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ManuelAlv

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Cancer

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"Although de Torres et al. demonstrated by using a well-characterized cohort of patients with COPD that the incidence of dense lung cancers decreased as the severity of the airflow obstruction at baseline increased [23] the severity of COPD in Japanese patients with newly diagnosed lung cancer was classified mainly as GOLD grade 1 and 2 rather than as GOLD grade 3. Furthermore our data showed that most patients were newly classified with COPD (84.4%; 124/147 cases) compatible with the incidence of the severity of COPD shown above or previously [1323]. It should be noted that in comparing patients undergoing thoracic surgery COPD patients had an average postoperative stay that was 61% higher and a 100% greater need of prolonged oxygen therapy than patients without COPD indicating the clinical impact of the coexistence of COPD [14]. The prevalence of COPD might increase in Japanese patients with lung cancer whereas the impact of COPD-related systemic comorbidities is also increasingly recognized in clinical aspects of COPD [7]. Thus whether or not the decision-making process involved in proposing the therapeutic management of lung cancer might be independently affected by COPD in patients with lung cancer remains elusive. To address this issue we evaluated whether or not completion of clinical staging and proposal of thoracic surgery with curative intent might be affected by the coexistence of COPD. The percentage of patients in whom clinical staging had been not completed was significantly higher in the COPD group than in the non-COPD group. More than half of these patients were referred to other hospitals for further support while the others were patients with disease recurrence. The proportion of patients with each classification in the clinical staging was compatible with that reported in previous studies about thoracic surgery [24]. Clinical guidelines recommend the assessment of spirometry to evaluate the optimum selection of surgical procedure in view of the risks of mortality and postoperative complications [6825]. Therefore we analyzed data from 185 patients with lung cancer at stage 1A to 3A because these patients are generally eligible for thoracic surgery with curative intent [1726]. Even among these surgical candidates however the number of surgeries performed was significantly lower in the COPD group (64.1%; 59/92 cases) than in the non-COPD group (81.7%; 76/93 cases) (). Furthermore our data showing that COPD-related systemic comorbidities might not be independent factors for proposing thoracic surgery with curative intent was supported by previous data as described above [14]. Thus these data indicate that the decision-making process for the therapeutic management of Japanese lung cancer patients might be affected by the prevalence and severity of COPD. Finally we evaluated whether or not the severity of COPD classified by GOLD grade might be an independent factor affecting the proposal of thoracic surgery with curative intent. Multivariate analysis indicated that severity of COPD was a critical and independent factor for proposing thoracic surgery with curative intent to Japanese patients with lung cancer who underwent bronchoscopy. This finding might be supported by our previous study showing that in comparing patients undergoing thoracic surgery COPD patients with an FEV1/FVC below 0.70 had an average postoperative stay that was 61% higher and a 100% greater need of prolonged oxygen therapy (POT) than patients without COPD [14]. Some limitations of the present study deserve mention. First the reversibility test was performed in only 62.2% of patients (168/270 cases) although COPD was defined as a postpronchodilator FEV1/FVC below 0.7 [16]. This limitation is present in other studies that have evaluated the prevalence of COPD [91027]. The other explanation might be the preoperative pulmonary assessment based on the clinical guidelines in which the need to perform a reversibility test for assessment of airflow obstruction is not mentioned [825]. Although a recent study suggests that some COPD patients show relatively high reversibility for a short-acting beta2-agonist [28] only 1.2% of 168 cases showed significant reversibility in the present study indicating that Japanese patients with both lung cancer and COPD might have different characteristics from that population [27]. Second the present study retrospectively analyzed 270 out of a total of 320 cases with lung cancer in a single institution and therefore might be subject to selection bias. However analyzing the data from 84.4% of all patients in a single institution who were sequentially registered and underwent bronchoscopy from 2010 to 2012 might minimize the possible contribution of the selection bias for patients with lung cancer. Although many studies suggest that COPD remains underdiagnosed in the patients with lung cancer [1314] Zang et al. suggest that awareness of COPD might contribute the conformity to GOLD treatment guideline for stable condition and acute exacerbation of COPD in lung cancer patients during hospitalization [13]. When spirometry was performed at bronchoscopy the median time from the date of spirometry to thoracic surgery was 50 days in the present study. Therefore comprehensive assessment of COPD at bronchoscopy might allow us to implement the optimum management for lung cancer patients [2930]. Conclusions In the present study the high prevalence of COPD among Japanese patients with newly diagnosed lung cancer was shown. Although further investigation into the validity of the assessment of COPD at bronchoscopy from studies of patients with lung cancer from other institutions is warranted we conclude that appropriate risk stratification and comprehensive management of patients with lung cancer and COPD might be made by assessment of the coexistence and severity of COPD at the time of bronchoscopy. Competing interests The authors have declared that no conflict of interest exists. Authors™ contributions NH AM and YH had full access to all of the data in the study and are responsible for the integrity of the data and the accuracy of the data analysis"

Lung_Cancer

"followed by intravenous tail vein injection of 1 — 106 MX-1 cells. Thirteen days after injection lungs were harvested and stained with Bouin's solution to visualise metastatic colonies. For survival analysis mice were monitored daily for signs of morbidity including body weight hydration level coat appearance mobility and behaviour. Mice that showed cumulative signs meeting the criteria of a moribund state were euthanised. All procedures using laboratory animals were done in accordance with all applicable institutional and government regulatory guidelines and policies were performed in an animal facility accredited by the Center for Accreditation of Laboratory Animal Care of the Japan Health Sciences Foundation. Statistical analysis Eribulin-treated vs control groups were analysed by the Dunnett multiple comparisons test using two-sided approaches. Values of P<0.05 were considered as statistically significant. Statistical analyses were performed using GraphPad Prism version 5.04 (GraphPad Software La Jolla CA USA) or R (v2.15.2 http://www.r-project./index.html) and SAS (v9.2 2M2 Cary NC USA) programs. Analyses of lung metastatic nodules and survival data were done using Kaplan“Meier and log-rank test methods and the Mann“Whitney U-test (GraphPad Prism version 6 La Jolla CA USA). Results Eribulin reverses EMT and induces MET in TNBC cells in vitro To investigate the effects of eribulin on EMT/MET balance three TNBC cell lines were treated with eribulin for 7 days (A). The doses of eribulin for MX-1 and Hs578T were IC50 and three times IC50 decided by 3 days proliferation assay result (Supplementary Figure S1A). For MDA-MB-157 half IC50 and IC50 of eribulin were selected because almost all cells died after 7 days treatment with three times IC50. Although eribulin strongly inhibited proliferation of these cells surviving cells were no longer spindle shaped like control cultures but instead had flat more epithelial-like morphologies (B). This conversion from mesenchymal to epithelial-like morphologies prompted us to examine alterations in EMT/MET-related genes. RNA from surviving TNBC cells treated with eribulin for 7 days was assayed by qPCR analysis. As shown in C (MX-1) D (MDA-MB-157) and E (Hs578T) eribulin treatment consistently upregulated mRNA expression levels of epithelial markers CDH1 and KRT18 while it downregulated mesenchymal markers CDH2 VIM TWIST1 SNAI2 ZEB1 and ZEB2 even though the pattern and degree of these alterations was somewhat different among the three TNBC cell lines (C D and E). Of the three eribulin effects on EMT/MET markers were most robust in MX-1 cells so this line was selected for further more detailed analyses. Thus using MX-1 cells western blotting analysis confirmed that protein levels of key EMT/MET markers behaved similarly to mRNA expression patterns: eribulin increased levels of E-cadherin protein while decreasing levels of N-cadherin and vimentin proteins (F and G). Taken together results of morphological observations and gene and protein expression patterns strongly point to eribulin-induced reversion of EMT and induction of MET in TNBC cells. Eribulin reverses EMT and induces MET in MX-1 TNBC tumour xenografts in mice Next eribulin's ability to induce a shift from EMT to MET phenotypes was tested in MX-1 tumour xenografts in vivo. MX-1 cells were transplanted into athymic mice to establish MX-1 xenografts followed by treatment of animals with eribulin using the schedule shown in A. Under these conditions eribulin showed a significant antitumour activity as determined by measurement of tumour weights on days 4 and 8 of the study (Supplementary ). Nevertheless even in tumours in the 3?mg?kg?1 eribulin group residual tumour tissues remained intact and showed evidence of tumour vasculature remodelling (Matsui et al 2013; Funahashi et al manuscript in preparation). Therefore an IHC analysis of EMT/MET-related proteins in the resected tumours was performed. B showed representative images of IHC staining for E-cadherin N-cadherin and ZEB1 protein in tumours from animals receiving each of the three eribulin treatment dose levels. As shown in all eribulin-treated groups the epithelial E-cadherin signal was increased whereas the mesenchymal N-cadherin and ZEB1 signals were decreased. Quantification of these IHC results for all animals in each group revealed that these alterations were significant at all doses tested when compared with the corresponding vehicle controls (C). Together with results from cell-based studies analysis of protein expression levels in in vivo MX-1 tumour xenografts provides strong evidence for eribulin-induced reversion of EMT and induction of MET in breast cancer cells. Eribulin regulates TGF-? signalling pathway via downregulation of Smad phosphorylation To examine the mechanism by which eribulin induces MET in cells the effect of eribulin on TGF-?/Smad signalling a key signalling pathway for induction of EMT was investigated. It is known that TGF-? enhances phosphorylation of receptor-regulated Smad2 and Smad3 proteins resulting in enhanced complexing with Smad4. Translocation of the resulting complex into the nucleus activates transcription of essential EMT-related genes including TWIST1 SNAI1 SNAI2 ZEB1 and others (Takano et al 2007; Gregory et al 2011). To evaluate the effect of eribulin on this pathway MCF10A normal mammary epithelial cells were utilised. These cells represent a non-cancerous triple negative basal cell type and as such are often used as an EMT model since they quickly undergo EMT in response to TGF-?. A shows the treatment scheme of the experiment. The EMT phenotype of MCF10A cells treated with TGF-? for 7 days was confirmed by cell morphology (B) as well as gene expression profiles of epithelial and mesenchymal markers (C). Next effects of eribulin on MCF10A cells already induced to the EMT phenotype by TGF-? treatment were examined. For this analysis eribulin treatment was begun after cells had been induced to the EMT phenotype by 7 days of TGF-? pretreatment and 7 days later cellular morphology and gene expression profiles were examined. The concentration of eribulin used in this experiment was ?0.5 — IC50 0.25?nM (see Supplementary Figure S1B). As shown in D on day 15 eribulin treatment reversed the observed phenotype from the previously induced spindle-like EMT morphology seen at day 8 to the original cuboidal morphology typical of TGF-?-untreated cells (compare D right with B left). Furthermore eribulin treatment also significantly upregulated mRNA expression levels of epithelial marker CDH1 while downregulating several mesenchymal markers (E). Finally phosphorylation levels of Smad2 and Smad3 were investigated. MCF10A cells were pretreated with eribulin for 1 day and the phosphorylation status of Smad2 and Smad3 was analysed 1?h after subsequent TGF-? stimulation. As shown in F eribulin pretreatment significantly decreased TGF-?-induced phosphorylation of Smad2 and Smad3 suggesting that the MET induced by eribulin was at least in part due to of downregulation of the TGF-?/Smad pathway. Eribulin decreases migration and invasion capacity of MX-1 TNBC cell in vitro One of the functional changes associated with EMT is an increase in migration and invasion capacities traits typically associated with mesenchymal phenotypes. To investigate whether decreases in cell migration and invasion capacities accompany the eribulin-induced shift from EMT to MET phenotypes seen above in vitro migration and invasion assays were conducted. For these studies MX-1 cells were treated for 7 days with 1 or 3?nM eribulin or 10??M 5-FU (active metabolite of capecitabine the comparator used in a recent phase III clinical trial of eribulin; see Kaufman et al 2012) followed by drug washout and evaluation of in vitro migration and invasion in the absence of drugs (A). The concentration of 5-FU used 10??M was approximately a 2 — IC50 growth inhibitory concentration (Supplementary Figure S1C). Treatment with eribulin significantly decreased both the migration (B and C) and invasiveness (D and E) capacities of MX-1 cells in vitro. In contrast although treatment with 5-FU also decreased both migration and invasiveness of MX-1 cells such effects were smaller than those seen with eribulin (B“E). Eribulin treatment decreases lung metastases and prolongs survival in MX-1 in vivo experimental metastasis model Finally we utilised an in vivo experimental metastasis model to investigate whether MX-1 cells pretreated with eribulin have a reduced capacity to generate metastatic lung nodules and whether the host mice would survive longer. Equivalent numbers of MX-1 cells pretreated in vitro with DMSO 1?nM eribulin or 3??M 5-FU for 7 days were injected into tail veins of mice followed 13 days later by assessment of the number of metastatic lung nodules. To exclude the possibility that pretreated cells had lost their viability at the time of injection proliferation rates of samples of pretreated cells (i.e. cells identical to those injected into mice) were measured after washout of the drugs. Results indicated that the post-washout proliferation rates of both eribulin- and 5-FU-pretreated cells were slightly slower at 2 days but had recovered to virtually identical rates as DMSO control cells by day 4 indicating that all post-washout cells injected into tail veins retained full viability and proliferative capacity (Supplementary Figure S2). Thirteen days after tail vein injection numbers of metastatic lung nodules were assessed. As shown in Figure 5A and B animals injected with MX-1 cells pretreated with eribulin showed dramatic reductions in the number of metastatic lung nodules compared with controls. Pretreatment of MX-1 cells with 5-FU also led to a reduction in numbers of metastatic lung nodules compared with controls (Figure 5A and B) albeit to a considerably lesser degree than seen with eribulin. Next we examined whether the observed reductions in lung metastases were associated with prolonged survival. As shown in Figure 5C control mice started to die around 13 days after DMSO-treated MX-1 cell injection with mice in the 5-FU group starting to die only a few days later. By day 21 all mice in both the DMSO and 5-FU groups had died. In marked contrast the first deaths in the eribulin-treated group were not seen until day 39 with the final survival rate at the conclusion of the study on day 80 being 60% (Figure 5C) linking the reduction in metastases to functional prolongation of survival. Discussion Recent advances in novel drug development strategies have improved treatment paradigms for hormone-sensitive and HER2 overexpressing breast cancers; however the most malignant and heterogeneous subtype TNBC remains largely intractable due to an aggressive metastatic character and rapid recurrence after treatment. Emerging results from many groups suggest that TNBC's resistance to chemotherapy may be explained in part by the EMT hypothesis. Epithelial“mesenchymal transition progression is characterised by a transition from epithelial to mesenchymal phenotype loss of proteins involved in cell junctions such as E-cadherin and increased expression of mesenchymal markers such as N-cadherin and vimentin. Moreover circulating tumour cells (CTCs) involved in breast cancer metastasis are reported to harbour mesenchymal characteristics (Yu et al 2013) and gene signatures of mesenchymal type cells induced by EMT are highly correlated with those of cancer stem cells (Shipitsin et al 2007; Mani et al 2008; Taube et al 2010). Thus the processes of tumour aggressiveness chemoresistance metastasis and invasion appear to be inextricably linked to EMT. The current preclinical studies represent an attempt to identify the scientific basis behind clinical observations of eribulin's enhancement of OS without corresponding increases in PFS. The studies described here have uncovered a potential new biology associated with eribulin treatment regulation of tumour EMT/MET balance which may add a new dimension to eribulin's known antimitotic antiproliferative effects on cancer cells. Results in support of this conclusion are several-fold. First in TNBC cells in vitro eribulin promoted a shift from EMT to MET states as shown by phenotypic shifts from mesenchymal to epithelial morphologies as well as changes in EMT/MET-related markers strongly favouring MET. Second eribulin treatment of mice bearing TNBC xenografts led to increased tumour expression of epithelial markers concurrent with decreased levels of mesenchymal markers. Third eribulin treatment of TNBC cells in vitro led to decreased cellular migration and invasiveness capacities an observation consistent with the known functional phenotype of MET. Finally eribulin pretreated TNBC cells had a significantly decreased capacity to colonise the lung in an in vivo experimental metastasis model findings that also correlated with significant prolongation of survival. It has been shown in several reports that the primary target of eribulin is tubulin and microtubules (Towle et al 2001; Kuznetsov et al 2004; Jordan et al 2005; Okouneva et al 2008; Smith et al 2010). However the relationship between microtubule regulation and EMT has received little attention. Smad proteins which are essential mediators of TGF-? signalling pathway normally bind microtubules in the absence of TGF-? but dissociate from them upon TGF-? stimulation (Dong et al 2000). Dissociated Smad2 and Smad3 become phosphorylated and then associate with Smad4 followed by translocation of the entire complex to the nucleus where it activates transcription. Eribulin inhibits the growth phase of microtubule dynamics (Jordan et al 2005) by binding to high affinity sites on microtubule plus ends (Smith et al 2010) possibly resulting in maintenance of the association between Smad proteins and microtubules with consequent inhibition of Smad phosphorylation. In fact the microtubule stabiliser paclitaxel even with a distinct mode of action from eribulin decreased Smad2 phosphorylation in gastric cancer (Tsukada et al 2013). On the other hand the microtubule destabiliser nocodazole enhances the release of Smad proteins from microtubules and thus increases their phosphorylation (Dong et al 2000). Although it cannot be excluded that eribulin may have other unique targets to evoke MET the precedents set by paclitaxel and nocodazole in altering Smad-related signalling suggests that eribulin binding to microtubules may at least partially explain its induction of MET in TNBC cells as observed in our studies. Involvement of EMT in drug resistance has been reported in several cancer types. For instance positive staining for the mesenchymal marker Vimentin appears in specimens from non-small cell lung cancer (NSCLC) patients who develop resistance to EGFR inhibitors suggesting that EMT has been triggered in such tumours (Uramoto et al 2010; Chung et al 2011; Sequist et al 2011). It will be interesting to determine whether eribulin reverses the EMT phenotype of lung cancer cells preclinically to reduce resistance to EGFR inhibitors. An important caveat to the studies presented here is that it is not currently known whether other tubulin-targeting agents such as the taxanes vinca alkaloids or epothilones have effects on EMT/MET balance similar to those described here for eribulin. Studies are currently ongoing to investigate this important question. In conclusion the preclinical studies presented here reveal that in addition to having a primary anticancer mechanism associated with classical antimitotic effects eribulin may also render residual tumours less aggressive and less likely to metastasise by triggering a shift from mesenchymal to epithelial phenotypes via reversal of the EMT state to the MET state. We thank Makoto Asano Naoko H Sugi Hajime Shimizu Taisuke Uehara and Hideki Watanabe for preparation of materials used in this study. We also thank Kentaro Matsuura and Kentaro Takahashi for statistical analysis Kishan Agarwala for helpful discussions and Bruce Littlefield for critical reading of the manuscript. Supplementary Information accompanies this paper on British Journal of Cancer website (http://www.nature.com/bjc) All authors are employees of Eisai Co. Ltd or Eisai Inc. Arteaga CL Sliwkowski MX Osborne CK Perez EA Puglisi F Gianni L 2012 Treatment of HER2-positive breast cancer: current status and future perspectives Nat Rev Clin Oncol 9 (1 16 32 22124364 Bonnomet A Syne L Brysse A Feyereisen E Thompson EW Noel A Foidart JM Birembaut P Polette M Gilles C 2012 A dynamic in vivo model of epithelial-to-mesenchymal transitions in circulating tumor cells and metastases of breast cancer Oncogene"

Lung_Cancer

"The DNA (1500-ng aliquots) was resolved by electrophoresis on a 1.5% agarose gel containing 0.5 µg/mL ethidium bromide and was visualized under ultraviolet light [23]. ROS Assay The generation of ROS was assessed in Huh-7 or SMMC-7721 cells with the 2?7?-dichlorofluorescein diacetate (DCFH-DA) (Invitrogen) probe which is hydrolyzed within cells to non-fluorescent 2?7?-dichlorodihydrofluorescin (DCFH). DCFH can be oxidized to the fluorescent 2?7?-dichlorofluorescein (DCF) by hydroxyl radicals peroxynitrite and nitric oxide. Briefly Huh-7 or SMMC-7721 cells were seeded in a 96-well plate. Overnight the cells were incubated with different concentration of luteoloside for 8 h then reacted with 10 µM DCFH-DA at 37°C for 20 min. Or the cells were incubated with NAC (10 mM) H2O2 (100 µM) diamide (10 mM) or BSO (100 µM) for 4 h followed by 50 µM luteoloside for 4 h [24]. DCF was determined at ?ex?=?490 and ?em?=?520 nm on a Synergy H4 microplate reader (BioTek Winooski VT). Furthermore ROS were measured with a Leica DMI4000B inverted fluorescence (Leica Wetzlar Germany). Protein Extraction and Western Blotting The proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad Hercules CA). The membrane was blocked with 5% non-fat milk and incubated with rabbit anti-LC3 polyclonal antibody (pAb) (Novus Biologicals) (2 µg/ml) rabbit anti-Beclin 1 pAb (Abcam) (3 µg/ml) rabbit anti-NLRP3 pAb (Novus Biologicals) (1?1000) rabbit anti-caspase-1 (p10) pAb (Santa Cruz Biotechnology) (1?1000) rabbit anti-IL-1? pAb (Santa Cruz Biotechnology) (1?1000) or rabbit anti-?-actin pAb (Bioworld Technology) (1?5000). The proteins were detected with enhanced chemiluminescence reagents (Pierce). Cell Proliferation Assay The cell proliferation assay was conducted as previously described by us [22]. Scratch-wound Assay Scratch-wound assay was conducted as previously described by us [22]. The migration of cells into the wound was monitored in multiple wells using a CellVoyager CV1000 confocal scanner system (Yokogawa Electronic Tokyo Japan) with an Olympus UPLSApo 10—2 10—/0.4 Dry ?/0.17/26.5 WD 3.1 plan super apochromat objective lens. The images were acquired every 0.5 hour for 48 hours (or every hour for 72 hours). The images shown represent 0 and 48 hour (or 0 and 72 hour). In Vitro Migration and Invasion Assays Assays were performed as described previously by Yao et al [25]. Xenograft Model and Treatments Two different mouse models were used to observe in vivo effect of Luteoloside on HCC cells. For the subcutaneous model the mice (male BALB/c-nu/nu 6 weeks old) were anesthetized using 1% sodium pentobarbital (0.2 ml/20 g body weight Sigma Chemical) as described by us previously [22]. The SMMC-7721 cells (2—106 cells) were suspended in 200 µl serum-free DMEM and subcutaneously injected into the right upper flank of each mouse. Two weeks after the cells were injected when tumors were observable the animals were equally divided into two groups (ten per group). The first group received only 0.2 ml of vehicle material by gavage daily and served as a control group. The second group of animals received luteoloside (2 mg/kg body weight; equivalent to a dose of 6.5 mg/m2 in patients) in vehicle respectively for 4 weeks. Body weight was measured every 4 days to adjust the drug dosage. The tumors were measured using digital calipers every 3 to 4 days after they reached a volume of 100 mm3 and tumor volumes were calculated as described: V (cm3)?=?Width2 (cm2)—Length (cm)/2. At the termination of the experiment the mice were sacrificed by cervical dislocation and the tumors were weighed immediately after dissection. For lung metastasis experiments 1—106 SMMC-7721 cells were suspended in 100 µl PBS and injected into the tail veins of each mouse (male BALB/c-nu/nu 6 weeks old) [26]. Then the animals were equally divided into two groups (ten per group). The first group received only 0.2 ml of vehicle material by gavage daily and served as a control group. The second group of animals received Luteoloside (2 mg/kg body weight) in vehicle respectively for 8 weeks. Body weight was measured every 4 days to adjust the drug dosage. At the termination of the experiment the mice were sacrificed by cervical dislocation and their lungs were removed and subjected to hematoxylin & eosin (H&E) staining. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Jiangsu Normal University (Permit Number: 13-0221). All surgery was performed under sodium pentobarbital anesthesia and all efforts were made to minimize suffering. Statistical Analysis Data are presented as means ± SEM and comparisons were made using Student™s t test. A probability of 0.05 or less was considered statistically significant. Results Luteoloside Inhibits the Proliferation of HCC Cells in vitro We first determined whether luteoloside inhibits the proliferation of human HCC cells. We found that luteoloside significantly inhibited cell proliferation in all six-cell lines in a dose- and time-dependent manner (Fig. 1B 1C). The results suggest that luteoloside has promising antihepatoma activity. Luteoloside Inhibits the Migration and Invasion of HCC Cells in vitro Luteoloside significantly decreased the migration of Huh-7 and SMMC-7721 cells compared with the control groups (Fig. 2A“2H; Supplementary Movies 1“4). Transwell assays without Matrigel demonstrated that luteoloside could significantly inhibit migration of Huh-7 cells when compared with control groups (Fig. 2I 2J 2M). Transwell assays with Matrigel showed that the invasive capacities of Huh-7 cells which were treated with luteoloside were significantly inhibited compared with the control cells (Fig. 3K 3L 3N). These results indicate that luteoloside can significantly inhibit HCC cells migration and invasion in vitro. .0089961.g002 Luteoloside inhibits migration and invasion of HCC cells. The migration of cells into the wound was monitored in multiple wells using a CellVoyager CV1000 confocal scanner system. The images were acquired every 0.5 hour for 48 hours (Huh-7 cells) or every hour for 72 hours (SMMC-7721 cells) (see Supplemental Movies 1“4). The images shown represent 0 hour (A B E F) 48 hours (C D) and 72 hours (G H). The distance between the two edges of the scratch in the luteoloside-treated cells (D or H) was greater than that of the control (C or G). (I“N) Transwell migration and invasion assays of Huh-7 cells. For the transwell migration assay 5—104 cells were placed on the top chamber of each insert with the noncoated membrane. For the invasion assay 1—105 cells were placed on the upper chamber of each insert coated with 150 µg Matrigel (BD Biosciences MA). Cells in both assays were trypsinized and resuspended in DMEM and 700“900 µL of medium supplemented with 10% fetal bovine serum was injected into the lower chambers. Representative images are shown on the left (I J K L) and the quantification of five randomly selected fields is shown on the right (M N). The values shown are expressed as the mean ± SEM. ** P<0.01 versus non-luteoloside-treated control group. Scale bar: 100 µm. .0089961.g003 Luteoloside decreases intracellular ROS. ROS levels were measured using the ROS assay with DCFH-DA fluorescence dye. (A“B) Cells were treated with luteoloside at the indicated concentration for 8 h then reacted with 10 µM DCFH-DA for 20 min. DCF fluorescence was determined on a Synergy H4 microplate reader. Cells were incubated with NAC (C) H2O2 (D) diamide (E) or BSO (F) for 4 h followed by 50 µM luteoloside for 4 h. DCF was determined on a microplate reader. (G) DCFH-DA fluorescence (green) imaging of ROS in Huh-7 cells. Scale bar: 25 µm. Luteoloside has no Apoptotic Effects on HCC Cells Huh-7 and SMMC-7721 cells were treated with luteoloside for 24 h and caspase-3/7 was measured. The results showed that caspase-3/7 activity was not significantly different between luteoloside-treated cells and control cells when added 5102050100 150 or 200 µM luteoloside respectively (Fig. S1A S1B). Similar results were obtained by analyzing changes in nuclear fragmentation (Fig. S1C) and condensation (Fig. S1D) in cells. These results indicated that luteoloside has no apoptotic effects on Huh-7 and SMMC-7721 cells. Luteoloside does not Affect Autophagy Autophagic cell death (also known as Type II programmed cell death to distinguish it from apoptosis or Type I programmed cell death) has been described as a distinct form of cell death that differs from other death mechanism such as apoptosis and necrosis. Next we investigated whether luteoloside can induce autophagy in HCC cells. Beclin 1 and LC3 (microtubule-associated protein 1A/1B-light chain 3) play a pivotal role in mammalian autophagy. Beclin 1 is involved in both the signaling pathway activating autophagy and in the initial step of autophagosome formation [27]. LC3 comprises both a soluble LC3-I and a lapidated form called LC3-II. LC3-II correlates with autophagy being recruited into autophagosomes. Various types of stressors up-regulate LC3 and promote the conjugation of its cytosolic form LC3-I to phosphatidylethanolamine to constitute the autophagosome-specific LC3-II which is so far considered the most reliable marker of autophagy [27] [28]. Huh-7 and SMMC-7721 cells were treated with luteoloside for 48 h and the levels of LC3 and Beclin 1 proteins of different treatment groups were determined. The results showed that LC3 protein level was not significantly different between luteoloside-treated cells and control cells when added 25 µM or 50 µM luteoloside respectively. Similar results were obtained by analyzing changes in levels of Beclin 1 (Fig. S2). These results indicated that luteoloside has no autophagic effects on Huh-7 and SMMC-7721 cells. Luteoloside Reduces Intracellular ROS Accumulation ROS and cellular oxidant stress have long been associated with cancer [29]. Flavonoids are well known as ROS scavengers. As luteoloside is a kind of flavonoid isolated from Chinese herb [30] we investigated whether the intracellular ROS is part of the mechanism by which luteoloside suppress the proliferation migration and invasion potential of HCC cells. We found that luteoloside could significantly decrease the ROS level of Huh-7 and SMMC-7721 cells in a dose-dependent manner (Fig. 3A 3B). N-acetyl-cysteine (NAC) is a ROS-specific inhibitor [31]. NAC was shown to be capable of suppressing the ROS production in Huh-7 and SMMC-7721 cells (Fig. 3C). When the cells were pretreated with 10 mM NAC for 4 h then treated with 50 µM luteoloside for 4 h the ROS level was significantly lower than the cells which treated with 10 mM NAC only (Huh-7 cells P?=?0.0208; SMMC-7721 cell P?=?0.0224). H2O2 diamide and BSO are all ROS inducers [4]. Treatment with 100 µM H2O2 10 mM diamide or 100 µM BSO showed similar effects resulted in an increase in ROS levels compared with control (Fig. 3D“3F). The results showed that H2O2 diamide and BSO could significantly increase the ROS level of Huh-7 and SMMC-7721 cells compared the control group (Fig. 3D“3F). However after a prolonged time when the cells were treated with 50 µM luteoloside for 4 h the amount of ROS could significantly decrease (Fig. 3D“3F). Furthermore the ROS in Huh-7 cells were monitored using a fluorescence microscope. We also found that luteoloside could significantly decrease the ROS level of Huh-7 cells (Fig. 3G). Luteoloside Downregulates the Expression Level of NLRP3 Caspase-1 (p10) and IL-1? The NLRP3 inflammasome functions as a positive regulator of tumor cells proliferation and metastasis [17] [32]. "

Lung_Cancer

"All the experiments were repeated three times. The data are presented as means ± SEM. Colony formation assay Cells were seeded in triplicate at 300 cells/well in a 6-well plate. After 7 days of culture the cells were washed twice with NaCl (0.9%) stained with 2% gentian violet for 20 min washed with water and air-dried. Foci were counted by microscopy. The experiments were repeated three times and data are presented as means ± SEM. Soft-agar assay Cells (1000) were seeded into 6-well plates in 2 mL of growth medium containing 0.3% agar and used to overlay 1.4-mL layers of growth medium containing 0.6% agar. After 21 days of culture the colonies were counted. All the experiments were repeated three times. The data are presented as means ± SEM. Cell cycle analysis Cells were harvested washed with cold PBS twice and fixed in 70% ethanol at “20°C overnight. The cells were then centrifuged (1500 rpm 10 min) and washed twice using phosphate-buffered saline (PBS). Next the cells were resuspended in 0.5 mL of PBS containing 50 µg/mL RNase A for 1 h at 37°C. The cells were then loaded with 65 ?g/mL PI for 30 min in the dark at 4°C. The percentage of cells in different phases of the cell cycle was measured by flow cytometry (Beijing Determination of Traditional Chinese Medicine Research Institute). The experiments were repeated three times. The data are presented as means ± SEM. Western blotting Cells were digested with trypsin and centrifuged. The cell pellet was washed twice with PBS. Next the cells were disrupted in lysis buffer (10 mM Tris-HCl pH 7.4 1 mM EDTA 0.1% Triton X-100 0.1% SDS and 1— protease inhibitor cocktail) on ice for 15 min and centrifuged at 12000 rpm for 20 min. Insoluble material was removed and protein concentrations were determined using a bicinchoninic acid kit. For Western blot analysis cell lysates (30 ?g/well) were subjected to SDS-PAGE and transferred to nitrocellulose filter membranes. The membranes were incubated with primary antibodies (anti-PAX6 -ERK1/2 p38 -pERK -pp38 -cyclin D1 -RB or -RB S780 phosphorylation) overnight at 4°C. Secondary antibodies conjugated with horseradish peroxidase were subsequently used. Signals were detected using ECL and exposed to Kodak X-OMAT film. The results were scanned and analyzed using Alpha View Analysis Tools. Statistical analysis All values are expressed as the mean ± SEM. Through real-time RT-PCR MTS assay colony formation soft-agar assays cell cycle analysis and western-blot assay for comparison between means of 2 groups statistical differences were tested with unpaired Student t-tests. Statistical significance was tested using SPSS Statistics version 13.0. P<0.05 (*) was considered different; P<0.01 (**) was considered significantly different. Results PAX6 mRNA expression was inhibited in cells infected with the PAX6 shRNA lentiviral vector PAX6 mRNA expression was determined in this study. As shown in A PAX6 was highly expressed in most lung cancer cell lines. In contrast MRC-5 a normal human fetal lung fibroblast cell line did not express PAX6 (A). .0085738.g001 PAX6 mRNA was highly expressed in lung cancer cells and its expression was suppressed by pax6-shRNA. A Real-time PCR analysis for the PAX6 mRNA expression level in H460 A2 95C 95D H1299 H446 801 D A549 and L lung cancer lines as well as in the normal human fetal lung fibroblast cell line MRC-5. B -C Confirmation of PAX6 mRNA knockdown by real-time RT-PCR assays performed on total RNA isolated from A549 (B) and H1299 (C) cells infected with pax6-shRNA or a random shRNA. The PAX6 mRNA expression levels in A549 and H1299 cells were measured by quantitative real-time RT-PCR. The y-axis represents the normalized PAX6 mRNA expression relative to A549 (B) or H1299 (C) cells. **P < <0.01. D The protein levels of PAX6 were determined by western-blot and GAPDH expression level was used as a control. Quantification was made by determining the gray level of PAX6 protein which was normalized against GAPDH levels. Data are expressed as mean ±SEM of independent experiments (times of the experiments are listed above the histograms). "

Lung_Cancer

"These results indicate the importance of RrmJ and the methylation of U2552. In our phylogenetic analysis in this study the RrmJ homologs clustered into the following three groups in Eukaryota: FTSJ1/Trm7p FTSJ2/Mrm2p and FTSJ3/Spb1p (). In a comparison of the divergent distance between RrmJ and the ancestral roots of each group FTSJ2/Mrm2p showed the closest relation to RrmJ. In the FTSJ2/Mrm2p protein group S. cerevisiae Mrm2p has been studied extensively. In the mitochondrial rRNA of S. cerevisiae only three nucleotides are modified including the U2791 of the 21S rRNA which is 2?-O-ribose-methylated by Mrm2p. It has been proposed that Mrm2p is the mitochondrial RrmJ ortholog in S. cerevisiae according to the equivalent catalytic positions of Um2791 in the S. cerevisiae mitochondrial 21S rRNA and Um2552 in the E. coli 23S rRNA [6] [12]. However mammalian FTSJ2 remains uncharacterized. Thus we performed a sequence alignment of human FTSJ2 with RrmJ Mrm2p and FtsJ2 in M. jannaschii and three typological invertebrate species () and we compared the 3D structures of RrmJ human FTSJ2 and porcine FTSJ2 (Figure S1). A previous study showed the highly conserved catalytic tetrad K-D-K-E in site-specific 2?-O-ribose MTases [10] and this catalytic tetrad was also present in our sequence alignment. Furthermore a sequence alignment revealed the highly conserved amino acids involved in SAM binding. However interestingly the 3D structure of human FTSJ2 showed a different orientation for the first residue of the catalytic tetrad (lysine) compared with RrmJ. This difference may indicate a different A-loop structure of the rRNA substrate or a different catalytic mechanism of the FTSJ2/Mrm2p protein in mammals. Because S. cerevisiae Mrm2p is localized in the mitochondria [12] we hypothesized that hFTSJ2 is a mitochondrial protein. We used immunofluorescence and Western blot analysis to verify that hFTSJ2 was predominantly located in the mitochondria but not in the nucleus or cytoplasm (Figures 3C and 3D). In addition E. coli RrmJ is well known as a heat shock protein. The rrmJ mRNA expression increases over 20-fold after heat shock [3] and the rrmJ deletion strain fails to adapt to heat shock temperatures [7]. In S. cerevisiae the growth of the mrm2 deletion strain at 37°C is slightly reduced on glucose-containing medium and severely reduced on glycerol-containing medium [12]. Thus to evaluate the heat shock response of the RrmJ ortholog in mammals we tested the heat shock response of piglets at 30°C or 35°C. The large intestine lung and bladder showed an up-regulated expression of Ftsj2 mRNA at temperatures of 30°C and 35°C but only the lung tissue demonstrated a simultaneous heat shock response with the up-regulation of Hsp70.2 mRNA (). This finding in the lung may have been caused by the direct exposure of this tissue to the increased temperature through the inhalation of hot air. However under these heat shock treatments for the piglets only 5 (small intestine muscle lung kidney and liver) of the 11 tissues showed an up-regulation tendency of Hsp70.2 expression possibly because of the systemic effect of the response of the warm-blooded piglets to the heat shock stress. Furthermore to eliminate this systemic effect and to confirm the FTSJ2 mRNA up-regulation in the lung a human lung adenocarcinoma cell line (A549) was subjected to heat shock for 1 hour and allowed to recover at 37°C. The results of this experiment showed a 1.5-fold increase in the hFTSJ2 expression at both 42°C and 45°C and then a gradual return to its normal level after the recovery period (). Although the exact role of FTSJ2 in the heat shock response in mammals is unknown these results indicate that FTSJ2 inherited the HSP characteristics of its orthologs in E. coli and S. cerevisiae. In the previous studies of HSPs such as HSP70 and HSP90 it has been demonstrated that the heat shock responses are highly conserved during evolution. From Prokaryota to Eukaryota and Protozoa to Metazoa the HSPs represented the universal protein structures and similar physiological functions and following evolution the HSPs diverged and translocated into different anelles [1] [2] [38]“[40]. These characteristics are in alignment with the results of the RrmJ phylogenic analysis and the conservation of the heat shock response properties in mammals. In addition to certain small HSPs (i.e. HSP32 HSP25 and HSP22) most of the HSPs are expressed in all types of tissues [2] [41] and our results showed that Ftsj2 was expressed in all of the 13 normal piglet tissues (Figure S2). These results indicated that FTSJ2 was not only involved in the heat shock response but also might be necessary for mitochondrial functions under normal condition according to the mitochondrial localization of FTSJ2. In addition previous functional studies have shown that the HSPs (i.e. HSP70 and HSP90) are involved in tumorigenesis and the inhibition of apoptosis in cancer cells [40] [42]“[44]. Similarly the amplification of the genetic locus of FTSJ2 has been discovered in several NSCLC clinical samples and was considered as a novel oncogenic locus [20]. In our study the expression of FTSJ2 was also shown in different cancer cells (hepatocarcinoma lung adenocarcinoma and rhabdomyosarcoma cells). However in the human lung adenocarcinoma cell sublines CL1-0 and CL1-5 [45] we found that hFTSJ2 mRNA was decreased in the more invasive CL1-5 cells compared with the less invasive CL1-0 cells. Moreover the TE671-hFTSJ2 cells which over-expressed the hFTSJ2 protein showed a decrease in cell migration and invasion (). These results indicate that the mitochondrial hFTSJ2 protein exhibits an additional function to suppress cancer cell metastasis. Previous reports have suggested that hFTSJ2 functions in the mitochondria. Thus it is reasonable that FTSJ2 is required for extensive ATP production through respiration in the mitochondria of proliferating cancer cells [46]“[48]. In contrast according to recent studies a mitochondrial complex I and NAD+/NADH imbalance enhances the metastasis of breast cancer cell lines [49] and the dynamics of mitochondrial fusion or fission also regulates cell migration and invasion [50] [51]. These results indicate that the invasiveness of cells is affected by the condition and state of their mitochondria. In we characterized FTSJ2 as an ortholog of the E. coli 23S rRNA 2?-O-ribose MTase and showed that it functions in the mitochondria. We also provided evidence that FTSJ2 is a novel heat shock protein that is over-expressed after heat shock treatment in both piglet lung and lung adenocarcinoma cells. Surprisingly FTSJ2 may also be involved in the inhibition of cancer cell migration and invasion by influencing the mitochondrial functions. Accession Numbers The GenBank accession numbers of the protein sequences which were used in the phylogenetic tree construction and the protein sequences alignment are labeled in and . The protein coding regions of the porcine Ftsj1 and Ftsj2 mRNA were first sequenced in this study and the GenBank accession numbers of the corresponding porcine Ftsj1 and Ftsj2 mRNA are EU694401 and EU694400 respectively and the porcine FTSJ1 and FTSJ2 proteins are ACH57153 and ACH57152 respectively. Supporting Information Figure S1 Three-dimensional protein structures of E. coli RrmJ"

Lung_Cancer

"11-years of experience. Surg Endosc23: 55“6118437482 7 CerfolioRJ BryantAS McCartyTP MinnichDJ (2011) A prospective study to determine the incidence of non-imaged malignant pulmonary nodules in patients who undergo metastasectomy by thoracotomy with lung palpation. Ann Thorac Surg91: 1696“1700 discussion 1700“1691.21619965 8 DownsSH BlackN (1998) The feasibility of creating a checklist for the assessment of the methodological quality both of randomised and non-randomised studies of health care interventions. J Epidemiol Community Health52: 377“3849764259 9 KondoH OkumuraT OhdeY NakagawaK (2005) Surgical treatment for metastatic malignancies. Pulmonary metastasis: indications and outcomes. Int J Clin Oncol10: 81“8515864692 10 PorterGA CantorSB WalshGL RuschVW LeungDH et al (2004) Cost-effectiveness of pulmonary resection and systemic chemotherapy in the management of metastatic soft tissue sarcoma: a combined analysis from the University of Texas M. D. Anderson and Memorial Sloan-Kettering Cancer Centers. J Thorac Cardiovasc Surg127: 1366“137215115994 11 PetersenRP PhamD BurfeindWR HanishSI TolozaEM et al (2007) Thoracoscopic lobectomy facilitates the delivery of chemotherapy after resection for lung cancer. Ann Thorac Surg83: 1245“1249 discussion 1250.17383320 12 PaulS AltorkiNK ShengS LeePC HarpoleDH et al (2010) Thoracoscopic lobectomy is associated with lower morbidity than open lobectomy: a propensity-matched analysis from the STS database. J Thorac Cardiovasc Surg139: 366“37820106398 13 WhitsonBA GrothSS DuvalSJ SwansonSJ MaddausMA (2008) Surgery for early-stage non-small cell lung cancer: a systematic review of the video-assisted thoracoscopic surgery versus thoracotomy approaches to lobectomy. Ann Thorac Surg86: 2008“2016 discussion 2016“2008.19022040 14 EckardtJ LichtPB (2012) Thoracoscopic versus open pulmonary metastasectomy: a prospective sequentially controlled study. Chest142: 1598“160222677347 15 AmbrogiV PaciM PompeoE MineoTC (2000) Transxiphoid video-assisted pulmonary metastasectomy: relevance of helical computed tomography occult lesions. Ann Thorac Surg70: 1847“185211156082 16 MargaritoraS PorziellaV D'AndrilliA CesarioA GalettaD et al (2002) Pulmonary metastases: can accurate radiological evaluation avoid thoracotomic approach? Eur J Cardiothorac Surg21: 1111“111412048094 17 ParsonsAM DetterbeckFC ParkerLA (2004) Accuracy of helical CT in the detection of pulmonary metastases: is intraoperative palpation still necessary? Ann Thorac Surg78: 1910“1916 discussion 1916“1918.15561000 18 KaytonML HuvosAG CasherJ AbramsonSJ RosenNS et al (2006) Computed tomographic scan of the chest underestimates the number of metastatic lesions in osteosarcoma. J Pediatr Surg41: 200“206 discussion 200“206.16410133 19 Long-term results of lung metastasectomy: prognostic analyses based on 5206 cases. The International Registry of Lung Metastases. J Thorac Cardiovasc Surg113: 37“499011700 20 MutsaertsEL ZoetmulderFA RutgersEJ (2001) Port site metastasis as a complication of thoracoscopic metastatectomy. Eur J Surg Oncol27: 327“32811373113 21 JohnstonePA RohdeDC SwartzSE FetterJE WexnerSD (1996) Port site recurrences after laparoscopic and thoracoscopic procedures in malignancy. J Clin Oncol14: 1950“19568656265 22 TreasureT (2007) Pulmonary metastasectomy: a common practice based on weak evidence. Ann R Coll Surg Engl89: 744“74817999813 23 RothJA PassHI WesleyMN WhiteD PutnamJB et al (1986) Comparison of median sternotomy and thoracotomy for resection of pulmonary metastases in patients with adult soft-tissue sarcomas. Ann Thorac Surg42: 134“1383741009 24 FloresRM IhekweazuUN RizkN DycocoJ BainsMS et al (2011) Patterns of recurrence and incidence of second primary tumors after lobectomy by means of video-assisted thoracoscopic surgery (VATS) versus thoracotomy for lung cancer. J Thorac Cardiovasc Surg141: 59“6421055770 25 SchaeffB PaolucciV ThomopoulosJ (1998) Port site recurrences after laparoscopic surgery. A review. Dig Surg15: 124“1349845574 26 ChenYR YeowKM LeeJY SuIH ChuSY et al (2007) CT-guided hook wire localization of subpleural lung lesions for video-assisted thoracoscopic surgery (VATS). J Formos Med Assoc106: 911“91818063512 27 MolnarTF GebitekinC TurnaA (2010) What are the considerations in the surgical approach in pulmonary metastasectomy? J Thorac Oncol5: S140“14420502249 28 NakajimaJ TakamotoS TanakaM TakeuchiE MurakawaT et al (2001) Thoracoscopic surgery and conventional open thoracotomy in metastatic lung cancer. Surg Endosc15: 849“85311443456 29 MutsaertsEL ZoetmulderFA MeijerS BaasP HartAA et al (2002) Long term survival of thoracoscopic metastasectomy vs metastasectomy by thoracotomy in patients with a solitary pulmonary lesion. Eur J Surg Oncol28: 864“86812477479 30 NakasA KlimatsidasMN EntwisleJ Martin-UcarAE WallerDA (2009) Video-assisted versus open pulmonary metastasectomy: the surgeon's finger or the radiologist's eye? Eur J Cardiothorac Surg36: 469“47419464921 31 CarballoM MaishMS JaroszewskiDE HolmesCE (2009) Video-assisted thoracic surgery (VATS) as a safe alternative for the resection of pulmonary metastases: a retrospective cohort study. J Cardiothorac Surg4: 1319239710 32 GossotD RaduC GirardP Le CesneA BonvalotS et al (2009) Resection of pulmonary metastases from sarcoma: can some patients benefit from a less invasive approach? Ann Thorac Surg87: 238“24319101304 33 ChaoYK ChangHC WuYC LiuYH HsiehMJ et al (2012) Management of lung metastases from colorectal cancer: video-assisted thoracoscopic surgery versus thoracotomy”a case-matched study. Thorac Cardiovasc Surg60: 398“40422228090 101150042 30118 Mol Cancer Res Mol. Cancer Res. Molecular cancer research : MCR 1541-7786 1557-3125 24202705 3946989 10.1158/1541-7786.MCR-13-0300 NIHMS538386 œNEDD9 Depletion Leads to MMP14 Inactivation by TIMP2 and Prevents Invasion and Metastasis. McLaughlin Sarah L. 5 * Ice Ryan J. 5 * Rajulapati Anuradha 5 Kozyulina Polina Y. 1 Livengood Ryan H. 4 Kozyreva Varvara K. 5 Loskutov Yuriy V. 5 Culp Mark V. 3 Weed Scott A. 2 5 Ivanov Alexey V. 1 5 Pugacheva Elena N. 1 5 # 1Department of Biochemistry West Virginia University School of Medicine Mantown WV 26506 2Department of Neurobiology and Anatomy West Virginia University School of Medicine Mantown WV 26506 3Department of Statistics West Virginia University School of Medicine Mantown WV 26506 4Department of Pathology West Virginia University School of Medicine Mantown WV 26506 5Mary Babb Randolph Cancer Center West Virginia University School of Medicine Mantown WV 26506 #Corresponding author: Elena N. Pugacheva Mailing address: Department of Biochemistry and Mary Babb Randolph Cancer Center PO Box 9142 1 Medical Center Drive West Virginia University School of Medicine Mantown WV 26506. Phone: (304) 293-5295; Fax: (304) 293-4667; epugachevahsc.wvu.edu S.L. McLaughlin* and R. J. Ice* contributed equally to this work. 17 12 2013 07 11 2013 1 2014 01 1 2015 12 1 69 81 The scaffolding protein NEDD9 is an established pro-metastatic marker in several cancers. Nevertheless the molecular mechanisms of NEDD9 driven metastasis in cancers remain ill defined. Here using a comprehensive breast cancer (BCa) tissue microarray it was show that increased levels of NEDD9 protein significantly correlated with the transition from carcinoma in situ to invasive carcinoma. Similarly it was shown that NEDD9 overexpression is a hallmark of highly invasive BCa cells. Moreover NEDD9 expression is crucial for the protease-dependent mesenchymal invasion of cancer cells at the primary site but not at the metastatic site. Depletion of NEDD9 is sufficient to suppress invasion of tumor cells in vitro and in vivo leading to decreased circulating tumor cells (CTCs) and lung metastases"

Lung_Cancer

"The LSCC tissue microarrays contain 75 matched pairs of specimens (Chaoxin Biotechnology Co. Shanghai China). Strict pathological diagnoses and post-operative follow-ups were performed for all patients. Thirty-two cases are from metastatic LSCC patients and 43 cases are from non-metastatic LSCC patients. IHC staining was performed to detect the expression of ALDOA. Sections were scored as positive if epithelial cells or tumor cells showed a staining reaction in the cytoplasm and/or the nucleus. A quantitative score was given by estimating the percentages of positive cells: 0 (0 to 5%); 1 (5% to 25%); 2 (25% to 50%); 3 (50% to 75%); and 4 (75% to 100%). The intensity of positive staining was given scores as negative (0) pallide-flavens grains (1) buffy grains (2) and brown-black grains (3) respectively. Finally total scores (0“12) for each samples were determined by combination of the quantitative scores time the intensity scores. The score 0 to 4 5 to 8 and 9 to 12 indicated ALDOA expression as negative positive and strongly positive. Cell Culture and Stable Knockdown of ALDOA in NCI-H520 The human lung squamous cell carcinoma NCI-H520 cell line was maintained in RPMI 1640 medium (Invitrogen USA) supplemented with 10% fetal bovine serum (FBS). The pGPU6/GFP/Neo vector containing shRNAs specific to human ALDOA were transfected into NCI-H520 cells using Lipofectamine 2000 (Invitrogen) and the resulting cells were selected with G418 (400 µg/ml) to establish clones of stable depletion of ALDOA. Cell Migration assays soft agar colony formation assays and xenografting nude mice Cells were seeded in 10-cm petri dishes at a density of 1—106 cells/ml and grown to approximately 90% confluency. The cells were cut with a sterile 200-µl pipette tip and the resulting cells were continually cultured in serum-free medium and were photographed at 0 8 and 24 hs post-scratching. Cell migration assays were performed in the transwell plates with an 8-µm pore size membrane (Corning New York NY). 5—105 cells were put into the upper chamber and medium containing 10% FBS as attractant was placed in the lower chamber. After 24 hours of incubation the cells on the upper surface of the filter were wiped out with cotton swabs and the migrated cells were fixed in 70% methanol and stained with 0.1% crystal violet. The assays were performed in triplicates and at least 6 unbiased fields were counted per filter. The mean cell numbers and standard deviations were calculated. Six weeks old male nude mice (Inspection No: 0002858) were purchased and allowed to acclimate one week in the animal facility. NCI-H520 cells and derivatives (107 cells in 200 µL medium) were prepared and injected subcutaneously into the left flank of the mice (three mice per group). The mice were checked daily for tumor growth and sacrificed 8 weeks after injection. The isografted tumors were thoroughly examined. Statistical analysis All statistical analyses were performed using SPSS 12.0 software. Data were presented as mean ± standard deviation (S.D.). The chi-square test and Fisher's exact test were used to analyze the association between ALDOA expression and clinicopathologic features of SCC. Kaplan-Meier methods with the log-rank test were used to estimate differences in survival curves among LSCC patients. Comparisons among all groups were performed with the one-way analysis of variance (ANOVA) test and Student Newman Keuls method. P-values less than 0.05 were considered as statistically significant. Results ALDOA is highly expressed in LSCC metastasis To identify differentially expressed proteins from LSCC strictly selected 7 pairs of the LSCC specimens from well documented patients were subjected to 2D-DIGE and MS proteomic analysis. Of the protein identified 63 proteins were found more than 1.5- or less than ?1.5-fold difference in metastatic or non-metastatic SCC tissues compared to the adjacent normal tissues. ALDOA was up-regulated 3.12-fold in metastatic LSCC tissues and 1.77-fold in non-metastatic LSCC tissues (A). The amino acid residues highlighted in red were those detected by MS analysis and counted for 36% sequence coverage of ALDOA (B). .0085804.g001 ALDOA is highly expressed in lung squamous carcinoma. (A) Proteomic analysis of the differentially expressed proteins from normal tissue and LSCC metastasis. Strictly selected 7 pairs of the matched LSCC specimens were subjected for 2D-DIGE and MS proteomic analysis. ALDOA was up-regulated 3.12-fold in metastatic LSCC tissues and 1.77-fold in non-metastatic LSCC tissues compared to adjacent normal tissues (the pointing arrow shows the ALDOA spots). (B) The amino acid residues highlighted in red were those detected by MS/MS analysis and counted for 36% sequence coverage of ALDOA. (C) Western blotting analysis of the ALDOA protein expression in 17 pairs of LSCC and matched adjacent normal tissues. Higher expression of ALDOA was observed in most of LSCC tissues examined. Data from 6 pairs of specimens were shown here. (D) Average of the ALDOA protein from 17 pairs of matched specimens. The relative expression values of ALDOA were 0.87±0.47 in carcinoma tissues and 0.54±0.27 in normal tissues. The level of Actin was used as control for normalization. We next carried out western blotting assays to examine the ALDOA protein expression in 17 pairs of LSCC and matched adjacent normal tissues. Higher expression of ALDOA was observed in most of LSCC tissues examined (C). The level of Actin was used as a control for normalization. The relative expression values of ALDOA were 0.87±0.47 in carcinoma tissues and 0.54±0.27 in normal tissues respectively (Fig. 1D). ALDOA is predominately localized in cytoplasm To examine the subcellular localization of the endogenous ALDOA we first performed immunofluorecence staining assays in NCI-H520 cells using antibody specific to ALDOA. Images taken by confocal microscopy showed that ALDOA was predominately distributed in cytoplasm and in the nucleus ALDOA formed sporadic bright dots (A). Next we performed IHC assays to examine the localization of ALDOA in LSCC tissues. Consistently strong staining of ALDOA was observed in the cytoplasm of the tumor cells (B). .0085804.g002 ALDOA is a major cytoplasmic protein. (A) Endogenous ALDOA was detected by immunofluorescence staining using antibody specific to ALDOA in NCI-H520 cells. Cellular ALDOA protein (red) and nuclear condensation (blue DAPI) were examined by using a Leica confocal SP5 microscopey. (B) IHC assays of ALDOA were performed in paired LSCC tissues vs adjacent normal tissues. High expression of ALDOA is correlated with LSCC metastasis tumor grades and differentiation status To analyze the clinical relevance between high expression of ALDOA and LSCC we performed immunochemistry staining to examine ALDOA protein expression in a LSCC tissue microarray (TMA) containing 75 pairs of matched LSCCs and adjacent normal tissues. Among which 32 pairs are from metastatic LSCC patients and 43 pairs are from non-metastatic LSCC patients. The intensity of the positive stain of TMA was measured and quantified as positive (+) or strong positive (++) (A). In normal tissues 22 cases (29.3%) were measured as positive (+) and 1 case (1.3%) was strong positive (++). Strikingly in LSCC tissues 25 cases (33.3%) were measured as positive (+) and 25 cases (33.3%) were strong positive (++) (B). .0085804.g003 High expression of ALDOA is highly expressed in LSCC metastasis. (A) Tissue microarray (TMA) analysis of ALDOA expression in 75 pairs of matched LSCCs and adjacent normal tissues. IHC staining assays were performed to examine ALDOA protein expression and three pairs were shown as representatives. (B) In normal tissues 22 cases (29.3%) were measured as positive (+) and 1 case (1.3%) was strong positive (++). In LSCC tissues 25 cases (33.3%) were measured as positive (+) and 25 cases (33.3%) were strong positive (++). We further analyzed if there are any correlations between the protein level of ALDOA in LSSC tissues and tumor metastasis grade and differentiation status. ALDOA shows a higher positive rate (84.4% 27 out of 32 cases) of expression in metastatic LSSC tissues than that of non-metastatic LSSC tissues (53.5% 23 out of 43 cases). Moreover 19 cases showed strong positive ALDOA stain in metastatic LSCC tissues while only 6 cases showed strong positive stain in non-metastatic LSCC tissues (Table 1). Similarly high ALDOA expression was observed with increasing tumor grades and differentiation status. .0085804.t001 Table 1 Summary of the TMA analysis of ALDOA expression. ALDOA level Metastasis Tumor grades Differentiation no yes I II III IV low middle high ? 20 5 15 6 4 0 7 13 3 + 17 8 9 5 6 0 2 22 3 ++ 6 19 3 17 8 2 4 17 4 Positive (%) 53.5 84.4 44.4 78.6 77.8 100 46.2 75 70 ?2 7.876 9.786 4.072 p value 0.005 0.02 0.13 Correlations of the protein level of ALDOA in LSSC tissues and tumor metastasis grade and differentiation status. Of the total 75 specimens on TMA examined 32 pairs are from metastatic LSCC patients and 43 pairs are from non-metastatic LSCC patients. The intensity of the positive stain of TMA was measured and quantified as positive (+) or strong positive (++). We next investigated the correlation of ALDOA expression with patients' prognosis. Thirty-two out of the 75 cases on TMA met the requirement for the Kaplan-Meier method analysis. The results indicated that patients having high expression of ALDOA displayed low survival rate (Figure 4). Taken together high expression of ALDOA is associated with LSCC metastasis and poor prognosis. .0085804.g004 Figure 4 Expression level of ALDOA protein is negatively correlated with survival rates and prognosis of LSCC patients. Thirty-two out of the 75 cases on TMA met the requirement for the Kaplan-Meier method analysis. ?: negative expression; +: positive expression; ++: strong positive expression. ALDOA is required for migration of the lung cancer cell NCI-H520 To examine if ALDOA is required for lung cancer cell migration we depleted ALDOA in NCI-H520 cells by stably expressing two independent shRNAs specific to ALDOA. Western blot assays showed that the two shRNAs efficiently decreased ALDOA protein level (Figure 5A) and concomitantly depletion of ALDOA resulted in an up-regulation of epithelial markers E-cadherin and ?-catenin and a down regulation of mesenchymal markers Fibronectin and Vimentin. Conversely overexpression of ALDOA in NCI-H520 cells decreased E-cadherin and ?-catenin and concomitantly increased Fibronectin and Vimentin (Figure 5B). These observations suggest that ALDOA may induce epithelial-mesenchymal transition and promote cell migration. .0085804.g005 Figure 5 ALDOA affects the expression of epithelial and mesenchymal markers in NCI-H520 cells. (A) Depletion of ALDOA in NCI-H520 cells results in reversal expression of epithelial and mesenchymal markers. Two independent shRNAs specific to ALDOA were stably expressed in NCI-H520 cells. Western blot assays showed that the two shRNAs efficiently decreased ALDOA protein level and concomitantly the epithelial markers E-cadherin and ?-catenin were increased while the mesenchymal markers Fibronectin and Vimentin were decreased. (B) Overexpression of ALDOA decreased E-cadherin and ?-catenin and concomitantly increased Fibronectin and Vimentin. ALDOA protein was expressed in NCI-H520 cells via transcient transfection of palsmids encoding human ALDOA. The parental vector was used as a control. The expression of various proteins was analyzed by western blot assays. We next carried out wound-healing assays in these established NCI-H520 cell derivatives stably expressing vector or shRNAs specific to ALODA. At 8 hrs after scratching NCI-H520-shVector cells apparently migrated to the wound area and at 24 hrs the wound area was well covered by the cells. In contrast no apparent cell migration was observed in NCI-H520-shALDOA cells at 8 hrs and the wound area was only partially covered by cells at 24 hrs (Figure 6A). .0085804.g006 Figure 6 ALDOA is required for migration of the lung cancer cell NCI-H520. (A) Wound-healing assays of the resulting NCI-H520 cells in cultured plates. Cell were grown in petri dishes to 90% confluency and cut with a sterile 200-µl pipette tip. The resulting cells were continually cultured in serum-free medium and were photographed at 0 8 and 24 hs post-scratching. (B) Cell migration assays using the transwell assays kits. 5—105 cells per well were plated in the upper chambers of transwell plates with an 8-µm pore size membrane and media containing 10% FBS was placed in the lower chamber. After 24 hours of culture the cells on the upper surface of the filter were removed. The migrated cells were fixed in 70% methanol and stained with 0.1% crystal violet. The assays were performed in triplicates and at least 6 unbiased fields were counted per filter. To further examine the effect of depletion of ALDOA on cell motility the migration potential of the resulting cells was assayed using transwell plates. Migrated cells were monitored using an inverted microscopy. Depletion of ALDOA apparently reduced NCI-H520 cells migration (Figure 6B). Taken together these data indicate high expression of ALDOA is required for NCI-H520 cells to migrate effectively. Depletion of ALDOA reduces the tumorigenicity of NCI-H520 cells To examine the effect of ALDOA on the tumorigenicity suspensions of single cancer cells of the NCI-H520-shALDOA cells and NCI-H520-shVector cells were cultured in soft agar to evaluate anchorage-independent colony formation. The colonies formed by these NCI-H520-shALDOA cells were significantly decreased in colony number compared with NCI-H520-shVector cells (Figure 7A B). Consistently when transplanted subcutaneously into the nude mice the NCI-H520-siALDOA cells did not grow or only grow into very small tumors compared with that of NCI-H520-shVector cells (Figure 7C D). .0085804.g007 Figure 7 Depletion of ALDOA reduces the tumorigenicity of NCI-H520 cells. (A) Soft agar assays. The NCI-H520-siALDOA cells and NCI-H520-vector cells were suspended in single cell and cultured in soft agar to evaluate anchorage-independent colony formation. The experiments were done in triplates and repeated twice. (B) Data shows the average of the colonies formed by these NCI-H520 cells in triplicates. (C) The NCI-H520-shALDOA cells did not grow or only grow into very small tumors in the nude mice. Discussion In the present study we identified the glycolytic enzyme ALDOA was highly expressed in metastatic LSCC and its express is highly correlated with LSCC metastasis tumor grade and differentiation status. We further demonstrated that depletion of ALDOA expression in NCI-H520 cells reduced the capabilities of cell motility and tumorigenesis. These data suggest that ALDOA could be a potential marker for LSCC metastasis and a potential therapeutic target for drug development. A typical feature of tumor cells is highly active glycolysis associated to an inhibition of apoptosis. As first stated by Warburg cancer cells need to activate glycolysis to proliferate despite the presence of oxygen because glycolysis provides most of the building blocks required for massive cell proliferation [21]. ALDOA is a ubiquitous glycolytic enzyme that drives the glycolytic metabolic pathway in mammalian cells and is predominantly expressed in adult muscle tissue. Overexpression of ALDOA is observed in various cancers including lung renal cell and hepatocellular carcinoma suggesting enhanced glycolysis in these cancer cells [17]“[20]. We also observed that depletion of ALDOA results in an up-regulation of epithelial markers and a down regulation of mesenchymal markers suggesting ALDOA is required for maintaining the mesenchymal morphology a characteristic of migrating cells. Accordingly our results indicate that overexpression of ALDOA was significantly relevant to high degree of metastasis and low degree of pathologic staging as well as low survival rate and poor prognosis. These findings suggest ALDOA could be a potential marker for LSCC metastasis prognosis prediction and as a target for clinical treatment of LSCC. References 1 HeistRS SequistLV EngelmanJA (2012) Genetic Changes in Squamous Cell Lung Cancer: A Review. J Thorac Oncol7: 924“3322722794 2 MayC BrosseronF ChartowskiP MeyerHE MarcusK (2012) Differential proteome analysis using 2D-DIGE. Methods Mol Biol893: 75“8222665295 3 KajitaE MoriwakiJ YatsukiH HoriK MiuraK et al (2001) Quantitative expression studies of aldolase A B and C genes in developing embryos and adult tissues of Xenopus laevis. Mech Dev102: 283“711287212 4 KusakabeT MotokiK HoriK (1997) Mode of interactions of human aldolase isozymes with cytoskeletons. Arch Biochem Biophys344: 184“939244396 5 CarrD KnullH (1993) Aldolase-tubulin interactions: removal of tubulin C-terminals impairs interactions. Biochem Biophys Res Commun195: 289“938103323 6 St-JeanM IzardT SyguschJ (2007) A hydrophobic pocket in the active site of glycolytic aldolase mediates interactions with Wiskott-Aldrich syndrome protein. J Biol Chem282: 14309“1517329259 7 ArnoldH PetteD (1968) Binding of glycolytic enzymes to structure proteins of the muscle. Eur J Biochem6: 163“715725503 8 WalshJL KnullHR (1988) Heteromerous interactions among glycolytic enzymes and of glycolytic enzymes with F-actin: effects of poly(ethylene glycol). Biochim Biophys Acta952: 83“913334856 9 ClarkeFM MastersCJ (1975) On the association of glycolytic enzymes with structural proteins of skeletal muscle. Biochim Biophys Acta381: 37“461111588 10 KnullHR WalshJL (1992) Association of glycolytic enzymes with the cytoskeleton. Curr Top Cell Regul33: 15“231499331 11 WalshJL KeithTJ KnullHR (1989) Glycolytic enzyme interactions with tubulin and microtubules. Biochim Biophys Acta999: 64“702553125 12 TochioT TanakaH NakataS HosoyaH (2010) Fructose-16-bisphosphate aldolase A is involved in HaCaT cell migration by inducing lamellipodia formation. J Dermatol Sci58: 123“920362419 13 LuM HollidayLS ZhangL DunnWAJr GluckSL (2001) Interaction between aldolase and vacuolar H+-ATPase: evidence for direct coupling of glycolysis to the ATP-hydrolyzing proton pump. J Biol Chem276: 30407“1311399750 14 KishiH MukaiT HironoA FujiiH MiwaS et al (1987) Human aldolase A deficiency associated with a hemolytic anemia: thermolabile aldolase due to a single base mutation. Proc Natl Acad Sci84: 8623“72825199 15 MiwaS FujiiH TaniK TakahashiK TakegawaS et al (1981) Two cases of red cell aldolase deficiency associated with hereditary hemolytic anemia in a Japanese family. Am J Hematol11: 425“377331996 16 YaoDC TolanDR MurrayMF HarrisDJ DarrasBT et al (2004) Hemolytic anemia and severe rhabdomyolysis caused by compound heterozygous mutations of the gene for erythrocyte/muscle isozyme of aldolase ALDOA(Arg303X/Cys338Tyr). Blood103: 2401“314615364 17 RhoJH RoehrlMH WangJY (2009) Glycoproteomic Analysis of Human Lung Adenocarcinomas Using Glycoarrays and Tandem Mass Spectrometry: Differential Expression and Glycosylation Patterns of Vimentin and Fetuin A Isoforms. Protein J28: 148“6019412661 18 PoschmannG SitekB SiposB UlrichA WieseS et al (2009) Identification of Proteomic Differences between Squamous Cell Carcinoma of the Lung and Bronchial Epithelium. Mol Cell proteomics8: 1105“1619176476 19 PfleidererG Th¶nerM WachsmuthED (1975) Histological examination of the aldolase monomer composition of cells from human kidney and hypernephroid carcinoma. Beitr Pathol1563: 266“79766744 20 ChaerkadyR HarshaHC NalliA GucekM VivekanandanP et al (2008) A quantitative proteomic approach for identification of potential biomarkers in hepatocellular carcinoma. J Proteome Res7: 4289“9818715028 21 DangCV (2012) Links between metabolism and cancer. Genes Dev26: 877“9022549953 Mol Cell Proteomics Mol. Cell Proteomics mcprot mcprot MCP Molecular & Cellular Proteomics : MCP 1535-9476 1535-9484 The American Society for Biochemistry and Molecular Biology 24520089 3977184 M113.036905 10.1074/mcp.M113.036905 Research A Targeted Quantitative Proteomics Strategy for Global Kinome Profiling of Cancer Cells and Tissues* Global Kinome Profiling of Cancer Cells and Tissues Xiao Yongsheng ¡ Guo Lei § Wang Yinsheng ¡ § ¶ From the ¡Department of Chemistry and §Environmental Toxicology Graduate Program University of California Riverside CA 92521-0403 ¶ To whom correspondence should be addressed: Department of Chemistry University of California Riverside CA 92521-0403. Tel.: (951) 827-2700; Fax: (951) 827-4713; E-mail: yinsheng.wang@ucr.edu. 4 2014 11 2 2014 1 4 2015 13 4 1065 1075 8 12 2013 8 12 2013 © 2014 by The American Society for Biochemistry and Molecular Biology Inc. 2014 Kinases are among the most intensively pursued enzyme superfamilies as targets for anti-cancer drugs. Large data sets on inhibitor potency and selectivity for more than 400 human kinases became available recently offering the opportunity to design rationally novel kinase-based anti-cancer therapies. However the expression levels and activities of kinases are highly heterogeneous among different types of cancer and even among different stages of the same cancer. The lack of effective strategy for profiling the global kinome hampers the development of kinase-targeted cancer chemotherapy. Here we introduced a novel global kinome profiling method based on our recently developed isotope-coded ATP-affinity probe and a targeted proteomic method using multiple-reaction monitoring (MRM) for assessing simultaneously the expression of more than 300 kinases in human cells and tissues. This MRM-based assay displayed much better sensitivity reproducibility and accuracy than the discovery-based shotgun proteomic method. Approximately 250 kinases could be routinely detected in the lysate of a single cell line. Additionally the incorporation of iRT into MRM kinome library rendered our MRM kinome assay easily transferrable across different instrument platforms and laboratories. We further employed this approach for profiling kinase expression in two melanoma cell lines which revealed substantial kinome reprogramming during cancer progression and demonstrated an excellent correlation between the anti-proliferative effects of kinase inhibitors and the expression levels of their target kinases. Therefore this facile and accurate kinome profiling assay together with the kinome-inhibitor interaction map could provide invaluable knowledge to predict the effectiveness of kinase inhibitor drugs and offer the opportunity for individualized cancer chemotherapy. "

Lung_Cancer

"The change of ADC value may be a sensitive indicator to predict early response to chemotherapy in lung cancer. Prediction ability could be improved by combining the change of ADC value and longest diameter. 1. Introduction Lung cancer is the most common and highly lethal cancer worldwide with poor prognosis [1]. About 75% of patients are diagnosed at advanced stage since there is no specific recognized symptom at early stage [2]. Surgery alone is not the appropriate treatment for those patients at terminal stage [3 4]. A large meta-analysis pooled 4584 patients suggested that the adjuvant chemotherapy had a 5.4% improvement of 5-year survival rate in non-small cell lung cancer [5]. Assessing the early response to chemotherapy in lung cancer is crucial because optimized chemotherapy regimen needs individualization to gain a preferable outcome and to avoid toxic effect and unnecessary expenditure. Currently computed tomography (CT) and magnetic resonance (MR) imaging are the regular methods to monitor the tumor changes in size to evaluate the effect of chemotherapy [6]. However response assessment with these morphologic imaging has limitations in reliable differentiation of residual tumor tissue from necrotic tumor or fibrotic scar. Moreover the tumor change in size which lags behind the biological and molecular changes may be not an early and sensitive indicator [7]. MR diffusion-weighted imaging (DWI) reflects the differences in the Brownian motion of water molecules between tissues [8]. As a surrogate marker of tissue cellularity by observing water mobility within the tumor the apparent diffusion coefficient (ADC) can be used to distinguish the highly cellular tumor from normal tissue or necrotic regions [8“10]. It has been used to differentiate pulmonary malignant tumors from solid benign lesions or to stage lung cancer [11 12]. Therefore the change of ADC value may be used to monitor the treatment response which manifested as change in cellularity of the tumor [8 13]. Previous studies have demonstrated that ADC values can be used as an indicator to evaluate the tumor response in tumor from many ans [13“20]. The purpose of this study is to observe the change of ADC value after chemotherapy and to determine the ability of the change of ADC value to predict treatment response in lung cancer at the early stage of chemotherapy. 2. Materials and Methods 2.1. Patients Research ethics committee approval and patient written informed consent were obtained. All the patients participated in the study were diagnosed as non-small cell lung cancers histologically. They were scheduled for chemotherapy and with no history of previous chemotherapy or other anticancer treatments. The patient selection was shown in the diagram (). Finally from 2010-12 to 2012-07 25 patients (17 male 8 female; median age: 61.4 ± 8.0 years) with advanced non-small cell lung cancers were consecutively enrolled. There were 2 patients with multiple lung lesions. Only the largest lung lesion was included in the MR image analysis. 14 squamous cell carcinomas 9 adenocarcinomas and 2 adenosquamous carcinomas were confirmed histologically. The initial staging protocol was evaluated by CT MR SPECT and PET-CT if available. There were 23 stage III tumors and 2 stage IV tumor. All patients underwent concurrent chemotherapy which consisted of gemcitabine or vinorelbine on days 1 and 8 and platinum-based pharmaceutical (75?mg/m2) for first 2 or 3 days in each 21-day cycle. The posttherapy MR imaging was performed at the end of the first cycle of chemotherapy. Patients remained on these treatment protocols until disease progression was detected. 2.2. MR All patients accepted MR imaging one week before and after 1 cycle of chemotherapy (). All the MR examinations were performed on a 3-T superconducting magnet (HDx; General Electric Medical Systems Milwaukee Wis) following the same scan protocol. Respiratory and electrocardiographically gated T2-weighted fast relaxation fast spin echo images with fat suppression images were obtained. The parameters were as follows: repetition time/echo time8000~8571?ms/86~96?ms; matrix size 256 — 160; field of view 42?cm; number of excitations 2; slice thickness 4?mm; gap 1?mm. Electrocardiographically gated T1-weighted dual inversion recovery fast spin echo images were also obtained with the following parameters: repetition time/echo time1120“1760?ms/4.1“6.2?ms; matrix size 256 — 160; field of view 42?cm; number of excitations 1; slice thickness 4?mm; gap 1?mm. These images were inspected initially to define locations of the pulmonary lesions for the DWI. DWI were acquired using a respiratory gated single-shot echo-planar imaging sequence and array spatial sensitivity encoding technique with b values of 0 and 1000?s/mm2. The parameters were as follows: repetition time/echo time5000“9230?ms/55?ms; matrix size 256 — 160; field of view 42?cm; number of excitations 4; slice thickness 4?mm; gap 1?mm; R factor 2; slice-select phase-encoding and frequency-encoding directions. The total MR imaging acquisition time in this study was about 15 minutes. 2.3. Image Analysis All MR images were transferred to a workstation (AW 4.3; GE Healthcare Milwaukee Wis) and analyzed by two experienced radiologists (A and B 20 and 11 years experience in reading chest imaging resp.) who were blinded to the therapeutic response and other data of patients. The following parameters were measured and recorded: lesion number location size and mean ADC value. This procedure was performed by the two radiologists then the mean values were calculated. The lesion size was reflected by tumor longest and shortest diameter (in perpendicular angle) measured with a caliper tool on axial T2-weighted images. The lesion location was observed on the T2- and T1-weighted images. Meanwhile T2- and T1-weighted images were used as a slice selection reference for ADC value measurement. During the MR image analysis DWI reconstructed images with b = 1000?s/mm2 were evaluated. The ADC map of each DWI image was produced on a pixel-by-pixel basis. An axial slice showing the largest tumor size corresponding to T2- and T1-weighted images was chosen. A polygonal region of interest was drawn manually encompassing the entire area of the target lesion on the ADC map (). The mean ADC values were calculated. 2.4. CT Imaging All patients underwent contrast enhanced CT scans before and after 2-3 cycles of chemotherapy conventionally () [21]. The images were obtained by using a 64-detector row CT (GE light speed VCT XT) with a 64 — 0.625?mm collimation 120?kVp 250?mA and 500?msec gantry rotation time in a spiral mode. The contrast enhanced CT images were acquired about 30 seconds after contrast material (Omnipaque 350 90?mL) administration. The images were obtained in the transverse plane and then reconstructed by 2.5?mm section thickness and 2.5?mm section interval. One radiologist (C 3 years experience in reading chest imaging) who was blinded to the results of MR images and other data of patients measured the longest diameter of each tumor on the mediastinal window of CT images. Furthermore the short diameters of lymph nodes (if lesions ?15?mm in short axis) will be recorded and included in the sum of lesions in calculation of the tumor response [22]. At last the sum of the longest tumor diameters and shortest diameters of lymph nodes was calculated. 2.5. PR and SD Groups According to Response Evaluation Criteria in Solid Tumors (RECIST version 1.1) [22 23] the responses to chemotherapy in lung cancer were classified by B: (1) complete response: disappearance of all target lesions and reduction of any pathological lymph nodes (<10?mm in short diameter); (2) partial response (PR): at least 30% decrease in the sum of diameters of target lesions taking as reference the baseline sum diameters; (3) stable disease (SD): neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for progressive disease; (4) progressive disease: at least 20% increase in the sum of diameters of target lesions taking as reference the smallest sum on the study. Furthermore a 5?mm absolute increase in sum of the target disease is needed. Based on the revised RECIST 1.1 the short diameters of lymph nodes (short diameter >15?mm) on axial CT images were included in the sum of target lesions in calculation of tumor response [23]. 2.6. Statistical Analysis Statistical analysis was performed using SPSS 17.0. Independent and paired student t-tests were used to analyze the difference of ADC value and tumor diameters between different groups or time periods. The frequencies of patient and tumor characteristics between PR and SD groups were tested by using X2 test. To combine the changes of ADC value and diameter a logistic regression model that allows the discrimination between PR and SD groups was employed. Receiver operator characteristic curves were generated to establish the cutoff value of the change of ADC value in order to differentiate PR lesion from SD lesion. A P value less than 0.05 was considered significant. 3. Results In the current study there were 8 and 17 patients in PR and SD group respectively. No patient belonged to complete response or progressive disease group. The patient and tumor demographics between PR and SD groups was shown (Table 1). Both longest and shortest diameter of the tumor had significant statistical differences before and after chemotherapy regardless in the SD or PR group (Table 2). "

Lung_Cancer

"A decrease in Hsp70 by RNAi promoted cisplatin-dependent activation of Bax. A549 cells treated with Hsp70 or control siRNA were incubated in presence or absence of cisplatin; each cell extract was immunoprecipitated with an anti-active Bax antibody followed by immunoblotting with anti-Bax antibody. The data are representative of three separate experiments Synergistic effect of Hsp70 suppression on the cisplatin-mediated activation of caspase-9. (a) A549 cells were treated with cisplatin and/or ibuprofen and cell extracts were immunoblotted with active caspase-9 antibody. The lower panel shows the measurement of each caspase-9. (b) A549 cells exposed to siRNA targeting Hsp70 or control siRNA were incubated with or without cisplatin and the active caspase-9 was detected by western blot using an anti-caspase-9 antibody. The quantity of each protein was estimated by densitometric analysis (lower panels). (c and d). Assay for enzymatic activity of caspase-9 using a fluorogenic substrate. (c) After the incubation of the A549 cells with cisplatin (10??M) and/or ibuprofen (400??M) the caspase-9 activity of each cell extract was measured as described in Materials and methods section. (d) A549 cells transfected with Hsp70 siRNA or control siRNA were exposed to cisplatin for 48?h. The caspase-9 activity was then assessed using an enzymatic assay as described earlier. The value of caspase-9 activity was presented relative to the activity in untreated cells set at 1.0. The data represent mean values of three separate experiments. Significances were determined by Student's t-test (*P<0.05) Effects of nonsteroidal anti-inflammatory drugs on the expression of Hsp70 in A549 cells NSAIDs Hsp70 expression (%) Ibuprofen (400??M) 22.7±2.8 Aspirin (2500??M) 95.1±7.8 Diclofenac (200??M) 97.2±5.6 Sulindac (15??M) 98.9±2.9 Piroxicam (60??M) 96.6±6.2 Indometacin (10??M) 95.0±15.1 Mefenamic acid (25??M) 100.5±6.0 Values are shown as means±S.D. The expression of Hsp70 was measured by immunoblotting with an anti-Hsp70 antibody. The quantity of Hsp70 protein was estimated by densitometric analysis using Scion Image. The values in parentheses are the highest non-toxic concentrations (approximately 90% viability) used for each NSAID on the A549 cells for 48?h Effects of nonsteroidal anti-inflammatory drugs on the expression of HSF-1 in A549 cells NSAIDs HSF-1 expression (%) Ibuprofen (400??M) 16.2±3.9 Aspirin (2500??M) 93.5±2.9 Diclofenac (200??M) 96.7±6.6 Sulindac (15??M) 99.8±3.6 Piroxicam (60??M) 96.3±4.7 Indometacin (10??M) 98.1±1.6 Mefenamic acid (25??M) 98.5±1.1 Values are shown as means±S.D. The expression of HSF-1 was measured by immunoblotting with anti-HSF-1 antibody. The quantity of HSF-1 protein was estimated by densitometric analysis using Scion Image. The values in parentheses are the highest non-toxic concentrations (approximately 90% viability) used for each of the NSAID on the A549 cells for 48?h PLoS One one 1932-6203 Public Library of Science San Francisco USA 24505298 3914822 PONE-D-13-39952 .0087629 Research Biology Population Biology Epidemiology Medicine Clinical Research Design Epidemiology Drugs and Devices Drug Research and Development Epidemiology Cancer Epidemiology Clinical Epidemiology Oncology Cancers and Neoplasms Lung and Intrathoracic Tumors Non-Small Cell Lung Cancer Cancer Treatment Radiology Nuclear Medicine PET imaging [18F]FDG Positron Emission Tomography within Two Weeks of Starting Erlotinib Therapy Can Predict Response in Non-Small Cell Lung Cancer Patients Theranostic Use of FDG-PET in NSCLC Patients Hachemi Mammar 1 2 Couturier Olivier 1 2 3 Vervueren Laurent 1 2 Fosse Pac´me 1 2 Lacœuille Franck 1 2 3 Urban Thierry 1 3 4 5 Hureaux Jos 1 3 4 5 * 1 LUNAM Universit 49 000 Angers France 2 Universit d'Angers CHU Angers P´le de Radiologie Service de Mdecine Nuclaire Angers France 3 INSERM UMR_S 1066 Micro et Nanomdecines Biomimtiques Angers France 4 Universit d'Angers CHU Angers P´le des Spcialits Mdicales et Chirurgicales Intgres Dpartement de Pneumologie Angers France 5 Universit d'Angers Equipe Pyver Angers France Singh Pankaj K. Editor University of Nebraska Medical Center United States of America * E-mail: JoHureauxchu-angers.fr Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: OC TU JH. Performed the experiments: MH OC LV PF FL JH. Analyzed the data: MH OC FL TU JH. Contributed reagents/materials/analysis tools: MH OC TU JH. Wrote the paper: MH OC JH. 2014 5 2 2014 9 2 e87629 29 9 2013 26 12 2013 2014 Hachemi et al This is an open-access distributed under the terms of the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original author and source are credited. Purpose The aim of this prospective study was to evaluate whether [18F]FDG-PET/CT performed within two weeks of starting erlotinib therapy can predict tumor response defined by RECIST 1.1 criteria after 8 weeks of treatment in patients with inoperable (stage IIIA to IV) non-small cell lung cancer patients. Patients and Methods Three [18F]FDG-PET/CT scans were acquired in 12 patients before (5±4 days) and after 9±3 days (early PET) and 60±6 days (late PET) of erlotinib therapy. Conventional evaluation including at least chest CT (baseline versus after 8 weeks of treatment) was performed according to RECIST 1.1 criteria. Change in [18F]FDG uptake was compared with conventional response progression-free survival (PFS) and overall survival (OS). Results By using ROC analysis the Area Under the Curve for prediction of metabolic non-progressive disease (mNP) by early PET was 0.86 (95% CI 0.62 to 1.1; P?=?0.04) at a cut-off of 21.6% reduction in maximum Standardized Uptake Value (SUVmax). This correctly classified 11/12 patients (7 with true progressive disease; 4 with true non-progressive disease; 1 with false progressive disease). Non-progressive disease after 8 weeks of treatment according to RECIST 1.1 criteria was significantly more frequent in patients classified mNP (P?=?0.01 Fisher's exact test). mNP patients showed prolonged PFS (HR?=?0.27; 95% CI 0.04 to 0.59; P<0.01) and OS (HR?=?0.34; 95% CI 0.06 to 0.84; P?=?0.03). Late PET analysis provided concordant results. Conclusion Morphologic response PFS and OS survival in non-small cell lung cancer patients can be predicted by [18F]FDG-PET/CT scan within 2 weeks after starting erlotinib therapy. The authors have no support or funding to report. Introduction Lung cancer is the leading cause of cancer-related death in both Europe[1] and the United States of America.[2] The most common forms of lung cancer are non-small cell lung cancer (NSCLC) histological subtypes. Systemic chemotherapy has contributed to a significant improvement in NSCLC therapy but progress appears to be stagnating.[3] [4] Over the last decade a better knowledge of cellular pathways has allowed the development of new therapies based on NSCLC-driving genetic abnormalities. Targeted therapies have been developed to block pathological cellular pathways involved in cancer cell survival proliferation and metastasis. Epidermal Growth Factor Receptor (EGFR) is overexpressed in NSCLC[5] and has been extensively studied as a potential therapeutic target. Two EGF Receptor blockers gefitinib and erlotinib have been demonstrated to be effective in front-line therapy in patients with inoperable NSCLC harboring EGFR-activating mutations.[6] [7] Erlotinib is also authorized after failure of previous chemotherapy and as maintenance therapy.[8] [9] In clinical practice evaluation of tumor response is based on changes in tumor size according to criteria proposed by the World Health anization[10] or RECIST criteria.[11] [12] This morphological evaluation may lead to underestimation of the efficacy of cytostatic therapeutic agents such as erlotinib that stabilize the disease in non-mutated patients whereas conventional cytotoxic drugs induce shrinkage of tumor dimensions in the case of tumor response. NSCLC tumor size evaluation can also be difficult due to atelectasis of normal lung. The major limitations to morphological imaging methods are their inability to assess response to therapy at an early stage and their inability to identify cancer in residual masses after treatment. In patients with NSCLC [18F]FDG-PET has been recognized as an adequate staging tool[13] [14] and several studies also suggest that the standardized uptake value (SUV) has a prognostic value in NSCLC.[15] [16] The value of SUV for evaluation of tumor response to targeted therapy is currently being investigated. We designed a preliminary study to evaluate tumor response in NSCLC patients eligible for erlotinib therapy. The aim of this prospective study was to determine whether [18F]FDG-PET/CT performed several days after starting erlotinib therapy could predict tumor response defined by RECIST 1.1 criteria and [18F]FDG-PET/CT after 8 weeks of treatment. Materials and Methods Patients Twelve consecutive eligible patients with stage IIIA to IV NSCLC (7 adenocarcinomas 3 large cell carcinomas 2 squamous cell carcinomas) in whom erlotinib therapy was indicated were studied at the Angers University Hospital France. Screening for EGF receptor mutations was carried out (patient characteristics are shown in ). Eligibility criteria were: histologically or cytologically proven NSCLC; unresectable stage III/IV disease or recurrent disease after surgery; age over 18 years; measurable disease according to RECIST 1.1 criteria; Eastern Cooperative Oncology Group (ECOG) performance status between 0 to 2; adequate bone marrow function liver function and renal function. Patients were not included if they had previous lung diseases such as interstitial pneumonitis or lung fibrosis identified by chest Computed Tomography (CT) scan or diabetes mellitus that could artefact PET imaging. Life expectancy was predicted to be longer than 12 weeks. Erlotinib was administered orally in a dosage of 150 mg/day on an empty stomach until clinical disease progression unacceptable toxicity or patient refusal. The medical ethics committee of the CHU of Angers approved the study protocol. All patients gave informed written consent before inclusion according to local medical ethical committee regulations and in accordance with the guidelines established by the World Medical Association Declaration of Helsinki. .0087629.t001 Clinical characteristics of the study population. Patients Male 6 (50) Female 6 (50) Total 12 (100) Histology Adenocarcinoma 7 (58) Large cell carcinoma 3 (25) Squamous cell carcinoma 2 (17) Clinical stage IIIA or IIIB 2 (17) IV 10 (83) Smoking status Current 5 (42) Former 2 (17) Never 5 (42) EGFR mutation status Presence 2 (17) Absence 10 (83) Previous chemotherapy Yes 10 (83) No 2 (17) Size of primary tumor (cm) 1.0€“2.0 4 (33) 2.1€“3.0 3 (25) 3.1€“5.0 5 (42) >5.1 1 (8) Metastasis Lymph nodes 12 (100) Lung 4 (33) Liver 2 (17) Bone 4 (33) Adrenal glands 0 Work Plan (study design) [18F]FDG PET/CT imaging Three [18F]FDG PET/CT scans were planned: PET1 before starting therapy PET2 within 2 weeks after starting therapy and a third [18F]FDG PET/CT scan (PET3) 8 weeks after starting erlotinib therapy. PET/CT examinations were obtained in 2D mode from the vertex to mid-thighs (5 minutes of emission scan per bed position with an average of 7 bed positions at 15 cm intervals) (Discovery-ST GE Healthcare France). Patients were instructed to fast for at least 6 hours prior to scanning. Unenhanced CT scan was performed from the skull base to the upper thighs. CT parameters were 120 kVp 100 mAs 0.8 second rotation 3.27 mm slice collimation and Pitch 1.5. CT data were used for attenuation correction and PET images were reconstructed by clinical standard 2D-iterative algorithm (ordered subset expectation maximization using 4 iterations and 16 subsets; zoom 100%; image matrix size: 128—128; and Gaussian post-smoothing of 5 mm in full width at half maximum). No corrections for partial volume effect lean body mass or blood glucose levels were applied. Conventional evaluation Conventional staging and follow-up were performed according to standards of care.[11] [12] Conventional evaluation included at least clinical examination plus CT scan performed before (CT1; 7±6 days) and 8 weeks after (CT2; 58±8 days) starting erlotinib therapy. None of the patients underwent additional CT scanning during the 2 weeks after starting erlotinib therapy. Chest abdomen and pelvis CT scans (Brillance 64 PHILIPS Medical System® France) were acquired from the lung apex to the symphysis pubis after an intravenous embolus of 130 mL of iodinated contrast agent (Xenetix350®). Helical scanning parameters were 130 kVp 120 mAs 1 second rotation 4 mm slice collimation 8 mm/s bed speed and 3 mm section width. Image analysis and response evaluation CT data were interpreted by two experienced physicians specialists in thoracic oncology blinded to PET/CT results according to the Response Evaluation Criteria in Solid Tumors (RECIST 1.1 criteria[12]) by comparison of baseline CT scan (CT1) and final CT scan (CT2). Therapeutic response evaluation was defined as: 1) complete response (CR: disappearance of all target lesions); 2) partial response (PR: at least 30% decrease in the sum of the longest diameter of five target lesions); 3) progressive disease (PD: at least a 20% increase in the sum of the longest diameter of five target lesions); and 4) stable disease (SD: neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD). Patients were then classified in the progressive disease (P) group or the non-progressive disease (NP) group including CR PR and SD therapeutic response. [18F]FDG PET interpretation was performed on an Imagys® workstation (Keosys Saint-Herblain France) qualitatively and semi-quantitatively by two experienced nuclear medicine physicians blinded to clinical and conventional evaluation results. Any focus of increased [18F]FDG uptake over background not located in areas of normal [18F]FDG uptake and/or [18F]FDG excretion was considered to be positive for tumor. For semi-quantitative analyses of [18F]FDG uptake 3D regions of interest (VOIs) were placed over all lesions considered to be positive for tumor by using Imagys® software (Keosys France). The maximum standardized uptake value (SUVmax) was calculated using the single hottest pixel inside the tumor VOI. SUV peak was also calculated using a 1.2 cm diameter spherical VOI containing the SUVmax. Bone lesions were not taken into account as they were considered to be non-measurable lesions. For patients with more than one tumor lesion the sum of SUVmax and SUVpeak were calculated and used for evaluation of changes between PET1 and PET2. PET measurements were performed in up to a maximum of five measurable target lesions. All SUVs were normalized to the injected dose and patient body weight. The percentage changes in SUV between PET1 and PET2 were finally calculated as follows: ?SUV?=?(SUV1?SUV2)/SUV1. The same protocol was used for PET1 and PET3. Statistical analysis Data are expressed as mean±SD excepted for survival data that were expressed as the median. The primary endpoint of the study was comparison of changes in tumor [18F]FDG uptake on PET2 versus PET1 PET3 versus PET1 and subsequent CT scan evaluation at 8 weeks after initiation of erlotinib therapy. Friedman test was used for non-parametric comparison of repeated measures. The secondary endpoints were to determine the Receiver Operating Characteristic (ROC) analysis for [18F]FDG changes with regard to predicting response to erlotinib therapy. The relationship between metabolic response (patients stratified according to the median value of SUV variations) and clinical response was analyzed by Fisher's exact test. Progression-free survival (PFS) and overall survival (OS) were determined by standard Kaplan-Meier survival analysis and between-group comparison was performed by log-rank test. PFS was defined as the time interval from the date of enrolment in the study until the first signs of progression. OS was calculated from the date of enrolment until death from any cause. All analyses were performed using Graphpad prism version 4.0 b 2004 (Graphpad Software San Diego CA). The limit of significance was set at 0.05. Results Population Twelve eligible patients with NSCLC 6 women (50%) and 6 men (50%) with a mean age of 60±13 years were included. Two patients presented tumors harboring an activating Epidermal Growth Factor Receptor mutation (2573T>G substitution (p.Leu858Arg) in exon 21 in one patient; deletion (L747_E749del) in exon 19 in the other patient). Patient characteristics are described in . The median duration of erlotinib therapy was 75 days. Due to rapid progression and death PET3 and CT3 could not be performed in 2 patients. Tumor 18F-FDG uptake The three [18F]FDG PET/CT scans were acquired as follows: PET1 5±4 days before starting therapy PET2 9±3 days after starting therapy and PET3 60±6 days after starting erlotinib therapy. Scanning started 68±17 min (PET1) 71±16 min (PET2) and 64±13 min (PET3) after [18F]FDG injection of 271±53 MBq (PET1) 270±61 MBq (PET2) and 263±54 MBq (PET3). Blood glucose level was less than 1.5 g/L for all PET examinations i.e. 1.1±0.1 g/L for PET1 1.1±0.2 g/L for PET 2 and 1.1±0.2 g/L for PET3. Non-parametric Friedman tests did not show any significant difference between PET1 PET2 and PET3 for FDG uptake time injected FDG dose or blood glucose. Fifty-five lesions were described on PET1 before treatment and 45 lesions were defined as target lesions for PET evaluation of response to treatment (up to five most hypermetabolic lesions per patient; mean 3.8 lesions/patient). The mean tumor SUVmax of the most [18F]FDG€“avid lesion (SUVmax) was 10.0±4.7 for PET1 and did not vary significantly over time with a mean of 10.1±6.6 for PET2 and a mean of 9.1±5.6 for PET3 (P?=?0.97). The SUVpeak was 8.6±4.3 for PET1 8.1±5.4 for PET2 and 7.1±4.6 for PET3 and did not vary over time (P?=?0.60). No variation over time was observed for the sums of SUV. The mean sum of tumor SUVmax of all target lesions was 30.1±19.5 for PET1 27.5±17.7 for PET2 and 28.3±22.4 for PET3 (P?=?0.83). Sums of SUVpeak of all target lesions were 22.7±14.3 for PET1 20.6±13.4 for PET2 and 22.2±18.6 for PET3 (P?=?0.44). [18F]FDG-PET response versus conventional evaluation CT scan data were interpreted by chest physicians blinded to PET/CT scan results (). Evaluation of response to treatment according to RECIST 1.1 criteria demonstrated 7 patients with progressive disease (group P) and 5 patients with non-progressive disease (group NP) including 4 cases of stable disease (SD) and 1 partial response (PR). .0087629.t002 CT and PET assessments of response rates OS and PFS. Patient PET2 versus PET1 PET3 versus PET1 RECIST 1.1 Evaluation PFS OS New lesion ? SUVmax * ? SUVpeak * ? SUVmax * ? SUVpeak * Response to Treatment Progressive (P) or not (NP) days days on PET3 #1 ?21.6 ?17.6 18.6 ?1.5 SD NP 267 915 ? #2 25.9 26.9 70.3 77.4 PD P 57 316 + #3 9.0 7.6 23.4 23.3 PD P 216 447 + #4 ?18.6 ?15.0 ?3.2 ?2.6 PD P 67 414 + #5 ?20.3 ?11.1 42.1 51.1 PD P 53 152 + #6 ?56.7 ?59.9 ?72.1 ?70.6 PR NP 190 296 ? #7 ?22.0 ?26.0 ?31.3 ?24.3 SD NP 727 1249 + #8 ?32.0 ?25.1 3.9 ?3.9 SD NP 317 1146 ? #9 16.4 7.8 ?5.4 ?10.8 SD NP 77 359 ? #10 2.1 4.4 MD MD PD P 37 92 MD #11 36.1 20.0 30.3 25.7 PD P 104 734 ? #12 ?7.2 ?10.5 MD MD PD P 61 71 MD * For patient with more than one tumor lesion the sum of SUVmax and of SUVpeak were calculated and used for the evaluation of changes between PET1 and PET2 (or between PET1 and PET3). Missing data are indicated as MD. On ROC analysis the AUC for prediction of non-progressive disease by PET2 was 0.86 (95% CI 0.62 to 1.1; P?=?0.04) corresponding to a maximum specificity of 0.80 and sensitivity of 0.86 for non-progressive disease at a cut-off of 21.6% reduction in SUVmax (Figure 1) and a positive predictive value (PPV) of 0.86 a negative predictive value (NPV) of 0.80 an accuracy of 0.83 and a maximum Youden index of 0.65. The use of this SUVmax cut-off value correctly classified 11/12 patients (7 with true progressive disease (Figures 2 and 3); 4 with true non-progressive disease (Figures 4 and 5); 1 with false progressive disease (). Non-progression after 2 months of treatment was significantly more frequent in patients with an early decrease in SUVmax of 21.6% or more (P?=?0.01 Fisher's exact test). The only misclassified patient (patient #9 false progressive disease on PET2 versus PET1) displayed a 16.4% increase of SUVmax but metabolic progression was not confirmed on PET3 with a 5.4% decrease of SUVmax compared to PET1. Similar results were observed for SUVpeak as non-progressive disease after 2 months of treatment was significantly more frequent in patients with a decrease in SUVpeak of at least 17.6% on PET2 (P?=?0.01 Fisher's exact test). Similar results were also obtained in terms of AUC sensitivity specificity PPV NPV and accuracy and with the same classification of patients (7 with true progressive disease; 4 with true non-progressive disease; 1 with false progressive disease). .0087629.g001 Figure 1 Percentage change in SUVmax on 18F-FDG PET/CT (cut-off: ?21.6%) within 2 weeks of starting erlotinib therapy in relation to conventional imaging response. Each red or green bar represents a patient NP or P respectively. .0087629.g002 Figure 2 Example of a progressive patient on PET (mP) and conventional imaging. Progressive patient with right upper lobe NSCLC associated with mdiastinal lymphadenopathy lung and bone metastases (patient #2). Sum of the SUVmax of the 5 most hypermetabolic lesions (2 lung lesions 2 mediastinal lymph nodes one hilar lesion) were 35.2 44.3 (+26%) and 59.9 (+70%) for PET1 PET2 (% versus PET1) and PET3 (% versus PET1) respectively. Based on a SUVmax cut-off value of ?21.6 the patient was classified as mP on PET2 in accordance with RECIST evaluation on CT scan (performed 57 days after starting erlotinib). mP was confirmed on PET3 with the appearance of a new lesion (subcarinal adenopathy) and a 70% increase of SUVmax. .0087629.g003 Figure 3 New subcarinal adenopathy on PET3 (same patient as Figure 2). .0087629.g004 Figure 4"

Lung_Cancer

"Tumor markers play a key role in patient management for many malignancies. The potential uses of serum tumor markers include aiding early diagnosis determining prognosis prospectively predicting response or resistance to specific therapies and monitoring therapy in patients with advanced disease. Kallikrein-related peptidases 11 (KLK11) is a member of the human kallikrein gene family which localized on chromosome 19q13.4 [5]. Recent studies have reported that KLK11 has been expressed in many cancers including prostate cancer [6] ovarian cancer [7] gastric cancer [8] as well as rectal carcinoma [9]. An immunofluorometric assay study demonstrated that KLK11 expression in ovarian cancer tissues is a marker of favorable prognosis since patients with KLK-positive tumors exhibit a longer progression-free survival (PFS) and overall survival (OS) [10]. Additionally Sasaki et al. [11] reported that lower KLK11 mRNA expression in lung cancer is an indicator of poor prognosis in patients with lung cancer. However there seems to be a paucity of research concerned with serum KLK11 expression in NSCLC. For this reason the goal of the present study was to investigate the baseline serum levels of KLK11 in patients with NSCLC to determine its potential diagnostic and prognostic roles. Materials and methods Patients A total of 138 patients with NSCLC were examined at the Nanjing Chest Hospital between January 2006 and May 2008. The cohort of patients included 80 (58.0 %) male and 58 (42.0 %) female subjects with a median age of 56 years (range 45“68 years). The clinical features of the patients are summarized in . Follow-up lasted through December 2012 with a median follow-up period of 22 months for living patients (range 3“80 months). PFS was defined as the time interval between the date of diagnosis and the date of disease relapse. OS was defined as the time interval between the date of diagnosis and the date of death.Clinical characteristics of NSCLC patients and controlsVariablesNSCLCControl P valueSubject no.13840Age year57.8?±?10.254.6?±?7.80.614Male/Female80/5826/140.325Histology?AC78?SCC60 AC adenocarcinoma SCC squamous cell carcinoma The diagnosis of lung cancer was made using various methods: sputum cytology fine-needle aspiration or bronchoscopy as dictated by the patient™s presentation. Pathologists interpreted the cytology or histology of tissue biopsy. Lung cancer was staged using a widely used classification system and the staging procedure included a clinical examination; CT of the chest abdomen and brain; abdominal ultrasonography; bone scanning; and positron emission tomography. The study protocol was approved by the ethics committee of Nanjing Chest Hospital. All patients provided written informed consent before enrollment. Measurement of serum KLK11 levels Serum samples from each individual were obtained at the time of diagnosis before any therapeutic measures were started (surgery chemotherapy or radiation). Samples were centrifuged at 1500—g for 10 min at ?4 °C. The supernatant was stored at ?80 °C for assessment of the levels of KLK11. The KLK11 concentration was determined by ELISA with the commercial KLK11 ELISA Ready-SET-Go kit (eBioscience San Diego CA). All samples were blinded to the technologists running the assays and the code was broken to the statisticians after the database was constructed. Statistical analysis Statistical software (SPSS for Windows version 18) was used for the analysis. Differences between independent groups were examined by the Mann“Whitney U test. To determine the diagnostic accuracy of KLK11 receiver operating characteristic (ROC) curves were retrieved from logistic regression analysis and the area under the curve (AUC) was calculated. Univariate survival analysis was performed using the Kaplan“Meier method and the log-rank test. Multivariate analysis was conducted to determine an independent impact on survival using the Cox proportional hazard method. P?<?0.05 was considered statistically significant. Results Comparison of serum KLK11 levels between NSCLC patients and controls As shown in Fig. 1 the concentration of KLK11 was significantly higher in patients with NSCLC (2.04?±?0.86 ng/ml) than in those with the controls (0.93?±?0.52 ng/ml) (P?<?0.01).Fig. 1Levels of KLK11 in NSCLC. Among 138 NSCLC patients the serum levels of KLK11 were 2.04?±?0.86 ng/ml which were significantly higher than 0.93?±?0.52 ng/ml in healthy controls (P?<?0.01) Diagnostic value of KLK11 in NSCLC A ROC curve analysis was carried out to assess the value of KLK11 in NSCLC. The area under the ROC curve was 0.892 (confidence interval (95 % CI) 0.841“0.942). With a cutoff point of 1.05 ng/ml which was defined as the normal value based on the mean value plus two standard deviation obtained from healthy controls serum KLK11 has a sensitivity of 65.9 % (91/138) a specificity of 82.5 % (33/40) an accuracy of 69.7 % (124/178) a positive predictive value of 92.9 % (91/98) and a negative predictive value of 41.3 % (33/80) (Fig. 2).Fig. 2ROC of KLK11 for the diagnosis of NSCLC. Serum levels of KLK11 among 138 NSCLC patients and 40 healthy controls were determined. The diagnostic potentials of KLK11 were assessed by ROC curves. The AUC value was 0.892 Relationship between serum KLK11 levels and clinicopathologic factors The relationships between KLK11 levels and clinicopathologic factors of lung cancer patients are shown in . The serum KLK11 levels did not differ significantly with age (P?=?0.569) sex (P?=?0.505) or histology (P?=?0.713). The levels of KLK11 were significantly correlated with tumor-node-metastasis (TNM) stage (P?=?0.000) lymph node metastases (P?=?0.000) and distant metastases (P?=?0.000).The clinicopathological factors of NSCLC and the association with KLK11 levelsFactorsnKLk11 (ng/ml) P- valueAge year0.569??60622.07?±?0.77?<60762.12?±?0.66Gender0.505?Male802.16?±?0.82?Female581.99?±?0.53Histology0.713?AC782.05?±?0.85?SCC602.01?±?0.53TNM stage0.000?I“II882.51?±?0.61?III“IV501.76?±?0.63Lymph node metastases0.000?Absent682.41?±?0.64?Present701.65?±?0.57Distant metastases0.000?Absent982.38?±?0.59?Present401.89?±?0.71 AC adenocarcinoma SCC squamous cell carcinoma Association of serum KLK11 levels with survival Finally we determined whether the baseline serum concentration of KLK11 would be a prognostic marker in NSCLC. The cutoff point of 1.05 ng/ml was selected to categorize patients as KLK11-high or low. Univariate analysis showed that serum KLK11 level was significantly correlated OS (P?=?0.002) and PFS (P?=?0.009) ().Univariate and multivariate analysis of KLK11 status with regard to PFS and OSVariablesPFSOSHR95 % CI P valueHR95 % CI P valueUnivariate analysis?KLK11 (Low vs. High)0.460.25“0.820.0090.360.19“0.690.002?Age (?60 vs. <60)1.230.67“2.280.5061.180.59“2.130.792?Gender (Male vs. Female)1.320.71“1.820.7821.190.69“1.980.673?Histology (AC vs. SCC)1.830.59“2.130.7921.340.65“1.980.546?Stage (I“II vs. III“IV)1.330.65“2.210.0010.931.09“3.440.025?Lymph node metastases (absent vs. present)1.421.04“1.940.2711.770.32“1.660.347?Distant metastases (absent vs. present)1.981.03“3.010.0391.871.04“2.990.075Multivariate analysis?KLK11 (low vs. high)0.530.29-0.970.0420.480.24-0.950.037?Age (?60 vs. <60)0.980.52-1.940.8341.061.28-3.010.128?Gender (male vs. Female)1.280.67-1.890.6721.140.46-2.140.542?Histology (AC vs. SCC)1.371.04-2.330.3151.260.64-2.560.424?Stage (I“II vs. III“IV)1.250.56-2.260.0011.961.02-3.770.043?Lymph node metastases (absent vs. present)1.130.81-1.570.1481.840.33-1.720.334?Distant metastases (absent vs. present)1.440.85-1.970.0981.890.99-2.350.051 HR hazard ratio CI confidence interval In multivariate analysis high KLK11 was found to be significantly associated with a longer PFS and OS (HR 0.53 and 0.48; P?=?0.042 and P?=?0.037 respectively). Kaplan“Meier survival curves (Fig. 3) further demonstrate that lung cancer patients with high KLK11 have substantially longer PFS and OS (P?<?0.05) compared to those with low KLK11 cancer. As expected disease stage was found to be strongly associated with decreased PFS and OS in both univariate and multivariate analyses (P?<?0.05).Fig. 3Kaplan“Meier survival curves for PFS and OS in patients with KLK11-high and -low NSCLC."

Lung_Cancer

"The categories were significantly associated with the sensitivity to Ad-REIC treatment (p<0.01). .0087900.t002 Ad-REIC sensitivity and categories based on predictive factors. (n) Category A (8) Category B (12) Category C (5) Highly sensitive (13) 8 5 0 Resistant in 20 MOI (12) 0 7 5 JNK and GRP78 expression in NSCLC cell lines treated with Ad-REIC A western blotting analysis demonstrated the significant expression of REIC/Dkk-3 protein in 14 NSCLC cell lines treated with Ad-REIC. In 9 cell lines infected with 20 MOI Ad-REIC treatment resulted in the phosphorylation of JNK and the up-regulation of GRP78 (b). In the other 8 cell lines which were relatively resistant to Ad-REIC the activation of JNK and GRP78 were observed at higher MOI values (100 and 200 MOI) (c). Effect of Ad-REIC on NSCLC tumors in a xenotransplantation model We investigated the effect of Ad-REIC on the growth of A549 cells in vivo. One week after transplantation when the tumor volume reached 50 to 100 mm3 1—109 plaque-forming units of Ad-REIC or Ad-LacZ in 100 µL of PBS or 100 µL of PBS alone were injected intratumorally. The tumors grew progressively in the PBS and Ad-LacZ treatment groups during the subsequent 24-day observation period. In contrast the tumor growth in the Ad-REIC treatment group was significantly (p<0.001 by repeated measurement ANOVA) suppressed during the observation period (ab). .0087900.g003 Anti-tumor effect of Ad-REIC treatment on A549 tumor growth in vivo. (a) The mean volume of the subcutaneous xenograft tumors was calculated for 5 mice in each group. A significant difference was observed between the results of Ad-REIC and Ad-LacZ treatment (*p<0.001 by repeated measurement of ANOVA). (b) Appearance of the tumors at the time of sacrifice after treatment with PBS Ad-LacZ and Ad-REIC. Discussion In the present study we found that Ad-REIC was directly effective in more than half of the NSCLC cell lines that were examined independent of its known driver alterations such as EGFR and KRAS mutations. An animal xenograft model also showed the therapeutic effect of Ad-REIC. The anti-tumor effect of Ad-REIC depends on ER-stress-mediated JNK activation loaded by the overproduction of REIC/Dkk-3 protein resulting in the induction of apoptosis [14] [20]. The activation of JNK which is an essential step in the induction of ER stress and apoptosis by Ad-REIC was observed at 20 MOI in NSCLC cell lines. On the other hand the anti-tumor effect of recombinant REIC/Dkk-3 protein was not observed as in other types of cancers that were previously examined. Originally REIC/Dkk-3 was identified as a secretory protein and was assumed to exert a physiological function but its cell surface receptor and its role as a secretory protein have not been identified. We defined 20 MOI as a low MOI value and 200 MOI as a high MOI value because the normal human fibroblast cell line OUMS-24 was not inhibited at 20 or 200 MOI of Ad-REIC whereas malignant cell lines were inhibited when the MOI value was elevated to 100 and 200 MOI in cell lines in which Ad-REIC had been ineffective at 20 MOI. In NSCLC Ad-REIC was effective at a low MOI value in more than half of the cell lines that were tested. Considering the result that 211H was inhibited only at a high MOI value Ad-REIC might be more effective in NSCLC than in mesothelioma. Patient selection based on the molecular characteristics of tumor cells is an important theme for maximizing the therapeutic benefit and minimizing adverse effects. For this purpose we focused on the GRP78 expression and CAR expression levels. GRP78 is a member of the Hsp70 family which serves as an ER stress-signaling regulator [21]. A previous study showed that the overexpression of GRP78 conferred resistance to a wide variety of chemotherapeutic agents in various kinds of cells [22]. We also showed that the acquired resistance clone of PC-3 cells to Ad-REIC established after repeated exposure to Ad-REIC exhibited a high expression level of GRP78 compared with parental PC-3 cells [13]. Theoretically Ad-REIC should be effective for tumor cells defined as Category A and not as effective for those defined as Category C. Although sensitive cells in Category B were identified all 8 cells in Category A responded to Ad-REIC treatment. These results suggested that the expression statuses of GRP78 and CAR in tumors might be useful as biomarkers for customized Ad-REIC therapy in NSCLC while further confirmation is needed by a large scaled investigation using various kinds of cell lines. As a recent topic of lung cancer treatment EGFR-TKIs have been shown to be effective for the treatment of EGFR-mutant NSCLCs. However acquired resistance to EGFR-TKIs after TKI treatment is a problem that needs to be overcome. In the current study our results showed that the effect of Ad-REIC against acquired EGFR-TKI-resistant cells was equal to that against the parental cells suggesting that Ad-REIC may be useful after the acquisition of resistance to EGFR-TKIs. Although adenovirus vectors carrying appropriate tumor suppressor genes such as REIC/Dkk-3 have great potential for cancer gene therapy they do not exhibit target specificity and therefore may also infect normal cells in the vicinity of cancer cells. The authors reported that the infection of normal human fibroblasts (NHF) with Ad-REIC did not cause the apoptosis of NHF itself but instead induced the production of interleukin (IL)-7. When Ad-REIC-infected NHF were mixed with untreated cancer cells and the mixture was transplanted into mice the growth of the cancer cells was significantly suppressed suggesting an indirect tumor-suppressive effect of Ad-REIC mediated by IL-7 [20]. These findings show that the mis-targeted infection of cancer stroma cells by Ad-REIC activates the immune system through the production of IL-7. In addition the authors reported that REIC/Dkk-3 protein played a cytokine-like role in monocyte differentiation into dendritic-cell-like features in vitro and that the infiltration of CD11c- and CD8-positive (dendritic and killer T cell markers respectively) cells was observed within the treated tumors in vivo. In the experiment using an orthotopic prostate tumor model with pre-established lung metastasis the number of metastatic lung tumors significantly decreased after the injection of Ad-REIC at the primary tumor site in addition to the inhibition of the growth of orthotopic prostate tumors suggesting that anti-cancer immune up-regulation by Ad-REIC treatment in primary tumor sites triggered anti-tumor effects even at distant tumor site [16]. These facts strongly suggest that REIC/Dkk-3 shows an indirect anti-tumor effect through the anti-tumor immune system that is an important factor in the treatment of metastatic disease. Because Ad-REIC has both direct and indirect effects on cancer therapy it may become a powerful therapeutic option as a œone-bullet two-arms anti-cancer agent especially for NSCLCs which often metastasize to other ans. In regards to clinical usage because our data suggest that CAR and GRP78 expression statuses in tumor cells predict the responsiveness of Ad-REIC treatment Ad-REIC treatment should be preferentially performed for patients who are categorized as high sensitive group in early phase of treatment with low dose Ad-REIC. For patients whose tumor cells reveal intermediate or poor effectiveness with low dose Ad-REIC it should be late phase in their treatment with high dose Ad-REIC. For these patients cost effectiveness for treatment and clinical outcome should be carefully considered. As for administration strategy local administration might be preferable rather than systemic administration to minimize the adverse effect in clinical situations. We previously confirmed in mouse model that Ad-REIC could be widely distributed in the bodies after intratumoral local administration and local administration was effective not only directly but also indirectly through the immune system effect [16] [23]. In addition intrapleural local administration could be another administration strategy for the patients with malignant pleural effusions. It has been reported that the intrapleural administration of adenoviral-mediated gene therapy is a useful approach for the generation of anti-tumor immune responses in malignant mesothelioma and metastatic pleural effusion in several clinical trials [24] [25]. In conclusion we demonstrated that Ad-REIC induced JNK activation and subsequent apoptosis in NSCLC cells irrespective of the type of known molecular alterations or the sensitivity to EGFR-TKI. The present study suggests that Ad-REIC has a therapeutic potential for NSCLC and the expression statuses of GRP78 and CAR may be a predictor of Ad-REIC therapy. Supporting Information Figure S1 The heatmap image of mRNA expression of REIC/Dkk-3 gene."

Lung_Cancer

"Chinese women. International Journal of Cancer40: 604“609 37 Wu-WilliamsAH DaiXD BlotW XuZY SunXW et al (1990) Lung cancer among women in north-east China. Br J Cancer62: 982“9872257230 38 ZhongL GoldbergMS GaoYT JinF (1999) Lung cancer and indoor air pollution arising from Chinese-style cooking among nonsmoking women living in Shanghai China. Epidemiology (Cambridge Mass)10: 488“494 PLoS One one 1932-6203 Public Library of Science San Francisco USA 24586842 3934888 PONE-D-13-30257 .0089518 Research Biology Genetics Genetic mutation Mutation types Cancer genetics Medicine Clinical research design Retrospective studies Diagnostic medicine Pathology Clinical pathology Test evaluation Oncology Cancer detection and diagnosis Cancer screening Cancer treatment Chemotherapy and drug treatment Clinical trials (cancer treatment) Cancers and neoplasms Lung and intrathoracic tumors Non-small cell lung cancer Oncology agents Clinical Validation of a PCR Assay for the Detection of EGFR Mutations in Non“Small-Cell Lung Cancer: Retrospective Testing of Specimens from the EURTAC Trial EGFR Mutation Testing in NSCLC in EURTAC Trial Benlloch Susana 1 * Botero Maria Luisa 1 Beltran-Alamillo Jordi 1 Mayo Clara 1 Gimenez-Capit¡n Ana 1 de Aguirre Itziar 2 Queralt Cristina 2 Ramirez Jose Luis 2 Cajal Santiago Ramn y. 1 6 Klughammer Barbara 3 Schlegel Mariette 3 Bordogna Walter 3 Chen David 4 Zhang Guili 5 Kovach Barbara 5 7 Shieh Felice 5 7 Palma John F. 5 Wu Lin 5 Lawrence H. Jeffrey 5 7 Taron Miquel 1 2 1 Pangaea Biotech SL Barcelona Spain 2 Medical Oncology Service-ICO Hospital Germans Trias i Pujol Badalona Spain 3 F. Hoffmann-La Roche Basel Switzerland 4 Genentech South San Francisco California United States of America 5 Roche Molecular Systems Pleasanton California United States of America 6 Pathology Department Vall d'Hebron University Hospital Universidad Autnoma de Barcelona Barcelona Spain 7 Roche Molecular Systems Pleasanton California United States of America Minna John D. Editor Univesity of Texas Southwestern Medical Center at Dallas United States of America * E-mail: sbenllochpangaeabiotech.com Competing Interests: BK MS WB DC GZ JFP LW are all current employees of Roche. BK DC JFP and LW have stock holdings in Roche. BK and HJL are former Roche employees and HJL has stock in Roche and has served as a paid consultant. FS was a paid consultant to Roche. SB JBA CM AGC are current employees of Pangaea Biotech. MLB is a former Pangaea employee. MT and SRyC have stock holdings in Pangaea Biotech. IdA CQ JLR are current employees of Medical Oncology Service-ICO Hospital Germans Trias i Pujol. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. Conceived and designed the experiments: MT HJL. Performed the experiments: CM AGC JBA IdA CQ JLR. Analyzed the data: SB DC GZ CM AGC JBA IdA CQ JLR FS LW JFP HJL MT. Contributed reagents/materials/analysis tools: SB MLB JBA CM AGC IdA CQ JLR SRyC BK MS WB DC GZ BK FS LW JFP HJL MT. Wrote the paper: SB MLB JBA CM AGC IdA CQ JLR SRyC BK MS WB DC GZ BK FS LW JFP HJL MT. 2014 25 2 2014 9 2 e89518 23 7 2013 21 1 2014 2014 Benlloch et al This is an open-access distributed under the terms of the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original author and source are credited. The EURTAC trial demonstrated that the tyrosine kinase inhibitor (TKI) erlotinib was superior to chemotherapy as first-line therapy for advanced non-small cell lung cancers (NSCLC) that harbor EGFR activating mutations in a predominantly Caucasian population. Based on EURTAC and several Asian trials anti-EGFR TKIs are standard of care for EGFR mutation-positive NSCLC. We sought to validate a rapid multiplex EGFR mutation assay as a companion diagnostic assay to select patients for this therapy. Samples from the EURTAC trial were prospectively screened for EGFR mutations using a combination of laboratory-developed tests (LDTs) and tested retrospectively with the cobas EGFR mutation test (EGFR PCR test). The EGFR PCR test results were compared to the original LDT results and to Sanger sequencing using a subset of specimens from patients screened for the trial. Residual tissue was available from 487 (47%) of the 1044 patients screened for the trial. The EGFR PCR test showed high concordance with LDT results with a 96.3% overall agreement. The clinical outcome of patients who were EGFR-mutation detected by the EGFR PCR test was very similar to the entire EURTAC cohort. The concordance between the EGFR PCR test and Sanger sequencing was 90.6%. In 78.9% of the discordant samples the EGFR PCR test result was confirmed by a sensitive deep sequencing assay. This retrospective study demonstrates the clinical utility of the EGFR PCR test in the accurate selection of patients for anti-EGFR TKI therapy. The EGFR PCR test demonstrated improved performance relative to Sanger sequencing. This study was funded by Roche. The funders contributed to the study design data collection analysis decision to publish and preparation of the manuscript. Introduction The efficacy of many novel targeted cancer therapies can be predicted by the detection of specific biomarkers in the tumor. The FDA has indicated that if the identification of a specific biomarker is required for the safe and efficacious administration of a drug a well-validated FDA approved companion diagnostic assay is required for that drug. The optimal approval path for a new targeted therapy and its companion diagnostic is a parallel clinical development process that involves clinical trials for the investigational agent where the investigational diagnostic test is used to either select patients for the trials or to predict response to treatment and ends ideally with simultaneous health authority approval of the drug and the companion diagnostic. Successful examples of this include the co-development (and co-approval) of the BRAF inhibitor vemurafenib and its companion diagnostic BRAF V600E mutation assay for BRAF-mutant metastatic melanoma[1] and the ALK inhibitor crizotinib and its companion diagnostic ALK fusion gene test in advanced ALK-fusion positive non-small cell lung cancer (NSCLC) patients.[2] [3] [4] However in some cases predictive biomarkers for a targeted therapy are not recognized until after the drug is first approved. As an example the anti-EGFR antibody cetuximab was first approved in the US for the treatment of metastatic colorectal cancer in 2004. Numerous retrospective and prospective trials subsequently revealed that tumors harboring KRAS mutations were very unlikely to respond to cetuximab. In July 2009 FDA required labeling changes for cetuximab and another anti-EGFR antibody panitumumab requiring that the indications and usage state there was no treatment benefit with the drugs for patients whose tumors had KRAS mutations in codon 12 or 13 at a time when there were no FDA-approved diagnostic assays for KRAS mutations.[5] Only later in July 2012 did a KRAS mutation assay receive FDA approval based on the results of a prospective randomized trial highlighting the challenges of retrospectively validating a companion diagnostic assay after the pivotal drug trials have been completed.[6] The anti-EGFR TKI erlotinib was initially approved for all patients with advanced NSCLC who had progressed on first-line chemotherapy. A number of subsequent studies determined that patients with EGFR-mutant NSCLC had a high likelihood of responding to these TKI leading to trials in the first-line setting for EGFR-mutant cancer.[7] [8] [9] [10] [11] [12] [13] Four prospective randomized clinical trials studied in Asian populations demonstrated that erlotinib and gefitinib resulted in improved progression-free survival compared to chemotherapy for first line therapy in NSCLC patients with EGFR mutations.[7] [8] [9] [13] Other clinical studies in mixed ethnicity cohorts have concluded with similar results.[10][12] The EURTAC trial was a randomized phase 3 trial to assess the safety and efficacy of erlotinib compared with standard platinum-based chemotherapy for first-line treatment of a patient population with advanced EGFR-mutation detected NSCLC in a largely Caucasian population of European patients. Erlotinib-treated patients experienced significant improvements in median PFS (9.7 months vs. 5.2 months) compared to chemotherapy. Patients on the erlotinib arm also had a considerably higher percentage of responses (58% vs. 15%) in the intent-to-treat population.[11] This trial has been submitted for first line indication of erlotinib in EGFR mutated NSCLC patients. The majority of activating EGFR mutations are located in exons 19 (45%) and 21 (40“45%).[14] [15] [16] [17] [18] [19] [20] Guidelines from organizations such as ASCO CAP/AMP and NCCN recommend the use of anti-EGFR TKIs as first-line therapy in patients with EGFR-mutant advanced NSCLC based on the results of these pivotal clinical trials. [21] [22] [23] Recent recommendations by CAP/IASLC/AMP advise the identification of EGFR mutations present at >1% of which exon 19 deletions and an exon 21 mutation (L858R) account for greater than 90% of all mutations.[24] None of the guidelines specify the testing method to be used however the cobas EGFR Mutation test is CE-IVD approved and is recently FDA approved.[25] Here we present the retrospective analysis of a clinical validation study of the EGFR PCR test on a subset of lung cancer specimens from patients screened for the EURTAC trial. The EGFR PCR test demonstrated improved sample workflow relative to the LDTs used in the EURTAC trial enabling EGFR mutation screening in a single assay with a one-day turn-around time. The EGFR PCR test showed superior sensitivity and specificity compared with conventional Sanger sequencing. Methods The major study objectives were 1) to correlate the clinical outcomes (PFS BORR) from the subgroup of available samples tested by the EGFR PCR test to the results from the entire EURTAC population and 2) to compare the analytic performance of the EGFR PCR test to that of the original LDT and Sanger sequencing using massively parallel pyrosequencing (MPP) to resolve discrepancies observed between the other 3 testing methods. In the EURTAC trial1044 patients from hospitals in France Italy and Spain were screened using the LDT. For this study all samples were retrospectively analyzed under IRB approval from Copernicus IRB (00001313). Site specific IRB approval from each clinical site and written consent from all patients was obtained prior to the study conduct phase of NCT00446225.[11] [26] In 487 cases residual specimens were available for retesting with the EGFR PCR test (Figure 1). A single 5 µm section with at least 10% tumor content from each of the 487 specimens was used for the EGFR PCR test. Genomic DNA from existing eluate or extracted from additional sections was tested on Sanger sequencing and MPP. Table 1 lists the demographics of the patients screened for the EURTAC trial by the LDT sub-categorized by patients tested or not tested by the EGFR PCR test. Patients enrolled in the EURTAC trial were selected using a laboratory-developed test validated by the Laboratory of Oncology (ICO-Hospital Germans Trias i Pujol Badalona Spain) consisting of three methodologies.[26] In this study a single PCR-based assay for detecting EGFR mutations was used. Details of the analytical performance of this assay have been described previously.[27] .0089518.g001 Figure 1 Flow of samples through the study."

Lung_Cancer

"The 24-week metrics (albeit with higher c-index point estimate) were not meaningfully better than the 12-week metrics. None of the metrics did particularly well for breast cancer. Conclusion Alternative cut points to RECIST standards provided no meaningful improvement in OS prediction. Metrics assessed at 12 weeks have good predictive performance. J Thorac Oncol J Thorac Oncol JTO Journal of Thoracic Oncology 1556-0864 1556-1380 Lippincott Williams & Wilkins 24787965 4132045 00005 10.1097/JTO.0000000000000157 Original s Translational Oncology A Comparison of Immunohistochemical Assays and FISH in Detecting the ALK Translocation in Diagnostic Histological and Cytological Lung Tumor Material Le Quesne John MA (Cantab) PhD MBBS FRCPath * Maurya Manisha PhD   Yancheva Slaveya G. FRCPath * O™Brien Mary MD ¡ Popat Sanjay FRCP PhD ¡ Wotherspoon Andrew C. MBBCh FRCPath § de Castro David Gonzalez PhD FRCPath   Nicholson Andrew G. MBBS DM FRCPath * *Department of Histopathology Royal Brompton and Harefield NHS Foundation Trust London;  Centre for Molecular Pathology The Royal Marsden Hospital Sutton Surrey; ¡Department of Oncology The Royal Marsden Hospital; and §Department of Histopathology Royal Marsden Hospital Chelsea London United Kingdom. Address for correspondence address: Andrew G. Nicholson MBBS DM FRCPath Department of Histopathology Royal Brompton Hospital Sydney St London SW3 6NP United Kingdom. E-mail: a.nicholsonrbht.nhs.uk. 6 2014 30 5 2014 9 6 769 774 Copyright 2014 by the International Association for the Study of Lung Cancer 2014 This is an open-access distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivitives 3.0 License where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially. Introduction: Detection of the ALK rearrangement in a solid tumor gives these patients the option of crizotinib as an oral form of anticancer treatment. The current test of choice is fluorescence in situ hybridization (FISH) but various cheaper and more convenient immunohistochemical (IHC) assays have been proposed as alternatives. Methods: Fifteen FISH-positive cases from patients seven with data on crizotinib therapy and clinical response were evaluated for the presence of ALK protein using three different commercially available antibodies: D5F3 using the proprietary automated system (Ventana) ALK1 (Dako) and 5A4 (Abcam). A further 14 FISH-negative and three uncertain (<15% rearrangement detected) cases were also retrieved. Of the total 32 specimens 17 were excisions and 15 were computed tomography-guided biopsies or cytological specimens. All three antibodies were applied to all cases. Antibodies were semiquantitatively scored on intensity and the proportion of malignant cells stained was documented. Cutoffs were set by receiver operating curve analysis for positivity to optimize correct classification. Results: All three IHC assays were 100% specific but sensitivity did vary: D5F3 86% ALK 79% 5A4 71%. Intensity was the most discriminating measure overall with a combination of proportion and intensity not improving the test. No FISH-negative IHC-positive cases were seen. Two FISH-positive cases were negative with all three IHC assays. One of these had been treated with crizotinib and had failed to show clinical response. The other harbored a second driving mutation in the EGFR gene. Conclusions: IHC with all three antibodies is especially highly specific (100%) although variably sensitive (71%-86%) specifically in cases with scanty material. D5F3 assay was most sensitive in these latter cases. Occasional cases are IHC-positive but FISH-negative suggesting either inaccuracy of one assay or occasional tumors with ALK rearrangement that do not express high levels of ALK protein. Pulmonary adenocarcinoma ALK Immunohistochemistry Fluorescence in situ hybridization Crizotinib OPEN-ACCESS TRUE Rearrangements of the anaplastic lymphoma kinase (ALK) gene drive the malignant phenotype in 3% to 7% of primary lung adenocarcinomas.1“5 The resulting fusion protein most often a fusion with echinoderm microtubule-associated protein-like 4 (ELM4) has a constitutively active tyrosine kinase domain. The small molecule drug crizotinib is a specific inhibitor of this kinase6 and cases with the rearrangement respond to crizotinib treatment.7 Therefore accurate rapid and inexpensive identification of tumors growing under the influence of translocated ALK is needed. Currently the only test approved by the FDA is fluorescence in situ hybridization (FISH) using œbreak-apart probes (Vysis Abbott Molecular Abbott Park IL). This test is regarded as the œgold standard for detection of re-arrangements and is recommended by CAP/International Association for the Study of Lung Cancer/AMP.8 However FISH is technically demanding expensive and many diagnostic laboratories lack either the expertise or the facilities to perform the test. Even in ideal circumstances the results are often difficult to interpret requiring the scrutiny of large numbers of individual cells by a highly experienced diagnostician. Furthermore there are rare circumstances (such as small intrachromosomal inversion) in which the FISH test is negative but the tumor nevertheless expresses EML4-ALK fusion protein.59“11 A cheaper and potentially more widely applicable method is immunohistochemistry (IHC); indeed overexpression of ALK protein has been used in the diagnosis of anaplastic large-cell lymphoma for many years. Although early studies in lung cancer lacked sensitivity45 more recent studies have shown greater specificity and sensitivity8“11 and recent international guidelines (CAP/International Association for the Study of Lung Cancer/AMP) have recommended that if clinically validated IHC may be used as a screening test for FISH testing.8 However there have been few comparative studies on the most appropriate antibody to use. The aim of this study was therefore to compare three different immunohistochemical assays two being routine methods using antibodies widely used in the diagnosis of lymphoma with the third being a proprietary system including signal amplification that is currently being promoted as an alternative to FISH (Ventana). We also evaluated the relationships between ALK rearrangement as detected by FISH IHC and patient response to therapy. MATERIALS AND METHODS Clinical Samples The diagnostic archives from the Royal Brompton and Harefield NHS Foundation Trust and Royal Marsden hospitals from 2007 onwards were reviewed to identify cases with a diagnosis of lung adenocarcinoma that tested positive for an ALK rearrangement (>15% positive cells) and a randomly selected complementary group of cases with a normal ALK locus for comparison. We had been testing all primary lung tumors regardless of stage as part of a feasibility study which led to a large number of early stage cases being included. More recently our current policy is only to test advanced cases of non-squamous non“small-cell carcinoma using IHC screening with confirmatory FISH as per recently published guidelines.8 The cases under study are summarized in . TABLE 1. Summary Data of All Cases Included in the Study Paraffin blocks from a total of 32 diagnostic cases were retrieved; 15 of these had tested positive for the ALK rearrangement by FISH three were uncertain (with <15% of cells showing rearrangement) and the remaining 14 cases were negative. All but two blocks dated from 2011 or later. Seventeen cases were blocks from tumor excisions (six of these were FISH positive) and the remainder were cytological or core biopsy/endobronchial ultrasound samples. Data on treatment with crizotinib and response were retrieved from patient records. Cases with at least partial response to treatment defined according to the Response Evaluation Criteria in Solid Tumors criteria12 (i.e. at least 30% decrease in the sum of the longest diameters of target lesions) were designated as œresponsive. The study was evaluated and classified as a service evaluation by the Imperial College Heads of Consortia and as such was exempt from Research Ethics Committee review. Fluorescent In Situ Hybridization Unstained 2 ?m FFPE sections were put through deparaffinization and protease pretreatment steps before being denatured and hybridized overnight with the commercially available Vysis ALK dual color break apart probe (Abbott Molecular). Tissue sections then underwent SSC washes and were mounted in 4'6-diamidino-2-phenylindole for nuclei counterstaining. Results were analyzed and interpreted in accordance with probe manufacturer™s instructions. Non-rearranged ALK showed as fused (yellow) signals. Rearranged ALK appeared as split 3? (red) and 5? (green) signals or an isolated 3? (red) signal. The recommended cutoff of 15% was used to interpret samples as positive or negative for ALK rearrangements in 200 nuclei. Immunohistochemistry An additional five sections were cut per case. Three were used for the immunohistochemical assays and the remaining two for negative controls. Immunohistochemical assays were optimized using the monoclonal antibodies D5F3 (Ventana) ALK1 (Dako) and 5A4 (Abcam). The D5F3 assay was performed using the Ventana autostainer and a tyramide amplification step as specified in the manufacturer™s protocol. The other assays were performed using a Dako autostainer with conventional polymer-based diaminobenzidine staining (no tyramide amplification). Details of the antibodies and conditions employed are given in Table 2. TABLE 2. Immunohistochemical Assay Conditions Used Scoring Immunohistochemically stained sections were examined without knowledge of FISH status by two pathologists independently. Scores for proportion and intensity of immunohistochemical staining were assigned by consensus. The predominant intensity of staining was recorded on a scale of 0“3 (0 = negative 1 = weak 2 = moderate 3 = strong). As the Ventana stain was more intense due at least partly to the signal amplification step the visual cutoffs for intensity scoring with this antibody were different (e.g. a œmoderate degree of intensity seen with the Ventana stain would usually be interpreted as œstrong on a section stained with 5A4). The proportion of malignant cells staining positive was recorded as per œAllred estrogen receptor scoring in breast cancer on a scale of 0“5 (0 = 0% 1 ? 1% 2 = 1“10% 3 = 11“33% 4 = 34“66% and 5 ? 66%). A composite score (intensity + proportion) was also derived. Statistical Analysis Statistical analyses were performed using the STATA/IC package. RESULTS Fluorescence In Situ Hybridization Slides were scored according to the manufacturer™s recommendations. Representative FISH images are shown in Figure 1A. The 15 positive cases all showed greater than 15% cells with rearranged ALK genes. Three cases were classified as œindeterminate; these were all scanty biopsy or cytological samples with 10% to 15% of positively rearranged FISH signals. Seventeen further cases were FISH negative. FIGURE 1. (A) Representative fluorescence in situ hybridization images showing normal fused signals (neg) and nuclei with multiple separated red signals (pos). (B) Three representative excision specimens of adenocarcinoma. Case 1 is negative with all three immunohistochemical assays; the D5F3 assay shows relatively high background presumably because of the tyramide signal amplification (TSA) step. Cases 2 and 3 are positive with all three immunohistochemical assays with clear cytoplasmic staining. The markedly reduced signal seen with the 5A4 and ALK1 assays in case 2 was typical and again probably related to the absence of tyramide amplification. Case 3 demonstrates that occasional cases show strong staining using the non-TSA assays. Immunohistochemistry No signal was observed in negative controls. The intensity of staining between the three antibodies varied (Fig. 1B). IHC was impossible to assess in three cases with very scanty material (two FISH negative and one FISH positive). "

Lung_Cancer

"Validation of samples The primary aim of this scheme was to develop a flexible scalable EQA scheme designed to assess issues related to techniques and minimum detection limits used in standard laboratory practice focusing exclusively on the analytical (that is sample processing genotyping) and reporting phases (interpretation of the results in relation to the clinical context). To enable this and to avoid the significant challenges of sample heterogeneity in real tissue samples 20 artificial materials were used composed of formalin-fixed paraffin-embedded (FFPE) cell line samples. These EQA materials were designed to mimic real tissue samples as closely as possible and contained homogenous mixtures of mutant vs wild-type cell lines at a range of different allelic ratios. The paraffin blocks were cut and 10??m sections placed in eppendorf tube at the Pathology department of the VU University Medical Centre in Amsterdam The Netherlands by Dr Erik Thunnissen. H&E (4??m) sections were used to estimate the number of tumour cells. In each EQA sample section at least 200 nuclei were present (usually >300) roughly mimicking the amount of cells from a small NSCLC biopsy. For each EQA sample one 10-?m-thick section was sent by EMQN to each of the three validating laboratories for mutational analysis in a blinded fashion. Different sections from the block were analysed for EGFR mutation status to ensure that the mutation was homogeneously represented within each block. The validating laboratories independently analysed the samples by using three different approaches: direct sequencing of the PCR product for exons 18“21 mutations; fragment analysis for exon 19 deletions and an allelic discrimination-based real-time PCR assay for the L858R mutation in exon 21; and the Therascreen EGFR RGQ kit (Qiagen Hilden Germany) reporting the results directly to the EMQN. The allelic ratios of mutations in each sample used in rounds 2 and 3 were accurately quantified by a commercial sponsor (Horizon Diagnostics Cambridge UK) using droplet digital PCR (ddPCR) on a BioRad QX100 (Hercules CA USA) platform. Genomic DNA (gDNA) was extracted from FFPE sections on the Promega (Madison WI USA) Maxwell System using the Maxwell 16 FFPE Plus LEV DNA purification kit according to the manufacturer's protocol. Quantification was performed using a Promega QuantiFluor dsDNA assay kit according to the manufacturer's protocol. ddPCR was performed using Taqman custom SNP 40 — primer/probe assays (Life Technologies Carlsbad CA USA) to assess the frequency of each mutation with the exception of the p.(E746_A750) assay which was designed in-house. DNA (40?ng) was added to each ddPCR reaction. Reactions were performed in quadruplicate and droplets were generated using a Droplet Generator according to the manufacturer's instructions. PCR was performed on a standard thermocycler using previously optimised assay-specific cycling conditions. Droplets were analysed using a QX100 Droplet Reader as described in the manufacturer's instructions. Data from at least 45?000 useable droplets were collected for each sample. Formalin-fixed paraffin-embedded reference standards (Horizon Diagnostics) were included as assay controls. Registration of participant laboratories and shipment of samples Laboratories that performed EGFR mutational analysis were invited to participate in the EQA via an open call from the EMQN in conjunction with the ESP ETOP and ESMO. Participating laboratories registered via the EMQN website (European Molecular Genetics Quality Network (EMQN) 2014) and were requested to perform DNA extraction and analysis using their routine method. In each round 10 samples (one 10-?m-thick section for each) with accompanying mock clinical referral information were sent to participating laboratory. Each laboratory was identified only by a unique EMQN ID code to avoid exchange of information between participants and minimise bias in the results' interpretation process. The laboratories were given 8 weeks to complete their analyses and to submit the results of genotyping to the EMQN website. The centres were requested to provide information on the technique used for mutational analysis and metrics relating to their experience of performing EGFR mutational analyses. Evaluation of results The scheme included three rounds: the first was restricted to a maximum of 30 labs to establish proof of principle and validate the materials. A subsequent second round of the scheme was anised with no restriction on participation. Laboratories that failed the second round were provided with another set of samples in a restricted third round. The steering group evaluated the results according to a pre-defined scoring system. The scoring system assigned two points to correct genotype and zero points to false-positive or -negative results (). Errors in mutation nomenclature that might lead to misinterpretation of the results (for example stating ˜deletion' without specifying the exon in which the deletion occurs) were assigned 1.50 points. This deduction was applied only once for each center generally to the first sample for which the error was found. One point was awarded for cases in which the genotype was mispositioned or miscalled: this error sometimes occurs with exon 19 deletions for which it might be difficult to define the precise base or amino acid in which the deletion starts or ends. If a test failed giving no result on the sample (analytical failure) then the lab received 1.00 point for that sample. The threshold to pass the EQA was set at a total score for the 10 samples of ?18 out of 20 (Thunnissen et al 2011) “ laboratories with a genotyping score <18 were classified as poor performers (applied to rounds 2 and 3 only). Performance in the assessment of clinical interpretation and reporting did not contribute to poor performance. Results Selection of the samples for the EQA The first step of the EQA scheme was the selection and the validation of the samples. Twenty materials were manufactured by Dr Thunnissen by mixing four lung cancer cell lines (A549 EGFR wild type) H1650 (EGFR p.(E746_A750del) H1975 (EGFR p.(T790M) p.(L858R)) and SW48 (p.G719S). Cell lines with mutations were serially diluted into A549 or SIHA cells at different ratios relevant to establishing the analytical sensitivity of the tests used by labs. Each material was validated in three different reference laboratories using different techniques to confirm the genotype and the results showed that the mutations were detectable at all the designated ratios dependent on the technology used (). A good yield of gDNA was obtained from all the samples. In addition there was complete concordance on the EGFR mutational status of the selected specimens and therefore all were selected for use in the quality assessment scheme with samples A1“A10 used for the pilot and B1“10 and C1“C10 in subsequent rounds 2 and 3. To accurately establish quantitative measurements of the allelic frequencies of the EGFR mutations all 10 EQA samples (; samples B/C1“B/C10) used in rounds 2 and 3 were analysed on a ddPCR platform (BioRad QX100). Three of the samples had allelic frequencies higher than expected (C3 C8 and C9) two were lower (C2 and C10) and in one (C5) it was not possible to establish the true value due to insufficient availability of sample material (). First round proof of principle pilot scheme Twenty-nine laboratories registered from 13 countries and 25 participated in the pilot EQA scheme (4 labs withdrew due to customs sample importation problems) which was run in fourth quarter of 2011. A set of 10 samples were sent to the laboratories (; samples A1“A10). All the participating laboratories submitted results within the 8-week time frame. The main methodology used by the participants was PCR/sequencing (n=10 laboratories; 34%) and real-time PCR (n=10; 34%) (). Two analytical errors (false-negative results) were observed. A further five laboratories made process errors (sample swaps) that resulted in an additional 24 genotype errors. In all cases the genotypes were correct but reported for the wrong sample. Therefore 92% of the false-negative results were concentrated in five laboratories. No false-positive results were reported. The materials performed well and there were no analytical test failures."

