrane progestin receptor α mPRα receptor binding mechanism in Purkinje cells Subsequently PACAP inhibits the activation of caspase3 that facilitates theapoptosis of cerebellar Purkinje cells Right panel The abnormal cerebellar development under the lightatnight condition during the first week after hatching Thelightatnight condition disrupts the diurnal rhythm in pineal allopregnanolone synthesis Decreased pineal allopregnanolone synthesis leads to decreased expressionof PACAP in Purkinje cells Consequently the active caspase3 level increases inducing the apoptosis of Purkinje cells in the cerebellumFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cHaraguchi and TsutsuiPineal Neurosteroidsin cerebellar Purkinje cells allopregnanolone action on theexpression of neuroprotectiveneurotoxic factors “ hasbeen investigated Pinealectomy decreases the mRNA levels ofpituitary adenylate cyclaseactivating polypeptide PACAP aneuroprotective factor in the cerebellum of juvenile birds It has been found that a daily injection of allopregnanolone inpinealectomized juvenile birds upregulates PACAP relative tothe levels in control birds These findings show that PACAPmediates the neuroprotective action of pineal allopregnanolonethrough mPRα receptor binding during cerebellar developmentFigure LIGHTATNIGHT AFFECTS THEDEVELOPMENT OF CEREBELLUMTHROUGH A MECHANISM MEDIATED BYPINEAL ALLOPREGNANOLONE ACTIONIt is known that environmental stimuli aï¬ect the developmentof animals including humans In vertebrate brain development anatural lightdark cycle promotes better brain development thanconstant conditions such as constant light or constant darkness“ Howeverthe molecular mechanisms that controlhow environmental light conditions aï¬ect brain developmentremain unclear The pineal gland is a photosensitive anTo investigate whether light conditions are involved in thesynthesis of allopregnanolone in the pineal gland the birdshave been incubated under either a h lightdark LDcycle or LD cycle with h light exposure during the darkperiod lightatnight Consequently it has been found that theallopregnanolone concentration and synthesis during the darkperiod are higher in the pineal glands of LD birds than in thoseof lightatnight birds Figure Furthermore the numberof cerebellar Purkinje cells is decreased by the lightatnightcondition Figure It is therefore considered that pinealallopregnanolone is a critical metabolite that aï¬ects cerebellardevelopment in vertebrates depending on the environmentallight conditionsREFERENCES Baulieu EE Neurosteroids of the nervous system by the nervous system forthe nervous system Rec Prog Hormone Res “ Tsutsui K Ukena K Takase M Kohchi C Lea RW Neurosteroidbiosynthesis in vertebrate brains Comp Biochem Physiol C “ 101016S0742841399000651 Compagnone NA Mellon SH Neurosteroids biosynthesis and functionthese novel neuromodulators Front Neuroendocrinol “of 101006frne19990188CONCLUSIONSthedatarecentreview summarizedThison pinealneurosteroids Studies have indicated that the pineal glandproduces neurosteroids from cholesterol in birds Pineal 7αhydroxypregnenolone regulates locomotion in response to lightstimuli in birds Pineal allopregnanolone prevents the death ofdeveloping Purkinje cells by suppressing neuronal apoptosisduring development In addition circadian disruption by lightexposure during nighttime leads to cell death of developingPurkinje cellsthrough pineal allopregnanolonedependentmechanisms in juvenile birds These observations suggest thatnighttime artificial light exposure in modern societies may alsoperturb the development of the human brainAlmost all animals have circadian rhythms However modernlife conditions chronically disrupt circadian rhythm throughartificial light exposure The disruption of circadian rhythm isassociated with a decline in mental and physical health “The most potent circadian rhythm disruption is inappropriatelytimed bright light exposure eg lightatnight To investigatethe eï¬ects of chronic circadian disruption in modern societieson mental and physical health which is efficiently modeled bythe lightatnight condition presented here many studies havebeen conducted on mice However it is important for us tobear in mind that laboratory mice are mainly nocturnal animalswhereas humans are diurnal Thus birds are excellent animalmodels to uncover the eï¬ect of lightatnight on diurnal animalsincluding humansAUTHOR CONTRIBUTIONSSH and KT wrote the manuscript All authors contributed to the and approved the submitted versionFUNDINGThis work wasJSPS GrantsinAid forScientific Research KAKENHI Grant Numbers JP15K18571and JP19K09033supported bysterol regulatory elementbinding protein Xboxbinding protein andheat shock factor pathways Proc Natl Acad Sci USA “ 101073pnas1015959108 Haraguchi S Hara S Ubuka T Mita M Tsutsui K Possible role of pinealallopregnanolone in Purkinje cell survival Proc Natl Acad Sci USA “ 101073pnas1210804109al Lightatnight Haraguchi S Kamata M Tokita T Tashiro KI Sato M Nozaki Mthroughaï¬ects brain developmenteLifeetallopregnanolonedependentpineal8e45306 107554eLife45306037mechanismsexposure Tsutsui K Matsunaga M Miyabara H Ukena K Neurosteroid biosynthesis in Reiter RJ Pineal melatonin cell biology of its synthesis and of its physiologicalthe quail brain J Exp Zool 305A733“ 101002jeza302interactions Endocr Rev “ 101210edrv122151 DoRego JL Seong JY Burel D Leprince J LuuThe V Tsutsui Ket al Neurosteroid biosynthesis enzymatic pathways and neuroendocrineregulation by neurotransmitters and neuropeptides Front Neuroendocrinol “ 101016jyfrne200905006 Hatori M Hirota T Iitsuka M Kurabayashi N Haraguchi S KokameK et al Lightdependent and circadian clockregulated activation of Fukada Y Okano T Circadian clock system in the pineal gland Mol Neurobiol “ 101385MN251019 Matsunaga M Ukena K Baulieu EE Tsutsui K 7αHydroxypregnenoloneacts as a neuronal activator to stimulate locomotor activity of breeding newtsby means of the dopaminergic system Proc Natl Acad Sci USA “ 101073pnas0407176101Frontiers in Endocrinology wwwfrontiersinAugust Volume 0cHaraguchi and TsutsuiPineal Neurosteroids Tsutsui KInoue K Miyabara H Suzuki S Ogura Y Haraguchi7αHydroxypregnenolone mediates melatonin action underlying“Sdiurnal 101523JNEUROSCI3562072008locomotorNeuroscirhythmsJ Haraguchi S Koyama T Hasunuma I Vaudry H Tsutsui K Prolactin increasesthe synthesis of 7αhydroxypregnenolone a key factor for induction oflocomotor activity in breeding male newts Endocrinology “ 101210en20091229 Haraguchi S Koyama T Hasunuma I Okuyama S Ubuka T Kikuyama S et alAcute stress increases the synthesis of 7αhydroxypregnenolone a new keyneurosteroid stimulating locomotor activity through corticosterone action innewts Endocrinology “ 101210en20111422 Haraguchi S Yamamoto Y Suzuki Y Hyung Chang J Koyama T Sato Met al 7αHydroxypregnenolone a key neuronal modulator of locomotionstimulates upstream migration by means of the dopaminergic system insalmon Sci Rep 101038srep12546 Fillenz M Volume transmission in the brain Novel mechanisms for neuronaltransmission In Fuxe K Agnati LF editors Advances in Neurosciences Vol New York NY Raven Press p “ Kilic E Hermann DM Isenmann S B¤hr M Eï¬ects of pinealectomy andmelatonin on the retrograde degeneration of retinal ganglion cells in a novelmodel of intraorbital optic nerve transection in mice J Pineal Res “ 101034j1600079x20021823x Tun§ AT Turgut M Aslan H Sahin B Yurtseven ME KaplantheS Neonatalcerebellum of“ 101016jbrainres200510011pinealectomythechick a stereologicalstudy Brain ResPurkinjeinduceslosscellin Pang Y Dong J Thomas P Characterization neurosteroid binding and braindistribution of human membrane progesterone receptors δ and ε mPRδand mPRε and mPRδ involvement in neurosteroid inhibition of apoptosisEndocrinology “ 101210en20121772 Schumacher M Mattern C Ghoumari A Oudinet JP Liere P LabombardaF et al Revisiting the roles of progesterone and allopregnanolone in thenervous system resurgence of the progesterone receptors Prog Neurobiol “ 101016jpneurobio201309004 Belelli D Lambert JJ Neurosteroids endogenous regulators of the GABAAreceptor Nat Rev Neurosci “ 101038nrn1703 Bernal J Thyroid hormone receptors in brain development and function NatClin Pract Endocrinol Metabol “ 101038ncpendmet0424 FalluelMorel A Vaudry D Aubert N Galas L Benard M Basille M et alPituitary adenylate cyclaseactivating polypeptide prevents the eï¬ects ofceramides on migration neurite outgrowth and cytoskeleton remodelingProc Natl Acad Sci USA “ 101073pnas04096 Koibuchi N Chin WW ThyroidEndocrinoldevelopment 101016S1043276000002381TrendshormoneMetabactionandbrain“ Vaudry D FalluelMorel A Leuillet S Vaudry H Gonzalez BJ Regulators ofcerebellar granule cell development act through specific signaling pathwaysScience “ 101126science1085260 Sasahara K Shikimi H Haraguchi S Sakamoto H Honda S Harada N et alMode of action and functional significance of estrogeninducing dendriticgrowth Spinogenesis and synaptogenesis in the developing Purkinje cell JNeurosci “ 101523JNEUROSCI0710072007 Bakkum BW Benevento LA Cohen RS Eï¬ects of lightdark and darkrearing on synaptic morphology in the superior colliculus and visualcortex ofJ Neurosci Res “ 101002jnr490280107the postnatal and adult rat Brooks E Waters E Farrington L Canal MM Diï¬erential hypothalamictyrosine hydroxylase distribution and activation by light in adult mice rearedunder diï¬erent light conditions during the suckling period Brain Struct Funct “ 101007s0042901103189 DulcisDSpitzerNCIlluminationcontrolsdopamineof“ 101038nature07569neuronsregulatingbehaviourdiï¬erentiationNature Li Y Komuro Y Fahrion JK Hu T Ohno N Fenner KB et al Lightstimuli control neuronal migration by altering ofinsulinlike growthfactor IGF1 signaling Proc Natl Acad Sci USA “ 101073pnas1111326109 Ohta H Mitchell AC McMahon DG ConstantPediatrthe“ 10120301pdr00002331141840366developing mousebiologicalclocklight disruptsRes Kantermann T Roenneberg TIsfactor or a health risk predictor Chronobiol lightatnightInthealthriska “ Wu J Dauchy RT Tirrell PC Wu SS Lynch DT Jitawatanarat P et alLight at night activates IGF1RPDK1 signaling and accelerates tumrowth in human breast cancer xenografts Cancer Res “ 10115800085472CAN103837 Smarr BL Grant AD Perez L Zucker I Kriegsfeld LJ Maternal andearlylife circadian disruption have longlasting negative consequenceson oï¬spring development and adult behavior in mice Sci Rep 101038s41598017034064Conflict of Interest The authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestCopyright Haraguchi and Tsutsui This is an openaccess distributedunder the terms of the Creative Commons Attribution License CC BY The usedistribution or reproduction in other forums is permitted provided the originalauthors and the copyright owners are credited and that the original publicationin this journal is cited in accordance with accepted academic practice No usedistribution or reproduction is permitted which does not comply with these termsFrontiers in Endocrinology wwwfrontiersinAugust Volume 0c'

Thyroid_Cancer

Is preoperative heart rate variability aprognostic indicator for overall survival andcancer recurrence in patients with primarycolorectal cancerM T A StrousID1 A M Daniels1 F M Zimmermann2 F N van Erning3 Y Gidron4 FJ Vogelaar1 Department of Surgery VieCuri Medical Centre Venlo The Netherlands Department of CardiologyCatharina Hospital Eindhoven The Netherlands Department of Research Netherlands ComprehensiveCancer anisation Utrecht The Netherlands Faculty of Welfare and Health University of Haifa HaifaIsrael maudstrousviecurinlAbstracta1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Strous MTA Daniels AM ZimmermannFM van Erning FN Gidron Y Vogelaar FJ Ispreoperative heart rate variability a prognosticindicator for overall survival and cancer recurrencein patients with primary colorectal cancer PLoSONE e0237244 101371journalpone0237244Editor Louise Emilsson University of OsloNORWAYReceived February Accepted July Published August Copyright Strous This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement Data cannot beshared publicly because of ethical concernsPatients were included on a no objection base toconduct retrospective data studies and publishfindings but were not asked for permission topublish full encrypted data Data are available fromthe VieCuri Institutional Data Access contact viawetenschapsbureauviecurinl for researcherswho meet the criteria for access to confidentialdataBackgroundHeart Rate Variability HRV represents efferent vagus nerve activity which is suggested tobe inversely related to fundamental mechanisms of tumorigenesis and to be a predictor ofprognosis in various types of cancer HRV is also believed to predict the occurrence andseverity of postoperative complications We aimed to determine the role of preoperativeHRV as a prognostic factor in overall and cancer free survival in patients with colorectalcancerMethodsRetrospective analysis was performed in a detailed dataset of patients diagnosed with primary colorectal cancer between January and December who underwent curative surgical treatment HRV was measured as timedomain parameters SDNN StandardDeviation of NNintervals and RMSSD Root Mean Square of Successive Differencesbased on preoperative second ECGs Groups were created by baseline HRV Low HRVSDNN 20ms or RMSSD 19ms and normal HRV SDNN �20ms or RMSSD �19msPrimary endpoints were overall and cancer free survivalResultsA total of patients were included in this study HRV was not significantly associated withoverall survival SDNN 20ms vs SDNN �20ms244 vs adjusted HR “ p RMSSD 19ms vs RMSSD �19ms270 vs adjustedHR “ p or cancer recurrence SDNN 20ms vs�20ms201 vs adjusted HR “ p RMSSD 19ms vs�19ms vs adjusted HR “ p There was noPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerFunding The authors received no specific fundingfor this workCompeting interests The authors have declaredthat no competing interests existsignificant association between HRV and CEAlevel at one year followup or between HRVand occurrence of a postoperative complication or the severity of postoperativecomplicationsConclusionsHeart rate variability was not associated with overall or cancer free survival in patients withprimary colorectal cancer who underwent curative surgical treatment These results do notalign with results found in studies including only patients with advanced cancer which suggests that there is only an association in the other direction cancer causing low HRVIntroductionIn there were over million newly diagnosed colorectal cancer patients worldwide andover in the Netherlands alone It is the fourth most common cause of death worldwide[] To improve survival it is of importance to get a better insight into modifiable prognosticfactors Emerging evidence suggests that vagal nerve activity indexed by heart rate variabilityHRV could be one of these prognostic factors [“]HRV is the physiological phenomenon of the fluctuation in time intervals between adjacentheartbeats and represents efferent vagus nerve activity to the heart [“] It has been suggestedthat efferent vagal activity is inversely related with fundamental mechanisms of tumorigenesisas inflammation oxidative stress and excessive sympathetic activity [] These mechanisms arebelieved to be controlled by the vagus nerve via a bidirectional braintoimmune pathwaysthrough the release of neurotransmitters via the cholinergic antiinflammatory pathway[] A higher vagal tone may reflect a more flexible topdown regulation of the immunesystem and physiological activity moderated by the brain [] Absence of vagus activity due tovagotomy has been shown to increase the risk of developing colorectal cancer []In addition to influencing development of cancer vagus nerve activity seems to be a predictor of prognosis in various types of cancer Recent studies show an association betweendecreased activity of the vagus nerve and worse survival in patients with cancer of the gastrointestinal tract liver pancreas lung prostate and breast among others [] Also patientswith normal HRV seem to live longer in different sorts of metastatic cancer independent ofconfounders [] In patients with colorectal cancer a low HRV at baseline has shown to beassociated with higher CEA levels at months after diagnosis which predicts a poorer prognosis []In patients undergoing curative treatment for colorectal cancer HRV does not only seemto influence cancer prognosis A recent study showed that patients with lower HRV have moreintraoperative blood loss and more and more severe postoperative complications []Identifying patients with low HRV is easy and noninvasive When its predictive value forthe prognosis of cancer patients is of satisfactory significance vagus nerve activation prior toor during cancer treatment could theoretically be beneficial in improving prognosis [] Alsoif we could predict the occurrence and severity of postoperative complications based on HRVimproving HRV before surgery could possibly accelerate postoperative recovery and indirectlyaffect patients™ prognosis Recent studies focussing on improving HRV by improving physicalfitness by means of physical exercise show promising results in both older men and woman[] However the only previous study on colon cancer and HRV including patients receiving curative treatment included a small sample and did not examine whether HRV predictsPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancersurvival in these patients [] To clarify the predictive value of HRV in prognosis of patientswith colorectal cancer further exploration is needed Current studies identifying HRV as aprognostic factor did not specifically focus on colorectal cancer have small study populationsdid not correct for confounders and mainly focused on metastatic disease [“]The aim of this study was to determine the role of preoperative HRV as a prognostic factorin overall and cancer free survival in patients with primary colorectal cancer who underwentcurative surgical treatmentMethodsData collectionData from the Netherlands Cancer Registry NCR were used The NCR collects data on allnewly diagnosed cancer patients in the Netherlands Information on patient and tumour characteristics diagnosis and treatment is routinely collected from the medical records by trainedadministrators of the cancer registry Anatomical site of the tumour is registered according tothe International Classification of Diseases Oncology The tumournodemetastasis TNMclassification is used for stage notification of the primary tumour according to the editionvalid at time of cancer diagnosis Quality of the data is high due to thorough training of theregistration team and consistency checks []For the study population additional data were collected from the medical records of thepatients This encompassed information on HRV CEAlevels ASA classification comorbidities identified at admission divided into groups cardiac disease hypertension diabetes mellitus thyroid disease pulmonary disease vascular disease neurological disease and otheroccurrence and severity of postoperative complications and cancer recurrence Groups ofcomorbidities were chosen based on matching features within these groups and their potentialinfluence on HRV or the endpoint being analysed Severity of the postoperative complicationsaccording to the ClavienDindo classification was also documented Medical records wereassessed between January and July and reevaluated for revision of this between the 20th and 25th of April This study was approved by the research committee and the Board of Directors of VieCuriMedical Centre Data was obtained under the law ˜scientific research and statistics in the interest of public health where asking for permission is not possible or appropriate for several reasons™ in the Netherlands unless patients objected to use of their personal medical record forscientific research Data was encrypted with an encryption key provided by the NCR Encryption was shortly lifted to access the patients™ number for accessing hisher medical record Following extraction data were encrypted againStudy populationThe study population included all consecutive patients diagnosed with primary colorectal adenocarcinoma between January and January at VieCuri Medical Centre who underwent curative surgical treatment Patients with metastatic disease at time of surgery orcarcinoma in situ were excluded as their treatment and prognosis differs from those receivingcurative treatment for colorectal cancer Metastasis found within months after surgery wereconsidered present at time of surgery and therefore excluded Other excluded patients werepatients with neuroendocrine tumours because of different tumour characteristics and prognosis patients with cardiac arrhythmias including atrial and ventricular extrasystole pacemakers patients taking betablockers as this enhances HRV indexes or patients withbradycardia heart rate bpm or tachycardia heart rate bpm as this precluded reliable calculation of HRV [] We did not exclude patients taking alphablockers calciumPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerinhibitors diuretics amiodarone ACEinhibitors or ARB™s as these types of medicationreduce central sympathetic functioning rather than peripheral and their influence on HRV istherefore less univocal and sometimes completely absent []Heart rate variabilityHeart rate variability was analysed using a 12lead 10second ECG 150Hz used for preoperative screening In case of multiple ECG™s per patient the most recent ECG before date of surgery was used for HRVanalysis In case of multiple ECG™s per patient on the same date theECG with the best quality was chosen meaning an ECG without motion artefacts In case ofmotion artefacts there was always an ECG without motion artefacts available recorded on thesame date Time between two consecutive Rpeaks was measured in lead II with an accuracy of02ms using MUSEECG HRV was presented using the timedomain HRV parameters SDNNStandard Deviation of NNintervals and RMSSD Root Mean Square of Successive Differences in milliseconds calculated using the following calculations []rSDNN ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffii¼1 ðRRi 00 RRmeanÞ2Pnn 00 RMSSD ¼rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffii¼1 ðRRiþ1 00 RRiÞ2Pn 00 n 00 ð1Þð2ÞSDNN and RMSSD obtained from 10s ECGs were found to correlate with results of ECGsof longer durations Power spectral analysis HRV parameters as LF and HF can only beobtained in longer recording ECGs and were therefore not implementable in this study[]SDNN and RMSSD were both analysed as continuous variables as well as binary variablesusing cutoffs of 20ms versus �20ms and 19ms versus �19ms respectively In case of anSDNN 20ms or RMSSD 19ms HRV was classified as low and in case of SDNN �20ms orRMSSD �19ms as normal These cutoff values were based on cutoff values used in otherstudies showing an association between lowHRV as SDNN 20ms and RMSSD 19ms andcolorectal cancer as there is no standardised definition of low and normal HRV []Endpoints and definitionsThe primary endpoints of this study were overall and cancer free survival Overall survival wasdefined as the time between the date of surgery to the date of death or last followup date inmonths Cancer free survival was defined as the time in months from the date of surgery untilthe date of cancer recurrence defined as the first date of either radiologic or pathologic diagnosis of metastases or tumour recurrence of colorectal cancer Patients dying without cancerrecurrence were censored on day of death Secondary endpoints were elevated CEAlevel ugl at oneyear followup occurrence of postoperative complications within daysafter surgery and severity of postoperative complications according to the ClavienDindoclassificationStatistical analysisIn this retrospective observational cohort study we utilized descriptive statistics to provide anoverview of control variables of the study population patient characteristics as age sex BMIcomorbidities and ASAclassification heart rate and tumour characteristics as TNMstagetumour localisation and tumour differentiation and their association with HRV andPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerprognosis Normal distribution of the continuous variables heart rate age and BMI as well asSDNN and RMSSD were tested with a KolmogorovSmirnov test Because of normal distribution heart rate age and BMI were compared between HRVgroups using unpaired ttest Allother variables were categorical and were compared between HRVgroups using Chisquarestatistics as groups were all of sufficient powerDifferences in overall survival and cancer free survival in months according to SDNN andRMSSD were visualized by means of KaplanMeier curves and statistically tested using the logrank test Multivariate coxregression analyses were conducted to calculate the prognosticassociation between HRV and overall and cancer free survival while adjusting for other prognostic variables Multivariate logistic regression was used to assess the independent effect ofSDNN and RMSSD on CEAlevels and the occurrence and severity of postoperative complications Variables included for adjustment were chosen by means of forward stepwise selectionbased on clinical judgment differences at baseline eg differences on any predictor betweenpatients who later died or not and database availability and depended on the analysed endpoint Those included patient demographics age sex bodymassindex comorbidities identified at admission divided into groups cardiac disease hypertension diabetes mellitus thyroiddisease pulmonary disease vascular disease neurological disease other including Crohn™sdisease hepatitis kidney failure disorders anaemia depression arthritis tumour characteristics localisation stage differentiation and the occurrence of postoperative complicationswhen the later was not an outcome Differences in CEAlevel at baseline and one year checkup between and within groups of low HRV and normal HRV were assessed with a repeatedmeasures linear model and tested using the tukey test To test the implication of a longer timebetween ECG and treatment all analyses were repeated after excluding patients with an ECGolder than months A twotailed pvalue � was considered significant in all analysesData were analysed using IBM SPSS Statistics version IBM Corp NY Armonk USAResultsOf colorectal cancer patients that underwent a surgical resection a total of patientswere included in this study Reasons for exclusion are presented in Fig Median SDNN andRMSSD were 204ms interquartile range IQR 115ms351ms and 175ms IQR 99ms299ms respectively Table shows descriptive data of the included patients by HRV groupsBaseline heart rate and age were negatively associated with HRV The group of patients withlow HRV contains more patients with a history of cardiac disease regardless of the HRV defining parameter When defining low HRV by RMSSD 19ms more patients in this group havehypertension as comorbidity This group also contains more patients with an ASA classification greater than oneDuring a median followup of months IQR “ all causedeath occurred in patients Cancer recurrence occurred in patients during a median followup of months IQR “To rule out any distort in results caused by a delay between ECG and treatment all analyseswere repeated after exclusion of ECG™s older than months This did not lead to any new significant results Therefore these results were not displayed in detail in this paperSurvivalIn low HRV groups slightly more patients died compared to normal HRV groups SDNN20ms versus �20ms versus respectively RMSSD 19ms versus�19ms versus respectively These observed differences between HRVgroups in overall survival were not significant Fig A SDNN p B RMSSDPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerFig Flowchart of the study101371journalpone0237244g001p After adjustment for some potential confounders these associations remained notsignificant SDNN 20ms versus �20ms HR “ p and RMSSD19ms versus �19ms HR “ p Tables and Age cardiac disease tumour stage and postoperative complications had a significant influence on overall survival Age also differed at baseline and was identified as a confounder When defining low andnormal HRVgroups by SDNN cardiac disease was identified as confounder When conducting sensitivity analyses with SDNN and RMSS as continuous variables results remained thesame There was no association between HRV and overall survival SDNN HR “ p and RMSSD HR “ p In low HRV groups slightly more patients had recurrence of cancer compared to normalHRV groups SDNN 20ms versus �20ms versus respectivelyRMSSD 19ms versus �19ms versus respectively These observed differences between HRV groups in cancer free survival were not significant Fig A SDNNp B RMSSD p As in overall survival after adjustment for some potentialconfounders these associations remained not significant SDNN 20ms versus �20msHR “ p and RMSSD 19ms versus �19ms HR “ p Tables and In SDNNbased groups baseline heart rate was identified asa confounding variable Sensitivity analyses with SDNN and RMSSD as continuous variablesPLOS ONE 101371journalpone0237244 August PLOS ONE 0cTable Descriptive data of included patients according to prespecified HRV groupsSDNN 20ms n SDNN �20ms n pRMSSD 19ms n RMSSD �19ms n pHRV and prognosis in colorectal cancerHeart Rate�Age�Age n year� yearSex nMaleFemale [“] [“] [“] [“] [“] [“] [“] [“] Mean BMI SD [“] [“] [“] [“]Comorbidities nCardiac diseaseHypertensionDiabetes MellitusThyroid diseasePulmonary diseaseVascular diseaseNeurological diseaseOtherASA nASA1ASA2ASA34Localisation tumour nRight colonLeft colonRectumTumour stage nIIIIII Differentiation grade nWellmoderate Poorundifferentiated � Non normaldistributed data presented as median with interquartile range101371journalpone0237244t001did not alter these results There was no association between HRV and cancer free survivalSDNN HR “ p and RMSSD HR “p CEAlevelCEAlevel at baseline and one year checkup was registered in patients and elevated in of these patients This was elevated at one year checkup in only patients Low HRV was notsignificantly associated with elevated CEAlevels at one year checkup as shown in Table Sensitivity analyses with SDNN and RMSSD as continuous variables did not alter these resultsSDNN HR “ p and RMSSD HR “PLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerFig KaplanMeier curves for overall survival in groups of low HRV and normal HRV presented as A SDNN andB RMSSD101371journalpone0237244g002PLOS ONE 101371journalpone0237244 August PLOS ONE 0cTable Multivariate analyses of associations of SDNN and covariates with overall and cancer free survivalHRV and prognosis in colorectal cancerSDNN20ms�20msHeart RateAgeBMICardiac diseaseNoYesHypertensionNoYesASAclassificationASA1ASA2ASA34LocalisationRight colonLeft colonRectumTumour stageIIIIIIOverall survivalHR CIpCancer free survivalHR CI “ “ reference “ “ “ reference “ “ “p reference reference “ “ reference reference “ “ reference “ “ reference “ “ reference “ “ “ “ reference “ “ reference “ “Postoperative complicationNoYes reference reference “ “101371journalpone0237244t002p Adjusting for covariates was not possible because of the small number of patientswith an elevated CEAlevelDifferences between CEAlevel at baseline and one year checkup were compared betweenand within HRVgroups Results were displayed in Fig There were no significant differencesin CEAlevel at baseline and one year checkup between the low HRV group and normal HRVgroup defined by SDNN and RMSSD p and p respectively Also there were nosignificant differences in CEAlevel at baseline and at one year checkup within the low HRVgroup and normal HRV group defined by SDNN and RMSSD p and p respectivelyPostoperative complicationsIn patients one or more postoperative complications occurred within days after surgeryThe occurrence of a postoperative complication was not significantly associated with lowHRV defined as SDNN 20ms or RMSSD 19ms even after adjustment for some potentialconfounders Table Heart rate age cardiac disease and hypertension were identified asconfounding covariates When conducting sensitivity analyses with SDNN and RMSS as continuous variables the association between occurrence of a postoperative complication withPLOS ONE 101371journalpone0237244 August PLOS ONE 0cTable Multivariate analyses of associations of RMSSD and covariates with overall and cancer free survivalHRV and prognosis in colorectal cancerRMSSD19ms�19msHeart RateAgeBMICardiac diseaseNoYesHypertensionNoYesASAclassificationASA1ASA2ASA34LocalisationRight colonLeft colonRectumTumour stageIIIIIIOverall survivalHR CIpCancer free survivalHR CI “ “ reference “ “ “ reference “ “ “p reference reference “ “ reference reference “ “ reference “ “ reference “ “ reference “ “ reference “ “ reference “ “ reference “ “Postoperative complicationNoYes reference reference “ “101371journalpone0237244t003baseline HRV remained not significant SDNN HR “ p andRMSSD “ p The same applied when postoperative complicationswere graded according to the ClavienDindo classification and the complication that is gradedthe highest for each patient is compared to the absence of postoperative complicationsTable DiscussionIn this observational cohortstudy we determined the Heart Rate Variability in preoperativeECGs of patients with primary colorectal cancer who underwent curative surgical treatment HRV refers to physiological variations in beattobeat intervals It was presented intimedomain parameters SDNN and RMSSD HRV was not significantly associated with overall survival or cancer free survival independent of some risk factors Also this study showedno significant differences in CEAlevels at one year checkup between patients with low HRVand patients with normal HRV Patients with low HRV did not have more or more severepostoperative complications compared to patients with normal HRVTumorigenesis has three fundamental mechanisms inflammation promoting oxidativestress and stimulating tumour growth oxidative stress causing DNAdamage andPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerFig KaplanMeier curves for cancer free survival in groups of low HRV and normal HRV presented as A SDNNand B RMSSD101371journalpone0237244g003PLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerTable Univariate analyses of low HRV and risk of elevated CEAlevel at one year checkupCEA μglOR CIpCEA μglOR CIIndependent of baseline CEAIndependent of baseline CEASDNN 20ms n “RMSSD 19ms n “SDNN �20ms n Normal baseline CEA � μglSDNN 20ms n SDNN �20ms n Elevated baseline CEA μglSDNN 20ms n reference “ reference “RMSSD �19ms n Normal baseline CEA � μglRMSSD 19ms n RMSSD �19ms n Elevated baseline CEA μglRMSSD 19ms n reference “ reference “SDNN �20ms n referenceRMSSD �19ms n referencep101371journalpone0237244t004interfering with subsequent repair mechanisms and sympathetic neurotransmitters stimulating tumour metastasis and progression [] It has been suggested that afferent fibres of thevagus nerve inform the brain about peripheral inflammation This is followed by a braintoimmune response via the efferent route of the vagus nerve that modulates the function ofimmuneregulatory cells through the release of neurotransmitters via the cholinergic antiinflammatory pathway Malfunctioning of this route may play part in the onset of cancer []This theory has been supported by studies of patients with gastric ulcers who underwent avagotomy who then showed an increased risk of developing lung and colorectal cancer [“] The vagus nerve is also believed to modulate tumour progression An active vagus nervecan inhibit inflammation oxidative stress and the release of sympathetic neurotransmittersthat stimulate tumour metastasis and progression [“] Absence of this inhibitory effect inturn results in metastasis and tumour progression as shown in a study of Erin showingthat mice who underwent chemical vagotomy developed more metastasis of breastcancer cellsthan controls [] Epidemiologically low HRV has been associated with worse overall survivalover time in patients with recurrent or metastatic cancer of various types even after correctionfor confounders [“] However the results of the present study do not support thesefindingsTo our knowledge this is the first study including only patients with colorectal cancer whoare eligible for curative treatment by partial bowel resection and not those receiving palliativetreatment De Couck studied the relationship between HRV and tumour burden in bothcurative and palliative patients with prostate cancer or nonsmall cell lung cancer Independent of confounders the hypothesised inverse relationship of HRV and the tumour markerPSA at and months after diagnosis was only significant in patients with stage IV prostatecancer not stage II and III [] In colorectal cancer Mouton found that low HRV definedas SDNN ms predicted significantly higher levels of the tumour marker CEA at months after diagnosis Again these results were only found in patients receiving palliativetreatment not curative [] Only one previous study showed a significant inverse relationshipbetween HRV and mortality in cancer in general independent of stage [] This study of Guo had a large study population of patients with various types of cancer Low HRV wasdefined as SDNN 70ms and was significantly associated with shorter survival times Thissuggests that HRV is a predictive indicator of survival time not only in palliative but also incurative patients However results were not controlled for cancer type which could affect bothHRV and survival and should therefore be interpreted with caution [] The fact that thepatients recruited in the present sample were only with less advanced cancer may partlyexplain the lack of prognostic role in HRV in this samplePLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerFig Bar chart for CEAlevel at baseline and one year checkup in groups of low HRV and normal HRV presented as ASDNN and B RMSSD101371journalpone0237244g004PLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerTable Low HRV and risk of occurrence of a postoperative complication within daysPostoperative complicationOR CIUnadjustedSDNN 20ms n “SDNN �20ms n referenceAdjusted�SDNN 20ms n “SDNN �20ms n referenceUnadjustedRMSSD 19ms n “RMSSD �19ms n referenceAdjusted�RMSSD 19ms n “RMSSD �19ms n reference�adjusted for heart rate age cardiac disease and hypertension101371journalpone0237244t005pSome of these previous studies suggest a bidirectional relationship between vagus nerveactivity and cancer [] However based on current evidence on this subject we cannot supportthis hypothesis The positive association between low HRV and worse prognosis found inpatients with colorectal cancer receiving palliative treatment but not in patients receivingcurative treatment suggest that this relation is not bidirectional but that advanced cancer isassociated with low HRV Midlatestage tumours are often accompanied by damage to autonomic nerves resulting in decreased HRV [] A study of De Couck showed that cancerpatients in general have a significantly lower HRV than healthy people [] The same resultswere found in studies of Bijoor where RMSSD was found to be significantly lower inpatients with early and advanced stage cancer compared to a healthy control group []When comparing patients with advanced stage cancer TNM III and IV to patients with anearly stage of cancer TNM I and II RMSSD was found to be significantly lower in patientswith advanced stages of cancer [] Thus though experimental studies in animals showthat vagal nerve functioning can causally slow tumorigenesis the human data suggests that themalignant tumour causes vagal nerve dysfunction and therewith decreased HRV []Besides the proposed influence of low HRV on survival of colorectal cancer patientsthrough development and increased progression of cancer Reimer suggested that lowHRV could influence survival of those undergoing surgical treatment due to more and moresevere postoperative complications [] However the results found in this study were notconcurrent with those of Reimer who included patients with ASA undergoingelective surgery Their analysis of HRV was through powerspectrum parameters based on longer ECGrecordings instead of the timedomain parameters based on 10s ECGs used in thisstudy But the difference in used parameters used in both studies is probably not the explanation for the differences in results between both studies since it has been demonstrated thatRMSSD and SDNN based on ECG recordings of 10s are in substantial agreement with those of45min and a 10s ECG therefore suffices to determine time domain HRV parameters HoweverReimer did not correct for possible confounders In their study patients with low HRVwere more likely to have diabetes a known risk factor for postoperative ileus and wound infections both found to be common postoperative complications in their low HRV group Correcting for this comorbidity may change the significance of their findings [] A study ofPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerTable Low HRV and severity of postoperative complications according to ClavienDindo classificationUnadjustedMinor grade I and IIOR CIpMajor grade III IV VOR CISDNN 20msn “n “SDNN �20msn referencen referenceAdjusted�SDNN 20msn “SDNN �20msn referenceUnadjustedRMSSD 19msn “RMSSD �19msn referenceAdjusted�n “n referencen “n referenceRMSSD 19msn “n “RMSSD �19msn referencen referencep�adjusted for heart rate age categories vs � and hypertension101371journalpone0237244t006Scheffler did not show any significant preoperative differences in HRV between patientswith or without postoperative complications or between patients with postoperative complications of different severity levels [] Thus also in relation to predicting postoperative complications results are not uniform

Thyroid_Cancer

"rebound effect after stopping treatment with denosumab may be associated with rapid loss ofthe gains in bone mineral density achieved with treatment high levels of bone remodeling markers the occurrenceof vertebral fractures and even hypercalcemiaCase presentation A 64yearold osteoporotic Caucasian woman suffered from a fracture of her second lumbarvertebra in From January she was treated with denosumab for years with good densitometry resultsfor her hip and lumbar areas and no fractures over the last years of treatment Ten months after the treatmentwith denosumab was stopped a cascade of vertebral fractures including some in unusual locations third thoracicvertebra and multiple rib fractures in a context of hypercalcemia suggested possible malignancy A completeevaluation including systemic biological and biopsy analyses ruled out this hypothesis The hypercalcemia wasassociated with normal plasma phosphate and vitamin D concentrations and a high parathyroid hormone levelwith an abnormal fixation of the lower lobe of the thyroid on sestamethoxyisobutylisonitrile scintigraphyHistological analysis of the excised parathyroid tissue revealed hyperplasia The associated thyroidectomy goiterled to the discovery of a thyroid papillary microcarcinomaConclusions We consider the consequences of this rebound effect not only in terms of the major loss of bonedensity return to basal values within years and the multiple disabling fracture episodes but also in terms of thehypercalcemia observed in association with apparently autonomous tertiary hyperparathyroidism Several cases ofspontaneous reversion have been reported in children but the intervention in our patient precluded anyassessment of the possible natural course The discovery of an associated thyroid neoplasm appears to befortuitous Better understanding of the various presentations of the rebound effect after stopping treatment withdenosumab would improve diagnostic management of misleading forms as in this case Bisphosphonates couldpartially prevent this rebound effectKeywords Osteoporosis Denosumab rebound Fracture Hypercalcemia Hyperparathyroidism Correspondence yvesmaugarschunantesfrRheumatology Department Nantes University Hospital place AlexisRicordeau Nantes Cedex France The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cMaugars Journal of Medical Case Reports Page of IntroductionSome warning signs need to be explored and are wellknown aftertreatment with denosumab A majorrebound effect after stopping denosumab can be responsible for rapid bone loss with vertebral crushes [“]Some other manifestations have been described such ashypercalcemia in both children [“] and adults []hyperparathyroidism [] and vertebral osteonecrosis[] A suspected increase in the number of cases ofprimary neoplasia has been reported in a recent metaanalysis [] We discuss a new observation with allthese manifestations together which poses diagnosisproblems and several explorations to eliminate a neoplasia This could have been avoided with better knowledge of these rebound manifestationsCase presentationA 64yearold Caucasian woman suffered from a firstvertebral fracture in the second lumbar vertebra L2 in following a fall from her bicycle She did not obtainany treatment Dual Xray absorptiometry DXA relumbar Tscore of ˆ’ standardvealed osteoporosisdeviation SD Our patient™s characteristics during follow up are summarized in Table Her phosphorus andcalcium levels were normal plasma calcium concentration mmoll normal range to mmollparathyroid hormone PTH concentration was normal ngl normal range to ngl and vitamin D levelwas low ngml normal range to ngl Shewas included in the FREEDOM protocol comparingdenosumab mg subcutaneously every monthsplus mg of calcium and IU of vitamin D dailywith placebo for the treatment of postmenopausal osteoporosis in January The unblinding of the trial years later showed that she had been randomized to thedenosumab group Several vertebral fractures occurredTable Patient™s characteristics during follow upduring this 3year period fifth thoracic vertebra T5eighth thoracic vertebra T8 and an aggravation of theL2 fracture She continued to participate in the extension protocol in mode for years and then withdrew of her own volition with a finalinjection ofdenosumab in July there were no new vertebralfractures during this entire period DXA in September demonstrated increased bone mineral densityBMD of in her lumbar region Tscore ˆ’ SD and of in her total left hip Tscore ˆ’ SD She was a former tobacco smoker and her medicalhistory included osteoarthritis of the knee a hiatus hernia hypertensionand colonicpolyps Calcaemia monitoring revealed a return to normal values until January nmoll normal range to mmoll Checkups while our patient wasstill on denosumab yielded values of nmoll in January and in September Fig amlodipineallergyIn May our patient complained of acute intensespinal pain that resisted standard painkillers and required treatment with opiates An evaluation was carriedout in hospital in June Spinal Xrays revealed fractures of the fourth thoracic vertebra T4 wedge grade T5 biconcave grade T8 wedge grade ninththoracic vertebra T9 crush grade tenth thoracic vertebra T10 wedge grade 11th thoracic vertebra T11crush grade first lumbar vertebra L1 biconcave grade L2 wedge grade and third lumbar vertebra L3 biconcave grade Fig Bone scintigraphy revealedhypersignals in all these vertebrae except L2 and T5 andin several ribs Magnetic resonance imaging MRI identified vertebra T4 in hypersignal on a T2weighted sequence and hyposignal on a T1weighted sequence withno signs of infiltration or suspected lysis Fig T9 T10T11 L1 and L3 also showed hypersignal and T5 T8 andL2 were older vertebral fractures with no bone marrowFracturesL2Tscore lumbartotalhip BMD SDˆ’ˆ’CalcaemiammollClinicalpresentationBicycle fallNo painFollow up of thepatient January FREEDOM 3yearevaluationJanuary FREEDOM extensionSeptember T5 T8 and aggravationof L2ˆ’ˆ’No painNo new fractureˆ’ˆ’TreatmentsInitiation denosumab mg semiannuallycalcium g daily vitamin D IU dailyPursuit denosumab mg semiannuallycalcium g daily vitamin D IU dailyAll treatments stoppedNoneZoledronate two infusions of mg annuallyvitamin D IU quarterlyHospitalizationJune Acute intensedorsal painNew evaluationSeptember Chronic dorsaland lumbar painT4 T9 T10 T11 L1 L3ˆ’ˆ’No new fractureˆ’ˆ’ˆ’ˆ’New evaluationAugust BMD bone mineral density L1 first lumbar vertebra L2 second lumbar vertebra L3 third lumbar vertebra SD standard deviation T4 fourth thoracic vertebra T5fifth thoracic vertebra T8 eighth thoracic vertebra T9 ninth thoracic vertebra T10 tenth thoracic vertebra T11 11th thoracic vertebraChronic lumbarpainTreatments stoppedNo new fracture 0cMaugars Journal of Medical Case Reports Page of Fig Calcemia bone mineral density and fracture events for a patient treated with denosumab for years who experienced a rebound effectwhen treatment was stopped with a combination of hypercalcemia related to hyperparathyroidism multiple fractures and rapid bone lossedema Lumbar and dorsal pain remained severe throughout this period of exploration justifying bed rest Biologicaltests revealed hypercalcemia with plasma concentrations of mmoll for calcium normal range to mmoll Fig and mmoll for phosphate normal range to mmoll hypercalciuria mmol24 hoursnormal range to mmol24 hours a 25OH vitaminD3 concentration of ngml normal range to ngla PTH concentration of pgml normal range to ngl a Creactive protein CRP concentration of mglnormal values mgl normal protein electrophoresiswith no Bence Jones proteinuria and a plasma creatinineconcentration of μmollrange to μmoll Blood formula and plasma concentrationsthyroidstimulating hormone TSH parathyroidofhormonerelated peptideand 25OH2D were normal Carboxyterminal collagen crosslinklevels were very high μgl normal valuesCTX μgl but were difficult to interpret in the context ofvertebral fracture DXA performed year after the last injection of denosumab revealed BMD losses of in our patient™s lumbar region and in her total hipPTHrpnormalcortisolThe association of fractures that are unusual for osteoporosis T4 acute and persistent back pain other rib fractures and hypercalcemia were suggestive of a potentialneoplasia which led to systemic explorations vertebralbiopsy and hyperparathyroidectomy and thyroidectomyknown goiter at the ultrasound exploration A thoracicabdominalpelvic computed tomography CT scan showedonly a heterogeneous multinodular goiter Sestamethoxyisobutylisonitrile MIBI scintigraphy revealed a small areaof fixation of the posterior lower right thyroid lobe and alower lobe nodule displaying clear uptake Fineneedleaspiration results were negative A biopsy of the T4 wascarried out under CT control and produced normal resultsWith hindsight a gassy image of the upper facet of the T4was suggestive of necrosis A parathyroid neoplasia couldhave been evoked too Surgery was performed at the end ofJuly to remove the right upper parathyroid gland × × mm weight g and histological analysissuggested nodular hyperplasia Associated total thyroidectomy led to the detection of a dystrophic goiter withmacrovesicular nodules and a mm isthmic papillarymicrocarcinoma with no associated adenopathy 0cMaugars Journal of Medical Case Reports Page of Fig Lumbar and thoracic Xray in January months before denosumab was stopped and in June months after denosumabwas stopped three old vertebral fractures L2 in and T5 T8 L2 aggravation during the period 20052008stars and new fractures T4 T9T10 T11 L1 L3arrows with denosumab rebound Stars are the old fractures and arrows the new oneHer pain was initially acute but of the mechanical typewith a generally favorable outcome Her calcaemia normalized the day after surgery mmoll with a plasma PTHconcentration of ngl normal range to ngl Anew bone densitometry evaluation was carried out in October at which time bone losses of for the lumbarregion Tscore ˆ’ SD and for the total left hipTscore ˆ’ SD were recorded Fig She continued tocomplain of disabling spinal pain Her phosphorus andcalcium evaluation results remained normal as did her vitamin D levels with the continuation of substitution treatment Given the considerable decrease in BMD she wasplaced on risedronate in September but this wasbadly tolerated She was then placed on zoledronate mgHer calcaemia remained stable at mmoll normalrange to mmoll After two infusions October and October a new DXA in August showed stabilization of the lumbar BMD and a significant loss in the total hip BMD ˆ’ Her plasma calcium levels remained normal mmoll and she did nothave any new vertebral or peripheral fracturesDiscussion and conclusionsThis patient who was included in the initial FREEDOMprotocol [] had benefited from denosumab treatmentwith no new vertebral fractures in the last years oftreatment and an increase in BMD to values exceedingˆ’ SD with no secondary effects However monthsafter stopping the treatment a cascade of vertebral fractures some in unusual locations T4 although this canbe possible when there are multiple lower vertebral fractures and multiple rib fracturesin a context of 0cMaugars Journal of Medical Case Reports Page of Fig Bone scintigraphy computed tomography scan and magnetic resonance imaging in June Magnetic resonance imaging and bonescintigraphy confirmed that there were six recent vertebral fractures fourth thoracic vertebra ninth thoracic vertebra tenth thoracic vertebra11th thoracic vertebra first lumbar vertebra and third lumbar vertebra small white arrows There were some costal fractures on bonescintigraphy sixth posterior on the right side and laterally tenth 11th and 12th on the left side black arrows A computed tomography scan alsoshowed old vertebral fractures fifth thoracic vertebra eighth thoracic vertebra and second lumbar vertebra white starssuggestedpossible malignancy Ahypercalcemiacomplete evaluation with systemic biological and biopsy T4 analyses ruled out this hypothesis Histological analysis ofthe tissue removed during theparathyroid intervention revealed hyperplasia but no adenoma The hypothesis of coincidental hyperparathyroidism had to be considered Before the treatment withdenosumab our patient™s calcaemia and PTH levels werenormal Based on retrospective analyses of calcaemia results we concluded that the increase in calcaemia became abnormal after years of denosumab treatmentHyperparathyroidism could have appeared during thedenosumab treatment phase with no obvious link between the two occurrences however there were no clearincreases in calcaemia during the first years and hypercalcemia was markedly aggravated by stopping denosumab The link between hyperparathyroidism anddenosumab was the subject of a recent publication []However the hyperparathyroidism described occurredrapidly after a single injection of denosumab with a fold increase in PTH levels at week normalization at months and normal calcaemia throughout [] This caseresolved within a year In our case calcaemia was highafter years of treatment No PTH determinations werecarried out in parallel during this period It is therefore difficult to determine the date of onset of the hyperparathyroidism as the denosumab treatment may have masked thehypercalcemia Similarly as our patient underwent surgery it is impossible to know what spontaneous course itwould have takenTwo similar cases of hypercalcemia during the rebound effect after stopping treatment with denosumabhave been reported one with low PTH levels and aspontaneously favorable outcome over several months[] and the other after a high dosage of denosumab mg quarterly [] The excessive bone remodelingobserved in the absence of associated fractures may havebeen due to major bone reabsorption potentially accounting for the hypercalcemia as in immobilizationrelated osteoporosis The high hypercalcemia observed isconsistent with this hypothesis Alendronate was administered and the patient™s plasma calcium concentrationsreturned to normal values within months In contrastour patient had a PTH concentration that was well 0cMaugars Journal of Medical Case Reports Page of nothighandanddisplayedcontrolledrapidnormalization within hours of calcaemia after theintervention Normal phosphate midupper calcaemiaand normal creatinine were not in favor of tertiaryhyperparathyroidism However the hyperparathyroidismdescribed occurred rapidly after a single injection ofdenosumab with a 22fold increase in intact PTHlevels at “ weeks and normal calcaemiaiPTHthroughout in a case report [] This dramatic increasein iPTH resolved spontaneously within a year Given thesurgical intervention carried out on our patient we cannot determine what the natural course of this hypercalcemia might have been in the same way that half thecases of hyperparathyroidism in kidney transplant recipients resolve within year for example [] The surgerywas carried out because we were concerned that our patient might be suffering from a parathyroid carcinomaThis rebound effect has been reported after stoppingdenosumab administered at an oncological dosage mg monthly for months [] Seven nonmalignantvertebral crushes were observed months after the lastinjection of denosumab At lower dosages denosumab mg halfyearly for years patients with breastcancer receiving aromatase inhibitors developed vertebral crushes after stopping denosumab [] The riskof vertebral fracture was higher if the treatments werelonger and if the patients had preexisting osteoporosistumorssometimesEight other cases of hypercalcemia have been reportedin children Denosumab was administered in these casesfor giantcellfibrous dysplasia brittle bonedisease and juvenile Paget™s disease [“] The hypercalcemia occurred early to months after the denosumab injection and wassevere plasmacalcium concentrations of up to mmoll but it occurred in a context of high doses and bone remodelingnot comparable with the contextin adults and therebound effect The hypercalcemia regressed over a fewmonths either spontaneously or on zoledronate Reactional bone hyperreabsorption was again suggestedThis hyperreabsorption seems to be related to therelease of receptor activator of nuclear factor kappaBligand RANKL with high crosslinked carboxyterminaltelopeptide of type collagen CTX1 and low Dickkopf1DKK1 and sclerostin [] Osteocytes are known to be theprincipal source of RANKL [] We have suggested thatosteocytes may undergo apoptosis during this reboundeffect when the treatment with denosumab is stoppedpotentially accounting for the necrosis and strong hyperreabsorption reported in certain patients presenting thisrebound effect []The discovery of a thyroid papillary microcarcinomaappears to have been fortuitous in this case Other studies have shown that denosumab may not only decreasethe frequency of bone tumor events but even have adirect or indirect antitumoral effect [] However ametaanalysis comparing denosumab and zoledronateand including four randomized trials patientsfound a significantly higher risk of primary neoplasiawith a cumulative annual incidence of on denosumab versus on zoledronate [] No particular cancer type profile was identified but the number of caseswas small n The cumulative doses in our patientamounted to approximately g of denosumab corresponding to months at the dose of mgmonth prescribed to prevent secondary bone tumor complicationsIt is not possible to draw any firm conclusions concerningour case as goiter is itself a risk factor for thyroid cancerwith a poorly defined incidence of between and forcancer in patients undergoing surgery for goiter []In conclusionit is important to be aware of thisrebound effect with strong bone hyperreabsorption incertain patients after stopping treatment with denosumab Several explorations to eliminate a neoplasm couldbe avoided with knowledge of these cases It can takeseveral different forms a simple loss of the BMD gainedon the treatment vertebral fractures or transient hypercalcemia which in this context may raise concerns of orsimulate malignant bone diseases or hyperparathyroidism as in our case The rebound after stopping treatment with denosumab makes it necessary to checkcalcaemia and CTX early before the risk of further vertebral fractures DXA will be carried out at the end ofthe denosumab sequence but an early new comparativeDXA would be less sensitive than CTX dosage We canpropose CTX and calcaemia months after the last injection of denosumab as a reference and then or months later A marked increase will require preventionThe most appropriate therapeutic approach remains unclear The effects of gradually decreasing the dose ofdenosumab have not yet been reported Bisphosphonatesseem to be only partially effective according to publishedpreliminary results []AbbreviationsBMD Bone mineral density CRP Creactive protein CT Computedtomography CTX Carboxyterminal collagen crosslink CTX1 Crosslinkedcarboxyterminal telopeptide of type collagen DKK1 Dickkopf1 DXA DualXray absorptiometry iPTH Intact parathyroid hormone L1 First lumbarvertebra L2 Second lumbar vertebra L3 Third lumbar vertebraMRI Magnetic resonance imaging PTH Parathyroid hormonePTHrp Parathyroid hormonerelated peptide RANKL Receptor activator ofnuclear factor kappaB ligand SD Standard deviation T4 Fourth thoracicvertebra T5 Fifth thoracic vertebra T8 Eighth thoracic vertebra T9 Ninththoracic vertebra T10 Tenth thoracic vertebra T11 11th thoracic vertebraTSH Thyroidstimulating hormone MIBI MethoxyisobutylisonitrileAcknowledgementsNAAuthors™ contributionsAll authors contributed to the work contribution to the observation YMCDL the discussion JMB BLG PG JG and writing the manuscript YMCDL All authors read and approved the final manuscript 0cMaugars Journal of Medical Case Reports Page of function Curr Drug Saf “ httpsdoi102174 Maugars Y Bart G Guillot P Multiple vertebral osteonecrosesKümmell's Disease after years on denosumab is osteocyte apoptosis toblame Calcif Tissue Int “ Chen F Pu F Safety of denosumab versus zoledronic acid in patients withbone metastases a metaanalysis of randomized controlled trials Oncol ResTreat “ Papapoulos S Lippuner K Roux C The effect of or years ofdenosumab treatment in postmenopausal women with osteoporosisresults from the FREEDOM Extension study Osteoporos Int “ Nakamura Y Kamimura M Ikegami S Changes in serum vitamin D andPTH values using denosumab with or without bisphosphonate pretreatment in osteoporotic patients a shortterm study BMC Endocr Disord“Torres A Lorenzo V Salido E Calcium metabolism and skeletal problemsafter transplantation J Am Soc Nephrol “ GonzalezRodriguez E AubryRozier B Stoll D Zaman K Lamy O Sixtyspontaneous vertebral fractures after denosumab discontinuation in women with earlystage breast cancer under aromatase inhibitorsBreast Cancer Res Treat “ httpsdoi101007s10549019054588Tyan A Patel SP Block S Hughes T McCowen KC Rebound VertebralFractures in a Patient with Lung Cancer After OncologyDose DenosumabDiscontinuation A Cautionary Tale Mayo Clin Proc Innov Qual Outcomes“Fassio A Adami G Benini C Vantaggiato E Saag KG Giollo A Lippolis IViapiana O Idolazzi L Orsolini G Rossini M Gatti D Changes in Dkk1sclerostin and RANKL serum levels following discontinuation of longtermdenosumab treatment in postmenopausal women Bone “ Bonewald LF The amazing osteocyte J Bone Miner Res “ de Groot AF AppelmanDijkstra NM van der Burg SH Kroep JR The antitumor effect of RANKL inhibition in malignant solid tumors A systematicreview Cancer Treat Rev “Sahbaz NA Tutal F Aksakal N Cancer frequency in retrosternal goiterAm Surg “Lamy O Stoll D AubryRozier B Rodriguez EG Stopping Denosumab CurrOsteoporos Rep “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsFundingNone for this workAvailability of data and materialsAll original data are available corresponding authorEthics approval and consent to participateInclusion in the FREEDOM protocol signedConsent for publicationWritten informed consent was obtained from the patient for publication ofthis case report and any accompanying images A copy of the writtenconsent is available for review by the EditorinChief of this journalCompeting interestsWe do not have any competing interestsReceived July Accepted May ReferencesAnastasilakis AD Polyzos SA Makras P AubryRozier B Kaouri S Lamy OClinical features of patients with reboundassociated vertebral fracturesafter denosumab discontinuation systematic review and additional cases JBone Miner Res “Dupont J Laurent MR Dedeyne L Luyten FP Gielen E Dejaeger MReboundassociated vertebral fractures after stopping denosumab Reportof four cases Joint Bone Spine “ httpsdoi101016jjbspin201907010Fernández Fernández E Benavent Núñez D Bonilla Hernán G Monjo HenryI García Carazo S Bernad Pineda M Balsa Criado A Aguado AP MultipleVertebral Fractures Following Discontinuation of Denosumab TreatmentTen Clinical Cases Report Reumatol Clin httpsdoi101016jreuma201811002 [Epub ahead of print]Florez H Ramírez J Monegal A Guañabens N Peris P Spontaneousvertebral fractures after denosumab discontinuation A case collection andreview of the literature Semin Arthritis Rheum “Popp AW Varathan N Buffat H Senn C Perrelet R Lippuner K Bone mineraldensity changes after year of denosumab discontinuation inpostmenopausal women with longterm denosumab treatment forosteoporosis Calcif Tissue Int “TriptoShkolnik L Rouach V Marcus Y RotmanPikielny P Benbassat CVered I Vertebral fractures following denosumab discontinuation inpatients with prolonged exposure to bisphosphonates Calcif Tissue Int“Koldkjær Sølling AS Harsløf T Kaal A Rejnmark L Langdahl BHypercalcemia after discontinuation of longterm denosumab treatmentOsteoporos Int “Kurucu N Akyuz C Ergen FB Denosumab treatment in aneurysmalbone cyst Evaluation of nine cases Pediatr Blood Cancer httpsdoi101002pbc26926 Epub Dec PubMed PMID Uday S Gaston CL Rogers L Osteonecrosis of the jaw and reboundhypercalcemia in young people treated with denosumab for giant celltumor of bone J Clin Endocrinol Metab “Setsu N Kobayashi E Asano N Severe hypercalcemia followingdenosumab treatment in a juvenile patient J Bone Miner Metab “ Gossai N Hilgers MV Polgreen LE Greengard EG Critical hypercalcemiafollowing discontinuation of denosumab therapy for metastatic giant celltumor of bone Pediatr Blood Cancer “ Boyce AM Chong WH Yao J Denosumab treatment for fibrousdysplasia J Bone Miner Res “Trejo P Rauch F Ward L Hypercalcemia and hypercalciuria duringdenosumab treatment in children with osteogenesis imperfecta type VI JMusculoskelet Neuronal Interact “ Roux S Massicotte MH Huot Daneault A BrazeauLamontagne L DufresneJ Acute hypercalcemia and excessive bone resorption following antiRANKLwithdrawal Case report and brief literature review Bone “ Mazokopakis EE Denosumabinduced normocalcemic hyperparathyroidismin in a woman with postmenopausal osteoporosis and normal renal 0c"

Thyroid_Cancer

ShortTerm Consequences ofPediatric Anticancer TreatmentRegarding Blood Pressure MotorPerformance Physical Activity andReintegration Into Sports StructuresTina Keiser Dominik Gaser Christiane Peters Renate OberhofferFritz Sabine Kesting   and Irene von Luettichau  Edited byKirsten K NessSt Jude Children™s Research Hospital Department of Sports Medicine and Exercise JustusLiebig University GieŸen GieŸen Germany Department of Sport andHealth Sciences Institute of Preventive Pediatrics Technical University of Munich Munich Germany Department ofPediatrics and Children™s Cancer Research Center Kinderklinik M¼nchen Schwabing TUM School of Medicine TechnicalUnited StatesReviewed bySeth E KarolSt Jude Children™s Research HospitalUnited StatesJacques GrillInstitut Gustave Roussy FranceCorrespondenceSabine KestingsabinekestingtumdeIrene von LuettichauIreneTeichertvonLuettichaumritumde These authors sharesenior authorshipSpecialty sectionThis was submitted toPediatric Oncologya section of the journalFrontiers in PediatricsReceived January Accepted July Published August CitationKeiser T Gaser D Peters COberhofferFritz R Kesting S and vonLuettichau I ShortTermConsequences of PediatricAnticancer Treatment RegardingBlood Pressure Motor PerformancePhysical Activity and ReintegrationInto Sports StructuresFront Pediatr 103389fped202000463University of Munich Munich GermanyBackground Cardiovascular diseases in childhood cancer survivors are knownlate sequelae following treatment Arterial stiffness pulse wave velocity PWV andcentral systolic blood pressure cSBP are potential predictors to assess the statusof cardiovascular health Frequent inpatient stays and reduced physical activity PAduring treatment can lead to noticeable impairments regarding motor skills and physicalperformance The present study examined parameters of cardiovascular health motorperformance and the status of integration into sports structures shortly after cessationof treatmentMethods A crosssectional monocentric study was conducted from April to June Participants “ yrs mixed cancer entities during maintenance therapy andfollowup care were recruited Peripheral and central systolicdiastolic blood pressurepSBP pDBP cSBP and PWV were assessed using the MobilOGraph® The MOONtest MOtor performance in pediatric ONcology was used to scale motor performancePA levels and status ofintegration into sports structures were assessed with aquestionnaire referring to the KiGGS study All measured data were compared topublished reference valuesResults Forty participants ± years female were recruited ± years posttreatment PSBP zscore ± p pDBP ± p and cSBP ‰¥ years ± p were significantly increasedcompared to reference values PWV was also elevated but not significantly Motorperformance was reduced in almost all motor abilities Thirtysix percent of the examinedgroup did not participate in physical education at school to the full extent Only reported hour of daily moderatetovigorous PA as recommended for children andadolescents by the World Health anization Half of the participants were active sportsclub members before treatment but one third did not resume their former membershipFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerConclusionIncreased cardiovascular parameters and impaired motor performanceshortly after cessation of treatment physical inactivity and low rates of integration intoregular sports programs highlight the support needed Young cancer patients shouldreceive early support in coping with physical limitations preferably soon after diagnosisMotor deficits could be reduced by applying targeted interventions Furthermorea regular sports therapy program during in and outpatient care could increaseengagement in PA to possibly counteract risk factors and improve cardiovascular healthKeywords childhood cancer cardiovascular health motor performance physical activity sports reintegrationblood pressure arterial stiffnessINTRODUCTIONExtensive research and optimized treatment regimens resulted inan increase of the 5year survival rate to in the USA and of the 15year survival rate to for patients under theage of in Germany However childhood cancer is a raredisease It contributes only around to all malignant diseasesin developed countries Worldwide children underthe age of and adolescents aged between and are diagnosed with cancer every year As a consequence ofthe success in the treatment of childhood cancer the importanceof survival quality and prevention of late sequelae have receivedmore attention during the last yearsKnown negativelongterm consequences ofintensivetreatment for childhood and adolescent cancer patients ofteninclude adverse eï¬ects on the cardiovascular system Cardiovascular diseases are the most frequently reported causesof death in childhood cancer survivors following secondarytumors Arterial stiï¬ness pulse wave velocity PWV andcentral systolic blood pressure cSBP are potential predictorsfor cardiovascular diseases frequently investigated in medicalresearch to evaluate the status of a patient™s cardiovascularhealth PWV describes the velocity of the pressure wave in the aortawhich spreads from the left ventricle through the arterial vascularsystem during systole Noninvasive investigation of the PWVvia ultrasound or oscillometric methods provides informationon the elasticity ofthe vascular system and enables earlyrecognition of damages in the vessels Thus in order to detectpotential structural modifications in the vascular system andindicators for arterial stiï¬ness at an early stage this subclinicalparameter should be surveyed continually Previous data indicatea positive correlation of PWV with arterial vascular stiï¬ness Moreover elevated PWV reflecting subclinical vasculardamage was shown in pediatric patients after hematopoieticstem cell transplantation On the contrary another studyinvestigated elevated blood pressure levels but no statisticallyAbbreviations PWV Pulse Wave Velocity WHO World Health anizationcSBP central systolic blood pressure pSBP peripheral systolic blood pressurepDBP peripheral diastolic blood pressure BMI Body mass index MOONMotor performance in pediatric oncology KiGGS German Health Interviews andExamination Survey for Children and Adolescentssignificant variation for PWV in pediatric cancer survivorscompared to healthy children and adolescents In addition to the abovementioned late sequelae severalproblems already arise during treatment and often persistthroughout survivorship For instancelongterminpatient stays and reduced physical activity during treatmentcan lead to noticeably reduced physical performance ofsurvivors and reintegration into sportschildhood cancerstructures mightberehabilitationprocess throughoutfrequentaï¬ectedIn healthy populations reduced physical activity leadsto negative consequencesfor cardiovascular health Additionally the necessary use of anthracyclines in almost ofapplied therapy regimens in childhood cancer increases the riskof cardiovascular morbidity and mortality eightfold comparedto agematched patients not receiving anthracyclines indicatingthe importance of reducing such longterm consequences Due to a poor state of health and impaired immune functionsports options such as physical education at school engagementin sports clubs or recreational sports are no longer feasibleduring therapy Consequently reintegration after cessation oftreatment is associated with even more barriers due to diseaseand treatmentrelated impairments Circumstances ofanticancer treatment can lead to inactivity resulting in deficitsof fine and gross motor skills reduced muscle strength andpoor physical fitness following treatment Especiallymotor performance in pediatric bone tumor patients oftenremains reduced until at least years after cessation of treatment Impairments of physical performance have been shown topersist throughout survivorship which may complicate thesurvivors™ reintegration into both social and sports structures aswell as the development of a longterm active lifestyle According to a questionnairebased study childhood cancersurvivors™ reintegration rate into physical education at school isvery low especially after treatment for bone tumors The lackof comprehensive oï¬ers of physical activity promotion and motordevelopment might exacerbate motor impairments and problemsof reintegration into sports structures Von Korn et al examined motor performance usingthe Fitnessgram Rcid13 as well as peripheral blood pressure centralblood pressure and PWV using the MobilOGraph Rcid13 in childrenafter treatment for childhood cancer n aged ± years ± years postdiagnosis Their results show reducedFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood Cancermotor performance of childhood cancer survivors compared toreference values of healthy children However no correlationcould be drawn regarding cardiovascular parameters and motorperformance The present crosssectional study aimed atinvestigatingvarious parameters of cardiovascular health motor performanceand status of physical activity in children and adolescentsshortly after cessation of anticancer treatment or during ongoingoral maintenance therapy The collection of such data isof considerable importance for the early detection of healthimplications related to both disease and treatment Moreoverthe findings will help to support the development of preventivestrategies regarding the health of children and adolescents treatedfor cancer Appropriate strategies during primary and secondaryprevention and following cancer treatment tertiary preventionneed improvementsMATERIALS AND METHODSDesignThe crosssectional monocentric study was performed over aperiod of months April“June at our institution Theassessment of cardiovascular parameters using the MobilOGraph Rcid13 was followed by the MOON test MOtor performance inpediatric ONcology to evaluate motor performance Finally theparticipants completed a standardized questionnaire referring tothe KiGGS study German Health Interview and ExaminationSurvey for Children and Adolescents to collect data regardingtheir current level of physical activity and status of integrationinto sports structures The Ethics Committee of the School ofMedicine of the Technical University of Munich approved thestudy project number SSR Participation was voluntaryand informed written consent was signed by each participant aswell as by his or her legal guardian All data was collected encodedpseudonym and in accordance with privacy policy standardsParticipantsPrior to addressing the participants all eligible children andadolescents were screened using the electronic patient recordSAP Rcid13 ERP Patients were recruited during routine followupvisits The following inclusion criteria were applied childrenand adolescents during maintenance therapy and followupcare of a pediatric oncological disease and currently agedbetween and years No restriction was applied regardingthe period posttreatment Exclusion criteria were medicalcontraindications such as fever acute infection orthopedicrestrictions and mental retardation insufficient knowledgeof the German language and absence of written informedconsent The attending physician confirmed participation forall recruited children and adolescents Following these inclusioncriteria children and adolescents were initially found eligibleFigure Outcome MeasuresAnamnestic and Anthropometric DataAnamnestic and clinical data ie type of cancer treatmentregime end of therapy was obtained from the electronic patientrecord SAP Rcid13 ERP The nursing staï¬ assessed anthropometricdata height and weight during routine medical examinationseca electronic column scaleseca mechanicalmeasuring rod Body mass index BMI was calculated as aratio of body weight kg per square body height m2 By usingthe reference values of a healthy German cohort BMI wasconverted into percentiles and classified in underweight 10thpercentile normal weight 10thˆ’90th percentile and overweight90th percentile Cardiovascular ParametersPrior to the measurement the participants had to rest for atleast min in a supine position PWV central blood pressureand peripheral blood pressure were assessed using the MobilOGraph Rcid13 IEM GmbH Stolberg Germany and HMS ClientServer Version an oscillometric noninvasive methodMeasurements were performed on the left upper arm The cuï¬was inflated twice with a rest of s in between Cuï¬ sizewas chosen according to the circumference of the participant™sleft upper arm An ARCSolver Algorithm calculated the cSBPindirectly as well as the PWV from recorded brachial pulseRaw data was transformed into zscores and compared by usingzscores of a healthy reference cohort PSBP and pDBPwere compared to references from the national cohort of children and adolescents KiGGS study For the assessmentof PWV and cSBP values references from Elmenhorst et al of healthy children and young adults were used To evaluatethe results of the parameters cSBP and PWV the examinedparticipants were separated into two groups years and‰¥ years According to the age distribution of the referencevalues participants years were compared to heightmatchedreference values and participants ‰¥ years were compared toagematched referencesThe measurement using the MobilOGraph Rcid13 was previouslyused several times in pediatric patients “ and was alsosuccessfully applied in childhood cancer survivors Motor PerformanceTo quantify motor performancethe MOON test MOtorperformance in pediatric ONcology was applied Thetest assesses motor abilities coordination speed flexibility andstrength and consists of eight test items eyehand coordinationinserting pins static balance static stand upper extremitycoordination throwing at a targetspeed reaction testmuscular endurance sittostand flexibility stand and reachhand grip strength handheld dynamometry and muscularexplosive strength medicine ball shot The test lasts min onaverage Data of each item was compared to published age andsexmatched reference values of a healthy population within anage range of to years Data of participants older than years was compared to reference values of healthy 17yearoldssince reference values of healthy 18yearolds are not availableCalculation of a total score is not possible within this toolInstead each item was analyzed individually and the percentagedeviation to reference values was computedFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerFIGURE Flow chart of recruitment Out of participants eligible could not be addressed due to several medical examinations in different departments of thehospital and resulting in missing time slots Longer periods posttreatment are associated with fewer appointments for followups and more medical examinationstake place in day This does not necessarily mean that the children who could not be included in the study due to missing time slots are medically more complexTen participants refused participation Thus the sample included participantsPhysical Activity and Reintegration Into SportsStructures After Acute TreatmentPhysical activity levels and status of integration into sportsstructures were assessed with a standardized selfreportingreferring to the KiGGS study Thequestionnairequestionnaire wassupplemented by several disease andtreatmentrelated aspects in accordance with the study ofKesting et al to investigate potential barriers regardingreintegration and participation in sports activities eg barrierswith respect to exemption from physical education at school ornonparticipation in sports clubs sports therapy oï¬ers duringtreatment The KiGGS study oï¬ers the reference values ofhealthy children and adolescents n for comparison ofour dataData AnalysisCardiovascular parameters were analyzed and compared to thehealthy reference population with the one sample ttest Motorperformance was analyzed using the Wilcoxon signedranktest in comparison to age and sexmatched reference valuesPearson correlation was applied to calculate possible associationsbetween motor performance and cardiovascular parametersBMI and the period posttreatment The MannWhitneyUtestwas performed to evaluate anthracyclinemediated eï¬ects oncardiovascular health as well as diï¬erences within subgroupsregarding diï¬erent entities and levels of physical activity motorperformance physical education at school and achievement ofphysical activity recommendationsExplorative twosided statistical tests were conducted andp ‰¤ was considered statistically significant No adjustmentfor multiple comparison was conducted Correlations coefficientρ were classified according to Cohen Descriptive statistics were calculated with Microsoft Excelversion for demographic characteristics and medicaldata GraphPad Prism version was used to perform allfurther statistical analyses Data analysis was performed inconsultation with the Institute of Medical Informatics Statisticsand Epidemiology of the Technical University of MunichRESULTSParticipantsOut of eligible children and adolescents who met the inclusioncriteria a total of participants female with variouscancer entities were recruited and examined Table Performed AssessmentsFor various reasons not alltests could be realized withall participants In participants cardiovascularparameters could not be evaluated due to missing time slotsFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerTABLE Anthropometric and medical characteristics of the participants n CharacteristicsN Mean ± SD MedianRangeAge at diagnosis years ± Age at assessment years ± ““ years‰¥ years Period postdiagnosis years ± Period posttreatment years ± ““ year“ years yearsLeukemiaLymphomaBone tumorBrain tumorOther solid tumors Body mass index¢ kgm2 ± “UnderweightNormal weightOverweightChemotherapyAnthracycline applicationCumulative dose mgm2RadiotherapyChestdirected radiation Anthracycline chest radiation Surgical tumor resectionRelapse ± “FIGURE Cardiovascular parameters shown in zscores and compared topublished reference values pSBP peripheral systolic blood pressurepDBP peripheral diastolic blood pressure PWV pulse wave velocity cSBPcentral systolic blood pressure Significant values p ‰¤ Results are given in Mean ± SD M median RangeOther solid tumors alveolar rhabdomyosarcoma n carcinoid tumor of the appendixn nephroblastoma n focal nodular hyperplasia liver n mature cysticteratoma ovary n thoracic ganglioneuroma n papillary thyroid carcinoman neuroblastoma n ¢BMI was converted into percentiles and classified in underweight 10th percentilenormal weight 10thˆ’90th percentile and overweight 90th percentile Thirteen participants received a combination of anthracyclines mainly the combination ofDoxorubicin and Daunorubicinduring routine appointment and a lack of willingness to prolongthe outpatient visit Six of the participants could not performthe MOON test due to medical limitations current orthopedicrestrictions n examinationrelated limitations ie a draintube in the crook of the arm for followup MRT n andabandonment due to lack of time n The central venousdevice has already been explanted in all participants prior toour studyOf the remaining participants not everyone performedevery test item Two participants could not perform the testitem speed due to a drain tube in the crook of the armFour participants could not accomplish the test item muscularexplosive strength due to orthopedic restrictions as well asexaminationrelated limitations Three participants did notperform the item muscular endurance of the legs n hadcrutches n severe muscular deficit in the legs n lackof time Two participants could not perform the test item handgrip strength with both hands due to lack of time n andinfusion needle in the crook of the arm n The test itemupper extremity coordination was measured in n participantsbecause reference values are provided for children between and years onlyCardiovascular HealthIn participants all parameters were assessed Based on theunderlying reference values for cSBP and PWV of Elmenhorstet al the group was separated into participants aged years heightmatched reference values and ‰¥ years agematched reference values PSBP zscore ± p pDBP zscore ± p as well as cSBP values‰¥ years zscore ± p were significantlyincreased compared to reference values of healthy children andadolescents Figure PWV was elevated but not significantly years zscore ± p ‰¥ years zscore ± p Comparison of cardiovascular parameters of participantswho received anthracyclines during intense therapy with acumulative dose of ± mgm2 and subjects whodid not receive cardiotoxic agents did not show statisticallysignificant diï¬erencesMotor PerformanceThe participants™ n motor performance wasreduced in almost all motor abilities compared to the referencevalues of healthy children and adolescents Table Significantimpairments became obvious in the following dimensionsmuscular explosive strength p upper extremitycoordination p muscular endurance of the legs p Frontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerTABLE Results of the MOONtest compared to reference values n Motor abilityTest itemEyehand coordinationStatic balanceφSpeedInserting pins timeStatic stand contactsReaction test timeUpper extremity coordinationθFlexibilityThrowing at a target pointsStand and reach cmMuscular explosive strengthMedicine ball shot meterMuscular endurance legsSittostand secHand grip strengthHandheld dynamometry kgRightLeftNMean ± SD of differenceMedian pvalueto reference values ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’All results were compared to the reference values of each single test item Speed could only be measured in participants muscular explosive strength in participants andhand grip strength in right and left participants due to lack of time orthopedic restrictions or drain tube in the crook of the armFor the test items static balance and flexibility the absolute differences were used as the measured values would have fluctuated around zero and would have givenoversized percentagesφ Static balance was assessed counting the contacts with a foot to the ground while balancing on a rail in this context a negative difference to reference values represents fewercontacts and therefore better resultsθ Although all participants completed this test item reference values are provided for children between and years onlyM median abs indicates absoluteBold numbers indicate significant values p ‰¤ TABLE Results of the MOONtest comparing participants treated for leukemialymphoma and participants treated for brain tumors compared to reference valuesLeukemiaLymphoma n Brain tumor n Motor abilityEyehand coordinationStatic balanceφSpeedFlexibilityMuscular explosive strengthMuscular endurance legsHand grip strength rightHand grip strength leftNMean ± SD Median ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’NMean ± SD Median pvalueˆ’ ± ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ All results were compared to the reference values of each single test item φ Static balance was assessed counting the contacts with a foot to the ground while balancing on a rail in this context a negative difference to reference values represents fewercontacts and therefore better resultsFor the test items static balance and flexibility the absolute differences were used as the measured values would have fluctuated around zero and would have givenoversized percentagesDue to insufficient number of participants the comparison regarding the test item upper extremity coordination was disregarded reference values only provided for children aged and yearsM median abs indicates absoluteBold numbers indicate significant values p ‰¤ and hand grip strength on the right hand p The performance of eyehand coordination speed flexibility andhand grip strength on the left hand were also reduced but notsignificantly In the test item static stand the study participantsperformed slightly better compared to the reference populationFor upper extremity coordination throwing at a target onlyreference values from children aged “ years are availableTherefore comparison of the collected data was only possiblewith the same age group n Data for olderparticipants was not collectedComparing motor performance of participants diagnosedwith leukemialymphoma n and participantsn diagnosed with brain tumorsrevealedsome diï¬erences Table Children and adolescents treatedfor brain tumors performed significantly worse in eyehandcoordination than participants treated for leukemialymphomap Moreover the performances in all other testedmotoric dimensions of participants diagnosed with braintumor were deteriorated compared to participants treated forleukemialymphomas but not significantlyTo determine influencing factors on motor performance thecorrelation between motor performance results and BMI as wellas the period posttreatment was performed With increasingBMI values of static balance deteriorated significantly ρ Frontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerFIGURE Pearson correlation between static balance and BMI kgm2¢ leukemialymphoma —¦ bone tumor –³ brain tumor cid7 other solid tumorsHorizontal line indicates the reference values The absolute difference fromreference values is given number of contacts to the ground A negativedifference means fewer ground contacts and therefore better performanceThirty four participants performed the testp which corresponds to a moderate to high correlationFigure A negative diï¬erence to reference values means fewercontacts to the ground and therefore better performance in staticbalance Furthermore some nonsignificant correlations werefound Deteriorated eyehand coordination ρ ˆ’ p and flexibility ρ ˆ’ p were also associatedwith a higher BMI However superior values in upper extremitycoordination ρ p muscular explosive strengthρ p hand grip strength right ρ p left ρ p and muscle endurance ofthe legs ρ p were associated with increasedBMI The test item speed showed no association with BMIρ p A longer period posttreatment was significantly associatedwith decreased eyehand coordination ρ ˆ’ p corresponding to a moderate correlation Figure Especially participants treated for a brain tumor with a longerperiod posttreatment showed deteriorated values in eyehandcoordination Speed performance was deteriorated in participantswith longer posttreatment period ρ ˆ’ p Physical Activity and Reintegration IntoSports StructuresAccording to the selfreported questionnaire n did not participate in physical education at school to full extend n were not admitted to school sports activitiesand n were partly excluded Table Neitherof the two participants treated for bone tumor was takingpart in physical education at school n whereaschildren with other tumors participated to a notably higherrate Treatmentrelated muscular deficits n andosteonecrosis n were the most common reasons forparticipants not taking part in physical education at schoolFIGURE Pearson correlation between eyehand coordination and periodposttreatment months ¢ leukemialymphoma —¦ bone tumor –³ brain tumorcid7 other solid tumors Horizontal line indicates the reference values Thirtyfour participants performed the test itemTABLE Participation in physical education at school subdivided into entitiesn Participation Partial exemption Full exemptionN N N Entire group n LeukemiaLymphoma n Bone tumor n Brain tumor n Other solid tumors n Abs N number Four out of children were still attending kindergarten and could notanswer the question regarding participation in physical education at schoolOnly n reported moderatetovigorous physicalactivity for min daily as generally recommended by the WHOfor healthy children and adolescents Table This percentageis comparable to the achievements in the healthy referencepopulation n Every second participant questioned was an active memberin a sports club whereas n did not return to asports club following cancer treatment Almost onethird n has never been a sports club member Reasons fornot engaging in sports club activities of participants wereno interestfun n physical weakness n no time n anxiety n andphysicianbased prohibition due to clear medical reasons n Nearly all participants n were active inrecreational sportsFurther analyses pointed toward diï¬erences in physicalactivity and sports club participation especially betweenparticipants with brain tumors and leukemialymphomas Thenumber of participants in recreational sports was reported highin both groups leukemialymphoma and brain tumor In contrast a diï¬erence was found in sports club activitySixtysix percent of leukemialymphoma patients were membersFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerTABLE Physical activity and engagement in sports club and recreationalsportsEntire groupReference populationn n Physical activity guidelinesWHO mindayDaily physical activityDaily walking distance kmDaily walking distance “ kmDaily walking distance kmSports club activity currentlyFormer membershipRecreational sports activityAbs N number Reference values derived from the national cohort of healthy childrenand adolescents in the KiGGS study German Health Interviews and Examination Surveyfor Children and Adolescents of a sports club whereas only of participants with a braintumor were active in a sports club On the other hand almosthalf of the children treated for brain tumor and of thechildren treated for leukemialymphoma were former membersConcerning possible correlations between motor abilities andphysical education at school participation in sports clubs orrecreational sports defined as activeinactive as well as meetingthe physical activity recommendations no significant associationscould be determined Likewise the comparison of motor abilitiesof participants receiving sports therapy during treatment didnot show any correlation Almost half of the participants n took part in a sports therapy programme duringtreatment which was mainly oï¬ered as care and varied greatlyin terms of training interventions without any standardizationDISCUSSIONtreatmentThe results of our study clearly present evidence for deterioratedcardiovascular function in children and adolescents shortlyafter cessation of cancerIncreased pSBP andincreased pDBP are risk factors for cardiovascular diseasesregarding guidelines for arterial hypertension Potentialcardiovascular consequences such as stroke sudden deathheart failure and peripheral artery disease due to elevatedblood pressure values are described in the aforementionedguidelines as well as in the literature Accordinglychildhood cancer survivors with elevated blood pressure are atrisk to experience such cardiovascular late eï¬ects Regarding10years survivors of childhood cancer a higher prevalenceof hypertension is assumed and cardiovascular diseaserelated deaths are eighttimes more likely in childhoodcancer survivors compared to the general population Our findings support previous study results which depictcomplications such as increased blood pressure prehypertensionand hypertension in children and adolescents treated for cancer This study aimed at investigating specific parameters thatcould serve as early predictors for potential damage to thecardiovascular system Recent evidence of cSBP as a suitableparameter to determine the elasticity of blood vessels suggeststhat cSBP is more closely related to cardiovascular events in thefuture than brachial blood pressure Increased cSBP inparticipants in our study may result from early changes in arterialwall stiï¬ness As a further parameter to detect early impairmentsin elasticity of the vascular system PWV was investigated Incontrast to prior studies no decisive change was observedin PWV While anthracycl

Thyroid_Cancer

lack of COVID19 diagnostic tests for the whole Spanish population thecurrent strategy is to identify the disease early to limit contagion in the communityAimTo determine clinical factors of a poor prognosis in patients with COVID19 infectionDesign and settingDescriptive observational retrospective study in three primary healthcare centres with anassigned population of MethodExamination of the medical records of patients with COVID19 infections confirmed by polymerase chain reaction Logistic multivariate regression models adjusted for age and sexwere constructed to analyse independent predictive factors associated with death ICUadmission and hospitalizationResultsWe included patients mean age years female aged � years were health workers doctors nurses auxiliaries Predictors of ICUadmission or death were greater age OR 95CI to male sex OR 95CI to autoimmune disease OR 95CI to bilateral pulmonary infiltrates OR 95CI to elevated lactatedehydrogenase OR 95CI to elevated Ddimer OR 95CI to and elevated Creactive protein OR 95CI to Myalgia or arthralgia OR 95CI to was protective factor against ICU admission andPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsdeath Predictors of hospitalization were chills OR 95CI to feverOR 95CI to dyspnoea OR 95CI to depressionOR 95CI to lymph ia OR 95CI to andelevated Creactive protein OR 95CI to Anosmia OR 95CI to was the only significant protective factor for hospitalization after adjusting forage and sexConclusionDetermining the clinical biological and radiological characteristics of patients with suspected COVID19 infection will be key to early treatment and isolation and the tracing ofcontactsIntroductionOn December the health authorities of Wuhan city Hubei Province China reporteda cluster of cases of pneumonia of unknown aetiology with onset of symptoms on December including severe cases with a common exposure identified in a city market [] whichwas closed on January On January the Chinese authorities identified a newCoronaviridae family virus initially named coronavirus 2019nCoV and later coronavirusSARSCoV2 as the causal agent [] The genetic sequence was shared by the Chinese authorities on January On January the first case was detected in the USA in Washingtonstate [] On January the World Health anization declared the SARSCoV2 outbreak in China a public health emergency of international concern [] Subsequently the outbreak has spread outside China with Europe especially affected []The first positive case diagnosed in Spain was confirmed on January on the islandof La Gomera while the first death occurred on February in Valencia city the date was confirmed twenty days later The first confirmed case in Barcelona was on February and fromthen until June there have been confirmed cases in Spain []The most common signs of infection are respiratory symptoms fever cough and shortnessof breath In more severe cases the infection may cause pneumonia severe acute respiratorysyndrome renal failure and death [] Transmission appears to be mainly persontopersonvia the airway through respiratory droplets measuring microns when the patient has respiratory symptoms cough and sneezing and contact with fomites [] Most estimates of theincubation period of COVID19 range from to days with most around five days Evidenceon the transmission of the virus before symptom onset is unclear There is currently no specifictreatment for COVID19 infections To date the most important scientific efforts have focusedon three areas strategies to contain the spread of the disease the initiation of clinical trialswith antivirals and multiple therapies and the design of a new vaccine which is still unclearThese strategies include some of a community nature where primary healthcare plays a centralrole in disease prevention and control [] Few studies have described the clinical characteristics of the disease fewer the predictive factors and virtually none have described the Mediterranean population compared with the rest of the world Therefore this study aimed todescribe the clinical biological and radiological manifestations the evolution treatments andmortality rate of patients with COVID19 infection in the population of Barcelona city anddetermine the most important predictors of a poor prognosisPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsMaterials and methodsA multicentre observational descriptive study was carried out in three urban primary healthcare centres serving an assigned population of with one reference hospital The studyincluded all consecutive adult patients with COVID19 confirmed by polymerase chain reaction PCR from nasal and pharyngeal samples during the study period of February to April Diagnostic confirmation was made in the hospital laboratories as PCR is not available in primary healthcare centres Signs and symptoms the main available haematologicaland biochemical data and the results of imaging tests were recorded as were comorbiditiesthe evolution the hospitalization rate intensive care unit ICU admission and the treatmentsreceived The study population was divided into four age groups “ years “ years“ years and � years Other variables recorded were the type of followup the need fortemporary work disability and the source of possible contacts The time to first medical visitwas defined as the difference in days between symptom onset and medical visit by a familyphysician The factors that determined a poor prognosis hospitalization ICU admissiondeath were collected The data were obtained from the electronic medical record Missingdata were collected by telephone interviews with patients when possible Patients from nursinghomes were excluded as the rate of infections and mortality has been shown to be muchhigher than in the noninstitutionalized population The study was approved by the EthicsCommittee of the Hospital Clinic of Barcelona registration number HCB20200525 Thestudy was conducted according to the Helsinki Declaration and Spanish legislation on biomedical studies data protection and respect for human rightsStatistical analysisCategorical variables are presented as absolute frequencies and percentages and continuous variables as means and standard deviations SD Predictors of death ICU admission andhospitalization were determined using the student™s t test for continuous variables and the chisquare test for categorical variables Logistic multivariate regression models adjusted for ageand sex were constructed to analyse independent predictive factors associated with death ICUadmission and hospitalization Odds ratios OR and their confidence intervals 95CIobtained in the adjusted regression analysis were calculated Forest plots were used to represent OR and 95CI Values of p005 were considered statistically significant The statisticalanalysis was performed using the R version for WindowsResultsClinical characteristics and comorbiditiesWe included patients mean age years female aged � yearsThe mean time from symptom onset to the medical visit was SD days Clinical characteristics are shown in Table Notably were health workers doctors nurses auxiliaries The most frequentclinical symptoms were cough fever general malaise fatigue myalgia or arthralgia dyspnoea diarrhoea headache anosmia and dysgeusia Physical examination in patients showed had auscultatory alterations tachypnoea and an oxygen saturation of � ICU admission and death were associated with a greater mean age years vs yearsp male sex vs p dyspnoea vs p fever vs p auscultatory alterations vs p and low oxygen saturation vs p Table Myalgia or arthralgia vs PLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsTable Clinical and exploratory factors predicting hospitalization and ICU admissiondeathVariablesTotalDeath or ICU admissionHospitalizationAge”yearsDistribution”no “ years“ years“ years� yearsMale”no Occupation”no n No n YesPAdjusted ORNoYesPAdjusted OR ± ± n ± [“][ CI]n ± n ± [“][ CI] [“] [“]Other type of exposure Health professionalOther health workersSmoking exsmokersmoker”nototal no Temperature at admission” ˚C Patients with fever �˚C”nototal no Time from symptom onset tomedical visit”days¡Symptoms”no ¶ ± ± ± ± ± ± NANANA [“] [“] [“] [“] ± ± ± [“] [“] ± [“] NANANA [“]CoughGeneral malaiseFatigue [“] [“] [“] [“] [“] [“]Myalgia or arthralgia [“] [“]DyspnoeaDiarrhoeaHeadacheAnosmiaDysgeusiaSore throatBlocked noseNausea or vomitingSputum productionChillsAstheniaChest painAlterations in physical examination”nototal no §Auscultatory alterationsTachypnoeaTachycardiaPharyngitis [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] NA [“]Continued PLOS ONE 101371journalpone0237960 August PLOS ONE 0cTable ContinuedVariablesTotalDeath or ICU admissionHospitalizationPrognostic factors in Spanish COVID19 patientsOxygen saturation �”nototalno n n No n YesPAdjusted ORNoYes[ CI]n n [“] PAdjusted OR[ CI] [“]In bold statistically significant independent predictive factors associated with hospitalization death or ICU admission logistic multivariate regression adjusted for ageand sex  Temperature distribution was ˚C “ËšC “ËšC and ˚C ¡ In patients™ data on period between symptom onset and medical visit were lacking¶ Symptoms with a frequency of patients were disorientation n conjunctivitis n haemoptysis n and cutaneous lesions n § patients had a physical examination The alterations with a frequency of patients were cutaneous lesions n and tonsillopharyngitis n Ref reference NA not applicable101371journalpone0237960t001p headache vs p dysgeusia vs p andanosmia vs p were less frequent in patients admitted to the ICU or whodied than the remaining patients Age OR 95CI to and male sexOR 95CI to were independent predictors of ICU admission and deathMyalgia or arthralgia OR 95CI to was the only significant protective factor against ICU admission and death after adjusting for age and sex Fig The best clinicalpredictors of hospitalization were chills OR 95CI to fever OR 95CI to and dyspnoea OR 95CI to Anosmia OR 95CI to was the only significant protective factor for hospitalization after adjusting for age and sex Table and Fig Comorbidities were presented by patients the most common were hypertension in diabetes mellitus in and obesity in Table Heartdisease vs p autoimmune disease vs p diabetes vs p hypertension vs p and chronic kidney disease vs p were the comorbidities significantly associated with ICUadmission and death Table Autoimmune disease was the only significant predictivecomorbidity for ICU admission and death after adjusting for age and sex OR CI to Fig Depression was the best predictor of hospitalization among allcomorbidities OR 95CI to Fig Having � comorbidity was associated with ICU admission and death OR 95CI to and hospitalizationOR 95CI to independently of age and sexImaging and laboratory testsChest Xray was necessary in patients and showed lobar pulmonary infiltrates in bilateral pulmonary infiltrates in and an interstitial pattern in Table Chest CT was required in patients and pulmonary ultrasound in Biologically of patients had lymph ia mm3 Likewise had a lactate dehydrogenase LDH Uml and liver test alterations were commonelevated ASTGOT in and ALTGPT in In of cases Ddimer waselevated 500mgL The most important factors for ICU admission and death were bilateralpulmonary infiltrates OR 95CI to elevated lactatedehydrogenaseOR 95CI to elevated Ddimer OR 95CI to andelevated Creactive protein OR 95CI to Fig Significant predictivePLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsFig Prognostic factors for death and ICU admission �The upper limits of the confidence intervals were restricted to in order not to mask the significant effects of other variables with smaller ranges101371journalpone0237960g001factors associated with hospitalization after adjusting for age and sex were lymph iaOR 95CI to and elevated Creactive protein OR 95CI to Fig Treatment complications and evolutionTreatment included hydroxychloroquine in patients azithromycin in lopinavirritonavir in glucocorticoids in and tocilizumab in among others Table and of patients required hospitalization Phone follow up was registered in patients patients were monitored at homePLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsFig Prognostic factors for hospitalization �The upper limits of the confidence intervals were restricted to inorder not to mask the significant effects of other variables with smaller ranges101371journalpone0237960g002 of the patients of working age sought work disability due to COVID19 TheICU admission rate was The evolution included pneumonia in patientsadult respiratory distress syndrome in severe renal failure in pulmonarythromboembolism in and sepsis in patients Occupational contact with persons with confirmed or suspected COVID19 infection was reported by patientswhile reported that contact occurred in the family setting Occupational contactwas a protective factor against hospitalization OR 95CI to ICU admission and death OR 95CI to after adjusting for age and sex The mortalityrate to date was PLOS ONE 101371journalpone0237960 August PLOS ONE 0cTable Comorbidities associated with hospitalization and ICU admissiondeathVariablesTotalDeath or ICU admissionHospitalizationn NoYesPAdjusted OR [NoYesPAdjusted OR [n n CI]n n CI]Prognostic factors in Spanish COVID19 patientsComorbidities”no  Any comorbidityHypertensionDiabetesObesityDyslipidaemiaCancer [“] [“] [“] [“] [“] [“] [“] [“] Chronic kidney disease Heart disease Autoimmune disease Chronic obstructive pulmonary diseaseDepressionCardiac arrhythmiaThyroid alterationsAsthmaLiver diseaseCerebrovascular diseaseAlzheimer disease [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“]In bold statistically significant independent predictive factors associated with hospitalization death or ICU admission logistic multivariate regression adjusted for ageand sex  Comorbidities with a frequency of patients were bronchiectasis n fibromyalgia n anaemia n arthritis n HIV n syphilis n andtuberculosis n 101371journalpone0237960t002DiscussionThis study summarizes the clinical biological and radiological characteristics evolution andprognostic factors of patients with COVID19 disease in primary and community healthcareTo date we are aware of three published Spanish studies [“] The first reported data from patients on ICU admissions in a region where the pandemic was reported early [] Thestudy by Borobia [] describes the first adult patients with COVID19 consecutivelyadmitted to a University Hospital in Madrid The third focuses on the differences by agedependent categories in the clinical profile presentation management and shortterm outcomes [] Although there have been two systematic reviews and metaanalysis that analysethe clinical characteristics of COVID19 they are limited to Chinese cohorts or case series [] and a large USA cohort [] that did not analyse clinical predictors of a poor prognosisClinically the same main symptoms of cough and fever are reported in all series Howeverin Barcelona city we have observed diarrhoea anosmia and dysgeusia which is hardlyreported in the Chinese series [] which unlike ours comes principally from hospitals diarrhoea occurred in of cases very similar to the in New York [] and clearly higherthan the reported in China Nearly of patients had anosmia and dysgeusia similar tothe results obtained in French patients [] In contrast expectoration was found in only compared with in the Chinese seriesPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsTable Analytical and radiological predictors of hospitalization and ICU admissiondeathVariablesTotalDeath or ICU admission n Hospitalization n n NoYesPAdjusted ORNoYesn n [ CI]n n PAdjusted OR[ CI]Alterations in chest Xray”nototalno  Bilateral pulmonary infiltratesInterstitialground glass patternLobar pulmonary infiltrateAlterations in chest CAT scan”nototal no ¡ [“] [“] [“] [“] [“] [“]Bilateral pulmonary infiltrates Interstitialground glass pattern Laboratory parameters”nototalno [“] [“] [“] [“]Leukocytes mm3 [“] [“]Lymphocytes mm3Platelets mm3 [“] [“] [“] [“]Haemoglobin gdl [“] [“]Creactive protein mglitreProcalcitonin ngmlLactate dehydrogenase UlitreAminotransferase aspartate UlitreAlanine aminotransferase UlitreTotal bilirubin mgdL [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“]Creatine kinase Ulitre [“] [“]Creatinine 15mgdL Ddimer mglitreSodium mEqlitrePotassium mEqlitre [“] [“] [“] [“] [“] [“] [“] [“]In bold statistically significant independent predictive factors associated with hospitalization death or ICU admission logistic multivariate regression adjusted for ageand sex  patients had a chest Xray The alterations with a frequency patients were pneumothorax n and pleural effusion n Chest Xray resultswere not available in patients¡ patients had a chest CAT scan Alterations with a frequency of patients were pulmonary thromboembolism n emphysema n lobarpulmonary infiltrates n pneumonia n atelectasis n and pleural effusion n CAT scan results were not available in five patients101371journalpone0237960t003PLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsTable Predictors of the evolution complications and treatment in patients hospitalized or with ICU admissiondeathVariablesTotalDeath or ICU admission n Hospitalization n n NoYesPAdjusted OR [NoYesPAdjusted OR [n n CI]n n CI]Complications”no  Any complicationPneumonia NA [“] [“] [“]Adult respiratory distress [“ [“]syndromeRenal failurePulmonary thromboembolismTreatments”no ¡HydroxychloroquineAzithromycinLopinavirRitonavir [“] [“] [“]NA] [“] [“] [“] [“] [“] [“]Oxygen therapy [“] [“]Intravenous antibiotics [“] [“]GlucocorticoidsTocilizumabCephalosporinsLow molecular weight heparin Remdesivir Covid19 infection”no [“] [“] [“] [“] [“] [“] [“] [“] [“] [“]Any cohabitant [“] [“]Any work colleague [“] [“]Any contact person in other [“] [“]settingsIn bold statistically significant independent predictive factors associated with hospitalization death or ICU admission logistic multivariate regression adjusted for ageand sex  Complications in patients were sepsis n multian failure n electrolyte alterations n hematologic alterations n and lung cancer n ¡ Treatments with a frequency of patients except remdesivir were amoxicillin n interferon n rituximab n darunavir n and entecavirn NA not applicable101371journalpone0237960t004Chinese patients had a mean age of years ten years lower than our series and ofour patients were aged � years compared with and in China Germany andthe USA respectively but in Italy [ “] Older age and male sex predisposed to ahigher mortality rate in our and all large series [ ] In our patients comorbidities werethree times higher than in the Chinese cohort [] and were similar to the findings of the NewYork study [] Any comorbidity was a risk factor for hospitalization ICU admission anddeath Depression was an independent risk factor for hospitalization which has not beenobserved in other cohorts studied Depression was often accompanied by a vulnerable socialsituation which may have justified hospitalization Likewise autoimmune diseases were independent risk factor for ICU admission and death Various hypotheses have been postulated onpossible autoimmune alterations in the pathogenic evolution of the disease With respect toPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientstreatment no drug has proved effective against Covid19 until now Moreover many treatments were unavailable in the outpatient setting Currently we are only certain that treatmentwith tocilizumab showed better survival rates in retrospective cohorts [] although its efficacy has not been tested in randomized clinical trials Therefore the results on the outcomesassociated with treatment should be interpreted with cautionThe same comorbidities were identified with hypertension and diabetes being the twomost common while in the USA and Italy obesity seems to be higher Our results show thatobesity was close to being an independent risk factor for hospitalization OR CI to Strikingly of our patients were healthcare workers compared with in Wuhanand in Germany [ ] Although these studies recognized an important degree ofunderreporting of cases in health workers the difference remains important There are at leasttwo possible explanations first the lack of personal protective equipment in the initial phaseof the epidemic a constant revindication of health professionals who felt undersupplied Secondly many cases were health professionals from primary healthcare or the reference hospitalwho reside in the same area where they workIn all reported series bilateral pneumonia was the most common radiological finding waspresent in more than half the cases [] and was a factor of a poor prognosis and mortality Incontrast an interstitial radiological pattern did not confer an increased risk of mortality TheWuhan study reported a CAT scan use of compared with in Barcelona In contrast chest Xrays were carried out in and respectively the availability of diagnostic means was higher in China A recent international consensus states that radiologicalassessment is not necessary in asymptomatic patients or those with mild disease but is requiredin patients with moderate or severe disease regardless of whether a definite diagnosis ofCOVID19 has been made [] In addition simple chest Xrays are preferable in a resourceconstrained environment with difficulties in accessing CAT scans [] The possible use of pulmonary ultrasound for the pointofcare diagnosis of COVID19 pneumonia has not been sufficiently analysed but might be an efficient alternative due to its portability and reliability []In fact the regional Catalan government has recently acquired ultrasound machines toenable family physicians to make doctors can make pointofcare home or nursing homediagnoses of pneumonia [] Biologically lymph ia and increased CRP LDH and Ddimer were usually constant and similar in all series and were associated with an increased riskof mortality A differential variable in our series is a greater number of alterations in liver testswhich was present in “ of patients data similar to the USA and Italian cohorts but different from the Chinese cohort where it was [] We also found hypokalaemia in of patients a factor not reported in other studiesWe found a hospitalization rate of compared with “ in the USA and inChina and an ICU admission rate of which was similar to the Chinese USA “ and German results While the protocols of action and admission are similarand depend on the level of clinical involvement the therapeutic protocols differ between hospitals cities and countries There remain many unknowns in the treatment of COVID19The only truth is that we do not have a vaccine an etiological treatment or a treatment withsufficient scientific evidence to generalize its use Currently the systematic review of antiretroviral treatments has not offered conclusive results [] and despite in vitro results for hydroxychloroquine COVID19 infections are currently intractable [ ]The mortality rate in our study was compared with in New York in hospitalized patients in China in Germany and in Italy Different informationand recording systems the availability of diagnostic tests and above all the anization ofnational health systems may have contributed to the differences observedPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsThe study had some limitations due to the observational retrospective design However itis sufficiently representative of the population with confirmed COVID19 to permit betteridentification of the factors of a poor prognosis of the disease from a clinical perspective Wecannot rule out some heterogeneity in data codification due to observers™ interpretations of themedical records However this bias is minimal as most clinical factors included are clearlydefined in the electronic medical record Another limitation of this study is the percentage ofpatients without laboratory parameters more than Even though in real clinical practicethese percentages may be expected the results corresponding to laboratory parameters shouldbe interpreted with cautionFour months after the declaration of the pandemic there is not a sufficiently reliable available and generalizable diagnostic test that can analyse the seroprevalence of COVID19 evenin the most industrialized countries Given this lack determining the clinical biological andradiological characteristics of probable cases of COVID19 infection will be key to the initiation of early treatment and isolation and for contact tracing especially in primary healthcareSupporting informationS1 DatasetXLSXAcknowledgmentsThe authors thank David Buss for editorial assistanceAuthor ContributionsConceptualization Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuData curation Antoni Siso´Almirall Belchin Kostov Minerva MasHeredia Sergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana August AnguitaGuimetJaume BenaventÃreuFormal analysis Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuInvestigation Antoni Siso´Almirall Belchin Kostov Minerva MasHeredia Sergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana August AnguitaGuimetJaume BenaventÃreuMethodology Antoni Siso´Almirall Belchin Kostov Minerva MasHeredia Sergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana August AnguitaGuimetJaume BenaventÃreuProject administration Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuResources Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuSupervision Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuValidation Antoni Siso´Almirall Belchin Kostov Minerva MasHeredia Sergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana August AnguitaGuimetJaume BenaventÃreuPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsVisualization Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuWriting “ original draft Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuWriting “ review editing Antoni Siso´Almirall Belchin Kostov Minerva MasHerediaSergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana AugustAnguitaGuimet Jaume BenaventÃreuReferences World Health anization Pneumonia of unknown cause”China wwwwhointcsrdon05january2020pneumoniaofunkowncausechinaen accessed April Centers for Disease Control and Prevention Novel Coronavirus 2019nCoV Situation Summarystackscdcgovviewcdc84806cdc_84806_DS1pdf accessed April Holshue ML DeBolt C Lindquist S Lofy KH Wiesman J Bruce H First Case of Novel Coronavirus in the United States N Engl J Med “ 101056NEJMoa2001191 PMID World Health anization Novel Coronavirus2019nCoV wwwwhointdocsdefaultsourcecoronavirusesituationreports20200130sitrep10ncovpdfsfvrsnd0b2e480_2 accessed AprilJohns Hopkins University CSSE COVID19 Dashboard by the Center for Systems Science and Engineering CSSE at Johns Hopkins University JHU gisanddatamapsarcgiscomappsopsdashboardindexhtmlbda7594740fd40299423467b48e9ecf6 accessed April Ministerio de Sanidad Gobierno de España Situacio´n del COVID19 en España covid19isciiies accessed April Guan WJ Ni ZY Hu Y Liang WH Ou CQ He JX Clinical Characteristics of Coronavirus Disease in China N Engl J Med 101056NEJMoa2002032 PMID Yeo C Kaushal S Yeo D Enteric involvement of coronaviruses is faecaloral transmission of SARSCoV2 possible Lancet Gastroenterol Hepatol “ 101016S2468 PMID Chang BB Chiu TY Ready for a long fight against the COVID19 outbreak an innovative model oftiered primary health care in Taiwan BJGP 103399bjgp 20X101068PMID Barrasa H Rello J Tejada S Martı´n A Balziskueta G Vinuesa C SARSCov2 in Spanish Intensive Care Early Experience with 15day Survival In Vitoria Anaesth Crit Care Pain Med 101016jaccpm202004001 PMID Borobia AM Carcas AJ Arnalich F A´ lvarezSala R MonserratVillatoro J Quintana M A Cohortof Patients with COVID19 in a Major Teaching Hospital in Europe J Clin Med Martı´nSa´nchez FJ Del Toro E Cardassay E Valls Carbo´ A Cuesta F Vigara M Clinical presentation and outcome across age categories among patients with COVID19 admitted to a Spanish Emergency Department Eur Geriatr Med 101007s41999020003592 PMIDFu L Wang B Yuan T Chen X Ao Y Fitzpatrick T Clinical characteristics of coronavirus disease COVID19 in China A systematic review and metaanalysis J Infect 101016jjinf202003041 PMID RodriguezMorales AJ CardonaOspina JA Gutie´rrezOcampo E VillamizarPeña R HolguinRiveraY EscaleraAntezana JP Clinical laboratory and imaging features of COVID19 A systematicreview and metaanalysis Travel Med Infect Dis 101016jtmaid2020101623PMID Richardson S Hirsch JS Narasimhan M Crawford JM McGinn T Davidson KW PresentingCharacteristics Comorbidities and Outcomes Among Patients Hospitalized With COVID19 inthe New York City Area JAMA 101001jama20206775 PMID Goyal P Choi JJ Pinh

Thyroid_Cancer

"Immune checkpoint inhibitors ICIs can induce immunerelated adverse events irAEs includingthyroid dysfunction There are only a few reports on Graves™ disease induced by ICIs We report a case of newonsetGraves™ disease after the initiation of nivolumab therapy in a patient receiving gastric cancer treatmentCase presentation The patient was a 66yearold Japanese man who was administered nivolumab mg every weeks as a thirdline therapy for stage IVb gastric cancer His thyroid function was normal before the initiation ofnivolumab therapy However he developed thyrotoxicosis before the third administration of nivolumab Elevatedbilateral and diffuse uptake of radioactive tracer was observed in the 99mTcpertechnetate scintigraphyFurthermore the thyroidstimulating hormone receptor antibody TRAb and thyroidstimulating antibody TSAbtest results which were negative before the first administration of nivolumab were positive after starting thetherapy The patient was diagnosed with Graves™ disease and the treatment with methimazole and potassiumiodide restored thyroid functionConclusions This is the first complete report of a case of newonset Graves™ disease after starting nivolumabtherapy confirmed by diffusely increased thyroid uptake in scintigraphy and the positive conversion of antibodiesagainst thyroidstimulating hormone receptor It is important to perform thyroid scintigraphy and ultrasonographyto accurately diagnose and treat ICIinduced thyrotoxicosis because there are various cases in which Graves™ diseaseis developed with negative and positive TRAb titresKeywords Graves™ disease Nivolumab Thyrotoxicosis Immune checkpoint inhibitor 99mTcpertechnetatescintigraphy Thyroidstimulating hormone receptor antibodyBackgroundImmune checkpoint inhibitors ICIs such as cytotoxicTlymphocyteassociated protein CTLA4 programmed cell death protein1 PD1 and programmeddeath ligand PDL1 inhibitors have been widely usedas a standard cancer treatment during recent yearsimmunerelatedHowever occasionallycauseICIs Correspondence yhiroshinmsacjp1Department of Endocrinology Diabetes and Metabolism Graduate Schoolof Medicine Nippon Medical School Sendagi Bunkyoku Tokyo JapanFull list of author information is available at the end of the adverse events irAEs which affect different anssuch as the lung gastrointestinal tract liver nervous system skin and endocrine glands The endocrine irAEsinclude hypophysitis thyroid dysfunction adrenal insufficiency and type diabetes While endocrine irAEs dueto CTLA4 inhibitors such as ipilimumab and tremelimumab mainly include pituitary dysfunction those dueto PD1 inhibitors such as nivolumab and pembrolizumab are mainly related to thyroid dysfunction [“]The PD1 inhibitorinduced thyroid dysfunction oftenincludes hypothyroidism rather than hyperthyroidism [ ] Thyrotoxicosis following ICI therapy is caused The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cYamada BMC Endocrine Disorders Page of spontaneouslyrecover withmostly by thyroiditis syndrome which has been reportedsubsequenttohypothyroidism in many cases[ ] HoweverGraves™ disease induced by ICI treatment has not beenextensively explored Here we present a case of Graves™disease shortly after the initiation of nivolumab therapyfor gastric cancerthecancerCase presentationA 66yearold man was diagnosed with stage IVbT4bN0M1 human epidermal growth factor receptor HER2positive gastricat Nippon MedicalSchool Chiba Hokusoh Hospital one and a half yearsbefore the onset of thyrotoxicosis After diagnosis hewas not referred for surgery because of liver metastasiswith a portal tumour thrombus rather the patient received cycles of first line chemotherapy with a combination of tegafurgimeraciloteracil S1 cisplatin andtrastuzumab However the patient presented with progressive disease assessed based on the computed tomography CT and oesophagogastroduodenoscopy OGDevaluations following the first line therapy Hence he received a second line chemotherapy with paclitaxel andramucirumab After cycles of the second line chemotherapy although there was a reduction in tumour sizeafter cycles the patient presented with progressivedisease as assessed by CT At this stage nivolumab mg every weeks was started The patient had anormal thyroid function before the first administrationHowever TSH suppression was observed before the second administration and thyrotoxicosis occurred beforethe third administration of the drug hence nivolumabtherapy was discontinued and the patient was referred toour departmentThe patient had complained of fatigue and shortnessof breath during exertion His height was cm bodyweight was kg heart rate was beats per minute and blood pressure was mmHg There wasno evidence of Graves™ orbitopathy or pretibial myxedema He and his family members had no history ofandTgAbantibody ngmL Thyroidthyroid diseases Thethyroidstimulating hormoneTSH free triiodothyronine FT3 and free thyroxineFT4 levels were μIUmL pgmL and ngdL respectively Table The titres of thyroidstimulating hormone receptor antibody TRAb andthyroidstimulating antibody TSAb were positive IUL and respectively whereas those of antithyroglobulinantithyroidperoxidase antibody TPOAb were negative IULrespectively The thyroglobulin Tgand IULlevel wasultrasonographyshowed slight goitre Fig 1a and rich blood flow in theparenchyma Fig 1b 99mTcpertechnetate scintigraphywhich was performed on the first consultation day of thepatient at our department showed elevated bilateraland diffuse uptake of the radioactive tracer Fig Wemeasured antithyroid autoantibodiesin preservedserum samples The titres of TRAb and TSAb werenegative before the first administration of nivolumabwhereas they were positive IUL and respectively before the second administration Thus we diagnosed his thyrotoxicosis as newonset Graves™ diseaseafter the initiation of nivolumab therapy The humanleukocyte antigen HLA typing of the patient showedthe following allelic variants A24022601 B510154 C01021502 DRB104051501 DQA1010203 DQB104010602 DPA10202 and DPB10501We treated the patient with methimazole MMI at adose of mgday and potassium iodide KI at a doseof mgday One month after the initiation of the therapy when the FT3 and FT4 levels of the patient werenormal we discontinued KI Gradually we reduced thedosage of MMI and the continued administration tillthe death of the patient of MMI at a dose of mg everyalternate day stabilised his thyroid function Fig Furthermore as nivolumab was found to be ineffectivebased on the CT and OGD evaluations the patient received irinotecan therapy However after cycles ofchemotherapy the patient was diagnosed with brain metastasis by magnetic resonance imaging MRIforTable TSH FT3 FT4 and Tg levels and TRAb and TSAb titres in our patientTSH μIUmLFT3 pgmLFT4 ngdLTRAb IULTSAb Tg ngmLDay of nivolumab administration NA NANA NANANANANADay first administration of nivolumab Day second administration of nivolumabThe normal range of the thyroid parameters is as follows TSH “ μIUmL FT3 “ pgmL FT4 “ ngdL TRAb IUL TSAb ‰ and Tg ‰ ngmL 0cYamada BMC Endocrine Disorders Page of Fig Thyroid ultrasonography of the patient a Slight swelling in isthmus b Rich blood flow in parenchymawhich he received gamma knife and steroid therapyThe patient died months after his first visit to ourdepartmentDiscussion and conclusionsWe present a case of newonset Graves™ disease after theinitiation of nivolumab therapy in a patient receivinggastric cancer treatment Thyrotoxicosisinduced byICIs is mainly a form of destructive thyroiditis Threecases of newonset Graves™ disease during nivolumabtherapy other than the present case have been reported[“] Table Iadarola [] reported a case ofGraves™ diseaselike hyperthyroidism after the second administration of nivolumab in a patient with left lungIn this case 99mTcpertechnetate scintigcarcinomaraphy in the patient with T3toxicosis showed diffusethyroid uptake of the radionuclide suggesting Graves™diseaselike hyperthyroidism whereas the TRAb testswere consistently negative Thyroid ultrasonographyshowed a multinodular goitre with a normoechoic pattern and normal vascularity ofthe parenchyma []Brancatella [] reported a case similar to that ofIadarola [] with diffuse thyroid uptake and negative TRAb titre In this case ultrasonography showed anenlargement of the thyroid with a hypoechoic patternand mild hypervascularity Kurihara [] reported acase of simultaneous development of Graves™ disease andtype diabetes mellitus during nivolumab therapy InFig 99mTcpertechnetate scintigraphy showing elevated bilateral and diffuse uptake of the radioactive tracer 0cYamada BMC Endocrine Disorders Page of Fig Clinical course of the patient MMI methimazole KI potassium iodide Day first administration of nivolumab Day secondadministration of nivolumabTable Comparison of case reports on newonset Graves™ disease during nivolumab therapyStudyTSHμIUmL FT3pgmLFT4ngdLTRAb IULBeforeNAIadarola []AfterNegativeTSAb BeforeNANAAfterNANANAUSNormalHypervascularNormalRAIU99mTcuptakeHigh99mTcHighRAIUNAHLANANADRB104Brancatella []NANegativeKurihara []NAPositive NAYamada presentcaseUS ultrasonography RAIU radioactive iodine uptake Before before the initiation of nivolumab therapy After after the initiation of nivolumab therapy at theonset of the thyrotoxicosisNegative NegativeHypervascular PositivePositiveHigh99mTcDPB105 0cYamada BMC Endocrine Disorders Page of this case thyrotoxicosis was detected after the sixth administration of nivolumab with positive TRAb titreHowever ultrasonography showed no enlargement ofthe thyroid and a normal vascularisation pattern Thispatient was clinically diagnosed as mild Graves™ diseaseand treated with MMI [] Unlike these cases our caseis important in terms of confirmation of both positiveTRAb titre and diffuse thyroid uptake in scintigraphyMoreover titres of TRAb and TSAb were convertedfrom negative to positive after starting nivolumab therapy It seems reasonable to presume that Graves™ diseasewas induced by nivolumab although there is a possibilityof coincidence Furthermore our patient had HLADPB10501 which has been reported to be associatedwith Japanese Graves™ disease [ ] Although the involvement of HLA cannot be argued based only on asingle case accumulating similar cases might help clarifythe mechanism of development of rare ICIinducedGraves™ diseaseGraves™ disease induced by ICIs other than nivolumabhas been rarely reported Azmat [] reported aipilimumabinduced thyrotoxicosis caused bycase ofGraves™ disease Gan et al[] reported a case oftremelimumabinduced Graves™ hyperthyroidism Yajima [] reported a case of Graves™ disease induced bypembrolizumab a PD1 inhibitor In this case TRAbwas positive after the fifth administration of pembrolizumab and thyroid ultrasonography showed a mild increase in the intrathyroidal blood flow A thyroidscintigraphy was not performed because of the iodinetreatment [] The cases of nivolumabinduced Graves™disease with negative TRAb titre suggest that performingthyroid scintigraphy and ultrasonography can help to accurately diagnose and treat ICIinduced thyrotoxicosisA relationship between thyroid antibodies and PD1inhibitorinduced thyroid dysfunction has not been explained Kimbara [] suggested that patients withpreexisting TgAb and an elevated TSH level at baselineare at a higher risk of thyroid dysfunction induced bynivolumab Osorio [] reported an association between positive thyroid antibodies antithyroglobulin orantimicrosomal antibodies and thyroid dysfunction induced by ICIs In the studies on newonset Graves™ disease during nivolumab therapy it is interesting to notein two caucasian patients with Graves™ diseasethatTRAb was negative [ ] Furthermore TRAb waspositive in two Japanese patients including our patient[] Table However additional evidence is requiredto reveal the role of TRAb in the pathogenesis of ICIinduced hyperthyroidismA limitation of our case was radioactive iodine uptakeRAIU was not performed Because imaging with99mTcpertechnetate reflects both blood flow and uptakevia the symporter and does not assess anificationmalignant nodules may appear hyperfunctioning in pertechnetate imaging but hypofunctioning in 123IimagingIn our study tumours were not detected although a partof 99mTc uptake was strongerIn conclusion we reported a case of Graves™ diseaseshortly after the initiation of nivolumab therapy for gastric cancer Our case presented a typical Graves™ diseasewith both positive TRAb titre and diffuse thyroid uptakein scintigraphy Moreover our case is valuable in termsof confirming the conversion of TRAb and TSAb fromnegative to positive titres after starting the therapy It isimportant to perform thyroid scintigraphy and ultrasonography because there are cases of nivolumabinducedGraves™ disease with negative TRAb titre as previouslyreported To revealthe pathogenesis of ICIinducedGraves™ disease it is necessary to study additional casesof similar natureAbbreviationsCT Computed tomography CTLA4 Cytotoxic Tlymphocyteassociated protein FT3 Free triiodothyronine FT4 Free thyroxine HER2 Humanepidermal growth factor receptor HLA Human leukocyte antigenICI Immune checkpoint inhibitor IrAE Immunerelated adverse eventKI Potassium iodide MMI Methimazole MRI Magnetic resonance imagingOGD Oesophagogastroduodenoscopy PD1 Programmed cell deathprotein1 PDL1 Programmed death ligand RAIU Radioactive iodineuptake Tg Thyroglobulin TgAb Antithyroglobulin antibody TPOAb Antithyroidperoxidase antibody TRAb TSH receptor antibody TSAb Thyroidstimulating antibody TSH Thyroidstimulating hormoneAcknowledgmentsNot applicableAuthors™ contributionsHY FO and NE interpreted the data drafted the manuscript andparticipated in the endocrinological treatment of the patient HS revised themanuscript TO and SF participated in the gastroenterological treatment ofthe patient All authors have read and approved the final version of themanuscript for publicationFundingNot applicableAvailability of data and materialsThe data that support the findings of this study are stored in NipponMedical School Chiba Hokusoh Hospital Inzai Chiba and available from thecorresponding author on reasonable requestEthics approval and consent to participateThis case report was approved by the ethics committee of Nippon MedicalSchool Chiba Hokusoh HospitalConsent for publicationWritten informed consent was obtained from the patient™s next of kin forpublication of this case report and any accompanying images A copy of thewritten consent is available for review by the editor of this journalCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Endocrinology Diabetes and Metabolism Graduate Schoolof Medicine Nippon Medical School Sendagi Bunkyoku Tokyo Japan 2Department of Gastroenterology Nippon Medical SchoolChiba Hokusoh Hospital Kamagari Inzai Chiba Japan 0cYamada BMC Endocrine Disorders Page of Received April Accepted August ReferencesGonzalezRodriguez E RodriguezAbreu D Spanish Group for CancerImmunoBiotherapy GETICA Immune checkpoint inhibitors review andmanagement of endocrine adverse events Oncologist “ Michot JM Bigenwald C Champiat S Collins M Carbonnel F PostelVinay S Immunerelated adverse events with immune checkpoint blockade acomprehensive review Eur J Cancer “Bertrand A Kostine M Barnetche T Truchetet ME Schaeverbeke T Immunerelated adverse events associated with antiCTLA4 antibodies systematicreview and metaanalysis BMC Med Faje A Immunotherapy and hypophysitis clinical presentation treatmentand biologic insights Pituitary “Topalian SL Hodi FS Brahmer JR Gettinger SN Smith DC McDermott DF Safety activity and immune correlates of antiPD1 antibody in cancerN Engl J Med “Robert C Schachter J Long GV Arance A Grob JJ Mortier L et alPembrolizumab versus ipilimumab in advanced melanoma N Engl J Med“Orlov S Salari F Kashat L Walfish PG Induction of painless thyroiditis inpatients receiving programmed death receptor immunotherapy formetastatic malignancies J Clin Endocrinol Metab “Kimbara S Fujiwara Y Iwama S Ohashi K Kuchiba A Arima H et alAssociation of antithyroglobulin antibodies with the development ofthyroid dysfunction induced by nivolumab Cancer Sci “Iadarola C Croce L Quaquarini E Teragni C Pinto S Bernardo A et alNivolumab induced thyroid dysfunction Unusual clinical presentation andchallenging diagnosis Front Endocrinol Lausanne Brancatella A Viola N Brogioni S Montanelli L Sardella C Vitti P et alGraves' disease induced by immune checkpoint inhibitors a case reportand review of the literature Eur Thyroid J “Kurihara S Oikawa Y Nakajima R Satomura A Tanaka R Kagamu H et alSimultaneous development of Graves' disease and type diabetes duringantiprogrammed cell death1 therapy a case report J Diabetes Investig“ Dong RP Kimura A Okubo R Shinagawa H Tamai H Nishimura Y et alHLAA and DPB1 loci confer susceptibility to graves™ disease Hum Immunol“ Ueda S Oryoji D Yamamoto K Noh JY Okamura K Noda M et alIdentification of independent susceptible and protective HLA alleles inJapanese autoimmune thyroid disease and their epistasis J Clin EndocrinolMetab “ Azmat U Liebner D JoehlinPrice A Agrawal A Nabhan F Treatment ofipilimumab induced graves™ disease in a patient with metastatic melanomaCase Rep Endocrinol Gan EH Mitchell AL Plummer R Pearce S Perros P Tremelimumab inducedgraves hyperthyroidism Eur Thyroid J “ Yajima K Akise Y A case report of Graves' disease induced by the antihuman programmed cell death1 monoclonal antibody pembrolizumab ina bladder cancer patient Case Rep Endocrinol Osorio JC Ni A Chaft JE Pollina R Kasler MK Stephens D Antibodymediated thyroid dysfunction during Tcell checkpoint blockade in patientswith nonsmallcell lung cancer Ann Oncol “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

Thyroid_Cancer

generate a map of the copy number variations CNV segregating in a population of Murciano‘Granadina goats the most important dairy breed in Spain and to ascertain the main biologi‘cal functions of the genes that map to copy number variable regionsResults Using a dataset that comprised Murciano‘Granadina goats genotyped with the Goat SNP50 Bead‘Chip we were able to detect and autosomal CNV with the PennCNV and QuantiSNP software respec‘tively By applying the EnsembleCNV algorithm these CNV were assembled into CNV regions CNVR of which of the total CNVR count were consistently called by PennCNV and QuantiSNP and used in subsequent analyses In this set of CNVR we identified gain loss and gainloss events The total length of all the CNVR Mb represented of the goat autosomal genome Mb whereas their size ranged from kb to Mb with an average size of kb Functional annotation of the genes that overlapped with the CNVR revealed an enrichment of pathways related with olfactory transduction fold‘enrichment q‘value — ˆ’ ABC transporters fold‘enrichment q‘value — ˆ’ and bile secretion fold‘enrichment q‘value — ˆ’Conclusions A previous study reported that the average number of CNVR per goat breed was CNVR50 breeds which is much smaller than the number we found here CNVR We attribute this difference to the fact that the previous study included multiple caprine breeds that were represented by small to moderate numbers of individuals Given the low frequencies of CNV in our study the average frequency of CNV is such a design would probably underestimate the levels of the diversity of CNV at the within‘breed level We also observed that functions related with sensory perception metabolism and embryo development are overrepresented in the set of genes that overlapped with CNV and that these loci often belong to large multigene families with tens hundreds or thousands of paralogous members a feature that could favor the occurrence of duplications or deletions by non‘allelic homologous recombinationCorrespondence marcelamillsuabcat Centre for Research in Agricultural Genomics CRAG CSIC‘IRTA‘UAB‘UB Universitat Aut²noma de Barcelona Bellaterra SpainFull list of author information is available at the end of the BackgroundCopy number variations CNV encompass genomic deletions or duplications with sizes ranging from base pairs bp to several megabases Mb and which display polymorphisms in terms of copy number among individuals of a particular species [“] In livestock a broad array of phenotypes related with among others morphology [ ] pigmentation [“] sexual development The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cGuan a0et a0al Genet Sel Evol Page of [] and susceptibility to disease [] is caused by the segregation of CNV Genome scans to detect structural variations in cattle have revealed that CNV regions CNVR are often enriched in genes that are involved in immunity [“] metabolism [ ] embryo development [ ] and sensory perception [ ] There is evidence that the dNdS ratios of genes that map to taurine CNV are generally higher than those of genes that do not overlap with CNV which indicates that CNV genes probably evolve under reduced selective constraint [] The analysis of gene networks has also shown that genes that colocalize with duplications tend to have fewer interactions with other genes than loci that do not overlap with CNV reinforcing the idea that genes mapping to duplicated regions have fewer essential housekeeping functions than nonCNV genes and also have reduced pleiotropy []Although structural chromosomal variations can have strong effects on gene expression and phenotypic variability technical limitations and the moderate quality of genome assemblies have hampered CNV mapping in livestock [] Until recently this has been particularly true for goats In Fontanesi et a0al [] published the first caprine CNV map by identifying with the Bovine a0k aCGH array CNVR including and copy loss and gain variants respectively Later on resequencing the genome of individuals from several caprine breeds made it possible to identify CNV that overlap with pigmentation genes and to detect an association between increased ASIP copy number and light pigmentation [] The first worldwide survey of copy number variation in goats was performed within the Goat ADAPTmap Project httpwwwgoata daptm ap and involved the genomewide genotyping of goats from breeds [] This study resulted in the identification of CNVR among which several overlapped with genes that are functionally related with local adaptation such as coat color muscle development metabolic processes and embryonic development [] Moreover the patterns of the diversity of CNV differed according to geographic origin which indicates that they have been influenced by population history [] In another study on individuals from East African goat breeds Nandolo et a0al [] detected CNVR More recently Henkel et a0al [] demonstrated the existence of complex patterns of structural variation in the regions containing the caprine ASIP and KIT genes with potential causal effects on pigmentation In spite of these efforts the description of structural chromosomal variation in goats is still lagging behind that of other domestic species Most of the CNV surveys in goats have analyzed large populations that represent a mixture of different breeds each with a limited number of individuals [ ] thus making it difficult to assess the magnitude of the CNV diversity at the withinbreed level Our goal was to fill this gap by analyzing a population of individuals from a single Spanish breed MurcianoGranadina and to investigate the functional roles of genes that map to CNVR and compare these results with data obtained in composite goat populationsMethodsGenomic DNA extraction and a0high‘throughput genotypingBlood samples from MurcianoGranadina female goats from farms that are connected through the use of artificial insemination were collected in EDTA K3 coated vacuum tubes and stored at ˆ’ a0 °C before processing Genomic DNA was isolated by a modified saltingout procedure [] Four volumes of red cell lysis solution Tris“HCl a0 mmolL pH EDTA a0mmolL Tween were added to a0mL of whole blood and this mixture was centrifuged at —g Pelleted cells were resuspended in a0mL lysis buffer Tris“HCl a0 mmolL pH EDTA a0 mmolL SDS NaCl a0 mmolL plus µL proteinase K a0 mgmL The resulting mixture was incubated at a0°C for h followed by centrifugation at —g in the presence of a0mL of ammonium acetate a0molL The supernatant a0mL was mixed with a0mL of isopropanol which was subsequently centrifuged at —g for a0min The supernatant was removed and the DNA pellet was washed with a0 mL of ethanol After centrifuging at —g for a0min the DNA precipitate was dried at room temperature and resuspended in a0mL of TE buffer a0mmolL Tris pH a0mmolL EDTA pH Highthroughput genotyping of the MurcianoGranadina DNA samples was carried out with the Goat SNP50 BeadChip [] according to the manufacturer™s instructions Illumina Signal intensity ratios ie log R Ratio or LRR the total probe intensity of a SNP referred to a canonical set of normal controls [] and B allele frequencies or BAF relative quantity of one allele compared to the other one [] were exported for each single nucleotide polymorphism SNP with the GenomeStudio software Illumina https emeaillum inacom Then SNP coordinates were converted to the latest version of the goat reference genome ARS1 [] After filtering out unmapped and nonautosomal SNPs and those with a call rate lower than a set of SNPs remained for CNV mappingCopy number variant calling with a0PennCNV and a0QuantiSNPBased on their excellent performance in comparative studies we selected two software packages PennCNV v105 [] and QuantiSNP v2 [] to call CNV in the MurcianoGranadina population [ ] The PennCNV software [] detects CNV by applying the default 0cGuan a0et a0al Genet Sel Evol Page of parameters of the HiddenMarkov model Population frequencies of B alleles were compiled based on the BAF of each SNP in the population We used the œ“gcmodelfile option to adjust œgenomic waves [] The number of goat chromosomes was set with the œ“lastchr  instruction The QuantiSNP analysis [] assumes an objective Bayes hiddenMarkov model to improve the accuracy of segmental aneuploidy identification and mapping This CNV calling software was run under default parameters by modifying the œ“chr  option The CNV that were supported by less than three SNPs were removed from the filtered set used hereDefinition and a0functional annotation of a0copy number variant regionsWe used the EnsembleCNV algorithm beta version [] to assemble CNVR All CNV called by PennCNV andor QuantiSNP were combined to generate a set of initial CNVR by using the heuristic algorithm threshold of minimum overlap described in [] Subsequently CNVR boundaries were refined by considering the local correlation structure of the LRR values of the SNPs mapping to CNVR [] Then we reassigned the CNV calls that were initially obtained with PennCNV and QuantiSNP to each refined CNVR so that the final set of CNVR comprised only those that were simultaneously detected by both callers The resulting CNVR were matched to gene features that are annotated in the National Center for Biotechnology Information NCBI https wwwncbinlmnihgov by using BEDTools v2250 [] In addition we performed gene ontology GO enrichment and pathway analyses using the DAVID Bioinformatics Resources [ ] based on human and goat background gene sets The statistical significance was set to a qvalue ‰¤ Confirmation of a0copy number variant regions by a0quantitative real‘time PCRIn order to evaluate the rate of false positives in our experiment we conducted quantitative realtime PCR qPCR experiments to obtain an independent estimate of the copy number of four putative CNVR CNVR_371_chr5 CNVR_506_chr6 CNVR_160_chr2 and CNVR_1229_chr21 Primers were designed with the Primer Express software Applied Biosystems to amplify specific regions of the ADAMTS20 BST1 NCKAP5 and TNFAIP2 genes see Additional file a0 Table a0 S1 As reference genes we used the melanocortin receptor MC1R and glucagon GCG genes see Additional file a0 Table a0S1 loci [ “] Quantitative PCR reactions contained a0ng genomic DNA µL — SybrSelect Master mix Applied Biosystems a0pmol of each forward and reverse primer and ultrapure water to a maximum final volume of µL Each sample was analyzed in triplicate in order to obtain averaged copy number estimates Reactions were loaded onto 384well plates and run in a QuantStudio a0K Flex RealTime PCR System instrument Applied Biosystems The specificity of the PCR reactions was evaluated with a melting curve analysis procedure and the efficiency was assessed with standard curves Thus relative copy number was inferred with the qbase software Biogazelle Ghent Belgium by using the ˆ’ΔΔCt approach [] Copy number values were calibrated by taking as a reference four samples which according to Goat SNP50 BeadChip data had two copies of the investigated genomic lociResultsDetection of a0copy number variation in a0Murciano‘Granadina goatsThe initial calling with PennCNV and QuantiSNP yielded and autosomal CNV respectively By using the EnsembleCNV tool [] we assigned these CNV into CNVR with refined boundaries of which of the total CNVR count were detected simultaneously by PennCNV and QuantiSNP The resulting CNVR included copy gain copy loss and copy gainloss variants Fig a0 and Table a0 and see Additional file a0 Table a0 S2 The total length of the CNVR covered a0Mb of the goat autosomal genome a0Mb whereas their individual size ranged from a0kb to a0Mb with an average of a0kb Fig a02a and Table a0 Moreover we found that of the CNVR showed minimum allele frequencies lower than with an average frequency of Fig a02b In addition CNVR with frequencies higher than were distributed over seven caprine chromosomes With a frequency of CNVR_1229_chr21 was the CNVR with the highest frequency in the whole dataset see Additional file a0 Table a0S2 By using the BEDTools v2250 program [] of the CNVR that we detected overlapped with unique CNVR published by Liu et a0al [] Fig a0 and see Additional file a0 Table a0S2 The CNVR that were detected in both studies are referred to as œshared CNVR whereas those that were identified in our study only are referred to as œnonshared CNVR Fig a0 Six of the ten œshared CNVR with frequencies higher than show positional concordance with six CNVR detected by Liu et a0al [] see Additional file a0 Table a0S2Functional annotation of a0the a0genes that a0are located in a0copy number variable regionsWithin the CNVR defined in our study we detected proteincoding genes according to the goat reference genome annotation ARS1 [] from the NCBI database see Additional file a0 Table a0S2 and Additional file a0 0cGuan a0et a0al Genet Sel Evol Page of Fig Genomic distribution of CNVR detected with the PennCNV and QuantiSNP software on the caprine autosomes Squares triangles and circles represent copy number gain loss and gainloss events respectively Red and black colors represent shared and non‘shared CNVR respectively Shared CNVR are those detected both in our study and in Liu et al [] while non‘shared CNVR are those identified only in our studyTable a0S3 In a survey of the diversity of CNV in goats with a worldwide distribution Liu et a0 al [] detected copy number variable genes of which were also identified in our study and are referred to as œshared copy number variable genes see Additional file a0 Table a0S3 Among the œshared copy number variable genes the ASIP and ADAMTS20 genes are particularly relevant they are involved in pigmentation [ “] and colocalize with selection signals detected in a worldwide sample of goats [] In addition we found that about of the annotated genes that colocalize with CNVR are olfactory receptors or olfactory receptorlike genes see Additional file a0 Table a0S3 Consistently the most significantly enriched pathway was œOlfactory transduction qvalue — ˆ’ Table a0 followed by œABC transporter qvalue — ˆ’ Table a0 A significant pathway related with immunity ie Fc epsilon RI signaling qvalue was also identified based on a human background gene set Table a0 Several overrepresented GO terms were related with embryonic skeletal system morphogenesis qvalue — ˆ’ and Gprotein coupled purinergic nucleotide receptor activity 0cGuan a0et a0al Genet Sel Evol Page of Table Main features of a0 copy number variation regions CNVR detected in a0 MurcianoGranadina goatsSummary statisticsTotalGainLossGainlossTotal length MbTotal number of CNVRNumber of CNVR kbNumber of CNVR “ kbNumber of CNVR “ kbNumber of CNVR “ kbNumber of CNVR kb“ MbNumber of CNVR ‰¥ MbAverage number of SNPs per CNVRMinimum size of CNVR kbMaximum size of CNVR kbAverage CNVR size kbStandard deviation of CNVR size kb qvalue — ˆ’ Table a0 Interestingly the copy number variable genes were also enriched in pathways with metabolic significance such as prolactin signaling and insulin signaling as well as GO terms related with feeding behavior but none of these pathways reached the significance threshold qvalue ‰¤ after correction for multiple testing see Additional file a0 Table a0S4 Several of the pathways outlined in Additional file a0 Table a0S4 play important roles in immunity eg chemokine signaling B cell receptor signaling and T cell receptor signaling cancer eg endometrial cancer proteoglycans in cancer thyroid cancer as well as in oncogenic signaling eg Ras and ErbB signaling see Additional file a0 Table a0S4 but most of them are not significant after correction for multiple testingFig Histograms displaying the distribution of CNVR according to their size a and frequency b CNVR that were longer than kb were included in the ‘kb bin whereas those with frequencies above were grouped in the bin The histograms were drawn by using the ggplot2 package httpggplo t2tidyv erse implemented in R https wwwr‘proje ct 0cGuan a0et a0al Genet Sel Evol Page of Table Functional enrichment of a0genes colocalizing with a0CNVR detected in a0 MurcianoGranadina goatsBackground gene setCategory IDTermNumber of a0genesFold enrichment P valueq‘valueGoatGoatGoatHumanHumanHumanHumanHumanHumanHumanKEGGKEGGKEGGKEGGGOBPGOBPGOBPGOCCGOMFGOMFOlfactory transductionABC transportersBile secretionFc epsilon RI signaling pathwaychx04740chx02010chx04976hsa04664GO0009952 Anteriorposterior pattern specificationGO0048704 Embryonic skeletal system morphogenesisGO0035589 G‘protein coupled purinergic nucleotide receptor signaling pathwayGO0016020 MembraneGO0003677 DNA bindingGO0045028 G‘protein coupled purinergic nucleotide receptor activity126Eˆ’ 161Eˆ’333Eˆ’ 427Eˆ’446Eˆ’ 570Eˆ’140Eˆ’ 176Eˆ’936Eˆ’ 161Eˆ’713Eˆ’ 122Eˆ’418Eˆ’ 716Eˆ’145Eˆ’ 198Eˆ’110Eˆ’ 160Eˆ’424Eˆ’ 622Eˆ’KEGG Kyoto Encyclopedia of Genes and Genomes pathway GOMF gene ontology GO term related with molecular function GOBP GO term related with biological process GOCC GO term related with cellular componentValidation of a0four copy number variants by a0real‘time quantitative polymerase chain reactionIn order to confirm our results we selected four CNVR ie CNVR_371_chr5 CNVR_506_chr6 CNVR_160_chr2 and CNVR_1229_chr21 that colocalized with the ADAMTS20 BST1 NCKAP5 and TNFAIP2 genes respectively the primers used to amplify these CNVR are listed in Additional file a0 Table a0 S1 As shown in Fig a0 the estimated copy numbers obtained by qPCR analysis of MurcianoGranadina goat samples were to copies relative to the calibrator ADAMTS20 to copies BST1 to copies NCKAP5 and to copies TNFAIP2 According to D™haene et a0 al [] copy number estimates between and most likely correspond to a normal copy number of whereas any number below or above these thresholds could represent a deletion or a duplication respectively Thus based on these values evidence of copy number variation was inferred for three of the four genes analyzed by qPCRFig Relative quantification of four copy number variation regions by real‘time quantitative polymerase chain reaction analysis a CNVR_371_chr5 ADAMTS20 b CNVR_506_chr6 BST1 c CNVR_160_chr2 NCKAP5 d CNVR_1229_chr21 TNFAIP2 The x and y axes represent sample ID and relative quantification of CNVR mean ± standard error with each sample analyzed in triplicate respectively As calibrator we used the average of four samples estimated to have two copies diploid status based on the Goat SNP50 BeadChip analysis 0cGuan a0et a0al Genet Sel Evol Page of DiscussionIn this work our aim was to characterize copy number variation in MurcianoGranadina goats a native Spanish breed used for milk production By genotyping MurcianoGranadina goats with a SNP array we were able to identify CNVR covering of the goat genome whereas Liu et a0 al [] identified CNVR that covered of the goat genome The latter higher percentage reported by Liu et a0al [] can be explained by the fact that they analyzed breeds with different geographical origins ie a composite population that is probably much more diverse than that used in our work Besides the pipeline that we used to identify CNVR is more stringent than that employed by Liu et a0al [] removing CNVR that were not consistently detected by PennCNV and QuantiSNP In the literature estimates of to for CNVR coverage in the human genome are reported [] Our results and those obtained by Liu et a0al [] are consistent with these valuesIndeed when Liu et a0 al [] calculated the CNVR length for each breed normalized by the goat genome size their results agreed well with our estimate of For instance this parameter reached values of in goats from Southeastern Africa and in goats from Northwestern Africa and Eastern Mediterranean whereas it was lowest for individuals from West Asia [] The number of CNV detected at the withinbreed level by Liu et a0 al [] was on average CNV per breed and ranged from to whereas the average number of CNVR was only per breed [] Since the number of detected CNVR is proportional to population size for most of the breeds investigated in [] the level of withinbreed CNV variation is probably underestimated In summary one important conclusion from our study is that the magnitude of CNV diversity at the withinbreed level is likely to be much larger than that previously reported in studies that analyzed multiple populations each represented by a small or moderate number of individualsMost of the CNVR that we report here ranged in size from to a0kb with a mean size of a0kb Similarly the average CNVR size reported by Liu et a0al [] was a0kb Both estimates are quite large and reflect that mediumdensity SNP arrays are not well suited to detect small CNVR in spite of their high abundance In cattle the average sizes of CNVR detected with the Illumina BovineHD Genotyping BeadChip a0K SNPs [] Illumina wholegenome sequencing and PacBio sequencing [] were and a0 kb respectively Another consistent feature of CNVR is that in general their frequencies are low or very low In our study approximately of the CNVR had frequencies lower than and the average frequency was Liu et a0al [] reported lower CNVR frequencies ranging from Alpine and Northern European goats to Northwestern African goats This decreased average CNVR frequency is not very significant and probably reflects differences in sampling size and the use of composite populations with multiple breeds each one with its specific CNVR frequenciesThe CNVR detected in our study covered proteincoding genes Pathway analyses reflected a substantial enrichment of genes that are involved in olfactory perception which is consistent with previous reports in cattle [ ] In this regard there is an important difference between our results and those by Liu et a0al [] Whereas in the study of Liu et a0al [] the term œsensory perception was underrepresented among the CNV genes fold enrichment in our work the terms œolfactory transduction fold enrichment and œGprotein coupled purinergic nucleotide receptor activity fold enrichment were overrepresented and many CNV genes were olfactory receptors The two terms mentioned before are closely related because a broad array of purinergic receptors are differentially expressed in the olfactory receptor neurons that modulate odor responsiveness [] Moreover purinergic nucleotides are important neuromodulators of peripheral auditory and visual sensory systems [] In cattle Keel et a0al [] reported that œsensory perception of smell and œGprotein coupled receptor signaling pathway were significantly overrepresented in the proteincoding genes that overlapped with CNVR Similarly Upadhyay et a0 al [] showed that œsensory perceptions of smell and œchemical stimuli are enriched in their set of CNV genes A potential explanation for the underrepresentation of the œsensory perception functional category among the genes overlapping CNV reported by Liu et a0al [] could be that in goats these genes are not well annotated yet so the majority of them are identified with a LOC prefix and a number and as a consequence of this they are not correctly detected by PANTHER [] thus biasing the results obtained in the gene ontology enrichment analysisLoci belonging to large multigene families might be more prone to colocalize with CNV because paralogous genes can act as templates in nonallelic homologous recombination events which promote increases or reductions in copy number [] It should be noted that olfactory receptor genes constitute the largest gene superfamily and in humans more than genes and pseudogenes have been identified [] In cattle olfactory receptor genes and pseudogenes are distributed in clusters across bovine chromosomes [] and similar numbers have been reported for pigs [] Moreover purifying selection against CNV is probably less 0cGuan a0et a0al Genet Sel Evol Page of intense in regions that contain olfactoryreceptor genes than in genomic regions that contain genes with essential functions [] Interestingly copy number changes in the olfactory receptor genes of wild and domestic mammals might have consequences on food foraging as well as on mate and predator recognition [ ]In the set of genes that colocalize with CNVR we also detected an enrichment of loci related with the multigene family of ATP binding cassette ABC transporters a result that agrees well with previous findings in humans [“] and cattle [ ] In mammals ABC transporters fulfill the mission of carrying a broad array of endogenous substrates such as amino acids peptides sugars anions and hydrophobic compounds and metabolites across lipid membranes At least ABC genes that belong to eight subfamilies have been identified in the human genome [] Copy number variation in the human ABCC4 and ABCC6 genes is associated with susceptibility to esophageal squamous cell carcinoma [] and to the rare autosomal recessive disease pseudoxanthoma elasticum [] respectively Moreover largescale deletions of the human ABCA1 gene are a causative factor for hypoalphalipoproteinemia [] a disease that is characterized by the complete absence of the apolipoprotein AI and extremely low levels of plasma highdensity lipoprotein HDL cholesterol We also found a highly significant enrichment of pathways related with embryo development anteriorposterior pattern specification embryonic skeletal system morphogenesis as previously reported [] These pathways are featured by genes that belong to the Hox multigene family of transcription factors possibly reflecting the genomic instability of certain homeobox gene clusters as evidenced by the existence of many syntenyparalogy breakpoints and assembly gaps as outlined in comparative studies []Although not significant after correction for multiple testing we detected an enrichment of pathways with metabolic significance such as prolactin and insulin signaling which could have an impact on milk production and growth [“] Interestingly the comparison of our work with that of Liu et a0al [] revealed proteincoding genes that colocalize with the set of shared CNVR One of the most relevant shared genes encodes ASIP a protein that increases the ratio of pheomelanin to eumelanin by binding to the melanocortin receptor and delivering an antagonist signal that blocks the downstream expression of eumelanogenic enzymes [] Mutations in the ASIP gene play critical roles in animal pigmentation [] For instance the causal factor of the white color typical of many sheep breeds is the ubiquitous expression of a duplicated copy of the ASIP coding sequence which is regulated by a duplicated promoter corresponding to the itchy E3 ubiquitin protein ligase gene [ ] Although some studies proposed that the ASIP CNV might be associated with different pigmentation patterns in goats [ ] no functional assay has verified an association of ASIP copy number with ASIP mRNA levels Another interesting shared copy number variable gene is ADAMTS20 which was also identified in two previous CNV surveys [ ] This gene encodes a metalloproteinase with an important role in melanoblast survival by mediating Kit signaling [] and in palatogenesis [] Bertolini et a0al [] performed a selection scan in white vs colored black and red goats and detected a selective sweep in the ADAMTS20 gene In the light of these results the potential involvement of a structural variation in ADAMTS20 in goat pigmentation should be explored further Moreover it is worthwhile to mention that several CNVR genes have functions related with production and reproduction traits For instance the NCKAP5 gene which colocalizes with CNVR_160_chr2 frequency is associated with milk fat percentage in cattle [] Taking the above evidence into account the implication of structural chromosomal variations in the genetic determinism of traits of economic interest with a complex inheritance deserves further exploration by designing tools that allow inferring CNVR genotypes with high confidenceConclusionsWith the PennCNV and QuantiSNP software we detected CNVR in the genome of the MurcianoGranadina breed In a a0 previous study [] that used a less stringent pipeline only PennCNV was used and included multiple populations with small to moderate sample sizes the average number of CNVR events per breed was One conclusion of our study is that CNV surveys which are based on a broad array of breeds represented by only a few individuals underestimate the true levels of the CNV diversity at the withinbreed level The main reason for this outcome is that since the majority of CNV have very low frequencies they cannot be detected efficiently when sample size is small and in consequence much of the existing variation is missed We have also found that genes that overlap with CNV are functionally related with olfactory transduction embryo development ABC transporters and Gprotein coupled purinergic nucleotide receptor activity Most of these genes belong to large multigene families encompassing tens hundreds or thousands of paralogous genes that could act as substrates in nonallelic homologous recombination events which is one of the main mechanisms generating duplications and deletions in humans and other species Finally we detected CNV that colocalize with the ASIP and ADAMTS20 pigmentation genes 0cGuan a0et a0al Genet Sel Evol Page of which according to previous studies have been subjected to positive selection for coat color in goatsSupplementary informationSupplementary information accompanies this paper at https doi101186s1271 ‘‘ ‘ Additional file a0 Table a0S1 List of primers used in the real‘time quanti

Thyroid_Cancer

molecular heterogeneity of renal cell carcinoma RCC complicates the therapeutic interventions for advancedmetastatic disease and thus its management remains a significant challenge This study investigates the role of thelncRNA CDKN2BAS1 and miR1413p interactions in the progression and metastasis of kidney cancer Human renalcancer cell lines ACHN and Caki1 normal RPTEC cells tissue cohorts and a series of in vitro assays and in vivo mousemodel were used for this study An overexpression of CDKN2BAS1 was observed in RCC compared to normal samplesin TCGA and our inhouse SFVAMC tissue cohorts Reciprocally we observed reduced expression of miR141 in RCCcompared to normal in the same cohorts CDKN2BAS1 shares regulatory miR141 binding sites with CCND1 andCCND2 genes Direct interactions of CDKN2BAS1miR141Cyclin D1“D2 were confirmed by RNA immunoprecipitationand luciferase reporter assays indicating that CDKN2BAS1miR141Cyclin D1“D2 acts as a ceRNA network in RCCFunctionally attenuation of CDKN2BAS1 andor overexpression of miR141 inhibited proliferation clonogenicitymigrationinvasion induced apoptosis in vitro and suppressed tumor growth in xenograft mouse model Furtheroverexpression of CDKN2BAS1 is positively correlated with poor overall survival of RCC patients Expression of miR141also robustly discriminated malignant from nonmalignant tissues and its inhibition in normal RPTEC cells induced procancerous characteristics CDKN2BAS1 attenuation or miR141 overexpression decreased CCND1CCND2 expressionresulting in reduced RAC1pPXN that are involved in migration invasion and epithelial“mesenchymal transition Thisstudy for the first time deciphered the role of CDKN2BAS1miR141Cyclin D axis in RCC and highlights this networkas a promising therapeutic target for the regulation of EMT driven metastasis in RCCIntroductionRenal cell carcinoma RCC is one of the most commoncancers in the USA accounting for nearly deathsand new cases in Surgery is the first line oftreatment resulting in successful resection and longtermdiseasefree status with an overall survival rate of moreCorrespondence Rajvir Dahiya rdahiyaucsfedu1Department of Urology Veterans Affairs Medical Center San Francisco andUniversity of California San Francisco San Francisco CA USA2Department of Surgery University of Miami Miller School of Medicine MiamiFL USAFull list of author information is available at the end of the These authors contributed equally Pritha Dasgupta Priyanka KulkarniEdited by E Candithan However in approximately of localizedRCC cases recurrence occurs with distant metastasis2The obstinate nature of RCC to current treatment regimens is a primary cause of poor prognosis in patients withmetastatic recurrence Lack of sensitivity to both chemotherapytherapeuticoptions difficult3“ It is therefore of utmost importanceto improve our understanding of RCC pathogenesis byidentifying new biomarkers that lead to better predictionand therapeutic intervention of aggressive RCC6and immunotherapy makesEmerging lines of evidence suggest that cancer aggressiveness is associated with epithelial“mesenchymal transition EMT7 It is a wellorchestrated process involved in The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate ifchanges were made The images or other third party material in this are included in the ™s Creative Commons license unless indicated otherwise in a credit line to the material Ifmaterial is not included in the ™s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this license visit httpcreativecommonslicensesby40Official journal of the Cell Death Differentiation Association 0cDasgupta Cell Death and Disease Page of tumor invasion and metastasis comprising characteristicphenotypic changes through transition from polarizedimmotile epithelial cells to motile mesenchymal cells8EMT changes in cellular morphology and migratoryproperties are governed by numerous factors9 Increase inmesenchymal properties accompanied by augmentedexpression of mesenchymal markers like Ncadherbronectinvimentin and matrix metalloproteinaseMMPs and decreased expression of epithelial markerslike Ecadherin αECatenin claudin etc10“ are common EMT phenomena Often the progression of cancerthrough EMT is significantly induced by the interaction ofCyclinD with its binding partner Cdk4 which act astranscriptional regulators controlling cell proliferationand migration14“ It is well known that CyclinD regulates the ratelimiting step in cell cycle progression fromG1 to S phase Accumulating evidence also suggest thatabnormal CyclinDCdk4overexpression promotestumor growth and metastasis17 but how this correlateswith tumor metastasis or controls cell adherence andinvasion is poorly understoodReports show that noncoding RNAs are involved in thefactors involved in EMT18 MicroRNAsregulation ofmiRNAs a naturally occurring class of small noncodingRNA molecules of nucleotides long19 are known toregulate gene expression via both translational inhibitionand mRNA degradation20 whereas long noncoding RNAslncRNAs with more than nucleotides can also actas regulators for tumorsuppressive miRNAs in differentcancers21“ Recently a class oflncRNAs have beencategorized as competing endogenous RNA ceRNAwhich involves crosstalk among lncRNAs mRNAs andtheir shared miRNAs Thus a novel regulatory mechanismis hypothesized suggesting that lncRNAs and mRNAscommunicate with each other by competing for commonmiRNA response elements24“In this context we describe the novel role of lncRNACDKN2BAS1 and miR1413p miR141 in the regulation of CyclinD to govern the metastatic progression ofRCC To our knowledge this is the first report to directlydemonstrate that CDKN2BAS1miR141 interaction is acrucial component in RCC progression and metastasisthrough the CyclinDRac paxillin pathwayMaterials and methodsCell lines and cell cultureThe normal RPTEC ATCC number CRL4031 andrenalcancer ACHN ATCC number CRL1611and Caki1 ATCC number HTB46 cell lines were purchased from the ATCC Manassas VA These humanderived celllines were authenticated by DNA shorttandem repeat analysis by ATCC Cell line experimentswere performed within “ months of their procurementresuscitation ACHN cells were cultured in MEM mediaOfficial journal of the Cell Death Differentiation AssociationCaki1 cells in and McCoy 5A medium and RPTEC cellsin DMEMF12 Medium ATCC® „¢ All mediawere supplemented with FBS and 1X antibioticspenicillin and streptomycin Cell lines were maintainedat °C and humidified atmosphere of CO2miRNAsiRNA transfectionsTo induce overexpression or knockdown cells were transiently transfected with either mirVana miRNA Mimic nmolL or antimiR miRNA inhibitor nmolLThermo Fisher Scientific and nmolL of siRNA SigmaAldrich using Lipofectamine RNAi Max Thermo FisherScientific according to the manufacturers™ protocol Toverify transfection efficiency mirVana miRNA MimicNegative Control miRNA inhibitor control and siRNAcontrol were used respectively in each transfection experiment at the same concentration All transfection experimentswere carried out for hClinical specimensFormaldehydefixedparaffinembedded FFPE tissue specimens from patients undergoing radical nephrectomy wereobtained from the San Francisco Veterans Affairs MedicalCenter SFVAMC Written informed consent was obtainedfrom all patients and the study was in accordance withinstitutional guidelines IRB approval no Allpatient samples were pathologically confirmed for clear cellRCC ccRCC and slides were reviewed by a boardcertifiedpathologist for the identification of tumor foci and adjacentnormal tissue Apart from SFVAMC cohort TCGAKIRCTCGAKICH TCGAKIRP ICGC and GEO cohorts forRCC from online databases were also used to check theexpression levelsRNAmiRNA extraction and quantitative realtime PCRqRTPCRTotal RNA was extracted from microdissected FFPEtissues and cell lines using miRNeasy FFPE and miRNeasymini kits Qiagen respectively in accordance to manufacturer™s instructions Mature miRNA and mRNAs wereassayed by qRTPCR using QuantStudio FlexReal TimePCR System Applied Biosystem using Fast SYBR®Green Master MixTaqMan universal PCR master mixprobes and primers Applied Biosystems Inc Foster CityCA USA following manufacturer™s protocol HumanGAPDH and RNU48 were used as endogenous controlsand relative expression of RNAmiRNA were calculatedusing comparative Ct threshold cycle Primer sequencesare provided in Supplementary Table T1DNA methylation analysis insilico in cell lines and 5AzaCdR treatmentDNA hypermethylation of the miR141 promoter innormal and RCC samples was first confirmed in the 0cDasgupta Cell Death and Disease Page of TCGA database using Wanderer software27 In order toconfirm the methylation status of the miR141 promoterin RCC cell lines we extracted DNA from ACHN andCaki1 using DNeasy tissue kit Qiagen Sodium bisulphite modification was done using EZ DNA methylationGold kit Zymo Research Orange CA USA followingthe manufacturer™s protocol Bisulfitetreated DNA wasanalyzed by methylationspecific quantitative polymerasechain reaction MSqPCR with primer pairs specific formethylated and unmethylated regions of the miR141promoter MSqPCR was performed as described earlier28 For each sample the percent of methylation wascalculated by the difference of Ct in methylated sampleCtM and Ct in unmethylated sample CtU The primers sequences are mentioned in Supplementary Table ACHN and Caki1 cells were treated daily with μmolL5AZAdeoxycytidine 5AzaCdRfor h29 and total RNA was isolated using a miRNeasy minikit Qiagen to check miR141 expressionSigmaAldrichCell viability clonability migratory invasion andapoptosis assaysCell viability was measured at and h using aCellTiter Aqueous Solution Cell Proliferation Assay kitPromega Madison WI following the manufacturer™sinstructions For colony formation assay cells were seeded at a low density cellsplate after h oftransfection and were allowed to grow until visible colonies were formed Plates were then stained with giemsafollowed by crystal violet and colonies were countedCulture inserts of 8µm pore size Transwell Costar wereused for migration and invasion assay Inserts were coatedwith Matrigel BD Biosciences µgwell for invasionBriefly h posttransfection cells were counted andplaced on inserts at × cellsml for migration and × for invasion in serumfree medium and wereallowed to migrateinvade for “ h at °C Cellsmigrated or invaded through the pores were fixed stainedwith crystal violet Crystal violet was solubilizedwith methanol and quantified at nm by a kineticmicroplate reader Spectra MAX Molecular DevicesFACS analysis for apoptosis was done h posttransfection using Annexin VFITC and 7AAD Kit Beckmanin accordance with the manufacturer™sCoulter Incinstructions Cold PBS washed cells were resuspended in1X binding buffer and stained with Annexin VFITC7AAD viability dye After min of incubation at roomtemperature in the dark stained cells were analyzed usingBD FACSVerse BD PharmingenDualluciferase reporter assayThe wild type WT and offtarget OT luciferasereporter constructs were made by ligating annealed custom oligonucleotides containing putative target bindingOfficial journal of the Cell Death Differentiation Associationsites and corresponding nontarget mutant sites into thepmiRGLO reporter vector Luciferase constructs µgwere cotransfected into ACHN and Caki1 cells along with nmolL miR141 mimic or controlmiR using transfection reagent JetPrime Polyplustransfection IllkirchFrance Luciferase activities were measured using theDualluciferase assay Promega Madison WI h posttransfection Relative luciferase activity was calculated bynormalizing Firefly luciferase to Renilla luminescenceRNA immunoprecipitation RIP assaySimultaneous binding of miR141 to lncRNA andmRNA was confirmed by RIP assay An imprint RIP kitwas used following the manufacturer™s protocol SigmaAldrich St Louis MO USA IgG control and Ago2antibodies were used forimmunoprecipitation Theimmunoprecipitated RNA fraction was reverse transcribed to cDNA using High capacity cDNA reversetranscription kit Thermo Fisher Fold enrichment oflncRNA and mRNA to Ago2 with respect to IgG wascalculated using quantitative RTPCRWestern blot and immunofluorescence analysisTotal protein extraction was performed as describedpreviously18 Proteins were then separated by NuPAGE“ BisTris Protein Gels Invitrogen and subsequentlytransferred onto nitrocellulose membraneResulting blots were blocked using Odyssey blockingbuffer LICOR and subsequently probed with primaryand secondary antibodies Blots were scanned using anOdyssey Infrared Imaging System Scan and quantificationwas carried out with the LICOR Odyssey® scanner andsoftware LICOR Biosciences The primary antibodiesused are listed in Supplementary Table For immunofluorescencetransfected ACHN andCaki1 cells were fixed in paraformaldehyde for minfollowed by blocking 1X PBS5 normal goat serum Triton X100 for h at room temperature Cellswere then incubated overnight in fold diluted primary antibody at °C Cells were then reprobed with fold diluted secondary antibody for h and counterstained with µgml of ²6diamidino2phenylindoleDAPI for min Cells were then mounted on a slideusing prolong gold antifade reagent Images were captured using Zeiss microscope model Axio ImagerD2transientlyRenal cancer xenograftsWe studied the antitumorigenic effects of miR141 inestablished tumors using a renal cancer xenograft nudemouse model as previously described630 Male nude mice“ weekold n Charles River Lab were subcutaneously injected with × Caki1 cells Oncepalpable tumors were formed mice were randomized intwo groups for the treatment and control groups five in 0cDasgupta Cell Death and Disease Page of each Synthetic miRNA miR141 mimicmiRCON of μg was complexed with μL siPORTamine transfection reagent Ambion in μL PBS and deliveredintratumorally in 3day intervals Tumor volume wascalculated according to the formula x2 y2 where x yx width y length Experiments were terminated days after the last treatment day Tumor measurements and statistical analysis were performed byresearchers who were blinded for the control and treatment groups All animal care was in accordance withinstitutional guidelines IACUC approval no Statistical analysisAll quantified data represents an average of at leastthree independent experiments or as indicated Statisticalanalyses were performed using GraphPad Prism andMedCalc Error bars represent ± standard error meanSEM The Mann“Whitney U test was used to assessthe difference between miRNA expressions in tumornormal adjacent tissue Significant differences betweenthe groups were determined using the Student™s ttest Alltests were performed either one tailed or two tailed andresults were considered statistically significant at P ‰¤ Receiver operating curves ROC were performed toevaluate the potential of miR141 to differentiate betweenmalignant and nonmalignant samples using MedCalcsoftware showing area under curve AUC and confidence interval Kaplan“Meier analyses for overall survival with respectto miR141 methylation levels inTCGAKIRC cohort were generated using softwareœEZRhttpsdoi101038bmt2012244 Tumormeasurements and statistical analyses for all experimentswere performed blindly for the control and treatment groupsResultslncRNA CDKN2BAS1 is oncogenic and is a direct target ofmiR141Initially we found CDKN2BAS1 is an oncogeniclncRNA in RCC based on TCGA Fig 1a ICGC andGEO databases Fig S1 TCGA cohort also revealed thathigh CDKN2BAS1 expression increases from lower gradeand stage to higher grade and stage Fig 1b Moreoverhigher expression is significantly p correlated tooverall survival Fig 1c In agreement with these datacohorts significantly higher CDKN2BAS1 expression wasalso seen in RCC cell lines ACHN Caki1 as compared tonormal RPTEC Fig 1d and SFVAMC cohort Fig 1ePatient and tumor characteristics are summarized inSupplementary Table T3 ROC analysis shows an AUC of P CI “ Fig 1f suggesting the diagnostic potential of CDKN2BAS1 to discriminate between normal and tumor tissues We usedcomputational algorithms and identified putative miR141binding sites in the CDKN2BAS1 sequence Fig 1g ToOfficial journal of the Cell Death Differentiation Associationexamine potential miR141CDKN2BAS1 interactionexperimentally we performed luciferase reporter assayBoth ACHN and Caki1 cells cotransfected with miR141and CDKN2BAS1 wild type binding site revealed a consistent reduction ofluciferase activity suggesting thatmiR141 directly interacts and regulates CDKN2BAS1Fig 1h Thus all these data suggest that clinicallyimportant CDKN2BAS1 is an oncogenic lncRNA in RCCand is a novel target of miR141CDKN2BAS1 inhibition by siRNA suppressestumorigenicity in RCCTransient transfection of ACHN and Caki1 cells withCDKN2BAS1 siRNAs for h showed significant reduction in CDKN2BAS1 expression Fig 2a CDKN2BAS1knockdown in both cell lines significantly inhibited cellproliferation Figs 2b S2A and clonogenic survivalFigs 2c S2B with a significant increase in apoptosis Fig2d e Decreased cell migrationinvasion Figs 2f S2C Dwith simultaneous changes in EMT markers such as anincrease in epithelial markers αECatenin and claudinand decrease in mesenchymal markers vimentin andfibronectin were also observed Figs 2g S3Expression of miR141 and its clinical importance in renalcarcinomaand samples LowerSince our results confirmed CDKN2BAS1 as a directtarget of miR141 we examined miR141 status andclinical importance in RCC Expression of miR141 wassignificantly downregulated in RCC cell lines Fig 3aand in tumor samples Fig 3b“e compared to normal celllinesignificantlydecreased with increasing grade stages and in metastaticcompared to nonmetastatic tumors Fig 3c“e Patientand tumor characteristics are summarized in Supplementary Table T3 ROC analysis showed an AUC of P CI “ Fig 3f suggesting thatmiR141 can be used as a potential diagnostic parameterto discriminate between normal and tumor tissuesexpressionsEpigenetic regulation of the miR141 locusWe identified a genomic site rich in CpG island locatedupstream of the miR141 in chromosome 12p13 In theTCGA cohort we observed hypermethylation of miR141promoter in tumor tissues as compared to normalFigs 3g S4A which is significantly associated with poorpatient survival Fig 3h Similarly RCC cell lines ACHNand Caki1 also showed hypermethylation compared tonormal RPTEC cells Fig 3i Further we treated ACHNand Caki1 cell lines with demethylating agent 5AzaCdRand observed decrease in methylation Fig S4B withconcomitant increase in miR141 expression Fig 3jindicating possible epigenetic regulation A significantdecrease in the expression of methylation regulatory 0cDasgupta Cell Death and Disease Page of Fig lncRNA CDKN2BAS1 is oncogenic in renal cancer and is a direct target of miR141 a Expression levels of CDKN2BAS1 among KIRCnormal tumor KICH normal tumor and KIRP normal tumor patient samples in TCGA cohort using Wanderersoftware Pvalue calculated by Mann“Whitney twotailed test b Expression of CDKN2BAS1 in TCGAKIRC cohort among different grades normal grade “ grade “ and stages normal stage I“II and stage III“IV c Overall survival in TCGAKIRC cohort as performedby Kaplan“Meier analysis using UALCAN software d Relative expression levels of lncRNA CDKN2BAS1 in RCC cell lines ACHN and Caki1 e ExpressionCDKN2BAS1 in matched pairs of RCC tissue samples from SFVAMC cohort Pvalue calculated by Mann“Whitney twotailed test f Receiver operatingcurve ROC analysis on SFVAMC cohort showing ability of lncRNA CDKN2BAS1 to differentiate between malignant and nonmalignant samples SFVAMCcohort g Predicted binding sites of miR141 in CDKN2BAS1 sequence h Luciferase assays showing decreased reporter activity after cotransfection witheither wildtype WT offtarget OT CDKN2BAS1 or luciferase control constructs EV with miRCONmiR141 in ACHN and Caki1 cellsgenes such as DNMTl DNMT3a and DNMT3b werealso noted after 5AzaCdR treatment compared to control DMSO in both ACHN and Caki1 cell lines31miR141 overexpression phenocopies functional effectsobtained with CDKN2BAS1 inhibition in vitro andsuppresses tumorigenicity and in vivoWe sought to determine if CDKN2BAS1 causes itsantitumorigenic effects through miR141 We checkedthe effect of miR141 overexpression in RCC cellsTransient transfection of miR141 mimic in ACHN andCaki1 cells for h led to over expression of miR141compared to control miRCON Fig 4a Also overexpression of miR141 significantly reduced CDKN2BAS1 expression Fig 4b indicating a reciprocal correlation between miR141 and CDKN2BAS1 A significant decrease in cell proliferation over time Fig 4cand marked decreaseFig 4din clonogenicityOfficial journal of the Cell Death Differentiation Association 0cDasgupta Cell Death and Disease Page of Fig Knockdown of lncRNA CDKN2BAS1 reduces tumorigenicity in renal cancer a Relative expression of CDKN2BAS1 in ACHN and Caki1 cellstransfected with CDKN2BAS1 siRNAs b Cell proliferation assessed by MTS assay after knockdown of CDKN2BAS1 in both cell lines with siRNA2 CGraphical representation showing knockdown of CDKN2BAS1 with siRNA2 significantly decreased colony formation in ACHN and Caki1 cellsd e ACHN and Caki1 cell lines showing significant induction of apoptosis early late compared to control after knockdown of CDKN2BAS1f Reduced migration and invasion in CDKN2BAS1 siRNA transfected cells compared to control treatment g Changes in EMT related proteins in bothACHN and Caki1after knockdown of CDKN2BAS1compared to controls were also observed Further westudied the therapeutic potential of miR141 in amouse xenograft model A significant decrease intumor growth was observed by intratumoral delivery ofmiR141 mimic compared to control over the course ofexperiment Average tumor volume in the controlgroup was mm3 compared to mm3 in micethat received miR141 mimic Fig 4e In additionmiR141 overexpression significantly induced apoptosis with a concomitant decrease in the viable population in both RCC celllines compared to controlFig 5a This proapoptotic role was supported by theinduction of cleaved caspase3 cleaved polyADPribose polymerase PARP an increase in BAX and adecrease in BCl2 at protein levels Fig 5b A significantdecrease in migration Fig 5c and invasion Fig 5dwas also observed in both RCC cell lines with miR141overexpression We also examined EMT markers aschange in migration and invasion are directly associated with EMT Our results showed an increase inepithelial markers αEcatenin and claudin with concomitant decrease in mesenchymal markers fibronectinand vimentin at both protein Fig 5e and mRNAOfficial journal of the Cell Death Differentiation AssociationFig S5 levels Taken together overexpression of miR phenocopies the functional effects of CDKN2BAS1 inhibition in vitro and tumor growth suppressioneffects in vivoLike CDKN2BAS1aremiR141CDKN2BAS1 interaction negatively regulatesCyclinD and its downstream effectors in RCCcyclinD1cyclinD2alsooncogenic in RCC Fig S6 and are direct targets of miR As discussed earlier lncRNAs can act as ceRNAs tocarry out their regulatory functions32“ We observedthat CDKN2BAS1 shared regulatory miR141 bindingsites with cyclinD1cyclinD2 Fig 6a and therebysponges miR141 allowing cyclinD1D2 to be expressedin tumors To determine potential miR141CDKN2BAS1CyclinD interaction experimentally we performedRIP assay Both ACHN and Caki1 cells over expressingmiR141 revealed significant enrichment of cyclinD1cyclinD2 and CDKN2BAS1 with Ago2 as compared toIgG control Fig 6b Moreover decreased luciferaseactivity also confirmed direct binding of miR141 tocyclinD in miR141overexpressing ACHN andCaki1 cells compared to controls Fig 6c We also found 0cDasgupta Cell Death and Disease Page of Fig Expression clinical significance and epigenetic regulation of miR141 in renal cancer a miR141 expression levels in ACHN Caki1 andRPTEC cells b Expression levels of miR141 in KIRCTCGA cohort normal and tumor c Expression levels of miR141 in normal n nonmetastatic n metastatic n KIRC patient samples in TCGA cohort d Expression levels of miR141 in KIRCTCGA cohort amongdifferent grades normal grade “ grade “ and stages normal stage I“II and stage III“IV e Relative miR expression in RCC tissue vs matched adjacent normal regions n among different grades normal grade grade “ andin different stages normal stage I“II stage III“IV as assessed by qRTPCR SFVAMC cohort f Receiver operating curve ROC analysisshowing ability of miR141 to differentiate between malignant and nonmalignant samples g Methylation for KIRC patient samples in TCGAKIRCcohort for probe cg02624246 h Overall survival with TCGAKIRC methylation as performed by Kaplan“Meier analysis i Methylation status of miR141promoter in RCC cell lines compared to nonmalignant RPTEC as assessed by MSqPCR j Expression of miR141 in 5AzaCdR treated and untreatedACHN and Caki1 cell lines Results are representative of three independent experiments P value calculated by Student t test Bar mean ± standarderror mean SEMthat overexpression of miR141 or inhibition of CDKN2BAS1 significantly decreased cyclinD1D2 expression atboth the mRNA Fig 6d e and protein levels Figs 6f“hS8A B This effect was significantly attenuated by miR inhibitor Fig S7 indicating that cyclinD expressionis dependent on the interaction between miR141 andCDKN2BAS1 We further observed a decrease in Rac1 asmall GTPase and a reduction in the phosphorylation ofpaxillin a focal adhesion protein at mRNA levelsFigs S3 S5 and protein levels Figs 6g I“j S8C“F inboth miR141 overexpressed and CDKN2BAS1 inhibitedRCC cell lines which in turn are involved in regulatingcellular migrationinvasion Cumulatively these resultsindicate that suppression of CDKN2BAS1 by miR141inhibits renal cell proliferation invasion and migration byinhibiting cyclinD Rac1 and phosphorylation of paxillinOfficial journal of the Cell Death Differentiation Association 0cDasgupta Cell Death and Disease Page of Fig miR141 overexpression mimics the knockdown effect of lncRNA CDKN2BAS1 in vitro and reduces tumorigenicity in vivo a Relativeexpression of miR141 in ACHN and Caki1 cells transfected with miR141 mimic and control b Significant decrease in CDKN2BAS1 expressioncompared to control in both cell lines overexpressed with miR141 c RCC cell proliferation after transfecting with miR141 mimic and control asassessed by MTS assay d Colony formation and its graphical representation in miR141 overexpressing ACHN and Caki1 cells compared to controlse Pictures of excised tumors are taken at the termination of experiment day Graph represents tumor volume after intratumoral injection ofcontrol or miR141 mimic into established tumors Injection was started at day and was followed for days Each mouse in both groups miRCONand miR1413pMimic received a total of eleven injections intermittently Data represent the mean of each group and error bars are SEM Results arerepresentative of three independent experiments P value calculated by Student t test Bar mean ± standard error mean SEMAttenuation of miR141 exerts tumorigenic attributes innormal RPTEC cellsWe next determined whether attenuation of miR141induces tumorigenic characteristics in normal RPTECcells by targeting CDKN2BAS1 and cyclinD Transienttransfection of miR141 inhibitor indeed showed a significant decrease in miR141 expression Fig 7a and anincrease in CDKN2BAS1 expression Fig 7b along withother procancerous phenotypes such as increased cellproliferation Fig 7c colony formation Fig 7d migration and invasion Fig 7e as compared to controlsAdditionally a significant increase in cyclinD1 cyclinD2 rac1 and paxillin Pxn expressions were observed inmiR141 inhibited RPTEC cells Fig 7f A noticeableincrease in prometastatic fibronectin and vimentin with aconcomitant decrease in antimetastatic claudin andαEcatenin genes were also observed in miR141 inhibited RPTEC cells compared to controls Fig 7gDiscussionPrior studies have shown the regulatory role of noncoding RNAs in tumorigenesis especially in the EMTOfficial journal of the Cell Death Differentiation Associationpathway leading to cancer aggressiveness CDKN2BAS1also known as ANRIL is located at chromosome 9p21CDKN2BAS1 is reported to be upregulated in tumortissues and function as an oncogenic lncRNA in pancreatic ovarian and laryngeal squamous cell carcinoma36“ Human miR141 is located at chromosome12p1331 and is transcribed from a miR200 familyclusterInterestingly expression of miR141 is controversial since it exhibits either oncogenic39“ or tumorsuppressive roles42“ in specific types of cancer Theprime goal of the present study was to understand therole of CDKN2BAS1miR141 interactions in regulatingRCC progression and metastasisIn this study we identify CDKN2BAS1 to be a crucialoncogenic lncRNA that plays an important role in renalcarcinogenesis CDKN2BAS1significantly overexpressed in RCC and the expression increases fromlower to higher grades and stages LncRNA CDKN2BAS1directly interacted with miR141 as it was found to be anovel target of miR141 In contrast to CDKN2BAS1 weobserved significant attenuation of miR141 expression inRCC cell lines and tumor samples compared to normalis 0cDasgupta Cell Death and Disease Page of Fig Ectopic miR141 expression induces apoptosis and inhibits migrationinvasion in renal cancer cells a Apoptosis assessed by flowcytometric analysis of annexinVFITC 7AAD“stained ACHN and Caki1 cells transfected with miRCONmiR141Mimic Graph represents totalapoptosis early late b Immunoblots showing apoptotic proteins in miRCONmiR141Mimic treated ACHN and Caki1 cells with GAPDH asendogenous control c Migration assay and d invasion assay as seen in pictures and graphical representation of both ACHN and Caki1 cells after miR overexpression compared to control e Immunoblot assay showing EMT related proteins in miRCONmiR141Mimic treated ACHN andCaki1 cells with βactin as endogenous control Graphs are average of three independent experiments P value calculated by Student t testBar mean ± standard error mean SEMcell line or matched normal samples As it is known thatextensive DNA hypermethylation of CpG islands is highlycorrelated to activation of cancerspecific genes45 wechecked the methylation status of miR141 in normal andRCC tissues Interestingly insilico analysis showed thepresence of CpG island in the promoter region of miR and we also found hypermethylation of miR141 inTCGA samples as compared to normal This hypermethylation is also found to be significantly associatedwith poor survival of patients Similar results were alsoobserved in RCC cell lines compared to a normal RPTECcell line Functionally inhibition of CDKN2BAS1 andoroverexpression of miR141 significantly inhibits thetumorigenic characteristics such as cell proliferationclonogenicity migration and invasion whereas inducesanticancer apoptotic phenotype in RCC in vitro In vivodata show suppression of tumor growth by miR141overexpression Conversely attenuation of miR141 innormal RPTEC cells induced precancerous characteristicsindicated by increased proliferation migration andinvasionFrom a clinical point of view noncoding RNAs signatures are powerful tools for early cancer diagnosisOfficial journal of the Cell Death Differentiation Associationmaking them attractive candidates as diagnostic andprognostic biomarkers184647 Our results revealed thathigher expression of CDKN2BAS1 is positively correlatedwith poor overall survival probability of RCC patientsindicating its prognostic capability CDKN2BAS1 canalso discriminate normal from tumor samples showing itsdiagnostic potential Similarly miR141 expression canalso robustly distinguish between cancerous from noncancerous samples and hence has potential to be

Thyroid_Cancer

thyroid stimulating hormone highlightspleiotropic effects and inverse association withthyroid cancerWei Zhouet alThyroid stimulating hormone TSH is critical for normal development and metabolism Tobetter understand the genetic contribution to TSH levels we conduct a GWAS metaanalysisat million genetic markers in up to individuals and identify genomewidesignificant loci for TSH of which are previously unreported Functional experiments showthat the thyroglobulin proteinaltering variants P118L and G67S impact thyroglobulin secretion Phenomewide association analysis in the UK Biobank demonstrates the pleiotropiceffects of TSHassociated variants and a polygenic score for higher TSH levels is associatedwith a reduced risk of thyroid cancer in the UK Biobank and three other independent studiesTwosample Mendelian randomization using TSH index variants as instrumental variablessuggests a protective effect of higher TSH levels indicating lower thyroid function on risk ofthyroid cancer and goiter Our findings highlight the pleiotropic effects of TSHassociatedvariants on thyroid function and growth of malignant and benign thyroid tumorsA list of authors and their affiliations appears at the end of the paperNATURE COMMUNICATIONS 101038s4146702017718z wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s4146702017718zNormal thyroid function is essential for proper growth anddevelopment and for metabolic functions Approximately million people in the United States are affected bythyroid disorders and of the population is expected todevelop thyroid conditions over their life span1 Thyroidstimulating hormone TSH is secreted by the pituitary glandand stimulates the growth of the thyroid gland and its synthesisand secretion of thyroid hormones These include thyroxine T4most of which is converted to its more bioactive form ²triiodothyronine T3 TSH levels are negatively regulated by T3and T4 and lower or higher levels than the reference rangerespectively usually suggest that the thyroid gland is overactive asin primary hyperthyroidism or underactive as in primary hypothyroidism The complex inverse relationship between TSH andthyroid hormones means TSH is a more sensitive marker ofthyroid status a feature that has been used to identify individualswith thyroid dysfunction2Thyroid disorders affect multiple ans and are associatedwith a range of clinical consequences including an increased riskof metabolic disorders and cardiovascular mortality3“ Over thepast few decades a steady increase of the incidence rates of nonmedullary thyroid cancer henceforth referred as thyroid cancerhas been observed in most areas of the world including in Europe7 Previous studies have led to inconsistent s on therelationship between TSH levels and thyroid cancer risk8 Severalstudies have observed an association between low TSH levelswhich can occasionally occur as a consequence of autonomousthyroid nodules and an increased risk of thyroid cancer8“ Incontrast several studies have indicated that TSH promotes thegrowth of thyroid cancers814“ which has led to the recommendation to lower TSH levels among people with thyroid cancerto reduce the risk of cancer recurrence Two initial genomewideassociation studies GWAS identified five significant loci forthyroid cancer in Europeans and the risk alleles of all five locihave been associated with decreased TSH levels1718 In contrast amore recent GWAS identified five additional loci associated withthyroid cancer none of which were even nominally associatedwith TSH19 A recenttwosample Mendelian randomizationstudy suggested a causal inverse association between TSH levelsand overall cancer risk including thyroid cancer20 Additionalstudies are needed to clarify the role of TSH and TSHassociatedvariants in thyroid cancerTwin studies have shown TSH levels are moderately heritablewith estimates up to Previous TSH GWAS studies haveidentified independent TSHassociated loci172223 accountingfor of TSH variance thus leaving a large proportion of theTSH heritability unexplained2324 With the goal of identifying themissing genetic components for TSH to further understand itsunderlying genetic architecture and impact on thyroid cancer weperform a GWAS metaanalysisfor TSH levels on thepopulationbased NordTr¸ndelag Health Study HUNT studyN Michigan Genomics Initiative26 MGI N consortium up to N and the ThyroidOmics samples23To investigate the genetic relationship between TSH andthyroid cancer and other human diseases we examine phenomewide associations in the UK Biobank UKBB27 for TSHassociated index variants We also conduct phenomewide association tests for the polygenic scores PGS of TSH in the UKBBand the FinnGen study We observe an association between highTSH PGS and low thyroid cancer risk and replicate that observation in two other study populations from Columbus USA andIceland19 To evaluate the potential causality of TSH on thyroidcancer we perform a twosample Mendelian Randomizationanalysis using the TSHassociated top association signals asinstrumental variables and the thyroid cancer GWAS results on individuals cases and controls from ametaanalysis of UKBB2728 MGI26 and results from a previousmetaanalysis for thyroid cancer based on a Icelandic data setfrom deCODE referred to as deCODE in this manuscript aswell as four other case“control data sets with European ancestryas reported in Gudmundsson et al19ResultsDiscovery of genetic loci for TSH We identified loci associated with TSH Table Supplementary Data and andSupplementary Fig in our metaanalysis of the HUNT studyN the MGI biobank N and the ThyroidOmics consortium23 up to N Twentyeight of the loci have not been previously reported for TSH172223Table To identify secondary independent association signalswe performed stepwise conditional analysis within each locususing GCTACOJO29 based on GWAS summary statistics fromthe metaanalysis of HUNT MGI and ThyroidOmics and thelinkage disequilibrium LD correlation between variants estimated in HUNT We observed additional associations in novelTSH locus B4GALNT3 and previously known TSH lociTable and Supplementary Data In total independent topvariants have been identified at the loci explaining ofthe variance of TSH levelsDespite having only moderate effect sizes top variants inseveral novel TSH loci point to nearby genes with a known orsuspected link to thyroid function Table and SupplementaryFig An intronic variant rs10186921 in the thyroid adenomaassociated gene THADA was identified to be associated with TSHTHADA has been identified as a somatic mutatedrearrangedgene in papillary thyroid cancer30 and observed to be truncated inthyroid adenoma31 Although THADA is known to play a role incold adaptation obesity and type diabetes its role in thyroidvariantfunction remainsrs145153320 in gene B4GALNT3 is associated with TSHminor allele frequency in HUNT MAFHUNT effectsizeHUNT standard deviation SD confidence intervalCI “ SD PvalueHUNT — ˆ’ and is times more frequent in the Norwegian HUNT samples than inother nonFinnish Europeans34 The WNK1B4GALNT3 genefusion has been identified in papillary thyroid carcinoma35elusive3233 A rare missenseTwo novel independent rare coding variants with effect sizeslarger than one SD were identified in the known TSH locus TSHRwhich encodes the TSH receptor Both variants were only observedin HUNT The rare missense variant TSHR pR609Q rs139352934MAFHUNT effect sizeHUNT SD CI “SD is the most significant variant in the locus PvalueHUNT — ˆ’ followed by pA553T rs121908872 MAFHUNT CI “ SDPvalueHUNT — ˆ’ TSHR pR609Q rs139352934is times more frequent in HUNT than in other nonFinnishEuropeans34 TSHR pR609Q has been reported to aggregate in afamily with nonautoimmune isolated hyperthyrotropinemia36 andTSHR pA553T has been previously detected in a family withcongenital hypothyroidism37sizeHUNT SDeffectAs singlevariant association tests may lack power for rarevariants MAF ‰ and to search for genes with multiple rareproteinaltering variants we performed exomewide genebasedSKATO38 tests as implemented in SAIGEGENE39 to identifyrare coding variants associated with TSH We grouped missenseand stopgain variants with MAF ‰ and imputation qualityscore ‰¥ within each gene and tested genes with at leasttwo variants This analysis identified two genes TSHR andB4GALNT3 as significantly associated with TSH Pvalue —ˆ’ Supplementary Table and Supplementary Fig RareNATURE COMMUNICATIONS 101038s4146702017718z wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s4146702017718zeulavPytienegoreteHcnoitceriDNPESbtceffEˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’E“E“Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’aqerFIRMCNKDCIFRMfroCPAHTKOBADAHTLXSASRECCFGEVDEDPPPLSNTCOLNDLCGARPCOMSTCADXNSSRFSTAEYTNLAGBTNLAGBCELOCCDCCKENSAHPDFNZBHSBARHOPAGNGCNILCSACGRDCNILPBPSHRPPPGNGIRMTNWmaertsnwoDicnegretniicnegretnIiicnortn_ANRcnicnortnIicnegretnIicnortnIicnortnIicnortnIicnegretnIicnortnIicnortnIicnortnIicnegretnIicnegretnIicnortnIicnortnIicnortnIicnortnIsuomynonysnoNicnegretnIsuomynonysnoNicnortnIsuomynonysnoNicnortnIiicnortn_ANRcnicnegretnIicnortnIicnegretnIicnegretnIGAAACTGGGAGCAATCGGATCCCTAGCGTCAGGGACTAAGCTGCATACGCTTTCGCTACTsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsisylanaatemscimOdioryhTIGMTNUHseneGtseraeNyrogetaCtlAfeRDIsrdliubnoitisoPemosomorhCxednIsucoLscimOdoryhTidnaIGMTNUHfosisylanaatemehtnidefiitnediHSThtiwdetaicossaicoltnednepednilevoninhtiwstnairavdaeLelbaTtesatadigndnopserrocehtnignissimsitnairavehtfisadetoNlyevitcepseriscmOdoryhTidnaIGMTNUHstesatadliaudvdniinieelllaetanretlaehtfoHSTnonoitceridslevelHSTfoDSfotinuehtnieelllaetanretlaehtottcepserhtiwdetropererasezistceffEbtceffEctesatadlsisyanaatemdenbmociehtnieelllaetanretlaehtottcepserhtiwdetropereraisecneuqerFaNATURE COMMUNICATIONS 101038s4146702017718z wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s4146702017718zvariants in both genes associated with TSH were also identifiedfrom singlevariant analysis After conditioning on the two rarevariants in TSHR that were genomewide significant in the singlevariantandrs139352934 the gene TSHR was still exomewide significantwith Pvalue — ˆ’ while B4GALNT3 was no longersignificantly associated with TSH with Pvalue afterconditioning on the top variant rs145153320Pvalue — ˆ’rs121908872analysisFinemapping for potentially causal variants among TSH lociTo identify potentially causal variants at TSH loci we conductedfinemapping using SuSiE40 which estimates the number ofcausal variants and obtains credible sets of variants with cumulative posterior probability through Iterative BayesianStepwise Selection41 Supplementary Data The LD matrixused in SuSiE was calculated based on HUNT We identified eightindependent causal variants at the TSHR locus by finemappingusing SuSiE40 and seven independent association signals by thestepwise conditional analysis Supplementary Data suggestingallelic heterogeneity at the TSHR locusIn addition finemapping by SuSiE40 and stepwise conditionalanalysis identified two association signals in the locus of thethyroglobulin gene TG TG encodes a highly specializedhomodimeric multidomain glycoprotein for thyroid hormonebiosynthesis27 it is the most highly expressed gene in the thyroidgland and its protein product represents roughly half the proteinof the entire thyroid gland4243 The TG locus has been reportedin a recent TSH GWAS23 The credible set for each causalassociation contains one missense variant that is in strong LDwith the most strongly associated intronic variant Supplementary Table and Supplementary Fig In the HUNT study themissense variant TG pG67S rs116340633 MAF effectsize SD CI “ SD Pvalue — ˆ’is in strong LD r2 with the most strongly associatedvariant rs117074997 intronic At the other association signalmissense variant TG pP118L rs114322847 MAF effectsize SD CI “ SD Pvalue — ˆ’is in strong LD r2 with the most strongly associatedvariant rs118039499 intronic Supplementary Table andSupplementary Fig TG pP118L has been previously detectedamong familial cases with congenital hypothyroidism44 TG pP118L rs114233847 is significantly associated with nontoxicnodular goiter odds ratio OR CI “Pvalue — ˆ’ in the UKBB2728 while the association ofTG pG67S rs116340633 with nontoxic nodular goiter isless significant OR CI Pvalue —ˆ’ TG pP118L has been previously detected in patients withsporadic congenital hypothyroidism in a Finnish cohort44Functional followup of missense variants in the gene TG Weperformed sitedirected mutagenesis studies to investigate theimpact on the protein expression of TG of the two independentmissense variants both located in the highly conserved Tg1domain of unclear function The protein encoded by the humanTG is conserved in mice with nearly perfect conservation of allcritical amino acid residues including those that maintain theprotein structure and hormone synthesis45 A cDNA encodingwildtype mouse Tg mTgWT expressed in 293T cells hasnormal synthesis and secretion of thyroid hormones46 We thenintroduced the observed human TG variants rs116340633 andrs114322847 into the mTg cDNA 293T cells were eitheruntransfected or transfected with pcDNA31 in which a cytomegalovirus promoter drives expression of mTgWT or the pP118L or pG67S Tg variants mature Tg numbering Then weexamined the intracellular vs secreted levels of the mTgWT andthese two human Tg variants TgpP118L and TgpG67STransfected cells were incubated overnight and the culturemedium and cell lysates were analyzed by SDSpolyacrylamide gelelectrophoresis PAGE and immunoblotting with antiTg antibody The experiment was independently repeated three timesand the results analyzed in a manner that is independent oftransfection efficiency On average of the total expressedWT form of mTg was recovered in the media and extracellular intracellular MC ratio of mTg was as expected between and the TgP118L variant showed a significant reduction in the MC ratio Pvalue and the TgG67S variant also showed asignificant reduction in the MC ratio Pvalue Fig Compared with the WTPrioritization of TSH genes pathways and tissues To furtherunderstand the biology underlying TSH associations we prioritized associated genes tissues and cell types in which TSH genesare likely to be highly expressed using Datadriven ExpressionPrioritized Integration for Complex Traits DEPICT47 based on loci with TSH association Pvalue cutoff — ˆ’ andclumped based on LD in HUNT As expected the membranesand thyroid gland are the most strongly associated tissues followed by tissues from the digestive system ileum gastrointestinaltract pancreas and colon respiratory system lung and accessory ans for eyes conjunctiva eyelids and anterior eyealthough none of the tissues reached the Bonferroni significantthreshold Pvalue or have false discovery rate FDR Supplementary Data Based on functional similarity toother genes among TSH loci genes at the TSHassociated lociwere prioritized by DEPICT with FDR ‰ SupplementaryData among which the prioritized genes ZFP36L2B4GALNT3 PPP1R3B FAM109A GNG12 GADD45A BMP2VEGFC LPP and MAL2 were at the novel TSH loci identified inour metaanalysis Table In addition among reconstituted gene sets gene sets were enriched among TSH lociwith FDR The most significantly enriched one is the CTSDPPI subnetwork followed by gene sets for regulation of phosphorylation Supplementary Data Pleiotropic effects of TSH loci To explore the pleiotropic effectsof the TSH loci we examined associations of the nonhumanleukocyte antigen HLA independent TSH top variants with human diseases PheCodes272848 and continuoustraits httpwwwnealelabisukbiobank in the UKBB variants and rs121908872 are not available in the UKBB Dueto the strong associations between HLA variants and autoimmune diseases49 we excluded two HLA variants associatedwith TSH rs1265091 and rs3104389 in the analysis for pleioPvalue — ˆ’tropic effects We identified significantpleiotropic association for out of nonHLA variants across disease phenotypes Supplementary Fig 5a and Supplementary Data including thyroid disorders diabetes cardiovasculardisease digestive system disorders asthma and cataractsIn addition nonHLA variants were significantly associatedPvalue — ˆ’ with one or more quantitativetraitsincluding body mass index lung function measurements metricsof bone density spherical powermeridian measurements andblood cell counts Supplementary Fig 5b and SupplementaryData TSHincreasing alleles at one or more loci were associated with an increased risk of cardiovascular disease smallerbody size reduced bone mineral density decreased lung functionand an increased risk of hypothyroidism and a decreased risk ofgoiter These results are generally consistent with previous studies23 We also examined the associations between the TSH indexin the ThyroidOmicsvariantsand freethyroxinelevelsNATURE COMMUNICATIONS 101038s4146702017718z wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s4146702017718zaTg kDabcUntransfectedmTgWTmTgP118LmTgG67SMCMCMCMCMCMCMCTg kDaTg kDaddnab nillubogoryhTeUA ytisnetniCellMediaWTG67SP118Loitar CMFig Both the TGP118L and TGG67S point mutants exhibit a secretion defect a“c Three independent replicate experiments Western blotting ofTG in 293T cells that were either untransfected a no detectable bands or transfected with constructs encoding mouse TG wild type WT or P118L 67S point mutants in the pcDNA31 background in which the CMV promoter drives the respective cDNA expression Serumfree media M werecollected overnight and the cells C were lysed Equal volumes of media and cells were analyzed by SDSPAGE electrotransfer to nitrocellulose andimmunoblotting with antiTgspecific antibodies Full scans of western blotting are presented in Supplementary Fig From scanning densitometryd shows the content of thyroglobulin and its variants intracellularly and in the secretion e The extracellular intracellular MC ratio of each constructd e Three independent replicate experiments All boxplots in d and e indicate median center line 25th and 75th percentiles bounds of box andminimum and maximum whiskersWTG67SP118Lconsortium23 Out of TSHassociated variantsfor whichassociation results with free thyroxine were available have TSHlowering alleles associated with higher free thyroxinelevels Supplementary Data Pvaluebinomial — ˆ’We further examined the association with thyroid cancer forTSH index variants We metaanalyzed UKBB2728 and a previousmetaanalysis of deCODE and four other case“control data sets19for thyroid cancer in thyroid cancer cases and controls and examined out of TSH nonHLA index variantsthat are available in the metaanalysis for thyroid cancerSupplementary Data The TSHincreasing alleles of outof TSHassociated variants were associated withreduced thyroid cancerrisk Supplementary Data andSupplementary Fig 6a and 6b Pvaluebinomial — ˆ’Eighteen out of the TSHassociated variants tested wereat least nominally associated with thyroid cancer P Pvaluebinomial — ˆ’ For out of the TSHassociatedvariants the TSHincreasing alleles were associated with reducedthyroid cancer risk Pvaluebinomial — ˆ’ SupplementaryData and Supplementary Fig 6c d Moreover when weexamined alleles that predisposed to thyroid cancer17“ out of had a consistent direction of effect towards lower TSH Pvaluebinomial Ofriskalleles that were at least nominally associated with TSH levelP all six variants were associated with lower TSHPvaluebinomial Supplementary Data and Supplementary Fig thyroidcancerthesixAssociations of polygenic scores of TSH with other phenotypesAlthough individual TSH variants may exhibit pleiotropic effectsit is also possible that the cumulative effects of TSHmodifyinggenetic variants may lead to disease Therefore we constructedcodesICDPGS from the independent nonHLA TSH top variantsrs1265091 and rs3104389 are HLA variants and rs121908872and were not in UKBB and examined their association with the human diseases constructed from International Classification of Diseasesin theUKBB272848 As in the pleiotropy analysis we excluded the twoHLA variants in the PGS calculation to study the cumulativegenetic effects of TSHassociated variants in nonHLA regionswith human diseases The TSH PGS was significantly associatedwith phenotypes Pvalue — ˆ’ Bonferroni correctionfor phenotypes including an increased hypothyroidism riskand decreased risk of goiter thyrotoxicosis and hyperhidrosisSupplementary Data and Supplementary Fig We alsoevaluated the phenotypic variance Nagelkerke™s r250 explainedby TSH PGS for phenotypes in the UKBB that have at least cases in unrelated white British samples Supplementary Data The phenotypes with highest r2 were nontoxicnodular goiter r2 secondary hypothyroidism r2 and thyrotoxicosis with or without goiter r2 InFinnGen we also observed that high TSH PGS was associatedwith high risk of hypothyroidism and low risk of goiter HighTSH PGS in FinnGen was marginally associated with an increasein risk of depression OR per SD of TSH PGS CI “ Pvalue — ˆ’ and a reduced risk of pregnancyhypertension OR per SD of TSH PGS CI “ Pvalue — ˆ’ The phenomewide associationresults for the TSH PGS in FinnGen are shown in SupplementaryData and Supplementary Fig Depressive symptomsand hypertension during pregnancy have been observed to beclinically associated with hypothyroidism and thyroid dysfunction51“ respectively However their genetic associations havenot been extensively studiedNATURE COMMUNICATIONS 101038s4146702017718z wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s4146702017718zaUKBBrecnac doryhit f oecneaverPlQuintiles of TSH PGSbdeCODEirecnac doryht fo ecneaverPlQuintiles of TSH PGSrecnac doryhit fo oitar sdrecnac doryht fo oitar sddOQuintiles of TSH PGSQuintiles of TSH PGScFinnGenirecnac doryht fo ecneaverPlirecnac doryht fo oitar sddOQuintiles of TSH PGSQuintiles of TSH PGSFig The risk of thyroid cancer is lower for individuals with genetically predicted higher TSH levels Plots of thyroid cancer prevalence by quintiles ofTSH PGS left and odds ratio of thyroid cancer in relation to the lowest quintile right in data sets UKBB a N case N control deCODEb N case N control and FinnGen c N case N control N sample size N case sample size of cases N controlsample size of controls Error bars represent confidence intervalsthyroid cancer CI “In the UKBB TSH PGS was significantly associated with aOR per SD ofdecreased risk ofPvalue — ˆ’TSH PGSSupplementary Data and Fig Compared with the rest ofthethyroid cancer ofindividuals with TSH PGS in the lowest quintile was “ and the OR for thyroid cancer of individuals withTSH PGS in the highest quintile was “ suggestingthe OR CIsamplesforthe protective effects of TSHincreasing genetic variants onthyroid cancer riskWe successfully replicated the association between high TSHPGS and low thyroid cancer risk in study populations fromColumbus USA19 OR CI “ Pvalue — ˆ’ and deCODE19 OR CI “Pvalue — ˆ’ We also observed the association inFinnGen OR CI “ Pvalue — ˆ’NATURE COMMUNICATIONS 101038s4146702017718z wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s4146702017718zalthough the evidence was much less strong The Columbusstudy19 is a case“control study of thyroid cancer cases and controls with much higher thyroid cancer prevalence thanthe three populationbased biobanks UKBB3031 cases and controls FinnGen cases and controls anddeCode19 cases and controls In Fig theprevalence of thyroid cancer left panel and OR of thyroid cancerright panel are plotted against the TSH PGS for the threepopulationbased cohorts results for Columbus are provided inSupplementary Fig Similar plots of hypothyroidism andgoiter are plotted for UKBB and FinnGen in SupplementaryFigs and Mendelian randomization for TSH thyroid cancer and goiterWe investigated a possible causal effect of TSH on thyroid cancerusing twosample Mendelian randomization Ninetyfour nonHLA genetic variants for TSH identified by our metaanalysis ofHUNT MGI and ThyroidOmics were used as instrumentalvariables Fstatistic for all single nucleotide polymorphismsSNPs rs1265091 and rs3104389 are HLA variants andthe summary statistics for thyroid cancer were not available forrs121908872 rs4571283 and To avoid sampleoverlap for the TSH and thyroid cancer GWASs we used effectson TSH estimated by metaanalyzing HUNT and ThyroidOmicsto construct the instrumental variable for TSH levels and wemetaanalyzed MGI deCODE and UKBB for thyroid cancer Wefound that a one SD increase in TSH SD mUL wasassociated with a decreased risk of thyroid cancer inversevariance weighted OR CI “ MREgger intercept Pvalue Sensitivity analyses using the penalizedweighted median method the weighted median method and theweighted mode method including all variants are presented inFig 3a and Supplementary Data Similar results were observedbetween methods with the exception of the weighted modewhich was strongly attenuated To reduce the possibility that theresults were influenced by occult thyroid dysfunction typicallyoccurring in older age we repeated the analysis using SNPTSHeffect estimates obtained among those younger than years ofage at the time of TSH measurement Supplementary Data Similar results were observed except for the weighted modewhich was again attenuated towards the null OR CI“ Supplementary Data Furthermorethere wasstrong evidence of heterogeneity suggesting some instrumentswere invalid Nevertheless when repeating the main analysisusing MRPRESSO which excluded nine variants due to thedetection of specific horizontal pleiotropic outlier variants54 thecausal association was similar MRPRESSO outlier corrected OR CIs “ Fig 3a and Supplementary Data Finally using only the proteincoding nonsynonymous variant pP118L in the TG gene Fstatistic we observed a protective effect of increased TSH on thyroid cancer Wald ratio OR CI “ To investigate if TSH may also influencethe risk of benign thyroid growth disorders we similarly performed atwosample Mendelian Randomization analysisbetween TSH and goiter The effects on TSH were estimated by ametaanalysis of HUNT and ThyroidOmics SupplementaryData and and the GWAS results for goiter from the UKBBwere used2728 A SD increase in TSH SD mULwas associated with a decreased risk of goiter inversevariance weighted OR CI “ MREgger interceptPvalue Fig 3b and Supplementary Data DiscussionMetaanalysis of the HUNT study the MGI biobank and theThyroidOmics consortium for TSH on up to individualsidentified TSH loci of which are previously unreported AllTSH loci reported by previous GWAS studies172223 are replicated in our metaanalysis Several novel loci pointed to nearbygenes with a known or suspected link to thyroid functionAdditional independent signals were identified among several locibased on GWAS results in the metaanalysis and LD informationin the HUNT study including two rare variants rs546738875 andrs145153320 at the B4GALNT3 locus and two rare missensevariants TSHR pA553T rs121908872 and TSHR pR609Qrs139352934 which have been observed to be associated withcongenital hypothyroidism in previous family studies3637 TSHRpR609Q rs139352934 is the most strongly associated with TSHin the TSH receptor gene TSHR with an effect size greater thanone standard deviation of TSH mUL As these rare variants were only imputed in HUNT not in MGI or ThyroidOmicsfurther followup to verify the associations is needed As individual GWAS was conducted on inversenormal transformed TSHlevels before metaanalysis it is challenging to convert the effectsizes reported by our metaanalysis to actual scales of TSH levelsFinemapping for potential causal variants among TSH locidetected two independent missense variants in the TG gene TGpG67S and pP118L The two variants have a similar frequency but pP118L shows stronger evidence for an associationwith goiter and with thyroid cancer Functional experimentsdemonstrated each of these defects in the TG gene pP118L andpG67S respectively causes defective secretion of TG Furtherstudies are needed to investigate how the proteinaltering variantsimpact TSH levelsAs expected membranes and thyroid gland were identified asthe tissue in which genes from TSHassociated loci are most likelyto be highly expressed Genes ZFP36L2 B4GALNT3 PPP1R3BFAM109A GNG12 GADD45A BMP2 VEGFC LPP and MAL2were prioritized as functional candidates among the novel TSHassociated loci based on functional similarity to other genes at allTSH loci using DEPICT47A PheWAS ofthe TSHassociated variants in the UKBBdemonstrated that TSHincreasing alleles are associated with anincreased risk of cardiovascular disease smaller body sizereduced bone mineral density decreased lung function and anincreased risk of hypothyroidism but were favorably associatedwith a decreased risk of goiter Our results suggest that thesevariants have pleiotropic effects although they tend to affect TSHthrough actions in the thyroid glandPhenomewide association tests in the UKBB and FinnGen forthe TSH PGS showed that genetically predicted increased TSH isassociated with a high risk of hypothyroidism and a low risk ofgoiter thyrotoxicosis hyperhidrosis and thyroid cancer Twosample Mendelian randomization analyses suggested that lowerTSH causes an increased risk of thyroid cancer and goiter This isan unexpected direction given that TSH promotes the growth ofthyroid cancers814“ Nonethelessit has previously beenspeculated that lower TSH levels may lead to less differentiationof the thyroid epithelium which could predispose to malignanttransformation17 Alternatively our genetic instrument for TSHmay represent a thyroid gland phenotype that influences bothTSH through the negative feedback of thyroid hormones on thepituitary gland and thyroid growth increasing the risk of thyroidcancer and goiter Supplementary Fig In that scenario theeffect on thyroid cancer would not be downstream of TSH andaltering TSH levels eg by medication would not be expected toalter thyroid cancer risk Tissue enrichment analyses of genes atTSHassociated loci and the observation that TSHloweringalleles were generally associated with higher free thyroxine levelsSupplementary Data suggest that most TSHassociated variants act primarily on the thyroid gland where effects on boththyroid function and growth have previously been described forNATURE COMMUNICATIONS 101038s4146702017718z wwwnaturecomnaturecommunications 0carecnac doryhit no tceff ePNSbretiog no tceffe PNSNATURE COMMUNICATIONS 101038s4146702017718z““““MethodInverse variance weightedMR EggerMRPRESSOPenalized weighted medianWeighted medianWeighted modeSNP effect on TSHMethodInverse variance weightedMR EggerMRˆ’PRESSOPenalized weighted medianWeighted medianWeighted modeSNP effect on TSHFig Twosample Mendelian randomization analysis for casual associations between TSH and thyroid cancer and between TSH and goiter a Twosample MR between TSH and thyroid cancer based on summary statistics from the metaanalysis of HUNT and ThyroidOmics n for TSH andfrom the metaanalysis of UKBB2728 MGI26 deCODE and four other case“control data sets with European ancestry as reported in Gudmundsson et al19for thyroid cancer association casescontrols b TSH and goiter based on summary statistics from the same TSH metaanalysis asabove and from the GWAS of UKBB2728 for goiter association N case N control The crosshairs on the plots represent the confidence intervals for each SNPTSH or SNPoutcome association The variant on the top left corner is the rare nonsynonymous variant B4GALNT3 pR724W rs145153320 N sample size N case sample size of cases N control sample size of controlsTSHR mutations55 Nonetheless the Mendelian randomizationfindings were robust to most of the sensitivity analyses performedto detect and correctfor pleiotropy Restricting the geneticinstrument to TSHlowering variants associated with lower freethyroxine levels could have helped resolve the causal question butthose variants were too few to yield meaningful estimat

Thyroid_Cancer

RNAs circRNAs are a novel and unique class of noncoding RNAs that are backspliced from premRNAs It hasbeen confirmed that circRNAs are involved in various malignant behaviors of hepatocellular carcinoma HCCHowever the role of circRNA in the regulation of ferroptosis and the underlying mechanism remain unknown HerecIARS hsa_circ_0008367 was found to be the most highly expressed circRNA after sorafenib SF treatment in HCCcells Small interfering RNA against cIARS sicIARS significantly suppressed the cellular sensitivity to SF or Erastinthrough inactivating ferroptosis which may be partially attributed to the inhibition of autophagy and ferritinophagyPrediction analysis and mechanistic identification revealed that cIARS physically interacted with RNA binding proteinRBP ALKBH5 which was a negative regulator of autophagic flux in HCC The dissociation of BCL2BECN1 complexmediated by ALKBH5 silencing was effectively blocked by sicIARS Furthermore the inhibition of ferroptotic eventsautophagic flux and ferritinophagy resulted from sicIARS were significantly rescued by ALKBH5 downregulationOverall cIARS may be an important circRNA positively regulating SFinduced ferroptosis through suppressing theALKBH5mediated autophagy inhibitionIntroductionHCC is a highly refractory and prevalent cancerworldwide with new cases and deathsevery year1 Rapid advances in diagnosis and treatmenthad improved patient outcomes However the survivalgains are stagespecific2 Liver resection transplantationablation ortransarterial chemoembolization benefitspatients in early or intermediate stages3 while treatmentfor latestage HCC has remained challenging The standard firstline systemic drug against advanced HCC issorafenib SF BAY Nexavar which is currentlyCorrespondence Xiaoqing Wei weixqrm163com orJie Li heplijie163com1Department of Hepatobiliary Surgery The First Affiliated Hospital ofShandong First Medical University Jinan Shandong China2Department of Hepatobiliary Surgery Shandong Provincial QianfoshanHospital Shandong University Jinan Shandong ChinaFull list of author information is available at the end of the These authors contributed equally Zhiqian Liu Qi WangEdited by Inna Lavrik The Authors known as a ferroptosis inducer4 exerting only limitedeffects on overall survival and time to tumor progression56 Therefore it is imperative to explore novel andeffective therapeutic target to improve the cellular sensitivity to SFˆ’ was targeted SubsequentlyIn recent years ferroptosis has been confirmed to be anessential mechanism in SF treatment7 It is a kind of finelycontrolled cell death featured with irondependent accumulation of lipid hydroperoxides8 When HCC cells wereexposed to SF the cystineglutamate antiporter systemsystem xcthe cystineuptake was blocked and the biosynthesis of glutathioneGSH was inhibited resulting in accumulation of lipidperoxidation products and eventually inducing ferroptosis Meanwhile SF also induces autophagic flux in cellsWhen autophagy flux is initially induced MTORC1 dissociates from the ULK12ATG13 complex leading todephosphorylation of the complex9 Then phagophorenucleation occursthe ATG14BECN1recruiting Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate ifchanges were made The images or other third party material in this are included in the ™s Creative Commons license unless indicated otherwise in a credit line to the material Ifmaterial is not included in the ™s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this license visit httpcreativecommonslicensesby40Official journal of the Cell Death Differentiation Association 0cLiu Cell Death Discovery Page of PIK3C3PIK3R4 complex10 Nexttwo ubiquitinlikeconjugation systems are involved in phagophore elongation11 When de novo autophagosome formation iscomplete they are delivered to the lysosome initiatinglysosomal degradationActually autophagy is a targetable pathway regulatingcellular sensitivity to ferroptosis12 The crosstalk betweenautophagy and ferroptosis has attracted more and moreattentions in the recent years Autophagy inhibition bybafilomycin A1 or chloroquine or lack of key autophagyassociated gene for example ATG5 ATG7 will partiallysuppressed ferroptotic eventsthrough ferritinophagyinactivation reducing the turnover of ironbinding ferritinand the iron release13“CircRNAs primarily originate from backsplicing eventsof premRNAs The covalently bonded loops are highlystable and abundant16 The deregulation of circRNAs isclosely related to various human diseases including cancer17“ In HCC several studies have revealed that circRNAs are involved in multiple malignant behaviors20However the functions and mechanisms of circRNAs inSF treatment are still not comprehensively understood Inthe current research we found a novel circRNA derivedfrom the IARS gene ID from circBase hsa_circ_0008367named cIARS cIARS was proven to be a promoter offerroptosis in HCC cells after SF treatment which waspartially attributed to the activation of autophagy andferritinophagy The underlying molecular mechanism ofcIARSmediated ferroptosis was also clarified in this studyResultsCircular transcript cIARS hsa_circ_0008367 is significantlyupregulated in SFtreated HCC cellsRNAseq was performed in three pairs of SFtreated anduntreated HCC celllines HepG2 SMMC7721 andHuh7 to screen differentially expressed circRNAs Cellswere treated with μM of SF for h uniquecircRNAs were found Among these circRNAs had already been reported in circBase21 and theother circRNAs were newly discoveredAmong the annotated circRNAs were ofextremely low abundance with fragments per kilobasemillion FPKM values in cellular samples and werethus excluded from this study The other circRNAscomprised two groups presented no significantchanges in expression levels after SF treatment and theother were differentially expressed circRNAs foldchange ‰¥ pvalue The volcano plots demonstrated that there were upregulated and downregulated transcripts Fig 1a The results of hierarchicalclustering are displayed in a heatmap Fig 1b generatedwith Heatmap Illustrator22 and suggest that there weretwo different clusters of transcripts The most highlyexpressed circRNA was cIARS hsa_circ_0008367 andOfficial journal of the Cell Death Differentiation Associationwas marked with black arrow in the volcano plotsAccording to circBase cIARS is an exonic circRNA nt in length originating from the exon and exon of the IARS gene on chr9 “ Fig1c qPCR showed that the relative expression levels ofcIARS were significantly higher in SFtreated cell linesthan in untreated ones Fig 1d Both convergent anddivergent primers of cIARS were applied for amplificationThe band of cIARS was only observed in cDNA samplenot the genomic DNA Fig 1e Sanger sequencing further validated that the sequence around the junction siteabout bp around the site was consistent with theresult of RNAseq and CircInteractome database23 Fig1f In addition cIARS was much more resistant to RNaseR Uμg which degrades linear but not circularthan IARS and GAPDH Fig 1g Whentranscriptsactinomycin D ActD a transcription inhibitor wasadded to HCC cells for the indicated time periods cIARSwas much more stable than its linear counterpart Fig1h These evidences suggested cIARS to be a highlyabundant and stable circular transcript in HCC cellscIARS is found to be a significant regulator of SFinducedferroptosisTo clarify the biological role of cIARS we first knockdown cIARS expression with a junction sitespecificsiRNA vector sicIARS The effects of the sicIARSwas shown in Fig 2a CCK8 assay showed that SFinduced growth inhibition was evidently weakened in sicIARS transfected cells Erastininduced growth inhibitionwas also attenuated by sicIARS Fig 2b c To determinethe underlying mechanism sicIARSintroduced HCCcells were treated with various cell death inhibitorsFerrostatin124 a specific ferroptosissignificantly undermined the therapeutic effects of either SFor Erastin in both sicIARS and NC transfected cellsHowever ZVADFMK an apoptosisinhibitor andNecrosulfonamide a necroptosis inhibitor exerted nosignificant influence on SF or Erastininduced growthinhibition Fig 2c Simultaneously malondialdehydeMDA and the level of Fe2 were significantly reducedwhile intracellular GSH obviously increased in the cIARSsilencing cells following SF or Erastin administration Fig2d These evidences suggested cIARS to be a positiveregulator of ferroptosis in HCC cellsinhibitorcIARS positively regulates SFinduced autophagy andferritinophagycIARS was also found to be an autophagy regulatorWestern blot WB assay showed that cIARS knockdownsignificantly decreased LC3 lipidation and increased p62accumulation Fig 3a Either autophagosomes or autolysosomes were observed via microscopic examinationafter AdmCherryGFPLC3 adenovirustransfection 0cLiu Cell Death Discovery Page of Fig See legend on next pageThis experiment is applied for concurrent observation ofautophagosome and autolysosome The signal of greenfluorescent protein will be quenched during fusion ofautophagosome and lysosome Thus the red signal ofmCherry indicates autolysosome and the merge of greenand red signals yellow puncta indicates autophagosomesicIARS significantly decreased the amount ofredautolysosome and yellow autophagosome puncta percell demonstrating an inhibition of autophagy fluxFig 3b TEM visuallyautophagicsuggested theOfficial journal of the Cell Death Differentiation Association 0cLiu Cell Death Discovery Page of see figure on previous pageFig Circular transcript cIARS hsa_circ_0008367 is significantly upregulated in SFtreated HCC cells RNAseq was performed in three pairsof SFtreated μM h and untreated HCC cell lines HepG2 SMMC7721 and Huh7 a The differentially expressed circRNAs were visualized withvolcano plots the ˆ’log10 pvalue and the log2 fold change are plotted on the y and x axes respectively The dashed lines signify the filteringcriteria p fold change ‰¥ Upregulated circRNAs are shown in red and downregulated circRNAs are shown in green The most highlyexpressed circRNA was marked by a black arrow b Hierarchical cluster analysis showing the differentially expressed circRNAs The six columnsrepresent the six different cellular samples Each row indicates a circular transcript and the colors represent the abundance of the transcriptsc Schematic diagram of the backsplicing transcript generated from linear IARS Exon and exon are colored red and green respectively dRelative expression of cIARS after SF treatment for h in the HCC cell lines e Divergent and convergent primers for cIARS were applied to amplifyboth cDNA and gDNA Agarose gel electrophoresis visualized the products f The sequence around the junction site was confirmed through Sangersequencing The empty triangle indicates the junction site g RNase R exoribonuclease Uμg °C min was applied to remove the lineartranscripts from cellular extracts leaving circRNAs behind qPCR was applied to assess the resistance of RNAs to RNase R h RNA decay assayevaluating the stability of cIARS and IARS by qPCR after ActD μgml administration The error bars represent the standard deviation SD of at leastthree independent experiments p compartments sicIARS decreased the number of doublemembraned vacuoles to a relatively low level Fig 3cThese results showed that cIARS is a positive autophagyregulator in SFtreated HCC cells Furthermoretheprotein levels of FTH1 and NCOA4 the substrate andcargo receptor of ferritinophagy were determined by WBassay sicIARS resulted in remarkable accumulation ofboth FTH1 and NCOA4 Fig 3d This finding indicatedthatthe ferroptotic events in SFtreated HCC cellspositively regulated by cIARS may be partially associatedwith autophagy and ferritinophagycIARS specifically interacted with RBP ALKBH5 AlkBHomolog RNA demethylaseRecent studies have revealed that circRNA“RBP interaction has important roles in diverse biological processesAccording to CircInteractome database we found six RBPsbearing at least two binding sites matching to cIARSincluding FMRP Fragile X Mental Retardation SFRS1Serine And Arginine Rich Splicing Factor ALKBH5HuR ELAV like RNA binding protein IGF2BP1 InsulinLike Growth Factor MRNA Binding Protein andLIN28A Lin28 Homolog A Fig 4a RBP immunoprecipitation RIP assay in both HepG2 and Huh7 cell linesdemonstrated that the relative levels of cIARS in ALKBH5enriched samples were much higher than in the other fiveRBPs Fig 4b RNA pulldown and RNA EMSA were performed to determine the interaction using an antisenseprobe spanning the junction site of cIARS The presence ofspecific bands demonstrated the physical binding of cIARSand ALKBH5 Fig 4c d Interestingly SF administrationhad no influence on the protein levels of ALKBH5 in bothHepG2 and Huh7 cells Fig 4e but remarkably increasedthe cIARS“ALKBH5 interaction Fig 4f which may be dueto SFinduced expression of cIARScIARS repressed the role of ALKBH5 in the regulation ofautophagyALKBH5 had been previously proven to be an autoprole inin cancer2526 Howeverhagy inhibitoritsOfficial journal of the Cell Death Differentiation AssociationSFtreated HCC cells remains unclear WB assay showedthat siRNA against ALKBH5 siALKBH5 significantlypromoted the transformation of LC3B I to II and degraded p62 Fig 5a This result suggested ALKBH5 to be anegative regulator of autophagy in HCC cells qPCR andWB showed that sicIARS failed to influence ALKBH5mRNA and protein levels Fig 5b c similarly siALKBH5 also had no impact on the relative expressionof cIARS Fig 5d A previous study revealed that siALKBH5 promoted the dissociation of BECN1 and BCL a key step during phagophore nucleation In SFtreated HCC cells we gained similar results via IP assayMore importantly this process can be effectively blockedby cIARS silencing Fig 5e These results demonstratedthat cIARS repressed the biological role of ALKBH5 inautophagycIARS regulates ferroptosis through ALKBH5mediatedautophagyTo explore whether cIARSregulated ferroptosis viaALKBH5 we performed several phenotyperescueexperiments First siALKBH5 successfully rescued theeffects of sicIARS in autophagic flux and ferritinophagyFig 6a b Second ALKBH5 knockdown effectively reintensified the SF cytotoxicity which was remarkablyimpaired by sicIARS Fig 6c Third sicIARSmediateddecrease of MDA Fe2 and increase of GSH can berescued by siALKBH5 Fig 6d“fTaken together a novel circRNA in HCC was revealedin our research We partially clarified its role andmechanism in ferroptosisDiscussionAs a novel class of noncoding RNA circular transcriptshave attracted widespread attention However circRNAregulated ferroptosis in human diseases has not beenwidely investigated A small portion of studies focused onthe circRNAmediated autophagy For instance Chenet al27 reported that circHIPK3 depletion significantlyvia miR1243pSTAT3PRKAAinduced autophagy 0cLiu Cell Death Discovery Page of Fig cIARS is found to be a significant regulator of SFinduced ferroptosis a The expression levels of cIARS after transfection of sicIARS or NCb The evaluation of growth inhibition induced by SF in sicIARS or NC transfected cells at the indicated concentrations for h c The evaluation ofcytotoxicity of SF μM h and Erastin μM h with or without several inhibitors of cell death including ferrostatin1 μM ZVADFMK μM or necrosulfonamide μM d The assessment of MDA Fe2 and GSH during SF treatment μM h p AMPKa axis and there was an antagonistic regulation onautophagy between circHIPK3 and linear HIPK3 Du WWet al28 showed that the oncogenic circDnmt1stimulatedautophagy flux in breast carcinoma via interaction withboth p53 and AUF1 These findings demonstrated thepotential of circRNAsin autophagy regulation andprompted us to explore the role of circRNA in ferroptosiswhich had been identified as an autophagic cell death12research we delineated a mechanism ofcircRNAmediated ferroptosis during SF treatment inHCC cells circRNA cIARS hsa_circ_0008367 wasIn ourOfficial journal of the Cell Death Differentiation Associationscreened from RNAseq analysis Phenotypically cIARSpositively regulated ferroptosis which may be partiallydependent on autophagy and ferritinophagy Mechanistically cIARS physically interacted with RBP ALKBH5and negatively regulated its role in autophagyitis essentialTo comprehensively investigate the complicated cirto deeply evaluate itscRNA networkbinding partners In this study cIARS is found to be aninteractor of RBP ALKBH5 which had previously beenreported to be a N6methyladenosine m6A eraser Itsrole in autophagy regulation is completely different in 0cLiu Cell Death Discovery Page of Fig cIARS positively regulates SFinduced autophagy and ferritinophagy a The expression levels of LC3B and p62 in sicIARSintroduced cellswith or without SF treatment b Microscopic observation of autophagy flux after transfection of AdmCherryGFPLC3 for h mCherryGFPLC3 wasvisualized by fluorescence microscopy The green fluorescent protein is acid sensitive and will be quenched in the acidic condition of lysosome while themCherry is stable at low pH Thus the red puncta represent autolysosomes and the yellow puncta generated from the merge of both green and red signalsrepresent autophagosomes The autophagosomal and autolysosomal abundance was measured by the number of puncta lower density of yellow and redpuncta suggested lower level of autophagic flux c Visualization of autophagic compartments via TEM The red arrowheads indicate doublemembranedvacuoles d The assessment of the protein levels of FTH1 and NCOA4 in sicIARS or NC transfected cells with or without SF treatment μM hlower m6A leveldifferent tissues and diseases252629“ In lung cancer25ALKBH5 upregulation stabilized UBE2C an autophagyinhibitor with maintenance ofInovarian cancer26 ALKBH5 inhibited autophagy throughactivating PI3KAktmTOR signaling pathway stabilizedBCL2 mRNA and promoted the interaction betweenBCL2 and BECN1 Herein cIARS was proven to be apivotal regulator of autophagy ferroptosis and ferritinophagy depending on negatively regulating the biologicalinHCC cellsrole of ALKBH5 an autophagy inhibitibco Carlsbad CA USA and penicillin Umlstreptomycin µgml solution in the medical researchcenter of Shandong Provincial Qianfoshan HospitalShandong First Medical University All of the cell linesmentioned in this research were cultured within tenpassages The transfection experiments were performedaided by Lipofectamine Life Technologies Carlsbad CA USA The RNA oligonucleotides in this workwere designed and constructed by GenePharma Shanghai China including siRNAs against cIARS or ALKBH5and the corresponding negative controls NC and NC™Sequences were shown as follows ²“² sicIARS GACUUU GAG GAG AUC AGA CAC siALKBH5 GGAUAU GCU GCU GAU GAA ATT NCNC² UUC UCCGAA CGU GUC ACG UIn this study the cIARS“ALKBH5 axis was demonstrated to be a key mechanism regulating ferroptosisduring SF treatment Further studies are still needed atthe clinical and mechanistic levelsMaterials and methodsCell culture and transfection assayUnder humidified conditions with CO2 at °C theHCC cell lines HepG2 SMMC7721 and Huh7 boughtfrom the National Infrastructure of Cell Line Resourcewere cultured in Dulbecco™s Modified Eagle™s mediumHyClone Logan UT USA with fetal bovine serumCell Counting Kit8 CCK8 assayThe growth inhibition rate of HepG2 and Huh7 cellswas assessed by the CCK8 Dojindo Laboratories Japanassay The blank or transfected cells × cells perwell were seeded into 96well plates with three replicatewells After the treatment with SF or Erastin or variouscell death inhibitorsFerrostatin1 ZVADFMK orOfficial journal of the Cell Death Differentiation Association 0cLiu Cell Death Discovery Page of Fig cIARS specifically interacts with ALKBH5 a Prediction analysis of the interaction between cIARS and RBPs through CircInteractome b RIPassay evaluating the physical interaction between cIARS and the six candidate RBPs in HCC cells c d RNA EMSA and RNA pulldown evaluating thephysical interaction between cIARS and ALKBH5 e The protein levels of ALKBH5 in HCC cells in the absence or presence of SF treatment f Theassessment of the physical binding of cIARS and ALKBH5 by RIP assay in the absence or presence of SF treatment p Necrosulfonamidefor h a µl volume CCK8reagent was added to each well Then measuring theabsorbance at nm after incubation with the CCK8solution at °C for hRNA extraction and analysisExpression profiles of genomewide circRNAs in threepairs of HCC cell lines before and after SF treatmentwere explored on an Illumina HiSeq platform byNovogene Beijing China The cDNA from divergentprimers was subjected to Sanger sequencing by Zhonghong Boyuan Biological Technology Jiangxi ChinaAgarose gel electrophoresis was used to detect the qPCRamplification of cDNA and genomic DNA after applyingwith the divergent and convergent primers of cIARSTherelative fold changes in expression were calculated withthe formula ˆ’ΔΔCt The sequences of all the primers werelisted as follows ²“² cIARS F AGC GAT GAC TTTGAG GAG ATC A R CCC AGT AGC ACA GGT CATTG IARS F CAT ATC CAG TTT CTC CAT CGG A RTGG ATT TTC CAG GAG CAA TAC T ALKBH5 FGCA AGG TGA AGA GCG GCA TCC R GTC CACCGT GTG CTC GTT GTA C U6 F CTC GCT TCGGCA GCA CAT A R ATT TGC GTG TCA TCC TTGCG GAPDH F CAG AAC ATC ATC CCT GCC TCTAC R ATG AAG TCA GAG GAG ACC ACC TGRibonuclease R RNase R assayRNase R assay R0301 Uµl Geneseed GuangzhouChina was used for the identification of circRNAAccording to the manufacturer™s guidance µl ×Reaction Buffer and U RNase Rµg RNA were mixedto the total RNA and then added RNaseFree Water toform a µl reaction solution system After digestion withRNase R for min at °C the enzyme then was inactivated at °C for min and then directly performOfficial journal of the Cell Death Differentiation Association 0cLiu Cell Death Discovery Page of Fig cIARS repressed the role of ALKBH5 in the regulation of autophagy a The expression levels of LC3B and p62 in siALKBH5 or NC²introduced cells with or without SF administration b c The relative expression of ALKBH5 mRNA and protein in sicIARS or NC introduced HCC cellsd The relative expression of cIARS in siALKBH5 or NC² transfected cells e The assessment of the role of sicIARS in ALKBH5mediated interactionbetween BECN1 and BCL2Official journal of the Cell Death Differentiation Association 0cLiu Cell Death Discovery Page of Fig See legend on next pageOfficial journal of the Cell Death Differentiation Association 0cLiu Cell Death Discovery Page of see figure on previous pageFig cIARS regulates ferroptosis through ALKBH5 a b The evaluation of the role of siALKBH5 in sicIARSregulated autophagy and ferritinophagyThe WB assay demonstrated that the effect of sicIARS on LC3 lipidation and p62 accumulation can be significantly reversed by siALKBH5 in SF μM h treated HCC cells a the effect of sicIARS on the accumulation of FTH1 and NCOA4 can be evidently rescued by siALKBH5 in either HepG2 or Huh7cells treated by SF μM h b c Identification of the role of ALKBH5 in cIARSregulated growth inhibition by CCK8 The results demonstrated thatsiALKBH5 significantly reversed sicIARSmediated growth inhibition in SF administered HCC cells d“f The rescue experiments evaluating the effects ofsiALKBH5 in the sicIARSmediated ferroptotic events The relative levels of MDA and iron were obviously decreased and the level of GSH increased insicIARS introduced HCC cells these impacts can be effectively reversed by ALKBH5 knockdown The black whiskers indicated the difference between theœsicIARS and œNC groups while the red ones indicated the difference between the œsicIARSNC™ and œsicIARSsiALKBH5 groups p reverse transcription reaction The qPCR assay was usedto determine the relative expression of cIARS IARS andGAPDH compared to the mock groupActinomycin D ActD assayActD assay HY17559 MedChem Express New JerseyUSA was used to detect the stability of RNA μgmlActD reagent was used to treat HepG2 and Huh7 cellsAfter or h of administration the total RNAwas extracted respectively to determine the relativeexpression of cIARS or IARS by qPCR assayWestern blot analysisRadioimmunoprecipitation assay buffer RIPA was appliedto lyse cells Total proteins were then harvested and quantified with bicinchoninic acid assays Beyotime ShanghaiChina The target proteins were separated through SDSPAGE and transferred to polyvinylidene fluoridemembranes Merck Millipore Burlington MA USA Themembranes were blocked with nonfat milk incubated withprimary antibodies and then incubated with secondaryantibody diluted at a ratio of Jackson ImmunoResearch West Grove PA USA The primary antibodieswere antiLC3B Cell Signaling Technology BeverlyMA USA antip62 Cell Signaling TechnologyantiHuR ab28660 Abcam Cambridge MA USA antiSFRS1 ab133689 antiFMRP ab17722 antiALKBH5ab195377antiLIN28Aab46020 antiFTH1 ab65080 antiNCOA4 ab86707antiBCL2 ab32124 antiBECN1 ab62557 and antiGAPDH ab9485 The protein signals were visualized withenhanced chemiluminescence detection reagentsECLMillipore Burlington MA USA and quantified with ImageLab software BioRad Hercules CA USAantiIGF2BP1ab82968based on the 3rd edition of the Guidelines for the Interpretation of Assays for Monitoring Autophagy32ImmunoprecipitationandImmunoprecipitation KitRIP and IP were performed to confirm the RNA“proteinand protein“protein interactions using RIP Kit Milliporeab206996according to the manufacturer™s guidance RIP related detailshad been described in our previous studies3334 The IP assayconsists of four steps including antibody binding beadspreparation bead capture and elution The volume of theantibody binding system was made up to µl with lysisbuffer containing the protease inhibitor cocktail and gentlymixed for h the protein AG sepharose µlreactionwas washed twice with wash buffer centrifuged at × gfor min and aspirated the supernatant between washesRNA pulldown assayPierce„¢ Magnetic RNAProteinPullDown KitThermo was applied to evaluate RNA“protein interaction using biotinlabeled junctionspecific probe and itsnegative control designed and synthesized by ViageneBiotech Jiangsu China Detailed procedure strictly followed the manufacturer™s guide cIARSprobe ²TGTAAA TTA GAG GAG TGT CTG ATC TCC TCA AAGTC3² Negative control ²GCA GCC TGA TCA CGACTG ACT TTA GTG TTT GCA TT3²RNA EMSAThe LightShift Chemiluminescent RNA EMSA KitThermoFisher Scientific was applied to evaluate theinteraction between cIARS and ALKBH5 according to themanufacturer™s guidance The details were described inour as previous research34Observation of autophagy fluxLipid peroxidation assayAdmCherryGFPLC3 adenovirus Servicebio Technology Wuhan China was transfected into HCC cellsAfter h incubation cells were observed and photographed using a fluorescence microscope OlympusFSX100 Tokyo Japan Autophagic compartments werefinely observed through transmission electron microscopyTEM HT7700 HITACHI Tokyo Japan The identification of autophagic vacuoles in TEM images was mainlyThe relative level of MDA was evaluated through Lipidab118970 according to the manuPeroxidation Kitfacturer™s guidanceIron assayThe relative level of intracellular iron was determinedby an Iron Assay Kit ab83366 according to the manufacturer™s instructionsOfficial journal of the Cell Death Differentiation Association 0cLiu Cell Death Discovery Page of Glutathione assayThe relative level of intracellular GSH was assessedthrough a GSH Colorimetric Detection Kit CS0260SigmaAldrich St Louis USA according to the manufacturer™s instructionsStatistical analysisThe statistical analyses in this work were carried outusing Prism The results from at least three independenttests are shown as the mean value ± standard deviationSD The mean values of two groups were compared viaunpaired Student ttests œpvalue  was defined assignificant p p AcknowledgementsThis work was supported by the National Natural Science Foundation of China[Grant No ] Shandong Natural Science Foundation [Grant NoZR201808200282] and Medical and Pharmacological TechnologyDevelopment plan Shandong [Grant Nos 2017WS188 and 2017WS284]Author details1Department of Hepatobiliary Surgery The First Affiliated Hospital ofShandong First Medical University Jinan Shandong China2Department of Hepatobiliary Surgery Shandong Provincial QianfoshanHospital Shandong University Jinan Shandong China 3Department ofReproduction Medicine Jinan Maternal and Child Health Care HospitalAffiliated to Shandong First Medical University Jinan Shandong ChinaAuthor contributionsStudy design ZL XW and JL data collection QW ZX and XW dataanalysis ZL QW and XW cellular experiments QW ZL XW and ZXmanuscript preparation ZL QW ZX and JL figures and manuscriptproofing ZL QW JL and XW project administration XW and JLConflict of interestThe authors declare that they have no conflict of interestPublisher™s noteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsReceived May Revised July Accepted July References Bray F Global cancer statistics GLOBOCAN estimates of incidenceand mortality worldwide for cancers in countries CA Cancer J Clin “ Siegel R L Miller K D Jemal A Cancer statistics CA Cancer J Clin “ Forner A Reig M Bruix J Hepatocellular carcinoma Lancet “ Li J Ferroptosis past present and future Cell Death Dis Llovet J M Sorafenib in advanced hepatocellular carcinoma N Engl JMed “ Cheng A L Efficacy and safety of sorafenib in patients in the AsiaPacificregion with advanced hepatocellular carcinoma a phase III randomiseddoubleblind placebocontrolled trial Lancet Oncol “ Official journal of the Cell Death Differentiation Association Dixon SJ Pharmacologicalinhibition of cystineglutamateexchange induces endoplasmic reticulum stress and ferroptosis Elife e02523 Stockwell B R Ferroptosis a regulated cell death nexus linking metabolism redox biology and disease Cell “ Jung C H ULKAtg13FIP200 complexes mediate mTOR signaling to theautophagy machinery Mol Biol Cell “ Itakura E Mizushima N Characterization of autophagosome formation siteby a hierarchical analysis of mammalian Atg proteins Autophagy “ Geng J Klionsky D J The Atg8 and Atg12 ubiquitinlike conjugation systems in macroautophagy ˜Protein modifications beyond the usual suspects™review series EMBO Rep “ Zhou B Ferroptosis is a type of autophagydependent cell death SeminCancer Biol httpsdoi101016jsemcancer201903002 Torii S An essential role for functional lysosomes in ferroptosis of cancercells Biochem J “ Gao M Ferroptosis is an autophagic cell death process Cell Res “ Hou W Autophagy promotes ferroptosis by degradation of ferritinAutophagy “ Memczak S Circular RNAs are a large class of animal RNAs with regulatory potency Nature “ Bi W CircRNA circRNA_102171 promotes papillary thyroid cancer progression through modulating CTNNBIP1dependent activation of betacateninpathway J Exp Clin Cancer Res Su Y circRIP2 accelerates bladder cancer progression via miR1305Tgfbeta2smad3 pathway Mol Cancer Chen J Circular RNA profile identifies circPVT1 as a proliferative factorand prognostic marker in gastric cancer Cancer Lett “ Wang M Yu F Li P Circular RNAs characteristics function and clinicalsignificance in hepatocellular carcinoma Cancers httpsdoi103390cancers10080258 Glazar P Papavasileiou P Rajewsky N circBase a database for circular RNAsRNA “ Deng W Wang Y Liu Z Cheng H Xue Y HemI a toolkit for illustratingheatmaps PLoS ONE e111988 Dudekula D B CircInteractome A web tool for exploring circular RNAsand their interacting proteins and microRNAs RNA Biol “ Dixon S J Ferroptosis an irondependent form of nonapoptotic celldeath Cell “ Guo J Deregulation of UBE2Cmediated autophagy repression aggravates NSCLC progression Oncogenesis Zhu H A

Thyroid_Cancer

"Nonsmall cell lung cancer is the most common cause of cancer death worldwide highlighting the need fornovel therapeutic concepts In particular there is still a lack of treatment strategies for the group of elderly and frail patientswho are frequently not capable of receiving standard therapy regimens Despite comprising the majority of lung cancerpatients this group is underrepresented in clinical trials This applies also to elderly and frail patients suffering fromunresectable stage III NSCLC who are unfit for chemotherapy and therefore cannot receive the standard therapycomprising of radiochemotherapy and the recently approved subsequent durvalumab consolidation therapy These patientsoften receive radiotherapy only which raises the concern of undertreatment The TRADEhypo trial aims at optimizingtreatment of this patient group by combining radiotherapy with concomitant durvalumab administration therebyemploying the immunepromoting effects of radiotherapy and determining safety feasibility and efficacy of this treatmentMethods design In this prospective phase II clinical trial durvalumab therapy will be combined with either conventionallyfractionated CONgroup or hypofractionated HYPOgroup thoracic radiotherapy A safety stopandgo leadin phase willassess safety of hypofractionated radiotherapy with respect to severe pneumonitis in small patient cohorts before ing fullenrollment Tumor tissue blood and stool samples will be collected before and during the study period to investigate theimmunological mechanisms responsible for checkpoint inhibitor efficacy and immunepromoting effects of radiotherapyContinued on next page Correspondence FarastukBozmehrmeduniheidelbergde1Department of Thoracic Oncology Thoraxklinik at University Hospital ofHeidelberg R¶ntgenstraŸe Heidelberg Germany2Translational Lung Research Center Heidelberg TLRCH Member of theGerman Center for Lung Research DZL Im Neuenheimer Feld Heidelberg GermanyFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cBozmehr BMC Cancer Page of Continued from previous pageDiscussion Preclinical data suggests that irradiationinduced immunogenicity can be further increased if applied in ahypofractionated setting potentially boosting the expected synergistic effect with immune checkpoint inhibition in restoringthe immune antitumor response If proven safe and efficient a hypofractionated radiation schedule can provide a considerablymore practicable option for the patient Taking into consideration the intend to develop a combination treatment strategy thatcan be made available to patients soon after proving to be efficient and the potentially elevated toxicity of a hypofractionatedradiotherapy approach this trial was designed as a twotrialsinone design An accompanying translational research program isplanned striving to gain insights into the tumorhost biology and to identify suitable biomarkers to predict therapy responseTrial registration Clinicaltrialsgov NCT04351256 Registered April EudraCT “ Registered October Keywords Nonsmall cell lung cancer NSCLC Radioimmunotherapy Immune checkpoint inhibition AntiPDL1 monoclonalantibody Hypofractionated radiation Geriatric risk profileBackgroundLung cancer is the most common cause of cancer deathworldwide with nonsmall celllung cancer NSCLCrepresenting “ of cases [ ] Improving therapeutic strategies is thus of imminent importance especially considering elderly and frail patients With a medianage of about years at diagnosis lung cancer clearly is adisease of the elderly yet this group is underrepresentedin clinical trials and these patients are frequently not capable of receiving standard treatment protocols due to ageand tobaccoassociated comorbidities [“]attracting both immunocompetentIn recent years the advent of immunotherapy has pavedthe way for novel therapeutic concepts including the combination of radiotherapy with immune checkpoint inhibitioneg Programmed cell death ligand PD1 PDL1 Thisapproach is of particular interest as it utilizes synergistic effects While immune checkpoint inhibitors can restore thepatients™ antitumor immunity through T cell activationradiotherapy may further boostimmunemediated anticancer mechanisms by exposing tumorassociated antigensand byantigenpresenting cells and tumoricidal effector cells [ ] Indeedfor patients with unresectable stage III NSCLC the PACIFICtrial has revealed a profound clinical benefit treatment withthe antiPDL1 monoclonal antibody durvalumab after chemoradiotherapy with remarkably low toxicities [ ] As aresult sequential treatment with durvalumab after chemoradiotherapy has become the new standard treatment for locally advanced unresectable NSCLC However about ofpatients do not receive chemotherapeutic agents presumablydue to significantly higher rates of age and comorbidityrelated adverse events AE under chemoradiotherapy [] Thus elderly and frail patients often receive radiotherapyalone raising the serious concern of undertreatment and theneed for new therapeutic concepts [ ]Considering the immunepromoting effects of radiotherapy a combination with durvalumab therapy mayimprove response rates in these potentially undertreatedpatients Moreoverif applied early concomitant localdatathatsuggestradiotherapy with systemic immunotherapy may particularly increase control of distant micrometastases Preclinicalirradiationinducedimmunogenicity can even be further increased if appliedin a hypofractionated setting with single doses ‰¥ GrayGy in line with a radiation dosedependent abscopal[“] While a hypofractionated radiationeffectschedule is also considerably shorter and more convenient for the patient safety of concurrent immunoradiotherapy is a concern as both therapy modalities maycause severe pneumonitisIn this prospective phase II clinical trial we thereforeaim to determine feasibility and treatment efficacy ofdurvalumab treatment combined with thoracic radiotherapy TRT in previously untreated NSCLC stage IIIpatients unable to receive radiochemotherapy Strivingto develop a combination treatment strategy thatifproven safe and efficient can be quickly made availableto patients a twotrialsinone design was chosen thatcombines durvalumab with either conventionally fractionated CONgroup or hypofractionated thoracicradiotherapy HYPOgroup This study not only aims toincrease the efficacy of radiotherapy by utilizing theimmunesensitizing effects elicited by PDL1 inhibitionbut will also provide biomaterials that will be analyzedwith respect to immunological mechanisms responsiblefor checkpoint inhibitor efficacy and immunepromotingeffects of radiotherapy as well as potential biomarkersMethodsdesignStudy designThe TRADEhypo trialis a prospective randomized label multicenter phase II trial with a safety stopandgo leadin phase Fig During the leadin phasepatients in the HYPOgroup who will receive durvalumab in combination with hypofractionated thoracicradiotherapy will be closely evaluated with regard totoxicity defined as pneumonitis ‰¥ grade within 0cBozmehr BMC Cancer Page of Fig Study design of the TRADEhypo trial Patients will be enrolled according to eligibility criteria and treated with either a hypofractionated TRTregimen HYPOgroup or conventionally fractionated TRT CONgroup in combination with durvalumab For the HYPOgroup a safety stopandgophase with a design precedes full enrollment Whenever this arm is for recruitment patients will be allocated to this arm until the cohort isclosed whenever HYPOarm is closed for Stop Go decision evaluation based on the toxicity assessment of this regimen weeks after the end of TRTpatients are allocated to the CONarm When the study proceeds to expansion phase patients will be allocated to treatment arms by randomizationusing œbiased coin algorithm An efficacy interim analysis will be performed after patients have been enrolled in each armweeks after radiotherapy in small cohorts n beforeproceeding with full enrollment into this arm Fig respectrelated biomarkersto treatmentinduced changes and immuneStudy settingThe TRADEhypo trial will recruit patients from participating centers across Germany over a period of months Start of recruitment was planned for April but was delayed to May due to the Covid19pandemic A full list of sites can be obtained at clinicaltrialsgov NCT04351256Study objectivesThe primary objective of this study is to evaluate safetyand tolerability of conventionally fractionated CONgroup and hypofractionated HYPOgroup TRT incombination with durvalumab in patients with unresectable stage III NSCLC unfit for chemotherapy Moreoverefficacies of the two modes of radiotherapy will be evaluated with respect to response rates Further parameterswill be determined in order to assess efficacy safety andquality of life QoL in both treatment arms by recordingincidence and severity of adverse events AEs as well asspecific laboratory abnormalitiesExploratory endpoints include assessment of vulnerability and analyses of tumor tissue blood and stoolsamples that are collected during the clinical trial withCharacteristics of participantsA total of patients will be included into this studyPatients potentially eligible for trialinclusion will beapproached and asked to participate as they present inthe clinic Before a patient™s participation in the clinicalstudy the investigator must obtain written informedconsentEach participant must be eligible regarding all inclusion and exclusion criteria set for this trial Table Key inclusion criteria include diagnosis of unresectablestage III NSCLC and nonfeasibility of sequential chemoradiotherapy due to increased oxygenindependentvulnerability as reflected by fulfilling at least one of thefollowing criteria i Performance status Eastern Cooperative Oncology Group [ECOG] scale ii ECOG and Charlson comorbidity index CCI ‰¥ or iii age ‰¥ Moreover participants must have at least one measurable site of disease as defined by RECIST as wellas adequate bone marrow hepatic and renal functionPatients having received prior immunotherapy other investigational agents or thoracic radiotherapy within thepast years will be excluded from the study Additionally participants must not have an active or recent history of a known or suspected autoimmune disease or 0cBozmehr BMC Cancer Page of Fig Cohort design of the safety stopandgo leadin phase HYPOgroup The safety leadin phase follows a design in order to carefullyevaluate the toxicity of the treatment in the HYPOgroup with respect of the occurrence of a grade pneumonitis œevent within weeksafter the end of TRT Two events in the first six patients two events in the first or two events in the first patients will result in terminationof the HYPOgroup œStop If no event is observed within the first two safety cohorts ie the first patients the HYPOarm will be ed forfull enrollment with close toxicity assessment with respect to pneumonitis grade and terminated as soon as two events are reported withinthe subsequent six patients œGo Full enrollment in the HYPOarm will only take place if the criteria for the nontoxicity scenario are met ie ‰¤ event in n patients œGoany other medical condition conflicting with the studyinterventions and not have used immunosuppressivemedication For a full list of the inclusion and exclusioncriteria see Table TreatmentDurvalumabPatients will be enrolled based on the inˆ’ exclusion criteria Two treatment groups will be evaluated Onegroup will receive durvalumab immunotherapy combined with conventionally fractionated TRT CONgroup and the other one with hypofractionated TRTHYPOgroup In both groups durvalumab will be administered intravenously at a fixed dose of mg onday and every weeks thereafter for a maximum of months maximum doses last dose at week untilconfirmed disease progressioninacceptable toxicitywithdrawal of consent or end of the study Fig andTable RadiotherapyAll patients are subjected to preparation of individualpositioning devices and CTbased planning Motionmanagement may comprise either 4DCT or midbreathing CT image acquisition Further imaging modalitiessuch as FDGPETCT may be used when deemed necessary Gross tumor volumes GTV will be contouredand expanded by adequate clinical CTV and planningPTV safety margins in order to account for subclinicaldisease and positioning errors No elective nodal irradiation will be performed As for ans at risk bothlungs spinal cord heart and esophagus must be contoured In the HYPOarm no more than of œbothlungs minus GTV should receive Gy in the CONarm no more than of œboth lungs minus GTVshould receive GyIn the HYPOarm fractions of Gy will be administered total dose Gy corresponding to GyBED αβ In the CONarm fractions of Gywill be administered total dose Gy corresponding to GyBED αβ Dose deviations of ± are acceptable when clinically indicated Radiotherapy isscheduled to start within h after the first administration of durvalumab Dose prescription will follow international reports ICRU and Both 3Dconformal and modulated photon radiation techniquessuch as IMRT and VMATRapdArc are acceptable Allparticipating institution are encouraged to perform regular if no daily positioning control using either portalimaging or onboardCT devicesStudy proceduresIn order to minimize the risk exposure of patients whensubjected to the hypofractionated radiation regimen a 0cBozmehr BMC Cancer Page of Table Complete list of inclusion and exclusion criteriaInclusion criteriacid129 Fullyinformed written consent and locally required authorization obtained from the patient legal representative prior to performing any protocolrelated procedures including screening evaluationscid129 Age ‰¥ yearscid129 Histologically documented diagnosis of unresectable stage III NSCLCcid129 Nonfeasibility of sequential chemoˆ’radiotherapy as determined by the site™s multidisciplinary tumor board if there is no tumor board then thisdecision will be made by the investigator in consultation with a radiation oncologist if the investigator is not a radiation oncologist or by the investigator in consultation with an oncologist if the investigator is not an oncologistcid129 Fulfills at least one of the following criteria—‹ ECOG —‹ ECOG and CCI ‰¥ —‹ Age ‰¥ yearscid129 Must have a life expectancy of at least weekscid129 FEV1 ‰¥ of predictedcid129 DLCO ‰¥ of predictedcid129 FVC or VC ‰¥ of predictedcid129 At least one measurable site of disease as defined by RECIST criteriacid129 Adequate bone marrow renal and hepatic functioncid129 Female patients with reproductive potential must have a negative urine or serum pregnancy test within days prior to start of trialcid129 Evidence of postmenopausal status or negative urinary or serum pregnancy test for female premenopausal patientscid129 The patient is willing and able to comply with the protocol for the duration of the study including hospital visits for treatment and scheduledfollowup visits and examinationsExclusion criteriacid129 Concurrent enrollment in another clinical study or enrollment within days prior to first dose of treatment unless it is an observational noninterventional clinical study or during the followup period of an interventional studycid129 Prior immunotherapy or use of other investigational agentscid129 History or current radiology suggestive of interstitial lung diseasecid129 Oxygendependent medical conditioncid129 Any concurrent chemotherapy investigational product IP biologic or hormonal therapy for cancer treatmentcid129 Prior thoracic radiotherapy within the past years before the first dose of study drugcid129 Major surgery within weeks prior to enrollment into the study patients must have recovered from effects of any major surgery Local nonmajorsurgery for palliative intent is acceptablecid129 Active or prior documented autoimmune or inflammatory disorders with the following exceptions Patients with vitiligo or alopecia patients withhypothyroidism stable on hormone replacement or any chronic skin condition that does not require systemic therapy Patients without activedisease in the last years may be included but only after consultation with the study physiciancid129 Active uncontrolled inflammatory bowel disease Patients in stable remission for more than year may be includedcid129 Uncontrolled intercurrent illness including but not limited to ongoing or active infection symptomatic congestive heart failure uncontrolledhypertension unstable angina pectoris uncontrolled cardiac arrhythmia interstitial lung disease gastrointestinal conditions associated with diarrheaor psychiatric illnesssocial situations that would limit compliance with study requirement substantially increase risk of incurring AEs or compromisethe ability of the patient to give written informed consentcid129 History of another primary malignancy except for a malignancy that has been treated with curative intent and was not active for ‰¥ years beforethe first dose of IP and of low potential risk for recurrence or adequately treated nonmelanoma skin cancer or lentigo maligna without evidence ofdisease or adequately treated carcinoma in situ without evidence of diseasecid129 History of leptomeningeal carcinomatosiscid129 History of active primary immunodeficiencycid129 History of allogenic an or tissue transplantationcid129 Clinical diagnosis of active tuberculosiscid129 Positive testing for hepatitis B virus surface antigen or hepatitis C virus RNA indicating acute or chronic infection or for human immunodeficiencyviruscid129 Current or prior use of immunosuppressive medication within days before the first dose of durvalumab The following are exceptions to thiscriterion Intranasal inhaled topical steroids or local steroid injections systemic corticosteroids at physiologic doses not to exceed mgday ofprednisone or its equivalent steroids as premedication for hypersensitivity reactionscid129 Receipt of live attenuated vaccine within days prior to the first dose of IPcid129 Female patients who are pregnant or breastfeeding or male or female patients of reproductive potential who are not willing to employ effectivebirth controlcid129 Known allergy or hypersensitivity to any of the IPs or any of the constituents of the productcid129 Any coexisting medical condition that in the investigator™s judgement will substantially increase the risk associated with the patient™s participationin the studycid129 Patient who has been incarcerated or involuntarily institutionalized by court order or by the authorities § Abs S Nr AMGcid129 Patients who are unable to consent because they do not understand the nature significance and implications of the clinical trial and thereforecannot form a rational intention in the light of the facts [§ Abs S Nr 3a AMG]safety leadin phase with stopandgo design will precedefull enrollment into the HYPOgroup Fig Toxicitywill be evaluated with a design that is based on thestatistical assumption that ‰¤ event in n patientsconforms to a nontoxicity scenario with œevent beingdefined as the occurrence of pneumonitis grade ‰¥ within weeks after end of TRT Consequently twoevents in the first six patients or two events in the first 0cBozmehr BMC Cancer Page of Table Schedule of assessmentsProcedure Point in timeInformed Consent eligibility criteria demographicsmedical and historyAllocation RandomizationPrior and Concomitant Medication ReviewDurvalumab administrationRadiotherapy CONa or HYPObAEsFull Physical ExaminationDirected Physical ExaminationVital Signs O2 Saturation and WeightPulmonary function tests12lead ECGECOG Performance StatusPregnancy Test CBC with Differential Serum ChemistryPanel Thyroid function testHBV HCVUrinanalysisTumor ImagingFACTL and G8 screening questionnaireTissueScreening Treatment Cycles Q4WScreening C1D1 C1D4 C2D1 C3D1 C4D1XC5D1C13D1EOT PostTreatmentEOT Safety FU FU Q12WXXXXXXXXXXXXXXXXXXXXXXXXXXXXXcXX cXXXXXXXXXXXXXtogether with staging XWhenever clinically indicatedXXXXXXXXXXXXXXXXWhenever clinically indicatedXdXdXeQ8WeXfXXXtogether with staging XXXgXhXXiOptional ReBiopsy at time of progressionXiXXBlood and stoolaCONgroup radiation scheme Patients receive conventional fractions of — Gy Gy within weeks of TRT to be started within h after start ofdurvalumab treatmentbHYPOgroup radiation scheme Patients receive hypofractionated thoracic radiotherapy consisting of — Gy Gy within weeks of TRT to be startedwithin h after start of durvalumab treatmentcTo be performed on C2D1 and C3D1 if in accordance with local standarddChest Xray to be performed on cycles and if in accordance with local standardeFirst onstudy CT imaging to be performed weeks after first durvalumab administration Further onstudy imaging to be performed Q8W days ± daysfOnly applicable if EOT not according to already detected disease progressiongIn patients with EOT not due to disease progression tumor imaging will be performed until the start of a new anticancer treatment disease progression deathwithdrawal of consent or the end of the studyhQuestionnaires will be collected until disease progression only and may be collected by telephone callsiBiomarker sample to be taken prior to first study drug medication either during screening or C1D1 visitX or two events in the first patients will result in termination of the HYPOgroup Fig During this safety stopandgo phase patients will beallocated to treatment arms asfollows While theHYPOarm is recruiting patients will exclusively enterthis treatment group During safety evaluation of the sixpatients of a HYPOcohort Stop Go decision patientswill be allocated to the CONgroup only If safety criteria in the HYPOcohort are met the HYPOarm willbe re ed to continue toxicity assessment and patients will solely be allocated to this arm Fig Inorder to avoid any bias patients will be allocated centrally by the study management during this phase If thenontoxicity criteria in the safety cohorts are met thestudy will proceed to theandremaining patients will be randomized into the twotreatment arms using an interactive web responseexpansion phasesystem integrated in the electronic case report formeCRF A possibly uneven distribution of patients between the treatment groups at this stage will be compensated by a randomization strategy based on the œbiasedcoin method [ ] In the randomization phase patients will be stratified according to tumor stage stageIIIA vs stage IIIBIIICIn total patients will be enrolled per group Aftern patients have been enrolled to the HYPO orCONtreatment arm respectively an interim efficacy analysis for the respective arm will be conducted based on theobjective response rate ORR at weeks after first durvalumab administration In case of insufficient efficacy ofone of the arms ie the number of patients who haveachieved a response is eight out of or lower this treatment arm will be terminated Recruitment will be halteduntil results of the interim efficacy analysis are available 0cBozmehr BMC Cancer Page of Tumor response will be assessed according to RECIST by CT and or MRI scans at baseline weeks afterdurvalumab initiation and then every weeks Safetymeasures willinclude physical examinations performance status ECOG clinical laboratory profiles and continuous assessments of AEs An overview of all studyprocedures is presented in Table An Independent Safety Monitoring Board ISMB willensure the continued safety of participants throughoutthe trial Data management and data quality assurancewill be conducted following the Standard OperationalProcedures of the Institute of Clinical Cancer ResearchIKF at Northwest Hospital GmbH Frankfurt GermanyAn eCRF will be carefully maintained for each participant for data collection also reporting serious and nonserious AEs following the common criteria for adverseevents CTCAE version throughout the entire trialAfter the end of the study participants will be proactively followed up regarding treatmentrelated AEsuntil resolved returned to baseline or considered irreversible until lost to followup or withdrawal of studyconsent All subjects will be followed for survival Patients who decline to return to the site for evaluationswill be offered a followup FU by phone every months as an alternative At the end of the study treatment the investigators are responsible for the furthertreatment of the patient and must ensure that he or shereceives appropriate standard of care or other appropriate therapiesSampling for translational researchIf patients participate in the translational research program blood samples will be collected prior to cycles and and at the time of disease progression or end oftreatment EOT along with stool samples Table Samples of untreated tumor lesions serving as baselinespecimens will be collected as paraffinembedded tissueIf rebiopsies are taken at the time of progression samples should also be also submitted for translationalresearchStudy endpointsThe primary endpoint of the study will be toxicity defined by the occurrence of treatmentrelated pneumonitis grade ‰¥ The ORR evaluated weeks after firstdurvalumab administration according to RECIST isset as the primary efficacy endpoint Secondary endpoints ofthe occurrence oftreatmentrelated AEs and serious AEs SAEsfrequency of prespecified abnormal laboratory parametersprogressionfree survival PFS and duration of clinicalbenefit metastasisfree survival overall survival OSand QoLcomprisethestudyPatient vulnerability and its association with survivaland outcome will be assessed as an exploratory endpoint To this end the G8screening questionnaire asimple and fast screening tool for identifying geriatricrisk profiles with a strong prognostic value for functionaldecline and OS in older populations with cancer will beused [] Furthermore biomaterials will be collectedduring the trial for correlation analyses on selected molecular parameters and clinical data in order to identifymolecular biomarkers predictive for response to therapyThisto obtainhypothesisgenerating data for future research due to theexplorative character of this trialapproach is deemed appropriateStatistical analysisSample size justificationSafety runin phase HYPOgroup With regard to thepneumonitis grade ‰¥ rate this phase is designed to distinguish between a toxicity scenario pTox and anontoxicity scenario pTox A sample size ofn will yield a probability of to correctly detectthe toxicity scenario while the nontoxicity scenario willcorrectly be detected with a probability of Theseprobabilities are based on the decision rule that if thenumber of patients with pneumonitis grade ‰¥ in thiscohort is ‰¥ recruitment to the HYPOgroup will beterminatedanalysisInterim efficacyregarding ORR andexpansion phase An interim efficacy analysis based onthe ORR will be conducted after n patients in eacharm have completed radiotherapy and the 18th patienthas undergone first radiographic assessment at weeksafter first durvalumab dosePreviously an ORR of after radiotherapy alonehas been reported in a Japanese population of elderly patients with unresectable stage III NSCLC [] Based onthis and the observation that Asian ethnicity is associated with a favorable prognosis we assume for theTRADEhypo trial that an ORR higher than in bothtreatment arms can be demonstrated ie the null hypotheses for arm HYPO and CON are defined as H0HYPO Ï HYPO ‰¤ and H0CON Ï CON ‰¤ where ÏHYPO and Ï CON denote the actual ORR in arm HYPOand CON respectively [ ] Under the alternativehypothesis it is assumed that both Ï HYPO and Ï CONamount to Using an optimal Simon™s twostage design with a onesided significance level of α and apower of 1β for each hypothesis test n patients per arm are required while the interim analysis isconducted after n patients per arm have been recruited to the trial After successfully passing the safetyanalysis in the HYPOgroup if among patients in the 0cBozmehr BMC Cancer Page of HYPO or CONarm the number of patients who haveachieved a response is eight or lower the respective armwill be closed Otherwise an additional number of patients will be enrolled into the respective arm Thenull hypothesis of the respective arm can be rejected ifat least of all patients per arm achieve a responseSample size calculation was done using the R packageœOneArmPhaseTwoStudy []To account for an estimated dropout rate of fourpatients will additionally be recruited to each treatmentarm Deviations from planned sample sizes will be handled as described by Englert Kieser allowing strictcontrol of the aspired type I error rate in each arm []Methods of statistical analysis The primary populationfor evaluating all efficacy endpoints and subject characteristics is defined as all patients enrolled according toinitial allocationrandomization intentiontotreat population IIT Secondary efficacy analyses will be carriedout on the perprotocol PP population The safetypopulation comprising all patients who received at leastone dose of the study medication will be used for allsafety endpoints and will be analyzed according to theactual treatment receivedResponse rates with confidence intervals CI and pvalues in both study arms will be estimated taking intoaccount the twostage nature of the design [ ] Secondary endpoints will be evaluated descriptively All toxicities will be summarized by relative and absolutefrequency and severity grade based on CTCAE V50 AEand SAE summary tables will provide the number andpercentage of patients with AEs and the ClopperPearson type CIs All analyses will be done using SASversion SAS Institute Cary NC or higherTrial statusAs of July 15th eight study sites are initiated Firstinitiation coincided with the beginning of the Covid19pandemic in Germany Therefore recruitment of patients was withheld On May 8th recruitment wasresumed after consultation with the ISMB The first patient was enrolled on July 13th renal carcinoma and NSCLCDiscussionIn recent years the concept of restoring the patients™ antitumor immunity by immune checkpoint inhibition hasrevolutionized cancer therapy especially in advancedmelanomaImmunecheckpoint molecules efficiently regulate T cell activation and thus enable tumor cells to evade the immunesystem for example by hijacking the PD1 PDL1 interaction to downregulate effector T cells [ ] To dateseveral human monoclonal antibodies pharmacologicallyblocking these interactions have been implemented incancer therapy such as the antiPD1 antibody pembrolizumab that has been approved in combination withchemotherapy for nonsquamous NSCLC irrespective ofPDL1 expression [ ]Several studies have shown that immune checkpointinhibition and radiotherapy in combination can act synergistically to further boost antineoplastic effects [“] Although in a large phase III trial no benefit ofblockade of cytotoxic T lymphocyteassociated antigen CTLA4 after radiotherapy was observed in metastaticprostate cancer [] clinical trials such as NICOLASNCT02434081 and DETERRED NCT02525757investigating concurrent PDL1directed immunotherapyand chemoradiot

Thyroid_Cancer

"enzymatic cascade for posttranslational protein modification It includes the ubiquitinactivatingenzyme E1 ubiquitinconjugating enzyme E2 and ubiquitin ligase E3 RINGtype E3 ubiquitin ligases catalyse theposttranslational proteolytic and nonproteolytic functions in various physiological and pathological processes such as‚ammationassociated signal transduction Resulting from the diversity of substrates and functional mechanisms RINGtypeligases regulate microbe recognition and ‚ammation by being involved in multiple ‚ammatory signalling pathways Theseprocesses also occur in autoimmune diseases especially ‚ammatory bowel disease IBD To understand the importance ofRINGtype ligases in ‚ammation we have discussed their functional mechanisms in multiple ‚ammationassociatedpathways and correlation between RINGtype ligases and IBD Owing to the limited data on the biology of RINGtype ligasesthere is an urgent need to analyse their potential as biomarkers and therapeutic targets in IBD in the future IntroductionUbiquitination is a crucial part of a diverse range of physiological and pathological processes such as protein degradationand ‚ammationassociated signalling [ ] It is a threestep enzymatic process that consists of ubiquitinactivatingenzyme E1 ubiquitinconjugating enzyme E2 and ubiquitin ligase E3 [] E3 ligases transfer activated ubiquitin fromE2 to specific substrates thereby forming mono or polyubiquitinated proteins to activate proteasomemediated proteolysis signal transduction endocytosis etc [] E3 ligases arecrucial as they catalyse target ubiquitination and enable theformation of polyubiquitin chains that enhance the complexity of ubiquitination in physiological and pathological processes Although dysregulated ubiquitination is involved inthe development of various types of immune pathologieseg systemic lupus erythematosus rheumatoid arthritisand ‚ammatory bowel disease [IBD] [ ] there is limitedknowledge regarding the role of RINGtype ligases in‚ammationassociated pathways In this review we havefocused on IBD owing to its complex pathogenesis involvement in a wide range of etiological factors including dysregulated ubiquitination The term IBD is used for a group ofchronic autoimmune gastrointestinal disordersincludingmainly Crohn™s disease CD and ulcerative colitis UC []Chronicity of IBD often causes intestinal complications hospitalisation steroid dependency and surgery in diagnosedpatients [] Although significant progress has been made inunderstanding the nature of IBD the underlying interactingmechanism involving ubiquitination RINGtype ligases andonset of IBD remains to be fully understood Ubiquitination Mediated by RINGType LigasesHuman cells express more than E3 ubiquitin ligases thatare classified into three types based on their catalyticdomains RING HECT homologous to the E6AP carboxylterminus and a recently identified RBR RINGbetweenRINGRING type of E3 ligases [ ] The RING domainhas a crossbraced structure with two atoms of zinc that catalyse the direct transfer of ubiquitin from the E2Ubiquitin 0cMediators of Inflammationthioester to the substrate [] Figure Apart from catalysing monoubiquitination RINGtype E3 ligases also elongatehomotypic polyubiquitin chains with varying linkage specificities such as that on Lys48 during the proteasomal targeting ofsubstrates and Lys63 in signal transduction thereby modulating proteins for proteolytic and nonproteolytic activity []However their roles in catalysing other less common typesof ubiquitination including atypical homotypic eg Lys6Lys11 Lys27 Lys29 Lys33 and Met1 [] heterotypic andbranched polyubiquitination remain ambiguous The efficiency of RINGtype E3 ligases in ubiquitination depends onmultiple factors such as substrate modification phosphorylation of E2E3 enzymes autoubiquitination by E3 ligases andpseudosubstrate competition [] The role of the RINGtype ligases and their sophisticated functional mechanism ofubiquitination will be discussed in the following sections Signalling Pathways Regulated by RINGType LigaseslectinlikereceptorsRLRs Ctype Pathogen Recognition Under physiological conditionspattern recognition receptors PRRs comprise tolllikereceptors TLRs retinoic acid inducible gene RIG Ilikereceptorsandnucleotidebinding oligomerisation domainlike receptorsPRRs recognise pathogenassociated molecular patternsPAMPs and trigger the activation of downstream eï¬ectorsin innate immune responses [] For ‚ammatory diseasesthat are closely associated with microbiome dysbiosis such asIBD [ ] dysregulation of PRRs and relevant RINGtypeligases may be involved in pathogeninduced ‚ammation TLR Signalling Upon recognising a wide range ofmicrobial components such as lipopolysaccharides flagellinand microbial nucleic acids activated TLRs expressed onantigenpresenting cells trigger eï¬ector T cell responses in‚ammatory diseases [“] RINGtype ligases modulatethe activation of PAMPinduced TLRs By directly bindingwith TLR3 ring finger protein RNF170 catalyses theLys48linked ubiquitination and proteasomal degradationof TLR3 thereby suppressing TLR3mediated innate immunity in macrophages [] On the other hand FcÎ receptorFcÎR IIb an inhibitory FcR on antibodydependent monocyte phagocytosis is targeted by MARCH3 RNF173 forubiquitination and degradation in lipopolysaccharideinduced TLR4 activation [] RIGI Signalling RIGI is a cytoplasmic PRR that recognises viral RNA and triggers the activation of downstreamimmune responses that are associated with both viral infections and noninfectious autoimmune diseases such asenterocolitis [] The deficiency of RIGI aggravates virusinduced cell death in intestinal epithelial cells and inducessusceptibility to chemically induced colitis in mice suggesting the importance of RIGI signalling in intestinal antiviralimmune response [ ] E3 ligaseslike RNF122 andRNF125 mediate Lys48linked RIGI ubiquitination andproteasomal degradation leading to the reduced expressionof infection inducedpro‚ammatory cytokines includingIL6 and type I interferons α and [ ] In contrastindependent of its E3 ligase activity RNF123 binds with theCARD domain of RIGI and melanoma diï¬erentiationassociated gene to compete with the mutual downstreamadaptor mitochondrial antiviral signalling protein MAVSand inhibit RLRmediated antiviral response [] Unlikethe aforementioned RINGtype ligases that directly targetRLRs RNF114 negatively regulates RLR signalling by polyubiquitinating and inducing the proteasomal degradation ofMAVS []is essential Pro‚ammatory Pathways Nuclear Factor Kappa B NFκB Signalling The NFκBpathway is one of the most wellstudied pro‚ammatorypathways regulated by ubiquitination [] TRAF6 RNF85ubiquitinates the evolutionarily conserved signalling intermediate in TLR activation thatfor TLR4dependent NFκB activation [] RNF183 promotes NFκBsignalling by inducing the ubiquitindependent degradationof IκBα [] TRAF2 RNF117 and TRAF3 RNF118induce Lys48linked ubiquitination and proteasomal degradation of cRel and interferon regulatory factor therebyprohibiting the synthesis of pro‚ammatory cytokines inmacrophages [] MKRN2 RNF62 mediates the polyubiquitination and degradation of the p65 subunit of NFκBthereby inhibiting NFκB signalling [] RNF114 negativelyregulates NFκB signalling and T cell activation by ubiquitinating and stabilising NFκB signalling inhibitors A20 andIκBα [ ] It has also been reported that RNF20 downregulation decreases histone H2B monoubiquitination and leadsto the NFκBdependent transcription of pro‚ammatorycytokines such as IL6 and IL8 [] Nevertheless despitethe formation of heterodimers of RNF40 with RNF20RNF40 alone activates NFκB signalling and upregulatesNFκBdependent transcription by promoting IκB kinaseIKK phosphorylation and p65 nuclear translocation indicating the involvement of NFκBdependent transcriptionin the ubiquitination of substrates other than H2B []MARCH3 RNF173 mediates Lys48linked ubiquitinationand lysosomal degradation of IL1 receptor I and therebyinhibits IL1triggered NFκB activation [] Independentof its E3 ligase activity RNF8 inhibits TNFαinduced NFκBactivation by directly binding with the kinase domain of IKKand interfering with IKKα phosphorylation [] RNF11also exerts a noncanonical role in negatively regulating NFκB signalling RNF11 has high affinity for the E2 enzymeUbc13 and minimal E3 ligase activity that subsequently outcompetes E1 enzymes and other E3 enzymes such as TRAF6[] and impedes the activation of NFκB signalling [] MitogenActivated Protein Kinase MAPK SignallingMAPKs are another family of proteins closely related to‚ammationassociated pathologies such as IBD []Upon stimulation by TNFα TRAF2 is autoubiquitinatedon the Lys63 residue that enables its translocation to thecytoskeletal fraction and activates JNK signalling [ ]In vitro experiments have shown that JNK signalling issuppressed and enhanced in cells overexpressing RNF213 0cMediators of InflammationUbUbE2LysSubstrateRING E3 ligasePolyubiquitinationchain initiationUbE2SubstrateRING E3 ligaseMonoubiquitinationE2SubstrateRING E3 ligaseMultiple monoubiquitinationUbUbE2SubstrateRING E3 ligaseHomotypic polyubiquitinationmodification sites of ubiquitin include Lys6 Lys11 Lys27 Lys29 Lys33 Lys48Lys63 and Met1 SubstrateUb Ub Ub Ub UbChain elongationSubstrateUbUb Ub Ub UbUbUbHeterotypic polyubiquitinationSubstrateUbUbUb Ub UbE2SubstrateRING E3 ligaseBranched polyubiquitinationSubstrateUb Ub Ub Ub UbUb Ub Ub UbFigure Functional mechanisms of RINGtype E3 ligase Acting as a scaï¬olding RINGtype E3 ligase recruits a E2ubiquitin thioester and asubstrate and allows the lysine of substrate to attack the thioester for ubiquitin transfer RINGtype E3 ligases catalyse monoubiquitination ormultiple monoubiquitination by transferring a single ubiquitin to one or several residues of the substrate For polyubiquitination ubiquitinsform eight diï¬erent linkage types including Met1 Lys6 Lys11 Lys27 Lys29 Lys33 Lys48 and Lys63 Apart from homotypic chains RINGtype E3 ligases also catalyse heterotypic chains and branched ubiquitin chains by adopting multiple linkage types and branched topology inthe formation of polyubiquitin chainsand RNF186 respectively [ ] however the mechanismsinvolved in this regulation remain to be understood TRAF7RNF119 upregulatesthe kinase activity of mitogenactivated protein kinase via the WD40 domain and potentiates cell apoptosis via the RING finger domain []Similarly RNF13 mediates endoplasmic reticulum ERstressinduced JNK activation and subsequent cell apoptosisby binding with and promoting the phosphorylation ofthe ER stress sensor endoplasmic reticulum to nucleussignalling [] Janus Kinase JAKSignal Transducer and Activator ofTranscription STAT3 Signalling JAKSTAT3 is one of themajor pro‚ammatory signalling pathways that orchestrate 0cRINGtype ligaseRNF11RNF183RNF2RNF135RNF125TRAF6cCblZNRF2Table RINGtype ligases targeted by noncoding RNAs and the relevant pathwaysMediators of InflammationNoncoding RNAmiR19b3pmiR7miR1395pmiR4853pmiR15bmiR124miR146miR216alncRNA TTN antisense RNA acts as a ceRNA for miR1533pRelevant pathwayNFκB signallingNFκB signallingER stressinduced apoptosisMAPK signallingMAPKERK signallingRIGI signallingTLR signallingTLR signallingPI3KAKT signallingPI3KAKT signallingReference[][][][][][][][][][]the progression of ‚ammatory and autoimmune diseases[] A number of RINGtype ligases modulate JAKSTAT3signalling RNF6 and RNF38 function in catalysing theubiquitinationinduced proteasomal degradation of SH2containing protein tyrosine phosphatase thattargetsphosphorylated STAT3 thereby maintaining STAT3 phosphorylation and activating STAT3 signalling [ ] Incontrast TRAF6 promotes Lys63linked ubiquitination ofSTAT3 and represses STAT3mediated transcription ofdownstream ‚ammationrelated genes such as Creactiveprotein [] Interestingly RNF41 modulates the cell surfaceexpression of JAK2associated cytokine receptors by blocking the cleavage of receptors and enhancing receptor shedding in a ubiquitinationdependent manner [] Phosphatidylinositol 3Kinase PI3K Signalling PI3K isanother classical pathway involved in ‚ammation whereinRINGtype ligases are of crucialimportance MKRN1RNF61 functions in the positivefeedback regulation of sustained PI3KAKT activation upon stimulation by epidermalgrowth factor AKT activation phosphorylates and stabilisesE3 ligase MKRN1 that further ubiquitinates and degradesphosphatase and tensin homologue a PI3KAKT inhibitor[] MKRN2 RNF62 induces the ubiquitindependent degradation of the p85α subunit of PI3K and downregulated AKTphosphorylation suggesting a negative regulatory role ofMKRN2 in PI3KAKT signalling [] Downregulation ofUHRF1 RNF106 represses the phosphorylation of PI3Kand AKT which reveals an underlying interaction betweenUHRF1 and PI3KAKT signalling [] Transforming Growth Factor TGF Signalling TGF signalling functions in immunosuppression and inhibitingthe activity of eï¬ector T cells maintaining Treg diï¬erentiation reducing B cell responsiveness and inducing macrophage anergy [] RNF11 plays a dualrole in themodulation of TGF signalling By competing with Smad7for Smurf2 RNF11 is a positive regulator for TGF signalling and reduces the formation of Smad7Smurf2 complexesthat degrade TGF receptors [] RNF11 is also responsiblefor the ubiquitinationmediated stabilisation of Smad4 thatenhances Smad4dependent TGF signalling by directinteraction [] Notably RNF11 may negatively regulatesignallingbytheenablingformationTGFofSmurf2RNF11 complexes and inducing the ubiquitinationand degradation of the associated molecule with the SH3domain of STAM that promotes TGF signalling []PRAJA RNF70 mediates the ubiquitinationinduced proteasomal degradation of embryonic liver fodrin a Smad4adaptor protein thereby negatively regulating TGF signalling [] Autophagy Accumulating evidence reveals that autophagy contributes extensively to immune cell developmentand cell death and its dysregulation has been implicated inmany autoimmune diseases [] TRAF6 catalyses Lys63linked ubiquitination of BECN1 and stimulates TLRinduced autophagy in macrophages upon pro‚ammatorystimulation [ ] RNF166 has a novel role in antibacterialhost defence owing to its function in inducing the Lys29 andLys33linked ubiquitination of autophagy adaptor p62which mediates the recruitment of p62 to bacteria and initiates bacteria engulfment [] Noncoding RNAs Since dysregulated noncoding RNAsare involved in the progression of ‚ammatory diseases[] the posttranscriptional regulation of RINGtype ligasesby noncoding RNAs may play critical roles in potential‚ammationrelevant signalling pathways Until now quitea few microRNAs have been proved to posttranscriptionallyregulate RINGtype ligases by hampering translation orinducing mRNA degradation Table [ “] Nevertheless although the other two major types of noncodingRNAs long noncoding RNAs and circular RNAs also havediverse functions in ‚ammatory diseases eg competingendogenous RNA [ceRNA] transcription regulation andRNAbinding protein sponges [ ] to what extent theymodulate RINGtype ligases awaits further analysis RINGType Ligases in IBD Pathogenesis of IBD The pathogenesis of IBD has beenelucidated over the past years More than loci have beenimplicated in increased genetic risk for IBD that correlate withthe functioning of cellular processes such as innateadaptiveimmune responseintestinal mucosal barrier homeostasis 0cMediators of InflammationTable Roles of RINGtype ligases in IBD patients and animal models of colitisRINGtypeligaseRoleIBD patientsAnimal model of colitisReferenceRNF186ControversialIncrease risk of UC and ERstressinduced apoptosisAttenuate ER stress and maintain intestinalpermeability in DSSinduced mouse model[“]of colitisRNF20RNF40Anti‚ammatoryPro‚ammatoryDecrease in the colonic tissueProtect mice from DSSinduced colitis andfrom UC patientsmaintain intestinal barrier”Activate NFκB signalling in DSSinducedmouse model of colitis[][][][“][ ]RNF183Pro‚ammatoryIncrease in the colonic tissuefrom CD and UC patientscorrelate with endoscopicindex of disease severityUHRF1Anti‚ammatory”TRAF2Anti‚ammatoryIncrease in the colonic tissuefrom CD and UC patientsTRAF3Anti‚ammatoryTRAF5Anti‚ammatoryIncrease in the plasma andcolonic tissue from CD andUC patientsIncrease in the plasma andcolonic tissue from CD andUC patientsIncrease in TNBSinduced mouse modelof colitisRegulate TNFα expression in mice withDSSinduced colitis and zebrafishModulate the proliferation anddiï¬erentiation of Treg cellsModulate colonic microbiotacomposition pro‚ammatorycytokine expression and immunecell ltration in mice withDSSinduced colitisRegulate pro‚ammatory cytokineexpression and mitigate ‚ammatorydamage in DSSinduced mousemodel of colitis[ ]Inhibit IL17mediated pro‚ammatorypathway in TNBSinduced mousemodel of colitisControl pro‚ammatory cytokineexpression and protect mice againstexperimental colitis[“]and autophagy suggesting the involvement of multiple factorsin shaping the procolitogenic environment during the development of IBD [ ] IBD patients manifest with abnormalities in the composition of gut microbiota such as decreasedbacterial diversity increased proportion of harmful bacterialstrains and decreased proportion of protective probioticswhich trigger pro‚ammatory intestinal pathogenic immuneresponses and contribute to the pathogenesis of IBD [ ]Elevated levels of pro‚ammatory cytokines eg IL1 IL6and IL23 and activation of adaptive eg Th1 Th2 Th9and Th17 cells and innate immune cells eg neutrophilsand NK cells constitute a synergistic ‚ammatory networkthat induces intestinal mucosal ‚ammation and sustainedactivation of multiple pro‚ammatory signalling pathways[ ] IL1 familyinduced NFκB IL6induced STAT3MAPK and PI3K signalling pathways are pivotal in intestinal‚ammation [ ] TGF signalling can mitigateimmune cell hyperactivation but also causes the formation ofintestinal strictures in chronic intestinal ‚ammation []Autophagy is involved in the regulation of immune cell function thus defective autophagy also plays an important role inIBD pathogenesis [] A dysfunctional gut barrier and subsequent increased intestinal permeability are also consideredimportant etiologic factors in the development of IBD thatresult in the uncontrolled exchange of materials between theintestinal lumen and internal environment A compromisedgut barrier is often attributed to the pro‚ammatory stimulation and subsequent downregulation of sealing tight junctionproteins eg claudin5 and claudin8 and upregulation ofporeforming tight junction proteins eg claudin2 [“]Since multiple etiologic factors function in the pathogenesisof IBD in a synergistic manner further research is warrantedto understand the dynamics between these players Role of RINGType Ligases in IBD Despite the limitedknowledge on RINGtype ligases in IBD research suggestsa correlation between RINGtype ligases and IBD pathogenesis Table Genomewide association studies have identified RNF186 as one of the genes associated with susceptibilityto UC the diseasecoding variant of RNF186 involves analtered RING domain [ ] The truncated RNF186 lacking the second transmembrane domain is associated withprotecting individuals against developing UC by inhibitingthe ER localisation of RNF186 and subsequent Lys29 andLys63linked polyubiquitination of proapoptotic BCL2 interacting protein under ER stress [ ] Notably RNF186functions diï¬erently in a dextran sulfate sodium DSSinduced mouse model of colitis RNF186deficient mice 0cMediators of Inflammationdevelop more severe colitis during DSS administration andtheir colonic epithelial cell exhibits enhanced signs of ER stressand apoptosis [] RNF186 also modulates intestinal barrierfunction by mediating the Lys48linked ubiquitination of tightjunction protein occludin and RNF186 deficiency increasesintestinal permeability in RNF186 knockout mice [] ThusRNF186 targets diï¬erent substrates and has a complex association with gut ‚ammationApart from the reduced expression of RNF20 in the colonsamples from UC patients homozygous RNF20knockoutmice die due to embryonic lethality and heterozygous miceare susceptible to DSSinduced colitis with increased intestinal permeability suggesting the anti‚ammatory role ofRNF20 [] RNF40 knockout mice exhibit mitigated gut‚ammation upon treatment with DSS this can be attributed to the attenuated activation of NFκB signalling []The upregulation of RNF183 in the colonic tissue from IBDpatients and 246trinitrobenzenesulfonic acid TNBSinduced mouse model of colitis indicates its pro‚ammatory function probably by promoting the ubiquitinationinduced degradation of IκBα []Because UHRF1catalysed histone H3 monoubiquitination recruits and stimulates DNA methyltransferase toDNA methylation sites and thereby maintains DNA methylation UHRF1 participates in the epigenetic control of multiple genes such as TNFα [ ] Mice with macrophagesdeficient for UHRF1 manifest with TNFα overexpressionand aggravated DSSinduced colitis Also the loss of functionin UHRF1 reduces the methylation of the TNFα promoterin macrophages indicating the regulatory role of UHRF1 inthe mouse model of colitis [] Similarly an in vivo studyin zebrafish has revealed that loss in function of UHRF1 leadsto defects in the epigenetic regulation of TNFα promotermethylation and elicits elevated TNFα expression in ‚ammatory processes including intestinal epithelial cell apoptosis neutrophil recruitment and weakened intestinal barrierfunction [] However UHRF1 may eï¬ect diï¬erentlyamong subtypes of regulatory T Treg cells as UHRF1 maintains the proliferation and maturation of colonic Treg cellsbut inhibits the diï¬erentiation of peripheral induced Tregcells in the development of colitis [ ]Studies have shown the diverse roles involved with theupregulation of TRAFs including TRAF1235 in the bloodor colonic mucosa of IBD patients [ ] DSSinducedcolitis models of TRAF2 and TRAF3deficient mice revealsimilar functions of TRAF2 and TRAF3 as negative regulators of experimental colitis by decreasing pro‚ammatorycytokines and reducing the ltration of immune cells inthe colon [] In another study TRAF2deficient micedevelop severe spontaneous colitis and exhibit altered colonicmicrobiota composition indicating the anti‚ammatoryrole of TRAF2 in controlling colonic microbiota [] TRAF3also acts as a colitis regulator by binding with the IL17receptor and interfering with the IL17mediated pro‚ammatory pathway in mice with TNBSinduced colitis []Although TRAF5 RNF84 promotes the ubiquitinationand stabilisation of the retinoic acidrelated orphan receptorÎt that mediates pro‚ammatory Th17 cell diï¬erentiationand IL17AIL17F expression [ ] TRAF5deficientmice exhibit aggravated experimental colitis and upregulation of pro‚ammatory cytokines [] The complex function of TRAF5 needs further analysis DiscussionAs the importance of RINGtype E3 ligases is graduallyunveiled there are still problems to be solved Firstly apartfrom the canonical role of RINGtype ligases in modulatingkey signalling pathways and their downstream adaptors asE3 ubiquitin ligases some RINGtype ligases interfere withthe ubiquitination cascade by competition or direct interaction with other E3 ligases [] Owing to the variety ofRINGtype ligases and substrate specificity of E3 ligases thepotential competition among RINGtype ligases in regulatingimmune response remains to be fully understood Secondlydiï¬erences in RING E3 ligasemediated target ubiquitinationcan also be attributed to the variance in length and linkagetype of ubiquitin chains Although RINGtype ligases function in proteolytic degradation and signal transduction bycatalysing Lys48linked and Lys63linked ubiquitinationrespectively their roles in catalysing less common linkagetypes of homotypic polyubiquitin chains such as Lys11linked ubiquitination [] and the outcome of such polyubiquitination are still obscureRINGtype ligases form a sophisticated but importantubiquitination network wherein the expression and functionof RINGtype ligases are also ‚uenced reciprocally in physiological and pathological processes Understanding themechanisms employed by RINGtype ligases in modulating‚ammationassociated pathways by catalysing atypicallinkages and aï¬ecting signaltransduction may furtherexplain the interaction between RINGtype ligases and IBDSimilarly research on heterotypic polyubiquitin chains isalso important to unveil these underlying mechanismsIn this review we have highlighted the roles of RINGtype ligases in PAMP recognition and modulation of‚ammationassociated pathways that are crucial etiologicalfactors in the development of autoimmune diseases Accumulating evidence shows that many RINGtype ligases areinvolved in ‚ammationassociated pathways such as pro‚ammatory NFκB MAPK JAKSTAT3 and PI3K signalling and anti‚ammatory TGF signalling Subsequentlywe have discussed the role of RINGtype ligases in the pathogenesis of IBD via ‚ammationrelated pathways Patientswith IBD exhibit the diï¬erential expression of specific RINGtype ligases such as TRAFs However there are limited studies on the potential clinical value of RINGtype ligases inpredicting or treating IBD Thus far there have been a fewattempts to use RINGtype ligases as predictive biomarkersand therapeutic targets in treating cancer RNF43 modulatesWnt signalling and has been used to target colorectal cancerand pancreatic ductal carcinoma [ ] Neverthelessthe potential of RINGtype ligases in autoimmune diseasesespecially in IBD needs to be understood in greater detailfuture research on the expression profile ofThereforeRINGtype ligases in the gastrointestinaltract and thedetailed mechanisms is warranted 0cMediators of InflammationConflicts of InterestThe authors have no conflict of interest to discloseAuthors™ ContributionsThe guarantor of the article is Shenghong Zhang ShenghongZhang designed the study Shenghong Zhang Liguo ZhuYing Li and Longyuan Zhou wrote and revised the manuscript Guang Yang Ying Wang Jing Han and Li Li revisedthe important intellectual content of the manuscript All theauthors approved the final version Liguo Zhu Ying L andLongyuan Zhou contributed equally to the workAcknowledgmentsThis study was supported by the National Natural ScienceFoundation of China under Grants and Guangdong Science and Technology Department under Grant 2017A030306021 and FundamentalResearch Funds for the Central Universities under Grant19ykzd11References[] D Popovic D Vucic and I Dikic œUbiquitination in diseasepathogenesis and treatment Nature Medicine vol no pp “ [] D Aki Q Li H Li Y C Liu and J H Lee œImmune regulation by protein ubiquitination roles of the E3 ligases VHLand itch Protein Cell vol no pp “ [] C M Pickart œMechanisms underlying ubiquitinationAnnual Review of Biochemistry vol no pp “[] H Hu and S C Sun œUbiquitin signaling in immuneresponses Cell Research vol no pp “ [] R Hodson œInflammatory bowel disease Nature vol no p S97 [] L PeyrinBiroulet E V Loftus Jr J F Colombel and W JSandborn œThe natural history of adult Crohnʼs disease inpopulationbased cohorts The American Journal of Gastroenterology vol no pp “ [] N Zheng and N Shabek œUbiquitin ligases structure function and regulation Annual Review of Biochemistryvol no pp “ [] F E Morreale and H Walden œTypes of ubiquitin ligasesCell vol no pp “248e1 [] C E Berndsen and C Wolberger œNew insights into ubiquitin E3 ligase mechanism Nature Structural MolecularBiology vol no pp “ [] R J Deshaies and C A P Joazeiro œRING domain E3 ubiquitin ligases Annual Review of Biochemistry vol no pp “ [] Y Kulathu and D Komander œAtypical ubiquitylation theunexplored world of polyubiquitin beyond Lys48 and Lys63linkages Nature Reviews Molecular Cell Biology vol no pp “ [] S Akira œInnate immunity to pathogens diversity in receptors for microbial recognition Immunological Reviewsvol no pp “ [] X C Morgan T L Tickle H Sokol œDysfunction ofthe intestinal microbiome in ‚ammatory bowel diseaseand treatment Genome Biology vol no p R79[] R B Sartor and G D Wu œRoles for intestinal bacteriaviruses and fungi in pathogenesis of ‚ammatory bowel disapproaches Gastroenterologyeasesvol no pp “339e4 therapeuticand[] T Kawai and S Akira œThe role of patternrecognition receptors in innate immunity update on tolllike receptors NatureImmunology vol no pp “ [] J Q Chen P Szodoray and M Zeher œTolllike receptorpathways in autoimmune diseases Clinical Reviews inAllergy and Immunology vol no pp “ [] J H Park L PeyrinBiroulet M Eisenhut and J I ShinœIBD immunopathogenesis a comprehensive review of‚ammatory molecules Autoimmunity Reviews vol no pp “ [] X Song S Liu W Wang Z Ma X Cao and M Jiang œE3ubiquitin ligase RNF170 inhibits innate immune responsesby targeting and degrading TLR3 in murine cells Cellular Molecular Immunology vol no pp “ [] K Fatehchand L Ren S Elavazhagan œTolllikereceptorIIbFcÎRIIb via MARCH3 proteinmediated ubiquitinationThe Journal of Biological Chemistry vol no pp “ ligands downregulate FcÎ receptor[] T Matsumiya and D M Staï¬orini œFunction and regulationof retinoic acidinducible geneI Critical Reviews in Immunology vol no pp “ [] Y Hirata A H Broquet L Menchén and M F Kagno﬜Activation of innate immune defense mechanisms by signaling through RIGIIPS1 in intestinal epithelial cellsJournal of Immunology vol no pp “[] W Wang M Jiang S Liu œRNF122 suppresses antiviraltype I interferon production by targeting RIGI CARDs tomediate RIGI degradation Proceedings of the NationalAcademy of Sciences ofthe United States of Americavol no pp “ [] K Arimoto H Takahashi T Hishiki H Konishi T Fujitaand K Shimotohno œNegative regulation of the RIGI signaling by the ubiquitin ligase RNF125 Proceedings of theNational Academy of Sciences of the United States of Americavol no pp “ [] S Wang Y K Yang T Chen œRNF123 has an E3ligaseindependentreceptormediated antiviral signaling EMBO Reports vol no pp “ in RIGIlikefunction[] B Lin Q Ke H Li N S Pheifer D C Velliquette and D WLeaman œNegative regulation of the RLH signaling by the E3ubiquitin ligase RNF114 Cytokine vol pp “ [] J Chen and Z J Chen œRegulation of NFκB by ubiquitination Current Opinion in Immunology vol no pp “ [] Y Min M J Kim S Lee E Chun and K Y Lee œInhibitionof TRAF6 ubiquitinligase activity by PRDX1 leads to inhibition of NFKB activation and autophagy activation Autophagy vol no pp “ [] Q Yu S Zhang K Chao œE3 ubiquitin ligasein ‚ammatory bowelRNF183 is a novelregulator 0cMediators of Inflammationdisease Journal of Crohn's Colitis vol no pp “ [] J Jin Y Xiao H Hu œPro‚ammatory TLR signallingis regulated by a TRAF2dependent proteolysis mechanismin macrophages Nature Communications vol no article [] C Shin Y Ito S Ichikawa M Tokunaga K SakataSogawa and T Tanaka œMKRN2 is a novel ubiquitin E3ligase for the p65 subunit of NFκB and negatively regulates ‚ammatory responses Scientific Reports vol no article [] M S Rodriguez I Egaña F LopitzOtsoa œThe RINGubiquitin E3 RNF114 interacts with A20 and modulatesNFκB activity and Tcell activation Cell Death Diseasevol no article e1399 [] K Heyninck and R Beyaert œA20 inhibits NFkappaB activation by dual ubiquitinediting functions Trends in Biochemical Sciences vol no pp “ [] O Tarcic I S Pateras T Cooks œRNF20 links histoneH2B ubiquitylation with ‚ammation and ‚ammationassociated cancer Cell Reports vol no pp “ [] R L Kosinsky R L Chua M Qui œLoss of RNF40decreases NFκB activity in colorectal cancer cells andreduces colitis burden in mice Journal of Crohn's Colitisvol no pp “ [] H Lin D Gao M M Hu œMARCH3 attenuates IL1triggered ‚ammation by mediating K48linked polyubiquitination and degradation of IL1RI Proceedings of theNational Academy of Sciences of the United States of America