Lung_Cancer

"Mesothelin; MTB: Mycobacterium tuberculosis; Hsp: Heat shock protein; i.p.: Intraperitoneal; i.d.: Intradermal; BMDCs: Bone marrow-derived dendritic cells; APCs: Antigen-presenting cells; PBMCs: Peripheral blood mononuclear cells; PBLs: Peripheral blood leukocytes; LPS: Lipopolysaccharide; H&E: Haematoxylin and eosin; PFA: Paraformaldehyde; DAB: Diaminobenzidine; mAb: monoclonal antibody. Competing interests The authors declare that they have no competing interests. Authors™ contributions JY played a role in the design of the experiments acquisition analysis and interpretation of the data and writing the manuscript. PR JN YY NHA MN GJ-M XT SK HC PU BF TC and PL participated in the performance of experiments. SK and TB were involved in design of the experiments. RB was involved in data analysis. ER was involved in setting up murine ovarian cancer model. SO provided the murine ovarian cancer model. NS provided the plasmid that encodes an scFv fragment specific to MSLN and the recombinant P4 scFv protein. GD NS and SO gave constructive input on experimental design and data analysis. JG played a role in conception and design of the fusion protein. MP and JG were involved in the conceptualization and design of the study analysis and interpretation of datasets and in writing the manuscript. All authors read and approved the final manuscript. Supplementary Material Additional file 1: Figure S1 scFvMTBHsp70 binds to 40L mesothelioma cells. 40L cells were stained with scFvMTBHsp70 or MTBHsp70 followed by mouse anti-MTBHsp70 and Donkey anti-mouse Alexa Fluor 594. Cells were observed using a Nikon Eclipse TiE fluorescence microscope. A Representative pictures from three independent experiments. Scale bar 10 ?m. B Images were analyzed using the NIS-Elements AR Microscope Imaging Software. Mean Fluorescence Intensity was analyzed using ImageJ. P values were determined using One-Way ANOVA followed by Turkey™s multiple comparison tests. ****p?<?0.0001. Click here for file Additional file 2: Figure S2 scFvMTBHsp70 or MTBHsp70 plus P4 scFv treatment does not lead to infiltration of inflammatory cells into abdominal or intestinal mesothelial tissues. Samples of abdominal wall and intestine were prepared from C57BL/6 mice that had previously received multiple i.p. injections of scFvMTBHsp70 MTBHsp70 plus P4 scFv or saline as described in the Methods section. Sections of these tissues were stained with H&E and images were acquired on a Zeiss Axio A1 microscope. Representative images from 3 animals per treatment group are shown. No detectable level of mononuclear cell or granulocyte infiltrate within mesothelial tissues was seen in any sampled tissues. Scale bar 20 ?m. Click here for file Additional file 3: Figure S3 scFvMTBHsp70 treatment does not affect numbers of tumor-infiltrating CD8+ or Foxp3+ T cells. (A) Representative images of intratumoral CD8+ and Foxp3+ T cells from saline (n?=?3) scFvMTBHsp70 (n?=?3) or MTBHsp70 plus P4 scFv (n?=?3) -treated mice. Mouse spleen sections were used as positive controls: CD8+ and Foxp3+ T cells are clearly evident in the sections. Scale bar 20 ?m. (B) Numbers of CD8+ and Foxp3+ cells were quantified from 3“5 randomized fields. Click here for file Additional file 4: Figure S4 Validation of in vivo depletion of CD8+ cells in FVB/NJ mice. Mice were injected i.p. with 200 ?g of anti-CD8 mAb or an isotype-matched irrelevant rat IgG2a as described in Methods. All the mice were bled from the tail vein and the depletion of CD8+ cells was examined by flow cytometry analysis of peripheral blood cells stained with fluorophore-conjugated anti-CD8 on days 7 and 28 after tumor inoculation. (A) Representative results of flow analyses on 10 mice per group and reported as the percentage of CD8+ cells in lymphocytes. (B) CD8+ cells in the mice treated with isotype IgG2a or anti-CD8 mAb were compared. ***p< 0.001. Click here for file Acknowledgments This manuscript is dedicated to the memory of Janet Gelfand a victim of ovarian cancer. The authors gratefully acknowledge the continuing support for this work from the Edmund C. Lynch Jr. Cancer Fund Arthur Luxenberg Esq. Perry Weitz Esq. and the VIC Mesothelioma Research and Resource Program at MGH and the Friends of VIC Fund. PU and NHA were supported by the Prof. Dulcie V. Coleman Studentship at Imperial College London. We thank Oliver Mitchell John Cao Lujia Zhou Rumbidzai Mushavi and Sayinthen Vivekanantham for their technical assistances Dr. Yuhui Huang for his useful comments Michael Waring Dr. Michael Santuosuosso and Dr. Ravi Mylvaganam for their technical advice Dr. Musie Ghebremichael for his advice in statistical analysis and Mahnoor Valibhoy for her assistance with the schematic figure. Banchereau J Palucka AK Dendritic cells as therapeutic vaccines against cancer Nature reviews Immunology 2005 5 296 306 10.1038/nri1592 15803149 Mellman I Coukos G Dranoff G Cancer immunotherapy comes of age Nature 2011 480 480 489 10.1038/nature10673 22193102 Topalian SL Weiner GJ Pardoll DM Cancer immunotherapy comes of age J Clin Oncol 2011 29 4828 4836 10.1200/JCO.2011.38.0899 22042955 Kantoff PW Higano CS Shore ND Berger ER Small EJ Penson DF Redfern CH Ferrari AC Dreicer R Sims RB Xu Y Frohlich MW Schellhammer PF IMPACT Study Investigators Sipuleucel-T immunotherapy for castration-resistant prostate cancer The New England journal of medicine 2010 363 411 422 10.1056/NEJMoa1001294 20818862 Chambers JD Neumann PJ Listening to provenge“what a costly cancer treatment says about future medicare policy The New England journal of medicine 2011 364 1687 1689 10.1056/NEJMp1103057 21470004 Chang K Pastan I Molecular cloning of mesothelin a differentiation antigen present on mesothelium mesotheliomas and ovarian cancers Proc Natl Acad Sci USA 1996 93 136 140 10.1073/pnas.93.1.136 8552591 Argani P Iacobuzio-Donahue C Ryu B Rosty C Goggins M Wilentz RE Murugesan SR Leach SD Jaffee E Yeo CJ Cameron JL Kern SE Hruban RH Mesothelin is overexpressed in the vast majority of ductal adenocarcinomas of the pancreas: identification of a new pancreatic cancer marker by serial analysis of gene expression (SAGE) Clinical cancer research: an official journal of the American Association for Cancer Research 2001 7 3862 3868 11751476 Ho M Bera TK Willingham MC Onda M Hassan R FitzGerald D Pastan I Mesothelin expression in human lung cancer Clinical cancer research: an official journal of the American Association for Cancer Research 2007 13 1571 1575 10.1158/1078-0432.CCR-06-2161 17332303 Tang Z Qian M Ho M The role of mesothelin in tumor progression and targeted therapy Anti-cancer agents in medicinal chemistry 2013 13 276 280 10.2174/1871520611313020014 22721387 Hassan R Bullock S Premkumar A Kreitman RJ Kindler H Willingham MC Pastan I Phase I study of SS1P a recombinant anti-mesothelin immunotoxin given as a bolus I.V. infusion to patients with mesothelin-expressing mesothelioma ovarian and pancreatic cancers Clinical cancer research: an official journal of the American Association for Cancer Research 2007 13 5144 5149 10.1158/1078-0432.CCR-07-0869 17785569 Kreitman RJ Hassan R Fitzgerald DJ Pastan I Phase I trial of continuous infusion anti-mesothelin recombinant immunotoxin SS1P Clinical cancer research: an official journal of the American Association for Cancer Research 2009 15 5274 5279 10.1158/1078-0432.CCR-09-0062 19671873 Feldhaus MJ Siegel RW Opresko LK Coleman JR Feldhaus JM Yeung YA Cochran JR Heinzelman P Colby D Swers J Graff C Wiley HS Wittrup KD Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library Nature biotechnology 2003 21 163 170 10.1038/nbt785 12536217 Bergan L Gross JA Nevin B Urban N Scholler N Development and in vitro validation of anti-mesothelin biobodies that prevent CA125/Mesothelin-dependent cell attachment Cancer"

Lung_Cancer

"disease? and treatment?related symptoms; pharmacokinetics (PK); pre? and posttreatment concentrations of the extracellular domains of HER2 and EGFR in serum; and genetic variation in HER family and KRAS genes from free tumor DNA in blood. This trial was conducted in compliance with the Declaration of Helsinki and with the International Conference on Harmonization Good Clinical Practice Guidelines protocol and was approved by the Institutional Review Boards and/or Independent Ethics Committees at each of the participating investigational centers. All patients provided written informed consent prior to study participation. Evaluation of Antitumor Activity Evaluation of antitumor activity per RECIST version 1.012 was by investigator review. Tumor assessments were performed at baseline and at the end of every even?numbered cycle or when progressive disease was suspected. Evaluation of Safety and Tolerability Safety and tolerability were assessed by standard methods from initiation of study treatment until??28 days after the last dose of study drug. Adverse events (AEs) were graded by National Cancer Institute Common Terminology Criteria for Adverse Events version 3.0. Pharmacokinetic Analyses Biomarker Determination and Pharmacodynamic Analyses Blood samples for PK analyses were collected up to 24 hours after dose on day 1 of cycle 1 before dose on days 27 and 14 of cycle 1 and day 1 of cycle 2. PK parameters for dacomitinib including the maximum concentration (Cmax) the time to Cmax (Tmax) and the area under the plasma concentration curve from 0 to 24 hours (AUC0?24) were analyzed using a noncompartmental approach. Tumor tissue from new biopsies obtained at enrollment or archival samples (which may have been pre? or post?erlotinib) was analyzed for EGFR and KRAS gene mutation status using Qiagen Scorpion ARMS (Amplified Refractory Mutation System) allele?specific polymerase chain reaction assay; HER2 mutation status was determined by DNA sequencing. EGFR and HER2 gene amplification were assessed by fluorescence in situ hybridization. EGFR amplification was defined as?>15 copies of EGFR gene signals in?>10% of analyzed cells; HER2 amplification was defined as a HER2 gene/centromere of chromosome 17 ratio of?>2. Blood samples for biomarker analysis were collected at baseline and prior to dosing on day 1 of each cycle. Concentrations of HER2 and EGFR extracellular domains were determined by enzyme?linked immunosorbent assay. Patient?Reported Outcomes PROs of HRQoL disease symptoms specific to lung cancer and side effects of treatment were assessed using the 30?question European anisation for Research and Treatment of Cancer Quality of Life Questionnaire core module (EORTC QLQ?C30)13 which includes functional symptom side effects and global health status scales and the 13?item Lung Cancer symptom?specific module (QLQ?LC13).14 The impact of dacomitinib on patients' skin condition was assessed using the 10?item Dermatology Life Quality Index (DLQI) questionnaire. Statistical Design and Analyses The trial used a Fleming single?stage design for each patient population (adenocarcinoma and nonadenocarcinoma respectively). The primary objective of this study was to test the null hypothesis (H0) at the 0.05 significance level with 80% power that the objective response rate (ORR) in patients with adenocarcinoma did not exceed 5%. At the end of the study if there were at least 6 objective responders in 44 response?evaluable patients then the null hypothesis would be rejected demonstrating that treatment with dacomitinib is associated with a true response rate that exceeds 5%. A secondary objective was to test the H0 at the 0.05 significance level with 80% power that the ORR in patients with nonadenocarcinoma did not exceed 1%. At least 2 objective responders among 22 response?evaluable patients were required to reject the null hypothesis and conclude that treatment with dacomitinib demonstrates a true response rate that exceeds 1%. Target enrollment of 49 and 25 patients with adenocarcinoma and nonadenocarcinoma respectively was required and accounted for a rate of nonevaluability for response of up to 10%. Baseline characteristics PFS PFS6M OS OS6M and OS12M were evaluated in the intent?to?treat population safety in the as?treated population and response was assessed in response?evaluable patients. Results Patient Characteristics and Disposition Sixty?six patients were enrolled between April 2008 and November 2009 50 with adenocarcinoma and 16 with nonadenocarcinoma. Patient disposition is shown in Fig. 1. Enrollment of the nonadenocarcinoma arm was closed prior to reaching the planned target of 25 due to few nonadenocarcinoma patients identified with prior erlotinib treatment. Patient characteristics are summarized in . The majority of patients had received 2 or 3 prior treatment regimens (n?=?26 [39%] each). In addition to erlotinib prior EGFR?directed therapies comprised gefitinib (n?=?4) cetuximab (n?=?3) and neratinib (n?=?1). Fifty?five percent of the enrolled population were current or former smokers. Wild?type KRAS NSCLC was either directly confirmed (n?=?54) or assumed from a known EGFR mutation (n?=?12; EGFR mutation status was known for a total of 26 patients). Mutation and gene amplification data were collected from EGFR and HER2 according to availability of sufficient tissue for analysis (). Six patients had EGFR T790M resistance mutation identified after treatment with erlotinib (Supporting ; see online supporting information). T790M status was unknown in 54 patients who had biopsies taken prior to progression on erlotinib. Overall 74% of patients started dacomitinib within 3 months of discontinuing erlotinib. Of the 26 patients who had EGFR?mutant tumors at baseline the interval from discontinuing erlotinib to starting dacomitinib ranged from 15 to 544 days with 69% starting dacomitinib within 3 months of discontinuing erlotinib. Study flow diagram shows patient disposition and analysis populations. image Patient Baseline Characteristics Characteristic Adenocarcinoma (n?=?50) Nonadenocarcinoma (n?=?16)e Total (N?=?66) Median age years (range) 60 (37?79) 61 (50?84) 60 (37?84) Sex n (%) Male 15 (30.0) 14 (87.5) 29 (43.9) Female 35 (70.0) 2 (12.5) 37 (56.1) Race n (%) Caucasian 35 (70.0) 11 (68.8) 46 (69.7) Asian 12 (24.0) 2 (12.5) 14 (21.2) Other 3 (6.0) 3 (18.8) 6 (9.1) Smoking status n (%) Never?smoker 27 (54.0) 3 (18.8) 30 (45.5) Current smoker 1 (2.0) 2 (12.5) 3 (4.5) Exsmoker 22 (44.0) 11 (68.8) 33 (50.0) ECOG performance status n (%) 0 18 (36.0) 5 (31.3) 23 (34.8) 1 27 (54.0) 8 (50.0) 35 (53.0) 2 5 (10.0) 3 (18.8) 8 (12.1) Prior treatment regimens n (%) 1 regimena 4 (8.0) 1 (6.3) 5 (7.6) 2 regimens 18 (36.0) 8 (50.0) 26 (39.4) 3 regimens 19 (38.0) 7 (43.8) 26 (39.4) >3 regimensb 9 (18.0) 0 9 (13.6) Mutational status n (%) KRAS WT or EGFR sensitizing mutation 50 (100.0) 16 (100.0) 66 (100.0) KRAS WT 39 (78.0) 15 (93.8) 54 (81.8) KRAS unknown 11 (22.0) 1 (6.3) 12 (18.2) EGFR WT 10 (20.0) 13 (81.3) 23 (34.8) EGFR sensitizing mutation 24 (48.0) 2 (12.5) 26 (39.4) Exon 19 or 21 18 (75.0) 1 (50.0) 19 (73.1) Other 6 (25.0) 1 (50.0) 7 (26.9) EGFR unknown 16 (32.0) 1 (6.3) 17 (25.8) EGFR T790M secondary resistance mutation 6 (12.0)c 0 6 (9.1)c T790M unknown 39 (78.0) 15 (93.8) 54 (81.8) HER2 mutation 0 0 0 HER2 WT 29 (58.0) 13 (81.3) 42 (63.6) HER2 mutation unknown 21 (42.0) 3 (18.8) 24 (36.4) HER2 amplification positive 2 (4.0) 1 (6.3) 3 (4.5) HER2 amplification negative 22 (44.0) 11 (68.8) 33 (50.0) HER2 amplification unknown 26 (52.0) 4 (25.0) 30 (45.5) Prior EGFR?directed treatment n (%) Erlotinib 50 (100.0) 16 (100.0) 66 (100.0) Gefitinib 3 (6.0) 1 (6.3) 4 (6.1) Neratinibd 1 (2.0) 0 1 (1.5) Cetuximabd 2 (4.0) 1 (6.3) 3 (4.5) Response to immediately prior EGFR?directed treatment n (%) CR 1 (2.0) 0 1 (1.5) PR 13 (26.0) 0 13 (19.7) SD 21 (42.0) 5 (31.3) 26 (39.4) PD 9 (18.0) 10 (62.5) 19 (28.8) Unknown 6 (12.0) 1 (6.25) 7 (10.6) Abbreviations: CR complete response; ECOG Eastern Cooperative Oncology Group; EGFR epidermal growth factor receptor; PD progressive disease; PR partial response; SD stable disease. a Four patients in Arm A and 1 patient in Arm B had prior systemic treatment and prior erlotinib entered as 1 regimen. b Patients with >3 prior regimens includes patients with neoadjuvant and/or adjuvant therapies and/or investigational treatment regimen(s). c T790M status was derived from archival biopsies for 4 patients and fresh baseline biopsies for 2 patients. Three of the 4 patients with T790M status ascertained from an archival biopsy initiated dacomitinib more than 90 days after discontinuing from erlotinib. d Patients previously treated with investigational EGFR?directed therapies were eligible to participate in the study and did not represent protocol deviations provided the study sponsor provided written agreement. e Squamous n?=?12. Efficacy Best Overall Response In the overall population the ORR for response?evaluable patients was 5.2% (3 partial responses [PRs] of durations 1224 and 66 weeks). The ORR for patients with adenocarcinoma was 4.8% (2 PRs; 1?sided P?=?.372). For patients with nonadenocarcinoma the ORR was 6.3% (1 PR). The 25 response?evaluable patients with EGFR mutation?positive tumors (from both arms) achieved an ORR of 8% (2 PRs) and 17 (68%) achieved a BOR of stable disease (SD)??6 weeks (Table 2). Further details of the patients with PRs are presented in Supporting Table 2. Six patients had known EGFR T790M; of these 3 had SD??6 weeks (912 and 12 weeks respectively) and 3 had progressive disease (PD). Table 2 Summary of Best Overall Response Per RECIST by Investigator Assessment PFS and OS Adenocarcinoma Arm A Nonadenocarcinoma Arm B Total Overall Patient Population No. of patients evaluable n 42 16 58 Objective response (CR?+?PR) n (%) [95% exact CI]a 2 (5) [1?16] 1 (6) [0?30] 3 (5) [1?14] P valueb 0.372 0.149 – Clinical benefit (CR?+?PR?+?SD???24 weeks) n (%) [95% exact CI]a 10 (24) [12?40] 3 (19) [4?46] 13 (22.4) [13?35] Duration of response weeks 24c 66d 12e NA No. of patients enrolled n 50 16 66 No. of PFS eventsf n (%) 40 (80) 14 (88) 54 (82) PFS weeks [95% CI] 12 [8?20] 11 [6?18] 12 [9?19] PFS6M % [95% CI] 24 [12?38] 8 [1?30] 20 [11?32] No. of deaths n (%) 33 (66) 14 (88) 47 (71) OS weeks [95% CI] 45 [29?73] 27 [10?36] 37 [28?57] OS6M % [95% CI] 72 [57?82] 50 [25?71] 66 [53?76] OS12M% [95% CI] 46 [31?60] 22 [6?45] 40 [28?52] Patients With EGFR?Mutant Tumors No. of patients evaluable n 23 2 25 Objective response (CR?+?PR) n (%) [95% exact CI]a 2g (9) [1?28] 0 2g (8) [1?26] Clinical benefit (CR?+?PR?+?SD ?24 weeks) n (%) [95% exact CI]a 7 (30) [13?53] 0 7 (28) [12?49] No. of patients n 24 2 26 No. of PFS eventsf n (%) 19 (79) 2 (100) 21 (81) PFS weeks [95% CI] 18 [6?30] 21 [17?24] 18 [9?29] PFS6M % [95% CI] 36 [16?57] – 32 [14?52] No. of deaths n (%) 17 (71) 1 (50) 18 (69) OS weeks [95% CI] 59 [42?76] – [24 –] 57 [42?75] OS6M % [95% CI] 83 [62?93] 50 [1?91] 81 [60?92] OS12M % [95% CI] 61 [38?77] – 59 [37?76] Patients With EGFR Wild?Type Tumors No. of patients evaluable n 7 13 20 Objective response (CR?+?PR) n (%) [95% exact CI]a 0 [0?41] 1h (8) [0?36] 1h (5) [0?25] Clinical benefit (CR?+?PR?+?SD ?24 weeks) n (%) [95% exact CI]"

Lung_Cancer

"unrestricted use distribution and reproduction in any medium provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons./publicdomain/zero/1.0/) applies to the data made available in this unless otherwise stated. Abstract Background Metastatic spread of tumor through lymphatic vasculature is an important adverse prognostic factor in a variety of human cancer and tumor lymphangiogenesis requires the interplay of several growth factors. Platelet-derived growth factor (PDGF)-BB and vascular endothelial growth factor (VEGF)-C are two important molecules involving in tumor metastasis and lymphangiogenesis. Therefore the aim of this study was to investigate the coexpression of PDGF-BB and VEGF-C in primary human non-small cell lung cancer (NSCLC) and its association with lymphangiogenesis. Methods Using immunohistochemical staining PDGF-BB and VEGF-C expression were detected in 109 primary NSCLC tissues while the lymphatic micro-vessel density (LMVD) was counted. Results Of 109 cases PDGF-BB and VEGF-C overexpression was 66.97% (73/109) and 65.14% (71/109) respectively. 52 (47.7%) had overexpression of both PDGF-BB and VEGF-C (P?+?V+) 21 (19.3%) overexpression of PDGF-BB but low expression of VEGF-C (P?+?V-) 19(17.4%) overexpression of VEGF-C but low expression of PDGF-BB (P-V+) and 17(15.6%) low expression of both PDGF-BB and VEGF-C (P-V-). PDGF-BB expression was positively related to that of VEGF-C (r?=?0.451 p?=?0.034). LMVD in cases with P?+?V?+?was much higher than those with P-V- (p?=?0.004). In addition the patients with P?+?V?+?were younger and also had larger tumor size more likely lymph node metastasis and worse histological differentiation than those with P-V-. Moreover the overall survival (OS) of patients with P?+?V?+?was shorter than those with P-V- (p?=?0.015). Conclusion Coexpression of both PDGF-BB and VEGF-C was associated with lymphangiogenesis and poor prognosis in NSCLC and might play a critical role in NSCLC progression. Virtual Slides The virtual slide(s) for this can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2261801312571320 Platelet-derived growth factor-BB Vascular endothelial growth factor-C Lymphatic micro-vessel density Non-small cell lung cancer Background Lung cancer is the leading cause of tumor-related mortality throughout the world of which 80% are non-small cell lung cancer (NSCLC). In 2008 lung cancer replaced liver cancer as the first cause of death among people with malignant tumors in China [1]. Despite all efforts in the field of early diagnosis and adjuvant therapy the morbidity and mortality of NSCLC trend to ascend straightly [2]. One of the most important factors with direct impact on prognosis and therapeutic strategy in NSCLC is lymphatic metastasis [34]. Lymphangiogenesis the formation of new lymphatic vessels is considered to be an important process in the development of lymphatic metastasis [5]. The status of lymphangiogenesis and lymphatic vessel remodeling has been estimated by lymphatic micro-vessel density (LMVD) [6]. D2-40 is the preferred lymphatic endothelium-specific monoclonal antibody (mAb) for investigating intra-tumoral and peri-tumoral lymphatic micro-vessels [7]. Increased amount of LMVD provides more opportunities for tumor cells to disseminate to the lymph nodes. The correlation between LMVD and prognosis was confirmed in a variety of human cancer including breast cancer melanoma and NSCLC [8-11]. The family of VEGFs is composed of VEGF-A VEGF-B VEGF-C VEGF-D VEGF-E VEGF-F and placental growth factor (PlGF). VEGF-A is directly linked to angiogenesis while VEGF-C is considered as a prime mediator of lymphangiogenesis and has been implicated in carcinogenesis and metastasis. VEGF-C is a ligand for the VEGF receptor (VEGFR)-3 a tyrosine kinase receptor that is expressed predominantly on lymphatic endothelial cells (LECs) [1213]. It is demonstrated that VEGF-C induces lymphangiogenesis by VEGFR-3 signaling [14]. Studies showed that VEGF-C expression is associated with lymphatic invasion LMVD lymph node metastasis and prognosis in some human tumors such as breast cancer gastric cancer and NSCLC [15-18]. Recent studies show that platelet-derived growth factors (PDGFs) also enable the process of functional lymphangiogenesis. They can connect the receptors on LECs to promote LECs™ proliferation migration and the formation of tubular structures which induce lymphangiogenesis [19]. PDGF family consists of five isoforms -AA -AB -BB -CC and “DD [20]. PDGF-BB is a direct lymphangiogenic factor [21]. Emerging evidences indicate that the tight communication between vascular endothelial cells and mural cells by platelet-derived growth factor (PDGF)-BB is essential for capillary stabilization during the angiogenic process [22]. It was reported that the expression of PDGF-BB was correlated with tumor growth lymph node metastasis and lymphatic invasion in human esophageal squmaous cell carcinomas and NSCLC [2324]. Based on these data PDGF-BB and VEGF-C may play an important role in the process of tumor growth and lymphangiogenesis. However it is still unknown about the significance of combination of PDGF-BB and VEGF-C i.e. expression of both PDGF-BB and VEGF-C compared with only PDGF-BB or VEGF-C expression in NSCLC. In this study we examined the expression of PDGF-BB and VEGF-C in primary NSCLC tissues and investigated the clinicopathological significance of their coexpression and association with lymphangiogenesis. Methods Patients™ characteristics Tumor specimens were obtained from 109 patients with primary NSCLC who underwent surgery at the Jinan Central Hospital Affiliated to Shandong University China during the period from October 2008 to September 2010. They did not receive radiation therapy or chemotherapy before biopsy or surgical resection. There were 78 men (72%) and 31 women (28%) with median age of 58 years (interquartile range: 50?~?65 years) at the time of diagnosis. We determined the cell differentiation degree according to the classification amended in 1999 [25] and found 81 cases of well and moderately differentiated cells and 28 cases of poorly differentiated cells. The tumors were staged according to the USA Cancer Union Guidelines [26]. 38 patients were diagnosed with early NSCLC (I-IIa) and 71 with advanced NSCLC (IIb-III). Other clinical features are summarized in Table 1. All patients were followed up for at least 3 years after surgery. The median follow-up period was 47 months (interquartile range: 42?~?50 months). Overall survival (OS) was calculated from the date of surgery to the last follow up. The work was conducted in accordance with the Declaration of Helsinki. Informed consent was obtained from all the patients in this study. All patients signed the informed consent for use of specimens and the study was approved by the Institutional Review Board (Medical Ethics Committee of Jinan Central Hospital). Correlations of both PDGF-BB and VEGF-C coexpression with clinicopathological factors in primary human NSCLC Factors P?+?V+ P-V- P1 P?+?V - P2 P-V+ P3 Gender Male 35 13 0.476 16 0.716 14 0.847 Female 17 4 5 5 Age >60 years 23 11 0.047 13 0.859 11 0.676 ?60 years 29 6 8 8 Histology SQC 28 6 0.184 6 0.539 5 0.559 ADC 24 11 15 14 Tumor size >5 cm 24 3 0.037 5 0.950 6 0.563 ?5 cm 28 14 16 13 differentiation WD MD 35 17 0.017 17 0.757 12 0.027 PD 17 0 4 7 TNM stage I-IIa 12 8 0.113 10 0.973 8 0.765 IIb-III 40 9 11 11 Nodal status Positive 29 3 0.006 5 0.471 7 0.362 Negative 23 14 14 12 Note: P1 P value between P?+?V?+?and P-V-; P2 P value between P?+?V- and P-V-; P3 P value between P-V?+?and P-V-. Abbreviations: WD well differentiated MD moderately differentiated PD poorly differentiated ADC adenocarcinoma SQC squamous cell carcinoma. Main reagents The main reagents were anti-podoplanin mouse monoclonal antibody D2-40 (Dako Co. Denmark) anti-PDGF-BB rabbit polyclonal antibody (abcam Cambridge UK) Anti-VEGF-C rabbit monoclonal antibody (Beijing Zhongshan Goldenbrige Biotechnology China) immunohistochemical SP reagent box and DAB colour reagent (Fuzhou Maixin Co. China.P.R). Immunohistochemistry Immunohistochemical staining was carried out using the DAKO Envision detection kit (Dako Carpinteria CA USA). In brief paraffin-embedded tissue blocks were sectioned (4 ?m-thick) dried deparaffinized and rehydrated. Antigen retrieval was performed in a microwave oven for 15 min in 10 mM citrate buffer (pH 6.0). For all samples endogenous peroxidase activity was blocked with a 3% H2O2-methanol solution. The slides were blocked with 10% normal goat serum for 10 min and incubated with an appropriately diluted primary antibody mouse monoclonal antibody D2-40 (diluted 1:50) anti-PDGF-BB rabbit polyclonal antibody (diluted 1:200) or anti-VEGF-C rabbit polyclonal antibody (diluted 1:100) overnight at 4°C. The slides were then probed with an HRP-labeled polymer conjugated to an appropriate secondary antibody for 30 min. Each step was followed by washing with PBS. Each batch of staining was accompanied by positive and negative control slides. Primary human NSCLC tissues which are demonstrated to exhibit high levels of PDGF-BB and VEGF-C protein were used as positive controls. Normal mouse IgG substituted for primary antibody was a negative control. Quantitation of immunohistochemistry Clinicopathological findings were evaluated simultaneously using a double-headed light microscope by two independent examiners in a blinded fashion and mean values were calculated. The percentage of stained cells was recorded in at least 5 fields at 400-fold magnification in randomly selected tumor areas. In tumor specimens analysis of staining was exclusively restricted to the NSCLC cell reactions. Staining of stromal cells was not considered. Because cancer cells showed heterogeneous staining the dominant pattern was used for scoring. A combined scoring method that accounts for the intensity of staining as well as the percentage of cells stained was used as described previously [27]. The intensity of staining was graded from 0 to 3 with strong moderate weak and negative staining intensities as grade 321 and 0 respectively. The scores indicating percentage of positive cancer cells and staining intensity were multiplied to get a weighted score for each sample. For example a sample with 10% weak staining 10% moderate staining and 80% strong staining would be assigned a score of 270 (10?×?1?+?10?×?2?+?80?×?3?=?270) out of a possible score of 300. For statistical analyses samples with weighted scores 0“100 were defined as negative otherwise as positive. LMVD was performed according to a modification of Weidner™s method [28]. The immunostained sections were scanned by light-microscopy at low magnification (40×) and the areas of tissue with the greatest number of distinctly highlighted microvessels (hot spots) were selected. LMVD was then determined by counting all immunostained vessels at a total magnification of (200×) from five areas for each case. Determination of the staining reaction was strictly confined to the hot spots and the mean number of the vessels in each case was evaluated. Statistical analysis Data were analyzed according to the Statistical Package for Social Sciences (SPSS. 18.0 Chicago IL USA). Spearman™s coefficient of correlation Chi-squared test and two-tailed Student t test were used as appropriate. Overall survival (OS) curves were delineated by the Kaplan-Meier method and compared with log-rank test. For all tests p-values less than 0.05 were considered to be significant. All p-values given were results of two-sided tests. Results PDGF-BB and VEGF-C coexpression in primary human NSCLC In primary human NSCLC tissues PDGF-BB (Figure 1A B) and VEGF-C (Figure 1C D) expression were mainly present in the cytoplasm of cancer cells. PDGF-BB was also found on cancer cell membrane. Occasional and weak expression of PDGF-BB and VEGF-C were found in both cancer stroma and paracancerous normal tissues. Among 109 cases PDGF-BB and VEGF-C overexpression was 66.97% (73/109) and 65.14% (71/109) respectively. A cohort of patients was classified into 4 groups according to the expression of PDGF-BB and VEGF-C in the same patient. As shown in Table 1 47.7% (52/109) had overexpressions of both PDGF-BB and VEGF-C ( P?+?V+); 19.3% (21/109) had overexpression of PDGF-BB but low expression of VEGF-C (P?+?V-); 17.4% (19/109) patients had overexpression of VEGF-C but low expression of PDGF-BB (P-V+); 15.6% (17/109) patients had low expressions of both PDGF-BB and VEGF-C (P-V-). PDGF-BB expression had a positive correlation with that of VEGF-C (r?=?0.451 p?=?0.034) ( Figure 2). Figure 1 Immunohistochemical staining for PDGF-BB VEGF-C and D2-40 in primary NSCLC tissues (×200). A: PDGF-BB overexpression in adenocarcinoma. B: PDGF-BB overexpression in squamous cell carcinoma. C: VEGF-C expression in adenocarcinoma. D: VEGF-C expression in squamous cell carcinoma. E: D2-40 expression in the lymphatic endothelial cells in adenocarcinoma. F: D2-40 expression in the lymphatic endothelial cells in squamous cell adenocarcinoma. Figure 2 Relationship between the expression of PDGF-BB and VEGF-C in all adenocarcinoma and squamous cell carcinomas in NSCLC patients. Among 44 specimens from cases with lymph node metastasis 29 had P?+?V+ 5P?+?V- 7 P-V+ and 3 P-V-. There was a significant association between P?+?V?+?and lymph node metastasis (p?=?0.006). In addition compared with the P-V- cases the cases with P?+?V?+?were younger (p?=?0.047) and also had larger tumor size (p?=?0.037) and worse histological differentiation (p?=?0.017). While the cases with P-V?+?patients had worse histological differentiation (p?=?0.027) no other clinicopathological factores were found to be related to P?+?V- or P-V?+?. Relationship between lymphangiogenesis and coexpression of both PDGF-BB and VEGF-C in primary human NSCLC D2-40 expression was strictly present in the lymphatic endothelial cells. D2-40 positive lymphatic vessels were almost exclusively found at the tumor™s invasion front within the tumor stroma. The peri-tumoral lymphatic vessels were dilated and occasional invasion of the cancer cells into the dilated lymph vessels was observed (Figure 1E F). The amount of LMVD (25.970 ± 14.9347) in specimens from cases with lymph node metastsis was much higher than those without lymph node metastasis (17.860 ± 6.5640) p?=?0.015 (Figure 3A). Figure 3 Comparison of LMVD between the patients (A) who had lymph node metastasis and who didn™t and among the patients (B) who had P?+?V+ P?+?V- P-V?+?and P-V-. LMVD was also observed to be linked to P?+?V+. The amount of LMVD was 24.727 ± 13.772 in specimens with P?+?V+ 19.860 ± 6.663 in P?+?V- 20.395 ± 10.137 in P-V+ and 13.453 ± 4.503 in P-V-. Compared with other three groups LMVD in P?+?V?+?was significantly increased p?=?0.004 (Figure 3B). Prognostic significance of PDGF-BB and VEGF-C coexpression in primary human NSCLC P?+?V?+?was correlated with poor overall survival (OS). The univariate survival analysis showed that cases with P?+?V?+?had shorter survival time (38.7 m ) compared with those with P-V- (45.8 m) p?=?0.015. However no significant relationship was observed between OS and P?+?V- or P-V?+?( Figure 4). Figure 4 Relationship between coexpression of VEGF-C and PDGF-BB and overall survival in primary NSCLC patients. Disscussion Today accumulating evidences show that tumor may establish not only their own new blood vessels supply but might also induce lymphangiogenesis to promote its spread [29]. So possible inhibition of those processes might be of benefit for cancer patients especially as recent data suggest that the process of lymphangiogenesis is not only limited to primary tumor but is also present in lymph node metastases resulting in further cancer cell spread [30]. In this study we found the disordered and dilated lymphatic vessels were almost exclusively in peri-tumoral lesions but not in intra-tumoral lesions. And the amount of LMVD in cases with lymph node metastasis was significantly higher than those without lymph node metastasis. The results showed lymphangiogenesis existed in NSCLC tissues and was associated with lymphatic metastasis which is consistent with previous reports [11] and might be explained by a rising interstitial pressure caused by an increase in the size of lesion or by the lack of intratumoral lymphangiogenesis in NSCLC [31]. Indicating that peri-tumoral lymphatic vessels are important for the process of metastatic spread while intra-tumoral lymphatic vessels are non-functional [3233]. Lymphangiogenesis may require the interaction of several tumor-derived growth factors. It is demonstrated that VEGF-C and PDGF-BB are both important growth factors contributing to lymphangiogenesis [22]. VEGF-C can activate the VEGFR-3 signaling pathway to induce the lymphatic enlargement and lymphangiogenesis [14]. A study demonstrated that PDGF-BB can promote lymphangiogenesis and lymphatic metastasis by a VEGFR-3 independent mechanism in the mouse cornea in vivo [19]. In this model the lymphangiogenesis induced by PDGF-BB could not be restricted by blocking interaction of VEGF-C with VEGFR-3 suggesting that PDGF-BB exerts its effect via an independent pathway that may involve PDGF receptors on lymphatic vessels [34]. Another study showed that VEGF-C is an essential regulator determining PDGF-BB expression for vascular stabilization via a paracrine mode of action [22]. The stimulation of proliferation of lymphatic endothelial cells by platelets seems to be induced in a time and dose dependent manner mainly by VEGF-C and PDGF-BB which are secreted by platelets. Blocking the experiments indicate a predominant role of VEGF-C in this process [35]. All those results suggested that both factors play complicated roles in tumor lymphangiogenesis. However the overlapping biological effects of these two factors have not been clarified clearly in human cancers. In this study overexpression of both PDGF-BB and VEGF-C significantly correlated. LMVD. Those cases were also younger and had larger tumor size more likely lymph node metastasis worse histological differentiation and poorer OS. In addition a significant association between VEGF-C overexpression alone and worse histological differentiation was found. For the rest however PDGF-BB or VEGF-C alone was not linked to any other clinical feature including LMVD. The results indicated NSCLC patients who had overexpression of both PDGF-BB and VEGF-C might present with more rapid growth and higher potential for invasion due to their lymphangiogenesis. Thereby these patients had poorer OS which was consistent with the results in patients with esophageal squamous cell carcinoma those with positive expressions of PDGF-BB and VEGF-C have been shown to possess a worse prognosis compared to those with negative expressions [23]. Also those results suggested that poorly differentiated cancer cells might be more capable to secrete VEGF-C and PDGF-BB which induced lymphangiogenesis thereby promoting disease progression in NSCLC. The secretion of VEGF-C or PDGF-BB by tumor could induce the activation of their receptors on the vascular endothelium and thereby inducing the formation of new lymphatic vessels [36]. However little is currently known about the interplay among these lymphangiogenic factors. In this study a significant positive correlation between PDGF-BB and VEGF-C protein expression of tumor cells was seen in NSCLC suggesting a lymphangiogenesis pathway that one factor (PDGF-BB or VEGF-C alone) may up-regulate the other factor expression in the same cells. Therefore we suspected that PDGF-BB and VEGF-C could synergistically promote NSCLC lymphangiogenesis and enhance the tumor growth and lymph node metastasis. Combined targeting both PDGF-BB and VEGF-C may become a promising strategy for the treatment of NSCLC. Conclusions We found for the first time that compared with the overexpression of PDGF-BB or VEGF-C alone both PDGF-BB and VEGF-C overexpression in primary human NSCLC was significantly associated with lymphangiogensis and poor outcome. Furthermore our data suggested that PDGF-BB and VEGF-C expression might have a correlative dependence and interplay not only in NSCLC lymphangiogenesis but also in cancer progression. Based on the expression of PDGF-BB and VEGF-C we speculated the therapy targeting VEGF-C expression in combination with targeting PDGF-BB might be an important approach for control the cancer growth in patients with NSCLC having high expression of both PDGF-BB and VEGF-C. Competing interests All authors declare they have no actual or potential competing financial interests. Authors™ contributions All authors read and approved the final manuscript. JL and CL designed the study analyzed the data and drafted the manuscript. LQ and JL assisted with the design of the study and collected clinical data. JL and PZ carried out the immunohistochemi- stry and collected clinical data. YS conceived and designed the study analyzed the data and edited the manuscript. Acknowledgements This work was supported by the Project of the National Natural Science Foundation of China (Grant no. 81372334) and the Project of Jinan Youth Team for Technological Innovation (Grant no 2010“1). 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N Kandem?r NO Yavuzer D Korkmaz T Gecmen G Kokturk F Inducible nitric oxide synthase expression in gastric adenocarcinoma: impact on lymphangiogenesis and lymphatic metastasis Diagn Pathol 2013 8 151 24044375 Schoppmann SF Bayer G Aumayr K Taucher S Geleff S Rudas M Kubista E Hausmaninger H Samonigg H Gnant M Prognostic value of lymphangiogenesis and lymphovascular invasion in invasive breast cancer Ann Surg 2004 240 2 306 15273556 Valencak J Heere-Ress E Kopp T Schoppmann S Kittler H Pehamberger H Selective immunohistochemical staining shows significant prognostic influence of lymphatic and blood vessels in patients with malignant melanoma Eur J Cancer 2004 40 3 358 364 14746853 Renyi-Vamos F Tovari J Fillinger J Timar J Paku S Kenessey I Ostoros G Agocs L Soltesz I Dome B Lymphangiogenesis correlates with lymph node metastasis prognosis and angiogenic phenotype in human non“small cell lung cancer Clin Canc Res 2005 11 20 7344 7353 Takanami I Lymphatic microvessel density using D2-40 is associated with nodal metastasis in non-small cell lung cancer Oncol Rep 2006 15 2 437 442 16391866 Zetto VA Silveira GG Oliveira-Costa JP Soave DF Soares FA Ribeiro-Silva A The relationship between lymphatic vascular density and vascular endothelial growth factor A (VEGF-A) expression with clinical-pathological features and survival in pancreatic adenocarcinomas Diagn Pathol 2013 8 1 170 24138811 Joukov V Pajusola K Kaipainen A Chilov D Lahtinen I Kukk E Saksela O Kalkkinen N Alitalo K A novel vascular endothelial growth factor VEGF-C is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases EMBO J 1996 15 2 290 8617204 Chen J-C Chang Y-W Hong C-C Yu Y-H Su J-L The role of the VEGF-C/VEGFRs axis in tumor progression and therapy Int J Mol Sci 2012 14 1 88 107 23344023 Kinoshita J Kitamura K Kabashima A Saeki H Tanaka S Sugimachi K Clinical significance of vascular endothelial growth factor?C (VEGF?C) in breast cancer Breast Canc Res Treat 2001 66 2 159 164 Cai X Ma S Gu M Zu C Qu W Zheng X Survivin regulates the expression of VEGF-C in lymphatic metastasis of breast cancer Diagn Pathol 2012 7 1 1 8 22217299 Kigure W Fujii T Sutoh T Morita H Katoh T Yajima R Yamaguchi S Tsutsumi S Asao T Kuwano H The Association of VEGF-C expression with tumor lymphatic vessel density and lymph node metastasis in patients with gastric cancer and gastrointestinal stromal tumor Hepatogastroenterology 2012 60 122 277 280 23574654 Arinaga M Noguchi T Takeno S Chujo M Miura T Uchida Y Clinical significance of vascular endothelial growth factor C and vascular endothelial growth factor receptor 3 in patients with nonsmall cell lung carcinoma Cancer 2003 97 2 457 464 12518370 Cao Y Direct role of PDGF-BB in lymphangiogenesis and lymphatic metastasis Cell Cycle 2005 4 2 231 233 15655362 Heldin C-H Eriksson U Östman A New members of the platelet-derived growth factor family of mitogens Arch Biochem Biophys 2002 398 2 284 290 11831861 Cao Y Emerging mechanisms of tumour lymphangiogenesis and lymphatic metastasis Nat Rev Canc 2005 5 9 735 743 Onimaru M Yonemitsu Y Fujii T Tanii M Nakano T Nakagawa K Kohno R-i Hasegawa M Nishikawa S-i Sueishi K VEGF-C regulates lymphangiogenesis and capillary stability by regulation of PDGF-B Am J Physiol Heart Circ Physiol 2009 297 5 H1685 H1696 19734356 Matsumoto S Yamada Y Narikiyo M Ueno M Tamaki H Miki K Wakatsuki K Enomoto K Yokotani T Nakajima Y Prognostic significance of platelet-derived growth factor-BB expression in human esophageal squamous cell carcinomas Anticancer Res 2007 27 4B 2409 2414 17695532 Donnem T Al-Saad S Al-Shibli K Busund L-T Bremnes R Co-expression of PDGF-B and VEGFR-3 strongly correlates with lymph node metastasis and poor survival in non-small-cell lung cancer Ann Oncol 2010 21 2 223 231 19628565 Fleming ID Phillips JL Menck HR Murphy GP Winchester DP The National Cancer Data Base report on recent hospital cancer program progress toward complete American Joint Committee on Cancer/TNM staging Cancer 1997 80 12 2305 2310 9404708 Hensing TA Clinical evaluation and staging of patients who have lung cancer Hematol Oncol Clin North Am 2005 19 2 219 235 15833404 Hasina R Whipple ME Martin LE Kuo WP Ohno-Machado L Lingen MW Angiogenic heterogeneity in head and neck squamous cell carcinoma: biological and therapeutic implications Lab Investig 2008 88 4 342 353 18283272 Weidner N Semple JP Welch WR Folkman J Tumor angiogenesis and metastasis”correlation in invasive breast carcinoma New Engl J Med 1991 324 1 1 8 1701519 Fidler IJ The pathogenesis of cancer metastasis: the™seed and soil™hypothesis revisited Nat Rev Canc 2003 3 6 453 458 Kerjaschki D Bago-Horvath Z Rudas M Sexl V Schneckenleithner C Wolbank S Bartel G Krieger S Kalt R Hantusch B Lipoxygenase mediates invasion of intrametastatic lymphatic vessels and propagates lymph node metastasis of human mammary carcinoma xenografts in mouse J Clin Investig 2011 121 5 2000 21540548 Padera TP Stoll BR Tooredman JB Capen D di Tomaso E Jain RK Cancer cells compress intratumour vessels Nature 2004 427 6976 695 14973470 Bono P Wasenius V-M Heikkilä P Lundin J Jackson DG Joensuu H High LYVE-1“positive lymphatic vessel numbers are associated with poor outcome in breast cancer Clin Canc Res 2004 10 21 7144 7149 Padera TP Kadambi A di Tomaso E Carreira CM Brown EB Boucher Y Choi NC Mathisen D Wain J Mark EJ Lymphatic metastasis in the absence of functional intratumor lymphatics Science 2002 296 5574 1883 1886 11976409 Cao R Björndahl MA Religa P Clasper S Garvin S Galter D Meister B Ikomi F Tritsaris K Dissing S PDGF-BB induces intratumoral lymphangiogenesis and promotes lymphatic metastasis Cancer Cell 2004 6 4 333 345 15488757 Schoppmann SF Alidzanovic L Schultheis A Perkmann T Brostjan C Birner P Thrombocytes correlate with lymphangiogenesis in human esophageal cancer and mediate growth of lymphatic endothelial cells in vitro PLoS One 2013 8 6 e66941 23840559 Shibuya M Vascular Endothelial Growth Factor (VEGF) and Its Receptor (VEGFR) Signaling in Angiogenesis A Crucial Target for Anti-and Pro"

Lung_Cancer

"days for untreated control and 59 days (p?=?0.03 Log rank test). B. A Kaplan-Meyer plot of time to pre-defined tumor volume endpoint for subcutaneous RS4 leukemia subcutaneous xenografts treated with thiaminase 850 units SC BIW or buffer control. The median time to endpoint was 16.5 days for the control group and not reached after 60 days of observation in the treated group (p<0.001 Log rank test). C. Primary ALL and AML specimens were thawed and plated in triplicate in two concentrations of thiaminase 0.4 units/ml and 4 units/ml and assessed for viability at 48 hours relative to untreated cells. The ALL specimen with the asterisk was used for the in vivo study shown in D. D. Primary ALL cells were injected IV on Day 1; three thiaminase treatments of 2000 units/kg SC were administered on days 17 20 and 24. The animals were sacrificed on Day 33 and bone marrow was examined by flow cytometry for human ALL cells (percent human CD45); Untreated n?=?4; vehicle treated n?=?10 native thiaminase n?=?8 (* p<0.01 Mann-Whitney test). The studies in along with previous reports demonstrate that the enzyme thiaminase has activity against breast and lymphoid leukemia cell lines. To first determine whether the activity of thiaminase was related to thiamine depletion we explored the ability of small molecule thiamine antagonists to potentiate the cytotoxicity of thiaminase. We screened thiamine analogs pyrithiamine oxythiamine and N3™ pyridylthiamine (N3PT) [13] [14] for activity as inhibitors of thiaminase and substrates of thiaminase. We found that oxythiamine and N3PT were thiaminase inhibitors and that pyrithiamine was a thiaminase substrate (data not shown). Therefore we hypothesized that oxythiamine and N3PT would reverse thiaminase toxicity if given simultaneous with thiaminase (because they would inhibit enzyme activity) but would act synergistically if given in sequence (because cells deprived of thiamine would become sensitive to small molecule thiamine antagonists due to TDE apoenzyme formation during thiamine starvation). In A simultaneous incubation of thiaminase and thiamine antagonists inhibits thiaminase activity while in B sequential administration of thiaminase followed by N3PT demonstrates synergistic cytotoxicity. Importantly the effect of pre-incubation in thiaminase is similar to the effect of pre-incubation in thiamine-free medium indicating that TDE apoenzyme formation is required for the cytotoxicity of small molecule TDE inhibitors and also demonstrating that extracellular thiamine starvation is sufficient to make small molecule TDE inhibitors cytotoxic. In order to further examine the interplay of N3PT and thiaminase in an in vivo model we administered PEGylated thiaminase in sequence with N3PT choosing PEGylated enzyme over the native enzyme shown in B to be able to separate extracellular thiamine deprivation caused by PEGylated thiaminase from the intracellular effect of N3PT [6]. In C the RS4 xenograft experiment shows that sequential administration of PEGylated thiaminase followed by N3PT produced an increased median TTE (55 days) compared to the untreated control (16.5 days) PEG-thiaminase (25 days) or N3PT (23 days) alone (p<0.01) confirming the in vitro data. .0085702.g002 Effect of thiamine antagonists on thiaminase activity. A. Growth inhibition of Reh and RS4 leukemia cells in the presence or absence of thiaminase and either N3PT or oxythiamine. B. Growth inhibition of RS4 and Reh leukemia cells incubated in normal medium thiamine-free medium or medium containing thiaminase prior to exposure to different concentrations of N3PT. C. RS4 subcutaneous xenografts showing untreated control N3PT alone 1k-PEGylated thiaminase and 1k-PEGylated thiaminase followed by N3PT. The time-to-endpoint was 16.5 days for control 23 days for N3PT 25 days for 1k-PEG thiaminase and 55 days for 1k-PEG thiaminase (p<0.01 Log rank test). D. Oxygen consumption rate of RS4 cells treated with thiaminase N3PT or thiaminase followed by N3PT (clear: control; thin-stripe: thiaminase; thick- stripe: N3PT; solid: thiaminase + N3PT). Since thiaminase should inhibit two key TDEs involved in Krebs cycle metabolism pyruvate dehydrogenase complex and 2-oxoglutarate dehydrogenase (2-OGDH aka alpha-ketoglutarate dehydrogenase) we examined respiration (as oxygen consumption rate; OCR) and the rate of extracellular acidification (a measure of lactate production; ECAR) in leukemia and breast cancer cell lines. In D the oxygen consumption rate is decreased in an additive fashion with the sequential administration of thiaminase followed by N3PT showing further evidence of interaction of thiaminase and a small molecule thiamine antagonist. In addition since rapamycin reversed thiaminase-mediated growth inhibition in leukemia cell lines [7] but not breast cancer cell lines (data not shown) we examined the effect of rapamycin alone and in combination with thiaminase. In A thiaminase consistently decreased OCR in two lymphoid leukemia cell lines RS4 and Reh in the breast cancer cell line MCF-7 and in the non-malignant breast cell line MCF-10A. Also as expected rapamycin similarly suppressed OCR in the four cell lines. However adding rapamycin resulted in an inhibition of the thiaminase-mediated decrease in OCR rather than resulting in an additive effect as was seen with N3PT in B. .0085702.g003 OCR and ECAR of leukemia and breast cell lines incubated for 48(clear: control; thin-stripe: thiaminase; thick- stripe: rapamycin; solid: thiaminase + rapamycin). A. Quantification of OCR parameters. The ATP-linked rate is the basal rate minus the rate measured after the addition of oligomycin. The maximal capacity is the rate measured after the addition of FCCP. The reserve capacity is the basal rate minus the FCCP rate. All data are the mean ± SEM of triplicate samples and are representative of 3 independent experiments ( p<0.05 *p<0.01 two way ANOVA (Newman Kruskal Wallis test). B. Quantification of ECAR parameters. Glycolysis is the rate determine from subtracting the basal rate from the rate after the addition of glucose. Glycolytic reserve is the rate determined by subtracting the rate following the addition of oligomycin from the rate following the addition of glucose. All data are the mean ± SEM of triplicate samples and are representative of 3 ( p<0.05 *p<0.01 two way ANOVA (Newman Kruskal Wallis test). Similarly thiaminase increased ECAR in the two breast cell lines: the MCF-7 cell line and the MCF-10A cell line (B). Although rapamycin alone did not have a significant effect on ECAR the addition of rapamycin to thiaminase once again blunted the effect of thiaminase in all four cell lines. The consistent inhibition of thiaminase effect on OCR and ECAR by rapamycin in all four cell lines demonstrates a previously unidentified activity of mTOR inhibition in relation to TDEs. However the observation that the rapamycin has similar effects on the leukemia cell lines where it reverses growth inhibition [7] and MCF-7 and MCF-10A cell lines where it has no effect on thiaminase cytotoxicity (data not shown) suggests that the effect of thiaminase on OCR and ECAR may not be the determinant of thiaminase anticancer activity. To rule out the possibility that rapamycin could directly inhibit thiaminase enzymatic activity which could explain the reversal of the effects of thiaminase by rapamycin we directly examined the ability of rapamycin to inhibit thiaminase enzyme activity and found no evidence that rapamycin was a thiaminase enzyme inhibitor (data not shown). To further understand how thiaminase alters cellular metabolism and how rapamycin reverses thiaminase effects we undertook a metabolomic analysis of RS4 leukemia cells and MCF-7 breast cancer cells exposed to thiaminase rapamycin or both. A shows internal validation of the effects of thiaminase in RS4 cells. As expected the levels of thiamine and thiamine diphosphate were decreased in the thiaminase treated conditions and thiazole (a catabolic product of thiaminase cleavage of thiamine) is increased in the thiaminase treated cells confirming the finding that rapamycin does not interfere with the enzymatic activity of thiaminase. In fact thiamine was below the limit of detection in thiaminase treated cells in the absence of rapamycin. Also rapamycin was detected only in the rapamycin-treated cells. Evidence for inhibition of the TDE transketolase was observed with significant increases in ribose and ribulose surrogates for transketolase substrates ribose 5-phosphate and ribulose 5-phosphate respectively in both RS4 and MCF-7 cells (B). These results demonstrate a common signature of transketolase inhibition in the two cell lines. Furthermore the accumulation is partly to completely reversed by rapamycin demonstrating an antagonistic effect of rapamycin on thiamine-induced transketolase inhibition in addition to its antagonistic effects on OCR and ECAR. .0085702.g004 Validation of thiaminase action in of RS4 leukemia cells and MCF-7 breast cancer cells by metabolomic analysis. Both cell lines were analyzed under six conditions: control for 24 hours (C-24); incubation in thiaminase for 24 hours (T-24); control for 48 hours (C-48); thiaminase for 48 hours (T-48); rapamycin for 48 hours (R-48); and both rapamycin and thiaminase for 48 hours (R+T-48). The median is indicated by the bar in the center of the rectangle the rectangle dimensions reflect the range of the two mid-quartile values and the outer bars represent the ranges of all of the values. The data represent four independent experiments. For C-48 vs T-48 and C-48 vs T+R-48 comparisons ** indicates p<0.05 and * indicates 0.05<p<0.1). A. Internal validation in RS4 cells showing the expected decrease in thiamine and thiamine diphosphate in thiaminase treated cells an increase in thiazole the product of thiaminase cleavage of thiamine in the thiaminase-treated cells and the appearance of rapamycin only in the rapamycin treated cells. B. Validation of thiaminase-mediated inhibition of transketolase by demonstration of the accumulation of substrate surrogates ribose and ribulose in both RS4 and MCF-7 cell lines at 48 hours. C. Immunoblot analysis of thiamine pyrophosphate kinase (TPK1) and thiamine triphosphatase (THTPA) in RS4 and MCF-7 cells treated with thiaminase rapamycin or both. D. Immunoblot analysis of pyruvate kinase isozymes M1 and M2 and carnitine palmitoyl transferase (CPT1) in RS4 and MCF-7 cells treated with thiaminase rapamycin or both. Although there was no evidence that rapamycin altered the level of thiaminase-induced thiamine depletion we sought to confirm this observation by examining the expression of enzymes that regulate thiamine phosphorylation. In C the expression of both thiamine pyrophosphokinase TPK1 an enzyme that phosphorylates thiamine and thiamine monophosphate into its active diphosphate form and thiamine triphosphatase (THTPA) an enzyme that dephosphorylates intracellular thiamine triphosphate into the active diphosphate form are shown. A modest increase in THTPA expression is seen in RS4 cells treated with both thiaminase and rapamycin which may be a result of feedback from intracellular thiamine diphosphate depletion but no other changes are seen in either RS4 or MCF-7 cells. In D the expression of enzymes involved in energy metabolism pyruvate kinase isoforms M1 and M2 and carnitine palmitoyl transferase 1 (CPT1A) were also examined by immunoblot. PK-M1 expression was decreased in MCF-7 cells relative to RS4 cells. In MCF-7 cells both PK-M2 and CPT1A expression levels were modestly reduced by rapamycin and this suppression was reversed by thiaminase in the case of CPT1A but not PK-M2. However similar findings were not observed in RS4 cell lines adding more evidence to the overall observation that different cell lines will respond differently to acute thiamine depletion. Figure 5 shows effects of thiaminase on the TDE branched-chain keto acid dehydrogenase (BCKDH) and demonstrates different metabolomic signatures between RS4 leukemia cells and MCF-7 breast cancer cells. In Figure 5A the metabolic pathways for the catabolism of branched chain amino acids is illustrated. The metabolomic profile for these metabolites is shown in Figure 5B and Figure S1. In RS4 cells thiaminase increases the levels of BCKDH substrates alpha-hydroxyisocaproate 2-hydroxy-3-methylvalerate and alpha-hydroxyisovalerate all metabolites that are increased in maple syrup urine disease the congenital form of BCKDH deficiency. In contrast a decrease in the products of BCKDH are observed in MCF-7 cells with decreased isobutyrylcarnitine 2-methybutyrylcarnintine and isovalerylcarnitine. Once again the effects of thiaminase were offset by rapamycin. These findings suggest that the substrate load for BCKDH is much higher in RS4 leukemia cells. Therefore we investigated the expression of the cytosolic form and the mitochondrial form of the branched chain aminotransferases cBCAT and mBCAT - the enzymes immediately upstream of BCKDH in both RS4 and MCF-7 cells. As shown in Figure 5C MCF-7 cells do not appear to express cBCAT providing a potential explanation for the lack of BCKDH substrate accumulation in this cell line after thiaminase treatment. Figure 5C also appears to show an apparent increase in phosphorylated BCKD-E1 after thiaminase treatment in MCF-7 cells which is reversed by co-treatment with rapamycin. Phosphorylation of BCKDH inactivates the enzyme and may contribute to the decrease of BCKDH products seen in the MCF-7 cells and suggests that there may be an interaction between thiaminase rapamycin and the enzymes that regulate BCKDH phosphorylation. To determine whether the accumulation of BCKDH substrates contributed to thiaminase-induced growth inhibition in RS4 cells we examined thiaminase growth inhibition in the presence of gabapentin a structural analog of leucine and an inhibitor of cBCAT [15]. As shown in Figure 5D gabapentin partially protected RS4 cells from thiaminase growth inhibition while having no effect on MCF-7 cells. .0085702.g005 Figure 5 Differential effects of thiaminase on branched chain amino acid catabolism. A. Schematic diagram showing branched chain amino acid catabolism. B. Different metabolomic signatures indicating inhibition of BCKDH by thiaminase. The top three panels show accumulation of BCKDH substrates after 48-7 cells after thiaminase treatment- most notably isovalerylcarnitine which is also reversed by rapamycin. C. An immunoblot of cytosolic and mitochondrial branched chain amino acid transferase (cBCAT and mBCAT) the enzymes that catalyzes the reactions that produce BCKDH substrates and total and phosphorylated BCKDH subunit E1 (BCKD-E1 and pBCKD-E1 respectively) in RS4 leukemia cells treated with thiaminase rapamycin or both. For C-48 vs T-48 and C-48 vs T+R-48 comparisons ** indicates p<0.05 and * indicates 0.05<p<0.1). D. Cytotoxicity assay of RS4 and MCF-7 cells treated with increasing concentrations of thiaminase (in milliunits mu) under control conditions or in medium that contains gabapentin. Thiaminase also affected the catabolism of aromatic amino acids in RS4 leukemia cells. As shown in Figure 6A the breakdown products of both phenylalanine (phenylpyruvate and phenyllactate) and tyrosine (4-hydroxyphenylpyruvate (HPP) and 3-(4-hydroxyphenyl)lactate (HPLA)) are increased in the thiaminase treated cells. Furthermore as shown in Figure 6B tryptophan catabolites are increased through one pathway which produces indolelactate but not through the kynurenine pathway. As in the approach we took with the alteration of branched chain amino acids we examined the possibility that the accumulation of these aromatic amino acid catabolites mediated the growth inhibitory effects of thiaminase. The drug nitisinone which inhibits 4-hydroxyphenylpyruvate dioxygenase an enzyme that catalyzes an alternative catabolic pathway for phenylalanine [16] and would be expected to increase the accumulation of aromatic amino acid catabolities did not however alter the dose-response curves of thiaminase in either cell line (data not shown). .0085702.g006 Figure 6 Effects of thiaminase on aromatic amino acid catabolism. A. Accumulation of the products of phenylalanine (phenylpyruvate and phenyllactate) and tyrosine (hydroxyphenylpyruvate and hydroxyphenyllactate) catabolism in RS4 cells after treatment with thiaminase for 48 hours. B. Accumulation of tryptophan catabolites after thiaminase treatment showing accumulation of indolelactate but not kynurenine. For C-48 vs T-48 and C-48 vs T+R-48 comparisons ** indicates p<0.05 and * indicates 0.05<p<0.1). Discussion Any systematic difference between cancer cells and normal cells provides a potentially exploitable therapeutic opportunity. The altered energy metabolism in cancer cells known as the Warburg effect represents such an opportunity. The vitamin thiamine is a key cofactor in two critical enzymes in energy metabolism: PDH and 2-OGDH. It is also a cofactor for two other critical enzymes; TK which is required for the formation of the biomass of a cell through the pentose phosphate shunt; and BCKDH which regulates the catalysis of branched chain amino acids. Thiamine requires active transport for cellular uptake. We have previously shown down-regulation of thiamine transporters in human tumors [2] [3] suggesting that tumors may have a nutritional vulnerability that could be exploited clinically analogous to the down-regulation of asparagine synthase in acute lymphoblastic leukemia which is exploited therapeutically by the bacterial enzyme asparaginase. The enzyme thiaminase catalyzes the cleavage of thiamine into two molecules: one is a pyrimidine (conjugated to a nucleophile for type I thiaminases) and the other is a thiazole that may be phosphorylated depending on the thiamine substrate that enters the reaction [5]. Several forms of thiaminase exist in nature including plant animal and bacterial forms of the enzyme thus serving as an example of convergent evolution even though the physiologic role of an enzyme that destroys an essential nutrient is not clear. We have developed methods to produce purify and modify Bacillus thiaminolyticus thiaminase I enzyme and have shown in preclinical xenograft models that therapy directed at thiamine dependent enzymes (TDEs) a completely novel concept and strategy has the potential to treat leukemia [7]and breast cancer [8] and despite its anti-metabolite role thiaminase can cause tumor responses at a systemically tolerable dose. To determine which TDE is responsible for the cytotoxic effects of thiaminase we examined the effect of the enzyme on cellular respiration and metabolism with the goal of identifying the critical TDEs responsible for cytotoxicity in the setting of acute thiamine deprivation. Since thiaminase is a bacterial enzyme it is less-than-ideal as a pharmaceutical product and the identification of the specific pathway disrupted by thiamine deprivation that leads to cytotoxicity could reveal an alternative pharmacologic approach. The metabolomic changes in the global biochemical profiles of RS4 leukemia cells and MCF-7 breast cancer"

Lung_Cancer

"The immunoreactivity was detected by a Vectastain ABC kit (Vector Laboratories Burlingame CA). Quantitative PCR Quantitative Real-time PCR was performed using SYBR Premix Ex Taq (Takara Bio Otsu Shiga Japan) in a CFX96TM Real-Time System and C1000 TM Thermal Cycler (BIO-RAD Hercules CA). The primers for quantitative PCR were as follows: Firefly luciferase F 5'-TCA AAG AGG CGA ACT GTG TG-3' R 5'-GGT GTT GGA GCA AGA TGG AT-3'. Immunofluorescent staining The miRZip and miRZip-182 stable expression H1299 cells were seeded onto coverslips (with a thickness of 0.17 mm) and incubated for 24 h. After fixation with 4% paraformaldehyde (Sigma-Aldrich) in PBS for 15 min and permeabilization with 1% Triton X-100 for 5 min cells on the coverslip were blocked with 1% bovine serum albumin (Sigma-Aldrich) for 1 h and stained with the antibody against F-Actin by using Alexa Fluor 568-conjugated phalloidin (Invitrogen) for 1 h at room temperature. Subsequently cells on the coverslip were washed with PBS three times. Finally cells were mounted in Prolong Gold antifade reagent with DAPI (Invitrogen) and examined using immunofluorescence microscope (Delta Vision Personal DV). The images were analyzed with softWoRx software (Applied Precision). Wound-healing assay The miRZip and miRZip-182 stable expression H1299 cells (1.5 x 106) were seeded in 6 cm dish and cultured for 24 h the linear wound of cellular monolayer was created by scratching confluent cell monolayer using a plastic pipette tip. The monolayer of Scratched cell was washed by PBS to remove debris. After incubation at 37? with 5% CO2 for 16 h area of migration was photographed under light microscope for evaluation. Transwell migration assay The cell migration assay was performed using Transwell system with an 8 mm pore size polycarbonate filter membrane (Corning Costar Cambridge MA). Cells were trypsinized and suspended in serum-free DMEM. Upper wells were filled with cell suspensions in serum-free DMEM and lower wells were filled with DMEM containing 10% fetal bovine serum. After incubation for 14 h at 37? with 5% CO2 the lower side of filter membrane was fixed with methanol and stained with DAPI. The migrated cells were counted under a fluorescent microscope and quantified by Image J software (National Institutes of Health Bethesda MD). In vivo metastasis assay The miRZip and miRZip-182 stable expression H1299 cells were trypsinized and suspended in PBS for tail vein injection. A total of 4 x 106 cells in 100 ml of PBS were injected into the lateral tail vein of SCID mice. Mice were killed after 8 weeks and the excised lungs were fixed with 4% formaldehyde for 48 h. Finally the number of pulmonary metastatic nodules on the surface of lung was counted. Microarray analysis Total RNA was extracted using TRIzol from miRZip and miRZip-182 stable expression H1299 cells. Microarray analysis was performed by the Phalanx Biotech Group (Hsinchu Taiwan). Microarray data were analyzed by using DAVID Bioinformatics Resources 6.7 [50 51]. SUPPLEMENTARY TABLES REFERENCES AND FIGURES This work was supported by the National Cheng Kung University project of the Program for Promoting Academic Excellence and Developing World Class Research Centers together with grants NSC 99-2320-B-006-031-MY3 and NSC 101-2321-B-006-004-MY3 obtained from the National Science Council Taiwan. SUPPLEMENTARY DATA Supplementary Data are available at Oncotarget online including Supplementary References [52-58]. REFERENCES 1. Halbeisen RE Galgano A Scherrer T Gerber AP Post-transcriptional gene regulation: from genome-wide studies to principles Cell Mol Life Sci 2008 65 5 798 813 18043867 2. Loya CM Van Vactor D Fulga TA Understanding neuronal connectivity through the post-transcriptional toolkit Genes Dev 2010 24 7 625 635 20360381 3. Jewer M Findlay SD Postovit LM Post-transcriptional regulation in cancer progression : Microenvironmental control of alternative splicing and translation J Cell Commun Signal 2012 6 4 233 248 23054595 4. Lai EC Micro RNAs are complementary to 3' UTR sequence motifs that mediate negative post-transcriptional regulation Nat Genet 2002 30 4 363 364 11896390 5. Bartel DP MicroRNAs: target recognition and regulatory functions Cell 2009 136 2 215 233 19167326 6. Suzuki H Maruyama R Yamamoto E Kai M DNA methylation and microRNA dysregulation in cancer Mol Oncol 2012 6 6 567 578 22902148 7. Chen PS Su JL Hung MC Dysregulation of microRNAs in cancer J Biomed Sci 2012 19 90 23075324 8. Kopp F Wagner E Roidl A The proto-oncogene KRAS is targeted by miR-200c Oncotarget 2013 9. Vosa U Vooder T Kolde R Vilo J Metspalu A Annilo T Meta-analysis of microRNA expression in lung cancer Int J Cancer 2013 132 12 2884 2893 23225545 10. Abd-El-Fattah AA Sadik NA Shaker OG Aboulftouh ML Differential MicroRNAs Expression in Serum of Patients with Lung Cancer Pulmonary Tuberculosis and Pneumonia Cell Biochem Biophys 2013 11. Jemal A Bray F Center MM Ferlay J Ward E Forman D Global cancer statistics CA Cancer J Clin 2011 61 2 69 90 21296855 12. Rosell R Bivona TG Karachaliou N Genetics and biomarkers in personalisation of lung cancer treatment Lancet 2013 382 9893 720 731 23972815 13. McFarlane C McFarlane S Paul I Arthur K Scheaff M Kerr K Stevenson M Fennell DA Johnston JA The deubiquitinating enzyme USP17 is associated with non-small cell lung cancer (NSCLC) recurrence and metastasis Oncotarget 2013 4 10 1836 1843 24123619 14. Shen J Stass SA Jiang F MicroRNAs as potential biomarkers in human solid tumors Cancer Lett 2013 329 2 125 136 23196059 15. Boeri M Pastorino U Sozzi G Role of microRNAs in lung cancer: microRNA signatures in cancer prognosis Cancer J 2012 18 3 268 274 22647364 16. Melo SA Kalluri R Molecular pathways: microRNAs as cancer therapeutics Clin Cancer Res "

Lung_Cancer

"We developed a novel immunotherapeutic agent that links a single-chain antibody variable fragment (scFv) targeting mesothelin (MSLN) which is overexpressed on ovarian cancer and mesothelioma cells to Mycobacterium tuberculosis (MTB) heat shock protein 70 (Hsp70) which is a potent immune activator that stimulates monocytes and DCs enhances DC aggregation and maturation and improves cross-priming of T cells mediated by DCs. Methods Binding of this fusion protein with MSLN on the surface of tumor cells was measured by flow cytometry and fluorescence microscopy. The therapeutic efficacy of this fusion protein was evaluated in syngeneic and orthotopic mouse models of papillary ovarian cancer and malignant mesothelioma. Mice received 4 intraperitoneal (i.p.) treatments with experimental or control proteins post i.p. injection of tumor cells. Ascites-free and overall survival time was measured. For the investigation of anti-tumor T-cell responses a time-matched study was performed. Splenocytes were stimulated with peptides and IFN?- or Granzyme B- generating CD3+CD8+ T cells were detected by flow cytometry. To examine the role of CD8+ T cells in the antitumor effect we performed in vivo CD8+ cell depletion. We further determined if the fusion protein increases DC maturation and improves antigen presentation as well as cross-presentation by DCs. Results We demonstrated in vitro that the scFvMTBHsp70 fusion protein bound to the tumor cells used in this study through the interaction of scFv with MSLN on the surface of these cells and induced maturation of bone marrow-derived DCs. Use of this bifunctional fusion protein in both mouse models significantly enhanced survival and slowed tumor growth while augmenting tumor-specific CD8+ T-cell dependent immune responses. We also demonstrated in vitro and in vivo that the fusion protein enhanced antigen presentation and cross-presentation by targeting tumor antigens towards DCs. Conclusions This new cancer immunotherapy has the potential to be cost-effective and broadly applicable to tumors that overexpress mesothelin. Mycobacterial Hsp70 Mesothelin Single chain variable fragment Cancer immunotherapy Murine tumor model Background The goal of cancer immunotherapy is to stimulate the immune system to destroy cancer cells. Numerous strategies that involve tumor antigen-specific and non-specific activation of the immune system have been developed. These include dendritic cell (DC) vaccines adoptive T-cell therapy and immune checkpoint blockade [1-3]. Antigen-specific active immunotherapy is expected to be the most attractive strategy because of its capacity to induce both therapeutic and protective T-cell immunity. Among various approaches DC vaccine is considered to be a promising treatment for advanced cancer based on the ability of DCs to orchestrate all of the elements of the immune system. DCs capture tumor antigens process these antigens into peptides as they move to the draining secondary lymphoid ans and present the peptides to na¯ve T cells thus inducing anti-tumor cellular immune responses. DCs can also activate B cells NK cells and NKT cells [1]. In pre-clinical and clinical studies that exploited DCs as a means to improve vaccine efficiency autologous DCs are loaded ex vivo with antigens and re-administered to the patient. For example Sipuleucel-T (Provenge) that consists of ex vivo activated autologous peripheral blood mononuclear cells (PBMCs) including antigen-presenting cells (APCs) has resulted in a significant survival benefit in Phase III trials for prostate cancer [4]. However the production and administration of these tailor-made DC vaccines are costly and labor-intensive [5]. As a next-step in the development of DC vaccines we designed a recombinant protein that contains a Mycobacterium tuberculosis heat shock protein 70 (MTBHsp70) fused to a single chain variable fragment (scFv) derived from human B cells that targets mesothelin. Mesothelin (MSLN) is a validated immunotherapy target that is highly overexpressed on the surface of common epithelial cancers including ovarian cancers epithelial malignant mesotheliomas ductal pancreatic adenocarcinomas and lung adenocarcinomas while expressed at relatively low levels only in mesothelial cells lining the pleura pericardium and peritoneum in healthy individuals [6-9]. Several therapeutic agents targeting MSLN are evaluated in preclinical and clinical studies such as the recombinant immunotoxin SS1P [9-11]. In our fusion protein the anti-MSLN scFv moiety was originally isolated from a yeast-display human scFv library [12] and demonstrated the ability to recognize both membrane-bound and soluble MSLNs and inhibit CA125/MSLN-dependent cell adhesion [13-15]. The recombinant MTBHsp70 protein provides immunostimulatory functions including the activation of monocytes and DCs to produce CC-chemokines that attract antigen processing and presenting DCs macrophages and effector T and B cells enhanced DC aggregation and maturation [1617] induction of the cytotoxic activity of natural killer cells [18] and improved cross-priming of T cells which is dependent on DCs [19]. The capabilities of MTBHsp70 as a potent immune adjuvant have been well characterized in cancer models including murine models of melanoma and lymphoma [1820-24]. While in these studies proteins or peptides fused with Hsp70 used for immunizations in mice were shown to generate humoral or cellular immune responses we expect that fusion of anti-MSLN scFv and MTBHsp70 takes advantage of the immune-activating action of MTBHsp70 and the tumor-targeting activity of the scFv which will yield anti-tumor responses against the broadest profile of tumor antigens. We evaluated the therapeutic efficacy of this MSLN-targeted fusion protein in syngeneic mouse models of ovarian cancer and mesothelioma and examined its mechanism of action in in vitro and in vivo cross-presentation assay systems. These studies demonstrate that this bifunctional fusion protein significantly enhances survival and slows tumor growth through the augmentation of tumor-specific cell-mediated immune responses. Results Expression of scFvMTBHsp70 fusion protein and MTBHsp70 The structure of scFvMTBHsp70 is shown in Figure 1A. VH and VL from anti-MSLN P4 scFv [13] are linked using a (G4S)3 linker and fused to full length MTBHsp70 with a (G4S)3 linker in between. As shown in Figure 1B only one protein band was observed with a molecular weight of approximately 100 kDa for scFvMTBHsp70 and one protein band with a molecular weight of 70 kDa for MTBHsp70 which match the expected molecular weights of these specific proteins. Endotoxin contamination levels in scFvMTBHsp70 and MTBHsp70 were found to be very low at less than 50 EU per mg of protein. Structure and analysis of scFvMTBHsp70 fusion protein. A anti-MSLN VH and VL are linked with a (G4S)3 linker and fused to full length MTBHsp70 with a (G4S)3 linker. B RAPIDstain based on Coomassie dye following purification and hIgG-Fc tag removal of MTBHsp70 and scFvMTBHsp70. C BR5FVB1 ovarian cancer cells and 40L mesothelioma cells were incubated with 40 ?g/ml scFvMTBHsp70 or 26 ?g/ml MTBHsp70 (blue line) or without either protein (solid) followed by anti-MTBHsp70 (IgG2a) biotinylated anti-IgG2a and Streptavidin-APC and then analyzed by flow cytometry. To confirm that the scFv portion of the fusion protein binds to MSLN on the surface of tumor cells scFvMTBHsp70 or MTBHsp70 was preincubated with 12 ?g/ml recombinant human MSLN for 30 min (red line) before being added to the cells. Data are representative of three independent experiments in duplicate tubes. D Median fluorescence intensity (MFI) values of cells stained with scFvMTBHsp70 or MTBHsp70 normalized to cells stained without either protein. Data are expressed as means?±?SEM in arbitrary units. P values were determined using One-Way ANOVA followed by Turkey™s multiple comparison tests. *p?<?0.05; **p?<?0.01;ns non-significant. E scFvMTBHsp70 binds with peritoneal mesothelial cells at a low level compared to ovarian cancer and mesothelioma cells. Binding of the fusion protein is at very low or undetectable levels on PBLs and splenocytes. Thick line with incubation of scFvMTBHsp70; solid without incubation of scFvMTBHsp70. Data are representative of three independent experiments. scFvMTBHsp70 binds to BR5FVB1 ovarian cancer cells and 40L mesothelioma cells through the interaction of scFv with MSLN on the surface of tumor cells Binding of scFvMTBHsp70 or MTBHsp70 to BR5FVB1 ovarian cancer cells or 40L mesothelioma cells as determined by flow cytometry is shown in Figure 1C and D. Binding of scFvMTBHsp70 to MSLN-expressing tumor cells was almost completely inhibited by preincubation of scFvMTBHsp70 with recombinant human MSLN. Although MTBHsp70 also binds to these MSLN-expressing tumor cells the level of binding is not significantly different from background (p?=?0.187 for BR5FVB1 cells and p?=?0.086 for 40L cells). Furthermore the binding of MTBHsp70 to cancer cells cannot be blocked by recombinant MSLN. These data support the view that binding of scFvMTBHsp70 to these tumor cells occurred via the interaction of the scFv portion of the fusion protein with MSLN on the surface of tumor cells. Binding of these proteins with 40L mesothelioma cells was further compared using fluorescence microscopy. scFvMTBHsp70 shows significantly stronger binding intensity as compared to MTBHsp70 (Additional file 1: Figure S1A and B). In order to determine if scFvMTBHsp70 also binds to normal tissue in addition to tumor cells we incubated the fusion protein with peripheral blood leukocytes (PBLs) splenocytes or peritoneal mesothelial cells from healthy FVB/NJ mice and stained the cells using the same method as was used for staining tumor cells. As shown in Figure 1E scFvMTBHsp70 binds with peritoneal mesothelial cells at a low level compared to ovarian cancer and mesothelioma cells. Binding of the fusion protein is at very low or undetectable levels on PBLs and splenocytes. Since scFvMTBHsp70 may potentially target peritoneal mesothelial cells we also explored whether it could induce inflammation in peritoneal mesothelial tissues. We injected na¯ve mice with saline scFvMTBHsp70 or MTBHsp70 plus P4 scFv at the same doses as those used for tumor therapy described in Method sacrificed the mice 7 days post final treatments and examined haematoxylin and eosin (H&E) stained sections prepared from abdominal and intestinal peritoneum. Light microscopic examination revealed no evidence of inflammation and no infiltration of inflammatory cells such as macrophages or granulocytic cells around the mesothelial cells lining the abdominal and intestinal peritoneum of the actively treated or control animals. Representative microscopic images are shown in Additional file 2: Figure S2. scFvMTBHsp70 significantly prolongs ascites-free survival and overall survival in ovarian cancer- or mesothelioma-bearing mice To determine whether scFvMTBHsp70 can prolong survival in tumor-bearing mice we first evaluated the protein in a syngeneic mouse model of papillary ovarian cancer using immune-competent FVB/NJ mice. As shown in Figure 2A scFvMTBHsp70 prolonged both ascites-free and overall survival time compared with saline or the equimolar mixture of MTBHsp70 plus P4 scFv. To further support the efficacy of this fusion protein in prolonging survival in MSLN-expressing tumor-bearing mice we evaluated this protein in a second syngeneic mouse model of mesothelioma using immune-competent C57BL/6 mice. Animals treated with scFvMTBHsp70 showed significantly prolonged ascites-free and overall survival time compared with saline- or MTBHsp70 plus P4 scFv- treated mice (Figure 2B). Figure 2 A and B Kaplan-Meier survival curves of tumor-bearing mice following treatment with scFvMTBHsp70 control proteins or normal saline. A In a syngeneic mouse model of papillary ovarian cancer in immune-competent FVB/NJ mice scFvMTBHsp70 prolonged ascites-free survival time compared with saline (n?=?10 per group representative of two independent experiments; median survival (Med. sur.)?=?47 days vs. 37.5 days) or the mixture of MTBHsp70 plus P4 scFv (Med. sur. = 39 days). scFvMTBHsp70 also prolonged overall survival time in the mice compared with saline (Med. sur. = 51.5 days vs. 43 days) or the mixture of MTBHsp70 plus P4 scFv (Med. sur. = 43 days). B In a syngeneic mouse model of mesothelioma in immune-competent C57BL/6 mice the fusion protein prolonged ascites-free survival time compared with saline-treated mice (n?=?20 per group pooled from two independent experiments; Med. sur. = 28 days vs. 26 days) or the mixture of MTBHsp70 plus P4 scFv (Med. sur. = 27 days). The fusion protein also prolonged overall survival time compared with saline (Med. sur. = 36 days vs. 31 days). P values were determined using the log-rank test. *p?<?0.05; **p?<?0.01; ***p?<?0.001. scFvMTBHsp70 enhances anti-tumor CD8+ T-cell responses in ovarian tumor-bearing mice To investigate whether the anti-tumor effects of scFvMTBHsp70 was associated with anti-tumor effector CD8+ T-cell responses we re-stimulated splenocytes from ovarian tumor-bearing FVB mice that received different treatments with the CD8+ T-cell Her2/neu epitope or MSLN Ld1 as a negative control ex vivo and analyzed the cells for production of IFN? and Granzyme B using flow cytometry. We previously showed that Her2/neu is expressed by BR5FVB1 cells [25]. Ld1 is an in-house designed H2d-restricted MSLN peptide that did not induce ovarian cancer specific T-cell response in H-2q FVB mice. We demonstrated significantly greater anti-Her2/neu CD8+ T-cell responses in splenocytes from scFvMTBHsp70-treated mice compared to mice treated with saline or a simple mixture of MTBHsp70 plus P4 scFv as measured by IFN? and Granzyme B production by CD8+ T cells (Figure 3A and B). This indicates that scFvMTBHsp70 enhances anti-tumor specific CD8+ T-cell responses in ovarian tumor-bearing mice. However no significant difference was seen in the number of tumor-infiltrating CD8+ T cells and no tumor-infiltrating Foxp3+ T cells were seen in tumors from mice in different treatment groups indicating that scFvMTBHsp70 may improve effector cell function rather than the number of intratumoral CD8+ T cells (Additional file 3: Figure S3A and B). Figure 3 Anti-tumor specific CD8+ T-cell functions in tumor-bearing mice following different treatments. A Splenocytes harvested from mice treated with scFvMTBHsp70 fusion protein equimolar mixture of MTBHsp70 plus P4 scFv or saline (n = 10 per group) were re-stimulated with Her2/neu peptide or MSLN Ld1 peptide. Results are reported as the difference between nonstimulated (media alone) and stimulated cells and expressed as the frequency of parent CD3+CD8+ cells. P values were determined using One-Way ANOVA followed by Dunnett™s multiple comparison tests. B Representative flow data are presented. C In vivo CD8+ T-cell depletion study. FVB/NJ mice were injected i.p. with anti-CD8 mAb or an isotype-matched irrelevant rat IgG2a and were treated with scFvMTBHsp70 or saline as described in the methods. CD8+ T-cell depletion significantly and negatively impacted ascites-free survival in the scFvMTBHsp70 treated BR5FVB1 tumor-bearing animals compared to non depleted actively treated (n = 10 per group representative of two independent experiments; Med. sur. = 32.5 days vs. 48 days) animals. After CD8+ T cells depletion scFvMTBHsp70 treatment did not delay onset of disease (clinically evident ascites) compared with saline (Med. sur. = 32.5 days vs. 31.5 days; p = 0.5938). P values were determined using log-rank test. *p< 0.05; **p < 0.01 ***p < 0.001. scFvMTBHsp70 is able to prime an adaptive tumor-specific immune response that has an absolute requirement for tumor-specific CD8+ T cells To determine whether CD8+ T cells play a major role in the protective anti-tumor effects observed in mice treated with scFvMTBHsp70 we conducted in vivo CD8+ T-cell depletion experiments using monoclonal antibodies. The absence of circulating CD8+ cells in peripheral blood following depletion was confirmed by flow cytometry (Additional file 4: Figure S4A and B). As shown in Figure 3C CD8+ T-cell depletion significantly and negatively impacted ascites-free survival in the scFvMTBHsp70-treated BR5FVB1 tumor-bearing animals compared to non-depleted actively-treated animals. Following CD8+ T-cell depletion scFvMTBHsp70 treatment did not delay onset of disease (clinically evident ascites) compared to saline treatment. Therefore our data suggest that the priming of an adaptive tumor-specific immune response by scFvMTBHsp70 treatment is chiefly mediated by tumor-specific CD8+ T cells. scFvMTBHsp70 stimulates maturation of murine bone marrow-derived dendritic cells In order to investigate immunological mechanisms involved in the scFvMTBHsp70-enhanced anti-tumor immune response we first examined if the scFvMTBHsp70 or MTBHsp70 proteins used in our study could stimulate maturation of bone marrow-derived dendritic cells (BMDCs) as shown in previous studies [1617]. We stimulated CD11c+ BMDCs with 2 ?g/ml of scFvMTBHsp70 or an equimolar amount of MTBHsp70 (1.3 ?g/ml). 1 ?g/ml lipopolysaccharide (LPS) was used as positive control. To determine whether the BMDC maturation was attributable to LPS contamination of the recombinant proteins used in this study we also incubated BMDCs with 0.1 ng/ml LPS which was the equivalent amount of endotoxin found in 2 ?g/ml scFvMTBHsp70. After a 24 h-incubation both scFvMTBHsp70 and MTBHsp70 induced DC maturation indicated by an increase in the expression of CD40 CD80 CD86 and MHC class II molecules in comparison to the control cultures in medium. The increased expression of these DC maturation markers were comparable to those on cells stimulated with 1 ?g/ml LPS. The contamination control showed that addition of 0.1 ng/ml LPS did not replicate the effects of scFvMTBHsp70 or MTBHsp70 allowing us to discriminate the scFvMTBHsp70- or MTBHsp70-specific effects from effects of LPS (Figure 4A and B). Figure 4 scFvMTBHsp70 induces DC maturation and promotes antigen presentation and cross-presentation. A CD11c+ BMDCs isolated form FVB/NJ mice were incubated for 24 h with 2 ?g/ml scFvMTBHsp70 1.3 ?g/ml MTBHsp70 1 ?g/ml LPS as positive control or 0.1 ng/ml LPS as contamination control (thick lines) or medium only (solid) stained for CD11c CD40 CD80 CD86 and MHC II and analyzed by flow cytometry. Histograms were gated on CD11c+ DCs. Data are representative of three independent experiments in duplicate wells. B Median fluorescence intensity (MFI) of LPS- or protein-stimulated BMDCs normalized to MFI of BMDCs maintained in medium. Data are expressed as means?±?SEM in arbitrary units. P values were determined using One-Way ANOVA followed by Dunnett™s multiple comparison tests. C BMDCs cultured from FVB/NJ mice were pulsed with BR5FVB1 cells alone (Column a) or BR5FVB1 cells pre-complexed with MTBHsp70 (Column b) or scFvMTBHsp70 (Column c) and then incubated with BR5FVB1 tumor cell-primed T cells. Intracellular granzyme B and IFN? expressions in CD3+CD4+ and CD3+CD8+ T cells were analyzed by flow cytometry. Data from three independent experiments in duplicate wells are pooled and analyzed using One-Way ANOVA followed by Turkey™s multiple comparison tests. Data are presented as mean?±?SEM. D Representative flow data are presented. E scFvMTBHsp70 enhanced tumor cell immunogenicity in vivo. Results are reported as the difference between nonstimulated (media alone) and stimulated cells and expressed as the frequency of parent CD3+CD4+ or CD3+CD8+ cells. P values were determined using One-Way ANOVA followed by Turkey™s multiple comparison tests. *p?<?0.05; **p?<?0.01; ***p?<?0.001; ****p?<?0.0001. The scFvMTBHsp70 fusion protein increases tumor antigen presentation and cross-presentation by DC in vitro In the current study we demonstrated that splenic CD8+ T cells from scFvMTBHsp70-treated tumor-bearing mice could produce cytokines upon specific tumor antigen stimulation ex vivo which was associated with their antitumor therapeutic efficacy in vivo. To determine whether scFvMTBHsp70 promotes tumor specific T-cell responses by enhancing antigen presentation and cross-presentation by antigen presenting cells we co-cultured BR5FVB1 tumor cell-primed T cells with DCs that had been pulsed with BR5FVB1 tumor cells in the presence of scFv-MTBHsp70 MTBHsp70 or PBS. The scFvMTBHsp70/tumor cell-pulsed DCs induced significantly higher production of IFN-? and Granzyme B from both CD4+ and CD8+ tumor cell-primed T cells as compared with MTBHsp70 or PBS indicating that scFvMTBHsp70 enhances tumor antigen presentation and cross-presentation by DCs (Figure 4C and D). scFvMTBHsp70 enhances tumor cell immunogenicity in vivo Having demonstrated in vitro that scFvMTBHsp70 enhances tumor antigen presentation and cross-presentation by DCs we next explored whether scFvMTBHsp70 enhances tumor antigen presentation and cross-presentation by DCs and consequently enhances tumor cell immunogenicity in vivo. It has been demonstrated that the high density of DCs at dermal sites facilitates the capture of tumor antigens and that local inflammation induces DC maturation and migration into draining lymph nodes where they present antigens to na¯ve T cells generating a tumor specific immune response [26]. We primed FVB mice with an intradermal (i.d.) injection of mitomycin C-treated BR5FVB1 tumor cells followed by a booster i.d. injection of BR5FVB1 tumor cells with or without scFvMTBHsp70 or MTBhsp70. After 20 days we dissociated skin-draining lymph nodes and re-stimulated lymph node lymphocytes with Her2/neu peptides mitomycin C-treated BR5FVB1 tumor cells or BR5FVB1 tumor cell lysate and performed flow cytometric analysis for the presence of Granzyme B-generating CD4+ and CD8+ T cells. As shown in Figure 4E we demonstrated that Granzyme B-generating CD4+ and CD8+ T cells were significantly enhanced in mice that were immunized with scFv-MTBHsp70-bound tumor cells as compared to those in the mice immunized with tumor cells alone MTBHsp70-bound tumor cells or saline. Discussion We have developed a novel protein-based immunotherapy consisting of a fusion of an anti-MSLN scFv of human origin and recombinant mycobacterial heat shock protein 70 that has the ability to adjuvant significant T-cell responses against specific tumor antigens. P4 scFv directed against MSLN a surface antigen overexpressed on several types of tumor cells is used as a means of targeting the immunotherapeutic agent. We have demonstrated that this bifunctional fusion protein effectively binds BR5FVB1 ovarian cancer cells or 40L mesothelioma cells through the interaction of scFv with MSLN on the surface of tumor cells. We found that the fusion protein significantly prolonged survival time in syngeneic mouse models of papillary ovarian cancer and malignant mesothelioma. Treatment with the fusion protein induced significant tumor-specific CD8+ T-cell immune responses in the splenocytes of ovarian tumor-bearing mice. Furthermore in vivo CD8+ T-cell depletion studies demonstrated that this protective antitumor effect is mainly mediated by tumor-specific CD8+ T cells."

Lung_Cancer

"Identifying somatic mutations is critical for cancer genome characterization and for prioritizing patient treatment. DNA whole exome sequencing (DNA-WES) is currently the most popular technology; however this yields low sensitivity in low purity tumors. RNA sequencing (RNA-seq) covers the expressed exome with depth proportional to expression. We hypothesized that integrating DNA-WES and RNA-seq would enable superior mutation detection versus DNA-WES alone. We developed a first-of-its-kind method called UNCeqR that detects somatic mutations by integrating patient-matched RNA-seq and DNA-WES. In simulation the integrated DNA and RNA model outperformed the DNA-WES only model. Validation by patient-matched whole genome sequencing demonstrated superior performance of the integrated model over DNA-WES only models including a published method and published mutation profiles. Genome-wide mutational analysis of breast and lung cancer cohorts (n = 871) revealed remarkable tumor genomics properties. Low purity tumors experienced the largest gains in mutation detection by integrating RNA-seq and DNA-WES. RNA provided greater mutation signal than DNA in expressed mutations. Compared to earlier studies on this cohort UNCeqR increased mutation rates of driver and therapeutically targeted genes (e.g. PIK3CA ERBB2 and FGFR2). In summary integrating RNA-seq with DNA-WES increases mutation detection performance especially for low purity tumors. cover-date 2014 INTRODUCTION Somatically acquired sequence mutations (nucleotide substitutions insertions and deletions) fuel the initiation and progression of cancer (1). Knowledge of mutations in patient specimens informs therapeutic management (23) and in large patient cohorts provides the basis to assess recurrently altered genes that may drive molecular pathogenesis (14“5). DNA whole exome sequencing (DNA-WES) is currently the popular technology to sequence cancer genomes and has led to an abundance of discoveries in many cancer types (46“8). However detecting somatic mutations by DNA-WES with high sensitivity and specificity remains a challenge (79“10) as evidenced by validation rates of 73% in repeated sequencing and by large inter-rater disagreement among different groups analyzing the same sequencing data (710). The biggest challenge is high quality mutation detection in low purity tumors (2911) which are prevalent in widespread cancer types such as breast and lung (12). Advances in somatic mutation detection could improve cancer genome characterization and lead to new diagnostic and therapeutic targets. Somatic mutation detection is dependent on tumor features the sequencing technology and the method of statistical modeling (8“913“17). To detect somatic mutations algorithms compare tumor and patient-matched germline sequencing based on a variety of models (46“7913“17). A tumor's degree of normal contamination and clonal heterogeneity decrease tumor purity. Low purity affects the fraction of mutated DNA observed out of all DNA at a genomic site the mutant allele fraction (MAF) (812). MAF is not often 100% can be slightly above zero in low purity tumors and varies across the genome depending on the prevalence of clones possessing a given mutation and on copy number alterations (7912). DNA-WES targets roughly 200 000 exonic regions and in practice can yield depths of 100X or greater over targeted regions (46). DNA-WES has limitations including variable capture-efficiency and incomplete exome coverage (718). In cases of high MAF mutation detection is straightforward as only a small number of reads are needed to detect the mutation with confidence. The combination of low depth and low MAF make mutation detection very difficult because of low statistical power a result of the scant sample size in which to observe and detect the low prevalence mutation. Increased mutation detection sensitivity and specificity could be achieved by statistical improvements by increasing sequencing quantity or by increasing sequencing quality. In cancer profiling projects such as The Cancer Genome Atlas (TCGA) (46) and in clinical sequencing (219) DNA-WES is utilized for mutation detection while RNA sequencing (RNA-seq) (20) is performed for gene expression fusion transcript and splicing analyses. Beyond those applications RNA-seq provides an observation of the underlying tumor DNA sequence via transcription and can be used to detect sequence variants (21). In fact we have previously used RNA-seq to confirm mutations from DNA-WES (4). A few earlier studies have used RNA-seq alone for genome-wide identification of somatic mutations (22“25) and germline variants (2627). However RNA-seq has challenges including dependency on gene expression which limits the genes that can be measured for sequence mutations and quality control requirements which when not considered result in abundant false positive variants (112128“30). For these reasons RNA-seq has not been the standard for somatic mutation detection. Herein we posed the original hypothesis that integrating patient-matched tumor RNA-seq and tumor DNA-WES would enable superior mutation detection versus DNA-WES alone. We developed a first-of-its-kind method UNCeqR that simultaneously analyzes DNA-WES and patient-matched RNA-seq to detect somatic mutations genome-wide. UNCeqR was applied to large breast and lung cancer cohorts and evaluated with respect to simulation and whole genome sequencing validation. Subsequently genome-wide analysis of UNCeqR mutations led to novel discoveries in tumor genomics. MATERIALS AND METHODS Data sources DNA-WES and RNA-seq alignments in BAM (31) format for 176 lung squamous cell carcinoma cases and for 695 breast cancer cases were acquired from TCGA at ://cghub.ucsc.edu (Supplementary Table S1). RNA-seq were paired 50 nt read from Illumina HiSeq aligned by MapSplice (432). DNA-WES were paired 76“100 nt reads from Illumina Genome Analyzer aligned by BWA (33). All lung and breast cancer cases had germline DNA-WES tumor DNA-WES and tumor RNA-seq and were referred to as the triplet cohorts. A subset of 12 lung and 91 breast tumors also had germline RNA-seq available and were referred to as the quadruplet cohorts. DNA whole genome sequencing (DNA-WGS) was acquired from TCGA for tumors in this cohort (breast: n = 43 lung: n = 17) which consisted of BWA alignments of paired 100 nt reads. Exonic coordinates were extracted from the TCGA Genome Annotation File (http://tcga-data.nci.nih.gov/docs/GAF/GAF.hg19.June2011.bundle/outputs/TCGA.hg19.June2011.gaf) and padded with 10 flanking positions for a total of 222 055 exons. Published mutations (lung: LUSC_Paper_v8.aggregated.tcga.somatic.maf breast: genome.wustl.edu_BRCA.IlluminaGA_DNASeq.Level_2.5.1.0.somatic.maf) expression subtypes DNA copy number calls and tumor purity calls (12) were obtained when available from TCGA. Numerical purity calls of 1 with an incongruent ˜Low purity™ categorical call were censored. Sequencing quality filtering The high quality data filter applies to alignments and genomic positions similar to earlier studies (914). High quality sequenced bases from tumor alignments had base quality ?20 and occurred in a parent alignment with the following properties: mapping quality ? 20 sum of reference mismatches insertions and deletions ?2 a proper pair orientation not a marked duplicate or qc-failure not within the terminal two bases and the singular best alignment. All bases from germline alignments were accepted. High quality genomic positions were those with germline depth ?10 tumor high quality depth ?5 in RNA or DNA no homopolymer > 4 on either side of the site proportion of high quality bases ?0.25 in RNA or DNA and without an insertion or deletion event at 10% allele fraction within 50 positions in germline sequencing. The high quality data filter was applied prior to detecting to tumor variant alleles. The high quality variant filter passes DNA or RNA variant alleles without significant strand bias compared to germline alleles (chi-square P < 0.01) with at least one read on both strands for indel variants with major variant allele prevalence (the proportion of major variant reads out of all variant reads) ?0.75 and a MAD of distance to the end of its aligned read sequence ?1. Somatic mutation detection The UNCeqR algorithm detected somatic mutations within exons based on input of tumor and patient-matched germline sequence alignments. The algorithm applied the following steps to each genomic site within exons: filter for high quality data;identify germline alleles from germline reads that have at least 2% allele prevalence; add population polymorphisms and mapping artifact alleles to germline alleles (see following section ˜Population polymorphisms and mapping artifacts™).Using tumor sequences: let g be the number of reads matching germline allelesdetermine most frequent allele that does not match germline alleleslet k be the number of reads with this major variant allelelet n = k + g.If major variant allele is insertion or deletion re-align nearby indel alleles: scan 20 neighboring sites to find site s with maximum k and same major variant alleleif current site is not s. Move major variant read count from current site to s by incrementing k at s and decrementing g at s by current site's major variant read count.Continue to next site.If high quality variant filter is passed apply statistical test otherwise P = 1 if k = 0 else P = NA.. A set of mutation detection models applied the algorithm with different inputs and statistical models. UNCeqRDNA takes tumor DNA-WES as input and models the corresponding read counts by a beta-binomial distribution. For a variant site with read count \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$k_{{\rm DNA}}$\end{document} the P-value to assess whether this variant allele is a somatic mutation was calculated by \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{} \begin{equation*} P_{{\rm DNA}} = 1 - \sum\limits_{i = 0}^{k - 1} {\left( {\begin{array}{*{20}c} {n_{{\rm DNA}} } \\ i \\ \end{array}} \right)} \frac{{B\left( {i + \alpha _{{\rm DNA}} n_{{\rm DNA}} - i + \beta _{{\rm DNA}} } \right)}}{{B(\alpha _{{\rm DNA}} \beta _{{\rm DNA}} )}} \end{equation*} \end{document}where B is the beta function and ?DNA and ?DNA are parameters of the null distribution where the variant allele is not a somatic mutation. Specifically ?DNA and ?DNA are estimated using randomly sampled sites until 50 000 have passed the high quality data filter in both tumor DNA-WES and tumor RNA-seq. In real data analysis these sampled sites may include real somatic mutations and thus the estimates of ? and ? are conservative which may lead to conservative P-value estimates. However based on mutation rates reported in prior studies (8 mutations per 1 000 000 sites (4)) less than one mutation is expected in these sampled sites and thus our estimates of ? and ? would be good approximations of the estimates from a set of non-somatic mutation sites. The UNCeqRRNA model is identical to UNCeqRDNA substituting tumor RNA-seq for tumor DNA-WES. The UNCeqRMETA model combines P-values from UNCeqRDNA and UNCeqRRNA if RNA and DNA have the same major variant allele irrespective of filtering; otherwise the UNCeqRMETA P-value is set to that of UNCeqRDNA. In effect this condition precludes sites with only RNA variant evidence that are suggestive of RNA-editing (3435) from being called somatic mutations. UNCeqRMETA combines P-values by the Stouffer method (36“38) with weights of the root of their sample size (read depth at the site) as follows: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{} \begin{eqnarray*} &&P_{{\rm META}} = 1 - \\ &&\varPhi\left({\frac{{\varPhi ^{ - 1} \left( {1 - P_{{\rm DNA}} } \right)\sqrt {n_{{\rm DNA}} } + \varPhi ^{ - 1} \left( {1 - P_{{\rm RNA}} } \right)\sqrt {n_{{\rm RNA}} } }}{{\sqrt {n_{{\rm DNA}}^{} + n_{{\rm RNA}}^{} } }}} \right) \end{eqnarray*}\end{document}where \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\varPhi$\end{document} is the standard normal cdf and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\varPhi ^{ - 1}$\end{document} is the inverse of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\varPhi$\end{document} i.e. the quantile function of the standard normal distribution. If the RNA major variant equals the DNA major variant and PDNA = NA PMETA is set to PRNA. DNA and RNA variant read counts among putative false positives were unassociated supporting the usage of Stouffer's method (Supplementary Figure S1). Due to possible ambiguity around insertions and deletions (˜indels™) between DNA and RNA alignments high quality variant sites with an insertion or deletion major variant allele in one alignment and with the same variant allele (insertion or deletion) occurring within 20 sites as the major variant allele in the other alignment were merged to have the same genomic position prior to statistical testing. This indel merge allowed indel variants sites between DNA and RNA that represent the same variant to be recorded at the same site and allowed UNCeqRMETA to combine this DNA and RNA evidence despite slightly different representation in the sequence alignments. UNCeqR software consisted of modified samtools (31) Perl R and VGAM (39). The total number of applied statistical tests is reported in UNCeqR output to provide interested users the possibility of multiple testing adjustment. Population polymorphisms and mapping artifacts Population-level polymorphisms were acquired from dbSNP common version 137 via the UCSC genome browser (40). Variant alleles caused by ambiguous mapping artifacts were calculated by BlackOps (41) using 2 × 50 paired-end reads aligned by MapSplice. UNCeqR was applied to 45 TCGA RNA-seq of matched normal tissue specimens (not part of the lung or breast cohorts) to detect non-reference sequence variants representing further germline polymorphic and alignment artifact alleles. These alleles always augmented germline genotype in UNCeqR thus preventing somatic mutation detections with these alleles even if unobserved in a given germline sequencing. Mutation annotation and analysis Sequence mutations were annotated with a gene a predicted transcript and protein alteration using Annovar (version 8/23/13) (42) and RefSeq gene models. Non-silent mutations referred to non-silent substitution insertion and deletion mutations within translated regions and splice-site mutations. MAFs were compared by one-sided Fisher's exact tests on mutant versus germline read counts with significant results having false discovery rate < 5%. Sequence alignments were visualized using the Integrative Genomics Viewer (43). Germline variant analysis Patient germline variants relative to the reference genome were detected in germline DNA-WES and patient-matched germline RNA-seq using UNCeqRMETA without population polymorphism or mapping artifact allele augmentation P ? 1.1e?9. Germline variant allele fractions were defined and compared between DNA and RNA using the procedure described for somatic mutations. Simulation analysis A novel simulation strategy was followed (diagrammed in Supplementary Figure S2). Using chromosome 2 simulated tumor genomes were generated by randomly sampling 500 sites from exons to define positive mutation sites while the remainder of exon sites served as negative mutations. For the positive sites mutant alleles (substitution insertion or deletion) were randomly sampled at rates 90 5 and 5%. For insertion and deletion alleles allele lengths of 1“6 were randomly sampled at rates 602095 5 and 1%. Positive mutations were spiked into germline DNA-WES and RNA-seq sequencing by editing a specified MAF of read alignments overlapping the site producing simulated tumor alignments. ˜V™ characters were used for substitutions and insertions to avoid overlap with germline genotype. Simulated tumor alignments contained a subset of the total positive mutations because the alignment may have minimal or zero depth at some positive sites reflecting reality that a sequencing technology does not cover every site in the genome at high depth and enabling simulated mutations to occur at RNA-seq and DNA-WES uniquely covered sites. Original tumor sequencing served as simulated germline sequencing. Simulated germline sequencing contained the original somatic mutations which had the effects of expanding germline genotype with additional alleles and not triggering variant detection. UNCeqR models were applied to these simulated data. Limiting to sites with at least a germline depth of 10 model detections were compared to the truth to define receiver operating characteristic (ROC) curves (44). A pair of models was compared by their difference in area under the curve over the false positive rate range of 0 to 1 × 10?5. A P-value was defined using a distribution of differences in area under the curve calculated from 100 permuted models in which the rank of the discrimination threshold (i.e. P-value) between the models at each genomic site was randomly shuffled. Mutation detection by other programs Strelka v2.0.8 (17) was executed on tumor and germline DNA-WES using recommended settings for BWA alignments (strelka_config_bwa_default.ini) DNA-WES (isSkipDepthFilter = 1) and filtering (passed). SNVMix2 (13) was executed upon RNA-seq using default settings. Validation analysis Within exonic regions true positive and false positive mutation detections were defined using patient-matched DNA-WGS alignments based on a published procedure for exome mutation validation (4). Tumor and germline DNA-WGS BAM files were downloaded from ://cghub.ucsc.edu. Specifically tumor and germline DNA-WGS were interrogated at each predicted mutation using samtools (31) with no filtering. True positive mutation predictions met one of two conditions: (1) germline depth ? 10 and read count of predicted mutant allele ?1 in tumor and zero in germline; or (2) germline depth ?10 proportion of mutant allele in germline sequencing not significantly > 2% (proportions test P > 0.25) and proportion of mutant allele in tumor significantly greater than in germline (proportions test P < 0.05). Otherwise false positive mutation predictions had germline DNA-WGS depth ?10 and had depth in tumor DNA-WGS providing ?80% power to detect the mutant allele based the predicted MAF. Power was estimated by a binomial distribution a null probability of 3 × 10?3 an alpha of 0.05 the "

Lung_Cancer

"Thus luteoloside exerts its inhibitory effect on proliferation invasion and metastasis of HCC cells through inhibition of NLRP3 inflammasome. Our results indicate that luteoloside can be a potential therapeutic agent not only as an adjuvant therapy for HCC but also in the control and prevention of metastatic HCC. This work was supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions the Natural Science Foundation for Colleges and Universities in Jiangsu Province (12KJB320001) the National Natural Science Foundation of China (8117101281271225 and 30950031). The funders had no role in study design data collection and analysis decision to publish or preparation of the manuscript. Introduction Hepatocellular carcinoma (HCC) is the third leading cause of cancer-induced death worldwide and patients have a very poor prognosis [1] [2]. Usually HCC is treated by surgical resection or liver transplantation which curative options for the patients when the disease is diagnosed at an early stage. However approximately 70% of patients are inoperable because of tumor metastasis [3]. The current therapeutic options for HCC are not very effective because it is resistant to chemotherapy. Furthermore many anti-cancer drugs have toxicity and side effects for the patients. Thus novel therapeutic strategies are needed to decrease the incidence and severity associated with this cancer [4]. Therefore there is a pressing need for new therapeutic drugs with increased efficacy and decreased toxicity. Natural products continue to provide promising lead compounds and drug candidates in modern antitumor drug discovery. Flavonoids are a heterogeneous group of polyphenolic compounds found ubiquitously in a wide variety of plants. Our recent reports show that they display a wide range of pharmacological properties e.g. anti-inflammatory and antioxidative activities [5] [6]. The anti-tumor activity of flavonoid has recently attracted much attention [7]“[9]. Luteoloside (luteolin-7-O-glucoside; cynaroside; CAS 5373-11-5) a flavone subclass of flavonoids possesses potential anti-inflammatory [10] free radical scavenging [11] and antibacterial [12]. Although it is reported that luteoloside could inhibit the proliferation of colon cancer cells [13] the exact mechanism remains unclear. Furthermore the precise impact of luteoloside on cancer migration and invasion is still unreported. NLR family pyrin domain containing 3 (NLRP3; also known as NALP3 or cryopyrin) is a member of the nucleotide-binding domain and leucine-rich repeat containing gene family of intracellular sensors. When activated NLRP3 forms a protein complex called the inflammasome [14]. The inflammasome combines NLRP3 with the adaptor molecule ASC/PYCARD/TMS/CARD5 Cardinal and pro-caspase-1 to form a multimer. The result is the proteolytic maturation of caspase-1 which cleaves and activates proIL-1? to mature and active IL-1? [14]“[16]. NLRP3 inflammasome plays an important role in the development of many cancer types including melanoma [17] intestinal cancer [18] nasopharyngeal carcinoma [19] skin cancer [20] colorectal cancer [21]. However Whether NLRP3 inflammasome plays an important role in the process of HCC to our knowledge is still unknown. In the present study we demonstrate that luteoloside is a potent agent against human hepatoma cells both in vitro and in vivo and NLRP3 inflammasome might be involved in the signaling of luteoloside-induced suppression of proliferation migration and invasion. Our data provide the mechanistic insight into the role of luteoloside in inhibition of HCC cell proliferation migration and invasion. Materials and Methods Cell Lines and Reagents The human HCC cell lines (Hep3B and SNU-449) were purchased from the American Type Culture Collection. Human hepatoma cells (Huh-7) was purchased from Japanese Collection of Research Bioresources (JCRB Tokyo Japan). The human hepatoma cell line SMMC-7721 was purchased from the Committee on Type Culture Collection of Chinese Academy of Sciences (Shanghai China). The human HCC cell lines (MHCC-LM3 and MHCC97-H) were obtained from the Liver Cancer Institute of Zhongshan Hospital Fudan University (Shanghai China). Luteoloside (Fig. 1A Batch Number: 025-120622 Purity?=?99.7% purchased from Chengdu Herbpurify Co. Ltd. Chengdu China) a naturally occurring flavonoid isolated from the medicinal plant Gentiana macrophylla was dissolved at a concentration of 20 mM in 100% DMSO as a stock solution stored at ?20°C and diluted with medium before each experiment. The final DMSO concentration did not exceed 0.1% throughout the study. .0089961.g001 Luteoloside inhibits proliferation of HCC cells. (A) HPLC analysis of the purity of luteoloside used in the present study. Insert: The chemical structure of luteoloside. (B)“(C) comparative dose- and time-dependent effect of luteoloside on the proliferation potential of HCC cells. The percentage of cell viability in different treatment groups was determined using Cell Counting Kit-8 assay. * P<0.05; ** P<0.01; *** P<0.001; NS not significant (P>0.05) versus non-luteoloside-treated control group. Caspase-3/7 Activity Assay The caspase-3/7 activity assay was conducted as previously described by us [22]. 2.3. DNA Fragmentation Assay DNA from Huh-7 and SMMC-7721 cells (2—106 cells) treated with 0 or 50 µM luteoloside for 24 hours was extracted by using a DNA extraction kit (Beyotime China). "

Lung_Cancer

"(2911) which are prevalent in widespread cancer types such as breast and lung (12). Advances in somatic mutation detection could improve cancer genome characterization and lead to new diagnostic and therapeutic targets. Somatic mutation detection is dependent on tumor features the sequencing technology and the method of statistical modeling (8“913“17). To detect somatic mutations algorithms compare tumor and patient-matched germline sequencing based on a variety of models (46“7913“17). A tumor's degree of normal contamination and clonal heterogeneity decrease tumor purity. Low purity affects the fraction of mutated DNA observed out of all DNA at a genomic site the mutant allele fraction (MAF) (812). MAF is not often 100% can be slightly above zero in low purity tumors and varies across the genome depending on the prevalence of clones possessing a given mutation and on copy number alterations (7912). DNA-WES targets roughly 200 000 exonic regions and in practice can yield depths of 100X or greater over targeted regions (46). DNA-WES has limitations including variable capture-efficiency and incomplete exome coverage (718). In cases of high MAF mutation detection is straightforward as only a small number of reads are needed to detect the mutation with confidence. The combination of low depth and low MAF make mutation detection very difficult because of low statistical power a result of the scant sample size in which to observe and detect the low prevalence mutation. Increased mutation detection sensitivity and specificity could be achieved by statistical improvements by increasing sequencing quantity or by increasing sequencing quality. In cancer profiling projects such as The Cancer Genome Atlas (TCGA) (46) and in clinical sequencing (219) DNA-WES is utilized for mutation detection while RNA sequencing (RNA-seq) (20) is performed for gene expression fusion transcript and splicing analyses. Beyond those applications RNA-seq provides an observation of the underlying tumor DNA sequence via transcription and can be used to detect sequence variants (21). In fact we have previously used RNA-seq to confirm mutations from DNA-WES (4). A few earlier studies have used RNA-seq alone for genome-wide identification of somatic mutations (22“25) and germline variants (2627). However RNA-seq has challenges including dependency on gene expression which limits the genes that can be measured for sequence mutations and quality control requirements which when not considered result in abundant false positive variants (112128“30). For these reasons RNA-seq has not been the standard for somatic mutation detection. Herein we posed the original hypothesis that integrating patient-matched tumor RNA-seq and tumor DNA-WES would enable superior mutation detection versus DNA-WES alone. We developed a first-of-its-kind method UNCeqR that simultaneously analyzes DNA-WES and patient-matched RNA-seq to detect somatic mutations genome-wide. UNCeqR was applied to large breast and lung cancer cohorts and evaluated with respect to simulation and whole genome sequencing validation. Subsequently genome-wide analysis of UNCeqR mutations led to novel discoveries in tumor genomics. MATERIALS AND METHODS Data sources DNA-WES and RNA-seq alignments in BAM (31) format for 176 lung squamous cell carcinoma cases and for 695 breast cancer cases were acquired from TCGA at ://cghub.ucsc.edu (Supplementary Table S1). RNA-seq were paired 50 nt read from Illumina HiSeq aligned by MapSplice (432). DNA-WES were paired 76“100 nt reads from Illumina Genome Analyzer aligned by BWA (33). All lung and breast cancer cases had germline DNA-WES tumor DNA-WES and tumor RNA-seq and were referred to as the triplet cohorts. A subset of 12 lung and 91 breast tumors also had germline RNA-seq available and were referred to as the quadruplet cohorts. DNA whole genome sequencing (DNA-WGS) was acquired from TCGA for tumors in this cohort (breast: n = 43 lung: n = 17) which consisted of BWA alignments of paired 100 nt reads. Exonic coordinates were extracted from the TCGA Genome Annotation File (http://tcga-data.nci.nih.gov/docs/GAF/GAF.hg19.June2011.bundle/outputs/TCGA.hg19.June2011.gaf) and padded with 10 flanking positions for a total of 222 055 exons. Published mutations (lung: LUSC_Paper_v8.aggregated.tcga.somatic.maf breast: genome.wustl.edu_BRCA.IlluminaGA_DNASeq.Level_2.5.1.0.somatic.maf) expression subtypes DNA copy number calls and tumor purity calls (12) were obtained when available from TCGA. Numerical purity calls of 1 with an incongruent ˜Low purity™ categorical call were censored. Sequencing quality filtering The high quality data filter applies to alignments and genomic positions similar to earlier studies (914). High quality sequenced bases from tumor alignments had base quality ?20 and occurred in a parent alignment with the following properties: mapping quality ? 20 sum of reference mismatches insertions and deletions ?2 a proper pair orientation not a marked duplicate or qc-failure not within the terminal two bases and the singular best alignment. All bases from germline alignments were accepted. High quality genomic positions were those with germline depth ?10 tumor high quality depth ?5 in RNA or DNA no homopolymer > 4 on either side of the site proportion of high quality bases ?0.25 in RNA or DNA and without an insertion or deletion event at 10% allele fraction within 50 positions in germline sequencing. The high quality data filter was applied prior to detecting to tumor variant alleles. The high quality variant filter passes DNA or RNA variant alleles without significant strand bias compared to germline alleles (chi-square P < 0.01) with at least one read on both strands for indel variants with major variant allele prevalence (the proportion of major variant reads out of all variant reads) ?0.75 and a MAD of distance to the end of its aligned read sequence ?1. Somatic mutation detection The UNCeqR algorithm detected somatic mutations within exons based on input of tumor and patient-matched germline sequence alignments. The algorithm applied the following steps to each genomic site within exons: filter for high quality data;identify germline alleles from germline reads that have at least 2% allele prevalence; add population polymorphisms and mapping artifact alleles to germline alleles (see following section ˜Population polymorphisms and mapping artifacts™).Using tumor sequences: let g be the number of reads matching germline allelesdetermine most frequent allele that does not match germline alleleslet k be the number of reads with this major variant allelelet n = k + g.If major variant allele is insertion or deletion re-align nearby indel alleles: scan 20 neighboring sites to find site s with maximum k and same major variant alleleif current site is not s. Move major variant read count from current site to s by incrementing k at s and decrementing g at s by current site's major variant read count.Continue to next site.If high quality variant filter is passed apply statistical test otherwise P = 1 if k = 0 else P = NA.. A set of mutation detection models applied the algorithm with different inputs and statistical models. UNCeqRDNA takes tumor DNA-WES as input and models the corresponding read counts by a beta-binomial distribution. For a variant site with read count \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$k_{{\rm DNA}}$\end{document} the P-value to assess whether this variant allele is a somatic mutation was calculated by \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{} \begin{equation*} P_{{\rm DNA}} = 1 - \sum\limits_{i = 0}^{k - 1} {\left( {\begin{array}{*{20}c} {n_{{\rm DNA}} } \\ i \\ \end{array}} \right)} \frac{{B\left( {i + \alpha _{{\rm DNA}} n_{{\rm DNA}} - i + \beta _{{\rm DNA}} } \right)}}{{B(\alpha _{{\rm DNA}} \beta _{{\rm DNA}} )}} \end{equation*} \end{document}where B is the beta function and ?DNA and ?DNA are parameters of the null distribution where the variant allele is not a somatic mutation. Specifically ?DNA and ?DNA are estimated using randomly sampled sites until 50 000 have passed the high quality data filter in both tumor DNA-WES and tumor RNA-seq. In real data analysis these sampled sites may include real somatic mutations and thus the estimates of ? and ? are conservative which may lead to conservative P-value estimates. However based on mutation rates reported in prior studies (8 mutations per 1 000 000 sites (4)) less than one mutation is expected in these sampled sites and thus our estimates of ? and ? would be good approximations of the estimates from a set of non-somatic mutation sites. The UNCeqRRNA model is identical to UNCeqRDNA substituting tumor RNA-seq for tumor DNA-WES. The UNCeqRMETA model combines P-values from UNCeqRDNA and UNCeqRRNA if RNA and DNA have the same major variant allele irrespective of filtering; otherwise the UNCeqRMETA P-value is set to that of UNCeqRDNA. In effect this condition precludes sites with only RNA variant evidence that are suggestive of RNA-editing (3435) from being called somatic mutations. UNCeqRMETA combines P-values by the Stouffer method (36“38) with weights of the root of their sample size (read depth at the site) as follows: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{} \begin{eqnarray*} &&P_{{\rm META}} = 1 - \\ &&\varPhi\left({\frac{{\varPhi ^{ - 1} \left( {1 - P_{{\rm DNA}} } \right)\sqrt {n_{{\rm DNA}} } + \varPhi ^{ - 1} \left( {1 - P_{{\rm RNA}} } \right)\sqrt {n_{{\rm RNA}} } }}{{\sqrt {n_{{\rm DNA}}^{} + n_{{\rm RNA}}^{} } }}} \right) \end{eqnarray*}\end{document}where \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\varPhi$\end{document} is the standard normal cdf and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\varPhi ^{ - 1}$\end{document} is the inverse of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\varPhi$\end{document} i.e. the quantile function of the standard normal distribution. If the RNA major variant equals the DNA major variant and PDNA = NA PMETA is set to PRNA. DNA and RNA variant read counts among putative false positives were unassociated supporting the usage of Stouffer's method (Supplementary Figure S1). Due to possible ambiguity around insertions and deletions (˜indels™) between DNA and RNA alignments high quality variant sites with an insertion or deletion major variant allele in one alignment and with the same variant allele (insertion or deletion) occurring within 20 sites as the major variant allele in the other alignment were merged to have the same genomic position prior to statistical testing. This indel merge allowed indel variants sites between DNA and RNA that represent the same variant to be recorded at the same site and allowed UNCeqRMETA to combine this DNA and RNA evidence despite slightly different representation in the sequence alignments. UNCeqR software consisted of modified samtools (31) Perl R and VGAM (39). The total number of applied statistical tests is reported in UNCeqR output to provide interested users the possibility of multiple testing adjustment. Population polymorphisms and mapping artifacts Population-level polymorphisms were acquired from dbSNP common version 137 via the UCSC genome browser (40). Variant alleles caused by ambiguous mapping artifacts were calculated by BlackOps (41) using 2 × 50 paired-end reads aligned by MapSplice. UNCeqR was applied to 45 TCGA RNA-seq of matched normal tissue specimens (not part of the lung or breast cohorts) to detect non-reference sequence variants representing further germline polymorphic and alignment artifact alleles. These alleles always augmented germline genotype in UNCeqR thus preventing somatic mutation detections with these alleles even if unobserved in a given germline sequencing. Mutation annotation and analysis Sequence mutations were annotated with a gene a predicted transcript and protein alteration using Annovar (version 8/23/13) (42) and RefSeq gene models. Non-silent mutations referred to non-silent substitution insertion and deletion mutations within translated regions and splice-site mutations. MAFs were compared by one-sided Fisher's exact tests on mutant versus germline read counts with significant results having false discovery rate < 5%. Sequence alignments were visualized using the Integrative Genomics Viewer (43). Germline variant analysis Patient germline variants relative to the reference genome were detected in germline DNA-WES and patient-matched germline RNA-seq using UNCeqRMETA without population polymorphism or mapping artifact allele augmentation P ? 1.1e?9. Germline variant allele fractions were defined and compared between DNA and RNA using the procedure described for somatic mutations. Simulation analysis A novel simulation strategy was followed (diagrammed in Supplementary Figure S2). Using chromosome 2 simulated tumor genomes were generated by randomly sampling 500 sites from exons to define positive mutation sites while the remainder of exon sites served as negative mutations. For the positive sites mutant alleles (substitution insertion or deletion) were randomly sampled at rates 90 5 and 5%. For insertion and deletion alleles allele lengths of 1“6 were randomly sampled at rates 602095 5 and 1%. Positive mutations were spiked into germline DNA-WES and RNA-seq sequencing by editing a specified MAF of read alignments overlapping the site producing simulated tumor alignments. ˜V™ characters were used for substitutions and insertions to avoid overlap with germline genotype. Simulated tumor alignments contained a subset of the total positive mutations because the alignment may have minimal or zero depth at some positive sites reflecting reality that a sequencing technology does not cover every site in the genome at high depth and enabling simulated mutations to occur at RNA-seq and DNA-WES uniquely covered sites. Original tumor sequencing served as simulated germline sequencing. Simulated germline sequencing contained the original somatic mutations which had the effects of expanding germline genotype with additional alleles and not triggering variant detection. UNCeqR models were applied to these simulated data. Limiting to sites with at least a germline depth of 10 model detections were compared to the truth to define receiver operating characteristic (ROC) curves (44). A pair of models was compared by their difference in area under the curve over the false positive rate range of 0 to 1 × 10?5. A P-value was defined using a distribution of differences in area under the curve calculated from 100 permuted models in which the rank of the discrimination threshold (i.e. P-value) between the models at each genomic site was randomly shuffled. Mutation detection by other programs Strelka v2.0.8 (17) was executed on tumor and germline DNA-WES using recommended settings for BWA alignments (strelka_config_bwa_default.ini) DNA-WES (isSkipDepthFilter = 1) and filtering (passed). SNVMix2 (13) was executed upon RNA-seq using default settings. Validation analysis Within exonic regions true positive and false positive mutation detections were defined using patient-matched DNA-WGS alignments based on a published procedure for exome mutation validation (4). Tumor and germline DNA-WGS BAM files were downloaded from ://cghub.ucsc.edu. Specifically tumor and germline DNA-WGS were interrogated at each predicted mutation using samtools (31) with no filtering. True positive mutation predictions met one of two conditions: (1) germline depth ? 10 and read count of predicted mutant allele ?1 in tumor and zero in germline; or (2) germline depth ?10 proportion of mutant allele in germline sequencing not significantly > 2% (proportions test P > 0.25) and proportion of mutant allele in tumor significantly greater than in germline (proportions test P < 0.05). Otherwise false positive mutation predictions had germline DNA-WGS depth ?10 and had depth in tumor DNA-WGS providing ?80% power to detect the mutant allele based the predicted MAF. Power was estimated by a binomial distribution a null probability of 3 × 10?3 an alpha of 0.05 the observed depth in DNA-WGS and an alternate probability of the predicted DNA MAF. The number of true positives and false positives were tabulated at each model discrimination threshold i.e. P-value or score. The step function of these points (number of false positives versus number of true positives) generated a performance curve in absolute counts that is equivalent to a ROC curve without the denominators of total positives and negatives which were constant and unknown for the validation cohort. Between models performance curves were compared by area under the curve from 0 to 3000 false positives and by the number of true positives (proportional to sensitivity) at fixed numbers of false positives (proportional to 1 ? specificities) of 250 500 and 1000). P-values were calculated to provide evidence for the change in area under the curve and sensitivity estimates using permutation (see ˜Simulation analysis™ methods). RESULTS Mutation detection models Existing methods to detect somatic mutations are based on either DNA sequencing alone or on RNA sequencing alone and do not integrate more than one type of sequencing (913“17). In order to test whether integrating DNA-WES and RNA-seq enables superior somatic mutation detection versus the current standard of DNA-WES alone a new method was developed called UNCeqR. UNCeqR contains different models for detecting somatic mutations based on different sequencing input and statistical modeling. Briefly UNCeqRMETA integrates tumor DNA-WES and RNA-seq UNCeqRDNA uses tumor DNA-WES and UNCeqRRNA uses tumor RNA-seq. UNCeqR software is available at http://lbg.med.unc.edu/tools/unceqr. Evaluation in simulated tumor sequencing To test our hypothesis that somatic mutation detection based on integrated RNA-seq and DNA-WES is superior to that based on DNA-WES alone simulated tumor genomes were generated so that the entire genome space is a completely defined truth of positive and negative somatic mutations. In brief for each patient's sequencing 500 mutant sites were sampled for each site a mutant allele was randomly sampled and then aligned reads in the real RNA-seq and DNA-WES were edited to have the mutant allele at a rate of a fixed MAF (Supplementary Figure S2). By using real sequencing as the basis of the simulation authentic sequencing depths random errors (sequencing and alignment) and patients™ germline variants were preserved. Sequencing from the lung cancer quadruplet cohort was used for simulation. Patients™ DNA-WES and RNA-seq had large and similar numbers of sequenced nucleotides (DNA-WES median: 10.6 billion RNA-seq median: 10.2 billion; Kruskal-Wallis P = 0.54) indicating no significant imbalance in total sequencing. UNCeqR models were applied to the simulated tumor sequencing and detected mutations were compared against the truth by receiver operating characteristic curves. In simulations with a 10% MAF (Figure 1A) the UNCeqRMETA model had significantly superior performance over UNCeqRDNA (difference in area under the curve P < 0.01); in other words UNCeqRMETA achieved a greater true positive rate (greater sensitivity) at the same false positive rate (same specificity) than UNCeqRDNA. In simulations with a 20% MAF (Figure 1B) UNCeqRMETA continued to be superior to UNCeqRDNA (difference in area under the curve P < 0.01) although the gain in 20% MAF simulations was less (roughly 50% less) than the gain in 10% MAF simulations. This demonstrates that adding RNA-seq improved sensitivity particularly when the mutation signal that is MAF was low. UNCeqRMETA and UNCeqRDNA had large and clear superior performance to UNCeqRRNA which incurred false positives at a higher rate. Alternative ways to integrate RNA and DNA (taking the union or intersection of UNCeqRDNA and UNCeqRRNA) were both inferior to UNCeqRMETA (Supplementary Figure S3). Therefore in simulation UNCeqRMETA achieved superior performance over UNCeqRDNA with the largest gains occurring in mutations with low MAF. Figure 1. Mutation detection performance in simulated tumor genomes. Model performance is displayed as receiver operating characteristic curves. Sensitivity plateaus below 1 because simulated mutations include sites with zero tumor sequencing depth in DNA and/or RNA (see ˜Simulation analysis™ methods). Validation by whole genome sequencing To validate the superior performance of integrated DNA-WES and RNA-seq mutation detection (UNCeqRMETA) over DNA-WES only detection (UNCeqRDNA) tumor and germline whole genome DNA sequencing (DNA-WGS) was used as an independent measure of truth for evaluating DNA-WES and RNA-seq mutation detections. Following a published validation procedure (4) mutation detections were interrogated in patient-matched DNA-WGS to determine if a mutation detection was a true positive that is present in the tumor specimen and absent from the germline specimen or false positive that is absent from the tumor specimen or present in the germline specimen. For each mutation model true positives and false positives were summed at each discrimination threshold (e.g. P-value) to generate a performance curve by which true positive rates could be compared at the same false positive rates (see methods for further description). These curves demonstrated that UNCeqRMETA achieved overall superior performance than UNCeqRDNA (difference in area under the curve P < 0.01) and at fixed false positive thresholds (250 500 and 1000) thus validating the result from simulated tumor genomes (Figure 2). Therefore in real tumor sequencing integrated DNA and RNA mutation detection by UNCeqRMETA outperformed DNA-only mutation detection. Figure 2. Validation of mutation detection by whole genome sequencing. The number of true positives and false positives of mutation detection models are plotted as step functions. At fixed false positive totals (250 500 or 1000) each pair of models was compared for differences in number of true positives (*). The published mutation set (46) did not include mutation rankings and was not amenable to rank-based statistical analysis. Other models displayed overall reduced performance relative to UNCeqRMETA and UNCeqRDNA. As another DNA-only control a leading (45) DNA-WES mutation caller from Illumina Strelka (17) was run on the same DNA-WES. Strelka exhibited inferior performance overall smaller true positive rates at fixed false positive rates and never achieved the sensitivity of UNCeqRMETA or UNCeqRDNA (Figure 2). Strelka had greater sensitivity than UNCeqRMETA or UNCeqRDNA at the highest extreme of specificity; however at UNCeqR's minimum false positive rate Strelka's sensitivity was only ?70% of either UNCeqR model. Providing another DNA-only control previously published mutations of this cohort made by heterogeneous pipelines (46915“16)"

Lung_Cancer

" For the agreement analyses the positive percent agreement (PPA) negative percent agreement (NPA) and overall percent agreement (OPA) with their corresponding 95% confidence intervals (CIs) were calculated. In addition 3-way analyses using MPP as a second reference method was performed to resolve the discrepancy results. Mutation testing methods EGFR PCR Test The EGFR PCR test (cobas EGFR Mutation Test Roche Molecular Systems Inc Branchburg NJ USA) is a CE-IVD marked multiplex allele-specific PCR-based assay designed to detect 41 mutations in exons 181920 and 21 in FFPET specimens of human NSCLC.[28] DNA is isolated using the cobas DNA Sample Preparation Kit (Roche Molecular Systems Branchburg NJ). [29] A minimum of 150 ng of genomic DNA is required for PCR amplification which can typically be isolated from a single 5 µm FFPET section. The EGFR PCR test software version used in this study was designed to detect 29 deletions in exon 19 and 2 L858R variants in exon 21. Macrodissection is only recommended if tumor content is less than 10%; laser capture microdissection is not required. The EGFR PCR test was performed per manufacturer's package insert and results were automatically analyzed and reported. The limit of detection has been validated to 5% mutant alleles. The workflow from DNA isolation to results reporting can be performed in one 8 hour period.[27] LDT Patients in the EURTAC study were screened using a combination of methods developed by Laboratory of Oncology ICO-Hospital Germans Trias i Pujol Barcelona Spain.[11] In short EGFR activating mutations in exons 19 and 21 were initially identified by Sanger sequencing and confirmed by fragment length analysis for exon 19 deletions (FAM-labelled primer in an ABI prism 3130 DNA analyser (Applied Biosystems Foster City CA USA) and by Taqman assay for exon 21 (L858R) mutation. All tumor specimens were from the original biopsy taken prior to any treatment and before randomization. Testing was performed on ? 2mm2 of tissue obtained from one to three slides of 4-micron tissue sections which were subjected to laser capture microdissection to enrich for the presence of tumor cells. DNA was extracted using a standard laboratory protocol and tested at a single site in Spain in Laboratory of Oncology for EGFR activating mutations in exon 19 and 21 using a previously described method. The average turnaround time was approximately 5 days.[26] Bi-directional Sanger sequencing All samples tested by the EGFR PCR test were also tested by Sanger sequencing using DNA from FFPET specimens prepared by the cobas DNA Sample Preparation Kit and sequenced with 2— bidirectional Sanger sequencing by a CLIA-certified laboratory (SeqWright Houston TX USA) using a validated protocol. Repeat Sanger sequencing was performed to compare the detection of EGFR mutations from adjacent sections of tissue to minimize any impact of tissue heterogeneity used for the EGFR PCR test relative to the original LDT results. Also sequencing protocols vary by laboratory in terms of the percent tumor content/sample that requires macrodissection. DNA isolated with the cobas DNA Sample Preparation Kit and used for sequencing required ?10% tumor content. Average turnaround time to results was 7 days. The estimated limit of detection is approximately 20% mutant alleles.[30] Massively parallel pyrosequencing (MPP) Samples with valid EGFR PCR test results with adequate DNA remaining from the initial extraction were tested by a MPP method (454 GS Titanium 454 Life Sciences Branford CT USA) by a CLIA-certified laboratory (SeqWright Houston TX USA) using a validated protocol.[31] This method is a 5“7 day process that involves amplicon generation pooling ligation emulsion PCR amplification and massively parallel pyrosequencing with manual data analysis. The estimated limit of detection for the assay is 1.25% mutant alleles. [27] The MPP method was used to demonstrate performance of the EGFR PCR test to a more sensitive method and as an arbiter for discrepant cases observed between the LDT or the repeat Sanger sequencing. In order to preserve patient privacy associated with tested clinical samples raw MPP sequencing results were anonymized and presented in Table S1. Results Specimen demographics 487 (47%) of 1044 specimens screened for the EURTAC trial using LDTs were available for testing using the EGFR PCR test. The flow of samples through the study is shown in . Patient demographics and baseline tumor characteristics for all patients by LDT status are shown in . There were no significant differences between subsets of patients tested and patients not tested by the EGFR PCR test (p>0.05) for each LDT status (mutation detected mutation not detected) with the exception of country of the screening clinic. Clinical outcomes for patients based on the EGFR PCR test results Of the 174 patients enrolled in EURTAC trial specimens from 134 (77%) patients were available for testing using the EGFR PCR test. Excluding 11 patients with invalid EGFR PCR test results and 7 patients with a result of EGFR mutation not detected a total of 116 (67%) patients were mutation detected by the EGFR PCR test and evaluable for clinical outcome analysis (57 patients in the chemotherapy arm and 59 in the erlotinib arm). Clinical outcomes (PFS BORR and OS) are presented in . Among EGFR PCR test positive patients those treated with erlotinib had a significantly prolonged PFS when compared to patients treated with chemotherapy (p-value <0.0001 log-rank test); the median PFS was 10.4 months (95% CI: 8.0 to 13.8 months) and 5.4 months (95% CI: 4.4 to 6.8 months) for patients treated with erlotinib or chemotherapy respectively (). The HR based on the Cox proportional hazards model was reduced by 66% (HR 0.34; [95% CI: 0.21 to 0.54]) for patients in the erlotinib versus chemotherapy arm. One year after randomization a higher percentage of patients in the erlotinib compared with the chemotherapy arm were event-free (45% [95% CI: 32% to 59% versus 6% [95% CI: 0% to 15%] respectively). .0089518.g002 Kaplan-Meier curves of progression-free survival (PFS) for different treatments in treatment-na¯ve patients with non“small-cell lung cancer and EGFR mutation detected by the EGFR PCR test and LDT. .0089518.t002 Summary of Clinical Outcome Analysis among EGFR PCR test positive patients in the EURTAC trial. Chemotherapy (N?=?57) Erlotinib (N?=?59) PFS (Investigator) Patients with event 37 (64.9%) 47 (79.7%) Patients without eventa 20 (35.1%) 12 (20.3%) ?Time to event (months) ?Medianb (95%CI) 5.4 [4.4; 6.8] 10.4 [8.0; 13.8] ?p-Value (Log-Rank Test) <0.0001 ?Hazard Ratio (95% CI) 0.34 [0.21; 0.54] ?1 year estimate ?Patients remaining at risk 2 24 ?Event-free Rateb (95%CI) 6% [0%; 15%] 45% [32%; 59%] Best Overall Analysis Response rates (95% CI) 14.0% [ 6.3%; 25.8%] 59.3%[ 45.7%; 71.9%] Difference in Response Rates (%) 45.29% [ 28.8%; 61.7%] ?p-Value (Chi-squared Test) <.0001 Odds Ratio (95% CI) 8.93 [3.59; 22.19] OS Patients with event 35 (61.4%) 36 (61.0%) Patients without eventa 22 (38.6%) 23 (39.0%) ?Time to event (months) ?Medianb (95%CI) 20.8 [17.3; 29.4] 25.8 [16.1; 30.0] ?p-Value (Log-Rank Test) 0.5381 ?Hazard Ratio (95% CI) 0.86 [0.54; 1.38] ?2 - year estimate ?Patients remaining at risk 16 23 ?Event-free Rateb (95% CI) 43% [29%; 57%] 51% [38%; 64%] Note: All eligible patients enrolled in study ML20650 were determined as EGFR mutation detected by the LDT. Among those patients with EGFR mutation confirmed by the EGFR PCR test were included in this table. Event ?=? Death or progression free whichever comes first for PFS analysis and event?=?death for OS analysis. a censored. b Kaplan-Meier estimates. C including censored observations. BORR were higher in patients in the erlotinib arm (59.3% [95% CI: 45.7% to 71.9%]) compared to the chemotherapy arm (14.0% [95% CI: 6.3% to 25.8%]). Patients in the erlotinib arm were much more likely to respond to therapy than patients in the chemotherapy arm (odds ratio of 8.93 [95% CI: 3.59 to 22.19]). There was no significant difference in OS between the treatment arms (25.8 months in the erlotinib arm (95% CI: 16.1 to 30.0) and 20.8 months in the chemotherapy arm (95% CI: 17.3 to 29.4) (log-rank test p-value ?=?0.5381)). PFS BORR and OS results for EGFR PCR test positive patients did not differ significantly from those obtained in all patients enrolled in the EURTAC trial which suggests that the EGFR PCR test positive patients are representative of all EURTAC enrolled patients. For the 7 cases where the EGFR PCR test result was mutation not detected and discrepant with the LDT two cases resolved in favor of the LDT by MPP three cases resolved in favor of the EGFR PCR test and one sample was invalid for both Sanger and MPP and the other was in agreement between the EGFR PCR test and Sanger but not MPP (Table S2). Anecdotally 6 of the 7 patients were treated with erlotinib and only one patient achieved greater than or equal to median PFS based on the LDT or the EGFR PCR test. Comparison of EGFR PCR test and LDT results Among 432 specimens with valid results from both the EGFR PCR test and LDT the PPA NPA and OPA were 94.2% (146/155 CI: 89.3% 96.9%) 97.5% (270/277 CI: 94.9% 98.8%) and 96.3% (416/432 CI: 94.1% 97.7%) respectively (Table 3). Thus there was a high concordance between the original LDT and EGFR PCR test results. Among sixteen specimens with discordant results the EGFR PCR test result was confirmed by MPP in 68.8% (11/16) cases (Table S3). .0089518.t003 Table 3 Agreement analysis between EGFR PCR test and LDT. SLCG LDT Total N?=?432 Mutation detected Mutation not detected EGFR PCR test Mutation detected 146 7 153 Mutation not detected 9 270 279 Total 155 277 432* ¢12 samples with inconclusive LDT results and 43 samples with invalid EGFR PCR test results were excluded. Positive percent agreement ?=?94.2% (95% CI [89.3“96.9%]). Negative percent agreement ?=?97.5% (95% CI [94.9“98.8%]). Overall percent agreement ?=?96.3% (95% CI [94.1“97.7%]). Comparison of the EGFR PCR test results with Sanger Sequencing Of 487 specimens tested using the EGFR PCR test and Sanger sequencing 406 gave valid results by both methods (38 were invalid by both methods five were invalid by EGFR PCR test and 38 were invalid by Sanger sequencing). The PPA NPA and OPA for EGFR PCR test compared with Sanger sequencing were 96.6% (112/116 CI: 91.7% 98.7%) 88.3% (256/290 CI: 84.1% 91.5%) and 90.6% (368/406 CI: 87.4% 93.1%; Table 4) respectively. Among 38 discordant results between the EGFR PCR test and Sanger sequencing MPP agreed with the EGFR PCR test result in 30 (78.9%) cases (Table S4). Sanger sequencing detected one L858R not detected by MPP and failed to detect 22 exon 19 deletions and 7 L858R mutations confirmed by MPP. Four MPP results were invalid and the remaining four results agreed with Sanger. The range of percent mutant alleles of the cases missed by Sanger was 3% to 60% with several specimens (n?=?16) under the estimated limit of detection for Sanger. .0089518.t004 Table 4 Agreement analysis between EGFR PCR test and Sanger sequencing. Sanger sequencing Total N?=?406 Mutation detected Mutation not detected EGFR PCR test Mutation detected 112 34 146 Mutation not detected 4 256 260 Total 116 290 406 *81 samples with invalid EGFR PCR test or Sanger sequencing results were excluded. Positive percent agreement ?=?96.6% (95% CI [91.5“98.7%]). Negative percent agreement ?=?88.3% (95% CI [84.1“91.5%]). Overall percent agreement ?=?90.6% (95% CI [87.4“93.1%]). Discussion This study supports the feasibility of performing a retrospective clinical validation of a companion diagnostic from prospective therapeutic clinical trials. The EGFR PCR test results were highly concordant (>96%) with the LDT results used to select patients for the EURTAC trial. As a consequence PFS and BORR of the subset of patients with EGFR mutations detected with the EGFR PCR test were comparable to the full cohort of patients enrolled in the EURTAC trial thus validating the use of the EGFR PCR test to select patients for treatment with anti-EGFR TKIs such as erlotinib. Median PFS survival was 9.7 versus 10.4 months for the erlotinib group and 5.2 versus 5.4 months for the LDTs and EGFR PCR test respectively. The BORR was 58% versus 59.3% months for the erlotinib group and 15% versus 14.0% for the LDTs and EGFR PCR test respectively. Among the 16 discordant specimens between the EGFR PCR test and LDTs a third mutation testing method agreed with the EGFR PCR test result in 11 cases. Of seven cases that were mutation detected by the EGFR PCR test and mutation not detected by the LDT 5 were confirmed by MPP. These patients could have potentially benefited from anti-EGFR TKI therapy. The EGFR PCR test had a number of technical advantages over the LDT used in the EURTAC trial. The LDT required laser capture microdissection of multiple tissue sections and involved 3 separate assays with a median turnaround time of 4.5 days. By comparison the EGFR PCR test required macrodissection only if the tumor content was <10% and can be performed in one day using a single 5 µm section. Furthermore the EGFR PCR test is a commercially available kit-based assay that provides an automated result rather than a manual process subject to interpretation and which can be performed by any qualified clinical laboratory. More than 80% of the specimens tested in this study were small biopsy specimens. The overall invalid rate for Sanger sequencing was 15.6% (76/487) compared to the EGFR PCR assay at 9% (43/487). However the invalid rate for the subset of specimens derived from resected specimens was 0% (0/109) likely because of sufficient tissue availability. Thus the assay is extremely robust when performed on resected tumor specimens and has an approximately 90% success rate on biopsy specimens which are often the only tumor sample available for testing in NSCLC. Sanger sequencing has been widely used to detect EGFR mutations.[30] [32] Similar to the overall invalid rates for the 134 EGFR mutation detected LDT samples enrolled in the EURTAC trial Sanger sequencing had a higher invalid rate (15.7%) compared to 8.2% for the EGFR PCR test. There were also 30 mutation not detected results for Sanger sequencing (22.4%) and 7 mutation not detected results for the EGFR PCR test (5.2%). With 21 invalid results and 30 mutation not detected results Sanger sequencing would have misclassified 38% of patients enrolled in the EURTAC trial. Similar invalid rates have been reported in three other studies suggesting that this methodology has limitations when applied to DNA from FFPET samples.[33] [34] [35] In addition Sanger sequencing has shown poor sensitivity in samples containing less than 20“25% mutant alleles.[35] [36] [37] When we compared the agreement between valid results for the EGFR PCR test with Sanger sequencing (n?=?406) there were 38 discordant cases of which 30 were confirmed by MPP. Twenty-nine of the 30 cases resulted in mutation detected status by the EGFR PCR test and would make these patients eligible for anti-EGFR therapy. Poor sensitivity of Sanger sequencing thus explains the relatively low NPA compared to EGFR PCR test observed in this study. Given the criticality of EGFR mutation testing in selecting specific therapies for life-threatening cancers such as advanced NSCLC robust and accurate assays with rapid turnaround time are preferred. Recent quality assurance studies to ascertain the mutation status of a standard panel of tumors have shown that different clinical laboratories do not correctly identify the mutation status of 100% of the panel members even when they are using the same or similar testing methodologies.[38] [39] For assays that involve mutation analysis of tumor samples important factors contributing to the assay performance include analytic standardization validation of reagents and methodology laboratory experience and the appropriate involvement of the pathologist. In conclusion results of the present study indicate that the cobas EGFR mutation test is a highly robust and highly accurate companion diagnostic assay to select patients for treatment with anti-EGFR therapies such as erlotinib. Supporting Information Table S1 Listing of MPP Result. (PDF) Click here for additional data file. Table S2 Outcome from samples discrepant between the cobas EGFR PCR test and LDT that were enrolled in the clinical trial (cobas MND/LDT MD). (PDF) Click here for additional data file. Table S3 Agreement results between discordant EGFR PCR and LDT tests. (PDF) Click here for additional data file. Table S4 MPP results from resolution analysis of discordant specimens between EGFR PCR test and Sanger sequencing. (PDF) Click here for additional data file. We would like to acknowledge Patrick O'Donnell and Karen Yu for their contributions to this study. References 1 ChapmanPB HauschildA RobertC HaanenJB AsciertoP et al (2011) Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med364: 2507“251621639808 2 OuSH BartlettCH Mino-KenudsonM CuiJ IafrateAJ (2012) Crizotinib for the treatment of ALK-rearranged non-small cell lung cancer: a success story to usher in the second decade of molecular targeted therapy in oncology. Oncologist17: 1351“1375 3 O'BryantCL WengerSD KimM ThompsonLA (2013) Crizotinib: a new treatment option for ALK-positive non-small cell lung cancer. "

Lung_Cancer

"Identifying somatic mutations is critical for cancer genome characterization and for prioritizing patient treatment. DNA whole exome sequencing (DNA-WES) is currently the most popular technology; however this yields low sensitivity in low purity tumors. RNA sequencing (RNA-seq) covers the expressed exome with depth proportional to expression. We hypothesized that integrating DNA-WES and RNA-seq would enable superior mutation detection versus DNA-WES alone. We developed a first-of-its-kind method called UNCeqR that detects somatic mutations by integrating patient-matched RNA-seq and DNA-WES. In simulation the integrated DNA and RNA model outperformed the DNA-WES only model. Validation by patient-matched whole genome sequencing demonstrated superior performance of the integrated model over DNA-WES only models including a published method and published mutation profiles. Genome-wide mutational analysis of breast and lung cancer cohorts (n = 871) revealed remarkable tumor genomics properties. Low purity tumors experienced the largest gains in mutation detection by integrating RNA-seq and DNA-WES. RNA provided greater mutation signal than DNA in expressed mutations. Compared to earlier studies on this cohort UNCeqR increased mutation rates of driver and therapeutically targeted genes (e.g. PIK3CA ERBB2 and FGFR2). In summary integrating RNA-seq with DNA-WES increases mutation detection performance especially for low purity tumors. cover-date 2014 INTRODUCTION Somatically acquired sequence mutations (nucleotide substitutions insertions and deletions) fuel the initiation and progression of cancer (1). Knowledge of mutations in patient specimens informs therapeutic management (23) and in large patient cohorts provides the basis to assess recurrently altered genes that may drive molecular pathogenesis (14“5). DNA whole exome sequencing (DNA-WES) is currently the popular technology to sequence cancer genomes and has led to an abundance of discoveries in many cancer types (46“8). However detecting somatic mutations by DNA-WES with high sensitivity and specificity remains a challenge (79“10) as evidenced by validation rates of 73% in repeated sequencing and by large inter-rater disagreement among different groups analyzing the same sequencing data (710). The biggest challenge is high quality mutation detection in low purity tumors (2911) which are prevalent in widespread cancer types such as breast and lung (12). Advances in somatic mutation detection could improve cancer genome characterization and lead to new diagnostic and therapeutic targets. Somatic mutation detection is dependent on tumor features the sequencing technology and the method of statistical modeling (8“913“17). To detect somatic mutations algorithms compare tumor and patient-matched germline sequencing based on a variety of models (46“7913“17). A tumor's degree of normal contamination and clonal heterogeneity decrease tumor purity. Low purity affects the fraction of mutated DNA observed out of all DNA at a genomic site the mutant allele fraction (MAF) (812). MAF is not often 100% can be slightly above zero in low purity tumors and varies across the genome depending on the prevalence of clones possessing a given mutation and on copy number alterations (7912). DNA-WES targets roughly 200 000 exonic regions and in practice can yield depths of 100X or greater over targeted regions (46). DNA-WES has limitations including variable capture-efficiency and incomplete exome coverage (718). In cases of high MAF mutation detection is straightforward as only a small number of reads are needed to detect the mutation with confidence. The combination of low depth and low MAF make mutation detection very difficult because of low statistical power a result of the scant sample size in which to observe and detect the low prevalence mutation. Increased mutation detection sensitivity and specificity could be achieved by statistical improvements by increasing sequencing quantity or by increasing sequencing quality. In cancer profiling projects such as The Cancer Genome Atlas (TCGA) (46) and in clinical sequencing (219) DNA-WES is utilized for mutation detection while RNA sequencing (RNA-seq) (20) is performed for gene expression fusion transcript and splicing analyses. Beyond those applications RNA-seq provides an observation of the underlying tumor DNA sequence via transcription and can be used to detect sequence variants (21). In fact we have previously used RNA-seq to confirm mutations from DNA-WES (4). A few earlier studies have used RNA-seq alone for genome-wide identification of somatic mutations (22“25) and germline variants (2627). However RNA-seq has challenges including dependency on gene expression which limits the genes that can be measured for sequence mutations and quality control requirements which when not considered result in abundant false positive variants (112128“30). For these reasons RNA-seq has not been the standard for somatic mutation detection. Herein we posed the original hypothesis that integrating patient-matched tumor RNA-seq and tumor DNA-WES would enable superior mutation detection versus DNA-WES alone. We developed a first-of-its-kind method UNCeqR that simultaneously analyzes DNA-WES and patient-matched RNA-seq to detect somatic mutations genome-wide. UNCeqR was applied to large breast and lung cancer cohorts and evaluated with respect to simulation and whole genome sequencing validation. Subsequently genome-wide analysis of UNCeqR mutations led to novel discoveries in tumor genomics. MATERIALS AND METHODS Data sources DNA-WES and RNA-seq alignments in BAM (31) format for 176 lung squamous cell carcinoma cases and for 695 breast cancer cases were acquired from TCGA at ://cghub.ucsc.edu (Supplementary Table S1). RNA-seq were paired 50 nt read from Illumina HiSeq aligned by MapSplice (432). DNA-WES were paired 76“100 nt reads from Illumina Genome Analyzer aligned by BWA (33). All lung and breast cancer cases had germline DNA-WES tumor DNA-WES and tumor RNA-seq and were referred to as the triplet cohorts. A subset of 12 lung and 91 breast tumors also had germline RNA-seq available and were referred to as the quadruplet cohorts. DNA whole genome sequencing (DNA-WGS) was acquired from TCGA for tumors in this cohort (breast: n = 43 lung: n = 17) which consisted of BWA alignments of paired 100 nt reads. Exonic coordinates were extracted from the TCGA Genome Annotation File (http://tcga-data.nci.nih.gov/docs/GAF/GAF.hg19.June2011.bundle/outputs/TCGA.hg19.June2011.gaf) and padded with 10 flanking positions for a total of 222 055 exons. Published mutations (lung: LUSC_Paper_v8.aggregated.tcga.somatic.maf breast: genome.wustl.edu_BRCA.IlluminaGA_DNASeq.Level_2.5.1.0.somatic.maf) expression subtypes DNA copy number calls and tumor purity calls (12) were obtained when available from TCGA. Numerical purity calls of 1 with an incongruent ˜Low purity™ categorical call were censored. Sequencing quality filtering The high quality data filter applies to alignments and genomic positions similar to earlier studies (914). High quality sequenced bases from tumor alignments had base quality ?20 and occurred in a parent alignment with the following properties: mapping quality ? 20 sum of reference mismatches insertions and deletions ?2 a proper pair orientation not a marked duplicate or qc-failure not within the terminal two bases and the singular best alignment. All bases from germline alignments were accepted. High quality genomic positions were those with germline depth ?10 tumor high quality depth ?5 in RNA or DNA no homopolymer > 4 on either side of the site proportion of high quality bases ?0.25 in RNA or DNA and without an insertion or deletion event at 10% allele fraction within 50 positions in germline sequencing. The high quality data filter was applied prior to detecting to tumor variant alleles. The high quality variant filter passes DNA or RNA variant alleles without significant strand bias compared to germline alleles (chi-square P < 0.01) with at least one read on both strands for indel variants with major variant allele prevalence (the proportion of major variant reads out of all variant reads) ?0.75 and a MAD of distance to the end of its aligned read sequence ?1. Somatic mutation detection The UNCeqR algorithm detected somatic mutations within exons based on input of tumor and patient-matched germline sequence alignments. The algorithm applied the following steps to each genomic site within exons: filter for high quality data;identify germline alleles from germline reads that have at least 2% allele prevalence; add population polymorphisms and mapping artifact alleles to germline alleles (see following section ˜Population polymorphisms and mapping artifacts™).Using tumor sequences: let g be the number of reads matching germline allelesdetermine most frequent allele that does not match germline alleleslet k be the number of reads with this major variant allelelet n = k + g.If major variant allele is insertion or deletion re-align nearby indel alleles: scan 20 neighboring sites to find site s with maximum k and same major variant alleleif current site is not s. Move major variant read count from current site to s by incrementing k at s and decrementing g at s by current site's major variant read count.Continue to next site.If high quality variant filter is passed apply statistical test otherwise P = 1 if k = 0 else P = NA.. A set of mutation detection models applied the algorithm with different inputs and statistical models. UNCeqRDNA takes tumor DNA-WES as input and models the corresponding read counts by a beta-binomial distribution. For a variant site with read count \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$k_{{\rm DNA}}$\end{document} the P-value to assess whether this variant allele is a somatic mutation was calculated by \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{} \begin{equation*} P_{{\rm DNA}} = 1 - \sum\limits_{i = 0}^{k - 1} {\left( {\begin{array}{*{20}c} {n_{{\rm DNA}} } \\ i \\ \end{array}} \right)} \frac{{B\left( {i + \alpha _{{\rm DNA}} n_{{\rm DNA}} - i + \beta _{{\rm DNA}} } \right)}}{{B(\alpha _{{\rm DNA}} \beta _{{\rm DNA}} )}} \end{equation*} \end{document}where B is the beta function and ?DNA and ?DNA are parameters of the null distribution where the variant allele is not a somatic mutation. Specifically ?DNA and ?DNA are estimated using randomly sampled sites until 50 000 have passed the high quality data filter in both tumor DNA-WES and tumor RNA-seq. In real data analysis these sampled sites may include real somatic mutations and thus the estimates of ? and ? are conservative which may lead to conservative P-value estimates. However based on mutation rates reported in prior studies (8 mutations per 1 000 000 sites (4)) less than one mutation is expected in these sampled sites and thus our estimates of ? and ? would be good approximations of the estimates from a set of non-somatic mutation sites. The UNCeqRRNA model is identical to UNCeqRDNA substituting tumor RNA-seq for tumor DNA-WES. The UNCeqRMETA model combines P-values from UNCeqRDNA and UNCeqRRNA if RNA and DNA have the same major variant allele irrespective of filtering; otherwise the UNCeqRMETA P-value is set to that of UNCeqRDNA. In effect this condition precludes sites with only RNA variant evidence that are suggestive of RNA-editing (3435) from being called somatic mutations. UNCeqRMETA combines P-values by the Stouffer method (36“38) with weights of the root of their sample size (read depth at the site) as follows: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{} \begin{eqnarray*} &&P_{{\rm META}} = 1 - \\ &&\varPhi\left({\frac{{\varPhi ^{ - 1} \left( {1 - P_{{\rm DNA}} } \right)\sqrt {n_{{\rm DNA}} } + \varPhi ^{ - 1} \left( {1 - P_{{\rm RNA}} } \right)\sqrt {n_{{\rm RNA}} } }}{{\sqrt {n_{{\rm DNA}}^{} + n_{{\rm RNA}}^{} } }}} \right) \end{eqnarray*}\end{document}where \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\varPhi$\end{document} is the standard normal cdf and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\varPhi ^{ - 1}$\end{document} is the inverse of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\varPhi$\end{document} i.e. the quantile function of the standard normal distribution. If the RNA major variant equals the DNA major variant and PDNA = NA PMETA is set to PRNA. DNA and RNA variant read counts among putative false positives were unassociated supporting the usage of Stouffer's method (Supplementary Figure S1). Due to possible ambiguity around insertions and deletions (˜indels™) between DNA and RNA alignments high quality variant sites with an insertion or deletion major variant allele in one alignment and with the same variant allele (insertion or deletion) occurring within 20 sites as the major variant allele in the other alignment were merged to have the same genomic position prior to statistical testing. This indel merge allowed indel variants sites between DNA and RNA that represent the same variant to be recorded at the same site and allowed UNCeqRMETA to combine this DNA and RNA evidence despite slightly different representation in the sequence alignments. UNCeqR software consisted of modified samtools (31) Perl R and VGAM (39). The total number of applied statistical tests is reported in UNCeqR output to provide interested users the possibility of multiple testing adjustment. Population polymorphisms and mapping artifacts Population-level polymorphisms were acquired from dbSNP common version 137 via the UCSC genome browser (40). Variant alleles caused by ambiguous mapping artifacts were calculated by BlackOps (41) using 2 × 50 paired-end reads aligned by MapSplice. UNCeqR was applied to 45 TCGA RNA-seq of matched normal tissue specimens (not part of the lung or breast cohorts) to detect non-reference sequence variants representing further germline polymorphic and alignment artifact alleles. These alleles always augmented germline genotype in UNCeqR thus preventing somatic mutation detections with these alleles even if unobserved in a given germline sequencing. Mutation annotation and analysis Sequence mutations were annotated with a gene a predicted transcript and protein alteration using Annovar (version 8/23/13) (42) and RefSeq gene models. Non-silent mutations referred to non-silent substitution insertion and deletion mutations within translated regions and splice-site mutations. MAFs were compared by one-sided Fisher's exact tests on mutant versus germline read counts with significant results having false discovery rate < 5%. Sequence alignments were visualized using the Integrative Genomics Viewer (43). Germline variant analysis Patient germline variants relative to the reference genome were detected in germline DNA-WES and patient-matched germline RNA-seq using UNCeqRMETA without population polymorphism or mapping artifact allele augmentation P ? 1.1e?9. Germline variant allele fractions were defined and compared between DNA and RNA using the procedure described for somatic mutations. Simulation analysis A novel simulation strategy was followed (diagrammed in Supplementary Figure S2). Using chromosome 2 simulated tumor genomes were generated by randomly sampling 500 sites from exons to define positive mutation sites while the remainder of exon sites served as negative mutations. For the positive sites mutant alleles (substitution insertion or deletion) were randomly sampled at rates 90 5 and 5%. For insertion and deletion alleles allele lengths of 1“6 were randomly sampled at rates 602095 5 and 1%. Positive mutations were spiked into germline DNA-WES and RNA-seq sequencing by editing a specified MAF of read alignments overlapping the site producing simulated tumor alignments. ˜V™ characters were used for substitutions and insertions to avoid overlap with germline genotype. Simulated tumor alignments contained a subset of the total positive mutations because the alignment may have minimal or zero depth at some positive sites reflecting reality that a sequencing technology does not cover every site in the genome at high depth and enabling simulated mutations to occur at RNA-seq and DNA-WES uniquely covered sites. Original tumor sequencing served as simulated germline sequencing. Simulated germline sequencing contained the original somatic mutations which had the effects of expanding germline genotype with additional alleles and not triggering variant detection. UNCeqR models were applied to these simulated data. Limiting to sites with at least a germline depth of 10 model detections were compared to the truth to define receiver operating characteristic (ROC) curves (44). A pair of models was compared by their difference in area under the curve over the false positive rate range of 0 to 1 × 10?5. A P-value was defined using a distribution of differences in area under the curve calculated from 100 permuted models in which the rank of the discrimination threshold (i.e. P-value) between the models at each genomic site was randomly shuffled. Mutation detection by other programs Strelka v2.0.8 (17) was executed on tumor and germline DNA-WES using recommended settings for BWA alignments (strelka_config_bwa_default.ini) DNA-WES (isSkipDepthFilter = 1) and filtering (passed). SNVMix2 (13) was executed upon RNA-seq using default settings. Validation analysis Within exonic regions true positive and false positive mutation detections were defined using patient-matched DNA-WGS alignments based on a published procedure for exome mutation validation (4). Tumor and germline DNA-WGS BAM files were downloaded from ://cghub.ucsc.edu. Specifically tumor and germline DNA-WGS were interrogated at each predicted mutation using samtools (31) with no filtering. True positive mutation predictions met one of two conditions: (1) germline depth ? 10 and read count of predicted mutant allele ?1 in tumor and zero in germline; or (2) germline depth ?10 proportion of mutant allele in germline sequencing not significantly > 2% (proportions test P > 0.25) and proportion of mutant allele in tumor significantly greater than in germline (proportions test P < 0.05). Otherwise false positive mutation predictions had germline DNA-WGS depth ?10 and had depth in tumor DNA-WGS providing ?80% power to detect the mutant allele based the predicted MAF. Power was estimated by a binomial distribution a null probability of 3 × 10?3 an alpha of 0.05 the "

Lung_Cancer

"TP53 17(22.4%) 7(41.2%) 1(16.7%) 9(17.3%) 0(0.0%) VHL 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) Missense mutation distribution in the exons and functional domains of EGFR Out of 76 sequenced lung cancer samples 36.1% of EGFR mutations were missense along exon 19 50.0% were missense along exon 21 5.6% along exon 20 and 8.3% along exon 18 (Fig. 2A). These mutations were in and around the tyrosine kinase domain of EGFR (Fig. 2B“3C). Activating mutations in the tyrosine kinase domain of the EGFR gene stimulates protein tyrosine kinase which leads to activation of signaling pathways associated with cell growth and survival. Mutations in the extracellular domain of EGFR is often associated with the amplification of genes in other cancers [12]. 57.7% of EGFR-associated lung cancers were adenocarcinomas () and 86.7% of EGFR mutations associated with ˜high differentiation™ cancers (). In our sample set 50% of EGFR-associated lung cancers metastasized to local regions 27.3% to lymphs and 46.2% of cancers metastasized to distant ans in our sample set (). .0095228.g002 Missense mutation distribution in the exons and function domains of EGFR. A. Frequencies of detected mutations in different exons. B. Mutation distribution in exons. C. Mutation distribution in functional domains. .0095228.g003 Missense mutation distribution in the exons and function domains of KRAS. A. Frequencies of detected mutations in different exons. B. Mutation distribution in exons. C. Mutation distribution in functional domains. Missense mutation distribution in the exons and functional domains of KRAS Out of 76 sequenced Lung cancer samples 100% of KRAS mutations were missense along exon 2 (Fig. 3A). The 34G>T mutations result in an amino acid substitution at position 12 in KRAS from a glycine (G) to a cysteine (C) or a valine (V). The 64C>A mutation results in an amino acid substitution at position 22 from a glutamine (Q) to a lysine (K) in KRAS. All of these amino acid substitutions occurred along the GTP binding domain of KRAS (Fig. 3A“C). KRAS binds to GTP in the active state and possesses an intrinsic enzymatic activity which cleaves the terminal phosphate of the nucleotide converting it to GDP. Upon conversion of GTP to GDP KRAS is turned off [13]. The result of these mutations is constitutive activation of KRAS signaling pathways. Once it is turned on it recruits and activates proteins necessary for the propagation of growth factor and other receptors' signal such as c-Raf and PI3-kinase [13]. 7.7% of KRAS-associated lung cancers were adenocarcinomas () and 6.1% of KRAS mutations associated with ˜low differentiation™ cancers and 7.4% of KRAS mutations were ˜mid differentiation™ cancers (). In our sample set 6.3% of KRAS-associated lung cancers metastasized to local regions 9.1% to lymphs and 5.1% of cancers metastasized to distant ans in our sample set (). Missense mutation distribution in the exons and functional domains of TP53 Abnormality of the TP53 gene is one of the most significant events in lung cancers and plays an important role in the tumorigenesis of lung epithelial cells. The p53 tumor suppressor gene is located on 17p13 chromosome and spans 20 kb genomic DNA encompassing 11 exons that encodes for a 53KD phosphoprotein [14]. Most TP53 mutations cluster in the TP53 DNA-binding domain which encompasses exons 5 through 8 and spans approximately 180 codons or 540 nucleotides and is not limited to a few particular sequences or codons along this gene [15]. TP53 incurred several deleterious mutations in our sample set of 76 lung cancers mostly along the DNA-binding domain encoded from exon 5 (27.8%) 6 (16.7%) 7 (33.3%) 8 (16.7%) and along the oligomerization domain encoded from exon 10 (15.6%) (Fig. 4A“C). Most TP53 missense mutations lead to the synthesis of a stable protein which lacks its specific DNA-binding and transactivation function and accumulates in the nucleus of cells. Such mutant proteins become inactive and lack the ability to transactivate the downstream target genes that regulate cell cycle and apoptosis [16]. Apart from these mutations affecting the role of TP53 as a tumor-suppressor protein TP53 mutations also endow the mutant protein with ˜gain-of-function™ (GOF) activities which can contribute actively to various stages of tumor progression including distant metastases and to increased resistance to anticancer treatments [17]“[19]. 50.0% of TP53-associated lung cancers were squamous cell carcinoma () and 20.0% of TP53 mutations associated with ˜high differentiation™ cancers and 25.9% of TP53 mutations were ˜mid differentiation™ cancers (). In our sample set 25.0% of TP53-associated lung cancers metastasized to local regions 54.5% to lymphs and 12.8% of cancers metastasized to distant ans in our sample set (). .0095228.g004 Missense mutation distribution in the exons and function domains of TP53. A. Frequencies of detected mutations in different exons. B. Mutation distribution in exons. C. Mutation distribution in functional domains. Multiple mutations and mutation hot spots in human lung cancers Clinical success with individualized combination therapy relies on the identification of mutational combinations and patterns for co-administration of a single or combination of target agents against the detected mutational combinations. Some of the mutations detected in our tumor group through sequencing analysis were not only recurrent and frequent but also occurred in combination with other mutations. Lung cancers in our sample set contained the following: 64.5% of samples had at least one or more missense mutations 19.7% had at least two or more missense mutations 3.9% had at least three or more missense mutations 1.3% had at least four or more missense mutations and 35.5% of samples incurred no deleterious mutations in any of the screened 13500 loci of the potential tumor suppressor and oncogenes (). .0095228.t007 Single and multiple missense mutations (including coding silent/deletion/insertion) in genes of 76 human lung cancer samples. Mutations combination (including Missense point mutations/deletion/insertion) Number of samples with mutation combination Percentage in all sequenced samples Single and more 49 64.50% Double and more 15 19.70% Three and more 3 3.90% Four and more 1 1.30% Five and more 0 0.00% No missense deletion insert or substitution-nonsense 27 35.50% Discussion As lung cancer is the most prevalent cancer and leading cause of cancer deaths worldwide ongoing efforts are aimed to improve prevention diagnosis and effective treatment options for patients with lung cancer. Currently there are a range of treatment options for lung cancer patients with surgery being the most effective for treatment of NSCLCs and chemotherapy with or without radiation therapies as the standard treatment for SCLCs. Because most SCLCs metastasize early to distant ans surgery is often ineffective in curing this cancer. NSCLCs on the other hand are more likely to remain localized during development and are thus are more effectively treated with surgical intervention. Additionally SCLCs are typically much more sensitive to chemotherapy and/or radiation therapy than are NSCLCs [20] [21]. One challenge in proper classification and treatment of lung cancer is the extreme heterogeneity caused by differing genetic biological and clinical properties including response to treatment with over 50 histological variants recognized by the WHO typing system [22] [23]. Because of this correct classification of lung cancer cases is necessary to assure that patients receive optimum management [24]. Due to of these various levels of heterogeneity generalized treatments may be less effective. Alternatively targeted therapy which involves the usage of specially designed drugs to selectively target molecular pathways correlated with the malignant phenotype of lung cancer cells may be more useful [25]. Several genes commonly found to be mutated in various lung cancers have been reported including ALK/ELM4 fusion K-ras EGFR VEGF and p53 yet the entire genetic profile of each form is still not been fully defined [3]. This indicates the necessity of sequencing individual human lung cancers in order to match the use of a single targeted drug or two or more targeted drugs in combination against individual lung cancer-specific mutations. In this study we have used Ion Ampliseq Cancer Panel to sequence 13500 loci in 45 cancer-related genes mainly oncogenes and tumor suppressor genes of 76 human lung cancer samples. We identified frequent mutations in a group of genes including EGFR KRAS and TP53 (). Although most of these genes were already known to be associated with lung cancers the mutated points and the associated mutations in other genes were different in our sample set (Tables 7). As there is increasing awareness about the changes in lung cancer cells in recent times newer drugs that specifically target these changes have been developed. These targeted drugs either work synergistically with the chemotherapy drugs or by themselves with much lesser toxicity due to a selective effect as an alternative to a more systemic modulation of proteins associated with oncogenesis. EGFR inhibitors (Afatinib Erlotinib and Gefitinib) and VEGF inhibitors (Bevacizumab) are currently used for target therapies for NSCLC patients with mutations in the VEGF and EGFR [26]. Erlotinib is a drug that blocks EGFR from signaling the cell to grow. It prevents the progression of lung cancer specifically in non-smoking women and is mostly used in advanced NSCLC treatment that was not responsive to chemotherapy. It is also used as the first treatment in patients whose cancers have a mutation in the EGFR gene [27]. Cetuximab is a monoclonal antibody that targets EGFR which is also used in advanced NSCLC in combination with standard chemotherapy as part of first-line treatment [28]. Like erlotinib afatinib is a drug that blocks the growth signal from EGFR and used for advanced NSCLCs that have mutations in the EGFR gene [29]. Some younger non-smokers with adenocarcinomas are found to have an ALK/EML4 fusion oncogene which is currently a target for the drug Crizotinib [30]. Other drugs currently used to treat lung cancers are not gene-specific and instead target general molecular pathways like folate anitmetabolites (methotrexate and pemetrexed) mitotic inhibitors (docetaxel piclitaxel and vinorelbine) topoisomerase inhibitors (etopophos and topotecan) and nucleoside analogs which interfere with DNA synthesis (carboplatin cisplatin and gemciabine) [31] [32]. Inhibitors of EGFR-directed tyrosine kinase are established to be an effective treatment option for advanced NSCLC not responding to chemotherapy. However EGFR-directed monoclonal antibodies in combination with platinum-based first-line chemotherapy cetuximab combined with cisplatin/vinorelbine and bevacizumab in combination with platinum-based chemotherapy resulted in better survival compared to chemotherapy alone in patients with advanced EGFR-positive NSCLC [33]. Other targeted therapies including dual and multi-kinase inhibitors are in earlier stages of clinical development [34]. With the accumulation of knowledge and experience in next generation technologies it is necessary to expand our understanding in the sensitivity of specific mutations to individualized therapies. "

Lung_Cancer

"Background Compared with FISH and qRT-PCR analyses immunohistochemistry (IHC) is the preferred screening test in most pathology practices for ALK-rearrangement detection. With 100% sensitivity and 98% specificity the VENTANA ALK (D5F3) IHC assay has been approved in the EU and some Asian countries for ALK-rearrangement detection. However an automated Ventana IHC platform is not available in most pathology labs. In this study we evaluated the applicability of conventional IHC with D5F3 antibody in routine pathological practice and proposed detection methods and procedures that ensure that patients with ALK+?are not missed. Methods FISH and IHC analyses were performed on 297 lung adenocarcinoma cases. VENTANA IHC and qRT-PCR assay were applied to evaluate ALK-fusion status in the discordant cases of FISH and IHC. The association of ALK+?with clinicopathological characteristics was statistically analyzed. Results IHC had 100% sensitivity and 81.8% specificity for detecting ALK+. Eight ALK-expressed cases were ALK- five of which had ALK fusion detected by qRT-PCR analysis. Three of these five cases showed ALK expression using VENTANA IHC assay. ALK+ was associated with younger age and lymph node metastasis in this Chinese lung adenocarcinoma patient cohort. Conclusions The advantages of low cost and 100% sensitivity allow conventional IHC to serve as a robust diagnostic tool for screening patients with ALK+ especially in pathology labs without a VENTANA IHC platform. For cases in which ALK is weakly expressed qRT-PCR is necessary as a diagnostic test for ALK-fusion detection. Virtual slides The virtual slide(s) for this can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2269448351088278. Immunohistochemistry Fluorescence in situ hybridization qRT-PCR ALK rearrangement D5F3 antibody Lung adenocarcinoma Introduction Lung cancer is the most common cause of cancer death worldwide estimated to be responsible for nearly 1.38 million cancer deaths per year [1]. Despite improvements in the prevention and treatment of lung cancer the overall 5-year survival rate remains at 15% [2]. Efforts have been made to develop new treatment strategies. In recent years rearrangements of the anaplastic large cell kinase (ALK) gene have been discovered in approximately 5% of lung adenocarcinomas resulting in the constitutive expression of a fusion protein - most commonly EML4-ALK - with oncogenic activity [3-7]. Crizotinib a potent and specific small molecule inhibitor of both ALK and c-MET tyrosine kinases [8-10] was approved by the Food and Drug Administration (FDA) for the treatment of non-small-cell lung cancer (NSCLC) patients with ALK gene rearrangement (ALK+). The FDA-approved Vysis ALK Break Apart FISH Probe Kit (Abbott Molecular) was mandated for ALK+?testing in crizotinib trials which in a sense indicates that FISH analysis has been clinically validated. However the FISH detection of ALK gene rearrangement in routine surgical pathology practice remains impractical due to financial and technical problems. Theoretically reverse transcriptase-polymerase chain reaction (RT-PCR) is a standard method for determining the fusion genes but the requirement of fresh frozen tissue samples for extracting RNA has limited its application in clinical practice. IHC is relatively inexpensive and faster and is performed routinely in most surgical pathology practices. Mutation-specific IHC has been demonstrated as a reliable prescreening test for detecting EGFR mutations in lung adenocarcinoma [11]. Recently a fully automated VENTANA ALK (D5F3) assay was developed using D5F3 primary antibody (commercialized by Cell Signaling Technology or CST) and VENTANA OptiView DAB detection for use with VENTANA automated platforms. Our group demonstrated that the sensitivity and specificity of the VENTANA ALK assay were 100% and 98% respectively [12]. The VENTANA ALK (D5F3) IHC assay was approved to detect ALK rearrangement in pathology practice in the EU and some Asian countries including China and Japan. However the application of the VENTAMA ALK IHC assay requires a VENTANA automated platform which is not available in most pathology labs. In this study we applied IHC analysis using CST™s D5F3 antibody to detect ALK rearrangement in a Chinese lung adenocarcinoma patient cohort to assess the sensitivity and specificity of IHC analysis. In the third detection method a qRT-PCR assay (Amoy Diagnostics Xiamen China) approved by European Conformity (CE marking) and the China Food and Drug Administration (CFDA) was applied on formalin-fixed paraffin embedded (FFPE) samples to analyze the discordant cases of IHC and FISH. Materials and method Clinical materials and tissue microarray (TMA) construction This study included 297 FFPE samples with lung adenocarcinoma diagnosed at the Cancer Institute and Hospital Chinese Academy of Medical Sciences (CICAMS) in Beijing between January 2009 and March 2012. Among the 297 cases 218 were unselected and 79 cases were not effectively treated using conventional treatment. Among the 218 unselected cases 178 (with enough tissue) were constructed onto seven TMAs to represent biopsies. A 1.5 mm diameter core was taken from the cancer area based on hematoxylin and eosin (H&E)-stained sections of each sample. The remaining 39 unselected cases (without enough tissue) and 79 selected cases were cut into tissue sections. In the cases where tissue sections/cores fell off the slides during FISH or IHC analysis tissue sections were re-cut. The collection of these specimens was approved by the National Cancer Center Ethics Committee. The patients™ medical records were reviewed to obtain their clinicopathological parameters including age at diagnosis sex smoking history tumor size histological classification and pathological TNM stage. IHC Immunohistochemical staining was performed on 4 ?m-thick FFPE tissue sections or TMAs. Briefly the slides were deparaffinized and antigen retrieval was then performed in a steam cooker for 1.5 minutes in 1 mM EDTA pH 9.0 (Maixin Biological Techology Co. Ltd. Fuzhou China). ALK (D5F3) rabbit monoclonal (Cell Signaling Technology Danvers MA USA) was applied at 1:150 in SigalStain antibody diluent (Cell Signaling Technology Danvers MA USA) for 1 h. Universal secondary antibody (DAKO) was applied for 15 min. Diaminobenzidine or 3-amino-9-ethylcarbazole was used as chromogens and slides were counterstained with haematoxylin before mounting. The criteria for scoring ALK were as follows. First the intensity was graded as 0 negative; 1 weak (light brown); 2 moderate (brown); and 3 strong (dark brown). Second the proportion of positive tumor cells was graded: 0 no positive cells; 1 <10%; 2 11%-30%; 3 31%-50%; 4 51-70%; and 5 >70%. A final score was derived by adding the two primary scores. Final scores of 0 were defined as œnegative expression (?); scores of 2“5 as œweakly positive expression (+); and scores of 6“8 as œstrongly positive expression (++). Fully automated VENTANA ALK (D5F3) IHC analysis was performed as previously described [12]. According to the manufacture™s scoring algorithm a binary scoring system (positive or negative for ALK status) was adopted to evaluate the staining results. The presence of strong granular cytoplasmic staining in tumor cells (any percentage of positive tumor cells) was considered to be ALK positive while the absence of strong granular cytoplasmic staining in tumor cells was deemed to be ALK negative. FISH FISH was performed on 3 ?m-thick FFPE tumor tissues using a break-apart probe specific to the ALK locus (Vysis LSI ALK Dual Color Break Apart Rearrangement Probe; Abbott Molecular Abbott Park Illinois USA) according to the manufacturer™s instructions. Tumor cells the nuclei of which had one or more FISH signals of each color were enumerated. A positive cell was defined as one in which the nucleus had split signals (two or more signal diameters apart) or a single orange signal (deleted green signal) in addition to fused and/or split signals. A sample was considered positive if >25 cells out of 50 were positive. If a sample had 5 to 25 positive cells (10 to 50%) another 50 tumor cells were counted. If the average percentage of positive cells in 100 tumor cells was <15% (<15/100) the sample was considered negative. If the average percentage of positive cells was ?15% (?15/100) the sample was considered positive. TMA cores with high backgrounds or very weak signals that affected the signal assessment were excluded from the analysis. Real-time quantitative reverse transcription PCR (qRT-PCR) The EML4-ALK fusion mRNA was detected by qRT-PCR using an AmoyDx EML4-ALK Fusion Gene Detection Kit (Amoy Diagnostics Xiamen China). Briefly total RNA was extracted with an AmoyDx FFPE RNA Kit (Spin Column) from 5“10 ?m-thick FFPE sections with over 70% tumor cells. For each sample 100“500 ng of extracted RNA was used for reverse transcription into cDNA at 42°C for 1 h. Real-time PCR was then carried out in each of the four reactions of the EML4-ALK Fusion Gene Detection Kit according to the manufacturer™s protocol. Reaction 1 amplifies EML4-ALK variants 12 3a and 3b (variants 1/2/3a/3b); reaction 2 amplifies EML4-ALK variants 4 and 4?; reaction 3 amplifies EML4-ALK variants 5a 5b 5? and 8 (variants 5a/5b/5?/8); and reaction 4 amplifies the reference gene beta-actin. All of the assays were performed on an Agilent Mx3000P QPCR instrument (Agilent Technologies Santa Clara CA). The following PCR procedure was used: an initial denaturation at 95°C for 5 min followed by 95°C for 25 s 64°C for 20 s and 72°C for 20 s to ensure the specificity and 31 cycles of 93°C for 25 s 60°C for 35 s and 72°C for 20 s to perform the data collection. The quantitative judgment was according to the fusion fluorescence signal. Assay reactions achieving Ct values of ?30 cycles were considered positive for one of the variants detected by that reaction mixture. A housekeeping gene (beta-actin) was used to control the integrity of the RNA. Statistical analysis The statistical analysis of the tumors™ size and age was carried out using Student™s t tests. The values are shown as mean?±?SD. The relationship between ALK+?and clinicopathological variables was analyzed with the chi-square test. Statistical significance was defined as p?<?0.05. Results Concordance of ALK IHC and FISH Using the newly developed antibody ALK (D5F3) we analyzed ALK expression in 297 lung adenocarcinoma cases. The cases with strongly or weakly positive ALK expression showed readily appreciable cytoplasmic staining (Figures 1A and 1B). In contrast the cases with negative expression did not show any discernable staining (Figure 1C). Strong ALK expression was identified in 32 cases weak expression in 12 cases and no expression in 253 cases (Table 1). Figure 1 Representative cases of IHC staining FISH and qRT-PCR analysis in lung adenocarcinoma. (A-C) ALK IHC staining using CST™s D5F3 antibody. (A) Cytoplasmic reactivity of strong intensity in tumor cells (original magnification x40). (B) Weak to moderate cytoplasmic reactivity in tumor cells (original magnification x100). (C) No staining in tumor cells (original magnification x200). (D-F) FISH analysis using Vysis ALK Break-Apart probes. (D) The ALK+ case in which the majority of cells contained more than one copy of a single green signal without a corresponding orange signal in addition to fused signals using FISH analysis. Green arrow represents more than one copy of a single green signal red arrow represents single red or split red-green signals indicative of ALK-rearrangement and yellow arrow represents touching red-green signals not indicative of ALK-rearrangement. (E)ALK+ case with split red-green signals. (F) NSCLC case without ALK rearrangement. (G-I) VENTANA ALK (D5F3) IHC assay revealed no expression in ALK- patients and strong expression in ALK+ patients. (G) Strong ALK expression (original magnification x20). (H) Unspecific staining (original magnification x40). (I) No ALK expression (original magnification x20). (G-L) Graphs from qRT“PCR showing change in the normalized reporter signal (delta Rn) against PCR cycle number. (J)ALK fusion was detected at around 14 cycles of qRT-PCR analysis in a case with strong ALK expression. (K)ALK fusion was detected at around 28 cycles in a case with weak ALK expression. (L) No ALK fusion was detected with endogenous control gene beta-actin expressed normally. Table 1 Correlation of IHC and FISH IHC Total ++ a + b - c FISH+ 31(96.9%) 5(41.7%) 0(0%) 36 FISH- 1(3.1%) 7(58.3%) 242(100%) 250 Total 32 12 242 286 astrongly positive ALK expression. b weakly positive ALK expression. cnegative ALK expression. "

Lung_Cancer

" Silent lunch and tea break 7. Taking care of yourself - Sitting meditation ending in choiceless awareness - Exercise on taking care of yourself by examining how to improve balance in life - Meditation without CD - Yoga or walking meditation - Reflect on training - 3-min breathing space 8. The rest of your life - Bodyscan - Reflection on training - Further sources of information - Short sitting meditation - Maintaining practice Outcome measures Primary outcome measure Psychological distress The primary outcome measure is the total score on the HADS [39-41] which is developed to measure psychological distress in somatic patient populations. It consists of a 7-item anxiety (HADS-A) and 7-item depression (HADS-D) subscale. The HADS shows good psychometric properties in the general medical population including oncology patients [42]. Internal consistency as measured with Cronbach™s ? varied from .84 to .90 [4042].Test-retest reliability was good as Pearson™s r > .80 were obtained [4043]. Though the cut-off scores of the HADS vary among populations [44] in lung cancer patients they have found to be <8 versus ?8 on the HADS-A or HADS-D [45]. The HADS has been shown to be highly correlated with the Beck Depression Inventory [42]. It has previously been used in intervention studies of mindfulness and shown to be sensitive to change (e.g. [46]). Secondary outcome measures Quality of life (only for patients) The European anisation for Research and Treatment of Cancer (EORTC) Core Quality of Life Questionnaire (QLQ-C30) [47] is included along with the supplemental Lung Cancer questionnaire module (QLQ-LC13) [48]. The QLQ-C30 is designed to use in clinical trials on physical treatments for cancer patients. It incorporates five functional scales (physical role cognitive emotional social) three symptom scales (fatigue pain nausea and vomiting) a global health and quality of life scale and an array of single-item symptom measures. After revisions in the role functioning global health and physical functioning scale internal consistency of the subscales varied between .65 and .94 except for the cognitive functioning scale with ? varying from .56 to .63 [474950]. Test-retest reliability varied from .63 to .86 [51]. The lung cancer questionnaire module is designed to supplement the core questionnaire and comprises specific symptoms associated with lung cancer (coughing haemoptysis dyspnoea pain) and side-effects from conventional chemo- and radiotherapy (hair loss neuropathy sore mouth dysphagia). While the multi-item dyspnoea scale showed high internal consistency the pain subscale did not. When combined with the dyspnoea and pain items of the core questionnaire both the dyspnoea (? = .86) and pain (? = .71) subscale showed high internal consistency. Since the QLQ-C30 and QLQ-LC13 are mainly focused on physical symptoms we added the items Social Interaction and Alertness Behavior of the Sickness Impact Profile (SIP) [52]. Internal consistency was .94 and test-retest reliability was .92. The SIP correlated with self-assessed sickness and dysfunction [52]. Caregiver appraisal (only for partners) We use the 9-item Self-Perceived Pressure from Informal Care (SPPIC) [53] to assess the extent to which caregiving is experienced as burdensome. To also measure positive aspects of caregiving the 9-item subscale Care-Derived Self-Esteem of the Caregiver Reaction Assessment (CRA-SE) [54] is included. Internal consistency of the SPPIC was .79 and of the CRA-SE was .73. The SPPIC and CRA-SE were unrelated to each other [55]. Relationship quality To measure relationship satisfaction we included the 10-item Satisfaction subscale of the Investment Model Scale (IMS-S) [56]. The IMS-S starts with 5 items that measure concrete examplars of satisfaction to enhance the comprehensibility of the global items which are utilized to form the construct. Internal consistency varied from .79 to .95 and the IMS-S was related to the Dyadic Adjustment Scale. Also the Mutual Interpersonal Sensitivity scale (MIS) [57] is included to measure communication between partners about the cancer. It contains 18 items and is divided into two scales: open communication and avoiding negative thoughts about the cancer. Spirituality is measured with the Spiritual Attitude and Involvement List (SAIL) [58] and consists of 26 items divided into the subscales meaningfulness trust acceptance caring for others connectedness with nature transcendent experiences and spiritual activities. The internal consistency varied from .74 to .88 and test-retest reliability varied from .77 to .92. All subscales except for connectedness with nature were related with the Functional Assessment of Chronic Illness Therapy “ Spiritual Well-Being Scale. Costs (only for patients) The cost-effectiveness evaluation is carried out from a societal perspective considering direct as well as indirect health costs. Data on costs are collected prospectively using a diary in which participants register a) health care utilization: the type of care and its duration and b) cancer-related absence from work. Unit cost estimates are derived from the national manual for cost prices in the health care sector [59]. Costs of reduced ability to work are estimated using the friction costs method which results in a more realistic estimate than the human capital approach [60]. Treatment costs of MBSR are calculated using activity-based-costing methods thus measuring actual resources (time of therapist time of patients facilities) used. All unit cost prices are adjusted to 2013 prices. Unit cost estimates are combined with resource utilization data to obtain a net cost per patient over the entire follow-up period. Process measures Mindfulness skills are examined with the 39-item Five Facet Mindfulness Questionnaire (FFMQ) [6162]. The FFMQ is based on an exploratory factor analysis of five mindfulness measures which allowed items from different instruments to form factors providing an empirical integration of these independent attempts to operationalize mindfulness. This led to the following five subscales: observing describing acting with awareness non-judging of inner experience and non-reactivity to inner experience. Internal consistency varied from .72 to .93 among the different subscales. Most subscales were related to meditation experience Psychological Well-Being scales and psychological symptoms including the Brief Symptom Inventory [61]. FFMQ is sensitive to change in mindfulness-based interventions and is found to mediate the relationship between mindfulness practice and improvements in psychological symptoms (e.g. [63]). Self-compassion is assessed with the Self Compassion Scale (SCS) [6465] which has 26 items and is divided into six subscales: self-kindness versus self-judgment common humanity versus isolation and mindfulness versus over-identification. Internal consistency of the different subscales varied from .75 to .81 and test-retest reliability varied from .80 to .93. SCS correlated moderately with self-esteem measures including the Rosenberg Self-Esteem Scale. Furthermore whereas the self-esteem measures correlated significantly with the Narcissistic Personality Inventory the SCS was unrelated to narcissism [64]. SCS is sensitive to change through mindfulness-based interventions and is found to mediate MBCT™s treatment effects [66]. To measure rumination we administered the extended version of the Ruminative Response Scale (RRS-EXT) [67] Raes and Hermans: The revised version of the Dutch Ruminative Response Scale unpublished instrument]. The RRS-EXT contains 26 items in which a more adaptive thinking style (i.e. reflection) is distinguished from a more maladaptive one (i.e. brooding). Internal consistency varied from .72 to .77 and test-retest reliability varied from .60 to .62 for the brooding and reflection subscales. The concept of rumination seems to be sensitive to change through mindfulness-based interventions and has been shown to mediate the effect of MBSR on depressive symptoms in oncology patients [68]. The psychological stress reaction is measured with the 15-item Impact of Event Scale (IES) [6970] which assesses two categories of responses: intrusive experiences and avoidance of thoughts and images associated with the event. Internal consistency varied from .65 to .92 [71] and test-retest reliability varied from .79 to .87 among the subscales [69]. IES correlated with anxiety and depression subscales of the General Health Questionaire. Adherence to MBSR is assessed during the entire study period with a calendar on which participants in the MBSR condition fill out on a daily basis whether they adhere to the mindfulness exercises: either formal practice (e.g. meditation exercise like the bodyscan) informal practice (e.g. activity with awareness) or no exercise. Adherence to MBSR has been shown to mediate the effects of MBCT on depressive symptoms [72]. Statistical analysis plan Sample size To determine the required sample size first the sample size was calculated that would be needed for a simple t-test and subsequently it was corrected for clustering repeated measurements and baseline. A two-sided t-test on the total HADS score [3940] (i.e. our primary outcome measure examining psychological distress (HADS-total) anxiety symptoms (HADS-A) and depressive symptoms (HADS-D)) would require 64 participants in each group to have 80% power to detect a medium-sized difference (effect size = 0.5) with alpha = 0.05. To correct for clustering we multiplied this sample size of 64 with the design factor (1 + (n ? 1) * ICC) where n denotes the cluster size and where ICC denotes the intra-cluster correlation. In our study the treatment groups will consist of 14 people of whom about 7 will be patients. With n = 7 and an estimated ICC = 0.01. [72] the correction factor equals 1.06. To correct for repeated measurements and the use of the baseline measurement as a covariate we multiplied the required sample size by the design factor 1+?/2??02 where ? denotes the correlation between the post-treatment HADS measurements and ?0 denotes the correlation between the baseline HADS with the post-treatment HADS measurements. With ? = 0.8 and ? = 0.5 as conservative estimates the second design factor equals 0.65. Consequently after correction for clustering and covariates we arrived at a required sample size of 0.65 * 1.06 * 64 = 44 patients per arm. So 88 patients with lung cancer would be required for the study. Based on our pilot study [van den Hurk Schellekens Molema Speckens and van der Drift in preparation] we expect a 20% drop-out rate. Therefore we intend to include 110 patients and 110 partners. Primary analyses The samples of lung cancer patients and partners will be analyzed separately. Baseline characteristics of the population will be compared between MBSR and control group to ensure that key variables were evenly distributed by randomization. First analyses will be based on the intention-to-treat approach. Next we will perform per-protocol analyses with the treatment-adherent sample (i.e. in the MBSR condition participants have to attend at least four of the eight MBSR sessions [73] and in the TAU condition participants do not attend a mindfulness-based programme). We will use linear mixed models to analyze all outcome variables (i.e. psychological distress quality of life (only for patient) caregiver appraisal (only for partner) relationship quality and spirituality) with treatment as fixed factor baseline measurement as covariate and a random intercept based on MBSR group. This procedure will use all observed data in our analyses. In addition Cohen™s d effect size [74] will be reported based on the difference between the group means on baseline and follow-up scores divided by the pooled standard deviation at baseline and follow-up. Secondary analyses Cost effectiveness The quality of life measures (i.e. QLQ-C30; QLQ-LC13) will be used to calculate Quality of Adjusted Life Years (QALYs) for each individual. Costs and effects (in terms of QALYs) will be combined in the incremental cost-effectiveness ratio (ICER). The ICER expresses cost-effectiveness in terms of incremental costs per QALY gained. To estimate confidence intervals for the mean of the ICER a non-parametric bootstrapping method will be used performing 1000 replications of the original data. In order to express the implications of the cost-effectiveness results more clearly a cost-acceptability curve will be constructed. In case of dominance a full cost analysis will be conducted to estimate the mean savings per patient per year. Mediation analyses To examine the possible underlying mechanisms of change in MBSR mediation analyses will be conducted. Only the data of the treatment-adherent sample will be included in these analyses. By means of a multiple mediation model suggested by Preacher and Hayes [75] we will test the mediating effect of mindfulness skills self-compassion rumination and adherence to MBSR on psychological distress quality of life (only in patients) caregiver appraisal (only in partners) relationship quality and spirituality. Discussion In the last ten years MBSR has not only proven to be a feasible and acceptable intervention in cancer patients [76] but it also seems to be effective in reducing psychological distress [30]. However the generalization of these results is limited because most participants were female patients with breast cancer. A large part of lung cancer patients already have advanced cancer at time of diagnosis and are confronted with a poor prognosis and low health status. Consequently they more often report psychological distress than patients with other diagnoses of cancer [89]. Hence it is not yet clear whether MBSR is a feasible acceptable and effective intervention in patients with lung cancer. Moreover little is known about the effectiveness of MBSR in partners of cancer patients [30] though they also often report psychological distress. Our pilot study of 19 lung cancer patients and 16 partners participating in an MBSR course provides preliminary evidence that MBSR is feasible and acceptable in this population (van den Hurk Schellekens Molema Speckens and van der Drift in preparation). The current trial will answer the question whether MBSR is effective in patients with lung cancer and their partners. We started enrolment of participants in February 2012. At the moment we think recruiting a sufficient number of patients and partners will be a challenge due to rapidly fluctuating health status and sudden changes in cancer treatment [77]. The main reasons for declining participation in patients is ˜being too ill™ or that it is ˜too much of a burden during chemo and/or radiotherapy™. Furthermore no perceived need or motivation for the training is commonly mentioned. Among partners participation is highly depending on whether the patient is willing to participate. Although partners can take part separately partners who are interested do often not participate when the patients decline participation. Considering the difficulty of studying lung cancer patients and their partners [77] our trial will offer valuable information on whether MBSR as one of the few available psychosocial care programmes contributes to the alleviation of their psychological distress. Abbreviations MBSR: Mindfulness-based stress reduction; RCT: Randomized controlled trial; RUNMC: Radboud University Nijmegen Medical Centre; MBCT: Mindfulness-based cognitive therapy; MMSE: Mini mental state examination; DT: Distress thermometer; HADS: Hospital anxiety and depression scale; QLQ-C30: Quality of life “ cancer; QLQ-LC13: Quality of life “ lung cancer; SIP: Sickness impact profile; SPPIC: Self-perceived pressure from informal care; CRA-SE: Caregiver reaction assessment “ care-derived self-esteem; IMS-S: Investment model scale-satisfaction; MIS: Mutuality and interpersonal sensitivity; SAIL: Spiritual attitude and involvement list; FFMQ: Five facet mindfulness questionnaire; SCS: Self-compassion scale; RRS-EXT: Rumination response scale “ extended version; IES: Impact of event scale. Competing interests The authors declare that they have no competing interests. Authors™ contributions All authors contributed to the design of the study. AS MD and JP are the principal investigators of the study. MS drafted the paper which was modified and supplemented by all other authors. DH MS and MD are involved in recruiting participants while MS and DH take care of the logistics of the study and data collection. RD contributed specifically to the statistical analysis plan and WW contributed specifically to the design of the cost-effectiveness evaluation. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2407/14/3/prepub Acknowledgements This research is funded by Foundation Alpe d™HuZes and the Dutch Cancer Society (Grant number KUN 2011“5077 awarded to Prof. dr. Anne E. M. Speckens Dr. Miep A. van der Drift and Prof. dr. Judith B. Prins). 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Williams JMG Teasdale JD Mindfulness-Based Cognitive Therapy for depression: a new approach to preventing relapse 2002 New York: Guilford Press Piet J Wurtzen H Zachariae R The effect of Mindfulness-Based Therapy on symptomps of anxiety and depression in adult cancer patients and survivors: a systematic review and meta-analysis J Consult Clin Psychol 2012 80 6 1007 1020 22563637 Birnie K Garland SN Carlson LE Psychological benefits for cancer patients and their partners participating in Mindfulness-Based Stress Reduction (MBSR) Psychooncology 2010 19 9 1004 1009 10.1002/pon.1651 19918956 Hagedoorn M Sanderman R Bolks HN Tuinstra J Coyne JC Distress in couples coping with cancer: a meta-analysis and critical review of role and gender effects Psychol Bull 2008 134 1 1 30 18193993 Folstein MF Folstein SE McHugh PR Mini-mental state: practical method for grading cognitive state of patiens for clinician J Psychiatr Res 1975 12 3 189 198 10.1016/0022-3956(75)90026-6 1202204 Roth AJ Kornblith 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Lung_Cancer

"Activation of the NLRP3 inflammasome is dependent on the generation of ROS [33]. Verifying the inhibitory effect of luteoloside on the proliferation and metastasis of HCC cells was accomplished by inhibiting the NLRP3 inflammasome; the levels of NLRP3 inflammasome protein of different treatment groups were determined. The results showed a significant decrease in the expression of NLRP3 of Huh-7 and SMMC-7721 cells treated with luteoloside (25 µM and 50 µM) compared with the non-treated control cells (Fig. 4A line 1; 4B). Caspase-1 is a family member of intracellular cysteine proteases and they are first synthesized as inactive pro-caspase-1. Upon stimulation pro-caspase-1 zymogen is self activated by proteolytic cleavage into the enzymatically active heterodimer composed of two 10- and 20-kDa subunits. Inflammasome elicits the proteolytic maturation and secretion of interleukin-1? (IL-1?) and IL-18 through caspase-1 activity [17] which was also assessed in luteoloside treated Huh-7 and SMMC-7721 HCC cells. The results from this experiment suggests that luteoloside decreases the proteolytic cleavage of pro-caspase-1 in both Huh-7 and SMMC-7721 HCC cells in a dose-dependent fashion compared with non-treated control cells (Fig. 4A line 2; 4C). Furthermore treatment of luteoloside decreased the expression level of IL-1? in both Huh-7 and SMMC-7721 HCC cells (Fig. 4A line 3; 4D). The results indicate that luteoloside suppresses the proliferation and metastasis of HCC cells by inhibition of NLRP3 inflammasome (Fig. 4E). .0089961.g004 Luteoloside suppresses the NLRP3 inflammasome activation. (A) Western blot analyses of NLRP3 Caspase-1 (p10) and IL-1? protein expression in Huh-7 and SMMC-7721 cells exposed two different concentrations of luteoloside for 48 h. (B“D) Relative quantitation of NLRP3 Caspase-1 (p10) and IL-1?. (E) A hypothetical cascade pathway of NLRP3 inflammasome suppressed by luteoloside. * P<0.05; ** P<0.01; *** P<0.001 versus non-luteoloside-treated control group. Luteoloside Inhibits in vivo Proliferation and Metastasis of HCC Cells The results obtained from in vitro studies showed that treatment of HCC cells with luteoloside inhibits the proliferation migration and invasion capacity of these cells. To determine the in vivo effects of luteoloside we performed in vivo proliferation and metastasis study. The average size and weight of xenografts in the luteoloside-treated group were dramatically smaller and lighter than those of the control group (P?=?0.0026 and P?=?0.0417 respectively). (Fig. 5c 5d). Therefore the luteoloside treatment significantly inhibited the growth of the xenograft with inhibition rates (versus the control volume and weight of the tumors) of 44.1 and 53.1% respectively. Furthermore we injected SMMC-7721 cells into the lateral tail veins of nude mice (n?=?10) and evaluated the metastatic growth of cells in the lung. After 8 weeks the luteoloside-treated mice displayed a statistically significantly lower number of lung metastases than the control group mice (P?=?0.0003) indicative of extravasation and tumor growth in the lung (Fig. 5e). When lungs underwent hematoxylin and eosin staining lung metastases were observed in all ten mice intravenously injected SMCC-7721 cells only whereas no obvious lung metastases were observed in the mice intravenously injected SMMC-7721 cells with luteoloside treated (Fig. 5f 5g). It is worth noting that no difference in mouse weight was observed between the treatment group and the control group suggesting that luteoloside has no adverse effects on mouse growth. .0089961.g005 Luteoloside inhibits tumorigenic and spontaneous lung metastatic capabilities of SMMC-7721 cells. (A) Subcutaneous injection of SMMC-7721 cells plus luteoloside treatment in nude mice inhibited tumor growth. (B) Tail vein injection of SMMC-7721 cells plus luteoloside treatment in nude mice inhibited the metastasis of SMMC-7721 cells. (a) 2—106 SMMC-7721 cells were subcutaneously injected into the right upper flank of each mouse. When tumors were observable the animals were equally divided into two groups (ten per group). The first group received only 0.2 ml of vehicle material by gavage daily and served as a control group. The second group of animals received luteoloside (2 mg/kg body weight) in vehicle respectively for 4 weeks. At the termination of the experiment the mice were sacrificed and the tumors were weighed immediately after dissection. The yellow arrow shows the tumor. (b) The photo of tumors isolated from killed nude mice of the indicated groups. (c-d) The volume and weight of the tumors. (e) Number of metastatic nodules on the surface of the lungs of mice injected with SMMC-7721 (n?=?10 mice in per group) are presented as the means and SEM. (f-g) Representative pictures of lungs with or without metastatic nodules are shown (H&E staining). * P<0.05; ** P<0.01; *** P<0.001 versus non-luteoloside-treated control group. Scale bar: 30 µm. Discussion HCC is a rapidly fatal disease with a life expectancy of about 6 months from the time of the diagnosis. Therapeutic strategies employed to date have significantly improved the prognosis for patients with unresectable HCC. This emphasizes the need for investigating the molecular mechanisms responsible for HCC development and seeking effective and non-cytotoxic chemical agents for chemoprevention and treatment. However few synthetic antineoplastic compounds have been identified to be effective for the treatment of this disease [3]. In this respect more and more researchers paid much attention to natural active compounds for cancer chemoprevention and treatment. In the present study luteoloside which was previously found to exert antineoplastic effect was clearly demonstrated to inhibit the proliferation of all six human hepatoma cell lines (Fig. 1B 1C). In in vivo experiments we obtained the same results (Fig. 5A). Invasion and metastasis two of the most important hallmarks of cancer are the leading lethal factors for malignant cancer especially for HCC [34]. The long-term survival of HCC patients after curative resection is still confronted by the major obstacle of a high recurrence rate which is mainly due to the spread of intrahepatic metastases [25]. Therefore the identification of metastatic factors and an understanding of the underlying molecular pathways that are involved in the progression of metastasis become critical issues. Evidences are accumulating that some flavonoids could significantly inhibit the invasion and metastasis of HCC cells [35] [36]. In this study luteoloside was shown to dramatically inhibit HCC cell migration invasion and metastasis both in vitro (Fig. 2; Movies S1“S4) and in vivo (Fig. 5B). ROS such as superoxide (O2?) and hydrogen peroxide (H2O2) are constantly produced during metabolic processes in all living species. Under normal physiological conditions cellular ROS generation is counterbalanced by the action of antioxidant enzymes and other redox molecules. The balance between O2? generation and elimination is important for maintaining proper cellular redox states. Recent evident suggests that a moderate increase in ROS can stimulate cell proliferation invasion and metastasis [37] [38]. However the precise molecular signaling events of such a regulation are not yet well characterized. In this study we found that luteoloside could significantly decrease the ROS level of HCC cells such as Huh-7 and SMMC-7721 cells (Fig. 3). NLRP3 was recently identified to form a cytoplasmic complex known as the NLRP3 inflammasome which potently modulates innate immune function by regulating the maturation and secretion of pro-inflammatory cytokines such as interleukin-1? (IL-1?) [39]. Activation of the NLRP3 inflammasome is dependent on the generation of ROS [40] [41]. In fact all known NLRP3 activators generate ROS and conversely inhibitors of ROS block inflammasome activation [33]. The NLRP3 inflammasome functions as a positive regulator of tumor cells proliferation and metastasis [17] [32] [42]. Several studies have demonstrated that some flavonoids were found to suppress NLRP3 inflammasome activation [43] [44]. Our results showed that luteoloside could significantly decrease the expression of NLRP3 protein of Huh-7 and SMMC-7721 HCC cells (Fig. 4A lane 1; 4B). Furthermore luteoloside also decreased the expression level of caspase-1 (p10) (Fig. 4A lane 2; 4C) and IL-1? (Fig. 4A lane 3; 4D). Based on the results of the present study the mechanisms by which luteoloside inhibits HCC cells is summarized in Fig. 4E. In addition we found that luteoloside had no significantly effect on the cell apoptosis (Figure S1). Earlier studies have shown that induction of autophagy could result in decreases in mitochondrial ROS generation NLRP3 protein level and pro-IL-1? processing [45]. However in this study we found that luteoloside had no significantly effect on the protein levels of LC3 and Beclin 1 (Figure S2) two important autophagy markers. The new classification of cell death established by the Nomenclature Committee on Cell Death (NCCD) was based on molecular features [46] [47]. According to this classification cell deaths can be roughly divided into: apoptosis (caspase dependent extrinsic apoptosis and caspase-independent intrinsic apoptosis) necrosis autophagy cell death and other tentative definitions of cell death modalities including anoikis entosis pyroptosis netosis and cornification. In HCC at least four types of cell death pathways have been observed and studied including apoptosis [48] necrosis [49] autophagy [50] anoikis [51] None of the above described cell deaths contribute to HCC proliferation and metastasis equally and HCC progression is not dependent entirely on any single cell death pathway. In this study we found that luteoloside had no significantly effects on the cell apoptosis or autophagy. Further study is underway to explore whether luteoloside has significantly effects on other kinds of cell death. Luteoloside significantly inhibited the proliferation of HCC cells in vitro and in vivo. But luteoloside had no significantly effect on the cell apoptosis or autophagy. J¸rgensen et al have shown that the predominant effect of nilotinib a kind of tyrosine kinase inhibitor is antiproliferative rather than proapoptotic. They further suggested that combining nilotinib with other drugs should be carefully considered from the point of view of merely inducing G0/G1 block without apoptosis [52]. Papeleu et al found Trichostatin A a drug candidate for cancer therapy could inhibit cell proliferation at different steps of the cell cycle. But they also found Trichostatin A did not induce apoptosis in cells. Their finding supports its use in the treatment of proliferative disorders [53]. So from another perspective perhaps the predominant effect of luteoloside is antiproliferative rather than an œexecutor of cell death. Further studies are required to explore this possibility. To the best of our knowledge this is the first to show that luteoloside a flavone subclass of flavonoids inhibits the proliferation invasion and metastasis of HCC cells through inhibition of NLRP3 inflammasome. Our findings provide an important basis for a further exploration towards understanding the action mechanisms of luteoloside and possibly its beneficial effect in the prevention of tumor proliferation invasion and metastasis. Supporting Information Figure S1 Luteoloside does not affect the apoptosis rate of Huh-7 and SMMC-7721 cells. (A“B) The effect of luteoloside on caspase activity. Cells were plated in a 96-well plate. Overnight the cells were incubated with different concentrations of luteoloside. After 24 hours caspase-3/7 activity was measured using the Caspase-Glo® 3/7 Assay (Promega Madison WI). The caspase-3/7 activity was proportionate to the produced luminescence intensity. (C) Detection of DNA ladder formation in Huh-7 and SMMC-7721 cells after treatment with luteoloside for 24 hours. (D) Hoechst 33342 staining. The cells treated with luteoloside and stained with Hoechst 33342. Arrows show apoptotic small bodies. NS not significant (P>0.05). Scale bars: 1 µm. (TIF) Click here for additional data file. Figure S2 Luteoloside does not affect autophagy. Western blot analyses of LC3 and Beclin 1 protein expression in Huh-7 and SMMC-7721 cells exposed two different concentrations of luteoloside for 48 h. (TIF) Click here for additional data file. Movie S1 The migration of non-luteoloside-treated SMMC-7721 cells into the wound was monitored using a CellVoyager CV1000 confocal scanner system. The images were acquired every hour for 72 hours. (MP4) Click here for additional data file. Movie S2 The migration of luteoloside-treated SMMC-7721 cells into the wound was monitored using a CellVoyager CV1000 confocal scanner system. The images were acquired every hour for 72 hours. (MP4) Click here for additional data file. Movie S3 The migration of non-luteoloside-treated Huh-7 cells into the wound was monitored using a CellVoyager CV1000 confocal scanner system. The images were acquired every 0.5 hour for 48 hours. (MP4) Click here for additional data file. Movie S4 The migration of luteoloside-treated Huh-7 cells into the wound was monitored using a CellVoyager CV1000 confocal scanner system. The images were acquired every 0.5 hour for 48 hours. (MP4) Click here for additional data file. We thank Dr. Sheng Zhao for assistance in the preparation of this manuscript. References 1 SiegelR NaishadhamD JemalA (2013) Cancer statistics 2013. CA Cancer J Clin63: 11“3023335087 2 El-SeragHB (2011) Hepatocellular carcinoma. N Engl J Med365: 1118“112721992124 3 LiS DongP WangJ ZhangJ GuJ et al (2010) Icariin a natural flavonol glycoside induces apoptosis in human hepatoma SMMC-7721 cells via a ROS/JNK-dependent mitochondrial pathway. Cancer Lett298: 222“23020674153 4 SahasrabuddheVV GunjaMZ GraubardBI TrabertB SchwartzLM et al (2012) Nonsteroidal anti-inflammatory drug use chronic liver disease and hepatocellular carcinoma. J Natl Cancer Inst104: 1808“181423197492 5 FanSH ZhangZF ZhengYL LuJ WuDM et al (2009) Troxerutin protects the mouse kidney from D-galactose-caused injury through anti-inflammation and anti-oxidation. Int Immunopharmacol9: 91“9619000936 6 FanSH ZhangZF WangYY ZhengYL LuJ et al (2012) Purple sweet potato color attenuates D-galactose-induced renal injury in mice by inhibiting the expression of NF-?B-dependent inflammatroy genes. J Med Plants Res6: 3694“3704 7 Zamora-RosR FedirkoV TrichopoulouA Gonz¡lezCA BamiaC et al (2013) Dietary flavonoid lignan and antioxidant capacity and risk of hepatocellular carcinoma in the European prospective investigation into cancer and nutrition study. Int J Cancer133: 2429“244323649669 8 WalterA Etienne-SelloumN BrasseD KhalloufH BronnerC et al (2010) Intake of grape-derived polyphenols reduces C26 tumor growth by inhibiting angiogenesis and inducing apoptosis. FASEB J24: 3360“336920442318 9 IorioF BosottiR ScacheriE BelcastroV MithbaokarP et al (2010) Discovery of drug mode of action and drug repositioning from transcriptional responses. Proc Natl Acad Sci U S A107: 14621“1462620679242 10 HuC KittsDD (2004) Luteolin and luteolin-7-O-glucoside from dandelion flower suppress iNOS and COX-2 in RAW264.7 cells. Mol Cell Biochem265: 107“11315543940 11 SunX SunGB WangM XiaoJ SunXB (2011) Protective effects of cynaroside against H2O2-induced apoptosis in H9c2 cardiomyoblasts. J Cell Biochem112: 2019“202921445859 12 XiongJ LiS WangW HongY TangK et al (2013) Screening and identification of the antibacterial bioactive compounds from Lonicera japonica Thunb. leaves. Food Chem138: 327“33323265495 13 BaskarAA IgnacimuthuS MichaelGP Al NumairKS (2011) Cancer chemopreventive potential of luteolin-7-O-glucoside isolated from Ophiorrhiza mungos Linn. Nutr Cancer63: 130“13821161823 14 van DeventerHW BurgentsJE WuQP WoodfordRM BrickeyWJ et al (2010) The inflammasome component NLRP3 impairs antitumor vaccine by enhancing the accumulation of tumor-associated myeloid-derived suppressor cells. Cancer Res70: 10161“1016921159638 15 AndersonOA FinkelsteinA ShimaDT (2013) A2E induces IL-1? production in retinal pigment epithelial cells via the NLRP3 inflammasome. PLoS One8: e6726323840644 16 HuaKF ChouJC LamY TasiYL ChenA et al (2013) Polyenylpyrrole Derivatives Inhibit NLRP3 Inflammasome Activation and Inflammatory Mediator Expression by Reducing Reactive Oxygen Species Production and Mitogen-Activated Protein Kinase activation. PLoS One8: e7675424116148 17 AhmadI MuneerKM TamimiIA ChangME AtaMO et al (2013) Thymoquinone suppresses metastasis of melanoma cells by inhibition of NLRP3 inflammasome. Toxicol Appl Pharmacol270: 70“7623583630 18 ChenGY Nº±ezG (2011) Inflammasomes in intestinal inflammation and cancer. Gastroenterology141: 1986“199922005480 19 ChenLC WangLJ TsangNM OjciusDM ChenCC et al (2012) Tumour inflammasome-derived IL-1? recruits neutrophils and improves local recurrence-free survival in EBV-induced nasopharyngeal carcinoma. EMBO Mol Med4: 1276“129323065753 20 ChowMT TschoppJ M¶llerA SmythMJ (2012) NLRP3 promotes inflammation-induced skin cancer but is dispensable for asbestos-induced mesothelioma. Immunol Cell Biol90: 983“98623010873 21 Ungerb¤ckJ BelenkiD Jawad ul-HassanA FredriksonM Frans©nK et al (2012) Genetic variation and alterations of genes involved in NF?B/TNFAIP3- and NLRP3-inflammasome signaling affect susceptibility and outcome of colorectal cancer. Carcinogenesis33: 2126“213422843550 22 FanS NiuY TanN WuZ WangY et al (2013) LASS2 enhances chemosensitivity of breast cancer by counteracting acidic tumor microenvironment through inhibiting activity of V-ATPase proton pump. Oncogene32: 1682“169022580606 23 SethmanCR HawigerJ (2013)"

Lung_Cancer

"Upon CT examination all lung nodules showed a similar appearance with heterogeneous density consisting of a hypoattenuating center (Fig. 2Fig. 2.CT images of the dog. In pre-contrast CT images (A D and G) at soft tissue window setting (window level=40 window width=400) all lung nodules (arrows) showed heterogeneous density consisting of hypoattenuating center (c). In pre-contrast CT images (B and E) at lung window setting (window level= ?600 window width=1200) the pleural indentations (arrows) were found in the right cranial and left caudal nodules (m). On post contrast CT images (C F and H) partial contrast enhancement (arrows) occurred from peripheral parenchyma of nodules (m) (window level=40 window width=400). The size of the right caudal nodule the largest one was 3.2 — 2.7 cm in diameter. In reconstructed dorsal planes note the obstruction of right caudal bronchus (arrow) by the right caudal nodule (m) in (I) and the cranial deviation of right cranial bronchus (arrow) by the right cranial nodule (m) in (J). The left side of the image is the right side of the dog.). The mean density of lung nodules was 27.28 ± 6.19 HU. The size of each nodule was 2.2 — 2.0 cm (right cranial nodule) 3.2 — 2.7 cm (right caudal nodule) 1.0 — 1.0 cm (left cranial nodule) and 2.7 — 2.0 cm (left caudal nodule) respectively. Only the peripheral parenchyma of nodules was partially enhanced after an iodine contrast injection (iohexol Omnihexol 300 Korea United Pharm Co. Seoul Korea). Thus most of the parenchyma was considered to have undergone an ischemic or necrotic lesion. PET-CT (Discovery 600 PET/CT system GE Healthcare Wauwatosa WI U.S.A.) using FDG was performed at 47 min after the administration of 11.1 MBq/kg of FDG intravenously. The increased FDG uptake was observed from the peripheral region of each nodule and the maximum standardized uptake values were as follows; 6.4 of the right cranial nodule 4.3 of the right caudal nodule 3.8 of the left cranial nodule and 4.7 of the left caudal nodule respectively (Figs. 3 and 4Fig. 3.Positron emission tomography (PET) images using 18F-fluorodeoxyglucose (FDG). Maximum intensity projection (MIP) view showed four hypermetabolic lesions in both lungs and no significant FDG uptake was found from other regions (including mammary gland) except for several physiologic uptakes (esp. brain heart kidneys intestine urinary bladder and so on). B=brain H=heart K=kidney I=intestine U=urinary bladder S=injection site. The left side of the image is the right side of the dog.Fig. 4.Axial view of positron emission tomography and computed tomography (PET-CT) fusion images using 18F-fluorodeoxyglucose (FDG). Each image corresponds to the dashed line of Fig. 3 in order from top to bottom. It shows the increased FDG uptake mainly from the peripheral region of each nodule (arrows) which probably resulted from central necrosis (A“C). H=heart L=liver. The left side of the image is the right side of the dog.). There was no evidence of FDG uptake from other regions including the mammary gland nodules. None other than four pulmonary nodules were detected on CT and PET-CT. Ultrasound guided tru-cut biopsy was performed for the right and left caudal lung nodules. However histopathological diagnosis was not possible due to extensive necrosis of the sample. Multiple lung nodules in the dog were tentatively diagnosed as a primary lung tumor because there was no evidence of an unknown primary tumor on the basis of the diagnostic imaging. "

Lung_Cancer

" Successful examples of this include the co-development (and co-approval) of the BRAF inhibitor vemurafenib and its companion diagnostic BRAF V600E mutation assay for BRAF-mutant metastatic melanoma[1] and the ALK inhibitor crizotinib and its companion diagnostic ALK fusion gene test in advanced ALK-fusion positive non-small cell lung cancer (NSCLC) patients.[2] [3] [4] However in some cases predictive biomarkers for a targeted therapy are not recognized until after the drug is first approved. As an example the anti-EGFR antibody cetuximab was first approved in the US for the treatment of metastatic colorectal cancer in 2004. Numerous retrospective and prospective trials subsequently revealed that tumors harboring KRAS mutations were very unlikely to respond to cetuximab. In July 2009 FDA required labeling changes for cetuximab and another anti-EGFR antibody panitumumab requiring that the indications and usage state there was no treatment benefit with the drugs for patients whose tumors had KRAS mutations in codon 12 or 13 at a time when there were no FDA-approved diagnostic assays for KRAS mutations.[5] Only later in July 2012 did a KRAS mutation assay receive FDA approval based on the results of a prospective randomized trial highlighting the challenges of retrospectively validating a companion diagnostic assay after the pivotal drug trials have been completed.[6] The anti-EGFR TKI erlotinib was initially approved for all patients with advanced NSCLC who had progressed on first-line chemotherapy. A number of subsequent studies determined that patients with EGFR-mutant NSCLC had a high likelihood of responding to these TKI leading to trials in the first-line setting for EGFR-mutant cancer.[7] [8] [9] [10] [11] [12] [13] Four prospective randomized clinical trials studied in Asian populations demonstrated that erlotinib and gefitinib resulted in improved progression-free survival compared to chemotherapy for first line therapy in NSCLC patients with EGFR mutations.[7] [8] [9] [13] Other clinical studies in mixed ethnicity cohorts have concluded with similar results.[10][12] The EURTAC trial was a randomized phase 3 trial to assess the safety and efficacy of erlotinib compared with standard platinum-based chemotherapy for first-line treatment of a patient population with advanced EGFR-mutation detected NSCLC in a largely Caucasian population of European patients. Erlotinib-treated patients experienced significant improvements in median PFS (9.7 months vs. 5.2 months) compared to chemotherapy. Patients on the erlotinib arm also had a considerably higher percentage of responses (58% vs. 15%) in the intent-to-treat population.[11] This trial has been submitted for first line indication of erlotinib in EGFR mutated NSCLC patients. The majority of activating EGFR mutations are located in exons 19 (45%) and 21 (40“45%).[14] [15] [16] [17] [18] [19] [20] Guidelines from anizations such as ASCO CAP/AMP and NCCN recommend the use of anti-EGFR TKIs as first-line therapy in patients with EGFR-mutant advanced NSCLC based on the results of these pivotal clinical trials. [21] [22] [23] Recent recommendations by CAP/IASLC/AMP advise the identification of EGFR mutations present at >1% of which exon 19 deletions and an exon 21 mutation (L858R) account for greater than 90% of all mutations.[24] None of the guidelines specify the testing method to be used however the cobas EGFR Mutation test is CE-IVD approved and is recently FDA approved.[25] Here we present the retrospective analysis of a clinical validation study of the EGFR PCR test on a subset of lung cancer specimens from patients screened for the EURTAC trial. The EGFR PCR test demonstrated improved sample workflow relative to the LDTs used in the EURTAC trial enabling EGFR mutation screening in a single assay with a one-day turn-around time. The EGFR PCR test showed superior sensitivity and specificity compared with conventional Sanger sequencing. Methods The major study objectives were 1) to correlate the clinical outcomes (PFS BORR) from the subgroup of available samples tested by the EGFR PCR test to the results from the entire EURTAC population and 2) to compare the analytic performance of the EGFR PCR test to that of the original LDT and Sanger sequencing using massively parallel pyrosequencing (MPP) to resolve discrepancies observed between the other 3 testing methods. In the EURTAC trial1044 patients from hospitals in France Italy and Spain were screened using the LDT. For this study all samples were retrospectively analyzed under IRB approval from Copernicus IRB (00001313). Site specific IRB approval from each clinical site and written consent from all patients was obtained prior to the study conduct phase of NCT00446225.[11] [26] In 487 cases residual specimens were available for retesting with the EGFR PCR test (). A single 5 µm section with at least 10% tumor content from each of the 487 specimens was used for the EGFR PCR test. Genomic DNA from existing eluate or extracted from additional sections was tested on Sanger sequencing and MPP. lists the demographics of the patients screened for the EURTAC trial by the LDT sub-categorized by patients tested or not tested by the EGFR PCR test. Patients enrolled in the EURTAC trial were selected using a laboratory-developed test validated by the Laboratory of Oncology (ICO-Hospital Germans Trias i Pujol Badalona Spain) consisting of three methodologies.[26] In this study a single PCR-based assay for detecting EGFR mutations was used. Details of the analytical performance of this assay have been described previously.[27] .0089518.g001 Flow of samples through the study. E1 samples: tumor block not available for analysis. E2 samples: tumor material insufficient for analysis. LDT ?=? laboratory-developed test. .0089518.t001 Demographics of the patient cohort screened for EURTAC trial. SLCG LDT MD SLCG LDT MND EGFR PCR tested EGFR PCRnot tested EGFR PCR tested EGFR PCR not tested Total 172 53 303 489 Age (years) mean ± SD 64.1±10.4 62.9±10.4 61.7±10.6 61.7±10.6 Sex n (%) Male 41 (23.8) 14 (26.4) 179 (59.1) 281 (57.5) Female 131 (76.2) 39 (73.6) 124 (40.9) 208 (42.5) Race/ethnicity n (%) Caucasian 168 (97.7) 52 (98.1) 296 (97.7) 481 (98.4) Other* 4 (2.3) 1 (1.9) 7 (2.3) 8 (1.6) Smoking status n (%) Never smoked 124 (72.1) 31 (58.5) 74 (24.4) 133 (27.2) Past/currentsmoker 47 (27.3) 22 (41.5) 219 (72.3) 339 (69.3) Unknown 1 (0.6) 0 (0.0) 10 (3.3) 17 (3.5) Stage IIIB 13 (7.6) 2 (3.8) 17 (5.6) 40 (8.2) Stage IV 157 (91.3) 50 (94.3) 277 (91.4) 432 (88.3) Other* 2 (1.2) 1 (1.9) 9 (3.0) 17 (3.5) Histology n (%) Adenocarcinoma 156 (90.7) 47 (88.7) 266 (87.8) 407 (83.2) BronchioalveolarCarcinoma 1 (0.6) 2 (3.8) 5 (1.7) 16 (3.3) Other* 15 (8.7) 4 (7.5) 32 (10.6) 66 (13.5) *Other includes subjects with no information available. LDT ?=? laboratory-developed test; MD ?=? mutation detected; MND ?=? mutation not detected. SLCG inconclusive (n?=?27) data not shown. Statistical considerations Mutation Detected (MD) was defined as the presence of either an exon 19 deletion or L858R mutation. Mutation Not Detected (MND) was defined as the absence of both exon 19 deletions and the L858R mutation. SAS/STAT® software was used for all data analysis. Clinical outcome study statistics Kaplan-Meier survival curves were used to assess the PFS by treatment method (chemotherapy or erlotinib) among patients who were enrolled in the EURTAC trial and screened with the LDT as well as the subset of patients who were determined to be mutation-positive by the EGFR PCR test. Nonparametric log-rank test was performed to assess PFS between patients who were randomized to chemotherapy or erlotinib. The hazard ratio (chemotherapy vs. erlotinib) relative to PFS was also calculated. Best overall response was the best response recorded from the start of treatment until disease progression and BORR (Best overall response rate) was summarized with 95% confidence limits according to Pearson-Clopper methods based on investigators assessment for each treatment arm. Analytical performance statistics For analytical performance an agreement analysis was performed between the EGFR PCR test result and the LDT test. Mutation detection of exon 19 deletions and L858R mutations were analyzed in aggregate. Separately the EGFR PCR test was also compared to Sanger sequencing and MPP by a CLIA-certified laboratory. For the agreement analyses the positive percent agreement (PPA) negative percent agreement (NPA) and overall percent agreement (OPA) with their corresponding 95% confidence intervals (CIs) were calculated. In addition 3-way analyses using MPP as a second reference method was performed to resolve the discrepancy results. Mutation testing methods EGFR PCR Test The EGFR PCR test (cobas EGFR Mutation Test Roche Molecular Systems Inc Branchburg NJ USA) is a CE-IVD marked multiplex allele-specific PCR-based assay designed to detect 41 mutations in exons 181920 and 21 in FFPET specimens of human NSCLC.[28] DNA is isolated using the cobas DNA Sample Preparation Kit (Roche Molecular Systems Branchburg NJ). [29] A minimum of 150 ng of genomic DNA is required for PCR amplification which can typically be isolated from a single 5 µm FFPET section. The EGFR PCR test software version used in this study was designed to detect 29 deletions in exon 19 and 2 L858R variants in exon 21. Macrodissection is only recommended if tumor content is less than 10%; laser capture microdissection is not required. The EGFR PCR test was performed per manufacturer's package insert and results were automatically analyzed and reported. The limit of detection has been validated to 5% mutant alleles. The workflow from DNA isolation to results reporting can be performed in one 8 hour period.[27] LDT Patients in the EURTAC study were screened using a combination of methods developed by Laboratory of Oncology ICO-Hospital Germans Trias i Pujol Barcelona Spain.[11] In short EGFR activating mutations in exons 19 and 21 were initially identified by Sanger sequencing and confirmed by fragment length analysis for exon 19 deletions (FAM-labelled primer in an ABI prism 3130 DNA analyser (Applied Biosystems Foster City CA USA) and by Taqman assay for exon 21 (L858R) mutation. All tumor specimens were from the original biopsy taken prior to any treatment and before randomization. Testing was performed on ? 2mm2 of tissue obtained from one to three slides of 4-micron tissue sections which were subjected to laser capture microdissection to enrich for the presence of tumor cells. DNA was extracted using a standard laboratory protocol and tested at a single site in Spain in Laboratory of Oncology for EGFR activating mutations in exon 19 and 21 using a previously described method. The average turnaround time was approximately 5 days.[26] Bi-directional Sanger sequencing All samples tested by the EGFR PCR test were also tested by Sanger sequencing using DNA from FFPET specimens prepared by the cobas DNA Sample Preparation Kit and sequenced with 2× bidirectional Sanger sequencing by a CLIA-certified laboratory (SeqWright Houston TX USA) using a validated protocol. Repeat Sanger sequencing was performed to compare the detection of EGFR mutations from adjacent sections of tissue to minimize any impact of tissue heterogeneity used for the EGFR PCR test relative to the original LDT results. Also sequencing protocols vary by laboratory in terms of the percent tumor content/sample that requires macrodissection. DNA isolated with the cobas DNA Sample Preparation Kit and used for sequencing required ?10% tumor content. Average turnaround time to results was 7 days. The estimated limit of detection is approximately 20% mutant alleles.[30] Massively parallel pyrosequencing (MPP) Samples with valid EGFR PCR test results with adequate DNA remaining from the initial extraction were tested by a MPP method (454 GS Titanium 454 Life Sciences Branford CT USA) by a CLIA-certified laboratory (SeqWright Houston TX USA) using a validated protocol.[31] This method is a 5“7 day process that involves amplicon generation pooling ligation emulsion PCR amplification and massively parallel pyrosequencing with manual data analysis. The estimated limit of detection for the assay is 1.25% mutant alleles. [27] The MPP method was used to demonstrate performance of the EGFR PCR test to a more sensitive method and as an arbiter for discrepant cases observed between the LDT or the repeat Sanger sequencing. In order to preserve patient privacy associated with tested clinical samples raw MPP sequencing results were anonymized and presented in Table S1. Results Specimen demographics 487 (47%) of 1044 specimens screened for the EURTAC trial using LDTs were available for testing using the EGFR PCR test. The flow of samples through the study is shown in . Patient demographics and baseline tumor characteristics for all patients by LDT status are shown in . There were no significant differences between subsets of patients tested and patients not tested by the EGFR PCR test (p>0.05) for each LDT status (mutation detected mutation not detected) with the exception of country of the screening clinic. Clinical outcomes for patients based on the EGFR PCR test results Of the 174 patients enrolled in EURTAC trial specimens from 134 (77%) patients were available for testing using the EGFR PCR test. Excluding 11 patients with invalid EGFR PCR test results and 7 patients with a result of EGFR mutation not detected a total of 116 (67%) patients were mutation detected by the EGFR PCR test and evaluable for clinical outcome analysis (57 patients in the chemotherapy arm and 59 in the erlotinib arm). Clinical outcomes (PFS BORR and OS) are presented in Table 2. Among EGFR PCR test positive patients those treated with erlotinib had a significantly prolonged PFS when compared to patients treated with chemotherapy (p-value <0.0001 log-rank test); the median PFS was 10.4 months (95% CI: 8.0 to 13.8 months) and 5.4 months (95% CI: 4.4 to 6.8 months) for patients treated with erlotinib or chemotherapy respectively (Figure 2). The HR based on the Cox proportional hazards model was reduced by 66% (HR 0.34; [95% CI: 0.21 to 0.54]) for patients in the erlotinib versus chemotherapy arm. One year after randomization a higher percentage of patients in the erlotinib compared with the chemotherapy arm were event-free (45% [95% CI: 32% to 59% versus 6% [95% CI: 0% to 15%] respectively). .0089518.g002 Figure 2 Kaplan-Meier curves of progression-free survival (PFS) for different treatments in treatment-naïve patients with non“small-cell lung cancer and EGFR mutation detected by the EGFR PCR test and LDT. .0089518.t002 Table 2 Summary of Clinical Outcome Analysis among EGFR PCR test positive patients in the EURTAC trial. Chemotherapy (N?=?57) Erlotinib (N?=?59) PFS (Investigator) Patients with event 37 (64.9%) 47 (79.7%) Patients without eventa 20 (35.1%) 12 (20.3%) ?Time to event (months) ?Medianb (95%CI) 5.4 [4.4; 6.8] 10.4 [8.0; 13.8] ?p-Value (Log-Rank Test) <0.0001 ?Hazard Ratio (95% CI) 0.34 [0.21; 0.54] ?1 year estimate ?Patients remaining at risk 2 24 ?Event-free Rateb (95%CI) 6% [0%; 15%] 45% [32%; 59%] Best Overall Analysis Response rates (95% CI) 14.0% [ 6.3%; 25.8%] 59.3%[ 45.7%; 71.9%] Difference in Response Rates (%) 45.29% [ 28.8%; 61.7%] ?p-Value (Chi-squared Test) <.0001 Odds Ratio (95% CI) 8.93 [3.59; 22.19] OS Patients with event 35 (61.4%) 36 (61.0%) Patients without eventa 22 (38.6%) 23 (39.0%) ?Time to event (months) ?Medianb (95%CI) 20.8 [17.3; 29.4] 25.8 [16.1; 30.0] ?p-Value (Log-Rank Test) 0.5381 ?Hazard Ratio (95% CI) 0.86 [0.54; 1.38] ?2 - year estimate ?Patients remaining at risk 16 23 ?Event-free Rateb (95% CI) 43% [29%; 57%] 51% [38%; 64%] Note: All eligible patients enrolled in study ML20650 were determined as EGFR mutation detected by the LDT. Among those patients with EGFR mutation confirmed by the EGFR PCR test were included in this table. Event ?=? Death or progression free whichever comes first for PFS analysis and event?=?death for OS analysis. a censored. b Kaplan-Meier estimates. C including censored observations. BORR were higher in patients in the erlotinib arm (59.3% [95% CI: 45.7% to 71.9%]) compared to the chemotherapy arm (14.0% [95% CI: 6.3% to 25.8%]). Patients in the erlotinib arm were much more likely to respond to therapy than patients in the chemotherapy arm (odds ratio of 8.93 [95% CI: 3.59 to 22.19]). There was no significant difference in OS between the treatment arms (25.8 months in the erlotinib arm (95% CI: 16.1 to 30.0) and 20.8 months in the chemotherapy arm (95% CI: 17.3 to 29.4) (log-rank test p-value ?=?0.5381)). PFS BORR and OS results for EGFR PCR test positive patients did not differ significantly from those obtained in all patients enrolled in the EURTAC trial which suggests that the EGFR PCR test positive patients are representative of all EURTAC enrolled patients. For the 7 cases where the EGFR PCR test result was mutation not detected and discrepant with the LDT two cases resolved in favor of the LDT by MPP three cases resolved in favor of the EGFR PCR test and one sample was invalid for both Sanger and MPP and the other was in agreement between the EGFR PCR test and Sanger but not MPP (Table S2). Anecdotally 6 of the 7 patients were treated with erlotinib and only one patient achieved greater than or equal to median PFS based on the LDT or the EGFR PCR test. Comparison of EGFR PCR test and LDT results Among 432 specimens with valid results from both the EGFR PCR test and LDT the PPA NPA and OPA were 94.2% (146/155 CI: 89.3% 96.9%) 97.5% (270/277 CI: 94.9% 98.8%) and 96.3% (416/432 CI: 94.1% 97.7%) respectively (Table 3). Thus there was a high concordance between the original LDT and EGFR PCR test results. Among sixteen specimens with discordant results the EGFR PCR test result was confirmed by MPP in 68.8% (11/16) cases (Table S3). .0089518.t003 Table 3 Agreement analysis between EGFR PCR test and LDT. SLCG LDT Total N?=?432 Mutation detected Mutation not detected EGFR PCR test Mutation detected 146 7 153 Mutation not detected 9 270 279 Total 155 277 432* ¢12 samples with inconclusive LDT results and 43 samples with invalid EGFR PCR test results were excluded. Positive percent agreement ?=?94.2% (95% CI [89.3“96.9%]). Negative percent agreement ?=?97.5% (95% CI [94.9“98.8%]). Overall percent agreement ?=?96.3% (95% CI [94.1“97.7%]). Comparison of the EGFR PCR test results with Sanger Sequencing Of 487 specimens tested using the EGFR PCR test and Sanger sequencing 406 gave valid results by both methods (38 were invalid by both methods five were invalid by EGFR PCR test and 38 were invalid by Sanger sequencing). The PPA NPA and OPA for EGFR PCR test compared with Sanger sequencing were 96.6% (112/116 CI: 91.7% 98.7%) 88.3% (256/290 CI: 84.1% 91.5%) and 90.6% (368/406 CI: 87.4% 93.1%; Table 4) respectively. Among 38 discordant results between the EGFR PCR test and Sanger sequencing MPP agreed with the EGFR PCR test result in 30 (78.9%) cases (Table S4). Sanger sequencing detected one L858R not detected by MPP and failed to detect 22 exon 19 deletions and 7 L858R mutations confirmed by MPP. Four MPP results were invalid and the remaining four results agreed with Sanger. The range of percent mutant alleles of the cases missed by Sanger was 3% to 60% with several specimens (n?=?16) under the estimated limit of detection for Sanger. .0089518.t004 Table 4 Agreement analysis between EGFR PCR test and Sanger sequencing. Sanger sequencing Total N?=?406 Mutation detected Mutation not detected EGFR PCR test Mutation detected 112 34 146 Mutation not detected 4 256 260 Total 116 290 406 *81 samples with invalid EGFR PCR test or Sanger sequencing results were excluded. Positive percent agreement ?=?96.6% (95% CI [91.5“98.7%]). Negative percent agreement ?=?88.3% (95% CI [84.1“91.5%]). Overall percent agreement ?=?90.6% (95% CI [87.4“93.1%]). Discussion This study supports the feasibility of performing a retrospective clinical validation of a companion diagnostic from prospective therapeutic clinical trials. The EGFR PCR test results were highly concordant (>96%) with the LDT results used to select patients for the EURTAC trial. As a consequence PFS and BORR of the subset of patients with EGFR mutations detected with the EGFR PCR test were comparable to the full cohort of patients enrolled in the EURTAC trial thus validating the use of the EGFR PCR test to select patients for treatment with anti-EGFR TKIs such as erlotinib. Median PFS survival was 9.7 versus 10.4 months for the erlotinib group and 5.2 versus 5.4 months for the LDTs and EGFR PCR test respectively. The BORR was 58% versus 59.3% months for the erlotinib group and 15% versus 14.0% for the LDTs and EGFR PCR test respectively. Among the 16 discordant specimens between the EGFR PCR test and LDTs a third mutation testing method agreed with the EGFR PCR test result in 11 cases. Of seven cases that were mutation detected by the EGFR PCR test and mutation not detected by the LDT 5 were confirmed by MPP. These patients could have potentially benefited from anti-EGFR TKI therapy. The EGFR PCR test had a number of technical advantages over the LDT used in the EURTAC trial. The LDT required laser capture microdissection of multiple tissue sections and involved 3 separate assays with a median turnaround time of 4.5 days. By comparison the EGFR PCR test required macrodissection only if the tumor content was <10% and can be performed in one day using a single 5 µm section. Furthermore the EGFR PCR test is a commercially available kit-based assay that provides an automated result rather than a manual process subject to interpretation and which can be performed by any qualified clinical laboratory. "

Lung_Cancer

"This phenomenon which we did not observe in our previous cohorts might relate to the average quality of chimeras as for this experiment we induced tumors in chimeric mice with a wide range (5“95%) of coat-color chimerism (supplementary Fig S8B). Monitoring of Luciferase expression in individual mice from the F1 cohort revealed that all Mycl1 expressing tumors initiated around the same time point and showed identical growth leftacteristics making this model very suitable for tumor intervention studies (Fig 4E). To proof that Mycl1 is the driver gene for the 4qD2.2 amplicon we verified Mycl1 amplification status in genomic DNA of tumors from control Rb1F/F ;Trp53F/F mice or from mice with either the invCAG-Luc or the invCAG-Mycl1-Luc2 transgene (both chimeras and F1) by low-coverage DNA sequencing and real-time PCR (Fig 4F). Mycl1 was amplified in 37.5“66.7% of the control tumors whereas it was only amplified in 6% of the Mycl1 transgenic tumors clearly validating Mycl1 as a driver for SCLC development. Discussion The GEMM-ESC procedure as presented here can be divided in two separate phases: a resource phase and an experimental phase (Fig 5). The resource phase starting from the selection of the original GEMM until cryogenic storage of quality controlled Col1a1-frt targeted GEMM-ESC clones is fairly laborious taking up to 6 months. The success rate is high and independent of the strain background. This can be largely attributed to the optimized ESC culture conditions which not only simplify procedures but also allow for better quality ESCs as compared to previous protocols. The 2i culture protocol also enables derivation of ESCs from mouse strains that were previously considered refractory (Ying et al 2008; Reinholdt et al 2012). In this study we derived 47 ESC clones from 364 embryos representing 13% derivation efficiency. Although this seems low most of our ESC derivation attempts were successful. The derivation efficiency could be further increased by expanding all early ESC clones instead of selecting them on the basis of their morphology and growth rate (). Efficiency of the GEMM-ESC approach.Schematic representation of the GEMM-ESC approach including the performance of the individual steps. The approach is divided in two phases: a resource phase and an experimental phase. The resource phase includes ESC's derivation and targeting with the Col1a1-frt vector performed once per GEMM and takes ˜6 months including the necessary quality controls. The experimental phase is mainly focused on introducing a transgene-coding plasmid in a validated GEMM-ESC clone using the Flp-in method that allows for consecutive manipulations and takes ˜4 months to obtain a chimeric cohort. Alternatively GEMM-ESC clones are also suitable for direct targeting of a specific gene or the introduction of mutant alleles using gene editing (arrows with dotted lines). The experimental phase also includes the option to follow an F1 route as almost all GEMM-ESC clones showed germline transmission (GLT). In practice we advise that for each model (i) multiple Col1a1-frt targeted GEMM-ESC clones are screened for their ability to efficiently generate high quality chimeras (ii) two of the best-performing clones are selected for the Flp-in procedure and (iii) at least two transgene-coding GEMM-ESC clones are used to generate cohorts. The final clones should originate from different Col1a1-frt targeted parental clones to minimize the chance of miss-interpreting phenotypes due to possible unwanted genetic alterations introduced by long-term culture. The selection of best-performing Col1a1-frt targeted GEMM-ESC clones is crucial for the efficiency to later generate experimental cohorts as the number of chimeras born per injected embryo is likely to decline after additional manipulations and propagation in culture. One issue we noted is that different genetic backgrounds of the original GEMMs require fine-tuning of the ESC injection procedure in order to achieve an optimal balance between quality and yield of the resulting chimeras. This balance is affected by several factors including the strain background and the developmental stage of the host embryos. Injection of ESCs into morulae instead of blastocysts (Plagge et al 2000) leads to very high quality chimeras but also causes an increase in birth problems still-born animals and pups with growth retardation in particular for FVB/n and FVB/n;129/Ola ESC clones (Fig 1D and supplementary Table S1). We therefore optimized the injection procedure for each ESC background. In practice ESCs derived from C57BL/6J strains were injected into FVB/n morulae whereas ESCs from FVB/n;129/Ola or FVB/n strains were injected into C57BL/6N blastocysts. Also other strategies are available to shift the balance between the quality and the yield of chimeras. For instance fully ESC-derived chimeras can be produced using tetraploid complementation techniques (Eakin & Hadjantonakis 2006). Alternatively lower numbers of ESCs can be injected per morula or blastocyst in order to improve the yield of life-born chimeras. The quality of GEMM-ESC clones remains stable over time even after genetic manipulation by targeting or Flp-mediated integration as 70“78% of newly modified ESC clones gave good chimeras and GLT (Fig 5). The genetic stability of ESCs cultured in 2i medium is comparable to ESCs cultured under classic conditions (Fig 3B; Liang et al 2008). The occurrence of CNVs highlights the need for thorough genetic screening of the Col1a1-frt targeted GEMM-ESC clones by e.g. aCGH deep sequencing or spectral karyotyping especially because a fraction of these CNVs will be transmitted from the chimeras to the F1 offspring (Fig 3C). The development of CNVs might be related to targeting or Flp-mediated integration; however we have also observed CNVs in freshly subcloned ESCs (supplementary Table S4). It is therefore more likely that CNVs arise spontaneously when cells are stressed for instance by single-cell cloning or antibiotic selection regimens and not by specific recombination or integration events. Occasionally we observed identical CNVs in independent subclones from the same GEMM-ESC implying that some CNVs are already present in a subpopulation of the derived GEMM-ESC clone (supplementary Table S4). This places the occurrence of some CNVs very early in the ESC derivation process. Thus far we did not observe any phenotypic changes in our chimeras or their offspring that could be attributed to a particular CNV; however this remains a possibility. Also aCGH screening may not allow for full identification of all aberrations that occur during in vitro culture as some apparently normal clones failed to give rise to chimeras indicating an underlying genetic or epigenetic defect. It should be noted that these issues are not specific for the approach described here but plays a role in any experiment using cloned ESCs (Liang et al 2008); in a conventional ESC approach these CNVs are likely bred out of the cohort which is not the case when chimeras are used directly although use of independent ESC clones can further reduce this risk. Further refinements in the ESC culture conditions might improve the (epi)genetic stability of the GEMM-ESC clones. The experimental phase of the GEMM-ESC procedure is based on the ˜plug-and-play™ principle which ensures optimal flexibility and short time frames. It takes typically <4 months to obtain an experimental cohort. Another advantage of the plug-and-play system is that it is compatible with a wide array of vectors for gain-of-function or loss-of-function studies. To this end we are developing additional Flp-in vector backbones for inducible expression of shRNAs to allow for specific knockdown of target genes. In the near future one only has to clone a cDNA or validated shRNA construct into one of these vectors and introduce that into the GEMM-ESC clone of choice after which an experimental cohort can be produced. This will permit the swift validation of candidate cancer genes identified in genomic sequencing efforts and functional genetic screens in a suitable mouse model as we have shown for Mycl1. Moreover in the case where full target gene silencing is required to observe a phenotype the highly efficient gene editing tools TALENS and CRISPR/Cas can be applied on GEMM-ESCs to generate a homozygous null allele that can be directly evaluated in chimeras (Fig 5). By using a GEMM instead of a xenograft model for in vivo validation also the effects of the target gene on tumor initiation tumor progression and tumor maintenance can be monitored including interactions between tumor cells and immune cells. The GEMM-ESC procedure allows for the direct use of chimeras for experimentation. We show that the tumor leftacteristics of chimeric mice are very similar to those of conventional mice (Fig 2) though the tumor latency can differ depending on the model and level of chimerism (Fig 4C supplementary Fig S2). Typically the level of chimerism is estimated on basis of coat-color contribution although this consistently results in an overestimation of the true chimerism in the various tissues (Fig 2C and D). In our experience 70“100% chimeras give a quite consistent reproducible tumor phenotype when the penetrance is high. In GEMMs with low penetrant phenotypes it is advisable to backcross the chimeras to the parental strain and use the F1 cohort. We feel that a more quantitative analysis on a particular tissue e.g. tail does not provide a substantial advantage above estimating chimerism on the basis of coat color as variations can also be found among different tissues. The chimeric approach is particularly useful for side-by-side comparison of multiple allelic variants of the same gene or for in vivo screening of multiple candidate cancer genes in one GEMM in a semi-high throughput fashion. Direct comparison of tumorigenesis in chimeras that differ only on basis of the expression of one candidate cancer gene will accelerate the identification of true driver genes within a large group of candidate cancer genes for instance Mycl1 in the SCLC model. Furthermore all tumors will have an identical genetic background excluding any variation caused by modifier alleles introduced via breeding. This simplifies subsequent deep sequencing efforts as all tumors can be compared to the same GEMM-ESC derived reference DNA. It is however advisable to use two independent ESC lines as also recombinase-mediated introduction of constructs into the ESCs can give rise to chromosomal changes that could influence the outcome. We realize that in most cases the chimeras will be crossed to the original GEMM to establish stable mouse strains. Indeed a single cross of the chimeras to the original strain will result in F1 mice with or without the introduced construct. These mice can be directly used as experimental and control cohorts respectively (Fig 4D). The feasibility of this approach depends on the efficiency of GLT of the chimeras. Based on two GEMMs we observed that twelve out of fourteen chimeras gave GLT in their first litter and one chimera in the second (supplementary Table S1)."

Lung_Cancer

"ssed during the entire study period with a calendar on which participants in the MBSR condition fill out on a daily basis whether they adhere to the mindfulness exercises: either formal practice (e.g. meditation exercise like the bodyscan) informal practice (e.g. activity with awareness) or no exercise. Adherence to MBSR has been shown to mediate the effects of MBCT on depressive symptoms [72]. Statistical analysis plan Sample size To determine the required sample size first the sample size was calculated that would be needed for a simple t-test and subsequently it was corrected for clustering repeated measurements and baseline. A two-sided t-test on the total HADS score [3940] (i.e. our primary outcome measure examining psychological distress (HADS-total) anxiety symptoms (HADS-A) and depressive symptoms (HADS-D)) would require 64 participants in each group to have 80% power to detect a medium-sized difference (effect size = 0.5) with alpha = 0.05. To correct for clustering we multiplied this sample size of 64 with the design factor (1 + (n ? 1) * ICC) where n denotes the cluster size and where ICC denotes the intra-cluster correlation. In our study the treatment groups will consist of 14 people of whom about 7 will be patients. With n = 7 and an estimated ICC = 0.01. [72] the correction factor equals 1.06. To correct for repeated measurements and the use of the baseline measurement as a covariate we multiplied the required sample size by the design factor 1+?/2??02 where ? denotes the correlation between the post-treatment HADS measurements and ?0 denotes the correlation between the baseline HADS with the post-treatment HADS measurements. With ? = 0.8 and ? = 0.5 as conservative estimates the second design factor equals 0.65. Consequently after correction for clustering and covariates we arrived at a required sample size of 0.65 * 1.06 * 64 = 44 patients per arm. So 88 patients with lung cancer would be required for the study. Based on our pilot study [van den Hurk Schellekens Molema Speckens and van der Drift in preparation] we expect a 20% drop-out rate. Therefore we intend to include 110 patients and 110 partners. Primary analyses The samples of lung cancer patients and partners will be analyzed separately. Baseline characteristics of the population will be compared between MBSR and control group to ensure that key variables were evenly distributed by randomization. First analyses will be based on the intention-to-treat approach. Next we will perform per-protocol analyses with the treatment-adherent sample (i.e. in the MBSR condition participants have to attend at least four of the eight MBSR sessions [73] and in the TAU condition participants do not attend a mindfulness-based programme). We will use linear mixed models to analyze all outcome variables (i.e. psychological distress quality of life (only for patient) caregiver appraisal (only for partner) relationship quality and spirituality) with treatment as fixed factor baseline measurement as covariate and a random intercept based on MBSR group. This procedure will use all observed data in our analyses. In addition Cohen™s d effect size [74] will be reported based on the difference between the group means on baseline and follow-up scores divided by the pooled standard deviation at baseline and follow-up. Secondary analyses Cost effectiveness The quality of life measures (i.e. QLQ-C30; QLQ-LC13) will be used to calculate Quality of Adjusted Life Years (QALYs) for each individual. Costs and effects (in terms of QALYs) will be combined in the incremental cost-effectiveness ratio (ICER). The ICER expresses cost-effectiveness in terms of incremental costs per QALY gained. To estimate confidence intervals for the mean of the ICER a non-parametric bootstrapping method will be used performing 1000 replications of the original data. In order to express the implications of the cost-effectiveness results more clearly a cost-acceptability curve will be constructed. In case of dominance a full cost analysis will be conducted to estimate the mean savings per patient per year. Mediation analyses To examine the possible underlying mechanisms of change in MBSR mediation analyses will be conducted. Only the data of the treatment-adherent sample will be included in these analyses. By means of a multiple mediation model suggested by Preacher and Hayes [75] we will test the mediating effect of mindfulness skills self-compassion rumination and adherence to MBSR on psychological distress quality of life (only in patients) caregiver appraisal (only in partners) relationship quality and spirituality. Discussion In the last ten years MBSR has not only proven to be a feasible and acceptable intervention in cancer patients [76] but it also seems to be effective in reducing psychological distress [30]. However the generalization of these results is limited because most participants were female patients with breast cancer. A large part of lung cancer patients already have advanced cancer at time of diagnosis and are confronted with a poor prognosis and low health status. Consequently they more often report psychological distress than patients with other diagnoses of cancer [89]. Hence it is not yet clear whether MBSR is a feasible acceptable and effective intervention in patients with lung cancer. Moreover little is known about the effectiveness of MBSR in partners of cancer patients [30] though they also often report psychological distress. Our pilot study of 19 lung cancer patients and 16 partners participating in an MBSR course provides preliminary evidence that MBSR is feasible and acceptable in this population (van den Hurk Schellekens Molema Speckens and van der Drift in preparation). The current trial will answer the question whether MBSR is effective in patients with lung cancer and their partners. We started enrolment of participants in February 2012. At the moment we think recruiting a sufficient number of patients and partners will be a challenge due to rapidly fluctuating health status and sudden changes in cancer treatment [77]. The main reasons for declining participation in patients is ˜being too ill™ or that it is ˜too much of a burden during chemo and/or radiotherapy™. Furthermore no perceived need or motivation for the training is commonly mentioned. Among partners participation is highly depending on whether the patient is willing to participate. Although partners can take part separately partners who are interested do often not participate when the patients decline participation. Considering the difficulty of studying lung cancer patients and their partners [77] our trial will offer valuable information on whether MBSR as one of the few available psychosocial care programmes contributes to the alleviation of their psychological distress. Abbreviations MBSR: Mindfulness-based stress reduction; RCT: Randomized controlled trial; RUNMC: Radboud University Nijmegen Medical Centre; MBCT: Mindfulness-based cognitive therapy; MMSE: Mini mental state examination; DT: Distress thermometer; HADS: Hospital anxiety and depression scale; QLQ-C30: Quality of life “ cancer; QLQ-LC13: Quality of life “ lung cancer; SIP: Sickness impact profile; SPPIC: Self-perceived pressure from informal care; CRA-SE: Caregiver reaction assessment “ care-derived self-esteem; IMS-S: Investment model scale-satisfaction; MIS: Mutuality and interpersonal sensitivity; SAIL: Spiritual attitude and involvement list; FFMQ: Five facet mindfulness questionnaire; SCS: Self-compassion scale; RRS-EXT: Rumination response scale “ extended version; IES: Impact of event scale. Competing interests The authors declare that they have no competing interests. Authors™ contributions All authors contributed to the design of the study. AS MD and JP are the principal investigators of the study. MS drafted the paper which was modified and supplemented by all other authors. DH MS and MD are involved in recruiting participants while MS and DH take care of the logistics of the study and data collection. RD contributed specifically to the statistical analysis plan and WW contributed specifically to the design of the cost-effectiveness evaluation. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2407/14/3/prepub Acknowledgements This research is funded by Foundation Alpe d™HuZes and the Dutch Cancer Society (Grant number KUN 2011“5077 awarded to Prof. dr. Anne E. M. Speckens Dr. Miep A. van der Drift and Prof. dr. Judith B. Prins). Jemal A Bray F Center MM Ferlay J Ward E Forman D Global cancer statistics CA Cancer J Clin 2011 61 2 69 90 10.3322/caac.20107 21296855 Akechi T Okamura H Nishiwaki Y Uchitomi Y Psychiatric disorders and associated and predictive factors in patients with unresectable nonsmall cell lung carcinoma: a longitudinal study Cancer 2001 92 10 2609 2622 10.1002/1097-0142(20011115)92:10<2609::AID-CNCR1614>3.0.CO;2-K 11745196 Uchitomi Y Mikami I Kugaya A Akizuki N Nagai K Nishiwaki Y Akechi T Okamura H Depression after successful treatment for nonsmall cell lung carcinoma: a 3-month follow-up study Cancer 2000 89 5 1172 1179 10.1002/1097-0142(20000901)89:5<1172::AID-CNCR27>3.0.CO;2-U 10964348 Montazeri A Milroy R Hole D McEwen J Gillis CR Anxiety and depression in patients with lung cancer before and after diagnosis: findings from a population in Glasgow Scotland J Epidemiol Community Health 1998 52 3 203 204 10.1136/jech.52.3.203 9616429 Hyodo I Eguchi K Takigawa N Segawa Y Hosokawa Y Kamejima K Inoue R Psychological impact of informed consent in hospitalized cancer patients: a sequential study of anxiety and depression using the Hospital Anxiety and Depression scale Support Care Cancer 1999 7 6 396 399 10.1007/s005200050299 10541981 Turner NJ Muers MF Haward RA Mulley GP Psychological distress and concerns of elderly patients treated with palliative radiotherapy for lung cancer Psychooncology 2007 16 8 707 713 10.1002/pon.1109 17115458 Hopwood P Stephens RJ Depression in patients with lung cancer: prevalence and risk factors derived from quality-of-life data J Clin Oncol 2000 18 4 893 903 10673533 Zabora J Brintzenhofeszoc K Curbow B Hooker C Piantadosi S The prevalence of psychological distress by cancer site Psychooncology 2001 10 1 19 28 10.1002/1099-1611(200101/02)10:1<19::AID-PON501>3.0.CO;2-6 11180574 Carlson LE Angen M Cullum J Goodey E Koopmans J Lamont L MacRae JH Martin M Pelletier G Robinson J High levels of untreated distress and fatigue in cancer patients Br J Cancer 2004 90 12 2297 2304 15162149 Temel JS Greer JA Muzikansky A Gallagher ER Admane S Jackson VA Dahlin CM Blinderman CD Jacobsen J Pirl WF Early Palliative Care for Patients with Metastatic Non-Small-Cell Lung Cancer N Engl J Med 2010 363 8 733 742 10.1056/NEJMoa1000678 20818875 Abernethy AD Chang HT Seidlitz L Evinger JS Duberstein PR Religious coping and depression among spouses of people with lung cancer Psychosomatics 2002 43 6 456 463 10.1176/appi.psy.43.6.456 12444228 Thielemann PA Conner NE Social support as a mediator of depression in caregivers of patients with end-stage disease J Hosp Palliat Nurs 2009 11 2 82 90 10.1097/NJH.0b013e31819974f9 Pinquart M Duberstein PR Optimism pessimism and depressive symptoms in spouses of lung cancer patients Psychol Health 2005 20 5 565 578 10.1080/08870440412331337101 Kim Y Duberstein PR Sorensen S Larson MR Levels of depressive symptoms in spouses of people with lung cancer: effects of personality social support and caregiving burden Psychosomatics 2005 46 2 123 130 10.1176/appi.psy.46.2.123 15774950 Mosher CE Jaynes HA Hanna N Ostroff JS Distressed family caregivers of lung cancer patients: an examination of psychosocial and practical challenges Support Care Cancer 2013 21 2 431 437 10.1007/s00520-012-1532-6 22797839 Mosher CE Bakas T Champion VL Physical health mental health and life changes among family caregivers of patients with lung cancer Oncol Nurs Forum 2013 40 1 53 61 10.1188/13.ONF.53-61 23269770 Ostlund U Wennman-Larsen A Persson C Gustavsson P Wengstrom Y Mental health in significant others of patients dying from lung cancer Psychooncology 2010 19 1 29 37 10.1002/pon.1433 19253315 Wennman-Larsen A Persson C Ostlund U Wengstrom Y Gustavsson JP Development in quality of relationship between the significant other and the lung cancer patient as perceived by the significant other Eur J Oncol Nurs 2008 12 5 430 435 10.1016/j.ejon.2008.07.004 18845476 Siminoff LA Wilson-Genderson M Baker S Jr Depressive symptoms in lung cancer patients and their family caregivers and the influence of family environment Psychooncology 2010 19 12 1285 1293 10.1002/pon.1696 20119935 Manne S Badr H Intimacy processes and psychological distress among couples coping with head and neck or lung cancers Psychooncology 2010 19 9 941 954 10.1002/pon.1645 19885852 Badr H Taylor CLC Effects of relationship maintenance on psychological distress and dyadic adjustment among couples coping with lung cancer Health Psychol 2008 27 5 616 627 18823188 Buccheri G Depressive reactions to lung cancer are common and often followed by a poor outcome Eur Respir J 1998 11 1 173 178 10.1183/09031936.98.11010173 9543289 Walker J Sawhney A Hansen CH Symeonides S Martin P Murray G Sharpe M Treatment of depression in people with lung cancer: a systematic review Lung Cancer 2013 79 1 46 53 10.1016/j.lungcan.2012.09.014 23102652 Follwell M Burman D Le LW Wakimoto K Seccareccia D Bryson J Rodin G Zimmermann C Phase II study of an outpatient palliative care intervention in patients with metastatic cancer J Clin Oncol 2009 27 2 206 213 10.1200/JCO.2008.17.7568 19064979 Jordhoy MS Fayers P Loge JH Ahlner-Elmqvist M Kaasa S Quality of life in palliative cancer care: results from a cluster randomized trial J Clin Oncol 2001 19 18 3884 3894 11559726 Gustafson DH DuBenske LL Namkoong K Hawkins R Chih M-Y Atwood AK Johnson R Bhattacharya A Carmack CL Traynor AM An eHealth system supporting palliative care for patients with non“small cell lung cancer Cancer 2013 119 9 1744 1751 10.1002/cncr.27939 23355273 Greer JA Pirl WF Jackson VA Muzikansky A Lennes IT Heist RS Gallagher ER Temel JS Effect of early palliative care on chemotherapy use and end-of-life care in patients with metastatic non-small-cell lung cancer J Clin Oncol 2012 30 4 394 400 10.1200/JCO.2011.35.7996 22203758 Kabat-zinn J Full catastrophe living: using the wisdom of your body and mind to face stress pain and illness 1990 New York: Delacourt Segal ZV Williams JMG Teasdale JD Mindfulness-Based Cognitive Therapy for depression: a new approach to preventing relapse 2002 New York: Guilford Press Piet J Wurtzen H Zachariae R The effect of Mindfulness-Based Therapy on symptomps of anxiety and depression in adult cancer patients and survivors: a systematic review and meta-analysis J Consult Clin Psychol 2012 80 6 1007 1020 22563637 Birnie K Garland SN Carlson LE Psychological benefits for cancer patients and their partners participating in Mindfulness-Based Stress Reduction (MBSR) Psychooncology 2010 19 9 1004 1009 10.1002/pon.1651 19918956 Hagedoorn M Sanderman R Bolks HN Tuinstra J Coyne JC Distress in couples coping with cancer: a meta-analysis and critical review of role and gender effects Psychol Bull 2008 134 1 1 30 18193993 Folstein MF Folstein SE McHugh PR Mini-mental state: practical method for grading cognitive state of patiens for clinician J Psychiatr Res 1975 12 3 189 198 10.1016/0022-3956(75)90026-6 1202204 Roth AJ Kornblith AB Batel-Copel L Peabody E Scher HI Holland JC Rapid screening for psychologic distress in men with prostate carcinoma Cancer 1998 82 10 1904 1908 10.1002/(SICI)1097-0142(19980515)82:10<1904::AID-CNCR13>3.0.CO;2-X 9587123 Tuinman MA Gazendam-Donofrio SM Hoekstra-Weebers JE Screening and referral for psychosocial distress in oncologic practice Cancer 2008 113 4 870 878 10.1002/cncr.23622 18618581 Kübler-Ross E On death and dying 1969 New York: Macmillan MBSR teacher certification pathway: complete checklist http://www.umassmed.edu/uploadedFiles/cfm2/training/MBSR%20Teacher%20Certification%20Pathway%20Complete%20Checklist[1].pdf Crane RS Kuyken W Williams JMG Hastings RP Cooper L Fennell MJV Competence in teaching mindfulness-based courses: concepts development and assessment Mindfulness 2012 3 76 84 10.1007/s12671-011-0073-2 23293683 Zigmond AS Snaith RP The H"

Lung_Cancer

"Sulindac is a Food and Drug Administration (FDA)-approved non-steroidal anti-inflammatory drug (NSAID) for the treatment of osteoarthritis ankylosing spondylitis gout or rheumatoid arthritis [20]“[23]. Its anti-inflammatory activity is due to its inhibition of the synthesis of prostaglandins [24] which cause inflammation and pain in the body. Sulindac has also been found to block cyclic guanosine monophosphate-phosphodiesterase an enzyme that inhibits the normal apoptosis signaling pathway and this inhibitory effect allows the apoptotic signaling pathway to proceed unopposed resulting in apoptotic cell death and reducing the incidence of various tumors including breast esophageal stomach prostate bladder ovary and lung cancers [25] [26]. In humans sulindac is reduced to the active anti-inflammatory metabolite sulindac sulfide undergoes a 2-step reoxidation to sulindac sulfone [27] [28]. All three compounds have been shown to have chemoprotective effects. In colon cancer sulindac has been used to increase the anticancer effects of some reagents or stresses including bortezomib [4] hydrogen peroxide [29] and oxidative stress [30]. Importantly sulindac and its metabolites modulate the expression of multioxidative enzymes including glutathione S-transferases and NQO1 the latter being the key regulator of ?-lapachone-induced cell death in cancer cells [28] [31] [32] and sulindac might therefore have a synergistic anti-tumor effect with ?-lapachone. Lung cancer the major cancer worldwide is now the leading cause of cancer-related deaths [33]“[35]. According to a report of the Department of Health Executive Yuan ROC (Taiwan) published in 2010 the mortality rate for lung cancer is 20% topping the list of all cancer-related deaths. The cost of health care for treatment of lung disease is increasing tremendously each year and threatens to overwhelm public health services [36]. In order to get a better target therapy researchers have tried to identify key differences between lung cancer cells and normal lung cells such as mutation or overexpression of genes including EGFR ras and VEGF [37]“[39]. Unfortunately current chemotherapies for lung cancer lack adequate specificity efficacy and treatment heterogeneity is also a big issue [40]. There is therefore an urgent need for new therapeutic drugs or new combinations of drugs to provide more efficient lung cancer therapy. Since NQO1 overexpression has been noted in both non-small cell lung cancer (NSCLC) cell line [41] [42] ?-lapachone could be a potential therapeutic drug for lung cancers. However some lung cancer cells show lower NQO1 expression or activity and might therefore be resistant to ?-lapachone toxicity. In this study we first investigated the relationship between ?-lapachone toxicity and NQO1 levels in NSCLC cell lines then determined the signaling pathway involved in the cell death caused by high concentrations of ?-lapachone. We also used lower concentrations of ?-lapachone to explore whether sulindac and its metabolites could facilitate the anticancer effect of ?-lapachone by increasing NQO1 expression or activity in lung cancer cell lines with low NQO1 levels and checked the importance of NQO1 in this combination therapy. We found that the toxicity of ?-lapachone was related to the level of NQO1 expression or activity in lung cancer cells and that high concentrations of ?-lapachone killed cells by decreasing phosphorylation of PI3K AKT and ERK and activating JNK. In addition the cytotoxicity of low concentrations of ?-lapachone was increased by combination with sulindac and its metabolites a process involving upregulation of expression or activity of NQO1. Materials and Methods Cell Culture The human lung cancer cell lines CL1-1 CL1-5 and A549 were cultured in 5% CO2 at 37°C in RPMI 1640 medium containing 10% fetal calf serum 100 Units/ml of penicillin and 100 mg/ml of streptomycin (all from Gibco). The cell lines were gifts from Dr. PC Yang National Taiwan University Hospital [43] in whose laboratory CL1-5 cells were selected from parental CL1-1 cells for greater metastatic potential using a transwell system. Cell Viability Assays CL1-1 CL1-5 or A549 cells (1x104) were seeded for 24 h at 37°C in a 96-well culture plate then were subjected to starvation for 14 h in RPMI 1640 medium containing 2% fetal calf serum 100 Units/ml of penicillin and 100 mg/ml of streptomycin. Following 6 h pretreatment with medium or the indicated concentration of sulindac or its metabolites (all from Sigma) the cells were incubated for 12 h with or without the indicated concentration of ?-lapachone in the continued presence of sulindac or its metabolites and then cell viability was evaluated. Two cell viability assays were used. In the crystal violet staining assay the cells were fixed with 4% paraformaldehyde for 15 min stained with 0.4% crystal violet for 15 min and washed with H2O then 50% acid alcohol was used to dissolve the bound crystal violet and the OD at 550 nm measured on an ELISA reader. In the MTT assay 10 µl of MTT (0.5 mg/ml) (Sigma) was added to each well and the plates incubated at 37°C for 4 h then the formazan product was dissolved in 100 µl of DMSO at 37°C for 30 min and the OD at 570 nm measured on a microplate reader. Acridine Orange (AO) Staining Cells (5x104) cultured on cover-slides in 24-well plates were incubated for 14 h in RPMI 1640 medium containing 2% fetal calf serum preincubated with sulindac sulfide for 6h and then treated with or without ?-lapachone for 24 h then were immediately fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min at room temperature (RT) and stained for 10 min with 0.5 ml of AO (10 mg/ml in PBS) (Sigma). After several PBS washes the cells were examined on an Olympus BH-2 inverted microscope equipped with a fluorescence attachment. Detection of Apoptosis and Measurement of Intracellular Calcium Levels To detect apoptosis cells (1x106) were treated for 36 or 9 h with 5 µM ?-lapachone then were washed with ice-cold PBS trypsinized with 0.05% trypsin-0.02% EDTA stained for 15 min at 37°C with Annexin V-FITC (10 µg/ml) (Strong Biotech Corporation AVK050 Taipei Taiwan) and analyzed by flow cytometry on a FACScan flow cytometer (Becton Dickinson). To measure intracellular calcium levels the cells were incubated for 10 min at 37°C with 2 mM Fluo-4/AM (Molecular Probes) washed with PBS trypsinized and analyzed by FACSan flow cytometry using the FL1H parameter. Western Blot Analyses Treated cells were lysed with RIPA buffer containing 10 µg/ml of protease inhibitor (Sigma) and then the lysate was centrifuged at 10000—g for 15 min at 4°C and the supernatant collected for immunoblotting. The protein concentration was measured by the Bradford assay and samples containing 20 µg of protein were separated by 10 or 12% SDS“PAGE and then transferred to Immobilon-P membranes for 2 h at 200 V (Millipore) in a Trans-Blot Electrophoretic Transfer cell. The membranes were blocked for 1 h at RT with 5% skim milk in PBS-0.2% Tween 20 (PBS-T) then incubated for 2 h at RT with antibodies against NQO1 (Cell Signaling) PI3 kinase or p-PI3 kinase (Millipore) AKT or p-AKT (Epitomics) ERK p-ERK JNK or p-JNK (Cell Signalling)GAPDH (Genetex) or ?-actin (Abcam) diluted 1?1000 in 1% BSA. After washing for 30 min at RT with PBST the membranes were incubated for 1 h at RT with horseradish peroxidase-conjugated secondary antibody (Perkin-Elmer Boston MA; 1?5000 dilution in PBST) then bound antibody was detected using the ECL Western blotting reagent (Amersham) chemiluminescence being detected using a Fuji Medical X-ray film (Tokyo Japan) and quantified by gel image analyses with Image Pro software. The intensity of the band of interest was divided by that for ?-actin or GAPDH (loading controls) and this value normalized to that seen with no treatment. "

Lung_Cancer

" Total patient questionnaire scores by the multidisciplinary team in the intervention group at baseline (pre) and at the end of the study (post). A low score indicates better experience. Each symbol represents the mean score for each trust in the intervention group. The maximum possible score for the questionnaire is 11. Quality improvement plan themes Quality improvement plan theme Number of plans Multidisciplinary team effectiveness 31 Diagnostic pathways 13 Treatment pathways 9 Access to clinical nurse specialists 8 Clinical trial recruitment 4 Patient experience 2 Baseline (2009) national lung cancer audit indicators Control ( n =47) Intervention ( n =31) Excluded ( n =67) P -value Mean (%) s.e.m. Mean (%) s.e.m. Mean (%) s.e.m. Control vs intervention vs non-participant control vs intervention Case ascertainment 158.1 38.6 122.0 7.2 107.4 3.6 0.220 0.455 Discussed at the MDT meeting 95.2 0.7 93.7 1.7 90.9 1.9 0.155 0.370 Histological confirmation rate 75.7 1.2 76.4 1.8 78.4 1.6 0.409 0.739 Active treatment 59.5 1.2 55.9 2.2 59.5 1.5 0.305 0.131 Surgery (all cases) 13.4 0.6 13.0 0.8 14.2 0.7 0.469 0.648 SCLC (chemo) 65.1 2.2 66.5 3.9 63.3 2.7 0.746 0.733 Seen by CNS 70.3 3.8 76.6 3.2 58.3 4.2 0.007 0.243 CNS present diagnosis 44.0 3.8 49.4 5.4 38.7 3.8 0.237 0.403 Abbreviations: CNS=clinical nurse specialist; MDT=mulitdisciplinary team; SCLC=small-cell lung cancer. Data are shown as mean and s.e. proportion of patients. BMC Cancer BMC Cancer BMC Cancer 1471-2407 BioMed Central 24386906 3893473 1471-2407-14-3 10.1186/1471-2407-14-3 Study Protocol Study protocol of a randomized controlled trial comparing Mindfulness-Based Stress Reduction with treatment as usual in reducing psychological distress in patients with lung cancer and their partners: the MILON study Schellekens Melanie PJ 1 Melanie.Schellekensradboudumc.nl van den Hurk Desiree GM 2 Desiree.vandenHurkradboudumc.nl Prins Judith B 3 Judith.Prinsradboudumc.nl Molema Johan 2 Johan.Molemaradboudumc.nl Donders A Rogier T 4 Rogier.Dondersradboudumc.nl Woertman Willem H 4 Willem.Woertmanradboudumc.nl van der Drift Miep A 2 Miep.vanderDriftradboudumc.nl Speckens Anne EM 1 Anne.Speckensradboudumc.nl 1Department of Psychiatry Radboud University Nijmegen Medical Centre Nijmegen The Netherlands 2Department of Pulmonary Diseases Radboud University Nijmegen Medical Centre Nijmegen The Netherlands 3Department of Medical Psychology Radboud University Nijmegen Medical Centre Nijmegen The Netherlands 4Department of Epidemiology Biostatistics and Health Technology Assessment Radboud University Nijmegen Medical Centre Nijmegen The Netherlands 2014 3 1 2014 14 3 3 28 6 2013 19 12 2013 Copyright 2014 Schellekens et al.; licensee BioMed Central Ltd. 2014 Schellekens et al.; licensee BioMed Central Ltd. This is an open access distributed under the terms of the Creative Commons Attribution License (http://creativecommons./licenses/by/2.0) which permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited. Background Lung cancer is the leading cause of cancer death worldwide and characterized by a poor prognosis. It has a major impact on the psychological wellbeing of patients and their partners. Recently it has been shown that Mindfulness-Based Stress Reduction (MBSR) is effective in reducing anxiety and depressive symptoms in cancer patients. The generalization of these results is limited since most participants were female patients with breast cancer. Moreover only one study examined the effectiveness of MBSR in partners of cancer patients. Therefore in the present trial we study the effectiveness of MBSR versus treatment as usual (TAU) in patients with lung cancer and their partners. Methods/Design A parallel group randomized controlled trial is conducted to compare MBSR with TAU. Lung cancer patients who have received or are still under treatment and their partners are recruited. Assessments will take place at baseline post intervention and at three-month follow-up. The primary outcome is psychological distress (i.e. anxiety and depressive symptoms). Secondary outcomes are quality of life (only for patients) caregiver appraisal (only for partners) relationship quality and spirituality. In addition cost-effectiveness ratio (only in patients) and several process variables are assessed. Discussion This trial will provide information about the clinical and cost-effectiveness of MBSR compared to TAU in patients with lung cancer and their partners. Trial registration ClinicalTrials.gov NCT01494883. Mindfulness-based stress reduction Lung cancer patients Partners Psychological distress Randomized controlled trial Background With an estimated 1.4 million deaths per year lung cancer is the leading cause of death by cancer worldwide. Even with the best available treatment five-year survival is merely 16% and about 60 to 70% of patients die within the first year after diagnosis [1]. This poor prognosis is often caused by a late diagnosis as the presentation usually occurs when the lung cancer is advanced. Patients may develop burdensome symptoms like pain dyspnoea fatigue and cough and they may undergo radical treatment including surgery chemo- and radiotherapy. Not surprisingly lung cancer has a major impact on the psychological wellbeing of patients and their family. Akechi and colleagues [2] showed that 19% of patients with advanced lung cancer meets the criteria of psychiatric disorders especially depressive and adjustment disorders. Of patients who had been successfully treated for lung cancer 15% met the criteria for a minor or major depressive disorder [3]. The prevalence rate of depressive and anxiety symptoms among lung cancer patients ranges from 20 to 47% [4-7]. Compared to patients with other cancer diagnoses lung cancer patients report the highest rates of distress (43 to 58%) [89] resulting in a lower quality of life [10]. Family friends and especially partners of patients with lung cancer also have to deal with its psychological impact [11-14]. Partners not only provide emotional and practical support they also have to cope with their own concerns including the uncertainty regarding the course of the illness and the fear of losing their partner [15]. More than 50% of partners of lung cancer patients report negative emotional effects of caregiving [16]. Around 40% of partners of patients with advanced lung cancer report high levels of distress [17]. The relationship between patient and partner can also be affected by the cancer. It has been shown that some partners report a lower quality of their relationship after the diagnosis of lung cancer [18]. Though numerous studies examined the psychological distress of lung cancer patients and their partners [2-22] not much research is done on how to alleviate distress in these groups [23]. In addition the available studies on managing the psychosocial care needs of cancer patients and their families have focused on care at the very end of life (e.g. [24-26]). Recently studies have demonstrated that palliative care initiated early in treatment improves the quality of life and depressive symptoms of lung cancer patients [1027]. This stresses the importance of integrating psychosocial care for lung cancer patients and their partners early in the treatment rather than instigating it once life-prolonging therapies fail. In the past ten years MBSR has become a promising psychosocial intervention for cancer patients. Mindfulness is defined as intentionally paying attention to moment-by-moment experiences in a non-judgmental way [28]. MBSR is an 8-week group-based training consisting of meditation practices such as the bodyscan gentle yoga sitting and walking meditation. By repeatedly bringing attention back to the current experience participants gradually learn to disengage from dysfunctional thoughts and directly experience the emotions and bodily sensations of the present moment. MBSR aims to provide participants with the ability to step back from ruminating about the past or worrying about the future and simply allow experiences to unfold [2829]. A recent meta-analysis [30] of 13 nonrandomized studies and 9 randomized controlled trials (RCT) concluded there is positive evidence for the use of mindfulness-based interventions in reducing psychological distress in cancer patients. Among the RCT€™s a reduction in symptom severity was found for both anxiety and depression corresponding to moderate pooled controlled effect sizes (Hedges€™s g = 0.37 and Hedges€™s g = 0.44 respectively) [30]. Though mindfulness-based interventions seem to be effective the authors note that across studies the majority of participants were women (85%) and diagnosed with breast cancer (77%). Compared to breast cancer patients patients with lung cancer are more often male older and have a poorer prognosis. Furthermore of these 22 studies only one study included the partners of the patients showing that partners also benefit from the MBSR training [31]. This is quite surprising since partners of cancer patients also report high levels of distress [32]. Aims The aim of the Mindfulness for Lung Oncology Nijmegen (MILON) study is to examine the effectiveness of MBSR compared to TAU in reducing psychological distress in patients with lung cancer and their partners. We hypothesize that patients in the MBSR group will report a lower level of psychological distress (i.e. anxiety and depressive symptoms) higher levels of quality of life quality of relationship and spirituality than those in the TAU group. Medical and societal costs will be lower in the MBSR versus TAU group. We expect partners in the MBSR group to report a lower level of psychological distress and higher levels of caregiver appraisal relationship quality and spirituality than their counterparts in the TAU group. With regard to the working mechanisms of the MBSR programme we will examine changes in mindfulness skills self-compassion rumination intrusion avoidance and adherence to MBSR. Methods/Design Study design The design of the €˜MILON€™ study is a parallel group randomized controlled trial with an embedded process study. Participants are randomized between MBSR and TAU. The study protocol has been approved by our ethical review board (CMO Arnhem-Nijmegen) and registered under number 2011€“519. Participants and procedure Patients and partners are recruited at the outpatient clinic of the Department of Pulmonary Diseases Radboud University Nijmegen Medical Centre (RUNMC) by a nurse practitioner and the attending physician. Patients and partners are invited to participate together but both are welcome to participate on their own if they do not have a partner or their partner is not willing to participate. Patients and/or partners who are interested are provided with an information leaflet. If they are willing to participate they are invited for a research interview in which in- and exclusion criteria are assessed and informed consent is taken. At other participating hospitals (Department of Pulmonary Diseases Canisius-Wilhelmina Hospital Nijmegen; Department of Pulmonary Medicine Rijnstate Arnhem; Department of Oncology Elkerliek Hospital Helmond; Department of Pulmonary Medicine Jeroen Bosch Hospital; Department of Pulmonary Diseases Maas hospital Pantein Boxmeer) patients and their partners will be sent a letter with the invitation to participate in the study. One week later the researcher calls the patients to answer possible questions and asks whether the patient and partner are interested in participation. If so they are invited for a research interview at the RUNMC. Eligibility We include patients and/or partners of patients who are (a) diagnosed with cytologically or histologically proven non-small cell lung cancer or small cell lung cancer and (b) have received or are still under treatment. Exclusion criteria for both patient and partner include: (a) being under 18 years of age (b) not being able to understand or use the Dutch language (c) former participation in MBSR or Mindfulness-Based Cognitive Therapy (MBCT) (d) current and regular treatment by psychologist or psychiatrist (e) current participation in other psychosocial programme and (f) physical or cognitive (<26 on the Mini-Mental State Examination (MMSE)) impairments hampering participation in MBSR training or completion of questionnaires. Baseline Patients and partners are interviewed to obtain demographics and clinical characteristics after which they are screened for cognitive impairments with the MMSE [33]. After that baseline questionnaires including the Distress Thermometer (DT) [3435] are administered followed by randomization. shows the assessment instruments and time points at which the questionnaires are administered to patients and partners. Measurements and corresponding time points for patient and partner Measure Target T0 T1 T2 pt pr pt pr pt pr MMSE Cognitive impairments x x DT General distress x x HADS Psychological distress x x x x x x QLQ-C30 Quality of life x x x QLQ-LC13 Quality of life x x x SIP Impact of sickness x x x SPPIC Caregiver burden x x x CRA-SE Caregiver self-esteem x x x IMS-S Relationship satisfaction x x x x x x MIS Communication about cancer x x x x x x SAIL Spirituality x x x x x x FFMQ Mindfulness skills x x x x x x SCS Self-compassion x x x x x x RRS-EXT Rumination x x x x x x IES Psychological stress reaction x x x x x x Diary Health care use work absence Monthly during study period for pt Calendar Mindfulness adherence Monthly during study period for pt and pr Note. T0 = Baseline measurement; T1 = Post-intervention measurement; T2= 3-month follow-up measurement; pt = Patient; pr = Partner; MMSE = Mini Mental State Examination; DT = Distress Thermometer; HADS = Hospital Anxiety and Depression Scale; QLQ-C30 = Quality of Life €“ Cancer; QLQ-LC13 = Quality of Life €“ Lung Cancer; SIP = Sickness Impact Profile; SPPIC = Self-Perceived Pressure from Informal Care; CRA-SE = Caregiver Reaction Assessment €“ Care-Derived Self-Esteem; IMS-S = Investment Model Scale-Satisfaction; MIS = Mutuality and Interpersonal Sensitivity; SAIL = Spiritual Attitude and Involvement List; FFMQ = Five Facet Mindfulness Questionnaire; SCS = Self-Compassion Scale; RRS-EXT = Rumination Response Scale €“ Extended Version; IES = Impact of Event Scale. Randomization Randomization is stratified according to setting and minimized for (a) stage of disease (curative versus palliative) (b) baseline level of anxiety and depressive symptoms (anxiety or depression subscale score of Hospital Anxiety and Depression Scale (HADS) <8 versus ?8) (c) treatment during MBSR (no treatment versus chemo- and/or radiotherapy) and (d) participation (patient alone versus partner alone versus patient and partner together). Randomization is computerized using a randomization website specifically designed for this study on which the researcher can fill out the required data. The researcher communicates treatment allocation to the nurse practitioner who informs the patient and/or partner. Follow-up assessments Follow-up assessments take place post intervention and at three-month follow-up. Participants who have access to the internet and have an email address receive the questionnaires online. If not they receive the questionnaires on paper along with a reply envelope. In case of drop-out the researcher tries to contact the participant by phone to complete a minimum set of outcome measures and to identify the main reason for drop-out. Intervention The MBSR curriculum used is primarily based on the Mindfulness-Based Stress Reduction programme as developed by Kabat-Zinn [28] but contains some elements of the MBCT programme by Segal Williams and Teasdale [29] like psycho-education on the interrelatedness of feelings and thoughts. Moreover some modifications have been made to make the intervention more suitable for patients with lung cancer and their partners such as psycho-education about grief [36]. In addition a mindful communication exercise in which partners talk with each other about the cancer was added. The programme consists of 8 weekly 2.5-hour sessions a silent day between session six and seven and home practice assignments of about 45 minutes 6 days per week. Participants receive a set of CDs with guided mindfulness meditation exercises for home practice and a folder with information and home practice instructions for the forthcoming week. shows the content of the MBSR programme per session. The MBSR courses are taught by mindfulness teachers with extensive training in MBSR. They all fulfil the advanced criteria of the Center for Mindfulness of the University of Massachusetts Medical School [37] and maintain a regular personal meditation practice. Teachers were trained supervised and assessed to ensure their competency levels met the qualification criteria to instruct the MBSR classes. During the trial teachers will receive weekly supervision and a number of sessions will be videotaped to evaluate competence and adherence with the Mindfulness-Based Interventions €“ Teaching Assessment Criteria [38]. Content of MBSR programme per session Theme of session Meditation exercise Didactic teaching Homework 1. Automatic pilot - Bodyscan - Intention of participating - Bodyscan - Raisin exercise - Eating one meal mindfully - Attention for routine activity 2. Mindfulness of the breath - Bodyscan - Imagery exercise to demonstrate relationship between thoughtsand feelings - Bodyscan - Sitting mediation with focus on breath - Attention for breath - Awareness of pleasant events - Attention for routine activity 3. Observing limits - Yoga while lying down - Seeing exercise to demonstrate difference between observation and interpretation - Bodyscan or yoga - 3-min breathing space - Sitting meditation - Awareness of unpleasant events - 3-min breathing space 4. Opening up to distress - Sitting mediation with focus on breath body and sound - Interrelatedness of feelings thoughts and bodily sensations - Bodyscan or yoga - Sitting meditation - 3-min breathing space - Psychoeducation about grief - Awareness of stress reactions - 3-min breathing space 5. Responding to distress - Sitting mediation with focus on breath body sound thoughts difficulty - Reacting versus responding - Meditation by choice - Coping with grief - Awareness of reaction in difficult situation - Walking meditation - Awareness of communication difficulties - 3-min breathing space - 3-min breathing space 6. Mindful communication - Yoga in standing position - Mindful communication exercise about effect of lung cancer with their own partner - Sitting meditation or yoga - 3-min breathing space - Awareness of communication - 3-min breathing space during stress Silent day - Varying meditation exercises "

Lung_Cancer

"MALAT1 also known as NEAT2 (nuclear-enriched abundant transcript 2) is a highly conserved nuclear lncRNA and a predictive marker for metastasis development in lung cancer [23]. In this study we found that the expression of another lncRNA BANCR was significantly downregulated in NSCLC tissues. Specifically BANCR expression was significantly lower at the later stages of tumor development and in tumors that had undergone extensive metastasis. Moreover the overall survival time of patients with lower BANCR expression levels was significantly shorter than that for patients with higher BANCR expression levels. Our results indicate that BANCR expression provided a significant independent predictive value for TNM stage (P?=?0.038). We demonstrated that upregulation of BANCR expression led to the significant inhibition of cell viability migration invasion and promotion of apoptosis. Knockdown of BANCR expression promoted cell migration and invasion. BANCR induced cell apoptosis may be partly via P53 which could contribute to the less cells in migration and invasion; however the impaired migration and invasion ability is the main reason which could be supported by wound-healing assay. Moreover increased BANCR expression levels resulted in a significant reduction in the number of metastatic nodules on the lungs in vivo. These findings suggest that BANCR plays a direct role in the modulation of cell metastasis and NSCLC progression and may be useful as a novel prognostic or progression marker for NSCLC. Tumor development and progression is precisely regulated by several subsets of genes that act by either silencing tumor suppressor genes or activating oncogenes [24]. Tumor suppressor genes can negatively regulate cell proliferation by inducing growth arrest and inhibiting cell invasion. In cancer cells tumor suppressor genes are usually silenced by genetic or epigenetic alterations [25]. Whether epigenetic regulatory factors such as histone acetylation or DNA methylation manipulate the expression of lncRNAs remains unclear. Hypermethylation of the promoter or the intergenic differentially methylated region has been found to contribute to reduced lncRNA MEG3 expression in tumors indicating that epigenetic regulation is also involved in the expression of these genes [2627]. Our findings highlight that histone acetylation is a key factor in controlling lncRNA BANCR expression. These results along with those from a recent study [28] highlight the role of epigenetics in regulating lncRNA transcription. To explore the molecular mechanism through which BANCR contributes to the invasion and metastasis of NSCLC we investigated potential target proteins involved in cell motility and matrix invasion. Hallmarks of EMT are the loss of E-cadherin expression and aberrant expression of N-cadherin and Vimentin [29-32]. Therefore we determined the protein levels of these EMT-induced markers following BANCR overexpression. Our results indicated that inhibitory effects on cell migration and invasion were associated with EMT. Matrix metalloproteases (MMPs) are also important to many aspects of biology ranging from cell proliferation differentiation and remodeling of the extracellular matrix (ECM) to vascularization and cell migration. Upregulation of BANCR expression in NSCLC cells led to a significant decrease in MMP2 protein levels. Our findings demonstrated that BANCR mediated NSCLC cell migration invasion and metastasis suppression which possibly also affected EMT. As a central differentiation process EMT allows for remodeling of tissues during the early stages of embryogenesis and is implicated in the promotion of tumor cell invasion and metastasis [733]. It has been proposed and supported by many studies that EMT could be a potent mechanism for promoting the detachment of cancer cells from primary tumors. A characteristic of cells that undergo EMT is increased expression levels of N-cadherin and Vimentin and a loss of E-cadherin expression. Importantly EMT has been reported to be associated with poor clinical outcome in NSCLC [3435]. Therefore lncRNAs as regulators of EMT might be suitable candidates for intervention in the treatment of cancer. Although only a small number of functional lncRNAs have been well characterized to date they have been shown to regulate gene expression at various levels including chromatin modification transcription and post-transcriptional processing. Hox transcript antisense intergenic RNA (HOTAIR) is one of the most studied lncRNAs involved in chromatin modification which can target PRC2 genome-wide to alter H3K27 methylation and gene expression patterns [22]. A muscle-specific lncRNA linc-MD1 may function as competing endogenous RNAs (ceRNAs) to sponge miRNAs thereby modulating the derepression of miRNA targets and impose an additional level of post-transcriptional regulation [36]. Here although we observed BANCR overexpression induced NSCLC cells apoptosis and regulate EMT phenotype the possible mechanisms that underlie such regulatory behaviors still remain to be fully understood. Further investigation of BANCR molecular and biological functions in controlling EMT will undoubtedly be important in understanding the molecular biology of NSCLC metastasis and progression. Conclusions The expression of BANCR was significantly decreased in NSCLC tissues suggesting that its downregulation may be a negative prognostic factor for NSCLC patients and indicative of poor survival rates and a higher risk for cancer metastasis. We showed that BANCR possibly regulates the invasive and metastatic ability of NSCLC cells partially through regulation of EMT. Our findings further the understanding of NSCLC pathogenesis and development and facilitate the development of lncRNA-directed diagnostics and therapeutics against cancers. However the molecular mechanisms through which BACNR regulates EMT requires further investigation."