Thyroid_Cancer

Circulating tumor cells CTCs derived from primary tumors andor metastatic tumors aremarkers for tumor prognosis and can also be used to monitor therapeutic efficacy andtumor recurrence Circulating tumor cells enrichment and screening can be automatedbut the final counting of CTCs currently requires manualintervention This not onlyrequires the participation of experienced pathologists but also easily causes artificialmisjudgment Medicalimage recognition based on machine learning can effectivelyreduce the workload and improve the level of automation So we use machinelearning to identify CTCs First we collected the CTC test results of patientsAfter immunofluorescence staining each picture presented a positive CTC cell nucleusand several negative controls The images of CTCs were then segmented by imagedenoising image filtering edge detection image expansion and contraction techniquesusing python™s CV scheme Subsequently traditional image recognition methodsand machine learning were used to identify CTCs Machine learning algorithms areimplemented using convolutional neural network deep learning networks for trainingWe took cells from patients for training and testing About cells wereused for training and the others were used for testing The sensitivity and specificity ofrecognition reached and respectively We will further revise our modelshoping to achieve a higher sensitivity and specificityKeywords circulating tumor cells CTCs imFISH machine learning image segmentation CNN networkINTRODUCTIONThe metastasis of cancers is a complex and multistage process The circulating tumor cells CTCsare the œseeds shed from the primary tumor andor metastatic lesions and rooted in a new œsoiltransferred by the circulatory system Paget Circulating tumor cell is an intermediate stageof cancer metastasis correlated with cancer aggressiveness and the likelihood of metastasis andtherefore can be used to predict disease progression and survival on a realtime basis by liquidbiopsy Lindsay Praharaj Anand and Roszik Baek Maly Marcuello Pan Riebensahm The molecular subtypesof CTCs not only the CTCs count are interrelated with the prognosis BanysPaluchowski Cristofanilli Dong Stefanovic What™s more the PDL1Edited byCheng GuoColumbia University United StatesReviewed byJuanying XieShaanxi Normal University ChinaKhanh N Q LeTaipei Medical University TaiwanCorrespondenceBinsheng Hehbscsmu163comQiliang Zhou13974942986163comYuebin LiangliangybgeneiscnGeng Tiantianggeneiscn These authors have contributedequally to this workSpecialty sectionThis was submitted toComputational Genomicsa section of the journalFrontiers in Bioengineering andBiotechnologyReceived March Accepted July Published August CitationHe B Lu Q Lang J Yu HPeng C Bing P Li S Zhou Q Liang Yand Tian G A New Methodfor CTC Images Recognition Basedon Machine LearningFront Bioeng Biotechnol 103389fbioe202000897Frontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine Learningexpression in CTCsis correlated with the response toimmunity inhibitors Kloten PDL1EMTCTCs were associated with significantly poorer survival aftercurative surgery showing that PDL1 expression and EpithelialMesenchymal Transition EMT of CTCs are negative survivalpredictors for Nonsmall celllung cancer NSCLC patientsJanning Manjunath Pretreatment PDL1 CTCs are usually associated with a bad prognosis in patientstreated with PD1 inhibitors in NSCLC such as nivolumabGuibert The liquid biopsies worked as an ongoing monitoring systemto assess tumor heterogeneity and make it possible to detect asingle CTC or clusters of cells Wan Merker Praharaj Asante The breakthroughfor CTCdetection is the application of immunomagnetic CTCenrichment combined with flow cytometry which is still theœgold standard of CTCdetection Racila Howeverthis method that lack of the cancer specific markers still remainslots of limitation Grover Ferreira Gabriel Keller Thusthe multimarker immunofluorescence staining is required for recognizeCTCs Antibodies against chromosome centromere duplicationCEP8chromosome centromere duplication CEP17 areused to mark the rapidly dividing tumor cells antibodies againstCD45 as typical leukocytes filaments as well as 4cid486diamidino2phenylindole DAPI for labeling nuclears Koudelakova Lu Liu Lee Although there are great advantages in enrichment technologythe automatic recognition of CTCs still remains problemsManual identification is very timeconsuming and unreliableWith the continuous deepening of the application of CTCsrecognition in various cancer diseases the demand for rapidand automatic identification and counting methods of CTCs isincreasing Several studies have reported the automated screeningprocess Nagrath Yang Kraeft performed a fluorescencebased automated microscope systemREIS for cell detection This scanning can quantify the numberof cells reliably and reproducibly and categorize positive cellsbased on the marker expression profile Ligthart redefined the CTCs by computer algorithms after the manualcounting The stricter definition with the standard deviationof the signal in the CKPE channel the peak signal value inboth the DNADAPI and CD45APC channels and the size ofthe objects used as classifier was well validated CTC by clinicaloutcome using a perfectly reproducing automated algorithmMingxing reported an automated CTC enumerationZhou Allimages with diï¬erent colors weretransferred to a grayscale image and the grayscale images wereused to identify the position and outline of cells Howeverdespite the widely accepted these classification methods stillremain subjective as the rules are set artificially The fixedconditions may not identify the morphologically heterogeneousCTCs integrally What™s more diï¬erent technologies usually usediï¬erent antibodies making comparison and standardizationacross diï¬erent platforms challenging Marcuello With the maturity of artificial intelligence AI recent yearsmachine learning become an exciting field for research TheUS Food and Drug Administration FDA has approved severalcommercial products using machinelearning algorithms in themedical diagnosis and research The cardiovascular MRI analysissoftware of Arterys was the world™s first internet platform formedical imaging AI powered and FDA cleared This software isable to analyze multiple multiperiod MR images to determineblood flow in heart and main vessels The cloud platformwill enable software to collect and analyze the vast amount ofcardiovascular data from MR scanners in real time which willspeed up doctors™ diagnosis This artificial machine is consistentand tireless and is able to identify characters beyond humanperception which provided a substantial interest in the fieldof medical research specifically medical images Dominguez Erickson Lundervold and Lundervold Maier Many algorithms are developed forselecting the best weights for features involving neural networksHornik decision trees Quinlan supportvector machines Cristianini and ShaweTaylor the na¯veBayes Lowd and Domingos knearest neighbors Zhouand Chen and deep learning McBee Wainberg Zou Deep learning as wellas deep neural network learning refers to the use of neuralnetworks with more than layers able to integrate vast datasetslearn arbitrarily complex relationships and incorporate existingknowledge Convolutional neural networks CNNs is a powerfulalgorithm for advancing biomedical image analysis as it assumesthat the input layer has a geometric relationship such as the rowsand columns of images Anthimopoulos Poplin It has been successfully applied in the cancer diagnosisand nuclei or tissue identification Le Le Xing present a novel method for automatednucleus segmentation powered by CNNs The features involvedin the images are considered as a part of the search processand there is no need to limit the features compared to thetraditional machine learning methods which will eliminate thebias created subjective Here we apply deep learning to therecognition of CTCs in order to reduce the artificial errors andimprove accuracyMATERIALS AND METHODSPatients and Samples PreparationA cohort of patients with cancers were enrolled inthis study during “ which was approved by theethics committee of Chifeng Municipal Hospital The clinicalpathological characteristics of patients including age genderCTC number and cancer type are summarized in Table Fourmilliliter of peripheral venous blood was routinely collectedfor every patient The first ml blood samples obtainedafter puncture was discarded in order to avoid the skinepithelial cells contamination Then the blood was placed inanticoagulation tubes and store at room temperature The testwas completed within hAll the patients were divided into two parts according tothe collecting date The earlier patients we collected wereused as the training data the others were used as the independentFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningTABLE Clinical pathological characteristicsClinicopathologic variableAgeGenderSamples typeCTC numberCancer typeCategoryMeanMaleFemaleUnknownPeripheral bloodMeanLung cancerLiver cancerGastrointestinal cancerBreast cancerCarcinoma of thyroidNPCOtherClinical level““testing data Thousand three hundred cells images in the earlierreceived patients were selected to build the CTC recognitionmodel which will be further tested by the cells images of thetest dataset There was no cross part between the two datasets inorder to avoiding the overfittingEnrichment and imFISH Identification ofCTCsThe Cyttel method was used to isolate and enumerate CTCsThe peripheral blood was first centrifuged at g for minto get the precipitation and then washed by CS1 buï¬er CyttelBiosciences Co Ltd Beijing China Then the red blood cellswere lysed by CS2 buï¬er Cyttel After centrifuged at gfor min the precipitate was washed by CS1 buï¬er Thenthe cells were incubated completely with antiCD45 monoclonalantibodyconjugated beads Cyttel for min Three milliliterseparation medium was used to separate the beads and the CTCsby gradient centrifugation at g for min Then the upper rarecell layer was centrifuged at g for min and resuspendedby CS1 The tube was put on a magnetic stand for min Aftersmeared fixed and dried cells were used to perform the imFISHThe slides were fixed dehydrated and then dried at roomtemperature µl CEP8CEP17 antibody was added to the cellsand the slides were placed in a hybridization and denatured for h at —¦C The probe was eluted and the slides were washedtwice in — SSC Then the CD45 fluorescent antibody was addedto the sample area and the slides were put in a wet box andincubate for h at —¦C After incubation CD45 fluorescentantibody was aspirated and µl mounting media containingDAPI was added to the sample area After mounted the cells canbe observed and counted under a fluorescence microscopeThe Manual Interpretation Standard ofCTCs CountingAfter imFISHlots ofimages were acquired with diï¬erentfluorescent colors Usually manual counting is the œgoldstandard but it™s a time consuming and exhausted processionThe Manual interpretation standard of CTCs counting is Eliminates the aggregation superposition and interference ofnucleus or impurity DAPI positive CD45 negative and Three or more than three CEP8CEP17 signal points Itwill be regarded as one signal point if the distance between twosignal points is smaller than the diameter of one pointThe Image Segmentation Method WasUsed to Segment Single Nucleus andGive Labels of Cells Instead of ManualSince the obtained microscopic image is very huge the algorithmwill be limited by the memory and cannot be executed normallyon a conventional computer We first selected part of the imagecontaining one CTC cell and several nonCTC cells around toperform the following test The chosen resolution is — The CV package of python was used to process theCTCs images including conversion of color and morphologicaltransformations The RGB image was converted to the gray image The derivatives were calculated using the CVfunction Sobel from an image Morphological transformations operations based on theimage shapeThe Morphological package of python was used to segmentthe images of CTCs by image denoising image filtering edgedetection image expansion and contractionNuclei were segmented in the blue channel DAPI and theproportion of red in the red channel was detected based onthe position of the nucleus The nucleus with proportion of redhigher than was defined as having a common leukocyteantigen The orange channel was used to detect the number ofCEP8 chromosomes and the green channel was used to detectthe number of centromere probes extracted by CEP17 Diï¬erentcell types were distinguished by diï¬erent colors Figure The CNN Deep Learning Method WasUsed for CTCs IdentificationWith the development of AI machine learning has been wildlyused in the procession of medical images Deep learning is a bigimprovement on artificial neural networks allowing higherlevelfeature extraction and better data prediction with more layersAfter segmentation CNN network were used to identify CTCcells in single nucleus Finally it enters the output layer andoutput the result ie CTCs or nonCTCsOur CNN model was built based on AlexNet which wasfirst introduced in Krizhevsky The networkconsists of eight weighted layers Figure the first five layersare convolution layers and the remaining three layers are fullconnection layers The output of the last full connection layeris the input of the dimensional softmax values which willgenerate the distribution network of two types of labelsThe fivefold cross validation was used to prevent overfittingand select hyperparameters of the model The best crossvalidation score was obtained by searching the hyperparameterspace round and round The final hyperparameters involved inFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningFIGURE The imFISH result and the segmentation of chromosome and nuclear A“C The imFISH result of CEP8 CD45 and DAPI D The merge of panelsA“C E The CTCs were identified by CV segmentation method and marked in red box a“c The CEP8 signal points were identified by CVsegmentation method and marked in red boxour model are activation function kernel regularizer type andregularization factor The workflow is shown belowSp The grid was defined on 3dimensions with eachofthese maps for hyperparameter sets eg hyperparameters activation function kernel regularizer typeregularization factor activation function œsoftmaxœReLU œtanh kernel regularizer type œl1 œl2regularization factor œ œ The range of possible values were defined of eachdimension Allestablishing the best onethe possibleconfigurations weresearched forEvaluation Criteria for ClassificationModelsAfter segmentation some performance evaluation criteria Xie were involved in to evaluate the performance of theclassification model such as sensitivity Se or recall specificitySp precision F1 score and area under the receiver operatingcharacteristic curve AUCSerecall TPTP FNTNTN FPTPprecision TP FPF1 — precision — recallprecision recallIn the equations TP stands for the number of positive CTCcells which are correctly recognized as positive CTC cells FPstands for the number of negative CTC cells that are incorrectlyrecognized as positive CTC cells FN stands for the numberof positive CTC cells incorrectly recognized as negative CTCcells TN stands for the number of negative CTC cells correctlyrecognized as negative CTC cells Table RESULTSPatient CharacteristicsA total of patients were enrolled in this study from January to June The average age is years old Patients withlung cancer count of all patients and the next is breastcancer and gastrointestinal cancer Table Frontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningFIGURE The layers of the CNN model The first five layers are convolution layers and the remaining layers are full connection layersThree SubImages Were Required forManual CountingWe performed imFISH for all the patients and required images of CTCs cells Every image was divided into or channels with diï¬erent color The orange channel representedthe chromosome with CEP8 Figure 1A the green channelthecentromereofthechromosomerepresentedCEP17 Supplementary Figure S1represented the whitethe blueFigure 1C The mergence wasWe then manually labeled all withred channelcell with CD45 Figure 1Brepresented the nuclei with DAPIshown in Figure 1Dthese subimages accordingchannelFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningTABLE Confusion matrix definitionsTABLE The confusion matrix of the models for test datasetConfusion MatrixPredictionMethodConfusion MatrixPredictionPositiveNegativePositiveNegativeTruePositiveNegativeTrue positive TPFalse positive FPFalse Negative FNTrue Negative TN CVALexNetTrueTruePositiveNegativePositiveNegativethetoare CTCs positivestandard AmongourresultspatientsTABLE Tuning of the hyperparameters of AlexNetActivation functionKernel regularizer typeRegularization factorThe Segmentation of Nuclear andIdentifying CTCs by CVSegmentation MethodIn order to avoid the artificial error and save costs we performedthe traditional image identification method for CTCs countingFigure The nucleus was separated in the blue channelDAPI Figure 1E and the red proportion of the red channelwas detected according to the location of the cell nucleus Theproportion higher than was defined as the number of theCEP8 chromosome detected by the common antigen orangechannel of white blood cells Figures 1A“C the number ofcentromeric probes detected by the green channel such as CEP17Supplementary Figure S1After segmentation of nuclear we used CV segmentationmethod to identify CTC cells from single nucleus regions in testing dataset by the manual interpretation standard ofCTCs counting After identification and judgment cells of negative nuclei were recognized as CTC negative About cells of positive nuclei were recognized as CTCnegative The sensitivity and specificity were and while the precision and F1 score reached and respectively Table We also applied the regionbased image segmentationalgorithm such as watershed algorithm in the segmentationprocess The watershed algorithm was implemented the bywatershed function in CV python and CV In this method optimal threshold value was used respectivelyin binaryzation process by setting THRESH_OTSU mode Thetraditional watershed algorithm was sensitive to noise and theaccuracy was lower than our segmentation method on CTCnegative data set in size of Supplementary Table S3The HyperParameters Selected forEvaluating the CNN MethodWe used GridSearchCV class in scikitlearn by providinga dictionary of hyperparameters to determine the hyperparameters of the model After the crossvalidation processactivation function was set to ReLU kernel regularizer type wasset to l2 and regularization factor was set to as shown inTable with the best performance Further the hyperparameterswe selected were used to construct the model on the wholetraining datasetsoftmaxReLUtanhl1l2l1l2l1l2The underline value shows the best result of AUC value in the tuning process of thehyperparameters of AlexNetThe Identification of CTCs by CNNMethodWe got nuclei of patients by segmentation processFigure showed the whole flowchart of the experiment About nuclei were used for training the left were usedfor testing We use the same images for testing cells of negative nuclei were recognized as CTC negative and cells of were recognized as CTC positive The sensitivityand specificity were and while the precisionand F1 score reached and respectively Table and Figure Before that we also compared the performance of AlexNetmodel with others such as ResNet and Xception All ofthem have close AUC values Figure butthe AlexNetwas less timeconsuming in the training and test processSupplementary Table S1DISCUSSIONThis study showed a method for CTC counting powered bymachine learning The use of machine learning for imageinterpretation can capture important image features reduceerrors caused by manually setting interpretation standardsand save time and labor costs Although this method showsa higher sensitivity and specificity in CTC countingisslightly worse than the first method for the data used inthis study Actually we have analyzed that the main reasonis that there are fewer positive samples for training and thealgorithm cannot extract features of more positive samplesin the group were excludedIn additiondue to quality problems Unfortunatelythe CTC imagesincluded in the group doesn™t cover the whole film but asome picturesitFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningFIGURE The flowchart of the whole experimentFIGURE The ROC curve of AlexNet ResNet and Xception modelpicture just focused on a certain CTCpositive cell under themicroscope which results in that the machine learning methodhas no advantage in recognition speed compared with thetraditional image recognition method Enlarging the scope ofimages and collected more samples is also that need to beimproved in the futureDeep learning has already been shown to be suitablefor detection of CTCs because of the high sensitivity andspecificity in CTC counting We had changed the filter sizeand number in all convolution layers in order to find thebest CNN parameters We found diï¬erent filter size andnumber will influence the results largely We changed filternumber from range to in our training process Wefound that the training result was not convergence when thenumber was less than It showed that the range of thefeature number of the image is about “ We tried toincrease the filter size from to but the result was notchanged a lot and the convergence speed even became slowerwhen the filter size higher than From this process wesummarized that the feature size in CTCs could not be greaterthan pixels Furthermore there are many appropriately AImodels such as VGG InceptionV14 We will apply themon the CTCs dataset to establish a more suitable model inthe later testingtumorsCirculating tumor cellis an important marker for earlyscreening and prognosis ofIn addition CTCsoriginating from the primary tumor may be more eï¬ectivefor tumor tissue tracing and molecular classification Imagerecognition can only obtain the characteristics ofthe cellsurface If strict tissue tracing is required other molecularbiological experimental data such as the isolation of CTCcells and single cell sequencing may be required Besidesin this study we also evaluated the performance of AlexNetmodel in variant types of cancers Supplementary Table S2and Figure S2 showed that our model presents a betterperformance in Lung cancer than Gastrointestinal cancer andBreast cancer One of the reasons may be that the trainingdata size of Lung cancer is much larger than those ofGastrointestinal cancer and Breast cancer Furtherpostoperative recurrence may occur in approximately of patients even after complete resection of NSCLC Yano These proteins especially epithelial proteinssuch as EpCAM PIK3CA AKT2 TWIST and ALDH1may have more activitiesHanssen whichwilllead more influence in the morphology of cells andaï¬ecting the recognition performance thereby Therefore themultiimage omicsincluding CT images HE staining andimmunohistochemical images as well as the sequencing datamay be urgently needed at this stageFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCONCLUSIONIn orderIn the present study we established a CTC cell recognitionsoftware based on deep learningto make itmore practical we collected samples from the real worldinstead of using the public databases We performed theCTC enrichment and imFISH experiments and screened thefluorescence images according to the figure™s quality In order toimprove the efficiency we used the machine instead of ngmanual screening First the python™s package was used to mage segmentation The obtained recognition sensitivity andspecificity are and respectively In addition therecognition sensitivity and specificity can also reach to and respectively using CNN instead of manual interventionIn the future studies we willfocus on the improvementof the accuracy and sensitivity with a more suitable deeplearning model promoting this technology to the clinic assoon as possibleDATA AVAILABILITY STATEMENTThe datasets generated for this study are available on request tothe corresponding authorCTCs Recognition by Machine Learninglegal guardiannextstudy was provided by the participants™of kin Written informed consent was obtained from theindividuals and minors™ legal guardiannext of kin for thepublication of any potentially identifiable images or data includedin this AUTHOR CONTRIBUTIONSGT YL BH and QZ conceived the concept of the work BH QLJL PB HY and SL performed the experiments QL and BH wrotethe manuscript CP and HY reviewed the manuscript All authorsapproved the final version of this manuscriptFUNDINGThis research was funded by Hunan Provincial Innovation2018RS3105Platform and Talents Program NotheNaturalNo Science Foundation of Chinathe Natural Science Foundation of Hunan Province No2018JJ3570 and the Project of Scientific Research Fundof Hunan Provincial Education Department Nos 19A060and 19C0185ETHICS STATEMENTSUPPLEMENTARY MATERIALThe studies involving human participants were reviewed andapproved by The Ethics Committee of Chifeng MunicipalHospital Written informed consentto participate in thisThe Supplementary Materialonline202000897fullsupplementarymaterialfor this can be foundhttpswwwfrontiersins103389fbioeatREFERENCESAnand K and Roszik J Pilot study of circulating tumor cells in earlystage and metastatic uveal melanoma Cancers Basel cancers11060856Anthimopoulos M Christodoulidis S Ebner L Christe A and MougiakakouS Lung pattern classification for interstitial lung diseases using a deepconvolutional neural network IEEE Trans Med Imaging “ 101109tmi20162535865Asante D B Calapre L Ziman M Meniawy T M and Gray E S Liquid biopsy in ovarian cancer using circulating tumor DNA and cellsready for prime time Cancer Lett “ 101016jcanlet2019Baek D H Kim G H Song G A Han I S Park E Y Kim H S Clinical potential of circulating tumor cells in colorectal cancer aprospective study Clin Transl Gastroenterol 10e00055 1014309ctgBanysPaluchowski M Schneck H Blassl C Schultz S MeierStiegen FNiederacher D Prognostic relevance of circulating tumor cellsin molecular subtypes of breast cancer Geburtshilfe Frauenheilkd “ 101055s00351545788Cristianini N and ShaweTaylor J An Introduction to Support VectorMachines and Other KernelBased Learning Methods Cambridge Cambridgeuniversity pressCristofanilli M Pierga J Y Reuben J Rademaker A Davis A A Peeters D J The clinical use of circulating tumor cells CTCs enumerationfor staging of metastatic breast cancer MBC International expert consensuspaper Crit Rev Oncol Hematol “ 101016jcritrevonc201812Dominguez C Heras J and Pascual V IJ CV combining ImageJand CV for processing images in biomedicine Comput Biol Med “ 101016jcompbiomed201703027Dong J Zhu D Tang X Qiu X Lu D Li B Detectionof circulating tumor cell molecular subtype in pulmonary vein predictingprognosis of stage IIII nonsmall cell lung cancer patients Front Oncol 103389fonc201901139Erickson B J Korfiatis P Akkus Z and Kline T L Machinelearning for medical imaging Radiographics “ 101148rg20171Ferreira M M Ramani V C and Jeï¬rey S S Circulating tumortechnologies Mol Oncol “ 101016jmolonc2016cellGabriel M T Calleja L R Chalopin A Ory B and Heymann D Circulating tumor cells a review of nonEpCAMbased approaches for cellenrichment and isolation Clin Chem “ 101373clinchem2015Grover P K Cummins A G Price T J RobertsThomson I C andHardingham J E Circulating tumour cells the evolving concept andthe inadequacy of their enrichment by EpCAMbased methodology for basicand clinical cancer research Ann Oncol “ 101093annoncmdu018Guibert N Delaunay M Lusque A Boubekeur N Rouquette I Clermont E PDL1 expression in circulating tumor cells of advanced nonsmallcell lung cancer patients treated with nivolumab Lung Cancer “ 101016jlungcan201804001Hanssen A Wagner J Ges T M Taenzer A Uzunoglu F G Driemel C Characterization of diï¬erent CTC subpopulations in nonsmallcell lung cancer Sci Rep 101038srep28010Frontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningHornik K Stinchcombe M and White H Multilayer feedforwardnetworks are universal approximators Neural Netw “ Janning M Kobus F Babayan A and Wikman H Determination ofPDL1 expression in circulating tumor cells of NSCLC patients and correlationwith response to PD1PDL1 inhibitors Cancers Basel cancers11060835Keller L Wernerand clinicalrelevance of EpCAM Cell Stress “ 1015698cst2019and Pantel K BiologySKloten V Lampignano R Krahn T and Schlange T Circulatingtumor Cell PDL1 expression as biomarker for therapeutic efficacy ofimmune checkpoint inhibition in NSCLC Cells 103390cells808Koudelakova V Trojanec R Vrbkova J Donevska S Bouchalova K KolarZ Frequency of chromosome polysomy in relation to CEP17copy number in a large breast cancer cohort Genes Chromosomes Cancer “ 101002gcc22337Kraeft S K Ladanyi A Galiger K Herlitz A Sher A C Bergsrud D E Reliable and sensitive identification of occult tumor cells usingthe improved rare event imaging system Clin Cancer Res “ 10115810780432ccr030361Krizhevsky A Sutskever I and Hinton G œImageNet classification withdeep convolutional neural networks in Paper Presented at the NIPS LakeTahoe Harrahs and HarveysLe N Q Ho Q T and Ou Y Y Incorporating deep learning withconvolutional neural networks and position specific scoring matrices 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Thyroid_Cancer

"Many cancerassociated single nucleotide polymorphisms SNPs are located in the genomic regionsof long noncoding RNAs lncRNAs Mechanisms of these SNPs in connection to cancer risk are not fullyunderstoodMethods Association of SNP rs140618127 in lncRNA LOC146880 with nonsmall cell lung cancer NSCLC wasevaluated in a casecontrol study of individuals The mechanism of the SNP™s biologic influence was exploredwith in vitro and in vivo experiments including plasmid transfection siRNA knockdown flow cytometry assessmentand assays of cell proliferation migration invasion and colony formationResults Association analysis showed that A allele of SNP rs140618127 was associated with low risk of NSCLC in theChinese population Lab experiments indicated that SNP rs140618127 contained a binding site for miR5395p andthe binding between miR5395p and LOC146880 resulted in declined phosphorylation of an oncogene ENO1 Thereduced phosphorylation of ENO1 led to decreased phosphorylation of PI3K and Akt which is further linked to thedecline in cell proliferation and tumor progressionConclusion The study demonstrates that SNP rs140618127 in lncRNA loc146880 provides an alternate binding sitefor microRNA miR5395p which affects the phosphorylation of ENO1 and activation of the PI3K and Akt pathwayKeywords NSCLC lncRNA SNP miRNA ENO1BackgroundLung cancer is the most commonly diagnosed cancer of the total cases and the leading cause of cancerdeath of the total cancer deaths in the world []The majority oflungcancer NSCLC which accounts for around of alllung cancer is nonsmall cell Correspondence qianbiyunsjtueducn Tienan Feng and Nannan Feng contributed equally to this work1Hongqiao International Institute of Medicine Shanghai Tongren HospitalClinical Research Institute Shanghai Jiao Tong University School of MedicineShanghai China7Second Affiliated hospital of Chengdu Medical College China NationalNuclear Corporation Hospital Chengdu Sichuan ChinaFull list of author information is available at the end of the lung cancer cases Genetic factors may play an importantrole in an individual™s susceptibility to NSCLC Longnoncoding RNAs lncRNAs are a class of noncodingtranscripts with nucleotides or more Increasingevidence suggests thatlncRNAs are involved in theoccurrence oflung cancer due to their functions asoncogenes or tumor suppressors [] Our previous studies indicated that a lncRNA on chromosome 17q243named LOC146880 was expressed higher in tumortissues than in adjacent normal tissues and high expression was associated with poor prognosis of NSCLC []Singlenucleotide polymorphisms SNPs in the noncoding regions of the genome have been shown to affect The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cFeng Journal of Experimental Clinical Cancer Research Page of rs140618127 the ˜A™ allelecancer risk via regulating the transcription andor changing the structure of lncRNA [“] A previous studyidentified SNPs in more than humanlncRNAs and a large number of SNPs were predicted tohave a potentialimpact on the microRNA miRNAlncRNA interaction [] Here we report the identificationof SNP rs140618127 in LOC146880 as a new susceptiblelocus to NSCLC Bioinformatics analysis predicts thatin LOC146880variantprovides an altered secondary structure which maycreate a binding site for microRNA miR5395p []sequestering its action on other molecules Shiraishi conducted a GWAS on lung adenocarcinoma andidentified SNP rs7216064 in BPTF 17q243 in association with the cancer risk OR p 7e11 []Seow confirmed that SNP rs7216064 was associated with the risk of lung cancer based on a GWASstudy of Asian female nonsmokers [] We found thatSNP rs140618127 was in strong linkage disequilibriumwith SNP rs7216064 LD r2 and this lncRNASNP was associated with lung cancer risk OR p in our casecontrol study of individualsToSNPrs140618127 in NSCLC development and progressionweconsequence ofLOC146880 and miR5395p interaction and found thatthe microRNA behaved like a tumor suppressor []which prevented LOC146880 from interacting withprotein ENO1 an oncogene product [] reducing itsphosphorylation As a resultthe phosphorylation ofPI3KAKT was also reduced after the suppression ofENO1 phosphorylation [] which further inhibitedtumor growth and metastasisleading to a betterprognosis of NSCLCthe molecular mechanism ofexploreevaluated thatthe biologicalMaterials and methodsStudy populationsSuspected NSCLC individuals had histopathological orcytologically confirmed diagnosis according to theWorld Health anization classification These studysubjects including suspected individuals diagnosed withlung cancer or normal were recruited from the ChinaMedical University CMU Distributions of the basiccharacteristics ofthe study subjects are provided inTable At recruitment an informed consent wasIlluminated Only ifthe subject agreed heshe wasincluded This study was approved by the InstitutionalReview Board at CMUSNP selection and genotypingSNPs with r2 were considered to be in the same LDblock With this criterion one SNP was selected in eachLD block and genotyped using the TaqMan genotypingmethod in the ABI RealTime PCR systemApplied Biosystems For quality control we implemented several measures in our genotyping assays including each plate contained both case and controlsamples technicians were blinded to the casecontrolstatus of the samples both positive and negativecontrol no DNA template samples were included ineach 384well plate and nearly of the sampleswere assayed in duplicate and the concordances werebetween and Cell linesHuman NSCLC cell lines A549 and PC9 and humanlung epithelial BEAS2B cells were purchased from theCell Bank of Type Culture Collection at the ChineseAcademy of Sciences Shanghai Institute of Biochemistryand Cell Biology These celllines were passaged forfewer than months All the cells were tested for mycoplasma and were found to be free from infection Thecells were maintained in DMEM supplemented with FBS and grown without antibiotics in an atmosphere of CO2 and relative humidity at °C² and ² RACE and coding prediction of LOC146880We used ² and ² RACE to determine the transcripinitiation and termination sites of LOC146880tionalwith a SMARTe RACE cDNA Amplification kit Clontech The Alignment File of a fulllength sequence ofLOC146880 obtained from ² and ² RACE is availableupon requestConstruction of reporter plasmids transient transfectionsand luciferase assaysA reporter plasmid in the psiCHECK2 vector Promegawas created which contains a 1000bp LOC146880 exonregion flanking rs140618127 [G] or rs140618127 [A]with the restriction enzymes XhoI and NotI FermentasA549 and PC9 cells were seeded at — cells per wellin 24well plates and ng of the reporter plasmid and pmol of miR5395p mimic Ambion were cotransfected into the cells h later using Lipofectamine Invitrogen These cells were collected h aftertransfection Renilla luciferase activity was measured andused to normalize the efficiency of transfectionRNA extraction and qRTPCR analysisTotal RNA from the NSCLC tissue specimens and celllines used in this study was extracted using the TRIzolreagent Firststrand cDNA was synthesized using theSuperScriptInvitrogenRelative RNA levels determined by qPCR were measuredon an ABI sequence detection system AppliedBiosystems using the SYBR Green method Βetaactinwas employed as an internal control for the quantification of LOC146880 and the mRNA levels of other genesreverse transcriptase kitII 0cFeng Journal of Experimental Clinical Cancer Research Page of For miRNA quantification small nuclear RNA U6 wasused as an endogenous control The relative expressionof RNA was calculated using the comparative CtmethodSubcellular fractionationCytosolic and nuclear fractions of A549 and BEAS2Bcells were prepared and collected according to theinstructions ofthe NuclearCytoplasmic Isolation kitBiovision LOC146880 was mainly detected in thenuclear fraction although it was also present in the cytoplasm Fig S1belowBrieflydescribedRNA pulldown and mass spectrometry analysisRNA pulldown assays were performed following theprotocolbiotinylatedLOC146880 or antisense LOC146880 was incubated withcellular protein extracts from A549 cells and streptavidin beads were then added Recovered proteins associated with LOC146880 or antisense LOC146880 wereexcised and proteomics screening was accomplished bymass spectrometry analysis on a MALDITOF instrumentIn vitro transcription ofLOC146880 and its deletion fragments were analyzedwith primers containing the T7 promoter sequenceBruker DaltonicsPlasmid construction and transfectionTo construct a lentiviral vector expressing humanLOC146880 NR_026899 a fulllength of LOC146880cDNA containing rs140618127 [G] or rs140618127 [A]was commercially synthesized GeneChem and subcloned into the AgeI and NheI sites of the GV367IRESPuromycin lentiviral expression vector GeneChem Toproduce lentivirus containing LOC146880 T cellswere cotransfected with the vector described above andlentiviral vector packaging system GeneChem using Lipofectamine Infectious lentiviruses were collectedat h after transfection and filtered through 045μmPVDF filters for analysis of genotype After conformation these lentiviruses were designated to LOC146880[G] or LOC146880 [A] We used the GV367IRESPuromycin empty vector as a negative control Theviruscontaining pellet was dissolved in DMEM and aliquots of the solution were stored at ˆ’ °C A549 andPC9 cells were infected with concentrated virus in thepresence of polybrene SigmaAldrich The supernatantwas replaced with complete culture medium after hfollowed by selection with puromycin and the expression of LOC146880 in infected cells was verified byqPCRRNA immunoprecipitation assaysRIP experiments were performed using the Magna RIPProteinRNABindingkitMillipore Antibodiesagainst ENO1 Abcam orcontrol proteins were diluted at Total RNA inputcontrol and precipitation with the isotype control IgGfor each antibody were assayed simultaneously The coprecipitated RNAs were detected by RTqPCRImmunoprecipitationCell lysis and immunoprecipitationCells were homogenized in — RIPA buffer supplemented with ProteasePhosphatase Inhibitor CocktailPierce Cell lysates were centrifuged and the supernatants were prepared for immunoblotting or immunoprecipitation withbelowImmunoblot signal was detected using Clarity WesternECL Substrate Thermo FisherantibodiesdescribedtheTable Characteristics of lung cancer patients and healthy controlsVariablesP valueControlN NSCLCN Age at dx year ‰¥ GendermalefemaleSmoking statusnoyesSNP rs140618127G alleleA allele adjusted by smokinggenderage OR valueP valueOR value “ “ “ “ “ “ “ “ 0cFeng Journal of Experimental Clinical Cancer Research Page of from cells orImmunoblot assaysProtein extractsimmunoprecipitationsamples were prepared using detergentcontaining lysisbuffer Total protein μg was subjected to SDSPAGE and transferred to PVDF membrane MilliporeAntibodies against ENO1 Abcam ab155102 ENO1phosphorylated at Cterminalinhibitory site Tyr44StressMarq Bioscience spc965D PI3K CST 13666SPI3K phosphorylated at Tyr458 CST 4228S AKTCST 2938S AKT phosphorylated at Ser473 CST9018S PCNA CST 2586S NFkB CST 8242SVimentin Abcam ab92547 βCatenin CST 8480S Ecadherin CST 14472S NCadherin Abcam ab18203and βActin SigmaAldrich A1978200UL were usedMembranes were incubated overnight at °C withprimary antibody diluted and proteins were detected with the Odyssey near infrared dualcolor laserimaging system LICORAnalysis of cell proliferation migration invasion cellcycle and colony formationCells were seeded in 96well flatbottom plates with cells in μl cell suspension in each well Afterculture cell viability was measured with the CCK8assay Each experiment with six replicates was repeatedthree times For cell cycle analysis cells were collectedand fixed in ethanol overnight at °C Singlecellsuspensions were labeled with μgml Propidium Iodide Sigma and analyzed by flow cytometry BeckmanCoulter For colony formation cells were seededin 65mm culture dishes and allowed to grow until visible colonies formed in complete growth medium weeks Cell colonies were fixed with methanol stainedwith crystal violet and counted Invasion assays wereperformed in Millicell chambers in triplicate The 8μmpore inserts were coated with μg of Matrigel BD Biosciences Cells — were added to the coated filtersin serumfree medium PMI1640 medium containing FBS was filled in the lower chambers as a chemo attractant After h at °C in an incubator suppliedwith CO2 cells that migrated through the filters werefixed with methanol and stained with crystal violet Cellnumbers in three random fields were counted The migration assay was conducted in a similar fashion withoutcoating the filters with MatrigelExperiments on xenograft animalsTen male BALBc mice weeks old were kept in aspecific pathogenfree grade environment All animalexperiments were approved by the Animal Care and UseCommittee of Shanghai Jiao Tong University School ofMedicine Shanghai China All applicable guidelines ofthe Animal Care and Use Committee of Shanghai JiaoTong University School of Medicine for the care and usethe hind flank regions ofof animals were followed PC9 cells of rs140618127 [A]and rs140618127 [G] type were collected and resuspended in PBS at a concentration of — cellsmLand mixed with Matrigel® at a ratio of respectivelyThe mixture mL was subcutaneously injected intotwo sides ofthe micers140618127 [A] and rs140618127 [G] cells in the samemouse Tumor size was measured once every daysusing a Vernier caliper across its two perpendicular diameters and tumor volume was calculated using the following formula V 12ab2 where V is the tumorvolume a is the largest diameter and b is the smallestdiameter After weeks oftreatment all mice weresacrificed and their tumors were collected and weighedHistological evaluation ofsamples wasperformedthe tumorHistopathological analysesTumor tissues from the animals were fixed in paraformaldehyde BOSTER Wuhan Chinafor h atroom temperature The fixed tissues were then dehydrated in a graded series of alcohol cleaned in xyleneand embedded in paraffin A rotary microtome was usedto section paraffin the blocks into 4μm thick sectionsThe sections were deparaffinized and stained withhematoxylin and eosin HE A light microscopeOlympus was used to examine the stained tissuesectionsStatistical analysisThe association between SNP rs140618127 and NSCLCrisk was analyzed under an additive model using the unconditionallogistic regression model adjusted for agesex and smoking status Results of laboratory experiments were presented as Means ± SD Student™s t testwas used to compare means between two groups andANOVA was employed for comparison of more thantwo groups Repeated ANOVA was employed for comparison of more than two groups which contained repeated measure data All the statistical analyses wereperformed using Statistical Product and Service Solutions SPSS software version and GraphPad PrismVersion GraphPad Software San Diego CA USAResultsSNP rs140618127 G A in LOC146880 and NSCLC riskSNP in LOC146880 rs140618127 is in strong linkagedisequilibrium with SNP rs7216064 r2 which is aGWASdiscovered risk allele for NSCLC We found thatSNP rs140618127 G A in the exon of LOC146880chr17 was associated with the risk of NSCLC the ˜A™ allele compared to ˜G™ had an adjusted oddsratio OR “ in a casecontrol study of subjects Table Stratified analyses suggested 0cFeng Journal of Experimental Clinical Cancer Research Page of that this effect was more evidence in those who were ‰¥female and nonsmokers Supplemental years oldTable S1 The minorfrequency of SNPrs140618127 is low globally but can be high as in some American populations see Suppl alleleEffects of LOC146880 with rs140618127 [a] on cellproliferation and behaviorsWe examined the effects of LOC146880 on cell proliferation by its allele at rs140618127 and found that overexpression of rs140618127 [A] in the NSCLC celllinesA549 and PC9 both with the G allele at rs140618127substantially reduced the rate of cell proliferation whencompared with rs140618127 [G]Fig 1A Colonyformation ability in both A549 and PC9 cells wasmarkedly suppressed by rs140618127 [A] when compared with rs140618127 [G] Fig 1B Overexpression ofrs140618127 [A] significantly suppressed the invasionand migration of NSCLC cells Fig 1C 1D Tumorsize in a xenograft animal model of PC9 was decreasedin both genotype groups but the decline in tumor sizewas greater for rs140618127 [A] than for rs140618127[G] P There was no significant difference intumor size between the vector control group and wildtype rs140618127 [G] Fig S2 HE staining showedthat tumors of rs140618127 [A] possessed less malignantmorphology Fig 1E Together these results indicatethat rs140618127 [A] can inhibit the growth of lung cancer more in vitro and in vivo compared to rs140618127[G]such a possibilityInteraction between LOC146880 and miR5395pEvidence suggests that SNPs in lncRNAs may generatenew interacting sites between lncRNAs and other transcripts such as miRNAs [] Using an online softwarelncRNASNP httpbioinfolifehusteducnlncRNASNP[] we found that several SNPs in LOC146880 werepredicted to haveand SNPrs140618127 was indicated to lie within a putative binding site for miR5395p The G A mutation atrs140618127 was predicted to change the local foldingstructures and free energy of LOC146880 which mightcreate a binding site for miR5395p Following this prediction we investigated whether miR5395p interactswith LOC146880 based on its genotype at rs140618127Luciferase reporter assays showed that in comparison tothe construct containing rs140618127 [G] the constructwith the ˜A™ allele had significantly reduced luciferase activity in the presence of miR5395p suggesting moreinteraction of miR5395p with LOC146880 [A] thanwith LOC146880 [G] Fig 2A The interaction betweenmiR5395p and LOC146880 [A] could be blocked bythe miR5395p inhibitor miR5395p is constitutivelyexpressed in both A549 and PC9 cells In cells stablyoverexpressing LOC146880 miR5395p only decreasedthe levels of LOC146880 with rs140618127 [A] not alleleG indicating that allele A is a target of miR5395p Fig2Bthe RNA pulldown assay weInteraction between LOC146880 and ENO1isolated aUsingLOC146880 withrs140618127[G]protein complexMass spectrometry analysis showed that there were threeproteins in this complex and the most abundant onecompared to antisense one was ENO1 Fig 3A Wethen selected ENO1 for validation detecting ENO1 inthree independent RNA pulldown assays RNA immunoprecipitation RIP assays also showed enrichment ofLOC146880 in the complexes precipitated with ENO1antibody as compared with IgG or another irrelevantantibody indicating that ENO1 may be a key target protein of LOC146880 Fig 3b Next we evaluated the consequences of the interaction between LOC146880 andENO1 We found that ENO1 mRNA expression andprotein level were not significantly different P see Fig S3 in the cells overexpressing LOC146880 withrs140618127[A] or rs140618127[G] in the presence ofmiR5395p Fig 3C 3D However HE staining ofxenograft tumors in mice showed that phosphorylatedENO1 was higher in rs140618127 [G] than in [A] Fig3E The expression of CMYC a downstream target ofENO1 was decreased remarkably when the cells weretransfected with a siRNA against ENO1 Fig 3FRegulation of PI3KAKT signal by LOC146880 via ENO1phosphorylationWe found that ENO1 phosphorylation was markedly decreased in cells overexpressing rs140618127 [A] as compared with those overexpressing rs140618127 [G] in thepresence of miR5395p mimics Fig 4A Fig S4 andFig S5 Using insilico prediction tools [ ] weidentified a phosphorylation site at Tyr44 in the proteinbased on the PDB database Fig S5 [] We next investigated the impact of altered LOC146880 levels on thedownstream signal of ENO1 Since our results describedaboveoverexpressionincreased cell proliferation migration and invasion wefocused our investigation on the PI3KAKTNFkB signaling The total amount of PI3K and AKT proteins wasnot significantly different between cells overexpressingrs140618127 [A] and [G] However we observed thatprotein phosphorylation levels affected the expression ofdownstream molecules in the PI3KAKT signaling inA549 Fig S4 Cells overexpressing LOC146880 withrs140618127 [A] showed substantial decreases in NFkB PCNA Vemintin and Ncadherin levels while theirβcatenin and Ecadherin levels weresignificantlyincreased when compared withthesamecellsLOC146880indicatedthat 0cFeng Journal of Experimental Clinical Cancer Research Page of Fig rs140618127[A] inhibits NSCLC cell proliferation and EMT process A proliferation assay of different samples which were compared byrepeated ANOVA B clone formation ability of different samples which were compared by ANOVA C wound healing assay of different sampleswhich were compared by ANOVA D cell invasion assay of different samples which were compared by ANOVA e cancer protective effect ofrs140618127[A] in xenograft animals which were illuminated by repeated ANOVA and ttest 0cFeng Journal of Experimental Clinical Cancer Research Page of Fig rs14061812[G][A] expression of different condition in A549 and PC cell lines a luciferase activity of different conditions which wereilluminated by ANOVA b LOC146880 expression level of different conditions which were compared by ttestoverexpressing LOC146880 with rs140618127 [G] Fig4B Fig 4C Fig S7 and Fig S8 Immunohistochemical staining of xenograft tumors showed that pPI3KpAKT TWIST NCadh and SNAIL were all significantly higher in rs140618127 [G] than in [A] Fig 4Dand Fig S9DiscussionIn this study we found that SNP rs140618127 inLOC144680 contained a binding site for miR5395pand the binding between miR5395p and LOC146880resulted in declined phosphorylation of an oncogeneENO1 which was found to be a downstream target ofLOC146880 Furthermore the reduced phosphorylationof ENO1 led to decreased phosphorylation of PI3K andAkt which was linked to the decline in tumor cell proliferation and progress The entire process of how SNPrs140618127 influences NSCLC is depicted in Fig Our casecontrol study supports the notion that SNPrs140618127 genotype [A] may have a protective effecton NSCLC compared to genotype [G] In a previousstudy we found that LOC146880 expression was significantly higher in NSCLC tumors than adjacent normaltissuesforLOC146880 []a possible oncogenicsuggestingroleThere has been an increasing interest in understandingthe mechanisms of rare genetic variants in lncRNAs inrelation to the complex traits and diseases [ ] Ingle suggested that genetic polymorphisms in lncRNAMIR2052HG offer a pharmacogenomic basis for the response of breast cancer patients to aromatase inhibitortherapy [] Tang indicated that SNP rs9839776in SOX2OT was significantly associated with breast cancer possibly via influencing the expression of SOX2OT[] Redis demonstrated that the GWASidentifiedSNP rs6983267 on 8q24 is in a lncRNA gene calledCCAT2 which regulates cancer cell metabolism in anallelespecific manner through binding to the cleavagefactor I complex This complex is implicated in anallelespecific regulatory mechanism of cancer metabolism orchestrated by alleles ofthe lncRNA [ ]Russell et alidentified a neuroblastoma susceptibilitylocus rs9295534 located in the upstream enhancer of atumor suppressor CASC15S The SNP could decreasethe transcriptional activity of CASC15S and be associated with the disease outcome [] Wang foundthat SNP rs965513 a locus on 9q22 in the FOXE1 geneand lncPTCSC2 was associated with the risk of papillary thyroid carcinoma []In this study we found that a lncRNA could regulatethe function of a protein via its phosphorylation with 0cFeng Journal of Experimental Clinical Cancer Research Page of Fig LOC146880 promotes ENO1 activation in a variantspecific manner A pulldown assaymass spectrum of identificationsilver staining BRIP assay with A549 and PC9 cell lines which were compared by ANOVA C D ENO1 mRNA expression and protein level of rs140618127[G][A]by overexpression plasmid transfection which were compared by ttest E HE staining of ENO1 phosphorylation in vivo which were comparedby ttest F ENO1 LOC146880 and cMYC expression level after ENO1siRNA transfection which were compared by ttestlittle influence on gene expression or protein concentration Similar findings have been reported before in whichthe phosphorylation site of a protein can be blocked by alncRNA leading to decreased phosphorylation For example NFkB can be inhibited by a long noncodingRNA which directly blocks IKB phosphorylation inbreast cancer [] LncRNA can also bind to proteinsincreasing or decreasing their phosphorylation viaanother protein LncRNA DANCR and PANDAR influence the phosphorylation of serine in RXRA and SFRS2via GSK3β and P53 in breast and ovarian respectively[ ] GSK3β™s phosphorylation in breast cancer wasreported to be reduced by lncRNA NLIPMT [] Thephosphorylation of ULK1 can be suppressed by lncRNAHOTAIR in NSCLC [] LINC00675 enhances the phosphorylation of vimentin on Ser83 to suppress gastriccancer progression [] Our finding of a lncRNA™s impact on the phosphorylation of a protein was quiteunique and interesting because it is achieved by a microRNA through a polymorphic site in LOC146880In our study we found that a G to A transition atrs140618127 in LOC146880 could turn into a bindingsite for a microRNA and miR5395p was indeed the target Interestingly the wildtype of LOC146880 had no 0cFeng Journal of Experimental Clinical Cancer Research Page of Fig LOC146880 regulates PI3KAKT signaling via ENO1 A549 PC9 A ENO1pENO1 PI3KpPI3K AKTpAKT protein level usingrs140618127[G][A] overexpression plasmid transfection B PCNA NFkB protein level using rs140618127[G][A] overexpression plasmidtransfection C βCatenin Vimentin NCadherin ECadherin protein level using rs140618127[G][A] overexpression plasmid transfection D HEstaining of pPI3K pAkt TWIST NCadhersin and SNAIL 0cFeng Journal of Experimental Clinical Cancer Research Page of Fig Diagrammatic sketch of rs14061812mediated NSCLC tumorigenesis LOC146880 rs14061812[G] increases ENO1™s phosphorylationresulting in activating PI3KAKT signaling pathway and NSCLC tumorigenesis while LOC146880rs14061812[A] binds to miR5395p decreasesENO1™s phosphorylation resulting in deactivating PI3KAKT signaling pathway and NSCLC tumorigenesisinteraction with the microRNA at all This SNP has notbeen reported before in any studies [] However miR is known to be a tumor suppressor [ ] Ourfinding of miR539™s binding to loc146880 provided newinsights into a possible mechanism that explains the biologic function of miR539 as a tumor suppressor Thiseffect takes place when a microRNA and lncRNA interact through a polymorphic site which results in changesin phosphorylation in a protein ENO1 that the lncRNAmay target on Low levels of LOC146880 did not influence the mRNA expression or protein levels of ENO1but suppressed the phosphorylation of ENO1 ENO1 is ametabolic enzyme involved in the synthesis of pyruvateIt also acts as a plasminogen receptor and mediates theactivation of plasmin and extracellular matrix degradation In tumor cells ENO1 is upregulated and supportsthe Warburg effect The protein is located on the cellsurface where it promotes cancerinvasion and issubjected to substantial posttranslational modificationsnamely acetylation methylation and phosphorylation[] Reduced phosphorylation of ENO1 lowers thePI3KAkt signal which results in slower cell migrationor invasion of NSCLCThe SNPbased interaction between miRNA andlncRNA in regulation of protein function has beenhypothesized and predicted by YaRu but fewstudied have provided evidence [] Our study was thefirst to show the interaction between LOC146880 andmir5395p in the NSCLC and to elucidate the downstream mechanism involving tumor growth and metastasis The modulation model of the lncRNA and miRNA isnot the classical competing endogenous RNAs ceRNAHow LOC146880 interacts with ENO1 to regulate itsphosphorylation and downstream signals remains to beelucidated Although the ˜A™ allele of rs140618127 is lowin general some racial groups still have a relatively highfrequency In some Caucasian populations the ˜A™ allelefrequency is close to ConclusionsWe found in a casecontrol study of Chinese thatSNP rs140618127 in LOC146880 was associated with therisk of NSCLC People with the G allele of rs140618127had higher risk than those with the A allele Our in vitroand in vivo experiments demonstrated that LOC146880was an oncogene and the G allele of rs140618127 hadstronger oncogenic effects on lung cancer cells than theA allele in LOC146880 This differential effect appearedto come from the binding of a microRNA miR539 tothe A allele but not the G allele at rs140618127 ThemicroRNA binding prevented the lncRNA™s interactionwith its downstream target ENO1 which led to the reduction of ENO1 phosphorylation and suppression ofthe PI3KAKT signaling resulting in lower tumor cellproliferation and less aggressive cell behaviors 0cFeng Journal of Experimental Clinical Cancer Research Page of Supplementary informationSupplementary information accompanies this paper at httpsdoi101186s13046020016525Availability of data and materialsThe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable requestAdditional file Fig S1 Locations of LOC146880 A549 BEAS2BLOC146880 locals mainly in cytoplasm The comparison between twogroups using ttestAdditional file Fig S2 Comparison of tumor size between vectorcontrol group and the wide type There was no significant difference oftumor size between vector control group and the wide typers14061812[G]Additional file Fig S3 Comparison of ENO1 protein level ofrs140618127[G][A] by overexpression plasmid transfection which werecompared by ttestAdditional file Fig S4 Comparison of ENO1pENO1 PI3KpPI3KAKTpAKT protein level of A549 cell lines using rs140618127[G][A]overexpression plasmid transfection which were compared by ttestAdditional file Fig S5 Comparison of ENO1pENO1 PI3KpPI3KAKTpAKT protein level of PC9 cell lines using rs140618127[G][A]overexpression plasmid transfection which were compared by ttestAdditional file Fig S6 Predicting site of phosphorylation of ENO1A predicting phosphorylation site of ENO1ENO1 chain A usingNetPhos B predicting phosphorylation site of ENO1ENO1 chain Ausing PhosphoELM BLAST C The predicting phosphorylation site ofENO1 chain A using data of PDB databaseAdditional file Fig S7 Comparison of PCNA and NHkB protein levelof using rs140618127[G][A] overexpression plasmid transfection whichwere compared by ttest A results of A549 cell lines B result of PC9cell linesAdditional file Fig S8 Comparison of βCatenin Vimentin NCadherin ECadherin protein level using rs140618127[G][A] overexpression plasmid transfection which were compared by ttest A results ofA549 cell lines B result of PC9 cell linesAdditional file Fig S9 Comparison of the HE staining of pPI3K pAkt TWIST NCadhersin and SNAIL which were compared by ttestAdditional file Distribution of

Thyroid_Cancer

pathogenesis of multiple myeloma MM is not completely known Uncovering the potential mechanism of MM initiation and progression is essential for identifying novel diagnostic and therapeutic targets Herein we explored the function and the working mechanism of circular RNA circ_0007841 in MM progressionMethods Quantitative realtime polymerase chain reaction qRTPCR was employed to detect the expression of circ_0007841 microRNA3383p miR3383p and bromodomain containing BRD4 Cell proliferation ability was analyzed through cell counting kit8 CCK8 assay colony formation assay and flow cytometry Transwell assays were conducted to measure the migration and invasion abilities of MM cells Cell apoptosis was also assessed by flow cytometry The interaction between miR3383p and circ_0007841 or BRD4 was confirmed by dualluciferase reporter assay and RNApull down assayResults Circ_0007841 was highly expressed in bone marrow BMderived plasma cells of MM patients and MM cell lines than that in healthy volunteers and normal plasma cell line nPCs Circ_0007841 promoted the proliferation cell cycle and metastasis and impeded the apoptosis of MM cells miR3383p was a direct target of circ_0007841 in MM cells and circ_0007841 accelerated the progression of MM through targeting miR3383p BRD4 could directly bind to miR3383p in MM cells and miR3383p exerted an antitumor role through targeting BRD4 Circ_0007841 promoted the activation of PI3KAKT signaling via miR3383pBRD4 axis Exosomes generated from mesenchymal stromal cells MSCs elevated the malignant behaviors of MM cells via circ_0007841Conclusion Circ_0007841 acted as an oncogene to promote the proliferation cell cycle and motility and restrain the apoptosis of MM cells through sequestering miR3383p to upregulate the expression of BRD4Keywords Multiple myeloma circ_0007841 miR3383p BRD4 ExosomeBackgroundMultiple myeloma MM is a kind of hematologic cancer featured by malignant proliferation of plasma cells [] The therapeutic strategies for MM patients include chemotherapy radiotherapy and targeted therapy [“] However MM is still incurable by current treatment Correspondence wy1782126com Department of Hematology The Fifth Affiliated Hospital of Zhengzhou University No3 Kangfuqian Street Zhengzhou Henan ChinaFull list of author information is available at the end of the methods Uncovering the molecular mechanism behind the progression of MM and intercellular interaction is important to find more effective treatment methods for MM patientsNoncoding RNAs ncRNAs are a class of RNAs that are unable to code proteins generally and they are abundant in human genome to regulate cellular processes including proliferation metastasis and apoptosis [] Circular RNAs circRNAs are a kind of ncRNAs that characterized by covalently closed loop structure [] CircRNAs are more stable than linear RNAs and they The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cWang a0et a0al Cancer Cell Int Page of are resistant to exonuclease due to their loop structure [] CircRNAs engaged in the pathogenesis of cancers through serving as microRNAs miRNAs sponges to modulate the abundance of downstream genes linked to proliferation metastasis and apoptosis [ ] The roles of circRNAs in hematological cancers have been reported before [ ] For instance circCBFB contributed to the proliferation ability while suppressed the apoptosis of chronic lymphocytic leukemia cells through targeting miR607FZD3Wntbetacatenin signaling [] However the functions of circRNAs in MM remain to be uncoveredMiRNAs belong to another class of ncRNAs that involved in the progression of cancers through inducing degradation or translational repression of target messenger RNAs mRNAs [] The dysregulation of miRNAs was involved in the pathogenesis of MM [ ] We concentrated on the role of miR3383p miR3383p suppressed the development of many cancers [“] As for MM Cao et a0al reported that miR3383p suppressed the proliferation and accelerated the apoptosis of MM cells via CDK4 [] Nevertheless the function of miR3383p in MM is largely unexploredBromodomain containing BRD4 is a crucial epigenetic protein and it has been reported to elevate the levels of oncogenic proteins and accelerate the progression of cancers [] Zheng et a0al claimed that H19 accelerated the development of MM through upregulating BRD4 via sponging miR1523p [] Here the direct interaction between miR3383p and BRD4 was first found in MM and the function of BRD4 in MM was investigatedIn this study circ_0007841 was found to be abnormally upregulated in MM Lossoffunction experiments revealed that circ_0007841 silencing blocked the proliferation cell cycle progression migration and invasion while induced the apoptosis of MM cells The underlying mechanisms behind the oncogenic role of circ_0007841 in MM were further exploredMaterials and a0methodsPatientsPlasma cells from MM patients n and healthy volunteers n in The Fifth Affiliated Hospital of Zhengzhou University were collected to detect the expression of circ_0007841 miR3383p and BRD4 via qRTPCR and Western blot assayCell cultureMM cell lines H929 and OPM2 and normal plasma cell line nPCs were purchased from BeNa Culture Collection Beijing China and maintained in Roswell Park Memorial Institute1640 RPMI1640 medium Gibco Carlsbad CA USA added with fetal bovine serum FBS Gibco unitsmL penicillin and a0μgmL streptomycin Cell culture plates were placed in a CO2 incubator at a0°C and cells were collected in the log phase of growthQuantitative real‘time polymerase chain reaction qRT‘PCRAfter measuring the concentration using NanoDrop Invitrogen Carlsbad CA USA RNA sample a0ng was used to synthesize complementary DNA cDNA with ReverTra Ace qPCR RT Kit for circ_0007841 BRD4 and U6 Takara Dalian China and AllinOne„¢ miRNA glyceraldehyde3phosphate dehydrogenase GAPDH First stand cDNA Synthesis Kit for miR3383p GeneCopoeia Rockville MD USA U6 served as the internal control for miR3383p while GAPDH acted as the internal reference for circ_0007841 and BRD4 PCR amplification reaction was conducted with SYBR Green PCR Master Mix Applied Biosystems Foster City CA USA on an ABI thermocycler Applied Biosystems The quantification of circ_0007841 miR3383p and BRD4 was carried out with the ˆ’ΔΔCt method The specific primers in this study were synthesized from Sangon Biotech Shanghai China and listed as below circ_0007841 ²CTA ACA TCT GTG AAA CCA TCGT3² Forward Reverse ²TCA TCA CAT ACA CGA TAG ACTGG3² miR3383p Forward ²UCC AGC AUC AGU GAU UUU GUUG3² Reverse ²CAA CAA AAU CAC UGA UGC UGGA3² BRD4 Forward ²GTG GTG CAC ATC ATC CAG TC3² Reverse ²CCG ACT CTG AGG ACG AGA AG3² U6 Forward ²CTC GCT TCG GCA GCACA² Reverse ²AAC GCT TCA CGA ATT TGC GT3² GAPDH Forward ²GCG ACA CCC ACT CCT CCA C3² Reverse ²TCC ACC ACC CTG TTG CTG TAG3²and interfering RNAs siRNAs including sicirc_00078411 Cell transfectiontargeting Three small ²UGU circ_0007841 sicirc_00078412 UAG UUG CAA UGA AGA GAG3² si²UAA UGA UCA UGC CAA AUA CUC3² circ_00078413 ²UCA CAU ACA CGA UAG ACU GGC3² its negative control siNC circ_0007841 overexpression plasmid circ_0007841 its control Vector BRD4 overexpression plasmid BRD4 its control pcDNA miR3383p mimics miR3383p its control miRNC miR3383p inhibitor inmiR3383p and its control inmiRNC were obtained from Genepharma Shanghai China MM cells were seeded into 24well plates at a density of × cellswell overnight and transfection was conducted with Lipofectamine Invitrogen 0cWang a0et a0al Cancer Cell Int Page of Cell counting kit‘ CCK8 assayMM cells were plated in 96well plates at the density of × cellswell and cultured overnight After transfection for indicated time points a0h a0h a0h or a0h MM cells were incubated with μL CCK8 Sigma St Louis MO USA for a0h The absorbance at a0 nm was detected by a microplate reader BioTek Winooski VT USAColony formation assayA total of cells were seeded onto the 6well plates to settle down The culture medium was replenished every d After 2week incubation the colonies were immobilized using poly methanol Sangon Biotech for a0 min followed by staining using crystal violet Sangon BiotechFlow cytometry for a0cell cycle and a0apoptosis detectionFor cell cycle analysis MM cells were collected using cold phosphate buffer saline PBS and then immobilized using cold ethanol solution overnight Prior to propidium iodide PI Solarbio Beijing China staining RNase was used to remove RNA in the samples The percentage of MM cells in different phases of cell cycle was detected on the FACSCalibur Becton“Dickinson San Jose CA USA and analyzed using Cell Quest software Becton“DickinsonFor apoptosis analysis after transfection for a0 h MM cells were collected with PBS and then these cells were suspended in binding buffer Annexin Vcombined fluorescein isothiocyanate Annexin VFITC Solarbio and PI Solarbio were added to the reaction mixture and MM cells were simultaneously incubated with Annexin VFITC and PI at a0°C for a0min in a dark room The apoptotic MM cells were identified by FACSCalibur Becton“Dickinson and analyzed using Cell Quest software Becton“DickinsonTranswell assaysIn transwell migration assay cell suspension MM cells suspended in μL serumfree medium was added into the upper chambers Costar Corning NY USA A total of μL culture medium with FBS was added into the lower chambers FBS acted as the chemotactic factor in this study After 24h incubation MM cells remained in the upper surface were removed with the cotton swab and the migrated MM cells were fixed with paraformaldehyde Sigma for a0 min and stained with crystal violet Sigma The number of migrated MM cells in five random visual fields was counted by the microscope Olympus Tokyo JapanIn transwell invasion assay the upper chambers were precoated with μL Matrigel Sigma to mimic the extracellular matrix The detection of cell invasion was conducted through using these precoated transwell chambers following the similar procedureBioinformatic prediction and a0dual‘luciferase reporter assayThe targets of circ_0007841 and miR3383p were predicted by circinteractome and targetscan software respectivelyThe wildtype partial sequence in circ_0007841 that predicted to bind to miR3383p along with the mutanttype sequence with miR3383p in circ_0007841 that was synthesized through using Sitedirected gene mutagenesis kit Takara Dalian China was amplified and cloned into pGL3 luciferase reporter vector Promega Madison WI USA termed as circ_0007841 WT or circ_0007841 MUT MM cells were cotransfected with a0 nM miRNC or miR3383p and a0 ng circ_0007841 WT or circ_0007841 MUT After 48h transfection MM cells were harvested and the luciferase activity was detected with the dualluciferase reporter assay system kit Promega using the luminometer Plate Chameleon V Hidex Finland according to the manufacturer™s instructions Firefly luciferase activity in each group was normalized to Renilla fluorescence intensityThe wildtype fragment of BRD4 ² untranslated region ²UTR that predicted to bind to miR3383p and the mutant type fragment of BRD4 ²UTR were also amplified and inserted into pGL3 luciferase reporter vector Promega to generate BRD4 ²UTR WT and BRD4 ²UTR MUT Cotransfection of MM cells with BRD4 ²UTR WT or BRD4 ²UTR MUT and miRNC or miR3383p was conducted following the similar procedureRNA‘pull down a0assayRNApull down assay was conducted to test the interaction between circ_0007841 and miR3383p Biotin RNA Labeling Mix Roche Shanghai China was used in this study The wildtype and mutanttype binding sites in circ_0007841 that were predicted to bind to miR3383p were biotinylated to obtain Biocirc_0007841 WT and Biocirc_0007841 MUT MM cells were disrupted and incubated with BioNC Biocirc_0007841 WT or Biocirc_0007841 MUT The abundance of miR3383p was measured by qRTPCRWestern blot assayProteins were obtained using whole cell lysis buffer Roche Basel Switzerland for a0min on the ice Protein samples were quantified using Pierce BCA Protein Assay kit Thermo Fisher Scientific Rockford IL USA Then a0 µg of proteins were run on sodium dodecyl sulfate 0cWang a0et a0al Cancer Cell Int Page of polyacrylamide gel electrophoresis SDSPAGE gel and transferred to the polyvinylidene fluoride PVDF membrane Millipore Billerica MA USA After blocking with wv nonfat dry milk for a0h primary antibodies were used to probe the indicated proteins followed by incubation with the secondary antibody ab205718 Abcam Cambridge MA USA The protein bands were measured using the enhanced chemiluminescent ECL system Beyotime Shanghai China according to the manufacturer™s instructions Gray analysis was conducted to quantify the expression of proteins using ImageJ software Primary antibodies including antiBRD4 ab128874 antiphosphorylatedphosphatidylinositol 3kinase antipPI3K ab70912 antiPI3K ab32089 antipAKT serinethreonine kinase pAKT ab38449 antiAKT ab64148 antiCD63 ab59479 antiCD81 ab79559 and antiβactin ab8226 were purchased from AbcamExosome isolationExosome isolation kit Qiagen Frankfurt Germany was used to extract exosomes from the culture medium of MM cells according to previous studies [ ]Statistical analysisAll statistical data in three independent experiments were shown as mean ± standard deviation SD Data were analyzed using GraphPad Prism The differences between two groups or among more than two groups were assessed through using Student™s t test or oneway analysis of variance ANOVA followed by Tukey™s test The comparison between groups was considered significant when P value less than Linear correlation was analyzed using Spearman™s correlation coefficientResultsCirc_0007841 elevates the a0malignant behaviors of a0MM cellsCirc_0007841 was abnormally upregulated in bone marrow BMderived plasma cells from MM patients compared with that in healthy individuals Fig a01a Meanwhile the level of circ_0007841 was higher in MM cell lines than that in normal plasma cell line nPCs Fig a01b The dysregulation of circ_0007841 in MM attached our attention Circ_0007841 specific small interfering RNAs were used to knockdown circ_0007841 to uncover its biological functions in MM cells As mentioned in Fig a0 1c and d the level of circ_0007841 was downregulated with the transfection of sicirc_00078411 sicirc_00078412 or sicirc_00078413 Among these three siRNAs sicirc_00078411 was chose for the following assays due to its highest knockdown efficiency Fig a01c d Cell proliferation was assessed through CCK8 assay colony formation assay and flow cytometry According to the results of CCK8 assay sicirc_00078411 transfection significantly inhibited the proliferation of MM cells Fig a0 1e f The number of colonies was markedly reduced with the knockdown of circ_0007841 compared with siNC group Fig a0 1g The cell cycle of MM cells was arrested in G1S transition in sicirc_00078411 group than that in siNC group Fig a01h These findings together demonstrated that circ_0007841 silencing hampered the proliferation ability in MM cells What™s more circ_0007841 interference notably suppressed the migration and invasion of MM cells via transwell migration and invasion assays Fig a01i j The apoptosis rate of MM cells was increased in sicirc_00078411 group compared with that in siNC group Fig a01k Overall circ_0007841 accelerated the proliferation cell cycle progression and metastasis and inhibited the apoptosis of MM cellsmiR‘‘3p could directly interact with a0circ_0007841 in a0MM cellsTo address the mechanism by which circ_0007841 functioned in MM cells circinteractome website was used to seek the targets of circ_0007841 As shown in Fig a0 2a miR3383p possessed the complementary sites with circ_0007841 The luciferase activity was dramatically reduced in circ_0007841 WT group when cotransfected with miR3383p suggesting the target relationship between circ_0007841 and miR3383p in MM cells Fig a02b c We also constructed mutant type luciferase plasmid circ_0007841 MUT to investigate if œUGC UGG  in circ_0007841 was the binding sequence with miR3383p The luciferase intensity remained unaffected in circ_0007841 MUT group with the cotransfection of miRNC or miR3383p Fig a02b c suggested that circ_0007841 bound to miR3383p via its œUGC UGG  sequence RNApull down assay revealed that miR3383p could be pulleddown when using Biocirc_0007841 WT proving the target relationship between miR3383p and circ_0007841 Fig a0 2d e An obvious decrease in the level of miR3383p was observed in BMderived plasma cells from MM patients in contrast to that in normal volunteers Fig a02f Additionally there was a prominent reduction in the expression of miR3383p in MM cell lines than that in nPCs cell line Fig a0 2g The expression of miR3383p was negatively correlated with the level of circ_0007841 in BMderived plasma cells from MM patients Fig a02h The overexpression efficiency of circ_0007841 was high in MM cells and circ_0007841 accumulation caused a notable decrease in the level of miR3383p in MM cells Fig a02i j In summary circ_0007841 could inversely regulate the expression of miR3383p through direct interaction 0cWang a0et a0al Cancer Cell Int Page of Fig Circ_0007841 elevates the malignant behaviors of MM cells a The enrichment of circ_0007841 was examined in BMderived plasma cells of MM patients and normal volunteers by qRTPCR b The expression of circ_0007841 was measured in MM cell lines and normal plasma cell line nPCs by qRTPCR c d The level of circ_0007841 was detected in H929 and OPM2 cells transfected with siNC sicirc_00078411 sicirc_00078412 or sicirc_00078413 by qRTPCR e“k MM cells were transfected with siNC or sicirc_00078411 e f CCK8 assay was employed to assess the proliferation ability of MM cells g Colony formation assay was performed for the determination of cell proliferation ability in transfected MM cells h Flow cytometry was carried out to detect the influence of circ_0007841 silencing on the cycle of MM cells i j The metastasis ability of MM cells was evaluated by transwell assays k The apoptosis of MM cells was analyzed by flow cytometry P P P P if circ_0007841 exerted Circ_0007841 plays an a0oncogenic role through a0targeting miR‘‘3p in a0MM cellsTo disclose its oncogenic role through targeting miR3383p we conducted rescue experiments through cotransfecting H929 and OPM2 cells with siNC sicirc_00078411 sicirc_00078411 inmiRNC or sicirc_00078411 inmiR3383p As mentioned in Fig a03a sicirc_00078411 transfection increased the level of miR3383p and the introduction of inmiR3383p reversed the influence of circ_0007841 silencing in the expression of miR3383p Sicirc_00078411mediated inhibitory effect on the proliferation of MM cells was counteracted by the interference of miR3383p via CCK8 assay Fig a03b c Circ_0007841 silencing restrained the colony formation ability while the addition of miR3383p inhibitor partly recovered the colony formation ability in MM cells Fig a0 3d Additionally cell cycle of MM cells was arrested at G1S transition in sicirc_00078411 group and this suppressive impact in the cell cycle of MM cells was attenuated by the addition of inmiR3383p Fig a03e f The migration and invasion of MM cells were suppressed by the knockdown of circ_0007841 and the metastasis ability was recovered in sicirc_00078411 and inmiR3383p cotransfected group Fig a0 3g h Sicirc_00078411induced apoptosis of MM cells was 0cWang a0et a0al Cancer Cell Int Page of Fig miR3383p could directly interact with circ_0007841 in MM cells a miR3383p was predicted as a candidate target of circ_0007841 by circinteractome software b c Dualluciferase reporter assay was conducted to verify whether miR3383p could bind to circ_0007841 in MM cells d e RNApull down assay was performed to confirm the target relationship between miR3383p and circ_0007841 in MM cells f g The expression of miR3383p was detected in BMderived plasma cells of MM patients and healthy volunteers MM cells and nPCs cells by qRTPCR h The correlation between the expression of miR3383p and circ_0007841 was analyzed using Spearman™s coefficient i j The abundance of circ_0007841 and miR3383p was examined in H929 and OPM2 cells transfected with Vector or circ_0007841 by qRTPCR P P P P attenuated by the addition of inmiR3383p Fig a0 3i Overall circ_0007841 could promote the malignant potential of MM cells through sponging miR3383pBRD4 is a0validated as a0a a0target of a0miR‘‘3p in a0MM cellsBRD4 was predicted as a direct target of miR3383p by targetscan database and the wild type or the mutant type binding sequence between miR3383p and BRD4 was shown in Fig a04a As exhibited in Fig a04b c the luciferase activity was markedly decreased in miR3383p and BRD4 ²untranslated region ²UTR WT cotransfected group while miR3383p transfection had no effect on the luciferase activity in BRD4 ²UTR MUT group compared with that in miRNC and BRD4 ²UTR MUT cotransfected group suggesting the interaction between BRD4 and miR3383p BRD4 was conspicuously upregulated in BMderived plasma cells of MM patients compared with that in healthy individuals Fig a0 4d Meanwhile BRD4 was also found to be upregulated in MM cell lines than that in nPCs cells Fig a0 4e The expression correlation between BRD4 and circ_0007841 or miR3383p was analyzed using Spearman™s correlation coefficient As shown in Fig a0 4f g there was an inverse correlation between the levels of BRD4 and miR3383p while the expression of BRD4 was positively correlated with the level of circ_0007841 miR3383p overexpression significantly downregulated the expression of BRD4 in MM cells suggesting the negative regulatory relationship between BRD4 and miR3383p in MM cells Fig a04h Circ_0007841 and miR3383p were cotransfected into MM cells to uncover the relationship among circ_0007841 miR3383p and BRD4 As presented in Fig a0 4i circ_0007841 overexpression upregulated the level of BRD4 and the expression of BRD4 was decreased in circ_0007841 and miR3383p cotransfected group Collectively BRD4 was a target of miR3383p and circ_0007841 could elevate the expression of BRD4 through sponging miR3383pBRD4 overexpression attenuates the a0effects of a0miR‘‘3p accumulation on a0MM cellsmiR3383p and BRD4 were cotransfected into MM cells to explore whether miR3383p exerted an antitumor role in MM cells through targeting BRD4 As shown in Fig a0 5a the addition of BRD4 overexpression plasmid recovered the expression of BRD4 in MM cells that was downregulated by the accumulation of miR3383p miR3383p overexpression inhibited the proliferation cell cycle and metastasis of MM cells and these inhibitory effects were attenuated by the 0cWang a0et a0al Cancer Cell Int Page of Fig Circ_0007841 plays an oncogenic role through targeting miR3383p in MM cells a“i MM cells were transfected with siNC sicirc_00078411 sicirc_00078411 inmiRNC or sicirc_00078411 inmiR3383p a The level of miR3383p was examined in MM cells by qRTPCR assay b c The proliferation of MM cells was measured through conducting CCK8 assay d The proliferation capacity in transfected MM cells was assessed by colony formation assay e f The percentage of MM cells in G0G1 S or G2M phase was analyzed using flow cytometry g h The migration and invasion abilities of MM cells were evaluated by transwell assays i The apoptosis rate of MM cells in different groups was analyzed by flow cytometry P P P addition of BRD4 overexpression plasmid Fig a0 5b“h The apoptosis of MM cells was induced by the transfection of miR3383p and the introduction of BRD4 overexpression plasmid recovered the viability of MM cells Fig a0 5i In conclusion miR3383p accumulation restrained the malignant behaviors of MM cells through targeting BRD4Circ_0007841 activates PI3KAKT signal pathway through a0targeting miR‘‘3pBRD4 axisThe activation of PI3KAKT signal pathway is linked to the promotion of cell proliferation and metastasis and the inhibition of cell apoptosis Herein we examined the phosphorylation levels of PI3K and AKT to illustrate the influence of circ_0007841miR3383pBRD4 axis in the See figure on next pageFig BRD4 is validated as a target of miR3383p in MM cells a The complementary sites between miR3383p and the ²UTR of BRD4 were predicted by targetscan software b c The luciferase activity was measured in H929 and OPM2 cells transfected with miRNC or miR3383p and BRD4 ²UTR WT or BRD4 ²UTR MUT d The protein level of BRD4 in BMderived plasma cells of MM patients and healthy volunteers was detected by Western blot assay e The level of BRD4 in H929 OPM2 and nPCs cells was evaluated by Western blot assay f g The linear relationship between BRD4 and miR3383p or circ_0007841 was analyzed using Spearman™s coefficient h The expression of BRD4 was detected in MM cells transfected with miRNC or miR3383p by Western blot assay i The protein level of BRD4 was detected in MM cells transfected with Vector circ_0007841 circ_0007841 miRNC or circ_0007841 miR3383p by Western blot assay P P P P 0cWang a0et a0al Cancer Cell Int Page of 0cWang a0et a0al Cancer Cell Int Page of Fig BRD4 overexpression attenuates the effects of miR3383p accumulation on MM cells a“i MM cells were transfected with miRNC miR3383p miR3383p pcDNA or miR3383p BRD4 a qRTPCR was employed to measure the expression of BRD4 in MM cells b c CCK8 assay was applied to assess the proliferation ability of MM cells d Colony formation assay was performed to analyze the influences of miR3383p and BRD4 on the proliferation of MM cells e f Flow cytometry was conducted to detect the cell cycle of MM cells g h Transwell assays were performed to detect the metastasis of MM cells i The apoptosis rate of MM cells was examined by flow cytometry P P P activation of PI3KAKT signaling Circ_0007841 silencing downregulated the level of BRD4 and the level of BRD4 was recovered in sicirc_00078411 and inmiR3383p cotransfected group Fig a06a b The activation of PI3KAKT signaling was suppressed with the silencing of circ_0007841 and the addition of inmiR3383p recovered the phosphorylation levels of PI3K and AKT Fig a06a c Meanwhile H929 and OPM2 cells were transfected with miRNC miR3383p miR3383p pcDNA or miR3383p BRD4 As mentioned in Fig a06d e miR3383p overexpression downregulated the level of BRD4 and the introduction of BRD4 overexpression plasmid regained the level of BRD4 in MM cells The addition of BRD4 alleviated the inhibitory influence of miR3383p overexpression on the activation of PI3KAKT signaling in MM cells Fig a0 6d f Taken together circ_0007841 accelerated the progression of MM through miR3383pBRD4PI3KAKT axisMesenchymal stromal cells MSCs‘generated exosomes accelerate the a0malignant potential of a0MM cells via a0circ_0007841MSCs exert crucial roles in the progression of MM Herein we explored whether exosomes derived from MSCs could regulate the proliferation cell cycle metastasis and apoptosis of MM cells via circ_0007841 MSCs were isolated from the adjacent tissues of MM and normal tissues The expression of circ_0007841 was higher in MSCs and MSCsderived exosomes from adjacent tissues than that in normal tissues Fig a07a b The markers of exosomes CD63 and CD81 were notably upregulated in exosomes of MSCs instead of cell lysate Fig a07c As mentioned in Fig a07d we established a working model as previously described to explore if MSCsderived exosomes could regulate the proliferation cell cycle motility and apoptosis of MM cells [] In this model only exosomes could be transmitted through the filter to the upper chambers As presented in Fig a07e“k sicirc_00078411 0cWang a0et a0al Cancer Cell Int Page of Fig Circ_0007841 activates PI3KAKT signal pathway through targeting miR3383pBRD4 axis a“c Western blot assay was performed to detect the levels of BRD4 and PI3KAKT signalingrelated proteins in MM cells transfected with siNC sicirc_00078411 sicirc_00078411 inmiRNC or sicirc_00078411 inmiR3383p and gray analysis was used to assess the abundance of these proteins d“f The expression of BRD4 and PI3KAKT signalingassociated proteins in MM cells transfected with miRNC miR3383p miR3383p pcDNA or miR3383p BRD4 was examined by Western blot assay P P P transfection inhibited the malignant behaviors of MM cells in Mock sicirc_00078411 group compared with that in Mock siNC group Besides MSCsderived exosomes MSCs siNC group promoted the proliferation cell cycle metastasis and inhibited the apoptosis of MM cells than that in Mock siNC group and these effects were attenuated by the silencing the circ_0007841 suggested that MSCsderived exosomes could promote the progression of MM via circ_0007841 What™s more the exosomes generated from MSCs accelerated the activation of PI3KAKT signaling while this effect was counteracted with the transfection of sicirc_00078411 Fig a0 7l Collectively MSCsderived exosomes could facilitate the progression of MM via circ_0007841DiscussionMM is an incurable cancer currently Because many MM patients were diagnosed at late stage the treatment outcomes of MM patients were unsatisfactory [] Therefore finding crucial markers in MM is urgent to improve the prognosis of MM patientsCircRNAs are featured by closely loop structure and they are widely distributed in human tissues Due to the stability and the universality of the distribution circRNAs are identified as ideal biomarkers for human cancers and other diseases [] For example the high expression of circ_0004277 was associated with the better prognosis of AML patients [] Xia et a0al claimed that circCBFB was highly expressed in chronic lymphocytic leukemia and circCBFB accelerated the proliferation and suppressed the apoptosis of chronic lymphocytic leukemia cells [] Circ_0007841 was found to be overexpressed in BMderived plasma cells of MM patients and MM cells Further studies suggested that circ_0007841 promoted the proliferation cell cycle metastasis and inhibited the apoptosis of MM cells These findings

Thyroid_Cancer

Genomics Score Based onGenomeWide Network Analysis forPrediction of Survival in GastricCancer A Novel PrognosticSignatureZepang Sun  Hao Chen  Zhen Han  Weicai Huang Yanfeng Hu Mingli Zhao Tian LinJiang Yu Hao Liu Yuming Jiang and Guoxin LiDepartment of General Surgery Nanfang Hospital Southern Medical University Guangzhou ChinaGCPurpose Gastric canceris a product of multiple genetic abnormalitiesincluding genetic and epigenetic modifications This study aimed to integrate variousbiomolecules such as miRNAs mRNA and DNA methylation into a genomewidenetwork and develop a nomogram for predicting the overall survival OS of GCMaterials and Methods A total of GC cases as a training cohort with a random of examples included as a validation cohort were screened from The Cancer GenomeAtlas database A genomewide network was constructed based on a combination ofunivariate Cox regression and least absolute shrinkage and selection operator analysesand a nomogram was established to predict and 5year OS in the trainingcohort The nomogram was then assessed in terms of calibration discriminationand clinical usefulness in the validation cohort Afterward in order to confirm thesuperiority of the whole gene network model and further reduce the biomarkers for theimprovement of clinical usefulness we also constructed eight other models accordingto the different combinations of miRNAs mRNA and DNA methylation sites and madecorresponding comparisons Finally Gene Ontology GO and Kyoto Encyclopedia ofGenes and Genomes KEGG analyses were also performed to describe the function ofthis genomewide networkResults A multivariate analysis revealed a novel prognostic factor a genomics scoreGS comprising seven miRNAs eight mRNA and DNA methylation sites In thevalidation cohort comparing to patients with low GS highGS patients HR P were significantly associated with increased allcause mortality Furthermoreafter stratification of the TNM stage I II III and IV there were significant differencesrevealed in the survival rates between the highGS and lowGS groups as wellP The and 5year Cindex of whole genomicsbased nomogram were and respectively The other models have comparable or relativelypoor comprehensive performance while they had fewer biomarkers Besides thatDAVID further revealed multiple molecules and pathways related to the genomewidenetwork such as cytomembranes cell cycle and adipocytokine signalingEdited byXin Maizie ZhouStanford University United StatesReviewed byHailin TangSun Yatsen University Cancer CenterSYSUCC ChinaJie TianInstitute of Automation CAS ChinaCorrespondenceYuming Jiangjiangymbest163comGuoxin Ligzliguoxin163com These authors have contributedequally to this workSpecialty sectionThis was submitted toComputational Genomicsa section of the journalFrontiers in GeneticsReceived April Accepted July Published August CitationSun Z Chen H Han Z Huang WHu Y Zhao M Lin T Yu J Liu HJiang Y and Li G GenomicsScore Based on GenomeWideNetwork Analysis for Predictionof Survival in Gastric Cancer A NovelPrognostic SignatureFront Genet 103389fgene202000835Frontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreConclusion We successfully developed a GS based on genomewide network whichmay represent a novel prognostic factor for GC A combination of GS and TNMstaging provides additional precision in stratifying patients with different OS prognosesconstituting a more comprehensive subtyping system This could potentially play animportant role in future clinical practiceKeywords gastric cancer genomewide network miRNA mRNA DNA methylation nomogramBACKGROUNDGastric cancer GC is one of the most common malignanthuman tumors and the third leading cause of cancerrelatedmortalities worldwide Reports estimate that nearly one millionnew cases and deaths occur each year across the worldTorre Despite the rapid research advancement GCrelated impacts on human life remain high around the globeAccording to the global cancer burden data hundreds of billionsof dollars in economic losses are incurred each year due to GCAt the same time stomach cancer has been reported to cause million disabilityadjusted life years with of these resultingfrom years of life lost and from years lived with disabilityGlobal Burden of Disease Cancer Collaboration Despite major breakthroughs in GC prevention diagnosisand treatment therapies reported over the past decade prognosisremains a challenge at diï¬erent TNM stages Jiang 2018aSun 2019ab Notably patients with similar clinicalfeatures and at the same tumor stage who receive uniformtreatment have exhibited varying clinical outcomes Bang Jiang 2018b Such evidence indicates the existingchallenges to traditional TNM staging Serra possiblydue to a lack of molecular tools for eï¬ectively predicting theprognosis and the therapeutic eï¬ect of GC patients Thereforemore rigorous and reliable systems that accurately reflect theheterogeneity of diï¬erent patients and guide the development oftreatment approaches are urgently needed Duarte Serra Tumors are a product of multiple genetic mutations includinggenetic gene expression and epigenetic DNA methylation andhistone modification modifications as well as deregulationsof tumorsuppressor genes and protooncogenes Anna Choi In addition changes in a set of geneticmaterials have been closely associated with cancer outcomesAnna Choi Therefore to eï¬ectivelypredict the prognosis of tumors such as GC a single biomarkeris insufficient necessitating the need for a gene networkA variety of mRNAs have been associated with GC prognosisCamargo with microRNAsmiRNAs alsoimplicated in tumor prediction in the recent years Li Ueda Camargo These smallnoncoding RNAs comprising nucleotides primarily functionto regulate protein translation by inhibiting the expressionoffrom geneticsepigenetics is currently receiving considerable research attentionDNA methylation isthe most common epigenetic eventassociated with cancer development and progression Anna Consequently numerous studies have implicated DNAtarget messenger RNAs mRNAs Apartmethylation in the diagnosis and the prognosis of various tumorsincluding GC Camargo Choi Althoughthese studies have revealed several biomarkers that have proved tobe prognostic predictors in GC only a handful have been adoptedin clinical therapies or are used to build predictive models forthe disease Anna Duarte Camargo Choi Serra Previous studies have identified and recommended numerousbiomarkers for GC However since malignant tumors ofteninvolve multiple layers and diï¬erent levels of genetic changesincluding the genome transcriptome and proteome or evenepigenetic content selecting reasonable candidate factors fromtens of thousands of biomarkers and comprehensively analyzingthem as an independent feature is imperative to eï¬ectivelydevelop a suitable prognostic target Therefore genetic networkscontaining a panel of abnormal factors from diï¬erent regulatorylevels represent the best chance for achieving prognostic valueThe whole genomewide network analysis is reported inseveral other cancers such as colorectal cancer breast cancer andlung cancer Hou Zhang and it showsgreat value in diï¬erentiating the prognosis of these patientsTherefore it is feasible and advantageous to apply genomewidenetwork analysis to GCIn the current study we performed a series of sophisticatedstatistical analyses and identified genetic molecules that werehighly correlated with the prognosis of GC Specifically wescreened The Cancer Genome Atlas TCGA a genome projectwith types of cancer including gene expression and DNAmethylation as well as other biological information Furthermorewe extended these independent prognostic factors to theœomics concept Then a genomewide network was constructedInterestingly the genomics score GS obtained herein couldsupplement TNM staging and enhance the prognostic value ofdiï¬erent patients Moreover we developed multiple prognosticmodels then validated and compared them to ascertain theirstrengths and weaknesses Finally we performed pathwayenrichment and gene oncology annotation analyses to elucidatethe function of this gene networkMATERIALS AND METHODSData Acquisition and PreprocessingLevel data were downloaded from the TCGA databaseusing TCGAAssembler Module Ain January whichwas then pretreated with Module B The dataset comprised ofclinical variables from patients including age sex stageFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics Scoreprimary site grade treatment and survival as well as associatedgenomewide data In addition the expression levels of miRNAs mRNA and DNA methylation sitesIllumina methylation were obtained from and patients respectively Afterward an intersection with a totalof samples was eventually retained Furthermore patientswith missing active followup data were excluded from theanalysis leaving patients in the final cohort Figure Moreover genomewide level data whose expression levelsfor miRNAs mRNA and DNA methylation sites were missingin more than of all samples were excluded from the finalanalysis Finally GC patients with miRNAs mRNA and DNA methylation sites were chosen forfurther analysisGenomeWide Network AnalysisGene expression and DNA methylation data were normalizedusing R package before subsequent processing Univariate andleast absolute shrinkage and selection operator LASSO Coxregression models were combined and used to identify themost useful prognostic factors in miRNAs mRNA and DNAmethylation sites associated with survival Firstly univariate Coxregression was performed on each candidate miRNA mRNA andDNA methylation site to elucidate its role in patient survivalthen signatures with P value less than were retained forsubsequent analysis Thereafter the LASSO Cox regression modelwas applied to select and shrink the data SupplementaryFigure S3 Tibshirani Finally a GS based on a genomewide network comprising seven miRNAs eight mRNAs and DNA methylation sites was constructed for predicting survivalA summary of the whole screening process is displayed inSupplementary Figure S1Development and Comparison ofIndividualized Prediction ModelsThe TCGA cohort with cases was used as the trainingset with a random cases from the total cohort includedas a validation group The random number is Firstlywe developed the original GS based on biomarkers sevenmiRNAs eight mRNAs and DNA methylation sites Thenconsidering the complexity of the original GS and difficultclinical applicationin order to obtain a more concise andFIGURE A Venn diagram displays the patients™ screening processFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics Scoreeï¬ective GS we also constructed eight other models accordingto the diï¬erent combinations of miRNAs mRNA and DNAmethylation and made corresponding comparisons Finally atotal of nine GS models based on the genomewide networkfrom LASSO were adopted to screen for the most appropriatemarkers These included the following models genomics sevenmiRNAs eight mRNA and DNA methylation sites miRNAsseven miRNAs mRNA eight mRNA methylation DNAmethylation sites miRNAs methylation seven miRNAsand DNA methylation sites miRNAs mRNA sevenmiRNAs and eight mRNA mRNA methylation eightmRNA and DNA methylation sites Coxmodel twomiRNAs six mRNA and nine DNA methylation sites andCoxmodel one miRNA one mRNA and seven DNAmethylation sites Among them markers from Coxmodel were separately detected from miRNAs mRNA or DNAmethylation sites using multivariate Cox regression analysis afterLASSO Supplementary Tables S2“S4 On the other handmarkers from Coxmodel depended on signatures from amultivariate Cox regression analysis combining the genomewide network and the clinical characteristics SupplementaryTable S5 Thereafter we constructed several nomograms byincorporating significant P GS variables and otherclinical features following multivariate Cox regression Iasonos and a clinical nomogram was built as a blank controlThe equations used for calculating the GS of these models arelisted in Supplementary Table S6To calculate the discrimination and the stability of diï¬erentCox regression models we applied Cstatistics and calibrationAdditionally we performed an analysis oftimedependentreceiver operator characteristics ROC based on the and 5year survival endpoints to assess the prognostic accuracyof the diï¬erent nomograms Furthermore we evaluated thepotential net benefit of diï¬erent predictive models using decisioncurve analysis DCA DCA compares the clinical usefulness ofdiï¬erent indicators by calculating the potential net benefit of eachdecision strategy at each threshold probability Thus DCA wasa significant novel approach for comparing the old and the newmodels Vickers and Elkin Screening for Potential miRNA TargetGenesWe predicted the potential target genes of the seven miRNAsfrom LASSO by screening the miRTarBase miRDB andTargetScan databases Common genes from each miRNA acrossthe three databases were then used for subsequent studies Morethan of the miRNAs showed negative regulation to targetgenes Consequently the expression data from TCGA were usedto perform a batch of correlation analysis of each miRNAwith corresponding target genes and the three genes with thelargest absolute negative correlation were retained as the mostlikely targets Additionally at least three potential target genesfrom miRTarBase which is coexpressed with miRNAs wereconsidered as equally important and were subjected to Cytoscapeversion for identification of miRNA“target genes coexpression network analysis Supplementary Figure S2Functional Enrichment AnalysisThe potential target genes that were negatively correlated withmiRNAs in TCGA as well as the coding sequences for mRNAand DNA methylation sites were used for functional enrichmentanalysis using the Kyoto Encyclopedia of Genes and GenomesKEGG pathway and Gene Ontology GO using DAVID Supplementary Figure S2 Functional enrichment analysisindicates why the gene network produces images on the survivalof GC from a molecular mechanism Visualization was then doneusing the œggplot2 package implemented in RStatistical AnalysisThe patients were divided into lowrisk and highrisk groupsby the median GS as the cutoï¬ point Survival estimates wereobtained according to the Kaplan“Meier method and comparedusing the logrank test Variables that reached significance withP were entered into the multivariable analyses usingthe Cox proportional hazards model with an entry stepwiseapproach to identify covariates associated with increased allcause mortality and then hazard ratio with confidenceintervals CIs of each variable was achieved All the statisticalsignificance values were set as twosided P LASSOCox regression was performed through the œglmnet packageTimedependent ROC analysis at diï¬erent followup times wasimplemented using the œtimeROC package of R project in orderto further expound the performance of diï¬erent GS modelsand DCA was used to compare their clinical use by œrmdapackage Finally nomogram based on the Cox regression modelwas constructed using the œrms package Cindex and calibrationto calculate the discrimination and the stability of these modelswere performed using cstatistics and Bootstrap sample Harrell™sconcordance index Cindex indicated a better prognostic modelif its value was closer to and the calibration diagram showedthat the better the prediction if the closer the correction line wasto the diagonal All statistical methods are applied to both thetraining group and the validation group Statistical analyses wereperformed using SPSS statistical software version and Rsoftware version RESULTSPatient CharacteristicsAmong the GC patients analyzed in this study were male whereas were female The average ageof the entire study population was ± years In termsof pathological stage cases were identified as stageI were stage II were stage III and were at stage IV With regards to treatment patients received surgery cases of R0 surgery R1 and nineR2 whereas were subjected to fluorouracilbasedchemotherapy The genomic nomogram classified samplesinto low GS GS ‰¤ and into high GS GS groups based on the median cutoï¬ Figure A detaileddescription of tumor location pathology grade and Laurenclassification is outlined in Supplementary Table S1 while aheat map of the genomic scores layered by clinicopathologicalFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreFIGURE Distribution of patient cases and density based on genomics score GS in the total The Cancer Genome Atlas cohort AB Scatter plots of genomicsscores regarding the classification of low and high GS CD and the bold line represents the medianfactors is illustrated in Supplementary Figure S4 The medianmean CI survival time for OS was “days in the total cohort “ days in the highGS group and mean “ days in the lowGSgroup Supplementary Figure S5 Toward the last followupa total of deaths and censoring were recorded Theestimated cumulative and 5year OS in the total cohortwere and respectively although these ratesincrease to and respectivelyin the lowGS group Conversely the and 5year OS decreased to and respectively in the highGS group Thebaseline information of the validation cohort is also listed inSupplementary Table S1 and Supplementary Figure S6Survival AnalysisWe identified a basic genomewide network comprisingseven miRNAs eight mRNAs and DNA methylationsites as the prognostic factor for OSfrom hundreds ofthousands of univariate Cox regression and LASSO analysesThis network was then classified as other models in thetraining and the validation groups Among the featuresidentified poor prognosis was significantly associated witha high expression of seven miRNAs hsamir100 hsamir hsamir136 hsamir193b hsamir22 hsamir653and hsamir6808 six mRNAs NRP18829 RNF144A9781ZNF227570 DUSP11843 CPNE8144402 MAGED19500and LOC9145091450 and seven DNA methylation sitesFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics Scorecg22813794cg26014401cg25361506cg07020967 cg08859156 cg12485556 cg15861578 cg15861578cg25161386 and cg22740006 Conversely poor prognosis wasstrongly associated with a low expression of SOX148403 and DNA methylation sites including cg02223323 cg00481239cg14791193 cg15486740 cg20100408 cg20350671 cg22395807cg24361571andcg26856948 Table Univariate analysis performed onclinical characteristics revealed a significant association betweenage pathological stage TNM and surgery with OS Table On the other hand results from multivariable Cox regressionshowed that age pathological stage and GS were significantlyassociated with allcause mortality in GC Table and Figure Furthermore stratification of the pathological stage I II IIIand IV revealed significant diï¬erences in survival rates betweenthe highGS and the lowGS groups Figure A similarresult was found when the data were stratified by demographicvariables sex and age clinical characteristics primary sitegrade and Lauren classification as well as treatments surgeryand chemotherapy Supplementary Figures S7 S8 Onthe other hand categorizing GS into high or low groupsusing the median value across diï¬erent modelsindicatedthe genomics nomogram had the highest HR valuethatInterestingly HR was almost equal to miRNAs methylationmRNA methylation Coxmodel and Coxmodel nomograms which contained fewer gene features Moreoverthe HR value showed a marked decrease in miRNAs mRNAmethylation and miRNAs mRNA nomograms which includedthe least characteristics Table Nomograms Based on GenomeWideNetworkA genomics nomogram was first constructed based on thegenomewide network comprising gene features Figure To obtain a more concise and eï¬ective nomogram we also builta Coxmodel gene features and Coxmodel nine genefeatures nomograms Supplementary Figures S9 S10 Nexta clinical nomogram based on stage and age was built as acontrol Supplementary Figure S11 Thereafter we performedinternal and external validation to evaluate the feasibility of allnomograms using a threegrouped random bootstrap samplingFigure and Supplementary Figures S9“S11 We observedgood predictive performance in the first three nomograms butnot in the simple clinical modelValidation of the Nomograms Using ROCand DCATo ensure a good comparison across diï¬erent GS nomogramswe performed a timedependent ROC at and years offollowup as well as DCA In the validation group genomicsnomogram revealed the best comprehensive performance with and 5year area under the curve AUC values of and respectively Table and Coxmodel miRNAs methylation and mRNA methylation nomogramshad a comparable performance with and 5year AUCvalues of “ “ and “ respectivelybut it had fewer biomarkers Table Although the Coxmodel nomograms had the least biomarkers including miRNA mRNAand DNA methylation sites it had a relatively poor performancewith and 5year AUC values of and respectively Besides that the miRNA mRNA methylationmiRNAs methylation and miRNAs mRNA nomogramsrecorded and 5year AUC values of “ “ and “ respectively Finally we found thatcompared to miRNA and and mRNAnomogram and methylation nomogramhad higher and 5year AUC values of and Nevertheless all of them showed better performance thanthe clinical nomogram which recorded and 5year AUCvalues of and respectively Figures 6AB andSupplementary Figure S12 The Cindex based on diï¬erentnomograms exhibited a similar eï¬ect Supplementary Table S8Additionally DCA showed that the genomics Coxmodel mRNA methylation and methylation nomograms had asignificant net benefit compared to other GS models and theclinical nomogram Figures 6CDPotential miRNA Target GenesA total of hsamir22 hsamir100 hsamir136 hsamir193b hsamir653 hsamir1304 and hsamir6808 potential target genes were identified from themiRTarBase miRDB and TargetScan databases SupplementaryFigure S14 We then performed a correlation analysis betweeneach target gene and miRNAs and finally generated a miRNA“potential target gene plot Supplementary Figure S15A as wellas a miRNA“target gene coexpression network SupplementaryFigure S15B using CytoscapeFunctional Analysis of GenomeWideNetworkWe imported the potentialtarget genes mRNA andDNA methylation sitecoding sequences identified above intoDAVID for KEGG and GO analyses and identified biologicalprocesses molecular functions as well as cellular componentsFigures 7A“C Their corresponding KEGG pathways were alsoplotted Figure 7DDISCUSSIONGC can be divided into two types or four main categoriesaccording to the Lauren and World Health anizationWHO classifications Lauren Nagtegaal although neither of these classifications is based on molecularmarkers In the last decade however three novel molecularbased classification systems have been suggested for GC TheSingaporeDuke Group was the first to describe a classificationwith two intrinsic genomic subtypes GINT and GDIF whichhad diï¬erent gene expression Tan Serra The subtypes have diï¬erent levels of resistance to variouschemotherapy drugs and show limited prognostic value LaterTCGA used molecular evaluation to propose a new classificationwith four subtypes EBV MSI GS and CIN The identification ofthese subtypes has provided a roadmap for patient stratificationFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreTABLE Univariable and multivariable analyses of the genomics score and the clinicopathological characteristics for overall survival in the training group and thevalidation groupVariablesTraining group n Validation group n Univariable analysisMultivariable analysisUnivariable analysisMultivariable analysisHR CIP valueHR CIP valueHR CIP valueHR CIP valueAge at diagnosis tears‰¥Pathological stageIIIIIIIVSurgeryR0R1R2UnknownGenomics scoreaLowHighT stagingT1T2T3T4N stagingN0N1N2N3M stagingM0M1SexFemaleMalePrimary siteCardiaFundusbodyAntrumUnknownPathology gradeI“IIIII“IVUnknownLauren classificationIntestinal typeDiffused typeUnknownChemotherapyYesNo“NANA““NA“NA“““ NA““ “““““““NANANA“““ NA““ “““““““NANANA“““ “NANANANA““““““NANANA““““““““““NANANANANA““““““NANANA“ “““““““““NANANANANAaBased on biomarkers seven miRNAs eight mRNAs and DNA methylation sitesFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreTABLE miRNAs mRNA and DNA methylation whose expression levels showed a significant association with overall survival in least absolute shrinkage andselection operatorMolecular probeID reference geneCoefficientmiRNAsmRNAcg02223323cg00481239cg07020967cg08859156cg12485556cg14791193cg15861578cg15486740cg20100408cg20350671cg22395807cg24361571cg25361506cg25622155cg25161386cg22740006cg22813794cg26014401cg26856948hsamir100hsamir1304hsamir136hsamir193bhsamir22hsamir653hsamir6808NRP18829RNF144A9781ZNF227570SOX148403DUSP11843CPNE8144402MAGED19500LOC9145091450MAP7D2SHC4EID1TMEM117RPS4XPREPC1orf144ZC3H10ACOT13TTRAPHLADPB1IL1RAPL1ATXN10MIR3652UnconfirmedUnconfirmedNUFIP2PCLRFN4STYXL1MDH2UnconfirmedGOLGA3ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’HR CI““““““““““““““““““““““““““““““““““SEz valuep valueˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’as well as targeted therapeutic trials Cancer Genome AtlasResearch However initial data on disease outcomes fromthis cohort did not show diï¬erences in survival among thefour groups A series of positive studies on prognosis basedon TCGA classification was also reported Sohn In addition the Asian Cancer Research group divided GCinto four subtypes MSI EMT MSSTP53 and MSS TP53based on gene expression data and found significantly diï¬erentsurvival outcomes across them Cristescu Serra Despite the significant milestones of these studiesthey are all mainly based on the analysis of gene expressionmRNA Besides that a study proposed a fivemiRNAmodel while it had a Cindex of only Zhang In the current study we included methylation data andperformed functional enrichment analysis making our workstronger The aforementioned classifications are also complicatedand need further optimization to increase clinical applicabilityFurthermore they focused on typing and finding new targetswhereas our study reports on prognostic analysisSome of the biomarkers we identified herein including hsamir22 hsamir100 hsamir136 hsamir193b hsamir1304NRP1 DUSP1 and MAP7D2 cg02223323 have previouslybeen reported in GC Grandclement and B Chen Mu Zuo Zheng Chen Kurata and Lin Liu KT Song Teng Liu Pan Wang Others such as CPNE8MAGED1 RNF144A SOX14 ACOT13 cg15486740 EID1cg00481239 RPS4X cg08859156 and TTRAP cg15486740have been identified in various tumors other than GC Kamio Zeng Zhou Kuang Stanisavljevic Liu X Tosic Nagasawa Yang The remainingbiomarkers including hsamir653 hsamir6808 LOC91450Frontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreFIGURE Kaplan“Meier curve of overall survival in all patients then stratified by genomics score GS pathological stage and age Survival analysis in the lowand highGS groups was further divided based on stages stages I“IVcg20100408 LRFN4cg14791193 GOLGA3ZNF22 C1orf144cg26856948HLADPB1cg22740006 MDH2cg22813794 MIR3652 cg24361571 NUFIP2 cg25161386PREPcg22813794 TMEM117cg07020967 ZC3H10 ZC3H10 IL1RAPL1 cg20350671 PCcg22740006 SHC4 cg00481239 and ATXN10 cg22395807have not been previously reportedcg12485556STYXL1Currently focus has been directed on identifying prognosticmiRNAs for GC Particularly one miRNA can regulate multipletargets while multiple miRNAs can regulate a single mRNATherefore a single miRNA may play an opposite role incancer progression by regulating diï¬erent target genes Forexample Mir22 and Mir100 were found to be tumorsuppressors in various cancers including GC Chen Zuo Similarly a high expression of Mir136 wasfound to promote proliferation and invasion in GC cell linesby inhibiting PTEN expression Chen while acontrasting result was reported when HOXC10 was targetedZheng Similarly Mir193b reportedly inducedGC proliferation or apoptosis by mediating diï¬erent mRNAexpressions Mu Song whereas a highMir1304 expression in GC was reported as a negative predictorfor prognosis of lung and thyroid cancers Kurata and Lin Liu Pan However the function of Mir653and Mir6808 has not been previously reported In the currentstudy we found an association between a high expression of allmiRNAs and poor survival Diï¬erent outcomes may be observedin our study relative to previous reports owing to the hugeFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreTABLE Comparison of different genomics score models based on the median value for overall survival in the training group and the validation groupVariablesTraining group n Validation group n Hazard ratio CIP valueHazard ratio CIP valueGenomics nomogramAgePathological stageGenomics scoreaClinical nomogramAgePathological stagemiRNAs nomogramAgePathological stageGenomics scorebmRNA nomogramAgePathological stageGenomics scorecMethylation nomogramAgePathological stageGenomics scoredmiRNAs methylation nomogramAgePathological stageGenomics scoreemiRNAs mRNA nomogramAgePathological stageGenomics scorefmRNA methylation nomogramAgePathological stageGenomics scoregCoxmodel nomogramAgePathological stageGenomics scorehCoxmodel nomogramAgePathological stageGenomics scorei““““““““““““““““““““““““““““““““““““““““““““““““““““““““““aBased on biomarkers seven miRNAs eight mRNA and DNA methylation sites bBased on seven miRNAs cBased on eight mRNA dBased on DNA methylationsites eBased on seven miRNAs DNA methylation sites fBased on seven miRNAs eight mRNA gBased on eight mRNA DNA methylation sites hBased ontwo miRNAs six mRNA nine DNA methylation sites iBased on one miRNAs one mRNA seven DNA methylation sitesnumber of corresponding miRNA target genes herein and lackof evidence on their role in GC developmentMessenger RNAs have been reported to play an essentialrole in GC cancer For example high NRP1 expression andhypermethylation were associated with poor GC prognosisWang whereas another study indicated thatit could be an antitumor target Grandclement and B In addition high DUSP1 expression levels were foundto promote progression drug resistance and poor prognosisof GC Teng On the other hand SOX14showed opposite prognostic values in cervical cancer andleukemia with antitumor and carcinogenic eï¬ects respectivelyStanisavljevic Tosic Studies have alsoimplicated CPNE8 MAGED1 and RNF144A in ovarian andbreast cancers Zeng Nagasawa Yang However LOC91450 and ZNF22 have not beenreported in cancerAccumulating evidence indicates that DNA methylation playsa significant role in cancer progression However only a handfulof studies have described the relationship between levels ofFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreFIGURE Genomics nomogram to predict the probability of and 5year overall survival OS in the training cohort A and the validation cohort B todetermine how many points for each variable to the probability of OS locate the variable on its axis draw a line straight upward to the point axis repeat this processfor each variable sum up the points achieved for each of the risk factors locate the final sum on the total point axis and draw a line straight down to find thepatient™s probability of OSsinglesite methylation and GC prognosis Particularly highexpressions of MAP7D2 ACOT13 EID1 RPS4X and TTRAPhave been associated with poor prognosis in gastric lung andpancreatic cancers as well as hepatic carcinoma respectivelywhile a high TTRAP expression reportedly inhibits the growthof osteosarcoma Kamio Zhou Kuang Liu KT Liu X Notably therelationship between methylation levels and corresponding geneexpression profiles is unknown necessitating further researchFurthermore the remaining DNA methylation sites and theircorresponding genes have not been reported Lastly no studyhas described the prognostic significance using a genomewide networkLast but not l

Thyroid_Cancer

"pharmacological therapies and treatments targeting pancreatic neuroendocrine tumorsPNETs have proven ineffective far too often Therefore there is an urgent need for alternative therapeuticapproaches Zyflamend a combination of antiinflammatory herbal extracts that has proven to be effective invarious in vitro and in vivo cancer platforms shows promise However its effects on pancreatic cancer in particularremain largely unexploredMethods In the current study we investigated the effects of Zyflamend on the survival of betaTC6 pancreaticinsulinoma cells TC6 and conducted a detailed analysis of the underlying molecular mechanismsResults Herein we demonstrate that Zyflamend treatment decreased cell proliferation in a dosedependent mannerconcomitant with increased apoptotic cell death and cell cycle arrest at the G2M phase At the molecular level treatmentwith Zyflamend led to the induction of ER stress autophagy and the activation of cJun Nterminal kinase JNK pathwayNotably pharmacological inhibition of JNK abrogated the proapoptotic effects of Zyflamend Furthermore Zyflamendexacerbated the effects of streptozotocin and adriamycininduced ER stress autophagy and apoptosisConclusion The current study identifies Zyflamend as a potential novel adjuvant in the treatment of pancreatic cancer viamodulation of the JNK pathwayKeywords Pancreatic neuroendocrine tumor cells Zyflamend JNK Apoptosis Autophagy ER stressPlain English summaryThrough investigating the effects of treating an experimental model of pancreatic neuroendocrine tumor cells withZyflamend we discovered a novel therapeutic potential ofthis polyherbal blend Findings from this study could helppioneer future advancements in our understanding of howphytochemicals and natural compounds could synergistically prove effective against pancreatic cancer by alteringcancer cell survival and proliferation Furthermore the evidence presented within promotes Zyflamend as an adjuvantprospect where it could enhance the effectiveness of standard cancer therapies In addition we believe that thesenovel findings will be of major interest to a broad spectrumof scientists and may pave the way towards more effectiveand translatable therapies Correspondence abettaieutkedu1Department of Nutrition University of Tennessee Knoxville CumberlandAvenue Jessie Harris Building Knoxville TN USA3Graduate School of Genome Science and Technology University ofTennessee Knoxville TN USAFull list of author information is available at the end of the BackgroundPancreatic cancer remains one of the deadliest types ofcancer in the United States with over new casesand deaths in accounting for of allcancer deaths [] Recent epidemiological studies predict The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cPuckett Cell Communication and Signaling Page of that in pancreatic cancer will be the third leadingcause of cancerrelated death [] Although advancements in science and health care have led to decreasedmortality from numerous forms of cancer pancreaticcancer survival rates have not improved significantlyover the past several decades leaving a desperate needfor more effective treatment options The risk of developing pancreatic cancer has been associated with numerous biological environmental pathological andgenetic factors These factors include variables such asfamilial history chronic pancreatitis smoking obesityand diabetes reviewed in [ ] In addition hereditaryfamilial factors and germline mutations could contributeto increased risk of cancer onset However the survivaloutcomehighlydependent upon the time of diagnosis While pancreaticductal adenocarcinoma PDAC is the most commonlycontracted and investigated subtype the pathological nature ofthe rarer pancreatic neuroendocrine tumorsPNETSs remains elusive []effectivenesstreatmentandarePNETs account for less than of all pancreatic cancers and are often diagnosed at a late stage in patientswith advanced metastasis making surgery a nonviabletreatment option [ ] Additionally because of theirheterogeneous clinical presentation and responses tochemotherapeutic agents current pharmacological therapies and treatment options targeting PNETs have toooften proven ineffective [] PNETs treatment optionsoften include the use of chemotherapeutic compoundssuch as streptozocin 5fluorouracil doxorubicin andcisplatin both alone or in combination reviewed in [] The effectiveness of these compounds often increases at higher doses but this directly exacerbates therisk for cytotoxicity and collateral side effects [] Inaddition adjunct therapy involving the combination ofvarious treatment approaches such as surgery and radiotherapy is often implemented [] In pursuit of survivaland improved quality of life patients often seek to enhancetherapiesthrough dietary and supplemental means [ ]effectiveness ofconventionaltheNew Chapter Brattleboro VT first launched Zyflamend based on the idea of combining extracts of ten different herbs to effectively reduce inflammation throughcyclooxygenase COX inhibition [] A large volume ofresearch has emerged over the last two decades that supports the antiinflammatory properties of Zyflamend andits ability to inhibit COX in various types of cancer including prostate [] melanoma [] and oral cancer[] Individually many of the extracted components ofZyflamend have proven to exhibit anticancer activity[ ] However the high doses required to optimizeeffectiveness against cancer could prove infeasible forthe majority In theory the combined effects generatedthrough integrating these unique and powerful herbscould grant superior benefit over their isolated form[] Additionally Zyflamend has shown the capabilityto interact with a variety of integral cellular signalingpathways beyond COX These signaling pathways andmechanisms of interaction include AMPactivated protein kinase AMPK [ ] nuclear factor kappalightchainenhancer of activated B cells NFκB [ ]mammalian target of rapamycin mTORC1 [] apoptosis cell growth [ ] endoplasmic reticulum ERstress [ ] and finally autophagy [] Whilethese studies show that Zyflamend could exhibit profound potential in the therapeutic application more research is required to elucidate the molecular basisunderlying its anticancer effects In the current studywe investigated the effects of Zyflamend on the survivalof betaTC6 pancreatic insulinoma cells TC6 anddeciphered the underlying molecular mechanismsMethodsChemicals and reagentsMedia sera and trypsin for cell culture were purchasedfrom Gibco Thermo Fisher Scientific Waltham MAPrimary antibodies and secondary antibodies were acquired from varying sources Supplementary Table General caspases inhibitor ZVADfmk was obtainedfrom Calbiochem La Jolla CA Zyflamend„¢whole bodywas purchased from New Chapter New Chapter IncBrattleboro VT Zyflamend composition is indicated inSupplementary Table Quality assurance is in full compliance with Good Manufacturing Practicing Standardsas mandated by CRF Part Additionally full description and characterization of Zyflamend and itspreparation have been previously described in detail[] Chemical reagents such as dithiothreitol DTTpercoll digitonin phenylmethylsulfonyl fluoride PMSFprotease inhibitors cocktail sodium deoxycholate Tritonglycolbis2aminoethylNNN€²NX100€²tetraacetic acid EGTA sodium fluoride NaF sodium phenylbutyrate 4PBA Hoechst propidium iodide streptozotocin STZ adriamycin ADRautophagy inhibitor 3methyladenine 3MA and JNKinhibitor SP600125 were acquired from MilliporeSigma Burlington MA Finally AMPK inhibitor BML aka compound C was purchased from Santa CruzBiotechnology Santa Cruz CAethyleneCell cultureMouse betaTC6 pancreatic insulinoma TC6 ATCC®CRL11506„¢ and rat pancreatic insulinoma RIN5FATCC® CRL2058„¢ cells were cultured as monolayersin Eagle€™s modified Dulbecco medium plus Lglutamine mM sodium pyruvate mM and fetal bovineserum FBS GibcoThermo Fisher Scientific WalthamMA Cells were maintained in tissue culture plates 0cPuckett Cell Communication and Signaling Page of Thermo Fisher Scientific Waltham MA at °C in ahumidified atmosphere of CO2 Medium was replaced with fresh medium h before experimentsZyflamend treatmentZyflamend was dissolved in dimethyl sulfoxide DMSOat a concentration of mgml Cells were treated withZyflamend at the indicated concentrations and for theindicated durations Treatments were terminated by twowashes with icecold phosphate buffer saline PBSPlates were then flashfrozen in liquid nitrogen andstored at ˆ’ °C until further analysesProliferation assayCell proliferation assay was performed using the sulforhodamine B SRB MilliporeSigma method as previously described [] with modification Briefly an equalnumber of TC6 cells X cells were seeded in well plates Six h later cells were treated with the indicated concentrations of Zyflamend and incubated at °C in an atmosphere of CO2 for the indicatedtime Treatment was stopped by two washes with icecold PBS and cells were fixed with trichloroaceticacid in PBS Intracellular proteins were stained for min at room temperature using SRB dissolved in acetic acid Excess SRB stain was removed by rinsingthe plates thoroughly with running tap water Plateswere airdried for at least h prior to dissolving the stainin mM Tris pH Intracellular proteins werequantified using the Synergy„¢ HTX MultiMode microplate reader BioTek Instruments Inc Winooski VT ata wavelength of nm The relative survival rates ofcells were determined by dividing the absorbance observed for a given treatment by the absorbance detectedin control cells treated with DMSO and expressed as afold changeCytotoxicity assayThe MTT [45dimethylthiazol2yl]25diphenyltetrazolium bromide cytotoxicity assay was performed aspreviously described with modification [] Briefly cells were plated in a 96well plate for h Then afreshly prepared solution of Zyflamend alone or in combination with ZVADfmk 10uM 4PBA 250uMSP600125 10uM STZ mM or adriamycin μMfor an additional h The experiment was terminatedby adding μl of the MTT solution mgml to eachwell for h then the cell culture medium was removedand the dye was dissolved in μl SDS solution overnight at °C Relative cytotoxicity was determinedby measuring the absorbance at nm using the Synergy„¢ HTX MultiMode microplate readerColognic testColonie formation assay was performed as previously described [ ] with modification Cells were seeded inthe presence of DMSO control Zyflamend alone or incombination with ZVADfmk uM 4PBA uM SP600125 uM STZ mM or adriamycin μM After h media was replaced with a freshlyprepared new cell culture media and plates were incubated for days at °C in an atmosphere of CO2After incubation the colonies were washed with icecoldPBS fixed and stained with a mixture of glutaraldehyde and crystal violet for min The plates werewashed with water dried and colonies with morethan cellscolony were counted The relative number of colonies in each condition was determined bydividing the number of colonies for a given treatmentby the total number of colonies in DMSO treatedcells control and expressed as a percentage relativeto DMSOtreated cells CtrlWestern blotting analysisCells were lysed in radioimmunoprecipitation assayRIPA buffer as previously described [] Lysates wereclarified by centrifugation at g for min andprotein concentrations was determined using bicinchoninic acid assay kit Pierce Chemical Dallas TX Proteins €“ μg were resolved by sodium dodecyl sulfatepolyacrylamide gel electrophoresisSDSPAGE andtransferred to polyvinylidene fluoride PVDF membranes Immunoblotting of lysates was performed withprimary antibodies Supplementary Table and afterincubation with secondary antibodies proteins were visualized using Luminata„¢ Forte Western Chemiluminescent HRP Substrate MilliporeSigma Pixel intensitiesofusingFluorChem Q Imaging software Alpha Innotech CorpSan Leandro CA Data for phosphorylated proteins arepresented as the intensity of phosphorylation normalizedto total protein expression while total protein expression was normalized to the loading control actinimmunoreactivebands werequantifiedMorphological analysis of apoptosisTC6 cells were exposed to Zyflamend for the indicated duration then washed with PBS and labeledwith Hoechst μgmlbluegreenfluorescence Hoechst binds to condensed nuclearchromatin [] and was used to visualize apoptoticcells green fluorescence by fluorescence microscopyLeica DMI8 Leica Microsystems Inc Buffalo GroveIL For each condition atleast cells werecounted Percentages of apoptotic cells were calculated relative to total cellsin PBS 0cPuckett Cell Communication and Signaling Page of MilliporeSigmaAnnexin V stainingQuantification of externalized phosphatidylserine anearly event in the apoptotic cascade was performedusing flow cytometry as previously described [] withmodification Briefly €“ confluent TC6 cellswere exposed to Zyflamend for h then washed withPBS and resuspended in μl of PBS containing FBS Immediately after an equal volume ofthe 2XGuava Nexin reagentcontainingAnnexin V Fluorescein isothiocyanate FITC and aminoactinomycin D 7AAD was added to each treatment and incubated for min at room temperatureunder lightprotected conditions Intensities of fluorescence emitted by Annexin V FITC and 7AAD weremeasured using the Guava® easyCyte Flow CytometerMilliporeSigma on PM1 and PM2 channels respectively Viable negative for both Annexin V and 7AADstaining and apoptotic cells both at early Annexin Vpositive 7AAD negative and late positive for bothAnnexin V and 7AAD stages were quantified using theInCyte„¢ and GuavaSuite Software package LuminexCorp Austin TXCell cycle analysisCell cycle analysis was conducted through assessing theDNA content of cells stained with propidium iodide aspreviously described [] with modification Briefly €“ confluent TC6 cells were starved in serummedia for h then complete growth media was addedto the cells along with various freshly prepared concentrations of Zyflamend h later cells were harvestedwashed twice with icecold with PBS and fixed overnight in ethanol at °C Next cells were washedtwice with icecold PBS and incubated in a freshly prepared RNase solution [ mM TrisHCl pH containing Uml of DNasefree RNase A AppliedBiosystems Austin TX] for min at °C Cells werewashed twice with icecold PBS and incubated in a solution of propidium iodide PI μgml in PBS overnightat °C under light protected conditions Fluorescenceintensity of PI was measured using the Guava® easyCyteflow cytometer on PM2 channel DNA histogram analysis was performed on cells using the InCyte„¢ andGuavaSuite Software package and the proportions ofcells with one or two copies of their chromosomal DNAwere calculatedStatistical analysisData were analyzed using JMP Pro program SASInstitute NC and presented as means standard errorof the mean SEM Unpaired heteroscedastic twotailStudent€™s t test was used for all statistical analyses anddifferences were considered significant at p Singlesymbol such as or €  was used to indicate a p valuethat is less than while double symbol such as or€ €  corresponds to a p value that is less than ResultsZyflamend decreases cell proliferation causes G2M cellcycle arrest and induces apoptotic cell death inpancreatic cancer cellsWe first examined the effects of varying doses of Zyflamend on the proliferation of pancreatic insulinoma TC6 cells Zyflamend caused a significant dose andtimedependent decrease in cell growth Fig 1a Additionally a Zyflamend dose of μgml was sufficient toinhibit cell proliferation by after h of treatmentwhile a dose of μgml completely abolished cell proliferation Fig 1a In line with these findings cell cycleanalysis demonstrated that Zyflamend alters cell cycledistribution in a dosedependent manner Indeed Zyflamend treatment resulted in the enrichment of the G2Mfraction with N DNA content which was accompaniedby a reduction in cell cycle progression through the G0G1 and S phases Fig 1bc These results suggest thatZyflamendinduced inhibition of cell proliferation is mediated at least in part through cell cycle arrest in theG2M phaseIn order to determine whether Zyflamendinduced inhibition of cell proliferation was associated with apoptotic cell death we determined changes in apoptosis inTC6 cells treated with increasing doses of Zyflamend and μgml for h usingtwo approaches the Guava Nexin Annexin V assay andHoechst stain Using the Annexin V assay thepercentages of both Annexin V positive7AAD negativecells reflective of early apoptotic cells and Annexin Vpositive7AAD positive cells reflective oflate apoptosis exhibited a dosedependent and significant increase in response to Zyflamend treatment Fig 1deConsistent with this observationthe number ofHoechstpositive cells was also higher in Zyflamendtreated cells compared to control cells Fig 1fgHoechst is a nucleic acid dye that binds to condensed chromatin in the nucleus of apoptotic cells thusgiving an assessment of overall apoptotic cell death []At a dose of and μgml the percentages ofapoptotic cells were ± ± and ± respectively further emphasizing the proapoptotic effects of Zyflamend on these cells Similarfindings were obtained using the MTT assay Fig 1hA human equivalent dose of Zyflamend induces apoptoticcell death in TC6 cellsTo further characterize the proapoptotic properties ofZyflamend we conducted a time course analysis using aphysiological relevant fixed dose of Zyflamend μgml [] This dose is representative of the maximum 0cPuckett Cell Communication and Signaling Page of Fig Zyflamend Reduces Cell Survival and Induces Cell Death of Pancreatic Cancer Cells in a Dose Dependent Manner a Effects of Zyflamendon cell survival and proliferation cells were treated with increasing doses of Zyflamend for h Line graphs represent the intensity of SRBstaining reflective of the cell number and presented as means SEM bc Cell cycle analysis and assessment of DNA content in TC6 cellstreated with DMSO control or the indicated concentration of Zyflamend for h Representative histogram distributions for each treatment areshown c Bar graphs represent the percentages of cells in each phase of the cell cycle which were estimated using the GuavaSuite Softwarepackage and are presented as means SEM from three independent experiments p p indicate significant difference betweenthe indicated concentration and control cells treated with the vehicle DMSO de Zyflamend treatment induces apoptosis in TC6 Cells confluent cells were treated with increasing concentrations of Zyflamend and then labeled with Annexin VFITC and 7AAD Representative dotplots are shown Annexin V positive and 7AAD negative cells lower right quadrants represent early stages of apoptosis whereas cells that arepositive for both Annexin V and 7AAD upper right quadrants are in late stages of apoptosis e Bar graphs represent live early and lateapoptotic cells are presented as means SEM of at least three independent experiments p p indicate significant differencebetween the indicated concentration of Zyflamend and control cells treated with the vehicle DMSO fg Chromatin condensation in cells treatedwith increasing doses of Zyflamend for h Representative images are shown Scale bar μm g Bar graphs represent the number of apoptoticcells Hoechst positive as means SEM of at least three independent experiments h Cell toxicity assay using the MTT method Bar graphsrepresent the intensity of formazan produced from MTT by viable cells staining reflective of the cell number and presented as means SEM ofat least three independent experiments In g and h p p indicate a significant difference between cells treated with Zyflamendand nontreated cellstreatmentplasma concentration of a primary ingredient of Zyflamend curcumin that was reported in humans after oraladministration [] At this dose a marked increase inchromatin condensation and apoptotic cell number wasobserved after h ofFig 2ab Subsequently markers of apoptosis and cell survival were investigated using Western blotting Zyflamend inducedcleavage of caspase3 and its downstream target polyADPribose polymerase PARP Fig 2cd In additionwe examined changes in the mitogenactivated proteinkinases MAP kinases pathways in response to Zyflamend Our data revealed that TC6 cells treated withZyflamend exhibited a marked decrease in the phosphorylation of protein kinase B AKT and extracellularsignalregulated kinases ERK particularly after h oftreatmentTo determine whether Zyflamendinduced cell deathwas associated with the caspase dependent pathwaysof apoptosis we tested whether blocking caspases€™activity usingcarbobenzoxyvalylalanylaspartyl[Omethyl]fluoromethylketone ZVADfmk could inhibit Zyflamendinduced chromatin condensation andapoptosis ZVADfmk is a potent cell permeable pancaspase inhibitor which acts by irreversibly bindingto the catalytic site of the caspase proteases and thusinhibiting their activities Our study shows that pretreatment with ZVADfmk caused a significant decreased in the levels of chromatin condensation inZyflamendtreated cells Fig 2ef Additionally ZVADfmk treatment alleviated Zyflamendinduced celltoxicity as judged by the MTT Fig 2g and the colony formation Fig 2hi assays Taken together ourfindings indicate that Zyflamend treatment reducescell viability and induces cell death through the induction of the apoptotic machinery in TC6 cellsZyflamend induces ER stress apoptosis and autophagyresponses in TC6 cellsA plethora of intrinsic and extrinsic pathways can leadto apoptosis in response to stressors including ER stressand autophagy among many more Therefore in orderto dissect the precise molecular mechanism mediatingthe proapoptotic effects of Zyflamend we examined theactivation of key signaling molecules related to thesepathways Zyflamend μgml significantly inducedER stressactivation of ERjudged byasthe 0cPuckett Cell Communication and Signaling Page of Fig See legend on next page 0cPuckett Cell Communication and Signaling Page of See figure on previous pageFig Zyflamend Induces Apoptotic Cell Death in TC6 Cells ab Effects of Zyflamend on chromatin condensation Cells were treated withZyflamend μgml for the indicated time and chromatin condensation was evaluated by fluorescence microscopy using Hoechst Representative images are shown Scale bar μm b Bar graphs represent the number of apoptotic cells Hoechst positive as means SEMp indicates a significant difference between cells treated with Zyflamend and nontreated cells cd Immunoblots of key proteins in cellsurvival and apoptosis markers in cells treated with μgml of Zyflamend for the indicated time d Bar graphs represent cleaved caspase3 CCasp 3actin cleaved PARP CPARPactin pAKTAKT and pERKERK as means SEM p p indicates a significant differencebetween cells treated with Zyflamend and nontreated cells ef Chromatin condensation in TC6 cells treated with μgml Zyflamend withand without the pancaspase inhibitor ZVADfmk Representative images are shown Scale bar μm f Bar graphs represent the number ofapoptotic cells Hoechst positive as means SEM g Cell toxicity assay using the MTT method Bar graphs represent the intensity of formazanstaining reflective of the cell number and presented as means SEM hi Colony formation assay i Bar graphs represent the relative number ofcolonies in each condition determined by dividing the number of colonies for a given treatment by the total number of colonies in DMSOtreated cells Ctrl and expressed as a percentage In g and i p p indicate a significant difference between cells treated withZyflamend and nontreated cells € p € € p indicate a significant difference between cells treated with ZVADfmk and nontreatedproteinCHOPby Westerntransmembrane sensors protein kinase RNAlike endoplasmic reticulum kinase PERK and inositolrequiringtransmembrane kinaseendoribonuclease 1α IRE1αalong with downstream targets such as eukaryotic translation initiation factor alpha EIF2α and CEBP homologousblottingZyflamend induced ER stress as evidenced by increasedPERK Thr980 EIF2α Ser51 and IRE1α Ser724 phosphorylation Fig 3a Furthermore the level of CHOPexpression was elevated a direct downstream target ofboth the PERK and IRE1 pathways The activation ofCHOP a potent inducer of apoptotic cell death [ ]in response to ER stress Fig 3ab strengthens our conclusions of Zyflamendinduced apoptosis in these cellsMoreover Zyflamend has been shown to activateAMPK and our results recapitulate these previous findings []The AMPK signaling pathway has been shown toinregulate autophagy and cell death [] Thereforeorder to assess whether Zyflamend induces autophagy inTC6 cells we immunoblotted for autophagyrelatedproteins We observed a time dependent increase inbeclin microtubuleassociated proteins 1A1B lightchain LC3I II and autophagyrelated proteins and ATG57 Fig 3cd The increase in the expressionof these proteins is indicative of elevated autophagy inthese cells Because ER stress inflammation and autophagy can all lead to apoptosis we used the pancaspaseinhibitor ZVADfmk to determine which pathway mightbe responsible for the proapoptotic effects of Zyflamend Our data shows that while there was a significantattenuation of Zyflamendinduced cleavage of caspase3and its downstream target PARP treatment with ZVADfmk had no effects on Zyflamendinduced activation of the AMPK autophagy and ER stress signalingcascades Fig 3ef These findings suggestthat ERstress autophagy and MAP kinases pathways are upstream of the apoptotic signaling cascade that might bemediating the proapoptotic effects of Zyflamend in TC6 cellsZyflamendinduced cell death is mediated through the ERstressJNKautophagy pathwayThe exact molecular mechanisms leading to apoptosisby Zyflamend in cancer cells hashave not been revealedyet although recent studies have supported the role ofAMPK in the regulation of cancer cell growth bioenergetics autophagy and cell death To investigate the potential role of AMPK in Zyflamendinduced apoptosiswe pretreated cells with the AMPK inhibitor compoundC CC μM for h prior to Zyflamend treatment foran additional h The dose and duration of exposurewere determined based on the ability of compound C toreverse AMPKdependent inhibitory phosphorylation ofacetylCoA carboxylase ACC data not shown Cellswere then examined for AMPK activation as well as activation of inflammation ER stress autophagy and celldeath Fig 4a While the level of phosphorylated AMPKwas reduced in cotreated cells pretreatment with compound C had no effects on Zyflamendinduced JNKphosphorylation ER stress autophagy or cell death Fig4ab These data suggest that Zyflamendinduced apoptosis in TC6 cells is independent of AMPK activationNext we sought to examine whether blocking autophagyusing 3MA could protect cells against Zyflamendinduced apoptosis 3MA inhibits autophagy by blockingautophagosome formation via the inhibition of class Iand class III phosphatidylinositol 3kinases PI3K []Cells were preincubated with 3MA nM for hprior to Zyflamend treatment Cotreatment with Zyflamend and 3MA significantly decreased the expressionof beclin LC3 and cleaved caspase3 but had no effects on ER stress markers nor on AMPK phosphorylation Fig 4cd These data suggest that autophagymediates Zyflamendinduced apoptosis and that bothAMPK and ER stress activation by Zyflamend occur upstream of autophagy and apoptosisThe relationship between these two fundamental processes ER stress and autophagy is complex and poorlyunderstood Recent literature demonstrates that bothpathways display dual roles in cell survival in multiple 0cPuckett Cell Communication and Signaling Page of Fig See legend on next page 0cPuckett Cell Communication and Signaling Page of See figure on previous pageFig Zyflamend Induces Inflammatory ER Stress and Autophagy Responses in TC6 Cells ab Total cell lysates from control and Zyflamendtreated cells for and h were immunoblotted for ER stress markers pPERK pEIF2α pIRE1α their respective unphosphorylatedproteins sXBP1 CHOP and actin as a loading control Representative immunoblots are shown b Bar graphs represent pPERKPERK pEIF2αEIF2α pIRE1IRE1 sXBP1actin and CHOPactin as means SEM p p indicate a significant difference between cells treatedwith Zyflamend and nontreated cells cd Markers of autophagy were examined in the same lysates using antibodies against Beclin LC3 IIIATG5 ATG7 and actin as a loading control d Bar graphs represent Beclin 1actin LC3actin ATG5actin and ATG7actin as means SEM p p indicate a significant difference between cells treated with Zyflamend and nontreated cells ef Immunoblots of keyproteins in autophagy AMPK ER stress and apoptosis signaling in TC6 cells treated with μgml Zyflamend with and without the pancaspase inhibitor ZVADfmk Representative immunoblots are shown f Bar graphs represent pAMPKAMPK pPERKPERK pEIF2αEIF2α pIRE1αIRE1α sXBP1actin CHOPactin pJNKJNK Beclin 1actin LC3IIIactin and cleaved caspase3actin as means SEM p p indicate a significant difference between cells treated with Zyflamend and nontreated cells € p € € p indicate a significant differencebetween cells treated with ZVADfmk and nontreated cellscancer celllines Similar to ER stress autophagy hasbeen shown to promote cell survival by clearing unwanted components from the cells Nonetheless a considerable body of evidence also indicates that bothautophagy and ER stress can lead to apoptosis in tumorcells In addition to this a growing body of literaturesupports existing crosstalk between the two pathways[ ] However which pathway is upstream of theother is yet to be determined Our data suggest thatZyflamendinduced autophagy is likely to be downstream of ER stress To test this hypothesis we pretreated TC6 cells with an ER stress inhibitor phenylbutyrate 4PBA mM for h prior to Zyflamend treatment and we examined changes in inflammation ER stress autophagy and cell death Fig 4ef PBA is a cell permeant chemical chaperone that hasbeen shown to inhibit ER stress and ER stressinducedapoptosis in many cancer cell types including pancreaticcancer cells [ ] Our findings show a profound decrease in ER stress autophagy and cell death markers inresponse to Zyflamend Fig 4ef when cells were pretreated with 4PBA Additionally 4PBA treatment alleviated the decrease in cell proliferation Fig 4g and colonycaused by ZyflamendFurthermore pretreatment of TC6 cells with 4PBAreduced Zyflamendinduced chromatin condensationFig 4jk Conversely 4PBA did not alter the activationof AMPK by Zyflamend Fig 4ef suggesting that ERstress occurs upstream of autophagy and apoptotic celldeathformation Fig 4hiPrevious studies have shown that the ER stress sensorIRE1 may promote autophagy through the TRAF2ASK1JNK pathway [ ] To test this hypothesis wetreated TC6 cells with SP600125 a selective JNK inhibitor and investigated changes in inflammation ERstress and proliferation in response to Zyflamend treatmentFig As expected cells pretreated withSP600125 exhibited a significant reduction in the phosphorylation of JNK and reduced expression of autophagyand cell death markers in response to Zyflamend Fig5ab Likewise JNK inhibition protected TC6 cellsfrom Zyflamendinduced reduction in cell survival Fig5c colo

Thyroid_Cancer

Adjunctive Therapy to Achieve Preoperative Euthyroidism in Graves™ Disease A Case Report Authors™ Contribution Study Design A Data Collection B Statistical Analysis C Data Interpretation D Manuscript Preparation E Literature Search F Funds Collection G ABDEF Noor Abdulghani AlghanimABDEF Shymaa M AlkahtaniAEF Fatimah S AssariAEF Sarah W AlnosaierAEF Reham M BaderAEF ABEF Mariam M HendazAEF Amal AlhefdhiIsra E Elmahi Corresponding Author Conflict of interest Noor Abdulghani Alghanim email nalghanimalfaisaleduNone declared College of Medicine Alfaisal University Riyadh Saudi Arabia Breast and Endocrine Surgery King Faisal Specialist Hospital and Research Center Riyadh Saudi ArabiaPatient Final Diagnosis Symptoms Medication ” Clinical Procedure Specialty Male 37yearoldGraves™ diseaseDifficulty breathing ¢ voice change ¢ weight gainTotal thyroidectomySurgery Objective Background Case Report Conclusions Unusual clinical courseGraves™ disease is an autoimmune disease of the thyroid gland and it is considered the most common cause of hyperthyroidism It is characterized by particular eye manifestations skin changes and pretibial myxedema in addition to the signs and symptoms of hyperthyroidism Graves™ disease can be diagnosed based on clinical presentation and low thyroid stimulating hormone TSH and elevated free T4 FT4 levels Presence of TSH receptor antibody TRAb in the serum confirms the diagnosis of Graves™ disease Imaging studies like radioactive iodine scan will show a high and diffuse uptake Graves™ disease can be managed with three different treatment modalities antithyroid medications radioactive iodine or surgical removal of the thyroid gland Whenever surgery is indicated careful preoperative management to achieve euthyroidism is needed to optimize the surgical outcomeThis is a case of a 37yearold Saudi male known to have Graves™ disease for years who presented to the endocrine surgery clinic with neck swelling difficulty breathing and change in voice After multiple attempts to control his fluctuating thyroid levels the team eventually managed to achieve a euthyroid state in the patient with the addition of saturated solution of potassium iodide SSKI and thus rendering him eligible for urgent surgeryWe report this case to show that SSKI can be used as adjunctive therapy to achieve a preoperative euthyroid state in refractory Graves™ disease MeSH Keywords Graves™ Disease ¢ Hyperthyroidism ¢ Hypothyroidism Fulltext PDF wwwamjcaserepcomindexidArt923342 ” e9233421Indexed in [PMC] [PubMed] [Emerging Sources Citation Index ESCI][Web of Science by Clarivate]This work is licensed under Creative Common AttributionNonCommercialNoDerivatives International CC BYNCND 0cAbdulghani Alghanim N Use of adjunctive therapy to achieve preoperative euthyroidism in Graves™ disease¦ Am J Case Rep e923342Figure Typical eye manifestations of Graves™ disease proptosis and periorbital edemaFigure Ultrasound showing an enlarged and hypervascular left thyroid lobe with no suspicious nodulesof the examination was unremarkable except for right scrotal swelling and delayed deep tendon reflexesThe patient then underwent ultrasound US of the thyroid which showed an enlarged and hypervascular gland compatible with Graves™ disease with no suspicious nodules Figure A computed tomography CT scan was done which revealed homogeneous diffuse swelling of bilateral thyroid lobes with no retrosternal extension along with bilateral proptosis Figures Therefore the patient was admitted to achieve a euthyroid state before proceeding for a total thyroidectomy The patient was managed by a multidisciplinary team with the goal of clearing him for surgery During the patient™s 3week hospital stay the dosage of methimazole was continuously altered because serial thyroid function tests showed a change from hyperthyroid to hypothyroid status His TSH and FT4 levels ranged from mUL to mUL and pmolL to pmolL respectively A few days after administration of saturated solution of potassium iodide SSKI was initiated drops three times daily the patient achieved a euthyroid state with TSH mUL and FT4 pmolL so urgent surgery was performed Intraoperatively the patient™s thyroid gland was found to be enlarged and vascular with each lobe measuring approximately to cm The gland was excised bilaterally along with the pyramidal lobe because it was also enlarged The postoperative BackgroundGraves™ disease is an autoimmune disease affecting the thyroid gland [] It is characterized by presence of autoantibodies that target thyroid stimulating hormone TSH receptors causing stimulation of the thyroid gland [] Patients with Graves™ disease usually present with signs and symptoms of hyperthyroidism that include fatigue heat intolerance sweating weight loss palpitations and tremor along with particular eye manifestations and sometimes skin changes [] It is considered the most common cause of hyperthyroidism accounting for approximately to of cases [] The diagnosis of Graves™ disease can be straightforward in the presence of typical signs and symptoms along with low thyroid stimulating hormone TSH and elevated free T4 FT4 levels [] Measuring TSH receptor antibody TRAb is helpful for confirming the diagnosis as it is present in of patients If the cause of hyperthyroidism remains uncertain a radioactive iodine uptake scan should be considered The scan helps to distinguish Graves™ disease from thyroiditis and other causes of hyperthyroidism In Graves™ disease iodine uptake is increased and diffuse [] Treatments of choice for hyperthyroidism include antithyroid medications radioactive iodine and surgical approaches [] The success rate for antithyroid medications is almost compared to and with radioactive iodine and surgery respectively [“] Whenever surgery is indicated careful preoperative management to achieve euthyroidism is needed to optimize the surgical outcome In most cases a euthyroid state is reached within a few weeks of conventional antithyroid medications however in certain conditions as in drug malabsorption and in cases of predominantly high T3 levels it cannot be easily achieved and adjunctive therapy should be considered []Case ReportA 37yearold Saudi male presented to the endocrine clinic with palpitation sweating and weight loss He was diagnosed with Graves™ disease and treated with methimazole mg orally twice daily When symptoms of hypothyroidism developed the dose was decreased to mg orally twice daily The patient was referred to the endocrine surgery clinic complaining of obstructive symptoms in the form of difficulty breathing and voice changes due to neck swelling weight gain of kg during the last month and easy fatigability along with the typical eye manifestations of proptosis and periorbital edema Figure He was otherwise healthy and the rest of his history was unremarkable On physical examination the patient had a hoarse voice fine tremor in both hands and his skin was warm with diaphoresis There was proptosis lid retraction and diplopia involving both eyes Neck examination showed a diffuse tender swelling with bilateral lumps measuring cm on the right and cm on the left along with positive Pemberton sign The rest e9233422Indexed in [PMC] [PubMed] [Emerging Sources Citation Index ESCI][Web of Science by Clarivate]This work is licensed under Creative Common AttributionNonCommercialNoDerivatives International CC BYNCND 0cAbdulghani Alghanim N Use of adjunctive therapy to achieve preoperative euthyroidism in Graves™ disease¦ Am J Case Rep e923342Figure CT scan showing diffuse enlargement of the thyroid with no retrosternal extension or invasion of surrounding structurespathology report showed diffuse hyperplasia consistent with Graves™ disease with no evidence of malignancy After the surgery the patient was moved to the Intensive Care Unit ICU where he was assessed and found to be stable with no signs and symptoms of thyrotoxicosis or hypocalcemia Three days later the patient was discharged with orders to take calcium carbonate mg orally three times daily for days acetaminophen mg orally as needed for days levothyroxine mcg orally daily for days and calcitriol mcg orally daily for daysWhen the patient presented to the clinic weeks later for followup he was found to be in good health with no active complaints He had lost weight and there were no voice changes His eye manifestations had decreased but not disappeared completely Laboratory results showed a euthyroid state with a normal calcium levelDiscussionFigure CT scan demonstrating that the distance from the anterior margin of the globe to the interzygomatic line exceeds mm indicating significant bilateral proptosisfor antithyroid medications is almost compared to with radioactive iodine therapy [] Surgical approaches are considered the most successful and definitive treatment with total thyroidectomy being the preferred choice [] A review of the literature done in showed that total thyroidectomy is times more successful than radioactive iodine therapy [] Another study concluded that the highest rates of longterm remission reaching up to are achieved with surgery [] Nonetheless there is no clear consensus on the best treatment modality for Graves™ disease and the choice should be individualized Choice of modality depends on several factors including age comorbidities size of the goiter and severity of thyrotoxicosis [] Surgery is recommended in certain conditions for example in patients with compression symptoms due to presence of a large goiter those with low radioactive iodine uptake suspected thyroid cancer moderate to severe Graves™ ophthalmopathy and patients who cannot tolerate antithyroid medications [] Whenever surgery is selected careful preoperative management is needed to optimize the surgical outcome Preparing a patient with antithyroid medications is recommended by the American Thyroid Association ATA to achieve a euthyroid state and thus lower risk of intraoperative complications []Graves™ disease can be managed with three different treatment modalities antithyroid medications radioactive iodine or surgical removal of the thyroid gland [] The success rate In most cases a euthyroid state is achieved within weeks of antithyroid treatment In certain conditions however that is difficult to achieve with conventional therapy and patients e9233423Indexed in [PMC] [PubMed] [Emerging Sources Citation Index ESCI][Web of Science by Clarivate]This work is licensed under Creative Common AttributionNonCommercialNoDerivatives International CC BYNCND 0cAbdulghani Alghanim N Use of adjunctive therapy to achieve preoperative euthyroidism in Graves™ disease¦ Am J Case Rep e923342Moreover the largest case series of patients with severe thyrotoxic Graves™ disease was published in The study involved patients who reached euthyroidism after days of an intensive treatment regimen The authors concluded that patients with severe hyperthyroid Graves™ disease can rapidly achieve preoperative euthyroidism with simultaneous administration of iopanoic acid dexamethasone betablocker and methimazole or propylthiouracil [] Another case of Graves™ disease resistant to antithyroid medications was reported in The patient was promptly managed preoperatively with both iopanoic acid and dexamethasone []Three scientific papers on resistant thyrotoxicosis due to Graves™ disease were published between to In all cases a euthyroid state could not be reached with the usual antithyroid medications and the patients received prednisolone andor lithium which resulted in complete normalization of thyroid function before surgery [“] Furthermore several refractory cases of Graves™ disease unresponsive to usual preoperative management were reported in and The patients were successfully prepared for surgery with use of plasmapheresis []ConclusionsPreoperative management of Graves™ disease can sometimes be challenging There have been many attempts to achieve a euthyroid state with different approaches In the patient described here Graves™ disease was resistant to conventional antithyroid medication for establishment of preoperative euthyroidism Our experience demonstrates that SSKI can be used in a case like ours to not only decrease vascularity of the thyroid gland but also as adjunctive therapy to achieve preoperative euthyroidismshould be prepared for surgery using adjunctive therapy [] Resistance to conventional antithyroid medications is not commonly encountered in clinical practice however there are few reported cases addressing the use of adjunctive therapy to rapidly restore normal thyroid function [] SSKI has been used for many years in management of Graves™ disease [] ATA hyperthyroidism management guidelines recommend preoperative administration of potassium iodide solutions KI for thyroidectomy [] The main rationale of using KI preoperatively is to decrease vascularity and blood loss during the surgery however a few studies suggest that when combined with antithyroid medications it can decrease thyroid hormone levels [“] In a retrospective study showed the effectiveness of adding KI as a rescue preoperative management in uncontrolled Graves™ disease In patients in the study use of KI was safe and effective as preoperative preparation for total thyroidectomy []Cholestyramine was first used to treat Graves™ disease in in Korea [] The patient in that study was a 22yearold female with severe refractory Graves™ disease who was initially managed with a maximal dose of methimazole and propranolol with no improvement She was admitted and treated with methimazole propranolol hydrocortisone and KI The next day cholestyramine was added which resulted in a rapid decline of FT4 Ten days after admission the patient underwent total thyroidectomy [] Several cases of refractory Graves™ disease were reported in the literature between to In all these cases the patients failed to achieve a preoperative euthyroid state with conventional antithyroid medications Within to weeks of administration of cholestyramine as adjunctive therapy they became euthyroid Two studies were published in and to evaluate the effectiveness of adding cholestyramine to the conventional treatment regimen in cases of resistant Graves™ disease The conclusion from these reports is that cholestyramine can be used to safely and rapidly achieve preoperative euthyroidism [“]References Pokhrel B Bhusal K Graves disease In StatPearls Treasure Island FL StatPearls Publishing Barbesino G Tomer Y Clinical review Clinical utility of TSH receptor antibodies J Clin Endocrinol Metab “ DeGroot LJ Graves™ disease and the manifestations of thyrotoxicosis In Feingold KR Anawalt B Boyce A eds Endotext South Dartmouth MA MDTextcom Inc Subekti I Pramono LA Current diagnosis and management of Graves™ disease Acta Med Indones Girgis CM Champion BL Wall JR Current concepts in Graves™ disease Ther Adv Endocrinol Metab “ Wiersinga WM Graves™ disease Can it be cured Endocrinol Metab Seoul “ Wong KK Shulkin BL Gross MD Avram AM Efficacy of radioactive iodine treatment of Graves™ hyperthyroidism using a single calculated I dose Clin Diabetes Endocrinol Piantanida E Preoperative management in patients with Graves™ disease Gland Surg “ Yang Y Hwang S Kim M Refractory Graves™ disease successfully cured by adjunctive cholestyramine and subsequent total thyroidectomy Endocrinol Metab Seoul “ Genovese BM Noureldine SI Gleeson EM What is the best definitive treatment for Graves™ disease A systematic review of the existing literature Ann Surg Oncol “ Bartalena L Diagnosis and management of Graves disease A global overview Nat Rev Endocrinol “ SebastianOchoa A QuesadaCharneco M FernandezGarcia D Dramatic response to cholestyramine in a patient with Graves™ disease resistant to conventional therapy Thyroid “ Calissendorff J Falhammar H Lugol™s solution and other iodide preparations Perspectives and research directions in Graves™ disease Endocrine e9233424Indexed in [PMC] [PubMed] [Emerging Sources Citation Index ESCI][Web of Science by Clarivate]This work is licensed under Creative Common AttributionNonCommercialNoDerivatives International CC BYNCND 0cAbdulghani Alghanim N Use of adjunctive therapy to achieve preoperative euthyroidism in Graves™ disease¦ Am J Case Rep e923342 Naafs MA Lugol™s solution in thyroid surgery A minireview Global Journal of Otolaryngology Muldoon BT Mai VQ Burch HB Management of Graves™ disease An overview and comparison of clinical practice guidelines with actual practice trends Endocrinol Metab Clin North Am “ Burch HB Cooper DS Management of Graves disease A review [published erratum appears in JAMA “] JAMA Calissendorff J Falhammar H Rescue preoperative treatment with Lugol™s solution in uncontrolled Graves™ disease Endocr Connect “ Chae SB Kim ES Lee YI Min BR A case of methimazoleresistant severe Graves™ disease Dramatic response to cholestyramine Int J Thyroidol “ Kadem SG Resistant hyperthyroidism responses dramatically to adjunctive oral cholestyramine Jourbnal of Diabetes and Endorinology “ Mercado M MendozaZubieta V BautistaOsorio R EspinozaDe Los Monteros AL Treatment of hyperthyroidism with a combination of methimazole and cholestyramine J Clin Endocrinol Metab “ Tsai WC Pei D Wang TF The effect of combination therapy with propylthiouracil and cholestyramine in the treatment of Graves™ hyperthyroidism Clin Endocrinol Oxf “ Panzer C Beazley R Braverman L Rapid preoperative preparation for severe hyperthyroid Graves™ disease J Clin Endocrinol Metab “ Pandey CK Raza M Dhiraaj S Rapid preparation of severe uncontrolled thyrotoxicosis due to Graves™ disease with Iopanoic acid “ a case report Can J Anaesth “ Saleem T Sheikh A Masood Q Resistant thyrotoxicosis in a patient with Graves disease A case report J Thyroid Res Nair GC C Babu MJ Menon R Jacob P Preoperative preparation of hyperthyroidism for thyroidectomy “ role of supersaturated iodine and lithium carbonate Indian J Endocrinol Metab “ Jude EB Dale J Kumar S Dodson PM Treatment of thyrotoxicosis resistant to carbimazole with corticosteroids Postgrad Med J “ Candoni A De Marchi F Vescini F Graves™ disease thyrotoxicosis and propylthiouracil related agranulocytosis successfully treated with therapeutic plasma exchange and GCSF followed by total thyroidectomy Mediterr J Hematol Infect Dis Ezer A Caliskan K Parlakgumus A Preoperative therapeutic plasma exchange in patients with thyrotoxicosis J Clin Apher “e9233425Indexed in [PMC] [PubMed] [Emerging Sources Citation Index ESCI][Web of Science by Clarivate]This work is licensed under Creative Common AttributionNonCommercialNoDerivatives International CC BYNCND 0c'

Thyroid_Cancer

case of a 14year old boy with tumorassociated refractory epilepsy Positron emission tomography imaging demonstrated a region with heterogeneous high 11Cmethionine uptake and a region with homogenous low 18Ffluorodeoxyglucose uptake within the tumor Histopathological and genomic analyses confirmed the tumor as BRAF V600Emutated polymorphous lowgrade neuroepithelial tumor of the young PLNTY Within the highmethionineuptake region we observed increased protein levels of Ltype amino acid transporter LAT1 a major transporter of methionine cMyc and constituents of the mitogenactivated protein kinase MAPK pathway We also found that LAT1 expression was linked to the BRAF V600E mutation and subsequent activation of MAPK signaling and cMyc Pharmacological and genetic inhibition of the MAPK pathway suppressed cMyc and LAT1 expression in BRAF V600Emutated PLNTY and glioblastoma cells The BRAF inhibitor dabrafenib moderately suppressed cell viability in PLNTY Collectively our results indicate that BRAF V600E mutationactivated MAPK signaling and downstream cMyc induces specific metabolic alterations in PLNTY and may represent an attractive target in the treatment of the diseaseKeywords PLNTY BRAF V600E mutation Methionine PET LAT1IntroductionPediatric lowgrade neuroepithelial tumors PLGNTs encompass a group of central nervous system neoplasms that longterm epilepsyassociated tumors LEATs such as ganglioglioma and dysembryoplastic neuroepithelial tumor DNT PLGNTs have different characteristics than their adult counterparts and includes Correspondence ktate12yokohamacuacjp Department of Neurosurgery Graduate School of Medicine Yokohama City University Fukuura Kanazawa Yokohama JapanFull list of author information is available at the end of the are commonly driven by genomic alterations in the Rasmitogenactivated protein kinase MAPK pathway such as mutations in BRAF and NF1 [ ] Recent largescale genomic studies and genomewide methylation analyses allowed a thorough characterization of PLGNTs [] and cIMPACTNOW the Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy currently classifies P“LGNTs as distinct disease entities [ ] In Huse et a0al described ten cases of polymorphous lowgrade neuroepithelial tumor of the young PLNTY which were histologically characterized by oligodendrogliomalike cellular components with intense CD34 immunopositivity According to previous publications PLNTYs are indolent tumors The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cTateishi a0et a0al acta neuropathol commun Page of that generally exhibit a benign clinical course and harbor either a BRAF V600E mutation or FGFR2FGFR3 fusion [] Based on its histological and genomic profiles cIMPACTNOW Update recommends PLNTY as a possible future classification for pediatrictype glialglioneuronal tumors However because of their rare etiology only a few PLNTYs have been described to date [ ] and it is unclear how genomic alterations promote the pathogenesis of the disease Herein we present a case of PLNTY with unique metabolic imaging features Using positron emission tomography PET we found regions of heterogeneous high 11Cmethionine uptake and homogenous low 18Ffluorodeoxyglucose FDG uptake within the tumor Activation of the MAPK pathway cMyc and expression of Ltype amino acid transporter LAT1 were increased in the highmethionineuptake area compared with the surrounding cortex lowmethionineuptake Glycolytic metabolites were expressed only weakly in tumor cells Pharmacological and genetic inhibition of the MAPK pathway suppressed cMyc and LAT1 and inhibited tumor cell viability suggesting that MAPK signaling and downstream cMyc activates methionine metabolism and inhibition of this pathway induces therapeutic vulnerability in PLNTYMaterials and a0methodsCell viability analysisAM38 and normal human astrocytes was purchased from JCRB Cell Bank and ScienCell Research Laboratories respectively Tumorsphere lines were cultured in serumfree neural stem cell medium as previously described [] Normal human astrocytes were cultured with astrocyte medium ScienCell To assess cell viability primary cultured cells were dissociated into single cells and seeded into 96well plates at a density of cellswell After a0h dabrafenib Selleck and trametinib Selleck were serially diluted and added to the wells Cell viability was measured using the CellTiterGlo Promega assay at day and the results were indicated as viability of the DMSO controlshRNA cell line generationTo knockdown BRAF 293T cells were transfected with lentiviral vector packaging plasmid DNA containing a0 μg of Human BRAF shRNA TRCN0000381693 GP and a0μg of a0pVSVgRev a0with Lipofectamine„¢ TRCN0000196844 Sigma Aldrich a0μg of a0pHIVThermo Fisher Scientific YMG62 and AM38 cells were infected with lentivirus in polybrene a0μgmL for a0h Two days later the cells were selected with puromycin a0μgmL for a0days and used for experiments GIPZ nonsilencing lentiviral shRNA Control RHS4348 Horizon Discovery was used as a nonsilencing NS controlImmunohistochemistryTumor tissue specimens were fixed in neutral buffered formalin and embedded in paraffin Hematoxylin and eosin staining was performed using standard procedures For immunohistochemical analysis 5µmthick sections were deparaffinized treated with H2O2 in methanol rehydrated and heated for a0min for antigen retrieval After blocking with serum tissue sections were incubated with primary antibodies against CD34 Novus Biologicals LAT1 Cell Signaling Technology phosphoMEK Cell Signaling Technology phosphoERK Bethyl Laboratories and cMyc Cell Signaling Technology at a0°C overnight The next day sections were washed with PBS incubated with biotinylated secondary antibody for a0 min at room temperature and then incubated with ABC solution PK6101 PK6102 Vector laboratories for a0 min at room temperature Finally the sections were incubated with DAB Dako and counterstained with hematoxylinWestern blottingcOmplete„¢ Mini EDTAfree Protease Inhibitor Cocktail Cells were lysed in RIPA buffer SigmaAldrich with a Roche Fifty micrograms of protein was separated by SDSPAGE gel and transferred to polyvinylidene difluoride membranes Millipore by electroblotting After blocking with or nonfat dry milk in TBST a0mM Tris [pH ] a0 mM NaCl Tween20 membranes were incubated at a0 °C overnight with primary antibodies After washing and incubation with horseradish peroxidaseconjugated secondary antibodies Cell Signaling Technology blots were washed and signals were visualized with chemiluminescent HRP substrate Millipore Primary antibodies against BRAF Gene Tex cMyc Cell Signaling Technology GAPDH Gene Tex LAT1 Cell Signaling Technology phosphoMEK Cell Signaling Technology a0phosphoERK Bethyl Laboratories and Vinculin Novus Biologicals were used for western blottingCase presentationThis study was performed in accordance with declaration of Helsinki and was approved by the Institutional Review Board Yokohama City University [YCU Yokohama Japan] IRB numbers A1711300006 and B190600002 Written informed consent was obtained from the patient and parents A 14year old boy presented with chronic medial temporal lobe epilepsy for a year Magnetic resonance imaging MRI indicated 0cTateishi a0et a0al acta neuropathol commun Page of See figure on next pageFig Characteristics of a patient with PLNTY a T2weighted left T1weighted middle and contrastenhanced right MR images b Computed tomography CT left 18FfluorodeoxyglucosePETCT middle and 11CmethioninePETCT right images c Video electroencephalography indicating ictal onset in the left temporal lobe with spread to the contralateral temporal lobe d PETCT and MRI merged intraoperative navigation image left and surgical image right showing the highmethionineuptake region and surrounding abnormal lesion on MRIhypointensity on T2weighted images and hyperintensity on T1weighted images with a cystic component in the left temporal lobe Contrastenhanced MRI showed no significant enhancement in the lesion Fig a01a while computed tomography revealed heavy calcification FDGPET showed lower FDG uptake in the tumor while 11CmethioninePET demonstrated increased methionine uptake in the same lesion SUVmax tumornormal tissue ratio Fig a01b Videoelectroencephalographic EEG monitoring indicated ictal onset in the left temporal lobe with subsequent spread to the contralateral temporal lobe Fig a01c We speculated that this abnormal lesion was a LEAT Since we considered this tumor to be completely resectable the patient underwent craniotomy and resection of the neoplasm including the highmethionineuptake region Fig a01d To achieve epileptic control electrocorticography was performed intraoperatively After removal of the highmethionineuptake and T2 hyperintense lesions the surrounding tissue was resected until interictal epileptiform discharge could no longer be detected by electrocorticography The patient became epilepsyfree after lesion removal and MRI indicated complete remission a0months after the surgeryTissue samples of the highmethionineuptake region and surrounding cortex low methionine uptake were collected Hematoxylin and eosin staining indicated diffusely infiltrating growth patterns and presence of oligodendroglialike cellular components Fig a02a Astrocytic and highgrade features were absent with a Ki67 index of less than Chicken wirelike branching capillaries and microcalcification were also found in region Despite lower cellularity oligodendroglialike cells were present in the surrounding tissue Immunohistochemistry revealed extensive CD34 expression and peripherally associated ramified neural elements in the tumor cells Fig a0 2a Targeted DNA sequencing identified a BRAF V600E mutation in the tumor without recurrent mutations in IDH1 IDH2 TERT promoter FGFR1 H3F3A or HIST3H1B Fig a0 2b Chromosome 1p19q codeletion was absent Fig a02c The above histological and genetic features fulfilled the diagnostic criteria for PLNTYTo assess the mechanisms underlying the methionineFDG uptake mismatch indicated by PET we compared the expression of LAT1 glucose transporter GLUT and hexokinase2 HK2 between tissue regions and Notably LAT1 which is a major methionine transporter was more highly expressed in than in Fig a0 3a In contrast GLUT1 and HK2 which is correlated with FDG uptake and lactate dehydrogenase A LDHA expression were weak in either region Additional file a0 Fig a0 S1 LAT1 expression is mediated by cMyc activation and BRAF V600E mutation activates the MAPK pathway and downstream cMyc [ ] Therefore we hypothesized that BRAF V600E mutation promotes LAT1 expression through MAPK signaling and consequent cMyc activation a0 in PLNTY Levels of phosphoMEK phosphoERK and cMyc were higher in tissue region than in Fig a03a suggesting activation of the MAPK pathway and cMyc within the highmethionineuptake lesion To verify whether the BRAF V600E mutation can induce the expression of LAT1 we exposed primary cultured YMG83 PLNTY cells to a BRAF inhibitor dabrafenib As expected the expression of phosphoMEK phosphoERK cMyc and LAT1 was suppressed after dabrafenib treatment in YMG83 cells Fig a0 3b Notably BRAF inhibitor dabrafenibtreated YMG83 cells had lower cell viability compared to normal human astrocytes Fig a03c To confirm the reproducibility of these molecular features we used patientderived YMG62 cells epithelioid glioblastoma with the BRAF V600E mutation which exhibited high 11Cmethionine uptake by PET imaging Additional file a0 Fig a0 S2 and AM38 glioblastoma cells BRAF V600E mutant We found that dabrafenib and a MEK inhibitor trametinib inhibited the expression of proteins in the MAPK pathway as well as cMyc and LAT1 Fig a03d and 3e Similarly BRAF knockdown suppressed the expression of proteins in the MAPK pathway as well as cMyc and LAT1 Fig a03f Collectively these findings indicated that activation of the MAPK pathway by the BRAF V600E mutation deregulates cMyc and promotes LAT1 expression This oncogenic signaling pathway increases methionine metabolism and tumor maintenance in PLNTYDiscussionThirty cases of PLNTY have been described to date with the first ten reported by Huse et a0al in [ ] BRAF V600E mutation was seen in of the patients and BRAF fusion in patient These BRAF alterations were mutually exclusive with other genomic events including FGFR3TACC3 fusion FGFR3 amplification FGFR2CTNNA3 fusion FGFR2INA fusion 0cTateishi a0et a0al acta neuropathol commun Page of 0cTateishi a0et a0al acta neuropathol commun Page of Fig Histopathologic and genomic features of a patient with PLNTY a Hematoxylin and eosin HE staining top and CD34 immunohistochemistry bottom in the highmethionineuptake and lowmethionineuptake region within tumor tissue Bars μm b Sanger sequencing for detection of BRAF V600E arrow left and IDH1 R132H arrow right mutations c Fluorescence in situ hybridization for detection of 1p311q25 left and 19q1319p13 right chromosomal deletionsFGFR2 KIAA1598 fusion FGFR2 rearrangement and NTRK2 disruption suggesting that the vast majority of PLNTYs are induced by BRAF mutation or FGFR fusion and subsequent MAPK activation Therefore targeting MAPK signaling may become a potential therapeutic strategy in PLNTY Indeed BRAF V600Emutated PLNTY cells were relatively vulnerable to dabrafenib and trametinib in the present study Thus targeted molecular therapy for the MAPK pathway may be particularly useful in PLNTY located in surgically unresectable regions In addition Koh et a0 al reported that the BRAF V600E mutation contributes to the intrinsic epileptogenicity in pediatric brain tumors and that inhibition of BRAF suppressed epileptic seizures [] Thus BRAFMEK inhibitors could exert antiepileptic as well as antitumor effects in PLNTYPET imaging revealed a region with increased methionine uptake and low FDG uptake within tumor tissue in our patient Consistent with this finding previous case reports demonstrated increased methionine uptake but only mild FDG uptake in patients with BRAF V600Emutated PLNTY [ ] Thus excessive 0cTateishi a0et a0al acta neuropathol commun Page of Fig Activating the MAPK pathway induces LAT1 expression in a patient with PLNTY a Immunohistochemistry of indicated proteins in the highmethionineuptake and lowmethionineuptake regions within tumor tissue Bars μm b Western blot analysis of phosphoMEK phosphoERK cMyc and LAT1 proteins in YMG83 PLNTY left cells treated with DMSO and μM BRAF inhibitor BRAFi dabrafenib for h GAPDH loading control c Relative cell viability of dabrafenibtreated left and trametinibtreated right YMG83 cells and immortalized normal human astrocytes NHA P DMSO versus dabrafenib left and trametinib right d Western blot analysis for indicated proteins in YMG62 epithelioid glioblastoma left and AM38 glioblastoma right cells treated with DMSO μM BRAF inhibitor BRAFi dabrafenib and μM MEK inhibitor MEKi trametinib for h GAPDH loading control e Western blot analysis of BRAF phosphoMEK phosphoERK cMyc and LAT1 proteins in YMG62 left and AM38 right cells treated with DMSO and dabrafenib at indicated concentrations Vinculin loading control f Western blot analysis for indicated proteins in nonsilencing NS and BRAF and transduced YMG62 and AM38 cells GAPDH loading controlmethionine uptake and low FDG uptake may be imaging features specific to PLNTY A preclinical study has demonstrated that high uptake of 18FFDG was correlated with increased Glut1 and HK2 expression in human cancers [] Although the diagnostic accuracy is insufficient FDGPET imaging is useful to differentiate highgrade from lowgrade gliomas [] In the present case low FDG uptake and weak expression of Glut1 HK2 and LDHA were observed in tumor tissue suggesting low glycolytic activity in PLNTY On the other hand due to a high signaltonoise ratio 11Cmethionine PET imaging is practical for brain tumors [ ] Several PET imaging studies have demonstrated that methionine uptake was higher in 0cTateishi a0et a0al acta neuropathol commun Page of highgrade adult gliomas than in lowergrade gliomas [ ] In epileptogenic brain tumors however all gangliogliomas and “ of DNT had increased methionine uptake although these tumors are classified as WHO grade I [ ] implying that methionine uptake may be irrespective of tumor grade in LEATsPrevious studies have reported that methionine uptake was correlated with LAT1 in gliomas [ ] LAT1 plays a major role in the transport of neutral essential amino acids including methionine and is driven by several cancerrelated genes such as MYC [] It has been demonstrated that cMyc which is partly mediated by the MAPK pathway regulates LAT1 expression and MEK inhibitor suppresses LAT1 SLC7A5 transcription [ ] thereby indicating a role of the MAPK pathway and cMyc in the regulation of LAT1 Since RASMAPK pathwayassociated genomic alterations are common in LEATs [] and that the BRAF V600E mutation has been identified in “ and of gangliogliomas and DNTs respectively [ ] there is a possibility that the BRAF V600E mutation and MAPK pathwayrelated genomic alterations may activate methionine metabolism in LEATs To investigate this hypothesis we evaluated the protein expression of LAT1 and the molecules that are involved in the MAPK pathway As expected levels of phosphoMEK phosphoERK cMyc and LAT1 were higher in the highmethionineuptake area than in the lowmethionineuptake area We also found that genetic andor pharmacological BRAF inhibition suppressed MAPK pathway activation and attenuated LAT1 expression in BRAF V600EmutatedPLNTY cells and glioblastoma cell lines These findings support the hypothesis that the BRAF V600E mutation may upregulate LAT1 and methionine metabolism through cMyc activation for cell survival In addition to LAT1 methionine uptake was correlated with microvascular density MVD in gliomas [] PLNTYs are considered benign brain neoplasms proposed as WHO grade I however in the present case a chicken wirelike MVD which is one of the histopathological characteristics of oligodendroglioma was also observed in the highmethionineuptake tissue region Intriguingly methionine uptake has been reported to be relatively higher in oligodendrogliomas than in astrocytomas [] Thus PLNTY which has an oligodendrogliomalike microvascular structure might show unique metabolic imaging features Further studies are warranted to validate this hypothesis Nonetheless our data indicated that the BRAF V600E mutation induced MAPK pathway activation and downstream cMyc promoted LAT1 expression and methionine metabolism with little effect on glycolytic pathway activation These findings may explain the unique metabolic imaging features of FDGmethionine mismatch in PLNTYSupplementary informationSupplementary information accompanies this paper at https doi101186s4047 Additional file a0 a0Figure S1 Low glycolysis activation in a patient with PLNTY Immunohistochemistry for glucose transporter hexokinase and lactate dehydrogenase A in the highmethionineuptake upper and lowmethionineuptake lower region within tumor tissue A Bars μm Figure S2 Images of the patient™s glioblastoma with the BRAF V600E mutation Contrastenhanced magnetic resonance left and 11Cmethionine positron emission tomography right images of the YMG62 patientAcknowledgementsWe thank Mrs Emi Hirata and Yasuko Tanaka YCU for technical and administrative assistance We also would like to thank Editage wwweditagecom for English language editingAuthors™ contributionsKT led the study collected samples designed experiments performed experiments interpreted data and wrote the manuscript JS TH and YM performed experiments NI HM provided tumor samples and associated clinical details TO RM and DU interrupted PET and MRI studies NU and SY performed the histological classification of tumor samples TY designed experiments and interpreted data All authors read and approved the final manuscriptFundingThis work was supported by GrantAid for Scientific Research 19K09488 Princess Takamatsu Cancer Research Fund Takeda Science Foundation SGH Cancer foundation Yokohama Foundation for Advancement of Medical Science and BristolMyers Squibb FoundationCompeting interestsThe authors declare that they have no competing interestsAuthor details Department of Neurosurgery Graduate School of Medicine Yokohama City University Fukuura Kanazawa Yokohama Japan Department of Pathology Yokohama City University Hospital Yokohama Japan Department of Radiology Graduate School of Medicine Yokohama City University Yokohama Japan Departmento of Radiology Division of Nuclear Medicine National Center for Global Health and Medicine Tokyo Japan Received June Accepted August References Borbely K Nyary I Toth M Ericson K Gulyas B Optimization of semiquantification in metabolic PET studies with 18Ffluorodeoxyglucose and 11Cmethionine in the determination of malignancy of gliomas J Neurol Sci “ https doi101016jjns200602015 Chappe C Padovani L Scavarda D Forest F NanniMetellus I Loundou A Mercurio S Fina F Lena G Colin C et al Dysembryoplastic neuroepithelial tumors share with pleomorphic xanthoastrocytomas and gangliogliomas BRAFV600E mutation and expression Brain Pathol “ https doi101111bpa12048 Chen Y Tian T Guo X Zhang F Fan M Jin H Liu D Polymorphous lowgrade neuroepithelial tumor of the young case report and review focus on the radiological features and genetic alterations BMC Neurol https doi101186s1288 Ellison DW Hawkins C Jones DTW OnarThomas A Pfister SM Reifenberger G Louis DN cIMPACTNOW update diffuse gliomas 0cTateishi a0et a0al acta neuropathol commun Page of characterized by MYB MYBL1 or FGFR1 alterations or BRAFV600E mutation Acta Neuropathol “ https doi101007s0040 Gupta VR Giller C Kolhe R Forseen SE Sharma S Polymorphous lowgrade neuroepithelial tumor of the young a case report with genomic findings World Neurosurg “ https doi101016jwneu201908221 Hafliger P Graff J Rubin M Stooss A Dettmer MS Altmann KH Gertsch J Charles RP The LAT1 inhibitor JPH203 reduces growth of thyroid carcinoma in a fully immunocompetent mouse model J Exp Clin Cancer Res https doi101186s1304 60180907z Hatakeyama T Kawai N Nishiyama Y Yamamoto Y Sasakawa Y Ichikawa T Tamiya T 11Cmethionine MET and 18Ffluorothymidine FLT PET in patients with newly diagnosed glioma Eur J Nucl Med Mol Imaging “ https doi101007s0025 Hayashi K Jutabha P Endou H Anzai N cMyc is crucial for the expression of LAT1 in MIA Paca2 human pancreatic cancer cells Oncol Rep “ https doi103892or20121878 Huse JT Snuderl M Jones DT Brathwaite CD Altman N Lavi E Saffery R SextonOates A Blumcke I Capper D et al Polymorphous lowgrade neuroepithelial tumor of the young PLNTY an epileptogenic neoplasm with oligodendrogliomalike components aberrant CD34 expression and genetic alterations involving the MAP kinase pathway Acta Neuropathol “ https doi101007s0040 Johnson DR Giannini C Jenkins RB Kim DK Kaufmann TJ Plenty of calcification imaging characterization of polymorphous lowgrade neuroepithelial tumor of the young Neuroradiology “ https doi101007s0023 y Kato T Shinoda J Oka N Miwa K Nakayama N Yano H Maruyama T Muragaki Y Iwama T Analysis of 11Cmethionine uptake in lowgrade gliomas and correlation with proliferative activity AJNR Am J Neuroradiol “ https doi103174ajnrA1242 Katsanos AH Alexiou GA Fotopoulos AD Jabbour P Kyritsis AP Sioka C Performance of 18FFDG 11Cmethionine and 18FFET PET for glioma grading a metaanalysis Clin Nucl Med “ https doi101097RLU00000 dysembryoplastic neuroepithelial tumors and other epileptogenic brain neoplasms with [11C]methionine PET Neuro Oncol “ https doi101093neuon cnou02 Riva G Cima L Villanova M Ghimenton C Sina S Riccioni L Munari G Fassan M Giangaspero F Eccher A Lowgrade neuroepithelial tumor unusual presentation in an adult without history of seizures Neuropathology “ https doi101111neup12504 Rosenberg DS Demarquay G Jouvet A Le Bars D Streichenberger N Sindou M Kopp N Mauguiere F Ryvlin P [11C]Methionine PET dysembryoplastic neuroepithelial tumours compared with other epileptogenic brain neoplasms J Neurol Neurosurg Psychiatry “ https doi101136jnnp200405160 Ryall S Tabori U Hawkins C Pediatric lowgrade glioma in the era of molecular diagnostics Acta Neuropathol Commun https doi101186s4047 z Ryall S Zapotocky M Fukuoka K Nobre L Guerreiro Stucklin A Bennett J Siddaway R Li C Pajovic S Arnoldo A et al Integrated molecular and clinical analysis of pediatric lowgrade gliomas Cancer Cell “583e565 https doi101016jccell Salisbury TB Arthur S The regulation and function of the ltype amino acid transporter LAT1 in cancer Int J Mol Sci https doi103390ijms1 Schindler G Capper D Meyer J Janzarik W Omran H HeroldMende C Schmieder K Wesseling P Mawrin C Hasselblatt M et al Analysis of BRAF V600E mutation in nervous system tumors reveals high mutation frequencies in pleomorphic xanthoastrocytoma ganglioglioma and extracerebellar pilocytic astrocytoma Acta Neuropathol “ https doi101007s0040 Sumdani H Shahbuddin Z Harper G Hamilton L Case report of rarely described polymorphous lowgrade neuroepithelial tumor of the young and comparison with oligodendroglioma World Neurosurg “ https doi101016jwneu201903181 Surrey LF Jain P Zhang B Straka J Zhao X Harding BN Resnick AC Storm PB Buccoliero AM Genitori L et al Genomic analysis of dysembryoplastic neuroepithelial tumor spectrum reveals a diversity of molecular alterations dysregulating the MAPK and PI3KmTOR pathways J Neuropathol Exp Neurol “ https doi101093jnennlz10 Kobayashi K Ohnishi A Promsuk J Shimizu S Kanai Y Shiokawa Y Nagane Tateishi K Nakamura T Yamamoto T Molecular genetics and M Enhanced tumor growth elicited by Ltype amino acid transporter in human malignant glioma cells Neurosurgery “ discussion “ https doi10122701neu00003 Koh HY Kim SH Jang J Kim H Han S Lim JS Son G Choi J Park BO Heo WD et al BRAF somatic mutation contributes to intrinsic epileptogenicity in pediatric brain tumors Nat Med “ https doi101038s4159 10180172x Kracht LW Friese M Herholz K Schroeder R Bauer B Jacobs A Heiss WD Methyl[11C] lmethionine uptake as measured by positron emission tomography correlates to microvessel density in patients with glioma Eur J Nucl Med Mol Imaging “ https doi101007s0025 Lelotte J Duprez T Raftopoulos C Michotte A Polymorphous lowgrade neuroepithelial tumor of the young case report of a newly described histopathological entity Acta Neurol Belg “ https doi101007s1376 Louis DN Wesseling P Aldape K Brat DJ Capper D Cree IA Eberhart C FigarellaBranger D Fouladi M Fuller GNet al cIMPACTNOW update new entity and diagnostic principle recommendations of the cIMPACTUtrecht meeting on future CNS tumor classification and grading Brain Pathol https doi101111bpa12832 Okubo S Zhen HN Kawai N Nishiyama Y Haba R Tamiya T Correlation of Lmethyl11Cmethionine MET uptake with Ltype amino acid transporter in human gliomas J Neurooncol “ https doi101007s1106 Ong LC Jin Y Song IC Yu S Zhang K Chow PK [18F]2deoxydglucose FDG uptake in human tumor cells is related to the expression of GLUT1 and hexokinase II Acta Radiol “ https doi10108002841 Rheims S Rubi S Bouvard S Bernard E Streichenberger N Guenot M Le Bars D Hammers A Ryvlin P Accuracy of distinguishing between therapeutic targets of pediatric lowgrade gliomas Brain Tumor Pathol “ https doi101007s1001 Tateishi K Tateishi U Nakanowatari S Ohtake M Minamimoto R Suenaga J Murata H Kubota K Inoue T Kawahara N 62Cudiacetylbis N4methylthiosemicarbazone PET in human gliomas comparative study with [18F]fluorodeoxyglucose and Lmethyl[11C]methionine PET AJNR Am J Neuroradiol “ https doi103174ajnrA3679 Wakimoto H Kesari S Farrell CJ Curry WT Jr Zaupa C Aghi M Kuroda T StemmerRachamimov A Shah K Liu TC et al Human glioblastomaderived cancer stem cells establishment of invasive glioma models and treatment with oncolytic herpes simplex virus vectors Cancer Res “ https doi10115800085472CAN083886 Yue M Jiang J Gao P Liu H Qing G Oncogenic MYC activates a feedforward regulatory loop promoting essential amino acid metabolism and tumorigenesis Cell Rep “ https doi101016jcelre p201712002 Zhang W Liu HT MAPK signal pathways in the regulation of cell proliferation in mammalian cells Cell Res “ https doi101038sjcr72901 Zhao C Zhang Y Wang J A metaanalysis on the diagnostic performance of 18FFDG and 11Cmethionine PET for differentiating brain tumors AJNR Am J Neuroradiol “ https doi103174ajnrA3718 Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 0c'

Thyroid_Cancer

coronavirus disease COVID19 pandemic access to surgical care for patients with head and neck cancer HNC is limited and unpredictable Determining which patients should be prioritized is inherently subjective and difficult to assess The authors have proposed an algorithm to fairly and consistently triage patients and mitigate the risk of adverse outcomes METHODS Two separate expert panels a consensus panel participants and a validation panel participants were constructed among international HNC surgeons Using a modified Delphi process and RAND CorporationUniversity of California at Los Angeles methodology with consensus rounds and meetings groupings of highpriority intermediatepriority and lowpriority indications for surgery were established and subdivided A pointbased scoring algorithm was developed the Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancer SPARTANHN Agreement was measured during consensus and for algorithm scoring using the Krippendorff alpha Rankings from the algorithm were compared with expert rankings of case vignettes using the Spearman rank correlation coefficient RESULTS A total of indications for surgical priority were rated Weights for each indication ranged from ˆ’ to scale range ˆ’ to The response rate for the validation exercise was The SPARTANHN demonstrated excellent agreement and correlation with expert rankings Krippendorff alpha [ CI ] and rho [ CI ] S The SPARTANHN surgical prioritization algorithm consistently stratifies patients requiring HNC surgical care in the COVID19 era Formal evaluation and implementation are required Cancer American Cancer Society LAY SUMMARY ¢ Many countries have enacted strict rules regarding the use of hospital resources during the coronavirus disease COVID19 pandemic Facing delays in surgery patients may experience worse functional outcomes stage migration and eventual inoperability¢ Treatment prioritization tools have shown benefit in helping to triage patients equitably with minimal provider cognitive burden¢ The current study sought to develop what to the authors™ knowledge is the first cancer“specific surgical prioritization tool for use in the COVID19 era the Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancer SPARTANHN This algorithm consistently stratifies patients requiring head and neck cancer surgery in the COVID19 era and provides evidence for the initial uptake of the SPARTANHN KEYWORDS coronavirus disease COVID19 delivery of health care head and neck cancer health priorities patient selection surgical procedures waiting listsCorresponding Author John R de Almeida MD MSc Division of Surgical Oncology Department of Otolaryngology“Head and Neck Surgery 8NU883 Toronto General Hospital University Health Network Elizabeth St Toronto ON M5G 2C4 Canada Johndealmeidauhnca Division of Surgical Oncology Department of Otolaryngology“Head and Neck Surgery Princess Margaret Cancer Center University Health Network University of Toronto Toronto Ontario Canada Institute of Health Policy Management and Evaluation Dalla Lana School of Public Health University of Toronto Toronto Ontario Canada Division of Otolaryngology“Head and Neck Surgery Dalhousie University Halifax Nova Scotia Canada Division of Otolaryngology“Head and Neck Surgery McMaster University Hamilton Ontario Canada Department of Otolaryngology“Head and Neck Surgery Western University London Ontario Canada Department of Otolaryngology“Head and Neck Surgery Memorial Sloan Kettering Cancer Center New York New York Head and Neck“Endocrine Oncology Moffitt Cancer Center Tampa Florida Department of Otolaryngology“Head and Neck Surgery Medical University of South Carolina Charleston South Carolina Department of Otolaryngology“Head and Neck Surgery Stanford University Palo Alto California Department of Otolaryngology“Head and Neck Surgery University of Michigan Ann Arbor Michigan Department of Otolaryngology“Head and Neck Surgery The University of Texas MD Anderson Cancer Center Houston Texas Head and Neck Unit The Royal Marsden Hospital London United Kingdom Department of Otolaryngology“Head and Neck Surgery Icahn School of Medicine at Mount Sinai New York New York Department of Otolaryngology“Head and Neck Surgery Sunnybrook Health Sciences Centre University of Toronto Toronto Ontario Canada Department of Otolaryngology“Head and Neck Surgery Sinai Health System University of Toronto Toronto Ontario CanadaThe first authors contributed equally to this Additional supporting information may be found in the online version of this 101002cncr33114 Received June Revised June Accepted June Published online Month in Wiley Online Library wileyonlinelibrarycomCancer Month 0cOriginal INTRODUCTIONOn March the World Health anization declared a global pandemic due to the novel coronavirus severe acute respiratory syndrome coronavirus SARSCoV2 and the resulting coronavirus disease COVID191 As a result in many jurisdictions operating room capacity has been limited to only emergent or urgent surgical procedures2 Several advisory bodies have issued recommendations to safeguard access to oncologic surgery while still acknowledging that treatment delays may be necessary The American College of Surgeons has recommended postponing elective surgery including for patients with lowrisk cancers while recommending that other urgent cancer surgeries proceed34 Cancer Care Ontario has issued similar guidance recommending that hospitals include cancer surgery in their care delivery plan5The time from the diagnosis of head and neck cancer HNC to surgery is a metric with prognostic importance with treatment delays portending poorer oncologic outcomes68 In a recent systematic review evaluating delays in time from diagnosis to treatment initiation of studies demonstrated a decrease in survival to be associated with treatment delays68 These data support the urgency of initiating treatment for patients with HNC but to our knowledge do not inform a stratification schema when operating room access is not available for all patientsAs a result of these new imposed constraints difficult decisions regarding prioritization for cancer surgery are obligatory and require the consideration of broader principles regarding scarce resource allocation9 Key among these is the need for consistency and transparency to achieve fairness and to avoid engendering disparities in both access and outcomes1011 Prioritization on a casebycase basis using expert clinical judgment can be logistically challenging carries a cognitive burden and is susceptible to the biases of practitionersSurgical prioritization tools or algorithms offer decisionmaking transparency and provide equitable and timesensitive access to care to the patients who need it most1213 Although tools for surgical prioritization in the era of COVID19 continue to emerge to our knowledge oncology patients have not been explicitly considered14 Herein we have presented the development and validation of a novel algorithm Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancer SPARTANHN for the prioritization of surgery for patients with HNCMATERIALS AND METHODSThe current study was granted a waiver from the research ethics board at the University Health NetworkParticipants and SettingFor instrument development a group of expert HNC surgeons JRD DPG RG JCI DBC DB AE DJE KMH EM and IJW from institutions University Health Network Sinai Health Systems and Sunnybrook Health Sciences Centre at the University of Toronto participated in the consensus process consensus panel At the time of the consensus process all institutions were operating under significant resource constraints with limited availability of operating room time For instrument validation a group of participants JRD CWN DF DPG and EM completed the scoring algorithm designed after the consensus process Fifteen external head and neck surgeons HZ ACN RJW MAC CM EMG VD AGS AJR CML EYH JM VP BM and EG from institutions across Canada institutions the United States institutions and the United Kingdom institution participated in a ranking exercise of clinical vignettes validation panelScopeThe scope of variables considered in the prioritization algorithm was established and vetted by the consensus panel see Supporting Information All indications for prioritization were presented to the consensus panel using an online survey platform Google Forms httpsdocsgooglecomforms With exceptions survey respondents were asked to consider each of the indications in isolation For wait times panel members were asked to also consider histologic grade Similarly for surgical site the panel was asked to simultaneously consider extent of surgery Related indications were presented sequentially to facilitate pairwise comparison eg stage I and II vs stage III and IV were presented in sequence AJCC 8th edition The list of indications was pilot tested by surgeons JRD DPG EM and RG for sensibility readability content validity language and comprehensibilityConsensus ProcessThe consensus panel participated in a Delphi consensus process with rounds of rating see Supporting Information The first rounds aimed to achieve consensus regarding the priority grouping high intermediate or low High priority was defined as an indication to Cancer Month 0cproceed to surgery within weeks The second rounds of rating involved ranking each indication less important neutral or more important within their respective priority grouping Two teleconference meetings were conducted between the first and second rounds and between the third and fourth rounds with anonymized results from the prior round presented for discussion and to address inconsistencies and misinterpretationsA modification of the RANDUniversity of California at Los Angeles UCLA method was used to achieve consensus15 This methodology typically is used to determine the appropriateness of an intervention but in this setting was used to determine surgical priority We used a scale ranging from to in rounds and to indicate the decision to not operate or low priority scores intermediate priority scores or high priority scores For rounds and we used a scale from to to rate each indication compared with other indications within each of the priority groupings as either less important neutral or more important Consensus was determined based on RANDUCLA criteria15 For the first rounds to determine surgical priority a hierarchical logic was adopted to determine consensus regarding whether surgery should be performed and to then determine the priority of surgery based on the given indication Agreement on the decision to not operate was defined as a minimum of of the panelists rating a given indication with a zero score If there was no agreement to avoid surgery agreement for surgical priority then was defined as ‰¤ panelists rating the indication outside the 3point range containing the median as per RANDUCLA guidelines15 For rounds and any indication that failed to achieve consensus was classified as being of intermediate priority and for rounds and any indication failing to achieve consensus was classified as neutral within the priority groupingDevelopment of the SPARTANHNThe algorithm uses a pointbased system to assign a total score based on the sum of the individual indication scores see Supporting Information with higher scores corresponding to higher priority Scoring weights were based on consensus from both sets of rounds such that highpriority indications were assigned scores ranging from to intermediatepriority indications were assigned scores ranging from ˆ’ to and lowpriority indications were assigned scores ranging from ˆ’ to ˆ’ Within each priority grouping 3point range the scores were assigned based on the consensus ratings from the third and fourth rounds For any patients with the same total score the SPARTANHNde Almeida et alpatient with the longer surgical wait time was assigned the higher priority rankClinical VignettesTwelve clinical vignettes were constructed see Supporting Information after the consensus rounds to validate the SPARTANHN The vignettes described a variety of clinical scenarios incorporating multiple prioritization indications and additional clinical information Experts were asked to consider only the patientlevel information provided to them and not their own unique clinical and community practice environments Twelve scenarios were selected for diversity of cases The number was considered appropriate while avoiding the excessive cognitive burden associated with ranking too many scenariosStatistical AnalysisAgreementAgreement between raters during the Delphi process was calculated at each round and within each priority grouping using the Krippendorff alpha Kalpha Because typical coefficients of reliability are not suitable for coded data agreement for the rank orders generated by coders JRD CWN DF EM and DPG applying the SPARTANHN algorithm to the clinical vignettes was assessed using Kalpha calculated with bootstrap samples16 The Kalpha allows for estimation of reliability for any number of raters and categories and may be used when there are missing data17Validity of the SPARTANHN AlgorithmConvergent validity of the median rankings from the coders of each of the vignettes using the SPARTANHN and the expert panel rankings were assessed using the Spearman rank correlation coefficient The strength of the correlation was considered weak if the rho was moderate if the rho was between and and strong if the rho was In addition to SPARTANHN a second algorithm using a decisionmaking flowchart was developed SPARTANHN2 The tool and associated performance characteristics are included in Supporting Information Sample Size ConsiderationsFor determination of an adequate sample size for the expert panel we assumed that for model validity there was a strong correlation between the model rank order and expert rank order ie rho ‰¥ an alpha of power of and a nonresponse rate of Therefore the calculated sample size requirement was participantsCancer Month 0cOriginal All analyses were 2sided and statistical significance was set at P\xa0‰¤\xa0 Analyses were conducted using SAS University Edition statistical software SAS Institute Inc Cary North CarolinaRESULTSEstablishing Consensus Priority Groupings First Consensus RoundsAfter the first rounds the panel failed to achieve consensus for any indications that would result in a decision to not operate More than respondents indicated that they would not operate for the following indications the availability of alternative nonsurgical treatment with a similar prognosis respondents poor performance status ie Eastern Cooperative Oncology Group [ECOG] performance status of respondents and very severe comorbidity as indicated by a non“cancerspecific survival rate of at year respondents In the first round consensus was achieved for indications for surgical prioritization of which were considered high priority of which were considered intermediate priority and of which were considered low priority After review of firstround results consensus was achieved for an additional indications indications were rated as being of intermediate priority and indications were rated as low priority Table Establishing Ranking Within Each Priority Grouping Second Consensus RoundsOf the lowpriority indications consensus for the importance of factors was achieved for scenarios both of which were deemed less important Table Of the intermediatepriority indications consensus for the importance of factors was achieved for of scenarios Of highpriority factors consensus for the importance of factors was achieved for scenarios all of which were deemed to be more importantAgreement during consensus rounds was found to be weak to moderate for all rounds ranging from to The agreement was similar when measured as per priority grouping in which the Kalpha ranged from to Table SPARTANHN Surgical Prioritization Scoring SystemPriority weights for each indication ranged from ˆ’ to spanning a 9point range and translated from the rounds of priority groupings into categories Four indications were assigned a weight of based on consensus that these factors were both high priority and more important Supporting Information Table All other highpriority indications were assigned a weighted score because there was no consensus that they were either less or more important For intermediatepriority indications a weighted score of was assigned for of the indications deemed to be more important by consensus The other indication deemed to be more important thyroid cancer with tracheal invasion was assigned a score of because of the fact that this indication can be associated with lowgrade histology which is assigned a negative weighted score Three intermediatepriority indications that were rated as more important were resource use indications which generally are colinear As such the decision was made to assign a maximum score of for the presence of any or all of these indications One intermediatepriority indication was deemed to be less important by consensus and was assigned a score of ˆ’ All other intermediatepriority indications were assigned scores of For the lowpriority indications those deemed to be less important were assigned a weight of ˆ’ and all other indications were assigned a weight of ˆ’ The total scale score ranged from ˆ’ to Fig Reliability and Validity AssessmentAgreement between the coders for the SPARTANHN was excellent Kalpha Agreement between the expert raters was moderate Kalpha Convergent validity was demonstrated by a strong correlation between the rank orders generated by the SPARTANHN and external experts rho CI [P\xa0 a0 ] Agreement between expert rankings and SPARTANHN rankings for the vignettes is shown in Figure DISCUSSIONIn the setting of the COVID19 pandemic in which the availability of operating room time as well as hospital and intensive care unit beds is limited the prioritization of surgical oncology cases is imperative to mitigate downstream adverse outcomes1920 The current methodology was adopted based on expert consensus In the current study we have proposed the SPARTANHN with the objective of providing transparency and facilitating surgical prioritization for treatment providersCreating COVID19“era allocation schemas that are ethically sound is both critical and challenging Emanuel et al have advocated ethical principles with which to Cancer Month 0cSPARTANHNde Almeida et ali ytidbrom lanoitcnufi tnacifings laitnetoP fi ytilibareponi rohtw romut fi tnemriapmi citem etaredom laitnetoPsoc ro lanoitcnuf htiw recnac doryhTiinosavni laehcarthtw romut inossergorp esaeisd citamotpmySENEtsil tiawno e lihwTR suoverPi dedeecxe emit tiaW dedeecxe emit tiaWihgh rof kw‰¥ yb liygootsh edargihgh rof kw yb liygootsh edarg igngami ro lacniilC hpmyl decnavdAegats gncnavdai ei inossergorpi cpocsorcam roN ge esaesd edoni esaesdV inoitceserI ot III egatSnoitceser enob htiw recnac ytivac larOnoitide ht CCJA yregrus fo htgneL latot ro latotraen gniriuqer recnac ytivac larO yats fo htgnel latipsoHh tinu erac evsnetni ioNtinu nwodpets rod yregrus larosnart htiw recnac laegnyrahporOymotcessogllymotoubdnam htiiw recnac laegnyrahporO ymotcegnyral latot htiw recnac laegnyrahpopyHymotcegnyral latot gniriuqer recnac laegnyraLi cpocsodne gniriuqer recnac laegnyrahposaNymotcegnyrahp laitrapdna odne gniriuqer recnac suns lasanarap roi lasaNymotolli xamgniriuqer recnac laegnyrahposaNnoitceser cpocsinoitceseri noitceser nks gniriuqer recnac nks decnavdAinoitcurtsnocer palf lanoger dnai edon hpmyl detimil htiw renac kcen dna daeH edon hpmyl on htiw recnac kcen dna daeHycnangilam enob laropmeTesaesdiII ot I egatSy egAy egAy egAesaesdi SPGOCE y ‰¥ egAycnangil amditorap edarghgHinoitcurtsnocer palfeerf gniriuqer recnac nks decnavdAi laitrapgniriuqeryregrus laegnyral recnac laegnyraL gniriuqer recnac laegnyrahpopyHnyrahpognyral latotymotcegriuqer recnac sunsi roiretna nepogn i lasanarap ro lasaN ‰¥i lacgrus lacafonarciili ygootsh edargwol rof dedeecxe emit tiaWkwnoitceser eussittfos htiw recnac ytivac larOi rof gnhcaorppa tubdedeecxe ton em it tiaW dedeecxe emit tiaWliygootsh edarghghiwoliygootsh edargl rof kw deecxe ton emit tiaWromoc ereves yreV SP GOCE ei sutats ecnamrofrep rooP ypareht evitanretlAlebaliava ni dehcaorppa tub edargwol rof kw ditorap edargwoLycnangilam edon hpmyl htiw recnac doryhTiesaesdilygootshi recnacnonge ytidb i ta i lavvrusy srotcaF ytiroirPhgHisrotcaF ytiroirPetademretniIsrotcaF ytiroirPwoLˆ’ˆ’ˆ’ˆ’i gnknaR fo sdnuoR retfA serocSdna snoitacdn iI noitazitiroirP ELBATCancer Month 0cOriginal TRj tnavuda dna esaesddecnavda hti iw tneitaPderiuqer tinu nwodpets ro tinu erac evsnetniIderiuqer ebut ymotsoehcart oNderiuqer palf eerf oNd yats fo htgnel latipsoHh yregrus fo htgneLd yats fo htgnel latipsoHderiuqer palf eerFh yregrus fo htgneLnoitpo na si SPGOCEˆ’ˆ’ˆ’ˆ’srotcaF ytiroirPhgHisrotcaF ytiroirPetademretniIsrotcaF ytiroirPwoLdeunitnoC ELBATyparehtodar iTRi nosnetxe ladonartxe ENE sutats ecnamrofreppu ygoocnO evitarepooCnretsaE l SPGOCE snoitaverbbAiTABLE Agreement Between Experts During the Delphi ProcessRoundOrdinal ScaleaLCL UCLPer Priority GroupLCL UCLAbbreviations LCL lower confidence limit UCL upper confidence limitThere were raters and agreement was measured using the Krippendorff alphaaœOrdinal scale refers to the scale of to used to rate priority of surgery and œPer Priority Group refers to the lowpriority mediumpriority and highpriority groups related to the scoring scaleguide the allocation of scarce resources maximizing the benefits produced by scarce resources treating people equally promoting and rewarding instrumental value and giving priority to those patients who are worst off9 These have been contextualized for cancer care more broadly and are manifest in the SPARTANHN algorithm21 The highpriority indications implicitly embrace an underlying premise of saving the most lives andor preserving the most lifeyears Many procedures for patients with HNC are aerosolgenerating and increase the risk to health care workers and other hospitalized patients22 Our process accounted for these by giving consideration to these factors during the consensus process although indications associated with potential exposure to health care workers did not emerge as lowpriority ones Indications associated with lower resource use did achieve consensus for higher importance This may help to avoid the opportunity cost of treating fewer patients with longer surgeriesAnecdotal and institutionspecific prioritization schemas for patients with HNC and general otolaryngology have been suggested213 These parallel similar efforts for general surgery cardiac surgery and orthopedic surgery12132328 In many of these patients are prioritized by the scoring of several criteria and summing of the scores to achieve a total patient score Many of these systems have been validated against expert rankings of surgical priority2728We used a methodology for developing a pointbased prioritization system similar to those previously described29 To the best of our knowledge pointbased surgical prioritization systems have been very well studied to date Hansen et al previously proposed a methodology for developing a pointbased prioritization system using the following steps ranking patient case vignettes Cancer Month 0cSPARTANHNde Almeida et alFIGURE The Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancer SPARTANHN scoring system ECOG indicates Eastern Cooperative Oncology Group ENE extranodal extensionusing clinical judgment drafting the criteria and categories within each criteria pretesting the criteria and categories consulting with patient groups and other clinicians determining point values for criteria and categories checking the testretest reliability and face validity and revising the points system as new evidence emerges29 Our approach to the development of the SPARTANHN was similar However given the relatively expedited nature of the process we did not directly involve patientsOne method proposed for establishing the priority of all indications in a pointbased scoring system is known as Potentially All Pairwise Rankings of all Alternatives PAPRIKA30 In the current study we chose to use the RANDUCLA process instead of pairwise comparison to minimize computational burden We established Cancer Month 0cOriginal FIGURE External validation rank results A total of experts were asked to rate the scenarios provided shown on the xaxis and the results were compared with the rank generated by models and shown on the yaxis Green shading reflects high priority ranked yellow shading indicates medium priority ranked and red shading indicates low priority ranked Asterisk denotes ties from the algorithm SPARTANHN indicates Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancerindications for surgical prioritization that would create an enormous computational burden using pairwise comparison methodology One problem inherent in the PAPRIKA methodology is the assumption that all indications are not equal and can be ranked However clinically certain indications may be equivalent in priority Furthermore pairwise comparisons assume mutual exclusivity of each of the indications which is not always the case Use of the RANDUCLA consensus process avoids the need for multiple pairwise comparison and allows for consideration of each factor in isolation The goal of the consensus rounds was not to establish a rank order for all indications but mainly to understand which indications result in high intermediate or low priorityThe SPARTANHN algorithm has demonstrated preliminary reliability and validity We demonstrated good agreement between raters and the SPARTANHN algorithm suggesting minimal interpretive error Many of the highpriority indications accounted for some component of interpretation because raters were forced to consider imminent disease progression that may result in an adverse outcome Despite the subjective decisions that must be made as part of SPARTANHN agreement remained high In fact true interrater reliability was found to be higher because the Kalpha is a conservative measure of reliability Other measures of reliability such as the Kendall coefficient of concordance tend to overestimate reliability and cannot be applied to missing data31 Perhaps most important the SPARTANHN correlated highly with expert rankings With established validity this algorithm may be ready for preliminary clinical use although further testing against realworld data to validate it with other cancer outcomes such as survival is neededThe results of the current study must be interpreted within the context of the study design Although externally validated by other surgeons across North America and the United Kingdom the criteria for which consensus was achieved for prioritization were not vetted by patients advocacy groups or other stakeholders such as medical or radiation oncologists The latter groups represent essential providers in the multidisciplinary care of patients with HNC and may have important insight into the availability and effectiveness of nonsurgical treatments1920 Nonetheless the actual prioritization of surgical waitlists remains the sole responsibility of surgeons and their practice partners In addition the SPARTANHN algorithm is intended to assist in making difficult prioritization decisions and is not intended to make recommendations for the time frame in which patients should receive treatment Instead established guidelines should be adhered to for treatment targets Patient wait times as they relate to those targets should be considered when using the SPARTANHN algorithm The validation process in the current study used expert opinion as the gold standard of prioritization which is potentially biased and reflected the opinions of surgeons practicing in academic medical centers from resourcerich nations Subsequently use of the SPARTANHN algorithm in other geographic regions Cancer Month 0cand health care systems requires additional investigation because local treatment paradigms and risk factors may vary substantiallyThe current study has presented the development and validation of a novel algorithm for the prioritization of surgery for patients with HNC Further evaluation of its implementation in various practice settings will be obligatory However the results of the current study have provided data with which to inform realworld use as the current pandemic has obviated our ability to more rigorously study the instrument prior to making necessary and difficult realtime allocation decisionsFUNDING SUPPORTNo specific funding was disclosedCONFLICT OF INTEREST DISCLOSURESEvan M Graboyes has received grants from the National Cancer Institute and the Doris Duke Charitable Foundation for work performed outside of the current study Vinidh Paleri offers his services as a proctor for a transoral robotic surgery proctoring program run by Intuitive Surgical and has been remunerated for his time Antoine Eskander has received a research grant from Merck and acted as a paid consultant for BristolMyers Squibb for work performed outside of the current study Ian J Witterick has stock in Proteocyte Diagnostics Inc and has received honoraria from Sanofi Genzyme and Medtronic Canada for work performed outside of the current study The other authors made no disclosuresAUTHOR CONTRIBUTIONSJohn R de Almeida Study idea and design writing and editing Christopher W Noel Study design writing data analysis and editing David Forner Study design writing data analysis and editing Han Zhang Data acquisition and editing Anthony C Nichols Data acquisition and editing Marc A Cohen Data acquisition and editing Richard J Wong Data acquisition and editing Caitlin McMullen Data acquisition and editing Evan M Graboyes Data acquisition and editing Vasu Divi Data acquisition and editing Andrew G Shuman Writing data acquisition and editing Andrew J Rosko Data acquisition and editing Carol M Lewis Data acquisition and editing Ehab Y Hanna Data acquisition and editing Jeffrey Myers Data acquisition and editing Vinidh Paleri Data acquisition and editing Brett Miles Data acquisition and editing Eric Genden Data acquisition and editing Antoine Eskander Data acquisition and editing Danny J Enepekides Data acquisition and editing Kevin M Higgins Data acquisition and editing Dale Brown Data acquisition and editing Douglas B Chepeha Data acquisition and editing Ian J Witterick Data acquisition

Thyroid_Cancer

"Pressure sores are sometimes refractory to treatment often due to malnutrition Small intestinalbacterial overgrowth SIBO obstructs absorption in the digestive tract and causes malnutrition However little isknown about the association between pressure sore wound healing and SIBO Here we report a case of a patientwith a refractory sacral pressure sore and SIBOCase presentation A 66yearold woman who was spinal cord injured years before visiting our hospitalpresented with the chief complaint of a sacral pressure sore — cm in size which was refractory totreatment Physical examination showed abdominal distension and emaciation with a body mass index of Further examination revealed elevated serum alkaline phosphatase UL bilateral tibial fracture multiple ribfracture and osteoporosis We diagnosed the patient with osteomalacia with vitamin D deficiency Despite oralsupplementation serum levels of calcium phosphorous and vitamin D remained low Also despite concentrativewound therapy for the sacral pressure sore by plastic surgeons no wound healing was achieved Due to asuspicion of disturbances in nutrient absorption we performed bacterial examination of collected gastric andduodenal fluid which showed high numbers of bacteria in gastric content E coli Streptococcus speciesand Neisseria species and duodenal content E coli Candida glabrata Therefore we diagnosed thepatient with SIBO and started selective decontamination of the digestive tract using polymyxin B sulfate andamphotericin B After starting treatment for SIBO the sacral pressure sore began to heal and was nearly healed after days The patient™s serum levels of calcium phosphorous vitamin D and other fatsoluble vitamins alsogradually increased after starting treatment for SIBOContinued on next page Correspondence 2m2hy4gmailcom1Department of Plastic Surgery Chiba University Inohana ChuokuChibacity Chiba JapanFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cKubota BMC Gastroenterology Page of Continued from previous pageConclusion We report a case of a patient with a refractory sacral pressure sore that healed after starting treatmentfor SIBO We conclude that SIBO may be an overlooked cause of malnutrition and poor wound healing in patientswith chronic pressure soresKeywords Pressure wound Small intestinal bacterial overgrowth Spinal cord injury Malnutrition Wound healingCase reportBackgroundPressure sores in patients with spinal cord injury SCIare sometimes refractory to treatment Chronic gastrointestinal symptoms are also frequently seen in patientswith SCI [ ] and malnutrition caused by decreasedgastrointestinal motility in SCI patients is a major causeand exacerbating factor of pressure sores Evaluation ofnutritional status in patients with pressure sores is essential [] as nutritional intervention can be a valuabletreatment option for pressure sores However small intestinal bacterial overgrowth SIBO is rarely consideredin the evaluation of malnutrition in SCI patients withpressure sores SIBO is defined as the presence of morethan — colony forming units CFUmL of bacteriaor any amount of E coli in the proximal small bowelcontent [] Here we report the case of an SCI patientwith a refractory sacral pressure sore that healed afterstarting treatment for SIBO To the best of our knowledge this is the first report of an association between apressure sore and SIBOCase presentationA 66yearold woman visited our hospital for the purpose of treating her sacral pressure sore day whichshe developed months prior due to bed rest duringtreatment of a left humeral fracture in another hospitalShe had paraplegia as well as bladder and rectal disturbance due to SCI at the fourth lumbar level L4 causedby a suicidal jump in response to paranoid delusions at years of age Spinal fusion surgery and cystostomywere performed early after SCI Otherwise she had ahistory of hysterectomy due to uterine cancer at yearsof age lymphaticovenular anastomosis as a treatment forposthysterectomy lymphedema in the bilateral lower extremities at years of age and cholecystectomy at years of ageWhen she visited our hospital she was taking the following oral medicines propiverine hydrochloride vitamin B12 etizolamflunitrazepam sodium bicarbonateanhydrous monobasic sodium phosphate mixture Clostridium butyricum tablets sodium risedronate hydraterebamipide sodium ferrous citrate fursultiamine hydrochloride alfacalcidol and potassium Lasparate She didnot take proton pump inhibitors PPI Her vital signswere as follows body temperature of °C low bloodpressure of mmHg pulse rate of bpm and respiratory rate of per min Physical examinationshowed abdominal distension emaciation with a bodymass index of and a sacral pressure sore — cm in size including a pocket entrance of — cmFig 1a Most of the surface of the pressure sore wascovered by granulation Our evaluation of the pressuresore with DESIGNR [] was D3 e3 s8 i0 g3 N3 P24with a total score of Table Bacterial culture examination ofthe pressure soreshowed Corynebacterium striatum and methicillinresistant Staphylococcus aureus Laboratory data showedan elevated serum alkaline phosphatase level of UL and low serum levels of hemoglobin gdL albumin gdL calcium mgdL and zinc μgdL Onday we observed a sudden decrease of hemoglobin to gdL with a positive fecal occult blood test bilateralpleural effusion on chest xray and serum albumin levelof gdL Upper gastrointestinal endoscopy showed agastric ulcer at H2 stageAs a result of searching for the cause of alkaline phosphatase elevation bilateral tibial fracture multiple ribfracture and osteoporosis were found Fig 1e and fFemoral bone density was of the young adult meanA low serum inanic phosphorous level was foundTable along with a low serum level of 25hydroxyvitamin D3 25OHVitD3 below the detection limit andelevated level of parathyroid hormone Table Levelsof other fatsoluble vitamins were also low vitamin A μIUdL vitamin K1 ngdL and vitamin E mgdL Examination using ultrasound and computedtomography showed normalthyroid and parathyroidglands Basing on these finding we diagnosed osteomalacia with vitamin D deficiencyOn day oral supplementation of calcium phosphorous and vitamin D was started Despite supplementationserum levels of calcium phosphorous and 25OHVitD3on day showed poor improvement calcium mgdLphosphorous mgdL and 25OHVitD3 below the detection limitOn day we performed bacterial examination ofcollected gastric and duodenal fluid with suspicion of adisturbance in absorption which showed elevated num E coli bers of bacteria in gastric contentStreptococcus species and Neisseria species and 0cKubota BMC Gastroenterology Page of Fig See legend on next page 0cKubota BMC Gastroenterology Page of See figure on previous pageFig Patient images a b c and d Sacral pressure sore a Day sore — cm in size with an entrance of — cm DESIGNR score wasD3 e3 s8 i0 g3 N3 P24 with a total score of b Day ie days after starting SDD for treating SIBO reduced size of sore DESIGNR scorewas D3 e3 s3 i0 g1 n0 p0 with a total score of c Day ie days after staring SDD healed sore DESIGNR score was d0 e0 s0 i0 g0 n0p0 with a total score of d Day ie days after staring SDD no recurrence of the sore e and f Osteoporosis and multiple fractures eXray showing left tibial fracture f Tc99 m bone scan showing accumulation in multiple ribs vertebrae and right ulna g h and i Endoscopicexamination and results of bacterial culture of the upper digestive tract All stomach duodenum and proximal jejunum samples were positive forE coli g Stomach Food residue can be seen Acid level was decreased to pH h Duodenum Food residue is evident i Proximal jejunum Flatvilli and a jejunal ulcer are observedTable DESIGNR assessment tool for pressure sore Reprinted with permission from John Wiley and Sons In Matsui et alDevelopment of the DESIGNR with an observational study an absolute evaluation tool for monitoring pressure ulcer woundhealing Wound Repair Regen “Depthd No particular skin lesion and no rednessD Lesion extends into the subcutaneous tissuePersistent rednessLesion extends into dermisExudatee NoneSlight does not require daily dressing change Moderate requires daily dressing changeSizes NoneSmaller than cm2 cm2 or larger but smaller than cm2 cm2 or larger but smaller than cm2 cm2 or larger but smaller than cm2 cm2 or larger but smaller than cm2Lesion extends to the muscle tendon and boneLesion extends into the articular or body cavityU It is impossible to measure the depthE Heavy requires dressing change more than twice a dayS cm2 or largerInflammationInfectioniNoneSigns of inflammation fever redness swelling and pain around thewoundIClear signs of local infection eg inflammation pus and foulsmellSystemic impact such as feverGranulation tissueg Granulation cannot be assessed because the wound is healed or tooshallowG Healthy granulation tissue occupies or more but lessthan Healthy granulation tissue occupies or moreHealthy granulation tissue occupies or more but less than Healthy granulation tissue occupies less than No healthy granulation tissue existsNecrotic tissuen NonePocketp NoneN Soft necrotic tissue existsHard and thick necrotic tissue is attached to the woundP Smaller than cm2 cm2 or larger but smaller than cm2 cm2 or larger but smaller than cm2 cm2 or larger 0cKubota BMC Gastroenterology Page of Table Laboratory data before starting supplementation withvitamin DWBCμL104μLgdL104μLgdLgdLULULULULULULULULmgdLmgdLmLmin173 m2mEqLmEqLmgdLmgdLmgdLμgdLμgdLμgdLμgdLμIUmLmgdLmgdLmgdLmgdLsecondpgmLμgdLμIUmLpgmLngdLof woundirrigationtreatmentdebridementstarting SDD the pressure sore was refractory to multiple methodsincludingdepressurizationointmentbasic fibroblast growth factor and negative pressurewound therapy After starting SDD the pressure sorebegan to heal On day ie days after startingSDD the pressure sore DESIGNR score was D3 e3 s3i0 g1 n0 p0 with a total score of Fig 1b Regardingthe nutritional status the serum albumin level increasedfrom gdL just before starting SDD to gdL at days after starting SDD Also the hemoglobin levelincreased to gdL and the serum zinc level increasedto μgdL Serum levels of calcium phosphorous vitamin D and other fatsoluble vitamins also gradually increased Fig and Table A repeat diagnosticbacterial examination of the upper digestive tract contents for SIBO was not performed because of obviousimprovements in most of the laboratory data Therewere no adverse effects of SDD such as antibioticassociated diarrhea In contrast the patient presentedwith constipation that was noted before starting SDDThe sacral pressure sore was completely healed on day ie days after starting SDD with a DESIGNRscore of d0 e0 s0 i0 g0 n0 p0 and total score of Fig1c In addition the patient showed improved nutritionalstatus and had a serum albumin level of gdL Wesuccessfully reduced the dose of polymyxin B from to million units daily similarly the dose of amphotericin B was reduced from to mg daily on day ie days after starting SDD without any signs ofSIBO recurrence There was no recurrence of the sacralpressure sore with a serum albumin level of gdL onday ie days after staring SDD Fig 1d Onday we successfully ended the use of amphotericinB however the use of polymyxin B at million unitsper day continued On day ie days afterstarting SDD while still using polymyxin B at million per day the serum albumin level was gdL thehemoglobin level was gdL and the serum zinc levelwas μgdL There were no signs of SIBO recurrenceor the sacral pressure soreDiscussion and conclusionsWe report the case of a patient whose sacral pressuresore and osteoporosis were improved by treatment forSIBO Although nutrition status is known to be important for the healing of pressure sores SIBO is rarelychecked as a cause of malnutrition in patients with pressure sores However SIBO is a potential cause of malnutrition in patients with SCI due to decreased intestinalmotility resulting from autonomic disturbances and reduced physical activity [] SCI is also a risk factor forpressure sores [] However to the best of our knowledge there are no previous reports of an associationRBCHbPltTotal ProteinAlbuminASTALTγGTPLDHALPChECKAmyBUNCreatinineeGFRNaKCaiPMgFeZnUIBCFerritinErythropoietinTotal CholesterolTriglycerideHDLCholesterolLDLCholesterolPTINRAPTTACTHCortisolTSHFT3FT4duodenal content E coli Candida glabrataFig 1g h and i Therefore we diagnosed SIBO Onday we started selective decontamination of the digestive tract SDD using oral administration of polymyxin B sulfate million units daily and oraladministration of amphotericin B mg daily Before 0cKubota BMC Gastroenterology Page of Table Vitamins and bone metabolism markers before starting supplementation of vitamin DVitamin AVitamin K1Vitamin K2Vitamin E125OH2 Vitamin D25OH Vitamin D3Retinol binding proteinVitamin B1Vitamin B12Nicotinic acidFolic acidTRACP5bNTxBone type ALPIntact P1NPOsteocalcinintact PTHPTHrPFGF23TRPTmPGFR Below the detection limitIUdLngmLngmLmgdLpgmLpgmLmgdLngmLpgmLμgmLngmLμUmLnmolBCELμgLμgLngmLpgmLpmolLpgmLmgdLnormal range““ “““““““ ““““““ “““between pressure sores and SIBO Thus our case drawsattention to the fact that SIBO can be an overlookedcause of poor wound healing during the treatment ofpressure soresSIBO was first reported by Vantrappen as an increased concentration of 14CO2 in a bile acid breath testfor patients with an absent interdigestive motor complex[] Today consensus diagnostic criteria for SIBO arethe presence of more than — CFUmL of bacteriaor any amount of E coli in the proximal small bowelcontent [] Relatively little is known about commensalsinhabiting the small intestine mainly due to the limitedaccessibility of this environment for microbiological analysis [] In the healthy state the numbers of intestinalbacteria range from to CFUmL and mainly include gramnegative and grampositive aerobes such asStreptococcus Lactobacillus and Bacteroidesspecies[] Regarding the amount of bacteria in proximal jejunal aspiration Khoshini report that normal subexceed — CFUmL and thereforejectsproposed more than — CFUmL coliform bacteriaas the threshold for SIBO [] In our case CFUmLE coli existed in duodenal content and CFUmL Ecoli existed in gastric content which met the traditionaldiagnostic criteria of SIBOrarelyOther diagnostic methods for SIBO are breath testsusing hydrogen or hydrogen methane with lactulose lucose [] Breath tests have clinical utility for diagnosing SIBO because they are less invasive than obtaining proximal small bowel content However there areno standardized criteria for diagnosing SIBO usingbreath tests [] We did not perform a breath test inour case studyDespite no previous reports of an association betweenunhealed pressure sores and SIBO nutritional status isknown to be important for the healing of pressure sores []In our case the sacral pressure sore which was initially refractory began to heal after starting treatment for SIBOAmong intrinsic factors related to the healing of pressuresores blood levels of hemoglobin albumin and zinc are especially important [] Our patient had anemia hypoalbuminemia and a low zinc concentration which graduallyimproved after starting treatment for SIBOSIBO is caused by multiple factors including disturbances in defense mechanisms of the digestive tractanatomical abnormalities surgical interventions and disturbed gastrointestinal motility [ ] Bures described several endogenous defense mechanisms thatprevent bacterial overgrowth [] including secretion ofgastric acidintestinal motility a properly functioning 0cKubota BMC Gastroenterology Page of Fig Bone metabolism markers After starting SDD levels of bone metabolism markers gradually improvedTable Vitamins and trace elements before and after supplementation and selective digestive decontamination SDDVitamin K1 ngmLVitamin K2 ngmLVitamin E mgmL125OH2 Vitamin D pgmL25OH Vitamin D3 pgmLNicotinic acid μgmLMg mgdLFe μgdLBefore supplementationAt the time SDD started days after starting SDD days after starting SDDNANANA 0cKubota BMC Gastroenterology Page of ileocecal valve production of secretory immunoglobulinson the surface of the gastrointestinal mucous membraneand the bacteriostatic properties of pancreatic juice andbile In our case the patient™s history of cholecystectomyand hysterectomy were possible causes or exacerbatingfactors of SIBO Disturbed gastrointestinal motilitycaused by paraplegia below the L4 level due to SCI is another possible cause of SIBO in our case as well as decreased physical activity due to fracture ofthe lefthumerus and bilateral tibiaChronic gastrointestinalinvolvement is seen in “ of patients with SCI [ ] SCI patients lackcentral nervous system control over the gastrointestinalsystem [] Liu report that bowel problems in SCIpatients are related to high levels of cord injury completeness of cord injury and postinjury durations of years or more [] Moderate or severe grade depressivestatus is also associated with neurologic bowel dysfunction in SCI patients Of these risk factors our patienthad complete cord injury that had occurred more than years ago Also many bowel symptoms appear in patients with SCI eg constipation distension incontinence abdominal pain bowel accidents nausea diarrheastrainingautonomichyperreflexia headaches or sweat relieved by a bowelmovement [ “] However our patient showedno appetite loss a sufficient amount of food intake andnonsevere bowel symptoms Thus the presence of malnutrition despite adequate food intake and low levels oflipidsoluble vitamins that were unresponsive to supplementation led us to suspect SIBOrectal bleeding hemorrhoidsAlthough gastrointestinal symptoms are frequently observed in patients with SCI there are few reports ofSIBO in SCI patients Cheng report that of of SCI patients were diagnosed with SIBO basedon the glucose hydrogenmethane breath test [] However the prevalence of SIBO among SCI patients as confirmed by the consensus diagnostic criteria ofthepresence of more than CFUmL bacteria or anyamount of E coli in upper digestive tract content is unknown In patients with SCI absent central nervous system innervation of the digestive tract can change theinhabiting environment of bacteria Gungor reportdifferences in gut microbial patterns between SCI patients and control individuals as measured by bacterialgenome sequencing [] Specifically they found thatbutyrateproducing bacteria were specifically reduced inSCI patients Thus it is possible that SIBO is overlookedin patients with SCI In our caseit is unclear whenSIBO occurred relative to the time of SCI but we suspect that it arose due to gastrointestinal motility disorder caused by autonomic disturbancesDisturbances in fat absorption and deficiency in fatsoluble vitamins ie vitamins A K E and D3 areobserved in patients with SIBO [] Excess bacteria inthe small intestine promotes a change from conjugatedbile acid into deconjugated bile acid which decreasesthe micellar solubilization of dietary fat Bacterial fermented short chain fatty acid causes osmotic watermovement to the intestinal lumen which results in diarrhea and malabsorption [] Intestinal epithelial damagein SIBO also interferes with fat absorption Mucosaldamage is caused by metabolites of aerobic bacteria endotoxins of anaerobic bacteria and lithocholic acidwhich is a bacterial degradation product of unconjugatedbile acid [“] Our patient however showed constipation rather than diarrhea in spite of SIBO Whether ornot diarrhea occurs in patients with SIBO is determinedby multiple factors Constipation frequently occurs inpatients with SCI due to decreased physical activity andautonomic dysfunction De Looze reported that therate of constipation in the patients with SCI is []A certain proportion of the patients with SCI show constipation in spite of the coexisting SIBO Cheng reported that in patients with both SCI and SIBO showed constipation [] We believe that the factorsleading to constipation in our patient were stronger thanthose leading to diarrhea Vitamin D deficiency in SIBOcauses osteomalacia Our patientshowed multiplefractures and osteoporosis with serum vitamin D3 levelsbelow thetosupplementationrefractorydetectionlimitandThere is no consensus on the choice dose or durationof antibiotics for treating SIBO [] In principle antibiotics should be chosen based on the results of an antimicrobial susceptibility test but this approach cannotaddress the great diversity in microbiota of the digestivetract [ ] Metronidazole is a firstline choice forSIBO [] with other choices being rifaximin ciprofloxacin norfloxacin amoxicillinclavulanate trimethoprimsulfamethoxazole cephalexin or their combination []However these antibodies are selected based on customrather than scientific evidence [] In our case we usedoral polymyxin B and amphotericin B in accordancewith SDD which was first reported as a method of preventing ventilationassociated pneumonia and microbialtranslocation of gramnegative rod bacteria and fungi incritically ill patients treated in the intensive care unit[“] Polymyxin B administered to the digestive tractis nonabsorbent into the human body and has strongbactericidal power against gramnegative rod bacteriaexcept for naturally polymyxinresistant bacteria such asProteus Providencia Manella Burkholderia and Serratia [] Amphoteric B is an antifungal drug that isalso nonabsorbent into the human body when administered to the digestive tract In our case after startingSDD fatsoluble vitamins were increased and osteoporosis was improved No obvious adverse effects of SDD 0cKubota BMC Gastroenterology Page of such as antibioticassociated diarrhea were observed inour caseWhen and how to stop antibiotherapy for the treatment of patients with SIBO are difficult problemsFew reports are available for the method and timingfor making a decision to stop antibiotherapy in SIBOLauritano reported that the recurrence rate at months after stopping antibiotherapy in SIBO patientsis [] They also showed that an older age history of appendectomy and chronic use of PPIs areassociated with SIBO recurrence Bures reportedthat cyclical gastrointestinal selective antibiotics areneeded for SIBO treatment [] These reports indicatethat in many patients with SIBO it is actually impossible to stop antibiotherapy because of the underlyingconditions that lead to SIBO Similarly in our case itwas difficult to ameliorate the underlying condition ofdecreased motility of the digestive tract due to SCIWe were compelled to continue SDD for a long duration We did however succeed in gradually reducingthe dose of polymyxin B and end the use of amphotericin B without signs of SIBO recurrence Withcareful consideration it may be possible and feasibleto stop SDD completelyProbiotics are also a treatment approach for SIBO assome species of bacteria are thought to protect againsthigh numbers of E coli and fungi in the digestive tract[] However the role and effects of probiotics are stillunclear The digestive tract microbiome has both pathogenic potential and a protective role in maintaininghealth However metagenomic analysis reveals that “ of microanisms in the digestive tract cannot becultured under laboratory conditions [] The effects ofSDD and probiotics on the digestive tract microbiome inpatients with SIBO should be investigated to furtherunderstand the pathogenesis of the diseaseIn conclusion we treated a patient with a sacral pressure sore who also had SCI multiple fractures withosteoporosis and malabsorption especially of fatsolublevitamins Based on culture of upper digestive tract content we diagnosed the patient with SIBO and startedSDD using polymyxin B and amphotericin B which effectively ameliorated the absorbency disturbance andallowed healing of the pressure sore In light of severalcommon risk factors between pressure sores and SIBOsuch as decreased physical activity our case providesadditional information on the associations among pressure sores malnutrition and SIBOAbbreviationsCFU Colony forming units SCI Spinal cord injury SDD Selectivedecontamination of the digestive tract SIBO Small intestinal bacterialovergrowth 25OHVitD3 25hydroxy vitamin D3AcknowledgementsWe thank the many personnel involved in this interdisciplinary diagnosticworkup as their effective technical assistance enabled a comprehensiveapproach to this difficult diagnosisAuthors™ contributionsYK and TT treated the patient conceived of and wrote the manuscript KISK and TK treated the patient and collected the data SA and NMinterpreted the data HN analyzed the data and created the figures andtables All authors read and approved the final manuscriptFundingNo funding was receivedAvailability of data and materialsData on this case not reported in the manuscript are available from thecorresponding author upon reasonable requestEthics approval and consent to participateEthical approval was not necessary for the reported investigations as theywere performed in a routine clinical setting with therapeutic intentionConsent for publicationThe patient provided written consent for reporting her case in aninternational published medical journal including clinical details and imagesCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Plastic Surgery Chiba University Inohana ChuokuChibacity Chiba Japan 2Department of Molecular DiagnosisChiba University Inohana Chuoku Chibacity Chiba Japan3Department of Plastic Surgery Chiba Emergency Medical Center Isobe Mihamaku Chiba JapanReceived July Accepted August ReferencesStone JM NinoMurcia M Wolfe VA Perkash I Chronic gastrointestinalproblems in spinal cord injury patients a prospective analysis Am JGastroenterol “Liu CW Huang CC Chen CH Yang YH Chen TW Huang MH Prediction ofsevere neurogenic bowel dysfunction in persons with spinal cord injurySpinal Cord “Eglseer D Hodl M Lohrmann C Nutritional management of olderhospitalised patients with pressure injuries Int Wound J “Bures J Cyrany J Kohoutova D Forstl M Rejchrt S Kvetina J Vorisek VKopacova M Small intestinal bacterial overgrowth syndrome World JGastroenterol “ Matsui Y Furue M Sanada H Tachibana T Nakayama T Sugama J Furuta KTachi M Tokunaga K Miyachi Y Development of the DESIGNR with anobservational study an absolute evaluation tool for monitoring pressureulcer wound healing Wound Repair Regen “Sachdev AH Pimentel M Gastrointestinal bacterial overgrowth pathogenesisand clinical significance Ther Adv Chronic Dis “Groah SL Schladen M Pineda CG Hsieh CH Prevention of pressure ulcersamong people with spinal cord injury a systematic review PM R “Vantrappen G Janssens J Hellemans J Ghoos Y The interdigestive motorcomplex of normal subjects and patients with bacterial overgrowth of thesmall intestine J Clin Invest “Zoetendal EG Raes J van den Bogert B Arumugam M Booijink CC TroostFJ Bork P Wels M de Vos WM Kleerebezem M The human small intestinalmicrobiota is driven by rapid uptake and conversion of simplecarbohydrates ISME J “ Miazga A Osinski M Cichy W Zaba R Current views on theetiopathogenesis clinical manifestation diagnostics treatment andcorrelation with other nosological entities of SIBO Adv Med Sci “ 0cKubota BMC Gastroenterology Page of Khoshini R Dai SC Lezcano S Pimentel M A systematic review ofdiagnostic tests for small intestinal bacterial overgrowth Dig Dis Sci “ Heintschel M Heuberger R The potential role of zinc supplementation onpressure injury healing in older adults a review of the literature Wounds“Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations Han TR Kim JH Kwon BS Chronic gastrointestinal problems and boweldysfunction in patients with spinal cord injury Spinal Cord “Ebert E Gastrointestinal involvement in spinal cord injury a clinicalperspective J Gastrointestin Liver Dis “ Gungor B Adiguzel E Gursel I Yilmaz B Gursel M Intestinal microbiota inpatients with spinal cord injury PLoS One 2016111e0145878 Harari D Sarkarati M Gurwitz JH McGlincheyBerroth G Minaker KLConstipationrelated symptoms and bowel program concerning individualswith spinal cord injury Spinal Cord “ Menter R Weitzenkamp D Cooper D Bingley J Charlifue S Whiteneck GBowel management outcomes in individuals with longterm spinal cordinjuries Spinal Cord “ De Looze D Van Laere M De Muynck M Beke R Elewaut A Constipationand other chronic gastrointestinal problems in spinal cord injury patientsSpinal Cord “Lynch AC Wong C Anthony A Dobbs BR Frizelle FA Bowel dysfunctionfollowing spinal cord injury a description of bowel function in a spinalcordinjured population and comparison with age and gender matchedcontrols Spinal Cord “Krogh K Nielsen J Djurhuus JC Mosdal C Sabroe S Laurberg S Colorectalfunction in patients with spinal cord lesions Dis Colon Rectum “ Chen CY Chuang TY Tsai YA Tai HC Lu CL Kang LJ Lu RH Chang FY LeeSD Loss of sympathetic coordination appears to delay gastrointestinaltransit in patients with spinal cord injury Dig Dis Sci “ Cheng X Zhang L Xie NC Xu HL Lian YJ Association between smallintestinal bacterial overgrowth and deep vein thrombosis in patients withspinal cord injuries J Thromb Haemost “Kirsch M Bozdech J Gardner DA Hepatic portal venous gas an unusualpresentation of Crohn's disease Am J Gastroenterol “Jones RM Neish AS Recognition of bacterial pathogens and mucosalimmunity Cell Microbiol “ Hoog CM Lindberg G Sjoqvist U Findings in patients with chronicintestinal dysmotility investigated by capsule endoscopy BMCGastroenterol Singh VV Toskes PP Small bowel bacterial overgrowth presentationdiagnosis and treatment Curr Treat Options Gastroenterol “ Quigley EM AbuShanab A Small intestinal bacterial overgrowth Infect DisClin N Am “ viiiix Melchior C Gourcerol G Bridoux V Ducrotte P Quinton JF Leroi AMEfficacy of antibiotherapy for treating flatus incontinence associated withsmall intestinal bacterial overgrowth a pilot randomized trial PLoS One2017128e0180835 VandenbrouckeGrauls CM Vandenbroucke JP Effect of selectivedecontamination of the digestive tract on respiratory tract infections andmortality in the intensive care unit Lancet “Silvestri L van Saene HK Casarin A Berlot G Gullo A Impact of selectivedecontamination of the digestive tract on carriage and infection due togramnegative and grampositive bacteria a systematic review ofrandomised controlled trials Anaesth Intensive Care “ Camus C Salomon S Bouchigny C Gacouin A Lavoue S Donnio PYJavaudin L Chapplain JM Uhel F Le Tulzo Y Shortterm decline in allcause acquired infections with the routine use of a decontaminationregimen combining topical polymyxin tobramycin and amphotericin Bwith mupirocin and chlorhexidine in the ICU a singlecenter experienceCrit Care Med “ Olaitan AO Morand S Rolain JM Mechanisms of polymyxin resistanceacquired and intrinsic resistance in bacteria Front Microbiol Lauritano EC Gabrielli M Scarpellini E Lupascu A Novi

Thyroid_Cancer

"Nonsmall cell lung cancer is the most common cause of cancer death worldwide highlighting the need fornovel therapeutic concepts In particular there is still a lack of treatment strategies for the group of elderly and frail patientswho are frequently not capable of receiving standard therapy regimens Despite comprising the majority of lung cancerpatients this group is underrepresented in clinical trials This applies also to elderly and frail patients suffering fromunresectable stage III NSCLC who are unfit for chemotherapy and therefore cannot receive the standard therapycomprising of radiochemotherapy and the recently approved subsequent durvalumab consolidation therapy These patientsoften receive radiotherapy only which raises the concern of undertreatment The TRADEhypo trial aims at optimizingtreatment of this patient group by combining radiotherapy with concomitant durvalumab administration therebyemploying the immunepromoting effects of radiotherapy and determining safety feasibility and efficacy of this treatmentMethods design In this prospective phase II clinical trial durvalumab therapy will be combined with either conventionallyfractionated CONgroup or hypofractionated HYPOgroup thoracic radiotherapy A safety stopandgo leadin phase willassess safety of hypofractionated radiotherapy with respect to severe pneumonitis in small patient cohorts before ing fullenrollment Tumor tissue blood and stool samples will be collected before and during the study period to investigate theimmunological mechanisms responsible for checkpoint inhibitor efficacy and immunepromoting effects of radiotherapyContinued on next page Correspondence FarastukBozmehrmeduniheidelbergde1Department of Thoracic Oncology Thoraxklinik at University Hospital ofHeidelberg R¶ntgenstraŸe Heidelberg Germany2Translational Lung Research Center Heidelberg TLRCH Member of theGerman Center for Lung Research DZL Im Neuenheimer Feld Heidelberg GermanyFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cBozmehr BMC Cancer Page of Continued from previous pageDiscussion Preclinical data suggests that irradiationinduced immunogenicity can be further increased if applied in ahypofractionated setting potentially boosting the expected synergistic effect with immune checkpoint inhibition in restoringthe immune antitumor response If proven safe and efficient a hypofractionated radiation schedule can provide a considerablymore practicable option for the patient Taking into consideration the intend to develop a combination treatment strategy thatcan be made available to patients soon after proving to be efficient and the potentially elevated toxicity of a hypofractionatedradiotherapy approach this trial was designed as a twotrialsinone design An accompanying translational research program isplanned striving to gain insights into the tumorhost biology and to identify suitable biomarkers to predict therapy responseTrial registration Clinicaltrialsgov NCT04351256 Registered April EudraCT “ Registered October Keywords Nonsmall cell lung cancer NSCLC Radioimmunotherapy Immune checkpoint inhibition AntiPDL1 monoclonalantibody Hypofractionated radiation Geriatric risk profileBackgroundLung cancer is the most common cause of cancer deathworldwide with nonsmall celllung cancer NSCLCrepresenting “ of cases [ ] Improving therapeutic strategies is thus of imminent importance especially considering elderly and frail patients With a medianage of about years at diagnosis lung cancer clearly is adisease of the elderly yet this group is underrepresentedin clinical trials and these patients are frequently not capable of receiving standard treatment protocols due to ageand tobaccoassociated comorbidities [“]attracting both immunocompetentIn recent years the advent of immunotherapy has pavedthe way for novel therapeutic concepts including the combination of radiotherapy with immune checkpoint inhibitioneg Programmed cell death ligand PD1 PDL1 Thisapproach is of particular interest as it utilizes synergistic effects While immune checkpoint inhibitors can restore thepatients™ antitumor immunity through T cell activationradiotherapy may further boostimmunemediated anticancer mechanisms by exposing tumorassociated antigensand byantigenpresenting cells and tumoricidal effector cells [ ] Indeedfor patients with unresectable stage III NSCLC the PACIFICtrial has revealed a profound clinical benefit treatment withthe antiPDL1 monoclonal antibody durvalumab after chemoradiotherapy with remarkably low toxicities [ ] As aresult sequential treatment with durvalumab after chemoradiotherapy has become the new standard treatment for locally advanced unresectable NSCLC However about ofpatients do not receive chemotherapeutic agents presumablydue to significantly higher rates of age and comorbidityrelated adverse events AE under chemoradiotherapy [] Thus elderly and frail patients often receive radiotherapyalone raising the serious concern of undertreatment and theneed for new therapeutic concepts [ ]Considering the immunepromoting effects of radiotherapy a combination with durvalumab therapy mayimprove response rates in these potentially undertreatedpatients Moreoverif applied early concomitant localdatathatsuggestradiotherapy with systemic immunotherapy may particularly increase control of distant micrometastases Preclinicalirradiationinducedimmunogenicity can even be further increased if appliedin a hypofractionated setting with single doses ‰¥ GrayGy in line with a radiation dosedependent abscopal[“] While a hypofractionated radiationeffectschedule is also considerably shorter and more convenient for the patient safety of concurrent immunoradiotherapy is a concern as both therapy modalities maycause severe pneumonitisIn this prospective phase II clinical trial we thereforeaim to determine feasibility and treatment efficacy ofdurvalumab treatment combined with thoracic radiotherapy TRT in previously untreated NSCLC stage IIIpatients unable to receive radiochemotherapy Strivingto develop a combination treatment strategy thatifproven safe and efficient can be quickly made availableto patients a twotrialsinone design was chosen thatcombines durvalumab with either conventionally fractionated CONgroup or hypofractionated thoracicradiotherapy HYPOgroup This study not only aims toincrease the efficacy of radiotherapy by utilizing theimmunesensitizing effects elicited by PDL1 inhibitionbut will also provide biomaterials that will be analyzedwith respect to immunological mechanisms responsiblefor checkpoint inhibitor efficacy and immunepromotingeffects of radiotherapy as well as potential biomarkersMethodsdesignStudy designThe TRADEhypo trialis a prospective randomized label multicenter phase II trial with a safety stopandgo leadin phase Fig During the leadin phasepatients in the HYPOgroup who will receive durvalumab in combination with hypofractionated thoracicradiotherapy will be closely evaluated with regard totoxicity defined as pneumonitis ‰¥ grade within 0cBozmehr BMC Cancer Page of Fig Study design of the TRADEhypo trial Patients will be enrolled according to eligibility criteria and treated with either a hypofractionated TRTregimen HYPOgroup or conventionally fractionated TRT CONgroup in combination with durvalumab For the HYPOgroup a safety stopandgophase with a design precedes full enrollment Whenever this arm is for recruitment patients will be allocated to this arm until the cohort isclosed whenever HYPOarm is closed for Stop Go decision evaluation based on the toxicity assessment of this regimen weeks after the end of TRTpatients are allocated to the CONarm When the study proceeds to expansion phase patients will be allocated to treatment arms by randomizationusing œbiased coin algorithm An efficacy interim analysis will be performed after patients have been enrolled in each armweeks after radiotherapy in small cohorts n beforeproceeding with full enrollment into this arm Fig respectrelated biomarkersto treatmentinduced changes and immuneStudy settingThe TRADEhypo trial will recruit patients from participating centers across Germany over a period of months Start of recruitment was planned for April but was delayed to May due to the Covid19pandemic A full list of sites can be obtained at clinicaltrialsgov NCT04351256Study objectivesThe primary objective of this study is to evaluate safetyand tolerability of conventionally fractionated CONgroup and hypofractionated HYPOgroup TRT incombination with durvalumab in patients with unresectable stage III NSCLC unfit for chemotherapy Moreoverefficacies of the two modes of radiotherapy will be evaluated with respect to response rates Further parameterswill be determined in order to assess efficacy safety andquality of life QoL in both treatment arms by recordingincidence and severity of adverse events AEs as well asspecific laboratory abnormalitiesExploratory endpoints include assessment of vulnerability and analyses of tumor tissue blood and stoolsamples that are collected during the clinical trial withCharacteristics of participantsA total of patients will be included into this studyPatients potentially eligible for trialinclusion will beapproached and asked to participate as they present inthe clinic Before a patient™s participation in the clinicalstudy the investigator must obtain written informedconsentEach participant must be eligible regarding all inclusion and exclusion criteria set for this trial Table Key inclusion criteria include diagnosis of unresectablestage III NSCLC and nonfeasibility of sequential chemoradiotherapy due to increased oxygenindependentvulnerability as reflected by fulfilling at least one of thefollowing criteria i Performance status Eastern Cooperative Oncology Group [ECOG] scale ii ECOG and Charlson comorbidity index CCI ‰¥ or iii age ‰¥ Moreover participants must have at least one measurable site of disease as defined by RECIST as wellas adequate bone marrow hepatic and renal functionPatients having received prior immunotherapy other investigational agents or thoracic radiotherapy within thepast years will be excluded from the study Additionally participants must not have an active or recent history of a known or suspected autoimmune disease or 0cBozmehr BMC Cancer Page of Fig Cohort design of the safety stopandgo leadin phase HYPOgroup The safety leadin phase follows a design in order to carefullyevaluate the toxicity of the treatment in the HYPOgroup with respect of the occurrence of a grade pneumonitis œevent within weeksafter the end of TRT Two events in the first six patients two events in the first or two events in the first patients will result in terminationof the HYPOgroup œStop If no event is observed within the first two safety cohorts ie the first patients the HYPOarm will be ed forfull enrollment with close toxicity assessment with respect to pneumonitis grade and terminated as soon as two events are reported withinthe subsequent six patients œGo Full enrollment in the HYPOarm will only take place if the criteria for the nontoxicity scenario are met ie ‰¤ event in n patients œGoany other medical condition conflicting with the studyinterventions and not have used immunosuppressivemedication For a full list of the inclusion and exclusioncriteria see Table TreatmentDurvalumabPatients will be enrolled based on the inˆ’ exclusion criteria Two treatment groups will be evaluated Onegroup will receive durvalumab immunotherapy combined with conventionally fractionated TRT CONgroup and the other one with hypofractionated TRTHYPOgroup In both groups durvalumab will be administered intravenously at a fixed dose of mg onday and every weeks thereafter for a maximum of months maximum doses last dose at week untilconfirmed disease progressioninacceptable toxicitywithdrawal of consent or end of the study Fig andTable RadiotherapyAll patients are subjected to preparation of individualpositioning devices and CTbased planning Motionmanagement may comprise either 4DCT or midbreathing CT image acquisition Further imaging modalitiessuch as FDGPETCT may be used when deemed necessary Gross tumor volumes GTV will be contouredand expanded by adequate clinical CTV and planningPTV safety margins in order to account for subclinicaldisease and positioning errors No elective nodal irradiation will be performed As for ans at risk bothlungs spinal cord heart and esophagus must be contoured In the HYPOarm no more than of œbothlungs minus GTV should receive Gy in the CONarm no more than of œboth lungs minus GTVshould receive GyIn the HYPOarm fractions of Gy will be administered total dose Gy corresponding to GyBED αβ In the CONarm fractions of Gywill be administered total dose Gy corresponding to GyBED αβ Dose deviations of ± are acceptable when clinically indicated Radiotherapy isscheduled to start within h after the first administration of durvalumab Dose prescription will follow international reports ICRU and Both 3Dconformal and modulated photon radiation techniquessuch as IMRT and VMATRapdArc are acceptable Allparticipating institution are encouraged to perform regular if no daily positioning control using either portalimaging or onboardCT devicesStudy proceduresIn order to minimize the risk exposure of patients whensubjected to the hypofractionated radiation regimen a 0cBozmehr BMC Cancer Page of Table Complete list of inclusion and exclusion criteriaInclusion criteriacid129 Fullyinformed written consent and locally required authorization obtained from the patient legal representative prior to performing any protocolrelated procedures including screening evaluationscid129 Age ‰¥ yearscid129 Histologically documented diagnosis of unresectable stage III NSCLCcid129 Nonfeasibility of sequential chemoˆ’radiotherapy as determined by the site™s multidisciplinary tumor board if there is no tumor board then thisdecision will be made by the investigator in consultation with a radiation oncologist if the investigator is not a radiation oncologist or by the investigator in consultation with an oncologist if the investigator is not an oncologistcid129 Fulfills at least one of the following criteria—‹ ECOG —‹ ECOG and CCI ‰¥ —‹ Age ‰¥ yearscid129 Must have a life expectancy of at least weekscid129 FEV1 ‰¥ of predictedcid129 DLCO ‰¥ of predictedcid129 FVC or VC ‰¥ of predictedcid129 At least one measurable site of disease as defined by RECIST criteriacid129 Adequate bone marrow renal and hepatic functioncid129 Female patients with reproductive potential must have a negative urine or serum pregnancy test within days prior to start of trialcid129 Evidence of postmenopausal status or negative urinary or serum pregnancy test for female premenopausal patientscid129 The patient is willing and able to comply with the protocol for the duration of the study including hospital visits for treatment and scheduledfollowup visits and examinationsExclusion criteriacid129 Concurrent enrollment in another clinical study or enrollment within days prior to first dose of treatment unless it is an observational noninterventional clinical study or during the followup period of an interventional studycid129 Prior immunotherapy or use of other investigational agentscid129 History or current radiology suggestive of interstitial lung diseasecid129 Oxygendependent medical conditioncid129 Any concurrent chemotherapy investigational product IP biologic or hormonal therapy for cancer treatmentcid129 Prior thoracic radiotherapy within the past years before the first dose of study drugcid129 Major surgery within weeks prior to enrollment into the study patients must have recovered from effects of any major surgery Local nonmajorsurgery for palliative intent is acceptablecid129 Active or prior documented autoimmune or inflammatory disorders with the following exceptions Patients with vitiligo or alopecia patients withhypothyroidism stable on hormone replacement or any chronic skin condition that does not require systemic therapy Patients without activedisease in the last years may be included but only after consultation with the study physiciancid129 Active uncontrolled inflammatory bowel disease Patients in stable remission for more than year may be includedcid129 Uncontrolled intercurrent illness including but not limited to ongoing or active infection symptomatic congestive heart failure uncontrolledhypertension unstable angina pectoris uncontrolled cardiac arrhythmia interstitial lung disease gastrointestinal conditions associated with diarrheaor psychiatric illnesssocial situations that would limit compliance with study requirement substantially increase risk of incurring AEs or compromisethe ability of the patient to give written informed consentcid129 History of another primary malignancy except for a malignancy that has been treated with curative intent and was not active for ‰¥ years beforethe first dose of IP and of low potential risk for recurrence or adequately treated nonmelanoma skin cancer or lentigo maligna without evidence ofdisease or adequately treated carcinoma in situ without evidence of diseasecid129 History of leptomeningeal carcinomatosiscid129 History of active primary immunodeficiencycid129 History of allogenic an or tissue transplantationcid129 Clinical diagnosis of active tuberculosiscid129 Positive testing for hepatitis B virus surface antigen or hepatitis C virus RNA indicating acute or chronic infection or for human immunodeficiencyviruscid129 Current or prior use of immunosuppressive medication within days before the first dose of durvalumab The following are exceptions to thiscriterion Intranasal inhaled topical steroids or local steroid injections systemic corticosteroids at physiologic doses not to exceed mgday ofprednisone or its equivalent steroids as premedication for hypersensitivity reactionscid129 Receipt of live attenuated vaccine within days prior to the first dose of IPcid129 Female patients who are pregnant or breastfeeding or male or female patients of reproductive potential who are not willing to employ effectivebirth controlcid129 Known allergy or hypersensitivity to any of the IPs or any of the constituents of the productcid129 Any coexisting medical condition that in the investigator™s judgement will substantially increase the risk associated with the patient™s participationin the studycid129 Patient who has been incarcerated or involuntarily institutionalized by court order or by the authorities § Abs S Nr AMGcid129 Patients who are unable to consent because they do not understand the nature significance and implications of the clinical trial and thereforecannot form a rational intention in the light of the facts [§ Abs S Nr 3a AMG]safety leadin phase with stopandgo design will precedefull enrollment into the HYPOgroup Fig Toxicitywill be evaluated with a design that is based on thestatistical assumption that ‰¤ event in n patientsconforms to a nontoxicity scenario with œevent beingdefined as the occurrence of pneumonitis grade ‰¥ within weeks after end of TRT Consequently twoevents in the first six patients or two events in the first 0cBozmehr BMC Cancer Page of Table Schedule of assessmentsProcedure Point in timeInformed Consent eligibility criteria demographicsmedical and historyAllocation RandomizationPrior and Concomitant Medication ReviewDurvalumab administrationRadiotherapy CONa or HYPObAEsFull Physical ExaminationDirected Physical ExaminationVital Signs O2 Saturation and WeightPulmonary function tests12lead ECGECOG Performance StatusPregnancy Test CBC with Differential Serum ChemistryPanel Thyroid function testHBV HCVUrinanalysisTumor ImagingFACTL and G8 screening questionnaireTissueScreening Treatment Cycles Q4WScreening C1D1 C1D4 C2D1 C3D1 C4D1XC5D1C13D1EOT PostTreatmentEOT Safety FU FU Q12WXXXXXXXXXXXXXXXXXXXXXXXXXXXXXcXX cXXXXXXXXXXXXXtogether with staging XWhenever clinically indicatedXXXXXXXXXXXXXXXXWhenever clinically indicatedXdXdXeQ8WeXfXXXtogether with staging XXXgXhXXiOptional ReBiopsy at time of progressionXiXXBlood and stoolaCONgroup radiation scheme Patients receive conventional fractions of — Gy Gy within weeks of TRT to be started within h after start ofdurvalumab treatmentbHYPOgroup radiation scheme Patients receive hypofractionated thoracic radiotherapy consisting of — Gy Gy within weeks of TRT to be startedwithin h after start of durvalumab treatmentcTo be performed on C2D1 and C3D1 if in accordance with local standarddChest Xray to be performed on cycles and if in accordance with local standardeFirst onstudy CT imaging to be performed weeks after first durvalumab administration Further onstudy imaging to be performed Q8W days ± daysfOnly applicable if EOT not according to already detected disease progressiongIn patients with EOT not due to disease progression tumor imaging will be performed until the start of a new anticancer treatment disease progression deathwithdrawal of consent or the end of the studyhQuestionnaires will be collected until disease progression only and may be collected by telephone callsiBiomarker sample to be taken prior to first study drug medication either during screening or C1D1 visitX or two events in the first patients will result in termination of the HYPOgroup Fig During this safety stopandgo phase patients will beallocated to treatment arms asfollows While theHYPOarm is recruiting patients will exclusively enterthis treatment group During safety evaluation of the sixpatients of a HYPOcohort Stop Go decision patientswill be allocated to the CONgroup only If safety criteria in the HYPOcohort are met the HYPOarm willbe re ed to continue toxicity assessment and patients will solely be allocated to this arm Fig Inorder to avoid any bias patients will be allocated centrally by the study management during this phase If thenontoxicity criteria in the safety cohorts are met thestudy will proceed to theandremaining patients will be randomized into the twotreatment arms using an interactive web responseexpansion phasesystem integrated in the electronic case report formeCRF A possibly uneven distribution of patients between the treatment groups at this stage will be compensated by a randomization strategy based on the œbiasedcoin method [ ] In the randomization phase patients will be stratified according to tumor stage stageIIIA vs stage IIIBIIICIn total patients will be enrolled per group Aftern patients have been enrolled to the HYPO orCONtreatment arm respectively an interim efficacy analysis for the respective arm will be conducted based on theobjective response rate ORR at weeks after first durvalumab administration In case of insufficient efficacy ofone of the arms ie the number of patients who haveachieved a response is eight out of or lower this treatment arm will be terminated Recruitment will be halteduntil results of the interim efficacy analysis are available 0cBozmehr BMC Cancer Page of Tumor response will be assessed according to RECIST by CT and or MRI scans at baseline weeks afterdurvalumab initiation and then every weeks Safetymeasures willinclude physical examinations performance status ECOG clinical laboratory profiles and continuous assessments of AEs An overview of all studyprocedures is presented in Table An Independent Safety Monitoring Board ISMB willensure the continued safety of participants throughoutthe trial Data management and data quality assurancewill be conducted following the Standard OperationalProcedures of the Institute of Clinical Cancer ResearchIKF at Northwest Hospital GmbH Frankfurt GermanyAn eCRF will be carefully maintained for each participant for data collection also reporting serious and nonserious AEs following the common criteria for adverseevents CTCAE version throughout the entire trialAfter the end of the study participants will be proactively followed up regarding treatmentrelated AEsuntil resolved returned to baseline or considered irreversible until lost to followup or withdrawal of studyconsent All subjects will be followed for survival Patients who decline to return to the site for evaluationswill be offered a followup FU by phone every months as an alternative At the end of the study treatment the investigators are responsible for the furthertreatment of the patient and must ensure that he or shereceives appropriate standard of care or other appropriate therapiesSampling for translational researchIf patients participate in the translational research program blood samples will be collected prior to cycles and and at the time of disease progression or end oftreatment EOT along with stool samples Table Samples of untreated tumor lesions serving as baselinespecimens will be collected as paraffinembedded tissueIf rebiopsies are taken at the time of progression samples should also be also submitted for translationalresearchStudy endpointsThe primary endpoint of the study will be toxicity defined by the occurrence of treatmentrelated pneumonitis grade ‰¥ The ORR evaluated weeks after firstdurvalumab administration according to RECIST isset as the primary efficacy endpoint Secondary endpoints ofthe occurrence oftreatmentrelated AEs and serious AEs SAEsfrequency of prespecified abnormal laboratory parametersprogressionfree survival PFS and duration of clinicalbenefit metastasisfree survival overall survival OSand QoLcomprisethestudyPatient vulnerability and its association with survivaland outcome will be assessed as an exploratory endpoint To this end the G8screening questionnaire asimple and fast screening tool for identifying geriatricrisk profiles with a strong prognostic value for functionaldecline and OS in older populations with cancer will beused [] Furthermore biomaterials will be collectedduring the trial for correlation analyses on selected molecular parameters and clinical data in order to identifymolecular biomarkers predictive for response to therapyThisto obtainhypothesisgenerating data for future research due to theexplorative character of this trialapproach is deemed appropriateStatistical analysisSample size justificationSafety runin phase HYPOgroup With regard to thepneumonitis grade ‰¥ rate this phase is designed to distinguish between a toxicity scenario pTox and anontoxicity scenario pTox A sample size ofn will yield a probability of to correctly detectthe toxicity scenario while the nontoxicity scenario willcorrectly be detected with a probability of Theseprobabilities are based on the decision rule that if thenumber of patients with pneumonitis grade ‰¥ in thiscohort is ‰¥ recruitment to the HYPOgroup will beterminatedanalysisInterim efficacyregarding ORR andexpansion phase An interim efficacy analysis based onthe ORR will be conducted after n patients in eacharm have completed radiotherapy and the 18th patienthas undergone first radiographic assessment at weeksafter first durvalumab dosePreviously an ORR of after radiotherapy alonehas been reported in a Japanese population of elderly patients with unresectable stage III NSCLC [] Based onthis and the observation that Asian ethnicity is associated with a favorable prognosis we assume for theTRADEhypo trial that an ORR higher than in bothtreatment arms can be demonstrated ie the null hypotheses for arm HYPO and CON are defined as H0HYPO Ï HYPO ‰¤ and H0CON Ï CON ‰¤ where ÏHYPO and Ï CON denote the actual ORR in arm HYPOand CON respectively [ ] Under the alternativehypothesis it is assumed that both Ï HYPO and Ï CONamount to Using an optimal Simon™s twostage design with a onesided significance level of α and apower of 1β for each hypothesis test n patients per arm are required while the interim analysis isconducted after n patients per arm have been recruited to the trial After successfully passing the safetyanalysis in the HYPOgroup if among patients in the 0cBozmehr BMC Cancer Page of HYPO or CONarm the number of patients who haveachieved a response is eight or lower the respective armwill be closed Otherwise an additional number of patients will be enrolled into the respective arm Thenull hypothesis of the respective arm can be rejected ifat least of all patients per arm achieve a responseSample size calculation was done using the R packageœOneArmPhaseTwoStudy []To account for an estimated dropout rate of fourpatients will additionally be recruited to each treatmentarm Deviations from planned sample sizes will be handled as described by Englert Kieser allowing strictcontrol of the aspired type I error rate in each arm []Methods of statistical analysis The primary populationfor evaluating all efficacy endpoints and subject characteristics is defined as all patients enrolled according toinitial allocationrandomization intentiontotreat population IIT Secondary efficacy analyses will be carriedout on the perprotocol PP population The safetypopulation comprising all patients who received at leastone dose of the study medication will be used for allsafety endpoints and will be analyzed according to theactual treatment receivedResponse rates with confidence intervals CI and pvalues in both study arms will be estimated taking intoaccount the twostage nature of the design [ ] Secondary endpoints will be evaluated descriptively All toxicities will be summarized by relative and absolutefrequency and severity grade based on CTCAE V50 AEand SAE summary tables will provide the number andpercentage of patients with AEs and the ClopperPearson type CIs All analyses will be done using SASversion SAS Institute Cary NC or higherTrial statusAs of July 15th eight study sites are initiated Firstinitiation coincided with the beginning of the Covid19pandemic in Germany Therefore recruitment of patients was withheld On May 8th recruitment wasresumed after consultation with the ISMB The first patient was enrolled on July 13th renal carcinoma and NSCLCDiscussionIn recent years the concept of restoring the patients™ antitumor immunity by immune checkpoint inhibition hasrevolutionized cancer therapy especially in advancedmelanomaImmunecheckpoint molecules efficiently regulate T cell activation and thus enable tumor cells to evade the immunesystem for example by hijacking the PD1 PDL1 interaction to downregulate effector T cells [ ] To dateseveral human monoclonal antibodies pharmacologicallyblocking these interactions have been implemented incancer therapy such as the antiPD1 antibody pembrolizumab that has been approved in combination withchemotherapy for nonsquamous NSCLC irrespective ofPDL1 expression [ ]Several studies have shown that immune checkpointinhibition and radiotherapy in combination can act synergistically to further boost antineoplastic effects [“] Although in a large phase III trial no benefit ofblockade of cytotoxic T lymphocyteassociated antigen CTLA4 after radiotherapy was observed in metastaticprostate cancer [] clinical trials such as NICOLASNCT02434081 and DETERRED NCT02525757investigating concurrent PDL1directed immunotherapyand chemoradiot

Thyroid_Cancer

performance demonstration of a hybrid compton camera with an active pinhole for wide‘band X‘ray and gamma‘ray imagingAkihisa omata1 Jun Kataoka1 Kazuya fujieda1 Shogo Sato1 eri Kuriyama1 Hiroki Kato2 Atsushi toyoshima3 takahiro teramoto3 Kazuhiro ooe2 Yuwei Liu2 Keiko Matsunaga2 takashi Kamiya2 tadashi Watabe2 eku Shimosegawa2 Jun Hatazawa2X‘ray and gamma‘ray imaging are technologies with several applications in nuclear medicine homeland security and highenergy astrophysics However it is generally difficult to realize simultaneous wideband imaging ranging from a few tens of keV to MeV because different interactions between photons and the detector material occur depending on the photon energies for instance photoabsorption occurs below keV whereas Compton scattering dominates above a few hundreds of keV Moreover radioactive sources generally emit both X‘ray and gamma‘ray photons in this study we develop a œhybrid compton camera that can simultaneously achieve X‘ray and gamma‘ray imaging by combining features of œcompton and œpinhole cameras in a single detector system Similar to conventional compton cameras the detector consists of two layers of scintillator arrays with the forward layer acting as a scatterer for highenergy photons keV and an active pinhole for lowenergy photons keV The experimental results on the performance of the hybrid camera were consistent with those from the Geant4 simulation We simultaneously imaged Am keV and Cs keV in the same field of view achieving an angular resolution of —¦ FWHM for both sources in addition imaging of At was conducted for the application in future nuclear medicine particularly radionuclide therapy the initial demonstrative images of the At phantom were reconstructed using the pinhole mode using keV and Compton mode using keV exhibiting significant similarities in sourceposition localization We also verified that a mouse injected with MBq of At can be imaged via pinhole‘mode measurement in an hourIn the field of nuclear medicine it is essential to visualize the distribution of radioisotopes in a patient™s body Particularly a radiological diagnosis that enables noninvasive visualization of the pathology from outside the body is important The general approach is to visualize nuclear gamma rays emitted from radioactive tracers Two common techniques”single photon emission computed tomography SPECT and positron emission tomography PET”play important roles in the diagnosis However they image a specific energy range of either Xrays or gamma rays SPECT can image gamma rays with energies less than a0keV with the use of the collimator whereas PET can image positron emitters that emit 511keV gamma rays These lead to a limited number of radioactive tracers that can be imaged only with current SPECT and PET scanners In this context a Compton camera12 that can conduct imaging in a wide energy band is reckoned to cause a breakthrough in nuclear medicine34 Attempts are being made to develop Compton cameras aiming the image of prompt gamma rays emitted from the patient™s body during proton therapy5“ In addition several works have aimed to realize tracer visualization using the Compton camera For example Fontana et a0al describe the Compton camera as an 1Graduate School of Advanced Science and Engineering Waseda University Tokyo Japan 2Graduate School of Medicine Osaka University Osaka Japan 3Institute for Radiation Science Osaka University Osaka Japan email omt22fujiwasedajpScientific RepoRtS 101038s41598020710195Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Comparison of the simulated energy response of the intrinsic efficiency left and angular resolution right with the results of actual measurementselectronically collimated SPECT device which is however optimized for higher energies above a few hundreds of keV9 Yoshida et a0al proposed whole gamma imaging WGI wherein a conventional PET scanner is converted by inserting an additional scatterer to create a Compton camera10 They succeeded in realizing the triple gamma ie PETCompton imaging of 44Sc which emits keV and a0keV gamma rays however the application to imaging SPECT tracer remains to be researched in the future In this context the simultaneous capture of SPECT and PET images has also been reported using a Compton camera consisting of SiCdTe semiconductors which is however limited both in terms of detection efficiency and angular resolution11“ We argue that such difficulties can be easily overcome with the hybrid camera proposed in this paper This increases the variety of radioactive tracers available for nuclear medicine14“ The emergence of new tracers may solve current problems such as manufacturing costs of tracers in addition to achieving improvements in the diagnosis quality A Compton camera also enables the simultaneous imaging of multiple tracers this significantly increases the information obtained from living anisms in a single diagnosisMoreover nuclear medicines involving radioactive sources are applied not only in the diagnosis but also in the treatment of diseases such as cancer Particularly radionuclide therapy RNT1920 which uses the targeted radionuclide by administering radioisotopes to patients is widely used because it damages cancer cells while limiting the exposure of healthy tissues to radiation For instance RNT with α ps is receiving significant attention because of its high therapeutic potential owing to the higher ionization power for damaging cancer cells2122 Nevertheless once administered into the body it is difficult to determine the distribution and pharmacokinetics of a radionuclide For the safety and effectiveness of RNT it is conceivable to visualize the characteristic Xrays and nuclear gammarays emitted simultaneously with the alpha decay Some of these characteristic X rays and nuclear gamma rays can be visualized by SPECT Furthermore the use of a Compton camera for the in a0vivo visualization of 223Ra an αp emitter used for RNT was suggested in our previous study23 However these radionuclides emit several Xrays and gamma rays with different branching ratios Some emit strong characteristic Xrays that can be imaged with a SPECT whereas others emit highenergy gamma rays that can be imaged only with a Compton camera see Table a0 Imperatively a simple and costeffective imaging system that is sensitive to both Xrays and gamma rays is highly desiredThis paper proposes a œhybrid Compton camera that realizes simultaneous wideband imaging from a few tens of keV to approximately an MeV combining some features of œCompton cameras and œpinhole cameras in a single detector system Although the hybrid camera consists of two layers of positionsensitive detectors similar to a Compton camera1323 the front detector has a hole in the center Compton and pinhole imaging are enabled using the front detector as a scatterer for highenergy photons a0 keV and an active pinhole for lowenergy photons a0 a0keV We developed a prototype of the hybrid camera Simulation and experimental results depicted resolutions better than —¦ full width at half maximum FWHM in the range of “ a0keV In addition the imaging of At is receiving attention as a source applicable to RNT with αps24“ We initially investigated the capability of our hybrid camera system with a simple phantom of At and thus conducted mouse imagingResultsperformance evaluation The imaging performance of the hybrid camera was initially evaluated by simulations using Geant428 The simulation configuration includes the scintillator arrays the MPPC arrays and the metal case which are detailed in the œMethods section A monochromatic point source placed a0cm from the camera and at the center of the field of view FOV was imaged The detector performance was investigated between and a0keV for both the pinhole and Compton modes Considering the energy and spatial resolution of the actual detectors we utilized a reconstruction flow as described in the œMethods section The simulation showed that pinhole imaging is difficult over a0keV whereas Compton imaging is active at approximately a0keV or higher Figure a0 left details the intrinsic detection efficiency that indicates the proportion of events detected either as the pinhole or the Compton mode to all radiation emitted towards the detector The intrinsic detection efficiency Ç«int is expressed by the following equation using the absolute detection efficiency Ç«absScientific RepoRtS 101038s41598020710195Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Upper The experimental configuration of the simultaneous measurement of Am and 137Cs Lower The MLEM reconstructed images of the Am a0keV source analyzed by the pinhole mode left and the Cs a0keV source analyzed by the Compton mode rightÇ«int 4Ï�ǫabswhere 01 denotes the solid angle of the detector viewed from the source Figure a0 right shows the angular resolution of the pinhole and Compton images as a function of energy For Compton imaging we use the angular resolution measure ARM that is commonly used to measure the angular resolution of Compton cameras For measuring the angular resolution of pinhole imaging the �θ value is obtained by geometrically converting the position resolution of the image to angular resolution Note that for the source placed at the center of the FOV the angular resolution �θ can be calculated as follows�θ arctan�x2lwhere 01x and l denote the position resolution FWHM of the pinhole image and the distance between the camera and the source respectively The angular resolution was better than —¦ in the range of “ keV exhibiting the higher efficiency of the proposed hybrid camera than those of the conventional camerasSubsequently the fundamental imaging performance of the hybrid camera was evaluated by performing experiments under the same geometry as in the simulation The measurements were recorded at energies of a0keV and a0keV for pinhole imaging and a0keV and a0keV for Compton imaging The obtained angular resolutions were consistent with the values predicted by the simulation as represented by the red plots in Fig a0 rightExperimental demonstration of wideband imaging To examine the performance of the hybrid camera we conducted simultaneous imaging of Am and Cs sources As shown in Fig a0 upper these sources were placed cm away from the camera and at —¦ and ˆ’ —¦ respectively from the center of the FOV The Am source was reconstructed in the pinhole mode a0keV whereas the Cs source was reconstructed in the Compton mode a0keV From Fig a0 lower each convergence indicated the correct positions depicting the potential of broadband imaging using the hybrid cameraAs a next step extended sources were measured to examine the validity of the camera system including the image reconstruction technique The extended a0mm × a0mm œLshaped sources were reproduced by moving on a stage that automatically moved at a constant speed a0mmmin The image of an œLshaped Am source Scientific RepoRtS 101038s41598020710195Vol0123456789wwwnaturecomscientificreports 0cFigure a0 The MLEM reconstructed images of œLshaped sources Pinhole reconstruction of the Am source left and Compton reconstruction of the Cs source right that were measured separatelyFigure a0 Energy spectrum of At obtained by a LaBr3 scintillator coupled to a PMTwas reconstructed by the pinhole mode using events with energies around a0keV Subsequently the image of an œLshaped Cs source a0keV was also reconstructed by the Compton mode MLEM reconstruction images of each measurement are presented in Fig a0 At imaging of a small bottle Furthermore we investigated the performance of the hybrid camera with the imaging of a At source which is an αps source for RNT Initially the energy spectrum of At was obtained by a LaBr3 scintillator coupled to a photomultiplier tube PMT Figure a0 shows peaks of characteristic Xrays mainly a0keV and gamma rays with energies of and a0keV Furthermore the intensity of Xrays is approximately three orders of magnitude higher than that of the gamma raysNext placing a0cm away from the hybrid camera at —¦ the center of the FOV and —¦ to the right a small bottle was imaged with μL of At a0MBq The measurement times were and a0min at —¦ and —¦ respectively The images were reconstructed using a0keV Xrays in the pinhole mode and a0keV gamma rays in the Compton mode The measurements resulted in pinhole events and Compton events at —¦ and pinhole events and Compton events at —¦ The pinhole images of the a0keV Xrays and the Compton images of the a0keV gamma rays are presented in Fig a0 At imaging of a mouse To investigate the capability of the hybrid camera for animal imaging imaging of a mouse [8weekold male ICR mouse SLC Japan Hamamatsu Japan] with a At tracer was conducted The mouse length a0mm weight a0g was injected with At a0kBq a0h before the measurement The mouse was euthanized by an overdose of isoflurane a0h after the injection The hybrid camera was positioned on the right side of the mouse a0mm from the body axis as shown in Fig a0 left The measurement time was a0h resulting in pinhole events and Compton events From Fig a0 center the pinhole image depicted that the distribution of the At converged on the thyroid stomach and bladder from events obtained after a0h of measurement Moreover although the Compton image confirms the concentration near the Scientific RepoRtS 101038s41598020710195Vol1234567890wwwnaturecomscientificreports 0cFigure a0 The pinhole MLEM reconstruction image left and the Compton MLEM reconstruction image right of a bottle with At at the center of the FOV upper and —¦ to the right lowerFigure a0 Left Experimental configuration of the measurement of the mouse administered with 211At Center The pinhole MLEM reconstructed image obtained by a0h of measurement Right The Compton MLEM reconstructed image obtained by a0h of measurementcenter accumulation is splitting possibly owing to the lack of event statistics as shown in Fig a0 right All the animal experiments were approved by the animal ethics committees of Osaka University and were performed according to the institutional guidelinesScientific RepoRtS 101038s41598020710195Vol0123456789wwwnaturecomscientificreports 0cRadionuclide211At213Bi225Ac223Ra212BiX a0ray keV Gamma ray keV absolute intensity Pinhole Comptoncid31cid31cid31cid31cid31cid31Table Examples of αp emitters for RNT and their main Xrays and gamma rays and their absolute intensities in percentage accompanied by the adaptability of the pinhole mode andor the Compton modeFigure a0 The configuration of the hybrid camera left Schematic view of the pinhole event center for the lower energy range and the Compton event right for the higher energy range that are used for the pinholeCompton reconstruction in the hybrid cameraDiscussionIn the imaging experiment with the At inside a small bottle the source position was obtained by pinhole reconstruction using the events accumulated in the first s The time taken to localize the convergence through the pinhole mode was of that thorough the Compton mode This is due to the intense Xray emission from the 211At as shown in Fig a0 a0keV Xrays are statistically advantageous over a0keV gamma rays for imaging 211At In this regard it is comprehensible that Compton events are not enough to reconstruct the distribution of the At tracer although it can be imaged through the pinhole mode There are several αemitting nuclides that can be used for RNT in the future2122 The distribution of these nuclides should be comprehensible and controlled by monitoring characteristic Xrays and nuclear gamma rays from outside the body Table a0 summarizes several properties of αemitting nuclides that are planned for clinical use Moreover Xray or gammaray energy suitable for a specific imaging application may use different nuclides and consequently different energies as the a0keV for At investigated here Although some nuclides are easy to image with lowenergy Xrays others are suitable for gammaray imaging for example Ac and 212Bi The wide scope of the hybrid cameras allows us to select the appropriate energy from lowenergy Xrays to highenergy gamma rays and thus potentially cover a wide energy range including the conventional SPECT a0keV PET a0keV and Compton camera subMeV to MultiMeV for various applications This leads to a reduction in the measuring time thus reducing the burden on the patient In addition wideband imaging can image multiple tracers simultaneously which significantly increases a patient™s complete medical information obtained from a single diagnosisTo improve the performance for further applications the hole size in the front detector should be adjusted as a tradeoff between the efficiency and the resolution of pinhole imaging Additionally the application of the depthofinteraction technique29“ to the backward detector may improve the resolution Therefore the energy range of the pinhole and Compton modes can be adjusted based on the density andor thickness of the detectors By revising the structure of the detector imaging in a wider band can be realized Particularly Compton cameras developed for gammaray astronomy have realized imaging in high energy bands such as “ a0MeV32 By applying the configuration of the hybrid camera as proposed in this paper these Compton cameras can perform pinhole imaging without compromising on the performance of the original Compton camera Currently we are developing a new hybrid camera that covers a0keV to a0MeV by adopting the configuration of the scintillator as described in Kishimoto et a0al and Hosokoshi et a0al MethodsConfiguration of the hybrid camera The configuration of the hybrid camera is detailed in Fig a0 left The hybrid camera consists of a pair of positionsensitive detectors capable of receiving the reaction position and the energy deposit of each event The detectors are composed of Cedoped Gd3Al2Ga3O12 GAGG scintilScientific RepoRtS 101038s41598020710195Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Left Energy spectrum of all the events detected in either detector black the events accumulated by with the front detector green the events obtained only with the rear detector blue and the events reacted with both detectors red accumulated from placing Am and Cs sources simultaneously in front of the detector Right 2D energy spectrum of coincidence events from the front detector scatterer and the rear detector absorber The area painted red corresponds to the energy cut range for a0keV Compton events The brightest area Efront ˆ¼ a0 a0keV corresponds to backscattering eventsModePinholeComptonCoincidence selectionAnticoincidence rearCoincidence front and rearEnergy cutRearSum and frontTable Factors of event selection for each reconstruction modelator arrays3334 coupled with multipixel photon counter MPPC arrays29 A × array of × × a0mm3 GAGG pixels is used as the front detector and a × array of × × a0mm3 GAGG pixels is used as the rear detector The front detector has an active × mm2 pinhole in its center The distance between the detectors is a0cm The energy resolutions of the front and rear detectors are and FWHM at a0keV respectively The position resolution for gammaray interaction is equal to the pixel size both for the pinhole and Compton mode The time range of the coincidence is set to be ± a0 a0μs and the count rate capability is ‰¤ a0 a0kHz The camera is enclosed in a mmthick metal mainly tungsten density a0gcm3 case except the front surfaceThe proposed hybrid camera enables imaging similar to that of a pinhole camera in addition to the conventional Compton camera Commonly the Compton camera which consists of a scatterer and an absorber uses events that undergo Compton scattering in the scatterer and photoabsorption in the absorber as shown in Fig a0 right For each event the scattering angle of Compton scattering is calculated using Compton kinematics restricting the arrival direction in the conical area called the Compton cone The position of the radiation source is identified by superimposing the Compton cones However photons with energy lower than several hundreds of keV cannot be imaged by the Compton camera owing to the increased probability of photoabsorption in the front scatterer The hybrid camera can also make use of such photoabsorption events We have devised a method to operate the camera as a Compton and a pinhole camera by utilizing the front scatterer with a hole in the center As shown in Fig a0 center the arrival directions of lowenergy photons are limited by analyzing the scatterer as an active pinhole Among lowenergy events detected in the rear detector the events that are not detected in the front detector can be considered to have passed through the hole in the front detector Note that Compton andor pinhole reconstruction can be selected analytically after the measurementReconstruction flow for each mode The selection of valid events for Compton and pinhole modes are parameterized with two factors coincidence selection and energy cut Coincidence is an event pattern hitting one or both of the detectors Specifically only anticoincidence events wherein only the rear detector is triggered are used for pinhole mode imaging whereas coincidences between the front and rear detectors are used for Compton mode reconstruction An energy cut is used to restrict the energy range of photons deposited in each detector The event selection criteria for each mode are summarized in Table a0 From Fig a0 left blue the spectrum from the rear detector in the anticoincidence mode simultaneously irradiated with Am and Cs sources shows that a0keV Xray is accurately masked in the front detector Therefore the pinhole events Scientific RepoRtS 101038s41598020710195Vol0123456789wwwnaturecomscientificreports 0cRadionuclide241Am137Cs211At211AtTarget keV ModePinholeComptonPinholeComptonEnergy cut keV Erear Efront Efront Erear Erear Efront Efront Erear Table The numbers for applied energy cutsare selected by the energy cuts with the corresponding energy range On the other hand Compton events are chosen from candidates of coincident events Figure a0 left red shows the spectrum from coincidence events obtained with mixed Am and Cs measurement Therefore Compton events are chosen from the region of interest reddiamond restricted from the total energy deposit a0keV a0 Efront Erear a0keV and energy deposit of front detector to reject the backscattering events Efront a0keV Furthermore the events whose scattering angles cannot be determined geometrically are deleted A red diamond in the 2D spectrum of the coincidence events shown in Fig a0 right corresponds to valid events for Compton image reconstruction The quantitative numbers applied for energy cut are summarized in Table a0 In image reconstruction maximum likelihoodexpectation maximization MLEM a statistical approximation method153536 is used to improve the image quality This method uses statistical iterations to locate sources with greater accuracy and better signaltonoise ratio alternate to the simple back projection method The number of iterations is for each mode except for the œLshaped Compton image which is In the pinhole mode the signaltonoise ratio is also improved by subtracting the background image that was reconstructed from the events next to the source energy rangeReceived May Accepted August References Schönfelder V et al The imaging Compton telescope COMPTEL on the gamma ray observatory IEEE Trans Nucl Sci “ 101109TNS198443333 Schöenfelder V et al Instrument description and performance of the imaging gammaray telescope COMPTEL aboard the Compton gammaray observatory Astrophys J Suppl Ser 10108619179 Todd R Nightingale J Everett D A proposed γ camera Nature “ 10103825113 2a0 Kataoka J et al Ultracompact Compton camera for innovative gammaray imaging Nucl Instrum Methods Phys Res Sec A Acceler Spectrom Detect Assoc Equip “ 101016jnima201709048 Koide A et al Precision imaging of MeV gamma rays using a 3D position sensitive Compton camera Sci Rep “ 101038s4159 Mochizuki S et al Highprecision Compton imaging of MeV prompt gammaray toward an online monitor for proton therapy Nucl Instrum Methods Phys Res Sect A Detect Assoc Equip “ 101016jnima201811032 Golnik C et al Tests of a Compton imaging prototype in a monoenergetic MeV photon field”a benchmark setup for prompt gammaray imaging devices J Instrum P06009 101088174802211106P0600 Hilaire E Sarrut D Peyrin F Maxim V Proton therapy monitoring by Compton imaging influence of the large energy spectrum of the promptγ radiation Phys Med Biol 101088003191556183127 Fontana M Dauvergne D Létang J M Ley JL Testa E Compton camera study for high efficiency SPECT and benchmark with Anger system Phys Med Biol 10108813616560aa926 a Yoshida E et al Whole gamma imaging a new concept of PET combined with Compton imaging Phys Med Biol Nakano T et al Imaging of 99mTcDMSA and 18FFDG in humans using a SiCdTe Compton camera Phys Med Biol 05LT01 10108813616560ab8e8 10108813616560ab33d 10108813616560aae1d Sakai M et al In a0vivo simultaneous imaging with 99m Tc and F using a Compton camera Phys Med Biol Sakai M et al Compton imaging with 99m Tc for human imaging Sci Rep “ 101038s4159 z Kishimoto A et al Development of a compact scintillatorbased highresolution Compton camera for molecular imaging Nucl Instrum Methods Phys Res Sect A Acceler Spectrom Detect Assoc Equip “ 101016jnima201606056 Kishimoto A et al First demonstration of multicolor 3D in a0vivo imaging using ultracompact Compton camera Sci Rep “ 101038s4159 w Motomura S et al Gammaray Compton imaging of multitracer in biological samples using strip germanium telescope IEEE Trans Nucl Sci “ 101109TNS200789420 Motomura S Kanayama Y Haba H Watanabe Y Enomoto S Multiple molecular simultaneous imaging in a live mouse using semiconductor Compton camera J Anal At Spectrom “ 101039B8029 64D Motomura S et al Improved imaging performance of a semiconductor Compton camera GREI makes for a new methodology to integrate biometal analysis and molecular imaging technology in living anisms J Anal At Spectrom “ 101039C3JA3 0185K Hamilton J G Soley M H Studies in iodine metabolism by the use of a new radioactive isotope of iodine Am J Physiol Legacy Content “ 101152ajple gacy19391273557 Odonoghue J Bardies M Wheldon T Relationships between tumor size and curability for uniformly targeted therapy with betaemitting radionuclides J Nucl Med “ Poty S Francesconi L C McDevitt M R Morris M J Lewis J S Alpha emitters for radiotherapy from basic radiochemistry to clinical studies”part J Nucl Med “ 102967jnume d11720465 Sgouros G et al MIRD Pamphlet No Abridged radiobiology and dosimetry of αp emitters for targeted radionuclide therapy J Nucl Med “ 102967jnume d10805865 Scientific RepoRtS 101038s41598020710195Vol1234567890wwwnaturecomscientificreports 0c Fujieda K et al First demonstration of portable Compton camera to visualize 223Ra concentration for radionuclide therapy Instrum Methods Phys Res Sect A Acceler Spectrom Detect Assoc Equip 101016jnima201916280 Brown I Astatine211 its possible applications in cancer therapy Int J Radiat Appl Instrum Part A Appl Radiat Isot “ 101016088328898690273 X Willhauck M J et al The potential of 211Astatine for NISmediated radionuclide therapy in prostate cancer Eur J Nucl Med Mol Imaging 101007s0025 Watanabe S et al Difference in thyroid uptake between Astatine211 and Iodine123 in normal rats a comparative study between oral and intravenous administration J Nucl Med “ Watabe T et al Enhancement of At uptake via the sodium iodide symporter by the addition of ascorbic acid in targeted αtherapy of thyroid cancer J Nucl Med “ 102967jnume d11822263 Agostinelli S et al GEANT4”a simulation toolkit Nucl Instrum Methods Phys Res Sect A Acceler Spectrom Detect Assoc Equip “ 101016S0168 Kataoka J et al Recent progress of MPPCbased scintillation detectors in high precision Xray and gammaray imaging Nucl Instrum Methods Phys Res Sect A Acceler Spectrom Detect Assoc Equip “ 101016jnima201411004 Kishimoto A et al Development of a dualsided readout PET module using largearea monolithic MPPCarrays IEEE Trans Nucl Sci “ 101109TNS201222332 Kataoka J et al Handy Compton camera using 3D positionsensitive scintillators coupled with largearea monolithic MPPC arrays Nucl Instrum Methods Phys Res Sect A Acceler Spectrom Detect Assoc Equip “ 101016jnima201307018 Hosokoshi H et al Development and performance verification of a 3D positionsensitive Compton camera for imaging MeV gamma rays Sci Rep “ 101038s4159 z Kamada K et al Composition engineering in ceriumdoped Lu Gd3Ga Al5O12 singlecrystal scintillators Cryst Growth Des Kamada K et al inch diameter single crystal growth and scintillation properties of CeGd3Al2Ga3O12 J Cryst Growth “ 101021cg200 694a “ 101016jjcrys gro201111085 Dimmock M R Nikulin D A Gillam J E Nguyen C V An CL implementation of pinhole image reconstruction IEEE Trans Nucl Sci “ 101109TNS201221977 Wilderman S a0J Clinthorne N a0H Fessler J a0A Rogers W a0L Listmode maximum likelihood reconstruction of Compton scatter camera images in nuclear medicine In IEEE Nuclear Science Symposium Conference Record IEEE Nuclear Science Symposium and Medical Imaging Conference Cat No 98CH36255 vol a0 “ 101109NSSMI C199877387 IEEE AcknowledgementsThis research was supported by JSPS KAKENHI Grant Number JP15H05720 20H00669 and JPMJER1905 ERATOFS The At was supplied through the Supply Platform of Shortlived Radioisotopes supported by JSPS GrantinAid for Scientific Research on Innovative Areas Grant Number 16H06278Author contributionsJK conceived the concept of this research AO developed the hybrid camera AO JK KF SS and EK conducted the experiments HK AT TT KO YL KM TK TW ES and JH conducted the experiments using 211At AO wrote the manuscriptcompeting interests The authors declare no competing interestsAdditional informationCorrespondence and requests for materials should be addressed to AOReprints and permissions information is available at wwwnaturecomreprintsPublisher™s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Authors Scientific RepoRtS 101038s41598020710195Vol0123456789wwwnaturecomscientificreports 0c'

Thyroid_Cancer

Deacetylases inSkeletal Muscle Physiology andSystemic Energy HomeostasisImplications for Metabolic Diseasesand TherapyHaili Tian1  Sujuan Liu2  Jun Ren3 Jason Kai Wei Lee456 Ru Wang1 and Peijie Chen1 School of Kinesiology Shanghai University of Sport Shanghai China Department of Anatomy and Histology Schoolof Basic Medical Sciences Tianjin Medical University Tianjin China Department of Cardiology Shanghai Instituteof Cardiovascular Diseases Zhongshan Hospital Fudan University Shanghai China Department of Physiology Yong LooLin School of Medicine National University of Singapore Singapore Singapore Global Asia Institute National Universityof Singapore Singapore Singapore N1 Institute for Health National University of Singapore Singapore SingaporeSkeletal muscle is the largest metabolic an in the human body and is able torapidly adapt to drastic changes during exercise Histone acetyltransferases HATs andhistone deacetylases HDACs which target histone and nonhistone proteins are twomajor enzyme families that control the biological process of histone acetylation anddeacetylation Balance between these two enzymes serves as an essential elementfor gene expression and metabolic and physiological function Genetic KOTG murinemodels reveal that HDACs possess pivotal roles in maintaining skeletal muscles™metabolic homeostasis regulating skeletal muscles motor adaptation and exercisecapacity HDACs may be involved in mitochondrial remodelinginsulin sensitivityregulationturn onoff of metabolic fuel switching and orchestrating physiologicalhomeostasis of skeletal muscles from the process of myogenesis Moreover manymyogenic factors and metabolic factors are modulated by HDACs HDACs areconsidered as therapeutic targets in clinicaltreatment of cancer‚ammation and neurological and metabolicrelated diseases This review will focuson physiological function of HDACs in skeletal muscles and provide new ideas for thetreatment of metabolic diseasesresearch forKeywords histone deacetylases exercise capacity skeletal muscle metabolism muscle physiologyINTRODUCTIONSkeletal muscle is the largest metabolic an in the human body consuming about of theentire body daily expenditure of energy Durnin It produces various secrete factors andparticipates in the interplay among multiple tissues and ans Pedersen and Febbraio Mizgier Muscular contraction is one of the main physiological functions of skeletalmuscles and plays a role in maintaining an and systemic metabolic homeostasis Guo Proper exercises help build up body defense to combat various diseases including obesitytype diabetes Alzheimer disease osteoarthritis and so on Luan These importantEdited byBrian James MorrisThe University of Sydney AustraliaReviewed bySean L McGeeDeakin University AustraliaViviana MoresiSapienza University of Rome ItalyCorrespondenceRu WangwangrutysuseducnPeijie Chenchenpeijiesuseducn These authors have contributedequally to this workSpecialty sectionThis was submitted toIntegrative Physiologya section of the journalFrontiers in PhysiologyReceived May Accepted July Published August CitationTian H Liu S Ren J Lee JKWWang R and Chen P Roleof Histone Deacetylases in SkeletalMuscle Physiology and SystemicEnergy Homeostasis Implicationsfor Metabolic Diseases and TherapyFront Physiol 103389fphys202000949Frontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise Capacityfunctions require delicate regulations in muscle from thelevel of genome to signal transduction establishing muscleplasticity and responses to environmental stress AcetylCoAas a central metabolite of amino acidfatty acidglucose notonly serves as fuel for energy expenditure but also bridgesthe gap between environmental stress and anismal responseLempradl Liu This control occursthrough posttranslational modification on nucleosomes histonelysine residues and other signal transducing proteins whichin turn controls accessibility of DNA to regulatory factorsand protein activity Acetylation one of the most prevalentmodifications of proteinis believed to function as a keymodulator of chromatin structure and signal transduction andprovides an avenue to couple extracellular stimuli with genomeduring muscle metabolism process by regulating acetylation anddeacetylation Haberland histone deacetylasesHDACs as a family of proteindeacetylases have been demonstrated to moderate physiologicalhomeostasis and development by deacetylation Haberland Table In humans there are types ofHDACs which are classified into four categories based onhomologous proteins in yeast namely Class I Rpd3like proteinHDAC1 Class II Hda1like proteins are classifiedas Class IIa HDAC4 and Class IIb HDAC6 Class III Sir2plike nicotinamide adenine dinucleotideNADdependent SIRT1 and ClassIV HDAC11 containing homologous domain with bothRpd3 and Hda1 Seto and Yoshida HDACs modulategene expression and protein activity through deacetylatingproteins When histone lysine εamino acid in the nucleosomeis acetylated it can neutralize positive charge before looseningchromatin structuretranscriptionfactors to DNA and expression of downstream target genesBy contrast histone deacetylation compresseschromatinstructure thereby inhibiting transcriptional gene expressionShahbazian and Grunstein to promote binding ofBiochemical properties of HDACs have been comprehensivelyreviewed Shahbazian and Grunstein Howeverthephysiological functions of HDACs have yet to be well examined inskeletal muscles A series of researches shed light on the potentialof drugs targeting HDACs to improve muscle fitness and cardiacmuscle disease which needs further clarification of HDACsphysiological function to understand the mechanisms Bai Galmozzi Xie Zhang and Ren Gaur Ample work has revealed the role of HDACin muscle physiology such as skeletal muscles me olism and thusexercise capacity McGee and Hargreaves Table Class IHDACs can interact with myocyte enhancer factor MEF2MyoD regulating myogenesis and exercise capacity Mal Puri Ohkawa Gregoire HDAC3 participates in myocytes diï¬erentiation and is linked toskeletal muscles metabolic fuel switching Gregoire Hong Class II HDACs can be phosphorylated byHDAC kinases and are transduced from nucleus to cytoplasmin response to cellular stress mediating muscle fiber switch andaï¬ecting the pathway of insulin sensitivity such as Glut4 andAKT Haberland McGee and Hargreaves Class III HDACs target a crucial mitochondrial biogenesis factorperoxisome proliferatoractivated receptor gamma coactivator alpha PGC1α in skeletal muscles liver and fat tissue Whiteand Schenk Therefore we will further discuss howthese HDACs ‚uence respective downstream targets exercisecapacity and therapeutic eï¬ects in human diseasesCLASS I HDAC HDAC123Regulation of Myogenesis by Class IHDACsIn previous studies HDAC123 was shown to be associated withthe process of myogenesis or myocyte diï¬erentiation Mal Puri Ohkawa Gregoire HDAC1 tightly binds to MyoD and deacetylates specific sites toinhibit the expression of musclespecific genes such as MHC andMCK in myoblasts Mimicking muscle diï¬erentiation conditionby serum deprivation HDAC1 protein gradually decreasesduring myocyte diï¬erentiation and transfers to bind to the tumorsuppressor pRb accompanied by isolation of HDAC1MyoD andtranscriptional activation of musclespecific genes Puri A HDAC1 H141A mutant incapable of binding withMyoD loses its inhibitory property on musclespecific genes inmyoblast state Mal In the late stage of myocytediï¬erentiation activating factor gradually switches from MyoDto myogenin occurring with a decrease in the MEF2D inhibitoryregulator HDAC2 Simultaneous overexpression of myogeninand MEF2D can enhance the expression of the musclespecificgene MHC in the absence of MyoD Ohkawa MEF2D a key factor that controls myocyte diï¬erentiationcan only be eï¬ectively deacetylated by HDAC3 rather thanHDAC128 Gregoire In addition HDAC3 caninhibit autoacetylation of acetyltransferases p300 and p300CBPassociated factor PCAF Thus HDAC3 impedes MyoDMEF2PCAF to form a multicomplex disturbing MEF2dependentmyogenic transcription Gregoire Figure Redundant Roles of HDAC12 inMaintaining Sarcomere Homeostasis inMuscleLoss of function of HDAC12 inhibits autophagy flux in skeletalmuscles tissue accompanied by decreased LC3 III ratio andp62 accumulation under fasting condition Moresi Toxic autophagy intermediates accumulate in muscle fibersin which class I HDACs deficiency led to impaired exercisecapacity Moresi This oï¬ers convincing evidenceon the role of HDAC12 in stabilizing basic structure andfacilitating development in muscles Genetic models suggestthat Class I HDACs control the physiological homeostasis ofskeletal muscles A germline deletion shows that wholebodyknockout of either HDAC1 or HDAC2 leads to mortalityin mice prior to the perinatal period Montgomery However a tissuespecific single deletion of HDAC1or HDAC2 in myocardium does not cause any phenotypeMontgomery Double knockout HDAC12 in theFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityTABLE The targets of HADCs in skeletal muscles and their physiological functionsClassMembersDown targetsGenetic KOTG phenotypeReferencesIHDAC1MyoD†“ PTEN†“ FoxOHDAC2HDAC3HDAC8HDAC4HDAC5HDAC9HDAC7HDAC6MyoD†“ MEF2D†“ NFκBp65†“MEF2D PCAF†“ Ampd3†“RCAN1 MEF2ACpt1b†“MEF2†“ Glut4†“ Myosin HeavyChain PGC1α and Hsc70“Pax7†“MEF2†“ Glut4†“ Baf60c†“Atg7 Beclin1 LC3†“Dach2Myog Gdf5†“MEF2†“Pax7 MFN1†“ Fam65bdysferlin and MAFbxHDAC10SIRT1“PGC1α STAT3†“IIAIIBIII“MnSOD Hexokinase II PDHsubunit E1αMalonyl CoA decarboxylase†““NFκBMstnSIRT2SIRT3SIRT4SIRT5SIRT6SIRT7Functionally redundant HDAC12 DKO mice causesmitochondrial abnormalities and sarcomeredegenerationPuri Ohkawa Beharry Cho Zhu Bin mKO mice Enhanced amino acid\\lipid metabolism andendurance exercise Causes IR under basal conditionGregoire Han Yuan Hong “Functionally redundant HDAC45 DKO HDAC59 DKOHDAC459 TKO increases type I fiber percentage andMcKinsey 2000a Choi Niu Luo oxidation metabolismYuan Meng Niu Macpherson Zhang Dressel “KO mice Causes mitochondrial oxidative damagemitofusion defect Protect against muscle wastingBalasubramanian Lee Ratti “mKO mice Mediates CR induced insulin sensitivity hasno effect on glucose homeostasis or exercise capacityTG mice no effect on glucose homeostasis or exercisecapacityKO mice Exacerbates obesity and IR in HFDKO mice Exacerbates obesity and IR in HFDKO mice Increased exercise tolerance and protectagainst HFDinduced obesity“Rodgers Schenk Boyle Jing Lantier Laurent KO mice Abnormal hypoglycemia TG mice Protectagainst HFD mKO mice Impaired insulin sensitivity lossKanfi Xiao Cui Samant of muscle mass““HDAC11IVActivation inhibition†“ unknown KO wholebody deletion TG wholebody overexpression DKO double knockout TKO triple knockout mKO musclespecificknockout CR caloric restriction HFD high fat diet IR insulin resistance““““heart leads to severe cardiomyopathy in mice indicating theobligatory role for HDAC12 in specific tissues Montgomery In skeletal muscle double knockout of HDAC12by myogeninCre causes mitochondrial abnormalities andsarcomere degeneration disrupting fundamental structural unitsof myofibers Moresi Therefore these findingsdemonstrate that HDAC12 redundantly maintains sarcomerehomeostasis in skeletal muscleHDAC3 Functions as a Fuel Switch inMuscle Energy Metabolismthe enzymatic core of nuclearHistone deacetylases isreceptor corepressorNCoR and silencing mediator ofretinoic acid and thyroid hormone receptors SMRT corepressor correspondingly NCoR and SMRT corepressoractivate HDAC3 through its SANT domain enzyme activityKaragianni and Wong Mice suï¬er from insulin resistanceafter specifically knocking out HDAC3 in skeletal musclesmKO while their endurance exercise abilities are enhancedHong It seems a selfcontradictory phenomenonbecause former studies pointed out that increased enduranceexercise capacity enhances insulin sensitivity The study showsthat insulin signaling cascades including pAKTAKT pIRS1S1101 and pGSKGSK have no change in HDAC3 mKO musclewhile glucose uptake and insulin sensitivity are impaired inglucose tolerance test GTT and insulin tolerance test ITTHong which is called the œdissociation eï¬ect byresearchers Lipid tends to be used as energy fuel in HDAC mKOmice which inhibits glucose absorption but does not ‚uenceinsulin signaling sensitivity Hong Further workfound that the HDAC3 knockout upregulates the expressionof AMP deaminase AMPD3 the first ratelimiting enzymein purine metabolism in skeletal muscles Fortuin Hong AMPD3 can deaminate AMP to form IMPfacilitating aspartic acid to transmit into fumarate and malate theintermediate metabolites of tricarboxylic acid cycle TCA cycleHong Research studies found that exercise inducedglucose labeled 13C6 expressed a lower glycolysis flux rate inmuscles of HDAC3 mKO but a higher expression in the TCAFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Controls of myogenesis by Class I HDACs The inhibitory effects of HDACs on myogenic factors MEF2 and MyoD maintain a primary myoblast stateDuring myocyte differentiation and myogenic process the inhibition is lifted by other factors facilitating MEF2MyoD complex formation to promote myogenesis“Ac deacetylationcycle intermediates Hong Gong Thus ageneral increase in TCA cycle metabolites activates the oxidationin mitochondria In vitro radioactive aspartic acid isotope tracertest showed that inhibiting HDAC3 or overexpressing AMPD3can increase amino acid metabolism rate and enhance fatty acidmetabolism thereby downregulating glucose metabolism Hong Figure In liver knockout HDAC3 causes severeliver steatosis and can be rescued by wildtype or catalyticallyinactive mutants of HDAC3 indicating an enzymatic activityindependent function Sun However the musclefuel switching in HDAC3 mKO mice cannot be rescued by anenzymatic inactivity mutant HDAC3 Song Thisfinding demonstrates that HDAC3 controls the fuel utilizationin skeletal muscles and is dependent on its enzymatic activityThe relationship between exercise and glucose uptake is far morecomplex than one could expectCLASS II HDACs HDAC45796Phosphorylation and NucleusCytoplasmShuttle of Class II HDACsThe MEF2 family is a class of transcription factors that areclosely associated with myogenic basic helixloophelix domainsincluding MyoD Taylor and Hughes MEF2 may interactwith Class IIa histone deacetylase HDAC45 to inhibit thetranscription of MEF2dependent genes thereby impeding thediï¬erentiation from myoblast to myotube McKinsey et al2000a During exercise Ca2 flows out from sarcoplasmicreticulum and activate calciumcalmodulindependent proteinkinase CaMK which phosphorylates HDAC45 and mediatesHDAC45 shuttle from the nucleus to the cytoplasm thusreleasing the repressive eï¬ect of HDAC45 on MEF2dependentgenes McKinsey 2000b Yuan Further researchdemonstrates that the nucleoplasmic shuttle of HDAC4 relies on binding while the shuttle of HDAC5 depends on CaMKphosphorylation at serine and sites first and thenexporting from the nucleus by binding with McKinsey 2000b As expected HDAC7 in Class IIa also possessesthe ability to be exported from nucleus to cytoplasm Gao suggesting that there may be redundant functions of ClassIIa HDACs Except for Class IIa knocking out Class IIb HDAC6in embryonic stem cells ESCs can promote ESCs to diï¬erentiateinto myoblast cells and transplantation of ESCs with HDAC6knockdown can also help the regeneration of wound skeletalmuscles Lee type I fiberthe proportion ofRegulation of Class II HDACs onFiberType Switch MitochondriaRemodeling and Energy Stress inSkeletal MuscleEvidence showsinthatskeletal muscles has no change in animal knocked out aloneany member of Class IIa Potthoï¬ Howeverthe proportion of type I fiber is significantly increased inHDAC45 DKO HDAC59 DKO HDAC459 TKO miceas well as exercise capacity of skeletal muscles Potthoï¬ Altogether the finding indicated a functionalredundant mechanism in Class II HDACs family Previousstudy found that Class IIa HDACs are closely associated withthe MEF2 family and can repress their downstream targetgenes McKinsey 2000a Therefore similar to Class IIaHDACs DKO the proportion of type I fiber is decreased in theMEF2C and MEF2D knockout mouse models overexpressingMEF2C results in increasing type I fiber and exercise capacityHDAC45 not only regulates exercise capacity but also governsmotor adaptation Several studies had shown that exerciseFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Class I HDACs regulates sarcomere structure and exercise capacity in mature muscle A schematic diagram shows Class I HDACs regulate exercisecapacity on different levels HDAC12 have redundant roles in modulating autophagy flux in muscle eliminating toxic factors in muscle HDAC3 serves as a fuelswitch and promotes muscle to use fatty acid to adapt endurance exercisepromotes phosphorylation of HDAC45 mediated by CaMK andadenosine monophosphateactivated protein kinase AMPKthen increasing a MEF2dependent transcription of Glut4 andmusclespecific genes which participates in the adaptationand plasticity of skeletal muscles McGee McGee and Hargreaves Besides exercise glucose can alsoactivate KATP channeldependent calcium signaling and CaMKwhich induce phosphorylationdependent nuclearcytoplasmtransportation of HDAC5 Meng The leavingHDAC5 releases its repressive eï¬ect on Baf60c and enhancinginsulinindependent AKT activation Meng Usingspecific inhibitor of class IIa HDAC activity myosin heavy chainPGC1α and heatshock cognate HSC70 are confirmed to beone of the substrates of HDAC4 in denervationinduced atrophymuscle in comparison with acetylated protein enrichment withuntreated group Luo Taken together Class IIaHDACs play critical roles in exercise capacity remodeling andnutrient sensing of skeletal muscles Figure For Class IIbit was revealed that glucose deprivationinduced mitochondrialfusion mediated by mitofusion1MFN1 is significantly disrupted in HDAC6 KO mice causingmitochondria degeneration which isreversed followingapplication of MFN1 acetylationresistant mutant Lee This study suggested that MFN1 deacetylation by HDAC6plays an important role in mitochondrial adaptive energyproduction and remodeling of skeletal musclesCLASS III HDACsSir2HOMOLOGSIRT1634NuclearLocated SIRT16Numerous studies have found that caloric restriction CRextends lifespan of mammals Caenorhabditis elegans and fruitflies while such eï¬ect is lost in SIR2 or NPT1 mutant C elegansstrains Imai Lin SIR2 encodes thesilencing protein Sir2p and NPT1 participates in the synthesis ofNAD NAD is an essential cofactor for Sir2pClass III HDACsto exert enzyme activity rather than Class I II and IV relayingon the zinc Imai Lin In mammalsSir2 has homologous proteins and these proteins are essentialto mitochondrial energy homeostasis antioxidant defense cellproliferation and DNA repair VargasOrtiz SIRT1 Mediates Energy Stress Adaptation in SkeletalMuscles reported that SIRT1 promotes theRodgers et altranscriptional activity of PGC1α by deacetylating the PGC1αat K13 site and mediates CRinduced gluconeogenesisrelatedgenes G6P PEPCK expression in liver Further research showedthat the NADNADH ratio and the enzymatic activity of Sir2 asensor of redox state were decreased in diï¬erentiating myocyteswhich relieved the inhibitory eï¬ect on MyoD and then promotedcell diï¬erentiation Resveratrol RSV a compound that maytarget SIRT1PGC1α can improve mitochondrial function andmetabolic homeostasis owning a potential to prolong lifespanRSV loses its activating eï¬ect on PGC1α in SIRT1 ˆ’ˆ’ MEFsLagouge Moreover RSVtreated mice at a dose of gkg increased their exercise capacity oxygen consumptionand improved insulin sensitivity against highfat induced obesityLagouge In addition RSV could enhance thedeacetylation of PGC1α in brown fat tissue BAT and skeletalmuscles which promotes mitochondrial production oxygenconsumption and thus improves metabolic syndrome Lagouge The function of SIRT1 in regulating skeletal musclesmetabolic homeostasis and exercise capacity has attracted greatattention from researchers Researchers further generated skeletalmusclespecific SIRT1mKO mice and found that mKO miceFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Control of muscle exercise adaption and mitochondrial function by Class II HDACs A Schematic of the Class IIa HDACs nucleoplasm shuttle Understress condition CaMK and AMPK could be activated and phosphorylate Class IIa HDACs Phosphorylated HDACs then bind with and shuttle to cytoplasmrelieving inhibitory effects on MEF2 and Baf60c B Class IIb HDAC6 can deacetylate MFN1 and facilitates its mitofusion function P phosphorylationlost the eï¬ect of CRinduced increasing insulin sensitivity andwere unable to deacetylate and inactivate STAT3 resulting inupregulating the phosphatidylinositol3kinase PI3K inhibitoryregulator p55αp50α expression Schenk Althoughknockout or overexpression of SIRT1 in skeletal muscles hasno eï¬ect on endurance capacity or glucose homeostasis in miceGurd Philp White Svensson some studies have shown that AMPK regulatesenergy metabolism of skeletal muscles partially mediated bySIRT1 and SIRT1 is significantly increased after enduranceexercise Suwa Canto Overexpressionof Sirt1 in skeletal muscles by adenoassociated virus AAV1promotes the expression of oxidationrelated genes includingPpargc1a Tfam Cpt1b and Pdk4 while it has no eï¬ect on insulinsensitivity of body Vila But overexpressing Sirt1in liver by AAV8 can protect from fatty liver induced by highcarbohydrate food HCD Vila Taken togetherthe aforementioned studies suggest that SIRT1 possesses limitedregulation capacity of skeletal muscles motility and SIRT1may modulate mitochondrial homeostasis and mediate skeletalmuscles adaptation under certain physiological conditions suchas CR aging and regeneration Gomes Ryall Figure In addition there may be more beneficial eï¬ectsof SIRT1 on metabolism in the liver or fat tissue Lagouge Vila Stefanowicz SIRT6 Improves Muscle Fitness andExercise CapacitySIRT6 is another Sir2like deacetylase located in the nucleusWhole body deletion of SIRT6 causes mice death around weeks owning to hypoglycemia Xiao Neverthelessthe skeletal musclespecific knockout of SIRT6 causes insulinresistance and impairs glucose homeostasis of mKO mice Cui Because the activity of AMPK is reduced inSIRT6mKO mice the glycolipids absorption and utilizationof skeletal muscles are impaired and the exercise capacity ofthe mice was also attenuated Cui Furthermoreresearchers constructed the overexpressing SIRT6 Sirt6BACmice and found that their body weight and fat content werenormal but the Sirt6BAC mice could protect from HCDinducedhyperglycemia via increasing pAKTAKT induced by insulinstimulation and glucose absorption Anderson Vitroexperiments further demonstrated that the ability of glucoseuptake was enhanced mainly in skeletal muscles not in brainiWAT eWAT tissues of Sirt6BAC mice Anderson These results shows the potential eï¬ects of SIRT6 targeted inskeletal muscles on improving exercise performance and treatingmetabolic diseases Figure MitochondriaLocated SIRT34SIRT34 Maintains Mitochondria Function in SkeletalMusclesSIRT34 are mitochondrialocalized deacetylases and responsiblefor regulating the deacetylation of proteins in mitochondria andmaintaining mitochondrial homeostasis After feeding highfatdiet HFD accelerated obesity attenuated insulin sensitivityworsened fatty liver and increased acetylation of proteins inmitochondria were observed in SIRT3 KO mice Hirschey Multitissue proteomics and physiological examinationreveals that SIRT3 is responsible for mitochondrial acetylatedFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Class III HDACs promote a stressinduced adaptation in muscle and regulate mitochondrial function Class III HDACs are NADdependentdeacetylases and may function as a redox sensor in muscle cell SIRT1 could activate PGC1alpha by directly deacetylasing K13 site and regulate insulin sensitivityby targeting STAT3 Mitochondrialocated Class III HDACs control mitochondrial function by deacetylating certain mitochondrial proteins modulating muscle fuelutilization and exercise capacityproteome regulation and metabolic fuel switching in brainheart kidneyliver and skeletal muscles DittenhaferReed Protein enrichment analysis discovers that itsregulatory proteins were mainly concentrated in lipid metabolismDittenhaferReed Knocking out SIRT3 leadsto reduction in the levels of deacetylation of manganesesuperoxide dismutase MnSOD mitochondrial complex II andpyruvate dehydrogenase PDH subunit E1α a downregulationof hexokinase II HK II binding with mitochondria resultingin impaired glucose and lipid metabolism of skeletal musclesLantier Interestingly SIRT3 liverspecific knockouthepˆ’ˆ’ and skeletal musclespecific knockoutskmˆ’ˆ’mice did not aï¬ect glucose homeostasis under chow or HFDconditions FernandezMarcos The above resultsindicate that SIRT3 has an important role in metabolic regulationbut in specific physiological processes such as redox stateexercise and aging and its regulatory mechanism is yet definedin skeletal muscles Kong Robin Williams A recent study found that SIRT4 knockoutcan resist HFDinduced obesity and increase endurance exercisein mice by repressing malonyl CoA decarboxylase which wasa key enzyme controlling fatty acid betaoxidation and wasreported to regulate muscle fuel switching between carbohydratesand fatty acids Koves Laurent Moreover knockdown of SIRT4 by adenoviral shRNA canincrease the mRNA and protein content of SIRT1 therebyenhancing the expression of fatty acid oxidation genes andmitochondrial oxidation capacity in hepatocytes and myotubesNasrin Table THERAPEUTIC TARGETS OF HDACsAND THEIR POTENTIAL FORMETABOLIC DISEASESA comparative analysis used HDAC paninhibitor SAHA aclass I HDAC selective inhibitor MS275 and a class IIHDAC selective inhibitor MC1568 to treat C2C12 separatelyIt was found that only MS275 could significantly stimulatemitochondria biogenesis and oxygen consumption Galmozzi In obese diabetic mice it was found that specificallyinhibiting Class I rather than Class II HDACs improvedGTT and insulin sensitivityincreased oxidative metabolismof skeletal muscles and adipose tissue and reduced bodyweight Galmozzi Similar to the eï¬ect of MS275knockdown HDAC3 in C2C12 could also increase Pgc1αGlut4 Tfam Idh3α transcription suggesting that HDAC3 andits target genes played an important role in the above eventsGalmozzi For Class II HDACs studies have found that scriptaid a ClassIIa HDAC inhibitor has similar eï¬ects to exercise Six weeksof scriptaid administration significantly improved enduranceperformance and significantly increased the wholebody energyexpenditure and the expression of lipid oxidation related genesPdk4 Cpt1b Pgc1α Pparδ etc of C57BL6 mice Gaur One possible explanation of above observation is thepotential target of scriptaid inhibition of titin a structure proteinof sarcomere deacetylation and downstream genes of Class IIaHDACs Gaur Huang Frontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityIn mice treated with SPT1720 another activator of SIRT1overtly enhanced endurance exercise ability was noted Moreoverthese mice were protected from HFDinduced obesity and insulinresistance owing to an upregulation of the oxidative metabolismin skeletal muscles liver and BAT tissues Pacholec In order to further explore the principle of SIRT1 activation theresearchers purified SIRT1 in vitro and added SRT1720 and itsanalogs SRT2183 SRT1460 and RSV and they found that theenzymatic activity of SIRT1 was not enhanced suggesting thatthese drugs may indirectly activate SIRT1 Pacholec The enzymatic activity of SIRT1 largely depends on the contentof cofactor NAD This implied that the enhancement of SIRT1activity may be achieved through indirect upregulation of NADStudy has discovered that knocking out poly ADPribosepolymerase1 PARP1 which is a NAD consuming enzymecould increase NAD content and enhance the activity of SIRT1in BAT and skeletal muscles Bai PARP1 inhibitorscan upregulate the proteins of mitochondrial respiratory chaincomplexes in mice enhance the aerobic oxidation capacity ofmitochondria and improve mitochondrial defects in the primarymyotubes of obese humans Bai Pirinen et alResveratrol was initially reported as an SIRT1 activatorthat improves mitochondrial function and exercise capacity inmice and resists HFDinduced obesity Lagouge However subsequent studies have discovered that administeringthe same dose of RSV to rats or mice does not increasemitochondrial protein content Higashida Bycontrast overexpression of SIRT1 in the triceps muscle of ratsdecreases the mitochondrial protein content Higashida In a doubleblind human trial healthy and obesemen were supplied for 30day RSV in which the data showedthat RSV can improve systolic blood pressure and homeostasismodel assessment HOMA indexindicating glucose metabolismability simulating the eï¬ect of CR Timmers However some researchers found that the overexpression ofSIRT1 alone cannot mimic the CR eï¬ect in transgenic mice andthe transcriptomic changes in various tissues were quite diï¬erentor even opposite Boutant Such an opposite situationmay explain that the genetic model and compound stimulationare not completely consistent RSV as a potential metabolicsyndrome treatment drug still needs largescale populationsample verificationCONCLUSIONThe very first mammalian histone deacetylase HDAC1 wascloned and isolated by Taunton and sabout HDACs have been published in the last years CurrentlyREFERENCESAnderson J G Ramadori G Ioris R M Galie M Berglund E D CoateK C Enhanced insulin sensitivity in skeletal muscle and liver byphysiological overexpression of SIRT6 Mol Metab “ 101016jmolmet201509003we know at least HDAC proteins They are responsible foreradicating epigenetic modifications establishing an epigeneticoï¬ chromatin state and regulating heritable gene expressionYang and Seto Haberland In these processeseach HDACs may play a diï¬erent role Table What kind ofgeneprotein is the specific downstream target of these HDACsIs its enzyme activity related to intracellular localization andthe formation of multiprotein complex It is still one of thekey and difficult issues in this field Based on previous researchexperience some techniques may be used to further experimentsas follows HDACs interacting proteinscomplex coIP Proteinacetylation western Histone acetylation target geneChIP High through put proteomicsacetylome with specificHDACs inhibitor etctherapeuticare potentialHistone deacetylasestargetsand clinical drugs such as SAHA Vorinostat and FK228romidepsin have been used for antitumor treatment Bolden However they have two disadvantages greattoxic and side eï¬ects and difficulty in specific inhibition ofHDACs activity With the development of computer simulationtechnology and structural biology it is believed that more specificHDACs inhibitorsactivators can be constructed And researchesshould attach importance to HDACs regulatory factors likePARP1 when direct targeting on HDACs fails to show its eï¬ectsFurther development of their inhibitors with more specificityand trials for the treatment of metabolic diseases may have greatpotential as well

Thyroid_Cancer

"rapeutic plasma exchange clears circulating soluble PD L1 and PD L1 positive extracellular vesicles Elizabeth Ann L Enninga2 Fabrice Lucien Matteoni3 Jacob J Orme Heather Dale4 Edwin Burgstaler4 Susan M Harrington3 Matthew K Ball4 Aaron S Mansfield1 Sean S Park5 Mathew S Block1 Svetomir N Markovic1 Yiyi Yan1 Haidong Dong3 Roxana S Dronca6 Jeffrey L Winters4To cite Orme a0JJ Enninga a0EAL Lucien Matteoni a0F et a0al Therapeutic plasma exchange clears circulating soluble PD L1 and PD L1 positive extracellular vesicles Journal for ImmunoTherapy of Cancer 20208e001113 101136jitc2020001113 –º Additional material is published online only To view please visit the journal online http dx jitc Accepted June Authors or their employers Re use permitted under CC BY NC No commercial re use See rights and permissions Published by BMJ1Division of Medical Oncology Mayo Clinic Rochester Minnesota USA2Department of Obstetrics and Gynecology Mayo Clinic Rochester Minnesota USA3Department of Urology Mayo Clinic Rochester Minnesota USA4Department of Laboratory Medicine and Pathology Mayo Clinic Rochester Minnesota USA5Department of Radiation Oncology Mayo Clinic Rochester Minnesota USA6Department of Hematology and Oncology Mayo Clinic Florida Jacksonville Florida USACorrespondence toDr Jacob J Orme orme jacob mayo eduBackground Trans acting programmed death ligand PD L1 derives from malignant cells in three known forms High levels of secreted splice variant PD L1 sPD L1 ADAM10ADAM17 shed sPD L1 and PD L1 positive extracellular vesicles evPD L1 each predict poor prognosis and limited response to PD L1 checkpoint inhibitors in cancer To our knowledge no clinical intervention has reduced any of these circulating forms of extracellular PD L1 Here we explore therapeutic plasma exchange TPE as a treatment to reduce circulating extracellular PD L1Results In patients with melanoma sPD L1 levels above ngmL predicted inferior overall survival In patients undergoing TPE for non malignant indications each TPE session removed a mean sPD L1 and evPD L1 detectable in plasma TPE also reduced total and ADAM10 positive extracellular vesiclesConclusion Here we report the first known clinical intervention to remove either sPD L1 or evPD L1 from plasma in vivo TPE reduces plasma sPD L1 and evPD L1 in vivo and may have a role in treatment with immunotherapy TPE may also prove useful in patients with other extracellular vesicle related conditionsINTRODUCTIONOvercoming initial or acquired resistance to programmed death ligand PD L1 immune checkpoint inhibitors is a major area of unmet need for many cancers1 Although the full scope of mechanisms of resistance to these therapies has yet to be determined different forms of tumor derived extracellular PD L1 have been linked to resistance in multiple clinical studies2“in Malignant cells produce trans acting extracellular PD L1 three distinct forms First tumor cells transcribe and secrete soluble PD L1 sPD L1 splice variants4 Second enzymes ADAM10 and ADAM17 shed sPD L1 ectodomain directly from the tumor cell surface6 Both forms of sPD L1 carry known homodimerization domains and can be detected by ELISA sPD L1 can outcompete PD L1 inhibitors kill CD8 T cells and limit the ability of healthy peripheral blood mononuclear cells to kill tumor cells in vitro6 In a third form tumors generate extracellular vesicles EVs bearing surface PD L18 PD L1 positive EVs evPD L1 exhibit similar properties to sPD L1 in systemic circulation9 Each type of trans acting extracellular PD L1 correlates with poor survival in multiple clinical trials online supplementary table While broad spectrum pharmacological inhibitors and genetic manipulation have been shown to reduce release of these forms of PD L1 in culture or animal models none are suitable for clinical use To date we know of no reported clinical intervention that safely and reliably eliminates any of these forms of immunosuppressive systemic extracellular PD L1Therapeutic plasma exchange TPE is a procedure in which blood is passed through an apheresis machine separating plasma from cellular components Removed plasma is discarded and replaced with either colloid eg albumin or crystalloid and colloid solutions Unlike dialysis which removes small ions by diffusion TPE removes plasma restricted substances like antibodies that are too large for rapid diffusion On average each TPE session removes approximately “ of large non cellular plasma restricted intravascular components10It is unknown whether sPD L1 approximately kDa or PD L1 positive EVs “ nm are plasma restricted non diffusing and unbound If so we hypothesized that TPE could efficiently remove these substances from patient blood Such an intervention could if effective improve response to immunotherapyOrme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c access Figure Soluble programmed death ligand PD L1 suppresses antitumor immunity and predicts overall survival in patients with melanoma A A model of three known tumor derived extracellular PD L1 forms” evPD L1 ADAM10ADAM17 cleaved soluble PD L1 sPD L1 ectodomain and secreted splice variant sPD L1”that downregulate antitumor immunity and prevent response to PDL1 inhibition B A Kaplan Meier plot shows significantly worse overall survival for patients with melanoma exhibiting high ‰¥ ngmL versus low ngmL plasma sPD L1 levels p0005 C Patients with melanoma exhibited a higher mean plasma sPD L1 level ngmL in comparison to healthy controls ngmL p0001RESULTSsPDL1 levels predict overall survival in patients with melanomaEach form of extracellular PD L1 acts in trans as a systemic immunosuppressant through PD1 signaling figure 1A online supplementary table “ To confirm the clinical impact of plasma sPD L1 we measured sPD L1 levels in a retrospective cohort of patients with melanoma Exploratory analysis of overall survival OS determined a working cut off value of sPD L1 ‰¥ ngmL and baseline characteristics at the time of entry into study were similar online supplementary table Patients with high plasma sPD L1 levels experienced inferior median OS compared with patients with low plasma sPD L1 levels figure 1B vs months p0005 In comparison to healthy age matched controls patients with melanoma exhibited higher mean plasma sPD L1 figure 1C ngmL vs ngmL p0001 In a multivariate Cox proportional hazards analysis high sPD L1 prior to treatment predicted worse survival HR CI to p0025 when accounting for advanced age not significant sex not significant late stage p0002 and high serum LDH p001 online supplementary table TPE significantly reduces plasma sPDL1 levelsWe hypothesized that TPE may remove extracellular PD L1 in its various forms figure 2A To address this question we prospectively enrolled patients undergoing planned TPE figure 2B Twenty eight patients met inclusion criteria of which provided informed consent Baseline patient characteristics are in table One patient was excluded for biotin containing supplement use as biotin interferes with the established sPD L1 detection assay The remaining patients underwent plasma exchange and sample collection before and after the procedure as described Discarded plasma samples from the TPE device waste bag for each session were also collected sPD L1 was measured in each sample and most patients undergoing TPE exhibited sPD L1 levels above the clinically relevant ngmL cut off from the retrospective melanoma studyMost patients undergoing TPE did not have an active cancer diagnosis Baseline sPD L1 levels in all patients were compared with matched normal controls and patients with melanoma online supplementary fig and some patients exhibited sPD L1 above the clinically significant cut off level determined in the retrospective melanoma cohort Patients with high baseline sPD L1 levels were significantly more anemic than patients with lower baseline sPD L1 even when controlling for the higher number of female subjects in the high sPD L1 group female only mean Hgb vs p004 male only mean Hgb vs p003 Groups were otherwise similar TPE significantly reduced plasma sPD L1 levels in patients receiving albumin only ie no Orme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c accessFigure Therapeutic plasma exchange TPE significantly reduces plasma soluble programmed death ligand sPD L1 levels A A model of the TPE procedure in which patient plasma is separated and replaced to extract non cellular substances confined to the plasma B A diagram of the present study in which patients undergo plasma exchange C All plasma levels of sPD L1 immediately prior to pre and after post TPE using albumin replacement fluid are plotted TPE significantly reduced sPD L1 levels in patient plasma by Wilcoxon signed rank test p00001 D In a typical timeline patient sPD L1 levels are reduced by each successive session of TPE gray bars See also table a0 online supplementary figures “FFP replacement fluid figure 2C p00001 Removed sPD L1 was detected in matching plasma samples from the TPE procedure waste bag Each TPE session removed a mean of detectable plasma sPD L1 mean regeneration of sPD L1 between sessions was table TPE sessions were usually separated by “ daysincluding sessions A representative individual patient treatment course showing sPD L1 reduction over four successive TPE sessions is also shown figure 2D All individual patient TPE courses involving donated human blood products eg fresh frozen plasma or FFP are shown in online supplementary fig Pre TPE and post TPE sPD L1 levels for all sessions are also shown online supplementary fig TPE significantly reduced plasma sPD L1 even when sessions requiring donated FFP were included p00001FFP is sometimes given during TPE for patients with increased risk of bleeding We observed that some patients receiving FFP with low baseline sPD L1 experienced rapid increases in sPD L1 levels after TPE presumably passively acquired from donor plasma as this was not observed in patients receiving albumin replacement alone sPD L1 was not detected in the discarded plasma from the procedure for these patients We observed a mild association between post FFP infusion rises in sPD L1 levels and the blood type of the recipient mainly in patients with O type blood Individuals with group Oˆ’ blood are universal recipients of FFP products and universal donors of cellular products due to a lack of ABO group antigens and the presence of preformed anti A and anti B antibodies respectively Recipients of FFP usually receive a mixture of compatible plasma from multiple donors To determine whether blood type in FFP donors is associated with FFP sPD L1 content we measured sPD L1 by ELISA in plasma from multiple FFP donors online supplementary fig O negative plasma donors showed higher sPD L1 levels than donors with most other blood typesTPE efficiently reduces plasma EV levels in vivoWe postulated that TPE may remove PD L1 positive EVs evPD L1 from patient blood To address this question we measured total EV levels and evPD L1 in each sample by flow cytometry We also determined the impact of TPE on platelet derived CD61 positive EVs one of the most abundant EV Orme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c access Table Patient baseline characteristicsCharacteristicHigh sPDL1 n17Low sPDL1 n7StatisticStarting sPD L1Age yearsGender FActive cancer YesImmunotherapyNoneAtezolizumabPembrolizumabPlasma exchange indicationCNS demyelination myelitis MS NMO myelopathyImmune encephalitisMyasthenia gravisParaneoplastic syndrome encephalitis neuropathy pemphigusParaproteinemia Waldenström cryoglobulinemia kappa gammopathySusac syndromeTransplant rejection heart kidneyPre TPE white cell countPre TPE hemoglobinPre TPE creatinine to to to to to to to to to to F1223619 p0001F122035 p0558X2070 p0404 X20 p0967 X2288 p0237  X2288 p0315  F122078 p0385F122860 p0008F122382 p0063Patients undergoing therapeutic plasma exchange TPE are compared by starting soluble programmed death ligand sPD L1 level above or below survival cut off established in patients with melanoma ngmL For categorical variables n is given For continuous variables mean quartiles is givenKruskal Wallis PearsonCNS central nervous system MS multiple sclerosis NMO neuromyelitis opticasubpopulations in blood CD61 is a platelet marker and ADAM10 positive low density EVs ADAM10 has been implicated in exosome loading and pathogenesis11“TPE significantly reduced total plasma particle concentration figure 3A average per exchange p00001 TPE sessions requiring FFP or other human blood product were excluded from analysis leaving session pairs PD L1 positive evPD L1 and ADAM10 positive EVs were Table Soluble programmed death ligand sPD L1 reduction and regeneration per exchange Reduction per exchangen44Mean SDMedian min max Regeneration between exchangesMean SDMedian min maxRegeneration per cycle pgmLMean SDMedian min max ˆ’ n44 ˆ’ ˆ’38k 154kFor each exchange not requiring FFP percent sPD L1 reduction and regeneration between each exchange is calculated n44FFP fresh frozen plasmasignificantly reduced by TPE figure 3BC p0028 and p00001 respectively and were detected in waste plasma data not shown Each TPE session using albumin based replacement fluid with pre TPE levels above one million removed a mean of detectable PD L1 positive EVs from patients online supplementary table Platelet derived CD61 positive EVs while abundant were not significantly reduced by plasma exchange figure 3DIndividual patient courses showing total plasma PD L1 positive ADAM10 positive and CD61 positive EV levels before and after each TPE session are shown in online supplementary fig with exemplary nanoflow plots in online supplementary fig Three successive TPE sessions consistently depleted total PD L1 positive and ADAM10 positive but not CD61 positive EVs These trends were less pronounced when sessions in which patients received donor FFP were included online supplementary fig In normal control FFP donors blood type did not correlate with plasma EV concentrations online supplementary fig DISCUSSIONExtracellular PD L1”in the form of splice variant sPD L1 ADAM10ADAM17 cleaved sPD L1 ectodomain or evPD L1 positive EVs evPD L1”mediates resistance to PD L1 inhibitors4“ These forms are resistant to clinically tested Orme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c accessFigure Plasma exchange efficiently reduces total programmed death ligand PD L1 positive and ADAM10 positive extracellular vesicle EV levels in vivo Plasma levels of total EVs immediately prior to pre and after post therapeutic plasma exchange TPE are plotted TPE significantly reduced A total p00001 B PD L1 positive p0028 and C ADAM10 positive p00001 but not D CD61 positive EVs p094 by Wilcoxon signed rank test See also online supplementary figures “ and online supplementary table combinations of chemotherapy and immunotherapies in vitro and in animal models and are associated with poor prognosis in many cancer types In the present study we found that TPE reliably reduces sPD L1 and evPD L1 This reduction was most pronounced over approximately three consecutive single plasma volume treatment sessions Given the dramatic reduction in sPD L1 and evPD L1 TPE may provide a novel approach to combating these mechanisms of resistanceWhile promising the present study was limited to patients receiving TPE mainly for non oncological indications over a short time horizon One patient in the study had melanoma receiving pembrolizumab and exhibited high pre TPE evPD L1 that was reduced on treatment Another patient had a uterine neuroendocrine tumor receiving atezolizumab and exhibited high pre TPE sPD L1 that was reduced on treatment The purpose of TPE in all cases however was to blunt paraneoplastic autoimmunity or treat some other coexisting autoimmune disorder”not the underlying malignancy Neither of these patients experienced improvement in their autoimmunity after TPE The source of sPD L1 and evPD L1 in these cases is uncertain as no assay currently differentiates tumor derived and non tumor derived PD L1 We observed that these forms of immunosuppressive extracellular PD L1 exist naturally although at lower levels in healthy subjects than in patients with cancer suggesting a potentially beneficial immunoregulatory role While we observed some regeneration for both sPD L1 and evPD L1 between TPE sessions it is unknown to what degree malignant cells may regenerate and maintain extracellular PD L1 homeostasis Relatedly it is uncertain how other plasma substances removed by TPE may affect response to immunotherapy or more broadly cancer immunity overall Nor is it known at what level sPD L1 andor PD L1 positive EV removal would become clinically relevant These facets will be tested in future studiesImmunotherapy resistance is widespread and costly In most instances PD L1 inhibitors such as pembrolizumab nivolumab atezolizumab durvalumab and avelumab are used in situations in which less than half of tumors are expected to respond Of patients that benefit many do not experience a sustained durable response These treatments represent a major investment the cost of PD L1 checkpoint blockade commonly reaches several hundred thousand dollars over the course of therapyTo our knowledge this is the first report of an intervention to achieve consistent rapid reduction in either sPD L1 or PD L1 positive EVs in a clinical setting TPE is safe and commonly prescribed Thus preimmunotherapy TPE may combat immunotherapy resistance In light of the heavy investment that anti PD L1 therapy entails the added cost of TPE in selected patients may be practical14 While the durability of extracellular PD L1 reduction in malignancy will be explored in future studies the present study suggests that this approach warrants further investigationOrme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c access Beyond evPD L1 this is also to our knowledge the first known intervention to reliably deplete EVs in a clinical setting EVs have been implicated in oncogenesis and metastasis through miRNA carriage and direct protein signaling independent of PD L115 Beyond cancer EVs have also been implicated in autoimmunity17 agingneurodegeneration18 infection19 obesity20 and heart disease21 The selective removal of ADAM10 positive ie likely immune derived versus CD61 positive ie likely platelet derived EVs in this study suggests flexible selective EV depletion may be both possible and expedient in other indications The present study is only a proof of concept and additional exploratory studies in these areas are necessaryIn summary TPE reduces extracellular forms of PD L1 associated with PD L1 checkpoint inhibitor resistance Future studies will explore the potential role of TPE in improving cancer immunotherapyMETHODSRetrospective melanoma outcomes study designIn a retrospective analysis baseline blood samples from patients with melanoma prior to treatment in one of three clinical trials by the North Central Cancer Treatment Group N057e22 N077523 and N087924 between and were tested for sPD L1 of patients were diagnosed with cutaneous melanoma and none received immunotherapy treatments Blood from healthy volunteers undergoing blood donation at Mayo Clinic was also testedProspective TPE study designIn an investigator initiated label single center observational study adults undergoing TPE were approached from December through March In consenting subjects samples of whole blood immediately prior to TPE and on completion of the procedure were collected in ACD vacutainers BD In each case the first mL of blood was discarded to avoid contamination after which an mL sample was obtained in sequence Plasma was isolated by centrifugation A postprocedure blood sample was obtained after completion of the procedure In addition matching samples from discarded plasma from the procedure waste bag were collected Samples were obtained from up to four consecutive procedures for each patient If a patient underwent fewer than four TPE procedures samples were obtained from as many procedures as possiblePatients included were adults able to give consent and undergoing TPE for a variety of hematological neurological and renal diseases as indicated by published guidelines from the American Society for Apheresis ASFA or according to the medical judgment of the referring physicians25 Patients taking biotin supplements were excluded from the study due to biotin interference with the sPD L1 ELISA assay Procedures were performed using centrifugation based cell separators either the Fenwal Amicus Fresenius KABI USA LLC Lake Zurich Illinois USA or the Spectra Optia Terumo BCT Lakewood Colorado USA For each patient a single plasma volume was exchanged using either peripheral intravenous preferred or central lines for vascular access For this study due to the possibility of sPD L1 or PD L1 positive EVs present in donor plasma only TPE sessions using no donor plasma ie fresh frozen plasma FFP in the replacement fluid were included in calculations Anticoagulation consisted of either mL of acid citrate dextrose solution A ACD A or mL of ACD A with units of unfractionated heparin Anticoagulant to blood ratios were when ACD A was used and when ACD Aheparin was used Patients did not receive routine electrolyte replacement but mL of calcium gluconate was administered by slow intravenous push for signs and symptoms of hypocalcemia related to the ACD A anticoagulant in one patientELISAELISA was performed as previously published26 Both secreted splice variant and shed sPD L1 are reliably detected by this ELISA In brief paired mouse IgG2 monoclonal antibody clones H1A and B11 against extracellular human PD L1 were utilized in a capture detection plate assay using biotinylation and HRP streptavidin detection This assay is specific for sPD L1 and does not exhibit cross reactivity to other B7 H homologues nor to evPD L1 Concentrations were determined by optical density measurements along a known standard curve of recombinant human PD L1 ELISAs were performed by team members who were blinded to the identity of the samplesFlow cytometryFlow cytometry for EVs was performed as previously published27 In brief plasma samples were centrifuged twice at 2000g to deplete platelets Resultant platelet free plasma were analyzed using an A60 Micro Plus Nanoscale Flow Cytometer Apogee FlowSystems gating for mid intensity light angle light scatter and markers of interest Anti PD L1 Genentech atezolizumab ADAM10 RD Systems clone and CD61 BioLegend clone VI PL2 antibodies were conjugated to fluorophores Life Technologies Alexa647 PE phycoerythrin and Alexa488 and titrated prior to use Nanoscale flow cytometer calibration was performed using a standard reference bead mix as previously published Flow cytometry was performed by team members blinded to the identity of the samplesStatistical analysisAll statistical analyses were performed using R Statistical Software R Foundation Retrospective progression free survival was analyzed using Kaplan Meier and Cox proportional hazards modeling Optimal cut off values for sPD L1 levels were determined using the greyzoneSurv package for R Wilcoxon signed rank test was used to compare paired pre TPE and post TPE patient sample sPD L1 and EV levels as indicated Baseline clinical characteristics for the study were compared by Kruskal Wallis test for continuous variables and Pearson™s χ2 test for discrete variables as indicated Orme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0cOtherwise groups were compared by unpaired two sided Student™s t test Figures comprising box plots show quartile values and individual data points Mean values and CI are indicated in corresponding online supplementary figures and tables P was considered statistically significant In figures p values are denoted with with and with Twitter Jacob J Orme JakeOrmeMDPhDAcknowledgements Statistical guidance was provided generously by Nathan Foster of the Mayo Clinic Center for Clinical and Translational Science Some illustrations were created using Servier Medical Art templates which are licensed under a Creative Commons Attribution Unported License https smart servier com Additional illustrations were provided by Mayo Clinic Media Services The authors thank Daniel Summerfield MD MS for use of his likeness in Fig 2AContributors JO originated hypotheses designed the study oversaw experiments performed analyses and wrote the article EALE performed retrospective melanoma cohort analysis FL M performed nanoflow cytometry HD and EB oversaw and performed TPE study enrollment sample collectionprocessing and blinding SMH performed ELISAs MB AM SP MB SNM YY HD RD and JLW helped develop hypotheses provided clinical samples and reagents and contributed support and oversightFunding R21 5R21CA19787802 Role of Bim and soluble B7 H1 in monitoring T cell responses to anti PD1 therapy in melanoma HD and RD L30 CA23154101 Soluble B7H1 as a PD1 Checkpoint œRemote Control in Cancer JJO U10 CA180790 EE K12 CA090628 YY Richard M Schulze Family Foundation HD and RDCompeting interests Intellectual property has been filed addressing discoveries disclosed in this manuscript The authors report no other relevant conflicts of interestPatient consent for publication ObtainedEthics approval All research protocols involving human subjects were approved by Mayo Clinic™s Institutional Review Board and all human subjects gave written informed consentProvenance and peer review Not commissioned externally peer reviewedData availability statement Data are available in a public access repository All data will be available for download at the Science Framework at https osf io qtskd access This is an access article distributed in accordance with the Creative Commons Attribution Non Commercial CC BY NC license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited appropriate credit is given any changes made indicated and the use is non commercial See http creativecommons licenses by nc ORCID iDJacob J a0Orme http orcid REFERENCES O'Donnell JS Long GV Scolyer RA et a0al Resistance to PD1PDL1 checkpoint inhibition Cancer Treat Rev “ Ando K Hamada K Watanabe M et a0al Plasma levels of soluble PD L1 correlate with tumor regression in patients with lung and gastric cancer treated with immune checkpoint inhibitors Anticancer Res “ Fan Y Che X Qu J et a0al Exosomal PD L1 retains immunosuppressive activity and is associated with gastric cancer prognosis Ann Surg Oncol “ Zhou J Mahoney KM Giobbie Hurder A et a0al Soluble PD L1 as a biomarker in malignant melanoma treated with checkpoint blockade Cancer Immunol Res “ access Mahoney KMet a0al œA secreted PD L1 splice variant that covalently 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Jaular L Trelis M et a0al Extracellular vesicles in parasitic diseases J Extracell Vesicles Huang Doran I Zhang CY Vidal Puig A œExtracellular Vesicles Novel Mediators of Cell Communication In Metabolic Disease Trends in Endocrinology and Metabolism Elsevier Inc “ Boulanger CM Loyer X Rautou PE et a0al œExtracellular vesicles in coronary artery disease Nature Reviews Cardiology Nature Publishing Group “ Kottschade LA Suman VJ Amatruda T et a0al A phase II trial of nab paclitaxel ABI007 and carboplatin in patients with unresectable stage IV melanoma a North Central Cancer Treatment Group Study N057E1 Cancer “ Kottschade LA Suman VJ Perez DG et a0al A randomized phase study of temozolomide and bevacizumab or nab paclitaxel carboplatin and bevacizumab in patients with unresectable stage IV melanoma a North Central Cancer Treatment Group study N0775 Cancer “ McWilliams RR Allred JB Slostad JA et a0al NCCTG N0879 Alliance A randomized phase cooperative group trial of carboplatin 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Thyroid_Cancer

poorest prognoses of all malignancies with little improvement in clinical outcome over the past years Pancreatic ductal adenocarcinoma is responsible for the vast majority of pancreatic cancer cases and is characterised by the presence of a dense stroma that impacts therapeutic efficacy and drives protumorigenic programs More specifically the inflammatory nature of the tumour microenvironment is thoughtto underlie the loss of antitumour immunity and development of resistance to current treatments Inflammatory pathways are largely mediated by the expression of and signallingthrough cytokines chemokines and other cellular messengers In recent years there hasbeen much attention focused on dual targeting of cancer cells and the tumour microenvironment Here we review our current understanding of the role of IL6 and the broader IL6cytokine family in pancreatic cancer including their contribution to pancreatic inflammationand various roles in pancreatic cancer pathogenesis We also summarise potential opportunities for therapeutic targeting of these pathways as an avenue towards combating poorpatient outcomesIntroductionPancreatic cancer is a devastating malignancy with a 5year relative survival rate of only “ dependenton the geographical location surveyed [“] with these statistics exhibiting only modest improvementover the last four decades [“] The median survival postdiagnosis ranges from just “ months forlocally advanced disease and “ months for metastatic disease [] It was estimated by the World Healthanisation that pancreatic cancer is currently the 7th leading cause of cancerrelated death being responsible for over deaths worldwide in [] With incidence increasing pancreatic cancerhas been predicted to be the third leading cause of cancerrelated death in the European Union by [] and the second leading cause of cancerrelated death in the United States of America and Germanyby []Several factors contribute to the poor survival of pancreatic cancer patients A current lack of reliablediagnostic markers that would enable early screening [] coupled with largely nonspecific symptoms ofdisease results in over of patients presenting with metastatic disease at diagnosis [] This subgroupof patients have limited therapeutic options and are thus typically administered palliative chemotherapyaimed at prolonging survival and reducing symptoms during endoflife care [“] Moreover whilstapproximately “ of patients present with localised disease that is eligible for potentially curativesurgery [] disease recurs in over of patients postresection [] Ultimately these factorsculminate in more than of patients diagnosed with pancreatic cancer succumbing to disease []These harrowing statistics highlight that despite research efforts there remains a lack of understandingof the pathogenesis of disease which in turn limits the development of new therapeuticsReceived March Revised July Accepted August Version of Record published August The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BYNCND 0cClinical Science “101042CS20191211Pancreatic ductal adenocarcinomaPancreatic ductal adenocarcinoma is the predominant pancreaticmalignancyPancreatic cancer can arise from either the endocrine or the exocrine region of the pancreas Tumours arising fromthe exocrine compartment are termed pancreatic ductal adenocarcinoma PDAC and account for over of allpancreatic cancers []PDAC develops via a stepwise progression from normal tissue through to invasive lesions which is associated withdistinct morphological characteristics [“] It has been proposed that this process begins with a phenomenontermed acinartoductal metaplasia ADM which is a normal homeostatic mechanism whereby acinar cells transdifferentiate into a ductal phenotype in response to particular stimuli [] However if compounded by an oncogenic˜hit™ cells are pushed towards a pathogenic phenotype that develops into pancreatic intraepithelial neoplasia PanIN[“] Disease progresses through preinvasive stages termed PanIN1A PanIN1B PanIN2 and PanIN3 priorto invasive PDAC [] This progression is associated with increasing nuclear atypia whereby the nuclei are no longerpositioned basally and loss of normal architecture as cells become more papillary in nature with PanIN3 lesionsdemonstrating increased mitoses [] As disease progresses to PDAC cells become invasive and breach the basement membrane growing through the extracellular matrix and metastasising to distant ans Figure Less common precursor lesions include intraductal papillary mucinous neoplasms IPMNs and mucinous cysticneoplasms MCNs that also develop through multistep processes [] Whilst they share some common featureseach lesion is morphologically and genetically distinct In contrast with PanINs that form within small ducts IPMNsdevelop within the primary or secondary branches of the main pancreatic duct whilst MCNs lack ductal involvement[]An ‚ammatory tumour microenvironment contributes to PDACpathogenesisAn archetypal feature of PDAC is the development of extensive stromal networks within the tumour microenvironment TME that can account for up to of the total tumour volume [“] This unique characteristic drives theinflammatory nature of PDAC that contributes to its aggressive phenotype [] Desmoplasia is driven by pancreaticstellate cells PSCs and cancerassociated fibroblasts CAFs that upon activation produce a range of extracellularmatrix ECM components such as collagen laminin and fibronectin which in turn form a physical barrier preventing the penetration of therapeutics [“] Though PSCs and CAFs have been shown to support cancer metastasis and drug resistance they interact with cancer cells in a bidirectional manner with each promoting the survivalgrowth and proliferation of the other [“] Quiescent fibroblasts are able to differentiate into two unique subtypes termed myofibroblastic CAFs myCAFs and inflammatory CAFs iCAFs [] These two subtypes are distinct whereby myCAFs express high levels of αsmooth muscle actin αSMA and are located adjacent to cancercells while iCAFs express low levels of αSMA and instead secrete high levels of inflammatory mediators including IL6 and are distributed distant from cancer cells within desmoplastic tumour regions [] Broadly myCAFsappear to have roles in epithelialtomesenchymal transition EMT and ECM remodelling whilst iCAFs appear tobe involved in inflammation and ECM deposition [] A third less abundant subtype termed antigenpresentingCAFs apCAFs has more recently been defined [] These cells express low levels of both αSMA and IL6 andinstead express high levels of major histocompatibility complex class II MHCII and related genes [] As such allthree subtypes are transcriptionally and functionally distinctThe wider TME contains a plethora of other cell types including endothelial cells tumourassociated macrophagesTAMs and neutrophils TANs mast cells regulatory Tcells myeloidderived suppressor cells MDSCs dendriticcells natural killer NK cells and nerve cells [] Interactions between various cells within the TME can driveeither proor antitumorigenic functions of others for example cancer cells can induce PSCs to secrete inflammatorycytokines that drive immune cells towards an immunosuppressive phenotype and also form a positive feedback loopby increasing the tumorigenic potential of cancer cells [] The ECM itself has also been suggested to modifyPSC behaviour in particular that ECM stiffness promotes the myCAF phenotype indicated by increased αSMAexpression [] This results in substantial complexity that ultimately determines tumour phenotype []The components of the microenvironment modify tumour behaviour through the production of cytokines growthfactors and other signalling molecules that predominantly drive a proinflammatory and immunosuppressive program that enhances PDAC tumour growth and progression [“] Figure The ability of the TME toinhibit therapeutic efficacy through both molecular mechanisms and physical fibrotic barriers contributes to the The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BYNCND 0cClinical Science “101042CS20191211Figure Our current understanding of the contribution of IL6 family cytokines to PanIN and PDAC developmentPreinvasive PanIN lesions develop from normal ductal epithelia through PanIN stages 1A 1B and to stage invasive andormetastatic PDAC This process is associated with acinartoductal metaplasia ADM early in disease combined with an accumulation of oncogenic mutations most common mutations are indicated A number of cells within the tumour microenvironment havebeen shown to secrete IL6 family cytokines which in turn results in the activation of a protumorigenic signalling cascade A betterunderstanding of the relationship between each of these cells within the tumour microenvironment may reveal new therapeuticopportunities to manage cancer progressionintrinsic resistance of disease [] Thus dual targeting of cancer cells and the TME may be required to induce afavourable therapeutic response although this poses a signficant scientific and clinical challenge [“]Molecular basis of pathogenesisPDAC development is associated with accumulation of mutationsThe progression of tumorigenesis through PanIN and PDAC stages is associated with the stepwise accumulation ofspecific genetic mutations that drive malignant transformation The most frequent genetic alteration is an activatingKRAS point mutation codon that occurs early on in tumour development [] and is detected in over ofPDAC tumours [“] Mutations in KRAS have been shown to drive development of precursor PanIN lesions andwhen combined with an appropriate tumour suppressor mutation these lesions progress to invasive or metastaticPDAC [] Figure Patient tumours harbouring wildtype WT KRAS often carry activating mutations indownstream effector molecules such as BRAF or PIK3CA [] The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BYNCND 0cClinical Science “101042CS20191211Inactivation of a range of tumour suppressor proteins is also common including mutations in TP53 CDKN2Aand SMAD4 in approximately and of tumours respectively [] Whilst each mutation has uniquemechanistic outcomes all three proteins are either directly or indirectly involved in the regulation of the G1S cellcycle checkpoint Analysis of patient tumours indicates that two or more of these mutations often occur together withCDKN2A mutation being combined with either TP53 SMAD4 or both usually in the background of KRAS mutation [] This suggests that by disrupting this checkpoint cancer cells are able to overcome inhibitory mechanismsallowing continued progression to invasive diseaseUnbiased sequencing efforts have also enabled identification of low prevalence PDAC mutations observed in lessthan of cases [] These include mutations in genes involved in chromatin modification KDM6A DNAdamage repair ATM and other tumourrelated processes such as growth TGFBR1 or TGFBR2 []Furthermore it is important to note that technical advances are continuously uncovering epigenetic mechanisms thatfurther modulate the PDAC transcriptional landscape and ultimately influence disease heterogeneity and tumourprogression []Molecular subtypesThe PDAC epithelial compartment is typically divided into two subtypes including a classical subtype exhibiting anepitheliallike expression profile and a squamous or mesenchymallike subtype [] An additional third exocrinesubtype is outlined in some analyses and is characterised by a gene expression profile related to digestive enzymeproduction [] The classical or epithelial subtype has also been further divided into a pancreatic progenitor andan immunogenic subtype whereby the immunogenic subtype is distinguished by significant immune infiltration andassociated gene programmes [] Though there is no consensus on which classification system will allow the mostvaluable stratification of patients the mesenchymal subtype is invariably associated with a poor prognosis []The stromal compartment has also been classified into either normal or activated subtypes reflecting the proandantitumorigenic capabilities of the TME with the activated subtype associated with reduced survival [] This isparticularly valuable as the extensive heterogeneity of PDAC complicates clinically relevant stratification of patientsThus the identification of molecularly unique subtypes may enable development of tailored therapeutic regimensthat will provide improved clinical outcomesCurrent treatment optionsRegardless of disease stage at time of diagnosis patients have relatively limited treatment options For the majorityof patients disease will be locally advanced or metastatic disqualifying them from undergoing potentially curativesurgery [] In these cases patients are typically offered chemotherapy with palliative intent [“]Surgery provides the only potentially curative treatmentSurgical resection remains the only potentially curative treatment option due to minimal efficacy of standardofcarechemotherapy and radiotherapy Due to its aggressive nature the majority of patients present to clinic with locallyadvanced or metastatic disease with only “ of patients presenting with localised tumours that are eligiblefor surgical resection [] Even for those able to undergo surgical intervention over of patients relapsepostresection [] with median survival improving to months and 5year relative survival rate increasingmodestly to “ [] The use of neoadjuvant therapy is generally reserved for borderline resectable disease inan effort to enable patients to become eligible for surgery [] However a range of recent trials have shown improved clinical outcomes including overall survival for neoadjuvant treatment of patients with resectable tumours[] Following surgical resection patients are typically treated with adjuvant gemcitabinebased chemotherapy []although a recent study showed improved diseasefree survival and overall survival with a modified FOLFIRINOXtherapy combination of oxaliplatin irinotecan leucovorin and fluorouracil []Radiotherapy provides variable clinical outcomeWhilst the use of radiotherapy and chemoradiotherapy combination chemo and radiotherapy in the neoadjuvantand adjuvant settings have been investigated there remains a lack of consensus regarding therapeutic benefit []This is due to issues such as insufficient radiation dose and low participant numbers as well as low uptake of moderntechniques [] In the neoadjuvant setting preliminary studies reported reduced lymph node positivity and rates oflocal recurrence for chemoradiotherapy compared to surgery with adjuvant chemotherapy [] However the useof radiotherapy in the palliative setting was reported to modestly reduce overall survival [] More recent studiesusing ablative doses of radiation have shown a survival benefit highlighting that technological advancements mayprovide an avenue for improved clinical outcomes following radiotherapy [] These contrasting results highlight the The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BYNCND 0cClinical Science “101042CS20191211need to determine which subset of patients may benefit from the inclusion of radiotherapy approaches in standardtreatment regimensChemotherapy remains the cornerstone of treatmentDespite modest improvements in overall survival palliative chemotherapy remains the standard treatment optionfor patients with locally advanced or metastatic disease Gemcitabine monotherapy has been the mainstay treatmentfor pancreatic cancer since when it was demonstrated to improve median survival by just over month compared with fluorouracil [] Within the last decade there have been some further improvements in clinical outcomewith combination chemotherapies gemcitabinenabpaclitaxel and FOLFIRINOX providing median overall survivalbenefits of and months respectively compared with gemcitabine alone [] Although FOLFIRINOX treatment resulted in a lower percentage of patients experiencing reduced quality of life it also had increased toxicity andadverse events thus preventing its administration to patients with multiple comorbidities []Therapeutic resistance remains a significant barrier to patient survivalDespite advances in chemotherapeutic options treatment efficacy and patient prognosis remain poor due to the inherent therapeutic resistance of pancreatic cancer It has been proposed that this drug resistance may be driven by theTME including changes to cytokine signalling and metabolic pathways [“] This intrinsic resistance is demonstrated by patients experiencing similar overall survival for chemotherapy treatment “ months compared withbest supportive care “ months which encompasses the use of palliative surgery psychological support painmanagement and other methods of symptom control [] Whilst a range of targeted treatments such as EGFR orcheckpoint inhibitors have been trialled with or without chemotherapy they have shown limited success [“]Emerging roles for the IL6 family of cytokines in PDACCytokines are soluble molecular messengers that enable efficient communication between a range of cell types andhave been recognised to be major contributors to the growth and metastasis of cancers [“] In pancreatic cancer cytokines mediate signalling between cancer cells and the cells of the TME including PSCs CAFs endothelialcells and a range of immune cells including macrophages mast cells neutrophils and regulatory Tcells [“]It is the specific signalling pathways active within this community of cells that dictates the balance of pro andantitumorigenic functions []The IL6 family of cytokinesThe interleukin IL6 family of cytokines includes IL6 IL11 leukaemia inhibitory factor LIF oncostatin MOSM ciliary neurotrophic factor CNTF cardiotrophin1 CT1 cardiotrophinlike cytokine CLC neuropoietin NP IL27 and IL31 [“] These cytokines are grouped as they share structural similarity forming a fourαhelical bundle termed helices AD with an upupdowndown topology []IL6 and IL11 utilise a hexameric signalling complex consisting of two molecules each of the cytokine αreceptoreither IL6R or IL11R respectively and βreceptor glycoprotein gp130 [“] IL6 and IL11 are ableto signal via two distinct mechanisms termed classic and transsignalling Classic signalling involves the formation of a complex including membranebound IL6R or IL11R with gp130 and the respective cytokine Converselytranssignalling utilises soluble IL6R or IL11R molecules which are able to form a signalling complex with gp130and the respective cytokine [“] In this way classic signalling relies on the responding cell™s intrinsic expressionof IL6R or IL11R whilst transsignalling is able to activate any cell expressing gp130 []LIF OSM IL27 and IL31 signal through trimeric complexes with a single cytokine molecule engagingthe respective receptor LIFR OSMR IL27R WSX1 or IL31R and either gp130 or OSMR for IL31[“] CNTF CT1 CLC and NP form tetrameric signalling complexes composed of one cytokinemolecule one LIFR one CNTFR and one gp130 receptor [] In each case the active signalling complex consists of two chains that are signalling competent with a combination of either gp130 LIFR OSMR IL27R or IL31R[] The requirement for multiple receptor subunits means that although gp130 is ubiquitously expressed the expression of other receptor subunits dictates the ability for any given cell to respond to cytokine as signalling initiationrequires the presence of cytokine and a compatible receptor complex [] Figure 2ASignalling complex assembly leads to transphosphorylation and activation of receptorassociated Janus tyrosinekinases JAKs largely JAK1 and to a lesser extent JAK2 and TYK2 [] In the case of gp130mediated signalling this results in phosphorylation of the cytoplasmic domain of gp130 at tyrosine Y and [] Phosphotyrosine pY and of gp130 provide docking sites for signal transducer and The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BYNCND 0cClinical Science “101042CS20191211Figure IL6 family cytokine signalling pathwayA Schematic representation of the stepwise binding process for the IL6 family members with IL6 as an example The site interaction involves cytokine binding to the respective receptor with the site interaction generally between the cytokine and thecommon gp130 receptor chain finally site interactions involve formation of the final active signalling complex in this case formation of the IL6IL6Rgp130 hexameric complex B General outline of the IL6 family cytokine signalling pathway Formation ofan active hexameric complex leads to activation of JAKs with subsequent activation of the STAT3 MAPK and PI3K pathways leftThis results in upregulation of the negative regulator SOCS3 as well as a range of inflammatory and protumorigenic moleculesThe pathway is inhibited by SOCS3 PIAS3 and PTPs via dephosphorylation ubiquitinmediated proteasomal degradation andSUMOylation right The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BYNCND 0cClinical Science “101042CS20191211activator of transcription STAT molecules leading to their subsequent phosphorylation by JAK1 and formation ofactive STAT dimers [“] Phosphorylated STAT pSTAT dimers then translocate to the nucleus and modulatetarget gene expression including upregulation of a range of genes involved in inflammatory and protumorigenicpathways [“] Figure 2B Broadly these STAT3regulated genes can be categorised into pathways associatedwith inhibition of apoptosis increased cell proliferation modulation of immunity and inflammation increased angiogenesis and increased invasive and metastatic potential [“]Although JAKSTAT signalling is the predominant pathway activated downstream of IL6 family cytokines themitogenactivated protein kinase MAPK and phosphoinositide 3kinase PI3K pathways can also be activated[] The MAPK pathway has been suggested to be activated by a Src homology domain 2containing phosphatase SHP2mediated mechanism whereby SHP2 is recruited to pY759 on gp130 allowing JAKmediated phosphorylation of SHP2 [] This promotes association with the adaptor protein growth factor receptor bound protein Grb2 leading to activation of the Gprotein Ras via son of sevenless SOS with a subsequent phosphorylationcascade including Raf MEK and ERK12 activity [] Following this a MAPKdependent phosphorylationevent leads to the recruitment of Grb2associated binding protein Gab1 to the plasma membrane where Gab1 issuggested to act as a scaffold or adaptor protein to allow binding of PI3K and SHP2 leading to activation of the PI3Kand MAPK pathways respectively [] Figure 2BThe suppressor of cytokine signalling SOCS3 is largely responsible for regulation of signalling and is directlyupregulated by STAT3 [] SOCS3 contains an SH2 domain allowing it to bind to pY residues within the gp130receptor [] with preferential binding to Y759 [] Once bound SOCS3 recruits an E3 ubiquitin ligasecomplex containing Cullin5 Rbx2 and adaptors Elongin B and C via its SOCS box domain [] This complexubiquitinates the gp130 receptor inducing its internalisation and targeting it for proteasomal degradation [“]and is also able to ubiquitinate JAK2 in vitro [] SOCS3 also mediates direct inhibition of the kinase activityof JAK12 via its kinase inhibitory region [“] Thus SOCS3 is able to downregulate IL6 family cytokinesignalling pathways through two distinct mechanismsThe phosphotyrosine phosphatases PTPs and protein inhibitors of activated STATs PIASs also limit the strengthand duration of cytokine signalling [] A range of PTPs including SHP2 are responsible for dephosphorylatingtyrosine phosphorylated substrates including JAKs STATs and other SHP2 molecules [] PIAS3 preferentially binds pSTAT3 and inhibits activity either by preventing STAT3 interaction with DNA by recruiting transcriptional repressors to STAT3 target genes or by SUMOylating STAT3 to prevent its activity [] Figure 2BInterleukin in PDACElevation of serum IL6 is a negative prognostic marker in human PDACSerum IL6 levels were increased in PDAC patients compared with healthy patients [“] or those withchronic pancreatitis [] and were also increased in patients with metastatic PDAC compared to thosewith locally advanced disease [“] Moreover elevated serum IL6 positively correlated with increased diseaseburden weight losscachexia and metastasis [““] however there are conflicting observations inthe literature regarding IL6 and cachexia [] Although increased serum IL6 levels correlate with increased disease stage and in metastatic patients correlates with poor overall survival [] As such it has been suggestedthat IL6 may be a superior marker for diagnostic and prognostic purposes compared with the standard Creactiveprotein CRP carcinoembryonic antigen CEA and carbohydrate antigen CA199 markers []IL6 is expressed within the TMElL6IL6 was overexpressed in human PDAC tumours in comparison with adjacent normal tissue [] Whilstthis tumourspecific elevation has been correlated with reduced survival in some studies [] othersshowed no significant correlation with survival [] similar to the data available in The Cancer Genome AtlasTCGA dataset for both IL6 and IL6R Figure 3AB The TCGA comprise aggregate sequencing data which doeshave limitations regarding interpretation of contributions of individual cell populations to disease outcome howeverit remains a widely used resource for exploratory investigations However overexpression of IL6 has been observedat the mRNA and protein level in the pancreata of PDAC mice [] with Il6 expression increasing with agewhich is indicative of disease stage in these models []Despite the presence of IL6 in tumours primary human and commercial pancreatic cancer cell lines have been reported to exhibit variable expression levels of IL6 and secreted cytokine albeit consistently higher than normal pancreatic ductal epithelial cells [] In an anoid model minimal IL6 was expressed by pancreaticcancer cells PCCs or PSCs in monoculture however in coculture PCCs expressed only Il6ra whilst iCAFs expressedhigh levels of IL6 with this activating STAT3 within PCCs [] iCAFs also demonstrate an upregulation of The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BYNCND 0cClinical Science “101042CS20191211Figure IL6 family cytokine expression in PDAC patientsOverall survival for patients with high top quartile and low bottom quartile level expression of A IL6 B IL6R C IL11 D IL11RE LIF F OSM G CNTF H CTF1 CT1 I CLCF1 CLC and J IL27 n per group Data and graphs obtained fromOncoLnc [] using data from The Cancer Genome Atlas TCGA Statistical significance determined by MantelCox Logranktest The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BYNCND 0cClinical Science “101042CS20191211the JAKSTAT pathway with expression of IL6 being dramatically increased in vitro when incubated with PCC conditioned media indicating that soluble factors trigger IL6 production [] More recently PCCderived IL1αhas been shown to induce autocrine LIF secretion and thereby promote the iCAF phenotype including activation ofthe JAKSTAT signalling pathway and IL6 production []In addition TAMs have been identified as producers of IL6 in pancreatic cancer by correlative immunohistochemistry and expression analysis of isolated cell populations [] Production of IL6 by TAMs was shownto influence tumour development via bonemarrow chimeras as mice reconstituted with IL6 knockout KO Il6myeloid cells developed lowgrade PanINs whilst those reconstituted with IL6 WT cells developed PanIN3 lesions[]IL6 is a driver of PDAC pathogenesisBoth in vitro and in vivo studies suggest that the presence of IL6 in the TME can drive activation of STAT3 []with IL6 inhibition reducing STAT3 phosphorylation [] This IL6STAT3 program has been proposed tobe a driver of PDAC pathogenesis by enhancing tumour initiation and progression angiogenesis regulation of cytokine expression and immune cell behaviour resistance to apoptosis and promotion of metastasis [“] In aninducible KRASdriven mouse model genetic deletion of Il6 resulted in a reduction of ADM and PanIN formationwhen KRAS mutation was initiated embryonically compared with controls suggesting a role for IL6 in tumour initiation [] This was also observed in a constitutive KRAS mutant model where genetic deletion of IL6 preventedtumour initiation in vivo with a reduction in the number of PanIN and lesions [] Interestingly oncogenicKRAS and hypoxic conditions both features of PDAC tumours [] were shown to induce IL6 production[] perhaps representing a feedforward pathway enhancing tumorigenesis [] However IL6 is notabsolutely required for PanIN formation as induction of KRAS mutation at weeks of age in conjunction with anexperimental pancreatitis model drove formation of PanIN lesions that were not significantly different between IL6WT and KO mice []Il6 mice exhibited reduced tumour progression with decreased proliferative capacity of both cancer and stromal cells enabling regression of precursor lesions [] Furthermore this inhibition of tumour progression by IL6deletion was due at least in part to the reversal of ADM with ductal cells reverting to an acinarlike phenotype[] Increased apoptosis of cancer and stromal cells was also shown to contribute to this reduced tumour progression as demonstrated by appropriate immunohistochemical analyses with upregulation of proapoptotic anddownregulation of antiapoptotic BCL2 family members [] This is mirrored by in vitro data whereby IL6 stimulation increased the expression of antiapoptotic BCL2BCL2 and BCL2L1BCLXL [] with blockade of IL6signalling or STAT3 activation inducing apoptosis [] Collectively these data suggest that whilst IL6 contributes it is not required for PDAC initiation and progressionThe process of angiogenesis supports tumour growth and progression by enabling adequate blood supply whichis enhanced by IL6 signalling Upon IL6 stimulation PDAC cell lines upregulate key angiogenic factors such asvascular endothelial growth factor VEGFVEGF and neurophilin1 NRP1NRP1 [] with significant correlation observed between the expression of IL6R and VEGF on human PDAC sections [] IL6inducedupregulation of VEGF correlated with a growth advantage in PCCs with both features inhibited by treatment witha JAK2 inhibitor []Another facet of the protumorigenic effects of IL6 is the regulation of cytokine expression that enables modulationof the immune system [] In particular it has been shown that IL6 is able to upregulate a type cytokine profile invitro that may inhibit antitumour immunity in disease [] IL6 suppressed the differentiation of human CD14cells into dendritic cells DCs in vitro whilst combination treatment with IL6 and granulocyte colonystimulatingfactor GCSF inhibited the ability of DCs to respond to alloantigen a process that is required for DC maturationand antigen presentation where these effects were reversed by blockade of IL6 andor GCSF [] IL6 has alsobeen implicated in driving increased apoptosis of type I conventional DCs cDC1s leading to cDC1 dysfunctionea

Thyroid_Cancer

"elucidate the molecular mechanism underlying the involvement of abnormal DNA methylation in the development of glioma and identify potential newtargets for glioma therapyMethods The GSE79122 chip achieved from the Gene Expression Omnibus GEOdatabase containing glioma samples and normal samples was analyzed Methylationspecific polymerase chain reaction MSPCR or MSP reverse transcriptionPCR andWestern blot analysis were used to confirm the methylation level and expression level ofthe interleukin receptorassociated kinase IRAK3 gene in glioma cells glioma samplesand the corresponding normal samples In vitro the proliferation apoptosis rate migrationand invasion abilities of glioma cells were detected by Cell Counting Kit8 assay Transwellassay enzymelinked immunosorbent assay and flow cytometry respectively Besides thexenograft assay of nude mice was used to confirm the effect of the IRAK3 on glioma in vivoResults Microarray analysis showed that the IRAK3 was one of the most hypermethylated genesin glioma and the related mitogenactivated protein kinase MAPK signaling pathway wasactivated More experiments supported the higher methylation level and lower expression levelof the IRAK3 in glioma tissues and cell lines The viability migration and invasion ability ofglioma cells significantly reduced and the apoptosis rate increased with the overexpression anddemethylation of the IRAK3 in vitro Besides treatment with the MAPK signaling pathwayinhibitor PD325901 alone or the overexpression or demethylation of the IRAK3 had a similareffect as the overexpression or demethylation of the IRAK3 alone in glioma cells In vivoxenotransplantation experiments in nude mice confirmed that the overexpression and demethylation of the IRAK3 and suppression of the MAPK signaling pathway inhibited the development ofgliomaConclusion IRAK3 inhibited the development of glioma progression through the MAPKsignaling pathwayKeywords glioma IRAK3 MAPK signaling pathway methylationIntroductionGlioma also known as glioblastoma is one of the most common primary malignant braintumors The average incidence rate of glioma is in individuals1 Despiteimprovements in neurosurgery and radiotherapychemotherapy most patients die within months of diagnosis1 and less than patients survive for years or even more2Recently the molecular mechanisms of glioma have gained increasing attention so as tofind some better methods to defeat this disease Previous studies evaluated that longtermsurvivors of glioma often displayed some favorable molecular characteristics such as thesubmit your manuscript wwwdovepresscomDovePresshttp102147CMARS252772Cancer Management and Research “ Wu This work is published and licensed by Dove Medical Press Limited The full terms of this license are available at wwwdovepresscomtermsphpand incorporate the Creative Commons Attribution “ Non Commercial unported v30 License httpcreativecommonslicensesbync30 By accessing the workyou hereby accept the Terms Noncommercial uses of the work are permitted without any further permission from Dove Medical Press Limited provided the work is properly attributed Forpermission for commercial use of this work please see paragraphs and of our Terms wwwdovepresscomtermsphp 0cWu et alDovepresshypermethylation of O6methylguanine DNA methyltransferase MGMT promoter3 which is known as a meaningfulpredictive biomarker for the favorable prognosis of the chemotherapeutic drugs4 Therefore this study proposed that epigenetic regulation might play a key role in the development ofglioma which deserves further research for understanding thiscancerDNA methylation isan epigenetic modificationinvolved in many biological processes especially geneexpression regulation5 The DNA methylation patterns ofnormal cells are controlled well by a balance betweenmethylation and demethylation However this balance isalways disturbed in cancer cells through ectopic deficientor excessive methylation leading to abnormal biologicalactivities6 Hypermethylation of CpG islands on specificpromoters inhibiting the transcription of downstreamtumor suppressor genes has been discovered in manycancers which may provide clinicians a new strategy forpatients with cancer7 For instance the promoter region ofSEPT9 is hypermethylated in colorectal cancer Hence theSEPT9 gene methylation assay might become a potentialoption for the early detection and screening of colorectalcancer8 CpG islands also display aberrant hypermethylation at a large number of loci and define the subgroup ofglioma910 However the underlying molecular events ofDNA methylation and glioma development remain poorlyunderstoodRecently technical advances in genomewide expression analysis have enabled an improved understanding ofthe diagnosis and prognosis of many types of tumors11Genomewide DNA methylation analysis allows comprehensive DNA methylation profiling of the whole genomehelping in the effective identification of novel genes thathave a potential value in clinical practice12 Previous studies have reported many specific methylation signaturegenes in differenttypes of cancers such as thyroidcancer13 lung squamous cell carcinoma14 hepatocellularcarcinoma15 prostate cancer16 and so on using DNAmethylation analysisThis study aimed to examine the genomewide DNAmethylation profiling of glioma tissuesrevealing thehypermethylation of several genes in glioma Then oneof these hypermethylated genes IRAK3 was selected toconductSubsequentlya relationship between IRAK3 and MAPK signaling pathway was demonstrated by using DNA methyltransferaseinhibitor overexpression of IRAK3 and MAPK signalingpathway inhibitor which can disrupt the development ofcomprehensiveaanalysisgliomas in vitro and in vivo The findings might providesome clues for the regulatory role of DNA methylationand the underlying application of targeted treatment ingliomaMethodsTissue SamplesGlioma tissues and adjacent normal tissues were collectedfrom patients with primary glioma n admitted to theZhangye People™s Hospital Affiliated to Hexi UniversitySample collection and use was performed according to theapproval of the ethics committee of the Affiliated Hospitalof Shandong University Written informed consent wasprovided by every patient with glioma All samples werefrozen in liquid nitrogen and stored at “°C All humanspecimens were obtained with the approval by theInstitutional Ethics Committee of Zhangye People™sHospital Affiliated to Hexi UniversityCell CultureHuman gliocyte cell line HEB and glioma cell linesSHG44 U251 GOS3 HS683 and SF539 wereobtained from Bena Culture Collection Beijing ChinaHEB U251 HS683 and SF539 cells were grown in highsugar Dulbecco™s modified Eagle™s medium DMEMwith fetal bovine serum FBS SHG44 cells weregrown in RPMI1640 medium containing NaHCO3 gL glucose gL and sodium pyruvate gLwith FBS The GOS3 cells were grown with minimum essential medium with Earle™s Balanced Saltswith FBS and mML glucose All cells were cultured at °C under a humidified atmosphere with CO2Cell TransfectionpcDNA31“interleukin receptor“associated kinase pcDNA31IRAK3 was obtained from GenePharma ShanghaiChina and 5azadC 5aza2ʹdeoxycytidine 5azadCand signaling pathway inhibitor PD325901 were obtainedfrom SigmaAldrich MO USA U251 cells were seeded insixwell plates at — cellswell and cultured at °C andthe cell confluence reached “ CO2 untilTransfections were executed using Lipofectamine Invitrogen CA USA following the manufacturer™s protocols The medium was changed with the complete mediumafter h of transfectionsubmit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressTable qRTPCR PrimersWu et alAccession NumberSequence 5ʹ3ʹForwardReverseForwardReverseForwardReverseForwardReverseACCCAAACTAACTGATTTTGCCAAGAGAAATTCCGAGGGCAGGCGACCTCCCATGGCAATTTTAACAGAGACAGGCATGGGAAGCCATCTCGACCAGTCCGTCTAGTTGGTCTGTCTCCGCTAAATACGGACTGCAGCCCTGAGGTCAATGAAGGGGTCGTGeneIRAK3MEK1CfosNM_007199NM_002755NM_005252GAPDHNM_008084GenomeWide DNA MethylationAnalysisFor DNA methylation profiling Infinium HumanMethylation450K BeadChip illuminaga_rnaseqv2100 was obtainedfrom the Gene Expression Omnibus GEO database TheDNA methylation data of chip number GSE79122 whichcontained glioma tissues glioblastomas diffuseastrocytomas and anaplastic astrocytomas and controlbrain tissues were analyzed The Infinium MethylationAbbiotec CA USA and Illumina BeadStudio softwareGenetech Biotech Taipei Taiwan were used to measurethe methylation profiles of modified DNA and loci CpGThe methylated signal intensity at particular CpG loci and450K BeadChip assay were presented as β values and percentage respectivelyQuantitative RTPCRTotal cellular RNA was extracted using PureLink Invitrogenfollowing the manufacturer™s protocol RT was performed on µg total cellular RNA using a HighCapacity cDNA ReverseTranscription Kit with RNase Inhibitor Applied Biosystemspurchased from Thermo Fisher Scientific MA USASubsequently quantitative reverse transcriptionpolymerasechain reaction RTPCR was performed using ArcturusParadise Plus qRTPCR Kit Applied Biosystems ThermoFisher Scientific Comparative expression values were calculated by the “ΔΔCt method GAPDH was used for internalreference All primer sequences involved are listed in Table converted into uracil without changing the state of methylated cytosine The IRAK3 methylation level in glioma wasidentified using methylationspecific PCR MSP The MSPprimers are listed in Table EnzymeLinked Immunosorbent AssayThe human interleukin IL6 enzymelinked immunosorbent assay kit Sangon Biotech Shanghai China was usedto test the IL6 level in the glioma cell culture medium Theglioma cell culture medium was centrifuged at rpm for min to remove cells and polymers The supernatant fluidstored at “°C was preserved in the supernatant fluid at “°C A normal glioma cell culture medium was used as thecontrolWestern Blot AnalysisLysis buffer RIPA Thermo Fisher Scientific and NPERThermo Fisher Scientific were used to extract proteinsfrom glioma cells and tissues respectively Then μg totalprotein per sample was separated using SDSPAGE andtransferred to polyvinylidene fluoride membranes ThermoFisher Scientific The membranes were probed with primaryantiIRAK3 antibody ab8116 Abcam MA USA antiMEK1 phospho S298 antibody [EPR3338] ab96379 antiERK1 ERK2 antibody [ERK7D8] ab54230 anticFosphospho T232 antibody ab17933 and antiGAPDH antibody [6C5] ab8245 Abcam as control The number ofTable MethylationSpecific PrimersDNA Methylation AssayGenomic DNA in glioma tissues and cells was extracted andtreated with bisulfite using CpGenome DNA ModificationKit Chemicon CA USA following the manufacturer™sprotocol All unmethylated cytosine residues in DNA wereGeneIRAK3 forwardIRAK3 reverseIRAK3 forwardIRAK3 reverseSequence 5ʹ3ʹ5ʹTCGGGATAGTGGTTAATATTTC3ʹ5ʹTTTTTTTCGTTTTTCGTAAAA3ʹ5ʹ AGTTTGGGATAGTGGTTAATATTTT 3ʹ5ʹ TTTTTTTCATTTTTCATAAAAAAA 3ʹCancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepressFigure Genomewide methylation data were obtained from the GEO database for available glioma9 adjacent mucosa A Density of methylated DNA intensity byeach sample B Type I and Type II assays showed variant β value distributions The differences between probe types were regulated by normalization procedures whichshowed that represented unmethylated sites while represented fully methylated sites C Multidimensional scaling plot showing differential clustering of control versustumor tissues D Dendrogram produced for probes in normal and tumor tissues E Heatmap of top differentially methylated imprinted CpG sitesAbbreviation MG malignant gliomasubmit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et alFigure Distribution of top differentially methylated imprinted CpG sites A Distribution of top differentially methylated imprinted CpG sites according toCpG islands island sea shelf and shore B Distribution of top differentially methylated imprinted CpG sites according to the position relative to genes 1stexon ² UTRs or ² UTRs body IGR TSS1500 and TSS200 C Combined genetic and epigenetic annotation information revealed the distribution of the top differentially methylated imprinted CpG sitesbinding proteins was measured using AlphaEaseFC softwareGenetic Technologies FL USACell Counting Kit8 AssayU251 cells were seeded in 96well plates and allowed toadhere for “ h at °C Then these cells were transfectedwith pcDNA31IRAK3 and treated with 5azadC orPD325901 After “ h Cell Counting Kit8 DojindoKumamoto Japan was used to test the cell viability at respective time points To summarize mL of the triazoliumsubstrate was added to each well and coincubated with cellsat °C for h The absorbance was measured at nm andCancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepressFigure DNA methylation analysis of tumor tissues and normal tissues and analysis of methylation degree for seven CpG sites of IRAK3 A The expression of the top candidate genes was analyzed IRAK3 was hypermethylated significantly in tumor tissues compared with normal tissues B The number of IRAK3“methylated CpG islands ineach isosite C“I Seven CpG sites for IRAK3 which are presented in the boxplot displayed a decreased methylation in the tumor group The boxplot for cg Ccg D cg E cg F cg G cg H and cg IAbbreviation MG malignant gliomasubmit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et alFigure Analysis of the IRAK3related signaling pathway A The top signaling pathways with the highest and lowest correlation with glioma B The top signalingpathway“related genes enriched in the glioma the MAPK signaling pathway was highly expressed C The MAPK signaling pathway was activated in glioma D The status ofthe top enriched signaling pathways in gliomaAbbreviation MG malignant gliomaCancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepressthe results were normalized to untreated cells at respectivetime pointsCell Migration and Invasion AssayBoth cell migration and invasion were performed using theTranswell assay For cell migration assay — U251cells were seeded in a serumfree medium in the upperchamber which contained a polyethylene terephthalatemembrane with mm in diameter and μm in poresize The bottom chamber was prepared with FBS asa chemoattractant After incubating at °C for hnonmigrated cells were scraped from the upper surfaceof the membrane with a cotton swab and the cells migrating to the bottom chamber were fixed with paraformaldehyde and stained with crystal violet Finally the stainedcells were counted under a microscope at — magnification in five randomly selected fields for quantificationinvasion assay — U251 cells weresuspended in mL of serumfree DMEM and thentreated using the same procedure as for the migrationassay following the manufacturer™s protocol but the chambers mm BD Biosciences San Jose CA USA wereplated with BD BioCoat MatrigelFor cellFlow CytometryEach treatment of U251 cells was washed with phosphatebuffered saline and resuspended in μL of AnnexinV binding buffer After staining with Alexa Fluor Annexin V and 7AAD Viability Staining Solution for min in the dark at room temperature these cells were analyzed using multicolor flow cytometer Data were analyzedusing Kaluza softwarethe mice were keptXenograft StudiesMale BALBc nude mice weeks were maintained underpathogenfree conditions Allina temperaturecontrolled airconditioned conventional animal house with a h light“dark cycle and given free accessto food and water Besides animal health and behaviour weremonitored every two days All experiments were approvedby the Ethics Committee of Zhangye People™s Hospital toguarantee that all studies involving experimental animalswere performed in full compliance with National Institutesof Health Guidelines for the Care and Use of LaboratoryAnimals The U251 — cells100 μL cells were transfected with pcDNA31IRAK3 and then transferred to micen12 Normal U251 cells were transferred to mice in the5azadC and PD325901 groups and then 5azadC andPD325901 were subcutaneously injected respectively intothe posterior flank of nude mice After and days™culture the mice were sacrificed and the tumor size wasmeasured The tumor volume was measured using a caliperfollowing the formula length — width22Statistical AnalysisAll data were collected from three independent experiments and presented as mean ± standard deviationStatistical analyses were performed using GraphPad software Differences between groups were analyzed usingStudent' ttest or chisquare test Statistical significancewas set at P ResultsGenome Methylation Profile in GliomaA total of glioma tissues and adjacent normal sampledata publicly available at GEO were analyzed to revealthe global DNA methylation patterns of glioma Firstdensity plots of β values generated from each samplewere used identifying a poor performance of methylatedsignals for raw data Figure 1A Infinium Type I and TypeII probe assays also showed somewhat different distribution of β value ranging from unmethylated sites to fully methylated sites Figure 1B Therefore these datawere regulated by normalization procedures to reduce thepotential impact later Multidimensional scaling MDSplot and dendrogram exhibited a differential clusteringbetween tumor and normal groups which distinguishedglioma from adjacent normal tissues Figure 1C and DFurther heatmap of the top differentially methylatedCpG sites indicated a visible difference in DNA methylation profiles between the tumor and normaltissueand general hypermethylation occurred insamplesglioma Figure 1EDistribution of Genomic Regions forDifferentially Methylated CpG SitesBased on the position relative to gene [1st exon ² untranslated region UTR ² UTRs body transcription start sites bp TSS1500 and TSS200] as well as CpG islands andneighborhood content shores shelves islands and sea the distribution of genomic regions for the differentiallymethylated CpG sites was exhibited More than and methylation differences occurred in bodyIGR and CpGisland sea respectively which was obviously higherthan that in other regions Figure 2A and B From thesubmit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et alFigure Analysis of the IRAK3related gene expression level A Based on the differential genes in the disease data the distribution of upregulated genes anddownregulated genes in the GOrelated pathway was enriched B The expression level of top MAPK signaling pathway“related genesAbbreviation MG malignant gliomaperspective of both genetics and epigenetics gene and CpGcontent regions were combined for more analyses whichshowed that CpG sites in genetic bodyisland sea andIGRisland sea were differentially methylated betweenglioma and adjacent normal samples Figure 2CIRAK3 Was Hypermethylated in GliomaThe heatmap was used to present the top hypermethylationgenes in glioma compared with normal tissues so as to figureout the most characteristic gene with methylation change intumors indicating that the IRAK3 was hypermethylated theCancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepressmost in glioma Figure 3A Then the methylation differentialdistribution of the IRAK3 was upregulated at each siteFigure 3B Furthermore the β value of CpG sitesIRAK3such as cg cg cg cg cg cg cg andso on on the IRAK3 was significantly higher in the tumorindicating that IRAK3group than in the normal groupwas hypermethylated in the tumor group due to CpGsFigure 3C“I The aforementioned results showed that theIRAK3 CpG sites were highly methylated in glioma tissuesthan in normal tissuestissues and the top signaling pathwayrelated genesenriched in the glioma were listed Figure 4A and B Theresults suggested that the MAPK signaling pathway washighly expressed and activated Figure 4C and D Based onthe differential genes in the disease data the distribution ofupregulated genes and downregulated genes in the GOrelatedpathway was enriched Figure 5A The expression level ofMAPK signaling pathwayrelated genes in glioma is shown inFigure 5B The results of Figures and manifested thatIRAK3related MAPK signaling pathway was highly activatedin glioma However whether any correction existed with thedevelopment of glioma remained to be verifiedMAPK Signaling Pathway Expression Leveland State in GliomaFor the identification of gliomarelated signaling pathwaysthat might be influenced by aberrant DNA methylation thetop IRAK3related signaling pathways in glioma and normalMethylation and Expression Level of theIRAK3 in Glioma Tissues and CellsThe impact of hypermethylation on the expression of IRAK3in glioma tissues and cells was elucidated The IRAK3 wasFigure Methylation level and expression level of the IRAK3 in glioma tissues and cells A The IRAK3 was highly methylated in glioma tissues compared with adjacent tissues BThe IRAK3 was less expressed in glioma tissues than in adjacent tissues C The IRAK3 in the five glioma cells SHG44 U251 HS683 SF539 and GOS3 was hypermethylatedcompared with U251 glioma cells D The IRAK3 was less expressed in the five glioma cells than in normal glioma cells The difference was significant P submit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et alFigure Effect on glioma cells after the overexpression and demethylation of the IRAK3 A The expression level of the IRAK3 was significantly higher in the glioma U251 cells of thepcDNA31IRAK3 and 5azadC groups than in the control group B and C The protein expression level of the IRAK3 significantly increased in the pcDNA31IRAK3 and 5azadC groupscompared with the control group D The expression level of the inflammatory factor IL6 significantly decreased in the pcDNA31IRAK3 and 5azadC groups compared with thecontrol group E The activity significantly decreased in the pcDNA31IRAK3 and 5azadC groups compared with the control group F and G The apoptosis rate significantlyincreased in the pcDNA31IRAK3 and 5azadC groups compared with the control group H and I The migration capability was significantly lower in the pcDNA31IRAK3 and 5azadC groups compared with the control group J and K The invasiveness capability was significantly lower in the pcDNA31IRAK3 and 5azadC groups than in the the control group Allthe mentioned differences were significant P The IRAK3 had a suppressive effect on glioma cells in vitroCancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepresshighly methylated and less expressed in glioma tissues than inadjacent normal tissues Figure 6A and B Next the IRAK3 infive glioma cell lines SHG44 U251 HS683 SF539 andGOS3 also showed hypermethylation and less expressioncompared with normal gliocytes Figure 6C and D U251cells were selected for subsequent experiments because themethylation level of the IRAK3 in U251 cells was the highestFigure 6COverexpression or Demethylation of theIRAK3 Inhibited the Development of GliomaCellsA systematic test with the overexpression or demethylation ofthe IRAK3 was conducted to understand whether the correctionof abnormal IRAK3 methylation and expression affected thedevelopment of glioma After using pcDNA31IRAK3 tooverexpress or 5azadC to demethylate the IRAK3 the proteinexpression level of the IRAK3 in glioma U251 cells increasedin the tumor group compared with the normal group Figure7A“C IL6 an inflammatory factor secreted by the MAPKsignaling pathway [PMID ] so that its level in theculture medium could be used to represent the status ofMAPK signaling pathway activation decreased in thepcDNA31IRAK3 and 5azadC groups compared with thecontrol groups Figure 7D Surprisingly the viability ofglioma cells significantly decreased while the apoptosis rateincreased Figure 7E“G and the migration and invasivenesscapability decreased in the pcDNA31IRAK3 and 5azadCFigure Effect on glioma cells after treatment with the MAPK signaling pathway inhibitor PD325901 pcDNA31IRAK3 and 5azadC A The expression level of MAPKsignaling pathway“related genes expression level was significantly lower in the PD325901 pcDNA31IRAK3 and 5azadC groups than in the control group B and C Theexpression level of MAPK signaling pathway“related proteins significantly decreased in the PD325901 pcDNA31IRAK3 and 5azadC groups compared with the controlgroup D The level of inflammatory factor IL6 was significantly lower in the PD325901 pcDNA31IRAK3 and 5azadC groups than in the control group E The activitysignificantly decreased in the PD325901 pcDNA31IRAK3 and 5azadC groups compared with the control group F and G The apoptosis rate significantly increased inthe PD325901 pcDNA31IRAK3 and 5azadC groups compared with the control group All the mentioned differences were significant P P submit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et algroups Figure 7H“K which elucidated the associationbetween IRAK3 and the development of glioma The aforementioned results verified that the overexpression or demethylation of the IRAK3 inhibited the development of glioma cellsin vitroMAPK Signaling Pathway SuppressionAlone or Combined with theOverexpression or Demethylation of theIRAK3 Inhibited the Development ofGlioma CellsThe MAPK signaling pathway inhibitors PD325901pcDNA31IRAK3 and 5azadC were used alone or incombination in vitro to confirm the influence of the IRAK3and MAPK signaling pathways on glioma In PD325901pcDNA31IRAK3PD325901 and 5azadCPD325901groups the mRNA and protein expression levels of theMAPK signaling pathwayrelated genes such as MEK1ERK and cFos as well as the level of IL6 decreased inglioma Figure 8A“D The cell viability significantlythe apoptosis rate increased Figure 8E“Gdecreasedand the migration and invasiveness capability significantlydecreased in PD325901 pIRAK3PD325901 and 5azadCPD325901 groups Figure 9A“C The aforementioned results verified that the MAPK signaling pathwaysuppression alone or combined with the overexpression ordemethylation of IRAK3MAPK Signaling Pathway SuppressionAlone or Combined with theOverexpression or Demethylation of theIRAK3 Inhibited Glioma in vivoFinally whether MAPK signaling pathway suppression aloneor combined with the overexpression or demethylation of theFigure Effect on the migration and invasiveness capability of glioma cells after treatment with the MAPK signaling pathway inhibitor PD325901 pcDNA31IRAK3 and5azadC A and B The migration capability was significantly lower in the PD325901 pcDNA31IRAK3 and 5azadC groups than in the control group A and C Theinvasiveness capability was significantly lower in the PD325901 pcDNA31IRAK3 and 5azadC groups than in the control group All the mentioned differences weresignificant P Cancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepressFigure Effect on mice after treatment with the MAPK signaling pathway inhibitor PD325901 pcDNA31IRAK3 and 5azadC A The tumor in the transplantationPD325901 p IRAK3 PD325901 and 5azadC PD325901 treatment groups after and days B The tumor volume was smaller in the PD325901pcDNA31IRAK3PD325901 and 5azadCPD325901 groups compared with the control group C The tumor weight followed the same trend as the volume D Theexpression level of the IRAK3 was lower in the transplantation PD325901 pcDNA31IRAK3 PD325901 and 5azadC PD325901 treatment groups compared with thetransplantation control group E and F The protein expression level of IRAK3 was lower in the transplantation PD325901 pcDNA31IRAK3 PD325901 and 5azadC PD325901 treatment groups than in the transplantation control group All the mentioned differences were significant P P IRAK3 had a suppressive effect on glioma in vivo was studiedAfter transplanting glioma U251 cells treated with PD325901pcDNA31IRAK3 PD325901 or 5azadC PD325901the tumorigenesis significantly decreased compared with thatin the control group mice Figure 10A“C Finally mRNAand protein expression levels ofthe MAPK signalingsubmit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et alpathwayrelated genes in xenograft glioma were detectedthey were found to be significantly decreased in thePD325901 pcDNA31IRAK3 PD325901 or 5azadC PD325901 group Figure 10D“FDiscussionAccording to the World Health anization the overallsurvival of patients with cancer has increased significantlyover the years with the improvement in diagnosis and treatment However glioma is still a highly fatal disease1Therefore this study aimed to elucidate the detail of itsprogression mechanisms and search for new therapeutic strategies One recent study reported that the use of MGMTpromoter methylation test in hospitals had a strong influenceon the prognosis of glioma17 suggesting a significant clinicalapplication prospect of DNA epigenetic regulation Hencethe main focus of the present study was on the relationshipbetween DNA methylation and glioma IRAK3IRAK3 belongs to the IL receptorassociated kinaseIRAK family involved in inhibiting Tolllike receptorsignaling by changing the activity of other members ofthe IRAK family to decrease inflammatory response1819Increasing evidence supported thatthe expression ofIRAK3 in tumorassociated macrophages led to compromised immune surveillance of cancer cells when it profitably prevented excessive inflammation contributing tocancer development Therefore the growth of transplantable cancer cells could be inhibited by enhancing hostimmune responses in IRAK3deficient mice20 A smallnumber oftumor cellintrinsicIRAK3 could also support the progression of tumor cellsin colorectal and lung cancers depending on the dysfunctional innate immune system indirectly Interestingly theexpression of IRAKM in colorectal and lung cancers correlated with poor prognosisin patients with thesecancers2122 However little is known about the molecularmechanism of the IRAK3 in glioma This study revealedthe downregulation of IRAK3 glioma tissues and cellsthrough DNA methylation analysis which seemed to becontrary to the previous findings on other cancers Thisstudy investigated whether a special signaling pathwayexisted that connected IRAK3 and glioma progressionshowed thatstudiesAn ongoing study has validated that all members of theIRAK family mediate the activation of MAPK and nuclearfactorκB NFκB signaling pathways23 MeanwhileIRAK3 a general negative regulator was confirmed toinhibit MAPK and NFκB activation2425 Likewisethese results identified that the MAPK signaling pathwaywas highly activated in glioma and its related geneexpression level was downregulated after overexpressionof the IRAK3 In fact upstream genomic events andordifferent extracellular stimuli could activate the MAPKsignaling pathway mediating a wide range of cellularprocesses26 The activation of the MAPK signaling pathway often led to abnormal cell growth and tumorigenesisThe predominant effect relied on the signal intensity andthe context or tissue in which the signal occurred2728Zhang revealed that aberrant DNA methylation inthe promoters of MMPTIMP axis genes upregulated theMAPK signaling pathway promoting the progression oftumor cells Correction of the abnormal DNA epigenotypes attenuated the migration and invasion of tumorcells in vitro and reduced tumorigenicity in vivo29Intriguingly these results were consistent with those ofthe present studyConsidering the reverse expression pattern of IRAK3between glioma and other cancers2122 different epigeneticmodification was regarded as the major reason causing thisdistinction In many cancers the DNA methylation patternsbecame aberrant with tissue specificity serving as diagnosticmarkers and therapeutic targets30“ Hence based on thelower expression and the tumorigenic function of the IRAK3in glioma its epigen

Thyroid_Cancer

Detecting ulcerative colitis from a0colon a0samples a0using a0efficient a0feature selection and machine learningHanieh Marvi Khorasani1 a0Hamid a0Usefi2 Lourdes pe±a‘castillo1Ulcerative a0colitis a0UC a0is a0one a0of a0the a0most a0common a0forms a0of a0inflammatory a0bowel a0disease a0IBD a0characterized a0by a0inflammation a0of a0the a0mucosal a0layer a0of a0the a0colon a0Diagnosis a0of a0UC a0is a0based a0on a0clinical a0symptoms a0and a0then a0confirmed a0based a0on a0endoscopic a0histologic a0and a0laboratory a0findings a0Feature a0selection a0and a0machine a0learning a0have a0been a0previously a0used a0for a0creating a0models a0to a0facilitate a0the a0diagnosis a0of a0certain a0diseases a0In a0this a0work a0we a0used a0a a0recently a0developed a0feature a0selection a0algorithm a0DRPT a0combined a0with a0a a0support a0vector a0machine a0SVM a0classifier a0to a0generate a0a a0model a0to a0discriminate a0between a0healthy a0subjects a0and a0subjects a0with a0UC a0based a0on a0the a0expression a0values a0of a0 a0genes a0in a0colon a0samples a0We a0validated a0our a0model a0with a0an a0independent a0gene a0expression a0dataset a0of a0colonic a0samples a0from a0subjects a0in a0active a0and a0inactive a0periods a0of a0UC a0Our a0model a0perfectly a0detected a0all a0active a0cases a0and a0had a0an a0average a0precision a0of a0 a0in a0the a0inactive a0cases a0Compared a0with a0results a0reported a0in a0previous a0studies a0and a0a a0model a0generated a0by a0a a0recently a0published a0software a0for a0biomarker a0discovery a0using a0machine a0learning a0BioDiscML a0our a0final a0model a0for a0detecting a0UC a0shows a0better a0performance a0in a0terms a0of a0average a0precisionInflammatory bowel disease IBD is a chronic inflammatory condition of the gut with an increasing health burden1 Ulcerative colitis UC and Crohn™s disease are the two most common forms of chronic IBD with UC being more widespread than Crohn™s disease2 There is no cure for UC3 and people with the disease alternate between periods of remission inactive and active inflammation2 The underlying causes of UC are not completely understood yet but it is thought to be a combination of genetic environmental and psychological factors that disrupt the microbial ecosystem of the colon34 Genomewide association studies GWAS have identified risk loci for IBD5 and risk loci specifically associated with UC6 However the lower concordance rate in identical twins of in UC compared with in Crohn™s disease indicates that genetic contribution in UC is weaker than in Crohn™s disease7 Thus using gene expression data for disease diagnostic might be more appropriate for UC than using GWAS data as it has been done for Crohn™s disease8There are several features used for clinical diagnosis of UC including patient symptoms and laboratory endoscopic and histological findings7 Boland et at9 carried out a proofofconcept study for using gene expression measurements from colon samples as a tool for clinical decision support in the treatment of UC The purpose of Boland et a0al™s study was to discriminate between active and inactive UC cases even though they only considered gene expression of eight inflammatory genes instead of assessing the discriminatory power of many groups of genes they concluded that mRNA analysis in UC is a feasible approach to measure quantitative response to therapyMachine learningbased models have a lot of potential to be incorporated into clinical practice10 specially in the area of medical image analysis1112 Supervised machine learning has already proved to be useful in disease diagnosis and prognosis as well as personalized medicine1314 In IBD machine learning has been used to classify IBD paediatric patients using endoscopic and histological data15 to distinguish UC colonic samples from control and Crohn™s disease colonic samples16 and to discriminate between healthy subjects UC patients and Crohn™s disease patients using transcriptional profiles of peripheral blood171Department of Computer Science Memorial University St John™s NL A1B3X5 Canada 2Department of Mathematics and Statistics Memorial University St John™s NL A1C5S7 Canada email usefimunca lourdesmuncaScientific RepoRtS 101038s41598020705830Vol0123456789wwwnaturecomscientificreports 0cAccession number of controls of UC cases Description of samplesGSE11521819GSE1122320GSE226192124GSE75214active22GSE75214inactive22Mucosal biopsies from uninflammed colonic tissuesBiopsies from uninflammed sigmoid colonMucosal colonic tissue from discordant twinsMucosal colonic biopsies from active UC patients and from controlsMucosal colonic biopsies from inactive UC patients from controlsPlatformAffymetrix Human Genome U133A Array and Affymetrix Human Genome U133B ArrayAgilent012391 Whole Human Genome Oligo Microarray G4112AAffymetrix Human Genome U133 Plus ArrayAffymetrix Human Gene ST ArrayAffymetrix Human Gene ST Array of genes features UsageModel selectionModel selectionModel selectionModel evaluationModel evaluationTable Summary of datasets used in this studyIn this study our goal was to investigate whether combining machine learning with a novel feature selection algorithm an accurate model using the expression profiles of few genes could be generated from transcriptomewide gene expression data To do this we apply a machine learning classifier on gene expression data to generate a model to differentiate UC cases from controls Unlike previous studies1617 to reduce the effect of technical conditions we combined a number of independent gene expression data sets instead of using a single data set to train our model Additionally by using feature selection we were able to identify genes out of thousands genes for which expression measurements were available The expression values of these genes is sufficient to generate a SVM model to effectively discriminate between UC cases and controls On a gene expression dataset not used during training our proposed model perfectly detected all active cases and had an average precision of in the inactive casesMethodsData a0gathering a0 We searched the NCBI Gene Expression Omnibus database GEO for expression profiling studies using colonic samples from UC subjects in active and inactive state and controls healthy donors We identified five datasets accession numbers GSE11521819 GSE1122320 GSE2261921 GSE7521422 and GSE945216 As healthy and Crohn™s disease subjects were used as controls in GSE945216 this data set was excluded from our study We used three of the datasets for model selection using 5fold crossvalidation and left one dataset for independent validation Table a0 We partitioned the validation dataset into two datasets Active UC vs controls and inactive UC vs controlsAll data sets were obtained from studies where the diagnoses of patients were either based on endoscopical findings GSE7521422 and GSE2261921 followed the criteria described by LennardJones23 GSE1122320 or based on clinical features as well as radiologic endoscopic and laboratory findings GSE115218 Disease state was either assessed during colonoscopy and classified into no signs of inflammation inactive low inflammation and moderatehigh inflammation active GSE22619 defined as active with a Mayo endoscopic subscore ‰¥ GSE75214 or graded by a gastroenterologist or gastrointestinal pathologist GSE11223 GSE1152 The control group had either normal mucosa at endoscopic level GSE75214 no significant pathological findings during endoscopic and histological examinations GSE22619 normal colonoscopies GSE1152 and GSE11223 or tissues abnormalities other than IBD GSE1152 and GSE11223For each dataset GEO2R25 was used to retrieve the mapping between probe IDs and gene symbols Probe IDs without a gene mapping were removed from further processing Expression values for the mapped probe IDs were obtained using the Python package GEOparse26 The expression values obtained were as provided by the corresponding authorsData a0pre‘processing a0 We performed the following steps for data preprocessing i Calculating expression values per gene by taking the average of expression values of all probes mapped to the same gene i Handling missing values with KNearest Neighbours KNN imputation method KNNImputer from the œmissingpy library in Python27 KNNImputer uses KNN to fill in missing values by utilizing the values from nearest neighbours We set the number of neighbours to nneighbours2 and we used uniform weightTo get our final training datasets we merged datasets GSE1152 GSE11223 and GSE22619 by taking the genes present in all of them The merged dataset has UC samples and controls and genes These same genes were selected from GSE75214 for validation As the range of expression values across all datasets were different we normalized the expression values of the final merged dataset and validation dataset by calculating Zscores per sampleModel a0 generation a0 To create a model to discriminate between UC patients from healthy subjects we selected the features genes using the dimension reduction through perturbation theory DRPT feature selection method28 Let D [A b] be a dataset where b is the class label and A is an m — n matrix with n columns genes and m rows samples There is only a limited number of genes that are associated with the disease and as such a majority of genes are considered irrelevant DRPT considers the solution x of the linear system Ax b with the smallest 2norm Hence b is a sum of xi Fi where Fi is the ith column of A Then each component xi of x is viewed as an assigned weight to the feature Fi So the bigger the xi the more important Fi is in connection with b DRPT then filters out features whose weights are very small compared to the average of local maximums over Scientific RepoRtS 101038s41598020705830Vol1234567890wwwnaturecomscientificreports 0cSubsetSubset Subset Subset Subset Subset Subset Subset Subset Subset Subset AP of FeaturesTable Ten top subsets of genes with the highest crossvalidated average APxi™s After removing irrelevant features DRPT uses perturbation theory to detect correlations between genes of the reduced dataset Finally the remaining genes are sorted based on their entropySelected features were assessed using 5fold crossvalidation and support vector machines SVMs as the classifier First we performed DRPT times on the training dataset to generate subsets of features Then to find the best subsets we performed repetitions of stratified 5fold crossvalidation CV on the training dataset We utilized average precision AP as calculated by the function average_precision_score from the Python library scikitlearn29 version as the evaluation metric to determine the best subset of genes among those generated subsets The four subsets with the highest mean AP over the crossvalidation folds were chosen for creating the candidate models For each of the four selected subset of features we created a candidate SVM model using all samples in the training dataset To generate the models we used the SVM implementation available in the function SVC with parameter kernel™linear™ from the Python library scikitlearn To evaluate the prediction performance of each of the ten models we validated it on the GSE75214active and GSE75214inactive datasets In this step we utilized the precisionrecall curve PRC to assess the performance of the candidate models on unseen data An additional candidate model was created using the most frequently selected genesBioDiscML a0 BioDiscML30 is a biomarker discovery software that uses machine learning methods to analyze biological datasets To compare the prediction performance of our models with BioDiscML we ran the software on our training dataset of the samples N52 were utilized for training and the remaining N25 for testing Since the software generates thousands of models and we required only one model we specified the number of best models as in the config file numberOfBestModels1 One best model out of all models was created based on the 10fold crossvalidated Area Under PrecisionRecall Curve numberOfBestModelsSortingMetric TRAIN10CVAUPRC on the train set We used Weka “ to evaluate the performance of the model generated by BioDiscML on the GSE75214active and GSE75214inactive datasets Selected features by BioDiscML are C3orf36 ADAM30 SLS6A3 FEZF2 and GCNT3 In order to be able to use the model in Weka we loaded the training dataset as it was created by BioDiscML which was one of the outputs of the software This dataset has six features including selected genes and class labels and samples We also modified our validation datasets by extracting BioDiscML selected features After loading the training and test dataset in Weka explorer we loaded the model and we entered the classifier configuration as œwekaclassifiersmiscInputMappedClassifier I trim W wekaclassifierstreesRandomTree “ K M V S  which is the classifier™s set up in the generated model by BioDiscMLUse a0of a0experimental a0animals a0and a0human a0participants a0 This research did not involve human participants or experimental animalsResultsFeature a0selection a0reduced a0significantly a0the a0number a0of a0genes a0required a0to a0construct a0a a0classifica‘tion a0model a0 We performed DRPT times on the training dataset to select subsets of features Then we performed 5fold crossvalidation to find the subsets with the highest mean average precision AP over the folds The range of AP for the subsets is between and with an average of ± Table a0 shows the ten subsets with the highest crossvalidated AP and the number of selected features genes on each subset On average DRPT selected ± genes per subsetTop a0 five a0 models a0 are a0 able a0 to a0 perfectly a0 discriminate a0 between a0 active a0 UC a0 patients a0 and a0 con‘trols a0 We selected the four top subsets with the highest mean AP which are subsets and Table a0 and created candidate models based on them Each candidate model was created using all samples on the training dataset and the features of the corresponding subset To identify the genes most relevant to discriminate between healthy and UC subjects we looked at the number of times each gene was selected by DRPT On DRPT runs genes were selected at least once The upper plot on Fig a0 shows the number of times each gene was selected and the lower plot shows the normal quantilequantile QQ plot Based on this plot we Scientific RepoRtS 101038s41598020705830Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Identifying the most frequently selected genes Top Number of times each gene was selected Genes were sorted based on the number of times they were selected by DRPT Bottom Normal QQplot Horizontal line at indicates the threshold selected to deem a gene as frequently chosensaw that the observed distribution of the number of times a gene was selected deviates the most from a Gaussian distribution above times We considered the genes selected by DRPT more than times as highly relevant and created a fifth model using genes selected by DRPT at least times over runsIn order to evaluate the prediction performance of the candidate models each model was tested on the validation datasets and PRC was plotted for model assessment Figs a0 and As the AP approximates the AUPRC34 we used AP to summarize and compare the performance of these five models All five candidate models achieved high predictive performance on the validation dataset GSE75214active with an average AP of ± while the average AP of these five models on the validation dataset GSE75214inactive was ± The models with the best performance were the model created with the most frequently selected genes and subset with an AP of and on GSE75214active and GSE75214inactive respectively However based on a Friedman test35 p ˆ’ value all five models have comparable performance on the validation datasets We chose the model generated with the most frequently selected genes as our final modelOur a0top a0models a0outperformed a0the a0model a0generated a0by a0BioDiscML a0 The average AUPRC achieved by the model created by BioDiscML on both GSE75214active and GSE75214inactive datasets was and respectively Comparing the performance of our candidate models and the model created by BioDiscML on the two validation datasets we observed that we achieved better AUPRC on both datasets AUPRC on the active dataset AUPRC on the inactive dataset In terms of running time subset selection by DRPT and final model creation and validation took minutes while the running time of BioDiscML to create all the models and output the best final model was minutesScientific RepoRtS 101038s41598020705830Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Precisionrecall curve of top selected subsets on GSE75214activeFigure a0 Precisionrecall curve of top selected subsets on GSE75214inactiveLinks a0between a0the a0most a0frequently a0selected a0genes a0and a0UC a0 We used Ensembl REST API Version to find the associated phenotypes with each gene belonging to the subset of the most frequently selected genes Table a0 Among these genes FAM118A is the only one with a known phenotypic association with IBD and its subtypes The evidence supporting the association of some of the other genes with UC based on phenotype is more indirect For example long term IBD patients are more susceptible to develop colorectal cancer37 and one of the genes TFRC is associated with colorectal cancer IBD patients are more prone to develop cardio vascular disease which is associated with blood pressure and cholesterol38 and four of the most frequently selected genes LIPF MMP2 DMTN and PPP1CB are associated with blood pressure and cholesterolWe looked at whether some of the most frequently selected genes contained any of the known IBDassociated SNPs5 To do this we utilized Ensembl™s BioMart39 website Ensembl Release version September to retrieve the genomic location of the genes We then used the intersectBed utility in BEDtools40 to find any overlap between the IDB risk loci and the genomic location of the genes None of the IBDassociated SNPs was located on our genes Similarly gene set enrichment analysis found no enriched GO term or pathway among these genes Additionally these genes are not listed as top differentially expressed genes in previous studies on UC4142We searched the literature for links between the genes and UC and we found the following MMP2 expression has been found significantly increased in colorectal neoplasia in a mouse model of UC43 and MMP2 levels are elevated in IBD44 TFRC has been found to have an antiinflammatory effect on a murine colitis model45 KRT8 genetic variants have been observed in IBD patients and it was suggested that these variants are a risk factor for IBD46 DUOXA2 has been shown to be critical in the production of hydrogen peroxide within the colon and to be upregulated in active UC47Scientific RepoRtS 101038s41598020705830Vol0123456789wwwnaturecomscientificreports 0cGene symbolCWF19L1FCER2MMP2PPP1CBRPL23AP32ZNF624REG1BTFRCFAM118ACFHR2KRT8PRELID1ZNF92ABHD2C16orf89CAB39LSPATC1LDUOXA2MESP1MAML3PITX2DMTNASF1BPGFBEX4ODF1PTGR1ZNF35LIPFSLC25A13BARX2C2orf42Associated phenotypesSpinocerebellar ataxia autosomal recessive depressive disorder MajorBlood protein levels post bronchodilator FEV1Multicentric OsteolysisNodulosisArthropathy MONA spectrum disorders cholesterol HDL lip and oral cavity carcinoma body height winchester syndromeNoonan Syndromelike disorder with loose anagen hair Heel bone mineral density Blood pressure basophils asopathy with developmental delay short stature and sparse slowgrowing hairAttention deficit disorder with hyperactivity body HeightNoneContrast sensitivity Body Mass IndexBreast ductal adenocarcinoma esophageal adenocarcinoma thyroid carcinoma clear cell renal carcinoma prostate carcinoma pancreatic cancer gastric adenocarcinoma hepatocellular carcinoma lung adenocarcinoma rectal adenocarcinoma basal cell carcinoma colorectal adenocarcinoma squamous cell lung carcinoma head and neck squamous cell carcinoma colon adenocarcinoma iron status biomarkers transferrin levels mean corpuscular hemoglobin concentration red cell distribution width combined immunodeficiency red blood cell traits high light scatter reticulocyte percentage of red cells reticulocyte fraction of red cells Immunodeficiency Chronic inflammatory diseases ankylosing spondylitis Crohn™s disease psoriasis primary sclerosing cholangitis ulcerative colitis Glucose Peanut allergy maternal genetic effects Heel bone mineral densityMacular degeneration blood protein levels feeling miserable alanine aminotransferase ALT levels after remission induction therapy in acute lymphoblastic leukaemia ALL asthmaCirrhosis familial cirrhosis hepatitis C virus susceptibility to cirrhosis cryptogenic cirrhosis noncryptogenic cirrhosis susceptibility to gamma glutamyl transferase levels cancer pleiotropyBody fat distribution heel bone mineral density activated partial thromboplastin timeNoneItch intensity from mosquito bite adjusted by bite size gut microbiota Obesityrelated traits coronary artery disease advanced age related macular degeneration squamous cell lung carcinoma pulse pressureNoneHemoglobin S erythrocyte count pancreatic neoplasmsNoneFamilial thyroid dyshormonogenesis thyroglobulin synthesis defectNoneSocial science traits intelligence MTAG chronic mucus hypersecretion borderline personality disorder congenital heart malformationAxenfeldRieger syndrome ring dermoid of cornea iridogoniodygenesis type peters anomaly familial atrial fibrillation rieger anomaly stroke ischemic stroke cataract PITX2related eye abnormalities phosphorus cognitive decline rate in late mild cognitive impairment creatinine intraocular pressure incident atrial fibrillation wolffparkinsonwhite pattern parkinson disease early onset atrial fibrillation anterior segment sygenesis Total cholesterol levels LDL cholesterolNoneMood instability blood protein levelsNoneBody weight body mass index glucose IgA nephropathy Chronic lymphocytic leukaemia type diabetes erythrocyte indicesBody height menarche monocyte count blood protein levelsNoneMaximal midexpiratory flow rate blood protein levels respiratory function tests blood pressureCitrullinemia type II neonatal intrahepatic cholestasis due to citrin deficiency citrin deficiency citrullinemia type I bone mineral densityType diabetes breast cancer night sleep phenotypes response to cyclophosphamide in systemic lupus erythematosus with lupus nephritis strokeNone of times selectedTable Phenotypes associated with the most frequently selected genes by DRPT as obtained from Ensembl REST API Version DiscussionIn this study we showed the feasibility of using machine learning and feature selection to identify a reduced number of genes from microarray data to aid in the diagnosis of UC One might argue that distinguishing UC patients from Crohn™s disease CD patients has more clinical relevance than distinguishing UC patients from controls However we were limited on the choice of groups to classify by data availability as we could only find three gene expression data sets obtained from colonic samples of UC and CD patients in GEO GSE1152 GSE75214 and GSE126124 As children samples were transcriptionally profiled for GSE12612448 instead of adults ones we decided that the age difference could introduce extra biological variation in the expression data unrelated to UC That left us with only two data sets which were not enough to train the model with multiple data sets and have at least one holdout data set for validationAnother limitation of this study is that we used gene expression profiles of colonic samples Further research is required to assess the accuracy of our 32gene model in gene expression profiles of blood samples A recent study48 found a similar transcriptional profile between blood and colon tissue from patients with IBD If indeed Scientific RepoRtS 101038s41598020705830Vol1234567890wwwnaturecomscientificreports 0cour 32gene model is found accurate in blood samples then a less invasive procedure such as a blood test could be used to diagnose UC instead of a colonoscopy or sigmoidoscopyIn a previous study where machine learning was employed to perform a risk assessment for CD and UC using GWAS data49 a twostep feature selection strategy was used on a dataset containing Crohn™s disease cases UC cases and controls with SNPs In that study Wei et a0al reduced the number of features by filtering out SNPs with pvalues greater than ˆ’ and then applied a penalized feature selection with L1 penalty to select a subset of SNPs We decided against filtering out genes based on an arbitrary pvalue of statistical significance of differential expression as researchers are strongly advised against the use of pvalues and statistical significance in relation to the nullhypothesis5051Our 32gene model achieved AP of and discriminating active UC patients from healthy donors and inactive UC patients from healthy donors respectively We found direct or indirect links to UC for about a quarter of the most frequently chosen genes The remaining genes should be further investigated to find associations with UC To put the performance of our 32gene model into perspective we looked at previous studies applying machine learning to create models for the diagnostic of UC Maeda et a0al52 extracted features from endocystoscopy images to train a SVM to classify UC patients as active or healing This approach achieve precision at recall which is lower than the one achieved by our 32gene model Figs a0 and Yuan et a0al17 applied incremental feature selection and a SMO classifier a type of SVM on gene expression data from blood samples to discriminate between healthy subjects UC patients and Crohn™s disease patients The 10fold crossvalidation accuracy of their best model using the expression values of genes to classify UC patients was while our method obtained better accuracy than this with substantially less number of genes In terms of potential for clinical translation of a machine learningbased model a model requiring to quantify the gene expression levels of fewer genes is more suitable for the development of a new diagnostic test than one requiring the quantification of the expression levels of thousands of genesUsing an efficient feature selection method such as DRPT and a SVMclassifier on gene expression data we generated a model that could facilitate the diagnosis of UC from expression measurements of genes from colonic samples To avoid systematic experimental bias on the training data we used three transcriptomic datasets from three separated studies Our top model was validated with promising results on a data set not used for training however additional research is required to evaluate the genes as potential biomarkers on a external set of subjectsReceived March Accepted July References Kaplan G G The global burden of IBD 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101038s4159 z Boland B S et al Validated gene expression biomarker analysis for biopsybased clinical trials in ulcerative colitis Aliment Pharmacol Ther “ 101111apt12862 Shah P et al Artificial intelligence and machine learning in clinical development a translational perspective NPJ Digit Med 101038s4174 Esteva A et al Dermatologistlevel classification of skin cancer with deep neural networks Nature “ McKinney S M et al International evaluation of an AI system for breast cancer screening Nature “ Molla M Waddell M Page D Shavlik J Using machine learning to design and interpret geneexpression microarrays AI 101038natur e2105 101038s4158 Mag perspectives Hum Genet “ 101038s4159 Xu J et al Translating cancer genomics into precision medicine with artificial intelligence applications challenges and future Mossotto E et al Classification of paediatric inflammatory bowel disease using machine learning Sci Rep Olsen J et al Diagnosis of ulcerative colitis before onset of inflammation by multivariate modeling of genomewide gene expression data Inflamm Bowel Dis “ 101002ibd20879 Yuan F Zhang YH Kong XY Cai YD Identification of candidate genes related to inflammatory bowel disease using minimum redundancy maximum relevance incremental feature selection and the shortestpath approach Biomed Res Int 101155201757419 Moehle C et al Aberrant intestinal expression and allelic variants of mucin genes associated with inflammatory bowel disease J Mol Med Berl “ 101007s0010 Zahn A et al Aquaporin8 expression is reduced in ileum and induced in colon of patients with ulcerative colitis World J Gas Noble C L et al Regional variation in gene expression in the healthy colon is dysregulated in ulcerative colitis Gut “ troenterol Scientific RepoRtS 101038s41598020705830Vol0123456789wwwnaturecomscientificreports 0c Lepage P et al Twin study indicates loss of interaction between microbiota and mucosa of patients with ulcerative colitis Gastroenterology “ Inflamm Bowel Dis “ Vancamelbeke M et al Genetic and transcriptomic bases of intestinal epithelial barrier dysfunction in inflammatory bowel disease LennardJones J E Classification of inflammatory bowel disease Scand J Gastroenterol Suppl “ 10310900365 discussion “ H¤sler R et al A functional methylome map of ulcerative colitis Genome Res “ Barrett T et al NCBI GEO archive for functional genomics data sets“update Nucleic Acids Res D991“D995 Gumienny R GEOparse pypiproje ctGEOpa rse Troyanskaya O et al Missing value estimation methods for DNA microarrays Bioinformatics “ 101093bioin forma tics176520 abs200212104 Afshar M Usefi H HighDimensional Feature Selection for Genomics Datasets KnowledgeBased Systems arxiv Pedregosa F et al Scikitlearn machine learning in Python J Mach Learn Res “ Leclercq M et al Largescale automatic feature selection for biomarker discovery in highdimensional omics data Front Genet Burlington Holmes G Donkin A Witten I a0H Weka A machine learning workbench In Proceedings of ANZIIS ™ Australian New Zealand Intelligent Information Systems Conference “ Hall M et al The weka data mining software an update ACM SIGKDD Explor Newsl “ Witten I H Frank E Hall M A Pal C J Data Mining Practical Machine Learning Tools and Techniques Man Kaufmann M¼ller A C et al Introduction to Machine Learning with Python A Guide for Data scientists O™Reilly Media Inc California DemÅ¡ar J Statistical comparisons of classifiers over multiple data sets J Mach Learn Res “ Yates A et al The Ensembl REST API Ensembl data for any language Bioinformatics “ Kim E R Chang D K Colorectal cancer in inflammatory bowel disease the risk pathogenesis prevention and diagnosis World J Gastroenterol diovasc Dis “ Schulte D et al Small dense LDL cholesterol in human subjects with different chronic

Thyroid_Cancer

"Previous evidence has suggested that lower gestational vitamin D levels might increase the risks ofadverse pregnancy and birth outcomes The results remain inconsistent and require further explorationMethods A total of Chinese motherinfant pairs were included in this retrospective cohort study Serumconcentrations of 25OHD were reviewed in early pregnancy ± weeks Outcomes of maternal gestationaldiabetes mellitus GDM cesarean section fetal distress preterm birth low birth weight LBW and macrosomiawere extracted from the medical records Cox regression analysis was used to explore these associationsResults In total of mothers were pregnant at an advanced age ‰¥ years and of pregnant womenhad vitamin D deficiency nmolL After adjusting for potential covariates the hazard ratio HR CI perstandard deviation SD increase of serum 25OHD concentrations was for GDM for preterm birth and for LBW Similar protective associations were found for GDMcesarean section and preterm birth for a better vitamin D status when compared with vitamin D deficiencyConclusion Higher early pregnancy vitamin D was associated with a lower risk of GDM cesarean section pretermbirth and LBWKeywords 25ohd Vitamin D Pregnancy Maternal outcome Infant outcomeBackgroundVitamin D is a secosteroid hormone that is well knownfor its physiological function in maintaining bone metabolism and health A high prevalence of vitamin D deficiencyusually defined as serum 25OHD levels nmolL hasbeen found in pregnant women globally especially in developing countries [] including China [ ] Increasing evidence has suggested that vitamin D sufficiency is important Correspondence liuzphlk81outlookcom Gengdong Chen and Tingting Pang contributed equally to this work1Foshan Institute of Fetal Medicine Department of Obstetrics AffiliatedFoshan Maternity Child Healthcare Hospital Southern Medical UniversityFoshan Guangdong ChinaFull list of author information is available at the end of the for the prevention of pregnancy complications in mothersand adverse fetal birth outcomes [ ] although divergentresults have been reported in other studies [ “] Severalsystematic reviews based on randomized clinical trials orobservational studies have suggested that lower vitamin Dstatus contribute to adverse outcomes such as preeclampsia[] gestational diabetes mellitus GDM [ ] low birthweight LBW [] and preterm birth [] However increasing evidence shows different associations [ ]and no definitive has yet been made [] Severalproblems remain to be solved by further studies the heterogeneity of associations from diverse areas and populations with different vitamin D status serve as evidencethewhen makingguidelinesregionsforspecific The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cChen BMC Pregnancy and Childbirth Page of observation or supplementation of vitamin D in the thirdtrimester in many studies might present the problem ofcausal inference or miss the practical period for the intervention In addition the results from early trimesters mightbe helpful and more important for the early prevention ofadverse outcomesIn additionit has been suggested that the risk ofadverse complications or birth outcomes increases asmaternal age increases [“] With the change ofpopulation policy in China the percentage of womenpregnant at an advanced age years has increasedand proper strategies are urgently needed for the prevention of adverse complications [] However whetherthe influence of vitamin D on maternal and infant outcomes remained the same for women pregnant at young years and advanced ages remains unclear andmore studies are needed to better illustrate the problemWe investigated the relationship between early gestational serum 25OHD concentrations and several adverse maternal and infant outcomes in a retrospectivecohort study including Chinese motherinfantpairs Our results provide further evidence for clinicalrecommendations on the early prevention of related adverse outcomes in this fieldMethodsThe study used data that were gathered from a largecenter Affiliated Foshan Maternity Child HealthcareHospital Southern Medical University Foshan CityGuangdong Province China from September toJuly The hospital is the largest gynecology andobstetrics center in Foshan City and covers a large population of million people The included subjects werewomen who underwent early pregnancy serum vitaminD measurement ‰ gestational weeks and deliveredtheir infants at the hospital The exclusion criteria included twin or higherorder multiple pregnancies seriousdiseases such as type diabetes mellitus cardiovasculardiseases thyroid disorder and cancer that occurred before pregnancy Ultimately a total motherinfantpairs were included in this study The study was approvedby the ethics committee of Affiliated Foshan Maternity Child Healthcare Hospital Southern Medical UniversityData collectionVitamin D data from the clinicallaboratory werereviewed Blood samples were collected during the regular obstetric checkups and immediately measured by aclinical laboratory without being frozen Serum concentrations of 25OHD 25OHD2 and 25OHD3 weredetected using colloidal gold immunochromatographyCommercial kits were obtained from Mei Ning KangCheng Bio Tec Inc The intraassay and interassay coefficients of variation were less than Outcomes including GDM cesarean section fetal distress preterm birth low birth weight and macrosomiawere extracted from medical records and reexamined bytwo independent staff members The disease diagnoseswere made by professional doctors with the samestandardization criteria and were extracted from themedical records Gestational hypertension preeclampsiaor eclampsia was not included because of the lack ofavailable cases An oral glucose tolerance test was performed from to gestational weeks and GDM wasdiagnosed if the subjects met any of the following criteria fasting blood glucose ‰¥ mmolL onehour bloodglucose postoral sugar ‰¥ mmolL or twohour bloodglucose postoral sugar ‰¥ mmolL Preterm birth wasdefined as delivery at ‰¥ but gestational weeksLBW was diagnosed if the neonatal birth weight g while a neonatal birth weight ‰¥ g was defined asmacrosomia Other variablesincluding the maternalage body mass index BMI gestational age parity season of blood collection and time of delivery were alsoextracted from the medical recordsStatistical analysesContinuous variables were represented by the mean ±standard deviation SD or median interquartile rangeand tested by Student™s ttest Categorical variables wererepresented by frequencies percentage and tested bythe chisquare test Cox regression analysis was performed to explore the associations between vitamin Dand maternal or infant outcomes Serum concentrationsof 25OHD 25OHD2 and 25OHD3 were first Zstandardized before being included in the regressionVitamin D deficiency was defined as serum 25OHDlevels of nmolL Two different models were testedwith Model as a univariate model without adjustmentand Model adjusted for maternal age BMI parity andseason the blood was collected and measured Stratifiedanalyses were performed according to the gestationalage young years advanced ‰¥ years All the analyses were performed using SPSS software version SPSS Inc Chicago IL USA A twosided P value ofless than was considered statistically significantResultsA total motherinfant pairs were included in thisstudy A high prevalence of gestational vitamin D deficiency was discovered in the mothers Even insubjects without vitamin D deficiency the highest serum25OHD concentration was only nmolL Compared with women pregnant at a younger age the subjects with advanced age of pregnancy ‰¥ years tendedto have a higher BMI parity higher percentage of vitamin D deficiency higher incidence of GDM cesareansection preterm birth and LBW but a lower gestational 0cChen BMC Pregnancy and Childbirth Page of age lower serum 25OHD and OHD3 concentrations lower incidence of fetal distress and lower incidence of macrosomia Table As shown in Table although the 25OHD concentrations were statistically higher in spring and summerthan those in autumn and winter only small differenceof 25OHD values to nmolL was observedbetween different seasons especially for 25OHD2 Significant protective associations were found between thevitamin D levels and GDM and preterm birth Afteradjusting for potential covariatesTable higher25OHD concentrations per one SD increase were associated with a HR CI decrease in the GDM risk a HR CI decrease in the preterm birth risk and a HR CI decrease in theLBW risk Similar protective results were also found for25OHD2 and 25OHD3 but the associations tended tobe more pronounced for 25OHD2 Null associationsbetween vitamin D and cesarean section fetal distressand macrosomia were observed in all the subjects Maternal serum 25OHD levels ‰¥ nmolL were associated with a decrease in the GDM risk a decrease in the cesarean section risk and a decrease in the preterm birth risk but the trend did nothold with the other outcomes compared with those withvitamin D deficiency Table After stratification by gestational age Table higherlevels of vitamin D were associated with a lowerrisk of GDM in those years In contrast a protectiveassociation of vitamin D with low birth weight wasfound for women pregnant at ‰¥ years but not years However no significant interactions were discovered Pinteraction Table Characteristic of subjectsAge yearsBMI kgcm2Gestational age weeksParity timesNeonatal birth weight kg25OHD nmolL25OHD2 nmolL25OHD3 nmolLVitamin D deficiency N YesNoGestational diabetes mellitus N YesNoCaesarean section N YesNoFetal distress in uterus N YesNoPreterm birth N YesNoLow birth weight N YesNoMacrosomia N YesNoTotal N ± years N ± ‰¥ years N ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± P 0cChen BMC Pregnancy and Childbirth Page of Table Seasonal difference between serum vitamin D indicatorsDetected Seasonspring summer ± ± ± autumn winter ± ± ± P 25OHD nmolL25OHD2 nmolL25OHD3 nmolLDiscussionIn this retrospective cohort study including Chinese motherinfant pairs protective associations werefound for higher serum 25OHD concentrations withGDM cesarean section postpartum hemorrhage preterm birth and low birth weight but not for the otheroutcomes The results for 25OHD2 tended to be morepronounced than those for 25OHD3Interestinglyhigher serum vitamin D contributes to a higher risk ofpostpartum anemia in women pregnant at a young agebut not in advanced yearsIn this population with a high prevalence of vitamin Ddeficiency better serum vitamin D status contributed toa lower risk of GDM This result was consistent withseveral other studies A to higher risk of GDMwas observed for pregnant women with insufficient ordeficient vitamin D status in three systematic reviewsand metaanalyses based on observational studies orclinical trials [ ] In a large randomized controltrial RCT based on Iranian women the intervention of 25OHD3 was associated with lower risk ofGDM [] The results were further supported by severalother prospective cohort studies [ ] However nullassociations were found between the vitamin D status orsupplements and GDM in a systematic review based onfive randomized trials including subjects [] anested casecontrol study of women [] and alarge prospective study including motherchildpairs [] Additionally two studies found detrimentalassociations of higher vitamin D levels with GDM butthe effects were tiny [] or restricted to specificethnicities Hispanic []Consistent with our results the protective associationof maternal vitamin D status with a lower risk of preterm or low birth weight has also been reported in severalstudies An inverse doseresponse relation ofvitamin D status was found for preterm birth in a systematic review and metaanalysis based on longitudinal studies[] Rostami reported that a 25OHD3 interventionof monthly IU could be beneficial for the preventionof preterm delivery in an RCT of Iranian women []In a large prospective cohort including motheroffspring pairs the risk of low birth weight was CI and CI among subjects with vitamin D deficiency and insufficiency respectively [] Higher maternal serum 25OHD was positivelyTable Associations between early pregnant serum vitamin D concentrations and maternal infant outcomes25OHD aHR95CIPa25OHD2HR95CIPa25OHD3HR95CIPGestational diabetes mellitusModel Model Caesarean sectionModel Model Fetal distressModel Model Preterm birthModel Model Low birth weightModel Model MacrosomiaModel Model Cox regression analysis were operated for exploration of associations Model without adjustment Model adjusted for age BMI parity season of bloodcollected a Per one SD increase 0cChen BMC Pregnancy and Childbirth Page of Table Associations between vitamin D status and maternal infant outcomesGestational diabetes mellitusCaesarean sectionFetal distressPreterm birthLow birth weightMacrosomia25OHD nmolL‰¥ nmolL nmolL‰¥ nmolL nmolL‰¥ nmolL nmolL‰¥ nmolL nmolL‰¥ nmolL nmolL‰¥ nmolLHR95CI PCox regression analysis were operated for exploration of associations and adjusted for covariates including age BMI parity season of blood collectedassociated with higher birth weight in a study of UnitedArab Emirates [] and supplementation of vitamin D at adose of IUd was optimal and safe for mothers andtheir infants in United Arab Emirates [] However nullor detrimental associations were also reported in otherstudies Although vitamin D increased the mean birthweight it did not significantly reduce the risk of low birthweight or preterm birth in a metaanalysis based on randomized trials [] these results were further supported byanother update study [] In addition higher 25OHDconcentrations were found to increase the riskof preterm delivery in a prospective study of pregnant Chinese women []Gestational vitamin D deficiency was found to be associated with a 2fold increased risk of cesarean section ina cohort of lowincome pregnant women []Within a cohort of women from the United Statesfor women with 25OHD concentrations lower than nmolL the risk of a primary cesarean section wasalmost times higher [] These results were consistent with ours However a metaanalysis of trials subjects found null associations of vitamin D supplements with cesarean section []Much heterogeneity exists in the previous evidence inthis field and there may be several factors that partly explain these differences First most of the randomized trials included had a small sample size and were of lowquality Most subjects included in the trials had sufficient vitamin D status ‰¥ nmolL and a further doseof a or IUd supplement might have attenuatedTable Subclass analysis of relationship between serum vitamin D concentrations and related outcomes stratified by gestational ageP bP bP baa25OHD aHR95CIP25OHD2HR95CIP25OHD3HR95CIPGestational diabetesmellitusCaesarean sectionFetal distressPreterm birth y ‰¥ y y ‰¥ y y ‰¥ y y ‰¥ y Low birth weight y ‰¥ y Macrosomia y ‰¥ y Cox regression analysis were operated for exploration of associations and adjusted for covariates including age BMI parity season of blood collecteda Per one SD increase b P for interaction 0cChen BMC Pregnancy and Childbirth Page of the detrimental influence of vitamin D deficiency or insufficiency in the reference group [] Increasing benefitsmight not exist for higher doses Second the vitamin Dreceptor is important for the utilization of vitamin D[ ] The difference in vitamin D receptor gene polymorphism might help explain the ethnic heterogeneity[] This requires further confirmation in the futureThird the studies were conducted during different trimesters of gestation which might increase the difficultyof making comparisons those studies conducted duringan early gestational period might be advantageous forcausal inference and early preventionBecause 25OHD3 contributes most to the 25OHDconcentrations it has been used to represent 25OHDin many studies and only a few studies have focused on25OHD2 In our study the results were generally consistent between these two subcomponents The associations tend to be more pronounced in 25OHD2 than in25OHD3 and mightindicate the prominence of25OHD2 over 25OHD3 for the prevention of relateddiseases This theory merits further confirmation Nevertheless our results and others provide further evidencefor the importance of a better early vitamin D status forthe prevention of GDM cesarean section preterm birthand low birth weight in a population with a high prevalence of vitamin D deficiency Our results indicated thatmore consideration should be given to distinguishing between the vitamin D subcomponents women pregnantat different ages and women with more pregnancy complications in this field Considering the high prevalenceof vitamin D deficiency during pregnancy observed inChina [ ] it is a matter of urgency to call for the supplementation of vitamin D and more efforts should bemade to improve the gestational vitamin D status ofChinese populationVitamin D deficiency was found to decrease vasculardiameter within the labyrinth region and dysregulateplacental development during early pregnancy in an animal experiment [] Vitamin D supplementation duringearly pregnancy might rescue this situation while furthersupplementation might be futile after missing this timepoint [] Moreover vitamin D supplementation duringearly pregnancy is likely to affect genetic information ofsystemic inflammation and immune responses involvedin the development of gestational comorbidities egGDM preeclampsia and infection as reviewed by Hollis [] These mechanisms help to explain the beneficialinfluences of vitamin D for maternal and infanthealth and emphasize the importance of improving vitamin D status during early pregnancyOurstudy had several advantages Firstserum25OHD concentrations were measured during an earlygestational period and with the retrospective cohort design we assured the temporal sequence and avoided thepossibility of causal inversion Second we studied multiple outcomes and conducted further analysis of thesubcomponent of vitamin D and a stratified analysis ofgestational age which provided a more comprehensiveunderstanding of this field There are also several limitations of our study First our study was limited by thelack of information on dietary vitamin D intake including by supplement and sunlight exposure during thepregnancy period The obtained data were difficult touse for a retrospective study while the use of blood indicators might be more precise than a dietary survey sowe adjusted the association for different seasons to attenuate their influence Second the 25OHD concentration was measured only once we could not monitor thedynamic changes afterward or determine whether theywere normalized after receiving vitamin D supplementation However the associations tended to be underestimated instead of overestimated Third Wagner et al[] and Mirzakhani [] indicated a 25OHDconcentration ‰¥ nmolL in early pregnancy was optimal for the prevention of preterm birth and preeclampsia however the highest 25OHD concentration in ourstudy is only nmolL Therefore we were unable toperform the analyses using a threshold of nmolLand assess the influences of higher 25OHD concentrations Finally residual confounding might still existthough we tried to control several potential covariatesConclusionsThis retrospective cohort study showed the beneficial associations of early gestational vitamin D with outcomesof GDM cesarean section preterm birth and low birthweight More welldesigned randomized clinical trialsare needed for further exploration and confirmation ofthese resultsAbbreviationsGDM Gestational diabetes mellitus LBW Low birth weight BMI Body massindex SD Standard deviationAcknowledgementsWe would like to thank Ye Shaoxin Yang Xiaoming Liu Haojing Wangdong and Huang Shaobing for their generous help in this studyAuthors™ contributionsGDC and ZPL devised the idea and designed the study GDC TTP PSLZXZ DXL DZF contributed to the primary data collection GDC and TTP reexamined the data GDC TT P PS L ZX Z DXL DZF contributedto the analysis of the data GDC and TTP wrote the original draft whichwas revised by XLG and ZPL XLG and ZPL supervised the study andZPL administered the project All authors have read and approved themanuscriptFundingThis work was supported by the Basic and Applied Basic ResearchFoundation of Guangdong Province No 2019A1515110163 GDC and theFoundation of Bureau of Science and Technology of Foshan City No GDC The funding sponsors had no role in the design ofthe study in the collection analyses or interpretation of data in the writingof the manuscript and in the decision to publish the results 0cChen BMC Pregnancy and Childbirth Page of Availability of data and materialsThe datasets used andor analyzed during the current study are availablefrom the corresponding author upon reasonable requestEthics approval and consent to participateThe study was approved by the ethics committee of Affiliated FoshanMaternity Child Healthcare Hospital Southern Medical University TheAffiliated Foshan Maternity Child Healthcare Hospital providedadministrative permissions for the research team to access and use the dataincluded in this research Data were extracted from medical records and theconsent to participate was unavailable due to the retrospective design of thestudy and difficulty in reconnection however the private information waswell protectedConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Foshan Institute of Fetal Medicine Department of Obstetrics AffiliatedFoshan Maternity Child Healthcare Hospital Southern Medical UniversityFoshan Guangdong China 2Department of Medical RecordsAffiliated Foshan Maternity Child Healthcare Hospital Southern MedicalUniversity Foshan Guangdong ChinaReceived February Accepted August ReferencesSaraf R Morton SM Camargo CA Jr Grant CC Global summary of maternaland newborn vitamin D status a systematic review Matern Child Nutr“Yun C Chen J He Y Mao D Wang R Zhang Y Yang C Piao J Yang XVitamin D deficiency prevalence and risk factors among pregnant Chinesewomen Public Health Nutr “Zhou J Su L Liu M Liu Y Cao X Wang Z Xiao H Associations between hydroxyvitamin D levels and pregnancy outcomes a prospectiveobservational study in southern China Eur J Clin Nutr “Agarwal S Kovilam O Agrawal DK Vitamin D and its impact on maternalfetal outcomes in pregnancy a critical review Crit Rev Food Sci Nutr “von Websky K Hasan AA Reichetzeder C Tsuprykov O Hocher B Impact ofvitamin D on pregnancyrelated disorders and on offspring outcome JSteroid Biochem Mol Biol “Nobles CJ Markenson G ChasanTaber L Early pregnancy vitamin D statusand risk for adverse maternal and infant outcomes in a biethnic cohort thebehaviors affecting baby and you BaBY study Br J Nutr “Roth DE Leung M Mesfin E Qamar H Watterworth J Papp E Vitamin Dsupplementation during pregnancy state of the evidence from a systematicreview of randomised trials BMJ 2017359j5237HautaAlus HH Viljakainen HT HolmlundSuila EM EnlundCerullo MRosendahl J Valkama SM Helve OM Hytinantti TK Makitie OM AnderssonS Maternal vitamin D status gestational diabetes and infant birth size BMCPregnancy Childbirth Khaing W Vallibhakara SA Tantrakul V Vallibhakara O Rattanasiri S McEvoyM Attia J Thakkinstian A Calcium and vitamin D supplementation forprevention of preeclampsia a systematic review and network metaanalysisNutrients 2017910E1141 Zhang Y Gong Y Xue H Xiong J Cheng G Vitamin D and gestationaldiabetes mellitus a systematic review based on data free of Hawthorneeffect BJOG “ Zhang MX Pan GT Guo JF Li BY Qin LQ Zhang ZL Vitamin D deficiencyincreases the risk of gestational diabetes mellitus a metaanalysis ofobservational studies Nutrients “ Aghajafari F Nagulesapillai T Ronksley PE Tough SC O'Beirne M Rabi DMAssociation between maternal serum 25hydroxyvitamin D level andpregnancy and neonatal outcomes systematic review and metaanalysis ofobservational studies BMJ 2013346f1169 Qin LL Lu FG Yang SH Xu HL Luo BA Does Maternal Vitamin D DeficiencyIncrease the Risk of Preterm Birth A MetaAnalysis of Observational StudiesNutrients 201685E301 Rodriguez A GarciaEsteban R Basterretxea M Lertxundi A RodriguezBernalC Iniguez C RodriguezDehli C Tardon A Espada M Sunyer J et alAssociations of maternal circulating 25hydroxyvitamin D3 concentrationwith pregnancy and birth outcomes BJOG “ Ogawa K Urayama KY Tanigaki S Sago H Sato S Saito S Morisaki NAssociation between very advanced maternal age and adverse pregnancyoutcomes a cross sectional Japanese study BMC Pregnancy ChildbirthKhalil A Syngelaki A Maiz N Zinevich Y Nicolaides KH Maternal age andadverse pregnancy outcome a cohort study Ultrasound Obstet Gynecol“Jolly M Sebire N Harris J Robinson S Regan L The risks associated withpregnancy in women aged years or older Hum Reprod “Li Q Deng D New medical risks affecting obstetrics after implementation ofthe twochild policy in China Front Med “ Poel YH Hummel P Lips P Stam F van der Ploeg T Simsek S Vitamin Dand gestational diabetes a systematic review and metaanalysis Eur J InternMed “ Rostami M Tehrani FR Simbar M Bidhendi Yarandi R Minooee S Hollis BWHosseinpanah F Effectiveness of prenatal vitamin D deficiency screeningand treatment program a stratified randomized field trial J Clin EndocrinolMetab “ Xu C Ma HH Wang Y Maternal early pregnancy plasma concentration of25Hydroxyvitamin D and risk of gestational diabetes mellitus Calcif TissueInt “ Boyle VT Thorstensen EB Mourath D Jones MB McCowan LM Kenny LCBaker PN The relationship between 25hydroxyvitamin D concentration inearly pregnancy and pregnancy outcomes in a large prospective cohort BrJ Nutr “Schneuer FJ Roberts CL Guilbert C Simpson JM Algert CS Khambalia AZTasevski V Ashton AW Morris JM Nassar N Effects of maternal serum hydroxyvitamin D concentrations in the first trimester on subsequentpregnancy outcomes in an Australian population Am J Clin Nutr “ Amegah AK Klevor MK Wagner CL Maternal vitamin D insufficiency andrisk of adverse pregnancy and birth outcomes a systematic review andmetaanalysis of longitudinal studies PLoS One 2017123e0173605 Chen YH Fu L Hao JH Yu Z Zhu P Wang H Xu YY Zhang C Tao FB XuDX Maternal vitamin D deficiency during pregnancy elevates the risks ofsmall for gestational age and low birth weight infants in Chinesepopulation J Clin Endocrinol Metab “ Amirlak I Ezimokhai M Dawodu A Dawson KP Kochiyil J Thomas LAbdulle AM Current maternalinfant micronutrient status and the effects onbirth weight in the United Arab Emirates East Mediterr Health J “ Dawodu A Saadi HF Bekdache G Javed Y Altaye M Hollis BW Randomizedcontrolled trial RCT of vitamin D supplementation in pregnancy in apopulation with endemic vitamin D deficiency J Clin Endocrinol Metab“ PerezLopez FR Pasupuleti V MezonesHolguin E BenitesZapata VA Thota PDeshpande A Hernandez AV Effect of vitamin D supplementation duringpregnancy on maternal and neonatal outcomes a systematic review and metaanalysis of randomized controlled trials Fertil Steril “1288e1274Scholl TO Chen X Stein P Maternal vitamin D status and delivery bycesarean Nutrients “ Merewood A Mehta SD Chen TC Bauchner H Holick MF Associationbetween vitamin D deficiency and primary cesarean section J ClinEndocrinol Metab “ Chun RF Peercy BE Orwoll ES Nielson CM Adams JS Hewison M VitaminD and DBP the free hormone hypothesis revisited J Steroid Biochem MolBiol Pt A132“ Bikle DD Gee E Halloran B Kowalski MA Ryzen E Haddad JG Assessmentof the free fraction of 25hydroxyvitamin D in serum and its regulation byalbumin and the vitamin Dbinding protein J Clin Endocrinol Metab “ Powe CE Evans MK Wenger J Zonderman AB Berg AH Nalls M Tamez HZhang D Bhan I Karumanchi SA Vitamin Dbinding protein and 0cChen BMC Pregnancy and Childbirth Page of vitamin D status of black Americans and white Americans N Engl J Med“Liu NQ Ouyang Y Bulut Y Lagishetty V Chan SY Hollis BW Wagner CEquils O Hewison M Dietary vitamin D restriction in pregnant female miceis associated with maternal hypertension and altered placental and fetaldevelopment Endocrinology “ Mirzakhani H Litonjua AA McElrath TF O'Connor G LeeParritz A Iverson RMacones G Strunk RC Bacharier LB Zeiger R Early pregnancy vitaminD status and risk of preeclampsia J Clin Invest “ Hollis BW Wagner CL Vitamin D supplementation during pregnancyimprovements in birth outcomes and complications through directgenomic alteration Mol Cell Endocrinol “ Wagner CL Baggerly C McDonnell SL Baggerly L Hamilton SA Winkler JWarner G Rodriguez C Shary JR Smith PG Posthoc comparison ofvitamin D status at three timepoints during pregnancy demonstrates lowerrisk of preterm birth with higher vitamin D closer to delivery J SteroidBiochem Mol Biol “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

Thyroid_Cancer

"MiRNAs play important roles in the development of ovarian cancer activation of primitive folliclesfollicular development oocyte maturation and ovulation In the present study we investigated the specific role ofmiR23a in cov434 cellsResults Downregulation of miR23a was observed in serum of PCOS patients compared with the healthy controlsuggesting the inhibitory effect of miR23a in PCOS MiR23a was positively correlated with Body Mass Index BMI andnegatively correlated with Luteinizing hormone LH Testostrone T Glucose Glu and Insulin INS of PCOS patientsMiR23a mimic inhibited the proliferation and promoted apoptosis of human cov434 cells In addition flow cytometryassay confirmed that miR23a blocked cell cycle on G0G1 phase MiR23a inhibitor showed opposite resultsFurthermore double luciferase reporter assay proved that miR23a could bind to the ™UTR of FGD4 directly throughsites predicted on Target Scan FGD4 level was significantly suppressed by miR23a mimic but was significantlyenhanced by miR23a inhibitor We further proved that miR23a increased the expression of activated CDC42 GTPbround and pPAK1 suggesting that miR23a induced cell cycle arrest through CDC42PAK1 pathwayConclusions In our study reveals that miR23a participates in the regulation of proliferation and apoptosisof cov434 cells through target FGD4 and may play a role in the pathophysiology of PCOSKeywords miR23a Polycystic ovary syndrome FGD4 Binding site Cell cycleBackgroundPolycystic ovary syndrome PCOS is the most common reproductive endocrine and metabolic disorder disease inwomen characterized by ovulation disorders hyperandrogenism and insulin resistance [ ] PCOS affects about “ of women of childbearing age accounting for ofanovulatory infertility and usually a lifelong disease Itscommon clinical manifestations include menstrual disorders subfertility acne vulgaris alopecia seborrheia obesity hirsutism and acanthosis [] Women with PCOS havean increased risk of insulin resistance hypertension type Correspondence linjinet163com3Gynaecology Mindong Hospital in Ningde City No Heshan Road FuanFujian ChinaFull list of author information is available at the end of the diabetes oxidative stress dyslipidemia cardiovasculardisease and endometrial cancer [] Therefore understanding the molecular mechanism of metabolic diseases underlying the pathophysiology of PCOS will help to identify newdiagnostic and therapeutic strategies In addition althoughthe exact etiology of PCOS remains to be understood ithas been clear that the survival and proliferation of granulosa cells are closely related to the pathogenesis of PCOS[]In recent years the role of microRNAs miRNAs inovarian physiology and pathology has attracted muchattention Some studies have shown that miRNAs playimportant roles in the development of ovarian canceractivation of primitive follicles follicular developmentoocyte maturation and ovulation [“] Several studies The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cLin Journal of Ovarian Research Page of have found a variety of differentially expressed microRNAs in ovarian granulosa cells of PCOS patients whichare closely related to the proliferation and apoptosis ofovarian granulosa cells and the production of progesterone estradiol and testosterone [ ]responsibleforinducing caspasedependentThe human miR23a gene is located on chromosome of the human genome and transcribed into a part of themiR23a27a242 cluster [] Mi23a27a242 clusterwhich encodes primicroRNA transcripts composed ofthree kinds of miRNAs miR23a miR27a and miR242isandcaspaseindependent apoptosis of embryonic kidney cellsHEK293Tthrough human cJun Nterminal kinasepathway [] In recent years more and more evidencehas shown that miR23a is essential for folliculogenesis Ithas been reported that the expression of circulating miR23a of patients with PCOS was downregulated comparedwith healthy women and proved that miR23a is a betterindicator for evaluation of PCOS than the miR23b []However as far as we know the specific role and mechanism of miR23a in PCOS have not been studiedStudies proved that miR23a issignificantly upregulated in premature ovarian insufficiency POI patients™ serum and poor ovarian response POR patients™ovarian granulosa cells [“] Compared with normalwomen miR23a was significantly upregulated in follicular cells of women receiving assisted reproductive technology ART due to oviduct and endometriosis []More critically miR23a can promote the apoptosis byaffecting the expression of multiple targetsincludingXIAP SMAD5 and Sirt1 [ ]Thereforein the present research we hypothesizedthat miR23a is involved in the development of PCOS byregulating downstream pathways related to cell survivalin ovarian cells The objective of this study was to confirm the regulatory effect and mechanism of miR23a onthe growth of cov434 cells We analyzed the expressionof miR23a in serum samples from PCOS patients andhealthy women and the correlation between miR23alevel and PCOS symptoms We focused on a new molecular mechanism by which miR23a induces apoptosisin granular cellsdisease smoking and using alcohol or drugs The serumof healthy women was collected as the control groupThe volunteers in the control group had normal menstruation normal ovaries and no history of reproductivesystem disease or appendicitis The control and PCOSgroup did nottake any medications in the past months including oral contraceptives or other hormonalmedications with no intrauterine devices or smokingPatients with reproductive system disease or appendicitishistory were excluded from the control group All volunteers had understood the purpose and requirements ofthis study and signed a written informed consent beforeparticipating in the study ml of elbow venous bloodfrom each sample was taken and stored in a refrigeratorat ˆ’ °C All the experiments involved in this studyhave obtained the ethical approval of Mindong hospitalin Ningde CityEvaluation of BMI and sex hormoneThe weight and height of the volunteers were measuredto calculate Body mass index BMIBMI weightheight2 Radioimmunoassay RigorBio Scientific andTechnology Co Beijing was used to measure the levelof total testosterone and other sex hormonesCell line and transfectionCell lines KGN derived from a granulosa cell tumorcov434 derived from a granulosa cell tumor and SVOGderived by immortalization of granulosaluteal cellsusing SV40 large T antigen were purchased from cellresource bank of Chinese Academy of Sciences — cells were seeded into well plates MiR23a micmic nM miR23a inhibitor nM and negative control NC nM mimic NC and nM inhibitor NCRuibo Biotechnology Co Ltd Guangzhou China weretransfected into cov434 cells by Lipofectamine„¢ Normal untreated cov434 cells were cultured as control Thesequence of siRNA used in this study is as follows miR23amimic ²CCTTTAGGGACCGTTACACTA3² mimicNC ²TTCTCCGAACGTGTCACGTTTC3² miR23ainhibitor ²TAGTGTAACGGTCCCTAAAGG3² inhibitor NC ²TTCTCCGAACGTGTCACGTTTC3²Materials and methodsSamplesThe serum of Chinese women with PCOS was collected in Mindong hospital Ningde City Fujian Provincefrom September to December According tothe revised PCOS diagnostic criteria published by theRotterdam consensus [] the PCOS group excluded patients with Cushing™s syndrome delayed congenital adrenal hyperplasia thyroid dysfunction hyperthyroidismhyperprolactinemia or androgen secreting tumor as wellas patients with diabetes hypertension chronic kidneyReal time fluorescence quantitative PCR qPCRTotal RNA were extract from samples or cells using Trizolreagent Related expression of target gene was calculatedusing 2ΔΔCt method This study involves the followingsequences miR23a3p Reverse transcription ² GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGGAAAT3² miR242 Reverse transcription ²GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTGTGT3² miR27a3P ²GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGCGGAA3² U6 Reverse transcription ²AAAATATG 0cLin Journal of Ovarian Research Page of GAACGCTTCACGAATTTG3² miR23a3p forward primer ²GCGATCACATTGCCAGGG3² and reverse primer²AGTGCAGGGTCCGAGGTATT3² miR242 forwardprimer ²CGCGTGCCTACTGAGCTGAA3² and reverseprimer ²AGTGCAGGGTCCGAGGTATT3² miR27a3Pforward primer ²GCGCGTTCACAGTGGCTAAG3² andreverse primer ²AGTGCAGGGTCCGAGGTATT3² U6forward primer ²CTCGCTTCGGCAGCACATATACT3²and reverse primer ²ACGCTTCACGAATTTGCGTGTC² FGD4 forward primer ²CCTGCCTCTGCTTCTTGTGTCTC3² and reverse primer ²TGGTTGTCAATCCATGCCTTCCTG3²Cell proliferation assayAfter h of transfection cells were seeded into a well plate at the density of — cells per well Eachgroup of cells was treated with replicates After incubation for the specified time and h μl of CCK8 reagent was added and incubated at °C for h The absorbance of each pore was measured at nm by an enzyme labeling instrumentFlow cytometry analysis for cell cycleAfter h of transfection the cell cycle was detected byflow cytometry The cells were fixed with ethanolovernight at °C The cells were resuspended with μl of binding buffer μl PI was added to the cellsuspension and incubated at room temperature for min The results were analyzed by ModFit and displayedby FL2w and FL2aFlow cytometry analysis for apoptosisAfter h of transfection the apoptotic cells were detected by flow cytometry μl of PI and FITC annein Vwere added into μl cell suspension and incubated atroom temperature for min Cell apoptosis was detected using a flow cytometerWestern blotThe total protein was extracted with RIPA buffer BCAmethod was used to detect the protein concentrationThe extracted protein was electrophoresis by SDSPAGEand transferred to PVDF membrane PVDF membranewas incubated in skimmed milk at room temperaturefor h and then primary antibody overnight at °Cfollowed by the secondary antibody at room temperaturefor h QUANTITY ONE software is used for resultanalysis The following antibodies were used in this research antiFGD4 Abcam ab97785 87KDaantiCDC42 Abcam ab155940 21KDa antiPAK1 Abcam ab223849 60KDa and βactinTransGen Biotech HC201 42KDaDouble luciferase reporting assayThe plasmids of wild type FDG4WT and mutant typeFDG4MUT luciferase reporter genes were constructedusing pcDNA31as the empty vector MiR23a mimicmimic NC FDG4WT and FDG4MUT plasmids were cotransfected into cov434 cells by LipofectamineTM Cells were divided into four groups FGD4WT ²UTR miR23a mimic NC FGD4Mut ²UTR miR23a mimicNC FGD4WT ²UTR miR23a mimic FGD4Mut ²UTR miR23a mimic After h of transfection FirelyLueiferase F and Renilla Luciferase R were detected byGLOMAX \\fluorescence detector and the relativeluciferase activity F R was calculatedStatistical analysesAll data were analyzed with SPSS SPSS Inc Chicago IL software and represented as mean ± SD Spearman method was used to analyze the relationshipbetween miRNA level and other indicators Independentsample ttest was used to evaluate the difference between two groups and Oneway ANOVA was used toanalyze the difference between three and more groupswith post hoc contrasts by Bonferroni test P wasconsidered statistically significantResultsMiR23a was downregulated in serum of PCOS patientsPeripheral blood was collected from local PCOS patients for the detection of miR23a level with healthywomen™s peripheral blood as the control Clinical information on age BMI and sex hormone levels of PCOSpatients and normal control samples are alllisted inTable As shown in Fig 1a the serum miR23a level inPCOS patients was significantly lower than that in thecontrol group P Then we detected the level ofmiR27a and miR242 using qPCR As shown in Fig 1amiR27a and miR242 also downregulated in peripheralblood of PCOS patients compared with healthy sampleTable The clinical information of PCOS and control groupsClinical indexPPCOSn ± Controln ± ± ± ± ± ± ± ± ± ± ± AgeE2 pgmLBMI Kgm2LH mIUmLFSH mIUmLPRL mIULT mIUmL ± ± ± Glu nmolmLINS μUmLE2 Estradiol BMI Body Mass Index LH Luteinizing hormone FSH Folliculestimulatinghormone PRL Prolactin T Testostrone Glu Glucose INS Insulin ± ± ± 0cLin Journal of Ovarian Research Page of Fig MiR23a was downregulated in serum of PCOS patients a qPCR was performed to detect the expression of miR23a in PCOS samplePCOS and healthy control Normal b miR27a and miR242 levels were detected using qPCR in PCOS and normal group Correlation betweenmiR23a level and BMI was analyzed in PCOS c and control d group Correlation between miR23a level and LH was analyzed in PCOS econtrol f group Correlation between miR23a and GLU level was analyzed in PCOS g and control h group Correlation between miR23a andINS level was analyzed in PCOS i control j group Correlation between miR23a and T level was analyzed in PCOS k and control l groupP P P The correlation between the expression of miR23a andclinical index of PCOS patientsWe further analyzed the correlation between the expression of miR23a and clinical index As shown in Table the BMI of PCOS patients were significantly higher thanthat of healthy controls P The correlation analysis showed that there was a positive correlation between serum miR23a level and BMI in PCOS patientsFig 1b P r but no correlation wasfound in healthy control group Fig 1c P r As shown in Table the serum LH concentration in PCOS patients was ± mIUmL whichwas significantly higher than that in healthy women ± mIUmL P Furthermore therewas a negative correlation between serum miR23a leveland LH concentration in PCOS patients Fig 1d P r but no correlation was found in healthy controlgroup Fig 1e P r The serum miR23alevel was also negative correlated with GLU Fig 1fP r INS Fig 1h P r and T Fig 1j P r concentration inPCOS patients but not in healthy control group GLUFig 1g P r INS Fig 1i P r and T Fig 1k P r MiR23a inhibits the proliferation of cov434 cellsIn this study the expression of miR23a in three humangranulosa cell lines was detected by qPCR As shown inFig 2a the expression level of miR23a was lowest incov434 cells and highest in KGN cells Therefore wechose cov434 cell line for subsequent experiments Subsequently miR23aspecificsiRNA or mimic was transfected into cov434 cells to explore the role of miR23aAs shown in Fig 2b the expression of miR23a in cells 0cLin Journal of Ovarian Research Page of Fig MiR23a inhibits the proliferation of human ovarian granulosa cells a The expression of miR23a in three human ovarian granulosa celllines KGN cov434 and SVOG was detected by qPCR b MiR23a was overexpressed by the transfection of miR23a mimics c MiR23a was knockeddown by the transfection of miR23a inhibitor d CCK8 was performed to detect the proliferation of cov434 cells P P was significantly increased by the transfection of miR23a mimic P Similarly the expression of miR23a in cells was significantly knocked down by the transfection of miR23a inhibitor Fig 2c P Then CCK8 assay was performed to detect the effectof miR23a on the proliferation of cov434 cells Asshown in Fig 2d compared with the control group thetransfection of miR23a mimic significantly inhibited theproliferation of cov434 cells P on the contrarythe transfection of miR23a inhibitor significantly promoted the proliferation of cov434 cells P Thesedata proved that the expression level of miR23a was involved in the regulation of cov434 cell proliferationMiR23a induced cell cycle arrest on G0G1 phase ofcov434 cellsNext flow cytometry was used to detect the effect ofmiR23a on the cell cycle of cov434 As shown in Fig cells stagnated in G0G1 phase after transfection ofmiR23a mimic P and the proportion of cells inS phaseand G2M phase decreased significantlyP The results were consistent with the inhibition of cell proliferation by overexpression of miR23asuggesting that miR23a induced cell cycle arrest andthus inhibit cell proliferation in cov434 cells On thecontrary the proportion of G2M phase cells increasedsignificantly in the miR23a inhibitor group P while that of G0G1 and S phase cells decreased P The results showed that low expression of miR23a promoted cell cycle progression and thus cell proliferationMiR23a promotes apoptosis of cov434 cellsFlow cytometry was performed to detect the effect of theexpression of miR23a on the apoptosis of cov434 cellsAs shown in Fig apoptotic cells increased significantlyP after the transfection of miR23a mimic anddecreased significantly P after the transfection ofmiR23a inhibitor These results suggested that overexpression of miR23a promoted apoptosis while low expression of miR23a inhibited apoptosisFGD4 is the bind target of miR23a in cov434 cellsThen we predicted six novel potential target of miR23avia the analysis on bioinformatics software Target ScanSubsequently the results of double luciferase reporterassay proved that only FGD4 could bind to miR23a directly through predicted sites The binding sites areshown in Fig 5a Cotransfection of miR23a mimicinhibited the luciferase activity of FGD4WT plasmidP but had no effect on the luciferase activity ofFGD4Mut plasmid Fig 5b The results showed thatmiR23a and FGD4 bind directly through predictive sitesThe effect of miR23a on the expression of FGD4 incov434 cells was investigated using qPCR and westernblot As shown in Fig 6a the expression of FGD4 wassignificantly decreased by the transfection of miR23amimic P whereas the transfection of miR23a 0cLin Journal of Ovarian Research Page of Fig MiR23a induced cell cycle arrest on G0G1 phase of cov434 cells a Flow cytometry was used to detect the effect of miR23a on the cellcycle of cov434 with transfection of miR23a mimics or inhibitor b Column diagram showed the analysis of cell cycle P inhibitor significantly increased the mRNA expression ofFGD4 in cov434 cells P As shown in Fig 6b andc the protein level of FGD4 was significantly decreasedby the transfection of miR23a mimic P whereasthe protein level of FGD4 was significantly increasedby miR23a inhibitor P Combining with thedouble Luciferase Report experiment these results indicated that miR23a physically bind to the ²UTRregion of FGD4 thereby regulating the level of FGD4in cov434 cellsMiR23a induces the activation of CDC42PAK1 signalingpathway in cov434 cellsCDC42 is a member of the Rho GTPase protein familyFGD4 is responsible for activating CDC42 through GTPexchange of GDP PAK1 a serinethreonine kinase wasinitially identified as a protein interacting with CDC42[] CDC42PAK1 signaling pathway involved in theregulation of cell proliferation apoptosis and cell cycle[] As shown in Fig 6d the protein expression of activated CDC42 GTP bround was significantly increasedby the transfection of miR23a mimic P and significantly decreased by the transfection of miR23a inhibitoreffect of miR23a on theexpression of pPAK1 protein was similar to that ofCDC42 protein Fig 6fP TheDiscussionIn this study we explored the differences in serum levelsof miR23a between PCOS patients and normal womenas well as the effects of miR23a on biological behaviorsuch as proliferation and apoptosis of cov434 cells andrelated specific molecular mechanisms in order to provide limited theoretical support and experimental datafor the application of miRNA in PCOS treatmentFirstly we found that compared with healthy womenthe serum level of miR23a in PCOS patients decreasedsignificantly According to previous reports the level ofmiR23a in patients with ovarian disease remains uncertain Yang reported that miR23a was highlyexpressed in the plasma from premature ovarian failure POF patients compared with controls with afold change [] However Dang et alfoundthat miR23a is downregulated in the plasma ofChinese patients with premature ovarian failure []This inconsistency may be caused by individual differences and low sample size MiR23a level in patientswith ovarian disease still needs to be verified in alarge number of samplesMoreover miR23a was positively correlated with BMIand negatively correlated with serum LH T Glu andINS concentration Hyperandrogenism and hyperinsulinemia in PCOS patients are the most important physiological changes exacerbating endocrine disorders [] 0cLin Journal of Ovarian Research Page of Fig MiR23a promotes apoptosis of human ovarian granulosa cells a Flow cytometry was used to detect the effect of miR23a on theapoptosis of cov434 with transfection of miR23a mimics or inhibitor b Column diagram showed the analysis of cell apoptosis P MiR23a is closely related to the changes of hormonelevels suggesting that it may be involved in the progression of PCOS and is a potential clinical treatment targetMurri also reported an inverse relationship betweenBMI and LH concentrations in patients with PCOS []Serum is composed of multiple components from a variety of tissues and ans Therefore the concentration ofmiR23a in serum is regulated by a variety of componentsand factors In addition the results also indicated that thedecrease in miR23a had a negative impact on the occurrence of PCOS and the increase in LHThen we investigated the role of miR23a in cov434cells We have found that miR23a can affect the proliferation of cov434 cells by regulating cell cycle and participate in the regulation of cell apoptosis through aseries of cell functional studies It has been shown thatmiR23a is closely related to apoptosis by inhibiting theexpression of Apaf1 and Bcl2 apoptotic proteins including Noxa Puma and Bax in neurons [] It hasalso been reported that miR23a protects differentiatedembryonic stem cells from apoptosis induced by bonemorphogenetic protein BMP4 by targeting SMAD5[] These data provide strong support for our resultssuggesting that miR23a may be closely related to granulosa cell apoptosis through a variety of pathwaysThese results suggest that miR23a may be closely related to the pathogenesis and development of PCOSTherefore we further study the molecular mechanism ofmiR23a involved in the proliferation and apoptosis ofcov434 cells The biological functions of miRNAs depend mainly on their effects on targets The same microRNAs may have hundreds oftarget proteins thosechange with cell type and cell state MiR23a can promote the apoptosis of cov434 cells by affecting the expression of multiple targets [ ] At presentmany targets have been found including Xlinked inhibitor of apoptosis protein XIAP SMAD5 and Sirt1 [] In this study we found FGD4 as a new target ofmiR23a The direct interaction between the ²UTR region of FGD4 mRNA and the expression of miR23awas demonstrated by double luciferase reporter assayThe results of qPCR and Western blot showed thatoverexpression of miR23a inhibited the expression ofFGD4 at the level of protein and mRNA while low expression of miR23a promoted the expression of FGD4at the level of protein and mRNAFGD4 is a Guanine Nucleotide Exchange Factor GEFspecific to CDC42 Rho GTPase and also an Factinbinding protein which is essential for maintaining myelin formation in Schwann cells [] FGD4 consists of N 0cLin Journal of Ovarian Research Page of Fig FGD4 binds to miR23a via the ™UTR in cov434 cells a the binding site of miR23a to ™UTR of FGD4 b Double luciferase reporter assaywas performed to confirm the binding between miR23a and FGD4™s ™UTR P terminal Factin bindingFAB domain Dbl homologyDH domain two pleckstrin homology PH domainand FYVE domain [] FGD4 has many functions including binding to Factin through FAB domain activating Rho GTPasetransduction pathway byincreasing the concentration of CD42 binding to GTPsignalThe structure domain of FGD4 indicates that it acts as acrosslinker between membrane structure and actincytoskeleton therefore the functional deletion mutationof FGD4 coding gene may result in truncated FGD4 expression and lead to motor sensory neuropathy orCharcotMarieToothCMTtype [ ] TheFig MiR23a induces the activation of CDC42PAK1 signaling pathway in cov434 cells a The expression of FGD4 was detected using qPCR incov434 cells with transfection of miR23a mimics or inhibitor b Western blot was performed to detect the levels of CDC42 and pPAK1 incov434 cells with transfection of miR23a mimics or inhibitor c Column diagram showed the expression level of FGD4 d Column diagramshowed the expression level of CDC42 e Column diagram showed the expression level of pPAK1 0cLin Journal of Ovarian Research Page of mutation is mediated by inhibiting guanine nucleotideexchange leading to the decrease of CDC42 activity andthe demyelination of peripheral nerves ultimately However in this study mir23a expression and function wereonly studied by using patients™ peripheral blood and celllines cultured in vitro The expression and function ofmir23a in vivo and patients™ ovarian cells still need further verificationIn addition recent studies have shown that FGD4 expression in prostate cancer clinical samples is significantly upregulated compared with the normal groupand downregulation expression of FGD4 in prostatecancer cell lines can cause cell cycle arrest and proliferation reduction [] It seems that FGD4 is also involvedin the tumorigenesis of nasopharyngeal carcinoma dueto its activation of CDC42 [] Studies have shown thatactivated CDC42 regulates downstream signals such asPAK1 WASP and ACK PAK1 as a serinethreoninekinase was originally identified as a protein that interacts with CDC42 and was subsequently found to serveas a downstream node for various oncogenic signalingpathways Studies have shown that the CDC42PAK1signaling pathway involved in cell cycle proliferationand apoptosis regulation [] Our study found thatmiR23a affects the expression of FGD4 as well as theprotein levels of activated CDC42 GTP bround and pPAK1 Therefore we hypothesized that miR23 regulated CDC42PAK1 signaling pathway by targetingFGD4 expression ultimately affecting apoptosis ofcov434 cellsIn our study reveals that the serum level ofmiR23a is significantly downregulated in PCOS patients and that miR23a participates in the regulation ofproliferation and apoptosis of cov434 cells through target FGD4 which may have potential for clinical treatment of PCOS patientsAcknowledgementsNot applicableAuthors™ contributionsJL and HH mainly performed the experiments and analyzed the data JL was amajor contributor in writing the manuscript LL helped with the data analysisand carried out the experiment design WL and JH helped with theexperiments and analysis All authors read and approved the final manuscriptFundingThis study was supported by Ningde medical technology improvementprojectAvailability of data and materialsAll data generated or analysed during this study are included in thispublished Ethics approval and consent to participateThis research study was approved by the Institutional Review Board of FujianMedical UniversityConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Graduate School Fujian Medical University Fuzhou China 2The 900thhospital of the Joint Service Support Force of the Chinese People™sLiberation Army Fuzhou China 3Gynaecology Mindong Hospital in NingdeCity No Heshan Road Fuan Fujian ChinaReceived December Accepted July ReferencesUtiger RD Insulin and the polycystic ovary syndrome Diabetes Res ClinPract Polak K Czyzyk A Simoncini T Meczekalski B New markers of insulinresistance in polycystic ovary syndrome J Endocrinol Invest “httpsdoi101007s4061801605238Trikudanathan S Polycystic ovarian syndrome Med Clin N Am “American College of Obstetricians and Gynecologists' Committee onPractice Bulletins”Gynecology ACOG Practice Bulletin No PolycysticOvary Syndrome Obstet Gynecol 20181316e157“ httpsdoi101097AOG0000000000002656Li X The role of androgen in autophagy of granulosa cells from PCOSGynecol Endocrinol “OvidiuLeonard B miRNA expression profiling in formalinfixedparaffinembedded endometriosis and ovarian cancer samples OncoTargets Ther “ Maalouf SW Liu WS Pate JL MicroRNA in ovarian function Cell Tissue Res“Kim SH Paeonol inhibits anaphylactic reaction by regulating histamineand TNFα Int Immunopharmacol “Naji M Differential Expression of miR93 and miR21 in Granulosa Cellsand Follicular Fluid of Polycystic Ovary Syndrome Associating with DifferentPhenotypes Sci Rep Bindu M miR122 Regulates LHR Expression in Rat Granulosa Cells byTargeting Insig1 mRNA Endocrinology Chhabra R Dubey R Saini N Gene expression profiling indicate role of ERstress in miR23a27a24“ cluster induced apoptosis in HEK293T cellsRNA Biol “ Xiong W Circulatory microRNA 23a and microRNA 23b and polycysticovary syndrome PCOS the effects of body mass index and sex hormonesin an Eastern Han Chinese population J Ovarian Res Guo Y Sun J Lai D Role of microRNAs in premature ovarian insufficiencyReprod Biol Endocrinol Yang X Role of microRNAs in premature ovarian insufficiency ReprodBiol Endocrinol “ Dang Y MicroRNA223p is downregulated in the plasma of HanChinese patients with premature ovarian failure Fertil Steril “807e1 Nie M Yu S Peng S Fang Y Wang H Yang X miR23a and miR27apromote human granulosa cell apoptosis by targeting SMAD5 Biol Reprod httpsdoi101095biolreprod115130690 Alford C Toloubeydokhti T AlKatanani Y The expression of microRNAmiRNA mir23a and 23b and their target gene CYP19A1 aromatase infollicular cells obtained from women undergoing ART[J] Fertil Steril 88suppS1 Nie M miR23a and miR27a Promote Human Granulosa CellApoptosis by Targeting SMAD5 Biol Reprod S¸rensen AE MicroRNA Species in Follicular Fluid Associating WithPolycystic Ovary Syndrome and Related Intermediary Phenotypes J ClinEndocrinol Metab “ jc20153588 Wu C Exercise activates the PI3KAKT signal pathway by decreasingthe expression of 5αreductase type in PCOS rats Sci Rep Murri M Effects of polycystic ovary syndrome PCOS sex hormonesand obesity on circulating miRNA21 miRNA27b miRNA103 and miRNA expression J Clin Endocrinol Metab 20139811E1835“Sabirzhanov B Downregulation of miR23a and miR27a followingExperimental Traumatic Brain Injury Induces Neuronal Cell Death throughActivation of Proapoptotic Bcl2 Proteins J Neurosci “ 0cLin Journal of Ovarian Research Page of Musto A miR23a miR24 and miR27a protect differentiating ESCsfrom BMP4induced apoptosis Cell Death Differ “ Nie MY Yang X Physiological and pathological effects of miR23a and miR27a in ovary Horn M Myelin is dependent on the CharcotMarieTooth Type 4Hdisease culprit protein FRABINFGD4 in Schwann cells Brain “Kondo D A novel mutation in FGD4 causes Charcot“Marie“Toothdisease type 4H with cranial nerve involvement Neuromuscul Disord “ Zis P A novel mutation in the FGD4 gene causing CharcotMarietooth disease J Peripher Nerv Syst “Edwards D PRL3 increases the aggressive phenotype of prostatecancer cells inVitro and its expression correlates with highgrade prostatetumors in patients Int J Oncol “Liu HP EpsteinBarr VirusEncoded LMP1 Interacts with FGD4 toActivate Cdc42 and Thereby Promote Migration of NasopharyngealCarcinoma Cells PLoS Pathog 201285e1002690Kumar R Gururaj AE Barnes CJ p21activated kinases in cancer Nat RevCancer “ httpsdoi101038nrc1892Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliat

Thyroid_Cancer

"deltalike protein DLL3 may be related with prognosis inpatients with small cell lung cancer SCLC However this finding remains controversial in small cell lung cancerThis metaanalysis was systematically performed to evaluate the prognostic value of DLL3 in SCLCMethods The PubMed EMBASE and Web of Science databases were retrieved to collect the eligible referencesThrough Stata software we pooled hazard ratios HR with confidence intervals CI by using random orfixedeffects models to evaluate the association between DLL3 and SCLC survival resultsResults A total of interrelated studies including patients were qualified After we removed study theremaining studies including patients were pooled to testify that high expression of DLL3 was an inferiorprognostic for patients with SCLC in Asian populations HR CI I2 p Thepooled results showed that DLL3 might be higher expression in advanced metastasis SCLC in Asian populations RR CI I2 p But the expression of DLL3 was not correlated with sex RR CI I2 p smoking history RR CI I2 p and tumour stage RR CI I2 p Conclusions Our metaanalysis confirms that in Asian populations high expression of DLL3 was a potential poorprognostic biomarker for SCLC and DLL3 highly expressed in advanced stage SCLC in Asian populationsKeywords Deltalike protein DLL3 Prognostic Small cell lung cancer SCLC MetaanalysisIntroductionLung cancer has the highest morbidity and mortality ofall malignant tumours which is one of the most common cancers worldwide [] The major histologic subtypes oflung cancerNSCLC and small cell lung cancer SCLC [] SCLC is an invasive highgrade malignancy withearly development and a bad prognosis Most patientswith SCLC are advanced with widespread metastasiswhen they are diagnosed and systemic chemotherapy islung cancer are nonsmall cell Correspondence ligaofenghl126comDepartment of Thoracic Surgery The Third Affiliated Hospital of KunmingMedical University Yunnan Cancer Hospital Kunming Yunnan Chinathe most effective therapy [] Few therapeutic methodsare available after SCLC relapse and the prognosis is especially poor Therapeutic treatments for patients withSCLC have essentially remained unchanged in recentyears [] Thus further research into the mechanism ofSCLC and the exploration of new therapeutic targets forSCLC is imperativeDeltalike protein DLL3 is a transmembrane protein that promotes the development of neuroendocrinetumours through its reciprocity with the Notch pathway[] It is expressed in cancer tissues in approximately of patients with SCLC and other neuroendocrineis not expressed in nonneuroendocrinecancer but The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cChen World Journal of Surgical Oncology Page of tumours or normal tissues [] Most studies have shownthat DLL3 is a latent treatment target point for SCLC[“] Recently many researches initiate to focus on theprognostic value of DLL3 in SCLC Some of the researchers demonstrated that DLL3 had no correlationwith prognosis in SCLC patients [ ] but other investigators showed that DLL3 was associated with thesurvival of patients with SCLC [] So previous studiesof the prognostic value of DLL3 in SCLC remain controversial we performed this systematic metaanalysis forresolving this controversialPublished s discussing the prognostic value ofDLL3 in SCLC were systematically reviewed in ourmetaanalysis We aimed to include all correlationalstudies to assess the prognostic value of DLL3 andattempted to identify an accurate biomarker to guideprognosis and treatment for SCLC in the futureMethodsSearch strategyThe PRISMA guidelines were followed for our metaanalysis see Additional file [] The literatures werecollected through retrieving the PubMed EMBASE andWeb of Science databases from the initial date to February The search strategy was œDLL3 OR deltalike protein  all fields AND œnonsmall cell lung cancer OR NSCLC OR small cell lung cancer OR SCLC ORLung Adenocarcinoma OR Lung Squamous cell carcinoma OR lung cancer OR lung tumour OR lung neoplasm OR lung carcinoma all fields There was nolimitation on the publication status All eligible studieswere retrieved and the headlines and s of all thereference lists of the reviews or studies were independently filtrated through three authors based on the criteria Differences between two authors were resolved bythe third author™s opinionsInclusion criteriaResearches were included by following the standards studies reported the prognostic value of DLL3 in SCLC studies were published as original s studiesreported the data of HR and CI or provided survivalcurve and studies in which the prognostic value wasinvestigated by survival analysis with overall survivalOS diseasefree survival DFS progressionfree survivalRFS ordiseasespecific survival DSS We excluded the animalresearches and other review literaturesPFS relapserecurrencefree survivalData extractionTwo researchers independently extracted the data included the author country edition year sample capacitypatients™ sex smoking history distant metastasis tumourstage histologic subtype biomarkers scoring methodsboth univariateand multivariatecutoffs value and the data of HR and CI We extracted the multivariate analysis outcome if one study includedanalysisoutcomes When we could not extract the data of HRand CI directly in the but the survival curveswere reported we extracted and digitized the survivalcurves by operating the Engauge Digitizer softwarehttpdigitizersourcefenet and we estimated thedata of HR and CI by the Excel programme files asexploited by Tierney [] When the data of HRand survival curves were not reported we contacted thecorresponding authors of eligible s by email to obtain the original data these s were excluded ifthere was no responseAssessment of study qualityTwo researchers independently used the Quality InPrognosis Studies QUIPS tool to assess risk of bias ofall the publications [] According to the criteria every was evaluated as low risk moderate risk or highrisk by following six different areas study participationstudy attrition prognostic factor measurement outcomemeasurement study confounding and statistical analysisand reporting [] Differences were settled throughdiscussionStatistical analysisTwo authors independently extracted the HR and CI from the original s The HR was used to describe the high DLL3 expression versus low DLL3 expression We took the reciprocal of the HR if the sshowed the low DLL3 expression vs high DLL3 expression We observed that high expression of DLL3 portended a worse prognosis when HR and that HR indicated preferable prognosis For the analysis results p was considered statistically significant Statisticalheterogeneity was assessed by calculating the I2 statistic[] The presence of heterogeneity was indicated whenI2 and then a randomeffects model was appliedto poolthe results [] a fixedeffects model wasemployed to pool the results when I2 [] Weperformed further subgroup or sensitivity analysis toanalyse the heterogeneity The publication bias was estimated by Begg™s and Egger™s tests when p indicates no publication bias [] Through Stata software we performed this metaanalysis and acquiredthe forest plotsResultsSearch results and study characteristicsUsing the searching strategy described above a total of original documents were identified from the databases with approximately studies remaining after excluding duplicates Afterandscreening thetitles 0cChen World Journal of Surgical Oncology Page of s of the publications publications werenot related to evaluating the prognosis role of DLL3in SCLC Finally we were left with eligible studiesafter screening the full text among which swere included in our final analysis Fig [“]Ten s were excluded for the following reasonsseven s were review researches and commentaries one did notreport hazard ratios andKaplanMeier curves and s were s anddid not report relevant outcomes The eligible s™ characteristics are shown in Table All theses came out from the initial date to February and the patients were diagnosed as SCLC byhistopathology Among these studies all studies investigated DLL3 byinparaffinembedded tissueimmunohistochemistryIHCQuality assessment of the included studiesWe performed quality evaluations of the s following the QUIPS tool and two authors independently evaluated allthe literature Differences were resolved bydiscussion After screening all included s we foundthat there were no researches that reported study attritionand there were no standardization of cutoffs value forevaluating DLL3 expression And then studies were evaluated as low risk were evaluated as moderate risk and study was evaluated as high risk Table This outcomeindicated that most of the studies we included were of amedium or high qualityPrognostic value of DLL3 in SCLCAs shown in Fig eligible s were pooled foranalysing the prognostic value of DLL3 in SCLC TheFig The flow diagram of studies selection 0cChen World Journal of Surgical Oncology Page of Table Study characteristics of the eligible sStudyYear Country No ofsamplesSexmalefemaleSmokinghistorynonyesYan [] ChinaDistantmetastasisnegativepositiveXie [] USATanaka [] JapanRegzedmaa [] ChinaHuang [] ChinaFuruta [] JapanNANANANANANANATumourstageIIIIIIIVHistologicsubtypeMethod Outcomes BiomarkersCutoffvalueNANASCLCSCLCSCLCSCLCSCLCSCLCIHCIHCIHCIHCIHCIHCOSOSOSOSDLL3TTF1DLL3DLL3DLL3CTLA4MSTNscore ‰¥ ‰¥ ‰¥ score ‰¥ OSPFSDLL3OSDLL3ASCL1score ‰¥ ‰¥ SCLC small cell lung cancer IHC immunohistochemistry OS overall survival PFS progressionfree survival DLL3 deltalike protein CTLA4 cytotoxic T lymphocyteassociated protein MSTN mesothelin ASCL1 achaetescute homologue1 NA not applicableresults showed that high expression of DLL3 indicatedno prognostic value in patients with SCLC HR CI I2 p Howeverprominent heterogeneity existed in the pooled results sowe conducted further subgroup analysis Among the sixstudies three s were from China two were fromJapan and one was from America We divided the studies into the Asian group and the American group forsubgroup analysis As shown in Additional file FigA1we found that the heterogeneity was in the Asiangroup and we believed that the main cause of the heterogeneity was the study from American So we decidedto eliminate the study from American and only analysedthe remained studies After we removed the Americanstudy the outcomes indicated that high DLL3 expressionwas a poor prognosis marker in SCLC HR CI I2 p Fig Then wedivided the studies into two groups according to thesize of the sample and the final outcomes indicated thathigh DLL3 expression was a poor prognosis marker inthe group with sample size greater than HR CI p Fig But there was nosignificant relationship between DLL3 expression andprognosis in the group with sample size less than HR CI I2 p Fig Begg™s test p and Egger™s test p there was nodemonstratedresultsthatpublication bias in our metaanalysis Fig Finallysensitivity analysis showed that allthe studies werestable see Additional file FigA6The clinical characteristics of patients with DLL3expression in SCLCThe pooled results showed that the expression of DLL3was not correlated with sex RR CI I2 p Table Additional file FigA2 smoking history RR CI I2 p Table Additional file FigA3 and tumour stage RR CI I2 p Table Additional file FigA4 But DLL3 was highly expressed in patients withdistant metastasis RR CI I2 p Table Additional file FigA5DiscussionMany studies have shown that DLL3 played a significantprognosis value in patients with cancer For examplehigh DLL3 expression was associated with shorter OSand PFS in small cell bladder cancer [] High DLL3expression was associated with bad OS and usuallyexpressed in older patients and advanced stage in endometrial cancer [] Xie reported that high DLL3expression predicted a better OS in patients with SCLC[] and other studiesforeboded that high DLL3Table Quality assessment of included studiesStudyStudyparticipation—‹Yan []Xie []Tanaka []Regzedmaa []Huang []—‹—‹—‹Furuta []œ—Ž indicates low risk of bias œStudyattrition——————Prognostic factormeasurement—‹—‹—‹—‹—‹—‹Outcomemeasurement—‹—‹—‹—‹—‹Study confounding—‹—‹—‹—Statistical analysisand reporting—‹—‹—‹—‹—‹—‹Total riskof biasLowModerateLowLowHighModerate moderate risk of bias and œ— high risk of bias 0cChen World Journal of Surgical Oncology Page of Fig Forest plots of prognostic value of DLL3 in SCLC patients DLL3 deltalike protein HR hazard ratios CI confidence intervals Isquared percentage heterogeneity between studies p test for heterogeneityexpression was an inferior prognostic marker for SCLC[ ] Thus the results of different studies remainedcontroversial We included a total of studies with patients with SCLC to assess the prognostic value ofDLL3 in SCLC by pooling the data of HR and CIFirst we performed this metaanalysis using studiesBut heterogeneity was observed in our pooled resultsThrough further subgroup analysis we found that astudy from the American was the main cause of heterogeneity As we removed the American study we foundFig Forest plots of prognostic value of DLL3 in Asia patients with SCLCDLL3 deltalike protein USA the United States of America HR hazardratios CI confidence intervals Isquared percentage heterogeneity between studies p test for heterogeneity 0cChen World Journal of Surgical Oncology Page of Fig Forest plots of prognostic value of DLL3 in different studies with different sample sizes DLL3 deltalike protein HR hazard ratios CI confidence intervals Isquared percentage heterogeneity between studies p test for heterogeneitythat high expression of DLL3 is a marker of poor prognosis in SCLC But we also noticed that the results ofthe American study were contrary to our conclusionsthey reported that high expression of DLL3 is a markerof good prognosis in SCLC One interpretation of thisresult was that DLL3 expression varies between differentpopulations The American study indicated that the highexpression of DLL3 was a marker of good prognosis[] and there was no significant correlation betweenDLL3 expression and prognosis in several Japan studies[ ] while the high expression of DLL3 is a markerof poor prognosis in China studies [ ] These results showed that the expression of DLL3 might be different in different populations However only one studyFig Funnel plot of DLL3 present on overall survival DLL3 deltalike protein HR hazard ratios 0cChen World Journal of Surgical Oncology Page of Table Summary of the correlation between DLL3 expression and the clinical characteristics of patients with SCLCClinical characteristicsSex malefemaleRR CI No of studiesI2 p Smoking historynonyesDistant metastasis negativepositive P valueTumour stage IIIIIIIVSCLC small cell lung cancer RR relative ratios CI confidence intervals DLL3 deltalike protein I2 percentage heterogeneity between studies p testfor heterogeneity researched the correlation with DLL3 expression andprognosis in SCLC outside of Asia so the results needto be treated with caution In addition more studieswere suggested for the future to further verify the existence of such differencesOur other explanation of this result was that the expression of DLL3 was the same in different populationsAmong the included studies the sample size of studieswas less than including the America and Japanstudies Therefore we speculated that insufficient sample size might cause bias in the results We conducted asubgroup analysis according to the sample size and theresults showed that the high expression of DLL3 in thestudies with large sample sizes N ‰¥ was associatedwith poor prognosis while the pooled results of thestudies with low sample sizes N showed no significant correlation between the expression of DLL3 andprognosis Therefore the sample size may be one of thereasons for the differences in DLL3 expression in eachstudy Moreover immunohistochemistry was used to detect DLL3 expression in all of the studies Immunohistochemical staining is a semiquantitative method and isevaluated with great subjectivity [] Different antibodies and different cutoff values for DLL3 expressionwere employed in all the included studies and thus couldalso be another cause of the differences in results TheAmerican study also explained their different results byclaiming that many of the other studies were performedusing mRNA expression instead of protein expression orused different cutoffs value of DLL3 expression []Researches havereported that DLL3 is highlyexpressed in SCLC [ ] which suggested that DLL3might promote the development of SCLC Therefore wealso discussed the correlation between DLL3 expressionand the clinical characteristics of patients with SCLCThe pooled results showed that DLL3 expression had nosignificant correlation with patients™ sex smoking statusand stage while DLL3 often highly expressed in metastasis patients of SCLC Our survival analysis outcomeswere consistent with this result which suggested thathigh expression of DLL3 might be one of the factorscontributing to poor prognosis in patients with advancedmetastatic SCLC in Asia However only a few studiesreported the correlation between the expression of DLL3and clinical characteristics and the methods and cutoffs values used to detect the DLL3 expression in eachstudy were not uniform Therefore more reliable studiesare needed to further verify our outcomes Our resultssuggest that it is valuable to further investigate the correlation between DLL3 and the clinical characteristics ofpatients with SCLCOur metaanalysis is the first to focus on the prognostic value of DLL3 in SCLC The significance of thismetaanalysis lies in providing a basic direction and evidence for further research into the mechanism of DLL3in SCLC For SCLC Notch1 over expression could induce G1 cell cycle arrest [] Previous studies reportedthat DLL3 downregulated the Notch receptor expression thereby the Notch signalling pathway was inhibitedwithin the cell [] Therefore high expression of DLL3can promote the development of SCLC by inhibiting theNotch signalling pathway Studies also have shown thatthe high expression of DLL3 may reduce the sensitivityof chemotherapy drugs [] These studies have demonstrated that DLL3 may be associated with the prognosisof SCLC and also consistent with our metaanalysis results Thus studies of the corresponding targeted drugsof DLL3 can effectively inhibit the expression of DLL3and thus improve the survival of SCLC Rovalpituzumabtesirine RovaT is a new antibodydrug conjugate directed against DLL3 in SCLC [] A phase I trial foundthat patients with high DLL3 expression in SCLCshowed a better response to RovaT than those with alow DLL3 expression [] However disappointingly thephase III TAHOE trial has been stopped because theRovaT group showed a worse OS compared to the control group [] But more clinical trials are recruitingparticipator to investigate RovaT as maintenance therapy in advanced stage SCLC The lack of progress withthis drug does not prevent us from making a breakthrough with other similar drugs Some researches foundthat the intratumoural and intertumoural distributionof DLL3 protein in SCLC is homogeneous [] supporting the conclusion that biopsy specimens are a reliablesource for DLL3 evaluation for targeted therapy Inaddition most studies have demonstrated that DLL3 is 0cChen World Journal of Surgical Oncology Page of highly expressed in SCLC while it is not or is lessexpressed in other types of lung cancer and normal tissues [] Therefore the expression of DLL3 can be detected by biopsy as an indicator for diagnosis predictingtherapeutic efficacy and monitoring recurrence or metastasis of SCLC in the futureAlthough our study fully explains the prognostic valueof DLL3 in SCLC our analysis still has several limitations Firstlarge heterogeneity was observed in thepooled results This is explained by the observation thatthe evaluation criteria for the expression of DLL3 areparticularly mixed and there are no international standards for cutoffs values to determine the expression ofDLL3 Thus the scoring methods and cutoffs values ofDLL3 should be unified to strengthen our conclusionsOtherwise the detection method of DLL3 in most studies is mainly immunohistochemistry at present which isa semiquantitative subjective and inaccurate detectionmethod Different studies show different prognosticvalues of DLL3 Therefore we need other more precisedetection methods to evaluate the expression of DLL3 inSCLC in the futureSecond the therapy method is also a key limitationThe current studies only focus on tissue specimens frompatients with SCLC after surgery or biopsy and fewstudies reported the treatment methods in their researches However the prognostic value of DLL3 may liein the therapeutic method Therefore every study shouldpay attention to the impact of patienttreatmentmethods on prognosis in the futureThird some of the original studies did not report thedata of HR and CI The HR and CI resultswere measured from survival curves an evaluationmethod with certain deviation and subjectivity whichmight influence the authenticity of the resultsConclusionIn summary our metaanalysis confirmed that high expression of DLL3 was a potential poor prognostic biomarker for SCLC in Asian populations moreover DLL3expression was correlated with advanced metastasisSCLC in Asian populations However the relationshipbetween DLL3 expression and the prognostic or clinicalcharacteristics of patients with SCLC in European andAmerican populations need to be further verified Thusdetecting the expression of DLL3 in tumour tissue willbe helpful to guide therapy in Asian patients of SCLCFor our research other highquality studies especiallyfrom European and American countries are required toconfirm our findings about the prognosis value of DLL3in SCLC in the future In view of the limitations of ouranalysisthe conclusions should be interpreted withcautionSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s12957020020045Additional file The PRISMA checklistAdditional file FigA1 Forest plots of prognostic value of DLL3 inSCLC SCLCsmall cell lung cancer HRhazard ratios CI confidence intervals DLL3deltalike protein I2percentageheterogeneity between studies ptest for heterogeneity FigA2 Forestplots of the correlation between DLL3 expression and sex of patientswith SCLC SCLCsmall cell lung cancer RRrelative ratios CI confidence intervals DLL3deltalike protein I2percentageheterogeneity between studies ptest for heterogeneity FigA3 Forestplots of the correlation between DLL3 expression and smoking history ofpatients with SCLC SCLCsmall cell lung cancer RRrelative ratios CI confidence intervals DLL3deltalike protein I2percentageheterogeneity between studies ptest for heterogeneity FigA4 Forestplots of the correlation between DLL3 expression and tumour stage ofpatients with SCLC SCLCsmall cell lung cancer RRrelative ratios CI confidence intervals DLL3deltalike protein I2percentageheterogeneity between studies ptest for heterogeneity FigA5 Forestplots of the correlation between DLL3 expression and metastasis ofpatients with SCLC SCLCsmall cell lung cancer RRrelative ratios CI confidence intervals DLL3deltalike protein I2percentageheterogeneity between studies ptest for heterogeneity FigA6Sensitivity analysis of all the studiesAbbreviationsCI Confidence intervals DLL3 Deltalike protein DFS Diseasefree survivalDSS Diseasespecific survival HR Hazard ratios IHC ImmunohistochemistryOS Overall survival PFS Progressionfree survival QUIPS Quality In PrognosisStudies RFS Relapserecurrencefree survival RR Risk ratio SCLC Small celllung cancerAcknowledgementsNot applicableAuthors™ contributionsAll authors contributed to the study conception and design Materialpreparation data collection and analysis were performed by Benchao ChenHeng Li and Chao Liu The first draft of the manuscript was written byBenchao Chen and all authors commented on previous versions of themanuscript All authors read and approved the final manuscriptAuthors™ informationNot applicableFundingThis work was supported by the National Natural Science Foundation ofChina [No ]Availability of data and materialsThe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsReceived July Accepted August ReferencesBray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancerstatistics GLOBOCAN estimates of incidence and mortality worldwide 0cChen World Journal of Surgical Oncology Page of Regzedmaa O Li Y Li Y Zhang H Wang J Gong H Prevalence ofDLL3 CTLA4 and MSTN expression in patients with small cell lung cancerOnco Targets Ther “ httpsdoi102147OTTS216362 Huang J Cao D Sha J Zhu X Han S DLL3 is regulated by LIN28B and miR518d5p and regulates cell proliferation migration and chemotherapyresponse in advanced small cell lung cancer Biochem Biophys ResCommun “ httpsdoi101016jbbrc201904130Furuta M SakakibaraKonishi J Kikuchi H Yokouchi H Nishihara HMinemura H Analysis of DLL3 and ASCL1 in surgically resected smallcell lung cancer HOT1702 Oncologist 20192411e1172“ httpsdoi101634theoncologist20180676Koshkin VS Elson P MagiGalluzzi C McKenney J Smith KS Shadrach B Prognostic value of DLL3 expression and clinicopathologic features insmall cell bladder cancer SCBC J Clin Oncol Juan Wang Kaishuo Zhang Zi Liu Upregulated deltalike protein expression is a diagnostic and prognostic marker in endometrial cancer aretrospective study Medicine Baltimore 20189751e13442 httpsdoi101097MD0000000000013442 Matos LLD Stabenow E Tavares MR Ferraz AR Capelozzi VL Pinhal MADSImmunohistochemistry quantification by a digital computerassistedmethod compared to semiquantitative analysis Clinics “httpsdoi101590S180759322006000500008 Hann CL Mensztern D Dowlati A Burns TF Jotte RM Pennell NA et alA study of rovalpituzumab tesirine in frontline treatment of patients withDLL3 expressing extensive small cell lung cancer J Clin Oncol httpsdoi101200JCO20173515_supplTPS2598Furuta M Sakakibara JK Shoji T Takashima Y Kikuchi H Kikuchi E et alDLL3 regulates migration and invasion of small cell lung cancer Cancer Res httpsdoi10115815387445Am20183158Sriuranpong V Bes MW Ravi RK Arnold DR Nelkin BD Baylin SB et alNotch signaling induces cell cycle arrest in small cell lung cancer cells CancerRes httpsdoi1010970000282020010400000012 Deng SM Yan XC Liang L Wang L Liu Y Duan JL The Notch liganddeltalike promotes tumor growth and inhibits Notch signaling in lungcancer cells in mice Biochem Biophys Res Commun “httpsdoi101016jbbrc201612117Komarnitsky P Lee H Shah M Wong S Gulbranson S Dziubinski J et alRovalpituzumab tesirine vs topotecan in patients with advanced small celllung cancer following 1st line chemotherapy J Thorac Oncol S1974“ Udagawa H Akamatsu H Tanaka K Takeda M Kanda S Kirita K Phase Isafety and pharmacokinetics study of rovalpituzumab tesirine in Japanesepatients with advanced recurrent small cell lung cancer Lung Cancer “ httpsdoi101016jlungcan201907025Komarnitsky P Lee HJ Shah M Wong S Gulbranson S Dziubinski J Aphase trial of rovalpituzumab tesirine vs topotecan in patients withadvanced small cell lung cancer following frontline platinumbasedchemotherapy Ann Oncol 201728v542 httpsdoi101093annoncmdx386009 Odashiro P Tomarchio G Gagne A Orain M Desmeules P Joubert P DLL3expression in small cell lung carcinomas SCLC Mod Pathol 202033SUPPL“Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsfor cancers in countries CA Cancer J Clin httpsdoi103322caac21492Amini A Byers LA Welsh JW Komaki RU Progress in the management oflimitedstage small cell lung cancer Cancer httpsdoi101002cncr28505 Mead GM Arnold AM Green JA Williams CJ Whitehouse JM SMALLCELLLUNG CANCER[J] Cancer Chemother Pharmacol httpsdoi101016S0140673680907333Powell HA Tata LJ Baldwin DR Potter VA Stanley RA Khakwani A et alTreatment decisions and survival for people with smallcell lung cancer Br JCancer “ httpsdoi101038bjc2013812Huang RSP Holmes BF Powell C Marati RV Tyree D Admire B Deltalike protein prevalence in small cell lung cancer and DLL3 SP347 assaycharacteristics Arch Pathol Lab Med “ httpsdoi105858arpa20180497OA Mensztern D Besse B Greillier L SantanaDavila R Ready N Hann CL Efficacy and safety of rovalpituzumab tesirine in thirdline and beyondpatients with DLL3expressing relapsedrefractory smallcell lung cancerresults from the phase II TRINITY study Clin Cancer Res “ httpsdoi10115810780432CCR191133Isobe Y Sato K Nishinaga Y Takahashi K Taki S Yasui H Near infraredphotoimmunotherapy targeting DLL3 for small cell lung cancerEBioMedicine httpsdoi101016jebiom2020102632Rossi A Rovalpituzumab tesirine and DLL3 a new challenge for smallcelllung cancer Lancet Oncol “ httpsdoi101016s1470Rudin CM Pietanza MC Bauer TM Ready N Mensztern D Glisson BS Rovalpituzumab tesirine a DLL3targeted antibodydrug conjugate inrecurrent smallcell lung cancer a firstinhuman firstinclass labelphase study Lancet Oncol “ httpsdoi101016s1470204516305654Fu X Tian T Liu M Ruan Z Liang X Nan K The expression andprognostic roles of PDL1 PAPR1 and DLL3 in small cell lung cancer JThorac Oncol 20191410S1024“ httpsdoi101016jjtho2019082267Lim S Hong M Kim SP Chung SH P2 Prevalence of DLL3 Expressionand its prognostic role in extensive stage small cell lung cancer J ThoracOncol 20191410S820 httpsdoi101016jjtho2019081763 Poirier JT Targeting DLL3 in smallcell lung cancer with novel modalities JThorac Oncol 2020152S8 httpsdoi101016jjtho201912024Liberati A Altman DG Tetzlaff J Mulrow C Gøtzsche PC Ioannidis JPA et alThe PRISMA statement for reporting systematic reviews and metaanalysesof studies that evaluate health care interventions explanation andelaboration J Clin 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Thyroid_Cancer

This study aimed to investigate serum matrix metalloproteinase MMP2 and MMP9levels in patients with papillary thyroid carcinoma PTCMethods Fortyone patients with PTC undergoing ultrasoundguided radiofrequency ablationRFA and controls were included Serum MMP2 and MMP9 levels were determined byenzymelinked immunosorbent assay before and after surgery Potential affecting factors wereevaluated by logistic regression analysisResults Serum MMP2 and MMP9 levels were significantly higher in PTC patients comparedwith controls and decreased significantly after surgery According to receiver operating characteristic curve analysis diagnostic values for preoperative serum MMP2 and MMP9 levels were and There was no contrastagent perfusion in the ablation zone in of lesionsand enhancement within or at the lesion edge in The volume reduction at months™followup was Age microcalcification irregular shape and lesion diameter and numberwere influencing factors for PTC Age and lesion diameter and number were independent riskfactors while calcification and morphology were protective factorsConclusion Serum MMP2 and MMP9 levels have important clinical values for the diagnosis andtreatment of PTC by RFA Preoperative serum MMP2 and MMP9 levels combined with otheraffecting factors contribute to disease prognosisDepartment of Ultrasound Beilun People™s Hospital ofNingbo Beilun Branch of the First Affiliated Hospital ofZhejiang University Ningbo Zhejiang ChinaThese authors contributed equally to this studyCorresponding authorQian Ding Department of Ultrasound Beilun People™sHospital of Ningbo Beilun Branch of the First AffiliatedHospital of Zhejiang University No East LushanRoad Ningbo Zhejiang ChinaEmail qianding02sohucomCreative Commons Non Commercial CC BYNC This is distributed under the terms of the CreativeCommons AttributionNonCommercial License creativecommonslicensesbync40 which permitsnoncommercial use reproduction and distribution of the work without further permission provided the original work is attributedas specified on the SAGE and Access pages ussagepubcomenusnam accessatsage 0cJournal of International Medical ResearchKeywordsPapillary thyroid carcinoma radiofrequency ablation matrix metalloproteinase2 matrix metalloproteinase9 disease prognosis serum levelDate received June accepted March IntroductionPapillary thyroid carcinoma PTCis acommon thyroid malignancy accountingfor about of systemic malignancies1PTC is currently characterized by a highincidence and low mortality2clinicalRecentimprovementsin highfrequencyultrasonic diagnostic technologies and theapplication of ultrasoundguided puncturetechniques have led to an apparent increasein the incidence of PTC year by year3“Regardingthyroidtumors the palpation detection rate forthyroid microtumors in the general population is about compared with as highas to for highfrequency ultrasound6 which has thus greatly improveddisease diagnosisdiagnosistheofSurgical resection is a routine treatmentfor thyroid tumors but the recurrence rateis usually high and the consequent reduction in thyroid function can seriously affectthe patient™s quality of life7 Associatedwith the increasing detection rate of thyroidtumors and the pursuit of minimally invasive treatments radiofrequency ablationRFA has been gradually applied in theclinic RFA uses local hyperthermia tocause tissue necrosis The thermal effectsdo not depend on the tissue type andmost lesions can be completely eliminatedby RFA89 RFA has thus become a novellocal treatment for tumorsThe clinical diagnosis of benign andmalignant thyroid tumors currently dependson the clinical manifestations and pathological examinations Howeverthe clinicalmanifestations are mostly derived frominvolvessubjective empirical analysis while a finalpathological diagnosisinvasiveprocedures with lesssatisfactory specificity It is therefore necessary to identifyappropriatepredictivetumor markersdiagnosticandforimplicationsThe collagenases matrix metalloproteinase MMP2 and MMP9 can degradetype IV collagen in the basement membranewith importanttumorangiogenesis and tumor cell invasion andmetastasis10 MMP2 and MMP9 expression levels were found to be upregulatedin thyroid cancer tissue11 however thesestudies mostly examined pathological tissuesafterinvasive surgery and lessinvasivemeasures such as serum levels of MMP2and MMP9 have been lesswell consideredIn this study we detected serum levels ofMMP2 and MMP9 in patients with PTCbefore and after ultrasoundguided RFAWe also determined the therapeutic effectsof RFA during the followup period andinvestigated the relevant prognostic factorsMaterials and methodsStudy subjectsPatients who underwent ultrasoundguidedRFA in our hospital from May toOctober were included in this studyThe inclusion criteria were as follows patients diagnosed by preoperative fineneedle aspiration cytology no historyof neck surgery and patients requiringminimally invasive treatment for aestheticreasons and because of neck oppression 0cPan et alwith anxiety The exclusion criteria were asfollows benign lesions confirmed by fineneedle aspiration cytology history ofneck surgery and severe coagulopathyPeripheral venous blood samples wereobtained from the included patients beforeand at and months after the operation and serum levels of MMP2 andMMP9 were determined Additional subjects with confirmed benign thyroid noduleswithout RFA were included as a controlgroup Prior written informed consent wasobtained from all patients and the studywas approved by the ethics review boardof our hospitalPreoperative preparationcalcificationThe number size nature echo boundarymorphologysurroundinghalo and nodular blood flow distributionof the tumors were assessed before the operation After skin disinfection local anesthesia was performed with lidocainesolution A total of mL Sonovue Bracco Milan Italy was injected via the elbowvein and the bloodsupply characteristicswere then evaluated by contrastenhancedultrasound CEUS of the ablationtargetedlesions using a Mylab90 ultrasonic devicewith 10MHz probe Esaote ShenzhenGuangdong ChinaAccording to the location of the thyroidnodules the thyroid and carotid space thyroid and tracheal space thyroid and esophageal space and posterior thyroid spacerecurrent laryngeal nerve were separatedusing a saline and lidocaine mixture basedon the intraoperative conditions to form aliquid separation zone to protectthesestructures from thermal damageAblation treatmentUnder ultrasound guidancethe tip ofthe RFA needle rated power Woutput frequency kHz was accuratelypenetrated into the nodule and RFA wasperformed using an OlympusCelon PowerRFA System Germany in mobile mode12following the fromdeeptoshallow principle The lesions were subjected to multipointed and multifaceted ablation untilthe thyroid tissue layer with the noduleswas completely covered by the strong echogenerated by heat accumulation The wholeprocess was carried out under continuousultrasound monitoring A highecho areawas produced in the ablation zone duringthe ablation treatment The position of theelectrode needle was gradually adjustedaccording to the lesion size After ensuringthat there was no residual enhancement inthe ablation zone the ablation needle wasremoved and the ablation was completedAfter the operation an ice compress wasapplied for h to avoid skin burnsSerum MMP determinationFor all subjects mL venous blood wascollected from the elbow vein under fastingconditions before and after the operationrespectively Blood samples in the controlgroup were collected after ultrasound contrast examination The blood samples wereplaced at room temperature for minutesand then subjected to centrifugation at 02 g for minutes The serum washarvested and serum levels of MMP3 andMMP9 were determined using enzymelinked immunosorbentELISAkitsBoster Bioengineering WuhanHubei ChinaassayFollowup and efficacy evaluationImmediately after the operation the ablation range was evaluated by CEUS If residualtissues were detected ablation wasrepeated immediately Ultrasound detectionwas performed at and monthsafter surgery to determine the nodule sizesand volumes The volumereduction rate 0cwas calculated according to the followingformula volume reduction rate¼ preoperative volume followup volumepreoperative volume 02 Echo and bloodflowchanges in the ablation zone were alsoobserved and analyzed The efficacy wasdetermined based on the criteria for RFAfor treating tumors13 disappearance ofnodules indicated by complete disappearance of blood flow confirmed by ultrasonography indicated complete cure noduleby 15 indicatedvolumemarked effect and nodule volume reducedby to indicated improvementreducedClinicopathological featuresInformation on ultrasoundbased clinicopathologicalincluding numbersize and calcification of the lesion wereobtainedfeaturesversion Statistical analysisData were expressed as mean 06 standarddeviation Statistical analysis was performed using IBM SPSS StatisticsforWindowsIBM CorpArmonk NY USA Comparisons betweengroups were performed using v2testsPotentially related factors were analyzedby univariate or multivariate logistic regression The prognostic predictive effects ofserum MMP2 and MMP9 levels wereevaluated by receiver operating characteristic ROC curve analysis P was considered statistically significantResultsPatientsJournal of International Medical Research men mean age 06 years range“ yearsSerum MMP2 and MMP9 levels beforeand after treatmentThe characteristics of the ultrasound images inthe included subjects are shown in Table Serum levels of MMP2 and MMP9 weremeasured before and after treatment Serumlevels of MMP2 and MMP9 were significantly higher in patients with PTC comparedwith the control subjects P Serumlevels of MMP2 and MMP9 had declinedat month after the operation comparedwith before surgery but the difference wasnot significant However serum levels ofMMP2 and MMP9 had declined significantly in the PTC patients at and monthsall P Table These results suggestthat changes in serum MMP2 and MMP9levels between before and after surgery mayhave significant implications for the therapyof PTCROC curve analysis of preoperative serumMMP2 and MMP9 levelsPreoperative serum MMP2 and MMP9levels were used as potential diagnostic indicators In the patients with PTC the predictive probability from theregressionmodel was used as the diagnostic resultsand the gold standard classification criteriawere used as the pathological results ROCcurves were obtained accordingly The areaundercurve AUC values for serum MMP and MMP9 levels were and respectively Figure These results suggestthat serum levels of MMP2 and MMP9could contribute to the disease diagnosisFortyone patients with PTC lesionswere enrolledincluding women and men mean age 06 years range to þ65 years The control group included patients with confirmed benignthyroid nodules including women andEvaluation of RFA efficacyWe also evaluated the efficacy of RFA CEUSof the lesions before ablation showed hypoenhancement in nodules isoenhancementin nodules and slight hyperenhancement 0cPan et alTable Characteristics of thyroid ultrasound imagesPTC patientsNormal controlLesion numberOneTwoMultipleLesion size 14 cm cmCalcificationMicrocalcificationCoarse calcificationMorphologyRegularIrregularAge years 15 yearsnP compared with the control groupnTable Serum matrix metalloproteinase2 and levels in controls and inpatients with papillary thyroid carcinoma before and after treatmentControlsPTC patientsBefore surgery month after surgery months after surgery months after surgery months after surgeryMMP2 06 06 06 06 06 06 P“MMP9P“ 06 06 06 06 06 06 MMP matrix metalloproteinase PTC papillary thyroid carcinomain nodules Ultrasound examination afterablation showed no contrastagent perfusionin the ablation zone in lesions and enhancement of different degrees at theedge or inside the lesion in the other lesions and the ablation area wasgradually reduced with prolonged ablationFigure There was no significant changein ablation volume in any patients at month after surgery compared with beforesurgery However the volume was reducedby at months offollowupcompared with before surgery P Table These results showed that RFAtreatmentthetumor volume in patients with PTCcould effectively reduceInfluence of relevant factors on diseaseprognosisTheclinical data of patients beforeRFA were retrospectively analyzed by 0cJournal of International Medical Researchirregularlogistic regression to identify factors thatmay affectthe disease prognosis Agemicrocalcificationshape anddiameter and number of lesions were significant influencing factors for PTC P The hazard ratios HRsfor age andlesion diameter and number were indicating that these represented independentrisk factors In contrast the HRs for microcalcification and irregular shape were negativeindicating that a greater degree ofcalcification and regular shape were associated with lower risks of developing the disease and were thus protective factorsTable Figure ROC analyses of serum MMP2 andMMP9 levels MMP matrix metalloproteinaseFigure Efficacy evaluation of radiofrequency ablation a Twodimensional 2D ultrasound showingobvious bloodflow signals around the tumor and fewer signals within the tumor b Preoperative contrastenhanced ultrasound showing no obvious enhancement in the lesion with lowperfusion performance c Inthe 2D imaging localization the ablation needle was inserted into the tumor for ablation d The tumor wascompletely ablated with no bloodflow signal at year after ablation 0cPan et alDiscussionitis difficultPTC is a type of thyroid tumor with a highincidence14 which has been increasingrapidly worldwide1516 Mostthyroidtumor cases are currently diagnosed by hiscytological detectiontopathological orHoweverto distinguishbetween benign and malignant papillaryhyperplastic nodules and it is therefore difficult to diagnose PTC There is also a lackof effective and specific prognostic molecular markers for PTC17 The relationshipbetween MMPs and tumors is a currenthotspot of modern cancer research MMPsplay important roles in pathophysiologicalprocesses such as the dynamic extracellularmatrix balance as well as in tissue remodeling and repair10 Tumor cells may inducethe matrix to secrete MMPs via a series ofsignalingprovidingpathwaysthusTable Volume reductions after radiofrequencyablation of papillary thyroid carcinomasReductionrate Lesionvolumecm3 06 06 06 06 months after surgery 06 P P compared with before surgeryBefore surgery month after surgery months after surgery months after surgery“favorable conditions for tumor cell invasionand metastasisalternatingthe extensive surgicalSurgical resection is a traditional methodthyroid nodules18for the treatment ofHowevertraumaunsightly neck scars and risks of laryngealnerve injuries postoperative recurrenceand multiple operations mean that increasing numbers of patients are opting for minimally invasive ablation methods RFA is athermal ablation therapy that uses highfrequencyelectromagneticwaves generated by the radiofrequencyinstrument inserted into the tumor tissueto accumulate heat by rapid friction of positive and negative ions within the cells causing local coagulation in the tumor tissuewhich isthen removed by the body™ssystem19 Reduction ratesimmuneforbenign thyroid nodules of to after month of ablation and to after months of ablation have beenreported20 In this study ultrasound performed immediately after ablation of lesions showed no perfusion of contrastagents in lesions and enhancementto varying degrees at the edge or inside thelesion in of lesions Although therewere no significant changes in lesion volumeat month after the operation the lesionvolumes were reduced by to at months after surgery Considering thatthe ablation effects might be associatedwith the heat and the ablation needle aTable Logistic regression analysis of relevant risk factors for disease prognosisAgeMicrocalcificationIrregular morphologyLesion diameterLesion numberB HRHR hazard ratio CI confidence interval95CILower limitUpper limitP 0cJournal of International Medical Researchfine needle is good for mobile conformalablationthe highfrequency alternatingelectromagnetic wave only circulates in theeffective region between the two needle tipsthus allowing accurate control of the ablation zone The ablation safety zone aroundthe PTC was relatively small in the currentstudy and the nodulereduction rate afterablation was thus relatively greaterWe analyzed the serum levels of MMP2and MMP9 in PTC patients by ELISAPreoperative serum levels of both enzymeswere significantly higher in patients withPTC compared with patients with benigninthyroid nodules Regarding changesserum MMP2 and MMP9 levelstheAUC values based on the ROC curveswere and for MMP2 andMMP9 respectively suggesting satisfactory clinical diagnostic and prognostic valuesIn the present study serum levels MMP2and MMP9 were lower in the first monthafter surgery compared with before surgerybut the difference was not significant Thismight be because before ablationthetumor parenchyma induced the tumorstroma to produce larger amounts ofMMP2 and MMP9 which were releasedinto the blood These results suggest thatfailure to completely ablate the tumor ortumor recurrence may result in the secretionof high levels of MMP2 and MMP9 intothe blood However serum levels of MMP and MMP9 were significantly decreasedat and months after surgery compared with before surgery suggesting thatserum MMP2 and MMP9 were secretedby the tumor The lesions disappearedafter PTC ablation thus reducing the secretion of MMP2 and MMP9 and therebyreducing the degradation and destructionof type IV collagen protecting the basement membrane of normaltissues andinhibiting the growth and metastasis oftumorresults alsoshowed that age microcalcification irregular shape and lesion diameter and numbercells Thecurrentwere risk factors for PTC Logistic regression analysis showed that age 14 yearswas an important risk factor for PTC inline with the findings of Yu et al21Microcalcification is caused by the deposition of calcium salts at the tip of the nippleor the secretion of calcium salts by thetumor itself and has been considered tobe the most specific sign of PTC In thisstudy the incidence of microcalcificationwas higher in PTC patients compared withthe control group and logistic regressionidentified it as an independent risk sign forthyroid PTC Our results also identifiedirregular morphology two nodules and anodule diameter 14 cm as danger signs forPTClargely consistent with previousfindings22This study had some limitations It was asinglecenter study with a relatively smallnumber of cases Moreover the relevantthyroid hormone analysis and other serumindicators could not be followed up for alonger periodtreating PTC FurthermoreIn summary the results of this studyshowed that RFA could shrink or eliminatethyroid lesions thus representing a minimally invasive safe and effective methodforserumlevels of MMP2 and MMP9 before RFAcould provide a valuable reference for thediagnosis of PTC These serological indexes combined with relevant risk factors mayalso help to predict the prognosis of PTCafter ablationAcknowledgementsThis work wasProvincial HealthCommission Project WJ2017F102supported by the HubeiPlanningand FamilyDeclaration of conflicting interestThe authors declare that there is no conflict ofinterest 0cPan et alFundingThis research received no specific grant from anyfunding agency in the public commercial ornotforprofit sectorsorcid000000026050ORCID iDQian DingReferences Zhang YB Zhang B Yan DG et al[Central compartment reoperation for recurrentpersistent differentiated thyroid cancer]Zhonghua Er Bi Yan Hou Tou Jing Wai KeZa Zhi “ [in Chinese] Pellegriti G Frasca F Regalbuto C et alWorldwide increasing incidence of thyroidcancer update on epidemiology and risk factors J Cancer Epidemiol DOI Brito JP Gionfriddo MR Al Nofal A et alThe accuracy of thyroid nodule ultrasoundto predict thyroid cancer systematic reviewand metaanalysis J Clin Endocrinol Metab “ DOI 101210jc2013 Zhao P Zheng D Dong X et al Clinicaldiagnosis and treatment of thyroid microcarcinoma a report of cases Chinese JGene Surg “ Anil G Hegde A and Chong FH Thyroidnodules risk stratification for malignancywith ultrasound and guided biopsy CancerImaging “ DOI Levine RA Current guidelines for the management of thyroid nodules Endocr Pract “ DOI 104158ep12071co Zhang Y Zhang MB Luo YK et al Effectof chronic lymphocytic thyroiditis on theefficacy and safety of ultrasoundguidedradiofrequency ablation for papillary thyroid microcarcinoma Cancer Med “ Liu Y Wang W Wang Y et al Ultrasoundguided percutaneous microwave ablation inthe treatment of recurrent thyroid nodulesJ Clin Otolaryngol Head Neck Surg “ Weslley Rosario P Franco Mourao GRegina Calsolari M et al Role of adjuvanttherapy with radioactive iodine in patientswith elevated serum thyroglobulin afterneck reoperation due to recurrent papillarythyroid cancer a monoinstitutional comparative study Endocrine “ Zhang WJ Song B and Yang T MMP2MMP9 TIMP1 and TIMP2 in theperipheral blood of patients with differentiated thyroid carcinoma Cancer Manag Res “Wu R Luo Y Tang J et al Ultrasoundguidedradiofrequency ablation for papillary thyroidmicrocarcinomaa retrospective analysis of patients Int J Hyperthermia “ Zhao CK Hu HX Lu F et al Factors associated with initial incomplete ablation forbenign thyroid nodules after radiofrequencyablation First results of CEUS evaluationClin Hemorheol Microcirc “ Faggiano A Ramundo V Assanti AP et alThyroid nodules treated with percutaneousradiofrequency thermal ablation a comparative study J Clin Endocrinol Metab “ DOI 101210jc20122251 Tomayko EJ Cachia AJ Chung HR et alResveratrol supplementation reduces aorticatherosclerosis and calcification and attenuates loss of aerobic capacity in a mousemodel of uremia J Med Food “ DOI 101089jmf20120219 Xhaard C Rubino C Clero E et alMenstrual and reproductive factors in therisk of differentiated thyroid carcinoma inyoung women in France a populationbased casecontrol study Am J Epidemiol “ DOI 101093ajekwu220 Tafani M De Santis E Coppola L et alBridging hypoxia inflammation and estrogen receptors in thyroid cancer progressionBiomed Pharmacother “ DOI101016jbiopha201310013 Bumber B Marjanovic Kavanagh MJacovcevic A et al Role of matrix metalloproteinases and their inhibitors in the development of cervical metastases in papillary 0cJournal of International Medical Researchthyroid cancer Clin Otolaryngol “ofAssociation Gharib H Papini E Paschke R et alAmericanClinicalEndocrinologists Associazione MediciEndocrinologiand European ThyroidAssociation medical guidelines for clinicalpractice for the diagnosis and managementof thyroid nodules executive summary ofrecommendations J Endocrinol Invest “ Baek JH LeeJH Valcavi Ret alThermal ablation for benign thyroid nodules radiofrequency and laser Korean JRadiol “ DOI kjr2011125525 Vuong NL Dinh LQ Radiofrequency ablation for benign thyroid nodules 1yearfollowup in patients World J Surg “ Lang BHH Woo YC and Chiu KWIdentifying predictive factors for efficacy inhigh intensity focused ultrasound HIFUablation of benign thyroid nodules a retrospective analysis Int J Hyperthermia “ Buryk MA Simons JP Picarsic J et al Canmalignant thyroid nodules be distinguishedfrom benign thyroid nodules in children andadolescents by clinical characteristics Areview of pediatric patients with thyroidnodules Thyroid “ 0c'

Thyroid_Cancer

EpidemiologyEPIDEMIOLOGICAL SCIENCERisk factors for hospital admissions related to COVID19 in patients with autoimmune inflammatory rheumatic diseasesDalifer D Freites Nu±ez1 Leticia Leon Arkaitz Mucientes1 Luis Rodriguez Rodriguez Judit Font Urgelles3 Alfredo Madrid Garc­a1 Jose I Colomer1 Juan A Jover34 Benjam­n Fernandez Gutierrez3 Lydia Abasolo1Handling editor Josef S Smolen1Rheumatology Department and IDISSC La Fundacion para la Investigacion Biomedica del Hospital Clinico San Carlos Madrid Spain2Department of Health and Education Universidad Camilo Jose Cela Villafranca del Castillo Madrid Spain3Rheumatology Department Hospital Clinico San Carlos Madrid Spain4Medicine Department Universidad Complutense de Madrid Madrid Comunidad de Madrid SpainCorrespondence toDr Leticia Leon IdISSC and Rheumatology La Fundacion para la Investigacion Biomedica del Hospital Clinico San Carlos Madrid Spain lleon hcsc salud madrid Received May Revised July Accepted July Objectives To describe patients with autoimmune inflammatory rheumatic diseases AIRD who had COVID19 disease to compare patients who required hospital admission with those who did not and assess risk factors for hospital admission related to COVID19Methods An observational longitudinal study was conducted during the pandemic peak of severe acute respiratory syndrome coronavirus March to April All patients attended at the rheumatology outpatient clinic of a tertiary hospital in Madrid Spain with a medical diagnosis of AIRD and with symptomatic COVID19 were included The main outcome was hospital admission related to COVID19 The covariates were sociodemographic clinical and treatments We ran a multivariable logistic regression model to assess risk factors for the hospital admissionResults The study population included patients with AIRD and COVID19 Of these patients required hospital admission related to COVID19 The mean age on admission was years and the median time from onset of symptoms to hospital admission was “ days The median length of stay was “ days A total of patients died during admission Compared with outpatients the factors independently associated with hospital admission were older age OR p000 and autoimmune systemic condition vs chronic inflammatory arthritis OR p001 No statistically significant findings for exposure to disease modifying antirheumatic drugs were found in the final modelConclusion Our results suggest that age and having a systemic autoimmune condition increased the risk of hospital admission whereas disease modifying antirheumatic drugs were not associated with hospital admission Authors or their employers No commercial re use See rights and permissions Published by BMJTo cite Freites Nu±ez a0DD Leon a0L Mucientes a0A et a0al Ann Rheum Dis Epub ahead of print [please include Day Month Year] 101136annrheumdis2020217984INTRODUCTIONSevere acute respiratory syndrome coronavirus SARS CoV2 causes a myriad of clinical signs and symptoms together with typical laboratory abnormalities that manifest as the disease COVID191Since the confirmation of the first patient infected with SARS CoV2 in Spain in January the current COVID19 outbreak has had a considerable impact especially in the Madrid region where the highest incidence of COVID19 cases has been Key messagesWhat is already known about this subject –º The epidemiological scenario is changing daily There is little evidence for risk factors of poor outcome with COVID19 specific to autoimmune inflammatory rheumatic diseasesWhat does this study add –º Patients with an autoimmune systemic condition have a higher risk of hospital admission related to COVID19 compared with those with chronic inflammatory arthritis –º Disease modifying agents were not associated with a higher risk of hospital admission related to COVID19How might this impact on clinical practice or future developments –º Our data show that in a real world setting a high percentage of patients with autoimmune inflammatory rheumatic diseases and COVID19 required hospital admission The patients were mainly elderly with comorbidities and a systemic autoimmune conditionrecorded with more than patients admitted to the hospital until the first week of May2The incidence and severity of COVID19 disease seem to be higher in patients with risk factors such as advanced age and associated comorbidities mainly hypertension diabetes heart disease and previous respiratory diseases3 It is not clear whether patients with rheumatic diseases are more susceptible to SARS CoV2 infection or when they are infected whether they have more severe disease or a poorer outcome Previous outbreaks caused by coronaviruses did not yield overwhelming evidence that patients with rheumatic diseases are at an increased risk4 although some patients are candidates for a higher number of infections owing to their rheumatic disease predominantly systemic or the treatment they are receiving for rheumatic diseases5 Preliminary experiences in patients with COVID19 show that those with chronic arthritis treated with synthetic conventional or targeted syntheticbiologic disease modifying antirheumatic Freites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cEpidemiologydrugs DMARDs do not seem to be at a greater risk of respiratory or life threatening complications from SARS CoV2 than the general population6 The epidemiological scenario is changing and evidence on the risk factors of poor outcome with COVID19 specific to inflammatory rheumatic disease is scarce In addition there are little data on how the hospital admissions of these patients with severe COVID19 infection have evolved8The aim of our study was to describe patients with autoimmune inflammatory rheumatic diseases AIRD who had COVID19 during the pandemic peak We also explored possible risk factors associated with hospital admission related to COVID19 disease in patients with AIRD from a tertiary hospital in Madrid SpainMETHODSSetting study design and patientsThe study was performed in a public tertiary hospital Hospital Cl­nico San Carlos HCSC in Madrid Spain The catchment area is home to almost peopleWe performed a prospective observational study from March when our health area had the first hospital admission related to COVID19 to April We preselected all patients attended at the rheumatology outpatient clinic of our centre during the study period whose data were recorded in the electronic clinical history of our department HCR Penelope The inclusion criteria were age years a medical diagnosis according to International Classification of Diseases ICD10 of inflammatory rheumatic disease and symptomatic COVID19 disease assessed by medical diagnosis or confirmed with a positive SARS CoV2 PCR diagnostic testPatient data were obtained during routine clinical practice The study was conducted in accordance with the Declaration of Helsinki and the principles of Good Clinical Practice and was approved by the HCSC Ethics Committee approval number E BSVariablesThe primary outcome was admission to hospital with a medical diagnosis of COVID19 andor a positive PCR result between March and April compared with outpatients with symptomatic COVID19 diseaseThe covariables recorded were as follows sociodemographic baseline characteristics including sex age and rheumatic disease duration Type of AIRD including systemic autoimmune conditions polymyalgia rheumatica mixed connective tissue disease systemic sclerosis Sjogren™s syndrome vasculitis Raynaud phenomenon polymyositis polychondritis sarcoidosis antiphospholipid syndrome autoinflammatory syndromes and systemic lupus erythematosus and chronic inflammatory arthritis rheumatoid arthritis inflammatory polyarthritis juvenile idiopathic arthritis psoriatic arthritis axial spondyloarthritis uveitis and inflammatory bowel disease Baseline comorbid conditions including hypertension dyslipidaemia depression diabetes mellitus smoking habit kidney disease chronic liver disease respiratory diseases chronic obstructive pulmonary disease and interstitial lung disease thyroid disease heart disease valve disease arrythmias cardiomyopathy heart failure and pericarditis ischaemic vascular disease stroke cardiovascular and peripheral vascular disease venous thrombosislung embolism and cancer Treatment for inflammatory rheumatic disease a glucocorticoids b non steroidal anti inflammatory drugs NSAIDs c conventional synthetic disease modifying antirheumatic drugs csDMARDs antimalarials hydroxychloroquine and chloroquine azathioprine cyclophosphamide cyclosporine colchicine leflunomide methotrexate mycophenolate mofetilmycophenolic acid and sulfasalazine d targeted syntheticbiologic DMARDs tsbDMARDs including antitumour necrosis factor TNF alpha drugs infliximab adalimumab etanercept certolizumab and golimumab other biologics anti interleukin IL6 tocilizumab and sarilumab rituximab abatacept belimumab anti IL1723 anti IL17 ustekinumab ixekizumab and secukinumab Janus kinase JAK inhibitors tofacitinib and baricitinibTreatment had to start at least month before the beginning of the study and continue during the study period until the end of the study or hospital admission for antimalarial therapy glucocorticoids sulfasalazine NSAIDs or colchicine Regarding csDMARDs and tsbDMARDs treatment had to start at least month before the beginning of the study and continue until at least 21st March the end of the study or hospital admission In the case of rituximab the last infusion had to be at least in JanuaryData sourcesPatient sociodemographic clinical laboratory and data on treatment of rheumatic disease were obtained through HCR PenelopePatients with COVID19 were detected by warning calls to our rheumatologists or nurses or via routine telephone consultation Other infected patients were detected through their sick leave forms for COVID19 The results of SARS CoV2 PCR diagnostic assays were obtained from the microbiologyinfectious service of HCSC In addition our Hospital Central Services registered all medical admissions to HCSC This information was provided from March to AprilThe researchers carried out an exhaustive review of the clinical histories of admitted patients to identify COVID19 cases and rule out patients admitted for other reasons Once the COVID19 cases were identified we collected clinical laboratory and treatment data during admission until the end of admission either discharge or death in order to describe the progress of the disease The review was performed until 24th April in order to include follow up data from patients admitted to the hospital with COVID19Statistical analysisPatient characteristics are expressed as mean and SD or median and IQR for continuous variables categorical variables are expressed as percentages Statistical tests were performed to compare characteristics between patients admitted with COVID19 and those without hospital admissions Continuous variables were analysed using the Mann Whitney test or t test and discrete variables were analysed using the χ2 or Fisher exact test Univariable logistic regression analyses were performed to assess differences between hospital admissions related to COVID19 risk and covariates Multivariable logistic regression models adjusted for age sex and comorbidity were run in a stepwise manner to examine the possible effect of sociodemographic clinical and therapeutic factors on hospital admissions related to COVID19 The model also included csDMARDs and all other variables with a p02 from the simple regression analysis The results were expressed as the OR with its respective CIAll analyses were performed in Stata V13 statistical software Stata Corp A two tailed p value was considered to indicate statistical significanceFreites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cRESULTSA total of patients with AIRD with symptomatic COVID19 disease were included in the study table The tests were performed as an exploratory measure of the association between a variable and the outcomeMost of the patients were women with a mean age of years and a mean time since diagnosis of years The main diagnosis was rheumatoid arthritis followed by axial spondyloarthritis Many patients had at least one baseline comorbid condition the most prevalent being hypertension dyslipidaemia and lung disease Most patients were taking csDMARDs Half of the patients were taking glucocorticoids a quarter were taking NSAIDs and were taking tsbDMARDs of which adalimumab was the most frequently prescribed followed by rituximab Only one patient was taking a JAK inhibitor Interestingly of the patients taking tsbDMARDs were taking the drug in combination with a csDMARDA total of patients had to be admitted to the hospital because of COVID19 Of these were evaluated in the HCSC Emergency Department were admitted to HCSC and were transferred to the Institucion Ferial de Madrid IFEMA support hospital owing to the lack of capacity in our hospital at that time The remaining three patients were evaluated and admitted to other hospitals in the Autonomous Community of Madrid Table presents data for the patients admitted to HCSCOf the patients admitted to our hospital were women with a mean age at admission of years and median lag time from the onset of symptoms to the admission of “ days The median length of stay was “ days table At admission the median haemoglobin was “ gdL and the median total lymphocyte count was “ ngmL The median D dimer value was “ ngmL In of patients median interleukin IL6 levels were “ pgmL Patients received various antibiotics mainly azithromycin levofloxacin and third generation cephalosporinsMost patients were treated with hydroxychloroquine during admission About half received glucocorticoids Eighteen were treated with lopinavirritonavir and received the anti IL 6R antibody tocilizumab table 2FEDERA total of patients developed relevant complications during admission the most frequent being myocarditis thrombosis and kidney failure Only two patients were admitted to the intensive care unit during admission The first was a patient in 50s with mixed connective tissue disease and associated comorbidities who developed acute respiratory insufficiency and bilateral pneumonia The patient was treated with antibiotic therapy lopinavirritonavir hydroxychloroquine and βinterferon Finally the patient was extubated days later and is recovering The other was a young adult patient with systemic lupus erythematosus treated with methotrexate rituximab hydroxychloroquine and glucocorticoids who days after being diagnosed with COVID19 PCR developed an erythematous rash and generalised urticaria requiring hospitalisation in the intensive care unit owing to general clinical and laboratory worsening elevated D dimer values The patient was treated with methylprednisolone heparin and a cephalosporin A few days later the patient™s condition improved and he recovered completely at dischargeOf the patients admitted to HCSC were sent to another care centre converted hotel hospitalIFEMA support hospital when their condition improved A further patients Epidemiologywere discharged home to continue self isolation after improvement At the end of the study five patients remained in hospital A total of patients died during admission men and women with a median age of “ years Of the patients who died had relevant comorbidity diabetes mellitus pulmonary disease ischaemic vascular disease hypertension venous thrombosislung embolism lung disease and or liver disease The main diagnoses were rheumatoid arthritis followed by spondyloarthritis polymyalgia rheumatica vasculitis and Sjogren™s syndrome The results of the univariable analysis are shown in table Older age systemic autoimmune conditions vs chronic inflammatory arthritis OR CI “ p0014 hypertension diabetes mellitus lung disease heart disease and glucocorticoids were associated with statistically significant greater risk of admission to the hospital Female sex NSAIDs and anti TNF drugs vs non use were associated with a statistically significant lower risk The differences reported for the remaining variables did not reach statistical significanceThe multivariable analysis was adjusted for gender age and comorbidities related to COVID19 These comorbidities were diabetes mellitus pulmonary disease ischaemic vascular disease hypertension venous thrombosislung embolism lung disease andor liver disease table Age and systemic autoimmune conditions had more probability of hospital admissions regardless of other factors Differences in exposure to glucocorticoids were not statistically significant The type of exposed DMARDs did not reach statistical significance in the multivariate model In fact long term treatment with antimalarials OR CI “ p066 other csDMARDs including methotrexate leflunomide and azathioprine OR CI “ p09 and NSAIDs OR CI “ p05 dropped from the final model The variable tsbDMARDs was also eliminated from the final model anti TNF vs none OR CI “ p016 and non anti TNF vs none OR CI “ p03DISCUSSIONOur study aims to shed light on rheumatologists™ concerns regarding their patients We found that in a real world setting of patients with AIRD and COVID19 required hospital admission These were mainly elderly patients with more comorbidities and systemic autoimmune conditions Our data show that patients exposed to disease modifying agents do not seem to be at higher risk of hospital admission related to COVID19Of the patients included in the study with COVID19 required hospital admission Comparison of the characteristics of patients admitted to hospital because of COVID19 and those who did not require hospital admission were as follows admitted patients had a median age close to years that is more than years older than patients who were not admitted Moreover those who were admitted more frequently had baseline comorbidities and systemic autoimmune conditions As for therapy admitted patients were less frequently exposed to antimalarial and anti TNF alpha agentsThe median lag time from onset of symptoms to admission was days and almost of patients had pneumonia at admission The baseline laboratory results for admitted patients in our study are consistent with those published elsewhere9“ and are characterised by lymphopenia and elevated acute phase reactants In fact of the patients had elevated D dimers normal and elevated IL6 normal pgmL Treatment during admission varied widely as the disease proved Freites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cEpidemiologyTable Baseline demographic and clinical characteristics of patients with AIRD and with COVID19 admitted vs no admitted at the hospitalAIRD“COVID19 patientsAIRD“COVID non admitted patientsAIRD“COVID admitted patientsVariableN123N69N54P value Positive Negative Not performed Women n Age years mean SDTime since diagnosis years mean SDPCR test n Smoking habit active vs noneDiagnosis AIRD n Rheumatoid arthritis Axial spondyloarthritis Polymyalgia rheumatica Psoriatic arthritis Systemic lupus erythematosus Mixed connective tissue disease Sjogren™s syndrome Vasculitis Uveitis Systemic sclerosis Inflammatory polyarthritis Polychondritis Polymyositis Raynaud phenomenon OtherComorbidities n NSAIDs n Glucocorticoids n csDMARDs n TsbDMARDs n JAKi n Others inflammatory bowel disease antiphospholipid syndrome juvenile idiopathic arthritis autoinflammatory syndromes and sarcoidosis Heart disease arrhythmiasvalve disease cardiomyopathy and heart failure Ischaemic vascular disease stroke cardiovascular and peripheral vascular diseaseAIRD autoimmune inflammatory rheumatic disease Anti TNF tumour necrosis factor alpha COPD chronic obstructive pulmonary disease csDMARD conventional synthetic disease modifying antirheumatic drug ILD interstitial lung disease JAKi JAK inhibitor tsbDMARDs target syntheticbiologic disease modifying antirheumatic drug Hypertension Dyslipidaemia Depression Diabetes mellitus Heart disease Vascular disease Liver disease Kidney disease Lung disease ILDCOPD Cancer Venous thrombosislung embolism Thyroid disease Anti TNF alpha agent Other biologics Abatacept Tocilizumab Belimumab Rituximab Methotrexate“leflunomide“azathioprine Sulfasalazine AntimalarialsFreites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cTable Hospital admissions related to COVID19 among patients with AIRDVariableValueTable OR of hospital admission related to COVID19 in patients with AIRD univariable analysisVariable CIORPEpidemiology Haemoglobin gdL D dimer ngmL Neutrophil count —109L Lymphocyte count —109L CRP mgdL LDH UL Platelet count —109L Creatinine mgdL Ferritine ngmLAdmissions nLag time from onset of symptoms to admission days median IQRPneumonia at admission n Systemic autoimmune conditions n Laboratory data at admission median IQR COVID19 related treatments during admission n Admitted by intensive care unit during hospital admission Length of stay days median IQRDischarge reason n Azithromycin Other antibiotics Glucocorticoids Lopinavirritonavir Remdesivir Darunavircobicistat Tocilizumab Interferon HCQ Immunoglobulin Improvement home isolation Other care centre medicalised hotelIFEMA hospital Death End of study no discharge No Yes “ Data for patients patients were treated in other support centres after referral or admission in other centresCRP C reactive protein HCQ hydroxychloroquine LDH lactate dehydrogenase challenging for specialists who prescribed various combinations of drugs based on little published evidence In this sense the anti IL 6R antibody tocilizumab has proven to be beneficial in patients with COVID1912 Treatment may also be successful in the early stages of cytokine release syndrome if it can effectively block the signal transduction pathway of IL6 therefore tocilizumab and sarilumab are likely to emerge as effective drugs for patients with moderate to severe COVID1913 In our study almost of the patients were treated with tocilizumabThe patients who eventually died had a median age of years This finding is in line with data for the general population where over of deaths occurred in persons years and more than of all deaths were in people aged ‰¥ years7The multivariable regression model showed that only age increasing by per year and systemic autoimmune conditions continued to be risk factors for hospital admission related to COVID19 “ “ “ “ “ “ “ “ “ ““““““““““““ Rheumatoid arthritis Inflammatory polyarthritis Systemic lupus erythematosus Psoriatic arthritis Spondyloarthritis MTCD Sjogren syndrome Hypertension Dyslipidaemia Depression Diabetes mellitus Heart disease Vascular disease Liver disease Kidney disease Lung disease ILDCOPD Cancer Venous thrombosislung embolism Thyroid diseaseGender womenAge yearsDiagnosis AIRD one category vs the rest Disease durationSmoking habit active vs noneComorbidities yes NSAIDsGlucocorticoidscsDMARDSs TsbDMARDs JAKisOther biologics anti IL6 tocilizumab sarilumab rituximab Rtx anti IL1723 anti IL17Othercategories could not be represented polymalgia rheumatica systemicsclerosis vasculitis Raynaud phenomenon polychondritis Beh§et diseasepolymyositis uveitis inflammatory boweldisease antiphospholipid syndrome juvenile idiopathic arthritis autoinflammatorysyndromes and sarcoidosisAIRD autoimmune inflammatory rheumatic disease anti TNF tumour necrosis factor csDMARD Conventional synthetic disease modifying antirheumatic drug IL6 interleukin6 JAKi JAK inhibitors tsbDMARDs target syntheticbiologic disease modifying antirheumatic drug““““““““““““ ““ Methotrexate“leflunomide“azathioprine Sulfasalazine Antimalarial agents““““““““““ None Anti TNF agents Other biologics““As for the association between sex and risk of hospital admission we did not find a higher risk of admission in women despite the fact that rheumatic diseases are more prevalent in this group The type of diagnosis seems to play an important role in the probability of hospital admission and patients with systemic autoimmune conditions seem to have the highest risk compared with chronic inflammatory arthritisAs it has been reported elsewhere comorbidities play an important role in the risk of hospital admission15 Clinical outcomes are poorer in patients with COVID19 with a comorbid condition than in those without and a greater number of comorbidities correlates with poorer clinical outcomes16 Diabetes is a major comorbidity in COVID19 and patient™s history of diabetes is an independent risk factor for morbidity and mortality in this condition17 Diabetes has been associated with admissions to Freites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cEpidemiologyTable Multivariable analysis risk factors for hospital admission related to COVID19 in patients with AIRDVariableOR CIP value“““““Gender womenAge yearsAIRD systemic autoimmune conditions vs chronic inflammatory arthritisCOVID comorbidities yesGlucocorticoidsSystemic autoimmune conditions polymyalgia rheumatica mixed connective tissue disease systemic sclerosis Sjogren™s syndrome vasculitis Raynaud polymyositis polychondritis sarcoidosis antiphospholipid syndrome autoinflammatory syndromes and systemic lupus erythematosus vs chronic inflammatory arthritis rheumatoid arthritis inflammatory polyarthritis juvenile idiopathic arthritis psoriatic arthritis axial spondyloarthritis uvetis and inflammatory bowel disease Comorbidities including the presence of at least one of the follows hypertension heart disease vascular disease diabetes mellitus venous thrombosislung embolism chronic kidney disease liver disease and lung disease ILDCOPDAIRD autoimmune inflammatory rheumatic disease COPD chronic obstructive pulmonary disease ILD interstitial lung diseasethe intensive care unit due to COVID19 in recent series19 and has been shown to increase mortality6 Therefore we adjusted for comorbidity in the multivariable analysisTreatment with glucocorticoids lost its statistical significance in the multiple regression model However the dose was not reported in our data and in the case of these agents the risk could be dose dependent In a recent publication from a European registry the authors found that exposure to mgday was associated with a greater probability of hospitalisation21The exposure to DMARDs regardless of whether they were biological or synthetic does not seem to be associated with a higher hospital admission related to COVID19 Although we have to consider the limited number of patients in our study our results are in concordance with data reported elsewhere8 Our results should be interpreted taking into account other limitations First patients were included from a single centre Second of all the patients with COVID19 who did not require admission one third contacted the rheumatology service to report the disease and the remainder were detected through the COVID19 discharge reports sent to their primary care physician Elderly persons and homemakers who did not contact us can be considered missing Consequently there may be some selection bias between those admitted and those not admitted although this problem was addressed by adjusting for confounders in the multivariable analysis Third while it is acknowledged that clinical suspicion must be confirmed by PCR assay almost of patients admitted did not undergo PCR owing to the lack of tests or the extreme healthcare overload Nevertheless all cases included were clinically compatible and managed as COVID19The key strength of our study is that it was performed in real life conditions during then pandemic peak with access to complete sociodemographic and clinical data from our rheumatology electronic clinical history including thorough hospital admission data such as laboratory abnormalities and COVID19 treatment data from the hospital computer services Consequently this has allowed us to analyse the risk of hospital admission related to COVID19 adjusted for confounders thus avoiding possible biasAlthough we are unable to modify the factors reported here knowing them can help rheumatologists to treat and advise their patients during this new and challenging period Results provided by our study are preliminary and should be corroborated with other real life studies but we consider our findings helpful to increase the knowledge in the management of patients with AIRD and COVID19Twitter Benjam­n Fernandez Gutierrez Fergutbe2001Acknowledgements The authors would like to thank Ana M Perez for her help with data collection They would like to say a special thanks to all the rheumatologists and nurses who contributed to the care of the patients in such an innovative and conscientious wayContributors BF G LL JAJ LR R and LA conceived and designed the study DDFN JFU AMG JIC and LL collected data LA and LL performed the data analysis and interpreted the data All of the authors were involved in the drafting andor revising of the manuscriptFunding This work was supported by the Instituto de Salud Carlos III ISCIII Ministry of Health Spain CP1600916 PI1801188 and RD1600120014 and cofunded by el Fondo Europeo de Desarrollo Regional FEDER The funders had no role in study design data collection analysis manuscript preparation or decision to publishCompeting interests None declaredPatient and public involvement Patients andor the public were not involved in the design or conduct or reporting or dissemination plans of this researchPatient consent for publication Not requiredEthics approval The study was approved by the Hospital Cl­nico San Carlos institutional ethics committee approval number E BS This study was conducted according to the principles of the Declaration of HelsinkiProvenance and peer review Not commissioned externally peer reviewedData availability statement Data are available upon reasonable requestThis article is made freely available for use in accordance with BMJ™s website terms and conditions for the duration of the covid19 pandemic or until otherwise determined by BMJ You may use download and print the article for any lawful non commercial purpose including text and data mining provided that all copyright notices and trade marks are retainedORCID iDsLeticia a0Leon http orcid Luis a0Rodriguez Rodriguez http orcid REFERENCES Fernandez Gutierrez B COVID19 with pulmonary involvement An autoimmune disease of known cause Reumatol Clin “ COVID19 Situaci³n actual en La Comunidad de Madrid Informe de situaci³n del de Mayo Available httpswww comunidad madrid sites default files doc sanidad 200508_ cam_ covid19 pdf [Accessed May ] Chen N Zhou M Dong X et a0al Epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in Wuhan China a descriptive study Lancet “ Figueroa Parra G Aguirre Garcia GM Gamboa Alonso CM et a0al Are my patients with rheumatic diseases at higher risk of COVID19 Ann Rheum Dis “ Favalli EG Ingegnoli F De Lucia O et a0al COVID19 infection and rheumatoid arthritis Faraway so close Autoimmun Rev Zhou F Yu T Du R et a0al Clinical course and risk factors for mortality of adult inpatients with COVID19 in Wuhan China a retrospective cohort study Lancet “ Monti S Balduzzi S Delvino P et a0al Clinical course of COVID19 in a series of patients with chronic art

Thyroid_Cancer

"conceptualization data curation investigation project administration supervision validation visualization writing review and editing Goneim I and Ibraheim A performed data curation Kamal NM was involved in literature review writingoriginal draft writing review and editing Alsofiani F and Alawur A performed literature review and writingoriginal draft All authors had read and approved the final manuscriptInformed consent statement Written informed consent in the patient™s native language was obtained from her fatherConflictofinterest statement The authors declare that they have no conflict of interestCARE Checklist statement The authors have read the CARE Checklist and the manuscript was prepared and revised according to the CARE Laila M Sherief Department of Pediatric Hematology and Oncology Faculty of Medicine Zagazig University Zagazig EgyptLaila M Sherief Amr Ibraheim Department of Pediatrics Faculty of Medicine Zagazig University Zagazig EgyptEsmael Goneim Department of Pediatric Oncology Tanta Cancer Institute Tanta EgyptNaglaa M Kamal Department of Pediatrics and Pediatric Hepatology Faculty of Medicine Cairo University Cairo EgyptNaglaa M Kamal Fuad A alsofiani Abdulraouf H Alawur Department of Pediatrics Alhada Armed Forces Hospital Taif Saudi ArabiaCorresponding author Naglaa M Kamal MD Full Professor Department of Pediatrics and Pediatric Hepatology Faculty of Medicine Cairo University Kasralainy Cairo Egypt naglakamalkasralainyeduegAbstractBACKGROUND thalassemia intermedia TI is one of the hemoglobinopathies It constitutes of thalassemia cases yet being associated with a better quality of life than thalassemia major TMCASE SUMMARY We recently reported the first case of acute lymphoblastic leukemia ALL from Egypt in a child with TM and we herein report the first case of ALL from Egypt in a child with TI In this report literature was reviewed for cases of malignancies associated with TI and the possible factors underling the relationship between the two entities We stress that physicians should have a high index of suspicion of malignancies in thalassemia patients if they present with any suggestive symptoms or signsKey words Acute lymphoblastic leukemia Thalassemia intermedia Children Malignancies Iron overload Hydroxyurea Case reportThe Authors Published by Baishideng Publishing Group Inc All rights reservedWJCPwwwwjgnetcomAugust Volume Issue 0cSherief LM ALL in a child with TIChecklist Access This is an access that was selected by an inhouse editor and fully peerreviewed by external reviewers It is distributed in accordance with the Creative Commons Attribution NonCommercial CC BYNC license which permits others to distribute remix adapt build upon this work noncommercially and license their derivative works on different terms provided the original work is properly cited and the use is noncommercial See httpcreativecommonslicensesbync40Manuscript source Unsolicited manuscriptReceived January Peerreview started January First decision April Revised May Accepted June in press June Published online August PReviewer Fujioka K Moschovi MA SEditor Dou Y LEditor A EEditor Li JHCore tip Cases have been reported for malignancies in patients of thalassemia major However rare case reports have been reported for malignancies in patients of thalassemiaintermedia as it is a nontransfusion dependent anemia Physicians should have high index of suspicion to diagnose malignancies in patients with thalassemiaintermediaCitation Sherief LM Goneim E Kamal NM Ibraheim A Alsofiani F Alawur A Acute lymphoblastic leukemia in a thalassemia intermedia child A case report World J Clin Pediatr URL wwwwjgnetcom22192808fullv9i11htm dx105409wjcpv9i11INTRODUCTIONThalassemia represents the most common singlegene disorder worldwide The total annual incidence of symptomatic individuals with thalassemia is estimated at in throughout the world of whom nearly have thalassemia intermedia TI which is intermediate in severity between the milder thalassemiaminor and the more severe transfusiondependent thalassemiamajor TM[]We herein report the first case from Egypt with TI who developed acute lymphoblastic leukemia ALLCASE PRESENTATIONChief complaintsThe reported patient is a 15yearold girl with TI who presented at the age of years with pallor decreased growth rate and decreased activity She had severe microcytic hypochromic anemia with hemoglobin Hb of gdLHistory of present illnessPediatric hematologist workup proved the diagnosis of TI Her Hb electrophoresis showed HbA HbF and HbA2 Genetic molecular testing revealed compound heterozygosity for cd27 GT and cd39 CT mutations Hydroxyurea at a dose of mgkg per day was started in addition to folic acidShe was then followed at the pediatric hematology unit at regular intervals to monitor her tolerance to drug therapy with special attention to hematological toxicity There were no significant side effects during seven years of therapy and the patient showed good response with occasional need for blood transfusions She underwent splenectomy during her late teensHistory of past illnessAt the age of years she developed generalized bone aches abdominal pain persistent fever and dyspnea and so she was referred to our hospitalPhysical examinationOn physical examination there was severe pallor tachypnea tachycardia and hepatomegalyLaboratory examinationsInitial complete blood picture showed a Hb level gdL white blood cell count of — 109L and platelets count of — 109LSerum electrolytes cerebrospinal fluid analysis and kidney and liver function tests were normal expect for mild elevation of total serum bilirubin which was mgdLSerum ferritin was ngdL Serological studies including EpsteinBarr virus cytomegalovirus human immunodeficiency virus hepatitis C virus and hepatitis B virus were negative Lactate dehydrogenase was UL and serum uric acid was mgdLWJCPwwwwjgnetcomAugust Volume Issue 0cSherief LM ALL in a child with TIImaging examinationsHer chest Xray was normal Abdominal ultrasonography revealed hepatomegaly with calcular cholecystitis and bilateral diffuse renal enlargement Echocardiography showed mitral valve prolapse with trivial mitral regurgitationMULTIDISCIPLINARY EXPERT CONSULTATIONThe pediatric haematologistoncologist assessment requested bone marrow biopsy which was carried by the hematopathologistBone marrow examination revealed blast cells in a hypercellular marrow with depressed erythropoiesis and granulopoiesies and normal thrombopoiesis Immunophenotyping showed lymphoblasts that are CD10 positive CD19 positive CD34 positive TDT positive HLADR positive CD13 positive and CD33 positiveCytogenetic examination showed a normal karyotype with a DNA index of and negative t1221 t119 BCRABL or 11q23 translocationsmutationsFINAL DIAGNOSISA final diagnosis of Bacutelymphoblasticleukemia ALL with aberrant expression of CD13 and CD33 was achievedTREATMENTInduction chemotherapy of the total XV protocol with prednisone vincristine Lasparaginase doxorubicin cyclophosamide cytarabine 6mercaptopurine and intrathecal chemotherapy was commencedShe received multiple packed red cell transfusions which eventually led to elevation of serum ferritin to ngdL Thus she was started on oral chelation therapy with deferasirox with no complicationsThe patient eventually went into complete remission She then received consolidation chemotherapy of standard risk of the total XV protocol with times of high dose methotrexate HDMTX 6mercaptopurine and intrathecal chemotherapyShe received multiple packed red blood cell transfusions and other supportive measures during the periods of induction and consolidation The transfusions therapy was given according to the guidelines of pediatric oncologists who usually transfuse if Hb level is less than gdL and if associated with pulmonary or cardiac comorbidities or exposed to invasive procedure and hemorrhage and they transfuse with Hb less than gdL The transfusions were not associated with any complications Deferasirox was stopped in consolidation phase during infusion of high dose methotrexateThe main problem observed during the periods of induction and consolidation therapy was increased requirement of blood transfusions as well as repeated infections as during this period the child received intensive chemotherapy which caused bone marrow suppressionOUTCOME AND FOLLOWUPThe child is still in complete remission while being now in the continuation phase for standard risk week fortyDISCUSSIONWe have recently reported the first case from Egypt with thalassemia major TM who developed ALL[] herein we report similarly the first report from Egypt for a patient with TI who developed ALL to highlight that the coexistence of malignancy and beta thalassemia is not rareA thorough look in literature for previously reported cases of malignancies in WJCPwwwwjgnetcomAugust Volume Issue 0cSherief LM ALL in a child with TIpatients with TI revealed only one report in from Turkey on a years old boy with TI who developed ALL[] Other previous reports on malignancies associated with TI described nonHodjkinlymphoma[] chronic myeloid leukemia[] Hodjkin lymphoma[] hepatocellular carcinoma[] and thyroid malignancies[] Table To our knowledge our patient is the second worldwide and the first from EgyptAlthough reported cases of malignancies associated with TI are few but it raises the attention of physicians to have high index of suspicion of malignancies in this group of patients when they present with unexplained new symptoms or proposed symptoms and signs of malignancySpecial concern about management plans in these patients as they usually require more frequent blood transfusions as the chemotherapy causes suppression of the bone marrow which adds to the base line chronic hemolysisIn spite that reported cases of malignancies in TI are scarce which makes our trial to find causal relationship between TI and cancer development beyond the scope of our report but we tried to search literature foe possible contributing factors Those factors can™t rise to the level of conclusions and definitely need to be proved and validated by larger prospective cohort with large control groups multicenter worldwide studies addressing all possible hypothesesIndeed the most practical logical thinking about that underlying factors for the development of malignancy in TI is being multifactorial[]In a large multicenter study on thalassemia patients from Iran the proportion of patients with cancer was higher in those with TI than those with TM and respectively[] They explained it by the fact that bone marrow in TM patients is suppressed by the regular transfusions while it is very active with high turnover in those with TI[] They suggested that this can lead to a higher rate of DNA repair faults and mutations with subsequent higher rate of hematological malignancies[]Another potential factor is the prolonged use of hydroxyurea Conflicting data are there regarding its carcinogenic potential Hydroxyurea as an antimetabolite interferes with both DNA synthesis and repair mechanisms with later accumulation of mutations and subsequent chromosomal damage Although no studies have yet investigated the relationship between hydroxyurea and the development of cancers in thalassemia but clinically concerns have been raised regarding its potential leukemogenic potential[] Other authors were against this assumption[] The BABYHUG clinical trial which compared hydroxyurea with placebo treated controls refuted this assumption and did not suggest any increased risk of genotoxicity[]Overall there is no evidence to suggest an increased risk of carcinogenesis in patients with thalassemia with hydroxyurea and further studies will need to be designed to establish any potential relationship[]One more probable factor is that patients with TI being having milder disease than those with TM with fewer blood transfusions might lead to delayed diagnosis and even if diagnosed usually there is underestimation of their iron overload problem and sometimes the deceiving relatively mildly elevated ferritin as compared to TM which has been shown to underestimate the true iron burden in TI patient with ultimate fate that these patients accumulate iron but it usually goes unnoticed unchelated and unmonitored Anemia hypoxia and ineffective erythropoiesis suppress the expression of hepcidin by increasing expression of growth differentiation factor and hypoxiainducible transcription factors with the resultant increased intestinal iron absorption and in turn adds to the problem of iron overload[]The longstanding iron overload with its deposition in different body ans with the wellknown association between excess iron and cancer development can be a predisposing factor for all types of malignancies through direct and indirect effects[]Iron can directly damage DNA by nontransferrinbound iron with the consequent inactivation of tumorsuppressor genes such as p53 or their products The indirect effects include the formation of reactive oxygen species ironinduced lipid peroxidation and altered immune system with decreased immune surveillance suppression of tumoricidal action of macrophages and alteration of cytokine activities TI patients usually survive longer than TM patients with enough time for iron overload to develop[]Some authors suggested that improved management protocols of thalassemia patients have led to increased survival with most of them reaching adult age with the consequent occurrence of diseases associated with long life span like malignancies[] This assumption can partially explain other reports in elder patients but it can™t work in our patient and the Turkish one who are teenagersMany authors suggested that the occurrence of malignancies in thalassemia patients could be a pure coincidence or a combination of genetic and environmental factors[]WJCPwwwwjgnetcomAugust Volume Issue 0cTable Previously reported cases of thalassemia intermedia who developed malignanciesSherief LM ALL in a child with TINumber of patientsType of malignancyAcute lymphoblastic leukemiaNonHodgkin lymphoma NHLNHL Hodgkin lymphoma HLNHL HL chronic myeloid leukemia CMLCMLHLHepatocellular carcinoma HCCHCCHCCHCCHCCThyroid cancerSome patients have thalassemia major and others have thalassemia intermedia in references and Ref[][][][][][][][][][][][]We can sum up to a clear message that whatever the pathogenesis of malignancies in thalassemias the most important message is to alarm physicians to have high index of suspicion for malignancies if their thalassemia patients develop suggestive symptoms and signs Worsening anemia leukocytosis fever boneache lymphadenopathy and splenomegaly are alarming to look for leukemias and other hematological malignanciesREFERENCES Galanello R Origa R Betathalassemia Orphanet J Rare Dis [PMID ]Sherief LM Kamal NM Abdelrahman HM Hassan BA Zakaria MM First report of acute lymphoblastic leukemia in an Egyptian child with thalassemia major Hemoglobin [PMID ]Tuğcu D Karakaş Z G¶k§e M Ağaoğlu L Un¼var A Sarıbeyoğlu E Ak§ay A Devecioğlu O Thalassemia Intermedia and Acute Lymphoblastic Leukemia Is it a Coincidental Double Diagnosis Turk J Haematol [PMID 104274tjh20140068]Chehal A Loutfi R Taher A Betathalassemia intermedia and nonHodgkin's lymphoma Hemoglobin [PMID 101081hem120015025]Benetatos L Alymara V Vassou A Bourantas KL Malignancies in betathalassemia patients a singlecenter experience and a concise review of the literature Int J Lab Hematol [PMID 101111j1751553X200700929x]Karimi M Giti R Haghpanah S Azarkeivan A Hoofar H Eslami M Malignancies in patients with betathalassemia major and betathalassemia intermedia a multicenter study in Iran Pediatr Blood Cancer [PMID 101002pbc22144]Alavi S Safari A Sadeghi E Amiri S Hematological malignancies complicating thalassemia syndromes a single center experience Blood Res [PMID 105045br2013482149]Jabr FI Aoun E Yassine H Azar C Taher A Betathalassemia intermedia and Hodgkin lymphoma Am J Hematol [PMID 101002ajh20478]BnaPignatti C Vergine G Lombardo T Cappellini MD Cianciulli P Maggio A Renda D Lai ME Mandas A Forni G Piga A Bisconte MG Hepatocellular carcinoma in the thalassaemia syndromes Br J Haematol [PMID 101046j13652141200304732x]Mancuso A Sciarrino E Renda MC Maggio A A prospective study of hepatocellular carcinoma incidence in thalassemia Hemoglobin [PMID ]Restivo Pantalone G Renda D Valenza F D'Amato F Vitrano A Cassar  F Rigano P Di Salvo V Giangreco A Bevacqua E Maggio A Hepatocellular carcinoma in patients with thalassaemia syndromes clinical characteristics and outcome in a long term single centre experience Br J Haematol [PMID 101111j13652141201008180x]Fragatou S Tsourveloudis I Manesis G Incidence of hepatocellular carcinoma in a thalassemia unit Hemoglobin [PMID ] WJCPwwwwjgnetcomAugust Volume Issue 0cSherief LM ALL in a child with TI Maakaron JE Cappellini MD Graziadei G Ayache JB Taher AT Hepatocellular carcinoma in hepatitisnegative patients with thalassemia intermedia a closer look at the role of siderosis Ann Hepatol [PMID ]Poggi M Sorrentino F Pascucci C Monti S Lauri C Bisogni V Toscano V Cianciulli P Malignancies in thalassemia patients first description of two cases of thyroid cancer and review of the literature Hemoglobin [PMID ]Halawi R Cappellini MD Taher A A higher prevalence of hematologic malignancies in patients with thalassemia Background and culprits Am J Hematol [PMID 101002ajh24682]McGann PT Flanagan JM Howard TA Dertinger SD He J Kulharya AS Thompson BW Ware RE BABY HUG Investigators Genotoxicity associated with hydroxyurea exposure in infants with sickle cell anemia results from the BABYHUG Phase III Clinical Trial Pediatr Blood Cancer [PMID 101002pbc23365] WJCPwwwwjgnetcomAugust Volume Issue 0cPublished by Baishideng Publishing Group Inc Koll Center Parkway Suite Pleasanton CA USA Telephone Email bpgofficewjgnetcom Help Desk wwwf6publishingcomhelpdesk wwwwjgnetcom Baishideng Publishing Group Inc All rights reserved\x0c"

Thyroid_Cancer

ORIGINAL Clinical Translational Immunology e1165 101002cti21165wwwwileyonlinelibrarycomjournalcti Elly Marcq1 Bianca von Scheidt2 Ashleigh S Davey2Novel combination immunotherapy for pancreatic cancerpotent antitumor effects with CD40 agonist andinterleukin15 treatmentJonas RM Van Audenaerde12Amanda J Oliver2 Jorrit De Waele1 Delphine Quatannens1 Jinthe Van Loenhout1Patrick Pauwels13 Geert Roeyen4 Filip Lardon1 Clare Y Slaney25Michael H Kershaw25  Phillip K Darcy25  Evelien LJM Smits17 1Center for Oncological Research CORE Integrated Personalized Precision Oncology Network IPPON University of AntwerpWilrijk Belgium2Cancer Immunotherapy and Immune Innovation Laboratory Peter MacCallum Cancer Centre Melbourne VIC Australia3Department of Pathology Antwerp University Hospital Edegem Belgium4Department of Hepatobiliary Endocrine and Transplantation Surgery Antwerp University Hospital Edegem Belgium5Sir Peter MacCallum Department of Oncology The University of Melbourne Parkville VIC Australia6Department of Oncology and Multidisciplinary Oncological Centre Antwerp Antwerp University Hospital Edegem Belgium7Center for Cell Therapy and Regenerative Medicine Antwerp University Hospital Edegem Belgium Marc Peeters16CorrespondenceELJM Smits Center for Oncological ResearchCORE Integrated Personalized PrecisionOncology Network IPPON University ofAntwerp CDE T431 Universiteitsplein Wilrijk BelgiumEmail eveliensmitsuantwerpenbe Contributed equally as senior authorsReceived May Revised July Accepted July 101002cti21165Clinical Translational Immunology e1165AbstractObjectives With the poorest 5yearsurvival of all cancersimproving treatment for pancreatic cancer is one of the biggestchallenges in cancer research We sought to explore the potentialof combining both priming and activation of the immune systemTo achieve this we combined a CD40 agonist with interleukin15and tested its potential in pancreatic cancer Methods Responseto this combination regimen was assessed in pancreatic ductaladenocarcinoma mouse models and a thorough analysis of thetumor microenvironment was performed Results We demonstratedprofound reduction in tumor growth and increased survival ofmice with the majority of mice being cured when both agentswere combined including an unprecedented 8fold dose reductionof CD40 agonist without losing any efficacy RNAseq analysisshowed involvement of natural killer NK cell and Tcellmediatedantitumor responses and the importance of antigenpresentingcell pathways This combination resulted in enhanced ltrationof tumors by both T cells and NK cells as well as a striking increaseT cells over Tregs We also observed ain the ratio of CD8significant increase in numbers of dendritic cells DCs in tumorDCs with crossdraining lymph nodes particularly CD103T cells andpresentation potential A criticalinvolvement of NK cells in the antitumor effect was highlightedImportantly strong immune memory was established with anT cells only when both interleukin15 andincrease in memory CD8the CD40 agonist were combined Conclusion These novelpreclinical data support initiation of a firstinhuman clinical trialrole for CD8ª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc Vol e1165Page 0cNovel immunotherapy for pancreatic cancerJRM Van Audenaerde et alwith this combination immunotherapy strategy in pancreaticcancerKeywords CD40 agonist combination immunotherapy interleukin natural killer cells pancreatic cancer T cellsINTRODUCTIONPancreatic ductal adenocarcinoma PDAC is thethird most lethal cancer worldwide with a 5yearsurvival of barely It is even projected tobecome the second leading cause of cancerrelated death by To date it remains one ofthe most aggressive and challenging malignanciesbecause of a complex tumor microenvironmentincluding a strong desmoplastic reaction4 lowimmunogenicity56 and a molecular signature infavor of the tumor driven by loss of multipletumor suppressor genes7 Despite all efforts madein the past almost no improvement in survival hasbeen achieved rendering PDAC a disease whichrepresents the very definition of an urgent unmetneed for novel therapeutic approaches to finallyimprove the outcome of patientsAbout of patients are not eligible for curativesurgical resection because of locally advanced ormetastatic disease at the time of diagnosis Hencepatients are treated with either FOLFIRINOX emcitabinenabpaclitaxel depending on theirphysical fitness Howeverthese treatments areassociated with majortoxicity issues and havelimited impact89 New promising approaches thatare successful in other cancer types such as antiPD1and antiCTLA4 have shown little improvementover treatment with gemcitabine1011 This highlightsthe need for other novel compounds to enter thebattle arena of PDACIn this context the use of CD40 agonists inPDAC has been explored during the past decadeboth as a single modality1213 and in combinationwith current firstline treatments1415 or othercompounds such as checkpoint inhibitors PD1and CTLA416 Antitumor responses have beenreported in both mice and humans and there is ageneral consensustherapyprovides the necessary immunepriming signals toconverttumormicroenvironmenthot‚ammatory microenvironment17 In addition allstudies demonstrated that combination therapyinvolving CD40 agonists provided more potentresults in terms of tumor growth suppression andthat CD40 agonistimmunogenicdesirablecoldthetoaextended survival1516 These data support furtherinvestigation of combination approaches withother promising candidates to unlock the fullpotentialSinceinterleukin IL15 is an essential cytokine foractivation and maintenance of immune effectorcells there is a strong rationale for combiningimmunepriming agents with this moleculetherapyagonistCD40ofbothTcellproliferationto malignantin maintainingWe have previously shown in vitro that IL15stimulated natural killer NK cells can kill bothPDAC tumor cells and stromal pancreatic stellatecells which are responsible for the poor responseto treatment18 IL15 is a versatile cytokine whichstimulatesandgeneration of cytotoxic T lymphocytes as well asactivation and expansion of natural killer NKcells Furthermore it has the capability to induceTcell memory cells thereby playing a crucialCD8immuneroleresponsespossibletheseprevention offeatures render IL15 a highly attractive cancerimmunotherapeutic as confirmed by its high rankin the NCI™s top immunotherapeutic drugs withthe greatest potential for broad usage in cancertherapy22 MoreoverIL15 needs to be transpresented by the IL15Rα on dendritic cells DCsto its target to be effective2023 Since it has beendemonstrated that CD40 agonists also increase theexpression of IL15Rα on DCs we hypothesisedthatinenhanced immune activation and increased antitumor effects24combining both agents mightlonglastingandrelapse19“ AlltumorresultcellsIn this we show for the first time in micewith pancreatic tumors that when CD40 agonistantibody and IL15 are combined they exhibitsynergistic effectsin terms of enhanced antitumor efficacy resulting in profound increases inlongterm survival with complete cure in themajority of cases Moreover an unprecedentedstriking dose reduction of CD40 agonist waspossible by the addition of IL15 The antitumoreffect was found to be mediated predominantlysupported byby CD8dendritic cells DCincreased amounts of CD103ThewithT cells and NK cellscrosspresentingcapacityunique Vol e1165Page ª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc 0cJRM Van Audenaerde et alNovel immunotherapy for pancreatic cancertumors by both celltypes wasltration ofcommensurate with a reduction in the amount ofregulatory T cells These noveltranslationalpreclinical data provide a solid rationale toinitiate a clinicalinvestigating this novelimmunotherapy combination strategy for patientswith one oftumorsnowadaysthe hardestto treattrialRESULTSCombined IL15 and CD40 agonist therapyresults in increased antitumor efficacyin vivosignificantreduction ofWe sought to investigate whether the combinedtreatment of IL15 and a CD40 agonist antibodymay lead to augmented antitumor responses inPDAC To investigate this C57BL6J mice bearingeither Panc02 or KPC tumors were treated with IL andor a CD40 agonist antibody deliveredintraperitoneally when tumors reached a size of“ mm2 Figure 1a We demonstrated that thecombination of these agents resulted in decreasedtumor volumes and increased survival of mice inboth the Panc02 and KPC tumor models In thefirsttumor model Panc02 we observed asignificant decrease in tumor volume when micewere treated with either single agent Howevertreatment with the combination regimen resultedin a furthertumorvolume and a significant increase in survival with out of mice being completely tumor freeFigure 1b d and f and Supplementary figure 1AC E and G In the second tumor model KPC weobserved very similar results with reduced tumorvolumes and increased survival of mice with thecombination being significantly betterthanuntreated control or either singleagent therapyIn this experiment seven out of mice werecompletely tumorfree following combinationtreatment Figure 1c e and g and Supplementaryfigure 1B D F and H Remarkably while weobserved similar responses between the Panc02and KPC tumor modelsthe dose of CD40agonist used in the Panc02 model five doses of µg was eight times lower than in the KPCfirst dose µg with four consecutivemodeldoses of µg In summary we show here thatthe combination ofIL15 and CD40 agonisttherapy has profound antitumor activities andcombining both agentsto significantsynergistic effectsleadsDistinct gene expression profiles pointtowards combined immune priming andimmune activation following therapyoftheclusteringTo unravel the effects caused by the single agentsRNA sequencing wasand their combination performed on the more resistant KPC tumorsharvested on day in the treatment scheme Firstprincipal component analysis PCA demonstrateda clear distinction between the isotype IL15 andcombination group while the CD40 agonisttreatment revealed a bigger overlap with theisotype control Figure 2a To investigate in moredetail the effect of therapy on immunerelatedgenes we performed a PCA using a verifiedNanostring immunerelated gene panel whichdemonstrateddifferenttreatment groups Figure 2b The top up anddownregulated immunerelated genesshowedthat genes involved in antigen presentation Thelper immune type responses and NK cellcytotoxicity were modulated Figure 2cToconfirm this gene set enrichment analysis GSEAwas performed showing that gene sets involved inNK cellmediated cytotoxicity Figure 2d and eand Supplementary figure 2A the IL122 pathwayFigure 2f and g and Supplementary figure 2Band the CD8 TCR downstream pathway Figure 2hand i and Supplementary figure 2C were clearlyamong the top upregulated pathwaysin thegroups when IL15 was administered while theCD40promotedantigen processing and presentation pathwaysFigure 2j and k and Supplementary figure 2DImportantlythese features of both IL15 andCD40 agonisttherapy were retained in thecombination grouptreatmentstronglyagonistT cells are responsible for antitumorCD8efficacy following therapyTo gain more insightinto the immune cellsresponsible for the observed antitumor responseswe depleted several immune cell populations intumorbearing mice in both tumor models usingspecific depletion antibodies We monitored theeffect the depletions had on both tumor growthkinetics and survival of mice Figure 3a“d andSupplementary figure Upon depletion of CD4T cells we observed in both PDAC models nosignificant difference in survival of mice betweenthe depleted and nondepleted groups indicatingT cells do not play a significant role inthat CD4ª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc Vol e1165Page 0cNovel immunotherapy for pancreatic cancerJRM Van Audenaerde et alFigure Combining IL15 with a CD40 agonist results in decreased tumor volume and increased survival C57BL6j mice were injected witheither × Panc02 or KPC cells subcutaneously When tumors reached a size of “ mm2 mice were randomised and treated withisotype control IL15 CD40 agonist or IL15 CD40 a Treatment scheme showing timing of dosing is indicated for IL15 µg with blackarrows and for CD40 agonist or the corresponding isotype with red arrows five doses of µg for Panc02 or first dose µg andconsecutive four doses µg for KPC b c Tumor growth kinetics are depicted [n or mice per group representative data of Panc02or KPC independent experiments] Oneway ANOVA with Bonferroni post hoc d e Survival of Panc02 n and KPC n micetreated as indicated Pooled data of Panc02 or KPC independent experiments Survival was determined by tumor size reaching mm2Logrank test f g Waterfall plots showing the change in tumor area relative to baseline after days Panc02 n or days KPCn Pooled data of Panc02 or KPC independent experiments All data represent mean 06 SEM P ˂ P ‰¤ P ‰¤ P ‰¤ Vol e1165Page ª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc 0cJRM Van Audenaerde et alNovel immunotherapy for pancreatic cancerFigure RNA profile of KPC tumors KPC tumors were harvested on day of the treatment schedule for subsequent RNA isolation andsequencing a PCA plot showing separation of samples based on all genes b PCA plot showing separation of samples based on immunerelated genes Nanostring mouse immunerelated genes c Volcano plot showing significantly differentially expressed immunerelated genesbetween isotype control group and the combination group d“k GSEA plots for differentially expressed genes are showing enrichment for genesinvolved in a n tumorsgroupª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc Vol e1165Page 0cNovel immunotherapy for pancreatic cancerJRM Van Audenaerde et alFigure Immune cell depletion C57BL6j mice bearing Panc02 or KPC tumors were treated with isotype control or the IL15 CD40 agonistcombination regimen alone or with depleting antibodies against CD4 CD8 asialoGM1 NK cell depletion a b Tumor growth kinetics ofPanc02 or KPC tumors either nontreated isotype treated with the combination regimen only no depletion or combination and depletionantibodies Oneway ANOVA with Bonferroni post hoc Data points represent mean 06 SEM n “ micegroup representative data of twoindependent experiments c d Waterfall plots showing the change in tumor area relative to baseline after days e“k Survival of Panc02or KPCbearing mice either nontreated isotype treated with the combination regiment no depletion or the combination and depletionantibodies against CD4 e f CD8 g h asialoGM1 i j or CD8 asialoGM1 k l Data pooled from two independent experiments withn or Panc02 or n “ KPC LogRank test ns P ‰¥ P P ‰¤ P ‰¤ P ‰¤ Vol e1165Page ª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc 0cJRM Van Audenaerde et alNovel immunotherapy for pancreatic canceroftheeffectantitumorthe antitumor response elicited by the IL15 andCD40 agonist combination treatment Figure 3eT and NK cells wereand f However when CD8depletedthecombination treatment was significantly reducedin both tumor models Figure 3k and l WhenT cells alone were depleted there was aCD8significant increase in tumor growth and reducedsurvival of mice Figure 3g and h Howeverdepletion of NK cells only led to a mixed responseIn the Panc02 tumor model we clearly observed asignificant decrease in survival of mice followingdepletion of NK cells although interestingly therewas no effect on tumor growth In the KPC tumormodelthere was a trend towards decreasedsurvival of mice following NK cell depletionalthough this was notsignificantP In conclusion theseT cells wereexperiments demonstrated that CD8the predominant effector cell type responsible forthe observed antitumor effects with a clearcontribution of NK cells in the Panc02 modelFigure 3i and jstatisticallyCombination therapy increases intratumoral ltration of immune cellsintheKPCutilisedto monitorTo further explore the anticancer effects of thecombination regimen multicolour flow cytometrytumorltratingwaslymphocytestumor modelSupplementary figure We observed strikingdifferences between the different treatment armsthat were tested Figure 4a The first observationwas that the antitumor effect was not due to ahigher total number of ltrating lymphocytesbut rather caused by significant differences inwhich immune cellsubsets were present Thefrequency of ltrating NK and NKT cells wassignificantly higher in the combination therapygroup compared to the isotype and CD40 agonisttreatment groupsIL15 alone demonstrated anincreased frequency of both cell types althoughthis was not statistically significant compared tocontroltreated miceIn terms of neutrophilnumbers although they were abundantly presentwethetreatment groups For T cells we observed anincreased number offollowingcombination therapy compared to the othertreatment groups This increased number of T cellsT cellscould be attributed to predominantly CD8which werethecombination therapytreated mice compared to alltotal T cellssignificantlydifferenceobservedbetweenhighernoincellsseveralon NK and NKTtreatment arms while there was nootherT cellsdifference in numbers of ltrating CD4Interestingly we also observed a significantdecrease in numbers of regulatory T cells Tregspresent in the tumor when CD40 agonist was partof the treatment We also measured expression ofCD69 on immune cells as an indicator ofactivation We observed clear upregulation of thismarkerfollowingTcombination therapy however not on the CD8cells The results were confirmed by geneexpression analysis ofrelevant genesFigure 4b Here FoxP3 transcription was indeeddownregulated when Tregs were less abundantand the combination regimen demonstratedthe strongest upregulation of NKT cellrelatedgenes such as granzyme A and B The analysisrevealed a strong immune activation signature asIFNγ and CD69 were upregulatedIL12Puttingthecombination treatment of CD40 agonist andIL15 had a more profound antitumor effect asit caused a significant increase in the amountof antitumor immune cells NK NK T and CD8T cells compared to control orsingleagenttreatmentcommensurate with adecrease in immunosuppressive Tregs resulting inan enhanced CD8Treg ratio within the tumorsobservationsthat wastogetherIL18theseCombination therapy results in increasedamounts of CD103crosspresenting DCsDendritic cells are known to play critical roles inantigen processing and presentation and are keyplayers in the activation of both NK and T cellsWe further explored their presence in the KPCtumor model Here we observed a significantincrease in number of DCs in the tumor only inthecombination therapy group Figure 5aDCs theFurthermore the amount of CD103subtype responsible for crosspresentation wasIL15 caused a significantdetermined HereDCs in theincrease in the number of CD103tumor while this was significantly lower in thegroups following treatment with CD40 agonistFigure 5b To further investigate how the DCsbehaved we analysed the presence of these cellsin the tumordraining lymph nodes TDLN andobserved a 3fold increase in number of DCs whenCD40 agonist was administered Figure 5c Thecrosspresenting DCsfrequencyincreasedconditionsFigure 5d suggesting that these DCs capturedoftwofoldCD103undertheseª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc Vol e1165Page 0cNovel immunotherapy for pancreatic cancerJRM Van Audenaerde et alFigure Characterisation of tumorltrating lymphocytes C57BL6j mice bearing KPC tumors were treated with isotype control IL15 CD40agonist or the combination of the latter a Tumors were harvested at day posttreatment initiation Singlecell suspensions were acquired afterenzymatic digestion for flow cytometry analysis Immune cell populations indicated as fold change of absolute number of cells and CD69T cells Data pooled from three independent experiments n “16group Oneway ANOVA withexpression MFI on NK NKT and CD8Bonferroni P P ‰¤ P ‰¤ P ‰¤ b Heatmap of gene expression of relevant genes for the quantified immunesubsets n tumorsgroup Vol e1165Page ª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc 0cJRM Van Audenaerde et alNovel immunotherapy for pancreatic cancerantigens at the tumor site and migrated to theTDLN to activate NK and T cells Furthermoregene signatures showed a higher expression ofCD80 CD83 and CD86 when the combinationtherapy was administeredindicating that likelyantigenpresenting cells like DCs are activated andmaturein mRNA encodingexpression of CXCR3 CXCL9 and CXCL10 notCXCL11 as chemotactic chemokines suggests theirinvolvementin the trafficking of antitumorimmune cells to the tumor site Figure 5eincreaseTheInduction of immune memorythe goals ofimmunotherapy isOne oftheinduction of strong immunological memory toprevent future relapse We observed in our studyan increased number of effector and centralT cells in KPC tumors when treatedmemory CD8with the combination regimencompared toisotypearm treatmentssingleFigure 6aTo investigate whetherfunctional immune memory was induced tumororand bcontrolFigure Characterisation of DCs in tumor and TDLN C57BL6j mice bearing KPC tumors were treated with isotype control IL15 CD40agonist or the combination of the latter Tumors or TDLN were harvested at day posttreatment initiation Singlecell suspensions were acquiredafter enzymatic digestion for flow cytometry analysis a b DCs or CD103DCs in TDLN Data pooledfrom three independent experiments n “16group Oneway ANOVA with Bonferroni P P ‰¤ P ‰¤ e Heatmapof gene expression of relevant genes for the quantified immune subsets n tumorsgroupDCs in tumors c d DCs or CD103ª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc Vol e1165Page 0cNovel immunotherapy for pancreatic cancerJRM Van Audenaerde et alfree mice from the combination treatment groupwere rechallenged with the same tumor cell typeas they were originally inoculated with Here weobserved clear induction of immune memory inboth PDAC models with out of micebecoming tumor free for the Panc02 tumor modeland all mice becoming tumor free for the KPCtumor model compared to naı¨ve control miceFigure 6c“fDISCUSSIONWith high resistance to current firstline treatmentand failure of numerous clinicaltrials PDACpatients are in desperate need for new treatmentoptions25 There is a general consensus that CD40agonist therapy triggers the necessary immunepriming signalling to convert immunogenic coldcancerenhancementto itstargetthe effects are ofofto hot However when applied intumorsshortpancreaticduration warrantingthistherapy1217 IL15 is a powerful stimulator of NKT cells and induces CD44hi memorycells and CD8T cells This cytokine needs to be transpresentedby the IL15Rα on DCsto beeffective2023 Since it had been demonstrated thatthe CD40 agonist also increases the expression ofIL15Rα on DCs we hypothesised that combiningboth agents may resultin enhanced immuneactivation and increased antitumor effects In thisstudy we demonstrated thatIL15 and CD40agonist administered alone can elicit a powerfulimmune response However when combinedenhanced andthesestronglyimportantly exceeded survivalrates of CD40agonist with gemcitabine and nabpaclitaxel oreffects wereFigure Rechallenge experiments C57BL6j mice cured from Panc02 or KPC tumors after treatment with IL15 CD40 agonist were reinjected with the same tumor type at the contralateral side of the abdomen a b Tumor kinetics and survivallogrank test of micerechallenged with Panc02 tumor cells n c d Tumor kinetics and survival of mice rechallenged with KPC tumor cells n e f FlowEffector or Memory T cells of KPC tumorbearing mice after days following treatment n cytometry quantification of intratumoral CD8Oneway ANOVA with Bonferroni P ‰¤ P ‰¤ P ‰¤ Vol e1165Page ª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc 0cJRM Van Audenaerde et alNovel immunotherapy for pancreatic cancerstudy26 A limitation ofCD40 agonist and gemcitabine and nabpaclitaxeltogether with PD1 and CTLA4 blockade in similarpancreatic mouse models1516 In addition whenCD40 agonist was combined with antiangiogenicdrugs blocking VEGFA and angiopoietin survival rates did not equal the ones observed inthese studiesthisincluding ours may be that the mouse models donot completely mimic the human situation sincetumorsubcutaneouslyNevertheless CD40 agonists given as a singlemodality have shown beneficial effect also inhuman clinical trials with PDAC patients131427 andour previous studies have demonstrated that uponIL15 stimulation human NK cells can kill bothpancreatic cancer and stromal stellate cells in anautologous human ex vivo setting18cells wereinjectedThe potential of this combination regimen isnot just limited to PDAC since IL15 and CD40agonist therapy has been tested by others in micebearing established CT26 and MC38 colorectaltumors The authors showed promising resultsalbeit with less surviving mice compared to ourstudy28 This might be due to the fact that wegave in total five doses of CD40 agonist instead offour as in the other studies Furthermore resultsof otherinvestigators using this combinationtherapy in a prostate cancer model TRAMPC2demonstrated similar numbers of surviving mice aswe found underscoring the enormous potentialof the combination approach24 Of note bothcolorectal cancer and prostate cancer have asignificant better 5year overall survival of andthesignificance of our findings in pancreatic cancerwith a 5year survival of barely Strikinglyin this study we also demonstrated that IL15potentiates CD40 agonist treatment causing an fold dose reduction in one of the PDAC mousemodels which has not been reported so far Thisimportant dose reduction could be of greattranslationalimportance as lower doses mightsignificantly decrease adverse events in patientsunderscoringrespectivelyWe observed thatthe combination therapy‚uenced several immune cell types in favor ofincreased antitumor efficacy As also observed byothers in prostate cancer there was an increase innumber of intratumoral effector immune cellsT cells that boththat included NK cells and CD8contributed to enhancing tumor control but thesestudies did not look beyond these immune cells24In our more extensive analysis we also observed areduction in number of Tregs known for theirCD8anTcellsfavorableestablishesreceptor activationimmunosuppressive potential and an increasedfrequency of DCsfor priming T cells andactivating NK cells Especially our finding ofDCs withstrongly increased amounts of CD103crosspresenting potentialin the tumordraininglymph nodes elucidates on the mechanism behindour observed antitumor effectscompriseimportantcompartment ofthe adaptive immune systemwith wellestablished antitumor effects Upon Tcellthey produce effectorcytokines like IFNγ and kill cancer cells in anantigenspecific manner via the granzymeperforinsystem3132 Increased numbers of CD8T cell incancer are therefore linked to better outcome andprognosis With our combination strategy weT cellsobserved an increased number of CD8whichtumormicroenvironment for an anticancer response Wefound these cells were the most important playerin our therapy models since depletion of thesecells virtually abrogated the antitumor responseBoth CD40 and IL15 already as single modalitiesTcell numbers butinduced an increase in CD8the combination therapy induced significantlyhigher intratumoral ltration of these effectorcells This might be due to certain chemokinessince our unique gene expression analysis showeda higher expression of CXCR3 and its ligandsCXCL9combinationtreatment These chemokines are known to beresponsible for chemoattraction of both T cellsand NK cells33 Moreover these two chemokinesareandchemotherapeutic efficacy in PDAC in patients34Interestinglyweredownregulated following combination therapyHowever the CCL5CCR5 axis in PDAC has beenshown to correlate with promotion of migrationand invasiveness of the pancreatic cancer cellsand thus downregulation could be actuallythebeneficialCX3CR1 axis is associated with early recurrenceafter surgery with poor patient prognosis36 Theactivation marker CD69 was not shown to beT cells This may be dueupregulated on the CD8to the fact that tumors were harvested at day posttreatment and CD69 is considered to be anearly activation marker which disappears after days37 Among the CD8subsets wememory T cellsfound higher presence of CD8which is linked to immune memoryIL15 hasbeen described to be important for the inductionin our models35 Furthermorecorrelated with increased survivalfollowingandCXCL10TcellCCL5andCX3R1aª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc Vol e1165Page 0cNovel immunotherapy for pancreatic cancerJRM Van Audenaerde et aland maintenance of these cells and combiningthis modality with for instance PDL1 blockademightincrease the number of CD8memory T cells38furtherNatural killer cells have become an increasinglyimportant target for cancer immunotherapy sincethey have demonstrated to mediate successful andefficient antitumor responses3940 They play animportant role in pancreatic cancer as we haveshown here and although depletion of NK cellsdid not have as drastic an effect on response toT cells it isCD40 and IL15 combination as CD8likely that they still play an important role in thistherapy given the increase in both their activationand numbers in the tumor This is an importantobservation since a higher frequency of NK cells isclearly linked to better survival41 IL15 is a strongstimulator of NK cells23 and its effect is stronglyincreased by the addition of CD40 agonists thatupregulate IL15Rα on DCs28 which is necessaryT cells andfor transpresentation of IL15 to CD8NK cells20 Moreover since expression of IL12 IL and IL18 is upregulated this may lead toin the tumorformation of NK memory cellsmicroenvironment which needsto be furtherexplored42ahighhaveTregsimmunesuppressivepotential43 When CD40 agonist was administeredwe observed a significantreduction in thenumber of Tregs within the tumor as confirmedby others in other solid tumor models2644 Themechanism by which CD40 agonists cause Tregreduction still needs to be elucidated althoughone study indirectly points towards the blockageofmyeloidderivedsuppressor cells and the Tregs45 In addition thehighlyfollowingcombination therapy was very encouraging sincemetaanalysis showed that this is associated withimproved overall survival in cancer patients46 andresponse to therapy4748CD8TreginteractionincreasedbetweenratioFinally reduction of Tregs in PDAC allows DCsimmuneto induce a more potent antitumorT cells49 Ourresponse largely mediated by CD8data demonstrated that DCs in general increasedby 3fold in the tumordraining lymph nodes as aofresultTheirtheimportancein cancer has been extensivelydemonstrated as they function as the generals ofour immune system by capturing tumor antigensand presenting them to T cells thereby elicitingspecific immune responses5051 A very importantDCs which aresubset of these DCs are the CD103combinationtherapyTbycellstheyactivateconsidered to reside in peripheral tissues Uponthey migrate to the lymph nodesactivationwhereantigenpresentation52 Moreover they have found to bethe only APCs to transport intact antigens totumordraining lymph nodes and prime tumorT cells53 Hence our novel dataspecific CD8DC in the tumorshowing a doubling in CD103draining lymph nodes support the increased antitumor responses we observ