Lung_Cancer

"Hypertension and renal insufficiency are two risk factors for RPLS. Twenty-four patients (57%) experienced hypertension (15 grade 3 but no grade 4) during the study 15 of whom had a history of hypertension and 12 had taken antihypertensive medications before entering the study. Eight patients with hypertension also experienced proteinuria. Fourteen patients (33%) experienced proteinuria (all grades 1 or 2 except a single grade 3) none of whom had a history of renal disease. Fourteen patients experienced CrCL decreases during treatment with six patients having CrCL decreases below 60?ml?min?1 after treatment cycle 4. Efficacy evaluation As the study was closed prematurely there was no statistical power to test the primary hypothesis. Of the 42 patients enrolled 4 patients discontinued early from the study due to AEs (2) consent withdrawal (1) and investigator decision (1). As they did not have a post-baseline tumour assessment they were excluded from the efficacy assessment per predefined statistical analysis plan. Of the 38 patients evaluable for efficacy the median PFS was 5 months (95% CI 4.3“7.1; A) and ORR was 26% (95% CI 12“40%) all of which (10/38) were PR. The disease control rate (PR+stable disease) was 89% (26%+63%). A mean reduction of 20% was observed in average percentage changes over time in tumour burden (sum of largest diameters of target lesions) from baseline. Of the 38 patients evaluable for efficacy 22 (58%) developed hypertension as AE during the study. Seven (7 out of 22=32%) had a PR compared with only three with no hypertension (3 out of 16=18%) suggesting that patients who developed hypertension may have had a higher likelihood of response to this treatment. Correlative studies Participation in the correlative studies was optional. Erythropoietin levels were obtained from 16 patients. No trend towards increase in haemoglobin or change in erythropoietin level was seen over time. Pharmacokinetic and antibody evaluation Twenty-three patients participated in blood sampling for PK analysis. Mean observed noncompartmental PK parameters for free ziv-aflibercept are presented in . The concentration“time profiles and PK of free ziv-aflibercept and adjusted bound ziv-aflibercept: VEGF were consistent with results in the phase I study (Diaz-Padilla et al 2012). Mean trough concentrations after the second ziv-aflibercept dose plateaued and remained at ?10?mg?l?1. The mean adjusted bound ziv-aflibercept:VEGF complex Cmax was 7.81?mg?l?1. Trough concentrations plateaued after day 42 and remained constant for the remainder of the study. Two patients had one sample each that was positive in the anti-drug antibody (ADA) assay. One was positive at baseline but did not have an antibody titre drawn at the end of treatment (EOT) visit. This patient completed all six cycles of combination treatment without dose delay/reduction or grade 3/4 AEs and had stable disease. The other one was negative at baseline but positive at the EOT visit. This patient experienced anaphylaxis 20?minutes after start of the ziv-aflibercept infusion on day 1 of the second cycle. Study drug was permanently withdrawn. This patient had PK parameters and a concentration“time profile different from ADA-negative patients probably because of the ADA formation. Discussion Ziv-aflibercept has been tested as a single agent and in combination with chemotherapy in the treatment of NSCLC (Leighl et al 2010; Ramlau et al 2012). On the basis of activity and safety profile we conducted the current phase II study to explore the efficacy of ziv-aflibercept in the first-line setting. Similar to ECOG 4599 AVAiL and PointBreak trials (Sandler et al 2006; Patel et al 2009a; Reck et al 2009) this study was designed to test a three-drug regimen including an anti-angiogenesis agent in this case ziv-aflibercept/cisplatin/pemetrexed. In addition maintenance therapy with single-agent ziv-aflibercept was intended to prolong the benefits and delay the development of resistance. This approach was first tested in a phase I dose-escalation study that used the same regimen of ziv-aflibercept/cisplatin/pemetrexed in 18 patients with advanced solid tumours (Diaz-Padilla et al 2012). Our study population was representative of patients with advanced NSCLC. Overall the median ziv-aflibercept dose intensity was similar to the planned intensity. The delivered dose intensities of pemetrexed/cisplatin in this study were over 98% higher than those in the pemetrexed/cisplatin arm (94.8% and 95.0% respectively) of the phase III trial (Scagliotti et al 2008). The PK of ziv-aflibercept in this study was characterised as nonlinear and similar to that observed in the phase I study. The mean observed terminal t1/2 was independent of ziv-aflibercept dose. Administration of ziv-aflibercept did not alter pemetrexed PK. Development of ADA was a rare event leading to reduced drug concentration in one patient who experienced anaphylaxis. As with all therapeutic proteins there is a potential for immunogenicity; however severe hypersensitivity reactions are rare. Although this study was terminated early the two co-primary end points ORR of 26% and median PFS of 5 months were in accordance with most historical first-line NSCLC studies (Schiller et al 2002; Scagliotti et al 2008) and slightly less than triplet regimens incorporating another anti-VEGF agent (Sandler et al 2006; Reck et al 2009). However there was no statistical power to test the primary hypothesis that ziv-aflibercept would enhance the efficacy of standard chemotherapy in NSCLC. Biomarkers that can reliably predict the degree of VEGF blockade in vivo are currently not available. Preclinical studies identified increased erythropoietin production and erythropoiesis as a possible surrogate marker of VEGF inhibition as animal data indicate that stringent VEGF inhibition including by ziv-aflibercept modulates erythropoiesis via increased hepatic erythropoietin synthesis (Tam et al 2006). Bevacizumab has also been associated with increased haemoglobin in NSCLC (Riess et al 2012) and reduced anaemia (Sher and Wu 2011). Therefore this study explored whether the increase in haemoglobin observed previously could be reproduced in the presence of chemotherapy and would correlate with anti-angiogenic activity. No trend towards increase in haemoglobin or change in erythropoietin level was found in a small subset of patients."

Lung_Cancer

"A pulmonary function test showed mild restrictive patterns. The patient underwent bronchoscopy and no endobronchial lesion was found. Bronchoalveolar lavage results were negative for malignant cells. Thick varicose septal veins and intraalveolar macrophages were identified on transbronchial lung biopsy. After seven months a follow-up chest CT scan revealed increased interstitial septal thickening (Fig. 1E F) new peribronchovascular cuffing and small amounts of bilateral pleural effusion (Fig. 1G). Finally the patient underwent video-assisted thoracoscopic surgery wedge resection of the right middle and lower lobes. In the surgical field abnormal hypervascularity was noted on the lung surface (Fig. 1H). Microscopic examination showed proliferation of thinwalled anastomosing vascular spaces lined by a single layer of endothelial cells lacking cytological atypia (Fig. 1I). Theses lesions were located along the lymphatic distribution and were highlighted by D2-40 (1:100 Dako Glostrup Denmark) immunohistochemical stain (Fig. 1J) characteristic of DPL. Presently the patient is alive without any symptoms and being observed without specific treatment such as low fat medium chain fat. DISCUSSION Diffuse pulmonary lymphangiomatosis is a rare condition in which diffuse proliferation of anastomosing lymphatic channels is observed. It manifests equally in both sexes mostly in children and young adults (3). Symptoms include dyspnea nonproductive cough bronchial casts milky sputum fever and recurrent pneumonia. However patients can present with the disease later in adulthood and often have symptoms dating back to their childhood (5). To the best of our knowledge only five cases of DPL in middle-aged patients have been reported in the English literature () (1-5) with the current report being the sixth. Ours is a unique one in the following aspects; although the disease progression revealed on serial follow-up CT required differentiation from lymphangitic metastasis the patient remained asymptomatic and did not need any treatment; also the extent of the disease markedly increased within one year and a neoplastic but possibly benign component was presumed. Making a preoperative diagnosis of DPL in adults is difficult. The most common radiologic finding is increased interstitial markings on chest radiography. CT demonstrates smooth or nodular interlobular septal and peribronchovascular thickenings. Patchy ground glass opacities are also seen. Pleural thickening pleural effusion and diffuse mediastinal soft tissue infiltration can also occur (5). Pulmonary edema pulmonary veno-occlusive disease Erdheim-Chester disease and lymphangiectasis can be considered possible diagnoses when smooth interlobular septal thickening is found. In this patient pulmonary edema was less likely since there was no evidence of congestive heart failure or pleural effusion. Prominent central pulmonary arteries were also not identified making pulmonary veno-occlusive disease less likely (6). The possibility of Erdheim-Chester disease characterized by proliferation of lipid-containing foamy histiocytes in the skeleton and other ans was ruled out by the absence of sclerotic changes in the diaphyses and metaphyses of long bones in the current case (7). The CT appearance of DPL is virtually identical to that of pulmonary lymphangiectasia (8). Pulmonary lymphangiectasia is a rare condition characterized by the diffuse dilatation of pulmonary lymphatics and classified as congenital or secondary (9). In this case there was no evidence of pulmonary hypertension or venous obstruction factors that can cause secondary lymphangiectasia. Congenital lymphangiectasia typically presents shortly after birth and is associated with high neonatal morbidity and mortality. Lymphangiomatosis typically presents in older children and is rarely seen in adults (10). Histopathologically lymphangiomatosis is characterized by an increased number of variable-sized lymphatic spaces. This should be distinguished from the findings of lymphangiectasia in which nonproliferative lymphatic channels are dilated (4). Other diseases with lymphatic distributions such as lymphangitic carcinomatosis sarcoidosis and pulmonary lymphoma can also be considered. In these diseases interlobular septal thickening tends to be nodular. There is no established treatment for DPL. "

Lung_Cancer

"B-mode ultrasound image after single high-intensity focused ultrasound insonation demonstrates a sharply demarcated and strongly hyperechoic sonolesion within the tumour. Macroscopic appearance of the zone of ablated tumour tissue (inside the highlighted circle) in the resected specimen. Arrows show the tumour margin of non-ablated adenocarcinoma. H&E staining showed coagulative necrosis with cytoplasmic eosinophilia disruption of cellular membranes with blurred cytoplasmic borders karyolysis and nuclear pyknosis. Insonated and non-insonated cancer tissues showed sharp demarcations. There was a small region between the area of coagulation necrosis and vital cancer tissue in which cells showed cytoplasmic vacuolization (). These characteristics were not found in the untreated areas of cancer tissue. NADPH-diaphorase staining produced a sharp demarcation between ablated and non-ablated cancer tissue and demonstrated that vital lung tissue was present immediately adjacent to ablated cancer tissue (). HIFU-treated tumour tissue did not show evidence of vitality by NADPH-diaphorase staining. Sonographic lesions were not detected in the tumour surrounding the lung tissue or in the direction of the HIFU irradiation. In contrast a non-treated metastatic tumour in the same lobe showed complete vitality. Histological examination of large cell neuroendocrine carcinoma immediately after ex vivo high-intensity focused ultrasound application. The border between ablated and non-ablated tumour is shown. There is a vital tumour in the upper left image (A). The thermal effect is visible as a small area which contains cells with cytoplasmic vacuolization (B) and as a coagulative necrosis with cytoplasmic eosinophilia disruption of cellular membranes with blurred cytoplasmic borders karyolysis and nuclear pyknosis (C). Arrows indicate the sonolesion boundary (H&E). Nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining. There is a sharp demarcation between vital lung tissue (A) and ablated tumour tissue (B). Immediately after ex vivo high-intensity focused ultrasound application NADPH-diaphorase staining showed a lack of uptake of the vitality stain in finger-shaped tumour extensions (B). In vivo examination A lung tumour could be simulated in the flooded lung of each of the three animals by sonography-guided injection of BioGlue®. The central tumour appeared hypoechoic by B-mode sonography and was sharply demarcated from the lung parenchyma with small inclusions of air. A temperature rise was monitored within each one-second application of HIFU irradiation. A section of 25 seconds of HIFU exposure in the ˜one second on one second off™ scheme is shown in . Repeated HIFU insonation caused an overall average temperature increase of up to 53.7°C (±4.5 n?=?3). Temperature profile inside the simulated target lesion (BioGlue®). The temperature profile is shown for the simulated lesion during transthoracic high-intensity focused ultrasound insonation after in vivo flooding of the left lung of a pig. Temperature increases were monitored for each one-second treatment with HIFU irradiation. A section of 25 seconds of HIFU exposure following the ˜one second on/off™ scheme is demonstrated. The repeated HIFU insonation caused a temperature increase up to 54°C. Arterial blood pressure oxygen and carbon dioxide partial pressure were normal in all three animals during and after HIFU insonation. No animals died during the procedure. Discussion Currently the best curative treatment for early-stage non-small cell lung cancer and lung metastases is surgical resection. However surgery is not appropriate for patients with reduced pulmonary function due to the loss of lung parenchyma. Minimally invasive technologies for local tumour control have been developed as alternatives to surgery. The most common method is percutaneous radiofrequency ablation. However this kind of thermal ablation is limited because the extent to which tissue has been successfully ablated cannot be accurately assessed. This could be a reason for the observed rate of local recurrence. Another risk factor for local tumour progression is the adherence of viable tumour tissue to needle applicators and subsequent spreading of tumour cells [12]. In addition pulmonary percutaneous radiofrequency ablation is invasive and associated with major complications and mortality. HIFU has been examined in animal experiments and clinical trials as a technique for localized tissue ablation for more than 60 years. HIFU is superior to other radiation beams because it penetrates deep and selectively destroys tumour tissue. In addition HIFU can be applied as many times as needed [13]. HIFU is currently used as a therapy for cancers of the abdominal ans breast and brain [14-18]. Non-invasive local therapies with curative intention are not available for lung tumours. HIFU has never been applied to lung tumours because the air content in ventilated lungs reflects acoustic intensities. We solved this problem with lung flooding. Lung flooding enables efficient lung sonography and tumour imaging in ex vivo human and in vivo porcine lung cancer models [11]. The cancerous tissue was visualized by replacing the alveolar gas with fluid facilitating ultrasound-guided HIFU. The aim of the present study was to explore the ability of therapeutic ultrasound to penetrate the overlying lung parenchyma without damage and to increase the temperature of the tumour tissue. A tissue temperature greater than 60°C will usually cause instantaneous and irreversible cell death within one second in most extrapulmonary tissues due to coagulation necrosis. Coagulation necrosis is the primary mechanism by which HIFU destroys tumour cells [6]. Our ex vivo studies showed that lung cancer tissue absorbed acoustic energy leading to an increase in tumour temperature of 52.1 K. Based on the temperature of a cooled lung lobe (15°C) a peak temperature of 67°C was created. The heat induced by HIFU was sufficient to generate coagulation necrosis in the lung cancer tissue. In contrast the same acoustic HIFU energy caused a minimal temperature increase (7.1 K) in flooded parenchyma. NADPH-diaphorase staining showed that HIFU was highly selective for tumour tissue in flooded lungs. The lung parenchyma directly adjacent to the ablated cancer tissue was undamaged and viable. This can be explained by the very low attenuation of saline which is the major mass component in flooded lungs [19]. Thus the risk of damaging the surrounding tissue or adjacent ans can be minimised as previously described in a study of HIFU in abdominal tumours [20]. A normal saline solution was injected into the abdominal cavity to reduce complications from HIFU therapy for abdominal tumours. This method does not have adverse effects on the efficacy of HIFU ablation [21]. "

Lung_Cancer

"The overfitting characterizing AIC-selected models in scenarios of simple exposure“lag“response dependencies does not seriously affect its performance a result in line with previous findings 18. However AIC-selected models also suffer from bias and undercoverage of confidence intervals to some extent. Part of this seems to be related to the limited flexibility of the functions applied in the simulation study and may be described as a smoothing problem rather that an inherent limitation of the estimators. It should also be noted that the simulation study only evaluates a limited set of exposure“response and lag“response shapes simulated under the assumption of independency. Different functions such as cubic splines and more complex exposure“lag“response surfaces will be assessed in future simulation studies. Also an extension of DLNMs with penalized splines characterized by higher flexibility can be explored as well exploiting previous research on bivariate smoothing techniques 3031. A related problem is about the inferential procedures being conditional on a posteriori selection of the best-fitting model. Previous studies on unidimensional models have proposed a correction for the inflation of type I errors in tests on a constant effect along lags 1727. However this approach is not easily extended to the bidimensional setting of exposure“lag“response associations and the definition of a hypothesis testing procedure for DLNMs is left to future developments. Although a posteriori selection may also be a source of undercoverage of confidence intervals its impact seems to be limited if compared with that associated with lack of fit at least in the simple scenarios investigated in the simulation study. Another limitation is the lack of a formal testing procedure on the hypothesis of independency. As suggested in Section 3.4 a graphical assessment of the proportionality of exposure“response and lag“response curves such as those in can help investigating the issue. Further research is needed to provide more consistent inferential procedures in this setting. The analysis of the temporal evolution of the risk associated with protracted time-varying exposures has straightforward applications in different research fields. For example the DLNM methodology may be used to characterize the risk of chronic exposures to occupational or environmental factors to differentiate the role of exposures sustained at different ages in life course studies or to define the temporal frame of beneficial or adverse effects of drugs in clinical trials and pharmaco-epidemiology. The development of this methodology and software implementation provide a promising analytical tool for biomedical research. 6. Software and data All the analyses presented in this paper were performed using the R software version 3.0.1 32. The DLNM modeling framework is fully implemented in the package dlnm 25 by using the expressly extended version 2.0.0. The permutational algorithm for simulating time-to-event data in the presence of time-varying exposures is implemented in the package PermAlgo 29 version 1.0. Both packages are available through R from its central repository. The data of the Colorado Plateau uranium miners cohort in the form of a comma-separated values file is included in the supporting information¡ together with the R scripts for the analysis performed in the example and the simulation study of Sections 3“4 which are entirely reproducible. In particular the script example.R provides a short illustration of the modeling framework. Versions of the scripts updated to future versions of the dlnm package will be available at http://www.ag-myresearch.com. Distributed lag non-linear models were originally conceived and developed for describing temperature“health associations in time series data by Ben Armstrong. The data from the Colorado Plateau uranium miners cohort were collected by the researchers of National Institute for Occupational Safety and Health. I am grateful to Bryan Langholz for kindly making data and documentation available. The simulation study was performed using the high-processing computing system at the London School of Hygiene and Tropical Medicine. The final version of this has been substantially improved following the comments of an unknown reviewer. This research was supported by a Methodology Research fellowship by Medical Research Council-UK (grant ID G1002296). References 1 Goodman PG Dockery DW Clancy L Cause-specific mortality and the extended effects of particulate pollution and temperature exposure Environmental Health Perspectives 2004 112 2 179 185 14754572 2 Elliott P Shaddick G Wakefield JC de Hoogh C Briggs DJ Long-term associations of outdoor air pollution with mortality in Great Britain Thorax 2007 62 12 1088 1094 17666438 3 Collet JP Sharpe C Belzile E Boivin JF Hanley J Abenhaim L Colorectal cancer prevention by non-steroidal anti-inflammatory drugs: effects of dosage and timing British Journal of Cancer 1999 81 1 62 8 10487613 4 Abrahamowicz M Bartlett G Tamblyn R du Berger R Modeling cumulative dose and exposure duration provided insights regarding the associations between benzodiazepines and injuries Journal of Clinical Epidemiology 2006 59 4 393 403 16549262 5 Checkoway H Pearce N Hickey JL Dement JM Latency analysis in occupational epidemiology Archives of Environmental Health 1990 45 2 95 100 2334237 6 Thomas DC Models for exposure-time-response relationships with applications to cancer epidemiology Annual Review of Public Health 1988 9 451 482 7 Breslow NL Day NE Statistical Methods in Cancer Research 1987 II Lyon International Agency for Reasearch on Cancer (IARC) 232 271 The desing and analysis of cohort studies chap. 6: Modelling the relationship between risk dose and time 8 Thomas DC Brown CC Chu KC Goldsmith DF Saracci R Proceedings of a symposium on time-related factors in cancer epidemiology Journal of Chronic Diseases 1987 40 Suppl. 2 1S 211S 9 Thomas DC Statistical Methods in Environmental Epidemiology 2009 New York Oxford University Press 279 300 chap. 13: Mechanistic models 10 Thomas DC Statistical methods for analyzing effects of temporal patterns of exposure on cancer risks Scandinavian Journal of Work Environment & Health 1983 9 4 353 366 11 Vacek PM Assessing the effect of intensity when exposure varies over time Statistics in Medicine 1997 16 5 505 513 9089959 12 Langholz B Thomas D Xiang A Stram D Latency analysis in epidemiologic studies of occupational exposures: application to the Colorado Plateau uranium miners cohort American Journal of Industrial Medicine 1999 35 3 246 256 9987557 13 Richardson DB Latency models for analyses of protracted exposures Epidemiology 2009 20 3 395 399 19262389 14 Hauptmann M Wellmann J Lubin JH Rosenberg PS Kreienbrock L Analysis of exposure-time-response relationships using a spline weight function Biometrics 2000 56 4 1105 1108 11129467 15 Hauptmann M Berhane K Langholz B Lubin J Using splines to analyse latency in the Colorado Plateau uranium miners cohort Journal of Epidemiology and Biostatistics 2001 6 6 417 424 11831677 16 Hauptmann M Pohlabeln H Lubin JH Jockel KH Ahrens W Bruske-Hohlfeld I Wichmann HE The exposure-time-response relationship between occupational asbestos exposure and lung cancer in two German case-control studies American Journal of Industrial Medicine 2002 41 2 89 97 11813213 17 Sylvestre MP Abrahamowicz M Flexible modeling of the cumulative effects of time-dependent exposures on the hazard Statistics in Medicine 2009 28 27 3437 3453 19708037 18 Abrahamowicz M Beauchamp ME Sylvestre MP Comparison of alternative models for linking drug exposure with adverse effects Statistics in Medicine 2012 31 11-12 1014 1030 22095719 19 Abrahamowicz M MacKenzie TA Joint estimation of time-dependent and non-linear effects of continuous covariates on survival Statistics in Medicine 2007 26 2 392 408 16479552 20 Berhane K Hauptmann M Langholz B Using tensor product splines in modeling exposure-time-response relationships: Application to the Colorado Plateau Uranium Miners cohort Statistics in Medicine 2008 27 26 5484 5496 18613262 21 Almon S The distributed lag between capital appropriations and expenditures Econometrica 1965 33 178 196 22 Schwartz J The distributed lag between air pollution and daily deaths Epidemiology 2000 11 3 320 326 10784251 23 Armstrong B Models for the relationship between ambient temperature and daily mortality Epidemiology 2006 17 6 624 631 17028505 24 Gasparrini A Armstrong B Kenward MG Distributed lag non-linear models Statistics in Medicine 2010 29 21 2224 2234 20812303 25 Gasparrini A Distributed lag linear and non-linear models in R: the package dlnm Journal of Statistical Software 2011 43 8 1 20 22003319 26 Thomas DC Statistical Methods in Environmental Epidemiology 2009 New York Oxford University Press chap. 6: Modelling exposure-time-response relationships 27 Mahmud M Abrahamowicz M Leffondré K Chaubey Y Selecting the optimal transformation of a continuous covariate in Cox's regression: Implications for hypothesis testing Communications in Statistics: Simulation and Computation 2006 35 1 27 45 28 Breslow NL Day NE Statistical Methods in Cancer Research 1987 II Lyon International Agency for Reasearch on Cancer (IARC) 178 231 The desing and analysis of cohort studies chap. 5: Fitting models to continuous data 29 Sylvestre MP Abrahamowicz M Comparison of algorithms to generate event times conditional on time-dependent covariates Statistics in Medicine 2008 27 14 2618 2634 17918753 30 Wood SN Generalized Additive Models: an Introduction with R 2006 Chapman & Hall/CRC 31 Eilers PHC Currie ID Durban M Fast and compact smoothing on large multidimensional grids Computational Statistics and Data Analysis 2006 50 1 61 76 32 R Development Core Team R: A Language and Environment for Statistical Computing Lung_Cancer Background The development of a rash has been retrospectively associated with increased response and improved survival when treated with erlotinib at the standard dose of 150 mg per day. The objective of this trial was to evaluate the association of the activity of erlotinib in the first-line setting in patients with advanced non-small-cell lung cancer (NSCLC) with the development of a tolerable rash via dose escalation of erlotinib or tumor characteristics. Methods Patients with advanced NSCLC without prior systemic therapy were treated with erlotinib 150 mg orally per day. The dose was increased by 25 mg every two weeks until the development of grade 2/tolerable rash or other dose limiting toxicity. Tumor biopsy specimens were required for inclusion. Results The study enrolled 137 patients 135 were evaluable for safety and 124 were eligible and evaluable for response. Only 73 tumor samples were available for analysis. Erlotinib dose escalation occurred in 69/124 patients. Erlotinib was well tolerated with 70% of patients developing a grade 1/2 rash and 10% developing grade 3 rash. Response rate and disease control rate were 6.5% and 41.1% respectively. Median overall survival was 7.7 months. Toxicity and tumor markers were not associated with response. Grade 2 or greater skin rash and low pMAPK were associated with improved survival. Conclusions Overall survival was similar in this trial compared to first-line chemotherapy in this unselected patient population. Dose escalation to the development of grade 2 skin rash was associated with improved survival in this patient population. Bioinformatics Bioinformatics bioinformatics bioinfo Bioinformatics 1367-4803 1367-4811 Oxford University Press 25161229 4147902 10.1093/bioinformatics/btu449 btu449 Eccb 2014 Proceedings Papers Committee Original Papers Pathways and Molecular Networks Personalized identification of altered pathways in cancer using accumulated normal tissue data Ahn TaeJin 1 2 3 Lee Eunjin 1 2 Huh Nam 1 * Park Taesung 3 4 * 1Samsung Advanced Institute of Technology130 Suwon-si Gyeonggi-do 443-803 Korea 2Samsung Genome Institute Seoul 135-710 Korea 3Interdisciplinary Program in Bioinformatics and 4Department of Statistics Seoul National University Seoul South Korea *To whom correspondence should be addressed. 01 9 2014 22 8 2014 22 8 2014 30 17 i422 i429 © The Author 2014. Published by Oxford University Press. 2014 This is an Open Access distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial re-use distribution and reproduction in any medium provided the original work is properly cited. For commercial re-use please contact journals.permissions@oup.com Motivation: Identifying altered pathways in an individual is important for understanding disease mechanisms and for the future application of custom therapeutic decisions. Existing pathway analysis techniques are mainly focused on discovering altered pathways between normal and cancer groups and are not suitable for identifying the pathway aberrance that may occur in an individual sample. A simple way to identify individual™s pathway aberrance is to compare normal and tumor data from the same individual. However the matched normal data from the same individual are often unavailable in clinical situation. Therefore we suggest a new approach for the personalized identification of altered pathways making special use of accumulated normal data in cases when a patient™s matched normal data are unavailable. The philosophy behind our method is to quantify the aberrance of an individual sample's pathway by comparing it with accumulated normal samples. We propose and examine personalized extensions of pathway statistics overrepresentation analysis and functional class scoring to generate individualized pathway aberrance score. Results: Collected microarray data of normal tissue of lung and colon mucosa are served as reference to investigate a number of cancer individuals of lung adenocarcinoma (LUAD) and colon cancer respectively. Our method concurrently captures known facts of cancer survival pathways and identifies the pathway aberrances that represent cancer differentiation status and survival. It also provides more improved validation rate of survival-related pathways than when a single cancer sample is interpreted in the context of cancer-only cohort. In addition our method is useful in classifying unknown samples into cancer or normal groups. Particularly we identified ˜amino acid synthesis and interconversion™ pathway is a good indicator of LUAD (Area Under the Curve (AUC) 0.982 at independent validation). Clinical importance of the method is providing pathway interpretation of single cancer even though its matched normal data are unavailable. Availability and implementation: The method was implemented using the R software available at our Web site: http://bibs.snu.ac.kr/ipas. Contact: tspark@stat.snu.ac.kr or namhuh@samsung.com Supplementary information: Supplementary data are available at Bioinformatics online. 1 INTRODUCTIONCancer arises from normal cells and can evolve to become malignant metastatic and/or resistant to therapy. The analysis of altered pathways in an individual cancer patient may help to understand the disease status and suggest customized anticancer therapies.It is straightforward to compare the molecular profile of an individual™s tumor and normal cells to discover molecular aberrances specific to his/her cancer. However it may not be feasible in the current clinical practice environment to perform a metastatic tumor biopsy at the time of treatment resistance in patients with advanced cancer (Dancey et al. 2012). A case study of custom-tailored medicine based on an individual™s genome and transcriptome highlights this limitation (Jones et al. 2010). A patient™s tumor had metastasized to the lung after surgery at the primary site. A biopsy from his lung tumor was analyzed by mutation and transcription profiling; however the patient™s normal lung tissue was not biopsied. Because there was no matched normal tissue messenger RNA (mRNA) expression in the patient™s own blood and information collected from various normal tissues were used to identify differentially expressed genes (DEGs). The results of pathway analysis based on DEGs integrated copy number variation and mutation information led the doctor to change the patient™s drug treatment and the disease was stabilized for 3 months.Although the personalized interpretation of pathways can be demanding most current pathway analyses have been developed to investigate deregulated pathways between two phenotype groups. Khatri et al. (2012) classified these methods into three types: overrepresentation analysis (ORA) functional class scoring (FCS) and a pathway topology (PT)-based approach.ORA approaches typically apply an arbitrary threshold value (e.g. fold change >2 or P < 0.05) on gene expression to assess whether the number of genes beyond threshold are significantly over- or underrepresented in the given pathway. There are two drawbacks to ORA. First it uses only the most significant genes and discards others thus resulting in information loss for marginally significant genes (Breitling et al. 2004). Second it considers only the number of genes and does not consider the magnitude of expression changes leading to information loss regarding the importance of genes (e.g. a gene with a fold change of 2.01 and a gene with a fold change of 4 are considered equally). Unlike ORA FCS methods do not discard genes with an arbitrary threshold but use all available genes which is an improvement over ORA (Tian et al. 2005). PT methods are essentially based on FCS methods with the addition that they consider network topology information. They compensate for the common limitation of ORA and FCS in reporting false-positive gene sets due to sets of overlapping genes. In our we focus on ORA and FCS methods extending and implementing each for personalized pathway analysis.There are two exceptional studies examining individualized pathway analysis (Drier et al. 2013; Vaske et al. 2010). PARADIGM is a tool that infers a pathway status by using known functional structures. The method models the functional structure of pathway as a set of interconnected variables where the variables are omic objects such as DNA mRNA and protein where the interaction between variables describes the functional status of a pathway. PARADIGM may perform better with multiple omics as it uses known functional relationships between a gene or inter-gene DNA and protein. Hence it might not perform well with single layer omic data such as from mRNA microarrays.Drier et al. (2013) proposed a personal pathway deregulation score (PDS) which represents the distance of a single cancer sample from the median of normal samples on the principal curve. To calculate PDS they reduced the dimensions by principal component analysis and found the best principal curve using entire cohort samples containing both normal and/or different stages of cancers. Drier™s method performs better than PARADIGM in the mRNA only datasets of brain and colon cancers. Calculating PDS requires data dependent preprocessing steps including selecting the number of principal components to be used and filtering out noisy gene data to obtain optimized principal curves. PDS fully uses whole cohort data to interpret an individual™s pathway which can be a drawback in that it requires a number of cohort data to extract principal curve to interpret a single patient data. It has a limitation to interpret a single sample such as a patient™s recurrent tumor that is not accompanied with cohort dataset to extract the principal curve.Our proposed method is based on the comparison of one cancer sample with many accumulated normal samples (we use ˜nRef™ to refer to the accumulated normal samples) that is different from the previous studies in following sense. The proposed method is suitable to adopt single-layer omics data and expendable to interpret a patient in the context of many published or user-defined pathway gene sets. PARADIGM has less freedom in terms of data and gene sets as it prefers multi-layered omics data and requires predefined functional structure among omics objects. Unlike PDS which extracts the principal curve from entire cohort data our method does not assume an individual sample belongs to a cohort. We introduce using accumulated normal tissue data as a reference. This is a simple and biologically intuitive guideline in such a case to interpret a single sample that lack cohort data.Our method provides a series of analysis steps which consists of four parts: data processing gene-level statistics individualized pathway aberrance score (iPAS) and a significance test. To discover the most feasible method for iPAS we extend existing pathway analysis techniques namely ORA and FCS to properly reflect the nature of testing one cancer to many normal samples.To demonstrate that iPAS captures biologically and clinically relevant information in a sensible valid and useful manner we apply it to samples of lung and colon adenocarcinoma. We show that our representation generates clinically relevant stratifications and outcome predictors which would not have been achieved when the same data are analyzed by the conventional method that does not use accumulated normal data.Our empirical study suggests two different strategies depending on the biological question that iPAS is focused on. In the case of cancer diagnosis a method that uses the inter-gene correlation structure of the accumulated normal samples performs best. In the case of cancer prognosis a simple averaging of all member genes™ standardized gene expression values performs best.2 METHODS AND MATERIALS2.1 Gene expression dataWe built nRef by the manual curation of data obtained from NCBI GEO (Barrett et al. 2012). Microarray data of adjacent normal tissues obtained from patients undergoing surgery were selected to serve as the nRef. Data from biopsied samples primary cultures of normal tissues and post-mortem donors were not included in the nRef. We collected 120 nRef for lung 60 from GSE19804 (Lu et al. 2011) 27 from GSE7670 (Su et al. 2007) and 33 from GSE10072 (Landi et al. 2008). Samples came from individuals with variable smoking histories and different ethnic backgrounds. We collected 101 nRef™ for colon concentrating on normal mucosa tissue samples from six datasets available at GEO. To evaluate the effectiveness of our method in survival analysis we used Beer™s data of 442 lung adenocarcinomas (LUADs) (Beer et al. 2002) to discover survival-related pathways and validated the associations of 61 LUAD samples of GSE8894 (Lee et al. 2008). The pathway based identification of LUAD were tested on 120 cancers and 120 normal samples of GSE19804 GSE7670 and GSE10071. Further validation was conducted with 48 cancers and 35 normal samples collected from GSE19188 (Hou et al. 2010) and GSE31547. For patient stratification by colon cancer differentiation status we used 566 microarrays of GSE39582 (Marisa et al. 2013) which provided in a separate manner 443 for discovery 123 for validation. GSE17536 (Smith et al. 2010) was also used for validation.2.2 Pathway dataInformation from gene sets representing biological pathways were obtained from REACTOME (Croft et al. 2011) which are also provided in the Molecular Signature Database (Subramanian et al. 2005). Pathways with small number of genes are more easily understood by human experts. We decided to filter out pathways of which gene set size is >97. The cutoff covers at least 80% of contents of each public pathway resources. Of 674 pathways in REACTOME 583 pathways (86.7%) remained after filtering by the gene set size.2.3 Individualized analysis using the nRefThe aim of our approach is to identify altered pathways in an individual by making use of the nRef. A schematic diagram of our method of individualized pathway analysis is described in Figure 1 and the following sections describe each step. Fig. 1.Schematic description of individualized pathway analysis using accumulated normal data (nRef). An individual™s tumor data are normalized with the nRef. Gene expression is standardized by mean and SD of the nRef. The iPAS is calculated from standardized gene expression values in the pathway. Null distribution calculated from the nRef provides significance2.3.1 Data preprocessing and gene-level statisticsExpression level was defined by using the robust multichip average (Irizarry et al. 2003). For datasets using different microarrays only those with probes in common from Affymetrix U133A to Affymetrix U133Plus 2.0 were used for further analysis. For individual tumor cases we performed quantile normalization (Bolstad et al. 2003) after combining the single tumor microarray with all nRef samples. In cases of genes with multiple probes gene expression level was summarized by averaging probe-level expression. Individual tumor sample gene expression was standardized using the mean and standard deviation of the reference.2.3.2 "

Lung_Cancer

"To further functionally validate this point we show two promoters where the NME2 target site was not occupied by NME2 and had positioned nucleosomes in A549 cells and NME2-depleted A549 cells but were available for NME2 binding following NME2 induction (Supplementary Figure S3). In case of 53 genes we found positioned nucleosomes on or near NME2 target sites on NME2 depletion relative to control A549 cells. Together this suggests that in contrast to the nucleosomal changes following increase in NME2 expression NME2 target sites remain nucleosome-occupied in most cases on depletion of NME2. Binding site occupancy is transcriptionally active when associated with nucleosome repositioning We found that occupancy of about a fifth (870 of 3956 NME2 target sites ?22%) of the transcription target sites was concurrent with repositioning of nucleosomes in the NME2-induced condition. Interestingly these repositioning events resulted in altered expression of all the 791 genes (D). In contrast we found 1175 genes where the NME2 binding site (unique to the induced condition) was co-occupied with nucleosomes”only 130 (11%) of these genes showed altered expression. As a third possibility we found 1990 genes with NME2 occupancy in the induced condition though no nucleosomes were present in the vicinity of the NME2 site either before or after induction”i.e. target sites appeared to be independent of nucleosome repositioning. Again out of 1990 only 179 (8.9%) genes were differentially expressed. On mapping the NFR between the ?1 and +1 nucleosome positions in each of the three situations described above we found repositioning of the ?1 nucleosome by ?40 bp in the first case when repositioning was linked to binding site occupancy whereas in the other two situations the NFR was minimally altered on inducing NME2 (). DISCUSSION Our findings suggest that TF binding when closely associated with nucleosome repositioning results in altered gene expression changes. Interestingly in most cases when TF binding did not impact local nucleosome reanization it was not associated with altered transcriptional state of target gene. As we used human cancer cells that are metastatic and expression of the TF NME2 decreased their metastatic potential these findings also help in understanding how TF binding-induced nucleosome level changes influence the transcriptome during metastasis. TF binding and transcriptional activity are linked through local nucleosome repositioning It was recently reported that repositioning of the +1 nucleosome resulted in changes to NFR in genes that were differentially regulated during meiotic development in yeast (9). Though this was noted as a result of change in possibly multiple regulatory factors involved in meiotic development it is consistent with our results. Furthermore our findings indicate that assignment of transcriptional function to genome-wide target site binding would require information on nucleosome reanization to be more precise. This helps explain the noted discrepancy in high throughput DNA binding studies where low overlap between experimentally determined binding sites and gene expression has been observed (3435). A recent study noted chromatin accessibility before and after binding of the receptors (androgen (AR) or estrogen (ESR1)) were significantly altered (19) and suggested that both AR and ESR1 binding are associated with changes in local nucleosome occupancy. This is in line with our findings and suggests a model that integrates factor binding and transcriptional activity of genes with local nucleosomal changes. Non-specific binding of NME2 in the induced condition could be a confounding factor. To address this first we checked and found that in NME2-depleted cells a large number of genes were oppositely expressed with respect to their status in NME2-induced cells; it is unlikely that non-specifically activated/repressed genes as a result of NME2 induction would be differentially expressed on depleting NME2 (Supplementary Figure S4). Second the differentially expressed genes in NME2-induced cells correlate significantly with transcriptome changes that are clinically relevant (Supplementary Figure S5). Therefore though all NME2 binding events do not lead to increase/decrease in transcription it is unlikely to be due to spurious binding”it is possible that many of these associations are required for functions other than transcription. Overall chromatin landscape in promoters is largely constant site-specific changes are associated with transcription We found only 11.4% of nucleosomes to be repositioned in promoter proximal regions as a result of NME2 induction. Therefore it is interesting to consider that overall chromatin level changes may be relatively small. On the other hand and perhaps more interestingly there may be shift in nucleosome occupancy on TF binding leading to site-specific ˜open™ or ˜closed™ regions that facilitate regulatory events. Our findings (discussed above) further support this: nucleosomes repositioning along with engagement of TF at specific sites were in almost all cases associated with transcriptional change in the corresponding gene. In addition in both cases before and after NME2 induction enriched promoter nucleosome occupancy correlated with decreased expression of genes. Together these support a model where nucleosome occupancy generally determines the suppressed state of the transcriptome and reanization induced by DNA binding factors (themselves or when associated with chromatin modifiers) results in transcriptional activation at specific loci. Although further studies using other TFs will be required to substantiate this it appears to be consistent with an earlier study which observed decreased presence of nucleosomes in promoters of genes that were expressed during heat shock in yeast (16). However others have also noted either unchanged nucleosome occupancy (yeast grown in different carbon sources (3637)) or found nucleosome positioning to correlate with the state of transcription (active or silent) and not the extent of gene expression (18). Epigenetic signaling directs the location of TFs to cognate sites in given chromatin territories. Following this TFs are believed to be one of the key recruiters of chromatin modification and remodeling machineries (38“40). Recent evidence suggests that even the general TFs such as subunit of TFIID (Transcription Factor II D) complexes may be functional component of these machineries (41). In agreement with this basic understanding of transcription through chromatin our results demonstrate TF binding to be transcriptionally competent when coupled with locally altered nucleosome positioning. Furthermore our findings for the first time underline the importance of these aspects of chromatin biology in suppression of cancer spread mediated by NME2."

Lung_Cancer

"Low magnification image of the tumor (HE staining) revealed a subpleural tumor consisting predominantly of lepidic growth with a small (<5 mm) focus of invasion. (iv) Middle magnification image of the invasive area of the tumor (HE staining) revealed acinar-type growth pattern. (c) A 74-year-old female patient with lepidic-predominant invasive adenocarcinoma. (i) Lung window CT showed a pure GGN 19.7 mm in size. The mean CT attenuation was ?618 HU. (ii) Mediastinal window CT showed no tumor components except for vessels. (iii) Low magnification image (HE staining) revealed a tumor consisting mostly of lepidic growth with a smaller area (8 mm) of acinar invasion. (iv) Middle magnification image of the invasion area of the tumor (HE staining) revealed acinar gland proliferation in the fibrous stroma. (d) A 76-year-old male patient with papillary-predominant invasive adenocarcinoma. (i) Lung window CT image showed a pure GGN 10.7 mm in size. The mean CT attenuation was ?509 HU. (ii) Mediastinal window CT showed no tumor components. (iii) Low magnification image of the tumor (HE staining) revealed that the tumor predominantly consisted of papillary proliferation. (iv) Middle magnification image of the tumor (HE staining) revealed cuboidal tumor cells growing along fibrovascular cores in a papillary configuration. Comparison of Demographic and Clinicopathological Characteristics The patient characteristics and tumor properties for all patients and for patients in each group are shown in . When comparing the 3 groups the tumor size and mean CT attenuation significantly differed between the groups (p?=?0.0005 and p<0.0001 respectively). Tumor size was significantly larger in the MIA and I-ADC groups than in the AIS group (p?=?0.0100 and 0.0025 respectively). Mean CT attenuation was significantly higher in the I-ADC group than in the AIS and MIA groups (p?=?0.0001 and 0.0129 respectively). Pearson™s Correlation Analyses between the Pathological Invasion Diameter and the Tumor Size/CT Attenuation The correlation coefficients for the pathological invasion diameter with tumor size and CT attenuation were 0.3740 and 0.4072 (p?=?0.0001 and p<0.0001 respectively). ROC Curve Analyses of Tumor Size and CT Attenuation for Predicting Invasive Adenocarcinoma With regard to tumor size as a predictor of invasive adenocarcinoma the highest odds ratio (20.2) was obtained at a cutoff value of 11.0 mm and the sensitivity and specificity were 95.8% and 46.8% respectively with an area under the curve (AUC) of 0.75 (). With regard to CT attenuation the highest odds ratio (12.4) was obtained at a cutoff value of “680 HU and the sensitivity and specificity were 95.8% and 35.1% respectively with an AUC of 0.77 (). A combined variable was constructed using these 2 cutoff values: .0097867.g002 Receiver operating characteristic curve analysis for invasive adenocarcinoma prediction: tumor size and computed tomography attenuation. The sensitivity and specificity of tumor size for predicting invasive adenocarcinoma were 95.8% and 46.8% respectively at a cutoff value of 11.0 mm with an area under the curve (AUC) of 0.75 (green curve). The sensitivity and specificity of the mean computed tomography (CT) attenuation were 95.8% and 35.1% respectively at a cutoff value of “680 HU with an AUC of 0.77 (yellow curve). The sensitivity and specificity of the combined variable (tumor size and mean CT attenuation) were 91.7% and 71.4% respectively at cutoff values of 11 mm and ?680 HU with an AUC of 0.82 (blue curve). Group A: Neither tumor size nor mean CT attenuation indicated invasive adenocarcinoma (i.e. tumor size ?11 mm and mean CT attenuation ?“680 HU) Group B: Either tumor size or mean CT attenuation indicated invasive adenocarcinoma (i.e. tumor size >11 mm or mean CT attenuation >“680 HU) Group C: Both tumor size and mean CT attenuation indicated invasive adenocarcinoma (i.e. tumor size >11 mm and mean CT attenuation >“680 HU) The sensitivity and specificity of the combined variable for predicting invasive adenocarcinoma at the cutoff values were 100% and 10.4% for group B and 91.7% and 71.4% for group C respectively with an AUC of 0.82 (). Discussion In this present study we showed that approximately half of all resected pulmonary pure GGNs displayed a pathological invasive area. Moreover approximately a quarter of the resected pure GGNs were diagnosed as invasive adenocarcinomas and we found that tumor size and mean CT attenuation were useful in predicting pathological invasiveness. We have previously reported a strong negative association between CT attenuation and retained air space in tumors in a study of small peripheral lung adenocarcinomas detected by CT screening [7]. We moreover found that the retained air space was larger in non-invasive adenocarcinomas than in invasive adenocarcinomas and that this was largely due to an increased tumor tissue component and thickening of the alveolar septa in invasive adenocarcinomas resulting in reduced air space. These findings suggested that the high CT attenuation of pure GGNs reflects the large number of tumor cells that grow along alveolar septa and in terms of a stepwise progression of adenocarcinoma indicates that the lesion is progressing to invasive adenocarcinoma [4]. In general an invasive area is thought to be nonaerated and is theoretically thought to appear as a solid component on high-resolution CT. "

Lung_Cancer

"Among the remaining fifty unamplified cases eight (14%) also showed enhanced SETDB1 expression that could be associated with other upstream regulatory events. Overall our results indicate that the histone methyltransferase SETDB1 undergoes gene amplification in the natural history of lung tumorigenesis in non-small and small cell lung cancers. The copy-number gain for SETDB1 is associated with overexpression of the transcript and protein in lung cancer cell lines and primary tumors. From a functional standpoint SETDB1 exerts growth enhancing activity in vitro and in vivo as we have shown by depletion and transfection experiments in cell culture and in the nude mice model. Lung cancer cells carrying a SETDB1 gene amplification event are also more sensitive to the antiproliferative action mediated by the antitumoral antibiotic mithramycin a proposed inhibitor of SETDB1 activity. Thus our results suggest an oncogenic role for SETDB1 in lung carcinogenesis and raise the possibility of exploring new targeted therapies for the subset of lung tumor patients harboring the SETDB1 gene amplification event. This work was supported by the European Community's Seventh Framework Programme (FP7/2007-2013) under Grant agreement number HEALTH-F2-2010-258677”CURELUNG project the Institute of Health Carlos III (ISCIII)”PI10/02992 Ministerio de Educacin Ciencia e Innovacin Grant SAF2010-14935 Kutxa-Fundacin INBIOMED and the Health and Science Departments of the Catalan Government (Generalitat de Catalunya). ME is an ICREA Research Professor. Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc) The authors declare no conflict of interest. Jones PA Baylin SB The epigenomics of cancer Cell 2007 128 683 692 17320506 Berdasco M Esteller M Aberrant epigenetic landscape in cancer: how cellular identity goes awry Dev Cell 2010 19 698 711 21074720 F¼llgrabe J Kavanagh E Joseph B Histone onco-modifications Oncogene 2011 30 3391 3403 21516126 Fraga MF Ballestar E Villar-Garea A Boix-Chornet M Espada J Schotta G Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer Nat Genet 2005 37 391 400 15765097 Seligson DB Horvath S Shi T Yu H Tze S Grunstein M Global histone modification patterns predict risk of prostate cancer recurrence Nature 2005 435 1262 1266 15988529 Schneider R Bannister AJ Kouzarides T Unsafe SETs: histone lysine methyltransferases and cancer Trends Biochem Sci 2002 27 396 402 12151224 Esteller M Epigenetics provides a new generation of oncogenes and tumour-suppressor genes Br J Cancer 2006 94 179 183 16404435 Yoshimi A Kurokawa M Key roles of histone methyltransferase and demethylase in leukemogenesis J Cell Biochem 2011 112 415 424 21268062 Rodr­guez-Paredes M Esteller M Cancer epigenetics reaches mainstream oncology Nat Med 2011 17 330 339 21386836 Chen H Tu SW Hsieh JT Down-regulation of human DAB2IP gene expression mediated by polycomb Ezh2 complex and histone deacetylase in prostate cancer J Biol Chem 2005 280 22437 22444 15817459 Yu J Cao Q Mehra R Laxman B Yu J Tomlins SA Integrative genomics analysis reveals silencing of beta-adrenergic signaling by polycomb"

Lung_Cancer

"Division of Anatomic and Molecular Pathology Division of Laboratory and Genomic Medicine 660 Euclid Ave. #8118 St. Louis MO 63110. eduncavagepath.wustl.edu 1 7 2015 7 2014 16 4 405 417 6 3 2014 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. 2014 American Society for Investigative Pathology and the Association for Molecular Pathology This document may be redistributed and reused subject to certain conditions. The identification of recurrent gene rearrangements in the clinical laboratory is the cornerstone for risk stratification and treatment decisions in many malignant tumors. Studies have reported that targeted next-generation sequencing assays have the potential to identify such rearrangements; however their utility in the clinical laboratory is unknown. We examine the sensitivity and specificity of ALK and KMT2A (MLL) rearrangement detection by next-generation sequencing in the clinical laboratory. We analyzed a series of seven ALK rearranged cancers six KMT2A rearranged leukemias and 77 ALK/KMT2A rearrangement“negative cancers previously tested by fluorescence in situ hybridization (FISH). Rearrangement detection was tested using publicly available software tools including Breakdancer ClusterFAST CREST and Hydra. Using Breakdancer and ClusterFAST we detected ALK rearrangements in seven of seven FISH-positive cases and KMT2A rearrangements in six of six FISH-positive cases. Among the 77 ALK/KMT2A FISH-negative cases no false-positive identifications were made by Breakdancer or ClusterFAST. Further we identified one ALK rearranged case with a noncanonical intron 16 breakpoint which is likely to affect its response to targeted inhibitors. We report that clinically relevant chromosomal rearrangements can be detected from targeted gene panel“based next-generation sequencing with sensitivity and specificity equivalent to that of FISH while providing finer-scale information and increased efficiency for molecular oncology testing. Biomed Res Int Biomed Res Int BMRI BioMed Research International 2314-6133 2314-6141 Hindawi Publishing Corporation 24524077 3913339 10.1155/2014/485067 Research Investigating the Feasibility of Rapid MRI for Image-Guided Motion Management in Lung Cancer Radiotherapy http://orcid./0000-0002-3275-4160 Sawant Amit 1 * Keall Paul 2 Pauly Kim Butts 3 Alley Marcus 3 Vasanawala Shreyas 3 Loo Jr. Billy W. 3 Hinkle Jacob 4 Joshi Sarang 4 1University of Texas Southwestern Medical Center Dallas TX 75235 USA 2University of Sydney Sydney NSW 2006 Australia 3Stanford University Stanford CA 95305 USA 4University of Utah Salt Lake City UT 84112 USA *Amit Sawant: amit.sawantutsouthwestern.edu Academic Editor: Jack Yang 2014 12 1 2014 2014 485067 17 4 2013 6 11 2013 7 11 2013 Copyright 2014 Amit Sawant et al. 2014 This is an open access distributed under the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited. Cycle-to-cycle variations in respiratory motion can cause significant geometric and dosimetric errors in the administration of lung cancer radiation therapy. A common limitation of the current strategies for motion management is that they assume a constant reproducible respiratory cycle. In this work we investigate the feasibility of using rapid MRI for providing long-term imaging of the thorax in order to better capture cycle-to-cycle variations. Two nonsmall-cell lung cancer patients were imaged (free-breathing no extrinsic contrast and 1.5?T scanner). A balanced steady-state-free-precession (b-SSFP) sequence was used to acquire cine-2D and cine-3D (4D) images. In the case of Patient 1 (right midlobe lesion ~40?mm diameter) tumor motion was well correlated with diaphragmatic motion. In the case of Patient 2 (left upper-lobe lesion ~60?mm diameter) tumor motion was poorly correlated with diaphragmatic motion. Furthermore the motion of the tumor centroid was poorly correlated with the motion of individual points on the tumor boundary indicating significant rotation and/or deformation. These studies indicate that image quality and acquisition speed of cine-2D MRI were adequate for motion monitoring. However significant improvements are required to achieve comparable speeds for truly 4D MRI. Despite several challenges rapid MRI offers a feasible and attractive tool for noninvasive long-term motion monitoring. 1. Introduction Respiratory motion causes significant uncertainties in tumor delineation radiotherapy (RT) dose calculations and delivery particularly in the case of thoracic tumors (e.g. lung liver) [1]. The management of respiratory motion has been an active area of research over the last decade. Several investigational as well as clinically implemented respiratory motion management strategies have been described in the literature [1]. However a common limitation of most of these strategies is that they rely on image-guidance techniques that make simplifying assumptions about respiratory motion and do not adequately capture cycle-to-cycle variations which invariably occur in all patients. Modern motion-managed radiotherapy typically uses four-dimensional computed tomography (4DCT) as the tool of choice for pretreatment anatomic imaging (also termed as œCT simulation or œCT-sim in the literature). In this technique Lung_Cancer CT projections are acquired over several respiratory cycles from successive œslabs in the body. At the same time an external surrogate (e.g. an optical marker) records the amplitude of respiration. Based on the surrogate motion trace the reconstructed slices are sorted into 6“10 volumes over a single respiratory average cycle where each volume represents a specific phase of respiration (inhalation through exhalation) [2“4]. This retrospectively reconstructed œmovie of a single respiratory cycle serves as the anatomical ground truth for all subsequent stages of radiotherapy (contouring treatment planning and dose delivery). It is well recognized however that respiratory motion is far more complex than can be characterized by a single average cycle. Cycle-to-cycle variations such as baseline shifts and changes in the amplitude and/or frequency of the respiratory waveform are inadequately accounted for in 4DCT-based planning and can lead to significant geometric and therefore dosimetric errors [5]. Furthermore binning CT projection data acquired over several cycles into a single cycle leads to severe image artifacts. For example Yamamoto et al. found that 45 of 50 patients had at least one artifact with mean magnitude of 11.6?mm (range: 4.4“56.0?mm) [6]. In a separate study Persson et al. found that 4DCT artifacts caused significant uncertainties in the delineation of the gross tumor volume (GTV) in 16 out of 19 patients [7]. Finally the equivalent dose for 4DCT is quite high (29“40?mSv) about 4 times higher than that for 3DCT (3“10?mSv) [8]. Such high imaging dose discourages long-term monitoring and frequent imaging. Due to these limitations 4DCT-based image guidance provides an incomplete picture of respiration-induced spatial and temporal changes in the thoracic anatomy. The aim of this work is to investigate the feasibility of using rapid magnetic resonance imaging (MRI) as a nonionizing imaging modality to capture long-term and/or frequent information about respiratory motion and its effects on the movement and deformation of lung tumors and surrounding critical ans. The fundamental difference and therefore advantage of cine MRI are that unlike 4DCT the MR image (i.e. slice or volume) is acquired prospectively thereby capturing an actual instance of the patient anatomy which is closer to reality compared to an average estimate of the anatomical state that is represented by 4DCT. Prospective acquisition also enables MRI to overcome the two main challenges that limit the utility of 4DCT images namely the ability to capture cycle-to-cycle variations and elimination of binning-related image artifacts. In addition due to the fact that MRI does not involve ionizing radiation there is no dose penalty for repeated imaging (as opposed to 4DCT). The use of rapid cine-2D as well as 4D MRI for radiotherapy guidance has been previously reported in the literature. In cine-2D MRI a slice of the anatomy is selected at arbitrary orientation and imaged repeatedly in time. 4D MRI is conceptually similar except that in this case an entire volume is selected and imaged. Plathow et al. have reported cine-2D imaging of lung cancer patients at ~3 frames per second (fps) [9] and 4D imaging of malignant pleural mesothelioma patients at ~1 volume/s [10] under slow-breathing conditions using a 1.5?T scanner. Von Siebenthal et al. have reported on a 4D MR imaging technique using retrospective stacking of cine-2D slices [11]. Biederer et al. report 4D MRI of a ventilated chest phantom that uses porcine lung with embedded agarose nodules to simulate tumors [12]. More recently Cai et al. have reported a 4D MRI study of a moving phantom using a technique that uses retrospective sorting of cine-2D slices [13]. To our knowledge there has been no systematic study of rapid lung MRI in the context of image-guided radiotherapy (IGRT) motion management under realistic (prospective acquisition free-breathing human subjects) conditions. In this work we present a pilot investigation of prospective rapid cine-2D and cine-3D (commonly termed as œ4D in radiotherapy and the MRI literature) MRI of two nonsmall-cell lung cancer (NSCLC) patients under free-breathing conditions without externally administered contrast. Subsequently we compute and analyze the motion trajectories of tumors and structures of interest. Our current goal is to demonstrate the feasibility and the utility of rapid MR imaging to monitor respiratory motion over multiple cycles and obtain guidance information about the motion deformation and the interplay between lung tumors and surrounding critical ans. Our long-term goal (beyond the current scope) is to use the information obtained from rapid MRI to augment and potentially correct 4DCT images. 2. Methods 2.1. Imaging of NSCLC Patients Two NSCLC patients were imaged following informed consent. Patient number 1 was a 67-year old female with an ~40?mm diameter right midlobe tumor. Patient number 2 was an 80-year old male with an ~60?mm diameter left upper-lobe tumor. Both patients were scanned on a 1.5?T scanner (GE Signa). Both patients were scanned in the supine position under free-breathing conditions and without externally administered contrast. For each patient a 4-channel cardiac coil was centered around the tumor. cine-2D time series in the coronal and sagittal planes were acquired using a balanced steady-state free precession (b-SSFP) sequence and the images were reconstructed using the vendor's in-built software. In all cases except one (Patient number 1 coronal series) half-Fourier acquisition was used in order to achieve higher imaging speed. In the case of Patient number 2 an additional 3D+t (4D) scan of a tumor-inclusive coronal slab (8 slices each 5?mm thick) was acquired using the b-SSFP sequence in the 3D mode and in conjunction with parallel imaging (acceleration = 4). The 4D images were reconstructed using the autocalibrating reconstruction for Cartesian imaging (ARC) algorithm [14]. Table 1 summarizes the image acquisition parameters for the cine-2D and the 4D acquisitions. 2.2. Motion Analysis For each time series from Table 1 the motion trajectories of the tumor and structures of interest were determined as follows. A fluid-flow-based deformable image registration previously validated for RT applications [15“17] was applied to each time series to compute deformation vector fields (DVFs) across the temporal dimension. In order to reduce errors and achieve high computation speed (i.e. fewer iterations) the registration was performed in two stages-rigid registration which accounted for gross translation and affine transformations of the tumor and ans followed by deformable registration which accounted mainly for tumor and an deformation. For each time series a reference image was selected (typically at mid-inhale) and ~15 points each on the tumor boundary and the diaphragm were manually selected. Subsequently the motion trajectory of each pixel on a contour was determined from the DVFs. The validity of using diaphragmatic motion as a surrogate for tumor motion was examined by calculating the correlation between the average motion trajectory of the pixels comprising the diaphragm boundary with the average trajectory of the pixels comprising the tumor boundary. The presence of complex motion such as tumor rotation and/or deformation was tested by comparing the motion trajectory of the tumor centroid with those of the selected points on the tumor boundary. 3. Results and Discussion Figure 1 shows MR images acquired from Patient number 1 (Figures 1(a) and 1(b)) and Patient number 2 (Figures 1(c) and 1(d)). The acquisition times per image ranged from ~0.15 to 0.27?s”speeds adequate for monitoring most respiratory motion. In each case the tumor mass (indicated by an arrow) can be clearly delineated against the background of lung parenchyma."

Lung_Cancer

"We have previously reported that the MOR is upregulated in lung tissue from patients with NSCLC [12] and that overexpression of MOR promotes tumor growth and metastasis in human NSCLC xenograft models [13]. Further data from Fujioka et al. indicate that the MOR regulates EGF-induced signaling events in NSCLC [35]. Considering there is very limited data on opioid regulation of epithelial mesenchymal transition (EMT a crucial process for cancer progression) and the molecular mechanisms integrating cancer cell proliferation migration and EMT this study examined the role of opioids and MOR in these processes [17] [18] [19] [20]. Here we present evidence that activation of growth factor receptors promotes complex formation with the mu opioid receptor (MOR) and Grb-2 as well as Src activation. These events induce recruitment of the scaffolding protein Gab1 to the plasma membrane and consequent recruitment/activation of PI3 kinase Akt and STAT3 signaling which are required for human lung cancer proliferation migration and EMT. Inhibition of MOR (siRNA shRNA and/or MOR antagonists) blocks morphine and DAMGO binding to the mu opioid receptor. In addition inhibiting MOR attenuates growth factor-induced phosphorylation/activation of Src Gab-1 PI3 kinase Akt and STAT3 with consequent inhibition of human lung cancer proliferation migration and EMT (see ). .0091577.g009 Schematic diagram illustrating mu opioid receptor (MOR) regulation of growth factor receptor signaling and human lung cancer proliferation migration and epithelial mesenchymal transition (EMT). Activation of growth factor receptors promotes complex formation with the mu opioid receptor (MOR) and Grb-2 as well as Src activation (1). These events induce recruitment of the scaffolding protein Gab1 to the plasma membrane (2) and consequent recruitment/activation of PI3 kinase Akt and STAT3 signaling (3) which are required for human lung cancer proliferation migration and epithelial mesenchymal transition (EMT) (4). Inhibition of MOR (siRNA shRNA and/or the peripheral MOR antagonist methylnaltrexone (MNTX)) blocks morphine and DAMGO binding to the mu opioid receptor (5). In addition inhibiting MOR attenuates growth factor-induced phosphorylation/activation of Src Gab-1 PI3 kinase Akt and STAT3 with consequent inhibition of human lung cancer proliferation migration and EMT (6). Our results are consistent with Fujioka et al. who demonstrated that morphine and EGF challenge of human lung cancer cells (H2009 NSCLC) induced Akt and MAPK/ERK activation as well as cell proliferation and invasion [35]. These effects were blocked with the MOR antagonist naloxone [35]. Further these authors and others have reported that agonists of MOR can transactivate the EGRF [35] [46] [47] [48]. While the current study did not look at opioid stimulation of EGFR phosphorylation our results indicate that blocking MOR with the peripheral MOR antagonist MNTX did not affect EGF-induced EGFR tyrosine phosphorylation. Our results further demonstrate that MNTX can block EMT transformation by another growth factor implicated in lung cancer progression insulin growth factor (IGF) [49] [50] [51]. Whether MOR can regulate IGF receptor phosphorylation/signaling in human NSCLC is currently being investigated in our laboratory. The current study presents the novel findings that the scaffolding protein Grb2-associated-binding protein 1 (Gab-1) regulates opioid and growth factor-mediated human lung cancer cell proliferation migration and EMT transformation. The adaptor protein Growth factor receptor-bound protein 2 (Grb-2) can directly bind tyrosine phosphorylated sites on the EGRF via its SH2 domain which promotes plasma membrane recruitment of Gab-1 and binding of proline-rich motifs on Gab-1 to SH3 domains on Grb-2 [37]. Further the proline-rich motifs on Gab-1 can bind to the non-receptor tyrosine kinase Src [37] [52]. We have previously demonstrated that MNTX is a potent inhibitor of Src activation [43] [53]. Our results that MNTX can also inhibit Grb-2/Gab-1 recruitment/activation could explain how MNTX regulates Src activity. Tyrosine phosphorylation of Gab-1 by Src and other tyrosine kinases promotes recruitment of several signaling molecules including PI3K PLCgamma RasGAP and SHP2 [37] [52] [54]. Our current results indicate that MOR antagonism by MNTX blocks activation (tyrosine phosphorylation) of the PI3K regulatory subunits p85 and p55. Further inhibition of PI3K activity attenuates opioid- and growth factor-induced human lung cancer cell proliferation migration and EMT. Whether PLCgamma RasGAP and/or SHP2 also play a role in MOR regulation of NSCLC progression is currently being investigated in our laboratory. The transcription factor Signal transducer and activator of transcription 3 (STAT3) is an important regulator of NSCLC progression [41] [55] [56] [57]. Tyrosine phosphorylation of STAT3 induces dimerization and nuclear translocation [57]. Recently it has been shown that PI3K can also regulate STAT3 activity [38] [39]. In this study we have demonstrated that STAT3 regulates opioid and growth factor-induced proliferation migration and EMT. Further MNTX inhibits STAT3 tyrosine phosphorylation implying a role for MOR in STAT3 activation. This is in partial agreement with Debruyer et al.2010 indicating that delta opioid receptor (DOR) agonists can differentially affect migration of HCT-8/E11 human colon cancer cells [58]. Another downstream effector of PI3K is the serine/threonine kinase Akt [42] [59]. Akt contains an SH2 domain that can bind to PIP2 and PIP3 as well as serine threonine and tyrosine phosphorylation sites [60] [61]. We have previously reported that MNTX inhibits opioid and VEGF-induced Akt activation in human endothelial cells and that overexpression of MOR in human NSCLC activates Akt [4] [13] [62] [63]. In this study using Akt inhibitor X (a selective inhibitor of Akt phosphorylation and activity) we demonstrate that Akt regulates opioid and EGF-induced human NSCLC cell proliferation migration and EMT transformation. EMT is a complex process in which there is a loss of cell-cell adhesion (characterized by a reduction of epithelial cell adhesion proteins including the tight junction proteins ZO-1 and claudin-1) followed by acquisition of mesenchymal characteristics including a loss of baso-apical polarization cytoskeletal remodeling and increased cell motility (characterized by increases in specific cytoskeletal proteins (i.e. vimentin) and transcription factors (i.e. Slug and Snail)[16] [17] [18] [19] [20] [22]. We have previously reported that overexpression of MOR1 (the most abundant MOR transcript that consists of exons 12 3 and 4)[64] in human H358 NSCLC cells induced elongated cellular projections suggestive of a migratory œmesenchymal phenotype [19]. This prompted us to examine MOR regulation of EMT in human NSCLC cells. While we used a panel of EMT markers several others exist including Twist N-cadherin E-cadherin ZEB1/2 beta-catenin fibronectin and desmoplakin [16] [17] [18] [19] [20] [22]. In addition to cancer EMT has been implicated in several fibrotic diseases by providing a source for myofibroblasts [22] [65]. Whether MOR is involved in the regulation of fibrotic diseases via EMT is currently unknown. Exogenous opioids are utilized clinically for their analgesic effects cough and diarrhea suppression and pain relief [66] [67]. Opioid relief of pain is indicated in various conditions such as acute pain after surgery injury or trauma as well as chronic pain from advanced cancer. Recently Garcia-Recio et al.2013 demonstrated that the pain-associated tachykinin Substance P and its GPCRs can transactivate HER2/EGFR signaling in human breast cancer cell lines [68]. This study provides a novel mechanism by which pain and inflammation can promote tumor progression. Substance P receptors can heterodimerize with mu opioid receptors and these receptors can transactivate each other [69] [70] [71] [72] [73] [74] [75]. Therefore it is possible that pain and inflammation could through Substance P transactivation of MOR promote EMT in cancer cells. This potential link could be ameliorated with MOR antagonists. Further research is warranted. Given our previous published data indicating the mu opioid receptor (MOR) is increased in human lung cancer [12] that lung cancer cells do not form visible tumors in MOR knockout mice [12] and that MOR overexpression promotes NSCLC primary tumor growth and metastasis [4] [43] we hypothesized that MOR regulation of EMT might be a plausible explanation for the differences in recurrence rates observed in the epidemiologic studies."

Lung_Cancer

" Bryant et al. Overall survivalAverage Za0.370.00011Beer (N = 442) Overall survivalAverage Zb0.620.00003Fisher0.500.00068GSEA0.650.00001Euclidean0.650.00001Mahalanobis0.670.00001GSE8894 (N = 61) Recurrent free survivalAverage Zb0.900.01163Fisher0.910.01076GSEA0.780.02899Euclidean0.870.01544Mahalanobis0.680.05485aDerived from mean and SD of all cancer samples in the dataset bDerived by mean and SD of the nRef.Prognostic gene expression signatures for Stages II and III colon cancers have been reported in seven papers yielding 207 genes in total (Bandres et al. 2007; Barrier et al.2006 2007; Eschrich et al. 2005; Kopetz and Abbruzzese 2009; Lin et al. 2007; Wang et al. 2004). The genes are enriched in 32 REACTOME pathways (False Discovery Rate (FDR) < 0.05 pathway size < 96). We assumed the 32 pathways were valid as ground truth to be identified and analyzed in the colon cancer dataset GSE39585 (Stages II and III were only considered). Average Z provided best performer (sensitivity = 0.88) with 28 pathways deemed as significant. GSEA Fisher Euclidean and Mahalanobis gave the following values0.780.66 0.06 and 0.03 respectively.These results satisfied us that our approach captures the fundamental knowledge of cancer thus it is reasonably considered as iPAS.To investigate which of the candidates for iPAS most robustly reflect phenotype association we evaluated the proposed methods by determining whether survival-associated pathways are validated in datasets never used for discovery using LUAD and colon cancer [LUAD: Beer™s set n = 442 for discovery GSE8894 (n = 61) GSE3141 (n = 58) for validation; colon cancer: GSE39582d (n = 443) for discovery GSE39582v (n = 123) and GSE17536 (n = 109) for validation logrank P < 0.05 comparing tumors in the top 50th percentile of aberrance scores to those in the bottom 50th percentile]. Validation rates varied depending on the dataset and these were possibly affected by the small sample size compared with that of the discovery set. In these cases we were not able to determine a superior method that outperformed the others. Average Z gave the highest validation rate in three of four dataset with validation rates of GSE8894 (43.6% 92/211) GSE3141 (13.3% 28/211) and GSE17536 (10.7% 24/224). When validation rates from four datasets are averaged Average Z gave the highest validation rate (21.9% Fig. 2 blue bars). Pathways validated as significantly associated with patient survival for each cancer are listed in the Supplementary Materials (Supplementary Tables S1 and S2). Fig. 2.Averaged validation rate of discovered survival-related pathway at four datasets. Proposed approach using nRef (blue) versus conventional approach that standardizes individual sample by mean and SD of entire cohort dataset (red)We also investigated the validation rate of iPAS candidates under the conditions where the same data are not standardized by the nRef but instead standardized by the mean and SD of the cohort dataset which consists of only cancers (Fig. 2 red bars). It is noteworthy that use of the nRef increased the validation rate for every iPAS candidate investigated. This implies that the strategy of using accumulated normal samples as a reference is beneficial in terms of pathway-based survival analysis.3.2 Identification of clinical importanceCluster analysis of using Average Z as the iPAS method on Beer™s data identified 12 pathway clusters (denoted by 1?12 in Fig. 3) and 3 sample clusters (S2?S4; S1 is from the nRef; Fig. 3). Sample clusters S2 and S4 represent well the differentiation status of LUAD (Fisher exact test P < 4.65 — 10?15). Well-differentiated adenocarcinoma resembles the normal glandular structure; therefore it is a reasonable result that cluster S2 is close to the nRef. The survival outcome of S2 and S4 are significantly different (P < 0.0028) and this assures us that unbiased clustering-based iPAS has enough sensitivity to capture clinically important associations. This finding is concordant with prior knowledge that well-differentiated LUAD patients are likely to have better prognosis (Barletta et al. 2010). Pathway cluster P9 is distinguished as commonly upregulated in tumor samples. The pathways are transfer RNA aminoacylation amino acid or purine synthesis DNA elongation and the extension of telomeres. Fig. 3.Clustered iPAS of LUAD dataset. Pathways (n = 583) and samples (n = 442) are clustered according to iPAS. Normal samples are clustered at left (S1). Tumors (S2?S4) deviate from normal in both up- and downregulated directions (darker red and blue respectively). Sample clusters are well-representing histopathological differentiation status (S2: for well-differentiated LUAD P < 4.65 — 10?15) and overall survivalUnbiased pathway-based clustering of colon cancer data also captures clinically important associations by revealing sample clusters that are survival related (S2 and S3 P = 0.0037 Supplementary Fig. S1). It is important to note that iPAS is not only sensitive enough to identify clinically meaningful substructure of patients but also reveals common characteristics of a cancer at the same time. For example pathways commonly up- or downregulated in all cancer samples for example P9 or P2 would have not been discovered if the analysis had been performed by a conventional approach that does not make use of ˜nRef™ (Supplementary Fig. S2).3.3 Pathway-based identification of cancerCancer develops unique mechanisms for malignancy. Therefore it is reasonable to believe that identifying the unique molecular aberrances of cancer will aid in cancer diagnosis. Our empirical study of iPAS-based clustering of LUAD revealed several pathways commonly up- or downregulated in all of the cancer samples. Further analysis was performed to determine whether iPAS could be successfully used in the accurate identification of cancer. We tested this in a simple unsupervised way by judging whether an unknown sample is significantly different against the nRef as a tumor if not as normal. We performed a 5-fold cross-validation one hundred times with the LUAD dataset which consisted of 120 cancers and 120 normal samples. Microarray data from the normal samples was randomly divided into five groups and four of the five served as the reference group. The remaining group was used as the true normal set for the test of pathway-based identification of cancer. To build true cancer set for the test the same number of cancer sample was randomly picked. We considered 583 pathways in REACTOME giving 293 500 (583 pathways — 5-fold — 100 repeats) AUCs and accuracy values. We averaged AUCs and accuracies from the five candidate methods for iPAS and used this as a representative AUC and accuracy of a given pathway.By ranking the pathways by AUC top pathways that marked averagely high performance by all iPAS candidates are listed (Supplementary Table S3). The ˜amino acid synthesis and interconversion and transamination™ pathway showed the highest classification performance. Unsurprisingly this pathway was one of the commonly upregulated pathways in the analysis of the Beer™s data (Fig. 3 pathway cluster P9). Among the tested iPAS candidates for this pathway Mahalanobis yielded the highest AUC (0.980) while Average Z gave 0.936 and Fisher™s exact test gave the lowest value (0.914). The standardized gene expression pattern for this pathway differed between tumor and normal. Many of the genes deviated from mean of the nRef by more than two orders of sigma contributing to its best performance out of all iPAS candidate methods including ORA method like Fisher™s exact test (Fig. 4a). Fig. 4.(a) Expression pattern of genes in the pathway. Each line represents sample. (gray: normal red: tumor). Dashed line represents expression value deviated 1.96? from the mean expression value of normal tissues. (b). Performance of classification of cancer by ˜amino acid synthesis and interconversion and transamination™. AUC of 0.980 has marked in discovery set (95% confidence interval provided as error bar) independent validation set results AUC of 0.982 (Validation 1: normal samples in validation set served as reference) and 0.982 (Validation 2: normal samples in discovery set served as reference)We also analyzed the influence of using the subset of normal samples as nRef. We compared the pathway-based cancer identification results using the full set of normal samples (n = 120) against 100 different runs using 75% (n = 90) 50% (n = 60) of randomly chosen normal samples. Among the pathways that marked averagely high performance in the identification of cancer the best and the second best pathways are considered ˜amino acid synthesis and interconversion and transamination™ and ˜unwind of DNA™ respectively. The result shows little loss of performance even though only a half of normal samples were used for the test (Fig. 5a and b). Fig. 5.Performance of pathway-based identification of cancer (AUC)"

Lung_Cancer

" MTBHsp70 at both flanks. Histopathology Abdominal walls and intestines from mice were fixed for at least 24 h in PBS-buffered 10% formalin. Tissues were routinely embedded in paraffin. 5 ?m thick sections were stained routinely with H&E. For staining tumor-infiltrating T cells mice were perfused with 4% paraformaldehyde (PFA) in PBS and tumor nodules were fixed in 4% PFA/PBS for additional 2 hours washed and infiltrated with 30% sucrose/PBS at 4°C. 6 ?m thick frozen sections were stained with rat anti-mouse CD8 (BD Biosciences 1:100 dilution) or rat anti-mouse Foxp3 (eBioscience 1:12 dilution) followed by polyclonal rabbit anti-rat immunoglobulin/HRP (Dako 1:750 dilution). Signal was developed with diaminobenzidine (DAB Dako). Images were acquired on a Zeiss Axio A1 microscope. All histopathological and immunohistochemical samples were reviewed and the quantitation of the cellular infiltrate was performed in a blinded manner to the observer. Statistical analysis Statistical differences between three or more experimental groups were analyzed using One-Way ANOVA followed by Turkey™s multiple comparison tests when mean of each group is compared with that of every other group or followed by Dunnett™s multiple comparison tests when mean of each group is compared with that of a control group. Statistical differences between two experimental groups were analyzed using Student™s t-test. Survival was analyzed with the Log-rank test. Prism 6.0 software (GraphPad Software) was used for all the statistical analysis. Abbreviations DC: Dendritic cell; scFv: Single-chain antibody variable fragment; MSLN: Mesothelin; MTB: Mycobacterium tuberculosis; Hsp: Heat shock protein; i.p.: Intraperitoneal; i.d.: Intradermal; BMDCs: Bone marrow-derived dendritic cells; APCs: Antigen-presenting cells; PBMCs: Peripheral blood mononuclear cells; PBLs: Peripheral blood leukocytes; LPS: Lipopolysaccharide; H&E: Haematoxylin and eosin; PFA: Paraformaldehyde; DAB: Diaminobenzidine; mAb: monoclonal antibody. Competing interests The authors declare that they have no competing interests. Authors™ contributions JY played a role in the design of the experiments acquisition analysis and interpretation of the data and writing the manuscript. PR JN YY NHA MN GJ-M XT SK HC PU BF TC and PL participated in the performance of experiments. SK and TB were involved in design of the experiments. RB was involved in data analysis. ER was involved in setting up murine ovarian cancer model. SO provided the murine ovarian cancer model. NS provided the plasmid that encodes an scFv fragment specific to MSLN and the recombinant P4 scFv protein. GD NS and SO gave constructive input on experimental design and data analysis. JG played a role in conception and design of the fusion protein. MP and JG were involved in the conceptualization and design of the study analysis and interpretation of datasets and in writing the manuscript. All authors read and approved the final manuscript. Supplementary Material Additional file 1: Figure S1 scFvMTBHsp70 binds to 40L mesothelioma cells. 40L cells were stained with scFvMTBHsp70 or MTBHsp70 followed by mouse anti-MTBHsp70 and Donkey anti-mouse Alexa Fluor 594. Cells were observed using a Nikon Eclipse TiE fluorescence microscope. A Representative pictures from three independent experiments. Scale bar 10 ?m. B Images were analyzed using the NIS-Elements AR Microscope Imaging Software. Mean Fluorescence Intensity was analyzed using ImageJ. P values were determined using One-Way ANOVA followed by Turkey™s multiple comparison tests. ****p?<?0.0001. Click here for file Additional file 2: Figure S2 scFvMTBHsp70 or MTBHsp70 plus P4 scFv treatment does not lead to infiltration of inflammatory cells into abdominal or intestinal mesothelial tissues. Samples of abdominal wall and intestine were prepared from C57BL/6 mice that had previously received multiple i.p. injections of scFvMTBHsp70 MTBHsp70 plus P4 scFv or saline as described in the Methods section. Sections of these tissues were stained with H&E and images were acquired on a Zeiss Axio A1 microscope. Representative images from 3 animals per treatment group are shown. No detectable level of mononuclear cell or granulocyte infiltrate within mesothelial tissues was seen in any sampled tissues. Scale bar 20 ?m. Click here for file Additional file 3: Figure S3 scFvMTBHsp70 treatment does not affect numbers of tumor-infiltrating CD8+ or Foxp3+ T cells. (A) Representative images of intratumoral CD8+ and Foxp3+ T cells from saline (n?=?3) scFvMTBHsp70 (n?=?3) or MTBHsp70 plus P4 scFv (n?=?3) -treated mice. Mouse spleen sections were used as positive controls: CD8+ and Foxp3+ T cells are clearly evident in the sections. Scale bar 20 ?m. (B) Numbers of CD8+ and Foxp3+ cells were quantified from 3“5 randomized fields. Click here for file Additional file 4: Figure S4 Validation of in vivo depletion of CD8+ cells in FVB/NJ mice. Mice were injected i.p. with 200 ?g of anti-CD8 mAb or an isotype-matched irrelevant rat IgG2a as described in Methods. All the mice were bled from the tail vein and the depletion of CD8+ cells was examined by flow cytometry analysis of peripheral blood cells stained with fluorophore-conjugated anti-CD8 on days 7 and 28 after tumor inoculation. (A) Representative results of flow analyses on 10 mice per group and reported as the percentage of CD8+ cells in lymphocytes. (B) CD8+ cells in the mice treated with isotype IgG2a or anti-CD8 mAb were compared. ***p< 0.001. Click here for file Acknowledgments This manuscript is dedicated to the memory of Janet Gelfand a victim of ovarian cancer. The authors gratefully acknowledge the continuing support for this work from the Edmund C. Lynch Jr. Cancer Fund Arthur Luxenberg Esq. Perry Weitz Esq. and the VIC Mesothelioma Research and Resource Program at MGH and the Friends of VIC Fund. PU and NHA were supported by the Prof. Dulcie V. Coleman Studentship at Imperial College London. We thank Oliver Mitchell John Cao Lujia Zhou Rumbidzai Mushavi and Sayinthen Vivekanantham for their technical assistances Dr. Yuhui Huang for his useful comments Michael Waring Dr. Michael Santuosuosso and Dr. Ravi Mylvaganam for their technical advice Dr. Musie Ghebremichael for his advice in statistical analysis and Mahnoor Valibhoy for her assistance with the schematic figure. 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10.1111/j.1349-7006.2012.02233.x 22320267 Li H Yu Y Sun L Wang H Zhang P Wei H Wang L Wan M Cao Z Wang Y Chen Y Dong B Wang L Vaccination with B16 tumor cell lysate plus recombinant Mycobacterium tuberculosis Hsp70 induces antimelanoma effect in mice Cancer biotherapy & radiopharmaceuticals 2010 25 185 191 10.1089/cbr.2009.0716 20423232 Cheng WF Hung CF Chai CY Hsu KF He L Rice CM Ling M Wu TC Enhancement of Sindbis virus self-replicating RNA vaccine potency by linkage of Mycobacterium tuberculosis heat shock protein 70 gene to an antigen gene J Immunol 2001 166 6218 6226 11342644 Uto T Tsujimura K Uchijima M Seto S Nagata T Suda T Chida K Nakamura H Koide Y A novel vaccine strategy to induce mycobacterial antigen-specific Th1 responses by utilizing the C-terminal domain of heat shock protein 70 FEMS immunology and medical microbiology 2011 61 189 196 10.1111/j.1574-695X.2010.00762.x 21204994 Rasoli M Omar AR Aini I Jalilian B Syed Hassan SH Mohamed M Fusion of HSP70 gene of 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microenvironment: official journal of the International Cancer Microenvironment Society 2010 4 39 50 21505561 Singh R Paterson Y Vaccination strategy determines the emergence and dominance of CD8+ T-cell epitopes in a FVB/N rat HER-2/neu mouse model of breast cancer Cancer research 2006 66 7748 7757 10.1158/0008-5472.CAN-05-4469 16885378 Huang Y Yuan J Righi E Kamoun WS Ancukiewicz M Nezivar J Santosuosso M Martin JD Martin MR Vianello F Leblanc P Munn LL Huang P Duda DG Fukumura D Jain RK Poznansky MC Vascular normalizing doses of antiangiogenic treatment reprogram the immunosuppressive tumor microenvironment and enhance immunotherapy Proc Natl Acad Sci USA 2012 109 17561 17566 10.1073/pnas.1215397109 23045683 Fields RC Shimizu K Mule JJ Murine dendritic cells pulsed with whole tumor lysates mediate potent antitumor immune responses in vitro and in vivo Proc Natl Acad Sci USA 1998 95 9482 9487 10.1073/pnas.95.16.9482 9689106 Millar DG Garza KM Odermatt B Elford AR Ono N Li Z Ohashi PS 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Lung_Cancer

"cells treated with thiaminase are consistent with inhibition of BCKDH complex pyruvate dehydrogenase complex and transketolase inhibition. However inhibition of the same enzyme was shown to result in different metabolic consequences in the case of BCKDH where inhibition in RS4 cells caused a demonstrable increase in BCKDH substrates whereas in MCF-7 cells inhibition led to a decrease in the products of the enzymatic reaction. This difference may then lead to different consequences on cell growth as the substrates for BCKDH are known to be toxic metabolites and result in the an toxicity in the metabolic syndrome maple syrup urine disease. We have previously shown that RS4 cells as well as other lymphoid leukemia cell lines have a biphasic dose response to thiaminase where there is an initial growth inhibition related to extracellular thiamine depletion and a cytotoxic response at higher concentrations related to intracellular thiamine depletion [7]. Gabapentin appears to be protective against the growth inhibitory effects of thiaminase in RS4 cells at the concentrations that produce extracellular thiamine depletion indicating that growth inhibition is not due solely to thiamine depletion but also to the accumulation of branched-chain amino acid catabolites. These studies demonstrate that the cell lines and presumable tumors have different metabolic consequences resulting from inhibition of the same enzymes and that these consequences may form the basis of sensitivity to TDE inhibition as well as identify potential biomarkers of response. In addition thiaminase treatment was associated with increased lipolysis and decreased polyamine synthesis (data not shown) both of which were reversed by co-treatment with rapamycin. These changes are consistent with altered energy production and reduced cellular proliferation as a result of thiamine depletion but also may be related to our previous observation that mammary tumor formation is delayed in mice fed a low-thiamine normal-fat diet but not in mice fed a low-thiamine high-fat diet [4]. Although fatty acid metabolism may be affected by inhibition of 2-hydroxyl-CoA lyase (HACL1) a peroxisomal enzyme that also requires thiamine pyrophosphate as a cofactor and mediates the catabolism of branched chain fatty acids and 2-hydroxyl straight chain fatty acids [17] we did not see any evidence for accumulation of these metabolites in either cell line. Conclusions Overall these studies demonstrate that TDE disruption leads to specific metabolic consequences that are predictable from its role as a cofactor for enzymes that catalyze important steps in energy production biomass generation and amino acid catabolism. Comparison of the changes observed with thiaminase treatment in RS4 cells versus MCF-7 cells reveals different biochemical signatures as a result of thiamine depletion that may reflect differences in the relative contributions of different pathways to energy production and anaplerotic contributions to the TCA cycle. The near-global reversal of the specific metabolic effects of thiaminase by rapamycin indicate a closer regulation of thiamine-dependent metabolism by mTOR than has been previously appreciated and further studies are needed to determine how mTOR signaling pathways regulates thiamine-dependent metabolism. The disruption of thiamine metabolism results in cytotoxicity in breast cancer and leukemia tumor model systems and has the potential to provide novel targets for therapies directed toward these malignancies. Supporting Information Figure S1 Results of metabolomic analysis for branched-chain amino acid metabolites in A.) RS4 cell line and B.) MCF7 cell cline analyzed under six conditions: control for 24 hours (C-24); incubation in thiaminase for 24 hours (T-24); control for 48 hours (C-48); thiaminase for 48 hours (T-48); rapamycin for 48 hours (R-48); and both rapamycin and thiaminase for 48 hours (R+T-48). The median is indicated by the bar in the center of the rectangle the rectangle dimensions reflect the range of the two mid-quartile values and the outer bars represent the ranges of all of the values. The data represent four independent experiments. For C-48 vs T-48 and C-48 vs T+R-48 comparisons ** indicates p<0.05 and * indicates 0.05<p<0.1). (TIF) Click here for additional data file. Table S1 Metabolomic pathway heat map data for RS4 and MCF-7 cells treated with thiaminase (T) rapamycin (R) or both (R+T) for 24 and 48 hours. (XLSX) Click here for additional data file. The authors thank the Research Communication Office of the Markey Cancer Center at the University of Kentucky for editorial and graphics assistance. This work was supported by DanceBlue an effort of the University of Kentucky student community to support pediatric oncology care and research. References 1 ZastreJA SweetRL HanberryBS YeS (2013) Linking vitamin B1 with cancer cell metabolism. Cancer & Metabolism1: 1“1424280107 2 LiuS HuangH LuX GolinskiM ComesseS et al (2003) Down-regulation of thiamine transporter THTR2 gene expression in breast cancer and its association with resistance to apoptosis. Mol Cancer Res1: 665“67312861052 3 LiuS StrombergA TaiHH MoscowJA (2004) Thiamine transporter gene expression and exogenous thiamine modulate the expression of genes involved in drug and prostaglandin metabolism in breast cancer cells. Mol Cancer Res2: 477“48715328374 4 Daily A Liu S Bhatnagar S Karabakhtsian RG Moscow JA (2013) Low thiamine diet increases mammary tumor latency in FVB/N-Tg(MMTVneu) mice. Int J Vitamin Nutrition Res In press. 5 CostelloCA KelleherNL AbeM McLaffertyFW BegleyTP (1996) Mechanistic studies on thiaminase I. 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Blood116: 5983“599020889920 10 EvansAM DeHavenCD BarrettT MitchellM MilgramE (2009) Integrated nontargeted ultrahigh performance liquid chromatography/electrospray ionization tandem mass spectrometry platform for the identification and relative quantification of the small-molecule complement of biological systems. Anal Chem81: 6656“666719624122 11 OhtaT MasutomiN TsutsuiN SakairiT MitchellM et al (2009) Untargeted metabolomic profiling as an evaluative tool of fenofibrate-induced toxicology in Fischer 344 male rats. Toxicol Pathol37: 521“53519458390 12 DehavenCD EvansAM DaiH LawtonKA (2010) anization of GC/MS and LC/MS metabolomics data into chemical libraries. J Cheminform2: 920955607 13 Le HuerouY GunawardanaI ThomasAA BoydSA de MeeseJ et al (2008) Prodrug thiamine analogs as inhibitors of the enzyme transketolase. 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Clinical cancer research : an official journal of the American Association for Cancer Research 1078-0432 24536073 3962777 10.1158/1078-0432.CCR-13-0583 NIHMS557917 Article NEW STRATEGIES FOR TRIPLE NEGATIVE BREAST CANCER “ DECIPHERING THE HETEROGENEITY Mayer Ingrid A. 1 3 Abramson Vandana G. 1 3 Lehmann Brian D. 2 3 Pietenpol Jennifer A. 2 3 1Department of Medicine Vanderbilt University School of Medicine Nashville TN 2Department of Cancer Biology Vanderbilt University School of Medicine Nashville TN 3Breast Cancer Research Program Vanderbilt-Ingram Cancer Center Vanderbilt University School of Medicine Nashville TN Corresponding author: Ingrid A. Mayer Div. Hematology/Oncology VUMC 2220 Pierce Avenue 777 PRB Nashville TN 37232-6307 Tel: 615 936 3524 Fax: 615 343-7602 ingrid.mayer@vanderbilt.edu 11 3 2014 15 2 2014 15 2 2015 20 4 782 790 Triple-negative breast cancer (TNBC) is a heterogeneous disease; gene expression (GE) analyses recently identified six distinct TNBC subtypes each displaying a unique biology. Exploring novel approaches to treatment of these subtypes is critical since less than 30% of women with metastatic breast cancer survive five years and virtually all women with metastatic TNBC will ultimately die of their disease despite systemic therapy. To date not a single targeted therapy has been approved for the treatment of TNBC and cytotoxic chemotherapy remains the standard treatment. We will discuss the current and upcoming therapeutic strategies being explored in an attempt to œtarget TNBC. J Womens Health (Larchmt) J Womens Health (Larchmt) jwh Journal of Women's Health 1540-9996 1931-843X Mary Ann Liebert Inc. 140 Huguenot Street 3rd FloorNew Rochelle NY 10801USA 24372085 4011422 10.1089/jwh.2013.4516 10.1089/jwh.2013.4516 Original Articles Perceived Versus Objective Breast Cancer Breast Cancer Risk in Diverse Women Fehniger Julia MD 1 2 Livaudais-Toman Jennifer PhD 1 Karliner Leah MD 1 3 Kerlikowske Karla MD 34 5 Tice Jeffrey A. MD 1 3 Quinn Jessica MS 1 Ozanne Elissa PhD 6 Kaplan Celia P. DrPH MA 1 3 1Department of Medicine Division of General Internal Medicine University of California San Francisco California. 2Department of Obstetrics and Gynecology University of California San Francisco California. 3Helen Diller Family Comprehensive Cancer Center University of California San Francisco California. 4General Internal Medicine Section Department of Veterans Affairs University of California San Francisco California. 5Departments of Medicine and Epidemiology and Biostatistics University of California San Francisco California. 6Institute for Health Policy Studies and Department of Surgery University of California San Francisco California. Address correspondence to: Celia P. Kaplan DrPH MA Helen Diller Family Comprehensive Cancer Center University of California San Francisco 3333 California Street Box 0856 San Francisco CA 94143 E-mail: celia.kaplan@ucsf.edu 01 5 2014 23 5 420 427 Copyright 2014 Mary Ann Liebert Inc. 2014 Abstract Background: Prior research suggests that women do not accurately estimate their risk for breast cancer. Estimating and informing women of their risk is essential for tailoring appropriate screening and risk reduction strategies. Methods: Data were collected for BreastCARE a randomized controlled trial designed to evaluate a PC-tablet based intervention providing multiethnic women and their primary care physicians with tailored information about breast cancer risk. We included women ages 40“74 visiting general internal medicine primary care clinics at one academic practice and one safety net practice who spoke English Spanish or Cantonese and had no personal history of breast cancer. We collected baseline information regarding risk perception and concern. Women were categorized as high risk (vs. average risk) if their family history met criteria for referral to genetic counseling or if they were in the top 5% of risk for their age based on the Gail or Breast Cancer Surveillance Consortium Model (BCSC) breast cancer risk model. Results: Of 1261 participants 25% (N=314) were classified as high risk. More average risk than high risk women had correct risk perception (72% vs. 18%); 25% of both average and high risk women reported being very concerned about breast cancer. Average risk women with correct risk perception were less likely to be concerned about breast cancer (odds ratio [OR]=0.3; 95% confidence interval [CI]=0.2“0.4) while high risk women with correct risk perception were more likely to be concerned about breast cancer (OR=5.1; 95%CI=2.7“9.6). Conclusions: Many women did not accurately perceive their risk for breast cancer. Women with accurate risk perception had an appropriate level of concern about breast cancer. Improved methods of assessing and informing women of their breast cancer risk could motivate high risk women to apply appropriate prevention strategies and allay unnecessary concern among average risk women. Clin Exp Immunol Clin. Exp. Immunol cei Clinical and Experimental Immunology 0009-9104 1365-2249 Blackwell Science Inc Oxford UK 24654775 4226594 10.1111/cei.12332 Original Articles Spontaneous antibodies against Engrailed-2 (EN2) protein in patients with prostate cancer Annels N E * Simpson G R * Denyer M * McGrath S E * Falgari G * Killick E   Eeles R   Stebbing J ¡ Pchejetski D ¡ Cutress R § Murray N § Michael A * Pandha H * * Oncology Faculty of Health and Medical Sciences University of Surrey Guildford Surrey UK   Institute of Cancer Research Sutton Surrey UK ¡ Charing Cross Hospital London Hampshire UK § University Hospital Southampton Southampton Hampshire UK H. Pandha Oncology Faculty of Health and Medical Sciences Leggett Building University of Surrey Guildford Surrey GU2 7WG UK. E-mail: h.pandha@surrey.ac.uk 8 2014 09 7 2014 177 2 428 438 16 3 2014 © 2014 British Society for Immunology 2014 We reported the expression of the homeodomain-containing transcription factor Engrailed-2 (EN2) in prostate cancer and showed that the presence of EN2 protein in the urine was highly predictive of prostate cancer. This study aimed to determine whether patients with prostate cancer have EN2 autoantibodies what the prevalence of these antibodies is and whether they are associated with disease stage. The spontaneous immunoglobulin (Ig)G immune response against EN2 and for comparison the tumour antigen New York Esophageal Squamous Cell Carcinoma 1 (NY-ESO-1) were tested by enzyme-linked immunosorbent assay (ELISA) in three different cohorts of prostate cancer patients as well as a group of men genetically predisposed to prostate cancer. Thirty-two of 353 (9·1%) of the SUN cohort representing all stages of prostate cancer demonstrated EN2 IgG responses 12 of 107 patients (11·2%) in the advanced prostate cancer patients showed responses while only four of 121 patients (3·3%) with castrate-resistant prostate cancer showed EN2 autoantibodies. No significant responses were found in the predisposed group. Anti-EN2 IgG responses were significantly higher in patients with prostate cancer compared to healthy control males and similarly prevalent to anti-NY-ESO-1 responses. While EN2 autoantibodies are not a useful diagnostic or monitoring tool EN2 immunogenicity provides the rationale to pursue studies using EN2 as an immunotherapeutic target. autoantibodies Engrailed-2 prostate cancer BMC Cancer BMC Cancer BMC Cancer 1471-2407 BioMed Central 24383403 3880410 1471-2407-14-1 10.1186/1471-2407-14-1 Research Article Prolyl-4-hydroxylase ? subunit 2 promotes breast cancer progression and metastasis by regulating collagen deposition Xiong Gaofeng 1 gaofeng.xiong@uky.edu Deng Lei 1 dnaxy@hotmail.com Zhu Jieqing 1 jzh254@g.uky.edu Rychahou Piotr G 1 2 piotr.rychahou@uky.edu Xu Ren 1 3 ren.xu2010@uky.edu 1Markey Cancer Center University of Kentucky Lexington KY 40536 USA 2Department of Surgery University of Kentucky Lexington KY 40536 USA 3Department of Molecular and Biomedical Pharmacology University of Kentucky 741 S. Limestone BBSRB Lexington KY 40536 USA 2014 2 1 2014 14 1 1 27 9 2013 26 12 2013 Copyright © 2014 Xiong et al.; licensee BioMed Central Ltd. 2014 Xiong et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons./licenses/by/2.0) which permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons./publicdomain/zero/1.0/) applies to the data made available in this article unless otherwise stated. Background Increased collagen deposition provides physical and biochemical signals to support tumor growth and invasion during breast cancer development. Therefore inhibition of collagen synthesis and deposition has been considered a strategy to suppress breast cancer progression. Collagen prolyl-4-hydroxylase ? subunit 2 (P4HA2) an enzyme hydroxylating proline residues in -X-Pro-Gly- sequences is a potential therapeutic target for the disorders associated with increased collagen deposition. However expression and function of P4HA2 in breast cancer progression are not well investigated. Methods Gene co-expression analysis was performed in the published microarray datasets to identify potential regulators of collagen I III and IV in human breast cancer tissue. Expression of P4HA2 was silenced by shRNAs and its activity was inhibited by 1 4-DPCA a prolyl-4-hydroxylase inhibitor. Three-dimensional culture assay was used to analyze roles of P4HA2 in regulating malignant phenotypes of breast cancer cells. Reduced deposition of collagen I and IV was detected by Western blotting and immunofluorescence. Control and P4HA2-silenced breast cancer cells were injected into fat pad and tail vein of SCID mice to examine effect of P4HA2 on tumor growth and lung metastasis. Results Using gene co-expression analysis we showed that P4HA2 was associated with expression of Col1A1 Col3A1 and Col4A1 during breast cancer development and progression. P4HA2 mRNA levels were significantly upregulated in breast cancer compared to normal mammary tissue. Increased mRNA levels of P4HA2 correlated with poor clinical outcome in breast cancer patients which is independent of estrogen receptor status. Silencing P4HA2 expression or treatment with the P4HA inhibitor significantly inhibited cell proliferation and suppressed aggressive phenotypes of breast cancer cells in 3D culture accompanied by reduced deposition of collagen I and IV. We also found that knockdown of P4HA2 inhibited mammary tumor growth and metastasis to lungs in xenograft models. Conclusion These results suggest the critical role of P4HA2 in breast cancer progression and identify P4HA2 as a potential therapeutic target and biomarker for breast cancer progression. Tumor microenvironment Breast cancer Collagen deposition Cancer progression Cell proliferation Background Extracellular matrix (ECM) is an important component of tumor microenvironment and plays critical roles in cancer development [1-3]. Collagens are the major structural ECM proteins and form fibers or networks in tumor tissue [4-6]. Cell-collagen interaction controls a variety of cellular activities including proliferation migration and invasion through integrin and discoidin domain receptor [7-9]. Enhanced expression and deposition of collagens are associated with tumor development and progression [10-12]. Recent studies demonstrate that increased collagen deposition and crosslinking enhance the stiffness and density of mammary tissue [51013] which is an important risk factor for breast cancer development. Type I collagen has been identified as a prognosis marker and is associated with cancer recurrence in human breast cancer patients [14]. Collagen VI knockout mice have reduced primary tumor formation and growth [12] while enhancing collagen deposition and inhibiting collagen degradation significantly enhances tumor initiation and tumor growth [510]. In addition cancer cell invasion usually occurs at tumor-stromal interfaces with oriented collagen fibers and aligned collagen fibers can facilitate cell migration and metastasis [5101115]. These results indicate that increased collagen expression and deposition promotes breast cancer development and progression by enhancing tumor growth and invasion. Therefore inhibiting collagen synthesis or deposition is a promising strategy to suppress breast cancer progression. Collagen biosynthesis is a multistep process that involves several post-transcription modification enzymes and one of the most important members of these enzymes is collagen prolyl-4-hydroxylase [16]. It catalyzes the formation of 4-hydroxyproline by hydroxylating proline residues in -X-Pro-Gly- sequences [17-20]. Collagen prolyl-4-hydroxylase resides within the lumen of the endoplasmic reticulum (ER) [18] and its expression is used as a marker for collagen synthesis [2122]. Because 4-hydroxyproline residues formed in this reaction are essential for triple helix formation and stabilization of collagen [22-24] inhibiting the prolyl-4-hydroxylases activity efficiently blocks collagen synthesis and deposition. All known vertebrate collagen prolyl-4-hydroxylases are ?2?2 tetramers consisting of two ? subunits and two ? subunits. Each ? subunit contains the peptide substrate binding domain and the two catalytic sites of the enzyme and the ? subunits have been identified as protein disulfide isomerases [171925]. Three types of collagen prolyl-4-hydroxylases ? isoforms (P4HA1 P4HA2 and P4HA3) have been identified in human tissue. P4HA1 is expressed in most cell types; P4HA2 is mainly expressed in chondrocytes osteoblasts and capillary endothelial cells; P4HA3 expression is detected in adult and fetal tissues at very low levels compared to P4HA1 and P4HA2 [1826]. Increased P4HA2 expression has been detected in many solid tumors including oral cavity squamous cell carcinoma papillary thyroid cancer and breast cancer [27-30] however the function of P4HA2 in cancer progression largely remains to be determined. Here we showed that expression of P4HA2 and collagen genes (Col1A1 Col3A1 and Col4A1) is significantly correlated during breast cancer development and progression and that increased mRNA levels of P4HA2 are associated with poor prognosis in breast cancer patients. Silencing P4HA2 or treatment with the P4HA inhibitor attenuates cell proliferation and suppresses aggressive 3D phenotypes tumor growth and cancer metastasis"

Lung_Cancer

" Materials and Methods We examined the anti-tumor effect of Ad-REIC on 25 NSCLC cell lines in vitro and A549 cells in vivo. Two of these cell lines were artificially established as EGFR-tyrosine kinase inhibitor (TKI) resistant sublines. Results Ad-REIC-treatment inhibited the cell viability by 40% or more in 13 (52%) of the 25 cell lines at multiplicity of infection (MOI) of 20 (20 MOI). These cell lines were regarded as being highly sensitive cells. The cell viability of a non-malignant immortalized cell line OUMS-24 was not inhibited at 200 MOI of Ad-REIC. The effects of Ad-REIC on EGFR-TKI resistant sublines were equivalent to those in the parental cell lines. Here we demonstrated that Ad-REIC treatment activated c-Jun N-terminal kinase (JNK) in NSCLC cell lines indicating the induction of ER stress with GRP78/BiP (GRP78) up-regulation and resulting in apoptosis. A single intratumoral injection of Ad-REIC significantly inhibited the tumorigenic growth of A549 cells in vivo. As predictive factors of sensitivity for Ad-REIC treatment in NSCLC we examined the expression status of GRP78 and coxsackievirus and adenovirus receptor (CAR). We found that the combination of the GRP78 and CAR expressional statuses may be used as a predictive factor for Ad-REIC sensitivity in NSCLC cells. Conclusion Ad-REIC induced JNK activation and subsequent apoptosis in NSCLC cells. Our study indicated that Ad-REIC has therapeutic potential against NSCLC and that the expression statuses of GRP78 and CAR may predict a potential therapeutic benefit of Ad-REIC. No current funding sources for this study. Introduction Lung cancer is the most common cause of death from cancer worldwide and the metastatic form is a major factor leading to mortality [1]. There are two major histological subtypes of lung cancer: non-small cell lung cancer (NSCLC) and small cell lung cancer. Recent intensive studies have identified causative molecular alterations that have directly led to the development of new therapeutic strategies and have improved patient prognosis [2]. For example mutations of the epidermal growth factor receptor gene (EGFR) are found in approximately 30% of NSCLCs especially in lung adenocarcinomas and EGFR-tyrosine kinase inhibitors (TKIs) are particularly effective in these tumors [3] [4]. More recently crizotinib has been shown to be effective for NSCLCs with an EML4-ALK fusion gene [5] [6]. However the number of patients with these alterations is limited and little improvement in prognosis has been obtained in NSCLCs without these drug-sensitive alterations. Furthermore acquired resistance eventually occurs in the majority of EGFR-mutant tumors which had previously responded to EGFR-TKI after an average of 10 months of treatment [7]. Thus a new therapeutic modality is needed to improve the clinical outcome of patients with lung cancer. REIC/Dkk-3 a member of the Dickkopf (Dkk) gene family is originally found in immortalized cells and has been reported to be a tumor suppressor; its expression is significantly down-regulated in a broad range of cancer cell types including lung cancer [8]. The heatmap image of messenger RNA (mRNA) expression of REIC/Dkk-3 gene from the UCSC Cancer Genome Browse which is freely available public database (://genome-cancer.ucsc.edu/) (we downloaded the data on July 16 2013) showed that REIC/Dkk-3 gene expression was reduced in majority of examined samples of both lung adenocarcinomas and squamous cell carcinomas compared with normal lung tissues (Figure S1). In addition it could be confirmed from a public database that expression of REIC/Dkk-3 was also low in many NSCLC cell lines (Gene Expression Omnibus repository [http://www.ncbi.nlm.nih.gov/geo GEO accession GSE4824]). REIC/Dkk-3 is known to interfere with Wnt signaling via Wnt receptors [9] [10] and was previously reported to play a distinct role in the induction of apoptosis and the inhibition of metastasis [11] [12]. The induction of apoptosis in cancer cells is mainly caused by endoplasmic reticulum (ER) stress induced by the overproduction of REIC/Dkk-3 in the cells. ER stress triggers the activation of c-Jun N-terminal kinase (JNK) which is a critical event in apoptosis induced by the overproduction of REIC/Dkk-3 using an adenovirus vector (Ad-REIC) [11] [13]. In our previous studies we found that Ad-REIC had a therapeutic effect on various types of human cancer including the prostate testis pleura and breast carcinomas [11] [13]“[15]. Ad-REIC infection and REIC/Dkk-3 protein are also known to up-regulate the anti-tumor immunosystem [16]. Based on preclinical data a clinical trial using Ad-REIC for human prostate cancer has been ongoing in Japan and the USA (NCT01197209). In this study we investigated the therapeutic effect of Ad-REIC on NSCLC cells in vitro and in vivo. We also examined factors related to the sensitivity of cell lines to Ad-REIC as a step toward the development of customized Ad-REIC therapy for patients with NSCLC. Materials and Methods Ethics Statement This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Animal Care and Use Committee of Okayama University (Permit Number: OKU-2012-549). All surgery was performed under ketamine and xylazine anesthesia and all efforts were made to minimize suffering. Cell lines Sixteen cell lines of human lung adenocarcinoma 3 squamous cell carcinoma 3 large cell carcinoma 1 adenosquamous cell carcinoma 2 EGFR-TKI-resistant sublines from HCC827 and PC-9 cells (HCC827-GR-high2 and RPC-9) the human mesothelioma cell line MSTO-211H (211H) and the normal human fibroblast cell line OUMS-24 were used in this study (). The details of cell lines used in this study are described in Method S1. The HCC827-GR-high2 and RPC-9 cell lines were established as described previously [17] [18]. OUMS-24 was established at our institution [19]. .0087900.t001 Characteristics and the inhibition rate of cell viability on NSCLC cell lines. Cell lines Histological subtypes Genetic alterations Inhibition rate (%) GRP78/Actin ratio (Low/High) CAR/Actin ratio (Low/High) Category 20 MOI 100 MOI 200 MOI H2009 AD KRAS mut 60 - - 0.13 (Low) 0.73 (High) A H2228 AD EML4-ALK fusion 60 - - 0.27 (High) 0.70 (High) B HCC827 AD EGFR mut 56 - - 0.25 (High) 0.66 (High) B HCC827-GR-high2 AD EGFR mut 55 - - 0.12 (Low) 2.12 (High) A H2087 AD BRAF mut 55 - - 0.14 (Low) 1.22 (High) A HCC4006 AD EGFR mut 54 - - 0.15 (Low) 0.60 (High) A HCC4011 AD EGFR mut 54 - - 0.22 (Low) 0.13 (Low) B H522 AD W/t 50 - - 0.27 (High) 1.55 (High) B H157 SQ KRAS mut 50 - - 0.13 (Low) 0.98 (High) A A549 AD KRAS mut 49 - - 0.07 (Low) 0.55 (High) A H838 AD W/t 47 - - 0.10 (Low) 1.33 (High) A H1299 LC NRAS mut 47 - - 0.08 (Low) 0.68 (High) A H661 LC W/t 40 - - 0.42 (High) 1.89 (High) B H1819 AD HER2 amp 23 46 63 0.46 (High) 1.55 (High) B H1993 AD W/t 22 32 40 0.69 (High) 0.09 (Low) C H441 AD KRAS mutation 18 49 61 0.21 (Low) 0.17 (Low) B H2170 SQ W/t 18 28 42 0.65 (High) 0.09 (Low) C HCC15 SQ HER4 mut 17 21 43 0.18 (Low) 0.12 (Low) B H460 LC KRAS PIK3CA mut 17 57 78 0.98 (High) 0.10 (Low) C PC-9 AD EGFR mut 16 24 55 0.20 (Low) 0.16 (Low) B H1975 AD EGFR mut 10 45 63 0.41 (High) 0.19 (Low) C HCC366 ADSQ W/t 8 42 54 0.39 (High) 0.08 (Low) C RPC-9 AD EGFR mut 4 15 40 0.24 (Low) 0.16 (Low) B H358 AD KRAS mut 6 59 73 0.57 (High) 1.21 (High) B H3255 AD EGFR mut 3 16 40 0.54 (High) 0.59 (High) B 211H MM - 5 13 46 - - - OUMS-24 NHF - 5 0 0 - - - NSCLC non-small cell lung cancer; AD adenocarcinoma; SQ squamous cell carcinoma; LC large cell carcinoma; ADSQ adeno-squamous cell carcinoma; MM malignant mesothelioma; NHF normal human fibroblast; mut mutation; W/t. wild type; MOI multiplicity of infection. Adenovirus vector carrying REIC/Dkk-3 REIC/Dkk-3 was overexpressed using an adenovirus (Ad-REIC) that we have previously generated [11]. A full-length cDNA of REIC/Dkk-3 was integrated into a cosmid vector pAxCAwt and transferred into an adenovirus vector by the COS-TPC method (Takara Bio Shiga Japan). An adenovirus vector carrying LacZ gene (Ad-LacZ) was also used as control [11]. Cell viability assay Cells were plated in 96-well plates at a density of 1.5×103 cells/well at 48 h after infection with Ad-LacZ or Ad-REIC at a multiplicity of infection (MOI) of 20100 or 200 MOI. Cell viability was evaluated 3 days later using an MTS assay with CellTiter 96 Aqueous One Solution Reagent (Promega Madison WI). Apoptosis assay To examine the in vitro induction of apoptosis after treatment we seeded the cells in 6-well plates and incubated them for 24 h. The cells were treated with Ad-LacZ or Ad-REIC at 20 MOI in serum-free medium (500 µL) for 2 h; the medium was then exchanged for fresh complete medium (2 mL). After an additional 48 h of incubation Hoechst 33342 dye (Sigma-Aldrich St. Louis MO) was added to the medium at a concentration of 2 µg/mL and the cells were incubated in the dark for 10 min. Hoechst 33342 is an intercalating dye that allows the determination of variations in the total chromatin quantity and the degree of chromatin condensation [15]. Using fluorescence microscopy we identified apoptotic cells by the presence of highly condensed or fragmented nuclei. Apoptotic cells were counted in 5 different fields under microscopic observation. Western blot analysis The detailed protocol for the Western blot analysis is described in Method S1. It was performed under conventional conditions using the following antibodies: rabbit anti-human REIC/Dkk-3 antibody raised in our laboratory [11]; rabbit anti-human GRP78/BiP (GRP78) (ab21685; Abcam Cambridge MA); rabbit anti-human SAPK/JNK (#9252) and rabbit anti-human phospho-SAPK/JNK (Thr183/Tyr185; #9251) (Cell Signaling Technology Beverly MA); rabbit anti-human coxsackievirus and adenovirus receptor (CAR) (HPA030411; Atlas antibodies Stockholm Sweden); and mouse anti-actin (MAB1501; Millipore Billerica MA). The following secondary antibodies were used: goat anti-rabbit or anti-mouse IgG-conjugated horseradish peroxidase (Santa Cruz Biotechnology Santa Cruz CA). To detect the specific signals the membranes were examined using ECL plus Western Blotting Detection Reagents (Amersham Biosciences UK Limited Buckinghamshire UK). In addition the band intensities for GRP78 CAR and actin representing their expression levels were measured using ImageQuant TL software (GE Healthcare Bioscience) and quantified by GRP78 or CAR/actin ratio. Tumor growth assay in vivo A549 cells (5×106 in 50 µL of phosphate buffered saline [PBS]) mixed with 50 µL of Matrigel (BD Biosciences San Jose CA) were subcutaneously injected into the right flank of adult female BALB/c nu/nu mice (CLEA Japan Tokyo Japan). The tumor volume was calculated using the empirical formula V?=?1/2×[(the shortest diameter)2×(the longest diameter)]. When the tumors had reached approximately 50“100 mm3 mice (n?=?15) were randomly divided into 3 treatment groups: (a) PBS; (b) Ad-LacZ; and (c) Ad-REIC. Viruses (1×109 pfu) in 100 µL of serum-free medium were administered intratumorally. At the end of experiments mice were sacrificed after 24-days after the viral injection and tumors were harvested measured and photographed. Statistical analyses All data were analyzed using STATA ver.12 (STATA Corp. College Station TX). Fisher's exact test was applied when appropriate. For a comparison of induction of apoptosis between Ad-REIC-treated and Ad-LacZ-treated A549 cells a Cochran-Mantel-Haenszel statistics was applied for comparing. Repeated measurement ANOVA was applied for the comparison of xenotransplanted NSCLC tumor sizes among PBS Ad-LacZ and Ad-REIC. P<0.05 was considered significant. All tests were two-sided. Results Effect of Ad-REIC on NSCLC cell lines We examined the inhibition of cell viability using Ad-REIC and an MTS assay. In 13 (52%) of 25 NSCLC cell lines Ad-REIC treatment at 20 MOI inhibited the cell viability (40%“60% inhibition) compared with Ad-LacZ treatment ( Figure 1). These cell lines were regarded as highly sensitive to Ad-REIC. In contrast 12 cell lines (48%) were not inhibited by Ad-REIC treatment at 20 MOI and were regarded as resistant cells. OUMS-24 was not inhibited at 20 or 200 MOI of Ad-REIC. Of note Ad-REIC treatment at 100 and 200 MOI improved the inhibition of cell viability (100 MOI: 15%“59% inhibition 200 MOI: 40%“78% inhibition) compared with Ad-LacZ treatment (). Thus we defined 20 MOI as a low MOI value and 200 MOI as a high MOI value. For comparison Ad-REIC treatment was also performed in the human mesothelioma cell line 211H which we previously reported to be Ad-REIC-sensitive [14]. The 211H was not inhibited at 20 MOI but was inhibited at 200 MOI of Ad-REIC (). The known molecular characteristics of each cell line are shown in . The 25 NSCLC cell lines consisted of 8 EGFR-mutant 6 KRAS-mutant 1 HER4-mutant 1 NRAS-mutant 1 PIK3CA-mutant 1 EML4-ALK fusion 1 HER2-amplified and 6 cell lines without gene alterations listed. Nine of the 17 EGFR-wild type cell lines were sensitive to Ad-REIC. HCC827 and its resistant subline HCC827-GR-high2 showed a similar degree of sensitivity to Ad-REIC. No trend in molecular genotype was seen between the sensitive and non-sensitive cell lines. These results suggested that the effect of Ad-REIC does not depend on a known molecular genotype. .0087900.g001 Figure 1 Sensitivity and predictive factors of sensitivity for Ad-REIC treatment in 25 NSCLC cell lines. The inhibition rates of 25 NSCLC cell lines transfected with Ad-REIC compared to Ad-LacZ are shown as black bar in 20 MOI and white bar in 200 MOI. Thirteen cell lines with over 40% inhibition rate in 20 MOI are defined as highly sensitive and 12 cell lines with lower inhibition rate in 20 MOI are defined as resistant. All the resistant cell lines shows over 40% inhibition rate in 200 MOI. The cell lines are classified into 3 categories based on the GRP and CAR protein expression level as follows; category A (low GRP/high CAR) category B (low GRP/low CAR or high GRP/high CAR) category C (high GRP/low CAR). All 8 highly sensitive cell lines were included in category A and all 5 resistant cell lines were included in category C. Sq; squamous cell carcinoma AD; adenocarcinoma LC; large cell carcinoma ADSQ; adenosquamous cell carcinoma MM; malignant mesothelioma NHF; normal human fibroblast. Hoechst 33342 staining was performed in A549 cells to examine the induction of apoptosis. Apoptotic cells were observed in Ad-REIC-treated A549 cells (Figure 2a). The mean rate of apoptosis was 22% and it was significantly (p<0.001 by Cochran-Mantel-Haenszel test) increased in comparison with the control Ad-LacZ treatment. .0087900.g002 Figure 2 Ad-REIC induced JNK activation and subsequent apoptosis in NSCLC cells. (a) Induction of apoptosis after in vitro Ad-REIC treatment as examined in A549 cells using Hoechst 33342 staining. The upper panel indicates the appearance of apoptotic cells after Ad-REIC treatment. The lower panel shows the apoptotic rate of A549 cells after the indicated treatment. A total of 5 different fields were examined under a microscope to determine the apoptotic rate. A significant difference was observed (*p<0.001) between the Ad-LacZ and the Ad-REIC treatment. (bar: 100 µm) (b) Western blot analysis for proteins involved in signal transduction triggered by Ad-REIC. Cells were harvested at 48 h after transfection with Ad-LacZ or Ad-REIC at 20 MOI. (c) H460 cells which are resistant to adenovirus transduction were harvested at 48 h after transfection with Ad-LacZ or Ad-REIC at 20100 and 200 MOI. The effect of recombinant REIC/Dkk-3 protein on NSCLC cell lines was examined in 7 randomly selected cell lines (NCI-H522 NCI-H611 NCI-H1299 NCI-H1819 NCI-H2009 PC-9 and A549). The MTS assay showed that REIC/Dkk-3 protein did not affect cell viability in the examined cell lines when administered at a concentration ranging from 1 to 200 µg/mL (data not shown). Expression of GRP78 and CAR in response to Ad-REIC therapy As predictive factors of Ad-REIC sensitivity in NSCLC we examined the expressions of GRP78 and CAR; these expression statuses were correlated with the inhibition of cell viability by Ad-REIC in 13 cell lines. A previous study reported that the overexpression of GRP78 inhibited ER-stress which may be oppositely correlated with the effect of Ad-REIC. CAR expression is tightly associated with the efficacy of adenovirus infection which may be positively correlated with the effect of Ad-REIC. Western blotting was performed and the expression level was quantified as shown in and Figure 1. The median (range) of GRP78 and CAR expressions were 0.24 (0.075“0.98) and 0.60 (0.080“2.1) respectively. Based on these data cells with a GRP78 expression level more than 0.25 were defined as High CRP78 expression while those with a GRP78 less than 0.24 were defined as Low GRP78 expression. Regarding the CAR 15 cell lines significantly high level of CAR expression (over 0.50) were defined as High CAR expression while 10 cell lines those with significantly low level of CAR expression (under 0.20) were defined as Low CAR expression. GRP78 expression was low in 8 of the 13 Ad-REIC-sensitive cells (62%) and in 4 of the 12 Ad-REIC-resistant cells (33%). CAR expression was high in 12 of the 13 Ad-REIC-sensitive cells (92%) and in 3 of the 12 Ad-REIC-resistant cells (25%). Next we classified the cell lines into three categories based on the GRP78 and CAR expression statuses; cells with a Low GRP78/High CAR expression were classified as Category A those with Low GRP78/Low CAR or High GRP78/High CAR expression were classified as Category B and those with High GRP78/Low CAR expression were classified as Category C. The high sensitive cell rates were 100% in Category A (8 out of 8 95% confidence interval [CI]: 63“100) 42% in Category B (5 out of 12 95% CI: 15“72) and 0% in Category C (0 out of 5 95% CI: 0“52) (Table 2). The categories were significantly associated with the sensitivity to Ad-REIC treatment (p<0.01). .0087900.t002 Table 2 Ad-REIC sensitivity and categories based on predictive factors. (n) Category A (8) Category B (12) Category C (5) Highly sensitive (13) 8 5 0 Resistant in 20 MOI (12) 0 7 5 JNK and GRP78 expression in NSCLC cell lines treated with Ad-REIC A western blotting analysis demonstrated the significant expression of REIC/Dkk-3 protein in 14 NSCLC cell lines treated with Ad-REIC. In 9 cell lines infected with 20 MOI Ad-REIC treatment resulted in the phosphorylation of JNK and the up-regulation of GRP78 (Figure 2b). In the other 8 cell lines which were relatively resistant to Ad-REIC the activation of JNK and GRP78 were observed at higher MOI values (100 and 200 MOI) (Figure 2c). Effect of Ad-REIC on NSCLC tumors in a xenotransplantation model We investigated the effect of Ad-REIC on the growth of A549 cells in vivo. One week after transplantation when the tumor volume reached 50 to 100 mm3 1×109 plaque-forming units of Ad-REIC or Ad-LacZ in 100 µL of PBS or 100 µL of PBS alone were injected intratumorally. The tumors grew progressively in the PBS and Ad-LacZ treatment groups during the subsequent 24-day observation period. In contrast the tumor growth in the Ad-REIC treatment group was significantly (p<0.001 by repeated measurement ANOVA) suppressed during the observation period (Figure 3ab). .0087900.g003 Figure 3 Anti-tumor effect of Ad-REIC treatment on A549 tumor growth in vivo. (a) The mean volume of the subcutaneous xenograft tumors was calculated for 5 mice in each group. A significant difference was observed between the results of Ad-REIC and Ad-LacZ treatment (*p<0.001 by repeated measurement of ANOVA). (b) Appearance of the tumors at the time of sacrifice after treatment with PBS Ad-LacZ and Ad-REIC. Discussion In the present study we found that Ad-REIC was directly effective in more than half of the NSCLC cell lines that were examined independent of its known driver alterations such as EGFR and KRAS mutations. An animal xenograft model also showed the therapeutic effect of Ad-REIC. The anti-tumor effect of Ad-REIC depends on ER-stress-mediated JNK activation loaded by the overproduction of REIC/Dkk-3 protein resulting in the induction of apoptosis [14] [20]. The activation of JNK which is an essential step in the induction of ER stress and apoptosis by Ad-REIC was observed at 20 MOI in NSCLC cell lines. On the other hand the anti-tumor effect of recombinant REIC/Dkk-3 protein was not observed as in other types of cancers that were previously examined. Originally REIC/Dkk-3 was identified as a secretory protein and was assumed to exert a physiological function but its cell surface receptor and its role as a secretory protein have not been identified. We defined 20 MOI as a low MOI value and 200 MOI as a high MOI value because the normal human fibroblast cell line OUMS-24 was not inhibited at 20 or 200 MOI of Ad-REIC whereas malignant cell lines were inhibited when the MOI value was elevated to 100 and 200 MOI in cell lines in which Ad-REIC had been ineffective at 20 MOI. In NSCLC Ad-REIC was effective at a low MOI value in more than half of the cell lines that were tested. Considering the result that 211H was inhibited only at a high MOI value Ad-REIC might be more effective in NSCLC than in mesothelioma. Patient selection based on the molecular characteristics of tumor cells is an important theme for maximizing the therapeutic benefit and minimizing adverse effects. For this purpose we focused on the GRP78 expression and CAR expression levels. GRP78 is a member of the Hsp70 family which serves as an ER stress-signaling regulator [21]. A previous study showed that the overexpression of GRP78 conferred resistance to a wide variety of chemotherapeutic agents in various kinds of cells [22]. We also showed that the acquired resistance clone of PC-3 cells to Ad-REIC established after repeated exposure to Ad-REIC exhibited a high expression level of GRP78 compared with parental PC-3 cells [13]. Theoretically Ad-REIC should be effective for tumor cells defined as Category A and not as effective for those defined as Category C. Although sensitive cells in Category B were identified all 8 cells in Category A responded to Ad-REIC treatment. These results suggested that the expression statuses of GRP78 and CAR in tumors might be useful as biomarkers for customized Ad-REIC therapy in NSCLC while further confirmation is needed by a large scaled investigation using various kinds of cell lines. As a recent topic of lung cancer treatment EGFR-TKIs have been shown to be effective for the treatment of EGFR-mutant NSCLCs. However acquired resistance to EGFR-TKIs after TKI treatment is a problem that needs to be overcome. In the current study our results showed that the effect of Ad-REIC against acquired EGFR-TKI-resistant cells was equal to that against the parental cells suggesting that Ad-REIC may be useful after the acquisition of resistance to EGFR-TKIs. Although adenovirus vectors carrying appropriate tumor suppressor genes such as REIC/Dkk-3 have great potential for cancer gene therapy they do not exhibit target specificity and therefore may also infect normal cells in the vicinity of cancer cells. The authors reported that the infection of normal human fibroblasts (NHF) with Ad-REIC did not cause the apoptosis of NHF itself but instead induced the production of interleukin (IL)-7. When Ad-REIC-infected NHF were mixed with untreated cancer cells and the mixture was transplanted into mice the growth of the cancer cells was significantly suppressed suggesting an indirect tumor-suppressive effect of Ad-REIC mediated by IL-7 [20]. These findings show that the mis-targeted infection of cancer stroma cells by Ad-REIC activates the immune system through the production of IL-7. In addition the authors reported that REIC/Dkk-3 protein played a cytokine-like role in monocyte differentiation into dendritic-cell-like features in vitro and that the infiltration of CD11c- and CD8-positive (dendritic and killer T cell markers respectively) cells was observed within the treated tumors in vivo. In the experiment using an orthotopic prostate tumor model with pre-established lung metastasis the number of metastatic lung tumors significantly decreased after the injection of Ad-REIC at the primary tumor site in addition to the inhibition of the growth of orthotopic prostate tumors suggesting that anti-cancer immune up-regulation by Ad-REIC treatment in primary tumor sites triggered anti-tumor effects even at distant tumor site [16]. These facts strongly suggest that REIC/Dkk-3 shows an indirect anti-tumor effect through the anti-tumor immune system that is an important factor in the treatment of metastatic disease. Because Ad-REIC has both direct and indirect effects on cancer therapy it may become a powerful therapeutic option as a œone-bullet two-arms anti-cancer agent especially for NSCLCs which often metastasize to other organs. In regards to clinical usage because our data suggest that CAR and GRP78 expression statuses in tumor cells predict the responsiveness of Ad-REIC treatment Ad-REIC treatment should be preferentially performed for patients who are categorized as high sensitive group in early phase of treatment with low dose Ad-REIC. For patients whose tumor cells reveal intermediate or poor effectiveness with low dose Ad-REIC it should be late phase in their treatment with high dose Ad-REIC. For these patients cost effectiveness for treatment and clinical outcome should be carefully considered. As for administration strategy local administration might be preferable rather than systemic administration to minimize the adverse effect in clinical situations. We previously confirmed in mouse model that Ad-REIC could be widely distributed in the bodies after intratumoral local administration and local administration was effective not only directly but also indirectly through the immune system effect [16] [23]. In addition intrapleural local administration could be another administration strategy for the patients with malignant pleural effusions. It has been reported that the intrapleural administration of adenoviral-mediated gene therapy is a useful approach for the generation of anti-tumor immune responses in malignant mesothelioma and metastatic pleural effusion in several clinical trials [24] [25]. In conclusion we demonstrated that Ad-REIC induced JNK activation and subsequent apoptosis in NSCLC cells irrespective of the type of known molecular alterations or the sensitivity to EGFR-TKI. The present study suggests that Ad-REIC has a therapeutic potential for NSCLC and the expression statuses of GRP78 and CAR may be a predictor of Ad-REIC therapy. Supporting Information Figure S1 The heatmap image of mRNA expression of REIC/Dkk-3 gene. The mRNA expression level of REIC/Dkk-3 gene was obtained from the UCSC Cancer Genome Browse which is freely available public database (://genome-cancer.ucsc.edu/) (we downloaded the data on July 16 2013) showed that REIC/Dkk-3 gene expression was reduced in majority of examined samples of both (a) lung adenocarcinomas and (b) squamous cell carcinomas compared with normal lung tissues. (PDF) Click here for additional data file. Method S1 Supporting information for cell lines and Western blot analysis. (DOC) Click here for additional data file. References 1 JemalA SiegelR WardE HaoY XuJ et al (2009) Cancer statistics 2009. CA Cancer J Clin59: 225“24919474385 2 LarsenJE CasconeT GerberDE HeymachJV MinnaJD (2011) Targeted therapies for lung cancer: clinical experience and novel agents. Cancer J17: 512“52722157296 3 ShigematsuH LinL TakahashiT NomuraM SuzukiM et al (2005) Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J Natl Cancer Inst97: 339“34615741570 4 Tokumo M Toyooka S Kiura K Shigematsu H Tomii K et al. (2005) The relationship between epidermal growth factor receptor mutations and clinicopathologic features in non-small cell lung cancers. Clin Cancer Res. 5 KwakEL BangYJ CamidgeDR ShawAT SolomonB et al (2010) Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med363: 1693“170320979469 6 ShawAT YeapBY SolomonBJ RielyGJ GainorJ et al (2011) Effect of crizotinib on overall survival in patients with advanced non-small-cell lung cancer harbouring ALK gene rearrangement: a retrospective analysis. Lancet Oncol12: 1004“101221933749 7 OxnardGR JanjigianYY ArcilaME SimaCS KassSL et al (2011) Maintained sensitivity to EGFR tyrosine kinase inhibitors in EGFR-mutant lung cancer recurring after adjuvant erlotinib or gefitinib. Clin Cancer Res17: 6322“632821831955 8 TsujiT MiyazakiM SakaguchiM InoueY NambaM (2000) A REIC gene shows down-regulation in human immortalized cells and human tumor-derived cell lines. Biochem Biophys Res Commun268: 20“2410652205 9 KrupnikVE SharpJD JiangC RobisonK ChickeringTW et al (1999) Functional and structural diversity of the human Dickkopf gene family. Gene238: 301“31310570958 10 MaoB WuW DavidsonG MarholdJ LiM et al (2002) Kremen proteins are Dickkopf receptors that regulate Wnt/beta-catenin signalling. Nature417: 664“66712050670 11 AbarzuaF SakaguchiM TakaishiM NasuY KuroseK et al (2005) Adenovirus-mediated overexpression of REIC/Dkk-3 selectively induces apoptosis in human prostate cancer cells through activation of c-Jun-NH2-kinase. Cancer Res65: 9617“962216266978 12 EdamuraK NasuY TakaishiM KobayashiT AbarzuaF et al (2007) Adenovirus-mediated REIC/Dkk-3 gene transfer inhibits tumor growth and metastasis in an orthotopic prostate cancer model. Cancer Gene Ther14: 765“77217599093 13 TanimotoR AbarzuaF SakaguchiM TakaishiM NasuY et al (2007) REIC/Dkk-3 as a potential gene therapeutic agent against human testicular cancer. Int J Mol Med19: 363“36817273781 14 KashiwakuraY OchiaiK WatanabeM AbarzuaF SakaguchiM et al (2008) Down-regulation of inhibition of differentiation-1 via activation of activating transcription factor 3 and Smad regulates REIC/Dickkopf-3-induced apoptosis. Cancer Res68: 8333“834118922905 15 KawasakiK WatanabeM SakaguchiM OgasawaraY OchiaiK et al (2009) REIC/Dkk-3 overexpression downregulates P-glycoprotein in multidrug-resistant MCF7/ADR cells and induces apoptosis in breast cancer. Cancer Gene Ther16: 65“7218654608 16 WatanabeM KashiwakuraY HuangP OchiaiK FutamiJ et al (2009) Immunological aspects of REIC/Dkk-3 in monocyte differentiation and tumor regression. Int J Oncol34: 657“66319212670 17 ShienK ToyookaS YamamotoH SohJ JidaM et al (2013) Acquired Resistance to EGFR Inhibitors Is Associated with a Manifestation of Stem Cell-like Properties in Cancer Cells. Cancer Res73: 3051“306123542356 18 KobayashiN"

Lung_Cancer

" (see Table IV and second column). This pattern is confirmed by the results in Table V showing the average df in each dimension and the empirical rejection rates for the hypotheses of linearity and constant risk. The AIC selection is affected by moderate overfitting sometimes suggesting flexible models in scenarios of linear and/or constant risk. In contrast BIC shows severe underfitting often selecting simple models for complex exposure“lag“response associations in particular regarding linearity. Table V Average df in each dimension for the best fitting models selected through AIC and BIC (left part) and empirical rejection rate for the AIC and BIC-based selection for the hypotheses of linearity and constant risk (right part) for the nine scenarios of exposure“lag“response associations. Results from m = 500 simulated data sets with ns = 400 subjects Average df Empirical rejection rate f(x) w(?) H0 : f(x) = x H0 : w(?) = c f(x) ? w(?) AIC BIC AIC BIC AIC BIC AIC BIC Linear-constant 1.50 1.03 1.57 1.02 0.29* 0.03* 0.23* 0.01* Linear-decay 1.26 1.00 3.60 3.17 0.18* 0.00* 1.00 1.00 Linear-peak 1.22 1.00 4.02 3.72 0.15* 0.00* 1.00 0.98 Plateau-constant 2.26 1.54 1.47 1.00 0.82 0.47 0.19* 0.00* Plateau-decay 2.53 1.55 3.49 3.10 0.97 0.54 1.00 1.00 Plateau-peak 2.18 1.21 4.01 3.56 0.85 0.19 1.00 0.93 Exponential-onstant 2.20 1.56 1.43 1.00 0.83 0.52 0.16* 0.00* Exponential-decay 2.36 1.81 3.58 3.12 0.99 0.80 1.00 1.00 Exponential-peak 2.15 1.29 4.05 3.69 0.90 0.27 1.00 0.93 * H0 is true The undercoverage of confidence intervals as shown in Table IV can be attributed to both lack of fit and a posteriori model selection. The latter as discussed in Section 2.5 may generate undercoverage through the underestimation of the true sampling (co)variance. A comparison of the importance of the two sources can be provided by the assessment of undercoverage in the first scenario where linear and constant functions are actually among the options of the selection procedure and the underlying simulated association can be potentially recovered with no lack of fit. In this scenario AIC-selected models affected by overfitting show a coverage of 91% very close to the nominal value as illustrated in Table IV. The under-coverage seems to be proportional to the bias as confirmed by with a lower coverage corresponding to sections of the bidimensional space characterized by worse fit. Empirical coverage across the risk surfaces for three scenarios of exposure“lag“response associations (linear-constant plateau-decay and exponential-peak in each column). Results from m = 500 simulated data sets with ns = 400 subjects. The simulated examples with ns = 200 and ns = 800 generate approximately 150 and 600 uncensored events respectively. The versions of Tables IV“V and for these examples are reported in Tables S2“S5 and Figures S9“S10 of the supporting information. The comparison suggests that varying the sample size does not dramatically affect the performance of the AIC-based test apart from the expected different power in identifying nonlinear and noncostant exposure“time“response associations. Consistently AIC-based selection seems to perform well across the range of number of subjects included in the analysis with a small bias and reasonable coverage. The results of this simulation study are consistent with previous findings on one-dimensional models for exposure“lag“response associations assuming a linear exposure“response relationship 18. 5. Discussion In this contribution I illustrate a statistical framework for modeling temporal dependencies with time-varying exposures defined here as exposure“lag“response associations. The approach is based on the extension of distributed lag non-linear models a modeling class previously proposed in time series analysis 2324. The extended DLNM methodology brings together and extends previous methodological developments on the topic as summarized in. Briefly it provides a unified framework for different study designs and regression methods and is applicable to time series cross-sectional case-control survival and longitudinal data. A major advantage is the possibility to describe the lag structure of either linear or nonlinear exposure“response relationships through the choice of two functions that define the association along the dimensions of the predictor and lags including most of the previous approaches as special cases. The example in illustrates how such flexibility is important for obtaining correct estimates of the association. Model specification easily accounts for previous knowledge on the association and incorporates assumptions on the phenomenon to be investigated through the choice of specific functions lag period and constraints. Interpretation of complex exposure“lag“response associations is aided by the definition of simple summary measures of effect and prediction and by graphical representation. The modeling framework is defined through a neat and compact algebraic representation including the derivation of measures of uncertainty such as standard errors and confidence intervals. Estimation is carried out with standard regression models which do not require specialized optimization procedures and may include terms for multiple exposure“lag“response dependencies as shown for radon and smoking here. The parameterization prediction and graphical representation are carried out with few general functions implemented in a freely available and documented software as discussed in. A key issue of the DLNM methodology is about selecting the appropriate model among different options for modeling the bidimensional exposure“lag“response relationship. The simulation study in indicates that AIC-based selection performs reasonably well over a range of 150“600 uncensored events while the strong penalty of BIC induces the selection of models too simple to recover the underlying dependency. The overfitting characterizing AIC-selected models in scenarios of simple exposure“lag“response dependencies does not seriously affect its performance a result in line with previous findings 18. However AIC-selected models also suffer from bias and undercoverage of confidence intervals to some extent. Part of this seems to be related to the limited flexibility of the functions applied in the simulation study and may be described as a smoothing problem rather that an inherent limitation of the estimators. It should also be noted that the simulation study only evaluates a limited set of exposure“response and lag“response shapes simulated under the assumption of independency. Different functions such as cubic splines and more complex exposure“lag“response surfaces will be assessed in future simulation studies. Also an extension of DLNMs with penalized splines characterized by higher flexibility can be explored as well exploiting previous research on bivariate smoothing techniques 3031. A related problem is about the inferential procedures being conditional on a posteriori selection of the best-fitting model. Previous studies on unidimensional models have proposed a correction for the inflation of type I errors in tests on a constant effect along lags 1727. However this approach is not easily extended to the bidimensional setting of exposure“lag“response associations and the definition of a hypothesis testing procedure for DLNMs is left to future developments. Although a posteriori selection may also be a source of undercoverage of confidence intervals its impact seems to be limited if compared with that associated with lack of fit at least in the simple scenarios investigated in the simulation study. Another limitation is the lack of a formal testing procedure on the hypothesis of independency. As suggested in Section 3.4 a graphical assessment of the proportionality of exposure“response and lag“response curves such as those in can help investigating the issue. Further research is needed to provide more consistent inferential procedures in this setting. The analysis of the temporal evolution of the risk associated with protracted time-varying exposures has straightforward applications in different research fields. For example the DLNM methodology may be used to characterize the risk of chronic exposures to occupational or environmental factors to differentiate the role of exposures sustained at different ages in life course studies or to define the temporal frame of beneficial or adverse effects of drugs in clinical trials and pharmaco-epidemiology. The development of this methodology and software implementation provide a promising analytical tool for biomedical research. 6. Software and data All the analyses presented in this paper were performed using the R software version 3.0.1 32. The DLNM modeling framework is fully implemented in the package dlnm 25 by using the expressly extended version 2.0.0. The permutational algorithm for simulating time-to-event data in the presence of time-varying exposures is implemented in the package PermAlgo 29 version 1.0. Both packages are available through R from its central repository. The data of the Colorado Plateau uranium miners cohort in the form of a comma-separated values file is included in the supporting information¡ together with the R scripts for the analysis performed in the example and the simulation study of Sections 3“4 which are entirely reproducible. In particular the script example.R provides a short illustration of the modeling framework. Versions of the scripts updated to future versions of the dlnm package will be available at http://www.ag-myresearch.com. Distributed lag non-linear models were originally conceived and developed for describing temperature“health associations in time series data by Ben Armstrong. The data from the Colorado Plateau uranium miners cohort were collected by the researchers of National Institute for Occupational Safety and Health. I am grateful to Bryan Langholz for kindly making data and documentation available. The simulation study was performed using the high-processing computing system at the London School of Hygiene and Tropical Medicine. The final version of this article has been substantially improved following the comments of an unknown reviewer. This research was supported by a Methodology Research fellowship by Medical Research Council-UK (grant ID G1002296). References 1 Goodman PG Dockery DW Clancy L Cause-specific mortality and the extended effects of particulate pollution and temperature exposure Environmental Health Perspectives 2004 112 2 179 185 14754572 2 Elliott P Shaddick G Wakefield JC de Hoogh C Briggs DJ Long-term associations of outdoor air pollution with mortality in Great Britain Thorax 2007 62 12 1088 1094 17666438 3 Collet JP Sharpe C Belzile E Boivin JF Hanley J Abenhaim L Colorectal cancer prevention by non-steroidal anti-inflammatory drugs: effects of dosage and timing British Journal of Cancer 1999 81 1 62 8 10487613 4 Abrahamowicz M Bartlett G Tamblyn R du Berger R Modeling cumulative dose and exposure duration provided insights regarding the associations between benzodiazepines and injuries Journal of Clinical Epidemiology 2006 59 4 393 403 16549262 5 Checkoway H Pearce N Hickey JL Dement JM Latency analysis in occupational epidemiology Archives of Environmental Health 1990 45 2 95 100 2334237 6 Thomas DC Models for exposure-time-response relationships with applications to cancer epidemiology Annual Review of Public Health 1988 9 451 482 7 Breslow NL Day NE Statistical Methods in Cancer Research 1987 II Lyon International Agency for Reasearch on Cancer (IARC) 232 271 The desing and analysis of cohort studies chap. 6: Modelling the relationship between risk dose and time 8 Thomas DC Brown CC Chu KC Goldsmith DF Saracci R Proceedings of a symposium on time-related factors in cancer epidemiology Journal of Chronic Diseases 1987 40 Suppl. 2 1S 211S 9 Thomas DC Statistical Methods in Environmental Epidemiology 2009 New York Oxford University Press 279 300 chap. 13: Mechanistic models 10 Thomas DC Statistical methods for analyzing effects of temporal patterns of exposure on cancer risks Scandinavian Journal of Work Environment & Health 1983 9 4 353 366 11 Vacek PM Assessing the effect of intensity when exposure varies over time Statistics in Medicine 1997 16 5 505 513 9089959 12 Langholz B Thomas D Xiang A Stram D Latency analysis in epidemiologic studies of occupational exposures: application to the Colorado Plateau uranium miners cohort American Journal of Industrial Medicine 1999 35 3 246 256 9987557 13 Richardson DB Latency models for analyses of protracted exposures Epidemiology 2009 20 3 395 399 19262389 14 Hauptmann M Wellmann J Lubin JH Rosenberg PS Kreienbrock L Analysis of exposure-time-response relationships using a spline weight function Biometrics 2000 56 4 1105 1108 11129467 15 Hauptmann M Berhane K Langholz B Lubin J Using splines to analyse latency in the Colorado Plateau uranium miners cohort Journal of Epidemiology and Biostatistics 2001 6 6 417 424 11831677 16 Hauptmann M Pohlabeln H Lubin JH Jockel KH Ahrens W Bruske-Hohlfeld I Wichmann HE The exposure-time-response relationship between occupational asbestos exposure and lung cancer in two German case-control studies American Journal of Industrial Medicine 2002 41 2 89 97 11813213 17 Sylvestre MP Abrahamowicz M Flexible modeling of the cumulative effects of time-dependent exposures on the hazard Statistics in Medicine 2009 28 27 3437 3453 19708037 18 Abrahamowicz M Beauchamp ME Sylvestre MP Comparison of alternative models for linking drug exposure with adverse effects Statistics in Medicine 2012 31 11-12 1014 1030 22095719 19 Abrahamowicz M MacKenzie TA Joint estimation of time-dependent and non-linear effects of continuous covariates on survival Statistics in Medicine 2007 26 2 392 408 16479552 20 Berhane K Hauptmann M Langholz B Using tensor product splines in modeling exposure-time-response relationships: Application to the Colorado Plateau Uranium Miners cohort Statistics in Medicine 2008 27 26 5484 5496 18613262 21 Almon S The distributed lag between capital appropriations and expenditures Econometrica 1965 33 178 196 22 Schwartz J The distributed lag between air pollution and daily deaths Epidemiology 2000 11 3 320 326 10784251 23 Armstrong B Models for the relationship between ambient temperature and daily mortality Epidemiology 2006 17 6 624 631 17028505 24 Gasparrini A Armstrong B Kenward MG Distributed lag non-linear models Statistics in Medicine 2010 29 21 2224 2234 20812303 25 Gasparrini A Distributed lag linear and non-linear models in R: the package dlnm Journal of Statistical Software 2011 43 8 1 20 22003319 26 Thomas DC Statistical Methods in Environmental Epidemiology 2009 New York Oxford University Press chap. 6: Modelling exposure-time-response relationships 27 Mahmud M Abrahamowicz M Leffondré K Chaubey Y Selecting the optimal transformation of a continuous covariate in Cox's regression: Implications for hypothesis testing Communications in Statistics: Simulation and Computation 2006 35 1 27 45 28 Breslow NL Day NE Statistical Methods in Cancer Research 1987 II Lyon International Agency for Reasearch on Cancer (IARC) 178 231 The desing and analysis of cohort studies chap. 5: Fitting models to continuous data 29 Sylvestre MP Abrahamowicz M Comparison of algorithms to generate event times conditional on time-dependent covariates Statistics in Medicine 2008 27 14 2618 2634 17918753 30 Wood SN Generalized Additive Models: an Introduction with R 2006 Chapman & Hall/CRC 31 Eilers PHC Currie ID Durban M Fast and compact smoothing on large multidimensional grids Computational Statistics and Data Analysis 2006 50 1 61 76 32 R Development Core Team R: A Language and Environment for Statistical Computing R Foundation for Statistical Computing Vienna Austria 2013. http://www.R-project.org/ Supplementary material 9005373 1697 Eur J Cancer Eur. J. Cancer European journal of cancer (Oxford England : 1990) 0959-8049 1879-0852 24246704 3991133 10.1016/j.ejca.2013.10.006 NIHMS541404 Article Dosing to Rash: A Phase II Trial of the First-Line Erlotinib for Patients with Advanced Non-Small-Cell Lung Cancer an Eastern Cooperative Oncology Group Study (E3503) Brahmer JR M.D. M.Sc. 1 Lee JW Ph.D. 2 Traynor AM M.D. 3 Hidalgo MM M.D. Ph.D. 4 Kolesar JM Pharm.D. 3 Siegfried JM Ph.D. 5 Guaglianone PP M.D. 6 Patel JD M.D. 7 Keppen MD M.D. 8 Schiller JH M.D. 9 1Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins Baltimore Maryland 2Dana-Farber Cancer Institute Boston Massachusetts 3University of Wisconsin Madison Wisconsin 4Spain 5University of Pittsburgh Pittsburgh Pennsylvania 6Decatur Memorial Hospital Decatur Illinois 7Northwestern University Chicago Illinois 8Sanford Cancer Center Sioux Falls South Dakota 9University of Texas Southwestern Medical Center Dallas Texas Corresponding Author Julie R. Brahmer M.D. M.Sc. Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins Bunting Blaustein Cancer Research Building Room G94 1650 Orleans Street Baltimore MD 21287-0013 Office phone: 410-502-7159; Fax 410-614-9334; brahmju@jhmi.edu 2 4 2014 15 11 2013 1 2014 01 1 2015 50 2 302 308 © 2013 Published by Elsevier Ltd. 2013 Background The development of a rash has been retrospectively associated with increased response and improved survival when treated with erlotinib at the standard dose of 150 mg per day. The objective of this trial was to evaluate the association of the activity of erlotinib in the first-line setting in patients with advanced non-small-cell lung cancer (NSCLC) with the development of a tolerable rash via dose escalation of erlotinib or tumor characteristics. Methods Patients with advanced NSCLC without prior systemic therapy were treated with erlotinib 150 mg orally per day. The dose was increased by 25 mg every two weeks until the development of grade 2/tolerable rash or other dose limiting toxicity. Tumor biopsy specimens were required for inclusion. Results The study enrolled 137 patients 135 were evaluable for safety and 124 were eligible and evaluable for response. Only 73 tumor samples were available for analysis. Erlotinib dose escalation occurred in 69/124 patients. Erlotinib was well tolerated with 70% of patients developing a grade 1/2 rash and 10% developing grade 3 rash. Response rate and disease control rate were 6.5% and 41.1% respectively. Median overall survival was 7.7 months."