Thyroid_Cancer

" new coronavirus SARSCoV2 has determined a pneumonia outbreak in China Wuhan Hubei Province in December called COVID19 disease In addition to the personto person transmission dynamic of the novel respiratory virus it has been recently studied the role of environmental factors in accelerate SARSCoV2 spread and its lethality The time being air pollution has been identified as the largest environmental cause of disease and premature death in the world It affects body™s immunity making people more vulnerable to pathogens The hypothesis that air pollution resulting from a combination of factors such as meteorological data level of industrialization as well as regional topography can acts both as a carrier of the infection and as a worsening factor of the health impact of COVID19 disease has been raised recently With this review we want to provide an update state of art relating the role of air pollution in particular PM25 PM10 and NO2 in COVID19 spread and lethality The Authors who first investigated this association often used different research methods or not all include confounding factors whenever possible In addition to date incidence data are underestimated in all countries and to a lesser extent also mortality data For this reason the cases included in the reviewed studies cannot be considered conclusive Although it determines important limitations for direct comparison of results and more studies are needed to strengthen scientific evidences and support firm s major findings are consistent highlighting the important contribution of PM25 and NO2 as triggering of the COVID19 spread and lethality and with a less extent also PM10 although the potential effect of airborne virus exposure it has not been still demonstrated Introduction A new coronavirus SARSCoV2 has determined a pneumonia outbreak in China Wuhan Hubei Province in December called COVID19 disease The scientific community has come together to implement pharmaceutical and nonpharmaceutical intervention measures designed to contain SARSCoV2 global spread Nevertheless on March 11th WHO™s Directeneral announced that COVID19 can be characterized as a pandemic SARSCoV2 is primarily transmitted from persontoperson through close contact approximately m by aerosol respiratory droplets smaller than μm in diameter wwwwhoint Indoor environments might be especially hazardous because of their reduced ventilation Morawska lack ultraviolet light which rapidly inactivates the virus and because it can become less diluted than it would in outdoor environments Schuit It is also known how the virus can survive and being infectious in aerosols for hours and on surface up to days van Doremalen et al similarly with transmission dynamics known for SARSCoV1 associated with nosocomial spread and superspreading events Chen et al 2020ab Beyond the causality it is uncertain even if certain demographics of the population are more vulnerable to SARSCoV2 infection Based on recent reports male gender advancing age and comorbidities seem to be correlated with death and severe illness Harris et al Furthermore COVID19 seems to be associated with an increasing rate of thromboembolic events in hospitalized patients Llitjos Mechanisms of social and economic interactions are additionally supposed to be involved in the diffusion dynamic of COVID19 in the diverse parts of the world or of the same country such as the living conditions the healthrelated behaviour KhalatbariSoltani et al Corresponding author Email address ccopatunictit C Copat 101016jenvres2020110129 Received July Received in revised form August Accepted August EnvironmentalResearch1912020110129Availableonline24August2020001393512020ElsevierIncAllrightsreserved 0cC Copat the commercial exchanges Bontempi 2020a or the migration scale index H Chen It seems that these diffusion dynamics have particularly affected the COVID19 spread at the early stage Among the environmental parameters some climate condition such as temperature humidity sunlight and wind revealed a reduction of the COVID19 spread S Chen Coccia 2020a and air pollution seems to have a role in airborne transmission of SARSCoV2 and severity of COVID19 Domingo Nevertheless to better understand COVID19™s diffusion patterns an interdisciplinary multidimensional approach should be encourage in order to develop firm s Bontempi Air pollution has been identified as the largest environmental cause of disease and premature death in the world GBD Ambient particulate matter PM induces its proinflammatory and thrombogenic effects through the generation of oxidative stress by its chemical compounds and metals Li Signorelli The recent identification of environmentally persistent free radicals EPFRs in the PM resulting from a mixture of combustion sources theorize its role in the increase of disease severity of lower respiratory tract infections LRTI Jaligama Scientific evidences support that short and longterm exposures to ambient air pollutants are associated with a broad of adverse health outcomes Ferrante and Conti Fiore such as higher mortality rates greater hospital admissions and increased outpatient visits Bremner Cohen Dehghani Dockery It has markedly detrimental consequences on asthma bronchitis pneumonia and COPD Dick Perng and Chen Raji Vignal Yarahmadi et al where bacteria and viruses are the most accepted causative factors that harm airway stability driving to infection exacerbation Furthermore air pollution represents an aggravating factor for infection diseases caused by some viral infections Domingo and Rovira such as respiratory syncytial virus RSV influenza A and B para influenza virus type pneumonia and influenzalike illness Carugno Croft Fukuda Huang Huh Liang Lin Silva Somayaji It determines an increase in the rate of hospitalizations and access to emergency department visits Studies related to the epidemic of SARSCoV coronavirus identified in November from the Guangdong province of southern China reported similar associations Cai Cui Kan Several Authors suggest that outdoor air pollution resulting from a combination of factors such as meteorological data level of industrialization as well as regional topography could operate both as a carrier of the infection and as a worsening factor of the COVID19 severity Conticini Frontera Isaifan Martelletti and Martelletti This association is getting stronger thanks to the results of the numerous studies that have been launched all over the world and summarized with this review Most of the reviewed studies support that chronic exposure to air pollution might led people more susceptible to COVID19 disease leading to widespread COVID19 spread and lethality Nevertheless as suggested by Bontempi 2020b the potential effect of airborne virus exposure due to PM10 remain unclear With this review we want to provide an updated state of art of the recently epidemiological studies dealing with understanding the role of air pollution in particular PM25 PM10 and NO2 in COVID19 spread and lethality Fig PRISMA Flow Diagram of identification screening and inclusion of studies EnvironmentalResearch19120201101292 0cC Copat Method We have conducted a systematic review of the literature concerning the relationship between some air pollutants PM25 PM10 and NO2 and COVID19 outbreak The research was performed in compliance with the PRISMA criteria Preferred Reporting Items for Systematic Reviews and MetaAnalyses and the Flow Diagram is showed in Fig The research was conducted between April and July 6th in PubMed database It was used the Advanced Search Builder and the keywords were searched in [Title OR Abstract] We have filtered only research articles published in English language and selected the following keywords Air pollution and COVID19 or SARSCOV2 Particulate matter or PM and COVID19 or SARSCOV2 Nitrogen dioxide or NO2 and COVID19 or SARSCOV2 We choose as inclusion criteria all the available epidemiological studies aimed to identify any temporal and spatial association between reported COVID19 cases andor deaths and air pollution data related to PM25 PM10 and NO2 thus excluding any Letter Opinion Commentary Review or nonrelevant articles We obtained a total of eligible published research articles in their final version and paper in its preprint version For some of them we chose to include only principal findings that clearly fit the aim this review Particulate Matter and COVID19 Atmospheric particulate matter PM is originated by a wide range of anthropogenic and natural sources Kim It consists of a heterogeneous mixture of solid and liquid particles suspended in air that varies continuously in size and chemical composition including nitrates sulphates elemental and anic carbon anic compounds biological compounds and metals WHO It has been associated with increased respiratory morbidity and mortality Liu especially in susceptible people due to cardiorespiratory events including asthma chronic obstructive pulmonary disease and thrombosis Li Rhee In vitro and in vivo studies highlighted its role in the exacerbation of respiratory viral infections Becker and Soukup Recently the research group of Setti gave first preliminary evidence that SARSCoV2 RNA can be present on outdoor particulate matter thus suggesting that in conditions of atmospheric stability and high concentrations of PM it could represent a potential early indicator of COVID19 although it does not give information regarding COVID19 progression or severity Several observations report a significant association between ambient concentrations of PM25 Adhikari and Yin Bashir Fattorini and Regoli Frontera Jiang Li VasquezApestegui Wu Yao Zhu Zoran 2020a and PM10 Bashir Coccia 2020b Fattorini and Regoli Jiang Li Yao Zhu Zoran 2020a with COVID19 pandemic across the most affected countries China Italy and USA see Table First evidences on the temporal association between air pollution and COVID19 were reported in China where the outbreak was first identified Zhu explored the relationship between particulate matter and the viral infection caused by the novel coronavirus in cities in China The Authors included over of dailyconfirmed new cases in the whole of China between January 23rd and February 29th They applied a Generalized Additive Model GAM to examine the effects of meteorological factors and air pollution on COVID19 incidence applying a movingaverage approach to capture the cumulative lag effect of ambient air pollution and considering population size and density as potential confounders They observed that the effect of PM25 on daily confirmed cases was greater than PM10 In particular they found that a 10μgm3 increase lag0“ in PM25 and PM10 was associated with a CI to and CI to increase in the daily counts of COVID19 confirmed cases respectively Jiang focused their attention on three most affected cities of China Wuhan XiaoGan and HuangGang collecting data of daily cases and ambient air pollutant from Jan 25th to Feb 29th The Authors by applying a multivariate Poisson regression revealed a significant temporal association between PM25 increased and COVID19 incidence in all the considered cities especially in HuangGang Wuhan RR CI “ XiaoGan RR CI “ HuangGang RR CI “ Conversely an increase in PM10 concentrations was associated with a decrease of COVID19 incidence These results were partially confirmed by findings of Li who conducted a simple linear regression to compare COVID19 incidence with PM concentrations in Wuhan and XiaoGan from Jan 26th to Feb 29th in They found that an increase in PM25 was correlated with an increase of COVID19 incidence in both cities Wuhan R2 p XiaoGan R2 p while for PM10 only in XiaoGan R2 p The spatial distribution of particulate matter and case fatality rate CFR of COVID19 was studied by Yao in cities of China including Wuhan collecting data up to March 22nd First they found a significantly positive global spatial autocorrelation of COVID19 CFR Global Moran™s index I p highlighting a high CFR clustering located in Hubei Province With a multiple linear regression they adjusted their results for several effect modifiers and confounder factors such temperature relative humidity gross domestic product GDP per capita hospital beds per capita local indicators of spatial association LISA map values city size and population or proportion of people older than years It was found that for every μgm3 increase in PM25 and PM10 the CFR increased by “ and “ respectively and the risk estimates increased to “ and “ with every μgm3 increase in average concentrations of PM25 and PM10 in “ respectively Some studies describe the association between air pollution and COVID19 across Italy the second country of the world where the infection spread significantly at the beginning of the pandemic and suddenly has reached many other European countries The 28th of July Italy recorded more than total confirmed cases and deaths WHO most of which were distributed in the regions of Northern Italy especially the Lombardy It is recognized as one the most air polluted areas of Europe EEA where the frequent PM10 annual exceedances of the WHO threshold of μgm3 are responsible for attributable deaths per year corresponding to attributable community rates of deaths per inhabitants per year Baccini Bontempi 2020bfocused the attention on two of the most affected regions of Northern Italy Lombardy and Piedmont The Authors based on PM10 daily exceedances and COVID19 confirmed cases on March 12th thus before the Italian sanitary crisis observed that PM10 concentration was exceeded only few times among the Lombard cities that at the beginning of the epidemic were most affected On the contrary among some Piedmont cities suffering of severe PM10 pollution events COVID19 incidence was lower Based on their results the Authors concluded that COVID19 diffusion by airborne PM10 is hard to demonstrate nevertheless several research article revealed how PM in particular PM25 could had a role in accelerate and vast diffusion of COVID19 in Northern Italy For example Coccia 2020b by analyzed data on Italian province capitals and data of infected individuals up to April 7th revealed a relationship between air pollution of cities measured with days exceeding the limits set for PM10 in previous years and COVID19 diffusion In particular cities with more than days of PM10 exceedances showed a very high average number of infected individual about infected individuals on 7th April whereas cities having less than days of PM10 exceedances showed a lower average number of infected about infected individuals Frontera gave also evidences on the role of PM25 as a contributing factor of COVID19 outbreak in Northern Italy where EnvironmentalResearch19120201101293 0cC Copat Table Summary table reporting reviewed results on the association between COVID19 casesdeaths and air pollution PM25 PM10 and NO2 References Zhu Data analysis Generalized Additive Model GAM Aim Temporal association between daily confirmed cases and air pollution PM25 PM10 and NO2 Temporal association between daily confirmed cases and air pollution PM25 PM10 and NO2 Temporal association between daily confirmed cases and air pollution PM25 PM10 and NO2 Spatial association between fatality rate and air pollution PM25 and PM10 Spatial association between deaths counts and air pollution NO2 Temporal association between total cases daily confirmed cases and total deaths and air pollution PM25 and PM10 Temporal association between total cases daily confirmed cases and total deaths and air pollution NO2 Spatial description of PM10 exceedances versus COVID19 cases Multivariate Poisson regression Simple linear regression Multiple linear regression Descriptive analysis percentage of deaths in three NO2 μmol m2concentration range “ “ “ Pearson coefficient correlation Pearson coefficient correlation Descriptive analysis Number of days of PM10 exceeding μgm3 and COVID19 incidence Area of Study cities of China Period From Jan 23rd to Feb 29th Jiang Li Yao Ogen Zoran 2020a Zoran 2020b Bontempi 2020b From Jan 25th to Feb 29th From Jan 26th to Feb 29th in Data up to March 22nd Data up to the end of Feb From Jan 1st to Apr 30th From Jan 1st to Apr 30th From Feb 10th to March 12th Wuhan XiaoGan and HuangGang China Wuhan and XiaoGan cities of China administrative regions in Italy Spain France and Germany Milan Italy Milan Italy provinces of Lombardy Italy provinces of Piedmont Italy Coccia 2020b Data up to April 7th Italian Provinces Fattorini and Regoli Data up to April 27th Italian provinces PM25 A 10μgm3 PM25 increase lag0“ was associated with a increase of daily confirmed new cases PM10 A 10μgm3 PM10 increase lag0“ was associated with a increase of daily confirmed new cases Wuhan RR CI1032“ XiaoGan RR CI “ HuangGang RR CI “ Wuhan R2 p XiaoGan R2 p Wuhan RR CI “ XiaoGan RR CI “ HuangGang RR CI “ Wuhan R2 p XiaoGan R2 p χ2 p A μgm3 increase in PM25 was associated with a “ increase in fatality rate χ2 p A μgm3 increase in PM10 was associated with a “ increase in fatality rate NO2 A 10μgm3 NO2 increase lag0“ was associated with a increase in daily confirmed new cases Wuhan RR CI “ XiaoGan RR CI “ HuangGang no association found Wuhan R2 p XiaoGan R2 p of fatality cases are associated with NO2 μmolm2 R cid0 R R cid0 R cid0 R R cid0 R cid0 R cid0 R cid0 Lombardy PM10 exceeding between and COVID19 incidence between and Piedmont PM10 exceeding between and COVID19 incidence between and COVID19 in North Italy has a high association with air pollution of cities measured with days exceeding the limits set for PM10 R2 p R2 p continued on next page Hierarchical multiple regression model Pearson regression coefficient analysis R2 p Spatial association between confirmed cases and air pollution PM10 Spatial association between total confirmed cases and air pollution PM25 PM10 and NO2 EnvironmentalResearch19120201101294 0cC Copat Table continued References Frontera Frontera Wu Adhikari and Yin Bashir Bashir VasquezApestegui VasquezApestegui VasquezApestegui Period Data up to 31st March Data up to 31st March Data up to April 04th From March 1st to Apr 20th From March 4th to April 24th From March 4th to April 24th Data up to June 12th Data up to June 12th Data up to June 12th Area of Study Italian regions Italian regions counties in the USA Queens county New York USA California California districts of Lima Perù districts of Lima Perù districts of Lima Perù Aim Spatial association between total confirmed cases and air pollution PM25 Spatial association between deaths and air pollution PM25 Prediction of risk of COVID19 deaths in the long term average exposure to fine particulate matter PM25 Temporal association between daily confirmed cases and total deaths and air pollution PM25 Association between confirmed cases and air pollution PM25 PM10 and NO2 Association between deaths and air pollution PM25 PM10 and NO2 Spatial association between total confirmed cases and air pollution PM25 Spatial association between deaths and air pollution PM25 Spatial association between case fatality rate and air pollution PM25 Data analysis Pearson regression coefficient analysis PM25 R2 p PM10 Pearson regression coefficient analysis R2 p Longterm exposure increase of μgm3 in PM25 is associated with a increase in the COVID19 death rate Estimate on cases values cid0 CI “ Estimate on deaths value cid0 CI “ Kendall r cid0 Spearman r cid0 Zeroinflated negative binomia models Negative binomial regression model Spearman and Kendall correlation tests Spearman and Kendall correlation tests NO2 Kendall r cid0 Spearman r cid0 Kendall r cid0 Spearman r cid0 Kendall r cid0 Spearman r cid0 Kendall r cid0 Spearman r cid0 Kendall r cid0 Spearman r cid0 Multivariate regression model Crude coefficient p Multivariate regression model Crude coefficient p Multivariate regression model Crude coefficient cid0 p mortality was found significantly higher than less polluted Italian regions By collecting data up to March 31st for all Italian regions and performing a Pearson correlation analysis they found a strong positive association both with the total number of confirmed cases R and deaths R other than with hospitalized cases R The Italian situation was further highlighted by the study of Fattorini and Regoli in Italian provinces They explored the spatial association between air pollution and COVID19 cases with data up to April 27th By applying the Pearson regression coefficient analysis they revealed a positive association both with PM25 and PM10 R2 p and R2 p respectively A focus on the most affected city of Italy Milan was conducted by Zoran 2020a This city is located in the Po valley basin known hotspot for atmospheric pollution at the continental scale EEA The Authors performed a temporal association between COVID19 Total cases Daily New positive cases and Total Deaths and particulate matter from Jan 1st and Apr 30th by applying a Person correlation In accordance with other studied they found a positive association between daily confirmed cases and PM25 R and PM10 R although they did not consider any delay time from infection to COVID19 onset Nevertheless they found a negative association between total cases and total deaths and particulate matter but the assumption of a temporal linear correlation may be inaccurate because the above mentioned variables could have more complex nonlinear relationships To date the USA have more than million confirmed cases and thousand deaths WHO Here ambient concentrations of PM and O3 were found responsible to cause between and premature deaths Fann The association between air pollutants and COVID19 cases and deaths was studied by Bashir in the state of California from March 4th to April 24th corresponding to the beginning of the COVID19 outbreak in USA Based on their significant correlation found the Authors state that a limited human exposure to these pollutants will contribute to defeating COVID19 This seems unclear because they found a negative correlation with PM25 and PM10 EnvironmentalResearch19120201101295 0cC Copat by applying both the Kendall rank correlation and Spearman™s one and it is not clear if they normalized COVID19 cases by population size and if they performed a day by day association or a spatial association across the country A focus on the Queen county New York USA was provided by Adhikari and Yin They retrieved data of PM daily concentrations from two ground monitoring stations and collected data of confirmed COVID19 cases and numbers of related deaths from USAFacts in the period from March to April The Authors elaborated their data with a negative binomial regression model and considered the cumulative lag effect of PM25 on disease outcomes over the past days They found a significant negative association among PM25 and new daily confirmed COVID19 cases cid0 CI “ and deaths cid0 CI “ Low PM concentrations in this area of study mean μgm3 are likely to have played a less central role in the spread of infection than in other areas such as Italy where PM25 monthly concentrations reached values higher than μgm3 Fattorini and Regoli Frontera or in China where PM25 monthly concentrations reached values higher than μgm3 Zhu Jiang As said by the Authors other gaseous pollutants such as NO2 and SO2 could have influenced transmission and pathogenesis of COVID19 In the United States Wu investigated whether longterm average exposure to fine particulate matter PM25 increases the risk of COVID19 deaths by considering approximately counties in the United States of the population With an exposure prediction model the Authors calculated the county level longterm exposure to PM25 averaged for to and collected COVID19 deaths counts up to April 04th They conducted a strong and robust statistical analysis with zeroinflated negative binomial mixed models adjusting their results by several potential confounders such as sociodemographic socioeconomic behavioural and meteorological factors They found that a small longterm exposure increase of only μgm3 in PM25 is associated with a increase in the COVID19 death rate confidence interval CI VasquezApestegui recently reported first evidences on the spatial relationship between particulate matter and COVID19 outbreak from Latin America The Authors described the situation occurred in districts of Lima located in the second most affected country of Latin America Peru In particular by applying a multivariate regression model they evaluated the association between the population exposure to PM25 concentrations in the previous years “ and cases deaths and casefatality rates of COVID19 with data up to June 12th A significant association has been found both with cases and deaths Crude coefficient with p and with p respectively but not with case fatality rate All these studies highlight the role of PM in triggers of the COVID19 disease and how government measures targeting to sustainable growth such as the reduction of urban and industrial emissions could have a positive impact on the prevention of health outcomes reducing mortality rate as well the burden on health care systems Nitrogen dioxide NO2 and COVID19 induced lung damage Hence viral infection becomes more common after exposure to NO2 Zhu Furthermore NO2 is associated with other several health effects such as elevated risks for asthma allergic rhinitis and eczema in children To increase of outpatient visits and hospitalizations due to bronchitis and asthma exacerbation Bahrami Asl Kowalska increase of chronic obstructive pulmonary disease COPD Ghanbari Ghozikali Pfeffer and increase of pulmonary heart disease related mortality Chen A recent study explored the possible role of NO2 in interference in Angiotensin converting enzyme ACE2 The expression of ACE2 is high on lung alveolar epithelial cells and it is the human cell receptor of virus agent of COVID19 Alifano First observations report an association between ambient concentrations of NO2 and COVID19 pandemic across Europe China and USA Bashir Fattorini and Regoli Jiang Li et al Ogen Zhu et al Zoran et al 2020b Conversely to the other papers findings of Zoran 2020b and Bashir provides different findings reporting no association or a negative one between NO2 and daily deaths counts In China Zhu by applying the same method explained for PM observed that a 10μgm3 increase lag0“ in NO2 is associated with a CI “ increase in the daily counts of COVID19 confirmed cases in cities of China These findings are confirmed by Jiang and Li et a who applied the same method described for PM Jiang revealed a significant positive association between NO2 and COVID19 both in Wuhan and XiaoGan Wuhan RR CI1053“ XiaoGan RR CI “ but did not found any significant association in HuangGang Li found a significant linear correlation both in Wuhan R2 p and XiaoGan R2 p Ogen presented evidences on the relationship between exposure to NO2 including the months of January and February shortly before the COVID19 spread in Europe and novel coronavirus fatality in the most affected European countries concluding that longterm exposure to NO2 may be a potential contributor to mortality caused by SARSCoV2 He collected data concerning the number of fatality cases from administrative regions in Italy Spain France and Germany and correlated mortality with tropospheric NO2 concentrations measured by the Sentinel5 Precursor spaceborne satellite The major tropospheric NO2 hotspot identified was located in the Northern Italy In all European regions considered gas concentrations ranged between and μmolm2 with airflows directed downwards Results showed that out of the fatality cases by March were in five regions located in north Italy and central Spain Furthermore by analysing mortality trends it was revealed that the highest percentage of deaths occurred in regions where the maximum NO2 concentration was above μmolm2 with a significant decrease where the maximum concentration was between and μmolm2 and below μmolm2 The methodology used by Ogen cannot support a longterm exposure investigation Surely a validation of the satellite measure with those of the ground ™ones the adjustment of the results according to the different population size of each country could have made their results more robust Nevertheless the study provide new insights for future investigation The Italian situation was further studied by Fattorini and Regoli who collected data of COVID19 incidence up to April 27th from Italian provinces They revealed a strong spatial corr

Thyroid_Cancer

"Effects of lowdose computed tomography LDCT screening on lung cancercontains a that is not consistent with the data presented With reference to the National Lung ScreeningTrial NLST there are several flaws in the methodology overlooked Also there is no significant reduction in deathsfrom all causes following the screening Therefore any claim that the LDCT screening is superior to usual care isinvalidKeywords Lung cancer screening Low dose computed tomography MethodologyMain textYou recently published a paper by Huang KL entitled œEffects of lowdose computed tomography on lungcancer screening a systematic review metaanalysis andtrial sequential analysis [] In that paper the authorsstate in their Conclusion that œLDCT screening hasshown statistically significant mortality benefits in highquality trials In the they further state thatœLDCT screening is superiority over usual care in lungcancer survivalYet in the Section Benefits and adverse outcomesthey state On the contrary LDCT screening demonstrated no statistically significant difference in allcausemortality RR CI “The authors need to explain how a screening technique that produces no statistically significant differencein allcause mortality between LDCT screening andusual care can be superior to usual careThis comment refers to the available at 101186s128900190883xCorrespondence donbenjaminbigpondcomCancer Information Support Society Chandos St St Leonards NSW AustraliaThe authors also assess the risk for the NLST trial asincludingbeing Good Green on allMethodologycriteriaPotential flaws in methodologyIn fact the NLST trial had several methodological flawsrelated to the randomisation process overlooked by theauthors of the paper The NLST trial compared LDCT screening of highrisk smokers with Chest Xray CXR screening andassumed that Chest Xray screening produced thesame outcome as usual care [] as suggested in theProstate Lung Colorectal and Ovarian PLCOTrial [] despite earlier trials showing it resulted inan increase in allcause mortality [] Anticipating the shortcoming in above theauthors of the NLST trial ensured that the PLCOtrial had in addition to comparing average risksmokers selected high risk smokers who wereoffered Chest Xray screening for comparison withhigh risk smokers offered usual care to validatethe assumption referred to in Yet this selection ofhigh risk smokers was done after randomisation so The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cBenjamin BMC Pulmonary Medicine Page of the comparison of deaths of high risk smokers afterChest Xray screening with deaths of those receiving usual care was invalid In addition the PLCOtrial published only lung cancer deaths for theNLSTeligible high risk smokers not deaths fromall causes This means the assumption in Point that Chest Xray screening of high risk smokersproduced the same outcome as usual care in termsof allcause mortality was invalidOther irregularitiesReich and Kim observed that the distribution of deathsover time from the NLSTeligible groups selected fromthe PLCO trial showed irregularities suggesting thatthere were some reporting errors in the PLCO trialThey also observed that there were no extra tumoursfound by the screening in the NLSTeligible groups selected from the PLCO trial [] casting further doubt onthis selection process suggesting another flaw in themethodology The PLCO trial identified less than more tumours by screening compared with about more in previous chest Xray trialsThe above potential flaws and irregularities suggestthat a ˜Red “˜ should be applied to the Randomizationprocess the Missing outcome data and the Overall riskrather than a ˜Green ™ On this basis a lower weightingshould be applied to the NLST trial for the purposes ofthe metaanalysisThe main shortcoming of the current metaanalysislike that of many other randomised controlled trialsRCTs is that the authors ignore the most importantoutcome Allcause Mortality and focus on the Deathsfrom Lung cancer If there is no reduction in overalldeaths following the screening it is not valid to claimthat LDCT screening is superior to usual careAs pointed out by Black WC Allcause Mortalityin Randomized Trials of Cancer Screening both trials ofChest XRay screening they reported on in showedan increase in allcause mortality following Chest XRayscreening that they attributed to the harm caused bypostscreening treatments of higher risk smokers Theypointed out that as œdiseasespecific mortality may missimportant harms or benefits of cancer screening because of misclassification in the cause of death this endpoint should only be interpreted in conjunction with allcause mortality In particular a reduction in diseasespecific mortality should not be cited as strong evidenceof efficacy when the allcause mortality is the same orhigher in the screened group []Other issuesThe NLST trial reported major complication rates following invasive procedures for the LDCT and CXRgroups The risk was higher among persons whounderwent LDCT compared with Chest Xray screening vs per screened The earlier CXR screening trials had shown an increase in deaths among thoseoffered screening compared to those not offered screening usual care This is strong evidence in support ofthe suggestion that some of the reduction in deaths fromlung cancer following LDCT screening could have beendue to deaths from other causes resulting from the treatment that as suggested by Black above shouldhave been classified as deaths from lung cancer Thereshould therefore be strong reservations made about anyclaim that the LDCT screening was superior to usualcareFrom the above one possible explanation for the apparently positive result claimed in the NLST trial is thatthe Chest Xray screening had in fact increased thenumber of deaths among those offered screening as hadbeen observed in previous trials [] the LDCT screeninghad reduced the number of deaths by a similar amountcompared to Chest Xray screening the net result beingthat there was no significant reduction in overall deathsas observed Some of the reduction in lung cancerdeaths could have been due to the methodological flawsoutlined aboveFinally the NLST trial is the only large trial to claimbenefits for cancer screening which would make lungcancer screening the only type of cancer screening toproduce significant benefits Randomised trials of breastbowel prostate and ovarian cancer screening have notproduced significant reductions in allcause mortality []and thyroid cancer screening has largely been discontinued due to much evidence suggesting no benefits butsignificant harm from overdiagnosis and overtreatmentAbbreviationsCXR Chest Xray LDCT Low dose computed tomography NLST NationalLung Screening Trial PLCO Prostate Lung Colorectal and Ovarian TrialRCT Randomised Controlled Trial RR Relative Risk CI Confidence IntervalAcknowledgementsNot applicableAuthor™s contributionsThe above letter is completely the work of the author DB The authors readand approved the final manuscriptAuthors™ informationDon Benjamin has previously published papers on the subject of evaluatingthe efficacy of cancer surgery and cancer screeningFundingThe research giving rise to the above letter is being funded by the author™semployer The Cancer Information Support Society Incorporated based ona recommendation from the Society™s Research Director the author Thisresearch is part of an ongoing fouryear project that has identified a flaw inclaims of benefits from lung cancer and other cancer screening The by Huang [] had supported the claim that LDCT lung cancer screeningproduces benefits contrary to the Society™s research findings The current letter commenting on this therefore uses data produced from the original research and funds for writing this letter come from the same project 0cBenjamin BMC Pulmonary Medicine Page of Availability of data and materialsNot applicableEthics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsThe author declares that he has no competing interestsReceived October Accepted July ReferencesHuang KL Effects of lowdose computed tomography on lung cancerscreening a systematic review metaanalysis and trial sequential analysisBMC Pulm Med National Lung Screening Trial Research Team Reduced lungcancermortality with lowdose computed tomographic screening N Engl J Med“ 101056NEJMoa1102873Oken MM for the PLCO Project Team Screening by chest radiographand lung cancer mortality The Prostate Lung Colorectal and OvarianPLCO Randomized Trial JAMA “ 101001jama20111591Black W Haggerstrom D Welch HG Allcause mortality in randomized trialsof cancer screening J Natl Cancer Inst “ Author™s responseto discussion June “Reich JM Kim JS The National Lung Screening Trial premise of null andchest radiography equivalence is to question Am J Roentgenol “Benjamin DJ The efficacy of surgical treatment of cancer “ years laterMed Hypotheses “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

Thyroid_Cancer

"analyse the quality of information included in websites aimed at the public on COVID19Methods Yahoo Google and Bing search engines were browsed using selected keywords on COVID19first websites from each search engine for each keyword were evaluated Validated tools wereThe used to assess readability [Flesch Reading Ease Score FRES] usability and reliability LIDA tool andquality DISCERN instrument Nonparametric tests were used for statistical analysesResults Eightyfour eligible sites were analysed The median FRES score was range 00 Themedian LIDA usability and reliability scores were range 00 and 37range14 00 respectively Alow overall LIDA score was recorded for n of the websites The median DISCERN score 14 was found in websites The DISCERNwas range “ The DISCERN score of score was significantly associated with LIDA usability and reliability scores p and the FRES scorep Conclusion The majority of websites on COVID19 for the public had moderate to low scores with regardsto readability usability reliability and qualityPractice Implications Prompt strategies should be implemented to standardize online health informationon COVID19 during this pandemic to ensure the general public has access to good quality reliableinformationï¾ Elsevier BV All rights reserved Introductionfor The coronavirus COVID19 pandemic has become the greatestglobal health crisis of the st century [] During this pandemicthe demand information on COVID19 has skyrocketedInformation such as latest news updates on the pandemic itssymptoms prevention and mechanism of transmission are highlysought by the public [] On the other hand free access toinformation especially through social media which is accessed bythe majority [] has led to an increase in misinformation and panicassociated with COVID19 [] Although high quality healthinformation is known to be related to lower stress levels andbetter psychological health [] previous studies have shown thatonline information on many medical disorders to be of substandard quality []A previous study done on websites related to COVID19 hasreported substandard quality information that could potentiallymislead the public [] However this study has used a limitedsearch strategy and had not assessed some important areasincluding usability and reliability of the information Thereforethis topic remains a knowledge gap in COVID19 research []Therefore we conducted this study to analyse the current COVID websites targeting the general public in terms of qualityusability readability and reliability using a wide search strategyand validated instruments MethodsAbbreviations USA United States of America FRES Flesch reading ease scoreHONcode Health on the net Code of Conduct SPSS Statistical package for socialsciences NICE National Institute for Health and Care Excellence WHO WorldHealth anization Corresponding author at Department of Surgery Faculty of MedicineUniversity of Colombo PO Box Kynsey Road Colombo Western ProvinceSri LankaEmail addresses ravindrijayasinghegmailcom R Jayasingheranasigmcgmailcom S Ranasinghe umeshejayagmailcom U Jayarajahsanjeewasrgcmbaclk S SeneviratneYahoo Google and Bing were searched using the keywordsœsevere acute respiratory synœnovel coronavirusSARSCoV2 and œcoronavirus The searchdrome coronavirus2 œCOVID19was performed during the first week of May The details ofthe search strategy and the piloting process are provided in thesupplementary material File S1 []Two independent investigators with previous experience ofconducting similar studies assessed the selected websites []Prior to the assessment a pilot run was conducted to ensure101016jpec20200800107383991ï¾ Elsevier BV All rights reservedPlease cite this in press as R Jayasinghe et al Quality of online information for the general public on COVID19 Patient Educ Couns 101016jpec202008001 0cModelGPEC No of Pages R Jayasinghe et al Patient Education and Counseling xxx xxx“xxxuniformity and accuracy The information on symptoms investigations public health measures and available treatmentmodalities were collected The accuracy of the content wasassessed using the national institute for health and care excellenceNICE guidelines on COVID19 [] A rating was given as all ornone based on congruence with the guidelinesValidated instruments were used to assess the quality ofwebsites Readability was assessed using the Flesch Reading EaseScore FRES [] The LIDA Instrument 2007Version12 wasused to analyse the content and the design of the websites usingthe usability and reliability domains [] The quality of thecontent was assessed using the DISCERN questionnaire which has questions in two separate groups [] The detailed assessmentcriteria and the scoring system is included in the supplementarymaterial File S1A website was classified as governmental if it was maintainedby the country™s public health authority If managed by privateinstitutions nongovernmental anizations or voluntary institutions independent from the government they were consideredas nongovernmental The online healthrelated websites arestandardized in terms of their credibility and reliability by onlinecertification sites We chose the Health on the Net code of conductHONcode which is the oldest and widely used out of the qualityevaluation tools available []Data analysis was performed using SPSS Version20 softwareand the associations were determined with non parametric testsA pvalue of was considered statistically significant ResultsOf the retrieved websites were excluded and thein the analysis Theremaining websites were characteristics of the websites are mentioned in Table included Half were governmental websites and only n were HON accredited websites The median FRES was range 00 10th12th grade readability level which is classified asfairly difficult to read Only three websites had a readabilityscore of above equivalent to 7th grade which is therecommended standardThe overall median LIDA score was range “ whilethe median LIDA usability and reliability scores were range 00 and range 00 respectively The median DISCERNscore was range “ which classifies websites as being ofœfair quality Excellent “ Good “ Fair “Poor “ Very poor “ However the top websitesTable A were of excellent qualityTable Website characteristicsWebsite Characteristics Frequency PercentageUsability Governmental websites Notforprofit and private websites HONcode accredited Readability score of above equivalent to 7th grade Readability Date of publication stated References mentioned Disclosure statement by authors Infographics Moderate Low Moderate 00 Low score Moderate 00 Low Used Textonly Reliability Table Correlation between DISCERN scores and other factorsDISCERN SCORELow Mean High 00 Mean Range 00 00 00 00 Range 00 00 00 00 N P valueP0001P0001P0001P P P LIDA Usability LIDA Reliability LIDA Overall FRES Score Government HON Certification No N No Yes Yes Significant correlations were observed between the DISCERNscore and the overall LIDA score as well as LIDA usability andreliability scores Table p HONcode certified websitessites obtained significantly higher DISCERN scores p Pertaining to the currency of information only publishers stated the date of the publication Most websites n did not declare the sources of evidence This was furtherestablished by the median reliability score of Nevertheless the authors have included a disclosure statement in mostn websitesœlowFigures A1 and A2 summarize the rating of websites onindividual criteria assessed by the DISCERN tool The specificinformation provided regarding COVID19 is shown in Fig More than half of the websites failed to discuss the treatmentoptions available n benefits or risks n and effects of no treatment n Furthermore potentialcomplications and prognosis were stated only in and websites respectively Discussion and conclusion DiscussionThis study has shown that still most of the websites on onlinehealth information on COVID19 are of suboptimal quality exceptfor a few credible sources of goodquality health informationNevertheless the websites ranked among the top according tothe DISCERN score Table A2 had high scores indicating thepotential for publishing credible highquality information onlinewhich would benefit the publicin turn causes panic which ranges Misinformation is a major concern during this pandemic aspeople fail to spend adequate time to critically analyse the onlineinformation This fromhoarding medical supplies to panic shopping and using drugswithout prescription with negative social and medical consequences [] Therefore measures implemented to ensure the qualityand accuracy of online information by the responsible authoritiesmay help negate these adverse consequencesinformation Stating the methods of content production with names of thecontributing authors may help increase the credibility of onlinehealth information while displaying the date of the publicationprovides an idea of the currency of the information Absence ofin over half of the websites was a majorsuch drawback especially for COVID19 where new information isgenerated almost daily Health authorities should therefore ensurethat the patient information websites provide the above information and certify websites based on such details so that the publiccan get information from trusted sources []Most users of the worldwide web only have an average level ofeducation and reading skills [] Guidance from the NationalPlease cite this in press as R Jayasinghe et al Quality of online information for the general public on COVID19 Patient Educ Couns 101016jpec202008001 0cModelGPEC No of Pages R Jayasinghe et al Patient Education and Counseling xxx xxx“xxx Fig Website characteristics evaluated outside the DISCERN toolInstitute of Health NIH had shown that the readability should bebelow the level of seventh grade for the lay public to adequatelyunderstand the content [] However the median readabilitylevel was found to be equivalent to 10th12th grade readabilitySuch complexities with the readability of information mayincrease the risk of misunderstandings or misinterpretation Usingshort sentences in writing using the active voice using 12point orlarger font size using illustrations and nontextual media asappropriate and accompanying explanations with examples wouldbe helpful to overcome this problem []So far only a limited number of studies have been done to assessthe quality of health information websites related to COVID19 Thestudy by CuanBaltazar et al prior to February reported poorquality information with approximately of included websiteswith low DISCERN scores [] Our study done three months latershows similar results with only a minimal improvement in thequality of information Furthermore the Cuan Baltazar study hadseveral limitations which includes the limited search strategy andnoninclusion of key quality parameters including readabilityFurthermore out of the sites they had includedwere online news sites that are not considered as patientinformation websites In that study the HONcode seal waspresent only in n websites whereas in our study ofthe sites were HONcode certifiedThere were several limitations in this study Although mostpopular search engines were used in this study under defaultsettings they may produce variable results depending on manyfactors including geographical location and popularity of websitesat a given point of time The algorithms unique to those searchengines are subjected to constant change and therefore the exactresults of our study may not be reproducible However we believethe general patterns observed in our study are validproviding health information to the general public are to be ofsubstandard quality Practice implicationsTo improve the credibility of the content the websites shouldstate methods of content production and display the date of thepublication to give an idea about the currency of the informationTo improve the readability of the content the websites shouldincorporate more nontextual media write in short sentencesusing the activevoice and use larger font sizes The patientinformation websites should display scores of reliability qualityand readability as a guidance for its users Furthermore it is vitalfor medical regulatory authorities and the government to imposeregulations to ensure quality and to prevent the spread ofmisinformationAvailability of data and materialsOn reasonable request from the corresponding author the dataused in the above study can be made availableEthics approval and consent to participateUnnecessary in this type of studyInformed consent and patient detailsNot applicable in this type of studyFundingNone ConclusionCRediT authorship contribution statementThis study has shown the quality readability usability andreliability of the information on COVID19 on majority of websitesRavindri Jayasinghe Conceptualization Methodology Datacuration Writing original draft Visualization InvestigationPlease cite this in press as R Jayasinghe et al Quality of online information for the general public on COVID19 Patient Educ Couns 101016jpec202008001 0cModelGPEC No of Pages R Jayasinghe et al Patient Education and Counseling xxx xxx“xxxValidation Formal analysis Resources Sonali RanasingheConceptualization Methodology Data curation Writing originaldraft Visualization Investigation Validation Formal analysisResources Umesh Jayarajah Conceptualization MethodologyData curation Writing original draft Visualization InvestigationValidation Formal analysis Resources Sanjeewa SeneviratneConceptualization Methodology Writing review editingSupervision Project administrationDeclaration of Competing InterestThe authors report no declarations of interestAcknowledgementsNone declaredAppendix A Supplementary dataSupplementary material related to this can be found in101016jversion at online the pec202008001References[] IJ Bes do Nascimento N Cacic HM Abdulazeem Novel Coronavirusinfection COVID19 in humans a scoping review and metaanalysis JClinical Med [] HT Le DN Nguyen AS Beydoun XTT Le TT Nguyen QT Pham NTK Ta QT Nguyen AN Nguyen MT Hoang Demand for health information onCOVID19 among Vietnamese Int J Environ Res Public Health [] C Wang R Pan X Wan Y Tan L Xu RS McIntyre FN Choo B Tran R Ho VK Sharma A longitudinal study on the mental health of general populationduring the COVID19 epidemic in China Brain Behav Immun [] CS Ho CY Chee RC Ho Mental health strategies to combat the psychologicalimpact of COVID19 beyond paranoia and panic Ann Acad Med Singapore “[] C Wang R Pan X Wan Y Tan L Xu CS Ho RC Ho Immediate psychologicalresponses and associated factors during the initial stage of the coronavirus disease COVID19 epidemic among the general population inChina Int J Environ Res Public Health [] RH Waidyasekera U Jayarajah DN Samarasekera Quality and scientificaccuracy of patientoriented information on the internet on minimallyinvasive surgery for colorectal cancer Health Policy Technol “[] R Jayasinghe S Ranasinghe U Jayarajah S Seneviratne Quality of patientoriented webbased information on oesophageal cancer J Cancer Educ In press[] JY CuanBaltazar MJ MuñozPerez C RobledoVega MF PÃrezZepeda ESotoVega Misinformation of COVID19 on the internet Infodemiology studyJMIR Public Health Surveill 2020e18444[] BX Tran GH Ha LH Nguyen GT Vu MT Hoang HT Le CA Latkin CS HoR Ho Studies of novel coronavirus disease COVID19 pandemic a globalanalysis of literature Int J Environ Res Public Health [] G Eysenbach C Köhler How do consumers search for and appraise healthinformation on the world wide web Qualitative study using focus groupsusability tests and indepth interviews Brit Med J “[] AS Prasanth U Jayarajah R Mohanappirian SA Seneviratne Assessment ofthe quality of patientoriented information over internet on testicular cancerBMC Cancer [] V Udayanga U Jayarajah SD Colonne SA Seneviratne Quality of thepatientoriented information on thyroid cancer in the internet Health PolicyTechnol [] National Institute for Health and Care Excellence Coronavirus COVID19 Accessed April wwwniceukcovid19[] Readable The Flesch Reading Ease and FleschKincaid Grade Level Accessed February readablecomblogthefleschreadingeaseandfleschkincaidgradelevel[] Minervation The Minervation Validation Instrument for Healthcare WebsitesLIDA Tool Accessed February httpwwwminervationcomwpcontentuploads201104MinervationLIDAinstrumentv12pdf[] Minervation Is the Lida Website Assessment Tool Valid Accessed February httpwwwminervationcomdoeslidawork[] Discern Online The DISCERN Instrument Accessed February httpwwwdiscernukdiscern_instrumentphp[] E Fahy R Hardikar A Fox S Mackay Quality of patient health information onthe Internet reviewing a complex and evolving landscape Australas Med J “[] J Kluger As Disinfectant Use Soars to Fight Coronavirus So Do AccidentalPoisonings Accessed April timecom5824316coronavirusdisinfectantpoisoning[] BX Tran AK Dang PK Thai HT Le XTT Le TTT Do TH Nguyen HQPham HT Phan GT Vu Coverage of health information by different sourcesin communities implication for COVID19 epidemic response Int J EnvironRes Public Health [] National Institutes of Health How to Write EasyToRead Health Materials Accessed April wwwscribdcomdocument261199628HowtoWriteEasyToReadHealthMaterialsMedlinePlus[] DM D™Alessandro P Kingsley J JohnsonWest The readability of pediatricpatient education materials on the world wide web Arch Pediatr AdolescMed “Please cite this in press as R Jayasinghe et al Quality of online information for the general public on COVID19 Patient Educ Couns 101016jpec202008001 0c"

Thyroid_Cancer

Coronavirus disease is caused by severe acute respiratory syndrome coronavirus SARSCoV2 and represents the causative agent of a potentially fatal disease The spread of the infection and the severe clinical disease have led to the widespread adoption of social distancing measures Special attention and efforts to protect or reduce transmission have been applied at all social levels including health care operators Hence this reports focuses on the description of a new protocol for the safe management of cytological samples processed by liquidbased cytology LBC with an evaluation of the changes in terms of morphology and immunoreactivity METHODS From March to April cytological cases suspicious for SARSCoV2 were processed with a new virusinactivating method suggested by Hologic Inc for all LBC specimens RESULTS The samples showed an increased amount of fibrin in the background A slight decrease in cellular size was also observed in comparison with the standard method of preparation Nonetheless the nuclear details of the neoplastic cells were well identified and the immunoreactivity of the majority of those cells was maintained The cell blocks did not show significant differences in morphology immunoreactivity or nucleic acid stability S Despite some minor changes in the morphology of the cells the results of this study highlight that the adoption of the new protocol for the biosafety of LBCprocessed samples in pathology laboratories is important for minimizing the risk for personnel trainees and cytopathologists without impairing the diagnostic efficacy of the technique Cancer Cytopathol American Cancer Society KEY WORDS coronavirus disease COVID19 cytology diagnosis liquidbased cytologyINTRODUCTIONCoronaviruses are enveloped viruses with a positivesense singlestranded RNA genome They infect birds and mammals and cause a variety of lethal diseases and they can also infect humans and cause disease to varying degrees ranging from upper respiratory tract infections resembling the common cold to lower respiratory tract infections such as bronchitis pneumonia and even severe acute respiratory syndrome Hubei Province China In late December several health facilities reported clusters of patients with pneumonia of unknown cause that were epidemiologically linked to a seafood and wet animal wholesale market in Wuhan Hubei Province China7The causative agent of this unidentified pneumonia has been confirmed to be a novel coronavirus by sequencing and etiological investigations by several independent laboratories in China8 The new coronavirus Corresponding Author Patrizia Straccia BD PhD Unit of Anatomic Pathology Agostino Gemelli University Polyclinic Foundation IRCCS Largo Francesco Vito Rome Italy stracciapatrizialiberoit Unit of Anatomic Pathology Agostino Gemelli University Polyclinic Foundation IRCCS Rome Italy Anatomic Pathology Section Department of Life Sciences and Public Health Catholic University of the Sacred Heart Rome ItalyWe thank Mrs Elena Visca for her invaluable technical support and expertiseReceived May Revised June Accepted July Published online Month in Wiley Online Library wileyonlinelibrarycom 101002cncy22341 wileyonlinelibrarycomCancer Cytopathology Month 0cOriginal originally called novel coronavirus 2019nCoV and officially renamed severe acute respiratory syndrome coronavirus SARSCoV2 by the International Committee on Taxonomy of Viruses and the disease it causes namely coronavirus disease COVID19 have quickly become of tremendous concern worldwide There have been significant outbreaks in many regions of China as well as global expansion to Asia Europe North America South America Africa and Oceania Persontoperson transmission occurs mostly through contact and respiratory transmission droplets but also by the fecaloral route9 For this reason there is an international push to contain the virus and prevent its spread The response to the COVID19 pandemic can be regarded at all social levels eg social community hospital laboratory and individual levelsBecause it is possible that infected samples may be submitted to pathology and cytopathology laboratories for diagnostic purposes it is important for us to take adequate precautions to protect ourselves and our staff The World Health anization recommends that all specimens collected for laboratory investigations be regarded as potentially infectious Health care workers who collect handle or transport any clinical specimens should adhere rigorously to the standard precautionary measures and biosafety practicesThe role of the cytology laboratory for a patient with known SARSCoV2 is limited although it may involve the examination of samples from the oropharyngeal and respiratory tract which is likely to host a significant amount of viruses Because the laboratory personnel might be exposed to contamination during the preparation and handling of fresh specimens from such patients a new procedure for the sterilization of material to be processed by liquidbased cytology LBC has been applied10 This study is focused on a description of this new procedure and on an evaluation of the changes in terms of morphology and cell immunoreactivity that this technique produces in cellular materialMATERIALS AND METHODSFrom March to April cytological cases considered to be possibly infected by SARSCoV2 were sent to the Cytopathology Laboratory of the Agostino Gemelli University Hospital of Rome IRCCSThe cytological material was processed in a dedicated highlevel biosafe hood by specialized technicians wearing adequate personal protective equipment eg mask face or eyes protection disposable medical gloves a disposable waterrepellent gown or coveralls with sleeves fully covering the forearms and shoe covers or dedicated shoes To each vial is added an amount of alcohol ethanol for at least the same amount of its volume to the material this is considered the safest way of handling cytological samples infected by SARSCoV2 The following is the modified method adopted at the study institution for all LBC specimens processed under the protocol suggested by Hologic Inc Marlborough Massachusetts Collect the sample in a ethyl alcohol solution Centrifuge it at 600g for minutes or at 1200g for Pour off the supernatant fluid and resuspend the cell Add mL of CytoLyt solution to reduce biological minutespelletcontamination Centrifuge at 600g for minutes Pour off the supernatant fluid Resuspend the cell pellet Evaluate the cell pellet if it is necessary repeat from step Add an appropriate amount of the specimen depending on the size of the cell pellet to the PreservCyt solution vial Allow it to stand in PreservCyt for minutes Run on a ThinPrep processor or a ThinPrep processorImmunohistochemistry was performed on either LBC slides or formalinfixed paraffinembedded cell blocks obtained from stored ThinPrep material All molecular testing was performed on cell block material only All patients consented to their procedure We received institutional Catholic University of the Sacred Heart ethical approval for this studyMolecular AnalysisA mutational analysis of epidermal growth factor receptor EGFR was performed with the Therascreen EGFR Rotene Q RGQ polymerase chain reaction kit Qiagen in the RGQ 5plex high resolution melt analyzer instrument according to the manufacturer™s protocol sensitivity The mutation nomenclature used in this work follows the guidelines indicated by the Human Genome Variation Society11Cancer Cytopathology Month 0cNew Protocol for Cytological SamplesStraccia et alTABLE Summary of Cytological Samples and Distribution of Molecular and Immunohistochemical AnalysesCytological SampleNumber of CasesMolecular Analysis NumberImmunohistochemical Analysis NumberThyroidUrineliquor in cerebrospinal fluidLungmediastinal FNABronchoalveolar washingsPleural effusionsPeritoneal effusionsPericardial effusionsAbbreviation FNA fineneedle aspirationTABLE Comparison of the Morphological Features of the New Method and the Standard MethodNew MethodSlightly smallerPresentNo changeFibrin mucusDecreaseStandard MethodNormalPresentNormalClearNormalFeatureCellular sizeNucleoliCytoplasmBackgroundCellularityRESULTSThe material processed according to the modified method consisted of specimens In all thyroid specimens urine specimens cerebrospinal fluid specimens lung aspiration specimens bronchoalveolar washings pleural effusions peritoneal effusions and pericardial effusions were evaluated Table The series included men and women and the median patient age was years range years The morphological features of the modified and standard methods were compared Table All cytological samples particularly the fineneedle biopsies showed an increased amount of fibrin in the background A decrease in cellularity in comparison with the standard method of preparation was also noted Figs In all cytological samples we observed that the cells were smaller and more scattered in comparison with samples processed with the original technique Therefore the distinction between normal reactive and atypical cells was slightly more difficult in the samples treated with the modified preparation in comparison with the standard method Nonetheless the nuclear details of the neoplastic cells were generally well identified and the immunoreactivity of the majority of the cells was maintained The cell blocks taken from the material processed by LBC did not show significant differences in morphology immunoreactivity or nucleic acid stability in comparison with the standard LBC method Molecular test data were available for lung fineneedle aspiration specimens Approximately ng of genomic DNA was FIGURE Urothelial cells suspicious for high grade urothelial carcinoma SHGUC ThinPrep Papanicolaou ×FIGURE Cluster of neoplastic cells adenocarcinoma ThinPrep Papanicolaou ×from a lung isolated from the samples quantified and amplified by polymerase chain reaction Sanger sequencing A molecular analysis for EGFR exons and was ordered for all specimens Mutations were identified in of the cases We found EGFRmutated non“small cell lung Cancer Cytopathology Month 0cOriginal FIGURE Same cells identified in the cell block taken from the sample shown in Figure FIGURE Group of neoplastic cells from a nodal metastasis of oropharyngeal carcinoma showing strong positivity for pancytokeratins Avidin Biotin Complex ThinPrep ×suggests that the morphological details and quality of the cellular component can be preserved to achieve the diagnostic efficacy of the original method Our results show that this modified technique might increase the amount of fibrin in the background especially for fineneedle aspiration biopsies this is probably related to the sudden fixation of the hemorrhagic material in a large volume of ethanol When we analyzed the efficacy of the cytological diagnosis only minimal differences from the standard procedure mostly concerning some nuclear details were noted In fact the degrees of nuclear hyperchromasia and nuclear atypia are more difficult to assess only if the cells are less preserved or show artifactual changesDespite the difficulty in diagnosing atypical cells due to these overlapping cytomorphological features the results of our study show that the morphological details combined with the use of immunohistochemical techniques whose quality is not affected by the procedure can lead to a definitive diagnosis of malignancy in the large majority of casesAs recently reported in the literature1214 during the COVID19 pandemic the adoption of strict protocols and guidelines is important for establishing and maintaining a safe work environment Because the pandemic will probably last for months from this point the adoption of protocols for the biosafety of the laboratory and the staff will enable the processing of cytological material until the end of the danger and can be useful for future critical situations Although the modification of the original FIGURE Clusters of neoplastic follicular cells of a papillary thyroid carcinoma ThinPrep Papanicolaou ×cancers case with short inframe deletions of exon and case with a singlenucleotide substitution in exon characterized by the missense mutation pL858RDISCUSSIONGiven the extraordinarily fast spread of the disease and the pace of change in the information and procedures concerning how to deal with the various aspects of fighting this infection one can give only general suggestions for a cytology laboratory™s response12In this study we report a series of cytological samples processed with a modified protocol that ensures effective biosafety in handling the samples for the staff exposed to the viral load The use of this protocol Cancer Cytopathology Month 0cprotocol results in limited changes in the morphology of cells the benefits in terms of laboratory biosafety during this COVID19 pandemic have to be considered significantly more importantFUNDING SUPPORTNo specific funding was disclosedCONFLICT OF INTEREST DISCLOSURESThe authors made no disclosuresAUTHOR CONTRIBUTIONSPatrizia Straccia Conceptualization data curation formal analysis investigation methodology project administration resources software supervision validation visualization writing“original draft and writing“review and editing Esther Diana Rossi Data curation formal analysis investigation methodology resources validation visualization writing“original draft and writing“review and editing Maurizio Martini Data curation investigation and resources Antonino Mulè Data curation investigation and resources Federica Cianfrini Data curation investigation and resources Mariangela Curatolo Data curation investigation and resources Alessandra Cancellieri Data curation investigation and resources Chiara Brunelli Data curation investigation and resources Gian Franco Zannoni Data curation investigation and resources Guido Fadda Data curation formal analysis investigation methodology resources validation and writing“review and editingREFERENCES Owusu M Annan A Corman VM et al Human coronaviruses associated with upper respiratory tract infections in three rural areas of Ghana PLoS One 20149e99782New Protocol for Cytological SamplesStraccia et al Van der Hoek L Human coronaviruses what do they cause Antivir Ther pt B651 Cui J Li F Shi ZL Origin and evolution of pathogenic coronaviruses Nat Rev Microbiol Fehr AR Perlman S Coronaviruses an overview of their replication and pathogenesis Methods Mol Biol De Wit E van Doremalen N Falzarano D Munster VJ SARS and MERS recent insights into emerging coronaviruses Nat Rev Microbiol Woo PC Lau SK Huang Y Yuen KY Coronavirus diversity phylogeny and interspecies jumping Exp Biol Med Wuhan City Health Committee WCHC Wuhan Municipal Health and Health Commission™s briefing on the current pneumonia epidemic situation in our city Accessed January httpwjwwuhangovcnfront webshowD etail Zhou P Yang XL Wang XG et al A pneumonia outbreak associated with a new coronavirus of probable bat origin Nature Kang S Peng W Zhu Y et al Recent progress in understanding novel coronavirus SARSCoV2 associated with human respiratory disease detection mechanisms and treatment Int J Antimicrob Agents Rossi ED Fadda G Mule A Zannoni GF Rindi G Cytologic and histologic samples from patients infected by the novel coronavirus SARSCoV2 an Italian institutional experience focusing on biosafety procedures Cancer Cytopathol Taschner PEM den Dunnen JT Describing structural changes by extending HGVS sequence variation nomenclature Hum Mutat Pambuccian SE The COVID19 pandemic implications for the cytology laboratory J Am Soc Cytopathol Published online March 101016jjasc202003001 Barbareschi M Facchetti F Fraggetta F Sapino A What are the priorities of pathologists™ activities during COVID19 emergency Pathologica Published online April 1032074 951X1520 Phua J Weng L Ling L et al Intensive care management of coronavirus disease COVID19 challenges and recommendations Lancet Respir Med Published online April 101016S2213 Cancer Cytopathology Month 0c'

Thyroid_Cancer

Purpose Pancreatic ductal adenocarcinomas exhibit a high degree of desmoplasia due to extensive extracellular matrix deposition Encasement of mesenteric vessels by stroma in locally advanced pancreatic cancer LAPC prevents surgical resection This study sought to determine if the addition of a monoclonal antibody to connective tissue growth factor pamrevlumab to neoadjuvant chemotherapy would be safe and lead to improved resectability in this surgically adverse patient populationMethods In this phase III trial patients with LAPC were randomised to gemcitabinenab paclitaxel plus Arm A n24 or minus Arm B n13 pamrevlumab Those who completed six cycles of treatment were assessed for surgical eligibility by protocol defined criteria Resection rates progression free and overall survival were evaluatedResults Eighteen patients in Arm A and seven in Arm B completed six cycles of therapy with similar toxicity patterns In Arms A and B carbohydrate antigen “ response as defined by ‰¥ decline from baseline occurred in and respectively Sixteen per cent of patients were radiographically downstaged by National Comprehensive Cancer Network criteria in Arm A and in Arm B Positron emission tomography normalised in vs of patients in Arm A vs Arm B respectively and correlated with surgical exploration Eligibility for surgical exploration was vs p00019 and resection was achieved in vs of patients in Arm A vs Arm B p01193 respectively Postoperative complication rates were not different between armsConclusions Neoadjuvant chemotherapy with pamrevlumab holds promise for enhancing resection rates in patients with LAPC without added toxicity This combination merits evaluation in a larger patient cohortIntRoduCtIonPancreatic cancer is currently the third leading cause of cancer death in the USA1 and by it will likely become the second leading cause of cancer related death after Key questionsWhat is already known about this subject –º Pamrevlumab is anti CTGF1 inhibitor highly safe when given to patients with pancreatic cancer and potentially adds to the activity of gemcitabine and erlotinib when given in metastatic diseaseWhat does this study add –º This study examines a the safety and activity of pamrevlumab when added to gemcitabine and nab paclitaxel in locally advanced pancreatic cancer b the impact of this regimen and surgical resection and postoperative safety c explores the utility of a novel study design for locally advanced pancreatic cancerHow might this impact clinical practice –º This study has the potential to lead to practice changing activity in locally advanced pancreatic cancer via the eventual approval of pamrevlumab for use in this situation the promulgation of a new study design for locally advanced pancreatic cancer and increased potential for surgical resection and thus prolonged OS curability in locally advanced pancreatic cancerlung cancer surpassing breast and colon cancer2 Surgical resection is generally necessary for treatment with curative intent or to extend life expectancy3 However only of patients have disease amenable to upfront curative resection at the time of diagnosis4 Approximately “ of patients are diagnosed with locally advanced disease5 determined surgically unresectable per National Comprehensive Cancer Network NCCN guidelines6 Patients with locally advanced pancreatic cancer LAPC have a prognosis similar to those with metastatic disease with a historical median overall survival OS of Picozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c access“ months with recent trials demonstrating median OS of months7 Recent single institution retrospective studies have reported the potential for resection of LAPC with neoadjuvant therapy irrespective of imaging findings with promising results8 However these are limited by significant selection bias lack of strict radiographic classification and chemotherapy standardisation Current prospective trials have documented resection rates of LAPC in the range of to therefore novel approaches are needed to improve patient outcomesThe tumour biology inherent to pancreatic ductal adenocarcinoma PDAC significantly contributes to the poor outcomes seen in this disease Notably PDAC exhibits a high degree of desmoplasia often in association with elevated connective tissue growth factor CTGF expression12 CTGF appears to play a central role in the biology of pancreatic cancer affecting both its cellular biology and its extracellular matrix composition This leads to many simultaneous biological effects that promote pancreatic cancer growth including increased cellular proliferation and differentiation increased cellular adherence and migration antiapoptosis vascular permeability angiogenesis and suppression of tumour immunological responses13 This stroma may also contribute to the radiographic imaging findings of mesenteric vessel involvement or encasement that is used to determine resectability of pancreatic tumours Executing a pharmacological intervention on the pancreatic cancer stromal environment is therefore a major goal of the development of novel pancreatic cancer therapeuticsPamrevlumab is a human monoclonal antibody that targets CTGF Preclinical studies showed that CTGF overexpression is associated with both desmoplasia and gemcitabine resistance in the KPC pancreatic cancer mouse model14 When pamrevlumab was used in combination with gemcitabine sensitivity to gemcitabine was enhanced which correlated with inhibition of XIAP an antiapoptotic protein15 When tested in patients with advanced pancreatic cancer Stage IV and locally advanced Stage III treated with gemcitabine and erlotinib in a phase III study n75 pamrevlumab displayed multiple favourable outcomes16We hypothesised that through inhibition of the downstream effects of CTGF overexpression on tissue adhesion and other mechanisms pamrevlumab may influence resectability of PDAC tumours With this in mind this novel phase III randomised multicentre trial was designed to explore the safety and efficacy of pamrevlumab in combination with gemcitabinenab paclitaxel in LAPC with special emphasis on surgical eligibility and safetyMetHodsstudy designThis was a phase III randomised trial of safety and efficacy in patients with LAPC who received gemcitabine and nab paclitaxel with or without pamrevlumab as neoadjuvant therapy The randomisation was preplanned and blinded to the investigator The study was approved by individual institutional review boards at nine US institutions and conducted according to the Declaration of Helsinki The trial was registered at clinicaltrials gov as NCT eligibilityKey protocol eligibility requirements included biopsy proven diagnosis of PDAC radiographic staging consistent with locally advanced unresectable disease as defined NCCN guidelines V2 clinical stage confirmed by diagnostic laparoscopy radiographically measurable disease per Response Evaluation Criteria in Solid Tumors RECIST V11 Eastern Cooperative Oncology Group ECOG performance status of or adequate haematological renal and hepatic function no prior therapy for PDAC and no concomitant cancer diagnosis within the past yearsstudy schemaEligible patients were randomised to Arm A or Arm B to receive a total of six treatment cycles “ weeks of therapy figure Patients in Arm A received pamrevlumab mgkg by intravenous infusion on Days and of each day cycle with an additional dose given on Day in the first cycle Patients in both Arms A and B received gemcitabine mgm2 by intravenous infusion on Days and of each day treatment cycle nab paclitaxel mgm2 by intravenous infusion on Days and of each day cycle Doses for gemcitabine and nab paclitaxel were modified for haematological and non haematological toxicity as per standard of care SOC15 Patients remained on therapy for six treatment cycles “ weeks unless they had disease progression an intolerable adverse event AE or toxicity withdrew consent or were withdrawn at the investigator™s discretion All patients were followed for drug toxicity until days after the last drug dose Patients undergoing surgery were followed for days following hospital discharge for surgical complications CTGF levels were obtained prior to treatment from all patients Plasma samples for pamrevlumab level determination were obtained from all patients receiving this drug After all protocol specified therapy was completed patients were followed for disease progression survival and additional oncological therapy Postoperative complications including day readmissions and day mortality were notedResponse assessmentPatients were evaluated for response by the following measures carbohydrate antigen CA “ measured at baseline first day of each cycle and end of treatment EOT RECIST V11 read based on full body CT imaging high resolution dual phase fine cut CT imaging at baseline and every weeks thereafter fluorodeoxyglucose FDG positron emission tomography PET imaging and NCCN V2 resectability criteria at baseline and EOTPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c accessFigure Patient flow and surgery outcomes In Arm A four of the eligible subjects had their surgeries cancelled 1portal vein thrombosis 3medical issues precluding surgery In Arm A four eligible subjects underwent surgery but resection was not achieved 3metastatic disease discovered 1extensive SMA encasement In Arm B one eligible subject underwent surgery but resection was not achieved 1extensive vascular encasement SMA superior mesenteric arterysurgical assessmentSubjects who finished six cycles of combination chemotherapy were evaluated for eligibility for surgical exploration per protocol PP defined criteria Given that patients included in the trial were determined to be initially unresectable by radiographic imaging and NCCN criteria objective criteria were developed to standardise attempts at surgical resectionPatients were deemed eligible for surgery if one or more of the following criteria were met reduction in plasma CA “ level by ‰¥ at EOT compared with baseline reduction in FDG PET maximum standardised uptake value SUVmax by ‰¥ at EOT compared with baseline radiological tumour response per RECIST of partial response PR or complete response CR at EOT or met the definition of resectable or borderline resectable per NCCN guidelines Subjects were classified as ineligible for surgical exploration if any of the following occurred development of distant metastases or local progression on CT scan tumour anatomy precluding vascular reconstruction unreconstructible local complications preventing surgery eg portal vein PVsplenic vein thrombosis pancreatitis or decline in performance status to a Karnofsky score ‰¤ or Picozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668absolute contraindication to surgery from comorbidity eg recovery from myocardial infarction or uncontrolled diabetes The final decision regarding whether resection was to be performed was made by the treating surgeonendpointsSafety endpoints included serious adverse events SAE during neoadjuvant therapy and surgical complications postresection The efficacy endpoints included surgical eligibility R0 resection R0R1 resection median OS progression free survival PFS and year survival rate All patients were followed and data analysis was stratified by PP population and intention to treat ITT cohortstatistical considerationsThe comparison between selected clinical characteristics toxicity profiles and eligibility for surgical exploration or completed surgical resection was performed using the χ² test Exact CIs for the point estimates as well as the treatment difference were obtained from the SAS PROC FREQ procedure with the EXACT option The two treatment arms were compared using the Cochran Mantel Haentzel test controlling for baseline factors TNM stage ECOG CA “ PET SUVmax 0c accesssuperior mesenteric artery SMA involvement coeliac abutment and so on as prespecified in the protocol All cause mortality was used in determining OS which was analysed by the Kaplan Meier method Survival status was updated within month before the data cut off date Data from patients who were alive at the cut off date were censored for survival analysis All statistical tests were performed at the significance level of α005 using two sided testsResultsPatient characteristics and dispositionThirty seven patients were randomised to study treatment to Arm A pamrevlumabgemcitabinenab paclitaxel and to Arm B gemcitabinenab paclitaxel alone Patient characteristics at baseline are summarised in table All patients enrolled were unresectable by NCCN criteria patients had tumour arterial involvement SMA encasement ° coeliac abutment Table Patient characteristicsBaseline demographics “ years “ years ‰¥ years Median Male FemaleAge group Sex BMI kgm2 Mean SD Median Min maxECOG Grade Grade TNM stage T3 N0 M0 T3 N1 M0 T4 N0 M0 T4 N1 M0 T4 NX M0Location of the tumour in the pancreas Non resectability per NCCN criterion Head Body Tail Median tumour size mm ° SMA encasement Any coeliac abutment Inferior vena cava invasion or encasement Unreconstructible SMVportal occlusion Aortic invasion and encasementArm AGNPPN24 Arm BGNPN13 TotalN37 to to to · OK as isNot mutually exclusiveBMI body mass index ECOG Eastern Cooperative Oncology Group G gemcitabine n number of subjects NCCN National Comprehensive Cancer Network NP nab paclitaxel P pamrevlumab PV portal vein SMA superior mesenteric artery SMV superior mesenteric veinPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c accesswithout gastroduodenal artery involvement or aortic involvement inferior vena cava invasion and unreconstructible PVsuperior mesenteric vein SMV occlusion A higher percentage of patients with SMA encasement ° were randomised to Arm A vs Arm B Patient disposition is summarised in figure Twenty four patients in Arm A received gemcitabinenab paclitaxel and pamrevlumab patients completed six treatment cycles Six patients discontinued treatment early due to progressive disease three patients AEs two patients or physician decision one patient Thirteen patients in Arm B received gemcitabinenab paclitaxel patients completed six treatment cycles Six patients discontinued treatment early due to progressive disease two patients AEs two patients or patientphysician decision two patientssafetySAEs are summarised in table Forty one per cent of patients had a treatment emergent SAE Arm A Arm B No individual toxicity category occurred with frequency except systemic infection patients There was no demonstrable increase in any toxicity with the addition of pamrevlumab to gemcitabinenab paclitaxel chemotherapyTable Summary of treatment emergent serious adverse eventsSystem organ classpreferred term Ascites Nausea Pancreatitis Vomiting Device occlusion Drug withdrawal syndrome FeverNo of patients with any treatment emergent SAEBlood and lymphatic disorders Haemolytic uremic syndrome LymphadenopathyCardiac disorders Cardiac failure Supraventricular tachycardiaGastrointestinal disorders General disorders and administrative site conditions Hepatobiliary disorders Infections Sepsis Cellulitis Urinary tract infectionInjury poisoning and procedural complications Respiratory thoracic and mediastinal disorders Skin and subcutaneous disorders Cholangitis Hyperbilirubinaemia Craniocerebral injury Pneumonitis Pulmonary embolism RashArm An24n Arm Bn13n Overalln37n · OK as isPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c accessResponse to therapyIn Arm A had ‰¥ CA “ decline at EOT response by RECIST PR ‰¥ decline in PET SUVmax and were radiographically downstaged by NCCN criteria During the treatment period the median CA “ decline was patients were non secretors Seven out of patients had best objective RECIST response CRPR Some patients had ˜exceptional™ responses defined as normalisation or ‰¥ decline of CA “ patients or normalisation PET SUVmax in In Arm B had ‰¥ CA “ decline at EOT response by RECIST PR ‰¥ decline in PET SUVmax and were radiographically downstaged by NCCN criteria Four out of patients had best objective RECIST response CR PR In Arm B of patients had an œexceptional CA “ response and had an ˜exceptional™ PET response as defined by either ‰¥ normalized Ca response normalized SUV max andorradiographic downstaging post therapy completion surgical evaluationOverall of the total study patients were eligible for surgical exploration using protocol defined criteria Arm A Arm B p00019 Resection was completed in of the patients Arm A Arm B p01193 Details of the nine resected patients are shown in table In Arm A of the patients were eligible for surgical exploration in the ITT population and of the patients were eligible in the PP population patients who completed six cycles of treatment In Arm A out of eligible patients ultimately underwent surgical exploration for resection five operations were cancelled four due to drug toxicitymedical comorbidity sepsis gallbladder perforation declined performance status and fever and one patient declined Eight out of patients in Arm A were resected R0 R1 The remaining four patients who were explored were not resected due to progression or unresectable disease intraoperatively In Arm B of the patients were eligible for surgical exploration in the ITT population and were eligible in the PP population Of the two subjects found to be surgically eligible only one was resected as the other patient had local progressionPredictors of resectionHigh CA “ response ‰¥ decline andor normalisation was contributive to surgical eligibility vs p03 Normalisation versus non normalisation of PET SUVmax was predictive of surgical eligibility vs p0013 and successful resection vs p0002 Combining these two criteria was highly predictive for surgical eligibility vs p0003 and completed vs p0002 resection All nine successful resections were identified by one or both of these criteria Table Summary of resected patientsSitesubject IDTreatmentarmResponse to treatmentNCCNbaselineNCCNend of treatmentResection status“““““““““AAAAAAAAB UnresectablecoeliacUnresectableSMA SMVUnresectablecoeliacUnresectablecoeliacUnresectableSMVUnresectableSMAUnresectableSMA SMV coeliacUnresectableSMAUnresectablecoeliacUnresectablecoeliacUnresectableSMA SMVUnresectablecoeliacBorderline resectableUnresectableSMVUnresectableSMAUnresectablecoeliacUnresectableSMAUnresectablecoeliacR0R1R0R0R1R1R1R0R0Protocol defined criteria CA “ decrease FDG PET SUVmax decrease ‰¥ RECIST V11 response PR or CR NCCN resectable or borderline resectable criteriaCA carbohydrate antigen CR complete response FDG fluorodeoxyglucose NCCN National Comprehensive Cancer Network PET positron emission tomography PR partial response RECIST Response Evaluation Criteria in Solid Tumors SMA superior mesenteric artery SMV superior mesenteric vein SUVmax maximum standardised uptake valuePicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0cConversely radiographic features of response did not correlate with operative potential Neither RECIST response nor radiographic downstaging per NCCN criteria statistically correlated with completed resectionsurgical complicationsPostoperative complications were summarised according to the Clavien Dindo classification posthoc analysis Ischaemic gastritis and ulceration and right lower lung lobe collapse were reported for one patient in Arm A Grade II There was one episode of clinically significant pancreatic leak in each arm Grade IIIA no reoperations and no day or day surgical mortality were noted One patient in Arm B had a delayed gastric perforation following a distal pancreatectomy with coeliac axis resection likely due to thermal injury and was treated non operatively Grade IIIB No wound complications or superficial site infections were noted in either group Four out of patients and out of patients in Arm A and B respectively were readmitted within days and the difference between treatment arms was not clinically or statistically significantsurvivalAs of the data cut off date of evaluable patients were known to be alive with a median length of follow up at approximately months PFS was months CI to and months CI to in Arm A and Arm B respectively One year survival and median OS were and months CI accessto in Arm A and and months CI NR in Arm B The median OS for all patients who were eligible for surgical exploration Arm A Arm B vs ineligible Arm A Arm B was months CI NR vs months CI to p00766 The median OS for resected Arm A Arm B vs non resected patients Arm A Arm B was not reached CI NR vs months CI to p00141 figure dIsCussIonThe treatment of LAPC with neoadjuvant therapy remains challenging and there is no established SOC Several high volume centres have reported their single centre experiences with varying neoadjuvant chemotherapy and chemoradiation strategies8 The combination of more active regimens delivered over an extended period and surgeons™ comfort with resecting and reconstructing major mesenteric vessels has led to an increase in resection rates A meta analysis of studies using FOLFIRINOX has demonstrated resection rates ranging from to in LAPC17 One of the larger studies including patients with LAPC reported a resection rate of that was more dependent on duration of therapy months than chemotherapy regimen FOLFIRINOX or gemcitabine based18 Recently a single institution and single arm prospective study of neoadjuvant FOLFIRINOX and losartan with selective use of radiation in patients with LAPC reported an R0 resection rate of Figure Overall survival Resected vs Non resected patientsPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c access However the lack of randomisation makes it difficult to determine what aspect of therapy was responsible for this effect and only of resected patients needed any type of vascular reconstruction perhaps suggesting more favourable outcome than usually seen in locally advanced disease These retrospective studies contain significant interpretative challenges including selection bias treatment variables including radiation and the use of different definitions of LAPCthe anti CTGF mechanism of action With respect to gemcitabine based therapy a recent large scale prospective trial of patients with LAPC treated with induction gemcitabine with or without erlotinib followed by radiation only patients were able to undergo resection10 Furthermore the addition of erlotinib to gemcitabine did not improve any downstaging capacity and there was no difference in survival with the addition of radiation More recently the LA PACT trial examining the role of induction gemcitabine nab paclitaxel found that only out of patients with LAPC were able to undergo surgery following neoadjuvant therapy with combination gemcitabine and nab paclitaxel for six cycles by investigator™s choice11 Last although FOLFIRINOX has been the most studied induction combination chemotherapy regimen in this population recent randomised data from European patients who received neoadjuvant FOLFIRINOX versus gemcitabinenab paclitaxel prior to resection20 showed no clear difference with respect to R0R1 to resection rate vs p0135 or OS vs months p0268Given for pamrevlumab its use in the neoadjuvant setting has the potential to impact tumour regression and modulate the desmoplastic niche and possibly affect tumour margins allowing for improved resection rates Previous studies have looked at the ability of gemcitabinenab paclitaxel to reduce cancer associated fibroblasts resulting in a ˜softening™ of tumours by endoscopic ultrasound elastography21 This stromal depletion also translated into a decrease of SUV uptake on PET22 In the study reported herein we combined gemcitabine and nab paclitaxel with pamrevlumab to explore its effect in terms of therapeutic response the impact on eligibility for surgical exploration and improved resection rates in locally advanced patientsThe protocol specified therapeutic response criteria CA “ PET SUVmax RECIST and NCCN criteria were used as criteria to determine eligibility for surgical exploration in LAPC This is a novel approach specific to the protocol and allows participating patients to be explored for resection when otherwise they may not qualify by current treatment standards NCCN criteria For example by NCCN conversion alone ie converted from unresectable to borderline resectable only of patients in Arm A would have been eligible for surgical exploration However by protocol criteria of patients in Arm A were eligible for surgical exploration A higher percentage of patients were eligible for surgical exploration by the above criteria in Arm A vs Arm B vs respectivelyOverall the rate of successful resections in the pamrevlumab treated group was higher than in the control group but this did not reach statistical significance most probably due to small sample size Of the nine subjects that were successfully resected in this trial only one was converted by NCCN criteria to borderline resectable prior to surgical exploration Despite this phenomenon the data support the hypothesis that pamrevlumab a human monoclonal antibody with anti CTGF mechanism of action could alter tumour characteristics allowing resection in otherwise unresectable patients This hypothesis needs to be confirmed and patients should be stratified by coeliac andor SMA involvementThe most common predictive factors for eligibility for surgical exploration and resection were CA “ decline and PET SUV max response which are indicators of tumour response to treatment The combination of these two factors proved to be a highly sensitive objective readout for prediction of potential surgical success Both the ability of CA “ response and the inability of radiographic response RECIST and NCCN criteria of resect ability to predict surgical outcome has been observed by others3 and these observations deserve further examination in subsequent clinical trials In the MPACT study both CA “ and PET response correlated to improved survival in metastatic patients treated with gemcitabine and nab paclitaxel23 Recent surgical series of patients with borderline resectable and LAPC have also corroborated their impact in the localised setting25 Correlation of clinical response with plasma levels of endogenous CTGF and pamrevlumab exposure as shown in the prior study by Picozzi et al16 may provide added prognostic and predictive insightWith regard to safety no major incremental toxicity in any category was noted with the addition of pamrevlumab to gemcitabinenab paclitaxel In addition a higher number of patients were able to complete six cycles of the three drug combination including pamrevlumab when compared with gemcitabinenab paclitaxel alone Pamrevlumab is well tolerated and considered safe compared with the SOC drugs for patients with PDAC These observations represent a very favourable attribute when considering potential neoadjuvant chemotherapy in a patient population with the frequency of medical problems typically seen in LAPC In addition there were no signals of increased surgical morbidity or wound healing problems with CTGF blockade by pamrevlumab In fact there were only two clinically significant pancreatic leaks one in each arm which is comparable to national outcome data from high volume pancreatic surgery centres Similarly readmissions following resection were comparable between arms and reflected the complexity of this challenging patient populationFinally while survival data are not yet mature both patients who were eligible for surgery and those that Picozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0cwere ultimately resected had longer PFS and OS highlighting the importance of surgical resection of the tumour Therefore more investigation into newer agents targeting LAPC and increased consideration of candidacy for surgery in those patients who do not progress on therapy or suffer toxicity should be of utmost importance to improve outcomes in this diseaseIn conclusion this is the first prospective randomised multicentre trial examining the role of neoadjuvant therapy in LAPC with prespecified criteria for surgical exploration The use of pamrevlumab in combination with gemcitabine and nab paclitaxel showed a potential to enhance tumour response and increase resection rates Further evaluation of this drug combination in the neoadjuvant treatment setting for LAPC is warranted and a larger phase III trial with resection and survival endpoints is ongoingContributors FibroGen Inc was the study sponsor that designed the study in consultation with the Principal Investigator VP and surgical co investigator FGR All authors except those of the sponsor contributed patients to the study FibroGen was responsible for data collection and analysis All authors reviewed the manuscript and signed off on its accuracyFunding The study was funded by FibroGen Inc San Francisco CAdisclaimer The corresponding author has the right to grant on behalf of all authors and does grant on behalf of all authors an exclusive licence or non exclusive for government employees on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article if accepted to be published in ESMO editions and any other BMJPGL products to exploit all subsidiary rights as set out in our licenceCompeting interests MC MZ SP EK and EC are employees of FibroGen and hold stock andor stock options

Thyroid_Cancer

"Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly citedAURKA a cell cycleregulated kinase is associated with malignant transformation and progression in many cancer types Weanalyzed the expression change of AURKA in pancancer and its eï¬ect on the prognosis of cancer patients using the TCGAdataset We revealed that AURKA was extensively elevated and predicted a poor prognosis in most of the detected cancer typeswith an exception in colon cancer AURKA was elevated in colon cancer but the upregulation of AURKA indicated betteroutcomes of colon cancer patients Then we revealed that undermethylation of the AURKA gene and several transcriptionfactors contributed to the upregulation of AURKA in colon cancer Moreover we demonstrated that AURKA overexpressionpromoted the death of colon cancer cells induced by Oxaliplatin whereas knockdown of AURKA significantly weakened thechemosensitivity of colon cancer cells to Oxaliplatin Mechanistically AURKA inhibited DNA damage response by suppressingthe expression of various DNA damage repair genes in a TP53dependent manner which can partly explain that ARUKA isassociated with a beneficial outcome of colon cancer This study provided a possibility to use AURKA as a biomarker to predictthe chemosensitivity of colon cancer to platinum in the clinic IntroductionAurora Kinase A AURKA is a cell cycleregulated kinaseinvolved in centrosome maturation mitotic entry bipolarspindle assembly and chromosome separation [] The elevated expression of AURKA is frequently reported in manycancer types [] AURKA alone or combined with otherfactors can trigger cell malignant transformation [ ] andpromote the malignant phenotype of cancer cells [ ]AURKA shows oncogenic activity by regulating multipleoncogenic and tumorsuppressive proteins [] Of theseproteinstumor suppressor TP53 has been intensivelystudied Phosphorylation of TP53 at Ser215 and Ser315 byAURKA results in TP53 degradation by MDM2mediatedubiquitination and abrogation of TP53 DNA bindingtransactivation activity respectively [ ] In turn TP53downregulation increases the expression of ARUKA atboth transcriptional and posttranslationallevels [ ]Negative feedback regulation between AURKA and TP53greatly promotes carcinogenesis and progressionMaintaining genome stability by transactivating theDNA damage response DDR genes is the critical mediatorof TP53dependent tumor suppression [ ] thus TP53deficiency causes the loss of various DDR mechanisms and 0cBioMed Research Internationalthereby facilitates genome instability and cancer development [] Meanwhile platinuminduced DNA damage cantrigger the DDR which is a major contributor to chemoresistance to platinum [] In view of the association betweenAURKA TP53 and DDR the upregulated AURKA in cancer might promote the cancer progression but meanwhileenhance the chemosensitivity of DNA damageinducingdrugs in the clinicIn this study we analyzed the expression state and regulation mechanism of AURKA in colon cancer We also testedthe eï¬ect of dysregulated AURKA on chemosensitivity to theplatinum drug and explored the underlying molecular mechanism in colon cancer These results provided a novel insightinto the function of AURKA in cancer Material and Methods Dataset and Processing The data of AURKA mRNAexpression in types of cancers and matched normal tissueswere downloaded from The Cancer Genome Atlas TCGAdatabase including Bladder Urothelial Carcinoma BLCABreast Invasive Carcinoma BRCA Cervical Squamous CellCarcinoma and Endocervical Adenocarcinoma CESCColon Adenocarcinoma COAD Head and Neck SquamousCell Carcinoma HNSC Kidney Renal Clear Cell Carcinoma KIRC Kidney Renal Papillary Cell CarcinomaKIRP Liver Hepatocellular Carcinoma LIHC Lung Adenocarcinoma LUAD Lung Squamous Cell CarcinomaLUSC Pancreatic Adenocarcinoma PAAD Prostate Adenocarcinoma PRAD Rectum Adenocarcinoma READSarcoma SARC Skin Cutaneous Melanoma SKCM Stomach Adenocarcinoma STAD Thyroid Carcinoma THCAand Uterine Corpus Endometrial Carcinoma UCEC Thecorrelation between the AURKA level and the overall survivalOS of cancer patients was also analyzed through the GEPIAhttpgepiacancerpkucnindexhtml The mRNA expression data are shown in Supplementary We compared theexpression level of AURKA mRNA by calculating the meanvalue and standard deviation The eï¬ect of AURKA copynumber variant CNV on AURKA expression level was alsoanalyzed based on the Colon Adenocarcinoma COAD datafrom the TCGA database The eï¬ect of methylation on theexpression of AURKA was assessed using the MEXPRESS datamexpressbe The transcription factor TF targeting ARUKA was screened based on the ChipSeq data in theUCSC databank httpgenomeucsceduENCODE Thetargeted regulatory capacity of TP53 on DDR genes wasassessed using the Cistrome Data Browser httpcistromedb Meanwhile the correlation between AURKAand TF genes was analyzed in colon cancer through theGEPIA httpgepiacancerpkucnindexhtml Cell Culture Two colon cancer cell lines SW1116 andHCT116 and 293TN cell line were used in this study Missense mutation presents in TP53 in SW1116 whereasHCT116 has a wildtype TP53 according to the Cancer CellLine Encyclopedia CCLEportalsbroadinstituteccleabout Additionally all the DDR genes involvedin our study are the wildtype but for BRCA2 which has aframeshift in HCT116 They were cultured using Dulbecco™sModified Eagle Medium DMEM HyClone Logan UTUSA with fetal bovine serum FBSInvitrogenCarlsbad CA USA μgml streptomycin and IUml°penicillin at C in a humidified atmosphere containing CO2 Cells used to detect phosphorylated TP53 were treatedwith a specific proteasome inhibitor MG132 Sigma StLouis MO USA μM for six hours The Construction of Stable Cell Lines Overexpression orknockdown of AURKA was achieved by using lentivirus ps to infect colon cancer cells described as before [] Inbrief the ORFs of AURKA cloned by PCR and synthesizedshRNA against AURKA were inserted into PlvxPuro andSHC201 vectors respectively The scramble sequences wereinserted into these vectors to be used as control Thesevectors were transfected into 293TN cells with the packingvectors System Bioscience Mountain View CA USA toget pseudo lentiviral ps After being filtered andconcentrated by PEG precipitation System Biosciencelentiviral ps were added to the culture medium toinfect colon cancer cells for h After routine culture for h the stable cells were selected and purified by puromycin μgml MTT Assay Colon cancer cells were seeded in 96wellplates at a density of cells per well and incubated overnight The culture medium was replaced with fresh culturemedium containing a diï¬erent concentration of Oxaliplatin and μgml with replicates each After h of incubation μl MTT gl was added to each wellfor h in the incubator The supernatant was removed and μl DMSO was added to each well After being vibratedfor min the plate was read on a microplate reader at nm to calculate the cell viability rate All assays werereplicated three times The result was analyzed using the cellviability percentage the total number of viable cells at eachdrug concentration relative to the number of viable cellstreated with the solvent control Western Blot Total proteins were extracted from coloncancer cells using the RIPA buï¬er Beyotime Institute ofBiotechnology Shanghai China μg protein was separated in SDSPAGE gel by electrophoresis and transferredonto PVDF membrane The blots were blocked by BSA°at C overnight The membrane was incubated with primaryantibodies AURKA rabbit polyclonal antibody ProteinTech Wuhan China No 102971AP diluted at TP53 rabbit polyclonal antibody Proteintech WuhanChina No104421AP diluted at phosphoTP53Ser315 mouse monoclonal antibody Santa Cruz Biotechnology Nosc135772 MDM2 rabbit polyclonal antibodyProteintech Wuhan China No 190581AP and GAPDHmouse monoclonal antibody ProteinTech Wuhan ChinaNo 600041Ig diluted at After washingthemembranes were incubated with peroxidaseconjugatedsecondary antibody Santa Cruz Biotechnology for h at°C The ECL system Thermo Scientific Rockford IL 0cBioMed Research InternationalUSA was used to visualize the blots All assays were replicated three times RealTime PCR Total RNA was extracted from coloncancer cells using TRIzol Invitrogen Carlsbad CA USAEasyScript® Reverse Transcriptase TransGen Biotech CoBeijing China was used to reverse RNA into cDNA Thelevel of DDR gene ATR XLF XRCC1 RPA1 BRCA2 andRAD51 was quantified using the SYBR Green PCR mixBioresearcher Beijing China through CFX96TM RealTime System BioRad The reaction mixture underwent° cycles consisting of denaturation for s at C and°annealing and prolongation for s each at C GAPDHwas used as the endogenous controls All assays were replicated three times The primers used for PCR are shown inSupplementary Statistics Analysis The expression of AURKA in a diï¬erent type of tumors and the diï¬erential expression of genesbetween two groups were analyzed by a twosided Student™sttest Survival analyses were conducted with the KaplanMeier method using the logrank test and the median valueseparation model based on the AURKA expression is presented The hazard ratio was calculated based on the CoxPH model The correlation between methylation statusand AURKA expression was analyzed using the Pearsoncorrelation and Wilcoxon ranksum test Pearson™s correlation and Z test were used to analyze the correlationbetween AURKA and TFs The eï¬ect of CNV on AURKAexpression was assessed by the KruskalWallis test MTTresults were analyzed using variance analysis ˆ—p ˆ—ˆ—p ˆ—ˆ—ˆ— p Results AURKA Was Upregulated and Predicted a BeneficialOutcome in Colon Cancer To explore the eï¬ect of AURKAon cancer progression and prognosis we firstly employedthe TCGA dataset to analyze the mRNA expression ofAURKA in types of tumors Compared with the matchednormal tissues AURKA was significantly upregulated incancer tissues in out of cancer types Figure 1a Nextwe assessed the correlation between the AURKA level andoverall survival OS in cancer types using the GEPIAWe showed that the AURKA level was adversely correlatedwith OS in of cancers including LUAD KIRP PAADSKCM and LIHC However a high level of AURKA wasassociated with a longer OS in COAD Figure 1b Theseresults suggested that AURKA overexpression might playan important role during the carcinogenesis and progressionof cancer however the elevated expression of AURKA predicted a beneficial outcome only in colorectal cancer DNA Undermethylation and Several TranscriptionFactors Might Contribute to the Elevated Expression ofAURKA in Colon Cancer To explore the mechanism bywhich AURKA was upregulated in colon cancer we firstlyanalyzed the eï¬ect of methylation status on AURKA expression By using the MEXPRESS there were methylationsites in the AURKA gene identified Of them methylationsites were significantly adverse correlated with the level ofAURKA Figure 2a Meanwhile we screened the potentialTFs activating AURKA expression based on the ChipSeqdata using the UCSC database and found that a total of TFs potentially regulate AURKA transcription Of themthe expression of TFs was positively correlated with thelevel of AURKA in colon cancer tissues according to theGEPIA correlation analysis Moreover of them have beenidentified to be overexpressed in colon cancer tissues compared with the matched normal tissues through the GEPIAexpression analysis Figure 2b The top four TFs highlycorrelated with AURKA r p were E2F1MYBL2 MYC and BRCA1 The expression and correlationwith AURKA of these four TFs are shown in Figures 2cand 2d We also analyzed the eï¬ect of AURKA CNV onthe expression level of AURKA The result indicated thatthe expression level of AURKA in the AURKA CNV gaingroup was much higher than that in the AURKA CNV neutral group in COAD whereas there was no diï¬erencebetween the AURKA CNV loss and CNV neutral groupFigure 2e But the incidence of CNV gain was lower incolon cancer patients These results indicated that undermethylation the elevated TFs and gene amplification mightcontribute to the elevated expression of AURKA in coloncancer AURKA Increased the Chemosensitivity of Colon CancerCells to Oxaliplatin We found that upregulated AURKAwas associated with the improved prognosis of colon cancerpatients thus we speculated that if AURKA increases chemosensitivity of platinum by increasing the genomic instability in colon cancer We firstly constructed the stable cell lineswith AURKA overexpression or knockdown Figure 3aand then assessed the eï¬ect of AURKA on the chemosensitivity of colon cancer cells The resultindicated thatAURKA overexpression promoted the death of HCT116and SW1116 colon cancer cells induced by Oxaliplatinwhereas knockdown of AURKA significantly weakened theresponse of colon cancer cells to Oxaliplatin Figures 3band 3c These results showed that AURKA may improvethe prognosis of colon cancer patients by increasing the chemosensitivity of colon cancer cells to the DNAdamagingdrug AURKA Downregulated the Expression of DDR Genes byInhibiting TP53 Previous research showed that AURKAinhibits the expression of TP53 which mediates the expression of DDR genes at the transcriptional level We detectedthe eï¬ect of AURKA on TP53 expression by immunoblotin colon cancer cells The result indicated that TP53 wasdownregulated when AURKA was overexpressed whereasupregulated when AURKA was knocked down in coloncancer cells Figures 4a and 4b Next we screened a setof DDR genes that play an important role in DNA damageinduced by chemotherapeutics Meanwhile most of themfunction after the activation of TP53 [] Using theCistrome Data Browser we assessed the transcriptionalregulatory potential of TP53 on these genes and found someof them had higher scores in two sets of data with high 0c AKRUA fo level evitaler ecid2ŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽBioMed Research InternationalŽŽŽŽŽŽŽŽŽCESCCOADHNSCKIRCKIRPLIHCLUADLUSCPAADPRADREADSARCSKCMSTADTHCAUCECTNBLCABRCATumorNormalCOAD Overall survivalLogrank p0034HRhigh06pHR0036nhigh135nlow135lavivrus tnecrePaLUAD Overall survivalLogrank p0047HRhigh13pHR0049nhigh238nlow239lavivrus tnecrePKIRP Overall survivalLogrank p0007HRhigh23pHR00088nhigh141nlow141MonthsMonthsMonthsLow AURKA TPMHigh AURKA TPMPAAD Overall survivalLogrank p00059HRhigh18pHR00068nhigh89nlow89Low AURKA TPMHigh AURKA TPMSKCM Overall survivalLogrank p0014HRhigh14pHR0014nhigh229nlow229lavivrus tnecrePlavivrus tnecrePLow AURKA TPMHigh AURKA TPMLIHC Overall survivalLogrank p000022HRhigh19pHR000028nhigh181nlow181lavivrus tnecrePlavivrus tnecrePMonthsLow AURKA TPMHigh AURKA TPMMonthsMonths Low AURKA TPMHigh AURKA TPMbLow AURKA TPMHigh AURKA TPMFigure AURKA was upregulated in colon cancer and predicted a benefit outcome a Compared with the matched normal tissuesAURKA was significantly upregulated in cancer tissues in out of cancer types b AURKA expression level was adversely correlatedwith OS in of cancers but positively correlated with OS in COADquality control Supplementary We then applied realtime PCR to verify the expression of six representative genesATR XLF XRCC1 RPA1 BRCA2 and RAD51 The resultsindicated that the six DDR genes were downregulated incolon cancer cells with AURKA overexpression but upregulated when knocking down AURKA in colon cancer cellsFigures 4c and 4d which implied that AURKAincreased the chemosensitivity of colon cancer cells to DNAdamageinducing drugs by inducing the degradation ofTP53 and then decreasing the expression of DDR genes 0cBioMed Research InternationalGpG islandAURKA chr2054967393chr2054944445 bpchr2054966401 chr2054967165 chr2054967495 chr2054967671 chr2054967718 r r r r r p p p p p TFE2F1MYBL2MYCBRCA1CBX3rapTFZNF217ELK1EZH2ZBTB33SMARCC1MYBLŽbrMYCŽpTFARID3ATCF3YY1FOXM1TEAD4rpBRCA1ŽE2F1ŽCOADnumT275 numN349COADnumT275 numN349COADnumT275 numN349COADnumT275 numN349cFigure Continued 0cBioMed Research International MPTFEgol MPTCYMgolp value r p value r MPTLBYMgollog2AURKA TPMlog2AURKA TPMp value r log2AURKA TPMŽŽp value r MPTACRBgollog2AURKA TPMdnoisserpxe AKRUACNV loss CNV neutral CNV gainCNV_TypeCNV_TypeCNV LossCNV NeutralCNV gaineFigure Undermethylation upregulation of TFs and gene amplification potentially contributed to the elevated expression of AURKAa Five methylation sites in AURKA DNA were significantly adversely correlated with the level of AURKA b Based on the public data analysisa total of TFs potentially regulated AURKA transcription The expression of TFs was positively correlated with the level of AURKAMoreover of them have been identified to be overexpressed in colon cancer tissues compared with the matched normal tissues c d Theexpression of the top four TFs highly correlated with AURKA was higher in colon cancer tissues compared with normal tissues e Theexpression level of AURKA in the AURKA CNV gain group was significantly higher than that in the AURKA CNV neutral group in COAD 0cBioMed Research InternationalCtrol AURKACtrolCtrol AURKACtrolshAURKAshAURKAAURKAGAPDH ytilibaiv lleC ytilibaiv lleCAURKAGAPDHSW1116aHCT116p00009Drug concentration 𝜇gmlControlAURKAIC50 𝜇gml 𝜇gml ytilibaiv lleCSW1116p00427Drug concentration 𝜇gmlControlAURKAIC50 𝜇gml 𝜇gmlb ytilibaiv lleCcHCT116HCT116p00029Drug concentration 𝜇gmlControlshAURKA1037shAURKA1184IC50 𝜇gml 𝜇gml 𝜇gmlSW1116p00043Drug concentration 𝜇gmlControlshAURKA1037shAURKA1124IC50 𝜇gml 𝜇gml 𝜇gmlFigure AURKA increased the chemosensitivity of colon cancer cells to Oxaliplatin a AURKA was upregulated or knocked down in twocancer cell lines b c AURKA overexpression promoted the death of HCT116 and SW1116 colon cancer cells induced by Oxaliplatinwhereas knockdown of AURKA significantly weakened the response of colon cancer cells to Oxaliplatin DiscussionIn this study we evaluated the expression of AURKA in types of tumor tissues and matched normal tissues Theresult indicated that AURKA was upregulated in most testedcancer types compared with their normal tissues OS analysisshowed that higher AURKA was correlated with a worse outcome of most of the cancer types whereas it only indicated afavorable outcome in colon cancer The prognostic role ofAURKA has ever been assessed in colorectal cancer patientsby a research team in [] Despite the lack of statisticalsignificance they still put forward that AURKA may have a 0cBioMed Research InternationalCtrolAURKACtrolCtrolAURKA CtrolshAURKAshAURKAAURKAMDM2TP53GAPDHpTP53GAPDHAURKAMDM2TP53GAPDHpTP53GAPDHGMGMANRm fo level evitaler ecid2ANRm fo level evitaler ecid2SW1116aŽŽŽŽŽŽŽATRBRCA2 RPA1 XRCC1 RAD51 NHEJ1SW1116 CtrolSW1116 AURKAŽŽŽŽŽŽATRBRCA2 RPA1 XRCC1 RAD51 NHEJ1HCT116 CtrolHCT116 AURKAANRm fo level evitaler ecid2cANRm fo level evitaler ecid2dGMGMHCT116bŽŽŽŽŽŽŽŽŽŽŽATRRPA1BRCA2SW1116 CtrolSW1116 shAURKA1024SW1116 shAURKA1037ŽŽŽŽXRCC1RAD51NHEJ1ŽŽŽŽŽŽŽ ŽŽŽŽŽŽŽATRRPA1BRCA2HCT116 CtrolHCT116 shAURKA1024HCT116 shAURKA1037XRCC1RAD51NHEJ1Figure AURKA downregulated the expression of DDR genes by inhibiting TP53 a b Overexpression of AURKA promoted thephosphorylation of TP53 and decreased the level of total TP53 whereas knockdown of AURKA reduced the phosphorylation of TP53 andincreased the level of total TP53 in colon cancer cells by immunoblot AURKA had no eï¬ect on the expression of MDM2 c d Sixrepresentative DDR genes were downregulated in colon cancer cells with AURKA overexpression but upregulated when knocking downAURKA in colon cancer cells by realtime PCRpositive eï¬ect on survival and emphasized the necessity tostudy the eï¬ect of AURKA on response to treatment []Further study showed that undermethylation and upregulation of TFs potentially contribute to the elevated expressionof AURKA in colon cancer at least partly Some studiesindicated that gene amplification is another contributor tothe elevated AURKA [ ] We also identified that geneamplification in colon cancer patients can result in AURKA 0cBioMed Research Internationalupregulation however its incidence rate was very low incolon cancer patients Finally we demonstrated that AURKAmight improve the prognosis of colon cancer patients byincreasing the chemosensitivity of colon cancer to Oxaliplatin via inhibiting the DDR Our results uncovered thedoubleedged sword eï¬ects of AURKA by inhibiting TP53in colon cancerThe genomic instability has been recognized as a hallmark of cancer and it is associated with carcinogenesis andprogression of cancer [ ] AURKA functions as an oncogene during the development of multiple malignant tumorsby inducing centrosome amplification and genomic instability [ ] In colon cancer the overexpressed AURKA is thecontributor to chromosomal instability [ ] MoreoverAURKA has been revealed to impair the function of DNAdamage repair through inhibiting the expression of DDRgenes such as RAD51 and BRCA12 [“] In additionto the DDR genes involved in Homologous RecombinationRepair HRR TP53 showed the transcriptional regulatorypotential on Mismatch Repair MMR genes according tothe binding scores from the ChipSeq data Supplementary The inhibitory eï¬ect of AURKA on TP53 which has beendemonstrated to transcriptionally activate many DDR genesenlarges the potential of AURKA facilitating DNA damage[] Some studies also indicate that TP53 is essential for chemoresistance rendered by AURKA [ ] In order to verifythe function of TP53 during this process we respectivelyassessed the correlation between AURKA level and OS inpatients with wildtype or mutant TP53 The results indicated that the patients with the higher AURKA had a longerOS time in TP53 wildtype groups although only a marginalsignificance was achieved due to the reduced number ofsamples But no diï¬erence was found in TP53 mutant groupsSupplementary The current research supports that AURKA is involved incolon carcinogenesis through promoting genomic instabilitybut the increased AURKA provides a good chance forenhancing the sensitivity of chemotherapy based on DNAdamageinducing drugs The eï¬ect of AURKA on chemosensitivity has been studied in diï¬erent cancer types Up to nowthey all concluded that AURKA impaired the chemosensitivity which is the exact opposite of our finding Forexample it was reported that inhibiting AURKA enhancesthe chemosensitivity of cancer cells to the taxane and paclitaxel [ ] cisplatin [] doxorubicin [ ] and5fluorouracil 5Fu [] In particular platinum chemosensitivity is inhibited by AURKA in various cancers includingovarian cancer [] hepatocellularcarcinoma [] medulloblastoma [] acute myeloid leukemia []as well as headand neck cancer [] Our finding that AURKA increasedthe platinum chemosensitivity in colon cancer was diï¬erentfrom the previous studies in other cancer types which coincided with our finding that higher AURKA indicated betterprognosis only in colon cancer but not in other cancersThough cancer stem cell is a small subpopulation of cancercells AURKA silencing sensitized the response of colorectalcancer stem cell CRCSC to Oxaliplatin by upregulatingantiapoptotic factors [] which is diï¬erent from our findings in colon cancer cells The diï¬erence might be associatedwith heterogeneity induced by tumor microenvironment andgenomic instability [] AURKAmediated TP53 inhibitionmight result in diï¬erent consequence in diï¬erent geneticcontexts However this hypothesis might be determined byfurther experiments ConclusionAURKA was upregulated in various cancer types but onlypositively correlated with the prognosis of colon cancerpatients The mechanism might be that AURKA improvesthe chemosensitivity of colon cancer cells to Oxaliplatin byinhibiting the expression of TP53regulated DDR genes andthen facilitating DNA damage This study provides a possibility to use AURKA as a biomarker to predict the chemosensitivity of colon cancer to platinum in the clinicData AvailabilityAll the data used to support the findings of this study areincluded within the Conflicts of InterestThe authors declare that there is no conflict of interestregarding the publication of this paperAuthors™ ContributionsBaocong Shan Ran Zhao Jian Zhou and Minghui Zhangcontributed equally to this workAcknowledgmentsThis work was partly supported by the National NaturalScience Foundation of China to Xiaobo Li Natural Science Foundation of Heilongjiang Province H2018009to Tianzhen Wang H2018010 to Yiqi Wu the FundamentalResearch Funds for the Provincial Universities to Ran Zhao to Yuanyuan Zhu and KYYWF0289 to Weiwei Yang and Heilongjiang Postdoctoral Fund LBHZ17136 to Weiwei YangSupplementary MaterialsSupplementary Figure The correlation of AURKA leveland OS in patients with wildtype TP53 or mutant TP53 AThe patients with the higher AURKA had a longer OS timein TP53 wildtype groups although only a marginal significance was achieved due to the reduced number of samplesB No diï¬erence was found in TP53 mutant groups C Mostof the mutations in TP53 were missense variant followed bystop gained and frameshift variant Supplementary Table We compared the expression level of AURKA mRNA by calculating the mean value and standard deviation The eï¬ect ofAURKA copy number variant CNV on AURKA expressionlevel was also analyzed based on the Colon adenocarcinomaCOAD data from the TCGA database SupplementaryTable The primers for PCRSupplementary Table Theassessment of transcriptional regulatory potential of p53 on 0cBioMed Research InternationalDDR genes based on ChipSeq data from cell lines Supplementary Table The assessment of transcriptional regulatory potential of p53 on Mismatch Repair MMR genesbased on ChipSeq data from celllines SupplementarymaterialsReferences[] B Goldenson and J D Crispino œThe aurora kinases in cellcycle and leukemia Oncogene vol no pp “[] A S Nikonova I Astsaturov I G Serebriiskii R L DunbrackJr and E A Golemis œAurora A kinase AURKA in normaland pathological cell division Cellular and Molecular Life Sciences vol 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isassociated with chromosomal instability phenotype in humancolorectal cancers Cancer Biology Therapy vol no pp “ [] Y Baba K Nosho K Shima œAuroraa expression isindependently associated with chromosomalinstability incolorectal cancer Neoplasia vol no pp “[] T Sourisseau D Maniotis A McCarthy œAuroraAexpressing tumour cells are deficient for homologydirectedDNA double strandbreak repair and sensitive to PARP inhibition EMBO Molecular Medicine vol no pp “ [] S Sankaran D E Crone R E Palazzo and J D ParvinœAuroraA kinase regulates breast cancer associated gene inhibition of centrosomedependent microtubule nucleationCancer Research vol no pp “ [] G Yang B Chang F Yang œAurora kinase A promotesovarian tumorigenesis through dysregulation of the cell cycleand suppression of BRCA2 Clinical Cancer Researchvol no pp “ [] H Yang L He P Kruk S V Nicosia and J Q ChengœAuroraA induces cell survival and chemoresistance by activation of Akt through a p53dependent manner in ovariancancer cells 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Thyroid_Cancer

Genetic alterations in the 3q263132 locus conferan aggressive prostate cancer phenotypeBenjamin S SimpsonSusan Heavey Jason Pitt5 Caroline M Moore6 Hayley C Whitakerœ‰ Niedzica Camacho234 Hayley J Luxton Hayley Pye Ron Finn1Largescale genetic aberrations that underpin prostate cancer development and progressionsuch as copynumber alterations CNAs have been described but the consequences ofspecific changes in many identified loci is limited Germline SNPs in the 3q2631 locus areassociated with aggressive prostate cancer and is the location of NAALADL2 a gene overexpressed in aggressive disease The closest gene to NAALADL2 is TBL1XR1 which is implicated in tumour development and progression Using publiclyavailable cancer genomic datawe report that NAALADL2 and TBL1XR1 gainsamplifications are more prevalent in aggressivesubtypes of prostate cancer when compared to primary cohorts In primary disease gainsamplifications occurred in CI“ and CI“ for NAALADL2 and TBL1XR1 respectively increasing in frequency in higherGleason grade and stage tumours Gainsamplifications result in transcriptional changes andthe development of a proproliferative and aggressive phenotype These results support apivotal role for copynumber gains in this genetic region Molecular Diagnostics and Therapeutics Group Research Department of Targeted Intervention Division of Surgery Interventional Science UniversityCollege London London UK Human Oncology and Pathogenesis Program Memorial Sloan Kettering Cancer Center New York NY USA MarieJoseand Henry R Kravis Center for Molecular Oncology Memorial Sloan Kettering Cancer Center New York NY USA Department of Pathology MemorialSloan Kettering Cancer Center New York New York for Genomics Research Discovery Sciences Biopharmaceutical RD AstraZeneca Cambridge UK Cancer Institute of Singapore National University of Singapore Singapore Singapore Department of Urology UCLH NHS Foundation Trust London UKœ‰email HayleyWhitakeruclacukCOMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xProstate cancer PCa is the most common noncutaneouscancer in developed countries12 and is defined by dynamicgenome alterations and both its pathological and geneticheterogeneity3 An important pathological predictor of prostatecancer aggressiveness is Gleason grade used to assess risk ofprogression and stratify patients for treatment however theunderlying genomic changes which accompany more aggressivetumours remains incompletely definedOverall copynumber alteration CNA burden has been linkedto poorer prognosis in prostate cancer associating with Gleasongrade biochemical recurrence and prostate cancer specific deathhowever the exact mechanism driving these prognostic changes isunknown and thought to be primarily driven by general chromosomal instability4“ Changes in specific loci have also beenlinked to aggressiveness in particular gains in proliferative geneseg MYC 8q24 and loss of tumour suppressors PTEN 10q23and NKX31 8p2178 Many genetic alterations have been linkedwith prostate cancer such as point mutations in SPOP FOXA1and IDH19 Largescale oncogenic structural rearrangementstranslocations and copynumber changes are also common oftenleading to the coordinated dysregulation of multiple elements forexample the loss of 21q which is associated with the TMPRSSERG fusion rearrangement and the subsequent rearrangement ofSMAD410 Improved understanding of the mechanisms governing disease pathogenesis and progression may allow for bettertherapeutic exploitation for example genetic alterations in theDNA repair machinery have been linked to susceptibility toPARP inhibitors in a range of tumour types and alterations in ARconfer sensitivity or resistance to androgen deprivation therapy inmetastatic castrate resistant prostate cancer mCRPC11NAALADL2 is located on 3q2631 and is a member of theglutamate carboxypeptidase II family along with the widely studied PCa marker PSMA NAALAD112 and its expression haspreviously been associated with prostate tumour stage andgrade13 with expression predicting poor survival following radicalprostatectomy13 A large genomewide association study GWASof prostate cancer cases found rs78943174 a SNP withinthe 3q2631 NAALADL2 locus was associated with high Gleasonsum score14 A further rs10936845 SNP was identified within aGATA2 motif that increases NAALADL2 expression in prostatecancer patients where increased expression also predicted biochemical reccurence15 The same study showed even higherbinding preference to HOXB13 and FOXA1 to this site suggestingcooccupancy by these important transcription factors both ofwhich have been shown to be involved in AR cistromereprogramming1516functionsAdjacent to NAALADL2 in the genome is TBL1XR1 a corecomponent of nuclear receptor corepressor NCoR complex thatacts as a coregulator of nuclear receptors ‚uencing severalcellularand‚ammation17 TBL1XR1 is also an androgen receptor AR coactivator18 Expression of TBL1XR1 has been associated withpoor prognosis in several cancers predicting poor overall survivaland lymph node metastasis in gastric19 and ovarian cancers20 andrecurrence in colorectal21 breast22 and liver cancers23antiapoptosisincludinggrowthHere we utilise largescale publicly available genomic data tobetter characterise the broad somatic copynumber changesoccurring within the 3q263132 locus particularly centredaround gainsamplifications in NAALADL2 and TBL1XR1 andlinking them to the clinical characteristics of aggressive prostatecancerResults3q263132 gain frequency is increased in aggressive PCaCopynumber alterations often alter the expression of the gene inwhich they occur with gene dosage known to correlate withmRNA expression Genetic structural variants are also known toalter transcriptional regulation by altering cisregulatory elementssuch as promotors and enhancers resulting in differentialexpression2425 Increased NAALADL2 and TBL1XR1 expressionhave previously been linked to poor prognosis in cancers leadingus to examine the frequency of somatic copynumber gains inthese genes across various prostate cancer subtypes19“Alteration frequency was assessed using data from cBioportalFig 1a and all study data was processed using a standardisedpipeline to ensure comparable results Alteration frequency wasassessed in a total of patients samples in nonoverlapping studies Appendix eleven studies focused onprimary prostate cancer four on metastatic prostate cancer andone on neuroendocrine and castrateresistant cancers Significantcopynumber increases above a derived background thresholdwere categorised as ˜gains™ and copynumber decreases as ˜deletions™ Overall the distribution of NAALADL2 and TBL1XR1alterations were significantly different between disease subtypesto that which is expected Chisquared goodnessoffittestFig Somatic alteration frequency of NAALADL2 and TBL1XR1 across prostate cancer subtypes in publically available genomic studies n a NAALADL2 genetic alteration frequency across different subtypes of prostate cancer b TBL1XR1 genetic alteration frequency across differentsubtypes of prostate cancer P primary prostate cancer M metastatic prostate cancer NE neuroendocrine prostate cancer and castrate resistantprostate cancer CRPC All annotations were assigned using Genome Nexus and CNAs are called using GISTIC or RAE algorithms Pvalues show theresults of a Chisquared goodnessoffit test to determine if the number of observed patients with each alteration type is different from that which isexpected across each cancer subtype Results detailed in Supplementary data COMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xp — ˆ’ and p — ˆ’ Fig 1a b with gains beingmost frequent in castrateresistant prostate cancer and respectively followed by neuroendocrine and metastatic and then primary prostatecancer and Fig 1a b3q263132 gains extend across an oncogenerich region ofChr3 As CNA™s are known to associate with more aggressivesubtypes of prostate cancer we investigated their association withclinical characteristics to establish if changes can be detected earlyin the life history of cancer predicting more aggressive diseaseWe utilised copynumber data from primary an confineddisease from both the UK and Canadian International CancerGenome Consortium ICGC cohorts and The Cancer GenomeAtlas TCGA These studies use intermediatehigh risk prostatecancer patients with no treatment prior to radical prostatectomyTo allow comparisons between the studies data were reanalysedusing the Genomic Identification of Significant Targets in Cancer GISTIC2 methodfavoured by the broad institute andTCGA27 as it distinguishes between lowlevel copy numberincreases gains and highlevel copynumber increases amplifications Within the three cohorts we found that copynumbergains across both genes were frequent with gains in NAALADL2ranging from Canada to UK and between“ for amplifications Table TBL1XR1 had an almostidentical CNA frequency of between UK to Canada Table We fitted a randomeffects model to more accurately estimatethe frequency of NAALADL2 and TBL1XR1 gainamplificationscombining the data from all three cohorts which estimated thetrue frequencies to be CI “ and CI “ for NAALADL2 and TBL1XR1 respectively Supplementary Fig Leaveone out analysis and adiagnostic plots revealed that the ICGC Canada study was asignificant source of heterogeneity thereforethe study wasremoved and the model refit The final estimated frequency ofNAALADL2 and TBL1XR1 gainsamplifications was CI “ and CI “ respectivelyin primary prostate cancerDue to their close proximity in the genome we investigated ifgainsamplifications in NAALADL2 and TBL1XR1 cooccurred inthe same patients using a genomewide Fisher™s exact test with afalse discovery rate correction NAALADL2 and TBL1XR1significantly coamplified in all three cohorts ICGC UK p — ˆ’ ICGC Canada p 158e — ˆ’ and TCGA p — ˆ’testingconfirmed that widespanning gainsamplifications occurred inneighbouring regions in the majority of patients In the ICGC UKSupplementary Fig Additionallycohort n there was a significant cooccurrence of somaticcopynumber gainsamplifications in NAALADL2 with TBL1XR1FDRcorrected Fisher™s exact test — ˆ’ Fig 2a Gainsin this region also significantly correlated with two regionsspanning chromosomes and both gains previously describedas being abundant in prostate cancer Supplementary Data The Canadian cohort n showed a similar pattern of cooccurrence with gainsamplifications spanning the region surrounding NAALADL2 and TBL1XR1 3p253 to 3q29 Fig 2bThere was also a significant cooccurrence with gains in thebeginning of chromosome as well as some sporadic cooccurrence across the genome Fig 2b Supplementary Data These results were supported by the outcome of the same analysisin the TCGA cohort n although several large spikes ofcooccurrence were also observed in regions nottoNAALADL2 and TBL1XR1 as these spikes were not present inthe other two cohorts they most likely represent artefacts Fig 2cSupplementary Data Overall across the three cohorts therewere was a consistent coamplification in region spanning genes between 3p141 and 3q29 While a number of patients hadmultiple CNAs we found no consistent cooccurrence withcommon CNAs such as MYC gain FGFR1 gain PTEN loss RB1loss or NKX31 loss FDRcorrected Fisher™s exact test p The 3q26 region where NAALADL2 and TBL1XR1 are locatedis rich in oncogenes such as PIK3CA SOX2 ECT2 and PRKCIwhich may act to drive tumorigenesis29 We determined thenumber of known oncogenes within this defined region bycomparing the overlapping genes that coamplified withNAALADL2 and TBL1XR1 in alltheNetwork of Cancer Genes database30 This revealed that of genes are known oncogenes including BCL6 ATRand PI3K family members Supplementary Data These resultsconfirm that a high proportion of prostate cancer patientsdevelop large copynumber gains across multiple oncogenes inthis genetic regionthree cohorts againstlocalGains in 3q263132 associate with adverse clinical featuresCommon prostate cancer CNAs such as those in MYC andPTEN are known to associate with higher Gleason grade31Consistent with these findings we also found NAALADL2 andTBL1XR1 amplifications were highly correlated with GradeGroup GG showing that the frequency of NAALADL2 andTBL1XR1 gains tripling between GG1 and GG2 lesions and morethan doubling between GG2 and Table A Chisquaredgoodnessoffit test showed that the distribution of gainsamplifications between Grade groups was significantly different to thedistribution of diploid patientsfor both NAALADL2 andTBL1XR1 p — ˆ’ and p — ˆ’ WhenTable Alteration frequency of NAALADL2 and TBL1XR1 called via the GISTIC2 method in three nonoverlapping primary anconfined radical prostatectomy cohorts from the International Cancer Genome Consortium ICGC and The Cancer GenomeAtlas TCGAICGC UKICGC CANADATCGANAALADL2TBL1XR1NAALADL2TBL1XR1NAALADL2TBL1XR1AlterationDeep DelShallow DelDiploidGainAmplificationTotalnnnnnnThe degree of copy number alteration is discretised into five categories amplification gain representing low and high level copy number increase diploid no significant CNA and shallow and deepdeletion representing low and high level copy number lossCOMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xand compared to those without and Moreover ofthe patients who had their lymph nodesexamined the percentage of patients with lymph node positivitydefined through positivity on haematoxylin and eosin stainingHE was more than double in patients with NAALADL2 orTBL1XR1 gains and compared to those withouta gain and Chisquared goodnessoffit test p and p Finally while only one man in the cohorthad evidence of positive findings in his bone scan we did observea significant between the number of equivocal bone scans inpatients with gains and compared to and in those patients without gainsChisquaredgoodnessoffit test p and p for NAALADL2and TBL1XR1 respectively however the number of expectedcases in each of these categories was less than adding someuncertainty to this result We found no significant difference inthe mean age between patients with different copynumbers ofNAALADL2 or TBL1XR1 KruskallWallis rank sum test p and As gainsamplifications in NAALADL2 and TBL1XR1 coincidewith a cluster of known oncogenes and coincide with clinicalvariables linked to more aggressive disease we also compareddiseasefree survival Comparing patients with gainsamplifications in NAALADL2 and TBLXR1 to those with diploid copies weobserved no significant association in the ICGC UK cohort n although there was a trend towards reduced diseasefreesurvival Supplementary Fig 4A In the larger TCGA cohortn there was a significant reduction in diseasefree survivalin patients with a gain in either NAALADL2 Logrank MantelCox p or TBL1XR1 Logrank MantelCox p Supplementary Fig 4BUnivariable Cox regression confirmed that carrying a gainamplification in NAALADL2 and TBL1XR1 in the TCGA cohortresulted in reduction in diseasefree survival hazard ratio HR CI “ p Forreference weperformed a similar analysis of patients with PTEN deletion orMYC gains two common copy number alterations with provenin prostate cancer3233association with diseasefree survivalWhen patients were stratified solely by CNA status and survivalcompared using the KaplanMeier method those patients withMYC gain or PTEN deletion homo or hemizygous showed nosignificant difference in diseasefree survival Logrank MantelCox p and p respectively while those stratified byNAALADL2 gain TBL1XR1 gain or both NAALADL2 andTBL1XR1 gain showed significant differences in survival Logrank MantelCox p Supplementary Fig 5A“E Univariable Cox regression estimated the hazard ratios for thesecopynumber alterations as CI “ CI “ and CI “ for MYCPTEN and NAALADL2TBL1XR1 respectively We also comparedthe diseasefree survival of patients with only a copynumberalteration in each of the four genes where each group wasmutually exclusive Supplementary Fig 5F G This showed thaton the whole patients with CNAs in NAALADL2TBL1XR1 hadreduced or equal diseasefree survival as those with either onlyMYC gain or only PTEN loss Patients with copy number gains inboth had a worse prognosis All clinical data is available inSupplementary Data Fig Genomewide cooccurrence with NAALADL2 and TBL1XR1 gainsamplifications The Y axis shows “log10 qvalues from a Fishers exact testbetween gainamplifications in NAALADL2 and cooccuring genes Thedotted line represents the threshold for statistical significance aftercorrection for multiple testing a Significantly cooccurring gains across thegenome in the ICGC UK cohort b Significantly cooccurring gains acrossthe genome in the ICGC Canada cohort c Significantly cooccurring gainsacross the genome in the TCGA cohort NAALADL2 and TBL1XR1 cytobandpositions are labelled All Fisher tests use NAALADL2 gain or amplificationas the altered group Full results are detailed in Supplementary Data compared to common CNAs such as PTEN loss and MYC gainthe alteration frequency of NAALADL2 and TBL1XR1 was morecorrelated with higher Gleason grade groups Spearmans rho was p p for NAALADL2 and TBL1XR1and p for PTEN and MYC respectivelySupplementary Fig 3AMoreover we also noted the same pattern ofincreasingfrequency of gains with T stage Chisquared goodnessoffit testp and p respectively Table Patients with gains exhibited differences in the location of thetumour within the prostate with and of thosewith NAALADL2 and TBL1XR1 gains having tumoursinoverlapping and multiple zones compared to just and for those without gains Chisquared goodnessoffit testp and p There was also an increased relativenumber of positive surgical margins Chisquared goodnessoffittest p and p in patients with gains As CNAs in NAALADL2 and TBL1XR1 were associated withclinical characteristics such as Gleason grade group and T stagewe used multivariable Cox regression models to confirm that anychanges in survival were driven by these associations and foundthat copy number gains in NAALADL2 and TBL1XR1 were nolonger significant once corrected for Gleason grade and T stagep Supplementary Data These results suggest thatthe differences in diseasefree survival seen when stratified byCOMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xTable The frequency of NAALADL2 and TBL1XR1 gainamplifications by Gleason Grade Group in the TCGA cohortGrade groupGG1ObservedExpected within GGGG2ObservedExpected within GGGG3ObservedExpected within GGGG4ObservedExpected within GGGG5ObservedExpected within GGTotal nNAALADL2DiploidGainTBL1XR1DiploidGainTotalGrade groups defined as Grade Group Gleason score ‰¤ Grade Group Gleason score Grade Group Gleason score Grade Group Gleason score Grade Group Gleason scores and Displayed are the numbers of patients observed with gain or without diploid a gainamplification in this region in each Grade Group Additionally the expected number ofpatients estimated to be within each category is also shown along with the percentage of each Grade Group which is made up by patients with or without a gain Bold values indicate the overallpercentage of the group with a given copynumber state All clinical data detailed in Supplementary Data Table The frequency of NAALADL2 and TBL1XR1 gainamplifications by T stage in the TCGA cohortT stageT2aObservedExpected within T stageT2bObservedExpected within T stageT2cObservedExpected within T stageT3aObservedExpected within T stageT3bObservedExpected within T stageT4ObservedExpected within T stageTotal nNAALADL2DiploidGainTBL1XR1DiploidGainTotalDisplayed are the numbers of patients observed with gain or without diploid a gainamplification in this region in each T stage Additionally the expected number of patients estimated to be withineach category is also shown along with the percentage of each T stage which is made up by patients with or without a gain Bold values indicate the overall percentage of the group with a given copynumber state All clinical data detailed in Supplementary Data COMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xgainamplification status are driven by strong association withthese clinical variablesIn the ICGC cohortsindividuals with somatic singlebasealterations in NAALADL2 also associated with reduced diseasefree survival in a combined ICGC cohort as well as associatingwith reduced diseasefree and overall survival in an early onsetprostate cancer cohort ICGC EOPC Denmark Singlebasesubstitutions in TBL1XR1 were only associated with diseasefreesurvival in the ICGC EOPC cohort Supplementary Fig Singlebase alterations did not occur with a frequency greater than onein any single base in NAALADL2 or TBL1XR13q263132 gains cooccur with proproliferative transcriptionTo determine the potentialfunctional consequences of gainswithin the NAALADL2 and TBL1XR1 amplicon mRNA expression profiles were explored using the TCGA RNAseq dataDESeq2 was used to determine differentially expressed genesbetween patients with copynumber gains for both NAALADL2and TBL1XR1 compared to those without For NAALADL2 therewere differentially expressed genes DEGs and DEGsfor TBL1XR1 when the two groups were compared FDR Supplementary Data Our previous study on NAALADL2identified nine genes which were reciprocally regulated by overexpression or knockdown of NAALADL226 Of these nine wefound that three cancer antigen XAGE1B adhesionmotiliy regulator SPON2 and AR regulator HN1 were significantly differentially expressed p in patients with a NAALADL2 gainand in the same direction as the overexpression model2634“When comparing the DEGs between patients with and withoutin either NAALADL2 or TBL1XR1 wegainsamplificationsobserved that of the DEGs overlapped between NAALADL2 and TBL1XR1 Fig 3a of the geneswere located within the locus we identified as coamplified withNAALADL2 and TBL1XR1 and were differentially expressedconsistent with a mechanism of selfregulating expression2425TBL1XR1 was one ofsignificant overlapping DEGsNAALADL2 was just on the boundary of statistical significanceFDR corrected Wald test p Supplementary Fig theNAALADL2 has been shown to be coexpressed with numberof androgen regulated proteins and contains a number of ARbinding sites and TBL1XR1 is an AR coactivator and may beinvolved in AR cistrome reprogramming18263738 We thereforelooked at overlap between androgen regulated genes with ARbinding sites full or partial and genes demonstrated to beandrogen regulated following R1881 stimulation in at least twoindependent studies3739 shared genes were differentiallyexpressed in patients with NAALADL2 and TBL1XR1 gainsamplifications that contained AR binding sites and demonstratedandrogen regulation by R1881 genes had either aAR binding motif were androgen regulated in two or morestudies or both Fig 3bOf the overlapping DEGs a total of were knownoncogenesSupplementary Data which may drive anaggressive clinical phenotype Of note was PI3K family membersPIK3C2G PIK3CA PIK3CB PIK3R4 Mucin family membersMUC1 MUC4 and MUC6 and other prostate cancer associatedgenes such as SMAD4 SOX9 and SPOP794041 Additionallyseveral genes which form commercial prognostic assays were alsodifferentially expressed such as the Decipher assay NFIB LASP1ZWILCH THBS2 COL1A2 and COL5A142 Oncotype DX assaySFRP4 COL1A1 KLK2 TPX24344 and the Prolaris assayASPM BUB1B CENPF and FOXM145We inspected of the top most significant shared DEGs usingunsupervised hierarchal clustering Fig 3b SupplementaryData DEGs mostly displayed upregulation consistent with agenedosage effect Fig 3b24 Enrichment for biological processes was assessed by Geneset enrichment analysis GSEA forNAALADL2 and TBL1XR1 gainsseparately and by overrepresentation analysis ORA on the shared DEG list usingWebGestalt46GSEA on the individual lists of DEGs showed that despite a largeoverlap the enriched biological processes did differ between the twogenes patients with a gain in NAALADL2 showed enrichment inprocesses related to NADH dehydrogenase complex assemblyFDR mitochondrial respiratory chain complex assemblyFDR translational initiation FDR cytochromecomplex assembly FDR protein localisation toendoplasmic reticulum FDR and cytoplasmic translationFDR Supplementary Data Patients with a gain inTBL1XR1 showed enrichment in mitotic cell cycle phase transitionchromosome segregation actin filamentbased movement microtubule cytoskeleton anisation involved in mitosis regulation ofcell cycle phase transition cell cycle G1S phase transition FDR as well as a number of other processes SupplementaryData To understand the combined effect of gainsamplification inthese genes we investigated overrepresentation of processes in theDEGs which were common to both NAALADL2 and TBL1XR1In the shared DEG list the significantly enriched Gene OntologyGO biological processes were all involved in the cell cycle cyclepathway including mitotic regulation and chromosome segregation Fig 3c Supplementary Data These findings support ahypothesis whereby gains in NAALADL2 and TBL1XR1 concomitantly bring about mRNA expression changes which supportan aggressive proproliferative phenotype in primary prostatecancerDiscussionIn this study we present evidence that somatic copynumber gainsin NAALADL2 and TBL1XR1 are more frequent in high gradeand aggressive forms of prostate cancer These results are bolstered by studies which have identified CNAs in this region inmCRPC however to our knowledge this is the first time thesegains have been reported in neuroendocrine disease47 We alsodemonstrate that NAALADL2 and TBL1XR1 gains occur in anearlier setting cooccurring with gains in neighbouring genes Amajor barrier to the adoption of CNA based tests in the clinic isthe reliance on expensive NGS approaches as well as sufficientsequencing depth and coverage to assess overall copynumberburden The discovery of smaller clinically significant loci couldallow for cheaper quicker targeted approaches particularly if asingle loci can elude to gainsamplifications in a larger regionsurvivalto diseasefreeIn primary prostate cancer Gainsamplifications in this regionassociated with Gleason grade tumour stage number of positivelymph nodes bone scan results and as these variables contributetostratified byNAALADL2TBL1XR1 status also have altered diseasefree survival times Our work is supported by previous studies that haveeluded to the clinical significance of this locus particularly asgermline SNPs within this locus have been associated with higherGleason grade tumours and more aggressive disease14 This alsosupports smaller studies such as those by HeselmeyerHaddadet al who identified two out of seven patients with gains inTBL1XR1 in recurrent prostate cancer48 However these studiesinvestigated these genes in isolation na¯ve to the larger context inwhich these alterations occur Here we have found that gainsamplifications atthis locus not only coamplify with otherdescribed oncogenes but associate with much larger transcriptional changes which are consistent with the observed aggressiveclinical phenotypetimepatientsCOMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xFig Transcriptomic changes in patients with NAALADL2TBL1XR1 gains a Venn diagram showing the number and percentage of overlapping DEGsbetween patients with NAALADL2 gainamplification and TBL1XR1 gainamplification overlap b Venn diagram showing the number ofNAALADL2 and TBL1XR1 DEGs and genes with identified AR binding sites determined through ChIPSeq and AR knockdown and genes shown to beandrogen regulated following R1881 stimulation c Unsupervised hierarchal clustering of the top most significant DEGs bar beneath upper dendrogramshows copynumber status of patients where red is patients with a gain in both NAALADL2 and TBL1XR1 and grey represents those without gainamplification in these genes Heatmap represents meancentred z scores derived from RKPM values d Chord diagram showing significantly overrepresented GO biological processes and key genes within these processes All clinical data detailed in Supplementary DataCOMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xOverall changes in copynumber burden have been shown to beindicative of genetically unstable tumours and predict prostatecancer relapse5 Many single CNAs have already been describedthat predict PSA recurrence after radical prostatectomy includingPTEN loss cooccurrence of PTEN FAS 10q2331 and PAPSS210q232“10q2331 loss a loss of 16q with or without a loss ofPTEN a loss within 6q 13q gains in MYC 11q1317 7q and aconcurrent loss of 8p22 with a gain of 8q2487“ Compared towellknown CNAs such as PTEN loss and MYC amplification wehave observed that Gainsamplifications in NAALADL2TBL1XR1equally or better segregate patients who will have reduced diseasefree survivalThe gainsamplifications in NAALADL2TBL1XR1 also corresponded to a significantincrease in both NAALADL2 andTBL1XR1 mRNA supporting previous studies that have described upregulation of these genes and linked them to poor prognosis in various cancers19“ This suggests that gains in thesegenes may cause increased expression of NAALADL2 andTBL1XR1 in cancers We also noted a number of the differentiallyexpressed genes between patients with and without a gainamplification in NAALADL2TBL1XR1 have been shown to beandrogen regulated however further work is required to determine if gainsamplifications in this region cause changes in ARtranscriptional regulation through cis regulatory elements or as adirect consequence of the genes altered in this region1837In those patients with these gains we noted transcriptionalchanges in several genes associated with aggressive prostatecancer including differential expression of genes appertaining toprognostic assays such as Decipher Oncotype DX and Prolaris aswell as families such as mucins50“ This may explain theaggressive clinical phenotype observed in these patients We alsoobserved that when weighted individually there were differencesin enrichment of biological processes between those with NAALADL2 gains and TBL1XR1 gains suggesting that each generesults in some unique cellular changesOur finding that gains in the 3q26 locus result in concurrentexpression of oncogenes located within this region and theirdownstream targets identifies multiple potential therapeutic avenues warranting further investigations This study centred aroundtwo genes NAALADL2 and TBL1XR1 both of which areattractive therapeutic targets with TBL1XR1 previously suggestedas a potential cancer target operating via the TGFβ signallingpathway and potentially regulating AR signalling5354 Additionally the tumour specificity of NAALADL2 and basal membranouslocalisation makes it potentially accessible using antibodydrugconjugates13 This approach may be feasible if like other familymembers such as PSMA antibody binding results in subcellularinternalisation12 Moreover several of the oncogenes in whichgains cooccur as well as the downstream oncogenes activatedfrom gains in the 3q26 region such as ATR PI3K family members PIK3C2G PIK3CA PIK3CB PIK3R4 MUC4 BCL6 SOX9can be therapeutically targeted or have been suggested as therapeutic targets in cancer5155“ In the PI3K pathway PIK3CBspecific inhibitors may have utility in patients with mutationsamplifications andor fusion of this gene59 These findings mayhave clinical relevence as it has been reported by de Bono et althat many individuals who had durable year responses toPIK3CBspecific inhibition harboured activating mutation oramplification in PIK3CB60 and phase II trials of ipatasertib anAkt inhibitor targeting the PI3KAkt axis has shown promise inlate stage mCRPC61 Together our results suggest that largescalegenomic gainsamplifications occur in the 3q26 region in a hi

Thyroid_Cancer

Radial shock waves prevent growth retardation caused by the clinically used drug vismodegib in ex vivo cultured bonesSowmya Ramesh123 Lars Svendahl245 Vrisha Madhuri135 farasat Zaman2In childhood medulloblastoma patients the hedgehog antagonist vismodegib is an effective anticancer treatment but unfortunately induces irreversible growth arrests and growth impairment limiting its use in skeletally immature patients We hypothesized that radial shock wave treatment rSWT may protect druginduced growth impairment owing to its osteogenic effects Fetal rat metatarsal bones were exposed to vismodegib day “ nM andor rSWT single session other bones from day were continuously exposed to a Gli1 antagonist GANT61 µM andor rSWT single session Control bones were untreated The bone length was measured at intervals histomorphometric analysis and immunostaining for PCNA Gli1 and Ihh were performed on the sectioned bones Bones treated with vismodegib showed impaired bone growth reduced height of the restingproliferative zone and reduced hypertrophic cell size compared to control In vismodegib treated bones a single session of rSWT partially rescued bone growth increased the growth velocity hypertrophic cell size and restored growth plate morphology Bones exposed to GANT61 showed impaired bone growth and disanized growth plate while when combined with rSWT these effects were partially prevented Locally applied rSWT had a chondroprotective effect in rat metatarsal bones and suggest a novel strategy to prevent growth impairment caused by vismodegibHedgehog Hh proteins are wellknown to be overexpressed in paediatric medulloblastoma1 Mutations that occur in the family of Hhpathway genes such as patched1 suppressor of fuse and smoothened leads to an increased level of the gliomaassociated oncogene Gli1 a downstream transcription factor of Hh2 In the clinic hedgehog inhibitors are used to decrease the Hhactivity and thereby impede tumor progression3“ However stable expression of the Hhgene is essential to maintain chondrocyte proliferation and hypertrophy during bone growth6 A recent study reported that prolonged exposure to vismodegib a Hhantagonist in children with medulloblastoma resulted in irreversible growth plate fusion causing growth arrest of long bones78 Preclinical studies in young mice exposed to a Hhantagonist also showed growth arrests and bone growth defects9 Mechanistic studies revealed that even brief exposure to a Hhinhibitor was enough to damage the growth plates by diminishing the numbers of reserve and proliferative chondrocytes9 These findings further imply that it may not be possible to arrive at a dose that selectively targets tumor growth with no sideeffects on bone development Therefore in children a protective strategy for growth plate shielding without interfering with the desired anticancer effects of vismodegib in the neural tissue is highly desiredIn vitro studies using cultured rat metatarsal bones10 and in a0vivo studies in rabbits11 and humans12 have shown that radial shock wave treatment rSWT a noninvasive modality used in the clinic have stimulatory effects on bone growth10 Furthermore the observed stimulation of chondrocyte proliferation and hypertrophy induced by rSWT was partially linked to local upregulation of Gli1 in cultured metatarsal bones10 Interestingly previous in a0vitro studies revealed that highenergy shock wave treatment increased the uptake of chemotherapy agents13 1Department of Paediatric Orthopaedics Christian Medical College Vellore India 2Division of Paediatric Endocrinology Department of Women™s and Children™s Health Karolinska Institutet Solna Sweden 3Centre for Stem Cell Research A Unit of inStem Bengaluru Christian Medical College Bagayam Vellore India 4Paediatric Endocrinology and Metabolism Astrid Lindgren Children²s Hospital Karolinska University Hospital Solna Sweden 5These authors contributed equally Lars Svendahl and Vrisha Madhuri email sowmyarameshkiseScientific RepoRtS 101038s41598020699040Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Rescuing effects of rSWT on vismodegib treated bones a Fetal rat metatarsal bones cultured exvivo were treated with the Hhinhibitor vismodegib a0nM for a0days n dotted line a single session of highenergy rSWT a0Hz a0mJ n or both n and thereafter followed for a0days The graph shows increases in bone length over time a0from day All error bars indicate SD Twoway ANOVA was applied b Graph shows the increase in the growth velocity on day of control vismodegib rSWT and vismodegib rSWT treated bones All error bars indicate SD Representative images of metatarsal bones stained with Alcian blue c untreated control d vismodegib e rSWT and f vismodegib rSWT Magnification 10x g height measurements of R P zone and h hypertrophic cell size Quantification of immunostaining for i Gli1 and j Ihh using the ImageJ software p p p thereby lowering the dose of the drug when applied to cell lines derived from human osteosarcoma14 human colorectal adenocarcinoma15 and anaplastic thyroid cancer16 The effect was mediated by a transient increase in cell membrane permeability allowing passage of a higher concentration of the drug16A recent report suggested that locally applied rSWT can promote longitudinal bone growth of rat metatarsal bones cultured exvivo in the absence of serumgrowth factors10 Based on these findings we hypothesized that rSWT may also have the capacity to prevent growth failure caused by Hhinhibitors We aimed to investigate the potential for rSWT to prevent growth retardation caused by two different Hhantagonists vismodegib and the Gli1 antagonist GANT61 in a wellestablished model of cultured fetal rat metatarsal bonesResultsEffect of vismodegib on bone growth and rescuing effects of rSWT To allow transient inhibition of Hhactivity cultured fetal rat metatarsal bones were treated with vismodegib for a0days whereafter growth was monitored for another a0days without any treatment Bones treated with vismodegib grew less than untreated controls and the difference was significant when measured on day ± vs ± bone length increase from day respectively p Fig a01aTo address the primary objective of this study if rSWT can prevent growth failure caused by hedgehog inhibition we first studied if a single exposure to rSWT can rescue metatarsal bone growth after transient exposure to a0days treatment with vismodegib Indeed bones treated with rSWT single exposure on day in combination with vismodegib day “ grew significantly better than bones treated with vismodegib alone when measured Scientific RepoRtS 101038s41598020699040Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Rescuing effects of rSWT on GANT61 treated bones a Fetal rat metatarsal bones cultured exvivo treated with the Hhinhibitor GANT61 a0µM n a single session of rSWT a0Hz a0mJ n or both n and followed for a0days The graph shows increases in bone length a0from day All error bars indicate SD Twoway ANOVA was applied Representative images of metatarsal growth plates stained with Alcian blue b untreated control c GANT61 d rSWT and e GANT61 rSWT Magnification 10x f Height measurements of R P zone and g hypertrophic cell size Quantification of immunostaining for h PCNA and i Gli1 using the ImageJ software p p on day ± vs ± respectively p Fig a01a Also the growth velocity tended to be higher in rSWT vismodegib compared to vismodegib alone Fig a01bGrowth plate morphology Histological evaluation revealed severe disruption in the growth plate morphology in the bones treated with vismodegib compared to control Fig a01c“d The disrupted growth plate morphology in vismodegib treated bones was found to be more normal in bones receiving rSWT alone Fig a01e and rSWT vismodegib Fig a01f When comparing vismodegib treated bones to controls we found reduced height of the resting proliferative R P zone ± a0µm vs ± a0µm respectively p Fig a01g The height of the R P zone tended to be increased in bones exposed to rSWT vismodegib when compared to vismodegib alone ± a0µm vs ± a0µm respectively p ns Fig a01g Histomorphometric analysis showed decreased size of hypertrophic chondrocytes in the bones treated with vismodegib compared to control ± a0µm vs ± a0µm respectively p Fig a01h In bones treated with rSWT vismodegib the hypertrophic chondrocytes were significantly larger compared to vismodegib alone and similar in size as in untreated control bones ± a0µm ± a0µm and ± a0µm respectively p vs vismodegib p ns vs control Fig a01h Immunostaining for Gli Fig a01i and Ihh Fig a01j did not show any significant effects of vismodegib andor rSWTShock wave treatment prevents bone growth retardation caused by GANT61 To study the effects of continuous inhibition of Hhactivity fetal rat metatarsal bones were treated with the Gli1 antagonist GANT61 from day of culture until the termination of the experiment on day Already on day GANT61 induced a pronounced suppression of the bone growth ± ± p vs control which remained at endpoint on day ± ± p vs control Fig a02aNext we asked if a single exposure to rSWT can rescue from growth retardation caused by continuous Hhinhibition induced by GANT61 Indeed metatarsal bones exposed to rSWT on day were rescued from GANT61induced growth retardation already from day of culture ± ± p vs GANT61 alone which remained until endpoint ± ± p vs GANT61 alone Fig a02aGrowth plate morphology Compared to control in the bones continuously treated with GANT61 growth plate morphology was found to be disturbed with disanized chondrocyte columns on day Fig a02b c Bones treated with rSWT alone showed anized growth plate morphology Fig a02d The growth rescuing Scientific RepoRtS 101038s41598020699040Vol0123456789wwwnaturecomscientificreports 0ceffect of rSWT in GANT61 treated bones was accompanied by a normalization of growth plate morphology including anized chondrocyte columns Fig a02eHistomorphometric analyses showed no significant differences in the height of the R P zone between GANT61 treated bones with or without rSWT ± a0µm ± a0µm p ns Fig a02f There was an increased size of hypertrophic chondrocytes in rSWT GANT61 group when compared to GANT61 alone ± a0µm ± a0µm p Fig a02g Furthermore the number of proliferative chondrocytes tended to be lower in GANT61 treated bones when compared to control ± cells ± cells p ns Fig a02h The number of proliferative PCNA positive cells tended to be higher in the rSWT GANT61 group compared to GANT61 alone ± cells ± cells p ns Fig a02h Immunoexpression of Gli1 tended to be reduced in GANT61 treated bones p ns vs control whereas bones treated with rSWT GANT61 tended to have higher Gli1 expression compared to GANT61 alone p ns Fig a02iDiscussionWe aimed to investigate the potential for locally applied rSWT to prevent bone growth impairment caused by the hedgehog inhibitor vismodegib a therapeutic investigational drug using a wellestablished model of ex a0vivo cultured fetal rat metatarsal bones Herein we report that a single session of rSWT partially prevented growth retardation caused by both transient and continuous Hhinhibition induced by vismodegib and GANT61 respectively The growth rescuing effects by rSWT were accompanied by preservation of growth plate morphology disrupted by the Hhinhibitors Altogether our data suggest that rSWT has the potential to noninvasively protect bones from growth retardation caused by vismodegibBone growth is majorly dependent on the preservation of a unique anization of chondrocytes in the growth plate17 Recent reports have demonstrated that long term exposure to vismodegib the first Hhantagonist approved in the US by FDA led to permanent growth impairment in children with medulloblastoma78 To date no successful strategy that targets tumor cells with no adverse effect on longitudinal bone growth has been described Previous reports have shown that rSWT a treatment modality that is already used in children for musculoskeletal indications18 can stimulate longitudinal bone growth locally in exvivo cultured metatarsal bones even in the absence of any systemic growth factors10 Proinflammatory cytokines are also known to impair bone growth19 and interestingly shockwave treatment has been shown to reduce inflammation and apoptosis while stimulating the regeneration of various tissues2021 These findings encouraged us to expand this knowledge and further investigate the potential for rSWT to prevent bone growth impairment caused by HhinhibitorsVismodegib at a0nM concentration has shown to impair bone growth in exvivo cultured metatarsal bones22 and decrease proliferation of the precursors of a0cerebellar granule neurons23 while in a0vivo studies in a model of medulloblastoma have also shown that vismodegib inhibits Gli1 at a IC50 of a0nM24 a similar range of concentration as used in the present study In young mice transient inhibition of the Hh pathway has been reported to cause permanent defects in bone and growth plate structure9 Similar to the previous in a0vivo observations in young mice9 our histomorphometric growth plate data suggest that partial loss of Hhactivity may result in the breakdown of chondrocyte columnar anization and reduced size of hypertrophic chondrocytes Also the disrupted growth plate ultrastructure caused by Hhinhibition explains the observed growth deficit in our study model system Besides undesired apoptosis of stemlike cells within the growth plate is another wellknown contributing factor linked to growth retardation caused by anticancer drugs2526Our key finding is that a single administration of rSWT not only prevented bone growth retardation caused by transient exposure to the Hhinhibitor vismodegib but also rescued bone growth under a condition of continuous Hhinhibition induced by another Hhinhibitor GANT61 Furthermore rSWT also improved growth velocity and restored growth plate morphology in bones exposed to vismodegib or GANT61 Thus our findings highlight the potential for shock wave technology to be developed as a new and safe treatment strategy to minimize deleterious effects of Hhinhibitors selectively in the growth plates of treated childrenHedgehog signaling drives chondrocyte proliferation and hypertrophy in the growth plate cartilage6 From in a0vitro studies we know that Hhinhibitors decrease the expression of Gli1 and induce cell cycle arrest in prostate cancer cells27 Despite rSWT rescued bones from Hhinhibitor impaired bone growth we did not see significant alterations in the expression of Gli1 and Ihh suggesting a crosstalk between hedgehog signaling and other pathways28 We speculate that the bone rescuing effect of rSWT is more evident if there is any ongoing disturbance within growth plate chondrocytes Indeed it was interesting to note that despite continuous exposure to a Hhinhibitor a single session of rSWT could partially rescue the bone growthOur study has several limitations Firstly the bone growth rescuing effects of rSWT were documented in an exvivo bone culture model and we do not know if this will be applicable under in a0vivo conditions Nevertheless in a0vivo studies in rats or mice are of limited value when it comes to exploring the potential for rSWT to rescue from vismodegib induced bone growth impairment as their growth plates do not normally fuse29 Secondly we only applied a single dose of rSWT while multiple sessions could potentially be even more efficient when it comes to preventing growth impairment caused by Hhinhibitors Thirdly the concentration of vismodegib used in the present study is different from the plasma concentration a0µM achieved in children30 Nevertheless mimicking a gradual decline in bone growth in order to test the rescuing effect of rSWT is more important in our experimental setting We therefore claim a protective effect and not a clinical effect which will require more rigorous testing Consequently our proof of concept finding s up a window of opportunity to explore the potential for locally applied rSWT to prevent bone growth impairment caused by vismodegib as it may not be possible to extrapolate the doses used for preclinical studies to a clinical setting3132In summary we here present a novel treatment strategy based on clinically used rSWT to locally prevent bone growth impairment caused by vismodegib a promising anticancer drug used in children with medulloblastoma Scientific RepoRtS 101038s41598020699040Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A hypothetical schematic diagram showing how extracorporeal radial shockwave treatment rSWT may be administered to lowhigh growth velocity areas a0encircled selectively to restore vismodegibinduced bone growth impairment in children with medulloblastomaFig a0 Before any clinical studies our promising ex a0vivo findings need to be validated in an in a0vivo animal model like the rabbit where growth plate fusion normally occurs just like in humansMethodsThe experiments were approved by the local institutional review board Min No and the institutional animal ethics committee Min No at Christian Medical College Vellore Animal care compiled with the Guide for the Care and Use of Laboratory Animals A a0radial shock wave machine from Radialspec Medispec Gaithersburg MD USA was used for the studyBone an culture system Metatarsal bones were microdissected from the hind limbs of Sprague Dawley rat fetuses sacrificed on day of gestation Ex a0vivo cultures were performed as previously reported33 Briefly each bone was transferred to a 24well plate and cultured in medium containing DMEMF12 a0mM betaglycerophosphate ascorbic acid a0µgml and gentamycin The medium was replenished every two days Figure a0 shows the experimental overviewExposure to Vismodegib Vismodegib was purchased from Selleck Chemicals Houston Texas USA The experimental setup consisted of four groups Metatarsal bones were cultured for a0days and were either a treated with vismodegib a0nM n from day to b a single exposure to rSWT impulses a0Hz a0mJ n on day c vismodegib rSWT n or d were left untreated control n The bone length was measured on days and Exposure to GANT61 GANT61 was purchased from Sigma Aldrich St Louis Missouri USA Metatarsal bones were treated with the a Hhinhibitor GANT61 a0µM from day to n b a single exposure Scientific RepoRtS 101038s41598020699040Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Experimental overview Middlethree metatarsals from the hind limb of embryonic rat at a 1920th day of gestation were dissected and b cultured exvivo c Bones were either exposed to vismodegibGANT61 a single session of highenergy radial shock wave treatment rSWT vismodegibGANT61 with rSWT or left untreated During this time d total bone length measurements at different intervals are performedto rSWT on day impulses a0Hz a0mJ n c GANT61 rSWT or d were left untreated control n The bone length was measured on days and Bone length measurement Digital images were captured for bone length measurements using an inverted microscope Leica Microsystems All measurements were performed by one of the investigators blinded to the nature of the group using an inbuilt ˜measurement tool™ Bone growth is expressed as percent bone length increase from day Bowed bones were measured in two parts added togetherQuantitative histology and immunostaining After termination of the culture the metatarsal bones were fixed with paraformaldehyde embedded in paraffin and fivemicrometer sections were cut along the longitudinal axis proximal to distal followed by staining with SafraninO and Alcian blue The microscopic description of the growth plate morphology included an assessment of the anization of the chondrocyte column Histomorphometric analysis was performed to measure the height of the restingproliferating zone at five different regions of the growth plate and the size of hypertrophic chondrocytes Hypertrophic cells were defined by a height along the longitudinal axis greater than a0µm Eight hypertrophic chondrocytes from the proximal and distal growth plate were measuredImmunostaining was performed as previously described34 Antigen retrieval was performed in citrate buffer at ° Celsius and endogenous peroxidase activity was quenched with H2O2 in methanol for a0min followed by a wash with PBS For immunostaining sections were blocked with bovine serum albumin for a0h incubated with primary antibodies dilution Gli1 Abcam Cambridge MA USA and Ihh mouse monoclonal antibody Santa Cruz Biotechnology Dallas TX USA overnight at ° Celcius After incubation for a0h with secondary polyclonal antimouse or antirabbit biotinylated antibody DakoCytomation Glostrup Denmark dilution sections were incubated with ABC solution and developed with diaminobenzidine Sections were counterstained with Alcian blue Nonimmune immunoglobin G IgG of the same species as the primary antibodies were used as negative controls Three to five bones per group were analyzed To quantify immunostaining the ImageJ software National Institutes of Mental Health Bethesda MD USA was used and the percentage of DAB positivity was calculated digitally using a plugin IHC profilerStatistical analysis All statistics were carried out using GraphPad Prism GraphPad Software Inc La Jolla CA USA Data were summarized using means ± SD for the bone length measurements and histomorphometric assessments A twoway ANOVA with Dunnett multiple comparisons test35 was performed to examine the change in bone length in terms of treatment and days Pairwise comparisons were done corrected for the alpha levels Margin plots with SD were presented to visualize the change in bone length Kruskal“Wallis and Man“Whitney U test were performed when the data were not normally disturbed A p value of was considered to indicate a significant differenceData availabilityAll data generated or analysed during this study are included in this manuscriptScientific RepoRtS 101038s41598020699040Vol1234567890wwwnaturecomscientificreports 0cReceived March Accepted July References Gerber N et al Recent developments and current concepts in medulloblastoma Cancer Treat Rev “ Gorlin R J Neurocutaneous Disorders Phakomatoses and Hamartoneoplastic Syndromes “ Springer Berlin Gajjar A J Robinson G W Medulloblastoma”translating discoveries from the bench to the bedside Nat Rev Clin Oncol Kool M et al Molecular subgroups of medulloblastoma an international metaanalysis of transcriptome genetic aberrations and clinical data of WNT SHH Group and Group medulloblastomas Acta Neuropathol “ Kieran M W Targeted treatment for sonic hedgehogdependent medulloblastoma Neurooncology “ Ohba S Hedgehog signaling in endochondral ossification J Dev Biol Robinson G W et al Irreversible growth plate fusions in children with medulloblastoma treated with a targeted hedgehog pathway inhibitor Oncotarget Kieran M W et al Phase I study of oral sonidegib LDE225 in pediatric brain and solid tumors and a phase II study in children and adults with relapsed medulloblastoma Neurooncology “ Kimura H Ng J M Curran T Transient inhibition of the Hedgehog pathway in young mice causes permanent defects in bone structure Cancer Cell “ Ramesh S Zaman F Madhuri V Svendahl L Radial extracorporeal shock wave treatment promotes bone growth and chondrogenesis in cultured fetal rat metatarsal bones Clin Orthop RelatRes “ Gollwitzer H et al Radial extracorporeal shock wave therapy rESWT induces new bone formation in a0vivo results of an animal study in rabbits Ultrasound Med Biol “ Kertzman P Cs¡sz¡r N B Furia J P Schmitz C Radial extracorporeal shock wave therapy is efficient and safe in the treatment of fracture nonunions of superficial bones a retrospective case series J Orthop Surg Res Sansone V et al Shockwave Medicine vol “ Karger Publishers Basel Palmero A et al High energy shock waves enhance the cytotoxic effect of doxorubicin and methotrexate to human osteosarcoma cell lines Oncol Rep “ Anticancer Res “ Canaparo R et al High energy shock waves HESW for sonodynamic therapy effects on HT29 human colon cancer cells Gambihler S Delius M In a0vitro interaction of lithotripter shock waves and cytotoxic drugs Br J Cancer “ Pines M Hurwitz S The role of the growth plate in longitudinal bone growth Poult Sci “ Speed C A systematic review of shockwave therapies in soft tissue conditions focusing on the evidence Br J Sports Med FernandezVojvodich P Zaman F Svendahl L Interleukin6 acts locally on the growth plate to impair bone growth Ann Ciampa A R et al Nitric oxide mediates antiinflammatory action of extracorporeal shock waves FEBS Lett “ “ Rheum Dis e24“e24 Chen YL et al Extracorporeal shock wave therapy effectively prevented diabetic neuropathy Am J Transl Res Zaman F et al Humanin is a novel regulator of Hedgehog signaling and prevents glucocorticoidinduced bone growth impairment FASEB J “ Sharpe H J et al Genomic analysis of smoothened inhibitor resistance in basal cell carcinoma Cancer Cell “ Wong H et al Pharmacokinetic“pharmacodynamic analysis of vismodegib in preclinical models of mutational and liganddependent Hedgehog pathway activation Clin Cancer Res “ Eriksson E et al Bortezomib is cytotoxic to the human growth plate and permanently impairs bone growth in young mice PLoS Zaman F Fadeel B Svendahl L Proteasome inhibition therapies in childhood cancer Leukemia Gonnissen A et al The hedgehog inhibitor GANT61 sensitizes prostate cancer cells to ionizing radiation both in a0vitro and in a0vivo ONE e50523 Oncotarget Zhong L Huang X Karperien M Post J N The regulatory role of signaling crosstalk in hypertrophy of MSCs and human articular chondrocytes Int J Mol Sci “ Roach H I Mehta G Oreffo R O Clarke N M Cooper C Temporal analysis of rat growth plates cessation of growth with age despite presence of a physis J Histochem Cytochem “ Gajjar A et al Phase I study of vismodegib in children with recurrent or refractory medulloblastoma a pediatric brain tumor consortium study Clin Cancer Res “ Singer T Appropriate Dose Selection”How to Optimize Clinical Drug Development “ Springer Berlin ReaganShaw S Nihal M Ahmad N Dose translation from animal to human studies revisited FASEB J “ Chagin A S Chrysis D Takigawa M Ritzen E Svendahl L Locally produced estrogen promotes fetal rat metatarsal bone growth an effect mediated through increased chondrocyte proliferation and decreased apoptosis J Endocrinol “ Bov©e J V van den Broek L J CletonJansen AM Hogendoorn P C Upregulation of PTHrP and Bcl2 expression characterizes the progression of osteochondroma towards peripheral chondrosarcoma and is a late event in central chondrosarcoma Lab Investig “ Vincent K et al Aging of mouse intervertebral disc and association with back pain Bone “ AcknowledgementSR would like to personally acknowledge Dr Sumith K Mathew Assistant Professor Clinical Pharmacology Christian Medical College for his input on calculating for clinically relevant dose This study was supported by internal grants received from Christian Medical College Vellore Centre for Stem Cell Research Swedish Research Council Swedish Childhood Cancer Foundation HKH Kronprinsessan Lovisas f¶rening Ake Wibergs Stiftelse and Karolinska Institutet Sweden access funding provided by Karolinska InstituteAuthor contributionsSR LS VM FZ designed the study SR carried out data collection and statistical analysis SR LS VM FZ analysed data SR wrote the initial draft of the manuscript FZ VM and LS contributed to the revision of the manuscript All authors contributed to the interpretation of resultsScientific RepoRtS 101038s41598020699040Vol0123456789wwwnaturecomscientificreports 0ccompeting interests The authors declare no competing interestsAdditional informationCorrespondence and requests for materials should be addressed to SRReprints and permissions information is available at wwwnaturecomreprintsPublisher™s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Access This article is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate if changes were made The images or other third party material in this article are included in the article™s Creative Commons license unless indicated otherwise in a credit line to the material If material is not included in the article™s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this license visit httpcreat iveco mmons licen sesby40 The Authors Scientific RepoRtS 101038s41598020699040Vol1234567890wwwnaturecomscientificreports 0c'

Thyroid_Cancer

Polyphenol induced improvements in glucose metabolism are associated with bile acid signaling to intestinal farnesoid X receptorKevin M Tveter1 Jose A Villa Rodriguez Alrick J Cabales1 Li Zhang2 Fiona G Bawagan1 Rocio M Duran1 Diana E Roopchand To cite Tveter a0KM Villa Rodriguez a0JA Cabales a0AJ et a0al Polyphenol induced improvements in glucose metabolism are associated with bile acid signaling to intestinal farnesoid X receptor BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 –º Additional material is published online only To view please visit the journal online http dx bmjdrc KMT and JAV R contributed equallyReceived March Revised June Accepted June Authors or their employers Re use permitted under CC BY NC No commercial re use See rights and permissions Published by BMJ1Food Science Rutgers The State University of New Jersey New Brunswick New Jersey USA2Key Laboratory of Genomic and Precision Medicine Beijing Institute of Genomics Chinese Academy of Sciences Beijing Branch Beijing ChinaCorrespondence toDr Diana E Roopchand roopchand sebs rutgers eduIntroduction Bile acid BA biotransformation by gut bacteria impacts BA profile and signaling to nuclear receptors such as the farnesoid X receptor FXR regulating glucose metabolism Altered BA FXR signaling was therefore investigated as a potential mechanism linking polyphenol induced gut bacterial changes and improved glucose metabolismResearch design and methods Diabetic dbdb were fed low fat diet LFD or LFD supplemented with a proanthocyanidin rich extract of grape polyphenols LFD GP for weeks Metabolic phenotypes serum BAs gut microbiota composition and gene expression markers relevant to gut barrier and glucose metabolism were assessed Gut anoids were used to investigate effects of individual BAs on ileal FXR activityResults Compared with LFD fed controls GP supplemented dbdb mice showed improved glucose metabolism decreased relative abundance of gut bacteria associated with production of secondary BAs SBAs and depleted serum levels of SBAs taurohyodeoxycholic acid THDCA ωmuricholic acid ωMCA and tauroωmuricholic acid TωMCA Serum levels of primary BAs PBAs increased consistent with higher gene expression of PBA synthesis enzyme Cyp7a1 GP induced BA changes associated with FXR inhibition as evidenced by reduced expression of FXR responsive genes Shp Fgf15 and Fabp6 in ileum tissue as well as hepatic Shp which negatively regulates PBA synthesis GP treatment did not affect expression of hepatic Fxr or expression of Abcb11 Slc51b and Obp2a genes controlling BA transport Ceramide biosynthesis genes Smpd3 Sptlc2 and Cers4 were decreased in liver and intestine suggesting lower tissue ceramides levels may contribute to improved glucose metabolism THDCA ωMCA and TωMCA behaved as FXR agonists in ileal anoid experiments therefore their depletion in serum of GP supplemented dbdb and wild type WT mice was consistent with FXR inhibitionConclusion These data suggest that by altering the gut microbiota GPs modify BA FXR signaling pathways to promote glucoregulationINTRODUCTIONDietary polyphenols in plant based foods contribute to improved glycemic control in Significance of this studyWhat is already known about this subject –º Dietary polyphenols such as proanthocyanidins alter the gut microbial community and are associated with improved metabolic resilience in humans and mice –º Bile acids BAs signal to farnesoid X receptor FXR a nuclear transcription factor that regulates hepatic BA synthesis and glucose metabolismWhat are the new findings –º Grape polyphenol GP supplementation decreased abundances of gut bacterial taxa associated with secondary BA SBA production concomitant with depletion of SBAs and increased primary BAs PBAs in murine serum –º GP supplementation suppressed expression of FXR regulated genes Fgf15 Fabp6 and Shp an inhibitor of PBA synthesis which was consistent with increased serum PBAs –º GP induced FXR inhibition was associated with decreased expression of genes required for biosynthesis of ceramides which impair glucose homeostasis –º The SBAs depleted in GP treated mice were revealed as FXR agonists in ileal gut anoidsHow might these results change the focus of research or clinical practice –º This study highlights BA FXR signaling pathways as an important mechanism for further investigation in human intervention studies of dietary polyphenols and metabolic healthmice and humans1“ Improved glucose metabolism in mice supplemented with berryfruit extracts was related to a proanthocyanidin PAC induced bloom of Akkermansia muciniphila1 a microbe shown to attenuate symptoms of metabolic syndrome MetS and type2 diabetes T2D in obese mice and humans5 We hypothesized that metabolic improvements also result from changes in BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cMetabolismhost derived bacterial metabolites regulating host energy metabolismBile acids BAs are signaling molecules linking the gut microbiota to host energy metabolism Primary BAs PBAs synthesized in the liver are conjugated with taurine mice or glycine humans7 Bacterial derived bile salt hydrolases deconjugate taurine or glycine from the sterol core of PBAs followed by bacterial transformations such as αdehydroxylation dehydrogenation and epimerization which generate secondary BAs SBAs7 PBAs and SBAs signal to key regulators of energy metabolism such as nuclear transcription factor farnesoid X receptor FXR and Takeda G protein coupled receptor TGR57 Fxrˆ’ˆ’ mice were protected from high fat diet HFD induced obesity and had altered BA and gut microbiota higher Bacteroidetes less Firmicutes profiles compared with wild type WT mice8 Transfer of cecal microbiota from HFD fed Fxrˆ’ˆ’ mice to germ free WT mice resulted in less adiposity and improved glucose metabolism compared with mice that received microbiota from HFD fed WT mice suggesting the altered gut microbial and BA profiles in FXR deficient mice contributed to metabolic improvements8 Studies using tissue specific FXR knockout mice showed that intestinal FXR activity was required to mediate HFD induced metabolic dysfunctions9 Pharmacological inhibition of intestinal FXR using tempol antibiotics metformin or glycinemuricholic acid Gly MCA led to gut microbial remodeling and improved glucose and lipid homeostasis9“In addition to the A muciniphila bloom mice fed HFD supplemented with PAC rich grape polyphenols GPs showed other profound bacterial community changes raising the possibility that altered BA profile and signaling could contribute to observed improvement in glucose homeostasis1 The present study provides compelling evidence in support of this hypothesisRESEARCH DESIGN AND METHODSDietsA complex of grape polyphenols and soy protein isolate GP SPI was prepared as previously described1 Nutritional profiles for SPI and GP SPI are provided in online supplementary table Isocaloric ingredient matched diets Research Diets New Brunswick New Jersey USA used in this study were previously described4 Mice were fed low fat diet LFD containing SPI LFD formulated with GP SPI delivering GP LFD GP HFD containing SPI or HFD formulated with GP SPI delivering GP HFD GP Online supplementary table provides diet formulation detailsAnimalsFour week old dbdb mice B6BKSD LeprdbJ stock no Jackson Laboratory Bar Harbor Maine USA were single housed on a hour lightdark cycle to hours light with ad libitum access to LFD and water in a controlled temperature room °C±°C for week for acclimation Mice were randomized to receive LFD n7 or LFD GP n7 for days Metabolic phenotyping included food intake body weight body composition EchoMRI in1 system Echo Medical Systems Houston Texas USA and oral glucose tolerance tests as previously described1 On day mice were euthanized by CO2 inhalation followed by cardiac puncture and collection of tissues as previously described1 Fecal and cecal samples were collected for microbial community profiling Wild type C57BL6J mice n10 aged weeks were acclimated on LFD for week and randomly divided into two groups and fed HFD or HFD GP n5 per group for weeks after which mice were euthanized by CO2 inhalationTissue gene expression analysisRNA was extracted from ileum jejunum colon and liver “ mg with RNeasy Plus Universal Mini Kit QIAGEN followed by RNA cleanup Machery Nagel RNA purification kit RNA µg was reverse transcribed to cDNA and qPCR was performed using TaqMan primers online supplementary table as previously described4 Data were analyzed using 2ΔCT method using hydroxymethylbilane synthase HMBS as housekeeping geneqPCR of A muciniphila in fecal and cecal samples was performed as previously described1 Briefly gDNA extracted from fecalcecal samples was diluted to ngµL for quantification of A muciniphila abundance relative to total bacteria and archaea by qPCR using A muciniphila AM1 AM2 and universal primer U341F U515R sets1 16S rRNA gene sequencingGenomic DNA was extracted from fecal and cecal samples collected from dbdb mice Illumina protocols were used to prepare V4 amplicons of the 16S rRNA gene for sequencing on a MiSeq system resulting in × reads Denoising and clustering were conducted using DADA2 algorithm to differentiate sequences into amplicon sequence variants ASVs for downstream analysis using QIIME Details are available in online supplementary materialsSerum biochemistrySerum for BA analysis was collected by cardiac puncture BAs were analyzed on a Water™s Alliance e2695 HPLC system Waters Milford Massachusetts USA coupled to a Water™s Acquity QDA mass spectrometer equipped with an electrospray interphase ESI Waters Milford Massachusetts USA and quantified by external calibration curves using pure standards Details are provided in online supplementary materials Serum leptin polypeptide YY PYY interleukin6 IL6 and insulin were determined using a MILLIPLEX MAP Mouse Metabolic Hormone Magnetic Bead kit Millipore with a MagPix instrument Luminex as previously described4anoid experimentsIntestinal crypts were isolated from WT C57BL6J mouse ileum according to established methods16 Crypts were counted and added to Matrigel five crypts per µL BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cCorning growth factor reduced and µL was added per well in well plates and allowed to polymerize °C for “ min followed by addition of mL of complete growth medium CGM see online supplementary materials CGM without Y27632 dihydrochloride monohydrate was replaced every “ days anoids were passaged every “ days ratio Mature anoids days postpassage were treated in triplicate n3 wells with methanol vehicle chenodeoxycholic acid CDCA µM alone or CDCA plus another BA µM in CGM for hours RNA was extracted for qPCR analysis using TaqMan primers as described above4Statistical analysesAnalyses were conducted using Prism GraphPad Software La Jolla California USA Significant differences two groups were assessed with a two tailed unpaired Student™s t test with Welch correction for unequal variance when needed or by one way or two way analysis of variance groups followed by Sidak™s or Tukey™s multiple comparison test Statistical analysis of alpha and beta diversity metrics was calculated using QIIME details in online supplementary materials ADONIS and permutation analysis were conducted using R Studio V342 R Studio Software Boston Massachusetts USA and Python RESULTSGPs improve glucose metabolism in dbdb mice independent of obesityLeptin receptor deficient dbdb mice develop obesity gut barrier dysfunction and hyperglycemia independent of HFD feeding17 Compared with LFD fed controls dbdb mice fed LFD containing PAC rich GPs LFD GP for weeks showed significantly improved oral glucose tolerance figure 1A4 Area under the curve remained stable over time for the LFD GP group but increased in the LFD group figure 1A Mice fed LFD GP initially exhibited a transient decrease in food intake presumably due to taste but at later time points LFD and LFD GP groups consumed similar amounts of food ± and ± gdaymouse respectively p005 online supplementary figure 1A The LFD GP group consumed ± mg of GPs per day Both groups had similar body weight gain body composition and liver weights online supplementary figure 1B“DGPs promote a bloom in A muciniphila without improving markers of metabolic endotoxemiaCompared with controls GP supplemented dbdb mice had decreased αdiversity as evidenced by richness Shannon index and Faith™s phylogenetic diversity index figure 2A Principal coordinate analysis showed that GPs significantly altered fecal community structure within days online supplementary figure As previously observed1 GPs promoted increased cecal mass online supplementary figure Metabolism1E a phenotype common to antibiotic treated and germ free mice and consistent with reported antibacterial properties of PACs18 GP supplemented mice had an increased relative abundance of phylum Verrucomicrobia at the expense of Firmicutes in fecal days “ and ceca samples day figure 2BC online supplementary figure 3AB Ceca of GP supplemented mice had higher relative abundance of Bacteroidetes 25LFD although except for day fecal Bacteroidetes remained similar between groups figure 2B online supplementary figure 3C GP supplementation did not consistently alter levels of Proteobacteria or Actinobacteria figure 2B online supplementary figure 3DE Consistent with increased Verrucomicrobia quantitative qPCR analyses confirmed that GPs promoted a bloom in Akkermansia muciniphila figure 2C at the expense of other taxa figure 2DReduced abundance of A muciniphila and metabolic endotoxemia characterized by gut dysbiosis compromised gut barrier integrity lipopolysaccharide LPS leakage and intestinal inflammation was associated with impaired glucose metabolism in obese mice and humans5 Oral administration of A muciniphila in obese diabetic mice and humans resulted in improved glucose homoeostasis and attenuated metabolic endotoxemia through improved gut barrier integrity5 GP induced improvement in glucose metabolism in HFD fed mice has therefore been considered a consequence of the A muciniphila bloom leading to reduced inflammation and increased gut barrier integrity1 Reduced metabolic endotoxemia could not however explain the improved glucose tolerance in GP supplemented dbdb mice despite increased A muciniphila in feces day and cecum figure 2C Relative to control GP supplementation did not change intestinal gene expression of markers of inflammation Tnf Il6 iNOS gut barrier integrity Tjp1 Ocln Muc2 peripheral lipid deposition Fiaf or glucose transport Glut2 online supplementary figure 4A“C GP supplemented dbdb mice had less Muc3 expression in jejunum and ileum online supplementary figure 4AB suggesting lower mucus secretion There were no differences in serum insulin IL6 or glucoregulatory hormones PYY and leptin online supplementary table These data suggested that other mechanisms were driving improved glucose metabolismGPs reduced gut bacterial taxa associated with production of SBAsAlthough total bacterial and archaeal abundance was not significantly different between LFD and LFD GP groups figure 2E GPs induced profound gut microbial changes online supplementary figure that would be expected to alter BA diversity abundance and signaling Targeted liquid chromatography mass spectrometry LC MS analysis revealed that GP supplemented dbdb mice had higher serum concentrations of BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cMetabolismFigure GPs improve glycemic control in obese dbdb mice A Oral glucose tolerance tests were performed after mice consumed LFD squares n7 or LFD GP closed squares n7 for and weeks Blood glucose concentrations mgdL expressed as mean±SD were measured at the indicated time points “ min following administration of glucose gkg Between group difference was determined by unpaired two tailed t test with Welch™s correction p01 p001 B Scatter plot of blood glucose AUC determined for individual mice at weeks and where mean AUC±SD are shown as horizontal and vertical bars Difference was determined using two way ANOVA followed by Tukey™s intragroup posthoc test different letters indicate significant difference p005 or by Sidak™s intergroup posthoc test p001 ANOVA analysis of variance AUC area under the curve GP grape polyphenol LFD low fat dietPBAs driven by increased cholic acid CA and taurocholic acid TCA figure 3A and C online supplementary figure 6A Concentrations of muricholic acid MCA tauroMCA TMCA tauroαMCA TαMCA taurochenodeoxycholic acid TCDCA were similar between groups αMCA was not detected in LFD GP group figure 3A online supplementary figure 6A Increased PBA pool correlated with an overall reduction in SBAs figure 3C where ωMCA TωMCA and taurohyodeoxycholic acid THDCA were undetectable in dbdb mice fed LFD GP although deoxycholic acid DCA was increased figure 3A Total serum BAs were similar between groups figure 3BThe GP induced depletion of SBAs was unrelated to the leptin receptor mutation in dbdb mice Compared with HFD fed controls wild type C57BL6J mice fed HFD supplemented with GP HFD GP for weeks also showed serum depletion of SBAs THDCA ωMCA and TωMCA reduced overall levels of serum SBAs and no difference in concentration of total serum BAs online supplementary figures 6B and Unlike dbdb mice GP supplemented WT mice had decreased tauro deoxycholic acid TDCA and no significant difference in CA or overall PBA pool although TCA concentration was increased online supplementary figures 6B and In dbdb mice multiple correlation analyses were performed to associate GP induced changes in serum BAs figure to changes in fecalcecal gut bacteria online supplementary figure The GP induced increase of CA TCA PBAs and DCA SBA was positively and significantly associated with increased abundance of Akkermansia Blautia Clostridium ASV59 and S247 figure 3EF Blautia and Clostridium possess BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cMetabolismFigure Supplementation of GPs remodeled gut microbial composition and diversity A Microbial αdiversity metrics of fecal samples collected from mice after consuming LFD squares n7 or LFD GP closed squares n7 for indicated number of days “ and of cecal Cec samples collected at endpoint day Difference was determined by two way ANOVA followed by Sidak™s test B Per cent relative abundance of main bacterial phyla based on Naïve Bayes taxonomic classifier non rarified data Phyla present at relative abundance were classified as ˜Other™ Percentage of C A muciniphila DNA and D nonA muciniphila DNA relative to total bacterial and archaeal DNA in fecal and cecal samples where relative abundance was determined by qPCR using primers specific for A muciniphila AM1AM2 and universal V4 primers 515F806R E Total 16S bacterial and archaeal gene countsng of gDNA extracted from fecal or cecal samples Group mean±SD at each time point are illustrated by horizontal and vertical lines Difference between diet groups over time panels C“E was determined using two way ANOVA followed by Sidak™s posthoc test intergroup comparison or Tukey™s posthoc test intragroup comparison Different letters a b and c indicate significant difference within diet groups p005 while the same letter indicates no difference Between group differences panels A C D E p005 p001 p0001 p00001 ANOVA analysis of variance ASV amplicon sequence variants AUC area under the curve GP grape polyphenol LFD low fat diet PD phylogenetic diversity7αdehydroxylating activity therefore taxa within these genera may be responsible for DCA production via dehydroxylation of CA21 GP treated mice had lower levels of taxa within the Clostridiales order ASV50 and Ruminococcaceae and Lachnospiraceae families ie ASV56 and and Clostridium genus ASV74 online supplementary figure which are reported to possess 7αdehydroxylating activity required for conversion of PBAs to SBAs21“ In agreement with evidence from mice and humans the GP induced decrease in these bacterial taxa encoding 7αdehydroxylation activity was highly correlated to the GP associated depletion of SBAs ωMCA and TωMCA figure 3EF21 Finally positive and significant associations were found between reduced αMCA TωMCA andor ωMCA and reductions in taxa belonging to RF39 Anaeroplasma Ruminococcus Butyricicoccus Dorea Dehalobacterium Christensenellaceae Lactococcus Streptococcus and Oscillospira figure 3EFGPassociated BA changes promote inhibition of FXR signaling and upregulation of classical BA synthesis pathwayTo investigate the consequences of GP induced serum BA changes gene expression of FXR TGR5 and their downstream targets were analyzed in tissues GP supplementation did not change ileal Fxr gene expression however FXR transcriptional activity was decreased as expression of its target genes fibroblast growth factor Fgf15 small heterodimer partner Shp and ileal BA binding protein I BABP gene Fabp6 were suppressed in ileum figure 4A Intestinal FXR signaling negatively regulates hepatic PBA synthesis through interaction of Fgf15 with hepatic fibroblast growth factor receptor 4Klotho receptor complex or by regulating BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cMetabolismFigure GPs increased PBAs and reduced SBAs in serum in association with depletion of bacterial ASVs related to SBA production A LC MS and pure standards were used to determine the mean concentration of individual PBAs and SBAs mean±SD in serum samples n7 samples collected from individual mice fed LFD white bars or LFD GP purple bars B Total serum BA concentration mean±SD was determined based on sum of individual PBA and SBA concentrations shown in panel A quantified for each mouse fed LFD n7 or LFD GP n7 C Serum PBA and SBA concentrations mean±SD in LFD versus LFD GP diet groups were calculated by summing the individual PBAs or SBAs shown in panel A D Using data from panels B and C pie charts illustrate pooled PBAs green and pooled SBA gray as a percentage of total serum BA concentration quantified for LFD and LFD GP groups For panels A“C significant difference was determined using unpaired two tailed t test followed by Welch™s correction p005 p001 Heatmap representation of the Spearman™s r correlation coefficient between serum BA profile and significantly changed bacterial ASVs at genera or family level of taxonomy in GP treated mice relative to control diet group from E day27 fecal samples or F day29 cecal samples Shades of red indicate serum BA and bacterial taxa are positively correlated to while shades of blue indicate a negative correlation to ˆ’ Significant positive or negative correlations are shown p005 p001 p0001 ASV amplicon sequence variant CA cholic acid DCA deoxycholic acid GP grape polyphenol LC MS liquid chromatography mass spectrometry LFD low fat diet MCA muricholic acid PBA primary bile acid SBA secondary bile acid TCA taurocholic acid TCDCA taurochenodeoxycholic acid TDCA tauro deoxycholic acid THCDA taurohyodeoxycholic acid TαMCA tauroαMCA TMCA tauroMCA TωMCA tauroωMCA TUDCA tauro ursodeoxycholic acidBMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cMetabolismFigure Expression of genes involved in FXR signaling ceramide synthesis and glucose metabolism in response to GP supplementation Scatter plot of relative mRNA levels of indicated genes expressed in A ileum and B liver tissues collected from individual mice fed LFD squares or LFD GP closed squares Group mean±SD n7 samples group is illustrated by horizontal and vertical lines Data represent qPCR of technical duplicates analyzed by ˆ’ΔCT method Between group difference was determined by unpaired two tailed t test with or without Welch™s correction for unequal variance p005 p001 p0001 FXR farnesoid X receptor GP grape polyphenol LFD low fat dietthe hepatic expression and gene repressive function of Shp via liver receptor homologue1 and hepatocyte nuclear factor4α26“In agreement with reduced ileal Fgf15 hepatic Shp expression was significantly decreased figure 4B Fgf15 and Shp negatively regulate BA synthesis therefore their reduced expression was consistent with hepatic upregulation of cytochrome P450 family subfamily A member Cyp7a1 the rate limiting enzyme in the classical BA synthesis pathway7 and a trending increase in downstream enzyme Cyp8b1 p006 figure 4B Gene expressions of Cyp27a1 and Cyp7b1 online BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cMetabolismsupplementary figure 4E involved in the alternative BA synthesis pathway were unaffected by GP treatment To determine if reduced hepatic Shp expression altered hepatic FXR activity we examined FXR target genes Abcb11 encoding bile salt export pump Bsep Slc51b encoding anic solute transporter Ost30 and Obp2a encoding lipocalin Lpn13 an acute phase protein32 Compared with control GP treatment did not affect hepatic expression of Fxr Abcb11 Slc51b or Obp2a figure 4B These data suggest that GP mediated effects on FXR activity are restricted to intestine resulting in suppression of ileal Fgf15 and Shp transcription and increased Cyp7a1 activity and PBA synthesis figure FXR activity regulates Tgr5 expression therefore we measured Tgr5 mRNA levels and downstream targets33 On activation by BAs TGR5 signals the release of incretin glucagon like peptide1 Glp1 to promote euglycemia33 Glp1 is produced when proglucagon protein Gcg is cleaved by prohormone convertase PC13 encoded by Pcsk134 GP supplemented mice showed reduced ileal expression of Tgr5 and no changes in Gcg or Pcsk1 figure 4A GPs did not change expression of Fxr Fgf15 or Tgr5 in colon tissue GPs induced an increase in colonic Gcg however Pcsk1 was unchanged indicating Glp1 levels were unaffected online supplementary figure 4D These data further support the idea that GPs inhibit ileal FXRGPmediated FXR inhibition is associated with downregulation of ceramide synthesis genes and improved markers of hepatic energy metabolismTissue accumulation of ceramides is linked to insulin resistance and diabetes which can be ameliorated by pharmacological or genetic inhibition of ceramide biosynthesis35 FXR activity positively upregulates genes required for ceramide synthesis in ileum which leads to impaired glucose metabolism and hepatic steatosis in mouse models of MetST2D10 Synthesis and accumulation of ceramides in liver contributes to hepatic insulin resistance steatohepatitis and metabolic disease35 We found that GP induced FXR inhibition was associated with lower expression of de novo ceramide synthesis genes specifically sphingomyelin phosphodiesterase Smpd3 in ileum figure 4A and serine palmitoyltransferase long chain base subunit Sptlc2 and ceramide synthase Cers4 in liver figure 4B Consistent with lower expression of ceramide biosynthesis genes GP supplemented mice showed improvements in markers of hepatic energy metabolism evidenced by lower hepatic expression of carbohydrate response element binding protein Chrebp a transcription factor that activates key enzymes of de novo lipogenesis38 glucose phosphatase G6Pase which catalyzes the final step in hepatic glucose production and Idh3a a subunit of the IDH3 isocitrate dehydrogenase heterotetramer complex that regulates fatty acid metabolism and whose inhibition is associated with hepatic glycogen synthesis figure 4B39 GP supplemented mice had reduced expression of Lbp encoding LPS binding protein suggesting decreased liver inflammation and insulin resistance40 Hepatic gene expression of gluconeogenesis enzyme phosphoenolpyruvate carboxykinase Pck1 and CEBP homologous protein Chop which are normally upregulated during hepatic endoplasmic reticulum stress41 were similar between groups online supplementary figure 4ESBAs depleted in GPsupplemented mice are FXR agonists that promote expression of ceramide synthesis genesThe majority of secreted BAs are reabsorbed in ileum and returned to the liver via the portal vein7 Cecum colon and feces have similar BA profiles while the serum BA profile is most closely related to that of ileum and portal vein as a minor fraction of reabsorbed BAs enter systemic circulation25 We sought to investigate how individual BAs altered by GP supplementation might affect ileal FXR signaling Gut anoids were cultured from ileal crypts isolated from WT mice figure 5A and treated with individual PBAs or SBAs that were differentially detected in serum of GP supplemented mice figure 5B To validate the system anoids were treated with CDCA a potent FXR agonist or CDCA in combination with the FXR antagonist TMCA As expected CDCA increased the expression of Fxr Fgf15 and Shp compared with vehicle treated anoids and this effect was attenuated by addition of TMCA figure 5B When anoids were treated with individual BA alone ie µM in the absence of CDCA activator FXR activity was unaffected as Fxr Fgf15 and Shp mRNA expression remained similar to untreated anoids online supplementary figure We therefore investigated individual BAs in the presence of FXR activator CDCA as previously reported11 to reveal agonistic or antagonistic effects on FXR signaling anoid cultures treated with CDCA in combination with ωMCA TωMCA THDCA or DCA increased CDCA induced expression of Fxr Fgf15 and Shp indicating that these SBAs are FXR agonists figure 5B In agreement with FXR agonistic activity anoids treated with CDCA in combination with TωMCA resulted in increased expression of Smpd3 Cers4 and Sptlc2 ceramide synthesis genes Cotreatment with CDCA and ωMCA THDCA or DCA only upregulated Sptlc2 figure 5B CA a PBA reported to be a weak FXR agonist and detected at higher concentration in GP supplemented dbdb but not WT mice increased CDCA induced activation of Fxr Fgf15 and Shp and increased expression of Cers4 and Sptlc243 TCA a PBA detected at higher concentration in GP supplemented dbdb and WT mice attenuated CDCA induced gene expression of Fxr and Fgf15 but not Shp and reduced CDCA induced gene expression of Cers4 figure 5B These anoid data suggest that GPs led to the depletion of FXR activators ωMCA TωMCA and THDCA and increase of an FXR antagonist TCA Consistent with in vivo data the net effect BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cMetabolismFigure Ileal anoids treated with BAs revealed agonistic or antagonistic effects on FXR and ceramide pathway genes A Ileal crypts were isolated and cultured in Matrigel medium detailed in methods Spheroids representative day photo matured into anoids representative day photo B Scatter plot of relative mRNA levels of indicated genes expressed in anoids after hours of treatment with vehicle methanol black diamonds µM CDCA closed red circles a combination of µM CDCA and known FXR inhibitor TMCA µM a combination of µM CDCA and indicated PBA µM red circles a combination of µM CDCA and indicated SBA µM blue squares Data shown were combined from two independent experiments and for each experiment three wells containing mature anoids were treated with indicated BAs Data represent qPCR of technical duplicates analyzed by ˆ’ΔCT method Group mean±SD n6 wells total per treatment group is illustrated by horizont

Thyroid_Cancer

" IBDFecal calprotectinEndoscopic activityIBD noninvasive managementThe term IBD is usually used for referring to a group of ‚ammatory gastrointestinal diseases mainly Crohn'sdisease and ulcerative colitis Accordingly IBD arises as a result of inappropriate immune response to intestinalcommensal anisms among genetically susceptible individuals Performing colonoscopy and histopathologicevaluation on an ‚amed bowel biopsy specimen are currently considered as gold standards for diagnosis andmanagement of IBD Correspondingly these techniques are known to be invasive and costly In recent decadesfecal calprotectin as a biomarker has received much attention for the diagnosis and noninvasive managementof IBD Up to now many studies have investigated the efficacy of fecal calprotectin in the areas of IBD diï¬erentiation from IBS prediction of endoscopic and histologic activities of IBD and prediction of disease recurrenceAlthough some of these studies have reported promising results some others have shown significant limitationsTherefore in this paper we reviewed the most interesting ones of these studies after a brief discussion of thelaboratory measurement of fecal calprotectin Moreover we attempted to provide an answer for the question ofwhether fecalcalprotectin could be considered as a potential surrogate marker for colonoscopy IntroductionInflammatory bowel disease IBD is a long life disease with remission and relapse periods IBD arises as a result of inappropriateimmune response to intestinal commensal anisms in individualswith genetic predisposition and consequently causes ‚ammation andintestinal ulcers [] In addition IBD has a complex pathogenesis andmany factors such as dysbiosis oxidative stress and epigenetics thatmay also be involved in disease pathogenesis [“] Ulcerative colitisUC and Crohn's diseases CD are known as two main forms of IBDAccordingly these diseases cause intestinal ulcers and some annoyingsymptoms such as diarrhea abdominal pain and rectal bleeding Occasionally the severity of these symptoms is very high which can leadpatients to be hospitalized In this regard therapeutic approaches totreat these diseases mainly focus on prolonging remission and are almost similar however diï¬erential diagnosis can also help to treat thedisease in a more eï¬ective way For example 5ASA which is acommon drug in the treatment of IBD is less eï¬ective on maintainingremission in CD patients On the other hand antibiotic therapy is notrecommended for the treatment UC but it can be eï¬ective on CD patients [][] Diï¬erential diagnosis is a serious challenge because CDand UC have significant similarities in terms of their clinical endoscopic and histological features However there are some diï¬erencesbetween UC and CD which are summarized in Table1 In addition tointestinal complications UC and CD also have significant extraintestinal manifestations For example it was shown that UC is significantly associated with Primary sclerosing cholangitis and CD is alsoassociated with cholelithiasis especially in cases that the ileum is involved [] Furthermore CD can cause fistulas to the urinary systemwhich leads intestinal bacteria to enter the urethra and recurrent urinary tract infections [] Both CD and UC can cause several disorderssuch as arthritis Erythema nodosum pyoderma gangrenosum andanemia which are known as the most important extraintestinal manifestations of IBD [][] The latest statistics showed that the globalŽ Corresponding author at Department of Clinical Biochemistry and Laboratory Medicine Faculty of Medicine Tabriz University of Medical Sciences DaneshgahStreet PO Box Tabriz IranEmail address vagharimtbzmedacir M VaghariTabari101016jcca202008025Received July Received in revised form August Accepted August Available online August Elsevier BV All rights reserved 0cF KhakiKhatibi et alTable1Clinical endoscopic and histological features of CD and UCClinical FeaturesFeaturesRectal bleedingAbdominal painFeverMucus defectionIntestinal obstructionPerineal diseasePostoperative recurrenceASCA positiveANCA positiveEndoscopic FeaturesCDOccasionallyFrequentlyFrequentlyOccasionallyYESYESYESFrequentlyNot commonUCFrequentlyOccasionallyNot commonFrequentlyNONONONot commonFrequentlyFeaturesCDUCLocationMucosal involvementDepth of ulcerationfistulaCobblestone appearanceAphthous ulcerationMucosal friabilityHistological featuresFeaturesGranulomasCrypt abscessesPatchinessAny part of GI tractDiscontinuousDeepYesYESFrequentlyNot commonCDFrequentlyNot commonFrequentlyColon and rectumContinuoussuperficialNONOOccasionallyFrequentlyUCRareFrequentlyNot commonprevalence of IBD currently is on the rise and it is not an exaggerationif we consider it as a global serious health problem [] According to areport published in IBD has the highest prevalence rate inEurope and its prevalence in the newly industrialized countries of AsiaAfrica and South America also appears to be increased over the pastthree decades []Unfortunately the peak of the disease is at the young age of“ years old [] therefore in addition to the suï¬ering from ‚icts on the patients it also has many negative eï¬ects on societyMoreover many financial burdens are annually imposed on countriesfor controlling and treating this chronic disease The invasive diagnosticand therapeutic measures are currently undertaken to diagnose andmanage IBD which are unpleasant for patients as well as having thehigh associated costs Now the gold standard method for diagnosingIBD and monitoring patient status is performing colonoscopy examination and histopathologic evaluation which are invasive measures[] Therefore in recent years many studies have been conducted tofind a suitable laboratory marker with sufficient sensitivity and specificity for the purpose of diagnosing and noninvasive management ofIBD A high proportion of these studies have investigated the efficacy offecal calprotectin in diagnosing and monitoring patients Althoughsome of these studies reported auspicious results there are still somedoubts on the eï¬ectiveness of fecal calprotectin on diagnosing andmonitoring IBD patients So in this review we addressed the advantages and limitations of fecal calprotectin for the diagnosis andmanagement of IBD The role of fecal calprotectin in diagnosis and management ofIBDThe efficacy of fecal calprotectin as an laboratory marker in various areas of IBD diagnosis and management have been studied including IBD diï¬erentiation from irritable bowel syndrome IBS evaluation of endoscopic activity of the disease evaluation of histologicalactivity of the disease and prediction of disease recurrence andClinica Chimica Acta “response to treatment In following after a brief introduction andmentioning the important points regarding laboratory measurement offecal calprotectin we reviewed the most interesting findings in all ofthe abovementioned areas Calprotectin A clinically valuable proteinCalprotectin is an antimicrobial protein mainly secreted by neutrophils This protein competes with bacteria over zinc thus kills thebacteria However this is not the only contribution that it has to antimicrobial activity Moreover this protein has many potential clinicalapplications such as the elevated serum levels that have been observedunder various immunological and immunopathological conditionsSerum calprotectin levels rapidly increase in response to bacterial infections in the kidney and heart or during transplant rejection At theearly stages of ‚ammation of the lung serum calprotectin can also beconsidered as a reliable marker besides plasma levels of calprotectinappear to be useful in reflecting disease activity in ‚ammation of thejoints [] In addition it has been demonstrated that serum calprotectin levels are increased in patients with bacterial sepsis so it can beconsidered as a reliable biomarker [] In Neonatal Sepsis the serumlevel of calprotectin increases as well as a sensitivity of and aspecificity of that have been reported for serum calprotectin indiagnosis of Neonatal Sepsis [] It has been recently shown thatserum calprotectin levels increase in patients with aneurysmal subarachnoid hemorrhage and higher levels in the first of onset areassociated with a poor prognosis at the first three months [] Serumcalprotectin levels also increase in patients with rheumatoid arthritisand even in patients with a moderate to high disease activity who havenormal or low CRP levels so they appear to be more efficient at reflecting disease activity []Some studies have also investigated the efficacy of serum calprotectin in the diagnosis of cancers Correspondingly in one of thesestudies it was shown that serum calprotectin levels significantly increased in patients with laryngeal carcinoma compared with healthyindividuals and those with benign laryngeal pathologies Moreover inthis study a direct relationship was also observed between serum levelsof calprotectin and stage of cancer [] Another study showed that theserum level of calprotectin increased in patients with papillary thyroidcarcinoma but it significantly decreased after operation [] Alsoregarding the efficacy of serum and saliva calprotectin for the diagnosisof IBD impressive results have been reported [][] A study onpatients with IBD both UC and CD have shown that serum calprotectinlevels were directly correlated with fecal calprotectin levels and weremore potent in IBD diagnosis compared to CRP and albumin This studyalso indicated that the combination of serum calprotectin with CRP oralbumin can be helpfulin the prediction of treatment escalationespecially in patients with CD [] However no significant correlationwas observed between serum calprotectin and fecal calprotectin levelsin patients with CD and UC as well as a slight correlation betweenserum calprotectin level and CRP that was observed only in patientswith UC [] Another study showed that the serum level of calprotectin was significantly higher in patients with CD compared to healthyindividuals In addition although a significant correlation was observedwith the clinical activity of the disease no significant correlation wasfound between the level serum calprotectin and endoscopic activity ofthe disease [] The efficacy of salivary calprotectin in the diagnosisof IBD has also been studied which showed that salivary calprotectinsignificantly increased in patients with IBD compared to healthy individuals In this study AUC values for unstimulated saliva and stimulated saliva to distinguish IBD patients from healthy individualswere reported to be and respectively [] However thepopularity of calprotectin is mainly due to the use of fecal calprotectinin the diagnosis and management of IBD that is discussed in the following 0cF KhakiKhatibi et alClinica Chimica Acta “ Laboratory measurement and reference intervalFecal calprotectin is a stable protein that remains stable for “ daysat room temperature [] This property is an excellent advantage for alaboratory marker Also it seems that keeping the specimen at refrigerated temperature °C can increase the stability of fecal calprotectin [] However evidence has been obtained regarding thatthe stability of this protein decreases after staying for three days atroom temperature On the other hand it is not also recommended tokeep samples in the refrigerator for more than days [] It seemsthat fecal calprotectin remains stable up to one year at ˆ’ °C []Measurement of fecal calprotectin can be done both qualitatively andquantitatively Accordingly in the qualitative measurement monoclonal antibodies are used to detect fecal calprotectin and the positiveresults are characterized by the appearance of colored lines on the testcassette However in the qualitative one only positive or negative results are reported and despite of sensitivity test specificity in theevaluation of disease activity was reported to be only It seems thatthe main application of this test is to diï¬erentiate healthy individualsfrom IBD patients rapidly however some studies have shown that it isnot accurate enough in this case as well [][] Nevertheless asignificant concordance has been reported between home test resultsIBDoc and fecal calprotectin laboratory measurement results whenQuantum Blue calprotectin ELISA kit was used Notably the agreements between results were and depending on the selectedcutoï¬s [] Several commercial kits are also available for fecal calprotectin qualitative test known as rapid calprotectin These tests reportpositive results ranged from to µgg There are also severalcommercial kits that can be used for the quantitative measurement offecal calprotectin These kits are usually designed in terms of the ELISAmethod and some have a measurement range between and µgg Moreover the chemiluminescence immunoassays CLIAmethod can also detect values between and µgg Fluoro enzyme immunoassays FEIA and particle enhanced turbidimetric immunoassays PETIA can also be used for the measurement of fecalcalprotectin In this regard one of the most serious challenges to thelaboratory evaluation of fecal calprotectin is the determination of theupper limit in healthy individuals Among healthy adults there is asignificant agreement on µgg as an upper limit One study suggested values up to µgg in people over years old and up to µgg in children aged between and years old as referenceranges of fecal calprotectin in healthy individuals []Fecal calprotectin levels appear to be higher in healthy infants andchildren under four years old than in adults and further studies areneeded in this regard to determine the acceptable upper limit for diagnosis of pediatric IBD [] Table lists the median levels of fecalcalprotectin in healthy individuals with diï¬erent ages reported in somestudies According to these reports age can aï¬ect fecal calprotectinlevels Fecal calprotectin and IBD diagnosisOnly a small percentage of patients complaining of abdominal painand diarrhea have IBD In many cases IBS as a functional gastrointestinal disorder is known as the cause of such clinical symptomsPatients with IBS have normal colonoscopy results while IBD patientsindicate abnormal colonoscopy results and have intestinal ulcersUnfortunately the significant prevalence of IBS and the overlap between clinical symptoms and IBD can increase the colonoscopy rateTherefore a noninvasive diagnostic marker can be very helpful in thisregard Notably the first evidence of the efficacy of fecal calprotectin inthe diagnosis of IBD was obtained in the 1990s Røseth et al in proposed a method for measuring Calprotectin in stool specimens []One of the first and most interesting studies regarding fecal calprotectinutility in IBD diagnosis was the study by Røseth et al published in In this study patients with ulcerative colitis were studied and according to their results fecal calprotectin levels are higher in patientswith ulcerative colitis compared to healthy controls This study havealso shown that even patients with low disease activities had higherlevels of fecal calprotectin compared to healthy individuals []Subsequent studies somehow confirmed and complemented the findings of this study In another study published in AUC values of CI “ were reported for fecal calprotectin in thediagnosis of colorectal ‚ammation [] Moreover in a study onchildren with IBD it was shown that the level of fecal calprotectin washigher in these patients compared to healthy children so it can beconcluded that it is also directly correlated with ESR levels [] In astudy published in Kolho et al reported AUC values of CI “ for fecal calprotectin in the diagnosis of pediatric IBD [] In a study on patients with Crohn disease a sensitivity of and a specificity of at cutoï¬ of μgg have been reportedfor fecal calprotectin in diagnosis of the disease [] The results of ourrecent study along with other studies showed that fecal calprotectin ispreferred over traditional ‚ammatory biomarkers such as CRP andESR in the diagnosis of IBD [][] Diamanti et al reported a sensitivity of and a specificity of for fecal calprotectin at a cutoï¬ of μgg in IBD diagnosis [] In our recent study a sensitivityof and a specificity of at a cutoï¬ of μgg were observed for fecal calprotectin in the diagnosis of IBD however oursample size was and the majority of patients were in the active phaseof the disease []In another study conducted on patients with ulcerative colitis asensitivity of and a specificity of at cutoï¬ of μgg havebeen reported in this regard [] In one study it was shown that fecalcalprotectin in cutoï¬ of μgg is able to distinguish patients withIBD from patients without IBD patients with diseases other than IBDpatients with IBS and healthy persons with sensitivity and specificity [] Caviglia et al in their study reported a sensitivity of and a specificity of at a cutoï¬ of μgg for fecalcalprotectin in diï¬erentiating between IBS and IBD [] Howeversome studies have reported significantly lower values Accordingly in astudy on patients with ulcerative colitis Kalantari et al reported asensitivity of and a specificity of at a cutoï¬ of μgg []Besides there is a considerable agreement between fecal calprotectinand capsule endoscopy findings in patients with Crohn's disease Asensitivity of and a specificity of have also been reported at acutoï¬ of mgkg for fecal calprotectin in predicting CE findings anddiagnosis of Crohn's disease [] In another study lower sensitivityand specificity rates sensitivity specificity were reportedfor fecal calprotectin in this regard [] Furthermore in one studythat examined the efficacy of fecal calprotectin in predicting wirelesscapsule endoscopy findings a sensitivity of and a specificity ofTable Reported median levels of fecal calprotectin in healthy individuals of diï¬erent agesAgesMedian levels of fecal calprotectin range µggNumber of subjectsUsed kitUp to monthChildren “ yearsChildren “ yearsAdultsOver years “ “ “ “ “Bühlmann ELISABühlmann ELISACALPRO® Calprotectin ELISA Test ALPPhiCalPhicalReference[][][][][] 0cF KhakiKhatibi et al were reported for this biomarker at μgg in the diagnosis ofsmall bowel ‚ammation in Crohn's disease [] Given these findings it seems that fecal calprotectin has no ideal sensitivity and specificity for the diagnosis of IBD where the small intestine is involvedBesides there are some preanalytical limitations which are explainedin the next sections Therefore optimistically speaking fecal calprotectin measurement can eliminate the need for colonoscopy Howeverin a metaanalysis performed to evaluate the efficacy of fecal calprotectin and some other ‚ammatory markers to diï¬erentiate betweenIBD and IBS the probability of IBD was less than at fecal calprotectin values lower than µgg or CRP values lower than mgdL[] Therefore it seems that fecal calprotectin can be helpful at leastin ruling out the possibility of IBD in patients with IBSlike symptoms aswell as reducing the rate of colonoscopy Moreover it should be notedthat although a systematic review has reported pooled sensitivity andspecificity above for fecal calprotectin to diï¬erentiate between IBDand IBS it emphasized more on the possibility of falsepositive resultsin low cutoï¬ points [] Hence performing extensive studies indiï¬erent countries on the healthy population and the IBD patient is beneeded to determine a suitable cutoï¬ with maximum sensitivity andspecificity and minimum falsepositive resultsTable summarizes the results of various clinical investigationsregarding fecal calprotectin utility in the diï¬erential diagnosis of IBDfrom IBS and Table4 summarizes some metaanalysis results in thisregard As shown in Table the most important limitation of the majority of clinical studies conducted to date is the small sample size Alarge global study may be helpful in providing a more precise evaluation of fecal calprotectin clinical value in discrimination between IBDand nonIBD diseases Fecal calprotectin and endoscopic and histologic activity evaluationUndoubtedly one of the most serious challenges in the managementof IBD is evaluating the endoscopic and histologic activities of thedisease Nowadays colonoscopy and histopathologic examinations arethe routine tools for the assessment of mucosal healing in patients withIBD As noted earlier several scoring systems have been devised toscore disease activity based on the findings of colonoscopy and histopathologic examinations In recent years many promising results havebeen reported regarding the correlation between these scores and fecalcalprotectin levels In addition many studies have been performed inthe last decade all of which cannot be reviewed in this article The firstevidence of a link between fecal calprotectin and disease endoscopicactivity was obtained in the late 1990s In one of the first studiesRoseth et al found a significant correlation between fecal calprotectinlevels and endoscopic and histologic activities in patients with ulcerative colitis [] Furthermore in another study they observed that IBDpatients who were in remission clinically and had normal fecal calprotectin levels less than mgL had normal colonoscopy results[] These interesting findings indicate that fecal calprotectin can beconsidered as a biomarker in the evaluation of endoscopic activity andClinica Chimica Acta “Table4summarized results of some metaanalysis regarding the utility of fecal calprotectin in discrimination between patients with IBD and without IBDSample sizePooled SensitivityPooled SpecificityReferences[][][][][]mucosal healing in IBD patients Also these studies were the startingpoint of extensive studies that have been conducted up to now In astudy conducted on patients with Crohn's disease Sipponen et alinvestigated the sensitivity and specificity of fecal calprotectin in predicting endoscopic activity of Crohn's disease [] Correspondinglythe researchers used the Crohn's Disease Endoscopic Index of SeverityCDEIS scoring system in their study to evaluate the endoscopic activity of Crohn's disease As a result they found that there was a significant correlation between the endoscopic activity of the disease andthe level of fecal calprotectin Besides the findings of this study demonstrated that fecal calprotectin at µgg cutoï¬ can predict theendoscopic activity of Crohn's disease with sensitivity and specificity In another study CDEIS and Mayo Disease Activity IndexMDAI were used to evaluate the endoscopic activity of Crohn's diseaseand ulcerative colitis respectively According to the results of thatstudy on IBD patients there was a significant correlation between fecalcalprotectin levels and disease endoscopic activity [] Another studyshowed that fecal calprotectin is more strongly correlated with theendoscopic activity of the disease in ulcerative colitis compared to theRachmilewitz clinical activity index In addition in this study theoverall accuracy of fecal calprotectin for endoscopically active diseaseidentification was obtained as []Some studies have also shown the superiority of fecal calprotectinover traditional ‚ammatory markers like CRP Besides one studyfound that fecal calprotectin was more strongly correlated with theSimple Endoscopic Score for Crohn's disease SESCD compared to theCRP and even Crohn's disease activity index CDAI [] The modifiedBaron Index was also used in another study to evaluate the endoscopicactivity of ulcerative colitis As a result it was shown that calprotectinis more strongly correlated with the endoscopic activity of ulcerativecolitis compared to CRP and clinical activity of the disease [] In thisregard similar results were also observed in our recent study in whichthe Ulcerative Colitis Endoscopic Index of Severity UCEIS and SESCDwere used [] Therefore fecal calprotectin appears to be superior totraditional ‚ammatory markers in the prediction of IBD endoscopicactivity The high values of sensitivity and specificity that were mentioned earlier have raised the hope that using fecal calprotectin canreduce colonoscopy rate for patients™ monitoring However severalrecent studies have reported some significantly lower values Accordingly in a recent study in which Mayo Endoscopic Score [MES] wasused to evaluate the endoscopic activity of ulcerative colitis aTable Summary of the results of some studies regarding the utility of fecal calprotectin in discrimination between patients with IBD and without IBDNumber of IBD patientsAge groupLocationCut oï¬SensitivitySpecificity CD and UC CD and UC CD and UC and unclassified68CD and UC CD and UC and unclassified CD and UC CD and UC UC CD UCAdultsAdultsAdultsBoth adult and pediatricpediatricAdultspediatricAdultsAdultsBoth adult and pediatricTaiwanChinaItalySpainFinlandIranItalyIranDenmarkIndia48µgg µgg150µgg150µgg595µgg784µgg160µgg164µgg150µgg188µggAUCReferences[][][]SPSrefidbib60[][][][][][][] 0cF KhakiKhatibi et alClinica Chimica Acta “Table Summary of the results of some studies regarding the correlation of fecal calprotectin with endoscopic activity in IBD patientsAge groupStudylocationUsedendoscopicactivity indexCorrelationcoefficientrReferenceNumberof IBDpatients CD UC UC CD UCAdultsAdultsAdultsAdultsAdultsFinlandIranSwitzerlandSwitzerlandSwitzerland ModifiedCDEISUCEISRachmilewitzSESCD UC CDAdultsAdults UC CD UC CD CD UC UCAdultsAdultsAdultsAdultsAdultsAdultsAdultsBaron ScoreRachmilewitzSESCDGermanyUSA andCanadaJapanItalyItalyBrazilFranceFranceSouth Korea UCEISMattsSESCDMayo scoreSESCDCDEISMayo score[][][][][][][][][][][][][][]sensitivity of and a specificity of were reported for fecalcalprotectin at µgg to diï¬erentiate active endoscopic from inactiveMES or from MES or [] In another study the sensitivityand specificity of fecal calprotectin at a cutoï¬ of µgg for diï¬erentiating MES ‰¤ in patients with ulcerative colitis were and respectively [] Overall as presented in Table several studiesperformed in diï¬erent countries reported the correlation between fecalcalprotectin and IBD endoscopic activity Although some of these studies reported a strong correlation some others reported a relativelyweak correlation As noted earlier there are significant diï¬erencesbetween the reports on the sensitivity and specificity of fecal calprotectin to predict the endoscopic activity of IBD Undoubtedly a widerange of factors from sample size and the inclusionexclusion criteriato preanalysis variables and indexes used to evaluate the endoscopicactivity may also contribute to these diï¬erences However fecal calprotectin does not appear to be a very reliable marker for the predictionof IBD endoscopic activity so currently it seems a bit optimistic toconsider fecal calprotectin as a reliable alternative for colonoscopy Inthis regard further studies are still needed However under some certain circumstances such as pregnancy or pandemics the use of fecalcalprotectin to evaluate IBD endoscopic activity can be helpfulPregnant patients with IBD have serious limitations for colonoscopyexamination and it has been recommended that colonoscopy should beonly performed in the second trimester of pregnancy and where there isa strong indication [] Therefore noninvasive markers such as fecalcalprotectin can be helpful during pregnancy In one study physicianglobal assessment [PGA] which is a clinical symptombased criterionwas used to evaluate IBD activity and subsequently the associationbetween fecal calprotectin and this criterion was investigated in pregnant women with IBD The results of this study showed a significantcorrelation between fecal calprotectin and PGA levels at prepregnancyduring pregnancy and postpartum stages [] In another study asignificant association was reported between fecal calprotectin levelsand clinical activity of IBD in pregnant women Moreover it was shownthat stool calprotectin at a cutoï¬ of mgkg had a sensitivity between and as well as a specificity between and in the assessment of IBD clinical activity at diï¬erent stages ofpregnancy [] A recently published systematic review has also confirmed the conclusions obtained from these studies [] According tothese results it seems that fecal calprotectin is not aï¬ected by physiological changes during pregnancy however it is significantly correlatedwith IBD clinical activity during pregnancy Therefore from the viewpoint of relatively acceptable sensitivity and specificity in predictingthe endoscopic activity of IBD fecal calprotectin may be considered as anoninvasive biomarker for the evaluation of IBD endoscopic activity inpregnant women In addition under pandemic conditions fecal calprotectin can be very helpful Following the COVID19 pandemicwhich began in late and is still ongoing healthcare systems indiï¬erent countries were forced to impose significant limitations oncolonoscopy Therefore noninvasive IBD management and fecal calprotectin as a noninvasive laboratory marker have become moreimportant than before The combination between disease clinical activity and fecal calprotectin has been recommended as a noninvasiveapproach that can help in making decisions on treatment duringCOVID19 pandemic [] Therefore it seems that fecal calprotectincan be considered as an alternative for colonoscopy used for IBD endoscopic activity evaluation during pandemic Fecal calprotectin appears to be associated with IBD histologic activity as well Given thedifficulty in the evaluation of the histologic activity of Crohn's disease[] some studies have been focused on the ulcerative colitis andmany scoring systems have been devised so far Correspondingly thesesystems score the disease's histologic activity based on histologic observationsTherefore for this purpose a biopsy of the intestinal tissue is required which can be prepared by colonoscopy and then sent to thelaboratory In this regard one of these histologic scoring systems isRobert™s score that was used in one of our recent studies where weobserved a significant correlation between the level of fecal calprotectinand the histologic activity of ulcerative colitis which was calculatedbased on the Robert™s scoring system [] Theede et al also used themodified Harpaz Index and performed some interesting studies in thisregard In one of their studies fecal calprotectin was found to be significantly associated with the histologic activity of the ulcerative colitisand it was shown that it could predict histological mucosal healingAUC CI95 “ Sensitivity Specificity andCutoï¬ mgkg [] In another study on patients with endoscopically inactive ulcerative colitis Mayo endoscopic score the researchers showed that patients with ulcerative colitis who were inendoscopic remission but had histologically active disease had higherlevels of fecal calprotectin compared to patients with no histologicallyactive disease versus mgkg P Also despite thehigh specificity the sensitivity of fecal calprotectin in theprediction of score of histological activity was achieved as at mgkg [] In a recent study the Geboes

Thyroid_Cancer

RANK are expressed in different cell types and tissues throughout thebody They were originally described for their essential roles in bone remodeling and theimmune system but have subsequently been shown to provide essential signals fromregulating mammary gland homeostasis during pregnancy to modulating tumorigenesisThe success of RANKLRANK research serves as a paragon for translational researchfrom the laboratory to the bedside The case in point has been the development ofDenosumab a RANKLblocking monoclonal antibody which has already helped millionsof patients suffering from postmenopausal osteoporosis and skeletal related events incancer Here we will provide an overview of the pathway from its origins to its clinicalrelevance in disease with a special focus on emerging evidence demonstrating thetherapeutic value of targeting the RANKLRANKOPG axis not only in breast cancer butalso as an addition to the cancer immunotherapy arsenalKeywords osteoimmunology RANKLRANKOPG malignant tumor targeted therapy DenosumabEdited byLinda ConnellyCalifornia University of Science andMedicine United StatesReviewed byDhivya R SudhanINTRODUCTIONUniversity of Texas SouthwesternMedical Center United StatesSophia HL GeeUniversity of Miami United StatesCorrespondenceJosef M PenningerjosefpenningerubccaSpecialty sectionThis was submitted toWomen™s Cancera section of the journalFrontiers in OncologyReceived February Accepted June Published August CitationMing J Cronin SJF and Penninger JM Targeting theRANKLRANKOPG Axis for CancerTherapy Front Oncol 103389fonc202001283In the œseed and soil theory was first proposed by Stephen Paget for tumor metastasesto distant ans When tumor cells œseeds leave their primary site of origin and spreador metastasize the microenvironment œsoil of the target an is usually favorable for tumorcell anchoring and expansion of metastatic cells Bone is not only the site for primary bonetumors such as giant cell tumors and osteosarcoma but is also one of the most common distantmetastatic sites for solid tumors such as multiple myeloma MM breast cancer prostate cancerand nonsmall cell lung cancer NSCLC suggesting that the bone environment can serve asœsoil for tumor development and might also serve as a œseed for further metastatic spread Recentresearch on the bone microenvironment and its involvement in cancer biology has focused on thefield of osteoimmunology which includes the crosstalk between bone stromal cells osteoblastsand osteoclasts and immune cells Identifying key players regulating bone homeostasis couldpave the way for potential therapeutic cancer targets in particular to break the vicious circle ofmetastasis to the bonesThe receptor activator of the nuclear factor kappaB ligand RANKL also known as TNFSF11together with its receptor RANK TNFRSF11A the decoy receptor osteoprotegerin OPGTNFRSF11B and the recently identified receptor Leucinerich repeatcontaining Gproteincoupled receptor LGR4 has been shown to play critical bottleneck functions not only inregulating bone metabolism but also in immunity and tumorigenesis In this review we will brieflyintroduce the key functions of the RANKLRANKOPG axis in maintaining bone homeostasisand regulating immunity Furthermore we will discuss the role of this pathway from primaryFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancertumorigenesis to cancer metastasis with particular attention tobreast cancer and the hormonal control of this pathway We willalso discuss recent data pointing to the RANKLRANK axis as anovel therapeutic target in BRCAmutated breast cancers and asa novel promising cancer immunotherapy agentRANKLRANKOPG AND BONEHOMEOSTASISBone provides strength and structure protects vital ans storesminerals such as calcium and is essential in the productionof hematopoietic cells Bone homeostasis is maintained by thebalance between mainly two types of cells osteoblasts derivedfrom mesenchymal cells which build bone and osteoclastsderived from bone marrow hematopoietic precursor cells whichresorb bone Figure Osteoblasts act as both mechanicalsensors together with osteocytes and coordinators for the boneremodeling process which is controlled by local growth factorsand systemic factors for example calcitonin or sex hormonessuch as estrogen The pathological imbalance between boneformation and resorption leads to the development of local orsystemic bone diseases such as osteopetrosis and osteoporosis The interaction and communication between osteoclasts andosteoblasts is intricately regulated in feedback loops to maintainbone homeostasis and this constant remodeling process of thebone matrix is critical for healthy bone strength and efficienthematopoiesis RANK TNFRSF11A OFE ODFR TRANCER ODARCD265 and RANKL TNFSF11 TRANCE ODF andOPGL “ are a receptorligand pair of the TNF receptorsuperfamily discovered at the end of the last millennium and wereidentified as key regulators of osteoclast development and bonemetabolism Figure Factors that can induce boneresorption such as the sex hormone progesterone vitamin D3PTHrP IL1 IL11 IL17 or TNFα “ act on osteoblaststo induce RANKL expression which then binds to its receptorRANK on the surface of osteoclast progenitor cells inducing preosteoclast diï¬erentiation into multinucleated fullyfunctionalosteoclasts RANKL also plays an important role in the continuedsurvival and function of osteoclasts “ Figure RANKLis produced as a membranebound protein which can alsobe shed as a soluble trimeric protein SheddaseresistantRANKL mice have been generated in which soluble RANKLis undetectable in the circulation bone mass or boneFIGURE Role of RANKLRANKOPG axis on bone homeostasis and immune system RANKL is secreted by osteoblasts and osteocytes when stimulated byparathyroid hormone PTH vitamin D andor prostaglandin PGE2 RANKL binds to RANK on the membrane of osteoclast progenitors preosteoclasts whichresults in bone resorption by mature osteoclasts Osteoprotegerin OPG binds to RANKL thus inhibiting RANK signaling and bone resorption RANKRANKL alsoplays a role in immune cell regulation and the crosstalk between both systems termed osteoimmunology T cells can also express RANKL which can both act onpreosteoclasts but can also act on dendritic cells DCs to promote their survival and to prolong T“DC interactions DCs can exhibit modulating effects onRANKmediated osteoclastogenesis through the secretion of OPG HSC hematopoietic stem cell MSC mesenchymal stem cellFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancerstructure was not aï¬ected during development in these mice butadult mice displayed reduced osteoclast numbers and increasedcancellous bone mass Importantly the bone loss caused byestrogen deficiency was unaï¬ected by the lack of soluble RANKLThus these data show that it is the membranebound formof RANKL which is largely responsible for the physiologicalfunctions of RANKL although the soluble form can contributeto bone remodeling in adult mice Osteoprotegerin OPG TNFRSF11B acts as a decoy receptorfor RANKL and is induced by estrogen IL4 or transforminggrowth factor beta TGF OPG competitively binds toRANKL thereby interfering with RANKL“RANK interactionsand blocking bone resorption “ The relative levels ofOPG and RANKL are precisely controlled to ensure healthybone During pathological conditions such as menopauserelatedosteoporosis decreased estrogen levels result in decreased OPGand subsequently increased RANKL resulting in enhancedosteoclast activation and bone loss Recently leucinerichrepeat G proteincoupled receptor LGR4 was identifiedas an additional receptor for RANKL Similar to RANKLGR4 is expressed on osteoclasts but unlike RANK LGR4 is anegative regulator for osteoclast diï¬erentiation Therefore bothOPG and LGR4 are endogenous inhibitors of RANKLRANKsignaling A recent study has shown that RANKL reversesignaling from osteoclasts to osteoblasts couples bone resorptionto bone formation processes This is achieved through thesecretion of small extracellular vesicles from osteoclasts thatcontain RANK The authors showed that these RANK vesiclesbind membranebound RANKL on the osteoblasts and therebypromote bone formation by triggering RANKL reverse signalingvia activation of Runtrelated transcription factor Runx2Targeting RANKL reverse signaling represents a novel strategyto avoid the reduced bone production associated with inhibitionof osteoclastogenesis As RANKL is an important regulator of bone loss in bonemetastases associated with cancers such as multiple myelomaand in postmenopausal osteoporosis a specific fully human IgG2monoclonal RANKL antibody mAb has been developed whichneutralizes the activity of RANKL which has been designated asDenosumab The efficacy of Denosumab has been confirmed inmultiple clinical trials and Denosumab therapy is now approvedand widely used for the treatment of various boneassociateddiseases “RANKLRANKOPG IN THE IMMUNESYSTEMApart from bone homeostasis the RANKLRANKOPG axis isalso involved in various physiological immune processes RANKwas originally discovered on dendritic cells DCs and RANKLmediates the survival of DCs The interaction betweenactivated T cellderived RANKL and RANK expressed on DCsincreases the antigenpresenting capabilities of the latter thusaugmenting the number and cell cycle of antigenspecific Tcells as well as enhancing the immune response of memory Tcells Interestingly phenotyping of rankl and rankdeficient micerevealed a complete absence of peripheral lymph nodes but intactspleen and Peyer™s plaque structures “ Subsequent studieshave found that during embryogenesis RANKL is expressedby hematopoietic lymphoid tissue inducing LTi cells andmesenchymallymphoid tissue anizer LTo cells “RANKL has been demonstrated to stimulate lymphotoxin LTexpression and regulate LTi cell accumulation FurthermoreRANKL also triggers the proliferation of adult lymph nodestroma indicating that RANKL may directly activate LTo cells“ In the thymus the RANKLRANK pathway is criticalfor CD80 AIRE medullary thymic epithelial cell mTECmaturation involved in central immune tolerance RANKdeficient mice display mild autoimmunity at an advancedage RANKLRANK activation in lymphatic endothelialcells LECs is important for the tissueresident macrophagesnamely sinusoidal macrophage maturation not only duringembryogenesis but also after inflammationinduced loss of thesecells Moreover group innate lymphoid cells ILC3s inthe intestine use RANKLRANK interactions to control theirown abundance and intestinal homeostasis Genetic ablation ofRANKL specifically in IL3C cells leads to an increased number ofthese cells with enhanced levels of proinflammatory cytokinessuch as interleukin17A IL17A and IL22 during intestinalinfection Human patients carry RANK mutations and mice lackingRANKL or RANK exhibit a defect in B cell developmentresulting in a significant reduction in B cell numbers however these eï¬ects might be indirect because in themousetissuespecific deletion of Rank in B cells showedno diï¬erence in function nor development of B cells andblocking RANKRANKL with Denosumab does not apparentlyaï¬ect B cell physiology in osteoporosis patients In addition reports using Bcellspecific rankldeficient micehave shown that B cellderived RANKL increases osteoclastnumbers and bone loss brought on by estrogen deficiency Overexpression of RANKL in keratinocytes results in functionalalterations of epidermal dendritic cells and systemic increases inregulatory CD4CD25 T cells Tregs numbers Thereforeenvironmental stimuli can rewire the local and systemic immunesystems via RANKL The RANKLRANK system is alsoinvolved in M microfoldcell development a specific antigensampling cellular subtype found in the intestine as mesenchymalcells produce RANKL that can directly interact with intestinalepithelial cells to regulate M cell diï¬erentiation “Inhibition of mesenchymal RANKL impairs M celldependentantigen sampling and B celldendritic cell interaction in thesubepithelial dome SED resulting in decreased IgA productionand microbial diversity In addition B cells are absentin cryptopatches CPs and isolated lymphoid follicle ILFsformation was abrogated in rankl null mice Whether B cells or T cells are essential for bone loss isstill controversial Ovariectomy has been shown to enhance Tcelldependent TNFalpha production in a bone loss mousemodel because of the enhanced macrophage colonystimulatingfactor MCSF and RANKL In contrast anotherstudy suggested T cells are not involved in ovariectomyinducedFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancertrabecular bone loss Nevertheless it has been reportedin postmenopausal women that increased T cell activity andincreased RANKL production by T cells are associated withosteoporosis Furthermore studies in conditionalknockout mice to specifically eliminate RANKL in B cells or Tcells have shown that RANKL produced by B cells but not T cellsleads to bone loss by the induction of osteoclastogenesis Thelack of mature B cells does not prevent bone loss suggestingthat RANKL is derived from immature B cells Moreover it hasbeen reported that deletion of rankl in T cells does not change thenumber of T cells but results in impaired mature B cell numbersin the bone marrow suggesting that RANKL might promote Bcell maturation via paracrine signaling RANKLRANKLOPG IN MAMMARYGLAND PHYSIOLOGY AND BREASTCANCERBreast cancer is the most prevalent female malignancy Studies based on large populations have shown that womenwho receive estrogen plus progesterone hormone replacementtherapy called combined HRT are more vulnerable to breastcancer compared to women who receive estrogen only “furthermore progesterone levels have been demonstrated to bean independent risk factor for increased breast cancer incidence In rankl knockout mice our group was the first to reportthat during pregnancy RANKL deficiency results in a totalblock in the development oflobuloalveolar milksecretingstructures Whereas estrogen triggers the expansion ofthe mammary epithelium in puberty progesterone drives theproliferation of mammary epithelial cells in the estrous cycleand in pregnancy induces the growth and diï¬erentiation of themammary epithelium into ultimately milksecreting acini Figure Mechanistically progesterone induces progesteronereceptor PRpositive mammary epithelial cells to expressRANKL resulting in the proliferation of neighboring RANKmammary epithelial progenitor cells in an autocrine and alsoparacrine fashion “ Moreover RANKL can induce theproliferation of RANKpositive ductal epithelial cells through theFIGURE RANKRANKL pathway in mammary gland physiology and breast cancer A RANK is constitutively expressed on the membrane of luminal and basalepithelial cells including mammary stem cells MaSCs Stimulation with progesterone induces RANKL expression and secretion in progesterone receptor PRpositiveluminal epithelial cells RANKL binds in an autocrine fashion to RANK on luminal epithelial cells which stimulates further RANKL expression and in a paracrine fashionto RANK on basal epithelial cells resulting in enhanced RANK expression on basal mammary epithelial cells and the activation of the IKKαNFκB“cyclin D1 signalingaxis to induce a variety of physiological responses necessary for mammary gland development B Heterozygous BRCA1 mutationcarrying women canspontaneously lose the remaining wildtype BRCA1 gene from somatic mutation or epigenetic silencing Subsequently loss of BRCA1 protein can result in increasedgenomic instability DNA damage and genetic mutations eg TP53 Progesterone as well as synthetic progestins upregulate RANKL expression in PR luminalbreast epithelial cells which stimulates RANKmediated cell proliferation of adjacent progenitor cells as discussed in A Altogether the genotoxic stress and amplifiedproliferation cues culminate in uncontrolled proliferation and the development of breast cancerFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancerinduction of Rspondin Therefore RANK and RANKL linksex hormones to mammary progenitor cell proliferation duringthe estrous cycle and in pregnancy Figure Clinically an increase in serum progesterone and RANKLlevels is associated with an increase in breast cancer risk inpostmenopausal women Higher concentrations of solubleRANKL are positively correlated with an increased risk ofestrogen receptorpositive but not estrogen receptornegativebreast cancer indicating that the RANKRANKLOPG axis maybe involved in the tumorigenesis of ER breast cancer Indeed in a hormoneinduced spontaneous mouse breast cancermodel RANKL is critical for the development of sex hormonedriven breast cancer Deletion of RANK and Ikkα akey downstream regulator of the RANK signaling pathway inmammary epithelial cells also significantly delayed progestinMPA and DNAmutation DMBAinduced mammary tumorformation further indicating that the RANKRANKL pathwaydrives breast cancer Furthermore the selective inhibitionof RANKL by RANKFc not only attenuated breast tumorprogression in a hormone and carcinogendriven mouse breastcancer model but also decreased the progression of breast cancerin a transgenic spontaneous tumor model BRCA1 and BRCA2 mutations are the most prevalent geneticdrivers for hereditary breast cancer in humans Interestinglywomen with germline BRCA12 mutations usually exhibithigher progesterone and estrogen levels during the gestationalphase ofthe estrous cycle compared to women withoutthese mutations Inversely decreased serum OPG levelsare associated with increased breast cancer incidence Moreover high levels of RANK expression were observed inbreast cancer samples from premalignant lesions and patientswith BRCA1 mutations SNP data analysis from theCooperative Tumor GeneEnvironmental Research iCOGSincluding approximately BRCA1 and BRCA2mutation carriers identified SNPs which were significantlyassociated with breast cancer risk at the TNFRSF11A locusencoding RANK Altogether these human data stronglysupport the idea that the RANKLRANKOPG axis is intimatelyinvolved in the tumorigenesis of BRCA12 mutationdrivenbreast cancerSubsequent animal studies provided direct evidence thatRANKL and RANK are critically involved in the oncogenesis ofBRCA1 mutationdriven hereditary breast cancer Figure Genetically engineered mice carrying Brca1 and Tp53 mutationsshowed hyperproliferation and malignancy in their mammaryglands at months of age the inactivation of the RANKLRANKpathway in these mice largely prevented the occurrence ofmalignanttumors and resulted in significantly prolongedsurvival Additionally the pharmacological blockade of RANKLusing RANKFc completely abolished the development ofprecancerousin the Brca1Tp53 doublemutatedbreast cancer model Amplification of RANKexpressingmammary duct progenitor cells can be found in the nontumorbreast tissue of BRCA1 mutant carriers and these cells havesimilar molecular characteristics as basallike breast cancercells RANKL inhibition also significantly suppressedthe proliferation oftumor anoids derived from BRCA1mutant human breast biopsy specimens and RANKLRANKlesionspathway blockade strongly reduced tumorigenesis in patientderived xenograft PDX breasttumor mouse model Thus independent work among diï¬erent laboratories usingdiï¬erent mouse models as well as studies using humanbreast epithelial progenitor assays hasled to the sameconclusion RANKLRANK aï¬ect mammaryprogenitorcells and are critically involved in the BRCA1mutation drivenmammary tumorigenesisTherefore we and others have proposed that the monoclonalantibody Denosumab which specifically inhibits RANKRANKLinteractions could potentially be used for the prophylactictreatment of breast cancer in BRCA12 carriers Indeed weposit that healthy women with BRCA1 mutation will benefit notexcluding an eï¬ect on other TNBCs In a pilot clinical studytermed BRCAD the proliferation marker Ki67 was significantlydownregulated in the breast biopsy of BRCA1 mutation carrierswho received shortterm treatment with Denosumab suggestingthat RANKL inhibition may be a feasible method for the chemoprevention of breast cancer in women with BRCA1 mutationsThis study requires additional patient data which is currentlyongoing Another clinical study DBEYOND which aimedto investigate whether neoadjuvant RANKL inhibition therapycan reduce tumor proliferation in premenopausal early breastcancer patients found no significant change in Ki67positivetumor cells in the breast cancer tissues treated with Denosumabbut the density of tumorinfiltrating lymphocytes TILs wasincreased in the stroma and tumor tissues upon Denosumabtreatment In addition to the now experimentally well validated role ofRANKLRANKOPG in the sex hormone and BRCA1 mutationdriven mammary cancer tumorigenesis it has also been reportedthat this pathway can induce epithelialmesenchymal transitionEMT in breast cancer cells as well as in prostate andendometrial cancers “ suggesting that RANKLRANKsupports tumorigenesis in various epithelial cancers Moreoverour group has recently reported on the role of RANKL andRANK in lung cancer We demonstrated that the inactivation ofrank in lung epithelial cells disrupts mitochondrial bioenergeticsand significantly reduceslung cancer development bothculminating in increased survival This genetic modeling inthe mouse supports findings in human clinical trials in whichRANK inhibition with the monoclonal antibody Denosumabresulted in prolonged survival especially in patients withnonsmall celllung cancer NSCLC adenocarcinomas andsquamous tumors Notably this Denosumabdependent survivaladvantage occurred in lung cancer patients irrespective of visceralmetastasis hinting that the underlying eï¬ects of RANKLRANKblockade in addition to those targeting the bone are involved Epidemiological reports have also uncovered genderdiï¬erences particularly in lung cancer with respect to etiologyprogression and treatment response believed to be due tosexrelated hormonal factors “ though the underlyingmolecular mechanisms are poorly understood We have recentlyshown in our experimental lung cancer model that by ablatingthe sex hormones in female mice we could eï¬ectively eliminatethe survival advantages brought about by loss of rank in the lungtumors Furthermore synthetic progesterone MPAdependentenhanced lung cancer initiation required RANK expressionFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in CancerTogether these data suggest that the sex hormone regulation ofRANKLRANK could also explain the gender diï¬erences seen inhuman lung cancerRANKRANKL AS REGULATORS OFMETASTASISStudies have now shown that the RANKLRANKOPG axis playsa role in the progression of malignant tumors by promotingtumor cell migration stimulating tumor neovascularizationand promoting distant metastasis of tumor cells Disseminated tumor cells are responsible for the earlymetastasis of tumors which frequently can be detected inthe bone marrow of patients with malignant tumors Thisœmicrometastases niche forms a favorable microenvironmentfor the development of metastatic spread protecting cancer cellsfrom various antitumor treatments and modulating anticancerimmune responses thereby allowing the tumor cells to escapeimmune surveillance The tumor microenvironment is acomplex milieu composed of distinct factors such as cytokinesextracellular matrix components and various cell types suchas fibroblasts endothelial cells and immune cells all ofwhich participate in cancer development progression andmetastasis In bone tissue the tumor microenvironmentincludes immune and tumor cells as well as osteoblasts andosteoclasts all of which participate in a œvicious cycle thataccelerates osteolysis and cancer cell proliferation through inpart the RANKRANKLOPG axis For instance cancercells can increase the expression of RANKL in osteoclastsby secreting parathyroid hormonerelated peptide PTHrP Tumor cells can also directly express RANKLand secrete cytokines such as interleukin IL1α TNFα macrophage colonystimulating factor MCSF orprostaglandin E2 PGE2 all of which promote osteoclastdiï¬erentiation and survival resulting in local osteolysis whichsupports metastatic growth “ Subsequently growthfactors released by the bone matrix such as insulinlike growthfactors IGFs fibroblast growth factor FGFs plateletderivedgrowth factor PDGF or bone morphogenetic proteins BMPspromote cancer cell proliferation “ In addition tocytotoxic drugs and endocrine disruptive drugstherapiestargeting the RANKRANKLOPG axis exhibit direct andorindirect antitumor eï¬ects by blocking the vicious cycle betweenbone and cancer cells “In a murine model of melanoma metastasis it was foundthat for malignant tumors with RANK expression RANKLproduced by osteoblasts and bone marrow stromal cells couldact as a chemical attractant and promote the migration andmetastasis of malignant tumors to these sites Similareï¬ects were also found in malignant tumors such as breastcancer “ prostate cancer “ and lung cancer The activation of phospholipase C PLC proteinkinase C PKC ERK and phosphatidylinositol3OH kinasePI3K pathways were involved in RANKinduced tumor cellmigration “ RANK engagement by RANKL inducestrimerization of the RANK receptor which then stimulates therecruitment and activation of the adapter protein TRAF6 viaTRAF6binding sites in the Cterminus of RANK™s cytoplasmictail TRAF6 in turn complexes with many other downstreamadapters and kinases to activate the aforementioned pathwaysMoreoverthe RANKLRANK pathway was also shown topromote the formation of new blood vessels and regulate thetumor microenvironment at the primary tumor site to promotethe migration of tumor cells into the bloodstream and formetastasis to distant ans “In breast cancer RANKL is also produced by Foxp3expressing Tregs and tumorassociated macrophages TAMsthat can aï¬ect tumor growth tumor cell dissemination andmetastasis RANKL expression on tumorinfiltratingregulatory T cells may also be involved in cancer metastasis TAMs are either M1 or M2 macrophages with M1 being antitumor and M2 TAMs promoting tumorigenesis ImportantlyM2 macrophages express RANK and are attracted by RANKLproduced by the tumor microenvironment The RANKLRANKpathway in M2 macrophages can regulate the production ofchemokines and promote the proliferation of Treg lymphocyteswhich supports the immunosuppressive milieu within the tumormicroenvironment Recently it has been reported that estrogenrelatedreceptoralpha ERRα an important factor of cancer cell invasivenesspromotes breast cancer cell dissemination from primarymammary tumors to the bone Intriguingly RANK hasbeen shown to be a target for ERRα Furthermore the metaexpression analysis of breast cancer patients has uncovereda positive association between metastases and ERRαRANKexpression as well as a positive correlation between ERRαand BRCA1 mutation carriers revealing a novel pathwaywhereby ERRα in primary breast cancer could promote earlydissemination of cancer cells to bone Moreover it wasrecently shown that RANKL serum levels are significantlyincreased in breast cancer patients who developed bonemetastases p and patients within the highest quartileof RANKL had a significantly increased risk of developing bonemetastases compared to those in the lowest HR 95CI“ p This study further suggests a role ofRANKL in breast cancer metastasis TARGETING RANKLRANK IN HUMANCANCERIn light of the diï¬erent roles of the RANKLRANK pathwayin bone metabolism and immune system functions therapytargeting this axis may not only control primary tumordevelopment such as in the case of breast cancer and reducebone metastasis which has been demonstrated in clinical trials but also exert a direct antitumor eï¬ect via regulatinglocal tumorassociated immune responses as observed in studiesusing the monoclonal RANKL antibody inhibitor Denosumab In randomized clinical trials Denosumab has shown rapideï¬ectiveness by directly impairing osteoclast activity andinducing osteoclast apoptosis Moreover Denosumabwassignificantly more eï¬ective in reducing urinary Nterminal peptides a biochemical marker for bone turnoverFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancerand more eï¬ective in delaying skeletalrelated events SREssuch as pathologic fractures spinal cord compression andhypercalcemia which greatly aï¬ect quality of life in patients withbreast cancer and castrationresistant prostate cancer CRPCbone metastases However the eï¬ect of Denosumab to delaySREs in patients with NSCLC and multiple myeloma MMpatients with bone metastases is comparable to bisphosphonatedrugs “ Moreover the benefit of Denosumab andbisphosphonates is not only restricted to osteolytic cancers suchas breast myeloma and NSCLC but also evident in osteoblasticcancers Recently it was demonstrated in osteoblastic cancerssuch as prostate cancer that Denosumab or bisphosphonate canaï¬ect the osteoclastosteoblast balance in the œvicious cycle ofbone destruction induced by metastasized cancer cells which highlights the potential rationale in treating osteoblasticcancer patients with Denosumab or bisphosphonatesIn a randomized phase III clinical trial comparing Denosumaband bisphosphonate zoledronic acid ZA in patients with solidtumors breast cancer prostate cancer multiple myeloma andbone metastases the results showed that Denosumab was similarto ZA in preventing or delaying the onset of primary SREs However in nonsmallcell lung carcinoma NSCLCn treatment with Denosumab showed a significantimprovement in overall survival In these patients nostatistically significant SRE delay was observed in Denosumabtreated patients suggesting that this survival advantage maybe independent of the bone system The result of arandomized phase III trial of multiple myeloma MM patientsn also demonstrated the eï¬ectivity of Denosumab toreduce the occurrence of primary SRE events moreover the useof Denosumab significantly improved progressionfree survivalPFS Whether this survival benefit is due to the decreasein the incidence of bone metastasis or whether Denosumab hasother antitumor eï¬ects requires further researchIn the randomized placebocontrolled phase III ABCSG18trial which enrolled postmenopausal female patients withearly hormone receptorpositive breast cancer the first clinicalfracture of the Denosumabtreated group was compared with theplacebo group and a significant protection of bone breaks wasdemonstrated hazard ratio [HR] · [ CI ·“·] p A median followup of months showeda significant improvement in the diseasefree survival DFS inthe Denosumabtreated group HR CI “Cox p These data suggest that adjuvant Denosumabcan significantly improve the DFS rate of HR postmenopausalbreast cancer patients However in another randomizedphase III clinical trial of breast cancer DCARE recent reportshave shown that adjuvant Denosumab does not reduce therisk of breast cancer recurrence or death in earlystage breastcancer patients receiving standard adjuvant therapy Theseinconsistencies which could be explained by diï¬erent cohortsfor patient stratifications eg more advanced early cases ofbreast cancer were included in the DCARE trials as comparedto the ABCSG18 study need to be further evaluated with largercohorts of patients and multiplecenter analysis Importantlya recent followup study of the ABCSG18 trial confirmed thethat blocking RANKL in an adjuvant breast cancer therapysetting not only markedly reduces the risk of breaking bones butalso significantly reduces the reoccurrence of the breast tumors It should be also noted that although there was nodiï¬erence in bonemetastasesfreesurvival in the DCARE trialDenosumab treatment significantly reduced the time to bonemetastasis at the site of first occurrence DENOSUMAB AS A NOVEL CANCERIMMUNOTHERAPYThe field of cancer immunotherapy has paved the way for a newparadigm to combat cancer by coaxing the body™s own immunesystem to seek out specifically target and destroy cancer cellsAmong the various approaches immunecheckpoint inhibitorsthattarget CTLA4 as

Thyroid_Cancer

"Cellular recognition of microbial DNA is an evolutionarily conserved mechanism by which the innate immunesystem detects pathogens Cyclic GMPAMP synthase cGAS and its downstream effector stimulator of interferongenes STING are involved in mediating fundamental innate antimicrobial immunity by promoting the release oftype I interferons IFNs and other inflammatory cytokines Accumulating evidence suggests that the activation ofthe cGASSTING axis is critical for antitumor immunity The downstream cytokines regulated by cGASSTINGespecially type I IFNs serve as bridges connecting innate immunity with adaptive immunity Accordingly a growingnumber of studies have focused on the synthesis and screening of STING pathway agonists However chronicSTING activation may lead to a protumor phenotype in certain malignancies Hence the cGASSTING signalingpathway must be orchestrated properly when STING agonists are used alone or in combination In this review wediscuss the dichotomous roles of the cGASSTING pathway in tumor development and the latest advances in theuse of STING agonistsKeywords cGASSTING Innate immunity Type I interferon STING agonists Antitumor response CancerdevelopmentIntroductionThe discovery of phagocytosis in advanced the understanding of innate immunity the first line of host defenses[]Protection againston patternrecognition receptors PRRs which recognize microbialproducts coordinate antimicrobial defenses and activateinfection dependsagainstinfection byvarious pathogens Correspondence zqliucsueducn Juyan Zheng and Junluan Mo contributed equally to this work1Department of Clinical Pharmacology Hunan Key Laboratory ofPharmacogenetics and National Clinical Research Center for GeriatricDisorders Xiangya Hospital Central South University Changsha People™s Republic of China2Institute of Clinical Pharmacology Engineering Research Center for appliedTechnology of Pharmacogenomics of Ministry of Education Central SouthUniversity Changsha People™s Republic of ChinaFull list of author information is available at the end of the adaptive immunity [] Abnormal RNA or DNA RNADNA hybridization and cyclic dinucleotides derived frommicrobes are usually considered pathogenassociated molecular patterns PAMPs [ ] Cells associated with innate immunity recognize different microbial PAMPsthrough specific PRRs thereby playing key roles in hostresistance to microbial infection [] The pathways governing RNA recognition such as retinoid acid induciblegene I RIGIlike receptors have been reviewed elsewhere and will not be covered herein In the case of DNArecognition one of the best known PRRs is Tolllike receptor TLR9 which senses extracellular CpG hypomethylated DNA that has entered the cytosol through thephagosomelysosome system [] In addition the AIM2like receptor AIM2 inflammasome can be triggered afterthe entry of doublestranded DNA dsDNA into the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cZheng Molecular Cancer Page of cytosolic compartment which induces the proteolyticmaturation of proinflammatory cytokines such as IL1and IL18 and the activation of gasdermin D leading topyroptosis [“] Nevertheless the most notable PRR iscGAS a direct cytosolic dsDNA sensor which was identified by Dr Chen™s group in [] Once cGAS bindsto dsDNA the cGASSTING pathway is activated to further induce the expression of type I IFNs and other inflammatory cytokinesthus triggering innate immuneresponses [] Mounting evidence suggests that cGASSTING signaling not only plays pivotal roles in the hostdefense against microbialinfection but also modulatestumorigenesis Hence in this review we summarize themechanism of cGASSTING activation and elaboratefindings regarding its dual effects on tumor developmentCurrent advances in the use of STING agonists as a novelstrategy for antitumor therapy are also reviewedInsights into the cGASSTING signal transductioncascadecGAS is an innate immune sensor that identifies variouscytosolic dsDNAincluding DNA with viral bacterialmitochondrial micronuclei and retroelement originswhich can be mainly divided into pathogenderivedDNA and selfDNA Table In the cytoplasm cGAS isactivated by interacting with dsDNA in a sequence[“]independent butStructural and biochemical analyses have revealed thatthe Cterminal lobe of cGAS contains a conserved zinclengthdependent mannerionbinding module that mediates DNA binding andcGAS dimerization [ ] DNA ligands promotecGAS activation primarily by inducing conformationalchanges around the catalytic site and in the DNAbinding structures of cGASthe GScontaining loopundergoes conformational change to maintain stabilitywhich is a major mechanism of cGAS activation byDNA [] In addition to the primary DNAbinding sitementioned above the secondary site located beside theprimary site is a helix formed between strands 78and several surfaceexposed loops [] The proximity ofthe two DNAbinding sites in cGAS leads to a cGASDNA complex assembly in which two cGAS moleculesembrace two molecules of dsDNA [ ] The cGASdimers are anized in œheadtohead alignment nextto the DNA [] and thus form stable œladderlike networks between one long curved dsDNA helix or two independent dsDNA strands [ ] In this way eachindividual cGASdsDNA complex can be cooperativelystabilized and can lead to stronger enzymatic activitywhich may provide a possible explanation for longerdsDNA as more likely to activate cGAS [] In additionlong DNA is more efficient than short DNA in drivingthe liquidliquid phase separation of cGAS and the formation ofcriticallydependent on the concentration of cGAS and DNA inthe cytoplasm [] cGAS and dsDNA are spatially concentratedcGASdimerization and activation [“] Once cGAS andcGAS liquidlike dropletsin liquiddropletsistofacilitateTable Classification of the cytosolic dsDNA that activates the cGASSTING signaling axisClassificationSelfDNASource of dsDNAMicronucleiPossible mechanismsRupture of the micronuclei membrane leads to exposureof chromatin DNA that is recognized by cGAS whichactivates the cGASSTING pathwayReferences[]MitochondrionNuclear RNAPathogenderived DNADNA virusHSV1 HSV2 KSHV adenovirus vacciniavirus cytomegalovirus papillomavirusmurine gammaherpesvirus RetrovirusHIV SIV murine leukemia virusRNA virusWest Nile virus dengue virus VSVSARSCOV2BacteriaListeria monocytogenes Mycobacteriumtuberculosis Listeria Shigella FrancisellaChlamydia and NeisseriaMitochondrial stress induces mtDNA leakage into thecytosol thus activating the STING pathway and inducingproduction of cytokinesFacilitated by endogenous retroelements nuclear RNAcan be reversely transcribed into DNA that activatescGASSTING signaling[][]DNA viruses invade host cells and release pathogenderivedDNA to induce STING activation[“]DNA intermediates generated from reverse transcription maybe recognized by cGAS to stimulate downstream STINGsignaling[]Infection with RNA viruses might cause cellular damage andcell death which results in the release of cellular DNA andfurther activation of the cGASSTING axis SARSCoV2 bindingto ACE2 can lead to excessive angiotensin II signaling thatactivates the STING pathway in mice[“]Bacteria produce CDNs such as cyclic diGMP and cyclicdiAMP which can directly bind to and activate STING[ “]HSV1 herpes simplex virus HSV2 herpes simplex virus KSHV Kaposi sarcoma“associated herpesvirus HIV human immunodeficiency virus SIV simianimmunodeficiency virus VSV vesicular stomatitis virus CDNs cyclic dinucleotides and SARSCOV2 severe acute respiratory syndrome coronavirus 0cZheng Molecular Cancer Page of dsDNA interacts structural switches rearrange the catalytic pocket to enable cGAS to catalyze the synthesis of²²cyclic GMPAMP ²²cGAMP with ATP andGTP as substrates The first step in this process is theformation of a linear dinucleotide ²pppG ²²pAwith ATP serving as the donor and ²OH on GTP serving as the acceptor Then the intermediate product flipsover in the catalytic pocket placing GTP at the donorposition and AMP at the acceptor position to form asecond ²² phosphodiester bond [ ] Notablyalthough dsRNA or singlestrand DNA ssDNA is ableto bind to cGAS neither can rearrange the catalyticpocket which may explain the exclusive activation ofcGAS by dsDNA Ultimately cGAMP acts as a secondmessenger to bind to and activate STING a small endoplasmic reticulum ERlocated protein KD withfour putative transmembrane domains [ ] Normally in a resting state STING is retained in the ER byinteracting with the Ca2 sensor stromalinteractionmolecule STIM1 [] The cytosolic ligandbindingdomain LBD of STING exists as the most functionalunit capable of integrating with ²² cGAMP or CDNscyclic dinucleotides such as cdiAMP cdiGMP or ²²cGAMP from bacteria Upon interaction the obviousclosure of the ligand binding pocket in the LBD is observed which is related to the activation of STING []Next STING transforms into a tetramer through a highorder oligomerization reaction and is translocated fromthe ER to the perinuclear area facilitated by cytoplasmiccoat protein complex II COPII and ADPribosylationfactor ARF GTPases [ ] In the Golgi STING ispalmitoylated atCys88 andCys91 a posttranslational modification necessary forSTING activation [] Modified STING recruits thekinase TANKbinding kinase TBK1 in turn the Cterminal domains of STING are phosphorylated byTBK1 and then phosphorylated STING recruits interferon regulatory factor IRF3 which is also phosphorylated by TBK1 and dimerizes ultimately dimerizedIRF3 enters the nucleus and exerts its function in thetranscription of type I IFNs and interferonstimulatedgenes ISGs [] In parallel STING can also bind toand stimulate IκB kinase IKK to mediate the production of nuclear factorκB NFκBdriven inflammatorygenes Upon signal transduction termination STING istransferred to endolysosomes for degradation [] Considering that cGAMP can be transferred through gapjunctions or delivered in viralexosome packages cGASSTING signaling may be activated in the cytoplasmwithout dsDNA [ ] Moreover newly produced typeI IFNs activate heterodimer interferon receptors IFNAR1 and IFNAR2 through paracrine signaling and thusinduce the transcription of ISGs [ ] In summaryonce virusderived DNA and selfDNA are located intwo cysteine residuesthe cytoplasm they can be sensed by cGAS and a cGASdsDNA complex is formed to catalyze the synthesis of ²²cGAMP with ATP and GTP Then ²²cGAMP and bacteriaderived CDNs induce STING activation and mediate the release of downstream type IIFNs TNFα and IL6 which are prerequisites for antimicrobial defense and antitumor effects The wholeprocess shows that the dsDNAcGASSTING axis canlead to the activation of both innate and adaptive immunity Fig The antitumor functions of the cGASSTINGsignaling pathwayRecent evidence has revealed the close association of thecGASSTING pathway with cancer development Thissignaling pathway is generally regarded as a potent regulator of cancer immunity A STINGmediated immunesupportive microenvironment can hamper malignancyoccurrence []stressbyTumor cell cytosolic dsDNA induces STING activationUnder normal circumstances DNA is strictly unaffiliatedwith the cytoplasm in eukaryotic cells to avoid autoimmunity [] However DNA leaks aberrantly in tumorcells [ ] Cancer cells share common features including genome instability tumor suppressor gene mutation or deletion oxidativeand vigorousmetabolism [] Under these intense states nuclear andmitochondrial DNA is fragile and easily damaged whichleads to eventual DNA leakage in the forms of micronuclei chromatin fragments andor free telomeric DNA[ ] Chromosomal instability CIN is the primary source of cytoplasmic DNA in malignant cells andis generally associated with tumor progression distantmetastasis and therapeutic tolerance [] Excessive proliferation of cancer cells results in unstable genomes [] usuallychromosomal missegregation during mitosis Due to defects in segregation lagging chromosomes generate micronuclei in a cellcycledependent manner [] The vulnerable membraneof micronuclei easily exposes the inner DNA to the cytoplasm and activates the cGASSTING signaling axis [] Exogenous stimuli such as chemotherapy and irradiation can also cause DNA damage In addition to leakednuclear DNA oxidative stressinduced mitochondrialDNA leakage is another crucial initiator of STING pathway activation Several anticancer treatments that precisely attack mitochondrial membranes result in effluxand cell death Therefore the permeabilization of mitochondria membranes provides a reasonable explanationfor mitochondrial DNA escape [ ] Other sourcessuch as apoptotic cellderived DNA exosomal DNAExoDNA and transposable elements have also beencharacterized 0cZheng Molecular Cancer Page of Fig The cGASSTING DNA sensing signaling pathway Various DNA derived from virus dying tumor cells or nucleus and mitochondria binds toand activates the cytosolic DNA sensor cGAS cGAS catalyzes the synthesis of ²²cGAMP in the presence of ATP and GTP then ²²cGAMP bindsto the ER adaptor STING which also can be activated by CDNs derived from bacteria Upon activation STING translocates from ER to Golgicompartments where it activates TBK1 and IKK which phosphorylate IRF3 and IκBα respectively Then IRF3 and IκBα dimerize and enter nucleusinitiating the transcription of Type I IFN TNF and IL6 The primary roles of these cytokines are reflected in host defense inflammation andantitumor immunitydemonstrated to evoke cGAS“STING activation intumor cells [ ]Type I IFNs mediators of STING and adaptive antitumoreffectscGASSTING signaling exerts antitumor functions incancer cells both in an autonomous and nonautonomousmanner On the one hand DNA damage can provokeacute STING signal transduction and induce cellularsenescence an irreversible cell cycle arrest state whichthwarts the aberrant proliferation of tumor cells throughacquisition ofsecretoryphenotype SASP which is associated with the releaseof abundantinflammatory mediators proteases andgrowth factors [ ] In contrast to undergoingsenescence tumor cells also directly propel apoptosisprocesses by upregulating proapoptosis protein BCL2associated X BAX and downregulating the BCL2 apoptosis[] On the other hand STINGsenescenceassociatedtheregulatoractivation in tumor cells not only facilitates the transcription of downstream type I IFNs to induce dendriticcell maturation but also recruits supportive immunecells for direct nonspontaneous tumor elimination []STING activation in nonmalignant cells causes tumorsuppressive effects as well STING signaling protectsagainst colitisassociated carcinomas CACs induced byazoxymethane AOM and dextran sulfate sodiumDSS which induce DNA damage in intestinal epithelialcells and further trigger STING activation Downstreamcytokines of STING signaling such as IL1 and IL18prevent neoplastic transformation by facilitating woundrepair More importantly STING signaling can also provoke cytotoxic T cell responses to control tumorigenesis[] Necrotic cancer cells are commonly engulfed byantigenpresenting cells especially the basic leucine zippertranscription factor ATFlike BATF3drivenlineage of dendritic cells DCs [] BATF3 DCs take intumorassociated antigens and migrate towardsthe 0cZheng Molecular Cancer Page of tumordraining lymph node via the lymphatic systemwhere they crossprime tumorspecific CD8 T cellsThen CD8 T cells undergo activation and clonal expansion in the lymph nodes and are trafficked throughblood vessels to kill tumor cells In turn damaged cancercells release more antigens that are further captured byDCs the whole process forms a positive feedback loopcalled the cancerimmunity cycle [] Tumor eradication can be achieved by multiple processes in thecancerimmunity cycle including tumor antigen captureand presentation and T cell priming and activation withtumor antigenspecific T cell priming and activationrelying on DCs and type I IFN release [] The involvement of type I IFNs in innate immune sensing and adaptive immunity provides a reasonable hypothesis forexploring candidate PRR pathways as potential immunomodulators Mice lacking TLR9 myeloid differentiationprimary response gene MyD88 cytosolic RNA sensor MAVS or the purinergic receptor P2X7R maintainintact antitumor immunity responses whereas mice deficient in STING or IRF3 present with impaired CD8 Tcell priming and activation [ ] In fact dying tumorcells can release multiple damageassociated molecularpatterns DAMPs to trigger innate immune responsesin DCs among these released stimuli tumor cellderivedDNA is a pivotal inducer In general the phagocytosis ofapoptotic cells causesimmune silence because ofDNasebased degradation [] Nevertheless tumor cellreleased DNA can be preserved in the DC endolysosomal compartment through an unknown mechanism [] cGAS recognizes DNA invading the cytoplasm andinduces the activation of STING cascades excretion oftype I IFNs and expression of ISGs Additionally undersome physiological conditions such as hypoxia andacidic environments nuclear or mitochondrial DNAmight be packaged in exosomes Exosomal DNAExoDNA animates STING signaling once it is absorbedby tumorinfiltrating DCs [] Finallytumor cellderived cGAMP can also be transferred to host DCs bythe folate transporter SLC19A1 and then directly bindsto STING activating it in DCs [] A recent study moredirectly demonstrated that cellautonomous STING promoted the maintenance of stem celllike CD8 T cellsand augmented antitumor T cell responses and mechanistically cGASSTINGmediated type I interferon signaling reinforced the stem cell“like CD8 T celldifferentiation program mainly by restraining Akt activity []Immune cellderived type I IFNs have crucial functions in antitumor immunity control On the one handtype I IFNs boost cross presentation by various mechanisms first they stimulate the maturation of DCs secondthey slow the endosomelysosome acidificationprocess to prevent engulfed tumor antigen clearance andelevate the expression of MHC I molecules on the cellsurface [ ] finally they accelerate DC migrationtowardslymph nodes where they can crossprimetumorspecific CD8 T cells [] On the other handtype I IFNs drive the expression of multiple chemokinessuch as CXCL9 and CXCL10 both of which are necessary for cytotoxic T lymphocyte CTL transfer and infiltration [] Similarly type I IFNs restrain the defaultimmune suppressive action of regulatory T Treg cellsby downregulating phosphodiesterase PDE4 and upregulating cyclic AMP cAMP [] Consequently typeI IFNs serve as bridges linking the cGASSTING pathway with CD8 T cellmediated antitumor immunityThe antitumor mechanisms of the cGASSTING signaling axis are illustrated in Fig Indeed previous studies revealed that STING activation can stimulate antitumor immune responses inleukemia melanoma glioma and hepatocellular carcinoma [“] Additionally STING expression is downregulated in a wide variety of tumor tissues and celllines according to a pancancer analysis with a smallproportion of tumors approximately bearing silent STING expression [] Lower STING expressionwas found in hepatic carcinoma and gastric cancer compared with its level in corresponding normal tissues andthis lower expression level was correlated with highertumor stage and poorer prognosis [ ] Consistentlycompared with that in the MCFG10A mammary epithelial cell line lower STING expression was detected inmalignant breast cancer cellincluding MCF7HBL100 and T47D cells as well as human melanomacell lines and colorectal adenocarcinoma lines [ ] Collectivelythat cGASSTING signaling might act as a tumor suppressor in certain types of cancersthese findings suggestlinesSTING pathway agonists as cancer therapeuticsThe immunostimulatory potential of the cGASSTINGpathway makes it an attractive pharmacological targetsince its activation in the tumor microenvironmentTME can induce efficient crosspriming oftumorspecific antigens and facilitate the infiltration of effectorT cells Recent drug research has focused on the development of STING agonists because of their potential inanticancer therapy [ ] To date various kinds ofSTING agonists have been discovered and they aremainly divided into the following categories cyclic dinucleotides and their derivates DMXAA and its analogsand small molecular agonists In addition some conventional antitumor therapeutics can also indirectly activateSTING such as chemotherapy radiotherapy RT andtargeted therapy [] In addition STING agonists areable to enhance the efficacy of other anticancer therapeutic agents when used in combination STING 0cZheng Molecular Cancer Page of Fig The antitumor immunity effect of the cGASSTING pathway DNA damage leads to the formation of dsDNA in tumor cells upon itsstimulation STING signaling is activated and promotes the release of Type I IFN which is crucial for DC maturation STING signaling activation inDCs is the core step of the whole cancerimmunity cycle which can be initiated through engulfment of dyingdamaged tumor cells exosometransfer and cGAMP gap junctions Then DCs migrate towards the tumordraining lymph node and crossprime tumor specific CD8 T cells withthe help of Type I IFNs Finally T cells undergo clonal expansion and traffic through the blood vessel to conduct tumor killingagonists and their synergistic use with other remedies isfurther explored in detail belowCyclic dinucleotides CDNsCDNs constitute a main type of STING agonist whichmainly originate from bacteria The known naturalCDNs consist of exogenous cyclic diGMP cdiGMPcdiAMP ²²cGAMP and endogenous ²²cGAMPAmong these cdiGMP cdiAMP and ²²cGAMPare synthesized by bacteria and identified as secondarymessengers that mediate STING signal transduction inprokaryotic cells while ²²cGAMP functions as theinitiator of STING in mammalian cells [] The antitumor potential of these natural dinucleotides was firstproven by the finding that cdiGMP could inhibit theproliferation of human colon cancer cells in vitro andbasal cell proliferation of human cecal adenocarcinomaH508 cells was inhibited with μM cdiGMP []Intraperitoneal ip injection of highdose cdiGMPdirectly activated caspase3 and triggered T1 tumoripcell apoptosis in vitro nmol of cdiGMP reduced thegrowth of T1 tumor cells in vitro by and nmreduced it by while lowdose cdiGMP nmol accelerated the adaptive T cell response by converting a subgroup of myeloidderived suppressor cellsMDSCs into immune stimulatory cells producing IL12injection of ²²cGAMP [] Consistentlymgkg expedited dramatic leukemic elimination in ElTCL1 transgenic mice bearing chronic lymphocyticleukemia CLL and promoted tumor shrinkage of multiple myeloma in vivo [] From the perspective of endogenous CDNs ²²cGAMP mgkg was alsoshown to restrain tumorigenesis and improve the survival rate of mice bearing CT26 colon adenocarcinomain a dosagedependent manner relying on DC activationand T cell crosspriming [] More recently OhkuriT further demonstrated that intratumoral it injection of ²²cGAMP μg25 μLdose on and days after the injection of tumor cells significantly mitigated tumor growth and prolonged the survival of breast 0cZheng Molecular Cancer Page of cancer T1luc squamous cell carcinoma mSCC1colon cancer CT26 and melanoma B16F10 mousemodels [] Notably the it injection of ²²cGAMPinhibited not only tumor growth but also lung metastases in mice bearing B16F10 cellderived tumors suggesting that cGAMPinduced CD8 Tcell priming can drivesystemic antitumor immunity to control local and distant tumor growth []termedvaccineSTINGVAXConsidering the superior properties of STING signaling in activating adaptive immunityit is rational toutilize STING agonists such as CDNs as cancer vaccineadjuvants to increase tumor immunogenicity [] Fu investigated the in vivo therapeutic efficacy of acancercomprisinggranulocytemacrophage colonystimulating factor GMCSF and bacteriaderived or synthetic CDNs Theyobserved that after it injection of STINGVAX with μg of CDNs per vaccine dose the volume of B16melanoma tumors was dramatically reduced in a dosedependent manner Compared to mice receiving GMCSF cancer vaccine alone STINGVAXtreated mice hadmore infiltrating CD8 IFNγ T cells in the tumormicroenvironment The in vivo antitumor effect of STINGVAX was also verified in models of colon carcinomaCT26 pancreatic carcinoma Panc02 and upper aerodigestive squamous cell carcinoma SCCFVII []Although natural CDNs are able to produce robust antitumor immunity their chemical features might hindertheir future application in the clinical setting First native CDNs are easily degraded by enzymes inside the cellor in the bloodstream Second their negatively chargedproperty hydrophilicity and phosphate moieties severelyimpede CDNs from penetrating cell membranes to activate cytosolic STING leading to low bioavailability andpoor retention of the CDNs in specific cells and tissuesThird unintentional toxicities and narrow therapeuticwindows are also unavoidable Thus new strategies toimprove therapeutic efficacy and reduce adverse effectsare urgently needed including drug delivery carrier engineering original structural modification and nonnucleotide agonist screening [] Regarding agonistdelivery Smith reported that biopolymer implantscodelivering cdiGMP μg and chimeric antigen receptor T CART cells resulted in significant tumor regression in mice bearing pancreatic tumors[]Moreoveriv administration of cdiGMPYSK05Lip equivalent to μg of cdiGMP aYSK05liposome delivery system encapsulating cdiGMP led to a tremendous decrease in metastatic lesionsin a B16F10 mouse melanoma model with nearly ofthe injected mice showing resistance against tumor relapsethe adaptive immune responsememory was successfully induced [] Chen alsofound thatliposomalindicating thatinjection ofintravenousintravenousivnanopdelivered cGAMP cGAMPNP could activate the STING axis more effectively than solublecGAMP and converted the immunosuppressive TME toa tumoricidal state in a transplanted B16F10 cell melanoma model and in a genetically engineered triplenegative breast cancer model [] Moreover a recentstudy creatively suggested that modified bacteria mightbe exploited as a selective carrier of STING agonistsIntroduction of a dinucleotide cyclasecoding gene intothe Escherichia coli Nissle strain was an attempt at realizing this effect however advancements to the systemare needed []tobysnakeApartdigestionresistancecompoundatoms The modifiedfrom improving delivery methods CDNswith superior properties are currently being synthesized and tested For instance to prevent enzymatichydrolysis of cGAMP the nonbridging oxygen atomsin cGAMP phosphodiester linkages were replaced by²²sulfurcGsAsMP showed resistance against degradation byENPP1 a major ²²cGAMP hydrolasetherebyleading to a longer halflife and sustained high affinity for human STING hSTING[] Syntheticdithio mixedlinkage CDNs with both Rp Rp R Rand Rp Sp R S dithio diastereomers possessed notonlyvenomphosphodiesterase but also enhanced affinityforSTING A novel superior modified product ML RRS2 CDA also termed ADUS100 had the potencyto activate all hSTING variants and mouse STINGmSTING ADUS100 had higher efficiency in activating STING signaling than endogenous or exogenous CDNs mainly because of its enhanced stabilityand lipophilicity Its powerful tumor elimination effect was extensively demonstrated in multiple murinemodelsincluding B16 melanoma T1 breast cancer and CT26 colon cancer with all treated animalsshowing significant and durable tumorregressionafter itinjection of ADUS100 three mg doseswhen tumor volumes reached mm3 [] TheremarkableforhSTING laid the foundation for its clinical use Related clinicaltrials of ADUS100 are outlined inTable In addition to ADUS100 some other novelSTING agonists have been well designed IACS8779and IACS8803 are two highly potent ²²thiophosphate CDN analogs that induced striking systemicantitumorin a B16 melanoma murineinjection μg on and daysmodel after itposttumor implantation compared with ADUS100or cGAMP [] The characteristics and preclinicalapplications of all these mentioned CNDs are summarized in Table Because of the structural modification and optimization of delivery strategiestheapplication range and efficacy of CDNs have beenand high affinityresponsescurativeeffect 0cZheng Molecular Cancer Page of Table Characteristics and preclinical applications of different STING agonistsClassificationCharacteristicsApplicationmodelsNatural CDNagonistscdiGMPPoor membrane permeabilitysuitable for various codeliverytechnologiesColon cancer H508cells T1 metastaticbreast cancerTreatmentinformation μM nmol ip nmol ip nmol ip²²cGAMP²²cGAMPHigher binding affinity formSTING than for hSTINGHigher affinity for hSTING thanits lineage isomers binds tovarious STING nucleotidepolymorphisms observed inhumans easily degraded byphosphodiesteraseimpermeable to the cellmembraneChronic lymphocyticleukemia mgkg ipmultiple myeloma mgkg ipCT26 colonadenocarcinoma mgkgbreast cancer T1lucsquamous cellcarcinomasmSCC1 μg25 μLdose it μg25 μLdose itcolon cancer CT26 μg25 μLdose itmelanoma B16F10 μg25 μLdose itTherapeutic effects References[ ][][ ]Inhibitsproliferation tumorregression tumorregressionAccelerates TcellresponseLeukemiaeliminationSuppressesgrowthRestrainstumorigenesisImproves survivalrateDelays tumrowthDelays tumrowthDelays tumrowthDelays tumrowthSTINGVAXSyntheticCDNagonistsPotent in vivo antitumor efficacyin multiple therapeutic modelsof established cancercGAMPNPsBiopolymer scaffolds cdiGMP and CAR T cellscdiGMPYSK05Lip²²cGsAsMPADUS100IACS8779IACS8803NonCDNagonistsFAALiposomal nanops NPsdeliver cGAMP intracellularlymore effectively than realizedwith soluble cGAMPEradicates tumors moreeffectively than systemicdeliveryYSK05 is a lipid that can efficientlydeliver cdiGMP to the cytosolpossesses high fusogenic activitywhich enhances endosomalescapeMore resistant to degradation byENPP1 tenfold more potent atinducing IFN secretion potentialuse as a cancer vaccine adjuvantImproves stability and lipophilicityhigher affinity for hSTING thannatural CDN agonists capable toactivate all hSTING variants andmSTINGStimulates a superior systemicantitumor response thanADUS100 and cGAMPCauses hemorrhagic necrosisfailed in a phase I clinical trialdue to species specificity μg CDNs itReduces tumorvolume[]B16 melanomacolon carcinomaCT26pancreaticcarcinoma Panc02B16F10 melanomaivTNBCCreates atumoricidal state[]Pancreatic cancer μg cdiGMPTumor regression[]B16F10 mousemelanoma μg cdiGMP ivDecreasesmetastasisTHP1 monocytesB16 melanomathree mg doses it T1 breast cancerthree mg doses itMC26 colon cancerthree mg doses itDurable tumorregressionDurable tumorregressionDurable tumorregression[][][]B16 melanoma μg on day and posttumor implantationAntitumorresponse[]Murine colontumorsExtensive tumorrejection[ ]DMXAAFirst discovered as a vascularRat mammary mgkg ipHigh anticancer[ 0cZheng Molecular Cancer Page of Table Characteristics and preclinical applications of different STING agonists ContinuedClassificationCharacteristicsApplicationmodelsTreatmentinformationInduces proinflammatory cytokinesin a STINGdependent mannerHuman fibroblastsAntiviral activity[]Selectively induces STINGdependentsynthesis and secretion of bioactiveIFNs no evidence of binding directlyto STINGActivates STING

Thyroid_Cancer

Construction of a novel prognosticpredicting modelcorrelated to ovarian cancerWeichun Tang12 Jie Li3 Xinxia Chang12 Lizhou Jia1 Qi Tang12 Ying Wang4 Yanli Zheng4 Lizhou Sun5 andZhenqing Feng121National Health Commission Key Laboratory of Antibody Technique Nanjing Medical University Nanjing People™s Republic of China 2Department of Pathology Nanjing MedicalUniversity Nanjing People™s Republic of China 3Department of Nursing The Second Affiliated Hospital of Nantong University Nantong People™s Republic of China 4Department ofGynaecology and Obstetrics The Second Affiliated Hospital of Nantong University Nantong People™s Republic of China 5Department of Obstetrics and Gynecology First AffiliatedHospital of Nanjing Medical University Nanjing People™s Republic of ChinaCorrespondence Zhenqing Feng fengzhenqingnjmueducn or Lizhou Sun lizhou sun163comBackground Ovarian cancer OC is one of the most lethal gynecological cancers worldwide The pathogenesis of the disease and outcomes prediction of OC patients remainlargely unclear The present study aimed to explore the key genes and biological pathwaysin ovarian carcinoma development as well as construct a prognostic model to predict patients™ overall survival OSResults We identified upregulated and downregulated differentially expressedgenes DEGs associated with OC Gene Ontology GO term enrichment showed DEGsmainly correlated with spindle microtubes For Kyoto Encyclopedia of Genes and GenomesKEGG pathways cell cycle was mostly enriched for the DEGs The protein“protein interaction PPI network yielded nodes and edges Top three modules and ten hub geneswere further filtered and analyzed Three candidiate drugs targeting for therapy were alsoselected Thirteen OSrelated genes were selected and an eightmRNA model was presentto stratify patients into high and lowrisk groups with significantly different survivalConclusions The identified DEGs and biological pathways may provide new perspective onthe pathogenesis and treatments of OC The identified eightmRNA signature has significantclinical implication for outcome prediction and tailored therapy guidance for OC patientsBackgroundOvarian cancer OC is the most lethal malignant disease in the female reproductive system with over new cases and deaths each year worldwide [] Epithelial OC accounts for “ ofovarian malignancies listed as the most common histological type Since the ovaries locate in the deeppelvis with mere symptoms emerging at the beginning of ovarian morbid change the early detectionfor the malignancy is truly difficult Hence when OC is detected the patient is usually at an advancedstage with invasion and metastasis accompanied [] For patients in the early stage the 5year survivalrate can reach “ whereas for advancedstage patients the number is mere ˆ¼ [] Thereforeit is imperative to explore the molecular mechanisms of malignant biological behavior of OC cells andto develop more reliable molecular markers for predicting recurrence and evaluating prognosis furtherguiding clinicians for therapyAt present various highthroughput microarrays and nextgeneration sequence genomic datasetswhich were deposited in the Gene Expression Omnibus GEO [] and The Cancer Genome Atlas TCGAdatabases have been widely analyzed for identifying differentially expressed genes DEGs which couldserve as candidate diagnostic or prognostic markers further effectively improving our understanding ofthe disease from genetic perspective Whereas since the existence of tissue or sample heterogeneity inThese authors contributedequally to this workReceived April Revised July Accepted July Accepted Manuscript online July Version of Record published August The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261each independent experiment as well as the discrepancy of different data processing methods and technology platforms the DEGs identified from a singlecohort study may generate false positives Herein the Robust Rank Aggregation RRA method which analyzes the significant probability of all elements by a probabilistic model is developedto identify statistically significant genes from multiple datasets and provide more accurate and valuable informationfor clinical use far beyond one gene list [] To date a bunch of novel prognostic markers have been discovered topotentially improve the efficacy of diagnosis and prognosis of OC [] However these identified markers were onlyeffective for partial stages or grades and were difficult to apply widely Hence a prognostic model which is basedon signature gene expression level with high discriminating power to effectively assist prognosis prediction for eachpatient is required in clinical practiceIn the present study we downloaded six primary microarray datasets from the GEO database which containeda total of samples with OC samples and normal samples The geneset and relative clinical information on ovary tissues of OC patients and healthy females from TCGA and GTEx portal were also downloadedIntegrated DEGs between cancerous and normal ovarian samples were screened using the ˜limma™ R package andRRA method Gene Ontology GO and Kyoto Encyclopedia of Genes and Genomes KEGG pathways enrichmentof DEGs were performed for nextstep functional analysis The Search Tool for the Retrieval of Interacting GenesDatabase STRING and the Connectivity Map CMap online database were then used to analyze the association ofDEGs and explore the molecular mechanisms as well as drugs involved in tumorigenesis Through survival analysisprognostic mRNAs were also selected By performing Cox regression analysis we identified an eightmRNA signature model with the ability to predict the prognosis of OC patients and independent from clinical factors Our studyprovides reliable molecular markers and prognostic models for early detection and outcome prediction as well aseffective drug targets for treating OCMethodsData collectionThrough searching on the GEO Repository with ˜ovarian cancer™ we downloaded the gene expression profiles ofGSE54388 GSE40595 GSE38666 GSE27651 GSE18520 and GSE14407 and the corresponding annotation files fromthe GPL570 [HGU133 Plus ] Affymetrix Human GenomeU133 Plus Array platform GSE54388 contains ovarian tissue samples with normal ovarian surface epithelium and tumor epithelial components from highgradeserous OC patients GSE40595 includes OCassociated stroma and epithelium samples which consist of cancerassociated stroma samples and epithelial tissues from highgrade serous OC patients along with stromal component and ovarian epitheliums from the normal ovary GSE38666 comprises stroma and matchedovarian epitheliums from healthy females as well as cancer stroma and matched cancer epitheliums from OCpatients GSE27651 incorporates normal ovarian surfaces epithelial and serous borderline ovarian tumors lowgrade serous ovarian carcinomas and highgrade serous ovarian carcinomas GSE18520 covers advancedstage highgrade primary OC specimens and normal ovarian surface epithelium tissues GSE14407 involves healthy ovarian surface epithelial samples and paired serous OC epithelium Note that all samples from these GEOdatasets are classified into the cancerous or normal part to be clear the normal stromal and surface epithelium is defined as normal ovarian tissues whereas the borderline tumors as well as cancerous stromal and epithelial tissues areconsidered as malignancies Besides we also downloaded the FPKM format gene expression data and relative clinicalinformation of OC patients™ samples and normal ovarian tissues from TCGA and GTEx portal respectivelyScreening for DEGs and integration of microarray dataWe used the ˜limma™ R package [] to integrate the expression profiles from TCGA and GTEx portal standardize the data from the integrated TCGA and GTEx expression matrix as well as six GEO datasets and furtherscreen the DEGs between ovarian cancerous and normal samples The list of DEGs obtained from six GEO microarray datasets by limma analysis was further integrated by the ˜RRA™ method to get prioritized commonly upor downregulated gene list The final overlapped DEGs for subsequent biological function analysis were the combination of prioritized jointly dysregulated genes from six GEO microarrays and the results from TCGA and GTExdatabases The cutoff criteria were set as FDR and log2fold change FC GO term and KEGG pathway enrichment analysisGO classified the known genes into three main biological progress Molecular Function MF Cellular ComponentCC and Biological Process BP [] KEGG provides researchers highlayer functions of the biological system frommolecular level information [] The Enrichr online tool amppharmmssmeduEnrichr allows for GO term The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The expression heatmap of the top significantly dysregulated genes in six GEO datasetsHierarchical clustering that shows the expression profiles of mRNAs from A GSE14407 B GSE18520 C GSE27651 DGSE38666 E GSE40595 F GSE54388annotation and KEGG pathway for a cluster of genes [] We explored the biological functions of overlappedDEGs and hub modules from our protein“protein interaction PPI network using Enrichr website Pvalue was considered as significant enrichment Likewise the functional biological pathways of the top ten hub genes fromPPI network were also analyzed by the FunRich tool version [] and the top five enriched pathways of up anddownregulated genes were displayed as bar charts respectively We set the Pvalue as statistically significant The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261PPI network construction and analysisPPI networks display the relationships of various proteins according to their physical or biochemical propertiesSTRING is a database that encompasses the interaction information between known proteins and potentially interacted proteins [] In order to explore the correlations between the DEGs we used the STRING database to constructa PPI network and visualize our results by Cytoscape software [] Confidence score was set as significantMolecular Complex Detection MCODE was utilized to select hub modules of PPI networks in Cytoscape [] Weset the degree cutoff node score cutoff kscore and max Depth was set as the criterion Thenthe significant modules were performed by GO and KEGG analyses Top ten genes were defined according to thehigh degree of connectivity in STRING network [] The coexpression analysis of ten hub genes was performed bySTRING eitherValidation of the hub genesWe downloaded the raw geneset of OC patients from TCGA to explore the expression differences of hub genes inlow and highgrade tumor tissues of OC and draw the survival plot using Kaplan“Meier plotter webtool [] Thegene and protein expression level of graderelated hub genes were then confirmed by Oncomine and The HumanProtein Atlas HPA database [] Meanwhile we explored the genetic alteration information of the selected tenhub genes in OC patients by plugin cBioPortal cBio Cancer Genomics Portal tool which deposits the genomicsprofiles of various cancer types and provides analysis and visualization of the genomics datasets []Identification of candidate small molecule drugsThe CMap database was able to predict potential drugs which might reverse or induce the biological state encoded incertain gene expression signatures in OC [] The DEGs from our study were used to query the CMap databaseThe enrichment scores which represent the similarity were calculated ranging fromˆ’ to The positive connectivity score means an inducing influence on the input signature whereas drugs with negative connectivity score presentreversion impact on the characteristic in human cell lines and are considered as candidate therapeutic molecules After sorting all instances the connectivity score of various instances was filtered by Pvalue Next we investigatethe structures of these candidate molecular drugs from the Pubchem database pubchemncbinlmnihgovEstablishment and evaluation of the prognostic modelThe OC patients from the TCGA project were randomly classified into the training cohort n188 and the testingcohort n186 OSrelated genes were determined by performing univariate Cox regression analysis in the trainingcohort with the ˜Survival™ R package and further selected for the nextstep screening Least Absolute Shrinkage andSelection Operator LASSO is a parameter selection algorithm which shrinks all highdimensional regression coefficients and generates the penalty regularization parameter λ via the crossvalidation routine by ˜glmnet™ R packageTo select the optimal prognostic mRNAs we adopted LASSO regression among the selected candidate genes and further perform multivariate Cox proportional hazards regression to evaluate their independent prognostic values Theriskscore model for predicting outcomes of OC patients was the sum of each optimal prognostic mRNA expressionlevel multiplying relative regression coefficient weight calculated from the multivariate Cox regression modelRisk score patient cid2Coefficient mRNAi × Expression mRNAiiAll training cohort patients were classified into high and lowrisk groups according to the median risk score TheKaplan“Meier curves of two diverse groups were plotted using ˜survfit™ function and the receiver operating characteristic ROC curve was unfolded for OS prediction to estimate the sensitivity and specificity of the prognostic modelCox multivariate analysis was also performed to examine whether the risk score was independent of the clinical characters such as age tumor stage and grade Next we used the testing group to check the efficacy of the prognostic riskmodel Each individual in the testing cohort was also categorized as high or lowrisk case by comparing the patient™srisk score with the cutoff value calculated from the training cohort Kaplan“Meier curve analysis timedependentROC analysis and Cox multivariate analysis were performed eitherSearching tumorinfiltrating immune cells associated with patients™prognostic signaturesThe TIMER webtool allows for systematical evaluations of the relationship between the six immune cell types inthe tumor microenvironment which are B cell CD4 T cell CD8 T cell neutrophil macrophage as well as dendritic The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The volcano plot of all gene expression distribution in six GEO datasetsVolcano plot of differentially expressed mRNAs of A GSE14407 B GSE18520 C GSE27651 D GSE38666 E GSE40595 FGSE54388cell and clinical impact in various cancer types via a novel statistical method [] To further explore the prognosticsignature we used the TIMER online tool to search the most significant tumorinfiltrating immune cells according tothe TCGA OC gene data To be clear the relative gene expression levels of six types of immune cells for each patientin high and lowrisk groups from training and testing cohort were measuredResultsThe DEGs among six GEO microarray datasetsThe top significantly up and downregulated genes from each microarray dataset were displayed in the heatmapsFigure 1A“F and the distribution of all gene expression was presented in volcano plots Figure 2A“F ThroughRRA analysis of expression microarrays we identified DEGs which consisted of upregulated and downregulated genes and displayed the top dysregulated genes by ˜pheatmap™ R package in Figure Next weanalyzed the expression profiles of TCGA and GTEx getting dysregulated genes Intriguingly when these DEGs were combined with the DEGs from GEO datasets we found that genes were commonly dysregulatedin these two databases with upregulated Figure 4A and downregulated genes Figure 4BGO term and KEGG pathway enrichment analysis of DEGsTo study the potential biological function of the DEGs we performed biological pathway analysis and identifiedsignificantly enriched pathways via Enrichr web tools In GO term Figure 5A for the BP group the DEGs weremostly enriched in ˜regulation of attachment of spindle microtubes to kinetochore™ ˜cellular response to laminar fluidshear stress™ and ˜microtubule cytoskeleton anization involved in mitosis™ In MF group the dysregulated geneswere highly correlated to ˜microtubulebinding™ ˜microtubule motor activity™ and ˜tubulinbinding™ As for CC group The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure Heatmap showing the top upregulated genes and top downregulated genes according to PvalueEach row represents one gene and each column indicates one dataset Red indicates upregulation and blue represents downregulation The numbers in the heatmap indicate log FC in each dataset calculated by the ˜limma™ R package Abbreviation log FClogarithmic fold changethe DEGs were closely related to ˜condensed nuclear chromosome kinetochore™ and ˜mitotic spindle™ KEGG pathwayanalysis showed DEGs highly enriched in ˜cell cycle™ and ˜Alanine aspartate and glutamate metabolism™ Figure5B The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The intersection of up and downregulated DEGs of GEO and TCGA datasetsA upregulated intersected DEGs in both datasets B downregulated intersected DEGs in both GEO and TCGA dataset Theintersected DEGs were defined as the significant DEGsPPI network construction and modules analysisUsing the STRING database and Cytoscape software a total of DEGs were mapped into the PPI network whichincluded nodes and edges Figure 6A The PPI enrichment Pvalue was 10e16 The top three key modules Figure 6C“E within PPI network were then selected Module MCODE score Module MCODEscore Module MCODE score and the biological function of Module which consisted of nodesand edges was further analyzed GO analysis indicated that Module1 was mainly associated with ˜regulation ofattachment of spindle microtubules to kinetochore™ ˜condensed nuclear chromosome kinetochore™ and ˜microtubulemotor activity™ KEGG analysis showed that ˜cell cycle™ and ˜oocyte meiosis™ were the most highly enriched pathwaysSupplementary Figure S1The screening of Hub genes and their characteristicsThe top ten hub genes with the highest degree of connectivity were CDC45 CDK1 TOP2A CDC20 CCNB1CEP55 UBE2C HMMR FOXM1 and TPX2 Figure 6B The coexpression analysis results of the hub genes demonstrated that these genes actively interacted with each other Supplementary Figure S2 Besides we established theinteraction network of ten hub genes with their related genes and explored the biological role Supplementary Figure S2AC“F of the network by FunRich The gene alteration type and frequency as well as the most frequentlyaltered neighbor genes were also exhibited Figure Gene alteration frequency of ten hub genes among TCGAOC samples was beyond with most genes showed amplified and multiple altered Figure 7AB The top threemost frequently altered genes were FOXM1 CDC20 and CCNB1 with FOXM1 and CDC20 largely amplified whileCCNB1 deep deleted Through analysis of OC patients™ geneset from TCGA we found that CCNB1 UBE2C CDK1CEP55 as well as FOXM1 expressed significantly higher in highgrade tumors and predicted worse outcomes sincepatients overexpressed above genes owned lower overall survival OS and diseasefree survival DFS rates Figure The Oncomine database showed results from various studies were consistent to our finding Supplementary Figure S3 The HPA website also demonstrate that proteins translated by such five hub genes were overexpressed in OCtissues Supplementary Figure S4 HMMR and TPX2 were also negatively correlated to patients™ prognosis while noexpression difference was observed in diverse tumor grades and CDC20 was positively associated with tumor gradebut not correlated to patients™ outcomesRelated small molecule drugs screeningIn total DEGs were analyzed in CMap to screen small molecule drugs and the candidate molecules with top tenconnectivity score are listed in Table Five of these molecules showed a negative correlation and suggested potentialin clinical applications Among them Trichostatin A pyrvinium and 8azaguanine showed a significantly negativecorrelation and the stuctures of such candidate molecule drugs was found in the PubChem database and shown inSupplementary Figure S5 The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure GO and KEGG functional annotation for the significant DEGsA The top ten enriched BP of the DEGs B The top ten enriched CC of the DEGs C The top ten enriched MF of the DEGs DThe top ten enriched KEGG pathways of the DEGs The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The PPI network and top hub genes as well as top three modules were constructedA The PPI network of the DEGs B The hub genes of the DEGs C“E Top three hub modules were identified by Cytoscapeplugin MCODE C module1 D module2 E module3Table The top ten OCrelated small molecules with highly significant correlations in results of CMap analysisRankCMap nametrichostatin A8azaguaninepyrviniumisoflupredonequinpirolevorinostatgenisteinantimycin AheptaminolmidodrineMeanˆ’ˆ’ˆ’ˆ’ˆ’NEnrichmentP valueˆ’ˆ’ˆ’ˆ’ˆ’ The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The gene mutation overview of ten hub genes in TCGA OC patientsA Ten hub genes are altered in of queried patients B The summary of mutation type of ten hub genes in OC patientsC The network of hub genes and the most frequently altered neighbor genesTable Univariate cox regression identified DEGs correlated to patients™ OSGene IDCCND1SYNE4CCDC80TMC4MCCFOXQ1KRTCAP3CXCR4IL4I1DEFB1CSGALNACT1KLHL14MCUR1HRAbbreviation HR hazard ratioHR95LHR95HPvalueConstruction of prognostic model and evaluation of its predictive abilityUnivariate Cox regression analysis revealed that of DEGs were significantly correlated to patients™ OS in thetraining cohort Table The OSrelated genes were listed as follows CCND1 SYNE4 CCDC80 TMC4 MCC The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The clinical characteristics of CCNB1 CEP55 CDK1 FOXM1 and UBE2C in OC patientsA Five genes were overexpressed in high grade G1 and G2 compared with low grade G3 and G4 in OC BC The OS time Band DFS time C of five genes in OC patients The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure Identification of prognosisrelated mRNAs using LASSO regression modelA LASSO coefficient profiles of the mRNAs associated with the OS of OC B Plots of the crossvalidation error rates Each dotrepresents a λ value along with error bars to give a confidence interval for the crossvalidated error rateTable Multivariate Cox regression selected eight DEGs correlated to patients™ OSGene IDTMC4KLHL14CXCR4CCDC80KRTCAP3DEFB1SYNE4FOXQ1HRHR95LHR95HPvalue845E08Abbreviation HR hazard ratioFOXQ1 KRTCAP3 CXCR4 IL4I1 DEFB1 CSGALNACT1 KLHL14 and MCUR1 Through LASSO Cox regression we narrowed the number of prognosisassociated genes to according to the minimum criteria Figure Next based on the multivariate Cox model of candidate mRNAs retained their prognostic significance and werefinally selected as independent remarkable prognostic factors which were TMC4 KLHL14 CXCR4 CCDC80 KRTCAP3 DEFB1 SYNE4 and FOXQ1 Table To predict patients™ outcomes we developed an individual™s risk scoremodel as follows risk score × expression value of TMC4 × expression value of KLHL14 ˆ’ × expression value of CXCR4 × expression value of CCDC80 ˆ’ × expression value of KRTCAP3 ˆ’ × expression value of DEFB1 × expression value of SYNE4 × expression value of FOXQ1 On the basis of the median risk score patients were divided into high orlowrisk groups Kaplan“Meier curve analysis showed that the OS time of the lowrisk group was significantly longerthan the highrisk group P1147e07 Figure 10E ROC curve analysis revealed the area under the ROC curveAUC of the prognostic model was Figure 10D Meanwhile the risk scores Figure 10A of OC patients inthe training group were ranked for displaying their distribution and the survival status Figure 10B was marked onthe dot plot The expression pattern of eight prognostic mRNAs between high and lowrisk groups was also shown inthe heatmap Figure 10C Univariate and multivariate Cox regression analyses concerning the risk score and clinicalfactors showed that the prognostic model was able to serve as an independent prognostic indicator Figure 11ABROC curve analysis also showed that the AUC value of the model was much significantly higher than the tumor stage AUC grade AUC and patients™ age AUC Figure 11C Interestingly when The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure Prognostic analysis of the TCGA training modelA The risk score B survival status C expression heatmap D timedependent ROC curves and E Kaplan“Meier survival ofthe prognostic model for the TCGA OC training cohort The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The clinical independency of the risk model in training cohortUnivariate A and multivariate B regression analyses as well as timedependent ROC curve analysis CD of the prognostic valuebetween the training model and OC patients™ OS status when compared with or combined with clinical factorscombined the risk score with clinical factors the ROC curve of combination model was much higher than each aloneFigure 11DAs for the testing cohort we divided the group into highrisk and lowrisk individuals based on the trainingcohort cutoff risk score The outcomes of low and highrisk groups™ patients of the testing cohort were also measuredand the OS time of the highrisk group was significantly shorter than the lowerrisk group P1721e02 Figure12E The AUC of the prognostic model was Figure 12D The risk scores distribution Figure 12A and survivalstatus Figure 12B of OC patients as well as the eightprognostic gene expression heatmap Figure 12C in the testinggroup were also present Meanwhile the independency of the prognostic model was confirmed in testing cohort sinceunivariate and multivariate Cox regression analyses showed the model correctly predicted high or lowrisk factroups patients™ outcomes without relying on any clinical factors Figure 13AB ROC curve analysis showed that theprognostic model exhibited better sensitivity and specificity when compared with tumor stage grade and patients™age for the AUC value of the model was much higher than later Figure 13C In accordance with results from trainingcohort the combination of risk score and clinical factors showed better OS prediction capability Figure 13DThe prognostic signature correlating to immune cells infiltrationThrough TIMER webtool we analyzed the relative gene expression levels of six types of immune cells for each patientand found that genes concerning macrophage fraction were expressing significantly higher in the highrisk groupP005 compared with the lowrisk group in training cohort Figure Interestingly same result was also observed in the testing cohort Figure The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure Prognostic analysis of the TCGA testing modelA The risk score B survival status C expression heatmap D timedependent ROC curves and E Kaplan“Meier survival ofthe prognostic model for the TCGA OC testing cohort The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The clinical independency of the prognostic risk signature in testing cohortUnivariate A and multivariate B regression analyses as well as timedependent ROC curve analysis CD of the prognostic valuebetween the testing model and OC patients™ OS status when compared with or combined to clinical factorsDiscussionIn the present study we used the RRA methods to jointly analyze six GEO OC microarrays which contained OC and normal samples identifying DEGs and overlapped them with dysregulated genes of OC cohort fromTCGA and GTEx portal finally getting upregulated and downregulated genes Functional analysis showedthat DEGs were significantly enriched in the cell division cycle to be clear in the process of the mitotic spindleSpindle microtubules have been proved to play crucial role in physiological and pathological processes As for celldivision only when all chromosomes linked to spindle microtubules through kinetochores and the spindle assemblycheckpoint is satisfied this process could step to anaphase [] Suraokar et al found that the mitotic spindle assemblycheckpoint and microtubule network were significantly altered in malignant pleural mesothelioma MPM whileusing epothiloneB a nontaxane small molecule inhibitor targeting the microtubules could greatly decrease theviability of MPM cell lines [] Rogalska et al compared the antiproliferative capacity of epothilone B with paclitaxelon OC cell line SKOV3 found that this effect of Epo B was greater than latter [] The researches above wereconsistent with our study that the mitotic spindle process was dysregulated in OC progression playing importantroles in OC cell proliferation and tumor developmentPPI network construction of DEGs included nodes and edges among which we identified three keymodules Interestingly the top1 module was also highly associated with spindle microtubules and chromosome kinetochore confirming the role of cell cycle in OC pathogenesis The top ten hub genes from the PPI network were alsorecognized which were CDC45 CDK1 TOP2A CDC20 CCNB1 CEP55 UBE2C HMMR FOXM1 and TPX2 The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The expression level of immune cells related genes in high and lowrisk groups of the training cohortA Bcell fraction B dendritic fraction C CD4 Tcell fraction D CD8 Tcell fract

Thyroid_Cancer

"Meningiomas are the most common primary central nervous system tumors Potential risk factorsinclude obesity height history of allergyatopy and autoimmune diseases but findings are conflicting This studysought to assess the role of the different risk factors in the development of meningioma in adolescentsyoungadultsMethods The cohort included Jewish men and women who had undergone compulsory physicalexamination between and at age to years prior to and independent of actual military enlistmentTo determine the incidence of meningioma the military database was matched with the Israel National CancerRegistry Cox proportional hazard models were used to estimate the hazard ratios for meningioma according to sexbody mass index BMI height and history of allergic or autoimmune diseaseResults A total of subjects females were diagnosed with meningioma during a followup of personyears Median age at diagnosis was ± years range “ On univariate analysis female sex p and height p were associated with risk of meningioma When the data were stratified by sex heightremained a significant factor only in men Spline analysis of the male subjects showed that a height of m wasassociated with a minimum disease risk and a height of meters with a significant riskConclusions This large population study showed that sex and adolescent height in males m wereassociated with an increased risk of meningioma in adulthoodKeywords Allergy Autoimmune disease Height Meningioma SexBackgroundMeningiomas are the most common primary centralnervous system tumors They originate from the meninges which are the membranous layers surrounding thebrain Most meningiomas “ are grade I benign“ are grade II atypical and “ are grade IIIanaplastic [] Benign meningiomas have a female predominance or which is not found in the more Correspondence matanbe4gmailcom1NeuroOncology Unit Davidoff Cancer Center Rabin Medical Center “Beilinson Hospital Petach Tikva IsraelFull list of author information is available at the end of the aggressive types [] In the USA meningiomas werefound to be more common in blacks than in whites witha ratio of [] The risk of acquiring a meningiomaincreases with age The median age at diagnosis is years []The only established external nongenetic risk factorfor brain tumors is exposure to ionizing radiation []An Israeli study revealed abnormally high rates of meningioma in patients treated with lowdose radiation tothe scalp for tinea capitis during the 1950s [] Otherpotential risk factors include obesity height history ofallergyatopy and history of autoimmune diseases but The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cBenZion Berliner BMC Cancer Page of the results are conflicting [“] Establishing risk factors for meningioma can help identify individuals whomight benefit from risk reduction strategies and possiblyearly screening methodsThe aim of the present study was to assess potentialrisk factors for the development of meningioma in adolescence and early adulthoodMethodsStudy populationIsraeli adolescents undergo a compulsory medicalexamination at age to assess their fitness for military service regardless of whether they are drafted ornot Arab and Orthodox Jewish females and malesand Druze females are exempted Together with thephysical examination sociodemographic and psychobehavioral data are collected and the medical historyis thoroughly reviewed using documents provided byeach subject™s primary care physician At the end ofthe process recruits are assigned a Functional Classification Code FCC that describes their medical status and occupational medical ranking The medicaldata and FCC are stored in the army™s main databasewhich was computerized in []The population for the present study was derived from subjects born in “ who underwentpreenlistment medical examination between whenthe database was computerized and at age “years Subjects with missing data on height and weightwere excluded n We also excluded subjects of North African and Asian origin born before many of whom had been exposed to radiation forthe treatment of tinea capitis after immigrating to Israelduring the 1950s [] These communities were laterfound to have a particularly high rate of meningioma []The final cohort consisted of subjects Fig Records were reviewed from the date of the initialmedical examination for military fitness to the date of afirst diagnosis of any cancer death or the predeterminedend of followup December Study variablesAtthe preenlistment medical examination demographic variables for each recruit were recorded in thearmy database as follows date of birth age at examination country of origin education socioeconomic status height weight and body mass index BMI Originwas defined by father™s country of birth or if the examinee™s father was born in Israel by paternal grandfather™scountry of birth and categorized as Europe includingNorth and South America Australia and SouthernAfrica Asia predominantly the Middle East Africaoverwhelmingly North Africa and Israel third or latergeneration Education was categorized as ‰ Fig Selection of the study populationand ‰¥ years of schooling Socioeconomic status wasdetermined by place of residence at the time of examination coded on a scale of “ and categorized intolow score “ middle score “ and high score “ [] Height and weight were measured by trainedmedics using a stadiometer and a beam balance with examinees barefoot and in underwear BMI was calculatedas weight in kilograms divided by height squared inmeters and categorized according to the WHO asunderweight kgm2 healthy weight “kgm2 overweight “ kgm2 and obese ‰¥kgm2 Height was categorized according to the Centersfor Disease Control and Prevention below 25th percentile 25th to 50th percentile 50th to 75th percentile and75th percentile and aboveCognitive function including language skills and intellectual performance [] was assessed by a general 0cBenZion Berliner BMC Cancer Page of intelligence test administered by trained personnel Thetest is scored on a 90point scale that is adjusted fromtime to time scores are categorized as low “medium “ and high “ []inflammatory bowel disease pemphigusMedical history was assessed according to the FCCslupus vascuAutoimmune diseases diabetes mellituslitisthyroiddisease celiac rheumatoid arthritis Addison disease andidiopathic thrombocyt ia purpura and allergic diseases asthma urticaria eczema allergic rhinitis atopicdermatitis allergic conjunctivitis and anaphylaxis weregrouped together for the present analysisAscertainment of meningioma incidenceTo determine the incidence of meningioma we matchedthe subjects who underwent the preenlistment medicalexamination during the study years to the Israel National Cancer Registry INCR a national populationbased registry established in In Israeli lawmandated the reportage of all diagnoses of malignant insitu and invasive borderline and certain benign brainand central nervous system tumors The estimated rateof reportage for solid tumors is which meets thestandards of the International Association of CancerRegistrieswwwhealthgovilPublicationsFilesICDC_365_EN_summarypd The INCR data includethe date of diagnosis site affected the InternationalClassification of Diseases code and the histologic description of the tumor according to the third edition ofthe International Classification of Diseases for OncologyICDO3 codes and At the time ofmatching the INCR had been updated until the end ofStatistical analysisCategorical variables are presented as number and percentage and continuous variables as mean and standarddeviation SD median 25th and 75th percentiles minimum and maximum were also calculated The association between risk factors and time to meningiomadiagnosis was assessed using Cox proportional hazardmodels hazard ratios HR confidence intervals CI and pvalues were calculated Log minus logfigures were inspected to confirm the proportionality ofthe hazard Crude rates were also determined Independent variables were initially entered individually into theCox model After sex and the interaction of sex andheight were found to be statistically significant separatemodels were established for men and women A Cox regression cubic spline function with three equally spacedknots positioned between the minimum and maximumvalues of height was fit to the data to estimate the heightvalue associated with minimum risk of meningioma inmen SASSTAT and SASGRAPH software version SAS Institute Inc Cary NC USA Other statistical analyses were performed with SPSS Statistics for Windowsversion IBM Armonk NY USA Twosided pvalues of ‰ were considered statistically significantResultsStudy populationThe baseline characteristics of the study population arepresented in Table The mean age at initial examination was ± years of the cohort was female The mean duration of followup was ± years median which represent in this study population a follow up of personyears The characteristics of the medical history of the subjects arepresented in the supplementary table Table 1SLinkage of the military database with the INCR yieldeda diagnosis of meningioma in of the subjects who underwent medical examination in to at age “ years grade I atypical anaplastic not specified and one patient with meningiomatosisTable The mean age at diagnosis ofmeningioma was ± years range “ andat the end of followup ± years median Univariate analysisOverall as expected meningiomas were more common infemales cases crude rate per personyears than in males cases crude rate per personyears p HR CI “ However there was no sex difference in the incidence for themore aggressive meningiomas atypical and anaplasticcrude rate per personyears for males andfemales On univariate analysis only sex and height weresignificantly associated with the risk of meningioma in thewhole study population p for both variables Afterstratification by sex height remained significant only inmales Table The risk of meningioma was minimalwhen height was up to m and statistically significantwhen height was greater than m Fig BMI was notassociated with an elevated risk of meningioma even whenanalyzed separately by sex Table Past medical history of asthma diabetes and otheratopic or autoimmune diseases was not associated withrisk of meningioma Even when autoimmune and allergic diseases were analyzed as a group there was no association with lower risk of meningioma Table andSupplemental Table When the subjects of African and Asian origin whowere excluded from the main analysis were included inthe cohort there was a significant interaction betweenperiod of birth “ vs “ and Asianand African origin representing the Middle East andNorth Africa as opposed to European and Israeli origin 0cBenZion Berliner BMC Cancer Page of Table Baseline characteristics of the study population total and by sexMaleCharacteristicsNumberBirth yearTotal““““LowMediumHigh years years years years“““ 25th percentile25th“50th percentile50th“75th percentile 75th percentileEuropeAsiaAfricaIsraelEuropeAsiaAfricaIsraelSocioeconomic statusEducationCognitive indexaBMI category Kgm2Height category CDC percentileCountry of birthOriginAge at time of medical examination yearsBMIHeight metersaRated on a 90point scaleFemaleNumberSDMeanSDTotalNumberMeanSDMeanThe conjoined effect of birth year and origin showedthat origin North Africa and Asia was significant onlyfor subjects born between and SupplementalTable DiscussionIn this nationwide populationbased study we analyzedthe association of the development of meningioma insubjects born between and with baseline variables obtained for the subjects at the average age of years As expected meningiomas were found to be associated with sex female and taller stature None of theother sociodemographic and medical variables assessedincluding BMI and a diagnosis of asthma or diabetes atage years was associated with an increased risk ofmeningioma 0cBenZion Berliner BMC Cancer Page of Table Meningioma type and rate total and by sexMeningioma typeMeningioma NOSMeningiomatosisGrade Atypical AnaplasticTotalPersonyearsNOS Not otherwise specifiedMalesNumberPer FemalesNumberPer TotalNumberPer It is well accepted that benign meningioma is morecommon in females than males but the sex predilectiondisappears with the more aggressive meningiomas []The female predominance might be explained by thefinding that meningiomas harbor receptors for estrogenand progesterone []We discovered an association between the risk ofmeningioma and height in men but not with BMI inmen or women The results of previous studies for thesetwo factors were conflicting A large Norwegian studyincluding million subjects found that height was associated with meningioma in both men and women butBMI was not [] whereas another study of postmenopausal females revealed an association of meningiomawith both BMI and height [] A metaanalysis of studies supported the correlation of BMI and meningioma It is worth noting that the Norwegian study exceeds the metaanalysis in size and power and that inthe Norwegian study a subgroup analyses for womenand men as well as different age groups was performedwithout finding convincing evidence of a strong association between overweight obesity and risk for meningioma [] In our study BMI was measured when thesubjects were years old much younger than theTable Univariate analysis association of potential risk factors with diagnosis of meningioma by sexVariablesMalesNCases Crude rate HR CILower UpperpCases Crude rate HR CILower UpperpFemalesNHeightHeight continuousBMIPercentile ““ Kgm2 Autoimmune diseasesa NoAllergic diseasesbAsthmaDiabetesYesNoYesNoYesNoYes aAutoimmune disease diabetes mellitus lupus vasculitis IBD pemphigus thyroid disease celiac rheumatoid arthritis Addison disease and idiopathic thrombocyt icpurpurabAllergic disease including asthma urticaria eczema allergic rhinitis atopic dermatitis allergic conjunctivitis and anaphylaxis 0cBenZion Berliner BMC Cancer Page of Fig Spline analysis in the men group showing the minimum risk for meningioma at a height of m and a statistically significant increase inthe risk for meningioma at heights taller than mstudies included in the metanalysis which might explainthe discordant results []Height has been associated with different types of cancer melanoma thyroid testis breast and lymphomaSuggested mechanism for the greater risk of meningioma in taller people is their higher levels of circulatinginsulinlike growth factors IGFs which may influencecell proliferation and tumor growth [] Moreoveroverexpression of IGFI and IGFII mRNA transcriptshas been demonstrated in meningioma [] Circulatinglevels of IGFs are highest during puberty They decreaserapidly in the third decade of life in the general population but seem to stay consistently higher in taller adults[] It is not clear why this association was evident onlyin males in our study maybe in women the influence ofthe hormonal status blurred the influence of the heightSeveral earlier studies reported an inverse associationbetween a history of allergic diseases including asthmaand meningioma [ ] However this finding wasnot supported by others [ ] We failed to demonstrate an association between meningiomas and allergicdiseases including asthma urticaria eczema allergicrhinitis atopic dermatitis conjunctivitis and anaphylaxisand allergy to beesSimilarly a recent study reported an inverse association between hyperglycemia and the risk of meningioma [] whereas another found a positive associationwith a history of diabetes mellitus [] In the presentstudy diabetes was not associated with the risk of meningioma This was true for other autoimmune diseasesas welllimitationThis analysis also has certain limitations The followup period in this study was limited to years such thatthe study population was still young when the studyended Subsequently the median age of those who developed meningioma in our study was younger than themedian age of patients with meningioma in the generalpopulation [] With a more extensive followup wemight find more latent tumor growths that could potentially increase or shift the incidence of intracranial neoplasms Anotherisunderreporting of meningiomas that are diagnosed onlyaccording to radiographic findings without histologicalfindings As it is well known that in some cases meningiomas diagnosed radiographically mayjust befollowed by repeat scanningcohortits prospectivepopulationbased design large sample size high degreeof completeness of the cancer registry data throughoutthe study period and the ability to carefully control forpotential confounders such as exposure to radiation Itshould be noted that in a study that was published recently and examined the same cohort the median heightremained almost stable during the study period theStrengths of ourofthestudyinclude 0cBenZion Berliner BMC Cancer Page of median height of males increased by cm and that offemales remained stable despite environmental socialand nutritional changes []ConclusionThis large populationbased study showed that sex female and tall stature in adolescent males was associatedwith an increased risk of meningioma in adulthoodSupplementary informationSupplementary information accompanies this paper at doi101186s12885020072924Additional file Supplementary Table Medical historycharacteristics of the study populationAdditional file Supplementary Table Univariate analysisassociation of potential risk factors with diagnosis of meningioma by sexAdditional file Supplementary Table Interaction between birthperiod and origin whole population adjusted for sexAbbreviationsCNS Central nervous system BMI Body mass index FCC FunctionalClassification INCR Israel National Cancer Registry ICDO InternationalClassification of Diseases for Oncology SD Standard deviation HR Hazardratio CI Confidence interval IGF Insulinlike growth factorAcknowledgmentsNot applicableAuthors™ contributionsMBZB and SYK analyzed the preliminary database extracted the relevantinformation to allow hypothesis testing and prepared the manuscript andtables Statistical analysis and figure preparation were performed by LHK andED HL LKB YL AH JM and GT participated in the preliminary preparationand conceptual design and revised the final manuscript ABA OG AK and TSreviewed the neurological proof of concept and revised the final manuscriptand supplementary materials All authors read and approved the finalmanuscriptFundingThe study was funded by the Israel Cancer Association by the Lillia andJacob Alther donation financial support without any role in the manuscriptpreparationAvailability of data and materialsThe datasets used during this study are available from the correspondingauthor on reasonable requestEthics approval and consent to participateAll procedures performed in studies involving human participants were inaccordance with the ethical standards of the institutional andor nationalresearch committee and with the Helsinki declaration and its lateramendments or comparable ethical standards The study was approved bythe IDF Israel Defense Forces Medical Corps Institutional Review Boardwhich waived the requirement for informed consent because the data usedwere obtained from medical records without patient participation referencenumber “Consent for publicationNot applicableAuthor details1NeuroOncology Unit Davidoff Cancer Center Rabin Medical Center “Beilinson Hospital Petach Tikva Israel 2Department of GastroenterologyHadassah University Hospital “ Ein Kerem Jerusalem Israel 3Sackler Facultyof Medicine Tel Aviv University Tel Aviv Israel 4Braun School of PublicHealth and Community Medicine Hadassah University Hospital “ Ein KeremJerusalem Israel 5Israel Center for Disease Control Israel Ministry of HealthRamat Gan Israel 6School of Public Health University of Haifa Haifa Israel7Department of Neurosurgery Rabin Medical Center “ Beilinson HospitalPetach Tikva Israel 8Medical Corps Israel Defense Forcesand Department ofMilitary Medicine Hebrew University of Jerusalem Faculty of MedicineJerusalem Israel 9Institute of Endocrinology and Talpiot Medical LeadershipProgramSheba Medical Center Tel Hashomer IsraelReceived April Accepted August ReferencesOstrom QT Gittleman H Fulop J Liu M Blanda R Kromer C Wolinsky YKruchko C BarnholtzSloan JS CBTRUS statistical report primary brain andcentral nervous system tumors diagnosed in the United States in NeuroOncology 201517Suppl 4iv1“iv62 doi101093neuoncnov189Claus EB Bondy ML Schildkraut JM Wiemels JL Wrensch M Black PMEpidemiology of intracranial meningioma Neurosurgery “doi10122701neu000018828191351b9Braganza MZ Kitahara CM Berrington de González A Inskip PD Johnson KJRajaraman P Ionizing radiation and the risk of brain and central nervoussystem tumors a systematic review NeuroOncology “doi101093neuoncnos208 Modan B Baidatz D Mart H Steinitz R Levin SG Radiationinduced headand neck tumours Lancet “ doi101016s0140 Wiedmann MKH Brunb C Di Ieva A Lindemann K Johannesen TBVatten L Helseth E Zwart JA Overweight obesity and height as risk factorsfor meningioma glioma pituitary adenoma and nerve sheath tumor alarge populationbased prospective cohort study Acta Oncol “ doi1010800284186X20171330554Johnson DR Olson JE Vierkant RA Hammack JE Wang AH Folsom ARVirnig BA Cerhan JR Risk factors for meningioma in postmenopausalwomen results from the Iowa Women's health study NeuroOncology“ doi101093neuoncnor081 Michaud DS Bové G Gallo V Schlehofer B Tjønneland A Olsen A OvervadK Dahm CC Teucher B Boeing H Steffen A Trichopoulou A Bamia CKyrozis A Sacerdote C Agnoli C Palli D Tumino R Mattiello A BuenodeMesquita HB Peeters PH May AM Barricarte A Chirlaque MD DorronsoroM José Sánchez M Rodríguez L Duell EJ Hallmans G Melin BS Manjer JBquist S Khaw KT Wareham N Allen NE Travis RC Romieu I Vineis PRiboli E Anthropometric measures physical activity and risk of glioma andmeningioma in a large prospective cohort study E Cancer Prev Res Phila“ doi10115819406207CAPR110014Niedermaier T Behrens G Schmid D Schlecht I Fischer B Leitzmann MFBody mass index physical activity and risk of adult meningioma andglioma a metaanalysis Neurology “ doi101212WNL0000000000002020Brenner AV Linet MS Fine HA Shapiro WR Selker RG Black PM Inskip PDHistory of allergies and autoimmune diseases and risk of brain tumors inadults Int J Cancer “ doi101002ijc10320 Wang M Chen C Qu J Xu T Lu Y Chen J Wu S Inverse associationbetween eczema and meningioma a metaanalysis Cancer Causes Control“ doi101007s1055201198086 Gal R The selection classification and placement process in a portrait ofthe Israeli soldier Westport CT Greenwood Press p “ YustKatz S Bar Oz A Derazne E Katz LH Levine H KeinanBoker L Amiel ACompeting interestsThe authors declare that they have no financial or nonfinancial competinginterestsKanner A Laviv Y Honig A Shelef I Siegal T Twig G Kark J Echoes fromthe past changing associations between brain tumors and ethnicity JNeurol Sci doi101016jjns2019116552 [Epubahead of print]Israel Central Bureau of Statistics Characterization and classification of localauthorities by the socioeconomic level of the population Jerusalem Israelcentral Bureau of Statistics 0cBenZion Berliner BMC Cancer Page of Twig G Gluzman I Tirosh A Gerstein HC Yaniv G Afek A Derazne E Tzur DKarasik A Gordon B Fruchter E Lubin G Rudich A CukiermanYaffe TCognitive function and the risk for diabetes among young men DiabetesCare Nov37112982“ doi102337dc140715 Guevara P EscobarArriaga E SaavedraPerez D MartinezRumayor A FloresEstrada D Rembao D Calderon A Sotelo J Arrieta O Angiogenesis andexpression of estrogen and progesterone receptors as predictive factors forrecurrence of meningioma J NeuroOncol “ doi101007s110600172662y Gunnell D Oliver SE Donovan JL Peters TJ Gillatt D Persad R Hamdy FCMeal DE Holly JMP Do heightrelated variations in insulinlike growthfactors underlie the associations of stature with chronic diseases J ClinEndocrinol Metab “ doi101210jc2003030507 Zumkeller W Westphal M The IGFIGFBP system in CNS malignancy MolPathol “ Crowe FL Key TJ Allen NE A crosssectional analysis of theassociations between adult height BMI and serum concentrations of IGFIand IGFBP1 and ˆ’ in the European prospective investigation intocancer and nutrition EPIC Ann Hum Biol “ doi BergBeckhoff G Schüz J Blettner M Münster E Schlaefer K Wahrendorf JSchlehofer B History of allergic disease and epilepsy and risk of glioma andmeningioma INTERPHONE study group Germany Eur J Epidemiol “ doi101007s1065400993556Schneider B Pülhorn H Röhrig B Rainov NG Predisposing conditions andrisk factors for development of symptomatic meningioma in adults CancerDetect Prev “ doi101016jcdp200507002Linos E Raine T Alonso A Michaud D Atopy and risk of brain tumors ametaanalysis J Natl Cancer Inst “ doi101093jncidjm170 Bernardo BM Orellana RC Weisband YL Hammar N Walldius G MalmstromH Ahlbom A Feychting M Schwartzbaum J Association betweenprediagnostic glucose triglycerides cholesterol and meningioma andreverse causality Br J Cancer “ doi101038bjcPublisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

Thyroid_Cancer

"deltalike protein DLL3 may be related with prognosis inpatients with small cell lung cancer SCLC However this finding remains controversial in small cell lung cancerThis metaanalysis was systematically performed to evaluate the prognostic value of DLL3 in SCLCMethods The PubMed EMBASE and Web of Science databases were retrieved to collect the eligible referencesThrough Stata software we pooled hazard ratios HR with confidence intervals CI by using random orfixedeffects models to evaluate the association between DLL3 and SCLC survival resultsResults A total of interrelated studies including patients were qualified After we removed study theremaining studies including patients were pooled to testify that high expression of DLL3 was an inferiorprognostic for patients with SCLC in Asian populations HR CI I2 p Thepooled results showed that DLL3 might be higher expression in advanced metastasis SCLC in Asian populations RR CI I2 p But the expression of DLL3 was not correlated with sex RR CI I2 p smoking history RR CI I2 p and tumour stage RR CI I2 p Conclusions Our metaanalysis confirms that in Asian populations high expression of DLL3 was a potential poorprognostic biomarker for SCLC and DLL3 highly expressed in advanced stage SCLC in Asian populationsKeywords Deltalike protein DLL3 Prognostic Small cell lung cancer SCLC MetaanalysisIntroductionLung cancer has the highest morbidity and mortality ofall malignant tumours which is one of the most common cancers worldwide [] The major histologic subtypes oflung cancerNSCLC and small cell lung cancer SCLC [] SCLC is an invasive highgrade malignancy withearly development and a bad prognosis Most patientswith SCLC are advanced with widespread metastasiswhen they are diagnosed and systemic chemotherapy islung cancer are nonsmall cell Correspondence ligaofenghl126comDepartment of Thoracic Surgery The Third Affiliated Hospital of KunmingMedical University Yunnan Cancer Hospital Kunming Yunnan Chinathe most effective therapy [] Few therapeutic methodsare available after SCLC relapse and the prognosis is especially poor Therapeutic treatments for patients withSCLC have essentially remained unchanged in recentyears [] Thus further research into the mechanism ofSCLC and the exploration of new therapeutic targets forSCLC is imperativeDeltalike protein DLL3 is a transmembrane protein that promotes the development of neuroendocrinetumours through its reciprocity with the Notch pathway[] It is expressed in cancer tissues in approximately of patients with SCLC and other neuroendocrineis not expressed in nonneuroendocrinecancer but The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cChen World Journal of Surgical Oncology Page of tumours or normal tissues [] Most studies have shownthat DLL3 is a latent treatment target point for SCLC[“] Recently many researches initiate to focus on theprognostic value of DLL3 in SCLC Some of the researchers demonstrated that DLL3 had no correlationwith prognosis in SCLC patients [ ] but other investigators showed that DLL3 was associated with thesurvival of patients with SCLC [] So previous studiesof the prognostic value of DLL3 in SCLC remain controversial we performed this systematic metaanalysis forresolving this controversialPublished s discussing the prognostic value ofDLL3 in SCLC were systematically reviewed in ourmetaanalysis We aimed to include all correlationalstudies to assess the prognostic value of DLL3 andattempted to identify an accurate biomarker to guideprognosis and treatment for SCLC in the futureMethodsSearch strategyThe PRISMA guidelines were followed for our metaanalysis see Additional file [] The literatures werecollected through retrieving the PubMed EMBASE andWeb of Science databases from the initial date to February The search strategy was œDLL3 OR deltalike protein  all fields AND œnonsmall cell lung cancer OR NSCLC OR small cell lung cancer OR SCLC ORLung Adenocarcinoma OR Lung Squamous cell carcinoma OR lung cancer OR lung tumour OR lung neoplasm OR lung carcinoma all fields There was nolimitation on the publication status All eligible studieswere retrieved and the headlines and s of all thereference lists of the reviews or studies were independently filtrated through three authors based on the criteria Differences between two authors were resolved bythe third author™s opinionsInclusion criteriaResearches were included by following the standards studies reported the prognostic value of DLL3 in SCLC studies were published as original s studiesreported the data of HR and CI or provided survivalcurve and studies in which the prognostic value wasinvestigated by survival analysis with overall survivalOS diseasefree survival DFS progressionfree survivalRFS ordiseasespecific survival DSS We excluded the animalresearches and other review literaturesPFS relapserecurrencefree survivalData extractionTwo researchers independently extracted the data included the author country edition year sample capacitypatients™ sex smoking history distant metastasis tumourstage histologic subtype biomarkers scoring methodsboth univariateand multivariatecutoffs value and the data of HR and CI We extracted the multivariate analysis outcome if one study includedanalysisoutcomes When we could not extract the data of HRand CI directly in the but the survival curveswere reported we extracted and digitized the survivalcurves by operating the Engauge Digitizer softwarehttpdigitizersourcefenet and we estimated thedata of HR and CI by the Excel programme files asexploited by Tierney [] When the data of HRand survival curves were not reported we contacted thecorresponding authors of eligible s by email to obtain the original data these s were excluded ifthere was no responseAssessment of study qualityTwo researchers independently used the Quality InPrognosis Studies QUIPS tool to assess risk of bias ofall the publications [] According to the criteria every was evaluated as low risk moderate risk or highrisk by following six different areas study participationstudy attrition prognostic factor measurement outcomemeasurement study confounding and statistical analysisand reporting [] Differences were settled throughdiscussionStatistical analysisTwo authors independently extracted the HR and CI from the original s The HR was used to describe the high DLL3 expression versus low DLL3 expression We took the reciprocal of the HR if the sshowed the low DLL3 expression vs high DLL3 expression We observed that high expression of DLL3 portended a worse prognosis when HR and that HR indicated preferable prognosis For the analysis results p was considered statistically significant Statisticalheterogeneity was assessed by calculating the I2 statistic[] The presence of heterogeneity was indicated whenI2 and then a randomeffects model was appliedto poolthe results [] a fixedeffects model wasemployed to pool the results when I2 [] Weperformed further subgroup or sensitivity analysis toanalyse the heterogeneity The publication bias was estimated by Begg™s and Egger™s tests when p indicates no publication bias [] Through Stata software we performed this metaanalysis and acquiredthe forest plotsResultsSearch results and study characteristicsUsing the searching strategy described above a total of original documents were identified from the databases with approximately studies remaining after excluding duplicates Afterandscreening thetitles 0cChen World Journal of Surgical Oncology Page of s of the publications publications werenot related to evaluating the prognosis role of DLL3in SCLC Finally we were left with eligible studiesafter screening the full text among which swere included in our final analysis Fig [“]Ten s were excluded for the following reasonsseven s were review researches and commentaries one did notreport hazard ratios andKaplanMeier curves and s were s anddid not report relevant outcomes The eligible s™ characteristics are shown in Table All theses came out from the initial date to February and the patients were diagnosed as SCLC byhistopathology Among these studies all studies investigated DLL3 byinparaffinembedded tissueimmunohistochemistryIHCQuality assessment of the included studiesWe performed quality evaluations of the s following the QUIPS tool and two authors independently evaluated allthe literature Differences were resolved bydiscussion After screening all included s we foundthat there were no researches that reported study attritionand there were no standardization of cutoffs value forevaluating DLL3 expression And then studies were evaluated as low risk were evaluated as moderate risk and study was evaluated as high risk Table This outcomeindicated that most of the studies we included were of amedium or high qualityPrognostic value of DLL3 in SCLCAs shown in Fig eligible s were pooled foranalysing the prognostic value of DLL3 in SCLC TheFig The flow diagram of studies selection 0cChen World Journal of Surgical Oncology Page of Table Study characteristics of the eligible sStudyYear Country No ofsamplesSexmalefemaleSmokinghistorynonyesYan [] ChinaDistantmetastasisnegativepositiveXie [] USATanaka [] JapanRegzedmaa [] ChinaHuang [] ChinaFuruta [] JapanNANANANANANANATumourstageIIIIIIIVHistologicsubtypeMethod Outcomes BiomarkersCutoffvalueNANASCLCSCLCSCLCSCLCSCLCSCLCIHCIHCIHCIHCIHCIHCOSOSOSOSDLL3TTF1DLL3DLL3DLL3CTLA4MSTNscore ‰¥ ‰¥ ‰¥ score ‰¥ OSPFSDLL3OSDLL3ASCL1score ‰¥ ‰¥ SCLC small cell lung cancer IHC immunohistochemistry OS overall survival PFS progressionfree survival DLL3 deltalike protein CTLA4 cytotoxic T lymphocyteassociated protein MSTN mesothelin ASCL1 achaetescute homologue1 NA not applicableresults showed that high expression of DLL3 indicatedno prognostic value in patients with SCLC HR CI I2 p Howeverprominent heterogeneity existed in the pooled results sowe conducted further subgroup analysis Among the sixstudies three s were from China two were fromJapan and one was from America We divided the studies into the Asian group and the American group forsubgroup analysis As shown in Additional file FigA1we found that the heterogeneity was in the Asiangroup and we believed that the main cause of the heterogeneity was the study from American So we decidedto eliminate the study from American and only analysedthe remained studies After we removed the Americanstudy the outcomes indicated that high DLL3 expressionwas a poor prognosis marker in SCLC HR CI I2 p Fig Then wedivided the studies into two groups according to thesize of the sample and the final outcomes indicated thathigh DLL3 expression was a poor prognosis marker inthe group with sample size greater than HR CI p Fig But there was nosignificant relationship between DLL3 expression andprognosis in the group with sample size less than HR CI I2 p Fig Begg™s test p and Egger™s test p there was nodemonstratedresultsthatpublication bias in our metaanalysis Fig Finallysensitivity analysis showed that allthe studies werestable see Additional file FigA6The clinical characteristics of patients with DLL3expression in SCLCThe pooled results showed that the expression of DLL3was not correlated with sex RR CI I2 p Table Additional file FigA2 smoking history RR CI I2 p Table Additional file FigA3 and tumour stage RR CI I2 p Table Additional file FigA4 But DLL3 was highly expressed in patients withdistant metastasis RR CI I2 p Table Additional file FigA5DiscussionMany studies have shown that DLL3 played a significantprognosis value in patients with cancer For examplehigh DLL3 expression was associated with shorter OSand PFS in small cell bladder cancer [] High DLL3expression was associated with bad OS and usuallyexpressed in older patients and advanced stage in endometrial cancer [] Xie reported that high DLL3expression predicted a better OS in patients with SCLC[] and other studiesforeboded that high DLL3Table Quality assessment of included studiesStudyStudyparticipation—‹Yan []Xie []Tanaka []Regzedmaa []Huang []—‹—‹—‹Furuta []œ—Ž indicates low risk of bias œStudyattrition——————Prognostic factormeasurement—‹—‹—‹—‹—‹—‹Outcomemeasurement—‹—‹—‹—‹—‹Study confounding—‹—‹—‹—Statistical analysisand reporting—‹—‹—‹—‹—‹—‹Total riskof biasLowModerateLowLowHighModerate moderate risk of bias and œ— high risk of bias 0cChen World Journal of Surgical Oncology Page of Fig Forest plots of prognostic value of DLL3 in SCLC patients DLL3 deltalike protein HR hazard ratios CI confidence intervals Isquared percentage heterogeneity between studies p test for heterogeneityexpression was an inferior prognostic marker for SCLC[ ] Thus the results of different studies remainedcontroversial We included a total of studies with patients with SCLC to assess the prognostic value ofDLL3 in SCLC by pooling the data of HR and CIFirst we performed this metaanalysis using studiesBut heterogeneity was observed in our pooled resultsThrough further subgroup analysis we found that astudy from the American was the main cause of heterogeneity As we removed the American study we foundFig Forest plots of prognostic value of DLL3 in Asia patients with SCLCDLL3 deltalike protein USA the United States of America HR hazardratios CI confidence intervals Isquared percentage heterogeneity between studies p test for heterogeneity 0cChen World Journal of Surgical Oncology Page of Fig Forest plots of prognostic value of DLL3 in different studies with different sample sizes DLL3 deltalike protein HR hazard ratios CI confidence intervals Isquared percentage heterogeneity between studies p test for heterogeneitythat high expression of DLL3 is a marker of poor prognosis in SCLC But we also noticed that the results ofthe American study were contrary to our conclusionsthey reported that high expression of DLL3 is a markerof good prognosis in SCLC One interpretation of thisresult was that DLL3 expression varies between differentpopulations The American study indicated that the highexpression of DLL3 was a marker of good prognosis[] and there was no significant correlation betweenDLL3 expression and prognosis in several Japan studies[ ] while the high expression of DLL3 is a markerof poor prognosis in China studies [ ] These results showed that the expression of DLL3 might be different in different populations However only one studyFig Funnel plot of DLL3 present on overall survival DLL3 deltalike protein HR hazard ratios 0cChen World Journal of Surgical Oncology Page of Table Summary of the correlation between DLL3 expression and the clinical characteristics of patients with SCLCClinical characteristicsSex malefemaleRR CI No of studiesI2 p Smoking historynonyesDistant metastasis negativepositive P valueTumour stage IIIIIIIVSCLC small cell lung cancer RR relative ratios CI confidence intervals DLL3 deltalike protein I2 percentage heterogeneity between studies p testfor heterogeneity researched the correlation with DLL3 expression andprognosis in SCLC outside of Asia so the results needto be treated with caution In addition more studieswere suggested for the future to further verify the existence of such differencesOur other explanation of this result was that the expression of DLL3 was the same in different populationsAmong the included studies the sample size of studieswas less than including the America and Japanstudies Therefore we speculated that insufficient sample size might cause bias in the results We conducted asubgroup analysis according to the sample size and theresults showed that the high expression of DLL3 in thestudies with large sample sizes N ‰¥ was associatedwith poor prognosis while the pooled results of thestudies with low sample sizes N showed no significant correlation between the expression of DLL3 andprognosis Therefore the sample size may be one of thereasons for the differences in DLL3 expression in eachstudy Moreover immunohistochemistry was used to detect DLL3 expression in all of the studies Immunohistochemical staining is a semiquantitative method and isevaluated with great subjectivity [] Different antibodies and different cutoff values for DLL3 expressionwere employed in all the included studies and thus couldalso be another cause of the differences in results TheAmerican study also explained their different results byclaiming that many of the other studies were performedusing mRNA expression instead of protein expression orused different cutoffs value of DLL3 expression []Researches havereported that DLL3 is highlyexpressed in SCLC [ ] which suggested that DLL3might promote the development of SCLC Therefore wealso discussed the correlation between DLL3 expressionand the clinical characteristics of patients with SCLCThe pooled results showed that DLL3 expression had nosignificant correlation with patients™ sex smoking statusand stage while DLL3 often highly expressed in metastasis patients of SCLC Our survival analysis outcomeswere consistent with this result which suggested thathigh expression of DLL3 might be one of the factorscontributing to poor prognosis in patients with advancedmetastatic SCLC in Asia However only a few studiesreported the correlation between the expression of DLL3and clinical characteristics and the methods and cutoffs values used to detect the DLL3 expression in eachstudy were not uniform Therefore more reliable studiesare needed to further verify our outcomes Our resultssuggest that it is valuable to further investigate the correlation between DLL3 and the clinical characteristics ofpatients with SCLCOur metaanalysis is the first to focus on the prognostic value of DLL3 in SCLC The significance of thismetaanalysis lies in providing a basic direction and evidence for further research into the mechanism of DLL3in SCLC For SCLC Notch1 over expression could induce G1 cell cycle arrest [] Previous studies reportedthat DLL3 downregulated the Notch receptor expression thereby the Notch signalling pathway was inhibitedwithin the cell [] Therefore high expression of DLL3can promote the development of SCLC by inhibiting theNotch signalling pathway Studies also have shown thatthe high expression of DLL3 may reduce the sensitivityof chemotherapy drugs [] These studies have demonstrated that DLL3 may be associated with the prognosisof SCLC and also consistent with our metaanalysis results Thus studies of the corresponding targeted drugsof DLL3 can effectively inhibit the expression of DLL3and thus improve the survival of SCLC Rovalpituzumabtesirine RovaT is a new antibodydrug conjugate directed against DLL3 in SCLC [] A phase I trial foundthat patients with high DLL3 expression in SCLCshowed a better response to RovaT than those with alow DLL3 expression [] However disappointingly thephase III TAHOE trial has been stopped because theRovaT group showed a worse OS compared to the control group [] But more clinical trials are recruitingparticipator to investigate RovaT as maintenance therapy in advanced stage SCLC The lack of progress withthis drug does not prevent us from making a breakthrough with other similar drugs Some researches foundthat the intratumoural and intertumoural distributionof DLL3 protein in SCLC is homogeneous [] supporting the conclusion that biopsy specimens are a reliablesource for DLL3 evaluation for targeted therapy Inaddition most studies have demonstrated that DLL3 is 0cChen World Journal of Surgical Oncology Page of highly expressed in SCLC while it is not or is lessexpressed in other types of lung cancer and normal tissues [] Therefore the expression of DLL3 can be detected by biopsy as an indicator for diagnosis predictingtherapeutic efficacy and monitoring recurrence or metastasis of SCLC in the futureAlthough our study fully explains the prognostic valueof DLL3 in SCLC our analysis still has several limitations Firstlarge heterogeneity was observed in thepooled results This is explained by the observation thatthe evaluation criteria for the expression of DLL3 areparticularly mixed and there are no international standards for cutoffs values to determine the expression ofDLL3 Thus the scoring methods and cutoffs values ofDLL3 should be unified to strengthen our conclusionsOtherwise the detection method of DLL3 in most studies is mainly immunohistochemistry at present which isa semiquantitative subjective and inaccurate detectionmethod Different studies show different prognosticvalues of DLL3 Therefore we need other more precisedetection methods to evaluate the expression of DLL3 inSCLC in the futureSecond the therapy method is also a key limitationThe current studies only focus on tissue specimens frompatients with SCLC after surgery or biopsy and fewstudies reported the treatment methods in their researches However the prognostic value of DLL3 may liein the therapeutic method Therefore every study shouldpay attention to the impact of patienttreatmentmethods on prognosis in the futureThird some of the original studies did not report thedata of HR and CI The HR and CI resultswere measured from survival curves an evaluationmethod with certain deviation and subjectivity whichmight influence the authenticity of the resultsConclusionIn summary our metaanalysis confirmed that high expression of DLL3 was a potential poor prognostic biomarker for SCLC in Asian populations moreover DLL3expression was correlated with advanced metastasisSCLC in Asian populations However the relationshipbetween DLL3 expression and the prognostic or clinicalcharacteristics of patients with SCLC in European andAmerican populations need to be further verified Thusdetecting the expression of DLL3 in tumour tissue willbe helpful to guide therapy in Asian patients of SCLCFor our research other highquality studies especiallyfrom European and American countries are required toconfirm our findings about the prognosis value of DLL3in SCLC in the future In view of the limitations of ouranalysisthe conclusions should be interpreted withcautionSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s12957020020045Additional file The PRISMA checklistAdditional file FigA1 Forest plots of prognostic value of DLL3 inSCLC SCLCsmall cell lung cancer HRhazard ratios CI confidence intervals DLL3deltalike protein I2percentageheterogeneity between studies ptest for heterogeneity FigA2 Forestplots of the correlation between DLL3 expression and sex of patientswith SCLC SCLCsmall cell lung cancer RRrelative ratios CI confidence intervals DLL3deltalike protein I2percentageheterogeneity between studies ptest for heterogeneity FigA3 Forestplots of the correlation between DLL3 expression and smoking history ofpatients with SCLC SCLCsmall cell lung cancer RRrelative ratios CI confidence intervals DLL3deltalike protein I2percentageheterogeneity between studies ptest for heterogeneity FigA4 Forestplots of the correlation between DLL3 expression and tumour stage ofpatients with SCLC SCLCsmall cell lung cancer RRrelative ratios CI confidence intervals DLL3deltalike protein I2percentageheterogeneity between studies ptest for heterogeneity FigA5 Forestplots of the correlation between DLL3 expression and metastasis ofpatients with SCLC SCLCsmall cell lung cancer RRrelative ratios CI confidence intervals DLL3deltalike protein I2percentageheterogeneity between studies ptest for heterogeneity FigA6Sensitivity analysis of all the studiesAbbreviationsCI Confidence intervals DLL3 Deltalike protein DFS Diseasefree survivalDSS Diseasespecific survival HR Hazard ratios IHC ImmunohistochemistryOS Overall survival PFS Progressionfree survival QUIPS Quality In PrognosisStudies RFS Relapserecurrencefree survival RR Risk ratio SCLC Small celllung cancerAcknowledgementsNot applicableAuthors™ contributionsAll authors contributed to the study conception and design Materialpreparation data collection and analysis were performed by Benchao ChenHeng Li and Chao Liu The first draft of the manuscript was written byBenchao Chen and all authors commented on previous versions of themanuscript All authors read and approved the final manuscriptAuthors™ informationNot applicableFundingThis work was supported by the National Natural Science Foundation ofChina [No ]Availability of data and materialsThe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsReceived July Accepted August ReferencesBray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancerstatistics GLOBOCAN estimates of incidence and mortality worldwide 0cChen World Journal of Surgical Oncology Page of Regzedmaa O Li Y Li Y Zhang H Wang J Gong H Prevalence ofDLL3 CTLA4 and MSTN expression in patients with small cell lung cancerOnco Targets Ther “ httpsdoi102147OTTS216362 Huang J Cao D Sha J Zhu X Han S DLL3 is regulated by LIN28B and miR518d5p and regulates cell proliferation migration and chemotherapyresponse in advanced small cell lung cancer Biochem Biophys ResCommun “ httpsdoi101016jbbrc201904130Furuta M SakakibaraKonishi J Kikuchi H Yokouchi H Nishihara HMinemura H Analysis of DLL3 and ASCL1 in surgically resected smallcell lung cancer HOT1702 Oncologist 20192411e1172“ httpsdoi101634theoncologist20180676Koshkin VS Elson P MagiGalluzzi C McKenney J Smith KS Shadrach B Prognostic value of DLL3 expression and clinicopathologic features insmall cell bladder cancer SCBC J Clin Oncol Juan Wang Kaishuo Zhang Zi Liu Upregulated deltalike protein expression is a diagnostic and prognostic marker in endometrial cancer aretrospective study Medicine Baltimore 20189751e13442 httpsdoi101097MD0000000000013442 Matos LLD Stabenow E Tavares MR Ferraz AR Capelozzi VL Pinhal MADSImmunohistochemistry quantification by a digital computerassistedmethod compared to semiquantitative analysis Clinics “httpsdoi101590S180759322006000500008 Hann CL Mensztern D Dowlati A Burns TF Jotte RM Pennell NA et alA study of rovalpituzumab tesirine in frontline treatment of patients withDLL3 expressing extensive small cell lung cancer J Clin Oncol httpsdoi101200JCO20173515_supplTPS2598Furuta M Sakakibara JK Shoji T Takashima Y Kikuchi H Kikuchi E et alDLL3 regulates migration and invasion of small cell lung cancer Cancer Res httpsdoi10115815387445Am20183158Sriuranpong V Bes MW Ravi RK Arnold DR Nelkin BD Baylin SB et alNotch signaling induces cell cycle arrest in small cell lung cancer cells CancerRes httpsdoi1010970000282020010400000012 Deng SM Yan XC Liang L Wang L Liu Y Duan JL The Notch liganddeltalike promotes tumor growth and inhibits Notch signaling in lungcancer cells in mice Biochem Biophys Res Commun “httpsdoi101016jbbrc201612117Komarnitsky P Lee H Shah M Wong S Gulbranson S Dziubinski J et alRovalpituzumab tesirine vs topotecan in patients with advanced small celllung cancer following 1st line chemotherapy J Thorac Oncol S1974“ Udagawa H Akamatsu H Tanaka K Takeda M Kanda S Kirita K Phase Isafety and pharmacokinetics study of rovalpituzumab tesirine in Japanesepatients with advanced recurrent small cell lung cancer Lung Cancer “ httpsdoi101016jlungcan201907025Komarnitsky P Lee HJ Shah M Wong S Gulbranson S Dziubinski J Aphase trial of rovalpituzumab tesirine vs topotecan in patients withadvanced small cell lung cancer following frontline platinumbasedchemotherapy Ann Oncol 201728v542 httpsdoi101093annoncmdx386009 Odashiro P Tomarchio G Gagne A Orain M Desmeules P Joubert P DLL3expression in small cell lung carcinomas SCLC Mod Pathol 202033SUPPL“Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsfor cancers in countries CA Cancer J Clin httpsdoi103322caac21492Amini A Byers LA Welsh JW Komaki RU Progress in the management oflimitedstage small cell lung cancer Cancer httpsdoi101002cncr28505 Mead GM Arnold AM Green JA Williams CJ Whitehouse JM SMALLCELLLUNG CANCER[J] Cancer Chemother Pharmacol httpsdoi101016S0140673680907333Powell HA Tata LJ Baldwin DR Potter VA Stanley RA Khakwani A et alTreatment decisions and survival for people with smallcell lung cancer Br JCancer “ httpsdoi101038bjc2013812Huang RSP Holmes BF Powell C Marati RV Tyree D Admire B Deltalike protein prevalence in small cell lung cancer and DLL3 SP347 assaycharacteristics Arch Pathol Lab Med “ httpsdoi105858arpa20180497OA Mensztern D Besse B Greillier L SantanaDavila R Ready N Hann CL Efficacy and safety of rovalpituzumab tesirine in thirdline and beyondpatients with DLL3expressing relapsedrefractory smallcell lung cancerresults from the phase II TRINITY study Clin Cancer Res “ httpsdoi10115810780432CCR191133Isobe Y Sato K Nishinaga Y Takahashi K Taki S Yasui H Near infraredphotoimmunotherapy targeting DLL3 for small cell lung cancerEBioMedicine httpsdoi101016jebiom2020102632Rossi A Rovalpituzumab tesirine and DLL3 a new challenge for smallcelllung cancer Lancet Oncol “ httpsdoi101016s1470Rudin CM Pietanza MC Bauer TM Ready N Mensztern D Glisson BS Rovalpituzumab tesirine a DLL3targeted antibodydrug conjugate inrecurrent smallcell lung cancer a firstinhuman firstinclass labelphase study Lancet Oncol “ httpsdoi101016s1470204516305654Fu X Tian T Liu M Ruan Z Liang X Nan K The expression andprognostic roles of PDL1 PAPR1 and DLL3 in small cell lung cancer JThorac Oncol 20191410S1024“ httpsdoi101016jjtho2019082267Lim S Hong M Kim SP Chung SH P2 Prevalence of DLL3 Expressionand its prognostic role in extensive stage small cell lung cancer J ThoracOncol 20191410S820 httpsdoi101016jjtho2019081763 Poirier JT Targeting DLL3 in smallcell lung cancer with novel modalities JThorac Oncol 2020152S8 httpsdoi101016jjtho201912024Liberati A Altman DG Tetzlaff J Mulrow C Gøtzsche PC Ioannidis JPA et alThe PRISMA statement for reporting systematic reviews and metaanalysesof studies that evaluate health care interventions explanation andelaboration J Clin 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Thyroid_Cancer

"Ethnopharmacological relevance Tetrastigma hemsleyanum Diels et Gilg Themsleyanum a rare herbal plant distributed in subtropical areas of mainland China has become a focus of scientific attention in recent years because of its high traditional value including uses for treatment of children with fever pneumonia asthma rheumatism hepatitis menstrual disorders scrofula and pharynx pain Aim This systematic review aims to provide an insightful understanding of traditional uses chemical composition pharmacological effect and clinical application of T hemsleyanum and lay a foundation for the further study and for the utilization of T hemsleyanum resource Materials and methods A domestic and overseas literature search in known databases was conducted for published s using the relevant keywords Results One hundred and fortytwo chemical constituents identified from T hemsleyanum have been reported including flavonoids phenolic acids polysaccharide anic acids fatty acids terpenoids steroids amino acid and others Among these components flavonoids and polysaccharides were the representative active ingredients of T hemsleyanum which have been widely investigated Modern pharmacological studies have shown that these components exhibited various pharmacological activities such as antiinflammatory antioxidant antivirus antitumor antipyretic antihepatic injury immunomodulatory antibacterial etc Moreover different toxicological studies indicated that the clinical dosage of T hemsleyanum was safe and reliable Conclusions Modern pharmacological studies have well supported and clarified some traditional uses and T hemsleyanum has a good prospect for the development of new drugs due to these outstanding properties However the present findings did not provide an indepth evaluation of bioactivity of the extracts the composition of its active extracts was not clear Moreover they were insufficient to satisfactorily explain some mechanisms of action Data regarding many aspects of T hemsleyanum such as links between the traditional uses and bioactivities pharmacokinetics quality control standard and the clinical value of active compositions is still limited which need more attention Introduction Tetrastigma hemsleyanum Diels et Gilg T hemsleyanum mostly known as œSan ye qing is a kind of folk plant Because of its slow growth it usually takes “ years to meet the requirements of commercial medicinal materials so it is a precious perennial medicinal resource It mainly grows in the eastern central southern and south western provinces of China such as Zhejiang Jiangsu Guangxi Fujian and Yunnan provinces Peng and Wang T hemsleyanum is known worldwide as sources of phytotherapeutics which have been used for the treatment of conditions related to inflammatory and immune response and been recorded based on clinical trials or the use of animal models Xu As an edible plant the leaves of T hemsleyanum consumed as a functional tea or dietary supplement for its health benefits such as improving the immune system of the body Sun while the aerial parts of T hemsleyanum developed as potential new traditional chinese medicine TCM preparations Guo Corresponding author Ningbo Research Institute of Zhejiang University Ningbo Zhejiang People™s Republic of China Email address px4142163com X Peng 101016jjep2020113247 Received May Received in revised form July Accepted August JournalofEthnopharmacology2642021113247Availableonline12August2020037887412020ElsevierBVAllrightsreserved 0cT Ji Abbreviations T hemsleyanum Tetrastigma hemsleyanum Diels et Gilg TCM UPLCESIQTOFMSMS Ultra high performance liquid Traditional Chinese Medicine chromatography tandem triple quadrupole time of flight mass spectrometry minimum inhibitory concentration glutathione malondialdehyde nuclear factorκB 5hydroxytryptamine norepinephrine dopamine prostaglandin E2 lipopolysaccharide tumor necrosis factoralpha interleukin1 beta interleukin MIC GSH MDA NFκB 5HT NE DA PGE2 MAPK mitogenactivated protein kinase LPS Celegans Caenorhabditis elegans TNFα IL1 IL6 IL12p40 interleukin subunit p40 sTNFR1 soluble TNF receptors IL10 IL1 IL4 iNOS TLR4 MD2 MyD88 myeloid differentiation protein JNK GPT GOT ALP SOD interleukin interleukin interleukin inducible NO synthase Tolllike receptor myeloid differentiation factor2 cJun Nterminal kinase glutamicpyruvic transaminase glutamicoxalacetic transaminase alkaline phosphatase superoxide dismutase and activities antiinflammatory The root tubers of T hemsleyanum are extensively used either alone or in combination with other herbal medicines in TCM clinics for the treatment of children with fever convulsion pneumonia asthma rheumatism hepatitis menstrual disorders scrofula and pharynx pain Sun Chen and Guo Therefore it was called as œnatural plant antibiotic according to its wide spectrum of prominent bactericidal In February T hemsleyanum was awarded as the new œeight famous kinds of TCM in Zhejiang province meant that it has become a key object of industrialization development of Zhejiang™s dominant large varieties of medicinal materials In COVID19 broke out and has caused more than deaths in China and infection cases have been reported in more than countries Hua Shi Xuan Fei mixture Approval number of Zhejiang medicine Z20200026000 which composed of T hemsleyanum has been approved by Zhejiang Provincial Drug Administration for clinical treatment of COVID19 Futhermore the modern pharmacological studies had shown that T hemsleyanum also had effects of antiinflammatory Ji antioxidant Hossain antivirus Ding antitumor Lin antipyretic Yang and Wang antihepatic injury Ma et al immunomodulatory Xu antibacterial Chen hypoglycemic Ru 2018ab etc Numerous reports have demonstrated that the biological activities of T hemsleyanum are attributed to its many chemical components Fu Wang has reported isolated alkaloids from the aerial parts of T hemsleyanum Wang Ru extracted a novel polysaccharide TDGP3 from is mainly alanine aminotransferase aspartate aminotransferase hyaluronan laminin total bilirubin total protein interferongamma immunoglobulin A secretory immunoglobulin A Epithelialmesenchymal transition ALT AST HA LN TBiLi TP IFNÎ IgA SIgA EMT MMPs matrix metalloproteinase TIMPs matrixmetallo proteinase Cytc CAT GSHPx glutathione peroxidase Tregs TGF COX2 Foxp3 PDL TAOC CCl4 CEF HVJ VSV A F S1 S2 PEF CFF EAF BAF Cytochrome c catalase regulatory T cells transforming growth factor beta cyclooxygenase forkheadwinged helix transcription factor gene population doubling time total antioxidant capacity carbon tetrachloride chicken embryo fibroblast Hemagglutinating virus of Japan vesicular stomatitis virus alkalicontaining extract of T hemsleyanum ketonecontaining extract of T hemsleyanum crude extract of T hemsleyanum crude extract of T hemsleyanum Petroleum ether extractions of T hemsleyanum ethanol extract Chloroform extractions of T hemsleyanum ethanol extract ethyl acetate extractions of T hemsleyanum ethanol extract nbutanol extractions of T hemsleyanum ethanol extract T hemsleyanum with a molecular weight of — Da by enzymolysisultrasonic assisted extraction method Ru 2019ab Large amounts of flavonoids were found in leaves aerial parts and root tubers of T hemsleyanum Xu 2014ab Deng Yu In addition T hemsleyanum also contains a variety of functional components such as anic acids Hu phenolic acids Liu minerals Fan amino acids Fu etc In recent years wild resources of T hemsleyanum have been overexploited and now are on the verge of extinction due to its multiple medicinal values coupled with the strict requirements of the growing environments In it was listed in the preferentially protected crop germplasm resources of Zhejiang province Based on our team™s preliminary research Peng Peng 2016ab Li we comprehensively summarized and analyzed the domestic and overseas research progress on traditional uses the bioactive components of T hemsleyanum pharmacological activities toxicology with the aim of providing guidance for indepth research and reference for its development and utilization Materials and methods The available information about the traditional uses phytochemicals and pharmacological properties of T hemsleyanum was searched via Web of Science Google Scholar PubMed Science Direct China National Knowledge Infrastructure CNKI and Springer search using Chinese or English as the retrieval languages The keywords used include T hemsleyanum root tubers of T hemsleyanum Radix Tetrastigma JournalofEthnopharmacology26420211132472 0cT Ji traditional uses phytochemistry bioactive components pharmacological activities toxicology and other related words All references were from experimental studies and published prior to April were reviewed All chemical structures were drawn using ChemDraw Pro software heatclearing were Botanical characteristics T hemsleyanum is a perennial grass climbing vine with longitudinal ribs glabrous or sparsely pilose It is usually grown in a cool and humid environment and the main soil type is yellow soil or yellow brown soil with rich humus The optimum pH is between and The root tubers are thick spindle shaped or elliptical and single or several are connected into a string of beads generally “ cm long and “ cm in diameter Fig The epidermis of the root tubers is tan and most of them are smooth a few of them have folds and lenticel like protuberances some of them have depressions in which there are residual tan roots hard and brittle with a flat and rough section The stem of T hemsleyanum is thin and weak with longitudinal rhombus rooting on the lower node Palmate compound leaves alternate leaflets are lanceolate oblong or ovate lanceolate The leaflets are “ cm long and “ cm wide with a tapered tip and a wedgeshaped or round base The flowers of T hemsleyanum are small yellow green and ovate The flowering stage of T hemsleyanum ranges from April to June and the fruit phase is normally from August to November When the flower withered it will form a small green round fruit with the size of millet When it is mature the fruit will turn from green to red the berries are spherical and soft spherical Traditional uses T hemsleyanum belonging to the family Vitaceae was firstly recorded in Ben Cao Gang Mu Ming Dynasty AD The aliases of Sanyeqing include Shi Hou Zi Shi Bao Zi Shi Lao Shu Lan Shan Hu Lei Dan Zi Po Shi Zhu Tu Jing Wan Sou Jia Feng San Ye Dui golden wire hanging gourd golden bell golden wire hanging potato etc The root tubers or whole grass of T hemsleyanum traditionally and ethnically used as a medicine for a long time it has been recorded in multiple hemsleyanum ancient books of TCM such as Zhi Wu Ming Shi Tu Kao Qing Dynasty Wu Jiangxi herbal medicine Common folk herbal medicine in Zhejiang All of these ancient works described the effects of toxicityremoving T dyspnearelieving promoting blood circulation and pain relief thus it can be applied to cure febrile convulsion pneumonia bronchitis pharyngitis sore throat acute and chronic hepatitis rheumatic arthralgia viral meningitis bruise eczema insect and snake bite poor joint flexure and extension irregular menstruation of women National compilation team of Chinese herbal medicine In the TCM culture the properties of T hemsleyanum was described as bitter and acrid in taste cool in nature which recorded in dictionaries of traditional Chinese medicine and Zhong Hua Ben Cao Shanghai Science and Technology Press The channel tropism was lung heart liver and kidney meridians Decocting with water or mashing for external application are the traditional possess methods of T hemsleyanum Considering its extensive traditional effects many prescriptions containing T hemsleyanum have been passed down from generation to generation and have been well supported and clarified by modern pharmacological studies Excitingly it has reported that Jinlian disinfection drink containing san ye qing combined with interferon can treat Covid19 He Jinqi Tablet made up of san ye qing astragalus and ginsenoside was used to treat cases of malignant tumor cases were completely relieved cases were partially relieved the total effective rate was Wei Moreover Zhonggan mixture including san ye qing could improve the quality of life and prolong the survival time of patients with stage III primary liver cancer Jiang and Gong In addition it has been used in the treatment of common gynecological diseases such as blood avalanche and leucorrhea Gao and it also has a good effect on measles complicated with pneumonia anal fissure chronic bronchitis and mosquito bites Ji Chemical compounds of Themsleyanum The chemical constituents of T hemsleyanum have been widely investigated Sun Sun Zeng Xu 2014ab Fu Fan Chen Ding 2015a Fig The aerial part A root tuber B and raw herb C of T hemsleyanum JournalofEthnopharmacology26420211132473 0cT Ji b Ding a total of one hundred and fortytwo compounds have been isolated and identified from T hemsleyanum until now The information about compound name molecular weight compound formula detection method analysis sample is summarized in Table Flavonoids and their glycosides Modern phytochemical studies have indicated that flavonoids are the representative and predominated class of constituents isolated from T hemsleyanum Lin Zhang Table To date fiftyone flavonoids and their glycosides have been extracted and identified from T hemsleyanum In this series compounds quercetin orientin vitexin isorhamnetin apigenin and kaempferol are the main types of skeleton some of their analogues can be identified from hydroxy moiety on C3² and C4™ on the B ring of flavonoid aglycone At present many modern analytical techniques have been used for qualitative and quantitative analysis of flavonoids Among them ultra high performance liquid chromatography tandem triple quadrupole time of flight mass spectrometry UPLCESIQTOFMS has become a powerful tool for identifying the complicated compounds due to its higher mass accuracy and resolution Our team used UPLCESIQTOFMS to identify chemical constituents from the aerial part of T hemsleyanum including flavonoids such as isoorientin quercetin kaempferol vitexin isovitexin kaempferol3glucoside etc Sun According to the report Liu total flavonoids of T hemsleyanum could protect the aged mice from acute lung injury through inhibiting the phosphorylation of mitogenactivated protein kinase MAPK and nuclear factorκB NFκB in lung tissue Moreover the flavonoids of T hemsleyanum had the activity of antilung cancer Wei Luteolin a flavonoid found in T hemsleyanum acted as an anticancer agent against various types of human malignancies such as lung breast glioblastoma prostate colon and pancreatic cancers Muhammad It is certain that T hemsleyanum flavonoids give a new vision for researchers to explore clinical anticancer drugs Polysaccharide Saccharide is another important active ingredient extracted from T hemsleyanum Shao Polysaccharide has great potential in clinical application because of its unique pharmacological activity However due to the complex structure of polysaccharide it is difficult and special to determine and synthesize their structures Guo Table The prescriptions and traditional uses of T hemsleyanum in China Prescriptions name Qingteng Fengshi Qufengshi Yaojiu Main composition Jiu Traditional use T hemsleyanum Parabarium chunianum Tsiang Zanthoxylum nitidum Roxb DC T hemsleyanum Deeringia amaranthoides Lam Merr Blumea aromatica Wall DC T hemsleyanum Deeringia amaranthoides Lam Merr Zanthoxylum nitidum Roxb DC Panax notoginseng Burk FH Chen T hemsleyanum Gypsum Lonicera japonica Thunb Houttuynia cordata Thunb Ophiopogon japonicus Linn f KerGawl T hemsleyanum T hemsleyanum Lysimachia christinae Hance Imperata cylindrica Citrus reticulata Blanco T hemsleyanum ginsenoside Astragalus propinquus Schischkin T hemsleyanum Nepeta cataria L Lonicera japonica Thunb Saposhnikovia divaricata Trucz Schischk Huatuo Fengtongbao capsule Sanyeqing Gypsum Decoction Sanyeqing Power Zhonggan mixture Jinqi Tablet Hua Shi Xuan Fei mixture extracted the polysaccharides from roots of T hemsleyanum RTP1 RTP2 and RTP3 were successively found by protein precipitation and purification Moreover further study indicated RTP31 was high purity polysaccharide with a molecular weight of kDa and it is mainly composed of kinds of monosaccharides arabinose galacturonic acid galactose and fructose the proportion is and respectively Ru 2018ab extracted a polysaccharide THP from T hemsleyanum with the average molecular weight estimated as kDa The results of study on the composition of polysaccharide showed that it was mainly composed of rhamnose arabinose mannose glucose galactose with the molar ratio of In Ru 2019ab successfully extracted polysaccharide THDP3 from T hemsleyanum with molecular weight of kDa which consists of rhamnose arabinose mannose glucose and galactose with molar ratio of Moreover TDGP3 mainly consists of †’4αDGalAp1†’ †’4DGalp1†’ and †’4αDGlcp1†’ residues as backbones and DManp1†’ †’36DManp1†’ and αDAraf1†’residues as branches Phenolic acids Phenolic acids refer to aromatic carboxylic acids with multiple phenolic groups substituted on one benzene ring As a secondary metabolite phenolic acids are widely found in many natural plants and have antiinflammatory antioxidant and lipid lowering effects Twenty three phenolic acids No52“ Table have been reported in the aerial parts of T hemsleyanum such as caffeic acid chlorogenic acid 1OgalloylDglucose protocatechol glucoside epigallocatechin 1caffeoylquinic acid 3caffeoylquinic acid 4caffeoylquinic acid 5caffeoylquinic acid 1pcoumaroylquinic acid 4pcoumaroylquinic acid and 5pcoumaroylquinic acid There were twentyone phenolic acids in the root tuber of T hemsleyanum some of which were the same as aerial parts Alkaloids Alkaloids are a group of basic anic compounds containing nitrogen that exist in nature Alkaloids are stored in small quantities in T hemsleyanum and the bioactivity investigations of those alkaloids are still rather rare Wang Fu extracted the aerial parts of T hemsleyanum with ethanol and then isolated ten alkaloids for the first time including seven indole alkaloids an amide a maleimide and Treatment of joint pain wind cold dampness arthralgia Treatment of arthralgia syndrome rheumarthritis rheumatoid arthritis scapulohumeral periarthritis Treatment of arthralgia syndrome rheumarthritis rheumatoid arthritis scapulohumeral periarthritis joint pain muscular constricture Treatment of infantile hyperpyretic convulsion Treatment of blood avalanche leucorrhea Treatment of liver cancer Treatment of malignant tumor Treatment of Covid19 Usage Oral administration “ mL once times a day Oral administration mL once times a day Oral administration capsules once times a day References Ministerial standard Ministerial standard Ministerial standard One dose a day decoct twice in water and take it “ times after mixing Oral administration Oral administration mL once times a day Oral administration capsules once times a day Oral administration mL once times a day Xu Gao Jiang and Gong Wei Zhejiang Provincial Drug Administration JournalofEthnopharmacology26420211132474 0cT Ji Detection Mode Analysis parts of sample Reference aerial part root tuber aerial part root tuber aerial part root tuber root tuber aerial part root tuber root tuber root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber root tuber root tuber aerial part root tuber root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber aerial part root tuber root tuber aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part root tuber aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber aerial part aerial part aerial part aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber Sun Sun Zeng Sun Sun Sun Zeng Zeng Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Zeng Sun Zeng Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Xu 2014b Sun Zeng Sun Zeng Sun Zeng Zeng Sun Sun Sun Sun Xu 2014b Sun Xu 2014b Sun Zeng Sun Xu 2014b Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Fu Sun Sun Xu 2014b Fan Xu 2014b Fan Sun continued on next page UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS Table Chemical constituents isolated from the different parts of T hemsleyanum Name Flavonoids and their glycosides quercetin quercitrin quercetin3Oglucoside quercetin3Orutinoside quercetin3galactoside quercetin3Oxylosylglucoside quercetin3Oxylosylglucose7Orhamnoside orientin orientin2²²Orhamnoside Isoorientin isoorientin2²²Orhamnoside isoorientin cid0 ²²Oxyloside vitexin vitexin2²²Orhamnoside vitexin2²²Oglucoside vitexin2²²Oarabinoside isovitexin isovitexin2²²Orhamnoside isovitexin2²²Oxyloside isorhamnetin isorhamnetin3rutinoside isorhamnetin3pyranoarabinose7glucosylrhamnoside apigenin apigenin7rhamnoside apigenin8Cxylosyl6Cglucoside apigenin6CαLarabinose8CDglucose eriodictyol eriodictyolOhexoside I eriodictyolOhexoside II luteolin luteolin6 8diChexoside catechin catechin glucopyranoside isomer epicatechin kaempferide kaempferol kaempferol3glucoside kaempferol3rutinoside kaempferol3sambubioside kaempferol3Oneohesperidin kaempferol3Orhamnoside kaempferol7Orhamnose3Oglucoside kaempferol3robinoside7rhamnoside kaempferol3rutinoside kaempferol3Ocarfuran7Orhamnosyl glucoside daidzein biochanin A procyanidin dimmer procyanidin B1 procyanidin B2 procyanidin trimer Phenolic acids and derivatives gallic acid protocatechuic acid caffeic acid dihydroxybenzoic acid hexoside 1caffeoylquinic acid 3caffeoylquinic acid 4caffeoylquinic acid 5caffeoylquinic acid 1pcoumaroylquinic acid 4pcoumaroylquinic acid 5pcoumaroylquinic acid phydroxybenzaldehyde pcoumaric acid ferulic acid hexoside salicylic acid chlorogenic acid neochlorogenic acid cryptochlorogenic acid protocatechualdehyde UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS 1HNMR13CNMR MS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS JournalofEthnopharmacology26420211132475 0cT Ji Table continued Name salicin2benzoate trihydroxycinnamoylquinic acid isomer protocatechuic acid hexoside apiosylglucosyl 4hydroxybenzoate 1OgalloylDglucose protocatechol glucoside epigallocatechin vanillic acid1Ofuran celery glucosyl ester protocatechuic acid1Ofuran celery glucosyl ester methoxyphenol1Ofuran glycosylOglucoside 2methoxy4methylbenzene1ofuracresyl glucoside oxyresveratrol dicaffeoylquinic acid 4hydroxycinnamic acid Alkaloids indole indole3carboxylic acid indole3propanoic acid 5hydroxyindole3carboxaldehyde 5hydroxyindole3carboxylic acid 6hydroxy3 4dihydro1oxocarboline hippophamide 4hydroxycinnamide pyrrole3propanoic acid Scid0 trolline Fatty acids trihydroxy octadecadienoic acid trihydroxy octadecenoic acid dihydroxy octadecenoic acid 9hydroxy1012octadecadienoic acid 9hydroxy octadecatrienoic acid hydroxyoctadecenoic acid hydroxyoctadecatrienoic acid Dihydroxyoctadecatrienoic acid dihydroartemisinin ethyl ether Trihydroxy octadecadienoic acid isomer hydroxyoxooctadecatrienoic acid octadecenedioic acid diMeester stearic acid linolenic acid linoleic acid palmitic acid oleic acid anic acids and derivatives malic acid quinic acid citric acid azelaic acid oxalic acid galactonic acid gallic acid succinic acid fumaric acid propanoic acid Terpenoids and steroids sitosterol daucosterol campesterol Stigmasterol 6Obenzoyl daucosterol ergosterol taraxerone Taraxerol αamyrine pteroside Z ganoderic acid H 3epipapyriferic acid oleanic acid Saponins Ginsenoside Rh1 Detection Mode UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS 1HNMR LCMS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS GCMS TCL HNMR CNMR MS GCMS GCMS IR HNMR EIMS IR HNMR MS IR HNMR MS IR HNMR MS IR EIMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS HNMR CNMR MS Analysis parts of sample root tuber root tuber root tuber root tuber aerial part aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber root tuber root tuber aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts root tuber aerial part root tuber aerial part root tuber root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber aerial part aerial part aerial part aerial part root tuber root tuber root tuber root tuber Reference Sun Sun Sun Sun Sun Sun Zeng Sun Xu 2014b Zeng Zeng Zeng Zeng Xu 2014b Xu 2014b Chen Fu Fu Fu Fu Fu Fu Fu Fu Fu Fu Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Chen Ding Sun Sun Guo Ru Ru Ru Ru Sun Sun Sun Ding UPLCESIQTOFMSMS root tuber Sun continued on next page JournalofEthnopharmacology26420211132476 0cT Ji Table continued Name Ginsenoside Rh2 Vinaginsenoside R1 Amino acid and derivatives Phenylalanine pyroglutamic acid glutimic acid hexose Tryptophan Lglutamic acid Detection Mode UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS Analysis parts of sample root tuber root tuber root tuber aerial part aerial part aerial part aerial part aerial part Reference Sun Sun Sun Sun Sun Sun Sun respectively a carboline By comparing with the spectral data of known compounds the alkaloids were indole3carboxylic acid indole3propanoic acid 5hydroxyindole3carboxaldehyde 5hydroxyindole3carboxylic acid 6hydroxy3 4dihydro1oxocarboline hippophamide 4hydroxycinnamide pyrrole3propanoic acid and Scid0 trolline The chemical structures were shown in Fig identified as indole a

Thyroid_Cancer

Molecular Medicine The role of selenium metabolism andselenoproteins in cartilage homeostasisand arthropathiesDonghyun Kang Jeeyeon Lee Cuiyan Wu3 Xiong Guo3 Byeong Jae Lee24 JangSoo Chun5 andJinHong Kim AbstractAs an essential nutrient and trace element selenium is required for living anisms and its beneficial roles in humanhealth have been well recognized The role of selenium is mainly played through selenoproteins synthesized by theselenium metabolic system Selenoproteins have a wide range of cellular functions including regulation of seleniumtransport thyroid hormones immunity and redox homeostasis Selenium deficiency contributes to various diseasessuch as cardiovascular disease cancer liver disease and arthropathy”Kashin“Beck disease KBD and osteoarthritisOA A skeletal developmental disorder KBD has been reported in lowselenium areas of China North Korea and theSiberian region of Russia and can be alleviated by selenium supplementation OA the most common form of arthritisis a degenerative disease caused by an imbalance in matrix metabolism and is characterized by cartilage destructionOxidative stress serves as a major cause of the initiation of OA pathogenesis Selenium deficiency and dysregulation ofselenoproteins are associated with impairments to redox homeostasis in cartilage We review the recently exploredroles of selenium metabolism and selenoproteins in cartilage with an emphasis on two arthropathies KBD and OAMoreover we discuss the potential of therapeutic strategies targeting the biological functions of selenium andselenoproteins for OA treatmentIntroductionSelenium Se is an essential trace element in humans12Selenium is generally taken up from the diet through foodor other forms of external supplementation Dietaryselenium is obtained in the form of selenomethionineSeMet selenocysteine Sec selenite and selenate Significant health benefits have been attributed to seleniummetabolic systems that play major physiological roles inthyroid hormone metabolism immunity and antioxidantdefense23 Selenium is required for the production ofthyroid hormonemetabolizing enzymes and seleniumCorrespondence JinHong Kim jinhkimsnuackr1Center for RNA Research Institute for Basic Science Seoul South Korea2Department of Biological Sciences College of Natural Sciences Seoul NationalUniversity Seoul South KoreaFull list of author information is available at the end of the These authors contributed equally Donghyun Kang Jeeyeon Leesupplementation is thought to improve the function ofthyrocytes and immune cells4 Selenium supplementationdemonstrated immunostimulant effects such as enhancedproliferation of activated T cells activation of naturalkiller cells and tumor cytotoxicity mediated by cytotoxiclymphocytes56 In contrast selenium deficiency is associated with the occurrence virulence and disease progression of viral infections7Selenium inadequacy can lead to various types ofdiseases most notably cardiovascular disease8“ cancer13“ hepatopathy1617 and arthropathy Cardiovascular diseases are associated with systemic seleniumlevel with a higher risk at or μgL seleniumconcentration in the blood10 A type of endemic cardiomyopathy Keshan disease is linked to selenium deficiency811 Keshan disease occurs in lowselenium areasin Chinasodium seleniteand is prevented by The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate ifchanges were made The images or other third party material in this are included in the ™s Creative Commons license unless indicated otherwise in a credit line to the material Ifmaterial is not included in the ™s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this license visit httpcreativecommonslicensesby40Official journal of the Korean Society for Biochemistry and Molecular Biology 0cKang Experimental Molecular MedicinePage of studiesEpidemiologicalsupplementation12 Lowselenium status is correlatedwith a significantly increased risk of cancer incidenceand mortality13“haveprovided evidence on the cancerpreventing effects ofselenium18“ Selenium deficiency is also characterizedby elevated levels of oxidative stress markers in the liver21which significantly contribute to liver injury17 The oxidative stress caused by selenium deficiency further plays adetrimental role in joint development Selenium deficiency is the main cause of endemic Kashin“Beck diseaseKBD which is mainly reported in lowselenium areas ofChina North Korea and the Siberian region of RussiaMoreover there is a growing body of evidence suggestingthat the pathogenesis of osteoarthritis OA the mostcommon form of arthritis may be associated with selenium deficiency by resulting in oxidative stress22“However it is noteworthy that excessive selenium intakecan also cause selenosis2930 which accompanies adversesymptoms including fatigue diarrhea nausea increasedheart rate necrosis in liver and kidney and neurologicaldamage Chroniccompromisesimmune and reproductive systems in humanseventuallyselenosisOA is characterized by progressive loss of cartilageextracellular matrix ECM and pathological changes inother joint tissues such as subchondral bone sclerosisosteophyte formation and synovial ‚ammation31 Cartilage destruction is considered a hallmark of OA and is aresult of increased production of catabolic effectors32“and reduced matrix biosynthesis by chondrocytes36 OA isassociated with multiple etiologies involving systemicfactors such as age37 as well as local factors such asmechanical stress38 driven by weightbearing and jointinstability Both OAcausing factors have been found tocause oxidative stress in chondrocytes Oxidative stressresults from the abnormal production of reactive oxygenspecies ROS and the loss of cellular antioxidant capacityMany preclinical and clinical studies have indicated theaccumulation of oxidative burden in chondrocytesundergoing osteoarthritic changes3940 Emerging evidence suggests that oxidative stress is mechanisticallylinked to the initiation of osteoarthritic changes inchondrocytes through the acquisition of senescent phenotypes36 Therefore restoring redox homeostasis canserve as a rational therapeutic strategy to alleviate OAprogression Here we review the role of selenium metabolism in cartilage and bone and the significance ofmaintaining its homeostasis in the context of joint diseases such as KBD and OAOverview of the selenium metabolic systemThe selenium metabolic system and the biosynthesis ofselenoproteinsSelenium metabolism is a systemic process that includesandtransformationtransportationabsorptiontheOfficial journal of the Korean Society for Biochemistry and Molecular Biologyexcretion of selenium Fig Selenium is obtained inanic forms”SeMet and Sec”and inanic forms”selenite and selenate”from diet Selenium is taken up bythe liver that synthesizes and exports SELENOP whicheventually circulates through the bloodstream SELENOPwith multiple Sec residues41 transports selenium to othertissues and ans42 and the transported selenium isconverted to selenophosphate by intracellular seleniummetabolic pathways Selenium is excreted through exhalation and urine in the form of smallmolecule metabolites formed by sequential methylation4344Selenium plays biological roles predominantly in theform of selenoproteins synthesized by the seleniummetabolic system Ingested inanic selenium is firstreduced to hydrogen selenide H2Se via glutathioneGSH and thioredoxin TXN systems Selenide is furtherconverted to Sec amino acids for incorporation intospecific sites of selenoproteins such as the catalytic sites ofa selenoenzyme Mechanistically selenophosphate synthetase SEPHS2 catalyzes the production of selenophosphate through the reduction of hydrogen selenideThe subsequent reaction with phosphoseryltRNA PSertRNA[Ser]Sec yields SectRNA[Ser]Sec Sec amino acids areincorporated into polypeptidethrough themachinery utilizing the UGA codon Selenocysteineinsertion sequence binding protein SBP2 binds toselenocysteine insertion sequence SECIS element whichis located in the ²untranslated region ²UTR of selenoprotein mRNA and mediates the transfer of SectRNA[Ser]Sec to the Asite of ribosome which recognizesthe UGA codon as the Sec integration codon Collectivelythe selenoprotein translation machinery consists of SECISelement SBP2 Secspecific eukaryotic elongation factorEEFSEC and aminoacylated SectRNA[Ser]Sec therebyenabling UGA to be recognized as a Sec codon and utilized for translation into the growing polypeptidechainsSelenoproteinssome ofSelenoprotein is defined as a protein containing Secamino acid residue The biological functions of seleniumare mostly exerted through selenoprotein domains thatcontain Sec residues Twentyfive selenoprotein geneshave been identified in the human genome45 In mice atotal of selenoproteins have been characterized46 andtargeted deletion ofthese selenoproteinsdemonstrated their essential roles in developmental processes and in disease pathogenesis Selenoproteins can beclassified into subfamilies based on their cellular functionssuch as those implicated in antioxidation GPX1 GPX2GPX3 GPX4 redox regulation TXNRD1 TXNRD2TXNRD3 MSRB1 SELENOH SELENOM SELENOWthyroid hormone metabolism DIO1 DIO2 DIO3 selenium transport and storage SELENOP selenophosphatesynthesis SEPHS2 calcium metabolism SELENOK 0cKang Experimental Molecular MedicinePage of Fig Selenium metabolic system in mammals Selenium is absorbed from the diet undergoes several conversion steps and is incorporated intopolypeptide chains completing selenoprotein synthesis Dietary sources of selenium uptake exist in inanic form such as selenate and selenite andanic form such as Sec and SeMet Inanic forms are reduced by TXNRDTRX or GRXGSH systems and anic forms are cleaved by SCLYforming selenide Selenophosphate is synthesized from selenide by SEPHS2 and the subsequent reaction with PSertRNA[Ser]Sec mediated by SEPSECSyields SectRNA[Ser]Sec SectRNA[Ser]Sec is transferred to the Asite of ribosome mediated by SBP2 which binds to SECIS located in the ²UTR of aselenoprotein mRNA Finally the UGA codon is recognized as the Sec integration codon Abbreviations SeMet selenomethionine Secselenocysteine GRX glutathione reductase TRX thioredoxin TXNRD thioredoxin reductase GSH glutathione MGL methionine gammalyase SCLYselenocysteine lyase SEPHS2 selenophosphate synthetase SARS seryltRNA synthetase PSTK phosphoserylSeptRNA kinase SEPSECS SeptRNASectRNA synthase EEFSEC Secspecific eukaryotic elongation factor SBP2 SECIS binding protein SELENOT myogenesis SELENON protein foldingSELENOF SELENOI SELENOS and protein AMPylation SELENOO4748 The functions of other selenoproteins such as GPX6 and SELENOV still remain unclearGlutathione peroxidases GPXs such as GPX1 cytosolicGPX GPX2 gastrointestinal GPX and GPX4 phospholipid hydroperoxide GPX catalyze the decompositionof a great variety of peroxides thus protecting cellsagainst oxidative damage4950 Thioredoxin reductasesTXNRDs employ NADPH as an electron donor to revertoxidized TXN to a reduced dithiol the oxidation status ofwhich is critically implicated in regulating various cellbehaviors including proliferation and apoptosis51 Thephysiological significance of TXNRDs is further supported by the embryonic lethality of Txnrd1 or Txnrd2knockout mice5253 Deiodinases DIOs regulate thyroidhormone metabolism by catalyzing the conversion ofthyroid hormones from precursor thyroxine T4 to biologically active triiodothyronine T3 or inactive reverseT3 rT354 The expression levels of several selenoproteinsOfficial journal of the Korean Society for Biochemistry and Molecular Biologyare ‚uenced by the extent of selenium uptake Forexample seleniumdeficient animals and human cell linesexhibit reduced transcription of selenoproteins such asGPX1 DIOs SELENOI and SELENOW55“ A subset ofselenoproteins such as GPX1 and SELENOW is moresensitive to selenium supplementation or deficiency Thehierarchy of selenoprotein expression is more apparentwhen the intracellular level of selenium is limited1Seleniumresponsive genesgenesareseleniumcontainingSeleniumresponsivethe genes whoseexpression patterns are ‚uenced by supplementationwith selenium orcompoundsTreatment of a cancer cell line with methylseleninic acidin genes58 Theseinduced expression changesresponsive genes were closely associated with annotationsrelated to cell cycle regulation androgenresponsive genesand phase II detoxification pathway Selenium supplementation of macrophages diminished the expression oflipopolysaccharide LPSinduced pro‚ammatory genes 0cKang Experimental Molecular MedicinePage of such as cyclooxygenase2 COX2 and tumor necrosisfactorα TNFα59 suggesting that selenium has anti‚ammatory effects on the immune system The CTDdatabase httpctdbase reports the effect of environmental chemicals including selenium on gene expression profiles in various human tissuesThe role of selenium and selenoproteins incartilage development and KBDSelenium levels and its role in joint tissuesJoints are composed of various types of connective tissues including cartilage bone synovium meniscus andligament Among these tissues cartilage is the maincomponent that absorbs mechanical stress cushioningbones from impacting each other during various weightbearing activities In the human knee joint the seleniumconcentration in cartilage is approximately μgkg dryweight whereas the selenium concentrations in ligamentand meniscus are and μgkg dry weight respectively6061 The requirement of adequate physiologicalselenium levels for maintaining cartilage homeostasis hasbeen recognized Selenium deficiency retards the growthand development of cartilage and bone62“ Growthretardation was observed in rats after two generations ofselenium deficiency62 Mice fed a diet deficient in selenium resulted in fibrocartilage formation at the articularsurface ultimately showing degeneration of articularcartilage63 Selenium deficiency induced the expression ofthe chondrocyte hypertrophy marker gene type X collagenCOLX in articular cartilage64 The expression of parathyroid hormonerelated protein PTHrP which controlschondrocyte maturation during endochondral ossification was enhanced in both articular cartilage andhypertrophic growth plate following selenium deficiencyThese changes were in line with the phenotypic changesobserved in the cartilage of KBD patients64 However itshould be noted that growth retardation caused by selenium deficiency may also be associated with the deregulation of bone metabolism65 In a study by Cao et alselenium deficiency severely compromised bone microarchitecture as a result of increased bone resorption66Abnormalities in selenium metabolism and skeletaldevelopment diseasesSelenium deficiency is regarded as one of the initiatingfactors of KBD which is an endemic osteoarthropathycaused by the premature closure of epiphyseal plate andthe impaired skeletal development Skeletal deformities inhands fingers knees and elbows and in severe casesdwarfism and movement disorders are the symptoms ofKBD22 The KBD area roughly coincides with lowselenium areas including a geological belt extendingfrom northeast to southwest China North Korea andeastern Siberia22 A metaanalysis showed that seleniumOfficial journal of the Korean Society for Biochemistry and Molecular Biologylevels in the water soil cereal and corn in KBD endemicregions were lower than they were in nonendemicregions supporting the fact that the level of selenium intissue is predominantly affected by dietary intake23 In linewith this finding selenium levels in the whole bloodserum hair and urine of KBD patients were markedlylower than those of healthy controls24Selenoprotein gene polymorphisms are associated withincreased susceptibility to KBD There were significantdifferences in the allelic frequency of GPX1 Pro198Leurs1050450 between the KBD and control group67 Inaddition the mRNA level of GPX1 and enzyme activity oftotal GPX in blood were lower in the KBD group thanthey were in the control group67 Haplotypes of TCCTTC and TTT of rs1050450 rs3811699 and rs1800668in GPX1 gene also had a significant link to KBD68 Asinglenucleotide polymorphism SNP in the promoterregion of SELENOS rs28665122 ˆ’105G A was relatedto the increased risk of KBD and upregulation of PI3KAktsignaling in patients with KBD69 In this study tertbutylhydroperoxide tBHPtreatmentinduced chondrocyteapoptosis was mitigated by selenium supplementation viasodium selenitetreatment which suppressed thePI3KAkt pathway The minor Aallele of SELENOFrs5859 was associated with a significantly higher incidenceof KBD70The animals fed a seleniumdeficient diet recapitulatedsome of the pathological manifestations of KBD stronglysupporting the notion that selenium deficiency is criticallyassociated with the development of this endemic arthropathy Selenium deficiency impaired bone and cartilagegrowth with the exhibition of premature chondrocytehypertrophy as evidenced by an increased expression ofCOLX compatible with the phenotypes in KBD cartilage64The lowselenium condition in combination with threemycotoxins deoxynivalenol DON nivalenol NIV and T yielded procatabolic changes and hypertrophic phenotype of chondrocytes as evidenced by the loss of aggrecanand type II collagen COLII and the increase in COLX andmatrix metalloproteinases MMPs expressionrespectively71 In contrast selenium supplementation partiallyalleviated these mycotoxininduced damages in chondrocytes71 In rats dietary selenium deficiency over twogenerations caused the onset of physiological seleniuminsufficiency72 In this condition pathological changes inthe epiphyseal plate were observed with the decreasedexpression of COLII and GPX1 in the chondrocytes suggesting a possible association of reduced chondrocyte anabolism and antioxidant capacity with the epiphyseal platelesions observed in KBD72 The relevance ofimpairedselenium metabolism to the onset of KBD was furthervalidated using a mouse genetic deletion model Targeteddeletion of SectRNA[Ser]Sec Trsp gene in osteochondroprogenitor cells from embryonic stage caused the 0cKang Experimental Molecular MedicinePage of depletion of selenoproteins in skeletal systems causinggrowth retardation abnormalities in the epiphyseal growthplate delayed endochondral ossificationand chondronecrosis which recapitulated the major pathologicalfeatures of KBD73As a prophylactic treatment selenium supplementationswere given to children living in a KBD area The supplemented group showed elevated physiological seleniumlevels in their hair samples and exhibited a substantiallylower prevalence of KBD74 A metaanalysis including fiverandomized controlled trials RCTs and ten prospectivenonRCTs statistically demonstrated the benefits of selenium supplementation in preventing KBD in children75Selenium metabolism and OAPhysiological significance of oxidative stress inchondrocytesOA is the most common form of arthritis and is primarilycharacterized by the loss of cartilagespecific ECM and otherpathological changes in joints including subchondral bonesclerosis osteophyte formation and synovial ‚ammation31Articular cartilage is composed of abundant proteoglycans inwhich sulfated glycosaminoglycan chains such as chondroitinsulfates are bound to a core protein such as aggrecan Loss ofcartilage matrix during OA progression is a combined resultof increased catabolic process in cartilage and reduced anabolic activity of chondrocytes The molecularlevel understanding of OA pathogenesis has led to the identification ofmajor catabolic enzymes ADAMTS576 MMP377 andMMP1378 which mediate the degradation of cartilagematrix Pro‚ammatory cytokines drive the expression ofthese catabolic factors in chondrocytes through the activationof transcription factors such as HIF2α32 and NFκB79Abnormalities in various metabolic pathways such as glucose80 or amino acid metabolic system81 in chondrocyteshave been implicated in activating catabolic cascades inosteoarthritic cartilage82 Moreover increased cellular uptakeof Zn2 through the upregulation of zinc transporter ZIP8activates metalregulatory transcription factor1 MTF1which in turn induces the expression of matrixdegradingenzymes in chondrocytes3383 Regulation of catabolism bythefurthershowed the association of metabolic abnormalities with thecatabolic process of OA34cholesterol“CH25H“CYP7B1“RORαaxisMeanwhile the upstream regulatory mechanism eliciting an imbalance in OA matrix homeostasis needs furtherinvestigation OAcausing factorssuch as age andmechanical stress lead to excessive oxidative stress inchondrocytes3738 Consistently clinical and preclinicalOA studies indicated a cumulative oxidative burden inosteoarthritic chondrocytes3940 Emerging evidence suggests that oxidative stress plays a significant role in OAdevelopment and the disease progression can be mitigatedby counteracting oxidative stress3684“In generalOfficial journal of the Korean Society for Biochemistry and Molecular Biologyoxidative stress results from the abnormal production ofROS and the loss of cellular antioxidant capacity Synovialfluid from patients with latestage OA who were undergoing knee joint replacement had a lower level of oxidoreductases than that from healthy controls87 In partthe increase in oxidative stress is attributable to mitochondrial dysfunction in OA chondrocytes8889 OAchondrocytes displayed reduced mitochondrial DNAcontent mitochondrial dysfunction and diminishedexpression of NRF2 which regulates the transcription ofoxidoreductase genes89 Similarly chondrocytes fromaged individuals exhibited increased ROS burden andmitochondrial and genomic DNA damage90“ Therefore the proper maintenance of redox homeostasis canpotentially serve as a rational therapeutic strategy toprotect against OA progressionPotential roles of selenium metabolism in OAThe protective effect of selenium in OA has beenexplored in a large number of epidemiological and geneticstudies Table The concentration of selenium in serumwas significantly lower in OA patients than that of normalcontrols25 Similarly the results from a populationbasedcohort study demonstrated the linkage between lowselenium levels in toenails with OAassociated pain anddisease severity2627 Several studies have indicated thatcartilage matrix homeostasis is impaired in seleniumdeficiency Lowselenium status diminished COLIIexpression level regulated by SOX9 which is known as amaster regulator required for maintaining cartilage matrixIn fact SOX9 was destabilized by thehomeostasisdownregulation ofseleniumresponsive PRMT5 thatsustains SOX9 stability via methylation93 In anotherstudy rats fed a seleniumdeficient diet exhibited lowsulfotransferase activity which resulted in diminishedforcontents ofmechanicalcartilagematrix28 In contrast selenium supplementation ameliorated the spontaneous degeneration of articular cartilagein STR1 N mice by increasing the expression of GPXs94In cultured chondrocytes pretreatment with SeMetmarkedly inhibited nitric oxide NO and prostaglandinE2 PGE2 production in response to pro‚ammatorycytokine IL1β95 Expression of SBP2 a factor recognizingSECIS element had a positive correlation with GPX1 andGPX4 expression and antioxidant capacity in chondrocytes96 Oxidation resistance mediated by SBP2 wasdiminished in response to IL1β treatment in vitro and indamaged regions of cartilage in OA patients96 Downregulation of selenoprotein mRNAs including GPX397GPX1 and GPX49698 and Selenop99 was observed inhuman and mouse OA chondrocytessulfated glycosaminoglycan essentialstressabsorbingpropertyofGenetic factors such as SNPs in selenoproteins wereidentified to be risk factors for OA development A GAG 0cKang Experimental Molecular MedicinePage of Table List of selenoproteins associated with the pathogenesis of arthropathies KBD and OAGeneGPX1GPX3GPX4DIO2DIO3SELENOFSELENOPSELENOSFunctionExpression in OASNPAntioxidantReduction of hydrogen peroxide and anic peroxidesDownregulatedPlasma antioxidantDetoxification of lipid hydroperoxidesMetabolism of lipidsActivation of hormonesDeiodination of T4 to T3Inactivation of hormonesConversion of T4 to rT3Protein foldingStorage and transport of SeAntioxidant propertiesProtein foldingERassociated protein degradationDownregulatedDownregulatedUpregulatedDownregulatedrs1050450 KBDrs3811699 KBDrs1800668 KBDrs225014 OArs12885300 OArs945006 OArs5859 KBDrs28665122 KBDRef“haplotype in SELENOS gene was significantly associatedwith increased levels of‚ammatory factors in OApatient plasma100 SNPs in DIO2 which converts precursor thyroid hormone T4 to its active form T3 were alsorelated to genetic susceptibility to OA developmentLevels of DIO2 mRNA and protein were markedly upregulated in OA cartilage101 A common DIO2 haplotypecomposed of the minor Callele of SNP rs225014 and thecommon Callele of SNP rs12885300 was significantlyassociated with advanced hip OA as indicated by a higherodds ratio101“ Locus rs225014 which confers risk toOA was associated with the differential methylation ofCpG located in the upstream region of DIO2 gene andwas correlated with upregulated DIO2 expression inOA104 Meanwhile DIO3 depletes the resources that canbe utilized for the production of active thyroid moleculesby catalyzing the conversion of T4 and T3 into inactivemetabolites The minor Gallele of the DIO3 variantrs945006 was associated with a protective effect againstOA development105However a few aspects regarding the relationshipbetween selenium and OA remain controversial Firstseveral studies indicate that there are no differences inselenium levels between OA and normal tissues Theselenium concentrations in synovial fluid and plasma of OA patients were not significantly different from thoseof healthy controls106 Similarly no significant difference in selenium concentration was noted between sixdogs with posttraumatic OA and six control dogs107Second the beneficial effect of selenium supplementationin alleviating OA symptoms has been debated The resultsfrom a controlled doubleblind trial of patientsOfficial journal of the Korean Society for Biochemistry and Molecular Biologyrevealed that the supplementation of a formulation containing selenium with vitamins A C and E SeACE didnot have any remarkable curative effect compared to aplacebo108 In a study with an independent cohort theprevalence of radiographic knee OA was not significantlyassociated with dietary selenium intake109Nonethelessit is apparent that selenium deficiencydysregulation of selenoproteins and genetic variations inselenoprotein genes serve as potential risk factors for OAThe vital role of selenium metabolism in maintainingcartilage homeostasis is expected considering its criticalinvolvementin regulating cellular processes such aschondrogenic differentiation of progenitor cells maintenance of redox homeostasis and DNA damage repair inchondrocytes which are covered in the next sectionIntracellular roles of selenium metabolism andselenoproteins in cartilageChondrogenic differentiation programs of progenitor cellsSelenium exerts various beneficial effects to promoteproliferation and differentiation of chondrogenic progenitorcells110111 Selenium supplementation stimulated the proliferation of ATDC5 chondrogenic cells even under serumdeprivation by inducing cyclin D1 expression110 Deficiencyof SELENOO interfered with the chondrogenic differentiation of ATDC5 cells by suppressing the expression ofchondrogenic genes SOX9 COLII and aggrecan anddecreasing the activity of alkaline phosphatase112 Knockdown of Gpx1 reduced the chondrogenic differentiation ofATDC5 cells by modulating intracellular GSHoxidizedGSH GSSG ratio113 Selenop was differentially upregulatedduring the chondrogenic differentiation of micromass 0cKang Experimental Molecular MedicinePage of culture of mesenchymal cells isolated from mouse limbbuds114 In line with the effects of selenium metabolism andselenoproteins in chondrogenic progenitor cells observedin vitro deficient uptake of selenium severely affectedchondrogenic differentiation of mesenchymal lineage cellsin mice64andOsteochondroprogenitorspecific deletion of Trsp genesignificantly impaired chondrogenic programs causingabnormalities in bone and cartilage development in mice73endochondralossificationthusAntioxidant defense and redox homeostasisfunction ofattributed to theThe protective effects of selenium on cartilage are primarilyantioxidantdefense115“ The metabolism and survival of chondrogenic progenitors and chondrocytes are greatly compromised by ROS including free radicals peroxides andsuperoxide anions118“ Recent studies strongly supportthe notion that mitochondrial dysfunction and oxidativestress are the main drivers of OA pathogenesis37Although ROS play essential roles in the maintenance ofbasal cellular activities such as chondrocyte proliferationand matrix remodeling in cartilage excessive oxidativestress causes detrimental events such as cellular senescence36121 dedifferentiation122 and apoptosis123 ROScause oxidative damage to various cellular componentsand disrupt the balance between ECM catabolism andanabolism119 ROS suppress mitochondrial oxidativephosphorylation and ATP production which are essentially required to sustain cartilage matrix synthesis124 Inaddition ROS induce matrix degeneration through theupregulation of matrixdegrading enzyme expressionwhile this effecttreatment123125 The detrimental effects of ROS on cartilagehomeostasis can be effectively alleviated by augmentingcellular antioxidant activity under stress conditions andseveral attempts have been made to treat OA by targetingthe regulators involved in oxidative stress production incartilage84“is abolished by antioxidantThe protective role of selenium metabolism is thoughtto be exerted through the neutralization of ROS viaantioxidant activities of selenoproteins including GPXsand TXNRDs Bone marrow stromal cells cultured inmedium supplemented with low selenite concentrationexhibited ROS accumulation along with the reducedexpression of GPXs TXNRDs and other seleniumindependentinmicronuclei generation which is an indication of chromosome damage126 Both GPX1 expression and activitywere substantially lower in mice fed a seleniumdeficientdiet than those in mice fed a normal dietleading todecreased trabecular number reduced femoral trabecularvolumetotal bone volume ratio and trabecular separation66 The rats exposed to a seleniumdeficient diet withT2 toxin showed increased lipid peroxidation level andoxidoreductaseenzymesresultingOfficial journal of the Korean Society for Biochemistry and Molecular Biologydecreased antioxidant GPX activity in their serum andcartilage127 A seleniumdeficient dietinduced theexpression of miR1385p which in turn suppressed theexpression of SELENOM that has antioxidant functionand caused mitochondrial dysfunction and apoptosis ofchondrocytes128 Lead Pbinduced oxidative stress andtoxicity reduced the expression of selenoprotein mRNAsand the effect was mitigated by selenium supplementation129 In summary the antioxidant properties of selenoproteins showed therapeutic potential by counteractingthe accumulation of damage induced by oxidative stress incartilageDNA damage repairIt is well known that DNA damage pathways play substantial roles in the progression of arthropathies119 Theexpression of genes related to DNA damage was changedin the cartilage of KBD patients130131 Chronic DNAdamage induces the initiation of apoptosis or cellularsenescence in chondrocytes36132133 Selenium has apotential to reduce DNA damage and increase DNArepair capacity134 In part the beneficial effect of seleniumon genomic stability is associated with the antioxidationeffect of selenoproteins such as GPXs and TXNRDswhich remove ROS before they cause DNA damage134Cancer cells supplemented with selenium nM sodiumselenite or μM SeMet showed elevated levels of GPX1and TXNRD1 enzyme activity effectively protectingagainst DNA strand breaks induced by ultraviolet A orH2O2induced oxidative stress135 SeMet reduced theextent of DNA damage and enhanced DNA repair capacity by inducing repair complex formation in DNAdamaged cells through U

Thyroid_Cancer

"unrestricted use distribution and reproduction in any medium provided the original work is properly citedPurpose The present study was aimed at determining the serum levels of actinin4 ACTN4 in cervical cancer CC andinvestigating the diagnostic and prognostic value of serum ACTN4 in CC Materials and Methods We included CC patients cervical intraepithelial neoplasia CIN patients and healthy women Serum ACTN4 levels were assessed using an ELISAmethod A receiver operating characteristic ROC curve was performed to evaluate the diagnostic value of serum ACTN4 Thesurvival curves were used to display the overall survival distributions Results Serum ACTN4 levels in CC patients were ± pgmL which is significantly higher than those in CIN patients ± pgmL P and those in healthycontrols ± pgmL P The ROC analysis demonstrated that the area under the curve AUC of ACTN4 was 95CI “ with sensitivity of and specificity of Serum ACTN4 levels were associated with theFIGO stage lymph node metastasis and lymphovascular space invasion of CC all P The survival curve suggested thathigh serum ACTN4 levels were related to poor prognosis Conclusion Our findings suggest that serum ACTN4 levels may bevaluable diagnostic and prognostic biomarkers for CC IntroductionCervical cancer CC is the second most common femalemalignancy globally and it is the most common femalemalignancy in developing countries which has high morbidity and mortality rates [] In recent years the incidence ofCC has increased greatly in young women under the age of [] Despite great advances in surgical and adjuvant therapy the overall survival of CC patients especially that ofadvanced patients is still very poor [] At present a Papsmear combined with an HPV test has been used for the earlyscreening of cervicalthe screeningmethods are invasive and costly leading to lower screeningcoverage in China [] Previous studies have reported thatthe human papillomavirus HPV screening results have arelatively high falsepositive rate and a relatively low specificity [ ] In addition the results of TCT interpretation byfilmreading doctors are uneven which might cause somelesions Howevermisleadingness in the choices of prevention measures andtreatment for CC [] Noteworthily when applying the sametreatment plan to patients with similar pathological types theefficacy and prognosis are quite diï¬erent Therefore it is necessary to identify new biomarkers directly related to the progression and prognosis of CCAlphaactinins ACTNs are actinbinding proteins inthe spectrin gene superfamily [] which are known to becrosslinked with filamentous actin Factin to maintainthe integrity of cytoskeleton and to control cell motility []The ACTN family has four members numbered ACTN1“which are present in humans and other mammals [“]ACTN4 is encoded by the ACTN4 gene and is widelyexpressed in many tissues especially in glomerular podocytes[] ACTN4 has an actinbinding domain at the Nterminus and ACTN4 monomers can form a homodimer throughreverse binding forming a dumbbellshaped structure []As an actinbinding protein ACTN4 is closely related to 0cDisease Markersenhancing cell viability and tumor invasion and metastasis[] Recent researches have reported that the expression ofACTN4 is significantly elevated in multiple cancers including breast cancer [] pancreatic cancer [] ovarian cancer[] and lung cancer [] In addition the ACTN4 levels aremarkedly associated with the poor prognosis of lung cancer[ ] thyroid cancer [] and salivary gland carcinoma[] An [] have found that the expression level ofACTN4 in human cervical tumors is dramatically higherthan that in normal cervical tissues Their finding demonstratedepithelialtomesenchymal transition and tumorigenesis by regulatingSnail expression and the Akt pathway in CC [] Thereforethe expression of ACTN4 in cervical tissues may be used inthe clinical diagnosis and prognosis prediction of CCthat ACTN4promotestheHowever up to now the significance of the serumACTN4 levels in CC has not been evaluated Hence in thecurrent study the serum levels of ACTN4 in patients withCC were measured In addition we estimated the potentialdiagnostic and prognostic value of serum ACTN4 expressionin CC Materials and Methods Study Population A retrospective study was designed toevaluate serum actinin4 as a biomarker for CC Between July and June newly diagnosed female CC patientsand newly diagnosed female cervical intraepithelial neoplasia CIN patients who received treatment at Huai™anMaternal and Child Health Care Hospital Huai™an JiangsuChina were recruited The diagnoses of all patients were verified by the histopathological examination The patients withother types of tumor or autoimmune atherosclerotic andhematologic diseases were excluded The mean age of CCpatients was years with a range of years Meanwhile healthy women with no evidence of neoplasmsand other serious diseases were enrolled from the physicalexamination center in the same hospital There was no significant diï¬erence in age among the CC CIN and healthy control groups This study was consistent with the Helsinkideclaration and was authorized by the Ethics Committee ofHuai™an Maternal and Child Health Care Hospital approvalnumber H20130504 All participantssigned writteninformed consent Clinicopathologic Feature Collection and FollowUp Byreviewing the medical records we collected the clinicopathologic characteristics of the patients including age at diagnosis pathological type FIGO stage tumor diï¬erentiationpelvic lymph node metastasis tumor size and lymphovascular space invasion The CC patients were classified based onthe revised FIGO staging system for CC in The tumorsize was the maximum tumor diameter determined by agynecologic oncologist during pelvic examination Thepatients in stage 1A1 received hysterectomy the patients instages IB1 and IIB received radical hysterectomy and pelviclymph node dissection the patients with ‰¥stage IIB receivedradiotherapy or radiotherapy combined with chemotherapyA regular telephone followup was conducted after treatmentto obtain the overall survival OS time of CC patients andthe OS was defined as the time from diagnosis to death orthe last followup The followup was in accordance withthe FIGO guidelines Blood Sample Collection and Detection of Serum Actinin and SCCA A mL peripheral blood sample from eachpatient was collected before receiving any treatment Afterstanding at room temperature for minutes the blood samples were centrifugated at gmin for min and then°the supernatant was stored at ˆ’C until further usageThe serum actinin4 concentration was measured by a quantitativeELISAmethod Uscn Life Science Inc Wuhan China The levelsof SCCA in serum were determined using an ELISA kitRD Systems Minneapolis MN The detection of all samples was strictly in accordance with the instructions providedby the manufacturer and was performed in duplicatesenzymelinked immunosorbentassay Statistical Analysis All statistical analyses were conducted by using SPSS and GraphPad Prism The continuous data following normal distribution were expressed asthe mean ± standard deviation°SDž A ttest was used tocompare serum ACTN4 levels between the two subgroupsof each clinicopathological parameters and the serumACTN4 levels of CC patients CIN patients and healthy controls were compared by the SNKq test Receiver operatingcharacteristic ROC curves were performed to assess thediagnostic value of serum ACTN4 levels for diï¬erentiatingCC patients from CIN patients and healthy controls TheKaplanMeier method and logrank test were used to plotsurvival curves The Cox proportional hazards models in univariate and multivariate analyses were used for evaluating theprognostic value of serum ACTN4 expression A twotailed Pvalue was considered to be statistically significant Results Serum ACTN4 Levels Are Higher in Patients with CCSerum concentrations of ACTN4 were detected to rangefrom to pgmL with a mean ±SD of ± pgmL for CC patientsto range from to ngmL with a mean ±SD of ± pgmL forCIN patients and to range from to ngmL witha mean ±SD of ± pgmL for healthy controlsSerum ACTN4 levels in CC patients were significantly higherthan those in CIN patients and healthy controls P However no significant diï¬erence in serum ACTN4 wasfound between CIN patientscontrolsP as shown in Figure and healthy The Diagnostic Value of Serum ACTN4 Levels for CC Wenext used ROC curve analysis to estimate the diagnostic valueof serum ACTN4 expression for CC The ROC curve showedthat the serum levels of ACTN4 were robust for discriminating CC patients from benign and healthy control subjectswith an area under the curve AUC value of 95CI “ as demonstrated in Figure index we usedAccordingto maximum Youden™s 0cDisease MarkersŽŽlymph node metastasis were the independent prognostic factors for CC all P Table Lmgp NTCAnsCCCINCON DiscussionCervical cancer is a heterogeneous disease with complicatedetiology Genetic and environmental factors play a crucialrole in the pathogenesis of CC [] Although the diagnosisand prognosis of CC have improved greatly over the pastfew decades it is necessary to improve early detection andscreening methods to determine additional promising circulating biomarkers for better patient selection and more personalized treatments [] As far as we know this studyrepresented the first eï¬ort to evaluate the serum expressionof ACTN4 as a new biomarker for CCAs an actinbinding protein ACTN4 can participate inregulating cell migration invasion and metastasis via regulating the actin filament flexibility at the leading edge ofinvading cancer cells [ ] ACTN4overexpressing cancercells have the potential to metastasize because the overexpression of ACTN4 protein in cancer cells can stimulate thedynamic reconstruction of the actin cytoskeleton [] Upto now numerous studies have reported the associationbetween ACTN4 and multiple cancers Okamoto []observed that ACTN4 is expressed in smallcell lung cancerNSCLC and it had a significant correlation with invasionand distant metastasis Additionally ACTN4 was reportedto be a potential predictive biomarker for the efficacy of adjuvant chemotherapy in patients with NSCLC [] Watabe [] revealed that the copy number increase of ACTN4is a novel indicator for poor overall survival of patients withsalivary gland carcinoma and the copy number variationwould aï¬ect the expression of protein A recent study demonstrated that serum ACTN4 levels were dramatically elevated in patients with breast cancer when compared tohealthy controls and serum ACTN4 may be an eï¬ective clinical indicator for diagnosing or predicting the clinical outcomes of breast cancer patients [] In addition ACTN4was proven to be associated with the pathogenesis of CCAn [] proposed a novel mechanism for epithelialtomesenchymal transition and tumorigenesis in CC whichcould be induced by ACTN4 through regulating Snail expression and βcatenin stabilization Hence it is significant toinvestigate the role of serum ACTN4 in CCIn the current study we observed that serum levels ofACTN4 in CC patients were statistically higher than thosein CIN patients and those in healthy controls Howeverserum ACTN4 levels were not significantly diï¬erent betweenthe CIN group and the control group It was shown thatserum ACTN4 expression could strongly diï¬erentiate CCpatients from CIN patients and healthy controls The ROCanalysis demonstrated that the AUC of ACTN4 was and at the optimal cutoï¬ of pgmL the sensitivity andspecificity were respectively and suggestingthat serum ACTN4 might be a potential diagnostic biomarker for CC In a recent study which included Chinesewomen Hu [] reported that the sensitivity and specificity of HPV screening in the diagnosis of CC were and The sensitivity of the HPV test was a litter higherFigure The serum ACTN4 levels in CC patients CIN patientsand healthy controls ˆ—P pgmL as the cutoï¬ value and the sensitivity and specificity were and respectively Association between Serum ACTN4 Levels andClinicopathological Parameters of CC Patients We furtherinvestigated the correlations between serum levels of ACTN4and clinical pathological data of CC patients and theresults are demonstrated in Table We observed that serumACTN4 levels were related to the FIGO stage lymph nodemetastasis and lymphovascular space invasion all P Nevertheless no significant association was found betweenserum ACTN4 levels and age pathological type diï¬erentiation degree and tumor size in CC patients all P Survival Analysis of Serum ACTN4 Levels in CC Duringthe followup period nine CC patients were lost and thefollowed up rate is Finally the prognostic value ofserum ACTN4 was assessed in patients The patients werefollowed up to December The range of followup timewas to months with the median time of months andmean time of months According to the median serumlevels of ACTN4 in CC patients pgmL the CCpatients were divided into the high ACTN4 level group pgmL N and low ACTN4 level group‰¥ pgmL N The estimated 5year OS of patientswith high serum ACTN4 levels and low serum ACTN4 levelswere and respectively The KaplanMeier survival curve and logrank test indicated that CC patients withhigh serum ACTN4 levels had a worse prognosis than thosewith low serum ACTN4 levels P Figure Univariate Cox regression analyses showed that theserum ACTN4 levels P FIGO stage P diï¬erentiation degree P lymph node metastasisP and lymphovascular space invasion P had significant prognostic value for OS Multivariate analysiswas further performed to evaluate the prognostic value ofserum ACTN4 as an independent factor for CC All the statistically significant factors from univariate analyses wereincluded and the results indicated that the FIGO stage and 0cDisease MarkersytivitisneS ˆ’ specificityFigure ROC curve analysis assessed the diagnostic performance of serum ACTN4 in CC The AUC was P Table Serum ACTN4 levels in CC patients according toclinicopathological parametersParametersAge years‰¤Pathological typeSquamous cell carcinomaAdenocarcinomaFIGO stageIA1IB1‰¥IB2Diï¬erentiationWell and moderatelydiï¬erentiatedPoorly diï¬erentiatedLymph node involvementNegativePositiveTumor size‰¤Lymphovascular space invasionNegativePositiveN ACTN4pgmLP ± ± ± ± ± ± ± ± ± ± ± ± ± ± than that of serum ACTN4 detection though the specificityof serum ACTN4 detection was well above that of the HPVtest Hence comparing with the HPV test in diagnosingCC detecting serum ACTN4 has some advantages Furthermore serum ACTN4 levels have been indicated to be a greatbiomarker for diagnosing multiple cancers Fang [] intheir study reported that serum ACTN4 was a promisingindicator for diagnosing breast cancer with the AUC of Wang [] used ACTN4 expression in peripheralblood to diï¬erentiate NSCLC patients from healthy individuals in two groups of participants and they obtained bothsatisfactory eï¬ects Furthermore we investigated the correlation between serum ACTN4 and clinical characteristics ofCC patients The serum ACTN4 levels were significantlyassociated with the FIGO stage lymph node metastasis andlymphovascular space invasion of CC which suggests thatACTN4 could contribute to the development invasion andmetastasis of CC In addition our results indicated that highACTN4 levels were associated with the poor survival of CCpatients In the multivariate analysis although ACTN4 levelsdid not reach the statistical significance it still seems to beable to influence the OSHowever several limitations in the present study should betaken into consideration First the sample size was relativelysmall which was likely to reduce the statistical power of ourresults Second we only explored the relationship betweenserum ACTN4 and OS and other prognostic indicators werenot examined due to the incomplete data which needs to beimproved in the future Third this study was a primary studyto determine the clinical significance of serum ACTN4 levelsfor the diagnosis and prognosis of CC but the specific molecular mechanisms remain unclear Hence further experimentsshould be conducted to elucidate the mechanismsIn conclusion our study showed that serum ACTN4levels were increased in CC patients and were related to the 0cDisease Markers lavivrus muCLog rank P Overall survival monthsLow ACTN4 groupHigh ACTN4 groupFigure KaplanMeier curve compared OS of CC patients with high serum ACTN4 levels versus those with low serum ACTN4 levelsTable Univariate and multivariate Cox regression analysis of OS in CC patientsUnivariate CIVariablesAge vs ‰¤ yearsPathological type squamous cell carcinoma vs adenocarcinomaFIGO stage ‰¥IB2 vs IA1IB1Diï¬erentiation poorly diï¬erentiated vs well and moderately diï¬erentiated “Lymph node involvement positive vs negativeTumor size vs ‰¤ cmLymphovascular space invasion positive vs negativeSerum ACTN4 levels high vs low levelsHR “ “ “ “ “ “ “ “ “ “ “ “P”””Multivariate CIPHRFIGO stage lymph node metastasis and lymphovascularspace invasion of CC patients In addition serum levels ofACTN4 have great diagnostic and prognostic value in CCNevertheless further studies with a larger sample size shouldbe carried out to confirm our resultsAcknowledgmentsWe thank all the patients and blood donors who participatedin our study This study was funded by grants from the Science and Technology Project of Traditional Chinese Medicine Bureau of Jiangsu province China YB2015128Data AvailabilityReferencesThe datasets used andor analyzed during the present studyare available from the corresponding author on reasonablerequestConflicts of InterestAll authors declare that they have no conflicts of interestAuthors™ ContributionsXigui Ma and Huiying Xue contributed equally to this workand should be considered as cofirst authors[] M H Forouzanfar K J Foreman A M Delossantos et alœBreast and cervical cancer in countries between and a systematic analysis The Lancet vol no pp “ [] E Pelkofski J Stine N A Wages P A Gehrig K H Kimand L A Cantrell œCervical cancer in women aged yearsand younger Clinical Therapeutics vol no pp “ [] Y Zhou W Wang R Wei œSerum bradykinin levels as adiagnostic marker in cervical cancer with a potential mechanism to promote VEGF expression via BDKRB2 International Journal of Oncology vol pp “ [] Y J Hu H P Zhang B Zhu H Y Chen L H Ma andY Wang œThe role of FH detection combined with HPV 0cDisease Markers[] N Miura M Kamita T Kakuya œEfficacy of adjuvantchemotherapy for nonsmall cell lung cancer assessed by metastatic potential associated with ACTN4 Oncotarget vol no pp “ [] N Tanaka T Yamashita S Yamamoto œHistologicalgrowth pattern of and alphaactinin4 expression in thyroidcancer Anticancer Research vol no pp “[] Y Watabe T Mori S Yoshimoto œCopy numberincrease of ACTN4 is a prognostic indicator in salivary glandcarcinoma Cancer Medicine vol no pp “ [] HT An S Yoo and J Ko œÎ±Actinin4 induces theepithelialtomesenchymal transition and tumorigenesis viaregulation of Snail expression and βcatenin stabilization incervical cancer Oncogene vol no pp “[] F Niu T Wang J Li œThe impact of genetic variants inIL1R2 on cervical cancer risk among Uygur females fromChina a casecontrol study Molecular Genetics GenomicMedicine vol no article e00516 [] W Li Y Zhao L Ren and X Wu œSerum human kallikrein represents a new marker for cervical cancer Medical Oncology vol no p [] H Shao J HC Wang M R Pollak and A Wells œÎ±Actinin4 is essential for maintaining the spreading motility andcontractility of fibroblasts PLoS One vol no articlee13921 [] K Honda T Yamada Y Hayashida œActinin4 increasescell motility and promotes lymph node metastasis of colorectalcancer Gastroenterology vol no pp “ [] D G Thomas and D N Robinson œThe fifth sense mechanosensory regulation of alphaactinin4 and its relevance forcancer metastasis Seminars in Cell Developmental Biologyvol pp “ screening on the diagnostic significance of cervical cancer andprecancerous lesions European Review for Medical and Pharmacological Sciences vol no pp “ [] KH Wang C J Lin C J Liu œGlobal methylationsilencing of clustered protocadherin genes in cervical cancerserving as diagnostic markers comparable to HPV CancerMedicine vol no pp “ [] T Li Y Li G X Yang œDiagnostic value of combination of HPV testing and cytology as compared to isolatedcytology in screening cervical cancer a metaanalysis Journal of Cancer Research and Therapeutics vol no pp “ [] K Honda T Yamada R Endo œActinin4 a novel actinbundling protein associated with cell motility and cancer invasion The Journal of Cell Biology vol no pp “ [] E de Almeida Ribeiro N Pinotsis A Ghisleni œThestructure and regulation of human muscle αactinin Cellvol no pp “ [] D Wang X W Li X Wang œAlphaactinin4 is a possible target protein for aristolochic acid I in human kidneycellsin vitro The American Journal of Chinese Medicinevol no pp “ [] I V Ogneva N S Biryukov T A Leinsoo and I M Larina œPossible role of nonmuscle alphaactinins in musclecell mechanosensitivity PLoS One vol no articlee96395 [] K Honda œThe biological role of actinin4 ACTN4 in malignant phenotypes of cancer Cell Bioscience vol no p [] X Zhao K S Hsu and J H Lim œÎ±Actinin potentiatesnuclear factor κlightchainenhancer of activated BcellNFκB activity in podocytes independent of its cytoplasmic actin binding function The Journal of BiologicalChemistry vol no pp “ [] H Shams J Golji K Garakani and M R Mofrad œDynamicRegulation of α Actinin's Calponin Homology Domains on FActin Biophysical Journal vol no pp “[] C Fang J J Li T Deng B H Li P L Geng and X TZeng œActinin4 as a diagnostic biomarker in serum ofbreast cancer patients Medical Science Monitor vol pp “ [] T Watanabe H Ueno Y Watabe œACTN4 copynumber increase as a predictive biomarker for chemoradiotherapy of locally advanced pancreatic cancer British Journal of Cancer vol no pp “ [] S Yamamoto H Tsuda K Honda œACTN4 gene amplification and actinin4 protein overexpression drive tumourdevelopment and histological progression in a highgrade subset of ovarian clearcell adenocarcinomas Histopathologyvol no pp “ [] M C Wang Y H Chang C C Wu œAlphaactinin is associated with cancer cell motility and is a potential biomarker in nonsmall cell lung cancer Journal of ThoracicOncology vol no pp “ [] N Okamoto H Suzuki K Kawahara œThe alternativelyspliced actinin4 variant as a prognostic marker for metastasisin smallcell lung cancer Anticancer Research vol no pp “ 0c"

Thyroid_Cancer

CD146 was originally identified as a melanoma cell adhesion molecule MCAM and highly expressed in many tumors andendothelial cells However the evidence that CD146 acts as an adhesion molecule to mediate a homophilic adhesion through thedirect interactions between CD146 and itself is still lacking Recent evidence revealed that CD146 is not merely an adhesionmolecule but also a cellular surface receptor of miscellaneous ligands including some growth factors and extracellular matrixesThrough the bidirectional interactions with its ligands CD146 is actively involved in numerous physiological and pathologicalprocesses of cells Overexpression of CD146 can be observed in most of malignancies and is implicated in nearly every step of thedevelopment and progression of cancers especially vascular and lymphatic metastasis Thus immunotherapy against CD146 wouldprovide a promising strategy to inhibit metastasis which accounts for the majority of cancerassociated deaths Therefore todeepen the understanding of CD146 we review the reports describing the newly identified ligands of CD146 and discuss theimplications of these findings in establishing novel strategies for cancer therapySignal Transduction and Targeted Therapy 101038s41392020002598INTRODUCTIONIn Johnson first found that a tumor antigen MUC18was expressed most strongly on metastatic lesions and advancedprimary melanoma with rare detection in benign lesions Due tothe high sequence homology between MUC18 with cell adhesionmolecules CAMs this melanoma antigen was given an officialname melanoma CAM MCAM1 With an increasing number ofdiscoveries about MCAM by various research groups more aliasnames were given to this protein including P1H12 MUC18 A32antigen SEndo1 MelCAM METCAM HEMCAM or CD1461“There are three forms of CD146 proteins in human mouse andchicken The two membraneanchored forms of CD146 areencoded by cd146 gene and soluble form of CD146 sCD146is generated by the proteolytic cleavage ofthe membraneforms11“ Soluble CD146 can be detected in cell culturesupernatants serum and interstitial fluids from either healthyor unhealthy subjects14“ Because sCD146 does not have eitherto cell or cellCAM is a kind of proteins located on the cell surface andmediates contacting and binding of celltoextracellular matrix ECM4 These dynamic interactions providesignals input into the cellular decisionmaking process such as cellgrowth survival migration and differentiation5 essentialforembryonic development and for maintaining the integrity oftissue architecture in adults67 Dependent on adhesion someCAMs can initiate the formation of complexes composed ofextracellular ligands kinases and cytoskeletal proteins8 Abnormalexpression of CAMs can cause various diseases such as cancer and‚ammatory disorders910transmembrane or cytoplasmic regionsit is not competent incellular adhesion1718 Therefore we will not describe sCD146 itsligands and its functions in this review although it is a potentialtarget in tumor microenvironment of CD146positive invasivetumors19Recent evidence has revealed that membranebound CD146may act as a cellsurface receptor to bind with various ligandsinvolved in cellular signaling transduction independent of theadhesion properties In order to deepen the understanding of thefunctions of CD146 in physiological and pathological processeswe summarize the various newly identified ligands of CD146and the ligandelicited roles in signal transduction and discussthe implications of CD146 in remodeling interactions betweenthe cancerous cells with the elements oftheir surroundingmicroenvironmentsTHE CD146 PROTEINMembrane CD146 protein has two isoforms long form CD146lhas a long cytoplasmic tail short form CD146s has a shortcytoplasmic tail1718 These two CD146 isoforms are produced fromdifferent exon splicing strategies and the premature moleculeshave a signal peptide located on the anterior region of the aminoterminal20 In human mature CD146 protein is composed of anextracellular sections with five distinct Iglike domains that exist ina V“V“C2“C2“C2 structural motif a hydrophobic transmembraneregion and a short cytoplasmic tail21 The cytoplasmic domain inboth isoforms contains two potential recognition sites for protein1Key Laboratory of Protein and Peptide Pharmaceuticals Institute of Biophysics Chinese Academy of Sciences Beijing China 2College of Life Science University ofChinese Academy of Sciences Beijing China 3Department of Gastrointestinal Hepatobiliary Tumor Surgery Beijing Shijitan Hospital Capital Medical University Beijing China 4Departments of Pathology Beijing Shijitan Hospital Capital Medical University Beijing China and 5Nanozyme Medical Center School of Basic MedicalSciences Zhengzhou University Zhengzhou ChinaCorrespondence Zhaoqing Wang clairezqwanghotmailcom or Xiyun Yan yanxyibpaccnThese authors contributed equally Zhaoqing Wang Qingji XuReceived March Revised June Accepted June The Authors 0cCD146 from a melanoma cell adhesion molecule to a signaling receptorWang et alkinases C PKC an ERM protein complex of ezrin radixin andmoesin binding site a motif with microvilli extension and adouble leucine motif for basolateral targeting21 The two isoformscoexist as monomers and dimers and the dimerization ismediated through a disulfide bond between cysteine residues inthe C2 domain most proximal to the membrane2022 However theinformation about CD146 crystal structureincluding secondaryand tertiary is still lackingCD146 is a highly glycosylated type I transmembrane proteinand belongs to the immunoglobulin superfamily Based onbioinformation analysis eight putative Nglycosylation sites arepresent in the extracellular fragment across species23 In clear cellrenal cell carcinoma and prostate cancer CD146 glycosylationlevels were upregulated2425 In it was reported that CD146glycosylation is favorably carried out by b13galactosylOglycosylglycoprotein b16Nacetylglucosaminyltransferase3 whichwas overexpressed in highly metastatic melanomas Suchglycosylations can extend CD146 protein stability upregulateCD146 protein levels and lead to elevation of CD146mediatedcellular motility in melanoma cells26 These observations suggestthat the degree of CD146 glycosylation may be directly relatedto malignant progression of tumors especially CD146positiveneoplasmsTHE EXPRESSION PROFILE OF CD146 PROTEINBased on literature metazoan CD146 has been detected inmajority of cell types including vessel constituting cells endothelium pericyte and smooth muscle cell epithelia fibroblastsmesenchymal stem cells and lymphocytes except erythrocytes21Under physiological conditions CD146 expression is restricted tolimited adult normal tissues and its adhesive strength is relativelyweakto most other CAMs which show wideexpression patterns in normal adult tissues and strong adhesionstrength2123 However CD146 expression is broadly and highlydetected in embryonic tissues compared to its abundance innormal adult tissues21 In quickly proliferating cellsincreasedexpression of CD146 may allow cells to actively interact with eachother and with the elements of the cellular microenvironmentpromoting cell proliferation and migrationin contrastUnder pathological conditions such as ‚ammation andtumorigenesis CD146 was upregulated in the related cells andhas been identified as a reliable marker for numerous types ofcancers Accumulating evidence shows that CD146 overexpressionhas been linked to either the initial development of the primarylesion or progression to metastases of most of cancer typesprimarily including melanoma127“ breast63031 ovarian32“lung3637 prostate38“ glioma41 kidney42 hepatic4344 and gastriccancers2145 In Nollet reported that TsCD146 mAb fortumor specific antiCD146 monoclonal antibody can specificallyrecognize CD146 expressed in cancer cells but not CD146 inphysiological vessels suggesting that structural features of cancerCD146 differ from those of physiological CD14628RECOGNITION OF CD146 LIGANDS IN HISTORYThe recognition of CD146 ligands and analysis of their functionswas undertaken over a prolonged period in history Because CD146is highly expressed in vessel cells and cancer cells it is likely thatCD146 within these cells contributes to cancer metastasis throughthe mediation of a homophilic adhesion between cancerous cellsand vascular endothelia a key part of the metastatic processHowever evidence of the direct interactions between CD146 anditself is stillit is possible that CD146mediated adhesion between cancerous cells with vascularendothelia as well as with their surrounding elements occursthrough the bidirectional heterophilic interactions between CD146with its ligands but not the homophilic interaction with itselflacking46“ AccordinglyIn the first CD146™s ligand was found using chickensmooth muscle cells Taniura discovered that neuriteoutgrowth factor NOF was a ligand of chicken CD146 Gicerinand that binding of NOF to CD146 is essentialthedevelopment of the chicken retina4950 However at that timedue to technological limitations the molecular characteristics ofNOF were not determined In Laminin was revealed asthe ligand of CD146 facilitating the entry of blood lymphocytesinto the central nervous system CNS In this report the authorsclaimed that Laminin is a major tissue ligand for CD146 onlymphocytes51 In Ishikawa finally determined theidentity of NOF Laminin which has the same α4 subunit asLaminin forIn our laboratory identified that CD146 can bind withvascular endothelial growth factor receptor VEGFR2 as a coreceptor required for the activation by vascular endothelial growthfactorA VEGFA53 Because VEGFA is a wellknown growth factorwith strong proangiogenesis effects this finding provided themechanism underlying the roles of CD146 in tumor angiogenesisespecially in sprouting stage Subsequently ourlaboratoryidentified an array of proangiogenetic growth factors includingWinglessintegrase Wnt5a54 Netrin155 fibroblast growth factorFGF456 VEGFC57 and Wnt158 as the ligands of CD146 In we further identified that CD146 on endothelia can directly bindreceptorβ PDGFRβ onwith plateletderived growth factorrequired for PDGFBinduced PDGFRβ activation59pericyteBecause PDGFBPDGFRβ plays crucial roles in recruiting adjacentpericytes to the endothelia this finding indicates that CD146 isrequired for vessel integrityUntil now there had been a total of molecules or complexesidentified as the CD146 ligands Table According to thecharacteristics of these ligands they can be categorized into threegroups components of the ECM proangiogenic factor receptorsand growth factors All these ligands have been sown to directlyinteract with CD146 in physiological and pathological processesare involved in the promotion of CD146mediated angiogenesisand tumor metastasis Here we will review the various CD146²heterophilic ligands and discuss the implications of these findingsin tumoral contextCD146 IS THE RECEPTOR OF PROTEINS IN RELATION TO THEECMOne of the critical features of malignant proliferation is cancermetastatic plasticity affected by its microenvironment Thisplasticity is a major reason for the failure of inhibition of cancermetastasis The metastatic process involves epithelial mesenchymaltransition EMT attachment of metastatic cells to theendothelium of the vascular or lymphatic vessels and invasioninto distant metastatic tissues60 It is well known that the aberranthigh expression of CD146 is involved in nearly every step ofdevelopment and progression in almost all types of malignantcancers21 The findings that several ECMrelated proteins including Laminin and Galectin1 and S100A8A9 andmatriptase are specific ligands of CD146 may elucidate themechanism underlying the function of CD146 in remodelingtumor microenvironments during tumor development especiallymetastasis via vascular and lymphatic vessels Fig Laminins and Laminins are a family of large heterotrimeric αβγ proteins with over different isoforms Five laminin α chains α1“α5 four laminin βchains β1“β4 and three laminin γ chains γ1“γ3 constitute αβγheterotrimers They are denominated according to their chaincomposition for example laminin α4β1γ1 is designated as Laminin Laminins are predominantly found in basement membranesthat compartmentalize different tissues and surround blood vesselsnerves and adipocytes6263 They play a crucial role in physiologicalSignal Transduction and Targeted Therapy 0cCD146 from a melanoma cell adhesion molecule to a signaling receptorWang et alTable CD146 ligandsLigandsLaminin Laminin Galectin1Galectin3S100A8A9MatriptaseVEGFR2PDGFRβWnt5aWnt1Netrin1FGF4VEGFCFunctionTime of discoveryReferencesFacilitates lymphocytes entry into CNSImproves cancer metastasis via vascular andor lymphatic vesselsInhibits cell apoptosisEnhances cell migration and secretion of prometastasis cytokinesHelps lung tropic metastasisPromotes neuron differentiationProangiogenesisControl of vascular vessel integrityEnables cell migrationPromotes fibroblast activationProangiogenesisPromotes cell polarity establishmentMediates sprouting during lymphangiogenesisand pathological remodeling of the ECM during angiogenesiswound healing embryogenesis and tumor metastasis Remodelingof the ECM during metastasis allows tumor cells to invade theirsurrounding ECM spread via the vascular or lymphatic circulationand extravasate into distant ansLaminin isoforms particularly the laminin α chain are expressedin a cell and tissue specific manner and are distinctly bound byalmost ten different integrins and other cellsurface receptors6263The α4laminins are mesenchymal laminins expressed by the cells ofmesenchymal origin such as vascular and lymphatic endothelialcells pericytes and leukocytes and are required for normaldevelopment of the cardiovascular and neurological system inmice64“ Under pathological conditions α4laminins are expressedand secreted by various tumor cells such as melanoma andglioma67“ orlymphatic and vascular vesselsnervous system697374tumor stromaLaminin Laminin is expressed along the vascular endothelium687375This laminin isoform is recognized by various integrins includingα6β1 α3β1 α6β4 and αVβ3 which promote the migration ofseveral cell types along vascular or nervous system tracks76“In Laminin on the vascular endothelia was discovered asa specific ligand for CD146 on a subset of human CD4 T helper Thcells51 This subset of human T cells expresses CD146 and can entertissues to promote pathogenic autoimmune responses To determinethe CAMs involved in the migratory capacity of Th17 cells into tissuesresearchers used purified Laminin to identify its receptor In thisstudy the authors demonstrated that purified CD146Fc binds toLaminin with high affinity nM and thatthis bindingdisappeared when the endogenous Laminin was specificallydeleted Correspondingly blocking this binding by CD146 antibodyin vivo also reduced Th17 lymphocyte ltration into the CNSTherefore the authors concluded that Laminin is a major tissueligand for CD146 lymphocyteHoweverthe role of Th17 cells in the pathogenesis ofmalignant tumors is still remains controversial Some studiesrevealed that increased percentage of Th17 lymphocytes amongcells ltrating ovarian cancer cells stimulate tumor progression81 whereas other studies showed that Th17 lymphocytes haveanticancer activity and can reduce tumor growth and metastasis82Therefore the roles of CD146 Th17 cells in cancer developmentmay be worthy of further investigationsLaminin CD146 is a reliable biomarker of endothelia and is concentrated atthe intercellular junctions of endothelial cells of vessel system21Signal Transduction and Targeted Therapy Most cancer cellsincluding melanoma migrate along theabluminal sides of vascular andor lymphatic vessels as theydisseminate throughout the body83 Laminin is major lamininsof along the tumordissemination tracks blood and lymphaticvessels nerves and tumor stroma84“To determine the mechanism of CD146 roles in metastasisresearchers used melanoma cells to test what laminin isoformsother than Laminin can bind with the melanoma marker ofCD146 Therefore they used all laminin α chains to examine thebinding affinity with human CD146 in a solidphase ligand bindingassay87 Finally they found that only Laminin of severallaminin isoformsreadily bound to CD146 suggesting thatLaminin is a primary ligand for CD146 in melanomaAccordingly a functionblocking mAb to CD146 inhibited tumorcell migration on Laminin but not on laminins or Inaddition this investigation determined that the identity of NOFpreviously identified as a ligand for chicken CD146 gicerin isactually Laminin In this study the authors also determined that Laminin andespecially Laminin are capable of stimulating migration of abroad panel of cancer cell lines through a filter This investigationis consistent with the observation that the α4laminins includingLaminins and expressed and secreted by variouscarcinoma cells have already emerged as œoncolaminins“Melanoma CD146 binds with Laminin but not whereaslymphocyte CD146 only binds with Laminin suggesting thatthe epitopes of CD146 on somatic cancer cells are different fromthose of CD146 on blood lymphocytes Therefore the ltrationof CD146 invasive cancers into tumordissemination tracks islikely dependent on the interaction between CD146 and Laminin and blocking their binding may affect the efficacy of cell“cellinteractions and interfere metastasisGalectin1 and CD146 is a highly glycosylated junctional CAM involved in thecontrol of vascular vessel integrity Sequence analysis predicts thepresence of eight putative Nglycosylation sites atresiduepositions and It has beenestimated that of the CD146 molecular mass is attributed toglycans88 The galactose residues in glycans can bind withgalectins and such binding can be inhibited by lactoseGalectins are a family of soluble carbohydratebinding lectinsthat modulate celltocell and celltoECM adhesions89 Up to now galectins have been identified in mammals and are foundin humans Among them Galectin1 and are three bestinvestigated galectins and Galectin1 and promote tumordevelopment progression and immune escape90 Galectin1 and 0cCD146 from a melanoma cell adhesion molecule to a signaling receptorWang can hamper antitumor responses and are considered multifunctional targets for cancer therapy9192 The underlying mechanisms include interfering with drug efficacydelivery or reducingthe antitumor effect of immune cells For instance Galectin1confers drug resistance via inducing the expression of multidrugresistance protein which in turn helps tumor cells to pump outcytotoxic drugs facilitating cancer cells to combat anticancerdrugs93 Regarding the immunosuppressive effects of Galectin1and on T cells in a mouse melanoma model targeted inhibitionof Galectin1 enhanced T cellmediated tumor clearance94Galectin3 can neutralize glycosylated IFNγ in tumor matricesablating the immune response to tumors95 To increase overallresponsiveness of tumors to chemo or immunetherapy inhibitors of Galectin19697 and have been used in combinationwith antiCTLA4 or antiPD1 to treat cancer patients in clinicaltrialsBecause both CD146 and galectins are involved in themodulation of angiogenesis researchers hypothesized that somegalectins may be the ligands of CD146 and the interactionsbetween them are required for functional CD146 in angiogenesisas well as in cancer metastasis To date two galectins and have been identified as the ligands of CD146It has been reported thatGalectin1Galectin1 prefers to bind with the branched Nglycans of cellsurface glycoproteins and mediates a glycosylationdependentangiogenesis91100“increasedsecretion of Galectin1 in the ECM facilitates cancer cellproliferation and resistance to cancertherapy in prostatecancer104 and Kaposi™s sarcoma105 Mechanistic investigation hasrevealed that Galectin1 can bind to Nglycans on VEGFR2 toactivate VEGFlike signaling in antiVEGFA refractory tumorspromoting tumor progression Accordingly disruption oftheGalectin1Nglycan axis inhibits tumor growth by promotingvascular remodeling101 This research highlights the importance ofGalectin1 in tumor angiogenesis and cancer metastasis Howeverthese studies cannot exclude the fact that other cellsurfaceproteins with branched Nglycans are also involved in thisglycosylationdependent proangiogenesis pathwayEarly in it was reported that the coexpression of Galectin and CD146 is required for tumor vascularization in a humanmesenchymal stem cell strain with significant angiogenic potential106 In Jouve reported that Galectin1 binds toCD146 on endothelial cells facilitating cell survival107 In thisthey explained that CD146 glycosylation is mainlyreportcomposed of branched Nglycans They showed thattheinteraction of CD146 with Galectin1 is carbohydratemediatedusing both an enzymelinked immunosorbent assay and surfaceplasmon resonance assays In addition they demonstrated thatthe interaction between Galectin1 and CD146 protects endothelial cells against apoptosis induced by Galectin1 Thusit istempting to speculate that CD146 could be a decoy receptor falectin1 preventing the Galectin1 from binding to proapoptotic receptors107 However whether this interaction affectstumor cell survival remains unknown In Yazawa thusfurther analyzed the functions of this interaction on melanomaand found that when Galectin1 binds to CD146 it helps maintainintrinsic malignant features108 The authors examined the expressionidentity and function of Galectin1 ligands in melanomaprogression and demonstrated that CD146 is the major Galectin1ligand on melanoma cellsThese findings provide a perspective on the interactionsbetween CD146 and its ligands such as Galectin1 as contributorsto cancer malignancy Indeed various membrane glycoconjugateshave been identified as binding partners of Galectin1 such as β1integrins CD2 CD3 CD4 CD43 CD45 and GM1 ganglioside Inaddition Galectin1 can bind to a number of ECM components ina dosedependent and βgalactosidedependent manner Forinstance laminin and fibronectin which are highly Nglycosylatedinteract with Galectin1109 Because it has been reported thatCD146 can interact with Laminin Laminin and β1integrin it is reasonable to speculate that CD146 may also interactwith all of those Galectin1 interactors within cancerous cellsSince the tumor vasculature is an easily accessible target forcancer therapy understanding how galectins ‚uence cancerangiogenesis is important for the translational development oftherapies intended to prevent tumor progression Based on thefact that VEGFtargeted therapies often fail when tumors receivecontinuedglycosylationdependentGalectin1receptorsuch as Galectin1CD146VEGFR2 may increase the efficacy of antiVEGF treatmenttreatment110interactionstargetingGalectin3Like Galectin1 Galectin3 can also bind to various galactoseterminated glycans of cellsurface receptors and proteins of ECMand is involved in many physiological and pathological processesfrom cell adhesion and migration to cell activation111112 In cancercells it modulates cell“cell and cell“microenvironment communications contributing to cancer development progression andmetastasis113“ Patients with metastatic diseases tend to havehigher concentrations of circulating Galectin3 than those withonly localized tumors121 Increased circulating Galectin3 promotes bloodborne metastasis due to the interaction of Galectin3with receptors on vascular endothelial cellsfurther causingendothelial secretion of several metastasispromoting cytokinesTo identify the Galectin3binding molecules on the endothelialcell surface using the Galectin3 affinity purification methodfound that CD146 was the major cellsurfaceColomb et alreceptorto strongly bind and colocalize with Galectin3compared with other glycosylated receptors CD31 CD144 andCD106 They also showed that Galectin3 bound to Nlinkedglycans on CD146 and induced CD146 dimerization andsubsequent activation of protein kinase B AKTsignalingCorrespondingly suppression of CD146 expression abolishesGalectin3induced secretion of metastasispromoting cytokinefrom the endothelial cells Thus they concluded that CD146 is thefunctional Galectin3binding receptor on the endothelial cellsurface responsible for Galectin3induced secretion of cytokinesand therefore ‚uences cancer progression and metastasis122Subsequently the binding moieties of CD146 by Galectin3have been further identified The authors demonstrated thatGalectin3 interacts with the highly glycosylated Domain in theCD146 extracellularthe presence orabsence of lactose These findings provide a better understandingof how Galectin3 interacts with cellsurface receptors to mediateendothelial cell migration and the secretion of cytokines123124regardless offragmentThe endothelial galectins are confined to four family membersie Galectin1 and which contribute to tumorangiogenesis92 Tumorinduced angiogenesis is a pathologiccondition in which tumor cells secrete growth factors such asVEGFs to promote the growth of new blood vessels125126 Thesegrowth factors activate quiescent endothelial cells in host tissue tofacilitate them to invade into the tumor stroma for growth of newcapillaries127 Endothelial galectins binding with glycoconjugateson tumors are involved in different processes during tumorinduced angiogenesis Because Galectin1 and binding ofglycoconjugates on tumor cells mediates many key processes inangiogenesis and elevated levels of Galectin1 and in theendothelium are correlated with tumor vascularization105128“the promotion of tumor vascular remodeling by tumor CD146 maybe due to the interactions between CD146 with Galectin1 and S100A8A9S100 proteinsIn humans there are at least members of theS100 protein family132 which have EFhand calciumbindingmotifs and are soluble in saturated ammonium sulfate133Signal Transduction and Targeted Therapy 0cS100 family members typically form homodimers as well asheterodimers trimers and tetramers etc132134 S100 proteins aretypically cytoplasmic proteins but several family members aresecreted by cells as extracellulartheycontribute to a broad array ofintracellular and extracellularfunctions134135 Upregulation of S100 proteins promotes pro‚ammatory responses that contribute to the development andprogression of cancer and autoimmune and chronic ‚ammatorydiseases138“factors134“ ThusincludingadvancedThe secreted S100 proteins bind with several cellsurfacereceptorsproductsRAGE142“ tolllike receptor TLR4147 CD36148 FGFR1149ALCAM150 CD68151 and ErbB4152 However how the cellsurfacereceptors mediate extracellular S100 signaling is lacking and howS100 protein secretion is dynamically regulated in biologicalprocesses also still remains unknownglycationendseem to require the release ofThe secreted S100A8A9 proteins are theS100A8A9 heterodimer‚ammatorybest characterized soluble S100 proteins Mostthe S100A8A9processesinto the ECM153“ Significant upregulation ofheterodimerS100A8A9 has been observed in many tumors including lunggastric esophageal colon pancreatic bladder ovarian thyroidbreast and skin cancers156157 The upregulation of S100A8A9 iscaused either by the ltrating immune cells oftumormicroenvironment158 or by the tumor itself156157 contributingto the establishment of a premetastatic niche in the tumormicroenvironment159Mechanistic investigations demonstrated that upregulatedS100A8A9 induces the expression of serum amyloid which inturn recruits myeloidderived suppressor cell MDSC producing apro‚ammatory environment during metastasis of aggressivedisease160“ In addition enhanced expression of S100A8A9 isalso associated with poor prognosis168S100A8A9 proteins mediate these effects by binding to plasmamembrane elementsincluding heparan sulfate proteoglycanHSPG169 Nglycans170 TLR4171 and RAGE172173 In a melanomalung metastasis model Hiratsuka clearly demonstrated thatlung S100A8A9 as a strong chemokine interacts with TLR4 onmelanoma to attract distant cancer cells to the lungs174 Recentlyit has also been shown that CD146 on melanoma and breastcancer can respond to lung S100A8A9 to induce lungspecificmetastasis of melanoma175176 and breast cancer177S100A8A9 as the ligand of CD146The expression levels ofS100A8 and S100A9 were higher in the lungs than in other ansand the higher expression levels were induced by the primarytumor itself162 In lungassociated MDSC and endothelial cellstumorderived transforming growth factorbeta TGFβ and VEGFA can upregulate the expression and secretion of S100A8A9162Thus it has been recognized that S100A8A9 plays a critical role inlung tropic metastasis and the subsequent growth of cancer cellsin the lungs26178 During metastasis lung S100A8A9 might act asa guiding protein for cancer cells that possess high expressionlevels of CD146162In Ruma revealed that S100A8A9 uses CD146 as areceptor during lungspecific metastasis of melanoma cells175 In thisstudythey demonstrated that S100A8A9 binding to CD146activates nuclear factorkappa B NFκB and induces reactive oxygenspecies formation significantly increasing cell adhesion growth andinvasion Notably this study proposed that CD146 governs cancerinvasion toward the lungs by sensing the cancer microenvironmentas a soil sensor receptor of lung S100A8A9175 Therefore the authorsconclude that S100A8A9 plays a crucial role in lung tropic cancermetastasis by helping to establish an immunosuppressive metastaticniche to which it then attracts remote cancer cells by interactingwith CD146 on the cancer cell surfacesIn Chen further determined the importance of theSignal Transduction and Targeted Therapy CD146 from a melanoma cell adhesion molecule to a signaling receptorWang et alS100A8A9CD146 axis in melanoma dissemination in a skinlesion a critical early step for metastasis of melanoma Thismechanistic study revealed that S100A8A9CD146 bindingactivates a cascade of functions it leads to significant activationof the transcription factor ETS translocation variant ETV4 andthe subsequent induction of matrix metalloproteinase25 Theactivation of MAP3K8ETV4 by S100A8A9CD146 binding finallyresults in lung tropic metastasis of melanoma176Breast cancer cells prefer the lung liver bone and brain astheir metastatic sites This antropic metastasis is known asthe œseed and soil theory179 This conclusion was reachedbecause CD146 was remarkably overexpressed in metastaticbreast cancer cells180“ In in breast cancer cells theS100A8A9CD146 axiselicited downstream signals that producethe driving force for distant metastasis were identified This studyrevealed how S100A8A9 binding to CD146 accelerates breastcancer growth and metastasis They found that S100A8A9 actsas an extracellular cytokine to activate the CD146ETV4 axiswhich upregulates a very high level of ZEB1 a strong EMTinducer ZEB1 in turn induced a mobile phenotype ie EMT incellsthe downregulation of CD146ETV4 axisrepressed S100A8A9induced EMT resulting in greatly weakened tumor growth and lung metastasis Thus this reportsuggested that S100A8A9 contributesto these signalingprocesses through CD146177In contrastSince metastasis accounts for the majority of cancerassociateddeaths studies on metastasis mechanisms are needed to establishinnovative strategies for cancer treatments These findings thatCD146 as a novel receptor for S100A8A9 mediates the transitionof malignant cancers to metastatic sites suggest that strategiesmodulating the interaction between CD146 and S100A8A9 maybe useful for interference with cancer metastasis especially in theprogression of premetastatic tumors to the lungsMatriptaseMatriptase is an epithelialspecific membraneanchored serineprotease that proteolytically degrades targets such as ECMcomponents and the proforms of growth factors183“ Becausemost of solid tumors are originated from epithelia matriptase isthus critically involved in cancerinvasive growth throughdegradation events related to breaching the basement membrane reanization of the ECM and activation of oncogenicsignaling pathways187During neurogenesis matriptase expressed on neural stemprogenitor NSP plays a critical role in cellcontact signalingbetween NSP and brain endothelial cells188 In the directbinding between brain endothelial CD146 and NSP matriptase wasidentified to be involved in the direct endotheliaNSP contact189Such binding can activate the downstream signaling cascades fromincluding p38 and canonical Wntβcatenin pathways inCD146endothelia leading to secretio

Thyroid_Cancer

Electronic health records EHRs contain rich documentation regarding disease symptomsand progression but EHR data is challenging to use for diagnosis prediction due to its highdimensionality relative scarcity and substantial level of noise We investigated how to bestrepresent EHR data for predicting cervical cancer a serious disease where early detectionis beneficial for the outcome of treatment A case group of patients with cervical cancerwere matched to ten times as many controls and for both groups several types of eventswere extracted from their EHRs These events included clinical codes lab results and contents of free text notes retrieved using a LSTM neural network Clinical events are describedwith great variation in EHR texts leading to a very large feature space Therefore an eventhierarchy inferred from the textual events was created to represent the clinical texts Overallthe events extracted from free text notes contributed the most to the final prediction and thehierarchy of textual events further improved performance Four classifiers were evaluatedfor predicting a future cancer diagnosis where Random Forest achieved the best resultswith an AUC of from a year before diagnosis up to one day before diagnosis Weconclude that our approach is sound and had excellent discrimination at diagnosis but onlymodest discrimination capacity before this point Since our study objective was earlier disease prediction than such we propose further work should consider extending patient histories through eg the integration of primary health records preceding referral to hospitalIntroductionInformation on disease progression documented in electronic health records EHRs is apotential source of valuable new knowledge which could lead to improved health care [“]Since EHR information is derived directly from health care there is a great interest on how tobest use this source for reallife applications by way of advanced medical informatics Applications that can benefit from EHR mining include clinical decision support adverse eventa1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Weegar R Sundstro¨m K Usingmachine learning for predicting cervical cancerfrom Swedish electronic health records by mininghierarchical representations e0237911 101371journalpone0237911Editor Sreeram V Ramagopalan University ofOxford UNITED KINGDOMReceived January Accepted August Published August Copyright Weegar Sundstro¨m This is an access distributed under the terms ofthe Creative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement The data set consistsof second party data and is part of Health Bank Swedish Health Record Research Bank which ismanaged by the Department of Computer andSystems Sciences at Stockholm University dsvsusehealthbank The ethical permissionsassociated with this data set does not allow for access to the data For interestedresearchers inquiries can be made to the directorof Health Bank herculesdsvsuse or to thecorresponding author to access the data in thesame way as the authors ie on site at DepartmentPLOS ONE 101371journalpone0237911 August PLOS ONE 0cof Computer and Systems Sciences at StockholmUniversityFunding Both authors RW and KS were fundedby the Nordic Information for Action eScienceCenter of Excellence in HealthRelated eSciencesNIASC project number wwwnordicehealthse The funders had no role in studydesign data collection and analysis decision topublish or preparation of the manuscriptCompeting interests The authors have declaredthat no competing interests existUsing machine learning for predicting cervical cancer from Swedish electronic health recordsdetection and risk prediction [] One important potential of mining of EHRs is to generatenew clinical hypotheses and since EHRs document large populations observed over time theyallow for investigations regarding the relationship between clinical events and outcomes []Compared to cohort studies where specific information about individuals is typically collected at predefined time intervals the use of EHR data has several distinct strengths but alsolimitations Some of the advantages of using EHR data are that data are collected continuouslythat a richer set of information types is included and that less resources may be required toacquire the data However since EHRs are not primarily used to collect data for research purposes EHR data are typically sparse and can contain a high level of noise making this type ofdata challenging to analyze []In this work the focus is prediction of a major human cancer ie cervical cancer usingEHRs as input Globally cervical cancer is one of the dominating cancer forms in womenwith half a million cases each year In Sweden due to prevention through anized cervicalscreening the disease is rarer but there are still about cases of cervical cancer per year andthe incidence has risen in the past two years The median age of women getting the diagnosisis years and thus it is a disease which strikes relatively early in life [] Improved predictionof risk could lead to interventions at an earlier stage where the illness may still exist as a precancerous lesions amenable to surgical removal For cancer diseases earlier diagnosis couldalso lead to improvements in the outcomes of treatment and quality of life as well as for survival [] and for cervical cancer relative survival is higher when the cancer is detected at anearly stage [] The latter improvement is termed downstaging and would be of value especially in a disease such as cervical cancer where higher stages are associated with very highmortality Machine learning models created from EHRs could if sufficiently accurate potentially lead to an earlier diagnosis and increased knowledge regarding the events preceding adiagnosis In this work the aim is therefore to apply machine learning to EHRs and to explorehow well classifiers can identify future cervical cancer cases To this end both the informativeness of different event types found in EHRs diagnosis codes drug codes free text proceduresand lab results was evaluated together with the issue of how to best represent such events fordiagnosis predictionMachine learning methods have been applied to EHRs for predicting a number of differentoutcomes both for specific diseases and also for the risk of mortality and hospitalization andthe number of studies using EHR data for creating risk prediction models is increasing []Zhao and Weng [] used variables known to be related to pancreatic cancer andweighted them using PubMed s The variables included symptoms labresults andcomorbidities and were extracted from EHRs for cases and controls Each selected variable was assigned a weight according to if the association to pancreatic cancer mined from thes was positive or negative and a Bayesian Network Inference model using the assignedweights gave a better predictive performance compared to not using the weights derived fromPubMedIn their study on using machine learning to develop risk prediction models from healthrecord data Mani [] aimed to identify patients at risk of type diabetes They extracted variables representing demographic information clinical findings and laboratory valuesfor over patients where of were patients with a diabetes type diagnosis and theremaining were controls Three data sets representing different time intervals were createdIn the first one all data up to the diagnosis date were included while in the second and thirdones data were included from over days and over days before the diagnosis A numberof classifiers were evaluated for predicting which patients would develop type diabetes andan AUC of was achieved for each of the three time intervalsPLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordsHuang [] aimed to predict future cases of depression the severity of the depressionand the response to treatment In brief patients with a future depression diagnosis andat least years of data before the diagnosis were matched to six times as many controlsDiagnosis codes medication codes demographics and free text from EHRs belonging to thecases and controls were used as input and their model could predict depression diagnoses months in advance with an AUC area under the curve of and when including all available data up to the diagnosis data the corresponding result was an AUC of Kop [] used structured data from EHRs to predict future cases of colorectal cancerCRC Six months of data for over patients whereof CRC cases were extractedfrom primary care health records The data included ICPC codes International Classificationof Primary Care ATC codes Anatomical Therapeutic Chemical Classification System andlaboratory values Temporal and cooccurrence patterns were mined from the data set andused as input for three different classifiers CART decision trees logistic regression and Random Forest An AUC of was achieved and the input features were ranked according tothe importance factor provided by the logistic regression model Most of the features with highranks corresponded to events known to be linked to colorectal cancer providing validation oftheir modelFurther promising examples of studies have used neural network models such denoisingautoencoders in combination with Random Forest convolutional networks and recurrentnetworks for risk prediction of subsequent clinically relevant diseases through the use of EPRdata [“]Materials and methodsEthics statementThe use of health record data in this project was approved by the Regional Ethical ReviewBoard in Stockholm Sweden which determined that informed consent from the study participants was not required The data was anonymized before being accessed by the researchersEthical permission number Feature extractionWhen health records are mined a first necessary step is feature extraction during which dataof interest are selected and extracted from the records There are two possible ways of selectingwhich features to include either a topdown approach is used where domain or expert knowledge guides the feature selection or a bottomup approach is applied with an datadriven feature selection [] A benefit of using the bottom up approach is that it allows fordetecting new previously unknown links between events and outcomes since no a prioriassumptions are made regarding which features are relevant to include in the model []EHRs contain several information types requiring different levels of preprocessing beforethey can be included in a machine learning model Often a distinction is made between structured and unstructured information where diagnosis codes drug codes and demographicinformation such as age or gender is considered as structured and therefore easier to representfor machine learning purposes Free text on the other hand is regarded as unstructured information as it is not possible to directly determine the value or meaning of an EHR free textnote This free text makes up a substantial part of the documentation in EHRs and since itdescribes a patient™s health status symptoms and treatments it is potentially valuable toinclude free text in risk prediction models Free text notes require a higher level of preprocessing and therefore Natural Language Processing methods may be applied to structure the freetext and extract relevant information from it []PLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordsThe level or degree of structure can vary also for the structured data for example even ifthe type of a lab test is coded the result of the test might be a in free text with different unitsused for the same test Such information can be regarded as semistructured and thereforeadditional preprocessing is required also for these types of eventsDataThe health records collected for this study comes from Karolinska University Hospital inStockholm from the years [] These records contain coded information such asdiagnosis codes drug codes and procedural codes semistructured information such as labtest results and free text From this data set the patients with an ICD10 diagnosis coderepresenting cervical cancer ie a code starting with C53 were selected as the case groupNext a control group was created where each patient in the C53 case group was matched onage to ten other control women who did not have any C53 diagnosis on record in the dataSince one aim of this work was to investigate the events leading up to a first diagnosis of cervical cancer only cases with EHR data before the diagnosis date of cancer were included andall patients with the code Z854C previous cervical cancer were also filtered out leaving cases and controls Five basic event types were extracted from the ERHs for both casesand controls clinical entities found in the free text notes diagnosis codes drug codes labresults and procedure codes The extracted data were then divided into intervals for the firstof which all data up to the day before diagnosis were included For the next interval only dataregistered at least days before diagnosis were included and so on in intervals of days upto a year before diagnosis The available data was divided into two parts a development setcontaining of the data and a test set with the remaining The development set was setaside for hyperparameter tuning of the classifiers and for feature selection The test set wasused to evaluate the selected classifiers through a process of 10fold crossvalidation wherebyten rounds of training and testing was performed with of the data used for training and for testing for each round The development set was not included in the final evaluationof the classifiersICD codesThe health records contain diagnosis codes from the Swedish version of the ICD10 codesInternational Statistical Classification of Diseases and Related Health Problems”Tenth Revision ICD10 codes are hierarchically arranged with chapters at the highest level thesechapters are further divided into sections subsections and full codes This makes it possible toinclude both very finegrained information in form of the full diagnosis codes as well as higherlevel information such as chapters and subsections Table gives an example of the ICD10Table Example from the ICD hierarchyLevelChapterSectionExampleC00D48 NeoplasmsC51C58 Malignant neoplasms of female genital ansSubsectionC53 Malignant neoplasm of cervix uteriCodeC530 Malignant neoplasm endocervixSizeIn dataThe top of the hierarchy consists of different chapters where chapter II comprises codes for neoplasms Furtherdown in the hierarchy more detailed information is included in the codes The column titled Size gives the numberof different codes for each level in the complete code hierarchy and the last column In data shows the number ofdifferent codes included in the current data sets101371journalpone0237911t001PLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordsFig ICD chapters and sections for the case group and the control group The left part corresponds to the case group and the right part the control group The innercircle represents the ICD chapters and the outer circle the ICD sections included in the chapter the labels show the sections with the most frequent codes101371journalpone0237911g001hierarchy for the code C530 Fig provides a visualization of the distribution of ICD10 codesfor the control group and the C53 case group Comparing cases and controls it can be notedthat many of the patients in the case groups have experienced other types of cancers ofcases and of controls had a diagnosis code from chapter II of ICD10 which is the chaptercontaining codes for tumorsneoplasms Additionally a large part of these codes came fromthe section representing codes for malignant neoplasms of the female genital ans C51C58other than cervical cancerATC codesThe next feature type are the ATC codes from the Anatomical Therapeutic Chemical Classification System representing drugs As with ICDcodes these codes are hierarchical and therefore it was possible to represent them with different levels of detail Table shows an exampleof the included ATC levelsClinical entities extracted from textMost of the data extracted from the EHRs were in the form of free text notes and differentapproaches have previously been used to create representations of the free text in EHRs OneTable Example from the ATC code hierarchyLevelMain group3rd level subgroup4th level subgroupFull codeExampleN Nervous systemN02B Other analgesics and antipyreticsN02BE AnilidesN02BE01 ParacetamolIn dataThe last column of the table shows how many different codes appear in the current data set for each code level101371journalpone0237911t002PLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordsFig Example of patient record text This example contains one multiword Disorder œsquamous cell carcinoma insitu101371journalpone0237911g002possibility is to map the text to some existing ontology or terminology Roque []matched free text to ICD codes to enrich coded information extracted from EHRs This hasthe advantage of getting a coded representation practical for interpretation and machinelearning purposes however mapping directly from clinical text to standard terminologies canlead to low recall [] as the language used in clinical text differs from the standardised language in terminologies Tools for matching text to terminologies such as MetaMap [] arenot available for Swedish Another approach which also reduces the size of the feature spacewas applied by Miotto [] They included free text in their model by extracting entitiesfrom clinical notes using the Biomedical Annotator and topic modeling this approachwas effective for predicting diagnoses but has the drawback of reduced interpretabilityHere named entity recognition NER was applied to the free text notes belonging to casesand controls Using NER allowed for extracting the most relevant parts of the text and compared to including the full texts in a bagofwords model named entities makes it possible torepresent multiword expressions such as diabetes typ diabetes type and cancer in situ Toextract these entities from the free text a bidirectional long shortterm memory biLSTMnetwork was trained on notes annotated by two medical experts The notes used as trainingdata for the network were annotated for the entity types Body part annotations Disorder annotations and Finding annotations For this work findings and disorderswere included as events and following the SNOMED definitions a œFinding represent bothnormal and abnormal observations regarding a patient while a œDisorder always is the resultof an underlying pathological process see Fig A large corpus of clinical texts was used to generate word2vec [] embeddings of the textsas input representation for the network This corpus contained GB of text from SwedishEHRs with a complete vocabulary of about words The properties of the LSTM network are described in detail in Weegar [] This network was applied to all clinical notesfor the cases and the controls to extract all mentions of findings and disorders from these textswhich contained on average tokens for each patient Next negation detection was used toidentify negated entities using a rulebased module NegEx adapted specifically to the domainof Swedish clinical text Swedish [] This was an important step because clinical notes oftencontain documentation of discussions and reasoning around a potential condition in text andit is thus particularly common that findings are negated Indeed in this material up to ten percent of findings and six percent of the extracted disorders were actually in the negated formSimilarly to mitigate the risk of including information regarding individuals other than theactual patient to whom the record belonged deriving from eg discussions on family historyof disease clinical notes with headings related to family members were excluded The use ofNER for event extraction resulted in events for the controls on average For the cases onlyevents occurring before the C53 diagnosis were included on average eventsThe extracted events were lemmatized which reduced the scarcity of the free text data aslemmatization maps inflected versions of a word to the same basic representation By usinglemmatization words such as hudirritationskin irritation singular and hudirritationerskinirritations plural will be considered as the same event Similarly the event hostadecoughedPLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordspast tense will be joined with hostarcoughs present tense But even after lemmatizationthe number of different events extracted from the texts is very large There were about different events appearing at least two times This is partially due to the possibility of describing the same event in many different ways in writing for example œfractured patella andœpatella fracture refers to the same type of event but will be considered as two different eventsas their surface forms are different Therefore to further reduce the feature space two additional preprocessing steps were applied to the textual events Firstly a spelling normalizationmodule was used to group different spelling variations of the same concept This grouping wasachieved through calculating the edit distance between each pair of events A Levenshtein editdistance of two strings is a measurement of how different the two strings are and is calculatedby counting the number of insertions deletions and substitutions of characters that is requiredto make two strings equal [] Groups of events were formed where each member in thegroup had at most an edit distance of one from some other member of the group and the samefirst letter as all other members in the group as it was found that allowing larger distancesintroduced errors For multiword expressions the distance was calculated per word and itwas also required that each included word had the same first letter The spelling differences inthe text data were mainly the result of misspellings but also of inflections or instances of writing the same concept as a either a compound word or a as two separate words After groupingthe events using edit distance all variations were exchanged with the most frequent surfaceform One example of such a group is the spelling variations for thyroid cancerthyreoideacancer tyreoideacancer thyroidecancer thyreoidacancer thyreoidcancer tyroideacancer tyreoidecancer thyroidea cancer thyroideacancer thyreoidecancerwhere each variation was mapped to the most frequently occurring form œthyreoideacancerThis normalization mapped about different surface forms of events into about different groupsNext a hierarchical representation of the events extracted from the free texts was createdThe hierarchy was constructed by first sorting the events extracted from the texts according totheir length and placing oneword entities at the top of the hierarchy Next for each level anyevent that contained all words of an event on the higher level was added as a child node of thatevent In this hierarchy the oneword event œleukaemia at the top level had the child nodesœacute leukaemia and œchronic leukaemia where œchronic leukaemia in turn had the childnodes œchronic myeloid leukaemia and œchronic lymphocytic leukaemia Events furtherdown in the hierarchy became gradually more detailed as they often contained some type ofmodifier to its parent node such as œnormal œserious or œmalignant capturing many relevant relationships between eventsA hierarchy of four levels was created with oneword entities at level one and events containing four or more words at the fourth level It is worth noting that when representing thetextual events for a single patient the longer lowlevel events had influence over higher levelrepresentations in the same way as for the ICD or ATC code hierarchies Using the ICD codehierarchy a diagnosis can be represented by the corresponding full lowlevel code or by thesection or chapter it belongs to In a similar way the lower level event œchronic myeloid leukaemia can be represented by the higher level event œleukaemiaThis hierarchy of textual events was however different from the ICD and ATC code hierarchies in two ways Firstly each child node could have more than one parent node an œinfectedwound had the parent œwound and the parent œinfected The second difference was thatwhile the top levels are the smallest in the code hierarchies the opposite is true for the textualevents There was a higher number of short textual events and the short events also make up alarger part of the total set of textual events The top level of the hierarchy contained PLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordsdifferent events followed by events at level two events at level three and events at level fourLab resultsThe included data for lab results consists of the type of test and the result of the test Includingthe value of test result can be too finegrained for the task of prediction since many tests arerare [] Therefore the result is represented as being either inside or outside the referencerange for a test value For example the result of a Hemoglobin test can either be in the normalrange for Hemoglobin above the range or below it Therefore each lab result in the includedhealth records will be represented by one of three different features depending on the outcomeof the testProcedure codesProcedure codes classify procedures including surgical procedures medical investigationspreventive measures and treatments for example the code AK044 corresponding to an ultrasound of the kidneys The codes are used in health records for statistical and administrativepurposes and have been included as an event type in this workA number of the events that were extracted from the EHRs were unique meaning that theyonly occurred for a single patient Since such events cannot contribute to the diagnosis prediction but only increase the scarcity of the data they were removed at this stage leaving in total different events in the data set Each case had on average different events and events in total before the diagnosis For the controls the corresponding numbers were on average different events and an average total of The reason for the larger number of eventsfor the control group is that only data up to a cervical cancer diagnosis is included for the casegroup and all events after the diagnosis were discarded For the controls on the other hand alldocumented events were kept even if they occurred after the time of their matched case™s diagnosis Since the objective of our study was correct classification based on EHR events withpotentially low expected contrast between cases and controls we opted for this choice to maximize the control information for the classifiers to be trained on minimize the risk ofmisclassification of disease status by ensuring to the best of our knowledge that no hospitalbased female controls were diagnosed with cervical cancer later during the study period and to increase generalizability to a reallife clinical situation where EHRs are available but diseasestatus of the individuals is not already known Fig gives an overview of the available data forthe different time intervals However some events will appear outside of the study periodleading to data censoring [ ] Important and informative events could have occurred beforethe start of data collection and this also means as in most casecontrol or other studies thatwe do not know whether some members of the control group develop cervical cancer after thestudy end point when no more EHR data was available to us However this is a wellknownfact resulting from this type of study design and does not invalidate the comparison made during the actual study period definedClassification experimentsFour different classifiers were used to evaluate the appropriateness of the different featuretypes for classification of future C53 cases These classifiers were Random Forest ComplementNaive Bayes Bernoulli Naive Bayes and Support Vector Machines all implemented in Scikitlearn [] The first classifier Random Forest is an ensemble classifier robust to noise andlarge feature spaces [] Complement Naive Bayes [] is capable in cases of data sets withclass imbalance Bernoulli Naive Bayes additionally takes absence of features into account forPLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordsFig Available data for the case group The xaxis denotes the number of days before a diagnosis and events only appearing onetime in the data set have been excluded At days before a diagnosis events were available for patients101371journalpone0237911g003classification [] and Support Vector Machines SVM is a classifier well suited for highdimensional spaces []This classification task can be understood as a text classification problem where each eventextracted from a patient record corresponds to a word and each patient is represented by a vector with the same dimension as the complete vocabulary of events For Random Forest andComplement Naive Bayes the input vectors consisted of the raw counts of events For Bernoulli Naive Bayes binary input vectors were used and for the SVM classifier normalized vector counts were used as input Binary vectors correspond to if a patient ever experienced anevent and count vectors also represent how many times each event occurred Another possibility is to use tfidf term frequencyinverse document frequency weights The idea behindtfidf is to give more weight to the events that are representative for individual patients However using tfidf did not improve classification resultsResultsEvent typesEach type of event was evaluated individually for its ability to correctly classify the patients Fig shows the average AUC using the four classifiers over time for each event type ICD codesATC codes Procedure codes Clinical entities and Lab results The average AUC was calculated as the sum of the scores AUCi for the individual classifiers divided by the number of classifiers AUCavg ¼ the classifiers were the clinical entities extracted from the text as the highest AUC scores wereachieved using only the text entities ðAUC1 þ AUC2 þ AUC3 þ AUC4Þ The most informative event type forPLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordsFig Average AUC for the different event types over time101371journalpone0237911g004Event levelsSince the data in EHRs typically are noisy high dimensional and sparse it is necessary toinvestigate data representation how the information included in the records should be presented to the model [] In this work both which types of feature to include and also the detailwith which those features should be represented was evaluatedIt has previously been found that including features derived from several levels of hierarchical clinical codes improves the performance for predicting adverse drug events from EHR data[] and as the input events can be represented with different levels of detail the next set ofexperiments aimed to evaluate the most suitable representation of the events in this regardUsing the most detailed levels such as a full ICD10 code gives very detailed information buta very sparse data set By including the higherlevel representations the sparsity of the data canbe reduced Fig shows the average AUC o

Thyroid_Cancer

Neurosteroids Biosynthesisand Physiological FunctionsShogo Haraguchi and Kazuyoshi Tsutsui Department of Biochemistry Showa University School of Medicine Tokyo Japan Graduate School of Integrated Sciencesfor Life Hiroshima University Hiroshima JapanSimilar to the adrenal glands gonads and placenta vertebrate brains also producevarious steroids which are known as œneurosteroids Neurosteroids are mainlysynthesized in the hippocampus hypothalamus and cerebellum however it has recentlybeen discovered that in birds the pineal gland a photosensitive region in the brainproduces more neurosteroids than other brain regions A series of experiments usingmolecular and biochemical techniques have found that the pineal gland producesvarious neurosteroids including sex steroids de novo from cholesterol For instanceallopregnanolone and 7αhydroxypregnenolone are actively produced in the pinealgland unlike in other brain regions Pineal 7αhydroxypregnenolone an upregulator oflocomotion enhances locomotor activity in response to light stimuli in birds Additionallypineal allopregnanolone acts on Purkinje cells in the cerebellum and prevents neuronalapoptosis within the developing cerebellum in juvenile birds Furthermore exposure tolight during nighttime hours can cause loss of diurnal variations of pineal allopregnanolonesynthesis during early posthatch life eventually leading to cerebellar Purkinje cell deathin juvenile birds In light of these new findings this review summarizes the biosynthesisand physiological functions of pineal neurosteroids Given that the circadian rhythms ofindividuals in modern societies are constantly interrupted by artificial light exposure thesefindings in birds which are excellent model diurnal animals may have direct implicationsfor addressing problems regarding the mental health and brain development of humansKeywords allopregnanolone 7αhydroxypregnenolone neurosteroid pineal gland cerebellum lightINTRODUCTIONSimilar to the gonads and placenta vertebrate brains actively also produce various steroidhormones These steroid hormones produced in the brain are named œneurosteroids Theproduction of neurosteroids was demonstrated firstly in mammals and then in other vertebrates“ Thus neurosteroid production appears to be a universal feature of the brain in vertebratesIt is known that neurosteroids are produced in glial cells and neurons of the centraland peripheral nervous systems However we have demonstrated thatthe pinealgland produces neurosteroids from cholesterol in birds during early posthatch period “Notably allopregnanolone also known as 3α5αtetrahydroprogesterone 3α5αTHP and 7αhydroxypregnenolone are the two major neurosteroids produced in the pineal gland Of thesetwo pineal allopregnanolone prevents the death of developing Purkinje cells and pineal 7αhydroxypregnenolone functions as an upregulator of locomotion regulating locomotor activity inresponse to light stimuli in birds Edited byVance L TrudeauUniversity of Ottawa CanadaReviewed byVincent M CassoneUniversity of Kentucky United StatesMaria Claudia Gonzalez DeniselleCONICET Instituto de Biolog­a yMedicina ExperimentalIBYME ArgentinaCorrespondenceShogo HaraguchishogoharaguchigmailcomSpecialty sectionThis was submitted toNeuroendocrine Sciencea section of the journalFrontiers in EndocrinologyReceived April Accepted July Published August CitationHaraguchi S and Tsutsui K Pineal Neurosteroids Biosynthesisand Physiological FunctionsFront Endocrinol 103389fendo202000549Frontiers in Endocrinology wwwfrontiersinAugust Volume 0cHaraguchi and TsutsuiPineal NeurosteroidsBIOSYNTHESIS OF PINEALNEUROSTEROIDSThe pineal glands of vertebrates respond to light stimuli andfulfillimportant functions in the anization of circadianrhythms The secretion of melatonin a major hormone producedby the pineal gland shows a clear daily rhythm with its peakconcentration occurring at night However it was notknown whether the pineal gland produces neurosteroids untilrecently We have recently demonstrated that the pineal gland isa newly found neurosteroidogenic an producing a variety ofneurosteroids from cholesterol αhydroxypregnenolone and allopregnanolone are activelyreleased Taken together these findings indicate that the pineal gland injuvenile birds produces various neurosteroids from cholesterolAccordingly this is the first demonstration of neurosteroidsynthesis in the pineal gland in a vertebratePHYSIOLOGICAL FUNCTION OF PINEAL7αHYDROXYPREGNENOLONE INLIGHTDEPENDENT LOCOMOTIONthatgene3hydroxysteroidPregnenolone is an anabolic intermediate of most endogenoussteroid hormones and is produced from cholesterol throughthe mitochondrial cholesterol side chain cleavage enzymecytochrome P450scc P450scc encoded by the Cyp11a geneWe have demonstrated by transcriptionpolymerase chainreaction RTPCR thatthe pineal gland in juvenile birdsexpresses P450scc mRNA Figure The proteinproduct of this mRNA is localized in the cells that form thefollicular structures in the pineal glands of birds We havedemonstrated by highperformance liquid chromatography3HHPLC with radioactive flow detector analysischolesterolis converted to radioactive pregnenolone whenincubated with pineal gland extract from juvenile birds This observation has confirmed the presence offunctionalP450scc in the pineal gland Figure which has also beendetected by gas chromatographymass spectrometry GCMS Subsequent RTPCR“based assessment has revealed thatkey steroidogenic enzymes cytochrome P450 7αhydroxylaseP4507α encoded by the Cyp7b gene 3αhydroxysteroiddehydrogenase 01 014isomerase 3αHSD encoded by thedehydrogenase 01 01Hsd3aisomerase 3HSD encoded by the Hsd3b gene 5αreductaseencoded by the Srd5a gene 5reductase encoded by theSrd5b gene cytochrome P450 17αhydroxylasec1720lyaseP45017αlyase encoded by the Cyp17 gene 17hydroxysteroiddehydrogenase 17HSD encoded by the Hsd17b gene andcytochrome P450 aromatase P450arom encoded by the Cyp19gene are expressed in the pineal gland of birds Figure We further demonstrated that steroid hormones are indeedpresent in the pineal gland Incubation of 3Hpregnenolonewith pineal glands from posthatch birds generates 7α andor7hydroxypregnenolone by the action of P4507α foundin the pineal glands Figure In addition to theseneurosteroid isomers progesterone allopregnanolone 3α 5αTHP andor epipregnanolone 3 5THP androstenedionetestosteroneandestradiol17 are also produced Figure These exvivo observations have confirmed that the pineal glands injuvenile birds have the biosynthetic machinery for majorsteroid hormones which have also been verified to be producedas neurosteroidsFigure Although HPLCanalysis has failed to resolve the isomers of these hormonesalloepipregnanolonesuch as 7αhydroxypregnenoloneand 5αdihydrotestosterone several sets ofisomers havebeen successfully isolated by GCMS analysis Especially5dihydrotestosteronein vivo andor5αanalysis hasactivation oftranscriptomicslightinduced transcriptionalfor studiesThe chick pineal gland is used as a modeltheon the lightdependent phaseshifting mechanism ofcircadian clock To search for genes involved in thisa diï¬erential GeneChip analysis has beenmechanismidentifiedperformed Thisthethefullset of genes in the pineal gland involved in cholesterolbiosynthesis When the pineal gland was exposed tolightit produced cholesterol and 7αhydroxypregnenoloneex vivo Interestingly this lightinduced production of 7αhydroxypregnenolone occurred only when the gland wasexposed to light at early night but not atlate night orduring the daytime During early night time the circadianclock is sensitive to light which causes phasedelay of theclock Thusthe lightsensitive pineal production of7αhydroxypregnenolone appearsto be regulated by thecircadian clockactivateslocomotorIn vertebratesan intracerebroventricularinjection of7αhydroxypregnenoloneactivities“ Thusthe intracerebroventricular injection of 7αhydroxypregnenolone was administered in a dosedependentmanner at early night in chicks After the injection chickswere placed individually for locomotor activity measurementin an open field apparatus for min Spontaneous locomotoractivities of chicks were stimulated by the intracerebroventricularinjection of 7αhydroxypregnenolone in a dosedependentmanner Furthermore when chicks are exposed to lightduring early nightreachthe daytime level These results suggest that pineal 7αhydroxypregnenolone reaches the target sites within the brain byvolume transmission upon light exposure at early nightlocomotor activitiestimetheirPHYSIOLOGICAL FUNCTION OF PINEALALLOPREGNANOLONE IN PURKINJECELL SURVIVAL DURING DEVELOPMENT7αHydroxypregnenolone and allopregnanolone are activelyreleased during early posthatch period compared with adulthood Therefore 7αhydroxypregnenolone and allopregnanolonemay play key roles in birds during early posthatch periodIn vertebrates pinealectomy decreases cell number in thedeveloping brain These findings suggest that these majorneurosteroids secreted from the pineal gland are involved in thedevelopment of brain cellsFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cHaraguchi and TsutsuiPineal NeurosteroidsFIGURE Biosynthetic pathways of pineal neurosteroids Allopregnanolone and 7αhydroxypregnenolone are the major neurosteroids produced in the pineal glandof birds P450scc cytochrome P450 sidechain cleavage enzyme P4507α cytochrome P450 7αhydroxylase 3HSD 3hydroxysteroiddehydrogenase 01 014isomerase 3αHSD 3αhydroxysteroid dehydrogenase 01 014isomerase 5αreductase 5reductase P45017αlyase cytochrome P45017αhydroxylasec1720lyase 17HSD 17hydroxysteroid dehydrogenase and P450arom cytochrome P450 aromataseFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cHaraguchi and TsutsuiPineal Neurosteroidstheincreasessupplementation oftheIn chicks pinealectomy decreases the concentration ofallopregnanolone and the number of cerebellar Purkinjeallopregnanolonecells whereasto pinealectomized birdsconcentration ofallopregnanolone and recovers the number of Purkinje cells Thus pineal allopregnanolone is considered to be an essentialfactor for the normal development of cerebellar Purkinje cellsIt thus appears that pineal allopregnanolone functions as anessential factor for Purkinje cells during posthatch periodIn addition pinealectomy in juvenile birds increases theexpression of active caspase3 in Purkinje cells whereasallopregnanolone supplementation decreases the expressionof active caspase3 during posthatch period Thus theneuroprotective action of pineal allopregnanolone on cerebellarPurkinje cells is exerted by suppressing the activation of caspase3Figure Allopregnanolone acts mainly as a ligand ofthe γaminobutyric acid type A GABAA receptor and may alsoact as an agonist of the membrane progesterone receptors αmPRα as well as the mPR and mPRγ “ Thereforeeither mPR siRNA orisoallopregnanolone an antagonistof allopregnanolone was delivered into the cerebellum ofposthatched chicks It was found that the silencing of mPRαincreases the number of Purkinje cells that express active caspase in the cerebellum of chicks Furthermore to uncoverthe mechanism of neuroprotective action of allopregnanoloneFIGURE A schematic model of the effect of pineal allopregnanolone on Purkinje cell survival immediately after hatching under a h lightdark cycle or with hlight exposure during the dark period lightatnight condition Left panel The normal cerebellar development under a h lightdark cycle during the first weekafter hatching Pineal allopregnanolone induces the expression of pituitary adenylate cyclaseactivating polypeptide PACAP a neuroprotective factor through themembrane progestin receptor α mPRα receptor binding mechanism in Purkinje cells Subsequently PACAP inhibits the activation of caspase3 that facilitates theapoptosis of cerebellar Purkinje cells Right panel The abnormal cerebellar development under the lightatnight condition during the first week after hatching Thelightatnight condition disrupts the diurnal rhythm in pineal allopregnanolone synthesis Decreased pineal allopregnanolone synthesis leads to decreased expressionof PACAP in Purkinje cells Consequently the active caspase3 level increases inducing the apoptosis of Purkinje cells in the cerebellumFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cHaraguchi and TsutsuiPineal Neurosteroidsin cerebellar Purkinje cells allopregnanolone action on theexpression of neuroprotectiveneurotoxic factors “ hasbeen investigated Pinealectomy decreases the mRNA levels ofpituitary adenylate cyclaseactivating polypeptide PACAP aneuroprotective factor in the cerebellum of juvenile birds It has been found that a daily injection of allopregnanolone inpinealectomized juvenile birds upregulates PACAP relative tothe levels in control birds These findings show that PACAPmediates the neuroprotective action of pineal allopregnanolonethrough mPRα receptor binding during cerebellar developmentFigure LIGHTATNIGHT AFFECTS THEDEVELOPMENT OF CEREBELLUMTHROUGH A MECHANISM MEDIATED BYPINEAL ALLOPREGNANOLONE ACTIONIt is known that environmental stimuli aï¬ect the developmentof animals including humans In vertebrate brain development anatural lightdark cycle promotes better brain development thanconstant conditions such as constant light or constant darkness“ Howeverthe molecular mechanisms that controlhow environmental light conditions aï¬ect brain developmentremain unclear The pineal gland is a photosensitive anTo investigate whether light conditions are involved in thesynthesis of allopregnanolone in the pineal gland the birdshave been incubated under either a h lightdark LDcycle or LD cycle with h light exposure during the darkperiod lightatnight Consequently it has been found that theallopregnanolone concentration and synthesis during the darkperiod are higher in the pineal glands of LD birds than in thoseof lightatnight birds Figure Furthermore the numberof cerebellar Purkinje cells is decreased by the lightatnightcondition Figure It is therefore considered that pinealallopregnanolone is a critical metabolite that aï¬ects cerebellardevelopment in vertebrates depending on the environmentallight conditionsREFERENCES Baulieu EE Neurosteroids of the nervous system by the nervous system forthe nervous system Rec Prog Hormone Res “ Tsutsui K Ukena K Takase M Kohchi C Lea RW Neurosteroidbiosynthesis in vertebrate brains Comp Biochem Physiol C “ 101016S0742841399000651 Compagnone NA Mellon SH Neurosteroids biosynthesis and functionthese novel neuromodulators Front Neuroendocrinol “of 101006frne19990188CONCLUSIONSthedatarecentreview summarizedThison pinealneurosteroids Studies have indicated that the pineal glandproduces neurosteroids from cholesterol in birds Pineal 7αhydroxypregnenolone regulates locomotion in response to lightstimuli in birds Pineal allopregnanolone prevents the death ofdeveloping Purkinje cells by suppressing neuronal apoptosisduring development In addition circadian disruption by lightexposure during nighttime leads to cell death of developingPurkinje cellsthrough pineal allopregnanolonedependentmechanisms in juvenile birds These observations suggest thatnighttime artificial light exposure in modern societies may alsoperturb the development of the human brainAlmost all animals have circadian rhythms However modernlife conditions chronically disrupt circadian rhythm throughartificial light exposure The disruption of circadian rhythm isassociated with a decline in mental and physical health “The most potent circadian rhythm disruption is inappropriatelytimed bright light exposure eg lightatnight To investigatethe eï¬ects of chronic circadian disruption in modern societieson mental and physical health which is efficiently modeled bythe lightatnight condition presented here many studies havebeen conducted on mice However it is important for us tobear in mind that laboratory mice are mainly nocturnal animalswhereas humans are diurnal Thus birds are excellent animalmodels to uncover the eï¬ect of lightatnight on diurnal animalsincluding humansAUTHOR CONTRIBUTIONSSH and KT wrote the manuscript All authors contributed to the and approved the submitted versionFUNDINGThis work wasJSPS GrantsinAid forScientific Research KAKENHI Grant Numbers JP15K18571and JP19K09033supported bysterol regulatory elementbinding protein Xboxbinding protein andheat shock factor pathways Proc Natl Acad Sci USA “ 101073pnas1015959108 Haraguchi S Hara S Ubuka T Mita M Tsutsui K Possible role of pinealallopregnanolone in Purkinje cell survival Proc Natl Acad Sci USA “ 101073pnas1210804109al Lightatnight Haraguchi S Kamata M Tokita T Tashiro KI Sato M Nozaki Mthroughaï¬ects brain developmenteLifeetallopregnanolonedependentpineal8e45306 107554eLife45306037mechanismsexposure Tsutsui K Matsunaga M Miyabara H Ukena K Neurosteroid biosynthesis in Reiter RJ Pineal melatonin cell biology of its synthesis and of its physiologicalthe quail brain J Exp Zool 305A733“ 101002jeza302interactions Endocr Rev “ 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Prolactin increasesthe synthesis of 7αhydroxypregnenolone a key factor for induction oflocomotor activity in breeding male newts Endocrinology “ 101210en20091229 Haraguchi S Koyama T Hasunuma I Okuyama S Ubuka T Kikuyama S et alAcute stress increases the synthesis of 7αhydroxypregnenolone a new keyneurosteroid stimulating locomotor activity through corticosterone action innewts Endocrinology “ 101210en20111422 Haraguchi S Yamamoto Y Suzuki Y Hyung Chang J Koyama T Sato Met al 7αHydroxypregnenolone a key neuronal modulator of locomotionstimulates upstream migration by means of the dopaminergic system insalmon Sci Rep 101038srep12546 Fillenz M Volume transmission in the brain Novel mechanisms for neuronaltransmission In Fuxe K Agnati LF editors Advances in Neurosciences Vol New York NY Raven Press p “ Kilic E Hermann DM Isenmann S B¤hr M Eï¬ects of pinealectomy andmelatonin on the retrograde degeneration of retinal ganglion cells in a novelmodel of intraorbital optic nerve transection in mice J Pineal Res “ 101034j1600079x20021823x Tun§ AT Turgut M Aslan H Sahin B Yurtseven ME KaplantheS Neonatalcerebellum of“ 101016jbrainres200510011pinealectomythechick a stereologicalstudy Brain ResPurkinjeinduceslosscellin Pang Y Dong J Thomas P Characterization neurosteroid binding and braindistribution of human membrane progesterone receptors δ and ε mPRδand mPRε and mPRδ involvement in neurosteroid inhibition of apoptosisEndocrinology “ 101210en20121772 Schumacher M Mattern C Ghoumari A Oudinet JP Liere P LabombardaF et al Revisiting the roles of progesterone and allopregnanolone in thenervous system resurgence of the progesterone receptors Prog Neurobiol “ 101016jpneurobio201309004 Belelli D Lambert JJ Neurosteroids endogenous regulators of the GABAAreceptor Nat Rev Neurosci “ 101038nrn1703 Bernal J Thyroid hormone receptors in brain development and function NatClin Pract Endocrinol Metabol “ 101038ncpendmet0424 FalluelMorel A Vaudry D Aubert N Galas L Benard M Basille M et alPituitary adenylate cyclaseactivating polypeptide prevents the eï¬ects ofceramides on migration neurite outgrowth and cytoskeleton remodelingProc Natl Acad Sci USA “ 101073pnas04096 Koibuchi N Chin WW ThyroidEndocrinoldevelopment 101016S1043276000002381TrendshormoneMetabactionandbrain“ Vaudry D FalluelMorel A Leuillet S Vaudry H Gonzalez BJ Regulators ofcerebellar granule cell development act through specific signaling pathwaysScience “ 101126science1085260 Sasahara K Shikimi H Haraguchi S Sakamoto H Honda S Harada N et alMode of action and functional significance of estrogeninducing dendriticgrowth Spinogenesis and synaptogenesis in the developing Purkinje cell JNeurosci “ 101523JNEUROSCI0710072007 Bakkum BW Benevento LA Cohen RS Eï¬ects of lightdark and darkrearing on synaptic morphology in the superior colliculus and visualcortex ofJ Neurosci Res “ 101002jnr490280107the postnatal and adult rat Brooks E Waters E Farrington L Canal MM Diï¬erential hypothalamictyrosine hydroxylase distribution and activation by light in adult mice rearedunder diï¬erent light conditions during the suckling period Brain Struct Funct “ 101007s0042901103189 DulcisDSpitzerNCIlluminationcontrolsdopamineof“ 101038nature07569neuronsregulatingbehaviourdiï¬erentiationNature Li Y Komuro Y Fahrion JK Hu T Ohno N Fenner KB et al Lightstimuli control neuronal migration by altering ofinsulinlike growthfactor IGF1 signaling Proc Natl Acad Sci USA “ 101073pnas1111326109 Ohta H Mitchell AC McMahon DG ConstantPediatrthe“ 10120301pdr00002331141840366developing mousebiologicalclocklight disruptsRes Kantermann T Roenneberg TIsfactor or a health risk predictor Chronobiol lightatnightInthealthriska “ Wu J Dauchy RT Tirrell PC Wu SS Lynch DT Jitawatanarat P et alLight at night activates IGF1RPDK1 signaling and accelerates tumrowth in human breast cancer xenografts Cancer Res “ 10115800085472CAN103837 Smarr BL Grant AD Perez L Zucker I Kriegsfeld LJ Maternal andearlylife circadian disruption have longlasting negative consequenceson oï¬spring development and adult behavior in mice Sci Rep 101038s41598017034064Conflict of Interest The authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestCopyright Haraguchi and Tsutsui This is an openaccess distributedunder the terms of the Creative Commons Attribution License CC BY The usedistribution or reproduction in other forums is permitted provided the originalauthors and the copyright owners are credited and that the original publicationin this journal is cited in accordance with accepted academic practice No usedistribution or reproduction is permitted which does not comply with these termsFrontiers in Endocrinology wwwfrontiersinAugust Volume 0c'

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patients who have diabetes suffer from Type DMT2DM Many studies suggest using the significant role of lncRNAs to improve thediagnosis of T2DM Machine learning and Data Mining techniques are tools that canimprove the analysis and interpretation or extraction of knowledge from the dataThese techniques may enhance the prognosis and diagnosis associated withreducing diseases such as T2DM We applied four classification models including Knearest neighbor KNN support vector machine SVM logistic regression andartificial neural networks ANN for diagnosing T2DM and we compared thediagnostic power of these algorithms with each other We performed the algorithmson six LncRNA variables LINC00523 LINC00995 HCG27_201 TPT1AS1 LY86AS1DKFZP and demographic dataResults To select the best performance we considered the AUC sensitivityspecificity plotted the ROC curve and showed the average curve and range Themean AUC for the KNN algorithm was with standard deviation SD themean sensitivity and specificity were and respectively After applying theSVM algorithm the mean AUC obtained after stratified 10fold crossvalidationand the SD obtained The mean sensitivity and specificity were and respectively The mean AUC for ANN and the SD were and also the meansensitivity and specificity were and At last for the logistic regressionalgorithm our results showed of mean AUC and the SD of the meansensitivity and specificity were and respectively According to the ROCs theLogistic Regression and SVM had a better area under the curve compared to theothersContinued on next page The Authors Access This is licensed under a Creative Commons Attribution International License whichpermits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit tothe original authors and the source provide a link to the Creative Commons licence and indicate if changes were made Theimages or other third party material in this are included in the 's Creative Commons licence unless indicated otherwisein a credit line to the material If material is not included in the 's Creative Commons licence and your intended use is notpermitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyrightholder To view a copy of this licence visit httpcreativecommonslicensesby40 The Creative Commons Public DomainDedication waiver httpcreativecommonspublicdomainzero10 applies to the data made available in this unlessotherwise stated in a credit line to the data 0cKazerouni BMC Bioinformatics Page of Continued from previous pageConclusion We aimed to find the best data mining approach for the prediction ofT2DM using six lncRNA expression According to the finding the maximum AUCdedicated to SVM and logistic regression among others KNN and ANN also had thehigh mean AUC and small standard deviations of AUC scores among the approachesKNN had the highest mean sensitivity and the highest specificity belonged to SVMThis study™s result could improve our knowledge about the early detection anddiagnosis of T2DM using the lncRNAs as biomarkersKeywords Data mining Gene expression Machine learning algorithms Type diabetes mellitusBackgroundDiabetes mellitus DM is one of the most prevalent chronic noncommunicable diseases NCD around the world about of the patients who have diabetes sufferfrom Type DM T2DM [] The risk of developing T2DM is strongly associated withmany predispositions behavioral and environmental risk factors and also genetic factors [“] Besides the genetic factors strong evidence indicates that factors such asobesity and physical inactivity are the main nongenetic determinants of the disease [] T2DM can range from predominant insulin resistance with relative insulin deficiency to dominant defective secretion with insulin resistance [] It is often related tometabolic syndrome problems Individuals who have impaired glucose tolerance arehighrisk subjects of type diabetes []Studies demonstrate a drastic increase of the disease in recent decades The trendsestimate that by more than million people will be affected by the disease []People who suffer from T2DM are susceptible to many forms of complications leadingto morbidity and mortality in these patients Many studies emphasize the genetic factors in the pathogenesis of T2DM [ ] Long noncoding RNAs long ncRNAslncRNA are subsets of RNA specified as being transcripts with lengths exceeding nucleotides that could not be translated into protein [] Long noncoding RNAslncRNAs belong to a heterogeneous class of regulatory lncRNAs with transcriptlengths nucleotides which have a positive role in the development and growth ofseveral various diseases including T2DM supporting the hypothesis that abnormal expression of LncRNAs is related to various diseases [] Besides considering the significant role of lncRNAs in disease pathogenesis increasing researches suggest using themto improve diagnosis prognosis and clinical management of T2DM Genomewide association studies GWAS have recently introduced several particular diabetesrelatedloci in the human genome [] Also many studies discovered the relationship betweenmore than susceptible loci and T2DM at a genomewide significant level [ ]Deregulation of genes located in GWAS defined loci may be risk factors for human diseases concerning which we applied the GWAS catalog to select six lncRNAsLINC00523 LINC00995 CG27_201 TPT1AS1LY86AS1 DKFZP as our gene targets for the present study [] Knowledge Discovery in Databases KDD or data miningare techniques for the computational process of discovering patterns in large datasetscontaining various approaches such as artificial intelligence machine learning statisticsand database systems [] These methods are applied to recognize patterns in dataprediction association and classification problems [ ] Considering the 0cKazerouni BMC Bioinformatics Page of importance of early detection of T2DM machine learning and Data Mining techniquesare tools that can improve the analysis and interpretation or extraction of knowledgefrom the data [ ] These techniques may enhance the prognosis and diagnosis associated with life quality reducing diseases such as T2DM [ ]To date several other studies tried to predict diabetes mellitus using outstandingdata mining techniques [“] Vijayan et al[] applied the expectationmaximization algorithm KNN algorithm Kmeans algorithm amalgam KNN algorithm and ANFIS algorithm to predict and diagnose Diabetes Mellitus They usedthe UCI dataset containing blood test and demographic variables and their resultsshowed that EM possessed the least classification accuracy and amalgam KNN andANFIS provided better classification accuracy of more than and respectively Another study conducted by Saravananathan [] used popular classifiincluding J48 Support Vector Machines SVM Classificationcation algorithmsand Regression Tree CART and kNearest Neighbor kNNfor diabetic dataTheir performance indicators were accuracy specificity sensitivity precision errorrate They found that the J48 technique™s performance was remarkably superior tothe other three techniques for the classification of diabetes data Meng []compared three data mining models of logistic regression ANN and decision treefor predicting diabetes mellitus or prediabetes by risk factors They gathered information about demographic characteristics family diabetes history anthropometricmeasurements and lifestyle risk The decision tree model C50 had the best classification performance with an accuracy of with a sensitivity of andspecificity of Another study performed by Saeidi [] used logistic regression to assess the diagnostic value of LY86AS1 and HCG27_201 as biomarkersfor T2DM They obtained a sensitivity of and specificity of Anotherstudy [] used two other lncRNAs including LINC00523 and LINC00994 expressions for the evaluation of their potential diagnostic value for T2DM They appliedlogistic regression and achieved a sensitivity of and specificity of Inour study we combined six lncRNAs as variables for the first time and appliedfour classification models including classification algorithms like Knearest neighbor KNN support vector machine SVM logistic regression and artificial neuralnetworks ANN for diagnosing T2DM and we compared the diagnostic power ofthese algorithms with each other In the present study we aimed to find the bestdata mining approach for the prediction of T2DM using six lncRNA expressionThe result of this study could improve our knowledge about the early detectionand diagnosis of T2DM using the lncRNAs as biomarkers []MethodsThe primary aim of the present study was to implement four models to predict DT2Mapplying data mining techniques based on the lncRNA variables The research objectives of our study wereImplementing data mining techniques for prediction of the DT2M Comparing the applied methodsselecting the best model for the T2DM prediction 0cKazerouni BMC Bioinformatics Page of We used the variables for predicting T2DM and comparing the performance of thevarious data mining techniques For the implementation of the algorithms we usedANACONDA3“ bit a free and source platform distribution of pythonprogramming language with a vast number of modules packages and rich libraries thatprovide various methods for classification problems For obtaining the best amount ofperformance in the models 10fold crossvalidation performed on the dataset In dealing with the small data sets crossvalidation is a prominent strategy for estimating theperformance CrossValidation is a performance evaluation technique commonly usedin practice Here the data set is repeatedly partitioned into two nonoverlapping partsa training set and a holdout set For each partitioning the holdout set is used fortesting while the remainder is used for training The two most popular variants aretenfold crossvalidation 10fold CV where the data is split into ten mutually disjointfolds []Since our samples were more than and to be sure that each fold contains thesame proportion of healthy and diabetic individuals we used the stratified 10foldcrossvalidation approach [] Therefore the results are reliable and more credibleWe applied four popular data mining approaches on the lncRNA variables regression knearest neighbors SVM and neural network classification algorithmsKNN algorithmThe knearest neighbor™s algorithm kNN is an algorithm for classifying variables regarding the closest training data in the feature space KNN uses an instancebasedlearning method which is one of the simplest algorithms among data mining techniques This method considers the nearest neighbors to each object and decides todedicate the object to classes [ ]SVM algorithmSupport Vector Machine SVM is a supervised algorithm which divides the featurespace called hyperplanes considering the target classes SVM computes classification bymaximizing the margin of the hyperplane that intercepts classes This algorithm plots amultidimensional hyperplane that divides classes and increases the margin betweenclasses to enhance the accuracy of classification We used different kernel functionsembedded in the SVM class of SVC library in python framework as a quadratic polynomial radial basis etc to classify the instance and to detect the best accuracy amongthem [“]Artificial neural networkArtificial Neural Network is a data processing algorithm that simulates the biologicalneural network in its computations A common problem in using ANN is that they actfundamentally as a black box and the parameters are set by the model so we cannotdemonstrate them [] we can just apply the model in our problems and obtain thehigh performance We used Multilayer Perceptron Neural Networks MLPNN Thestructure of a multilayer perceptron neural network has been demonstrated in Fig It maps a set of input data into a set of appropriate output classes It includes threelayers input layer hidden layer output layer The principal function of neurons of the 0cKazerouni BMC Bioinformatics Page of Fig Artificial Neural Network structureinput layer is to divide input Xi into neurons in the hidden layer The neuron of thehidden layer adds the appropriate weights of Wij to the input variables The output formula isYj ¼ fWji XiX 10 11Where f is a simple threshold function that we considered sigmoid and hyperbolictangent function []In the present study a Multilayer Perceptron Neural Networks MLPNN was performed The structure of MLPNN is as shown in Fig It makes a map of input dataonto a set of suitable output dataThe RBF networks are another type of neural network In MLP each neuron considers the weighted sum of its input values in which each input value is multiplied by acoefficient and the results are the sum of values RBF is a more intuitive approach toMLP An RBFN classifies the inputs by calculating the input™s similarity to examplesfrom the training set Each RBFN neuron stores one of the examples from the trainingset as a œprototype for classification of new input in each neuron the Euclidean distance between the input and its prototype is calculated The input is dedicated to aclass when it has more similar to that class than the other classesLogistic regressionLogistic regression is a common approach for predictive modeling practices The functionpX provides probability output between and for all values of X where X1“Xp are thepredictors The coefficients β0“βp are estimated using maximum likelihood estimationþβp Xð Þ ¼ eβ þ eβ1X1 þ‹¯Ã¾Î²Ã¾Î²pXp1X1 þ‹¯Ã¾Î²pXpDatasetThis study was based on the data obtained from three previous research conducted bySaeidi and Mansoori [ ] and the research of Parvizi and colleagues which 0cKazerouni BMC Bioinformatics Page of is not published yet We integrated these three studies and our data mining analysiswas implemented in their studies The data were collected from unrelated Iraniansubjects T2DM patients and healthy individuals matched for age and sexT2DM patients were recruited from individuals who referred to the Diabetic Clinic atShohada Hospital Tehran Iran In the current study we applied six lncRNAs expression and also six demographic variables including sex age weight height BMI andFBS for analysis and inputs of algorithms For the preprocessing phase we normalizedthe data inputs for KNN SVM and ANN models We also had low missing variablesand we replaced them with zero Table lncRNA extraction and selectionIncreasing evidence has suggested several lncRNAs are implicated in T2DM pathogenesis Recently human βcell transcriptome analysis showed lncRNAs dynamic regulation and abnormal expression of lncRNAs in T2DM [] However the extent oflncRNA deregulation in T2DM has yet to be determined To date more than100 susceptibility loci have been identified as being associated with T2DM at a genomewidesignificant level [ ] Considering this into account and by querying the GWAS catalog we candidated lncRNAs LY86AS1 HCG27_201 LINC00523 LINC00994TPT1AS1and DKFZP as target genes for this studyThe large scale GWAS have recognized approximately SNPs that were susceptibleto T2DM [] From there we used the GWAS catalog access in June to create alist of SNPs associated with T2DM In the current study we selected six lncRNA forexpression analysis according to the scan carried out in the study of Mansoori []and Saeedi [] We selected variants that had associations with increased risk ofT2DM We applied a quantitative PCR analysis of lncRNA expression levels in the samples We calculated the respective amount of each lncRNAs applying the 2ΔΔct asmeans of duplicate measurementsTable The lncRNAs as inputs of algorithmsnumberLncRNA VariablesDemographic VariablesVariablesLINC00523LINC00995HCG27_201TPT1AS1LY86AS1DKFZPSexAgeWeightHeightBMIFBS 0cKazerouni BMC Bioinformatics Page of Analysis and evaluation criteriaTo select the best performance data mining algorithms in predicting diabetic patientswe considered AUC sensitivity specificity and plotted ROC curve for the folds we ranand showed the average curve and its range [ ]ResultsTable shows the significant downregulation of PBMC expressions of the variables inthe T2DM group compared with the control group The AUC of each classificationtechnique has been demonstrated in Table AUC stands for œArea under the ROC Curve AUC computes the entire twodimensional area under the whole ROC curve According to the finding the maximumAUC dedicated to SVM and logistic regression among others knn also had the highestmean AUC and minimum standard deviation of AUC scores among the approachesThe mean and standard deviation for AUC sensitivity and specificity of each algorithmis given in Table Apart from classification AUC sensitivity and specificity the Receiver Operating Characteristic ROC with stratified crossvalidation is shown for eachapproach in Figs and ROC curves generally plot true positive rate on the Yaxis and false positive rate onthe Xaxis In other words a false positive rate of zero and a true positive rate of onein the top left corner of the plot is called the ideal point It means that a larger areaunder the curve AUC is usually better According to the demonstrated ROCs theKNN and SVM have a better area under the curve in comparison with the othersDiscussionFor a medical diagnosis optimized approaches to gain useful and accurate outcomesare essential Applying machine learning and data mining methods to automate theprocess of diagnosis may assist practitioners to enhance the quality of their clinical decisions [ ]Since T2DM is one of the prevalent diseases with severe consequences [] developing efficient methods for early detection of the disease was the primary purpose of ourresearchRegardless of high number of lncRNAs in the RNA profile of human a few numbersof them has been proved to be biologically active The role of the few lncRNAs hasbeen identified but several studies discussed the significant impact of lncRNAs in diabetic people which may represent the role of abnormal expression of lncRNAs in thelncRNAs in theincidence of T2DM [] According to the possible function ofTable Relative expression of the variablesVariablesLINC00523LINC00995HCG27_201TPT1AS1LY86AS1DKFZPDiabetesΔCT ± SEM “ “ “ “ “ “ControlΔCT ± SEM “ “ “ “ “ “pvalue 0cKazerouni BMC Bioinformatics Page of Table The AUC of algorithms for each iterationNumberof foldsAUCKNNSVMANNLogistic Regressiondevelopment of T2DM we considered the expression levels of six lncRNAs in additionto the demographic data in diabetic and healthy individuals for our study Tomeasure the expression of the lncRNAs we applied PBMCs which demonstrate an extensive proportion of the genes encoded in the human genome [] Several studies haveinvestigated different machine learning and data mining methods to predict differentdiseases [ ] such as heart diseases thyroid tumors and also diabetestype diabetes prediction In the present study we combined four commonly used datamining algorithms KNN SVM neural networks and regression to predict type diabetes using Long noncoding RNAs expression and the demographic variables for thefirst time because most of the previous studies used blood test variables or the demographic data for their analysis Receiver operating characteristic ROC analysis AUCsensitivity and specificity measure was used to assess the diagnostic value of the sixbiomarkers for T2DM The mean AUC for the KNN algorithm was obtained andwith standard deviation and we obtained the highest sensitivity with thestandard deviation of among other approaches After applying the SVM algorithm the mean AUC obtained after 10folds with the standard deviation of and the highest specificity among other approaches obtained with the standarddeviation of For the ANN we applied a multilayer perceptron with five hiddenlayers and the mean AUC of folds was and the standard deviation was Atlast for the logistic regression algorithm our results showed of mean AUC andthe standard deviation of The lower standard deviations in the AUC scores ofcomputed folds means the algorithm has worked with more performance [ ]Other studies investigated data mining algorithms for several diseases Saravananathanand Velmurugan [] applied several classification algorithms in their study to analyzeincluding KNN Sadri Sa™di [] compared three data miningdiabetes dataTable The mean and standard deviation of AUC sensitivity and specificity of algorithmsAlgorithmKNNMean AUC std ˆ’ ˆ’ ˆ’ ˆ’ Mean sensitivity ˆ’ std ˆ’ ˆ’ ˆ’ ˆ’ Mean specificity ˆ’ std ˆ’ ˆ’ ˆ’ ˆ’ SVMANNLogistic Regression 0cKazerouni BMC Bioinformatics Page of Fig The ROC for KNNalgorithms to predict T2DM and gained precision for ANN Sidiq [] reported about accuracy for KNN and accuracy for SVM algorithms applyingfor the Diagnosis of Various Thyroid Ailments In another study for the heart diseasesThe data mining algorithms indicated more than accuracy The investigated studies are in line with the findings of our study that these algorithms have a strong powerfor prediction and early detection of many diseases including T2DM and we obtainedremarkably better accuracy for prediction for example the SVM and logistic regressionaccuracy were In our study we also obtained a better accuracy for logistic regression that was and in comparison with other studies is a strong point for exampleSaeidi [] conducted a study to review two Long noncoding RNA expressions intype diabetes mellitus and with applying regressions reported about accuracyAnother research [] used two different Long noncoding RNA expressions in type Fig The ROC for SVM 0cKazerouni BMC Bioinformatics Page of Fig The ROC for MLPdiabetes mellitus and found of accuracy with the regression algorithm In thepresent study for the first time we performed four data mining algorithms on six Longnoncoding RNAs and compared their power with each other We demonstrated thatLong noncoding RNAs are effective biomarkers for data mining algorithms and have afeasible power to be applied for prediction of T2DM Also in this research we optimized the parameters of every algorithm and used stratified 10fold crossvalidation togain the best performance To be mentioned in the nearest neighbor™s algorithm theparameter k was varied between one and nine to find the bestoptimized method andwe selected k to have the best performance and the lowest standard deviation in theaccuracy of the folds In addition in choosing the parameters of the artificial neuralnetwork the number of hidden layer neurons significantly affects the accuracy of thenetwork so we set the parameters with two hidden layers with five and three neuronsFig The ROC for logistic regression 0cKazerouni BMC Bioinformatics Page of respectively to yield the best accuracy Considering the standard deviation of scores foreach algorithm the KNN had the lowest std Moreover the highest accuracy amongthe algorithms was the SVM algorithm and Logistic regression which had the maximum accuracy in folds among others We should mention that the strong points ofour study are using demographic data and six Long noncoding RNAs and combiningthem to get the best detection power of T2DM and performing four outstanding datamining algorithms and comparing their performances As the limitations of this studywe should account for the limited number of samples which is due to the high costs ofmeasuring the Long noncoding RNAs No doubt the higher number of samples wouldlead to higher performance and more reliable resultsConclusionIn this paper the performance of conventional data mining classification techniqueshas been calculated and compared for a dataset of patients referred for the screeningof type diabetes to the Shohada Hospital Iran The biomarker applied in this studydemonstrated high diagnostic value and the diagnostic process is suitable which couldhelp in the diagnosis of prediabetes and T2DMThe classification techniques compared were support vector machine artificial neuralnetwork decision tree nearest neighbors and logistic regression In data mining it isnot possible to say one classification technique will always work best and it often depends on the number of samples their distribution and the choosing of the right algorithm In this research work SVM and Logistic Regression had the best Area UnderCurve among methods of classification with the mean AUC of KNN and ANNalso had the high mean AUC and small standard deviations of AUC scores among theapproaches KNN had the highest mean sensitivity and the highest mean specificitybelonged to SVMFor future works performing other data mining and machine learning methods andusing higher numbers of samples are recommended to enhance the performanceAcknowledgmentsNot applicableAuthors™ contributionsAB and FA designed the study FA and AB collected the data and performed the statistical analysis AB and NPinterpreted the data FK ZM and LS wrote and revised the paper All authors read and approved the final manuscriptFundingNot applicableAvailability of data and materialsThe datasets used andor analyzed during the current study are available from the corresponding author onreasonable requestEthics approval and consent to participateEthical approval was obtained from the Shahid Beheshti University of Medical Sciences Ethics CommitteeIRSBMURETECHREC13951036 We informed all participants that their participation was voluntary and the study didnot state any potential risk and their identities will be private Informed written consent forms were taken from allparticipants before participationConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interests 0ccoding RNAs LINC00523 and LINC00994 in type diabetes in an Iranian cohort Mol Biol Rep “Saeidi L Ghaedi H Sadatamini M Vahabpour R Rahimipour A Shanaki M Mansoori Z Kazerouni F Long noncodingRNA LY86AS1 and HCG27_201 expression in type diabetes mellitus Mol Biol Rep “Petersmann A Nauck M MüllerWieland D Kerner W Müller UA Landgraf R Freckmann G Heinemann L Definitionclassification and diagnosis of diabetes mellitus Exp Clin Endocrinol Diabetes “Armoon B Karimy M Epidemiology of childhood overweight obesity and their related factors in a sample of preschoolchildren from Central Iran BMC Pediatr Tuomilehto J Lindström J Eriksson JG Valle TT Hämäläinen H IlanneParikka P KeinänenKiukaanniemi S Laakso MLouheranta A Rastas M Prevention of type diabetes mellitus by changes in lifestyle among subjects with impairedglucose tolerance N Engl J Med “Guariguata L Whiting DR Hambleton I Beagley J Linnenkamp U Shaw JE Global estimates of diabetes prevalence for and projections for Diabetes Res Clin Pract “Leti F DiStefano J Long noncoding RNAs as diagnostic and therapeutic targets in type diabetes and relatedcomplications Genes Heydari M Teimouri M Heshmati Z Alavinia SM Comparison of various classification algorithms in the diagnosis of type diabetes in Iran International Journal of Diabetes in Developing Countries “Kazerouni BMC Bioinformatics Page of Author details1Department of Laboratory Medicine School of Allied Medical Sciences Shahid Beheshti University of MedicalSciences Tehran Iran 2Department of Health Information Technology and Management School of Allied MedicalSciences Shahid Beheshti University of Medical Sciences Tehran Iran 3Department of Clinical Biochemistry School ofMedicine Tehran University of Medical Sciences Tehran Iran 4Department of Genetics Faculty of Medicine BabolUniversity of Medical Sciences Babol IranReceived December Accepted August ReferencesLi X Zhao Z Gao C Rao L Hao P Jian D Li W Tang H Li M The diagnostic value of whole blood lncRNAENST00000550337 for prediabetes and type diabetes mellitus Exp Clin Endocrinol Diabetes “ Mansoori Z Ghaedi H Sadatamini M Vahabpour R Rahimipour A Shanaki M Kazerouni F Downregulation of long non Perkel JM Visiting œnoncodarnia In Future Science Kapranov P Cheng J Dike S Nix DA Duttagupta R Willingham AT Stadler PF Hertel J Hackermüller J Hofacker IL RNAmaps reveal new RNA classes and a possible function for pervasive transcription Science “ Cornelis F Martin M Saut O Buy X Kind M Palussiere J Colin T Precision of manual twodimensional segmentations oflung and liver metastases and its impact on tumour response assessment using RECIST European radiologyexperimental Liao M Liu Q Li B Liao W Xie W Zhang Y A group of long noncoding RNAs identified by data mining can predict theprognosis of lung adenocarcinoma Cancer Sci Deshpande S Thakare V Data mining system and applications a review International Journal of Distributed and Parallelsystems IJDPS “ Umar Sidiq D Aaqib SM Khan RA Diagnosis of various thyroid ailments using data mining classification techniques IntJ Sci Res Coput Sci Inf Technol “ Zou Q Qu K Luo Y Yin D Ju Y Tang H Predicting diabetes mellitus with machine learning techniques Front Genet Daghistani T Alshammari R Diagnosis of diabetes by applying data mining classification techniques InternationalJournal of Advanced Computer Science and Applications IJACSA “ Meng XH Huang YX Rao DP Zhang Q Liu Q Comparison of three data mining models for predicting diabetes orprediabetes by risk factors Kaohsiung J Med Sci “ Wu H Yang S Huang Z He J Wang X Type diabetes mellitus prediction model based on data mining Informatics inMedicine Unlocked “ Vijayan V Ravikumar A Study of data mining algorithms for prediction and diagnosis of diabetes mellitus Internationaljournal of computer applications Saravananathan K Velmurugan T Analyzing diabetic data using classification algorithms in data mining Indian J SciTechnol “ Nahar N Ara F Liver disease prediction by using different decision tree techniques International Journal of Data Mining Knowledge Management Process IJDKP Vol Airola A Pahikkala T Waegeman W De Baets B Salakoski T An experimental comparison of crossvalidation techniquesfor estimating the area under the ROC curve Computational Statistics Data Analysis “ Purushotham S Tripathy B Evaluation of classifier models using stratified tenfold cross validation techniques InInternational Conference on Computing and Communication Systems Springer “ Abdar M Kalhori SRN Sutikno T Subroto IMI Arji G Comparing Performance of Data Mining Algorithms in PredictionHeart Diseases International Journal of Electrical Computer Engineering “ Sambyal RS Javid T Bansal A Performance analysis of data mining classification algorithms to predict diabetesInternational Journal of Scientific Research in Computer Science Engineering and Information Technology “ Pradhan M Kohale K Naikade P Pachore A Palwe E Design of classifier for detection of diabetes using neural networkand fuzzy knearest neighbor algorithm International Journal of Computational Engineering Research “Tzeng FY Ma KL ing the black boxdata driven visualization of neural networks IEEE Morán I Akerman Ä° Van De Bunt M Xie R Benazra M Nammo T Arnes L Nakić N GarcíaHurtado J RodríguezSeguí SHuman β cell transcriptome analysis uncovers lncRNAs that are tissuespecific dynamically regulated and abnormallyexpressed in type diabetes Cell Metab “ 0cKazerouni BMC Bioinformatics Page of Voight BF Scott LJ Steinthorsdottir V Morris AP Dina C Welch RP Zeggini E Huth C Aulchenko YS Thorleifsson GTwelve type diabetes susceptibility loci identified through largescale association analysis Nat Genet Imamura M Maeda S Genetics of type diabetes the GWAS era and future perspectives Endocr J “Soni J Ansari U Sharma D Soni S Predictive data mining for medical diagnosis an overview of heart diseaseprediction International Journal of Computer Applications “ Asadi F Paydar S Presenting an evaluation model of the trauma registry software Int J Med Inform “ Dangare CS Apte SS Improved study of

Thyroid_Cancer

"evaluate the clinicopathologic characteristics of Lymph Node metastasisbetween investing layer of Cervical fascia and deep fascia of infrahyoid strap Muscles LNCM in papillary thyroidcarcinoma PTCMethods Retrospective review of patients with PTC who underwent thyroidectomy and central compartment neckdissection CND from January to January was performed in two tertiary referral academic medicalcenters A total of consecutive patients with PTC who underwent thyroidectomy and CND were included inthe retrospective review The LNCM was resected as a separate specimen by the surgeon and the clinicopathologiccharacteristics of the patients were recorded Multivariate logistic regression analysis was performed to identify riskfactors for LNCM metastasisResults Of PTC patients patients had lymph nodes in the LNCM Among them caseswere confirmed to be positive in the LNCM In total the metastasis rate of LNCM in PTC patients was Univariate analysis revealed that the metastasis of LNCM were more likely to have a primary site in theinferior pole extrathyroidal extension ETE central cervical metastasis level III and level IV metastasis Multivariateanalysis further showed tumor location in the inferior pole ETE level III and level IV metastasis conferred asignificantly increased odds ratio for LNCM metastasisConclusion Attention should be paid to the lymph tissue in the LNCM for PTC patients especially in presence of aprimary site in the inferior pole ETE level III and level IV metastasisKeywords Thyroid carcinoma Surgery Central compartment neck dissection Recurrence Suprasternal spaceIntroductionPatients with papillary thyroid carcinoma PTC have afavorable prognosis with central neck locoregional recurrence varying from to [] The goal of a prophylacticor therapeutic central compartment neck dissection pCNDor tCND is to decrease the incidence of local recurrenceby removing all lymphatic tissue within the levels VI and Correspondence wugaosongwhueducn1Department of Thyroid and Breast Surgery Zhongnan Hospital of WuhanUniversity Donghu Road Wuhan Hubei People™s Republic of ChinaFull list of author information is available at the end of the VII compartments which are generally the first and themost commonly involved with metastasis [] For patientswithout evidence of lymph node metastasis on preoperativeevaluation the additive value of a pCND at the time ofthyroidectomy is controversial Some authors advocatepCND considering high rate “ of occult metastaticnodal disease in cN0 PTC [] while other authors considerthat there is no highlevel evidence in favor of pCND []The performance of pCND is dependent on the weightgiven to the risks and benefits of pCND [] Consideringthe oncologic benefits of CND and the risks of a repeat The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of neck operation performing pCND is recommended toevery patient in China [ ]Although American Thyroid Association ATA guideline has defined the boundary of central neck compartment there is also significant variability in terms of theextent of CND In routine clinical practice CND canrange from sampling a few nodes in the paratrachealregion to a complete clearance from left carotid artery toright carotid artery and down to and including the uppermediastinum [] Owing to the variant extent of CNDsome central compartments are easily to be neglectedFor thyroid carcinoma patients with specific clinicopathologic characteristics incomplete lymph node dissectionmay result in increased recurrence reoperation andreoperationassociated complications [] Lymph Nodebetween investing layer of Cervical fascia and deep fasciainfrahyoid strap Muscles LNCM has not beenofreported The LNCM compartment is defined as followssuperiorly by the hyoid bonelaterally by the carotidarteries anteriorly by the investing layer of cervicalfascia and posteriorly by the deep fascia of infrahyoidstrap muscles LNCM space includes suprasternal spaceand intrainfrahyoid strap muscle spaceAnatomically LNCM is located anterior to the strapmuscles We consider that what is special about the concept of the LNCM is that it is belong to level VI but is aneasily overlooked anatomical area by a strap musculatureinvolving the sternohyoid and sternothyroid musclesduring selective or modified neck dissection Although themetastasis in LNCM was seldom it did occur in somePTC patients with regional recurrence As part of LCNMsuprasternal space metastasis for thyroid cancer wereinvestigated in three studies [“] Thus we routinelydetected the suprasternal space and intrainfrahyoid strapmuscle space Fig This study was performed to identify the clinicopathologic characteristics and indication forlymph node metastasis dissection in the LNCMMaterials and methodsPatientsA retrospective review from the clinical and histopathologydatabase of two tertiary referral academic medical centersTongji Hospital of Huazhong University of Science andTechnology and Zhongnan Hospital of Wuhan Universityfrom January to January were conducted In theinstitutions preoperative examinations consisted of athorough physical examination neck ultrasound a clinicalevaluation of thyroid nodules and neck lymph nodes Fineneedle aspiration cytology FNAC were performed in patients who were suspected thyroid nodules or lymph nodeWith a pathological confirmation of PTC all the patientsreceived a thyroidectomy with CND Accordingly a pCNDwas performed for cN0 patients and a therapeutic CNDwas performed for cN1 patients The inclusion criteria forthe patients were as follows the clinical history completely recorded the LNCM was resected as a separatespecimen by the surgeon PTC patients who underwentthyroidectomy plus CND with or without lateral neckdissection A total of consecutive PTC patients wereenrolled The medical ethic committee of ZhongnanHospital of Wuhan University approved the procedure andinformed written consent was obtained from all patientsSurgical approachAll the operations were performed by the same seniorsurgeon Gaosong Wu with the patients under generalFig Four subdivisions Level VI 1st Level VI 2nd Level VI 3rd and Level VI 4th of central neck compartment are divided by deep fascia ofinfrahyoid muscles pretracheal visceral fascial and right recurrent laryngeal nerve 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of anesthesia Thyroidectomy was performed with a standard technique of fine capsular en bloc dissection andresection from inferior pole to superior pole [“]Intraoperative neuromonitoring was employed for all ofthe thyroidectomies [] Superior parathyroid glandswere identified and preserved in situ inferior parathyroidglands were protected in situ or autotransplanted in thesternocleidomastoid muscle according to three certaintypes based on their blood supply and location [ ]After the incision of the investing layer of cervicalfascia the interval between sternohyoid and sternothyroid muscles and the space anterior to the sternohyoidmuscle above the clavicle and the sternum weredetected If there was fibrofatty tissue Instead of the enbloc removal of the entire centrallymph nodes theLNCM was resected as a separate specimen if occurredThe presence or absence of lymph node metastasis wasdefined according to postoperative pathological reportsWhile dissecting paratracheal lymph nodes intraoperative neuromonitoring was employed to detect RLN fromdistally to proximally minimizing morbidity from injuryto RLN during compartment nodal dissection LNCMand other compartment lymphatic tissue were processedfor routine hematoxylin and eosine HE separatelyThe pathologic results were independently determinedby two qualified pathologists without any prior knowledge of the patients™ clinical dataData collection and statistics analysisTo determine the relation between LNCM metastasis andclinicopathologic factors such as age sex primary tumorsite lateral cervical lymph node metastasis level VI metastasis the chisquare test and Fisher™s exact test were usedas appropriate Multivariate logistic regression analysiswas performed to identify risk factors for LNCM metastasis of PTC P was considered statistically significantAll calculations were performed using SPSS for windows Postthyroidectomy hypocalcemia lasting for morethan months was considered as permanent VCP All patients were followed up every “ months postoperativelyResultsPatients detected with LNCMAfter reviewing patients who underwent thyroidectomy plus CND with or without lateral neck dissectionfrom January to January patients were detected with LNCM and patients wereabsent of LNCM The average tumor size of LNCM was cm and the mean number of lymph nodes sampledfrom LNCM was ranging from to Table showsthe comparison of clinicopathologic characteristics between the present LNCM group and the absent groupIn univariate analysis Hashimoto™s disease p multifocality leisions p the tumor located ininferior portion p extrathyroidal extensionETE p central cervical metastasis p level III and level IV metastasis p were significantly associated with high prevalence of LNCMPatients with metastatic LNCMAmong a total of patients with LNCM metastaticLNCM was found in patients Table compares the clinicopathologic characteristics between themetastatic LNCM group and the nonmetastatic LNCMgroup Three hundred eightythree patients were confirmed free of LNCM metastasis of themwith clinically negative node performed pCND and of them with clinically positive performed tCND All thepatients in the metastatic LNCM group performedtCND Lateral neck dissection was performed in cases in the metastatic LNCM group and cases in the nonmetastatic group all lateral neckdissection wastherapeutically performed Univariateanalysis was performed for the patients with and patients without metastatic LNCM Age at diagnosisgender and tumor size coexistent thyroid disease tumorfocality and level II metastasis were not correlated withLNCM metastasis Univariate analysis identified tumorlocated in the inferior pole central cervical metastasisETE level III and level IV metastasis as significant predictors of LNCM metastasis in our study population Multivariate analysis further showed that tumor location ETElevel III and level IV metastasis conferred a significantlyincreased odds ratio for LNCM metastasis Table ComplicationsThe median followup time was months range “ of patients had voice changes all of whomrecovered within “ months Temporary vocal cord paralysis was confirmed in patients by laryngoscope andthirteen permanent hypocalcemia was observed aftersurgeryDiscussionIn order to achieve the best chance of cure and effectivedisease control thoroughness of dissection has to betaken into account We prospectively performed comprehensive CND for PTC patients who underwent thyroidectomy and CND In addition data were analyzed for PTC patients to investigate the clinicopathologic characteristics for LNCM metastasis The occurrence rate ofLNCM was and of the patients harbored metastatic LNCM In total the positiverate of the LNCM was In this studymultivariate analysis revealed that a primary site in the inferior pole ETE level III and level IV metastasis were ofhigher LNCM metastasis rate which was consistent withthe findings by the previous report oflymph node 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of Table Univariate analysis of demographic and clinicopathologic factors for patients who had lymph nodes in LNCM compared tothose who did notVariablesAge mean ± SDAbsent n Present n P value‰¤ GenderFemale MaleTumor size cm ‰¥ cmCoexistent thyroidNodular goiterYes NoToxic goiterYes NoHashimoto™s diseaseYes NoTumor focalityMultifocality UnifocalityTumor locationInferior portionUpperMiddle portionExtrathyroidal extensionYes NoCentral cervical metastasisYes NoLateral cervical metastasisLevel IIYes NoLevel IIIYes NoLevel IVYes No LNCM Lymph Node between superficial layer of deep Cervical fascia and deep fascia of infrahyoid Musclesmetastasis between sternocleidomastoid and sternohyoid muscle []Several studies have emphasized the importance of similar compartment in neck dissection for thyroid carcinomaSun pioneered the confirmation of the significantinvolvement of lymph node metastasis between sternocleidomastoid and sternohyoid muscle LNSS in lateralneck dissection [] which anatomically classified as part ofthe space of Burns They concluded that the positive rate ofLNSS was in clinically nodepositive cN PTCwhich was correlated with a primary site in the inferiorpole the lateral nodal metastasis level III and level IV nodalmetastasis [] Then Homma [] reported two casesof PTC patients with level III and IV lymph node metastases as well as metastasis in the suprasternal space Yu et al[] investigated the clinical significance of the suprasternalspace lymph node SSLN in pathological nodepositivepN PTC patients They concluded that metastasis rate ofSSLN was and the high SSLN metastasis of PTC wascorrelated with primary cancer site in the inferior thyroidpole strap muscle invasion level IV metastasis and LNSSmetastasis In our experience LNCM was rarely occurredin the central neck compartment and the positiveLNCM in PTC patients was infrequent as well Notably among the patients with pN PTC themetastasis rate of LNCM was which was muchlower than the metastasis incidence of SSLN described by Yu [] According to their results onefifth patients with pN PTC were performed incompleteCND and remained metastatic lymph nodes 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of Table Univariate analysis of demographic and clinicopathologic factors for positive LNCMVariablesAge mean ± SDMetastasis n Nonmetastasis n ‰¤ GenderFemale MaleTumor size cm ‰¥ cmCoexistent thyroidNodular goiterYes NoToxic goiterYes NoHashimoto™s diseaseYes NoTumor focalityMultifocality UnifocalityTumor locationInferior portionUpperMiddle portionExtrathyroidal extensionYes NoCentral cervical metastasisYes NoLateral cervical metastasisLevel IIYes NoLevel IIIYes NoLevel IVYes NoLNCM Lymph Node between superficial layer of deep Cervical fascia and deep fascia of infrahyoid MusclesP value The total number of lymph nodes in the central neckcan range from to [] There is no consensus on thenumber of nodes removed or examined that would constitute an adequate dissection Aimed to allow surgeons tomore accurately report the extent of lymphadenectomyTable Multivariate analysis of predictors for LNCM positivityVariablesTumor locationp valueCentral cervical metastasisExtrathyroidal extensionLevel III metastasisLevel IV metastasisOR CI “ “ “ “ “LNCM Lymph Node between superficial layer of deep Cervical fascia and deepfascia of infrahyoid Musclesvisceralwe divide the central neck compartment into four subdivisions by deep fascia of infrahyoid strap muscles pretrachealfascial and right RLN Fig Theproposed LNCM compartment is bounded superiorly bythe hyoid bone laterally by the carotid arteries anteriorlyby the investing layer of the cervical fascia and posteriorlyby the deep fascia of infrahyoid strap muscles which is defined as Level VI 1st In the current study suprasternalspace composed part of the LNCM Fig Compared toSSLN reported by Yu [] LNCM encompasseslymph nodes in the suprasternal space and lymph nodesbetween sternohyoid and sternothyroid muscles Figs and LNCM can fall under the normal subdivisions ofthe central compartment Subdivisions can actually recordthe extent of the CND which is able to provide detailedinformation for the possible second operation 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of Fig LNCM coverage area in vivo Lymph nodes between sternohyoid and sternothyroid muscles a and lymph nodes in the suprasternal space bIncluding LNCM as an anatomical part of the centralneck allows for removal of previously unrecognized micrometastatic disease in of PTC patients with the inferiorportion lesions ETElevel III and level IV metastasisDissection of the LNCM space is less invasive and easy toachieve and is not timeconsuming It is at the entrance ofcentral neck compartment which is easy to expose and haslow risk of damaging RLN or parathyroid With the application ofintraoperative neuromonitoring and in situpreservation or autotransplantation of parathyroid theoccurrence of vocal cord paralysis and permanenthypoparathyroidism in the current study were lowerin this study [ ] Therefore in cases where LNCMspace metastasis is suspected or preoperative ultrasoundand CT suggests LNCM metastasis greater attentionshould be paid to the nodal tissue in the LNCM space inthyroid carcinoma patients These patients might benefitfrom a reduced risk of regional recurrence central neckreoperative morbidity and improved decision making inrelation to the use of radioiodine ablationThere are severallimitations in the present studyThe retrospective design is a limitation of the studyAnd this was two tertiary referral centers retrospective review and routine prophylactic nodal surgerywas offered in China however it is not standard elsewhere in the world which is a major limitation Aprospective randomized trial with a long time followup period may help to further evaluate the clinicalsignificance of LNCM in PTC patientsConclusionsIn summary additional dissection of nodes in theLNCM were accessible and might not increase morbidity Therefore attention should be paid to the lymphtissue between investing layer of cervical fascia and deepfascia ofinfrahyoid strap muscles for PTC patientsespecially in presence of inferior portion lesions ETElevel III and level IV metastasisAcknowledgementsThe authors thank the studied patients for their willingness to cooperatewith our studyAuthors™ contributionsGaosong Wu Study concepts and design Qianqian Yuan Study designmanuscript preparation and editing Jinxuan Hou Data analysis andmanuscript editing Yiqin Liao Data acquisition Lewei Zheng Manuscriptpreparation Fang Lu Data acquisition Kun Wang Quality control of dataand algorithms The authors read and approved the final manuscriptFundingThe authors have no support or funding to reportAvailability of data and materialsNot applicableEthics approval and consent to participateThis research was comprised of human participants and was approved byMedical Ethics Committee of Wuhan University Zhongnan Hospital Informedconsent was obtained from all individual participants included in the studyConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Thyroid and Breast Surgery Zhongnan Hospital of WuhanUniversity Donghu Road Wuhan Hubei People™s Republic of China 2Department of Thyroid and Breast Surgery Tongji Hospital of TongjiMedical College of Huazhong University of Science and Technology Jiefang Avenue Wuhan Hubei People™s Republic of China Received April Accepted August ReferencesLang BH Ng SH Lau LL Cowling BJ Wong KP Wan KY A systematic reviewand metaanalysis of prophylactic central neck dissection on shorttermlocoregional recurrence in papillary thyroid carcinoma after totalthyroidectomy Thyroid “So YK Seo MY Son Y Prophylactic central lymph node dissection forclinically nodenegative papillary thyroid microcarcinoma influence onserum thyroglobulin level recurrence rate and postoperative complicationsSurgery “Hughes DT Rosen JE Evans DB Grubbs E Wang TS Solórzano CCProphylactic central compartment neck dissection in papillary thyroid 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of Cancer and effect on Locoregional recurrence Ann Surg Oncol “ McHenry CR Is prophylactic central compartment neck dissection indicatedfor clinically nodenegative papillary thyroid Cancer the answer isdependent on how the data are interpreted and the weight given to therisks and benefits Ann Surg Oncol “Selberherr A Riss P Scheuba C Niederle B Prophylactic œfirststep centralneck dissection level does not increase morbidity after Totalthyroidectomy Ann Surg Oncol “Hartl DM Leboulleux S Al Ghuzlan A Baudin E Chami L Schlumberger M Optimization of staging of the neck with prophylactic central andlateral neck dissection for papillary thyroid carcinoma Ann Surg “ McAlister ED Goldstein DP Rotstein LE Redefining classification ofcentral neck dissection in differentiated thyroid cancer Head Neck“ Miller JE AlAttar NC Brown OH Shaughness GG Rosculet NP Avram AM Location and causation of residual lymph node metastasis aftersurgical treatment of regionally advanced differentiated thyroid CancerThyroid “Sun G Wang Y Zhu Y Wang Y Xu K Wei W Lymph node metastasisbetween sternocleidomastoid and sternohyoid muscle in clinically nodepositive papillary thyroid carcinoma Head Neck “ Homma A Hatakeyama H Mizumachi T Furusawa J Kano S Sakashita T Lymph node metastasis in the suprasternal space from thyroidpapillary cancer Int Cancer Conf J “ Yu S Ge J Sun B Wei Z Lei S Lymph node metastasis in suprasternal spacein pathological node“positive papillary thyroid carcinoma Eur J Surg Oncol“ Wu G Kong D Thyroidectomy with Wu Gaosong's ProcedureVideoEndocrinologr httpsdoi101089ve20150050 Wu G Wang K Intraoperative Neuromonitoring and Protection of theSuperior Laryngeal Nerve with Wu Gaosong's ProcedureVideoEndocrinology httpsdoi101089ve20160070 Wu G Cui Q Wang K Carbon Nanops for Identifying Lymph Nodesand Protecting Parathyroid Glands in Thyroid Lobectomy with IpsilateralCentral Compartment Lymph Nodes Dissection VideoEndocrinology httpsdoi101089ve20160064Kong D Cui Q Gaosong W A Novel Classification of Parathyroid Glands andTheir Preservation in Thyroidectomy VideoEndocrinology httpsdoi101089ve20170093 Wang K Wu G Intraoperative Neuromonitoring in Selective Neck Dissectionfor Thyroid Cancer SND IIa Vb with Wu Gaosong's ProcedureVideoEndocrinology httpsdoi101089ve20160082 Yuan Q Wu G Hou J Liao X Liao Y Chiang F Correlation betweenelectrophysiological changes and outcomes of vocal cord function in recurrent laryngeal nerves with visual integrity during thyroidectomyThyroid “ Cui Q Li Z Kong D Wang K Wu G A prospective cohort study of novelfunctional types of parathyroid glands in thyroidectomy Medicine 9552e5810Tavares MR Cruz JA Waisberg DR Toledo SP Takeda FR Cernea CR et alLymph node distribution in the central compartment of the neck ananatomic study Head Neck “ Wang K Cai H Kong D Cui Q Zhang D Wu G The identificationpreservation and classification of the external branch of the superiorlaryngeal nerve in thyroidectomy World J Surg “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

Thyroid_Cancer

EndocrinesyPostoperative vocal fold dysfunction in covid19 era are we still intime for a recoveryElena Bonati Elena Giovanna Bignami2 Paolo Del Rio1Received May Accepted July Springer ScienceBusiness Media LLC part of Springer Nature To the EditorThe novel coronavirus COVID19 is a highly contagious zoonosis produced by SARSCoV2 which arose inChina and spread all over the world transmitting from manto man through respiratory secretions In March itwas defined by the World Health anization WHO as apandemic to underline its spread and severityHealthcare professionals are one of the categories most atrisk of contracting the infection in particular when theiractivity involves the direct management of the patient™sairways Among these categories we can count anesthetistshead and neck surgeons otolaryngologists maxillofacialsurgeons ophthalmologists and dentists For these reasonsthe latest evidencebased recommendations for otolaryngology and head and neck surgery practice suggest thathealthcare facilities should prioritize urgent and emergencyvisits and procedures until this condition stabilizes ceasingelective care []Nevertheless oncological surgical activity althoughslowed down did not stop in most hub hospitals Regardingthyroid cancer thyroid surgery is complex and the rate ofnerve damage is still considerable Immediate postoperativevocal fold rate is “ in our case study and decrease to“ after months Postoperative dysphonia can becaused by several factors other than nerve damage such astracheal intubation or scarring in the thyroid lodge It istherefore important to identify the cause of vocal corddysfunction and treat it correctly at the right time If anunilateral vocal fold paresisparalysis is diagnosedthetreatment consist in improving the speech while in case of Elena Bonatiebonati86gmailcom General Surgery Unit Department of Medicine and SurgeryParma University Hospital Parma Italy Unit of Anesthesiology Department of Medicine and SurgeryParma University Hospital Parma Italybilateral vocal fold paresisparalysis respiratory obstructionalso needs to be urgently treated Fortunately we havebroughtthe incidence of this last and most dangerouscomplication to at our Clinic since the introduction in of the routine use of intraoperative neuromonitoringduring thyroidectomyThe latest guidelines published by the Americanin March Association of Endocrine Surgeonsrecommend laryngeal examination in patients with knownor suspected new recurrent laryngeal nerve dysfunctionafter thyroidectomy for additional evaluation and possibletreatment with a speech pathologist According to theAmerican Academy of Otolaryngology”Head and NeckSurgery they assert that early referral “ weeks postsurgery to a laryngologistin combination with earlyintervention results in superior voice outcomes since theideal time for vocal fold augmentation is months afterthyroidectomy []A metaanalysis about therapy for vocal fold paresisparalysis after thyroidectomy concluded that the timingof therapy for unilateral vocal fold paralysis after thyroidectomy has a significant impact on the effect sizebeing significantly greater if therapy is performed within months This may be explained by progressive atrofolds and disappearance of nervephy offunction so that vocalfold movements cannot berecovered []the vocalPatients who underwent thyroid surgery from February and who had experienced a vocal fold disfunctionVFD were unable to undergo a laryngoscopy nor muchless a speech therapy according to health measuresnecessary to contain the spread of the virus This unfortunately causes a progressively reduced possibility ofrecovery increasing the specific morbidity related to surgery for thyroid cancer in this period The only indicationthat we can give to patients is the rest of the voice to avoidthe establishment of compensation mechanisms worseningthe clinical picture waiting to be able to resume the correcttreatment 0cTherapeutic diagnostic pathways in the COVID19 erahave become difficult and dangerous logarithms that mustconsider the need for patient care and the possibility oftreatments delay in safety but also the risk of contagion ofthe patientsleast protection ofhealthcare personnel The hospital setup has been significantly changed and much of the economic structuraland human health resources have been dedicated to themanagement of the COVID pandemicthemselves and notIn parallel with the COVID19 emergency we areexperiencing another health emergencythe one thatinvolves the management of nonCOVID19 patients Evenin the second phase of the pandemic only urgent healthservices are provided A reanizing effort within theindividual healthcare companies is required to guaranteetreatment even for nonCOVID19 patientsMoreoverThe COVID19 pandemic highlighted the limits andweaknesses of our health system and now that the correctprotocols for the protection of healthcare personnel havebeen described allthe all healthcare companies shouldequip their staff with the appropriate materials such as N95masks hair cover protective coverall gown gloves faceshields goggles and shoe covers In the face of higherexpenses this would allow the resumption of activitiesminimizing the risk of an increase in the rate of infectionroutine health practices must be reconsidered preferring less invasive techniques in order toscreen patients who need secondlevel examination Evenif not used yet in our hospital transcutaneous laryngealultrasonography is a valid noninvasive and painlessalternative method in the assessment of vocal cords It hasbeen demonstrated in a recent prospective multicentricstudy that it has concordance with laryngoscopy in themajority of cases and so it can be a valid alternative asfirstline exam for vocalfold examination pre andpostoperativelyEndocrineFinally the growing use of virtual platforms for the needof social distancing could encourage their application evenin healthcare services that can be performed by teleconference such as speech therapyWe can assess that COVID19 pandemic is causingdirect morbidity and mortality and even a related one dueto missed or delayed treatment of multiple nonCOVID19diseases The delivery of the health service should beimproved and the health system itself must be modernizedto adapt to new needsCompliance with ethical standardsConflict of interest The authors declare that they have no conflict ofinterestPublisher™s note Springer Nature remains neutral with regard tojurisdictional claims in published maps and institutional affiliationsReferences LP Kowalski A Sanabria JA Ridge WT Ng R de BreeA Rinaldo RP Takes A A Mkitie AL Carvalho CR Bradford V Paleri DM Hartl V Vander Poorten IJ Nixon C PiazzaPD Lacy JP Rodrigo O GuntinasLichius WM MendenhallA D™Cruz AWM Lee A Ferlito COVID19 pandemic effectsand evidencebased recommendations for otolaryngology and headand neck surgery practice Head Neck 101002hed26164 KN Patel L Yip CC Lubitz EG Grubbs BS Miller W ShenP Angelos H Chen GM Doherty TJ Fahey 3rd E KebebewVA Livolsi ND Perrier JA Sipos JA Sosa D StewardRP Tufano CR McHenry SE Carty The American Associationof Endocrine Surgeons Guidelines for the definitive surgical management of thyroid disease in adults Ann Surg e21“e93 X Chen P Wan Y Yu M Li Y Xu P Huang Z Huang Typesand timing of therapy for vocal fold paresisparalysis after thyroidectomy a systematic review and metaanalysis J Voice “ 0c'

Thyroid_Cancer

patients who have diabetes suffer from Type DMT2DM Many studies suggest using the significant role of lncRNAs to improve thediagnosis of T2DM Machine learning and Data Mining techniques are tools that canimprove the analysis and interpretation or extraction of knowledge from the dataThese techniques may enhance the prognosis and diagnosis associated withreducing diseases such as T2DM We applied four classification models including Knearest neighbor KNN support vector machine SVM logistic regression andartificial neural networks ANN for diagnosing T2DM and we compared thediagnostic power of these algorithms with each other We performed the algorithmson six LncRNA variables LINC00523 LINC00995 HCG27_201 TPT1AS1 LY86AS1DKFZP and demographic dataResults To select the best performance we considered the AUC sensitivityspecificity plotted the ROC curve and showed the average curve and range Themean AUC for the KNN algorithm was with standard deviation SD themean sensitivity and specificity were and respectively After applying theSVM algorithm the mean AUC obtained after stratified 10fold crossvalidationand the SD obtained The mean sensitivity and specificity were and respectively The mean AUC for ANN and the SD were and also the meansensitivity and specificity were and At last for the logistic regressionalgorithm our results showed of mean AUC and the SD of the meansensitivity and specificity were and respectively According to the ROCs theLogistic Regression and SVM had a better area under the curve compared to theothersContinued on next page The Authors Access This is licensed under a Creative Commons Attribution International License whichpermits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit tothe original authors and the source provide a link to the Creative Commons licence and indicate if changes were made Theimages or other third party material in this are included in the 's Creative Commons licence unless indicated otherwisein a credit line to the material If material is not included in the 's Creative Commons licence and your intended use is notpermitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyrightholder To view a copy of this licence visit httpcreativecommonslicensesby40 The Creative Commons Public DomainDedication waiver httpcreativecommonspublicdomainzero10 applies to the data made available in this unlessotherwise stated in a credit line to the data 0cKazerouni BMC Bioinformatics Page of Continued from previous pageConclusion We aimed to find the best data mining approach for the prediction ofT2DM using six lncRNA expression According to the finding the maximum AUCdedicated to SVM and logistic regression among others KNN and ANN also had thehigh mean AUC and small standard deviations of AUC scores among the approachesKNN had the highest mean sensitivity and the highest specificity belonged to SVMThis study™s result could improve our knowledge about the early detection anddiagnosis of T2DM using the lncRNAs as biomarkersKeywords Data mining Gene expression Machine learning algorithms Type diabetes mellitusBackgroundDiabetes mellitus DM is one of the most prevalent chronic noncommunicable diseases NCD around the world about of the patients who have diabetes sufferfrom Type DM T2DM [] The risk of developing T2DM is strongly associated withmany predispositions behavioral and environmental risk factors and also genetic factors [“] Besides the genetic factors strong evidence indicates that factors such asobesity and physical inactivity are the main nongenetic determinants of the disease [] T2DM can range from predominant insulin resistance with relative insulin deficiency to dominant defective secretion with insulin resistance [] It is often related tometabolic syndrome problems Individuals who have impaired glucose tolerance arehighrisk subjects of type diabetes []Studies demonstrate a drastic increase of the disease in recent decades The trendsestimate that by more than million people will be affected by the disease []People who suffer from T2DM are susceptible to many forms of complications leadingto morbidity and mortality in these patients Many studies emphasize the genetic factors in the pathogenesis of T2DM [ ] Long noncoding RNAs long ncRNAslncRNA are subsets of RNA specified as being transcripts with lengths exceeding nucleotides that could not be translated into protein [] Long noncoding RNAslncRNAs belong to a heterogeneous class of regulatory lncRNAs with transcriptlengths nucleotides which have a positive role in the development and growth ofseveral various diseases including T2DM supporting the hypothesis that abnormal expression of LncRNAs is related to various diseases [] Besides considering the significant role of lncRNAs in disease pathogenesis increasing researches suggest using themto improve diagnosis prognosis and clinical management of T2DM Genomewide association studies GWAS have recently introduced several particular diabetesrelatedloci in the human genome [] Also many studies discovered the relationship betweenmore than susceptible loci and T2DM at a genomewide significant level [ ]Deregulation of genes located in GWAS defined loci may be risk factors for human diseases concerning which we applied the GWAS catalog to select six lncRNAsLINC00523 LINC00995 CG27_201 TPT1AS1LY86AS1 DKFZP as our gene targets for the present study [] Knowledge Discovery in Databases KDD or data miningare techniques for the computational process of discovering patterns in large datasetscontaining various approaches such as artificial intelligence machine learning statisticsand database systems [] These methods are applied to recognize patterns in dataprediction association and classification problems [ ] Considering the 0cKazerouni BMC Bioinformatics Page of importance of early detection of T2DM machine learning and Data Mining techniquesare tools that can improve the analysis and interpretation or extraction of knowledgefrom the data [ ] These techniques may enhance the prognosis and diagnosis associated with life quality reducing diseases such as T2DM [ ]To date several other studies tried to predict diabetes mellitus using outstandingdata mining techniques [“] Vijayan et al[] applied the expectationmaximization algorithm KNN algorithm Kmeans algorithm amalgam KNN algorithm and ANFIS algorithm to predict and diagnose Diabetes Mellitus They usedthe UCI dataset containing blood test and demographic variables and their resultsshowed that EM possessed the least classification accuracy and amalgam KNN andANFIS provided better classification accuracy of more than and respectively Another study conducted by Saravananathan [] used popular classifiincluding J48 Support Vector Machines SVM Classificationcation algorithmsand Regression Tree CART and kNearest Neighbor kNNfor diabetic dataTheir performance indicators were accuracy specificity sensitivity precision errorrate They found that the J48 technique™s performance was remarkably superior tothe other three techniques for the classification of diabetes data Meng []compared three data mining models of logistic regression ANN and decision treefor predicting diabetes mellitus or prediabetes by risk factors They gathered information about demographic characteristics family diabetes history anthropometricmeasurements and lifestyle risk The decision tree model C50 had the best classification performance with an accuracy of with a sensitivity of andspecificity of Another study performed by Saeidi [] used logistic regression to assess the diagnostic value of LY86AS1 and HCG27_201 as biomarkersfor T2DM They obtained a sensitivity of and specificity of Anotherstudy [] used two other lncRNAs including LINC00523 and LINC00994 expressions for the evaluation of their potential diagnostic value for T2DM They appliedlogistic regression and achieved a sensitivity of and specificity of Inour study we combined six lncRNAs as variables for the first time and appliedfour classification models including classification algorithms like Knearest neighbor KNN support vector machine SVM logistic regression and artificial neuralnetworks ANN for diagnosing T2DM and we compared the diagnostic power ofthese algorithms with each other In the present study we aimed to find the bestdata mining approach for the prediction of T2DM using six lncRNA expressionThe result of this study could improve our knowledge about the early detectionand diagnosis of T2DM using the lncRNAs as biomarkers []MethodsThe primary aim of the present study was to implement four models to predict DT2Mapplying data mining techniques based on the lncRNA variables The research objectives of our study wereImplementing data mining techniques for prediction of the DT2M Comparing the applied methodsselecting the best model for the T2DM prediction 0cKazerouni BMC Bioinformatics Page of We used the variables for predicting T2DM and comparing the performance of thevarious data mining techniques For the implementation of the algorithms we usedANACONDA3“ bit a free and source platform distribution of pythonprogramming language with a vast number of modules packages and rich libraries thatprovide various methods for classification problems For obtaining the best amount ofperformance in the models 10fold crossvalidation performed on the dataset In dealing with the small data sets crossvalidation is a prominent strategy for estimating theperformance CrossValidation is a performance evaluation technique commonly usedin practice Here the data set is repeatedly partitioned into two nonoverlapping partsa training set and a holdout set For each partitioning the holdout set is used fortesting while the remainder is used for training The two most popular variants aretenfold crossvalidation 10fold CV where the data is split into ten mutually disjointfolds []Since our samples were more than and to be sure that each fold contains thesame proportion of healthy and diabetic individuals we used the stratified 10foldcrossvalidation approach [] Therefore the results are reliable and more credibleWe applied four popular data mining approaches on the lncRNA variables regression knearest neighbors SVM and neural network classification algorithmsKNN algorithmThe knearest neighbor™s algorithm kNN is an algorithm for classifying variables regarding the closest training data in the feature space KNN uses an instancebasedlearning method which is one of the simplest algorithms among data mining techniques This method considers the nearest neighbors to each object and decides todedicate the object to classes [ ]SVM algorithmSupport Vector Machine SVM is a supervised algorithm which divides the featurespace called hyperplanes considering the target classes SVM computes classification bymaximizing the margin of the hyperplane that intercepts classes This algorithm plots amultidimensional hyperplane that divides classes and increases the margin betweenclasses to enhance the accuracy of classification We used different kernel functionsembedded in the SVM class of SVC library in python framework as a quadratic polynomial radial basis etc to classify the instance and to detect the best accuracy amongthem [“]Artificial neural networkArtificial Neural Network is a data processing algorithm that simulates the biologicalneural network in its computations A common problem in using ANN is that they actfundamentally as a black box and the parameters are set by the model so we cannotdemonstrate them [] we can just apply the model in our problems and obtain thehigh performance We used Multilayer Perceptron Neural Networks MLPNN Thestructure of a multilayer perceptron neural network has been demonstrated in Fig It maps a set of input data into a set of appropriate output classes It includes threelayers input layer hidden layer output layer The principal function of neurons of the 0cKazerouni BMC Bioinformatics Page of Fig Artificial Neural Network structureinput layer is to divide input Xi into neurons in the hidden layer The neuron of thehidden layer adds the appropriate weights of Wij to the input variables The output formula isYj ¼ fWji XiX 10 11Where f is a simple threshold function that we considered sigmoid and hyperbolictangent function []In the present study a Multilayer Perceptron Neural Networks MLPNN was performed The structure of MLPNN is as shown in Fig It makes a map of input dataonto a set of suitable output dataThe RBF networks are another type of neural network In MLP each neuron considers the weighted sum of its input values in which each input value is multiplied by acoefficient and the results are the sum of values RBF is a more intuitive approach toMLP An RBFN classifies the inputs by calculating the input™s similarity to examplesfrom the training set Each RBFN neuron stores one of the examples from the trainingset as a œprototype for classification of new input in each neuron the Euclidean distance between the input and its prototype is calculated The input is dedicated to aclass when it has more similar to that class than the other classesLogistic regressionLogistic regression is a common approach for predictive modeling practices The functionpX provides probability output between and for all values of X where X1“Xp are thepredictors The coefficients β0“βp are estimated using maximum likelihood estimationþβp Xð Þ ¼ eβ þ eβ1X1 þ‹¯Ã¾Î²Ã¾Î²pXp1X1 þ‹¯Ã¾Î²pXpDatasetThis study was based on the data obtained from three previous research conducted bySaeidi and Mansoori [ ] and the research of Parvizi and colleagues which 0cKazerouni BMC Bioinformatics Page of is not published yet We integrated these three studies and our data mining analysiswas implemented in their studies The data were collected from unrelated Iraniansubjects T2DM patients and healthy individuals matched for age and sexT2DM patients were recruited from individuals who referred to the Diabetic Clinic atShohada Hospital Tehran Iran In the current study we applied six lncRNAs expression and also six demographic variables including sex age weight height BMI andFBS for analysis and inputs of algorithms For the preprocessing phase we normalizedthe data inputs for KNN SVM and ANN models We also had low missing variablesand we replaced them with zero Table lncRNA extraction and selectionIncreasing evidence has suggested several lncRNAs are implicated in T2DM pathogenesis Recently human βcell transcriptome analysis showed lncRNAs dynamic regulation and abnormal expression of lncRNAs in T2DM [] However the extent oflncRNA deregulation in T2DM has yet to be determined To date more than100 susceptibility loci have been identified as being associated with T2DM at a genomewidesignificant level [ ] Considering this into account and by querying the GWAS catalog we candidated lncRNAs LY86AS1 HCG27_201 LINC00523 LINC00994TPT1AS1and DKFZP as target genes for this studyThe large scale GWAS have recognized approximately SNPs that were susceptibleto T2DM [] From there we used the GWAS catalog access in June to create alist of SNPs associated with T2DM In the current study we selected six lncRNA forexpression analysis according to the scan carried out in the study of Mansoori []and Saeedi [] We selected variants that had associations with increased risk ofT2DM We applied a quantitative PCR analysis of lncRNA expression levels in the samples We calculated the respective amount of each lncRNAs applying the 2ΔΔct asmeans of duplicate measurementsTable The lncRNAs as inputs of algorithmsnumberLncRNA VariablesDemographic VariablesVariablesLINC00523LINC00995HCG27_201TPT1AS1LY86AS1DKFZPSexAgeWeightHeightBMIFBS 0cKazerouni BMC Bioinformatics Page of Analysis and evaluation criteriaTo select the best performance data mining algorithms in predicting diabetic patientswe considered AUC sensitivity specificity and plotted ROC curve for the folds we ranand showed the average curve and its range [ ]ResultsTable shows the significant downregulation of PBMC expressions of the variables inthe T2DM group compared with the control group The AUC of each classificationtechnique has been demonstrated in Table AUC stands for œArea under the ROC Curve AUC computes the entire twodimensional area under the whole ROC curve According to the finding the maximumAUC dedicated to SVM and logistic regression among others knn also had the highestmean AUC and minimum standard deviation of AUC scores among the approachesThe mean and standard deviation for AUC sensitivity and specificity of each algorithmis given in Table Apart from classification AUC sensitivity and specificity the Receiver Operating Characteristic ROC with stratified crossvalidation is shown for eachapproach in Figs and ROC curves generally plot true positive rate on the Yaxis and false positive rate onthe Xaxis In other words a false positive rate of zero and a true positive rate of onein the top left corner of the plot is called the ideal point It means that a larger areaunder the curve AUC is usually better According to the demonstrated ROCs theKNN and SVM have a better area under the curve in comparison with the othersDiscussionFor a medical diagnosis optimized approaches to gain useful and accurate outcomesare essential Applying machine learning and data mining methods to automate theprocess of diagnosis may assist practitioners to enhance the quality of their clinical decisions [ ]Since T2DM is one of the prevalent diseases with severe consequences [] developing efficient methods for early detection of the disease was the primary purpose of ourresearchRegardless of high number of lncRNAs in the RNA profile of human a few numbersof them has been proved to be biologically active The role of the few lncRNAs hasbeen identified but several studies discussed the significant impact of lncRNAs in diabetic people which may represent the role of abnormal expression of lncRNAs in thelncRNAs in theincidence of T2DM [] According to the possible function ofTable Relative expression of the variablesVariablesLINC00523LINC00995HCG27_201TPT1AS1LY86AS1DKFZPDiabetesΔCT ± SEM “ “ “ “ “ “ControlΔCT ± SEM “ “ “ “ “ “pvalue 0cKazerouni BMC Bioinformatics Page of Table The AUC of algorithms for each iterationNumberof foldsAUCKNNSVMANNLogistic Regressiondevelopment of T2DM we considered the expression levels of six lncRNAs in additionto the demographic data in diabetic and healthy individuals for our study Tomeasure the expression of the lncRNAs we applied PBMCs which demonstrate an extensive proportion of the genes encoded in the human genome [] Several studies haveinvestigated different machine learning and data mining methods to predict differentdiseases [ ] such as heart diseases thyroid tumors and also diabetestype diabetes prediction In the present study we combined four commonly used datamining algorithms KNN SVM neural networks and regression to predict type diabetes using Long noncoding RNAs expression and the demographic variables for thefirst time because most of the previous studies used blood test variables or the demographic data for their analysis Receiver operating characteristic ROC analysis AUCsensitivity and specificity measure was used to assess the diagnostic value of the sixbiomarkers for T2DM The mean AUC for the KNN algorithm was obtained andwith standard deviation and we obtained the highest sensitivity with thestandard deviation of among other approaches After applying the SVM algorithm the mean AUC obtained after 10folds with the standard deviation of and the highest specificity among other approaches obtained with the standarddeviation of For the ANN we applied a multilayer perceptron with five hiddenlayers and the mean AUC of folds was and the standard deviation was Atlast for the logistic regression algorithm our results showed of mean AUC andthe standard deviation of The lower standard deviations in the AUC scores ofcomputed folds means the algorithm has worked with more performance [ ]Other studies investigated data mining algorithms for several diseases Saravananathanand Velmurugan [] applied several classification algorithms in their study to analyzeincluding KNN Sadri Sa™di [] compared three data miningdiabetes dataTable The mean and standard deviation of AUC sensitivity and specificity of algorithmsAlgorithmKNNMean AUC std ˆ’ ˆ’ ˆ’ ˆ’ Mean sensitivity ˆ’ std ˆ’ ˆ’ ˆ’ ˆ’ Mean specificity ˆ’ std ˆ’ ˆ’ ˆ’ ˆ’ SVMANNLogistic Regression 0cKazerouni BMC Bioinformatics Page of Fig The ROC for KNNalgorithms to predict T2DM and gained precision for ANN Sidiq [] reported about accuracy for KNN and accuracy for SVM algorithms applyingfor the Diagnosis of Various Thyroid Ailments In another study for the heart diseasesThe data mining algorithms indicated more than accuracy The investigated studies are in line with the findings of our study that these algorithms have a strong powerfor prediction and early detection of many diseases including T2DM and we obtainedremarkably better accuracy for prediction for example the SVM and logistic regressionaccuracy were In our study we also obtained a better accuracy for logistic regression that was and in comparison with other studies is a strong point for exampleSaeidi [] conducted a study to review two Long noncoding RNA expressions intype diabetes mellitus and with applying regressions reported about accuracyAnother research [] used two different Long noncoding RNA expressions in type Fig The ROC for SVM 0cKazerouni BMC Bioinformatics Page of Fig The ROC for MLPdiabetes mellitus and found of accuracy with the regression algorithm In thepresent study for the first time we performed four data mining algorithms on six Longnoncoding RNAs and compared their power with each other We demonstrated thatLong noncoding RNAs are effective biomarkers for data mining algorithms and have afeasible power to be applied for prediction of T2DM Also in this research we optimized the parameters of every algorithm and used stratified 10fold crossvalidation togain the best performance To be mentioned in the nearest neighbor™s algorithm theparameter k was varied between one and nine to find the bestoptimized method andwe selected k to have the best performance and the lowest standard deviation in theaccuracy of the folds In addition in choosing the parameters of the artificial neuralnetwork the number of hidden layer neurons significantly affects the accuracy of thenetwork so we set the parameters with two hidden layers with five and three neuronsFig The ROC for logistic regression 0cKazerouni BMC Bioinformatics Page of respectively to yield the best accuracy Considering the standard deviation of scores foreach algorithm the KNN had the lowest std Moreover the highest accuracy amongthe algorithms was the SVM algorithm and Logistic regression which had the maximum accuracy in folds among others We should mention that the strong points ofour study are using demographic data and six Long noncoding RNAs and combiningthem to get the best detection power of T2DM and performing four outstanding datamining algorithms and comparing their performances As the limitations of this studywe should account for the limited number of samples which is due to the high costs ofmeasuring the Long noncoding RNAs No doubt the higher number of samples wouldlead to higher performance and more reliable resultsConclusionIn this paper the performance of conventional data mining classification techniqueshas been calculated and compared for a dataset of patients referred for the screeningof type diabetes to the Shohada Hospital Iran The biomarker applied in this studydemonstrated high diagnostic value and the diagnostic process is suitable which couldhelp in the diagnosis of prediabetes and T2DMThe classification techniques compared were support vector machine artificial neuralnetwork decision tree nearest neighbors and logistic regression In data mining it isnot possible to say one classification technique will always work best and it often depends on the number of samples their distribution and the choosing of the right algorithm In this research work SVM and Logistic Regression had the best Area UnderCurve among methods of classification with the mean AUC of KNN and ANNalso had the high mean AUC and small standard deviations of AUC scores among theapproaches KNN had the highest mean sensitivity and the highest mean specificitybelonged to SVMFor future works performing other data mining and machine learning methods andusing higher numbers of samples are recommended to enhance the performanceAcknowledgmentsNot applicableAuthors™ contributionsAB and FA designed the study FA and AB collected the data and performed the statistical analysis AB and NPinterpreted the data FK ZM and LS wrote and revised the paper All authors read and approved the final manuscriptFundingNot applicableAvailability of data and materialsThe datasets used andor analyzed during the current study are available from the corresponding author onreasonable requestEthics approval and consent to participateEthical approval was obtained from the Shahid Beheshti University of Medical Sciences Ethics CommitteeIRSBMURETECHREC13951036 We informed all participants that their participation was voluntary and the study didnot state any potential risk and their identities will be private Informed written consent forms were taken from allparticipants before participationConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interests 0ccoding RNAs LINC00523 and LINC00994 in type diabetes in an Iranian cohort Mol Biol Rep “Saeidi L Ghaedi H Sadatamini M Vahabpour R Rahimipour A Shanaki M Mansoori Z Kazerouni F Long noncodingRNA LY86AS1 and HCG27_201 expression in type diabetes mellitus Mol Biol Rep “Petersmann A Nauck M MüllerWieland D Kerner W Müller UA Landgraf R Freckmann G Heinemann L Definitionclassification and diagnosis of diabetes mellitus Exp Clin Endocrinol Diabetes “Armoon B Karimy M Epidemiology of childhood overweight obesity and their related factors in a sample of preschoolchildren from Central Iran BMC Pediatr Tuomilehto J Lindström J Eriksson JG Valle TT Hämäläinen H IlanneParikka P KeinänenKiukaanniemi S Laakso MLouheranta A Rastas M Prevention of type diabetes mellitus by changes in lifestyle among subjects with impairedglucose tolerance N Engl J Med “Guariguata L Whiting DR Hambleton I Beagley J Linnenkamp U Shaw JE Global estimates of diabetes prevalence for and projections for Diabetes Res Clin Pract “Leti F DiStefano J Long noncoding RNAs as diagnostic and therapeutic targets in type diabetes and relatedcomplications Genes Heydari M Teimouri M Heshmati Z Alavinia SM Comparison of various classification algorithms in the diagnosis of type diabetes in Iran International Journal of Diabetes in Developing Countries “Kazerouni BMC Bioinformatics Page of Author details1Department of Laboratory Medicine School of Allied Medical Sciences Shahid Beheshti University of MedicalSciences Tehran Iran 2Department of Health Information Technology and Management School of Allied MedicalSciences Shahid Beheshti University of Medical Sciences Tehran Iran 3Department of Clinical Biochemistry School ofMedicine Tehran University of Medical Sciences Tehran Iran 4Department of Genetics Faculty of Medicine BabolUniversity of Medical Sciences Babol IranReceived December Accepted August ReferencesLi X Zhao Z Gao C Rao L Hao P Jian D Li W Tang H Li M The diagnostic value of whole blood lncRNAENST00000550337 for prediabetes and type diabetes mellitus Exp Clin Endocrinol Diabetes “ Mansoori Z Ghaedi H Sadatamini M Vahabpour R Rahimipour A Shanaki M Kazerouni F Downregulation of long non Perkel JM Visiting œnoncodarnia In Future Science Kapranov P Cheng J Dike S Nix DA Duttagupta R Willingham AT Stadler PF Hertel J Hackermüller J Hofacker IL RNAmaps reveal new RNA classes and a possible function for pervasive transcription Science “ Cornelis F Martin M Saut O Buy X Kind M Palussiere J Colin T Precision of manual twodimensional segmentations oflung and liver metastases and its impact on tumour response assessment using RECIST European radiologyexperimental Liao M Liu Q Li B Liao W Xie W Zhang Y A group of long noncoding RNAs identified by data mining can predict theprognosis of lung adenocarcinoma Cancer Sci Deshpande S Thakare V Data mining system and applications a review International Journal of Distributed and Parallelsystems IJDPS “ Umar Sidiq D Aaqib SM Khan RA Diagnosis of various thyroid ailments using data mining classification techniques IntJ Sci Res Coput Sci Inf Technol “ Zou Q Qu K Luo Y Yin D Ju Y Tang H Predicting diabetes mellitus with machine learning techniques Front Genet Daghistani T Alshammari R Diagnosis of diabetes by applying data mining classification techniques InternationalJournal of Advanced Computer Science and Applications IJACSA “ Meng XH Huang YX Rao DP Zhang Q Liu Q Comparison of three data mining models for predicting diabetes orprediabetes by risk factors Kaohsiung J Med Sci “ Wu H Yang S Huang Z He J Wang X Type diabetes mellitus prediction model based on data mining Informatics inMedicine Unlocked “ Vijayan V Ravikumar A Study of data mining algorithms for prediction and diagnosis of diabetes mellitus Internationaljournal of computer applications Saravananathan K Velmurugan T Analyzing diabetic data using classification algorithms in data mining Indian J SciTechnol “ Nahar N Ara F Liver disease prediction by using different decision tree techniques International Journal of Data Mining Knowledge Management Process IJDKP Vol Airola A Pahikkala T Waegeman W De Baets B Salakoski T An experimental comparison of crossvalidation techniquesfor estimating the area under the ROC curve Computational Statistics Data Analysis “ Purushotham S Tripathy B Evaluation of classifier models using stratified tenfold cross validation techniques InInternational Conference on Computing and Communication Systems Springer “ Abdar M Kalhori SRN Sutikno T Subroto IMI Arji G Comparing Performance of Data Mining Algorithms in PredictionHeart Diseases International Journal of Electrical Computer Engineering “ Sambyal RS Javid T Bansal A Performance analysis of data mining classification algorithms to predict diabetesInternational Journal of Scientific Research in Computer Science Engineering and Information Technology “ Pradhan M Kohale K Naikade P Pachore A Palwe E Design of classifier for detection of diabetes using neural networkand fuzzy knearest neighbor algorithm International Journal of Computational Engineering Research “Tzeng FY Ma KL ing the black boxdata driven visualization of neural networks IEEE Morán I Akerman Ä° Van De Bunt M Xie R Benazra M Nammo T Arnes L Nakić N GarcíaHurtado J RodríguezSeguí SHuman β cell transcriptome analysis uncovers lncRNAs that are tissuespecific dynamically regulated and abnormallyexpressed in type diabetes Cell Metab “ 0cKazerouni BMC Bioinformatics Page of Voight BF Scott LJ Steinthorsdottir V Morris AP Dina C Welch RP Zeggini E Huth C Aulchenko YS Thorleifsson GTwelve type diabetes susceptibility loci identified through largescale association analysis Nat Genet Imamura M Maeda S Genetics of type diabetes the GWAS era and future perspectives Endocr J “Soni J Ansari U Sharma D Soni S Predictive data mining for medical diagnosis an overview of heart diseaseprediction International Journal of Computer Applications “ Asadi F Paydar S Presenting an evaluation model of the trauma registry software Int J Med Inform “ Dangare CS Apte SS Improved study of

Thyroid_Cancer

construct a circRnAmiRnAmRnA regulatory network to explore potential pathogenesis and therapy options of clear cell renal cell carcinomaShuheng Bai1 YinYing Wu3 Yanli Yan1 Shuai Shao1 Jiangzhou Zhang2 Jiaxin Liu2 Beina Hui1 Rui Liu1 Hailin Ma1 Xiaozhi Zhang1 Juan Ren1clear cell renal cell carcinoma ccRcc is the most representative subtype of renal cancer circRnA acts as a kind of ceRnA to play a role in regulating microRnA miRnA in many cancers However the potential pathogenesis role of the regulatory network among circRnAmiRnAmRnA is not clear and has not been fully explored CircRNA expression profile data were obtained from GEO datasets and the differentially expressed circRNAs DECs were identified through utilizing R package Limma firstly Secondly miRNAs that were regulated by these circRNAs were predicted by using Cancerspecific circRNA database and Circular RNA Interactome Thirdly some related genes were identified by intersecting targeted genes which was predicted by a web tool miRWalk and differentially expressed genes which was obtained from TCGA datasets Function enrichment was analyzed and a PPI network was constructed by Cytoscape software and DAVID web set Subsequently ten hubgenes were screened from the network and the overall survival time in patients of ccRcc with abnormal expression of these hubgenes were completed by GEPIA web set In the last a circRNAmiRNAmRnA regulatory network was constructed and potential compounds and drug which may have the function of anti ccRCC were forecasted by taking advantage of CMap and PharmGKB datasets Six DECs hsa_circ_0029340 hsa_circ_0039238 hsa_circ_0031594 hsa_circ_0084927 hsa_circ_0035442 hsa_circ_0025135 were obtained and six miRNAs miR1205 miR657 miR587 miR637 miR miR548p which are regulated by three circRNAs hsa_circ_0084927 hsa_circ_0035442 hsa_circ_0025135 were also predicted Then overlapped genes regulated by these six miRNAs above had been predicted and function enrichment analysis revealed these genes are mainly linked with some regulation functions of cancers Ten hubgenes PTGER3 ADCY2 APLN CXCL5 GRM4 MCHR1 NPY5R CXCR4 ACKR3 MTNR1B have been screened from a PPI network PTGER3 ADCY2 CXCL5 GRM4 and APLN were identified to have a significant effect on the overall survival time of patients with ccRCC Furthermore one compound josamycin and four kinds of drugs capecitabine hmgcoa reductase inhibitors ace Inhibitors and bevacizumab were confirmed as potential therapeutic options for ccRcc by cMap analysis and pharmacogenomics analysis this study implies the potential pathogenesis of the regulatory network among circRnAmiRnAmRnA and provides some potential therapeutic options for ccRccRenal cancer is common cancer and the incidence rates in males and females are and respectively1 Clear cell renal cell carcinoma ccRCC accounts for “ of renal cancer which is the most representative 1Department of Radiotherapy Oncology Department First Affiliated Hospital of Xi™an Jiaotong University Xi™an Shaanxi Province China 2Medical School Xi™an Jiaotong University Xi™an Shaanxi Province China 3Department of Chemotherapy Oncology Department First Affiliated Hospital of Xi™an Jiaotong University Xi™an Shaanxi Province China email 869491533qqcomScientific RepoRtS 101038s41598020704842Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Flowchart of this study about constructing a circRNAmiRNAmRNA network and predicting some potential therapeutic options of ccRCCsubtype and the incidence rate increased year by year Compared with other cancers kidney cancerrelated clinical symptoms and biomarkers are less so early diagnosis is difficult Moreover ccRCC has poor responses to conventional chemotherapy and radiation therapy leading to a low 5year survival rate of advanced patients which is only “ Nowadays VEGF tyrosine kinase inhibitor monotherapy had been one type of standard therapy Moreover with the advances in immunotherapy and the more newly discovered therapeutic target a combination of the immunotherapy and the targeted therapies could be the next standard of treatment4 Therefore it is especially necessary to explore the internal mechanism of ccRCC to find some new therapeutic targetsCircular RNA circRNA derived from the exon or intron region of a gene is a particular type of noncoding RNA molecule that is different from linear RNA Compared with linear RNA structure circRNA has no ²“² polarity and no polyA tail making it a closed circular structure Therefore it is more stable than linear RNA and it is not easily degraded by RNA exonuclease or RNase5 CircRNAs™ function can generalize as below miRNA can regulate the posttranscriptional expression of target genes and circRNA can act as a competing endogenous RNA ceRNA to bind to miRNA like a sponge to regulate the function of miRNA thus indirectly regulating the expression of genes It can affect gene expression through interacting with RNA binding protein and modulating the stability of mRNAs It also can function as protein scaffolds and encode functional proteins in some cancer cells lines6“ Recently some studies have demonstrated that circRNA not only acts as a molecular marker but can also participate in cancer proliferation and invasion by regulating miRNA in colorectal cancer lung cancer and bladder cancer10In this study some novel circRNAs may act as ceRNA to regulate gene expression in ccRCC and their potential mechanisms have been investigated by utilizing gene chip and bioinformatics methods The process digraph is showed in Fig a0 Firstly circRNAs related microarray datasets of ccRCC were obtained from GEO database and differential expressed circRNAs DECs were also acquired Then to demonstrate whether the DECs function as ceRNAs in ccRCC their related miRNAs and miRNA target genes have been collected and a circRNAmiRNAmRNA network also has been constructed Furthermore a protein“protein interaction PPI network was successfully built and the hubgenes were also obtained Functional enrichment and pathway enrichment analyses were performed to reveal the potential pathogenesis of ccRCC Furthermore connectivity map CMap analysis Scientific RepoRtS 101038s41598020704842Vol1234567890wwwnaturecomscientificreports 0cand pharmacogenomics analysis were conducted to predict bioactive compounds and potential drugs for the treatment of ccRCC which may provide a new method in the latent therapeutic capacity of circRNAs in ccRCCMethodsData obtained and DECs acquired Gene Expression Synthesis GEO wwwncbinlmnihgovgeo is a public functional genomic database that allows users to query locate review and download research and gene expression profiles11 Microarray datasets that provide circRNA expression profile data in clear cell renal cell carcinoma ccRCC were acquired from the GEO database All raw expression data were normalized and log2transformed Then Limma a Bioconductor package in R was applied for differential analysis of microarray data to determine DECs in microarray dataset with the criteria of log2 fold change and P value prediction of MRes Cancerspecific circRNA database CSCD gbwhueducnCSCD was constructed to understand the functional effects of circRNAs through predicting the miRNA response element MRE sites and RNA binding protein RBP sites for each circRNA13 Circular RNA Interactome CircInteractome is also a web tool to map RNAbinding proteins RBP and miRNA response element MRE sites on human circRNAs by searching some public databases of circRNA miRNA and RBP It also provides bioinformatic analyses of binding sites on circRNAs and additionally analysis of miRNA and RBP sites14 DIANAmiRPath v30 wwwmicro rnagrmiRPa thv3 is an online software that is committed to assessing miRNAs regulatory roles and forecasting the related regulation pathways15 The miRNA response elements MREs of those selected DECs were predicted with these two web tools CSCD and CircInteractome Overlapped miRNAs of the two algorithms were predicted as potential target miRNAs of the DECs DIANAmiRPath also predicted these overlapped miRNA™s functions Then these overlapped miRNAs were selected for further mRNA predictionsforecasting of miRnA targeted genes MiRWalk is a web tool to predict miRNA“mRNA interactions It involves predicted algorithms miRWalk Microt4 mirbridge Targetscan RNAhybrid RNA22 PITA Pictar2 miRNAMap miRDB miRanda and miRMap to ensure the correctness of forecast results16 Then targeted genes forecasted by at least seven algorithms were selected to overlapped with differentially expressed genes DEGs in ccRCC from TCGA databaseCollecting DEGs of ccRCC and obtaining the overlapped genes The Cancer Genome Atlas TCGA is a public database that demonstrated major cancer related genomic alterations Differentially expressed genes DEGs were determined by the edgeR package in Bioconductor with the screening criteria of log2 fold change and FDR 00517Then the overlapped genes between the predicted miRNA target genes and the DEGs were obtained through the Venn diagramfunctional enrichment analysis of overlapped genes The database for annotation visualization and integrated discovery DAVID V68 david abccncifc rfgov is a freely accessed webbased online bioinformatics resource that provides tools for the functional interpretation of large lists of genesproteins18 It was used to perform Gene Ontology GO analysis and Kyoto Encyclopedia of Genes and Genomes KEGG pathway enrichment analysis about the overlapped genes with a setting P and counts Establishment of PPI network and identification of hubgenes The Search Tool for the Retrieval of Interacting Genes database STRING a0provides credible information on interactions between proteins and supplies detailed annotation19 In this study the interactions between proteins which has a combined score was considered as a statistically significant interaction Then a PPI network of the overlapped genes was constructed by the STRING version Cytoscape is a generalpurpose source software environment for the broad integration of molecular interaction network data In molecular and biology fields it can load molecular and genetic interaction data integrate global datasets and functional annotations establish powerful visual mappings across these data A wide variety of Cytoscape Apps can enhance their capabilities20 Molecular Complex Detection MCODE is an app of Cytoscape to find densely connected regions of a vast molecular interaction network as PPI protein“proteininteraction network based on nodeweighting arithmetic effectively So PPI networks were drawn using Cytoscape version and essential modules consisting of hubgenes and several relatively essential modules in the PPI networks identified by MCODE with selected criteria as follows degree cutoff node score cutoff Max depth and kscore overall survival oS analysis of the hub genes The Gene Expression Profiling Interactive Analysis GEPIA is a webbased tool to deliver fast and customizeable functionalities based on TCGA and GTEx data It provides key interactive and customizable functions including differential expression analysis profiling plotting correlation analysis patient survival analysis21 In this study overall survival OS analysis was used to explore the influence on OS by differentially expressed hubgenes between ccRCC tissues and normal onesconstruction of circRnA“miRnA“mRnA network A circRNA“miRNA“mRNA regulatory network was constructed by using Cytoscape software to draw the regulatory networkScientific RepoRtS 101038s41598020704842Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Acquire DECs of ccRCC a Volcano plots for DECs the green points and red points represent up and down expressed circRNAs respectively b A heatmap for differentially expressed circRNAs we selected the change in color represents the difference in expressionconnectivity Map cMap analysis The Connectivity Map CMap is a gene expression profiling database and is great potential for discovering new therapeutic drugs for the disease It is a database of biological applications in which gene expression is linked to disease helping researchers quickly use data from gene expression profiles to compare small molecule compounds or drugs that are highly associated with disease22 With the help of CMap candidate compounds would be discovered to treat the ccRCC Negatively related drugs P and connectivity score for anticcRCC were screened These compounds™ structures and some annotations were obtained from the website PubChem pubch emncbinlmnihgov which is an essential chemical information resource containing million substance descriptions million unique chemical structures and million bioactivity test results from million biological assays23pharmacogenomics analysis for hub genesThe Pharmacogenomics and Pharmacogenetics Knowledge Base PharmGKB wwwpharm gkb is committed to Collecting and classifying information about how genetic variation affects drug response24 It is a comprehensive resource for pharmacogenetics including their variations pharmacodynamic pathways and their effects on drugrelated phenotypes25 Furthermore researchers can freely get this knowledge about potential worked drugs from this web In this research the network database was used to predict drugs that might act on hubgenesResultsAcquiring DECs in clear cell renal cell carcinoma ccRCC In order to explore the potential function of circRNAs and construct the interaction network between circRNAs and miRNAs in ccRCC DECs were confirmed at the first step A microarray dataset GSE100186 was obtained from GEO database which includes four clear cell renal cell carcinoma tissues and four matched nontumor tissues The gene chip was from the platform of Agilent074301 Arraystar Human CircRNA microarray V2 By applying Limma package a string of circRNAs was considered as significant difference points and six circRNAs which shows the most credible in different expression P and LogFC have been selected as research objects in this study a0as the volcano Fig a02a showing These six circRNAs™ different expressions between clear cell renal cell carcinoma tissues and matched nontumor tissues present as Fig a02b The basic characteristics of the circRNAswere displayed in Table a0Identification of circRNA“miRNA interactions Increasing evidence has demonstrated that some circRNAs play critical roles in tumors by functioning as œsponge to trap miRNAs So some miRNAs which are related to these six DECs we got were predicted based on this ceRNA theory The basic structural patterns of the circRNAs were illustrated in Fig a0 which all have these structures of MRE RBP and ORF To explore whether all these circRNAs had the function as ceRNA in ccRCC two online databases CSCD and CircInteractome were used to collect and explore potential target miRNAs for them A total of circRNA“miRNA interactions including hsa_circ_0029340miR1205miR657 hsa_circ_0025135miR587miR637 and hsa_circ_0039238Scientific RepoRtS 101038s41598020704842Vol1234567890wwwnaturecomscientificreports 0cCircRNA IDhsa_circ_0039238hsa_circ_0031594hsa_circ_0029340hsa_circ_0084927hsa_circ_0035442hsa_circ_0025135Aliashsa_circRNA_039238hsa_circRNA_101341hsa_circRNA_101202hsa_circRNA_104651hsa_circRNA_101529hsa_circRNA_101001CircRNA typeexonicexonicexonicexonicexonicexonicPositionChr1647162235“chr1434398281“chr12125292306“chr895676924“chr1558284902“chr126458115“StrandBest transcriptGene symbolRegulationˆ’ˆ’ˆ’ˆ’ˆ’NM_018092NETO2NM_022073EGLN3NM_005505SCARB1UpUpUpNM_017697ESRP1DownNM_001206897 ALDH1A2DownNM_001159576SCNN1ADownTable Basic characteristics of these circRNAsFigure a0 Basic structures of the circRNAs The different colors in the outer and inner ring represent the different exons and the positions of MRE RBP and ORFmiR1278miR548p were identified DIANAmiRPath was then used to probe the signaling pathways in that the miRNAs may be involved As shown in Fig a0 these RNAs are involved in some pathways in the development of tumorigenesisobtaining the overlapped genes Six miRNAs linked with circRNAs had been obtained To explore these miRNAs™ function in ccRCC overlapped genes will be obtained in this work RNAsequencing RNAseq data contained clear cell renal cell carcinoma tissue samples and normal controls that were obtained Scientific RepoRtS 101038s41598020704842Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Significant signaling pathways about the miRNAs by utilizing the DIANAmiRPathfrom the TCGA Total DEGs in clear cell renal cell carcinoma showing in the Volcano Fig a05a which were gained by running edgeR package Moreover target genes of the six miRNAs mentioned above were obtained by the miRWalk Furthermore overlapped genes were identified by intersecting the miRNA targeted genes and DEGs from TCGA as Fig a05b showingfunction enrichment analyses To understand the biological roles and potential functions of the overlapped genes GO analysis which including biological process BP molecular function MF and cellular component CC and KEGG signal pathway enrichment analysis had been performed GO analysis showed that in BP terms these overlapped genes were mainly enriched in positive regulation of transcription from RNA polymerase II promoter negative regulation of transcription from RNA polymerase II promoter transcription from RNA polymerase II promoter and positive regulation of transcription DNAtemplated For MF overlapped genes were mainly enriched in protein homodimerization activity transcription factor activity sequencespecific DNA binding calcium ion binding and sequencespecific DNA binding For CC these overlapped genes were mainly enriched in integral component of membrane plasma membrane integral component of plasma membrane and extracellular region The KEGG signal pathway enrichment analysis found that DEGs are mainly enriched in pathways such as Pathways in cancer Neuroactive ligandreceptor interaction PI3KAkt signaling pathway Rap1 signaling pathway and Ras signaling pathway The results are shown in Fig a0Identification of hubgenes The functions of these overlapped genes had been analyzed above To further explore the effect of circRNAmiRNA regulatory networks on gene expression levels in ccRCC a PPI network was constructed and some hubgenes which screened from this PPI network by applying some bioinformatics algorithms were obtained By utilizing STRING and Cytoscape a PPI network consisting of Scientific RepoRtS 101038s41598020704842Vol1234567890wwwnaturecomscientificreports 0cFigure a0 a Volcano plots showing DEGs obtained from TACG dataset by utilizing edgeR package b Venn graph showing the overlapped genes by intersecting the miRNA targeted genes and DEGs from TCGAnodes and edges to display the interactions among the mRNAs Fig a07a Then a subnetwork MCODE score90 with ten nodes and edges was selected which revealed the critical roles of the ten genes PTGER3 ADCY2 APLN CXCL5 GRM4 MCHR1 NPY5R CXCR4 ACKR3 MTNR1B in clear cell renal cell carcinoma Fig a07b After this a network about the association between these circRNA miRNAs and hubgenes were built Fig a0 7c circRNAmiRNAmRNA regulatory axis including hsa_circ_0029340hasmiR657GRM4 axis hsa_circ_0025135hasmiR587PTGER3 axis hsa_circ_0025135hasmiR587ADCY2 axis hsa_circ_0025135hasmiR587ACKR3 axis hsa_circ_0025135hasmiR587NPY5R axis hsa_circ_0025135hasmiR637APLN axis hsa_circ_0025135hasmiR637GRM4 axis hsa_circ_0025135hasmiR637MTNR1B hsa_circ_0039238hasmiR548pPTGER3 axis hsa_circ_0039238hasmiR548pCXCL5 axis hsa_circ_0039238hasmiR548pMCHR1 axis hsa_circ_0039238hasmiR548pCXCR4 axis extracted from this networkaxis hsa_circ_0039238hasmiR548pHAPLN1 Effects of hubgenes on overall survival The hubgenes and it™s predicted regulated pathways had been found out And now the overall survival time of patient™s with ccRCC will be displayed which is the most concerning thing by all clinicians The OS of these hubgenes are exhibited in Fig a0 excluding MTNR1B which did not have sufficient data to go on survival analysis Total five genes were found to have significant effects on Scientific RepoRtS 101038s41598020704842Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Dot plot of function enrichment analysis a Biological process analysis b Cellular component analysis c Molecular function analysis d KEGG pathway analysis The color intensity of the nodes shows the degree of enrichment of this analysis The enrichfactor is defined as the ratio of the differential genes in the entire genome The dot size represents the count of genes in a pathwayoverall survival The overexpression of these genes PTGER3 ADCY2 and APLN can prolong the overall survival and may act as a protective factor but the downregulated genes CXCL5 and GRM4 have adverse effects on OS may act as risk factorsconstruction of a circRnA“miRnA“mRnA network To present the relationship between circRNA miRNA and mRNA a circRNAmiRNAmRNA network was constructed through combining the circRNAmiRNA interactions and the miRNAmRNA interactions by using Cytoscape shown as Fig a0 It provided an expression about the connections between the DECs hsa_circ_0029340 hsa_circ_0025135 hsa_circ_0039238 the miRNAs miR1205 miR657 miR587miR637 miR1278miR548p and the mRNAscandidate compounds from cMap The regulated network of circRNAs miRNAs mRNAs had been constructed and now some candidates or drugs which may have effects on ccRCC will be forecasted The candidate compounds were predicted by CMap showing in Table a0 The enrichment correlation coefficient calculated by the correlation coefficient in all instances ranges from to The score is positive indicating that a small molecule compound or drug has a similar or codirectional relationship with a particular biological process score is negative indicating that a small molecule compound or drug has an opposite or antagonistic link to a particular anism The P value was evaluated for the significance of the enrichment correlation coefficient and the smaller the value the greater the credibility25Pharmacogenomics analysis for hub genes to find potential drugBy exploring the website PharmGkb the above ten hubgenes were selected for pharmacogenomic analysis to find some potential drugs and complement the results of CMap The results are showing in Table a0 Bevacizumab can treat Colorectal Neoplasms efficiency which may be related to the expression of CXCR4 And capecitabine can also work with colorectal cancer reduced progressionfree survival but slightly These two drugs mentioned above are all associated with cancer but hmg coa reductase inhibitors and Ace Inhibitors Plain are not associated with cancer as the annotation from the website introducesDiscussionAn increasing number of researches have demonstrated that circRNAs and miRNAs play important roles in cancer biological recently Experiments had demonstrated that miRNAs are closely related to the proliferation migration and invasion and circRNAs can regulate these processes via miRNAs in ccRCC “ Some Scientific RepoRtS 101038s41598020704842Vol1234567890wwwnaturecomscientificreports 0cFigure a0 a A network of overlapped genes™ interaction Gradual changes in color represent differences in the expression levels of different genes of ccRCC b A network of hubgenes Gradual changes in color represent differences in the expression levels of different genes of ccRCC c A network showing the interactions of circRNAsmiRNAshubgenes circRNAs miRNAs hubgenes are shown in red yellow and green respectivelyScientific RepoRtS 101038s41598020704842Vol0123456789wwwnaturecomscientificreports 0cFigure a0 The overall survival time of hubgenesstudies also demonstrated that circRNAmiRNA has a strong association with diseases especially with cancer by utilizing advanced computational methods such as KATZ algorithm LocalityConstrained Linear Coding algorithm inductive matrix completion decision tree29“ Therefore circRNAs and miRNAs can apply as a therapeutic method or a biomarker of diagnosis In this study DECs hsa_circ_0029340 hsa_circ_0039238 hsa_circ_0031594 hsa_circ_0084927 hsa_circ_0035442 hsa_circ_0025135 were selected at the first step To our knowledge hsa_circ_0084927 had been demonstrated that it is involved in the development of lung adenocarcinomaassociated malignant pleural effusion but the other circRNAs had not been studied yet36 These five circular RNAs were first discovered that are abnormally expressed in ccRCC and they have the potential to be excellent biomarkers or potential therapeutic targets As a kind of highly conserved endogenous RNA ceRNA circRNA has been confirmed to have a function as sponge to absorb corresponding miRNA by interacting with miRNA binding sites and thus playing a role in regulating genes expression indirectly Among the DECs above circRNAs hsa_circ_0029340 hsa_circ_0025135 hsa_circ_0039238 were ascertained as ceRNA to regulate the expression of miRNAs miR1205 miR657 miR587 miR637 miR1278 miR548p However based on the ceRNA theory no miRNAs related to the other three circRNAs were found which may play other roles such as coding protein interaction with RNA binding protein or modulating the stability of mRNAs so Scientific RepoRtS 101038s41598020704842Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A network of circRNAmiRNAmRNA Octagon represents circRNA Diamond represents miRNA Oval represents mRNA Gradual changes in color represent differences in the expression levels of different genes of ccRCCCmap namejosamycinjosamycinjosamycinEnrichment score Dose μm Cellˆ’ ˆ’ ˆ’ MCF7HL60PC3Up score Down scoreˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ Table A potential compound identified by Cmap for ccRCCon Among the miRNAs identified miR657 miR587 was confirmed to act as tumorpromoting molecules in some cancer types37“ Some research have found that miR657 overexpressed in lung cancer cervical squamous cell carcinoma larynx carcinoma hepatocellular carcinoma and it can promote carcinoma cells abilities of tumorigenesis proliferation and invasion by some complex targeting pathways3740 And For miR587 it can antagonize 5FUinduced apoptosis and confers drug resistance by regulating PPP2R1B expression in colorectal cancer and it is related to the survival time of glioblastoma multiforme patients4142 In contrast mir1205 miR637 and miR548p act as tumor suppressor molecules MiR1205 can target some downstream gene sits Scientific RepoRtS 101038s41598020704842Vol0123456789wwwnaturecomscientificreports 0cGenesSNPDrugSignificantP value AssociationADCY2rs4702484CapecitabineADCY2rs4702484CapecitabineYesNoCXCL5rs352046Hmg coa reductase inhibitorsYesPTGER3rs11209716 Ace Inhibitors PlainCXCR4rs2228014BevacizumabYesYesIn patients receiving capecitabine monotherapy CC carriers showed slightly reduced progressionfree survival CC vs CT a0months P Analyzing the entire cohort of capecitabine monotherapy N and Combination therapy N no association for genetic markers with progressionfree survival was foundGenotype CC is associated with increased response to hmg coa reductase inhibitors in people with Acute coronary syndrome as compared to genotypes CG GGAllele C is associated with decreased risk of Cough when treated with Ace Inhibitors Plain in people with Hypertension as compared to allele TGenotypes AA AG is associated with decreased progressionfree survival when treated with bevacizumab in people with Colorectal Neoplasms as compared to genotype GGReferencesPMID PMID PMID PMID PMID Table Four potential drugs identified by PharmGKB for ccRCCto inhibited and promote cell proliferation and invasion in some cancers “ For miR637 many studies suggest it act as a protective factor to suppress the cancer cells proliferation invasion and migration by targeting on regulating the expression of AKt1 RING1 or NUPRI in hepatocellular carcinoma colorectal cancer glioma and cervical cancer45“ MiR548p decreases Hepatic Apolipoprotein B Secretion and Lipid Synthesis acting at HBxHNF4AmiR548pHBXIP pathway that controls hepatoma cell growth and tumorigenesis of hepatocellular carcinoma50 But there are no studies having found that miR1278 plays an important regulatory role in tumors In general the findings of this study about these miRNAs are similar to other studies and these miRNAs may play an important role in the development of ccRCCCircRNAs affect the expression of genes by acting at miRNA as shown above To further explore the effects of circRNA on gene expression by acting at miRNA overlapping genes were collected to go for function enrichment analyses These genes were enriched mainly in the biological process about the regulation about transcription and cell proliferation Meanwhile KEGG analysis showing that these genes were mainly enriched in some cancerrelated pathways such as Pathways in cancer PI3KAkt signaling pathway Rap1 signaling pathway and Ras signaling pathway Furthermore these pathways have been shown to have an interaction in some cancers which may indicate that these circRNAs explored in this study may exert the same or related regulatory functions by acting on the circRNAmiRNAmRNA axis PI3KAkt signaling pathway is an important pathway to regulate cancer proliferation adhesion migration invasion and angiogenesis It acts at downstream targets as Forkhead box O transcription factors FoxO mammalian target of rapamycin mTOR to stimulate expression of death receptor ligands and enhance Vascular endothelial growth factor VEGF secretion5152 Meanwhile PI3KAkt signaling pathway can be activated by RAS or Rap1 pathway and it can interact with gene P53 to promote the function of enabling gene repair and maintaining gene stability53 Moreover Rap1 is activated by upstream signaling molecules as calmodulin cAMP and tyrosine kinase as PKA PKC acting on downstream molecular markers such as Braf PAPL to regulate gene expression cell proliferation adhesion etc5455 In summary the function and signaling pathways of these genes are related to the occurrence and development of tumors which have been confirmed in other studies Therefore these genes which are regulated by circRNAs indirectly play an essential role in the signal pathway of ccRCCIn this study hubgenes PTGER3 ADCY2 APLN CXCL5 GRM4 MCHR1 NPY5R CXCR4 ACKR3 MTNR1B were obtained and some circRNAmiRNAmRNA regulatory axis about these hubgenes were constructed which may help researchers build a more systematic more profound about the regulatory network Among these hubgenes PTGER3 CDCT2 APLN CXCL5 and GRM4 show significant effects on overall survival time between ccRCC tissue group and normal tissue group PTGER3 CDCT2 and APLN have positive effects on overall survival time but CXCL5 and GRM4 have negative effects on it Research demonstrated that APLN could interact with APLN receptor which is a Gproteincoupled receptor which may influence the aggressive of ccRCC and the effect of immune therapy56 And CXC chemokine receptor CXCR4 is the major chemokine receptor in solid tumors Increased CXCR4 expression was associated with more aggressive tumor behavior in RCC patients especially in ccRCC subtypes due to their more metastatic behavior57 However to our best knowledge the rest hubgenes in ccRCC had not been investigatedClear cell renal cell carcinoma ccRCC is one of the most drugresistant malignancies Exploring potential compounds or drugs that may have a therapeutic effect on ccRCC is necessary Cmap and Pharmacogenomics analysis had been exploited to find potential compounds and drugs Josamycin is a naturally produced antibiotics that have a 16membered macrocyclic lactone ring predicted by Cmap A study had demonstrated that Josamycin can suppress the development of altered liver cell foci but not indicted this compound has anticancer function58 Meanwhile a study demonstrated that 14membered ring macrolide antibiotics roxithromycin and clarithromycin have a significant inhibitory effect to mouse B16 melanoma cell on tumor angiogenesis tumor growth and metastasis However t a 16membered ring macrolide as josamycin did not show any inhibitory effect on these ways when at the same dose59 So more studies are needed to illustrate whether josamycin can act as antitumor function and its mechanism of action Capecitabine is targeting to the SNPs of rs