Lung_Cancer

"materials and marking with radio-opaque materials. Examples of directly visible materials are hook wire methylene blue and indocyanine green. Ethiodized oil (lipiodol) barium and iodine contrast agents are used for radio-opaque markers. Each marking method has strong and weak points. Localization with a hook wire is easy to perform but carries a high risk of pneumothorax and a propensity to dislodge during transport and surgical preparation (6 7). Methylene blue and indigo carmine have a tendency to diffuse over a large area by the time the operation is done and render localization features inadequate (8 9). The use of a radio-opaque marker (such as barium or lipiodol) requires an intraoperative fluoroscopy to confirm an adequate excision as well as lead to increased radiation exposure (10-13). The use of mixture has been reported to make up for the weakness of marking materials. For example the problem of dye diffusion has led to attempts to use a mixture of dye with various materials such as cyanoacrylate adhesive or collagen or autologous blood (14-16). However they have not been widely used for localization due to difficulties in making and manipulation. Lipiodol and methylene blue are commonly used materials for localization (17-20). We hypothesized that lipiodol reduces the spread of methylene blue and provides additional localization opportunities by its radio-opacity. The use of a mixture of lipidol and methylene blue (MLM) for a percutaneous injection material requires a high success rate for appropriate localization and a low complication rate. To our knowledge there have been no reports that evaluate the availability of MLM as a percutaneous injection material in human lungs. This study compared MLM with methylene blue as a percutaneous injection material for pulmonary localization in rabbit lungs. MATERIALS AND METHODS Animal preparation This study was performed after approval by the Institutional Animal Care and Use Committee (IACUC) in Seoul National University Hospital biomedical research institute (IACUC approval No. 11-0356). Twenty-four adult New Zealand White rabbits were used. We recorded their weight before the procedures. The animals were randomly divided into two groups: Group A (n=12) and Group B (n=12) each sacrificed at about 6 hr and 24 hr after percutaneous injections respectively (Fig. 1). Six hours after percutaneous injections were same day operations of the preoperative localization; and 24 hr after percutaneous injections were next day operations of the preoperative localization. The injection of each material was done in all 24 subjects because we injected methylene blue and MLM at two different lung sites for each subject. Percutaneous injection materials: mixture of lipiodol and methylene blue versus methylene blue A pilot study was performed to decide the optimal amount of materials for percutaneous injections. Methylene blue (1% 100 mg/mL TERA Pharmaceuticals Buena Park CA USA) of 0.3 to 0.9 mL was used for human lung localization in previous studies by Wicky et al. (18) and Vandoni et al. (19). In the pilot study with rabbit lungs we injected 0.1 mL and 0.05 mL of methylene blue and MLM in four subjects. We found that staining was extensive (more than half height of one lobe) with 0.1 mL and localized (about 1 cm of staining diameter) with 0.05 mL for both methylene blue and MLM. Extensive dispersion made it difficult to find exact injecting sites; subsequently 0.05 mL of methylene blue was administered. We made variable mix ratios of lipiodol and methylene blue in vitro; 1:1 1:2 1:3 1:4 and 1:5 in order to find an appropriate mixing ratio of lipiodol (480 mg Iodine/mL Andre Guerbet Aulnay-sous-Bois France) and methylene blue. The separation of two materials occurred instantly after mechanical blending to the fat-soluble character of lipiodol and the water-soluble character of methylene blue. A higher concentration of lipiodol in MLM resulted in increased uneven blending and rapid separation. A mixture with a 1:6 (or lower) mixing ratio contained a minimal amount of lipiodol and it might make it difficult to be detected on the fluoroscopy; subsequently we decided that 1:5 was an appropriate mixing ratio for injection. A total of 0.06 mL of MLM (0.01 mL of lipiodol plus 0.05 mL of methylene blue) was administrated in each subject to avoid the effect of different volumes of methylene blue to the diffusion extent of the materials. CT guided percutaneous injections Percutaneous injection was performed with computed tomography (CT) guidance (Discovery CT750 HD; GE Healthcare Waukesha WI USA). We performed pre-procedural CT scans in order to determine an appropriate skin entry site for the successful placement of a needle in the desired location. The desired location was the basal portion of both caudal lobes around the mid-scapula line. We tried to situate the needle tip at 5 mm depth from the visceral pleura and avoid passing through the pulmonary vessels. We placed the needle of 20 gauze and 3.5 cm length in the lung parenchyma after marking the appropriate skin entry site. The parameters of CT used in our study were: tube voltage of 120 kV tube current of 25 mA slice thickness of 2 mm thickness and gantry rotation speed of 350 milliseconds. We connected 1 mL syringe to the needle hub and retracted the syringe piston to confirm that no blood was aspirated after the needle tip was accurately located within the desired location. We then injected the materials and immediately removed the needle. On the procedural CT scan we measured the distance from the skin-entry to the needle tip and the depth from visceral pleura to the needle tip. A post-procedural CT scan identified procedure-related complications that included the leakage of injecting materials and pneumothorax; in addition we recorded the extent shape and density of radio-opacity of MLM after injection. The extent of MLM was defined as a maximum diameter of the radio-opacities. The shape of radio-opacity was categorized into 3 groups (small faint nodular scattered nodular and discrete compact nodular). We recorded the injection time to measure the time interval between injection and sacrifice. Fluoroscopic examinations A successful localization of lipiodol was determined by fluoroscopic examination; subsequently we evaluated the radio-opacity of MLM using the fluoroscopy X-ray unit (BV Pulsera; Philips Medical Systems Best The Netherlands) at the immediate post-procedure session and the follow up session at 6 hr in Group A and 24 hr in Group B. The parameters of fluoroscopy were: tube voltage of 59 kV and tube current of 946 mA. We obtained anteroposterior fluoroscopic images of the thorax of the rabbit with a 17 cm of field of view. A radio-opaque ruler of 5 cm was located near the rabbit in order to estimate the exact size of lipiodol opacity. We recorded the time of the fluoroscopic examinations and the radiographic findings of MLM (size and shape of the radio-opacity). Evaluation of the staining and radio-opacity We assessed the directly visible staining on the freshly excised lung surface and radio-opacity of MLM on the fluoroscopic examinations using 4-point scoring in order to compare the localization ability of MLM and methylene blue as a percutaneous injection material. A blind reviewer who was unaware of the injection materials assessed the staining ability. In order to evaluate the staining ability the blind reader reviewed the photographic images of the freshly excised lung specimens obtained before formalin fixations and rated the staining by 4-point scores: 0=non-visualization of staining 1=inappropriate; extensive dispersion made it difficult to find accurate injecting locations 2=acceptable; available to estimate injecting locations in spite of the dispersion and 3=excellent definitely localized staining (Fig. 2). The maximum diameter of the staining extent on the lung surface was measured. We calculated and compared scores and extent of staining between two materials. For the fluoroscopic findings the radio-opacity of MLM was evaluated using 4-point scoring: 0=no detectable radio-opacity 1=inappropriate minimally increased opacity 2=acceptable low density of increased opacity 3=excellent compact nodular increased opacity (Fig. 3). We compared the average scores of initial and follow up fluoroscopic examinations. We considered a score of 0 or 1 as inappropriate and a score of 2 or 3 as appropriate for localization for both staining and radio-opacity. We compared the number of appropriate or excellent localization between MLM and methylene blue. Sacrifice and histopathologic examinations Both freshly excised entire lungs were used as final specimens. The lung tissues were fixed in 10% neutral formalin embedded in paraffin and cut into 5 µm thick slices after we took photographs to record staining on the lung surface. We made 4 axial slices that covered the center of the staining. The slices were subjected to hematoxylin-eosin (H-E) stain to the evaluate lung parenchymal change. We evaluated the presence or absence of neutrophil infiltration vasculitis necrosis hemorrhage and foam cell in alveolus. The extent of each histopathologic finding was estimated using visual grading scores as 0 (no) 1 (focal) or 2 (diffuse). Localized parenchymal change (<50% of total area) surrounded by normal lung was defined as focal. Extensive lung parenchymal change (?50% of total area) that replaced normal lung was defined as diffuse. An experienced pathologist with eight years of experience reviewed all slices. The overall severity of the lung parenchymal change was defined as a total score by adding visual grading scores for each histopathologic finding. We compared the overall severity score between MLM and methylene blue as well as between Group A and Group B. Statistical analysis All data are expressed as mean±standard deviation (SD) unless otherwise stated. Comparisons of the average scores were performed by two-tailed unpaired Student's t-test or Mann-Whitney test. We used a Fisher's exact test to compare the number of subjects in the subgroups. Linear by linear association evaluated the association of the extent of lung parenchymal change and materials or groups. Null hypotheses of no difference were rejected if the P values were less than 0.05. The statistical analysis was performed with commercially available statistical software IBM SPSS Statistics version 20.0 (IBM Corp. in Armonk NY USA). RESULTS Subject characteristics procedural records time interval of injection and examinations Among the 24 subjects included in our study successful CT-guided percutaneous injections into the desired location of the lung were achieved in 21 subjects (11 in Group A and 10 in Group B). Three subjects died during anesthesia. Mean weight was 3.2±0.2 kg for Group A and 3.3±0.2 kg for Group B. Injection depth from visceral pleura to needle tip was 0.4±0.1 cm (range: 0.3-0.6 cm) for MLM and 0.4±0.1 cm (range: 0.3-0.7 cm) for methylene blue (P=0.43). Distance from skin to needle tip was 2.8±0.6 cm (range: 2.1-5.0 cm) for MLM and 2.8±0.3 cm (range: 2.2-3.5 cm) for methylene blue (P=0.83). Of 42 CT-guided percutaneous injections total number of procedure related complications was 10 (24%) including 7 leakage (all in MLM) and 3 pneumothorax (2 in MLM 1 in methylene blue). The complication rate in MLM was significantly higher than methylene blue (43% vs 5%) (P=0.004). On post-procedural CT images the extent of the radio-opacity of MLM was 1.3±0.4 cm (range: 0.7-2.0 cm) for Group A and 0.6±0.3 cm (range: 0.3-1.1 cm) for Group B. Discrete compact nodular opacity was achieved in 15 subjects (72%) scattered nodular opacities in 3 (14%) and small faint opacity in 3 (14%) (Fig. 4). The average value of radio-opacity of MLM was 1415±856 HU (range: 307-2768 HU). The interval between injection and sacrifice was 7.9±0.1 hr (range: 7.8-8.0 hr) for Group A and 23.5±0.1 hr (range: 23.4-23.7 hr) for Group B. Time from injection to initial and follow up fluoroscopy was 3.4±0.5 hr (range: 2.5-4.2 hr) and 6.8±0.4 hr (range: 6.3-7.7 hr) for Group A and 1.5±0.4 hr (range: 0.9-2.1 hr) and 22.6±0.4 hr (range: 21.9-23.2 hr) for Group B respectively. Scores and extent of staining and radio-opacity Table 1 demonstrates the staining extent and localization ability of MLM and methylene blue. In total groups the staining extent of MLM was significant smaller than methylene blue (0.6 cm vs 1.0 cm P<0.001). MLM showed a significantly higher staining ability score than methylene blue (2.8 vs 2.2 P=0.010). Radio-opacity in the initial fluoroscopy was not significantly different from the follow up (2.0 vs 1.9 P=0.49). Table 2 showed the number of subjects in each score of localization ability of staining or radio-opacity. In Group A appropriate staining was 100% for both MLM and methylene blue. In Group B appropriate staining was 90% for MLM and 70% for methylene blue. Appropriate staining of MLM was not significantly different from that of methylene blue (95% vs 86% P=0.61); however excellent staining in MLM was significantly higher than methylene blue (81% vs 38% P=0.011) (Table 3). Table 4 shows the localization ability of MLM regarding both staining ability and radio-opacity. There was no subject with a score of 0 or 1 in both radio-opacity and staining. MLM achieved appropriate staining or radio-opacity in 21 subjects (100%) with a dual localization feature. Histopathologic findings Table 5 demonstrates the results of the histopathologic findings. In all lung specimens both methylene blue and MLM showed acute lung parenchymal change that included neutrophil infiltration hemorrhage and foam cell in alveolus (Fig. 4). Comparing the two materials the number of specimen having neutrophil infiltration vasculitis necrosis hemorrhage and foam cell in alveolus was similar in each extent. In terms of all features the number of specimen that showed diffuse extent was more in Group B than Group A for both MLM and methylene blue. The extent of the histopathologic findings was not significantly associated with the materials for all histopathologic features (Table 5). Among the histopathologic findings the extent of vasculitis was significantly associated with Group for both MLM and methylene blue (P=0.002 for both MLM and methylene blue). Focal or diffuse extent of vasculitis was more frequently found in Group A than Group B (P=0.001 for both MLM and methylene blue). The overall severity of lung parenchymal change was not different between MLM and methylene blue (5.6±1.6 vs 5.7±1.5 P=0.839); in addition Group B showed a significantly higher overall severity score of lung parenchymal change than Group A (6.6±1.6 vs 4.7±0.9 P=0.005). DISCUSSION The results of this study show that MLM is a useful percutaneous injection material for a successful localization in the lung. The average staining score of MLM was significantly higher than methylene blue (2.8±0.5 vs 2.2±0.7 P=0.010). In terms of staining the appropriate localization rate (acceptable or excellent staining) in our study was 95% using MLM. The result was in close agreement with previous studies that showed a high success performance rate of lipiodol localization (99%-100%) (21-23). An appropriate localization rate (acceptable or excellent staining) of methylene blue injection was 86% in our study. This is lower than the results found in previous studies where the success rate of methylene blue injection was 96%-100% (18 20). We found that an acceptable (or excellent staining rate) of MLM and methylene blue was not significantly different (95% vs 86% P=0.610). However MLM showed excellent staining for localization in 17 (81%) of 21 subjects and was significantly higher than methylene blue (38%) (P=0.011). The results indicate that lipiodol reduced the spread of methylene blue. This is the first study to indicate that MLM is an available percutaneous injection material for localization with superior staining ability compared to methylene blue. The complication rate was 43% in MLM and 5% in the methylene blue (P=0.004). Possible complications after percutaneous injection for pulmonary localization include pneumothorax leakage hemorrhage pain hemoptysis hemothorax and embolism. Previous studies reported that the complication rate was 17-29% for lipiodol and 33% for methylene blue (2023 24). The complication rate of MLM in the current study was higher than the results of previous studies mainly due to the leakage of MLM into the pleural cavity (n=9). This difference was probably because the distance from the pleura to the injecting needle tip (0.4±0.1 cm for MLM) was inadequate to avoid leakage into the pleural cavity. In the previous studies of lipiodol marking for localization the mean distance from the pleura to the target nodule was 1.0-1.9 cm (22-24) more than twice our study. The results indicate that the high complication rate of our study is associated with the inserting procedure of the needle rather than MLM itself. The dispersion of methylene blue throughout the lung parenchyma may lead to unnecessarily large wedge resections; in addition some have reported instances of the dispersion of methylene blue throughout the entire pleural surface or intraoperative identification failure due to severe anthracosis of the visceral pleura. The failure rate was reported to be 0%-13% with the use of methylene blue (1819 25). The results are similar to our study and indicate that inappropriate staining on the lung surface was 14% in methylene blue. In this study we found that the dispersion of methylene blue in MLM through the lung parenchyma was significantly smaller than methylene blue (0.6±0.3 cm vs 1.0±0.4 cm P<0.05). The result implies that lipiodol reduces the spread of methylene blue in lung parenchyma. Regarding the score of radio-opacity 38% of MLM showed non-visualization or minimally increased opacity on the fluoroscopic examinations. It means the proportion of lipiodol in MLM at the time of the percutaneous injection was too small to be detected. Post-procedural CT images also revealed that 3 subjects had small faint radio-opacity after the injection of MLM. It suggests that the uneven blending of lipiodol and methylene blue occurred during the preparation of MLM. Water-insolubility of lipiodol would result in the uneven mixing of water soluble methylene blue after mechanical blending of the two materials. Further research is required to reduce non-homogeneity of MLM at the time of injection. Previous studies reported the availability of a mixture of methylene blue with other materials such as collagen or autologous blood (15 16). They performed VATS resection on the same day as localization. In our study we evaluated the localization ability of MLM on the same day of localization (6 hr) as well as 24 hr after injection. Localization is usually performed on the day of surgery. This requires the simultaneous use of the CT and the operating room which is not always available. Surgeries on the next day of localization were reported in several published articles (26 27). MLM shows a prolonged localization ability of up to 24 hr in terms of staining ability and radio-opacity. Stable localization ability is the advantage of MLM in our study. Due to uneven blending of MLM one subject (10%) showed inappropriate staining and appropriate radio-opacity and required an intraoperative fluoroscopic examination to detect MLM. Possible radiation exposure is a drawback of MLM. We would like to justify the use of intraoperative fluoroscopy because the operator can avoid radiation exposure with a lead apron. In regards to the risk-benefit for patients lowering the risk of detection failure is thought to be more important than radiation exposure. Histopathologic examinations showed lung parenchymal changes in all specimens. Both methylene blue and MLM induced acute lung injury that included neutrophil infiltration vasculitis necrosis hemorrhage and foam cell in alveolus (Table 5). The results of our study are similar to those of a previous study by Kwon et al. (28) that showed that lipiodol led to acute lung injury. They described that lipiodol creates the histopathologic feature of acute lung injury such as peripheral endothelial cell damage neutrophil infiltration necrosis hemorrhage alveolar wall destruction vasculitis emboli (or thrombi in arteriole) and macrophages in the alveolar space (28). In our results the extent of lung parenchymal change was not associated with the materials for all histopathologic features. In addition the overall severity score of lung parenchymal change in MLM was not different from methylene blue (5.6 and 5.7 P=0.839). This suggests that MLM shows similar histopathologic effects in the lung parenchyma to methylene blue. The overall severity score of parenchymal change was higher in Group B (follow up interval of 24 hr) than Group A (follow up interval of 6 hr) (6.6 vs 4.7 P=0.005). The extent of lung parenchymal change depends on the time interval. Acute lung injury after the percutaneous injection of lipiodol or methylene blue was reported in animal studies (28 29); however there are no clinical results that show the adverse effect of acute lung injury in human lungs. Injection material (such as barium) can potentially complicate the pathologic diagnosis of the target lesion due to acute inflammation (29 30). To our knowledge no study has indicated that lipiodol or methylene blue hinders the histopathologic diagnosis of target lesions in human lungs. The small amount of material injection in human lungs might not create a significant parenchymal change or disrupt underlying lung disease. It is necessary to avoid directly injecting materials into the target lesion in human lungs in order to avoid the adverse effect of injection materials on underlying lung disease (especially ground glass opacity nodule or potential benign lesion). There were several limitations in our study. First we included only a small number of subjects. Second the overall localization success rate was low and the complication rate was high (compared to the results of previous studies) due to the difficulty in an accurate percutaneous injection at the desired location and depth in the small sized rabbit lung. Third we used a 1 mL syringe with manual administration to inject materials in the lung parenchyma and there were possible individual difference in the administering volume of materials. Fourth we could not evaluate complications such as intractable pain material related anaphylaxis or embolism. Fifth we could not evaluate if the histopathologic changes had any effect on underlying lung disease because the lung parenchyma of the experimental rabbits were normal. Finally we did not evaluate a successful localization for the true target lesion in lung parenchyma. The criteria for appropriate staining and radio-opacity were subjective. We expect that further clinical studies might provide an answer to if MLM can be a useful percutaneous injection material for localization in the human lung. In conclusion MLM is available for percutaneous injection for the pulmonary localization. The results of this study showed that MLM provides superior ability for appropriate localization than that of methylene blue. Further research on human lungs can clarify the availability of MLM as a CT guided percutaneous injection material. This study was supported by grant from the Seoul National University College of Medicine Research Fund 2012 (800-20120036). We have no potential conflicts of interest or commercial "

Lung_Cancer

"IGFBP3 is known to block IGF-1 induced activation of IGF-1R1617. Overall these studies show that the IGF-1R signaling pathway is activated by multiple mechanisms in H3122 XR cells. We evaluated the effects of IGF-1R inhibition in ALK TKI resistant cells. The combination of X-376 with either MAb391 or OSI-906 (Fig. 4de) partially restored X-376 sensitivity in H3122 XR cells. Apoptosis was also enhanced in H3122 XR cells treated with X-376 and the IGF-1R TKI AEW-54118 (Fig. 4f). In accord with these data combination treatment with ALK and IGF-1R inhibitors in H3122 XR cells inhibited AKT phosphorylation to a greater extent than either inhibitor alone (Fig. 4g). The addition of OSI-906 also partially restored the sensitivity of H3122 CR cells to the growth inhibitory effects of crizotinib (Supplementary Fig. 4c). Since IRS-1 levels were increased in H3122 XR cells (Fig. 4c) we examined whether IRS-1 knock-down would also affect signaling and proliferation in the resistant cells (Fig. 4hi). We transfected H3122 XR cells with IRS-1 or control siRNAs and then treated cells with X-376. IRS-1 knockdown sensitized these cells to the anti-proliferative effects of ALK inhibition (Fig. 4h) and resulted in a further decline in phosphorylation of downstream targets compared to X-376 alone or IRS-1 knockdown alone (Fig. 4i). Previous studies have suggested that the IGF-1R pathway can drive EGFR inhibitor resistance1920. We tested the efficacy of combined EGFR/IGF-1R inhibition in 4 different isogenic pairs of EGFR TKI- sensitive and -resistant cell lines2122 (Supplementary ). The addition of the IGF-1R TKI OSI-906 was not synergistic with the EGFR TKI erlotinib in any of these cells (Supplementary Fig. 5a“d). Furthermore in contrast to the ALK TKI-resistant cells there was no increase in IGF-1R or IRS-1 in the EGFR TKI-resistant cell lines (Supplementary Fig. 5e). These data suggest that the effects seen with the ALK/IGF-1R inhibitor combinations in the ALK cell lines are true differences. Increased IGF-1R in tumor samples at the time of resistance To validate the clinical implications of our in vitro findings we evaluated phospho-IGF-1R (pIGF-1R) and IRS-1 levels in patient tumor biopsy samples. Three sets of paired pre-/post-crizotinib tumor samples as well as two post-crizotinib tumor samples from five different patients were examined by immunohistochemistry (IHC) for pIGF-1R and blindly evaluated by pathologists. As a control we also performed pIGF-1R IHC on lung cancer tissue microarrays (TMAs); representative examples are shown in Supplementary Fig. 6a“c. Four of five tumor biopsies taken at the time of acquired resistance displayed increased levels of pIGF-1R (Fig. 5a patients 1“4). For two of the paired tumor samples we had sufficient tissue to examine IRS-1 levels by IHC (Fig. 5b); one post-treatment sample (patient 2) had increased IRS-1 expression. These five samples were also assessed for ALK kinase domain mutations associated with crizotinib resistance (Supplementary ). Patient 4's post-crizotinib tumor harbored an ALK G1202R mutation. As an orthogonal approach we performed mRNA expression analysis for IGF-1R and IRS-1 using Nanostring23 on matched patient samples and on isogenic pairs of ALK TKI-sensitive and resistant cell lines. In the one case with enough pre- and post-treatment tissue for available for analysis IGF-1R and IRS-1 (Fig. 5cd) mRNA levels were increased in the post-crizotinib relative to the pre-crizotinib tumor sample. Similar results were obtained with the cell lines. In contrast Nanostring analysis of 11 matched pairs of EGFR mutant lung tumor biopsies revealed no significant change in IRS-1 levels post EGFR TKI therapy (Supplementary Fig. 6d“f) suggesting that the changes observed in IRS-1 were specific to ALK+ lung cancer. Overall these data validate our pre-clinical findings showing that levels of IGF-1R and IRS-1 can be increased post ALK TKI therapy in patient-derived samples. LDK-378 inhibits phosphorylation of ALK and IGF-1R in vitro The second-generation ALK TKI LDK-378 (ceritinib) has demonstrated a 56% ORR in patients with ALK+ lung cancer who have progressed on crizotinib24. Yet only a minority of patients had ALK alterations suggesting the possibility of alternative ˜bypass™ mechanisms which are sensitive to LDK-378. Interestingly LDK-378 and the structurally related ALK inhibitor TAE-684 can inhibit both ALK and IGF-1R in vitro25. We hypothesized that the efficacy of LDK-378 may be due to this drug's ability to simultaneously block both ALK and IGF-1R. LDK-378 was more potent than crizotinib in H3122 (Fig. 6a) STE-1 (Supplementary Fig. 7a) and H3122 XR cells (Supplementary Fig. 7b). LDK-378 was also more potent at inducing apoptosis in H3122 cells (Supplementary Fig. 7c). Furthermore LDK-378 was significantly more effective at delaying the growth of H3122 xenografts compared to the equivalent dose crizotinib (Fig. 6b). Next we tested LDK-378's ability to inhibit IGF-1R phosphorylation. H3122 (Fig. 6c) and H2228 (Fig. 6d) cells were treated with LDK-378 alone or in combination with IGF-1. Importantly LDK-378 treatment inhibited ALK phosphorylation and was also able to overcome the IGF-1 ligand induced increase in IGF-1R phosphorylation in both ALK+ cell lines (Figures 6cd compare lanes 4 and 6). These data suggest that LDK-378's potency in vivo may be due to this agent's combined ability to block both ALK and IGF-1R. Discussion We report that ALK and IGF-1R inhibitors have cooperative anti-proliferative effects. IGF-1R inhibitors sensitized tumor cells to the effects of ALK inhibition. The therapeutic synergism between ALK and IGF-1R inhibitors was observed in both the ALK TKI-sensitive and ALK TKI-resistant settings. Chronic ALK inhibition was associated with enhanced IGF-1R signaling. The ALK TKI resistant cells utilized numerous mechanisms to activate IGF-1R signaling. Importantly the addition of an IGF-1R inhibitor sensitized the resistant cells to the effects of ALK blockade. We propose drug combinations co-targeting ALK and IGF-1R as a novel therapeutic approach in patients with ALK+ lung cancer. This rationally selected combination of targeted therapies should be effective in both the ALK TKI-na¯ve and TKI-resistant setting. Our data may also in part explain the surprising 56% ORR for the ˜second generation™ ALK TKI LDK-378 in patients with ALK+ lung cancer who had progressed on crizotinib24. Since responses to LDK-378 were observed in both patients with and without ˜second-site™ ALK mutations the increased ˜on-target™ potency of LDK-378 towards ALK is alone not enough to explain all of the responses seen to this agent. We hypothesize that the potency of this agent is due to its ability to simultaneously inhibit both ALK and IGF-1R and our in vitro experiments confirm that LDK-378 does inhibit phosphorylation of both ALK and IGF-1R. Further studies will be necessary to validate this hypothesis in clinical samples. Mechanisms of acquired resistance to ALK inhibitors are just beginning to be understood. Target alterations have only been found in a minority of resistant tumors examined to date. ˜Bypass™ signaling has also been reported3-6. Interestingly target alterations and bypass signaling do not appear to be mutually exclusive."

Lung_Cancer

"were a fraction of those needed for the photon plans (). This translates to a beam on time per field of between 5 and 10 seconds for the PT-SABR plans compared to 75 to 90 seconds for photon plan. This 5 to 10 second time estimate is based on a conservative 1 nC/sec dose rate however new proton centers may be able to achieve greater than 2 nC/sec thereby reducing this time by a factor of 2. By decreasing the treatment time to less than 10 seconds per field breath-hold techniques may be better tolerated in greater number of lung cancer patients with suboptimal lung function. Breath-hold technique would minimize tumor motion (i.e. ITV) leading to a smaller overall irradiation volume and interplay would not be a significant issue [8]. Spot scanning proton therapy that utilizes the Bragg peak would require a larger planning volume due to the various uncertainties that need to be taken into account; and it would require a longer treatment time due to the use of multiple proton energies. Each change in energy requires several seconds (2 to 7) and at least 5 to 10 energies would be required for these treatments. Volumetric modulated arc therapy (VMAT) with photons may decrease treatment times compared to multiple static-gantry beams. However VMAT comes at the cost of larger volumes of normal tissue receiving low doses of radiation since the beam is continuously on as it rotates about the patient. The use of four to five proton transmission beams achieves both shorter treatment times as well as a lower integral dose to the body. The dosimetric data of the normal tissues in the photons plans met the constraints of RTOG 0915. The dosimetric gains of protons over these plans may be considered modest and the statistical analysis comparing plans is limited by the small sample size. However in some plans the dose to particular critical ans can be avoided completely without compromising target coverage by choosing beam arrangements appropriately. This may be beneficial in treating patients with tumors in challenging locations [9] or recurrent tumors that have had prior radiotherapy. The interim analysis of RTOG 0617 reported local failure rates of 25% and 34% in the standard and high dose RT arms [10] and therefore re-irradiation may play a role in this subset of patients who fail after definitive chemoradiotherapy. For these patients keeping dose at or near zero to the spinal cord heart lungs or other critical structures is feasible with protons. Planning with PT-SABR using only transmission beams without the Bragg peak is feasible. This proof of principle as described in our study eliminates the uncertainty of proton dose distribution in lung tumors which has the potential to underdose the target and overdose surrounding normal tissues. Proton therapy planning with this technique also demonstrates better sparing of normal tissues and fast treatment times than photon plans. Further study of this novel approach to proton SABR is warranted. The authors thank Katy Nelson for maintaining the SABR database. References 1 GeD HillbrandM StockM DieckmannK PotterR (2008) Can protons improve SBRT for lung lesions? Dosimetric considerations. Radiotherapy and oncology: journal of the European Society for Therapeutic Radiology and Oncology88: 368“37518405986 2 HoppeBS HuhS FlampouriS NicholsRC OliverKR et al (2010) Double-scattered proton-based stereotactic body radiotherapy for stage I lung cancer: a dosimetric comparison with photon-based stereotactic body radiotherapy. Radiotherapy and oncology: journal of the European Society for Therapeutic Radiology and Oncology97: 425“43020934768 3 MacdonaldOK KruseJJ MillerJM GarcesYI BrownPD et al (2009) Proton beam radiotherapy versus three-dimensional conformal stereotactic body radiotherapy in primary peripheral early-stage non-small-cell lung carcinoma: a comparative dosimetric analysis. International journal of radiation oncology biology physics75: 950“958 4 WestoverKD SecoJ AdamsJA LanutiM ChoiNC et al (2012) Proton SBRT for medically inoperable stage I NSCLC. Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer7: 1021“1025 5 PaganettiH (2012) Range uncertainties in proton therapy and the role of Monte Carlo simulations. Physics in medicine and biology57: R99“11722571913 6 SecoJ PanahandehHR WestoverK AdamsJ WillersH (2012) Treatment of non-small cell lung cancer patients with proton beam-based stereotactic body radiotherapy: dosimetric comparison with photon plans highlights importance of range uncertainty. International journal of radiation oncology biology physics83: 354“361 7 VideticGM HuC SinghA ChangJY ParkerW et al (2013) Radiation Therapy Oncology Group (RTOG) Protocol 0915: A Randomized Phase 2 Study Comparing 2 Stereotactic Body Radiation Therapy (SBRT) Schedules for Medically Inoperable Patients With Stage I Peripheral Non-Small Cell Lung Cancer. International journal of radiation oncology biology physics87: S3 8 KeallPJ MagerasGS BalterJM EmeryRS ForsterKM et al (2006) The management of respiratory motion in radiation oncology report of AAPM Task Group 76. Medical physics33: 3874“390017089851 9 RegisterSP ZhangX MohanR ChangJY (2011) Proton stereotactic body radiation therapy for clinically challenging cases of centrally and superiorly located stage I non-small-cell lung cancer. International journal of radiation oncology biology physics80: 1015“1022 10 BradleyJD PaulusR KomakiR MastersGA ForsterK et al (2013) A randomized phase III comparison of standard-dose (60 Gy) versus high-dose (74 Gy) conformal chemoradiotherapy with or without cetuximab for stage III non-small cell lung cancer: Results on radiation dose in RTOG 0617. Journal of Clinical Oncology31: 7501 Cancer Cancer cncr Cancer 0008-543X 1097-0142 BlackWell Publishing Ltd Oxford UK 24752945 4140446 10.1002/cncr.28714 Original s A phase 2 cooperative group adjuvant trial using a biomarker-based decision algorithm in patients with stage I non-small cell lung cancer (SWOG-0720 NCT00792701) Bepler Gerold MD PhD 1 Zinner Ralph G MD 2 Moon James MS 3 Calhoun Royce MD 4 Kernstine Kemp MD 5 Williams Charles C MD 6 Mack Philip C PhD 4 Oliveira Vasco PhD 1 Zheng Zhong MD PhD 6 Stella Philip J MD 7 Redman Mary W PhD 2 Gandara David R MD 4 1 Karmanos Cancer Institute Detroit Michigan 2 The University of Texas MD Anderson Cancer Center Houston Texas 3 SWOG Statistical Center Seattle Washington 4 University of California at Davis Sacramento California 5 City of Hope Duarte California 6 H. Lee Moffitt Cancer Center Tampa Florida 7 Michigan Cancer Research Consortium Community Clinical Oncology Program Ann Arbor Michigan Corresponding author: Gerold Bepler MD PhD Karmanos Cancer Institute 4100 John R Detroit MI 48201; Fax: (313) 576-8628; beplergkarmanos. 01 8 2014 18 4 2014 120 15 2343 2351 10 2 2014 17 3 2014 18 3 2014 2014 The Authors. Cancer published by Wiley Periodicals Inc. on behalf of American Cancer Society 2014 This is an open access under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License which permits use and distribution in any medium provided the original work is properly cited the use is non-commercial and no modifications or adaptations are made. BACKGROUND This cooperative group adjuvant phase 2 trial in patients with completely resected stage I non-small cell lung cancer with tumor diameters measuring ??2 cm was designed to assess the feasibility and preliminary efficacy of assigning patients to therapy or observation using a molecularly based decision algorithm. METHODS At least a lobectomy and sampling of recommended mediastinal lymph node stations good Zubrod performance status adequate an function and a formalin-fixed and paraffin-embedded tumor specimen were required. Excision repair cross-complementing group 1 (ERCC1) and ribonucleotide reductase M1 (RRM1) were analyzed using immunofluorescence-based in situ automated quantitative image analysis and categorized as high or low using prespecified cutoff values. Patients with high ERCC1 and RRM1 were assigned to observation and all others to 4 cycles of cisplatin and gemcitabine. Feasibility was defined as treatment assignment within 84 days from surgery in >?85% of patients. Secondary objectives were to estimate the 2-year survival. RESULTS Treatment assignment met the feasibility criteria in 88% of eligible patients (71 of 81 patients). The collective 2-year disease-free and overall survival rates were 80% and 96% respectively. Protein levels for RRM1 fell within the previously established range ERCC1 levels were slightly lower than expected and they were significantly correlated (correlation coefficient 0.4). "

Lung_Cancer

"BronchioalveolarCarcinoma 1 (0.6) 2 (3.8) 5 (1.7) 16 (3.3) Other* 15 (8.7) 4 (7.5) 32 (10.6) 66 (13.5) *Other includes subjects with no information available. LDT ?=? laboratory-developed test; MD ?=? mutation detected; MND ?=? mutation not detected. SLCG inconclusive (n?=?27) data not shown. Statistical considerations Mutation Detected (MD) was defined as the presence of either an exon 19 deletion or L858R mutation. Mutation Not Detected (MND) was defined as the absence of both exon 19 deletions and the L858R mutation. SAS/STAT® software was used for all data analysis. Clinical outcome study statistics Kaplan-Meier survival curves were used to assess the PFS by treatment method (chemotherapy or erlotinib) among patients who were enrolled in the EURTAC trial and screened with the LDT as well as the subset of patients who were determined to be mutation-positive by the EGFR PCR test. Nonparametric log-rank test was performed to assess PFS between patients who were randomized to chemotherapy or erlotinib. The hazard ratio (chemotherapy vs. erlotinib) relative to PFS was also calculated. Best overall response was the best response recorded from the start of treatment until disease progression and BORR (Best overall response rate) was summarized with 95% confidence limits according to Pearson-Clopper methods based on investigators assessment for each treatment arm. Analytical performance statistics For analytical performance an agreement analysis was performed between the EGFR PCR test result and the LDT test. Mutation detection of exon 19 deletions and L858R mutations were analyzed in aggregate. Separately the EGFR PCR test was also compared to Sanger sequencing and MPP by a CLIA-certified laboratory. For the agreement analyses the positive percent agreement (PPA) negative percent agreement (NPA) and overall percent agreement (OPA) with their corresponding 95% confidence intervals (CIs) were calculated. In addition 3-way analyses using MPP as a second reference method was performed to resolve the discrepancy results. Mutation testing methods EGFR PCR Test The EGFR PCR test (cobas EGFR Mutation Test Roche Molecular Systems Inc Branchburg NJ USA) is a CE-IVD marked multiplex allele-specific PCR-based assay designed to detect 41 mutations in exons 181920 and 21 in FFPET specimens of human NSCLC.[28] DNA is isolated using the cobas DNA Sample Preparation Kit (Roche Molecular Systems Branchburg NJ). [29] A minimum of 150 ng of genomic DNA is required for PCR amplification which can typically be isolated from a single 5 µm FFPET section. The EGFR PCR test software version used in this study was designed to detect 29 deletions in exon 19 and 2 L858R variants in exon 21. Macrodissection is only recommended if tumor content is less than 10%; laser capture microdissection is not required. The EGFR PCR test was performed per manufacturer's package insert and results were automatically analyzed and reported. The limit of detection has been validated to 5% mutant alleles. The workflow from DNA isolation to results reporting can be performed in one 8 hour period.[27] LDT Patients in the EURTAC study were screened using a combination of methods developed by Laboratory of Oncology ICO-Hospital Germans Trias i Pujol Barcelona Spain.[11] In short EGFR activating mutations in exons 19 and 21 were initially identified by Sanger sequencing and confirmed by fragment length analysis for exon 19 deletions (FAM-labelled primer in an ABI prism 3130 DNA analyser (Applied Biosystems Foster City CA USA) and by Taqman assay for exon 21 (L858R) mutation. All tumor specimens were from the original biopsy taken prior to any treatment and before randomization. Testing was performed on ? 2mm2 of tissue obtained from one to three slides of 4-micron tissue sections which were subjected to laser capture microdissection to enrich for the presence of tumor cells. DNA was extracted using a standard laboratory protocol and tested at a single site in Spain in Laboratory of Oncology for EGFR activating mutations in exon 19 and 21 using a previously described method. The average turnaround time was approximately 5 days.[26] Bi-directional Sanger sequencing All samples tested by the EGFR PCR test were also tested by Sanger sequencing using DNA from FFPET specimens prepared by the cobas DNA Sample Preparation Kit and sequenced with 2— bidirectional Sanger sequencing by a CLIA-certified laboratory (SeqWright Houston TX USA) using a validated protocol. Repeat Sanger sequencing was performed to compare the detection of EGFR mutations from adjacent sections of tissue to minimize any impact of tissue heterogeneity used for the EGFR PCR test relative to the original LDT results. Also sequencing protocols vary by laboratory in terms of the percent tumor content/sample that requires macrodissection. DNA isolated with the cobas DNA Sample Preparation Kit and used for sequencing required ?10% tumor content. Average turnaround time to results was 7 days. The estimated limit of detection is approximately 20% mutant alleles.[30] Massively parallel pyrosequencing (MPP) Samples with valid EGFR PCR test results with adequate DNA remaining from the initial extraction were tested by a MPP method (454 GS Titanium 454 Life Sciences Branford CT USA) by a CLIA-certified laboratory (SeqWright Houston TX USA) using a validated protocol.[31] This method is a 5“7 day process that involves amplicon generation pooling ligation emulsion PCR amplification and massively parallel pyrosequencing with manual data analysis. The estimated limit of detection for the assay is 1.25% mutant alleles. [27] The MPP method was used to demonstrate performance of the EGFR PCR test to a more sensitive method and as an arbiter for discrepant cases observed between the LDT or the repeat Sanger sequencing. In order to preserve patient privacy associated with tested clinical samples raw MPP sequencing results were anonymized and presented in Table S1. Results Specimen demographics 487 (47%) of 1044 specimens screened for the EURTAC trial using LDTs were available for testing using the EGFR PCR test. The flow of samples through the study is shown in . Patient demographics and baseline tumor characteristics for all patients by LDT status are shown in . There were no significant differences between subsets of patients tested and patients not tested by the EGFR PCR test (p>0.05) for each LDT status (mutation detected mutation not detected) with the exception of country of the screening clinic. Clinical outcomes for patients based on the EGFR PCR test results Of the 174 patients enrolled in EURTAC trial specimens from 134 (77%) patients were available for testing using the EGFR PCR test. Excluding 11 patients with invalid EGFR PCR test results and 7 patients with a result of EGFR mutation not detected a total of 116 (67%) patients were mutation detected by the EGFR PCR test and evaluable for clinical outcome analysis (57 patients in the chemotherapy arm and 59 in the erlotinib arm). Clinical outcomes (PFS BORR and OS) are presented in . Among EGFR PCR test positive patients those treated with erlotinib had a significantly prolonged PFS when compared to patients treated with chemotherapy (p-value <0.0001 log-rank test); the median PFS was 10.4 months (95% CI: 8.0 to 13.8 months) and 5.4 months (95% CI: 4.4 to 6.8 months) for patients treated with erlotinib or chemotherapy respectively (). The HR based on the Cox proportional hazards model was reduced by 66% (HR 0.34; [95% CI: 0.21 to 0.54]) for patients in the erlotinib versus chemotherapy arm. One year after randomization a higher percentage of patients in the erlotinib compared with the chemotherapy arm were event-free (45% [95% CI: 32% to 59% versus 6% [95% CI: 0% to 15%] respectively). .0089518.g002 Kaplan-Meier curves of progression-free survival (PFS) for different treatments in treatment-na¯ve patients with non“small-cell lung cancer and EGFR mutation detected by the EGFR PCR test and LDT. .0089518.t002 Summary of Clinical Outcome Analysis among EGFR PCR test positive patients in the EURTAC trial. Chemotherapy (N?=?57) Erlotinib (N?=?59) PFS (Investigator) Patients with event 37 (64.9%) 47 (79.7%) Patients without eventa 20 (35.1%) 12 (20.3%) ?Time to event (months) ?Medianb (95%CI) 5.4 [4.4; 6.8] 10.4 [8.0; 13.8] ?p-Value (Log-Rank Test) <0.0001 ?Hazard Ratio (95% CI) 0.34 [0.21; 0.54] ?1 year estimate ?Patients remaining at risk 2 24 ?Event-free Rateb (95%CI) 6% [0%; 15%] 45% [32%; 59%] Best Overall Analysis Response rates (95% CI) 14.0% [ 6.3%; 25.8%] 59.3%[ 45.7%; 71.9%] Difference in Response Rates (%) 45.29% [ 28.8%; 61.7%] ?p-Value (Chi-squared Test) <.0001 Odds Ratio (95% CI) 8.93 [3.59; 22.19] OS Patients with event 35 (61.4%) 36 (61.0%) Patients without eventa 22 (38.6%) 23 (39.0%) ?Time to event (months) ?Medianb (95%CI) 20.8 [17.3; 29.4] 25.8 [16.1; 30.0] ?p-Value (Log-Rank Test) 0.5381 ?Hazard Ratio (95% CI) 0.86 [0.54; 1.38] ?2 - year estimate ?Patients remaining at risk 16 23 ?Event-free Rateb (95% CI) 43% [29%; 57%] 51% [38%; 64%] Note: All eligible patients enrolled in study ML20650 were determined as EGFR mutation detected by the LDT. Among those patients with EGFR mutation confirmed by the EGFR PCR test were included in this table. Event ?=? Death or progression free whichever comes first for PFS analysis and event?=?death for OS analysis. a censored. b Kaplan-Meier estimates. C including censored observations. BORR were higher in patients in the erlotinib arm (59.3% [95% CI: 45.7% to 71.9%]) compared to the chemotherapy arm (14.0% [95% CI: 6.3% to 25.8%]). Patients in the erlotinib arm were much more likely to respond to therapy than patients in the chemotherapy arm (odds ratio of 8.93 [95% CI: 3.59 to 22.19]). There was no significant difference in OS between the treatment arms (25.8 months in the erlotinib arm (95% CI: 16.1 to 30.0) and 20.8 months in the chemotherapy arm (95% CI: 17.3 to 29.4) (log-rank test p-value ?=?0.5381)). PFS BORR and OS results for EGFR PCR test positive patients did not differ significantly from those obtained in all patients enrolled in the EURTAC trial which suggests that the EGFR PCR test positive patients are representative of all EURTAC enrolled patients. For the 7 cases where the EGFR PCR test result was mutation not detected and discrepant with the LDT two cases resolved in favor of the LDT by MPP three cases resolved in favor of the EGFR PCR test and one sample was invalid for both Sanger and MPP and the other was in agreement between the EGFR PCR test and Sanger but not MPP (Table S2). Anecdotally 6 of the 7 patients were treated with erlotinib and only one patient achieved greater than or equal to median PFS based on the LDT or the EGFR PCR test. Comparison of EGFR PCR test and LDT results Among 432 specimens with valid results from both the EGFR PCR test and LDT the PPA NPA and OPA were 94.2% (146/155 CI: 89.3% 96.9%) 97.5% (270/277 CI: 94.9% 98.8%) and 96.3% (416/432 CI: 94.1% 97.7%) respectively (Table 3). Thus there was a high concordance between the original LDT and EGFR PCR test results. Among sixteen specimens with discordant results the EGFR PCR test result was confirmed by MPP in 68.8% (11/16) cases (Table S3). .0089518.t003 Table 3 Agreement analysis between EGFR PCR test and LDT. SLCG LDT Total N?=?432 Mutation detected Mutation not detected EGFR PCR test Mutation detected 146 7 153 Mutation not detected 9 270 279 Total 155 277 432* ¢12 samples with inconclusive LDT results and 43 samples with invalid EGFR PCR test results were excluded. Positive percent agreement ?=?94.2% (95% CI [89.3“96.9%]). Negative percent agreement ?=?97.5% (95% CI [94.9“98.8%]). Overall percent agreement ?=?96.3% (95% CI [94.1“97.7%]). Comparison of the EGFR PCR test results with Sanger Sequencing Of 487 specimens tested using the EGFR PCR test and Sanger sequencing 406 gave valid results by both methods (38 were invalid by both methods five were invalid by EGFR PCR test and 38 were invalid by Sanger sequencing). The PPA NPA and OPA for EGFR PCR test compared with Sanger sequencing were 96.6% (112/116 CI: 91.7% 98.7%) 88.3% (256/290 CI: 84.1% 91.5%) and 90.6% (368/406 CI: 87.4% 93.1%; Table 4) respectively. Among 38 discordant results between the EGFR PCR test and Sanger sequencing MPP agreed with the EGFR PCR test result in 30 (78.9%) cases (Table S4). Sanger sequencing detected one L858R not detected by MPP and failed to detect 22 exon 19 deletions and 7 L858R mutations confirmed by MPP. Four MPP results were invalid and the remaining four results agreed with Sanger. The range of percent mutant alleles of the cases missed by Sanger was 3% to 60% with several specimens (n?=?16) under the estimated limit of detection for Sanger. .0089518.t004 Table 4 Agreement analysis between EGFR PCR test and Sanger sequencing. Sanger sequencing Total N?=?406 Mutation detected Mutation not detected EGFR PCR test Mutation detected 112 34 146 Mutation not detected 4 256 260 Total 116 290 406 *81 samples with invalid EGFR PCR test or Sanger sequencing results were excluded. Positive percent agreement ?=?96.6% (95% CI [91.5“98.7%]). Negative percent agreement ?=?88.3% (95% CI [84.1“91.5%]). Overall percent agreement ?=?90.6% (95% CI [87.4“93.1%])."

Lung_Cancer

"Our method was first applied to two published lung cancer microarray gene expression data sets. As shown in gene sets like the proteasome and BCR signaling pathways were identified by our method. These gene sets were not identified in the previous study [8] since the differential gene expression among these gene sets were relatively weak. However the concordant enrichment of these gene sets was detected by our method. This comparison illustrated the advantage of our proposed concordant integrative gene set enrichment analysis. The analysis results from our second application (a concordant integrative analysis of three data sets) also showed that many gene sets could be identified with low false discovery rates. A consistency between both results was also observed. A further exploration based on the KEGG cancer pathway collection demonstrated the practical usefulness of our proposed method. Overall this study illustrates that we can improve detection power and discovery consistency through a concordant integrative analysis of multiple large-scale two-sample gene expression data sets. There are several advantages for our proposed method. The genome-wide concordance can be statistically tested before the integrative analysis. The mixture model is estimated based on the maximum likelihood estimation procedure. Furthermore our integrative analysis of gene sets is based on a probabilistic framework which can be conveniently used for the calculation of false discovery rates. However there are also limitations. Our proposed mixture model is simple and it contains only three components. Normal distributions are assumed for these components. Furthermore we assume that different genes behave independently (Gold et al. [32] have showed that the independence assumption can be acceptable in practice). These limitations should be considered when our method is used in practice. For our future research it will be useful to extend our proposed method for an integrative analysis of data with multiple sample groups. This will be particularly useful for studying diseases with different progression stages. Although a major proportion of gene expression data have been collected for binary outcomes (e.g. normal vs. abnormal) data with other types of responses (e.g. survival data) have also been collected. It will also be useful to extend our method for these data. Furthermore when our proposed method is used for an integrative analysis of more than 3 data sets it is desirable to simplify the mixture model so that the number of model parameters (particularly for ) can be reduced to achieve statistical efficiency. Furthermore we would also like to consider more robust approaches "

Lung_Cancer

"In vivo transthoracic HIFU application in pigs caused a mean peak temperature increase up to 53.7°C in a simulated lesion (BioGlue®) deep inside the flooded lung. HIFU energy penetrated through the pleura and flooded lung into the target lesion. The simulated lesion which was located at a 6 cm depth below the transducer was heated by HIFU. The temperature increase was highly variable and inconstant. The mechanism of heat generation in the BioGlue®-simulated lesion was unclear. The lesion consisted of purified BSA and glutaraldehyde. Although this simulated lesion was not a true human tumour the acoustic and thermal properties are similar to those of human cancerous tissue due to similar densities and high protein contents. In addition movements of the heart and mediastinum may be transmitted to the target lesion bringing them out of the focal zone. Lung flooding seems to be ideal for HIFU application because sonographic imaging is possible and because of other advantages. Compared to other human tissues a flooded lung has an ideally suitable beam path because water has a very low attenuation. In contrast to flooded lung tissue tumour tissue converts acoustic energy into a therapeutic thermal dose and enables selective heating of the cancer mass. Consequently damage to healthy lung tissue is minimised. In addition to acoustic advantages there are other favourable conditions for tumour ablation in a flooded lung. There is no pulmonary blood flow in a flooded lung [22]. Perfusion reduces heat and is not desirable with locoregional thermal therapy. In addition ischaemia-related acidosis sensitises tumour tissue to heat. In comparison to a ventilated lung lung flooding reduces tumour movements caused by breathing. The following limitations existed in the current study. The risk of lung flooding in patients with limited lung function is unclear. Further studies on patients with limited lung function should be performed to examine the influence of unilateral lung flooding on haemodynamics and gas exchange. Ongoing studies in pigs showed that lung flooding of only one lobe is feasible. Transcutaneous application was not applied for the in vivo or ex vivo studies. One rib had to be resected to apply the HIFU applicator which consisted of a HIFU transducer and sector-array probe to the chest of a pig. Technical improvements in the application system are necessary to ensure that the HIFU focal zone is aimed precisely at the target lesion with the guidance of sonography. Transthoracic HIFU application is difficult despite resection of the ribs because the intercostal spaces in the animal model are narrow. A method and transducers have been developed to avoid the shielding of therapeutic ultrasound by the human rib cage [23]. Different HIFU exposure schemas (ten seconds ex vivo versus ˜one second on/off™ in vivo) and transducers were applied during this study. Ultrasound imaging is disturbed if HIFU is continuously on. Therefore the intermittent ˜one second on/off™ in vivo schema was needed to control the focal alignment with the thermocouple during the ˜one second off™ interval. For HIFU application to human tumours resected lung lobes were used. The gas-free filling occurs at 71.4% of the resected lobes. This limitation is due to residual air in non-collapsed bronchi that can only solved by resorption under in vivo conditions [11]. Lung flooding was performed with cooled saline at 15°C for the ex vivo HIFU application to ensure that hypoxic damage did not occur within the tumour and lung tissues. This was important because the NADPH-diaphorase staining method is based on mitochondrial vitality which is very sensitive to ischaemia. A hyperechoic area was found within the tumour tissue immediately following HIFU exposure. Grayscale changes have been shown to be a useful marker for HIFU-induced tissue destruction [8]. However it is unclear whether the area of grayscale changes corresponds with the ablated area such that a hyperechoic sonolesion represents irreversibly damaged tissue. B-mode ultrasound imaging is probably not the best method for monitoring tissue response. Currently the most important problem associated with ultrasound-guided HIFU ablation is the lack of reliable thermometry and lesion production monitoring [6]. In addition to ultrasound imaging real-time magnetic resonance thermometry could be important for ascertaining the extent of tumour destruction [24-26]. Conclusions In combination with lung flooding high-intensity focused ultrasound produced a thermal effect in an ex vivo model of human lung carcinoma and in simulated lung tumours in an in vivo porcine model. High-intensity focused ultrasound is a potential strategy for treating lung cancer. Further studies will examine HIFU therapy in animal models of lung cancer. Abbreviations BSA: Bovine serum albumin; FIO2: Fraction of inspired oxygen; HIFU: High-intensity focused ultrasound; H&E: Hematoxylin and eosin; NSCLC: Non-small cell lung cancer; NADPH-diaphorase: Nicotinamide adenine dinucleotide phosphate-diaphorase. Competing interests The authors declare that they have no financial or non-financial competing interests. Authors™ contributions FW was responsible for HIFU technique and temperature measurement. He collected and evaluated the data and wrote the manuscript. HS and SB performed the anaesthesia. CB performed the histopathological and enzyme histochemistry examinations. TGL co-wrote and revised the manuscript and discussed the results with the authors. All authors read and approved the manuscript. Acknowledgments This work was funded by the SRH Foundation Heidelberg Germany. The funder had no role in the study design or in the collection analysis and interpretation of data. The authors would also like to thank Uwe Leder for his anizational support and Mrs. Petra Dobermann for active help with the animal experiments. DKFZ Atlas of Cancer Mortality 2007 Heidelberg: German Center of Cancer Research Drings P Dienemann H Wannenmacher M Management of Lung cancer 2002 Berlin Heidelberg New York: Springer Colice GL Rubins J Unger M Follow-up and surveillance of the lung cancer patient following curative-intent therapy Chest"

Lung_Cancer

"About half (49%) of UNCeqRMETA mutations had no RNA evidence and were based only on DNA evidence. Surprisingly among UNCeqRMETA expressed somatic mutations (those with RNA and DNA mutant read evidence) the MAF in RNA was often significantly greater than in DNA (lung: 21% of expressed mutations breast: 17% fdr < 0.05) (A and Supplementary Figure S6A). This increase was often >2-fold (lung: 12% of expressed mutations breast: 11%). In contrast DNA MAF was significantly greater than RNA MAF at much lower frequency (lung: 2% of expressed mutations breast: 3% fdr < 0.05). As a control germline variants were detected in germline DNA-WES and patient-matched germline RNA-seq relative to the reference genome by UNCeqRMETA under the same settings as somatic mutation detection (B and Supplementary Figure S6B). In contrast to expressed somatic mutations expressed germline variants displayed rare significant differences in allele fraction (RNA greater than DNA: lung: 0.8% breast: 0.7%; DNA > RNA: lung 0.1% breast: 0.3%). Therefore the prevalent increased mutation signal in RNA-seq was cancer-specific. . Mutation signal in RNA versus DNA. Mutant allele fraction distributions of UNCeqRMETA expressed mutations from the lung triplet cohort tumor sequencing (A). Germline variant allele fraction distributions of expressed germline variants from lung quadruplet cohort germline sequencing (B). Diagonal lines indicate equal allelic fraction between DNA and RNA with points above the diagonal having greater allelic fraction in RNA below the diagonal greater allelic fraction in DNA. Breast cancer somatic mutation and germline allele distributions in Supplementary Figure S6. Distributions of MAF difference among driver genes having a significant difference in MAF over all mutations (C). MAF distributions for all TP53 UNCeqRMETA mutations expressed and unexpressed (C and D). In addition to the genome-wide phenomenon the increased mutation signal in RNA versus DNA might additionally be frequent in cancer driver genes. Lung and breast cancer's driver genes (46) with at least 10% prevalence were analyzed for differences in RNA to DNA MAF across all mutations whether expressed or not. Eight driver genes had significantly different MAF between DNA and RNA (Wilcoxon signed rank test fdr < 0.05; C). All of these genes had greater median MAF in RNA than in DNA including an oncogene PIK3CA and tumor suppressors such as TP53. The TP53 MAF distributions of lung and breast cancer had remarkable similarities (D) in that nonsynonymous and splice site mutations had extremely high RNA MAF relative to DNA MAF often 2-fold greater. Stop-gain and frameshift mutations in TP53 had greater MAF in DNA versus RNA but these decreases were less common and had a smaller magnitude in MAF difference. The TP53 results extend an earlier report in lung cancer using direct sequencing of TP53 RNA transcripts which found mutant transcript predominant expression (46). In summary expressed mutations tend to have larger mutation signal in RNA than in DNA. Importantly this effect was common among driver genes suggesting that integrating DNA and RNA for mutation detection provides the best opportunity to identify cancer causing mutations. Because DNA copy number can affect the quantity of tumor versus germline DNA at a locus tumor DNA copy number alterations were compared among mutations with a significantly greater MAF in RNA versus DNA and vice versa. Mutations with greater MAF in RNA exhibited a small (roughly 5%) relative increase in DNA copy number deletions (Supplementary Figure S7) suggesting that RNA is beneficial to detect mutations in regions of genome deletion. MAF differences in TP53 mutations did not associate with either DNA amplifications or DNA deletions (Supplementary Figure S7). Large gains in low purity tumors Because low tumor purity (caused by normal contamination and multiple clones) can affect mutation detection (28) the outcome of integrating RNA-seq and DNA-WES in mutation detection was compared among tumors by their purity. The rate of mutation gain after adding RNA-seq to DNA-WES was non-uniform both in the breast and lung triplet cohorts such that the greatest gains occurred in tumors having the lowest purity. Specifically tumors™ total mutation ratio (the number of mutations detected by UNCeqRMETA over UNCeqRDNA) had significant negative correlation with tumor purity in both lung and breast cancer (A). Mutation gains were largest among tumors with purity <40%. In addition tumors™ average difference in mutation signal between RNA and DNA (the mean difference of RNA MAF to DNA MAF across all expressed UNCeqRMETA mutations) also had significant negative correlation with tumor purity both in lung and breast cancer (B). Therefore tumors with low purity had the largest RNA-seq mutation signal and gained the most new mutations after incorporation of RNA-seq evidence. . Tumor purity effects on mutation detection. Lines summarize breast and lung triplet cohorts displaying total mutation ratios (A) or mean mutant allele fraction difference within expressed mutations (B) among tumors binned by tumor purity quintile and plotted at midpoint. Pearson's correlation tests compared the association of mutation ratio and MAF associations among triplet cohort tumors (P). MAF distributions from two exemplar low purity tumors™ mutations (C and D). Diagonal lines indicate equal MAF in DNA-WES and RNA-seq with mutations above the diagonal having greater MAF in RNA below the diagonal greater MAF in DNA. Unexpressed mutations are marked along the horizontal axes in (C and D). Examples of low purity tumors with large mutation gains include a low purity breast tumor that had 1.8 total mutation ratio and a mean 0.18 difference in mutation signal among expressed mutations. Two of this tumor's mutations with much larger signal in RNA than DNA occurred in PIK3CA (p.H1047R) and GATA3 (p.S412fs) (C). These mutations occur in major mutational hotspots (47) and are also characteristic molecular drivers for the Luminal A expression subtype (648) of which this tumor is a member. Incorporation of RNA-seq evidence was essential to identify these two driving mutations; e.g. there was only 1 DNA read with the PIK3CA mutation but 29 mutant reads in RNA-seq (). An example lung tumor had a 1.2 total mutation ratio and an average 0.22 difference in mutation signal among expressed mutations including CDKN2A (p.H98P) and TP53 (p.R273H) which exhibited very large RNA MAF (at 100 and 84%) relative to DNA MAF (at 43 and 46%) (D). These PIK3CA GATA3 and TP53 mutations were not detected by earlier studies utilizing DNA-WES alone (46) emphasizing the advantage of RNA integration. "

Lung_Cancer

" Silent lunch and tea break 7. Taking care of yourself - Sitting meditation ending in choiceless awareness - Exercise on taking care of yourself by examining how to improve balance in life - Meditation without CD - Yoga or walking meditation - Reflect on training - 3-min breathing space 8. The rest of your life - Bodyscan - Reflection on training - Further sources of information - Short sitting meditation - Maintaining practice Outcome measures Primary outcome measure Psychological distress The primary outcome measure is the total score on the HADS [39-41] which is developed to measure psychological distress in somatic patient populations. It consists of a 7-item anxiety (HADS-A) and 7-item depression (HADS-D) subscale. The HADS shows good psychometric properties in the general medical population including oncology patients [42]. Internal consistency as measured with Cronbach™s ? varied from .84 to .90 [4042].Test-retest reliability was good as Pearson™s r > .80 were obtained [4043]. Though the cut-off scores of the HADS vary among populations [44] in lung cancer patients they have found to be <8 versus ?8 on the HADS-A or HADS-D [45]. The HADS has been shown to be highly correlated with the Beck Depression Inventory [42]. It has previously been used in intervention studies of mindfulness and shown to be sensitive to change (e.g. [46]). Secondary outcome measures Quality of life (only for patients) The European anisation for Research and Treatment of Cancer (EORTC) Core Quality of Life Questionnaire (QLQ-C30) [47] is included along with the supplemental Lung Cancer questionnaire module (QLQ-LC13) [48]. The QLQ-C30 is designed to use in clinical trials on physical treatments for cancer patients. It incorporates five functional scales (physical role cognitive emotional social) three symptom scales (fatigue pain nausea and vomiting) a global health and quality of life scale and an array of single-item symptom measures. After revisions in the role functioning global health and physical functioning scale internal consistency of the subscales varied between .65 and .94 except for the cognitive functioning scale with ? varying from .56 to .63 [474950]. Test-retest reliability varied from .63 to .86 [51]. The lung cancer questionnaire module is designed to supplement the core questionnaire and comprises specific symptoms associated with lung cancer (coughing haemoptysis dyspnoea pain) and side-effects from conventional chemo- and radiotherapy (hair loss neuropathy sore mouth dysphagia). While the multi-item dyspnoea scale showed high internal consistency the pain subscale did not. When combined with the dyspnoea and pain items of the core questionnaire both the dyspnoea (? = .86) and pain (? = .71) subscale showed high internal consistency. Since the QLQ-C30 and QLQ-LC13 are mainly focused on physical symptoms we added the items Social Interaction and Alertness Behavior of the Sickness Impact Profile (SIP) [52]. Internal consistency was .94 and test-retest reliability was .92. The SIP correlated with self-assessed sickness and dysfunction [52]. Caregiver appraisal (only for partners) We use the 9-item Self-Perceived Pressure from Informal Care (SPPIC) [53] to assess the extent to which caregiving is experienced as burdensome. To also measure positive aspects of caregiving the 9-item subscale Care-Derived Self-Esteem of the Caregiver Reaction Assessment (CRA-SE) [54] is included. Internal consistency of the SPPIC was .79 and of the CRA-SE was .73. The SPPIC and CRA-SE were unrelated to each other [55]. Relationship quality To measure relationship satisfaction we included the 10-item Satisfaction subscale of the Investment Model Scale (IMS-S) [56]. The IMS-S starts with 5 items that measure concrete examplars of satisfaction to enhance the comprehensibility of the global items which are utilized to form the construct. Internal consistency varied from .79 to .95 and the IMS-S was related to the Dyadic Adjustment Scale. Also the Mutual Interpersonal Sensitivity scale (MIS) [57] is included to measure communication between partners about the cancer. It contains 18 items and is divided into two scales: open communication and avoiding negative thoughts about the cancer. Spirituality is measured with the Spiritual Attitude and Involvement List (SAIL) [58] and consists of 26 items divided into the subscales meaningfulness trust acceptance caring for others connectedness with nature transcendent experiences and spiritual activities. The internal consistency varied from .74 to .88 and test-retest reliability varied from .77 to .92. All subscales except for connectedness with nature were related with the Functional Assessment of Chronic Illness Therapy “ Spiritual Well-Being Scale. Costs (only for patients) The cost-effectiveness evaluation is carried out from a societal perspective considering direct as well as indirect health costs. Data on costs are collected prospectively using a diary in which participants register a) health care utilization: the type of care and its duration and b) cancer-related absence from work. Unit cost estimates are derived from the national manual for cost prices in the health care sector [59]. Costs of reduced ability to work are estimated using the friction costs method which results in a more realistic estimate than the human capital approach [60]. Treatment costs of MBSR are calculated using activity-based-costing methods thus measuring actual resources (time of therapist time of patients facilities) used. All unit cost prices are adjusted to 2013 prices. Unit cost estimates are combined with resource utilization data to obtain a net cost per patient over the entire follow-up period. Process measures Mindfulness skills are examined with the 39-item Five Facet Mindfulness Questionnaire (FFMQ) [6162]. The FFMQ is based on an exploratory factor analysis of five mindfulness measures which allowed items from different instruments to form factors providing an empirical integration of these independent attempts to operationalize mindfulness. This led to the following five subscales: observing describing acting with awareness non-judging of inner experience and non-reactivity to inner experience. Internal consistency varied from .72 to .93 among the different subscales. Most subscales were related to meditation experience Psychological Well-Being scales and psychological symptoms including the Brief Symptom Inventory [61]. FFMQ is sensitive to change in mindfulness-based interventions and is found to mediate the relationship between mindfulness practice and improvements in psychological symptoms (e.g. [63]). Self-compassion is assessed with the Self Compassion Scale (SCS) [6465] which has 26 items and is divided into six subscales: self-kindness versus self-judgment common humanity versus isolation and mindfulness versus over-identification. Internal consistency of the different subscales varied from .75 to .81 and test-retest reliability varied from .80 to .93. SCS correlated moderately with self-esteem measures including the Rosenberg Self-Esteem Scale. Furthermore whereas the self-esteem measures correlated significantly with the Narcissistic Personality Inventory the SCS was unrelated to narcissism [64]. SCS is sensitive to change through mindfulness-based interventions and is found to mediate MBCT™s treatment effects [66]. To measure rumination we administered the extended version of the Ruminative Response Scale (RRS-EXT) [67] Raes and Hermans: The revised version of the Dutch Ruminative Response Scale unpublished instrument]. The RRS-EXT contains 26 items in which a more adaptive thinking style (i.e. reflection) is distinguished from a more maladaptive one (i.e. brooding). Internal consistency varied from .72 to .77 and test-retest reliability varied from .60 to .62 for the brooding and reflection subscales. The concept of rumination seems to be sensitive to change through mindfulness-based interventions and has been shown to mediate the effect of MBSR on depressive symptoms in oncology patients [68]. The psychological stress reaction is measured with the 15-item Impact of Event Scale (IES) [6970] which assesses two categories of responses: intrusive experiences and avoidance of thoughts and images associated with the event. Internal consistency varied from .65 to .92 [71] and test-retest reliability varied from .79 to .87 among the subscales [69]. IES correlated with anxiety and depression subscales of the General Health Questionaire. Adherence to MBSR is assessed during the entire study period with a calendar on which participants in the MBSR condition fill out on a daily basis whether they adhere to the mindfulness exercises: either formal practice (e.g. meditation exercise like the bodyscan) informal practice (e.g. activity with awareness) or no exercise. Adherence to MBSR has been shown to mediate the effects of MBCT on depressive symptoms [72]. Statistical analysis plan Sample size To determine the required sample size first the sample size was calculated that would be needed for a simple t-test and subsequently it was corrected for clustering repeated measurements and baseline. A two-sided t-test on the total HADS score [3940] (i.e. our primary outcome measure examining psychological distress (HADS-total) anxiety symptoms (HADS-A) and depressive symptoms (HADS-D)) would require 64 participants in each group to have 80% power to detect a medium-sized difference (effect size = 0.5) with alpha = 0.05. To correct for clustering we multiplied this sample size of 64 with the design factor (1 + (n ? 1) * ICC) where n denotes the cluster size and where ICC denotes the intra-cluster correlation. In our study the treatment groups will consist of 14 people of whom about 7 will be patients. With n = 7 and an estimated ICC = 0.01. [72] the correction factor equals 1.06. To correct for repeated measurements and the use of the baseline measurement as a covariate we multiplied the required sample size by the design factor 1+?/2??02 where ? denotes the correlation between the post-treatment HADS measurements and ?0 denotes the correlation between the baseline HADS with the post-treatment HADS measurements. With ? = 0.8 and ? = 0.5 as conservative estimates the second design factor equals 0.65. Consequently after correction for clustering and covariates we arrived at a required sample size of 0.65 * 1.06 * 64 = 44 patients per arm. So 88 patients with lung cancer would be required for the study. Based on our pilot study [van den Hurk Schellekens Molema Speckens and van der Drift in preparation] we expect a 20% drop-out rate. Therefore we intend to include 110 patients and 110 partners. Primary analyses The samples of lung cancer patients and partners will be analyzed separately. Baseline characteristics of the population will be compared between MBSR and control group to ensure that key variables were evenly distributed by randomization. First analyses will be based on the intention-to-treat approach. Next we will perform per-protocol analyses with the treatment-adherent sample (i.e. in the MBSR condition participants have to attend at least four of the eight MBSR sessions [73] and in the TAU condition participants do not attend a mindfulness-based programme). We will use linear mixed models to analyze all outcome variables (i.e. psychological distress quality of life (only for patient) caregiver appraisal (only for partner) relationship quality and spirituality) with treatment as fixed factor baseline measurement as covariate and a random intercept based on MBSR group. This procedure will use all observed data in our analyses. In addition Cohen™s d effect size [74] will be reported based on the difference between the group means on baseline and follow-up scores divided by the pooled standard deviation at baseline and follow-up. Secondary analyses Cost effectiveness The quality of life measures (i.e. QLQ-C30; QLQ-LC13) will be used to calculate Quality of Adjusted Life Years (QALYs) for each individual. Costs and effects (in terms of QALYs) will be combined in the incremental cost-effectiveness ratio (ICER). The ICER expresses cost-effectiveness in terms of incremental costs per QALY gained. To estimate confidence intervals for the mean of the ICER a non-parametric bootstrapping method will be used performing 1000 replications of the original data. In order to express the implications of the cost-effectiveness results more clearly a cost-acceptability curve will be constructed. In case of dominance a full cost analysis will be conducted to estimate the mean savings per patient per year. Mediation analyses To examine the possible underlying mechanisms of change in MBSR mediation analyses will be conducted. Only the data of the treatment-adherent sample will be included in these analyses. By means of a multiple mediation model suggested by Preacher and Hayes [75] we will test the mediating effect of mindfulness skills self-compassion rumination and adherence to MBSR on psychological distress quality of life (only in patients) caregiver appraisal (only in partners) relationship quality and spirituality. Discussion In the last ten years MBSR has not only proven to be a feasible and acceptable intervention in cancer patients [76] but it also seems to be effective in reducing psychological distress [30]. However the generalization of these results is limited because most participants were female patients with breast cancer. A large part of lung cancer patients already have advanced cancer at time of diagnosis and are confronted with a poor prognosis and low health status. Consequently they more often report psychological distress than patients with other diagnoses of cancer [89]. Hence it is not yet clear whether MBSR is a feasible acceptable and effective intervention in patients with lung cancer. Moreover little is known about the effectiveness of MBSR in partners of cancer patients [30] though they also often report psychological distress. Our pilot study of 19 lung cancer patients and 16 partners participating in an MBSR course provides preliminary evidence that MBSR is feasible and acceptable in this population (van den Hurk Schellekens Molema Speckens and van der Drift in preparation). The current trial will answer the question whether MBSR is effective in patients with lung cancer and their partners. We started enrolment of participants in February 2012. At the moment we think recruiting a sufficient number of patients and partners will be a challenge due to rapidly fluctuating health status and sudden changes in cancer treatment [77]. The main reasons for declining participation in patients is ˜being too ill™ or that it is ˜too much of a burden during chemo and/or radiotherapy™. Furthermore no perceived need or motivation for the training is commonly mentioned. Among partners participation is highly depending on whether the patient is willing to participate. Although partners can take part separately partners who are interested do often not participate when the patients decline participation. Considering the difficulty of studying lung cancer patients and their partners [77] our trial will offer valuable information on whether MBSR as one of the few available psychosocial care programmes contributes to the alleviation of their psychological distress. Abbreviations MBSR: Mindfulness-based stress reduction; RCT: Randomized controlled trial; RUNMC: Radboud University Nijmegen Medical Centre; MBCT: Mindfulness-based cognitive therapy; MMSE: Mini mental state examination; DT: Distress thermometer; HADS: Hospital anxiety and depression scale; QLQ-C30: Quality of life “ cancer; QLQ-LC13: Quality of life “ lung cancer; SIP: Sickness impact profile; SPPIC: Self-perceived pressure from informal care; CRA-SE: Caregiver reaction assessment “ care-derived self-esteem; IMS-S: Investment model scale-satisfaction; MIS: Mutuality and interpersonal sensitivity; SAIL: Spiritual attitude and involvement list; FFMQ: Five facet mindfulness questionnaire; SCS: Self-compassion scale; RRS-EXT: Rumination response scale “ extended version; IES: Impact of event scale. Competing interests The authors declare that they have no competing interests. Authors™ contributions All authors contributed to the design of the study. AS MD and JP are the principal investigators of the study. MS drafted the paper which was modified and supplemented by all other authors. DH MS and MD are involved in recruiting participants while MS and DH take care of the logistics of the study and data collection. RD contributed specifically to the statistical analysis plan and WW contributed specifically to the design of the cost-effectiveness evaluation. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2407/14/3/prepub Acknowledgements This research is funded by Foundation Alpe d™HuZes and the Dutch Cancer Society (Grant number KUN 2011“5077 awarded to Prof. dr. Anne E. M. Speckens Dr. Miep A. van der Drift and Prof. dr. Judith B. Prins). 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Lung_Cancer

"The proband™s father (II-5) and sister (III-5) were both unaffected and peripheral blood samples were obtained from these individuals. Some family members who were not considered as critical for this study were excluded from the pedigree chart to preserve confidentiality. Whole-exome sequencing was performed for individuals II-4 II-5 III-4 and III-5. After obtaining permission from the Institutional Review Board at Okayama University Hospital and informed consent from the patients and other family members we performed a whole-exome sequencing study. Tumor DNA samples from II-4 tumor and peripheral blood DNA samples from III-4 and peripheral blood DNA samples from two unaffected family members (II-5 and III-5) were used for the analysis. The candidate germline alterations were restricted to 29 variants by comparing the whole-exome sequencing results between the patients and the unaffected family members. Among them we focused on a point mutation in the human epidermal growth factor receptor 2 (HER2/neu) gene (NM_004448 G660D GGC to GAC) which was located in exon 17 encoding the transmembrane domain of HER2 (Supplementary Tables 1“3). This alteration was confirmed by direct sequencing (A). We also confirmed that there was no copy number gain of HER2 in the examined tumors based on the degree of read-depth in the whole-exome sequencing results. Of note no mutations in genes known to cause lung cancers were detected for tumors from III-4 and II-4. . DNA and amino acid sequences in the transmembrane domain of HER2. A) Direct Sanger sequencing of the proband (III-4) her affected mother (II-4) and her unaffected sister (III-5). The results indicated that G660D was a germline mutation. B) Direct sequencing of a sporadic lung adenocarcinoma with a HER2 V659E mutation. V659E was found to be of somatic origin based on the sequencing results of the peritumoral lung tissue from the same specimen. All the sequence variants were confirmed by independent polymerase chain reaction amplifications and were sequenced in both directions. C) Interspecies conservation of the transmembrane domain of HER2 (UCSC Genome Browser http://genome.ucsc.edu accessed September 12 2013). The yellow highlight indicates the N-terminal glycine zipper motif Thr652-X3-Ser656-X3-Gly660 a tandem variant of a GG4-like motif of human HER2. Codons 659 and 660 in human HER2 are highly conserved among the listed vertebrate species (shown in red). X. tropicalis = Xenopus tropicalis. We considered that somatic mutations in the HER2 transmembrane domain might act as driver mutations in lung cancer. Hence we sequenced exon 17 of the HER2 in the tumor samples of 315 sporadic non“small cell lung cancer patients of which 253 were adenocarcinomas. Although the HER2 G660D mutation was not detected a novel nonsynonymous mutation V659E (GTT to GAA) next to codon 660 was identified in one of these patients. This patient was histologically diagnosed as nonmucinous adenocarcinoma in situ and the patient had neither smoking history nor apparent family history of lung cancer. This V659E mutation was certainly a somatic mutation because it was not identified in the peritumoral lung tissue of the same patient (B). The alignment of HER2 amino acid sequences showed high conservation of valine 659 and glycine 660 among vertebrates (C). HER2 somatic mutations have been reported in 2% to 4% of lung adenocarcinomas (5“7). However all reported mutations were restricted to its tyrosine kinase domain (67). According to the cBioPortal for Cancer Genomics (http://www.cbioportal./public-portal/ accessed September 12 2013) the same genetic mutation in the HER2 has not been reported in any type of cancer. Interestingly a previous study reported that a mutation in the transmembrane domain (V664E) of the rat neu gene which corresponds to V659E in its human homolog HER2 induced oncogenic transformation (8). In addition in vivo experiments showed that the HER2 V659E mutation contributed to the stability of HER2 dimers resulting in the dysregulated receptor activation and subsequent cell transformation (910). Furthermore the novel mutations were located within the glycine zipper motif Thr652-X3-Ser656-X3-Gly660 a tandem variant of the GG4-like motif at the N-terminal portion of the transmembrane domain which was critically related to the dimerization of HER2 (C) (911). Accordingly we performed a functional analysis of the mutant HER2 proteins. We found that the degradation of HER2 protein after the administration of cycloheximide was slower in G660D and V659E mutants as compared with wild-type (Supplementary A) indicating the higher stability of the mutant proteins than wild-type protein. In addition results of a phospho-mitogen“activated protein kinase array indicated the activation of Akt and p38? (data not shown). Indeed Akt is known to be activated by HER2 by phosphatidylinositol 3-kinase and leads to increased cell growth and survival (1213). Also the activation of p38 was shown to contribute to the viability of lung adenocarcinoma cells derived from never or light smokers (1415). A western blot analysis for Akt and p38 successfully confirmed the upregulation of both phospho-Akt and phospho-p38 expression in the mutant HER2 transfectants (Supplementary B). Because the G660D alteration in HER2 might have been the cause of the lung cancer in the pedigree studied we investigated whether familial aggregation of cancer in other ans could be seen in this pedigree. We found that II-1 and II-6 developed renal and gastric cancers respectively; however both of them also had lung cancer. The reason why other types of clinically apparent malignances were rarely found in this pedigree is unclear. The G660D germline mutation may be tolerated in ans other than the lung. This study had some limitations. First the carcinogenic potential of the HER2 mutation at the transmembrane domain should be confirmed in other models such as transgenic mice. Second the rarity of these mutations in sporadic lung cancers may be the limitation for generalizability to other cases even if targeting therapies for similar types of HER2 mutation were developed. In we identified a novel germline mutation in the transmembrane domain of the HER2 in familial lung adenocarcinomas. Somatic mutation in the HER2 transmembrane domain may be a possible cause of sporadic lung adenocarcinomas. Funding This study was supported by a Grant-in Aid for Scientific Research from the Ministry of Education Culture Sports Science and Technology of Japan (25293302 to ST). H. Yamamoto J. Soh S. Miyoshi and S. Toyooka conceived the project. K. Higasa M. Sakaguchi K. Shien and K. Ichimura performed the experiments. H. Yamamoto J. Soh M. Furukawa S. Hashida N. Takigawa K. Kiura K. Tsukuda and S. Toyooka collected the samples and assisted with the experiments. H. Yamamoto K. Higasa K. Shien and K. Matsuo analyzed the data. H. Yamamoto K. Higasa M. Sakaguchi F. Matsuda and S. Toyooka prepared the manuscript with input from the other authors. S. Miyoshi F. Matsuda and S. Toyooka supervised the project. The authors declared no conflicts of interest. References 1. BellDWGoreIOkimotoRA Inherited susceptibility to lung cancer may be associated with the T790M drug resistance mutation in EGFR. Nat Genet. 2005;37(12):1315“131616258541 2. IkedaKNomoriHMoriTSasakiJKobayashiT Novel germline mutation: EGFR V843I in patient with multiple lung adenocarcinomas and family members with lung cancer. Ann Thorac Surg. 2008;85(4):1430“143218355544 3. OhtsukaKOhnishiHKuraiD Familial lung adenocarcinoma caused by the EGFR V843I germ-line mutation. 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Cancer. 2012;118(20):5015“502522415779 Oncotarget Oncotarget ImpactJ Oncotarget 1949-2553 Impact Journals LLC 24519909 3996653 Research Paper Sp1-mediated microRNA-182 expression regulates lung cancer progression Yang Wen-Bin 1 Chen Ping-Hsin 2 Hsu Tsung-I 3 Fu Tzu-Fun 4 Su Wu-Chou 5 Liaw Hungjiun 6 Chang Wen-Chang 7 Hung Jan-Jong 1 2 3 7 1 Institute of Bioinformatics and Biosignal Transduction College of Bioscience in Biotechnology National Cheng Kung University Tainan 701 Taiwan 2 Department of Pharmacology College of Medicine National Cheng Kung University Tainan 701 Taiwan 3 Center for Infectious Disease and Signal Transduction Research National Cheng Kung University Tainan 701 Taiwan 4 Department of Medical Laboratory Science and Biotechnology College of Medicine National Cheng Kung University Tainan 701 Taiwan 5 Department of Internal Medicine College of Medicine and Hospital National Cheng Kung University Tainan 701 Taiwan 6 Department of Life Sciences College of Bioscience in Biotechnology National Cheng Kung University Tainan 701 Taiwan 7 Graduate Institute of Medical Sciences College of Medicine and Center for Neurotrauma and Neuroregeneration Taipei Medical University Taipei 110 Taiwan Correspondence to:petehung petehung@mail.ncku.edu.tw 2 2014 25 1 2014 5 3 740 753 18 11 2013 24 11 2014 Copyright: © 2014 Yang et al. 2014 This is an open-access article distributed under the terms of the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original author and source are credited. Our recent study indicated that overexpression of Sp1 enhances the proliferation of lung cancer cells while represses metastasis. In this study we found that the transcriptional activity of FOXO3 was increased but its protein levels decreased following Sp1 expression. Sp1 increased expression of miR-182 which was then recruited to the 3'-untranslated region of FOXO3 mRNA to silence its translational activity. Knockdown of miR-182 inhibited lung cancer cells growth but enhanced the invasive and migratory abilities of these cells through increased N-cadherin expression. Repression of FOXO3 expression in the miR-182 knockdown cells partially reversed this effect suggesting that miR-182 promotes cancer cell growth and inhibits cancer metastatic activity by regulating the expression of FOXO3. The expression of several cancer metastasis-related genes such as ADAM9 CDH9 and CD44 was increased following miR-182 knockdown. In in the early stages of lung cancer progression Sp1 stimulates miR-182 expression which in turn decreases FOXO3 expression. This stimulates proliferation and tumor growth. In the late stages Sp1 and miR-182 decline thus increasing FOXO3 expression which leads to lung metastasis. Sp1 miR-182 FOXO3 Lung cancer INTRODUCTION Post-transcriptional regulation plays an important role in diverse cellular processes such as development neurogenesis and cancer progression [1-3]. MicroRNAs (miRNAs) have emerged as important post-transcriptional regulators that inhibit mRNA translation or induce mRNA cleavage by base pairing with a seed region in the 3'-untranslated region (3'-UTR) of target genes [4 5]. Recent studies have shown that dysregulation of miRNAs contributes to the initiation progression metastasis and drug resistance of cancer [6 7]. For example miR-200c targets Kras to regulate Kras expression during tumorigenesis [8]. Furthermore several upregulated and downregulated miRNAs have been identified in lung cancer the most frequently diagnosed cancer and the most common cause of cancer-related death worldwide [9-11]. Identification of early-detection biomarkers and precise diagnosis are necessary if lung cancer patients are to receive efficacious therapeutic treatment quickly. Several factors such as USP17 have been identified as potential biomarkers for lung cancer [12 13]. Circulating miRNAs could also serve as useful clinical biomarkers for the screening of high-risk populations and the detection solid tumors in the early stages of cancer progression [14 15]. miRNAs offer new targets for cancer therapy [16 17]. Therefore a detailed understanding of the mechanisms underlying miRNA production and function is important. Identification of miRNA target genes and the use of gene set enrichment analysis have clarified the function role of miRNAs. However the molecular mechanisms that regulate of miRNA biogenesis are still largely unknown. Recent studies have shown that transcription factors (TFs) regulate not only the expression of protein-encoding genes but also miRNA biogenesis through RNA polymerase II-dependent transcription [18]. Several TFs including p53 c-myc and HIF1? that directly recognize miRNA promoters and regulate miRNA transcription have been reported [19-21]. Specificity protein 1 (Sp1) which belongs to the specificity protein/ Krüppel-like family was the first TF identified in mammalian cells. Sp1 contains three Cys2His2-type zinc finger DNA binding motifs that recognize GC-rich promoter sequences [22]. Sp1 regulates thousands of coding genes such as those encoding cyclin A2 p21cip1/waf1 E-cadherin and Sp1 itself. These genes are involved in a variety of physiological processes including cell cycle progression and cell migration [23-26]. Sp1 also regulates the expression of noncoding genes. Sp1 forms a complex with NF-?B to downregulate miR-29b expression through the recruitment of histone deacetylase (HDAC) 1 and HDAC3 in leukemia and thereby contributes to the growth of leukemia cells [27]. Sp1 also forms a complex with HDAC4 to downregulate miR-200a expression in hepatocellular carcinoma and contributes to cell proliferation and migration [28]. In addition Sp1 is an activator of miR-34c miR-132 and miR-365 expression [29-31]. However no studies have assessed whether Sp1 regulates the expression of miRNAs involved in lung tumorigenesis. Because the accumulation of Sp1 is required for lung tumor growth further investigation of Sp1-mediated miRNA regulation is needed. In this study we showed that Sp1 suppressed FOXO3 expression via post-transcriptional regulation. To elucidate whether miRNAs were involved in this process we used a systematic screening approach to identify Sp1-regulated miRNAs. We identified a novel Sp1-regulated miRNA miR-182 in lung cancer cells and demonstrated that Sp1 downregulated FOXO3 expression by upregulating miR-182 expression. Our results show that miR-182 functions as an oncomiR to enhance cancer cell proliferation and acts as a tumor suppressor to inhibit cancer metastasis. RESULTS Sp1 regulates miR-182 expression Our previous studies demonstrated that Sp1 is involved in KrasG12D-induced lung tumorigenesis [23 32]. Using cDNA microarray analysis we found that Sp1 increased oncogene expression and decreased tumor suppressor gene expression. In the present study we initially used software to analyze the promoters of all identified miRNAs. According to the miRBase database the human genome contains 1600 miRNA genes. We investigated whether Sp1 participates in the regulation of intergenic miRNAs. First we screened the upstream (-1 kb) flanking sequences of intergenic miRNAs. Using the TFSEARCH program we identified 205 intergenic miRNAs that contained potential binding sites for Sp1. Because Sp1 is upregulated in lung cancer and the expression of its target genes is altered we next examined the expression of these miRNAs in lung cancer. According to previous studies the expression patterns of 22 miRNAs differed significantly in lung cancer tissue and normal lung tissue (Supplementary Table S1). In most of these studies miR-182 which contains two putative Sp1 binding sites within its upstream region was upregulated in lung cancer. When we examined miR-182 expression we found that miR-182 was decreased in Sp1-knockdown cells but increased in IMR-90 cells that overexpressed GFP-Sp1 (Figure 1A and 1B) suggesting that Sp1 positively regulates miR-182 expression."

Lung_Cancer

"MB was supported by the European Regional Development Fund grant number FKZ:005-111-0027. GVM was supported by the Victorian Cancer Agency grant TS10_01. KKW is supported by the NIH CA122794 CA140594 CA163896 CA166480 and CA154303 grants. PKP was supported by a Uniting Against Lung Cancer grant. RKT is supported by the EU-Framework Programme CURELUNG (HEALTH-F2-2010-258677) by the Deutsche Forschungsgemeinschaft through TH1386/3-1 and through SFB832 (TP6) by the German Ministry of Science and Education (BMBF) as part of the NGFNplus program (grant 01GS08100) and by the Deutsche Krebshilfe as part of the Oncology Centers of Excellence funding program. SP was supported by a grant from the Rudolph Becker Foundation. JW was supported by the German Cancer Aid (CIO K¶ln Bonn) the Federal Ministry of Education and Research (NGFNplus) and the Ministry of Economy Energy Industry and Craft of North Rhine-Westfalia in the PerMed. NRW framework program. ZZ was supported by the NIH R01LM011177. We thank J. Sosman and C. Arteaga for their critical review of this manuscript C. Liang for providing X-376 and A. Nashabi for administrative assistance. Australian specimens were processed by the Victorian Cancer Biobank. The human anaplastic lymphoma cell line SUDHL-1 was a generous gift from Dr. S. Morris of St. Jude Children's Research Hospital. Author Contributions: C.M.L. and W.P. conceived the project and wrote the manuscript. C.M.L. N.T.M. Y.Y. H.J. and M.R.B performed the molecular biology experiments. H.C. P.L. X.C. and R.S. performed the statistical analysis. S.C.O. L.C.H. A.F. and R.K.T. performed all the IGF-1R and IRS-1 immunohistochemistry experiments. S.C.O. L.O. P.K.P. R.B. S.A. S.P. M.B. M.B. J.W. M.G. G.V.M. B.S. P.A.R. T.M.R. and R.K.T. provided clinical samples. D.H.J. and L.H. provided clinical care for the index patient. Z.C. and K.K.W. provided the EML4-ALK E13;A20 transgenic mice. D.L. L.W. Y.S. and M.L. performed all the FISH and nanostring experiments. R.T. and E.D.S. performed the xenograft studies. Q.W. and Z.Z. analyzed the whole-genome sequencing data. Conflicts of Interest: CML and WP have filed a provisional patent describing the improved efficacy of combined treatment of ALK inhibitors plus IGF-1R inhibitors compared to ALK inhibitors alone (serial number 61/768072). CML has served on an Advisory Board for Pfizer and has served as a speaker for Abbott and Qiagen. WP has done consulting for MolecularMD AstraZeneca Bristol-Myers Squibb Symphony Evolution Clovis Oncology Exelixis Clarient Champions Clarient (MDOutlook) and WebMD. WP has received research funding from Enzon Xcovery AstraZeneca Symphogen Clovis Oncology and Bristol-Myers Squibb. Rights to EGFR T790M testing were licensed on behalf of WP and colleagues to MolecularMD. RKT is a founder and shareholder of Blackfield AG a company focused on cancer genome diagnostics and cancer genomics-based drug discovery. LCH received lecture fees (Roche Novartis Pfizer Qiagen). RKT received consulting and lecture fees (Sanofi-Aventis Merck Roche Lilly Boehringer Ingelheim Astra-Zeneca Atlas-Biolabs Daiichi-Sankyo Blackfield AG) as well as research support (Merck EOS and AstraZeneca). RB is a cofounder and owner of Targos Molecular Inc. and served on advisory boards for Pfizer Roche Boehringer Ingelheim Merck Serono Novartis and Lilly. JW has served as a Consultant or Advisory Role for Roche Novartis Pfizer Boehringer-Ingelheim AstraZeneca Bayer Pharmaceuticals Eli Lilly and Merck. GVM has served on an Advisory board for Pfizer. BS has served on Advisory Boards for Pfizer and Novartis. KKW received sponsored research grants from Takeda AstraZeneca Roche and Infinity Pharmaceuticals. He is also a consultant for G1 Therapeutics. PKP has served on an Advisory Board for Bristol-Myers-Squibb. SP received consulting and lecture fees (Novartis Curagita Roche Abbot Definiens) as well as research support (Ventana-Roche). JW has served as a Consultant or Advisory Role for Roche Novartis Pfizer Boehringer-Ingelheim AstraZeneca Bayer Pharmaceuticals Eli Lilly Merck. GVM has served on an Advisory board for Pfizer. BS has served on Advisory Boards for Pfizer and Novartis. LH has served on Advisory Boards for Bristol-Myers-Squibb (compensated) PUMA (uncompensated) and HelixBio (uncompensated). LH has also received research grants from Astellas and has served as a speaker for Boehringer Ingelheim. References 1 Grande E Bolos MV Arriola E Targeting oncogenic ALK: a promising strategy for cancer treatment. Mol Cancer Ther 2011 10 569 579 21474455 2 Camidge DR Activity and safety of crizotinib in patients with ALK-positive non-small-cell lung cancer: updated results from a phase 1 study. Lancet Oncol 2012 3 Katayama R Mechanisms of acquired crizotinib resistance in ALK-rearranged lung Cancers. Sci Transl Med 2012 4 120ra117 4 Doebele RC Mechanisms of Resistance to Crizotinib in Patients with ALK Gene Rearranged Non-Small Cell Lung Cancer. Clin Cancer Res 2012 5 Lovly CM Pao W Escaping ALK inhibition: mechanisms of and strategies to overcome resistance. Sci Transl Med 2012 4 120ps122 6 Tanizaki J Activation of HER family signaling as a mechanism of acquired resistance to ALK inhibitors in EML4-ALK-positive non-small cell lung cancer. Clin Cancer Res 2012 7 Iyer G Genome sequencing identifies a basis for everolimus sensitivity. Science 2012 338 221 22923433 8 Shaw AT Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK. J Clin Oncol 2009 27 4247 4253 19667264 9 Boik JC Newman RA Boik RJ Quantifying synergism/antagonism using nonlinear mixed-effects modeling: a simulation study. Stat Med 2008 27 1040 1061 17768754 10 Mulvihill MJ Discovery of OSI-906: a selective and orally efficacious dual inhibitor of the IGF-1 receptor and insulin receptor. Future Med Chem 2009 1 1153 1171 21425998 11 Metz HE Houghton AM Insulin receptor substrate regulation of phosphoinositide 3-kinase. Clin Cancer Res 2011 17 206 211 20966354 12 Yang X Using tandem mass spectrometry in targeted mode to identify activators of class IA PI3K in cancer. Cancer Res 2011 71 5965 5975 21775521 13 Chen Z Inhibition of ALK PI3K/MEK and HSP90 in murine lung adenocarcinoma induced by EML4-ALK fusion oncogene. Cancer Res 2010 70 9827 9836 20952506 14 Lovly CM Insights into ALK-driven cancers revealed through development of novel ALK tyrosine kinase inhibitors. Cancer Res 2011 71 4920 4931 21613408 15 Katayama R Therapeutic strategies to overcome crizotinib resistance in non-small cell lung cancers harboring the fusion oncogene EML4-ALK. Proc Natl Acad Sci U S A 2011 108 7535 7540 21502504 16 Guix M Acquired resistance to EGFR tyrosine kinase inhibitors in cancer cells is mediated by loss of IGF-binding proteins. J Clin Invest 2008 118 2609 2619 18568074 17 Cortot AB Resistance to irreversible EGF receptor tyrosine kinase inhibitors through a multistep mechanism involving the IGF1R pathway. Cancer Res 2013 73 834 843 23172312 18 Garcia-Echeverria C In vivo antitumor activity of NVP-AEW541-A novel potent and selective inhibitor of the IGF-IR kinase. Cancer Cell 2004 5 231 239 15050915 19 Millo F Implication of the insulin-like growth factor-IR pathway in the resistance of non-small cell lung cancer cells to treatment with gefitinib. Clin Cancer Res 2007 13 2795 2803 17473213 20 Vazquez-Martin A IGF-1R/epithelial-to-mesenchymal transition (EMT) crosstalk suppresses the erlotinib-sensitizing effect of EGFR exon 19 deletion mutations. Scientific reports 2013 3 2560 23994953 21 Chmielecki J Optimization of dosing for EGFR-mutant non-small cell lung cancer with evolutionary cancer modeling."

Lung_Cancer

"Background We aimed to evaluate the clinical significance of microvessel density (MVD) lymphatic vessel density (LVD) and cancer-associated fibroblasts (CAFs) in relation to tumor location in advanced colorectal cancer (CRC). Methods Using immunohistochemistry we examined 181 advanced CRC patients for CD31 and D2-40 to measure MVD and LVD respectively ?-smooth muscle actin (SMA) and desmin to identify CAFs and PTEN to examine genetic changes of CAFs. To evaluate the regional heterogeneity of these properties we examined tissue from four sites (the center and periphery of the primary cancer a distant metastasis and a lymph node metastasis) in each patient. Results MVD LVD and CAFs showed significant heterogeneity with respect to the tumor location. LVD was the greatest in the center of the primary cancers and the amount of CAFs was the lowest in distant metastases. In distant metastases those from the lung had higher LVD and MVD but fewer CAFs than those from the liver peritoneum or ovary. Patients with low MVD and LVD in the center of the primary cancer had worse outcomes and patients with few CAFs in distant metastases and in the primary tumor had a lower survival rate. PTEN expression in CAFs in distant metastases was lost in 11 of 181 CRC patients (6.1%) which was associated with a worse prognosis. Conclusions The microenvironment including cancer-associated microvasculature and fibroblasts is heterogeneous with respect to the tumor location in CRC patients. Therefore heterogeneity of microenvironments should be taken into account when managing CRC patients. This study was supported by grant number 03-2011-012 from the Seoul National University Bundang Hospital Research Fund. The funder had no role in study design data collection and analysis decision to publish or preparation of the manuscript. Introduction Although the mortality rates of colorectal cancer (CRC) patients have decreased in most western countries and in several developing countries in Asia advanced CRC patients who initially present with stage IV disease or those who develop distant metastases several months after diagnosis still have a lower five-year survival rate [1] [2]._ENREF_4 Recently the range of systemic chemotherapy has expanded and targeted therapy including epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) inhibitor therapies have been used in advanced CRC patients increasing patient survival [3]. However some CRC patients respond poorly to targeted therapy despite presenting positive results in targeted therapy-specific mutation studies [4]. One possible explanation for this therapeutic failure is tumor heterogeneity; several studies have reported that CRCs possess a heterogenic genotype or phenotype including KRAS p53 and BRAF [5]“[7]. Therefore the differing characteristics of the primary tumor site and the corresponding metastatic an need to be clarified to improve the management of CRC patients with metastatic diseases. Furthermore understanding the clinicopathological characteristics of advanced CRC is important for the development and improvement of systemic therapies. Since Paget et al. first described the cancer microenvironment by the œseed and soil theory [8] there has been growing evidence that cancer-associated stroma might affect the cancer cells themselves and contribute to cancer progression [9]. The main components of the cancer microenvironment are microvasculature (microvessels and lymphatic vessels) inflammatory cells and cancer-associated fibroblasts (CAFs) [10]“[12]. The current method of verifying angiogenetic and lymphangiogenetic activity in cancer tissue is to assess microvessel density (MVD) and lymphatic vessel density (LVD) respectively. MVD has been proposed as a surrogate marker of cancer-associated angiogenesis to identify patients with a high risk of recurrence or those with poor prognoses for various cancers including CRC [13] [14]; however the prognostic correlation of angiogenesis in CRC is still controversial [15] [16]. Similar to angiogenesis LVD has received interest as a means of lymphatic metastasis and survival [17] [18] but its role in tumor progression is still unclear [19]. The other prominent component of stroma CAFs are consistently activated and affect many aspects of tumor initiation invasion and progression [9]. While some studies have suggested that CAFs may inhibit tumor progression [20] [21] other studies have proposed that CAFs may promote progression in prostate breast and skin cancers [22]“[24]. In the context of CRC Tsujino et al. have suggested that ?-smooth muscle actin (SMA)-expressing CAFs might be a useful indicator of poor prognosis. However these results were restricted to stage II and III CRCs [25]. In addition to cancer cells genetic alterations in CAFs have demonstrated including the loss of heterozygosity microsatellite instability and genetic mutations [26] [27]. Recently genetic inactivation of PTEN in CAFs was reported in breast cancer patients [28]. Trimboli et al. identified that PTEN loss in stromal fibroblasts resulted in extensive extracellular matrix remodeling and angiogenesis which characteristic of tumor progression [28]. However expression loss of PTEN and its clinical significance have not been investigated in colorectal cancer patients. The aim of this study was to investigate the characteristics of microenvironments including microvasculatures and CAFs in advanced CRC patients. Additionally we assessed the intratumoral heterogeneity in the primary tumor and the discordance between primary tumor and distant metastasis microenvironments. Materials and Methods Patient selection A total of 181 advanced CRC patients with synchronous or metachronous metastases who were treated at Seoul National University Bundang Hospital (Seongnam-si South Korea) between 2003 and 2009 were enrolled in this study. Synchronous metastases were defined as distant metastases occurring within six months of the primary diagnosis of CRC and metachronous metastases were those occurring after that time point [29]. The cancer tissue used in this study was received from patients that had surgical resection of both the primary tumor and related metastases. None of the patients had received chemo- or radiotherapy before the resection of the primary tumor. Medical charts and pathology reports were reviewed to record clinical and pathological data. Glass slides were reviewed to determine the histological type according to the WHO classification [30]. Follow-up information including the patient outcome and the time interval between the date of surgical resection and death was collected. The cases lost to follow-up and deaths from causes other than CRC were considered censored data for the survival analysis. The median follow-up period was 37.9 months (range 0.8“104.6 months). Ethical statement All human specimens were obtained from the files of surgically resected cases examined at the Department of Pathology Seoul National University Bundang Hospital for the pathologic diagnosis. The retrospective study was performed using the stored samples after the pathologic diagnosis and all of the samples were anonymized before the study. The participants did not provide written informed consent in this study. The study was approved by the Institutional Review Board of Seoul National University Bundang Hospital under the condition of anonymization (reference: B-1109/136-302). Tissue array methods To evaluate the regional stromal differences samples were taken from each patient from four areas: the center and periphery of the primary cancer distant metastases and lymph node metastases. The distant metastatic sites for the tissue arrays were as follows: liver in 83 cases (45.9%) lung in 38 cases (21.0%) seedings in 38 cases (21.0%) distant lymph nodes in 6 cases (3.3%) and ovary in 16 cases (8.3%). The representative core tissue specimens (2 mm in diameter) were taken from individual paraffin blocks and rearranged in new tissue array blocks using a trephine apparatus (Superbiochips Laboratories Seoul South Korea) [31]. Immunohistochemistry Array slides were labelled by immunohistochemistry using antibodies for CD31 (1?100 DAKO Glostrup Denmark) D2-40 (1?100 DAKO) SMA (1?1000 Neomarkers Fremont CA USA) desmin (1?300 DAKO) and PTEN (1?80 Epitomics Burlingame CA USA) after a microwave antigen retrieval procedure except SMA. Non-reactive sites were blocked using 1% horse serum in Tris-buffered saline (pH 6.0) for 3 min. Primary antibodies were applied and antibody binding was detected with diaminobenzidine (DAB). Sections were counterstained with hematoxylin. The reactivity of PTEN in each tissue section was scored as negative faint or strong and the percentage of PTEN-positive fibroblasts was quantified. For the statistical analysis the sample was deemed PTEN-positive if 5% or more CAFs were scored as strong positives. Calculation of LVD MVD and CAFs using digital pathology Slides were concurrently evaluated by two pathologists (H.E.L and H.S.L) using light microscopy to improve the accuracy of the results (Fig. 1). CRC cells were considered as internal negative controls. Medium- to large-sized vessels were considered as internal positive controls for CD31 and D2-40. Intestinal muscular layer or medium- to large-sized vessels were considered as internal positive controls for desmin and SMA. Samples showing inappropriate staining in internal negative or positive controls were considered non-informative and were excluded from the analysis. Slides were scanned using an Aperio ScanScope® CS instrument (Aperio Technologies Inc. Vista CA) at 20× magnification. Subsequently they were analyzed in ImageScope„¢ using the Microvessel Analysis v1 algorithm (Aperio Technologies) and MVD and LVD were calculated. Because desmin-positive muscularis mucosa and propria are positive for SMA immunostaining the area of CAFs (mm2) was calculated by subtracting the areas of desmin staining from that of SMA staining (SMA - desmin). 10.1371/journal.pone.0091811.g001 Figure 1 Representative sections from four tumor locations stained with CD31 D2-40 SMA or desmin antibodies (×100). Statistical analysis A chi-squared test or Fisher's exact test (2-sided) for non-continuous variables and Mann-Whitney or Kruskal-Wallis analysis for continuous variables were used to compare each parameter with respect to the CRC site and according to its clinicopathologic features. The correlation between continuous variables was analyzed using the Pearson correlation coefficient. To determine the best cut-offs of continuous variables for predicting patient survival the maximal chi-squared method was performed using R program (http://cran.r-project./). The overall survival curves were plotted using the Kaplan-Meier product-limit method and the significance of the differences between these curves was determined using the log-rank test. A univariate and multivariate regression analysis was performed using the Cox's proportional hazards model to determine hazard ratios (HRs). P-values of less than 0.05 were considered statistically significant. All statistical analysis excluding the maximal chi-squared test was performed with the IBM SPSS statistics 20 (Armonk NY USA). Results 1. Heterogeneity of cancer-associated stroma according to examined tumor locations The clinicopathological characteristics of the advanced CRC patients are described in Table 1. The CRC patients with synchronous metastases had aggressive features including larger tumor size more advanced pT and pN stage and the presence of perineural and venous invasion than the patients with metachronous metastasis (p<0.05). 10.1371/journal.pone.0091811.t001 Table 1 Clinicopathologic characteristics of advanced colorectal cancers. Parameters Total Metachronous Synchronous P value (n?=?181) (n?=?57) (n?=?124) Age (median range) 60.00 (28“93) 62.00 (36“79) 60.00 (28“93) 0.241 Sex 0.007 Male 97 39 (68.4%) 58 (46.8%) Female 84 18 (31.6%) 66 (53.2%) Location 0.055 Right colon 37 6 (10.5%) 31 (25.0%) Left colon 75 29 (50.9%) 46 (37.1%) Rectum 69 22 (38.6%) 47 (37.9%) Size of primary tumor 5.30 (2.0“13.0) 4.20 (2“9) 5.50 (2.5“13) <0.001 Histologic grade 0.227 Low grade 157 52 (91.2%) 105 (84.7%) High grade 24 5 (8.8%) 19 (15.3%) T stage <0.001 T1 0 0 0 T2 5 3 (5.3%) 2 (1.6%) T3 107 45 (78.9%) 62 (50.0%) T4 69 9 (15.8%) 60 (48.4%) N stage <0.001 N0 35 23 (40.4%) 12 (9.7%) N1 58 23 (40.4%) 35 (28.2%) N2 88 11 (19.3%) 77 (62.1%) Perineural invasion 0.011 Absent 89 36 (63.2%) 53 (42.7%) Present 92 21 (36.8%) 71 (57.3%) Venous invasion 0.028 Absent 126 46 (80.7%) 80 (64.5%) Present 55 11 (19.3%) 44 (35.5%) The heterogeneous values for LVD MVD and CAF area are shown in Fig 2. LVD was the highest in center of the primary cancers (median interquartile range (IQR); 37.00 10.50“81.00) than any other site (5.00 1.00“23.75 at the periphery; 2.50 1.00“15.00 in lymph node metastases; 3.00 1.00“20.00 in distant metastases). MVD was lower in distant metastases (median IQR; 641.50 428.00“1006.75) than at the periphery of the primary cancer (731.00 508.25“1049.75) and lymph node metastases (893.50 520.25“1275.25). The area occupied by CAFs was the lowest in the distant metastases (median IQR; 0.91 0.68“1.18) than any other site (1.12 0.88“1.41 in the center; 1.22 0.96“1.54 in the periphery1.4 1.00“1.71 in lymph node metastases). In addition the stromal characteristics varied in relation to the metastatic an examined. MVD and LVD were the higher in lung metastases than those in the liver peritoneum or lymph nodes (p<0.001; Fig. 3). However the amounts of CAFs were consistent among the different metastatic ans (p?=?0.180). 10.1371/journal.pone.0091811.g002 Figure 2 Heterogeneity of lymphatic vessel density (LVD) microvessel density (MVD) and amount of cancer-associated fibroblasts (CAFs) with respect to tumor location. The LVD (A) MVD (B) and CAF area (C) was significantly different according to each tumor location. 10.1371/journal.pone.0091811.g003 Figure 3 LVD MVD and CAF area at different distant metastasis sites. The characteristics of cancer-associated stroma differed with respect to the metastatic site. LVD (A) and MVD (B) were greater in the metastatic tumor samples collected from the lung than in samples collected from other metastatic sites (p<0.001). However the amount of CAFs was not significant different between metastatic sites (C). Despite the heterogeneity of stromal characteristics CRC cases with higher LVD MVD and CAFs in center of the primary cancers had a tendency of higher LVD MVD and CAFs in periphery (p<0.05; Table S1). However LVD in center and periphery of primary cancer were not correlated with LVD in related distant metastasis (Table S1). In addition the amount of microvasculature was significantly correlated with the amount of CAFs (Table S2). 2. Clinical significance of cancer-associated stroma in advanced CRCs The MVD LVD and amount of CAFs present at each tumor location were compared according to their clinicopathologic features (Table 2). High grade CRCs were associated with lower CAFs in samples taken from the central cancer site (p?=?0.041). When compared with synchronous metastases the patients with metachronous metastases had higher LVD in center and periphery of the primary cancer and had higher MVD in lymph node metastases. Most patients with metachronous metastases were treated by adjuvant chemotherapy before metastasectomy. LVD and MVD in the distant metastases were significantly higher in the patients who had received chemotherapy before metastasectomy than those who did not (p?=?0.011 and 0.048 respectively). 10.1371/journal.pone.0091811.t002 Table 2 Clinicopathologic factor and LVD MVD and CAFs. Center (median) Periphery (median) LN metastasis (median) Distant metastasis (median) LVD MVD CAFs LVD MVD CAFs LVD MVD CAFs LVD MVD CAFs Total 39 717 1.13 5 740 1.22 3 888 1.42 3 648 0.91 Histologic grade Low grade 40 717 1.15* 5 741 1.23 3 895 1.43 3 665 0.92 High grade 34 683.5 0.94* 6 643.5 1.18 2 656 1.32 6 498 0.82 pT stage pT2 34 758 1.15 16 870 1.48 6 772 0.73 pT3 47 737 1.19 5 803 1.22 2.5 884 1.43 3 724 0.92 pT4 33 639 1.09 4 630 1.22 3 895 1.41 3 520 0.93 LN metastasis Absent 49 602 1.15 8 712 1.42 4 772 0.94 Present 39 737.5 1.12 4 740 1.21 3 884 1.41 3 617 0.91 Perineural invasion Absent 41 738 1.12 6 772 1.32 5.5 931.5 1.42 4 687 0.94 Present 39 672 1.13 4 702 1.2 2 796 1.39 3 548.5 0.86 Metastasis Synchronous 34* 717.5 1.11 3.0* 741 1.21 3 797* 1.39 3 617 0.93 Metachronous 55* 716 1.21 8.0* 712 1.23 2 1117* 1.63 5 698 0.91 Chemotherapy  Not done 2.0* 597.5* 0.93 Done 10.0* 684* 0.91 * p<0.05; ** p<0.01;   chemotherapy prior to metastatectomy of distant metastasis. 3. Expression loss of PTEN in CAFs PTEN was expressed in cytoplasm and sometimes the nucleus of both cancer and non-neoplastic cells when examined using immunohistochemistry. Expression of PTEN was lost in 8 cases in the center 2 cases in the periphery 4 cases in lymph node metastases and 11 cases in distant metastases (Table S3). In all 11 distant metastases with PTEN loss PTEN expression was intact in both the center and periphery of primary cancer (data not shown). PTEN loss in distant metastasis was correlated with synchronous metastasis (p?=?0.018). 4. Cancer-associated stroma and patient prognosis By using the obtained cut-offs lower LVD MVD and CAFs in the center LVD and CAFs in the periphery and MVD and CAFs in distant metastases were all significantly correlated with lower survival (p<0.05; Fig. S1). Among other clinicopathologic features synchronous metastasis old age larger size high histologic grade advanced pT and pN stage and presence of perineural invasion were associated with a worse prognosis (Table 3). By multivariate Cox regression analysis the hazard ratio of synchronous versus metachronous was the highest (4.029) with the lowest p value (p<0.001). CAFs in distant metastasis LVD and MVD in the center LVD in the periphery age and perineural invasion also independently predicted patient survival. In addition loss of PTEN expression in CAFs in distant metastases was associated with a worse prognosis (p?=?0.042; Fig S2) but not in primary cancer or lymph node metastasis. 10.1371/journal.pone.0091811.t003 Table 3 Univariate and multivariate survival analysis according to clinicopathologic features. Univariate survival analysis Multivariate survival analysis Factors HR (95% CI) P value HR (95% CI) P value Synchronous vs. Metachronous 4.617 (2.472“8.624) <0.001 3.762 (1.838“7.701) <0.001 Age 1.023 (1.004“1.044) 0.020 1.033 (1.011“1.056) 0.003 Sex (female vs. male) 1.428 (0.920“2.218) 0.113 ” ” Location (left vs. right) 0.503 (0.314“0.806) 0.004 0.700 (0.413“1.188) NS (0.186) Size 1.073 (1.005“1.146) 0.036 1.040 (0.903“1.198) NS (0.584) Histologic grade (high vs. low) 1.862 (1.061“3.269) 0.030 1.491 (0.763“2.912) NS (0.243) pT stage (pT4 vs. pT2/3) 2.341 (1.503“3.645) <0.001 1.137 (0.674“1.921) NS (0.630) pN stage (pN1/2 vs. pN0) 3.848 (1.760“8.411) 0.001 1.773 (0.758“4.146) NS (0.186) Perineural invasion 2.628 (1.640“4.211) <0.001 2.108 (1.265“3.513) 0.004 Venous invasion 1.217 (0.757“1.956) 0.418 ” Center LVD (high vs. low) 0.364 (0.158“0.836) 0.017 0.298 (0.118“0.753) 0.010 Center MVD (high vs. low) 0.391 (0.233“0.655) <0.001 0.437 (0.238“0.801) 0.007 Center CAFs (high vs. low) 0.579 (0.352“0.954) 0.032 1.038 (0.607“1.773) NS (0.892) Periphery LVD (high vs. low) 0.235 (0.086“0.644) 0.005 0.279 (0.096“0.809) 0.019 Periphery MVD (high vs. low) 1.456 (0.911“2.327) 0.117 ” Periphery CAFs (high vs. low) 0.524 (0.336“0.817) 0.004 0.813 (0.499“1.326) NS (0.406) LN LVD (high vs. low) 1.646 (0.874“3.100) 0.123 ” LN MVD (high vs. low) 0.597 (0.294“1.213) 0.154 ” LN CAFs (high vs. low) 0.717 (0.423“1.217) 0.218 ” Metastasis LVD (high vs. low) 0.569 (0.314“1.032) 0.063 ” Metastasis MVD (high vs. low) 0.579 (0.364“0.921) 0.021 1.262 (0.720“2.211) NS (0.417) Metastasis CAFs (high vs. low) 0.492 (0.271“0.894) 0.020 0.290 (0.144“0.582) 0.001 Metastasis PTEN (intact vs. loss) 0.454 (0.208“0.993) 0.048 0.575 (0.239“1.383) NS (0.217) Discussion Carcinoma cells in different tissue areas have distinct characteristics [32]. In central areas of the tumor carcinoma cells maintain an epithelial cell phenotype but carcinoma cells in the invasive front acquire a more malignant and mesenchymal phenotype and are thought to have an increased migratory capacity and contribute to metastatic diseases. These metastatic cells may restore the epithelial phenotype at metastatic sites [33]. In addition to carcinoma cells themselves microenvironment is suggested to be uneven within a given tumor because tumor formation and progression involve the co-evolution of cancer cells and microenvironments [34]. The present study demonstrated that the cancer-associated microenvironment also had distinct characteristics in different areas. Of the sites examined LVD was highest in the center of the primary cancer. MVD was slightly higher in center than at the periphery of the primary cancer but this difference was not statistically significant. Interestingly the amount of CAFs in distant metastases was significantly lower than in center and periphery of the primary cancer. We show that the stromal microenvironment has regional heterogeneity both within the primary tumor and between the primary site and its related metastases. Furthermore our data suggests that the stromal heterogeneity might be attributable to tumor heterogeneity. Therefore it would be beneficial to consider both stromal and tumor cell heterogeneity in order to manage CRC patients better. We evaluated the MVD LVD and amount of CAFs in metastatic tissues of various ans including the liver lung peritoneal seeding distant lymph nodes and ovary. Of the metastatic ans we examined both LVD and MVD were the highest in lung. In our previous study the KRAS discordance rate was also significantly higher in matched lung metastases than in other matched metastatic ans [35]. The underlying mechanism is not known. It could be that primary CRCs with high LVD and MVD have a tendency to produce lung metastases; however our results indicated that LVD and MVD in the center and at the periphery of the primary cancers were lower in the patients with lung metastases (data not shown). Alternatively it may be due to the physiological characteristics of metastatic ans interactions between cancer cells and microenvironment within the metastatic an or the characteristics of the cancer cell clones prone to lung metastasis. However technical or sampling errors also may be possible thus further large-scale studies are required. Although numerous studies have attempted to demonstrate an association between tumor microenvironment characteristics and survival the prognostic impacts of MVD and LVD are still controversial. Some studies have been presented that active angiogenesis and lymphangiogenesis represented by high MVD and LVD are associated with poor prognosis and aggressive clinicopathologic factors [36] [37]. Recent meta-analysis has demonstrated that LVD was significantly associated with disease-free survival but not overall survival [38]. Other studies have reported no statistical significance of MVD and LVD on survival [39]. Prall et al. has reported that high MVD and LVD are related with better survival in a consecutive series and liver metastases [40]. Our results were based on patients with advanced disease with distant metastasis and we showed that high MVD and LVD were related with improved survival. This might be because all the patients in this study had confirmed to have distant metastasis and microvasculatures could influence even delivery of the chemotherapeutic drug into the tumor. However our study had some limitations in terms of the survival analysis. We enrolled the CRC patients with available surgically resected cancer tissues from both primary tumors and corresponding metastatic tumors. Not all advanced CRC patients with metastatic diseases were included and far advanced cases were not enrolled because of their inoperability. Therefore unrecognized biases might have influenced our survival results. Some studies have demonstrated an anti-tumorigenic effect of fibroblasts [20] [21]. However it has become clear that CAFs contribute to the progression of cancer and their prognostic significance in various cancers also has been raised [41] and furthermore several studies have observed genetic alterations in CAFs [26] [27]. PTEN loss of CAFs has been observed in breast cancer and prognostic association of it has been suggested [27] [28]. We observed PTEN loss of CAFs in CRC patients and it was more frequently observed in the corresponding distant metastases. It is suggested that CAFs not only cancer cells have altered gene expression. Moreover loss of PTEN expression of CAFs in distant metastases was significantly correlated with the survival of patients. To our knowledge these are the first results showing PTEN loss in CAFs in CRC patients. Although more research is required we expect that it might be a prognostic factor in CRC patients. In our large cohort of advanced CRC patients with synchronous and metachronous distant metastasis we demonstrated the regional heterogeneity of stromal microenvironment factors according to the tumor location. The amount of microvasculature measured by LVD and MVD was also heterogeneous in relation to the metastatic an examined. By Cox regression analysis center LVD and MVD periphery LVD and CAFs in distant metastasis were independently associated with patients' prognosis in addition to synchronous distant metastasis age and perineural invasion. Heterogeneity of microenvironment not only of cancer cells is suggested to contribute to tumor heterogeneity and biologic complexity thus it should be considered in managing CRC patients. In addition our results showed that PTEN expression was altered in CAFs of CRCs suggesting that CAFs might have altered gene expression and play an active role in cancer progression. Supporting Information Figure S1 The prognostic association of stromal characteristics as it relates to tumor location. The analysis was performed by using cut-off values obtained by maximal chi-squared methods. (TIF) Click here for additional data file. Figure S2 Representative PTEN antibody stainings of stromal cells and the prognostic association of PTEN expression. (A) Intact expression of PTEN in CAFs (×400) and (B) loss of PTEN expression in CAFs (×400). (C“F) Kaplan-Meier survival curves for the center (C) and periphery (D) of the primary tumor lymph node metastases (E) and distant metastases (F) according to CAF PTEN expression status. (TIF) Click here for additional data file. Table S1 Pearson's correlation coefficients among center periphery lymph node metastasis and distant metastasis. (DOCX) Click here for additional data file."