Thyroid_Cancer

Bone metastasis classification using wholebody images from prostate cancer patientsbased on convolutional neural networksapplicationNikolaos Papandrianos1 Elpiniki PapageiouID23 Athanasios Anagnostis34Konstantinos Papageiou4 General Department University of Thessaly Lamia Greece Faculty of Technology Dept of EnergySystems University of Thessaly Geopolis Campus Larisa Greece Institute for Bioeconomy and Agritechnology Center for Research and Technology Hellas Greece Department of Computer Science andTelecommunications University of Thessaly Lamia Greece npapandrianosuthgrAbstractBone metastasis is one of the most frequent diseases in prostate cancer scintigraphy imaging is particularly important for the clinical diagnosis of bone metastasis Up to date minimalresearch has been conducted regarding the application of machine learning with emphasison modern efficient convolutional neural networks CNNs algorithms for the diagnosis ofprostate cancer metastasis from bone scintigraphy images The advantageous and outstanding capabilities of deep learning machine learning™s groundbreaking technologicaladvancement have not yet been fully investigated regarding their application in computeraided diagnosis systems in the field of medical image analysis such as the problem of bonemetastasis classification in wholebody scans In particular CNNs are gaining great attention due to their ability to recognize complex visual patterns in the same way as human perception operates Considering all these new enhancements in the field of deep learning aset of simpler faster and more accurate CNN architectures designed for classification ofmetastatic prostate cancer in bones is explored This research study has a twofold goal tocreate and also demonstrate a set of simple but robust CNN models for automatic classification of wholebody scans in two categories malignant bone metastasis or healthy usingsolely the scans at the input level Through a meticulous exploration of CNN hyperparameter selection and finetuning the best architecture is selected with respect to classificationaccuracy Thus a CNN model with improved classification capabilities for bone metastasisdiagnosis is produced using bone scans from prostate cancer patients The achieved classification testing accuracy is whereas the average sensitivity is approximately Finally the bestperforming CNN method is compared to other popular and wellknown CNN architectures used for medical imaging like VGG16 ResNet50 GoogleNetand MobileNet The classification results show that the proposed CNNbased approach outperforms the popular CNN methods in nuclear medicine for metastatic prostate cancer diagnosis in bonesa1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Papandrianos N Papageiou EAnagnostis A Papageiou K Bonemetastasis classification using whole body imagesfrom prostate cancer patients based onconvolutional neural networks application PLoSONE e0237213 101371journalpone0237213Editor Jeonghwan Gwak Korea NationalUniversity of Transportation REPUBLIC OF KOREAReceived November Accepted July Published August Copyright Papandrianos This is an access distributed under the terms ofthe Creative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement The data which areanonymized bone scintigraphy images without anyfurther information are available only after requestfor research purposes Data availability Thedataset from Diagnostic Medical CenterœDiagnosticoIatriki AE was used under thelicense of the Board Committee Director of theDiagnostic Medical Center œDiagnosticoIatriki AE Dr Vasilios Parafestas for the current studyand is not publicly available The dataset may beavailable only under request from the Director ofPLOS ONE 101371journalpone0237213 August PLOS ONE 0cthe Diagnostic Center vparafestasyahoogr andthe relevant Doctor of Nuclear Medicine DrNikolaos Papandrianos npapandrianosuthgrnikpapandrgmailcomFunding The authors received no specificfunding for this workCompeting interests The authors have declaredthat no competing interests existBone metastasis classification using convolutional neural networks IntroductionMost common tumors such as those of the breast lung and prostate frequently metastasize tothe bone tissue and so the skeleton seems to be a site with the most significant tumor burdenin cancer patients with advanced disease Statistical analysis results have shown that of allbone metastases originate from the breast in women and from the prostate in men Theremaining emanates from thyroid lung and kidney cancers [] In the case of metastaticprostate cancer diagnosis has a significant impact on the quality of patient™s life [] In mostmen the metastatic prostate cancer mainly sites on the bones of the axial skeleton causingsevere lesions that can cause pain debility andor functional impairment [] As this type ofcancer has great avidity for bone and could cause painful and untreatable effects an early diagnosis is crucial for the patient Reviews on clinical evidences and diagnostic assessments ofbone metastases in men with prostate cancer can be found in []The implementation of a properly selected diagnostic imaging can reveal the number ofmetastatic foci in the skeletal system [ ] Rapid diagnosis of bone metastases can be achievedusing modern imaging techniques such as scintigraphy Positron Emission TomographyPET and wholebody Magnetic Resonance Imaging MRIThe primary imaging method in the diagnosis of metastases that offers the highest sensitivity among all imaging methods is Bone Scintigraphy BS [“] Through the depictionof the entire skeleton in one medical examination nuclear doctor is able to detect bone abnormalities in areas where intensive radionuclide activity is present However low specificityseems to be the main drawback of this method as it cannot tell whether the causes of boneturnovers are different than those of metastatic origin leukaemia healing fracture etc Atthe same time PET has been recognized as an efficient method for detecting cancer cellsbased on recent technological advancements in medical imaging PET and Computed Tomography CT combination can produce highresolution images [“]Although PET and PETCT are the most efficient screening techniques for bone metastasisBS remains the most common imaging procedure in nuclear medicine [ ] [] Asreported in European Association of Nuclear Medicine EANM guidelines [] BS is particularly important for clinical diagnosis of metastatic cancer both in men and women At presentwhen other imaging or examination methods are unable to provide a reliable diagnosis BSimaging becomes the proper modality for making a final diagnosis of bone metastasis []To address the considerable problem of bone metastasis diagnosis artificial intelligentmethods for medical image analysis implemented with deep learning algorithms have beenadequately investigated In this direction a recent survey reveals the entire penetration of deeplearning techniques into the field of medical image analysis detection segmentation classification retrieval image generation and enhancement registration and successful application ofdeep learning to medical imaging tasks are thoroughly examined [“]Implementation of deep learning in medical imaging is mainly conducted by ConvolutionalNeural Networks CNNs [ ] a relatively new and powerful way to learn useful representations of images and other structured data Before the application of CNNs these featurestypically had to be created by less powerful machine learning models or even handcraftedWith the introduction of CNNs such features could be learned directly from the provideddata since they include certain preferences in their structure that make them powerful deeplearning models for image analysis [ ] Typical CNNs have a similar structure withArtificial Neural Networks ANN and consist of one or more filters ie convolutional layers followed by aggregationpooling layers in order to extract features for classification tasks[] Gradient descent and backpropagation are both used as learning algorithms the sameway they are used in a standard ANN Their main difference lies in the fact that CNNs havePLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networkslayers of convolutions along with pooling layers in the beginning of their architecture Thefinal outputs are computed via fully connected layers located at the end of the network architecture []In recent years CNNs have gained wider recognition in medical image analysis domain aswell as in vision systems [“ ] Due to their enormous popularity several applications ofCNNs were investigated in the field of medical image analysis Two recent review studies []and [] gather all the important and most interesting applications of deep learningThe application of CNNs in medical imaging ranges from plain radiograph CT MRI andmicroscopy images to clinical photos dermatology capsule endoscopy and visual recognition[“] In addition a CNN was investigated in [] that regards automatic detection of tuberculosis on chest radiographs while in [] a brain tumor segmentation in magnetic resonanceimages was made possible with the use of a CNN More examples of CNNs™ successful application in medical domain include automated cardiac diagnosis [] detection of lesions and prediction of treatment response by PET [ ] as well as dynamic contrast agentenhancedcomputed tomography where CNN showed high diagnostic performance in the differentiation of liver masses [] Furthermore CNNs have shown outstanding performance in radiology and molecular imaging []Some models with major impact in the context of deep learning and medical image processing were introduced in several s the Unet model for biomedical data semanticsegmentation [] the GoogLeNet model introducing the inception module [] the ResNetmodel introducing the residual learning building block for extremely deep convolutional networks [] and also Deeplab which deals with the inclusion of many convolutional layersatrous for semantic segmentation of images in deep convolutional neural networks []In medical image analysis the most widely used CNN methods are the following i AlexNet [] This network has a quite deep architecture similar to GoogLeNet by YannLeCun [] incorporates more filters per layer and includes stacked convolutional layersIt attaches ReLU activations after every convolutional and fullyconnected layer ii ZFNet [] Being a rather slight modification of AlexNet this network won the ILSVRCcompetition iii VGGNet16 [ ] It consists of convolutional layers having avery uniform architecture similar to AlexNet iv GoogleNet [] It is a convolutional neuralnetwork with a standard stacked convolutional layer having one or more fully connected layers called inception modules able to extract various levels of features on the same time vResNet [] It is another efficient CNN architecture that introduced the œidentityshortcut connection to solve the notorious problem of the vanishing gradients of the deepnetworks vi DenseNet [] Being another important CNN architecture DenseNetoffers the main advantage of alleviating the gradient vanishment problem with the direct connection of all the layers Related work in nuclear medical imaging for metastatic prostate cancerdiagnosis in bonesReviewing the relevant literature for diagnosis of bone metastasis using bone scintigraphyscans the authors notice that only a couple of previous works have been adequately conductedfor metastatic prostate cancer classification using CNNs while the others are devoted to ANNsand their application in ComputerAided Diagnosis CAD These works have investigated theuse of Bone Scan Index BSI which was introduced to assess the bone scanning process andestimate the extent of bone metastasis [ ] Specifically it serves as a clinical quantitativeand reproducible parameter that can measure metastatic prostate cancer bone involvement[] The software developed for the BSIbased ANN approach was EXINI bone EXINIPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksDiagnostics AB Lund Sweden and afterwards a revised version of this software calledBONENAVI FUJIFILM Toyama Chemical Co Ltd Tokyo Japan was engineered using alarge number of Japanese multicenter training databases []The first of the reported studies was devoted to the development of a classification algorithm based on CNNs for bone scintigraphy image analysis [] It was carried out as a masterthesis in Lund University and is focused mainly on classification problems without considering any identification and segmentation tasks The used dataset was provided by Exini Diagnostics AB in the form of image patches of already found hotspots The process in whichhotspots were segmented cropped and collected from bone scans was implemented using asoftware developed at Exini BSI was calculated for wholebody bone scans by segmenting theentire skeleton from the background in both the anterior and posterior views Due to timeframe restrictions only the hotspots found in the spine have been used to train the CNN sincethey were considered to be the easiest to classify A shape model based on a mean shape of several normal wholebody scans was fitted to the skeleton using an image analysis algorithmcalled Morphon registration The outcomes of the aforementioned thesis [] have shown thatthe calculated accuracy of the validation set was whereas the calculated accuracy of thetesting set was The second study explored CNNs for classification of prostate cancer metastases usingbone scan images [] The tasks of this master project appeared to have a significant potentialon classifying bone scan images obtained by Exini Diagnostics AB too including BSI The twotasks were defined as i classifying anterior posterior pose and ii classifying metastatic nonmetastatic hotspots The outcome of this study is that the trained models produce highlyaccurate results in both tasks and they outperform other methods for all tested body regions inthe case of metastatic nonmetastatic hotspots classification The evaluation indicator of thearea under Receiver Operating Characteristic ROC score was equal to which is significantly higher than the respective ROC of obtained by methods reported in the literature for the same test setThe remaining research that concerns the same imaging modality BS is devoted to theintroduction of CAD systems with the use of ANN and other Machine Learning ML methods for bone metastasis detection in bone scintigraphy images Sadik were the first todevelop an automated CAD system as a clinical quality assurance tool for the interpretation ofbone scans [“] This bone scan CAD software was trained to interpret bone scans usingtraining databases that consist of bone scans from European patients who have the desiredimage interpretation metastatic disease or not The results showed a sensitivity of at aspecificity of These works result in certain outcomes that refer to the development of atotally automated computerassisted diagnosis system that can identify metastases after examining bone scans applying multilayer perceptron ANN techniques involving a small databaseof wholebody bone scans patients The highest sensitivity that was achieved from all thestudies and accomplished during this thesis was approximately []Horikoshi compared the diagnostic accuracy of two CAD systems one based on aEuropean and another on a Japanese training database in a group of bone scans from Japanesepatients [] The Japanese CAD software showed a higher specificity and accuracy comparedto the European Comparing the sensitivities the Japanese CAD software achieved whereas the European CAD software reached []In another study conducted by Tokuda the diagnostic capability of a completely automated CAD system which detects metastases in the images of bone scans by focusing on twodifferent patterns was investigated [] The first pattern was devoted to the detection ofmetastases per region the second one detects metastases per patient The investigated systemwas called œBONENAVI version  The produced results have shown that the new CADPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networkssystem is able to decrease the number of false positive findings which depends on the primarylesion of cancerIn Aslantas proposed œCADBOSS as a fully automated diagnosis system forbone metastases detections using wholebody images [] The proposed CAD system combines an active contour segmentation algorithm for hotspots detection an advanced methodof image gridding to extract certain characteristics of metastatic regions as well as an ANNclassifier for identifying possible metastases The calculated accuracy sensitivity and specificity of CADBOSS were and respectively outperforming other state of theart CAD systemsAdditionally ML methods have been exploited and applied in CAD systems for bonemetastasis detection in bone scintigraphy images A parallelepiped classification method wasspecifically deployed in [] to assist physicians in bone metastases detection of cancer Decision Trees DT and Support Vector Machines SVM were exploited for predicting skeletalrelated events in cancer patients with bone metastases achieving higher accuracies with asmaller number of variables than the number of variables used in Linear Regression LR MLtechniques can be also used to build accurate models to predict skeletalrelated events in cancer patients with bone metastasis providing an overall classification accuracy of ± []As far as PET and PETCT imaging techniques in nuclear medicine are concerned thereare some recent and prominent studies that apply the advantageous features of CNNs In []deep learning has been applied for classification of benign and malignant bone lesions in [F]NaF PETCT images The authors in this work followed the VGG19 architecture for theirnetwork by employing × convolutional layers followed by fully connected layers and asoftmax layer as final activation The ImageNet database of natural images was further used topretrain the network™s weights In this way the network first is trained on general image features and later is tuned using the lesion images and the physician™s scores Taking a closer lookat the results it can be concluded that network™s performance was improved when it wastrained to differentiate between definitely benign score and definitely malignantscore lesions The values of prediction metrics were and concerningthe accuracy sensitivity specificity and positive predictive value respectivelyAlso a CNNbased system was examined in a recent retrospective study which included sequential patients who underwent wholebody FDG PETCT [] The main purpose ofthe study was to detect malignant findings in FDG PETCT examinations while a neural network model equivalent to ResNet24 was built Additionally GradCAM was employed toidentify the part of the image on which the neural network used the largest information Thefindings of the study showed that GradCAM reasonably highlighted the area of malignantuptake allowing physicians to make a diagnosis The same research team recently developed a CNNbased system that predicts the location of malignant uptake and furtherevaluated predictions accuracy [] A network model with configuration equivalent toResNet24 was used to classify wholebody FDG PET imagesIn the research work [] a simple CNNbased system that predicts patient sex from FDGPETCT images was proposed Specifically consecutive patients have participated in thestudy and underwent wholebody FDG PETCT The CNN system was used for classifyingthese patients by sex Another CNNbased diagnosis system for wholebody FDG PETCT wasdeveloped in [] that predicts whether physician™s further diagnosis is required or not Athorough analysis of the results shows that the accuracy considering images of patients presenting malignant uptake and images of equivocal was ± and ± respectivelyThe task of segmentation with the use of deep learning models in skeletal scintigraphyimages has been discussed in more research studies For example in [] the authors followedPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksdifferent approaches to convert convolutional neural networks designed for classificationtasks into powerful pixelwise predictors Moreover in [] a deeplearning based segmentation method was developed using prostatespecific membrane antigen PSMA PET imagesand showed significant promise towards automated delineation and quantification of prostatecancer lesions However this research domain that involves bone scintigraphy segmentationwith the inclusion of advanced deep neural networks has not been yet well establishedAlthough bone scintigraphy is extremely important for the diagnosis of metastatic cancer itis clear that a small number of research works have been carried out which presented onlysome preliminary results while the advantageous and outstanding capabilities of CNNs havenot been fully investigated Reviewing the relevant literature it appears that CNNs have notbeen sufficiently applied for the diagnosis of prostate cancer metastasis from whole bodyimages Aim and contribution of this research workCNN is an efficient deep learning network architecture that has recently found great applicability in the medical domain It has shown excellent performance on medical image applications including bone scintigraphy and nuclear medical imaging and can offer a positiveimpact on diagnosis tasksNowadays the main challenge in bone scintigraphy as being one of the most sensitiveimaging methods in nuclear medicine is to build an algorithm that automatically identifieswhether a patient is suffering from bone metastasis or not based on patient™s whole bodyscans It is of utmost importance that the algorithm needs to be extremely accurate due to thefact that patients™ lives could be at stake Deep learning algorithms whose potential lies in thefact that they can improve the accuracy of cancer screening have been recently investigatedfor nuclear medical imaging analysis Recent studies in BS and PET have shown that a deeplearningbased system can perform as well as nuclear physicians do in standalone mode andimprove physicians™ performance in support mode Even though BS is extremely importantfor the diagnosis of metastatic cancer there is currently no research paper regarding the diagnosis of prostate cancer metastasis from whole body scan images that applies robust and moreaccurate deep CNNsThis research study investigates the application of a deep learning CNN to classify bonemetastasis using whole body images of men who were initially diagnosed with prostate cancerThe proposed method employs different CNNbased architectures with data normalizationdata augmentation and shuffling in the preprocessing phase In the training phase the backpropagation technique has been used for updating the weights as part of the optimization process Finally the network architecture is finetuned and the configuration that offers the bestperformance is selected to train the CNN modelThe scope of the current work entails the following two main components First this studyintroduces the CNN method into the diagnosis of bone metastasis disease based on wholebody images In the second phase the paper deals with the improvement of the existing CNNmethod in terms of both network architecture and hyperparameter optimization Thedeployed process seemingly improves the diagnostic effect of the deep learning method making it more efficient compared to other benchmark and wellknown CNN methods such asResNet50 VGG16 GoogleNET Xception and MobileNet [ ]The innovations and contributions of this paper are well summarized as follows¢ The development and demonstration of a simple fast robust CNNbased classification toolfor the identification of bone metastasis in prostate cancer patients from wholebody scansPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networks¢ The rigorous CNN hyperparameter exploration for determining the most appropriatearchitecture for an enhanced classification performance¢ A comparative experimental analysis utilizing popular image classification CNN architectures like ResNet50 VGG16 GoogleNET Xception and MobileNetThis paper is structured in the following fashion Section presents the material and methods related to this research study Section presents the proposed solution based on CNNmethod for bone metastasis diagnosis for prostate cancer patients using whole body imagesSection shows the results of exploration analysis of CNNs in Red Green and Blue RGBmode for different parameters and configurations thus providing the best CNN model forthis case study Furthermore all the performed experiments with representative results aregathered in section whereas section provides a thorough discussion on analysis of resultsSection concludes the paper and outlines future steps Materials and methods Patients and imagesA retrospective review of consecutive whole body scintigraphy images from differentmale patients who visited Nuclear Medicine Department of Diagnostic Medical Center œDiagnostico AE in Larisa Greece from June to June was performed The selection criterion was prostate cancer patients who had undergone wholebody scintigraphy because ofsuspected bone metastatic diseaseDue to the fact that whole body scan images contain some artifacts and other nonrelatedto bone uptake such as urine contamination and medical accessories ie urinary catheters[] as well as the frequent visible site of radiopharmaceutical injection [] a preprocessingapproach was accomplished to remove these artifacts and nonosseous uptake from the original images This preprocessing method was accomplished by a nuclear medicine physicianbefore the use of the dataset in the proposed classification approachThe initial dataset of images contained not only bone metastasis presence and absencepatient™s cases suffering from prostate cancer but also degenerative lesions [] Due to thisfact as well as aiming to cope with a twoclass classification problem in this study a preselection process concerning images of healthy and malignant patients was accomplished In specific out of consecutive wholebody scintigraphy images of men from differentpatients were selected and diagnosed accordingly by a nuclear medicine specialist with years of experience in bone scan interpretation Out of bone scan images bone scansconcern male patients with bone metastasis and male patients without bone metastasis Anuclear medicine physician classified all the patient cases into categories metastasis absentand metastasis present which was used as a gold standard see Fig The metastatic imageswere confirmed by further examinations performed by CTMRI Wholebody scintigraphy Bone scansA Siemens gamma camera Symbia S series SPECT System by dedicated workstation and software Syngo VE32B with two heads and low energy highresolution collimators was used forpatients scanning The speed of scanning was cmmin with no pixel zooming Two types ofradionuclide were used for bone scintigraphy 99mTcHDP TechneScan1 and 99TcMDPPoltechMDP 5mg Whole body scintigraphy was acquired approximately hours after intravenous injection of “ MBq of radiopharmaceutical agent depending on the patientbody type The common intravenous injection was MBq of radiopharmaceutical agentPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksFig Image samples in the dataset Label a Metastasis is present or b absent101371journalpone0237213g001In total planar bone scan images from patients with known prostate cancer werereviewed retrospectively The wholebody field was used to record anterior and posteriorviews digitally with resolution × pixels Images represent counts of detected gammadecays in each spatial unit with 16bit grayscale depthThe image data acquired were originally in DICOM files a commonly used protocol forstorage and communication in hospitals These image data were extracted from these DICOMfiles to create new images in JPEGformat instead A novel dataset of bone scintigraphyimages containing men patients suffering from prostate cancer with metastasis present andmetastasis absent two distinct classes of healthy and malignant cases was prepared for experimentation This dataset consisting of wholebody scans is available for research use afterrequestThis study was approved by the Board Committee Director of the Diagnostic Medical Center œDiagnosticoIatriki AE and the requirement to obtain informed consent was waived bythe Director of the Diagnostic Center due to its retrospective nature All procedures in thisstudy were in accordance with the Declaration of Helsinki MethodologyThe problem of classifying bone metastasis images is a complex procedure and so effectivemachine learning methods need to be exploited to cope with this diagnosis task Deep learningmethods such as CNNs are applied in order to train a classifier to distinguish images of prostate cancer patients with bone metastasis and metastasis absent on healthy patients The effective CNN method for bone metastasis classification proposed in this paper includes threeprocessing steps data preprocessing for the collected scan data normalization a trainingphase for CNN learning and validation and testing which includes the evaluation of the classification results as illustrated in Fig The proposed methodology is thoroughly presented inthe following sections Data preprocessing Step Load images to RGB The original images were saved inRGB mode All images are stored in a respective folder before loaded into the computer memory for CNN training In each image a suitable prefix was defined according to the patient™scategory for example œmalignant_ and œhealthy_ Next a small script was set up in order toPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksFig Flowchart of the proposed CNN methodology101371journalpone0237213g002assign a numerical value as label to each image according to its prefix In our case the value ˜™was assigned for œmalignant_ prefixes whereas the value ˜™ for œhealthy_ prefixesStep Data normalization It is common to follow a normalization process feature scalingin most machine learning algorithms [] The minmax normalization processnormalizes thedataset values within the to ensure that all feature data are in the same scale for trainingand testing The data normalization process also assists the convergence of the backpropagation algorithmStep Data shuffle To avoid or eliminate unbiased sampling in machine learning anappropriate shuffling method is needed to be defined More specifically a randomnumbergenerator is used to reorder the images An image sample chosen randomly is meant to be animpartial representation of the total images An unbiased random sample is important formachine learning to provide reliable conclusions In this study Python™s randomshufflemethod is used for dataset shufflingStep Data augmentation Data augmentation is used as a method to artificially increasethe diversity of training data by a large margin by manipulating the existing data instead ofcreating new Data augmentation techniques such as cropping padding and horizontal flipping are commonly used to train large neural networks [] In this research the number ofimages used for learning processes was followed by an image augmentation processing such asrotation enlargementreduction range and flip Note that the original images used for the testwere not subjected to such an augmentation processStep Data split The dataset was split into three sections a training portion a validationportion that would allow the training process to improve and a testing holdout portionwhich is part of the dataset that is completely hidden from the training process The first datasplit takes place by removing of the total dataset and saving for later use as testing Theremaining of the dataset is then split again into an ratio where the small po

Thyroid_Cancer

" Despite several efforts the development of an effective vaccine for COVID19 may take a much longer timeTraditionalnatural medicine already experienced by humans could be an earlier solution Considering the researchteam™s experience in using nanoclays as highaffinity material for cancer metastasis melanoma treatment andbone regeneration we propose to use these nanoclays for the preventiontreatment of COVID19 Owing to highaffinity nanoclays would capture the viruses before the latter get engaged with human hACE2 In this studymolecularlevel simulations and modeling of the interaction of coronavirus spike and hACE2 proteins wereperformed with and without nanoclays The results showed a very high level of affinitycohesiveness among SARSCoV2 spike and nanoclays as compared to the one between the former and hACE2 We premise that these nanoclays since already being used as drug carriers could also be injected as œclaysalone medicine Recommendationshave also been provided for future in vitro and in vivo studiesBackgroundThe sudden emergence and rapid spread of novel coronavirus SARSCoV2 have significantly affected thehealth and lives of human beings in addition to criticallyaffecting the world economy SARSCoV2 spike S bindswith high affinity to human angiotensinconverting enzyme hACE2 and uses it as an entry receptor to invade target cells Fig 1a b [] The virussurface spikeprotein mediates coronavirus entry into host cellsSARSCoV2 spike protein contains a receptorbindingdomain RBD that recognizes explicitly as its receptorhACE2 [ ] The surface of hACE2 contains two virusbinding hotspots that are criticalfor SARSCoV2 Sbinding Several naturally selected mutations in SARSCoV2 RBD surround these hotspots and regulate theinfectivity pathogenesis and crossspecies and humantohuman transmissions of SARSCoV2 [ ]At present there are no clinically approved vaccinesor drugs that specifically target SARSCoV2 Followingthe real protocol of developing a vaccine it may takemuch longer time to come up with an effective vaccine Correspondence habibrehmankfupmedusa3Engineering Research International ERI Riyadh Saudi ArabiaFull list of author information is available at the end of the There is a lot of interest in the development of therapeutic antibodies against SARSCoV2 Despite many efthese antibodies have not yet beenforts howeverdiscovered [] exceptin a few trials [] One trialshowed the potent neutralization of SARSCoV2 bybinding to the RBD of its S glycoprotein [] In this trial[] antibody cocktails a mixture of different antibodiesis recommended due to the increased neutralization effect it has on SARSCoV2 However use of antibodiesin the past from convalescent patients of SARSCoV totreat SARSCoV infection has shown adverse reactionsin the patients such as AntibodyDependent Enhancement ADE causing increased viral infectivity and otherharmful immune responses [] Moreover based on theexperience with the vaccine development efforts forSARSCoV and MERS chances of the materialization ofthe efforts being made for SARSCoV2 seems quitethin Therefore naturaltraditional medicines that have ahistory of safe consumptioningestion by humans couldbe considered as one of the treatment options for SARSCoV2 Being a natural material and a history of humanuseconsumption we suggest œhighly charged nanoclays to be used as coronavirus blockers and inhibitorsof the spikemediated entry into the human cells The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40 0cAbduljauwad Nanoscale Research Letters Page of Fig Schematics of the SARSCoV2 attack on human hACE2 and the subsequent immune system response a b RBD binding hACE2 withoutan interference c RBD complexed with the antibody at receptor attachment site hence competing with hACE2 d RBD complexed with RBD at asite other than where receptor attaches resulting in the alteration of RBD structure and interruption of lock and key binding of RBD to hACE2Nanoclays nanosized natural materials originatingfrom minerals of the sedimentary rocks have got avery high affinity to bacteria and viruses [] Due toisomorphous substitution in their molecular structurethese nanoclays exhibit charge deficiency on theirsurfaces This charge deficiency on their surfaces isneutralized by the water molecules and the dissolvedcations Fig The charged structure and large surface area of clay nanops give them an affinityfor charged entities as found on bacterial surfacesand bacterial toxins Their distinct biomedical properties include high absorption the ability to engulf microbes and no toxicity Each of the electrically activeclay minerals has its distinct morphology characteristics and interaction behavior The most studied biomedical application of nanoclays includes serving ascarriers and complexes for anticancer drugs such as5fluorouracil and trastuzumab [“] They havetherefore been a potential alternate medicine for several diseases [“] Clay nanops due to theiradhesive nature have also been used as carriers forsustainedrelease medicine [ ] Nanoclays havealso successfully been used to adsorb and treat bovinerotavirus and bovine coronavirus [] Researchers[] intercalated methotrexate MTX an anticanceragentinto the anionic clay to create a nanohybriddrug They used the coprecipitation and subsequenthydrothermal methodology to prepare this chemicallystructurally and morphologically welldefined twodimensional drugclay nanohybrid The researchers[] discovered that due to the biocompatibility andhigh loading capacity bentonite nanoclay could beused for the preparation of the drugdelivery vehiclesthey prepared doxorubicinbentoniteIn thisto form ananoclay complex DOXBent complexsustainedreleaseintradrugdeliverystudysystem for 0cAbduljauwad Nanoscale Research Letters Page of Fig a SEM image and b the corresponding molecular structure of Namontmorillonite showing the configuration isomorphous substitutioncharge deficiency and interlayer cations from []tumoral chemotherapy of melanoma As montmorillonite clay is recently being studied to be used as anadditive and drug carrier materialthese nanoclaycomposites appeal their use in various dosing formmainly for controlled release of the drug [] The researchers [] also discovered that nanoclays can beused into recent dual functional drug delivery systemsDDSs to have efficiency in the drug delivery and soreduce the toxicity of doxorubicin DOX that is being used for thyroid cancer treatment Using a libraryof single“single type photo cleavable amphiphilicJanus dendrimers researchers [] developed a selfassembling lightresponsive dendrimersomes vesicleplatform Similar to the nanoclays surface modifiedbioactive virusmimicking anic nanovesicles fromglycodendrimersomes have structural modificationsthat contribute to manifest SARSCoV2 and hostpathogenic molecular interactions that help the virusto escape from the human immune system []Through considerable previous research we developedbasic characterization and behavior modeling ofthecharged clay minerals [“] and their applications in thecontrol of cancer metastasis [] in vitro and in vivo studies on melanoma treatment [] and the calcium depositionbone regeneration studies [] In a previous study bythe authors [] it was demonstrated that clay nanops had got a high affinity to the charged surfaces Thehigh attraction affinity of the nanoclays and the increasednonspecific adhesion attraction of the cancer cells makenanoclays favorable candidates to control cancer metastasis In that study we demonstrated the possible use of twocharged clay minerals to control the metastasis of thecancer cells Namontmorillonite SWy3 and palygorskitePFll Further to the findings of the authors™ previous research [] on the use of these nanoclays for the control ofcancer metastasis we also through in vitro and in vivostudies established that these nanoclays have inhibitory effects on melanoma cancer cells mainly on cell proliferationand viability [] In these previous studies in addition tolaboratory experiments molecularlevel simulations werealso performed on the nanoclay and cells™ interactionsThese simulations provided the assessment of the relativelevel of cohesivenessaffinity in the interactions with andwithout clay nanopsperceptionthroughthisestablishingBased on all the above experience of the authors onthe highaffinity potential of nanoclays we propose thatthe nanoclays could be mimicked as antibodies and canthus attract and engulf coronaviruses before they get engaged with human hACE2 This paper is a first steptowardsamolecularlevelsimulation and modeling approachBased on the results of the molecularlevel simulationsan outline of the recommendations for the next phasesof in vitro and in vivo research is also provided As thesenanoclays are also successfully being used as medicinecarriers we also premise that they can also be injectedingested as œclaysalone medicine and thus we haveproposed a tentative nanoclays administration methodology for this purposeMaterials”MoleculesSelection and Formulation of SARSCoV2 and hACE2Molecules of SARSCoV2 spike S and hACE2 were acquired from the protein data bank website RCSB [“] 0cAbduljauwad Nanoscale Research Letters Page of The molecular models of SARSCoV2 spike S andhACE2 formulated in Materials Studio software [] arerespectively shown in Fig 3a b Before being subject tothe simulations these molecules were charged using thecharge equilibration method QEq of the softwareSelection and Formulation of Nanoclay CrystalliteNamontmorillonite one of the most active members ofthe smectite group of clay minerals was selected for thestudy Namontmorillonite is a layered phyllosilicate claysmectite Fig In the colloidal form the space between neighboring layers can contain free sodium calcium or magnesium cations that are electrostaticallyattracted to external negatively charged surfaces [] Inits dry powdered state Namontmorillonite exists asequidimensionalflakessheets with dimensions of approximately × × microns Fig 2a Thesenegative charges on their interlayer surfaces are balancedby the cations As colloids the interlayer cations get dissociated from the clay ps and associate themselveswith the other negatively charged surfaces These ps also have positively charged edges due to the presence of the broken bonds at their ends Morphology andfurther characteristics of these nanoclays are providedin Table while formulation of their crystallites in Materials Studio software are explained belowIn the software Namontmorillonite crystallites wereformulated based on fundamental properties such asCEC exchangeable cations and interlayer charges Table The size of the molecularcrystallite size was selectedbased on the results of the p size analysis usingthe dynamic light scattering DLS technique [] Thefinal form of clay crystallite created in the software istheseshown in Fig 3c Afterthe preparation ofcrystallites in the design mode of the software using theinherent properties these were charged using the chargeequilibration method QEq of the softwareMethods”MolecularLevel SimulationsThis part of the study consisted of the simulation andassessment of the interactions of the SARSCoV2 spikeS with clay crystallites and with hACE2 Although thesemodels may not be the complete replication of the actual in vitro conditions these have been incorporatedwith all the essentialinteractions and are quite wellsuited for the intended relative and comparative studyIn the software the sorption and simulations of theformulated configurations of SARSCoV2 S Namontmorillonite crystallites and hACE2 were carriedout using Monte Carlo MC and molecular mechanicsMM techniques The enhancement of affinity in all thesimulated configurations was assessed in terms of thecalculated cohesive energy density CED”CED beingconsidered as a measurement of the cohesiveness of themolecular system Due to the largesized computationsinvolved in the simulationsthese calculations werecarried out using the highperformance computing facilities HPC at KFUPM KSA The overall methodologyand the choice of particular methods and the simulationparameters were based on authors™ previous research[“] while it is detailed in the subsequent sectionSARSCoV2 Spike S Interactions with hACE2 and ClayCrystallitesTo simulate the interaction of SARSCoV2 S with claycrystallites various numbers of the crystallites of Namontmorillonite clay were sorbed on SARSCoV2 Smodel For these sorption simulations the MetropolisFig Molecularlevel models of a SARSCoV2 spike b hACE2 and c Namontmorillonite crystallite formulated in Materials Studio software 0cAbduljauwad Nanoscale Research Letters Page of lnoitauccoFlnoitcaretnInoitcaretnInoitcaretnIninoisrepsidretawnoisrepsiDygrenelatotygreneWdvˆ’ygreneBAlraopcilihpordyHˆ’ˆ’ˆ’ˆ’aCaNretaWytiniffasγlaitnetopVmPZateZreyalretnIegrahclardeharteTlardehatcOlebaegnahcxEegrahcegrahcsnoitacqemgCECecafruSNaeragmslarenmirehtOliacmehClaumrofacilisOiSgMlAaCaNecruoSytnuoCASUYWkoorC][yWSyacletinolliromtnomaNfonoitaziretcarahcliacmehcdnalacisyhpfoyrammuSelbaT 0cAbduljauwad Nanoscale Research Letters Page of Monte Carlo method was selected in the Sorption module of the software In each sorption step clay crystallitesoccupy spaces around the spike S model to lower theoverall energy of the complex The required number ofcrystallites were sorbed in a maximum of stepsand then the energy of the system was minimized usingthe Forcite module of the software based on the MDprinciples The similar sorption process was repeated forthe interaction modeling of the SARSCoV2 spike molecule with hACE2 In this process hACE2 moleculeswere sorbed around the RBD of the spike S of SARSCoV2 After the completion of the sorption process theenergy of the formulation was minimized using MDbased module of the softwareThe Forcite module ofthe software incorporatingNPT constant number of ps pressure andtemperature ensemble was used for MD simulationswith a modified universal force field [] The simulations were run for to ps with an interval of 05fs ortill a constant volume is obtained A Berendsen thermostat with a decay constant of ps was used to controlthe temperature during the simulation During the MDsimulations the assumed temperature was kept constantat K °C with an atmospheric pressure kPaA Berendsen barostat with a decay constant of pswas used to control the pressure of the system TheBerendsen methodology was considered as the most appropriate for the single crystallites after several trials involving other thermostats and barostats available in thesoftware In the Monte Carlo method the parametersfor the ratios of exchange conformer rotate translateand regrow were selected as and respectively with the corresponding probabilities as and Amplitudes adapted for rotationand translation were ° and respectivelyCohesive Energy Density CED MeasurementIn this study the assessment of the affinitybindinglevelin the SARSCoV2clay crystallites and SARSCoV2hACE2 complexes was measured through thechanges in the CED After the sorption of clay crystallites and the subsequent performance of moleculardynamics of each of the configurations the CED wasdetermined using the cohesive energy density optionof the Forcite module of the software The authorshave experienced that the CED concept consisting ofthe total van der Waals and electrostatic CEDs canquite closely explain the various molecularlevel processes and interactions and simulate the extent of affinitybinding created among the simulated complexes[“] Quantitatively CED is defined as the amountof energy needed for the transition of mol of material from the liquid to the gaseous phase It is also ameasureofaffinityattractivenessthe mutualofmolecules and is expressed both as electrostatic andvan der Waalsan NPTensembleaveraged overforcesIn the Forcite module van der Waals energies wereevaluated using atombased cutoffsIn this methodnonbond interactions are simply calculated to a cutoffdistance and interactions beyond this distance are ignored To avoid the discontinuities caused by direct cutoffs most simulations use a switching function to turnoff nonbond interactions over a range of distancessmoothly An effective potential is created by multiplyingthe actual potential by the smoothing function Thechoice of the function in the intermediate range is crucial and should be continuously differentiable in this region so that forces can be calculated In this study acubic spline smoothing function was used with a splinewidth of and a cutoff distance of Results and DiscussionsThe final configuration of the SARSCoV2 ShACE2complex is shown in Fig 4a while the complexes between SARSCoV2 spike and different numbers of clayNamontmorillonite crystallites are respectively shownin Fig 4b c For comparison purposes total CEDs ofvarious proportionsnumbers of the clay crystallites onthe SARSCoV2 spike and the interaction of the laterwith hACE2 are plotted in Fig Based on our experience we have hypothesized thatnanoclays due to their high adhesive properties couldalso act as SARSCoV2 inhibitors They can do it bystrongly associating with the spike S present on SARSCoV2 The results obtained from the molecularlevelsimulations of the interactions indicate that due to veryhigh CED between SARSCoV2 and the nanoclays ascompared to the former and hACE2 Fig they couldinhibit SARSCoV2 from getting engaged with hACE2Moreover it could also be concluded from Fig thatthe extent of inhibition due to nanoclays is increased inquantity dosagedependent wayNanoclay Interactions with SARSCoV2 Spike SAuthors in their earlier research have demonstrated therole of nanoclays in promoting adhesion among thecancer cells and their microenvironment and hence controlling metastasis [] Adhesion measurements of mix of Namontmorillonite and palygorskite showedan increase in adhesion by among cancer cells andthe extracellular matrix proteins Fig 6a A corresponding SEM of the nanoclays binding the Raji cells and thefibronectin proteins is shown in Fig 6b Sample imagingwas performed in SEM mode in an FEI ESEMFEG XL atthe Miller School of Medicine University ofMiami Florida Authors also discovered in their previousresearch that electrostatic van der Waals and ZP 0cAbduljauwad Nanoscale Research Letters Page of Fig Molecularlevel simulation results in Materials Studio Software a SARSCoV2 S and hACE2 CED Jcm3 b SARSCoV2 S modelinteracting with twelve crystallites of Namontmorillonite CED Jcm3 and c SARSCoV2 S model interacting with twentyfour crystallites ofNamontmorillonite CED Jcm3”obtained using Sorption technique implemented in the softwareattractions seem to be dominating in the adhesion processes [] We conclude that the same mechanismswould have also facilitated the binding of the adhesivesurfaces of the nanoclays to the spike of SARSCOV2Fig ZP is a measure of the dispersion or flocculationtendency in the colloidal form including the interactions 0cAbduljauwad Nanoscale Research Letters Page of Fig Variation of cohesive energy density CED for SARSCoV2 ShACE2 and the complexes of the former with different numbers ofNamontmorillonite crystalliteswith the other constituents present in the suspensionmedium As a general rule a zeta potential greater than mV either positive or negative indicates dispersiontendency while a zeta potential of less than mV generally results in agglomeration Higher dispersion tendencies ZP of the clay nanops used in the study ˆ’ to ˆ’ mV lead to higher dispersion tendency andhence in the generation of higher surface area amplifyingthe interactions with the SARSCoV2 spike Althoughbased on their ZP Namontmorillonite nanopshave hydrophilic nature they in the presence of saltsalso promote secondary adhesion mechanisms betweenhydrophobic and hydrophilic surfaces [] It should alsobe noted that these clay nanops have high dispersion tendency due to their hydrophilic nature and relatively higher repulsive acidbase AB interactions Table High dispersion in turn results in the generation ofhigh surface area for increasing the attractive interactions Higher surface areas promote larger attractionsdue to the van der Waal attractions and the electrostaticforces among oppositely charged surfaces Besides although of relatively lesser degree positively chargededges of Namontmorillonite ps also get electrically attracted to the spike SThe results of the molecularlevel simulations for theinteraction of SARSCoV2 spike S with the clay crystallites Fig also confirm the above interaction behaviors It has been observed that the sorption of the claynanops results in the formation of closely interacting strong van der Waals attraction fields These van derWaals attraction fields create higher CED of the claySARSCoV2 configuration Substantial increase in totalCED after the addition of clay crystallites Fig is alsoa testimony of a very high affinity of SARSCoV2 withthese ps as compared to the affinity of the formerwith hACE2systemagglutinationNanoclays as PseudoantibodiesBased on all the current and past research by the authors establishing the highaffinity potential of nanoclays we premise that nanoclays could be mimicked asantibodies and can thus attract and engulf coronavirusesbefore they get engaged with human hACE2 Antibodiesare glycoproteins synthesized by plasma cells as part ofthe adaptive immune response to assist in the clearanceof infection from the body Antibodies aid in infectionclearance in multiple ways such as opsonization of pathogens to facilitate phagocytosis activation of the complementandneutralization of viruses and toxins When bound to theviral surface proteins antibodies prevent the entry of theviruses into the cell by preventing the attachment of viruses to their target receptor on the cell Antibody binding can occur at different sites on the surface proteinleading to various mechanisms that cause the same effect In the case of SARSCoV2 two viral neutralizationmechanisms by antibodies have been observed [ ]and shown in Fig 1c d One of the mechanisms involvesdirect binding of antibodies to the attachment site of theSARSCoV2RBD resulting in the antibody competingwith the target receptor hACE2 Another mechanism involves the binding of antibodies to the other sites onRBD without any competition with the target receptorThe latter is shown to be involved in neutralization byof microbes 0cAbduljauwad Nanoscale Research Letters Page of Fig a Summary of adhesion force measurements among RajiRajiFN assembly using AFM before and after treatment with various proportionsof Namontmorillonite and palygorskite clay nanops [] Error bars represent the variations in the trials b SEM image of the binding of Rajicells and Fibronectin proteins produced by nanoclaysthe most potent Monoclonal Antibody mAb discoveredin the study [ ] Analogous to the antibodies interaction with SARSCoV2 RBD inhibiting the latter toengage with hACE2 a similar molecularlevel model isprepared for nanoclays resulting in a similar inhibitionof the coronaviruses and shown in Fig Owing to theirvery high affinity nanoclays would get attracted tospikes of SARSCoV2 and thus restrict engagement ofRBDs of these spikes with hACE2Proposed Nanoclay Administration MethodologyClay use as drug carriers has been tested multiple timesyielding promising results of little to no cytotoxicity tocells of the human body Kaolinite clay mineral wastested for use in a potential drug delivery system andwas shown to have high biocompatibility and very lowas[]negligiblecytotoxicity [] Poly DLlactidecoglycolidemontmorillonite nanop cytotoxicity in vitro was alsodemonstratedPalygorskitepolyethyleneiminefluorescein isothiocyanate nanocomposites also exhibited almost no cytotoxicity in vitro[] Authors have also experienced injecting nanoclayssubcutaneously for the treatment of melanoma duringin vivo studies [] Based on the use of clay as a cancerdrug carrier and in other sustainedrelease medicine[“] we propose that nanoclays may be injected asœclayalone medicine subject to the verification in vivoand clinical trialsAlthough nanoclays are nonbiodegradable a comprehensive understanding of the design of the similar inanic nanops with their metabolic performancein the body carried out in the study [] could also 0cAbduljauwad Nanoscale Research Letters Page of Fig Three possible mechanisms of interactions of montmorillonite nanoclay with the SARSCoV2 spike S Electrostatic attraction amongpositively charged nanop edges and NaCa ions with negatively charged virus surfaces Van der Waals attractions ZPelectrostatic interactionscategorize these nanoclays as human body clearable inanic agentsConclusions and RecommendationsBased on all the current and past research by the authors establishing the highaffinity potential of nanoclays these could be mimicked as antibodies and canthus attract and engulf coronaviruses before they get engaged with human hACE2The results of the molecularlevel simulations for theinteraction of SARSCoV2 spike S with the clay crystallites result in the formation of closely interacting strongvan der Waals attraction fields These van der Waals attraction fields create higher CED of the claySARSCoV configuration Substantial increase in total CED afterFig Interaction mechanism of nanoclay ps with SARSCoV2 spike S inhibiting the interaction of the later with hACE2 0cAbduljauwad Nanoscale Research Letters Page of addition of clay crystallites is also a testimony of a veryhigh affinity of SARSCoV2 with these ps as compared to the affinity of the former with hACE2We propose to continue the research by carrying outin vitro interaction studies between SARSCoV2 anddifferent percentage of nanoclays Based on theoptimum dose of nanoclay developed in the in vitrophase we suggest to carry out in vivo studies on the animals The animal study should be carried out both withand without nanoclay to finalize the nanoclay dose andshould lay the foundation for the clinical trialsAcknowledgementsThe authors highly acknowledge KFUPM for providing highperformancecomputing research facilitiesAuthors™ ContributionsAll the authors equally participated at all the research levels The authorsread and approved the final manuscriptFundingNo fundingAvailability of Data and MaterialsAll data generated or analyzed during this study are included in thispublished Ethics Approval and Consent to ParticipateNot applicableConsent for PublicationNot applicableCompeting InterestsThe authors declare that they have no competing interestsAuthor details1Civil Environmental Engineering department King Fahd University ofPetroleum Minerals KFUPM Dhahran Saudi Arabia 2Royal College ofSurgeons in Ireland RCSI Bahrain campus Busaiteen Bahrain 3Engineering Research International ERI Riyadh Saudi ArabiaReceived July Accepted August ReferencesEwen Callaway and Nik Spencer The race for coronavirus vaccines agraphical guide eight ways in which scientists hope to provide immunityto SARSCoV2 News Feature Nature vol April Li F Li W H Farzan M Harrison S C Structure of SARS coronavirusspike receptorbinding domain complexed with receptor Science httpsdoi101126science1116480 Li WH Angiotensinconverting enzyme is a functional receptorfor the SARS coronavirus Nature “ httpsdoi101038nature02145Li F Structural analysis 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Committee for Medicinal Products for HumanUse Eur J Cancer “Sahel N Abduljauwad and HabiburRehman Ahmed Enhancing cancer celladhesion with clay nanops for countering metastasis Nature ScientificReports April httpsdoi101038s4159801942498y Zhang Y Long M Huang P Yang H Chang S Hu Y Mao L Intercalated 2D nanoclay for emerging drug delivery in cancer therapyNano Res “ Chianelli R R Das S US Patent No Washington DC US Patent and Trademark Office Han S Liu F Wu J Zhang Y Xie Y Wu T Y Targeting of fluorescentpalygorskite polyethyleneimine nanocomposite to cancer cells Appl ClaySci “Sun B Ranganathan B Feng SS Multifunctional poly D Llactidecoglycolide montmorillonite PLGAMMT nanops decorated byTrastuzumab for targeted chemotherapy of breast cancer Biomaterials“Lin FH Lee YH Jian CH Wong JM Shieh MJ Wang CY A study ofpurified montmorillonite intercalated with 5fluorouracil as drug carrierBiomaterials “ Bothiraja C Thorat UH Pawar AP Shaikh KS Chitosan coated layeredclay montmorillonite nanocomposites modulate oral delivery of paclitaxel incolonic cancer Mater Technol 29sup3B120“B126Kevadiya BD Thumbar RP Rajput MM Rajkumar S Brambhatt H Joshi GVBajaj HC Montmorillonitepolyεcaprolactone composites asversatile layered material reservoirs for anticancer drug and controlledrelease property Eur J Pharm Sci “ Guo MY Wang AF Muhammad F Qi WX Ren H Guo YJ Zhu GS Halloysite nanotubes a multifunctional nanovehicle for anticancer drugdelivery Chin J Chem “ Martínez C D Cationic clays upon cancer therapy Virtual MultidisciplinaryConference QUAESTI Konta J Clay and man clay raw materials in the service of man ApplClay Sci “ Murray HH Traditional and new applications for kaolin smectite andpalygorskite a general overview Appl Clay Sci “ Volzone C Retention of pollutant gases comparison between clayminerals and their modified products Appl Clay Sci “ Dong Y Feng SS Poly dllactidecoglycolidemontmorillonitenanops for oral delivery of anticancer drugs Biomaterials “ Clarka KJ Sarrb AB Grantb PG Phillipsb TD Woodea GN In vitrostudies on the use of clay clay minerals and charcoal to adsorb bovinerotavirus and bovine coronavirus Vet Microbiol “ Choi G Huiyan P Alothman Z Vinu A Yun C Choy J Anionic clay asthe drug delivery vehicle tumor targeting function of layered doublehydroxidemethotrexate nanohybrid in C33A orthotopic cervical cancermodel International Journal of nanomedicine DOI httpsdoi102147IJNS95611 Hosseini F Hosseini F Jafari S M and Taheri A Bentonite nanoclaybaseddrugdelivery systems for treating melanoma Clay Minerals DOI httpsdoi101180clm201842018 Inamuddin Asiri A M and Mohammad Ali Applications of nanocompositematerials in drug delivery DOI httpsdoi101016C20160050751 Avolume in Woodhead Publishing Series in Biomaterials Zhang Y Long M Huang P Yang H Chang S Hu Y Tang A and MaoL Emerging integrated nanoclayfacilitated drug delivery system forpapillary thyroid cancer therapy doi 101038srep33335 Sci Rep Li S Xia B Javed B Hasley W D MelendezDavila A Liu M Kerzner MAgarwal S Xiao Q Torre P Bermudez J G Rahimi K Kostina N YMöller M RodriguezEmmenegger C Klein M Percec V and Good M CDirect visualization of vesicle disassembly and reassembly usingphotocleavable dendrimers elucidates cargo release mechanisms ACS 0cAbduljauwad Nanoscale Resear

Thyroid_Cancer

THYROID development of thyroid hormone TH analogues was prompted by the attempt to exploit the effects of THon lipid metabolism avoiding cardiac thyrotoxicosis Analysis of the relative distribution of the a and bsubtypes of nuclear TH receptors TRa and TRb showed that TRa and TRb are responsible for cardiac andmetabolic responses respectively Therefore analogues with TRb selectivity were developed and four differentcompounds have been used in clinical trials GC1 sobetirome KB2115 eprotirome MB07344VK2809and MGL3196 resmetirom Each of these compounds was able to reduce lowdensity lipoprotein cholesterolbut a phase trial with eprotirome was interrupted because of a significant increase in liver enzymes and thecontemporary report of cartilage side effects in animals As a consequence the other projects were terminatedas well However in recent years TRb agonists have raised new interest for the treatment of nonalcoholic fattyliver disease NAFLD After obtaining excellent results in experimental models clinical trials have beenstarted with MGL3196 and VK2809 and the initial reports are encouraging Sobetirome turned out to beeffective also in experimental models of demyelinating disease Aside TRb agonists TH analogues includesome TH metabolites that are biologically active on their own and their synthetic analogues ¢triiodothyroacetic acid has already found clinical use in the treatment of some cases of TH resistance due toTRb mutations and interesting results have recently been reported in patients with the Allan“Herndon“Dudleysyndrome a rare disease caused by mutations in the TH transporter MCT8 35diiodothyronine T2 has beenused with success in rat models of dyslipidemia and NAFLD but the outcome of a clinical trial with a syntheticT2 analogue was disappointing 3iodothyronamine T1AM is the last entry in the group of active TH metabolites Promising results have been obtained in animal models of neurological injury induced by bamyloidor by convulsive agents but no clinical data are available so farKeywords TH analogues sobetirome eprotirome resmetirom triac 35diiodothyronine 3iodothyronamineIntroductionT he term thyroid hormone TH analogue is used withregard to compounds that have a similar molecularstructure as TH and can therefore interact with at least someof its molecular targetsIt should be kept in mind that TH signaling is particularlycomplex Canonical TH signaling is based on the interactionwith nuclear TH receptors TRs leading to either activation orrepression of the transcription of a large number of genes Twogene subtypes exist TRa and TRb and different isoformswhich are designated by numerical subscripts can be producedby alternative splicing TRa1 is the major TR isoform in theheart and it is also expressed in other tissues including skeletal muscle brain and bone TRa2 lacks part of the THbinding domain so it is unable to bind TH and is thought tomediate constitutive repression of transcription TRb1 iswidely expressed in most tissues while TRb2 expression ismore circumscribed and it is particularly relevant in the brainpituitary retina and inner ear TRb3 has been identified in ratadipose tissue but it does not appear to be present in humansWith regard to the topic of this review the main functionalconsequence of TR subtype distribution is that the metaboliceffects of TH are largely mediated by TRb1 while cardiacTH actions depend on TRa1In addition to canonical signaling TH may activate anumber of noncanonical signaling pathways The latterinvolves TRs that do not bind DNA directly extranuclearDepartment of Pathology University of Pisa Pisa Italyª Riccardo Zucchi Published by Mary Ann Liebert Inc This Access is distributed under the terms of the CreativeCommons Attribution Noncommercial License httpcreativecommonslicensesbync40 which permits any noncommercial usedistribution and reproduction in any medium provided the original authors and the source are cited 0cZUCCHITRs that activate the phosphatidylinositol 3kinase pathwayand membrane receptors belonging to the integrin family In addition some TH metabolites have been suggested tobe potentially active although their physiological role is stillunclear By definition these compounds should be regardedas TH analogues although they are endogenous moleculesThey have also been used as templates to develop novelclasses of synthetic analogues Active TH metabolites include35diiodothyronine T2 3iodothyronamine T1AM andseveral thyroacetic acids ¢¢tetraiodothyroacetic acid orTetrac ¢triiodothyroacetic acid or Triac 3iodothroaceticacid or TA1 They have been the object of several recentreviews “ and their properties are briefly recalled in thesubsequent paragraphs only in as much as this is relevant to theobject of the present succinct reviewTH analogues have been developed for clinical and therapeutic purposes namely to exploit some aspects of THsignaling to produce beneficial effects in the diseaseTherefore in the following paragraphs these analogues arediscussed with the perspective of their potential therapeuticuse Special emphasis is placed on clinical investigationsalthough some results obtained in experimental models ofdisease are mentioned when appropriateTH Analogues with Selective TRb Activityin DyslipidemiaThe development of selective TRb agonists was promptedby the aim of treating dyslipidemia particularly hypercholesterolemia avoiding cardiac thyrotoxicosis The ligandbinding pocket is more flexible in TRb than in TRa and one ofthe amino acids involved in triiodothyronine T3 binding isdifferent since TRa serine is replaced by asparagine in TRb These differences have been exploited to synthesizeselective TRb agonists The analogues that have reached the clinical field areshown in Figure In GC1 now called sobetirome the iodine atoms and the oxygen linking the two aromatic rings arereplaced by alkyl groups and the amino acid side chain isreplaced by an oxoacetic chain The ratio of TRb to TRaaffinity is 10fold higher for GC1 than for T3 and an additional useful property is represented by selective liver uptakeIn KB2115 now referred to as eprotirome iodine is replaced by bromine in the tyrosyl ring or isopropyl in thephenolic ring and an amidoacetic side chain is presentyielding nearly 20fold TRb selectivityA different strategy was followed by researchers at MetabasisTheir compound known as MB07811 and now renamedVK2809 is a prodrug selectively taken up by the liver where it isconverted into the active principle MB07344 which differsfrom GC1 only for the presence of a phosphoryl group in the sidechainAnother compound that has been used in human is MGL also called resmetirom whose more complex chemicalstructure a substituted pyridazinone ring replaces the phenolicring and a heterocyclic cyanoazauracil group is included in theside chain allows nearly 30fold TRb selectivityAll these compounds have been used in animal models ofhypercholesterolemia where they reduced total and lowdensity lipoprotein LDL cholesterol without significantchanges in heart rate Based on these preclinical findingsclinical trials were started In both sobetirome andeprotirome were reported to produce a significant reduction in total and LDL cholesterol after “ weeks oftreatment in small groups of patients affected by hypercholesterolemia Similar results were obtained with MGL3196which reduced serum triglycerides and LDL cholesterol after weeks of treatment in hypercholesterolemic patients In patients treated with statins the addition of eprotirome caused further reduction in serum LDL cholesterol andtriglycerides No significant side effects were reported in any of theseinvestigations and therefore a phase trial was undertakento compare eprotirome at the daily doses of and lgversus placebo in patients with familial hypercholesterolemia After weeks total cholesterol LDL cholesterolFIG Chemical structureof the synthetic TRb analogues that have been used inclinical trials In the leftlower panel please note thatMB07811 now known asVK2809 is a prodrug in theliver it is converted into theactive principle formerlyknown as MB07344 by thehydroxylase CYP3A in a reaction requiring glutathioneGSH See text for furtherdetails 0cTHYROID HORMONE ANALOGUESand triglycerides were significantly reduced in both treatmentgroups without any change in HDL cholesterol However the trial was interrupted it was originally planned to last“ weeks because parallel experimental investigationsfound that eprotirome caused cartilage damage in dogs Notably during the study period a significant increase in liverenzymes was detected which determined treatment interruption in four patientsResulting in a partial domino effect termination of theeprotirome project caused parallel projects to be terminatedas well In particular the sobetirome project was interruptedand Metabasis announced that a phase investigation withMB07344 was aborted after observing increased liver enzymes in some patients Additional reasons probably contributed to these decisions While selective TRb agonistswere devised and tested excellent clinical results were obtained with statins in the primary and secondary preventionsof major cardiac events At the same time investigationsperformed with other experimental drugs showed that LDLcholesterol reduction per se was not necessarily associatedwith reduced cardiovascular risk In aggregate selective TRbstimulation was no longer regarded as a promising strategy totreat hypercholesterolemiaTH Analogues with Selective TRb Activityin Liver DiseaseDespite the disappointing results obtained with TRb agonists in hypercholesterolemia in recent years new potentialuses have been proposed for these agents The most attractivefield is probably represented by nonalcoholic fatty liver disease NAFLDThis is a highly prevalent condition since it is estimated toaffect “ of the adult population in the United Statesand Europe Its clinical presentation is quite variableMost NAFLD patients show a simple increase in liver enzymes with a histological pattern of increased hepatocytetriglyceride steatosis Symptoms may be minimal or absentand the clinical picture may be stable over time However asignificant fraction of NAFLD patients up to in someseries develop histological evidence of lobular inflammationand hepatocyte ballooning defining a variant of the diseaseknown as nonalcoholic steatohepatitis NASH NASH is aserious condition since it is associated with a high risk about“ of evolution into cirrhosis that is derangement inliver architecture leading to hepatic insufficiency Patientswith NASH andor cirrhosis are also prone to develop hepatocellular carcinoma HCCNAFLD is frequently associated with insulin resistancehypercholesterolemia or other components of the socalledmetabolic syndrome and patients are usually treated forthese underlying conditions but no specific treatment toprevent liver damage has been approved so far In principleTH should be beneficial in NAFLD due to reduced fatty acidsynthesis increased triglyceride and fatty acid breakdownand enhanced hepatocyte regeneration The liver effects of TH are mediated by TRb so selective TRb agonistshave been tested in experimental models and several compounds including sobetirome eprotirome and MB07811were able to reduce liver steatosis “ Sobetirome wasalso reported to prevent the development of HCC induced byactivation of the catenin pathway The study of TRb agonists in NAFLD has just entered theclinical field and the results of two clinical trials have recently been reported In patients with biopsyconfirmedNASH treated with MGL3196 resmetirom mg dailyfor weeks liver fat as assessed by magnetic resonanceimaging was significantly reduced versus with placebo if expressed as percentage of absolute livermass versus if expressed as percentage ofbaseline liver fat The effect was retained after weeksand in a subgroup of patients biopsy revealed a reduction ofhistological markers of inflammationSimilarresults were reported with VK2809 aliasMB07811 in patients with NAFLD treated for weeksat the dosages of mg every other day or mg daily In this trial liver fat content assessed by magnetic resonanceimaging decreased by “ versus with placebo“ vs if expressed as percentage ofbaseline liver fat While these results have raised a greatinterest and discussions have already started aboutthecomparison of these two drugs it should be stressed that thefinal results of the latter trial have not been published yetTH Analogues with Selective TRb Activity in CentralNervous System DiseaseIn recent years theoretical arguments have been developed suggesting the potential usefulness of TH analogues indemyelinating disease since TH favors both oligodendrocytedifferentiation and myelin sheet synthesis AlthoughTRa1 is widely expressed in the central nervous systemefforts have been focused on TRb agonists because of thenecessity to avoid cardiac side effects To increase bioavailability an ethanolamine ester of sobetirome has beensynthesized which acts as a prodrug since it crosses thebloodbrain barrier and is converted into sobetirome withinthe central nervous system This compound has beenrecently tested in different experimental models of demyelination with good results biochemical evidence of remyelination was confirmed by morphological findingsobtained by nuclear magnetic imaging and it was associatedwith improved functional recovery Clinical tests may be imminent for a specific diseaseknown as Xlinked adrenoleukodystrophy ALD This is arare congenital disease due to mutations in the ALD protein atransporter for verylongchain fatty acids ie with ¡carbon atoms physiologically located in the peroxisomalmembrane and encoded by the ABCD1 gene The consequence is the accumulation of verylongchain fatty acids andtheir derivatives which are eventually incorporated in cellular membranes whose structure and function are derangedAffected males develop adrenal insufficiency in childhoodand progressive myelopathy occurs in adulthood Demyelinating lesions in cerebral white matter also appear since theage of years ALD patients may benefit from hematopoietic stem celltransplantation but this treatment is effective only if performed in the early stages of the disease and it carries asignificant risk of mortality “ No specific pharmacological therapy is available for ALD Notably TH inducesthe expression of ABCD2 coding for an additional peroxisomal transporter and in a transgenic mouse model of ALDsobetirome administration reduced the brain and adrenal 0cZUCCHIcontent of verylongchain fatty acids Based on theseobservations a clinical trial with sobetirome in XlinkedALD has been posted in the NIH database NCT01787578The trial is presently labeled as withdrawn and the allegedreason is the need for revisions to the original protocolTriac in Syndromes of Reduced Sensitivity to THReduced sensitivity to TH is diagnosed when symptoms ofhypothyroidism occur despite normal or increased serum THThis finding may be the consequence of mutations in TRstransporters or metabolizing enzymes The expression˜˜resistance to TH™™ is usually reserved for syndromes causedby TR mutations Most cases are associated with TRb mutations Since TRb is involved in the inhibition of thyrotropin TSH secretion by T3 and thyroxine T4 circulatinglevels of T4 and T3 are usually high TSH is normal or highand goiter may be present The clinical picture is quitevariable and includes signs and symptoms of both hypothyroidism in TRbdependent ans and thyrotoxicosisin TRbdependent ans The most common findings aredelayed growth delayed bone maturation cognitive impairment and tachycardia Treating TH resistance is not easy Exogenous T4 or T3administration may improve some symptoms but it mayworsen others and symptomatic therapy is often prescribedfor example betablockers to reduce heart rate The idealtreatment would be represented by a T3 analogue able toactivate the mutated receptorIn some patients this can occur with Triac Fig Thelack of the amine group does not prevent Triac from activating TRs and it has similar affinity as T3 for the wildtypereceptor Apparently the different shape and charge of theTriac molecule allow several classes of mutated TRb to beactivated as well In these patients chronic Triac administration at dosages on the order of “ lg per kg of bodyweight daily represents the best therapeutic regimen Ingeneral Triacsensitive cases of TH resistance are caused bymutations in the carboxyterminal region of the T3 bindingFIG Chemical structure of some active thyroid hormone metabolites that have been used in patients or in animal models of human disease See text for further detailsdomain while mutations located close to the hinge region donot respond to Triac No positive response to Triac has beenreported in TRa mutations so farA different cause of reduced sensitivity to TH is represented by mutations in MCT8 a T3T4 transporter that isthe major pathway mediating T4 and T3 uptake in the centralnervous system The clinical syndrome associated withMCT8 mutations is known as the Allan“Herndon“Dudleysyndrome The MCT8 gene is located on the X chromosome and therefore virtually all patients are male Diagnosis is suspected in the presence of high T3 with low tonormal T4 and low to normal TSH associated with congenital brain hypothyroidism The phenotype is variable depending on the location and type of the mutationSymptoms usually include cognitive impairment associated with congenital hypotonia and weakness which mayprogress to spasticity Paroxysmal dyskinesias and seizuresmay also occur Peripheral hyperthyroidism often causestachycardia muscle wasting and progressive body weightreduction “Since it has been observed that cellular Triac uptake doesnot depend on MCT8 a clinical trial has been undertakenwith this endogenous TH analogue in patients with amedian age of years The results obtained after monthsof treatment at the dosage of “ lg daily have recently been published The treatment was highly effective in reducing serum T3 and Triac dosage was actuallytitrated on T3 reduction Signs of peripheral hypothyroidismnotably tachycardia and body weight reduction were significantly attenuated On the contrary the effects on theneurological symptoms were limited and improvement inthe indices of motor function was limited to patients youngerthan years It seems therefore that once the neurologicalphenotype is fully developed it is hardly reversibleOn this basis another phase trialis underwayNCT02396459 in which Triac treatment will be started asearly as possible in postnatal lifeSynthetic TH analogues might also be useful Notably in asmall clinical study diiodothyropropionic acid DITPA administration “ mgkg per day was able to normalizeT3 in four children affected by MCT8 deficiency aged “months Heart rate decreased in three patients and weightgain occurred in two Although DITPA was introduced over years ago as a relatively weak and poorly selective TRagonist its mode of interaction with TRs has not been specifically investigated so farPotential Uses of T2 and T1AMWhile Triac is basically a thyromimetic other active THmetabolites Fig can interact with different moleculartargets T2 has direct mitochondrial actions allegedly onrespiratory chain complex IV subunit Va and it stimulatesmitochondrial respiration and fatty acid oxidation These metabolic responses are further supported by genomiceffects whose molecular basis is unclear since they appear tobe different from those elicited by T3 In any case in ratstreated with highfat diet exogenous T2 decreased serumtriglycerides and LDL cholesterol as well as liver fat andbiochemicalindices of liver injury suggesting potentialtherapeutic value for either dyslipidemia or NAFLD However it is still controversial whether these positive 0cTHYROID HORMONE ANALOGUESeffects may be achieved without inducing tachycardia orcardiac hypertrophy since cardiac thyrotoxicosis has beenreported in mice A single pilot investigation was performed in two humanvolunteers taking T2 lgkg for weeks A slightbut significant decrease in body weight was reported without any change in serum T3 T4 TSH and cardiac function More recently a synthetic T2 analogue TRC150094was used in patients with metabolic syndrome but theresults were rather disappointing since no significant effect on insulin sensitivity and plasma lipid profile was observed The last entry into the group of active TH metabolites isrepresented by T1AM It does not interact with canonical ornoncanonical TH targets and it was discovered as a highaffinity agonist of TAAR1 a membrane G proteincoupledreceptor GPCR that is expressed in the brain and manyother tissues T1AM is a biogenic amine and sharesseveral properties of this class of compounds includingthe ability to interact with multiple targets namely otherGPCRs eg a2A adrenergic receptor membrane ionicchannels eg TRPM8 and monoamine transporters In experimental animals the administration of exogenousT1AM induced many different functional effects Neurological and metabolic effects are particularly interesting sincethey appear to be elicited at relative low doses and mighthave physiological relevance The former includesmodulation of feeding behavior and sleepwake cycle reduction of pain threshold prolearning and antiamnestic responses The major metabolic effects consistin thestimulation of triglyceride and fatty acid catabolism but antiinsulin effects on glucose metabolism are also elicited atslightly higher dosesAlthough the administration of exogenous T1AM has notbeen tested in humans some experimental results obtainedin murine models of disease have raised interest aboutpotential therapeutic applications T1AM reduced serumcholesterol in spontaneously obese mice as well asliver triglycerides in a mouse model of polycystic ovarysyndrome Neuroprotective effects appear even more promising Intracerebral T1AM rescued longterm potentiation and behavioral evidence of cognitive dysfunction in an in vivomodel of bamyloid toxicity namely transgenic mice overexpressing humanmutated amyloid precursor protein In addition the intracerebral injection of T1AM metabolite iodothyroacetic acid protected from the convulsive effect ofpentylenetetrazole and from kainate toxicity while exogenous T1AM reduced apoptosis and functional injury in amouse model of spinal cord clamp Because of the multitude of T1AM effects an active research line is focused on the development of synthetic derivatives with more favorable biodistribution andor receptorselectivity In conclusion the development TH analogues was initially prompted by the attempt to exploit the effects of THon lipid metabolism while avoiding unwanted cardiac effects TRb agonists have provided good results in a fewsmall clinical trials performed in hypercholesterolemic patients but these projects have been terminated after the report of potential side effects In recent years TRb agonistshave raised new interest for the treatment of NAFLD and acouple of clinical trials have provided encouraging initialresults Triac has already found clinical use in the treatmentof selected cases of TH resistance due to TRb mutationsand interesting results have recently been reported in theAllan“Herndon“Dudley syndromeOther TH analogues are under consideration for neurological diseases although human results are not yetavailable In particular sobetirome derivatives have beensuccessfulin animal models of multiple sclerosis andT1AM has been beneficial in an animal model of bamyloidtoxicityOverall research on TH analogues is experiencing a shiftin its focus but it still appears to be an active and promisingfieldAuthor Disclosure StatementNo competing financial interests existFunding InformationThis work was supported by a grant from Pisa UniversityPRA to RZReferences OrtigaCarvalho TM Sidhaye AR Wondisford FE Thyroid hormone receptors and resistance to thyroid hormone disorders Nat Rev Endocrinol “ Flamant F Cheng SY Hollenberg AN Moeller LC Samarut J Wondisford FE Yen PM Refetoff S Thyroidhormone signaling pathways time for a more precise nomenclature Endocrinology “ Davis PJ Goglia F Leonard JL Nongenomic actionsof thyrid hormone Nat Rev Endocrinol “ Senese R de Lange P Petito G Moreno M Goglia FLanni A 35diiodothyronine a novel thyroid hormone metabolite and a potent modulator of energy metabolism Front Endocrinol Lausanne Groeneweg S Peeters RP Visser TJ Visser WE Triiodothyroacetic acid in health and disease J Endocrinol234R99“R121 Hoefig CS Zucchi R Ko¨ hrle J Thyronaminesand derivatives physiological relevance pharmacological actions and future research directions Thyroid “ Ko¨hrle J Biebermann H 3iodohyronamine”a thyroid hormone metabolite with distinct target profiles andmodes of action Endocrine Rev “ Zucchi R Rutigliano G Saponaro F Novel thyroidhormones Endocrine “ Joharapurak AA Dhote VV Jain MR Selectivethyromimetics using receptor and tissue selectivity approaches prospects for dyslipidemia J Med Chem “ Mondal S Mugesh G Novel thyroid hormone analogues enzyme inhibitors and mimetics and their actionMol Cell Endocrinol “ Tancevski I Rudling M Eller P Thyromimetics ajourney from bench to bedside Pharmacol Ther “ Lin VW Klepp HM Hanley RM Sobetirome is aTRb and liverselective thyromimetic that can affectsubstantial LDLC lowering without significant changes inheart rate or the thyroid axis in euthyroid men [abstract] 0cZUCCHISan FranciscoENDO OR3633the Endocrine Society Annual Meeting Berkenstam A Kristensen J Mellstro¨m K Carlsson BMalmJ Rehnmark S Garg N Andersson CM Rudling MSjo¨berg F Angelin B Baxter JD The thyroid mimeticcompound KB2115 lowers plasma LDL cholesterol andstimulates bile acid synthesis without cardiac effects inhumans Proc Natl Acad Sci U S A “ Taub R Chiang E ChabotBlanchet M Kelly MJ ReevesRA Guertin MC Tardif JC Lipid lowering in healthyvolunteers treated with multiple doses of MGL3196 alivertargeted thyroid hormone receptorb agonist Atherosclerosis “ Ladenson PW Kristensen JD Ridgway EC Olsson AGCarlsson B Klein I Baxter JD Angelin B Use of thethyroid hormone analogue eprotirome in statintreateddyslipidemia N Engl J Med “ Sjouke B Langslet G Ceska R Nicholls SJ Nissen SEOhlander M Ladenson PW Olsson AG Hovingh GKKastelein JJ Eprotirome in patients with familial hypercholesterolaemia the AKKA trial a randomizeddoubleblind placebocontrolled phase study LancetDiabetes Endocrinol “ European Association for the Study of the Liver EASLEuropean Association for the Study of Diabetes EASDEuropean Association for the Study of Obesity EASO ESALEASDEASO clinical practice guidelines forthe management of nonalcoholic fatty liver diseaseJ Hepatol “ Coppola M Glinni D Moreno M Cioffi F Silvestri EGoglia F Thyroid hormone analogues and derivativesactions in fatty liver World J Hepatol “ Sinha RA Singh BK Yen PM Direct effects of thyroid hormones on hepatic lipid metabolism Nat Rev Endocrinol “ Martagon AJ Lin JZ Cimini SL Webb P Phillips KJ The amelioration of hepatic steatosis by thyroid hormonereceptor agonists is insufficient to restore insulin sensitivityin obob mice PLoS One 10e0122987 Puliga E Min Q Tao J Zhang R PradhanSundd TPoddar M Singh S Columbano A Yu J Momga SP Thyroid hormone receptorb agonist GC1 inhibits metbcatenindriven hepatocellular cancer Am J Pathol “ Harrison SA Bashir MR Guy CD Zhou L Moylan CAFrias JP Alkhouri N Bansal MB Baum S NeuschwanderTetri BA Taub R Moussa SE Resmetirom MGL for the treatment of nonalcoholic steatohepatitis amulticenter randomized doubleblind placebocontrolledphase trial Lancet “ Loomba R Neutel J Bernard D Severance R Mohseni RDao M Saini S Margaritescu C Homer K Tran B Mancini M Masamune H Lian B VK2809 a novel liverdirected thyroid receptor beta agonist significantly reducesliver fat in patients with nonalcoholic fatty liver disease aphase randomized placebocontrolled trial [abstract]Hepatology 681447A Zhang M Ma Z Qin H Yao Z Thyroid hormonepotentially benefits multiple sclerosis via facilitating remyelination Mol Neurobiol “ Placzek AT Ferrara SJ Hartley MD SanfordCrane HSMeining M Scanlan TS Sobetirome prodrug esterswith enhanced bloodbrain barrier permeability BioMed Chem “ Hartley MD Banerji T Tagge IJ Kirkemo LL ChaudharyP Calkins E Galipeau D Shokat MD DeBell MJ VanLeuven S Miller H Myelin repair stimulated by CNSselective thyroid hormone action JCI Insights 4e126329 Kemp S Huffnagel IC Linthorst GE Wanders RJ EngelenM Adrenoleukodystrophy”neuroendocrine pathogenesis and redefinition of natural history Nat Rev Endocrinol “ Hartley MD Kirkemo LL Banerji T Scanlan TS Athyroid hormonebased strategy for correcting the biochemical abnormality in Xlinked adrenoleukodystrophyEndocrinology “ Refetoff S Dumitrescu AM Syndromes of reducedsensitivity to thyroid hormone genetic defects in hormonereceptors cell transporters and deiodination Best PractClin Endocrinol Metabolism “ Groeneweg S Peeters RP Visser TJ Visser WE Therapeutic applications of thyroid hormone analogues inresistance to thyroid hormone RTH syndromes Mol CellEndocrinol “ Friesema ECH Visser WE Visser TJ Genetics andphenomics of thyroid hormone transport by MCT8 MolCell Endocrinol “ Schwartz CE Stevenson RE The MCT8 thyroidhormone transporter and AllanHerndonDudley Syndrome Best Pract Clin Endocrinol Metab “ Groeneweg S Peeters RP Moran C Stoupa A Aurial FTonduti D Dica A Paone L Rozenkova K Malikova Jvan der Walt A de Coo IFM McGowan A Lyons GAarsen FK Barca D van Beynum IM van der Knoop MMJansen J Manshande M Lunsing RJ Nowak S den UilCA Zillikens MC Visser FE Vrijmoeth P de Wit MCYWolf NI Zandstra A Ambegaonkar G Singh Y de RijkeYB Medici M Bertini ES Depoorter S Lebl J Cappa MDe Meirleir L Krude H Craiu D Zibordi F Oliver Petit IPolak M Chatterjee K Visser TJ Visser WE Effectiveness and safety of the triiodothyronine analogue Triacin children and adults with MCT8 deficiency an international single arm label phase trial Lancet Diabetes Endocrinol “ Verge CF Konrad D Cohen M Di Cosmo C DumitrescuAM Marcinkowski T Hameed S Hamilton J Weiss RERefetoff S Diiodothyropionic acid DITPA in thetreatment of MCT8 deficiency J Clin Endocrinol Metab“ Goglia F The effects of 35diiodothyronine on energybalance Front Physiol Jonas W Lietzow J Wohlgemuth F Hoefig CS WiedmerP Schweizer U Ko¨hrle J Schurmann A 35DiiodoLthyronine 35T2 exertsthyromimetic effects onhypothalamuspituitarythyroid axis body compositionand energy metabolism in male dietinduced obese miceEndocrinology “ Antonelli A Fallahi P Ferrari SM Di Domenicantonio AMoreno M Lanni A Goglia F 35diiodoLthyronineincreases resting metabolic rate and reduces body weightwithout undesirable side effects J Biol Regul HomeostAgents “ Van der Val F Hassing C Visser M Thakkar P MohananA Pathak K Dutt C Chauthaiwale V Ackermans MNederveen A Serlie M Nieuwdorp M Stroes E Theeffect of a diodothyronine mimetic on insulin sensitivity inmale cardiometabolic patients a doubleblind randomizedcontrolled trial PLoS One 9e86890 0cTHYROID HORMONE ANALOGUES Zucchi R Chiellini S Scanlan TS Grandy DK Traceamineassociated receptors and their ligands Br J Pharmacol “ Rutigliano G Accorroni A Zucchi R The case forTAAR1 as a modulator of central nervous system functionFront Pharmacol Zucchi R Accorroni A Chiellini G Update on iodothyronamine and its neurological and metabolic actions Front Physiol AssadiPorter FM Reiland H Sabatini M Lorenzini LCarnicelli V Rogowski M Selen Alpergin ES Tonelli MGhelardoni S Saba A Zucchi R Chiellini G Metabolic reprogramming by 3iodothyronamine T1AM anew perspective to reverse obesity through regulation ofsirtuin and expression Int J Mol Sci 19e1535 Selen Alpergin ES Bolandnazar Z Sabatini M RogowskiM Chiellini G Zucchi R AssadiPorter FM Metabolic profiling reveals reprogramming of lipid metabolicpathways in treatment of polycystic ovary syndrome with3iodothyronamine Physiol Rep 5e13097 Accorroni A Rutigliano G Sabatini M Frascarelli S BorsoM Novelli E Bandini L Ghelardoni L Saba A Zucchi ROriglia N Exogenous 3iodothyronamine rescues theentorhinal cortex from bamyloid toxicity Thyroid “ Laurino A Landucci E Resta F De Siena F PellegriniGiampietro DE Masi A Mannaioni G Raimondi L Anticonvulsant and neuroprotective effects of the thyroidhormone metabolite 3Iodothyroacetic Acid Thyroid “ Lv J Liao J Tan W Yang L Shi X Zhang H ChenL Wang S Li Q 3Iodothyronamine acting throughan antiapoptotic mechanism is neuroprotective againstspinal cord injury in rats Ann Clin Lab Sci “

Thyroid_Cancer

Neuropilin1 regulated by miR320a participates in the progression of cholangiocarcinoma by serving as a coreceptorthat activates multiple signaling pathways The present study sought to investigate upstream lncRNAs that control theexpression of miR320aneuropilin1 axis and dissect some of the underlying mechanisms Here we report lncRNATTNAS1 titinantisense RNA1 acts as a sponging ceRNA to downregulate miR320a and is highly expressed in humancholangiocarcinoma tissues and cells The expression of the above three molecules is correlated with theclinicopathologic parameters of cholangiocarcinoma patients In this study multiple bioinformatics tools anddatabases were employed to seek potential lncRNAs that have binding sites with miR320a and TTNAS1 wasidentified because it exhibited the largest folds of alteration between cholangiocarcinoma and normal bile ductepithelial cells The regulatory role of TTNAS1 on miR320a was further evaluated by luciferase reporter and RNApulldown assays coupled with in situ hybridization and RNA immunoprecipitation analyses which showed that TTNAS1 bound to miR320a through an argonaute2dependent RNA interference pathway in the cytoplasm ofcholangiocarcinoma cells Knockdown and overexpression assays showed that the regulatory effect between TTNAS1and miR320 was in a oneway manner TTNAS1 promoted the proliferation and migration of cholangiocarcinomacells via the miR320a neuropilin1 axis The function of TTNAS1 on tumor growth and its interaction with miR320awere confirmed in animal models Further mechanistic studies revealed that TTAAS1 through downregulating miR320a promoted cell cycle progression epithelial“mesenchymal transition and tumor angiogenesis by upregulatingneuropilin1 which cointeracted with the hepatocyte growth factorcMet and transforming growth factor TGFTGF receptor I pathways In the present results demonstrate that lncRNA TTAAS1 is a sponging ceRNAfor miR320a which in turn downregulates neuropilin1 in cholangiocarcinoma cells indicating these three moleculesrepresent potential biomarkers and therapeutic targets in the management of cholangiocarcinomaIntroductionCholangiocarcinoma CCA arises from the epithelialcells facing the lumen of the biliary trees and is the secondmost frequent primary hepatic tumor after hepatocellularCorrespondence Jun Lu lujunsd126com orXueying Sun sunxueyinghrbmueducn1Department of Hepatobiliary Surgery Shandong Provincial Hospital Affiliatedto Shandong First Medical University Jinan China2The Hepatosplenic Surgery Center the First Affiliated Hospital of HarbinMedical University Harbin ChinaFull list of author information is available at the end of the Edited by A Stephanoucarcinoma globally12 CCA is usually diagnosed atadvanced incurable stages due to the absence of priorrecognizable clinical manifestations coupled with thecurrent unavailability of specific tumor biomarkers3Despite the latest progress in the development of molecular targeted therapies the prognosis for this devastatingcancer remains grim3 Pemigatinib has recently beenapproved for “ of CCA patients harboring a fusionor rearrangement of growth factor receptor gene4 andivosidenib has been shown to significantly improve theprogressionfree survival of patients with isocitrate The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate ifchanges were made The images or other third party material in this are included in the ™s Creative Commons license unless indicated otherwise in a credit line to the material Ifmaterial is not included in the ™s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this license visit httpcreativecommonslicensesby40Official journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of dehydrogenase1 mutant advanced CCA in a phase clinical trial5 However CCA is a heterogeneous malignancy and bears a high mutation burden6 thus thepotential druggable genome alterations in a small proportion of CCAs are not ideal therapeutic targets owing tosignaling pathways7the anticipated redundancy ofTherefore there is great urgency in further elucidating themolecular mechanisms and pathways underpinning thisdisease so that the clinical outcome of CCA patients couldbe improvedNeuropilin1 NRP1 is a nontyrosine kinase transmembrane molecule overexpressed in gastrointestinalcancers89 and serves as a coreceptor for several cellularsignaling pathways involved in cancer progression10“We have recently demonstrated that human CCA tissuesexpressed higher levels of NRP1 which coactivates thevascular endothelial growth factor VEGF epidermalgrowth factorEGF and hepatocyte growth factorHGFmediated pathways involved in the progression ofCCA14 It is known that microRNAs miRNAs regulatemultiple cellular functions and have emerged as potentialtargetsIn exploring themiRNAmediated mechanisms that lead to the overexpression of NRP1 we have shown that miR320anegatively regulates NRP1 by binding to the ²UTR of itspromoter and is expressed at low levels in CAA tissuesand cells14 MiR320a is regarded as a tumorsuppressivemiRNA16 and inhibits the proliferation and metastasis ofCCA cells in vitro and in vivo through downregulatingNRP114 However its upstream regulatory mechanismsremain unknownin anticancer campaign15Long noncoding RNAs lncRNAs are a group of noncoding RNAs ncRNAs with over nucleotides inlength and comprise of ncRNAs Emerging studiesprovide strong evidence that lncRNAs exert pivotal rolesin regulating gene expression in many diseases17 One ofthe main regulatory functions of lncRNAs is to act ascompeting endogenous RNAs ceRNAs to sponge miRNAs leading to the loss of the ability to degrade silenceor hamper translation of their downstream genes17 ManylncRNAs have been shown to regulate key factorsinvolved in cancer cells18 and some of them representpotential diagnostic markers and therapeutic targets forCCA1920 Therefore we carried out the present study toexplore potential upstream lncRNAs that can regulate themiR320aNRP1 axis in CCAResultsIdentification of lncRNA TTNAS1 as a potential targetin CCAThe overexpression of NRP1 in clinical CCA tissueswas confirmed by using immunohistochemistry of tissuemicroarrays Supplementary Fig S1 A panel of CCA celllines expressed different levels of NRP1 where the orderOfficial journal of the Cell Death Differentiation Associationof cell lines with the highest to lowest expression was RBEHCCC9810 QBC939 CC262 and FRH0201 but allexpressed higher levels of NRP1 than normal humanbiliary epithelial HIBEC cells Supplementary Fig S2A BRBE cells expressed the highest levels of NRP1 proteinand mRNA which were and fold higher thanHIBEC cells respectively and expressed the lowest level ofmiR320a which was of that of HIBEC cells FigS2C A negative correlation was found between expression levels of miR320a and NRP1 mRNA Fig S2DLncRNAs that have binding sites with miR320a werescreened by using multiple bioinformatics tools anddatabasestarbasesysueducn DIANATarBasewwwlncrnadb LncBase Experimental v2 lncactdb20omictoolscom and httpbioinfolifehusteducnand potential candidates were selected based on thecriteria of free energy ‰ kcalmol and score Supplementary Table S1 We then detected their expressionlevels in RBE and HIBEC cells by quantitative reversetranscription polymerase chain reaction qRTPCR withspecific primers Supplementary Table S2 Among the candidates TTNAS1 titinantisense RNA1 was shownto have the largest folds of alteration between RBE andHIBEC cells Supplementary Fig S3A B Notably TTNAS1 is a novel lncRNA derived from the opposite strandoftitin TTN gene and has partial sequence complementarity with TTN gene21 LncRNA TTNAS1 hasbeen shown to promote the progression of several cancertypes including esophageal squamous cell carcinomaESCC21 lung adenocarcinoma22 and papillary thyroidcancer23 The expression of TTNAS1 was also detected inall the available CCA cell lines and showed a positivecorrelation with NRP1 but a negative correlation withmiR320a Fig S3CEAssociation of TTNAS1 expression with clinicopathologicparameters of CCA patientstissuesThe qRTPCR analyses revealed that CCA tumor tissuesexpressed significantly higher levels of TTNAS1 Fig 1aand NRP1 mRNA Fig 1b and significantly lower levelsof miR320a Fig 1c compared with adjacent normal bileductIn CCA tissues an inverse correlationbetween expression levels of TTNAS1 and miR320aFig 1d and between miR320a and NRP1 mRNAFig 1e and a positive correlation between TTNAS1 andNRP1 mRNA Fig 1f were found by using Pearsoncorrelation analyses Based on the expression levels ofTTNAS1 we divided CCA cases into the high meanand low ‰mean groups and analyzed the associationbetween TTNAS1 expression and clinicopathologicparameters The results showed that the expression ofTTNAS1 was significantly correlated with tumor differentiation and lymph node metastasis and marginallycorrelated with portal vein invasion while not with gender 0cZhu Cell Death and Disease Page of Fig The expression of lncRNA TTNAS1 miR320a and NRP1 and their correlation in CCA tissues The expression of TTNAS1 a NRP1mRNA b and mature miR320a c in pairs of human CCA tissues and corresponding adjacent normal biliary tissues was detected by qRTPCRn number of samples examined Statistical analyses were performed by a Student™s t test d“f The correlation between miR320aTTNAS1NRP1mRNAmiR320a and TTNAS1NRP1 mRNA expression was analyzed with a Pearson testage tumor location or TNM staging Table NamelyCCA patients with poor tumor cell differentiation positivelymph metastasis and portal vein invasion had higherexpression of TTNAS1 Table By using the sameanalyses based on expression levels of NRP1 mRNA andmiR320a we found that both were correlated with tumordifferentiationlymph node metastasis and portal veininvasion Further NRP1 mRNA expression levels alsocorrelated with TNM staging Table TTNAS1 functions as a ceRNA to sponge miR320aFor examining the regulatory effects between TTNAS1and miR320a we first showed that transfection of miR320a mimics had little effect on TTNAS1 expression butdepletion of TTNAS1 significantlyincreased theexpression of miR320a in RBE and HCCC9810 cellsSupplementary Fig S4A Bimplying that TTNAS1might negatively regulate miR320 in CCA cells Based onthe putative binding sites between TTNAS1 and miR320a luciferase reporter and RNA pulldown assays wereemployed to examine their direct binding SupplementaryFig S5 The luciferase intensity was decreased by cotransfected miR320a mimics and wildtype TTNAS1reporter vector but not the mutant reporter vector lackingthe miR320a binding site Consistently miR320a wasprecipitated by wildtype TTNAS1 but not TTNAS1mutant and TTNAS1 was pulled down by biotinlabeledwildtype miR320a but not miR320a mutant Fig S5TTNAS1 regulates miR320a in an argonaute2dependentmannerThe above results indicate that miR320a binds tolncRNATTNAS1 without causing TTNAS1 degradation TTNAS1 and miR320a were both located in thecytoplasm of CCA cells as detected by In situ hybridization Fig 2a“c suggesting that TTNAS1 may bind tomiR320a through the argonaute2 Ago2dependentRNA interference pathway24 As expected RNA immunoprecipitation RIP assay showed levels of miR320aand TTNAS1 precipitated by an antiAgo2 Ab weremarkedly increasedresulting in a and 3foldenrichment compared with controlIgG respectivelyFig 2d e Meanwhile endogenous TTNAS1 pulldownby the antiAgo2 Ab was specifically enriched uponectopic overexpression of miR320a Fig 2f These datasuggest that TTNAS1 binds to miR320a in the cytoplasm in an Ago2dependent mannerIn addition the expression of miR320a was downregulated by TTNAS1 overexpression and upregulatedby TTNAS1 knockdown and these effects could beabolished by miR320a mimics and antagomiR320arespectively Supplementary Fig S6A B However nosignificant difference in TTNAS1 expression was detected by transfection of miR320a mimics or antagomiR320a Fig S6C D These results indicate that the regulatory effects between TTNAS1 and miR320a are in aoneway mannerOfficial journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of Table Correlations of the expression of TTNAS1 NRP1 mRNA or miR320a with clinicopathological parameters ofCCA patientsParametersTotaln TTNAS1P value NRP1 mRNAP value miR320aP valueLown Highn Lown Highn Lown Highn GenderMaleFemaleAge years‰¥Tumor differentiationWellmoderatePoorTumor locationIntrahepaticPerihilarTumor size mm‰¥ mmTNM stagebIIIIIIIVLymph metastasisNegativePositivePortal vein invasionNoYes00042a00182a001823a001543a002709a003307aaindicates a significant differencebAccording to the 8th UICC Union for International Cancer ControlTNM staging system P value was estimated by a χ2 testCCA cholangiocarcinoma TTNAS1 lncRNA titinantisense RNA1 NRP1 neuropilin10009076a00194a004106aTTNAS1 promotes the proliferation of CCA cellsvia miR320aNRP1We have previously reported that NRP1 depletion andectopic expression of miR320a inhibited the proliferationof CCA cells14 In accord we confirmed that depletion ofNRP1 significantly reduced cell viability while miR320amimics showed a similar effect by downregulating NRP1expression Supplementary Fig S7 We could furthershow that knockdown of TTNAS1 significantly reducedcell viability while antagomiR320a partially restored cellviability Supplementary Fig S8A Mechanistically TTNAS1 knockdown led to a significant downregulation ofNRP1 cyclindependent kinase CDK2 and cyclin E asignificant upregulation of p27 but had little effect on theexpression of cyclin D1 and p21 The above molecules arekey factors involved in cell proliferation and cycle progression25 AntagomiR320a counteracted the effect ofTTNAS1 knockdown Fig S8B Cell cycle distributionassays showed that knockdown of TTNAS1 led to morecells arrested at the G0G1 phase while antagomiR320apartially abolished this effect of TTNAS1 knockdownSupplementary Fig S9On the other hand exogenous overexpression of TTNAS1 increased the viability of FRH0201 cells while miROfficial journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of Fig TTNAS1 regulates the expression of miR320a in the cytoplasm of CCA cells in an Ago2dependent manner a RBE cells weresubjected to in situ hybridizations of miR320a ²DIG tagged probe identified with Cy3conjugated Ab in red and TTNAS1 ²DIG tagged probeidentified with FITCconjugated Ab in green and stained with DAPI blue Three images from the same cells were merged Scale bar μmb c Total RNA was extracted from nuclear NU and cytoplasmic CY fractions of RBE cells and the expression of TTNAS1 b and miR320a c wasmeasured by qRTPCR and normalized U1 and U6 were used as internal nuclear controls for TTNAS1 and miR320a respectively and GAPDH as aninternal cytoplasmic control d e RBE cells were subjected to RNA immunoprecipitation RIP assays The fold enrichment of miR320a d andTTNAS1 e by an antiAgo2 Ab was normalized to a nonspecific IgG acting as a negative control f RBE cells transfected with negative control NC ormiR320a mimics were subjected to RIP to measure relative enrichment of TTNAS1 by the antiAgo2 Ab P indicates a significant differencefrom respective controls320a mimics partially abolished this effect Fig S8CTTNAS1 overexpression resulted in the upregulation ofNRP1 cyclin E and CDK2 and downregulation of p27while miR320a mimics could neutralize the effect ofTTNAS1 overexpression Fig S8DTTNAS1 promotes the migration of CCA cells via the miR320aNRP1 axisKnockdown of TTNAS1 significantly reduced theability of RBE cells to migrate while antagomiR320aFig 3a“d CCA cellspartially abolished this effectacquire the migratory and invasive properties through acritical process known as epithelialmesenchymal transition EMT26 Therefore we examined the effects ofTTNAS1 knockdown on the expression of decisive facinvolved in the process of EMT27 TTNAS1torsknockdown significantly downregulated the expressionof NRP1 Snail Ncadherin matrix metalloproteinaseMMP2 and MMP9 and upregulated the expression ofEcadherin Fig 3e The results were supported bygelatin zymography assays which showed that TTNAS1knockdown significantly reduced activities of MMP2 andOfficial journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of Fig Knockdown of TTNAS1 inhibits cell migration via regulating miR320a RBE cells were transfected with scrambled shRNA controlshRNATTNAS1 or shRNATTNAS1 antagomiR320a for h Cells were subjected to transwell migration a b and scratch c d assays a Migratedcells were visualized using Giemsa staining Scale bar μm a and µm c b Numbers of migrating cells were counted c Scratch areas wererecorded d Scratch distances were quantified at indicated time points e f Cells were immunoblotted for detecting key EMT proteins and the densityof each band was normalized to actin g Cells were subjected to gelatin zymography assays for analyzing the gelatinolytic activity of MMP9 andMMP2 P vs controls and P and P vs shRNATTNAS1MMP9 while antagomiR320a partially counteracted thiseffect Fig 3f On the other hand overexpression ofTTNAS1ofFRH0201 cells while miR320a mimics partially abolishedthis effect Supplementary Fig S10the migratoryincreasedabilityTTNAS1 contributes to the growth of CCA tumors inanimal modelsThe functional role of TTNAS1 was also confirmed inCCA tumors in vivo Subcutaneous RBE tumors wereestablished in mice which were randomly assigned todifferent treatments when tumors reached mm3Tumors treated with shRNATTNAS1 were significantlysmaller ± mm3 than control tumors ± mm3 however cotreatment of antagomiR320aOfficial journal of the Cell Death Differentiation Associationcould partially restore the growth of tumors ± mm3 as measured days after treatment commencement Fig 4a The results of tumor volume correlatedwith the weight of tumors Fig 4b Treatment of shRNATTNAS1 led to TTNAS1 downregulation and miR320aupregulation in tumors harvested days after treatmentsby in situ hybridization and downregulation of NRP1 byimmunohistochemistryofantagomiR320a partially abolished the effects of shRNATTNAS1 on miR320a upregulation and NRP1 downregulation but had little effect on TTNAS1 expressionFig 4c Treatment of shRNATTNAS1 significantlyinhibited cell proliferation in situ Fig 4d e and reducedtumor vasculature while antagomiR320a neutralized theeffects of shRNATTNAS1 Fig 4d f In agreement with4c CotreatmentFig 0cZhu Cell Death and Disease Page of Fig Knockdown of TTNAS1 inhibits the growth and angiogenesis of CCA tumors in vivo Subcutaneous CCA tumors were established inmice by inoculation of RBE cells and received respective treatments as described in Supplementary Information a The growth curve of RBE tumorswas recorded b RBE tumors were resected weighed and photographed at the end of experiments c Two mice were killed from each group toharvest tumors days after treatments and the expression of TTNAS1 and miR320a was examined by in situ hybridization magnification × Scalebar μm and NRP1 expression by immunohistochemistry Magnification × Scale bar μm Tumors harvested at the end of experimentsd Illustrated are representative tumor sections immunostained by Abs against Ki67 and CD31 respectively Magnification × Scale bar μm Insitu cell proliferation index e and tumoral microvessel density f were quantified g Tumor tissue homogenates were immunoblotted for detectingthe expression of key proliferation proteins P vs controls P and P vs shRNATTNAS1the in vitro results Fig S8A B immunoblotting analysisof tumor homogenates showed that shRNATTNAS1treatment led to downregulation of NRP1 cyclin E andCDK2 and upregulation of p27 while antagomiR320acounteracted the effects of shRNATTNAS1 Fig 4gOn the other hand by adopting another subcutaneousCCA tumor mouse model with FRH0201 cells whichwere shown to express a lower level of TTNAS1Fig S2A we demonstrated that exogenous overexpression of TTNAS1 promoted tumor growth bypromoting in situ cell proliferation and tumor angiogenesis while miR320a mimics partially abolished theseeffects Supplementary Fig S11TTNAS1 regulates the cMet and TGF pathways via NRP1We have previously demonstrated that NRP1 coactivates the HGFcMet pathway in CCA cells14 Controland shRNATTNAS1transfected RBE cells were incubated with recombinant human HGF protein in the presence or absence of tivantinib a cMet inhibitor and anOfficial journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of Fig TTNAS1 regulates the cMet and TGF pathways via NRP1 RBE and cells were transfected with negative control or shRNATTNAS1 andthen incubated for h in the presence or absence of recombinant HGF protein ngml and tivantinib μgml a or TGF protein ngmland LY2157299 μgml b Cell lysates were immunoblotted to determine the expression of key proteins involved in the above pathways asindicated The density of each band was normalized to actin P and P indicate a significant difference P indicates asignificant difference from negative control cells treated with vehicleanticancer drug used in CCA clinical trial28 TTNAS1knockdown led to downregulation of NRP1 expressionresulting in downregulation of phosphorylated cMetpcMet and sequential downregulation of phosphorylated Akt pAkt and upregulation of p27 Fig 5aIncubation of HGF protein did not affect NRP1 expression but could activate the cMet pathway evidenced byupregulation of pcMet and pAkt and downregulationof p27 and incubation of tivantinib showed the oppositeeffects to HGF ligand Fig 5aNRP1 cointeracts transforming growth factor TGFpathway29 which is crucial for EMT of cancer cells30Therefore we examined the effect of TTNAS1 knockdown on this pathway in CCA cells Control and shRNATTNAS1transfected RBE cells were incubated withrecombinant human TGF protein orand LY2157299 aspecific TGF receptor TGFR inhibitor31 Incubationof TGF protein or LY2157299 did not affecttheexpression of NRP1 or TGFRI Fig 5b HoweverTGF induced the upregulation while LY2157299expression of pTGFRI TTNAS1reduced theknockdown had little effect on TGFRI expression butsignificantly inhibited its phosphorylation Fig 5b TheOfficial journal of the Cell Death Differentiation Associationactivation of TGF pathway by TGF protein increasedthe sequential expression of pSmad23 and Snail whileLY2157299 and TTNAS1 knockdown demonstratedopposite effects and abolished the activating effects ofTGF protein Fig 5bDiscussioninotherrole wasconfirmedLncRNA TTNAS1 was initially reported to participatein the progression and metastasis of ESCC21 Later itsfunctionalcancertypes22233233 Importantly TTNAS1 exerts regulatoryeffects via acting as a ceRNA to sponge different miRNAsin different cancers For instance TTNAS1 regulated themiR133bactinbinding protein fascin homolog axis inESCC cells21 while promoted the migration and EMT oflung adenocarcinoma cells by sponging miR1425p toregulate CDK522 As schematically summarized in Fig we have in the present study found that TTNAS1 servesas a ceRNA to sponge miR320a through complementarybinding sites in an Ago2dependent manner in CCA cellsLncRNAs exhibit different functions depending on theirsubcellular localization This study showed that TTNAS1was mainly localized in the cytoplasm of CCA cells while 0cZhu Cell Death and Disease Page of dependent way and in a oneway manner in CCA cells Inaccord it has been reported that TTNAS1 was locatedmainly in the cytoplasm and acted as a ceRNA spongingmiRNAs in ESCC and papillary thyroid cancer cells2123MiR320a is one of the two most highly downregulatedmiRNAs in clinical CCA tissues and is closely associatedwith the progression and severity of CCA35 MiR320aalso represents a critical suppressor component of theprogression of other cancers163637 We have previouslyreported that miR320a negatively regulated the expression of NRP1 by binding to the ²UTR of NRP1 promoter and inhibited cell proliferation and migration ofCCA cells14NRP1 functions as versatile coreceptors that bind to anumber of growth factors and couple with cognatereceptor tyrosine kinase signaling pathways involved incancer progression111438 In the present study we havefurther demonstrated that NRP1 acts as a coreceptor forthe activation of HGFcMet pathway which induces thephosphorylation of Akt39 a downstream of cMet signaling40 Akt activation leads to the sequential downregulation of p2741 which inactivates the CDK2cyclinE complex resulting in cell cycle arrest41 Howevertivantinib a specific cMet inhibitor can block NRP1induced activation of the HGFcMet pathway Fig Onthe other hand NRP1 cointeracts with TGF29 leadingto the activation of the TGFTGFRI pathway whichin turn increases the expression of phosphorylated Smad2and Smad3 The latter two combine with Smad4 to form atrimeric SMAD complex that upregulates the expressionof Snail which conveys TGFinduced repression ofEcadherin and stimulation of Ncadherin42 thus promoting EMT of CCA cells However LY2157299 a specific TGFR inhibitor31 can block NRP1inducedactivation of the TGFTGFRI pathway Fig Asdemonstrated previously14 but not investigated in thisstudy the above signaling pathways may also crosstalkwith each other and contribute to the proliferation andmetastasis of cancer cells43In summary to the best of our knowledge this is thefirst study that reports the functional role of TTNAS1 asa sponging ceRNA for miR320a its high expression inCCA tissues and a significant association with clinicopathologic parameters of CCA TTNAS1 displays itsregulatory activity by binding to miR320a through theAgo2dependent RNA interference pathway and in a oneway manner in the cytoplasm of CCA cells Throughdownregulating miR320a TTAAS1 promotes cell cycleprogression EMT and angiogenesis via NRP1 which cointeracts HGFcMet and TGFTGFRI pathways inCCA cells Taken together the present study has unveileda novel axis consisting of TTNAS1miR320aNRP1which may also represent a therapeutic target and biomarkers in the management of CCAFig Schematic diagram of the TTNAS1miR320aNRP1 axiscontributing to the progression of CCA LncRNA TTNAS1 serves asa ceRNA to sponge miR320a through complementary binding sites inan Ago2dependent manner in CCA cells On the other hand miR320a downregulates the expression of NRP1 by binding to its ²UTRAn NRP1 protein molecule is composed of five extracellular domainsa1 a2 b1 b2 and c one transmembrane domain and a shortcytosolic tail and acts as a coreceptor for ligands HGF and TGF tostimulate the activation of respective cMet and TGF signalingpathways œ†’ indicates promotion positive regulation or activationœŠ¥ indicates inhibition negative regulation or blockade œp indicatesphosphorylation of proteins Ago2 argonaute2 CCAcholangiocarcinoma CDK2 cyclindependent kinase HGFhepatocyte growth factor NRP1 neuropilin1 ORF readingframe TGF transforming growth factor TGFR TGF receptorUTR untranslated regionmiR320a was located in both nuclear and cytoplasmsubcellular compartments It is well established that thematuration of miRNAs occurs in the cytoplasm wherethey execute posttranscriptional gene silencing via anRNAinduced silencing complex pathway34 Intriguinglythe ectopic expression of miR320a reduced the luciferaseactivities of the wildtype TTNAS1 reporter but theexpression of TTNAS1 remained unchanged uponoverexpression of miR320a Moreover endogenousTTNAS1 and miR320a could be pulled down by an antiAgo2 Ab These data suggest that miR320a recognizesand binds with TTNAS1 without triggering the degradation of TTNAS1 which plays a posttranscriptionalregulatory role in downregulating miR320a via an Ago2Official journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of Materials and methodsClinical CCA tissuesA total of pairs of CCA and matched adjacent normalbile duct tissues were collected at the Department ofHepatobiliary Surgery Shandong Provincial HospitalAmong them pairs have been described previously14while new pairs of tissues were collected between April and September Of the cases were perihilar CCA and were intrahepatic CCA The criteria ofthe included specimens were consistent with our previousstudy14 The current study has been approved by the EthicsCommittee of Shandong Provincial Hospital Jinan Chinaand informed consent was obtained from all subjectsCells antibodies and reagentsHuman CCA celllines HCCC9810 RBE QBC939CC262 and FRH0201 and normal human biliary epithelialHIBEC cells were obtained from the Cell Bank of theChinese Academy of Sciences Shanghai China1444 Cellswere routinely cultured at °C in RPMI1640 mediumsupplemented with vv fetal bovine serum in ahumidified atmosphere of CO2 Cell lines were confirmed to be negative for mycoplasma infection by using aPCRbased Universal Mycoplasma Detection kit AmericanType Culture Collection Manassas VA USA Relevantinformation regarding antibodies Abs reagents and kitsare described in detail in Supplementary InformationAnimal experimentsThe experimental protocol has been described previously1444 and approved permit SYXK20020009 by theInstitute Animal Ethics CommitteeImmunodeficientnude BALBc mice H2b were housed in the AnimalResearch Center the First Affiliated Hospital of HarbinMedical University China Two sets of experiments weredesigned to examine the effects of TTNAS1 knockdownand overexpression on tumor growth Detailed information for animal experiments is included in SupplementaryInformation Briefly cells were injected subcutaneouslyinto mice and palpable tumors were monitored Around“ weeks later mice bearing tumors with a volume ofˆ¼ mm3 were randomly assigned to different groupsn The TTNAS1 knockdown study had threegroups of animals which received intratumoral injectionsof control shRNATTNAS1 or shRNATTNAS1 antagomiR320a respectively while the TTNAS1 overexpression study comprised three groups of animalswhich received injections of either control TTNAS1 orTTNAS1 miR320a mimics respectively Two micefrom each group were killed days after injection fordetecting gene expression The remaining mice werefurther monitored and euthanized days after treatments commencedOfficial journal of the Cell Death Differentiation AssociationImmunohistochemistry Tissue microarrays Establishment of stable transfectants depleted of NRP1 Assays ofcell viability cell cycle Transwell migration Cell scratchqRTPCR western blot and Gelatin zymography Cellfraction isolation In situ hybridization RNA pulldownand RIP assays Transfection of miR320a mimicsantagomiR320a and TTNAS1 expression vectors Plasmid constructs and luciferase assay In situ Ki67 proliferation index and Assessment of tumor vascularityThe detailed description for these methods is includedin Supplementary Information and has also been described previously1114Statistical analysisGraphPad Prism GraphPad Software San DiegoCA USA was employed for performing statistical analyses Data are expressed as mean values ± standarddeviation Multiple comparisons were made with a oneway analysis of variance ANOVA followed by a Tukeyposthoc test Comparisons between two groups weremade by a ttest Correlations of TTNAS1 NRP1mRNA or miR320a with clinicopathological parameters were estimated by a χ2 test The relationshipbetween two variables was analyzed by using Pearson™scorrelation coefficient P was considered statistically significantAcknowledgementsThis study was supported in part by the grants from the National Key Researchand Development Program of China 2017YFC1308602 the Supportive Fundby Heilongjiang Provincial Department of Science and TechnologyGX18C010 Natural Scientific Foundation of Shandong ProvinceZR2019MH089 Natural Scientific Foundation of Heilongjiang ProvinceH2018028 and LH2019H018 and Research Projects from the Fourth AffiliatedHospital of Harbin Medical University HYDSYXH201904 an

Thyroid_Cancer

test the hypothesis that levobupivacaine has anti‘tumour effects on breast cancer cellsResults Colony formation and transwell assay were used to determine breast cancer cells proliferation Flow Cytom‘etry annexin V and PI staining was used to investigate breast cancer cells apoptosis The effects of levobupivacaine on cellular signalling and molecular response were studied with Quantitative Polymerase Chain Reaction and western blot Induction of apoptosis was confirmed by cell viability morphological changes showed cell shrinkage rounding and detachments from plates The results of the western blot and Quantitative Polymerase Chain Reaction indicated activation of active caspase‘ and inhibition of FOXO1 The results of the flow Cytometry confirmed that levobupiv‘acaine inhibited breast cancer cell proliferation and enhanced apoptosis of breast cancer cells Quantitative Polymer‘ase Chain Reaction and Western blot analysis showed increased p21 and decreased cyclin D Quantitative Polymerase Chain Reaction and western blot analysis showed that levobupivacaine significantly increased Bax expression accom‘panied by a significant decreased Bcl‘ expression and inhibition of PI3KAktmTOR signalling pathway These findings suggested that levobupivacaine inhibits proliferation and promotes breast cancer cells apoptosis in vitroKeywords Levobupivacaine Proliferation Invasion Apoptosis Breast cancerIntroductionBreast cancer is one of the most recorded cancer illness among women [] In the United States it is estimated that more than women die every year from breast cancerrelated illness despite the advance in chemotherapy and targeted treatments []Correspondence yanqiu63126com wqp89163com Department of Anaesthesiology Dalian Medical University Dalian China Department of Biochemistry and Molecular Biology Dalian Medical University Dalian ChinaFull list of author information is available at the end of the Molecular signalling pathways that are involved in breast cancer transformation have become targets for treatment [] The mechanisms of the PI3KAktmTOR signalling pathway have present some promising targets for cancer treatments This signalling pathway hinders the functions of several tumour suppressor genes such as Bad GSK3 FOXO transcription factors and tuberinhamartin complex which control cell survival proliferation and growth [“] Suppressing this signalling pathway may inhibit cancer cells proliferation and also stimulate them toward cell deathThe growing evidence of local anaesthetics inhibiting cancer cell growth seems promising though limited [] The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cKwakye a0et a0al BMC Res Notes Page of At the tissue level administration of a certain amount of local anaesthetics topical or local has shown to have a direct inhibitory effect on the action of epidermal growth factor receptor EGFR which is a potential target for antiproliferation in cancer cells [] Evidence also shows that ropivacaine and lidocaine hinder cancer cells growth invasion migration and enhance apoptosis of lung cancer cells [“] To the best of our knowledge the effect of levobupivacaine on breast cancer cells is yet to be determined The present study therefore aimed to investigate the antitumour effects of levobupivacaine on breast cancer cellsMain textMaterials and a0methodsEthics statementThe ethical committee of the Dalian Medical University First Affiliated Hospital approved for this study to be carried outCell cultureWe purchased MCF7 and MDAMB231 breast cancer cells from the ATCC Beijing Zhongyuan limited China We maintained the MCF7 and MDAMB231 cells with highglucose DMEM or DMEMF12 Gibco USA medium The medium was supplemented with fetal bovine serum FBS Gibco USA penicillin a0unitsml and streptomycin a0µgml TransGen Biotech China to maintain the cells The MCF7 and MDAMB231 cells were then maintained in an incubator at a0ºC humidified air with CO2 atmospheric condition The cells were routinely subcultured subsequentlyAntibodies and a0reagentsEPR17671 Akt monoclonal Antibody Abcam China Y391 mTOR Polyclonal Antibody Abcam China A2845 Bcl2 Polyclonal Antibody ABclonal Technology A11550 Bax Polyclonal Antibody ABclonal Technology A0265 PIK3CA Polyclonal Antibody ABclonal Technology A2934 FOXO1 Polyclonal Antibody ABclonal Technology EPR21032 Active caspase monoclonal Antibody Abcam China AFO931 Cyclin D1 Polyclonal Antibody Affbiotech China AF6290 p21 Polyclonal Antibody Affbiotech China AntimTOR phospho S2448 Antibody Abcam China PA517387 PhosphoPI3K p85p55 Tyr458 Tyr199 Polyclonal Antibody ThemoFisher Scientific PosphopanAKT123 Ser473 Antibody Affbiotech China Peroxidaseconjugated goat antirabbit IgG Proteintech China PRAP antibodies Proteintech China and GAPDH antibodies Proteintech ChinaCell viability assay and a0IC50We determined the MCF7 and MDAMB cells viability using CCK8 assay Levobupivacaine at a concentration of or a0mM was used to treat MCF7 and MDAMB cells plated in 96well plates — a0cellswell and then incubated for or a0h respectively in an incubator at the atmospheric condition of a0 °C with CO2 The rest of the procedures used for the CCK8 assay were the same as described elsewhere []Flow cytometryAnnexin V and propidium iodide PI staining assay were used to investigate the apoptosis of MCF7 and MDAMB cells following levobupivacaine treatment After treating the cells for a0h trypsin was used to harvest the treated cells and centrifugation at rcf for a0min The MCF7 and MDAMB treated cells were again suspended with — Binding Buffer and then a0 µl of fluorochromeconjugated annexin V SigmaAldrich Saint Louis USA was added into a0µl of the cell suspension to stain intracellular phosphatidylserine PS The cells were then incubation in a dark under room temperature The cells were again suspended and a0 µl propidium iodide staining solution SigmaAldrich Saint Louis USA added into a0µl of the cell suspension We detected the percentage of the apoptotic cells via FlowJo software Treestar Ashland USA and Flow cytometry FACS Calibur Becton Dickinson and Sunnyvale CA USAQuantitative polymerase chain reaction qPCRThe procedures used for the qPCR were the same as previously described [] The primers sequences were BAX 5TGG CAG CTG ACA TGT TTT CTG3 F 5TCC CGG AGG AAG TCC AAT G3 BCL2 5ACG GTG GTG GAG GAG CTC TT3 F 5GCC GGT TCA GGT ACT CAG TCAT3 R p21 5GCG ACT GTG ATG CGC TAA TG3 F 5GAA GGT AGA GCT TGG GCA GG3 R GAPDH ²CAT GTT CGT CAT GGG TGT GAA² F ²GGC ATG GAC TGT GGT CAT GAG3² RR Western blotAt the log phase of treated MCF7 and MDAMB cells growth we harvested the cells and then washed twice with icecold PBS The rest of the procedures used for the western blot were the same as described elsewhere []Colony formation assayThe procedures used for the colony formation assay were the same as previously described [] 0cKwakye a0et a0al BMC Res Notes Page of Transwell assayThe MCF7 and MDAMBA231 cells — that were pretreated with different dose of Levobupivacaine a0mM for a0h and resuspended in culture medium with the same concentrations of levobupivacaine were seeded onto the coated membrane in the upper chamber of the transwell 24well millicell cell culture insert a0mm diameter a0μm pores Merck KGaA P18P01250 China The procedures used for the Transwell assay were the same as previously describe []Data analysisValues were expressed as the mean ± SD Statistical analysis was performed with GraphPad Prism version 501GraphPad Software La Jolla CA US Oneway ANOVA was used to measure significance p Dunnett™s post hoc tests were used to test the difference between groupsResultsLevobupivacaine decreases breast cancer cell invasionTranswell assay analysis showed significantly decreased in the invasion ability of MCF7 and MDAMB231 cells in a dosedependent manner compared with the untreated cells Additional file a0 Fig S1a b Levobupivacaine inhibits proliferation in a0breast cancer cellsThe MCF7 and MDAMBA231 cell viability decreased as the concentrations of levobupivacaine or a0mM increased The MCF7 cells showed a cytotoxic effect while the MDAMB231 cells showed a similar cytotoxic effect of Fig a01a Under a fluorescence microscope cells treated with levobupivacaine showed morphological changes including cell rounding cell shrinkage and cells detachment from the plates Additional file a0 Fig S2a b The viability of breast cancer cells decreased in a dosedependent manner The results showed significantly decreased in the number of clones of the treated cells compared with the untreated cells Fig a01b c The data showed that the mRNA level of p21 significantly increased following levobupivacaine treatment Fig a0 1d e Western blot analysis showed a similar increased in p21 and decreased in FOXO1 and cyclin D1 expressions in a dosedependent manner compared with the untreated cells Fig a01f g Additional file a03f gLevobupivacaine promote apoptosis in a0breast cancer cellsLevobupivacaine significantly reduced the number of cells showing nuclear staining when compared with the untreated cells Fig a0 2a b The qPCR data showed a decreased in Bcl2 and increased in Bax expressions in MCF7 and MDAMB231 cells compared with the untreated cells Fig a0 2c d Western blot analysis also showed a similar decreased in Bcl2 and increased expressions of active caspase and Bax compared with the untreated cells Fig a02e f Additional file a03e fLevobupivacaine inhibits proliferation and a0promotes apoptosis in a0breast cancer through a0PI3KAktmTOR signalling pathwayWestern blot analysis showed a significant decreased in the expression of the nuclear localization of pPI3K pAkt and pmTOR compared with the untreated cells Fig a03a b Additional file a03a bDiscussionBreast cancer remains a common cause of mortality among women worldwide Though current orthodox drugs have demonstrated promise in breast cancer therapy its treatment options remain limited These therefore supports the concept that effective therapeutic approaches for breast cancer are critically needed Several retrospective studies have demonstrated that regional anaesthesia is associated with a decreased risk of recurrence or metastasis of multiple carcinomas including breast prostate and cervical cancers [“] Recent growing evidence demonstrates that local anaesthetics have an antitumour effect and may suppress the motility of cellular function and invasiveness more likely via voltagegated sodium channel inhibition [] A study report indicates that lidocaine inhibits the growth of human hepatocellular carcinoma cells HCC by increasing the Caspase activity whereas ropivacaine inhibits the growth of HCC cells by stopping the cell cycle in G2 phase [] Lee et a0al demonstrated that local anaesthetics potentiate TNFα mediated apoptosis in HK2 cells [] The cellular modification of treated cells is likely dependent on the duration of exposure and the dose of the local See figure on next pageFig Levobupivacaine inhibits proliferation in breast cancer cells MCF‘ and MDA‘MB‘ cells were treated with different concentrations of levobupivacaine a Cell viability was measured by CCK‘ assay IC50 results of levobupivacaine on MCF‘ and MDA‘MB cells b c Colony formation of MCF‘ and MDA‘MB cells treated with various concentrations of Levobupivacaine and stained with crystal violet d e The mRNA expression levels of p21 and GAPDH were analysed by qPCR f g Protein expression assessment of MCF‘ and MDA‘MB‘ cells by western blot against antibodies FOXO1 p21 Cyclin D1 and GAPDH used as control The data was statistically significant at indicates P indicates P indicates P compared with untreated cells This data corresponds to the mean ± SEM of three independent experiments 0cKwakye a0et a0al BMC Res Notes Page of 0cKwakye a0et a0al BMC Res Notes Page of Fig Effects of levobupivacaine on apoptosis of breast cancer cells a b MCF‘ and MDA‘MB cells were treated with different concentrations of levobupivacaine for h The cells were then stained with fluorescein‘conjugated annexin V and PI and analysed by flow cytometry Error bars represent standard error of the mean P versus the control c d Relative gene expression of Bax and Bcl‘ following the treatment of breast cancer cells with different concentrations of levobupivacaine for h and analysed by qPCR e f MCF‘ and MDA‘MB cells were treated with different concentrations of levobupivacaine for h and the activities of Bax Bcl‘ and Active caspase were examined by Western blot analysis using specific antibodies GAPDH was used as internal controls The data was statistically significant at indicates P indicates P compared with control The data correspond to the mean ± SEM of three independent experiments 0cKwakye a0et a0al BMC Res Notes Page of Fig MCF‘ and MDA‘MB cells were treated with different concentrations of levobupivacaine for h a b The cells were lysed and subjected to SDS‘PAGE and analysed by western blotting and probed with specific antibodies p‘PI3K p‘Akt and p‘mTOR The results showed a decrease in the expressions of p‘PI3K p‘Akt and p‘mTOR proteins GAPDH was used as internal controls The data represent the mean ± SD of three independent experimentsanaesthetic [“] In this study we employed MCF and MDAMB231 cells as models and found that different concentrations of levobupivacaine could effectively inhibit breast cancer cell proliferation and promote apoptosis in a0vitro The antiproliferation and apoptosis effects observed in this study suggest that levobupivacaine may have therapeutic effects on breast cancerPI3KAktmTOR signalling pathway plays a vital role in cell proliferation survival development metabolism motility and regulation of the immune response Breast cancer cell resistance to therapies can result from the activation of PI3KAktmTOR signalling pathway [“] This has made the PI3KAktmTOR signalling pathway an important object of study for understanding the development and progression of breast cancer In patients with breast cancer PI3KAktmTOR signalling pathway can be a target for diagnostic prognostic and treatment purposes [“] Akt plays a role in the activation and inactivation of many transcription factors Activation of Akt correlated with the activation of mTOR Phosphorylation of the FOXO proteins by Akt may results in cytoplasmic retention by interacting with other proteins thereby isolating them from their targeted genes Cyclin D1 classified as a pro oncogene is often overexpressed in several human malignancies including breast colon lung and prostate cancers [] Reports show that overexpression of cyclin D1 and underexpression of tumour suppressor p21 is required for cancer initiation as it is confirmed that downregulation of cyclin D1 and overexpression of p21 in xenograft model discontinues the formation of cancer in the early stages [] Datta et a0al reported that Akt can phosphorylate the proapoptotic Bcl2 family member Bad causing its isolation from the mitochondrial membrane by other proteins [] Local anaesthetics modify the protein levels of key members of the Bcl2 family in a manner that presents an increase in the ratio of BaxBcl2 which may contribute to the response of cancer cells to apoptosis In the present study the role of levobupivacaine on the expression of PI3K Akt and mTOR was investigated to illustrate the potential molecular mechanism We observed a significantly decreased expression of pAkt pPI3K pmTOR and subsequent decreased expression of FOXO Cyclin D1 and Bcl2 following levobupivacaine treatment which correlated with decreased breast cancer cells proliferation and increased apoptosis These emerging pieces of evidence suggest that levobupivacaine may inhibit proliferation and promote apoptosis by suppressing PI3KAktmTOR signalling pathway which demonstrated an antitumour effect on breast cancer cells in this studyConclusionlevobupivacaine has the potency of reducing breast cancer cell viability proliferation and also causes cell death by suppressing the PI3KAktmTOR signalling pathway These findings could lead to clinical studies which will seek to examine the anticancer effects of levobupivacaine and may also increase the benefits in cancer patient as well as improve patient care 0cKwakye a0et a0al BMC Res Notes Page of LimitationsNumerous studies have reported on the antitumour effects of local anaesthetics on various cancer cells [“] However our work is not without limitations In a0vivo and clinical studies on the antitumour effects of levobupivacaine are neededBiology Dalian Medical University Dalian China Department of Anaesthesia and Critical Care School of Medicine University of Health and Allied Sciences Ho Ghana Department of Biochemistry and Molecular Medicine School of Medicine and Health Sciences University for Development Studies Tamale Ghana Departments of Anaesthesia and Critical Care Ridge Hospital Accra Ghana Department of Medicine Princefied University Ho Ghana Received June Accepted July Supplementary informationSupplementary information accompanies this paper at https doi101186s1310 ‘‘ ‘Additional file a0 Figure S1 Levobupivacaine decreases breast cancer cell invasionAdditional file a0 Figure S2 Effect of levobupivacaine on the morphology of MCF‘ and MDA‘MB cellsAdditional file a0 Original gelsblots scan used in Fig 1f g Fig 2e f and Fig 3a b for MCF‘ and MDA‘MB‘ cellsAbbreviationsEGFR Epidermal growth factor receptor HCC Hepatocellular carcinoma cells NC Nitrocellulose PI Propidium iodide PS Phosphatidylserine qPCR quanti‘tativepolymerase chain reactionAcknowledgementsWe thank the First Affiliated Hospital and The Department of Biochemistry of Dalian Medical University for making available all the necessary materials needed for this work We also thank the Key Laboratory of Liaoning Provincial Education Department Grant NO LZ2016002 and Liaoning Natural Science Foundation Grant NO of China for supporting this work Our thanks also go to the China Scholarship Council and the Government of the Republic of Ghana for giving financial aid to some of the authors to study at Dalian Medical UniversityAuthors™ contributionsAKK SK QY and QPW conceived and designed this study QPW and QY were responsible for the supervision and coordination of this study AKK SK JL MNR QY and QPW conducted the data collections SK led the data analysis with inputs from AKK QY and QPW AKK and SK wrote the first draft of the manuscript and JL MNR SAR AAF JA and EAN contributed to revising and reviewing the manuscript All authors read and approved the final manuscriptFundingThis study was supported by the Key Laboratory of Liaoning Provincial Education Department Grant NO LZ2016002 and Liaoning Natural Science Foundation Grant NO Availability of data and materialsThe data used andor analysed in this study are available from the correspond‘ing author upon reasonable requestEthics approval and consent to participateThe ethical committee of the First Affiliated Hospital of Dalian Medical Univer‘sity approved the study protocol and because this study used breast cancer cells consent to participate was not applicable for the studyConsent for publicationsNot applicableCompeting interestsAuthors declare that they have no competing interestsAuthor details Department of Anaesthesiology Dalian Medical University Dalian China Department of Anaesthesiology First Affiliated Hospital of Dalian Medi‘cal University Dalian China Department of Biochemistry and Molecular References American Cancer Society Breast Cancer Facts and Figures “ Atlanta American Cancer Society American Cancer Society Cancer Facts and Figures Atlanta Ameri‘ can Cancer Society Siegel R Naishadham D Jemal A Cancer statistics CA Cancer J Clin “ Chang YC Hsu YC Liu CL Huang SY Hu MC Cheng SP Local anaesthetics induce apoptosis in human thyroid cancer cells through the mitogen‘activated protein kinase pathway PLoS ONE 20149e89563 Gomez‘Gutierrez JG Souza V Hao HY de Montes Oca‘Luna R Dong YB Zhou HS McMasters KM Adenovirus‘mediated gene transfer of FKHRL1 triple mutant efficiently induces apoptosis in melanoma cells Cancer Biol Ther “Sunters A de Fern¡ndez Mattos S Stahl M Brosens JJ Zoumpoulidou G Saunders CA Coffer PJ Medema RH Coombes RC Lam EW FoxO3a transcriptional regulation of Bim controls apoptosis in paclitaxel‘treated breast cancer cell lines J Biol Chem “Fu Z Tindall DJ FOXOs cancer and regulation of apoptosis Oncogene “ Barnes DM Gillett CE Cyclin D1 in breast cancer Breast Cancer Res Treat “Sherr CJ Roberts JM CDK inhibitors positive and negative regulators of G1‘phase progression Genes Dev “ Pelengaris S Khan M Evan G c‘MYC more than just a matter of life and death Nat Rev Cancer “ Di Padova M Barbieri R Fanciulli M Arcuri E Florida A Effect of local anaesthetic ropivacaine on the energy metabolism of Ehrlich ascites tumour cells Oncol Res 199810491e8 Xing W Chen DT Pan JH Chen YH Yan Y Li Q Xue RF Yuan YF Zeng WA lidocaine induces apoptosis and suppresses tumour growth in human hepatocellular carcinoma cells in vitro and in a xenograft model in vivo Anesthesiology “ Drasner K Lidocaine spinal anaesthesia a vanishing therapeutic index Anesthesiology “ Wang HW Wang LY Jiang L Tian SM Zhong TD Fang XM Amide‘linked local anaesthetics induce apoptosis in human non‘small‘cell lung cancer J Thorac Dis “ https doi1021037 jtd20160966 Piegeler T Votta‘Velis EG Liu G Place AT Schwartz DE Beck‘Schimmer B Minshall RD Beat A Anti‘metastatic potential of amide‘linked local anaesthetics inhibition of lung adenocarcinoma cell migration and inflammatory Src signalling independent of sodium channel blockade Anesthesiology “ Lirk P Berger R Hollmann MW Feigl H Lidocaine time and dose‘depend‘ently demethylates deoxyribonucleic acid in breast cancer cell lines in vitro Br J Anaesth “ Villar‘Garea A Fraga MF Espada J Esteller M Procaine is a DNA‘demethyl‘ating agent with growth‘inhibitory effects in human cancer cells Cancer Res 4984e634984e9 Kampo S Ahmmed B Zhou T Owusu L Anabah TW Doudou NR Kuug‘bee ED Cui Y Lu Z Yan Q Wen Q‘P Scorpion venom analgesic peptide BmK AGAP inhibits stemness and epithelial‘mesenchymal transition by down‘regulating PTX3 in breast cancer Front Oncol Hirata M Sakaguchi M Mochida C Sotozono C Kageyama K Yoshihiro K Munetaka H Lidocaine inhibits the tyrosine kinase activity of the epidermal growth factor receptor and suppresses proliferation of corneal epithelial cells Anesthesiology “ 0cKwakye a0et a0al BMC Res Notes Page of Grouselle M Tueux O Dabadie P Geescaud D Mazat JP Effect of local anaesthetics on mitochondrial membrane potential in living cells Biochem J “ Fraser SP Diss JKJ Chioni AM Mycielska ME Pan H Yamaci RF Pani F Siwy Z Krasowska M Grzywna Z Brackenbury WJ Theodorou D Koyuturk M Kaya H Battaloglu E De Tamburo Bella M Slade MJ Tolhurst R Palmieri C Jiang J Latchman DS Coombes RC Djamgoz MBA Voltage‘gated sodium channel expression and potentiation of human breast cancer metastasis Clin Cancer Res “ Le Gac G Angenard G Clement B Laviolle B Coulouarn C Beloeil H Local anaesthetics inhibit the growth of human hepatocellular carcinoma cells Anesth Analg “ Lee HT Xu H Siegel CD Krichevsky IE Local anaesthetics induce human renal cell apoptosis Am J Nephrol “ Unami A Shinohara Y Ichikawa T Baba Y Biochemical and microarray analyses of bupivacaine‘induced apoptosis J Toxicol Sci “ Villar‘Garea A Fraga MF Espada J Esteller M Procaine is a DNA‘demethyl‘ating agent with growth‘inhibitory effects in human cancer cells Cancer Res “ Sakaguchi M Kuroda Y Hirose M The antiproliferative effect of lidocaine on human tongue cancer cells with inhibition of the activity of epidermal growth factor receptor Anesth Analg “ Chang YC Liu CL Chen MJ Hsu YW Chen SN Lin CH Chen CM Yang FM Hu MC Local anaesthetics induced apoptosis in human breast tumour cells Anesth Analg “ Kawasaki C Kawasaki T Ogata M Sata T Chaudry IH Lidocaine enhances apoptosis and suppresses mitochondrial functions of human neutrophil in vitro J Trauma “ Hodgkin AL Huxley AFA quantitative description of membrane current and its application to conduction and excitation in nerve J Physiol “ Besson P Driffort V Bon ‰ Gradek F Chevalier S Roger S How do voltage‘gated sodium channels enhance migration and invasiveness in cancer cells Biochim Biophys Acta “ Roger S Gillet L Le Guennec JY Besson P Voltage‘gated sodium channels and cancer is excitability their primary role Front Pharmacol Roger S Potier M Vandier C Besson P Le Guennec JY Voltage‘gated sodium channels new targets in cancer therapy Curr Pharm Des “ Driffort V Gillet L Bon E Marionneau‘Lambot S Oullier T Joulin V Collin C Pag¨s JC Jourdan ML Chevalier S Bougnoux P Le Guennec JY Besson P Roger S Ranolazine inhibits NaV15‘mediated breast cancer cell invasive‘ness and lung colonization Mol Cancer Nelson M Yang M Dowle AA Thomas JR Brackenbury WJ The sodium channel‘blocking antiepileptic drug phenytoin inhibits breast tumour growth and metastasis Mol Cancer Yuan T Li Z Li X Yu G Wang N Yang X Lidocaine attenuates lipopoly‘saccharide‘induced inflammatory responses in microglia J Surg Res “ Brocco MC Paulo DNS Almeida CED Carraretto AR Cabral SA Silveira AC Gomez RS Baptista JF A study of interleukin IL‘ and tumour necrosis factor‘alpha TNF‘α serum levels in rats subjected to faecal peritonitis and treated with intraperitoneal ropivacaine Acta Cirurgica Brasileira “ Piegeler T Schl¤pfer M Dull RO Schwartz DE Beat A Minshall RD Beck‘Schimmer B Clinically relevant concentrations of lidocaine and ropivacaine inhibit TNFα‘induced invasion of lung adenocarcinoma cells in vitro by blocking the activation of Akt and focal adhesion kinase Br J Anaesth “ Shankar S Chen Q Srivastava RK Inhibition of PI3KAKT and MEKERK pathways act synergistically to enhance antiangiogenic effects of EGCG through activation of FOXO transcription factor J Mol Signal Qian J Zou Y Rahman JSM Lu B Massion PP Synergy between phosphatidylinositol kinaseAkt pathway and Bcl‘xL in the control of apoptosis in adenocarcinoma cells of the lung Mol “ Ortega MA Fraile‘Mart™Ä±nez O As™Unsolo A Buj™an J Garc™Ä±a‘Honduvilla N Coca S Signal transduction pathways in breast cancer the important role of PI3KAktmTOR J Oncol Royds J Khan AH Buggy DJ Update on existing ongoing prospective trials evaluating the effect of anaesthetic and analgesic techniques during primary Cancer surgery on Cancer recurrence or metastasis Int Anesthesiol Clin 2016544e76“ Burgering BMT Medema RH Decision on life and death FOXO forkhead transcription factors are in command when PKBAkt is off duty J Leukoc Biol “ Chen Q Ganapathy S Singh KP Shankar S Srivastava RK Resveratrol Induces Growth Arrest and Apoptosis through Activation of FOXO Tran‘scription Factors in Prostate Cancer Cells PLoS ONE 20105e15288 Arnold A Papanikolaou A Cyclin D1 in breast cancer pathogen‘esis J Clin Oncol “ Santarius T Shipley J Brewer D Stratton MR Cooper CS A census of amplified and overexpressed human cancer genes Nat Rev Cancer “ Datta SR Brunet A Greenberg ME Cellular survival a play in three Akts Genes DAev “ Lirk P Hollmann MW Fleischer M Weber NC Feigl H Lidocaine and ropivacaine but not bupivacaine demethylate deoxyribonucleic acid in breast cancer cells in vitro Br J Anaesth 2014113Suppl 1i32“i3838 Li K Yang J Han X Lidocaine sensitizes the cytotoxicity of cisplatin in breast cancer cells via the up‘regulation of RARβ2 and RASSF1A demeth‘ylation Int J Mol Sci “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims in pub‘lished maps and institutional affiliations¢ fast convenient online submission ¢ thorough peer review by experienced researchers in your field¢ rapid publication on acceptance¢ support for research data including large and complex data types¢ gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research over 100M website views per year ¢ At BMC research is always in progressLearn more biomedcentralcomsubmissionsReady to submit your research Choose BMC and benefit from 0c'

Thyroid_Cancer

molecular heterogeneity of renal cell carcinoma RCC complicates the therapeutic interventions for advancedmetastatic disease and thus its management remains a significant challenge This study investigates the role of thelncRNA CDKN2BAS1 and miR1413p interactions in the progression and metastasis of kidney cancer Human renalcancer cell lines ACHN and Caki1 normal RPTEC cells tissue cohorts and a series of in vitro assays and in vivo mousemodel were used for this study An overexpression of CDKN2BAS1 was observed in RCC compared to normal samplesin TCGA and our inhouse SFVAMC tissue cohorts Reciprocally we observed reduced expression of miR141 in RCCcompared to normal in the same cohorts CDKN2BAS1 shares regulatory miR141 binding sites with CCND1 andCCND2 genes Direct interactions of CDKN2BAS1miR141Cyclin D1“D2 were confirmed by RNA immunoprecipitationand luciferase reporter assays indicating that CDKN2BAS1miR141Cyclin D1“D2 acts as a ceRNA network in RCCFunctionally attenuation of CDKN2BAS1 andor overexpression of miR141 inhibited proliferation clonogenicitymigrationinvasion induced apoptosis in vitro and suppressed tumor growth in xenograft mouse model Furtheroverexpression of CDKN2BAS1 is positively correlated with poor overall survival of RCC patients Expression of miR141also robustly discriminated malignant from nonmalignant tissues and its inhibition in normal RPTEC cells induced procancerous characteristics CDKN2BAS1 attenuation or miR141 overexpression decreased CCND1CCND2 expressionresulting in reduced RAC1pPXN that are involved in migration invasion and epithelial“mesenchymal transition Thisstudy for the first time deciphered the role of CDKN2BAS1miR141Cyclin D axis in RCC and highlights this networkas a promising therapeutic target for the regulation of EMT driven metastasis in RCCIntroductionRenal cell carcinoma RCC is one of the most commoncancers in the USA accounting for nearly deathsand new cases in Surgery is the first line oftreatment resulting in successful resection and longtermdiseasefree status with an overall survival rate of moreCorrespondence Rajvir Dahiya rdahiyaucsfedu1Department of Urology Veterans Affairs Medical Center San Francisco andUniversity of California San Francisco San Francisco CA USA2Department of Surgery University of Miami Miller School of Medicine MiamiFL USAFull list of author information is available at the end of the These authors contributed equally Pritha Dasgupta Priyanka KulkarniEdited by E Candithan However in approximately of localizedRCC cases recurrence occurs with distant metastasis2The obstinate nature of RCC to current treatment regimens is a primary cause of poor prognosis in patients withmetastatic recurrence Lack of sensitivity to both chemotherapytherapeuticoptions difficult3“ It is therefore of utmost importanceto improve our understanding of RCC pathogenesis byidentifying new biomarkers that lead to better predictionand therapeutic intervention of aggressive RCC6and immunotherapy makesEmerging lines of evidence suggest that cancer aggressiveness is associated with epithelial“mesenchymal transition EMT7 It is a wellorchestrated process involved in The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate ifchanges were made The images or other third party material in this are included in the ™s Creative Commons license unless indicated otherwise in a credit line to the material Ifmaterial is not included in the ™s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this license visit httpcreativecommonslicensesby40Official journal of the Cell Death Differentiation Association 0cDasgupta Cell Death and Disease Page of tumor invasion and metastasis comprising characteristicphenotypic changes through transition from polarizedimmotile epithelial cells to motile mesenchymal cells8EMT changes in cellular morphology and migratoryproperties are governed by numerous factors9 Increase inmesenchymal properties accompanied by augmentedexpression of mesenchymal markers like Ncadherbronectinvimentin and matrix metalloproteinaseMMPs and decreased expression of epithelial markerslike Ecadherin αECatenin claudin etc10“ are common EMT phenomena Often the progression of cancerthrough EMT is significantly induced by the interaction ofCyclinD with its binding partner Cdk4 which act astranscriptional regulators controlling cell proliferationand migration14“ It is well known that CyclinD regulates the ratelimiting step in cell cycle progression fromG1 to S phase Accumulating evidence also suggest thatabnormal CyclinDCdk4overexpression promotestumor growth and metastasis17 but how this correlateswith tumor metastasis or controls cell adherence andinvasion is poorly understoodReports show that noncoding RNAs are involved in thefactors involved in EMT18 MicroRNAsregulation ofmiRNAs a naturally occurring class of small noncodingRNA molecules of nucleotides long19 are known toregulate gene expression via both translational inhibitionand mRNA degradation20 whereas long noncoding RNAslncRNAs with more than nucleotides can also actas regulators for tumorsuppressive miRNAs in differentcancers21“ Recently a class oflncRNAs have beencategorized as competing endogenous RNA ceRNAwhich involves crosstalk among lncRNAs mRNAs andtheir shared miRNAs Thus a novel regulatory mechanismis hypothesized suggesting that lncRNAs and mRNAscommunicate with each other by competing for commonmiRNA response elements24“In this context we describe the novel role of lncRNACDKN2BAS1 and miR1413p miR141 in the regulation of CyclinD to govern the metastatic progression ofRCC To our knowledge this is the first report to directlydemonstrate that CDKN2BAS1miR141 interaction is acrucial component in RCC progression and metastasisthrough the CyclinDRac paxillin pathwayMaterials and methodsCell lines and cell cultureThe normal RPTEC ATCC number CRL4031 andrenalcancer ACHN ATCC number CRL1611and Caki1 ATCC number HTB46 cell lines were purchased from the ATCC Manassas VA These humanderived celllines were authenticated by DNA shorttandem repeat analysis by ATCC Cell line experimentswere performed within “ months of their procurementresuscitation ACHN cells were cultured in MEM mediaOfficial journal of the Cell Death Differentiation AssociationCaki1 cells in and McCoy 5A medium and RPTEC cellsin DMEMF12 Medium ATCC® „¢ All mediawere supplemented with FBS and 1X antibioticspenicillin and streptomycin Cell lines were maintainedat °C and humidified atmosphere of CO2miRNAsiRNA transfectionsTo induce overexpression or knockdown cells were transiently transfected with either mirVana miRNA Mimic nmolL or antimiR miRNA inhibitor nmolLThermo Fisher Scientific and nmolL of siRNA SigmaAldrich using Lipofectamine RNAi Max Thermo FisherScientific according to the manufacturers™ protocol Toverify transfection efficiency mirVana miRNA MimicNegative Control miRNA inhibitor control and siRNAcontrol were used respectively in each transfection experiment at the same concentration All transfection experimentswere carried out for hClinical specimensFormaldehydefixedparaffinembedded FFPE tissue specimens from patients undergoing radical nephrectomy wereobtained from the San Francisco Veterans Affairs MedicalCenter SFVAMC Written informed consent was obtainedfrom all patients and the study was in accordance withinstitutional guidelines IRB approval no Allpatient samples were pathologically confirmed for clear cellRCC ccRCC and slides were reviewed by a boardcertifiedpathologist for the identification of tumor foci and adjacentnormal tissue Apart from SFVAMC cohort TCGAKIRCTCGAKICH TCGAKIRP ICGC and GEO cohorts forRCC from online databases were also used to check theexpression levelsRNAmiRNA extraction and quantitative realtime PCRqRTPCRTotal RNA was extracted from microdissected FFPEtissues and cell lines using miRNeasy FFPE and miRNeasymini kits Qiagen respectively in accordance to manufacturer™s instructions Mature miRNA and mRNAs wereassayed by qRTPCR using QuantStudio FlexReal TimePCR System Applied Biosystem using Fast SYBR®Green Master MixTaqMan universal PCR master mixprobes and primers Applied Biosystems Inc Foster CityCA USA following manufacturer™s protocol HumanGAPDH and RNU48 were used as endogenous controlsand relative expression of RNAmiRNA were calculatedusing comparative Ct threshold cycle Primer sequencesare provided in Supplementary Table T1DNA methylation analysis insilico in cell lines and 5AzaCdR treatmentDNA hypermethylation of the miR141 promoter innormal and RCC samples was first confirmed in the 0cDasgupta Cell Death and Disease Page of TCGA database using Wanderer software27 In order toconfirm the methylation status of the miR141 promoterin RCC cell lines we extracted DNA from ACHN andCaki1 using DNeasy tissue kit Qiagen Sodium bisulphite modification was done using EZ DNA methylationGold kit Zymo Research Orange CA USA followingthe manufacturer™s protocol Bisulfitetreated DNA wasanalyzed by methylationspecific quantitative polymerasechain reaction MSqPCR with primer pairs specific formethylated and unmethylated regions of the miR141promoter MSqPCR was performed as described earlier28 For each sample the percent of methylation wascalculated by the difference of Ct in methylated sampleCtM and Ct in unmethylated sample CtU The primers sequences are mentioned in Supplementary Table ACHN and Caki1 cells were treated daily with μmolL5AZAdeoxycytidine 5AzaCdRfor h29 and total RNA was isolated using a miRNeasy minikit Qiagen to check miR141 expressionSigmaAldrichCell viability clonability migratory invasion andapoptosis assaysCell viability was measured at and h using aCellTiter Aqueous Solution Cell Proliferation Assay kitPromega Madison WI following the manufacturer™sinstructions For colony formation assay cells were seeded at a low density cellsplate after h oftransfection and were allowed to grow until visible colonies were formed Plates were then stained with giemsafollowed by crystal violet and colonies were countedCulture inserts of 8µm pore size Transwell Costar wereused for migration and invasion assay Inserts were coatedwith Matrigel BD Biosciences µgwell for invasionBriefly h posttransfection cells were counted andplaced on inserts at × cellsml for migration and × for invasion in serumfree medium and wereallowed to migrateinvade for “ h at °C Cellsmigrated or invaded through the pores were fixed stainedwith crystal violet Crystal violet was solubilizedwith methanol and quantified at nm by a kineticmicroplate reader Spectra MAX Molecular DevicesFACS analysis for apoptosis was done h posttransfection using Annexin VFITC and 7AAD Kit Beckmanin accordance with the manufacturer™sCoulter Incinstructions Cold PBS washed cells were resuspended in1X binding buffer and stained with Annexin VFITC7AAD viability dye After min of incubation at roomtemperature in the dark stained cells were analyzed usingBD FACSVerse BD PharmingenDualluciferase reporter assayThe wild type WT and offtarget OT luciferasereporter constructs were made by ligating annealed custom oligonucleotides containing putative target bindingOfficial journal of the Cell Death Differentiation Associationsites and corresponding nontarget mutant sites into thepmiRGLO reporter vector Luciferase constructs µgwere cotransfected into ACHN and Caki1 cells along with nmolL miR141 mimic or controlmiR using transfection reagent JetPrime Polyplustransfection IllkirchFrance Luciferase activities were measured using theDualluciferase assay Promega Madison WI h posttransfection Relative luciferase activity was calculated bynormalizing Firefly luciferase to Renilla luminescenceRNA immunoprecipitation RIP assaySimultaneous binding of miR141 to lncRNA andmRNA was confirmed by RIP assay An imprint RIP kitwas used following the manufacturer™s protocol SigmaAldrich St Louis MO USA IgG control and Ago2antibodies were used forimmunoprecipitation Theimmunoprecipitated RNA fraction was reverse transcribed to cDNA using High capacity cDNA reversetranscription kit Thermo Fisher Fold enrichment oflncRNA and mRNA to Ago2 with respect to IgG wascalculated using quantitative RTPCRWestern blot and immunofluorescence analysisTotal protein extraction was performed as describedpreviously18 Proteins were then separated by NuPAGE“ BisTris Protein Gels Invitrogen and subsequentlytransferred onto nitrocellulose membraneResulting blots were blocked using Odyssey blockingbuffer LICOR and subsequently probed with primaryand secondary antibodies Blots were scanned using anOdyssey Infrared Imaging System Scan and quantificationwas carried out with the LICOR Odyssey® scanner andsoftware LICOR Biosciences The primary antibodiesused are listed in Supplementary Table For immunofluorescencetransfected ACHN andCaki1 cells were fixed in paraformaldehyde for minfollowed by blocking 1X PBS5 normal goat serum Triton X100 for h at room temperature Cellswere then incubated overnight in fold diluted primary antibody at °C Cells were then reprobed with fold diluted secondary antibody for h and counterstained with µgml of ²6diamidino2phenylindoleDAPI for min Cells were then mounted on a slideusing prolong gold antifade reagent Images were captured using Zeiss microscope model Axio ImagerD2transientlyRenal cancer xenograftsWe studied the antitumorigenic effects of miR141 inestablished tumors using a renal cancer xenograft nudemouse model as previously described630 Male nude mice“ weekold n Charles River Lab were subcutaneously injected with × Caki1 cells Oncepalpable tumors were formed mice were randomized intwo groups for the treatment and control groups five in 0cDasgupta Cell Death and Disease Page of each Synthetic miRNA miR141 mimicmiRCON of μg was complexed with μL siPORTamine transfection reagent Ambion in μL PBS and deliveredintratumorally in 3day intervals Tumor volume wascalculated according to the formula x2 y2 where x yx width y length Experiments were terminated days after the last treatment day Tumor measurements and statistical analysis were performed byresearchers who were blinded for the control and treatment groups All animal care was in accordance withinstitutional guidelines IACUC approval no Statistical analysisAll quantified data represents an average of at leastthree independent experiments or as indicated Statisticalanalyses were performed using GraphPad Prism andMedCalc Error bars represent ± standard error meanSEM The Mann“Whitney U test was used to assessthe difference between miRNA expressions in tumornormal adjacent tissue Significant differences betweenthe groups were determined using the Student™s ttest Alltests were performed either one tailed or two tailed andresults were considered statistically significant at P ‰¤ Receiver operating curves ROC were performed toevaluate the potential of miR141 to differentiate betweenmalignant and nonmalignant samples using MedCalcsoftware showing area under curve AUC and confidence interval Kaplan“Meier analyses for overall survival with respectto miR141 methylation levels inTCGAKIRC cohort were generated using softwareœEZRhttpsdoi101038bmt2012244 Tumormeasurements and statistical analyses for all experimentswere performed blindly for the control and treatment groupsResultslncRNA CDKN2BAS1 is oncogenic and is a direct target ofmiR141Initially we found CDKN2BAS1 is an oncogeniclncRNA in RCC based on TCGA Fig 1a ICGC andGEO databases Fig S1 TCGA cohort also revealed thathigh CDKN2BAS1 expression increases from lower gradeand stage to higher grade and stage Fig 1b Moreoverhigher expression is significantly p correlated tooverall survival Fig 1c In agreement with these datacohorts significantly higher CDKN2BAS1 expression wasalso seen in RCC cell lines ACHN Caki1 as compared tonormal RPTEC Fig 1d and SFVAMC cohort Fig 1ePatient and tumor characteristics are summarized inSupplementary Table T3 ROC analysis shows an AUC of P CI “ Fig 1f suggesting the diagnostic potential of CDKN2BAS1 to discriminate between normal and tumor tissues We usedcomputational algorithms and identified putative miR141binding sites in the CDKN2BAS1 sequence Fig 1g ToOfficial journal of the Cell Death Differentiation Associationexamine potential miR141CDKN2BAS1 interactionexperimentally we performed luciferase reporter assayBoth ACHN and Caki1 cells cotransfected with miR141and CDKN2BAS1 wild type binding site revealed a consistent reduction ofluciferase activity suggesting thatmiR141 directly interacts and regulates CDKN2BAS1Fig 1h Thus all these data suggest that clinicallyimportant CDKN2BAS1 is an oncogenic lncRNA in RCCand is a novel target of miR141CDKN2BAS1 inhibition by siRNA suppressestumorigenicity in RCCTransient transfection of ACHN and Caki1 cells withCDKN2BAS1 siRNAs for h showed significant reduction in CDKN2BAS1 expression Fig 2a CDKN2BAS1knockdown in both cell lines significantly inhibited cellproliferation Figs 2b S2A and clonogenic survivalFigs 2c S2B with a significant increase in apoptosis Fig2d e Decreased cell migrationinvasion Figs 2f S2C Dwith simultaneous changes in EMT markers such as anincrease in epithelial markers αECatenin and claudinand decrease in mesenchymal markers vimentin andfibronectin were also observed Figs 2g S3Expression of miR141 and its clinical importance in renalcarcinomaand samples LowerSince our results confirmed CDKN2BAS1 as a directtarget of miR141 we examined miR141 status andclinical importance in RCC Expression of miR141 wassignificantly downregulated in RCC cell lines Fig 3aand in tumor samples Fig 3b“e compared to normal celllinesignificantlydecreased with increasing grade stages and in metastaticcompared to nonmetastatic tumors Fig 3c“e Patientand tumor characteristics are summarized in Supplementary Table T3 ROC analysis showed an AUC of P CI “ Fig 3f suggesting thatmiR141 can be used as a potential diagnostic parameterto discriminate between normal and tumor tissuesexpressionsEpigenetic regulation of the miR141 locusWe identified a genomic site rich in CpG island locatedupstream of the miR141 in chromosome 12p13 In theTCGA cohort we observed hypermethylation of miR141promoter in tumor tissues as compared to normalFigs 3g S4A which is significantly associated with poorpatient survival Fig 3h Similarly RCC cell lines ACHNand Caki1 also showed hypermethylation compared tonormal RPTEC cells Fig 3i Further we treated ACHNand Caki1 cell lines with demethylating agent 5AzaCdRand observed decrease in methylation Fig S4B withconcomitant increase in miR141 expression Fig 3jindicating possible epigenetic regulation A significantdecrease in the expression of methylation regulatory 0cDasgupta Cell Death and Disease Page of Fig lncRNA CDKN2BAS1 is oncogenic in renal cancer and is a direct target of miR141 a Expression levels of CDKN2BAS1 among KIRCnormal tumor KICH normal tumor and KIRP normal tumor patient samples in TCGA cohort using Wanderersoftware Pvalue calculated by Mann“Whitney twotailed test b Expression of CDKN2BAS1 in TCGAKIRC cohort among different grades normal grade “ grade “ and stages normal stage I“II and stage III“IV c Overall survival in TCGAKIRC cohort as performedby Kaplan“Meier analysis using UALCAN software d Relative expression levels of lncRNA CDKN2BAS1 in RCC cell lines ACHN and Caki1 e ExpressionCDKN2BAS1 in matched pairs of RCC tissue samples from SFVAMC cohort Pvalue calculated by Mann“Whitney twotailed test f Receiver operatingcurve ROC analysis on SFVAMC cohort showing ability of lncRNA CDKN2BAS1 to differentiate between malignant and nonmalignant samples SFVAMCcohort g Predicted binding sites of miR141 in CDKN2BAS1 sequence h Luciferase assays showing decreased reporter activity after cotransfection witheither wildtype WT offtarget OT CDKN2BAS1 or luciferase control constructs EV with miRCONmiR141 in ACHN and Caki1 cellsgenes such as DNMTl DNMT3a and DNMT3b werealso noted after 5AzaCdR treatment compared to control DMSO in both ACHN and Caki1 cell lines31miR141 overexpression phenocopies functional effectsobtained with CDKN2BAS1 inhibition in vitro andsuppresses tumorigenicity and in vivoWe sought to determine if CDKN2BAS1 causes itsantitumorigenic effects through miR141 We checkedthe effect of miR141 overexpression in RCC cellsTransient transfection of miR141 mimic in ACHN andCaki1 cells for h led to over expression of miR141compared to control miRCON Fig 4a Also overexpression of miR141 significantly reduced CDKN2BAS1 expression Fig 4b indicating a reciprocal correlation between miR141 and CDKN2BAS1 A significant decrease in cell proliferation over time Fig 4cand marked decreaseFig 4din clonogenicityOfficial journal of the Cell Death Differentiation Association 0cDasgupta Cell Death and Disease Page of Fig Knockdown of lncRNA CDKN2BAS1 reduces tumorigenicity in renal cancer a Relative expression of CDKN2BAS1 in ACHN and Caki1 cellstransfected with CDKN2BAS1 siRNAs b Cell proliferation assessed by MTS assay after knockdown of CDKN2BAS1 in both cell lines with siRNA2 CGraphical representation showing knockdown of CDKN2BAS1 with siRNA2 significantly decreased colony formation in ACHN and Caki1 cellsd e ACHN and Caki1 cell lines showing significant induction of apoptosis early late compared to control after knockdown of CDKN2BAS1f Reduced migration and invasion in CDKN2BAS1 siRNA transfected cells compared to control treatment g Changes in EMT related proteins in bothACHN and Caki1after knockdown of CDKN2BAS1compared to controls were also observed Further westudied the therapeutic potential of miR141 in amouse xenograft model A significant decrease intumor growth was observed by intratumoral delivery ofmiR141 mimic compared to control over the course ofexperiment Average tumor volume in the controlgroup was mm3 compared to mm3 in micethat received miR141 mimic Fig 4e In additionmiR141 overexpression significantly induced apoptosis with a concomitant decrease in the viable population in both RCC celllines compared to controlFig 5a This proapoptotic role was supported by theinduction of cleaved caspase3 cleaved polyADPribose polymerase PARP an increase in BAX and adecrease in BCl2 at protein levels Fig 5b A significantdecrease in migration Fig 5c and invasion Fig 5dwas also observed in both RCC cell lines with miR141overexpression We also examined EMT markers aschange in migration and invasion are directly associated with EMT Our results showed an increase inepithelial markers αEcatenin and claudin with concomitant decrease in mesenchymal markers fibronectinand vimentin at both protein Fig 5e and mRNAOfficial journal of the Cell Death Differentiation AssociationFig S5 levels Taken together overexpression of miR phenocopies the functional effects of CDKN2BAS1 inhibition in vitro and tumor growth suppressioneffects in vivoLike CDKN2BAS1aremiR141CDKN2BAS1 interaction negatively regulatesCyclinD and its downstream effectors in RCCcyclinD1cyclinD2alsooncogenic in RCC Fig S6 and are direct targets of miR As discussed earlier lncRNAs can act as ceRNAs tocarry out their regulatory functions32“ We observedthat CDKN2BAS1 shared regulatory miR141 bindingsites with cyclinD1cyclinD2 Fig 6a and therebysponges miR141 allowing cyclinD1D2 to be expressedin tumors To determine potential miR141CDKN2BAS1CyclinD interaction experimentally we performedRIP assay Both ACHN and Caki1 cells over expressingmiR141 revealed significant enrichment of cyclinD1cyclinD2 and CDKN2BAS1 with Ago2 as compared toIgG control Fig 6b Moreover decreased luciferaseactivity also confirmed direct binding of miR141 tocyclinD in miR141overexpressing ACHN andCaki1 cells compared to controls Fig 6c We also found 0cDasgupta Cell Death and Disease Page of Fig Expression clinical significance and epigenetic regulation of miR141 in renal cancer a miR141 expression levels in ACHN Caki1 andRPTEC cells b Expression levels of miR141 in KIRCTCGA cohort normal and tumor c Expression levels of miR141 in normal n nonmetastatic n metastatic n KIRC patient samples in TCGA cohort d Expression levels of miR141 in KIRCTCGA cohort amongdifferent grades normal grade “ grade “ and stages normal stage I“II and stage III“IV e Relative miR expression in RCC tissue vs matched adjacent normal regions n among different grades normal grade grade “ andin different stages normal stage I“II stage III“IV as assessed by qRTPCR SFVAMC cohort f Receiver operating curve ROC analysisshowing ability of miR141 to differentiate between malignant and nonmalignant samples g Methylation for KIRC patient samples in TCGAKIRCcohort for probe cg02624246 h Overall survival with TCGAKIRC methylation as performed by Kaplan“Meier analysis i Methylation status of miR141promoter in RCC cell lines compared to nonmalignant RPTEC as assessed by MSqPCR j Expression of miR141 in 5AzaCdR treated and untreatedACHN and Caki1 cell lines Results are representative of three independent experiments P value calculated by Student t test Bar mean ± standarderror mean SEMthat overexpression of miR141 or inhibition of CDKN2BAS1 significantly decreased cyclinD1D2 expression atboth the mRNA Fig 6d e and protein levels Figs 6f“hS8A B This effect was significantly attenuated by miR inhibitor Fig S7 indicating that cyclinD expressionis dependent on the interaction between miR141 andCDKN2BAS1 We further observed a decrease in Rac1 asmall GTPase and a reduction in the phosphorylation ofpaxillin a focal adhesion protein at mRNA levelsFigs S3 S5 and protein levels Figs 6g I“j S8C“F inboth miR141 overexpressed and CDKN2BAS1 inhibitedRCC cell lines which in turn are involved in regulatingcellular migrationinvasion Cumulatively these resultsindicate that suppression of CDKN2BAS1 by miR141inhibits renal cell proliferation invasion and migration byinhibiting cyclinD Rac1 and phosphorylation of paxillinOfficial journal of the Cell Death Differentiation Association 0cDasgupta Cell Death and Disease Page of Fig miR141 overexpression mimics the knockdown effect of lncRNA CDKN2BAS1 in vitro and reduces tumorigenicity in vivo a Relativeexpression of miR141 in ACHN and Caki1 cells transfected with miR141 mimic and control b Significant decrease in CDKN2BAS1 expressioncompared to control in both cell lines overexpressed with miR141 c RCC cell proliferation after transfecting with miR141 mimic and control asassessed by MTS assay d Colony formation and its graphical representation in miR141 overexpressing ACHN and Caki1 cells compared to controlse Pictures of excised tumors are taken at the termination of experiment day Graph represents tumor volume after intratumoral injection ofcontrol or miR141 mimic into established tumors Injection was started at day and was followed for days Each mouse in both groups miRCONand miR1413pMimic received a total of eleven injections intermittently Data represent the mean of each group and error bars are SEM Results arerepresentative of three independent experiments P value calculated by Student t test Bar mean ± standard error mean SEMAttenuation of miR141 exerts tumorigenic attributes innormal RPTEC cellsWe next determined whether attenuation of miR141induces tumorigenic characteristics in normal RPTECcells by targeting CDKN2BAS1 and cyclinD Transienttransfection of miR141 inhibitor indeed showed a significant decrease in miR141 expression Fig 7a and anincrease in CDKN2BAS1 expression Fig 7b along withother procancerous phenotypes such as increased cellproliferation Fig 7c colony formation Fig 7d migration and invasion Fig 7e as compared to controlsAdditionally a significant increase in cyclinD1 cyclinD2 rac1 and paxillin Pxn expressions were observed inmiR141 inhibited RPTEC cells Fig 7f A noticeableincrease in prometastatic fibronectin and vimentin with aconcomitant decrease in antimetastatic claudin andαEcatenin genes were also observed in miR141 inhibited RPTEC cells compared to controls Fig 7gDiscussionPrior studies have shown the regulatory role of noncoding RNAs in tumorigenesis especially in the EMTOfficial journal of the Cell Death Differentiation Associationpathway leading to cancer aggressiveness CDKN2BAS1also known as ANRIL is located at chromosome 9p21CDKN2BAS1 is reported to be upregulated in tumortissues and function as an oncogenic lncRNA in pancreatic ovarian and laryngeal squamous cell carcinoma36“ Human miR141 is located at chromosome12p1331 and is transcribed from a miR200 familyclusterInterestingly expression of miR141 is controversial since it exhibits either oncogenic39“ or tumorsuppressive roles42“ in specific types of cancer Theprime goal of the present study was to understand therole of CDKN2BAS1miR141 interactions in regulatingRCC progression and metastasisIn this study we identify CDKN2BAS1 to be a crucialoncogenic lncRNA that plays an important role in renalcarcinogenesis CDKN2BAS1significantly overexpressed in RCC and the expression increases fromlower to higher grades and stages LncRNA CDKN2BAS1directly interacted with miR141 as it was found to be anovel target of miR141 In contrast to CDKN2BAS1 weobserved significant attenuation of miR141 expression inRCC cell lines and tumor samples compared to normalis 0cDasgupta Cell Death and Disease Page of Fig Ectopic miR141 expression induces apoptosis and inhibits migrationinvasion in renal cancer cells a Apoptosis assessed by flowcytometric analysis of annexinVFITC 7AAD“stained ACHN and Caki1 cells transfected with miRCONmiR141Mimic Graph represents totalapoptosis early late b Immunoblots showing apoptotic proteins in miRCONmiR141Mimic treated ACHN and Caki1 cells with GAPDH asendogenous control c Migration assay and d invasion assay as seen in pictures and graphical representation of both ACHN and Caki1 cells after miR overexpression compared to control e Immunoblot assay showing EMT related proteins in miRCONmiR141Mimic treated ACHN andCaki1 cells with βactin as endogenous control Graphs are average of three independent experiments P value calculated by Student t testBar mean ± standard error mean SEMcell line or matched normal samples As it is known thatextensive DNA hypermethylation of CpG islands is highlycorrelated to activation of cancerspecific genes45 wechecked the methylation status of miR141 in normal andRCC tissues Interestingly insilico analysis showed thepresence of CpG island in the promoter region of miR and we also found hypermethylation of miR141 inTCGA samples as compared to normal This hypermethylation is also found to be significantly associatedwith poor survival of patients Similar results were alsoobserved in RCC cell lines compared to a normal RPTECcell line Functionally inhibition of CDKN2BAS1 andoroverexpression of miR141 significantly inhibits thetumorigenic characteristics such as cell proliferationclonogenicity migration and invasion whereas inducesanticancer apoptotic phenotype in RCC in vitro In vivodata show suppression of tumor growth by miR141overexpression Conversely attenuation of miR141 innormal RPTEC cells induced precancerous characteristicsindicated by increased proliferation migration andinvasionFrom a clinical point of view noncoding RNAs signatures are powerful tools for early cancer diagnosisOfficial journal of the Cell Death Differentiation Associationmaking them attractive candidates as diagnostic andprognostic biomarkers184647 Our results revealed thathigher expression of CDKN2BAS1 is positively correlatedwith poor overall survival probability of RCC patientsindicating its prognostic capability CDKN2BAS1 canalso discriminate normal from tumor samples showing itsdiagnostic potential Similarly miR141 expression canalso robustly distinguish between cancerous from noncancerous samples and hence has potential to be

Thyroid_Cancer

"At present the relationship between hypothyroidism and the risk of breast cancer is still inconclusive Thismetaanalysis was used to systematically assess the relationship between hypothyroidism and breast cancer riskand to assess whether thyroid hormone replacement therapy can increase breast cancer riskMethods The relevant s about hypothyroidism and the risk of breast cancer were obtained on the electronicdatabase platform Relevant data were extracted and odd ratios OR with corresponding confidence intervalsCI were merged using Stata SE softwareResults A total of related studies were included in the metaanalysis including cohort studies and casecontrol studies The results show that hypothyroidism was not related to the risk of breast cancer odd ratios CI “ In the European subgroup we observed that patients with hypothyroidism have a lower risk ofbreast cancerodd ratios CI “ Furthermore no significant correlation was observed betweenthyroid hormone replacement therapy and the risk of breast cancer odd ratios CI “Conclusion Hypothyroidism may reduce the risk of breast cancer in the European population and no significantcorrelation was observed between hypothyroidism and breast cancer risk in nonEuropean populations Due to thelimited number of studies included more largescale highquality longterm prospective cohort studies areneededKeywords Hypothyroidism Thyroid hormone replacement therapy Breast cancer MetaanalysisBackgroundAs a global public health problem breast cancer has anincreasing incidence on a global scale [] According tothe US cancer statistics breast cancer has becomethe most common malignant tumour in women withabout new cases each year accounting for of new malignant tumours in women [] Therefore theidentification of risk factors for breast cancer and the Correspondence Yanhuangdr163com Ruobaolidr163com2Department of Oncology Affiliated Hospital of Weifang Medical UniversityWeifang China3School of Basic Medicine Weifang Medical University Weifang ChinaFull list of author information is available at the end of the adoption of effective early prevention and interventionmeasures are of great significance for patients withbreast cancerThe physiology and pathology of the breast are closelyrelated to the endocrine of the body [] As the largestendocrine an in the human body the thyroid glandhas specific regulation effects on various hormone levelsand cell growth and development in the body whichbrings new enlightenment to the research of breast cancer [“] In Kapdi et alfirst proposed thathypothyroidism maybe increase the risk of breast cancer[] Since then many scholars have studied the relationship between hypothyroidism and the risk of breast The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cWang BMC Cancer Page of [“]cancer However the relationship between the two diseases remains controversial [“] Some studies haveshown that hypothyroidism increases the risk of breastcancerthathypothyroidism reduces the risk of breast cancer []Besides some studies have found no correlation betweenthyroid disease and breast cancer risk [] Thereforewhether hypothyroidism can increase the risk of breastcancer is worthy of further studystudiesshownSomehaveTwo metaanalyses have previously been studied forhypothyroidism and breast cancer risk [ ] Based onprevious research we have included more prospectivestudies and Asian population studies to assess the relationship between hypothyroidism and breast cancer risksystematically Besides the impact of thyroid hormonereplacement therapy on breast cancer risk was exploredin this metaanalysisMethodsSearch strategyRelevant clinical literature was extracted by systematicretrieval of PubMed Medline EMBASE Springer Webof Science and Cochrane Library electronic databasesup to date to October Our search strategy includedorœhypothyroidism or œHT and œthyroid diseases orœbreast cancer or œBC or œbreast neoplasms or œmammarmy cancer and œrisk orœincidence At the sametime we manually screened out the relevant potentialliterature in the references extracteddysfunctionœthyroidtermsforInclusion and exclusion criteria The inclusion criteria Types of studies Published studies exploring therelationship between hypothyroidism and breastcancer risk Subject Female Exposure factors Primary hypothyroidism thediagnosis needs to be based on the detection ofthyroid function Outcome indicators the occurrence of primarybreast cancerThe exclusion criteria Nonprimary hypothyroidism due to other causes Non observational studiesInsufficient information was provided or no fulltext Unable to obtain full text or quality assessment ofthe literature Studies were repeated or publications overlappedData extraction and quality assessmentTwo researchers separately conducted literature screening data extraction and literature quality evaluationand any differences could be resolved through discussionor a third inspector Information secured from the enrolled literature included first author™s surname year ofpublication country ofthe population sample sizefollowup time and data on the relationship betweenhypothyroidism and the risk of breast cancerThe NewcastleOttawa Scale NOS was used to assessthe quality of the study from three aspects cohort selection cohort comparability and outcome evaluation []NOS scores of at least six were considered highqualityliterature Higher NOS scores showed higher literaturequalityStatistical analysisAll data analysis was performed using Stata120 softwareMetaanalysis was performed according to the PRISMAguidelines The OR and 95CI from included studieswere treated with the combined effect size After thatthe heterogeneity test was conducted When P ‰¥ orI2 was performed it mean that there was no apparent heterogeneity and the fixedeffect model shouldbe applied for a merger When P or I2 ‰¥ indicated high heterogeneity the randomeffect model wasapplied Combined effect size if OR indicates thathypothyroidism is an unfavorable factor for breast cancer If OR is the opposite Publication bias Begg funnel plot and Egger test linear regression test were usedto research publication bias detection of the literatureincluded If P indicates obvious publication biasResultsProcess of study selection and description of qualifiedstudiesA total of studies were identified on our online databases After exclusion of duplicate references129 s were considered After screening the andtitle s were excluded After careful review ofthe full texts studies have been excluded because ofthem did not provide relevant data and s didnot have fulltext Nineteen s published between and met the inclusion criteria Fig A total of samples from studies involvingwere enrolled in this metaanalysis [ “ “] Sixcohort studies and casecontrol studies were includedin the study Twelve s were studied in the European population five in the North American populationand two in the Asian population All s are of highquality because of NOS score no less than The chiefcharacteristics of the enrolled materials are detailed inTable 0cWang BMC Cancer Page of Fig Flow chart of search strategy and study selectionRelationship between hypothyroidism and breast cancerriskThere were studies reported the relationship betweenhypothyroidism and breast cancer risk With obviousheterogeneity I p among these studies so a random effect model was used for assessmentThe pooled analysis suggested that was not related tothe risk of breast cancer OR CI “P 0001Fig explorethefurtherrelationshipSubgroup analysis of hypothyroidism and risk of breastcancerTobetweenhypothyroidism and breast cancer risk subgroup analysis was conducted from three aspects study typepopulation distribution and followup time The resultsof subgroup analysis were shown in Table In theEuropean subgroup we observed that patients withhypothyroidism have a lower risk of breast cancer OR CI “ P In the subgroup witha followup date of more than four years patients withhypothyroidism can reduce the risk of breast cancerwith borderline significance OR CI “In otherP found thathypothyroidism was not related to the risk of breastcancersubgroups weRelationship between thyroid hormone replacementtherapy and breast cancer riskA total of studies reported the relationship betweenthe use of thyroid hormone replacement therapy and therisk of breast cancer [ ] Asobvious heterogeneity observed the fixedeffect modelwas usedI p The result suggestedthat patients who received thyroid hormone replacementtherapy was not related to the risk of breast cancerOR CI “109P Fig Publication biasFigure 4a shows the results of publication bias for the relationship between hypothyroidism and breast cancerrisk which were evaluated by funnel plots and Eggerstest The Begg test Pr and the Egger testP were used to detecting publication bias showedthat there was no possibility of publication bias Asshown in Fig 4b there were no publication biases in the 0cWang BMC Cancer Table Main characteristics of the included studies in ouranalysisStudySampleYearRegionAdamiKalacheHoffmanBrintonMosesonSmythSheringTalaminiSimonTurkenKuijpensCristofanilliSandhuHellevikDitschGraniSøgaardWengKimSwedenUKSwedenUSACanadaIrelandIrelandItalyUSAPragueNetherlandsUSACanadaNorwegianGermanyItalyDanishUSAKoreaMedianMean ageyearsNANA ± NANA ± ± ‰¥ ± ‰¥Page of NOSFollowupyearsNANANAStudydesignCasecontrolCasecontrolCohortCasecontrolCasecontrolCasecontrolCasecontrolCasecontrolCasecontrolCasecontrolCohortCasecontrolCohortCohortCasecontrolCasecontrolCohortCasecontrolCohortStudyIDAdami Kalache Hoffman Brinton Moseson Smyth Shering Talamini Simon Turken Kuijpens Cristofanilli Sandhu Hellevik Ditsch Grani Sogaard Weng Kim Overall Isquared p ES CIES CI WeightWeightNOTE Weights are from random effects analysisFig Relationship between hypothyroidism and breast cancer risk 0cWang BMC Cancer Table Stratiedanalysis of the relationship between hypothyroidism and breast cancer riskVariableOR95CINoofstudiesPHeterogeneityI2RegionEur orth AmericaAsiaStudy designCasecontrolCohortFollowup date ‰¤ “ “ “ “ “ “ “ Page of ModelusedFixedRandomedFixedRandomedFixedFixedRandomedPhStudyIDHoffman Kuijpens Sandhu Ditsch Cristofanilli Simon Moseson Brinton Adami Weng ES CIES CI WeightWeightOverall Isquared p NOTE Weights are from random effects analysisFig Relationship between thyroid hormone replacement therapy and breast cancer risk 0cWang BMC Cancer Page of A ]rr[golB ]rh[golBegg's funnel plot with pseudo confidence limitsEgger's publication bias plotse of log[rr]Begg's funnel plot with pseudo confidence limitstceffe idezdradnatstceffi edezdradnatsprecisionEgger's publication bias plotse of log[hr]precisionFig Publication bias assessment a hypothyroidism b thyroid hormone replacement therapy Metaanalysis estimates given named study is omitted Lower CI Limit Estimate Upper CI Limit Adami Kalache Hoffman Brinton Moseson Smyth Shering Talamini Simon Turken Kuijpens Cristofanilli Sandhu Hellevik Ditsch Grani Sogaard Weng Kim Fig Sensitivity analysis for relationship between hypothyroidism and breast cancer risk 0cWang BMC Cancer Page of s on the study of thyroid hormone replacementtherapy The Egger test was P and the Begg testwas Pr Sensitivity analysisThe results of sensitivity analysis are generally stableand the primary source of heterogeneity is in the research of Cristofanilli []Fig So we excludedthe literature of Cristofanilli and analyzed the otherstudies The results revealed that the hypothyroidismcould reduce the risk of breast cancer was borderlineOR096 95CI092“ P andsignificantthere was no heterogeneityI2 P cohortstudy ofDiscussionMore than years ago Beatson used thyroid extracts to treat patients with metastatic advanced breastcancer The condition was significantly alleviated sparkinginterest in exploring the relationship between thyroid andbreast cancer [] Subsequently a prospective study enrolled women and women with earlier diagnosisof hypothyroidism observed the occurrence of breast cancer during followup showed that low serum free thyroxine levels increased the risk of breast cancer [] In aprospective women withhypothyroidism and hyperthyroidism found thathypothyroidism slightly reduced the risk of breast cancer[] However a prospective cohort study of women with autoimmune hypothyroidism and women with normal thyroid function indicated that autoimmune hypothyroidism was not associated with breastcancer risk [] Besides some animal experiments alsoreflect the relationship between the two [ ] Animalexperiments by López Fontanafound thathypothyroidism mice inhibit the development of breastcancer and promote the apoptosis of breast cancer cellsdue to the low expression of βchain protein and activation of the apoptotic pathway on the tumour cell membrane [] Due to the inconsistency ofthe aboveconclusions we performed a metaanalysis to evaluate therelationship between hypothyroidism and breast cancerrisketalA total of studies were included in this metaanalysis and the results showed that patients withhypothyroidism not related to the risk of breast cancerHowever there was significant heterogeneity among theincluded studies After subgroup analysis and sensitivityanalysis we found that Cristofanilli™s research may causeheterogeneity [] Cristofanilli™s research is a retrospective study and the diagnosis of hypothyroidism patientswas based on the information recorded in the medicalrecords which may lead to the bias risk of misclassification and have a positive impact on the positive results ofthis study [] After excluding Cristofanilli™s researchwe found that patients with hypothyroidism had a lowerrisk of breast cancer with borderline significance [] Theresults of the metaanalysis are inconsistent with the findings of Hardefeldt and Angelousi [ ] Perhaps because our study included more prospective studiesand Asian population cohort study In addition we evaluated the risk of breast cancer in thyroid hormone replacement therapy and show that patients who received thyroidhormone replacement therapy was not related to the riskof breast cancerIn the analysis of the European population the resultsshow that hypothyroidism may reduce the risk of breastcancer We also found that patients with hypothyroidismcan reduce the risk of breast cancer was borderline significance in the subgroup with more longer followupdate However the relationship between the two was notobserved in North American and Asian populationsThe possible reasons for these disparities may be as follows First followup time may be the main contributorsto this difference A longer followup is required to demonstrate the relationship between hypothyroidism andbreast cancer risk In the metaanalysis five studies provided North American population data and two reported Asian population data However only one ofseven nonEuropean studies™ followup time for morethan years Second the differences may be attributedto different ethnicities sharing different genegene andgeneenvironmental backgrounds Third social and environmental factors are another critical cause for thisdifference With these in mind our findings suggest thathypothyroidism may reduce the risk of breast canceronly in the European population and more largescalehighqualitylongterm prospective cohort studies arestill needed to study on different human populationsThe following may explain the potential relationshipbetween hypothyroidism and the risk of breast cancerHealthy mammary epithelial cells can express a largenumber of T3 receptors and breast cancer cells have asimilar ability to bind to T3 [] T3 has an estrogenlike effect that promotes the growth of mammary glandlobes and stimulates normal breast tissue differentiation[ ] Therefore T3 can mimic the effect of estrogenon the proliferation of breast cancer cells When theconcentration of T3 is low in vivo it may inhibit theproliferation of breast cancer cells Hypothyroidism mayreduce the risk of breast cancer by affecting T3concentrationSome basic experiments support this theory In GonzalezSancho studied the relationship betweenT3 and breast cancer [] It was found that there is anoverexpressed T1 gene in human breast cancer cellsand T3 inhibits the proliferation of mammary epithelialcells by inhibiting the expression of cyclin D1 and T1thereby inhibiting the proliferation of breast cancer cells 0cWang BMC Cancer Page of Author details1School of Clinical Medicine Weifang Medical University Weifang China 2Department of Oncology Affiliated Hospital of Weifang MedicalUniversity Weifang China 3School of Basic Medicine WeifangMedical University Weifang ChinaReceived December Accepted July foundthat MartinezIglesias[] Afterthathypothyroidism can inhibit the growth of breast cancercells [] In Tosovic conducted a prospectivestudy of T3 levels associated with breast cancer risk andfound that T3 levels in postmenopausal women werepositively correlated with breast cancer risk in a doseresponse mannerthathypothyroidism through lower levels of T3 could reducethe incidence of breast cancer Our metaanalysis resultsalso confirm the above conjecture[] Therefore we suspectHowever this conclusion needs to be taken with caution as this study has several limitations First the studies that have been included do not adjust for importantrisk factors for breast cancer Second in subgroup analysis for example there are only two s in Asianstudies and we should be cautious about the results ofAsian analysis Third the results of this metaanalysis indicate that there is a large heterogeneity between studiesFourth followup time at different endpoints cannot beuniform Finally publication bias cannot be avoidedentirelyConclusionHypothyroidism may reduce the risk of breast cancer inthe European population and no significant correlationwas observed between hypothyroidism and breast cancerrisk in nonEuropean populations Furthermore therewas no obvious correlation between thyroid hormone replacement therapy and breast cancer risk It is necessaryto conduct a large sample size strictly controlled prospective study of hypothyroidism patients further todemonstrate the relationship between hypothyroidismand breast cancer riskAbbreviationsOR Odd ratios CI Confidence intervals NOS NewcastleOttawa ScaleAcknowledgementsNot applicableAuthors™ contributionsStudy design BW ZL RLYH and TL Data extraction BW ZL TL and YH Dataanalysis BW ZL RLand YH Manuscript writing BW and RL Manuscriptedition RL and YH All authors have read and approved the manuscriptFundingNo sources of funding were used to conduct this study or prepare this letterAvailability of data and materialsAll the published s and data were available onlineEthics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsNoneReferencesSiegel RL Miller KD Jemal A Cancer statistics CA Cancer J Clin “ httpsdoi103322caac21442Praestegaard C Kjaer SK Andersson M StedingJensen M Frederiksen KMellemkjaer L Risk of skin cancer following tamoxifen treatment in morethan breast cancer patients a cohort study Breast cancer “ httpsdoi101007s1228201506605 Mittra I Hayward JL Hypothalamicpituitarythyroid axis in breast cancerLancet “ httpsdoi101016s0140673674903444Adami HO Rimsten A Thoren L Vegelius J Wide L Thyroid disease andfunction in breast cancer patients and nonhospitalized controls evaluatedby determination of TSH T3 rT3 and T4 levels in serum Acta Chir Scand“Dargent M Berger M Lahneche B Thyroid function in patients with Cancerof the breast Acta “ Mustacchi P Greenspan F Thyroid supplementation for hypothyroidism Anlatrogenic cause of breast cancer JAMA “Kapdi CC Wolfe JN Breast cancer Relationship to thyroid supplements forhypothyroidism JAMA “ httpsdoi101001jamaKuijpens JL Nyklictek I Louwman MW Weetman TA Pop VJ Coebergh JWHypothyroidism might be related to breast cancer in postmenopausalwomen Thyroid “ httpsdoi101089thy200515 Weng CH Chen YH Lin CH Luo X Lin TH Thyroid disorders and breastcancer risk in Asian population a nationwide populationbased casecontrolstudy in Taiwan BMJ 201883e020194 httpsdoi101136bmj 2017020194Sogaard M Farkas DK Ehrenstein V Jensen JO Dekkers OM SorensenHT Hypothyroidism and hyperthyroidism and breast cancer risk anationwide cohort study Eur J Endocrinol “ httpsdoi101530EJE150989 Angelousi AG Anagnostou VK Stamatakos MK Geiopoulos GAKontzoglou KC Mechanisms in endocrinology primary HT and risk forbreast cancer a systematic review and metaanalysis Eur J Endocrinol “ httpsdoi101530EJE110838 Hardefeldt PJ Eslick GD Edirimanne S Benign thyroid disease is associatedwith breast cancer a metaanalysis Breast Cancer Res Treat “ httpsdoi101007s1054901220193Stang A Critical evaluation of the NewcastleOttawa scale for theassessment of the quality of nonrandomized studies in metaanalyses Eur JEpidemiol “ httpsdoi101007s106540109491zKalache A Vessey MP McPherson K Thyroid disease and breast cancerfindings in a large casecontrol study Br J Surg “ httpsdoi101002bjs1800690731 Hoffman DA McConahey WM Brinton LA Fraumeni JF Jr Breast cancer inhypothyroid women using thyroid supplements JAMA “ Brinton LA Hoffman DA Hoover R Fraumeni JF Jr Relationship of thyroiddisease and use of thyroid supplements to breast cancer risk J Chronic Dis“ httpsdoi1010160021968184900626 Moseson M Koenig KL Shore RE Pasternack BS The influence of medicalconditions associated with hormones on the risk of breast cancer Int JEpidemiol “ httpsdoi101093ije2261000Shering SG Zbar AP Moriarty M McDermott EW O'Higgins NJ Smyth PPThyroid disorders and breast cancer Eur J Cancer Prevent “Smyth PP Smith DF McDermott EW Murray MJ Geraghty JG O'Higgins NJA direct relationship between thyroid enlargement and breast cancer J ClinEndocrinol Metab “ httpsdoi101210jcem813Talamini R Franceschi S Favero A Negri E Parazzini F La Vecchia CSelected medical conditions and risk of breast cancer Br J Cancer “ httpsdoi101038bjc1997289 0cWang BMC Cancer Page of Simon MS Tang MT Bernstein L Norman SA Weiss L Burkman RT DalingJR Deapen D Folger SG Malone K Marchbanks PA McDonald JA Strom BLWilson HG Spirtas R Do thyroid disorders increase the risk of breast cancerCancer Epidemiol Biomarkers Prevent “Turken O NarIn Y DemIrbas S Onde ME Sayan O KandemIr EG Yaylac IMOzturk A Breast cancer in association with thyroid disorders Breast CancerRes 200355R110“ httpsdoi101186bcr609 Cristofanilli M Yamamura Y Kau SW Bevers T Strom S Patangan M Hsu LKrishnamurthy S Theriault RL Hortobagyi GN Thyroid hormone and breastcarcinoma Primary hypothyroidism is associated with a reduced incidenceof primary breast carcinoma Cancer “ httpsdoi101002cncr20881 Hellevik LR Vierendeels J Kiserud T Stergiopulos N Irgens F Dick ERiemslagh K Verdonck P An assessment of ductus venosus tapering andwave transmission from the fetal heart Biomech Model Mechanobiol “ httpsdoi101007s1023700901554Sandhu MK BrezdenMasley C Lipscombe LL Zagorski B Booth GLAutoimmune hypothyroidism and breast cancer in the elderly BreastCancer Res Treat “ httpsdoi101007s10549008 Ditsch N Liebhardt S Von Koch F Lenhard M Vogeser M Spitzweg CGallwas J Toth B Thyroid function in breast cancer patients Anticancer Res“ Grani G Dicorato P Dainelli M Coletta I Calvanese A Del Sordo M DeCesare A Di Matteo FM D'Andrea V Fumarola A Thyroid diseases inwomen with breast cancer La Clin Terapeut 20121636e401“Kim EY Chang Y Lee KH Yun JS Park YL Park CH Ahn J Shin H Ryu SSerum concentration of thyroid hormones in abnormal and euthyroidranges and breast cancer risk a cohort study Int J Cancer “ httpsdoi101002ijc32283 Beatson GT On The Treatment Of Inoperable Cases Of Carcinoma Of TheMamma Suggestions For A New Method Of Treatment With IllustrativeCases1 Lancet “Lopez Fontana CM Zyla LE Santiano FE Sasso CV CuelloCarrion FDPistone Creydt V Fanelli MA Caron RW Hypothyroidism reduces mammarytumor progression via Betacateninactivated intrinsic apoptotic pathway inrats Histochem Cell Biol “ httpsdoi101007s004180171544x MartinezIglesias O GarciaSilva S Regadera J Aranda A Hypothyroidismenhances tumor invasiveness and metastasis development PLoS One 47e6428 httpsdoi101371journalpone0006428 Nogueira CR Brentani MM Triiodothyronine mimics the effects of estrogenin breast cancer cell lines J Steroid Biochem Mol Biol ““httpsdoi101016s0960076096001173 Alyusuf RH Matouq JA Taha S Wazir JF The pattern of expression and roleof triiodothyronine T3 receptors and type I ²deiodinase in breastcarcinomas benign breast diseases lactational change and normal breastepithelium Appl Immunohistochem Mol Morphol “httpsdoi101097PAI0b013e3182a20917 Pereira B Rosa LF Safi DA Bechara EJ Curi R Control of superoxidedismutase catalase and glutathione peroxidase activities in rat lymphoidans by thyroid hormones J Endocrinol “ httpsdoi101677joe01400073 GonzalezSancho JM Figueroa A LopezBarahona M Lopez E Beug HMunoz A Inhibition of proliferation and expression of T1 and cyclin D1genes by thyroid hormone in mammary epithelial cells Mol Carcinog “ httpsdoi101002mc10046Tosovic A Bondeson AG Bondeson L Ericsson UB Malm J Manjer JProspectively measured triiodothyronine levels are positively associatedwith breast cancer risk in postmenopausal women Breast Cancer Res 123R33 httpsdoi101186bcr2587Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

Thyroid_Cancer

" IBDFecal calprotectinEndoscopic activityIBD noninvasive managementThe term IBD is usually used for referring to a group of ‚ammatory gastrointestinal diseases mainly Crohn'sdisease and ulcerative colitis Accordingly IBD arises as a result of inappropriate immune response to intestinalcommensal anisms among genetically susceptible individuals Performing colonoscopy and histopathologicevaluation on an ‚amed bowel biopsy specimen are currently considered as gold standards for diagnosis andmanagement of IBD Correspondingly these techniques are known to be invasive and costly In recent decadesfecal calprotectin as a biomarker has received much attention for the diagnosis and noninvasive managementof IBD Up to now many studies have investigated the efficacy of fecal calprotectin in the areas of IBD diï¬erentiation from IBS prediction of endoscopic and histologic activities of IBD and prediction of disease recurrenceAlthough some of these studies have reported promising results some others have shown significant limitationsTherefore in this paper we reviewed the most interesting ones of these studies after a brief discussion of thelaboratory measurement of fecal calprotectin Moreover we attempted to provide an answer for the question ofwhether fecalcalprotectin could be considered as a potential surrogate marker for colonoscopy IntroductionInflammatory bowel disease IBD is a long life disease with remission and relapse periods IBD arises as a result of inappropriateimmune response to intestinal commensal anisms in individualswith genetic predisposition and consequently causes ‚ammation andintestinal ulcers [] In addition IBD has a complex pathogenesis andmany factors such as dysbiosis oxidative stress and epigenetics thatmay also be involved in disease pathogenesis [“] Ulcerative colitisUC and Crohn's diseases CD are known as two main forms of IBDAccordingly these diseases cause intestinal ulcers and some annoyingsymptoms such as diarrhea abdominal pain and rectal bleeding Occasionally the severity of these symptoms is very high which can leadpatients to be hospitalized In this regard therapeutic approaches totreat these diseases mainly focus on prolonging remission and are almost similar however diï¬erential diagnosis can also help to treat thedisease in a more eï¬ective way For example 5ASA which is acommon drug in the treatment of IBD is less eï¬ective on maintainingremission in CD patients On the other hand antibiotic therapy is notrecommended for the treatment UC but it can be eï¬ective on CD patients [][] Diï¬erential diagnosis is a serious challenge because CDand UC have significant similarities in terms of their clinical endoscopic and histological features However there are some diï¬erencesbetween UC and CD which are summarized in Table1 In addition tointestinal complications UC and CD also have significant extraintestinal manifestations For example it was shown that UC is significantly associated with Primary sclerosing cholangitis and CD is alsoassociated with cholelithiasis especially in cases that the ileum is involved [] Furthermore CD can cause fistulas to the urinary systemwhich leads intestinal bacteria to enter the urethra and recurrent urinary tract infections [] Both CD and UC can cause several disorderssuch as arthritis Erythema nodosum pyoderma gangrenosum andanemia which are known as the most important extraintestinal manifestations of IBD [][] The latest statistics showed that the globalŽ Corresponding author at Department of Clinical Biochemistry and Laboratory Medicine Faculty of Medicine Tabriz University of Medical Sciences DaneshgahStreet PO Box Tabriz IranEmail address vagharimtbzmedacir M VaghariTabari101016jcca202008025Received July Received in revised form August Accepted August Available online August Elsevier BV All rights reserved 0cF KhakiKhatibi et alTable1Clinical endoscopic and histological features of CD and UCClinical FeaturesFeaturesRectal bleedingAbdominal painFeverMucus defectionIntestinal obstructionPerineal diseasePostoperative recurrenceASCA positiveANCA positiveEndoscopic FeaturesCDOccasionallyFrequentlyFrequentlyOccasionallyYESYESYESFrequentlyNot commonUCFrequentlyOccasionallyNot commonFrequentlyNONONONot commonFrequentlyFeaturesCDUCLocationMucosal involvementDepth of ulcerationfistulaCobblestone appearanceAphthous ulcerationMucosal friabilityHistological featuresFeaturesGranulomasCrypt abscessesPatchinessAny part of GI tractDiscontinuousDeepYesYESFrequentlyNot commonCDFrequentlyNot commonFrequentlyColon and rectumContinuoussuperficialNONOOccasionallyFrequentlyUCRareFrequentlyNot commonprevalence of IBD currently is on the rise and it is not an exaggerationif we consider it as a global serious health problem [] According to areport published in IBD has the highest prevalence rate inEurope and its prevalence in the newly industrialized countries of AsiaAfrica and South America also appears to be increased over the pastthree decades []Unfortunately the peak of the disease is at the young age of“ years old [] therefore in addition to the suï¬ering from ‚icts on the patients it also has many negative eï¬ects on societyMoreover many financial burdens are annually imposed on countriesfor controlling and treating this chronic disease The invasive diagnosticand therapeutic measures are currently undertaken to diagnose andmanage IBD which are unpleasant for patients as well as having thehigh associated costs Now the gold standard method for diagnosingIBD and monitoring patient status is performing colonoscopy examination and histopathologic evaluation which are invasive measures[] Therefore in recent years many studies have been conducted tofind a suitable laboratory marker with sufficient sensitivity and specificity for the purpose of diagnosing and noninvasive management ofIBD A high proportion of these studies have investigated the efficacy offecal calprotectin in diagnosing and monitoring patients Althoughsome of these studies reported auspicious results there are still somedoubts on the eï¬ectiveness of fecal calprotectin on diagnosing andmonitoring IBD patients So in this review we addressed the advantages and limitations of fecal calprotectin for the diagnosis andmanagement of IBD The role of fecal calprotectin in diagnosis and management ofIBDThe efficacy of fecal calprotectin as an laboratory marker in various areas of IBD diagnosis and management have been studied including IBD diï¬erentiation from irritable bowel syndrome IBS evaluation of endoscopic activity of the disease evaluation of histologicalactivity of the disease and prediction of disease recurrence andClinica Chimica Acta “response to treatment In following after a brief introduction andmentioning the important points regarding laboratory measurement offecal calprotectin we reviewed the most interesting findings in all ofthe abovementioned areas Calprotectin A clinically valuable proteinCalprotectin is an antimicrobial protein mainly secreted by neutrophils This protein competes with bacteria over zinc thus kills thebacteria However this is not the only contribution that it has to antimicrobial activity Moreover this protein has many potential clinicalapplications such as the elevated serum levels that have been observedunder various immunological and immunopathological conditionsSerum calprotectin levels rapidly increase in response to bacterial infections in the kidney and heart or during transplant rejection At theearly stages of ‚ammation of the lung serum calprotectin can also beconsidered as a reliable marker besides plasma levels of calprotectinappear to be useful in reflecting disease activity in ‚ammation of thejoints [] In addition it has been demonstrated that serum calprotectin levels are increased in patients with bacterial sepsis so it can beconsidered as a reliable biomarker [] In Neonatal Sepsis the serumlevel of calprotectin increases as well as a sensitivity of and aspecificity of that have been reported for serum calprotectin indiagnosis of Neonatal Sepsis [] It has been recently shown thatserum calprotectin levels increase in patients with aneurysmal subarachnoid hemorrhage and higher levels in the first of onset areassociated with a poor prognosis at the first three months [] Serumcalprotectin levels also increase in patients with rheumatoid arthritisand even in patients with a moderate to high disease activity who havenormal or low CRP levels so they appear to be more efficient at reflecting disease activity []Some studies have also investigated the efficacy of serum calprotectin in the diagnosis of cancers Correspondingly in one of thesestudies it was shown that serum calprotectin levels significantly increased in patients with laryngeal carcinoma compared with healthyindividuals and those with benign laryngeal pathologies Moreover inthis study a direct relationship was also observed between serum levelsof calprotectin and stage of cancer [] Another study showed that theserum level of calprotectin increased in patients with papillary thyroidcarcinoma but it significantly decreased after operation [] Alsoregarding the efficacy of serum and saliva calprotectin for the diagnosisof IBD impressive results have been reported [][] A study onpatients with IBD both UC and CD have shown that serum calprotectinlevels were directly correlated with fecal calprotectin levels and weremore potent in IBD diagnosis compared to CRP and albumin This studyalso indicated that the combination of serum calprotectin with CRP oralbumin can be helpfulin the prediction of treatment escalationespecially in patients with CD [] However no significant correlationwas observed between serum calprotectin and fecal calprotectin levelsin patients with CD and UC as well as a slight correlation betweenserum calprotectin level and CRP that was observed only in patientswith UC [] Another study showed that the serum level of calprotectin was significantly higher in patients with CD compared to healthyindividuals In addition although a significant correlation was observedwith the clinical activity of the disease no significant correlation wasfound between the level serum calprotectin and endoscopic activity ofthe disease [] The efficacy of salivary calprotectin in the diagnosisof IBD has also been studied which showed that salivary calprotectinsignificantly increased in patients with IBD compared to healthy individuals In this study AUC values for unstimulated saliva and stimulated saliva to distinguish IBD patients from healthy individualswere reported to be and respectively [] However thepopularity of calprotectin is mainly due to the use of fecal calprotectinin the diagnosis and management of IBD that is discussed in the following 0cF KhakiKhatibi et alClinica Chimica Acta “ Laboratory measurement and reference intervalFecal calprotectin is a stable protein that remains stable for “ daysat room temperature [] This property is an excellent advantage for alaboratory marker Also it seems that keeping the specimen at refrigerated temperature °C can increase the stability of fecal calprotectin [] However evidence has been obtained regarding thatthe stability of this protein decreases after staying for three days atroom temperature On the other hand it is not also recommended tokeep samples in the refrigerator for more than days [] It seemsthat fecal calprotectin remains stable up to one year at ˆ’ °C []Measurement of fecal calprotectin can be done both qualitatively andquantitatively Accordingly in the qualitative measurement monoclonal antibodies are used to detect fecal calprotectin and the positiveresults are characterized by the appearance of colored lines on the testcassette However in the qualitative one only positive or negative results are reported and despite of sensitivity test specificity in theevaluation of disease activity was reported to be only It seems thatthe main application of this test is to diï¬erentiate healthy individualsfrom IBD patients rapidly however some studies have shown that it isnot accurate enough in this case as well [][] Nevertheless asignificant concordance has been reported between home test resultsIBDoc and fecal calprotectin laboratory measurement results whenQuantum Blue calprotectin ELISA kit was used Notably the agreements between results were and depending on the selectedcutoï¬s [] Several commercial kits are also available for fecal calprotectin qualitative test known as rapid calprotectin These tests reportpositive results ranged from to µgg There are also severalcommercial kits that can be used for the quantitative measurement offecal calprotectin These kits are usually designed in terms of the ELISAmethod and some have a measurement range between and µgg Moreover the chemiluminescence immunoassays CLIAmethod can also detect values between and µgg Fluoro enzyme immunoassays FEIA and particle enhanced turbidimetric immunoassays PETIA can also be used for the measurement of fecalcalprotectin In this regard one of the most serious challenges to thelaboratory evaluation of fecal calprotectin is the determination of theupper limit in healthy individuals Among healthy adults there is asignificant agreement on µgg as an upper limit One study suggested values up to µgg in people over years old and up to µgg in children aged between and years old as referenceranges of fecal calprotectin in healthy individuals []Fecal calprotectin levels appear to be higher in healthy infants andchildren under four years old than in adults and further studies areneeded in this regard to determine the acceptable upper limit for diagnosis of pediatric IBD [] Table lists the median levels of fecalcalprotectin in healthy individuals with diï¬erent ages reported in somestudies According to these reports age can aï¬ect fecal calprotectinlevels Fecal calprotectin and IBD diagnosisOnly a small percentage of patients complaining of abdominal painand diarrhea have IBD In many cases IBS as a functional gastrointestinal disorder is known as the cause of such clinical symptomsPatients with IBS have normal colonoscopy results while IBD patientsindicate abnormal colonoscopy results and have intestinal ulcersUnfortunately the significant prevalence of IBS and the overlap between clinical symptoms and IBD can increase the colonoscopy rateTherefore a noninvasive diagnostic marker can be very helpful in thisregard Notably the first evidence of the efficacy of fecal calprotectin inthe diagnosis of IBD was obtained in the 1990s Røseth et al in proposed a method for measuring Calprotectin in stool specimens []One of the first and most interesting studies regarding fecal calprotectinutility in IBD diagnosis was the study by Røseth et al published in In this study patients with ulcerative colitis were studied and according to their results fecal calprotectin levels are higher in patientswith ulcerative colitis compared to healthy controls This study havealso shown that even patients with low disease activities had higherlevels of fecal calprotectin compared to healthy individuals []Subsequent studies somehow confirmed and complemented the findings of this study In another study published in AUC values of CI “ were reported for fecal calprotectin in thediagnosis of colorectal ‚ammation [] Moreover in a study onchildren with IBD it was shown that the level of fecal calprotectin washigher in these patients compared to healthy children so it can beconcluded that it is also directly correlated with ESR levels [] In astudy published in Kolho et al reported AUC values of CI “ for fecal calprotectin in the diagnosis of pediatric IBD [] In a study on patients with Crohn disease a sensitivity of and a specificity of at cutoï¬ of μgg have been reportedfor fecal calprotectin in diagnosis of the disease [] The results of ourrecent study along with other studies showed that fecal calprotectin ispreferred over traditional ‚ammatory biomarkers such as CRP andESR in the diagnosis of IBD [][] Diamanti et al reported a sensitivity of and a specificity of for fecal calprotectin at a cutoï¬ of μgg in IBD diagnosis [] In our recent study a sensitivityof and a specificity of at a cutoï¬ of μgg were observed for fecal calprotectin in the diagnosis of IBD however oursample size was and the majority of patients were in the active phaseof the disease []In another study conducted on patients with ulcerative colitis asensitivity of and a specificity of at cutoï¬ of μgg havebeen reported in this regard [] In one study it was shown that fecalcalprotectin in cutoï¬ of μgg is able to distinguish patients withIBD from patients without IBD patients with diseases other than IBDpatients with IBS and healthy persons with sensitivity and specificity [] Caviglia et al in their study reported a sensitivity of and a specificity of at a cutoï¬ of μgg for fecalcalprotectin in diï¬erentiating between IBS and IBD [] Howeversome studies have reported significantly lower values Accordingly in astudy on patients with ulcerative colitis Kalantari et al reported asensitivity of and a specificity of at a cutoï¬ of μgg []Besides there is a considerable agreement between fecal calprotectinand capsule endoscopy findings in patients with Crohn's disease Asensitivity of and a specificity of have also been reported at acutoï¬ of mgkg for fecal calprotectin in predicting CE findings anddiagnosis of Crohn's disease [] In another study lower sensitivityand specificity rates sensitivity specificity were reportedfor fecal calprotectin in this regard [] Furthermore in one studythat examined the efficacy of fecal calprotectin in predicting wirelesscapsule endoscopy findings a sensitivity of and a specificity ofTable Reported median levels of fecal calprotectin in healthy individuals of diï¬erent agesAgesMedian levels of fecal calprotectin range µggNumber of subjectsUsed kitUp to monthChildren “ yearsChildren “ yearsAdultsOver years “ “ “ “ “Bühlmann ELISABühlmann ELISACALPRO® Calprotectin ELISA Test ALPPhiCalPhicalReference[][][][][] 0cF KhakiKhatibi et al were reported for this biomarker at μgg in the diagnosis ofsmall bowel ‚ammation in Crohn's disease [] Given these findings it seems that fecal calprotectin has no ideal sensitivity and specificity for the diagnosis of IBD where the small intestine is involvedBesides there are some preanalytical limitations which are explainedin the next sections Therefore optimistically speaking fecal calprotectin measurement can eliminate the need for colonoscopy Howeverin a metaanalysis performed to evaluate the efficacy of fecal calprotectin and some other ‚ammatory markers to diï¬erentiate betweenIBD and IBS the probability of IBD was less than at fecal calprotectin values lower than µgg or CRP values lower than mgdL[] Therefore it seems that fecal calprotectin can be helpful at leastin ruling out the possibility of IBD in patients with IBSlike symptoms aswell as reducing the rate of colonoscopy Moreover it should be notedthat although a systematic review has reported pooled sensitivity andspecificity above for fecal calprotectin to diï¬erentiate between IBDand IBS it emphasized more on the possibility of falsepositive resultsin low cutoï¬ points [] Hence performing extensive studies indiï¬erent countries on the healthy population and the IBD patient is beneeded to determine a suitable cutoï¬ with maximum sensitivity andspecificity and minimum falsepositive resultsTable summarizes the results of various clinical investigationsregarding fecal calprotectin utility in the diï¬erential diagnosis of IBDfrom IBS and Table4 summarizes some metaanalysis results in thisregard As shown in Table the most important limitation of the majority of clinical studies conducted to date is the small sample size Alarge global study may be helpful in providing a more precise evaluation of fecal calprotectin clinical value in discrimination between IBDand nonIBD diseases Fecal calprotectin and endoscopic and histologic activity evaluationUndoubtedly one of the most serious challenges in the managementof IBD is evaluating the endoscopic and histologic activities of thedisease Nowadays colonoscopy and histopathologic examinations arethe routine tools for the assessment of mucosal healing in patients withIBD As noted earlier several scoring systems have been devised toscore disease activity based on the findings of colonoscopy and histopathologic examinations In recent years many promising results havebeen reported regarding the correlation between these scores and fecalcalprotectin levels In addition many studies have been performed inthe last decade all of which cannot be reviewed in this article The firstevidence of a link between fecal calprotectin and disease endoscopicactivity was obtained in the late 1990s In one of the first studiesRoseth et al found a significant correlation between fecal calprotectinlevels and endoscopic and histologic activities in patients with ulcerative colitis [] Furthermore in another study they observed that IBDpatients who were in remission clinically and had normal fecal calprotectin levels less than mgL had normal colonoscopy results[] These interesting findings indicate that fecal calprotectin can beconsidered as a biomarker in the evaluation of endoscopic activity andClinica Chimica Acta “Table4summarized results of some metaanalysis regarding the utility of fecal calprotectin in discrimination between patients with IBD and without IBDSample sizePooled SensitivityPooled SpecificityReferences[][][][][]mucosal healing in IBD patients Also these studies were the startingpoint of extensive studies that have been conducted up to now In astudy conducted on patients with Crohn's disease Sipponen et alinvestigated the sensitivity and specificity of fecal calprotectin in predicting endoscopic activity of Crohn's disease [] Correspondinglythe researchers used the Crohn's Disease Endoscopic Index of SeverityCDEIS scoring system in their study to evaluate the endoscopic activity of Crohn's disease As a result they found that there was a significant correlation between the endoscopic activity of the disease andthe level of fecal calprotectin Besides the findings of this study demonstrated that fecal calprotectin at µgg cutoï¬ can predict theendoscopic activity of Crohn's disease with sensitivity and specificity In another study CDEIS and Mayo Disease Activity IndexMDAI were used to evaluate the endoscopic activity of Crohn's diseaseand ulcerative colitis respectively According to the results of thatstudy on IBD patients there was a significant correlation between fecalcalprotectin levels and disease endoscopic activity [] Another studyshowed that fecal calprotectin is more strongly correlated with theendoscopic activity of the disease in ulcerative colitis compared to theRachmilewitz clinical activity index In addition in this study theoverall accuracy of fecal calprotectin for endoscopically active diseaseidentification was obtained as []Some studies have also shown the superiority of fecal calprotectinover traditional ‚ammatory markers like CRP Besides one studyfound that fecal calprotectin was more strongly correlated with theSimple Endoscopic Score for Crohn's disease SESCD compared to theCRP and even Crohn's disease activity index CDAI [] The modifiedBaron Index was also used in another study to evaluate the endoscopicactivity of ulcerative colitis As a result it was shown that calprotectinis more strongly correlated with the endoscopic activity of ulcerativecolitis compared to CRP and clinical activity of the disease [] In thisregard similar results were also observed in our recent study in whichthe Ulcerative Colitis Endoscopic Index of Severity UCEIS and SESCDwere used [] Therefore fecal calprotectin appears to be superior totraditional ‚ammatory markers in the prediction of IBD endoscopicactivity The high values of sensitivity and specificity that were mentioned earlier have raised the hope that using fecal calprotectin canreduce colonoscopy rate for patients™ monitoring However severalrecent studies have reported some significantly lower values Accordingly in a recent study in which Mayo Endoscopic Score [MES] wasused to evaluate the endoscopic activity of ulcerative colitis aTable Summary of the results of some studies regarding the utility of fecal calprotectin in discrimination between patients with IBD and without IBDNumber of IBD patientsAge groupLocationCut oï¬SensitivitySpecificity CD and UC CD and UC CD and UC and unclassified68CD and UC CD and UC and unclassified CD and UC CD and UC UC CD UCAdultsAdultsAdultsBoth adult and pediatricpediatricAdultspediatricAdultsAdultsBoth adult and pediatricTaiwanChinaItalySpainFinlandIranItalyIranDenmarkIndia48µgg µgg150µgg150µgg595µgg784µgg160µgg164µgg150µgg188µggAUCReferences[][][]SPSrefidbib60[][][][][][][] 0cF KhakiKhatibi et alClinica Chimica Acta “Table Summary of the results of some studies regarding the correlation of fecal calprotectin with endoscopic activity in IBD patientsAge groupStudylocationUsedendoscopicactivity indexCorrelationcoefficientrReferenceNumberof IBDpatients CD UC UC CD UCAdultsAdultsAdultsAdultsAdultsFinlandIranSwitzerlandSwitzerlandSwitzerland ModifiedCDEISUCEISRachmilewitzSESCD UC CDAdultsAdults UC CD UC CD CD UC UCAdultsAdultsAdultsAdultsAdultsAdultsAdultsBaron ScoreRachmilewitzSESCDGermanyUSA andCanadaJapanItalyItalyBrazilFranceFranceSouth Korea UCEISMattsSESCDMayo scoreSESCDCDEISMayo score[][][][][][][][][][][][][][]sensitivity of and a specificity of were reported for fecalcalprotectin at µgg to diï¬erentiate active endoscopic from inactiveMES or from MES or [] In another study the sensitivityand specificity of fecal calprotectin at a cutoï¬ of µgg for diï¬erentiating MES ‰¤ in patients with ulcerative colitis were and respectively [] Overall as presented in Table several studiesperformed in diï¬erent countries reported the correlation between fecalcalprotectin and IBD endoscopic activity Although some of these studies reported a strong correlation some others reported a relativelyweak correlation As noted earlier there are significant diï¬erencesbetween the reports on the sensitivity and specificity of fecal calprotectin to predict the endoscopic activity of IBD Undoubtedly a widerange of factors from sample size and the inclusionexclusion criteriato preanalysis variables and indexes used to evaluate the endoscopicactivity may also contribute to these diï¬erences However fecal calprotectin does not appear to be a very reliable marker for the predictionof IBD endoscopic activity so currently it seems a bit optimistic toconsider fecal calprotectin as a reliable alternative for colonoscopy Inthis regard further studies are still needed However under some certain circumstances such as pregnancy or pandemics the use of fecalcalprotectin to evaluate IBD endoscopic activity can be helpfulPregnant patients with IBD have serious limitations for colonoscopyexamination and it has been recommended that colonoscopy should beonly performed in the second trimester of pregnancy and where there isa strong indication [] Therefore noninvasive markers such as fecalcalprotectin can be helpful during pregnancy In one study physicianglobal assessment [PGA] which is a clinical symptombased criterionwas used to evaluate IBD activity and subsequently the associationbetween fecal calprotectin and this criterion was investigated in pregnant women with IBD The results of this study showed a significantcorrelation between fecal calprotectin and PGA levels at prepregnancyduring pregnancy and postpartum stages [] In another study asignificant association was reported between fecal calprotectin levelsand clinical activity of IBD in pregnant women Moreover it was shownthat stool calprotectin at a cutoï¬ of mgkg had a sensitivity between and as well as a specificity between and in the assessment of IBD clinical activity at diï¬erent stages ofpregnancy [] A recently published systematic review has also confirmed the conclusions obtained from these studies [] According tothese results it seems that fecal calprotectin is not aï¬ected by physiological changes during pregnancy however it is significantly correlatedwith IBD clinical activity during pregnancy Therefore from the viewpoint of relatively acceptable sensitivity and specificity in predictingthe endoscopic activity of IBD fecal calprotectin may be considered as anoninvasive biomarker for the evaluation of IBD endoscopic activity inpregnant women In addition under pandemic conditions fecal calprotectin can be very helpful Following the COVID19 pandemicwhich began in late and is still ongoing healthcare systems indiï¬erent countries were forced to impose significant limitations oncolonoscopy Therefore noninvasive IBD management and fecal calprotectin as a noninvasive laboratory marker have become moreimportant than before The combination between disease clinical activity and fecal calprotectin has been recommended as a noninvasiveapproach that can help in making decisions on treatment duringCOVID19 pandemic [] Therefore it seems that fecal calprotectincan be considered as an alternative for colonoscopy used for IBD endoscopic activity evaluation during pandemic Fecal calprotectin appears to be associated with IBD histologic activity as well Given thedifficulty in the evaluation of the histologic activity of Crohn's disease[] some studies have been focused on the ulcerative colitis andmany scoring systems have been devised so far Correspondingly thesesystems score the disease's histologic activity based on histologic observationsTherefore for this purpose a biopsy of the intestinal tissue is required which can be prepared by colonoscopy and then sent to thelaboratory In this regard one of these histologic scoring systems isRobert™s score that was used in one of our recent studies where weobserved a significant correlation between the level of fecal calprotectinand the histologic activity of ulcerative colitis which was calculatedbased on the Robert™s scoring system [] Theede et al also used themodified Harpaz Index and performed some interesting studies in thisregard In one of their studies fecal calprotectin was found to be significantly associated with the histologic activity of the ulcerative colitisand it was shown that it could predict histological mucosal healingAUC CI95 “ Sensitivity Specificity andCutoï¬ mgkg [] In another study on patients with endoscopically inactive ulcerative colitis Mayo endoscopic score the researchers showed that patients with ulcerative colitis who were inendoscopic remission but had histologically active disease had higherlevels of fecal calprotectin compared to patients with no histologicallyactive disease versus mgkg P Also despite thehigh specificity the sensitivity of fecal calprotectin in theprediction of score of histological activity was achieved as at mgkg [] In a recent study the Geboes

Thyroid_Cancer

"Oral cancer is one of the most common noncommunicable diseases worldwide This paper presentsan evaluation of the trends and geographical distributions of oral cancers in the Saudi Arabian populationMethods Data from Saudi Cancer Registry reports were used in this analysis which assessed the period between and All cancer cases are recorded in these reports as well as the age gender region and histologicalcancer sites for each patient Agestandardised and agespecific incidence rates were calculated in these reportsFor the purposes of this paper only cancers of the lips tongue and mouth were considered oral cancersResults Between and the Saudi Cancer Registry identified cancer cases in total Of these were oral cancer The mean agestandardised rate of oral cancer for the study period was per peoplefor females it was and for males it was The incidence of oral cancer varied by region with Jazan displayingthe highest agestandardised rate and Hail displaying the lowest A positive correlation was observed between oralcancer incidence and ageConclusion The overall trend of the agestandardised rate for both sexes remained constant from to However the oral cancer incidence in Saudi Arabia varies by region Studying this variation in more detail will helpto guide awareness programmes in the regions that are most in needKeywords Cancer epidemiology Cancer prevention and control Oral neoplasmBackgroundCancer is an intractable global health problem and theleading cause of death in the developed world in the developing worldit is the secondleading cause [] In the most recent year for which information fromthe International Agency for Research on Cancer IARCis available approximately million new cancer caseswere diagnosed and million people died from cancerworldwide [] In this same year new cases oflip and oral cavity cancers were reported representing of all cancer casesA review of the global prevalence of oral cancer revealsa wide variation in distribution among countries []Correspondence bmalshehrinuedusaDepartment of Clinical Laboratory Faculty of applied Medical SciencesNajran University PO Box Najran Kingdom of Saudi ArabiaTwothirds of the estimated incidence of oral cancer occurred in developing countries with up to of allnew oral cancer cases in Sri Lanka India Pakistan andBangladesh [] Converselyin France which has thehighest rate of oral cancer incidence in the EuropeanUnion only oral cancer cases were reported in representing just of all cancer cases [] Inthe USA the American Cancer Society estimated that in approximately people were diagnosed withoral cavity or oropharyngeal cancer and will dieof these cancers [] In Arab countries the prevalence oforal cancer is concentrated between western and southeast Asia [] While this type of cancer is relatively uncommon across Arab gulf countries Saudi Arabia andYemen are notable exceptions [] No studies have beenpublished discussing the epidemiological parameters andgeographic distribution of oral cancer cases or any of its The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cAlshehri World Journal of Surgical Oncology Page of subtypes in the Saudi Arabian population Thereforethis study analysed and discussed oral cancer trends inthe Saudi population by using the most recent dataavailableAccording to the International Classification of Diseases 10th revision ICD10 oral cancer is classifiedinto six sites mucosal lip ICD10 C00 tongue ICD C02 gum ICD10 C03 mouth floor ICD10C04 palate ICD10 C05 and mouth ICD10 C06However examining trends in oral cancer incidencerates that include all oral sites can be misleading Thedata analysed in this study only include cancers of thelip tongue and mouth ICD10C00“C06 which formthe majority of oral cancers moreover they have severalrisk factors in common and share a similar biology []Thus those accounting for a minority of oral cancercases were excludedMaterials and methodsDataThis retrospective descriptive epidemiological study analysed oral cancer cases in a Saudi population that hadbeen diagnosed from January through December The study used a method of analysis similar tothat used by Alshehri [] Their analyses incorporated male and female data on lip tongue and mouthICD10C00“C06 cancer cases to evaluate disease patterns in the Saudi population Data for the present studywere obtained from the Saudi Cancer Registry SCR apopulationbased registry established in by theMinistry of Health in Saudi Arabia This data can onlybe obtained from the reports published by the SCRSince the SCR has been publishing reports oncancer in Saudi Arabia with the primary objective of defining populationbased cancer incidences The presentstudy was conducted using these reports to derive a descriptive epidemiology of oral cancer in Saudi Arabia InSCR reports agestandardised ASR and agespecificAIR incidence rates were calculated with a focus ongenderspecific and regional differencesThe analysis included cases recorded in the SCR filesfrom January to December totalling cancer cases overall approximately of which wereoral cancerData analysisThe GraphPad Prism6 software was used to analyse thedata Descriptive analyses of epidemiological data wereconducted by calculating the mean of the percentagesand ASR stratified by age sex region and year of diagnosis The ASR was calculated in the SCR reports byadjusting all Saudi regions™ populations mathematicallyto have the same age structure On the other hand theAIR was calculated by summation of the number ofcancer cases occurring during the year in a region™spopulation among specific age and sex groups dividedby the midyear population of these age and sex groupsUsing these two standardised rates is important because age is a basic element of the risk of developingcancer globally [] Using summary measure tools suchas the ASR and AIR which represent the schedule ofagespecific rates in different regions and across timewill give us a more representative picture of the characteristics in question and enable comparisons of cancerincidences between several populations of Saudi regionsthat differ with respect to ageResultsIncrease in the number of oral cancer casesThe total number of cancer cases identified by the SCRfrom to was with males and females Of this total cases were oral cancer The number of registeredoral cancer cases increased gradually from MF in to a peak of MF in however only cases were reported in MF Table Table Number of oral cancer cases in Saudi Arabia for theperiod from to YearNumber of female casesNumber of male casesTotal 0cAlshehri World Journal of Surgical Oncology Page of The percentage of cases representing oral cancers was for females and for males Fig in These percentages decreased to for females and for males in Fig The percentage curve fororal cancer out of all cancer types for males and femalescorrelated with increases and decreases over the studyperiod apart from the years and Fig ASR of oral cancer fluctuated over the study periodBetween and the ASR per male casesfluctuated in it was trending downwards to alow of in and peaking at in beforedropping again to in Fig The female ASRper increased from in to a peak of in decreasing again to in Fig For bothsexes ASR curves like oral cancer percentages correlateto increases and decreases over the study period apartfrom the years and Fig and generally remained constant from to ASR of oral cancer varies by regionThe ASR data for oral cancer cases of all persons demonstrated a wide variation across Saudi regions TheASR means per people for the period from to ranged from in Hail to in Jazan with anational average of per Fig The Jazan region had the highest male ASR mean at followed by the Najran and Tabuk regions at each Fig Conversely Qassim Baha Hail and theNorthern province reported the lowest ASR averages at and per respectively Fig Male and female ASR data were generally equivalentin terms of region rankings with the Jazan region posting the highest overall ASR of as an average valueof both genders followed by the Makkah region at and the Najran region at Fig Similarly the HailBaha Qassim and Madinah regions posted the lowestASR averages at and respectively FigAIR of oral cancer increases with ageThe AIR data from to showed a positive correlation between oral cancer incidence and age withmost cancer cases occurring in the older age groups Figure shows the AIR of oral cancer increasing noticeablywith age up until age More than of cases werediagnosed after the age of Some AIR differences were found between the sexesacross age groups From ages to rates of oral cancer were higher in females than in males however thistrend had reversed to favour males in the 75andoverage group The overall AIR per showed onlyslight differences between the sexes at for femalesand for males Fig DiscussionA review of oral cancer data in Saudi Arabia for theperiod from to showed an overall increasingtrend in the numbers of oral cancer patients Despitethis rise ASR data trends for oral cancer remained constant from to Fig This curve stabilised inthe face of a substantial Saudi Arabian population increase from million in to million in [] Many accumulative factors could be contributed tothis stability First the significant increased access tohealth services in Saudi regions has contributed to thedissemination of oral health awareness and early diagnosis of some cases of metaplasia that were discovered before they could develop into cancerous tumours SecondFig Consistency in percentage curves for oral cancer out of all cancer types from to The percentage curve for oral cancer out of allcancer types for males and females are correlated with overall increases and decreases over the period from to with the exception ofyears and 0cAlshehri World Journal of Surgical Oncology Page of Fig Agestandardised incidence rates ASR of oral cancer fluctuated over the study period Between and the male ASR was per in and dropped to in The female ASR fluctuated between and per the increased level of public health in the Kingdom isusually linked to an increase in the economic level of thecountry and individuals may have contributed to thisconstancy as many infectious factors such as virusesand fungi have been linked to oral cancers Third SaudiArabia is a majority Islamic country wherein many oralcancer risk factors such as alcohol consumption andcigarette smoking are forbidden by Islamic law Islamiclaw may thus mediate the lower number of oral cancercases in Saudi Arabia compared to the rest of the world[] Thus based on the IARC data for eight ofthe nine world regions whose ASR of oral cancer isabove the global rate [ ] were located in nonMuslimcountries [] with Melanesian regions having the highest rate [] In contrast most of the regions locatedwithin Muslim countries were ranked below the globalASR [] Further investigation of this aspect could therefore be valuable to cancer prevention effortsThe ASR data revealed that more females than maleswere diagnosed with oral cancer in Saudi Arabia at for men and for women This finding is in contrastwith global data showing that men are more likely to develop oral cancer than women [] In the most recent year for which IARC information is available theglobal ASR of oral cancer was for men and forwomen [] While these rates do not match the globalFig Agespecific incidence rates AIR of oral cancer increases with age The total AIR of oral cancer increased noticeably with up until patientswere and over More than of the cases were diagnosed after the age of 0cAlshehri World Journal of Surgical Oncology Page of Fig Agestandardised incidence rates ASR of oral cancer varies by region in Saudi Arabia For all persons the ASR means per peoplefor the period from to ranged from in the Hail region to in the Jazan regionsex distribution oral cancer in Saudi Arabia has a relatively low overall ASR when compared to the globalaverage as discussed aboveResults also revealed consistency in the ASR oral cancer curve for both sexes Fig potentially due to thepresence of common risk factors for oral cancer in malesand females This finding could be used as a startingthreshold for studying the risk factors of oral cancer inthe Saudi population through studying the common factors between the sexesAs with many other types of cancer the present studyfound a correlation between the occurrence of oral cancer and age with of cases diagnosed after the age of years In the USA the average age at diagnosis of oralcancer is years and twothirds of individuals with thisdisease are over the age of [] Ageing is accompaniedby increased susceptibility to cancercausing geneticmaturations due to accumulated exposures to environmental and behavioural risk factors Avoiding these riskfactors could greatly reduce the role that ageing plays incancerThis study found a wide variation in the incidence oforal cancers among Saudi regions Such differencescould indicate that regional environmental factors andlifestyle habits affect oral cancer incidence The resultsreviewed above found that the Jazan region possessedthe highest ASR of people with oral cancer In contrastthe Northern province presented the lowest ASR Severalstudies have focused on investigating why the Jazan region has such a high incidence of oral cancer [“]Ibrahim and others focused on the association ofcertain eating habits and lifestyle behaviours with the development of oral cancer especially the abuse ofshamma a form of smokeless tobacco and the chewingof khat Catha edulis leaves These substances havebeen classified as carcinogens especially in relation tooral cancer Studies by these researchers found that consuming shamma increased the odds of developing oralcancer 29fold suggesting a strong link between oralcancer and diet and lifestyle choicesAccording to the above poor dietary habits related totobacco use and its derivatives are one of the main reasons for the high incidence of oral cancer in some citiesand not others Other factors such as variations in thegenetic background of the Saudi regions™ citizens cannotbe excluded especially because most of the populationin the Kingdom™s regions is tribal so consanguineousmarriages are highly common Thus genomic sequencing can provide information on genetic variants thatmay be present in citizens of these regions and that maybe linked with increased or decreased rates of oral cancer development Populationbased genetic testing issuggestedConclusionDespite the presence of yeartoyear changes in the incidence of oral cancer in the Saudi population there wasoverall no noticeable change in the incidence of oralcancer in the Saudi Arabian population for the periodbetween and In contrast to some international findings females were somewhat more likelythan males to be diagnosed with oral cancer in SaudiArabia The positive correlation between ageing and theincidence of oral cancer for both males and femalesdemonstrates that oral cancer is mainly a disease of theelderly both in Saudi Arabia and across the globe Thewide variation in the incidence rates among Saudi regions raises an important research question concerning 0cAlshehri World Journal of Surgical Oncology Page of World Bank World Development Indicators Washington DC WorldBank Access online via httpwwwirieduarpublicaciones_irianuariocd_anuario_2014Economia4bpdf Albar MA Islamic teachings and cancer prevention J Family CommunityMed “Ibrahim EM Satti MB Al Idrissi HY Higazi MM Magbool GM Al QA Oralcancer in Saudi Arabia the role of alqat and alshammah Cancer DetectPrev “ Alsanosy RM Mahfouz MS Gaffar AM Khat chewing among students ofhigher education in Jazan region Saudi Arabia prevalence pattern andrelated factors Biomed Res Int Quadri MFA Alharbi F Bajonaid AMS Moafa IHY Al Sharwani A AlamirAHA Oral squamous cell carcinoma and associated risk factors in JazanSaudi Arabia a hospital based case control study Asian Pacific J CancerPrev “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationspotential causes that need to be investigated furtherThe knowledge produced by this study must be translated into interventions by performing indepth analysesof regional differences This will contribute to the effortsof preventing oral cancer in Saudi ArabiaAbbreviationsAIR Agestandardised incidence rates ASR Agestandardised specific ratesIARC International Agency for Research on Cancer SCR Saudi CancerRegistry ICD10 International Classification of Diseases 10th revisionAcknowledgementsI express my thanks and gratitude to the Saudi Ministry of Health forproviding me with the Saudi Cancer Registry reportsAuthor™s contributionsI certify that I have participated sufficiently in the intellectual contentconception and design of this work analysis and interpretation of the dataas well as the writing of the manuscript to take public responsibility for itand have agreed to have my name listed as a contributor The author readand approved the final manuscriptFundingThis research received no specific grant from any funding agency in thepublic commercial or notforprofit sectorsAvailability of data and materialsThe data that support the findings of this study are available from SaudiMinistry of Health but restrictions apply to the availability of these datawhich were used under authorization for the current studyEthics approval and consent to participateThis study was approved by the Research Ethics Committee at NajranUniversity The ethical document reference No ETConsent for publicationA secondary data analysis was conducted in this retrospective study byusing a published dataCompeting interestsThe author declares that he is the only author for this work No other authorcontributed to this work He is also in agreement with the content of themanuscript He declares no conflict of interestReceived June Accepted August ReferencesJemal A Bray F Center MM Ferlay J Ward E Forman D Global cancerstatistics CA Cancer J Clin “Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancerstatistics GLOBOCAN estimates of incidence and mortality worldwidefor cancers in countries CA Cancer J Clin “ Warnakulasuriya S GloWarnakulasuriya S Global epidemiology of oral andoropharyngeal cancer Oral Oncology ““ httpsdoi101016joraloncology200806002bal epidemiology of oral andoropharyngeal cancer Oral Oncol Rick A Afsaneh B Cancer Facts Figures American Cancer Society Access online via httpswwwcancercontentdamcancerresearchcancerfactsandstatisticsannualcancerfactsandfigures2017cancerfactsandfigures2017pdfAlJaber A AlNasser L ElMetwally A Epidemiology of oral cancer in Arabcountries Saudi Med J “Ariyawardana A Johnson NW Trends of lip oral cavity and oropharyngealcancers in Australia overall good news but with rising rates inthe oropharynx BMC Cancer Alshehri B Descriptive epidemiological analysis of thyroid cancer in the Saudipopulation Asian Pacific J Cancer Prev “Armitage P Doll R The age distribution of cancer and a multistage theoryof carcinogenesis Br J Cancer “ 0c"

Thyroid_Cancer

Despite the proven benefits of iron chelation therapy ICT in the management of chronic iron overload and related complications compliance to longterm ICT is challenging Results from the ECLIPSE study an label randomized multicenter 2arm phase study evaluated the safety of deferasirox dispersible tablet and filmcoated tablet FCT formulations in patients with transfusiondependent thalassemia TDT or very low low or intermediate risk myelodysplastic syndrome MDS treated over weeksMethods The aim of the current study a 2year label multicenter singlearm phase study is to evaluate the longterm safety and efficacy of deferasirox FCT in a subset of patients with TDT or lowerintermediaterisk MDS treated for years after the completion of weeks of treatment with deferasirox in the ECLIPSE phase studyResults Of patients enrolled completed treatment and study Adverse events AEs reported in most patients were of mild to moderate severity Headache and diarrhea were the most frequently reported AEs None of the serious AEs including death were considered treatment related No new safety signal was identified and longterm safety of deferasirox FCT was consistent with the known safety profile of deferasirox No major concerns associated with gastrointestinal tolerability renal safety or hematological abnormalities thrombocyt ianeutr ia were reported during the years Patients receiving deferasirox FCT had a treatment compliance by pill count of and persistence continuous use for ‰¥ days of Reduction in serum ferritin level was almost consistent starting from week across all postbaseline time points relative reduction month month Correspondence giovanbattistaruffoarnascivicoit UOC Ematologia e Talassemia AO CivicoDi CristinaBenfratelli Piazza Nicola Leotta Palermo ItalyFull list of author information is available at the end of the The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cTartaglione a0et a0al Exp Hematol Oncol Page of Conclusions The results from this 2year interventional study suggest that the recommended dosing of deferasirox FCT with better tolerability palatability and compliance offers a favorable option of ICT for longterm management of iron overload and associated complications in TDTTrial registration ClinicalTrialsgov NCT02720536 Registered March wwwclini caltr ialsgovct2showNCT02 Keywords Deferasirox Chelation Filmcoated tablet Iron overload Longterm Safety Transfusiondependent thalassemia Myelodysplastic syndromeIntroductionIron overload in chronic anemias represents a serious consequence of impaired hematopoiesis and repeated blood transfusions leading to endan damage reduced quality of life and decreased survival Iron chelation therapy ICT can be a lifelong requirement in chronic transfusiondependent refractory anemias including thalassemia sicklecell disease and myelodysplastic syndrome MDS [ ] Despite the proven benefit of ICT patient compliance to longterm ICT is challenging [] Compliance with ICT is reported to influence the frequency and severity of iron overload“related complications [“] and a substantial increase in morbidity mortality and treatment cost has been seen among patients who are noncompliant to ICT []Currently main iron chelators are available for clinical use deferoxamine deferiprone and deferasirox Parenterally administered deferoxamine was the mainstay of chelation therapy until the availability of oral chelators in [] Oncedaily oral administration of deferasirox dispersible tablet DT formulation presented a better option with greater compliance and quality of life over parenteral deferoxamine [] Currently there are no direct comparison studies for the oral chelators however oncedaily simpler use of deferasirox has been projected to be a more costeffective option than the thricedaily administration of oral deferiprone in the management of longterm complications [ ] The deferasirox filmcoated tablet FCT formulation with a simpler oral administration and improved palatability and gastrointestinal GI tolerability compared with deferasirox DT offers a better option for optimal patient acceptance and improved compliance to longterm therapy [ ] Thus an appropriate choice of ICT plays an important role in patient compliance to chelation therapyThe deferasirox FCT used in this study contained the same active substance of the iron chelator deferasirox and was strengthadjusted to achieve comparable exposure to the currently approved deferasirox DT Deferasirox FCT is available in dose strengths a0mg a0mg and a0mg and is dosed based on body weight Unlike the DT deferasirox FCT can be administered once daily orally without any need for dispersion either on an empty stomach or with a light meal [ ]Results from the ECLIPSE study an label randomized multicenter 2arm phase study that evaluated the safety of deferasirox DT and FCT formulations in patients with transfusiondependent thalassemia TDT or MDS very low low or intermediate risk treated over a0weeks have been published previously [] The purpose of the current study was to collect data on the longterm safety and efficacy of deferasirox FCT in a subset of patients with TDT or very low low or intermediaterisk MDS who had the possibility to continue treatment with deferasirox FCT after completion of a0 weeks of treatment in the ECLIPSE study The study also aimed to collect efficacy data for deferasirox FCT in the reduction or maintenance of iron burden as measured by the serum ferritin levelMethodsKey inclusion and a0exclusion criteriaPatients recruited at European sites [Austria n Greece n Italy n ] who had completed the 24week treatment in the ECLIPSE study with tolerance to deferasirox and fulfilled all eligibility criteria were included in this study Patients were male or female aged ‰¥ a0 years with TDT or very low low or intermediaterisk MDS who had received deferasirox DT at doses ‰¥ a0mgkgday or ‰¥ a0mgkgday respectively as per clinical judgement Patients had a transfusion history of ‰¥ packed red blood cell units were anticipated to be transfused with ‰¥ a0unitsyear during the study and had serum ferritin a0ngmL at screening Key exclusion criteria in the study included patients with creatinine clearance CrCl below contraindication limit as per local label serum creatinine SCr — upper limit of normal ULN alanine aminotransferase ALT — ULN unless liver iron concentration was confirmed as a0mg Fedry weight within a0months prior to screening urine protein to creatinine ratio UPCR a0mgmg impaired GI function that may significantly alter the absorption of oral deferasirox or clinicallaboratory evidence of active hepatitis Bhepatitis C infection 0cTartaglione a0et a0al Exp Hematol Oncol Page of Study designThis was a 2year label multicenter singlearm phase study NCT02720536 aimed to provide additional efficacy safety and drug exposure data following the ECLIPSE study []Patients who were assigned to either the deferasirox DT or the deferasirox FCT arm and had completed the study period of a0weeks with tolerance to deferasirox in the ECLIPSE study were allowed to participate in this study It was planned that any patient continuing directly from the ECLIPSE study would receive the same dose of deferasirox FCT or an equivalent FCT dose at the start of this study corresponding to their DT dose at the end of the ECLIPSE study As all patients had a lag period between the completion of the ECLIPSE study and the start of this study ie patients who completed the ECLIPSE study switched to commercially available deferasirox DT or another ICT they entered the current study following a washout period with a starting dose that was based on clinical judgment For each patient the daily dose was calculated based on the patient™s actual body weight and then rounded up or down to the nearest whole tablet according to available strengths of deferasirox FCT tablets a0mg a0mg and a0mg Dose adjustments were allowed every a0months based on serum ferritin levels and clinical judgment with ± to a0mgkgday up to a maximum dose of a0mgkgdayThe study was conducted in accordance with the Good Clinical Practice guidelines and the Declaration of Helsinki and was approved by independent ethics committees at participating sites The study is registered at wwwclini caltr ialsgovct2showNCT02 Patients or parentsguardians provided written informed consent or assent prior to enrollmentOutcomesThe primary objective of the study was to evaluate the overall safety of the deferasirox FCT formulation measured by the frequency and severity of adverse events AEs and changes in laboratory values of interest such as SCr and CrCl The secondary objective was to evaluate the efficacy of deferasirox FCT with respect to serum ferritin levels decreased or maintained according to individual therapeutic goal measured by the absolute and relative changes in serum ferritin levels over timeStatistical evaluationsNo formal sample size was calculated A maximum of patients aged ‰¥ a0years were originally planned to be enrolled from the ECLIPSE study patients were enrolled into this study There were no screening failuresData from all centers participating in this study were for analyses The statistical software SAS® version collected using electronic case record forms and pooled was used for analysis The analyses were descriptive in nature no hypothesis was tested Data were summarized with respect to demographic and baseline characteristics primary and secondary assessments along with safety observations All analyses were based on data collected as per protocolscheduled assessments according to or including clinical judgment of the investigatorPatient compliance to deferasirox FCT was evaluated using the count of deferasirox FCT by the relative consumed tablet count Descriptive statistics including confidence intervals CIs for the mean relative consumed tablet counts were provided Persistence defined as continuous use of deferasirox FCT without a gap for ‰¥ or ‰¥ a0days over a fixed time interval of interest was summarized at month a0 month month and month The incidence of any treatmentemergent AEs ie AEs from the start of study treatment to a0days after the last intake of study drug overall and by maximum grade severity mild moderate or severe as reported by the clinician were summarized using frequency counts and percentages of patients For each of the laboratory parameters of interest SCr CrCl ALT and aspartate aminotransferase [AST] the worst postbaseline values were summarized as shift tables based on notableextended ranges For serum ferritin and hematological parameters red blood cells [RBCs] platelets total white blood cells hemoglobin and hematocrit the absolute mean and the relative mean changes from baseline were summarized at each postbaseline visitResultsOverall patients were enrolled from countries across Europe of whom discontinued early from treatment Fig a0Of the patients enrolled the majority patients [] including patients aged a0 years had TDT and patients had MDS all aged ‰¥ a0years Most patients were Caucasians and females comprised of the study population All patients had a history of prior ICT Demographics and other baseline characteristics are described in Table a0In most patients deferasirox monotherapy was the last ICT before study treatment The majority of patients had received prior medication or therapy The most common prior medications by the Anatomical Therapeutic Chemical classification system class included vitamin D and vitamin D analogues patients [] proton pump inhibitors PPIs patients [] sequential preparations of progestogens 0cTartaglione a0et a0al Exp Hematol Oncol Page of Patients enrolled N53Countries Austria Greece Italy Disease TDT MDS Completed treatment and studyaDiscontinued from treatment Withdrewconsent Pregnancy Unsatisfactorytherapeuticeffect Death Adverseevents Abnormallaboratoryvalues Unwillingnessto comply withprocedures Fig Patient disposition MDS myelodysplastic syndrome TDT transfusiondependent thalassemia aThe patients who completed the study had received treatment for at least monthsand estrogens patients [] fixed combinations of progestogens and estrogens patients [] and thyroid hormones patients []Almost all patients [] received concomitant medication or significant nondrug therapies on or after the start of study treatment The most common concomitant medications by ATC class included vitamin D and vitamin D analogues patients [] anilides patients [] antibiotics patients [] other agents for local oral treatment patients [] PPIs patients [ and other antibiotics for topical use patients []All patients received at least transfusions during the study with a maximum of transfusions received by patient The majority of patients [] including the MDS patients received to transfusions Additional file a0 Table a0S1Exposure to a0treatment and a0complianceThe mean standard deviation [SD] duration of exposure to treatment was days with most patients [] being treated for at least a0weeks The majority of patients receiving deferasirox FCT were in the longest exposure category ‰¥ a0weeks The sum of each patient™s treatment exposure to deferasirox FCT was patientyears Almost all patients [] received deferasirox FCT at an average dose of ‰¥ a0 mgkgday with mgkgday as the mean SD average actual deferasirox FCT dose received Table a0 MDS patients n received an average dose of ‰¥ a0mgkgday with mgkgday as the mean SD average actual deferasirox FCT dose receivedPatients had a mean relative consumed tablet count of CI “ The proportions of patients with continuous use of deferasirox FCT with no interruption for ‰¥ a0days and ‰¥ a0days were n and n respectively at a0monthsEfficacyA decrease in mean serum ferritin levels was observed from week a0 onward except for month a0 though the median serum ferritin levels showed a consistent decrease across all postbaseline time points The mean SD actual decrease in serum ferritin level was µgL from baseline to month a0 µgL from baseline to month a0 and µgL from baseline to month a0 Fig a0 The decrease relative change in [SD] was higher at month a0 than at month a0 and month a0 [] vs [] vs [] The mean SD actual decrease in serum ferritin level from baseline to month a0 was reported as a0µgL relative change in percentage in patient at month a0 0cTartaglione a0et a0al Exp Hematol Oncol Page of Table Demographics and a0other baseline characteristicsDemographic variableAge years mean SDAge category years n ‰¥ to ‰¥ to ‰¥ 65aSex n Male FemaleBMI kgm2 mean SDPredominant race n Caucasian Asian OtherbMain underlying disease n MDS IPSSR risk stratification Very low risk Low risk Intermediate risk TDTTime since diagnosis years mean SD MDS TDTPrior ICT received during the ECLIPSE study n Deferasirox DT Deferasirox FCTLast ICT received before deferasirox FCT in the current study n Deferiprone Deferoxamine and deferiprone Deferasirox monotherapyTransfusion history Total number of transfusions received over the past months mean SD Time since last blood transfusion days mean SDBaseline serum ferritin µgL mean SDDeferasirox FCT N BMI body mass index DT dispersible tablet FCT filmcoated tablet ICT iron chelation therapy IPSSR Revised International Prognostic Scoring System MDS myelodysplastic syndrome SD standard deviation TDT transfusiondependent thalassemiaa The oldest patient was years oldb Other included two patients who selfidentified as whiteSafetyAdverse eventsOf the patients almost all [] reported at least AE regardless of study drug relationship Additional file a0 Table a0S2 provides an overview of all AEsThe most common AEs by preferred term were headache diarrhea pyrexia nausea vomiting cough and upper abdominal pain The most common AEs reported in of patients by system an class SOC during the treatment with deferasirox FCT were related to infections and infestations [] influenza rhinitis gastroenteritis pharyngitis and urinary tract infection occurred in of patients by preferred term followed by GI disorders [] diarrhea nausea vomiting upper abdominal pain and abdominal pain occurred in of patients by preferred term Table a0 None of the AEs in the infections and infestations SOC were treatment related Of the AEs that were suspected to be treatment related in patients increased UPCR 0cTartaglione a0et a0al Exp Hematol Oncol Page of Table Exposure to a0treatment and a0complianceDeferasirox FCT N Duration of exposure days mean SDDuration of exposure categories weeks n to to to ‰¥ Patient treatment yearsAverage actual dose mgkgday mean SDAverage actual dose category mgkgday n to to ‰¥ Compliance Relative consumed tablet count mean SD Persistence Continuous use of deferasirox FCT with no interruption for ‰¥ days n Up to months n Up to months n Up to months n Up to months n Continuous use of deferasirox FCT with no interruption for ‰¥ days n Up to months n Up to months n Up to months n Up to months n FCT filmcoated tablet SD standard deviation diarrhea increased blood creatinine gastritis and proteinuria were the most common reported in of patients AEs by preferred term Of these suspected treatmentrelated AEs increased blood creatinine and diarrhea both of mild severity were reported in MDS patientsThe maximum grade of severity of AEs was reported as mild moderate and severe in and of patients respectively Table a0 Severegrade AEs reported by preferred term irrespective of study drug relationship included increased UPCR splenomegaly atrial fibrillation cardiac failure goiter cholestasis hepatic failure gastroenteritis lower respiratory tract infection lymph gland infection urosepsis femur fracture spinal fracture lumbar vertebral fracture rib fracture ulna fracture transfusion reaction arthralgia back pain malignant melanoma papillary thyroid cancer headache device failure renal colic calculus urinary hydronephrosis and ureterolithiasis each None of the patients had severe GI AEs Moderate and severegrade AEs by maximum severity grade were reported in patients and patients with deferasirox as prior chelation therapy and patients and patients with other ICT as prior chelation therapy respectively Of the patients moderategrade AEs were reported in patients in the to a0mgkgday dailydose group and patients in the ‰¥ a0 mgkgday dailydose group while severegrade AEs were reported in patients each in the to a0 mgkgday and ‰¥ a0 mgkgday dailydose groups respectivelySerious AEs SAEs regardless of study drug relationship were reported in patients MDS n TDT n and none of these were considered treatment related SAEs reported in patients with MDS included cardiac failure device failure femur fracture cholestasis hepatic failure and malignant melanoma reported in patient who died lumbar vertebral fracture in patient and urosepsis in patient Other SAEs reported in patients with TDT included spontaneous abortion atrial fibrillation biliary colic urinary calculus cholecystitis diverticulitis fracture goiter hydronephrosis lower respiratory tract infection lymph gland infection ovarian adenoma panic attack papillary thyroid cancer renal colic rib fracture ulna fracture and ureterolithiasis None of these SAEs were reported in more than patient each Death not suspected to be treatment related occurred in a 72yearold male patient with very low“risk MDS as per the Revised International Prognostic Scoring System [IPSSR] The cause of this ontreatment death according to clinical judgment was malignant melanoma with multiple metastasis in the liver and spleen with unknown primary origin Another contributing factor for the death was liver failure and intrahepatic cholestasisAEs leading to discontinuation of study treatment were reported in patients of which drug ineffective AE in patient and serum ferritin abnormal AE in patient were considered treatment related Treatment was discontinued due to an AE moderate in severity not suspected to be treatment related in MDS patient AEs that led to dose adjustmentinterruption occurred in patients with the most frequently reported being increased UPCR patients [] and vomiting patients [] Treatmentrelated AEs that led to dose adjustmenttemporary interruption were reported in patients and included increased UPCR patients [] increased blood creatinine patients [] gastritis glycosuria proteinuria upper abdominal pain patients [] each and diarrhea gastric ulcer increased serum 0cTartaglione a0et a0al Exp Hematol Oncol Page of morfnitirrefmuresni egnahcLgµenilesabDSnaeMˆ’ˆ’ˆ’ˆ’ˆ’W2n44 M1n51M2n48M3n50 M4n43M5n46 M6n44 M7n40M8n37M9n42M10n36M11n41M12n36M13n43M14n39 M15n28M16n39 M17n32M18n31M19n31M20n33M21n26M23n28 M24n25M22n33M25n21M27n1M26n22Fig Change in serum ferritin from baseline µgL by time point M month SD standard deviation W week Error bars represent the ± SD values for the respective mean valuesNumber of observations at each timepointTime pointTable Common adverse events reported by a0maximum grade of a0severityAEs N a0System an class a0 a0Preferred termOverall n Mild n Moderate n Severe n Number of patients with at least one event Infections and infestations Rhinitis Gastroenteritis Pharyngitis Urinary tract infection Gastrointestinal disorders Diarrhea Nausea Vomiting Upper abdominal pain Abdominal pain General disorders and administration site conditions Asthenia Influenza Pyrexia Respiratory thoracic and mediastinal disorders Cough Oropharyngeal pain Investigationsa Urine proteincreatinine ratio increased Musculoskeletal and connective tissue disorders Musculoskeletal pain Nervous system disorders Headache Proportion of patients with AEs reported by preferred term and grouped by system an classAE adverse eventa Abnormal laboratory values reported as AEs 0cTartaglione a0et a0al Exp Hematol Oncol Page of ferritin hypertransaminasemia and increased transaminases patient [] each AEs required additional treatment in patients with TDT macular edema and skin ulcer in patient [] diarrhea radius fracture and breast discomfort in patient [] each and none were suspected to be treatment relatedAdverse events of a0special interestOverall patients reported AEs of special interest of which AEs suspected to be treatment related were reported in patients Common AEs of special interest incidence included increased UPCR patients [] severe and suspected to be treatment related patients [] proteinuria patients [] increased blood creatinine patients [] MDS n and hypertransaminasemia patients [] Hepatic failure due to metastatic liver disease MDS n and transfusion reaction TDT n of severe grade but not suspected to be treatment related were reported in patient each One patient with MDS discontinued the treatment due to decreased creatinine renal clearance moderate in severity but not suspected to be treatment related Additional file a0 Table a0S3Laboratory parametersWorst postbaseline elevations in SCr of ULN at consecutive measurements at least a0days apart occurred in patients MDS n TDT n One patient with MDS had worst postbaseline UPCR a0 mgmmol at consecutive measurements at least a0 days apart notable range Two patients with MDS had worst postbaseline CrCl value within the notable range a0 mLmin at consecutive measurements at least a0days apart and patients had worst postbaseline CrCl a0mLmin value Two patients with TDT had a worst postbaseline ALT level in the notable range — ULN and — baseline value Elevations of transaminases AST or ALT — ULN were Table Shift tables of a0laboratory values based on a0notableextended rangesALT UL‰¤ ULN ULN to ‰¤ — ULNTotalAST UL‰¤ ULN ULN to ‰¤ — ULNTotalSerum creatinine‰¤ ULNTotalCreatinine clearance‰¥ ‰¥ to MissingTotalUrinary proteinurinary creatinine ratio mgmmol‰¤ MissingTotalBaselinen n n n n ‰¤ ULN n ‰¤ ULN n ‰¥ n ‰¤ n Worst postbaseline value‰¤ ULN n ULN to a0‰¤ —ULN n ULN to a0‰¤ —ULN n — ULN n — ULN n Notable range n Extended range n Notable range n Extended range n Two consecutive ULN n Notable range n One value ‰¥ to a0 n Notable range n Missing n One value n Extended range n One value n Two values n Notable range n Missing n ALT alanine aminotransferase AST aspartate aminotransferase ULN upper limit of normal 0cTartaglione a0et a0al Exp Hematol Oncol Page of uncommon only patient with TDT had a postbaseline increase in AST and ALT values — ULN and — baseline value Table a0Hematological parametersThe majority of patients had low RBC of hematocrit of and hemoglobin of values at baseline Among patients with a baseline platelet count ‰¥ — 109L only patients had worst postbaseline values in the notable range ‰¥ to — 109L Similarly among patients with a baseline absolute neutrophil count ‰¥ — 109L the worst postbaseline values were found to be in the notable range ‰¥ to — 109L in patient and extended range — 109L in patient Mean ± SD change mean relative percentage change from baseline to month and month in key hematological parameters is represented in Additional file a0 Table a0S4DiscussionThis 2year phase interventional study evaluated the efficacy and safety of deferasirox FCT in adult and pediatric patients mean age a0 years with MDS n and TDT n who had previously completed a0weeks of deferasirox treatment in the ECLIPSE study over a mean duration of further a0days Almost of patients received an average actual deferasirox FCT dose of ‰¥ a0mgkgday during the study The average actual dose in MDS patients was comparatively less than that in TDT patients reflecting the notion of physicians using lower doses to treat MDS patients [] None of the patients were administered with higher than the maximum recommended doses of deferasirox FCT a0mgkgday []Various studies have demonstrated that compliance with ICT significantly improves morbidity and mortality [“ ] Compliance mean relative consumed tablet count and persistence “ rates with deferasirox FCT during this study were found to be on the higher side Persistence rates proportion of patients with continuous use of deferasirox FCT with no interruption for ‰¥ a0days or ‰¥ a0days in this study are comparable to the realworld data on persistence in patients who switched from deferasirox DT “ to deferasirox FCT “ []Common AEs reported with deferasirox FCT in this study headache diarrhea pyrexia nausea vomiting cough and upper abdominal pain were consistent with the known profile of deferasirox [ ] AEs reported in of the patients were of mild to moderate in severity none of the GI AEs were of severe grade nor led to discontinuation of treatment These results were in line with those of previous studies which reported that GI tolerability profile may be improved with the FCT compared with the DT Lack of excipients that cause GI irritation and ease of administration along with a light meal could have contributed to the better GI tolerability of FCT [ ] It is noted that the concomitant use of PPIs during the study was higher than reported prior use This study was not designed to investigate the reasons behind the increased use of PPIs but we assume that they might have been prescribed as a general prophylaxis to avoid GI complications from other concomitant therapies analgesics antibiotics etc prescribed in comorbid conditions infections or proceduresSAEs regardless of study drug relationship were reported in of patients and none including the death due to malignant melanoma with multiple metastasis in the liver and spleen were considered treatment related Notably of SAEs reported in patients were reported in MDS patients aged ‰¥ a0 years reflecting the considerable burden of the underlying hematological disorder together with the comorbidities of the affected elderly patients [“] Discontinuation of treatment due to treatmentrelated AEs drug ineffective and abnormal serum ferritin during the a0years was observed in only TDT patients The low rate of SAEs and absence of treatmentrelated deaths in this study could have been a result of the greater compliance and persistence “ rates with deferasirox FCTThe ECLIPSE study reported that abnormal renal parametersrenal AEs were more common in patients receiving deferasirox FCT than in those receiving deferasirox DT due to an intake of higher than the recommended dose [] Most of the renal abnormalitiesAEs reported during this 2year study were mild and reversible with dosage adjustment or temporary interruption of deferasirox FCT Only patient discontinued the treatment due to decreased creatinine renal clearance which was moderate in severity and not suspected to be treatment related Increases in SCr and liver function tests in the notableextended ranges were observed in some patients and those increases were consistent with the known safety profile of deferasirox FCT [] No substantial difference in severity of all AEs was noted based on dosing groups to a0mgkgday and ‰¥ a0mgkgday as well Administration of the recommended doses of deferasirox FCT with constant dosage adjustment as per serum ferritin levels might have resulted in fewer renal and liver abnormaliti

Thyroid_Cancer

EpidemiologyEPIDEMIOLOGICAL SCIENCERisk factors for hospital admissions related to COVID19 in patients with autoimmune inflammatory rheumatic diseasesDalifer D Freites Nu±ez1 Leticia Leon Arkaitz Mucientes1 Luis Rodriguez Rodriguez Judit Font Urgelles3 Alfredo Madrid Garc­a1 Jose I Colomer1 Juan A Jover34 Benjam­n Fernandez Gutierrez3 Lydia Abasolo1Handling editor Josef S Smolen1Rheumatology Department and IDISSC La Fundacion para la Investigacion Biomedica del Hospital Clinico San Carlos Madrid Spain2Department of Health and Education Universidad Camilo Jose Cela Villafranca del Castillo Madrid Spain3Rheumatology Department Hospital Clinico San Carlos Madrid Spain4Medicine Department Universidad Complutense de Madrid Madrid Comunidad de Madrid SpainCorrespondence toDr Leticia Leon IdISSC and Rheumatology La Fundacion para la Investigacion Biomedica del Hospital Clinico San Carlos Madrid Spain lleon hcsc salud madrid Received May Revised July Accepted July Objectives To describe patients with autoimmune inflammatory rheumatic diseases AIRD who had COVID19 disease to compare patients who required hospital admission with those who did not and assess risk factors for hospital admission related to COVID19Methods An observational longitudinal study was conducted during the pandemic peak of severe acute respiratory syndrome coronavirus March to April All patients attended at the rheumatology outpatient clinic of a tertiary hospital in Madrid Spain with a medical diagnosis of AIRD and with symptomatic COVID19 were included The main outcome was hospital admission related to COVID19 The covariates were sociodemographic clinical and treatments We ran a multivariable logistic regression model to assess risk factors for the hospital admissionResults The study population included patients with AIRD and COVID19 Of these patients required hospital admission related to COVID19 The mean age on admission was years and the median time from onset of symptoms to hospital admission was “ days The median length of stay was “ days A total of patients died during admission Compared with outpatients the factors independently associated with hospital admission were older age OR p000 and autoimmune systemic condition vs chronic inflammatory arthritis OR p001 No statistically significant findings for exposure to disease modifying antirheumatic drugs were found in the final modelConclusion Our results suggest that age and having a systemic autoimmune condition increased the risk of hospital admission whereas disease modifying antirheumatic drugs were not associated with hospital admission Authors or their employers No commercial re use See rights and permissions Published by BMJTo cite Freites Nu±ez a0DD Leon a0L Mucientes a0A et a0al Ann Rheum Dis Epub ahead of print [please include Day Month Year] 101136annrheumdis2020217984INTRODUCTIONSevere acute respiratory syndrome coronavirus SARS CoV2 causes a myriad of clinical signs and symptoms together with typical laboratory abnormalities that manifest as the disease COVID191Since the confirmation of the first patient infected with SARS CoV2 in Spain in January the current COVID19 outbreak has had a considerable impact especially in the Madrid region where the highest incidence of COVID19 cases has been Key messagesWhat is already known about this subject –º The epidemiological scenario is changing daily There is little evidence for risk factors of poor outcome with COVID19 specific to autoimmune inflammatory rheumatic diseasesWhat does this study add –º Patients with an autoimmune systemic condition have a higher risk of hospital admission related to COVID19 compared with those with chronic inflammatory arthritis –º Disease modifying agents were not associated with a higher risk of hospital admission related to COVID19How might this impact on clinical practice or future developments –º Our data show that in a real world setting a high percentage of patients with autoimmune inflammatory rheumatic diseases and COVID19 required hospital admission The patients were mainly elderly with comorbidities and a systemic autoimmune conditionrecorded with more than patients admitted to the hospital until the first week of May2The incidence and severity of COVID19 disease seem to be higher in patients with risk factors such as advanced age and associated comorbidities mainly hypertension diabetes heart disease and previous respiratory diseases3 It is not clear whether patients with rheumatic diseases are more susceptible to SARS CoV2 infection or when they are infected whether they have more severe disease or a poorer outcome Previous outbreaks caused by coronaviruses did not yield overwhelming evidence that patients with rheumatic diseases are at an increased risk4 although some patients are candidates for a higher number of infections owing to their rheumatic disease predominantly systemic or the treatment they are receiving for rheumatic diseases5 Preliminary experiences in patients with COVID19 show that those with chronic arthritis treated with synthetic conventional or targeted syntheticbiologic disease modifying antirheumatic Freites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cEpidemiologydrugs DMARDs do not seem to be at a greater risk of respiratory or life threatening complications from SARS CoV2 than the general population6 The epidemiological scenario is changing and evidence on the risk factors of poor outcome with COVID19 specific to inflammatory rheumatic disease is scarce In addition there are little data on how the hospital admissions of these patients with severe COVID19 infection have evolved8The aim of our study was to describe patients with autoimmune inflammatory rheumatic diseases AIRD who had COVID19 during the pandemic peak We also explored possible risk factors associated with hospital admission related to COVID19 disease in patients with AIRD from a tertiary hospital in Madrid SpainMETHODSSetting study design and patientsThe study was performed in a public tertiary hospital Hospital Cl­nico San Carlos HCSC in Madrid Spain The catchment area is home to almost peopleWe performed a prospective observational study from March when our health area had the first hospital admission related to COVID19 to April We preselected all patients attended at the rheumatology outpatient clinic of our centre during the study period whose data were recorded in the electronic clinical history of our department HCR Penelope The inclusion criteria were age years a medical diagnosis according to International Classification of Diseases ICD10 of inflammatory rheumatic disease and symptomatic COVID19 disease assessed by medical diagnosis or confirmed with a positive SARS CoV2 PCR diagnostic testPatient data were obtained during routine clinical practice The study was conducted in accordance with the Declaration of Helsinki and the principles of Good Clinical Practice and was approved by the HCSC Ethics Committee approval number E BSVariablesThe primary outcome was admission to hospital with a medical diagnosis of COVID19 andor a positive PCR result between March and April compared with outpatients with symptomatic COVID19 diseaseThe covariables recorded were as follows sociodemographic baseline characteristics including sex age and rheumatic disease duration Type of AIRD including systemic autoimmune conditions polymyalgia rheumatica mixed connective tissue disease systemic sclerosis Sjogren™s syndrome vasculitis Raynaud phenomenon polymyositis polychondritis sarcoidosis antiphospholipid syndrome autoinflammatory syndromes and systemic lupus erythematosus and chronic inflammatory arthritis rheumatoid arthritis inflammatory polyarthritis juvenile idiopathic arthritis psoriatic arthritis axial spondyloarthritis uveitis and inflammatory bowel disease Baseline comorbid conditions including hypertension dyslipidaemia depression diabetes mellitus smoking habit kidney disease chronic liver disease respiratory diseases chronic obstructive pulmonary disease and interstitial lung disease thyroid disease heart disease valve disease arrythmias cardiomyopathy heart failure and pericarditis ischaemic vascular disease stroke cardiovascular and peripheral vascular disease venous thrombosislung embolism and cancer Treatment for inflammatory rheumatic disease a glucocorticoids b non steroidal anti inflammatory drugs NSAIDs c conventional synthetic disease modifying antirheumatic drugs csDMARDs antimalarials hydroxychloroquine and chloroquine azathioprine cyclophosphamide cyclosporine colchicine leflunomide methotrexate mycophenolate mofetilmycophenolic acid and sulfasalazine d targeted syntheticbiologic DMARDs tsbDMARDs including antitumour necrosis factor TNF alpha drugs infliximab adalimumab etanercept certolizumab and golimumab other biologics anti interleukin IL6 tocilizumab and sarilumab rituximab abatacept belimumab anti IL1723 anti IL17 ustekinumab ixekizumab and secukinumab Janus kinase JAK inhibitors tofacitinib and baricitinibTreatment had to start at least month before the beginning of the study and continue during the study period until the end of the study or hospital admission for antimalarial therapy glucocorticoids sulfasalazine NSAIDs or colchicine Regarding csDMARDs and tsbDMARDs treatment had to start at least month before the beginning of the study and continue until at least 21st March the end of the study or hospital admission In the case of rituximab the last infusion had to be at least in JanuaryData sourcesPatient sociodemographic clinical laboratory and data on treatment of rheumatic disease were obtained through HCR PenelopePatients with COVID19 were detected by warning calls to our rheumatologists or nurses or via routine telephone consultation Other infected patients were detected through their sick leave forms for COVID19 The results of SARS CoV2 PCR diagnostic assays were obtained from the microbiologyinfectious service of HCSC In addition our Hospital Central Services registered all medical admissions to HCSC This information was provided from March to AprilThe researchers carried out an exhaustive review of the clinical histories of admitted patients to identify COVID19 cases and rule out patients admitted for other reasons Once the COVID19 cases were identified we collected clinical laboratory and treatment data during admission until the end of admission either discharge or death in order to describe the progress of the disease The review was performed until 24th April in order to include follow up data from patients admitted to the hospital with COVID19Statistical analysisPatient characteristics are expressed as mean and SD or median and IQR for continuous variables categorical variables are expressed as percentages Statistical tests were performed to compare characteristics between patients admitted with COVID19 and those without hospital admissions Continuous variables were analysed using the Mann Whitney test or t test and discrete variables were analysed using the χ2 or Fisher exact test Univariable logistic regression analyses were performed to assess differences between hospital admissions related to COVID19 risk and covariates Multivariable logistic regression models adjusted for age sex and comorbidity were run in a stepwise manner to examine the possible effect of sociodemographic clinical and therapeutic factors on hospital admissions related to COVID19 The model also included csDMARDs and all other variables with a p02 from the simple regression analysis The results were expressed as the OR with its respective CIAll analyses were performed in Stata V13 statistical software Stata Corp A two tailed p value was considered to indicate statistical significanceFreites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cRESULTSA total of patients with AIRD with symptomatic COVID19 disease were included in the study table The tests were performed as an exploratory measure of the association between a variable and the outcomeMost of the patients were women with a mean age of years and a mean time since diagnosis of years The main diagnosis was rheumatoid arthritis followed by axial spondyloarthritis Many patients had at least one baseline comorbid condition the most prevalent being hypertension dyslipidaemia and lung disease Most patients were taking csDMARDs Half of the patients were taking glucocorticoids a quarter were taking NSAIDs and were taking tsbDMARDs of which adalimumab was the most frequently prescribed followed by rituximab Only one patient was taking a JAK inhibitor Interestingly of the patients taking tsbDMARDs were taking the drug in combination with a csDMARDA total of patients had to be admitted to the hospital because of COVID19 Of these were evaluated in the HCSC Emergency Department were admitted to HCSC and were transferred to the Institucion Ferial de Madrid IFEMA support hospital owing to the lack of capacity in our hospital at that time The remaining three patients were evaluated and admitted to other hospitals in the Autonomous Community of Madrid Table presents data for the patients admitted to HCSCOf the patients admitted to our hospital were women with a mean age at admission of years and median lag time from the onset of symptoms to the admission of “ days The median length of stay was “ days table At admission the median haemoglobin was “ gdL and the median total lymphocyte count was “ ngmL The median D dimer value was “ ngmL In of patients median interleukin IL6 levels were “ pgmL Patients received various antibiotics mainly azithromycin levofloxacin and third generation cephalosporinsMost patients were treated with hydroxychloroquine during admission About half received glucocorticoids Eighteen were treated with lopinavirritonavir and received the anti IL 6R antibody tocilizumab table 2FEDERA total of patients developed relevant complications during admission the most frequent being myocarditis thrombosis and kidney failure Only two patients were admitted to the intensive care unit during admission The first was a patient in 50s with mixed connective tissue disease and associated comorbidities who developed acute respiratory insufficiency and bilateral pneumonia The patient was treated with antibiotic therapy lopinavirritonavir hydroxychloroquine and βinterferon Finally the patient was extubated days later and is recovering The other was a young adult patient with systemic lupus erythematosus treated with methotrexate rituximab hydroxychloroquine and glucocorticoids who days after being diagnosed with COVID19 PCR developed an erythematous rash and generalised urticaria requiring hospitalisation in the intensive care unit owing to general clinical and laboratory worsening elevated D dimer values The patient was treated with methylprednisolone heparin and a cephalosporin A few days later the patient™s condition improved and he recovered completely at dischargeOf the patients admitted to HCSC were sent to another care centre converted hotel hospitalIFEMA support hospital when their condition improved A further patients Epidemiologywere discharged home to continue self isolation after improvement At the end of the study five patients remained in hospital A total of patients died during admission men and women with a median age of “ years Of the patients who died had relevant comorbidity diabetes mellitus pulmonary disease ischaemic vascular disease hypertension venous thrombosislung embolism lung disease and or liver disease The main diagnoses were rheumatoid arthritis followed by spondyloarthritis polymyalgia rheumatica vasculitis and Sjogren™s syndrome The results of the univariable analysis are shown in table Older age systemic autoimmune conditions vs chronic inflammatory arthritis OR CI “ p0014 hypertension diabetes mellitus lung disease heart disease and glucocorticoids were associated with statistically significant greater risk of admission to the hospital Female sex NSAIDs and anti TNF drugs vs non use were associated with a statistically significant lower risk The differences reported for the remaining variables did not reach statistical significanceThe multivariable analysis was adjusted for gender age and comorbidities related to COVID19 These comorbidities were diabetes mellitus pulmonary disease ischaemic vascular disease hypertension venous thrombosislung embolism lung disease andor liver disease table Age and systemic autoimmune conditions had more probability of hospital admissions regardless of other factors Differences in exposure to glucocorticoids were not statistically significant The type of exposed DMARDs did not reach statistical significance in the multivariate model In fact long term treatment with antimalarials OR CI “ p066 other csDMARDs including methotrexate leflunomide and azathioprine OR CI “ p09 and NSAIDs OR CI “ p05 dropped from the final model The variable tsbDMARDs was also eliminated from the final model anti TNF vs none OR CI “ p016 and non anti TNF vs none OR CI “ p03DISCUSSIONOur study aims to shed light on rheumatologists™ concerns regarding their patients We found that in a real world setting of patients with AIRD and COVID19 required hospital admission These were mainly elderly patients with more comorbidities and systemic autoimmune conditions Our data show that patients exposed to disease modifying agents do not seem to be at higher risk of hospital admission related to COVID19Of the patients included in the study with COVID19 required hospital admission Comparison of the characteristics of patients admitted to hospital because of COVID19 and those who did not require hospital admission were as follows admitted patients had a median age close to years that is more than years older than patients who were not admitted Moreover those who were admitted more frequently had baseline comorbidities and systemic autoimmune conditions As for therapy admitted patients were less frequently exposed to antimalarial and anti TNF alpha agentsThe median lag time from onset of symptoms to admission was days and almost of patients had pneumonia at admission The baseline laboratory results for admitted patients in our study are consistent with those published elsewhere9“ and are characterised by lymphopenia and elevated acute phase reactants In fact of the patients had elevated D dimers normal and elevated IL6 normal pgmL Treatment during admission varied widely as the disease proved Freites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cEpidemiologyTable Baseline demographic and clinical characteristics of patients with AIRD and with COVID19 admitted vs no admitted at the hospitalAIRD“COVID19 patientsAIRD“COVID non admitted patientsAIRD“COVID admitted patientsVariableN123N69N54P value Positive Negative Not performed Women n Age years mean SDTime since diagnosis years mean SDPCR test n Smoking habit active vs noneDiagnosis AIRD n Rheumatoid arthritis Axial spondyloarthritis Polymyalgia rheumatica Psoriatic arthritis Systemic lupus erythematosus Mixed connective tissue disease Sjogren™s syndrome Vasculitis Uveitis Systemic sclerosis Inflammatory polyarthritis Polychondritis Polymyositis Raynaud phenomenon OtherComorbidities n NSAIDs n Glucocorticoids n csDMARDs n TsbDMARDs n JAKi n Others inflammatory bowel disease antiphospholipid syndrome juvenile idiopathic arthritis autoinflammatory syndromes and sarcoidosis Heart disease arrhythmiasvalve disease cardiomyopathy and heart failure Ischaemic vascular disease stroke cardiovascular and peripheral vascular diseaseAIRD autoimmune inflammatory rheumatic disease Anti TNF tumour necrosis factor alpha COPD chronic obstructive pulmonary disease csDMARD conventional synthetic disease modifying antirheumatic drug ILD interstitial lung disease JAKi JAK inhibitor tsbDMARDs target syntheticbiologic disease modifying antirheumatic drug Hypertension Dyslipidaemia Depression Diabetes mellitus Heart disease Vascular disease Liver disease Kidney disease Lung disease ILDCOPD Cancer Venous thrombosislung embolism Thyroid disease Anti TNF alpha agent Other biologics Abatacept Tocilizumab Belimumab Rituximab Methotrexate“leflunomide“azathioprine Sulfasalazine AntimalarialsFreites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cTable Hospital admissions related to COVID19 among patients with AIRDVariableValueTable OR of hospital admission related to COVID19 in patients with AIRD univariable analysisVariable CIORPEpidemiology Haemoglobin gdL D dimer ngmL Neutrophil count —109L Lymphocyte count —109L CRP mgdL LDH UL Platelet count —109L Creatinine mgdL Ferritine ngmLAdmissions nLag time from onset of symptoms to admission days median IQRPneumonia at admission n Systemic autoimmune conditions n Laboratory data at admission median IQR COVID19 related treatments during admission n Admitted by intensive care unit during hospital admission Length of stay days median IQRDischarge reason n Azithromycin Other antibiotics Glucocorticoids Lopinavirritonavir Remdesivir Darunavircobicistat Tocilizumab Interferon HCQ Immunoglobulin Improvement home isolation Other care centre medicalised hotelIFEMA hospital Death End of study no discharge No Yes “ Data for patients patients were treated in other support centres after referral or admission in other centresCRP C reactive protein HCQ hydroxychloroquine LDH lactate dehydrogenase challenging for specialists who prescribed various combinations of drugs based on little published evidence In this sense the anti IL 6R antibody tocilizumab has proven to be beneficial in patients with COVID1912 Treatment may also be successful in the early stages of cytokine release syndrome if it can effectively block the signal transduction pathway of IL6 therefore tocilizumab and sarilumab are likely to emerge as effective drugs for patients with moderate to severe COVID1913 In our study almost of the patients were treated with tocilizumabThe patients who eventually died had a median age of years This finding is in line with data for the general population where over of deaths occurred in persons years and more than of all deaths were in people aged ‰¥ years7The multivariable regression model showed that only age increasing by per year and systemic autoimmune conditions continued to be risk factors for hospital admission related to COVID19 “ “ “ “ “ “ “ “ “ ““““““““““““ Rheumatoid arthritis Inflammatory polyarthritis Systemic lupus erythematosus Psoriatic arthritis Spondyloarthritis MTCD Sjogren syndrome Hypertension Dyslipidaemia Depression Diabetes mellitus Heart disease Vascular disease Liver disease Kidney disease Lung disease ILDCOPD Cancer Venous thrombosislung embolism Thyroid diseaseGender womenAge yearsDiagnosis AIRD one category vs the rest Disease durationSmoking habit active vs noneComorbidities yes NSAIDsGlucocorticoidscsDMARDSs TsbDMARDs JAKisOther biologics anti IL6 tocilizumab sarilumab rituximab Rtx anti IL1723 anti IL17Othercategories could not be represented polymalgia rheumatica systemicsclerosis vasculitis Raynaud phenomenon polychondritis Beh§et diseasepolymyositis uveitis inflammatory boweldisease antiphospholipid syndrome juvenile idiopathic arthritis autoinflammatorysyndromes and sarcoidosisAIRD autoimmune inflammatory rheumatic disease anti TNF tumour necrosis factor csDMARD Conventional synthetic disease modifying antirheumatic drug IL6 interleukin6 JAKi JAK inhibitors tsbDMARDs target syntheticbiologic disease modifying antirheumatic drug““““““““““““ ““ Methotrexate“leflunomide“azathioprine Sulfasalazine Antimalarial agents““““““““““ None Anti TNF agents Other biologics““As for the association between sex and risk of hospital admission we did not find a higher risk of admission in women despite the fact that rheumatic diseases are more prevalent in this group The type of diagnosis seems to play an important role in the probability of hospital admission and patients with systemic autoimmune conditions seem to have the highest risk compared with chronic inflammatory arthritisAs it has been reported elsewhere comorbidities play an important role in the risk of hospital admission15 Clinical outcomes are poorer in patients with COVID19 with a comorbid condition than in those without and a greater number of comorbidities correlates with poorer clinical outcomes16 Diabetes is a major comorbidity in COVID19 and patient™s history of diabetes is an independent risk factor for morbidity and mortality in this condition17 Diabetes has been associated with admissions to Freites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cEpidemiologyTable Multivariable analysis risk factors for hospital admission related to COVID19 in patients with AIRDVariableOR CIP value“““““Gender womenAge yearsAIRD systemic autoimmune conditions vs chronic inflammatory arthritisCOVID comorbidities yesGlucocorticoidsSystemic autoimmune conditions polymyalgia rheumatica mixed connective tissue disease systemic sclerosis Sjogren™s syndrome vasculitis Raynaud polymyositis polychondritis sarcoidosis antiphospholipid syndrome autoinflammatory syndromes and systemic lupus erythematosus vs chronic inflammatory arthritis rheumatoid arthritis inflammatory polyarthritis juvenile idiopathic arthritis psoriatic arthritis axial spondyloarthritis uvetis and inflammatory bowel disease Comorbidities including the presence of at least one of the follows hypertension heart disease vascular disease diabetes mellitus venous thrombosislung embolism chronic kidney disease liver disease and lung disease ILDCOPDAIRD autoimmune inflammatory rheumatic disease COPD chronic obstructive pulmonary disease ILD interstitial lung diseasethe intensive care unit due to COVID19 in recent series19 and has been shown to increase mortality6 Therefore we adjusted for comorbidity in the multivariable analysisTreatment with glucocorticoids lost its statistical significance in the multiple regression model However the dose was not reported in our data and in the case of these agents the risk could be dose dependent In a recent publication from a European registry the authors found that exposure to mgday was associated with a greater probability of hospitalisation21The exposure to DMARDs regardless of whether they were biological or synthetic does not seem to be associated with a higher hospital admission related to COVID19 Although we have to consider the limited number of patients in our study our results are in concordance with data reported elsewhere8 Our results should be interpreted taking into account other limitations First patients were included from a single centre Second of all the patients with COVID19 who did not require admission one third contacted the rheumatology service to report the disease and the remainder were detected through the COVID19 discharge reports sent to their primary care physician Elderly persons and homemakers who did not contact us can be considered missing Consequently there may be some selection bias between those admitted and those not admitted although this problem was addressed by adjusting for confounders in the multivariable analysis Third while it is acknowledged that clinical suspicion must be confirmed by PCR assay almost of patients admitted did not undergo PCR owing to the lack of tests or the extreme healthcare overload Nevertheless all cases included were clinically compatible and managed as COVID19The key strength of our study is that it was performed in real life conditions during then pandemic peak with access to complete sociodemographic and clinical data from our rheumatology electronic clinical history including thorough hospital admission data such as laboratory abnormalities and COVID19 treatment data from the hospital computer services Consequently this has allowed us to analyse the risk of hospital admission related to COVID19 adjusted for confounders thus avoiding possible biasAlthough we are unable to modify the factors reported here knowing them can help rheumatologists to treat and advise their patients during this new and challenging period Results provided by our study are preliminary and should be corroborated with other real life studies but we consider our findings helpful to increase the knowledge in the management of patients with AIRD and COVID19Twitter Benjam­n Fernandez Gutierrez Fergutbe2001Acknowledgements The authors would like to thank Ana M Perez for her help with data collection They would like to say a special thanks to all the rheumatologists and nurses who contributed to the care of the patients in such an innovative and conscientious wayContributors BF G LL JAJ LR R and LA conceived and designed the study DDFN JFU AMG JIC and LL collected data LA and LL performed the data analysis and interpreted the data All of the authors were involved in the drafting andor revising of the manuscriptFunding This work was supported by the Instituto de Salud Carlos III ISCIII Ministry of Health Spain CP1600916 PI1801188 and RD1600120014 and cofunded by el Fondo Europeo de Desarrollo Regional FEDER The funders had no role in study design data collection analysis manuscript preparation or decision to publishCompeting interests None declaredPatient and public involvement Patients andor the public were not involved in the design or conduct or reporting or dissemination plans of this researchPatient consent for publication Not requiredEthics approval The study was approved by the Hospital Cl­nico San Carlos institutional ethics committee approval number E BS This study was conducted according to the principles of the Declaration of HelsinkiProvenance and peer review Not commissioned externally peer reviewedData availability statement Data are available upon reasonable requestThis article is made freely available for use in accordance with BMJ™s website terms and conditions for the duration of the covid19 pandemic or until otherwise determined by BMJ You may use download and print the article for any lawful non commercial purpose including text and data mining provided that all copyright notices and trade marks are retainedORCID iDsLeticia a0Leon http orcid Luis a0Rodriguez Rodriguez http orcid REFERENCES Fernandez Gutierrez B COVID19 with pulmonary involvement An autoimmune disease of known cause Reumatol Clin “ COVID19 Situaci³n actual en La Comunidad de Madrid Informe de situaci³n del de Mayo Available httpswww comunidad madrid sites default files doc sanidad 200508_ cam_ covid19 pdf [Accessed May ] Chen N Zhou M Dong X et a0al Epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in Wuhan China a descriptive study Lancet “ Figueroa Parra G Aguirre Garcia GM Gamboa Alonso CM et a0al Are my patients with rheumatic diseases at higher risk of COVID19 Ann Rheum Dis “ Favalli EG Ingegnoli F De Lucia O et a0al COVID19 infection and rheumatoid arthritis Faraway so close Autoimmun Rev Zhou F Yu T Du R et a0al Clinical course and risk factors for mortality of adult inpatients with COVID19 in Wuhan China a retrospective cohort study Lancet “ Monti S Balduzzi S Delvino P et a0al Clinical course of COVID19 in a series of patients with chronic art

Thyroid_Cancer

catalytic activity of human Telomerase Reverse Transcriptase TERT compensates for the loss of telomere length eroded during each cell cycle to ensure a correct division of stem and germinal cells In human tumors ectopic TERT reactivation most frequently due to hotspot mutations in the promoter region TERTp ie c1124 C T c1146 C T confers a proliferative advantage to neoplastic cells In gliomas TERTp mutations TERTpmut mainly occur in oligodendroglioma and glioblastoma We screened for TERTp hotspot mutations adult patients with gliomas and identified heterozygous mutations in cases of oligodendroglioma of glioblastoma and of diffuseanaplastic astrocytoma Besides the recurrent c1124 C T and c1146 C T two cases of glioblastoma harbored novel somatic TERTp variants which consisted of a tandem duplications of nucleotides ie a TERTp c1100_179dup and TERTp c1110_189 both located downstream c1124 C T and c1146 C T In silico analysis predicted the formation of and new transcription factor™s recognition sites for TERTp c1100_179dup and TERTp c1110_189 respectively TERTp duplications TERTpdup mainly affected the binding capacity of two transcription factors™ families ie the members of the Etwentysix and the Specificity ProteinKr¼ppelLike Factor groups In fact these new TERTpdup significantly enhanced the Etwentysix transcription factors™ binding capacity which is also typically increased by the two c1124 C Tc1146 C T hotspot TERTpmut On the other hand they were distinguished by enhanced affinity for the Kr¼ppel proteins The luciferase assay confirmed that TERTpdup behaved as gainoffunction mutations causing a fold increase of TERT transcription The present study provides new insights into TERTp mutational spectrum occurring in central nervous system tumors with the identification of new recurrent somatic gainoffunction mutations occurring in of glioblastoma IDHwildtypeKeywords TERT Gliomas Gainoffunction mutation ETS and Kr¼ppel transcription factorsIntroductionThe abnormal reactivation of human Telomerase Reverse Transcriptase TERT is a common hallmark of human solid tumors Although it may be caused by Correspondence cristinamecucciunipgit robertalastarzaunipgit Cristina Mecucci Roberta La Starza have equally contributed to this work Molecular Medicine Laboratory Centro di Ricerche EmatoOncologiche CREO S Maria della Misericordia Hospital University of Perugia Ple Menghini Perugia ItalyFull list of author information is available at the end of the several mechanisms ie methylation mutations rearrangementsfusions and DNA copy number amplifications TERT promoter TERTp methylation and gainoffunction mutations are the most frequent [ ] In particular two recurrent hotspot mutations are respectively located at TERTp124 and TERTp146 base pairs bp from the TERT ATG start site [ “ ] Both mutations generated from a cytidine to thymidine dipyrimide transition C T are usually heterozygous mutually exclusive and produce The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cPierini a0et a0al acta neuropathol commun Page of an identical a0bp ˜CCC CTT CCGGG™ sequence resulting in the creation of de novo consensus binding motifs for Etwentysix ETS transcription family members These new binding sites recruit a larger number of ETS factors enhancing the transcription of TERT []TERT promoter mutations TERTpmut typically occur in tumors that arise from low selfrenewal tissue such as melanomas thyroid hepatobiliary carcinoma and central nervous system CNS tumors with a variable frequency that range from to of cases in diverse histological subtypes [ ] In CNS tumors TERTpmut are typically associated with glioblastoma GBM “ and oligodendroglioma ODG “ whereas their frequency decreases in other glioma subtypes such as diffuseanaplastic astrocytoma DAAA “ medulloblastoma and meningioma about [ ] Although the clinical value of TERTpmut in refining the diagnostic classification of gliomas is widely accepted [] its role as prognosticpredictive biomarker is still largely debated TERTpmut have been associated with a poor disease outcome in GBM IDHwildtype GBM IDHwt but there is no full agreement on its impact on DAAA [ ] It is worth noting however that DAAA IDHwildtype DAAA IDHwt harboring genomic abnormalities typically associated with GBM ie TERTp mutations or EGFR amplification or gain of whole chromosome in combination with monosomy of chromosome have a clinical outcome similar to or only slightly longer than GBM [] Thus the cIMPACT NOW Update recommended to use one of these molecular criteria to classify this subgroup of astrocytomas as œdiffuse astrocytic glioma IDHwildtype with molecular features of glioblastoma WHO grade IV and to revise the classification of DAAA IDHwt accordingly []Herein we report two new TERTp mutations that were identified in two patients with GBM IDHwt Both these new variants originated from the duplication of a stretch of nucleotides at TERTp TERTpdup and although slightly different shared an overlapping sequence of nucleotides We demonstrated the somatic nature of one of these TERTpdup and that enhancing the binding affinity for ETS transcription factors TFs they both elicit the TERT transcription thus widening the spectrum of recurrent gainoffunction mutations of TERTp in GBMCase presentationCohortThe study was carried out on a cohort of patients affected by primary CNS tumours and referred to our laboratory during the last a0years Table a0 There were males and females ratio with a median age of range age According to the WHO the diagnosis was grade II DA IDHwt cases and DA IDHmutant DA IDHmut cases grade III AA IDHwt cases and AA IDHmut grade IV GBM IDHwt and GBM IDHmut grade IIIII ODG Three patients had a diagnosis of uncommon glioma Table a0 The study was approved by Institutional Bioethics Committee University of Perugia and Santa Maria della Misericordia Hospital of PerugiaItaly Protocol no284316 all patients gave informed consent for sample collection and molecular analyses in agreement with the Declaration of HelsinkiIndex casesA 71yearold male UPN131 had a left frontal lesion of a0mm diameter partially infiltrating the corpus callosum the second case UPN171 a male of a0years presented with a right frontal lesion Histopathology and immunohistochemistry were consistent with a diagnosis of GBM IDHwt in both patients In case UPN131 neoplastic cells showed marked cytoplasmic and nuclear pleomorphism there was a discrete number of atypical mitotic figures widespread necrosis a diffuse GFAP positivity and few neoplastic elements with strong nuclear TP53 stain Case UPN171 was characterized by striking atypia of neoplastic cells diffuse necrosis vascular proliferation strong and diffuse positivity for GFAP and nuclear TP53 Fig a0 No IDH1IDH2 hotspot mutations were detected while both cases showed MGMT promoter methylation Monosomy of chromosome cooccurred with EGFR amplification UPN131 or with gain of the whole chromosome UPN171Materials and a0methodsTERT promoter mutational analysisGenomic DNA was extracted from FormalinFixed ParaffinEmbedded FFPE tumor tissue and from peripheral blood PB by QIAamp DNA FFPE and AllPrep DNARNA kits respectively following the manufacturer™s instructions QIAGEN Milan Italy Hotspot TERTpmut were investigated by Sanger sequencing using ABI Genetic analyzer instrument Applied Biosystems Monza Italy Primers were reported in Table a0S1 Additional file a0 Table a0S1 and referred to GRCh37 genomic coordinate system NM_0000059 for regulatory core promoter a0 bp wwwncbinlmnihgovgene [] wwwensem blHomo_sapie ns [] Sequences™ alignments and their analyses were supported by Clustal wwwebiacukTools msaclust alo Ensembl Omega httpwwwensem blHomo_sapie ns and [] 0cPierini a0et a0al acta neuropathol commun Page of Table Epidemiological and a0clinical features of a0our cohort of a0patientsEpidemiologicalclinical dataTotal cohortGenderAge yearsDiagnosis WHO Common GliomasUncommon GliomasAnatomic locationMaleFemaleMFRangeMedian years‰¥ yearsDiffuse astrocytoma IDHwt grade IIDiffuse astrocytoma IDHmut grade IIAnaplastic astrocytoma IDHwt grade IIIAnaplastic astrocytoma IDHmut grade IIIGlioblastoma IDHwt grade IVGlioblastoma IDHmut grade IVOligodendroglioma IDHmut and 1p19qcodeleted grade IIAnaplastic oligodendroglioma IDHmut and 1p19qcodeleted grade IIIPilocytic astrocytoma grade IPleomorphic xanthoastrocytoma grade IIAnaplastic pleomorphic xanthoastrocytoma grade IIIFrontalFrontalparietalFrontaltemporalParietalParietaloccipitalTemporalTemporalparietalTemporaloccipitalOccipitalCerebellar hemisphereCorpus callosumThalamusPituitary glandInsularMulticentric pts pts pts pts pts patients wt wildtype mut mutantCOSMIC https cance rsange racukcosmi c websites []In silico TERTpmut functional analysis JASPAR toolThis bioinformatic tool estimates the binding affinity and the number of TFs binding sites for the input sequence provided in FASTA format A relative threshold score of and Δ relative score ‰¥ mutant™s relative score”wildtype™s relative score were chosen to define the statistically significant changes induced by TERTpmut as previously reported [] The JASPAR CORE predicted the effects of the four different TERTpmut that we detected in our patients ie the two new TERTpdup the TERTp and the TERTp146 on TFs binding capacity JASPAR CORE Collection httpjaspa rgener egnet 8th version [ ] JASPAR was also used to analyze two TERTpdup which have been previously reported in a case of MDS c1110_1101dup and in a case of thyroid cancer c1104_183dup [ ] According to JASPAR data we used the Venn diagram to plot TFs for which a significant enhanced probability of binding capacity or an 0cPierini a0et a0al acta neuropathol commun Page of Fig Histological and immunohistochemical analysis in patient UPN171 a HematoxylinEosin staining original magnification 200X enlarged neoplastic cells with multiple often bizarre hyperchromatic nuclei and high number of mitoses Vascular proliferation as seen in these œglomeruloids lower half of the image is a specific pattern of microvascular growth b HematoxylinEosin staining original magnification 400X multiple mitotic figures are evident in the middle field œGeographic pattern of necrosis detail in insert panel b c Positive GFAP staining highlights high neoplastic cells with astrocytic differentiation d Intense and diffuse nuclear TP53 stainingincrease of the number of binding sites was predicted httpbioin forma ticspsbugent bewebto olsVennIn vitro TERTpmut functional study luciferase assayTo study the effect of TERTpmut on the expression of TERT a luciferase assay was done for the TERTpdup detected in case UPN171 the TERTp146 UPN205 and the TERTp124 UPN216 The TERTdup of case UPN131 could not be studied due to lack of material A TERTp wildtype TERTpwt construct already available in the laboratory was also used as reference Additional file a0 Table a0S2 [] TERT core promoter a0bp was amplified with specific primers reported in Table a0 S3 Additional file a0 Table a0 S3 introducing cleavage sites for BglII forward and HindIII reverse restriction enzymes Then TERTpmut constructs were inserted in pGEMT easy plasmid Promega Madison WI USA and cloned in Electromax DH10BT1 cells Invitrogen Milan Italy to increase the amount of mutant DNA Finally the inserts were subcloned in pGL410[luc2] vectors Promega Madison WI USA upstream of LUC2 gene encoding for luciferase enzyme of Photinus Pyralis and resequenced An empty pGL410[luc2] vector was also used as negative control Luciferase assay was performed using the GBM U87MG cell line maintained in Dulbecco™s Modified Eagle Medium Thermo Fisher Scientific Monza Italy with fetal bovine serum and streptomycinpenicillin at a0°C5 CO2 U87MG cells were seeded in a 6multiwell plate — cellsml cotrasfected with a0 µg of modified pGL410[luc2] plasmids and with of pGL474[hRlucTK] a vector containing the luciferase gene of Renilla Reniformis by Viafect Transfection Reagent Promega Madison WI USA After 24h incubation cells were lysed and fluorescence emission was assessed using DualGlo Luciferase assay kit Promega following manufacturer™s instructions All experiments were performed in triplicate in three independent experiments 0cPierini a0et a0al acta neuropathol commun Page of ResultsNew somatic TERT promoter variantsTERTpmut were detected in cases including ODG DAAA and GBM Additional file a0 Table a0 S4 In GBM and DAAA TERTpmut were prevalent in IDHwt cases GBM IDHwt vs GBM IDHmut DAAA IDHwt vs DAAA IDHmut Chi square P Additional file a0 Table a0S5 Thus in agreement with the diagnostic criteria recommended by the cIMPACTNOW Update the DAAA IDHwt with TERTpmut were referred to as œdiffuse astrocytic glioma IDHwildtype with molecular features of glioblastoma WHO grade IV []In GBM TERTpmut there was a significant enrichment of cases harbouring EGFR amplification vs Chi square P andor monosomy 10PTEN deletions vs Chi square P Likewise EGFR amplification or gain of whole chromosome in combination with monosomy occurred in of TERTpmut DAAA IDHwtThe most common variant TERTp124 was detected in cases while the TERTp146 was found in cases TERTpmut were mutually exclusive heterozygous and equally distributed among the different histological subtypes Additional file a0 Table a0S5 Besides the TERTp124 and TERTp146 we uncovered two new TERTp variants in two cases of GBM IDHwt UPN131 and UPN171 These novel TERTpmut consisted of a nucleotide tandem duplication occurring in a genomic region starting at and a0bp from the ATG starting site ie c1100_179dup TERTp10079 in case UPN131 and c1110_189dup TERTp11089 in case UPN171 Fig a02a b wwwncbinlmnihgovgene wwwensem blHomo_sapie ns cancersangeracukcosmic [ ] They shared a region of duplication of nucleotides from “ to “ nucleotides from the ATG start site The absence of TERTp10079 in the PB DNA demonstrated the somatic origin of this variant in case UPN131In silico analysis predicts TERTpmut effectsIn silico analysis predicted that both TERTpdup created new binding sites ie for TERTp10079 and for TERTp11089 which were respectively recognized by and TFs Instead TERTp124 and TERTp146 were predicted to increase the binding affinity for and sites and to enhance the probability of binding for and TFs respectively Additional file a0 Table a0S6 Although all TERTpmut affected the binding sites for diverse families of TFs the ETS group emerged as one of the most frequently involved in TERTp10079 for TERTp11089 in TERTp124 and in TERTp146 Fig a0 2c Additional file a0 Table a0 S7 Other recurrently involved TFs in TERTpdup variants were the Specificity ProteinKr¼ppelLike Factor SpKLF family ie in TERTp10079 and in TERTp11089 and the More than adjacent zinc finger factors family in TERTp10079 and TERTp11089 Additional file a0 Table a0S7The Venn diagram showed a close interrelationship between all TERTp mutations Namely all TERTp mutations shared an increase of the binding affinity or the number of binding motifs for common TFs Fig a03a including ETS members ETS1 ETS2 ERG ELK1 ETV6 FLI1 ELK4 SPIB ELF1 ELF3 ETV4 ETV1 FEV EHF ETV5 ELF5 SPI1 and GABPA and TEAD1 Fig a03a Additional file a0 Table a0S8 The Venn diagram also showed that the new TERTpdup were characterized by the exclusive involvement of common TFs Specifically there were SpKLF members ie KLF2 KLF3 KLF4 KLF5 KLF10 KLF11 KLF14 KLF15 KLF16 SP1 SP2 SP3 SP4 SP8 SP9 and EGR1 Fig a03a Additional file a0 Table a0 S8 and TFs that belong to different families Fig a03a Additional files and Tables S7 and S8 Matching our TERTpdup with the two cases of TERTpdup previously reported Additional files and Tables S9 and S10 [ ] JASPAR predicted that all variants determined an increase of binding sites for common TFs and confirmed that the SpKLF family was the most frequently involved Fig a0 3b Additional file a0 Table a0S11In vitro analysis confirms the a0increasing of a0TERT transcriptional activity induced by a0its promoter mutationsIn vitro luciferase assay was carried out to evaluate whether the new TERTp11089 variant induced an increase of TERT transcriptional activity enhancing its expression similarly to TERTp124 and TERTp146 [ ] In Table a0S12 Additional file a0 Table a0S12 we reported raw data referred to the fluorescence emission values expressed in Relative Luciferase Activity RLA of both Photinus Pyralis and Renilla Reniformis luciferase enzymes for all samples Our experiments demonstrated that all three variants caused a significant increase of TERT transcription by fold than wildtype TERTp11089 vs TERTpwt P TERTp124 vs TERTpwt P TERTp146 vs TERTpwt P Mann“Whitney U test Fig a0 On the other hand no differences on the levels of TERT expression were present between the diverse TERTp variants indicating they may all behave as gainoffunction mutations likely exerting the same consequences on TERT transcription 0cPierini a0et a0al acta neuropathol commun Page of Fig Schematic representation of TERTp mutations a TERT promoter electropherogram in case UPN131 The arrow indicates the start point of the c1100_179dup b TERT promoter electropherogram in case UPN171 The arrow indicates the start point of the c1110_189dup c Overview of all TERTp variants detected in our cases Upper arrow wildtype TERT core promoter with the normal location of ETS binding sites The vertical black lines indicate the genomic positions of TERTp variants Lower arrow positions and types of TERTp variants and their predicted effects on transcription factors binding sitesDiscussionAbnormal genomic events that alter telomere elongation are common in gliomas Particularly mutually exclusive mutations affect the TERT or the ATRX chromatin remodeler ATRX genes a critical regulator of telomere homeostasis by chromatin remodeling []Our studies on a cohort of patients confirmed previous data on the incidence and distribution of TERTpmut in diverse subtypes of CNS tumors As expected we found that TERTpmut were highly recurrent in ODG and GBM and less frequent in DAAA Additional file a0 Table a0 S4 TERTpmut were significantly enriched in GBM IDHwt cases Chi square P Additional file a0 Table a0S5 where they mainly occurred together with EGFR amplification Chi square P andor monosomy 10PTEN deletions Chi square P Similarly in DAAA TERTpmut were highly recurrent in IDHwt cases thus allowing the reclassification of of these subgroup of astrocytomas as œdiffuse astrocytic glioma 0cPierini a0et a0al acta neuropathol commun Page of ODG [] Afterwards TERTpdup were found in a case of myelodysplastic syndrome MDS c1110_1101dup and in a case of papillary thyroid carcinoma c1104_183dup [ ] Published TERTpdup as well as our cases are located in the same core promoter region that span a0 bp from the ATG start site Furthermore they are all located downstream TERTp124 and TERTp ie at “ nucleotides from TERTp124 and nucleotides from TERTp146 in a region that contains the binding sites for the TFs modulating TERT transcription Interestingly in silico analysis predicted these new TERTdup affect the transcriptional regulation of the gene through the creation of new binding sites for TFs that mainly belong to the ETS family Fig a02c Additional file a0 Table a0S7 Likewise an increased number of binding sites or an enhanced affinity for the ETS TFs has been previously reported in a thyroid cancer harbouring a TERTp c1104_183dup variant and in cases bearing TERTp124 or TERTp146 mutations [ ] Bioinformatic analyses were consistent with the luciferase data showing a significant increase of TERT expression in cells transfected with the new TERTp11089 variant as well as with the two recurrent TERTpmutThen we sought to assess the possible interrelationship between the four diverse TERTp mutations using the Venn diagram Fig a03a All four TERTp variants were predicted to share an increase binding capacity for ETS members Fig a03a Additional file a0 Table a0S8 which included GABPA a putative oncogene in GBM Namely in a0vitro studies on GBM cell lines have demonstrated that this transcription factor is needful in mediating the transcriptional reactivation of TERT dependent from TERTp or TERTp146 [ ] Besides ETS TFs all TERTp variants affected the binding capacity for TEAD1 a protein that belongs to TEF1related factors family and that has been demonstrated to act as a putative oncogene in GBM favoring cell infiltration in a0 vitroin vivo models []Although TERTp124 and TERTp146 and the new TERTp10079 and TERTp11089 variants shared the same effects on the binding capacity for ETS members the latters were characterized by the exclusive involvement of TFs mainly belonging to SpKLF family Fig a0 3a Additional files and Tables S7 and S8 SpKLF TFs are involved in a plethora of cellular processes ranging from proliferation and differentiation pluripotency and apoptosis in normal and tumoral tissues []Fig The Venn diagrams show all possible relations among a four TERTp variants reported in our cases refer to Additional file Table S8 and b TERTpdup described in this study c1100_179dup and c1110_189dup and those reported in literature c1104_183dup and c1110_1101dup refer to Additional file Table S11IDHwildtype with molecular features of glioblastoma WHO grade IV []Besides the two known TERTp124 and TERTp146 variants we uncovered two new TERTp variants in two cases of GBM IDHwt UPN131 and UPN171 These novel TERTpmut consisted of a nucleotide tandem duplication sharing a duplicated region of nucleotides from “ to “ from the ATG start site Hitherto somatic TERTpdup has been reported in three human tumors The first one a duplication of nucleotides in the TERT core promoter was detected in a case of Altogether these data support the hypothesis that the recruitment of ETS family TFs plays a pivotal role in mediating the reactivation of TERT transcription in human tumors bearing different types of TERTpmut However they also indicate that slight differences mark TERTpdup variants whose activities appear to be 0cPierini a0et a0al acta neuropathol commun Page of Fig Luciferase assay The histogram reports the relative luciferase activities RLA of TERTp wildtype and for the variants c1110_189dup c1124 C T and c1146 C T p value refers to probability obtained using Mann“Whitney U testalso dependent from Kr¼ppelrelated factors Indeed among the TFs shared by all TERTpdup Fig a03b belonged to SpKLF family as reported in Tables S10 and S11 Additional files and Hence the precise definition of mutationspecific profiles would strengthen the definition of TERTdependent oncogenesis mechanismsOur study contributes to enrich the spectrum of recurrent somatic TERTpdup variants reporting for the first time two new gainoffunction mutations ie TERTp10079 and TERTp11089 in of GBM IDHwt cases These new mutations can be reliably detected by diagnostic assays used to investigate hotspot TERTp and TERTp146 Although the assessment of TERTp mutational status is not an essential diagnostic criterion it can be a relevant information to assist histological diagnosis [] As a matter of fact the status of TERTp together with IDH mutations and 1p19q codeletion classify gliomas in distinct subcategories ie triple negative triple positive cases with IDHTERT mutations and cases with a unique mutation either IDH or TERT that are typified by unique demographic clinical and biological characteristics [] Moreover TERTpmut has been proposed as one of the most relevant molecular marker to stratify DAAA IDHwt [] Thus we consider that molecular testing of TERTp mutations should be included in the clinical workup of GBM and DAAA in order to provide a precise diagnosis prospective multicentric studies on large cohort of patients will clarify the value of TERTp mutations as prognostic markerSupplementary informationSupplementary information accompanies this paper at https doi101186s4047 Additional file a0 Table a0S1 Primer set used for Sanger sequencing Additional file a0 Table a0S2Samples used for in vitro luciferase assay Additional file a0 Table a0S3 Primer set used to create constructs for luciferase assay Additional file a0 Table a0S4 Incidence and distribution of TERTp variants in the main glioma subgroups Additional file a0 Table a0S5 Incidence and distribution of TERTp variants in glioma subtypes according to WHO guidelines Additional file a0 Table a0S6 JASPAR analysis for the TERTp c1124 CT c1 CT and the new TERTpdupc1100_179dup c1110_189dup Additional file a0 Table a0S7 Transcription Factors predicted to be involved in TERTp variants Additional file a0 Table a0S8 Transcription Factors predicted to be involved in different TERTp variants Additional file a0 Table a0S9 JASPAR analysis for the two published TERTp duplications c1110_1101dup and c1104_183dup [ref ] Additional file a0 Table a0S10 Transcription factors predicted to be involved in the TERTpdup c1110_1101dup and c1104_183dup [ref ] Additional file a0 Table a0S11 Transcription factors predicted to be involved in all TERTp duplications Additional file a0 Table a0S12 Luciferase assay raw dataAbbreviationsTERT Telomerase Reverse Transcriptase TERTp TERT promoter TERTpmut TERT promoter mutation TERTpdup TERT promoter duplication TERTp124 c1124 TERT promoter mutation TERTp146 c1146 TERT promoter mutation bp base pair ETS Etwentysix transcription factor CNS central nervous system GBM glioblastoma ODG oligodendroglioma DA diffuse astrocytoma AA anaplastic astrocytoma GBM IDHwt glioblastoma IDHwildtype DA IDHwt diffuse 0cPierini a0et a0al acta neuropathol commun Page of astrocytoma IDHwildtype AA IDHwt anaplastic astrocytoma IDHwildtype TFs transcription factors DA IDHmut diffuse astrocytoma IDHmutant AA IDHmut anaplastic astrocytoma IDHmutant GBM IDHmut glioblastoma IDHmutant FFPE formalinfixed paraffinembedded PB peripheral blood MDS myelodysplastic syndrome TERTpwt TERTp wildtype TERTp10079 c1100_179dup TERTp11089 c1110_189dup SpKLF Specificity ProteinKr¼ppelLike Factor RLA relative luciferase activity ATRX ATRX chromatin remodelerAcknowledgementsNot applicable Authors™ contributionsTP RLS conceived the study planned the experiments and wrote the paper TP carried out and evaluated mutational analysis and in vitro functional studies CN made in silico analysis AGLF contributed in the analysis of in vitro luciferase assay MM and SA performed DNA extraction and FISH experiments FP VN and PG performed sequencing analysis PG SA and MEL provided the diagnosis and the tissue sections for molecularcytogenetic studies CC and RC ML GM and CM provided all clinical data VP GR and CM were involved in drafting the manuscript All the authors read and approved the final manuscript FundingThe project was supported by Comitato per la vita œDaniele Chianelli Perugia Italy Sergio Luciani Association Fabriano Italy and Fondazione Cassa di Risparmio Perugia Italy Grant numbers to RLS Availability of data and materialsAll data generated or analyzed during this study are included in this published [and in its supplementary information files]Ethics approval and consent to participateThis study was approved by the local ethic committee CEAS code number August 8th Consent for publicationAll participants signed an institutional informed consentCompeting interestsThe authors declare that they have no competing interestsAuthor details Molecular Medicine Laboratory Centro di Ricerche EmatoOncologiche CREO S Maria della Misericordia Hospital University of Perugia Ple Menghini Perugia Italy Hematology and Center of Bone Marrow Transplants Medicine and Surgery Department University and Hospital of Parma Via Gramsci Parma Italy Diagnostic Cytology and Histology Unit S Maria della Misericordia Hospital Ple Giio Menghini Perugia Italy Division of Radiotherapy S Maria della Misericordia Hospital Ple Giio Menghini Perugia Italy Medical Oncology S Maria della Misericordia Hospital Ple Giio Menghini Perugia Italy Division of Neurosurgery S Maria della Misericordia Hospital Ple Giio Menghini Perugia Italy Pathology Unit S Maria Hospital V Tristano di Joannuccio Terni Italy Received June Accepted August References Allory Y Beukers W Sagrera A Fl¡ndez M Marqu©s M M¡rquez M et al Telomerase Reverse Transcriptase promoter mutations in bladder cancer high frequency across stages detection in urine and lack of association with outcome Eur Urol “ Barthel FP Wei W Tang M MartinezLedesma E Hu X Amin SB et al Systematic analysis of telomere length and somatic alterations in cancer types Nat Genet “ Bell RJ Rube HT Kreig A Mancini A Fouse SD Nagarajan RP et al The transcription factor GABP selectively binds and activates the mutant TERT promoter in cancer Science “ Brat DJ Aldape K Colman H Holland EC Louis DN Jenkins RB et al cIMPACTNOW update recommended diagnostic criteria for œDiffuse astrocytic glioma IDHwildtype with molecular features of glioblastoma WHO grade IV Acta Neuropathol “ COSMIC Catalogue of Somatic Mutations in Cancer Database Wellcome Sanger Institute Cambridge UK https cance rsange racukcosmi c Accessed May EckelPassow JE Lachance DH Molinaro AM Walsh KM Decker PA Sicotte H et al Glioma groups based on 1p19q IDH and TERT promoter mutations in tumors N Engl J Med “Ensembl DatabaseHomo Sapiens European Molecular Biology Laboratory™s European Bioinformatics Institute Cambridge UK httpwwwensem blHomo_sapie ns Accessed May Fornes O CastroMondragon JA Khan A van der Lee R Zhang X Richmond PA et al JASPAR update of the access database of transcription factor binding profiles Nucleic Acids Res 48D87“D92 https doi101093

Thyroid_Cancer

Computed Tomography Features andClinicopathological Characteristicsof Gastric Sarcomatoid CarcinomaYiyang Liu1 Pan Liang1 Kaixiang Feng2 Kuisheng Chen3 Songwei Yue1 Jiang Ji4Weiwei Li1 Xitong Zhao1 and Jianbo Gao1 Department of Radiology The First Affiliated Hospital of Zhengzhou University Zhengzhou China Departmentof Thyroid Surgery The First Affiliated Hospital of Zhengzhou University Zhengzhou China Department of PathologyThe First Affiliated Hospital of Zhengzhou University Zhengzhou China Department of Radiology General Hospital NingxiaMedical University Yinchuan ChinaPurpose Gastric sarcomatoid carcinoma GSC is a very rare malignant tumor Thepurpose of this study is to describe the clinical computed tomography CT andpathologic features of GSC to increase awareness of this entityEdited byZiwen LiuPeking Union Medical CollegeHospital CAMS ChinaMethods The CT features and clinical data of five patients with pathologicallydocumented GSC were retrospectively analyzed and compared with the correspondingdata of gastric adenocarcinoma and lymphomaReviewed bySavio Gee BarretoMedanta The Medicity IndiaHaruhiko SugimuraHamamatsu University Schoolof Medicine JapanXiuying XiaoShanghai Jiao Tong University ChinaCorrespondenceJianbo GaofccyisunshinegszzueducnSpecialty sectionThis was submitted toGastrointestinal Cancersa section of the journalFrontiers in OncologyReceived May Accepted July Published August CitationLiu Y Liang P Feng K Chen KYue S Ji J Li W Zhao X and Gao J Computed TomographyFeatures and ClinicopathologicalCharacteristics of GastricSarcomatoid CarcinomaFront Oncol 103389fonc202001611Results Among the patients were male and was female The median agewas years Of the cases of GSC were in the gastric fundus and cardia was in the gastric body and was in the gastric fundus The gastric wall had localthickening in cases and mass formation in case with stenosis and deformationof the adjacent gastric cavity The longaxis diameter of the lesions ranged from to cm mean cm and was cm in cases and cm in case The tumorshowed predominantly inhomogeneous density with radiodensity values ranging from to HU In addition ulcers with an irregular base and slightly raised borders wereobserved in of cases After an injection of contrast material heterogeneous n or homogeneous n enhancement was observed After contrast medium injectionobvious enhancement was seen in cases and moderate enhancement was seen in cases the peak tumor signal was observed in the portal phase Two of the patientsdemonstrated evidence of lymph node involvement and in one patient the boundarybetween the lesion and the left lobe of the liver was unclear with low attenuation in theright lobe of the liver with circular enhancement The remaining two patients showed noevidence of metastasisConclusion Although GSC is extremely rare it should be considered in the differentialdiagnosis of gastric adenocarcinoma and lymphoma CT findings combined withpatient age and sex can provide support for the diagnosis of GSC However the finaldiagnosis must be confirmed with histopathologyKeywords sarcomatoid carcinoma stomach gastric cancer tomography Xray computed diagnosisFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alINTRODUCTIONSarcomatoid carcinomas SCs are extremely rare aggressivemalignant tumors characterized by distinct cellular morphology The features of this tumor were first described in by Snover SCs can occur in a wide variety of sitesincluding the respiratory tract digestive tract genitourinary tractbreast and thyroid glands However these tumors are rarein the digestive tract especially in the stomach As of April there are only six cases of gastric sarcomatoid carcinomaGSC reported in the English medical literature These previousreports focused on the pathological and clinical manifestationsthem have not systematically described the radiologic appearanceof the tumor Due to the more invasive nature and poorerprognosis of GSC than pure gastric adenocarcinoma GACand gastric lymphoma GL it is clinically beneficial to narrowdown the diï¬erential diagnoses by understanding the computedtomography CT characteristics of GSC The present studyanalyzed our experience in diagnosing five patients with GSCin terms of the imaging findings and clinical features To thebest of our knowledge our study represents the largest seriesof GSCs to dateIn addition due to the rarity of GSC the diï¬erential diagnosisbetween GSC and other types of malignant gastric tumors hasnot received much attention so we also initially explored thediï¬erential diagnosis of GSC from GAC and GLMATERIALS AND METHODSThe protocol was approved by the Medical Ethics Committeeof Zhengzhou UniversityInformed consent was obtainedfrom all patientsPatient SelectionFrom August to January we searched the pathologyrecords and the Picture Archiving and Communication SystemsPACS of our hospital The search terms included stomachand sarcomatoid carcinomas A total of five patients wereidentified as having SC and were enrolled in the present study Weretrospectively reviewed all clinical data demographic featureslaboratory findings clinical interventions and the histologicfindings of the five biopsy or operation specimensCT EvaluationFive GSC patients underwent CT examinations The CTscans were acquired with a 64row multidetector deviceDiscoveryCT750HD GE Healthcare Waukesha WIUnited States Conventional axial scanning was performedbefore and after an intravenous iv injection of nonioniciohexol iopromide mgmL GE Medical Systems mLkgand mLs through a dualhead pump injector MedradWarrendale PA United States The imaging parameters wereas follows tube voltage kV tube current mA fieldof view FOV mm matrix — mm and sectionthickness mm Finally a 20mL saline flush was performedat a rate of mLsGastric Sarcomatoid CarcinomaContrastenhanced CT scans were acquired with scanningdelays of s arterial phase AP and s portal venous phasePP after the iv injection of the contrast agent started The CTdose index volume for the three phases was mSvImage AnalysisTwo experienced radiologists and years of abdominal CTexperience performed a retrospective analysis of the CT imagesAll analyses were performed with an AW47 workstation GEHealthcare and the radiologists were blinded to the clinicalinformation of the patients The evaluated parameters includedthe tumor location gastric cardia gastric fundus gastric bodygastric angle and gastric antrum longaxis diameter shapegrowth pattern serosa condition attenuation and enhancementcharacteristics such as the enhancement pattern and degreeof enhancement The enhancement pattern of the tumor wasclassified as homogeneous or heterogeneous based on the APimage The degree of enhancement of the tumor was based ondynamic CT imaging using HU attenuation where œobviousenhancement was defined as HU œmoderate enhancementas HU and œmildly enhancement as HUThe GSCs were staged with the Union for InternationalCancer Control UICC TNM staging standard All imagingfindings were compared with the postoperative pathologicalfindings The accuracy rate the number of CTs coincidentwith the pathological diagnosisthe number of actual pathologicaldiagnoses — Pathological EvaluationThree patients underwent gastrectomy and two underwentendoscopic biopsy The three gastrectomy specimens measured cm — cm — cm cm — cm — cmcm — cm — cm respectively in two of theseand tumors the mucosal surface of the excised specimen showedulcerative masses of approximately cm — cm — cmand cm — cm The remaining specimen was a soft massmeasuring cm — cm — cm For biopsy multiple sampleswere acquired and the diameter of each sample was cmAccording to the relevant literature the diagnostic criteria fSC were generally as follows the tumor originated fromthe stomach and the tumor consisted of both carcinomatousand sarcomatoid components and the sarcomatoid componentaccounted for more than of the tissue In addition if biopsywas performed the sarcomatoid component can be seen in everysample Furthermore sarcomatoid regions express epithelialmarkers such as CK or EMAThe specimens were fully stretched fixed and soaked in formaldehyde solution for h All biopsy specimenswere analyzed The specimens underwent routine dehydrationparaffin embedding sectioning into µm thick sectionsand hematoxylin eosin HE staining Immunohistochemicalstaining was performed using a Roche BenchMark XT automaticimmunohistochemical detector The antibodies used in thisstudy included AE1AE3 CKL CK818 epithelial membraneantigen EMA vimentin P40 P63 and antigen KI67 Ki67All antibodies listed above were purchased from DAKO DakoGlostrup DenmarkFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alGastric Sarcomatoid CarcinomaTABLE Comparison between GSC and GAC GLAge median age rangeMain symptomsEpigastric discomfortpainIntermittent vomitingAcute hematemesisBloody stoolDysphagiaLocationCardia and FundusBodyAntrumThe longaxis diameter median size rangeShapeFocal thickeningDiffuse thickeningMassSerosal surfacebare areaClearUnclearUlcersYesNoDensity characteristicsHeterogeneousHomogeneousEnhancement patterHeterogeneousHomogeneousLymph node involvementYesNoLiver involvementYesNoTherapyResectionChemotherapyResection and ChemotherapyNeoadjuvant chemotherapyRadiation therapyGSCGACGL “ years “ years “ years “ cm “ cm “ cm Comprehensive Comparative AnalysisEach patient with GSC was matched by age ± years year ofdiagnosis and sex to four patients with GAC GL patients witheach disease were retrieved from PACS Patients with GSC werecompared with those with GAC GL in terms of demographicclinical and CT characteristics Table RESULTSPatient CharacteristicsThe patients included four men and one woman ranging inage from to years with a median age of years Theclinical and CT features of these patients are summarized inTables All patients had nonspecific symptoms includingabdominal discomfort epigastric discomfort nausea or vomitingThe other presenting symptoms included hematemesis or weightloss Three patients underwent radical resectionin whichonly one patient was treated with adjuvant chemotherapyafter surgery And two patients chose to deny treatment Inaddition we also reviewed the upper gastrointestinal radiographyresults Figure The laboratory findings revealed that patient was positivefor tumor abnormal protein TAP and patient was positivefor carbohydrate antigen CA125 Before treatmenthemoglobin and erythrocyte count decreased in three patientsFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alGastric Sarcomatoid CarcinomaTABLE Clinical and pathological factors of the five GSC patientsCaseSexAge yearsComplaintLocationMaximumdiameterTumor markercmAnemiaTherapyMetastasisMMFMMSuddenhematemesisEpigastricdiscomfortIntermittentvomitingEpigastric painEpigastric painEpigastric painLesser curvatureRemnant stomachCardia andFundusCardia and FundusFundusCardia and FundusNormalTAP CA125 NormalNA““RRnNoneNoneRC“““œ yespresentpositive œ“ noabsentnegative F female M male age in years R Radical gastrectomy Rn Remnant gastrectomy C chemotherapyNA not availableTABLE Computed tomography features of the five GSC patientsCaseGross features of the tumorUlcersGrowth modeDensity characteristicsEnhancement patterFocal thickeningFocal thickeningMassFocal thickeningFocal thickening“IntracavityIntracavityIntracavityIntracavityIntracavityHeterogeneousHeterogeneousHomogeneousHeterogeneousHeterogeneousHeterogeneousHeterogeneousHomogeneousHeterogeneousHeterogeneousMarginUnclearUnclearUnclearClearUnclearœ yespresentpositive œ“ noabsentnegativeFIGURE Characteristics of Xray examinations of a 65yearold male patient with GSC AB Reveals that there is a huge niche with irregular shapes at the smallcurvature of the stomach the niche is located inside the outline of the stomach the niche is surrounded by transparent bands with different widths that is ringembankments with irregular outlines The surrounding mucosa is thickened interrupted and the local gastric cavity is narrowedpatients and and platelet count was elevated in fourpatients patients and Pathological FeaturesMicropathologically the gastric tumor cells showed infiltrativegrowth The cytological characteristics ofthe tumor cellsshowed obvious malignant characteristics Microscopicallythe spindle cell structure and the nucleus were obviouslyatypical pleomorphic and enlarged Mitotic figures were visibleFigures 2AB On immunohistochemical examination thetumor cells showed positive staining for AE1AE3 CKLCK818 EMA P40 vimentin The Ki67 index was higher than Figures 2C“I All five tumors were diagnosed as GSCIn addition the sarcomatoid component showed spindle cellsarcomatoid morphologyCT FindingsOf the cases of GSC were in the gastric fundus and cardiaFigure was in the gastric body and was in the gastricfundus of these tumors one was a recurrence in the remnantstomach The CT manifestations of this tumor included localthickening n mass formation n The longaxisFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alGastric Sarcomatoid CarcinomaFIGURE Histological and immunohistochemical features of GSC AB Hematoxylineosin HE staining showing tumor cells demonstrated spindleshapedstructures significant atypical nuclei pleomorphic nuclei and giant nuclei Mitotic figures visible Tumor cells showed infiltrative growth Cells were stained withhematoxylin and eosin stain magnification A — B — By immunohistochemistry the tumor cells were positive for AE1AE3 C CKL D CK818 E EMAF P40 G and vimentin H Moreover of them were positive for Ki67 I The final diagnosis was SC [magnification C“I —]diameter of the lesions ranged from to cm mean size cm In addition ulcers with an irregular base and slightlyraised borders were observed in of cases Among the threepatients who underwent surgery two lesions invaded the gastricserosa and the remaining lesion invaded the gastric bare areaAmong the two patients with biopsyproven GSC one patientexhibited tumor invasion of the gastric bare area The majorchanges in the CT imaging characteristics were an irregularouter layer of the gastric wall and obscuration of the perigastricfat Initially the CT findings were interpreted as GAC in fourcases and GL in The tumor showed predominantly inhomogeneous densityand the radiodensity values were “ HU in the noncontrastphase Heterogeneous enhancement was seen in four casesdue to necrotic or cystic areas and the other tumor revealedhomogeneous enhancement The radiodensity values on the APimages ranged from to HU and to HU in thevenous phase After contrast medium injection two tumorsshowed obvious enhancement and moderate enhancementwas seen in the other three tumors the peak tumor valuewas observed in the portal phase One of the three patientswho underwentlymphsurgery demonstrated evidence ofin one patientthe boundary betweennode involvementthe lesion and the left lobe of the liver was unclear andthe area with low attenuation was confirmed by pathologythe liver withas a metastatic lesion in the rightcircular enhancement The remaining patientshowed noevidence of metastasis Among the two patients with biopsyspecimens one patient was identified as having lymph nodemetastasis on CTlobe ofCT Staging Versus Pathological Stagingof GSCNone of the GSCs were staged as T1T2 by CT or pathologyThe accuracy of CT staging T3 and T4 GSC was and respectively The overall diagnostic accuracy of CTfor determining the T stage of GSC was None of the GSCs were staged as N2N3 by CT or pathologyThe accuracy of CT in staging N3 and N4 GSC was and respectively The overall diagnostic accuracy of CT fordetermining the N stage of GSC was The comparison of TN staging based on CT and pathology isshown in Table Frontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alGastric Sarcomatoid CarcinomaFIGURE Sarcomatoid carcinoma of the stomach in 62yearold women A Unenhanced CT image of stomach reveals an intraluminal mass of homogeneousattenuation with an irregular surface at the gastric fundus and cardiac region B“D Contrastenhanced CT image shows obvious homogeneous enhancement ofmass with the peak value of the tumor on the portal phase In perigastric lymph nodes an enlarged and significantly enhancement lymph node can be seenB Arterial phase of contrast enhancement image C Portal phase of contrast enhancement image D Portal phase of contrast enhancement coronal imageDISCUSSIONTABLE CT and pathological TN staging for comparisonSarcomatoid carcinoma is an extremely rare and complicatedmalignant tumor composed of malignant epithelial componentsand atypical spindle cells However the spindle cells of SCsappear to show evidence of epithelial diï¬erentiationforexample showing epithelial markers or epithelial ultrastructuralinstead of a specific line of mesenchymalcharacteristicsdiï¬erentiation Moreoverliteratureemphasizes that the sarcomatous components occupy ofthe elements involved In the present study our patients™tumor cells displayed atypical spindle shapes that expressed theepithelial phenotypethe currentsome ofSarcomatoid carcinomas can occur in almost any an wherecarcinoma can occur In the digestive system the incidencesof SCs in the esophagus and liver are relatively high but SCsare exceedingly rare in the stomach we could find only sixprevious reports in the English literature Table Between and patients with SC confirmed by pathologywere retrospectively analyzed with only five tumors occurringin the stomach The average age of the reported patientswas years range “ and that in our series was years range “ A previous study reported that the sexCaseNO NO NO NO NO CTT4aN0T3N0T3N1T3N0T4aN1NA not available T tumor N nodePathological stageT4aN1T3N0NANAT4aN0distribution of male to female GSC patients was and thecorresponding proportion in our patients was “ Ithas been noticed that SCs are more common in male patientsand sex is a probable risk factor GSC patients may present withepigastric pain or discomfort dysphagia nausea and vomitinghematemesis and emaciation Due to thickening of the gastricwall pain or discomfort in the upper abdomen is common Thesymptoms can last from a few days to several years withoutobvious specificityIn the present study of the cases of GSC were recognizedin the proximal stomach and the remaining tumor was founddistal to the stomach Four cases of GSC in the present study hadFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alGastric Sarcomatoid CarcinomaTABLE Clinical and imaging features of six previously reported cases of GCSCaseGenderAgeyearsLocationSize cmShapeUlcersEnhanceappearanceRecurrenceMetastasisTherapyPrognosis MMFFMMRemnant stomachGreater curvatureLesser curvatureGastroesophageal junctionRemnant stomachDistal stomachPolypoidPolypoidPolypoidUlceratedPolypoidMass“““NENENENENEHyperNE“““““NoneSurgerySurgerySurgeryEndoscopySurgeryNA Mo D Mo D Mo D Mo D Mo DThe patient succumbed to heart failure before the surgical treatment An autopsy was performed œ yespresentpositive œ“ noabsentnegative Hyper hyperdenseNE no evaluate Mo Month D Dieareregarded asa longaxis diameter less than cm and the remaining tumorhad the largest longaxis diameter among our patients cmThe location distribution and longaxis diameters of the GSCs inour patients were similar to those in previous reports “in the actual diagnosis processThe diagnosis of SC has always been difficult for cliniciansand pathologists especially the diï¬erential diagnosis fromcarcinosarcoma Carcinosarcomastrulybiphasic neoplasms composed of distinct malignant epithelialcarcinomatous and mesenchymal sarcomatous componentsThe sarcoma components show typical specialized diï¬erentiation Howeverthe termsœsarcomatoid carcinoma and œcarcinosarcoma have been usedinterchangeably in some cases Therefore the understanding ofthese tumors has been hampered Nevertheless we can try tofocus on whether there is a diï¬erence between these tumors froma new perspective The CT finding SC in the stomach have notbeen previously scientific reported or even detailed descriptionThere are only four simple descriptions ChunChao reported that a patient with a giant SC presented a mass witha cm diameter in the antrum and body of stomach whichinfiltrated the gastric serosa The hepatic flexure of the colon andgallbladder were also involved on CT Contrastenhanced CTimages showed obvious enhancement of the two lesions Sato reported a patient with SC of the remnant stomachand the radiographic examination showed an elevated lesionwith a large ulcer at the gastric cardiac lesser curvature thatmeasured cm in diameter The other two reports only describeda soft tissue mass or a large tumor in the dilated stomach On the other hand within in the upper gastrointestinal tractalthough there are fewer reports of carcinosarcoma localized inthe stomach this type of tumor is still more common than SC Gastric carcinosarcoma showed an elevated lesion or thickenedgastric walls in “ of all reviewed cases “ Tomoaki reported a 79yearold man with gastric carcinosarcomaand his veins showed severe invasion Enhanced abdominalCT showed irregular thickening and slight enhancement of thegastric wall on the side of the lesser curvature with suspiciousbulky lymph nodes Yoshiyuki reported a 70yearoldJapanese woman who presented with a soft tissue mass adjacentto the lesser curvature of the stomach that was lobulated andCT revealed an ulcer on the lesion The contrastenhanced CTimages showed heterogeneous enhancement of the mass Thefinal pathological diagnosis was gastric carcinosarcoma Inthe present study we found that GSC showed local thickeningof the gastric wall and mass formation often accompaniedby ulcers The site of the disease was mostly in the proximalpart of the stomach but these tumors can also occur in theremnant stomach The signal of the tumor was homogeneousor heterogeneous on plain CT scans After contrast mediuminjection of tumors demonstrated heterogeneousenhancement on AP images due to cystic areas or necrosis inthe lesions In this study the enhancement degree of all tumorsreached a peak in the PP after contrast enhancement For thesetumors the enhancement degree in the delayed phase wasnot significantly reduced The overall enhancement mode wasdelayed enhancement In addition CT showed that four patientshad invasion into the gastric serosal region or gastric bare areatwo patients had the characteristics of enlarged perigastric orretroperitoneal lymph nodes and uneven enhancement and onepatient had invasion into the adjacent liver tissue These findingsreflect the metastatic and highly invasive characteristics of GSCOverall CT and contrastenhanced CT can clearly show theprimary lesion infiltration range lymph node metastasis anddistant metastasis of GSCTomographic diagnosis of GSC has not been attemptedbecause of the rarity of this entity According to the findingsof our study GSC needs to be diï¬erentiated from GAC andGL on CT Adenocarcinoma is the most common pathologicaltype of gastric tumor and is mainly distributed in the antrumseldomly in the body and fundus of the stomach The incidenceof GAC is high in men and the median patient age is years The most common CT signs of GAC are localor extensive thickening of the gastric wall mass formationincluding fungoidestype polypoidtype masses rough orsmooth serous surfaces and continuous interruption of themucosal layer Tumors involving the mucosal surface can appearenhanced “ s after injecting a contrast agent The peakvalue for tumors invading the muscular layer usually appearsafter “ s and after the mucosal surface is strengthenedthe duration is longer Primary GL accounts for “ ofmalignant gastric tumors and is predominantly situated in thegastric antrum gastric body and gastric fundus The incidenceof GL is high among males with a median patient age of yearsThe clinical symptoms included epigastric pain bleeding earlysatiety and fatigue The most common CT manifestations ofGL are diï¬use thickening of the gastric wall or a homogeneousFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alGastric Sarcomatoid Carcinomatissue mass with slight attenuation or an appearancesoftsimilar to that of the normal gastric wall For GL becauseof hemorrhage necrosis submucosal edema or infarction thegastric wall may be heterogenous on CT GL originates froma submucosal process and gastric mucosa is commonly intactin the early stage but shows interruptions or ulceration in thelater stage After contrast medium injection most GL showedhomogeneous and slight enhancement in the delayed phase Lymphoma is considered when distant structures the mesenteryretroperitoneum or other parts of the abdomen have lymphnode metastasis The CT findings may only reflect features of GSC but cannotaccurately diagnose GSC let alone explore the origin of thesarcomatous portion Immunohistochemistry IHC also failedto conclusively establish the origin of GSC Rodrigues usedfluorescence in situ hybridization FISH to confirm that SC andadenocarcinoma have a common origin that is the epithelium while primary GL originated from gastric submucosallymphoid tissueThe main treatment for localized lymphomas is eradicationof Helicobacter pylori and surgical treatment whereas advanceddisease often requires radiation or chemotherapy alone Surgery is the only treatment option for patients with GACAdjuvant chemotherapy and chemoradiotherapy are also oftenused Targeted therapy isin the exploration stage However there are currently no specific National ComprehensiveCancer Network guidelines for the treatment of only GSCbecause the tumor is relatively rare although complete surgicalresection is the most important treatment method For examplewhile chemotherapy is considered in clinical practice whetherchemotherapy can be applied for GSC and the efficacy ofchemotherapy remain controversial Domblides firstevaluated the efficacy of immune checkpoint inhibitors ICIs forSC and found that lung SC patients exhibited high response ratesand prolonged overall survival OS with ICIs This studyprovides a new idea for the treatment of GSCBecause GL tends to be confined to the gastric wall forprolonged periods before tumor spread its prognosis is betterthan that of GAC Previous literature has found that SCin the parotid gland lung hypopharynx liver and pancreashave poor prognoses due to metastasis or recurrence with asurvival period of a few months “ Similarly GSCpatients also died or developed metastasis or recurrence withina few months or it was already in the advanced stage at thefirst diagnosis All these clinical manifestations suggest that GSChas a poorer prognosis than GAC and GL In additionGSC can metastasize through the blood and lymph nodesand the most common sites of metastasis are the local lymphnodes and liver This conclusion is consistent with ourresearch resultsCONCLUSIONThe incidence rate of GSC is extremely low so clinicians andradiologists are not familiar with the features of this tumorBased on systematic research of this rare tumor and comparisonswith common gastric cancers we found that GSC is morecommon in men who are approximately years old and isoften accompanied by ulcers The disease is mostly located in theproximal part of the stomach and can also occur in the remnantstomach with delayed enhancement on contrastenhanced CTimages These characteristics can provide a reference for furtherresearch on GSCs in the future However an accurate diagnosisof GSC depends on the combination of clinical imaging andhistopathological features Due to the aggressive nature and poorprognosis of the tumor rapid clinical intervention and detailedfollowup with CT are essentialDATA AVAILABILITY STATEMENTThe original contributions presented in the study are includedin the supplementary material further inquiries can bedirected to the corresponding authorETHICS STATEMENTThe studies involving human participants were reviewed andapproved by the Medical Ethical Committee of the ZhengzhouUniversity The patientsparticipants provided their writteninformed consent to participate in this study Written informedconsent was obtained from the individuals for the publication ofany potentially identifiable images or data included in this AUTHOR CONTRIBUTIONSYL manuscript preparationliterature research and dataanalysis PL literature research and data analysis KF manuscriptreview and data collection KC guidance of pathologicalknowledge SY guidance of imaging knowledge JJ imaging datacollection and analysis WL and XZ manuscript editing JGstudy conception and design manuscript review and guarantor ofintegrity of the entire study All authors have read and approvedthe final manuscriptFUNDINGThis work was supported by the National Natural and ScienceFund of China No REFERENCES Zhu CC Li MR Lin TL Zhao G Sarcomatoid carcinoma of the stomach acase report and literature review Oncol Lett “ ol20153460 Snover DC Levine GD RosaiJ Thymic carcinoma Five distinctivehistological variants Am J Surg Pathol “ 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Jiang L He Q Wei W Wang Y Zhang X The prognostic significa

Thyroid_Cancer

generate a map of the copy number variations CNV segregating in a population of Murciano‘Granadina goats the most important dairy breed in Spain and to ascertain the main biologi‘cal functions of the genes that map to copy number variable regionsResults Using a dataset that comprised Murciano‘Granadina goats genotyped with the Goat SNP50 Bead‘Chip we were able to detect and autosomal CNV with the PennCNV and QuantiSNP software respec‘tively By applying the EnsembleCNV algorithm these CNV were assembled into CNV regions CNVR of which of the total CNVR count were consistently called by PennCNV and QuantiSNP and used in subsequent analyses In this set of CNVR we identified gain loss and gainloss events The total length of all the CNVR Mb represented of the goat autosomal genome Mb whereas their size ranged from kb to Mb with an average size of kb Functional annotation of the genes that overlapped with the CNVR revealed an enrichment of pathways related with olfactory transduction fold‘enrichment q‘value — ˆ’ ABC transporters fold‘enrichment q‘value — ˆ’ and bile secretion fold‘enrichment q‘value — ˆ’Conclusions A previous study reported that the average number of CNVR per goat breed was CNVR50 breeds which is much smaller than the number we found here CNVR We attribute this difference to the fact that the previous study included multiple caprine breeds that were represented by small to moderate numbers of individuals Given the low frequencies of CNV in our study the average frequency of CNV is such a design would probably underestimate the levels of the diversity of CNV at the within‘breed level We also observed that functions related with sensory perception metabolism and embryo development are overrepresented in the set of genes that overlapped with CNV and that these loci often belong to large multigene families with tens hundreds or thousands of paralogous members a feature that could favor the occurrence of duplications or deletions by non‘allelic homologous recombinationCorrespondence marcelamillsuabcat Centre for Research in Agricultural Genomics CRAG CSIC‘IRTA‘UAB‘UB Universitat Aut²noma de Barcelona Bellaterra SpainFull list of author information is available at the end of the BackgroundCopy number variations CNV encompass genomic deletions or duplications with sizes ranging from base pairs bp to several megabases Mb and which display polymorphisms in terms of copy number among individuals of a particular species [“] In livestock a broad array of phenotypes related with among others morphology [ ] pigmentation [“] sexual development The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cGuan a0et a0al Genet Sel Evol Page of [] and susceptibility to disease [] is caused by the segregation of CNV Genome scans to detect structural variations in cattle have revealed that CNV regions CNVR are often enriched in genes that are involved in immunity [“] metabolism [ ] embryo development [ ] and sensory perception [ ] There is evidence that the dNdS ratios of genes that map to taurine CNV are generally higher than those of genes that do not overlap with CNV which indicates that CNV genes probably evolve under reduced selective constraint [] The analysis of gene networks has also shown that genes that colocalize with duplications tend to have fewer interactions with other genes than loci that do not overlap with CNV reinforcing the idea that genes mapping to duplicated regions have fewer essential housekeeping functions than nonCNV genes and also have reduced pleiotropy []Although structural chromosomal variations can have strong effects on gene expression and phenotypic variability technical limitations and the moderate quality of genome assemblies have hampered CNV mapping in livestock [] Until recently this has been particularly true for goats In Fontanesi et a0al [] published the first caprine CNV map by identifying with the Bovine a0k aCGH array CNVR including and copy loss and gain variants respectively Later on resequencing the genome of individuals from several caprine breeds made it possible to identify CNV that overlap with pigmentation genes and to detect an association between increased ASIP copy number and light pigmentation [] The first worldwide survey of copy number variation in goats was performed within the Goat ADAPTmap Project httpwwwgoata daptm ap and involved the genomewide genotyping of goats from breeds [] This study resulted in the identification of CNVR among which several overlapped with genes that are functionally related with local adaptation such as coat color muscle development metabolic processes and embryonic development [] Moreover the patterns of the diversity of CNV differed according to geographic origin which indicates that they have been influenced by population history [] In another study on individuals from East African goat breeds Nandolo et a0al [] detected CNVR More recently Henkel et a0al [] demonstrated the existence of complex patterns of structural variation in the regions containing the caprine ASIP and KIT genes with potential causal effects on pigmentation In spite of these efforts the description of structural chromosomal variation in goats is still lagging behind that of other domestic species Most of the CNV surveys in goats have analyzed large populations that represent a mixture of different breeds each with a limited number of individuals [ ] thus making it difficult to assess the magnitude of the CNV diversity at the withinbreed level Our goal was to fill this gap by analyzing a population of individuals from a single Spanish breed MurcianoGranadina and to investigate the functional roles of genes that map to CNVR and compare these results with data obtained in composite goat populationsMethodsGenomic DNA extraction and a0high‘throughput genotypingBlood samples from MurcianoGranadina female goats from farms that are connected through the use of artificial insemination were collected in EDTA K3 coated vacuum tubes and stored at ˆ’ a0 °C before processing Genomic DNA was isolated by a modified saltingout procedure [] Four volumes of red cell lysis solution Tris“HCl a0 mmolL pH EDTA a0mmolL Tween were added to a0mL of whole blood and this mixture was centrifuged at —g Pelleted cells were resuspended in a0mL lysis buffer Tris“HCl a0 mmolL pH EDTA a0 mmolL SDS NaCl a0 mmolL plus µL proteinase K a0 mgmL The resulting mixture was incubated at a0°C for h followed by centrifugation at —g in the presence of a0mL of ammonium acetate a0molL The supernatant a0mL was mixed with a0mL of isopropanol which was subsequently centrifuged at —g for a0min The supernatant was removed and the DNA pellet was washed with a0 mL of ethanol After centrifuging at —g for a0min the DNA precipitate was dried at room temperature and resuspended in a0mL of TE buffer a0mmolL Tris pH a0mmolL EDTA pH Highthroughput genotyping of the MurcianoGranadina DNA samples was carried out with the Goat SNP50 BeadChip [] according to the manufacturer™s instructions Illumina Signal intensity ratios ie log R Ratio or LRR the total probe intensity of a SNP referred to a canonical set of normal controls [] and B allele frequencies or BAF relative quantity of one allele compared to the other one [] were exported for each single nucleotide polymorphism SNP with the GenomeStudio software Illumina https emeaillum inacom Then SNP coordinates were converted to the latest version of the goat reference genome ARS1 [] After filtering out unmapped and nonautosomal SNPs and those with a call rate lower than a set of SNPs remained for CNV mappingCopy number variant calling with a0PennCNV and a0QuantiSNPBased on their excellent performance in comparative studies we selected two software packages PennCNV v105 [] and QuantiSNP v2 [] to call CNV in the MurcianoGranadina population [ ] The PennCNV software [] detects CNV by applying the default 0cGuan a0et a0al Genet Sel Evol Page of parameters of the HiddenMarkov model Population frequencies of B alleles were compiled based on the BAF of each SNP in the population We used the œ“gcmodelfile option to adjust œgenomic waves [] The number of goat chromosomes was set with the œ“lastchr  instruction The QuantiSNP analysis [] assumes an objective Bayes hiddenMarkov model to improve the accuracy of segmental aneuploidy identification and mapping This CNV calling software was run under default parameters by modifying the œ“chr  option The CNV that were supported by less than three SNPs were removed from the filtered set used hereDefinition and a0functional annotation of a0copy number variant regionsWe used the EnsembleCNV algorithm beta version [] to assemble CNVR All CNV called by PennCNV andor QuantiSNP were combined to generate a set of initial CNVR by using the heuristic algorithm threshold of minimum overlap described in [] Subsequently CNVR boundaries were refined by considering the local correlation structure of the LRR values of the SNPs mapping to CNVR [] Then we reassigned the CNV calls that were initially obtained with PennCNV and QuantiSNP to each refined CNVR so that the final set of CNVR comprised only those that were simultaneously detected by both callers The resulting CNVR were matched to gene features that are annotated in the National Center for Biotechnology Information NCBI https wwwncbinlmnihgov by using BEDTools v2250 [] In addition we performed gene ontology GO enrichment and pathway analyses using the DAVID Bioinformatics Resources [ ] based on human and goat background gene sets The statistical significance was set to a qvalue ‰¤ Confirmation of a0copy number variant regions by a0quantitative real‘time PCRIn order to evaluate the rate of false positives in our experiment we conducted quantitative realtime PCR qPCR experiments to obtain an independent estimate of the copy number of four putative CNVR CNVR_371_chr5 CNVR_506_chr6 CNVR_160_chr2 and CNVR_1229_chr21 Primers were designed with the Primer Express software Applied Biosystems to amplify specific regions of the ADAMTS20 BST1 NCKAP5 and TNFAIP2 genes see Additional file a0 Table a0 S1 As reference genes we used the melanocortin receptor MC1R and glucagon GCG genes see Additional file a0 Table a0S1 loci [ “] Quantitative PCR reactions contained a0ng genomic DNA µL — SybrSelect Master mix Applied Biosystems a0pmol of each forward and reverse primer and ultrapure water to a maximum final volume of µL Each sample was analyzed in triplicate in order to obtain averaged copy number estimates Reactions were loaded onto 384well plates and run in a QuantStudio a0K Flex RealTime PCR System instrument Applied Biosystems The specificity of the PCR reactions was evaluated with a melting curve analysis procedure and the efficiency was assessed with standard curves Thus relative copy number was inferred with the qbase software Biogazelle Ghent Belgium by using the ˆ’ΔΔCt approach [] Copy number values were calibrated by taking as a reference four samples which according to Goat SNP50 BeadChip data had two copies of the investigated genomic lociResultsDetection of a0copy number variation in a0Murciano‘Granadina goatsThe initial calling with PennCNV and QuantiSNP yielded and autosomal CNV respectively By using the EnsembleCNV tool [] we assigned these CNV into CNVR with refined boundaries of which of the total CNVR count were detected simultaneously by PennCNV and QuantiSNP The resulting CNVR included copy gain copy loss and copy gainloss variants Fig a0 and Table a0 and see Additional file a0 Table a0 S2 The total length of the CNVR covered a0Mb of the goat autosomal genome a0Mb whereas their individual size ranged from a0kb to a0Mb with an average of a0kb Fig a02a and Table a0 Moreover we found that of the CNVR showed minimum allele frequencies lower than with an average frequency of Fig a02b In addition CNVR with frequencies higher than were distributed over seven caprine chromosomes With a frequency of CNVR_1229_chr21 was the CNVR with the highest frequency in the whole dataset see Additional file a0 Table a0S2 By using the BEDTools v2250 program [] of the CNVR that we detected overlapped with unique CNVR published by Liu et a0al [] Fig a0 and see Additional file a0 Table a0S2 The CNVR that were detected in both studies are referred to as œshared CNVR whereas those that were identified in our study only are referred to as œnonshared CNVR Fig a0 Six of the ten œshared CNVR with frequencies higher than show positional concordance with six CNVR detected by Liu et a0al [] see Additional file a0 Table a0S2Functional annotation of a0the a0genes that a0are located in a0copy number variable regionsWithin the CNVR defined in our study we detected proteincoding genes according to the goat reference genome annotation ARS1 [] from the NCBI database see Additional file a0 Table a0S2 and Additional file a0 0cGuan a0et a0al Genet Sel Evol Page of Fig Genomic distribution of CNVR detected with the PennCNV and QuantiSNP software on the caprine autosomes Squares triangles and circles represent copy number gain loss and gainloss events respectively Red and black colors represent shared and non‘shared CNVR respectively Shared CNVR are those detected both in our study and in Liu et al [] while non‘shared CNVR are those identified only in our studyTable a0S3 In a survey of the diversity of CNV in goats with a worldwide distribution Liu et a0 al [] detected copy number variable genes of which were also identified in our study and are referred to as œshared copy number variable genes see Additional file a0 Table a0S3 Among the œshared copy number variable genes the ASIP and ADAMTS20 genes are particularly relevant they are involved in pigmentation [ “] and colocalize with selection signals detected in a worldwide sample of goats [] In addition we found that about of the annotated genes that colocalize with CNVR are olfactory receptors or olfactory receptorlike genes see Additional file a0 Table a0S3 Consistently the most significantly enriched pathway was œOlfactory transduction qvalue — ˆ’ Table a0 followed by œABC transporter qvalue — ˆ’ Table a0 A significant pathway related with immunity ie Fc epsilon RI signaling qvalue was also identified based on a human background gene set Table a0 Several overrepresented GO terms were related with embryonic skeletal system morphogenesis qvalue — ˆ’ and Gprotein coupled purinergic nucleotide receptor activity 0cGuan a0et a0al Genet Sel Evol Page of Table Main features of a0 copy number variation regions CNVR detected in a0 MurcianoGranadina goatsSummary statisticsTotalGainLossGainlossTotal length MbTotal number of CNVRNumber of CNVR kbNumber of CNVR “ kbNumber of CNVR “ kbNumber of CNVR “ kbNumber of CNVR kb“ MbNumber of CNVR ‰¥ MbAverage number of SNPs per CNVRMinimum size of CNVR kbMaximum size of CNVR kbAverage CNVR size kbStandard deviation of CNVR size kb qvalue — ˆ’ Table a0 Interestingly the copy number variable genes were also enriched in pathways with metabolic significance such as prolactin signaling and insulin signaling as well as GO terms related with feeding behavior but none of these pathways reached the significance threshold qvalue ‰¤ after correction for multiple testing see Additional file a0 Table a0S4 Several of the pathways outlined in Additional file a0 Table a0S4 play important roles in immunity eg chemokine signaling B cell receptor signaling and T cell receptor signaling cancer eg endometrial cancer proteoglycans in cancer thyroid cancer as well as in oncogenic signaling eg Ras and ErbB signaling see Additional file a0 Table a0S4 but most of them are not significant after correction for multiple testingFig Histograms displaying the distribution of CNVR according to their size a and frequency b CNVR that were longer than kb were included in the ‘kb bin whereas those with frequencies above were grouped in the bin The histograms were drawn by using the ggplot2 package httpggplo t2tidyv erse implemented in R https wwwr‘proje ct 0cGuan a0et a0al Genet Sel Evol Page of Table Functional enrichment of a0genes colocalizing with a0CNVR detected in a0 MurcianoGranadina goatsBackground gene setCategory IDTermNumber of a0genesFold enrichment P valueq‘valueGoatGoatGoatHumanHumanHumanHumanHumanHumanHumanKEGGKEGGKEGGKEGGGOBPGOBPGOBPGOCCGOMFGOMFOlfactory transductionABC transportersBile secretionFc epsilon RI signaling pathwaychx04740chx02010chx04976hsa04664GO0009952 Anteriorposterior pattern specificationGO0048704 Embryonic skeletal system morphogenesisGO0035589 G‘protein coupled purinergic nucleotide receptor signaling pathwayGO0016020 MembraneGO0003677 DNA bindingGO0045028 G‘protein coupled purinergic nucleotide receptor activity126Eˆ’ 161Eˆ’333Eˆ’ 427Eˆ’446Eˆ’ 570Eˆ’140Eˆ’ 176Eˆ’936Eˆ’ 161Eˆ’713Eˆ’ 122Eˆ’418Eˆ’ 716Eˆ’145Eˆ’ 198Eˆ’110Eˆ’ 160Eˆ’424Eˆ’ 622Eˆ’KEGG Kyoto Encyclopedia of Genes and Genomes pathway GOMF gene ontology GO term related with molecular function GOBP GO term related with biological process GOCC GO term related with cellular componentValidation of a0four copy number variants by a0real‘time quantitative polymerase chain reactionIn order to confirm our results we selected four CNVR ie CNVR_371_chr5 CNVR_506_chr6 CNVR_160_chr2 and CNVR_1229_chr21 that colocalized with the ADAMTS20 BST1 NCKAP5 and TNFAIP2 genes respectively the primers used to amplify these CNVR are listed in Additional file a0 Table a0 S1 As shown in Fig a0 the estimated copy numbers obtained by qPCR analysis of MurcianoGranadina goat samples were to copies relative to the calibrator ADAMTS20 to copies BST1 to copies NCKAP5 and to copies TNFAIP2 According to D™haene et a0 al [] copy number estimates between and most likely correspond to a normal copy number of whereas any number below or above these thresholds could represent a deletion or a duplication respectively Thus based on these values evidence of copy number variation was inferred for three of the four genes analyzed by qPCRFig Relative quantification of four copy number variation regions by real‘time quantitative polymerase chain reaction analysis a CNVR_371_chr5 ADAMTS20 b CNVR_506_chr6 BST1 c CNVR_160_chr2 NCKAP5 d CNVR_1229_chr21 TNFAIP2 The x and y axes represent sample ID and relative quantification of CNVR mean ± standard error with each sample analyzed in triplicate respectively As calibrator we used the average of four samples estimated to have two copies diploid status based on the Goat SNP50 BeadChip analysis 0cGuan a0et a0al Genet Sel Evol Page of DiscussionIn this work our aim was to characterize copy number variation in MurcianoGranadina goats a native Spanish breed used for milk production By genotyping MurcianoGranadina goats with a SNP array we were able to identify CNVR covering of the goat genome whereas Liu et a0 al [] identified CNVR that covered of the goat genome The latter higher percentage reported by Liu et a0al [] can be explained by the fact that they analyzed breeds with different geographical origins ie a composite population that is probably much more diverse than that used in our work Besides the pipeline that we used to identify CNVR is more stringent than that employed by Liu et a0al [] removing CNVR that were not consistently detected by PennCNV and QuantiSNP In the literature estimates of to for CNVR coverage in the human genome are reported [] Our results and those obtained by Liu et a0al [] are consistent with these valuesIndeed when Liu et a0 al [] calculated the CNVR length for each breed normalized by the goat genome size their results agreed well with our estimate of For instance this parameter reached values of in goats from Southeastern Africa and in goats from Northwestern Africa and Eastern Mediterranean whereas it was lowest for individuals from West Asia [] The number of CNV detected at the withinbreed level by Liu et a0 al [] was on average CNV per breed and ranged from to whereas the average number of CNVR was only per breed [] Since the number of detected CNVR is proportional to population size for most of the breeds investigated in [] the level of withinbreed CNV variation is probably underestimated In summary one important conclusion from our study is that the magnitude of CNV diversity at the withinbreed level is likely to be much larger than that previously reported in studies that analyzed multiple populations each represented by a small or moderate number of individualsMost of the CNVR that we report here ranged in size from to a0kb with a mean size of a0kb Similarly the average CNVR size reported by Liu et a0al [] was a0kb Both estimates are quite large and reflect that mediumdensity SNP arrays are not well suited to detect small CNVR in spite of their high abundance In cattle the average sizes of CNVR detected with the Illumina BovineHD Genotyping BeadChip a0K SNPs [] Illumina wholegenome sequencing and PacBio sequencing [] were and a0 kb respectively Another consistent feature of CNVR is that in general their frequencies are low or very low In our study approximately of the CNVR had frequencies lower than and the average frequency was Liu et a0al [] reported lower CNVR frequencies ranging from Alpine and Northern European goats to Northwestern African goats This decreased average CNVR frequency is not very significant and probably reflects differences in sampling size and the use of composite populations with multiple breeds each one with its specific CNVR frequenciesThe CNVR detected in our study covered proteincoding genes Pathway analyses reflected a substantial enrichment of genes that are involved in olfactory perception which is consistent with previous reports in cattle [ ] In this regard there is an important difference between our results and those by Liu et a0al [] Whereas in the study of Liu et a0al [] the term œsensory perception was underrepresented among the CNV genes fold enrichment in our work the terms œolfactory transduction fold enrichment and œGprotein coupled purinergic nucleotide receptor activity fold enrichment were overrepresented and many CNV genes were olfactory receptors The two terms mentioned before are closely related because a broad array of purinergic receptors are differentially expressed in the olfactory receptor neurons that modulate odor responsiveness [] Moreover purinergic nucleotides are important neuromodulators of peripheral auditory and visual sensory systems [] In cattle Keel et a0al [] reported that œsensory perception of smell and œGprotein coupled receptor signaling pathway were significantly overrepresented in the proteincoding genes that overlapped with CNVR Similarly Upadhyay et a0 al [] showed that œsensory perceptions of smell and œchemical stimuli are enriched in their set of CNV genes A potential explanation for the underrepresentation of the œsensory perception functional category among the genes overlapping CNV reported by Liu et a0al [] could be that in goats these genes are not well annotated yet so the majority of them are identified with a LOC prefix and a number and as a consequence of this they are not correctly detected by PANTHER [] thus biasing the results obtained in the gene ontology enrichment analysisLoci belonging to large multigene families might be more prone to colocalize with CNV because paralogous genes can act as templates in nonallelic homologous recombination events which promote increases or reductions in copy number [] It should be noted that olfactory receptor genes constitute the largest gene superfamily and in humans more than genes and pseudogenes have been identified [] In cattle olfactory receptor genes and pseudogenes are distributed in clusters across bovine chromosomes [] and similar numbers have been reported for pigs [] Moreover purifying selection against CNV is probably less 0cGuan a0et a0al Genet Sel Evol Page of intense in regions that contain olfactoryreceptor genes than in genomic regions that contain genes with essential functions [] Interestingly copy number changes in the olfactory receptor genes of wild and domestic mammals might have consequences on food foraging as well as on mate and predator recognition [ ]In the set of genes that colocalize with CNVR we also detected an enrichment of loci related with the multigene family of ATP binding cassette ABC transporters a result that agrees well with previous findings in humans [“] and cattle [ ] In mammals ABC transporters fulfill the mission of carrying a broad array of endogenous substrates such as amino acids peptides sugars anions and hydrophobic compounds and metabolites across lipid membranes At least ABC genes that belong to eight subfamilies have been identified in the human genome [] Copy number variation in the human ABCC4 and ABCC6 genes is associated with susceptibility to esophageal squamous cell carcinoma [] and to the rare autosomal recessive disease pseudoxanthoma elasticum [] respectively Moreover largescale deletions of the human ABCA1 gene are a causative factor for hypoalphalipoproteinemia [] a disease that is characterized by the complete absence of the apolipoprotein AI and extremely low levels of plasma highdensity lipoprotein HDL cholesterol We also found a highly significant enrichment of pathways related with embryo development anteriorposterior pattern specification embryonic skeletal system morphogenesis as previously reported [] These pathways are featured by genes that belong to the Hox multigene family of transcription factors possibly reflecting the genomic instability of certain homeobox gene clusters as evidenced by the existence of many syntenyparalogy breakpoints and assembly gaps as outlined in comparative studies []Although not significant after correction for multiple testing we detected an enrichment of pathways with metabolic significance such as prolactin and insulin signaling which could have an impact on milk production and growth [“] Interestingly the comparison of our work with that of Liu et a0al [] revealed proteincoding genes that colocalize with the set of shared CNVR One of the most relevant shared genes encodes ASIP a protein that increases the ratio of pheomelanin to eumelanin by binding to the melanocortin receptor and delivering an antagonist signal that blocks the downstream expression of eumelanogenic enzymes [] Mutations in the ASIP gene play critical roles in animal pigmentation [] For instance the causal factor of the white color typical of many sheep breeds is the ubiquitous expression of a duplicated copy of the ASIP coding sequence which is regulated by a duplicated promoter corresponding to the itchy E3 ubiquitin protein ligase gene [ ] Although some studies proposed that the ASIP CNV might be associated with different pigmentation patterns in goats [ ] no functional assay has verified an association of ASIP copy number with ASIP mRNA levels Another interesting shared copy number variable gene is ADAMTS20 which was also identified in two previous CNV surveys [ ] This gene encodes a metalloproteinase with an important role in melanoblast survival by mediating Kit signaling [] and in palatogenesis [] Bertolini et a0al [] performed a selection scan in white vs colored black and red goats and detected a selective sweep in the ADAMTS20 gene In the light of these results the potential involvement of a structural variation in ADAMTS20 in goat pigmentation should be explored further Moreover it is worthwhile to mention that several CNVR genes have functions related with production and reproduction traits For instance the NCKAP5 gene which colocalizes with CNVR_160_chr2 frequency is associated with milk fat percentage in cattle [] Taking the above evidence into account the implication of structural chromosomal variations in the genetic determinism of traits of economic interest with a complex inheritance deserves further exploration by designing tools that allow inferring CNVR genotypes with high confidenceConclusionsWith the PennCNV and QuantiSNP software we detected CNVR in the genome of the MurcianoGranadina breed In a a0 previous study [] that used a less stringent pipeline only PennCNV was used and included multiple populations with small to moderate sample sizes the average number of CNVR events per breed was One conclusion of our study is that CNV surveys which are based on a broad array of breeds represented by only a few individuals underestimate the true levels of the CNV diversity at the withinbreed level The main reason for this outcome is that since the majority of CNV have very low frequencies they cannot be detected efficiently when sample size is small and in consequence much of the existing variation is missed We have also found that genes that overlap with CNV are functionally related with olfactory transduction embryo development ABC transporters and Gprotein coupled purinergic nucleotide receptor activity Most of these genes belong to large multigene families encompassing tens hundreds or thousands of paralogous genes that could act as substrates in nonallelic homologous recombination events which is one of the main mechanisms generating duplications and deletions in humans and other species Finally we detected CNV that colocalize with the ASIP and ADAMTS20 pigmentation genes 0cGuan a0et a0al Genet Sel Evol Page of which according to previous studies have been subjected to positive selection for coat color in goatsSupplementary informationSupplementary information accompanies this paper at https doi101186s1271 ‘‘ ‘ Additional file a0 Table a0S1 List of primers used in the real‘time quanti

Thyroid_Cancer

CD146 was originally identified as a melanoma cell adhesion molecule MCAM and highly expressed in many tumors andendothelial cells However the evidence that CD146 acts as an adhesion molecule to mediate a homophilic adhesion through thedirect interactions between CD146 and itself is still lacking Recent evidence revealed that CD146 is not merely an adhesionmolecule but also a cellular surface receptor of miscellaneous ligands including some growth factors and extracellular matrixesThrough the bidirectional interactions with its ligands CD146 is actively involved in numerous physiological and pathologicalprocesses of cells Overexpression of CD146 can be observed in most of malignancies and is implicated in nearly every step of thedevelopment and progression of cancers especially vascular and lymphatic metastasis Thus immunotherapy against CD146 wouldprovide a promising strategy to inhibit metastasis which accounts for the majority of cancerassociated deaths Therefore todeepen the understanding of CD146 we review the reports describing the newly identified ligands of CD146 and discuss theimplications of these findings in establishing novel strategies for cancer therapySignal Transduction and Targeted Therapy 101038s41392020002598INTRODUCTIONIn Johnson first found that a tumor antigen MUC18was expressed most strongly on metastatic lesions and advancedprimary melanoma with rare detection in benign lesions Due tothe high sequence homology between MUC18 with cell adhesionmolecules CAMs this melanoma antigen was given an officialname melanoma CAM MCAM1 With an increasing number ofdiscoveries about MCAM by various research groups more aliasnames were given to this protein including P1H12 MUC18 A32antigen SEndo1 MelCAM METCAM HEMCAM or CD1461“There are three forms of CD146 proteins in human mouse andchicken The two membraneanchored forms of CD146 areencoded by cd146 gene and soluble form of CD146 sCD146is generated by the proteolytic cleavage ofthe membraneforms11“ Soluble CD146 can be detected in cell culturesupernatants serum and interstitial fluids from either healthyor unhealthy subjects14“ Because sCD146 does not have eitherto cell or cellCAM is a kind of proteins located on the cell surface andmediates contacting and binding of celltoextracellular matrix ECM4 These dynamic interactions providesignals input into the cellular decisionmaking process such as cellgrowth survival migration and differentiation5 essentialforembryonic development and for maintaining the integrity oftissue architecture in adults67 Dependent on adhesion someCAMs can initiate the formation of complexes composed ofextracellular ligands kinases and cytoskeletal proteins8 Abnormalexpression of CAMs can cause various diseases such as cancer and‚ammatory disorders910transmembrane or cytoplasmic regionsit is not competent incellular adhesion1718 Therefore we will not describe sCD146 itsligands and its functions in this review although it is a potentialtarget in tumor microenvironment of CD146positive invasivetumors19Recent evidence has revealed that membranebound CD146may act as a cellsurface receptor to bind with various ligandsinvolved in cellular signaling transduction independent of theadhesion properties In order to deepen the understanding of thefunctions of CD146 in physiological and pathological processeswe summarize the various newly identified ligands of CD146and the ligandelicited roles in signal transduction and discussthe implications of CD146 in remodeling interactions betweenthe cancerous cells with the elements oftheir surroundingmicroenvironmentsTHE CD146 PROTEINMembrane CD146 protein has two isoforms long form CD146lhas a long cytoplasmic tail short form CD146s has a shortcytoplasmic tail1718 These two CD146 isoforms are produced fromdifferent exon splicing strategies and the premature moleculeshave a signal peptide located on the anterior region of the aminoterminal20 In human mature CD146 protein is composed of anextracellular sections with five distinct Iglike domains that exist ina V“V“C2“C2“C2 structural motif a hydrophobic transmembraneregion and a short cytoplasmic tail21 The cytoplasmic domain inboth isoforms contains two potential recognition sites for protein1Key Laboratory of Protein and Peptide Pharmaceuticals Institute of Biophysics Chinese Academy of Sciences Beijing China 2College of Life Science University ofChinese Academy of Sciences Beijing China 3Department of Gastrointestinal Hepatobiliary Tumor Surgery Beijing Shijitan Hospital Capital Medical University Beijing China 4Departments of Pathology Beijing Shijitan Hospital Capital Medical University Beijing China and 5Nanozyme Medical Center School of Basic MedicalSciences Zhengzhou University Zhengzhou ChinaCorrespondence Zhaoqing Wang clairezqwanghotmailcom or Xiyun Yan yanxyibpaccnThese authors contributed equally Zhaoqing Wang Qingji XuReceived March Revised June Accepted June The Authors 0cCD146 from a melanoma cell adhesion molecule to a signaling receptorWang et alkinases C PKC an ERM protein complex of ezrin radixin andmoesin binding site a motif with microvilli extension and adouble leucine motif for basolateral targeting21 The two isoformscoexist as monomers and dimers and the dimerization ismediated through a disulfide bond between cysteine residues inthe C2 domain most proximal to the membrane2022 However theinformation about CD146 crystal structureincluding secondaryand tertiary is still lackingCD146 is a highly glycosylated type I transmembrane proteinand belongs to the immunoglobulin superfamily Based onbioinformation analysis eight putative Nglycosylation sites arepresent in the extracellular fragment across species23 In clear cellrenal cell carcinoma and prostate cancer CD146 glycosylationlevels were upregulated2425 In it was reported that CD146glycosylation is favorably carried out by b13galactosylOglycosylglycoprotein b16Nacetylglucosaminyltransferase3 whichwas overexpressed in highly metastatic melanomas Suchglycosylations can extend CD146 protein stability upregulateCD146 protein levels and lead to elevation of CD146mediatedcellular motility in melanoma cells26 These observations suggestthat the degree of CD146 glycosylation may be directly relatedto malignant progression of tumors especially CD146positiveneoplasmsTHE EXPRESSION PROFILE OF CD146 PROTEINBased on literature metazoan CD146 has been detected inmajority of cell types including vessel constituting cells endothelium pericyte and smooth muscle cell epithelia fibroblastsmesenchymal stem cells and lymphocytes except erythrocytes21Under physiological conditions CD146 expression is restricted tolimited adult normal tissues and its adhesive strength is relativelyweakto most other CAMs which show wideexpression patterns in normal adult tissues and strong adhesionstrength2123 However CD146 expression is broadly and highlydetected in embryonic tissues compared to its abundance innormal adult tissues21 In quickly proliferating cellsincreasedexpression of CD146 may allow cells to actively interact with eachother and with the elements of the cellular microenvironmentpromoting cell proliferation and migrationin contrastUnder pathological conditions such as ‚ammation andtumorigenesis CD146 was upregulated in the related cells andhas been identified as a reliable marker for numerous types ofcancers Accumulating evidence shows that CD146 overexpressionhas been linked to either the initial development of the primarylesion or progression to metastases of most of cancer typesprimarily including melanoma127“ breast63031 ovarian32“lung3637 prostate38“ glioma41 kidney42 hepatic4344 and gastriccancers2145 In Nollet reported that TsCD146 mAb fortumor specific antiCD146 monoclonal antibody can specificallyrecognize CD146 expressed in cancer cells but not CD146 inphysiological vessels suggesting that structural features of cancerCD146 differ from those of physiological CD14628RECOGNITION OF CD146 LIGANDS IN HISTORYThe recognition of CD146 ligands and analysis of their functionswas undertaken over a prolonged period in history Because CD146is highly expressed in vessel cells and cancer cells it is likely thatCD146 within these cells contributes to cancer metastasis throughthe mediation of a homophilic adhesion between cancerous cellsand vascular endothelia a key part of the metastatic processHowever evidence of the direct interactions between CD146 anditself is stillit is possible that CD146mediated adhesion between cancerous cells with vascularendothelia as well as with their surrounding elements occursthrough the bidirectional heterophilic interactions between CD146with its ligands but not the homophilic interaction with itselflacking46“ AccordinglyIn the first CD146™s ligand was found using chickensmooth muscle cells Taniura discovered that neuriteoutgrowth factor NOF was a ligand of chicken CD146 Gicerinand that binding of NOF to CD146 is essentialthedevelopment of the chicken retina4950 However at that timedue to technological limitations the molecular characteristics ofNOF were not determined In Laminin was revealed asthe ligand of CD146 facilitating the entry of blood lymphocytesinto the central nervous system CNS In this report the authorsclaimed that Laminin is a major tissue ligand for CD146 onlymphocytes51 In Ishikawa finally determined theidentity of NOF Laminin which has the same α4 subunit asLaminin forIn our laboratory identified that CD146 can bind withvascular endothelial growth factor receptor VEGFR2 as a coreceptor required for the activation by vascular endothelial growthfactorA VEGFA53 Because VEGFA is a wellknown growth factorwith strong proangiogenesis effects this finding provided themechanism underlying the roles of CD146 in tumor angiogenesisespecially in sprouting stage Subsequently ourlaboratoryidentified an array of proangiogenetic growth factors includingWinglessintegrase Wnt5a54 Netrin155 fibroblast growth factorFGF456 VEGFC57 and Wnt158 as the ligands of CD146 In we further identified that CD146 on endothelia can directly bindreceptorβ PDGFRβ onwith plateletderived growth factorrequired for PDGFBinduced PDGFRβ activation59pericyteBecause PDGFBPDGFRβ plays crucial roles in recruiting adjacentpericytes to the endothelia this finding indicates that CD146 isrequired for vessel integrityUntil now there had been a total of molecules or complexesidentified as the CD146 ligands Table According to thecharacteristics of these ligands they can be categorized into threegroups components of the ECM proangiogenic factor receptorsand growth factors All these ligands have been sown to directlyinteract with CD146 in physiological and pathological processesare involved in the promotion of CD146mediated angiogenesisand tumor metastasis Here we will review the various CD146²heterophilic ligands and discuss the implications of these findingsin tumoral contextCD146 IS THE RECEPTOR OF PROTEINS IN RELATION TO THEECMOne of the critical features of malignant proliferation is cancermetastatic plasticity affected by its microenvironment Thisplasticity is a major reason for the failure of inhibition of cancermetastasis The metastatic process involves epithelial mesenchymaltransition EMT attachment of metastatic cells to theendothelium of the vascular or lymphatic vessels and invasioninto distant metastatic tissues60 It is well known that the aberranthigh expression of CD146 is involved in nearly every step ofdevelopment and progression in almost all types of malignantcancers21 The findings that several ECMrelated proteins including Laminin and Galectin1 and S100A8A9 andmatriptase are specific ligands of CD146 may elucidate themechanism underlying the function of CD146 in remodelingtumor microenvironments during tumor development especiallymetastasis via vascular and lymphatic vessels Fig Laminins and Laminins are a family of large heterotrimeric αβγ proteins with over different isoforms Five laminin α chains α1“α5 four laminin βchains β1“β4 and three laminin γ chains γ1“γ3 constitute αβγheterotrimers They are denominated according to their chaincomposition for example laminin α4β1γ1 is designated as Laminin Laminins are predominantly found in basement membranesthat compartmentalize different tissues and surround blood vesselsnerves and adipocytes6263 They play a crucial role in physiologicalSignal Transduction and Targeted Therapy 0cCD146 from a melanoma cell adhesion molecule to a signaling receptorWang et alTable CD146 ligandsLigandsLaminin Laminin Galectin1Galectin3S100A8A9MatriptaseVEGFR2PDGFRβWnt5aWnt1Netrin1FGF4VEGFCFunctionTime of discoveryReferencesFacilitates lymphocytes entry into CNSImproves cancer metastasis via vascular andor lymphatic vesselsInhibits cell apoptosisEnhances cell migration and secretion of prometastasis cytokinesHelps lung tropic metastasisPromotes neuron differentiationProangiogenesisControl of vascular vessel integrityEnables cell migrationPromotes fibroblast activationProangiogenesisPromotes cell polarity establishmentMediates sprouting during lymphangiogenesisand pathological remodeling of the ECM during angiogenesiswound healing embryogenesis and tumor metastasis Remodelingof the ECM during metastasis allows tumor cells to invade theirsurrounding ECM spread via the vascular or lymphatic circulationand extravasate into distant ansLaminin isoforms particularly the laminin α chain are expressedin a cell and tissue specific manner and are distinctly bound byalmost ten different integrins and other cellsurface receptors6263The α4laminins are mesenchymal laminins expressed by the cells ofmesenchymal origin such as vascular and lymphatic endothelialcells pericytes and leukocytes and are required for normaldevelopment of the cardiovascular and neurological system inmice64“ Under pathological conditions α4laminins are expressedand secreted by various tumor cells such as melanoma andglioma67“ orlymphatic and vascular vesselsnervous system697374tumor stromaLaminin Laminin is expressed along the vascular endothelium687375This laminin isoform is recognized by various integrins includingα6β1 α3β1 α6β4 and αVβ3 which promote the migration ofseveral cell types along vascular or nervous system tracks76“In Laminin on the vascular endothelia was discovered asa specific ligand for CD146 on a subset of human CD4 T helper Thcells51 This subset of human T cells expresses CD146 and can entertissues to promote pathogenic autoimmune responses To determinethe CAMs involved in the migratory capacity of Th17 cells into tissuesresearchers used purified Laminin to identify its receptor In thisstudy the authors demonstrated that purified CD146Fc binds toLaminin with high affinity nM and thatthis bindingdisappeared when the endogenous Laminin was specificallydeleted Correspondingly blocking this binding by CD146 antibodyin vivo also reduced Th17 lymphocyte ltration into the CNSTherefore the authors concluded that Laminin is a major tissueligand for CD146 lymphocyteHoweverthe role of Th17 cells in the pathogenesis ofmalignant tumors is still remains controversial Some studiesrevealed that increased percentage of Th17 lymphocytes amongcells ltrating ovarian cancer cells stimulate tumor progression81 whereas other studies showed that Th17 lymphocytes haveanticancer activity and can reduce tumor growth and metastasis82Therefore the roles of CD146 Th17 cells in cancer developmentmay be worthy of further investigationsLaminin CD146 is a reliable biomarker of endothelia and is concentrated atthe intercellular junctions of endothelial cells of vessel system21Signal Transduction and Targeted Therapy Most cancer cellsincluding melanoma migrate along theabluminal sides of vascular andor lymphatic vessels as theydisseminate throughout the body83 Laminin is major lamininsof along the tumordissemination tracks blood and lymphaticvessels nerves and tumor stroma84“To determine the mechanism of CD146 roles in metastasisresearchers used melanoma cells to test what laminin isoformsother than Laminin can bind with the melanoma marker ofCD146 Therefore they used all laminin α chains to examine thebinding affinity with human CD146 in a solidphase ligand bindingassay87 Finally they found that only Laminin of severallaminin isoformsreadily bound to CD146 suggesting thatLaminin is a primary ligand for CD146 in melanomaAccordingly a functionblocking mAb to CD146 inhibited tumorcell migration on Laminin but not on laminins or Inaddition this investigation determined that the identity of NOFpreviously identified as a ligand for chicken CD146 gicerin isactually Laminin In this study the authors also determined that Laminin andespecially Laminin are capable of stimulating migration of abroad panel of cancer cell lines through a filter This investigationis consistent with the observation that the α4laminins includingLaminins and expressed and secreted by variouscarcinoma cells have already emerged as œoncolaminins“Melanoma CD146 binds with Laminin but not whereaslymphocyte CD146 only binds with Laminin suggesting thatthe epitopes of CD146 on somatic cancer cells are different fromthose of CD146 on blood lymphocytes Therefore the ltrationof CD146 invasive cancers into tumordissemination tracks islikely dependent on the interaction between CD146 and Laminin and blocking their binding may affect the efficacy of cell“cellinteractions and interfere metastasisGalectin1 and CD146 is a highly glycosylated junctional CAM involved in thecontrol of vascular vessel integrity Sequence analysis predicts thepresence of eight putative Nglycosylation sites atresiduepositions and It has beenestimated that of the CD146 molecular mass is attributed toglycans88 The galactose residues in glycans can bind withgalectins and such binding can be inhibited by lactoseGalectins are a family of soluble carbohydratebinding lectinsthat modulate celltocell and celltoECM adhesions89 Up to now galectins have been identified in mammals and are foundin humans Among them Galectin1 and are three bestinvestigated galectins and Galectin1 and promote tumordevelopment progression and immune escape90 Galectin1 and 0cCD146 from a melanoma cell adhesion molecule to a signaling receptorWang can hamper antitumor responses and are considered multifunctional targets for cancer therapy9192 The underlying mechanisms include interfering with drug efficacydelivery or reducingthe antitumor effect of immune cells For instance Galectin1confers drug resistance via inducing the expression of multidrugresistance protein which in turn helps tumor cells to pump outcytotoxic drugs facilitating cancer cells to combat anticancerdrugs93 Regarding the immunosuppressive effects of Galectin1and on T cells in a mouse melanoma model targeted inhibitionof Galectin1 enhanced T cellmediated tumor clearance94Galectin3 can neutralize glycosylated IFNγ in tumor matricesablating the immune response to tumors95 To increase overallresponsiveness of tumors to chemo or immunetherapy inhibitors of Galectin19697 and have been used in combinationwith antiCTLA4 or antiPD1 to treat cancer patients in clinicaltrialsBecause both CD146 and galectins are involved in themodulation of angiogenesis researchers hypothesized that somegalectins may be the ligands of CD146 and the interactionsbetween them are required for functional CD146 in angiogenesisas well as in cancer metastasis To date two galectins and have been identified as the ligands of CD146It has been reported thatGalectin1Galectin1 prefers to bind with the branched Nglycans of cellsurface glycoproteins and mediates a glycosylationdependentangiogenesis91100“increasedsecretion of Galectin1 in the ECM facilitates cancer cellproliferation and resistance to cancertherapy in prostatecancer104 and Kaposi™s sarcoma105 Mechanistic investigation hasrevealed that Galectin1 can bind to Nglycans on VEGFR2 toactivate VEGFlike signaling in antiVEGFA refractory tumorspromoting tumor progression Accordingly disruption oftheGalectin1Nglycan axis inhibits tumor growth by promotingvascular remodeling101 This research highlights the importance ofGalectin1 in tumor angiogenesis and cancer metastasis Howeverthese studies cannot exclude the fact that other cellsurfaceproteins with branched Nglycans are also involved in thisglycosylationdependent proangiogenesis pathwayEarly in it was reported that the coexpression of Galectin and CD146 is required for tumor vascularization in a humanmesenchymal stem cell strain with significant angiogenic potential106 In Jouve reported that Galectin1 binds toCD146 on endothelial cells facilitating cell survival107 In thisthey explained that CD146 glycosylation is mainlyreportcomposed of branched Nglycans They showed thattheinteraction of CD146 with Galectin1 is carbohydratemediatedusing both an enzymelinked immunosorbent assay and surfaceplasmon resonance assays In addition they demonstrated thatthe interaction between Galectin1 and CD146 protects endothelial cells against apoptosis induced by Galectin1 Thusit istempting to speculate that CD146 could be a decoy receptor falectin1 preventing the Galectin1 from binding to proapoptotic receptors107 However whether this interaction affectstumor cell survival remains unknown In Yazawa thusfurther analyzed the functions of this interaction on melanomaand found that when Galectin1 binds to CD146 it helps maintainintrinsic malignant features108 The authors examined the expressionidentity and function of Galectin1 ligands in melanomaprogression and demonstrated that CD146 is the major Galectin1ligand on melanoma cellsThese findings provide a perspective on the interactionsbetween CD146 and its ligands such as Galectin1 as contributorsto cancer malignancy Indeed various membrane glycoconjugateshave been identified as binding partners of Galectin1 such as β1integrins CD2 CD3 CD4 CD43 CD45 and GM1 ganglioside Inaddition Galectin1 can bind to a number of ECM components ina dosedependent and βgalactosidedependent manner Forinstance laminin and fibronectin which are highly Nglycosylatedinteract with Galectin1109 Because it has been reported thatCD146 can interact with Laminin Laminin and β1integrin it is reasonable to speculate that CD146 may also interactwith all of those Galectin1 interactors within cancerous cellsSince the tumor vasculature is an easily accessible target forcancer therapy understanding how galectins ‚uence cancerangiogenesis is important for the translational development oftherapies intended to prevent tumor progression Based on thefact that VEGFtargeted therapies often fail when tumors receivecontinuedglycosylationdependentGalectin1receptorsuch as Galectin1CD146VEGFR2 may increase the efficacy of antiVEGF treatmenttreatment110interactionstargetingGalectin3Like Galectin1 Galectin3 can also bind to various galactoseterminated glycans of cellsurface receptors and proteins of ECMand is involved in many physiological and pathological processesfrom cell adhesion and migration to cell activation111112 In cancercells it modulates cell“cell and cell“microenvironment communications contributing to cancer development progression andmetastasis113“ Patients with metastatic diseases tend to havehigher concentrations of circulating Galectin3 than those withonly localized tumors121 Increased circulating Galectin3 promotes bloodborne metastasis due to the interaction of Galectin3with receptors on vascular endothelial cellsfurther causingendothelial secretion of several metastasispromoting cytokinesTo identify the Galectin3binding molecules on the endothelialcell surface using the Galectin3 affinity purification methodfound that CD146 was the major cellsurfaceColomb et alreceptorto strongly bind and colocalize with Galectin3compared with other glycosylated receptors CD31 CD144 andCD106 They also showed that Galectin3 bound to Nlinkedglycans on CD146 and induced CD146 dimerization andsubsequent activation of protein kinase B AKTsignalingCorrespondingly suppression of CD146 expression abolishesGalectin3induced secretion of metastasispromoting cytokinefrom the endothelial cells Thus they concluded that CD146 is thefunctional Galectin3binding receptor on the endothelial cellsurface responsible for Galectin3induced secretion of cytokinesand therefore ‚uences cancer progression and metastasis122Subsequently the binding moieties of CD146 by Galectin3have been further identified The authors demonstrated thatGalectin3 interacts with the highly glycosylated Domain in theCD146 extracellularthe presence orabsence of lactose These findings provide a better understandingof how Galectin3 interacts with cellsurface receptors to mediateendothelial cell migration and the secretion of cytokines123124regardless offragmentThe endothelial galectins are confined to four family membersie Galectin1 and which contribute to tumorangiogenesis92 Tumorinduced angiogenesis is a pathologiccondition in which tumor cells secrete growth factors such asVEGFs to promote the growth of new blood vessels125126 Thesegrowth factors activate quiescent endothelial cells in host tissue tofacilitate them to invade into the tumor stroma for growth of newcapillaries127 Endothelial galectins binding with glycoconjugateson tumors are involved in different processes during tumorinduced angiogenesis Because Galectin1 and binding ofglycoconjugates on tumor cells mediates many key processes inangiogenesis and elevated levels of Galectin1 and in theendothelium are correlated with tumor vascularization105128“the promotion of tumor vascular remodeling by tumor CD146 maybe due to the interactions between CD146 with Galectin1 and S100A8A9S100 proteinsIn humans there are at least members of theS100 protein family132 which have EFhand calciumbindingmotifs and are soluble in saturated ammonium sulfate133Signal Transduction and Targeted Therapy 0cS100 family members typically form homodimers as well asheterodimers trimers and tetramers etc132134 S100 proteins aretypically cytoplasmic proteins but several family members aresecreted by cells as extracellulartheycontribute to a broad array ofintracellular and extracellularfunctions134135 Upregulation of S100 proteins promotes pro‚ammatory responses that contribute to the development andprogression of cancer and autoimmune and chronic ‚ammatorydiseases138“factors134“ ThusincludingadvancedThe secreted S100 proteins bind with several cellsurfacereceptorsproductsRAGE142“ tolllike receptor TLR4147 CD36148 FGFR1149ALCAM150 CD68151 and ErbB4152 However how the cellsurfacereceptors mediate extracellular S100 signaling is lacking and howS100 protein secretion is dynamically regulated in biologicalprocesses also still remains unknownglycationendseem to require the release ofThe secreted S100A8A9 proteins are theS100A8A9 heterodimer‚ammatorybest characterized soluble S100 proteins Mostthe S100A8A9processesinto the ECM153“ Significant upregulation ofheterodimerS100A8A9 has been observed in many tumors including lunggastric esophageal colon pancreatic bladder ovarian thyroidbreast and skin cancers156157 The upregulation of S100A8A9 iscaused either by the ltrating immune cells oftumormicroenvironment158 or by the tumor itself156157 contributingto the establishment of a premetastatic niche in the tumormicroenvironment159Mechanistic investigations demonstrated that upregulatedS100A8A9 induces the expression of serum amyloid which inturn recruits myeloidderived suppressor cell MDSC producing apro‚ammatory environment during metastasis of aggressivedisease160“ In addition enhanced expression of S100A8A9 isalso associated with poor prognosis168S100A8A9 proteins mediate these effects by binding to plasmamembrane elementsincluding heparan sulfate proteoglycanHSPG169 Nglycans170 TLR4171 and RAGE172173 In a melanomalung metastasis model Hiratsuka clearly demonstrated thatlung S100A8A9 as a strong chemokine interacts with TLR4 onmelanoma to attract distant cancer cells to the lungs174 Recentlyit has also been shown that CD146 on melanoma and breastcancer can respond to lung S100A8A9 to induce lungspecificmetastasis of melanoma175176 and breast cancer177S100A8A9 as the ligand of CD146The expression levels ofS100A8 and S100A9 were higher in the lungs than in other ansand the higher expression levels were induced by the primarytumor itself162 In lungassociated MDSC and endothelial cellstumorderived transforming growth factorbeta TGFβ and VEGFA can upregulate the expression and secretion of S100A8A9162Thus it has been recognized that S100A8A9 plays a critical role inlung tropic metastasis and the subsequent growth of cancer cellsin the lungs26178 During metastasis lung S100A8A9 might act asa guiding protein for cancer cells that possess high expressionlevels of CD146162In Ruma revealed that S100A8A9 uses CD146 as areceptor during lungspecific metastasis of melanoma cells175 In thisstudythey demonstrated that S100A8A9 binding to CD146activates nuclear factorkappa B NFκB and induces reactive oxygenspecies formation significantly increasing cell adhesion growth andinvasion Notably this study proposed that CD146 governs cancerinvasion toward the lungs by sensing the cancer microenvironmentas a soil sensor receptor of lung S100A8A9175 Therefore the authorsconclude that S100A8A9 plays a crucial role in lung tropic cancermetastasis by helping to establish an immunosuppressive metastaticniche to which it then attracts remote cancer cells by interactingwith CD146 on the cancer cell surfacesIn Chen further determined the importance of theSignal Transduction and Targeted Therapy CD146 from a melanoma cell adhesion molecule to a signaling receptorWang et alS100A8A9CD146 axis in melanoma dissemination in a skinlesion a critical early step for metastasis of melanoma Thismechanistic study revealed that S100A8A9CD146 bindingactivates a cascade of functions it leads to significant activationof the transcription factor ETS translocation variant ETV4 andthe subsequent induction of matrix metalloproteinase25 Theactivation of MAP3K8ETV4 by S100A8A9CD146 binding finallyresults in lung tropic metastasis of melanoma176Breast cancer cells prefer the lung liver bone and brain astheir metastatic sites This antropic metastasis is known asthe œseed and soil theory179 This conclusion was reachedbecause CD146 was remarkably overexpressed in metastaticbreast cancer cells180“ In in breast cancer cells theS100A8A9CD146 axiselicited downstream signals that producethe driving force for distant metastasis were identified This studyrevealed how S100A8A9 binding to CD146 accelerates breastcancer growth and metastasis They found that S100A8A9 actsas an extracellular cytokine to activate the CD146ETV4 axiswhich upregulates a very high level of ZEB1 a strong EMTinducer ZEB1 in turn induced a mobile phenotype ie EMT incellsthe downregulation of CD146ETV4 axisrepressed S100A8A9induced EMT resulting in greatly weakened tumor growth and lung metastasis Thus this reportsuggested that S100A8A9 contributesto these signalingprocesses through CD146177In contrastSince metastasis accounts for the majority of cancerassociateddeaths studies on metastasis mechanisms are needed to establishinnovative strategies for cancer treatments These findings thatCD146 as a novel receptor for S100A8A9 mediates the transitionof malignant cancers to metastatic sites suggest that strategiesmodulating the interaction between CD146 and S100A8A9 maybe useful for interference with cancer metastasis especially in theprogression of premetastatic tumors to the lungsMatriptaseMatriptase is an epithelialspecific membraneanchored serineprotease that proteolytically degrades targets such as ECMcomponents and the proforms of growth factors183“ Becausemost of solid tumors are originated from epithelia matriptase isthus critically involved in cancerinvasive growth throughdegradation events related to breaching the basement membrane reanization of the ECM and activation of oncogenicsignaling pathways187During neurogenesis matriptase expressed on neural stemprogenitor NSP plays a critical role in cellcontact signalingbetween NSP and brain endothelial cells188 In the directbinding between brain endothelial CD146 and NSP matriptase wasidentified to be involved in the direct endotheliaNSP contact189Such binding can activate the downstream signaling cascades fromincluding p38 and canonical Wntβcatenin pathways inCD146endothelia leading to secretio

Thyroid_Cancer

Growing evidence has demonstrated that glutathione peroxidases GPXs family genes play critical roles in onset and progression of human cancer However a systematic study regarding expression diagnostic and prognostic values and function of GPXs family genes in breast cancer remains absentMaterials and methods Several databases were employed to perform in silico analyses for GPXs family genes qRTPCR western blot and immunohistochemistry staining were introduced to validate GPX3 expression in breast cancer The functions of GPX3 in breast cancer cells were successively determinedResults By combination of receiver operating characteristic ROC curve analysis survival analysis and expression analysis GPX3 was considered as a potential tumor suppressor and a promising diagnosticprognostic biomarker in breast cancer Next low expression of GPX3 was confirmed in breast cancer cells and tissues when compared with corresponding normal controls Overexpression of GPX3 markedly suppressed proliferation colony formation migration and invasion of breast cancer in vitro Moreover two potential mechanisms responsible for GPX3 downregulation in breast cancer including hypermethylation of GPX3 promoter and release of hsamiR3245p inhibitionConclusions Collectively we demonstrate that GPX3 is markedly downregulated in breast cancer possesses significant diagnostic and prognostic values and attenuated in vitro growth and metastasis of breast cancerKeywords Glutathione peroxidase GPX3 Breast cancer Diagnosis Prognosis BiomarkerBackgroundBreast cancer is the most common diagnosed women™s malignant tumor and also the second leading cause of cancerrelated deaths in women worldwide [ ] Despite a variety of advancements have been achieved in diagnosis and therapy the total outcome of patients with breast cancer remains unsatisfactory Thus developing effective therapeutic targets and promising biomarkers for Correspondence 11718264zjueducn Peifen_Fu163comDepartment of Breast Surgery First Affiliated Hospital College of Medicine Zhejiang University QingChun Road Hangzhou Zhejiang Chinadiagnosis and prognosis prediction is very meaningful to improve prognosis of breast cancerGlutathione peroxidases GPXs consisting of eight members GPX18 are ubiquitously expressed proteins that catalyze the reduction of hydrogen peroxides and anic hydroperoxides by glutathione [] GPX family members have been well demonstrated to be frequently aberrantly expressed and are also closely linked to progression of diverse types of human cancer including kidney cancer [] pancreatic cancer [] hepatocellular carcinoma [] cervical cancer [] and gastric cancer [] However a comprehensive study about expression The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cLou a0et a0al Cancer Cell Int Page of function diagnostic and prognostic values of GPXs family in breast cancer remain absentIn this study we first assessed the roles of GPXs family genes in predicting diagnosis and prognosis of breast cancer and then determined the mRNA and protein expression of GPXs family genes in breast cancer using bioinformatic analysis Next the low expression of GPX3 was detected in breast cancer cells and tissues Subsequently the function of GPX3 in breast cancer cell growth and metastasis was also investigated Finally we explored the potential detailed mechanisms responsible for GPX3 downregulation in breast cancerMaterials and a0methodsROC curve analysisUsing TCGA breast cancer and normal breast expression data the diagnostic values of GPXs family genes were evaluated by ROC curve as we previously described [] Pvalue was considered as statistically significantKaplan“Meier‘plotter database analysisKaplan“Meierplotter database httpkmplo tcomanaly sis which is capable to access the effect of genes on survival in cancer types including breast cancer was employed to perform survival analysis for GPXs family genes and miRNAs in breast cancer [] Logrank Pvalue was considered as significantGEPIA database analysisGEPIA database httpgepia cance rpkucnindex html a newly developed interactive web server for analyzing the RNA sequencing expression data of tumors and normal samples from the TCGA and GTEx projects was used to determine mRNA expression profile of GPXs family genes in breast cancer [] Pvalue was considered as statistical significanceOncomine database analysisOncomine database wwwoncom ine which is a cancer microarray database and integrated datamining platform was also utilized to analyze mRNA expression of GPXs family genes in breast cancer [ ] Fold change FC Pvalue and a gene rank in top were set as the thresholds for selecting the included datasetsUALCAN database analysisThe protein expression levels of GPXs family genes in breast cancer were assessed using UALCAN database httpualca npathuabeduindex html which is a comprehensive userfriendly and interactive web resource for analyzing cancer OMICS data [] UALCAN database was also introduced to determine the promoter methylation level of GPX3 in breast cancer Pvalue of statistical analysis was considered to have significant differencesstarBase database analysisstarBase database tarb asesysueducnindex php an source platform for investigating miRNAassociated studies was used to predict the upstream binding miRNAs of GPX3 [ ] The correlation of GPX3 with miRNA in breast cancer and miRNA expression level in breast cancer were also assessed by starBase database Pvalue was considered as statistical significanceCell lines and a0clinical tissuesThe human breast cancer cell lines MCF7 and MDAMB231 and normal breast cell line MCF10A were purchased from Shanghai Institute of Biological Science Chinese Academy of Sciences Shanghai China breast cancer tissues and matched normal tissues were obtained from patients with breast cancer who received surgical resection in the First Affiliated Hospital of Zhejiang University College of Medicine Hangzhou China This study was approved by the ethics committee Table Correlation of a0 GPX3 expression with a0 various clinicopathological features in a0breast cancerFeaturesCasesBreast cancerLow expressionHigh expressionP‘valueAge ‰ Tumor size ‰ Lymph node metastasis Present AbsentHistopathological grade I“II IIIER status Positive NegativePR status Positive NegativeHER2 status Positive Negative 0cLou a0et a0al Cancer Cell Int Page of of the First Affiliated Hospital of Zhejiang University College of MedicineRNA isolation and a0qRT‘PCRTotal RNA was isolated from breast cancer cells and tissues by Trizol reagent Invitrogen USA qRTPCR was employed to detect GPX3 mRNA expression in breast cancer as we previously described [] GPX3 expression was normalized to GAPDH by the method of ˆ’ddCt The sequences of primers used in this study GPX3 forward primer ²GAG CTT GCA CCA TTC GGT CT3² GPX3 reverse primer ²GGG TAG GAA GGA TCT CTG AGTTC3² GAPDH forward primer ²AAT GGA CAA CTG GTC GTG GAC3² GAPDH reverse primer ²CCC TCC AGG GGA TCT GTT TG3²Protein extraction and a0western blotProtein of breast cancer cells was extracted using RIPA buffer Beyotime China supplemented with protease and phosphatase inhibitors Thermo Scientific USA Western blot was performed as previously described [] The primary antibodies of GPX3 and GAPDH were purchased from Abcam and antirabbit peroxidase conjugated secondary antibody was purchased from Sigma GPX3 band density was normalized to GAPDH and quantified by ImageJ softwareImmunohistochemistry IHC analysisIHC was utilized to analyze the protein expression of GPX3 in breast cancer tissues and matched normal breast tissues as we previously reported []Establishment of a0stably‘overexpressed cellFull length of GPX3 was first amplified after which the PCR product was cloned into pcDNA31PURO vector digested with BamH1 and XhoI GPX3overexpressed Lipofectamine„¢ Invitrogen USA according to the plasmid was transfected into breast cancer cells using manufactures™ instruction Then stablyoverexpressed cell was screened using puromycin a0μgmLCCK‘ assay stablyoverexpressed cells were seeded into 96well plates and cultured for varied period and a0h At the culture end of each time point a0μl CCK8 solution was added into each well and incubated for another a0 h at a0 °C Finally the optical density OD value at a0nm of each well was determined by a microplate readerColony formation assay stablyoverexpressed cells were seeded into sixwell plates and cultured for a0weeks At the end of culture the plates were washed using phosphate buffered saline PBS for two times Next the plates were fixed in methanol for a0min and stained with crystal violet solution for another a0 min Finally the visible colonies of each well were countedWound healing assayWound healing assay was introduced to detect the migrated ability of breast cancer cells × stablyoverexpressed cells were seeded into sixwell plates When the cells were grown to confluence a wound cross was made using a micropipette tip Photographs were then taken through a microscopy immediately or a0h after woundingTranswell invasion assayCell invasion was determined by Transwell invasion assay Briefly transwell inserts were firstly coated with Matrigel BD USA Then × stablyoverexpressed cells suspended in a0 mL serumfree medium were added into inserts And a0mL medium containing FBS was added to the lower compartment as a chemoattractant After culturing for a0h the cells on the upper membrane were carefully removed using a cotton bud and cells on the lower surface were fixed with methanol for a0 min and successively stained with crystal violet solution for a0min Photographs were then taken through a microscopyStatistical analysisStatistical analysis of bioinformatic analysis was performed by online databases as mentioned above The results of experimental data were shown as mean ± SD Student™s ttest was used to assess differences between two groups The diagnostic value was determined by ROC curve analysis A twotailed value of P was considered as statistically significantResultsThe diagnostic and a0prognostic values of a0GPXs family genes in a0breast cancerTo explore if the expression of GPXs family genes possesses significant diagnostic values in patients with breast cancer receiver operating characteristic ROC curve analysis was employed based on breast cancer data from TCGA database Fig a0 As shown in Fig a0 four GPXs family genes had the significant ability to distinguish breast cancer tissues from normal breast tissues including GPX2 GPX3 GPX4 and GPX8 However the other four GPXs family genes GPX1 GPX5 GPX6 and GPX7 showed no statistical diagnostic values in breast cancer Notably these findings suggested that GPX3 was the most potential diagnostic biomarker for patients 0cLou a0et a0al Cancer Cell Int Page of Fig The diagnostic values of GPXs family genes in breast cancer using ROC curve analysis a GPX1 b GPX2 c GPX3 d GPX4 e GPX5 f GPX6 g GPX7 h GPX8with breast cancer with the Area Under Curve AUC value being equal to Next we investigated the prognostic values of GPXs family genes in breast cancer using Kaplan“Meierplotter database Fig a0 Increased expression of GPX1 Fig a02a indicated poor prognosis of breast cancer Breast cancer patients with higher expression of GPX2 Fig a02b GPX3 Fig a02c or GPX5 Fig a02e had better prognosis GPX4 GPX6 and GPX7 had no significant predictive values for prognosis of breast cancer All these findings together indicated that only GPX2 and Fig The prognostic values of GPXs family genes in breast cancer determined by Kaplan“Meier plotter database a GPX1 b GPX2 c GPX3 d GPX4 e GPX5 f GPX6 g GPX7 h GPX8 0cLou a0et a0al Cancer Cell Int Page of GPX3 possessed significant diagnostic and prognostic values for breast cancerThe expression levels of a0GPXs family genes in a0breast cancerNext we further studied the expression levels of GPXs family genes in breast cancer First of all TCGA and GTEx databases were introduced to mine the mRNA expression of GPXs family genes in breast cancer The mRNA expression profile of GPXs family was shown in Fig a03a TCGA tumor tissues compared with TCGA normal tissues and Fig a03b TCGA tumor tissues compared with TCGA normal tissues and GTEx normal tissues We found that GPX2 and GPX3 were significantly downregulated in breast cancer Fig a0 3c“f Next Oncomine database was used to further analyze mRNA expression of GPXs family genes in breast cancer Fig a04a We performed metaanalysis for included studies about GPX3 and found that GPX3 mRNA expression was markedly decreased in breast cancer Fig a04b The downregulation of GPX3 mRNA expression in breast cancer of the GPX3associated studies was presented in Fig a04c“q However we found that GPX2 was not significantly downregulated in breast cancer Subsequently CPTAC database was utilized to assess the protein expression of GPXs family genes in breast cancer Fig a0 The results revealed that GPX1 GPX2 GPX3 and GPX4 protein levels were markedly decreased in breast cancer when compared with normal controls GPX7 protein expression in breast cancer was significantly increased GPX8 showed no statistical difference between breast cancer tissues and normal tissues And GPX5 and GPX6 were not found in CPTAC Taken together GPX3 was the most potential one among all GPXs family genes in breast cancer and was selected for following research Fig a0The expression level of a0GPX3 was a0confirmed in a0breast cancer and a0negatively correlated with a0tumor progressionTo further validate the results from in silico analysis we detected the mRNA and protein expression levels of GPX3 in breast cancer cells and tissues As presented in Fig a0 7a b GPX3 mRNA and protein were significantly downregulated in two breast cancer cells MCF7 and MDAMB231 when compared with normal cell MCF10A We also found that GPX3 mRNA expression in breast cancer tissues was much lower than that in adjacent matched normal tissues Fig a07c The protein expression of GPX3 was also detected using immunohistochemistry IHC analysis The results showed that GPX3 protein expression was significantly decreased in breast cancer tissues Fig a07d Collectively GPX3 mRNA and protein expression levels were significantly downregulated in breast cancer which was identical with the bioinformatic analytic results Furthermore Chi square test revealed that low expression of GPX3 was significantly negatively correlated with ERPR expression and positively linked to tumor size histopathological grade and lymph node metastasis Table a0 All these findings showed that GPX3 was negatively correlated with progression of breast cancer and might function as a tumor suppressor in breast cancerGPX3 overexpression suppressed proliferation and a0colony formation of a0breast cancer cellsGiven the low expression of GPX3 in breast cancer overexpression technology was used to study GPX3²s functions We then constructed the overexpressed plasmid of GPX3 After transfection of GPX3overexpressed plasmid GPX3 mRNA and protein expression levels were significantly upregulated in breast cancer cells Fig a0 8a b Firstly we explored the effect of GPX3 on growth of breast cancer cells CCK8 assay demonstrated that overexpression of GPX3 markedly suppressed in a0vitro proliferation of breast cancer cells MCF7 and MDAMB231 Fig a0 8c d Furthermore colony formation assay also revealed that GPX3 upregulation led to the inhibition of clonogenic capacity of breast cancer cells Fig a08e f These findings indicated that GPX3 overexpression significantly suppressed in a0 vitro proliferation and colony formation of breast cancer cellsGPX3 overexpression inhibited migration and a0invasion of a0breast cancer cellsMetastasis is another hallmark of malignant tumors including breast cancer We intended to ascertain if GPX3 affects metastasis of breast cancer Wound healing assay was first employed to investigate GPX3²s function in controlling migration of breast cancer cells and the result demonstrated that overexpression of GPX3 obviously attenuated the migrated ability of breast cancer cells Fig a09a b Moreover increased expression of GPX3 could also suppressed invasion of breast cancer cells which was detected by transwell invasion assay Fig a09c“f Taken together overexpression of GPX3 suppressed in a0vitro migration and invasion of breast cancer cellsThe potential mechanisms responsible for a0GPX3 downregulation in a0breast cancerFinally we preliminarily probed the possible molecular mechanisms that accounted for GPX3 downregulation in breast cancer Promoter hypermethylation may be responsible for expression suppression of tumor suppressors Intriguingly we found that the promoter methylation level of GPX3 was significantly upregulated in breast cancer tissues compared with normal controls Fig a010a Gene expression was also frequently negatively regulated by miRNAs at posttranscriptional 0cLou a0et a0al Cancer Cell Int Page of Fig The mRNA expression of GPXs family genes in breast cancer determined by GEPIA database a The mRNA expression profile of GPXs family genes in breast cancer tissues compared with TCGA normal breast tissues b The mRNA expression profile of GPXs family genes in breast cancer tissues compared with TCGA and GTEx normal breast tissues c d GPX2 was significantly downregulated in breast cancer e f GPX3 was significantly downregulated in breast cancer P level The miRNAs that potentially bind to GPX3 were predicted by starBase database and miRNAs were finally found For better visualization miRNAGPX3 network was established Fig a0 10b Based on the action mechanism of miRNA there should be negative correlation between miRNA and target gene We performed expression correlation analysis for miRNAGPX3 pairs As listed in Table a0 four potential miRNAs hsamiR3245p hsamiR3283p hsalet7a5p and hsamiR449b5p which were inversely associated 0cLou a0et a0al Cancer Cell Int Page of Fig The mRNA expression of GPXs family genes in breast cancer determined by Oncomine database a The mRNA expression of GPXs family genes in breast cancer b Metaanalysis for the included GPX3associated datasets in breast cancer c“q The mRNA expression of GPX3 was markedly downregulated in breast cancer in included GPX3assocaited datasets 0cLou a0et a0al Cancer Cell Int Page of Fig The protein expression of GPXs family genes in breast cancer detected by UALCAN database a GPX1 b GPX2 c GPX3 d GPX4 e GPX7 f GPX8 P 0cLou a0et a0al Cancer Cell Int Page of Fig The visual flowprocess diagram of this studywith GPX3 expression in breast cancer were identified The prognostic values of the four miRNAs in breast cancer were also evaluated by Kaplan“Meierplotter database Fig a0 10c d Survival analysis revealed that among the four miRNAs only high expression of hsamiR3245p indicated poor prognosis for patients with breast cancer Fig a0 10c The expression levels of four miRNAs in breast cancer was subsequently determined by starBase Fig a010g“j and showed that miR3245p and hsamiR449b5p were significantly upregulated whereas hsamiR3283p and hsalet7a5p were markedly downregulated in breast cancer compared with normal controls By combination of survival and expression analysis miR3245p was considered as the most potential upstream miRNA of GPX3 in breast cancer The above results implied that promoter hypermethylation and miR3245pmediated suppression were two potential mechanisms that may be responsible for GPX3 downregulation in breast cancer Fig a010lDiscussionBreast cancer is the most common cancer type in women The molecular mechanism of carcinogenesis of breast cancer is still unclear and need to be further investigated Increasing findings have showed that GPXs are critical regulators in onset and progression of human cancer However the knowledge of GPXs in breast cancer is still limitedROC curve and survival analysis for GPXs family revealed that some of them might serve as promising diagnostic and prognostic biomarkers for breast cancer especially GPX2 and GPX3 Expression analysis demonstrated the significant low expression of GPX3 in breast cancer GPX3 was reported to act as a tumor suppressor 0cLou a0et a0al Cancer Cell Int Page of Fig The expression levels of GPX3 in breast cancer cells and tissues The mRNA a and protein b expression of GPX3 in breast cancer cells was significantly lower than that in normal breast cell c The mRNA expression of GPX3 was markedly decreased in breast cancer tissues compared with matched normal breast tissues d IHC analysis of GPX3 expression levels in normal breast tissues and breast cancer tissues Bar scale um P in human cancer For example Cai et a0al indicated that GPX3 prevented migration and invasion of gastric cancer by targeting NFkBWnt5aJNK signaling [] Lee et a0al suggested that GPX3 arrested cell cycle and functioned as a tumor suppressor in lung cancer [] Hua et a0 al showed that silencing GPX3 expression promoted tumor metastasis in human thyroid cancer [] Caitlyn et a0 al revealed that plasma GPX3 limited the development of colitis associated carcinoma [] However the function and mechanism of GPX3 in breast cancer have not been reported and need to be further elucidatedNext we confirmed the low expression of GPX3 in breast cancer cells and tissues using qRTPCR western blot and IHC which supported the results of bioinformatic analysis Functional experiments revealed that overexpression of GPX3 significantly inhibited in a0 vitro proliferation colony formation migration and invasion of breast cancer cellsPrevious studies have showed the effect of promoter methylation level in regulating gene expression [] Thus we preliminarily evaluated the promoter methylation level of GPX3 in breast cancer and found that it was significantly upregulated in breast cancer compared with normal breast tissues Moreover Mohamed et a0 al also demonstrated the link between promoter hypermethylation of GPX3 and inflammatory breast carcinogenesis [] The report together with our finding revealed that hypermethylation of GPX3 promoter might be a potential mechanism responsible for GPX3 downregulation in breast cancermiRNAs are involved in multiple biological processes by suppressing gene expression [ “] We also explored the upstream regulatory miRNAs of GPX3 By combination of correlation analysis survival analysis and expression analysis for these miRNAs miR3245p was regarded as the most potential miRNA which was overexpressed negatively correlated with GPX3 expression and possessed poor prognosis in breast cancer Numerous studies have demonstrated that miR3245p served as an oncogenic miRNA in human cancer For example miR3245p promoted progression of papillary thyroid carcinoma via microenvironment alteration [] miR3245p facilitated progression of colon cancer by activating Wntbetacatenin pathway [] Moreover the 0cLou a0et a0al Cancer Cell Int Page of Fig Overexpression of GPX3 inhibited proliferation and colony formation of breast cancer cells in vitro a“b The overexpression effect of GPX3overexpressed plasmid in breast cancer cells c“d Overexpression of GPX3 inhibited proliferation of MCF7 and MDAMB231 cells e“f Overexpression of GPX3 inhibited colony formation of MCF7 and MDAMB231 cells P relationship between GPX3 and miR3245p has already been reported in lung cancer [] Thus overexpressed miR3244p might be another mechanism that accounted for GPX3 downregulation in breast cancer In the future the oncogenic roles of miR3245p need to be further investigated by in a0vitro and in a0vivo assaysConclusionsIn summary our current findings indicate that GPX3 is markedly downregulated in breast cancer promotes in a0 vitro growth and metastasis of breast cancer cells and servers as a promising diagnostic or prognostic biomarker for patients with breast cancer Moreover we also elucidate that promoter hypermethylation and miR3245pmediated suppression may be two 0cLou a0et a0al Cancer Cell Int Page of Fig Overexpression of GPX3 suppressed migration and invasion of breast cancer cells in vitro a b Increased expression of GPX3 attenuated migration of MCF7 and MDAMB231 cells c d Increased expression of GPX3 attenuated invasion of MCF7 cell e f Increased expression of GPX3 attenuated invasion of MDAMB231 cell Bar scale um P See figure on next pageFig The potential mechanisms responsible for GPX3 downregulation in breast cancer a The promoter methylation level of GPX3 was increased in breast cancer compared with normal controls b The miRNAGPX3 network c“f The prognostic values of four miRNAs in breast cancer g“j The expression levels of four miRNAs in breast cancer k The intersection analysis of survival analysis and expression analysis l The model of GPX3²s function and dysregulated mechanism in breast cancer P was considered as statistically significant 0cLou a0et a0al Cancer Cell Int Page of 0cLou a0et a0al Cancer Cell Int Page of Table The expression correlation of a0GPX3 with a0predicted miRNAs using TCGA breast cancer datamiRNAhsamiR3245phsamiR3283phsalet7a5phsamiR449b5phsamiR6295phsamiR47565phsamiR642a5phsalet7d5phsamiR449ahsamiR5895phsamiR181d5phsamiR21145phsamiR34a5phsamiR449c5phsamiR23a3phsamiR3150a3phsamiR47315phsamiR23b3phsamiR4915phsamiR4739hsamiR181c5phsamiR3612hsamiR5823phsamiR650hsalet7b5phsamiR3383phsamiR2278hsamiR1225phsamiR181a5phsamiR181b5phsamiR3139hsamiR4644hsamiR5013phsamiR26825phsamiR4306hsamiR95phsamiR620hsamiR1321hsamiR985phsalet7e5phsamiR1855phsamiR1270hsalet7 g5phsamiR2055phsamiR371a5phsamiR8735phsamiR34b5phsamiR5325phsamiR2963pRˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ P‘valueTable continuedmiRNAhsamiR7085phsamiR35295phsamiR5745phsamiR8765phsamiR2965phsamiR5023phsamiR1365phsamiR285phsamiR3615phsamiR520 hhsamiR6683phsamiR520 g3phsamiR376b3phsamiR6753phsalet7f5phsamiR34c5phsamiR665hsamiR1385phsamiR146a5phsamiR376a3phsamiR223phsamiR2995phsalet7i5phsamiR4953phsamiR1433phsamiR8893phsamiR146b5phsamiR3795phsamiR2233phsalet7c5pRP‘valuepotential mechanisms responsible for GPX3 downregulation in breast cancer These results provide key clues for developing effective therapeutic targets and biomarkers for breast cancerAcknowledgementsNot applicableAuthors™ contributionsWL and PF designed this work performed experiments analyzed data and draft the manuscript BD performed some experiments SW revised the manuscript All authors read and approved the final manuscriptFundingNot applicableAvailability of data and materialsThe data in this work are available from the corresponding author on reasonable request 0cLou a0et a0al Cancer Cell Int Page of Lou W Ding B Fan W High expression of pseudogene PTTG3P indicates a poor prognosis in human breast cancer Mol Therapy Oncolytics “ Lou W Liu J Ding B Jin L Xu L Li X Chen J Fan W Five miRNAsmediated PIEZO2 downregulation accompanied with activation of Hedgehog signaling pathway predicts poor prognosis of breast cancer Aging “ Chen D Si W Shen J Du C Lou W Bao C Zheng H Pan J Zhong G Xu L et al miR27b3p inhibits proliferation and potentially reverses multichemoresistance by targeting CBLBGRB2 in breast cancer cells Cell Death Dis Cai M Sikong Y Wang Q Zhu S Pang F Cui X Gpx3 prevents migration and invasion in gastric cancer by targeting NFsmall ka CyrillicBWnt5aJNK signaling Int J Clin Exp Pathol “ An BC Choi YD Oh IJ GPx3mediated redox signaling arrests the cell cycle and acts as a tumor suppressor in lung cancer cell lines Plos One 2018139e0204170 Zhao H Li J Li X Han C Zhang Y Zheng L Guo M Silencing GPX3 expression promotes tumor metastasis in human thyroid cancer Curr Prot Peptide Sci “ Barrett CW Ning W Chen X Smith JJ Washington MK Hill KE Coburn LA Peek RM Chaturvedi R Wilson KT et al Tumor suppressor function of the plasma glutathione peroxidase gpx3 in colitisassociated carcinoma Cancer Res “ Kulis M Esteller M DNA methylation and cancer Adv Genet “ Mohamed MM Sabet S Peng DF Nouh MA ElShinawi M Promoter hypermethylation and suppression of glutathione peroxidase are associated with inflammatory breast carcinogenesis Oxid Med Cell Lou W Liu J Ding B Chen D Xu L Ding J Jiang D Zhou L Zheng S Fan W Identification of potential miRNAmRNA regulatory network contributing to pathogenesis of HBVrelated HCC J Transl Med Lou W Liu J Gao Y Zhong G Chen D Shen J Bao C Xu L Pan J Cheng J et al MicroRNAs in cancer metastasis and angiogenesis Oncotarget “ Lou W Liu J Gao Y Zhong G Ding B Xu L Fan W MicroRNA regulation of liver cancer stem cells Am J Cancer Res “ Yang Y Xia S Zhang L Wang W Chen L Zhan W MiR3245pPTPRDCEBPD axis promotes papillary thyroid carcinoma progression via microenvironment alteration Cancer Biol Therapy “ Yan D Liu W Liu Y Luo M LINC00261 suppresses human colon cancer progression via sponging miR3243p and inactivating the Wntbetacatenin pathway J Cell Physiol “ Lin MH Chen YZ Lee MY Weng KP Chang HT Yu SY Dong BJ Kuo FR Hung LT Liu LF et al Comprehensive identification of microRNA arm selection preference in lung cancer miR3245p and3p serve oncogenic functions in lung cancer Oncol Lett “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsEthics approval and consent to participateThis study was approved by the ethics committee of the First Affiliated Hospital of Zhejiang University College of MedicineConsent for publicationNot applicableCompeting interestsThe authors state that they have no conflicts of interestReceived June Accepted July References Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA Cancer J Clin “Lou W Liu J Ding B Xu L Fan W Identification of chemoresistanceassociated miRNAs in breast cancer Cancer Manag Res “ Matouskova P Hanouskova B Skalova L MicroRNAs as potential regulators of glutathione peroxidases expression and their role in obesity and related pathologie

Thyroid_Cancer

Radial shock waves prevent growth retardation caused by the clinically used drug vismodegib in ex vivo cultured bonesSowmya Ramesh123 Lars Svendahl245 Vrisha Madhuri135 farasat Zaman2In childhood medulloblastoma patients the hedgehog antagonist vismodegib is an effective anticancer treatment but unfortunately induces irreversible growth arrests and growth impairment limiting its use in skeletally immature patients We hypothesized that radial shock wave treatment rSWT may protect druginduced growth impairment owing to its osteogenic effects Fetal rat metatarsal bones were exposed to vismodegib day “ nM andor rSWT single session other bones from day were continuously exposed to a Gli1 antagonist GANT61 µM andor rSWT single session Control bones were untreated The bone length was measured at intervals histomorphometric analysis and immunostaining for PCNA Gli1 and Ihh were performed on the sectioned bones Bones treated with vismodegib showed impaired bone growth reduced height of the restingproliferative zone and reduced hypertrophic cell size compared to control In vismodegib treated bones a single session of rSWT partially rescued bone growth increased the growth velocity hypertrophic cell size and restored growth plate morphology Bones exposed to GANT61 showed impaired bone growth and disanized growth plate while when combined with rSWT these effects were partially prevented Locally applied rSWT had a chondroprotective effect in rat metatarsal bones and suggest a novel strategy to prevent growth impairment caused by vismodegibHedgehog Hh proteins are wellknown to be overexpressed in paediatric medulloblastoma1 Mutations that occur in the family of Hhpathway genes such as patched1 suppressor of fuse and smoothened leads to an increased level of the gliomaassociated oncogene Gli1 a downstream transcription factor of Hh2 In the clinic hedgehog inhibitors are used to decrease the Hhactivity and thereby impede tumor progression3“ However stable expression of the Hhgene is essential to maintain chondrocyte proliferation and hypertrophy during bone growth6 A recent study reported that prolonged exposure to vismodegib a Hhantagonist in children with medulloblastoma resulted in irreversible growth plate fusion causing growth arrest of long bones78 Preclinical studies in young mice exposed to a Hhantagonist also showed growth arrests and bone growth defects9 Mechanistic studies revealed that even brief exposure to a Hhinhibitor was enough to damage the growth plates by diminishing the numbers of reserve and proliferative chondrocytes9 These findings further imply that it may not be possible to arrive at a dose that selectively targets tumor growth with no sideeffects on bone development Therefore in children a protective strategy for growth plate shielding without interfering with the desired anticancer effects of vismodegib in the neural tissue is highly desiredIn vitro studies using cultured rat metatarsal bones10 and in a0vivo studies in rabbits11 and humans12 have shown that radial shock wave treatment rSWT a noninvasive modality used in the clinic have stimulatory effects on bone growth10 Furthermore the observed stimulation of chondrocyte proliferation and hypertrophy induced by rSWT was partially linked to local upregulation of Gli1 in cultured metatarsal bones10 Interestingly previous in a0vitro studies revealed that highenergy shock wave treatment increased the uptake of chemotherapy agents13 1Department of Paediatric Orthopaedics Christian Medical College Vellore India 2Division of Paediatric Endocrinology Department of Women™s and Children™s Health Karolinska Institutet Solna Sweden 3Centre for Stem Cell Research A Unit of inStem Bengaluru Christian Medical College Bagayam Vellore India 4Paediatric Endocrinology and Metabolism Astrid Lindgren Children²s Hospital Karolinska University Hospital Solna Sweden 5These authors contributed equally Lars Svendahl and Vrisha Madhuri email sowmyarameshkiseScientific RepoRtS 101038s41598020699040Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Rescuing effects of rSWT on vismodegib treated bones a Fetal rat metatarsal bones cultured exvivo were treated with the Hhinhibitor vismodegib a0nM for a0days n dotted line a single session of highenergy rSWT a0Hz a0mJ n or both n and thereafter followed for a0days The graph shows increases in bone length over time a0from day All error bars indicate SD Twoway ANOVA was applied b Graph shows the increase in the growth velocity on day of control vismodegib rSWT and vismodegib rSWT treated bones All error bars indicate SD Representative images of metatarsal bones stained with Alcian blue c untreated control d vismodegib e rSWT and f vismodegib rSWT Magnification 10x g height measurements of R P zone and h hypertrophic cell size Quantification of immunostaining for i Gli1 and j Ihh using the ImageJ software p p p thereby lowering the dose of the drug when applied to cell lines derived from human osteosarcoma14 human colorectal adenocarcinoma15 and anaplastic thyroid cancer16 The effect was mediated by a transient increase in cell membrane permeability allowing passage of a higher concentration of the drug16A recent report suggested that locally applied rSWT can promote longitudinal bone growth of rat metatarsal bones cultured exvivo in the absence of serumgrowth factors10 Based on these findings we hypothesized that rSWT may also have the capacity to prevent growth failure caused by Hhinhibitors We aimed to investigate the potential for rSWT to prevent growth retardation caused by two different Hhantagonists vismodegib and the Gli1 antagonist GANT61 in a wellestablished model of cultured fetal rat metatarsal bonesResultsEffect of vismodegib on bone growth and rescuing effects of rSWT To allow transient inhibition of Hhactivity cultured fetal rat metatarsal bones were treated with vismodegib for a0days whereafter growth was monitored for another a0days without any treatment Bones treated with vismodegib grew less than untreated controls and the difference was significant when measured on day ± vs ± bone length increase from day respectively p Fig a01aTo address the primary objective of this study if rSWT can prevent growth failure caused by hedgehog inhibition we first studied if a single exposure to rSWT can rescue metatarsal bone growth after transient exposure to a0days treatment with vismodegib Indeed bones treated with rSWT single exposure on day in combination with vismodegib day “ grew significantly better than bones treated with vismodegib alone when measured Scientific RepoRtS 101038s41598020699040Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Rescuing effects of rSWT on GANT61 treated bones a Fetal rat metatarsal bones cultured exvivo treated with the Hhinhibitor GANT61 a0µM n a single session of rSWT a0Hz a0mJ n or both n and followed for a0days The graph shows increases in bone length a0from day All error bars indicate SD Twoway ANOVA was applied Representative images of metatarsal growth plates stained with Alcian blue b untreated control c GANT61 d rSWT and e GANT61 rSWT Magnification 10x f Height measurements of R P zone and g hypertrophic cell size Quantification of immunostaining for h PCNA and i Gli1 using the ImageJ software p p on day ± vs ± respectively p Fig a01a Also the growth velocity tended to be higher in rSWT vismodegib compared to vismodegib alone Fig a01bGrowth plate morphology Histological evaluation revealed severe disruption in the growth plate morphology in the bones treated with vismodegib compared to control Fig a01c“d The disrupted growth plate morphology in vismodegib treated bones was found to be more normal in bones receiving rSWT alone Fig a01e and rSWT vismodegib Fig a01f When comparing vismodegib treated bones to controls we found reduced height of the resting proliferative R P zone ± a0µm vs ± a0µm respectively p Fig a01g The height of the R P zone tended to be increased in bones exposed to rSWT vismodegib when compared to vismodegib alone ± a0µm vs ± a0µm respectively p ns Fig a01g Histomorphometric analysis showed decreased size of hypertrophic chondrocytes in the bones treated with vismodegib compared to control ± a0µm vs ± a0µm respectively p Fig a01h In bones treated with rSWT vismodegib the hypertrophic chondrocytes were significantly larger compared to vismodegib alone and similar in size as in untreated control bones ± a0µm ± a0µm and ± a0µm respectively p vs vismodegib p ns vs control Fig a01h Immunostaining for Gli Fig a01i and Ihh Fig a01j did not show any significant effects of vismodegib andor rSWTShock wave treatment prevents bone growth retardation caused by GANT61 To study the effects of continuous inhibition of Hhactivity fetal rat metatarsal bones were treated with the Gli1 antagonist GANT61 from day of culture until the termination of the experiment on day Already on day GANT61 induced a pronounced suppression of the bone growth ± ± p vs control which remained at endpoint on day ± ± p vs control Fig a02aNext we asked if a single exposure to rSWT can rescue from growth retardation caused by continuous Hhinhibition induced by GANT61 Indeed metatarsal bones exposed to rSWT on day were rescued from GANT61induced growth retardation already from day of culture ± ± p vs GANT61 alone which remained until endpoint ± ± p vs GANT61 alone Fig a02aGrowth plate morphology Compared to control in the bones continuously treated with GANT61 growth plate morphology was found to be disturbed with disanized chondrocyte columns on day Fig a02b c Bones treated with rSWT alone showed anized growth plate morphology Fig a02d The growth rescuing Scientific RepoRtS 101038s41598020699040Vol0123456789wwwnaturecomscientificreports 0ceffect of rSWT in GANT61 treated bones was accompanied by a normalization of growth plate morphology including anized chondrocyte columns Fig a02eHistomorphometric analyses showed no significant differences in the height of the R P zone between GANT61 treated bones with or without rSWT ± a0µm ± a0µm p ns Fig a02f There was an increased size of hypertrophic chondrocytes in rSWT GANT61 group when compared to GANT61 alone ± a0µm ± a0µm p Fig a02g Furthermore the number of proliferative chondrocytes tended to be lower in GANT61 treated bones when compared to control ± cells ± cells p ns Fig a02h The number of proliferative PCNA positive cells tended to be higher in the rSWT GANT61 group compared to GANT61 alone ± cells ± cells p ns Fig a02h Immunoexpression of Gli1 tended to be reduced in GANT61 treated bones p ns vs control whereas bones treated with rSWT GANT61 tended to have higher Gli1 expression compared to GANT61 alone p ns Fig a02iDiscussionWe aimed to investigate the potential for locally applied rSWT to prevent bone growth impairment caused by the hedgehog inhibitor vismodegib a therapeutic investigational drug using a wellestablished model of ex a0vivo cultured fetal rat metatarsal bones Herein we report that a single session of rSWT partially prevented growth retardation caused by both transient and continuous Hhinhibition induced by vismodegib and GANT61 respectively The growth rescuing effects by rSWT were accompanied by preservation of growth plate morphology disrupted by the Hhinhibitors Altogether our data suggest that rSWT has the potential to noninvasively protect bones from growth retardation caused by vismodegibBone growth is majorly dependent on the preservation of a unique anization of chondrocytes in the growth plate17 Recent reports have demonstrated that long term exposure to vismodegib the first Hhantagonist approved in the US by FDA led to permanent growth impairment in children with medulloblastoma78 To date no successful strategy that targets tumor cells with no adverse effect on longitudinal bone growth has been described Previous reports have shown that rSWT a treatment modality that is already used in children for musculoskeletal indications18 can stimulate longitudinal bone growth locally in exvivo cultured metatarsal bones even in the absence of any systemic growth factors10 Proinflammatory cytokines are also known to impair bone growth19 and interestingly shockwave treatment has been shown to reduce inflammation and apoptosis while stimulating the regeneration of various tissues2021 These findings encouraged us to expand this knowledge and further investigate the potential for rSWT to prevent bone growth impairment caused by HhinhibitorsVismodegib at a0nM concentration has shown to impair bone growth in exvivo cultured metatarsal bones22 and decrease proliferation of the precursors of a0cerebellar granule neurons23 while in a0vivo studies in a model of medulloblastoma have also shown that vismodegib inhibits Gli1 at a IC50 of a0nM24 a similar range of concentration as used in the present study In young mice transient inhibition of the Hh pathway has been reported to cause permanent defects in bone and growth plate structure9 Similar to the previous in a0vivo observations in young mice9 our histomorphometric growth plate data suggest that partial loss of Hhactivity may result in the breakdown of chondrocyte columnar anization and reduced size of hypertrophic chondrocytes Also the disrupted growth plate ultrastructure caused by Hhinhibition explains the observed growth deficit in our study model system Besides undesired apoptosis of stemlike cells within the growth plate is another wellknown contributing factor linked to growth retardation caused by anticancer drugs2526Our key finding is that a single administration of rSWT not only prevented bone growth retardation caused by transient exposure to the Hhinhibitor vismodegib but also rescued bone growth under a condition of continuous Hhinhibition induced by another Hhinhibitor GANT61 Furthermore rSWT also improved growth velocity and restored growth plate morphology in bones exposed to vismodegib or GANT61 Thus our findings highlight the potential for shock wave technology to be developed as a new and safe treatment strategy to minimize deleterious effects of Hhinhibitors selectively in the growth plates of treated childrenHedgehog signaling drives chondrocyte proliferation and hypertrophy in the growth plate cartilage6 From in a0vitro studies we know that Hhinhibitors decrease the expression of Gli1 and induce cell cycle arrest in prostate cancer cells27 Despite rSWT rescued bones from Hhinhibitor impaired bone growth we did not see significant alterations in the expression of Gli1 and Ihh suggesting a crosstalk between hedgehog signaling and other pathways28 We speculate that the bone rescuing effect of rSWT is more evident if there is any ongoing disturbance within growth plate chondrocytes Indeed it was interesting to note that despite continuous exposure to a Hhinhibitor a single session of rSWT could partially rescue the bone growthOur study has several limitations Firstly the bone growth rescuing effects of rSWT were documented in an exvivo bone culture model and we do not know if this will be applicable under in a0vivo conditions Nevertheless in a0vivo studies in rats or mice are of limited value when it comes to exploring the potential for rSWT to rescue from vismodegib induced bone growth impairment as their growth plates do not normally fuse29 Secondly we only applied a single dose of rSWT while multiple sessions could potentially be even more efficient when it comes to preventing growth impairment caused by Hhinhibitors Thirdly the concentration of vismodegib used in the present study is different from the plasma concentration a0µM achieved in children30 Nevertheless mimicking a gradual decline in bone growth in order to test the rescuing effect of rSWT is more important in our experimental setting We therefore claim a protective effect and not a clinical effect which will require more rigorous testing Consequently our proof of concept finding s up a window of opportunity to explore the potential for locally applied rSWT to prevent bone growth impairment caused by vismodegib as it may not be possible to extrapolate the doses used for preclinical studies to a clinical setting3132In summary we here present a novel treatment strategy based on clinically used rSWT to locally prevent bone growth impairment caused by vismodegib a promising anticancer drug used in children with medulloblastoma Scientific RepoRtS 101038s41598020699040Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A hypothetical schematic diagram showing how extracorporeal radial shockwave treatment rSWT may be administered to lowhigh growth velocity areas a0encircled selectively to restore vismodegibinduced bone growth impairment in children with medulloblastomaFig a0 Before any clinical studies our promising ex a0vivo findings need to be validated in an in a0vivo animal model like the rabbit where growth plate fusion normally occurs just like in humansMethodsThe experiments were approved by the local institutional review board Min No and the institutional animal ethics committee Min No at Christian Medical College Vellore Animal care compiled with the Guide for the Care and Use of Laboratory Animals A a0radial shock wave machine from Radialspec Medispec Gaithersburg MD USA was used for the studyBone an culture system Metatarsal bones were microdissected from the hind limbs of Sprague Dawley rat fetuses sacrificed on day of gestation Ex a0vivo cultures were performed as previously reported33 Briefly each bone was transferred to a 24well plate and cultured in medium containing DMEMF12 a0mM betaglycerophosphate ascorbic acid a0µgml and gentamycin The medium was replenished every two days Figure a0 shows the experimental overviewExposure to Vismodegib Vismodegib was purchased from Selleck Chemicals Houston Texas USA The experimental setup consisted of four groups Metatarsal bones were cultured for a0days and were either a treated with vismodegib a0nM n from day to b a single exposure to rSWT impulses a0Hz a0mJ n on day c vismodegib rSWT n or d were left untreated control n The bone length was measured on days and Exposure to GANT61 GANT61 was purchased from Sigma Aldrich St Louis Missouri USA Metatarsal bones were treated with the a Hhinhibitor GANT61 a0µM from day to n b a single exposure Scientific RepoRtS 101038s41598020699040Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Experimental overview Middlethree metatarsals from the hind limb of embryonic rat at a 1920th day of gestation were dissected and b cultured exvivo c Bones were either exposed to vismodegibGANT61 a single session of highenergy radial shock wave treatment rSWT vismodegibGANT61 with rSWT or left untreated During this time d total bone length measurements at different intervals are performedto rSWT on day impulses a0Hz a0mJ n c GANT61 rSWT or d were left untreated control n The bone length was measured on days and Bone length measurement Digital images were captured for bone length measurements using an inverted microscope Leica Microsystems All measurements were performed by one of the investigators blinded to the nature of the group using an inbuilt ˜measurement tool™ Bone growth is expressed as percent bone length increase from day Bowed bones were measured in two parts added togetherQuantitative histology and immunostaining After termination of the culture the metatarsal bones were fixed with paraformaldehyde embedded in paraffin and fivemicrometer sections were cut along the longitudinal axis proximal to distal followed by staining with SafraninO and Alcian blue The microscopic description of the growth plate morphology included an assessment of the anization of the chondrocyte column Histomorphometric analysis was performed to measure the height of the restingproliferating zone at five different regions of the growth plate and the size of hypertrophic chondrocytes Hypertrophic cells were defined by a height along the longitudinal axis greater than a0µm Eight hypertrophic chondrocytes from the proximal and distal growth plate were measuredImmunostaining was performed as previously described34 Antigen retrieval was performed in citrate buffer at ° Celsius and endogenous peroxidase activity was quenched with H2O2 in methanol for a0min followed by a wash with PBS For immunostaining sections were blocked with bovine serum albumin for a0h incubated with primary antibodies dilution Gli1 Abcam Cambridge MA USA and Ihh mouse monoclonal antibody Santa Cruz Biotechnology Dallas TX USA overnight at ° Celcius After incubation for a0h with secondary polyclonal antimouse or antirabbit biotinylated antibody DakoCytomation Glostrup Denmark dilution sections were incubated with ABC solution and developed with diaminobenzidine Sections were counterstained with Alcian blue Nonimmune immunoglobin G IgG of the same species as the primary antibodies were used as negative controls Three to five bones per group were analyzed To quantify immunostaining the ImageJ software National Institutes of Mental Health Bethesda MD USA was used and the percentage of DAB positivity was calculated digitally using a plugin IHC profilerStatistical analysis All statistics were carried out using GraphPad Prism GraphPad Software Inc La Jolla CA USA Data were summarized using means ± SD for the bone length measurements and histomorphometric assessments A twoway ANOVA with Dunnett multiple comparisons test35 was performed to examine the change in bone length in terms of treatment and days Pairwise comparisons were done corrected for the alpha levels Margin plots with SD were presented to visualize the change in bone length Kruskal“Wallis and Man“Whitney U test were performed when the data were not normally disturbed A p value of was considered to indicate a significant differenceData availabilityAll data generated or analysed during this study are included in this manuscriptScientific RepoRtS 101038s41598020699040Vol1234567890wwwnaturecomscientificreports 0cReceived March Accepted July References Gerber N et al Recent developments and current concepts in medulloblastoma Cancer Treat Rev “ Gorlin R J Neurocutaneous Disorders Phakomatoses and Hamartoneoplastic Syndromes “ Springer Berlin Gajjar A J Robinson G W Medulloblastoma”translating discoveries from the bench to the bedside Nat Rev Clin Oncol Kool M et al Molecular subgroups of medulloblastoma an international metaanalysis of transcriptome genetic aberrations and clinical data of WNT SHH Group and Group medulloblastomas Acta Neuropathol “ Kieran M W Targeted treatment for sonic hedgehogdependent medulloblastoma Neurooncology “ Ohba S Hedgehog signaling in endochondral ossification J Dev Biol Robinson G W et al Irreversible growth plate fusions in children with medulloblastoma treated with a targeted hedgehog pathway inhibitor Oncotarget Kieran M W et al Phase I study of oral sonidegib LDE225 in pediatric brain and solid tumors and a phase II study in children and adults with relapsed medulloblastoma Neurooncology “ Kimura H Ng J M Curran T Transient inhibition of the Hedgehog pathway in young mice causes permanent defects in bone structure Cancer Cell “ Ramesh S Zaman F Madhuri V Svendahl L Radial extracorporeal shock wave treatment promotes bone growth and chondrogenesis in cultured fetal rat metatarsal bones Clin Orthop RelatRes “ Gollwitzer H et al Radial extracorporeal shock wave therapy rESWT induces new bone formation in a0vivo results of an animal study in rabbits Ultrasound Med Biol “ Kertzman P Cs¡sz¡r N B Furia J P Schmitz C Radial extracorporeal shock wave therapy is efficient and safe in the treatment of fracture nonunions of superficial bones a retrospective case series J Orthop Surg Res Sansone V et al Shockwave Medicine vol “ Karger Publishers Basel Palmero A et al High energy shock waves enhance the cytotoxic effect of doxorubicin and methotrexate to human osteosarcoma cell lines Oncol Rep “ Anticancer Res “ Canaparo R et al High energy shock waves HESW for sonodynamic therapy effects on HT29 human colon cancer cells Gambihler S Delius M In a0vitro interaction of lithotripter shock waves and cytotoxic drugs Br J Cancer “ Pines M Hurwitz S The role of the growth plate in longitudinal bone growth Poult Sci “ Speed C A systematic review of shockwave therapies in soft tissue conditions focusing on the evidence Br J Sports Med FernandezVojvodich P Zaman F Svendahl L Interleukin6 acts locally on the growth plate to impair bone growth Ann Ciampa A R et al Nitric oxide mediates antiinflammatory action of extracorporeal shock waves FEBS Lett “ “ Rheum Dis e24“e24 Chen YL et al Extracorporeal shock wave therapy effectively prevented diabetic neuropathy Am J Transl Res Zaman F et al Humanin is a novel regulator of Hedgehog signaling and prevents glucocorticoidinduced bone growth impairment FASEB J “ Sharpe H J et al Genomic analysis of smoothened inhibitor resistance in basal cell carcinoma Cancer Cell “ Wong H et al Pharmacokinetic“pharmacodynamic analysis of vismodegib in preclinical models of mutational and liganddependent Hedgehog pathway activation Clin Cancer Res “ Eriksson E et al Bortezomib is cytotoxic to the human growth plate and permanently impairs bone growth in young mice PLoS Zaman F Fadeel B Svendahl L Proteasome inhibition therapies in childhood cancer Leukemia Gonnissen A et al The hedgehog inhibitor GANT61 sensitizes prostate cancer cells to ionizing radiation both in a0vitro and in a0vivo ONE e50523 Oncotarget Zhong L Huang X Karperien M Post J N The regulatory role of signaling crosstalk in hypertrophy of MSCs and human articular chondrocytes Int J Mol Sci “ Roach H I Mehta G Oreffo R O Clarke N M Cooper C Temporal analysis of rat growth plates cessation of growth with age despite presence of a physis J Histochem Cytochem “ Gajjar A et al Phase I study of vismodegib in children with recurrent or refractory medulloblastoma a pediatric brain tumor consortium study Clin Cancer Res “ Singer T Appropriate Dose Selection”How to Optimize Clinical Drug Development “ Springer Berlin ReaganShaw S Nihal M Ahmad N Dose translation from animal to human studies revisited FASEB J “ Chagin A S Chrysis D Takigawa M Ritzen E Svendahl L Locally produced estrogen promotes fetal rat metatarsal bone growth an effect mediated through increased chondrocyte proliferation and decreased apoptosis J Endocrinol “ Bov©e J V van den Broek L J CletonJansen AM Hogendoorn P C Upregulation of PTHrP and Bcl2 expression characterizes the progression of osteochondroma towards peripheral chondrosarcoma and is a late event in central chondrosarcoma Lab Investig “ Vincent K et al Aging of mouse intervertebral disc and association with back pain Bone “ AcknowledgementSR would like to personally acknowledge Dr Sumith K Mathew Assistant Professor Clinical Pharmacology Christian Medical College for his input on calculating for clinically relevant dose This study was supported by internal grants received from Christian Medical College Vellore Centre for Stem Cell Research Swedish Research Council Swedish Childhood Cancer Foundation HKH Kronprinsessan Lovisas f¶rening Ake Wibergs Stiftelse and Karolinska Institutet Sweden access funding provided by Karolinska InstituteAuthor contributionsSR LS VM FZ designed the study SR carried out data collection and statistical analysis SR LS VM FZ analysed data SR wrote the initial draft of the manuscript FZ VM and LS contributed to the revision of the manuscript All authors contributed to the interpretation of resultsScientific RepoRtS 101038s41598020699040Vol0123456789wwwnaturecomscientificreports 0ccompeting interests The authors declare no competing interestsAdditional informationCorrespondence and requests for materials should be addressed to SRReprints and permissions information is available at wwwnaturecomreprintsPublisher™s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Access This article is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate if changes were made The images or other third party material in this article are included in the article™s Creative Commons license unless indicated otherwise in a credit line to the material If material is not included in the article™s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this license visit httpcreat iveco mmons licen sesby40 The Authors Scientific RepoRtS 101038s41598020699040Vol1234567890wwwnaturecomscientificreports 0c'

Thyroid_Cancer

pathogenesis of multiple myeloma MM is not completely known Uncovering the potential mechanism of MM initiation and progression is essential for identifying novel diagnostic and therapeutic targets Herein we explored the function and the working mechanism of circular RNA circ_0007841 in MM progressionMethods Quantitative realtime polymerase chain reaction qRTPCR was employed to detect the expression of circ_0007841 microRNA3383p miR3383p and bromodomain containing BRD4 Cell proliferation ability was analyzed through cell counting kit8 CCK8 assay colony formation assay and flow cytometry Transwell assays were conducted to measure the migration and invasion abilities of MM cells Cell apoptosis was also assessed by flow cytometry The interaction between miR3383p and circ_0007841 or BRD4 was confirmed by dualluciferase reporter assay and RNApull down assayResults Circ_0007841 was highly expressed in bone marrow BMderived plasma cells of MM patients and MM cell lines than that in healthy volunteers and normal plasma cell line nPCs Circ_0007841 promoted the proliferation cell cycle and metastasis and impeded the apoptosis of MM cells miR3383p was a direct target of circ_0007841 in MM cells and circ_0007841 accelerated the progression of MM through targeting miR3383p BRD4 could directly bind to miR3383p in MM cells and miR3383p exerted an antitumor role through targeting BRD4 Circ_0007841 promoted the activation of PI3KAKT signaling via miR3383pBRD4 axis Exosomes generated from mesenchymal stromal cells MSCs elevated the malignant behaviors of MM cells via circ_0007841Conclusion Circ_0007841 acted as an oncogene to promote the proliferation cell cycle and motility and restrain the apoptosis of MM cells through sequestering miR3383p to upregulate the expression of BRD4Keywords Multiple myeloma circ_0007841 miR3383p BRD4 ExosomeBackgroundMultiple myeloma MM is a kind of hematologic cancer featured by malignant proliferation of plasma cells [] The therapeutic strategies for MM patients include chemotherapy radiotherapy and targeted therapy [“] However MM is still incurable by current treatment Correspondence wy1782126com Department of Hematology The Fifth Affiliated Hospital of Zhengzhou University No3 Kangfuqian Street Zhengzhou Henan ChinaFull list of author information is available at the end of the methods Uncovering the molecular mechanism behind the progression of MM and intercellular interaction is important to find more effective treatment methods for MM patientsNoncoding RNAs ncRNAs are a class of RNAs that are unable to code proteins generally and they are abundant in human genome to regulate cellular processes including proliferation metastasis and apoptosis [] Circular RNAs circRNAs are a kind of ncRNAs that characterized by covalently closed loop structure [] CircRNAs are more stable than linear RNAs and they The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cWang a0et a0al Cancer Cell Int Page of are resistant to exonuclease due to their loop structure [] CircRNAs engaged in the pathogenesis of cancers through serving as microRNAs miRNAs sponges to modulate the abundance of downstream genes linked to proliferation metastasis and apoptosis [ ] The roles of circRNAs in hematological cancers have been reported before [ ] For instance circCBFB contributed to the proliferation ability while suppressed the apoptosis of chronic lymphocytic leukemia cells through targeting miR607FZD3Wntbetacatenin signaling [] However the functions of circRNAs in MM remain to be uncoveredMiRNAs belong to another class of ncRNAs that involved in the progression of cancers through inducing degradation or translational repression of target messenger RNAs mRNAs [] The dysregulation of miRNAs was involved in the pathogenesis of MM [ ] We concentrated on the role of miR3383p miR3383p suppressed the development of many cancers [“] As for MM Cao et a0al reported that miR3383p suppressed the proliferation and accelerated the apoptosis of MM cells via CDK4 [] Nevertheless the function of miR3383p in MM is largely unexploredBromodomain containing BRD4 is a crucial epigenetic protein and it has been reported to elevate the levels of oncogenic proteins and accelerate the progression of cancers [] Zheng et a0al claimed that H19 accelerated the development of MM through upregulating BRD4 via sponging miR1523p [] Here the direct interaction between miR3383p and BRD4 was first found in MM and the function of BRD4 in MM was investigatedIn this study circ_0007841 was found to be abnormally upregulated in MM Lossoffunction experiments revealed that circ_0007841 silencing blocked the proliferation cell cycle progression migration and invasion while induced the apoptosis of MM cells The underlying mechanisms behind the oncogenic role of circ_0007841 in MM were further exploredMaterials and a0methodsPatientsPlasma cells from MM patients n and healthy volunteers n in The Fifth Affiliated Hospital of Zhengzhou University were collected to detect the expression of circ_0007841 miR3383p and BRD4 via qRTPCR and Western blot assayCell cultureMM cell lines H929 and OPM2 and normal plasma cell line nPCs were purchased from BeNa Culture Collection Beijing China and maintained in Roswell Park Memorial Institute1640 RPMI1640 medium Gibco Carlsbad CA USA added with fetal bovine serum FBS Gibco unitsmL penicillin and a0μgmL streptomycin Cell culture plates were placed in a CO2 incubator at a0°C and cells were collected in the log phase of growthQuantitative real‘time polymerase chain reaction qRT‘PCRAfter measuring the concentration using NanoDrop Invitrogen Carlsbad CA USA RNA sample a0ng was used to synthesize complementary DNA cDNA with ReverTra Ace qPCR RT Kit for circ_0007841 BRD4 and U6 Takara Dalian China and AllinOne„¢ miRNA glyceraldehyde3phosphate dehydrogenase GAPDH First stand cDNA Synthesis Kit for miR3383p GeneCopoeia Rockville MD USA U6 served as the internal control for miR3383p while GAPDH acted as the internal reference for circ_0007841 and BRD4 PCR amplification reaction was conducted with SYBR Green PCR Master Mix Applied Biosystems Foster City CA USA on an ABI thermocycler Applied Biosystems The quantification of circ_0007841 miR3383p and BRD4 was carried out with the ˆ’ΔΔCt method The specific primers in this study were synthesized from Sangon Biotech Shanghai China and listed as below circ_0007841 ²CTA ACA TCT GTG AAA CCA TCGT3² Forward Reverse ²TCA TCA CAT ACA CGA TAG ACTGG3² miR3383p Forward ²UCC AGC AUC AGU GAU UUU GUUG3² Reverse ²CAA CAA AAU CAC UGA UGC UGGA3² BRD4 Forward ²GTG GTG CAC ATC ATC CAG TC3² Reverse ²CCG ACT CTG AGG ACG AGA AG3² U6 Forward ²CTC GCT TCG GCA GCACA² Reverse ²AAC GCT TCA CGA ATT TGC GT3² GAPDH Forward ²GCG ACA CCC ACT CCT CCA C3² Reverse ²TCC ACC ACC CTG TTG CTG TAG3²and interfering RNAs siRNAs including sicirc_00078411 Cell transfectiontargeting Three small ²UGU circ_0007841 sicirc_00078412 UAG UUG CAA UGA AGA GAG3² si²UAA UGA UCA UGC CAA AUA CUC3² circ_00078413 ²UCA CAU ACA CGA UAG ACU GGC3² its negative control siNC circ_0007841 overexpression plasmid circ_0007841 its control Vector BRD4 overexpression plasmid BRD4 its control pcDNA miR3383p mimics miR3383p its control miRNC miR3383p inhibitor inmiR3383p and its control inmiRNC were obtained from Genepharma Shanghai China MM cells were seeded into 24well plates at a density of × cellswell overnight and transfection was conducted with Lipofectamine Invitrogen 0cWang a0et a0al Cancer Cell Int Page of Cell counting kit‘ CCK8 assayMM cells were plated in 96well plates at the density of × cellswell and cultured overnight After transfection for indicated time points a0h a0h a0h or a0h MM cells were incubated with μL CCK8 Sigma St Louis MO USA for a0h The absorbance at a0 nm was detected by a microplate reader BioTek Winooski VT USAColony formation assayA total of cells were seeded onto the 6well plates to settle down The culture medium was replenished every d After 2week incubation the colonies were immobilized using poly methanol Sangon Biotech for a0 min followed by staining using crystal violet Sangon BiotechFlow cytometry for a0cell cycle and a0apoptosis detectionFor cell cycle analysis MM cells were collected using cold phosphate buffer saline PBS and then immobilized using cold ethanol solution overnight Prior to propidium iodide PI Solarbio Beijing China staining RNase was used to remove RNA in the samples The percentage of MM cells in different phases of cell cycle was detected on the FACSCalibur Becton“Dickinson San Jose CA USA and analyzed using Cell Quest software Becton“DickinsonFor apoptosis analysis after transfection for a0 h MM cells were collected with PBS and then these cells were suspended in binding buffer Annexin Vcombined fluorescein isothiocyanate Annexin VFITC Solarbio and PI Solarbio were added to the reaction mixture and MM cells were simultaneously incubated with Annexin VFITC and PI at a0°C for a0min in a dark room The apoptotic MM cells were identified by FACSCalibur Becton“Dickinson and analyzed using Cell Quest software Becton“DickinsonTranswell assaysIn transwell migration assay cell suspension MM cells suspended in μL serumfree medium was added into the upper chambers Costar Corning NY USA A total of μL culture medium with FBS was added into the lower chambers FBS acted as the chemotactic factor in this study After 24h incubation MM cells remained in the upper surface were removed with the cotton swab and the migrated MM cells were fixed with paraformaldehyde Sigma for a0 min and stained with crystal violet Sigma The number of migrated MM cells in five random visual fields was counted by the microscope Olympus Tokyo JapanIn transwell invasion assay the upper chambers were precoated with μL Matrigel Sigma to mimic the extracellular matrix The detection of cell invasion was conducted through using these precoated transwell chambers following the similar procedureBioinformatic prediction and a0dual‘luciferase reporter assayThe targets of circ_0007841 and miR3383p were predicted by circinteractome and targetscan software respectivelyThe wildtype partial sequence in circ_0007841 that predicted to bind to miR3383p along with the mutanttype sequence with miR3383p in circ_0007841 that was synthesized through using Sitedirected gene mutagenesis kit Takara Dalian China was amplified and cloned into pGL3 luciferase reporter vector Promega Madison WI USA termed as circ_0007841 WT or circ_0007841 MUT MM cells were cotransfected with a0 nM miRNC or miR3383p and a0 ng circ_0007841 WT or circ_0007841 MUT After 48h transfection MM cells were harvested and the luciferase activity was detected with the dualluciferase reporter assay system kit Promega using the luminometer Plate Chameleon V Hidex Finland according to the manufacturer™s instructions Firefly luciferase activity in each group was normalized to Renilla fluorescence intensityThe wildtype fragment of BRD4 ² untranslated region ²UTR that predicted to bind to miR3383p and the mutant type fragment of BRD4 ²UTR were also amplified and inserted into pGL3 luciferase reporter vector Promega to generate BRD4 ²UTR WT and BRD4 ²UTR MUT Cotransfection of MM cells with BRD4 ²UTR WT or BRD4 ²UTR MUT and miRNC or miR3383p was conducted following the similar procedureRNA‘pull down a0assayRNApull down assay was conducted to test the interaction between circ_0007841 and miR3383p Biotin RNA Labeling Mix Roche Shanghai China was used in this study The wildtype and mutanttype binding sites in circ_0007841 that were predicted to bind to miR3383p were biotinylated to obtain Biocirc_0007841 WT and Biocirc_0007841 MUT MM cells were disrupted and incubated with BioNC Biocirc_0007841 WT or Biocirc_0007841 MUT The abundance of miR3383p was measured by qRTPCRWestern blot assayProteins were obtained using whole cell lysis buffer Roche Basel Switzerland for a0min on the ice Protein samples were quantified using Pierce BCA Protein Assay kit Thermo Fisher Scientific Rockford IL USA Then a0 µg of proteins were run on sodium dodecyl sulfate 0cWang a0et a0al Cancer Cell Int Page of polyacrylamide gel electrophoresis SDSPAGE gel and transferred to the polyvinylidene fluoride PVDF membrane Millipore Billerica MA USA After blocking with wv nonfat dry milk for a0h primary antibodies were used to probe the indicated proteins followed by incubation with the secondary antibody ab205718 Abcam Cambridge MA USA The protein bands were measured using the enhanced chemiluminescent ECL system Beyotime Shanghai China according to the manufacturer™s instructions Gray analysis was conducted to quantify the expression of proteins using ImageJ software Primary antibodies including antiBRD4 ab128874 antiphosphorylatedphosphatidylinositol 3kinase antipPI3K ab70912 antiPI3K ab32089 antipAKT serinethreonine kinase pAKT ab38449 antiAKT ab64148 antiCD63 ab59479 antiCD81 ab79559 and antiβactin ab8226 were purchased from AbcamExosome isolationExosome isolation kit Qiagen Frankfurt Germany was used to extract exosomes from the culture medium of MM cells according to previous studies [ ]Statistical analysisAll statistical data in three independent experiments were shown as mean ± standard deviation SD Data were analyzed using GraphPad Prism The differences between two groups or among more than two groups were assessed through using Student™s t test or oneway analysis of variance ANOVA followed by Tukey™s test The comparison between groups was considered significant when P value less than Linear correlation was analyzed using Spearman™s correlation coefficientResultsCirc_0007841 elevates the a0malignant behaviors of a0MM cellsCirc_0007841 was abnormally upregulated in bone marrow BMderived plasma cells from MM patients compared with that in healthy individuals Fig a01a Meanwhile the level of circ_0007841 was higher in MM cell lines than that in normal plasma cell line nPCs Fig a01b The dysregulation of circ_0007841 in MM attached our attention Circ_0007841 specific small interfering RNAs were used to knockdown circ_0007841 to uncover its biological functions in MM cells As mentioned in Fig a0 1c and d the level of circ_0007841 was downregulated with the transfection of sicirc_00078411 sicirc_00078412 or sicirc_00078413 Among these three siRNAs sicirc_00078411 was chose for the following assays due to its highest knockdown efficiency Fig a01c d Cell proliferation was assessed through CCK8 assay colony formation assay and flow cytometry According to the results of CCK8 assay sicirc_00078411 transfection significantly inhibited the proliferation of MM cells Fig a0 1e f The number of colonies was markedly reduced with the knockdown of circ_0007841 compared with siNC group Fig a0 1g The cell cycle of MM cells was arrested in G1S transition in sicirc_00078411 group than that in siNC group Fig a01h These findings together demonstrated that circ_0007841 silencing hampered the proliferation ability in MM cells What™s more circ_0007841 interference notably suppressed the migration and invasion of MM cells via transwell migration and invasion assays Fig a01i j The apoptosis rate of MM cells was increased in sicirc_00078411 group compared with that in siNC group Fig a01k Overall circ_0007841 accelerated the proliferation cell cycle progression and metastasis and inhibited the apoptosis of MM cellsmiR‘‘3p could directly interact with a0circ_0007841 in a0MM cellsTo address the mechanism by which circ_0007841 functioned in MM cells circinteractome website was used to seek the targets of circ_0007841 As shown in Fig a0 2a miR3383p possessed the complementary sites with circ_0007841 The luciferase activity was dramatically reduced in circ_0007841 WT group when cotransfected with miR3383p suggesting the target relationship between circ_0007841 and miR3383p in MM cells Fig a02b c We also constructed mutant type luciferase plasmid circ_0007841 MUT to investigate if œUGC UGG  in circ_0007841 was the binding sequence with miR3383p The luciferase intensity remained unaffected in circ_0007841 MUT group with the cotransfection of miRNC or miR3383p Fig a02b c suggested that circ_0007841 bound to miR3383p via its œUGC UGG  sequence RNApull down assay revealed that miR3383p could be pulleddown when using Biocirc_0007841 WT proving the target relationship between miR3383p and circ_0007841 Fig a0 2d e An obvious decrease in the level of miR3383p was observed in BMderived plasma cells from MM patients in contrast to that in normal volunteers Fig a02f Additionally there was a prominent reduction in the expression of miR3383p in MM cell lines than that in nPCs cell line Fig a0 2g The expression of miR3383p was negatively correlated with the level of circ_0007841 in BMderived plasma cells from MM patients Fig a02h The overexpression efficiency of circ_0007841 was high in MM cells and circ_0007841 accumulation caused a notable decrease in the level of miR3383p in MM cells Fig a02i j In summary circ_0007841 could inversely regulate the expression of miR3383p through direct interaction 0cWang a0et a0al Cancer Cell Int Page of Fig Circ_0007841 elevates the malignant behaviors of MM cells a The enrichment of circ_0007841 was examined in BMderived plasma cells of MM patients and normal volunteers by qRTPCR b The expression of circ_0007841 was measured in MM cell lines and normal plasma cell line nPCs by qRTPCR c d The level of circ_0007841 was detected in H929 and OPM2 cells transfected with siNC sicirc_00078411 sicirc_00078412 or sicirc_00078413 by qRTPCR e“k MM cells were transfected with siNC or sicirc_00078411 e f CCK8 assay was employed to assess the proliferation ability of MM cells g Colony formation assay was performed for the determination of cell proliferation ability in transfected MM cells h Flow cytometry was carried out to detect the influence of circ_0007841 silencing on the cycle of MM cells i j The metastasis ability of MM cells was evaluated by transwell assays k The apoptosis of MM cells was analyzed by flow cytometry P P P P if circ_0007841 exerted Circ_0007841 plays an a0oncogenic role through a0targeting miR‘‘3p in a0MM cellsTo disclose its oncogenic role through targeting miR3383p we conducted rescue experiments through cotransfecting H929 and OPM2 cells with siNC sicirc_00078411 sicirc_00078411 inmiRNC or sicirc_00078411 inmiR3383p As mentioned in Fig a03a sicirc_00078411 transfection increased the level of miR3383p and the introduction of inmiR3383p reversed the influence of circ_0007841 silencing in the expression of miR3383p Sicirc_00078411mediated inhibitory effect on the proliferation of MM cells was counteracted by the interference of miR3383p via CCK8 assay Fig a03b c Circ_0007841 silencing restrained the colony formation ability while the addition of miR3383p inhibitor partly recovered the colony formation ability in MM cells Fig a0 3d Additionally cell cycle of MM cells was arrested at G1S transition in sicirc_00078411 group and this suppressive impact in the cell cycle of MM cells was attenuated by the addition of inmiR3383p Fig a03e f The migration and invasion of MM cells were suppressed by the knockdown of circ_0007841 and the metastasis ability was recovered in sicirc_00078411 and inmiR3383p cotransfected group Fig a0 3g h Sicirc_00078411induced apoptosis of MM cells was 0cWang a0et a0al Cancer Cell Int Page of Fig miR3383p could directly interact with circ_0007841 in MM cells a miR3383p was predicted as a candidate target of circ_0007841 by circinteractome software b c Dualluciferase reporter assay was conducted to verify whether miR3383p could bind to circ_0007841 in MM cells d e RNApull down assay was performed to confirm the target relationship between miR3383p and circ_0007841 in MM cells f g The expression of miR3383p was detected in BMderived plasma cells of MM patients and healthy volunteers MM cells and nPCs cells by qRTPCR h The correlation between the expression of miR3383p and circ_0007841 was analyzed using Spearman™s coefficient i j The abundance of circ_0007841 and miR3383p was examined in H929 and OPM2 cells transfected with Vector or circ_0007841 by qRTPCR P P P P attenuated by the addition of inmiR3383p Fig a0 3i Overall circ_0007841 could promote the malignant potential of MM cells through sponging miR3383pBRD4 is a0validated as a0a a0target of a0miR‘‘3p in a0MM cellsBRD4 was predicted as a direct target of miR3383p by targetscan database and the wild type or the mutant type binding sequence between miR3383p and BRD4 was shown in Fig a04a As exhibited in Fig a04b c the luciferase activity was markedly decreased in miR3383p and BRD4 ²untranslated region ²UTR WT cotransfected group while miR3383p transfection had no effect on the luciferase activity in BRD4 ²UTR MUT group compared with that in miRNC and BRD4 ²UTR MUT cotransfected group suggesting the interaction between BRD4 and miR3383p BRD4 was conspicuously upregulated in BMderived plasma cells of MM patients compared with that in healthy individuals Fig a0 4d Meanwhile BRD4 was also found to be upregulated in MM cell lines than that in nPCs cells Fig a0 4e The expression correlation between BRD4 and circ_0007841 or miR3383p was analyzed using Spearman™s correlation coefficient As shown in Fig a0 4f g there was an inverse correlation between the levels of BRD4 and miR3383p while the expression of BRD4 was positively correlated with the level of circ_0007841 miR3383p overexpression significantly downregulated the expression of BRD4 in MM cells suggesting the negative regulatory relationship between BRD4 and miR3383p in MM cells Fig a04h Circ_0007841 and miR3383p were cotransfected into MM cells to uncover the relationship among circ_0007841 miR3383p and BRD4 As presented in Fig a0 4i circ_0007841 overexpression upregulated the level of BRD4 and the expression of BRD4 was decreased in circ_0007841 and miR3383p cotransfected group Collectively BRD4 was a target of miR3383p and circ_0007841 could elevate the expression of BRD4 through sponging miR3383pBRD4 overexpression attenuates the a0effects of a0miR‘‘3p accumulation on a0MM cellsmiR3383p and BRD4 were cotransfected into MM cells to explore whether miR3383p exerted an antitumor role in MM cells through targeting BRD4 As shown in Fig a0 5a the addition of BRD4 overexpression plasmid recovered the expression of BRD4 in MM cells that was downregulated by the accumulation of miR3383p miR3383p overexpression inhibited the proliferation cell cycle and metastasis of MM cells and these inhibitory effects were attenuated by the 0cWang a0et a0al Cancer Cell Int Page of Fig Circ_0007841 plays an oncogenic role through targeting miR3383p in MM cells a“i MM cells were transfected with siNC sicirc_00078411 sicirc_00078411 inmiRNC or sicirc_00078411 inmiR3383p a The level of miR3383p was examined in MM cells by qRTPCR assay b c The proliferation of MM cells was measured through conducting CCK8 assay d The proliferation capacity in transfected MM cells was assessed by colony formation assay e f The percentage of MM cells in G0G1 S or G2M phase was analyzed using flow cytometry g h The migration and invasion abilities of MM cells were evaluated by transwell assays i The apoptosis rate of MM cells in different groups was analyzed by flow cytometry P P P addition of BRD4 overexpression plasmid Fig a0 5b“h The apoptosis of MM cells was induced by the transfection of miR3383p and the introduction of BRD4 overexpression plasmid recovered the viability of MM cells Fig a0 5i In conclusion miR3383p accumulation restrained the malignant behaviors of MM cells through targeting BRD4Circ_0007841 activates PI3KAKT signal pathway through a0targeting miR‘‘3pBRD4 axisThe activation of PI3KAKT signal pathway is linked to the promotion of cell proliferation and metastasis and the inhibition of cell apoptosis Herein we examined the phosphorylation levels of PI3K and AKT to illustrate the influence of circ_0007841miR3383pBRD4 axis in the See figure on next pageFig BRD4 is validated as a target of miR3383p in MM cells a The complementary sites between miR3383p and the ²UTR of BRD4 were predicted by targetscan software b c The luciferase activity was measured in H929 and OPM2 cells transfected with miRNC or miR3383p and BRD4 ²UTR WT or BRD4 ²UTR MUT d The protein level of BRD4 in BMderived plasma cells of MM patients and healthy volunteers was detected by Western blot assay e The level of BRD4 in H929 OPM2 and nPCs cells was evaluated by Western blot assay f g The linear relationship between BRD4 and miR3383p or circ_0007841 was analyzed using Spearman™s coefficient h The expression of BRD4 was detected in MM cells transfected with miRNC or miR3383p by Western blot assay i The protein level of BRD4 was detected in MM cells transfected with Vector circ_0007841 circ_0007841 miRNC or circ_0007841 miR3383p by Western blot assay P P P P 0cWang a0et a0al Cancer Cell Int Page of 0cWang a0et a0al Cancer Cell Int Page of Fig BRD4 overexpression attenuates the effects of miR3383p accumulation on MM cells a“i MM cells were transfected with miRNC miR3383p miR3383p pcDNA or miR3383p BRD4 a qRTPCR was employed to measure the expression of BRD4 in MM cells b c CCK8 assay was applied to assess the proliferation ability of MM cells d Colony formation assay was performed to analyze the influences of miR3383p and BRD4 on the proliferation of MM cells e f Flow cytometry was conducted to detect the cell cycle of MM cells g h Transwell assays were performed to detect the metastasis of MM cells i The apoptosis rate of MM cells was examined by flow cytometry P P P activation of PI3KAKT signaling Circ_0007841 silencing downregulated the level of BRD4 and the level of BRD4 was recovered in sicirc_00078411 and inmiR3383p cotransfected group Fig a06a b The activation of PI3KAKT signaling was suppressed with the silencing of circ_0007841 and the addition of inmiR3383p recovered the phosphorylation levels of PI3K and AKT Fig a06a c Meanwhile H929 and OPM2 cells were transfected with miRNC miR3383p miR3383p pcDNA or miR3383p BRD4 As mentioned in Fig a06d e miR3383p overexpression downregulated the level of BRD4 and the introduction of BRD4 overexpression plasmid regained the level of BRD4 in MM cells The addition of BRD4 alleviated the inhibitory influence of miR3383p overexpression on the activation of PI3KAKT signaling in MM cells Fig a0 6d f Taken together circ_0007841 accelerated the progression of MM through miR3383pBRD4PI3KAKT axisMesenchymal stromal cells MSCs‘generated exosomes accelerate the a0malignant potential of a0MM cells via a0circ_0007841MSCs exert crucial roles in the progression of MM Herein we explored whether exosomes derived from MSCs could regulate the proliferation cell cycle metastasis and apoptosis of MM cells via circ_0007841 MSCs were isolated from the adjacent tissues of MM and normal tissues The expression of circ_0007841 was higher in MSCs and MSCsderived exosomes from adjacent tissues than that in normal tissues Fig a07a b The markers of exosomes CD63 and CD81 were notably upregulated in exosomes of MSCs instead of cell lysate Fig a07c As mentioned in Fig a07d we established a working model as previously described to explore if MSCsderived exosomes could regulate the proliferation cell cycle motility and apoptosis of MM cells [] In this model only exosomes could be transmitted through the filter to the upper chambers As presented in Fig a07e“k sicirc_00078411 0cWang a0et a0al Cancer Cell Int Page of Fig Circ_0007841 activates PI3KAKT signal pathway through targeting miR3383pBRD4 axis a“c Western blot assay was performed to detect the levels of BRD4 and PI3KAKT signalingrelated proteins in MM cells transfected with siNC sicirc_00078411 sicirc_00078411 inmiRNC or sicirc_00078411 inmiR3383p and gray analysis was used to assess the abundance of these proteins d“f The expression of BRD4 and PI3KAKT signalingassociated proteins in MM cells transfected with miRNC miR3383p miR3383p pcDNA or miR3383p BRD4 was examined by Western blot assay P P P transfection inhibited the malignant behaviors of MM cells in Mock sicirc_00078411 group compared with that in Mock siNC group Besides MSCsderived exosomes MSCs siNC group promoted the proliferation cell cycle metastasis and inhibited the apoptosis of MM cells than that in Mock siNC group and these effects were attenuated by the silencing the circ_0007841 suggested that MSCsderived exosomes could promote the progression of MM via circ_0007841 What™s more the exosomes generated from MSCs accelerated the activation of PI3KAKT signaling while this effect was counteracted with the transfection of sicirc_00078411 Fig a0 7l Collectively MSCsderived exosomes could facilitate the progression of MM via circ_0007841DiscussionMM is an incurable cancer currently Because many MM patients were diagnosed at late stage the treatment outcomes of MM patients were unsatisfactory [] Therefore finding crucial markers in MM is urgent to improve the prognosis of MM patientsCircRNAs are featured by closely loop structure and they are widely distributed in human tissues Due to the stability and the universality of the distribution circRNAs are identified as ideal biomarkers for human cancers and other diseases [] For example the high expression of circ_0004277 was associated with the better prognosis of AML patients [] Xia et a0al claimed that circCBFB was highly expressed in chronic lymphocytic leukemia and circCBFB accelerated the proliferation and suppressed the apoptosis of chronic lymphocytic leukemia cells [] Circ_0007841 was found to be overexpressed in BMderived plasma cells of MM patients and MM cells Further studies suggested that circ_0007841 promoted the proliferation cell cycle metastasis and inhibited the apoptosis of MM cells These findings

Thyroid_Cancer

Health related quality of life functional impairment and comorbidity in people with mild to moderate chronic kidney disease a cross sectional studySimon DS Fraser Jenny Barker1 Paul J Roderick1 Ho Ming Yuen1 Adam Shardlow2 James E Morris1 Natasha J McIntyre2 Richard J Fluck2 Chris W McIntyre3 Maarten W Taal To cite Fraser a0SDS Barker a0J Roderick a0PJ et a0al Health related quality of life functional impairment and comorbidity in people with mild to moderate chronic kidney disease a cross sectional study BMJ 202010e040286 101136bmj 2020040286 –º Prepublication history and additional material for this paper are available online To view these files please visit the journal online http dx bmj Received May Revised June Accepted July Authors or their employers Re use permitted under CC BY Published by BMJ1School of Primary Care Population Sciences and Medical Education Faculty of Medicine University of Southampton Southampton UK2The Department of Renal Medicine Royal Derby Hospital NHS Foundation Trust Derby UK3Department of Medical Biophysics University of Western Ontario London Ontario Canada4Division of Medical Sciences and Graduate Entry Medicine University of Nottingham Derby UKCorrespondence toDr Simon DS Fraser S Fraser soton ac ukObjectives To determine the associations between comorbidities health related quality of life HRQoL and functional impairment in people with mild to moderate chronic kidney disease CKD in primary careDesign Cross sectional analysis at year follow up in a prospective cohort studySetting Thirty two general practitioner surgeries in EnglandParticipants participants with CKD stage of people recruited at baseline in the Renal Risk in Derby study who survived to years and had complete follow up data for HRQoL and functional status FSPrimary and secondary outcome measures HRQoL assessed using the level EQ 5D version EQ 5D 5L with domains of mobility self care usual activities paindiscomfort and anxietydepression and index value using utility scores calculated from the English general population and FS using the Karnofsky Performance Status scale functional impairment defined as Karnofksy score ‰ Comorbidity was defined by self reported or doctor diagnosed condition disease specific medication or blood resultResults Mean age was years The numbers reporting some problems in EQ 5D 5L domains were for mobility for self care for usual activities for paindiscomfort and for anxietydepression Only reported no problems in any domain HRQoL index values showed greater variation among those with lower FS eg for those with Karnofsky score of the median IQR EQ 5D index value was to compared with to for those with Karnofsky score of Overall had functional impairmentIn multivariable logistic regression models functional impairment was independently associated with experiencing problems for all EQ 5D 5L domains mobility OR CI to p0001 self care OR CI to p0001 usual activities OR CI to p0001 paindiscomfort OR CI to p0001 anxietydepression CI to p0001 Higher comorbidity count and obesity were independently associated with problems in mobility self care usual activities and paindiscomfort for three or Strengths and limitations of this study –º This study involved a large cohort of people with chronic kidney disease CKD recruited from primary care a setting in which patients with mild to moderate CKD are typically managed in the UK –º A broad range of comorbidities were included but they were identified at baseline only not at follow up by which time the number of comorbidities may have changed –º Health related quality of life and functional status were measured in the same patient group and the use of the EQ 5D 5L index measure and data from the Health Survey for England enabled comparison with a general population –º Health related quality of life and functional status measures were taken at year follow up but not at baseline and we were therefore unable to identify change over time –º This was a cross sectional study of survivors and we are therefore not able to draw causative linksmore comorbidities versus none mobility OR CI to p for trend self care OR CI to p for trend usual activities OR CI to p for trend paindiscomfort OR CI to p for trend and for obese body mass index BMI ‰¥ kgm2 versus BMI kgm2 mobility OR CI to p for trend self care OR CI to p for trend usual activities OR CI to p for trend paindiscomfort OR CI to p for trend Female sex lower FS and lower educational attainment were independently associated with anxietydepression ORs CI to p CI to p0001 and CI to p respectively Older age higher comorbidity count albuminuria ‰¥ mgmmol vs mgmmol lower educational attainment no formal qualifications vs degree level and obesity were independently associated with functional impairment ORs CI to p0001 CI to p for trend Fraser a0SDS et a0al BMJ 202010e040286 101136bmj 2020040286 0c access CI to p for trend CI to p for trend and CI to respectivelyConclusions The majority of persons with mild to moderate CKD reported reductions in at least one HRQoL domain which were independently associated with comorbidities obesity and functional impairmentTrial registration number National Institute for Health Research Clinical Research Portfolio Study Number INTRODUCTIONChronic kidney disease CKD is common globally affecting about of the general adult population with CKD stage the most prevalent category1 Current treatment guidelines for CKD are disease specific and focus on reducing progression and preventing complications such as cardiovascular disease3 However in the UK most people with CKD stage are managed in primary care and in this context only a minority evidence progression over years4 The risk of end stage kidney disease ESKD is extremely low Conversely comorbidities additional chronic diseases are common in individuals with CKD and can worsen clinical outcomes and health related quality of life HRQoL5 Ninety six per cent of people with stage disease have at least one comorbidity around have a comorbidity count of two or more6A significant body of research has explored HRQoL and the functional status FS of people with ESKD or following kidney transplant but these factors are not well explored in those with less severe CKD Among people with high risk CKD in the Renal Impairment In Secondary Care study reported problems in one or more of the EQ5D domains7 This is a clinically important knowledge gap because mild to moderate reductions in glomerular filtration rate GFR are usually asymptomatic so improved understanding of the comorbidities and symptoms that affect HRQoL and FS in this group of people is important to facilitate a holistic approach to management The objective of this study was therefore to evaluate HRQoL and determine the associations between comorbidities HRQoL and functional impairment in people with mild to moderate CKD in primary careMATERIALS AND METHODSA detailed description of the Renal Risk in Derby RRID study methodology has been published elsewhere9 In summary approximately people with CKD stage were identified from renal registers at primary care clinics in Derbyshire UK between and and invited to participate in the study Of these people attended initial baseline visits and met eligibility criteria age ‰¥ years two estimated GFR eGFR results derived from the Modification of Diet in Renal Disease study MDRD equation of “ mLmin173 m2 at least days apart9 People with a life expectancy of less than year who were unable to attend study visits or who had a solid an transplant were excludedHealthrelated quality of lifeHRQoL was assessed at year follow up using the EQ 5D 5L a widely used validated measure of health status that can be standardised to different populations EQ 5D 5L consists of two aspects a descriptive system in which participants are asked to rate their health state from to against five domains mobility self care usual activities paindiscomfort anxietydepression and the EQ visual analogue scale EQ VAS in which participants rate their health on a scale ranging from ˜the best health you can imagine™ to ˜the worst health you can imagine™ An EQ 5D 5L value set has previously been published for England11 However concerns have been raised about the quality and reliability of the data collected in the valuation study such that the English National Institute for Health and Care Excellence NICE recommend ˜If data were gathered using the EQ 5D 5L descriptive system utility values in reference case analyses should be calculated by mapping the L descriptive system data onto the L value set™ For these analyses individual health states were therefore converted using the EuroQol EQ 5D 5L Crosswalk Index Value Calculator into a single 3L index value a preference based score that typically ranges from states worse than dead to full health with dead at using utility scores calculated from the English general population13 The index value and the EQ VAS score were used to graphically display the relationship between HRQoL and FSFunctional statusFS defined in this paper as the physical ability to perform normal activities and independently self care was assessed at year follow up using the Karnofsky Performance Status KPS scale The KPS is a clinician assessed score originally developed in oncology and was used for assessing prognosis and management in patients with cancer15 The scale ranges from ˜normalno complaints™ to ˜dead™ Theoretically the scale can take any whole number value within the range but in practice results are commonly recorded as multiples of therefore KPS was treated as an ordinal variable in this study The original continuous KPS score is defined as being ˜able to carry on normal activity and to work with no special care needed™ a score of between and inclusive is defined as ˜unable to work able to live at home and care for most personal needs varying amount of assistance needed™ and a score of less than or equal to is defined as ˜unable to care for self requiring the equivalent of institutional or hospital care™ Functional impairment was analysed as a binary outcome due to the small number of patients with low KPS score A KPS score of ‰ versus was chosen to compare those able to carry on normal life with those experiencing some functional impairment as has been used in evaluation of FS in patients with lung cancer16Fraser a0SDS et a0al BMJ 202010e040286 101136bmj 2020040286 0cComorbidities identified at baselineThe methods for defining comorbidities in participants have been described in detail elsewhere6 In brief eleven comorbidities were pragmatically identified at baseline using information from a combination of sources and agreed by consensus between three clinicians SF MWT and PJR patient questionnaires in which patients were asked to list chronic medications followed by verbal confirmation with verification of repeat prescriptions where possible blood pressure measurement at the time of baseline study visit and self reported clinical diagnoses Self reported comorbidities included heart failure ischaemic heart disease peripheral vascular disease defined as peripheral arterial revascularisation or amputation and cerebrovascular disease stroke or transient ischaemic attack Diagnoses of chronic respiratory disorder depression painful condition hypertension diabetes and thyroid disorders were made according to medication history or patient report Anaemia was defined according to Kidney Disease Improving Global Outcomes KDIGO guidelines as haemoglobin gdL gL in men and gdL gL in women at baseline17 Hypertension was defined either by medication history or by a systolic blood pressure mm Hg or diastolic mm Hg at baselineKidney functionKidney function was assessed at year follow up eGFR was calculated using the Chronic Kidney Disease Epidemiology Collaboration equation and was treated as a continuous variable The urine albumin to creatinine ratio uACR from three consecutive early morning specimens was used for analysis uACR was categorised into three levels according to KDIGO guidelines and fitted as a discrete variable in regression analysesMethods for defining CKD progression have also been detailed elsewhere4 In summary progression of CKD was defined as a decline in GFR coupled with a worsening of GFR category or an increase in albuminuria category CKD remission was defined as the presence of both eGFR mLmin173 m2 and uACR mgmmol at any study visit in an individual who had previously met KDIGO diagnostic criteria for CKDOther baseline measuresBody mass index BMI was calculated from weight in kilograms divided by square of height in metres and was treated as a categorical variable18 Smoking status was categorised as never smoked ex smoker and current smoker Socioeconomic status was assessed using self reported educational attainment categorised into no formal qualifications school or equivalent qualifications and degree or equivalent qualifications as well as the Index of Multiple Deprivation IMD score categorised in quintiles19 The IMD is a measure of relative deprivation for small areas of residence in England and combines information from seven domains income employment education skills and training health and disability crime accessbarriers to housing and services and living environment Self reported ethnicity status was also collectedStatistical analysesDescriptive statistics were used to show the characteristics of the study participants at year follow up Descriptive statistics were also used to show the distribution of functional impairment KPS ‰ among those reporting problems in the five EQ 5D 5L domains Associations between the patient reported EQ 5D 5L domains and FS was assessed using the χ2 test Ratings from the five participant reported EQ 5D 5L domains were also compared between the RRID cohort and those reported by people aged years and over in the Health Survey for England HSE”which is representative of the England population20 A comparison of basic characteristics was also made between those with and without complete year follow up dataUnivariable logistic regression models were used to assess the relationships between having ˜some problems™ in each EQ 5D 5L domain and each predictor variable including comorbidity count and year five eGFR Variables considered to be clinically relevant and where p01 on univariable analysis were subsequently included in multivariable logistic regression models This process was then repeated for the relationship with the outcome variable functional impairment Due to the small number of non white participants ethnicity was not included in the modelsIn the regression models interactions between the individual and area measures of socioeconomic status were also tested because of the potential for the relationship between individual socioeconomic status indicated by educational attainment and HRQoL to vary by area deprivation particularly for older people21 The level of significance was set at All analyses were performed using StataIC V150Patient and public involvementThe RRID study design was discussed with a patient and two feedback meetings for participants and their families were anised after the year visits which were well attended In addition a web page provides updates and information for participants httpswww uhdb nhs uk renal risk in derby rrid studyRESULTSOf participants recruited survived to years and of these participants of survivors had complete year follow up data for HRQoL and FS figure The mean age of the cohort was years SD and the majority n621 were female table Approximately half n506 reported having had no formal education just under half n497 lived in areas of lower deprivation IMD quintiles four or five and the majority n994 were white The Fraser a0SDS et a0al BMJ 202010e040286 101136bmj 2020040286 0c access Figure Flow chart of study participants CKD chronic kidney disease KDIGO Kidney Disease Improving Global Outcomes GP general practitioner QoL quality of lifemean eGFR at follow up was mLmin173 m2 SD and almost half n460 had had stable CKD over the preceding year period Only had no comorbidities and about a third n344 had three or more comorbidities For comparison of basic characteristics of those with and without complete year follow up data see online supplementary table S1 A slightly higher proportion of those with incomplete follow up data had three or more comorbidities and only a very small proportion had functional impairment online supplementary table S1The majority reported some impairment in HRQoL overall with a median score of out of IQR “ on the EQ VAS A minority n378 had an EQ 5D 3L index score higher than the agesex matched median and only of people n191 reported no problems across any of the individual HRQoL domains Furthermore a majority of participants reported some problems with mobility n582 and paindiscomfort n712 table When comparing the self reported HRQoL domains with HSE data the proportion of people in the RRID population reporting problems with mobility or paindiscomfort was higher vs and vs respectively than in the HSE population table For clinician assessed FS only two participants had performance status assessed as KPS ‰ ˜unable to care for self requiring the equivalent of institutional or hospice care™ and were assessed as KPS “ ˜unable to work able to live at home and care for most personal needs varying amount of assistance needed™The association between clinician assessed FS and patient reported HRQoL was complex either when based on the index score figure 2A or the VAS scale figure 2B HRQoL was generally higher among those with better FS However the spread of HRQoL scores using either of the HRQoL metrics was broader among those with lower FS suggesting a greater degree of variation in HRQoL among those with lower FS than among those with higher FS figure A higher proportion of people with clinician assessed functional impairment KPS ‰ reported having some degree of problems in each of the five EQ 5D 5L domains than people without functional impairment online supplementary table S2Using the mobility domain as an example table on univariable analysis older age greater area deprivation level higher number of comorbidities poorer FS lower eGFR higher level of albuminuria lower educational attainment and higher BMI were associated with having some problemsIn the fully adjusted multivariable model these associations remained for older age higher number of comorbidities poorer FS and higher BMI table A summary of the main independent associations identified in the multivariable logistic regression models for usual activities self care paindiscomfort and anxietydepression is shown in table and the full analyses in online supplementary tables S3 to S6Fraser a0SDS et a0al BMJ 202010e040286 101136bmj 2020040286 0cTable Characteristics of patients at year follow up in the Renal Risk in Derby study n1008 unless otherwise statedVariableCategoryAge in years mean SDAge group n years Sex  n “ years yearsMaleFemaleEducational attainment  n n1007Index of Multiple Deprivation IMD quintile relative to England  n n1006Ethnicity  n WhiteOther¡No formal qualificationsGCSE A level NVQ “First or higher degree NVQ “Quintile most deprivedQuintile Quintile Quintile Quintile least deprivedNormal or underweight kgm2Overweight “ kgm2Obese ‰¥ kgm2Never smokedEx smokerCurrent smokerBody mass index  n Smoking status  n eGFR in mLmin173 m2 mean SD n1007uACR in mgmmol median IQR n1007KDIGO uACR categories n KDIGO eGFR categories eGFR in mLmin173 m2Progression of kidney disease n Number of comorbidities  n A1A2A3G1 eGFR ‰¥G2 eGFR “G3a eGFR “G3b eGFR “G4 eGFR “G5 eGFR StableProgressionRemissionNone CKD onlyOneTwoThree or moreDescriptive statistics “ ContinuedTable ContinuedVariableCategory accessDescriptive statisticsIndividual comorbidities  n Quality of life domains any problems reported in each EQ 5D 5L domain n Functional status KPS score n HypertensionPainful conditionAnaemiaIschaemic heart diseaseDiabetesThyroid disorderCerebrovascular diseaseChronic respiratory disorderDepressionPeripheral vascular diseaseHeart failureMobility problemsSelf care problemsUsual activity problemsPaindiscomfortAnxietydepressionNo problems in any domainFunctional impairment KPS ‰KPS able to carry on normal activity and to work no special care needed Where variable category percentages sum to less than or more that this is due to roundingVariables assessed at year follow up Variables assessed at baseline¡Includes mixed Asian Cypriot and otherCKD chronic kidney disease eGFR estimated glomerular filtration rate GCSE General Certificate of Secondary Education KDIGO Kidney Disease Improving Global Outcomes KPS Karnofsky Performance Status A level advanced level NVQ National Vocational Qualifications uACR urine albumin to creatinine ratioFactors associated with a lower FS on univariable analysis included older age lower socioeconomic status assessed by both IMD score and educational attainment higher number of comorbidities obesity reduced eGFR and greater degree of albuminuria Other than reduced eGFR all of these factors remained significant after adjustment table No interactions were identified in any analysesDISCUSSIONIn this cross sectional study of people with mild to moderate CKD who survived to year in a UK primary care cohort overall patient reported HRQoL was relatively high Fraser a0SDS et a0al BMJ 202010e040286 101136bmj 2020040286 0c access Table Comparison of the EQ 5D 5L quality of life domains between the Health Survey for England HSE and the Renal Risk in Derby RRID cohortHSE cohort n258nRRID CKD cohort n1008nMobility no problems in walking about “ some problems Self care no problems washing or dressing “ some problemsUsual activities no problems ng usual activities “ some problems Paindiscomfort no pain or discomfort “ some pain or discomfort Anxietydepression not anxious or depressed “ some anxiety or depression All participants were aged years or aboveCKD chronic kidney diseasethough a substantial proportion of participants reported problems in each HRQoL domain A majority reported problems with mobility and paindiscomfort Although most people had a clinician assessed FS suggesting that they were able to carry on normal activity and to work with no special care needed about a quarter were assessed as having functional impairment being unable to work but able to live at home and care for most personal needs with a varying amount of assistance needed HRQoL was generally higher among those with better FS but there was more variation in HRQoL among those with lower FS and low FS was independently strongly associated with low HRQoL in regression analyses Higher number of comorbidities and obesity were independently associated with problems in most EQ 5D 5L domains and with functional impairment Functional impairment was independently associated with experiencing some problems across all EQ 5D 5L domainsFigure Relationship between quality of life and functional status A Functional status by Karnofsky score and quality of life by EQ 5D 3L Index score B Functional status by Karnofsky score and quality of life by EQ 5D self reported visual analogue scale VASThis study had several strengths including the large size of the cohort and recruitment from primary care a setting in which patients with mild to moderate CKD are typically managed The RRID cohort is pragmatic and likely to represent a population of typical patients with mild to moderate CKD in the UK22 We were able to identify a broad range of comorbidities but they were identified at baseline only The number of comorbidities may therefore have changed by the time of follow up assessment meaning that our comorbidity prevalence data were likely underestimates of the true prevalence in some patients Similarly certain other exposures were assessed at baseline and could potentially have changed by the time of follow up We recognise these as important limitations but consider that they are unlikely to significantly alter the main findings of our study with regard to HRQoL and FS A further strength is that we were able to measure HRQoL and FS in the same patient group and the HRQoL and FS data were relatively complete The use of the EQ 5D 5L index measure and data from the HSE enabled comparison with a general population However the index values for HRQoL required conversion to 3L values as reliable 5L index values are not yet available for all standard populations Evidence from a previous RRID analysis on prior renal function change provided depth to our analyses for this cross sectional study However there were also several important limitations”this was a Fraser a0SDS et a0al BMJ 202010e040286 101136bmj 2020040286 0cTable Logistic regression models examining associations between lower quality of life EQ 5D 5L mobility domain categorised as ˜no problems™ vs ˜any problems™ and patient characteristics accessUnivariableOR CIP valueMultivariableOR CIP value“   to “  to to Quintile most deprived Quintile Quintile Quintile Age yearsFemale sex vs maleIndex of Multiple Deprivation IMD quintile relative to England vs quintile least deprived n1006 Number of comorbidities vs no comorbidities Functional status KPS score vs KPS eGFR mLmin173 m2 N1007 to to to to to to to One Two Three or more Functional impairment KPS ‰ to   to to to to to to to to to to to to A2 “ mgmmol A3 ‰¥ mgmmol No formal qualifications GCSE A level or NVQ “uACR KDIGO categories vs category A13 mgmmol n1007 Educational attainment vs first or higher degree or NVQ “ n1007 BMI vs kgm2 Smoking status vs never smoked Overweight BMI “ kgm2 Obese BMI ‰¥ kgm2 to to to to to to Current smoker Ex smoker     to to to to    to to  “““n1008 in univariable models unless otherwise stated n1005 for final multivariable logistic regression modelsAdjusted for age deprivation level number of comorbidities functional status estimated glomerular filtration rate eGFR at year follow up urinary albumin to creatinine ratio uACR at year follow up educational attainment and body mass index BMI P value for trendGCSE General Certificate of Secondary Education KDIGO Kidney Disease Improving Global Outcomes KPS Karnofsky Performance Status A level advanced level NVQ National Vocational Qualificationscross sectional study of survivors and we were therefore not able to draw causative links HRQoL and FS measures were taken at year follow up but not at baseline and we were therefore unable to identify change over time The RRID cohort predominantly comprises people of white ethnicity limiting generalisability of our findings Comparison with HSE data was undertaken only via univariable analyses such that potential confounding factors may have influenced the differences observed between the two groups We also did not have sufficient numbers to allow for reliable exploration of associations between specific comorbidities and HRQoL or FS We also recognise the need for caution in the interpretation of the associations between functional impairment and problems in individual domains due to small numbers in some individual categories leading to wide CIs A further limitation is that one inclusion criterion was the ability to attend study visits which would have resulted in some selection bias by excluding the very frailPeople with CKD are likely to have multiple comorbidities due both to the nature of the disease process and the relationship between CKD and older age We have identified that comorbidity count was an independent determinant of both HRQoL and FS highlighting the importance of a holistic approach that includes attention to comorbidities in the management of people with Fraser a0SDS et a0al BMJ 202010e040286 101136bmj 2020040286 0c access Table Summary matrix of the independent associations with ˜some problems™ in each domain of the EQ 5D 5L from multivariable logistic regression analysesMobility Self care Usual activitiesPaindiscomfortAnxietydepressionIncreasing ageFemale sexGreater area deprivation levelHigher number of comorbiditiesFunctional impairmentLower eGFRHigher level of albuminuriaLower educational attainmentHigher BMISmokingΟ ΟΟ Ο ΟΟ ΟΟBMI body mass index eGFR estimated glomerular filtration rate ΟΟ Ο ΟΟ Ο Ο Ο Ο mild to moderate CKD As reported previously of people in this cohort with stage disease had at least two comorbidities6 There are clearly shared risk factors for several of the included comorbidities It is therefore perhaps unsurprising that in a large cohort of over half a million Canadian patients with CKD comorbidities such as hypertension and diabetes were common and respectively However a substantial number of patients also had ˜discordant™ comorbidities such as chronic pulmonary disease and of patients had chronic pain5 Comorbidities were all associated with an increased risk of hospitalisation5 It is striking that the majority of people in our cohort reported problems with mobility and chronic paindiscomfort and that both were more prevalent than in a nationally representative sample of the English general population of similar age About of our cohort were taking analgesic medication but about reported pain or discomfort in the EQ 5D 5L This likely reflects the association of CKD with comorbidities since mild to moderate reductions in GFR are unlikely to cause poor mobility or pain Nevertheless this observation further highlights the need to pay attention to mobility issues and pain management in order to improve quality of life in people with stage CKDThe prevalence of diabetes in this population of people with CKD stage was This is lower than the prevalence of noted in analyses of CKD prevalence in the HSE2 It is possible that this relates to this study population comprising survivors at years in a cohort study and those with diabetes may have been more likely to die prior to these analyses than those without The study population was also predominantly white and those ethnic groups with greater diabetes prevalence were therefore under representedMental health problems are common with of adults in England reporting a diagnosis at some point in time24 In our study about of people were classified as having a depressive condition defined pragmatically based on current antidepressant use or patient self report although about reported some anxiety or depression in the EQ 5D 5L so this was probably an underestimate In a large meta analysis approximately of adults with CKD stage “ had symptoms suggestive of depression25 This appears to persist even in milder forms of the disease and a large study from showed the prevalence of depression in people with CKD whose eGFR was ‰¥ mLmin173 m2 was These data imply that careful attention to mental health problems including screening for depression may also be key interventions to improve HRQoL in

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" COVID19 pathways for brain andheart injury in comorbidity patients A role of medical imaging and artificial intelligencebased COVIDseverity classification A review Computers in Biology and Medicine 101016jcompbiomed2020103960 0cThis is a PDF file of an that has undergone enhancements after acceptance such as the additionof a cover page and metadata and formatting for readability but it is not yet the definitive version ofrecord This version will undergo additional copyediting typesetting and review before it is publishedin its final form but we are providing this version to give early visibility of the Please note thatduring the production process errors may be discovered which could affect the content and all legaldisclaimers that apply to the journal pertain Published by Elsevier Ltd 0cCOVID19 Pathways for Brain and Heart Injury in Comorbidity Patients A role of Medical Imaging and Artificial Intelligencebased COVID severity Classification A Review Jasjit S Suri PhD MBA FAIMBE FAIUM FAPVS1 Anudeep Puvvula MBBS FCD12 Mainak Biswas PhD3 Misha Majhail14 Luca Saba MD5 Gavino Faa MD6 Inder M Singh MD7 Ronald Oberleitner BS MBA8Monika Turk MD PhD9 Paramjit S Chadha BE1 Amer M Johri MD FASE10 J Miguel Sanches PhD11 Narendra N Khanna MD DM FACC12 Klaudija Viskovic MD PhD13 Sophie Mavrogeni MD PhD FESC14 John R Laird MD FACC FACP15 Gyan Pareek MD16 Martin Miner MD17 David W Sobel MD16 Antonella Balestrieri MD5 Petros P Sfikakis MD18 Gee Tsoulfas MD19 Athanasios Protogerou MD PhD20 Durga Prasanna Misra MD FRCP21 Vikas Agarwal MD FRCP21 Gee D Kitas MD PhD FRCP22 Puneet Ahluwalia MS MCh24 Raghu Kolluri MD25 Jagjit Teji MD26 Mustafa AlMaini MD27 Ann Agbakoba MD28 Surinder K Dhanjil MSc FSVU29 Meyypan Sockalingam MBBS FRCR30 Ajit Saxena MD12 Andrew Nicolaides MS PhD FRCS31 Aditya Sharma MD32 Vijay Rathore MD33 Janet N A Ajuluchukwu MD34 Mostafa Fatemi PhD FAIMBE FIEEE FASA FAIUM35 Azra Alizad MD FAIMBE FAIUM36 Vijay Viswanathan MD PhD37 P K Krishnan MD38 Subbaram Naidu PhD Life FIEEE39 1Stroke Monitoring and Diagnostic Division AtheroPoint„¢ Roseville CA USA 2Annu™s Hospitals for Skin and Diabetes Nellore AP INDIA 3JIS University Kolkata West Bengal INDIA 4Oakmont High School and AtheroPoint„¢ Roseville CA USA 5Department of Radiology Azienda Ospedaliero Universitaria Cagliari ITALY 6Department of Pathology AOU of Cagliari Italy 7Stroke Monitoring and Diagnostic Division AtheroPoint„¢ Roseville CA USA 8Behavior Imaging USA 9The HanseWissenschaftskolleg Institute for Advanced Study Delmenhorst GERMANY 10Department of Medicine Division of CardiologyQueen™s University Kingston Ontario CANADA 11Institute of Systems and Robotics Instituto Superior Tecnico Lisboa PORTUGAL 12Department of Cardiology Indraprastha APOLLO Hospitals New Delhi INDIA 13University Hospital for Infectious Diseases Zagreb CROTIA 14Cardiology Clinic Onassis Cardiac Surgery Center Athens GREECE 15Heart and Vascular Institute Adventist Health St Helena St Helena CA USA 16Minimally Invasive Urology Institute Brown University Providence Rhode Island USA 17Men™s Health Center Miriam Hospital Providence Rhode Island USA 18Rheumatology Unit National Kapodistrian University of Athens GREECE 19Aristoteleion University of Thessaloniki Thessaloniki GREECE 20National Kapodistrian University of Athens GREECE 21Sanjay Gandhi Postgraduate Institute of Medical Sciences Lucknow UP INDIA 22Academic Affairs Dudley Group NHS Foundation Trust Dudley UK 23Arthritis Research UK Epidemiology Unit Manchester University Manchester UK 24Max Institute of Cancer Care Max Superspeciality Hospital New Delhi INDIA 25OhioHealth Heart and Vascular Ohio USA 26Ann and Robert H Lurie Children™s Hospital of Chicago Chicago USA 27Allergy Clinical Immunology and Rheumatology Institute Toronto CANADA 28University of Lagos NIGERIA 29AtheroPoint LLC CA USA 30MV Center of Diabetes Chennai INDIA 31Vascular Screening and Diagnostic Centre and University of Nicosia Medical School CYPRUS Journal Preproof 0c32Division of Cardiovascular Medicine University of Virginia Charlottesville VA USA 33Nephrology Department Kaiser Permanente Sacramento CA USA 34Department of Medicine Lagos University Teaching Hospital Lagos NIGERIA 35Dept of Physiology Biomedical Engg Mayo Clinic College of Medicine and Science MN USA 36Dept of Radiology Mayo Clinic College of Medicine and Science MN USA 37MV Hospital for Diabetes and Professor M Viswanathan Diabetes Research Centre Chennai INDIA 38Neurology Department Fortis Hospital Bangalore INDIA 39Electrical Engineering Department University of Minnesota Duluth MN USA Corresponding Author Dr Jasjit S Suri PhD MBA FAIMBE FAIUM FAPVS Fellow American Institute of Medical and Biological Engineering Fellow American Institute of Ultrasound in Medicine Fellow Asia Pacific Vascular Society Stroke Monitoring and Diagnosis Division AtheroPoint„¢ Roseville Roseville CA USA Phone Email jasjitsuriatheropointcom Journal Preproof 0cCOVID19 Pathways for Brain and Heart Injury in Comorbidity Patients A role of Medical Imaging and Artificial Intelligencebased COVID severity Classification A Review Abstract Artificial intelligence AI has penetrated the field of medicine particularly the field of radiology Since its emergence the highly virulent coronavirus disease COVID19 has infected over million people leading to over deaths as of July 1st Since the outbreak began almost s about COVID19 have been published pubmedncbinlmnihgov however few have explored the role of imaging and artificial intelligence in COVID19 patients”specifically those with comorbidities This paper begins by presenting the four pathways that can lead to heart and brain injuries following a COVID19 infection Our survey also offers insights into the role that imaging can play in the treatment of comorbid patients based on probabilities derived from COVID19 symptom statistics Such symptoms include myocardial injury hypoxia plaque rupture arrhythmias venous thromboembolism coronary thrombosis encephalitis ischemia inflammation and lung injury At its core this study considers the role of imagebased AI which can be used to characterize the tissues of a COVID19 patient and classify the severity of their infection Imagebased AI is more important than ever as the pandemic surges and countries worldwide grapple with limited medical resources for detection and diagnosis We conclude that imaging and AIbased tissue characterization when considered alongside COVID19 symptoms and their pretest probabilities offer a compelling solution for assessing the risk of comorbid patients These methods show the potential to become an integral part of tracking and improving the healthcare system both during the pandemic and beyond Keywords COVID19 comorbidity pathophysiology heart brain lung imaging artificial intelligence risk assessment Journal Preproof 0c Introduction In December a novel coronavirus referred to as œsevere acute respiratory distress syndrome coronavirus  SARSCoV2 [] appeared in Wuhan the capital of Hubei Province in PR China The disease caused by the virus was initially named œnovel coronavirus pneumonia NCP by the Chinese government but was subsequently renamed œcoronavirus disease  COVID19 by the World Health anization WHO On January 30th it was declared a public health emergency of international concern PHEIC [] It is believed that SARSCoV2 is primarily transmitted through saliva droplets or nasal discharge [] The first evidence of humantohuman transmission was found by Jasper FukWoo Chan et al in their study at The University of Hong KongShenzhen Hospital [] Due to its contagiousness Ro27 the virus has reached epidemic levels affecting countries and causing over million infections and more than deaths as of July 1st [] shown in Figure Recent literature suggests that patients with preexisting diseases are likely to experience severe complications from COVID19 [] In one study on admitted diabetic and nondiabetic COVID19 patients the mortality rate vs and the rate of admission to the intensive care unit ICU vs were significantly higher for diabetic patients Diabetic patients also experienced severe inflammatory responses and cardiac hepatic and renal coagulopathy [] The prevalence of heart and brain injuries was also higher in COVID19 patients with concomitant chronic conditions like diabetes kidney disease dyslipidemia hypertension [] chronic obstructive pulmonary disease COPD and cardiovascular diseases [] Recent studies have shown that SARSCoV2 invades the thin lining of the epithelial cells of the arteries leading to atherosclerosis [] and arterial inflammatory disease”one of the major causes of cardiovascular diseases CVDs which also causes heart and brain injuries [ ] This could be due to a reduced expression of angiotensinconverting enzyme ACE2 causing endothelial dysfunction which in turn aggravates existing Journal Preproof 0catherosclerosis [ ] It has also been observed that comorbid patients when subjected to imagescreening show mild to severe pretest probability PTP for COVID19 [] The conventional cardiovascular risk factors CCVRF in these comorbid patients appear strongly correlated either to their heart imaging or to surrogate biomarkers of coronary artery disease such as carotid artery disease Both imaging and biomarkers could be helpful in severity predictions for COVID19 [] Figure illustrates the associations between SARSCoV2 and other comorbidities such as diabetes as well as the comparative survival rates for COVID19 patients with and without diabetes Figure World map showing COVID19 spread over countries courtesy John Hopkins University ACE2 expression causes scars in the vessels and can even rupture the walls of the arteries [] For this reason CCVRF should be considered alongside imaging in patients who present with COVID19 and many comorbidities [] The second stage is the one at which a patient is most severely affected by Journal Preproof 0cCOVID19 and has the highest probability of cardiac injury or release of troponin T TnT Imaging has been shown to offer benefits in monitoring the tissue scars caused by COVID19 [] Figure a Association of SARSCoV2 with other comorbidities and b comparison of the mortality rate of diabetic and nondiabetic COVID19 patients reproduced with permission [] Multiple modalities can be utilized to determine whether a patient has the sequelae of COVID19 including magnetic resonance imaging [] computed tomography [] and ultrasound [] The advantage of these imaging modalities is the visual access they provide to the scar tissue caused by the disease A disadvantage however is their inability to provide a œrisk assessment The application of artificial intelligence AI can enhance the information provided by these imaging modalities resulting in a more accurate characterization of the tissue and the disease process [] In fact the combination of AI and medical imaging has been shown to improve diagnosis and risk stratification speed up patient evaluation enhance disease monitoring and accelerate early intervention [ ] Thus this review will focus on the use of AIbased tissue characterization of images of comorbid patients affected by COVID19 The layout of this paper is as follows Section presents the pathophysiology of the four pathways leading to brain and heart injury Section summarizes the evidence related to the use of Journal Preproof 0cimaging during the COVID19 pandemic Section elaborates on the use of AIbased tissue characterization for risk assessment Finally the paper concludes with a critical discussion The Pathophysiology of SARCoV2 Leading to Brain and Heart Injury Several studies suggest that SARSCoV2 uses the ACE2 receptor to gain access to cells by binding to the SPIKE protein ˜S™ protein on their surface [] see Figure ACE2 and angiotensinconverting enzyme ACE1 are homolog carboxypeptidase enzymes that have different vital functions in the reninangiotensinaldosterone system RAAS pathway [] ACE2 is widely expressed in myocardial cells [] type pneumocytes enterocytes and astrocytes in the brain [ ] Thus it is recognized as a cause of extrapulmonary complications Figure shows the overall picture of how SARSCoV2 causes brain and heart injuries via four different pathways These include i the neuronal pathway ii the hypoxia pathway iii the RAAS pathway and iv the immune pathway We will discuss these pathways and the injuries they lead to which may manifest as viral encephalitis infectious toxic encephalopathy or acute cerebrovascular disease i The Neuronal Pathway Figure the pathway I Recent epidemiological studies have demonstrated similarities at the genomic level between SARSCoV1 MERS and SARSCoV2 [ ] Meanwhile previous experimental studies have shown that beta coronaviruses in general”such as SARSCoV1 and MERS”can spread into and directly infect the brain when inhaled as droplets via the nasal epithelium [ ] Figure depicts the olfactory nerve and the olfactory bulb []”labeled as œa and œb respectively”on the image of the sagittal brain in the neuronal pathway Based on recent reports we are aware that patients infected by SARSCoV2 show symptoms of dysgeusia loss of taste and anosmia loss of smell [ ] Bohmwald et al further validate that coronaviruses that infect through the olfactory nerve and bulb can reach the brain and cerebrovascular fluid CSF within seven days Additionally these viruses have been observed to cause inflammation and demyelination [] The Journal Preproof 0cauthors demonstrated in an experimental study of mice that removing the olfactory bulb from the pathway can lead to the restriction of CoV in the central nervous system CNS [] Based on this evidence we believe that the neuronal pathway is one possible track for SARSCoV2 ii The Hypoxia Pathway Figure pathway II In this pathway decreased levels of ACE2 proliferate in the lung parenchyma cells after the coronavirus has passed through causing exaggerated neutrophils accumulation enhanced vascular permeability and the formation of diffuse alveolar and interstitial exudates This ultimately leads to pulmonary edema and acute respiratory distress syndrome ARDS [] ARDS is characterized by severe abnormalities in blood gas composition resulting from an oxygen and carbon dioxide mismatch which leads to low blood oxygen levels [ ] This ongoing hypoxia can lead to myocardial ischemia and heart injury [ ] see Figure pathway IIA Hypoxia in the brain increases anaerobic metabolism in the mitochondria of the brain cells [] leading to cerebral vasodilatation edema and impaired flow This can result in cerebral ischemia and acute cerebrovascular diseases such as acute ischemic stroke [ ] see Figure pathway IIB iii The RAAS Pathway after SARSCoV2 Figure pathway III The RAAS pathway is crucial in regulating blood pressure as well as the balance of fluid and electrolytes Any disturbance in this pathway can trigger the pathogenesis of cardiovascular diseases [] Before a SARSCoV2 infection triggers the RAAS Angiotensin I Ang I cleaves to Angiotensin II Ang II via ACE1 Ang II causes vasospasm It is also a proinflammatory agent with prothrombotic and proliferative effects that are detrimental to vascular tone and hemostasis [ ] Thus as a counterregulatory mechanism ACE2 degrades Ang II and generates Ang which counteracts the negative impacts of Ang II [ ] Both ACE2 and Ang have cardiocerebral vascular protective effects [] After the triggering of SARSCoV2 infection it results are in the deregulation of RAAS causing heart and brain injury in two different pathways The main culprit is an increase in Ang II which is caused by the decrease in ACE2 levels Figure pathway IIIA First an increase in Ang II levels stimulates the adrenal cortex of the kidney resulting in an increased production of aldosterone Aldosterone is a steroid hormone that causes sodium and water Journal Preproof 0creabsorption to increase at the distal tubule and collecting duct of the nephron [] This reabsorption increases blood volume and causes an elevation in blood pressure which results in the endothelial dysfunction that causes brain and heart injury [] The second effect of an increase in Ang II levels ie as a consequence of decreased ACE2 levels is endothelial dysfunction leading to intimal damage in the arterial walls [] which can be seen during the imaging of the arterial wall see Figure pathway IIIB This pathway can also trigger a cytokine storm as high levels of Ang II can cause an increase in proinflammatory cytokines see the bridge line between the RAAS and immune pathways iv The Immune Pathway Figure pathway IV Several recent studies have reported SARSCov2 viral pneumonia [ ] having an exaggerated inflammatory response known as a œcytokine storm This response appears to present at advanced stages of severe COVID19 with increased levels of inflammatory cytokines leading to multiplean failure [] The rise in inflammatory markers”including IL6 IL7 IL12 IL15 IL22 TNFα and CXCL10”results in the destabilization of plaque This in turn can cause plaque rupture resulting in heart and brain injury [ ] The Role of Imaging in Comorbid Patients with COVID19 As the previous section discussed COVID19 uses four pathways ie neuronal hypoxia RAAS and immune to cause critical heart and brain injuries in patients with comorbidities The prevalence of myocardial injury and brain injury caused by COVID19 [ ] points to a need for increased use of medical imaging to expedite assessments differential diagnoses and patient management [ ] with proper safety measures [] The seriousness of a patient™s COVID19 symptoms helps to determine which imaging modality is appropriate portable or nonportable and invasive or noninvasive BMode ultrasound imaging is portable and can be used for lowrisk patients Meanwhile Xray magnetic resonance imaging [] and computed tomography [] are nonportable and can be used for mediumrisk patients Intravascular ultrasound IVUS [] and ventriculography are invasive imaging modalities used in highly critical cases [ ] Amongst all the imaging modalities ultrasound is noteworthy because it is radiationfree portable quick repeatable inexpensive Journal Preproof 0cand can be performed in isolation thus lowering the chance of spreading the COVID19 infection [ ] There are several examples of medical imaging that have led to proper treatment and healthcare management during the pandemic ultimately reducing the mortality rate Xray imaging of the chest has demonstrated irregular patchy hazy reticular and widespread groundglass opacities indicating the progression of COVID19 at various stages this information can support the healthcare team in developing the most appropriate treatment plan [] Chest CT scans of COVID19 patients revealed in almost of the patients that the disease was affecting at least one of the five lobes of their lungs [] Chest MRI scans of COVID19 patients showed pulmonary tissue consolidation in six diffusionrestricted regions in six and lung damage in seven [] Meanwhile heart MRI studies of recovered patients showed that of the patients had myocardial edema At the same time late gadolinium enhancement was found in implying that COVID19“related cardiac injury is longstanding and requires frequent monitoring even after recovery [] In a different study MR scans of a COVID19 patient revealed myocardial inflammation signifying myocardial injury due to a cytokine storm related to the SARSCoV2 infection as discussed in Section Pathway IV [] Several studies have also evaluated the effects of COVID19 on the brain In one MRI scans revealed hemorrhagic rim enhancing lesions within the bilateral thalami medial temporal lobes and subinsular regions [] shown in Figure In another brain MRI scans were completed for patients of which produced abnormal findings [] Additionally evidence of liver injury and gall bladder abnormality was found in a joint CT and ultrasound study of the abdomen [] Recent MRI scans of COVID19 patients™ olfactory bulbs have revealed the cause of olfactory function loss to be the interaction between SARSCoV2 and the ACE2 protein expressed by the olfactory epithelium which leads to inflammatory obstruction [] Journal Preproof 0cFigure We have shown in four pathways how COVID19 can cause Brain and heart injury Brain image in pathway I httpdebugliescom20200123olfactorydisturbanceshaveimplicationsinmentalandemotionalwellbeinghealth Courtesy of Debug Lies Invasive imaging is another option for diagnosing COVID19 patients who have critical comorbidities In one such study IVUS along with stenting was performed with precautions on a COVID19 patient with myocardial infarction [] shown in Figure A detailed discussion of precautions is included in section In another study takotsubo syndrome a form of myocardial injury triggered by COVID19 was detected using ventriculography [] In various studies the medical imaging of COVID19 patients had been crucial to ascertaining the extent of tissue damage and critical infection although there were no visible symptoms [ ] Therefore medical imaging is the preferred way to ascertain the extent of cardiac and brain tissue damage throughout the lifetime of COVID19 patients COVID19 Journal Preproof 0cpatients with comorbidities are especially vulnerable and so they need to be screened through medical imaging from the first day of their diagnosis Medical imaging can help patients with deep vein thrombosis DVT as they are highly susceptible to severe tissue damage from COVID19 A study showed that COVID19 patients suffering from DVT had a worse prognosis than patients without DVT Patients with DVT were admitted to the ICU more frequently discharged less frequently and suffered more deaths than those without DVT [] Figure MRI scan of COVID19 patient showing hemorrhage MRI images demonstrate T2 FLAIR hyperintensity within the bilateral medial temporal lobes and thalami A B E F with evidence of hemorrhage indicated by hypointense signal intensity on susceptibilityweighted images C G and rim enhancement on postcontrast images D H reproduced with permission [] Journal Preproof 0c Figure Application of chest CT and IVUS for a COVID19 patient suffering from myocardial infarction a Chest CT scan with viral pneumonia showing fibrinous focal exudative changes b when the patient complained of chest pain the ECG report showed the STsegments elevations in V1V5 lead c d CAG radiology that the proximal segment of LAD was occluded e f The blood flow of LAD restoration after DESs was implanted g the dissection distal shown by IVUS to the stent in LAD from “ o'clock h the low echogenic shadow with scattered higher echogenic flicker indicating a thrombus i after a DES was implanted and the stent was well expanded the dissection could not be seen j The thrombus disappearance after the intervention reproduced with permission [] Journal Preproof 0c Although medical imaging can be very useful to patients and doctors alike the exponential pandemic curve inadequate medical facilities [] and a limited number of radiologists make the assessment diagnosis and management processes challenging tedious and errorprone Therefore although medical imaging can make diagnoses faster as stated above it will be of limited use Given this fact new age techniques such as artificial intelligence AI [] applications in medical imaging for tissue characterization can make computeraided assessments and diagnoses faster The main reason for this is that the AI can be scaled up to match the pandemic curve thereby meeting the immediate demands of medical image diagnoses during the COVID19 pandemic AIbased tests can categorize the nature of a patient™s risk in one of the categories namely norisk low lowmedium LM highmedium HM lowhigh LH or highhigh HH risk depending on the patient™s symptoms and their severity [ ] as shown in Figure The imaging modality also varies with the degree of risk as follows no imaging for norisk portable imaging for low and LM risk nonportable imaging for HM and LH and invasive imaging for HH A probability PTP is performed to accurately interpret diagnostic results to categorize the patient into one of the four groups [] After that for norisk patients nonimaging biomarkers can be collected for risk assessment using AIbased data protocols For lowrisk patients portable 2D3D imaging such as ultrasound is used whereas nonportable and invasive 2D3D imaging such as MRICTXRayechocardiography can be used for LM patients For HH patients invasive imaging techniques such as IVUS and ventriculography can be used Based on the data provided by various 2D3D scans AIbased medical imaging is applied for risk assessment Further treatment is then planned based on this imaging process In the next section deep learning DLbased medical imaging is proposed for medical imaging scans particularly ultrasounds for COVID19 patients Machine Learning and Deep Learning for Tissue Characterization Using AI and associated technologies in healthcare can significantly slow down diagnosis times Journal Preproof 0cespecially during the COVID19 pandemic as patient numbers are continually growing and there are few specialists available [] Figure Role of AIbased risk assessment on COVID19 patients having comorbidity Journal Preproof 0cAlthough some caution must be exercised regarding its fullscale deployment [] its overall usefulness in healthcare management during times of crisis cannot be ignored [ ] In general AI in healthcare refers to all artificial intelligencebased technologies that make educated decisions regarding a patient™s diagnosis monitoring treatment and management The importance of AI has specifically increased many folds when imaging comes into play mainly because of large volumetric data sizes and the extensive need to characterize and quantify the disease via lesion images [] Tissue imaging and its characterization is of prime importance since it has a direct influence on decisions related to COVID19 severity for a patient [] The main benefit of AI methods is that the machines can be used to train by mimicking the physician™s cognitive actions and such trained models can be used to predict the disease™s severity in asymptomatic patients Within a short period several machine learning MLbased techniques used the power of AI to manage COVID19 [ ] ML and DL Architectures ML Architecture ML is a twostage process In stage I different features are extracted from the lesion COVID images the extractions are then operated on by an ML statistical model called a training system to generate offline coefficients These coefficients are then transformed by the test lesion images which yield an intelligent classification or inference A typical ML system for predicting risk class is shown in Figure CUSIP is an imagebased phenotype that uses the event equivalent gold standard EEGS [ ] model DL Architecture DL refers to a visual cortex that imitates multiple layers of a neural network applied directly to tissue images to extract features and for characterization purposes [] The convolution neural network CNN [] shown in Figure is one such DL network architecture that is widely used to characterize medical images It performs a series of convolution maxpooling operations to extract features and perform characterizations ML and DL both follow the supervised learning Journal Preproof 0capproach by which models are trained using offline data Figure Typical lowcost machine learning architecture utilizing EEGS model As discussed in previous sections there are four pathways through which a COVID19 infection leads to heart and brain injuries AI can be used via medical imaging to detect the extent of tissue damage in these pathways and help medical professionals to develop an effective treatment plan for patients There are several instances in which the AI paradigm has been used for tissue characterization based on Journal Preproof 0cmedical images during the pandemic as well as during standard times Some uses of AI are described anwise following a proposed model for characterizing DLbased tissues Figure A convolution neural network courtesy of AtheroPoint„¢ CA USA ML Architecture used for Tissue Characterization for Stroke Risk Stratification There are two types of tissue characterization that can be carried out using AI i MLbased [ ] and ii DLbased [] Various MLbased technologies have been developed to classify symptomatic and asymptomatic plaque from ultrasound images For example an MLbased technique based on support vector machines SVM was developed to characterize the symptomatic and asymptomatic plaques of carotid scans that indicated the presence of plaque [ ] SVM classifiers work by determining the maximum margin between two data clusters First a texture analysis [] is used to extract the features ie standard deviation entropy symmetry and run percentage in

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IFNBased Biotherapeutics toHarness the Host AgainstFootAndMouth DiseaseGisselle N Medina Teresa de los Santos and Fayna DiazSan Segundo Plum Island Animal Disease Center PIADC ARS USDA Orient Point NY United States Kansas State University Collegeof Veterinary Medicine Manhattan KS United StatesFootandmouth disease FMD is a highly contagious vesicular disease of clovenhoofedanimals that severely constrains international trade of livestock and animal productsCurrently disease control measures include broad surveillance enforcement of sanitarypolicy and use of an inactivated vaccine While use of these measures has contributedto eliminating footandmouth disease virus FMDV from a vast area of the worldthe disease remains endemic in three continents and outbreaks occasionally appearin previously declared FMDfree zones causing economic and social devastationAmong others a very fast rate of viral replication and the need for days to achievevaccineinduced protection are the main limitations in controlling the disease Newfastacting antiviral strategies targeted to boost the innate immunity of the host to blockviral replication are needed Here we review the knowledge on the multiple strategiesFMDV has evolved to block the host innate immunity with particularly focus on the pastand current research toward the development of interferon IFNbased biotherapeuticsin relevant livestock speciesKeywords footandmouth disease virus FMDVIFNλ IFNωinterferon IFN antivirals biotherapeutics IFNα IFNÎINTRODUCTIONThe Disease FootAndMouth DiseaseFootandmouth disease FMD is one the most serious livestock diseases that aï¬ects clovenhoofedanimals including cattle swine sheep and goats as well as numerous species of wild species The disease displays high morbidity but is usually not lethal except when it aï¬ects young animalsthat may develop myocarditis Infected animals secrete copious amounts of virus ps beforethe onset of the clinical phase of the disease Typical FMD clinical signs include fever and theappearance of vesicular lesions on the tongue mouth feet and teats Among ruminants thatrecovered from the disease a relatively large number become asymptomatic virus carriers although it is not clear what is the contribution of these carrier animals to disease transmissionin nature The World anization for Animal Health OIE lists FMD as a reportable diseaseand therefore by law participating nations are required to inform the anization about all FMDoutbreaks OIE member nations with reported cases of FMD are forbidden to engage in tradingof FMDsusceptible animals or their products Thus the presence of FMD in a country can havesevere economic consequencesDiï¬erent interventions to control an FMD outbreak include restriction of susceptible animalmovement slaughter of infectedcontact animals decontamination of infected and surroundingEdited byMariano PrezFilgueiraNational Agricultural TechnologyInstitute ArgentinaReviewed byMargarita S¡izSevero Ochoa Molecular BiologyCenter CSICUAM SpainKenneth James GenoveseAgricultural Research ServiceUnited States Department ofAgriculture United StatesCorrespondenceGisselle N MedinagissellemedinausdagovTeresa de los SantosteresadelossantosusdagovFayna DiazSan SegundofaynadiazsansegundousdagovSpecialty sectionThis was submitted toVeterinary Infectious Diseasesa section of the journalFrontiers in Veterinary ScienceReceived April Accepted June Published August CitationMedina GN de los Santos T andDiazSan Segundo F Use ofIFNBased Biotherapeutics to Harnessthe Host Against FootAndMouthDisease Front Vet Sci 103389fvets202000465Frontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVpremises and vaccination Vaccination is an option used mostlyin countries in which FMD is endemic but diseasefree nationsprefer to abstain from such practice In general FMDfreecountries that occasionally opted to vaccinate to better containthe outbreak did slaughter all vaccinated animals to regaincommerce rights faster as occurred in the outbreak inthe UK and the Netherlands The current approvedFMD vaccine consists of purified chemically inactivated virus[binary ethylenimine BEItreated] formulated with oilbased oraluminum adjuvants that induces serotypespecific protection inapproximately days and it is applied with a boosting protocolfor ensuring longterm protection While this vaccine hasbeen successfully used for many decades leading to diseaseeradication of a vast area of our planet challenges remainFMD is endemic in most of Africa and Asia and occasionallyepizootics appear in South America or in nations that havebeen diseasefree for many years as it happened in the UKthe Netherlands South Korea Taiwan and Japan Novelvaccine technologies have been developed but to this end noneof them has fully addressed the limitations of the commerciallyavailable vaccine or is currently approved for massive use Alternatives or additional therapeutics that could complementor in some instances substitute for vaccination protocols includethe use of antivirals and biotherapeutics that act quickly priorto induction of vaccineinduced immunity The development ofsuch molecules requires a thorough understanding of the biologyof the virus and its intricate interactions particularly with theinnate immune molecular and cellular mechanisms evolved bythe hostThe AgentFootandmouth disease virus FMDV is a member of theAphthovirus genus within the Picornaviridae family and it is theetiologic agent of FMD The virus contains a singlestrandedRNA of positive polarity Its genome of ˆ¼ nucleotidesconsists of a long reading frame ORF flanked by a ² anda ²untranslated region UTR The ORF encodes a polyproteinof about amino acids which is processed by virusencodedproteases Processing results in the generation of precursors andmature protein products including four structural [1A VP4 1BVP2 1C VP3 1D VP1] and ten nonstructural NS proteins[Lpro 2A 2B 2C 3A three distinct copies of 3B VPg 3Cproand 3Dpol] Due to high genetic variability FMDV is categorizedin seven distinct serotypes A Asia1 C O and Southern AfricanTerritories “ SAT “ and numerous subtypes or topotypesUpon infection the virus spreads very rapidly usually achieving morbidity Depending on the route of entry less than tissue culture infectious doses are required to infect andcause disease in animals In fact FMDV is one of thefastest replicating RNA viruses in nature taking as little as “ h to induce cytopathic eï¬ects in susceptible tissue culture cellsOne could envisage that during FMDV replication almost everycomponent of the virus must play a role in dampening interferingcellular responses to allow such rapid virus replicationInnate Immunity and Interferon ActivationEarly protection against viralfundamentallymediated by the action of interferons IFNs the pillar moleculesof the innate immune system “ Expression of IFN isinfection istriggered by the recognition of molecular signatures collectivelynamed pathogenassociated molecular patterns PAMPs viacellular receptors pattern recognition receptors PRRs that candistinguish œself from nonself  molecules Figure Bindingof PAMPs to PRRs triggers a series of signal transduction eventsand posttranslational modifications PTMs phosphorylationubiquitination ISGylation etc that ultimately activate latenttranscription factors to induce IFN transcription Subsequentlysecreted IFN proteins bind to specific receptors on the plasmamembrane to activate in an autocrine and paracrine mannerdiscrete and overlapping cellular signal transduction pathwaysDepending on the cell type and aï¬ected tissue over specificIFNstimulated genes ISGs may be induced many of whichdisplay antiviral activity to control the viral infection There are three families of IFNs based on the specific receptorusage types I II and III Table “ Type I IFNsie IFNα and IFN signal through a heterodimeric receptorcomplex formed by IFNAR1IFNAR2 type II IFN IFNÎsignals through the complex IFNÎR1IFNÎR2 and type IIIIFNs bind the receptor complex IL28RαIL10R Despitethe receptor diï¬erencesthe three IFN families transducesignals through the Janus kinase JAK“signal transducer andactivator of transcription STAT pathway and type I and typeIII IFNs induce redundant responses Figure Overall therapid production of IFN helps to limit viral replication whilemodulating other immune functionsFOOTANDMOUTH DISEASE VIRUSIMPAIRS INNATE IMMUNITY MOLECULARINTERACTIONSRecognition of FMDV RNA by the host cell results in theestablishment of a rapid antiviral state to limit and controlinfection This selective pressure has allowed FMDV to evolvemany strategiesto ensure enhanced virulence and rapidinfectivity In general RNA viruses can bypass the IFN responseby blocking i global cellular transcription and translationii IFN induction and iii IFN signaling Similarly to otherRNA viruses FMDV can also target IFNindependent antiviralresponses mostly associated with cellular metabolic functionsie autophagy apoptosis stress granule formation etc thathave been extensively described elsewhere In thissection we will summarize the current literature on studiesconducted in vitro that explain how FMDV counteracts the hostinnate immune response at the molecular level including RNAsensing activation of adaptoreï¬ector proteins and regulation ofsignaling pathways by specific PTMsBlock on Cellular Transcription andTranslationFMDV inhibition of cellular gene expression and proteinsynthesis during infection is mainly driven by the viralencoded proteases Leader Lpro and 3C FMDV Lpro isa papainlike protease PLP thatthetranslation initiation factor eIF4G including eIF4GI and eIF4GII to disable capdependent protein synthesis AlsoFMDV Lpro causes degradation of the transcription factorinduces cleavage ofFrontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVFIGURE Antiviral signaling pathways induced during viral infection Cellular detection of microbial molecules known as pathogenassociated molecular patternsPAMPs ie viral RNA is mediated by pattern recognition receptors PRRs including cytosolic RNA sensors ie RIGI MDA5 or LGP2 andor membraneboundTLRs PAMPPRR interaction activates signal transduction cascades black arrows that result in the production of IFN and inflammatory cytokines RIGI and MDA5contain two caspase recruitment domains CARD and an RNA helicase domain In the case of RIGI ubiquitination green circles is required for its effective activationActivated signals from either RIGI or MDA5 are transmitted downstream via the mitochondrial adaptor MAVS resulting in the formation of MAVS filaments At thisstage different PTMs such as ubiquitination or ISGylation black circles can regulate their functions Endosomal RNAs are detected by TLR3 or TLR78 which signalthrough adaptor proteins TRIF and MyD88 respectively MyD88 uses other adaptors IRAK14 to allow for interaction with TRAF proteins In addition to their role asadaptor proteins TRAFs also serve as E3 ubiquitin Ub ligases to regulate signaling TRAFmediated induction of polyUb is sensed by NEMO thus recruitingdownstream effector kinases such as TBK1 or IKK These proteins form different signaling complexes ie NEMOTBK1 and NEMOIKK leading to phosphorylationblue arrows of transcription factors IRF37 to a lesser extent IRF1 and IRF5 are also phosphorylated IRF phosphorylation triggers dimerization and translocationContinuedFrontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVFIGURE orange arrows to the nucleus where they bind mainly to IFN promotersenhancers Alongside with this pathway TRAF6E3 ligases can activate MAPK3and other kinases including ERK12 and JNK which phosphorylate the components of the AP1 heterodimer allowing for translocation to the nucleus and binding tothe IFN promoterenhancer to activate transcription Activated IKK also phosphorylates IκB releasing NFκB which then translocates to the nucleus and binds at theIFN promoter AP1 activating protein CARD caspase activation and recruitment domain DUB deubiquitinase ER endoplasmic reticulum IκB inhibitor of KBkinases IKK IκB kinase IL interleukin IRAK interleukin1 receptorassociated kinase IRF IFN regulatory factor LGP2 laboratory of genetics protein MAPKmitogenactivated protein kinase MAVS mitochondrial antiviral signaling protein MDA5 melanoma differentiationassociated gene MyD88 myeloid differentiationprimary response protein 88d NEMO NFκB essential modulator NFκB nuclear factorκB PKR protein kinase R PTM posttranslational modification RIGIretinoic acidinducible gene I TANK TRAF family memberassociated NFκB activator TBK TANK binding kinase TLR Tolllike receptor TRAF TNF receptorassociated factor TRIF TIRdomaincontaining adapterinducing interferonTABLE Use of IFNbased therapies against FMDVTypeReceptSignalSubtypeSpeciesMilestoneType IIFNAR1IFNAR2JAK1 TYK2IFNαPorcinebovine ¢ Recombinant bacterial expressed IFNα is a potent biotherapeutic againstIFNαPorcine¢ Ad5 delivered poIFNα protects swine against different serotypes of FMDVFMDV in vitro IFNIFNδIFNω7IFNαωIFNτType IIIFNÎ R1 IFNÎ R2JAK1 JAK2IFNÎType IIIIFNλR1IL10R2JAK2 TYK2IFNλ1IFNΓ¢ poIFNαprotection correlates with enhanced tissuespecific innate immune cellinfiltration in swine ¢ poIFNα protection correlates with upregulation of essential ISGs in vitro ¢ Ad5 delivered porcine poIFN protects swine against FMDV ¢ Bacterially expressed poIFNδ8 significantly inhibits FMDV replication in vitro ¢ E coli produced poIFNω7 protects cells against FMDV ¢ Bacterially expressed IFNαω added prior to infection resulted in a significantreduction in FMDV replication in vitro ¢ Ovine IFNτ has antiviral effect against FMDV in vitro ¢ Recombinant bovine IFNÎ reduced FMDV replication in BTY cell culture ¢ High dose of Ad5poIFNÎ protects swine against FMD ¢ Replication of FMDV in IBRS2 cells is inhibited by treatment with the purifiedrecombinant poIFNλ1 PorcinePorcinePorcinePorcineOvineBovinePorcinePorcineIFN CombosOtherIFNvaccinecombosIFNλ3Bovine¢ Inoculation with Ad5boIFNλ3 resulted in the induction of several ISGs in tissuesof the upper respiratory tract and protected cattle against challenge withFMDV PorcinePorcineIFNαIFN΢ Ad5poIFNλ3 protects swine against challenge with FMDV ¢ Use of a combination of Ad5poIFNÎ and Ad5poIFNα or Ad5poIFNαΠshowed an enhancement of the antiviral activity against FMDV in swinePoly ICPorcine¢ Double stranded ds RNA poly ICLC in combination with Ad5poIFNαprotected swine against FMDV siRNAPorcine¢ Combination of Ad5poIFNαΠwith Ad3siRNA targeting FMDV NS codingregions blocked replication of all serotypes of FMDV in vitro IRF73Porcine¢ Inoculation with Ad5IRF735D resulted in induction of IFNα and fully protectedmice and swine challenged with FMDV day after treatment IRESPorcine¢ Use of synthetic IRES in combination with adjuvanted typeO FMD improvedimmune response and protection against FMDV challenge IFNαPorcine¢ Use of a combination of Ad5poIFNα and Ad5A24 in swine resulted incomplete protection after challenge IFNαÎPorcine¢ Ad5poIFNαΠcoadministered with Ad5siRNA targeting NS regions of FMDVand a commercial inactivated FMD vaccine partially protected swine IFNλ3Bovine¢ Use of a combination of Ad5bovIFNÎ3 and AdtO1M in cattle resulted incomplete protection after aerosol challenge nuclear factor NFκB and results in blockage of specificdownstream signaling eï¬ectors Studies in porcine cellsdemonstrated that FMDV Lpro can promote its selfbindingto the transcription factor activitydependent neuroprotectiveprotein ADNP and negatively regulate the activity of the IFNα promoter In contrast chromatin changes that favor theupregulation of IFN and ISGs can inhibit FMDV replication Interestingly deletion or mutations in diï¬erent domainsof Lpro result in viral attenuation in vitro and in vivo “ Furthermore these studies have shown a strong type I IFNactivity upon infection with diï¬erent versions of FMDV Lpromutants Frontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVFIGURE Type I II and III interferon IFNmediated signaling All type I and type III IFN subtypes bind to respective receptors IFNAR1IFNAR2 and IFNLR1IL10R2These interactions trigger the phosphorylation of JAK1 and TYK2 kinases which in turn phosphorylate STAT1 and STAT2 JAK2 mediates type III IFNdependent STATphosphorylation Phosphorylated heterodimers of STAT1STAT2 bind to IRF9 forming the ISGF3G complex which then translocates to the nucleus and binds toIFNresponsive elements ISREs present in the promoters of over ISGs Type II IFN binds to the heterodimeric IFNÎR1IFNÎR2 receptor also inducingphosphorylation of JAK1JAK2 kinases In turn mostly STAT1 is phosphorylated Phosphorylated homodimers of STAT1 translocate to the nucleus and induce theexpression of genes controlled by gammaactivated sequence GASdependent promoter sequences IFNAR12 IFN alpha receptor12 IFNÎR12 IFNgammareceptor12 IFNALR1 IFNlambda receptor IL10R2 IL10 receptor ISGs IFNstimulated genes ISGF3G ISG factor gamma JAK12 Janus kinase STATsignal transducer and activator of transcriptionInterruption of cellular translation during infection can alsobe mediated by FMDV 3Cpro a chymotrypsinlike cysteineprotease that similarly to Lpro targets eIF4G and the capbindingcomplex eIF4A for cleavage although these events occur later inthe infection 3Cpro can also participate in the inhibitionof host“cell transcription by cleaving histone H3 upon FMDVinfection Block on Interferon InductionDuring infection the initial event that leads to the productionof IFN and proinflammatory cytokines is the recognition ofviral RNA Figure Sensing of FMDVRNA is mediated byMDA5 a protein that belongs to a family of helicasesknown as retinoic acidinducible geneI RIGIlike receptorsRLRs Recent studies have shown that the interaction betweenRLRs RIGI and LGP2 and the FMDV proteins Lpro 2Band 3A interferes with the induction of type I IFN “Indeed overexpression of either FMDV 2B or 3A resulted in thedownregulation of RIGI and MDA5 mRNA expression In contrast upregulation of LGP2 transcripts has been observedduring FMDV infection in porcine cells despite a detectablereduction of LGP2 protein levels presumably due to FMDVFrontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVLproinduced cleavage The apparent inconsistencybetween the levels of LGP2 mRNA and protein during FMDVinfection may be explained by LGP2™s ability to serve as a positiveand negative regulator of RIGI and MDA5 signaling presumablyaï¬ecting multiple steps of the IFN induction pathway Inaddition to RLRs nucleotidebinding oligomerization domainNODlike receptors NLRs NOD1 and NOD2 also participatein the recognition of RNA A study by Liu et al describedthe association of NOD2 with FMDV 2B 2C and 3Cpro to blockinnate immunity activation Protein kinase R PKR is anotherrecognized PRR that acts as an RNA sensor Binding ofRNA to PKR induces a conformational change that leads toautophosphorylation and activation The primary target ofactivated PKR is the eukaryotic initiation factor α subuniteIF2α whose phosphorylation results in the blockage of cellularprotein synthesis a relatively common process during viralinfection Although no direct interaction between FMDVRNA and PKR has been demonstrated it has been reportedthat PKR activity modulates FMDV infectivity In fact in tissueculture experiments depletion of endogenous levels of PKR usingsiRNA resulted in increased FMDV titers Furthermoreit has been recently shown that overexpression of autophagyrelated ATG5ATG12 proteins induces transcription of PKR andsubsequent reduction of FMDV replication These resultssuggest that PKR has a complex role as an RNA sensor but also asan antiviral agent during FMDV infectionIt has been demonstrated that FMDV also targets DExDHbox RNA helicases formally accepted as PRRs and modulatorsof the antiviral signaling pathway In vitro experimentsintending to analyze protein“protein interactions revealed theassociation between the RNA helicase DDX1 and FMDV 3D Interestingly these studies indicated that during FMDVinfection in porcine cells cleavage of DDX1 was detected whileoverexpression of DDX1 resulted in the upregulation of IFNand other ISG mRNAs which correlated with virus inhibition Other DExDHbox RNA helicases such as RNA helicaseH RHA are hijacked during FMDV infection and interact withFMDV ™UTR 2C and 3A to facilitate virus replication Signaling pathways downstream from RNA sensing involvethe activation of diï¬erent adaptor and eï¬ector proteins Oneof the pathways that lead to signal activation requires theformation of specific complexes such as NFκB essentialmodulator NEMO and the kinase IKK which bridges theactivation of NFκB and IFN regulatory factor IRF signalingpathways It has been demonstrated that FMDV 3Cpro interactswith NEMO and induces its cleavage resulting in impairedinnate immune signaling IRFmediated signals driven byIRF3 and IRF7 can also be targeted by FMDV proteinsSpecifically overexpression of Lpro in PK15 cells resulted inthe downregulation of IRF3 and IRF7 protein levels andinactivation of IFN and IFNλ1 promoter Other factors involved in the activation of IFN includeconventional PTMs such as phosphorylation and ubiquitinationwhich ensure eï¬ective regulation of these signaling pathways Also diï¬erent cellular deubiquitinases DUBs can reverseubiquitination to control the intensity of the immune signalingresponse Interestingly it has been shown that FMDV Lprocan remove ubiquitin Ub molecules from several proteinsrequired for IFN mRNA expression and those involved in theactivationrepression of the IFN loop This role became moreevident by the observation that during infection FMDV Lprocan cleave cellular substrates modified with the Ublike moleculeISG15 Furthermore mutation of Lpro thatimpairsdeISGylaseDUB function results in viral attenuation Inthis regard identification of FMDV targets for deubiquitinationand deISGylation may contribute to elucidate the role ofthose factors in counteracting the innate response and developnovel countermeasuresBlock on Interferon SignalingThe ligandmediated association of the specific IFN receptorspromotes a signaling cascade that results in the phosphorylationof the receptor by the action of JAKs These events result inthe generation of docking sites for downstream adaptor andeï¬ector proteins including signaltransducer and activatoroftranscription STAT proteins that associate with otherfactors and translocate to the nucleus inducing transcriptionof a plethora of ISGs described above and in Figure Although blockage of the JAK“STAT signaling pathway hasnot been reported during FMDV infection overexpressionof either FMDV 3Cpro or VP3 can inhibit this response Forinstance IFNtreated HeLa cells overexpressing FMDV 3Cprosuppressed IFNstimulated promoter activities and inducedproteasome and caspaseindependent protein degradationof karyopherin α1 KPNA1 the nuclear localization signalreceptor Thisinteraction inhibited the nuclear translocation of STAT1STAT2impeding maximal ISG promoter activity In another studyin HEK293T cells overexpression of VP3 followed by coimmunoprecipitation revealed the association between VP3 andJAK1 FMDV VP3 also inhibited virustriggered activation of theIFN promoter leading to the decrease in transcription of ISGspresumably due to lysosomalinduced degradation of JAK1 A yeast twohybrid screen identified FMDV 2C in complex withNmyc and STAT interactor Nmi a protein known to augmentimmune function dependent on STATmediated transcriptionInterestingly such interaction resulted in the recruitment ofNmi to vesicular compartments followed by the induction ofapoptosis in BHK21 cells tyrosinephosphorylated STAT1forEvidently FMDV proteins can also target crosstalk pathwaysinduced by JAKSTAT signaling and due to this versatilityunderstanding of these signaling events during FMDV infectionis challengingFOOTANDMOUTH DISEASE VIRUSIMPAIRS INTERFERONMEDIATEDCELLULAR INNATE IMMUNE RESPONSESSimilarly to what happens in vitro FMDV manipulates theearly innate immune response in vivo to ensure a windowof opportunity that favors viral replication and spread beforeFrontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVthe onset of eï¬ective adaptive immunity required for virusclearance During infection FMDV interacts with a range of hostcells including natural killer NK cells dendritic cells DCsmonocytesMφ and Îδ T cells All these cells play an importantrole in innate immune responses that trigger the productionof large quantities of IFN and other cytokines which serve asautocrine agents “Shortly after FMDV infection in swinethe number ofcirculating NK cells transiently decreases and the remaining NKcells show a dysfunctional lytic activity against target cells and areduction of IFNÎ production In parallel FMDV blocks theability of porcine DCs to mature into conventional DCs cDCs dampening their response against Tolllike receptor TLRligands Another subset of porcine DCs plasmacytoid DCspDCs also referred to as the major professional systemic IFNαproducers are also aï¬ected by FMDV During infectionpartial depletion of pDCs in the peripheral blood has beendetected and the remaining pDCs are less capable of producingIFNα in response to ex vivo stimulation by TLR ligands or virus Similar to pDCs FMDV infection reduces the productionof IFNα on Langerhans cells LCs a distinct subset oftissueresident DCs of the skin It has also been suggestedthat porcine Îδ T cells and Mφ can serve as targets for FMDVinfection in swine although the interplay betweenthese cells and FMDV remains unclearComparably to swine FMDV infection in cattle triggersseveral early events in the innate immune system althoughthe eï¬ects are not exactly the same For instance bovine NKcells originated from FMDVinfected cows have an elevatedcytotoxic function against bovine target cells in vitro In addition some subsets of cDCs are significantly decreasedduring the peak of viremia while the expression of majorhistocompatibility complex MHC class II molecules on allbovine cDCs is reduced and the processing of exogenous antigenis impaired Furthermore during FMDV infection thenumber of systemic mature bovine pDCs characterized bythe expression of CD4 and MHC class II is increasedpresumably to intensify a humoral response and T cell activationwhile levels ofimmature CD4 MHC class IIpDCs aredeclined Examination of bovine Îδ T cells revealed thatthese cells with the surface expression marker WC1 showa transient activated phenotype and increased expression ofIFNÎ FMDV also aï¬ects the innate immune response at the cytokinelevel in the natural host In vivo cytokine profile analysis duringthe clinical phase of disease shows a systemic decrease of proinflammatory cytokines [IL1 IL6 and tumor necrosis factorTNFα] and an increase of the antiinflammatory cytokine IL and IFNα Most likely these changesare related to the early T cell unresponsiveness and lymph iadescribed in swine and cattle during FMDV infection Interestingly a significant induction of inflammatoryand antiviral factors at the local level is detected in cattle in sitesof abundant viral amplification such as the nasaloropharynx orvesicular lesions “ A consistent upregulation of IFNα Î and λ mRNA in distinct microanatomical compartmentsof the nasopharyngeal mucosa concurrent with occurrence ofviremia has also been detected in cattle In contrast studiesin swine demonstrated that IFN expression in infected swineskin is inhibited These diï¬erences may be due to theanalysis of follicleassociated epithelium of the nasopharyngealmucosa in cattle vs skin in swine or to the specific samplingtechnique used in each experiment While in the cattle studylasercapture microscopy was used to focus only in areas of highFMDV replication in the swine study RNA was extracted froma piece of skin without discriminating between microanatomicalcompartments Evidently more studies are needed to elucidatethe intricate interactions between FMDV and the innate immunesystem of specific animal hostsEFFECTIVE USE OF INTERFERONAGAINST FOOTANDMOUTH DISEASEVIRUS IN VITROType I InterferonThe role of IFN in controlling FMDV replication was firstproposed in when Dinter and Philipson demonstrated thatcalf kidney cells exposed to FMDV could become persistentlyinfected and proposed this was a consequence of the inductionof an IFNlike inhibitor present in the supernatant of infectedcells Later studies also suggested that swine leukocytestreated with phytohemagglutinin produced an inhibitor ofFMDV replication with properties similar to IFN It wasnot until that new studies demonstrated that the abilityof FMDV to form plaques in cell culture correlated with thesuppression of type I IFN α protein expression Theseresults were further supported by detection of IFN protein andantiviral activity in the supernatants of primary porcine ovineand bovine kidney cells infected with an attenuated FMDVmutant leaderless as compared to the supernatants of cellsinfected with wildtype WT virus Later studies by the samegroup provided proof of concept on the use of recombinantbacterial expressed IFNα as a potent biotherapeutic againstFMDV This approach was further developed by deliveringrecombinant porcine IFNα using a replicationdefectivehuman Adenovirus vector Ad5poIFNα Infection ofIBRS2 cells with Ad5poIFNα resulted in secreted poIFNα IFN protein detected as early as h postinfection hpiand lasting for at least h Most important expressed IFNprotein displayed strong biological antiviral activity againstFMDV Followup studies by the same group showed that allFMDV serotypes are very sensitive to Ad5delivered poIFNαand sterile protection could be achieved in vivo highlightingthe potential of this approach for the development into abroad biotherapeutic strategy to control FMDV replicationIn the last years advancements is genomics have ledto the characterization of almost all type I IFN subtypes inthe porcine and bovine genome “ which are morenumerous than those identified in primates and mice This hasrevealed diï¬erent functional genes and pseudogenes with diverseexpression profiles and antiviralfunctions against diï¬erentviruses mostly in swine In fact a recent studyFrontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVdemonstrated that poIFNω7 known for its ability to inducethe highest levels of antiviral activity when compared to otherpoIFNω subtypes elicits an antiviral state against FMDV inIBRS2 cells treated with the recombinant form of poIFNω7produced in Escherichia coli Other subclasses of type IIFN known to be produced in swine and cattle include IFNalphaomega IFNαω also known as IFNµ and IFN delta IFNδ Significant reduction in FMDV replication has been observedupon treatment of porcine cells with bacterially expressed IFNαω or IFNδ8 prior to viral infection Recently another member of type I IFN family IFNτ whichis only produced in ruminants has been evaluated as an antiviralagainst FMDV IFNτ is a paracrine reproductive hormonesecreted constitutively by trophoblasts and endometrial cells toincrease the life span of the corpus luteum however productionis not induced upon viral infection While its secretionis restricted to ruminantsit has a broadspectrum activityagainst various crossspecies viruses Interestingly IFNτ has homology with the amino acids of IFNα which allows forbinding to type I IFN receptors The property of IFNτ thatmakes it an interesting therapeutic candidate for the treatmentof various viral diseases is its significantly lower toxicity ascompared to other type I IFNsType II InterferonIn contrast to type I IFN the type II IFN family is composedof only one member IFNÎ which exerts its actions througha specific receptor IFNGR1IFNGR2 IFNÎ is weakly resistantto heat and acid and it is able to activate leukocytes suchas macrophages and granulocytes also exerting regulatoryfunctions on T and B lymphocytes Indeed productionof IFNÎ is used as a tool to measure cellmediated immuneresponses against FMDV in vaccinated cattle “ andin swine Interestingly IFNÎ responses as measured b

Thyroid_Cancer

" The advent of new cancer therapies alongside expected growth and ageing of the population better survival rates and associated costs of care is uncovering aneed to more clearly define and integrate supportive care services across the whole spectrum of the disease The current focus of cancer care is on initialdiagnosis and treatment and end of life care The Multinational Association of Supportive Care in Cancer defines supportive care as ˜the prevention andmanagement of the adverse effects of cancer and its treatment™ This encompasses the entire cancer journey and necessitates involvement and integration ofmost clinical specialties Optimal supportive care can assist in accurate diagnosis and management and ultimately improve outcomes A national strategy toimplement supportive care is needed to acknowledge evolving oncology practice changing disease patterns and the changing patient demographic“ The Royal College of Radiologists Published by Elsevier Ltd All rights reservedKey words Beyond cancer chronic cancer definition living with supportive careStatement of Search Strategies UsedA series of searches were constructed and carried out viaPubMed EMBASE and MEDLINE This generally consisted ofusing phrase searching due to the specificity of the subjectOnce concepts were established the authors used Booleanoperators to combine the concepts together and retrieve themost relevant papers Once a set of results were retrieved theauthors scanned each of the s using and titlefields to identify key papers Fulltext access to papers weresourced via the Christie Library and Knowledge ServiceIntroductionSupportive Care Makes Excellent Cancer Care PossibleMultinational Association of Supportive Care in CancerwwwmasccAdvances in diagnosis surgery radiotherapy and newdrugs have led to improvements in cancer survival PeopleAuthor for correspondence R Berman The Christie NHS FoundationTrust Wilmslow Road Manchester M20 4BX UK Tel ¾447710509402Email address RichardBermanchristienhsuk R Bermannow live nearly six times longer after their cancer diagnosisthan was the case years ago [] Half of people diagnosedwith cancer in England and Wales survive their disease for years or more [] Currently in England around million people are living with a diagnosis of cancer and thisnumber is increasing by over a year The total figure is setrise to over million by []Many more cancer patients are being treated closer todeath with novel less toxic high efficacy anticancer therapeutic agents developing with increasing pace within thelast decade The advent of molecular targeted agents forexample has brought new benefits as well as challenges tomodern cancer therapy potentially blurring the distinctionbetween active and palliative interventions []Yet despite this significant progress a large proportion ofpatients with cancer still experience morbidity and symptoms resulting from the cancer andor its treatment []Increases in cancer incidence [] emergency care hospitalisations [] earlier intensive care unit admissions [] andtreatment costs [] have all added to the global burden ofcancer care The disease is becoming a major economicexpenditure for all developed countries [] In the UK andin the USA cancer care costs are substantial and expected to101016jclon20200702009366555“ The Royal College of Radiologists Published by Elsevier Ltd All rights reservedPlease cite this as Berman R Supportive Care An Indispensable Component of Modern Oncology Clinical Oncology 101016jclon202007020 0cR Berman Clinical Oncology xxx xxxx xxxrise significantly in the future due to growth and aging ofthe population and improvements in survival as well astrends in treatment patterns and costs of care followingcancer diagnosis []cancerandcancerManagingtreatmentrelatedmorbidity is therefore a significant public health andeconomic challenge The coronavirus pandemic has deepened this challenge with many cancer outpatient visitsbeing replaced by telephone consultations and deferral ofsome routine therapy tests and procedures This has placedadditional pressures on an already fragile and vulnerablepopulation [] Patients and carers are experiencing moreuncertainty and anxiety associated with COVID19 A recentstudy found that although patients continue to feel wellsupported by their healthcare teams they have concernsabout the longerterm impact of changes to aspects of theirtreatment Patients and carers are no longer able to accessother support services in the way that they had previouslysuch as hospices and peer support groups []There is a growing body of evidence that timely access tosupportive treatments can lead to improvements in qualityof life and survival as well as benefitting the health economy [15e17] The development of a broad multiprofessionalbasis for the study and expansion of supportive carethrough the Multinational Association of Supportive Care inCancer MASCC has been an important step in fostering thegrowth of an evidence base [] MASCC's success has undoubtedly been underpinned by successful integration ofoncological and nononcological specialties []However variations in the definition of supportive careallocation of resources and a lack of clarity on who shouldlead onprovide services means that a clinical model forsupportive care in cancer does not yet exist [] Most specialties whilst they overlap other specialties are based on acore of knowledge or skill that is specific to that specialty[] Supportive care is currently provided by a patchwork ofdifferent medical specialties and is unique because it traverses the entire spectrum of the disease Figure fromdiagnosis through to survivorship or end of life care Theneed for ˜supportive oncology™ to become a specialty in itsown right is borne out not just by the progress in its development in the UK and abroad but by the unmet supportivecare need [] amplified by the rising incidence of cancerworldwide with many patients living longer with incurableillness because of more effective cancer treatments [] Asignificant next step would be to produce an evidencebasednational strategy for supportive care implemented throughappointment of supportive care lead clinicians within eachUK cancer centre This alongside support from the medicalRoyal Colleges and NHS England would be fundamental indeveloping a sustainable clinical modelPerhaps working as a distinct branch of oncology ˜specialists™ in supportive care medicine should have the skillsand resources to manage a broad range of effects associatedwith longterm cancers and cancer survival This paperexplores areas that are showing promise in this development and identifies key next steps needed to recognisesupportive care as an indispensable component of modernoncologyDefinition of Supportive CareThe Inuit may or may not have words for snow butsupportive care seems to have that number of definitions orconnotations [] Supportive care has been used as aeuphemism for palliative care and ˜early palliative care™[] and research suggests that a change in name frompalliative care to supportive care results in more and earlierreferrals to hospitalbased services [] Palliative care is anintegral component of supportive care but supportive careis much more than palliative care or even ˜early palliativecare™MASCC defines supportive care as ˜the prevention andmanagement of the adverse effects of cancer and its treatment This includes management of physical and psychological symptoms and side effects across the continuum ofthe cancer experience from diagnosis through treatment toposttreatment care Enhancing rehabilitation secondarycancer prevention survivorship and endoflife care areintegral to supportive care™ []Strategy for Implementation of SupportiveCare Within Cancer CareThe potential benefits of supportive care includedecreased morbidity improved quality of life and potentially decreased mortality ie secondary to optimal cancertreatment the potential benefits for healthcare servicesinclude decreased utilisation of healthcare resources andimproved treatment outcomes [] Indeed supportivecare offers patients more than many ˜palliative™ oncologicaltreatments and should be considered an essential not justan optional extraCurrently many cancer centres in the UK have supportivecare services either as a result of NHS England's EnhancedSupportive Care ESC Programme discussed below andFig The supportive care umbrellaPlease cite this as Berman R Supportive Care An Indispensable Component of Modern Oncology Clinical Oncology 101016jclon202007020 0cR Berman Clinical Oncology xxx xxxx xxxrelated Commissioning for Quality and Innovation CQUIN[] or as a result of local initiatives However the format ofthese teams is variable as is the patient cohort ie restrictedto specific cancer diagnoses and the interventions offeredie often restricted to symptom controlThus a national strategy is required to standardise supportive care services in relevant settings This needs to beevidencebased and ensure equity of care for all cancer patients irrespective of their cancer diagnosis or stage Thestrategy needs to address the current situation but alsoacknowledge evolving oncology practice ie new treatmentswith new toxicities changing disease patterns ie cancer as˜chronic disease™ and changing patient characteristicsIt needs to address education and training discussedbelow and be supported by benchmarking of servicesincluding inspections of clinical services incorporatingpatient feedback Investment will be required to standardise supportive care services and research fundingshould be allocated to determine the optimal model of careas well as the effectivenesscost effectiveness of the individual components of the servicesImplications for TrainingSupportive care encompasses the entire cancer journeyand so necessitates the involvement of most clinical specialties and many nonclinical services Figure Indeedmodern supportive care cannot be provided by a singleclinical specialty alone However as with other cancermultidisciplinary teams a dedicated ˜core team™ is neededto manage everyday problems with timely input from an˜extended team™ if the need arises Importantly the coreteam needs specificongoing education and training inprinciples of supportive careIt is also important to recognise that although manysupportive care services may have evolved from palliativecare services palliative care healthcare professionalsgenerally have limited formal training in supportive careand it is often not appropriate to extrapolate dataexperience from patients with advanced cancer to patientsreceiving anticancer treatment or cancer survivors Forexample the management of nausea and vomiting inadvanced cancer [] is very different from the management of chemotherapyinduced nausea and vomiting []The development of specialist supportive care servicesmust be supported by the educationtraining of the wideroncology workforce in the principles of supportive careand the management of common symptomsproblemsIndeed specialist supportive care services will only ever beable to see the ˜tip of the iceberg™ and so will need to focuson more complex problems and ones requiring specialistinterventions Moreover for example it is much moreappropriate for the team that gives the oncological intervention to manage the adverse effects of that oncologicalinterventionThus supportive care needs to be incorporated into thecurricula of all healthcare professionals involved in cancercare including primary care physicians AppropriateFig The extended supportive care teamcontinuing professional development opportunities need tobe developed for these groups Patients and their familiesneed access to appropriate educational resources in order tofacilitate rapidsuccessful treatment of the complications ofthe cancer andor the cancer treatmentEnhanced Supportive Care Programme eNHS EnglandNHS England promoted early development of supportivecare within some cancer centres via the ESC CQUIN programme CQUIN is the framework supporting improvements in the quality of services and the creation of newimproved patterns of care [] ESC CQUIN was developedby The Christie NHS Foundation Trust and was based uponsix key principles for the implementation and delivery ofsupportive care Figure [] The programme developedthrough recognition of what specialist palliative care professionals working alongside other cancer care disciplinescould offer across the whole cancer pathway e and throughrecognition of barriers to achieving earlier involvement[] Palliative care and supportive care are often differentlyanised across locations on the basis of resources andtraditions In some centres the two are anised as oneservice whereas in others they are completely separate[] The ESC programme required rebranding a closercollaboration with oncology and referral within weeks ofdiagnosis of incurable cancerNHS England's Specialised Commissioning ImprovingValue Team worked with commissioners and clinical teamsin ESC development Fourteen cancer centres took part inthe ESC CQUIN over a 3year period 2016e2019 Aninterim evaluation of the scheme took place in OctoberPlease cite this as Berman R Supportive Care An Indispensable Component of Modern Oncology Clinical Oncology 101016jclon202007020 0cR Berman Clinical Oncology xxx xxxx xxx The programme was associated with a variety ofpositive outcomes including timelier referral of patientswith supportive care needs improved symptom controlimproved quality of life reduced 30day mortality fromchemotherapyimproved overall survival and reducedhealthcare costs [] ESC's principles of early referral andintervention may have impacted positively on these outcomes by better supporting patients who decide to proceedwith chemotherapy as well as those who decide not toproceedA limitation of the ESC CQUIN related to variation inservice delivery model across the centres Further robustresearch needs to be undertaken to determine the ˜optimal™approach for delivery of supportive care services withincancer centres and in other settingsDeveloping the Research and Evidence inSupportive CareWhen the American Society for Clinical Oncology ASCOcelebrated its 50th anniversaryit listed the five topachievements in oncology over that period Prominentlylisted was the development of highly effective antiemetictreatment [] What has been the impact of this keyadvancement in cancer supportive care and how did we getthere Does this progress guide us in improving other areasin supportive careThe impact of preventing emesis is broad and largeNausea and vomiting affect all aspects of daily living thequality of life benefits of antiemetic prevention have beendocumented Economically this advance allowed nearly allchemotherapy to be given on an outpatient basis ratherthan requiring hospitalisation This also allows people tohave less disruption and to remain with their families whilepursuing normal activitiesThese improvements are the result of thoughtful andlogical research Principles of this research included thefollowing which can be applied to many supportive caresettings i an understanding of appropriate physiology[] ii establishment of good clinical methodology []and iii evidence that affecting specific neurotransmitterpathways resulted in major clinical benefit [] As aresult of this work 80e90 of patients can be spared emesisin difficult settings as opposed to in the pastAs we enter an era where chemotherapy is progressivelyless used new areas for supportive care emerge Are weprepared to understand in depth unanticipated challengesin supportive care Can we prevent dermatological toxicities with tyrosine kinase inhibitormediated molecularlytargeted approaches through better understanding of themechanisms of these agents and skin physiology Can wepredict who is likely to have autoimmune sideeffects withcheck point inhibitors []Skills in caring for patients with cancer and methods oftreating malignancy continue to improve The advancesmade in preventing chemotherapyinduced nausea andvomiting provide a model that can influence approaches tomany other aspects of supportive care in cancerInterface with Acute Oncology eAmbulatory Supportive CareAdvances in cancer management continue to improvepatient outcomes This has expectedly been associatedwith an increase in emergency presentations with disease or treatmentrelated complications The challengesof emergency oncology presentations have led to an interest in developing optimal care models for meetingpatients' needs [] Cancer patients seeking emergencycare generally have higher admission rateslongerFig NHS England ESC CQUIN programme principles of ESCPlease cite this as Berman R Supportive Care An Indispensable Component of Modern Oncology Clinical Oncology 101016jclon202007020 0cR Berman Clinical Oncology xxx xxxx xxxlengths of stay and higher mortality than noncancerpatients []Ambulatory care is recognised as a key tenet in ensuringthe safety and sustainability of acute care services Thefundamental basis for ambulatory care is that patientspresenting with acute illnesses can be stratified as low riskfor developing complications and therefore do not requiretraditional inpatient care []Individualised management of acute cancer presentations is a key issue for emergency oncology services sothat it can mirror routine cancer care [] There are anincreasing number of acute cancer presentations that can berisk assessed for care in an emergency ambulatory settingThese include lowrisk febrile neutropeniacancerincidental pulmonaryassociated deep vein thrombosisembolism chemotherapyrelated acute kidney injurychemotherapyinduced nausea and vomiting indwellingline infections acute management of pain crises malignanthypercalcaemiaabnormalitiesasymptomatic brain metastases and malignant pleuraleffusion [43e46]and otherelectrolyteAmbulatory models offer the opportunity to integratepalliative care and supportive care with oncology and acuteservices This facilitates improved access for patients toexpertise in cancer care and immediate management of thecomplications of cancer treatment with the goal of preventing downstream complications and future emergencypresentations For example ambulatory enhanced supportive care models have shown utility in the managementof lowrisk febrile neutropenia []Modelling of ambulatory emergency oncology serviceswithin integrated supportive care services is therefore keyin the provision of highquality personalised and sustainable emergency oncology careThe Importance of Supportive Care inExperimental Cancer MedicineExperimentalcancer medicine trialsECMTs arefundamental to the development of novel cancer therapiesThe primary aims of ECMTs are to identify treatmentrelated toxicities and determine the recommended drugdose [] These trials are increasingly complex []intensive with risks of toxicity for patients but there is agrowing recognition that they are a valid therapeutic option []ECMTs have strict eligibility criteria with the need forpatients to have a performance status of or indicatinghigh levels of day to day functioning [] However thesepatients typically have advanced disease multiple previouslines of treatment and therefore a high associated symptom burden [] Hui [] found that patients referredfor ECMTs have a similar symptom burden to those whowere not despite the perception of higher levels of fitness Ahigh symptom burden has also been associated with earlydiscontinuation from trials [] highlighting the potentialrole for supportive care Br 13edart [] suggested thatthis patient group is more likely to accept increased toxicityto facilitate continued access to trial drugs In one studyECMT patients stated that they would still participate in atrial despite the potential risk of serious toxicities and a chance of death []Research suggests that ECMT patients are less inclined toaccept traditional palliative care due to a general andsometimes unrealistic optimism regarding trial participation[] alongside the perception that palliative care is onlyapplicable at the end of life [] However supportive carepractices within the early phase trials setting have the potential to reduce the impact of symptom burden and adverseevents on patients [] potentially increasing trial recruitment and the length of time patients spend on an experimental therapy Evidence in an ongoing study by Ferrell et al[] indicates that additional support can improve thequality of life for this patient group On top of the benefit topatients of access to additional therapies prolonged exposure to trial drugs supports research through increasednumbers of evaluable patients aiding efficient and accurateassessment of novel therapies Thus there is growing evidence for the role of supportive care for ECMT patients withthe need for increased research to assess potential benefitsand identify optimal routes for its deliveryLearning from Other CountriesImplementation of Supportive Care inFranceWith the aim of increasing and improving communityinvestment in supportive care MASCC is promoting severaldifferent approaches to engage countries such as 0f The creation of accreditation for hospitals withdedicated supportive care units 0f Promotion of MASCC and collaboration with localassociations at MASCC meetings 0f Special links with these associations such as jointmembershipsFrance committed to the supportive care approach at theend of the 1990s and as part of its first cancer plan in The French Speaking Association for Supportive Care inCancer AFSOS affiliated to MASCC was created in with the objectives ofaccompaniment 0f Promoting knowledge and execution of supportivecare in oncology 0f Sharing experience with all professionals involved insymptomsthethroughout all phases of the disease 0f Identifying and understanding the impact of thetransferability and interdependency between disciplines facilitating key aspects obstacles interestsand limitations of work 0f Heightening ethical awareness among medical staffand careofAFSOS has set up a research committee with four strategic priority directions healthcare anisation crossPlease cite this as Berman R Supportive Care An Indispensable Component of Modern Oncology Clinical Oncology 101016jclon202007020 0cR Berman Clinical Oncology xxx xxxx xxxdisciplinary meetings and departments supportive careunits dedicated teams management of cancer symptomsand treatments health behaviour and human and socialsciencesIts actions are targeted towards institutions eg TheFrench National Cancer Institute Ministry of Health professionals guidance and symposia on specific topics such asemesis or nutritional disorders as well as patients andtheir specific associations through a patientfacing website a roadshow truck crisscrossing France and an inventoryof supportive care resources AFSOS has developed nationalmeetings devoted to physicians and nurses physiotherapists or other health caregivers Guideline resources with atoolkit app are discussed during a specific 2day event andupdated every years AFSOS is involved in promoting international collaboration with other MASCCaffiliated societies eg Network Italiano Cure di Supporto in Oncologia[NICSO] and the Japanese Association of Supportive Care inCancer [JASCC]This French national mobilisation has led many regionalteams to get involved in cancer safety management projectsfor the benefit of patients and their relatives and can becopied in other countriesof these is poor in the UK [] and there are a number ofreasons why this may be even lower in an oncology setting[] The risk of poor bone health and fracture is increasingly recognised across a number of malignancies forexample a recent large Danish registry study showedincreased risk of fragility fracture in adults with haematological malignancy with the largest risk in the first 2e4years following initiation of treatment [] Given thedevastating nature of fractures there is much supportivecare work to be done to identify and treat at risk patientsand manage fragility fractures effectively across the spectrum of the cancer journeyEndocrinologists have had a traditional role in cancersurvivorship [] For example managing the longtermeffects of brain radiotherapy on the pituitary gland inchildhood brain tumour survivors As the prognosis foradult brain tumour survivors improves similar issues mayarise [] More recently endocrine toxicities such ashypophysitis and insulindeficient diabetes caused byimmunotherapy treatments are also keeping endocrinologists busy [] in collaboration with acute oncology []This will be become an even more complex issue asimmunotherapy moves into the adjuvant arena with expertinput into decision making algorithms crucial []Interface with Other Specialities egEndocrinology and DiabetesDiscussionOptimal supportive care of cancer patients requires inputfrom a range of specialties outside of oncology to assistaccurate diagnosis and management and ultimatelyimprove outcomesUp to of inpatients with cancer have diabetes or areat risk of diabetes from the treatments they receive []The importance of this is increasingly recognised patientswith diabetes and cancer have an increased length of hospital stay [] and mortality [] Although there iscurrently a lack of data demonstrating that improving glycaemic control reduces mortality for cancer patients it iscertainly true that effective and timely management ofhyperglycaemia improves quality of life and reduces inpatient length of stay but this requires specialist input from adiabetes teamSimilarly up to of inpatients with cancer experiencehyponatremia commonly secondary to syndrome of inappropriate antidiuretic hormone secretion although in theera of immunotherapy cortisol deficiency is an importantand increasing cause which can be fatal if missed []Untreated hyponatremia can delay oncology treatmentsand extend the length of hospital stay [] Diagnosis andmanagement of hyponatremia is poorly managed in general and the oncology population are no exception [] Weconsider expert supportive care input into the managementof hyponatremia in oncology patients to be essential inimproving this situationFractures particularly those of the hip and spine aredevastating with up to mortality at year following hipfracture and significant ongoing morbidity Vertebral fractures are highly predictive of further fracture but reportingThe current focus of cancer care is on initial diagnosisand treatment and the last year of life end of life care []However a large proportion of patients with cancer experience debilitating morbidity and complex symptomsresulting from cancer andor its treatment across the entirecancer journey Supportive care has been shown to improvequality of life symptom burden and survival as well asbenefitting the health economy [15e17] Thus supportivecare should be an integral component of modern oncologymanagement and should involve input from a range ofspecialties within and outside of oncology Furthermore itscontinued development perhaps most effectively as a subspecialty of oncology is essential in supporting advances inoncology and the changing demographic of the cancerpopulationConflicts of interestR Berman is a director of Supportive Care UK Ltd Thisis outside the scope of the submitted workReferences[] Macmillan Cancer Support Living after diagnosis mediancancer survival times Available at wwwmacmillanukdocumentsaboutusnewsroomlivingaftercancermediancancersurvivaltimespdf[] Cancer Research UK Cancer survival statistics Available atwwwcancerresearchukhealthprofessionalcancerstatisticssurvivalheadingZeroPlease cite this as Berman R Supportive Care An Indispensable Component of Modern Oncology Clinical Oncology 101016jclon202007020 0cR Berman Clinical Oncology xxx xxxx xxx[] National Cancer Survivorship Initiative NCSI Living withand beyond cancer taking action to improve outcomesAvailableassetspublishingservicegovukgovernmentuploadssystemuploadsattachment_datafile1810549333TSO2900664NCSI_Report_FINALpdfat[] Clarke G Johnston S Corrie P Kuhn I Barclay S Withdrawal ofanticancer therapy in advanced disease a systematic literature review BMC Cancer [] Klastersky J Supportive care do we need a model Curr OpinOncol [] Global Burden of Disease Cancer Collaboration The GlobalBurden of Cancer JAMA Oncol 20151505e527[] National Audit Office Delivering the cancer reform strategyAvailable at wwwnaoukreportdeliveringthecancerreformstrategy[] Mokart D Pastores SM Darmon M Has survival increased incancer patients admitted to the ICU Yes Intens Care Med2014401570e1572[] National Institutes of Health Cancer trends progress report e update National Institutes of Health [] Sullivan R Peppercorn J Sikora K Zalcberg J Meropol NJAmir E Delivering affordable cancer care in highincomecountries Lancet Oncol 201112933e980[] Yabroff Y Lund J Kepka D Mariotto A Economic burden ofcancer in the US estimates projections and future CancerEpidemiol Biomarkers Prev 201120102006e2014[] Aggarwal A Sullivan R Affordability of cancer care in theUnited Kingdom e is it time to introduce user chargesJ Cancer Policy 2014231e39[] Saini K Heras B Castro J Venkitaraman R Poelman MSrinivasan G Effect of the COVID19 pandemic on cancertreatment and research Lancet Haem 202076e432ee435[] Radcliffe E Khan A Wright D Berman R Demain S RestorickBanks S Understanding the importance of selfmanagement support in people living with cancer reportReport on the impact of COVID19 in press[] Monnery D Benson S Griffiths A Cadwallader C HamptonMatthews J Coackley A Multiprofessionaldeliveredenhanced supportive care improves quality of life for patientswith incurable cancer Int J Palliat Nurs 20182410510e514[] Basch E Deal AM Dueck AC Scher HI Kris MG Hudis C et alOverall survival results of a trial assessing patientreportedoutcomes for symptom monitoring during routine cancertreatment JAMA 20173182197e198[] Cooksley T Campbell G AlSayed T LaMola L Berman RA novel approach to improving ambulatory outpatient management of low risk febrile neutropenia an Enhanced Supportive Care ESC clinic Support Care Cancer 2937e2940[] Klastersky J Christel F Editorial Supportive care in cancerpatients a constantly evolving field Curr Opin Oncol 314257e258[] Cooksley T Rice T Emergency oncology development current position and future direction in the USA and UK SupportCare Cancer 2017253e7[] Guly H Preface A history of accident and emergency medicine1948e2004 London Palgrave Macmillan [] Hui D Hannon BL Zimmerman C Bruera E Improving patientand caregiver outcomes in oncology teambased timely andtargeted palliative care CA Cancer J Clin 201868356e376[] Whelan TJ Mohide EA Willan AR Arnold A Tew M Sellick S The supportive care needs of newly diagnosed cancerpatients attending a regional cancer center Cancer 8081518e1524[] Hui D De La Cruz M Mori M Parsons HA Kwon JH TorresVigil I Concepts and definitions for œsupportive careœbest supportive care œpalliative care and œhospice care inthe published literature dictionaries and textbooks SupportCare Cancer 201321659e685[] Boyd K Moine S Murray SA Bowman D Brun N Shouldpalliative care be rebranded B

Thyroid_Cancer

Adjunctive Therapy to Achieve Preoperative Euthyroidism in Graves™ Disease A Case Report Authors™ Contribution Study Design A Data Collection B Statistical Analysis C Data Interpretation D Manuscript Preparation E Literature Search F Funds Collection G ABDEF Noor Abdulghani AlghanimABDEF Shymaa M AlkahtaniAEF Fatimah S AssariAEF Sarah W AlnosaierAEF Reham M BaderAEF ABEF Mariam M HendazAEF Amal AlhefdhiIsra E Elmahi Corresponding Author Conflict of interest Noor Abdulghani Alghanim email nalghanimalfaisaleduNone declared College of Medicine Alfaisal University Riyadh Saudi Arabia Breast and Endocrine Surgery King Faisal Specialist Hospital and Research Center Riyadh Saudi ArabiaPatient Final Diagnosis Symptoms Medication ” Clinical Procedure Specialty Male 37yearoldGraves™ diseaseDifficulty breathing ¢ voice change ¢ weight gainTotal thyroidectomySurgery Objective Background Case Report Conclusions Unusual clinical courseGraves™ disease is an autoimmune disease of the thyroid gland and it is considered the most common cause of hyperthyroidism It is characterized by particular eye manifestations skin changes and pretibial myxedema in addition to the signs and symptoms of hyperthyroidism Graves™ disease can be diagnosed based on clinical presentation and low thyroid stimulating hormone TSH and elevated free T4 FT4 levels Presence of TSH receptor antibody TRAb in the serum confirms the diagnosis of Graves™ disease Imaging studies like radioactive iodine scan will show a high and diffuse uptake Graves™ disease can be managed with three different treatment modalities antithyroid medications radioactive iodine or surgical removal of the thyroid gland Whenever surgery is indicated careful preoperative management to achieve euthyroidism is needed to optimize the surgical outcomeThis is a case of a 37yearold Saudi male known to have Graves™ disease for years who presented to the endocrine surgery clinic with neck swelling difficulty breathing and change in voice After multiple attempts to control his fluctuating thyroid levels the team eventually managed to achieve a euthyroid state in the patient with the addition of saturated solution of potassium iodide SSKI and thus rendering him eligible for urgent surgeryWe report this case to show that SSKI can be used as adjunctive therapy to achieve a preoperative euthyroid state in refractory Graves™ disease MeSH Keywords Graves™ Disease ¢ Hyperthyroidism ¢ Hypothyroidism Fulltext PDF wwwamjcaserepcomindexidArt923342 ” e9233421Indexed in [PMC] [PubMed] [Emerging Sources Citation Index ESCI][Web of Science by Clarivate]This work is licensed under Creative Common AttributionNonCommercialNoDerivatives International CC BYNCND 0cAbdulghani Alghanim N Use of adjunctive therapy to achieve preoperative euthyroidism in Graves™ disease¦ Am J Case Rep e923342Figure Typical eye manifestations of Graves™ disease proptosis and periorbital edemaFigure Ultrasound showing an enlarged and hypervascular left thyroid lobe with no suspicious nodulesof the examination was unremarkable except for right scrotal swelling and delayed deep tendon reflexesThe patient then underwent ultrasound US of the thyroid which showed an enlarged and hypervascular gland compatible with Graves™ disease with no suspicious nodules Figure A computed tomography CT scan was done which revealed homogeneous diffuse swelling of bilateral thyroid lobes with no retrosternal extension along with bilateral proptosis Figures Therefore the patient was admitted to achieve a euthyroid state before proceeding for a total thyroidectomy The patient was managed by a multidisciplinary team with the goal of clearing him for surgery During the patient™s 3week hospital stay the dosage of methimazole was continuously altered because serial thyroid function tests showed a change from hyperthyroid to hypothyroid status His TSH and FT4 levels ranged from mUL to mUL and pmolL to pmolL respectively A few days after administration of saturated solution of potassium iodide SSKI was initiated drops three times daily the patient achieved a euthyroid state with TSH mUL and FT4 pmolL so urgent surgery was performed Intraoperatively the patient™s thyroid gland was found to be enlarged and vascular with each lobe measuring approximately to cm The gland was excised bilaterally along with the pyramidal lobe because it was also enlarged The postoperative BackgroundGraves™ disease is an autoimmune disease affecting the thyroid gland [] It is characterized by presence of autoantibodies that target thyroid stimulating hormone TSH receptors causing stimulation of the thyroid gland [] Patients with Graves™ disease usually present with signs and symptoms of hyperthyroidism that include fatigue heat intolerance sweating weight loss palpitations and tremor along with particular eye manifestations and sometimes skin changes [] It is considered the most common cause of hyperthyroidism accounting for approximately to of cases [] The diagnosis of Graves™ disease can be straightforward in the presence of typical signs and symptoms along with low thyroid stimulating hormone TSH and elevated free T4 FT4 levels [] Measuring TSH receptor antibody TRAb is helpful for confirming the diagnosis as it is present in of patients If the cause of hyperthyroidism remains uncertain a radioactive iodine uptake scan should be considered The scan helps to distinguish Graves™ disease from thyroiditis and other causes of hyperthyroidism In Graves™ disease iodine uptake is increased and diffuse [] Treatments of choice for hyperthyroidism include antithyroid medications radioactive iodine and surgical approaches [] The success rate for antithyroid medications is almost compared to and with radioactive iodine and surgery respectively [“] Whenever surgery is indicated careful preoperative management to achieve euthyroidism is needed to optimize the surgical outcome In most cases a euthyroid state is reached within a few weeks of conventional antithyroid medications however in certain conditions as in drug malabsorption and in cases of predominantly high T3 levels it cannot be easily achieved and adjunctive therapy should be considered []Case ReportA 37yearold Saudi male presented to the endocrine clinic with palpitation sweating and weight loss He was diagnosed with Graves™ disease and treated with methimazole mg orally twice daily When symptoms of hypothyroidism developed the dose was decreased to mg orally twice daily The patient was referred to the endocrine surgery clinic complaining of obstructive symptoms in the form of difficulty breathing and voice changes due to neck swelling weight gain of kg during the last month and easy fatigability along with the typical eye manifestations of proptosis and periorbital edema Figure He was otherwise healthy and the rest of his history was unremarkable On physical examination the patient had a hoarse voice fine tremor in both hands and his skin was warm with diaphoresis There was proptosis lid retraction and diplopia involving both eyes Neck examination showed a diffuse tender swelling with bilateral lumps measuring cm on the right and cm on the left along with positive Pemberton sign The rest e9233422Indexed in [PMC] [PubMed] [Emerging Sources Citation Index ESCI][Web of Science by Clarivate]This work is licensed under Creative Common AttributionNonCommercialNoDerivatives International CC BYNCND 0cAbdulghani Alghanim N Use of adjunctive therapy to achieve preoperative euthyroidism in Graves™ disease¦ Am J Case Rep e923342Figure CT scan showing diffuse enlargement of the thyroid with no retrosternal extension or invasion of surrounding structurespathology report showed diffuse hyperplasia consistent with Graves™ disease with no evidence of malignancy After the surgery the patient was moved to the Intensive Care Unit ICU where he was assessed and found to be stable with no signs and symptoms of thyrotoxicosis or hypocalcemia Three days later the patient was discharged with orders to take calcium carbonate mg orally three times daily for days acetaminophen mg orally as needed for days levothyroxine mcg orally daily for days and calcitriol mcg orally daily for daysWhen the patient presented to the clinic weeks later for followup he was found to be in good health with no active complaints He had lost weight and there were no voice changes His eye manifestations had decreased but not disappeared completely Laboratory results showed a euthyroid state with a normal calcium levelDiscussionFigure CT scan demonstrating that the distance from the anterior margin of the globe to the interzygomatic line exceeds mm indicating significant bilateral proptosisfor antithyroid medications is almost compared to with radioactive iodine therapy [] Surgical approaches are considered the most successful and definitive treatment with total thyroidectomy being the preferred choice [] A review of the literature done in showed that total thyroidectomy is times more successful than radioactive iodine therapy [] Another study concluded that the highest rates of longterm remission reaching up to are achieved with surgery [] Nonetheless there is no clear consensus on the best treatment modality for Graves™ disease and the choice should be individualized Choice of modality depends on several factors including age comorbidities size of the goiter and severity of thyrotoxicosis [] Surgery is recommended in certain conditions for example in patients with compression symptoms due to presence of a large goiter those with low radioactive iodine uptake suspected thyroid cancer moderate to severe Graves™ ophthalmopathy and patients who cannot tolerate antithyroid medications [] Whenever surgery is selected careful preoperative management is needed to optimize the surgical outcome Preparing a patient with antithyroid medications is recommended by the American Thyroid Association ATA to achieve a euthyroid state and thus lower risk of intraoperative complications []Graves™ disease can be managed with three different treatment modalities antithyroid medications radioactive iodine or surgical removal of the thyroid gland [] The success rate In most cases a euthyroid state is achieved within weeks of antithyroid treatment In certain conditions however that is difficult to achieve with conventional therapy and patients e9233423Indexed in [PMC] [PubMed] [Emerging Sources Citation Index ESCI][Web of Science by Clarivate]This work is licensed under Creative Common AttributionNonCommercialNoDerivatives International CC BYNCND 0cAbdulghani Alghanim N Use of adjunctive therapy to achieve preoperative euthyroidism in Graves™ disease¦ Am J Case Rep e923342Moreover the largest case series of patients with severe thyrotoxic Graves™ disease was published in The study involved patients who reached euthyroidism after days of an intensive treatment regimen The authors concluded that patients with severe hyperthyroid Graves™ disease can rapidly achieve preoperative euthyroidism with simultaneous administration of iopanoic acid dexamethasone betablocker and methimazole or propylthiouracil [] Another case of Graves™ disease resistant to antithyroid medications was reported in The patient was promptly managed preoperatively with both iopanoic acid and dexamethasone []Three scientific papers on resistant thyrotoxicosis due to Graves™ disease were published between to In all cases a euthyroid state could not be reached with the usual antithyroid medications and the patients received prednisolone andor lithium which resulted in complete normalization of thyroid function before surgery [“] Furthermore several refractory cases of Graves™ disease unresponsive to usual preoperative management were reported in and The patients were successfully prepared for surgery with use of plasmapheresis []ConclusionsPreoperative management of Graves™ disease can sometimes be challenging There have been many attempts to achieve a euthyroid state with different approaches In the patient described here Graves™ disease was resistant to conventional antithyroid medication for establishment of preoperative euthyroidism Our experience demonstrates that SSKI can be used in a case like ours to not only decrease vascularity of the thyroid gland but also as adjunctive therapy to achieve preoperative euthyroidismshould be prepared for surgery using adjunctive therapy [] Resistance to conventional antithyroid medications is not commonly encountered in clinical practice however there are few reported cases addressing the use of adjunctive therapy to rapidly restore normal thyroid function [] SSKI has been used for many years in management of Graves™ disease [] ATA hyperthyroidism management guidelines recommend preoperative administration of potassium iodide solutions KI for thyroidectomy [] The main rationale of using KI preoperatively is to decrease vascularity and blood loss during the surgery however a few studies suggest that when combined with antithyroid medications it can decrease thyroid hormone levels [“] In a retrospective study showed the effectiveness of adding KI as a rescue preoperative management in uncontrolled Graves™ disease In patients in the study use of KI was safe and effective as preoperative preparation for total thyroidectomy []Cholestyramine was first used to treat Graves™ disease in in Korea [] The patient in that study was a 22yearold female with severe refractory Graves™ disease who was initially managed with a maximal dose of methimazole and propranolol with no improvement She was admitted and treated with methimazole propranolol hydrocortisone and KI The next day cholestyramine was added which resulted in a rapid decline of FT4 Ten days after admission the patient underwent total thyroidectomy [] Several cases of refractory Graves™ disease were reported in the literature between to In all these cases the patients failed to achieve a preoperative euthyroid state with conventional antithyroid medications Within to weeks of administration of cholestyramine as adjunctive therapy they became euthyroid Two studies were published in and to evaluate the effectiveness of adding cholestyramine to the conventional treatment regimen in cases of resistant Graves™ disease The conclusion from these reports is that cholestyramine can be used to safely and rapidly achieve preoperative euthyroidism [“]References Pokhrel B Bhusal K Graves disease In StatPearls Treasure Island FL StatPearls Publishing Barbesino G Tomer Y Clinical review Clinical utility of TSH receptor antibodies J Clin Endocrinol Metab “ DeGroot LJ Graves™ disease and the manifestations of thyrotoxicosis In Feingold KR Anawalt B Boyce A eds Endotext South Dartmouth MA MDTextcom Inc Subekti I Pramono LA Current diagnosis and management of Graves™ disease Acta Med Indones Girgis CM Champion BL Wall JR Current concepts in Graves™ disease Ther Adv Endocrinol Metab “ Wiersinga WM Graves™ disease Can it be cured Endocrinol Metab Seoul “ Wong KK Shulkin BL Gross MD Avram AM Efficacy of radioactive iodine treatment of Graves™ hyperthyroidism using a single calculated I dose Clin Diabetes Endocrinol Piantanida E Preoperative management in patients with Graves™ disease Gland Surg “ Yang Y Hwang S Kim M Refractory Graves™ disease successfully cured by adjunctive cholestyramine and subsequent total thyroidectomy Endocrinol Metab Seoul “ Genovese BM Noureldine SI Gleeson EM What is the best definitive treatment for Graves™ disease A systematic review of the existing literature Ann Surg Oncol “ Bartalena L Diagnosis and management of Graves disease A global overview Nat Rev Endocrinol “ SebastianOchoa A QuesadaCharneco M FernandezGarcia D Dramatic response to cholestyramine in a patient with Graves™ disease resistant to conventional therapy Thyroid “ Calissendorff J Falhammar H Lugol™s solution and other iodide preparations Perspectives and research directions in Graves™ disease Endocrine e9233424Indexed in [PMC] [PubMed] [Emerging Sources Citation Index ESCI][Web of Science by Clarivate]This work is licensed under Creative Common AttributionNonCommercialNoDerivatives International CC BYNCND 0cAbdulghani Alghanim N Use of adjunctive therapy to achieve preoperative euthyroidism in Graves™ disease¦ Am J Case Rep e923342 Naafs MA Lugol™s solution in thyroid surgery A minireview Global Journal of Otolaryngology Muldoon BT Mai VQ Burch HB Management of Graves™ disease An overview and comparison of clinical practice guidelines with actual practice trends Endocrinol Metab Clin North Am “ Burch HB Cooper DS Management of Graves disease A review [published erratum appears in JAMA “] JAMA Calissendorff J Falhammar H Rescue preoperative treatment with Lugol™s solution in uncontrolled Graves™ disease Endocr Connect “ Chae SB Kim ES Lee YI Min BR A case of methimazoleresistant severe Graves™ disease Dramatic response to cholestyramine Int J Thyroidol “ Kadem SG Resistant hyperthyroidism responses dramatically to adjunctive oral cholestyramine Jourbnal of Diabetes and Endorinology “ Mercado M MendozaZubieta V BautistaOsorio R EspinozaDe Los Monteros AL Treatment of hyperthyroidism with a combination of methimazole and cholestyramine J Clin Endocrinol Metab “ Tsai WC Pei D Wang TF The effect of combination therapy with propylthiouracil and cholestyramine in the treatment of Graves™ hyperthyroidism Clin Endocrinol Oxf “ Panzer C Beazley R Braverman L Rapid preoperative preparation for severe hyperthyroid Graves™ disease J Clin Endocrinol Metab “ Pandey CK Raza M Dhiraaj S Rapid preparation of severe uncontrolled thyrotoxicosis due to Graves™ disease with Iopanoic acid “ a case report Can J Anaesth “ Saleem T Sheikh A Masood Q Resistant thyrotoxicosis in a patient with Graves disease A case report J Thyroid Res Nair GC C Babu MJ Menon R Jacob P Preoperative preparation of hyperthyroidism for thyroidectomy “ role of supersaturated iodine and lithium carbonate Indian J Endocrinol Metab “ Jude EB Dale J Kumar S Dodson PM Treatment of thyrotoxicosis resistant to carbimazole with corticosteroids Postgrad Med J “ Candoni A De Marchi F Vescini F Graves™ disease thyrotoxicosis and propylthiouracil related agranulocytosis successfully treated with therapeutic plasma exchange and GCSF followed by total thyroidectomy Mediterr J Hematol Infect Dis Ezer A Caliskan K Parlakgumus A Preoperative therapeutic plasma exchange in patients with thyrotoxicosis J Clin Apher “e9233425Indexed in [PMC] [PubMed] [Emerging Sources Citation Index ESCI][Web of Science by Clarivate]This work is licensed under Creative Common AttributionNonCommercialNoDerivatives International CC BYNCND 0c'