Lung_Cancer

"which was then transferred to a polyvinylidene difluoride membrane (Millipore Billerica MA) by using a transfer apparatus according to the manufacturer's protocols. Membranes were blocked with 3% nonfat milk in TBST buffer (10 mM Tris-HCl pH 8.0 150 mM NaCl and 0.05% Tween 20) for 1 h washed in the same buffer and incubated with antibodies against Sp1 (Millipore) GFP (Clontech Palo Alto CA) FOXO3 (Genetex Hsinchu Taiwan) tubulin (Sigma-Aldrich St. Louis MO) N-cadherin (Cell Signaling Technology Beverly MA) ?-catenin (Cell Signaling Technology) and vimentin (Epitomics Burlingame CA) at 4? overnight. Membranes were washed three times for 10 min and incubated with the secondary antibody (goat anti-rabbit or anti-mouse immunoglobulin G linked with horse radish peroxidase (Millipore)) for 1 h at room temperature. After three more washes the protein bands were detected with the ECL Western blotting Detection System (Millipore) and recorded with the FluorChem image analysis system (Alpha Innotech San Leandro CA). Band intensities were quantified with Scion image software (Scion Frederick MD). Luciferase reporter assay Cells were transiently cotransfected with reporter plasmids (pGL2-miR-182 pGL2-miR-182 mutants pGL3-FOXO3-3'UTR or pGL2-FOXO3) and expression plasmids of interest using Lipofectamine 2000. Luciferase activity in cell lysates was determined by luminometer (LB9506; Berthold Technologies Bad Wildbad Germany) and normalized to total protein concentration. For construction genomic DNA of A431 cells were prepared. The miR-182 promoter (-1000/+50) was produced by PCR using primers: F 5'-GGG CAG GCA GCC TGC ACC CT-3' and R 5'-CAC CAG TGT GAG TTC TAC CAT TGC-3'. The FOXO3 (-1000/+50) promoter was produced by PCR using primers: F 5'-ACG CGT CGA GCT GAC AGG CGG TTC-3' and R 5'-AGA TCT CGC CCC CCG GCC AGG CCG-3'. After amplification and purification the DNA fragments were ligated to pGL2 vector using restriction enzymes KpnI and BglII for pGL2-miR-182 MluI and BglII for pGL2-FOXO3 (New England Biolabs Ipswich MA). For construction of pGL3-FOXO3-3'UTR cDNA of H1299 cells was prepared. The FOXO3 3'UTR was produced by PCR using primers: F 5'-TCT AGA AGG ATC ACT GAG GAA GGG-3' and R 5'-TCT AGA TCT GCA AAG CAA AAC AGG-3'. After amplification and purification the DNA fragments were ligated to pGL3 vector using restriction enzymes XbaI. For construction of pGL2-miR-182 mutants the pGL2-miR-182 plasmid was used as the template for mutagenesis of Sp1-binding sites. Primers for mutations of site 1 (-433C/-434G to?433A/-434A): F 5'-CTT AGT AAA TAG CAA AAC CCA AAC CAC ATT AGC CAT CTC TTC CC-3' and R 5'- GGG AAG AGA TGG CTA ATG TGG TTT GGG TTT TGC TAT TTA CTA AG-3'; and for site 2 (-398C/-399G to?398A/-399A): F 5''-CCA GCG CCC AGG GAA AGG GCT CTC TGG C-3' and R 5'- GCC AGA GAG CCC TTT CCC TGG GCG CTG G?3'. Mutagenesis was performed by PCR using plaque-forming unit DNA polymerase (Agilent Technologies Santa Clara CA). Chromatin immunoprecipitation (ChIP) assay The protocol for ChIP was performed as described previously [23]. Briefly formaldehyde-fixed DNA“protein complex was immunoprecipitated with 5 mg of normal rabbit IgG anti-acetyl-Histone H3 (Millipore) or anti-Sp1 antibodies. Immunoprecipitated DNA was analyzed by PCR. The primer sequences for promoter of miR-182 in PCR analyses were as follows: F 5'-ACT TCC CTC TCT CCC TTT GG-3' and R 5'-CAC CTG ACA GCA GGG ACT CA-3'. The primer sequences for promoter of miR-212 in PCR analyses were as follows: F 5'-AGC GGA GCT GTC CTC TCA G-3' and R 5'-CCG GGC AGT AAG CAG TCT A-3'. The primer sequences for promoter of p21 in PCR analyses were as follows: F 5'- ACC AAC GCA GGC GAG GGA CT-3' and R 5'- CCG GCT CCA CAA GGA ACT GA?3'. DNA affinity precipitation assay (DAPA) The DNA oligonucleotides (miR-182 5'-AAA ACC CAG CCC ACA TTA GCC ATC TCT TCC CCA GCG CCC AGG GGC AGG GCT CT-3'; miR-212 5'-GAC CGG GGG GGC GGG GCC TCC CAG GTC CCG CCC CGC CCC CAC GCC CCC GCC GG-3'; and p21 5'- CCC GCC TCC TTG AGG CGG GCC CGG GCG GGG CGG-3') were biotinylated at 5' end and then annealed with their complementary strands. The assay was performed by incubating 1 ?g of biotin-labeled probe with cell extract in 1 ml of DAPA buffer (60 mM KCl 12 mM HEPES pH 7.9 4 mM Tris-HCl 5% glycerol 0.5 mM EDTA and 1 mM dithiothreitol). After incubation for 1 h at 4? DNA“protein complexes were then incubated with 20 ?l of streptavidin-agarose (Sigma-Aldrich) for 1 h at 4?. DNA“protein complexes were then washed three times in the DAPA buffer. Clinical specimens of patients with lung adenocarcinoma Human clinical specimens used in this study were approved by the Clinical Research Ethics Committee at the Medical Center of National Cheng Kung University (Tainan Taiwan). After surgical resection at National Cheng Kung University Hospital specimens of patients with lung adenocarcinomas were collected for immunohistochemical analysis RT-PCR or Western blotting. shRNA lentivirus production We purchased scramble Sp1 and FOXO3 shRNA from National RNAi Core Facility in Academia Sinica of Taiwan (Taipei Taiwan) and miRZip and miRZip-182 from SBI (System Biosciences CA). The lentiviruses were obtained from RNAi Core of Research Center of Clinical Medicine National Cheng Kung University Hospital. (The protocol is described below-293T cells were cotransfected with 5 ?g of packaging plasmid (pCMV?R8.91) 0.5 ?g of envelop plasmid (pMD.G) and 5 ?g of pLKO.1 shRNA using Lipofectamine 2000 for 6 h. After 24 h incubation the supernatants containing viral ps were harvested and filtered through 0.45 mm filters.) Fluorescence-activated cell sorting (FACS) The miRZip and miRZip-182 stable expression H1299 cells were washed with PBS and fixed in cold 70% ethanol overnight at 4 Cells were then washed with cold PBS and permeabilized with 0.1% Triton X-100 for 10 min. After treatment with 10 ?g/ml RNase A (Qiagen Germantown MD) at 37 for 1 h cells were stained with 50 ?g/ml propidium iodide (Sigma-Aldrich) at room temperature for 2 h. Finally cells were analyzed by flow cytometer on the FACSCalibur (BD Biosciences Franklin Lakes NJ). Xenograft study The animal experiment was approved by the Institutional Animal Care and Use Committee at National Cheng Kung University. Female SCID mice were purchased from National Laboratory Animal Center in Taiwan. The miRZip and miRZip-182 stable expression H1299 cells (106 cells) were suspended in 100 ?l of PBS and implanted into the back of SCID mice. Immunohistochemistry (IHC) The experimental process of IHC was performed as described in our previous study [32]. Briefly blocked histological sections were stained with the anti-Sp1 or anti-FOXO3 antibodies. The immunoreactivity was detected by a Vectastain ABC kit (Vector Laboratories Burlingame CA). Quantitative PCR Quantitative Real-time PCR was performed using SYBR Premix Ex Taq (Takara Bio Otsu Shiga Japan) in a CFX96TM Real-Time System and C1000 TM Thermal Cycler (BIO-RAD Hercules CA). The primers for quantitative PCR were as follows: Firefly luciferase F 5'-TCA AAG AGG CGA ACT GTG TG-3' R 5'-GGT GTT GGA GCA AGA TGG AT-3'. Immunofluorescent staining The miRZip and miRZip-182 stable expression H1299 cells were seeded onto coverslips (with a thickness of 0.17 mm) and incubated for 24 h. After fixation with 4% paraformaldehyde (Sigma-Aldrich) in PBS for 15 min and permeabilization with 1% Triton X-100 for 5 min cells on the coverslip were blocked with 1% bovine serum albumin (Sigma-Aldrich) for 1 h and stained with the antibody against F-Actin by using Alexa Fluor 568-conjugated phalloidin (Invitrogen) for 1 h at room temperature. Subsequently cells on the coverslip were washed with PBS three times. Finally cells were mounted in Prolong Gold antifade reagent with DAPI (Invitrogen) and examined using immunofluorescence microscope (Delta Vision Personal DV). The images were analyzed with softWoRx software (Applied Precision). Wound-healing assay The miRZip and miRZip-182 stable expression H1299 cells (1.5 x 106) were seeded in 6 cm dish and cultured for 24 h the linear wound of cellular monolayer was created by scratching confluent cell monolayer using a plastic pipette tip. The monolayer of Scratched cell was washed by PBS to remove debris. After incubation at 37? with 5% CO2 for 16 h area of migration was photographed under light microscope for evaluation. Transwell migration assay The cell migration assay was performed using Transwell system with an 8 mm pore size polycarbonate filter membrane (Corning Costar Cambridge MA). Cells were trypsinized and suspended in serum-free DMEM. Upper wells were filled with cell suspensions in serum-free DMEM and lower wells were filled with DMEM containing 10% fetal bovine serum. After incubation for 14 h at 37? with 5% CO2 the lower side of filter membrane was fixed with methanol and stained with DAPI. The migrated cells were counted under a fluorescent microscope and quantified by Image J software "

Lung_Cancer

"or paclitaxel (200 mg/m2)/carboplatin (area under the curve 6.0) on day 1 every 3 weeks. Chemotherapy was continued for at least three cycles. Gefitinib was administered until the disease progressed intolerable toxicities developed or consent was withdrawn. The protocol recommended that the crossover regimen be used as a second-line treatment. Clinical Assessments The antitumor response to treatment was assessed using computed tomography every 2 months. Unidirectional measurements were adopted on the basis of the Response Evaluation Criteria in Solid Tumors (version 1.0).23 PFS was evaluated from the date of randomization to the date when disease progression was first observed or death occurred. The treatment response and PFS were determined by an external review of computed tomography scans by experts who were not aware of the treatment assignments. Overall survival (OS) was evaluated from the date of randomization to the date of death. Statistical Analysis To assess prognostic factors for OS we used univariate and multivariate Cox proportional hazards models. Kaplan“Meier survival curves were constructed for PFS and OS and differences between groups were identified using the log-rank test. Differences in response rates were identified using Fisher™s exact test. Each analysis was two sided with a 5% significance level and a 95% confidence interval. All analyses were performed using SAS for Windows software (release 9.1; SAS Institute Cary NC). RESULTS Patient Population A total of 230 chemonaive patients were enrolled in the NEJ002 study: 115 patients were assigned to receive gefitinib and 115 were assigned to receive carboplatin-paclitaxel (Fig. 1). To evaluate the efficacy of gefitinib in NSCLC patients with uncommon EGFR mutations we analyzed the data of 114 patients in the gefitinib group and 111 patients in the carboplatin-paclitaxel group. We identified five patients who had uncommon EGFR mutations in each group. Two patients who had common mutations and were treated with first-line chemotherapy consisting of carboplatin-paclitaxel were excluded from the PFS analysis in the NEJ002 study. However both were treated with gefitinib and were included in this post-hoc analysis. The demographic and disease characteristics of the patients with uncommon EGFR mutations were similar to those of patients with common EGFR mutations (). The characteristics of each patient with uncommon EGFR mutations are shown in supplementary Table S1 (Supplemental Digital Content 1 http://links.lww.com/JTO/A494). FIGURE 1. Enrollment randomization and follow-up of the study patients. TABLE 1. Patient Characteristics Survival Factors In the univariate analysis of 225 patients who received gefitinib at any point uncommon EGFR mutations had a significant detrimental effect on survival (). We also identified performance statuses 1 and 2 distant metastasis brain metastasis stable disease and progressive disease as significant predictors of worse prognosis for standard chemotherapy and stable disease and progressive disease as significant predictors of worse prognosis for gefitinib. When these variables were included in the Cox proportional hazards model we found that uncommon EGFR mutations performance statuses 1 and 2 stable disease and progressive disease for standard chemotherapy and stable disease and progressive disease for gefitinib had significant hazard ratios (). TABLE 2. Univariate and Multivariate Analysis by Cox Proportional Hazards Model Uncommon EGFR Mutations and Survival The Kaplan“Meier curve for OS for uncommon versus common EGFR mutations is shown in A. The OS was significantly shorter among patients with uncommon EGFR mutations compared with OS of those with common EGFR mutations in the overall population (12 versus 28.4 months; p = 0.002). A significantly shorter survival time was observed in patients with uncommon EGFR mutations compared with survival time in those with common EGFR mutations in the gefitinib group (11.9 versus 29.3 months; p < 0.001) (Fig. 2B). However a similar survival time was observed between the subgroups of uncommon and common EGFR mutations in the carboplatin-paclitaxel group (22.8 versus 28 months; p= 0.358) (Fig. 2C). FIGURE 2. The overall survival curves of patients with common mutations and uncommon mutations in the entire population (A) the gefitinib group (B) and the carboplatin-paclitaxel group (C). To examine whether the sequence of platinum doublet and gefitinib affected OS we performed a further subgroup analysis. The survival time tended to be shorter among patients receiving first-line gefitinib compared with the survival time among those receiving first-line carboplatin-paclitaxel in the uncommon EGFR mutation group (11.9 versus 22.8 months; p = 0.102). Consistent with previous publications a similar survival time was observed between patients receiving first-line gefitinib and those receiving first-line carboplatin-paclitaxel in the common EGFR mutation group (29.3 versus 28 months; p = 0.378). Uncommon EGFR Mutations PFS and Response In the gefitinib group the median PFS was significantly shorter for patients with uncommon EGFR mutations compared with median PFS of those with common EGFR mutations (2.2 versus 11.4 months; p < 0.001) (Fig. 3A). By contrast the median PFS did not differ significantly between patients with uncommon EGFR mutations and those with common EGFR mutations in the carboplatin-paclitaxel group (5.9 versus 5.4 months; p = 0.847) (Fig. 3B). The objective response rate was significantly lower in patients with uncommon EGFR mutations compared with the objective response rate in those with common EGFR mutations when treated with gefitinib (20% versus 76%; p = 0.017) (supplementary Table S2 Supplemental Digital Content 1 http://links.lww.com/JTO/A494). By contrast similar objective response rates were observed for patients with uncommon EGFR mutations and those with common EGFR mutations in the carboplatin-paclitaxel group (20% versus 32%; p = 0.336) (supplementary Table S2 Supplemental Digital Content 1 http://links.lww.com/JTO/A494). FIGURE 3. Progression-free survival curves in the gefitinib group (A) and the carboplatin-paclitaxel group (B) according to the type of epidermal growth factor receptor mutation. DISCUSSION Recent studies suggest that NSCLC patients with uncommon EGFR mutations are less responsive to EGFR-TKIs compared with patients with L858R and exon 19 deletions.9“20 However the efficacy of EGFR-TKIs in NSCLC patients with uncommon mutations has not been fully elucidated. We conducted a post-hoc analysis of the NEJ002 study to evaluate the effectiveness of gefitinib against NSCLC with G719X or L861Q. The NEJ002 study comparing gefitinib and standard carboplatin-paclitaxel chemotherapy as the first-line treatment for patients with EGFR mutations demonstrated no significant difference in OS between gefitinib and carboplatin-paclitaxel.6 In contrast to other phase 3 trials investigating EGFR-TKIs for patients with common EGFR mutations of exon 19 deletion and L858R the NEJ002 is the only study that included uncommon EGFR mutations of G719X and L861Q. The current study clearly demonstrated that NSCLC patients with the uncommon EGFR mutations G719X and L861Q had shorter survival than the survival of those with an exon 19 deletion or L858R mutation (Fig. 2). Our results are consistent with other clinical studies on EGFR-TKIs in patients with uncommon EGFR mutations (supplementary Table S3 Supplemental Digital Content 1 http://links.lww.com/JTO/A494). The overall response rate to EGFR-TKIs in patients with uncommon EGFR mutations was 41% which is lower than the response rate to TKIs (62%“83%) of patients with common EGFR mutations.7824 In the NEJ002 study G719X included G719C and G719S. No patients harbored G719A. To investigate the effectiveness of gefitinib on each uncommon EGFR mutations we evaluated the difference in OS between patients with uncommon EGFR mutations (G719C versus G719S and G719X versus L861Q). There was no significant difference between these subgroups (data not shown). This study showed that the PFS and OS tended to be shorter among patients treated with first-line gefitinib compared with PFS and OS among those treated with first-line carboplatin-paclitaxel in the uncommon EGFR mutation group (supplementary Table S2 Supplemental Digital Content 1 http://links.lww.com/JTO/A494). We also found poor disease control rate with gefitinib in patients with uncommon mutations. Three of five patients with uncommon mutations in the gefitinib group had progressive disease. By contrast no patients with uncommon mutations had progressive disease in the carboplatin-paclitaxel group. Although the number of patients with uncommon mutations in each treatment group was small platinum-doublet therapy might be a better choice than gefitinib for first-line therapy in patients with uncommon EGFR mutations. Because some of patients with uncommon mutations showed good clinical response to gefitinib in this study and they seemed to be heterogeneous in terms of response to gefitinib administration of gefitinib should be considered for patients with uncommon mutations when disease progression was observed after first-line chemotherapy. In vitro studies have indicated that the affinity of gefitinib for EGFR proteins with uncommon EGFR mutations is lower than the affinity of gefitinib for EGFR proteins with common EGFR mutations.25 A sixfold or 14-fold higher concentration of gefitinib was required to inhibit the growth of cells expressing G719X or L861Q respectively compared with cells expressing L858R.26 These results may explain the lack of response to gefitinib in patients with uncommon EGFR mutations. The authors also examined the sensitivity of G719X and L861Q mutations to erlotinib and irreversible TKIs.27 Cells expressing G719X were less resistant to erlotinib than gefitinib in vitro; however L861Q was resistant to both erlotinib and gefitinib. In contrast to erlotinib irreversible TKIs inhibited the growth of cells with G719X or L861Q at a lower concentration than those with wild-type EGFR. Indeed Sequist et al.28 reported that the effectiveness of an irreversible pan-ErbB receptor TKI neratinib on NSCLC patients with G719X. Niratinib induced partial responses in three of four patients with G719X and the fourth had durable stable disease for 40 weeks. It may be beneficial to evaluate erlotinib as a treatment for NSCLCs with G719X and irreversible EGFR-TKIs as treatments for NSCLCs with G719X and L861Q. Because previous phase 3 trials that investigated erlotinib or irreversible TKIs for NSCLC with EGFR mutations did not include uncommon EGFR mutations further clinical studies may need to be performed.7829 Another possible strategy for the treatment of uncommon EGFR mutations is the combination of EGFR-TKIs and cytotoxic agents. Our group has undertaken a randomized phase 3 trial to compare gefitinib plus carboplatin plus pemetrexed with gefitinib monotherapy for patients with NSCLC with an exon 19 deletion or an L858R G719X or L861Q EGFR mutation (NEJ009; University Hospital Medical Information Network Clinical Trials Registry [UMIN-CTR] number UMIN000006340). The data from this study will advance the treatment of NSCLC with uncommon EGFR mutations. In conclusion our post-hoc analysis clearly demonstrated shorter survival of TKI-treated patients with uncommon EGFR mutations compared with survival of those with common EGFR mutations. Furthermore the data suggest that the first-line chemotherapy may be relatively effective for NSCLC with uncommon EGFR mutations. ACKNOWLEDGMENTS Special thanks to Hiromi Odagiri for her expert assistance with data collection and management. This study was supported by the Tokyo Cooperative Oncology Group. The first two authors contributed equally to this work. Disclosure: Dr. Yoshizawa received grants and lecture fees from AstraZeneca; Dr. Maemondo received lecture fees from AstraZeneca and Chugai; Dr. Inoue received lecture fees from AstraZeneca and Chugai; Dr. Gemma received grants and lecture fees from AstraZeneca; Dr. Hagiwara received patent fees from Mitsubishi Chemical Medience consulting fees and lecture fees from AstraZeneca; Dr. Kobayashi received grants from Novartis Nihon Kayaku Chugai Shionogi Kyowa Kirin Yakult Taiho and AstraZeneca and lecture fees from AstraZeneca Chugai and Bristol-Myers Squibb. The remaining authors declare no conflict of interest. REFERENCES 1. Kim ES Hirsh V Mok T Gefitinib versus docetaxel in previously treated non-small-cell lung cancer (INTEREST): a randomised phase III trial. Lancet 2008 372 1809 1818 19027483 2. Shepherd FA Rodrigues Pereira J Ciuleanu T National Cancer Institute of Canada Clinical Trials Group Erlotinib in previously treated non-small-cell lung cancer. N Engl J Med 2005 353 123 132 16014882 3. Lynch TJ Bell DW Sordella R Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004 350 2129 2139 15118073 4. Paez JG EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004 304 1497 1500 15118125 5. Mitsudomi T Morita S Yatabe Y West Japan Oncology Group Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): an open label randomised phase 3 trial. 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Mitsudomi T Yatabe Y Mutations of the epidermal growth factor receptor gene and related genes as determinants of epidermal growth factor receptor tyrosine kinase inhibitors sensitivity in lung cancer. Cancer Sci 2007 98 1817 1824 17888036 10. Pallis AG Voutsina A Kalikaki A ˜Classical™ but not ˜other™ mutations of EGFR kinase domain are associated with clinical outcome in gefitinib-treated patients with non-small cell lung cancer. Br J Cancer 2007 97 1560 1566 18000506 11. Maheswaran S Sequist LV Nagrath S Detection of mutations in EGFR in circulating lung-cancer cells. N Engl J Med 2008 359 366 377 18596266 12. Sequist LV Martins RG Spigel D First-line gefitinib in patients with advanced non-small-cell lung cancer harboring somatic EGFR mutations. J Clin Oncol 2008 26 2442 2449 18458038 13. Costa DB Nguyen KS Cho BC Effects of erlotinib in EGFR mutated non-small cell lung cancers with resistance to gefitinib. Clin Cancer Res 2008 14 7060 7067 18981003 14. Masago K Fujita S Irisa K Good clinical response to gefitinib in a non-small cell lung cancer patient harboring a rare somatic epidermal growth factor gene point mutation; codon 768 AGC > ATC in exon 20 (S768I). Jpn J Clin Oncol 2010 40 1105 1109 20522446 15. Rizvi NA Rusch V Pao W Molecular characteristics predict clinical outcomes: prospective trial correlating response to the EGFR tyrosine kinase inhibitor gefitinib with the presence of sensitizing mutations in the tyrosine binding domain of the EGFR gene. Clin Cancer Res 2011 17 3500 3506 21558399 16. De Pas T Toffalorio F Manzotti M Activity of epidermal growth factor receptor-tyrosine kinase inhibitors in patients with non-small cell lung cancer harboring rare epidermal growth factor receptor mutations. J Thorac Oncol 2011 6 1895 1901 21841502 17. Wu JY Yu CJ Chang YC Effectiveness of tyrosine kinase inhibitors on œuncommon epidermal growth factor receptor mutations of unknown clinical significance in non-small cell lung cancer. Clin Cancer Res 2011 17 3812 3821 21531810 18. Ong M Kwan K Kamel-Reid S Neoadjuvant erlotinib and surgical resection of a stage iiia papillary adenocarcinoma of the lung with an L861Q activating EGFR mutation. Curr Oncol 2012 19 e222 e226 22670114 19. Ackerman A Goldstein MA Kobayashi S Costa DB EGFR delE709_T710insD: a rare but potentially EGFR inhibitor responsive mutation in non-small-cell lung cancer. J Thorac Oncol 2012 7 e19 e20 22982663 20. Sharma A Tan TH Cheetham G Rare and novel epidermal growth factor receptor mutations in non-small-cell lung cancer and lack of clinical response to gefitinib in two cases. J Thorac Oncol 2012 7 941 942 22722798 21. Nagai Y Miyazawa H Huqun Genetic heterogeneity of the epidermal growth factor receptor in non-small cell lung cancer cell lines revealed by a rapid and sensitive detection system the peptide nucleic acid-locked nucleic acid PCR clamp. Cancer Res 2005 65 7276 7282 16105816 22. Tanaka T Matsuoka M Sutani A Frequency of and variables associated with the EGFR mutation and its subtypes. Int J Cancer 2010 126 651 655 19609951 23. Therasse P Arbuck SG Eisenhauer EA New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer National Cancer Institute of the United States National Cancer Institute of Canada. J Natl Cancer Inst 2000 92 205 216 10655437 24. Mitsudomi T Yatabe Y Epidermal growth factor receptor in relation to tumor development: EGFR gene and cancer. FEBS J 2010 277 301 308 19922469 25. Yun CH Boggon TJ Li Y Structures of lung cancer-derived EGFR mutants and inhibitor complexes: mechanism of activation and insights into differential inhibitor sensitivity. Cancer Cell 2007 11 217 227 17349580 26. Kancha RK von Bubnoff N Peschel C Duyster J Functional analysis of epidermal growth factor receptor (EGFR) mutations and potential implications for EGFR targeted therapy. Clin Cancer Res 2009 15 460 467 19147750 27. Kancha RK Peschel C Duyster J The epidermal growth factor receptor-L861Q mutation increases kinase activity without leading to enhanced sensitivity toward epidermal growth factor receptor kinase inhibitors. J Thorac Oncol 2011 6 387 392 21252719 28. Sequist LV Besse B Lynch TJ Neratinib an irreversible pan-ErbB receptor tyrosine kinase inhibitor: results of a phase II trial in patients with advanced non-small-cell lung cancer. J Clin Oncol 2010 28 3076 3083 20479403 29. Miller VA Hirsh V Cadranel J Afatinib versus placebo for patients with advanced metastatic non-small-cell lung cancer after failure of erlotinib gefitinib or both and one or two lines of chemotherapy (LUX-Lung 1): a phase 2b/3 randomised trial. Lancet Oncol 2012 13 528 538 22452896 Clin Orthop Relat Res Clin. Orthop. Relat. Res Clinical Orthopaedics and Related Research 0009-921X 1528-1132 Springer US Boston 24249538 3971232 3385 10.1007/s11999-013-3385-9 Clinical Research Does Intensity of Surveillance Affect Survival After Surgery for Sarcomas? Results of a Randomized Noninferiority Trial Puri Ajay MS docpuri@gmail.com Gulia Ashish MS Hawaldar Rohini PhD Ranganathan Priya MD Badwe Rajendra A. MS Orthopaedic Oncology Tata Memorial Hospital Room No. 45 E Borges Road Mumbai India Tata Memorial Hospital Mumbai India Anaesthesiology Critical Care and Pain Tata Memorial Hospital Mumbai India Surgical Oncology Tata Memorial Hospital Mumbai India 19 11 2013 5 2014 472 5 1568 1575 30 7 2013 8 11 2013 © The Association of Bone and Joint Surgeons® 2013 Background Whether current postoperative surveillance regimes result in improved overall survival (OS) of patients with extremity sarcomas is unknown. Questions/purposes We hypothesized that a less intensive followup protocol would not be inferior to the conventional followup protocol in terms of OS. We (1) assessed OS of patients to determine if less intensive followup regimens led to worsened survival and asked (2) whether chest radiograph followup group was inferior to CT scan followup group in detecting pulmonary metastasis; and (3) whether less frequent (6-monthly) followup interval was inferior to more frequent (3-monthly) followup in detecting pulmonary metastasis and local recurrence. Methods A prospective randomized single-center noninferiority trial was conducted between January 2006 and June 2010. On the basis of 3-year survival of 60% with intensive more frequent followup 500 nonmetastatic patients were randomized to demonstrate noninferiority by a margin (delta) of 10% (hazard ratio [HR] 1.36). The primary end point was OS at 3 years. The secondary objective was to compare disease-free survival (DFS) (time to recurrence) at 3 years. At minimum followup of 30 months (median 42 months; range 30“81 months) 178 deaths were documented. Results Three-year OS and DFS for all patients was 67% and 52% respectively. Three-year OS was 67% and 66% in chest radiography and CT groups respectively (HR 0.9; upper 90% confidence interval [CI] 1.13). DFS rate was 54% and 49% in chest radiography and CT groups respectively (HR 0.82; upper 90% CI 0.97). Three-year OS was 64% and 69% in 6-monthly and 3-monthly groups respectively (HR 1.2; upper 90% CI 1.47). DFS was 51% and 52% in 6-monthly and 3-monthly groups respectively (HR 1.01; upper 90% CI 1.2). Almost 90% of local recurrences were identified by patients themselves. Conclusions Inexpensive imaging detects the vast majority of recurrent disease in patients with sarcoma without deleterious effects on eventual outcomes. Patient education regarding self-examination will detect most instances of local recurrence although this was not directly assessed in this study. Although less frequent visits adequately detected metastasis and local recurrence this trial could not conclusively demonstrate noninferiority in OS for a 6-monthly interval of followup visits against 3-monthly visits. Level of Evidence Level I therapeutic study. See Guidelines for Authors for a complete description of levels of evidence. issue-copyright-statement © The Association of Bone and Joint Surgeons® 2014 101528555 37539 Nat Commun Nat Commun Nature communications 2041-1723 24572595 3982882 10.1038/ncomms4365 NIHMS562641 Article Characterizing the genetic basis of methylome diversity in histologically normal human lung tissue Shi Jianxin 1 Marconett Crystal N. 2 3 Duan Jubao 4 Hyland Paula L. 1 Li Peng 1 Wang Zhaoming 1 Wheeler William 5 Zhou Beiyun 6 Campan Mihaela 2 3 Lee Diane S. 2 3 Huang Jing 7 Zhou Weiyin 1 Triche Tim 8 Amundadottir Laufey 1 Warner Andrew 9 Hutchinson Amy 1 Chen Po-Han 2 3 Chung Brian S.I. 2 3 Pesatori Angela C. 10 Consonni Dario 10 Bertazzi Pier Alberto 10 Bergen Andrew W. 11 Freedman Mathew 12 13 Siegmund Kimberly D. 8 Berman Benjamin P. 8 14 Borok Zea 3 6 Chatterjee Nilanjan 1 Tucker Margaret A. 1 Caporaso Neil E. 1 Chanock Stephen J. 1 Laird-Offringa Ite A. 2 3 Landi Maria Teresa 1 1Division of Cancer Epidemiology and Genetics National Cancer Institute NIH DHHS Bethesda MD 20892 USA 2Department of Surgery USC/Norris Comprehensive Cancer Center Keck School of Medicine Los Angeles CA 90089 USA 3Department of Biochemistry and Molecular Biology USC/Norris Comprehensive Cancer Center Keck School of Medicine Los Angeles CA 90089 USA 4Center for Psychiatric Genetics Department of Psychiatry and Behavioral Sciences North Shore University Health System Research Institute University of Chicago Pritzker School of Medicine Evanston IL 60201 USA 5Information Management Services Inc. Rockville MD 20852 USA 6Will Rogers Institute Pulmonary Research Center and Division of Pulmonary Critical Care and Sleep Medicine USC Keck School of Medicine Los Angeles CA 90089 USA 7Laboratory of Cancer Biology and Genetics Center for Cancer Research National Cancer Institute NIH DHHS Bethesda MD 20892 USA 8Bioinformatics Division Department of Preventive Medicine University of Southern California Los Angeles CA 90089 USA 9Pathology/Histotechnology Laboratory Laboratory Animal Sciences Program Frederick National Laboratory for Cancer Research Frederick Maryland21702 USA 10Unit of Epidemiology IRCCS Fondazione Ca™ Granda Ospedale Maggiore Policlinico and Department of Clinical Sciences and Community Health University of Milan Milan20122 Italy 11Molecular Genetics Program Center for Health Sciences SRI Menlo Park CA 94025 USA 12Program in Medical and Population Genetics The Broad Institute Cambridge MA 02142 USA 13Department of Medical Oncology The Center for Functional Cancer Epigenetics Dana-Farber Cancer Institute Boston MA 02215 14USC Epigenome Center and USC/Norris Comprehensive Cancer Center Los Angeles CA 90089 USA Correspondence: landim@mail.nih.gov 4 4 2014 27 2 2014 27 8 2014 5 3365 3365 The genetic regulation of the human epigenome is not fully appreciated. Here we describe the effects of genetic variants on the DNA methylome in human lung based on methylation-quantitative trait loci (meQTL) analyses. We report 34304 cis- and 585 trans-meQTLs a genetic-epigenetic interaction of surprising magnitude including a regulatory hotspot. These findings are replicated in both breast and kidney tissues and show distinct patterns: cis-meQTLs mostly localize to CpG sites outside of genes promoters and CpG islands (CGIs) while trans-meQTLs are over-represented in promoter CGIs. meQTL SNPs are enriched in CTCF binding sites DNaseI hypersensitivity regions and histone marks. Importantly 4 of the 5 established lung cancer risk loci in European ancestry are cis-meQTLs and in aggregate cis-meQTLs are enriched for lung cancer risk in a genome-wide analysis of 11587 subjects. Thus inherited genetic variation may affect lung carcinogenesis by regulating the human methylome. Introduction DNA methylation plays a central role in epigenetic regulation. Twin studies have suggested that DNA methylation at specific CpG sites can be heritable12; however the genetic effects on DNA methylation have been investigated only in brain tissues34 adipose tissues56 and lymphoblastoid cell lines (LCL)7. Most studies were based on the Illumina HumanMethylation27 array which has a low density and mainly focuses on CpG-sites mapping to gene promoter regions. While the functional role of DNA methylation in non-promoter or non-CpG Island (CGI) regions remains largely unknown evidence shows roles in regulating gene splicing8 and alternative promoters9 silencing of intragenic repetitive DNA sequences10 and predisposing to germline and somatic mutations that could contribute to cancer development1112. Notably a recent study13 suggests that most DNA methylation alterations in colon cancer occur outside of promoters or CGIs in so called CpG island shores and shelves and the Cancer Genome Project has reported high mutation rates in CpG regions outside CGI in multiple cancers14. Although expression QTLs (eQTLs) have been extensively studied in different cell lines and tissues15 the minimal overlap observed between cis-acting meQTLs and eQTLs (?5“10%)347 emphasizes the necessity of mapping meQTLs that may function independently of nearby gene expression. This might reveal novel mechanisms for genetic effects on cancer risk particularly since many of the established cancer susceptibility SNPs map to non-genic regions. Lung diseases constitute a significant public health burden. About 10 million Americans had chronic obstructive pulmonary disease in 201216 and lung cancer continues to be the leading cancer-related cause of mortality worldwide17. To provide functional annotation of SNPs particularly those relevant to lung diseases and traits we systematically mapped meQTLs in 210 histologically normal human lung tissues using Illumina Infinium HumanMethylation450 BeadChip arrays which provide a comprehensive platform to interrogate the DNA methylation status of 485512 cytosine targets with excellent coverage in both promoter and non-promoter regions (Fig. 1a) CGI and non-CGI regions (Fig. 1b) and gene and non-gene regions. Thus our study enables the characterization of genetic effects across the methylome in unprecedented detail. Moreover since DNA methylation exhibits tissue specific features18 we investigated whether similar meQTLs could be identified in other tissues. Results Identification of cis-acting meQTLs We profiled DNA methylation for 244 fresh-frozen histologically normal lung samples from non-small cell lung cancer (NSCLC) patients from the Environment and Genetics in Lung cancer Etiology (EAGLE) study19. A subset of 210 tissue samples that passed quality control and had germline genotype data from blood samples20 was used for meQTL analysis. The analysis was restricted to 338456 autosomal CpG probes after excluding those annotated in repetitive genomic regions or that harbored genetic variants. The distribution of methylation levels differed strongly across distinct types of genomic regions (Supplementary Fig. 1ab). Consistent with previous studies21 CpG sites in promoter or CGI regions were largely unmethylated while those in other regions were largely methylated (Supplementary Fig. 1ab). We performed cis-meQTL analysis for each methylation trait by searching for SNPs within 500kb of the target CpG-site in each direction (1Mb overall). The genetic association was tested under an additive model between each SNP and each normalized methylation probe adjusting for sex age plate population stratification and methylation-based principal component analysis (PCA) scores. Controlling FDR at 5% (P=4.0×10?5) we detected cis-meQTLs for 34304 (10.1% of 338456) CpG probes (Supplementary ) mapping to 9330 genes. A more stringent threshold (P=6.0×10?6) at FDR=1% detected cis-meQTLs for 27043 CpG probes mapping to 8479 genes. Moreover with a 200kb window (100kb from both sides) instead than 1Mb we detected 40650 cis-meQTLs (P=2.0×10?4) controlling for FDR=5%. The methylation distribution in CpG sites detected with meQTLs differed substantially from those without meQTLs (Supplementary Fig. 1ab). The peak SNPs were equally distributed on either side of the target CpG-sites with a median distance (?) of 11.8 kb. The proportion of explained phenotypic variance (h2) ranged from 7.7% to 79.8% (Supplementary Fig. 1c) "

Lung_Cancer

" DLA and TTN contributed equally to this manuscript. # AJW and MVG share senior position authorship 15 7 2014 02 12 2013 1 2014 01 1 2015 12 1 111 118 Activated ALK and ROS1 tyrosine kinases through gene fusions has been found in lung adenocarcinomas and are highly sensitive to selective kinase inhibitors. This study aimed at identifying the presence of these rearrangements in human colorectal adenocarcinoma (CRC) specimens using a 4-target 4-color break-apart fluorescence in situ hybridization (FISH) assay to simultaneously determine the genomic status of ALK and ROS1. Among the clinical CRC specimens analyzed rearrangement-positive cases for both ALK and ROS1 were observed. The fusion partner for ALK was identified as EML4 and the fusion partner for one of the ROS1-positive cases was SLC34A2 the partner for the other ROS1-positive case remains to be identified. A small fraction of specimens presented duplicated or clustered copies of native ALK and ROS1. In addition rearrangements were detected in samples that also harbored KRAS and BRAF mutations in two of the three cases. Interestingly the ALK-positive specimen displayed marked intra-tumoral heterogeneity and rearrangement was also identified in regions of high-grade dysplasia. Despite the additional oncogenic events and tumor heterogeneity observed elucidation of the first cases of ROS1 rearrangements and confirmation of ALK rearrangements support further evaluation of these genomic fusions as potential therapeutic targets in CRC. Implications ROS1 and ALK fusions occur in colorectal cancer and may have substantial impact in therapy selection. J Vet Med Sci J. Vet. Med. Sci JVMS The Journal of Veterinary Medical Science 0916-7250 1347-7439 The Japanese Society of Veterinary Science 24389742 4064154 13-0434 10.1292/jvms.13-0434 Internal Medicine Note CT and PET-CT of a Dog with Multiple Pulmonary Adenocarcinoma KIM Jisun 1 KWON Seong Young 2 CENA Rohani 1 PARK Seungjo 1 OH Juyeon 1 OUI Heejin 1 CHO Kyoung-Oh 1 MIN Jung-Joon 2 CHOI Jihye 1 * 1)College of Veterinary Medicine Chonnam National University Gwangju 500“757 Korea 2)Department of Nuclear Medicine Chonnam National University Hwasun Hospital Jeonnam 519“763 Korea *Correspondence to: Choi J. College of Veterinary Medicine Chonnam National University Yongbong-ro Buk-gu Gwangju 500“757 South Korea. e-mail: imsonochonnam.ac.kr 31 12 2013 4 2014 76 4 615 620 28 8 2013 21 12 2013 2014 The Japanese Society of Veterinary Science 2014 This is an open-access distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives (by-nc-nd) License. A 10-year-old intact female Yorkshire terrier had multiple pulmonary nodules on thoracic radiography and ultrasonography with no lesions elsewhere. Computed tomography (CT) and positron emission tomography and computed tomography (PET-CT) using 18F-fluorodeoxyglucose (FDG) were performed to identify metastasis and undetected primary tumors. On CT examination pulmonary nodules had a hypoattenuating center with thin peripheral enhancement suggesting ischemic or necrotizing lesion. In PET-CT at 47 min after intravenous injection of 11.1 MBq/kg of FDG the maximum standardized uptake value of each pulmonary nodule was about from 3.8 to 6.4. There were no abnormal lesions except for four pulmonary nodules on the CT and PET-CT. Primary lung tumor was tentatively diagnosed and palliative therapy using 2 mg/kg tramadol and 2.2 mg/kg carprofen twice per day was applied. After the dog™s euthanasia due to deteriorated clinical signs and poor prognosis undifferentiated pulmonary adenocarcinoma was diagnosed through histopathologic and immunochemistry examination. To the best of the authors™ knowledge this is the first study of CT and PET-CT features of canine pulmonary adenocarcinoma. In this case multiple pulmonary adenocarcinoma could be determined on the basis of FDG PET-CT through screening the obvious distant metastasis and/or lymph node invasions and excluding unknown primary tumors. computed tomography fluorodeoxyglucose multiple pulmonary nodules positron emission tomography pulmonary adenocarcinoma The etiology of lung masses including nodules (spots on the lung that are 3 cm in diameter or less) can be assumed on the basis of morphological characteristics and especially the number of masses can act as an important clue for differential diagnosis. Solitary masses may be primary lung tumor abscess granuloma or hematoma whereas multiple masses may be metastatic tumors septic emboli or parasitic granulomas [19]. However primary lung tumor can metastasize to lymph nodes distant ans or to other lung regions through hematogeneous or lymphatic routes or it can spread to adjacent lung tissues through local invasion [2 3]. Therefore primary lung tumor is not necessarily presented as a solitary mass but may also present itself as multiple masses or as in a disseminated form [414 15]. Surgical excision is the primary option for primary lung tumor regardless of whether solitary or multiple types are present and the goal of the surgery is to remove all the gross lesions. Therefore the primary lung tumor with multiple masses should be differentiated with metastatic lung tumors. When multiple masses are presented the largest one is usually considered to be the primary lesion. However if the masses™ sizes are equal it is impossible to determine which one is the origin [13]. Diagnostic imaging with high anatomic resolution such as computed tomography (CT) can be applied to evaluate the tumors in more detail to determine the treatment plan and prognostic factors through estimation of the tumor size lymph node involvement and pulmonary or distant metastasis [14]. Positron emission tomography (PET) a functional imaging modality can be used to determine the tumor or metastasis in equivocal situations in which enlarged lesions or those which have altered their shape due to metastasis are not identified with anatomic imaging during early stages or according to tumor type [18]. PET-CT provides the combined trans-sectional images which consist of functional information of PET superimposed on anatomic information obtained via CT scanning. PET-CT is considered to be one of the most sensitive diagnostic modalities for evaluating metastasis tumor staging and responses to therapy [9]. In veterinary medicine the physiologic values of 18F-fluorodeoxyglucose (FDG) uptake in normal dogs and a few cancer cases studied via PET-CT were reported in spite of the limited availability and cost of this modality [2710 11]. In this study we described the application of CT and PET-CT to distinguish primary lung tumor from metastatic lung tumor in a dog presenting multiple lung masses. A 10-year-old intact female Yorkshire terrier weighing 3.3 kg was presented at the Chonnam National University Veterinary Teaching Hospital for a general check-up without any specific clinical signs. Physical examination revealed small sized tumors (about 3 mm diameter) at the left 2nd and 4th mammary glands. Complete blood count revealed leukocytosis (40.14 K/?l; reference range 5.05“16.76 K/?l). There was no abnormal finding in serum chemistry. Thoracic radiographs revealed a total of four nodules on the left and right cranial and caudal lobes (Fig. 1Fig. 1.Canine thoracic radiographs (right lateral and ventrodorsal views) showing four nodules (long arrows) with uniform soft tissue density on the left and right cranial and caudal lobes. Enlarged teats (short arrows) were superimposed on the radiographs.). All nodules had soft tissue density with definite contour. The largest one located at the right caudal lobe was 4 cm in diameter and the others were about 2“3 cm. On ultrasonography all pulmonary nodules had slightly heterogeneous internal hypoechotexture confined by an echogenic border. The blood flow signal was not identified within all nodules on color Doppler mode. There was no remarkable finding on abdominal radiography and ultrasonography. Multiple pulmonary nodules were suspected as metastatic lesions however the primary tumor was not identified. Thus computed tomography (CT) and positron emission tomography-computed tomography (PET-CT) were performed under general anesthesia of a combination of 2.5 mg/kg zolazepam/tiletamine (Zoletil Virbac France) and 0.05 mg/kg medetomidine (Domitor Orion Corp. Espoo Finland) to identify the undetected primary tumor and additional metastasis which may have been present in other ans."

Lung_Cancer

"Moreover niclosamide could enhance antitumor immunity and inhibit lung metastasis by reducing the number of Gr1+/CD11b+ (MDSCs) in tumors. Therefore our studies provided strong evidence that niclosamide as a candidate of antibreast cancer drug is worth being further investigated. References 1 SiegelR NaishadhamD JemalA (2013) Cancer statistics 2013. CA Cancer J Clin63: 11“3023335087 2 DeSantisC SiegelR BandiP JemalA (2011) Breast cancer statistics 2011. CA Cancer J Clin61: 408“418 3 WiebeJP ZhangG WelchI Cadieux-PitreHAT (2013) Progesterone metabolites regulate induction growth and suppression of estrogen-and progesterone receptor-negative human breast cell tumors. Breast Cancer Res15: R3823663549 4 AndersonBO YipCH RamseySD BengoaR BraunS et al (2006) Breast cancer in limited-resource countries: health care systems and public policy. Breast J. 12: S54“S69 5 LiuMR CasimiroMC WangCG ShirleyLA JiaoXM et al (2009) p21CIP1 attenuates Ras-and c-Myc-dependent breast tumor epithelial mesenchymal transition and cancer stem cell-like gene expression in vivo. Proc Natl Acad Sci U S A106: 19035“1903919858489 6 Ottenhoff-KalffAE RijksenG Van BeurdenE HennipmanA MichelsA et al (1992) Characterization of protein tyrosine kinases from human breast cancer: involvement of the c-src oncogene product. Cancer Res52: 4773“47781380891 7 BrennanK BrownAM (2003) Is there a role for Notch signalling in human breast cancer? Breast Cancer Res5: 69“7512631384 8 CaiJC GuanHY FangLS YangY ZhuX et al (2013) MicroRNA-374a activates Wnt/?-catenin signaling to promote breast cancer metastasis. J Clin Invest123: 566“57923321667 9 HarrisonH SimµesBM RogersonL HowellSJ LandbergG et al (2013) Oestrogen increases the activity of oestrogen receptor negative breast cancer stem cells through paracrine EGFR and Notch signaling. Breast Cancer Res15: R2123497505 10 ZhangXL YuePB PageBD LiTS ZhaoW et al (2012) Orally bioavailable small-molecule inhibitor of transcription factor Stat3 regresses human breast and lung cancer xenografts. Proc Natl Acad Sci U S A109: 9623“962822623533 11 PageC HuangM JinX ChoK LiljaJ et al (2000) Elevated phosphorylation of AKT and Stat3 in prostate breast and cervical cancer cells. Int J Oncol17: 23“3110853013 12 CataldoL ChenNY YuanQ LiW RamamoorthyP et al (2000) Inhibition of oncogene STAT3 phosphorylation by a prolactin antagonist hPRL-G129R in T-47D human breast cancer cells. Int J Oncol17: 1179“126411078803 13 YuH JoveR (2004) The STATs of cancer-new molecular targets come of age. Nat Rev Cancer4: 97“10514964307 14 RenXM DuanL HeQ ZhangZ ZhouY et al (2010) Identification of Niclosamide as a New Small-Molecule Inhibitor of the STAT3 Signaling Pathway. ACS Med Chem Lett1: 454“459 15 HiranoT IshiharaK HibiM (2000) Roles of STAT3 in mediating the cell growth differentiation and survival signals relayed through the IL-6 family of cytokine receptors. Oncogene19: 2548“255610851053 16 SrivastavaK Kundumani-SridharanV ZhangBL BajpaiAK RaoGN (2007) 15 (S)-hydroxyeicosatetraenoic acid-induced angiogenesis requires STAT3-dependent expression of VEGF. Cancer Res67: 4328“433617483346 17 BollrathJ PhesseTJ von BurstinVA PutoczkiT BenneckeM et al (2009) gp130-mediated Stat3 activation in enterocytes regulates cell survival and cell-cycle progression during colitis-associated tumorigenesis. Cancer cell15: 91“10219185844 18 XinH HerrmannA ReckampK ZhangW PalS et al (2011) Antiangiogenic and antimetastatic activity of JAK inhibitor AZD1480. Cancer Res. 71: 6601“6610 19 LiR YouS HuZL ChenZG SicaGL et al (2013) Inhibition of STAT3 by niclosamide synergizes with erlotinib against head and neck cancer. PloS One8: e7467024019973 20 GarinJ DespeignesJ BillerauM (1964) Present treatment of taeniasis with niclosamide."

Lung_Cancer

"Clin Cancer Res 2012 18 6599 6608 23052255 24 Shaw AT Ceritinib in ALK-rearranged non-small-cell lung cancer. N Engl J Med 2014 370 1189 1197 24670165 25 Galkin AV Identification of NVP-TAE684 a potent selective and efficacious inhibitor of NPM-ALK. Proc Natl Acad Sci U S A 2007 104 270 275 17185414 26 Morris SW Fusion of a kinase gene ALK to a nucleolar protein gene NPM in non-Hodgkin's lymphoma. Science 1994 263 1281 1284 8122112 27 Li H Durbin R Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 2009 25 1754 1760 19451168 28 Li H The Sequence Alignment/Map format and SAMtools. Bioinformatics 2009 25 2078 2079 19505943 29 McKenna A The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome research 2010 20 1297 1303 20644199 30 Gainor JF ALK Rearrangements Are Mutually Exclusive with Mutations in EGFR or KRAS: An Analysis of 1683 Patients with Non-Small Cell Lung Cancer. Clin Cancer Res 2013 31 Dunning MJ Smith ML Ritchie ME Tavare S beadarray: R classes and methods for Illumina bead-based data. Bioinformatics 2007 23 2183 2184 17586828 32 Su Z A platform for rapid detection of multiple oncogenic mutations with relevance to targeted therapy in non-small-cell lung cancer. J Mol Diagn 2011 13 74 84 21227397 Exceptional response to an IGF-1R inhibitor prior to ALK TKI therapy in a patient with ALK+ lung cancer Representative images from serial CT scans of the chest in a 50 year-old female with ALK+ lung cancer documenting responses to the indicated therapies. Images are labeled a“f in temporal sequence. The red circles in a“c represent a new lesion in the right lung that developed after 1 month of erlotinib and then responded to erlotinib plus an IGF-1R antibody. The scale bar in a indicates 4 cm and is representative for all images. Combination therapy with an IGF-1R inhibitor plus an ALK inhibitor promotes cooperative inhibition of cell growth in TKI sensitive ALK+ lung cancer cells (a) H3122 (EML4-ALK E13;A20) lung cancer cells were treated with crizotinib or crizotinib + MAb391. Soft agar assays were performed to assess growth inhibition. Each point represents hextuplicate biological replicates. Data are presented as the percentage of viable cells compared to control (vehicle only) cells and are representative of three independent experiments. P values were determined with the Student's T-test. (b“d) H3122 (EML4-ALK E13;A20) (b) H2228 (EML4-ALK 6a/b;A20) (c) and STE-1 (EML4-ALK E13;A20) (d) lung cancer cells were treated with increasing amounts of crizotinib OSI-906 or the combination for 72h. Cell titer blue assays were performed to assess growth inhibition. Each point represents hextuplicate biological replicates. Data are presented as the percentage of viable cells compared to control (vehicle only) cells and are representative of three or more independent experiments. (e) STE-1 cells were treated with one dose of 1 ?M crizotinib 2 ?M OSI-906 or the combination for a total of 72h prior to harvest. Cells were stained with propidium iodide (PI) and counted on a FACSCalibur machine. (f) H3122 cells were treated with crizotinib OSI-906 or the combination for 2h prior to harvest. Lysates were subjected to immunoblotting with antibodies specific for the indicated proteins. Select images were quantified using a Bio-Rad Gel Doc XR and Image Lab software (Supplementary Fig. 1i and Supplementary Fig. 2b). IRS-1 knock-down impairs downstream signaling and blocks proliferation of ALK+ lung cancer cells (a) H3122 cells were treated with crizotinib or crizotinib + IGF-1 for 72h. Cell titer blue assays were performed to assess growth inhibition. Each point represents hextuplicate biological replicates. Data are presented as the percentage of viable cells compared to control. (b) H3122 cells were serum starved overnight and then treated with the indicated TKIs for 6h. As indicated cells were then stimulated with IGF-1 for 10min. Lysates were subjected to immunoblotting with antibodies specific for the indicated proteins. (c) H3122 cells were treated with vehicle or crizotinib. Lysates were subjected to immunoprecipitation (IP) for IRS-1 and western blotting for the indicated antibodies. (d) Tumor containing lung tissue from two different EML4-ALK E13;A20 transgenic mice were pulverized lysed and subjected to immunoprecipitation (IP) for IRS-1 and western blotting for the indicated antibodies. (e) STE-1 cells were transfected with the non-targeting siRNA (œNT) or with two distinct pools of IRS-1 siRNA and treated with 500nM crizotinib for 72h . Lysates were subjected to immunoblotting with antibodies specific for the indicated proteins. (f) STE-1 cells were transfected with the indicated siRNAs and treated with 500 nM crizotinib for 72h. Triplicate biological replicates for each sample were counted on Coulter Counter. P values were determined with the Student's T-test. Data are representative of three independent experiments. (g) Western blot showing IRS-1 knockdown in the experiment shown in Fig. 3f. The IGF-1R pathway is activated in models of ALK TKI resistance Isogenic pairs of H3122 parental (i.e. TKI sensitive) crizotinib-resistant (œCR) or X-376-resistant (œXR) cells were treated with crizotinib (a) or X-376 (b). Cell titer blue assays were performed with hextuplicate biological replicates. Data shown are representative of ? 3 independent experiments. (c) H3122 XR cells were treated with X-376 for 4h. Lysates were subjected to immunoblotting with antibodies specific for the indicated proteins. (d) H3122 XR cells were treated with X-376 or X-376 + MAb391. Soft agar assays were performed using hextuplicate biological replicates. Data are representative of two independent experiments. (e) H3122 XR cells were treated with X-376 or X-376 + OSI-906 for 72h. Cell titer blue assays were performed with hextuplicate biological replicates. Data are representative of three independent experiments. (f) H3122 XR cells were treated with X-376 AEW-541 or the combination daily for 72h. Cells were stained with propidium iodide (PI) and counted on a FACSCantoII machine. (g) H3122 XR cells were treated with the indicated inhibitors for 4h. Lysates were subjected to immunoblotting with antibodies specific for the indicated proteins. (h) H3122 XR cells were transfected with the indicated siRNAs and treated with 500 nM X-376 for 72h. Quadruplicate biological replicates for each sample were counted on Coulter Counter. Data are representative of three independent experiments. (i) Western blots confirming IRS-1 knockdown in the experiment shown in Fig. 4h. All P values shown were determined with the Student's T-test. Increased IGF-1R and IRS-1 in patient biopsy samples at the time of acquired resistance to crizotinib (a“b) Tumor samples taken before and at the time of resistance to ALK TKI therapy were analyzed for IGF-1R pY1161 expression (a) and for IRS-1 expression (b) by immuno-histochemistry. All images viewed correspond to a magnification of 40x. The scale bar indicates 200 micometers. (c“d) RNA was extracted from formalin-fixed paraffin embedded tumor biopsy samples prior to and at the time of progressive disease on crizotinib and run on the NanoString assay. Expression levels of IGF-1R (c) and IRS-1 (d) are compared pre- and post- crizotinib. NanoString target sequences for IGF-1R have been previously reported23. The colored dots within each box plot represent distinct pairs of matched pre- and post- crizotinib samples. The black dots indicate the patient sample. The red dots and the green dots represent H3122 parental (TKI sensitive cells) compared with H3122 CR cells at 1— crizotinib resistance (1 ?M final concentration of crizotinib red dot) or H3122 CR cells at 2— crizotinib resistance (2 ?M final concentration of crizotinib green dot). The blue dots represent H3122 parental compared with H3122 XR cells. P values were determined with a modified paired T-test using the limma package. The second generation ALK inhibitor LDK-378 blocks phosphorylation of both ALK and IGF-1R (a) H3122 lung cancer cells containing the EML4-ALK E13;A20 fusion were treated with increasing amounts of crizotinib LDK-378 or TAE-684 for 72h. Cell titer blue assays were performed to assess growth inhibition. Each point represents hextuplicate biological replicates. Data are presented as the percentage of viable cells compared to control (vehicle only treated) cells and are representative of three or more independent experiments. (b) Athymic nu/nu female mice were injected subcutaneously with H3122 lung cancer cells harboring the EML4-ALK E13;A20 fusion. When tumors reached an average volume of 100mm3 mice were randomized to receive crizotinib alone (50 mg kg?1 p.o. daily — 5 days) LDK-378 alone (50 mg kg?1 p.o. daily — 5 days) or vehicle control (n = 5 for crizotinib and LDK-378 n = 4 for vehicle control). Tumor volumes were assessed every 3-4 days. *P = 0.0159 based on the Wilcoxon rank sum test. (c“d) H3122 (c) and H2228 cells (d) were grown overnight in the presence or absence of serum and then treated with LDK-378 for 1 hour. As indicated cells were then stimulated with IGF-1 for 10min and harvested. Lysates were subjected to immunoblotting with antibodies specific for the indicated proteins. 0372741 3058 Clin Pharmacol Ther Clin. Pharmacol. Ther. Clinical pharmacology and therapeutics 0009-9236 1532-6535 24781527 4180036 10.1038/clpt.2014.93 NIHMS612008 Article Erlotinib in African Americans with Advanced Non-Small Cell Lung Cancer: A Prospective Randomized Study with Genetic and Pharmacokinetic Analysis Phelps Mitch A. 1 2 * Stinchcombe Thomas E. 3 * Blachly James S. 2 Zhao Weiqiang 2 Schaaf Larry J. 1 2 Starrett Sherri L. 4 Wei Lai 5 Poi Ming 2 Wang Danxin 2 Papp Audrey 2 Aimiuwu Josephine 1 Gao Yue 1 Li Junan 6 Otterson Gregory A. 2 Hicks William J. 2 Socinski Mark A. 3 Villalona-Calero Miguel A. 2 1College of Pharmacy The Ohio State University 2College of Medicine The Ohio State University 3University of North Carolina College of Medicine 4Wexner Medical Center The Ohio State University 5Center for Biostatistics The Ohio State University 6College of Public Health The Ohio State University Corresponding Author: Miguel Villalona-Calero MD Professor College of Medicine Director Division of Medical Oncology The Ohio State University A455A Starling Loving Hall 320 W 10th Avenue Columbus OH 43210 614-366-5068 Miguel.Villalona@osumc.edu * Contributed equally 6 9 2014 29 4 2014 8 2014 01 8 2015 96 2 182 191 Prospective studies focusing on EGFR inhibitors in African Americans with NSCLC have not been previously performed. In this phase II randomized study 55 African Americans with NSCLC received erlotinib 150mg/day or a body weight adjusted dose with subsequent escalations to the maximum allowable 200mg/day to achieve rash. Erlotinib and OSI-420 exposures were lower compared to previous reports consistent with CYP3A pharmacogenetics implying higher metabolic activity. Tumor genetics revealed only two EGFR mutations EGFR amplification in 17/47 samples 8 KRAS mutations and 5 EML4-ALK translocations. Although absence of rash was associated with shorter time to progression (TTP) disease control rate"

Lung_Cancer

"The Creative Commons Public Domain Dedication waiver (http://creativecommons./publicdomain/zero/1.0/) applies to the data made available in this unless otherwise stated. Background Complement receptor 1 (CR1) the receptor for C3b/C4b complement peptides plays a crucial role in carcinogenesis. However the association of genetic variants of CR1 with susceptibility to lung cancer remains unexplored. Methods This case-control study included 470 non-small cell lung cancer (NSCLC) patients and 470 cancer-free controls. Based on the Chinese population data from HapMap database we used Haploview 4.2 program to select candidate tag SNPs. Odds ratios (ORs) and 95% confidence intervals (CIs) were computed by logistic regression to evaluate the association of each tag SNP with NSCLC. Results Multivariate regression analysis indicated that the rs7525160 CC genotype was associated with an increased risk of developing NSCLC (OR?=?1.52 95% CI?=?1.02-2.28; P?=?0.028) compared with the GG genotype. When stratified by smoking status the risk of NSCLC was associated with the rs7525160 C allele carriers in smokers with OR (95% CI) of 1.72 (1.15-2.79) but not in non-smokers with OR (95% CI) of 1.15 (0.81-1.65). When the interaction between smoking status and rs7525160 G?>?C variant was analyzed with cumulative smoking dose (pack-year). Similarly GC or CC genotype carriers have increased risk of NSCLC among heavy smokers (pack-year???25) with OR (95% CI) of 2.01 (1.26-3.20) but not among light smokers (pack-year <25) with OR (95% CI) of 1.32 (0.81-2.16). Conclusion CR1 rs7525160 G?>?C polymorphism was associated with an increased risk of developing NSCLC in Chinese population. The association displays a manner of gene-environmental interaction between CR1 rs7525160 tagSNP and smoking status. CR1 Polymorphism Tag SNPs Lung cancer Background The complement system plays a critical role in the process of carcinogenesis. Despite of significant research controversial viewpoints remain on the exact relationship of complement system with cancer. Classically the complement system fights against cancer by exerting the effects of immunosurveillance in the immunologic microenvironment of tumors [1]. Recently it was found that complement may contribute to tumor growth by a wide variety of mechanisms including dysregulation of mitogenic signaling pathways sustained cellular proliferation angiogenesis insensitivity to apoptosis invasion and migration and escape from complement cytotoxicity [2]. This suggested complement just like a double-edged sword plays a dual role in carcinogenesis. In particular component C3 and its receptors have been demonstrated to be a key link between innate and adaptive immunity [3]. Complement receptor type 1 (CR1 CD35) is a multifunctional polymorphic glycoprotein which binds to C3b fragment of C3 and to C4b with lower affinity [45]. CR1 belongs to the regulators of complement activation (RCA) family of proteins and is expressed in a wide spectrum of cells and involved in T-cell and B-cell mediated immune regulation [67]. CR1 also modulates the complement cascade activation by preventing formation of classical and alternative pathway convertases and by acting as a cofactor for factor I mediated inactivation of C3b and C4b [89]. It has been demonstrated that chronic inflammation can predispose to cancer development and spread [10] as a fundamental component of innate immunity the complement cascade consists of potential proinflammatory molecules especially C3 and C5. Moreover complement activation and abnormal expression in tumor tissues has been demonstrated [11]. Considering the important role of CR1 in complement activation innate immunity and chronic inflammation CR1 has emerged as a molecule of immense interest in gaining insight into the susceptibility to cancer. CR1 gene is located on the Chromosome 1 at the locus 1q32 [12]. Various polymorphisms have been studied including the intronic and exonic density polymorphism for their ability to alter the density of erythrocyte CR1 on the cell membranes [13-15]. There are also the molecular weight variants due to insertion-deletion polymorphisms [16]. Up to now there have been very few studies on the association of genetic variants of CR1 with susceptibility to autoimmune and inflammatory diseases. It has been proposed that genetic variant at CR1 gene (rs6656401) might influence the susceptibility to late-onset Alzheimer™s disease [17]. CR1 expression in Peripheral Blood Mononuclear Cells (PBMCs) may be a new biomarker for prognosis of nasopharyngeal carcinoma and a potential therapeutic target [18]. Recently it has been indicated that CR1 A3650G (His1208Arg) polymorphism plays a critical role in conferring genetic susceptibility to gallbladder cancer in north Indian population [19]. However the association of genetic variants of CR1 with risk of lung cancer remains unexplored. Worldwide lung cancer is the most common cancer in terms of both incidence and mortality [20]. NSCLC is the most common subtype of lung cancer and less aggressive and metastic than SCLC. Although cigarette smoking is the predominant risk factor for lung cancer inherited genetic characteristics are presumed to account in part for this interindividual variation in lung cancer susceptibility. Recently several genome-wide association studies have demonstrated the common genetic variations associated with susceptibility to lung cancer [21-24]. Given the involvement of the complement system in coordinating innate immunity and inflammatory response [25] further examination of the potential association between genetic variation of CR1 genes and lung cancer is warranted. In the current study we conducted a case-control study to investigate the association of tag SNPs in CR1 gene with the risk of NSCLC and effect of the interaction of gene-environment on the risk of NSCLC. Results Subject characteristics The frequency distributions of select characteristics in cases and control subjects were shown in . The mean age (±SD) was 59.6?±?10.5 years for the cancer patients and 57.2?±?13.3 years for the controls. No significant difference was found in the mean age between cases and controls (P?=?0.470). There was no significant difference in proportion of sex and smoking status between cases and controls (P?=?0.832 and P?=?0.321 respectively). However there was significant difference between cases and controls when compared by pack-year smoked (P = 0.001). The heavy smokers (?25 pack-year) accounted for 61.5% in cases but only 45.5% in controls which suggested that cigarette smoking was a prominent contributor to the risk of lung cancer. Of the 470 case patients 178 (37.9%) were diagnosed as adenocarcinoma 238 (50.6%) as squamous cell carcinoma and 100 (%) as other types including large cell carcinoma (n?=?49) and mixed cell carcinoma (n?=?5). Distributions of select characteristics in cases and control subjects Variables ???Cases (n?=?470) ???Controls (n?=?470) No (%) No (%) P a ???Sex 0.832 ???Male 324 68.9 328 69.8 ???Female 146 31.1 142 30.2 ???Age 0.470 ???<50 84 17.9 96 20.4 ???50-59 177 37.7 187 39.8 ???60-69 129 27.4 111 23.6 ????70 80 17.0 76 16.2 ???Smoking status 0.321 ???Non-smoker 265 56.4 281 59.8 ???Smoker 205 43.6 189 40.2 ???Pack-year smoked 0.001 ???<25 75 36.6 96 50.8 ????25 130 63.4 93 49.2 aTwo-sided ?2 test. Association of CR1 tag SNP with NSCLC risk Total 13 selected tag SNPs of CR1 in HapMap database among Chinese population were analyzed. Except for rs9429782 polymorphism the genotype distributions of other SNPs in controls were consistent to Hardy-Weinberg equilibrium. Therefore we excluded the rs9429782 from further analysis. In order to screen the genetic variants that confer the susceptibility to lung cancer 12 candidate tagSNPs were genotyped in a case-control study consisting of 470 lung cancer patients and 470 cancer-free controls as shown in . Importantly genotype frequency of one intronic SNP (rs7525160 G?>?C) in cases was found to be significantly different from those of controls (?2?=?6.339 P=0.042). Further multivariate regression model with adjustment for age gender and smoking status was used to assess the association between rs7525160 G?>?C polymorphism and the risk of NSCLC. The results indicated that the rs7525160 CC genotype was associated with an increased risk of developing NSCLC with OR (95% CI) of 1.52 (1.02-2.28) compared with the GG genotype. Other tagSNPs of CR1 were not significantly associated with the risk of NSCLC in our study population (P >0.05). Genotype frequencies of CRI among cases and controls and their association with non-small cell lung cancers CRI Genotypes ??Controls (n?=?470) ??Cases (n?=?470) OR (95% CI ) * P No (%) No (%) rs7525160 ??GG 176 37.5 139 29.6 1.00 (ref.) ??CG 228 48.5 256 54.5 1.38 (1.04-1.85) 0.041 ??CC 66 14.0 75 15.9 1.52 (1.02-2.28) 0.028 rs3886100 ??GG 117 24.9 105 22.4 1.00 (ref.) ??AG 223 47.4 253 53.8 1.33 (0.97-1.81) 0.078 ??AA 130 27.7 112 23.8 1.06 (0.73-1.54) 0.755 rs11118167 ??TT 348 74.1 353 75.1 1.00 (ref.) ??CT 111 23.6 102 21.7 0.89 (0.65-1.21) 0.457 ??CC 11 2.3 15 3.2 1.35 (0.61-3.01) 0.461 rs9429782 ??GG 250 53.2 261 55.5 1.00 (ref.) ??GT 220 46.8 209 44.5 0.89 (0.69-1.16) 0.388 rs10494885 ??CC 178 37.9 164 34.9 1.00 (ref.) ??CT 224 47.6 232 49.4 1.11 (0.83-1.47) 0.490 ??TT 68 14.5 74 15.7 1.20 (0.81-1.78) 0.365 rs7542544 ??CC 128 27.2 108 23.0 1.00 (ref.) ??AC 223 47.5 252 53.6 1.21 (0.88-1.67) 0.239 ??AA 119 25.3 110 23.4 0.90 (0.62-1.30) 0.897 rs6691117 ??AA 324 68.9 327 69.6 1.00 (ref.) ??AG 131 27.9 128 27.2 0.98 (0.73-1.31) 0.888 ??GG 15 3.2 15 3.2 0.96 (0.46-2.02) 0.923 rs6656401 ??GG 436 92.8 447 95.1 1.00 (ref.) ??AG 34 7.2 23 4.9 0.68 (0.39-1.18) 0.174 ??AA 0 0.0 0 0.0 NC§ rs2296160 ??CC 185 39.4 194 41.3 1.00 (ref.) ??CT 226 48.1 220 46.8 0.91 (0.69-1.21) 0.521 ??TT 59 12.5 56 11.9 0.90 (0.59-1.37) 0.606 rs9429942 ??TT 452 96.2 457 97.2 1.00 (ref.) ??CT 18 3.8 13 2.8 0.77 (0.37-1.61) 0.482 ??CC 0 0.0 0 0.0 NC§ rs4844600 ??GG 171 36.4 179 38.1 1.00 (ref.) ??AG 230 48.9 228 48.5 0.92 (0.70-1.22) 0.571 ??AA 69 14.7 63 13.4 0.87 (0.58-1.31) 0.513 rs3818361 ??CC 187 39.8 188 40.0 1.00 (ref.) ??CT 224 47.7 224 47.7 0.98 (0.74-1.29) 0.868 ??TT 59 12.5 58 12.3 0.96 (0.63-1.46) 0.848 rs17048010 ??TT 301 64.0 286 60.8 1.00 (ref.) ??CT 154 32.8 164 34.9 1.09 (0.82-1.43) 0.556 ??CC 15 3.2 20 4.3 1.40 (0.70-2.79) 0.343 *Adjusted by age sex and smoking status; §NC not calculated. Table 3 Summary of MDR gene-gene interaction results Models Training bal. acc. (%) Testing bal. acc. (%) P value Cross-validation consistency rs7525160 54.03 50.53 0.828 7/10 rs4844600 rs10494885 55.45 49.32 0.989 3/10 rs4844600 rs10494885 rs7525160 57.60 48.48 0.623 6/10 Generalized Multifactor Dimensionality Reduction (GMDR) was used to evaluate gene-gene interaction. The summary of gene-gene interaction models is listed in Table 3. The SNP rs7525160 in CR1 had the highest testing balanced accuracy among 12 SNPs. The three-way interaction model among rs4844600 rs10494885 and rs7525160 showed high testing balance accuracy and cross validation consistency but the testing balanced accuracy was lower than the two-way gene-gene interaction in NSCLC. For each model the interaction was not significant (P?>?0.05). Table 4 Risk of CR1 genotypes with NSCLC by smoking status Smoking status CR1 genotype GG * OR (95% CI) § P value CG?+?CC * OR (95% CI) § P value Non-smoker 84/99 1.00 (reference) 181/182 1.15 (0.81-1.65) 0.440 Smoker 55/77 0.86 (0.54-1.38) 0.528 150/112 1.72 (1.15-2.59) 0.009 <25 pack-years 19/41 0.59 (0.31-1.10) 0.099 56/55 1.32 (0.81-2.61) 0.266 ?25 pack-years 36/36 1.18 (0.67-2.08) 0.562 94/57 2.01 (1.26-3.20) 0.003 *Number of cases/number of controls. §Data were calculated by logistic regression and adjusted for age and gender. Interaction of CR1 SNP with smoking Cigarette smoking is a well-known risk for lung cancer so stratification by smoking status was performed to investigate the association of rs7525160 G?>?C variant with the risk of NSCLC. As shown in Table 4 the risk of NSCLC was associated with the rs7525160 C allele carriers in smokers with OR (95% CI) of 1.72 (1.15-2.59) but not in non-smokers with OR (95% CI) of 1.15 (0.81-1.65) suggesting that the CR1 rs7525160 G?>?C polymorphism is a smoking-modifying risk factor for susceptibility to NSCLC. When the interaction between smoking status and rs7525160 G?>?C variant was analyzed with cumulative smoking dose (pack-year) consistently GC or CC genotype carriers have increased risk of NSCLC among heavy smokers (pack-year???25) with OR (95% CI) of 2.01 (1.26-3.20) but not among light smokers (pack-year <25) with OR (95% CI) of 1.32 (0.81-2.16). The P value for heterogeneity of the stratification analysis by smoking status is 0.015. However the P value for interaction between rs7525160 polymorphism and smoking is 0.172 and the power for the interaction is 0.49. Discussion The chronic airway inflammation and dysfunctional immune system might promote pulmonary carcinogenesis. Implicated in the immune and inflammatory responses the complement cascade plays a pivotal role in the development of cancer. Thus it is likely that the genetic variants of CR1 in the complement system confer the susceptibility to lung cancer. In this study we have for the first time demonstrated that one intronic SNP (rs7525160 G?>?C) out of 13 tag SNPs of CR1 was associated with the risk of NSCLC in Chinese population. Notably the rs7525160 CC genotype was associated with an increased risk of developing NSCLC (OR?=?1.52 95% CI?=?1.02-2.28; P?=?0.028) compared with the GG genotype. MDR analysis also showed that there was no gene-gene interaction among 12 tag SNPs in CR1 gene. Moreover the risk of NSCLC was associated with the rs7525160 C allele carriers in smokers with OR (95% CI) of 1.72 (1.15-2.59) but not in non-smokers with OR (95% CI) of 1.15 (0.81-1.65) indicating this SNP is a smoking-modifying risk factor for susceptibility to NSCLC. To the best of our knowledge this study shed new insight into the interplay of genetic variation of CR1 with lung cancer risk. More importantly it highlights the potential gene-environmental interaction influences the susceptibility to lung cancer. The complement system has been proposed to get involved in innate immunity with the ability to œcomplement antibody-mediated elimination of immune complex and foreign pathogens [26]. Upon complement activation the biologically active peptides C5a and C3a elicit a lot of pro-inflammatory effects and could be closely associated with tumorigenesis [27]. Complement proteins play a dual role in the tumor microenvironment. On one hand they exert a defensive effect against tumor through complement or antibody-dependent cytotoxicity [128]. On the other hand they may escape from immunosurveillance and facilitate carcinogenesis [2]. Specifically a number of experimental evidence has suggested an association between complement activation and tumor growth [2930] which provides a strong biologically link between the abnormal expression and activity of complement cascade and carcinogenesis. Till now a few studies have been carried out to demonstrate the association of genetic variants in complement proteins with susceptibility to cancer. A significant association of CR2 SNP (rs3813946) with the development of nasopharyngeal carcinoma was indicated in Cantonese population [31] and the genetic variations of complement system genes C5 and C9 plays a potential role in susceptibility to non-Hodgkin lymphoma (NHL) [32]. Recently it has been shown that complement factor H Y402H polymorphism interact with cigarette smoking to confer the susceptibility to lung cancer [33]. Furthermore it has been indicated that CR1 A3650G (His1208Arg) polymorphism plays a critical role in conferring genetic susceptibility to gallbladder cancer in north Indian population [19]. However whether the genetic variants of CR1 are related to the risk of lung cancer remains unknown. In this case-control study we found an intronic SNP (rs7525160 G?>?C) with CC genotype was significantly associated with an increased risk of NSCLC. Consistently our results were in accordance with the study that genetic polymorphisms in innate immunity genes may play a role in the carcinogenesis of lung cancer [34]. It is likely that some genetic variations in strong link disequilibrium with this intronic SNP (rs7525160 G?>?C) are functional which provides a new insight into the hallmarks in susceptibility to lung cancer and further functional experiments are warranted to address the proposal. Functionally human CR1 exists on the surface of almost all peripheral blood cells and plays a key role in immune complex clearance and complement inhibition at the cell surface by binding to activated products C3b and C4b [435]. CR1 also possesses cofactor activity for the serum protease factor I and is thus involved in the generation of further fragments of C3/4b with the activation of complement cascade and the cellular immune response [4]. In our study the association of CR1 polymorphism with lung cancer is biologically plausible in that the intronic polymorphism could affect the density of CR1 molecules on the cell surface thereby contributing to autoimmune disorders and neoplasm. Tobacco smoking is an established risk factor for susceptibility to lung cancer. However not all people who suffer from lung cancer are smokers. Lung cancer in non-smokers can be induced by second hand smoke air pollutants and diesel exhaust [36-39]. Our present data showed significant difference of pack-year smoked but not smoking status between NSCLC cases and controls which suggested the important role of other environmental factors in the development of NSCLC. Tobacco could induce chronic and sustained inflammation in lung microenvironment contributing to pulmonary carcinogenesis in smokers [40]. Support also comes from the epidemiologic data regarding inflammation and lung cancer [41]. CR1 an important molecule implicated in immunity and inflammation could protect the host from invasion of exogenous chemicals derived from cigarette smoking. Genetic variant of CR1 could alter gene function and result in deregulation of the inflammatory and immune responses thereby modulating the susceptibility to lung cancer. More importantly we observed a potential interaction of this SNP (rs7525160 G?>?C) with smoking status suggesting the gene-environmental interaction plays a prominent role in the susceptibility to lung cancer. Our present study has its limitation. Our patients may not be representative of total NSCLC patients at large because they were recruited from only one hospital. In addition due to the relatively small sample size further case-control studies are still needed to replicate and extend our findings. Conclusion We conducted a case-control study in Chinese subjects and found an intronic SNP (rs7525160 G?>?C) of CR1 was significantly associated with lung cancer risk. To the best of our knowledge this study provides the first evidence that genetic variant of CR1 (rs7525160 G?>?C) was a smoking-modifying contributor to the development of lung cancer. Methods Study subjects This case-control study consisted of 470 patients with histopathologically confirmed NSCLC and 470 cancer-free controls. All subjects were genetic unrelated ethnic Han Chinese. Patients were recruited between January 2008 and December 2012 at Tangshan Gongren Hospital (Tangshan China). There were no age gender or stage restrictions however patients with previous malignancy or metastasized cancer from other ans were excluded. The response rate for patients was 94%. The controls were randomly selected from a pool of a cancer-free population from a nutritional survey conducted in the same region. The selection criteria for control subjects included: i) no individual history of cancer; ii) frequency matched to cases according to gender age (±5 years); iii) the residential region; and iv) the time period for blood sample collection. At recruitment informed consent was obtained from each subject and each participant was then interviewed to collect detailed information on demographic characteristics. This study was approved by the institutional review board of Hebei United University. Tag SNPs selection and genotyping Based on the Chinese population data from HapMap database we used Haploview 4.2 program to select candidate tag SNPs with an r2 threshold of 0.80 and minor allele frequency (MAF) greater than 1%. Furthermore we also added two potential functional polymorphisms rs9429942 and rs6691117 [4243]. Therefore we included 13 SNPs in our study which represents common genetic variants in Chinese population. Genotyping was performed at Bomiao Tech (Beijing China) using iPlex Gold Genotyping Asssy and Sequenom MassArray (Sequenom San Diego CA USA). Sequenom™s MassArray Designer was used to design PCR and "

Lung_Cancer

"Background We aimed to evaluate the clinical significance of microvessel density (MVD) lymphatic vessel density (LVD) and cancer-associated fibroblasts (CAFs) in relation to tumor location in advanced colorectal cancer (CRC). Methods Using immunohistochemistry we examined 181 advanced CRC patients for CD31 and D2-40 to measure MVD and LVD respectively ?-smooth muscle actin (SMA) and desmin to identify CAFs and PTEN to examine genetic changes of CAFs. To evaluate the regional heterogeneity of these properties we examined tissue from four sites (the center and periphery of the primary cancer a distant metastasis and a lymph node metastasis) in each patient. Results MVD LVD and CAFs showed significant heterogeneity with respect to the tumor location. LVD was the greatest in the center of the primary cancers and the amount of CAFs was the lowest in distant metastases. In distant metastases those from the lung had higher LVD and MVD but fewer CAFs than those from the liver peritoneum or ovary. Patients with low MVD and LVD in the center of the primary cancer had worse outcomes and patients with few CAFs in distant metastases and in the primary tumor had a lower survival rate. PTEN expression in CAFs in distant metastases was lost in 11 of 181 CRC patients (6.1%) which was associated with a worse prognosis. Conclusions The microenvironment including cancer-associated microvasculature and fibroblasts is heterogeneous with respect to the tumor location in CRC patients. Therefore heterogeneity of microenvironments should be taken into account when managing CRC patients. This study was supported by grant number 03-2011-012 from the Seoul National University Bundang Hospital Research Fund. The funder had no role in study design data collection and analysis decision to publish or preparation of the manuscript. Introduction Although the mortality rates of colorectal cancer (CRC) patients have decreased in most western countries and in several developing countries in Asia advanced CRC patients who initially present with stage IV disease or those who develop distant metastases several months after diagnosis still have a lower five-year survival rate [1] [2]._ENREF_4 Recently the range of systemic chemotherapy has expanded and targeted therapy including epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) inhibitor therapies have been used in advanced CRC patients increasing patient survival [3]. However some CRC patients respond poorly to targeted therapy despite presenting positive results in targeted therapy-specific mutation studies [4]. One possible explanation for this therapeutic failure is tumor heterogeneity; several studies have reported that CRCs possess a heterogenic genotype or phenotype including KRAS p53 and BRAF [5]“[7]. Therefore the differing characteristics of the primary tumor site and the corresponding metastatic an need to be clarified to improve the management of CRC patients with metastatic diseases. Furthermore understanding the clinicopathological characteristics of advanced CRC is important for the development and improvement of systemic therapies. Since Paget et al. first described the cancer microenvironment by the œseed and soil theory [8] there has been growing evidence that cancer-associated stroma might affect the cancer cells themselves and contribute to cancer progression [9]. The main components of the cancer microenvironment are microvasculature (microvessels and lymphatic vessels) inflammatory cells and cancer-associated fibroblasts (CAFs) [10]“[12]. The current method of verifying angiogenetic and lymphangiogenetic activity in cancer tissue is to assess microvessel density (MVD) and lymphatic vessel density (LVD) respectively. MVD has been proposed as a surrogate marker of cancer-associated angiogenesis to identify patients with a high risk of recurrence or those with poor prognoses for various cancers including CRC [13] [14]; however the prognostic correlation of angiogenesis in CRC is still controversial [15] [16]. Similar to angiogenesis LVD has received interest as a means of lymphatic metastasis and survival [17] [18] but its role in tumor progression is still unclear [19]. The other prominent component of stroma CAFs are consistently activated and affect many aspects of tumor initiation invasion and progression [9]. While some studies have suggested that CAFs may inhibit tumor progression [20] [21] other studies have proposed that CAFs may promote progression in prostate breast and skin cancers [22]“[24]. In the context of CRC Tsujino et al. have suggested that ?-smooth muscle actin (SMA)-expressing CAFs might be a useful indicator of poor prognosis. However these results were restricted to stage II and III CRCs [25]. In addition to cancer cells genetic alterations in CAFs have demonstrated including the loss of heterozygosity microsatellite instability and genetic mutations [26] [27]. Recently genetic inactivation of PTEN in CAFs was reported in breast cancer patients [28]. Trimboli et al. identified that PTEN loss in stromal fibroblasts resulted in extensive extracellular matrix remodeling and angiogenesis which characteristic of tumor progression [28]. However expression loss of PTEN and its clinical significance have not been investigated in colorectal cancer patients. The aim of this study was to investigate the characteristics of microenvironments including microvasculatures and CAFs in advanced CRC patients. Additionally we assessed the intratumoral heterogeneity in the primary tumor and the discordance between primary tumor and distant metastasis microenvironments. Materials and Methods Patient selection A total of 181 advanced CRC patients with synchronous or metachronous metastases who were treated at Seoul National University Bundang Hospital (Seongnam-si South Korea) between 2003 and 2009 were enrolled in this study. Synchronous metastases were defined as distant metastases occurring within six months of the primary diagnosis of CRC and metachronous metastases were those occurring after that time point [29]. The cancer tissue used in this study was received from patients that had surgical resection of both the primary tumor and related metastases. None of the patients had received chemo- or radiotherapy before the resection of the primary tumor. Medical charts and pathology reports were reviewed to record clinical and pathological data. Glass slides were reviewed to determine the histological type according to the WHO classification [30]. Follow-up information including the patient outcome and the time interval between the date of surgical resection and death was collected. The cases lost to follow-up and deaths from causes other than CRC were considered censored data for the survival analysis. The median follow-up period was 37.9 months (range 0.8“104.6 months). Ethical statement All human specimens were obtained from the files of surgically resected cases examined at the Department of Pathology Seoul National University Bundang Hospital for the pathologic diagnosis. The retrospective study was performed using the stored samples after the pathologic diagnosis and all of the samples were anonymized before the study. The participants did not provide written informed consent in this study. The study was approved by the Institutional Review Board of Seoul National University Bundang Hospital under the condition of anonymization (reference: B-1109/136-302). Tissue array methods To evaluate the regional stromal differences samples were taken from each patient from four areas: the center and periphery of the primary cancer distant metastases and lymph node metastases. The distant metastatic sites for the tissue arrays were as follows: liver in 83 cases (45.9%) lung in 38 cases (21.0%) seedings in 38 cases (21.0%) distant lymph nodes in 6 cases (3.3%) and ovary in 16 cases (8.3%). The representative core tissue specimens (2 mm in diameter) were taken from individual paraffin blocks and rearranged in new tissue array blocks using a trephine apparatus (Superbiochips Laboratories Seoul South Korea) [31]. Immunohistochemistry Array slides were labelled by immunohistochemistry using antibodies for CD31 (1?100 DAKO Glostrup Denmark) D2-40 (1?100 DAKO) SMA (1?1000 Neomarkers Fremont CA USA) desmin (1?300 DAKO) and PTEN (1?80 Epitomics Burlingame CA USA) after a microwave antigen retrieval procedure except SMA. Non-reactive sites were blocked using 1% horse serum in Tris-buffered saline (pH 6.0) for 3 min. Primary antibodies were applied and antibody binding was detected with diaminobenzidine (DAB). Sections were counterstained with hematoxylin. The reactivity of PTEN in each tissue section was scored as negative faint or strong and the percentage of PTEN-positive fibroblasts was quantified. For the statistical analysis the sample was deemed PTEN-positive if 5% or more CAFs were scored as strong positives. Calculation of LVD MVD and CAFs using digital pathology Slides were concurrently evaluated by two pathologists (H.E.L and H.S.L) using light microscopy to improve the accuracy of the results (Fig. 1). CRC cells were considered as internal negative controls. Medium- to large-sized vessels were considered as internal positive controls for CD31 and D2-40. Intestinal muscular layer or medium- to large-sized vessels were considered as internal positive controls for desmin and SMA. Samples showing inappropriate staining in internal negative or positive controls were considered non-informative and were excluded from the analysis. Slides were scanned using an Aperio ScanScope® CS instrument (Aperio Technologies Inc. Vista CA) at 20× magnification. Subsequently they were analyzed in ImageScope„¢ using the Microvessel Analysis v1 algorithm (Aperio Technologies) and MVD and LVD were calculated. Because desmin-positive muscularis mucosa and propria are positive for SMA immunostaining the area of CAFs (mm2) was calculated by subtracting the areas of desmin staining from that of SMA staining (SMA - desmin). 10.1371/journal.pone.0091811.g001 Figure 1 Representative sections from four tumor locations stained with CD31 D2-40 SMA or desmin antibodies (×100). Statistical analysis A chi-squared test or Fisher's exact test (2-sided) for non-continuous variables and Mann-Whitney or Kruskal-Wallis analysis for continuous variables were used to compare each parameter with respect to the CRC site and according to its clinicopathologic features. The correlation between continuous variables was analyzed using the Pearson correlation coefficient. To determine the best cut-offs of continuous variables for predicting patient survival the maximal chi-squared method was performed using R program (http://cran.r-project./). The overall survival curves were plotted using the Kaplan-Meier product-limit method and the significance of the differences between these curves was determined using the log-rank test. A univariate and multivariate regression analysis was performed using the Cox's proportional hazards model to determine hazard ratios (HRs). P-values of less than 0.05 were considered statistically significant. All statistical analysis excluding the maximal chi-squared test was performed with the IBM SPSS statistics 20 (Armonk NY USA). Results 1. Heterogeneity of cancer-associated stroma according to examined tumor locations The clinicopathological characteristics of the advanced CRC patients are described in Table 1. The CRC patients with synchronous metastases had aggressive features including larger tumor size more advanced pT and pN stage and the presence of perineural and venous invasion than the patients with metachronous metastasis (p<0.05). 10.1371/journal.pone.0091811.t001 Table 1 Clinicopathologic characteristics of advanced colorectal cancers. Parameters Total Metachronous Synchronous P value (n?=?181) (n?=?57) (n?=?124) Age (median range) 60.00 (28“93) 62.00 (36“79) 60.00 (28“93) 0.241 Sex 0.007 Male 97 39 (68.4%) 58 (46.8%) Female 84 18 (31.6%) 66 (53.2%) Location 0.055 Right colon 37 6 (10.5%) 31 (25.0%) Left colon 75 29 (50.9%) 46 (37.1%) Rectum 69 22 (38.6%) 47 (37.9%) Size of primary tumor 5.30 (2.0“13.0) 4.20 (2“9) 5.50 (2.5“13) <0.001 Histologic grade 0.227 Low grade 157 52 (91.2%) 105 (84.7%) High grade 24 5 (8.8%) 19 (15.3%) T stage <0.001 T1 0 0 0 T2 5 3 (5.3%) 2 (1.6%) T3 107 45 (78.9%) 62 (50.0%) T4 69 9 (15.8%) 60 (48.4%) N stage <0.001 N0 35 23 (40.4%) 12 (9.7%) N1 58 23 (40.4%) 35 (28.2%) N2 88 11 (19.3%) 77 (62.1%) Perineural invasion 0.011 Absent 89 36 (63.2%) 53 (42.7%) Present 92 21 (36.8%) 71 (57.3%) Venous invasion 0.028 Absent 126 46 (80.7%) 80 (64.5%) Present 55 11 (19.3%) 44 (35.5%) The heterogeneous values for LVD MVD and CAF area are shown in Fig 2. LVD was the highest in center of the primary cancers (median interquartile range (IQR); 37.00 10.50“81.00) than any other site (5.00 1.00“23.75 at the periphery; 2.50 1.00“15.00 in lymph node metastases; 3.00 1.00“20.00 in distant metastases). MVD was lower in distant metastases (median IQR; 641.50 428.00“1006.75) than at the periphery of the primary cancer (731.00 508.25“1049.75) and lymph node metastases (893.50 520.25“1275.25). The area occupied by CAFs was the lowest in the distant metastases (median IQR; 0.91 0.68“1.18) than any other site (1.12 0.88“1.41 in the center; 1.22 0.96“1.54 in the periphery1.4 1.00“1.71 in lymph node metastases). In addition the stromal characteristics varied in relation to the metastatic an examined. MVD and LVD were the higher in lung metastases than those in the liver peritoneum or lymph nodes (p<0.001; Fig. 3). However the amounts of CAFs were consistent among the different metastatic ans (p?=?0.180). 10.1371/journal.pone.0091811.g002 Figure 2 Heterogeneity of lymphatic vessel density (LVD) microvessel density (MVD) and amount of cancer-associated fibroblasts (CAFs) with respect to tumor location. The LVD (A) MVD (B) and CAF area (C) was significantly different according to each tumor location. 10.1371/journal.pone.0091811.g003 Figure 3 LVD MVD and CAF area at different distant metastasis sites. The characteristics of cancer-associated stroma differed with respect to the metastatic site. LVD (A) and MVD (B) were greater in the metastatic tumor samples collected from the lung than in samples collected from other metastatic sites (p<0.001). However the amount of CAFs was not significant different between metastatic sites (C). Despite the heterogeneity of stromal characteristics CRC cases with higher LVD MVD and CAFs in center of the primary cancers had a tendency of higher LVD MVD and CAFs in periphery (p<0.05; Table S1). However LVD in center and periphery of primary cancer were not correlated with LVD in related distant metastasis (Table S1). In addition the amount of microvasculature was significantly correlated with the amount of CAFs (Table S2). 2. Clinical significance of cancer-associated stroma in advanced CRCs The MVD LVD and amount of CAFs present at each tumor location were compared according to their clinicopathologic features (Table 2). High grade CRCs were associated with lower CAFs in samples taken from the central cancer site (p?=?0.041). When compared with synchronous metastases the patients with metachronous metastases had higher LVD in center and periphery of the primary cancer and had higher MVD in lymph node metastases. Most patients with metachronous metastases were treated by adjuvant chemotherapy before metastasectomy. LVD and MVD in the distant metastases were significantly higher in the patients who had received chemotherapy before metastasectomy than those who did not (p?=?0.011 and 0.048 respectively). 10.1371/journal.pone.0091811.t002 Table 2 Clinicopathologic factor and LVD MVD and CAFs. Center (median) Periphery (median) LN metastasis (median) Distant metastasis (median) LVD MVD CAFs LVD MVD CAFs LVD MVD CAFs LVD MVD CAFs Total 39 717 1.13 5 740 1.22 3 888 1.42 3 648 0.91 Histologic grade Low grade 40 717 1.15* 5 741 1.23 3 895 1.43 3 665 0.92 High grade 34 683.5 0.94* 6 643.5 1.18 2 656 1.32 6 498 0.82 pT stage pT2 34 758 1.15 16 870 1.48 6 772 0.73 pT3 47 737 1.19 5 803 1.22 2.5 884 1.43 3 724 0.92 pT4 33 639 1.09 4 630 1.22 3 895 1.41 3 520 0.93 LN metastasis Absent 49 602 1.15 8 712 1.42 4 772 0.94 Present 39 737.5 1.12 4 740 1.21 3 884 1.41 3 617 0.91 Perineural invasion Absent 41 738 1.12 6 772 1.32 5.5 931.5 1.42 4 687 0.94 Present 39 672 1.13 4 702 1.2 2 796 1.39 3 548.5 0.86 Metastasis Synchronous 34* 717.5 1.11 3.0* 741 1.21 3 797* 1.39 3 617 0.93 Metachronous 55* 716 1.21 8.0* 712 1.23 2 1117* 1.63 5 698 0.91 Chemotherapy  Not done 2.0* 597.5* 0.93 Done 10.0* 684* 0.91 * p<0.05; ** p<0.01;   chemotherapy prior to metastatectomy of distant metastasis. 3. Expression loss of PTEN in CAFs PTEN was expressed in cytoplasm and sometimes the nucleus of both cancer and non-neoplastic cells when examined using immunohistochemistry. Expression of PTEN was lost in 8 cases in the center 2 cases in the periphery 4 cases in lymph node metastases and 11 cases in distant metastases (Table S3). In all 11 distant metastases with PTEN loss PTEN expression was intact in both the center and periphery of primary cancer (data not shown). PTEN loss in distant metastasis was correlated with synchronous metastasis (p?=?0.018). 4. Cancer-associated stroma and patient prognosis By using the obtained cut-offs lower LVD MVD and CAFs in the center LVD and CAFs in the periphery and MVD and CAFs in distant metastases were all significantly correlated with lower survival (p<0.05; Fig. S1). Among other clinicopathologic features synchronous metastasis old age larger size high histologic grade advanced pT and pN stage and presence of perineural invasion were associated with a worse prognosis (Table 3). By multivariate Cox regression analysis the hazard ratio of synchronous versus metachronous was the highest (4.029) with the lowest p value (p<0.001). CAFs in distant metastasis LVD and MVD in the center LVD in the periphery age and perineural invasion also independently predicted patient survival. In addition loss of PTEN expression in CAFs in distant metastases was associated with a worse prognosis (p?=?0.042; Fig S2) but not in primary cancer or lymph node metastasis. 10.1371/journal.pone.0091811.t003 Table 3 Univariate and multivariate survival analysis according to clinicopathologic features. Univariate survival analysis Multivariate survival analysis Factors HR (95% CI) P value HR (95% CI) P value Synchronous vs. Metachronous 4.617 (2.472“8.624) <0.001 3.762 (1.838“7.701) <0.001 Age 1.023 (1.004“1.044) 0.020 1.033 (1.011“1.056) 0.003 Sex (female vs. male) 1.428 (0.920“2.218) 0.113 ” ” Location (left vs. right) 0.503 (0.314“0.806) 0.004 0.700 (0.413“1.188) NS (0.186) Size 1.073 (1.005“1.146) 0.036 1.040 (0.903“1.198) NS (0.584) Histologic grade (high vs. low) 1.862 (1.061“3.269) 0.030 1.491 (0.763“2.912) NS (0.243) pT stage (pT4 vs. pT2/3) 2.341 (1.503“3.645) <0.001 1.137 (0.674“1.921) NS (0.630) pN stage (pN1/2 vs. pN0) 3.848 (1.760“8.411) 0.001 1.773 (0.758“4.146) NS (0.186) Perineural invasion 2.628 (1.640“4.211) <0.001 2.108 (1.265“3.513) 0.004 Venous invasion 1.217 (0.757“1.956) 0.418 ” Center LVD (high vs. low) 0.364 (0.158“0.836) 0.017 0.298 (0.118“0.753) 0.010 Center MVD (high vs. low) 0.391 (0.233“0.655) <0.001 0.437 (0.238“0.801) 0.007 Center CAFs (high vs. low) 0.579 (0.352“0.954) 0.032 1.038 (0.607“1.773) NS (0.892) Periphery LVD (high vs. low) 0.235 (0.086“0.644) 0.005 0.279 (0.096“0.809) 0.019 Periphery MVD (high vs. low) 1.456 (0.911“2.327) 0.117 ” Periphery CAFs (high vs. low) 0.524 (0.336“0.817) 0.004 0.813 (0.499“1.326) NS (0.406) LN LVD (high vs. low) 1.646 (0.874“3.100) 0.123 ” LN MVD (high vs. low) 0.597 (0.294“1.213) 0.154 ” LN CAFs (high vs. low) 0.717 (0.423“1.217) 0.218 ” Metastasis LVD (high vs. low) 0.569 (0.314“1.032) 0.063 ” Metastasis MVD (high vs. low) 0.579 (0.364“0.921) 0.021 1.262 (0.720“2.211) NS (0.417) Metastasis CAFs (high vs. low) 0.492 (0.271“0.894) 0.020 0.290 (0.144“0.582) 0.001 Metastasis PTEN (intact vs. loss) 0.454 (0.208“0.993) 0.048 0.575 (0.239“1.383) NS (0.217) Discussion Carcinoma cells in different tissue areas have distinct characteristics [32]. In central areas of the tumor carcinoma cells maintain an epithelial cell phenotype but carcinoma cells in the invasive front acquire a more malignant and mesenchymal phenotype and are thought to have an increased migratory capacity and contribute to metastatic diseases. These metastatic cells may restore the epithelial phenotype at metastatic sites [33]. In addition to carcinoma cells themselves microenvironment is suggested to be uneven within a given tumor because tumor formation and progression involve the co-evolution of cancer cells and microenvironments [34]. The present study demonstrated that the cancer-associated microenvironment also had distinct characteristics in different areas. Of the sites examined LVD was highest in the center of the primary cancer. MVD was slightly higher in center than at the periphery of the primary cancer but this difference was not statistically significant. Interestingly the amount of CAFs in distant metastases was significantly lower than in center and periphery of the primary cancer. We show that the stromal microenvironment has regional heterogeneity both within the primary tumor and between the primary site and its related metastases. Furthermore our data suggests that the stromal heterogeneity might be attributable to tumor heterogeneity. Therefore it would be beneficial to consider both stromal and tumor cell heterogeneity in order to manage CRC patients better. We evaluated the MVD LVD and amount of CAFs in metastatic tissues of various ans including the liver lung peritoneal seeding distant lymph nodes and ovary. Of the metastatic ans we examined both LVD and MVD were the highest in lung. In our previous study the KRAS discordance rate was also significantly higher in matched lung metastases than in other matched metastatic ans [35]. The underlying mechanism is not known. It could be that primary CRCs with high LVD and MVD have a tendency to produce lung metastases; however our results indicated that LVD and MVD in the center and at the periphery of the primary cancers were lower in the patients with lung metastases (data not shown). Alternatively it may be due to the physiological characteristics of metastatic ans interactions between cancer cells and microenvironment within the metastatic an or the characteristics of the cancer cell clones prone to lung metastasis. However technical or sampling errors also may be possible thus further large-scale studies are required. Although numerous studies have attempted to demonstrate an association between tumor microenvironment characteristics and survival the prognostic impacts of MVD and LVD are still controversial. Some studies have been presented that active angiogenesis and lymphangiogenesis represented by high MVD and LVD are associated with poor prognosis and aggressive clinicopathologic factors [36] [37]. Recent meta-analysis has demonstrated that LVD was significantly associated with disease-free survival but not overall survival [38]. Other studies have reported no statistical significance of MVD and LVD on survival [39]. Prall et al. has reported that high MVD and LVD are related with better survival in a consecutive series and liver metastases [40]. Our results were based on patients with advanced disease with distant metastasis and we showed that high MVD and LVD were related with improved survival. This might be because all the patients in this study had confirmed to have distant metastasis and microvasculatures could influence even delivery of the chemotherapeutic drug into the tumor. However our study had some limitations in terms of the survival analysis. We enrolled the CRC patients with available surgically resected cancer tissues from both primary tumors and corresponding metastatic tumors. Not all advanced CRC patients with metastatic diseases were included and far advanced cases were not enrolled because of their inoperability. Therefore unrecognized biases might have influenced our survival results. Some studies have demonstrated an anti-tumorigenic effect of fibroblasts [20] [21]. However it has become clear that CAFs contribute to the progression of cancer and their prognostic significance in various cancers also has been raised [41] and furthermore several studies have observed genetic alterations in CAFs [26] [27]. PTEN loss of CAFs has been observed in breast cancer and prognostic association of it has been suggested [27] [28]. We observed PTEN loss of CAFs in CRC patients and it was more frequently observed in the corresponding distant metastases. It is suggested that CAFs not only cancer cells have altered gene expression. Moreover loss of PTEN expression of CAFs in distant metastases was significantly correlated with the survival of patients. To our knowledge these are the first results showing PTEN loss in CAFs in CRC patients. Although more research is required we expect that it might be a prognostic factor in CRC patients. In our large cohort of advanced CRC patients with synchronous and metachronous distant metastasis we demonstrated the regional heterogeneity of stromal microenvironment factors according to the tumor location. The amount of microvasculature measured by LVD and MVD was also heterogeneous in relation to the metastatic an examined. By Cox regression analysis center LVD and MVD periphery LVD and CAFs in distant metastasis were independently associated with patients' prognosis in addition to synchronous distant metastasis age and perineural invasion. Heterogeneity of microenvironment not only of cancer cells is suggested to contribute to tumor heterogeneity and biologic complexity thus it should be considered in managing CRC patients. In addition our results showed that PTEN expression was altered in CAFs of CRCs suggesting that CAFs might have altered gene expression and play an active role in cancer progression. Supporting Information Figure S1 The prognostic association of stromal characteristics as it relates to tumor location. The analysis was performed by using cut-off values obtained by maximal chi-squared methods. (TIF) Click here for additional data file. Figure S2 Representative PTEN antibody stainings of stromal cells and the prognostic association of PTEN expression. (A) Intact expression of PTEN in CAFs (×400) and (B) loss of PTEN expression in CAFs (×400). (C“F) Kaplan-Meier survival curves for the center (C) and periphery (D) of the primary tumor lymph node metastases (E) and distant metastases (F) according to CAF PTEN expression status. (TIF) Click here for additional data file. Table S1 Pearson's correlation coefficients among center periphery lymph node metastasis and distant metastasis. (DOCX) Click here for additional data file."

Lung_Cancer

"This combination has two possible explanations. Firstly they might represent tumors which are not expressing ALK protein at detectable levels because of a false-positive FISH result or an absence of addiction to rearranged ALK protein despite presence of recombined ALK DNA. These tumors are unlikely to respond to crizotinib. Secondly they might represent a failure of the IHC assay because of poor preservation of antigen insufficient material or another technical error. In this case crizotinib therapy would still be likely to be effective. The study includes seven cases with positive FISH results with data on response to crizotinib. Six showed at least a partial response to crizotinib therapy as assessed by Response Evaluation Criteria in Solid Tumors criteria. A single case showed no response and this was one of the two œfalse negatives. Thus in this one case the IHC test would have correctly predicted response. This was a scanty cytological specimen in which FISH interpretation was difficult and only 20% of 515 cells showed rearranged ALK signals (fusion plus split red/green probes and fusion plus isolated red signal). Therefore it seems possible that this represents a technical failure of the FISH assay. The other case an excision specimen showed 39% of 626 cells with rearranged signals. It was also shown to harbor a driving mutation in a second gene; PCR testing demonstrated the loss of exon 19 of endothelial growth factor receptor. This has been described in another study that characterized two such œfalse-negative cases.15 Thus these tumors may well escape œoncogene addiction to the ALK kinase activity which would be consistent with indetectable ALK protein expression. Again IHC would be expected to be the best predictor of response to tyrosine kinase inhibitor therapy in such cases. It is essential to identify and molecularly characterize other œfalse-negative cases that have received crizotinib therapy. In addition it seems likely that IHC should guide treatments in œfalse-positive cases that express high levels of ALK from genetic lesions that are invisible to the current FISH assay. Although we identified no œfalse positives i.e. FISH-negative IHC-positive cases our sensitivity may be an overestimate (as judged by FISH) because of the small number of FISH-negative cases under study. Several studies have identified rare cases with rearrangements that are indetectable by FISH but detectable by IHC and confirmed by reverse-transcriptase PCR.5910 Such cases would be expected to respond to crizotinib and a recent study shows that at least one novel œFISH-indetectable rearrangement does indeed drive the malignant phenotype.5 One limitation of this study is the small number of cases under study although 15 FISH-positive cases is comparable to most other studies. The relatively small number of FISH-negative cases may have affected our ability to identify FISH-negative IHC-positive cases. However the study design does permit an assessment of the sensitivity of the IHC assay which is the most important consideration for a possible screening test. Our comparison of the immunohistochemical assays was not directly equivalent as the D5F3 assay included a proprietary tyramide signal amplification step whereas the ALK1 and 5A4 assays were conducted using our routine diagnostic detection system. However our study design also has several strengths. In particular the use of archival diagnostic paraffin blocks and FISH testing conducted in the course of routine diagnosis make the results of the study directly relevant to clinical practice. In summary we find IHC to be a highly sensitive (86%) and specific (100%) test for ALK rearrangement in lung adenocarcinoma. We find a slight advantage of a proprietary amplified assay (D5F3 Ventana) over two other antibodies with conventional DAB staining (ALK1 Dako and 5A4 Abcam) but only in scanty samples. Intensity of staining was the most discriminating measure and the proportion of cells staining did not contribute. We identified two cases that were positive for the ALK rearrangement by FISH but negative by all immunohistochemical assays and suggest that in discordant cases the IHC test result may be more predictive of treatment response than FISH. Further discordant cases need to be examined to help guide the treatment of these cases. Immunohistochemical testing is clearly at least a useful adjunct to FISH and we feel that it is reasonable in routine practice to use a sensitive IHC assay as a screening test. The danger of missing treatable cases using this method (i.e. FISH-positive IHC-negative crizotinib-sensitive tumors) appears very small especially when specimens contain adequate material. In difficult cases further investigations such as re-biopsy and repeated IHC/FISH may be helpful. Disclosure: This project was supported by the National Institute of Health Research Respiratory Disease Biomedical Research Unit at the Royal Brompton and Harefield NHS Foundation Trust and Imperial College London and the NIHR RM/ICR Biomedical Research Center. All other authors declare no conflict of interest. REFERENCES 1. Inamura K Takeuchi K Togashi Y EML4-ALK lung cancers are characterized by rare other mutations a TTF-1 cell lineage an acinar histology and young onset. Mod Pathol 2009 22 508 515 19234440 2. Koivunen JP Mermel C Zejnullahu K EML4-ALK fusion gene and efficacy of an ALK kinase inhibitor in lung cancer. Clin Cancer Res 2008 14 4275 4283 18594010 3. Shinmura K Kageyama S Tao H EML4-ALK fusion transcripts but no NPM- TPM3- CLTC- ATIC- or TFG-ALK fusion transcripts in non-small cell lung carcinomas. Lung Cancer 2008 61 163 169 18242762 4. Soda M Choi YL Enomoto M Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature 2007 448 561 566 17625570 5. To KF Tong JH Yeung KS Detection of ALK rearrangement by immunohistochemistry in lung adenocarcinoma and the identification of a novel EML4-ALK variant. J Thorac Oncol 2013 8 883 891 23625156 6. McDermott U Iafrate AJ Gray NS Genomic alterations of anaplastic lymphoma kinase may sensitize tumors to anaplastic lymphoma kinase inhibitors. Cancer Res 2008 68 3389 3395 18451166 7. Kwak EL Bang YJ Camidge DR Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med 2010 363 1693 1703 20979469 8. Lindeman NI Cagle PT Beasley MB Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists International Association for the Study of Lung Cancer and Association for Molecular Pathology. J Thorac Oncol 2013 8 823 859 23552377 9. Murakami Y Mitsudomi T Yatabe Y A Screening Method for the ALK Fusion Gene in NSCLC. Front Oncol 2012 2 24 22655265 10. Rodig SJ Mino-Kenudson M Dacic S Unique clinicopathologic features characterize ALK-rearranged lung adenocarcinoma in the western population. Clin Cancer Res 2009 15 5216 5223 19671850 11. Peled N Palmer G Hirsch FR Next-generation sequencing identifies and immunohistochemistry confirms a novel crizotinib-sensitive ALK rearrangement in a patient with metastatic non-small-cell lung cancer. J Thorac Oncol 2012 7 e14 e16 22895149 12. Eisenhauer EA Therasse P Bogaerts J New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 2009 45 228 247 19097774 13. Selinger CI Rogers TM Russell PA Testing for ALK rearrangement in lung adenocarcinoma: a multicenter comparison of immunohistochemistry and fluorescent in situ hybridization. Mod Pathol 2013 26 1545 1553 23743928 14. Conklin CM Craddock KJ Have C Laskin J Couture C Ionescu DN Immunohistochemistry is a reliable screening tool for identification of ALK rearrangement in non-small-cell lung carcinoma and is antibody dependent. J Thorac Oncol 2013 8 45 51 23196275 15. Sholl LM Weremowicz S Gray SW Combined use of ALK immunohistochemistry and FISH for optimal detection of ALK-rearranged lung adenocarcinomas. J Thorac Oncol 2013 8 322 328 23407557 16. Savic S Bode B Diebold J Detection of ALK-positive non-small-cell lung cancers on cytological specimens: high accuracy of immunocytochemistry with the 5A4 clone. J Thorac Oncol 2013 8 1004 1011 23689429 Br J Cancer Br. J. Cancer British Journal of Cancer 0007-0920 1532-1827 Nature Publishing Group 24292447 3915116 bjc2013735 10.1038/bjc.2013.735 Clinical Study A phase II multicentre study of ziv-aflibercept in combination with cisplatin and pemetrexed in patients with previously untreated advanced/metastatic non-squamous non-small cell lung cancer Ziv-aflibercept/cisplatin/pemetrexed in NSCLC Chen H 1 * Modiano M R 2 Neal J W 3 Brahmer J R 4 Rigas J R 5 Jotte R M 6 Leighl N B 7 Riess J W 3 Kuo C J 3 Liu L 8 Gao B 8 DiCioccio A T 8 Adjei A A 1 Wakelee H A 3 1Department of Medicine Roswell Park Cancer Institute Elm & Carlton Streets Buffalo NY 14263 USA 2Arizona Oncology/Arizona Clinical Research Center 1620W. St Mary's Rd Tucson AZ 85745 USA 3Department of Medicine Stanford University School of Medicine and Cancer Institute 875 Blake Wilbur Dr Stanford CA 94305 USA 4Department of Oncology The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins Bunting/Blaustein CRB 1650 Orleans St. G94 Baltimore MD 21231 USA 5Department of Medicine Norris Cotton Cancer Center Geisel School of Medicine at Dartmouth 1 Medical Center Drive Lebanon NH 03756 USA 6Rocky Mountain Cancer Centers 1800 Williams Street Suite 200 Denver CO 80218 USA 7Department of Medicine Princess Margaret Hospital and University of Toronto 610 University Avenue Toronto ON M5G 2M9 Canada 8Regeneron Pharmaceuticals Inc. 777 Old Saw Mill River Road Tarrytown NY 10591 USA *E-mail: hongbin.chen@roswellpark.org 04 02 2014 28 11 2013 110 3 602 608 29 08 2013 27 10 2013 30 10 2013 Copyright © 2014 Cancer Research UK 2014 Cancer Research UK From twelve months after its original publication this work is licensed under the Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License. To view a copy of this license visit http://creativecommons.org/licenses/by-nc-sa/3.0/ Background: This study evaluated the efficacy and safety of ziv-aflibercept in combination with cisplatin and pemetrexed in non-small cell lung cancer (NSCLC). Methods: This single arm multicentre phase II trial enrolled patients with previously untreated locally advanced or metastatic non-squamous NSCLC. Patients received intravenous ziv-aflibercept 6?mg?kg?1 pemetrexed 500?mg?m?2 and cisplatin 75?mg?m?2 every 21 days for up to six cycles. Maintenance administration of ziv-aflibercept was to continue until disease progression intolerable toxicity or other cause for withdrawal. The co-primary end points were objective response rate (ORR) and progression-free survival (PFS). Planned sample size was 72 patients. Results: The study was closed prematurely because of three confirmed and two suspected cases of reversible posterior leukoencephalopathy syndrome (RPLS). A total of 42 patients were enrolled. Median age was 61.5 years; 55% were male 86% Caucasian and 50% had Eastern Cooperative Oncology Group performance status (ECOG PS)=0. A median of four cycles of ziv-aflibercept was administered. The most common treatment-emergent adverse events (TEAEs) of any grade were nausea (69%) and fatigue (67%) with hypertension (36%) as the most common grade 3/4 TEAE. Of the 38 evaluable patients ORR was 26% and median PFS was 5 months. Conclusion: Cases of RPLS had been observed in other studies in the ziv-aflibercept clinical development programme but the rate observed in this study was higher than previously observed. This might be related to declining renal function and/or hypertension. Although ORR and PFS were in accordance with most historical first-line NSCLC studies this combination of ziv-aflibercept/cisplatin/pemetrexed will not be further explored in NSCLC. ziv-aflibercept non-small cell lung cancer reversible posterior leukoencephalopathy syndrome anti-angiogenesis Cancer growth is dependent upon angiogenesis to maintain a source of nutrition and oxygen (Folkman 1995) and vascular endothelial growth factor (VEGF) has a key role in tumour angiogenesis (Ferrara and Davis-Smyth 1997). Non-small cell lung cancer (NSCLC) produces VEGF and high serum levels of VEGF are correlated with poor prognosis (Korpanty et al 2010). Anti-angiogenic therapy thus aims to disrupt blood supply to tumours and has proven clinical benefit in non-squamous NSCLC (Jain 2001). Combination chemotherapy is used for the first-line treatment of advanced/metastatic NSCLC (Schiller et al 2002). The addition of the anti-VEGF antibody bevacizumab to carboplatin/paclitaxel in this setting improved response rate progression-free survival (PFS) and overall survival (OS; Sandler et al 2006). Similarly bevacizumab improved PFS when added to cisplatin/gemcitabine although OS was not significantly prolonged as a secondary end point in this case (Reck et al 2009). For non-squamous histology cisplatin/pemetrexed is a very active combination chemotherapy (Scagliotti et al 2008) and thus combinations of platinum/pemetrexed with bevacizumab or other anti-angiogenics are of strong interest for the first-line treatment of advanced/metastatic non-squamous NSCLC (Patel et al 2009b). Ziv-aflibercept (ZALTRAP Sanofi Bridgewater NJ USA and Regeneron Pharmaceuticals Tarrytown NY USA) is a recombinant fusion protein consisting of portions of human VEGF receptor extracellular domains fused to the Fc portion of human immunoglobulin (Gaya and Tse 2012). Ziv-aflibercept binds VEGF-A by acting as a high-affinity ligand trap to prevent binding to its endogenous receptor VEGFR-2 thereby inhibiting VEGF-induced angiogenesis in preclinical models (Lassoued et al 2010). Endothelial cells expressing high levels of VEGFR-2 were highly susceptible to blockade by ziv-aflibercept (Sitohy et al 2011). In addition ziv-aflibercept binds PIGF (placental growth factor) and VEGF-B which could potentially inhibit cancer invasion (Dowlati 2010). Studies have investigated ziv-aflibercept as a single agent or in combination with other chemotherapeutic agents in treatment of various types of cancers (Lockhart et al 2010; Tew et al 2010; de Groot et al 2011; Isambert et al 2012). In August 2012 ziv-aflibercept was approved by the US FDA for use in metastatic colorectal cancer based on the results of VELOUR trial (Van Cutsem et al 2012). A phase II study using ziv-aflibercept as monotherapy demonstrated objective responses in heavily pretreated patients with advanced adenocarcinoma of the lung (Leighl et al 2010) and improvement in response and PFS (but not OS) was observed in combination with docetaxel as second-line treatment of NSCLC (Ramlau et al 2012). We report the results of a phase II trial of ziv-aflibercept in combination with cisplatin and pemetrexed in patients with advanced or metastatic non-squamous NSCLC. This study was conducted after a phase I trial using the same regimen of ziv-aflibercept/cisplatin/pemetrexed (Diaz-Padilla et al 2012). That phase I trial determined the recommended dose of ziv-aflibercept (6?mg?kg?1 every 21 days) to be used in the current phase II trial which aimed to evaluate the efficacy and safety of ziv-aflibercept in combination with cisplatin and pemetrexed in the first-line treatment of advanced/metastatic NSCLC. Materials and methods Eligibility Patients eligible for this study had histologically/cytologically confirmed untreated locally advanced/metastatic NSCLC and they had to have measurable disease as per the Response Evaluation Criteria in Solid Tumors (RECIST) criteria (Therasse et al 2000). Patients with squamous histology and/or cavitating lesions were excluded. Patients were 18 years of age or older and had an Eastern Cooperative Oncology Group performance status (ECOG PS) of 0 or 1 with adequate bone marrow renal and hepatic functions and calculated creatinine clearance (CrCL) ?60?ml?min?1. Patients were excluded from the study if they had brain or central nervous system metastases; systolic blood pressure (BP) >150?mm?Hg and/or diastolic blood pressure >100?mm?Hg; bleeding diathesis or evidence of active bleeding; or recent significant cardiovascular cerebrovascular or thromboembolic conditions. The protocol was approved by the Institutional Review Boards at each participating institution. Informed consent was obtained from each patient. Study design This is a single arm open label multicentre phase II study (ClinicalTrials.gov identifier: NCT00794417). Patients received the three-drug combination intravenously on day 1 of every 21 days with ziv-aflibercept (6?mg?kg?1) first followed by pemetrexed (500?mg?m?2) and cisplatin (75?mg?m?2). Premedications consisted of folic acid vitamin B12 and dexamethasone as a prophylactic measure to reduce pemetrexed-related toxicities and standard anti-emetics. Patients could receive up to six cycles of combination therapy. For patients who completed the combined chemotherapy maintenance ziv-aflibercept every 21 days was to continue until disease progression intolerable toxicity or withdrawal from the study. End points and assessments The two co-primary end points were objective response rate (ORR) and PFS. The ORR was defined as the proportion of patients with complete response plus partial response (CR+PR). The PFS was defined as the time interval from the first dose of combination chemotherapy to tumour progression or death whichever occurred first. Secondary variables were the determination of the adverse events (AEs) pharmacokinetics (PK) and pharmacodynamic profiles (including anti-ziv-aflibercept antibody and hematopoiesis). Pharmacokinetic end points included the area under the concentration curve maximum concentration (Cmax) clearance and terminal half-life (t1/2). Tumour imaging (CT or MRI) was performed at screening on day 21 (±7 days) of every even numbered cycle (every 6 weeks) and when disease progression was suspected. Responses were assessed using RECIST version 1.0 (Therasse et al 2000). Safety and tolerability were assessed at baseline and at least every 21 days as evaluated by AEs and changes in laboratory parameters graded according to the Common Terminology Criteria for Adverse Events (CTCAE) version 3.0 (National Cancer Institute 2006). Ziv-aflibercept (free or bound to VEGF) in plasma samples was quantified using a validated direct enzyme-linked immunosorbent assay. A validated non-quantitative titre-based bridging assay was used to detect anti-ziv-aflibercept antibodies in serum samples. Correlative studies The exploratory objective of the correlative studies was to evaluate changes in erythropoiesis in response to VEGF inhibition. It was hypothesised that VEGF inhibition would result in an increase in haemoglobin via increased hepatic erythropoietin production. Statistical analysis Statistical testing was done to determine whether the ORR was larger than 20% or whether the PFS was greater than 4.5 months. Exact test (one-sided) was used to test the null hypothesis that ORR was ?20% versus the alternative hypothesis that ORR was ?35%. Assuming type I error was not >2.5% a sample of 72 patients would provide >80% power to test the hypothesis using exact binomial test. The calculated sample size of 72 patients would also provide >90% power to test the null hypothesis that median PFS was ?4.5 months versus the alternative hypothesis that PFS was ?6.5 months at one-sided alpha of 2.5% using one sample log-rank test. Safety data were to be summarised. Concentrations of free ziv-aflibercept and adjusted bound ziv-aflibercept: VEGF complex were to be summarised every 21 days over the duration of the study by nominal time point. Noncompartmental parameters were calculated using WinNonlin (version 5.3 Pharsight Corporation Mountain View CA USA) and model 202 (constant infusion) using nominal time points after a single dose of ziv-aflibercept. The noncompartmental analysis was performed over the dosing interval 21 days following the first dose. All analyses used statistical software SAS (version 9.1.3 Cary NC USA). Results Patients This study was closed prematurely because of three confirmed and two suspected but unconfirmed cases of reversible posterior leukoencephalopathy syndrome (RPLS). A total of 42 patients were enrolled from 17 participating sites across the United States and Canada between January 2009 and December 2010. Table 1 summarises the patient demographics. Median age was 61.5 years; 55% were male 86% were Caucasian and 50% had ECOG PS of 0. Safety evaluation Treatment exposure and dose modifications All 42 patients received at least one dose of each of the three study drugs with a median of 92 days of treatment (range 21“288 days). Twenty-seven (64%) patients completed four or more cycles of the combination treatment. A median of 4.5 (range 1“6) cycles of pemetrexed and 4 (range 1“6) cycles of cisplatin were administered. The median dose intensity was 163.9 (range 110.1“175.5)?mg?m?2 per week for pemetrexed and 24.6 (range 15.3“26.3)?mg?m?2 per week for cisplatin. The delivered dose intensities were 98.3% for pemetrexed and 98.5% for cisplatin. Seventeen (40%) completed six or more cycles of ziv-aflibercept. The median dose intensity of ziv-aflibercept was 1.97?mg?kg?1 per week close to the planned intensity of 2?mg?kg?1 per week. Reasons for treatment discontinuation were disease progression (33%) AEs (33%) and others (34% including withdrawal of consent and investigator request). Seventeen patients had at least one cycle delayed. Eleven patients (26%) had at least 1 dose modification of ziv-aflibercept 6 (14%) of pemetrexed and 11 of cisplatin. Adverse events Thirty-five patients (83%) experienced a treatment-emergent adverse event (TEAE) of grade 3 or 4 (3/4) and 16 patients (38%) experienced a serious TEAE. The most common TEAEs were nausea (69%) fatigue (67%) and hypertension (57%). Hypertension neutropaenia and hypokalaemia were the most common grade 3/4 TEAEs in 36%0.14 and 10% of patients respectively. Table 2 summarised the most common TEAEs. Thirty-nine patients (93%) experienced at least one haematologic abnormality with grade 3/4 in eight patients (19%) mostly neutropaenia. Every patient experienced at least one abnormal chemistry value with grade 3/4 in 15 (36%) most commonly hyponatraemia. Seven patients (17%) died before clinical cutoff of the study. Five died due to disease progression and two due to TEAEs: 1 pneumonia and 1 sepsis. Neurological toxicities Between 26 September 2010 and 30 December2010 five patients experienced neurological symptoms including altered mental status in four slurred speech in one seizure in two and headache in four patients. Three patients had a brain MRI that was consistent with RPLS (Figure 1B). Brain MRI was negative for RPLS in two other patients. The three patients diagnosed with RPLS were all Caucasian women aged 3851 and 72 years respectively. One of the three patients with RPLS entered the study with a history of hypertension. All three patients experienced elevated BP and two patients had reduced CrCL during the therapy: one patient's CrCL decreased by 45% from baseline (141 to 78?ml?min?1) after one cycle and the other's decreased by 20% (64 to 51?ml?min?1) after four cycles. They were diagnosed with RPLS after one two and five cycles of ziv-aflibercept respectively. Two patients recovered from RPLS and one died due to disease progression before RPLS resolution. Pharmacokinetic information was available for two of the three RPLS patients and for one suspected case: systemic concentrations of free ziv-aflibercept were within the range of other patients in the treatment cohort. In the phase I study using the same regimen (N=18) five patients experienced a mild neurocognitive disturbance but no RPLS was diagnosed (Diaz-Padilla et al 2012). Rare cases of RPLS have been observed in the ziv-aflibercept clinical development programme but the rate observed in this study (3 out of 42=7%) was much higher than previously reported (i.e. 0.5% of 3795 patients treated with ziv-aflibercept as monotherapy or in combination with chemotherapy ZALTRAP product insert). As a result this study was permanently closed to enrolment. Patients who remained on study were re-consented with updated safety information regarding RPLS in addition to continued close monitoring. Hypertension and renal insufficiency are two risk factors for RPLS. Twenty-four patients (57%) experienced hypertension (15 grade 3 but no grade 4) during the study 15 of whom had a history of hypertension and 12 had taken antihypertensive medications before entering the study. Eight patients with hypertension also experienced proteinuria. Fourteen patients (33%) experienced proteinuria (all grades 1 or 2 except a single grade 3) none of whom had a history of renal disease. Fourteen patients experienced CrCL decreases during treatment with six patients having CrCL decreases below 60?ml?min?1 after treatment cycle 4. Efficacy evaluation As the study was closed prematurely there was no statistical power to test the primary hypothesis. Of the 42 patients enrolled 4 patients discontinued early from the study due to AEs (2) consent withdrawal (1) and investigator decision (1). As they did not have a post-baseline tumour assessment they were excluded from the efficacy assessment per predefined statistical analysis plan. Of the 38 patients evaluable for efficacy the median PFS was 5 months (95% CI 4.3“7.1; Figure 1A) and ORR was 26% (95% CI 12“40%) all of which (10/38) were PR. The disease control rate (PR+stable disease) was 89% (26%+63%). "

Lung_Cancer

"Although de Torres et al. demonstrated by using a well-characterized cohort of patients with COPD that the incidence of dense lung cancers decreased as the severity of the airflow obstruction at baseline increased [23] the severity of COPD in Japanese patients with newly diagnosed lung cancer was classified mainly as GOLD grade 1 and 2 rather than as GOLD grade 3. Furthermore our data showed that most patients were newly classified with COPD (84.4%; 124/147 cases) compatible with the incidence of the severity of COPD shown above or previously [1323]. It should be noted that in comparing patients undergoing thoracic surgery COPD patients had an average postoperative stay that was 61% higher and a 100% greater need of prolonged oxygen therapy than patients without COPD indicating the clinical impact of the coexistence of COPD [14]. The prevalence of COPD might increase in Japanese patients with lung cancer whereas the impact of COPD-related systemic comorbidities is also increasingly recognized in clinical aspects of COPD [7]. Thus whether or not the decision-making process involved in proposing the therapeutic management of lung cancer might be independently affected by COPD in patients with lung cancer remains elusive. To address this issue we evaluated whether or not completion of clinical staging and proposal of thoracic surgery with curative intent might be affected by the coexistence of COPD. The percentage of patients in whom clinical staging had been not completed was significantly higher in the COPD group than in the non-COPD group. More than half of these patients were referred to other hospitals for further support while the others were patients with disease recurrence. The proportion of patients with each classification in the clinical staging was compatible with that reported in previous studies about thoracic surgery [24]. Clinical guidelines recommend the assessment of spirometry to evaluate the optimum selection of surgical procedure in view of the risks of mortality and postoperative complications [6825]. Therefore we analyzed data from 185 patients with lung cancer at stage 1A to 3A because these patients are generally eligible for thoracic surgery with curative intent [1726]. Even among these surgical candidates however the number of surgeries performed was significantly lower in the COPD group (64.1%; 59/92 cases) than in the non-COPD group (81.7%; 76/93 cases) (). Furthermore our data showing that COPD-related systemic comorbidities might not be independent factors for proposing thoracic surgery with curative intent was supported by previous data as described above [14]. Thus these data indicate that the decision-making process for the therapeutic management of Japanese lung cancer patients might be affected by the prevalence and severity of COPD. Finally we evaluated whether or not the severity of COPD classified by GOLD grade might be an independent factor affecting the proposal of thoracic surgery with curative intent. Multivariate analysis indicated that severity of COPD was a critical and independent factor for proposing thoracic surgery with curative intent to Japanese patients with lung cancer who underwent bronchoscopy. This finding might be supported by our previous study showing that in comparing patients undergoing thoracic surgery COPD patients with an FEV1/FVC below 0.70 had an average postoperative stay that was 61% higher and a 100% greater need of prolonged oxygen therapy (POT) than patients without COPD [14]. Some limitations of the present study deserve mention. First the reversibility test was performed in only 62.2% of patients (168/270 cases) although COPD was defined as a postpronchodilator FEV1/FVC below 0.7 [16]. This limitation is present in other studies that have evaluated the prevalence of COPD [91027]. The other explanation might be the preoperative pulmonary assessment based on the clinical guidelines in which the need to perform a reversibility test for assessment of airflow obstruction is not mentioned [825]. Although a recent study suggests that some COPD patients show relatively high reversibility for a short-acting beta2-agonist [28] only 1.2% of 168 cases showed significant reversibility in the present study indicating that Japanese patients with both lung cancer and COPD might have different characteristics from that population [27]. Second the present study retrospectively analyzed 270 out of a total of 320 cases with lung cancer in a single institution and therefore might be subject to selection bias. However analyzing the data from 84.4% of all patients in a single institution who were sequentially registered and underwent bronchoscopy from 2010 to 2012 might minimize the possible contribution of the selection bias for patients with lung cancer. Although many studies suggest that COPD remains underdiagnosed in the patients with lung cancer [1314] Zang et al. suggest that awareness of COPD might contribute the conformity to GOLD treatment guideline for stable condition and acute exacerbation of COPD in lung cancer patients during hospitalization [13]. When spirometry was performed at bronchoscopy the median time from the date of spirometry to thoracic surgery was 50 days in the present study. Therefore comprehensive assessment of COPD at bronchoscopy might allow us to implement the optimum management for lung cancer patients [2930]. Conclusions In the present study the high prevalence of COPD among Japanese patients with newly diagnosed lung cancer was shown. Although further investigation into the validity of the assessment of COPD at bronchoscopy from studies of patients with lung cancer from other institutions is warranted we conclude that appropriate risk stratification and comprehensive management of patients with lung cancer and COPD might be made by assessment of the coexistence and severity of COPD at the time of bronchoscopy. Competing interests The authors have declared that no conflict of interest exists. Authors™ contributions NH AM and YH had full access to all of the data in the study and are responsible for the integrity of the data and the accuracy of the data analysis"

Lung_Cancer

"The protocol was approved by the Institutional Review Boards at each participating institution. Informed consent was obtained from each patient. Study design This is a single arm open label multicentre phase II study (ClinicalTrials.gov identifier: NCT00794417). Patients received the three-drug combination intravenously on day 1 of every 21 days with ziv-aflibercept (6?mg?kg?1) first followed by pemetrexed (500?mg?m?2) and cisplatin (75?mg?m?2). Premedications consisted of folic acid vitamin B12 and dexamethasone as a prophylactic measure to reduce pemetrexed-related toxicities and standard anti-emetics. Patients could receive up to six cycles of combination therapy. For patients who completed the combined chemotherapy maintenance ziv-aflibercept every 21 days was to continue until disease progression intolerable toxicity or withdrawal from the study. End points and assessments The two co-primary end points were objective response rate (ORR) and PFS. The ORR was defined as the proportion of patients with complete response plus partial response (CR+PR). The PFS was defined as the time interval from the first dose of combination chemotherapy to tumour progression or death whichever occurred first. Secondary variables were the determination of the adverse events (AEs) pharmacokinetics (PK) and pharmacodynamic profiles (including anti-ziv-aflibercept antibody and hematopoiesis). Pharmacokinetic end points included the area under the concentration curve maximum concentration (Cmax) clearance and terminal half-life (t1/2). Tumour imaging (CT or MRI) was performed at screening on day 21 (±7 days) of every even numbered cycle (every 6 weeks) and when disease progression was suspected. Responses were assessed using RECIST version 1.0 (Therasse et al 2000). Safety and tolerability were assessed at baseline and at least every 21 days as evaluated by AEs and changes in laboratory parameters graded according to the Common Terminology Criteria for Adverse Events (CTCAE) version 3.0 (National Cancer Institute 2006). Ziv-aflibercept (free or bound to VEGF) in plasma samples was quantified using a validated direct enzyme-linked immunosorbent assay. A validated non-quantitative titre-based bridging assay was used to detect anti-ziv-aflibercept antibodies in serum samples. Correlative studies The exploratory objective of the correlative studies was to evaluate changes in erythropoiesis in response to VEGF inhibition. It was hypothesised that VEGF inhibition would result in an increase in haemoglobin via increased hepatic erythropoietin production. Statistical analysis Statistical testing was done to determine whether the ORR was larger than 20% or whether the PFS was greater than 4.5 months. Exact test (one-sided) was used to test the null hypothesis that ORR was ?20% versus the alternative hypothesis that ORR was ?35%. Assuming type I error was not >2.5% a sample of 72 patients would provide >80% power to test the hypothesis using exact binomial test. The calculated sample size of 72 patients would also provide >90% power to test the null hypothesis that median PFS was ?4.5 months versus the alternative hypothesis that PFS was ?6.5 months at one-sided alpha of 2.5% using one sample log-rank test. Safety data were to be summarised. Concentrations of free ziv-aflibercept and adjusted bound ziv-aflibercept: VEGF complex were to be summarised every 21 days over the duration of the study by nominal time point. Noncompartmental parameters were calculated using WinNonlin (version 5.3 Pharsight Corporation Mountain View CA USA) and model 202 (constant infusion) using nominal time points after a single dose of ziv-aflibercept. The noncompartmental analysis was performed over the dosing interval 21 days following the first dose. All analyses used statistical software SAS (version 9.1.3 Cary NC USA). Results Patients This study was closed prematurely because of three confirmed and two suspected but unconfirmed cases of reversible posterior leukoencephalopathy syndrome (RPLS). A total of 42 patients were enrolled from 17 participating sites across the United States and Canada between January 2009 and December 2010. summarises the patient demographics. Median age was 61.5 years; 55% were male 86% were Caucasian and 50% had ECOG PS of 0. Safety evaluation Treatment exposure and dose modifications All 42 patients received at least one dose of each of the three study drugs with a median of 92 days of treatment (range 21“288 days). Twenty-seven (64%) patients completed four or more cycles of the combination treatment. A median of 4.5 (range 1“6) cycles of pemetrexed and 4 (range 1“6) cycles of cisplatin were administered. The median dose intensity was 163.9 (range 110.1“175.5)?mg?m?2 per week for pemetrexed and 24.6 (range 15.3“26.3)?mg?m?2 per week for cisplatin. The delivered dose intensities were 98.3% for pemetrexed and 98.5% for cisplatin. Seventeen (40%) completed six or more cycles of ziv-aflibercept. The median dose intensity of ziv-aflibercept was 1.97?mg?kg?1 per week close to the planned intensity of 2?mg?kg?1 per week. Reasons for treatment discontinuation were disease progression (33%) AEs (33%) and others (34% including withdrawal of consent and investigator request). Seventeen patients had at least one cycle delayed. Eleven patients (26%) had at least 1 dose modification of ziv-aflibercept 6 (14%) of pemetrexed and 11 of cisplatin. Adverse events Thirty-five patients (83%) experienced a treatment-emergent adverse event (TEAE) of grade 3 or 4 (3/4) and 16 patients (38%) experienced a serious TEAE. The most common TEAEs were nausea (69%) fatigue (67%) and hypertension (57%). Hypertension neutropaenia and hypokalaemia were the most common grade 3/4 TEAEs in 36%0.14 and 10% of patients respectively. summarised the most common TEAEs. Thirty-nine patients (93%) experienced at least one haematologic abnormality with grade 3/4 in eight patients (19%) mostly neutropaenia. Every patient experienced at least one abnormal chemistry value with grade 3/4 in 15 (36%) most commonly hyponatraemia. Seven patients (17%) died before clinical cutoff of the study. Five died due to disease progression and two due to TEAEs: 1 pneumonia and 1 sepsis. Neurological toxicities Between 26 September 2010 and 30 December2010 five patients experienced neurological symptoms including altered mental status in four slurred speech in one seizure in two and headache in four patients. Three patients had a brain MRI that was consistent with RPLS (B). Brain MRI was negative for RPLS in two other patients. The three patients diagnosed with RPLS were all Caucasian women aged 3851 and 72 years respectively. One of the three patients with RPLS entered the study with a history of hypertension. All three patients experienced elevated BP and two patients had reduced CrCL during the therapy: one patient's CrCL decreased by 45% from baseline (141 to 78?ml?min?1) after one cycle and the other's decreased by 20% (64 to 51?ml?min?1) after four cycles. They were diagnosed with RPLS after one two and five cycles of ziv-aflibercept respectively. Two patients recovered from RPLS and one died due to disease progression before RPLS resolution. Pharmacokinetic information was available for two of the three RPLS patients and for one suspected case: systemic concentrations of free ziv-aflibercept were within the range of other patients in the treatment cohort. In the phase I study using the same regimen (N=18) five patients experienced a mild neurocognitive disturbance but no RPLS was diagnosed (Diaz-Padilla et al 2012). Rare cases of RPLS have been observed in the ziv-aflibercept clinical development programme but the rate observed in this study (3 out of 42=7%) was much higher than previously reported (i.e. 0.5% of 3795 patients treated with ziv-aflibercept as monotherapy or in combination with chemotherapy ZALTRAP product insert). As a result this study was permanently closed to enrolment. Patients who remained on study were re-consented with updated safety information regarding RPLS in addition to continued close monitoring."