Thyroid_Cancer

"Pancreatic cancer PC is one of the most aggressive cancers and has an extremely poor prognosisworldwide Long noncoding RNA lncRNA has been reported to be a potential prognostic biomarker in theinitiation and prognosis of PC Nevertheless the biological functions and the detailed molecular mechanism ofLINC00514 in PC remain unclearMethods We measured the expression level of LINC00514 in PC tissues and cell lines by quantitative realtime PCRGain and lossoffunction experiments were performed to explore the bioeffects of LINC00514 on PC developmentboth in vitro and in vivo Subcellular fractionation luciferase reporter assay RNA immunoprecipitation assay pulldown assay and western blotting were performed to investigate the oncogenic molecular mechanisms ofLINC00514Results In this study LINC00514 was shown to be upregulated in PC tissues and cell lines Increased LINC00514expression was significantly associated with the clinical progression and prognosis of PC patients In additionsilencing LINC00514 inhibited PC cell proliferation migration and invasion while LINC00514 overexpressionpromoted these processes Moreover LINC00514 knockdown remarkably inhibited PC development and metastasisin vivo Deeper investigations indicated that LINC00514 acted as a sponge for microRNA285p miR285p in PCand that Rap1b was a downstream target of miR285p Furthermore the positive correlation of LINC00514 andRap1b and the negative correlation between miR285p and LINC00514 or Rap1b were revealed Based on therescue assays Rap1b inhibition partially suppressed the oncogenic effect of LINC00514 overexpression on PC cellproliferation migration and invasionConclusions This study is the first to characterize the oncogenic function of the long noncoding RNA LINC00514in pancreatic cancer progression by acting as a competing endogenous RNA ceRNA of miR285p to upregulateRap1b expression Understanding this molecular mechanism might contribute to further discoveries of betterdiagnostic and therapeutic options for pancreatic cancerKeywords LINC00514 Pancreatic cancer Proliferation Invasion miR285p Rap1b Correspondence songzhiwangtongjieducnDepartment of Oncology the First Affiliated Hospital of Nanchang University Yongwaizheng Street Nanchang Jiangxi People™s Republic ofChina The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cHan Journal of Experimental Clinical Cancer Research Page of Table Primers involved in the studyGeneLINC00514Rap1bMiR285pGAPDHForward primerGAGGCAGGAGAATCGCTTGAACCACAGCAATGAGGGATTTATACTGGTGTCGTGGGTCGACGCTCTCTGCTCCTCCTGTTCU6Abbreviation LINC00514 long intergenic nonprotein coding RNA GAPDH glyceraldehyde 3phosphate dehydrogenaseCTCGCTTCGGCAGCACAReverse primerGAGGCAGGAGAATCGCTTGAACCTGACCTTGTTCCTTCCCTACCTCGCTTCGGCAGCACAATCCGTTGACTCCGACCTTCACAACGCTTVACGAATTTGCGTFig LINC00514 was upregulated in PC and predicted a poor prognosis a LINC00514 expression was detected in PC tissue and adjacentnormal tissue by qRTPCR bd Associations between LINC00514 expression and tumor size Lymph node metastasis or clinical stage weredetected by qRTPCR e qRTPCR was applied to confirm the expression level of LINC00514 in PC cell lines and normal pancreatic epithelial cellline f KaplanMeier analysis was used to assess the relation between LINC00514 expression level and overall survival in PC patients p05p01 p001 All experiments were repeated at least for three times and mean ± SD was used to represent the final result PC pancreaticcancer qRTPCR quantitative realtime polymerase chain reaction SD standard deviation 0cHan Journal of Experimental Clinical Cancer Research Page of BackgroundAs an extremely aggressive cancer worldwide pancreatic cancer PC has shown an increasing incidencerate in recent years [] Due to its high level of malignancy PC has become the fourth leading cause ofdeath from malignanttumors with poor prognosisand the 5year survival rate is less than [ ]Early diagnosis of PC has been a considerable challenge due to its complicated pathological process andintricate molecular mechanism While some advancesin imaging and clinical treatment have improved diagnosis and therapy [] the outcome of PC patients remains unsatisfactory Hence a better understandingofthe underlying molecular mechanism is essentialfor seeking a novel therapeutic target for PChavestudiesfunctions HoweverLong noncoding RNA lncRNA is a ribonucleotidechain with a coding length of more than nucleotides[] In the past it was thought that since lncRNAs didnot have the ability to encode proteins they lacked biologicalin recent years scientistshave found that lncRNAs execute their biological effectsin epigenetics [] at the histone modification [] tranlevels [] Accumuscriptional and posttranscriptionallatedelucidatedtheextraordinarysignificance of lncRNAs in the progression of a widerange of diseases such as cardiovascular diseases []diabetes [] neurodegenerative diseases [] and human cancers For instancelncRNA SNHG1 whichcan be positively regulated by miR21 activates theAKT pathway to promote sorafenib resistance in hepatocellular carcinoma cells [] lncRNA EPB41L4AAS1 suppresses the Warburg effect and plays a significant role in metabolic reprogramming of cancer[]intestinalstem cells and promote tumorigenesis of colorectalcancer []lncGata6 could maintain stemness ofLINC00514 is a newly identified lncRNA and very fewreports about it are found in the literature Research byLi [] proved that LINC00514 could be an inhibitor of malignant behaviors of papillary thyroid cancer Inaddition another study has also shown a relationship between LINC00514 and neuroendocrine prostate cancer[] In our study we explored the function and mechanism of LINC00514 in PC We discovered thatLINC00514 expression was increased in PC tissue andPC celllines and that the upregulated expression ofLINC00514 was associated with PC cell proliferationmigration and invasion in vitro and tumor growth andmetastasis in vivo Mechanistically LINC00514 accelerated pancreatic cancer progression via the miR285pRap1b axis AllthatLINC00514 might act as a potential prognostic biomarker of PC occurrence and provide a novel target forPC therapythe evidence above suggestsMethodsClinical samplesPC tissue and adjacent normal tissue were collectedfrom the First Affiliated Hospital of Nanchang University with the informed content of the enrolled patientsin this research Patients received neither chemotherapynor radiotherapy before surgery Our study was approved by the Human Research Ethics Committee ofNanchang UniversityQuantitative realtime PCRRNA was extracted by TRIzol reagent Invitrogen fromtissue samples and cells Extracted RNA was later reverse transcribed into complementary DNA cDNA byPrimeScript RT Reagent Takara Japan A SYBR GreenKit Takara Japan was utilized to perform RTPCRGAPDH and actin were used as internal controlsGene expression levels were calculated by the ˆ’ΔΔCtmethod The primer sequences are shown in Table Cell lines and cell cultureThe normal pancreatic epithelial cell line HPDE andPC celllines BxPC3 SW1990 PANC1 AsPC1Capan2 and MIAPaCa2 were purchased from ATCCCells were cultured in Dulbecco™s modified Eagle™sTable Correlation between LINC00514 expression level andclinical featuresCharacteristicsN2P valueAllAge years ‰¥ GenderMaleFemaleTumor size cm ‰¥ DifferentiationPoorModerateWellLymph node metastasisAbsentPresentClinical stage AJCCIIIIIIIVLINC00514 expressionHighLowAbbreviation LINC00514 long intergenic nonprotein coding RNA p was considered statistically significant 0cHan Journal of Experimental Clinical Cancer Research Page of Fig See legend on next page 0cHan Journal of Experimental Clinical Cancer Research Page of See figure on previous pageFig LINC00514 promoted PC cell proliferation migration and invasion ab Transfection efficiency of LINC00514 overexpression plasmids andshRNA in BxPC3 and SW1990 cells were evaluated by qRTPCR cd CCK8 assay was performed to detect cell BxPC3 SW1990 proliferationability with LINC00514 overexpression and LINC00514 silencing e Colony formation assay was carried out to further detect cell proliferationcapacity fg Transwell migration and invasion assay were carried out to detect cell migration and invasion under LINC00514 overexpression andknockdown h Western blot was conducted to evaluate the impact of LINC00514 on EMT progression p05 p01 p001 All experimentswere repeated at least for three times and mean±SD was used to represent the final result PC pancreatic cancer qRTPCR quantitative realtimepolymerase chain reaction SD standard deviation CCK8 cell counting kit8 EMT epithelialmesenchymal transitionmedium DMEM containing fetal bovine serumFBS at °C with CO2 in humidified airCell transfectionLINC00514 overexpression plasmid and shRNAs againstLINC00514 and Rap1b were purchased from GenePharma Shanghai China with scramble plasmid andshRNA used as negative controls MiR285p mimics andmiR285p inhibitors were acquired from Gene Pharma aswell All of the above reagents were transfected into cellsvia TF3000 Transfection Reagent Invitrogen accordingto the manufacturer™s recommendationsColony formation assayCells × cells per well were seeded in 6well platesand then incubated for days After being washed withPBS three times colonies were stained with hematoxylinand countedViability assayTo evaluate cell viability a CCK8 assay was carried outCells × cells per well were plated in 96well platesfor h h h and h The cell growth rate was analyzed by Cell Counting Kit8 Solarbio China reagentaccording to the manufacturer™s instructions The opticaldensity value was measured by a microplate reader at nmMigration and invasion assaysA transwell chamber Corning Tewksbury MA wasused to detect cell migration and invasion capacitiesCells × were seeded on the upper chamber covered with Matrigel Corning Tewksbury MA whileDMEM with FBS was placed on the lower chamberAfter h of transfection cells that passed from theupper chamber onto the lower chamber were fixed withmethanol stained with crystal violet and imaged under alight microscopeIn vivo analysisFiveweekold female nude mice were purchased fromthe National Laboratory Animal Center Beijing Chinaand maintained under specific pathogenfree conditionsSubsequently the mice were randomly separated intotwoLINC00514groups Cells × oftheoverexpression group and NC group were subcutaneously injected into the right axillary of nude miceTumor volume was measured every days and weightwas measured at the end of the experimentTo further evaluate the effects of LINC00514 we carried out pulmonary metastasis analysis PC cells × were injected into nude mice via the caudal vein After days the mice were euthanized The lungs of micewere removed to observe tumor metastasis All experiments were approved by the Animal Research EthicsCommittee of Nanchang UniversitySubcellular fractionation assayThe PARIS Kit Life Technologies was used to isolatenuclear and cytoplasmic RNAs according to the manufacturer™s protocol Reverse transcription of extractedRNAs and RTPCR were conducted as described beforeLuciferase reporter assayThe online software StarBase30 httpstarbasesysueducnwas used to predict the binding sites of LINC00514to miRNA285p Wildtype LINC00514 and mutantLINC00514 of the putative binding sites were clonedinto a luciferase vector Promega and cotransfected withmiR285p mimics into PC cells via LF3000 transfectionreagent After h cells were harvested for luciferase activity analysisPulldown assayWtmiR285p and NCmiRNA were labeled with biotinand transfected into BxPC3 and SW1990 cells The celllysates were incubated with streptavidin magnetic beads at °C for h After that the beads were rinsed with precooled lysis buffer and salt buffer The pulldown RNAswere extracted to detect LINC00514 levelsRNA immunoprecipitation assayThe Magna RIP RNABinding Protein ImmunoprecipitationKit Millipore MA was used to conduct the RIP assay according to the manufacturer™s protocol The cells were lysedand incubated with Ago2 and IgG Then cell lysates weremixed with antiAgo2 and antiIgG in RIP buffer MilliporePrecipitated RNAs were collected for RTPCR analysis 0cHan Journal of Experimental Clinical Cancer Research Page of Fig See legend on next page 0cHan Journal of Experimental Clinical Cancer Research Page of See figure on previous pageFig LINC00514 promoted tumor growth and pulmonary metastasis in vivo ab The images of subcutaneous tumors were obtained on Day cf The tumor volumes and weights of shLINC00514 group compared with NC group and LINC00514 overexpression group compared withempty group were quantified Tumor volumes were analyzed by ANOVA gh qRTPCR was used to assess the transfection efficiency ij Theimage of pulmonary metastasis was photographed at the endpoint kl Pulmonary metastasis of LINC00514 silencing and LINC00514overexpression compared with their control groups were evaluated p05 p01 p001 The mean±SD was used to represent the finalresults of experiments repeated at least three times PC pancreatic cancer qRTPCR quantitative realtime polymerase chain reaction SDstandard deviation NC negative control ANOVA analysis of variancePVDF membranesWestern blotProtein was extracted from cells and transferred topolyvinylidene difluorideafter sodium dodecyl sulfate polyacrylamide gel electrophoresis After that membranes were blocked with nonfat milk and incubated overnight at °C withprimary antibodies After rinsing the membranes threetimes with PBS a secondary antibody labeled withhorseradish peroxidase was used to incubate membranesroom temperature Antibodiesagainst ECadherin NCadherin and Vimentin wereall purchased from CST company and actin andRap1b antibodies were purchased from Abcam Thedilution ratio was determined accordingto theinstructionsfor h atStatistics analysisAll data are presented as the mean ± standard deviationAll experiments were repeated at least three times Student™s t test ANOVA Spearman™s rank correlation testand χ2 test were used for statistical analysis A value ofp was considered statistically significantResultsLINC00514 was upregulated in PC and predicted a poorprognosisFirst we analyzed the LINC00514 profile in PC Wefound that LINC00514 was remarkably increased in PCtissues compared with the corresponding normal tissuesFig 1a The upregulated expression of LINC00514 wassignificantly associated with the LINC00514 level andtumor sizelymph node metastasis and clinical stageFig 1bd while no significant correlation was foundbetween LINC00514 expression and age gender ortumor differentiation Table Additionally LINC00514expression was increased in PC cell lines compared withthe normal pancreatic epithelial cell line Fig 1e Furthermore Kaplan“Meier survival curves revealed thathigh LINC00514 expression was related to a lower overallthe lowLINC00514 level group Fig 1f Overall LINC00514was increased in PC and might be associated with clinical progression and a poor prognosis of PC patientsrate compared with that ofsurvivalcellandpromotedLINC00514 promoted cell proliferation migration andinvasionTo investigate whether LINC00514 is involved in cellproliferation migration and invasion we carried outgain and lossoffunction assays The LINC00514 overexpression plasmid and LINC00514 shRNA were stablytransfected into BxPC3 and SW1990 cells with ascramble plasmid and shRNA used as negative controlsFig 2ab According to the results of CCK8 and colony formation assays LINC00514 overexpression significantlySW1990proliferation capacity while suppression of LINC00514remarkably inhibited these processes Fig 2ce Moreovertranswell assays were utilized to prove thatLINC00514 increased cell migration and invasion capabilities Fig 2fg For further confirmation westernblotting was performed to measure the expression ofEMT markers in both BxPC3 and SW1990 cells As expected Ecadherin was observed to be strikingly downregulated by LINC00514 overexpression whereas Ncadherin and Vimentin were obviously upregulated andthe shLINC00514 group showed the opposite resultsFig 2h In summary LINC00514 promoted PC cellproliferative migratory and invasive capacitiesBxPC3LINC00514 knockdown inhibited tumor growth andpulmonary metastasis in vivoTo further identify the bioeffects of LINC00514 ontumor growth we constructed a subcutaneous xenografttumor model BxPC3 cells transfected with LINC00514shRNA compared with NC shRNA or transfected withthe LINC00514 overexpression plasmid and comparedwith the empty plasmid were subcutaneously injectedinto nude mice The tumors were measured every daysafter injection After euthanizing the mice we obtainedimages of the tumors Fig 3ab Compared with thoseof the NC group the volume and weight of tumors inthe LINC00514 shRNA group were significantly reducedwhileresults were observed in theLINC00514 overexpression group Fig 3cf QRTPCRwas used to assess the transfection efficiency Fig 3ghThen we further investigated the role of LINC00514 inPC metastasis in vivo Nude mice were injected withBxPC3 cells transfected with LINC00514 shRNA compared with NCLINC00514the oppositeshRNA orthe 0cHan Journal of Experimental Clinical Cancer Research Page of Fig See legend on next page 0cHan Journal of Experimental Clinical Cancer Research Page of See figure on previous pageFig LINC00514 was a sponge for miR285p ab Subcellular fractionation assay was used to determine the subcellular localization ofLINC00514 a BxPC3 cells b SW1990 cells C Sequence of WTLINC00514 MutLINC00514 and miR285p were conducted dg Luciferasereporter assay and RIP assay was performed to demonstrate that miR285p was a downstream target of LINC00514 h Pulldown assay wasconducted to detect the reaction between miR285p and WTLINC00514 or MutLINC00514 ij Relative miR185p expression level in BxPC3and SW1990 were determined by qRTPCR K The expression of miR285p in PC tissue and normal tissue were detected by qRTPCR LSpearman™s rank correlation test was utilized to analyze the correlation between the levels of LINC00514 and miR285p MN The miR285pexpression levels under LINC00514 silencing and LINC00514 overexpression were evaluated in vivo op CCK8 assay was performed to detectproliferation of cells transfected with LINC00514 shRNA and cells cotransfected with LINC00514 and miR285p inhibitor qr Transwell migrationand invasion assay were carried out to detect cell migration and invasion abilities p05 p01 p001 All experiments were repeated atleast for three times and mean±SD was used to represent the final result PC pancreatic cancer qRTPCR quantitative realtime polymerase chainreaction RIP RNA immunoprecipitation SD standard deviation WTlINC00514 wild type LINC00514 MutLINC00514 mutant LINC00514 CCK8cell counting kitoverexpression plasmid compared with the empty plasmid into the tail vein Images of pulmonary metastasiswere acquired at the endpoint Fig 3ij There was anobviously lower incidence of pulmonary metastasis and asmaller number of metastatic tumors per lung in the shLINC00514 group compared with the NC groupwhereas the LINC00514 overexpression group showedthe opposite results Fig 3klin the celldetected the expression level of miR285p in the tumorswe collected before and the results showed that miR285p expression was higherlines withLINC00514 knockdown while miR285p was lower inthe cells with LINC00514 overexpression Fig 4mnLINC00514 promoted cell proliferation migration andinvasion at least partially by sponging miR285p Fig4or In summary LINC00514 accelerates PC progression by sponging miR285pLINC00514 acted as a sponge for miR285pTo explore the underlying molecular mechanism of theoncogenic effects of LINC00514 on PC we determinedthe subcellular localization of LINC00514 The resultsshowed that LINC00514 was mostly distributed in thecytoplasm Fig 4ab which suggested that LINC00514might exert its biological function by sponging miRNAStarBase30 was utilized to identify a candidate microRNA miR285p and predict the potential downstreamtargets of LINC00514 Fig 4c The luciferase reporterassay results confirmed that the luciferase activity ofWTLINC00514 was clearly decreased by miR285pmimics while the luciferase activity of MutLINC00514did not change significantly Fig 4de In addition theRIP assay further revealed that LINC00514 and miR285p were enriched in beads conjugated to Ago2 comparedwith the IgG group Fig 4fg Furthermore overexpression of WTLINC00514 but not MutLINC00514 decreased miR285p expression in BxPC3 and SW1990cells Fig 4h Additionally overexpressing LINC00514dramatically decreased miR285p levels in both BxPC3and SW1990 cells while silencing LINC00514 increasedmiR285p levels with NC shRNA used as an internalreference Fig 4ij Then we further detected miR285p expression in tumor tissue The results revealed alower level of miR285p in PC tissue than in normal tissue and a negative correlation between LINC00514 expression and miR285p levels Fig 4kl To obtainmore evidence in vivo experiments were performed WeRap1b was a downstream target of miR285p in PCThe posttranscriptional function of miRNAs is usually toinhibit protein synthesis by base pairing with the ²untranslated region [] Next to ascertain the detailed regulatory mechanism of LINC00514 in PC we searchedStarBase30 and observed that Rap1b was predicted to bea downstream target of miR285p Fig 5a MutRap1bor WTRap1b and miR285p or NCmiRNA were transfected into BxPC3 and SW1990 cells A luciferase reporter assay and RIP assay were used to confirm thehypothesis that Rap1b is a direct target of miR285p Fig5be Then we found that Rap1b expression was downregulated by LINC00514 silencing while cotransfectingmiR285p and shLINC00514 inhibited the effect ofLINC00514 knockdown on Rap1b at both the transcriptional and translational levels Fig 5fgThen we detected Rap1b levels in tumor tissue Rap1bwas obviously increased in PC tissue compared with adjacent normal tissue and there was a positive relationshipbetween Rap1b expression and LINC00514 levels while anegative correlation was observed between Rap1b expression and miR285p levels Fig 5hj In addition we alsodetected the expression of Rap1b in vivo and the transfection efficiency was examined previously As expected theexpression of Rap1b was clearly decreased in BxPC3 cellsby LINC00514 knockdown while increased expressionwas observed in LINC00514overexpressing cells Fig 5kl Thus far we have proven that Rap1b is a direct target 0cHan Journal of Experimental Clinical Cancer Research Page of Fig See legend on next page 0cHan Journal of Experimental Clinical Cancer Research Page of See figure on previous pageFig Rap1b was a downstream target of miR285p in PC a The sequence of WTRap1b MutRab1b and miR285p were conducted efLuciferase reporter assay and RIP assay were performed to determine the association between miR285p and Rap1b fg QRTPCR and westernblot were used to detect Rap1b expression in cells of LINC00514 silencing and cells of cotransferring miR285p and LINC00514 shRNA attranscription and translation level h Relative Rap1b expression in tumor tissue and normal tissue were detected by qRTPCR ig Thecorrelation between Rap1b and LINC00514 as well as the correlation between Rap1b and miR285p were analyzed by Spearman™s rankcorrelation test kl The Rab1b expression levels under LINC00514 silencing and LINC00514 overexpression were evaluated in vivo mn CCK8assay was performed to detect proliferation of cells transfected with miR285p inhibitor and cells cotransfected with miR285p inhibitor andRap1b shRNA OP Transwell migration and invasion assay were carried out to detect cell migration and invasion abilities p05 p01p001 All experiments were repeated at least for three times and mean±SD was used to represent the final result PC pancreatic cancer qRTPCR quantitative realtime polymerase chain reaction RIP RNA immunoprecipitation SD standard deviationof miR285p Thereafter functional experiments werecarried out to investigate the bioeffects of Rap1b The resultsdemonstrated that Rap1b silencing remarkably suppressedthe promoting effects of the miR285p inhibitor on cell proliferation migration and invasion capacities Fig 5mppatients which indicated that LINC00514 might be involved in PC progression In addition it was determinedthat LINC00514 facilitated PC cell proliferation migration and invasion in vitro and tumor growth and metastasis in vivo The underlying molecules however havenot yet been revealedRap1b inhibition inhibited the tumorigenesis effects ofLINC00514Finally we explored the role of LINC00514 mediatedby Rap1b in promoting tumor growth We knockeddown Rap1b in BxPC3 and SW1990 cells to determine whether Rap1b inhibition can reverse the oncogenic effects of LINC00514 Based on the rescueassays Rap1b inhibition partially inhibited the effectof LINC00514 overexpression on cell proliferationmigration and invasion Fig 6adIn conclusionthesethatLINC00514 acted as a key tumor promotor of PC bycompetitively binding to miR285p and then upregulating the expression of Rap1bdemonstratedcollectivelyresultsthrough the miR1883pBRD4 axisDiscussionIn recent years PC has received increasing attention dueto its high incidence and extremely poor prognosis []Accumulated studies have shown that lncRNAs play animportant role in the initiation and development of cancers including PC [] For instance LINC00346 accelerated PC progression and gemcitabineresistancepartially[]lncRNA GLSAS mediated the feedback loop of Mycand GLS and provided a potential therapeutic strategyfor metabolic reprogramming in PC [] AFAP1AS1was shown to exert inhibitory effects on the stemness ofPC cells and ultimately PC tumorigenicity in vivo via themiR384ACVR1 axis [] LINC00514 has been previously reported in papillary thyroid cancer [] and neuroendocrine prostate cancer [] butthere are noreports in PC Our study revealed that LINC00514 expression was markedly elevated in PC tissues and PC celllines and that increased expression of LINC00514 wasassociated with the progression and prognosis of PCIn recent years increasing evidence has proven the hypothesis that lncRNAs exert their biological impact by acting as competitive endogenous RNAs ceRNAs to affectthe development of cancers [] There has been considerable progress in the study of ceRNAs in PC For examplelncRNAPVT1 promotes PC cell proliferation and migration by sponging miR448 [] cucurbitacin B inhibits PCcell proliferation both in vitro and in vivo throughlncRNAAFAP1AS1 binding with miR146b5p [] andPXNAS1 acts as a ceRNA of miR3064 which upregulates PIP4K2B expression and suppresses the progressionof pancreatic cancer [] In our research subcellular fractionation assays indicated that LINC00514 was mostly located in the cytoplasm which provided a basis forLINC00514 to act as a ceRNA in the initiation and progression of PC Then the online software StarBase30 wasutilized to predict the possible downstream target miR285p for LINC00514 Luciferase reporter assay RIPassay and pulldown assay were used to confirm the interaction between LINC00514 and miR285p Overexpression of LINC00514suppressed miR285p whileLINC00514 silencing upregulated miR285p expressionFurther investigation was carried out to demonstrate themigration and invasion promoting effect of miR285p inthe initiation and development of PC which suggested atumorpromotingeffectthat wasdependent on miR285pLINC00514ofAccording to the ceRNA hypothesis mRNA expression is upregulated due to lncRNA competitively bindingto miRNA Rap1b was first reported in the study of Chajut [] and was found to be related to various cancers such as thyroid cancer [] breast cancer []gastric cancer [] and colorectal cancer [] Howeverthere is only a limited number of reports about Rap1b inPC [] In our current study Rap1b was predicted to be 0cHan Journal of Experimental Clinical Cancer Research Page of Fig Rap1b inhibition restrained the tumorigenesis effects of LINC00514 ab CCK8 assay was performed to detect proliferation of cellstransfected with LINC00514 overexpression plasmids and cells cotransfected with LINC00514 overexpression plasmids and Rap1b shRNA cdTranswell migration and invasion assay were carried out to detect cell migration and invasion abilities p05 p01 p001 All experimentswere repeated at least for three times and mean±SD was used to represent the final result PC pancreatic cancer qRTPCR quantitative realtimepolymerase chain reaction SD standard deviation CCK8 cell counting kit8a direct target of miR285p by StarBase30 Luciferase reporter assay and RIP assay confirmed thedirect binding of Rap1b with miR285p Rap1bacting as a cancerpromoting gene had a positivecorrelation with LINC00514 while there was anegative relationship between Rap1b and miR285p Moreover silencing Rap1b partially abolishedthe tumorigenic effects of LINC00514 based on therescue assayIn conclusion ourstudy provides evidence thatLINC00514 promotes PC development by spongingmiR285p and increasing Rap1b expression Thishighlights the LINC00514miR285pRap1b axis as anovel diagnostic and therapeutic strategy for PCpatientsresults highlighted the significantConclusionsOurtheLINC00514miR285pRap1b axis in PC progressionsuggesting that LINC00514 may serve as a potential biomarker and therapeutic target in PCrole ofAbbreviationsPC Pancreatic cancer LncRNA Long noncoding RNA LINC00514 Longnoncoding RNA00514 MiR285p MicroRNA285p CeRNA Competingendogenous RNA EMT Epithelialmesenchymal transition QRTPCR Quantitative realtime PCR RIP RNA immunoprecipitation 0cHan Journal of Experimental Clinical Cancer Research Page of AcknowledgmentsNot applicableAuthors™ contributionsQH and ZWS designed the study QH JHL and JPX collated the data carriedout data analyses and produced the initial draft of the manuscript QH andZWS contributed to drafting the manuscript All authors have read andapproved the final submitted manuscriptFundingThis study was funded by Jiangxi Provincial Education Fund Project youth Science Foundation of Jiangxi Province20202BAB216027Availability of data and materialsAll the data and materials supporting the conclusions were included in themain paperEthics approval and consent to participateThe study was conducted in accordance with the Declaration of Helsinkiprinciples It was approved by the Ethics Committee of the First AffiliatedHospital of Nanchang UniversityConsent for publicationNot applicableCompeting interestsThe authors declare no competing interestsReceived May Accepted July ReferencesPei X Song F Wang Z Emerging incidence trends and application ofcurative treatments of pancreatic cancer in the USA Medicine e17175Ansari D Tingstedt B Andersson B Holmquist F Sturesson C Williamsson CSasor A B D Bauden M Andersson R Pancreatic cancer yesterdaytoday and tomorrow Future Oncol “Shin SJ Park H Sung YN Yoo C Hwang DW Park JH Kim KP Lee SS RyooBY Seo DW Prognosis of pancreatic Cancer patients with synchronousor Metachronous malignancies from other ans is better than those withpancreatic Cancer only Cancer Res Treat “Halbrook CJ Lyssiotis CA Employing metabolism to improve the diagnosisand treatment of pancreatic Cancer Cancer Cell “Beermann J Piccoli MT Viereck J Thum T Noncoding RNAs indevelopment and disease background mechanisms and therapeuticapproaches Physiol Rev “ Wei

Thyroid_Cancer

Activation by NaturalPhytochemicals An OverviewConcetta Iside Marika Scafuro Angela Nebbioso  and Lucia Altucci Department of Precision Medicine University of Campania œLuigi Vanvitelli Naples ItalySirtuins are class III histone deacetylases whose enzymatic activity is dependent on NADas a cofactor Sirtuins are reported to modulate numerous activities by controlling geneexpression DNA repair metabolism oxidative stress response mitochondrial functionand biogenesis Deregulation of their expression andor action may lead to tissuespecificdegenerative events involved in the development of several human pathologies includingcancer neurodegeneration and cardiovascular disease The most studied member of thisclass of enzymes is sirtuin SIRT1 whose expression is associated with increasinginsulin sensitivity SIRT1 has been implicated in both tumorigenic and anticancerprocesses and is reported to regulate essential metabolic pathways suggesting thatits activation might be beneficial against disorders of the metabolism Via regulation of p53deacetylation and modulation of autophagy SIRT1 is implicated in cellular response tocaloric restriction and lifespan extension In recent years scientific interest focusing on theidentification of SIRT1 modulators has led to the discovery of novel small moleculestargeting SIRT1 activity This review will examine compounds of natural origin recentlyfound to upregulate SIRT1 activity such as polyphenolic products in fruits vegetablesand plants including resveratrol fisetin quercetin and curcumin We will also discuss thepotential therapeutic effects of these natural compounds in the prevention and treatmentof human disorders with particular emphasis on their metabolic impactKeywords sirtuin natural compounds oxidative stress human disorders polyphenolsEdited byCecilia BattistelliSapienza University of Rome ItalyReviewed byNarasimham L ParinandiThe Ohio State UniversityUnited StatesCarmen JeronimoPortuguese Oncology InstitutePortugalCorrespondenceAngela NebbiosoangelanebbiosounicampaniaitLucia Altucciluciaaltucciunicampaniait These authors share last authorshipINTRODUCTIONSpecialty sectionThis was submitted toTranslational Pharmacologya section of the journalFrontiers in PharmacologyReceived April Accepted July Published August CitationIside C Scafuro M Nebbioso A andAltucci L SIRT1 Activation byNatural Phytochemicals An OverviewFront Pharmacol 103389fphar202001225Epigenetic modifications are associated with genome stability gene transcription and metabolicregulation Acetylation is one of the most characterized histone modifications Histoneacetyltransferase HAT and histone deacetylase HDAC enzymes control the levels of histoneacetylation modulating gene expression Cavalli and Heard Sirtuins SIRT “ are enzymes classified as class III HDACs They exhibit different subcellularlocalizations SIRT1 SIRT6 and SIRT7 are nuclear although SIRT1 isoforms were also identified inAbbreviations SIRT1 Sirtuin HATs Histone acetyl transferases HDACs Histone deacetylases ROS Reactive oxygenspecies PPAR Receptor peroxisome proliferatoractivated receptor NRF Nuclear respiratory factor TFAM Mitochondrialtranscription factor A SOD Superoxide dismutase TNFa Tumor necrosis factor a IAP Apoptosis protein inhibitor Bcl2Bcell lymphoma2 family MnSOD Manganese superoxide dismutase RSV Resveratrol Que Quercetin oxLDL OxidizedLDL BBR Berberine Cur Curcumin COX Cytochrome c oxidase T2D Type II diabetes NAFLD Nonalcoholic fatty liverdisease CRM Caloric restriction mimeticFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 Activatorsthe cytoplasm SIRT2 is mainly cytosolic SIRT3 SIRT4 andSIRT5 are mitochondrial and can shuttle to the nucleus Changand Guarente The enzymatic activity of SIRTs is dependent on NAD as acofactor and plays an important role in controlling geneexpression DNA repair metabolism oxidative stress responsemitochondrial function and biogenesis Deregulation of theiractivity may lead to tissuespecific degenerative events thatunderlie several human pathologiesincluding cancerdiabetes and cardiovascular diseases Haigis and Sinclair O™Callaghan and Vassilopoulos Waldman The most studied member of this enzymatic class isSIRT1 SIRT1 regulates metabolic pathways cell survivalcellular senescence and ‚ammation and acts in thepathogenesis of chronic conditions such as diabetes as well aspulmonary neurodegenerative and cardiovascular diseasesIndeed SIRT1 has been reported to play a key role intumorigenesis as an oncogene or tumor suppressor dependingon the context specificity BiasonLauber It is able tocontrol these processes via deacetylation of lysine groups ofhistone and nonhistone proteins including known transcriptionfactors FOXO MyoD p53 PGC1a Kupis Chronic ‚ammation caused by oxidative damage increasesthe risk of many chronic disordersincluding heartcardiovascular and neurodegenerative diseases obesity insulinresistance and type diabetes T2D Geto Oxidative stress plays a key role in the pathogenesis of theseconditions The overproduction of reactive oxygen speciesROS including free radicals and reactive nitrogen speciesRNS can lead to damage of cellular components such aslipids proteins and DNA The imbalance between oxidantsand antioxidants can result in cellular dysfunction apoptosisand necrosis Liguori SIRT1 guards against oxidative stress by activating genetranscription of PGC1a via deacetylation and by regulatingtranscription of factors such as the nuclear receptor peroxisomeproliferatoractivated receptor PPAR nuclear respiratory factorNRF and mitochondrial transcription factor A TFAMinvolved in modulation of biogenesis and mitochondrialfunction Ren and metabolism of glucose and lipidsRodgers SIRT1 is also able to regulate the expressionof superoxide dismutase SOD and glutathione peroxidase Sun In addition since mitochondrial dysfunction leads tothe activation of apoptosis SIRT1 can directly regulate theapoptotic process by modulating acetylation of PGC1a Zhang SIRT1 also regulates ‚ammatory responseKauppinen By modulating the acetylation level ofNFkB p65 SIRT1 is able to control transcription of genes such asIL interleukin1 tumor necrosis factor a TNFa IL8 IL6and other ‚ammatory factors Rodgers Ren Yeung Through NFkB SIRT1 also regulatesthe expression of genes such as inhibitor of apoptosis proteinIAP and Bcell lymphoma2 Bcl2 and tumor necrosis factorreceptor TNFR Ren SIRT1 protects against oxidative stress via regulation ofFOXO protein acetylation which is involved in antioxidantprocesses apoptosis and cell proliferation Wong andWoodcock By activating FOXOMsSOD pathwaySIRT1 increases the expression of manganese superoxidedismutase MnSOD and catalase counteracting oxidativestress and promoting damage repair Gu SIRT1also increases the expression of MnSOD by deacetylating p53thus enhancing cellular antioxidant capacity Brunet Zhang Ren Over the past few years the evergrowing awareness that goodhealth goes hand in hand with a healthy and balanced diet hasencouraged people to eat more fruit and vegetables and to takesupplements to make up for any deficiency D™Angelo Bioactive compounds in the diet can act as antioxidantand anti‚ammatory agents thereby reducing the negativeeffects of oxidative stress and the incidence of chronicdiseases such as obesity diabetes and cardiovascular disordersWang Several moleculesincluding naturalphytochemical compounds can modulate SIRT1 activityMiceli Numerous studies have provided evidenceof the protective effects of natural polyphenolic substances suchas resveratrol quercetin curcumin and fisetin and ofnatural nonpolyphenolic substances such as berberineMcCubrey Natural polyphenols are the largestgroup of phytonutrients and are considered potential agents forthe prevention and treatment of stressrelated oxidative diseasesThey are found in many plants and foods such as fruitsvegetables tea cereals and wine and longterm intake isassociated with health benefits Mediterranean diets are in factlinked to a reduced risk of chronic diseases due to theconsumption of olive oil and red wine which contain highamounts of polyphenols Romagnolo and Selmin Most of the evidence supporting the beneficial effects ofphytochemical compounds comes from in vitro or animalstudies while human studies evaluating the longterm impact ofphytomolecules are particularly few or inconsistent Interventionalstudies are in fact limited by issues of bioavailability andmetabolism However in vitro studies aimed at identifyingcellular targets linked to the beneficial actions of phytonutrientrich foods at concentrations ranging from nM to µM challenge thetranslatability of data After ingestion these compounds are in factdetected as phase II metabolites and their blood level does notexceed concentrations in the nM range Substantial amounts of thecompounds and their metabolites are degraded in the colon byintestinal microbiota giving rise to small phenolic acids andaromatic catabolises which are absorbed by the circulatorysystem Del Rio Interesting studies showed thatthese natural polyphenol and nonpolyphenol substances couldaffect SIRT1 expressionactivity Table de Boer Themain mechanisms of action common to polyphenol and nonpolyphenol molecules that lead to antioxidant and anti‚ammatory effects via SIRT1 activation are reported in Figure Here we focus on the natural molecules resveratrolquercetin fisetin curcumin and berberine and elucidate theireffect on SIRT1 activation and their potential to treat andorprevent several human pathologies mainly associated withmetabolic disorders Figure Frontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsTABLE Classification of nutraceuticals based on their action and food sourceNaturalSIRT1activatorsEffectSourceReferencesResveratrol Positive effect on bloodlipid profile antioxidantDark grapesraisins peanutsQuercetinBerberineCurcuminFisetinAnticancer positiveeffect on blood lipidprofile antioxidant anti‚ammatoryAntioxidant anti‚ammatoryAnticancer antioxidantanti‚ammatoryAnticancercardiovascularpreventive anti‚ammatoryantioxidantFruits vegetablesnutsNatural componentof traditionalChinese herbCoptidis rhizomaActive componentin Curcuma longaApplespersimmonsgrapes onionskiwi kalestrawberriesD™Angelo Zordoky Hung Nabavi Nabavi Wu Hung Zendedel Kim Chen NATURAL COMPOUNDS ENHANCINGSIRT1 EXPRESSION AND ACTIVITYResveratrolResveratrol RSV a nonflavonoid polyphenol found in grapesand grape products such as red wine exerts an antioxidant actionwith reported cancer preventive properties KrisEtherton RSV also has anti‚ammatory anticancer andantineurodegenerative effects Piotrowska The roleof RSV as an immune response modulator was demonstrated inboth in vitro and in vivo studies where it reversed immunesenescence in older rats reduced ‚ammatory responses inrodents and improved immunological activity against cancercells Malaguarnera RSV was shown to be involved in theactivation of macrophages T cells and natural killer cells as wellas in the suppression processes of CD4 CD25 regulatory T cellsYang Svajger and Jeras All these effects aredue to its ability to remove ROSinhibit cyclooxygenaseCOX and trigger anti‚ammatory pathways via SIRT1activation Miceli Malaguarnera ActivatedSIRT1 interrupts TLR4NFkBSTAT axis reduces cytokineproduction by inactivated immune cells and inhibits pro‚ammatory factors derived from macrophagesmast cellssuch as plateletactivating factor and TNFa Capiralla RSVSIRT1 interaction modifies SIRT1 structure andpromotes binding activity with its substrates including p65RelA Yeung a component of the NFkB complexwhich regulates activation of leukocytes and ‚ammatorycytokines SIRT1 activated by RSV inhibits acetylation of RelAby reducing the expression of ‚ammatory factors such as TNFa IL1b IL6 metalloprotease MMP1 MMP3 and NFkBmediated Cox2 Malaguarnera AMP activatedprotein kinase AMPK is also a target of RSV as it controlsSIRT1 activity via regulation of cellular levels of NAD thusacting as an energy sensor Price Cyclicadenosine monophosphate cAMP levels activate proteinkinase A resulting in phosphorylation and activation of SIRT1FIGURE Basic mechanisms and effects of SIRT1 activation by polyphenol and nonpolyphenol moleculesFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsFIGURE Nutraceutical action on SIRT1 expression Natural substances have beneficial effects on human health by regulating SIRT1 action in different cellularprocesses wwwpubchemncbinlmnihgovWan Activated SIRT1 catalyzes the deacetylationand activation of PGC1a thereby promoting beneficial effectsin the metabolism Ren In different anisms S cerevisiae C elegans and Dmelanogaster expressing SIRT1 or its homologous genesRSV treatment is able to extend life span In mammaliansRSV administration can improve SIRT1dependent cellularprocesses such as axonal protection Araki fatmobilization Chaplin and inhibition of NFkBdependent transcription Yeung these effects areabolished in SIRT1 knockdown models Numerous studiesinvestigated the beneficial effects of RSV in cardiovasculardiseases including hypertension Theodotou cardiac ischemia Fourny and atherosclerosisChassot RSV has an effect on blood vesselsreduces ‚ammation and prevents thrombus formation andplatelet oxidation Zordoky It can also reducecardiac dysfunction oxidative stress fibrosis and apoptosis inthe heart Gupta Yamagata In addition RSVwas found to improve heart and kidney damage in rats Li et al2020a The protective effect of RSV is associated with anincrease in SIRT1 activity which deacetylates FOXO1 andactivates MnSOD downstream RSVinduced MnSOD alsoreduces oxidative stress Li 2020a A recent in vitrostudy showed that RSV reduces hypoxiainduced apoptosis inH9C2 cells through activation of SIRT1miR30d5pNFkB axisHan RSV treatment decreased cortical andhippocampal malondialdehyde levels while increasing SODactivity and SIRT1 expression in a diabetic rat model Ma RSV was shown to activate SIRT1 and improve endothelialfunction in obese mice via upregulation of PPARd expressionactivity in PPARd mutant mice Cheang It hadpreviously been observed that Akt activation together withPPARd is involved in vascularization of dbdb mice Tian RSV was subsequently reported to increasephosphorylation of Akt and transcriptional activity of PPARdin the aorta of wildtype mice thus supporting the hypothesis ofSIRT1PPARd interaction and to strongly decrease LPCinduced mitochondrial ROS in the aortic endothelium ofC57BL6 mice Cheang Taken together thesefindings highlight the beneficial effects of RSV against oxidativestress which is involved in major pathologies such as heart andmetabolic disorders Although RSV is beneficialin manycontexts its pleiotropic actions need to be better studied inorder to understand which of its described activities are directlydue to SIRT1 modulation and whether this effect is always directBecause of the pleiotropic actions of RSV clinical trials arecurrently testing its therapeutic potential in a wide range ofhuman diseases However of all the mechanisms described in invitro and in vivo studies only a few have been confirmed inhumans such as gene and protein regulation in blood or musclecells and Akt signaling pathways Ghanim Brasnyo Many clinical studies conducted in healthy patientsand volunteers using both high and low doses of RSV highlightits potential cardioprotective benefit through improvement ofendothelial function‚ammatory markers and glucosemetabolism Nevertheless the mechanisms of action are notyet well defined Despite clinical evidence of its effects thepoor bioavailability and rapid metabolism of RSV severelylimit the potential use of this molecule in the clinic Futurescientific research should focus on identifying actual metabolitesor mediators of these observed effectsTo date clinical trials have tested the efficacy safety andpharmacokinetics of RSV in the prevention and treatment of different pathological conditions wwwclinicaltrialsgovFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsRestricting the search to interventional phase studiescompleted and terminated clinical trials addressed theability of RSV to improve the pathological conditions ofpatients affected by several diseases Most of these studiestested RSVmediated effects in central nervous systemdisorders Friedreich ataxia Alzheimer™s disease Parkinson™sdisease metabolic disorders [T2Dinsulin resistancedyslipidemia hypercholesterolemia metabolic syndrome Xnonalcoholic fatty liver disease NAFLD] A phase clinicaltrial NCT01640197 tested the effects of chronic resveratrolsupplementation mg daily for days in healthy humansand found considerable improvements in cognitive performancecerebral blood flow subjective sleep mood health and bloodpressure A list of completed and terminated clinical trials inwhich RSV was tested for metabolic disorders is reported inTable Focusing on completed clinical trials with availableresults NCT02114892 evaluated the effect of RSV on metabolicsyndrome demonstrating that when administered three timesper day mgdie before meals RSV was able to treat andprotect from obesity and diabetes with beneficial effects onglucose and lipid metabolism blood pressure and bodyweight Another phase study NCT02095873 evaluated theeffects of a formulation composed of RSV and hesperetin inobese subjects and found that these molecules are dietaryinducers of glyxalase improving metabolic and vascularhealth of obese subjects Xue QuercetinThe flavonoid polyphenol quercetin Que ²²pentahydroxyflavone is a natural safe dietary supplementfound in a glycoside form in fruits vegetables and nuts whichhas antioxidant and anti‚ammatory properties Nabavi Wu In recent years the scientific community has focused on thepotential antiproliferative chemopreventive and anticarcinogenicactivities of Que as well as on its role as a modulator of geneexpression However Que was also found to have potentially toxiceffects including mutagenicity prooxidant activity mitochondrialtoxicity and inhibition of enzymes involved in hormonalmetabolism Li Due to its poor solubility short halflife and low bioavailability its medical use is limited Konrad andNieman In humans Que bioavailability is very low and absorption varies from to in subjects receiving mgdie Costa Que may reduce infection Li hepatic lipemicoxidative damage Cui Zhang et al2016b and antioxidant risk Xu In addition Que isknown to exert a modulating action on immunity Galleggiante As regards its mechanism of action in some cell linesQue was able to inhibit the production of TNF in macrophagesTang IL8 in A549 lung cells induced bylipopolysaccharide LPS Geraets and TNFa andIL1a mRNA levels in glial cells causing a decrease in neuronal celldeath induced by microglial activation Li MainlyTABLE Resveratrol in clinical trials for metabolic disordersStatusStudy TitleConditionsInterventionPhaseNCT NumberCompleted Effects of Resveratrol in Patients With TypeType Diabetes DiabetesTerminated Effect of Administration of Resveratrol onType Diabetes Mellitus mg to a maximum dose of g daily mg times dailyPhase NCT01677611Phase NCT02549924Glycemic Variability in Individuals With Type Diabetes MellitusCompleted Effect of Resveratrol on Agerelated InsulinResistance and Inflammation in HumansCompleted Regulation of Intestinal and HepaticLipoprotein Secretion by ResveratrolType Diabetes Mellitus InsulinResistanceDyslipidaemia Insulin ResistanceCompleted Effects of Dietary Antioxidants to PreventHypercholesterolemia HealthyCardiovascular DiseaseHealthy Aging Through Functional FoodCompletedwith resultsCompleted Effects of Resveratrol on Inflammation inType Diabetic PatientsCompletedwith resultsEffect of Resveratrol Administration onMetabolic Syndrome Insulin Sensitivity andInsulin SecretionCompleted Resveratrol for the Treatment of NonAlcoholic Fatty Liver Disease and InsulinResistance in Overweight AdolescentsGlucose Intolerance Aortic Stiffness VasodilationType Diabetes Mellitus Inflammation Insulin Resistance Other Disorders ofBone Density and StructureMetabolic Syndrome XNAFLD Type Diabetes MetabolicSyndromeCompleted A Study of Resveratrol as Treatment forFriedreich AtaxiaFriedreich AtaxiaCompleted Effect of Banaba Lagerstroemia Speciosaon Metabolic Syndrome Insulin Secretionand Insulin SensitivityMetabolic Syndrome X mg twice daily for daysPhase NCT01354977 mg for week followed by g for weekDietary Supplement red wine for monthDietary Supplementresveratrol for monthTransresveratrol mg hesperetin mg combination months mg daily then months mg daily mg times daily beforemeals with a total dose of mg daily mg twice daily for a total dailydose of mg for days g daily mg twice daily for weeks then g daily gtwice daily for weeksBanaba capsules mg times daily before meals for daysPhase NCT01451918Phase NCT02409537Phase NCT02095873Phase NCT02244879Phase NCT02114892Phase NCT02216552Phase NCT01339884Phase NCT02767869Frontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 Activatorsin immunity and ‚ammation Que acts on leukocytes and targetsmany intracellular signaling kinases and phosphatases as well asenzymes and membrane proteins Li Theimmunostimulating effect of Que is due to induction of theexpression of interferong IFNg derived from Th1 andinhibition of IL4 derived from Th2 in normal peripheral bloodmononuclear cells Nair In addition Que reduces T cellproliferation by blocking IL12induced tyrosine phosphorylationof JAK2 TYK2 STAT3 and STAT4 Muthian and Bright Nabavi In ‚ammation Que inhibits the enzymesCOX and lipoxygenase Lee and Min Savikin Inthe RAW cell line Que was also shown to counteract LPSinduced ‚ammation by phosphorylation of tyrosinephosphatidylinositol3kinase PI3Kp85 and complexformation of tolllike receptor TLR4MyD88PI3K Endale Oxidative stress occurs following an imbalance of the body™santioxidant defence mechanisms and excessive generation of freeradicals and is involved in various pathologies such as diabetesatherosclerosis hypertension neurodegenerative diseases‚ammation and cancer Oboh Que is apowerful ROS scavenger and its antioxidant action is due tothe presence of two pharmacophores within the molecularstructure which confer a favorable configuration for freeradical elimination Costa Generally Que reducesthe effects of free radicals by transferring the hydrogen atom andstabilizing the radicals a feature that has a structurefunctionrelationship Oboh Que can also act as both an antioxidant and prooxidant agentAt low concentrations “ µM Que displayed a protective effectagainst oxidative DNA damage in vitro in human lymphocytes Li At concentrations between µM and µM Que wasable to directly eliminate ROS in vitro Costa Howeverits effect in vivo is very likely not direct but due to its ability tomodulate the cell™s antioxidant defense mechanisms moderateoxidative stress can in fact increase the cell™s antioxidant defensesresulting in general cytoprotection Halliwell Recentresearch showed that oxidized LDL oxLDL induces oxidativestress LaraGuzman Oxidative injuries promote ROSgeneration in human endothelial cells and SIRT1 regulatesendothelial function Therefore enhancement of SIRT1 activityand SIRT1AMPK axis upregulation inhibits oxidative injuryinducing endothelial dysfunction Chen Shentu Que may reduce oxLDLinduced oxidative damageby upregulating SIRT1 and AMPK Hung thereforepotentially preventing oxLDLimpaired SIRT1 inhibition linked toendothelial dysfunction These findings indicate that SIRT1 canfunction as a regulator to improve AMPK activity under oxLDLstimulation Hung It was very recently shown that Que mgkg can reduceinsulin resistance and improve glucose metabolism by reducingsensitivity to T2Dinsulin resistance in obob mice via SIRT1activation Hu In this context another study showedthat in streptozotocininduced diabetic rats Que mgkginhibits oxidative damage by increasing SIRT1 expression anddecreasing levels of NFkB a SIRT1 substrate Iskender In recent years the scientific community has focused on therole of apoptosis in cardiovascular disease showing thatoxidative stress myocardial ischemia hypoxia and ischemiareperfusion injury may induce myocardial apoptosis Donniacuo Tang and colleagues evaluated the effects of Que inimproving myocardialischemiareperfusion injury MIRinduced cell apoptosis both in vitro and in vivo SIRT1 andPGC1a expression levels were decreased in rat MIR groups butwere significantly increased after treatment with Que Tang Furthermore activation of SIRT1PGC1a pathwayupregulated Bcl2 expression and downregulated Bax exertingantiapoptotic effects The authors hypothesized that Que mightimprove MIRinduced myocardial damage via regulation ofSIRT1PGC1a and Bcl2Bax pathways Tang Que is also reported to regulate ROS generation and mitigatemitochondrial dysfunction by promoting their biogenesisSpecifically in a study to develop a therapeutic strategy forosteoarthritis Que was shown to increase expression levels ofSIRT1 PGC1a NRF1 and NFR2 TFAM and phosphoAMPKa in osteoarthritis rats confirming the hypothesis that Quemight act via the AMPKSIRT1 signaling pathway Qiu Overall these findings suggest that Que may counteractcardiovascular disease and oxidative damageThe growing body of evidence supporting the beneficial effects ofQue has led to its clinical use as demonstrated by the number ofclinical trials studies on ClinicalTrialsgov A list of completedstudies using Que in different metabolic and ‚ammatoryconditions is reported in Table Specifically a phase clinicaltrial NCT01839344 measured the effect of Que on glucosetolerance and postprandial endothelial function in subjects withT2D compared to the effect of an alphaglusidase inhibitor acarboseThe administration of g of Que led to a decrease in postprandialblood glucose NCT01839344 Given its antioxidative and anti‚ammatory capacities this flavonoid was considered a goodcandidate for antioxidant therapy in mucositis NCT01732393hepatitis C NCT01438320 idiopathic pulmonary fibrosisNCT02874989 osteoporosis NCT00330096 uric acidmetabolism NCT01881919 cytokine release NCT01106170and chronic obstructive pulmonary disease NCT01708278 Inthe latter study Que supplementation was safely tolerated bypatients with mildtosevere chronic obstructive pulmonarydisease opening the way towards the potential use of Que as atherapeutic agent for this conditionHowever as for RSV and nutraceuticals in general the resultsof molecular studies on Que obtained from in vitro investigationsand animal models are often inconsistent with data from clinicaltrials Concentration factor dose and timing of administrationand bioavailability are the two main issues that require furtherclarification Additional studies are needed to identify theoptimal concentration of Que for it to exert a beneficial effectfor example on insulin sensitivityBerberineBerberine BBR is an isoquinoline alkaloid reported to haveanalgesic anticancer anti‚ammatory and myocardialprotective properties Cicero and Baggioni It was foundFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsTABLE Quercetin in clinical trials for metabolic and ‚ammatory disordersStatusStudy TitleConditionsInterventionPhaseNCT NumberCompleted Effect of Quercetin in Prevention andTreatment of Oral MucositisBeneficial Effects of Quercetin in ChronicObstructive Pulmonary Disease COPDCompletedwith resultsCompleted QTrial in Patients With Hepatitis CCompleted Effects of Quercetin on Blood Sugar andChemotherapy Induced OralMucositisChronic ObstructivePulmonary DiseaseChronic Hepatitis CDiabetes Mellitus Type mg daily for weeksPhase NCT01732393 to mg daily for weekPhase NCT01708278 days mg oral single dose of mgPhase Phase NCT01438320NCT01839344Blood Vessel Function in Type DiabetesCompleted Effect of Quercetin Supplements onCompletedHealthy Males a 4Week RandomizedCrossOver TrialTargeting ProInflammatory Cells inIdiopathic Pulmonary Fibrosis a HumanTrialCompleted Efficacy of Provex CV Supplement toReduce Inflammation Cytokines andBlood PressureHyperuricemia Gout KidneyCalculi DiabetesCardiovascular DiseaseIdiopathic Pulmonary FibrosisIPFBlood Pressure mg tablet for days with meal breakfastpreferredEarly PhaseNCT01881919 doses administered over consecutive days in consecutive weeks oral administration ofquercetin mg daily mg of Provex CV supplement by mouth perday for weeksPhase NCT02874989Phase NCT01106170Completed Effects of Hesperidin on Bone MineralOsteoporosis OsteopeniaPhase NCT00330096Density and Bone Metabolism ofPostmenopausal Womento exert protective antioxidative effects in different physiologicand pathologic conditions Huang Li 2020bHowever the mechanisms underlying these effects remainunclear BBR was described as a potential antitumor agent thatinduces cell cycle arrest in G0G1 phase increases Cipp21 andKipp27 protein expression decreases expression of cyclin D1D2 and DE and the cyclindependent kinases Cdk2 Cdk4 andCdk6 promoting apoptosis in HL60 human leukemia cellsLi BBR can also deregulate telomerase activityand promote mitochondriadependent apoptosis in HepG2human hepatocarcinoma cells through caspase and caspase activation PARP cleavage induction increased expression ofthe proapoptotic protein Bax through activation of FOXOtranscription factors and inhibition of Bcl2 and Bclx antiapoptotic protein expression Hwang BBR wasobserved to exert an apoptotic effect by inducing ROSproduction and increasing MAPK and JNK activity of p38 inSW620 human colon carcinoma cells and by increasing Ca andcytochrome C release in HSC3 squamous cells Song In addition BBR inhibits the proliferation of cancer cellsthrough an anti‚ammatory pathway In oral carcinoma celllines and in SCC4 cells BBR inhibits expression of COX2 andAP1 bond decreases prostaglandin E2 PGE2 production andsuppresses NFkB IKK ERK and JNK activities FurthermoreBBR can inhibit colon cancer cell growth by activating retinoid Xreceptor a RXRa which binds RXRa and promoting bcatenin degradation Ruan However some studieshighlighted the potential ability of BBR to prevent oxidativestressinduced senescence by activating AMPK and restoringNAD levels Song Initial research revealed a significant role of SIRT1 signalingin mediating the antioxidant effect of BBR in diabetes Pang and in lipid metabolism Hasanein Thelipidlowering activity mediated by cotreatment with BBR andRSV was investigated in mice exposed to a high fat diet Zhu In vivo data showed that BBR combined with RSVlowered total cholesterol triglyceride and LDL cholesterol levelsin mice These findings were also confirmed in vitro with 3T3L1adipocytes treated with BBR or RSV alone Specifically BBR andRSV cotreatment was able to reduce lipid accumulation morerobustly than single treatments BBR in combination with RSVdisplayed hypolipidemic effects likely mediated by SIRT1expression regulation Moreover BBR pretreatment seemed tocounteract SIRT1 downregulation Zhu The antioxidant and anti‚ammatory effects of BBR werealso investigated in heart Yu BBRmediated SIRT1activation reduced MIR injury by affecting oxidative damageand ‚ammation signaling Specifically BBR exerted anantioxidant effect by decreasing the generation of cardiacsuperoxide and gp91phox expression and by increasing SODlevels Yu A previous study had also shown thatSIRT1 activation promotes antioxidant molecule production anddecreases proapoptotic proteins through FOXO1 activationthus protecting against MIR lesions Hsu As well as activating SIRT1 BBR is also able to decreaseFOXO1 acetylation triggering antiapoptotic signaling pathwaysvia Bcl2 expression and Bax and caspase3 downregulation Yu A very recent report described the protective effect of BBRagainst doxorubicininduced cardiovascular damage Wu This effect is

Thyroid_Cancer

Drug repurposing identifying novel indications for drugs bypasses common drug development pitfalls to ultimately deliver therapies to patients faster However most repurposingdiscoveries have been led by anecdotal observations eg Viagra or experimentalbasedrepurposing screens which are costly timeconsuming and imprecise Recently more systematic computational approaches have been proposed however these rely on utilizing theinformation from the diseases a drug is already approved to treat This inherently limits thealgorithms making them unusable for investigational molecules Here we present a computational approach to drug repurposing CATNIP that requires only biological and chemicalinformation of a molecule CATNIP is trained with diverse small molecules and uses different drug similarity features such as structural target or pathway based similarityThis model obtains significant predictive power AUC Using our model we createda repurposing network to identify broad scale repurposing opportunities between drugtypes By exploiting this network we identified literaturesupported repurposing candidatessuch as the use of systemic hormonal preparations for the treatment of respiratory illnessesFurthermore we demonstrated that we can use our approach to identify novel uses fordefined drug classes We found that adrenergic uptake inhibitors specifically amitriptylineand trimipramine could be potential therapies for Parkinson™s disease Additionally usingCATNIP we predicted the kinase inhibitor vandetanib as a possible treatment for Type Diabetes Overall this systematic approach to drug repurposing lays the groundwork tostreamline future drug development effortsa1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Gilvary C Elkhader J Madhukar NHenchcliffe C Goncalves MD Elemento O Amachine learning and network framework todiscover new indications for small moleculesPLoS Comput Biol e1008098 101371journalpcbi1008098Editor Avner Schlessinger Icahn School ofMedicine at Mount Sinai UNITED STATESReceived September Accepted June Published August Copyright Gilvary This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement Data is available atthe following URL wwwgithubcomcoryandarCATNIPFunding JE is supported by NLM of the NationalInstitutes of Health under award numberF31LM013058 The content is solely theresponsibility of the authors and does notnecessarily represent the official views of theNational Institutes of Health OE and his laboratoryare supported by NIH grants 1R01CA1945471U24CA210989 P50CA211024 UL1TR002384PLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingThe funders had no role in study design datacollection and analysis decision to publish orpreparation of the manuscriptCompeting interests OE is cofounder and equityholder in OneThree Biotech and Volastra twocompanies that use data science and machinelearning to develop novel therapies In addition OEis an advisor and equity holder in Freenome andOwkin NM is cofounder and equity holder inOneThree Biotech a company that use datascience and machine learning to develop noveltherapies CG is cofounder and equity holder inOneThree Biotech a company that use datascience and machine learning to develop noveltherapies This does not alter our adherence to allPLOS Computational Biology policies on sharingdata and materialsAuthor summaryCurrently clinical approval of a drug is an arduous process that results in an overwhelming number of compounds failing due to safety or efficacy concerns which leaves patientswithout novel lifesaving treatments The idea of drug repurposing is to take approveddrugs or compounds that were shelved due to reasons other than safety and identify newdiseases for them to treat This would allow drugs if they are sufficiently effective toquickly go through the FDA approval process and be available to patients quicker whichalso cuts the ever growing cost of novel compound research and development Here weintroduce CATNIP a computational model that can predict novel indications for specificdrugs or entire drug classes This approach analyzes drug similarity across a wide range ofbiological chemical and clinical features giving a complete picture of each drug™s mechanism and possible indications Interestingly CATNIP can be used for drugs that not onlyare previously approved but also shelved compounds which are often overlooked in previous repurposing analyses Most importantly CATNIP successfully identified noveltreatments for both Parkinson™s disease and Type Diabetes which are currently undergoing preclinical validationIntroductionWith over million spent bringing a single drug to market over the course of yearsdrug development has remained a costly and timeconsuming affair[] In response there hasbeen an increase in interest in drug repurposing the identification of novel indications forknown safe drugs Successes in this area have been seen in the past most notably in sildenafileg Viagra which was originally intended to treat hypertension and angina pectoris but waslater repurposed to treat erectile dysfunction Other examples of compounds repurposed fornew therapeutic applications include minoxidil[] and raloxifene[] which are now used totreat androgenic alopecia and osteoporosis respectively However most of these repurposingopportunities were discovered through inefficient approaches including anecdotal observations or hypothesisdriven investigations and a more efficient approach could lead to manymore repurposing opportunitiesComputational approaches for repurposing drugs are appealing in that they can be systematically and quickly applied to many drugs at a low cost compared to their experimental counterparts One computational approach that has proven to be invaluable in other areas of thedrug development pipeline is machine learning Machine learning is the use of computationalalgorithms to learn from available data to make novel predictions and gain new insight Usingthis technique one can create unbiased algorithms to match seemingly disparate drugs bycomparing their common features[] such as clinical indication toxicity profile[] or therapeutic target[ ] Previously our lab used a ˜similarity™ approach leveraging the principlethat similar drugs tend to have similar characteristics to predict a drug™s target by investigatingthe known targets of other drugs that were predicted to be œsimilar to the investigated drugbased on shared features[] We found that DRD2 a dopamine receptor was the predicted target for the compound ONC201 After identifying and experimentally validating this targetclinical trials were shifted to focus on gliomas which are now successfully completing phasetwo trials at the time of this publication[] The approach of leveraging drug similarity couldimmensely aid drug repurposing efforts with the appropriate dataPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingOthers have successfully used this ˜similarity™ approach to repurpose drugs and demonstrated high predictive power when tested against FDA approved drugdiseases[] However these methods have primarily linked drugs together using a diseasecentric approachinstead of using features related to the drug itself ie drugcentric These repurposingopportunities are identified by predicting diseases similar to the diseases a drug is alreadyknown to treat Disease similarities can be based on semantic pathophysiological or clinicalsimilarities related to the drug™s clinical indication For example PREDICT a repurposingmethod developed by Gottlieb et al[] exploits the semantic similarity of disease terms asa form of diseasedisease similarity Such approaches while reliable limit the scope of therepositioning effort in several ways First the vast majority of small molecules never reachclinical approval and would be overlooked in this type of analysis Second the use of a diseasecentric approach biases repurposing predictions toward exclusively similar clinical diseases ie cancer drugs to other cancer types [] We postulated that using solely druginformation such as chemical and biological features would be a more effective andbroader approach to drug repurposingHere we propose a novel approach to drug repurposing which operates by a platform wecall Creating A Translational Network for Indication Prediction CATNIP CATNIP is amachinelearning algorithm that learns to predict whether two molecules share an indicationbased solely on the drug™s chemical and biological features using unique drugs The systematic application of CATNIP to molecule pairs creates a network with million nodesthat can then be used to identify potential drug repurposing opportunities By identifying feature importance through the use of chemical structure and target information to make broadscale predictions CATNIP is able to effectively bridge between different therapeutic indications to advance methods of drug repurposing In this report we have identified various candidate drug classes that are predicted to have therapeutic activity outside of their intendedindication in diseases such as Parkinson™s disease and Type DiabetesResultsVariance in drug indication nomenclature can be standardizedWe collected a wide variety of drugs N including both approved and investigationalmolecules with a diverse set of indications to ensure that our drug network covered a largeportion of the known chemical space A subset of these drugs FDA approved drugs and indications taken from DrugBank [] were used as a goldstandard of drugindicationassociations in the training set for the model Disease names are often not standardized whichcan lead to many diverse names for the same disease This problem leads to many drug pairsappearing to not have shared indications when they are associated with two different namesfor the same disease To address inconsistencies in nomenclature for drug indications such asœprostate carcinoma and œcarcinoma of the prostate the MetaMap tool [] was applied tomap disease names to UMLS concepts Methods This standardization of medical terminologies allowed us to reconcile various variations in the database allowing us to confirm thatdrugs did in fact treat the same disease Examples of these variations and their mappingsmay be seen in Table Using MetaMap we clustered the DrugBank indications into standardized indications A multitude of indication types were included in this standardization including but not limited to oncological mental health and neurological diseasesS1A Fig Our rigorous standardization of drug indications ensured an accurate training setallowing for the discovery and modeling of drugindication relationshipsPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cTable Indication nomenclatures and their mappingsMetamap MappedIndicationIndicationDrugBankIndication IDDrugBankNumber of unique drugsassociated with IndicationUnique drugs associated with Indication IDMachine learning approach to drug repurposingProstateCarcinomaAcne VulgarisAdvanced ProstateCarcinomaAdvanced carcinomaof the prostateSevere AcneAcneDBCOND0070333DBCOND0020265DBCOND0077433DBCOND0019842DementiaVascularIdiopathicPulmonaryFibrosisPaget DiseaseModerate AcnevulgarisMild VascularDementiaDementia VascularDementiasIdiopathic PulmonaryFibrosis IPFMild IdiopathicPulmonary FibrosisPaget™s DiseasePaget™s Disease ofBoneDBCOND0022329DBCOND0022662DBCOND0029264DBCOND0060453DBCOND0031843DBCOND0093824DBCOND0038793DBCOND0030189101371journalpcbi1008098t001IDCyproterone acetate Esterified estrogensGoserelinCyproterone acetate Doxycycline TetracyclineAloe Vera Leaf Benzoyl peroxide Chloramphenicol ClioquinolGlycolic acid Linoleic acid Octasulfur Salicylic acid SilverSpironolactoneEthinylestradiol Minocycline Nestimate TazaroteneMemantineDonepezilGalantamine Trazodone TrifluoperazineNintedanib PrednisolonePirfenidoneAlendronic acid Pamidronic acid Risedronic acid ZoledronicacidEtidronic acidDrug pairs sharing indications have other similar characteristicsWe hypothesized that pairs of drugs that shared at least one indication would have other similar drug characteristics S1 Table To test this hypothesis we integrated the similarity of twodrugs across chemical and biological drug properties and created a computational model topredict if two drugs will share an indication Fig All of the drug similarity features S1Table collected could significantly distinguish between drug pairs known to share an indication and those not known to share an indication S2“S5 Figs For example we found thatdrug pairs with a shared clinical indication according to their listed DrugBank indicationstended to have significant overlap in targets Dstatistic pvalue S2A FigThe feature which best discriminated between drug pairs that shared a clinical indication versus drug pairs that do not was the similarity between the KEGG pathways that each drug™s targets are involved in Dstatistic p S4C Fig Pathway similarity was calculatedas the Jaccard Index between the KEGG pathways that contain each drug™s gene targets Methods The difference in effect size between the target similarity and the pathway similarity Dstatistic vs respectively indicates that the drugs do not necessarily have to targetthe same exact genes but rather the same biological pathway in order to share a clinical indication Additionally we found that drug pairs that share an indication had a more similarchemical structure than drug pairs that did not share an indication Dstatistic pvalue S5A Fig A biological network containing both physical and nonphysicalinteractions was curated containing proteincoding genes drugs and TFsThis curated network provided another feature for our model allowing us to utilize previouslyestablished interactions between proteins to aid with distinguishing drug pairs that share anindication Overall these features seem to indicate sufficient power in differentiating drugsPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingFig Schematic of CATNIP repurposing approach A The use of drug similarity properties to predict if two drugs will share an indication using agradient boosting model the model is referred to as CATNIP B Schematic showing the use of CATNIP output scores to create a network with the scoresused as edge weights The colors of each drug represent the known disease and this demonstrates how one could identify novel indications for drugsthrough the network101371journalpcbi1008098g001PLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingFig CATNIP model accurately predicts drugs that share an indication and can be used for repurposing A Receiveroperating characteristic curve for CATNIP the performance for drug pairs with high and low structural similarity is alsoshown B A network of all drug pairs with a CATNIP score higher than Nodes drugs are colored based on ATCclassification and a specific example of repurposing between ATC classifications is highlighted C A graph of all ATCclassification and the median CATNIP score between the drugs belonging to each of them only including drug pairswith CATNIP score The edges between ATC Classifications with the highest median CATNIP scores are colored red101371journalpcbi1008098g002that share and do not share indications which we hypothesized can then be leveraged to createa predictive modelDrug pairs that share indications can be predicted by modelUsing these diverse drug properties as features we trained a Gradient Boosting model to predict if two drugs share a clinical indication A Gradient Boosting model showed superiorresults when compared with other algorithms Methods S2 Table The model output is adrug similarity score hereby referred to as a œCATNIP score which allows us to classifydrug pairs that share clinical indications We performed a 5fold crossvalidation analysis andachieved significant predictive performance with an areaunderthereceiveroperator curveAUC of Fig 2A We confirmed the statistical significance of our model with aPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingprecisionrecall curve PRC because of the class imbalance in our dataset between drug pairsthat share indications against those that do not Shared Not Shared Whencompared to random predictions our model showed significant improvement vs areaunder PRC S6 Fig We retained a low percent of false positive predictions at various cutoffs false positives and false positives at a model prediction probability oftwo drugs sharing an indication of and respectively providing extra confidencethat our predictions can lead to strong repurposing candidatesWe found that the predictive model greatly benefited from the addition of diverse datatypes While structure similarity showed the highest feature importance of any single featureS11 Fig when used as a single feature within a gradient boosting model it only achieved anAUC of S12 Fig Interestingly when only supplying the model with ontology featuresa Jaccard index for the GO terms of the known targets of each drug within a drug pairachieved an AUC of However even at the highest AUC of any single feature typeit is significantly below the performance when combining all feature typesIn certain cases a high predictive performance is expected such as when two drugs arestructurally similar or share targets It has been shown before that structurally similar drugshave a high probability of treating the same indication[] However there continue to bedrug pairs known to treat the same indication that are not structurally similar For exampletamoxifen[] and anastrozole[] are structurally dissimilar compounds Dice similarity that treat the same indication Metathesaurus term Cancer Breast We recalculated our performance metrics to evaluate how our model performed in classifying drug pairsthat shared indications when only exposed to drug pairs with low structure similarityDice High performance was retained under with an AUC Fig 2A Additionally we found that our model performed similarly well when only exposed to drug pairs thatdid not have any known shared targets AUC Fig 2A These performance metricsconfirm that our model is robust enough to predict if a drug pair will share an indication evenfor more difficult prediction tasksNetwork clusters identify drugs with similar clinical characteristicsWe constructed a repurposing network by calculating a CATNIP score for all possible drugpairs found within DrugBank and assigning the drugs as nodes and the CATNIP score as theedge weight We pruned the network using a cutoff value of for the CATNIP scoresFig 2B which included different drug pairs This cutoff is equivalent to a predictedprobability of to share an indication and allowed for a balance between confidencewithin our predictions and drug diversity and availabilityWe hypothesized that drugs sharing at least one indication would cluster together in ournetwork To confirm this theory we classified each drug per its 1st order Anatomical Therapeutic Chemical ATC classification This identification is a method of distinguishing theclinical use of a drug that is widely used in European and North American chemoinformaticsdatabases[] Using ATC we observed clearly defined clusters within the repurposing network Fig 2B Many clusters featured multiple ATC classifications suggesting potential repurposing opportunities For example one cluster included the thiazolidinediones rosiglitazoneand pioglitazone ATC classification ˜Alimentary Tract and Metabolism™ and the fibratesfenofibrate and bezafibrate ATC classification ˜Cardiovascular system™ These two clusteredATC classifications were connected by a high CATNIP score between bezafibrate andpioglitazone an antidiabetic drug a relationship driven by the shared targeting of PPARa andPPARg resulting in the improvement of lipid and glucose metabolism Bezafibrate has shownefficacy in the treatment of Type Diabetes in numerous retrospective and preclinical studiesPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingATC Code ReferenceRespiratory SystemRespiratory SystemRespiratory System[“][“][“][ ]Table Literature Support for ATC Repurposing PredictionsATC Code DermatologicalsAlimentary Tract and MetabolismSensory ansSystemic Hormonal Preparations Excluding Sex Hormones AndRespiratory SystemInsulinsSensory ans101371journalpcbi1008098t002Alimentary Tract and[“]Metabolismincluding Phase trials[“] however is still not an approved antidiabetic The identification of bezafibrate as a potential diabetes treatment is a key example of how CATNIP can beused to identify repurposing opportunitiesWe reasoned that the connections between ATC classifications across all the drug clusterscould provide additional aid for drug repurposing purposes Using the pruned network CATNIP Score we collected all the scores between drugs of differing ATC classificationsFrom this collection we were able to determine the median score associated between each pairof ATC classifications The ATC classifications with the highest median CATNIP scores hadliterature support for numerous repurposing efforts between them Table For exampledrugs with the ATC classifications of œRespiratory System and œSystemic Hormonal Preparations excluding sex hormones and insulins were strongly connected to each other median CATNIP score This connection was driven by highly scored pairs of drugs includingrimexolone to mometasone CATNIP score and prednisone to triamcinolone CATNIP score These connections are supported by the fact that hormonal agents like glucocorticoids and beta adrenergic agonists have been used for decades to relax the airway musculature in patients with reactive airways disease and chronic obstructive pulmonary disease[]Interestingly our analysis identified glucagon a peptide hormone that increases blood glucoselevels as a candidate for œRespiratory System repurposing and this use already has clinicalsupport[][] Additionally drugs classified as œRespiratory System and œDermatologicalwere also observed to be highly associated because of interactions such as the one betweenciclesonide and hydrocortisone CATNIP score Ciclesonide and hydrocortisone do infact share a clinical indication œAsthma Bronchial giving added confidence to our findingsThese types of network observations are important in laying the groundwork for suggestingnovel clinical repurposing strategies for FDAapproved drugsCATNIP identifies novel disease areas for drug classesWe investigated the ability to leverage CATNIP scores to identify repurposing opportunitiesby evaluating specific drug classes Drug classes are predefined in DrugBank In order to identify actionable repurposing possibilities we narrowed this list down to classes containinginhibitors antagonists or agonists of specific gene or protein families We focused our attention on specific disease areas that are attractive for drug repurposing opportunities due to alack of current treatments or high rates of acquired resistance The specific disease areas wereœmental disorders œneurological diseases œdiabetes and œcancer cancer was furtherdivided into specific cancer types due to the large variance in disease pathology between typesMethodsPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingWe hypothesized that CATNIP scores could be used to identify specific drug classes thatwould be efficacious for a new disease area For each drug class and disease area we foundthe statistical difference in the CATNIP score distribution between two sets of drug pairsThe first set included pairs that had one drug within the drug class and the other drugapproved for the disease in question while the other set included drug pairs that had onedrug within the drug class and the other drug not approved for the disease in questionMethods We compared the effect size estimated by the Wilcoxon location shift for alldrug classdisease pairs that had a significant difference in distribution compared to drugclassnondisease pairs FDR Supplementary Data By using CATNIP scores wefound that many wellknown drug classdiseases associations could be recovered For example œmuscarinic antagonists were highly ranked for œneurological diseases and many suchagents are FDAapproved for this indication[] In addition we found that œkinase inhibitors were closely associated with the treatment of cancer and œdopamine antagonists forthe treatment of œmental disorders[ ] Wilcoxon Location Shift “ forœkinase inhibitors and select cancer types Location Shift for œdopamine antagonists and œmental disorders pvalue S7 Fig In fact almost all drug classdiseaseassociations contained at least one FDAapproved drug for the respective disease giving usadded confidence in our model Of note each drug was allowed to be categorized intonumerous drug classes leading to unexpected yet easily explained results for exampleœdopamine antagonists appearing as a top drug class for œneurological diseases This isdue to risperidone a drug traditionally used for schizophrenia and mood disorders alsohaving a secondary indication of Alzheimer™s type severe dementiaOur method reached significant levels of predictive power for predicting both drug class”disease associations and individual drugdisease association When predicting drug classdisease associations under our most lenient conditions calling cases where at least one drugwithin the class was known to treat the disease a true positives our method achieved a sensitivity of greater than However this improved to a sensitivity of when we implementedstricter cutoffs ie only calling drug classdisease associations true positives if of drugswithin the class treated that disease S10 Fig We additionally compared our method™s abilityto predict individual predictions to that of a previously highlighted method Gottlieb et al™sPREDICT[] We found our method had a slightly higher AUPRC vs andhigher sensitivity vs S4 Table S1 Methods While these results indicate modest improvements over PREDICT it is important to note that unlike in PREDICT diseaseinformation is not a required feature in CATNIP™s machine learning approach This meansthat CATNIP can be applied towards investigational molecules with no previously knownindications Additionally by not using disease information as a feature repositioning of drugswith known indications using CATNIP is not directly biased by the associated disease indication and instead uses mechanistic features chemical structure and properties targets etc aspart of the repositioning strategyNext we further interrogated the drug classes associated with œneurological diseases andœdiabetes specifically CATNIP scores could correctly identify drug classes known to treatthese diseases Table To identify possible repurposing candidates we focused our attentionon drug classes shown to have a large positive effect size with this CATNIP analysis but are notcurrently approved for treatment For œneurological diseases the use of adrenergic uptakeinhibitors traditionally used as antidepressants was the top repurposing candidate for œdiabetes alpha antagonists and kinase inhibitors were identified as possible novel treatmentsTable We believe further investigation into these drug classes and diseases could lead tosuccessful clinical applicationsPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingTable Top Predictions of Drug Class Repurposing OpportunitiesClassDiabetesDiseasePrediction RankAlpha1 AntagonistsKinase InhibitorProtein Kinase InhibitorsProtein Synthesis InhibitorsCytochrome P450 CYP2E1 InhibitorsMonoamine Oxidase InhibitorsNeurologicalAdrenergic Uptake InhibitorsAdrenergic alpha AgonistsProtease Inhibitors101371journalpcbi1008098t003CATNIP interpretability reveals reasoning for repurposing candidatesFrom our list of repurposing candidates we chose two novel drug classdisease associations tofurther investigateAdrenergic uptake inhibitors applied to Parkinson™s disease First we evaluated therelationship between œneurological diseases and œadrenergic uptake inhibitors We focusedon the drug pairs with the highest CATNIP scores ie those predicted with the highest confidence to share at least one indication Fig 3A Of all the adrenergic uptake inhibitors wefound that amitriptyline and trimipramine two antidepressants had the highest CATNIPscores with the œneurological diseases drugs The drugs that shared the strongest connectionswith amitriptyline and trimipramine were drugs approved for Parkinson™s disease PD Specifically metixene atropine pergolide and benzatropine were associated with amitriptylineaccording to CATNIP and trimipramine was associated to benzatropine and rotigotine Trimipramine was also strongly connected with orphenadrine which is sometimes used off labelin PD but will not be included in the following analysesUsing the CATNIP model we evaluated which features contributed towards the predictionof amitriptyline and trimipramine to share an indication with PD drugs We found that targetgene ontology and pathway similarity all strongly contributed to the predictions for both amitriptyline and trimipramine Fig 3B S8 Fig Since target similarity and distance between targets in a proteinprotein interaction network were among the top contributing features weinvestigated which gene targets were shared amongst these drug pairs We found that amitriptyline targets three specific gene classes that are also targeted by at least one of the PD drugsmuscarinic acetylcholine receptors Gcoupled protein receptors GPCRs and alpha adrenergic receptor Trimipramine also targets muscarinic acetylcholine receptors alphaadrenergicreceptors and dopamine transporters which is similar to benzatropine a PD drug All thesereceptors have welldefined relationships with PD and other neurological diseases[ ]which adds support for repurposing amitriptyline andor trimipramineAmitriptyline may be an ideal candidate for use in PD patients We evaluated the sharedmolecular function gene ontology terms shared between amitriptyline and all four PD drugsGPCR activity was once again identified S1“S4 Files We then interrogated the biologicalpathways these drug targets are involved in and found many broad GPCR pathways overlapping between amitriptyline and the PD drugs S9 Fig including the Reactome pathway œGASTRIN_CREB_SIGNALLING PATHWAY VIA PKC AND MAPK Several recent studiessupport the link between gastrinreleasing peptide signaling to brain function[] ThroughCATNIP we have identified œadrenergic uptake inhibitor

Thyroid_Cancer

Hepatitis B virus HBV infection has been associated with the risk andprognosis of many malignancies Nevertheless the association between HBV and theprognosis of breast cancer is unclear The objectives of this study were to investigate theprognostic role of hepatitis B surface antigen HBsAg and to integrate HBsAg to establishnomograms for better prognostic prediction of very young patients with breast cancerMethods This analysis was performed retrospectively in a cohort of consecutivevery young ‰ at diagnosis patients who received curative resection for breastcancer The significance of HBsAg in the prognosis of these patients was investigatedUnivariate and multivariate analyses were used to identify independent variables fordiseasefree survival DFS and overall survival OS Nomograms were built based onthose identified variablesResults Overall of the patients were seropositive for HBsAgThe median followup was CI “ months forthe entirepopulation The HBsAgpositive cohort had significantly inferior DFS HR CI “ P and OS HR CI “ P as compared with the HBsAgnegative cohort The rates of 10year DFS andOS were and in the HBsAgpositive group and and in the HBsAgnegative group respectively In multivariable analysis HBsAg statuswas identified as an independent significant unfavorable prognostic factor for DFSP and OS P in very young patients with breast cancer Nomogramswere established and displayed good calibration and acceptable discrimination TheCindex values for DFS and OS were CI “ and CI “ respectively Based on the total prognostic scores TPSof the nomograms different prognosis groups were identified for DFS and OSEdited byImtiaz Ahmad SiddiquiUniversity of Colorado AnschutzMedical Campus United StatesReviewed byAbhinit NagarUMass Memorial Medical CenterUnited StatesNidhi JainBeckman Coulter IndiaCorrespondenceLu YangyanglusysucccnWeiDong Weiweiwdsysucccn These authors have contributedequally to this workSpecialty sectionThis was submitted toCancer Epidemiology and Preventiona section of the journalFrontiers in OncologyReceived March Accepted July Published August CitationLi N Zhong QQ Yang XRWang QC Zhang DT Zheng SYang L and Wei WD Prognostic Value of Hepatitis B VirusInfection in Very Young Patients WithCuratively Resected Breast CancerAnalyses From an Endemic Area inChina Front Oncol 103389fonc202001403Frontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerConclusions HBsAg is an independent unfavorable prognostic factor for DFS andOS in very young patients with curatively resected breast cancer and nomogramsintegrating HBsAg provide individual survival prediction to benefit prognosis evaluationand individualized therapyKeywords HBV young breast cancer prognosis nomogram survivalINTRODUCTIONGlobally breast cancer is the most common cancer and theleading cause of cancer death for women accounting for of total cancer cases and of total cancer deaths Breast cancer has also been the top one malignancy in termsof incidence in Chinese women constituting of newlydiagnosed cases and of all deaths from breast cancer in theworld Although breast cancer occurs at a lower incidence inChinese women than in western women this disease occurs at ayounger age in China than in highincome countries and China™scontribution to global breast cancer rate is increasing rapidly The disparities between young and old breast cancer include ahigher mortality rate higher risk of recurrence poorer treatmentresponse and more aggressive phenotypes “ Thereforeunderstanding the etiology and identifying novel prognosticfactors are essential for early diagnosis prognosis evaluationearly intervention and personalized therapy in young patientswith breast cancerHepatitis B virus HBV infection is a serious public healthdilemma with ˆ¼ million chronic carriers worldwide China account for about a third of infectionassociated cancerglobally driven by high prevalence of HBV and H pylori infection Although China has made tremendous eï¬orts in controllingHBV over the past years and the prevalence of HBV ininfants and children has remarkably declined the hepatitisB surface antigen HBsAg prevalence is still high in Chineseadults ranging from to “ HBV is the leading causeof hepatocellular carcinoma and cholangiocarcinoma Inaddition there is also accumulating evidence that HBV infectionis associated with many extrahepatic malignancies includingnonHodgkin™s lymphoma pancreatic cancer gastriccancer nasopharyngeal carcinoma lung cancer esophageal cancer and ovarian cancer Thus it seemsreasonable that HBV is an important factor in the developmentof extrahepatic malignancies in endemic areasDespite the facts that HBsAg status is one of the routineexaminations in patients with operable breast cancer and severalstudies have showed that HBV is not associated with therisk of breast cancer the impact of HBV on theclinicopathological characteristics and prognosis of very youngpatients with breast cancer remains to be determined Giventhat both early breast cancer and HBV are endemic in China itis possible that HBV infection is associated with the prognosisof early breast cancer even though the precise mechanismsare yet to be determined It is crucial to address this issuesince HBV has been reported to be found in breast cancertissue We therefore performed this study to investigate theHBsAg prevalence in very young breast cancer and the impactof HBsAg on the survival of these patients and to establishnomograms to better predict prognosis for very young patientswith breast cancerMATERIALS AND METHODSPatient SelectionA retrospective review was conducted in a cohort of consecutive breast tumor women who were aged ‰ years oldand received curative resection for breast cancer at Sun Yatsen University Cancer Center between May and July This study was conducted according to the ethicalstandards of the Declaration of Helsinki Institutional ReviewBoard approval was obtained from the Medical Ethics Committeeof this cancer center All patients were restaged by the eighthinternational classification system for breast cancer Dueto the retrospective nature of this studyinformed consentwas waivedInformation was collected from electronic patient recordsand survival data were obtained from the followup registryofthis center The information collected included HBsAgstatus laterality type of breast surgery type of axillary surgeryhistological type tumor grade tumornodemetastasis TNMstage dates of surgeryrelapsedeath status of estrogen receptorER progesterone receptor PR human epidermal growthfactor receptor HER2 and Ki67 Breast cancers wereclassified as luminal Alike ER PR‰¥ HER2“ andKi6715luminal Blike ER andor PR HER2“HER2enriched ER“ PR“ HER2 or triplenegative ER“PR“ HER2“ subtypesPotentially eligible patients had to have curatively resectedbreast cancer without previous therapy other than neoadjuvanttherapy be aged years old or below and have definiteinformation of HBsAg The main exclusion criteria includedbenign tumor not having surgery having incomplete resectionprevious malignant disease hepatitis viral infections other thanHBV men patients and insufficient data of survival or HBsAgStatistical AnalysisThe main objectives of this study were to compare diseasefreesurvival DFS and overall survival OS between HBsAgpositivepatients and HBsAgnegative patients DFS was defined as theinterval from the date of being diagnosed to the date of diseaserecurrencemetastasis or death from any cause OS was defined asthe interval from the date of being diagnosed to the date of deathfrom any cause Median followup was estimated by KaplanMeier analysis with reversed meaning of status indicator Frontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancerforindependent variablesDFS and OS were estimated by the Kaplan“Meier methodand diï¬erences were compared by the logrank test Univariateand multivariate analyses with a Cox proportional hazardsmodel were used to testforDFS and OS Covariates included laterality left vs righttype of surgery breastconserving surgery vs mastectomytype of axillary surgery sentinellymph node dissection vsaxillary lymph node dissection histological type ductal vsotherstumor grade grade III vs grade III T stageT34 vs T12 N stage N23 vs N1 HER2statustriplenegativeHER2enriched vs luminal All variables reaching asignificance of in univariate analyses were included inmultivariate analysispositive vs negative molecularsubtypesThe nomograms for predicting and 10year DFSand OS were formulated based on the results of multivariateanalysis by the œrms package of R The discriminationofthe nomogram models was estimated by the Harrell™sconcordance index Cindex The value of the Cindex rangesfrom to with implying a random chance and indicating a perfect prediction Calibration curves of thenomogram models for DFS OS were plotted to assess thepredictive value of the model In addition patients weredivided into three diï¬erent risk groups highintermediatelow according to total prognostic scores TPS The totalprognostic scores of patients were transformed into categoricalvariables based on cutoï¬ points which were determined by theminimum Pvalue from logrank × statistics with the Xtileprogram Pearson™s chisquare test was used to compare categoricaldata All Pvalues were twosided and P was consideredstatistically significant Statistical analyses were performed by theSPSS software SPSS Inc version Chicago IL USA and Rfor Windows version httpwwwrprojectData AvailabilityThe authenticity of this has been validated by uploadingthe key raw data onto the Research Data Deposit public platformwwwresearchdatacn with the approval RDD numberas RDDA2020001410 The data that support the findings ofthe study are available from the corresponding authors uponreasonable requestRESULTSPatient CharacteristicsA total of very young ‰ at diagnosis breasttumor patients were screened of whom were excludedbecause of benign tumor n not having surgeryn not having R0 resection n or unknownHBsAg status n With further exclusions forinsufficientfollowup data a total of patients withcurative resection for breast cancer and aged years orbelow were included in this study Figure The patients™FIGURE The process of patient selectionFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerTABLE Patient characteristics by HBsAg statusCharacteristicsHBsAgpositiveN No HBsAgnegativeN No Pvalue FIGURE Number of recurrences by year of entire patientsLateralityLeftRightBilateralType of breast surgeryMastectomyBreastconserving surgeryType of axillary surgeryALNDSLNDHistological typeDuctalInvasive lobularOtherTumor gradeIIIIIIUnknownT StageT1T2T3T4N StageN0N1N2N3HER2 statusPositiveNegativeUnknownMolecular subtypesLuminal ALuminal BHER2enrichedTriple negativeUnknown HBV Hepatitis B Virus ALND axillary lymph node dissection SLND sentinelnode dissectionHER2 human epidermal growth factor receptor2lymphcharacteristics are shown in Table Overall ofthe patients were seropositive for HBsAg HBsAgpositive group and HBsAgnegative group were wellmatchedfor basic characteristics including laterality type of breast andaxillary surgery histological type tumor grade T stage Nstage and molecular subtypes About in patients receivedmastectomy and most patients received axillary lymph nodedissection ALNDAssociations Between the Status of HBsAgand SurvivalThe median followup was CI “ months forthe entire population By the time of analysis December instances of disease recurrence had occurred Thenumber of recurrences in each year of the followup is shown inFigure Of note although HBsAgpositive patients had a higherfrequency of extrahepatic metastasis vs P thanHBsAgnegative patients they had a comparable frequency ofliver metastasis vs P when compared withHBsAgnegative patientsDFS was significantly shorter among those who were HBsAgpositive than among those who were HBsAgnegative HR CI “ P The rates of 10yearDFS were in the HBsAgpositive group and inthe HBsAgnegative group respectively Figure 3A A totalof death events had occurred by the data cutoï¬ HBsAgpositive group had significantly inferior OS compared withHBsAgnegative group HR CI “ P with a 10year OS of and respectivelyFigure 3B The association of HBsAg status and survival ineach molecular subtype was further analyzed As expectedDFS and OS were significantly longer among those in theluminal A subgroup and the HER2enriched and triplenegativegroups had significantly shorter DFS and OS Figures 4ABNotably HBsAgpositive status was associated with shorterDFS P and OS P in the luminalB cohort HBsAgpositive status was also associated with aslightly shorter DFS in the triplenegative cohort and shorterOS in the HER2enriched cohort but statistical significancewas not reached Supplementary Figures There was nosignificant diï¬erence in DFS between HBsAgpositive patientsand HBsAgnegative patients in luminal A and HER2enrichedcohorts and in OS in luminal A and triplenegative cohortsSupplementary Figures In the univariate analysis type of breast surgery tumor gradeT stage N stage molecular subtype and HBsAg status wereFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancerindividual patient Figures 6AB The model™s explanatorycovariables consisted of HBsAg status T stage N stage andmolecular subtype Patients with higher scores correspondedto inferior survival The scatter plots for the TPS of DFSand OS and percentage of patient number were presentedin Figures 6CD The Cindex values for DFS and OS were CI “ and CI “ respectively The calibration curves for the probabilityof DFS and OS at or year presented an optimalagreement between the prediction by nomogram and actualobservation Supplementary Figure Next we divided thepatients into the following groups based on the TPS ofthe nomogram modelfor DFS using the Xtile programlowrisk group TPS “ patients intermediateriskgroup TPS “ patients and highrisk group TPS patients The 10year DFS for lowrisk groupintermediaterisk group and highrisk group were and respectively Survival analyses for DFS demonstratedsignificant discrimination between these three groups P Figure 7A Same procedures were performed for OS in theentire population and patients were divided into the following groups based on the TPS of the nomogram model for OS withthe Xtile program lowrisk group TPS “ patientsintermediaterisk group TPS “ patients and highrisk group TPS patients OS diï¬erences were alsoobserved among three subgroups with a 10year OS of and for lowrisk intermediaterisk and highrisk groupsP Figure 7BDISCUSSIONIn this study we investigated the association of HBsAg statusand very young breast cancer and to our knowledge reportfor the firsttime that HBsAgpositive status is associatedwith inferior DFS and OS from a population with a highprevalence of both HBV infection and young breast canceridentified as a significant unfavorableHBsAg status wasprognostic predictor for DFS and OSindependent of anyother clinicopathological features of breast cancer includingT stage N stage and molecular subtype We also integratedHBsAg to build nomograms to better predict prognosis foryoung patients with breast cancer The results of our studydemonstrated that the prevalence of HBsAg in young patientswith breast cancer in southern China was which wasin accordance with the “ reported in the populationof this endemic area This result suggests that unlikecervical cancer young breast cancer is not correlatedwith an increased prevalence of HBV infection Indeed breastcancer patients with HBsAg did not demonstrate a diï¬erentpattern of characteristics It is noteworthy that in our studyHBsAg did not increase the rate of liver metastases for veryyoung patients with breast cancer This results are comparableto those reported in previous studies for esophageal cancerand colorectal cancer which suggested that HBVinfection is associated with decreased risk of liver metastasis inthese malignanciesFIGURE Kaplan“Meier curves for A diseasefree survival and B overallsurvival stratified by HBsAg status in very young patients with breast canceridentified as significant prognostic factors for DFS Figure 5AWhen those variables were further analyzed in the multivariateanalysis we found that T stage P N stage P molecular subtype P and HBsAg status P remained statistically significant indicating that theyare significant independent predictors for DFS Figure 5ABy the same methods for OSthe results showed that Tstage P N stage P molecular subtype and HBsAg status P were independentprognostic factors for OS Figure 5B HBsAgpositive status isan independent negative prognostic factor for survival in veryyoung breast cancerPrognostic Nomograms For Very YoungBreast Cancer PatientsTo better assess the DFS and OS of very young breastcancer patients prognostic nomograms for DFS and OS wereestablished respectively All the independent predictors of DFSand OS in the multivariate analysis were integrated into thenomogram models and and 10year survivals weregraphically computed according to the characteristics of anFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE Kaplan“Meier curves for A diseasefree survival and B overall survival stratified by molecular subtype in very young patients with breast cancerOur study shows that HBsAgpositive status is associatedwith inferior DFS and OS in young patients with curativelyresected breast cancer decreasing the 10year DFS and OS by and respectively The association between HBsAgpositive status and poor prognosis is in keeping with thatdemonstrated in nasopharyngeal carcinoma lung cancer and ovarian cancer However some studies regardingother cancers indicate that HBsAgpositive cancer patientshad a favorable survival as compared with HBsAgnegativepatients This discrepancy can be partly explainedby the diversity and heterogeneity of diï¬erent malignanciesThe genetic or biological mechanisms underlying the inferiorFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE Univariate and multivariate analysis for diseasefree survival A and overall survival B for very young patients with breast cancerprognosis remain to be elucidated butthis may largelyrelate to the presence of hepatitis B Xinteracting proteinHBXIP which has been welldocumented to function asan oncoprotein in breast cancer HBXIP can act as atransactivator by activating certain genes including cMyc E2F1STAT4 and Sp1 to play a crucial role in the progression ofbreast cancer HBXIP is associated with controlling cellapoptosis and promoting cell proliferation by mTOC1 activation HBXIP can also act as a modulation factor of cellularoxidative stress by competitively binding KEAP1 to enhancethe progression of breast cancer Previously studies showedthat HBV is not associated with risk of breast cancer These results combined with the data of our study suggestthat HBV is not a risk factor but a prognostic factor forbreast cancerAnother reason for this might relate to the HBV reactivationin HBsAgpositive patients with breast cancer who werereceiving chemotherapy HBV reactivation occurs frequentlyin breast cancer patients who are HBV carriers while receivingcytotoxic chemotherapy HBV reactivation can result inliver failure and interruption of the chemotherapy scheduleOther potential mechanisms underlyingassociationtheFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE AB Nomograms predicting and 10year A diseasefree survival and B overall survival for very young patients with breast cancer CD Thescatter plots of percentage of patient number and groups of C total prognostic score for DFS and D total prognostic score for OSbetween the HBsAg and patient survival include the immunesuppression Chronic HBV infection is characterized by thefailure to elicit an eï¬ective adaptive immune response andthe immune modulation of key innate immune response Chronic HBV infection can lead to immune anergy andimpair the function of the immune system which has longbeen deemed to protect the host against the developmentof nonviral cancerstogether couldpartially explain the positive association between HBsAgpositive status and the poor prognosis in young patients withbreast cancer These reasonsInthisstudy wecharacteristicscombined HBsAgstatus withclinicopathologicaleï¬ectiveprognostic nomogram models of DFS and OS for very youngpatients with breast cancer Both nomograms showed goodcalibration and acceptable discrimination These nomogrammodels can be used for prognosis evaluation at diagnosis forvery young patients with breast cancer and may benefit patientestablishtocounseling and personalized therapy for these patients Weadopted Xtile program to divide these patients into three riskgroups based on TPS from nomograms for DFS and OS Thesurvival curves for DFS and OS separated very well Thusspecial attention should be paid to and active surveillanceshould be conducted over patients with high risk group for DFSand OSThis study nevertheless has certain limitations that shouldbe noted First this study was retrospective in nature andwe cannot rule outthe impact of selection bias Secondthe sample size was relatively small and the sample sizesof the two cohorts were unequal as only patients wereHBsAgpositive The small sample size may be insufficientto allow us to perform subgroup analysis by each molecularsubtype Anotherthat Cantonese constitutemost of our study population The monotonicity ofthestudy population confines the universality of our resultsFurthermore the information was insufficient to perform otherlimitation isFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE Kaplan“Meier curves for A diseasefree survival and B overall survival stratified by risk groups based on total prognostic scores fromnomogram modelsanalysis such as that of hepatic function and HBV DNAcopy number Nevertheless the results of our study providewhat to our knowledge is the first evidence of the impactof HBsAg on the prognosis of very young patients withbreast cancerIn conclusion we have demonstrated in a retrospectivestudy that HBsAg is an independent unfavorable prognosticfactor for patients with very young breast cancer Furtherprospective studies involving varied ethnic populations arewarranted to confirm the prognostic value of HBsAg status invery young breast cancer and simultaneously other potentialclinicopathologic factors for breast cancer and HBV infection arerequired to be taken into account The mechanisms of the impactof HBV infection on the progression of breast cancer also need tobe elucidatedDATA AVAILABILITY STATEMENTThe datasets generated for this study are available on request tothe corresponding authorETHICS STATEMENTThe studiesinvolving human participants were reviewedand approved by the Medical Ethics Committee of SunYatsen University Cancer Center WritteninformedFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancerconsentin accordance with the nationalinstitutional requirementsfor participation was not required for this studylegislation and theAUTHOR CONTRIBUTIONSNL QQZ LY and WDW designed the study NL QQZXRY QCW DTZ and SZ collected the data NL QQZ LYand WDW interpreted and analyzed the data All authors wereinvolved in writing the and approved the final versionREFERENCES Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Globalcancer statistics GLOBOCAN estimates of incidence and mortalityworldwide for cancers in countries CA Cancer J Clin “ 103322caac21492 Fan L StrasserWeippl K LiJJ St LouisJ Finkelstein DM Yu 15e279“KD et al Breast cancer 101016S1470204513705679in China Lancet Oncol Colleoni M Rotmensz N Robertson C Orlando L Viale G Renneet al 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lymphoma a systematic review and metaanalysisInternal Med J “ 101111j14455994200902060x Luo G Hao NB Hu CJ Yong X Lu MH Cheng BJ et al HBV infectionincreases the risk of pancreatic cancer a metaanalysis Cancer Causes Control “ 101007s1055201201442FUNDINGThis work was supported by the National Natural ScienceFoundation of China No SUPPLEMENTARY MATERIALThe Supplementary Materialonline202001403fullsupplementarymaterialfor this can be foundhttpswwwfrontiersins103389foncat Wei XL Qiu MZ Jin Y Huang YX Wang RY Chen WW et al Hepatitis Bvirus infection is associated with gastric cancer in China an endemic area ofboth diseases Br J Cancer “ 101038bjc2014406 Ye YF Xiang YQ Fang F Gao R Zhang LF Xie SHHepatitis B virusin southern China Cancer Epidemiol Biomarkers Prevent“ 10115810559965EPI150344et alinfection and risk of nasopharyngeal carcinoma Peng JW Liu DY Lin GN Xiao JJ Xia ZJ Hepatitis B virusinfection is associated with poor prognosis in patients with advancednon small cell“ 107314APJCP201516135285lung cancer Asian Pacific J Cancer Prevent Zou J Chen J Xie X Liu Z Cai X Liu Q et al Hepatitis B virus infectionis a prognostic biomarker for better survival in operable esophageal canceranalysis of patients from an endemic area in China Cancer EpidemiolBiomarkers Prevent “ 10115810559965EPI181095 Wong L Cheung TH Yim SF Lao TT Prevalence and impact of hepatitis Bvirus infection in ovarian cancer patients in an endemic areaA retrospectivecohort study J Viral Hepat “ 101111jvh13250 Song C Lv J Liu Y Chen JG Ge Z Zhu J et al Associations between hepatitisB virus infection and risk of all cancer types JAMA Netw Open 2e195718 101001jamanetworkopen20195718 Su FH Chang SN Chen PC Sung FC Su CT Yeh CC Associationrisk abetween chronic viral hepatitisinfection and breast cancernationwide populationbased casecontrol study BMC Cancer Qin B Zhao K Wei J Wang X Xu M Lang J et al Novel evidence indicatesthe presence and replication of hepatitis B virus in breast cancer tissue OncolRep “ 103892or20197393 Te HS Jensen DM Epidemiology of hepatitis B and C viruses a globaloverview Clinics Liver Dis “ vii 101016jcld200911009 Siu SS Cheung TH Chan PK Lin CK Lo KW Patients with malignant or premalignant cervical lesion have increased risk of becoming hepatitis B carrierJ Exp Clin Cancer Res “in patients with colorectal Zhao Y Lin J Peng J Deng Y Zhao R Sui Q et al Hepatitis B virus infectionpredicts better survivalliveronly metastasesundergoing liver resection J Cancer “ 107150jca24544 Liu X Li X Jiang N Lei Y Tang LL Chen L et al Prognostic value ofchronic hepatitis B virus infection in patients with nasopharyngeal carcinomaanalysis of patients from an endemic area in China Cancer “ 101002cncr28377 Zhao Y Li H Zhang Y Li L Fang R Li Y et al Oncoprotein HBXIPmodulates abnormal lipid metabolism and growth of breast cancer cells byactivating the LXRsSREBP1cFAS signaling cascade Cancer Res “ 10115800085472CAN151734 Zhang Y Zhao Y Li H Li Y Cai X Shen Y et al The nuclear import ofoncoprotein hepatitis B Xinteracting protein depends on interacting with cFos and phosphorylation of both proteins in breast cancer cells J Biol Chem “ 101074jbcM113458638 BarPeled L Schweitzer LD Zoncu R Sabatini DM Ragulator is a GEFfor the rag GTPases that signal amino acid levels to mTORC1 Cell “ 101016jcell201207032 Zhou XL Zhu CY Wu ZG Guo X Zou W The oncoproteinHBXIP competitively binds KEAP1 to activate NRF2 and enhanceFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancercancerbreast“ 101038s4138801906985growthcelland metastasis Oncogene Liu Z Jiang L Liang G Song E Jiang W Zheng Y et al Hepatitis B virusreactivation in breast cancer patients undergoing chemotherapy a reviewand metaanalysis of prophylaxis management J Viral Hepat “ 101111jvh12672 Yuen MF Chen DS Dusheiko GM Janssen HLA Lau DTY LocarniniSA et al Hepatitis B virus infection Nat Rev Dis Primers 101038nrdp201835 Dunn GP Old LJ Schreiber RD Theimmunobiology ofimmunosurveillance“ 101016jimmuni200407017immunoeditingandImmunitycancer Giuliano AE Edge SB Hortobagyi GN Eighth edition ofthe AJCCcancer staging manual breast cancer Ann Surg Oncol “ 101245s1043401864866 Schemper MSmith TL A note on quantifyingstudies 101016019724569600075Xfailuretime Control ClinofTrialsfollowup in“ Vickers AJ Elkin EB Decision curve analysis a novel method for “evaluating prediction models Med Decision Making 1011770272989X06295361 Camp RL DolledFilhart M Rimm DL Xtile a new bioinformatics toolfor biomarker assessment and outcomebased cutpoint optimization ClinCancer Res “ 10115810780432CCR040713Conflict of Interest The authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestCopyright Li Zhong Yang Wang Zhang Zheng Yang and Wei This is anopenaccess distributed under the terms of the Creative Commons AttributionLicense CC BY The use distribution or reproduction in other forums is permittedprovided the original authors and the copyright owners are credited and that theoriginal publication in this journal is cited in accordance with accepted academicpractice No use distribution or reproduction is permitted which does not complywith these termsFrontiers in Oncology wwwfrontiersinAugust Volume 0c'

Thyroid_Cancer

Association of variant on the promoter of cluster ofdifferentiation in graves disease and gravesophthalmopathyYuHuei Liu123 ChiouYuan Shen1 and FuuJen Tsai3451Graduate Institute of Integrated Medicine China Medical University Taichung Taiwan 2Drug development center China Medical University Taichung Taiwan 3Department ofMedical Genetics and Medical Research China Medical University Hospital Taichung Taiwan 4Department of Pediatrics China Medical University Hospital Taichung Taiwan5School of Chinese Medicine China Medical University Taichung TaiwanCorrespondence YuHuei Liu yuhueiliumailcmuedutwThe macrophage migration inhibitory factor MIFcluster of differentiation CD74plays a role in immunological functions The present study aims to investigate whethersinglenucleotide polymorphisms SNPs in the MIF and CD74 are risk factors for developing Graves ophthalmopathy GO in patients with Graves disease GD A case“controlstudy enrolled patients with GD with and without GO and healthy individuals SNPs were discriminated using realtime polymerase chain reaction Hardy“Weinbergequilibrium as well as frequencies of allele and genotype between GD patients with andwithout GO were estimated using the Chisquare test The effects of CD74 on adipocyteproliferation and differentiation were evaluated using 3T3L1 preadipocytes QuantitativeDNAimmunoprecipitation was used to detect the binding capacity of NR3C1 and FOXP3to AG oligonucleotides The results showed that individuals carrying the GG genotype atrs2569103 in the CD74 had a decreased risk of developing GD P3390 — ˆ’ oddsratio OR confidence interval CI “ however patients with GDcarrying the AG genotype at rs2569103 in the CD74 had an increased risk of developing GOP0009 OR CI “ The knockdown of CD74 reduced adipocyteproliferation and differentiation NR3C1 had a higher affinity for A whereas FOXP3 had ahigher affinity for G of rs2569103 The results suggested the existence of a link between thegenetic variation of CD74 promoter and the risk for developing GD and GO which shouldbe considered in clinical practiceBackgroundGraves disease GD a complex autoimmune disorder that occurs more often in women is characterized by the presence of autoantibodies and thyroidstimulating immunoglobulins targeting thethyroidstimulating hormone receptor to stimulate both thyroid hormone synthesis and thyroid glandgrowth and results in hyperthyroidism and its accompanying features [“] Graves ophthalmopathyGO is one common anspecific complication affecting “ of patients with GD [] Activation oforbital fibroblasts through proliferation and differentiation into adipocytes and myofibroblasts is thoughtto play a major role in the generation of the extracellular matrix During inflammatory cell infiltrationand edema the activation augments the volume of tissues surrounding the eyes which in turn leads to anincrease in intraocular pressure []Genetic predispositions epigenetic regulations and environmental factors are risk factors for GD andGO [“] Representative studies shed new light on the pathogenesis of GD such as thyroid antigensthyroidstimulating hormone receptor and human leukocyte antigen HLA class I and II regions []However the genomewide approaches to determining the relative risks of developing GO are relativelyReceived June Revised July Accepted July Accepted Manuscript online August Version of Record published August The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072limited [] Candidate gene approaches revealed that polymorphisms of genes involved in immune response andinflammation might be linked to the development of GO [“]Cluster of differentiation CD74 encoded by CD74 is an HLA class II histocompatibility antigen gamma chainalso known as HLADR antigenassociated invariant chain and a signaltransducing receptor of macrophage migration inhibitory factor MIF that maintains cell proliferation and survival [] The singlenucleotide polymorphisms SNPs in HLA class II and MIF play a role in the development of GD [“] Conversely the chromosome5q3133 region where CD74 is located 5q32 may play a pivotal role in the development of GD and could be thesusceptibility region for developing GD [] Results from mRNASeq also reveal CD74 as a novel signature fD However to our knowledge there is no study on the putative impact of CD74 locus variations on the risk ofGD or GO In an attempt to contribute to the understanding of the pathogenic processes underlying GD and GO acase“control study was designed to evaluate the association between SNPs in the upstreamdownstream regulatoryregion of the MIFCD74 axis and the risk of developing GD and GOMethodsPatients healthy individuals and DNA isolationThe study followed the Declaration of Helsinki and was approved by the Medical Ethics Committee of China MedicalUniversity Hospital DMR100IRB144 CMUH103REC2071 A total of patients with GD females100males mean age y range “ y at enrollment from the China Medical University Hospital and patients had GO and did not All participants provided written informed consent Detailed descriptions of theinclusionexclusion criteria blood drawing and handling genomic DNA storage and quality assurance have beendescribed [] SNP data for ethnicitymatched healthy individuals were obtained from the Taiwan biobankSNP selection and genotypingSNPs were selected based on the following criteria i a threshold minor allele frequency MAF in the Asian population of ii primerprobe set passed by the manufacturer criteria to ensure a high genotyping success rate andiii SNP data for healthy individuals could be obtained without imputation from the Taiwan biobank Four SNPsnamely rs476240 and rs507715 in the downstream region of MIF which is also the upstream region of MIF antisense RNA [MIFAS1] as well as rs13175409 and rs2569103 in the upstream region of CD74 were analyzedGenotyping using specific primerprobe sets have been described previously []Cell cultureThe human HEK293 cells and mouse 3T3L1 preadipocytes were obtained from Bioresource Collection and Research Center BCRC Hsinchu Taiwan and maintained in Dulbecco™s modified Eagle™s medium DMEM Thermo Fisher Scientific Waltham MA USA with fetal bovine serum Uml penicillin and μgml streptomycin and mM Lglutamine at —¦C in a humidified atmosphere of CO2CD74 knockdownShort hairpin RNAs shRNAs obtained from the RNAi core Academia Sinica Taipei Taiwan were used in CD74knockdown experiments For CD74 knockdown confluent 3T3L1 preadipocytes in sixwell dishes were incubated inOptiMEM Thermo Fisher Scientific and transfected with either CD74 shRNA or nonspecific shRNA using Lipofectamine Thermo Fisher Scientific according to the manufacturer™s protocol After h the medium was replacedwith complete DMEM with a differentiation cocktail μM 3isobutyl1methylxanthine μM dexamethasoneand μM insulin to induce differentiation into mature adipocytes day Western blottingEqual amounts of protein lysates were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis andthen transferred to polyvinylidene fluoride membranes After blocking with skim milk the membranes wereincubated with primary antibodies and subsequently with appropriate peroxidaseconjugated secondary antibodiesPrimary antibodies including targets catalog numbers dilutions and suppliers were as follows antibodies specific toCD74 GTX110477 were from GeneTex Hsinchu Taiwan and antibodies specific to actin MAB1501 were from MilliporeSigma St Louis MI USA The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Adipocyte differentiationThe 2day postconfluency preadipocytes were cultured in complete DMEM with a differentiation cocktail μM3isobutyl1methylxanthine μM dexamethasone and μM insulin On day of differentiation cells wereswitched to complete DMEM with μM insulin for the remaining duration of differentiationCell counting3T3L1 cells were detached from sixwell plates using trypsin Thermo Fisher Scientific resuspended in complete DMEM and counted using a cell counter Millipore every day from day “Oil Red O stainingDifferentiated adipocytes were fixed in formalin and stained for min with Oil Red O MilliporeSigma working solution Oil Red O dye in isopropanol Oil Red O was extracted using isopropanol and theabsorbance was measured at nm using a spectrophotometerCell culture and extraction of nuclear proteins from established NR3C1FOXP3 and CD74 transformantsCells were transfected with the pCMV3ˆ’Cˆ’Mycˆ’NR3C1 pCMV3ˆ’Cˆ’Mycˆ’FOXP3 or pCDNA4CD74 usingthe Lipofectamine kit Thermo Fisher Scientific according to the manufacturer™s protocol The nuclear proteinswere extracted using NEPER nuclear and cytoplasmic extraction reagents Thermo Fisher Scientific supplementedwith protease inhibitor cocktail and phosphatase inhibitors Roche Basel Switzerland according to the manufacturer™s protocolQuantitative DNA immunoprecipitation qDNAIP assayqDNA“IP assays were performed on nuclear extracts from established FOXP3 and NR3C1 transformantsDNA binding of FOXP3 or NR3C1 was assessed using the annealed double strand oligonucleotides 5cid3biotinlabeled rs2569103A probes 5cid3CCAAATGGCTGGTTTCAGGGCTGGAGATGGGGG3cid3 and 5cid3CCCCCATCTCCAGCCCTGAAACCAGCCATTTGG3cid3 as well as 5cid3biotinlabeled rs2569103G probes 5cid3CCAAATGGCTGGTTTCGGGGCTGGAGATGGGGG3cid3 and 5cid3CCCCCATCTCCAGCCCCGAAACCAGCCATTTGG3cid3 PURIGOBiotechnology Taipei Taiwan For the binding reactions μg of nuclear proteins were incubated with or without labeled oligonucleotides in binding buffer [ mM Tris“HCl pH mM NaCl mM MgCl2 mMEDTA mM DTT mgml polydI“dC and glycerol] for min at —¦C in a final volume of μl FOXP3“ or NR3C1“nucleotide complexes were crosslinked with formaldehyde final concentration for min at room temperature followed by immunoprecipitation with antibodies specific to Myc tag GTX115046 GeneTex and Protein AG magnetic beads GE Healthcare Immunoprecipitated DNA was detected usinghorseradish peroxidaseconjugated streptavidin The reaction was developed with the 33cid355cid3tetramethylbenzidinereagent Sigma and read at nm with a Microplate reader BioRad Hercules CA USAStatistical analysesThe statistical analyses were performed using the PASW Statistics software from IBM Armonk NY USAA ttest was used to evaluate the associations between GO and age A Chisquare test was used to evaluate the associations between polymorphisms and GD or GO Screening for linkage disequilibrium LD was performed usingHaploview ver [] A twotailed Pvalue less than with Bonferroni correction was considered statistically significant [] Logistic regression with a confidence interval CI was used to estimate odds ratiosORsResultsDemographic data clinical characteristics and their correlations withGO in patients with GDThe frequency distributions of clinical characteristics such as goiter nodular hyperplasia myxedema vitiligo andage in male and female groups were compared between the patients with GD with or without GO As demonstratedin Table gender and age were significantly associated with GO in patients with GD Even myxedema was associatedwith GO in patients with GD however due to a limited number of cases the association needs further investigation The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Table Demographic data and clinical characteristics of graves disease patients with or without graves ophthalmopathyCharacteristicGDnonGO N GDGO N PNumber of patientsFemale genderAge of diagnosis Year Mean ˆ’ SD[Range]Presence of goiterNo1a1bPresence of nodular hyperplasiaPresence of myxedemaPresence of vitiligoWith radioiodine therapy historyWith thyroid surgery historyWith smoke historyFree T3 pgmlFree T4 ngdlT3 ngdlT4 μgdlTSH μIUmlTRAb positive ˆ’ [ˆ’] ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ [ˆ’] ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ Abbreviations GD graves disease GO graves ophthalmopathy N numberaFrequencies of genotypes were determined by the chisquare test using — or — contingency tablesbSignificance of age were evaluated by t testP005P00010039a — 105b 0165a0539a0039a 0743a0273a0227a0527a0900a0692a0146a0310a0479a0482aThese results adhered to other epidemiological results that GO occurred more commonly in the middleaged femalepopulationLD among SNPs of MIF and CD74Four SNPs of the MIF and CD74 were genotyped to determine whether polymorphisms in these genes influencethe development of GO in patients with GD The distribution of the four SNPs fit the Hardy“Weinberg equilibriumHWE in patients with GD and healthy individuals However the strong r208 LD r2 values calculated for thetwo SNPs at the CD74 in healthy individuals were not observed in patients with GD with or without GO suggestingthat there is more variation in the extent of LD within CD74 in patients with GD Figure Allele and genotype distributions of CD74 contribute to GDGOdevelopmentNo significant association was found in the examined SNPs of MIF nor was a significant association found betweenthe polymorphisms and the clinical features or the indicators of thyroid function including free triiodothyronineT3 free thyroxine T4 thyroid stimulating hormone TSH and thyrotropin receptor antibodies TRAbs in patients with GD However allele frequencies showed that individuals carrying a G allele at rs2569103 in the CD74 hada reduced risk of developing GD P0005 OR CI “ Table Genotype frequenciesfurther showed that individuals carrying the GG genotype at rs2569103 in the CD74 had a reduced risk of developing GD P3390 — ˆ’ OR CI ˆ’ which was consistent with results from allelefrequencies however the patients with GD carrying the AG genotype at rs2569103 in the CD74 had an increasedrisk of developing GO P0009 OR CI ˆ’ Table The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Figure Linkage disequilibrium LD values between the two polymorphisms rs13175409 and rs2569103 in the CD74region in a TaiwaneseChinese populationThe color scale reflects the strength of LD between the two single nucleotide polymorphisms SNPs A Healthy individuals BPatients with Graves disease GD with and without Graves ophthalmopathy GO C Patients with GD without GO D Patientswith GD with GOTable Allele distributions of MIF and CD74GenotypesControl N GDnonGO NGDGO N Control vs GDPaControl vs GDOR 95CINonGO vsGO PaNonGO vsGO OR95CIMIF rs476240AGMIF rs507715ACCD74 rs13175409CTCD74 rs2569103AG ˆ’0929bAbbreviations CI confidence interval GD graves disease GO graves ophthalmopathy N number OR odds ratiosaFrequencies of genotypes were determined by the chisquare test using — or — contingency tablesbOdds ratios and CI per genotype were estimated by applying unconditional logistic regressionP005 with Bonferroni correction OR with significanceKnockdown of the expression of CD74 inhibits 3T3L1 adipocytedifferentiationThe swelling of extraocular orbital fat is one reason that the development of GO is triggered [] To understand thepossible regulation between CD74 and adipocyte differentiation 3T3L1 cells were chosen as an experimental modelThe expression of CD74 in CD74 knockdown CD74KD cells by shRNA was confirmed as compared with those withcontrol of shRNA Figure 2A Cell numbers of CD74KD and control cells were counted every day The knockdownof CD74 decreased cell proliferation from “ days after induction Figure 2B In addition the degree of Oil Red The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Table Genotype distributions of MIF and CD74GenotypesControl N GDnonGO NGDGO N Control vs GDP aControl vs GDOR 95CINonGO vsGO P aNonGO vsGO OR95CIMIF rs476240AAAGGGMIF rs507715AAACCCCD74 rs13175409CCCTTTCD74 rs2569103AAAGˆ’2495cGG — ˆ’ bˆ’0154bˆ’2467bˆ’Abbreviations CI confidence interval GD graves disease GO graves ophthalmopathy N number OR odds ratiosaFrequencies of genotypes were determined by the chisquare test using — or — contingency tablesbOR and CI per genotype were estimated by applying unconditional logistic regressioncOR and CI per genotype were estimated by adjusting with gender age and myxedemaP005 with Bonferroni correctionOR with significanceFigure Changes in adipocyte differentiation and proliferation after knockdown of CD74A Endogenous expression of CD74 protein in 3T3L1 cells was examined and knockdown of CD74 was examined by Westernblotting Actin was used as an internal control B The downregulation of CD74 inhibits cell growth 3T3L1 cells were detachedfrom sixwell plates and counted P001 P0001 CD74 knockdown vs control cells C Cells were stained with Oil Red Oafter inducing differentiation Quantitative analyses were performed by measurement of optical density OD at nm in extractsfrom Oil Red Ostained cells transfected with CD74 short hairpin RNA shRNA and control shRNA P0001 CD74 knockdownvs control cellsO staining was weaker in CD74KD cells than in control cells on day and on day respectively forCD74 shRNA vs control cells Figure 2C The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Different binding affinities of NR3C1 and FOXP3 for CD74 promoterdepends on SNP rs2569103The CD74 SNP rs2569103 was located within the upstream region of CD74 and showed the strongest associationwith the disease making it a possible target for transcription factors Indeed the putative transcription factorbindingsites were predicted using PROMO [] At SNP rs2569103 the A allele generates motifs for nuclear receptorsubfamily group C member NR3C1 TCAGG whereas the G allele generates a motif for forkhead box P3FOXP3 GTTTCG Bulk RNAseq analysis of NR3C1 and FOXP3 in thyroid and fat tissues from public datasetsPRJEB4337 were demonstrated Figure 3A To interpret the possible regulatory mechanisms of these moleculespublished mRNA expression results were explored The mRNA expression of NR3C1 only showed a negative correlation with that of CD74 in thymoma samples Pearson™s correlation ˆ’ Spearman™s correlation ˆ’ Figure3B whereas the mRNA expression of FOXP3 showed a positive correlation with that of CD74 Pearson™s correlation Spearman™s correlation in thymoma samples welldifferentiated papillary thyroidcarcinoma and welldifferentiated thyroid cancer respectively Figure 3C“E The qDNAIP results supported thatNR3C1 tends to bind to probes with promoter sequence containing AA at rs2569103 whereas FOXP3 tends to bindto probes with promoter sequence containing GG at rs2569103 Figure 3F These results suggested that the CD74expression may be orchestrated by complex transcription factor networks The AA genotype may play a role in response to NR3C1induced CD74 downregulation whereas the GG genotype on rs2569103 on the CD74 promotermay play an additional role in response to FOXP3induced CD74 upregulationDiscussionEnvironmental factors and genetic loci have been thought to be associated with immune regulation [] Here weidentified new candidates CD74 alleles and genotypes for the susceptibility of GD and GO in a TaiwaneseChinesepopulation CD74 is involved in adipocyte differentiation through its differential promoter binding affinity for transcription factors To the best of our knowledge this is the first study to demonstrate novel CD74 polymorphisms inassociation with the development of GD and GO Our results support wholegenome screening studies in that thechromosome 5q32 may play a role in generating GD and GO in humansThe thyroid gland of patients with GD revealed marked enlargement of the gland due to autoantibodies Patientswith accompanying GO exhibited enlargement of the retroorbital connective tissue and extraocular muscles inpart due to the inflammatory deposition of glycosaminoglycans collagen and fat [] Indeed genes involved inthe regulation of cell survival DNA transcription and protein synthesis have been considered risk factors for GDand GO [] Overexpression of CD74 plays a crucial role in preventing hyperreactivity between immature antigens and major histocompatibility complex class II as well as cell growth and survival whereas downregulation ofCD74 is often correlated with autoimmunity and cell apoptosis [] Upon expression of surface CD74 the cellsmay transduce survival signaling through extracellular signalregulated kinase or cJun Nterminal kinase JNKmitogenactivated protein kinase MAPK pathways or AKT pathways in a MIFdependent manner thereby improving cell survival and proliferation [] Due to the limitation to find identical cells expressed GG or AA genotypeon rs2569103 current results we did not show the direct impact of these transcription factors to the CD74 expression Further evidence such as RNAseq as secondary data was warranted The results showed that GD patients withor without GO although loss the protective GG genotype most of them hold AG heterogenous genotype insteadsuggested the lossofprotect effect on the disease In the present study cellbased experiments showed that CD74 isinvolved in adipocyte differentiation but the link toward GO development remained to be investigated On the otherhand the GG genotype on rs2569103 with a higher frequency in healthy individuals Table increased the bindingof FOXP3 to the CD74 promoter Figure 3F thereby increasing CD74 upregulation and protecting autoimmuneresponses Conversely the AA genotype on rs2569103 increases the binding of NR3C1 to the CD74 promoter whichdownregulates CD74 and increases autoimmune response and manifestations of GDGO Due to the limitation tofind identical cells expressed GG or AA genotype on rs2569103 current results we did not show the direct impactof these transcription factors to the CD74 expression Further evidence such as RNAseq as secondary data was warranted The results showed that GD patients with or without GO although they lost the protective genotype mostof them hold the AG heterogenous genotype instead suggesting the lossofprotection effect of the disease Furtherstudies on the detailed mechanisms through CD74derived adipocyte differentiation are warrantedConversely the ligand of CD74 MIF has previously been reported to be counterregulatory to glucocorticoid secretion [“] The glucocorticoidinduced MIF secretion was noted at min after dexamethasone administration[] In addition nonsteroidal antiinflammatory drugs such as aspirin ibuprofen and naproxen have been used torelieve the pain and inflammation of GO This evidence further supports the crucial role of CD74 in the transduction The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Figure Different binding affinities of NR3C1 and FOXP3 for CD74 promoter depends on singlenucleotide polymorphismSNP rs2569103A RNAseq analysis of NR3C1 and FOXP3 in thyroid and fat tissues from public datasets PRJEB4337 B“E Bioinformaticanalysis of mRNA expression correlation between NR3C1 and CD74 or FOXP3 and CD74 The mRNA expression of NR3C1 andCD74 in thymoma samples B and the mRNA expression of FOXP3 and CD74 in thymoma samples C welldifferentiated papillarythyroid carcinoma D and welldifferentiated thyroid cancer E F Probe with promoter sequence containing rs2569103 probe Ahas a higher affinity for NR3C1 whereas G at rs2569103 probe G has a higher affinity for FOXP3 as shown by quantitative DNAimmunoprecipitation qDNAIP assay P001 P0001 probe A vs probe G The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072of MIF signaling However due to the limited population of the minor polymorphism the present study is unable toreach the interactions among cells and molecules in the orbital microenvironment and their association toward thetarget polymorphism due to the inaccessibility of the orbital tissues The current finding may have further implications for understanding the link between the polymorphismexpression of CD74 and current treatments for GO”atherapeutic effect issue that might be of value for future treatment strategies targeting MIF or CD74In conclusion the current study identified new SNPs in the CD74 that were found to be associated with GD and GOin a TaiwaneseChinese population Biological studies provide insights into the genetic information that influencesthe development of GD and GO via adipocyte proliferation and differentiationPerspectives¢The impact of genetic factors on the orbital microenvironment cannot be closely monitored due tothe inaccessibility of the orbital tissue Studies on feasible cellbased models may help elucidate howgenetic factors such as CD74 SNPs modulate the target gene expression¢¢The present study combined clinical observations and cell models to investigate how CD74 polymorphisms affect adipocyte proliferation and differentiationThe present clinical observations suggest that the genetic factors of CD74 should be considered inclinical practiceCompeting InterestsThe authors declare that there are no competing interests associated with the manuscriptFundingThis work is supported by Ministry of Science and Technology Taiwan [grant numbers MOST 1042815C039002B and MOST1072320B039032MY3] the peak project and thematic project of Academia Sinica Taiwan the higher education sproutproject by the Ministry of Education MOE Taiwan via œDrug Development Center of China Medical University from The FeaturedAreas Research Center Program and China Medical University [grant numbers CMU105S33 and CMU106S46] TaichungTaiwanAuthor ContributionYHL proposed the concept designed the experiment anized the study wrote and reviewed the manuscript CYS performed the experiments FJT coordinated patient enrollment collected the clinical samples and applied official applicationAcknowledgementsWe thank Taiwan Biobank for providing related data all anonymous for our research The sponsorfunding anization had norole in the design or conduct of this researchAbbreviationsCD74 cluster of differentiation CI confidence interval FOXP3 forkhead box P3 GD graves disease GO graves ophthalmopathy HLA human leukocyte antigen HWE Hardy“Weinberg equilibrium JNK cJun Nterminal kinase LD linkagedisequilibrium MAPK mitogenactivated protein kinase MIF macrophage migration inhibitory factor NR3C1 nuclear receptorsubfamily group C member OR odds ratio PCR polymerase chain reaction qDNAIP quantitative DNA immunoprecipitation SNP singlenucleotide polymorphism T3 triiodothyronine T4 free thyroxine TRAb thyrotropin receptor antibody TSHthyroid stimulating hormoneReferences Smith TJ and Hegedus L Graves™ Disease N Engl J Med “ 101056NEJMra1510030 Brent GA Clinical practice Graves™ disease N Engl J Med “ 101056NEJMcp0801880 Ginsberg J Diagnosis and management of Graves™ disease CMAJ Canadian Med Assoc J J de l™Assoc Med Canadienne “ The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BY 0cBioscience Reports BSR20202072101042BSR20202072 McIver B and Morris JC The pathogenesis of Graves™ disease Endocrinol Metab Clin North Am “101016S0889852905702991 Bednarczuk T Gopinath B Ploski R and Wall JR Susceptibility genes in Graves™ ophthalmopathy searching for a needle in a haystackClin Endocrinol Oxf “ 101111j13652265200702854x Anvari M Khalilzadeh O Esteghamati A Esfahani SA Rashidi A Etemadi A et al Genetic susceptibility to Graves™ ophthalmopathy therole of polymorphisms in proinflammatory cytokine genes Eye Lond “ 101038eye2009244 Bahn RS Understanding the immunology of Graves™ ophthalmopathy Is it an autoimmune disease Endocrinol Metab Clin North Am “ vi Manji N CarrSmith JD Boelaert K Allahabadia A Armitage M Chatterjee VK et al Influences of age gender smoking and familyhistory on autoimmune thyroid disease phenotype J Clin Endocrinol Metab “ 101210jc20061402 Stan MN and Bahn RS Risk factors for development or deterioration of Graves™ ophthalmopathy Thyroid Official J Am Thyroid Assoc “ 101089thy20101634 Bahn RS and Heufelder AE Pathogenesis of Graves™ ophthalmopathy N Engl J Med “ Tomer Y Barbesino G Greenberg DA Concepcion E and Davies TF Mapping the major susceptibility loci for familial Graves™ andHashimoto™s diseases evidence for genetic heterogeneity and gene interactions J Clin Endocrinol Metab “ Tomer Y Ban Y Concepcion E Barbesino G Villanueva R Greenberg DA et al Common and unique susceptibility loci in Graves andHashimoto diseases results of wholegenome screening in a data set of multiplex families Am J Hum Genet “101086378588 Gianoukakis AG and Smith TJ Recent insights into the pathogenesis and management of thyroidassociated ophthalmopathy Curr OpinEndocrinol Diabetes Obesity “ 101097MED0b013e32830eb8ab Shiina T Ota M Shimizu S Katsuyama Y Hashimoto N Takasu M et al Rapid evolution of major histocompatibility complex class I genesin primates generates new disease alleles in humans via hitchhiking diversity Genetics “101534genetics106057034 Liu YH Chen YJ Wu HH Wang TY and Tsai FJ Single nucleotide polymorphisms at the PRR3 ABCF1 and GNL1 genes in the HLA class Iregion are associated with Graves™ ophthalmopathy in a genderdependent manner Ophthalmology “101016jophtha201404027 Wang S Sun H Chen HY Zhao ZF Yang Y Zhao YJ et al Intercellular adhesion molecule gene polymorphisms do not contribute toGraves™ disease in Chinese patients Endocrine “ 101007s1202000700329 Liu YH Chen RH Chen WC Tsai Y Wan L and Tsai FJ Disease association of the CD103 polymorphisms in Taiwan Chinese Graves™ophthalmopathy patients Ophthalmology “ 101016jophtha200912037 Bednarczuk T Hiromatsu Y Seki N Ploski R Fukutani T Kurylowicz A et al Association of tumor necrosis factor and human leukocyteantigen DRB1 alleles with Graves™ ophthalmopathy Hum Immunol “ 101016jhumimm200402033 Khalilzadeh O Anvari M Esteghamati A Mahmoudi M Tahvildari M Rashidi A et al Graves™ ophthalmopathy and gene polymorphisms ininterleukin1alpha interleukin1beta interleukin1 receptor and interleukin1 receptor antagonist Clin Exp Ophthalmol “ Siegmund T Usadel KH Donner H Braun J Walfish PG and Badenhoop K Interferongamma gene microsatellite polymorphisms inpatients with Graves™ disease Thyroid Official J Am Thyroid Assoc “ 101089thy199881013 Wong KH Rong SS Chong KK Young AL Pang CP and Chen LJ Genetic Associations of Interleukinrelated Genes with Graves™Ophthalmopathy a Systematic Review and Metaanalysis Sci Rep 101038srep16672 Bucala R and Shachar I The integral role of CD74 in antigen presentation MIF signal transduction and B cell survival and homeostasis MiniRev Med Chem “ 1021741389557515666150203144111 Leng L Metz CN Fang Y Xu J Donnelly S Baugh J et al MIF signal transduction initiated by binding to CD74 J Exp Med “ 101084jem20030286 Liu YH Chen CC Yang CM Chen YJ and Tsai FJ Dual effect of a polymorphism in the macrophage migration inhibitory factor gene isassociated with newonset Graves disease in a Taiwanese Chinese population PLoS ONE e92849 101371journalpone0092849 Nakabayashi

Thyroid_Cancer

The effects of tissue fixation on sequencingand transcript abundance of nucleic acidsfrom microdissected liver samples ofsmallmouth bass Micropterus dolomieuHeather L WalshID Adam J Sperry Vicki S BlazerUS Geological Survey National Fish Health Research Laboratory Leetown Science Center KearneysvilleWest Virginia United States of Americaa1111111111a1111111111a1111111111a1111111111a1111111111 hwalshusgsgovAbstractThere is an increasing emphasis on effectsbased monitoring to document responses associated with exposure to complex mixtures of chemicals climate change pathogens parasitesand other environmental stressors in fish populations For decades aquatic monitoring programs have included the collection of tissues preserved for microscopic pathology Consequently formalinfixed paraffinembedded FFPE tissue can be an important reservoir ofnucleic acids as technologies emerge that utilize molecular endpoints Despite the crosslinking effects of formalin its impact on nucleic acid quality and concentration amplification andsequencing are not well described While freshfrozen tissue is optimal for working withnucleic acids FFPE samples have been shown to be conducive for molecular studies Lasercapture microdissection LCM is one technology which allows for collection of specificregions or cell populations from fresh or preserved specimens with pathological alterationspathogens or parasites In this study smallmouth bass Micropterus dolomieu liver was preserved in three different fixatives including neutral buffered formalin NBF ZFix®ZF and PAXgene® PG for four time periods hr hr seven days and days Controls consisted of pieces of liver preserved in RNALater® or ethanol Smallmouth basswere chosen as they are an economically important sportfish and have been utilized as indicators of exposure to endocrine disruptors and other environmental stressors Small liversections were cut out with laser microdissection and DNA and RNA were purified and analyzed for nucleic acid concentration and quality Sanger sequencing and the NanoStringnCounter® technology were used to assess the suitability of these samples in downstreammolecular techniques The results revealed that of the formalin fixatives NBF samples fixedfor and hr were superior to ZF samples for both Sanger sequencing and the NanostringnCounter® The nonformalin PAXgene® samples were equally successful and they showedgreater stability in nucleic acid quality and concentration over longer fixation times This studydemonstrated that small quantities of preserved tissue from smallmouth bass can be utilizedin downstream molecular techniques however future studies will need to optimize the methods presented here for different tissue types fish species and pathological conditions ACCESSCitation Walsh HL Sperry AJ Blazer VS The effects of tissue fixation on sequencing andtranscript abundance of nucleic acids frommicrodissected liver samples of smallmouth bassMicropterus dolomieu e0236104 101371journalpone0236104Editor Rajakumar Anbazhagan National Instituteof Child Health and Human Development NICHDNIH UNITED STATESReceived April Accepted June Published August Copyright This is an access free of allcopyright and may be freely reproduceddistributed transmitted modified built upon orotherwise used by anyone for any lawful purposeThe work is made available under the CreativeCommons CC0 public domain dedicationData Availability Statement All relevant data arewithin the paper and its Supporting InformationfilesFunding This was fully funded internally by theUSGS Environmental Health Contaminant BiologyProgram and the USGS EcosystemsEnvironments and Fisheries Program MissionAreas There are no actual grant numbersPLOS ONE 101371journalpone0236104 August PLOS ONE 0cCompeting interests The authors have declaredthat no competing interests existEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostIntroductionGlobally environmental monitoring programs are increasingly used to identify adverse effectsof human activities on aquatic resources [“] The recognition that there are numerouschemical contaminants environmental stressors as well as new and emerging pathogensparasites cooccurring has led to an emphasis on biological environmental effectsbased assessments utilizing resident indicator fish species or caged model species [“] Histopathologyhas been used for many years to assess the health of wild fishes both for specific studies as wellas part of effectsbased monitoring programs [“] More recently genomic endpoints arealso being incorporated into environmental monitoring and risk assessment [“] Whenboth histopathology and molecular analyses are part of an assessment pieces of a tissue arecommonly preserved in buffered formalin or a similar preservative and adjacent separatepieces are preserved in RNAlater1 ethanol or frozen for molecular analyses [“]However for alterations not visible the tissue piece chosen for gene expression may not contain the same cellular components or alterations as those within the histology sectionThe use of formalin fixed paraffinembedded FFPE tissue has been regarded as a valuablereservoir of preserved nucleic acids in mammalian studies [“] Although FFPE tissuesprovide a vast source of pathologically diverse types of genetic material there are drawbackscompared to other tissue preservation methods Formalin fixation causes nucleic acids to fragment degrade and crosslink [] Frozen tissues or tissues specifically preserved for downstream nucleic acid applications do not experience the type of degradation observed fromformalin fixation Despite these setbacks nucleic acids extracted from FFPE tissue have provento be suitable for use in endpoint PCR [] realtime qPCR [ ] and Nextgenerationsequencing [ ] Optimization of FFPE tissues for downstream nucleic acid applicationshas been attempted in multiple studies by evaluation of different fixation methods [ ] tissue handling and processing times [ ] and extraction methods [ ]Laser capture microdissection LCM utilizes a microscope equipped with a laser to targetand isolate specific cells from a heterogeneous population of cells [] Single cells foci of cellpopulations within a tissue or pathogens and parasites can be microdissected Hence nucleicacids from specific cell populations of interest can be analyzed for gene expression studiestranscriptome development or molecular identification of pathogens and parasites Thisallows for a more direct connection between the histopathology and molecular analyses LCMhas been previously utilized in fishrelated studies [ “] with frozen sections Snapfrozen tissue is optimal for use with LCM for the downstream recovery of nucleic acids However the use of snapfrozen tissue is not always feasible particularly in wild fish studies whereremoval and fixation of the ans occurs in the field and it can be days before tissues arereturned to the laboratory and processed LCM of FFPE tissue can bridge the gap betweenmicroscopy and molecular analyses [] As with other species there is a vast amount of archival FFPE or similarly preserved fish tissue that could be useful for molecular analysesThe aim of this study was to determine how fixative type and fixation time affects nucleicacids in FFPE smallmouth bass liver tissue dissected with LCM Smallmouth bass Micropterusdolomieu are utilized in ongoing monitoring and assessment studies as an indicator species ofexposure to endocrinedisrupting and other contaminants Additionally they are a nonmodel but economically important species To address the utility of paraffinembedded fishtissue for molecular studies smallmouth bass liver was sampled and preserved for four timeperiods hr hr seven days and days in neutral buffered formalin NBFZFix1 ZF and the nonformalin fixative PAXgene1 PG The PAXgene1 Tissue Systemwas designed to improve tissue quality for parallel molecular and morphological analyses []Similarly ZF a zincbased formalin solution was chosen as it has been shown to producePLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleosthigher yields of DNA and RNA when compared to samples fixed in NBF [] In addition toDNA and RNA quantification downstream molecular techniques including Sanger sequencing and the Nanostring nCounter1 digital multiplexed gene expression assay [] were usedto determine if nucleic acids extracted from LCM tissue sections would have utility in futurestudies To the best of our knowledge this study provides novel research on the optimizationof fixative type and fixation time for the use of fish tissue extracts with Nanostring nCounter1technologyMaterials and methodsEthics statement and smallmouth bass sample collectionAll procedures including the handling and euthanasia of fish were approved by the US Geological Survey™s Leetown Science Center™s Institutional Animal Care and Use CommitteeIACUC protocol Five smallmouth bass approximately years old were sampledfrom a flowthrough tank at the US Geological Survey Fish Health Laboratory in Kearneysville West Virginia Fish were placed in a lethal dosage mgL of tricaine methanesulfonate TricaineS Syndel Ferndale WA for euthanasia An incision from the anus tooperculum was made the liver was excised dissected into five equal pieces and placed into fixatives consisting of NBF ZF Product Anatech Ltd Battle Creek MI and PG Product QIAGEN Valencia CA Pieces of liver from each fish were also placed into RNALater1 Product AM7021 Thermo Fisher Scientific Waltham MA and ethyl alcoholETOH to serve as controls Samples in RNALater1 were stored at ˚C for hr prior tostorage at ˚C and samples in ETOH were stored at room temperature RT until extractionswere completedHistological preparation and laser capture microdissectionSamples were fixed for hrs hrs seven days and days at RT for NBF and ZF Tissues preserved in PG were removed from the PAXgene1 Tissue FIX Product QIAGEN after hrs at RT placed in the PAXgene1 Tissue STABILIZER solution Product QIAGEN and stored at ˚C for hrs hrs seven and days Tissue processing was performed on a Shandon CitadelTM Tissue Processor Thermo FisherScientific as follows hrs in alcohol hr in alcohol hr in alcohol 2x hr in alcohol 3x hr in a solution of alcohol and histoclear 2x Product HS200 National Diagnostics Atlanta GA hr in histoclear 2x and hr in paraffin 2x at ˚C Upon completion tissues were embedded into paraffin wax and cooled tohardenTissues were cut at a thickness of μm using a new sterile razor for each sample and sections placed onto Leica Microsystems UVsterilized polyethylene napthalate PEN membraneslides Product NC0496333 Thermo Fisher Scientific Sterilized diethyl pyrocarbonateDEPC Product Millipore Sigma Burlington MA water was used in the water bathand slides were allowed to air dry after sections were placed on the PEN membrane slideUnstained tissue sections were deparaffinized with Anatech Ltd ProPar Clearant Product NC9537734 Thermo Fisher Scientific for min 2x and allowed to air dry prior to lasermicrodissection Liver sections were cut at 5x magnification with a Leica LMD6500 microscope Leica Microsystems at a pulse rate of “ nm Sections x mm2were cut and dropped into the cap of a sterile microcentrifuge tube by gravity Fig and subsequently extracted for RNA or DNAPLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostFig Laser capture microdissection of a smallmouth liver section A Liver section prior to microdissection BLiver section after microdissection C Liver section floating in buffer in the cap of a microcentrifuge tub prior tonucleic acid extraction101371journalpone0236104g001Nucleic acid extractions and downstream analysesFor RNA purification the EZNA1 FFPE RNA Kit Product R695401 Omega BioTekNorcross GA was used according to manufacturer™s protocols for the xylene extractionmethod Extraction began with the addition of GPL Buffer skipping the beginning of the protocol since the tissues were already deparaffinized Samples were digested with proteinase Kfor min and eluted in μl DEPC water As part of the assay protocol DNA contaminationwas removed with a step involving DNA Clearance Columns that binds genomic DNA andallows RNA to pass through the spin column For the controls preserved in RNALater1approximately “ mg liver was extracted with an EZNA1 Total RNA Kit I Product R683402 Omega BioTek according to manufacturer™s protocols and eluted in μl DEPCwater DNA contamination was also removed from these samples with the use of HiBind1RNA Mini Columns and RNasefree DNase Product E109102 Omega BioTek All samples were quantified with a Qubit1 Fluorometer Invitrogen Carlsbad CA using theQubit1 RNA HS Assay Kit Product Q32852 Thermo Fisher Scientific To analyze degradation RIN values were obtained with the Agilent RNA Pico Kit Product “Agilent Technologies Santa Clara CA on an Agilent Bioanalyzer Agilent Technologies Following quantification samples were stored at ˚C prior to use on the NanostringnCounter1DNA was extracted with a proteinase K digestion buffer 50mM TrisHCl pH mMEDTA Tween mgml proteinase K as described in Lehmann and Kreipe []Each sample was extracted in μl of proteinase K digestion buffer and incubated overnight at˚C The tubes were vortexed centrifuged and incubated at ˚C for min to deactivateproteinase K and stored at ˚C Approximately “ mg of control liver samples preservedin ETOH were extracted with a Qiagen DNeasy Blood Tissue Kit Product QIAGEN according to manufacturer™s protocols It is worth mentioning that in initial trialsfor this study the Qiagen DNeasy Blood Tissue Kit was also used to extract DNA from LCMsamples however no quantifiable DNA could be obtained which was why a single tube extraction method was subsequently utilized DNA was quantified with the Qubit1 dsDNA HSAssay Kit Product Q32851 Thermo Fisher Scientific Samples were analyzed for mean fragment size and distribution on an Agilent Bioanalyzer with the Agilent High SensitivityDNA Kit Product “ Agilent TechnologiesFor all LCM samples the final concentration of purified RNA and DNA was standardizedby dividing the total concentration by the total amount of tissue collected μgmm3 Since agreater amount of tissue was extracted from control samples the concentration of purifiedRNA and DNA was standardized to the amount of tissue collected for LCMTo assess the suitability of LCM samples for downstream molecular analyses Sangersequencing and the NanoString nCounter1 Technology were used For endpoint PCRPLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostprimers EF1α5F ™GAG CCC CCT TAC AGC CAG AAG3™ and EF1α5R ™TTCACC TCA GTG GTC AGG CA3™ were designed with NCBI Primer BLAST [] to amplifya bp amplicon of the smallmouth bass elongation factor alpha EF1α accession HQ4248721 gene This housekeeping transcript was chosen since it has been used in othersmallmouth bass studies [ ] and sequence data was available for both smallmouth bassand the closely related largemouth bass accession KT8277941 PCR amplification was conducted under the following conditions denaturation at ˚C for min followed by cyclesof ˚C for s ˚C for s and ˚C for min s with a final extension at ˚C for min Each reaction consisted of μl clear Go Taq Master Mix Product M7132 PromegaMadison WI μl of each primer at 10μM and μl template for LCM samples approximately “ ng and μl template for ETOH samples approximately “ ng Uponcompletion samples were analyzed on an agarose gel with a bp ladder Amplicons werecleaned with a QIAquick1 PCR purification kit Product QIAGEN and eluted in μl of Buffer EB Purified amplicons were used as template in cycle sequencing reactionswith the Applied Biosystems BigDye Terminator v31 Cycle Sequencing Kit Product Thermo Fisher Scientific for cycles of ˚C for min ˚C for s ˚C for sand ˚C for min Cycle sequencing reactions were purified with an Agencourt CleanSEQKit Product A29151 Beckman Coulter Brea CA and sequenced on an ABI 3130xl GeneticAnalyzer Applied Biosystems Foster City CA Sequences were analyzed with Geneious wwwgeneiouscom and quality was assessed by the percentage of bases with aquality score of or higher Q40 NCBI BLASTn was used to determine sequence similarityto the Micropterus spp EF1α gene HQ4248721 or KT8277941NanoString nCounter1 Technology was used with a Custom CodeSet that targeted transcripts expressed in the liver of smallmouth bass as described in Hahn et al [] The previous establishment and availability of this CodeSet was the reason liver was chosen as the tissue of focus in this study The liver is also the principal an for many chemical detoxificationpathways metabolic pathways and the production of vitellogenin In brief the nCounter1platform provides the capability to quantify up to RNA DNA or protein targets called aCodeSet in a multiplex fashion providing results similar to quantitative PCR qPCR [] Itis a costeffective method to analyze specific mRNA targets unlike RNAsequencing whichproduces a vast amount of data and captures all mRNA in a sample Sample setup for hybridizations was carried out according to manufacturer™s protocols with ng of total RNA forevery sample The limit of detection LOD was calculated as the mean of the negative controls � the standard deviation of the negative controls and was transcriptsStatisticsSignificant differences in nucleic acid concentrations and transcript abundance between fixatives for each fixation time were determined with a nonparametric KruskalWallis onewayANOVA followed by a Dunn™s multiple comparison posthoc analysis with a Bonferroni correction in the statistical program R [] Normalized transcript abundance data was used forthe analysis Transcript abundance data was normalized in nSolver Analysis Software Nanostring Technologies Seattle WA where the geometric mean of the negative controlswas subtracted to estimate background and the normalization factor was computed from thegeometric mean of the positive controls and the housekeeping transcripts Housekeeping transcripts were log2 transformed and analyzed for stability with NormFinder [] in R A KruskalWallis test was also used to identify differences amongst each fixative for each fixationtime and the template concentration used for PCR the Q40 score and sequence lengthobtained with Sanger sequencing Finally Spearman™s rank correlation analyses werePLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostconducted in R to determine if the concentration of DNA samples used for PCR sequencelength and the Q40 score were associated the number of sequences with similarity to theMicropterus spp EF1α gene with Sanger sequencing Pvalues � were considered statistically significantResultsNucleic acid concentrationsBoth RNA and DNA were recovered from samples of all fixatives and fixation times Fig Liver samples preserved in RNALater1 had more than times greater RNA concentrationsthan samples preserved in NBF ZF or PG with a mean concentration of ± ngmm3 mean ± standard error The highest concentration of RNA from LCM samples wasobtained from PG samples at hr ± ngmm3 Fig 2A The lowest concentrationsFig Nucleic acid auantification A Mean standard error of RNA and B DNA concentrations μgmm3 ofmicrodissected smallmouth bass liver samples fixed in neutral buffered formalin NBF ZFix1 ZF andPAXgene1 PG for hours hours and days Samples preserved in alcohol ETOH and RNALater1were included as controls101371journalpone0236104g002PLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostwere from NBF preserved tissues at two weeks ± ngmm3 The concentration ofRNA was significantly greater in RNALater1 samples than in NBF Pvalue and PGPvalue at hr NBF Pvalue and ZF Pvalue at hr NBF Pvalue and ZF Pvalue at seven days and NBF Pvalue � ZF Pvalue and PG Pvalue at days Mean concentrations of RNA in samplesfixed in NBF and ZF decreased at seven and days while those fixed in PG remained stablethroughout the time course Fig 2AThe amount of DNA recovered was less than RNA with all mean concentrations of LCMsamples below μgmm3 Samples fixed in ETOH had more than times greater concentrations of DNA than samples fixed for LCM with a mean concentration of ± ngmm3 The concentration of DNA was significantly greater in ETOH samples than in PG at hr Pvalue NBF Pvalue and PG Pvalue at hr NBF Pvalue and PG Pvalue at seven days and NBF Pvalue and PG Pvalue at days There was little variation in DNA concentrations over time for anyof the fixatives although for all fixatives the lowest concentration was at days Fig 2BThe quality of RNA varied among fixatives Mean RIN values of samples fixed in RNALater1 were at least twice as great as samples fixed in NBF ZF and PG The highest RIN valuesfor LCM samples were observed in NBF fixed tissue at hrs seven and days Fig 3A RINvalues were significantly greater in RNALater1 samples than in PG Pvalue and ZFPvalue samples at hr PG Pvalue and ZF Pvalue samples atFig Nucleic acid quality A Mean RIN values of RNA and B fragment size bp of DNA from samples fixed in neutral buffered formalin NBF ZFix ZF and PAXgene PG for hours hours and days Samplespreserved in alcohol ETOH and RNAlater were included as controls101371journalpone0236104g003PLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleost hr PG Pvalue and ZF Pvalue samples at seven days and PG Pvalue � and ZF Pvalue samples at days There were no significant differences in RIN values between LCM samplesMean fragment size of DNA varied over time within each fixative group but was not significantly different than samples fixed in ETOH Fig 3B Fragment size was only significantlygreater in NBF samples than in PG samples P value at hrDownstream analysesEndpoint PCR and Sanger sequencing were successful for the amplification and sequencingof the smallmouth bass EF1α gene although differences in sequencing success were apparentWithout trimming the ™ or ™ ends samples preserved in ETOH produced sequences with amean percentage of bases with a Q40 score or greater of while samples fixed for LCMproduced lower quality sequences Fig Of the samples fixed for LCM PG preserved samplesproduced the highest quality sequences At days there were no samples fixed in NBF or ZFthat were successful for sequencing the EF1α gene For NBF and ZF the best quality sequenceswere generated by samples fixed for hr conversely PG had the lowest quality sequencesfrom samples fixed for hr Fig It should be noted that of the five PG samples sequencedat hr two samples had much lower quality sequences than the other three samples whichmay have contributed to the decrease in the mean percentage of high quality sequences at hr Additionally multiple samples failed to sequence These included one of the ETOH samples forward and reverse sequences three NBF day samples forward sequences oneNBF seven day sample forward and reverse sequences two PG seven day samples forwardsequences one PG day sample forward sequence one PG hr sample reversesequence one ZF seven day sample reverse sequence four ZF day samples three forwardand one reverse sequence and one ZF hr sample forward sequence In order to calculatethe percentage of sequences with similarity to the Micropterus spp EF1α gene failed sequenceswere not included ie of sequences with similarity to Micropterus spp EF1α ofsequences with similarity to Micropterus spp EF1α total of sequences that were successfullysequenced � For LCM fixed samples NBF samples fixed for and hr produced theFig Sanger sequencing quality Mean SEM percentage of bases with a Q40 score or above indicative of highquality sequencing Samples preserved in ethanol ETOH were included as controls routinely used for DNApreservation101371journalpone0236104g004PLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostgreatest percentage of sequences with similarity to the EF1α gene In ZF samplesthe greatest percentage of samples with similarity to the EF1α gene was at hr and in PG samples it was at seven days Although PG samples produced the leastamount of sequences with similarity to the EF1α gene at and hr it produced the greatestnumber of sequences at seven and daysA Spearman™s rank correlation analysis with all samples revealed that sequence length pvalue � rho and template concentration pvalue rho were significantly associated with the number of sequences with similarity to the EF1α gene Fig Although PCR primers were estimated to produce an amplicon size around bp manysequences were longer than bp This could be due to the high degree of fragmentation inthe samples which may have resulted in the annealing of small fragments to the original template molecules in overlapping regions [] Fragment length and the percentage of bases witha Q40 score or greater were not significantly correlated with the number of sequences withsimilarity to the EF1α gene The correlations were also examined excluding the ETOH controlssince the DNA concentration of the controls was significantly greater than those of many fixedsamples and to examine the differences amongst the fixatives only Sequence length remainedsignificant pvalue � rho however template concentration was not significantpvalue rho Fragment length and the percentage of bases with a Q40 scoreFig Sanger sequencing and NCBI blastn results A Spearman™s rank correlation analysis of sequence length bpand the number of NCBI blastn matches to the Micropterus spp elongation factor alpha EF1α gene B Meansequence length bp of the EF1α gene obtained with Sanger sequencing and the percentage of sequences withsimilarity to the Micropterus spp EF1α gene101371journalpone0236104g005PLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostTable KruskalWallis nonparametric ANOVA results Significant differences in template concentration usedin PCR and Sanger sequencing results for each fixative between fixation timesFixativeNBFZFPGFixativeNBFNBFZFPGFixativeNBFNBFZFZFPGTemplate ConcentrationpvalueZ scoreFixation TimeNo significant differencesNo significant differences hr vs days of Bases with � Q40 ScorepvaluepvalueZ scoreFixation Time hr vs days hr vs hrNo significant differencesNo significant differencesSequence LengthZ scoreFixation Time days vs hr days vs hr days vs hr days vs hrNo significant differencesSignificant differences pvalue � in template concentration of bases with a Q40 score or greater andsequence length between samples fixed in neutral buffered formalin NBF ZFix1 ZF and PAXgene PG101371journalpone0236104t001or greater remained not significantly correlated A KruskalWallis test was used to identify significant differences between fixation times and template concentration percentage of baseswith a Q40 score or greater and sequence length for each fixative Table The Nanostring nCounter1 results revealed multiple occurrences of samples fixed forLCM with similar or greater transcript abundance compared to RNALater1 samples S1Table In samples fixed for LCM there was significantly greater transcript abundance in PGsamples than in NBF andor ZF samples for one transcript at seven days and eight transcriptsat days Interestingly there were multiple significant differences in housekeeping transcriptabundances between samples preserved in RNALater1 and samples fixed for LCM MeanEF1α transcript abundance was significantly greater in RNALater1 samples than in samplesfixed for LCM at all fixation times Conversely at seven and days 40S ribosomal protein S12transcripts were higher in NBF and ZF samples when compared to RNALater1 and ribosomalprotein L8 was higher in the PG samples Table Significant differences were also identified between fixation times for each fixative typeused to preserve LCM samples Table All significant differences were between fixationtimes hr and seven or days and hr and seven or days In some instances sampleswith longer fixation times had transcripts with significantly greater transcript abundance thansamples fixed for or hr Once again significant differences were identified amongsthousekeeping transcripts There were no significant differences in PG samples over timeNormFinder results ranked the housekeeping transcripts according to stability For all fixatives and all fixation times including RNALater1 samples the most stable housekeepingtranscript was Ribosomal Protein L8 stability followed by EF1α stability Eukaryotic Translation Initiation Factor 3D stability and 40S ribosomal protein S12stability PLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostTable KruskalWallis nonparametric ANOVA results Significant differences in transcript abundance betweenfixatives for each fixation timeTranscript NameElongation Factor Alpha�Heat Shock Protein Elongation Factor Alpha�Thyroid Hormone Receptor BetaTranscript NameElongation Factor Alpha�Heat Shock Protein Elongation Factor Alpha�Heat Shock Protein Transcript Name40S Ribosomal Protein S12�ArginaseBetacateninElongation Factor Alpha�Heat Shock Protein Transforming Growth Factor Beta40S ribosomal protein S12�C Reactive ProteinlikeElongation Factor Alpha�Eukaryotic Translation Initiation Factor 3D�Transforming Growth Factor BetaApolipoprotein Elongation Factor Alpha�Ribosomal Protein L8�Superoxide DismutaseThyroid Hormone Receptor BetaThyroid Hormone Receptor BetaTranscript Name40S ribosomal protein S12�ArginaseElongation Factor Alpha�40S ribosomal protein S12�ArginaseC Reactive ProteinlikeElongation Factor Alpha�Ribosomal Protein L8�Transforming Growth Factor BetaApolipoprotein Aryl Hydrocarbon ReceptorElongation Factor Alpha�Heat Shock Protein Ribosomal Protein L8� Hrpvalue Hrpvalue Dayspvalue DayspvalueZ scoreZ scoreZ scoreZ scorePLOS ONE 101371journalpone0236104 August FixativesRNALater vs NBFRNALater vs NBFRNALater vs PGRNALater vs PGFixativesRNALater vs PGRNALater vs NBFRNALater vs ZFRNALater vs ZFFixativesRNALater vs NBFRNALater vs NBFRNALater vs NBFRNALater vs NBFRNALater vs NBFRNALater vs NBFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs PGRNALater vs PGRNALater vs PGRNALater vs PGRNALater vs PGPG vs NBFFixativesRNALater vs NBFRNALater vs NBFRNALater vs NBFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs PGRNALater vs PGRNALater vs PGRNALater vs PGRNALater vs PGContinued PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostTable ContinuedAryl Hydrocarbon ReceptorEstrogen Receptor Beta Hepcidin Thyroid Hormone Receptor BetaType II Iodothyronine DeiodinaseAryl Hydrocarbon ReceptorHepcidin Thyroid Hormone Receptor BetaType II Iodothyronine DeiodinasePG vs NBFPG vs NBFPG vs NBFPG vs NBFPG vs NBFPG vs ZFPG vs ZFPG vs ZFPG vs ZFSignificant differences pvalue � in transcript abundance between RNALater1 samples and samples fixed in neutral buffered formalin NBF ZFix1 ZF and PAXgene PG� Indicates housekeeping transcripts101371journalpone0236104t002Table KruskalWallis nonparametric ANOVA results Significant differences in transcript abundance betweenfixation times for each fixativeTranscript Name40S ribosomal protein S12�ZFixpvalueCytochrome P450 family subfamily AElongation Factor Alpha�Eukaryotic Translation Initiation Factor 3D�Ribosomal Protein L8�TataBox Binding ProteinThyroid Hormone Receptor BetaTransforming Growth Factor BetaTranscript Name40S ribosomal protein S12�Cytochrome P450 family subfamily ABeta

Thyroid_Cancer

coronary arterybypass grafting thrombosis fibrin fibrinogen mutationIntroduction Intraoperative thrombosis of saphenous veins SV during harvesting is very rareCase Report We present a case of a 60yearold male patient with multivesselcoronary artery disease and a history of a nonST elevation acute coronary syndromeand type2 diabetes mellitus admitted for coronary artery bypass grafting in whombilateralintraoperative SV thrombosis occurred during graft harvesting Routinethrombophilia screening showed no abnormalities and cancer was excluded Compared with healthy controls we observed prolonged fibrin clot lysis time and increasedthrombin generation reflected by endogenous thrombin potential Scanning electronmicroscopy of the thrombosed material revealed compact and thick fibrin layer on theclot surface with a solid mass of unusually compressed platelets and erythrocytesunderneath The patient was tested for fibrinogen and factor F XIII polymorphismsand was found to be heterozygous for fibrinogen HaeIII 455G A and FXIIIVal34Leu 100G TConclusion fibrinogen HaeIII and FXIII Val34Leu polymorphisms are reflected inreduced clot permeability and susceptibility to lysis and might contribute to intraoperative SV thrombosis during vascular grafting procedures Carriers of those are atrisk of primary venous graft failure after bypass proceduresIntroductionCoronary artery bypass grafting CABG is a method of choicefor revascularization in patients with multivessel disease anddiabetes mellitus DM Although arterial grafts are preferredin selected scenarios the common practice is to use leftinternal thoracic artery LITA to bypass the left anteriordescending artery LAD and to place venous conduits toother target vessels An often chosen vascular graft the greatsaphenous vein SV offers decent durability and is easy toharvest SV graft occlusion may occur in up to of caseswithin the first months and as many as may occludewithin first to weeks1 SV harvesting dramaticallychanges the vein™s environment with disruption of bloodflow in vasa vasorum damage to the adventitia hypoxia andhyponutrition of the vessel wall along with focal endothelialdisruption2 Acute SV graft failure is usually a result of graftthrombosis which among other factors like technical failuregrafttarget vessel disproportion etc may be caused byhypercoagulabilityreceivedMarch acceptedJuly 101055s00401715657ISSN Ge Thieme Verlag KGStuttgart · New York 0ce198Bilateral Saphenous Vein Thrombosis during CABG Mazur et alCase ReportA 60yearold male patient with multivessel coronary arterydisease who suffered from a nonST elevation acute coronarysyndrome NSTEACS month prior to admission a nonsmoker with type2 DM on metformin peptic ulcer diseaseand a history of alcohol abuse was admitted to our institutionfor CABG Just after the NSTEACS a left ventricle LV thrombus was seen on one echocardiographic examination but itwas absent during followup There was no deep venousthrombosis or bleeding diathesis history On admission thepatient was on aspirin mg once daily and enoxaparin mg once daily Routine laboratory tests were withinnormal ranges –ºTable There were no abnormalities onphysical examination apart from obesity body mass index kgm2 when the patient was admitted The lower extremities appeared normal There were no varicose veins nosigns or symptoms of venous insufficiency and the pastmedical history was negative for both personal and familyhistory of chronic venous insufficiency or varicose veins Thepatient was operated on following the standard proceduresDuring LITA harvest a cardiac surgery resident harvested theright SV using the technique The wall of the SV lookedgrossly normal Upon dissection the side branches were tiedoff and clipped and a needle was placed at the distal end whilethe proximal end was still not separated An attempt was madeTable Results of initial and followup laboratory testingVariableCoagulation testsRed blood count 103µLHemoglobin gdLWhite blood count 103µLPlatelet count 103µLAPTT sPT sPT INRPlatelet aggregation mmolL arachidonic acid µmolL ADP Thrombophilia screeningFibrinogen gLAntithrombin III Ddimer µgLantiXa IUmLHomocysteine µmolLProtein C Protein S Factor VIII Leiden c1601G A 0397G AProthrombin cfibrinogen 455G AFactor XIII 100G TLupus anticoagulant ratioLupus anticoagulant ratio APTTAnticardiolipin IgGAnticardiolipin IgMAnti2glycoprotein I IgG antibodyAnti2glycoprotein I IgM antibodyNormal rangesPreoperativePostoperative day Postoperative day “““““““““““““““““““““““““““““““““““““““““““ GG no mutationGG no mutation““ GPL MPL SGU SMU““““““““““““GG no mutationGG no mutationGA heterozygoteGT heterozygote GPL MPL SGU SMUAbbreviations APTT activated partial thromboplastin time GPL IgG phospholipid unit Ig immunoglobulin INR international normalized ratioMPL IgM phospholipid unit PT prothrombin time SGU standard IgG 2 glycoprotein unit SMU standard IgM 2 glycoprotein unitTH Vol No 0cBilateral Saphenous Vein Thrombosis during CABG Mazur et ale199to flush the vein with a solution containing blood mLheparin IU and normal saline mL while the distalend was closed with an atraumatic vascular clamp and veinthrombosis was noted Upon the separation of the distal end aluminal thrombus was visible The left SV was then taken downusing the same protocol by an experienced staff cardiacsurgeon with the same result Presence of a luminal thrombuswas confirmed upon separation of the proximal end Systemicheparin was administered and normal LITA outflow wasconfirmed Concerns regarding safety of cardiopulmonarybypass use were raised due to suspected thrombotic issueand the approach was modified The LITA“LAD anastomosiswas completed offpump on a beating heartThe postoperative course was uneventful On postoperative day the patient received dual antiplatelet therapy withaspirin and clopidogrel and was discharged on day with nosigns of thrombosis or myocardial ischemia Elective angioplasty of nongrafted vessels was scheduled and a completethrombophilia screening was done –ºTable On the and12month followup the patient did wellDiagnostic ApproachBecause a thrombophilia was suspected screening wasinitiated showing no abnormalities –ºTable Cancer was excluded as a cause of thrombosis Positiveantibodies against neutrophil cytoplasm antigens pANCAand cANCA were excluded as a cause of vasculitis We thenproceeded to analyze fibrin phenotype using the previouslydescribed methodology34 Briefly plasma fibrin clot permeability was determined in a hydrostatic pressure systemTubes containing fibrin clots formed from adding mmolLcalcium chloride and UmL human thrombin Sigma tocitrated plasma were connected through plastic tubing to abuffer reservoir M TrisHCl M NaCl pH Thevolume flowing through the gel was measured within minutes A permeation coefficient Ks reflecting poresize was calculated from equation Ks ¼ Q 02 L ηt 02 A 02 Δpwhere Q is the flow rate in time t L is the length of a fibrin gelη is the viscosity of liquid A is the cross section area and Δp isa differential pressure in dynecm2 Lower Ks values indicated reduced permeability Fibrinogen was determined usingthe Clauss method Even though the followup fibrinogenlevel was normal we identified strongly decreased fibrin clotpermeability Ks ¼ 06 02 9cm2 compared withhealthy controls from our previous report n ¼ Ks ¼ 9cm23 samples collected during late follow[“] up appointment on postoperative day Compared withhealthy controls n ¼ we observed prolonged clot lysistime CLT 06 vs 06 minutes and increasedthrombin generation reflected by endogenous thrombinpotential ETP in the studied subject ETP ¼ 06 vs 06 nM 02 min respectively measurement ofthe thrombin generation was done with calibrated automated thrombography thrombinoscope BV Maastricht theNetherlands according to the manufacturer™s instructionsin the 96well plate fluorometer Ascent Reader Thermolabsystems OY Helsinki Finland equipped with the filter set at a temperature of °C Briefly microliters of plateletpoor plasma were diluted with µL of the reagent containing pmolL recombinant tissuefactor micromolar phosphatidylserinephosphatidylcholinephosphatidylethanolamine vesicle and µL of FluCasolution Hepes pH nmolL CaCl2 mgmL bovinealbumin and mmolL ZGlyGlyArg7amino4methylcoumarin Each plasma sample was analyzed in duplicateFor analysis the maximum concentration of thrombin generated was used3Cryosectioned tissue sections were fixed in icecold methanol“acetone mixture peroxidase activity was quenchedwith H2O2 and unspecific background was blocked with bovine albumin BSA Sigma Co St Louis Missouri UnitedStates Primary adequate antibodies against fibrin or tissuefactor TF were applied both Abcam Cambridge UnitedKingdom Primary antibodies were followed by thecorresponding secondary antibodies conjugated with fluorochrome Abcam as previously described5 Images were analyzed using Olympus BX microscope SVs immunostainingrevealed thick layer of fibrin directly on the vessel endothelium–ºFig 1A and high TF –ºFig 1B activity Within the thrombuswe found abundant blood nuclear cells nuclei stained usingDAPI suggesting the presence of proinflammatory monocyteswhich are known source of TF Unfortunately we were not ableto immunostain CD68 due to high unspecific backgroundresulting from large amounts of fibrin The microscopic analysisshowed abundant adventitial vessels –ºFig 1C D Withinalmost every single vessel we found thrombi rich in bothprothrombin –ºFig 1C and TF –ºFig 1DProthrombotic fibrin clot phenotype reflected by reducedKs and prolonged CLT along with enhanced thrombin generation and unusualimages obtained from the immunostaining of the SVs prompted us to perform analysis ofwhole blood clot morphology using scanning electron microscopy SEM as previously described6 After washing thethrombus was fixed with glutaraldehyde phosphatebuffered saline solution Specimens were dehydrated goldcoated and photographed digitally with a JEOL JSM JEOL Tokyo Japan The analysis revealed compact and thickfibrin layer on the clot surface with a solid massof unusually compressed platelets and erythrocytes underneath This observation suggested veryhigh contractileforces during clot formation in a plateletdriven fibrinmediated mechanism of clot contraction and prompted usto study common fibrinogen and factor F XIII polymorphisms The patient was heterozygous for fibrinogen HaeIII455G A and FXIII Val34Leu 100G TDiscussionA dramatic intraoperative SV thrombosis provoked by graftharvesting for CABG lead to change in revascularizationstrategy but its cause remained unknown following thestandard thrombophilia screening The cases of acute SVgraft thrombosis in the perioperative period are very rareand as few as of grafts occlude within first to weeks17TH Vol No 0ce200Bilateral Saphenous Vein Thrombosis during CABG Mazur et alFig Representative images of SV graft immunostaining after massive thrombosis A“D prothrombin stained red TF stained green nucleistained blue using DAPI and scanning electron microscopic images E F of the surface of whole blood clot formed in vitro from citrated bloodobtained from the patient undergoing CABG Box and arrow represent magnification of the fragment in the box Arrows show pertinent stainedfragments see text CABG Coronary artery bypass grafting SV saphenous veins TF tissue factorA normal SV is composed of the intima the media and theadventitia8 The intima is built of the layer of endothelial cellson the luminal side the media consists of smooth musclecells and the adventitia forms the outer part8 In a normalsetting the endothelium is crucial for vein integrity andprevention of thrombosis9 and its focal disruption maypredispose to vessel thrombosis2 SV manipulation andimplantation leads to loss of endothelial integrity and elicitsan inflammatory response with platelet adhesion and leukocyte recruitment Notwithstanding an overt thrombosis isextremely rare in the operating room SV dissection results inblood flow disruption in vasa vasorum and causes adventitial damage hypoxia and vessel wall hyponutrition10 Acuteperioperative saphenous vein graft failure is almost always aresult of graft thrombosis but this very uncommonly occursprior to graft placement Surgical factorslike technicalanastomotic failure or severe disproportion between thetarget vessel and the graft may lead to thrombosis butvessel injury and hypercoagulability are among potentialcauses as well11There was no evident inflammatory process in microscopy inour patient but even if an inflammatory process was presentTH Vol No 0cBilateral Saphenous Vein Thrombosis during CABG Mazur et ale201preoperatively in our patient™s SVs the inflammatory background alone could not explain the dramatic intraoperativethrombosis We hypothesized that increased thrombin generation and prothrombotic fibrin clot phenotype were responsiblefor the clinical presentation Conversion of fibrinogen to fibrinfacilitated by thrombin is a concluding step of coagulation Ithas been shown that fibrin clots with small pores betweentightly packed thin fibrin fibers are relatively lysis resistant12Such clot phenotype has been evidenced in multiple thromboticpathologies such as myocardial infarction6 ischemic stroke13and venous thromboembolism4 The prothrombotic clotphenotype reflected by a tendency to form dense fibrin clotsresistant to lysis has been previously reported in patients withinstent thrombosis14 While routine thrombophilia screeningresults in a high almost detection rate of commonhypercoagulable states15 there are prothrombotic conditionsthat escape routine diagnostic approach The overall microscopic clot appearance and prothrombotic fibrin properties lead tothe discovery of two mutations in our patient that are notroutinely tested during thrombophilia screening namely fibrinogen 455G A and FXIII100G TElevated fibrinogen was postulated as one of the riskfactors for early graft failure after CABG1116 Epidemiologicalstudies have established that elevated fibrinogen is stronglyassociated with cardiovascular diseases17 A metaanalysis of individual records of participants from prospective studies revealed that age and sexadjustedhazard ratio per gL increase in usual fibrinogen level forcoronary heart disease was confidence interval [CI]“ while for stroke the hazard ratio was as high as 95CI “ Risk of coronary disease progression wasalso linked to genetic polymorphisms of the fibrinogen geneDe Maat et al found that A allele of fibrinogen 455G Awas associated with more severe progression of coronarydisease as documented angiographically18 Gu and colleagues in a metaanalysis of studies with patientsfound that A allele of the fibrinogen 455G A is associated with susceptibility to coronary disease and also withischemic stroke odds ratio for stroke ¼ [ CI “]for AA þ GA vs GG19 In a recent study of patients with atrialfibrillation Hu and colleagues found that the A allele of fibrinogen 455G A was a risk factor for cardioembolicstroke probably by elevating the level of plasma fibrinogen20 On the other hand in a metaanalysis of studies involving cases and controls FXIIIVal34Leu polymorphism was shown to be associated withrisk myocardial infarction21 FXIII is crucial to thrombusstabilization and changes of its plasma concentration reflectnonspecifically the extent of thrombosis as shown by Li et alin a study on patients with cerebral venous thrombosis22Interesting associations of FXIII Val34Leu polymorphism andthrombotic disorders have been reported Jung et al reportedin a metaanalysis of studies that FXIII Val34Leu polymorphism is associated with recurrent pregnancy loss23Although no association with the incidence of ischemicstroke was found for this polymorphism24 apparentlywhen the stroke occurs Val34Leu polymorphism of FXIIIaffects the severity of its outcome25 Furthermore Kreutzand colleagues suggested in that FXIII Val34Leu polymorphism may increase risk of recurrent MI and death inpatients with angiographically established coronary arterydisease26 In our group has shown in a study of patients that in patients undergoing CABG FXIII Leu34 alleleis associated with decreased fibrin clot permeability andefficiency of fibrinolysis27ConclusionOur extensive workup showed that fibrinogen HaeIII andFXIII Val34Leu polymorphisms are reflected in reduced clotpermeability and susceptibility to lysis These mutationslikely contributed to intraoperative saphenous graft thrombosis Further studies are needed to elucidate the role ofthese polymorphisms in early graft failure after bypassgrafting procedures however their contributory role seemsevidentFundingThis study was funded by a grant from the JagiellonianUniversity Medical College no KZDS007961 to PMConflict of InterestNone declaredReferences Bourassa MG Fate of venous grafts the past the present and thefuture J Am Coll Cardiol “ Roubos N Rosenfeldt FL Richards SM Conyers RA Davis BBImproved preservation of saphenous vein grafts by the use ofglyceryl trinitrateverapamil solution during harvesting Circulation 19959209II31“II36 Mazur P Sokołowski G HubalewskaDydejczyk A PłaczkiewiczJankowska E Undas A Prothrombotic alterations in plasma fibrinclot properties in thyroid disorders and their posttreatmentmodifications Thromb Res “ Undas A Zawilska K CieslaDul M et al Altered fibrin clotstructurefunction in patients with idiopathic venous thromboembolism and in theirrelatives Blood “ Natorska J Marek G Hlawaty M Sadowski J Tracz W Undas AFibrin presence within aortic valves in patients with aorticstenosis association with in vivo thrombin generation and fibrinclot properties Thromb Haemost “ Undas A Szułdrzynski K Stepien E et al Reduced clot permeability and susceptibility to lysis in patients with acute coronarysyndrome effects of inflammation and oxidative stress Atherosclerosis “ Fitzgibbon GM Kafka HP Leach AJ Keon WJ Hooper GD BurtonJR Coronary bypass graft fate and patient outcome angiographicfollowup of grafts related to survival and reoperation in patients during years J Am Coll Cardiol “ Kim FY Marhefka G Ruggiero NJ Adams S Whellan DJ Saphenous vein graft disease review of pathophysiology preventionand treatment Cardiol Rev “ Allaire E Clowes AW Endothelial cell injury in cardiovascularsurgery the intimal hyperplastic response Ann Thorac Surg“ McGeachie JK Meagher S Prendergast FJ Veintoartery graftsthe longterm development of neointimal hyperplasia and itsTH Vol No 0ce202Bilateral Saphenous Vein Thrombosis during CABG Mazur et alrelationship to vasa vasorum and sympathetic innervation Aust NZ J Surg “ Harskamp RE Lopes RD Baisden CE de Winter RJ Alexander JHSaphenous vein graft failure after coronary artery bypass surgerypathophysiology management and future directions Ann Surg“ Undas A Fibrin clot properties and their modulation in thrombotic disorders Thromb Haemost “ Undas A Podolec P Zawilska K et al Altered fibrin clotstructurefunction in patients with cryptogenic ischemic strokeStroke “ Undas A Zalewski J Krochin M et al Altered plasma fibrin clotproperties are associated with instent thrombosis ArteriosclerThromb Vasc Biol “ GoldmanMazur S Wypasek E Karpiński M Stanisz A Undas AHigh detection rates of antithrombin deficiency and antiphospholipid syndrome in outpatients aged over years using thestandardized protocol for thrombophilia screening Thromb Res“ Moor E Hamsten A Blombäck M Herzfeld I Wiman B Rydén LHaemostatic factors and inhibitors and coronary artery bypassgrafting preoperative alterations and relations to graft occlusionThromb Haemost “ Danesh J Collins R Appleby P Peto R Association of fibrinogen Creactive protein albumin or leukocyte count with coronary heartdisease metaanalyses of prospective studies JAMA “ de Maat MP Kastelein JJ Jukema JW et al 455GA polymorphismof the betafibrinogen gene is associated with the progression ofcoronary atherosclerosis in symptomatic men proposed role foran acutephase reaction pattern of fibrinogen REGRESS groupArterioscler Thromb Vasc Biol “ Gu L Liu W Yan Y et al Influence of the fibrinogen455GApolymorphism on development of ischemic stroke and coronaryheart disease Thromb Res “ Hu X Wang J Li Y et al The fibrinogen gene 455GA polymorphism associated with cardioembolic stroke in atrial fibrillationwith low CHA2DS2VaSc score Sci Rep Jung JH Song GG Kim JH Seo YH Choi SJ Association of factor XIIIVal34Leu polymorphism and coronary artery disease a metaanalysis Cardiol J “ Li B Heldner MR Arnold M et al Coagulation Factor XIIIin Cerebral Venous Thrombosis TH e227“e229 Jung JH Kim JH Song GG Choi SJ Association of the F13A1Val34Leu polymorphism and recurrent pregnancy loss a metaanalysis Eur J Obstet Gynecol Reprod Biol “ Shemirani AH Pongrácz E Antalfi B Adány R Muszbek L FactorXIII A subunit Val34Leu polymorphism in patients sufferingatherothrombotic ischemic stroke Thromb Res “ Shemirani AH Antalfi B Pongrácz E Mezei ZA Bereczky Z Csiki ZFactor XIIIA subunit Val34Leu polymorphism in fatal atherothrombotic ischemic stroke Blood Coagul Fibrinolysis “ Kreutz RP Bitar A Owens J et al Factor XIII Val34Leu polymorphism and recurrent myocardialinfarction in patients withcoronary artery disease J Thromb Thrombolysis “ Stepień E Plicner D Kapelak B Wypasek E Sadowski J UndasA Factor XIII Val34Leu polymorphism as a modulator of fibrinclot permeability and resistance to lysis in patients withsevere coronary artery disease Kardiol Pol 2009678A“TH Vol No 0c'

Thyroid_Cancer

"Marek™s disease MD is a chicken neoplastic disease which brings huge economic losses to theglobal poultry industry The wild type p53 a tumor suppressor gene plays a key role in blocking cell cyclepromoting apoptosis and maintaining the stability of the genome However the mutant p53 losses its tumorinhibitory role and become an oncogene when a mutation has happenedResults The mutation rate of p53 was in the experimentally and naturally infected chickens The mutationsincluded pointmutations and deletions and mostly located in the DNAbinding domain The mutated p53 wasexpressed in various tumor tissues in an infected chicken The mutant P53 proteins were notably accumulated inthe cytoplasm due to the loss in the function of nuclear localization Unlike the study on human cancer theconcentrations of P53 in the serums of MD infected chicken were significantly lower than the control groupConclusions The p53 mutations were apparent in the development of MD P53 and P53 antibody level in serumcould be a useful marker in the diagnosis and surveillance of MDKeywords Marek™s disease p53 P53 antibodyBackgroundMarek™s disease MD is a lymphoproliferative neoplasticdisease caused by the chicken Marek™s disease virusMDV or named Gallid alphaherpesvirus The infection caused by this virus may lead to lymphocyte proliferation tumor formation immunosuppression paralysisand mononuclear cell infiltration in peripheral nervesgonads and immune ans [ ] As one of the mosthighly contagious tumor diseases in chickens the data ofOIE [] reported by about half of the world has shownthat this disease accounts for the loss of up to “ billionUS dollars annually in the global poultry industry [] Correspondence liusidsdaueducn1College of Animal Science and Veterinary Medicine Shandong AgriculturalUniversity Daizong Street Taian Shandong ChinaFull list of author information is available at the end of the Described often as the œguardian of the genome [] andthe œcellular gatekeeper [] p53 is the most relevantand important tumor suppressor gene with the highestmutation frequency in human and animal tumor diseases [] The chicken p53 gene has a fulllength reading frame ™ and ™ untranslated regions and a polyadenylation signal which encodes amino acidsThese amino acids share a homology to the aminoacids of human P53 []p53 is divided into the wild type and the mutant typeThe wild type is a normal tumor suppressor gene whilethe mutant p53 is an oncogene transformed from atumor suppressor gene due to spatial conformationalchange As a result the mutant P53 protein losses itsability in regulating cell growth apoptosis and DNA repair [] which often a prerequisite for tumorigenesis and The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cZhang BMC Veterinary Research Page of disease progression [] The proportion of p53 mutations in tumor tissue varies between “ []The wild type P53 has a very short halflife while themutant P53 protein is prolonged This abnormality ofP53 can lead to the accumulation of P53 antibody inserum as well as the P53 protein in tumor tissue []Several studies have demonstrated that the P53 antibodyplays a predictive role in tumorigenesis and its manifestation in serum is an early event in the development ofmalignant tumors in humans [“] The level of P53antibody in serum correlates significantly with commonclinical neoplastic diseases but it has been barely detectable in the serum of healthy subjects [] This correlation may also exist in chicken and currently there is aknowledge gap concerning the role of mutant p53 inMarek™s disease in chicken Therefore this study aimedto investigate the role of p53 as a tumor marker inassisting the clinical diagnosis and prognosis of MDResultsHistopathological and immunohistochemical analysis ofp53 expressionHistopathological examination revealed the evidence ofliver cells undergoing necrosis in the infected SPFchicken Such liver tissues demonstrated focal infiltration and hyperplasia of lymphoid tumor cells Fig 1aIn addition the lymphoid follicle in the bursa of Fabricius was atrophied diffuse infiltration and proliferationof lymphoid tumor cells between the follicles were observed Fig 1b In the livers of the clinically infectedchickens the cytoplasm of the lymphoid tumor cellsunderwent multifocal proliferation which was stainedbrown by the immunohistochemicalFig 2aPositive staining was also detected in the tumor cells inIHCthe spleen and the bursa of Fabricius tissues Fig 2b and cIn contrast no positive stained cells were observed in thecontrol group Fig 2dMutations in the p53The mutation rate of p53 was in the infectedpoultry There were two types of p53 mutations deletions and point mutations detected which was consistent with the results reported in a previous study []Among the mutated p53 genes the base sites withhigh mutation frequency were and There was no mutation found in the control groupMost of the mutations were located in the core domainand the Cterminal domain The mutation analyses wereshown in Table P53 antigen and P53 antibody levels for MDThe concentrations of the P53 antigen of the clinicaland experimental MDVinfected group were significantlylower than the control group However the P53 antibody levels in the experimental infection group were significantly higher than the control group These analyseswere shown in Fig DiscussionThe human P53 is widely acknowledged as an intranuclear phosphorylated protein The wide type P53 normally exists in the nucleus for an extremely short period[] On the other hand the mutated P53 has a prolonged halflife to “ h as it is not digested quicklyand therefore accumulates inside tumor cells [] Thisallows the detection of mutant P53 protein via IHC forFig Histopathological observation of diseased chickens infected with MDV a Different sizes and shapes of focal infiltration and hyperplasia oflymphoid tumor cells were observed in the liver tissues HE — b Diffuse infiltration and proliferation of lymphoid tumor cells between thefollicles were observed in the bursa of Fabricius HE — 0cZhang BMC Veterinary Research Page of Fig Immunohistochemical staining of p53 in infected chickens a Liver lymphoid tumor cells with cytoplasm expression HE — b Spleen lymphoid tumor cells with cytoplasm expression HE — c Bursa of Fabricius lymphoid tumor cells with cytoplasm expression HE —d Liver Negative controls were incubated with PBS HE —which IHC has now become an important modality forthe detection of various tumor biomarkers P53 proteinrepresents an effective substitute marker for TP53 mutation status [] but at present P53 IHC is mainly applied for tumor diseases in humans P53 IHC allows theassessment of the stage and grade of cancers with only afew studies reported in poultry tumor diseasesIn this study the immunohistochemical analysis revealed that P53 was present in the liver spleen and thebursa of Fabricius of infected chicken with MDV Interestingly P53 was notably expressed in the cytoplasmThe potential explanation of this phenomenon could bethat the mutated P53 protein lost its function in nuclearlocalizationtheaccumulatedinandeventuallycytoplasm Furthermore p53 has been regarded as theœhotspots given that p53 mutations have been frequently detected in a variety of tumors [] In thisstudy several types of p53 mutations were demonstratedin natural and experimental infections of MDV The fivemost frequent mutation sites were and The altered codons of mutant p53 comparedwith the wildtype were ACGACA GCAGCC CGCCGG GCCGCA and ACCACA but these were all synonymous mutationsIn our study a short form of p53 transcript was detected in a clinical case The deleted sequence was located at the “ bp in the reading frame of thereference sequence resulting in missense mutations fromTable Primers used toamplify chicken p53 cDNAPrimerp531Nucleotide sequence™GTGGCCGTCTATAAGAAATCAGA3™™AAAAAGGGGGCGTGGTCAGT3™p532Annealing temperature „ƒSize of fragments amplifiedlocation bp 0cZhang BMC Veterinary Research Page of Fig Levels of p53 antigen and antibody in the serum of study groups The Yaxis represented the concentrations of antigen or antibody andthe Xaxis represented the different groups of samples Each group consisted of seven samples Statistical significance was designated as p or p the position to the termination The short form ofp53 transcript was also found in the cases infected byavian leukosis virus [] A series of missense mutationsfrom the position to the termination due to a base deletion was found in an experimentally infected chicken Inaddition this study found that the p53 was mutated in of poultry oncology with the mutation region concentrated mainly in the DBD The DBD allows the specificrecognition of target sequences [] Once the p53 is deleted or point mutations in this region occursit mayaffect the formation of tetramers which in turn leads tothe conversion of wild type P53 into a mutant type resulting in the loss of normal functionThe conformation of mutant P53 extends its halflifeto several hours in humans The accumulated mutantP53 then acts as a target antigen which elicits an autoimmune response [] However in our study the levelsof serum P53 antigen in clinical and experimentalMDVinfected groups were significantly lower than thecontrol group contrary to the findings in human cancerresearch Only the serum P53 antibody concentrationsof the experimentalinfected group were significantlyhigher than the control group This might be that P53as a tumor suppressor was largely consumed in response to the occurrence of MD Moreover tumorigenesis promoted mutation of p53 and further reduced P53concentrationConclusionsOur study revealed p53 was mutated and expressed inMDV infection which suggested that these mutationswere playing an important role in the development ofMD The mutant p53 was expressed in the tumor cellsof various tissues of the infected chicken Mutant P53protein lost its nuclear localization function and transferred from the nucleus to the cytoplasm Unlike studiesin human cancer the concentration of P53 was significantly lower in the natural and experimental MDV infectionnovelinnovations for the diagnosis and monitoring of MD inpoultryresults maygroup OurprovideMethodsNatural infection of MDV in chickensSeven 160dayold egglaying hens were obtained from alocal flock These hens were confirmed to have acquiredMDV infection naturally with the absence of other common viral diseases by PCR detection Their serums werecollected for the detection of the P53 antigen and antibody Their tissue samples including liver spleen pancreas and bursa of Fabricius were collected and fixedwith formaldehyde for h before histopathologicaland immunohistochemical studiesExperimental infection of chickens with MDVThe number of experimental animal was referred to thenumber of clinical samples Twenty 1dayold SPF chickens were obtained from the Shandong Academy of Agricultural Sciences Poultry Institute SPF Chicken ResearchCenter Jinan China They were randomly divided intotwo equal groups as an infection group and a controlgroup The two groups of chickens were raised separately in isolators with filtered air under positive pressureOn the first day of the experiment each of the chickenin the infection group was inoculated intraabdominallyia with plaqueforming units PFU of vvMDV 0cZhang BMC Veterinary Research Page of GX0101 which normally cause tumors at the tenthweek The chickens in the control group were inoculatedwith PBS In the tenth week serums were collected fromseven chickens in each group The experiment endedafter ten weeks and all chickens were put into a euthanasia box Thirty percent of carbon dioxide CO2 by volume was infused into the euthanasia box per minuteuntil all chickens lost their consciousness Then theCO2 flow rate was increased to for one minuteand the euthanasia box was kept airtight for ten minutesWhen all chickens were confirmed dead they were dissected and their livers and spleens were collected forhistopathology and immunohistochemistry studyHistopathology and ImmunohistochemistryThe fixed liver spleen pancreas and bursa of Fabriciuswere dehydrated waxed and cutinto µm slicesfollowed by Haematoxylin and eosin HE staining for ahistopathology examination In IHC processing tissueswere cut into sections of µm thickness and mountedon microslides treated with polyLlysine The sections were deparaffinized in xylene and rehydrated in agraded series of ethanol solutions into PBS then werepretreated in citrate buffer molL pH °Cfor antigen retrieval by microwaving and cooled at roomtemperature for min [] Endogenous alkaline peroxidase was quenched with hydrogen peroxide solutionin methanol for min [] Nonspecific antibody binding sites were obliterated by incubating the sections with fetal bovine serum for min at room temperatureFollowing this the antiP53 polyclonal antibody BOSTER China as a primary antibody was diluted into and immersed overnight at °C in a black humidchamber The secondary antibodies were HRP horseradish peroxidaseconjugated goatIgGCWBIO China Immunoreactivity was then visualizedwith diaminobenzidine DAB staining The sectionswere counterstained with hematoxylin and mountedNegative controls were incubated with PBS instead ofthe primary antibody in the immunohistochemicalanalysisantimouseTotal RNA isolation and reverse transcriptionThe total RNA ofliver and spleen tissue samples mgtissue were isolated by using TRIzol reagentTakara Japan according to the manufacturer™s instructions Extracted total RNA 1ug was reverse transcribedto cDNA with PrimeScript„¢ RT Reagent Kit RocheSwitzerland According to the published complementaryDNA cDNA sequence of the chicken p53 GeneBankaccession number nm205264 specific primers were designed to amplify the DNAbinding domain DBD ofp53 as shown in Table Using PCR Polymerase ChainReaction techniques p53 cDNA genes sequences wereamplified The PCR reaction was performed by using athermal cycler Takara Japan under the following conditions „ƒ for min followed by cycles of „ƒfor s „ƒ for s „ƒ for s and a final „ƒTable Mutations of p53 genes in MDSample InfoNCMutation analysisBase mutation sitesNDMD CMD CMD CMD CMD CMD CMD EMD EMD EMD EMD EMD E TC AC C G AG AC gA CG CA AC CG CA CG CA delete TC TC AC CG CG CA“ delete CA CA TG AC CG CA gA CA CA AG AC CG CA CT gA AC CG CA gA gT CT CG CA gANC was SPF chicken without diseases E was experimental chicken C was clinical chickenAmino acid mutationsSite from toNDMutation area Y A E G R HNDND Stop StopNDND N S T I G DND R L V M E Y H YCore domainCterminal domainCore domainCore domainCore domainCore domainCterminal domainCore domainC terminal domain 0cZhang BMC Veterinary Research Page of for min extension cycle DNA templates that were acquired from the MDV positive chickens were amplifiedusing primer pair p5312 The SPF chickens wereregarded as negative controls DNA fragments were successfully amplified with sizes of bp The target fragment was gel extracted and connected to the pEASYT1vector Then the recombinant plasmid was transformedinto bacteria and sequencing analysis was made Finallythe sequencing results were compared with the wild typep53 cDNA sequences reported previouslyEnzymelinked immunosorbent assay ELISA for P53 andP53 antibodyThe P53 antigen and antibody levels of chicken weremonitored by ELISA Mlbio China In order to eliminate subjective interference the assessors were blinded tothe background of the samples The detection rangeswere “ pgml for P53 antigen and pgmlfor P53 antibody Samples were diluted five time with aspecial diluent and all processes were implemented inaccordance with the instructions The absorbance ODof each sample was finally measured at a wavelength of nm The sample concentration was calculated depending on the standard curveStatistical analysisStatistical analyses were conducted with GraphPadPrism Version San Diego CA USA The differences in the levels of P53 antigen and antibody were antwotailed Student™s Ttestalyzed by usingStatistical significance was designated as p or p theAbbreviationscDNA Complementary DNA DAB Diaminobenzidine DBD DNAbindingdomain ELISA Enzymelinked immunosorbent assay HE Haematoxylin andeosin IHC Immunohistochemical HRP Horse radish peroxidasePCR Polymerase chain reaction PFU Plaque forming unitsAcknowledgementsNot applicableAuthors™ contributionsHXZ carried out laboratory work wrote the manuscript and performed thedata analyses MDL designed the experiment wrote the manuscript andperformed the data analyses HZ SLC YL SNJ and YNS carried out laboratorywork SDL conceived and supervised this work revised manuscript All authorsread and approved the final manuscriptFundingThe work was supported by grants from the National natural sciencefoundation project NO The funding body was solely involved infunding and had no role in the design of the study the collection analysisand interpretation of the data or in writing the manuscriptEthics approval and consent to participateThe sample were collected and handled in accordance with the goodanimal practices required by the Animal Ethics Procedures and Guidelines ofthe People™s Republic of China Informed consent to participate wasobtained from the chicken farmer owner All animal protocols andprocedures were performed according to the Chinese Regulations ofLaboratory Animals and were approved by the Animal Ethics Committee ofShandong Agricultural UniversityConsent for publicationNot applicableAvailability of data and materialsThe data supporting the findings are included in the manuscriptCompeting interestsThe authors declare that they have no competing interestsAuthor details1College of Animal Science and Veterinary Medicine Shandong AgriculturalUniversity Daizong Street Taian Shandong China 2China AnimalHealth and Epidemiology Center Nanjing Road QingdaoShandong ChinaReceived January Accepted August ReferencesJarosinski KW Tischer BK Trapp S Marek™s disease virus lytic replicationoncogenesis and control Expert Rev Vaccines “Reddy MS Book Review Marek™s Disease An Evolving Problem Vet Pathol“Boodhoo N Gurung A Sharif S Behboudi S Marek™s disease in chickens areview with focus on immunology Vet Res Lane DP p53 guardian of the genome Nature “Bertzbach LD Kheimar A Ali FAZ Kaufer BB Viral Factors Involved inMarek™s Disease Virus MDV Pathogenesis Curr Clin Microbiol Rep “Levine AJ p53 the cellular gatekeeper for growth and division Cell “Soussi T B¨gue A Kress M Stehelin D May P Nucleotide sequence of acDNA encoding the chicken p53 nuclear oncoprotein Nucleic Acids ResZilfou JT Lowe SW Tumor suppressive functions of p53 Review ColdSpring Harb Perspect Biol 20091a001883Stiewe T Haran TE How mutations shape p53 interactions with thegenome to promote tumorigenesis and drug resistance Drug Resist Updat“K¶bel M Piskorz AM Lee S Lui S LePage C Marass F Rosenfeld N MesMasson AM Brenton JD Optimized p53 immunohistochemistry is anaccurate predictor of TP53 mutation in ovarian carcinoma J Pathol Clin Res“ Zekiye H B¼lent T Taner E p53 antibody is it an indicator ofdedifferentiated thyroid cancer Ann Nucl Med “ Balogh GA Mailo D Nardi H Corte MM Vincent E Barutta E Lizarraga GLizarraga P Montero H Gentili R Serological levels of mutated p53 proteinare highly detected at early stages in breast cancer patients Exp Ther Med“Sabapathy K Lane DP Understanding p53 functions through p53antibodies J Mol Cell Biol “Kunizaki M Fukuda A Wakata K Tominaga T Nonaka T Miyazaki TMatsumoto K Sumida Y Hidaka S Yasutake T Sawai T Hamamoto RNanashima A Nagayasu T Clinical Significance of Serum p53 Antibody inthe Early Detection and Poor Prognosis of Gastric Cancer Anticancer Res“ Yue Q Yulong G Liting Q Shuai Y Delong L Yubao L Lili J Sidang LXiaomei W Mutations in and Expression of the Tumor Suppressor Gene p53in EggType Chickens Infected With Subgroup J Avian Leukosis Virus VetPathol “ Bykov VJN Eriksson SE Bianchi J Wiman KG Targeting mutant p53 forefficient cancer therapy Nat Rev Cancer “ Yemelyanova A Vang R Kshirsagar M Lu D Marks MA Shih IeM Kurman RJImmunohistochemical staining patterns of p53 can serve as a surrogatemarker for TP53 mutations in ovarian carcinoma an immunohistochemicaland nucleotide sequencing analysis Mod Pathol “ 0cZhang BMC Veterinary Research Page of Takagi M Ohashi K Morimura T Sugimoto C Onuma M The presence ofthe p53 transcripts with truncated reading frames in Marek™s diseasetumorderived cell lines Leuk Res “ Arlt C Ihling CH Sinz A Structure of fulllength p53 tumor suppressorprobed by chemical crosslinking and mass spectrometry Proteomics “Thierry S Analysis of p53 Gene Alterations in Cancer A Critical View Years of p53 Research “Stiasny A Freier CP Kuhn C Schulze S Mayr D Alexiou C Janko C Wiest IDannecker C Jeschke U Kost BP The involvement of E6 p53 p16 MDM2and Gal3 in the clinical outcome of patients with cervical cancer OncolLett “Shen FX Ma GP Cheng AC Wang MS Li CF Sun KF Chang H Zhu DK JiaRY Chen XY Sun T Development and application of an indirectimmunohistochemical method for the detection of duck plague virusvaccine antigens in paraffin sections and localization in the vaccinatedduckling tissues Poult Sci “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

Thyroid_Cancer

genes of papillary thyroidcarcinoma by integrated bioinformatics analysisGang Xue11Department of Otorhinolaryngology Head and Neck Surgery The First Affiliated Hospital of Hebei North University Zhangjiakou China 2Department of Histology andEmbryology Hebei North University Zhangjiakou ChinaJingFang Wu2 Da Pei1 DongMei Wang2 Jing Zhang2 and WenJing Zhang2Xu Lin2Correspondence JingFang Wu wjfxg163comBackground Papillary thyroid carcinoma PTC is one of the fastestgrowing malignant tumor types of thyroid cancer Therefore identifying the interaction of genes in PTC is crucialfor elucidating its pathogenesis and finding more specific molecular biomarkersMethods Four pairs of PTC tissues and adjacent tissues were sequenced using RNASeqand differentially expressed genes were screened P005 logFC1 The enrichment analysis indicated that the vast majority of differentially expressed genes DEGs mayplay a positive role in the development of cancer Then the significant modules were analyzed using Cytoscape software in the protein“protein interaction network Survival analysisTNM analysis and immune infiltration analysis of key genes were analyzed And the expression of ADORA1 APOE and LPAR5 genes were verified by qPCR in PTC compared withmatching adjacent tissuesResults Twentyfive genes were identified as hub genes with nodes greater than Theexpression of genes were verified by the GEPIA database and the overall survival anddiseasefree survival analyses were conducted with Kaplan“Meier plotter We found onlythree genes were confirmed with our validation and were statistically significant in PTCnamely ADORA1 APOE and LPAR5 Further analysis found that the mRNA levels andmethylation degree of these three genes were significantly correlated with the TNM staging of PTC And these three genes were related to PTC immune infiltration Verification ofthe expression of these three genes by RTqPCR and Western blot further confirmed thereliability of our resultsConclusion Our study identified three genes that may play key regulatory roles in the development metastasis and immune infiltration of papillary thyroid carcinomaThese authors contributedequally to this work and shouldbe considered as cofirstauthorsReceived May Revised August Accepted August Accepted Manuscript online August Version of Record published August IntroductionThyroid carcinoma is presently the malignancy with the most rapidly increasing incidence in the worldand is the most widely recognized endocrine carcinoma in the Western world [] Thyroid cancers derivedfrom follicular thyroid cells can be sorted into papillary thyroid carcinoma PTC follicular thyroid carcinoma FTC and anaplastic thyroid carcinoma ATC according to the histological subtype [] Clinicalresults vary across these subtypesThe annual rate of thyroid cancer has more than doubled within the past two decades with the vast majority of this increase being ascribed to PTC which accounts for “ of all thyroid carcinomas [] Inaddition patients with PTC suffer from cervical lymph nodes metastasis or remote metastasis which leadsto unfavorable results and approximately “ of cases may progress to a potentially fatal recurrentailment [] Due to these reasons uncovering the causes of PTC and its fundamental mechanisms andfinding molecular biomarkers for early diagnosis and customized treatment are significant and important The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201555101042BSR20201555tasksWith the advancement and continuous improvement of gene sequencing and geneediting technology it is nowconvenient to recognize the hub biomarkers related to neoplasm metastasis and survival status using a large amountof information available by applying bioinformatics [] Currently there are no effective sensitive biomarkers for earlydiagnosis treatment and prevention of lymph node metastasis of PTC An examination of differentially expressedgenes DEGs between tumor and paracarcinoma tissue may help identify critical biomarkers of papillary thyroidcarcinoma As a form of molecular marker mRNA containing the most abundant genetic information is necessaryfor protein translation and it is separate from the pathological process of cancer at various stages [] Some studiesutilized public databases such as The Cancer Genome Atlas TCGA and the Gene Expression Omnibus GEO toidentify significant biomarkers of papillary thyroid carcinoma However these investigations were only founded onsingle datasets with constrained sample sizes or just based on online databases used to screen out the DEGsIn the present study we analyzed the DEGs in PTC tissues versus the matched adjacent tissues by RNASeq andbioinformatics methods to obtain the DEGs Then we screened out the key modules and extracted the key genes inthose modules by constructing DEGs interaction network Then the possible role of differentially expressed geneswas analyzed using GO annotation and KEGG pathway enrichment analysis The expression validation survivalanalysis and functional enrichment analysis of key genes were conducted by using relevant databases Finally wefound that the three genes ADORA1 APOE and LPAR5 were highly expressed in PTC and were associated withPTC methylation TNM staging and immune infiltrationMethodsTissue samplesThirty pairs of PTC and adjacent tissues were collected from January to July at the First Affiliated Hospitalof Hebei North University This experiment was approved by the Ethical Committee of the First Affiliated Hospitaland all patients provided informed consent All tissues were frozen in liquid nitrogen after surgical resectionRNA library construction and sequencingTotal RNA was isolated from four adjacent normal and cancerous thyroid samples utilizing TRIzol reagent QiagenValencia CA USA as indicated by the manufacturer™s guidelines RNAs of PTC tissues and paracancerous tissuessample numbers 1C 1P 2C 2P 3C 3P 4C 4P the number represents different samples the œC indicates a cancersample and the œP represents a matched paracancerous tissue sample were used Six libraries were built utilizingan Illumina standard kit as indicated by the manufacturer™s protocol All sequencing was carried out on an IlluminaHiseq LC Bio ChinaDifferentially expressed genes screeningThe level of expression of mRNAs was evaluated using StringTie by calculating FPKM [] The DEGs between PTCand paracancerous tissue were screened with log2 fold change1 and P005 was regarded as statistically significant the analyses were conducted using the R package Ballgown []Functional enrichment analysis and pathway analysisTo reveal the functional roles of the DEGs the Annotation Visualization and Integrated Discovery function annotation tool DAVID httpdavidabccncifcrfgovhomejsp was used to perform Gene Ontology GO enrichmentanalysis and Kyoto Encyclopedia of Genes and Genomes KEGG pathway enrichment analysis P values less than were considered as cutoff criteriaPPI network construction and identification of hub genesPPI networks were constructed successively using STRING database tringdb [] The interactions ofDEGs with a combined score were set as significant and Cytoscape software version was utilized tovisualize and analyze the results of the STRING database To find key hub genes in this PPI network the significantmodule was analyzed by using the plugin MCODE of Cytoscape software The criteria for selection were set to thedefault The key genes were chosen with degrees ‰¥ Subsequently genes in that module were used to analyse theirfunctional roles with FunRich software The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201555101042BSR20201555Table PCR primersGene symbolADORA1ActinAPOELPAR5bp base pair F forward primer R reverse primerPrimer sequenceF5cid3CCACAGACCTACTTCCACACC3cid3R5cid3TACCGGAGAGGGATCTTGACC3cid3F5cid3CACTCTTCCAGCCTTCCTTCC3cid3R5cid3AGGTCTTTGCGGATGTCCAC3cid3F5cid3 GTTGCTGGTCACATTCCTGG 3cid3R5cid3 GCAGGTAATCCCAAAAGCGACcid3F5cid3 CACTTGGTGGTCTACAGCTTG3cid3R5cid3 GCGTAGTAGGAGAGACGAACG3cid3Data validation and analysisTo verify the accuracy of our RNAseq results we used the Gene Expression Profiling Interactive Analysis databaseto verify the expression of key genes in PTC and adjacent tissues The overall survival and diseasefree survivalanalyses were performed by Kaplan“Meier plots for these PTCrelated hub genes Genetic alterations of hub genesin PTC and their correlations with other genes were analyzed utilizing the cBioPortal for Cancer Genomics Hub genesrelated to clinicopathological features were analyzed using the online database UALCAN httpualcanpathuabedu[] The correlation of ADORA1 APOE and LPAR5 expression with the immune infiltration level in PTC and theexpression of these three genes in different kinds of cancers was performed using the Tumor Immune EstimationResource database []For qRTPCR analysis total RNA was isolated from normal and cancerous papillary thyroid samples utilizingTRIzol reagent Qiagen Valencia CA USA cDNA was synthesized with RNA reverse transcription kit TIANGENBIOTECH Beijing China qRTPCR was performed with an ABI RealTime PCR System Applied Biosystems Life Technologies USA The expression of the genes of interest was normalized to actin The primers forADORA1 APOE LPAR5 and actin are shown in Table For Western blot RIPA buffer was used to extract protein from four pairs of tissue from PTC patients and theprotein concentrations were measured via BCA methods Briefly the SDSPAGE gel was used for electrophoresis andPDVF membrane was used for transmembrane transfer PDVF membrane was blocked and then incubated with primary antiADORA1 antibodies dilution Bioss bs6649R APOE dilution Bioss bs4892R LPAR5antibodies dilution Bioss bs15366R and actin dilution Bioss bs0061R at —¦C overnight followed by incubation with secondary antibodies Zhongshanjinqiao dilution at —¦C for h The signal wasdetected using ECL methodStatistical analysisAll the data were analyzed by R and SPSS SPSS Inc USA Kaplan“Meier method was used to estimate thesignificant difference in survival between the overexpression group and the lowexpression group of key genes inpapillary thyroid carcinoma The statistical difference was set at P ResultsDifferentially expressed genes screening based on RNASeqTo screen out the genes or modules that may play a role in promoting cancer in papillary thyroid carcinoma weperformed RNASeq experiments on four pairs of thyroid cancer tissues and their matched paracancerous tissues toobtain differentially expressed genes After RNASeq we acquire “ million reads for each sample The fold changesbetween PTC cancer tissues and matched paracancerous samples were calculated Setting the cutoff criterion as Pvalue and a fold change there were upregulated and downregulated genes These DEGswere considered to be candidate genes for subsequent study Figure 1A showed the expression of the top genes inPTC versus matched paracancerous tissuesFunctional enrichment analysis and pathway analysisConsidering that there were many falsepositive genes among these DEGs we verified our results one by onethrough the TCGA database We found that only genes in our data were consistent with the gene expression of the The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201555101042BSR20201555Figure Identification of DEGs by RNAseqThe heat map A and PPI network of the DEGs B C The volcano plots of the DEGs D The most significant module was selectedby MCODE in Cytoscape Red represents the upregulated genes and blue represents the downregulated genesFigure GO and KEGG pathway enrichment analysis of DEGs through RNASeqA Bubble plot of Gene Ontology enrichment analysis of DEGs B Bubble plot of Kyoto Encyclopaedia of Genes and Genomespathway enrichment analysis of DEGsTCGA database To investigate the potential function of these DEGs in PTC genes functional enrichment was conducted by using GO and KEGG pathway analyses For the biological process category the DEGs were significantly involved in the regulation of axonogenesis regulation of cell morphogenesis extracellular structure anization extracellular matrix anization synapse anization cellsubstrate adhesion and urogenital system development Thecellular component category results showed PTCrelated DEGs were enriched in collagencontaining extracellularmatrix synaptic membrane cell“cell junction glutamatergic synapse neurontoneuron synapse postsynaptic membrane basolateral plasma membrane DEGs in molecular function were mainly involved in cell adhesion moleculebinding passive transmembrane transporter activity extracellular matrix structural constituent glycosaminoglycanbinding growth factor binding transmembrane receptor protein kinase activity and transmembrane receptor proteintyrosine kinase activity Figure 2AAs Figure 2B showed the KEGG pathway results showed DEGs were enriched in cytokine“cytokine receptorinteraction MAPK signaling pathway proteoglycans in cancer Rap1 signaling pathway axon guidance Cushing The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201555101042BSR20201555Figure GO enrichment analysis and KEGG analysis for the key genesA Top elements involved in biological processes B Top elements involved in molecular function C Top elementsinvolved in cellular components D Top pathways related to the key genes through KEGG analysissyndrome parathyroid hormone synthesis secretion and action AGERAGE signaling pathway in diabetic complications growth hormone synthesis secretion and action salivary secretion circadian entrainment cholinergic synapse p53 signaling pathway ECM“receptor interaction arrhythmogenic right ventricular cardiomyopathyARVC endocrine resistance renin secretion Type II diabetes mellitus bladder cancer nicotinate and nicotinamidemetabolism and apoptosismultiple speciesPPI network construction and module analysisPPI networks were constructed successively by the database by loading the PTC related DGEs into the STRINGdatabase Figure 1BC Using Cytoscape we analyzed the most significant module in the PPI network Figure 1DThe PPI network consisted of nodes and edges Following the use of MCODE in Cytoscape the significantmodule was selected The top hub genes ADCY8 ADORA1 ADRA2C APOE C5AR1 CCL13 CCL20 CDH2CHGB CXCL12 EVA1A FAM20A FN1 GNAI1 GPC3 GRM4 LPAR5 MELTF or MFI2 MFGE8 NMU OPRM1SERPINA1 SSTR3 TIMP1 and TNC were evaluated by degree in the PPI network Figure 1D The resultsshowed that the functions of the key genes were mainly concentrated in signal transduction cell communicationGprotein coupled receptor activity cell adhesion molecule activity and GPCR ligand binding Figure Data analysis and validationAfter the key genes were selected the expression of key genes in PTC and its adjacent tissues were verified by theGEPIA database Figure ADORA1 APOE EVA1A LPAR5 MFGE8 OPRM1 SERPINA1 SSTR3 and TIMP1were positively related to the overall survival analysis of PTC patients while C5AR1 and GNAI1 were negativelyrelated Figure ADCY8 ADORA1 CHGB FN1 LPAR5 NMU and TNC showed positive associations withdiseasefree survival analysis of PTC patients but not APOE Figure Next we analyzed the alterations of the key genes by using the cBioPortal database Figure The key geneswere changed in of queried samples Figure 7B Figure 7A showed the frequency of alterations of eachPTC related key gene SSTR3 FN1 and ADORA1 were altered the most and respectively Figure 7Dshowed the network of the genes and their altered neighbouring genes in PTC patients out of a total of Among these genes only ADORA1 APOE and LPAR5 genes simultaneously showed statistical significance foroverall survival analysis and diseasefree survival analysis of PTC patients The qPCR experiments and Western blotdata verified that these three survivalrelated genes were all overexpressed in PTC Figure Then based on UAL The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201555101042BSR20201555Figure Validation of the key DEGs in the GEPIA databaseADORA1 APOE CCL13 CDH2 CXCL12 EVA1A FAM20A FN1 GNAI1 LPAR5 MFGE8 NMU SERPINA1 TIMP1 and TNC areoverexpressed in PTC tissues compared with paracancerous tissue while GNAI and GPC3 are downregulated The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201555101042BSR20201555Figure Overall survival analysis of key genes in PTC using Kaplan“Meier plotsExpression levels of ADORA1 APOE C5AR1 EVA1A FAM20A GNAI1 LPAR5 MFGE8 OPRM1 SERPINA1 SSTR3 and TIMP1are related to the overall survival of patients with PTCCAN the clinical features and degree of methylation of these three genes were analyzed The transcription levels ofADORA1 APOE and LPAR5 were significantly higher in PTC patients than normal tissues according to subgroupsof sample types individual stages and nodal metastasis status Figure In addition ADOR1 and LPAR5 exhibiteda hypomethylation state in the cancer group but APOE showed a hypermethylation state in PTC samples Figure10ATo further clarify the role of these genes we conducted an analysis of immune infiltration The ADOR1 expression level was positively corelated with infiltrating levels of B cells r0111 P151e2 CD8 T cells r0246P396eˆ’ neutrophils r0162 P331eˆ’ and DCs r0232 P232eˆ’ The expression of APOE was The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201555101042BSR20201555Figure Diseasefree survival analysis of key genes in PTC using Kaplan“Meier plotsExpression levels of ADCY8 ADORA1 APOE CHGB FN1 LPAR5 NMU and TNC are significantly related to the diseasefreesurvival of patients with PTCpositively associated with B cells r0228 P439eˆ’ CD8 T cells partialcor P930eˆ’ neutrophils r0197 P114eˆ’ and DCs partialcor P358eˆ’ LPAR5 expression level was positively related to B cells r0259 P815eˆ’ CD4 T cells r0238 P103eˆ’ macrophages r0175P105eˆ’ neutrophils r027 P142eˆ’ and DCs r0256 P104eˆ’ and negatively related to Purity r ˆ’ P294eˆ’ and CD8 T cells r ˆ’ P618eˆ’ Figure 10B These findings stronglysuggested that LPAR5 ADOR1 and APOE may play specific roles in immune infiltration in PTC especially those ofDCs Finally we examined the expression of these three genes in common cancer tissues and adjacent tissues and wefound that these three genes were highly expressed in most cancer tissues Figure The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201555101042BSR20201555Figure The key genes expression and mutation analysis in PTC by the cBioPortal for Cancer GenomicsA The genetic alterations of key genes of PTC samples Queried genes are altered in of queried patientssamplesB The expression heatmap of key genes C The alteration frequency of key genes in PTC D Network of key genesmutations and their frequently altered neighboring genes in PTC The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201555101042BSR20201555Figure The mRNA and protein expressions of ADORA1 APOE and LPAR5 in PTC tissuesA“C Validation of expression levels of ADORA1 APOE and LPAR5 by RTqPCR in cases of PTC and matched adjacent tissuesD ADORA1 APOE and LPAR5 protein levels are increased in four cases of PTC and matched adjacent tissues as measured byWestern Blot P0001DiscussionPTC is a common cancer with great heterogeneity in morphological features and prognosis [] Although most papillary thyroid carcinomas exhibit low biological activity there are still a few patients with higher invasive and metastaticclinical features [] Activation of oncogene expression and loss of function of tumor suppressor genes may lead tothe development or progression of PTC To better clarify the molecular mechanism of PTC occurrence development and metastasis we identified key genes related to PTC progression through comprehensive bioinformaticsmethods and we screened three of the PTC prognosisrelated genes for a comprehensive analysisIn the present study we identified differentially expressed genes by RNASeq with GO enrichment analysis showing that the DEGs were enriched in the regulation of the axonogenesis regulation of cell morphogenesis extracellular structure anization extracellular matrix anization synapse anization cell“substrate adhesion urogenital system development collagencontaining extracellular matrix synaptic membrane cell“cell junction glutamatergic synapse neuron to neuron synapse postsynaptic membrane basolateral plasma membranecell adhesion molecule binding passive transmembrane transporter activity extracellular matrix structural constituent glycosaminoglycan binding growth factor binding transmembrane receptor protein kinase activity andtransmembrane receptor protein tyrosine kinase activity And KEGG pathway results showed DEGs were enrichedin cytokine“cytokine receptor interaction MAPK signaling pathway proteoglycans in cancer Rap1 signaling pathway axon guidance Cushing syndrome parathyroid hormone synthesis secretion and action AGERAGE signalingpathway in diabetic complications growth hormone synthesis secretion and action salivary secretion circadian entrainment cholinergic synapse p53 signaling pathway ECM“receptor interaction arrhythmogenic right ventricularcardiomyopathy ARVC endocrine resistance renin secretion Type II diabetes mellitus bladder cancer nicotinateand nicotinamide metabolism and apoptosismultiple speciesTo further explore the interrelationship of differentially expressed genes in papillary thyroid carcinoma we constructed a PPI regulatory network A total of DEGs with nodes greater than were screened out in the networkThe key genes were ADCY8 ADORA1 ADRA2C APOE C5AR1 CCL13 CCL20 CDH2 CHGB CXCL12 EVA1AFAM20A FN1 GNAI1 GPC3 GRM4 LPAR5 MELTF MFGE8 NMU OPRM1 SERPINA1 SSTR3 TIMP1 andTNC Biological process and molecular function analyses of these key DEGs indicated that they were significantly The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201555101042BSR20201555Figure Relative expression of ADORA1 APOE and LPAR5 in normal thyroid tissues and PTC tissues individual cancerstages and nodal metastasis status respectively UALCANP0001involved in cancer regulation processes such as adjustment of cell growth or maintenance cell immune response celladhesion molecular activity and extracellular matrix structural constituentTo verify the credibility of the experiments and data the DEGs screened were verified by the GEPIA databaseAmong the selected genes genes showed expression differences consistent with our RNASeq data Among the The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201555101042BSR20201555Figure Methylation level and immune infiltration level of ADORA1 APOE and LPAR5A Relative methylation level of ADORA1 APOE and LPAR5 based on normal thyroid tissues and PTC tissues individual cancerstages and nodal metastasis status respectively UALCAN B The correlation between the three genes and TIICs TIMER TIICstumor infiltrating immune cells genes ADORA1 APOE CCL13 CDH2 EVA1A FAM20A FN1 LPAR5 MFGE8 NMU SERPINA1 TIMP1and TNC levels were overexpressed in PTC tissues while GPC3 and GNAI1 were downregulatedADORA1 belongs to the Gprotein coupled receptor family and protects human tissues and cells under physiological conditions [] Lin et al suggested that ADORA1 may promote the proliferation of breast cancer cellsby positively regulating oestrogen receptoralpha in breast cancer cells [] Similarly Jayakar indicated that knockdown of APOE expression can reduce the level of MMPs by regulating the AP1 signaling pathway and thus reducethe invasion and metastasis of oral squamous cell carcinoma [] Bioinformatics predictions were that APOE mRNAshows a significant increase in PTC [] CCL13 is a coding gene involved in immune regulation and inflammatoryresponses and it has been reported that CCL13 has a role in promoting the proliferation of tumorforming volumein nude mice [] CDH2 is overexpressed in various cancers Some research results indicate that overexpression ofCDH2 can increase the invasive ability and induce EMT in lung cancer cells [] Qiu et al confirmed CDH2 actsas an oncogene in papillary thyroid carcinoma which is consistent with our findings [] EVA1A acts as a regulatorof programmed cell death and Shen et al indicates that EVA1A can inhibit the proliferation of GBM cells by inducing autophagy and apoptosis via inactivating the mTORRPS6KB1 signaling pathway [] FAM20A may play a keyrole in haematopoiesis There are few reports on the relationship between FAM20A and cancer and our experimentfound that FAM20A is more highly expressed in papillary thyroid carcinoma than in other cancers FN1 is involvedin regulating cell adhesion cell movement wound healing and maintaining cell morphology [] Some researchersindicated that FN1 participates in regulating many types of cancer progression such as gastric cancer [] skin squamous cell carcinoma [] and papillary thyroid carcinoma [] It has been shown that LPAR5 is related tothe pathogenesis of pancreatic cancer [] Consistent with our study Zhang et al believes that LPAR5 may be involved in the development of papillary thyroid carcinoma [] According to previous reports MFGE8 is involvedin the progression of various malignancies such as breast cancer melanoma bladder tumors and ovarian cancer The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201555101042BSR20201555Figure The expression of ADORA1 APOE and LPAR5 in thyroid cancer tissues and normal thyroid tissuesThe three genes expression were analyzed in different kind of cancer tissues and normal tissues via the TIMER database P005P001 P001 The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201555101042BSR20201555[“] MFGE8 is considered to be a potential therapeutic target for ovarian cancer owing to its carcinogenic effect[] Consistent with our data Zhang et al indicate that NMU is one of the DEGs of papillary thyroid carcinoma[] Recently a researcher has shown that abnormal expression of NMU is associated with a variety of cancers []For SERPINA1 there are currently six s pointing out that SERPINA1 may be a key gene for PTC consistentwith our results [“] Clinical studies have shown that high expression of TIMP1 is positively correlated witha poor prognosis of colon brain prostate breast lung and several other cancers [] TNC is a component of theextracellular matrix ECM and is closely related to the malignant biological behavior of cancer In particular TNCoverexpression is positively associated with liver cancer oral squamous cell carcinoma and lymph node metastasisof breast cancer [] GPC3 belongs to the glypicans family It has been reported that overexpression of GPC3can promote the metastasis of hepatocellular carcinoma [] but we found that it is expressed at low levels in PTCSimilar to GPC3 some scholars believe that GNAI1 is a tumorpromoting gene and reported upregulated GNAI1mRNA in human glioma which is inconsistent with our data []Only the ADORA1 APOE and LPAR5 genes simultaneously showed statistical significance for overall survivaland diseasefree survival of PTC patients Considering that the occurrence and metastasis of cancer is a complexand multiregulated process we further analyzed the regulatory mechanisms of these three genes We found thatthe mRNA and methylation levels of these three genes were significantly correlated with TNM staging In additionADORA1 APOE and LPAR5 were all related to immune infiltration especially to dendritic cells Finally we foundthat these three genes were more highly expressed in cancer tissues than matched adjacent tissuesHowever our research has certain limitations First only four pairs of cancer and adjacent tissues were analyzedusing RNAseq in this experiment so further research requires a larger sample size Second further experiments areneeded to validate the specific mechanisms of these key genesData AvailabilityThe data used to support the findings of this study are available from the corresponding author upon requestCompeting InterestsThe authors declare that there are no competing interests associated with the manuscriptFundingThis study was supported by grants from the Hebei Provincial Department of Finance Specialist Capacity Building and SpecialistLeadership Program [grant number ] the Hebei Provincial Natural Science Foundation Project [grant number H201840505]and the Hebei North University Basic Research Business Expenses Project [grant number JYT2019015]Author ContributionXu Lin conducted the bioinformatics analysis Xu Lin and Gang Xue contributed as first authors Xu Lin wrote the manuscriptJingFang Wu and Gang Xue critically revised the Gang Xue and Da Pei obtained clinical specimens and the others contributed to verification of the RNAseq resultsAbbreviationsATC anaplastic thyroid carcinoma ECM extracellular matrix FTC follicular thyroid carcinoma PTC papillary thyroid carcinomaReferences Kitahara CM and Sosa JA The changing incidence of thyroid cancer Nat Rev Endocrinol “101038nrendo2016110 AschebrookKilfoy B Ward MH Sabra MM and Devesa SS Thyroid cancer incidence patterns in the United States by histologic type Thyroid “ 101089thy20100021 Pourseirafi S Shishehgar M Ashraf MJ and Faramarzi M Papillary Carcinoma of Thyroid with Nasal Cavity Metastases A Case Report IranJ Med Sci “ Ullmann TM Gray KD Moore MD Zarnegar R and Fahey III TJ Current controversies and future directions in the diagnosis andmanagement of differentiated thyroid cancers Gland Surg “ 1021037gs20170908 Jin X Deng B Ye K et al Comprehensive expression profiles and bioinformatics analysis reveal special circular RNA expression and potentialpredictability in the peripheral blood of humans with idiopathic membranous nephropathy Mol Med Rep “103892mmr201910671 Rapisuwon S Vietsch EE and Wellstein A Circulating biomarkers to monitor cancer progression and treatment Comput Struct BiotechnolJ “ 101016jcsbj201605004 The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BY 0cBioscience Reports BSR20201555101042BSR20201555 Pertea M Pertea GM Antonescu CM et al StringTie enables improved reconstruction of a transcriptome from RNAseq reads NatBiotechnol “ 101038nbt3122 Frazee AC Pertea G Jaffe AE et al Ballgown bridges the gap between transcriptome assembly and expression analysis Nat Biotechnol “ 101038nbt3172 Von Mering C Huynen M Jaeggi D et al STRING a database of predicted functional associations between prot

Thyroid_Cancer

American Joint Committee on Cancer AJCC Cancer Staging Manual 8th edition we explored the characteristics of central lymph node metastasis CLNM of papillary thyroid microcarcinoma PTMC in elderly patients ‰¥ years of age Our goal was to provide references for establishing a lymph node dissection scheme in such patientsMethods We retrospectively analyzed the clinical data of thyroid cancer patients admitted to the Head and Neck Surgery Center of Sichuan Cancer Hospital Chengdu China from January to September Then we screened and analyzed eligible PTMC cases in strict accordance with our inclusion and exclusion criteriaResults The study included patients including men and women Median age was ± years The maximum diameter range of the cancer foci was “ mm and the median was ± mm Unilateral lobectomy had been performed in cases total thyroidectomy in cases and lateral cervical lymph node dissection in cases There were cases of CLNM and cases of lateral cervical lymph node metastasis The sensitivity of preoperative ultrasound in predicting CLNM was but its accuracy was only Multivariate logistic regression analysis showed that multiple cancer foci area under the curve [AUC] extrathyroidal expansion of cancer focus AUC and irregular nodules AUC were independent risk factors for CLNM of PTMC in elderly patients P Overall predictability for PTMCCLNM was Conclusion Preoperative color Doppler ultrasound is not recommended as the basis for cervical lymph node dissection in PTMC patients For multiple cancer foci irregular nodules and elderly patients with PTMC extrathyroidal expansion we recommend a prophylactic central lymph node dissecting Nonsurgical observation of PTMC in elderly patients with low risk should be carefully selectedKeywords elderly patients thyroid cancer papillary carcinoma microcarcinoma central lymph node metastasisIntroductionIn the World Health anization defined thyroid cancer TC with a maximum tumor diameter of mm as microcarcinoma Up to now doctors in different countries and regions of the world still differ significantly on how to treat such diseases including on whether to perform preventive lymph node dissection in the central region of the cancer As the AJCC raised the age factor in the clinical staging of TC from to in it can be seen that the medical community Cancer Management and Research “ Fu This work is published and licensed by Dove Medical Press Limited The full terms of this license are available at wwwdovepresscomtermsphp and incorporate the Creative Commons Attribution “ Non Commercial unported v30 License httpcreativecommonslicensesbync30 By accessing the work you hereby accept the Terms Noncommercial uses of the work are permitted without any further permission from Dove Medical Press Limited provided the work is properly attributed For permission for commercial use of this work please see paragraphs and of our Terms wwwdovepresscomtermsphp 0cFu Dovepresstends to be more conservative in general on surgical treatment of TC Based on clinical staging of TC in the AJCC Cancer Staging Manual 8th edition this study aimed to explore the characteristics of CLNM of PTMC in the elderly population ‰¥ years so as to provide some references for developing clinical treatment plans for such patients mm underwent concurrent total thyroidectomy and bilateral central lymph node dissection Centralregion lymph nodes were dissected in the following areas upper boundary“lower hyoid margin lower boundary“superior sternal fossa upper margin of the unknown artery and external“lateral margin of the carotid sheath and lower boundary“anterior vertebral fasciaMethodsPatientsWe retrospectively analyzed the data of patients with PTMC admitted to the Head and Neck Surgery Center of Sichuan Cancer Hospital Chengdu China from January to September Eligible patients were screened in strict accordance with our inclusion and exclusion criteria for relevant data statistics and analysis Inclusion criteria were as follows Patient age was ‰¥ years All of the patients had been newly diagnosed and newly treated with no previous history of thyroid surgery Postoperative pathological diagnosis based on paraffinembedded sections was papillary carcinoma Benign nodules with or without pathology and mm in maximum diameter suggested by preoperative color ultrasound Patients™ complete medical records were available All of the surgeons had years™ experience in thyroid surgery Patients had undergone surgical resection of glandular lobes and isthmus on the affected side as well as lymph node dissection in the central region with or without lateral cervical lymph node dissection Exclusion criteria were as follows Postoperative pathology indicated mixed carcinoma with papillary and other types of carcinoma Patient had refused lymph node dissection in the central area on the affected side There were multiple cancer lesions with the sum of the maximum diameter mmSurgical TechniqueAll of the patients had been operated on according to the œat least  principle namely lymph node dissection at least in the central area on the cancerous side Total thyroidectomy bilateral centralarea lymph node dissection with or without lateral cervical lymph node dissection were performed on patients with preoperative cytological confirmation of bilateral multilobed carcinoma or lateral cervical lymph node metastasis Those with papillary microcarcinoma in one glandular lobe and benign nodules in the other glandular lobe ie multiple nodules with maximum diameter of Statistical AnalysisWe used SPSS software version SSPS Inc Chicago Illinois US to statistically analyze all of the data In our analysis of risk factors for lymph node metastasis in the central region we performed singlefactor analysis using χ2 test We ran multivariate logisticregression analysis on statistically significant positive univariate influencing factors as well as univariate and multivariate receiver operating curve ROC analysis on the previously analyzed risk factors to predict lymph node metastasis in the central regionResultsWe screened a total of PTMC cases Of these met the inclusion criteria including men and women with a maletofemale ratio of Patients™ age range was “ years old with a median of ± years The maximum diameter range of the cancer lesion was “ mm median ± mm There were cases with singleleaf singlefocus with singleleaf multifocus and with multileaf multifocus Unilateral lobectomy was performed in cases and total thyroidectomy in cases and lateral cervical lymph node dissection in cases In terms of staging of cases were in stage T1 in stage T3 and in stage T4 Postoperative pathology showed CLNM in cases and lateral cervical lymph node metastasis in cases Evaluation of Central Lymph Nodes by Color Doppler UltrasoundPreoperative ultrasound examination indicated stage cN1a cases and stage cN0 cases Postoperative pathology confirmed stage pN1a cases and stage pN0 cases which were significantly different Predictive sensitivity specificity positive predictive value PPV and negative predictive value NPV were and respectively Table submit your manuscript wwwdovepresscom DovePress Cancer Management and Research 0cDovepress Fu et alTable Comparison of Preoperative Ultrasound Predictions of CentralRegion Lymph Node MetastasisNTotalUltrasonic StagingcN1acN0Pathological stagepN1apN0TotalSensitivity Specificity PPV NPV Matching rate Abbreviations PPV positive predictive value NPV negative predictive valueUnivariate AnalysisIn this study singlefactor analysis of patients with PTMC age ‰¥ years showed that distribution nodule morphology calcification and extrathyroidal expansion of cancer focus significantly influenced centralregion lymph node metastasis P However patients™ gender thyroid stimulating hormone TSH levels thyroglobulin Tg levels nodular goiter Hashimoto™s thyroiditis maximum diameter of cancer focus nodular boundary and nodular blood flow had no statistical significance on such metastasis Table Multivariate LogisticRegression AnalysisFactors that had been statistically significant in univariate analysis results were further included in multivariate logisticregression analysis variables that might be clinically relevant but had been negative in univariate analysis were also included We found that distribution morphology and extrathyroidal expansion of cancer focus were independent risk factors for CLNM P while gender TSH Tg nodular goiters Hashimoto™s thyroiditis nodular boundary blood flow calcification and maximum diameter had no predictive significance Table ROC Curve AnalysisWe performed ROC curve analysis according to the independent risk factors obtained in our multivariate logistic regression analysis of CLNM as discussed above and we calculated areas under the curve AUCs Figures and DiscussionDisease Status of TCThe incidence of TC has been on the rise globally over the past years which has been confirmed by most current to International Agency studies1“ According for Research on Cancer IARC data for a total of new cases malefemale and deaths malefemale were reported in countries around the world respectively accounting for and of all new cancer cases and deaths4 On the one hand due to the great influence of medical ultrasound and cytologicalpuncture diagnosis the proportion of PTMC in new cases of TC has increased significantly According to the data the overall incidence of PTMC in the United States has increased by over the past years with average annual new cases accounting for about “ On the other hand PTMC incidence in the elderly is significantly higher than that in the general adult population Some studies have shown that the average annual growth rate of PTMC in patients years old is times that in adults years old8“ These results indicate that we should pay sufficient attention to elderly PTMC patients As the 8th edition of the AJCC Cancer Staging Manual raise PTC staging age from to years old it further confirms this viewTherapeutic ControversiesAs we all know diagnosis and treatment of PTMC have always been controversial especially in elderly patients In Japan™s Kuma hospital Ito defined the maximum diameter of thyroid cancer foci ‰¤10cm no cervical and distant lymph node metastasis and cytological biopsy of thyroid cancer foci as nonhighly malignant and no invasion of the trachea and recurrent laryngeal nerve as the judgment criteria for lowrisk PTMC After analyzing the data of patients they concluded that immediate surgical treatment of all PTMC patients was more harmful than beneficial so they suggested that lowrisk PTMC patients should choose active observation Among them elderly PTMC patients over years old were considered to be the most suitable group for observation1112 Conversely Megwalu UC of the National Cancer Center Plainview New York US reviewed cases in which senile PTMC patients age ‰¥ received nonoperative therapy His data analysis shows that the overall 5year survival rate was and the surgery was P which suggests that surgery for such patients has a survival advantage although more highquality investigative studies are necessary1 œ American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated Thyroid Cancer suggested that suspicious malignant thyroid nodules with a maximum diameter of cm be followed up to cm for cytological Cancer Management and Research submit your manuscript wwwdovepresscom DovePress 0cFu DovepressTable SingleFactor Analysis of CentralRegion Lymph Node MetastasisFactorsGenderMaleFemaleTSH levelsNormalAbnormalTg levelsNormalAbnormalNodular goiterYesNoHashimoto™s thyroiditisYesNoDistribution of carcinomaUnilateral glandular lobes single fociUnilateral glandular lobes multiple fociBilateral glandular lobes multiple fociMaximum diameter‰¤ mm mm x ‰¤ mmBoundaryClearUnclearEchoLow or noStrong or mixedExtrathyroidal expansionYesNoCalcificationYesNoBlood flowRichNot richNumber n107Central Lymph Node Metastasis CLNMχ2PYes n No n “““Abbreviations TSH thyroidstimulating hormone Tg thyroglobulinpuncture and other treatments but immediate surgical treatment is still recommended for highrisk patients In this study we performed surgical treatment on all PTMC patients with clear diagnoses including stage T1 T3 and T4 Although the study sample size needs to be further expanded we still believe that microcarcinoma is not equal to early cancer The percentage of differentiated tumor cells in elderly PTC patients is relatively higher than in youth and children leading to shorter life expectancy Choosing followup for middleaged and elderly submit your manuscript wwwdovepresscom DovePress Cancer Management and Research 0cDovepress Fu et alTable Multivariate LogisticRegression Analysis of Lymph Node Metastasis in the Central RegionFactorsGenderDistribution of carcinomaMaximum diameterTumor formation patternBoundaryExtrathyroidal expansionCalcificationBlood flowβˆ’ˆ’SEWaldPOR CI OR““““““““Abbreviations SE Standard error OR odds ratio CI confidence intervalPTMC patients may be feasible but for those with longer life expectancy early surgery can significantly reduce future progress of tumors may not only but also reduced the forward of surgery as a result of basic diseases such as cardiovascular increase intolerance Therefore for PTMC patients age ‰¥ with good survival expectations we believe surgical intervention is still necessary which is also consistent with Shindo et al13Risk FactorsIn the past there have been many studies analyzing PTMCCLNM but few such reports address elderly patients with PTMC Due to air interference in the tracheal cavity it is relatively difficult to diagnose CLNM of the neck using ultrasound which has a high falsenegative rate In this study the accuracy of ultrasound in predicting CLNM was Therefore it is questionable whether dissection of such lymph nodes can be performed only by preoperative ultrasound Chung et al14 found that in young PTMC patients multiple cancer foci enlarged nodules extrathyroidal expansion of cancer focus and vascular invasion are independent risk factors for PTMCCLNM and lateral cervical lymph node metastasis but they did not clearly identify calcified nodules as an independent risk factor By analyzing the data of PTMC patients Oh et al15 showed that the rate of lymph node metastasis in patients with calcified nodules was higher than in patients whose nodules were not calcified they concluded that calcified nodules were an important risk factor for PTMC cervical lymph node metastasis Haugen et al16 have a similar view In this study the metastasis rates of the central cervical region and lateral cervical lymph nodes were and respectively The χ2 test showed that distribution nodular morphology calcification and extrathyroidal expansion of cancer focus were risk factors for centralregion lymph nodes P Figure Areas under the curve AUC Distribution of carcinoma AUC extrathyroidal expansion AUC tumor formation pattern AUC Cancer Management and Research submit your manuscript wwwdovepresscom DovePress 0cFu DovepressAcknowledgmentsAll authors made substantial contributions to conception and design acquisition of data or analysis and interpretation of data took part in drafting the article or revising it critically for important intellectual content gave final approval of the version to be published and agree to be accountable for all aspects of the work We thank LetPub for its linguistic assistance during the preparation of this manuscriptDisclosureThe authors report no conflicts of interest for this workFigure Multiple independent risk factors predicted lymph node metastasis in the central region Areas under the curve AUC which was consistent with Liu et al17 However our multivariate logisticregression analysis found that only distribution of cancer lesions χ2 P nodule morphology χ2 P and extra thyroidal expansion of cancer focus χ2 P were independent risk factors for such metastasis The AUCs of these factors were and respectively and overall predictability was In summary we believe that active followup and observation should be carefully selected for elderly patients with PTMC especially for those with multiple cancer foci extrathyroidal expansion of cancer focus and irregular morphology preventive centralarea lymph node dissection is also appropriate Although we did not find nodular calcification maximum tumor diameter Hashimoto™s thyroiditis or other variables to be independent risk factors we believe this result may have a certain relationship with the small sample size which we will further expand in the future for related studies and supplementsEthical ApprovalThis study was approved by the Institutional Review Board of Sichuan Cancer Hospital and Institutional Ethics Committee and performed according to the ICH GCP principleInformed ConsentWe obtained written informed consent from all of the individual participants included in the studyReferences Megwalu UC Observation versus thyroidectomy for papillary thyroid microcarcinoma in the elderly J Laryngol Otol “ doi101017S0022215116009762 Davies L Welch HG Current thyroid cancer trends in the United States JAMA Otolaryngol Head Neck Surg “ doi101001jamaoto20141 Kilfoy BA Zheng T Holford TR et al International patterns and trends in thyroid cancer incidence “ Cancer Causes Control “ doi101007s1055200892604 Bray F Ferlay J Soerjomataram I et al Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA Cancer J Clin “ doi103322caac21492 Hughes DT Haymart MR Miller BS et al The most commonly occurring papillary thyroid cancer in the United States is now a microcarcinoma in a patient older than years Thyroid “ doi101089thy20100137 Davies L Welch HG Increasing incidence of thyroid cancer in the JAMA “ United States “ doi101001jama295182164 Kuo EJ Goffredo P Sosa JA Aggressive variants of papillary thyroid microcarcinoma are associated with extrathyroidal spread and lymphnode metastases a populationlevel analysis Thyroid “ doi101089thy20120563 Hay ID Hutchinson ME GonzalezLosada T Papillary thyroid microcarcinoma a study of cases observed in a 60year period Surgery “ discussion “ doi101016j surg200808035 Simard EP Ward EM Siegel R et al Cancers with increasing incidence trends in the United States through CA Cancer J Clin “ doi103322caac20141 Cramer JD Fu P Harth KC Analysis of the rising incidence of thyroid cancer using the surveillance epidemiology and end results national cancer data registry Surgery “ doi101016jsurg201010016 Ito Y Miyauchi A Kudo T et al Trends in the implementation of active surveillance for lowrisk papillary thyroid microcarcinomas at Kuma Hospital gradual increase and heterogeneity in the acceptance of this new management option Thyroid “ doi101089thy20170448 Ito Y Miyauchi A Kihara M Patient age is significantly related to the progression of papillary microcarcinoma of the thyroid under observation Thyroid “ doi101089thy20130367 Shindo M Wu JC Park EE The importance of central compartment elective lymph node excision in the staging and treatment of thyroid cancer Arch Otolaryngol Head Neck Surg papillary “ doi101001archotol1326650submit your manuscript wwwdovepresscom DovePress Cancer Management and Research 0cDovepress Fu Liu LS Liang J Li JH et al The incidence and risk factors for central lymph node metastasis in cN0 papillary thyroid microcarcinoma a metaanalysis Eur Arch Otorhinolaryngol “ doi101007s0040501643020 Chung YS Kim JY Bae JS Lateral lymph node metastasis in papillary thyroid carcinoma results of therapeutic lymph node dissection Thyroid “ doi101089thy20080244 Oh EM Chung YS Song WJ The pattern and significance of the calcifications of papillary thyroid microcarcinoma presented in preoperative neck ultrasonography Ann Surg Treat Res “ doi104174astr2014863115 Haugen BR Alexander EK Bible KC American Thyroid Association Management Guidelines for adult patients with thyroid nodules and differentiated thyroid cancer the American Thyroid Association Guidelines task force on thyroid nodules and differenthyroid cancer Thyroid “ doi101089 tiated thy20150020Cancer Management and Research Publish your work in this journal Cancer Management and Research is an international peerreviewed access journal focusing on cancer research and the optimal use of preventative and integrated treatment interventions to achieve improved outcomes enhanced survival and quality of life for the cancer patient The manuscript management system is completely online and includes a very quick and fair peerreview system which is all easy to use Visit httpwwwdovepresscomtestimonialsphp to read real quotes from published authors Dovepress Submit your manuscript here wwwdovepresscomcancermanagementandresearchjournalCancer Management and Research submit your manuscript wwwdovepresscom DovePress 0c'

Thyroid_Cancer

"Severe COVID19 has a high mortality rate Comprehensive pathological descriptions of COVID19 are scarce and limited in scope We aimed to describe the histopathological findings and viral tropism in patients who died of severe COVID19Methods In this case series patients were considered eligible if they were older than years with premortem diagnosis of severe acute respiratory syndrome coronavirus infection and COVID19 listed clinically as the direct cause of death Between March and April full postmortem examinations were done on nine patients with confirmed COVID19 including sampling of all major ans A limited autopsy was done on one additional patient Histochemical and immunohistochemical analyses were done and histopathological findings were reported by subspecialist pathologists Viral quantitative RTPCR analysis was done on tissue samples from a subset of patientsFindings The median age at death of our cohort of ten patients was years IQR “ Thrombotic features were observed in at least one major an in all full autopsies predominantly in the lung eight [] of nine patients heart five [] and kidney four [] Diffuse alveolar damage was the most consistent lung finding all ten patients however anisation was noted in patients with a longer clinical course We documented lymphocyte depletion particularly CD8positive T cells in haematological ans and haemophagocytosis Evidence of acute tubular injury was noted in all nine patients examined Major unexpected findings were acute pancreatitis two [] of nine patients adrenal microinfarction three [] pericarditis two [] disseminated mucormycosis one [] of ten patients aortic dissection one [] of nine patients and marantic endocarditis one [] Viral genomes were detected outside of the respiratory tract in four of five patients The presence of subgenomic viral RNA transcripts provided evidence of active viral replication outside the respiratory tract in three of five patientsInterpretation Our series supports clinical data showing that the four dominant interrelated pathological processes in severe COVID19 are diffuse alveolar damage thrombosis haemophagocytosis and immune cell depletion Additionally we report here several novel autopsy findings including pancreatitis pericarditis adrenal microinfarction secondary disseminated mucormycosis and brain microglial activation which require additional investigation to understand their role in COVID19Funding Imperial Biomedical Research Centre Wellcome Trust Biotechnology and Biological Sciences Research CouncilCopyright The Authors Published by Elsevier Ltd This is an Access under the CC BY licenseIntroductionIn the UK the death toll from severe COVID19 is among the highest worldwide1 Severe COVID19 is characterised by respiratory failure with socalled cytokine storm occurring in some patient subsets2 Pathological correlates are required to understand the pathophysiology of COVID19 Autopsybased histopathological analysis is crucial in this respect In anticipation of the COVID19 pandemic our group produced national guidelines for autopsy performance in suspected COVID19 cases3COVID19 is caused by infection with severe acute respiratory syndrome coronavirus SARSCoV245 Although SARSCoV2 and its predecessor SARSCoV causing severe acute respiratory syndrome [SARS] are toll similar on a molecular and clinical level COVID19 has a lower death rate for COVID19 vs for SARS and a substantially higher death deaths worldwide from COVID19 as of Aug vs from SARS than SARS due to a higher basic reproduction number1 The postmortem findings in patients with SARSCoV infection included diffuse alveolar damage DAD splenic and nodal lymphocyte depletion haemophagocytosis renal acute tubular injury cerebral oedema microthrombosis and adrenalitis with necrosis with intracellular SARSCoV detected in the lungs kidney brain and haematological ans6 Various autopsy series on COVID19 have begun to emerge in the literature7“ Here we document the major pathological Lancet Microbe Published Online August 101016 S2666524720301154Department of Cellular Pathology Northwest London Pathology B Hanley MBBCh Prof K N Naresh MD C Roufosse PhD J Weir FRCPath Prof R Goldin MD P Viola MD M Osborn FRCPath and Department of Hepatology P Manousou PhD Imperial College London NHS Trust London UK Centre for Haematology B Hanley Prof K N Naresh and Centre for Inflammatory Diseases C Roufosse Department of Immunology and Inflammation Department of Infectious Disease Prof G S Cooke FRCP A Abdolrasouli PhD O C Swann MRes L Baillon BSc R Penn MSc Prof W S Barclay PhD and Department of Metabolism Prof M Thursz MD Faculty of Medicine Imperial College London London UK Department of Histopathology Royal Brompton and Harefield NHS Foundation Trust and National Heart and Lung Institute Imperial College London London UK Prof A G Nicholson DM Renal and Transplant Centre Hammersmith Hospital Imperial College Healthcare NHS London UK R Corbett PhD Department of Neuropathology Kings College Hospital London UK Prof S AlSarraj FRCPath Death Investigation Committee Royal College of Pathologists London UK M Osborn and Nightingale NHS Hospital London UK M Osborn Correspondence to Dr Brian Hanley Department of Cellular Pathology Northwest London Pathology Charing Cross Hospital campus London W6 8NA UK bhanleyimperialacukwwwthelancetcommicrobe Published online August 101016S2666524720301154 s 0cResearch in contextEvidence before this studyCOVID19 is a new disease and comprehensive descriptions of the histopathological findings at autopsy are scarce We reviewed the literature available on COVID19 autopsy findings up to and including May For this we searched PubMed and Google Scholar databases with no language restrictions using the search terms œCOVID19 œSARSCoV2 œhistology œautopsy and œpostmortemAdded value of this studyOur series focused on providing a comprehensive description of the histopathological findings in patients with severe fatal COVID19 and correlating these findings with data on viral tropism The most prominent findings included diffuse alveolar damage thrombosis haemophagocytosis and immune cell depletion Several novel autopsy findings in patients with COVID19 were also described including pancreatitis pericarditis adrenal microinfarction secondary disseminated mucormycosis and brain microglial activationImplications of all the available evidenceOur study supports the existing clinical and autopsy literature that identified diffuse alveolar damage thrombosis immune cell depletion and macrophage activation as the most prominent pathological features in COVID19 Other factors including acute kidney injury pancreatitis pericarditis secondary fungal infections and preexisting liver disease require further investigation The presence of ongoing viral replication in late stage COVID19 supports the continued use of antiviral therapy even at a point in illness when immunopathology is dominantSee Online for appendixfindings of ten postmortem examinations done on patients with clinically confirmed COVID19MethodsPatient selectionFor this study eligible patients were older than years with premortem SARSCoV2 infection and COVID19 listed clinically as the direct cause of death under part on the Medical Certificate of Cause of Death [MCCD] Consent was obtained for all included patients according to the Human Tissue Authority codes of practice by a member of the Trust Core PostMortem Consent Team Consent rate was · ten of patients Exclusion criteria included extended postmortem interval before autopsy days and patients with COVID19 contributing but not directly leading to death under part of the MCCD Patients were from Imperial College National Health Service NHS Trust nine patients London UK and Royal Brompton Harefield Foundation NHS Trust one patient London UK Premortem SARSCoV2 infection was identified using the Coronavirus Typing multiplextandem PCR HighPlex System Aus Diagnostics Chesham UK Ethical approval for this project was provided by the Imperial College Healthcare Tissue Bank R20012Autopsy proceduresFull autopsies were done on nine patients PM1“ and one patient underwent percutaneous biopsy sampling heart lungs pancreas kidneys and liver using percutaneous biopsy under ultrasound guidance PM10 Full postmortem examinations included standard sampling and were done according to Royal College of Pathologists guidelines3 Eight different regions of the brain were sampled for each full neuropathological examination All tissue samples were fixed in formalin for a minimum of h before embedding Histochemical stains and immunohistochemistry were applied according to local protocols appendix p ans were reviewed by subspecialist pathologists in lung AGN and PV haem ato pathology and immune pathology KNN liver RG gastrointestinal MO neuropathology SAS and renal pathology CR Integrated interpretation was done by a subspecialty autopsy pathologist BH and MO All cases were reviewed independently by at least two pathologistsPCR proceduresFresh tissue for quantitative RTPCR qRTPCR analysis was processed within the biosafety level facilities at St Mary™s Hospital London UK approved by the UK Health and Safety Executive and in accordance with local rules at Imperial College London Total RNA was obtained from fresh tissue samples by use of TRIzolchloroform extraction followed by precipitation and purification using the RNeasy kit Qiagen Hilden Germany qRTPCR against E gene RdRp and subgenomic RNA was done as described elsewhere1617 In patient PM5 total fungal genomic DNA was extracted from four to five ribbon slices of a formalinfixed paraffinembedded lung tissue block Purified DNA was amplified with PCR for panfungal and Mucoralesspecific targetsStatistical analysisAll data was analysed using SPSS software version and expressed using median IQR and percentageRole of the funding sourceThe funder of the study had no role in study design data collection data analysis data interpretation or writing of the The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publicationResultsBetween March and April ten patients were included in the study The median age at death was years wwwthelancetcommicrobe Published online August 101016S2666524720301154s 0cIQR “ Seven of ten patients were men three were women and most patients were White or Asian nine [] Hypertension four [] patients and chronic obstructive pulmonary disease three [] were the most common contributing factors to death according to MCCD All ten patients developed fever and had at least two respiratory symptoms or signs cough shortness of breath reduced oxygen saturations or pleuritic chest pain during their early presentation Of eight patients assessed for inflammatory markers all had elevated inflammatory markers These features were either apparent upon presentation to hospital eight [] of ten patients or developed in an inpatient two [] patients PM8 and PM9 Most patients died within weeks of symptom onset seven [] patients and were not intubated or ventilated six [] patients Four patients were intubated during their presentation PM2 for days PM5 for days PM6 for less than day and PM7 for days The median bodymass index BMI was in the obese range · IQR ·“· and more patients were obese according to BMI at post mortem five [] of nine than indicated on the MCCD one [] of ten The median interval between death and postmortem examination was days IQR ·“· although the limited post mortem had a shorter interval less than h after death Detailed clinical case vignettes are available in the appendix p and clinical data are summarised also in the appendix p All patients had DAD six showed purely exudative phase DAD and four showed a mixture of exudative and anising DAD appendix p figure Three of four patients with anisingphase DAD had spent a substantial period on a ventilator days days and days Florid acute bronchopneumonia and ventilatorassociated pneumonia were not noted in this series although mild interstitial neutrophilic inflammation three [] of ten patients and patchy acute bronchopneumonia three [] patients were observed Interstitial macrophages were prominent Macrophages were accompanied by scattered plasma cells Mild or moderate lymphocyte inflammation was present in all ten patients although focal lymphocyte cuffing of small vessels was noted in six patients We noted that lymphocytes in the lung were predominantly CD4positive T cells CD56positive natural killer cells were rarely found Occasionally a patient had small aggregates of small B cells Chronic bronchiolitis was seen in most patients nine [] of ten No granulomas or viral inclusion were seen Invasive mucormycosis was noted in one patient PM5 figure and confirmed with Mucoralesspecific PCR The mucormycosis was vasculocentric and disseminated involving the hilar lymph nodes heart brain and kidney in the same patientMacroscopic two [] of nine patients and microscopic eight [] of nine pulmonary thromboemboli were frequent observations appendix p figure Both fibrinrich and plateletrich thrombi were identified in smallsized and mediumsized vessels and within the capillaries in alveolar septa figure External examination findings of deep venous thrombosis were not noted Very focal lymphocytic vasculitis was identified in one patientThrombotic features were universal in this cohort and all nine patients who underwent a full autopsy had at least one microthrombosis or macrothrombosis in a major an One of nine patients had a macroscopic acute coronary thrombosis in the right coronary artery whereas five patients had thrombi in the microcirculation of the heart on histological analysis Coronary artery disease was negligible or mild in most patients seven [] of nine Acute myocardial ischaemic damage h old was noted in the patient with an acute coronary artery thrombus figure 2A PM1 A mottled myocardium and subendocardial contraction band necrosis was noted in a ACEBDF 03m µm µmFigure Pulmonary pathological findings in patients with COVID19A Macroscopic subpleural petechial haemorrhage in a 24yearold man PM6 B Hyaline membranes indicative of exudative phase diffuse alveolar damage in a 79yearold woman PM9 at 20x magnification C CD61 immunohistochemical staining indicating plateletrich microthrombosis in alveolar capillaries PM6 D Squamous metaplasia in a 61yearold man PM1 with exudative phase diffuse alveolar damage at × magnification E Interstitial multinucleated giant cells in a 79yearold man PM7 with anising phase diffuse alveolar damage at × magnification the top right insert is of multinucleated giant cells showing positive CD68 staining indicative of macrophage lineage the bottom left insert shows absence of staining for cytokeratins F Periodic acid Schiff staining indicating wide irregular aseptate and ribbonlike hyphae with angle branching and a vasculocentric pattern indicative of mucormycosis in a 22yearold man PM5 the insert is a Grocott silver stain highlighting mucormycosis at 20x magnificationwwwthelancetcommicrobe Published online August 101016S2666524720301154 s 0csecond patient PM2 whether the contraction band necrosis was related to ischaemia or inotropic medication received in the intensive care unit is uncertain appendix p PM1 and PM2 were the two patients with the highest active viral load detected in the heart A single patient had a right atrial thrombus Pericarditis was ACEGBDFH µm µm 03m µmFigure Thrombotic features identified at autopsy in patients with COVID19A Macroscopic right coronary artery thrombosis arrow in a 61yearold man PM1 with exudative phase diffuse alveolar damage B Macroscopic pulmonary thromboembolism arrow in a 97yearold man PM8 C Thrombus in the lung of a 79yearold woman PM9 on haematoxylin and eosin staining at 20X magnification the insert shows CD61 immunohistochemistry indicating moderate staining for platelets D Plateletrich thrombus in the mediumsized vessels surrounding the heart in a 61yearold man PM1 the insert shows strong CD61 staining for platelets Periodic acid Schiff staining showing a glomerular microaneurysm arrow E and microthrombi within glomerular capillary loops arrow F at 40X magnification indicative of thrombotic microangiopathy in a 97yearold man PM8 Macroscopic splenic G and hepatic H infarction in a 22year old man PM5identified in two patients one patient showed florid fibrinous pericarditis containing fungal hyphae PM5 while the other showed only microscopic acute pericarditis appendix p figure The median heart weight was high g and four of nine patients had left ventricular hypertrophy Nonbacterial thrombotic marantic endocarditis was noted in one patient PM5 with no known history or autopsy findings consistent with malignancy or chronic disorder associated with nonbacterial thrombotic marantic endocarditis appendix p figure PM5 had disseminated mucormycosis and numerous other thrombotic features appendix p Cardiac amyloidosis and right atrial thrombosis were identified in one of ten patients PM8 appendix p Lymphocyte depletion involving specific compartments and increased phagocytosis were prominent findings appendix p figure Increased phagocytosis of other cells was identified in the sinusoidal macrophages of the red pulp of the spleen in four [] of seven patients sinus histiocytes of hilar lymph nodes in three [] of six and bone marrow four [] of eight Phagocytosis was identified in at least one of these ans in six of nine patients Bone marrow haemophagocytosis was prominent in two patients PM4 and PM8 and focal in two patients PM7 and PM9 Depletion of periarteriolar Tcell sheaths within the white pulp was observed figure Red pulp was generally congested showing reduced numbers of CD8positive T cells Plasma cells were variably prominent and sinusoidal histiocytes showed phagocytosis of red blood cells and other cells to varying extents Both IgMpositive and IgGpositive plasma cells were identified and they were polytypic for lightchain expression figure Lymph nodes showed preservation of follicles and relative depletion of paracortical areas Medullary areas showed prominence of plasma cells and histiocytes were prominent in the sinuses Bone marrow samples showed reactive changes with trilineage hyperplasia and prominence of plasma cells and histiocytes were a common finding A necrotising granuloma was noted in a single hilar lymph node in one patient and acidfast bacilli were noted on Ziehl Neelson staining appendix p All spleen and lymphoid material examined with immuno histochemistry were negative for EpsteinBarr virus and cytomegalovirusPancreatitis was noted in two of eight patients PM5 was a 22yearold man with frank necrotichaemorrhagic pancreatitis and secondary mucor mycosis figure No fungal hyphae were noted in the pancreas PM8 was a 97yearold man who showed no substantial macroscopic pancreatitis although micro scopic acute inflammation within the pancreas and periadrenal fat necrosis was noted figure A third of patients three [] of nine showed patchy areas of infarcttype adrenocortical necrosis with one patient showing anising microthrombi in adrenal vessels figure No wwwthelancetcommicrobe Published online August 101016S2666524720301154s 0cadrenalitis was noted Two of nine patients showed chronic inflammation in the thyroid with follicular epithelial cell disruption however the significance of this finding is uncertainMedian combined kidney weight was within normal range at g IQR “ Salient renal pathology findings were acute tubular injury in all nine patients underlying moderate cortical scarring of uncertain cause in one patient glomerular microaneurysm and thrombi in one patient figure and rare thrombi in interlobular arteries in four patients PM6 24yearold man of arterial intimal thickening than expected for that age appendix p We observed no evidence of focal and segmental glomerulosclerosis diabetic glomerulopathy or glomerulonephritisdegree had a higher Large droplet fatty change was seen in most patients seven [] of eight Cirrhosis or bridging hepatic fibrosis were noted in three patients No liver thrombosis was identified histologically but one patient showed macroscopic liver infarction figure The median liver weight was g IQR “ and three of nine patients showed hepatomegaly liver weighing g Two patients PM4 and PM7 showed marked autolysis and were not included in analysisModerate to intense microglial activation was the most prominent pathological feature in the CNS five [] of five patients Mild Tcell infiltration was noted around blood vessels and capillaries in all five patients but B cells were absent We found ischaemic changes of variable extent in the neurons of the cortex and in the white matter detected by BAPP β amyloid precursor protein stain However no necrosis of brain tissue or extensive infiltration of inflammatory cells in brain parenchyma or meninges was observed on histological examination although one of nine patients showed macroscopic haemorrhagic transformation in a large recent cerebral infarction in the distribution of the middle cerebral arteryTissues from five patients were analysed for presence of viral genomes against E gene and indications of viral replication against subgenomic RNA transcripts by qRTPCR Viral RNA was present in respiratory tract samples including lung of all five cases analysed In addition two of three patients had detectable viral RNA in the nasal epithelium and four of five patients in the trachea Evidence of viral genomes outside the respiratory tract was found for all five patients but the distribution and viral loads varied case by case figure 5A Viral genomes were also detected using a different qRTPCR targeted at RdRp gene and patterns were consistent between the two sets of primers data not shown A third primer set that detected subgenomic RNA indicated virus replication in all tissues examined with variation between patients in levels and distribution figure 5BACE µmBDF µm µmFigure Other notable autopsy findings in patients with COVID19A Contained aortic dissection green arrow and fibrinous pericarditis red arrow in a 22yearold man PM5 insert is a haematoxylin and eosin stain image of the pericardium showing fibrinous pericarditis 10X magnification B Adrenocortical microinfarcts in a 79yearold woman PM9 with reendothelialising thrombus in small adrenal vessels highlighted by CD34 insert bottom left and haematoxylin and eosin insert top right Marantic endocarditis C highlight with haematoxylin and eosin staining bottom left at 10x magnification and necrotising haemorrhagic pancreatitis D in a 22yearold man PM5 with COVID19 and a secondary fungal lung infection E Periodic acid Schiff staining showing a granular cast arrow indicative of acute tubular injury in a 24yearold man PM6 20X magnification F Microscopic acute pancreatitis on haematoxylin and eosin staining in a 97yearold man PM8 20X magnificationDiscussionIn this series we have described the major pathological findings identified at autopsy in ten patients who died of severe COVID19 The most consistent findings were DAD thrombosis haemophagocytosis and immune cell depletion although unexpected pathologies that are probably related to SARSCoV2 infection were also identifiedDAD was the most consistent and prominent feature in our series and others78 The specific phase of DAD probably represents the degree and chronicity of the offending insult SARSCoV2 infection in relation to the time of death This is similar to previous coronavirus epidemics6 The conclusion by Copin and colleagues7 that COVID19related lung injury œis not diffuse alveolar damage might relate to their sampling strategy and wwwthelancetcommicrobe Published online August 101016S2666524720301154 s 0cADBC µm µm µm µm µmEF µm µm µmFigure Pathological findings in haematological ans in patients with COVID19Tcell depletion in the spleen of a 79yearold woman PM9 with COVID19 haematoxylin and eosin staining of the spleen at 10X magnification A CD20 staining of spleen indicating presence of B cells B 10X magnification with the insert showing the same region at higher power 20X magnification and CD3 staining of spleen indicating depletion of T cells C 10X magnification with the insert showing the same region at higher power 20X magnification Bone marrow phagocytosis in a 97yearold man PM8 with COVID19 haematoxylin and eosin staining of a well preserved bone marrow with an arrow indicating presence of phagocytosis D 40X magnification and CD68PGM1 staining of bone marrow indicating presence of phagocytosis 20X [E] and 40x [F] magnificationchronicity five patients had spent approximately weeks on a ventilator Barton and colleagues8 described prominent acute bronchopneumonia as the major finding in one of two patients although the authors acknowledge that this was probably affected by aspiration in their patient with muscular dystrophy Reports of lung histology in early COVID19 also suggest a degree of lymphocytic pneumonia although DAD is probably superimposed on this over time in the majority of fatal cases7 Pulmonary macrophage infiltration and multinucleated giant cell reactions are prominent similar to other series8“ Definite evidence of in COVID19 will require quantitative analysis comparing tissues from COVID19 patients with DAD associated with other conditions and unaffected tissues Several cases of invasive pulmonary aspergillosis have been reported in patients with severe COVID19 pneumonia18 To our knowledge this is the first description of histologically proven mucormycosis in patients with COVID19 and suggests that other human fungal pathogens including members of Mucoromycotina can complicate COVID19associated infectionslymphocyte depletion tissuerelated Numerous clinical features including raised serum Ddimer concentrations raised procalcitonin concentrations and imaging findings suggest thrombosis is prominent in patients with COVID192 Thrombotic features were universal among patients who underwent full autopsies all nine patients had thrombi in at least one major an and have been noted to be prominent in other COVID19 autopsy series15 In a retrospective study of autopsies in patients with acute respiratory distress syndrome and DAD of various causes only showed thrombi within the small vessels of the lung despite sampling of every lobe of the lung19 Another study used postmortem angiography and identified thrombi in nearly all cases of acute respiratory distress syndrome from various causes20 Whether thrombosis in COVID19 is more common than in other causes of DAD remains uncertain however our data support thrombosis as being a striking feature in these patients A study suggested endotheliitis as a prominent feature in patients with severe COVID1910 but this was not a prominent feature in our patients Importantly limited post mortem or postmortem crosssectional imaging are likely to underrepresent the true extent of thrombosis particularly microthrombosis and its impact on patient death The extent of cardiomegaly fibrointimal thickening of renal blood vessels and obesity in our series supports a contribution of hypertension beyond that noted clinically only four patients had hypertension documented on the MCCDA raised cytokine profile has been documented in a subset of patients with severe COVID192 Consistent with this haematological ans in our series showed prominent phagocytosis in several patients which has not been documented in previous series21 Of the four patients with bone marrow haemophagocytosis one patient PM7 showed mild transaminitis hyperbilirubinaemia elevated serum ferritin concentrations and fever of ·°C however most clinical data were insufficient to assess the presence of haemophagocytic lympho histiocytosis wwwthelancetcommicrobe Published online August 101016S2666524720301154s 0cA substantial feature in COVID19 is lymphocyte depletion and this is supported in our series by the spleen and lymph node findings When compared with those with mild disease patients with severe COVID19 tend to have a higher neutrophil to lymphocyte ratio and higher CD4positive to CD8positive Tcell ratio22 Additionally a negative correlation exists between peripheral blood lymphocyte count and viral copy number22 We have corroborated this evidence by documenting a low number of T cells especially CD8positive T cells and FOXP3positive regulatory T cells in the spleen and lymph nodes in severe fatal COVID19 Notably normal plasma cell both IgM and IgG positive response was present in haematological ans in most patientsThe extent to which anspecific pathologies relate to direct viral replication or consequent immunological and cardiovascular complications is of clinical relevance We report here evidence of viral genomic RNA outside the respiratory tract This finding is in agreement with several previous studies that have identified viral genomes by qRTPCR in postmortem tissues including the colon14 spleen14 liver1423 skin24 heart23 and brain25 We also report detection of subgenomic RNA a product that is only produced in actively infected cells A report identified low viral load in the brain of three of patients with COVID19 but could not detect the virus in subsequent immunohistochemistry and concluded that the viral genomes might have been present in the blood25 Although we cannot exclude that the RNAs detected in our series were similarly carried to the site of sampling in blood the distribution of RNA in different tissues varied widely between postmortem casesPM3 and PM4 appear to have died earlier in the disease course days after symptom onset and had higher viral loads in the respiratory tract than other patients whereas PM3 and PM4 died after long stays in intensive care units and had either lower overall viral RNA PM5 or higher viral RNA outside the respiratory tract PM2 PM1 and PM2

Thyroid_Cancer

CASE REPORTpublished August 103389fbioe202000929SecondGeneration RTQuIC Assayfor the Diagnosis ofCreutzfeldtJakob Disease Patientsin BrazilBreno Jos Alencar Pires Barbosa1 Bruno Batitucci Castrillo1 Ricardo Pires Alvim1Marcelo Houat de Brito1 Helio R Gomes1 S´nia M D Brucki1 Jerusa Smid1Ricardo Nitrini1 Michele C Landemberger2 Vilma R Martins2 Jerson L Silva3 andTuane C R G Vieira3 Department of Neurology Hospital das Cl­nicas University of S£o Paulo Medical School S£o Paulo Brazil Tumor Biologyand Biomarkers Group International Research Center AC Camargo Cancer Center S£o Paulo Brazil National Centerof Nuclear Magnetic Resonance Jiri Jonas Institute of Medical Biochemistry Leopoldo de Meis National Institute of Scienceand Technology for Structural Biology and Bioimaging Federal University of Rio de JaneiroUFRJ Rio de Janeiro BrazilThe recent development of IQCSF the second generation of realtime quakinginducedconversion RTQuIC using cerebrospinal fluid CSF for the diagnosis of CreutzfeldtJakob Disease CJD represents a major diagnostic advance in the field Highly accurateresults have been reported with encouraging reproducibility among different centersHowever availability is still insufficient and only a few research centers have access tothe method in developing countries In Brazil we have had suspected cases of CJDsince when surveillance started Of these were undiagnosed This lack ofdiagnosis is due among other factors to the lack of a reference center for the diagnosisof these diseases in Brazil resulting in some of these samples being sent abroad foranalysis The aim of this research study is to report the pilot use of IQCSF in a smallcohort of Brazilian patients with possible or probable CJD implementing a referencecenter in the country We stored CSF samples from patients with possible probable enetic CJD one case during the time frame of December through June AllCSF samples were processed according to standardized protocols without access tothe clinical data Eight patients presented to our team with rapidly progressive dementiaand typical neurological signs of CJD We used CSF samples from seven patientswith other neurological conditions as negative controls Five out of seven suspectedcases had positive tests two cases showed inconclusive results Among controls therewas one falsepositive a CSF sample from a 5yearold child with leukemia undertreatment The occurrence of a false positive in one of the negative control samplesraises the possibility of the presence of interfering components in the CSF sample frompatients with nonneurodegenerative pathologies Our pilot results illustrate the feasibilityof having CJD CSF samples tested in Brazilian centers and highlight the importance ofinterinstitutional collaboration to pursue a higher diagnostic accuracy in CJD in Braziland Latin AmericaKeywords CreutzfeldtJakob disease prionconversion biomarkersrapidly progressive dementiarealtime quakinginducedEdited byMaria Dos Anjos PiresUniversity of Tr¡sosMontes and AltoDouro PortugalReviewed byAssia AngelovaGerman Cancer Research CenterDKFZ GermanySumit GhoshThe Research Institute at NationwideChildren™s Hospital United StatesCorrespondenceBreno Jos Alencar Pires BarbosabrenojbgmailcomTuane C R G VieiratuanebioqmedufrjbrSpecialty sectionThis was submitted toBiosafety and Biosecuritya section of the journalFrontiers in Bioengineering andBiotechnologyReceived May Accepted July Published August CitationBarbosa BJAP Castrillo BBAlvim RP de Brito MH Gomes HRBrucki SMD Smid J Nitrini RLandemberger MC Martins VRSilva JL and Vieira TCRG SecondGeneration RTQuIC Assayfor the Diagnosis of CreutzfeldtJakobDisease Patients in BrazilFront Bioeng Biotechnol 103389fbioe202000929Frontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cBarbosa et alINTRODUCTIONtogroupTSEsconditionsencompasstransmissibleasecondaryspongiformPrion diseases also known asofrareencephalopathiesneurodegenerativeabnormalconversion of a constitutively expressed cellular glycoproteinthe prion protein PrPC into an abnormally folded isoformPrPsc Geschwind Zanusso SporadicCreutzfeldtJakob disease sCJD is the most common priondisease in humans and usually presents as rapidly progressivedementia in combination with variable degrees of multisystemneurologicalimpairment Zerr and Hermann Sinceits clinical and molecular manifestations are heterogeneousand nonspecific early diagnosis of prion diseases remainschallenging in clinical practice Geschwind and Murray Brain histopathological evaluation andor detection of PrPScare still the standard criteria for establishing a definitive diagnosisfor CJD CDC However invasiveness when performingthis type of analysis antemortem brings very little benefit to thepatient since these diseases are still incurable Clinical signs andparaclinical tests are the most commonly used approaches duringthe course of the disease and can be used to classify it as a possibleor probable prion disease Brown CDC Among the paraclinical tests brain diï¬usion weightedMRIDWMRI and cerebrospinal fluid CSF analysis have increaseddiagnostic accuracy Eisenmenger Staï¬aroni Bizzi The presence of protein Tauprotein neuronspecific enolase NSE the astroglial proteinS100B and PrPSc itself are used as biomarkers in CFS for TSEdiagnosis Schmitz Connor Overallthe only protein that is a specific biomarker for TSE is PrPScPrPSc can be detected in CSF based on its selfpropagating abilityconverting and seeding the aggregation of the nonpathogenicPrPC into PrPScThe realtime quakinginduced conversion RTQuIC assayits experimentalwas developed in and since thenconditions have been improved and tested on a large number ofsamples in several laboratories worldwide Wilham Green It ultrasensitively detects limited amounts of PrPScin CSF and other tissue samples Wilham The recentdevelopment of the secondgeneration IQCSF RTQuIC assayusing CSF for the diagnosis of CJD represents a major diagnosticadvance in the field Wilham Orrº In humans RTQuIC analysis showed a sensitivity of and specificity of with encouraging reproducibility amongdiï¬erent centers Franceschini RTQuIC started to be clinically used in and becamea criterion of the Centers for Disease Control and PreventionCDC to diagnose CJD as probable CDC Its highsensitivity and specificity make it an important clinical laboratorytestits global availability is stillinsufficient only a few research centers have access to the methodespecially in developing countriesfor widespread use butSurveillance of TSE cases has been compulsory in Brazil since Martins and suspected CJD cases werereported up to June Of these were undiagnosed deOliveira Cardoso Ministrio da Saºde RTQuIC for the Diagnosis of Brazillian PatientsinimagingperformingexaminationsandDifficultiesneuropathological analyses are found in several medicalunits in the country making diagnosis problematic Somediagnosed patients had samples sent to centers outside thecountry for biomarker analysis allowing for greater coverageof the diagnostic criteria The implementation of a specifictest in Brazil such as RTQuIC which can provide diagnosisfor diï¬erent TSEs with ease and confidence is urgent to havenotification and diï¬erential diagnosis for these diseases Thisis also important to guide medical decisions Here we reportthe pilot use ofthe secondgeneration IQCSF RTQuICassay in a small cohort of Brazilian patients with possibleor probable CJD to implement a reference center for thisanalysis in the countryMATERIALS AND METHODSClinical InvestigationPatients with suspected CJD were admitted for investigationunder a protocol for rapidly progressive dementia RPD aspreviously reported Studart Neto Following a fullneurological examination all patients underwent complete bloodcell count serum electrolytes blood glucose acute Creactiveprotein liver renal and thyroid function tests antithyroid andantinuclear antibody testing as well as treponemal and HIVserology Patients also underwent brain magnetic resonanceimaging [T1 T2 fluidattenuated inversion recovery FLAIRgradient echo and diï¬usion weighted imaging sequences] EEGand CSF cell count biochemistry and g globulin levels CSF protein was assessed in cases of suspected prion diseasesas required by Brazilian norms Selected patients underwentchest and abdomen computed tomography mammography forwomen testicular ultrasound for men and thyroid ultrasoundto rule out paraneoplastic RPD In addition we obtainedonconeural andor neuronal surface antigen antibody testingwhen paraneoplastic or autoimmune encephalitis was suspectedA flowchart for patient inclusion is provided in Figure Three cases had access to molecular analysis of the PRNP genefor polymorphisms in the codon Samples were analyzedusing denaturing highperformance liquid chromatographyTechnical details about this procedure as well as amplificationreactions and DNA extraction have been previously describedCastro Smid CSF SamplesWe analyzed eight CSF samples from seven patients withpossible probable or genetic CJD referred to the Departmentof Neurology at University of S£o Paulo from December to June One patient named ABT had her CSF testedtwice at diï¬erent times We used CSF samples from sevenpatients with other neurological conditions as negative controlsCSF samples mL were collected by lumbar puncture LPfollowing a standard procedure Two milliliters of the CSFsample were then centrifuged at — g for min andstored in polypropylene tubes at ˆ’—¦C until blind analysis byresearcher TCRGV at UFRJFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cBarbosa et alRTQuIC for the Diagnosis of Brazillian Patientsof CJD The patient named ABT had her CSF tested twice atdiï¬erent times ABT1 and ABT2 samples therefore renderinga total of eight CSF samples ABT patient results will be betterreported in Case descriptionWe randomly used CSF samples from seven patients withother neurological conditions as negative controls Five out ofeight suspected samples had positive RTQuIC results in ourhands Figure 2A Clinical diagnosis CSF analysis and RTQuICresults are summarized in Table Samples from patients LRC and DAS were used as positivecontrols given that they were previously analyzed using RTQuICin a reference center outside the country The NationalPrion Disease Pathology Surveillance Center Cleveland OHUnited States Our RTQuIC analyses were also positivecorroborating this result Figure 2A Patient ASM was a geneticCJD positive for the E200K mutation and in our analysis thispatient was also positive presenting a very short lag phase and ahigh fluorescence signal Figure 2AAmong the negative controls there was one false positivesample CT2 Figure 2A The falsepositive case was a5yearold child with leukemia who was receiving intrathecalchemotherapy GBTLI protocol with methotrexate aracitincitrabin dexamethasone during the time frame of the studyIn the following two cases are presented in which the resultsobtained with RTQuIC show interesting aspects related toRTQuIC sensitivity and disease progressionCase ABT was a 72yearold woman with years of formal educationShe had an atypical presentation characterized by a 4monthhistory of rapidly progressive cognitive impairment associatedwith visual hallucinations and gait disturbances with repeatedfalls She had no relevant past medical history or family history ofany neurological conditions On neurological examination shescored on the MiniMental State Examination MMSEand physical tests revealed only a prominent axial syndrome asshe could not sit or stand up without bilateral support There wereno pyramidal signs parkinsonism or visuospatial impairmentA comprehensive investigation with metabolic inflammatoryparaneoplastic and infectious tests was unremarkable Brainmagnetic resonance imaging MRI revealed symmetric diï¬usionweighted image DWI hyperintensities in the basal gangliaFigure a finding that raised the suspicion of CDJ TheCSF analysis was unremarkable with elevated tau protein levelsand negative The RTQuIC results were inconclusiveonce from wells only two crossed the threshold ABT1sample in Figure 2B Electroencephalogram EEG revealeddisanized electrical brain activity with no periodic wavesThree months later she developed a significant worsening withthe need for aid for most activities of daily living severe cognitiveimpairment she could not be tested with the MMSE myoclonusand the presence of prominent frontal reflexes on neurologicalexamination At this point a prolonged EEG eventually showedbilateral periodic sharp waves and another lumbar puncturewas performed with a second sample sent for RTQuIC analysiswith a positive result with a high fluorescence ABT2 samplein Figure 2B She eventually died of clinical complicationsFIGURE Flowchart of samples included for RTQuIC analysisRTQuICThe RTQuIC assay was performed using the improvedQuICCSF IQCSF conditions as published Orrº Briefly µL of CSF was added to µL of reaction mixturein each well of a black well plate with a clear bottomNunc The final solution contained mM phosphate buï¬er atpH mM ethylenediaminetetraacetic acid tetrasodium saltdihydrate EDTA at pH mM NaCl µM thioflavinTThT sodium dodecyl sulfate SDS and mgmLrecombinant Syrian hamster truncated form of prion proteinHa rPrP “ Samples were tested in quadruplicate threeor four times independently generating a total of or wellsevaluated for each sample The plates were sealed and incubatedin a FLUOstar OMEGA plate reader BMG Labtech Germanyat —¦C with intermittent cycles of shaking s doubleorbital rpm and rest s ThT fluorescence was collected every min using ± nm excitation and ± nmemission wavelengths The threshold was defined as the averagefluorescence for all samples within the first h of incubationplus standard deviations SD A sample was consideredpositive when at least two of four replicate wells crossed thisthreshold All IQCSF RTQuIC analyses were performed at theFederal University of Rio de JaneiroUnseeded reaction not shown and random CSF samplesfrom patients with other neurological conditions Table wereselected as negative controls Given the descriptive nature of thisstudy no statistical analyses were performedRESULTSIn the referred time frame seven patients presented to our teamwith rapidly progressive dementia and typical neurological signsFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cBarbosa et alRTQuIC for the Diagnosis of Brazillian PatientsTABLE Results of diagnostic investigations in the tested patient cohortSample ageDiagnosisaCSF analysisRTQuIC statusWBCµ LProt mgdLGlu mgdLTaub pgmLCT1 CT2 CT3 HCS HBV RMC ABS LRC DAS ABT1 ABT2 ASM NAJJN GMT TN Control chronic meningitisControl leukemiaControl cranial nerves syndromeControl multiple sclerosisControl HIVControl SAHventriculitisControl chronic meningitisRPD probable CJDRDP probable CJDRPD possible CJDRPD possible CJDRPD genetic CJDRPD probable CJDRPD possible CJDRPD probable CJD1cNA“““““““NANA“““““““NegativePositiveNegativeNANANANegativeNegativePositiveNegativeNegativeNegativeNegativeNegativePositivedPositivedNegativePositivePositivePositiveNegativePositiveWBC white blood cells Prot protein levels Glu glucose levels RPD rapidly progressive dementia SAH subarachnoid hemorrhage NA not available aControls andtheir suspected diagnosisinvestigation bReference value for total tau levels was pgmL cThis sample had red blood cells dThese patients also had their CSFsamples sent abroad with positive results The National Prion Disease Pathology Surveillance Center Cleveland OH United States months after the first symptoms appeared PRNP gene analysisrevealed codon heterozygous MVin hisCase JJN was a 65yearold male who presented to our clinicwith an 8month history of behavioral changes He had years of education and a medical history of hypertensionand gouty arthritis There was no family history of anyneurological conditions His wife described the first symptomsas prominent changesfood preferences with anunusual demand for rice and chicken Two months laterhe developed visual hallucinations mostly described as thepresence of spiders in the ceiling In the first evaluationhe was independent of instrumental activities of daily livingNeurological examination revealed an MMSE of withan unremarkable physical examination A laboratory workupincluding metabolicinflammatory and serology studies wasnegative At this point we were surprised by the finding ofbrain MRI diï¬usion weighted imaging revealing marked bilateralhypersignal in the frontotemporoparietooccipital cortex basalganglia thalamus and “ less markedly “ in the hippocampusraising the suspicion of sporadic CreutzfeldtJakob DiseasesCJD Figures 4AB The patient was hospitalized for furtherinvestigation The EEG study revealed bilateral and synchronousslow waves A brain 18FDG PET showed hypometabolism in thetemporal and frontal lobes caudate nuclei and temporoparietalcortex Figure 4C The CSF study was initially unremarkableexcept for the high total tau protein levels whereas ptau and betaamyloid values were within the normal rangeA CSF sample was sentfor autoantibodies and RTQuICtesting The patient was empirically treated with intravenousmethylprednisolone for days and was discharged for outpatientfollowup Atthis point we received CSF results negativefor autoantibodies with an inconclusive RTQuIC responsefrom wells only two crossed the threshold Figure 2CIn the following months he eventually developed cognitivedeterioration and parkinsonism with the presence of morecomplex visual hallucinations He eventually died of clinicalcomplications approximately months after initial symptomsPRNP gene analysis revealed codon heterozygous MVDISCUSSIONsyndromes ofencephalopathiesThe present study reports the results of pilot secondgenerationreferred forRTQuIC testing in a small patient cohortrapidly progressive neurologicalsuspectedprion nature Our center previously reported cases ofrapidly progressive dementia among patients in a 3yearintervalrepresentedthe majority of the samples followed by CJD Studart Neto All cases from that study werediagnosed with CJD according to the University of CaliforniaSan Francisco Modified Criteria Vitali which doesnot include CSF testingImmunemediatedAs we described here IQCSF RTQuIC proved to be anextremely important tool in the diagnosis of ABT and JJN casesCases and Both patients presented with rapidly progressiveneurological syndromes and could not initially be classified withpossible or probable CJD according to the most recent criteriaCDC Both ABT and JJN showed slightly elevated levelsof total Tau protein but although this biomarker has “sensitivity it is only “ specific for CJD Connor protein was not elevated in both cases despitetheir genetic subtype MV which is often associated with positive protein levels Manix Frontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cBarbosa et alRTQuIC for the Diagnosis of Brazillian PatientsFIGURE Blind RTQuIC analysis of CSF samples A RTQuIC responses from reactions seeded with µl of CSF from suspected CJD cases LRC DAS ASMJJN GMT TN The RTQuIC responses from reactions seeded with CSF samples from patients with other neurological disorders CT1 CT2 CT3 HCS HBV RMCABS were used as a negative control Each curve represents the mean of four replicate readings of three or four repetitions B Singlewell analysis of CSF samplesfrom patient ABT ABT1 and ABT2 refer to the first and second collections respectively C Singlewell analysis of CSF samples from patient JJNIn the case of ABT patient the clinical presentation ofrapid cognitive impairment with visual hallucinations and gaitimpairment was considered atypical In this case the brain MRIwas an important diagnostic clue with DWI hyperintensitiesin the basal ganglia leading to a diï¬erential diagnosis withmetabolic encephalopathies hypoxicischemic lesions or toxicFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cBarbosa et alRTQuIC for the Diagnosis of Brazillian Patientsdisease progression To our knowledge this approach performedwith samples of a patient collected at diï¬erent times has not yetbeen tested and this is the first report suggesting that RTQuICresults change according to disease evolution A more feasiblehypothesis would be that the first sample had some interferencesuch as blood that could mask the positive result Cramm despite a only modest presence of red blood cells in thisparticular sample cellsµL see Table Atypical features for sCJD in JJN Case included a presentation with mild behavioral changes the initialsparing of motor systems with evidence of motor findingsin the neurological examination only almost months afterfirst symptoms an unremarkable EEG with “ months ofevolution and the presence of a bilateral T2 hypersignalin the hippocampus The RTQuIC was inconclusive buttogether with MRIit could suggest early diagnosis ofprobable CJD Perhaps with disease progression JJN™s CSFsample would obtain a positive RTQuIC result as observedfor ABT patientThe occurrence of one falsepositive case in our RTQuICtest weakens our diagnostic accuracy and underscores the needfor improvements in the protocol Despite having an extremelyhigh specificity IQCSF RTQuIC falsepositive results have beenreported in the literature Hayashi reported thecase of a 61yearold man who presented with rapid cognitiveimpairment myoclonus and recurrent seizures A brain MRIrevealed cortical hyperintensities and the CSF analysis showedelevated and tau levels with a positive RTQuIC Despiteaggressive treatment with corticotherapy the patient died andpostmortem assessment revealed only pathological changes afterconvulsion with no signs of prion disease The authors concludethat convulsion may cause falsepositive RTQUIC results andthat a postmortem evaluation remains the gold standard fordiagnosing similar cases The shaking eï¬ect was analyzed in vitroby Orrº and they showed that long shaking periodsreduced scrapieseeded reaction times but continuous shakingpromoted falsepositive reactionsFIGURE Diffusionweighted images DWI from ABT case revealingenlarged ventricles and symmetric hyperintensities in the basal ganglia Ayellow arrows B shows corresponding apparent diffusion coefficient ADChypointensities in the same territorylesions ie carbon monoxide intoxication Finelli and DiMario A careful clinical assessment made all possibilities lesslikely and CJD became our main hypothesis Despite aninconclusive RTQuIC result for the first collected sample thepatient eventually evolved with a more typicalimpairmentand the second CSF sample with a 3month interval yieldedpositive RTQuICThe diï¬erent results obtained with the ABT patient sampleswere very intriguing We hypothesize that this diï¬erence could beattributed to higher loads of PrPsc following disease progressionAlthough disease duration does not seem to be related toRTQuIC results McGuire it is not yet clear whetherthere is a change in the presence of seeds in CSF according toFIGURE A MRI diffusionweighted images from JJN case revealing hyperintensities on frontal temporoparietal and posterior cingulate cortical areas Ayellow arrow B MRI apparent diffusion coefficient maps with corresponding hypointensities in the same regions above B yellow arrow C 18FDG PETCTshows hypometabolism on the bilateral temporoparietal cortex posterior cingulate precuneus and caudate nucleusFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cBarbosa et alRTQuIC for the Diagnosis of Brazillian PatientsRegarding our falsepositive case we did not find any similarcases reporting the use of intrathecal medications as a possiblereason for a positive RTQuIC result A positive case in a5yearold child without any neurological symptoms wouldnever be expected an 18yearold woman was the youngestperson diagnosed with probable sCJD using RTQuIC Yao Therefore this sample was selected as a negative controlalthough infant CSF was never RTQuIC analyzed Despiteoptimized protocols sample processing issues can always be apossibility for such unexpected resultsThe present study is limited by its pilot nature with a modestsample size Improvements in the establishment of the protocolare necessary requiring a greater number of analyses The useof nasal brushes to collect patient samples for RTQuIC analysisis also valid to improve protocol sensitivity and specificityHowever this is the first study to our knowledge to report specificbiomarkerbased feasible results performed in a developingcountry for prion diagnosis in addition to pointing out newpossible interferences in the protocol and the need to understandhow the diï¬erent current diagnostic approaches can revealdisease progressionCONCLUSIONThe cases reported here illustrate the importance of usingRTQuIC for patients with neurological syndromes enabling thediagnosis of probable CJD while no other method was sufficientto support this diagnosis even with atypical clinical presentationIn addition the identification of a false positive in a sample froma leukemic pediatric patient undergoing intrathecal treatmentwith chemotherapy suggests new possible interferences in themethod This will require future investigation of the eï¬ect of thesechemotherapeutic agents for inclusion or not as a limitation forcarrying out the assayCSF samples are often evaluated only once due to theinvasiveness of CSF collection and the absence of curativetreatment for TSEs in addition to rapid disease progressionOne of the cases we report points out the importance ofcarrying out studies that evaluate the progression of the diseaseand that RTQuIC is a useful approach for such studies Inthis case the use of nasal brushes might be prioritized overCSF analysis since this provides greater sensitivity and enablesmore frequent sample collection given the less invasive natureof the procedureFinally our study illustrates the feasibility of having CJDCSF samples tested in Brazilian centers and highlights theimportance of interinstitutional collaboration in order to pursuea greater diagnostic accuracy for CJD in developing countriesREFERENCESBizzi A Pascuzzo R Blevinsetal Evaluation ofM E Mdetecting prion disease with diï¬usion magneticJAMA Neurol101001jamaneurol20201319print]J Grisoli M Lodi R Moscatellia new criterion forimagingofresonance[EpubaheadIt also demonstrates that RTQuIC can be clinically availablefor testing patients from Brazil and other Latin Americancountries and points to the need and feasibility of establishinga national reference center for the diagnosis of these rare butdevastating diseasesDATA AVAILABILITY STATEMENTAll datasets generated for this study are included in thesupplementary materialETHICS STATEMENTEthical review and approval was not required for the studyon human participants in accordance with the local legislationand institutional requirements Written informed consent toparticipate in this study was provided by the participants™ legalguardiannext of kin Written informed consent was obtainedfrom the individuals and minors™ legal guardiannext of kinfor the publication of any potentially identifiable images or dataincluded in this AUTHOR CONTRIBUTIONSBB study design patient data collection and manuscript writingBB BC RA MB HG and SB patient data collection andinterpretation JS and RN study design data interpretation andmanuscript critical revision ML and VM genetic analysis JLSand TV RTQuIC facility implementation TV study designRTQuIC evaluations RTQuIC data analysis and manuscriptwriting All authors contributed to the and approved thesubmitted versionFUNDINGThis work was supported by research grants from the CarlosChagas Filho Foundation for Research Support in the State of Riode Janeiro FAPERJ and the National Council of Technologicaland Scientific Development CNPq to JLS and TVACKNOWLEDGMENTSWe would like to acknowledge all the patients and their familiesfor their 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101002ana23589Ministrio da Saºde Protocolfor Notification and Investigation ofCreutzfeldtJakob Disease with a Focus on the Identification of the New Variant[Internet] Brazil Ministrio da SaºdeOrrº C D Bongianni M Tonoli G Ferrari S Hughson A GGroveman B R A test for CreutzfeldtJakob disease usingnasal brushings N Engl J Med “ 101056nejmoa131Orrº C D Groveman B R Hughson A G Zanusso G Coulthart M Band Caughey B Rapid and sensitive RTQuIC detection of humancreutzfeldtjakob disease using cerebrospinal fluid mBio 6e0245114Orrº C D Hughson A G Groveman B R Campbell K J Anson K J MancaM Factors that improve RTQuIC detection of prion seedingactivity Viruses 103390v8050140Schmitz M Ebert E Stoeck K Karch A Collins S Calero M Validation of protein as a marker in sporadic creutzfeldtjakob diseasediagnostic Mol Neurobiol “Smid J Landemberger M C Bahia V S Martins V R Nitrini R SmidJ Codon polymorphism of prion protein gene in is nota risk factor for Alzheimer™s disease Arquivos Neuro Psiquiatr “ 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