Lung_Cancer

"The rates of assignment of patients to observation (22%) and chemotherapy (78%) were as expected. S Gene expression analysis for treatment assignment is feasible. Survival results are encouraging and require future validation. Real-time performance of quantitative in situ ERCC1 and RRM1 analysis requires further development. lung cancer adjuvant therapy personalized medicine ERCC1 (excision repair cross-complementing group 1) RRM1 (ribonucleotide reductase M1) INTRODUCTION After publication of the International Adjuvant Lung Cancer Trial in 2004 adjuvant chemotherapy containing a platinum agent has become the standard of care for patients with a complete surgical resection of American Joint Committee on Cancer stage II to III (version 6) non-small cell lung cancer (NSCLC).1 The trial included patients with stage I to III disease and demonstrated an absolute 4.1% improvement in overall survival (OS) and a subgroup analysis indicated that the OS benefit increased with stage: the hazards ratio (HR) for death among patients receiving adjuvant chemotherapy compared with controls was approximately 0.98 for patients with stage I disease 0.88 for patients with stage II disease and 0.79 for patients with stage III disease.1 The data were confirmed by the National Cancer Institute of Canada Clinical Trials Group JBR.10 trial in 2005 which included patients with stage IB and stage II disease.2 A third trial Cancer and Leukemia Group B (CALGB) 9633 which included only patients with stage IB disease was terminated early and also reported a therapeutic benefit for adjuvant chemotherapy.3 However a final analysis of mature data revealed no statistically significant OS benefit (HR 0.83) but demonstrated a benefit for patients with tumor diameters of ??4 cm (HR 0.69).4 During the same time period an increasing number of correlative biomarker analyses demonstrated that the efficacy of platinum agents was associated with intratumoral levels of the excision repair cross-complementing group 1 (ERCC1) gene with high levels indicating resistance.5“9 Similarly high intratumoral levels of the regulatory subunit of ribonucleotide reductase M1 (RRM1) were reported to be predictive of resistance to gemcitabine.9“13 Finally both biomarkers had also been reported to be prognostic of survival in patients who had not received chemotherapy or radiation with high levels indicating longer survival.814“16 Based on these data we designed an adjuvant trial in 2007. The underlying hypothesis was that patients with high intratumoral levels of ERCC1 and RRM1 would not benefit from chemotherapy and would have a good prognosis because of a less aggressive tumor phenotype. In contrast patients with low levels of ERCC1 and RRM1 would have tumors that were sensitive to chemotherapy but with a more aggressive phenotype. Because a biomarker-driven adjuvant chemotherapy selection trial had not been performed in patients with NSCLC we focused on demonstrating the feasibility of such an approach before launching a phase 3 trial. In addition because adjuvant chemotherapy had quickly become the standard of care for patients with stage II/IIIA disease we focused our efforts on patients with stage I disease. After discussions within the SWOG (formerly the Southwest Oncology Group) lung cancer working group and the National Cancer Institute (NCI)'s Cancer Therapy Evaluation Program and after peer review by a National Institutes of Health study section the consensus was to focus this feasibility trial on patients with stage I disease and tumor diameters of ?2 cm. MATERIALS AND METHODS Trial Design and Treatment Plan The trial (NCT00792701 SWOG-0720) complied with the Declaration of Helsinki and was approved by the Institutional Review Boards of the study institutions. Eligibility criteria included a diagnosis of NSCLC; stage I disease (according to version 6 of the American Joint Committee on Cancer staging manual) with a tumor diameter ??2?cm; a complete surgical resection by lobectomy bilobectomy or pneumonectomy; surgical staging of the mediastinum through sampling of at least 2 lymph node stations; a positron emission tomography scan; a computed tomographic scan of the chest and abdomen; adequate bone marrow liver and renal function; a Zubrod performance status of 0 or 1; and willingness to provide a smoking history. Patients with a prior malignancy prior radiation to the chest or other significant illnesses according to good medical practice were excluded. Patients had to be registered on the trial within 35 days of surgery. Tumor specimens were then retrieved and shipped to a central laboratory. They were analyzed for in situ tumor levels of ERCC1 and RRM1 using an immunofluorescence-based automated quantitative analysis method.17 Prespecified cutoff levels that had been determined in 187 patients with stage I disease (??65 for ERCC1 and ??40 for RRM1) were used to categorize specimens as high or low expressors for each marker (Fig. 1).16 The appropriate therapeutic assignment was then passed on to the statistical center and the participating therapeutic center; however specific protein levels were not communicated to the treatment center. Therapeutic assignment was based solely on biomarker categories and no other stratification parameters were used. CONSORT (Consolidated Standards Of Reporting Trials) diagram of the trial is shown. Patients with high levels of both biomarkers received active surveillance and patients with low levels of one or both biomarkers received 4 cycles of cisplatin (at a dose of 80 mg/m2 on day 1) and gemcitabine (at a dose of 1 g/m2 on days 1 and 8) every 21 days. The protocol included provisions for dose reductions or treatment delays. The addition of other targeted or cytotoxic agents during therapy or as maintenance was not permitted. Specimen Collection Processing and Gene Expression Analysis The study required the collection and shipment of formalin-fixed and paraffin-embedded tumor blocks before therapy. However if local policies did not permit submission of a tissue block 10 serial unstained sections could be submitted. Processing was done in a reference laboratory by 1 of 2 investigators (V.O. and Z.Z.). Sections measuring 5 ?m in thickness were placed on frosted glass slides and in situ quantification was performed by the automated quantitative analysis method (PM-2000 [version 1] HistoRx Inc New Haven CT) as previously described.91618 The primary antibody for the detection of ERCC1 was clone 8F1 (product code NB500-704 lots G412 and H347 from Novus Biologicals [Littleton Colo]) and the antiserum for RRM1 was R1AS-6 (generated in a rabbit in 2003 against a keyhole limpet hemocyanin [KLH]-conjugated 21-aminoacid peptide specific to the N-terminal of RRM1 column purification lot 09-2008). Slides were scanned with SpotGrabber (HistoRx New Haven Conn.) and image data were captured with a digital camera and fluorescence microscope and analyzed. Scores were adjusted to range from 1 to 255. Because full sections were evaluated for each specimen multiple spots with diameters of 0.6 mm were analyzed to obtain a representative level of protein expression. The number of spots was dependent on suitable areas with tumor cells and it ranged from 5 to 25 spots (median 10 spots) for both targets. Runs included a tissue microarray of 15 control specimens in triplicate for control purposes. Statistical Analysis The primary objective of the current study was the feasibility of a biomarker-based treatment assignment in the cooperative group setting. If the true success rate were ??75% then a biomarker-based treatment assignment would not be considered feasible but if the true success rate were ??90% it would be feasible. If ??47 of 55 eligible patients (85%) were successfully assigned to treatment or active monitoring within 84 days from surgery this would be considered evidence of feasibility. The design had 91% power using an exact binomial test with a 1-sided type I error of 5%. Secondary objectives included estimating the collective 2-year disease-free survival (DFS) for patients who accepted their treatment assignment and in the subset of patients who received adjuvant chemotherapy. However there would be no comparison made between treatment arms. "

Lung_Cancer

" In short EGFR activating mutations in exons 19 and 21 were initially identified by Sanger sequencing and confirmed by fragment length analysis for exon 19 deletions (FAM-labelled primer in an ABI prism 3130 DNA analyser (Applied Biosystems Foster City CA USA) and by Taqman assay for exon 21 (L858R) mutation. All tumor specimens were from the original biopsy taken prior to any treatment and before randomization. Testing was performed on ? 2mm2 of tissue obtained from one to three slides of 4-micron tissue sections which were subjected to laser capture microdissection to enrich for the presence of tumor cells. DNA was extracted using a standard laboratory protocol and tested at a single site in Spain in Laboratory of Oncology for EGFR activating mutations in exon 19 and 21 using a previously described method. The average turnaround time was approximately 5 days.[26] Bi-directional Sanger sequencing All samples tested by the EGFR PCR test were also tested by Sanger sequencing using DNA from FFPET specimens prepared by the cobas DNA Sample Preparation Kit and sequenced with 2— bidirectional Sanger sequencing by a CLIA-certified laboratory (SeqWright Houston TX USA) using a validated protocol. Repeat Sanger sequencing was performed to compare the detection of EGFR mutations from adjacent sections of tissue to minimize any impact of tissue heterogeneity used for the EGFR PCR test relative to the original LDT results. Also sequencing protocols vary by laboratory in terms of the percent tumor content/sample that requires macrodissection. DNA isolated with the cobas DNA Sample Preparation Kit and used for sequencing required ?10% tumor content. Average turnaround time to results was 7 days. The estimated limit of detection is approximately 20% mutant alleles.[30] Massively parallel pyrosequencing (MPP) Samples with valid EGFR PCR test results with adequate DNA remaining from the initial extraction were tested by a MPP method (454 GS Titanium 454 Life Sciences Branford CT USA) by a CLIA-certified laboratory (SeqWright Houston TX USA) using a validated protocol.[31] This method is a 5“7 day process that involves amplicon generation pooling ligation emulsion PCR amplification and massively parallel pyrosequencing with manual data analysis. The estimated limit of detection for the assay is 1.25% mutant alleles. [27] The MPP method was used to demonstrate performance of the EGFR PCR test to a more sensitive method and as an arbiter for discrepant cases observed between the LDT or the repeat Sanger sequencing. In order to preserve patient privacy associated with tested clinical samples raw MPP sequencing results were anonymized and presented in Table S1. Results Specimen demographics 487 (47%) of 1044 specimens screened for the EURTAC trial using LDTs were available for testing using the EGFR PCR test. The flow of samples through the study is shown in . Patient demographics and baseline tumor characteristics for all patients by LDT status are shown in . There were no significant differences between subsets of patients tested and patients not tested by the EGFR PCR test (p>0.05) for each LDT status (mutation detected mutation not detected) with the exception of country of the screening clinic. Clinical outcomes for patients based on the EGFR PCR test results Of the 174 patients enrolled in EURTAC trial specimens from 134 (77%) patients were available for testing using the EGFR PCR test. Excluding 11 patients with invalid EGFR PCR test results and 7 patients with a result of EGFR mutation not detected a total of 116 (67%) patients were mutation detected by the EGFR PCR test and evaluable for clinical outcome analysis (57 patients in the chemotherapy arm and 59 in the erlotinib arm). Clinical outcomes (PFS BORR and OS) are presented in . Among EGFR PCR test positive patients those treated with erlotinib had a significantly prolonged PFS when compared to patients treated with chemotherapy (p-value <0.0001 log-rank test); the median PFS was 10.4 months (95% CI: 8.0 to 13.8 months) and 5.4 months (95% CI: 4.4 to 6.8 months) for patients treated with erlotinib or chemotherapy respectively (). The HR based on the Cox proportional hazards model was reduced by 66% (HR 0.34; [95% CI: 0.21 to 0.54]) for patients in the erlotinib versus chemotherapy arm. One year after randomization a higher percentage of patients in the erlotinib compared with the chemotherapy arm were event-free (45% [95% CI: 32% to 59% versus 6% [95% CI: 0% to 15%] respectively). .0089518.g002 Kaplan-Meier curves of progression-free survival (PFS) for different treatments in treatment-na¯ve patients with non“small-cell lung cancer and EGFR mutation detected by the EGFR PCR test and LDT. .0089518.t002 Summary of Clinical Outcome Analysis among EGFR PCR test positive patients in the EURTAC trial. Chemotherapy (N?=?57) Erlotinib (N?=?59) PFS (Investigator) Patients with event 37 (64.9%) 47 (79.7%) Patients without eventa 20 (35.1%) 12 (20.3%) ?Time to event (months) ?Medianb (95%CI) 5.4 [4.4; 6.8] 10.4 [8.0; 13.8] ?p-Value (Log-Rank Test) <0.0001 ?Hazard Ratio (95% CI) 0.34 [0.21; 0.54] ?1 year estimate ?Patients remaining at risk 2 24 ?Event-free Rateb (95%CI) 6% [0%; 15%] 45% [32%; 59%] Best Overall Analysis Response rates (95% CI) 14.0% [ 6.3%; 25.8%] 59.3%[ 45.7%; 71.9%] Difference in Response Rates (%) 45.29% [ 28.8%; 61.7%] ?p-Value (Chi-squared Test) <.0001 Odds Ratio (95% CI) 8.93 [3.59; 22.19] OS Patients with event 35 (61.4%) 36 (61.0%) Patients without eventa 22 (38.6%) 23 (39.0%) ?Time to event (months) ?Medianb (95%CI) 20.8 [17.3; 29.4] 25.8 [16.1; 30.0] ?p-Value (Log-Rank Test) 0.5381 ?Hazard Ratio (95% CI) 0.86 [0.54; 1.38] ?2 - year estimate ?Patients remaining at risk 16 23 ?Event-free Rateb (95% CI) 43% [29%; 57%] 51% [38%; 64%] Note: All eligible patients enrolled in study ML20650 were determined as EGFR mutation detected by the LDT. Among those patients with EGFR mutation confirmed by the EGFR PCR test were included in this table. Event ?=? Death or progression free whichever comes first for PFS analysis and event?=?death for OS analysis. a censored. b Kaplan-Meier estimates. C including censored observations. BORR were higher in patients in the erlotinib arm (59.3% [95% CI: 45.7% to 71.9%]) compared to the chemotherapy arm (14.0% [95% CI: 6.3% to 25.8%]). Patients in the erlotinib arm were much more likely to respond to therapy than patients in the chemotherapy arm (odds ratio of 8.93 [95% CI: 3.59 to 22.19]). There was no significant difference in OS between the treatment arms (25.8 months in the erlotinib arm (95% CI: 16.1 to 30.0) and 20.8 months in the chemotherapy arm (95% CI: 17.3 to 29.4) (log-rank test p-value ?=?0.5381)). PFS BORR and OS results for EGFR PCR test positive patients did not differ significantly from those obtained in all patients enrolled in the EURTAC trial which suggests that the EGFR PCR test positive patients are representative of all EURTAC enrolled patients. For the 7 cases where the EGFR PCR test result was mutation not detected and discrepant with the LDT two cases resolved in favor of the LDT by MPP three cases resolved in favor of the EGFR PCR test and one sample was invalid for both Sanger and MPP and the other was in agreement between the EGFR PCR test and Sanger but not MPP (Table S2). Anecdotally 6 of the 7 patients were treated with erlotinib and only one patient achieved greater than or equal to median PFS based on the LDT or the EGFR PCR test. Comparison of EGFR PCR test and LDT results Among 432 specimens with valid results from both the EGFR PCR test and LDT the PPA NPA and OPA were 94.2% (146/155 CI: 89.3% 96.9%) 97.5% (270/277 CI: 94.9% 98.8%) and 96.3% (416/432 CI: 94.1% 97.7%) respectively (). Thus there was a high concordance between the original LDT and EGFR PCR test results. Among sixteen specimens with discordant results the EGFR PCR test result was confirmed by MPP in 68.8% (11/16) cases (Table S3). .0089518.t003 Agreement analysis between EGFR PCR test and LDT. SLCG LDT Total N?=?432 Mutation detected Mutation not detected EGFR PCR test Mutation detected 146 7 153 Mutation not detected 9 270 279 Total 155 277 432* ¢12 samples with inconclusive LDT results and 43 samples with invalid EGFR PCR test results were excluded. Positive percent agreement ?=?94.2% (95% CI [89.3“96.9%]). Negative percent agreement ?=?97.5% (95% CI [94.9“98.8%]). Overall percent agreement ?=?96.3% (95% CI [94.1“97.7%]). Comparison of the EGFR PCR test results with Sanger Sequencing Of 487 specimens tested using the EGFR PCR test and Sanger sequencing 406 gave valid results by both methods (38 were invalid by both methods five were invalid by EGFR PCR test and 38 were invalid by Sanger sequencing). The PPA NPA and OPA for EGFR PCR test compared with Sanger sequencing were 96.6% (112/116 CI: 91.7% 98.7%) 88.3% (256/290 CI: 84.1% 91.5%) and 90.6% (368/406 CI: 87.4% 93.1%; Table 4) respectively. Among 38 discordant results between the EGFR PCR test and Sanger sequencing MPP agreed with the EGFR PCR test result in 30 (78.9%) cases (Table S4). Sanger sequencing detected one L858R not detected by MPP and failed to detect 22 exon 19 deletions and 7 L858R mutations confirmed by MPP. Four MPP results were invalid and the remaining four results agreed with Sanger. The range of percent mutant alleles of the cases missed by Sanger was 3% to 60% with several specimens (n?=?16) under the estimated limit of detection for Sanger. .0089518.t004 Table 4 Agreement analysis between EGFR PCR test and Sanger sequencing. Sanger sequencing Total N?=?406 Mutation detected Mutation not detected EGFR PCR test Mutation detected 112 34 146 Mutation not detected 4 256 260 Total 116 290 406 *81 samples with invalid EGFR PCR test or Sanger sequencing results were excluded. Positive percent agreement ?=?96.6% (95% CI [91.5“98.7%]). Negative percent agreement ?=?88.3% (95% CI [84.1“91.5%]). Overall percent agreement ?=?90.6% (95% CI [87.4“93.1%]). Discussion This study supports the feasibility of performing a retrospective clinical validation of a companion diagnostic from prospective therapeutic clinical trials. The EGFR PCR test results were highly concordant (>96%) with the LDT results used to select patients for the EURTAC trial. As a consequence PFS and BORR of the subset of patients with EGFR mutations detected with the EGFR PCR test were comparable to the full cohort of patients enrolled in the EURTAC trial thus validating the use of the EGFR PCR test to select patients for treatment with anti-EGFR TKIs such as erlotinib. Median PFS survival was 9.7 versus 10.4 months for the erlotinib group and 5.2 versus 5.4 months for the LDTs and EGFR PCR test respectively. The BORR was 58% versus 59.3% months for the erlotinib group and 15% versus 14.0% for the LDTs and EGFR PCR test respectively. Among the 16 discordant specimens between the EGFR PCR test and LDTs a third mutation testing method agreed with the EGFR PCR test result in 11 cases. Of seven cases that were mutation detected by the EGFR PCR test and mutation not detected by the LDT 5 were confirmed by MPP. These patients could have potentially benefited from anti-EGFR TKI therapy. The EGFR PCR test had a number of technical advantages over the LDT used in the EURTAC trial. The LDT required laser capture microdissection of multiple tissue sections and involved 3 separate assays with a median turnaround time of 4.5 days. By comparison the EGFR PCR test required macrodissection only if the tumor content was <10% and can be performed in one day using a single 5 µm section. Furthermore the EGFR PCR test is a commercially available kit-based assay that provides an automated result rather than a manual process subject to interpretation and which can be performed by any qualified clinical laboratory. More than 80% of the specimens tested in this study were small biopsy specimens. The overall invalid rate for Sanger sequencing was 15.6% (76/487) compared to the EGFR PCR assay at 9% (43/487). However the invalid rate for the subset of specimens derived from resected specimens was 0% (0/109) likely because of sufficient tissue availability. Thus the assay is extremely robust when performed on resected tumor specimens and has an approximately 90% success rate on biopsy specimens which are often the only tumor sample available for testing in NSCLC. Sanger sequencing has been widely used to detect EGFR mutations.[30] [32] Similar to the overall invalid rates for the 134 EGFR mutation detected LDT samples enrolled in the EURTAC trial Sanger sequencing had a higher invalid rate (15.7%) compared to 8.2% for the EGFR PCR test. There were also 30 mutation not detected results for Sanger sequencing (22.4%) and 7 mutation not detected results for the EGFR PCR test (5.2%). With 21 invalid results and 30 mutation not detected results Sanger sequencing would have misclassified 38% of patients enrolled in the EURTAC trial. Similar invalid rates have been reported in three other studies suggesting that this methodology has limitations when applied to DNA from FFPET samples.[33] [34] [35] In addition Sanger sequencing has shown poor sensitivity in samples containing less than 20“25% mutant alleles.[35] [36] [37] When we compared the agreement between valid results for the EGFR PCR test with Sanger sequencing (n?=?406) there were 38 discordant cases of which 30 were confirmed by MPP. Twenty-nine of the 30 cases resulted in mutation detected status by the EGFR PCR test and would make these patients eligible for anti-EGFR therapy. Poor sensitivity of Sanger sequencing thus explains the relatively low NPA compared to EGFR PCR test observed in this study. Given the criticality of EGFR mutation testing in selecting specific therapies for life-threatening cancers such as advanced NSCLC robust and accurate assays with rapid turnaround time are preferred. Recent quality assurance studies to ascertain the mutation status of a standard panel of tumors have shown that different clinical laboratories do not correctly identify the mutation status of 100% of the panel members even when they are using the same or similar testing methodologies.[38] [39] For assays that involve mutation analysis of tumor samples important factors contributing to the assay performance include analytic standardization validation of reagents and methodology laboratory experience and the appropriate involvement of the pathologist. In conclusion results of the present study indicate that the cobas EGFR mutation test is a highly robust and highly accurate companion diagnostic assay to select patients for treatment with anti-EGFR therapies such as erlotinib. Supporting Information Table S1 Listing of MPP Result. (PDF) Click here for additional data file. Table S2 Outcome from samples discrepant between the cobas EGFR PCR test and LDT that were enrolled in the clinical trial (cobas MND/LDT MD). (PDF) Click here for additional data file. Table S3 Agreement results between discordant EGFR PCR and LDT tests. (PDF) Click here for additional data file. Table S4 MPP results from resolution analysis of discordant specimens between EGFR PCR test and Sanger sequencing. (PDF) Click here for additional data file. We would like to acknowledge Patrick O'Donnell and Karen Yu for their contributions to this study. References 1 ChapmanPB HauschildA RobertC HaanenJB AsciertoP et al (2011) Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med364: 2507“251621639808 2 OuSH BartlettCH Mino-KenudsonM CuiJ IafrateAJ (2012) Crizotinib for the treatment of ALK-rearranged non-small cell lung cancer: a success story to usher in the second decade of molecular targeted therapy in oncology. Oncologist17: 1351“1375 3 O'BryantCL WengerSD KimM ThompsonLA (2013) Crizotinib: a new treatment option for ALK-positive non-small cell lung cancer. Annals Pharmacotherapy47: 189“197 4 SunJM ChoiYL WonJK HirschFR AhnJS et al (2012) A dramatic response to crizotinib in a non-small-cell lung cancer patient with IHC-positive and FISH-negative ALK. J Thorac Oncol7: e36“3823154564 5 Administration USFaD (2010) Class Labeling Changes to anti-EGFR monoclonal antibodies cetuximab (Erbitux) and panitumumab (Vectibix): KRAS Mutations. 6 HarbisonCT HorakCE LedeineJM MukhopadhyayP MaloneDP et al (2012) Validation of Companion Diagnostic for Detection of Mutations in Codons 12 and 13 of the KRAS Gene in Patients with Metastatic Colorectal Cancer: Analysis of the NCIC CTG CO.17 Trial. Arch Pathol Lab Med137: 820“82723030695 7 MaemondoM InoueA KobayashiK SugawaraS OizumiS et al (2010) Gefitinib or chemotherapy for non“small-cell lung cancer with mutated EGFR. N Engl J Med362: 2380“2388 8 MokTS WuYL ThongprasertS YangCH ChuDT et al (2009) Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med361: 947“95719692680 9 ZhouC WuYL ChenG FengJ LiuXQ et al (2011) Erlotinib versus chemotherapy as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer (OPTIMAL CTONG-0802): a multicentre open-label randomised phase 3 study. Lancet Oncol12: 735“74221783417 10 HirschFR KabbinavarF EisenT MartinsR SchnellFM et al (2011) A randomized phase II biomarker-selected study comparing erlotinib to erlotinib intercalated with chemotherapy in first-line therapy for advanced non-small-cell lung cancer. J Clin Oncol29: 3567“3573 11 RosellR CarcerenyE GervaisR VergnenegreA MassutiB et al (2012) Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre open-label randomised phase 3 trial. Lancet Oncol13: 239“24622285168 12 SequistLV MartinsRG SpigelD GrunbergSM SpiraA et al (2008) First-line gefitinib in patients with advanced non-small-cell lung cancer harboring somatic EGFR mutations. J Clin Oncol26: 2442“244918458038 13 MitsudomiT MoritaS YatabeY NegoroS OkamotoI et al (2010) Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): an open label randomised phase 3 trial. Lancet Oncol11: 121“12820022809 14 KosakaT YatabeY EndohH YoshidaK HidaT et al (2006) Analysis of epidermal growth factor receptor gene mutation in patients with non-small cell lung cancer and acquired resistance to gefitinib. Clin Cancer Res12: 5764“576917020982 15 LynchTJ BellDW SordellaR GurubhagavatulaS OkimotoRA et al (2004) Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med350: 2129“213915118073 16 PaezJG JannePA LeeJC TracyS GreulichH et al (2004) EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science304: 1497“1500 17 PaoW MillerV ZakowskiM DohertyJ PolitiK et al (2004) EGF receptor gene mutations are common in lung cancers from œnever smokers and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci U S A101: 13306“1331115329413 18 PaoW MillerVA PolitiKA RielyGJ SomwarR et al (2005) Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS2: 225“235 19 RielyGJ PolitiKA MillerVA PaoW (2006) Update on epidermal growth factor receptor mutations in non-small cell lung cancer. Clin Cancer Res12: 7232“7241 20 SharmaSV BellDW SettlemanJ HaberDA (2007) Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer7: 169“18117318210 21 GarridoP de CastroJ ConchaA FelipE IslaD et al (2012) Guidelines for biomarker testing in advanced non-small-cell lung cancer. A national consensus of the Spanish Society of Medical Oncology (SEOM) and the Spanish Society of Pathology (SEAP). Clin & Transl Oncol14: 338“349 22 KeedyVL TeminS SomerfieldMR BeasleyMB JohnsonDH et al (2011) American Society of Clinical Oncology provisional clinical opinion: epidermal growth factor receptor (EGFR) Mutation testing for patients with advanced non-small-cell lung cancer considering first-line EGFR tyrosine kinase inhibitor therapy. J Clin Oncol29: 2121“212721482992 23 EttingerDS AkerleyW BeplerG BlumMG ChangA et al (2010) Non-small cell lung cancer. J Natl Compr Canc Netw8: 740“801 24 LindemanNI CaglePT BeasleyMB ChitaleDA DacicS et al (2013) Molecular Testing Guideline for Selection of Lung Cancer Patients for EGFR and ALK Tyrosine Kinase Inhibitors: Guideline from the College of American Pathologists International Association for the Study of Lung Cancer and Association for Molecular Pathology. J Mol Diagn15: 415“45323562183 25 Administration USFaD (2013) FDA approves first companion diagnostic to detect gene mutation associated with a type of lung cancer. 26 RosellR MoranT QueraltC PortaR CardenalF et al (2009) Screening for epidermal growth factor receptor mutations in lung cancer. N Engl J Med361: 958“96919692684 27 O'Donnell PF Jane; Shyu Johnny; Current Robert; Rehage Taraneh; Tsai Julie; Christensen Mari; Tran Ha Bich; Chien Sean; Wei Wen; Lawrence H. Jeffrey; Soviero Steven; Wu Lin. A real-time PCR assay for detecting EGFR mutations in formalin-fixed paraffin-embedded tissue (FFPET) specimens of non-small cell lung cancer (NSCLC). 2012; Chicago IL. 28 O'Donnell PFJ ShyuJ CurrentR RehageT TsaiJ Christensen M. Bich TranH Shih-ChangC WeiW LawrenceHJ WuL SovieroS (2013) A Real-Time PCR Assay for Detecting EGFR Mutations in Formalin-Fixed Paraffin-Embedded Tissue Specimens of Non-Small Cell Lung Cancer. BMC Cancer13: 21023621958 29 (2011) cobas EGFR Mutation Test CE-IVD Package Insert Roche Molecular Systems Inc. USA. 30 CondeE AnguloB TangM MorenteM Torres-LanzasJ et al (2006) Molecular context of the EGFR mutations: evidence for the activation of mTOR/S6K signaling. Clin Cancer Res12: 710“71716467080 31 MarguliesM EgholmM AltmanWE AttiyaS BaderJS et al (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature437: 376“38016056220 32 AnguloB Garcia-GarciaE MartinezR Suarez-GauthierA CondeE et al (2010) A commercial real-time PCR kit provides greater sensitivity than direct sequencing to detect KRAS mutations: a morphology-based approach in colorectal carcinoma. J Mol Diagn12: 292“299 33 Gallegos RuizMI FloorK RijmenF GrunbergK RodriguezJA et al (2007) EGFR and K-ras mutation analysis in non-small cell lung cancer: comparison of paraffin embedded versus frozen specimens. Cell Oncol29: 257“26417452778 34 OginoS KawasakiT BrahmandamM YanL CantorM et al (2005) Sensitive sequencing method for KRAS mutation detection by Pyrosequencing. J Mol Diag7: 413“421 35 AndersonS BloomKJ ValleraDU RueschoffJ MeldrumC et al (2012) Multisite Analytic Performance Studies of a Real-Time Polymerase Chain Reaction Assay for the Detection of BRAF V600E Mutations in Formalin-Fixed Paraffin-Embedded Tissue Specimens of Malignant Melanoma. Arch Pathol Lab Med136: 1385“139122332713 36 TanYH LiuY EuKW AngPW LiWQ et al (2008) Detection of BRAF V600E mutation by pyrosequencing. Pathology40: 295“29818428050 37 KotoulaV CharalambousE BiesmansB MalousiA VrettouE et al (2009) Targeted KRAS mutation assessment on patient tumor histologic material in real time diagnostics. PLoS One4: e774619888477 38 Beau-FallerM DegeorgesA RollandE MounawarM AntoineM et al (2011) Cross-Validation "

Lung_Cancer

"Supplementary Material Supplementary Data Click here for additional data file. Determination of SETDB1 gene amplification and its association with RNA and protein overexpression in lung cancer cell lines. (a) Assessment of SETDB1 copy-number by quantitative genomic PCR. Amplification frequency of SETDB1 (evaluated with SYBR Green Bio-Rad Hercules CA USA) was calculated by the standard curve method using the 7900HT SDS program. To define an internal control gene we chose chromosome 1p36.23 because it is the least aneuploid region among our cell lines (PEX19 gene). Primers are available upon request. DNA from normal lung was used as the reference standard. Results are reported as n-fold copy-number increase relative to the PEX19 gene. (b) Fluorescence in situ hybridization for the SETDB1 gene. The UCSC genome browser (http://www.genome.ucsc.edu) was used to select the bacterial artificial chromosome (BAC) clone spanning the 1q21 region for the SETDB1 gene: RP11-42A12. A telomeric BAC clone located in the telomeric 1p36.23 region was used as a control. The BACs were obtained from the BACPAC Resource Center at the Children's Hospital Oakland Research Institute (Oakland CA USA). SETDB1 and telomeric probes were labeled with Spectrum Green and Red dUTP (Abbott Wiesbaden Germany) respectively using a CGH Nick Translation Reagent Kit (Abbott Molecular Inc. Des Plaines IL USA). The samples were counterstained with 4'6-diamidino-2-phenylindole in Vectashield antifade solution (Burlingame CA USA). Gene amplification was observed in the interphases of NCI-H1437 NCI-H1395 and DMS-273. Probes were verified to give a single signal on normal commercial lymphocyte metaphase slides (CGH Reagents Abbott). Quantitative reverse transcription“PCR (c) and western blot (d) demonstrate higher levels of SETDB1 mRNA and protein (ab12317 Abcam Cambridge UK) respectively in amplified cancer cell lines (H1437 NCI-H1395 and DMS-273) than that in unamplified cells. PCR primers are available upon request. Growth-promoting effects of SETDB1 in lung cancer. (a) Stable downregulation of the SETDB1 gene by short hairpins using two different target sequences for DMS-273 (clones A30/A31 and clone B32-63) and NCI-H1437 (clones A56-B and B46-9). SETDB1 shRNA sequences are available upon request. (b) The short hairpin SETDB1-depleted cells were less viable in the 3-(45-dimethyl-2-thiazolyl)-25-diphenyl-2H-tetrazolium bromide (MTT) assay than in the untransfected or scrambled shRNA-transfected cells (P-values obtained by the analysis of variance (ANOVA) test). (c) The colony formation assay showed that DMS-273 and NCI-H1437 cells stably transfected with the shRNA against SETDB1 formed significantly fewer colonies than scrambled shRNA-transfected cells (P-values obtained by the ANOVA test). Data shown are means±s.d. n=3. (d) Effect of SETDB1 shRNA-mediated depletion on the growth of DMS-273 and NCI-H1437 xenografts in nude mice. Tumor volume was monitored over time and the tumor was excised and weighed at 30 days. There was a significant decrease in tumor weight in the SETDB1 shRNA-stably transfected cells (P-values obtained by the ANOVA test). Data shown are means±s.d. n=10. Impact of SETDB1 on invasiveness and chemosensitivity. (a) Effect of SETDB1 on the invasion potential of A549 cells determined by the matrigel invasion assay. Cells were transfected with 3??g of Flag-SETDB1 or empty vector in 60?mm dishes. After 24?h cells were stimulated or not with phorbol myristate acetate (PMA) plus ionomycin (Io) for 30?min. Then cells were trypsinized and 5 — 104 cells were resuspended in serum-free media and added to the upper compartment of a transwell coated with 1?mg/ml Matrigel (BD Biosciences Lexington KY USA). Media with 10% fetal bovine serum was added in the lower compartment and cells were incubated at 37?°C for 42?h. Invasive cells were fixed with phosphate-buffered saline 4% paraformaldehide stained with 0.5% violet crystal and visualized and photographed under a — 10 magnification objective with a microscope. Invasive cells were counted using ImageJ 1.45s (Wayne Rasband National Institutes of Health Bethesda MD USA) and percentage of invasive cells were represented. Results are the mean of at least three experiments by duplicate and the significance was determined using analysis of variance test. *<P=0.05. (b) Cancer cells harboring the SETDB1 gene amplification are sensitive to the decrease in cell viability caused by mithramycin a SETDB1-interfering drug. 3-(45-dimethyl-2-thiazolyl)-25-diphenyl-2H-tetrazolium bromide (MTT) assays in control-scrambled shRNA DMS-273-transfected cells in comparison with three shRNA-stable downregulated SETDB1 clones (A21 A30 and A31) show enhanced inhibition of viability in cells with SETDB1 gene amplification-mediated overexpression. Detection of SETDB1 gene amplification and its associated overexpression in primary tumors from lung cancer patients. (a) Fluorescence in situ hybridization for the SETDB1 gene shows gene amplification in the primary lung tumors 1 2 and 3. SETDB1 unamplified tumors are shown in the cases 4 and 5. The UCSC genome browser (http://www.genome.ucsc.edu) was used to select the bacterial artificial chromosome (BAC) clone RP11-42A12 spanning the 1q21 region of SETDB1 gene. A telomeric BAC clone located in the telomeric 1p36.23 region was used as a control. (b) Immunohistochemistry for SETDB1 (HPA018142 Sigma-Aldrich St Louis MO USA) shows overexpression of the protein in the above shown three primary lung tumors harboring SETDB1 gene amplification. Minimal expression is detected in the unamplified cases (4 and 5). Magnification — 100. (c) Association between SETDB1 gene amplification and overexpression in the studied fifty-nine cases is shown. Fisher's test two-tailed P-value<0.0001. 0135054 6265 Oncology Oncology Oncology 0030-2414 1423-0232 24925190 4105702 10.1159/000360703 NIHMS595992 Distribution and timing of distant metastasis after local therapy in large cohort of patients with esophageal and esophagogastric junction cancer Shiozaki Hironori Sudo Kazuki Xiao Lianchun Wadhwa Roopma Elimova Elena Hofstetter Wayne L. Skinner Heath D. Lee Jeffrey H. Weston Brian Bhutani Manoop S. Blum Mariela A. Maru Dipen M. Ajani Jaffer A. U. T. M. D. Anderson Cancer Center (UTMDACC) Houston Texas USA All correspondence to: Jaffer A. Ajani Department of Gastrointestinal Medical Oncology University of Texas M. D. Anderson Cancer Center 1515 Holcombe Blvd (FC10.3022) Houston TX 77030 jajanimdanderson.; Phone: 713-792-2828; Fax: 793-745-1163 13 6 2014 07 6 2014 2014 07 6 2015 86 0 336 339 Background Patients with localized esophageal and esophagogastric junction cancer (EAC) receive chemoradiation then surgery (trimodality; TMT) or definitive chemoradiation (bimodality; BMT). Since distant metastases (DMs) are common but the details of the DM distribution and timing in a large cohort have not been described. Methods 629 patients with localized EAC who had TMT or BMT were analyzed. Standard statistical methods were used to define the endpoints. Results The median follow-up time was 37.2 months (interquartile range: 17.8“65.0). Among 356 TMT patients 33% (119) developed DM as their first relapse and among 273 patients with BMT 40% (109) developed DM. 91% (TMT) and 96% (BMT) of DMs were diagnosed within 2 years of local therapy. The most common sites of DMs were: lung distant nodes liver peritoneal cavity bone brain and pleura in the order of frequency. The median overall survival of TMT patients with DM was 10.2 months (95% CI: 7.8“12.7) and that for BMT patients with DM was 7.8 months (95% CI: 5.7“9.9). Conclusions Following TMT or BMT ?33% of patients developed DMs and most DM occurred within 2 years (>90%) of local therapy. A clinical model that highly associates with high-risk for DM in TMT-eligible patients prior to surgery is desirable. Risk of metastases Adenocarcinoma Chemoradiation Chemotherapy Esophageal cancer gastroesophageal cancer Metastasis Cell Death Dis Cell Death Dis Cell Death & Disease 2041-4889 Nature Publishing Group 24625970 3973193 cddis201456 10.1038/cddis.2014.56 Original Harnessing the lysosome-dependent antitumor activity of phenothiazines in human small cell lung cancer Antitumor activity of phenothiazines in SCLC Zong D 1 2 * Zielinska-Chomej K 1 Juntti T 1 M¶rk B 1 Lewensohn R 1 H¥¥g P 1 Viktorsson K 1 * 1Department of Oncology-Pathology Karolinska Biomics Center Karolinska Institutet Stockholm Sweden *Department of Oncology-Pathology Karolinska Biomics Center Karolinska Institutet Z5:01 S-171 76 Stockholm Sweden. Tel: +1 301 435 5764; Fax: +1 301 402 0711; E-mail: dali.zongnih.gov (DZ) or Tel: +46 8 517 701 77; Fax: +46 8 517 710 00; E-mail: kristina.viktorssonki.se (KV) 2 Current address: Laboratory of Genome Integrity National Cancer Institute National Institute of Health Bethesda MD USA 03 2014 13 03 2014 1 3 2014 5 3 e1111 24 09 2013 23 12 2013 14 01 2014 Copyright © 2014 Macmillan Publishers Limited 2014 Macmillan Publishers Limited This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license visit http://creativecommons./licenses/by-nc-nd/3.0/ Phenothiazines are a family of heterocyclic compounds whose clinical utility includes treatment of psychiatric disorders as well as chemotherapy-induced emesis. Various studies have demonstrated that these compounds possess cytotoxic activities in tumor cell lines of different origin. However there is considerable confusion regarding the molecular basis of phenothiazine-induced cell death. Lung cancer (LC) remains one of the most prevalent and deadly malignancies worldwide despite considerable efforts in the development of treatment strategies especially new targeted therapies. In this work we evaluated the potential utility of phenothiazines in human LC. We show that phenothiazines as single treatment decreased cell viability and induced cell death preferentially in small cell lung carcinoma (SCLC) over non small cell lung carcinoma (NSCLC) cell lines. Sensitivity to phenothiazines was not correlated with induction of apoptosis but due to phenothiazine-induced lysosomal dysfunction. Interestingly the higher susceptibility of SCLC cells to phenothiazine-induced cell death correlated with an intrinsically lower buffer capacity in response to disruption of lysosomal homeostasis. Importantly this effect in SCLC occurred despite mutation in p53 and was not influenced by intrinsic sensitivity/resistance toward conventional chemotherapeutic agents. Our data thus uncovered a novel context-dependent activity of phenothiazines in SCLC and suggest that phenothiazines could be considered as a treatment regimen of this disease however extended cell line analyses as well as in vivo studies are needed to make such conclusion. small cell lung cancer phenothiazines lysosomal dysfunctions Phenothiazines are a family of heterocyclic compounds whose clinical utility includes treatment of psychiatric disorders for example schizophrenia.1 In this setting the therapeutically relevant targets of phenothiazines are believed to be neurotransmitter receptors in particular the dopamine D2 receptor (D2R).2 However an array of putative cellular targets have additionally been described for phenothiazines ranging from calmodulin (CaM) to lipid membranes suggesting that the effect of these compounds likely extend beyond their impact on neuroendocrine signaling.3 Notably various studies have demonstrated that phenothiazines possess cytotoxic activities especially in established tumor cell lines.4 However there is considerable confusion regarding the molecular basis of phenothiazine-induced cell death and numerous hypotheses including CaM antagonism5 membrane fluidization6 and disruption of mitosis7 have been proposed. Lung cancer (LC) remains one of the most deadly malignancies worldwide8 despite considerable effort in the development of treatment strategies especially new targeted therapies. In parallel there has been a recent revival of interest in the use of ˜old' FDA-approved drugs for new indications. In this work we evaluated the potential utility of phenothiazines in LC. We show that phenothiazines induced cell death preferentially in small cell lung carcinoma (SCLC) over non small cell lung carcinoma (NSCLC) cell lines. In this context cytotoxicity was due to phenothiazine-induced lysosomal dysfunction and the higher susceptibility of SCLC cells correlated with an intrinsically lower buffer capacity in response to disruption of lysosomal homeostasis. Importantly this effect in SCLC occurred despite mutation in p53 and was not influenced by intrinsic sensitivity/resistance toward the conventional chemotherapeutic agents for example cisplatin etoposide or gemcitabine. Our data thus uncovered a novel context-dependent antitumor action of phenothiazines in SCLC. Results Single-drug treatment with phenothiazines induced cytotoxicity preferentially in human SCLCs Phenothiazines have been reported to induce apoptosis in tumor cells of different origin.34 9 Here we evaluated the potential therapeutic utility of phenothiazines in a panel of human SCLC and NSCLC cell lines. At a concentration of 10??M which is in the range of concentration achievable in the plasma of patients without eliciting significant adverse side effects10 phenothiazines were overtly cytotoxic analyzed as reduction in cell viability in most of the tested SCLC cell lines while NSCLC cell lines were generally less responsive (Figures 1a“c Table 1). The hyper-responsiveness of SCLC to phenothiazines was not recapitulated when the chemotherapeutic drugs cisplatin etoposide and gemcitabine were used11 as comparable decrease in cell viability was observed in SCLC and NSCLC cells (Supplementary Figure S1). Moreover our results clearly show that the sensitivity to phenothiazine-induced decrease in cell viability in SCLC is not substantially influenced by growth pattern site of isolation prior exposure to conventional chemotherapies and occurs despite mutation in the tumor suppressor p53 (Supplementary Table S1). To further verify the preferential activity of phenothiazines in SCLC we compared the sensitivity of four phenothiazine compounds (trifluoperazine dihydrochloride (TFP) fluphenazine dihydrochloride (FPZ) triflupromazine hydrochloride (TFPZ) and promazine hydrochloride (PZ)) over a range of concentrations in the SCLC cell line H82 and in the NCSLC cell line U-1810 (b upper panel). We also assessed the dose-dependent cytotoxicity of TFP analyzed as decrease in cell viability in several additional SCLC (H69 U-1285 U-1906 and U-2020) and NSCLC (A549 H125 and H1299) cell lines (b lower panel). These analyses confirmed that SCLC cells were more sensitive to phenothiazines than NSCLC cells (b). Importantly the cell viability of primary fetal lung WI-38 fibroblast was less affected by phenothiazines at concentrations that caused significant cytotoxicity in SCLC cells illustrating a potential therapeutic window for phenothiazines in SCLC (Figures 1a and c). The increased sensitivity of SCLC cells to phenothiazines was also evidenced by increased cell death and growth arrest after treatment with these agents (). Thus 10??M TFP induced a prominent impairment in cell division capacity in SCLC cells while in NSCLC cells 20??M TFP was needed to significantly affect proliferation (Figures 2a and b). Analysis of cell cycle kinetics of SCLC cells (H69 and H82) revealed that 10??M TFP caused a time-dependent cell cycle arrest in late S and G2/M phases whereas such changes in cell cycle were not observed for NSCLC (U-1810; c). Taken together our data demonstrate that phenothiazines preferentially decrease the viability and growth of human SCLC."

Lung_Cancer

"For example in protein“protein interaction (PPI) networks nodes represent proteins and edges represent physical interactions. In genetic interaction (GI) networks nodes represent genes and edges represent the anism fitness for double-knockout perturbations yielding two major types of edges: alleviating GIs and aggravating GIs. In alleviating GIs also called positive GIs the anism fitness after the double-knockout perturbation is better than expected based on the single-knockout results. In aggravating or negative GIs the fitness is worse than expected. In gene co-expression networks nodes represent genes and edges score the correlation in expression between the two genes (1011). In gene differential correlation (DC) networks edges score the change in gene pairwise correlation between one set of samples to another (e.g. cases and controls) (12“14). With the growing use and number of types of biological networks computational methods that exploit these rich data are of great importance.Computational methods that make use of several networks are often better than methods that analyze only a single network (47815“19). For example combined analysis of PPI networks and gene co-expression networks was used to detect gene sets that are co-expressed and are connected in the PPI network. Such analysis outperformed standard clustering algorithms and was successfully used for gene function prediction (581619). Alleviating and aggravating GI data were used to find epistasis among and within gene sets. Under the premise that negative GIs tend to occur between compensatory pathways and positive GIs occur within pathways (or complexes) analysis of GIs was used to suggest a map of epistatic relations among functional gene modules (151720“23). A marked improvement was reported after adding a connectivity constraint in a PPI network of the modules (1517). The ability to construct a summary map of several networks allows identifying associations among discovered modules thus improving the interpretability of the results compared with standard clustering of a single network.Building on prior studies of specific pairs of networks we introduce and study the fundamental problem of constructing a summary map of two biological networks H and G where the nodes of both are the same genes or proteins and the edges in each represent a distinct type of relations (see D). The map nodes are gene sets that are strongly connected in H and pairs of sets are connected by links. A link represents strong connection between two gene sets in G. The goal is to find gene modules in H simultaneously with finding module-to-module interactions according to G by optimizing a specific objective function. We call this computational problem the ˜module map problem™. .Module map: example and simulation results. (A and B) Performance of module map algorithms on 500-node graphs. (A) Unweighted graphs. (B) Weighted graphs. Each simulated pair of graphs contained an embedded module map of six modules in a tree structure. In addition two random cliques and two bicliques were embedded in the graphs as decoys. Module clique and biclique size was chosen uniformly at random between 10 and 20. In the unweighted model (A) each edge was replaced by a non-edge with probability P and vice versa. In the weighted model (B) edge weights are sampled from the normal distribution N(1?) and non-edge weights are sampled from the normal distribution N(?1 ?). Results are averages of 10 simulations for each data point. The four top performing algorithms for each simulation are presented using radar plots. MBC-DICER with global improvement is denoted as ModMap. The Jaccard coefficient between the modules produced by each algorithm and the true modules is shown as the distance from the center. Consecutive spokes from the top anticlockwise show increasing values of P in A and of ? in B. (C) Comparison of module map algorithms on unweighted graphs with 1000 nodes containing a map of 10 modules and five decoys and P = 0.15. (D) A toy example of the module map problem; left: the two networks. Nodes are genes H edges are black and G edges are blue; right: the module map. Nodes are modules and edges are links. Colors and numbers are the same on the left and right. The map contains three modules: module 2 is linked to modules 1 and 3 whereas module 1 and 3 are not linked. Black nodes are not part of the module map. The graph H (black edges) contains a clique that is not linked in G to another module and thus is not a part of the map. The example also demonstrates the difference between the local and global approaches. The local approach identifies modules 1 and 2 as linked whereas the global approach also identifies module 3 as linked to module 2. See text.Most algorithms for the module map problem to date were used to find a summary map of epistatic interactions among pathways (151720“23). Kelley and Ideker (15) proposed a method that is based on local searches in the graphs to find pairs of connected modules. Ulitsky et al. (17) used a clustering of H as a starting point and then improved the solution by merging modules. An algorithm akin to (15) has been recently proposed for analyzing gene co-expression and DC networks. The joint analysis of these networks revealed gene groups that are much more (or much less) correlated in one class of individuals (24). Although previous algorithms for the module map problem proved valuable a thorough analysis of the problem and of the merits and weaknesses of these algorithms in different scenarios is required.The problem of finding an optimal module map is NP hard under most formulations as it contains the clustering of H as a subproblem. Hence heuristics are used. These algorithms usually contain two phases. We call the first phase ˜initiators™: algorithms for finding an initial solution that may contain many small modules. The second phase uses ˜improvers™: algorithms for improving an initial solution according to a predefined objective function. A variety of algorithms can be formed from different combinations of initiators and improvers.Here we study novel and extant initiators and improvers. We show that a new initiator based on maximal bicliques in G together with a statistically formulated global improver strategy performs consistently better or equal to extant methods on synthetic and real networks of several types. We call the resulting algorithm ModMap. We apply ModMap to experimental data in three biological scenarios: (i) using yeast PPIs and negative GIs we find epistatic relations among protein complexes (ii) using yeast PPIs and DNA damage-specific positive GIs we detect emerging connections among protein complexes involved in DNA damage response and (iii) using DC analysis of gene expression profiles of non“small-cell lung cancer (NSCLC) tissues we identify disease-specific loss of correlation between immune activation processes and detect disease-specific microRNAs.MATERIALS AND METHODSDefinition of the module map problemThe input to the problem is a pair of networks H = (VEHWH) and G = (VEGWG) defined on the same set of vertices. These networks can be weighted or unweighted. The goal is to find a module map that summarizes both networks. A module map is a graph F = (ML) where M is a collection of disjoint node sets called modules M = {M1 ¦ Mp} Mi V Mi Mj = and L is a set of module pairs {(U1V1) ¦ (UpVp)} where each Ui and Vi are in M. These pairs are called the map links. In addition each module must be linked to at least one other module. Roughly speaking our goal is to find a module map such that each module corresponds to a heavy subgraph of H and each link represents a heavy bipartite subgraph in G between a pair of modules. A formal notion of heavy subgraphs will be introduced later. D shows a toy example of two unweighted networks and their module map.Previous algorithms for constructing module maps vary in the way they define the objective function and the links."

Lung_Cancer

"We also studied the oncogenic potential of SETDB1 by evaluating its ability to enhance cell invasion in a lung carcinoma cell line without SETDB1 gene amplification (A549; a). To this end we transfected the plasmids driving the expression of SETDB1 and performed a matrigel invasion assay. We observed a significant increase in the number of invasive cells upon SETDB1 transfection in comparison with empty vector-transfected cells (a). We also wondered about gene targets in the amplified lung cancer cells whose expression could be regulated by SETDB1-mediated H3-K9 promoter methylation and that could further explain the above observed impact in cell growth and invasiveness. To find downstream targets of SETDB1 in the amplified lung cancer cell lines DMS-273 and NCI-H1437 we have developed expression microarray analyses (Agilent G4851B 60K Santa Clara CA USA) of both SETDB1 shRNA-depleted cell lines in comparison with their corresponding shRNA-scrambled control cell lines. The microarray expression data obtained are freely available at the Gene Expression Omnibus database: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=tdexdacygiayejy&acc=GSE45175. Using this approach we have identified eighteen common genes repressed in both SETDB1-amplified lung cancer cell lines that become upregulated upon SETDB1 shRNA-mediated downregulation (Supplementary Table S2 and Supplementary Figure S4). We have also confirmed the expression changes of the candidate genes by quantitative reverse transcription“PCR and the shift in H3-K9 methylation status in their respective promoters by quantitative chromatin immunoprecitation (Supplementary Figure S4). Related to function gene ontology analysis of these genes determined ˜regulation of cell proliferation' as the most significantly enriched biological process (false discovery rate=0.0006). Representative examples included the tumor suppressor roles of delta/notch-like epidermal growth factor-related receptor34 and insulin-like growth factor-binding protein 7.35 The observation that the presence of the SETDB1 gene amplification with associated overexpression was critical for the tumorigenesis of these lung cancer cells prompted us to examine whether drugs targeting this pathway might find a therapeutic ˜niche' for their use in the described subset of cases with extra copies of this gene. Similar scenarios have been described for inhibitors of another histone methyltransferase DOT1L36 and the BET family of acetyl-lysine-recognizing chromatin ˜adaptor' proteins3738 39 in which hematological malignancies carrying gene-activating events involving targets of these pathways are more sensitive to these drugs. No highly specific inhibitor for SETDB1 has been described in the publicly available literature but to the best of our knowledge one agent ”mithramycin” could be important in targeting SETDB1.40 Mithramycin is a clinically approved antitumor antibiotic that binds to DNA by interacting with the minor groove and displacing transcriptional activators that bind to GC-rich binding sites.41 Most importantly it has been shown to suppress basal SETDB1 promoter activity in a dose-dependent manner by inhibiting the binding of Sp transcription factors.40 Thus we tested whether a putative growth inhibitory effect of this drug in lung cancer cells was dependent on SETDB1 expression. First we developed western blot analyses for the SETDB1 protein in the SETDB1-amplified lung cancer cell lines DMS-273 NCI-H1437 and NCI-H1395 upon the use of the drug. We found that mithramycin was able to inhibit SETDB1 expression in the three cell lines (Supplementary Figure S5). Using the small lung cancer cell line DMS-273 harboring the previously identified SETDB1 gene amplification in comparison with three stable short hairpin SETDB1-depleted clones we observed that the scramble shRNA DMS-273 cells were significantly more sensitive to the growth inhibitory effect mediated by mithramycin than any of the depleted clones (A30 A31 and B32-63; and Supplementary Figure S5). Experiments for each clone were performed in triplicate. Furthermore we extended the cell viability experiments using the MTT assay to the other two SETDB1-amplified lung cancer cell lines (NCI-H1437 and NCI-H1395) and to three SETDB1 non-amplified lung cancer cell lines (DMS-114 A549 and NCI-H1299). The determination of the corresponding EC50 values further confirmed that SETDB1-amplified cell lines (EC50=13.6?nM for NCI-H1437 and EC50=14.7?nM for NCI-H1395) are more sensitive to the drug than the non-amplified cell lines (EC50=347.5?nM for DMS-114 EC50=122?nM for A549 and EC50=32?nM for NCI-H1299; analysis of variance P<0.001). Thus at least in vitro the presence of SETDB1 gene amplification could ˜mark' lung cancer cells that are more sensitive to the inhibition of cell viability associated with the use of mithramycin for which previous data also suggested SETDB1 as a likely candidate target gene of the drug.40 Finally we sought to demonstrate that the presence of SETDB1 gene amplification was not a specific feature of in vitro grown lung cancer cell lines and that it also occurred in primary tumors of lung cancer patients. In this regard the 1q chromosome arm undergoes gains (trisomic or tetrasomic) in lung cancer4243 44 that are also associated with overrepresentation of the 1q21 region.4546 47 Recent genomic data48 49 using single-nucleotide polymorphism microarrays confirm the gain of the SETDB1-1q21 chromosomic region in primary lung tumors. Herein we performed fluorescence in situ hybridization analyses for the SETDB1 locus using a collection of 59 primary lung tumors corresponding to 40 non-small cell lung tumors (20 squamous cell carcinoma and 20 adenocarcinomas) and 19 small cell carcinomas (a). We identified SETDB1 gene amplification in nine tumors corresponding to 20% (4 of 20) 20% (4 of 20) and 5% (1 of 19) of adenocarcinoma squamous and small cell lung cancer cases respectively. Most importantly we also demonstrated that the presence of extra copies of SETDB1 in these nine primary tumors was associated with overexpression of the SETDB1 protein as determined by immunohistochemistry in all cases (b). "

Lung_Cancer

" The map is constructed based on both networks simultaneously and thus can capture and reveal structures that are not identifiable when analyzing each data type separately. Our novel algorithms recovered the planted map structure in simulated data even when the noise level in the data was high. We tested our methods in three biological applications: (i) yeast PPIs and negative GIs (ii) yeast PPIs and DNA damage-specific positive GIs and (iii) DC analysis of human disease expression profiles. In all cases certain parts of our maps are supported by prior biological knowledge whereas other parts reveal novel structure and suggest new biological findings. The module map paradigm can be applied in principle on any two types of networks with underlying common nodes.Our analysis of the yeast PPI and negative GI data constructed a large map describing epistatic relations among complexes. Our findings are in agreement with previous studies and show a complex map of interactions among chromatin modification-related complexes but also provide interactions with other functions such as protein modification-related complexes. The analysis of the yeast PPIs and DNA damage-specific positive GIs produced a smaller map which contains a DNA repair module as a central hub. The interactions of this module suggest that several mechanisms emerge simultaneously in response to MMS including double strand repair damaged replication fork repair and exosome complex activity. In the map constructed based on human NSCLC blood expression profiles modules represent gene sets that are highly co-expressed both in cases and in healthy controls whereas the map links correspond to specific rewiring of the co-expression network in NSCLC patients. In particular we identified two modules enriched with immune activation genes manifesting a sharp drop in correlation in the NSCLC patients suggesting diminished coordination between the T-cell and the B-cell enriched modules.The concept of a module map can be viewed as a higher level combination of clustering and biclustering. Each of those problems has been extensively studied and was applied successfully to numerous single-type genomic and proteomic studies (157“68). By performing joint analysis on two different data types we allow some relaxation of the objective function in each of the networks for the sake of obtaining an overall clearer structure. Therefore the new analysis can yield results when clustering or biclustering of one data type fails. One of the difficulties in clustering and biclustering is that module (or module-pair) sizes must be large enough to obtain highly significant sets. As our analysis demonstrates the added power of the module map approach can identify relatively small precise groups that are beyond the detection ability of those prior methods.Only a handful of studies have addressed the module map problem to date and most of them focused on joint analysis of yeast PPI and GI networks. Ulitksy et al. (17) and Bandyopadhyay et al. (69) developed clustering methods that seek a map in which the likelihoods of the edge weights of PPIs and GIs within clusters or of GIs between linked clusters are higher than a given background distribution. Leiserson et al. (2236) sought local maximum cuts in the weighted graph of the GIs by a greedy incremental approach producing a collection of linked pairs of modules. Kelley and Ideker (20) developed a clustering algorithm that is based on graph compression where the original GI graph is compressed to a module map. Hence both (2236) and (20) look for approximate bicliques that connect gene modules. In contrast we enumerate the maximal bicliques of GIs analyze them by taking into consideration the two interaction types to ensure that the initial solution contains dense strongly connected modules and improve the solution using our global improver. Because our approach is generic it does not exploit the specific probabilistic nature of the GI data as other methods do (2236). Nevertheless we show that our method outperforms these and other extant methods in several criteria on GI data. In addition because our algorithm is not limited by the type of the input data we are able to combine many heterogeneous data sets (e.g. using all GIs of BioGRID) in our analysis.When dissecting human expression profiles of disease patients and healthy controls DC analysis was proposed as a way to discover gene modules whose inter-module correlation levels are altered in disease (12142470). We previously developed DICER (24) which uses a local approach to detect module pairs. Here we go beyond it by finding maximal bicliques in the DC graph and by concurrently constructing a global map of modules. As we showed here in most cases the map links are highly significant. However we also observed cases where the absolute correlation change of modules might be mild even though the DC of the module pair is significant. A possible remedy is to give more emphasis to high absolute DC of map links so as to see the DC signal better. Another possible improvement is to enumerate bicliques using established heuristics [e.g. (68)].A key factor in the performance of the ModMap algorithm is the objective function optimized. Here we chose to maximize the sum of weights within modules plus the sum of weights of module links and assigned these weights based on a probabilistic model. On unweighted networks such as the PPI and GI yeast networks we set the weight of an edge to 1 and the weight of a non-edge to ?1 thereby promoting strongly connected modules and links. This setting produced good results and revealed functional interactions among protein complexes. By setting different weights to non-edges in the graphs future analyses can promote modules that are sparser thus enabling better detection of interactions among complete pathways.SUPPLEMENTARY DATASupplementary Data are available at NAR Online.FUNDINGIsrael Science Foundation [802/08 and 317/13]; Israel Cancer Research Fund; Lee Perlstein Kagan Charitable Trust (in parts). Azrieli Fellowship from Azrieli Foundation Edmond J. Safra Center for Bioinformatics at Tel Aviv University Israeli Center of Research Excellence (I-CORE) Gene Regulation in Complex Human Disease Center No 41/11 (to D.A.); The funders had no role in study design data collection and analysis decision to publish or preparation of the manuscript. Funding for open access charge: Israel Science Foundation and I-CORE.Conflict of interest statement. None declared. Supplementary Material Supplementary "

Lung_Cancer

"Finally because there is no head-to-head clinical trial comparing maintenance gefitinib with other maintenance drugs (eg erlotinib) after the standard chemotherapy of four chemotherapeutic cycles we have not conducted a cost-effectiveness analysis of gefitinib in comparison with other maintenance therapies. Although the current estimates were derived from just one study which is also the only phase III trial compared maintenance gefitinib treatment in patients with locally advanced/metastatic NSCLC according to our literature search we believe that the analysis of our study based on a current Chinese phase III trial and the justifiable extrapolation approach can provide important reference information for decision makers in China. First of all the clinical study itself is a multicentre double-blind randomized controlled-trial (RCT) which represents the best evidence available and is deemed to be the most accepted scientific method of determining the benefit of a drug or a therapeutic procedure. Second the analysis method applied in our study was reliable and widely used in economic evaluations especially in the field of medical and health care. In addition the Log-logistic and two parameters Weibull model matched the survival curves of the clinical trial satisfactorily () which shows that the model we constructed can mirror the effectiveness data of the trial commendably. And then direct medical costs related to each strategy were estimated including maintenance gefitinib therapy treatment of major adverse events routine follow-up treatment for patients without progression follow-up treatment in PS state and terminal-phase cost. Although the costs originated from our previous study [26] the published literature [27] or estimates according to local charges based on expert opinion all of them stemmed from a Chinese health care system perspective as well as in view of patients with advanced NSCLC which echoed the purpose of the current study. Last but not least to reflect substantial uncertainty of the input parameters the sensitivity analyses (including OSA and PSA) were conducted for each key parameter and all sensitivity analyses revealed that the model we applied was robust to the results. In according to the recommended WTP threshold (3—per-capita GDP) of cost-effectiveness guidelines from WHO maintenance gefitinib therapy after the standard chemotherapy of four chemotherapeutic cycles in locally advanced/metastatic NSCLC patients with unknown EGFR mutations is likely to be not cost-effective for Chinese mainland from the Chinese health care system perspective. Local governments with different economic level however could take fully into account covering maintenance gefitinib treatment. Because for rich regions (the per-capita GDP> $8767) the new strategy seems to be a reasonable option and if the per-capita GDP ranges from $5900 to $8767 the maintenance therapy may be favourable in terms of the different cost-effective probabilities. Decreasing the price of gefitinib the most significant parameter that could reduce the ICER should be considered to as a preferential factor for meeting widely treatment demands in China. Prof. L.B. Peng and J.H. Li are the guarantors for the overall content. The authors greatly thank many clinicians and the data managers who have recorded the initial data diligently of medicines over the years. In particular they thank Ouyang Lihui Wang Siying Zhao Ziying and Qiu Zhenhua for their help in the data collection and valuable discussions and advices. References 1 JemalA BrayF (2011) Center MM Ferlay J Ward E et al (2011) Global cancer statistics. CA Cancer J Clin61: 69“9021296855 2 FathiAT BrahmerJR (2008) Chemotherapy for advanced stage non-small cell lung cancer. Semin Thorac Cardiovasc Surg20: 210“21619038730 3 GovindanR PageN MenszternD ReadW TierneyR et al (2006) Changing epidemiology of small-cell lung cancer in the United States over the last 30 years: analysis of the surveillance epidemiologic and end results database. J Clin Oncol24: 4539“454417008692 4 Nation Comprehensive Cancer Network (2013) Non“small cell lung cancer (version 2.2014). Available: http://www.nccn./professionals/physician_gls/pdf/nscl.pdf Accessed 21 January 2014. 5 AzzoliCG BakerJS TeminS PaoW AliffT et al (2009) American Society of Clinical Oncology Clinical Practice Guideline update on chemotherapy for stage IV non-small-cell lung cancer. "

Lung_Cancer

"We split cis-meQTL SNPs into five categories according to the meQTL association strength (P>10?7 10?7>P>10?10 10?10>P>10?15 10?15>P>10?20 P<10?20). A SNP is determined to be related with a regulatory region if the SNP or any LD-related SNP (r2 ? 0.8) resides in the ChIP-Seq peaks of the regulatory regions. Regulatory elements include CTCF binding sites DNaseI hypersensitive sites and histone marks from small airway epithelial cells (SAEC) from ENCODE and human alveolar epithelial cells (hAEC) from our laboratory. For each p-value category we calculated the proportions of cis-meQTL SNPs related with regulatory regions. The figures show that the proportions of cis-meQTL SNPs related with regulatory regions increase with the significance of meQTL associations except for the repressive mark H3K27me3. DNA methylation regional associations for lung cancer GWAS SNPs in subjects of European ancestry (a b f and g) Symbols represent the association between established lung cancer GWAS genetic loci in four regions and methylation levels in nearby CpG probes. Y-coordinate P-value for association; x-coordinate genomic location. For each SNP the red solid circle or square represents the methylation probe with the strongest association whereas other methylation probes are colored on the basis of their correlation (measured as r2) to the most-associated probe. For the most-associated probes the P-values in EAGLE discovery set (n=210) and TCGA lung replication data (n=65) are shown. SNP locations are marked by a blue triangle. (c“e and h“j) show the associations between genotypes and methylation levels of the most associated CpG probes. The box plots show the distribution of the methylation levels in each genotype category with error bars representing the 25% and 75% quantiles. Enrichment of cis-meQTL SNPs for lung cancer risk Analysis based on NCI lung cancer GWAS data (5739 cases and 5848 controls). P-values were produced based on 10000 permutations. AD SQ and SC represent adenocarcinoma squamous cell carcinoma and small cell carcinoma. (a) Enrichment was tested using all cis-meQTL SNPs after LD pruning. (b and c) Strong enrichments were observed for cis-meQTL SNP associated with CpG probes annotated to north shores (b) and gene body (c) regions for SQ. (d) The enrichment in (c) was driven by the cis-meQTLs SNPs impacting CpG probes in non-CpG islands. (e) The enrichment in (d) is driven by the SNPs (or their LD SNPs with r2 > 0.95) overlapping with CTCF binding sites or H3K27me3 mark regions. Replication of EAGLE lung meQTLs in TCGA histologically normal tissue samples. Tissue N All cis associations in EAGLE(34304 associations P<4.0—10?5) Strong cis associations in EAGLE(12083 associations P<1.0—10?10) All trans associations in EAGLE(585 associations P<2.5—10?10) Consistentdirection FDR<0.05 Consistentdirection FDR<0.05 Consistentdirection FDR<0.05 Lung 65 32128 (93.7%) 22441 (65.4%) 11250 (99.3%) 11229 (92.9%) 556 (95.2%) 467 (79.8%) Breast 87 30391 (88.6%) 18762 (54.7%) 11640 (96.3%) 9987 (82.7%) 561 (96.1%) 488 (83.4%) Kidney 142 30975 (90.3%) 23984 (70.0%) 11634 (96.3%) 10783 (89.2%) 558 (95.5%) 506 (86.4%) N is the sample size in replication studies. FDR was calculated based on single-sided p-values. Chromatin marks are enriched on meQTL SNPs. control cis only trans only cis + trans cell line mark proportion proportion fold change proportion fold change proportion fold change SAEC CTCF 11.8% 35.3% 3.0 29.6% 2.5 45.4% 3.8 DnaseI 25.4% 54.0% 2.1 45.8% 1.8 59.6% 2.3 H3K27me3 20.4% 34.1% 1.7 25.4% 1.2 42.9% 2.1 H3K4me3 4.8% 29.7% 6.2 18.0% 3.8 39.9% 8.3 H3K36m3 13.4% 36.8% 2.7 22.8% 1.7 45.4% 3.4 HAEC H3K27me3 17.5% 25.3% 1.4 15.6% 0.9 33.2% 1.9 H3K4me3 7.6% 37.0% 4.9 25.0% 3.3 54.9% 7.2 H3K9-14Ac 17.3% 47.6% 2.8 32.3% 1.9 65.3% 3.8 meQTL SNPs were enriched in chromatin marks including CTCF binding sites DNaseI hypersensitive sites and histone marks from small airway epithelial cells (SAEC) from ENCODE and human alveolar epithelial cells (hAEC) from our laboratory. A SNP is determined to be related with a regulatory region if the SNP or any LD-related SNP (r2?0.8) resides in the ChIP-Seq peaks of the regulatory regions. Enrichment for cis-meQTL SNPs without trans effects (œcis only) trans-meQTL SNPs without cis effects (œtrans only) and SNPs with both trans and cis effects (œcis+trans). The baseline proportion (control set) was calculated based on SNPs not associated with meQTLs and with minor allele frequencies and local CpG probe density matching to the meQTL SNPs. The fold changes were calculated using the control set as baseline. PLoS One one 1932-6203 Public Library of Science San Francisco USA 24454925 3893268 PONE-D-13-29217 .0085738 Research Biology Genetics Gene Expression RNA interference Cancer Genetics Molecular Cell Biology Gene Expression RNA interference Medicine Oncology Cancers and Neoplasms Lung and Intrathoracic Tumors Non-Small Cell Lung Cancer Basic Cancer Research Downregulation of PAX6 by shRNA Inhibits Proliferation and Cell Cycle Progression of Human Non-Small Cell Lung Cancer Cell Lines PAX6 in NSCLC Zhao Xiaoting Yue Wentao * Zhang Lina Ma Li Jia Wenyun Qian Zhe Zhang Chunyan Wang Yue Department of Cellular Biology Beijing TB and Thoracic Tumor Research Institute/Beijing Chest Hospital Capital Medical University Beijing China Addison Christina Lynn Editor Ottawa Hospital Research Institute Canada * E-mail: yue.wentaogmail.com Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: WY. Performed the experiments: XZ WJ ZQ CZ YW. Analyzed the data: LZ LM. Contributed reagents/materials/analysis tools: CZ YW. Wrote the paper: WY XZ. 2014 15 1 2014 9 1 e85738 16 7 2013 1 12 2013 2014 Zhao et al This is an open-access distributed under the terms of the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original author and source are credited. Background The transcription factor PAX6 is primarily expressed in embryos. PAX6 is also expressed in several tumors and plays an oncogenic role. However little is known about the role of PAX6 in lung cancer. Methods The function of PAX6 in lung cancer cells was evaluated by small interfering RNA-mediated depletion of the protein followed by analyses of cell proliferation anchorage-independent growth and cell cycle arrest. The changes of cyclin D1 pRB ERK1/2 p38 expression caused by PAX6 inhibition were detected using western-blotting. The PAX6 mRNA level in 52 pairs of tumors and corresponding matched adjacent normal tissues from non-small cell lung cancer patients and lung cancer cell lines was detected by real-time PCR. Results Suppression of PAX6 expression inhibited cell growth and colony formation in A549 and H1299 cells. The percentage of cells in G1-phase increased when PAX6 expression was inhibited. The cyclin D1 protein level as well as the pRB phosphorylation level decreased as a result of PAX6 down-regulation. The activity of ERK1/2 and p38 was also suppressed in PAX6 knock-down cells. The PAX6 mRNA was highly expressed in lung cancer tissue and lung cancer cell lines. In most patients (about 65%) the relative ratio of PAX6 mRNA in primary NSCLC versus adjacent tissues exceeded 100. Conclusions Our data implicated that PAX6 accelerates cell cycle progression by activating MAPK signal pathway. PAX6 mRNA levels were significantly elevated in primary lung cancer tissues compared to their matched adjacent tissues. This work was supported by Beijing Novel Program grant (No. 2006B34); Beijing Research Foundation for Excellent Talents (No. 20061D03); Beijing Cultivation Project for Key Technical and Medicine Product (No. Z101100055610030); the Scientific Research Common Program of Beijing Municipal Commission of Education (No. KM201210025024). The funders had no role in study design data collection and analysis decision to publish or preparation of the manuscript. Introduction A recent overview on global cancer statistics showed that lung cancer was the most commonly diagnosed cancer as well as the leading cause of cancer death [1]. Early detection and targeted therapy is a potential method for lung cancer prevention and therapy [2]. It is important to find which pathways or proteins are active in lung tumor progression [3]. On the basis of the ""cancer stem cell hypothesis tumors are thought to originate through tissue-specific stem cell expression [4]“[6]; in other words tumors are attributed to stem cell factor overexpression [3] [5] [7]. Paired-box 6 (Pax6) is an important transcription factor during embryogenesis and a stem cell factor [3]. Hence PAX6 may play an important role in tumorigenesis. PAX6 belongs to the PAX gene family which encodes a group of nine paired-box transcription factors with important roles in development and disease [3]. PAX6 is an important transcription factor in development of the eyes pancreas and central nervous system [3] [8]. PAX6 expression was recently found in tumors suggesting an oncogenic role [9]. PAX6 is frequently expressed in retinoblastoma pancreatic tumors and intestinal tumors [6] [10] [11]. PAX6 is also highly expressed in brain and breast cancer cell lines [9]. In pancreatic carcinoma cell lines the inhibition of PAX6 expression leads to a decrease in cell growth and survival [12]. PAX6 is also a regulator of MET tyrosine kinase receptor expression in pancreatic carcinoma cell lines [12]. MET is a potential biomarker and therapeutic target for tumors which confirms the oncogenic role of PAX6 in tumorigenesis [13]. It was previously reported that PAX8 and PAX5 are highly expressed in non-small cell lung cancer (NSCLC) and small cell lung cancer cell lines respectively [14]; but little is known regarding PAX6 expression and function in lung cancer. In this study we investigated whether PAX6 regulated cell proliferation of NSCLC. Our findings show that PAX6 promotes G1-S progression by activating the MAPK signal pathway. PAX6 mRNA was frequently expressed in lung cancer tissue as compared to corresponding adjacent non-neoplastic tissue. This suggests that PAX6 is a new potential target in lung cancer. Materials and Methods RPMI 1640 fetal bovine serum (FBS) and Trizol Reagent were purchased from Invitrogen (Carlsbad CA); M-MLV reverse transcription CellTiter 96® aqueous non-radioactive cell proliferation assay oligo-dT and dNTP were obtained from Promega (Madison WI); SYBR® Green PCR Master Mixture was from Applied Biosystems (Carlsbad CA); anti-PAX6 antibodies were purchased from Abnova (Taibei Taiwan) anti-pRB -ERK1/2 p38 -pERK -pp38 -cyclin D1 and -pRB (S780 phosphorylation) antibodies were obtained from Abcam (Cambridge England UK); and enhanced chemiluminescence (ECL) reagent was obtained from Pierce (Rockford IL). Propidium iodide (PI) RNase A and protease inhibitor cocktail were purchased from Sigma (St. Louis MO). Samples Fifty-two NSCLC specimens were obtained from patients undergoing surgical resection at Beijing Chest Hospital. Primary lung cancer samples and matched adjacent normal tissues were used. The study and use of specimens was reviewed and approved by Research Ethic Committee in Beijing Chest Hospital Capital Medical University (Beijing China). Written informed consent was obtained from all patients. The clinical characteristics of the patients are listed in . .0085738.t001 Patients and Clinical Characteristics. Characteristics Number of Patients Patient Age Years 0 “ 60 27 >60 25 Gender Male 40 Female 12 Smoke Status Nonsmoker 21 Smoker 31 Histologic Type SCC* 31 Adenocarcinoma 21 Histological Grade III 21 II 31 Tumor Size 0 “ 3 cm 14 >3 cm 38 Lymph Node Status Negative 27 Positive 25 Distant Metastasis Negative 44 Positive 8 TNM Stage Stage I 17 Stage II 10 Stage III 19 Stage IV 6 : Squamous Cell Carcinoma. Cell culture Human lung adenocarcinoma cell lines A549 and NCI-H1299 human large cell lung carcinoma cell lines NCI-H460 small cell lung cancer cell line NCI-H446 human embryo lung fibroblasts (MRC-5) were obtained from the National Platform of Experimental Cell Resources Sci-Tech. Human large cell lung carcinoma cell lines 95C 95D and 801D were obtained from the tumor center of Chinese Academy of Medical Sciences. Human lung adenocarcinoma cell line A2 and squamous cell carcinoma cell line L were isolated and established by our lab. The lung cancer cell lines were cultured in RPMI 1640 medium (Invitrogen Carlsbad CA USA) supplemented with 10% fetal bovine serum (FBS; Gibco Los Angeles CA USA). MRC-5 were maintained in MEM-EBSS supplemented with 10% FBS. Construction of a PAX6 shRNA lentiviral vector and infection into cells Four RNA interference (RNAi) candidate target sequences were designed based on the human pax6 mRNA sequence and cloned into the pGCSIL-GFP vector (GeneChem Shanghai China). The RNAi sequence GAGTAGCGACTCCAGAAGT was the most effective at suppressing PAX6 mRNA in H1299 and A549 cells and was used in subsequent experiments to knock down endogenous PAX6. Nonsilencing (NS)-small interfering RNA (shRNA) (TTCTCCGAACGTGTCACGT) was also cloned into the pGCSIL-GFP vector and used as a control (GeneChem). The recombinant virus was packaged in 293T cells using a Lentivector Expression System (GeneChem). For cellular infection H1299 and A549 cells were subcultured at 5000 cells/well in 96-well culture plates and infected with lentivirus-mediated pax6-shRNA or NS-shRNA. The GFP expression level was detected via fluorescence microscopy (Nikon Tokyo Japan) to determine the infection efficiency. RNA isolation and real-time PCR Total RNA from tissue and cells was isolated with Trizol Reagent according to the manufacturer™s protocol. The total RNA concentration was calculated by measuring the OD260 and the samples were stored at “80°C. Total RNA (2 µg) was reverse-transcribed using an M-MLV Reverse Transcriptase Kit according to the manufacturer™s protocol. The cDNA (20 ng) was mixed with SYBR® Green Master Mix and genes were amplified with appropriate primers using a real-time PCR detection system (ABI7500; Life Technologies Carlsbad CA). The relative expression levels of PAX6 mRNA were calculated by normalization to the ?-actin mRNA level. The PCR primers used were as follows: PAX6 forward 5'-TTCAGCACCAGTGTCTACCA-3'; PAX6 reverse 5'-GCTGTAGGTGTTTGTGAGGG-3'; ?-actin forward 5'-TTAGTTGCGTTACACCCTTTC-3'; and ?-actin reverse 5'-GCTGTCACCTTCACCGTTC - 3'. Cell proliferation assay A proliferation assay was carried out using Non-Radioactive Cell Proliferation Assay according to the manufacturer™s protocol. Briefly5000 cells/well were seeded into 96-well culture plates in RPMI 1640 containing 10% FBS. The cells were cultured for 5 days then 20 µL of 3-(45-dimethyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium (MTS) was added to each well and the cells were incubated at 37°C for 3 h every 24 h. The absorbance was recorded at 490 nm with a universal microplate reader (Bio-Rad Hercules CA). All the experiments were repeated three times. The data are presented as means ± SEM. Colony formation assay Cells were seeded in triplicate at 300 cells/well in a 6-well plate. After 7 days of culture the cells were washed twice with NaCl (0.9%) stained with 2% gentian violet for 20 min washed with water and air-dried. Foci were counted by microscopy. The experiments were repeated three times and data are presented as means ± SEM. Soft-agar assay Cells (1000) were seeded into 6-well plates in 2 mL of growth medium containing 0.3% agar and used to overlay 1.4-mL layers of growth medium containing 0.6% agar. After 21 days of culture the colonies were counted. All the experiments were repeated three times. The data are presented as means ± SEM. Cell cycle analysis Cells were harvested washed with cold PBS twice and fixed in 70% ethanol at “20°C overnight. The cells were then centrifuged (1500 rpm 10 min) and washed twice using phosphate-buffered saline (PBS). Next the cells were resuspended in 0.5 mL of PBS containing 50 µg/mL RNase A for 1 h at 37°C. The cells were then loaded with 65 ?g/mL PI for 30 min in the dark at 4°C. The percentage of cells in different phases of the cell cycle was measured by flow cytometry (Beijing Determination of Traditional Chinese Medicine Research Institute). The experiments were repeated three times. The data are presented as means ± SEM. Western blotting Cells were digested with trypsin and centrifuged. The cell pellet was washed twice with PBS. Next the cells were disrupted in lysis buffer (10 mM Tris-HCl pH 7.4 1 mM EDTA 0.1% Triton X-100 0.1% SDS and 1— protease inhibitor cocktail) on ice for 15 min and centrifuged at 12000 rpm for 20 min. Insoluble material was removed and protein concentrations were determined using a bicinchoninic acid kit. For Western blot analysis cell lysates (30 ?g/well) were subjected to SDS-PAGE and transferred to nitrocellulose filter membranes. The membranes were incubated with primary antibodies (anti-PAX6 -ERK1/2 p38 -pERK -pp38 -cyclin D1 -RB or -RB S780 phosphorylation) overnight at 4°C. Secondary antibodies conjugated with horseradish peroxidase were subsequently used. Signals were detected using ECL and exposed to Kodak X-OMAT film. The results were scanned and analyzed using Alpha View Analysis Tools. Statistical analysis All values are expressed as the mean ± SEM. Through real-time RT-PCR MTS assay colony formation soft-agar assays cell cycle analysis and western-blot assay for comparison between means of 2 groups statistical differences were tested with unpaired Student t-tests. Statistical significance was tested using SPSS Statistics version 13.0. P<0.05 (*) was considered different; P<0.01 (**) was considered significantly different. Results PAX6 mRNA expression was inhibited in cells infected with the PAX6 shRNA lentiviral vector PAX6 mRNA expression was determined in this study. As shown in Figure 1A PAX6 was highly expressed in most lung cancer cell lines. In contrast MRC-5 a normal human fetal lung fibroblast cell line did not express PAX6 (Figure 1A). .0085738.g001 Figure 1 PAX6 mRNA was highly expressed in lung cancer cells and its expression was suppressed by pax6-shRNA. A Real-time PCR analysis for the PAX6 mRNA expression level in H460 A2 95C 95D H1299 H446 801 D A549 and L lung cancer lines as well as in the normal human fetal lung fibroblast cell line MRC-5. B -C Confirmation of PAX6 mRNA knockdown by real-time RT-PCR assays performed on total RNA isolated from A549 (B) and H1299 (C) cells infected with pax6-shRNA or a random shRNA. The PAX6 mRNA expression levels in A549 and H1299 cells were measured by quantitative real-time RT-PCR. The y-axis represents the normalized PAX6 mRNA expression relative to A549 (B) or H1299 (C) cells. **P < <0.01. D The protein levels of PAX6 were determined by western-blot and GAPDH expression level was used as a control. Quantification was made by determining the gray level of PAX6 protein which was normalized against GAPDH levels. Data are expressed as mean ±SEM of independent experiments (times of the experiments are listed above the histograms). PAX6 expression was obviously weakened in A549 PAX6 KD and H1299 PAX6 KD cells. To elucidate whether PAX6 expression has any effect on the growth of lung cancer cells RNAi was used to generate pax6 knock-down (PAX6 KD) cell lines. We selected two target cell lines: H1299 which showed high levels of PAX6 expression and A549 which showed low levels of expression. In the present study pGCSIL-pax6 shRNA-GFP was infected into H1299 and A549 cells. Cells were also infected with pGCSIL-NS shRNA-GFP (PAX6 NS) as a negative control (NC). To determine the function of PAX6 H1299 H1299NC A549 or A549NC cells were used as controls in all assays. The PAX6 mRNA level in H1299 PAX6 KD and A549 PAX6 KD cells was determined by real-time PCR to confirm whether PAX6 expression was specifically inhibited through RNAi in A549 and H1299 cells. As shown in Figure 1B PAX6 expression in A549 PAX6 KD cells was inhibited by 80“90% compared to cells infected with lentivirus-mediated NS-shRNA. We found similar results in H1299 PAX6 KD cells. PAX6 mRNA expression in these cells was also inhibited by 90“95% as compared to NC cells (**P<0.01; Figure 1C). PAX6 protein expression in these cells was detected by Western blotting. As shown in Figure 1D PAX6 protein in H1299 PAX6 KD and A549 PAX6 KD cells was not readily detected whereas a clear PAX6 protein band was evident in the control cells. Inhibition of PAX6 expression leads to a decline in cell proliferation PAX6 is a critical transcription factor that plays an important role in regulating proliferation and differentiation during human embryonic development [3]. A cell proliferation assay was performed to determine whether PAX6 plays a role in cellular growth. A549 PAX6 KD H1299 PAX6 KD and control cells were seeded in 96-well plates and cell proliferation activity was measured using a Cell Proliferation Assay kit. A549 and H1299 cell growth was obviously suppressed when PAX6 expression was inhibited by RNAi (Figure 2A and B). As shown in Figure 2A and B the decrease in cell growth caused by the inhibition of PAX6 expression in H1299 was much stronger than that in A549 cells. These different results may be attributable to the different PAX6 expression levels between H1299 and A549 cells displayed in Figure 1A and D. .0085738.g002 Figure 2 The lentivirus-mediated pax6-shRNA knockdown of PAX6 expression could suppress lung cancer cell growth. A -B A549 PAX6 KD H1299 PAX6 KD cells and control cells were seeded in 96-well plates and an MTS assay was performed. The absorbance at 490 nm (y -axis) was measured at 24-h intervals up to 120 h. C -D Colony formation efficiency in A549 PAX6 KD H1299 PAX6 KD cells and control cells. The y -axis represents the normalized colony formation rate relative to A549 (C) or H1299 (D) cells. E -F A soft -agar assay was performed to investigate the effects of PAX6 on tumorigenesis in vitro. The y -axis represents the normalized soft -agar colony formation rate relative to A549 (E) or H1299 (F) cells. The data are expressed as the means ± SEM from three separate experiments. Times of the experiments are listed above the graph.*P <0.05 **P <0.01. Reduced colony formation and soft-agar colony formation in PAX6 KD cells Colony formation represents a loss of contact inhibition or the ability to maintain cell growth and movement despite contact with surrounding cells. To clarify whether PAX6 could confer a loss of contact inhibition cells infected with pGCSIL-pax6 shRNA-GFP as well as their control cells were seeded into 6-well plates and cultured for 7 days. After 2% gentian violet staining colonies containing more than 50 cells were counted under a light microscope. As displayed in Figure 2C and D the inhibition of PAX6 expression in A549 and H1299 cells led to an obvious decrease in the number of foci generated compared to the control cells (**P<0.01). To further study the function of PAX6 soft-agar colony formation was analyzed to determine whether PAX6 contributed to anchorage-independent colony formation in lung cancer cells. The rate of soft-agar colony formation declined in A549 PAX6 KD and H1299 PAX6 KD cells compared to NC cells (**P<0.01 *P<0.05; Figure 2E and F). PAX6 expression increased cell growth by promoting faster progression into S phase of the cell cycle To detect the effect of PAX6 on cell cycle progression the cell cycle progression of A549 PAX6 KD H1299 PAX6 KD A549 PAX6 NC H1299 PAX6 NC A549 and H1299 cells was analyzed by flow cytometry. As displayed in Figure 3A the percentage of cells entering S phase was decreased in the A549 PAX6 KD cell line along with an increase in the population of G0-G1 phase cells. A similar result was observed in H1299 PAX6 KD H1299 PAX6 NC and H1299 cells (Figure 3B). In these experiments PAX6 expression led to cell growth by inducing cell cycle progression. .0085738.g003 Figure 3 PAX6 expression promoted cell cycle progression. A B Cell cycle analysis. Cells were stained with propidium iodide (PI) and analyzed for cell cycle phase distribution. The histogram was the statistical data from three independent experimental replicates. *P <0.05 **P <0.01. In this study the expression level of cyclin D1 a relevant cyclin regulating G1-S progression [15] [16] was detected in A549 PAX6 KD and H1299 PAX6 KD cells. As indicated in A cyclin D1 expression was decreased in A549 PAX6 KD cell lines compared to control cells. We found a similar result in H1299 PAX6 KD cells (A). Another relevant cyclin regulating G1/S progression is cyclin E [17]. We also determined whether cyclin E was regulated by PAX6 expression. As a result cyclin E expression was not affected by the stable shRNA-mediated knockdown of PAX6 in lung cancer cells (data not shown). This demonstrates that PAX6 might promote cell growth by inducing cyclin D1 expression. .0085738.g004 Cyclin D1 expression and pRB phosphorylation was inhibited while PAX6 expression was suppressed. A B The expression of cyclin D1 pRB and the phosphorylated pRB in A549 PAX6 KD A549 NC A549 cells as well as H1299 PAX6KD H1299NC H1299 cells was determined by Western blotting. ?-actin and GAPDH expression level was measured as internal loading controls respectively. Cyclin D1 and pRB levels were measured by the gray level and were normalized by internal loading controls. Data are expressed as mean ±SEM. Times of the experiments are listed above the histograms. *P <0.05 **P <0.01. The major substrate of cyclin D1-CDK4/6 complexes is retinoblastoma protein (pRB) [18]. Thus pRB S780 protein phosphorylation was also detected by Western blotting (B). The S780 phosphorylation of pRB was decreased when PAX6 expression was inhibited in A549 cells. A similar result was obtained when H1299 PAX6 KD cells were used (B). MAPK signal pathway was suppressed by the inhibition of PAX6 The MAPK (mitogen activated protein kinase) pathway has been implicated in the regulation of G1/S transitions and cell mitosis [19]. In our study some central regulatory molecules of MAPK pathways were examined using western blot analysis. As shown in the phosphorylation levels of ERK1/2 and p38 were decreased both in A549 PAX6 KD and H1299 PAX6 KD cells. It indicated that the MAPK signal was weakened resulted from the RNAi interference of PAX6. .0085738.g005 The phosphorylation levels of ERK1/2 and p38 were suppressed by the inhibiton of PAX6 expression. Western-blot analysis of A549 A549 NC A549 PAX6 KD H1299 H1299NC H1299 PAX6 KD with antibodies to ERK1/2 (A) p38(B) and their phosphorylated forms were shown in the figure. GAPDH and ?-actin was used as internal loading controls respectively. ERK1/2 and p38 levels were normalized by GAPDH and ?-actin respectively. Data are expressed as mean ±SEM. All the experiments were repeated three times. *P <0.05 **P <0.01. PAX6 was highly expressed in lung cancer tissue Pax6 mRNA in lung cancer tissue as well as matched adjacent tissue was detected to confirm the role of PAX6 in lung cancer. The clinical characteristics of the 52 patients are listed in . As shown in Figure 6A PAX6 mRNA was abundantly expressed in tumor tissue as compared to adjacent normal tissues. The expression of PAX6 represented by a cancer-to-adjacent nontumorous tissue ratio for each individual was indicated in B. PAX6 expression in lung cancer tissue was higher than that in each matched adjacent normal tissue in all but three cases (Figure 6B). The statistic results were listed in table 2 and the ratio (tumor/adjacent tissue) of 65% patients (34 samples) exceeded 100. That is to say in most cases PAX6 was mainly expressed in lung cancer tissues. .0085738.g006 Figure 6 PAX6 mRNA was highly expressed in primary lung cancer tissues and lung cancer cell lines. A Real-time PCR analysis of the PAX6 expression level in lung cancer tissues as well as the matched adjacent tissues from 52 patients. The PAX6 mRNA level was normalized by ?-actin expression level. B Each column represents the relative ratio of PAX6 mRNA in primary NSCLC versus adjacent lung tissue and the line across the graph represents the value 1 and 10 respectively. All the experiments were repeated three times. **P <0.01. .0085738.t002 The relative ratio of PAX6 mRNA in primary NSCLC versus adjacent nontumorous lung tissue. PAX6 mRNA level (Tumor/adjacent tissue) 0“1 1“100 100“10000 10000“100000 Number of patients 3 15 19 15 Discussion In our study the function of PAX6 in lung cancer cells was investigated. The growth ability of A549 and H1299 cells was declined when PAX6 expression was inhibited by specific PAX6 shRNA. We suggest that PAX6 promotes G1-S progression by activating the MAPK signal pathway. And PAX6 was highly expressed in lung cancer tissues and lung cancer cell lines. The transcription factor PAX6 plays different roles in different tumors. It is frequently expressed in pancreatic cancer and retinoblastoma cells implicating an oncogenic function while PAX6 is recognized as a tumor suppressor in gliomas and prostate cancer [6] [10] [11] [20] [21].PAX6 expression is significantly reduced in glioblastomas and the expression level is correlated with longer patient survival [22]. PAX6 suppresses glioblastoma cell growth anchorage-independent growth and glioma angiogenesis as well as invasiveness of glioblastoma cell via inhibition of matrix metalloproteinase-2 (MMP2) expression and vascular endothelial growth factor A (VEGFA) expression [20] [23] [24]. In prostate cancer PAX6 expression was lower in cancer tissues and cancer cell lines than normal epithelial cells [21]. Overexpression of PAX6 suppressed the proliferation and colony formation of prostate cancer cells [8]."

Lung_Cancer

"Therefore in depth studies should be still needed to enhance its antitumor and antimetastatic activities in vivo. Fortunately the in vivo results also exhibited that niclosamide can reduce expression of Ki67 and increase expression of cleaved caspase-3 in tumor cells compared with vehicle treated group. Furthermore accumulating evidence suggests that Stat3 plays an important role in up-regulating VEGF gene expression and inducing tumor angiogenesis under both physiological and pathological conditions [16] [42]. Inhibition of Stat3 can result in the suppression of tumor angiogenesis which was also observed in tumor tissues treated with niclosamide in this study. These results suggested that niclosamide may have a role in the treatment of angiogensis. Breast cancer is progressing toward increasingly malignant behavior in tumorigenic and metastatic stages. In the process of metastasis tumor cells will leave the primary tumor in breast and metastasize to distant sites (lung liver and lymph node) where they establish secondary tumors [36] [43]. Moreover it has been reported that Src/FAK/MMP (Matrix metalloproteinase) involved pathway is critical for breast cancer cell migration and invasion [34] [44]. Therefore inhibition of the step is a promising approach to antitumor and antimetastasis treatment [34]. In this study our observations indicated that niclosamide can inhibit breast cancer migration and invasion in vitro by down-regulating FAK phosphorylation at tyrsion residue 925 and Src- phosphorylation at tyrsion residue 416. Furthermore in our animal experiments administrations of niclosamide at the dose of 20 mg/kg significantly inhibited breast tumor metastasis to lung (D). Overall these results suggested that niclosamide may be a potential candidate for treating breast cancer metastasis. A recent studies showed that Stat3 is frequently activated not only in diverse cancer cells by common oncogenic pathways but also in tumor endothelial and myeloid cells including Gr1+/CD11b+ (MDSCs) and tumor-associated macrophages mediating immune suppression [34] [45]. Meanwhile myeloid cells and other immune cells are critical components of the tumor microenvironment and an excess of MDSCs can promote tumor angiogenesis and influence antitumor immune responses. Therefore MDSCs play a central role in carcinoma progression in tumor-bearing mice and cancer patients [46]. In this study our data showed that the treatment of mice with niclosamide caused a significant decrease in the number of MDSCs in tumors compared with that of vehicle treated group. It is therefore conceivable that blocking Stat3 signaling with niclosamide in vivo can induce immune-mediated antitumor effects. In the results presented here are to our knowledge the first study to demonstrate that niclosamide can inhibit breast cancer cell growth by inducing apoptosis and block cell migration and invasion. In addition niclosamide suppressed the breast tumor growth without significant toxicity."

Lung_Cancer

"Ibuprofen suppresses the expression of Hsp70 in lung adenocarcinoma cells. (a) Upregulation of Hsp70 in lung cancer cell lines. Each cell extract was separated by SDS-PAGE and immunoblotted with an anti-Hsp70 or actin antibody (upper panel). The quantity of each protein was estimated by densitometric analysis using Scion Image (Scion Frederick MD USA). The Hsp70/actin ratios are shown in the lower panel. (b) Effect of ibuprofen on the expression of Hsp70 protein and mRNA in A549 cells. Top: expression of Hsp70 and Hsc70 proteins in A549 cells treated with ibuprofen at the specified concentrations for 48?h examined as described in a (left panel). Bottom: densitometric analysis of each protein level in arbitrary unit (arb-u). The alternation of each mRNA expression after ibuprofen treatment was analyzed by semiquantitative RT-PCR (top right panel). These results are representative of three separate experiments. (c) Effect of ibuprofen on the expression of Hsp70 protein in H358 cells. Expression of Hsp70 proteins in H358 cells treated with ibuprofen at the specified concentrations for 48?h (upper panel). The quantity of each protein was estimated by densitometric analysis (lower panel) Ibuprofen increased the antitumoural activity of cisplatin by suppressing Hsp70. (a) The viability of A549 (upper panel) and H358 (lower panel) cells treated with ibuprofen for 48?h was analyzed by MTT assay. The value is represented as the percentage of cell viability without ibuprofen set at 100%. (b) Synergistic effect of ibuprofen on cisplatin-induced apoptosis in A549 (upper panel) or H358 (lower panel) cells. The cells were treated with the specified concentrations of cisplatin in absence or presence of 400??M ibuprofen for 48?h and the cell viability was assessed by MTT assay. The results are shown as means±S.D. from triplicated experiments. The results shown are representative of three separate experiments. *P<0.05; **P<0.01 (by Student's t-test). (c) Time course of cisplatin-mediated cell death with ibuprofen. A549 cells were treated with 400??M ibuprofen alone or A549 cells with 10??M cisplatin were cultured in absence or presence of 400??M ibuprofen and the cell viability was analyzed by TUNEL staining. The results shown as means±S.D. **P<0.01 (by Student's t-test). (d) The silencing efficiency of Hsp70 determined by immunoblotting. (e) Effect of Hsp70 RNAi on the cisplatin-mediated death of A549 cells. The cells exposed to siRNA targeting Hsp70 or control siRNA were treated with 10??M cisplatin for 48?h and MTT assay was used to determine the cell viability. Data are presented as means±S.D. from triplicated experiments. The results shown are representative of three separate experiments. *P<0.05; **P<0.01 (by Student's t-test). (f) Cytofluorimetric dot plot analysis of the CF488A-Annexin V versus propidium iodide (PI) staining performed in 10??M cisplatin-treated or -untreated A549 cells in absence or presence of 400??M ibuprofen for 48?h. A representative experiment out of three performed with similar results is shown. (g) Effect of cisplatin on the expression of Hsp70. The data are representative of three separate experiments Ibuprofen inhibits the expression of Hsp70 by transcriptional inactivation. (a) ChIP assay for the association of HSF-1 with Hsp70 gene in A549 cells treated with or without ibuprofen. The DNA in the product immunoprecipitated by anti-HSF-1 or non-immune IgG was followed by PCR with a primer specific to the Hsp70 promoter. The immunoprecipitates with antibody against HSF-1 were confirmed by immunoblotting (bottom left). The actin signal is a control of DNA input (bottom right). (b) Effect of ibuprofen on the expression of HSF-1. Cell extracts from A549 cells with ibuprofen for 48?h were separated by SDS-PAGE and immunoblotted with a HSF-1-specific antibody (upper panel). The quantity of each protein was estimated by densitometric analysis (middle panel). The data are representative of three separate experiments. The effect of ibuprofen on the mRNA level of HSF-1 was confirmed by RT-PCR (lower panel). (c) HSF1-mediated inhibition of Hsp70 expression. A549 cells were treated with 10?nM siRNAs against HSF-1 or non-code siRNA. The HSF-1 silencing efficiency and its effect on the expression of Hsp70 were examined by immunoblotting using appropriate antibodies. Three separate siRNAs oligo against HSF-1 were used for its knock-down. The results shown are representative of three separate experiments Ibuprofen exposure enhanced the cisplatin-dependent mitochondrial membrane depolarization and cytochrome c release. (a) A549 cells were treated for 48?h with 10??M cisplatin 400??M ibuprofen or both and subjected to JC-1 staining to study the changes in mitochondrial membrane potential. The percentages indicate the green fluorescence intensity of JC-1 measuring with FACSCalibur. A representative experiment out of three performed with similar results is shown. (b) A549 cells treated as described earlier were fractionated into cytosol and the release of cytochrome c was analyzed by western blot using anti-cytochrome c antibody. The expression of Erk was monitored as an internal control of cytosol protein. The quantity of each protein was estimated by densitometric analysis. The results are means of three separate experiments from cells in different cultures The downregulation of Hsp70 increased the cisplatin-mediated activation of Bax and its translocation to the mitochondria. (a) Detection of active Bax. A549 cells were treated with cisplatin (10??M) and/or ibuprofen (400??M) for 48?h. Active Bax was immunoprecipitated with an active conformation-specific monoclonal antibody and revealed by immunoblotting with an anti-Bax polyclonal antibody. The quantity of active Bax was estimated by densitometric analysis. (b) A549 cells treated as described earlier were lysed and fractionated by differential centrifugation to separate the mitochondria from the cytosol. The translocation of Bax to the mitochondria was visualized by the immunoblot of mitochondrial fractions using an anti-Bax antibody. VDAC-1 was used as a loading control to ensure the use of equal amounts of mitochondria. (c) A decrease in Hsp70 by RNAi promoted cisplatin-dependent activation of Bax. A549 cells treated with Hsp70 or control siRNA were incubated in presence or absence of cisplatin; each cell extract was immunoprecipitated with an anti-active Bax antibody followed by immunoblotting with anti-Bax antibody. The data are representative of three separate experiments Synergistic effect of Hsp70 suppression on the cisplatin-mediated activation of caspase-9. (a) A549 cells were treated with cisplatin and/or ibuprofen and cell extracts were immunoblotted with active caspase-9 antibody. The lower panel shows the measurement of each caspase-9. (b) A549 cells exposed to siRNA targeting Hsp70 or control siRNA were incubated with or without cisplatin and the active caspase-9 was detected by western blot using an anti-caspase-9 antibody. The quantity of each protein was estimated by densitometric analysis (lower panels). (c and d). Assay for enzymatic activity of caspase-9 using a fluorogenic substrate. (c) After the incubation of the A549 cells with cisplatin (10??M) and/or ibuprofen (400??M) the caspase-9 activity of each cell extract was measured as described in Materials and methods section. (d) A549 cells transfected with Hsp70 siRNA or control siRNA were exposed to cisplatin for 48?h. The caspase-9 activity was then assessed using an enzymatic assay as described earlier. The value of caspase-9 activity was presented relative to the activity in untreated cells set at 1.0. The data represent mean values of three separate experiments. Significances were determined by Student's t-test (*P<0.05) Effects of nonsteroidal anti-inflammatory drugs on the expression of Hsp70 in A549 cells NSAIDs Hsp70 expression (%) Ibuprofen (400??M) 22.7±2.8 Aspirin (2500??M) 95.1±7.8 Diclofenac (200??M) 97.2±5.6 Sulindac (15??M) 98.9±2.9 Piroxicam (60??M) 96.6±6.2 Indometacin (10??M) 95.0±15.1 Mefenamic acid (25??M) 100.5±6.0 Values are shown as means±S.D. The expression of Hsp70 was measured by immunoblotting with an anti-Hsp70 antibody. The quantity of Hsp70 protein was estimated by densitometric analysis using Scion Image. The values in parentheses are the highest non-toxic concentrations (approximately 90% viability) used for each NSAID on the A549 cells for 48?h Table 2 Effects of nonsteroidal anti-inflammatory drugs on the expression of HSF-1 in A549 cells NSAIDs HSF-1 expression (%) Ibuprofen (400??M) 16.2±3.9 Aspirin (2500??M) 93.5±2.9 Diclofenac (200??M) 96.7±6.6 Sulindac (15??M) 99.8±3.6 Piroxicam (60??M) 96.3±4.7 Indometacin (10??M) 98.1±1.6 Mefenamic acid (25??M) 98.5±1.1 Values are shown as means±S.D. The expression of HSF-1 was measured by immunoblotting with anti-HSF-1 antibody. The quantity of HSF-1 protein was estimated by densitometric analysis using Scion Image. The values in parentheses are the highest non-toxic concentrations (approximately 90% viability) used for each of the NSAID on the A549 cells"

Lung_Cancer

"Thus mathematical models that can estimate long-term cost-effectiveness of alternative strategies is a helpful technique to support economic analyses of health care resource ulitization [26] [27]. In current study a semi-Markov model along with two-parametric Weibull and Log-logistic distribution were used for measuring the time-dependency transition probabilities and calculating the direct medical costs LYGs and QALYs gained of the practice presented in the trial [15]. A cost-effectiveness evaluation was performed to analysis the economic impact of maintenance gefitinib therapy for patients with locally advanced/metastatic NSCLC with unknown EGFR mutations. Base case analyses of 1- 3- 6- and 10-year time horizon showed an unfavorable ICER of $184829 $19214 $19328 and $21308 per QALY gained respectively. OSA and PSA all revealed that the model we applied was robust to the results. Monte Carlo simulations of 1000 cases suggested that all ICERs for maintenance gefitinib therapy were higher than the recommended WTP threshold (3—per-capita GDP) of cost-effectiveness guidelines from Word Health anization (WHO). There are 31 province-level administrative units in Chinese mainland the per-capita GDP of which differs significantly. In 2011 for example it ranged from $2495 in Guizhou province to $13392 in Tianjin city [31]. According to the recommended threshold of WHO [25] the WTP threshold of different province-level administrative units extended from $7485 (3—$2495) to $40176 (3—$13392) per QALY gained which exceeded the sensitivity range of the WTP (about $17700 to $26300) obtained from PSA of the current study. Obviously local government could take fully into account covering maintenance gefitinib treatment following first-line platinum-based chemotherapy for locally advanced/metastatic NSCLC with unknown EGFR mutations in accordance with local economic development level. Cost-effective probability for different economic level provinces displayed in could supply available information for local governments when gefitinib is approved by local governments™ finance before it has access to the directory of drugs for national basic medical insurance in China. .0088881.t004 The cost-effective probabilities of gefitinib arm for 31 provinces of Chinese mainland. Region Per-capita GDP ($) WTP (3—Per-capita GDP $) Cost-effective Probability Mainland China 5449.71 16349 0 More affluent regionsa >8767 >26300 1.00 Guangdong 7819 23457 0.932 Liaoning 7795 23385 0.926 Fujian 7344 22032 0.717 Shandong 7273 21819 0.655 Less affluent regionsb <5900 <17700 0 a Consist of Tianjin Shanghai Beijing Jiangsu Zhejiang and Inner Mongolia. b Consist of Jilin Chongqing Hubei Hebei Shanxi Ningxia Heilongjiang Shangxi Xinjiang Hunan Qinghai Henan Hainan Jiangxi Sichuan Guangxi Anhui Tibet Gansu Yuannan and Guizhou. A number of different survival models such as Weibull Exponential Log-logistic Gompertz et al can be used to perform extrapolation according to the observed trial data [32]. It is therefore very vital to choose the justifiable extrapolation approach to ensure the associated results of economic analysis confident to decision makers. In the current study after the deviance information criterion test (reported by Jackson et al [33] to alternative models introduced by Latimer [32] we chose Weibull and Log-logistic for PFS and OS respectively instead of Weibull for extrapolating both PFS and OS curves like the previous study undertaken by Zhu J et al [23]. In addition a hazard ration (HR) of PFS was applied to derive the PFS curve for the gefitinib strategy in the previous study [23]. Latimer however in the resent published paper pointed out that the HR used may cause bias because of the requirement of the assumptions“that is the HR was from a related model and was constant over time [34]. Obviously the bias should be considered especially if the HR impacts the results markedly. Unfortunately the HR of PFS was one of the two most influential parameters on the basis of one-way sensitivity analyses performed by Zhu J et al [23]. In view of the above cases independent parametric models were fitted to both control and experimental groups in our study. Utility of PFS played a great role in the results not only in the resent study [23] but also in the current study. Nafees et al [28] reviewed that all toxicities (diarrhoea rash nausea and vomiting neutropenia fatigue and hair loss) were related to pulling utility down significantly. Of the toxicities rash and diarrhoea were associated with maintenance gefitinib strategy as reported the clinical trial [15]. For higher accuracy we weighted the utility of PFS according to the risks of the rash and diarrhoea which were displayed in . In particularly one point revealed by one-way sensitivity analysis () should be highlighted that the price of gefitinib would be the most significant parameter that could reduce the ICER. With the gefitinib price reduction of 20% discount the ICER decreased to $16731 per QALY gained which is very close to the WTP threshold of $16349 per QALY. Therefore if the price of gefitinib decreases >20% maintenance gefitinib therapy after the standard chemotherapy in patients with locally advanced/metastatic NSCLC may be a cost-effectiveness strategy. There are some limitations in the present study. First using Weibull and Log-logistic distribution to extrapolate the survival curves beyond the time scope of the trial was an unavoidable limitation of this process. There is not enough survival data provided by the short follow-ups of the clinical trial to compare the long-term outcomes estimated by the model. Our results should be updated when long-term survival data are available. Another important limitation is that the utility weight parameters originated from the published literature that may not reflect Chinese patients™ trait. It is an inevitable limitation of the current analysis because utilities data are not yet available for China. Fortunately opinions from Chinese oncologists suggested that quality of life of locally advanced or metastatic NSCLC patients in China should not be of significant difference from abroad patients. "

Lung_Cancer

"assessed by ROC curves. The AUC value was 0.892 Relationship between serum KLK11 levels and clinicopathologic factors The relationships between KLK11 levels and clinicopathologic factors of lung cancer patients are shown in . The serum KLK11 levels did not differ significantly with age (P?=?0.569) sex (P?=?0.505) or histology (P?=?0.713). The levels of KLK11 were significantly correlated with tumor-node-metastasis (TNM) stage (P?=?0.000) lymph node metastases (P?=?0.000) and distant metastases (P?=?0.000).The clinicopathological factors of NSCLC and the association with KLK11 levelsFactorsnKLk11 (ng/ml) P- valueAge year0.569??60622.07?±?0.77?<60762.12?±?0.66Gender0.505?Male802.16?±?0.82?Female581.99?±?0.53Histology0.713?AC782.05?±?0.85?SCC602.01?±?0.53TNM stage0.000?I“II882.51?±?0.61?III“IV501.76?±?0.63Lymph node metastases0.000?Absent682.41?±?0.64?Present701.65?±?0.57Distant metastases0.000?Absent982.38?±?0.59?Present401.89?±?0.71 AC adenocarcinoma SCC squamous cell carcinoma Association of serum KLK11 levels with survival Finally we determined whether the baseline serum concentration of KLK11 would be a prognostic marker in NSCLC. The cutoff point of 1.05 ng/ml was selected to categorize patients as KLK11-high or low. Univariate analysis showed that serum KLK11 level was significantly correlated OS (P?=?0.002) and PFS (P?=?0.009) ().Univariate and multivariate analysis of KLK11 status with regard to PFS and OSVariablesPFSOSHR95 % CI P valueHR95 % CI P valueUnivariate analysis?KLK11 (Low vs. High)0.460.25“0.820.0090.360.19“0.690.002?Age (?60 vs. <60)1.230.67“2.280.5061.180.59“2.130.792?Gender (Male vs. Female)1.320.71“1.820.7821.190.69“1.980.673?Histology (AC vs. SCC)1.830.59“2.130.7921.340.65“1.980.546?Stage (I“II vs. III“IV)1.330.65“2.210.0010.931.09“3.440.025?Lymph node metastases (absent vs. present)1.421.04“1.940.2711.770.32“1.660.347?Distant metastases (absent vs. present)1.981.03“3.010.0391.871.04“2.990.075Multivariate analysis?KLK11 (low vs. high)0.530.29-0.970.0420.480.24-0.950.037?Age (?60 vs. <60)0.980.52-1.940.8341.061.28-3.010.128?Gender (male vs. Female)1.280.67-1.890.6721.140.46-2.140.542?Histology (AC vs. SCC)1.371.04-2.330.3151.260.64-2.560.424?Stage (I“II vs. III“IV)1.250.56-2.260.0011.961.02-3.770.043?Lymph node metastases (absent vs. present)1.130.81-1.570.1481.840.33-1.720.334?Distant metastases (absent vs. present)1.440.85-1.970.0981.890.99-2.350.051 HR hazard ratio CI confidence interval In multivariate analysis high KLK11 was found to be significantly associated with a longer PFS and OS (HR 0.53 and 0.48; P?=?0.042 and P?=?0.037 respectively). Kaplan“Meier survival curves (Fig. 3) further demonstrate that lung cancer patients with high KLK11 have substantially longer PFS and OS (P?<?0.05) compared to those with low KLK11 cancer. As expected disease stage was found to be strongly associated with decreased PFS and OS in both univariate and multivariate analyses (P?<?0.05).Fig. 3Kaplan“Meier survival curves for PFS and OS in patients with KLK11-high and -low NSCLC. Log-rank test determined that the PFS and OS in high KLK11 group were significantly longer than those in the low KLK11 group (P?=?0.003; P?=?0.018) Discussion During the last few years numerous studies have been published which attempt to refine our understanding of determinants of prognosis in lung cancer by analyzing tumor-associated markers thought to be of biologic relevance in the carcinogenic process. Proteolytic enzymes of several catalytic classes have emerged as important prognostic factors in cancer [12]. Among these enzymes are many members of human tissue kallikrein family of secreted serine proteases including KLK11 a promising biomarker for lung cancer diagnosis and prognosis [1113]. In the present study serum KLK11 levels were significantly elevated in patients with lung cancer compared with control subjects making them potential adjunctive tools for diagnosis of lung cancer. Furthermore at a cutoff point of 1.05 ng/ml KLK11 had a sensitivity of 91.3 % and a specificity of 72.5 % for the prediction of lung cancer. Importantly the serum KLK11 levels did not differ significantly with age gender and histology. The levels of KLK11 were significantly correlated with TNM stage the presence of lymph node and distant metastases. Several previous studies have reported an association between kallikrein mRNA expression and cancer prognosis [14“16]. KLK5 and KLK4 have been associated with poor prognosis in ovarian cancer and KLK5 has also been shown to be associated with poor prognosis in breast cancer [1718]. In contrast KLK8 and KLK9 expression have been reported to be favorable prognosis in ovarian cancer [1920]. In addition KLK12 is reported to be an independent and favorable prognostic marker for breast cancer [21]. Sasaki et al. [11] have indicated that there is a significant correlation between decreased KLK11 mRNA expression level and poor prognosis in lung cancer. This study supports the increasing body of literature demonstrating the expression of kallikrein family gene involvement in the prognosis of human cancers. The most striking association we observed in NSCLC patients was a significant correlation between increased KLK11 level and favorable prognosis. We have demonstrated that high KLK11 was significantly associated with an increased PFS and OS in univariate analysis. This relationship was further illustrated in the Kaplan“Meier survival curves. Multivariate analysis also indicated that KLK11 was an independent indicator of PFS and OS. In our data suggest that serum KLK11 may be a useful diagnostic biomarker and shows a promising potential as prognostic marker in NSCLC patients. More large-scale prospective studies are warranted to confirm the findings. Conflicts of interest None. References 1. Chen Z Wang T Cai L Su C Zhong B Lei Y Clinicopathological significance of non-small cell lung cancer with high prevalence of Oct-4 tumor cells J Exp Clin Cancer Res 2012 31 10 10.1186/1756-9966-31-10 22300949 2. Smith RA Cokkinides V Brawley OW Cancer screening in the United States 2009: a review of current American Cancer Society guidelines and issues in cancer screening CA Cancer J Clin 2009 59 27 41 10.3322/caac.20008 19147867 3. Oguz A Unal D Tasdemir A Karahan S Aykas F Mutlu H Lack of any association between blood groups and lung cancer independent of histology Asian Pac J Cancer Prev. 2013 14 453 456 10.7314/APJCP.2013.14.1.453 23534772 4. Jemal A Siegel R Xu J Ward E Cancer statistics 2010 CA Cancer J Clin 2010 60 277 300 10.3322/caac.20073 20610543 5. Sano A Sangai T Maeda H Nakamura M Hasebe T Ochiai A Kallikrein 11 expressed in human breast cancer cells releases insulin-like growth factor through degradation of IGFBP-3 Int J Oncol 2007 30 1493 1498 17487371 6. Luo LY Shan SJ Elliott MB Soosaipillai A Diamandis EP Purification and characterization of human Kallikrein 11 a candidate prostate and ovarian cancer biomarker from seminal plasma Clin Cancer Res 2006 12 742 750 10.1158/1078-0432.CCR-05-1696 16467084 7. McIntosh MW Liu Y Drescher C Urban N Diamandis EP Validation and characterization of human Kallikrein-11 as a serum marker for diagnosis of ovarian carcinoma Clin Cancer Res 2007 13 4422 4428 10.1158/1078-0432.CCR-06-2224 17671125 8. Unal D Tasdemir A Oguz A Eroglu C Cihan YB Turak EE Is human Kallikrein-11 in gastric cancer treated with surgery and adjuvant chemoradiotherapy associated with survival? Pathol Res Pract 2013 209 779 783 10.1016/j.prp.2013.09.004 24169449 9. Yu X Tang HY Li XR He XW Xiang KM Overexpression of human kallikrein 11 is associated with poor prognosis in patients with low rectal carcinoma Med Oncol 2010 27 40 44 10.1007/s12032-009-9167-2 19184568 10. Diamandis EP Bo±o CA Scorilas A Harbeck N Dorn J Schmitt M Human kallikrein 11: an indicator of favorable prognosis in ovarian cancer patients Clin Biochem 2004 37 823 829 10.1016/j.clinbiochem.2004.04.009 15329323 11. Sasaki H Kawano O Endo K Suzuki E Haneda H Yukiue H Decreased Kallikrein 11 messenger RNA expression in lung cancer Clin Lung Cancer 2006 8 45 48 10.3816/CLC.2006.n.032 16870045 12. Lei KF Liu BY Zhang XQ Jin XL Guo Y Ye M Development of a survival prediction model for gastric cancer using serine proteases and their inhibitors Exp Ther Med 2012 3 109 116 10.1084/jem.20110399 22969854 13. Planque C Li L Zheng Y Soosaipillai A Reckamp K Chia D A multiparametric serum kallikrein panel for diagnosis of non-small cell lung carcinoma Clin Cancer Res 2008 14 1355 1362 10.1158/1078-0432.CCR-07-4117 18316555 14. Alexopoulou DK Papadopoulos IN Scorilas A Clinical significance of kallikrein-related peptidase (KLK10) mRNA expression in colorectal cancer Clin Biochem 2013 46 1453 1461 10.1016/j.clinbiochem.2013.03.002 23499583 15. Talieri M Alexopoulou DK Scorilas A Kypraios D Arnogiannaki N Devetzi M Expression analysis and clinical evaluation of kallikrein-related peptidase 10 (KLK10) in colorectal cancer Tumour Biol 2011 32 737 744 10.1007/s13277-011-0175-4 21487810 16. Patsis C Yiotakis I Scorilas A Diagnostic and prognostic significance of human kallikrein 11 (KLK11) mRNA expression levels in patients with laryngeal cancer Clin Biochem 2012 45 623 630 10.1016/j.clinbiochem.2012.03.005 22429520 17. Xi Z Kaern J Davidson B Klokk TI Risberg B Trop© C Kallikrein 4 is associated with paclitaxel resistance in ovarian cancer Gynecol Oncol 2004 94 80 85 10.1016/j.ygyno.2004.03.044 15262123 18. Yousef GM Scorilas A Kyriakopoulou LG Rendl L Diamandis M Ponzone R Human kallikrein gene 5 (KLK5) expression by quantitative PCR: an independent indicator of poor prognosis in breast cancer Clin Chem 2002 48 1241 1250 12142380 19. Kountourakis P Psyrri A Scorilas A Markakis S Kowalski D Camp RL Expression and prognostic significance of kallikrein-related peptidase 8 protein levels in advanced ovarian cancer by using automated quantitative analysis Thromb Haemost 2009 101 541 546 19277417 20. Bo±o CA Kishi T Scorilas A Harbeck N Dorn J Schmalfeldt B Human kallikrein 8 protein is a favorable prognostic marker in ovarian cancer Clin Cancer Res 2006 12 1487 1493 10.1158/1078-0432.CCR-05-2106 16533772 21. Talieri M Devetzi M Scorilas A Pappa E Tsapralis N Missitzis I Human kallikrein-related peptidase 12 (KLK12) splice variants expression in breast cancer and their clinical impact Tumour Biol 2012 33 1075 1084 10.1007/s13277-012-0347-x 22351561 9502500 8794 Clin Cancer Res Clin. Cancer Res. Clinical cancer research : an official journal of the American Association for Cancer Research 1078-0432 24423612 4136748 10.1158/1078-0432.CCR-13-2195 NIHMS556385 Article HEDGEHOG-GLI signaling inhibition suppresses tumor growth in squamous lung cancer Huang Lingling 1 Walter Vonn 2 Hayes D. Neil 2 Onaitis Mark 1 1Duke University Department of Surgery 2University of North Carolina Department of Medicine Corresponding Author: Mark Onaitis DUMC Box 3305 Durham NC 27710 mwo@duke.edu phone: 919-684-6974 fax: 919-684-8508 4 4 2014 14 1 2014 15 3 2014 15 3 2015 20 6 1566 1575 Purpose Lung squamous cell carcinoma (LSCC) currently lacks effective targeted therapies. Previous studies reported overexpression of HEDGEHOG (HH)-GLI signaling components in LSCC. However they addressed neither the tumor heterogeneity nor the requirement for HH-GLI signaling. Here we investigated the role of HH-GLI signaling in LSCC and studied the therapeutic potential of HH-GLI suppression. Experimental Design Gene expression datasets of two independent LSCC patient cohorts were analyzed to study the activation of HH-GLI signaling. Four human LSCC cell lines were examined for HH-GLI signaling components. Cell proliferation and apoptosis were assayed in these cells after blocking the HH-GLI pathway by lentiviral-shRNA knockdown or small molecule inhibitors. Xenografts in immunodeficient mice were used to determine the in vivo efficacy of GLI inhibitor GANT61. Results In both cohorts activation of HH-GLI signaling was significantly associated with the classical subtype of LSCC. In cell lines genetic knockdown of SMO produced minor effects on cell survival while GLI2 knockdown significantly reduced proliferation and induced extensive apoptosis. Consistently the SMO inhibitor GDC-0449 resulted in limited cytotoxicity in LSCC cells whereas the GLI inhibitor GANT61 was very effective. Importantly GANT61 demonstrated specific in vivo anti-tumor activity in xenograft models of GLI-positive cell lines. Conclusion Our studies demonstrate an important role for GLI2 in LSCC and suggest GLI inhibition as a novel and potent strategy to treat a subset of LSCC patients. Squamous cell lung cancer HEDGEHOG GLI J Korean Med Sci J. Korean Med. Sci JKMS Journal of Korean Medical Science 1011-8934 1598-6357 The Korean Academy of Medical Sciences 24431917 3890464 10.3346/jkms.2014.29.1.129 Original Article Medical Imaging Computed Tomography Guided Percutaneous Injection of a Mixture of Lipiodol and Methylene Blue in Rabbit Lungs: Evaluation of Localization Ability for Video-Assisted Thoracoscopic Surgery Jin Kwang Nam 1 Lee Kyung Won 2 Kim Tae Jung 2 Song Yong Sub 3 Kim Dong Il 4 1Department of Radiology Seoul Metropolitan Government-Seoul National University Boramae Medical Center Seoul Korea. 2Department of Radiology Seoul National University Bundang Hospital Seongnam Korea. 3Department of Radiology Seoul National University Hospital Seoul Korea. 4Department of Pathology Green Cross Laboratories Yongin Korea. Address for Correspondence: Kyung Won Lee MD. Department of Radiology Seoul National University Bundang Hospital 82 Gumi-ro 173beon-gil Bundang-gu Seongnam 463-707 Korea. Tel: +82.31-787-7604 Fax: +82.31-787-4011 lkwrad@radiol.snu.ac.kr 1 2014 26 12 2013 29 1 129 136 13 5 2013 22 10 2013 © 2014 The Korean Academy of Medical Sciences. 2014 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons./licenses/by-nc/3.0/) which permits unrestricted non-commercial use distribution and reproduction in any medium provided the original work is properly cited. Preoperative localization is necessary prior to video assisted thoracoscopic surgery for the detection of small or deeply located lung nodules. We compared the localization ability of a mixture of lipiodol and methylene blue (MLM) (0.6 mL 1:5) to methylene blue (0.5 mL) in rabbit lungs. CT-guided percutaneous injections were performed in 21 subjects with MLM and methylene blue. We measured the extent of staining on freshly excised lung and evaluated the subjective localization ability with 4 point scales at 6 and 24 hr after injections. For MLM radio-opacity was evaluated on the fluoroscopy. We considered score 2 (acceptable) or 3 (excellent) as appropriate for localization. The staining extent of MLM was significantly smaller than methylene blue (0.6 vs 1.0 cm P<0.001). MLM showed superior staining ability over methylene blue (2.8 vs 2.2 P=0.010). Excellent staining was achieved in 17 subjects (81%) with MLM and 8 (38%) with methylene blue (P=0.011). An acceptable or excellent radio-opacity of MLM was found in 13 subjects (62%). An appropriate localization rate of MLM was 100% with the use of the directly visible ability and radio-opacity of MLM. MLM provides a superior pulmonary localization ability over methylene blue. Lung Ethiodized Oil Methylene Blue Tomography X-Ray Computed Radiology Interventional Seoul National University College of Medicine 800-20120036 INTRODUCTION Preoperative localization is necessary for video-assisted thoracoscopic surgery (VATS) when pulmonary nodules are too small or distant from the visceral pleura to be detected (1-3). A failure to localize nodules disturbs the success of the thoracoscopic resection and leads to conversion to thoracotomy (4 5). There are two kinds of localizing procedures: marking with thoracoscopically directly visible "

Lung_Cancer

"Rabbit anti-MOR antibody was purchased from GeneTex (San Antonio TX). Rabbit anti-EGFR rabbit anti-phosphotyrosine-EGFR (pY845 pY992 pY1045 pY1068) rabbit anti-Grb-2 rabbit anti-Gab-1 rabbit anti-phosphotyrosine-Gab-1 (pY307 pY627) rabbit anti-Src rabbit anti-phosphotyrosine-Src (pY416) rabbit anti-p85 PI3 kinase rabbit anti-p55 PI3 kinase rabbit anti-phosphotyrosine-p85/p55 PI3 kinase (pY458 pY199) rabbit anti-STAT3 rabbit anti-phosphotyrosine-STAT3 (pY705) rabbit anti-vimentin rabbit anti-ZO-1 rabbit anti-claudin-1 rabbit anti-Snail and rabbit anti-Slug antibodies were purchased from Cell Signaling Technologies (Danvers MA). Mouse anti-?-actin antibody was purchased from Sigma (St. Louis MO). Secondary horseradish peroxidase-labeled antibodies were purchased from Amersham Biosciences (Piscataway NJ). N-methylnaltrexone bromide or methylnaltrexone was purchased from Mallinckrodt Specialty Chemicals (Phillipsburg NJ). The PI3 kinase inhibitor LY294002 Akt Inhibitor X the Src family kinase inhibitor PP2 and the STAT3 inhibitor Stattic were purchased from EMD Biosciences (Billerica MA). Immunoblotting Immunoblotting was performed as we have previously described. Cellular materials from treated or untreated human NSCLC cells were incubated with lysis buffer (50 mM HEPES (pH 7.5) 150 mM NaCl 20 mM MgCl2 1% Triton X-100 0.1% SDS 0.4 mM Na3VO4 40 mM NaF 50 µM okadaic acid 0.2 mM phenylmethylsulfonyl fluoride 1?250 dilution of Calbiochem protease inhibitor mixture 3). The samples were then run on SDS-PAGE in 4“15% polyacrylamide gels transferred onto Immobilon„¢ membranes and developed with specific primary and secondary antibodies. Visualization of immunoreactive bands was achieved using enhanced chemiluminescence (Amersham Biosciences Piscataway NJ). In some instances immunoreactive bands were quantitated using computer-assisted densitometry. Small Interfering RNA Transfection in Human NSCLC Cells Stable Control and either MOR Gab-1 or Src siRNA (Santa Cruz Biotechnology Santa Cruz CA) were transfected into H358 cells as we have previously described [12]. Cells (?40% confluent) were serum-starved for 1 hour followed by incubated with siRNA for 6 hours in serum-free media. Serum-containing media was then added (10% serum final concentration) for 42 hours. Inhibition of protein expression was confirmed by immunoblot analysis with specific antibodies. Stable Control and MOR Small Hairpin RNA Transfection in Human NSCLC Cells Stable Control and MOR shRNA (Santa Cruz Biotechnology Santa Cruz CA) were stably transfected into H358 cells as we have previously described [12]. Cells (?40% confluent) were serum-starved for 1 hour followed by incubated with shRNA for 6 hours in serum-free media. Serum-containing media was then added (10% serum final concentration) for 42 hours and puromycin selection reagent was added. Inhibition of protein expression was confirmed by immunoblot analysis with anti-MOR antibody (GeneTex San Antonio TX). Stable Vector Control and MOR1 Overexpression in Human NSCLC Cells Myc-DDK-tagged ORF clone of Homo sapiens opioid receptor mu 1 (OPRM1) transcript variant MOR-1 (OriGene Technologies Inc MD) was amplified using Platinum Taq DNA polymerase high fidelity enzyme (Invitrogen CA) and subsequently cloned into a pCR8/GW/Topo entry vector (Invitrogen CA) according to manufacturer's instructions. Plasmid DNA was extracted from selected clones by QIAquick Plasmid Mini kit (Qiagen CA). ORF integrity and fragment orientation were confirmed by sequencing. The MOR1-Myc fusion product was then transferred to pcDNA3.2/v5 DEST vector (Invitrogen CA) by LR reaction. The resulting construct (pcDNA3.2-MOR1-Myc) was transfected into H358 cells using FuGENE HD„¢ as the transfection reagent (Roche Applied Sciences) according to the protocol provided by Roche as we have previously described. Cells (?40% confluent) were serum-starved for 1 hour followed by incubation with pcDNA3.2-MOR1-Myc for 6 hours in serum-free media. Serum-containing media was then added (10% serum final concentration) for 42 hours and neomycin selection reagent was added. Overexpression was confirmed by immunoblot analysis with anti-MOR antibody (GeneTex San Antonio TX). Human NSCLC Cell Proliferation Assay Measurement of in vitro NSCLC cell growth was performed as we have previously described. Control or siRNA pretreated H358 cells (5—103 cells/well) were incubated with 0.2 ml of serum-free media containing either vehicle (control) methylnaltrexone (MNTX 100 nM) the PI3 kinase inhibitor LY294002 (10 uM) Akt Inhibitor X (5 uM) the Src family kinase inhibitor PP2 (100 nM) or the STAT3 inhibitor Stattic (10 uM) for 72 h at 37°C in 5%CO2/95% air in 96-well culture plates. The in vitro cell proliferation assay was analyzed by measuring increases in cell number using the CellTiter96„¢ MTS assay (Promega Madison WI) and read at 492 nm. Each assay was set up in triplicate and repeated at least five times. Human NSCLC Cell Migration Assay Measurement of in vitro NSCLC cell migration was performed as we have previously described. Twenty-four transwell units with 8 µM pore size (Millipore Billerica MA) were used for monitoring in vitro cell migration as we have previously described [12]. Control or siRNA pretreated H358 cells (5—103 cells/well) were incubated with 0.2 ml of serum-free media containing either vehicle (control) methylnaltrexone (MNTX 100 nM) the PI3 kinase inhibitor LY294002 (10 uM) Akt Inhibitor X (5 uM) the Src family kinase inhibitor PP2 (100 nM) or the STAT3 inhibitor Stattic (10 uM) were plated on the upper chamber and media with serum was added to the lower chamber. Cells were allowed to migrate through the pores for 18 hours. Cells from the upper and lower chamber were quantitated using the CellTiter96„¢ MTS assay (Promega San Luis Obispo CA) and read at 492 nm. % migration was defined as the # of cells in the lower chamber divided by the number of cells in both the upper and lower chamber. Each assay was set up in triplicate and repeated at least five times. Statistical Analysis Results are expressed as mean ± standard deviation of three independent experiments. For data analysis experimental samples were compared to controls by unpaired Student's t-test. For multiple-group comparisons a one-way variance analysis (ANOVA) and post hoc multiple comparisons tests were used. Differences between groups were considered statistically significant when P value was less than 0.05. All statistical analyses were performed using the GraphPad Prism program (GraphPad Software Inc. USA). Results Our results in indicate that inhibiting MOR with the peripheral MOR antagonist MNTX attenuates EGF-induced proliferation (-A) and migration (-B) of human H358 NSCLC cells in a dose-dependent manner. These data suggest a link between MOR and the EGFR in H358 cells. To mechanistically evaluate the role of MOR on EGF-induced EGFR dynamics we treated H358 cells with EGF at various times and immunoprecipitated the EGFR to determine potential MOR association. -A demonstrates that EGF induces a complex formation between the EGFR and MOR which peaks at 5 to 15 minutes after EFG challenge. Based on our results that a MOR/EGFR complex can occur with EGF stimulation of H358 cells we next examined whether MOR can regulate EGFR phosphorylation. Utilizing a panel of anti-phospho-EGFR antibodies -B demonstrates that pretreatment of H358 human NSCLC cells with the peripheral MOR antagonist MNTX failed to attenuate EGF-induced EGFR tyrosine phosphorylation. .0091577.g001 The peripheral mu opioid receptor antagonist methylnaltrexone (MNTX) inhibits epidermal growth factor (EGF)-induced proliferation and migration of human lung cancer cells in a dose-dependent manner. Panel A: Human H358 non-small cell lung cancer (NSCLC) cells were analyzed for methylnaltrexone (MNTX) inhibition of EGF-mediated proliferation using a MTS proliferation assay. Cells were growth in the presence of 100 ng/ml EGF and/or 0“250 nM MNTX for 72 hours. MNTX There is a statistically significant difference (p<0.05 indicated by an asterisks) between control and MNTX (1050100 250 nM) treatment with n?=?3 per condition and error bars?=?standard deviation. See the Methods section for experimental details. Panel B: Human H358 non-small cell lung cancer (NSCLC) cells were analyzed for methylnaltrexone (MNTX) inhibition of EGF-mediated migration using a transwell assay (8 uM pore size). Cells were allowed to migrate in the presence of 100 ng/ml EGF and/or 0“250 nM MNTX for 18 hours. There is a statistically significant difference (p<0.05 indicated by an asterisks) between control and MNTX (50100 250 nM) treatment with n?=?3 per condition and error bars?=?standard deviation. See the Methods section for experimental details. .0091577.g002 The mu opioid receptor (MOR) is recruited to the EGF receptor with EGF stimulation but does not regulate EGF receptor phosphorylation. Panel A: Human H358 non-small cell lung cancer (NSCLC) cells were treated with no (control) or 100 ng/ml EGF for 515 or 30 minutes. Cell lysates were obtained and immunoprecipitated with anti-EGFR antibody. Immunoblots were performed on total cell lysates (left) and immunoprecipitated material (right) using anti-MOR (a) and anti-EGFR (b) antibodies. The mu opioid receptor is recruited to the EGFR with EGF stimulation. Panel B: Human H358 non-small cell lung cancer (NSCLC) cells were either untreated (control) or treated with 100 nM MNTX alone 10 ng/ml EGF for 5 15 or 30 minutes or 100 nM MNTX and 10 ng/ml EGF for 515 or 30 minutes. Cell lysates were obtained and immunoblotted using anti-pY845 EGFR (a) anti-pY992 EGFR (b) anti-pY1045 EGFR (c) anti-pY1068 EGFR (d) anti-EGFR (e) and anti-actin (e) antibodies. MNTX does not inhibit EGF-induced EGFR tyrosine phosphorylation. We next examined whether MOR can regulate EGFR downstream signaling molecules. We first examined the adaptor protein Growth factor receptor-bound protein 2 (Grb-2)"

Lung_Cancer

"Although there has limited evidence to confirm the rest genes to be molecular targets for targeted therapy these genes provide useful clues for targeted therapy. By gene ontology analysis the biomarkers inferred in gene-subtype lower logic relationships were significantly enriched in biological processes of ˜cell adhesion™ (GO: 0007155) and ˜epidermis development™ (GO: 0008544). The identified biological processes had nonrandom probability values and enrichment scores and they were also significant biological processes which were important for tumorigenesis of NSCLC. The discovered biomarkers in the biological processes ˜cell adhesion™ and ˜epidermis development™ (i.e. DST CLCA2 DSG3 PKP1 FAT2 DSC3 PVRL1 KRT5 GJB5 BNC1) account for more than a half of all discovered biomarkers. The expression of these genes were all sufficient and necessary conditions of the presence of SCC as well as the absence of AC. It indicates that genes annotated to epidermis development and cell adhesion may be differently regulated between AC and SCC. In previous research several genes involved in ˜cell adhesion™ as well as ˜epidermis development™ were significantly up-regulated in SCC compared to normal and AC [26] which is in accordance with our results. The majority of cell adhesion genes (predominantly desmosomal genes) and epidermis development genes have been found to be significantly up-regulated in SCC compared to normal tissue and the AC subtype. For example desmosomal genes (DSC3 and DSG3) and epidermis development genes (KRT5) were increased in SCC compared to the AC subtype. Our results strengthen the importance of ˜cell adhesion™ and ˜epidermis development™ in distinguishing AC from SCC. It indicates that cell adhesion genes and epidermis development genes play central roles in the drug delivery and are promising targets for novel therapies. In biomarkers identified in this paper could be used to classify patients for the treatment of NSCLC. A classification based on the discovered biomarkers could help to supply potential information in clinical decision making. The identified gene-subtype logic relationships and GO terms may extend perception to disease mechanisms for NSCLC. In addition the targeted therapy agents may also be designed to interfere with the discovered biomarkers. However several biomarkers and GO terms have been less well understood yet which needs further experimental research. Materials and Methods Data source and data processing We use the specimens of GSE10245 (a Gene Expression Omnibus accession number for microarray data) GSE37745 GSE18842 and GSE28571 to form a microarray expression data which are available from National Center for Biotechnology Information (NCBI http://www.ncbi.nlm.nih.gov/). Each specimen is annotated with a phenotype property (AC SCC and Normal) (). The microarray expression data (see Appendix S2) contains the expression data of probes in specimens. The microarray expression data is converted into a binary probe data using the Microarray Suite 5 (Mas5) algorithm [42]. The Mas5 algorithm generates a p-value which assesses the reliability of the expression level for each probe and a detection call which is a three-valued discrete data of a p-value. Specifically if a p-value is less than then the detection call is ˜Present™; if a p-value is greater than and less than then the detection call is ˜Marginal™; if a p-value is greater than then the detection call is ˜Absent™. Probes are flagged ˜Marginal™ or ˜Absent™ when the detection of probes is not considered to be significantly reliable. Hence it is reasonable to consider that the probes with flag ˜Marginal™ or ˜Absent™ are not significantly detected. In this work we turn ˜Marginal™ and ˜Absent™ flags to ˜™s and turn ˜Present™ flags to ˜™s. A ˜0™ in the th row and th column of the binary probe data mean the th probe is not detected in the th specimen while a ˜1™ indicates the probe is detected. Once converted the binary probe data is supplemented with an additional phenotype profile data. The phenotype profile data has three rows and columns. The st nd and rd rows correspond to AC SCC and Normal specimens respectively (Appendix S2). The phenotype profile data represents the properties of phenotypes where a ˜1™ in the th row and th column of the phenotype profile data means the th specimen belongs to the th phenotype while a ˜0™ means not. The probes are associated to genes according to the information of GPL570 (a microarray chip)(see Table S7). According to the number of genes that a probe detects probes can be classified into three categories: probes detecting a single gene probes detecting more than one gene and probes detecting no genes. In Table S7 there are probes associated to a single gene probes associated to more than one gene and probes associated to no genes. We are focused on the probes associated to a single gene. The binary probe data contains rows describing the detection patterns of probes. Current relationship-inference method Calculating uncertainty coefficient The vector describes the vector via either Type or Type lower logic function (see ) i.e. and constitute a logic pair. A logic combination of the vectors and describes the vector via one of the eight higher logic functions (see ) i.e. and compose a logic triplet. Uncertainty coefficient for a vector pair or a vector triplet is a measure to describe to what extent a vector or a combination of two vectors predicts another vector [22]. .0094644.t003 Lower logic function of vector . Type Symbol Lower logic function Logic statement The value of is iff the value of is The value of is iff the value of is ˜™ denotes the function symbol of type of lower logic relationships where and represents the sign for the lower logic relationships. .0094644.t004 Higher logic function of vectors and . Type Symbol Higher logic function Logic statement The value of is iff the values of both and are The value of is iff the value of is or that of is The value of is iff the value of or that of is The value of is iff the values of both and are The value of is iff the value of is and that of is The value of is iff the value of is and that of is The value of is iff the value of is or that of is The value of is iff the value of is or that of is The value of is iff either the value of or that of is The value of is iff the values of both and are or ˜™ denotes function symbol of type of higher logic relationships where and represents the sign for the higher logic relationships. The value of represents how well for the vector is described by the vector under a lower logic function where and is the symbol for lower logic functions. The value of is calculated as follows (Matlab codes available in Appendix S3):(1)where is the entropy of and where is the probability of occurrence of and is either or . is the entropy of the vector . is the joint entropy of and and where is the probability of occurrence of . The uncertainty coefficient for given which is denoted by is the maximum of and . Referring from and we got . The value of ranges from to where means that is independent of and means that is completely determined by . We calculate the degree to which the logic combination of the vectors and (e.g. ) describes a third vector as follows (Matlab codes available in Appendix S3):(2)where ; and are the entropy of and respectively; is the symbol for higher logic functions; is the joint entropy of and . As similar with we have . The uncertainty coefficient for predicted by a logic combination of and is denoted by . is equal to the maximum of the following five values: . The value of ranges from to . A well known measure the confidence is used to select the greatest possible rules by which probes related with phenotypes from the set of all possible rules [43]. Here the set of all possible rules are lower/higher logic functions corresponding to the maximum lower/higher uncertainty coefficients. Suppose the vectors and follow the lower logic function where . The confidence of is calculated as: where and refer to the joint probability of occurrence of and for the vectors and respectively. Suppose vectors and follow the rule where then the confidence of the rule () is also the ratio of to and and refer to the joint probability of occurrence of and for the vector and vector . We calculate the confidence for two lower (or higher) logic functions with the same value of (or ). The higher the confidence of a logic function the higher the probability that vectors follow the logic type corresponding to the logic function. The value of measures how well approximates a sufficient condition for and the value of measures how well the combination of and approximates a sufficient condition for . We improve the logic analysis by taking the reverse uncertainty coefficients into consideration. That is given the and to be the final lower and higher logic functions respectively we calculate the value of and as follows (Matlab codes available in Appendix S3):(3)where is either or and and are the same as those in e.q (1).(4)where ; and are the entropy of vector and respectively; is the joint entropy of vector and . Calculating random uncertainty coefficient Let be the histogram of the vector . Suppose is the set of distinct elements of . For each is the number of times appears in where and is the number of elements in [44]. Given the vectors and the random uncertainty coefficient and is calculated using the following steps: Generate random vectors and . and maintain the same distribution of the vectors and (i.e. ). Compute where is the uncertainty coefficient for given in a trial. Compute where is the uncertainty coefficient for given in a trial. The calculation of and involves the following three steps: Generate random vectors and maintaining the individual distribution and pairwise distribution. The vector retains the position of its elements unchangeably. Note that could determine and . We generate and maintaining and . Compute where is the uncertainty coefficient for given the combination of and in a trial. Compute where is the uncertainty coefficient for the combination of and given in a trial. Identification of probe-phenotype lower and higher logic relationships Thresholds are defined here to separate lower or higher logic relationships from logic pairwise or triplet combinations. Let and be the thresholds of lower and higher logic relationships respectively. We calculate the random uncertainty coefficients of all probe-phenotype pairwise (i.e. a probe and a phenotype) and triplet combinations (i.e. a probe pair and a phenotype). is the maximum uncertainty coefficient of all probe-phenotype pairwise combinations and is the maximum uncertainty coefficient of all probe-phenotype triplet combinations. If the values of both and are higher than then approximates a necessary and sufficient condition for . There exists a lower logic relationship between and . If the values of both and are higher than and there are no lower logic relationships between either or and then the logic combination of vector and approximates a necessary and sufficient condition for . There is a higher logic relationship between the combination of and and . Statistical analysis A p-value is defined as a measure to reflect how well vectors related in the form of discovered logic pairwise or triplet combinations compared to chance relations. Here an actual uncertainty coefficient is compared to the random one in a random trial. The p-value of a discovered logic pairwise or triplet combination is equal to the number of random trials in which either one of the two random uncertainty coefficients of pairwise or triplet combination of random vectors in both directions is higher than the actual one divided by the total number of random trails. Specifically we compare and to the distribution of and where and are the random vectors of and . For each pair of and we calculate the random uncertainty coefficients and in random trails. We have the p-value of the discovered logic pairwise combination: where means the number of random trials in which either one of the following two items and is tenable. Similarly the p-value of the discovered logic triplet combination is where means the number of random trials in which either or is tenable. False discovery rate In order to evaluate a global significance value of the actual discovered logic pairwise or triplet combinations we measure a false discovery rate (FDR) [45]. Given the threshold of lower logic relationships we estimate the number of discovered random logic pairwise combinations with the mean uncertainty coefficients larger than the threshold by chance. We generate random independent data and extract discovered random logic pairwise combinations from each random data. The estimated number of false discovered logic pairwise combinations (denoted as ) is calculated as the mean number of discovered random logic pairwise combinations obtained from these random independent data. If is the number of actual discovered logic pairwise combinations then is a simple estimated positive FDR for the given threshold. We can scan all probe-phenotype pairwise combinations but it take too much time to scan all triplet combinations. Therefore we randomly select a fixed number of triplet combinations (e.g. of all possible triplet combinations) and extract higher logic relationships with respect to actual and random (denoted as and ) respectively. This process is repeated for times and the FDR is the mean value of . The Matlab codes are available in Appendix S3. Cross validation In a random trial a fixed number of columns corresponding to each phenotype are selected from the original probe binary data and phenotype data to form the random probe binary data and random phenotype data. We check whether a logic relationship could be obtained in the random trial. The above processes are repeated for times where represents the number of all random trials."

Lung_Cancer

"But PAX6 plays an oncogenic role in pancreatic cancer and retinoblastoma [4] [22].In pancreatic adenocarcinoma and pancreatic cancer cell lines down-regulation of PAX6 by specific siRNA leads to a decline in cell growth and cell apoptosis [12]. Methylation of PAX6-promoters is increased in early bladder cancer and methylated PAX6-promoters could be a represent biomarker for this disease [25]. And the suppression of PAX6 mRNA expression resulted in an inhibited growth and an increased apoptosis of cultured human retinoblastoma cells [26]. Our findings also reveal that PAX6 implicates an oncogenic function in lung cancer. In our results PAX6 mRNA was highly expressed in both lung cancer tissues and lung cancer cell lines. A549 and H1299 cell growth was inhibited by specific PAX6 shRNA. Suppression of PAX6 expression led to decreased cell growth and colony formation as well as anchorage-independent colony formation. But our findings indicate that cell apoptosis was not affected by the inhibition of PAX6 (data not shown). And cell migration was also not affected by the suppression of PAX6 mRNA (data not shown). PAX6 is cancer-dependent and has different signaling pathways in different tumors [13] [20]“[24] [26]. In HeLa cells PAX6 regulates cell-cycle progression by eliciting the expression of human RFPL1 (hRFPL1) which down-regulates cyclin B1 and Cdc2 expression and leads to the accumulation of cells in G2-M phase [27]. We also focused on the role of PAX6 in regulating cell cycle progression in lung cancer. In our cell cycle analysis cyclin D1 was suppressed in A549 PAX6 KD and H1299 PAX6 KD cells. It indicated that PAX6 expression promoted cell cycle progression by transitioning cells from G1 to S phase. Consistent with these findings cell cycle analysis showed a significant reduction of G0/G1 arrest and a significant induction of G2/M arrest in PAX6 overexpression human retinoblastoma cells [28]. Cyclin D1-CDK4 and cyclin D1-CDK6 complexes in the early to mid-G1 phase phosphorylate and inactivate pRB [29] [30]. Our findings in this study implicate that the pRB S780 phosphorylation level was weakened when PAX6 expression was inhibited. Thus we demonstrated that PAX6 increased the expression of cyclin D1 and enhanced cell growth by promoting the G1-S transition. Mitogen-induced Ras signaling promotes transcription of the cyclin D1 gene and it depends on the MAPK signal pathway [31]. Our findings indicate that inhibition of PAX6 decreases the phosphorylation level of ERK1/2 and p38. These studies suggest that PAX6 regulates cell G1/S progression via MAPK signal pathway in lung cancer cells. However the regulatory mechanism of PAX6 in lung cancer is still unclear. A recent study showed that PAX6 promotes cell growth by activating the MET tyrosine kinase receptor gene in pancreatic carcinoma [13]. In lung cancer cells ERK1/2 signal pathway is involved in the MET pathway [32]. Our finding indicated that ERK1/2 was activated by PAX6 expression. So that we suppose that PAX6 activates MAPK signaling and promotes cell cycle progression via MET gene transcription in lung cancer. PAX6 is primarily expressed during embryogenesis; little or no PAX6 protein is detected in adult tissues [3]. As PAX6 is frequently expressed in tumors [9] we determined the PAX6 level in primary lung cancer tissues. The PAX6 expression level in matched adjacent tissues was measured as a control. Similar to pancreatic tumors PAX6 expression was stronger in lung cancer tissues than in adjacent tissues. The cancer-to-adjacent nontumorous tissue ratio of PAX6 mRNA expression for each individual was calculated. Only 3 ratios were less than 1 and most of the ratios were much more than 100. All these findings demonstrated that PAX6 functioned as an oncogenic factor in lung cancer. Conclusions In this study we report that increased expression of PAX6 was noted in primary lung cancer tissues. PAX6 promoted cell growth by activating MAPK signalling and accelerating cell cycle progression. Moreover PAX6 regulated G1-S progression by inducing cyclin D1 expression and pRB phosphorylation. Our data suggests that PAX6 is a new potential target in lung cancer. The authors would like to thank Meng Gu for assistance with samples collection. References 1 JemalA BrayF (2011) Center MM Ferlay J Ward E et al (2011) Global cancer statistics. CA Cancer J Clin61: 69“9021296855 2 PaoW GirardN (2011) New driver mutations in non-small-cell lung cancer. Lancet Oncol12: 175“18021277552 3 OsumiN ShinoharaH Numayama-TsurutaK MaekawaM (2008) Concise review: Pax6 transcription factor contributes to both embryonic and adult neurogenesis as a multifunctional regulator. Stem Cells26: 1663“167218467663 4 SellheyerK (2011) Basal cell carcinoma: cell of origin cancer stem cell hypothesis and stem cell markers. Br J Dermatol164: 696“71121128907 5 MaB LeiX GuanY MouLS YuanYF et al (2011) Maintenance of retinal cancer stem cell-like properties through long-term serum-free culture from human retinoblastoma. Oncol Rep26: 135“14321573498 6 LangD MascarenhasJB PowellSK HalegouaJ NelsonM et al (2008) PAX6 is expressed in pancreatic adenocarcinoma and is downregulated during induction of terminal differentiation. Mol Carcinog47: 148“15617849422 7 ShahiMH SchiapparelliP AfzalM SinhaS ReyJA et al (2011) Expression and epigenetic modulation of sonic hedgehog-GLI1 pathway genes in neuroblastoma cell lines and tumors. Tumour Biol32: 113“12720830616 8 YoshizakiK OsumiN (2010) [Molecular mechanism and mental function of postnatal neurogenesis]. Brain Nerve62: 1315“132221139184 9 MuratovskaA ZhouC HeS GoodyerP EcclesMR (2003) Paired-Box genes are frequently expressed in cancer and often required for cancer cell survival. Oncogene22: 7989“799712970747 10 ZhongX LiY PengF HuangB LinJ et al (2007) Identification of tumorigenic retinal stem-like cells in human solid retinoblastomas. Int J Cancer121: 2125“213117565741 11 BerkhoutM NagtegaalID CornelissenSJ DekkersMM van de MolengraftFJ et al (2007) Chromosomal and methylation alterations in sporadic and familial adenomatous polyposis-related duodenal carcinomas. Mod Pathol20: 1253“126217873900 12 MascarenhasJB YoungKP LittlejohnEL YooBK SalgiaR et al (2009) PAX6 is expressed in pancreatic cancer and actively participates in cancer progression through activation of the MET tyrosine kinase receptor gene. J Biol Chem284: 27524“2753219651775 13 SalgiaR HensingT CampbellN SalamaA MaitlandM et al (2011) Personalized treatment of lung cancer. Semin Oncol38: 274“28321421117 14 Kanteti R Nallasura V Wang C Hasan R Loganathan S et al. (2007) The role of pax transcription factors in lung carcinogenesis: relationship to c-Met receptor tyrosine kinase: P2-126. Journal of Thoracic Oncology 2: S541 510.1097/1001.JTO.0000283590.0000209867.0000283563. 15 EwenME (2000) Where the cell cycle and histones meet. Genes Dev14: 2265“227010995383 16 KleinEA AssoianRK (2008) Transcriptional regulation of the cyclin D1 gene at a glance. J Cell Sci121: 3853“385719020303 17 GladdenAB DiehlJA (2003) Cell cycle progression without cyclin E/CDK2: breaking down the walls of dogma. Cancer Cell4: 160“16214522248 18 KnudsenES WangJY (1997) Dual mechanisms for the inhibition of E2F binding to RB by cyclin-dependent kinase-mediated RB phosphorylation. Mol Cell Biol17: 5771“57839315635 19 TalarminH RescanC CariouS GlaiseD ZanninelliG et al (1999) The mitogen-activated protein kinase kinase/extracellular signal-regulated kinase cascade activation is a key signalling pathway involved in the regulation of G(1) phase progression in proliferating hepatocytes. Mol Cell Biol19: 6003“601110454547 20 ZhouYH WuX TanF ShiYX GlassT et al (2005) PAX6 suppresses growth of human glioblastoma cells. J Neurooncol71: 223“22915735909 21 ShyrCR TsaiMY YehS KangHY ChangYC et al (2010) Tumor suppressor PAX6 functions as androgen receptor co-repressor to inhibit prostate cancer growth. Prostate70: 190“19919790232 22 ZhouYH TanF HessKR YungWK (2003) The expression of PAX6 PTEN vascular endothelial growth factor and epidermal growth factor receptor in gliomas: relationship to tumor grade and survival. Clin Cancer Res9: 3369“337512960124 23 ZhouYH HuY MayesD SiegelE KimJG et al (2010) PAX6 suppression of glioma angiogenesis and the expression of vascular endothelial growth factor A. J Neurooncol. 96: 191“200 24 MayesDA HuY TengY SiegelE WuX et al (2006) PAX6 suppresses the invasiveness of glioblastoma cells and the expression of the matrix metalloproteinase-2 gene. Cancer Res66: 9809“981717047041 25 HellwinkelOJ KediaM IsbarnH BudausL FriedrichMG (2008) Methylation of the TPEF- and PAX6-promoters is increased in early bladder cancer and in normal mucosa adjacent to pTa tumours. BJU Int101: 753“75718070176 26 BaiSW LiB ZhangH JonasJB ZhaoBW et al (2011) Pax6 regulates proliferation and apoptosis of human retinoblastoma cells. Invest Ophthalmol Vis Sci52: 4560“457021169528 27 BonnefontJ LaforgeT PlastreO BeckB SorceS et al (2011) Primate-specific RFPL1 gene controls cell-cycle progression through cyclin B1/Cdc2 degradation. Cell Death Differ18: 293“30320725088 28 LiL LiB ZhangH BaiS WangY et al (2011) Lentiviral vector-mediated PAX6 overexpression promotes growth and inhibits apoptosis of human retinoblastoma cells. Invest Ophthalmol Vis Sci52: 8393“840021948554 29 SherrCJ (1993) Mammalian G1 cyclins. Cell73: 1059“10658513492 30 WeinbergRA (1995) The retinoblastoma protein and cell cycle control. Cell81: 323“3307736585 31 SherrCJ RobertsJM (1999) CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev13: 1501“151210385618 32 MaPC TretiakovaMS NallasuraV JagadeeswaranR HusainAN et al (2007) Downstream signalling and specific inhibition of c-MET/HGF pathway in small cell lung cancer: implications for tumour invasion. Br J Cancer97: 368“37717667909 8406521 6573 Pharm Res Pharm. Res. Pharmaceutical research 0724-8741 1573-904X 23884568 3864614 10.1007/s11095-013-1134-0 NIHMS509609 Article Pharmacological Modulation of Cytotoxicity and Cellular Uptake of Anti-cancer Drugs by PDE5 Inhibitors in Lung Cancer Cells LI QING 1 2 SHU YAN 1 * 1Department of Pharmaceutical Sciences School of Pharmacy University of Maryland at Baltimore Maryland USA 2Institute of Clinical Pharmacology Central South University Hunan 410078 China *Correspondence to: Yan Shu M.D. Ph.D. Department of Pharmaceutical Sciences School of Pharmacy University of Maryland at Baltimore 20 Penn Street HSFII Room 555 Baltimore MD 21201 USA Phone: +01 410“706“7358 Fax: +01 410“706“7015 yshu@rx.umaryland.edu 28 8 2013 25 7 2013 1 2014 01 1 2015 31 1 10.1007/s11095-013-1134-0 Purpose Previous research has led to the recognition of a cGMP signaling pathway governing drug transport. This study is to investigate whether inhibitors of phosphodiesterase type 5 (PDE5) which increase intracellular cGMP levels modulate the cytotoxicity and uptake of anti-cancer drugs in cancer cells. Methods The experiments were conducted with and without PDE5 inhibitors: dipyridamole vardenafil and/or sildenafil. The cytotoxicity of doxorubicin cisplatin and oxaliplatin was determined in multiple cancer cell lines derived from different tissues. The cellular uptake of structurally diverse compounds was further examined in lung cancer cells with and without various endocytotic inhibitors. The tumor accumulation and the anti-tumor effect of trastuzumab were examined in a lung cancer xenograft mouse model. Results Dipyridamole could modulate the cytotoxicity of doxorubicin cisplatin and oxaliplatin in cancer cells. Particularly PDE5 inhibitors increased cellular uptake of structurally diverse compounds into lung cancer cells both in vitro and in vivo. The effect of vardenafil on drug uptake could be blocked by endocytotic inhibitors. The growth of lung cancer xenograft in nude mice was significantly suppressed by addition of vardenafil to trastuzumab treatment. Conclusion PDE5 inhibitors may increase the efficacy of anti-cancer drugs by increasing endocytosis-mediated cellular drug uptake and thus serve as adjuvant therapy for certain cancers such as lung cancer. phosphodiesterase type 5 (PDE5) inhibitor doxorubicin trastuzumab endocytosis lung cancer National Institute of General Medical Sciences : NIGMS R01 GM099742 || GM BMC Pulm Med BMC Pulm Med BMC Pulmonary Medicine 1471-2466 BioMed Central 24885608 4027997 1471-2466-14-83 10.1186/1471-2466-14-83 Case Report Extraordinary response to crizotinib in a woman with squamous cell lung cancer after two courses of failed chemotherapy Wang Qiushi 1 wqsdpbl@163.com He Yong 2 heyong8998@126.com Yang Xin 1 32317432@qq.com Wang Yubo 2 wangyubo_110@163.com Xiao Hualiang 1 dpbl_xhl@126.com 1Department of Pathology Daping Hospital and Research Institute of Surgery Third Military Medical University Chongqing 400042 China 2Department of Respiration Daping Hospital and Research Institute of Surgery Third Military Medical University Chongqing 400042 China 2014 15 5 2014 14 83 83 20 3 2014 8 5 2014 Copyright © 2014 Wang et al.; licensee BioMed Central Ltd. 2014 Wang et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0) which permits unrestricted use distribution and reproduction in any medium provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article unless otherwise stated. Background The discovery of the fusion gene echinodermmicro tubule associated proteinlike 4-anaplastic lymphoma kinase EML4-ALK in patients with non-small-cell lung cancer has led to the remarkable development of anaplastic lymphoma kinase inhibitors such as crizotinib. Consequently the clinical outcomes of these patients have improved dramatically. Herein we report the case of a woman with ALK gene translocation-squamous cell lung cancer who experienced a remarkable tumor response to crizotinib after two courses of failed chemotherapy. Case presentation A 55-year-old Chinese woman was diagnosed with cervical lymph node metastatic squamous carcinoma. Chest computed tomography scan showed the primary tumor in the lower lobe of the right lung. The patient had received two successive courses of first-line chemotherapy without tumor response. Tumor cells were negative for wild-type of epidermal growth factor receptor/K-RAS variants; thus she was not eligible for tyrosine kinase inhibitor therapy. Unfortunately increased levels of interleukin-6 and carcinoembryonic antigen and computed tomography scan results indicated cancer progression. Once crizotinib was approved by the China Food and Drug Administration and the ALK gene translocation was identified in tumor cells by fluorescent in situ hybridization the patient commenced treatment with crizotinib. Remarkably the tumor response to crizotinib was classified as partial response after only 26 days of treatment commencement. The partial response status has been maintained to date (23 weeks). Conclusion Considering this remarkable response to crizotinib we can safely conclude that patients with squamous cell lung cancer should have the option of undergoing ALK testing to determine if there is indication for crizotinib treatment even after they have failed chemotherapy. ALK Crizotinib Squamous cell lung cancer Chemotherapy Background Treatment of EML4-ALK fusion-positive non-small-cell lung cancer (NSCLC) with the anaplastic lymphoma kinase (ALK)-targeted agent crizotinib offers significant improvement of clinical outcomes [1]. Herein we report the successful case of a patient with squamous cell lung cancer and ALK gene translocation that experienced a remarkable response to crizotinib treatment after two courses of failed chemotherapy. Case presentation A 55-year-old woman presented in May 2013 with cough sputum and annihilation after activities of daily living for more than 20 days. She had no history of smoking or alcoholism but had undergone total hysterectomy because of hysteromyoma in 2010. She was diagnosed with hypertension three years earlier. On physical examination an enlarged right cervical lymph node was palpated. Chest computed tomography (CT) scan (Figure 1A) indicated the presence of a mass in the lower lobe of the right lung and mediastinal lymph node enlargement. The patient was then accepted and treated by the Department of Respiration for lung cancer stage IV with cervical lymph node metastasis (T4N3M1). Figure 1 Chest computed tomography (CT) scans. Before the first chemotherapy treatment (May 2013) (A). After the second course of chemotherapy (B). After 26 days (C) and 11 weeks (D) of crizotinib treatment. The whole enlarged right cervical lymph node was resected and followed by biopsy and histologic examination. Hematoxylin and eosin (H & E) staining showed the typical morphology of squamous cell carcinoma cells (Figure 2A and B). Immunohistochemistry (IHC) analysis demonstrated that tumor cells were positive for cytokeratin (CK) 5/6 (Figure 2C) and P63 (Figure 2D) and negative for CK7 CK20 TTF-1 and Napsin A. The positive rate of Ki-67 was 30%. Altogether these results confirmed the diagnosis of metastatic squamous cell carcinoma. The patient requested treatment with epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) because dozens of patients with squamous cell carcinoma and EGFR mutations responded well to TKIs at our institute. Amplification refractory mutation system (ARMS) was used to assess EGFR and K-RAS gene profiles to determine the presence of mutated variants. However evidence of a normal genotype excluded the patient from receiving EGFR-TKI treatment. Figure 2 Right cervical lymph node analysis. H & E staining (A and B). IHC staining of CK5/6 (C) P63 (D) and ALK (1A4) (E). ALK gene translocation (FISH arrows: split-apart signals for ALK gene translocation) (F). As first-line chemotherapy the patient was initially administered 135 mg/m2 (210 mg) of paclitaxel and 80 mg/m2 (120 mg) of nedaplatin. During treatment the patient™s condition did not seem to improve; thus after a 20-day treatment a second round of chemotherapy was administered with 75 mg/m2 (120 mg) of docetaxel and 80 mg/m2 (120 mg) of nedaplatin. Unfortunately increasing levels of interleukin-6 (IL-6) (25.41 vs 16.03 pg/mL) and carcinoembryonic antigen (CEA) (23.43 vs 7.13 ng/mL) indicated cancer progression which was confirmed by the presence of multiple metastases in both lungs on CT scan images (Figure 1B). Although not initially indicated the patient was then administered oral treatment with the EGFR-TKI erlotinib (1-month trial). However this treatment showed no efficacy. After the use of crizotinib was approved by the China Food and Drug Administration (CFDA) in July 2013 the patient underwent ALK testing. IHC staining (Clone 1A4 Origene 1:200) showed tumor cell positivity for ALK protein (Figure 2E). Then ALK break-apart fluorescent in situ hybridization (FISH) was performed on 4-?m formalin-fixed paraffin-embedded tissue sections. Slides were hybridized with the LSI ALK Break Apart Rearrangement Probe (Vysis Abbott Molecular Des Plaines IL USA) and read on an epifluorescence microscope (BX41 Olympus Tokyo Japan). The lung cancer cell line NCI-H2228 (American Type Culture Collection-ATCC) was used as positive control. At least 50 tumor cell nuclei were analyzed and at least 15% of interpretable tumor cells harboring break-apart signals were used as the cutoff value [2]. "

Lung_Cancer

"Here we report a case of solitary lung metastasis of eyelid sebaceous carcinoma and discuss the clinical implication of surgery for a solitary pulmonary metastasis from sebaceous carcinoma. Case presentation A 77-year-old woman underwent left upper lid resection in April 2006 for sebaceous carcinoma of the eyelid. The surgical margin was negative for cancer cells. In January 2008 she had developed a recurrence in the left upper eyelid and underwent radiotherapy with a total dose of 57.6 Gy of proton beam therapy followed by orbital exenteration of the left eye [1112]. In July 2012 positron emission tomography“computed tomography (PET-CT) revealed a solitary pulmonary nodule 0.5 cm in size in the right upper lobe of the patient™s lung which had increased to 1.1 cm by September 2013 (A). PET-CT revealed a focus of increased uptake in that nodule with a standardized uptake value of 3.7 (B). There was no evidence of other metastatic disease on PET-CT scans. In September 2013 the patient underwent video-assisted thoracoscopic wedge resection of the pulmonary nodule. Frozen sections using oil red O stain revealed accentuation of lipid and presences of foamy cytoplasm in tumor cells which was positive for lipid staining (). Permanent histology demonstrated tumor cells with foamy cytoplasm and atypical nuclei accompanying numerous lipid globules within the cytoplasm () consistent with metastasis of eyelid sebaceous carcinoma. At the last follow-up 7 months after resection there was no loco-regional recurrence or distant metastasis of the tumor after surgery. Computed tomography (CT) and positron emission tomography of the tumors. (A) Chest CT showed a 1.1 cm nodule in the anterior segment of the right upper lobe (arrow). (B) PET-CT showed fluorodeoxyglucose accumulation with a Standardized uptake value (SUV) of 3.7 (arrowhead). Accentuation of lipid by staining. The lipid globules have a red color (frozen sections oil red O magnification?—?100). Sebaceous carcinoma cells. Foamy and frothy cytoplasm and atypical nuclei occurred with numerous lipid globules within the cytoplasm of the tumors cells seen as clear spaces (hematoxylin and eosin magnification?—?100). Discussion Sebaceous carcinoma of the eyelid refers to a group of carcinomas derived from sebaceous gland cells that occur in the ocular adnexa. It can be invasive in the eyelid and conjunctiva and can metastasize to regional lymph nodes and distant ans [81314]. Treatment strategies for primary eyelid sebaceous carcinoma are surgery radiotherapy and chemotherapy [15-17]. Distant hematogenous metastases to the lung liver and brain have a mortality rate as high as 30% [1618]. However few reports demonstrated the surgical treatment of metastatic eyelid sebaceous carcinoma. Standard treatment strategy for pulmonary metastatic sebaceous carcinoma has not yet been established because of the limited number of cases. Chemotherapy regimens in existing reports are largely based on the combination regimens commonly used in the treatment of other forms of poorly differentiated carcinomas of the head and neck region [1920]. Husain et al. reported combined chemotherapy of carboplatin and docetaxel for the patient who had multiple lung and lymph node metastases which resulted in a 30% decrease in tumor size but the efficacy of this regimen for sebaceous carcinoma has not yet been fully evaluated [21]. Radiotherapy for primary eyelid sebaceous carcinoma was described in several reports; however there have been no reports describing radiotherapy for pulmonary metastatic eyelid sebaceous carcinoma [2223]. Resection of pulmonary metastases in patients with sebaceous carcinoma is controversial. However our case suggests that a surgical approach to lung metastasis of eyelid sebaceous carcinoma could prolong survival in certain subgroups of patients namely those with a limited number of metastatic nodules or a significant disease-free interval. The possibility of metastasis from eyelid sebaceous carcinoma or primary lung cancer cannot be predicted only on the basis of radiologic findings or disease-free interval. In the present case we could successfully differentiate solitary lung metastasis of eyelid sebaceous carcinoma from primary lung cancer using oil red O stain which stains lipid has a red color on frozen sections. Conclusion We report a rare case of solitary lung metastasis of eyelid sebaceous carcinoma which was successfully resected and differentiated from primary lung cancer using oil red O stain on frozen sections. Pulmonary resection is a good option for the treatment and diagnosis of metastatic eyelid sebaceous carcinoma. Consent Written informed consent was obtained from the patient for the publication of this case presentation and accompanying images. A copy of the written consent is available for the review by the Editor-in-Chief of this journal. Abbreviations CT: Computed tomography; FDG: Fluorodeoxyglucose; PET: Positron emission tomography. Competing interests The authors declare that they have no competing interests. Authors™ contributions KK and TO wrote the manuscript. KK TO KA and IK performed surgery. YH and KE carried out the pathological examination. MK and TG were involved in the final editing. All authors approved the final manuscript. Cook BE Jr Bartley GB Cook BE Jr Bartley GB Treatment options and future prospects for the management of eyelid malignancies: an evidence-based update Ophthalmology 2001 108 2088 2209 quiz 2099“2100 2121 10.1016/S0161-6420(01)00796-5 11713084 Lai TF Huilgol SC Selva D James CL Eyelid sebaceous carcinoma masquerading as in situ squamous cell carcinoma Dermatol Surg 2004 30 222 225 10.1111/j.1524-4725.2004.30069.x 14756656 Leibovitch I Selva D Huilgol S Davis G Dodd T James CL Intraepithelial sebaceous carcinoma of the eyelid misdiagnosed as Bowen™s disease J Cutan Pathol 2006 33 303 308 10.1111/j.0303-6987.2006.00423.x 16630181 Pereira PR Odashiro AN Rodrigues-Reyes AA Correa ZM de Souza Filho JP Burnier MN Jr Histopathological review of sebaceous carcinoma of the eyelid J Cutan Pathol 2005 32 496 501 10.1111/j.0303-6987.2005.00371.x 16008694 Sinard JH Immunohistochemical distinction of ocular sebaceous carcinoma from basal cell and squamous cell carcinoma Arch Ophthalmol 1999 117 776 783 10.1001/archopht.117.6.776 10369589 Chao AN Shields CL Krema H Shields JA Outcome of patients with periocular sebaceous gland carcinoma with and without conjunctival intraepithelial invasion Ophthalmology 2001 108 1877 1883 10.1016/S0161-6420(01)00719-9 11581065 Shields JA Demirci H Marr BP Eagle RC Jr Shields CL Sebaceous carcinoma of the eyelids: personal experience with 60 cases Ophthalmology 2004 111 2151 2157 10.1016/j.ophtha.2004.07.031 15582067 Shields JA Demirci H Marr BP Eagle RC Jr Shields CL Sebaceous carcinoma of the ocular region: a review Surv Ophthalmol 2005 50 103 122 10.1016/j.survophthal.2004.12.008 15749305 Yen MT Tse DT Wu X Wolfson AH Radiation therapy for local control of eyelid sebaceous cell carcinoma: report of two cases and review of the literature Ophthal Plast Reconstr Surg 2000 16 211 215 10.1097/00002341-200005000-00008 10826762 Wang JK Liao SL Jou JR Lai PC Kao SC Hou PK Chen MS Malignant eyelid tumours in Taiwan Eye (Lond) 2003 17 216 220 10.1038/sj.eye.6700231 12640409 Zenda S Kawashima M Nishio T Kohno R Nihei K Onozawa M Arahira S Ogino T Proton beam therapy as a nonsurgical approach to mucosal melanoma of the head and neck: a pilot study Int J Radiat Oncol Biol Phys 2011 81 135 139 10.1016/j.ijrobp.2010.04.071 20950948 Zenda S Kohno R Kawashima M Arahira S Nishio T Tahara M Hayashi R Kishimoto S Ogino T Proton beam therapy for unresectable malignancies of the nasal cavity and paranasal sinuses Int J Radiat Oncol Biol Phys 2011 81 1473 1478 10.1016/j.ijrobp.2010.08.009 20961697 Ginsberg J Present Status of Meibomian gland carcinoma Arch Ophthalmol 1965 73 271 277 10.1001/archopht.1965.00970030273022 14237799 Rao NA Hidayat AA McLean IW Zimmerman LE Sebaceous carcinomas of the ocular adnexa: a clinicopathologic study of 104 cases with five-year follow-up data Hum Pathol 1982 13 113 122 10.1016/S0046-8177(82)80115-9 7076199 Gardetto A Rainer C Ensinger C Baldissera I Piza-Katzer H Sebaceous carcinoma of the eyelid: a rarity worth considering Br J Ophthalmol 2002 86 243 244 10.1136/bjo.86.2.243 11815355 Kass LG Hornblass A Sebaceous carcinoma of the ocular adnexa Surv Ophthalmol 1989 33 477 490 10.1016/0039-6257(89)90049-0 2658172 Lan MC Lan MY Lin CZ Ho DM Ho CY Sebaceous carcinoma of the eyelid with neck metastasis Otolaryngol Head Neck Surg 2007 136 670 671 10.1016/j.otohns.2006.08.019 17418274 Boniuk M Zimmerman LE Sebaceous carcinoma of the eyelid eyebrow caruncle and orbit Trans Am Acad Ophthalmol Otolaryngol 1968 72 619 642 5706692 Midena E Angeli CD Valenti M de Belvis V Boccato P Treatment of conjunctival squamous cell carcinoma with topical 5-fluorouracil Br J Ophthalmol 2000 84 268 272 10.1136/bjo.84.3.268 10684836 Yeatts RP Engelbrecht NE Curry CD Ford JG Walter KA 5-Fluorouracil for the treatment of intraepithelial neoplasia of the conjunctiva and cornea Ophthalmology 2000 107 2190 2195 10.1016/S0161-6420(00)00389-4 11097594 Husain A Blumenschein G Esmaeli B Treatment and outcomes for metastatic sebaceous cell carcinoma of the eyelid Int J Dermatol 2008 47 276 279 10.1111/j.1365-4632.2008.03496.x 18289332 Hata M Koike I Omura M Maegawa J Ogino I Inoue T Noninvasive and curative radiation therapy for sebaceous carcinoma of the eyelid Int J Radiat Oncol Biol Phys 2012 82 605 611 10.1016/j.ijrobp.2010.12.006 21300468 Howrey RP Lipham WJ Schultz WH Buckley EG Dutton JJ Klintworth GK Rosoff PM Sebaceous gland carcinoma: a subtle second malignancy following radiation therapy in patients with bilateral retinoblastoma Cancer 1998 83 767 771 10.1002/(SICI)1097-0142(19980815)83:4<767::AID-CNCR20>3.0.CO;2-P 9708943 101274235 33311 J Thorac Oncol J Thorac Oncol Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer 1556-0864 1556-1380 24736085 4271824 10.1097/JTO.0000000000000082 NIHMS648380 Article A Randomized Placebo-Controlled Multicenter Biomarker-Selected Phase 2 Study of Apricoxib in Combination with Erlotinib in Patients with Advanced Non“Small-Cell Lung Cancer Gitlitz Barbara J. MD * Bernstein Eric MD   Santos Edgardo S. MD ¡ Otterson Greg A. MD § Milne Ginger PhD ? Syto Mary MS ¶ Burrows Francis PhD ¶ Zaknoen Sara MD ¶ *University of Southern California Keck School of Medicine Norris Comprehensive Cancer Center Los Angeles California  Providence Cancer Center Portland Oregon ¡University of Miami Sylvester Comprehensive Cancer Center Miami Florida §Ohio State University Columbus Ohio ?Vanderbilt University Nashville Tennessee ¶Tragara Pharmaceuticals San Diego California Address for correspondence: Barbara Gitlitz MD University of Southern California Keck School of Medicine Norris Comprehensive Cancer Center 1441 Eastlake Avenue Suite 3400 Los Angeles CA 90089. gitlitz@usc.edu 13 12 2014 4 2014 19 12 2014 9 4 577 582 Copyright © 2013 by the International Association for the Study of Lung Cancer 2013 Cyclooxygenase-2 (COX-2) overexpression is associated with a poor prognosis in non“small-cell lung cancer (NSCLC) and may promote resistance to epidermal growth factor receptor inhibitors. This randomized phase 2 trial evaluated apricoxib a novel COX-2 inhibitor in combination with erlotinib in biomarker-selected patients. Patients with stage IIIB/IV NSCLC previously treated with platinum-based chemotherapy were randomized (2:1) to 400 mg/day apricoxib plus 150 mg/day erlotinib (AP/E) or placebo plus erlotinib (P/E) in 21-day cycles until disease progression or unacceptable toxicity. The primary endpoint was time to progression (TTP). A decrease of 50% or more from baseline urinary prostaglandin E2 metabolite after a 5-day open-label run-in period was used to select eligible patients. One hundred twenty patients (median age 64 years) were randomized (78 to AP/E and 42 to P/E). Overall median TTP was 1.8 months in the AP/E group and 2.1 months in the P/E group with a 12% objective response rate in both groups (intent-to-treat analysis). A subgroup analysis in patients aged 65 years or younger demonstrated a statistically significant TTP benefit for AP/E (hazard ratio 0.5 [95% confidence interval: not applicable“0.9]; p=0.018) and overall survival advantage at minimum 1-year follow-up (median 12.2 versus 4.0 months; hazard ratio=0.5; p=0.021). The most common adverse events were rash diarrhea fatigue and nausea. Toxicity contributed to early discontinuations in patients aged more than 65 years treated with AP/E. This is the first randomized placebo-controlled study of a COX-2 inhibitor in NSCLC to use a prospective patient-selection strategy. Although AP/E seemed to improve TTP and overall survival in a subset of patients aged 65 years or younger the primary endpoint of the trial was not met. Non“small-cell lung cancer Apricoxib Erlotinib Cyclooxygenase-2 inhibitor Prostaglandin E2 metabolite 0413066 2830 Cell Cell Cell 0092-8674 1097-4172 24630729 4040459 10.1016/j.cell.2014.02.031 NIHMS573682 Article Genetic and Clonal Dissection of Murine Small Cell Lung Carcinoma Progression by Genome Sequencing McFadden David G. 1 5 Papagiannakopoulos Thales 1 5 Taylor-Weiner Amaro 3 5 Stewart Chip 3 5 Carter Scott L. 3 5 Cibulskis Kristian 3 Bhutkar Arjun 1 McKenna Aaron 3 Dooley Alison 1 Vernon Amanda 1 Sougnez Carrie 3 Malstrom Scott 1 Heimann Megan 1 Park Jennifer 1 Chen Frances 1 Farago Anna F. 1 Dayton Talya 1 Shefler Erica 3 Gabriel Stacey 3 Getz Gad 3 4 * Jacks Tyler 1 2 * 1Koch Institute for Integrative Cancer Research and Department of Biology Massachusetts Institute of Technology Cambridge MA 02142 USA 2Howard Hughes Medical Institute Massachusetts Institute of Technology Cambridge MA 02142 USA 3Cancer Program Broad Institute of MIT and Harvard Cambridge MA 02142 USA 4Cancer Center and Department of Pathology Massachusetts General Hospital Boston MA 02114 USA *Correspondence: gadgetz@broadinstitute. (G.G.) tjacks@mit.edu (T.J.) 5 Co-first author 6 5 2014 13 3 2014 13 3 2015 156 6 1298 1311 ©2014 Elsevier Inc. 2014 Summary Small cell lung carcinoma (SCLC) is a highly lethal smoking-associated cancer with few known targetable genetic alterations. Using genome sequencing we characterized the somatic evolution of a genetically engineered mouse model (GEMM) of SCLC initiated by loss of Trp53 and Rb1. We identified alterations in DNA copy number and complex genomic rearrangements and demonstrated a low somatic point mutation frequency in the absence of tobacco mutagens. Alterations targeting the tumor suppressor Pten occurred in the majority of murine SCLC studied and engineered Pten deletion accelerated murine SCLC and abrogated loss of Chr19 in Trp53; Rb1; Pten compound mutant tumors. Finally we found evidence for polyclonal and sequential metastatic spread of murine SCLC by comparative sequencing of families of related primary tumors and metastases. We propose a temporal model of SCLC tumorigenesis with implications for human SCLC therapeutics and the nature of cancer-genome evolution in GEMMs. J Natl Cancer Inst J. Natl. Cancer Inst jnci jnci.j JNCI Journal of the National Cancer Institute 0027-8874 1460-2105 Oxford University Press US 24317180 3906987 10.1093/jnci/djt338 Brief Communication Novel Germline Mutation in the Transmembrane Domain of HER2 in Familial Lung Adenocarcinomas Yamamoto Hiromasa Higasa Koichiro Sakaguchi Masakiyo Shien Kazuhiko Soh Junichi Ichimura Koichi Furukawa Masashi Hashida Shinsuke Tsukuda Kazunori Takigawa Nagio Matsuo Keitaro Kiura Katsuyuki Miyoshi Shinichiro Matsuda Fumihiko Toyooka Shinichi Affiliations of authors:Department of Thoracic Breast and Endocrinological Surgery (HY KS JS MF SH KT SM ST) Department of Clinical Genomic Medicine (KS ST) Department of Cell Biology (MS) Department of Pathology (KI) and Department of Hematology Oncology and Respiratory Medicine (KK) Okayama University Graduate School of Medicine Dentistry and Pharmaceutical Sciences Okayama Japan; Center for Genomic Medicine Kyoto University School of Medicine Kyoto Japan (KH FM); Department of General Internal Medicine 4 Kawasaki Medical School Okayama Japan (NT); Department of Preventive Medicine Kyushu University Faculty of Medical Sciences Fukuoka Japan (KM). Correspondence to: Shinichi Toyooka MD PhD Okayama University Graduate School of Medicine Dentistry and Pharmaceutical Sciences Clinical Genomic Medicine/Thoracic Breast and Endocrinological Surgery 2-5-1 Shikata-cho Kita-ku Okayama Okayama 700“8558 Japan (e-mail: toyooka@md.okayama-u.ac.jp). 1 2014 7 12 2013 7 12 2013 106 1 djt338 7 7 2013 14 10 2013 16 10 2013 © The Author 2013. Published by Oxford University Press. 2013 This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons./licenses/by-nc-nd/3.0/) which permits non-commercial reproduction and distribution of the work in any medium provided the original work is not altered or transformed in any way and that the work is properly cited. For commercial re-use please contact journals.permissions@oup.com We encountered a family of Japanese descent in which multiple members developed lung cancer. Using whole-exome sequencing we identified a novel germline mutation in the transmembrane domain of the human epidermal growth factor receptor 2 (HER2) gene (G660D). A novel somatic mutation (V659E) was also detected in the transmembrane domain of HER2 in one of 253 sporadic lung adenocarcinomas. Because the transmembrane domain of HER2 is considered to be responsible for the dimerization and subsequent activation of the HER family and downstream signaling pathways we performed functional analyses of these HER2 mutants. Mutant HER2 G660D and V659E proteins were more stable than wild-type protein. Both the G660D and V659E mutants activated Akt. In addition they activated p38 which is thought to promote cell proliferation in lung adenocarcinoma. Our findings strongly suggest that mutations in the transmembrane domain of HER2 may be oncogenic causing hereditary and sporadic lung adenocarcinomas. Familial lung cancers are rare among human malignancies. Recent studies have reported that germline mutations in the epidermal growth factor receptor (EGFR) gene predispose the development of lung cancer. Reported familial lung adenocarcinomas with a germline EGFR mutation such as T790M carry secondary somatic EGFR mutations including exon 19 deletion and exon 21 L858R mutation (1“4). We encountered a family of Japanese descent in which multiple members developed lung cancer (). The proband (III-4) was a 53-year-old woman with multiple lung adenocarcinomas in bilateral lungs. She was a light smoker with a 1.2-pack-year history of smoking. She had undergone a left lower lobectomy for multiple lung adenocarcinomas at the age of 44 years. Her mother (II-4) a never smoker also had multiple lung adenocarcinomas. Partial pulmonary resections of two tumors were performed for II-4 for the purpose of diagnosis after pleural dissemination was found during surgery and multiple lesions were removed in a lobectomy or partial resections in III-4. A histological examination of the resected tumors in II-4 revealed nonmucinous adenocarcinoma in situ and nonmucinous minimally invasive adenocarcinoma whereas the histological findings of pleural dissemination indicated mucus-containing adenocarcinoma. Those of III-4 contained various subtypes of adenocarcinoma including nonmucinous and mucinous adenocarcinoma in situ and invasive mucinous adenocarcinoma. In addition normal-appearing lung parenchyma obtained from a lobectomy in III-4 revealed innumerable small preinvasive lesions implying the presence of precancerous changes throughout the lung (Supplementary available online). Sequencing analyses of EGFR exons 18 to 21 and KRAS as well as an immunohistochemical staining for ALK protein in the resected tumors indicated no genetic alterations in these genes. The pedigree chart suggested that lung cancer was inherited in an autosomal dominant manner. . Pedigree chart of a Japanese family in which multiple members developed lung cancer. The boxes and circles indicate men and women respectively. The numbers at the bottom of each member indicate the age at the time of death or the time of the analysis. An oblique line shows deceased family members. The proband (III-4) had multiple lung adenocarcinomas (arrow). Tumor tissue nonmalignant lung tissue and peripheral blood samples were obtained from III-4. The proband™s mother (II-4) also had multiple lung adenocarcinomas and tumor and nonmalignant lung tissue samples were available. "

Lung_Cancer

"To further functionally validate this point we show two promoters where the NME2 target site was not occupied by NME2 and had positioned nucleosomes in A549 cells and NME2-depleted A549 cells but were available for NME2 binding following NME2 induction (Supplementary Figure S3). In case of 53 genes we found positioned nucleosomes on or near NME2 target sites on NME2 depletion relative to control A549 cells. Together this suggests that in contrast to the nucleosomal changes following increase in NME2 expression NME2 target sites remain nucleosome-occupied in most cases on depletion of NME2. Binding site occupancy is transcriptionally active when associated with nucleosome repositioning We found that occupancy of about a fifth (870 of 3956 NME2 target sites ?22%) of the transcription target sites was concurrent with repositioning of nucleosomes in the NME2-induced condition. Interestingly these repositioning events resulted in altered expression of all the 791 genes (D). In contrast we found 1175 genes where the NME2 binding site (unique to the induced condition) was co-occupied with nucleosomes”only 130 (11%) of these genes showed altered expression. As a third possibility we found 1990 genes with NME2 occupancy in the induced condition though no nucleosomes were present in the vicinity of the NME2 site either before or after induction”i.e. target sites appeared to be independent of nucleosome repositioning. Again out of 1990 only 179 (8.9%) genes were differentially expressed. On mapping the NFR between the ?1 and +1 nucleosome positions in each of the three situations described above we found repositioning of the ?1 nucleosome by ?40 bp in the first case when repositioning was linked to binding site occupancy whereas in the other two situations the NFR was minimally altered on inducing NME2 (). DISCUSSION Our findings suggest that TF binding when closely associated with nucleosome repositioning results in altered gene expression changes. Interestingly in most cases when TF binding did not impact local nucleosome reanization it was not associated with altered transcriptional state of target gene. As we used human cancer cells that are metastatic and expression of the TF NME2 decreased their metastatic potential these findings also help in understanding how TF binding-induced nucleosome level changes influence the transcriptome during metastasis. TF binding and transcriptional activity are linked through local nucleosome repositioning It was recently reported that repositioning of the +1 nucleosome resulted in changes to NFR in genes that were differentially regulated during meiotic development in yeast (9). Though this was noted as a result of change in possibly multiple regulatory factors involved in meiotic development it is consistent with our results. Furthermore our findings indicate that assignment of transcriptional function to genome-wide target site binding would require information on nucleosome reanization to be more precise. This helps explain the noted discrepancy in high throughput DNA binding studies where low overlap between experimentally determined binding sites and gene expression has been observed (3435). A recent study noted chromatin accessibility before and after binding of the receptors (androgen (AR) or estrogen (ESR1)) were significantly altered (19) and suggested that both AR and ESR1 binding are associated with changes in local nucleosome occupancy. This is in line with our findings and suggests a model that integrates factor binding and transcriptional activity of genes with local nucleosomal changes. Non-specific binding of NME2 in the induced condition could be a confounding factor. To address this first we checked and found that in NME2-depleted cells a large number of genes were oppositely expressed with respect to their status in NME2-induced cells; it is unlikely that non-specifically activated/repressed genes as a result of NME2 induction would be differentially expressed on depleting NME2 (Supplementary Figure S4). Second the differentially expressed genes in NME2-induced cells correlate significantly with transcriptome changes that are clinically relevant (Supplementary Figure S5). Therefore though all NME2 binding events do not lead to increase/decrease in transcription it is unlikely to be due to spurious binding”it is possible that many of these associations are required for functions other than transcription. Overall chromatin landscape in promoters is largely constant site-specific changes are associated with transcription We found only 11.4% of nucleosomes to be repositioned in promoter proximal regions as a result of NME2 induction. Therefore it is interesting to consider that overall chromatin level changes may be relatively small. On the other hand and perhaps more interestingly there may be shift in nucleosome occupancy on TF binding leading to site-specific ˜open™ or ˜closed™ regions that facilitate regulatory events. Our findings (discussed above) further support this: nucleosomes repositioning along with engagement of TF at specific sites were in almost all cases associated with transcriptional change in the corresponding gene. In addition in both cases before and after NME2 induction enriched promoter nucleosome occupancy correlated with decreased expression of genes. Together these support a model where nucleosome occupancy generally determines the suppressed state of the transcriptome and reanization induced by DNA binding factors (themselves or when associated with chromatin modifiers) results in transcriptional activation at specific loci. Although further studies using other TFs will be required to substantiate this it appears to be consistent with an earlier study which observed decreased presence of nucleosomes in promoters of genes that were expressed during heat shock in yeast (16). However others have also noted either unchanged nucleosome occupancy (yeast grown in different carbon sources (3637)) or found nucleosome positioning to correlate with the state of transcription (active or silent) and not the extent of gene expression (18). Epigenetic signaling directs the location of TFs to cognate sites in given chromatin territories. Following this TFs are believed to be one of the key recruiters of chromatin modification and remodeling machineries (38“40). Recent evidence suggests that even the general TFs such as subunit of TFIID (Transcription Factor II D) complexes may be functional component of these machineries (41). In agreement with this basic understanding of transcription through chromatin our results demonstrate TF binding to be transcriptionally competent when coupled with locally altered nucleosome positioning. Furthermore our findings for the first time underline the importance of these aspects of chromatin biology in suppression of cancer spread mediated by NME2."

Lung_Cancer

"however excellent staining in MLM was significantly higher than methylene blue (81% vs 38% P=0.011) (). shows the localization ability of MLM regarding both staining ability and radio-opacity. There was no subject with a score of 0 or 1 in both radio-opacity and staining. MLM achieved appropriate staining or radio-opacity in 21 subjects (100%) with a dual localization feature. Histopathologic findings demonstrates the results of the histopathologic findings. In all lung specimens both methylene blue and MLM showed acute lung parenchymal change that included neutrophil infiltration hemorrhage and foam cell in alveolus (Fig. 4). Comparing the two materials the number of specimen having neutrophil infiltration vasculitis necrosis hemorrhage and foam cell in alveolus was similar in each extent. In terms of all features the number of specimen that showed diffuse extent was more in Group B than Group A for both MLM and methylene blue. The extent of the histopathologic findings was not significantly associated with the materials for all histopathologic features (). Among the histopathologic findings the extent of vasculitis was significantly associated with Group for both MLM and methylene blue (P=0.002 for both MLM and methylene blue). Focal or diffuse extent of vasculitis was more frequently found in Group A than Group B (P=0.001 for both MLM and methylene blue). The overall severity of lung parenchymal change was not different between MLM and methylene blue (5.6±1.6 vs 5.7±1.5 P=0.839); in addition Group B showed a significantly higher overall severity score of lung parenchymal change than Group A (6.6±1.6 vs 4.7±0.9 P=0.005). DISCUSSION The results of this study show that MLM is a useful percutaneous injection material for a successful localization in the lung. The average staining score of MLM was significantly higher than methylene blue (2.8±0.5 vs 2.2±0.7 P=0.010). In terms of staining the appropriate localization rate (acceptable or excellent staining) in our study was 95% using MLM. The result was in close agreement with previous studies that showed a high success performance rate of lipiodol localization (99%-100%) (21-23). An appropriate localization rate (acceptable or excellent staining) of methylene blue injection was 86% in our study. This is lower than the results found in previous studies where the success rate of methylene blue injection was 96%-100% (18 20). We found that an acceptable (or excellent staining rate) of MLM and methylene blue was not significantly different (95% vs 86% P=0.610). However MLM showed excellent staining for localization in 17 (81%) of 21 subjects and was significantly higher than methylene blue (38%) (P=0.011). The results indicate that lipiodol reduced the spread of methylene blue. This is the first study to indicate that MLM is an available percutaneous injection material for localization with superior staining ability compared to methylene blue. The complication rate was 43% in MLM and 5% in the methylene blue (P=0.004). Possible complications after percutaneous injection for pulmonary localization include pneumothorax leakage hemorrhage pain hemoptysis hemothorax and embolism. Previous studies reported that the complication rate was 17-29% for lipiodol and 33% for methylene blue (2023 24). The complication rate of MLM in the current study was higher than the results of previous studies mainly due to the leakage of MLM into the pleural cavity (n=9). This difference was probably because the distance from the pleura to the injecting needle tip (0.4±0.1 cm for MLM) was inadequate to avoid leakage into the pleural cavity. In the previous studies of lipiodol marking for localization the mean distance from the pleura to the target nodule was 1.0-1.9 cm (22-24) more than twice our study. The results indicate that the high complication rate of our study is associated with the inserting procedure of the needle rather than MLM itself. The dispersion of methylene blue throughout the lung parenchyma may lead to unnecessarily large wedge resections; in addition some have reported instances of the dispersion of methylene blue throughout the entire pleural surface or intraoperative identification failure due to severe anthracosis of the visceral pleura. The failure rate was reported to be 0%-13% with the use of methylene blue (1819 25). The results are similar to our study and indicate that inappropriate staining on the lung surface was 14% in methylene blue. In this study we found that the dispersion of methylene blue in MLM through the lung parenchyma was significantly smaller than methylene blue (0.6±0.3 cm vs 1.0±0.4 cm P<0.05). The result implies that lipiodol reduces the spread of methylene blue in lung parenchyma. Regarding the score of radio-opacity 38% of MLM showed non-visualization or minimally increased opacity on the fluoroscopic examinations. It means the proportion of lipiodol in MLM at the time of the percutaneous injection was too small to be detected. Post-procedural CT images also revealed that 3 subjects had small faint radio-opacity after the injection of MLM. It suggests that the uneven blending of lipiodol and methylene blue occurred during the preparation of MLM. Water-insolubility of lipiodol would result in the uneven mixing of water soluble methylene blue after mechanical blending of the two materials. Further research is required to reduce non-homogeneity of MLM at the time of injection. Previous studies reported the availability of a mixture of methylene blue with other materials such as collagen or autologous blood (15 16). They performed VATS resection on the same day as localization. In our study we evaluated the localization ability of MLM on the same day of localization (6 hr) as well as 24 hr after injection. Localization is usually performed on the day of surgery. This requires the simultaneous use of the CT and the operating room which is not always available. Surgeries on the next day of localization were reported in several published s (26 27). MLM shows a prolonged localization ability of up to 24 hr in terms of staining ability and radio-opacity. Stable localization ability is the advantage of MLM in our study. Due to uneven blending of MLM one subject (10%) showed inappropriate staining and appropriate radio-opacity and required an intraoperative fluoroscopic examination to detect MLM. Possible radiation exposure is a drawback of MLM. We would like to justify the use of intraoperative fluoroscopy because the operator can avoid radiation exposure with a lead apron. In regards to the risk-benefit for patients lowering the risk of detection failure is thought to be more important than radiation exposure. Histopathologic examinations showed lung parenchymal changes in all specimens. Both methylene blue and MLM induced acute lung injury that included neutrophil infiltration vasculitis necrosis hemorrhage and foam cell in alveolus (). The results of our study are similar to those of a previous study by Kwon et al. (28) that showed that lipiodol led to acute lung injury. They described that lipiodol creates the histopathologic feature of acute lung injury such as peripheral endothelial cell damage neutrophil infiltration necrosis hemorrhage alveolar wall destruction vasculitis emboli (or thrombi in arteriole) and macrophages in the alveolar space (28). In our results the extent of lung parenchymal change was not associated with the materials for all histopathologic features. In addition the overall severity score of lung parenchymal change in MLM was not different from methylene blue (5.6 and 5.7 P=0.839). This suggests that MLM shows similar histopathologic effects in the lung parenchyma to methylene blue. The overall severity score of parenchymal change was higher in Group B (follow up interval of 24 hr) than Group A (follow up interval of 6 hr) (6.6 vs 4.7 P=0.005). The extent of lung parenchymal change depends on the time interval. Acute lung injury after the percutaneous injection of lipiodol or methylene blue was reported in animal studies (28 29); however there are no clinical results that show the adverse effect of acute lung injury in human lungs. Injection material (such as barium) can potentially complicate the pathologic diagnosis of the target lesion due to acute inflammation (29 30). To our knowledge no study has indicated that lipiodol or methylene blue hinders the histopathologic diagnosis of target lesions in human lungs. The small amount of material injection in human lungs might not create a significant parenchymal change or disrupt underlying lung disease. It is necessary to avoid directly injecting materials into the target lesion in human lungs in order to avoid the adverse effect of injection materials on underlying lung disease (especially ground glass opacity nodule or potential benign lesion). There were several limitations in our study. First we included only a small number of subjects. Second the overall localization success rate was low and the complication rate was high (compared to the results of previous studies) due to the difficulty in an accurate percutaneous injection at the desired location and depth in the small sized rabbit lung. Third we used a 1 mL syringe with manual administration to inject materials in the lung parenchyma and there were possible individual difference in the administering volume of materials. Fourth we could not evaluate complications such as intractable pain material related anaphylaxis or embolism. Fifth we could not evaluate if the histopathologic changes had any effect on underlying lung disease because the lung parenchyma of the experimental rabbits were normal. Finally we did not evaluate a successful localization for the true target lesion in lung parenchyma. The criteria for appropriate staining and radio-opacity were subjective. We expect that further clinical studies might provide an answer to if MLM can be a useful percutaneous injection material for localization in the human lung. In conclusion MLM is available for percutaneous injection for the pulmonary localization. The results of this study showed that MLM provides superior ability for appropriate localization than that of methylene blue. Further research on human lungs can clarify the availability of MLM as a CT guided percutaneous injection material. This study was supported by grant from the Seoul National University College of Medicine Research Fund 2012 (800-20120036). We have no potential conflicts of interest or commercial involvement to disclose. 1 Nakashima S Watanabe A Obama T Yamada G Takahashi H Higami T Need for preoperative computed tomography-guided localization in video-assisted thoracoscopic surgery pulmonary resections of metastatic pulmonary nodules Ann Thorac Surg 2010 89 212 218 20103238 2 Chen S Zhou J Zhang J Hu H Luo X Zhang Y Chen H Video-assisted thoracoscopic solitary pulmonary nodule resection after CT-guided hookwire localization: 43 cases report and literature review Surg Endosc 2011 25 1723 1729 21181200 3 Ciriaco P Negri G Puglisi A Nicoletti R Del Maschio A Zannini P Video-assisted thoracoscopic surgery for pulmonary nodules: rationale for preoperative computed tomography-guided hookwire localization Eur J Cardiothorac Surg 2004 25 429 433 15019673 4 Suzuki K Nagai K Yoshida J Ohmatsu H Takahashi K Nishimura M Nishiwaki Y Video-assisted thoracoscopic surgery for small indeterminate pulmonary nodules: indications for preoperative marking Chest 1999 115 563 568 10027460 5 Seo JM Lee HY Kim HK Choi YS Kim J Shim YM Lee KS Factors determining successful computed tomography-guided localization of lung nodules J Thorac Cardiovasc Surg 2012 143 809 814 22104686 6 Gossot D Miaux Y Guermazi A Celerier M Friga J The hook-wire technique for localization of pulmonary nodules during thoracoscopic resection Chest 1994 105 1467 1469 8181339 7 Pittet O Christodoulou M Pezzetta E Schmidt S Schnyder P Ris HB Video-assisted thoracoscopic resection of a small pulmonary nodule after computed tomography-guided localization with a hook-wire system: experience in 45 consecutive patients World J Surg 2007 31 575 578 17318707 8 Chen W Chen L Yang S Chen Z Qian G Zhang S Jing J A novel technique for localization of small pulmonary nodules Chest 2007 131 1526 1531 17494801 9 Bernard A Resection of pulmonary nodules using video-assisted thoracic surgery: the Thorax Group Ann Thorac Surg 1996 61 202 204 8561553 10 Martin AE Chen JY Muratore CS Mayo-Smith WW Luks FI Dual localization technique for thoracoscopic resection of lung lesions in children J Laparoendosc Adv Surg Tech A 2009 19 S161 S164 18999984 11 Kawanaka K Nomori H Mori T Ikeda K Ikeda O Tomiguchi S Yamashita Y Marking of small pulmonary nodules before thoracoscopic resection: injection of lipiodol under CT-fluoroscopic guidance Acad Radiol 2009 16 39 45 19064210 12 Yamagami T Miura H Yoshimatsu R Tanaka O Ono S Iehara T Hosoi H Nishimura T Experience of fluoroscopy-aided thoracoscopic resection of pulmonary nodule localised with Lipiodol in a child J Med Imaging Radiat Oncol 2011 55 401 403 21843175 13 Iwasaki Y Nagata K Yuba T Hosogi S Kohno K Ohsugi S Kuwahara H Takemura Y Yokomura I Fluoroscopy-guided barium marking for localizing small pulmonary lesions before video-assisted thoracic surgery Respir Med 2005 99 285 289 15733503 14 Yoshida J Nagai K Nishimura M Takahashi K Computed tomography-fluoroscopy guided injection of cyanoacrylate to mark a pulmonary nodule for thoracoscopic resection Jpn J Thorac Cardiovasc Surg 1999 47 210 213 10402768 15 Nomori H Horio H Colored collagen is a long-lasting point marker for small pulmonary nodules in thoracoscopic operations Ann Thorac Surg 1996 61 1070 1073 8607658 16 McConnell PI Feola GP Meyers RL Methylene blue-stained autologous blood for needle localization and thoracoscopic resection of deep pulmonary nodules J Pediatr Surg 2002 37 1729 1731 12483642 17 Hu J Zhang C Sun L Localization of small pulmonary nodules for videothoracoscopic surgery ANZ J Surg 2006 76 649 651 16813634 18 Wicky S Mayor B Cuttat JF Schnyder P CT-guided localizations of pulmonary nodules with methylene blue injections for thoracoscopic resections Chest 1994 106 1326 1328 7956378 19 Vandoni RE Cuttat JF Wicky S Suter M CT-guided methylene-blue labelling before thoracoscopic resection of pulmonary nodules Eur J Cardiothorac Surg 1998 14 265 270 9761435 20 Lenglinger FX Schwarz CD Artmann W Localization of pulmonary nodules before thoracoscopic surgery: value of percutaneous staining with methylene blue AJR Am J Roentgenol 1994 163 297 300 7518642 21 Ikeda K Nomori H Mori T Kobayashi H Iwatani K Yoshimoto K Kawanaka K Impalpable pulmonary nodules with ground-glass opacity: success for making pathologic sections with preoperative marking by lipiodol Chest 2007 131 502 506 17296654 22 Nomori H Horio H Naruke T Suemasu K Fluoroscopy-assisted thoracoscopic resection of lung nodules marked with lipiodol Ann Thorac Surg 2002 74 170 173 12118752 23 Watanabe K Nomori H Ohtsuka T Kaji M Naruke T Suemasu K Usefulness and complications of computed tomography-guided lipiodol marking for fluoroscopy-assisted thoracoscopic resection of small pulmonary nodules: experience with 174 nodules J Thorac Cardiovasc Surg 2006 132 320 324 16872957 24 Kim YD Jeong YJ I H Cho JS Lee JW Kim HJ Lee SH Kim DH Localization of pulmonary nodules with lipiodol prior to thoracoscopic surgery Acta Radiol 2011 52 64 69 21498328 25 Mayo JR Clifton JC Powell TI English JC Evans KG Yee J McWilliams AM Lam SC Finley RJ Lung nodules: CT-guided placement of microcoils to direct video-assisted thoracoscopic surgical resection Radiology 2009 250 576 585 19188326 26 Lee NK Park CM Kang CH Jeon YK Choo JY Lee HJ Goo JM CT-guided percutaneous transthoracic localization of pulmonary nodules prior to video-assisted thoracoscopic surgery using barium suspension Korean J Radiol 2012 13 694 701 23118567 27 Kamiyoshihara M Ishikawa S Morishita Y Pulmonary cryptococcosis diagnosed by video-assisted thoracoscopic surgery with CT-guided localization: report of a case Kyobu Geka 2000 53 795 797 10935411 28 Kwon WJ Kim HJ Jeong YJ Lee CH Kim KI Kim YD Lee JH Direct lipiodol injection used for a radio-opaque lung marker: stability and histopathologic effects Exp Lung Res 2011 37 310 317 21574876 29 Jang HS Effect of drugs for preoperative localization of thoracoscopy to histopathologic change in rabbit lung Seoul the Catholic University of Korea 2000 27 Dissertation 30 Okumura T Kondo H Suzuki K Asamura H Kobayashi T Kaneko M Tsuchiya R Fluoroscopy-assisted thoracoscopic surgery after computed tomography-guided bronchoscopic barium marking Ann Thorac Surg 2001 71 439 442 11235684 Fig. 1 Overview of the experimental design. Animals were randomly divided into two groups: Group A (n = 12) was sacrificed 6 hr after percutaneous injection and Group B (n = 12) was sacrificed 24 hr after a CT guided percutaneous injection of MLM and methylene blue. Fig. 2 Examples of evaluation of staining on the lung surface. Photographs show (A) the extensive staining (score 1) (B) localized dispersion of staining (score 2) and (C) minimal dispersion of staining (score 3). The white lines on the bottom of the figure are markings of the ruler. The distance between two lines is one centimeter. Fig. 3 Examples of assessment of radio-opacity on the fluoroscopic examinations. The fluoroscopic images show (A) a minimally increased opacity (arrow) (score 1) (B) a low density of increased opacity (arrow) (score 2) and (C) a compact nodular increased opacity (arrow) (score 3). Fig. 4 CT and corresponding photomicrograph of lung specimen. MLM in Group B (A-D); (A) discrete and compact nodular opacity (arrowheads) (B) focal neutrophil infiltration necrosis and hemorrhage (arrowheads) (H&E ×12.5) (C) scattered small nodular opacities of lipiodol (long arrows) and faint nodular opacity (arrowheads) (D) focal hemorrhage and necrosis (arrowheads) with diffuse neutrophil infiltration (short arrows) (H&E ×12.5). MLM in Group A (E F); (E) faint nodular lipiodol opacity (arrows) (F) focal hemorrhage (arrows) with diffuse neutrophil infiltration (arrowheads) (H&E ×12.5). Methylene blue in Group A (G H); (G) faint nodular opacity (arrowheads) and (H) focal extent of neutrophil infiltration necrosis and hemorrhage (arrowheads) (H&E ×12.5). Table 1 Staining extent and localization ability of MLM versus methylene blue Data are mean±standard deviation. Numbers in parentheses are ranges. N/A indicates not available. *Non-parametric Mann-Whitney test was performed to compare the average score of MLM and methylene blue. MLM mixture of lipiodol and methylene blue. Table 2 Localization ability score of staining and radio-opacity for MLM as well as methylene blue Data are numbers of subjects. Numbers in parentheses are percentages. MLM mixture of lipiodol and methylene blue. Comparison of localization ability between MLM and methylene blue in total subjects (n = 42) We considered a score of 2 or 3 as appropriate and 3 as excellent for localization respectively. Numbers in parentheses are percentages. *Fisher's exact test compared the proportion of appropriate or excellent staining between the mixture and methylene blue. MLM mixture of lipiodol and methylene blue. Localization ability of MLM: Evaluation of radio-opacity and staining score Data are given as numbers of subjects. Numbers in parentheses are percentages. MLM mixture of lipiodol and methylene blue. Histopathologic findings of lung specimens after percutaneous injections Data are numbers of subjects. Numbers in parentheses are percentages. N/A indicates not available. *Linear by linear association was performed between material and the extent of the histopathologic findings. € Linear by linear association was performed between groups and the extent of the histopathologic findings. MLM mixture of lipiodol and methylene blue. PLoS One PLoS ONE plos plosone PLoS ONE 1932-6203 Public Library of Science San Francisco USA 24819391 4018408 PONE-D-13-46027 10.1371/journal.pone.0096911 Research Biology and Life Sciences Biochemistry Biomarkers Genetics Heredity Medicine and Health Sciences Diagnostic Medicine Epidemiology Biomarker Epidemiology Cancer Epidemiology Health Care Environmental Health Oncology Cancer Risk Factors Environmental Causes of Cancer Pathology and Laboratory Medicine Public and Occupational Health Pulmonology Environmental and Occupational Lung Diseases Single Nucleotide Polymorphism in ATM Gene Cooking Oil Fumes and Lung Adenocarcinoma Susceptibility in Chinese Female Non-Smokers: A Case-Control Study ATM Polymorphism and Risk of Lung Adenocarcinoma Shen Li 1 2 Yin Zhihua 1 2 Wu Wei 1 2 Ren Yangwu 1 2 Li Xuelian 1 2 Zhou Baosen 1 2 * 1 Department of Epidemiology School of Public Health China Medical University Heping District Shenyang Liaoning Province China 2 Key Laboratory of Cancer Etiology and Intervention University of Liaoning Province China Chang Jeffrey S. Editor National Health Research Institutes Taiwan * E-mail: bszhou@mail.cmu.edu.cn Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: LS. Performed the experiments: LS YR XL. Analyzed the data: LS WW ZY. Contributed reagents/materials/analysis tools: LS ZY XL BZ. Wrote the paper: LS. Obtained informed consent from subjects: Baosen Zhou. 2014 12 5 2014 9 5 e96911 3 11 2013 12 4 2014 2014 Shen et al This is an open-access distributed under the terms of the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original author and source are credited. Background The ataxia-telangiectasia mutated (ATM) gene plays an important role in the DNA double-strand breaks repair pathway. Single nucleotide polymorphisms (SNPs) of DNA repair genes are suspected to influence the risk of lung cancer. This study aimed to investigate the association between the ATM -111G>A (rs189037) polymorphism environmental risk factors and the risk of lung adenocarcinoma in Chinese female non-smokers. Methods A hospital-based case-control study of 487 lung cancer patients and 516 matched cancer-free controls was conducted. Information concerning demographic and environmental risk factors was obtained for each case and control by a trained interviewer. After informed consent was obtained 10 ml venous blood was collected from each subject for biomarker testing. Single nucleotide polymorphism was determined by using TaqMan method. Results This study showed that the individuals with ATM rs189037 AA genotype were at an increased risk for lung adenocarcinoma compared with those carrying the GA or GG genotype (adjusted odds ratios (OR) 1.44 95% confidence interval (CI) 1.02€“2.02 P?=?0.039). The stratified analysis suggested that increased risk associated with ATM rs189037 AA genotype in individuals who never or seldom were exposed to cooking oil fumes (adjusted OR 1.89 95%CI 1.03€“3.49 P?=?0.040). Conclusions ATM rs189037 might be associated with the risk of lung adenocarcinoma in Chinese non-smoking females. Furthermore ATM rs189037 AA genotype might be a risk factor of lung adenocarcinoma among female non-smokers without cooking oil fume exposure. This study was supported by grant no. 81272293 from National Natural Science Foundation of China grant no. 81102194 from National Natural Science Foundation of China and grant no. 00726 from China Medical Board. The funders had no role in study design data collection and analysis decision to publish or preparation of the manuscript. Introduction Lung cancer is the leading cause of cancer-related deaths both worldwide and in China. Although cigarette smoke is the major risk factor for lung cancer only a fraction of smokers develop this disease [1] suggesting that host genetic susceptibility may play an important role in the development of lung cancer. Recent genetic susceptibility studies of lung cancer have focused on single nucleotide polymorphisms (SNPs) in candidate genes among which DNA repair genes are increasingly studied because of their critical role in maintain genome integrity. Genetic variations in DNA repair genes are thought to affect DNA repair capacity and deficits in DNA repair capacity may lead to genetic instability and carcinogenesis [2] [3]. "

Lung_Cancer

"Acute phase reactants are plasma proteins that are synthesized by hepatocytes as a nonspesific response against tissue damage infection inflammation trauma or cancer. Acute phase reactants are frequently used in the evaluation of chronic inflammation in diseases such as inflammatory bowel disease or rheumatoid arthritis. Particularly C-reactive protein fibrinogen haptoglobin ferritin ceruloplasmin copper and ?1-antitrypsin can be mentioned among the proteins that show notable increases during acute phase response. On the other hand proteins such as albumin and transferrin demonstrate decreases during the response and are called as œnegative acute phase reactants. Different diseases associated with asbestos may present with different levels of oxidative stress or inflammation. The importance of oxidative stress and inflammation can be assessed by various markers and modalities targeting this system can be established in the treatment and follow-up. In this study we aim to evaluate oxidative markers including TOL TAC OSI and inflammatory indicators and compare their relationship with each other in patients with asbestos exposure having no disease and in patients with asbestos exposure and MM. 2. Material and Methods 2.1. Study Subjects and Area This cross-sectional study was conducted at the Pulmonology Department of Dicle University Diyarbakir Southeastern Turkey. Environmental asbestos exposure is common in the southeast region of Turkey. In this region asbestos-containing soil is used to purpose of thermal insulation and waterproofing on roof and wall (material of whitewash-wall plaster). Environmental exposure occurs through inhalation of asbestos-laden soil. Exposure with asbestos begins at birth in rural areas and the exposure is continuous. Thus patient's age was accepted exposure duration time in our study. We enrolled eighty villagers who have more than 20 years of environmental asbestos exposure were included in the study. Asbestos was detected in village in soil analysis. This village also was in an area that MM patients occure. There was not any finding for MM and other diseases in Chest X-ray in villagers. Forty-six patients with MM who were registered and followed up in our Department of Pulmonology were enrolled the study. The control group was created 50 healthy people with a mean of similar age and gender and who living in an area which not detected asbestos in soil analysis and without any disease. The patients with chronic kidney failure chronic heart failure liver failure and chronic obstructive pulmonary disease and those who have got active infection were excluded the study. The patients with malignancies other than MM were excluded from the study. The study protocol was carried out in accordance with the Helsinki Declaration as revised in 1998 and approved by the local research committee for ethics. All subjects were informed about the study protocol and written consents were obtained from all inhabitants. 2.2. MM Diagnosis Thoracentesis and closed pleural needle biopsy were performed in patients with pleural effusion for pathological and cytological examination. Ramel needle biopsy set was used in closed pleural biopsy. Surgical biopsy was performed when closed pleural biopsy is not appropriate The ultrasound-guided biopsy was performed in patients with small amount of pleural effusion. Tissue samples were immediately placed in 10% formol and sent for histopathological examination. Hematoxylin and eosin staining was used as standard in histopathological evaluation. Histochemical or immunohistochemical staining were used if necessary. The patients with confirmed MM diagnosis histopathologically were included in the study. Certain laboratory clinical and radiographic variables which measured at the time of diagnosis. were defined as potential prognostic factors. 2.3. Blood Sampling To measure TOL and TAC two 10?mL samples of blood were drawn from antecubital veins and were collected in empty tubes. Samples were separated from cells by centrifugation at 4000?rpm for 5?min and then serum was stored at ?80 C until analysis. 2.4. Measurement of TOL TOL of serum was determined using an automated measurement method (Rel assay diagnostics kits MegaTip Gaziantep Turkey) developed by Coussens and Werb [8]. Oxidants present in the sample oxidize the ferrous ion-o-dianisidine complex to ferric ion. The oxidation reaction is enhanced by glycerol molecules which are abundantly present in the reaction medium. The ferric ion makes a colored complex with xylenol orange in an acidic medium. The color intensity which can be measured spectrophotometrically is related to the total amount of oxidant molecules present in the sample. The assay was cali-brated with hydrogen peroxide and the results are expressed as micromoles of hydrogen peroxide equivalents per litre (mmol H2O2?equiv/L). 2.5. Measurement of TAC Serum TAC levels were determined using a novel automated measurement method (Rel assay diagnostics kits Mega Tip Gaziantep Turkey)"

Lung_Cancer

"Therefore the development of novel approaches for the treatment of NSCLC including targeted gene treatment as a radiosensitizer to treat this lethal disease is urgently needed to enhance the survival rate in patients. microRNAs (miRNAs) [11] are a class of short noncoding RNAs that function as a regulation for gene expression via targeting mRNA for degradation or inhibition of translation [12]. miRNAs are new factors implicated in regulating the expression of genes involved in tumorigenic processes such as inflammation cell cycle regulation stress response differentiation apoptosis and invasion and over the past decade they have been found to have key roles in cancers [13“15] including lung cancer [16]. Moreover recent studies have suggested a link between expression of some miRNAs and radiotherapy particularly in lung cancer [17“19]. microRNA-21 (miR-21) is a miRNA which has been reported to be overexpressed in many human malignancies including NSCLC [20“22]. Interestingly miR-21 was found to be upregulated in radiotherapy resistant NSCLC cells relative to radiosensitive counterparts [18]. In addition Wang et al. also reported that comparing with radiotherapy resistant NSCLC patients miR-21 was greatly downregulated in radiotherapy sensitive group [23]. Considering miR-21 as a putative regulator of NSCLC radiotherapy resistance we explore the role of miR-21 in radiotherapy resistance of NSCLC A549 cells and the potential molecular mechanism in the present study. 2. Materials and Methods 2.1. Cell Culture The NSCLC cell line A549 was cultured in Dulbecco's modified Eagle's medium (Invitrogen Carlsbad CA) supplemented with 10% fetal bovine serum 100?U/mL penicillin and 100??g/mL streptomycin. Cell cultures were incubated in a humidified atmosphere of 5% CO2 at 37°C. 2.2. Transfection Anti-miR-21 (5?-UCAACAUCA-GUCUGAUAAGCUA-3?) and the negative control oligonucleotides (NC 5?-CAGUACUUUUG-UGUAGUACAA-3?) were obtained from Ambion Inc. (Austin TX USA). The transfection was performed using LipofectamineTM 2000 (Invitrogen USA) according to the instructions provided by the manufacturer. The transfected cells were resuspended and cultured in regular culture medium for 48?h before analysis. 2.3. Detection of miR-21 by TaqMan Real-Time PCR PCR-based detection of miR-21 was performed by the TaqMan miRNA assays (ABI Forest City CA) as described previously [24 25]. The real-time PCR results recorded as threshold cycle numbers (Ct) were normalized against an internal control (U6 RNA) and then expressed as fold changes [25]. 2.4. Ionizing Radiation 48?h after anti-miR-21 or anti-miR-NC transfection subconfluent cell monolayers were treated with ?-ray ionizing radiation (IR) from a 60Co source (PLA General Hospital Beijing China) at a rate of 2.4?Gy/min. 2.5. Clonogenic Survival Analysis After exposure to various doses of IR cells were trypsinized washed and replated at 200 cells per 10-cm dishes. Cells were grown for 14 days fixed with ethanol and stained with Giemsa to detect colonies. The number of colonies containing at least 50 cells was determined and surviving fractions were calculated. 2.6. MTT Assay Twenty-four hours before IR 200??L cells were seeded to 96-well microtiter plate at 5 — 104 cells/mL. Three days after IR 10??L MTT reagent was added to each well followed by incubation for 4?h at 37°C. The supernatants were aspirated and the reaction was terminated by adding 100??L DMSO. The contents of the plates were mixed for 10?min and the absorbance was read at 490?nm. All experiments were performed three times and the average results were calculated. 2.7. Flow Cytometry Attached cells were harvested at 48?h after IR for apoptosis detection using the annexin V-FITC apoptosis detection kit (Sigma Louis MO). Briefly the cells were washed twice with DPBS and then were resuspended in 1— binding buffer at a concentration of 1 — 106 cells/mL. 5??L of annexin V-FITC conjugate and 10??L of propidium iodide solution were added to 500??L of each cell suspension in a plastic 12?mm — 75?mm test tube followed by incubation at room temperature for 15?min and protection from light. The fluorescence of the cells was determined immediately with a flow cytometer. 10?ng/mL of PI3K activator IGF-1 (Prospec-Tany Rehovot Israel) was used in the apoptosis assay. 2.8. Western Blot Analysis Cells were lysed in lysis buffer (20?mM Tris-HCl pH 7.4 150?mM?NaCl 1% Triton X-100 0.1?mM?EDTA 1?mM?EGTA 2?mM sodium orthovanadate 2?mM?NaF and Complete TM Protease Inhibitor Mix [Roche Applied Science Mannheim Germany]) for 20?min on ice and cleared by centrifugation at 12000?rpm and 4°C. Proteins were resolved on a 10% SDS PAGE gel transferred onto nitrocellulose membranes and blocked with 5% nonfat dry milk in TBST (10?mM Tris-HCl pH 7.5 100?mM?NaCl and 0.05% Tween 20) followed by incubation with a primary antibody [total and anti-phosphorylated-Akt (Ser473) antibody (Cell Signaling Biotechnology Beverly MA USA)]. Blots were washed and incubated with horseradish peroxidase-conjugated secondary antibody. Antibody complexes were visualized using an enhanced chemiluminescence-Western blotting detection system (Thermo Fisher Scientific Inc. Rockford IL USA). 2.9. Statistical Analysis Statistical analysis was performed using SPSS 13.0. The results from three independent experiments were presented as the means ± standard deviation. Statistical analyses were done by Student's t-test. P value < 0.05 was considered statistically significant. 3. Results 3.1. miR-21 Expression Was Knocked down in A549 Cells by Anti-miR-21 Transfection To confirm knockdown efficiency of anti-miR-21 transfection the relative of miR-21 expression level was detected by real-time quantitative RT-PCR. Compared with anti-miR-NC-transfected A549 cells the level of miR-21 expression in anti-miR-21-transfected cells was significantly decreased by about 64% (). 3.2. Downregulation of miR-21 Inhibited Survival Capacity of A549 Cells after IR To assess whether miR-21 downregulation could sensitize NSCLC A549 cells to IR the A549 cells transfected with either anti-miR-NC or anti-miR-21 were irradiated and their response was analysed. In clonogenic survival analysis we observed the expected decreased survival capacity of A549 cells transfected with anti-miR-21 14 days after IR (). Forty-eight hours after transfection A549 cells were treated with various doses of IR (0246 or 8?Gy) and the survival fractions upon IR were detected. As shown in after IR at 46 or 8?Gy the survival fraction of A549 cells in anti-miR-21-transfected group (0.61 ± 0.06 0.43 ± 0.08 and 0.27 ± 0.07 resp.) was significantly lower than that in anti-miR-NC-transfected group (0.83 ± 0.08 0.76 ± 0.11 and 0.65 ± 0.10 resp.) indicating that downregulation of miR-21 could significantly enhance the sensitivity of A549 cells to IR. 3.3. Downregulation of miR-21 Suppressed Proliferation of A549 Cells after IR To confirm the increased IR sensitivity of A549 cells the effect of miR-21 on cell proliferation was further analysed at 72?h after IR (). Downregulation of miR-21 expression was found to reduce cell proliferation as demonstrated by the decreased proliferation index of cells transfected with anti-miR-21 compared with anti-miR-NC (75.6 ± 18.96% versus 100% P < 0.05). Importantly a more pronounced growth inhibition of A549 cells was found when miR-21 was knocked down in combination with IR. This inhibition of cell growth in the combined treatment (anti-miR-21 + IR) was found to be significantly higher compared with that in the sole IR treatment group (proliferation index: 36.1 ± 8.48% versus 73.2 ± 21.37% P < 0.05 ). This indicates that knockdown of miR-21 sensitizes radioresistant NSCLC A549 cells to radiation. 3.4. Downregulation of miR-21 Enhanced Apoptosis of A549 Cells Induced by IR We next explored the role of miR-21 in the apoptosis of NSCLC A549 cells induced by IR. Anti-miR-21 or anti-miR-NC was transfected into A549 cells and was exposed (or sham exposed) to 8?Gy of IR. As shown in the percentage of apoptosis cells in miR-21 knockdown group (anti-miR-21) was significantly higher than that of negative control group (anti-miRNA-NC) at the dose 8?Gy (61.5 ± 15.62 versus 21.2 ± 5.35 P < 0.05) indicating that miR-21 knockdown may enhance radiosensitivity of A549 cells by promoting apoptosis and thus confirm a role for miR-21 in the regulation of radiotherapy response of NSCLC. 3.5. Downregulation of miR-21 Inactivated PI3K/Akt Signaling Pathway Induced by IR Because the PI3K/Akt signaling pathway is associated with apoptosis we subsequently examined the potential effects of miR-21 on the activation of PI3K/Akt pathways by IR to explore the potential molecular mechanisms. The activation of PI3K/Akt signaling pathways was measured by Akt phosphorylation on Ser473. By Western blot we found that the endogenous level of phospho-Akt expression (Ser473) in anti-miR-21-transfected A549 cells was downregulated compared to that in anti-miR-NC-transfected A549 cells after IR (Figure 5). Interestingly phospho-Akt (Ser473) expression was significantly increased in the case of being treated with IGF-1 a PI3K activator in anti-miR-NC-transfected A549 cells and even in anti-miR-21-transfected A549 cells after IR (Figure 5). This suggested that activation of PI3K/Akt signaling pathway by IR in A549 cells was suppressed by knockdown of miR-21 and the suppression was reversed by PI3K activator IGF-1. 3.6. miR-21 Knockdown Caused Promotion on Apoptosis Induced by IR Was Mediated by PI3K/Akt Signaling Pathway To further confirm the molecular mechanisms of radiosensitization by miR-21 knockdown in NSCLC A549 cells we next treated the cells with or without PI3K activator IGF-1 and then examined the effects of miR-21 downregulation on cell apoptosis induced by IR. As shown in Figure 6 without IGF-1 treatment the cell apoptosis induced by IR was significantly increased in anti-miR-21-transfected A549 cells (61.5 ± 15.62%) compared with that in anti-miR-NC-transfected A549 cells (21.2 ± 5.35% P < 0.05). However after activation of PI3K/Akt signaling pathway the cell apoptosis induced by IR was inhibited in either anti-miR-21-transfected or anti-miR-NC-transfected A549 cells. The percentage of cell apoptosis was not significantly different between these two groups (18.1 ± 5.55% versus 18.3 ± 5.15% P > 0.05). These data showed that in the condition of PI3K/Akt activation knockdown of miR-21 did not promote the apoptosis of A549 cells induced by IR suggesting that PI3K/Akt signaling pathway was the downstream target of miR-21 and the promotive effects of miR-21 knockdown on apoptosis induced by IR were mediated by PI3K/Akt signaling pathway. 4. Discussion It is well known that the acquisition of resistance to radiotherapy which greatly increases patient morbidity and mortality is a significant problem in the treatment of NSCLC. Effective treatment which can sensitize the radioresistant NSCLC to radiotherapy is always being sought. Recently some miRNAs were found to be related to radioresistance. Among them miR-21 is reported to play a role in radioresistance of cancer including glioblastoma [26 27] breast cancer [28] and rectal cancer [29]. But up to now few researches have studied the correlations between miR-21 expression and radiotherapy sensitivity of NSCLC. Liu et al. reported that miR-21 expression promotes radioresistance in NSCLC but the related molecular mechanisms were not revealed [30]. The roles of miR-21 in the radiotherapy response of NSCLC are not fully understood and remain to be elucidated. Thus in the current study we investigated whether miR-21 could affect the radiosensitivity of NSCLC A549 cells and found that downregulation of miR-21 significantly enhanced the sensitivity of A549 cells to radiotherapy through inhibition of PI3K/Akt signaling pathway. Our data showed that following the transfection of anti-miR-21 into A549 cells the inhibition of survival fraction caused by various doses of IR was enhanced compared with radiotherapy alone. This result suggests that miR-21 is closely associated with the therapeutic efficiency of IR on radioresistant A549 cells and downregulation of miR-21 may sensitize A549 cells to IR. It is reported that miR-21 could stimulate growth in NSCLC [30 31]. Accordingly we also found that the proliferation of A549 cells was inhibited after miR-21 knockdown. Moreover the inhibition of cell proliferation induced by combination of miR-21 knockdown and IR was more pronounced compared with either miR-21 knockdown or IR treatment indicating that miR-21 knockdown plays a crucial role in the combined inhibition of cell proliferation and silencing miR-21 may increase the sensitivity of A549 cells to IR. Cell apoptosis induced by IR is one of the most important effects of tumor radiotherapy. Furthermore miR-21 is reported to be an antiapoptotic factor in lung cancer [32 33]. So we hypothesized that it is possible that miR-21 could affect the apoptosis of NSCLC induced by IR. Our current results demonstrated that miR-21 knockdown promoted apoptosis of A549 cells induced by IR indicating that the expression of miR-21 could affect radiosensitivity of NSCLC cells which might be associated with inhibition of apoptosis. This is also in agreement with the previous report [30]. To explore the potential molecular mechanisms of radiosensitization by miR-21 knockdown in NSCLC A549 cells we focused on analysis of PI3K/Akt signaling pathway because the influence of PI3K/Akt signaling pathway on IR-induced apoptotic propensity is well documented [34 35]. We examined whether downregulation of miR-21 could affect Akt phosphorylation at Ser473 and/or its total expression and found that miR-21 knockdown suppressed the activation of PI3K/Akt signaling pathway by IR in A549 cells. In addition the apoptosis induced by IR was enhanced in A549 cells after miR-21 knockdown. This data indicates that the stimulative effects of miR-21 knockdown on A549 cell apoptosis induced by IR are related to the inactivation of PI3K/Akt signaling pathway. Furthermore with the treatment of PI3K activator IGF-1 we found that the apoptosis of A549 cells induced by IR was not promoted even if miR-21 was downregulated. Our results suggest that the promotive effects of miR-21 knockdown on A549 cell apoptosis induced by IR depend on the inactivation of PI3K/Akt signaling pathway. In the current study how miR-21 interplays with PI3K/Akt signaling pathway under our experimental conditions is not clear. However it is reported that molecules such as PTEN have been proposed to be involved in NSCLC cells' radioresistance [36 37] and miR-21 is related to PTEN with high possibility [30 38]. In addition since PTEN PI3K and Akt are closely related it is one of the possible mechanisms that PTEN may play a role in PI3K/Akt signaling pathway mediated radiosensitization of A549 cells by miR-21 knockdown but this still needs further comfirmation in future studies. In summary the present study found that downregulation of miRNA-21 sensitized radioresistant NSCLC A549 cells to IR by inhibiting cell proliferation and enhancing apoptosis through inhibition of PI3K/Akt signaling pathway. This information may be useful to develop new treatments for the clinical therapy of NSCLC patients. Further analysis on targets of miR-21 is still of considerable interest as they may reveal novel radiotherapy sensitization strategies for radioresistant NSCLC. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. 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2-s2.0-79956276306 21378080 7 Danesi R Pasqualetti G Giovannetti E Pharmacogenomics in non-small-cell lung cancer chemotherapy Advanced Drug Delivery Reviews 2009 61 5 408 417 2-s2.0-67349279836 19292993 8 R¶del F Hoffmann J Distel L Survivin as a radioresistance factor and prognostic and therapeutic target for radiotherapy in rectal cancer Cancer Research 2005 65 11 4881 4887 2-s2.0-19644370614 15930309 9 Lee S Lim MJ Kim MH An effective strategy for increasing the radiosensitivity of Human lung Cancer cells by blocking Nrf2-dependent antioxidant responses Free Radical Biology & Medicine 2012 53 807 816 22684019 10 Provencio M S¡nchez A Garrido P Valc¡rce F New molecular targeted therapies integrated with radiation therapy in lung cancer Clinical Lung Cancer 2010 11 2 91 97 2-s2.0-77952511165 20199974 11 Lee RC Feinbaum RL Ambros V The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14 Cell 1993 75 5 843 854 2-s2.0-0027751663 8252621 12 Iorio MV Croce CM MicroRNAs in cancer: small molecules with a huge impact Journal of Clinical Oncology 2009 27 34 5848 5856 2-s2.0-73349125465 19884536 13 White NMA Fatoohi E Metias M Jung K Stephan C Yousef GM Metastamirs: a stepping stone towards improved cancer management Nature Reviews Clinical Oncology 2011 8 2 75 84 2-s2.0-79551602747 14 Farazi TA Spitzer JI Morozov P Tuschl T MiRNAs in human cancer Journal of Pathology 2011 223 2 102 115 2-s2.0-78650034475 21125669 15 Croce CM Causes and consequences of microRNA dysregulation in cancer Nature Reviews Genetics 2009 10 10 704 714 2-s2.0-70349320158 16 Ortholan C Puissegur M-P Ilie M Barbry P Mari B Hofman P MicroRNAs and lung cancer: new oncogenes and tumor suppressors new prognostic factors and potential therapeutic targets Current Medicinal Chemistry 2009 16 9 1047 1061 2-s2.0-65649152994 19275611 17 Oh J-S Kim J-J Byun J-Y Kim I-A Lin28-let7 modulates radiosensitivity of human cancer cells with activation of K-ras International Journal of Radiation Oncology Biology Physics 2010 76 1 5 8 2-s2.0-72049101611 18 Salim H Akbar NS Zong D miRNA-214 modulates radiotherapy response of non-small cell lung cancer cells through regulation of p38MAPK apoptosis and senescence British Journal of Cancer 2012 107 1361 1373 22929890 19 Yan D Ng WL Zhang X Targeting DNA-PKcs and ATM with miR-101 sensitizes tumors to radiation PLoS ONE 2010 5 7 article e11397 2-s2.0-77958133262 20 Markou A Sourvinou I Vorkas PA Yousef GM Lianidou E Clinical evaluation of microRNA expression profiling in non small cell lung cancer Lung Cancer 2013 81 388 396 23756108 21 Li S Liang Z Xu L Zou F MicroRNA-21: a ubiquitously expressed pro-survival factor in cancer and other diseases Molecular and Cellular Biochemistry 2012 360 1-2 147 158 2-s2.0-83555164788 21909994 22 Yang M Shen H Qiu C High expression of miR-21 and miR-155 predicts recurrence and unfavourable survival in non-small cell lung cancer European Journal of Cancer 2013 49 604 615 23099007 23 Wang X-C Du L-Q Tian L-L Expression and function of miRNA in postoperative radiotherapy sensitive and resistant patients of non-small cell lung cancer Lung Cancer 2011 72 1 92 99 2-s2.0-79952195451 20728239 24 Lao K Xu NL Yeung V Chen C Livak KJ Straus NA Multiplexing RT-PCR for the detection of multiple miRNA species in small samples Biochemical and Biophysical Research Communications 2006 343 1 85 89 2-s2.0-33645106895 16529715 25 Chen C Ridzon DA Broomer AJ Real-time quantification of microRNAs by stem-loop RT-PCR Nucleic Acids Research 2005 33 20 article e179 2-s2.0-29144470346 26 Griveau A Bejaud J Anthiya S Avril S Autret D Garcion E Silencing of miR-21 by locked nucleic acid-lipid nanocapsule complexes sensitize human glioblastoma cells to radiation-induced cell death International Journal of Pharmaceutics 2013 454 765 774 23732394 27 Li Y Zhao S Zhen Y A miR-21 inhibitor enhances apoptosis and reduces G2-M accumulation induced by ionizing radiation in human glioblastoma U251 cells Brain Tumor Pathology 2011 28 3 209 214 2-s2.0-80955180085 21618027 28 Anastasov N Hofig I Vasconcellos IG Radiation resistance due to high expression of miR-21 and G2/M checkpoint arrest in breast cancer cells Radiation Oncology 2012 7 article 206 29 Drebber U Lay M Wedemeyer I Altered levels of the onco-microRNA 21 and the tumor-supressor microRNAs 143 and 145 in advanced rectal cancer indicate successful neoadjuvant chemoradiotherapy International Journal of Oncology 2011 39 2 409 415 2-s2.0-79959960546 21567082 30 Liu ZL Wang H Liu J Wang ZX MicroRNA-21 (miR-21) expression promotes growth metastasis and chemo- or radioresistance in non-small cell lung cancer cells by targeting PTEN Molecular and Cellular Biochemistry 2013 372 35 45 22956424 31 Zhang J-G Wang J-J Zhao F Liu Q Jiang K Yang G-H MicroRNA-21 (miR-21) represses tumor suppressor PTEN and promotes growth and invasion in non-small cell lung cancer (NSCLC) Clinica Chimica Acta 2010 411 11-12 846 852 2-s2.0-77956061393 32 Hatley ME Patrick DM Garcia MR Modulation of K-Ras-dependent lung tumorigenesis by MicroRNA-21 Cancer Cell 2010 18 3 282 293 2-s2.0-77956501846 20832755 33 Seike M Goto A Okano T MiR-21 is an EGFR-regulated anti-apoptotic factor in lung cancer in never-smokers Proceedings of the National Academy of Sciences of the United States of America 2009 106 29 12085 12090 2-s2.0-67749110399 19597153 34 Qiu W Leibowitz B Zhang L Yu J Growth factors protect intestinal stem cells from radiation-induced apoptosis by suppressing PUMA through the PI3K/AKT/p53 axis Oncogene 2010 29 11 1622 1632 2-s2.0-77949655884 19966853 35 Lei Y Li HX Jin WS The radiosensitizing effect of Paeonol on lung adenocarcinoma by augmentation of radiation-induced apoptosis and inhibition of the PI3K/Akt pathway International Journal of Radiation Biology 2013 89 12 1079 1086 23875954 36 Kim EJ Jeong JH Bae S Kang S Kim CH Lim YB mTOR inhibitors radiosensitize PTEN-deficient non-small-cell lung cancer cells harboring an EGFR activating mutation by inducing autophagy Journal of Cellular Biochemistry 2013 114 1248 1256 23592446 37 Jung IL Kang HJ Kim KC Kim IG PTEN/pAkt/p53 signaling pathway correlates with the radioresponse of non-small cell lung cancer International Journal of Molecular Medicine 2010 25 4 517 523 2-s2.0-77749289244 20198299 38 Roy S Yu Y Padhye SB Sarkar FH Majumdar AP Difluorinated-curcumin (CDF) restores PTEN expression in colon cancer cells by down-regulating miR-21 PLoS One 2013 8 article e68543 miR-21 expression was knocked down by transfecting NSCLC A549 cells with anti-miR-21. miR-21 expression in A549 cells at 48?h after transfection with anti-miR-NC or anti-miR-21 was detected by TaqMan real-time quantitative RT-PCR. The mean and standard deviation of expression levels relative to U6 expression levels are shown and are normalized to the expression in A549 cells transfected with anti-miR-NC. All experiments were performed at least in triplicate. *P < 0.05 versus cells transfected with anti-miR-NC. Clonogenic survival of NSCLC A549 cells after varying doses of ionizing radiation. A549 cells were transfected with either anti-miR-21 or anti-miR-NC and 48?h later were irradiated followed by a further incubation for 24?h at 37°C before trypsinization and plating for clonogenic survival. After 14-day incubation colonies were stained and the surviving fractions were determined. *P < 0.05 versus cells transfected with anti-miR-NC. Each value represents the means ± SD for three independent experiments. Proliferation of NSCLC A549 cells after ionizing radiation (IR). A549 cells were transfected with either anti-miR-21 or anti-miR-NC and 48?h later were exposed to 8?Gy of IR and the growth characteristics of A549 cells were determined by MTT assay 72 hours after IR. The anti-miR-NC-transfected sample was normalized to 100% cell viability. The data represent the means ± SD of three separate experiments. Student's t-test was used to analyze the statistics (*P < 0.05)."

Lung_Cancer

"The Hsp70/actin ratios are shown in the lower panel. (b) Effect of ibuprofen on the expression of Hsp70 protein and mRNA in A549 cells. Top: expression of Hsp70 and Hsc70 proteins in A549 cells treated with ibuprofen at the specified concentrations for 48?h examined as described in a (left panel). Bottom: densitometric analysis of each protein level in arbitrary unit (arb-u). The alternation of each mRNA expression after ibuprofen treatment was analyzed by semiquantitative RT-PCR (top right panel). These results are representative of three separate experiments. (c) Effect of ibuprofen on the expression of Hsp70 protein in H358 cells. Expression of Hsp70 proteins in H358 cells treated with ibuprofen at the specified concentrations for 48?h (upper panel). The quantity of each protein was estimated by densitometric analysis (lower panel) Ibuprofen increased the antitumoural activity of cisplatin by suppressing Hsp70. (a) The viability of A549 (upper panel) and H358 (lower panel) cells treated with ibuprofen for 48?h was analyzed by MTT assay. The value is represented as the percentage of cell viability without ibuprofen set at 100%. (b) Synergistic effect of ibuprofen on cisplatin-induced apoptosis in A549 (upper panel) or H358 (lower panel) cells. The cells were treated with the specified concentrations of cisplatin in absence or presence of 400??M ibuprofen for 48?h and the cell viability was assessed by MTT assay. The results are shown as means±S.D. from triplicated experiments. The results shown are representative of three separate experiments. *P<0.05; **P<0.01 (by Student's t-test). (c) Time course of cisplatin-mediated cell death with ibuprofen. A549 cells were treated with 400??M ibuprofen alone or A549 cells with 10??M cisplatin were cultured in absence or presence of 400??M ibuprofen and the cell viability was analyzed by TUNEL staining. The results shown as means±S.D. **P<0.01 (by Student's t-test). (d) The silencing efficiency of Hsp70 determined by immunoblotting. (e) Effect of Hsp70 RNAi on the cisplatin-mediated death of A549 cells. The cells exposed to siRNA targeting Hsp70 or control siRNA were treated with 10??M cisplatin for 48?h and MTT assay was used to determine the cell viability. Data are presented as means±S.D. from triplicated experiments. The results shown are representative of three separate experiments. *P<0.05; **P<0.01 (by Student's t-test). (f) Cytofluorimetric dot plot analysis of the CF488A-Annexin V versus propidium iodide (PI) staining performed in 10??M cisplatin-treated or -untreated A549 cells in absence or presence of 400??M ibuprofen for 48?h. A representative experiment out of three performed with similar results is shown. (g) Effect of cisplatin on the expression of Hsp70. The data are representative of three separate experiments Ibuprofen inhibits the expression of Hsp70 by transcriptional inactivation. (a) ChIP assay for the association of HSF-1 with Hsp70 gene in A549 cells treated with or without ibuprofen. The DNA in the product immunoprecipitated by anti-HSF-1 or non-immune IgG was followed by PCR with a primer specific to the Hsp70 promoter. The immunoprecipitates with antibody against HSF-1 were confirmed by immunoblotting (bottom left). The actin signal is a control of DNA input (bottom right). (b) Effect of ibuprofen on the expression of HSF-1. Cell extracts from A549 cells with ibuprofen for 48?h were separated by SDS-PAGE and immunoblotted with a HSF-1-specific antibody (upper panel). The quantity of each protein was estimated by densitometric analysis (middle panel). The data are representative of three separate experiments. The effect of ibuprofen on the mRNA level of HSF-1 was confirmed by RT-PCR (lower panel). (c) HSF1-mediated inhibition of Hsp70 expression. A549 cells were treated with 10?nM siRNAs against HSF-1 or non-code siRNA. The HSF-1 silencing efficiency and its effect on the expression of Hsp70 were examined by immunoblotting using appropriate antibodies. Three separate siRNAs oligo against HSF-1 were used for its knock-down. The results shown are representative of three separate experiments Ibuprofen exposure enhanced the cisplatin-dependent mitochondrial membrane depolarization and cytochrome c release. (a) A549 cells were treated for 48?h with 10??M cisplatin 400??M ibuprofen or both and subjected to JC-1 staining to study the changes in mitochondrial membrane potential. The percentages indicate the green fluorescence intensity of JC-1 measuring with FACSCalibur. A representative experiment out of three performed with similar results is shown. (b) A549 cells treated as described earlier were fractionated into cytosol and the release of cytochrome c was analyzed by western blot using anti-cytochrome c antibody. The expression of Erk was monitored as an internal control of cytosol protein. The quantity of each protein was estimated by densitometric analysis. The results are means of three separate experiments from cells in different cultures The downregulation of Hsp70 increased the cisplatin-mediated activation of Bax and its translocation to the mitochondria. (a) Detection of active Bax. A549 cells were treated with cisplatin (10??M) and/or ibuprofen (400??M) for 48?h. Active Bax was immunoprecipitated with an active conformation-specific monoclonal antibody and revealed by immunoblotting with an anti-Bax polyclonal antibody. The quantity of active Bax was estimated by densitometric analysis. (b) A549 cells treated as described earlier were lysed and fractionated by differential centrifugation to separate the mitochondria from the cytosol. The translocation of Bax to the mitochondria was visualized by the immunoblot of mitochondrial fractions using an anti-Bax antibody. VDAC-1 was used as a loading control to ensure the use of equal amounts of mitochondria. (c) A decrease in Hsp70 by RNAi promoted cisplatin-dependent activation of Bax. A549 cells treated with Hsp70 or control siRNA were incubated in presence or absence of cisplatin; each cell extract was immunoprecipitated with an anti-active Bax antibody followed by immunoblotting with anti-Bax antibody. The data are representative of three separate experiments Synergistic effect of Hsp70 suppression on the cisplatin-mediated activation of caspase-9. (a) A549 cells were treated with cisplatin and/or ibuprofen and cell extracts were immunoblotted with active caspase-9 antibody. The lower panel shows the measurement of each caspase-9. (b) A549 cells exposed to siRNA targeting Hsp70 or control siRNA were incubated with or without cisplatin and the active caspase-9 was detected by western blot using an anti-caspase-9 antibody. The quantity of each protein was estimated by densitometric analysis (lower panels). (c and d). Assay for enzymatic activity of caspase-9 using a fluorogenic substrate. (c) After the incubation of the A549 cells with cisplatin (10??M) and/or ibuprofen (400??M) the caspase-9 activity of each cell extract was measured as described in Materials and methods section. (d) A549 cells transfected with Hsp70 siRNA or control siRNA were exposed to cisplatin for 48?h. The caspase-9 activity was then assessed using an enzymatic assay as described earlier. The value of caspase-9 activity was presented relative to the activity in untreated cells set at 1.0. The data represent mean values of three separate experiments. Significances were determined by Student's t-test (*P<0.05) Effects of nonsteroidal anti-inflammatory drugs on the expression of Hsp70 in A549 cells NSAIDs Hsp70 expression (%) Ibuprofen (400??M) 22.7±2.8 Aspirin (2500??M) 95.1±7.8 Diclofenac (200??M) 97.2±5.6 Sulindac (15??M) 98.9±2.9 Piroxicam (60??M) 96.6±6.2 Indometacin (10??M) 95.0±15.1 Mefenamic acid (25??M) 100.5±6.0 Values are shown as means±S.D. The expression of Hsp70 was measured by immunoblotting with an anti-Hsp70 antibody. The quantity of Hsp70 protein was estimated by densitometric analysis using Scion Image. The values in parentheses are the highest non-toxic concentrations (approximately 90% viability) used for each NSAID on the A549 cells for 48?h Effects of nonsteroidal anti-inflammatory drugs on the expression of HSF-1 in A549 cells NSAIDs HSF-1 expression (%) Ibuprofen (400??M) 16.2±3.9 Aspirin (2500??M) 93.5±2.9 Diclofenac (200??M) 96.7±6.6 Sulindac (15??M) 99.8±3.6 Piroxicam (60??M) 96.3±4.7 Indometacin (10??M) 98.1±1.6 Mefenamic acid (25??M) 98.5±1.1 Values are shown as means±S.D. The expression of HSF-1 was measured by immunoblotting with anti-HSF-1 antibody. The quantity of HSF-1 protein was estimated by densitometric analysis using Scion Image. The values in parentheses are the highest non-toxic concentrations (approximately 90% viability) used for each of the NSAID on the A549 cells for 48?h PLoS One PLoS ONE plos plosone PLoS ONE 1932-6203 Public Library of Science San Francisco USA 24505298 3914822 PONE-D-13-39952 10.1371/journal.pone.0087629 Research Article Biology Population Biology Epidemiology Medicine Clinical Research Design Epidemiology Drugs and Devices Drug Research and Development Epidemiology Cancer Epidemiology Clinical Epidemiology Oncology Cancers and Neoplasms Lung and Intrathoracic Tumors Non-Small Cell Lung Cancer Cancer Treatment Radiology Nuclear Medicine PET imaging [18F]FDG Positron Emission Tomography within Two Weeks of Starting Erlotinib Therapy Can Predict Response in Non-Small Cell Lung Cancer Patients Theranostic Use of FDG-PET in NSCLC Patients Hachemi Mammar 1 2 Couturier Olivier 1 2 3 Vervueren Laurent 1 2 Fosse Pacôme 1 2 Lacœuille Franck 1 2 3 Urban Thierry 1 3 4 5 Hureaux José 1 3 4 5 * 1 LUNAM Université 49 000 Angers France 2 Université d'Angers CHU Angers Pôle de Radiologie Service de Médecine Nucléaire Angers France 3 INSERM UMR_S 1066 Micro et Nanomédecines Biomimétiques Angers France 4 Université d'Angers CHU Angers Pôle des Spécialités Médicales et Chirurgicales Intégrées Département de Pneumologie Angers France 5 Université d'Angers Equipe Pyver Angers France Singh Pankaj K. Editor University of Nebraska Medical Center United States of America * E-mail: JoHureaux@chu-angers.fr Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: OC TU JH. Performed the experiments: MH OC LV PF FL JH. Analyzed the data: MH OC FL TU JH. Contributed reagents/materials/analysis tools: MH OC TU JH. Wrote the paper: MH OC JH. 2014 5 2 2014 9 2 e87629 29 9 2013 26 12 2013 2014 Hachemi et al This is an open-access article distributed under the terms of the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original author and source are credited. Purpose The aim of this prospective study was to evaluate whether [18F]FDG-PET/CT performed within two weeks of starting erlotinib therapy can predict tumor response defined by RECIST 1.1 criteria after 8 weeks of treatment in patients with inoperable (stage IIIA to IV) non-small cell lung cancer patients. Patients and Methods Three [18F]FDG-PET/CT scans were acquired in 12 patients before (5±4 days) and after 9±3 days (early PET) and 60±6 days (late PET) of erlotinib therapy. Conventional evaluation including at least chest CT (baseline versus after 8 weeks of treatment) was performed according to RECIST 1.1 criteria. Change in [18F]FDG uptake was compared with conventional response progression-free survival (PFS) and overall survival (OS). Results By using ROC analysis the Area Under the Curve for prediction of metabolic non-progressive disease (mNP) by early PET was 0.86 (95% CI 0.62 to 1.1; P?=?0.04) at a cut-off of 21.6% reduction in maximum Standardized Uptake Value (SUVmax). This correctly classified 11/12 patients (7 with true progressive disease; 4 with true non-progressive disease; 1 with false progressive disease). Non-progressive disease after 8 weeks of treatment according to RECIST 1.1 criteria was significantly more frequent in patients classified mNP (P?=?0.01 Fisher's exact test). mNP patients showed prolonged PFS (HR?=?0.27; 95% CI 0.04 to 0.59; P<0.01) and OS (HR?=?0.34; 95% CI 0.06 to 0.84; P?=?0.03). Late PET analysis provided concordant results. Conclusion Morphologic response PFS and OS survival in non-small cell lung cancer patients can be predicted by [18F]FDG-PET/CT scan within 2 weeks after starting erlotinib therapy. The authors have no support or funding to report. Introduction Lung cancer is the leading cause of cancer-related death in both Europe[1] and the United States of America.[2] The most common forms of lung cancer are non-small cell lung cancer (NSCLC) histological subtypes. Systemic chemotherapy has contributed to a significant improvement in NSCLC therapy but progress appears to be stagnating.[3] [4] Over the last decade a better knowledge of cellular pathways has allowed the development of new therapies based on NSCLC-driving genetic abnormalities. Targeted therapies have been developed to block pathological cellular pathways involved in cancer cell survival proliferation and metastasis. Epidermal Growth Factor Receptor (EGFR) is overexpressed in NSCLC[5] and has been extensively studied as a potential therapeutic target. Two EGF Receptor blockers gefitinib and erlotinib have been demonstrated to be effective in front-line therapy in patients with inoperable NSCLC harboring EGFR-activating mutations.[6] [7] Erlotinib is also authorized after failure of previous chemotherapy and as maintenance therapy.[8] [9] In clinical practice evaluation of tumor response is based on changes in tumor size according to criteria proposed by the World Health Organization[10] or RECIST criteria.[11] [12] This morphological evaluation may lead to underestimation of the efficacy of cytostatic therapeutic agents such as erlotinib that stabilize the disease in non-mutated patients whereas conventional cytotoxic drugs induce shrinkage of tumor dimensions in the case of tumor response. NSCLC tumor size evaluation can also be difficult due to atelectasis of normal lung. The major limitations to morphological imaging methods are their inability to assess response to therapy at an early stage and their inability to identify cancer in residual masses after treatment. In patients with NSCLC [18F]FDG-PET has been recognized as an adequate staging tool[13] [14] and several studies also suggest that the standardized uptake value (SUV) has a prognostic value in NSCLC.[15] [16] The value of SUV for evaluation of tumor response to targeted therapy is currently being investigated. We designed a preliminary study to evaluate tumor response in NSCLC patients eligible for erlotinib therapy. The aim of this prospective study was to determine whether [18F]FDG-PET/CT performed several days after starting erlotinib therapy could predict tumor response defined by RECIST 1.1 criteria and [18F]FDG-PET/CT after 8 weeks of treatment. Materials and Methods Patients Twelve consecutive eligible patients with stage IIIA to IV NSCLC (7 adenocarcinomas 3 large cell carcinomas 2 squamous cell carcinomas) in whom erlotinib therapy was indicated were studied at the Angers University Hospital France. Screening for EGF receptor mutations was carried out (patient characteristics are shown in ). Eligibility criteria were: histologically or cytologically proven NSCLC; unresectable stage III/IV disease or recurrent disease after surgery; age over 18 years; measurable disease according to RECIST 1.1 criteria; Eastern Cooperative Oncology Group (ECOG) performance status between 0 to 2; adequate bone marrow function liver function and renal function. Patients were not included if they had previous lung diseases such as interstitial pneumonitis or lung fibrosis identified by chest Computed Tomography (CT) scan or diabetes mellitus that could artefact PET imaging. Life expectancy was predicted to be longer than 12 weeks. Erlotinib was administered orally in a dosage of 150 mg/day on an empty stomach until clinical disease progression unacceptable toxicity or patient refusal. The medical ethics committee of the CHU of Angers approved the study protocol. All patients gave informed written consent before inclusion according to local medical ethical committee regulations and in accordance with the guidelines established by the World Medical Association Declaration of Helsinki. 10.1371/journal.pone.0087629.t001 Clinical characteristics of the study population. Patients Male 6 (50) Female 6 (50) Total 12 (100) Histology Adenocarcinoma 7 (58) Large cell carcinoma 3 (25) Squamous cell carcinoma 2 (17) Clinical stage IIIA or IIIB 2 (17) IV 10 (83) Smoking status Current 5 (42) Former 2 (17) Never 5 (42) EGFR mutation status Presence 2 (17) Absence 10 (83) Previous chemotherapy Yes 10 (83) No 2 (17) Size of primary tumor (cm) 1.0€“2.0 4 (33) 2.1€“3.0 3 (25) 3.1€“5.0 5 (42) >5.1 1 (8) Metastasis Lymph nodes 12 (100) Lung 4 (33) Liver 2 (17) Bone 4 (33) Adrenal glands 0 Work Plan (study design) [18F]FDG PET/CT imaging Three [18F]FDG PET/CT scans were planned: PET1 before starting therapy PET2 within 2 weeks after starting therapy and a third [18F]FDG PET/CT scan (PET3) 8 weeks after starting erlotinib therapy. PET/CT examinations were obtained in 2D mode from the vertex to mid-thighs (5 minutes of emission scan per bed position with an average of 7 bed positions at 15 cm intervals) (Discovery-ST GE Healthcare France). Patients were instructed to fast for at least 6 hours prior to scanning. Unenhanced CT scan was performed from the skull base to the upper thighs. CT parameters were 120 kVp 100 mAs 0.8 second rotation 3.27 mm slice collimation and Pitch 1.5. CT data were used for attenuation correction and PET images were reconstructed by clinical standard 2D-iterative algorithm (ordered subset expectation maximization using 4 iterations and 16 subsets; zoom 100%; image matrix size: 128×128; and Gaussian post-smoothing of 5 mm in full width at half maximum). No corrections for partial volume effect lean body mass or blood glucose levels were applied. Conventional evaluation Conventional staging and follow-up were performed according to standards of care.[11] [12] Conventional evaluation included at least clinical examination plus CT scan performed before (CT1; 7±6 days) and 8 weeks after (CT2; 58±8 days) starting erlotinib therapy. None of the patients underwent additional CT scanning during the 2 weeks after starting erlotinib therapy. Chest abdomen and pelvis CT scans (Brillance 64 PHILIPS Medical System® France) were acquired from the lung apex to the symphysis pubis after an intravenous embolus of 130 mL of iodinated contrast agent (Xenetix350®). Helical scanning parameters were 130 kVp 120 mAs 1 second rotation 4 mm slice collimation 8 mm/s bed speed and 3 mm section width. Image analysis and response evaluation CT data were interpreted by two experienced physicians specialists in thoracic oncology blinded to PET/CT results according to the Response Evaluation Criteria in Solid Tumors (RECIST 1.1 criteria[12]) by comparison of baseline CT scan (CT1) and final CT scan (CT2). Therapeutic response evaluation was defined as: 1) complete response (CR: disappearance of all target lesions); 2) partial response (PR: at least 30% decrease in the sum of the longest diameter of five target lesions); 3) progressive disease (PD: at least a 20% increase in the sum of the longest diameter of five target lesions); and 4) stable disease (SD: neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD). Patients were then classified in the progressive disease (P) group or the non-progressive disease (NP) group including CR PR and SD therapeutic response. [18F]FDG PET interpretation was performed on an Imagys® workstation (Keosys Saint-Herblain France) qualitatively and semi-quantitatively by two experienced nuclear medicine physicians blinded to clinical and conventional evaluation results. Any focus of increased [18F]FDG uptake over background not located in areas of normal [18F]FDG uptake and/or [18F]FDG excretion was considered to be positive for tumor. For semi-quantitative analyses of [18F]FDG uptake 3D regions of interest (VOIs) were placed over all lesions considered to be positive for tumor by using Imagys® software (Keosys France). The maximum standardized uptake value (SUVmax) was calculated using the single hottest pixel inside the tumor VOI. SUV peak was also calculated using a 1.2 cm diameter spherical VOI containing the SUVmax. Bone lesions were not taken into account as they were considered to be non-measurable lesions. For patients with more than one tumor lesion the sum of SUVmax and SUVpeak were calculated and used for evaluation of changes between PET1 and PET2. PET measurements were performed in up to a maximum of five measurable target lesions. All SUVs were normalized to the injected dose and patient body weight. The percentage changes in SUV between PET1 and PET2 were finally calculated as follows: ?SUV?=?(SUV1?SUV2)/SUV1. The same protocol was used for PET1 and PET3. Statistical analysis Data are expressed as mean±SD excepted for survival data that were expressed as the median. The primary endpoint of the study was comparison of changes in tumor [18F]FDG uptake on PET2 versus PET1 PET3 versus PET1 and subsequent CT scan evaluation at 8 weeks after initiation of erlotinib therapy. Friedman test was used for non-parametric comparison of repeated measures. The secondary endpoints were to determine the Receiver Operating Characteristic (ROC) analysis for [18F]FDG changes with regard to predicting response to erlotinib therapy. The relationship between metabolic response (patients stratified according to the median value of SUV variations) and clinical response was analyzed by Fisher's exact test. Progression-free survival (PFS) and overall survival (OS) were determined by standard Kaplan-Meier survival analysis and between-group comparison was performed by log-rank test. PFS was defined as the time interval from the date of enrolment in the study until the first signs of progression. OS was calculated from the date of enrolment until death from any cause. All analyses were performed using Graphpad prism version 4.0 b 2004 (Graphpad Software San Diego CA). The limit of significance was set at 0.05. Results Population Twelve eligible patients with NSCLC 6 women (50%) and 6 men (50%) with a mean age of 60±13 years were included. Two patients presented tumors harboring an activating Epidermal Growth Factor Receptor mutation (2573T>G substitution (p.Leu858Arg) in exon 21 in one patient; deletion (L747_E749del) in exon 19 in the other patient). Patient characteristics are described in . The median duration of erlotinib therapy was 75 days. Due to rapid progression and death PET3 and CT3 could not be performed in 2 patients. Tumor 18F-FDG uptake The three [18F]FDG PET/CT scans were acquired as follows: PET1 5±4 days before starting therapy PET2 9±3 days after starting therapy and PET3 60±6 days after starting erlotinib therapy. Scanning started 68±17 min (PET1) 71±16 min (PET2) and 64±13 min (PET3) after [18F]FDG injection of 271±53 MBq (PET1) 270±61 MBq (PET2) and 263±54 MBq (PET3). Blood glucose level was less than 1.5 g/L for all PET examinations i.e. 1.1±0.1 g/L for PET1 1.1±0.2 g/L for PET 2 and 1.1±0.2 g/L for PET3. Non-parametric Friedman tests did not show any significant difference between PET1 PET2 and PET3 for FDG uptake time injected FDG dose or blood glucose. Fifty-five lesions were described on PET1 before treatment and 45 lesions were defined as target lesions for PET evaluation of response to treatment (up to five most hypermetabolic lesions per patient; mean 3.8 lesions/patient). The mean tumor SUVmax of the most [18F]FDG€“avid lesion (SUVmax) was 10.0±4.7 for PET1 and did not vary significantly over time with a mean of 10.1±6.6 for PET2 and a mean of 9.1±5.6 for PET3 (P?=?0.97). The SUVpeak was 8.6±4.3 for PET1 8.1±5.4 for PET2 and 7.1±4.6 for PET3 and did not vary over time (P?=?0.60). No variation over time was observed for the sums of SUV. The mean sum of tumor SUVmax of all target lesions was 30.1±19.5 for PET1 27.5±17.7 for PET2 and 28.3±22.4 for PET3 (P?=?0.83). Sums of SUVpeak of all target lesions were 22.7±14.3 for PET1 20.6±13.4 for PET2 and 22.2±18.6 for PET3 (P?=?0.44). [18F]FDG-PET response versus conventional evaluation CT scan data were interpreted by chest physicians blinded to PET/CT scan results (). Evaluation of response to treatment according to RECIST 1.1 criteria demonstrated 7 patients with progressive disease (group P) and 5 patients with non-progressive disease (group NP) including 4 cases of stable disease (SD) and 1 partial response (PR). 10.1371/journal.pone.0087629.t002 CT and PET assessments of response rates OS and PFS. Patient PET2 versus PET1 PET3 versus PET1 RECIST 1.1 Evaluation PFS OS New lesion ? SUVmax * ? SUVpeak * ? SUVmax * ? SUVpeak * Response to Treatment Progressive (P) or not (NP) days days on PET3 #1 ?21.6 ?17.6 18.6 ?1.5 SD NP 267 915 ? #2 25.9 26.9 70.3 77.4 PD P 57 316 + #3 9.0 7.6 23.4 23.3 PD P 216 447 + #4 ?18.6 ?15.0 ?3.2 ?2.6 PD P 67 414 + #5 ?20.3 ?11.1 42.1 51.1 PD P 53 152 + #6 ?56.7 ?59.9 ?72.1 ?70.6 PR NP 190 296 ? #7 ?22.0 ?26.0 ?31.3 ?24.3 SD NP 727 1249 + #8 ?32.0 ?25.1 3.9 ?3.9 SD NP 317 1146 ? #9 16.4 7.8 ?5.4 ?10.8 SD NP 77 359 ? #10 2.1 4.4 MD MD PD P 37 92 MD #11 36.1 20.0 30.3 25.7 PD P 104 734 ? #12 ?7.2 ?10.5 MD MD PD P 61 71 MD * For patient with more than one tumor lesion the sum of SUVmax and of SUVpeak were calculated and used for the evaluation of changes between PET1 and PET2 (or between PET1 and PET3). Missing data are indicated as MD. On ROC analysis the AUC for prediction of non-progressive disease by PET2 was 0.86 (95% CI 0.62 to 1.1; P?=?0.04) corresponding to a maximum specificity of 0.80 and sensitivity of 0.86 for non-progressive disease at a cut-off of 21.6% reduction in SUVmax (Figure 1) and a positive predictive value (PPV) of 0.86 a negative predictive value (NPV) of 0.80 an accuracy of 0.83 and a maximum Youden index of 0.65. The use of this SUVmax cut-off value correctly classified 11/12 patients (7 with true progressive disease (Figures 2 and 3); 4 with true non-progressive disease (Figures 4 and 5); 1 with false progressive disease (). Non-progression after 2 months of treatment was significantly more frequent in patients with an early decrease in SUVmax of 21.6% or more (P?=?0.01 Fisher's exact test). The only misclassified patient (patient #9 false progressive disease on PET2 versus PET1) displayed a 16.4% increase of SUVmax but metabolic progression was not confirmed on PET3 with a 5.4% decrease of SUVmax compared to PET1. Similar results were observed for SUVpeak as non-progressive disease after 2 months of treatment was significantly more frequent in patients with a decrease in SUVpeak of at least 17.6% on PET2 (P?=?0.01 Fisher's exact test). Similar results were also obtained in terms of AUC sensitivity specificity PPV NPV and accuracy and with the same classification of patients (7 with true progressive disease; 4 with true non-progressive disease; 1 with false progressive disease). 10.1371/journal.pone.0087629.g001 Figure 1 Percentage change in SUVmax on 18F-FDG PET/CT (cut-off: ?21.6%) within 2 weeks of starting erlotinib therapy in relation to conventional imaging response. Each red or green bar represents a patient NP or P respectively. 10.1371/journal.pone.0087629.g002 Example of a progressive patient on PET (mP) and conventional imaging. Progressive patient with right upper lobe NSCLC associated with médiastinal lymphadenopathy"

Lung_Cancer

"Study for Epidermal Growth Factor Receptor and KRAS Mutation Detection in 74 Blinded Non-small Cell Lung Carcinoma Samples: A Total of 5550 Exons Sequenced by 15 Molecular French Laboratories. J Thorac Oncol6: 1006“101521532509 39 BellonE LigtenbergMJ TejparS CoxK de HertoghG et al (2011) External quality assessment for KRAS testing is needed: setup of a European program and report of the first joined regional quality assessment rounds. Oncologist16: 467“478 Br J Cancer Br. J. Cancer British Journal of Cancer 0007-0920 1532-1827 Nature Publishing Group 24921918 4119981 bjc2014308 10.1038/bjc.2014.308 Epidemiology Possible pro-carcinogenic association of endotoxin on lung cancer among Shanghai women textile workers Pro-carcinogenic association of endotoxin on lung cancer Checkoway H 1 * Lundin J I 2 Costello S 3 Ray R 4 Li W 4 Eisen E A 3 Astrakianakis G 5 Seixas N 2 Applebaum K 6 Gao D L 7 Thomas D B 4 1Department of Family and Preventive Medicine University of California San Diego La Jolla CA 92093 USA 2Department of Environmental and Occupational Health Sciences University of Washington Seattle WA 98195 USA 3Department of Environmental Health Sciences University of California Berkeley CA 94720 USA 4Division of Public Health Sciences Fred Hutchinson Cancer Research Center Seattle WA 98109 USA 5School of Population and Public Health University of British Columbia Vancouver BC V6T1Z4 Canada 6Department of Environmental and Occupational Health Gee Washington University Washington DC 20052 USA 7Zhong Shan Hospital Cancer Center Shanghai 200030 China *E-mail: hcheckowayucsd.edu 29 07 2014 12 06 2014 111 3 603 607 20 02 2014 05 05 2014 11 05 2014 Copyright 2014 Cancer Research UK 2014 Cancer Research UK From twelve months after its original publication this work is licensed under the Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License. To view a copy of this license visit http://creativecommons./licenses/by-nc-sa/3.0/ Background: Endotoxin (lipopolysaccharide) is a widespread contaminant in many environmental settings. Since the 1970s there has been generally consistent evidence indicating reduced risks for lung cancer associated with occupational endotoxin exposure. Methods: We updated a case“cohort study nested within a cohort of 267?400 female textile workers in Shanghai China. We compared exposure histories of 1456 incident lung cancers cases diagnosed during 1989“2006 with those of a reference subcohort of 3022 workers who were free of lung cancer at the end of follow-up. We applied Cox proportional hazards modelling to estimate exposure“response trends adjusted for age and smoking for cumulative exposures lagged by 010 and 20 years and separately for time windows of ?15 and >15 years since first exposure. Results: We observed no associations between cumulative exposure and lung cancer irrespective of lag interval. In contrast analyses by exposure time windows revealed modestly elevated but not statistically significant relative risks (?1.27) at the highest three exposure quintiles for exposures that occurred >15 years since first exposure. Conclusions: The findings do not support a protective effect of endotoxin but are suggestive of possible lung cancer promotion with increasing time since first exposure. endotoxin lipopolysaccharide lung cancer epidemiology textile industry occupational health Br J Cancer Br. J. Cancer British Journal of Cancer 0007-0920 1532-1827 Nature Publishing Group 24651386 3992504 bjc2014146 10.1038/bjc.2014.146 Clinical Study A multicentre randomised controlled trial of reciprocal lung cancer peer review and supported quality improvement: results from the improving lung cancer outcomes project Improving lung cancer outcomes project results Russell G K 1 Jimenez S 1 Martin L 1 Stanley R 2 Peake M D 1 3 Woolhouse I 1 4 * 1Clinical Standards Department Royal College of Physicians London NW14LE UK 2Clinical Audit Support Unit NHS Information Centre for Health and Social Care Leeds LS16AE UK 3Department of Respiratory Medicine Glenfield Hospital Leicester LE39QP UK 4Department of Respiratory Medicine Queen Elizabeth Hospital Birmingham Birmingham B152WB UK *E-mail: ian.woolhouseuhb.nhs.uk 15 04 2014 20 03 2014 110 8 1936 1942 19 12 2013 11 02 2014 24 02 2014 Copyright 2014 Cancer Research UK 2014 Cancer Research UK From twelve months after its original publication this work is licensed under the Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License. To view a copy of this license visit http://creativecommons./licenses/by-nc-sa/3.0/ Background: Results from the National Lung Cancer Audit demonstrate unexplained variation in outcomes. Peer review with supported quality improvement has been shown to reduce variation in other areas of health care but has not been formally tested in cancer multidisciplinary teams. The aim of the current study is to assess the impact of reciprocal peer-to-peer review visits with supported quality improvement and collaborative working on lung cancer process and outcome measures. Methods: English lung cancer teams were randomised to usual care or facilitated reciprocal peer review visits followed by 12 months of supported quality improvement. The primary outcome was change in the following national audit indicators; mulitdisciplinary team discussion histological confirmation active treatment surgical resection small-cell chemotherapy and specialist nurse review. Patient experience was measured using a new lung cancer patient questionnaire in the intervention group. Results: Thirty teams (31 trusts) entered the intervention group and 29 of these submitted a total of 67 quality improvement plans. Active treatment increased in the intervention group (n=31) by 5.2% compared with 1.2% in the control group (n=48 mean difference 4.1% 95% CI ?0.1 to 8.2% P=0.055). The remaining audit indicators improved similarly in all groups. Mean patient experience scores in the intervention group did not change significantly during the study but a significant improvement was seen in the scores for the five teams with the worst baseline scores (0.86 to 0.22 P<0.001). Conclusions: Reciprocal peer review with supported quality improvement was feasible and effective in stimulating quality improvement activity but resulted in only modest improvements in lung cancer treatment rates and patient experience. lung cancer multidisciplinary quality improvement peer Lung cancer is the commonest cause of cancer death in England and Wales with around 38?000 cases diagnosed each year and ?35?000 deaths. Data from the National Lung Cancer Audit (NLCA) demonstrate significant variation in process and outcome measures across England. In 2009 there was a three-fold difference in survival and active treatment rates which persisted following case mix adjustment (Beckett et al 2012). Furthermore reported lung cancer outcomes in the UK are worse than other comparable European countries (Walters et al 2013) and have improved little in recent years (Khakwani et al 2013). It has been estimated that if survival rates were increased to that of the best in Europe around 1300 lives could be saved each year in the United Kingdom (Abdel-Rahman et al 2009). Variation in health care is not unique to lung cancer and addressing unwarranted variation is challenging (Wise 2010). Although external regulation may have a role in some areas this approach is more difficult to apply to the complex pathways involved in lung cancer diagnosis and treatment. Peer review with supported quality improvement offers a promising alternative but the evidence for its effectiveness is limited. The Washington State's Surgical Care and Outcomes Assessment Program utilised a peer support programme to share the best practice which led to a significant reduction in post-operative complications (Kwon et al 2012). Within the United Kingdom the national COPD resources and outcomes project demonstrated that reciprocal peer-to-peer review led to only limited quantitative differences in the quality of services offered (Roberts et al 2012). A qualitative analysis of this study identified a number of barriers to improvement including difficulties in establishing effective working relationships funding changes and service re-design. In 2003 the Institute for Healthcare Improvement described the collaborative model to achieve a breakthrough improvement (Institute for Healthcare Improvement 2003). Collaboratives allow teams working on the same issue to share good practice and innovation permitting others to take these ideas and implement them in the context of their own anisation resources and case mix. Pronovost et al (2006) successfully employed this collaborative approach together with supported quality improvement to implement five evidence-based interventions on the intensive care unit resulting in the reduction in catheter-related bloodstream infections to zero. These studies offer a persuasive proof of concept but the absence of a control group or of patient-specific outcomes measures limits their implementation in other disease areas such as cancer. The aim of the current study is to determine whether a programme of reciprocal peer-to-peer review visits with supported quality improvement and collaborative working can significantly improve lung cancer process and outcome measures and thus reduce unwarranted variation in outcomes. Materials and methods Study design We conducted a prospective randomised controlled trial. Study population One hundred and sixty-two English NHS trusts were identified from the 2008 NLCA annual report. Centres only providing treatment (not diagnostics) orthopaedic hospitals and ambulance trusts were excluded. Invitations to participate were sent to the remaining 152 trusts. Trusts who agreed to participate and who had 2008 NLCA case ascertainment rates of > 50% expected were paired before randomisation on the basis of contrasting results for four key indicators from the NLCA. The indicators were active treatment rates surgical resection rates median survival and the proportion of patients assessed by a clinical nurse specialist. Each trust was colour coded for each indicator red if below the national average and green if above. By placing each trust with its colour-coded indicators on a map we were able to pair trusts on the basis of a contrasting mixture of red and green indicators and a travel time between centres of around 2?h. On the basis of data from the national COPD resources and outcomes project we determined that we would be able to complete 30 peer review visits during the lifetime of the project thus allowing 30 lung cancer multidisciplinary teams (15 pairs) to be randomised into the intervention arm. Randomisation was performed in a blinded fashion by assigning a random number to each pair of trusts and then allocating pairs numbered 1“15 to the intervention group. The remaining trusts formed either the control group (if they had agreed to participate) or the non-participant group and had no further contact with the study team but continued to submit data to the NLCA as usual. Intervention The study timeline is shown in Figure 1. Following introductory workshops the multidisciplinary teams within each pair undertook facilitated reciprocal site visits. The visits consisted of observation of the host team's multidisciplinary team meeting three discussion sessions focusing on the functioning of the mulitdisciplinary team meeting the host team's NLCA data and patient experience questionnaire results. The final session aimed to identify the focus of improvement work to be undertaken by the host team. The quality improvement facilitator introduced a structured template for the quality improvement plans and provided a short introduction to using the model of improvement to guide implementation of the plans. Over the next 12 months the quality improvement facilitator provided support via electronic mail telephone and follow-up visits where required. Teams within the intervention group supported each other via mini-collaboratives in the form of web-based teleconferences and two face-to-face workshops. Outcomes Changes in process and outcome were assessed using data from local quality-improving plans and the following indicators from the NLCA: the proportion of patients discussed at a multidisciplinary team meeting histological confirmation rate active treatment rate surgical resection rate the proportion of patients with small-cell lung cancer receiving chemotherapy and the proportion of patients seen by a lung cancer nurse specialist. Patient experience was assessed in the intervention group using a new lung cancer-specific patient experience questionnaire designed in collaboration with the Roy Castle Lung Cancer Foundation. The questionnaire included 11 questions selected with permission from the previously validated 2004 national cancer patient survey. The questions covered the following domains: communication privacy respect and dignity and three free text questions (see Appendix I). Participating teams were asked to distribute 30 questionnaires to patients recently seen in their services. The clinical nurse specialists distributed the questionnaires to patients who anonymously returned them to the Royal College of Physicians. An independent qualitative ethnographic evaluation of the study was undertaken by the Social Science Applied to Healthcare Improvement Research Group at the University of Leicester. Statistical methods Data were tested for normality using the Shapiro“Wilk test. Baseline NLCA indicators were taken from the 2009 NLCA report and the intervention control and non-participant groups were compared using a ?2- test. The changes in NLCA indicators from 2009 to 2011 were compared using an independent t-test. Patient experience questionnaire responses for each question were labelled and re-coded to separate them into the worst patient experience category (score 1) vs all other responses (score 0). These scores were then summated to create a domain and a total patient experience score with a possible range of 0“11 whereby a higher score indicates a worse patient experience. Analyses were performed using the statistical software package SPSS (International Business Machines Corp. Armonk NY USA). Funding and ethics The study was funded by a ˜Closing the Gap' grant from the Health Foundation. The National Research Ethics Service confirmed that the study was service evaluation and quality improvement and did not require ethical review. Results One hundred trusts (66%) replied to the invitation to participate and 91 (61%) agreed to participate in the study. Eighty-one trusts had 2008 NLCA data of sufficient quality to allow pairing. Two trusts provided a joint multidisciplinary team allowing 40 pairs of multidisciplinary teams to be created. One pair agreed to act as a pilot and was excluded from further analysis. Of the remaining 39 pairs 15 pairs (31 trusts) were randomised to the intervention group. The remaining 24 pairs formed the control group. During the study two trusts in the control group amalgamated to form one trust so the total number of trusts in the control group was 47 (Figure 2). Quality improvement plans Two hundred and thirty medical professionals from 31 trusts participated in the review visits. Twenty-nine teams submitted a total of 67 quality improvement plans. The issues identified in the quality improvement plans are shown in Table 1. Eighteen teams collected local data to measure impact. An example of such data is shown in Figure 3. This trust identified small-cell lung cancer chemotherapy as an area for improvement. They introduced a number of changes to their diagnostic and treatment pathways including prioritisation of small-cell pathology reporting faxing of the results to the multidisciplinary team coordinator and lung nurse specialist to allow early booking of oncology appointments. These changes were monitored using a run chart that demonstrated a reduction in the time from multidisciplinary team meeting to chemotherapy treatment and an increase in the proportion of small-cell lung cancer patients receiving chemotherapy from 60% in 2009 to 71% in 2011. National lung cancer audit indicators Baseline (2009) NLCA indicators for the intervention control and non-participant groups were similar (Table 2). The mean change for each NLCA indicator from baseline to 2011 in the intervention and control group is shown in Figure 4. The proportion of patients receiving active anti-cancer treatment in the intervention group increased by 5.2% compared with 1.2% in the controls (mean difference 4.1% 95% CI ?0.1 to 8.2% P=0.055). The remaining NLCA indicators improved similarly both in the intervention and control groups. Patient experience In the intervention group patient experience questionnaires were returned by 438 patients from 30 multidisciplinary teams at baseline (return rate 49%) and 372 patients from 27 trusts following the intervention (return rate 41%). Baseline total scores were low (0“1.31) indicating high levels of patient satisfaction with the care received although there was a statistically significant (P<0.001) variation in results by the multidisciplinary team (Figure 5). In particular the proportion of patients responding yes to the question ˜did you find that the person who told you about your diagnosis did so with sufficient sensitivity/care?' varied significantly by 57%“100% (P<0.001). The total questionnaire scores did not change significantly during the study (0.22“0.17 P=0.377) however the variation by the multidisciplinary team reduced (Figure 5). Given that the study aimed to bring the standard of the lower performing trusts to that of the best we performed a post hoc analysis for the five trusts with the worst baseline patient experience scores. This demonstrated that the mean total score improved significantly for these trusts from 0.86 to 0.22 P<0.001. The biggest improvement in this group was seen in the proportion of patients responding yes to the question ˜did you find that the person who told you about your diagnosis did so with sufficient sensitivity/care?' which increased from 75% to 90% (P=0.05). One multidisciplinary team in this group achieved this improvement by using their baseline questionnaire results as a lever to encourage attendance at an advanced communications skills course. The questionnaire domain-specific scores did not change significantly during the study. Of the individual questions a significant improvement was seen in the rating of the quality of information provided as excellent which rose from 53%“59% P<0.05. Qualitative evaluation Participants' experiences were overwhelmingly positive. The reciprocal peer-to-peer visits with supported quality improvement were seen as a strong driver to change. The method of pairing multidisciplinary teams was important. In particular pairing teams with different results not just ˜good' with ˜bad' and allowing teams to visit each other's sites to ensure a two-way sharing of best practice. The independent quality improvement facilitator role was seen as crucial to ensure the visits remained focussed and that the engagement with quality improvement plans was maintained. Finally the involvement of senior managers was crucial to the successful implementation of the quality improvement plans. The detailed findings from the independent evaluation of this project have been reported elsewhere (Aveling et al 2012). Discussion Lung cancer outcomes remain relatively poor and reducing unexplained variation is an attractive proposition to promote improvement. There are a number of ways that clinical teams may share best practice and innovative service delivery models however studies formally evaluating their impact are limited. To our knowledge this is the first study to formally test a national quality improvement strategy which aimed to bring the standard of all lung cancer teams to that of the best. We have demonstrated that reciprocal peer-to-peer review with supported quality improvement is both feasible and effective at stimulating local quality improvement activity but had a relatively modest and somewhat disappointing impact on process and outcome measures as measured by NLCA indicators and a new lung cancer patient experience questionnaire. The facilitated reciprocal visits represented a new and unique opportunity for all members of a lung cancer team to exchange ideas in a supported environment and to formally design then implement quality improvement plans. Nearly two-thirds of lung cancer multidisciplinary teams in England agreed to take part in the study and reassuringly baseline NLCA indicators did not differ significantly between participants and non-participants suggesting that the willingness to participate in quality improvement activity is not related to baseline performance. There were a wide range of areas identified for improvement but nearly half of the teams identified multidisciplinary team meeting effectiveness as a key issue. This is not surprising given that these meetings are pivotal in the lung cancer pathway. Live observation of each multidisciplinary team meeting followed by facilitated feedback proved to be a strong driver to improve on problems such as ensuring weekly presence of all the treatment specialists as well as more simple issues such as room layout. The need to streamline diagnostic and treatment pathways was also identified as a common problem. Recent NICE guidance on the management of lung cancer (National Institute for Health and Care Excellence 2011) recommended a paradigm shift in the diagnostic algorithm from performing multiple diagnostic and staging investigations to performing a single test that will provide both diagnostic and staging information. A number of teams within our study were able to introduce such pathways and demonstrate impressive reductions in diagnostic times and more prompt treatment. This together with more effective multidisciplinary team working may have led to the small increase in the active anti-cancer treatment rates seen within the intervention group. However an alternative explanation for the improvement is regression to the mean given that treatment rates in the intervention group were lower at baseline and overall the lack of significant improvement across the range of NLCA indicators in the intervention group was disappointing. One possible explanation for this is the challenge that some participating teams encountered converting enthusiastic quality improvement plans into tangible improvements for patients over a relatively short time period. The qualitative evaluation confirmed that participants often underestimated the time and energy required to implement and sustain change and highlighted the importance of early engagement with hospital managers to maintain momentum (Aveling et al 2012). Alternatively other national lung cancer initiatives implemented at the time of the study may have driven coexistent improvements in the control group. For example the drive to encourage all lung cancer patients to be referred for clinical nurse specialist support has subsequently been shown to increase the probability that a lung cancer patient receives active treatment. Although even small improvements in lung cancer treatment rates are very welcome it is recognised that undergoing investigation for suspected lung cancer generates high levels of patient anxiety and many patients will remain too unwell to benefit from currently available drugs. The assessment of patient experience is therefore of particular importance in lung cancer. This has proved challenging in detailed national cancer surveys owing to the advance in age poor health and short median survival of lung cancer patients. The response rate to our short questionnaire was relatively high at 41“49% compared with the 2011 national survey in which only 7% of lung cancer patients responded (Department of Health 2012) but still represents the views of less than half of lung cancer patients and is a relative limitation in terms of generalisability of the results. It was reassuring to note that at entry to the study patients in the intervention group generally rated their experience as highly satisfactory. This may explain the low number of teams who specifically identified patient experience as an area for quality improvement. In terms of assessing the impact of the reciprocal peer-to-peer review visits and supported quality improvement on patient experience it is likely that this high-baseline satisfaction and the lack of patient experience data for the control group limited our ability to detect a significant change. However our results suggest that those teams with poor scores may be able to use patient experience data to promote significant improvements particularly in areas such as communication skills. Further work is required to develop a lung cancer patient experience measure that is both acceptable to patients and able to detect small but clinically important changes in experience. Although similar in name to the national cancer peer review process there are a number of important differences between the reciprocal peer-to-peer review and supported quality improvement process employed in the current study and national cancer peer review. The latter predominantly performs a quality assurance role ensuring that cancer teams meet a minimum standard via compliance with a number of process measures. Support with quality improvement is not provided and site visits are now rarely performed. The qualitative evaluation of our study highlighted the importance of an independent quality improvement facilitator to the success of the peer review visits and the subsequent implementation of the quality improvement plans. Integration of facilitated reciprocal peer-to-peer review and supported quality improvement into national cancer peer review both for lung cancer and other tumour sites is an attractive proposition and requires further study. However our results suggest that this strategy alone is unlikely to have a major impact on lung cancer treatment rates. This phenomenon is not new in lung cancer for example the introduction and NICE approval of gefitinib treatment for the first-line treatment of lung cancer in 2010 was associated with only a 1% increase in active anti-cancer treatment rates over the following year (Health and Social Care Information Centre 2012). Achieving a stepwise increase in lung cancer treatment rates and survival is likely to require a multi-targeted approach including earlier diagnosis streamlined lung cancer pathways new treatments and a reduction in unexplained variation via supported quality improvement programmes. This project was funded by a Health Foundation Closing the Gap award. (grant number: 7797/5557). Appendix I Improving lung cancer outcomes project: patient experience questionnaireWhat is this survey about? This questionnaire asks about your experience of lung cancer treatment and care at the hospital. It was developed in 2010 and it has been used by Lung Cancer Nurse Specialists in 30 hospital across participating in the ˜Improving Lung Cancer Outcomes Project' led by the Royal College of Physicians and several other anisations. The project aims to improve the quality of services and care for people affected by lung cancer. Why should I complete the survey? We need to know your opinion of the current services and care to help improve these for people affected by lung cancer. Your participation in this survey is voluntary and your answers will be treated in confidence. If you choose not to take part in this survey it will not affect the care you receive from the NHS in any way. Please do not write your name and address anywhere on the questionnaire as this information is not required. No information you give in this questionnaire will be shared in a way that allows you to be identified. How to complete the survey and how long it will take. The questionnaire is short and will take 5“10?min to complete. Please try to answer every question. Please return your questionnaire even if you have not answered every question. If English is not your first language or if you if you have difficulty understanding the questions then please ask a relative or carer to help you complete the questionnaire. Questions or help? If you have any questions please contact your local lung clinical nurse specialist team. Please select one answer to each question by placing a in the appropriate box. There is space at the end of the survey for you to write any comments. This work is published under the standard license to publish agreement. After 12 months the work will become freely available and the license terms will switch to a Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License. Abdel-Rahman M Stockton D Rachet B Hakulinen T Coleman MP 2009 What if cancer survival in Britain were the same as in Europe: how many deaths are avoidable Br J Cancer 101 (Suppl 2 S115 S124 19956155 Aveling EL Martin G JimÃnez García S Martin L Herbert G Armstrong N Dixon-Woods M Woolhouse I 2012 Reciprocal peer review for quality improvement: an ethnographic case study of the Improving Lung Cancer Outcomes Project BMJ Qual Saf 21 1034 1041 Beckett P Woolhouse I Stanley R Peake MD 2012 Exploring variations in lung cancer care across the UK-the ˜story so far' for the National Lung Cancer Audit Clin Med 12 14 18 22372213 Department of Health2012National Cancer Patients' Experience Survey Programme 2012/13. England. Health And Social Care Information Centre2012National Lung Cancer Audit Report. Institute for Healthcare Improvement2003The Breakthrough Series: IHI's Collaborative Model for Achieving Breakthrough Improvement. Boston. Khakwani A Rich AL Powell HA Tata LJ Stanley RA Baldwin DR Duffy JP Hubbard RB 2013 Lung cancer survival in England: trends in non-small-cell lung cancer survival over the duration of the National Lung Cancer Audit Br J Cancer 109 (8 2058 2065 24052044 Kwon S Florence M Grigas P Horton M Horvath K Johnson M Jurkovich G Klamp W Peterson K Quigley T Raum W Rogers T Thirlby R Farrokhi E Flum D 2012 Creating a learning healthcare system in surgery: Washington State's Surgical Care and Outcomes Assessment Program (SCOAP) at 5 years Surgery 151 146 152 22129638 National Institute for Health and Care Excellence 2011 The Diagnosis And Treatment Of Lung Cancer (Update Of Nice Clinical Guideline 24) Clinical guidelines CG121 London UK Pronovost P Needham D Berenholtz S Sinopoli D Chu H Cosgrove S Sexton B Hyzy R Welsh R Roth G Bander J Kepros J Goeschel C 2006 An intervention to decrease catheter-related bloodstream infections in the ICU N Engl J Med 355 2725 2732 17192537 Roberts CM Stone RA Buckingham RJ Pursey NA Lowe D Potter JM 2012 A randomized trial of peer review: the UK National Chronic Obstructive Pulmonary Disease Resources and Outcomes Project: three-year evaluation J Eval Clin Pract 18 (3 599 605 21332611 Walters S Maringe C Coleman MP Peake MD Butler J Young N Bergström S Hanna L Jakobsen E Kölbeck K Sundstrøm S Engholm G Gavin A Gjerstorff ML Hatcher J Johannesen TB Linklater KM McGahan CE Steward J Tracey E Turner D Richards MA Rachet B ICBP Module 1 Working Group 2013 Lung cancer survival and stage at diagnosis in Australia Canada Denmark Norway Sweden and the UK: a population-based study 2004-2007 Thorax 68 551 564 23399908 Wise J 2010 Health atlas shows large variations in care in England BMJ 341 c6809 c6809 Figure 1 Study timelines. Figure 2 Consort diagram disposal of eligible trusts including screening randomisation and follow-up. Figure 3 Run chart showing the waiting times from the multidisciplinary team meeting to the first treatment for 10 consecutive small-cell lung cancer patients following the implementation of the quality improvement plan at one trust in the intervention group. Figure 4 Mean change in national lung cancer audit metrics from baseline (2009) to 2011. P=0.055 active treatment”intervention vs controls. Intervention n=31 trusts control n=47 trusts and non-intervention (control and non-participants combined) n=66 trusts. Abbreviations: CNS clinical nurse specialist; MDT multidisciplinary team; SCLC small-cell lung cancer."

Lung_Cancer

"Although de Torres et al. demonstrated by using a well-characterized cohort of patients with COPD that the incidence of dense lung cancers decreased as the severity of the airflow obstruction at baseline increased [23] the severity of COPD in Japanese patients with newly diagnosed lung cancer was classified mainly as GOLD grade 1 and 2 rather than as GOLD grade 3. Furthermore our data showed that most patients were newly classified with COPD (84.4%; 124/147 cases) compatible with the incidence of the severity of COPD shown above or previously [1323]. It should be noted that in comparing patients undergoing thoracic surgery COPD patients had an average postoperative stay that was 61% higher and a 100% greater need of prolonged oxygen therapy than patients without COPD indicating the clinical impact of the coexistence of COPD [14]. The prevalence of COPD might increase in Japanese patients with lung cancer whereas the impact of COPD-related systemic comorbidities is also increasingly recognized in clinical aspects of COPD [7]. Thus whether or not the decision-making process involved in proposing the therapeutic management of lung cancer might be independently affected by COPD in patients with lung cancer remains elusive. To address this issue we evaluated whether or not completion of clinical staging and proposal of thoracic surgery with curative intent might be affected by the coexistence of COPD. The percentage of patients in whom clinical staging had been not completed was significantly higher in the COPD group than in the non-COPD group. More than half of these patients were referred to other hospitals for further support while the others were patients with disease recurrence. The proportion of patients with each classification in the clinical staging was compatible with that reported in previous studies about thoracic surgery [24]. Clinical guidelines recommend the assessment of spirometry to evaluate the optimum selection of surgical procedure in view of the risks of mortality and postoperative complications [6825]. Therefore we analyzed data from 185 patients with lung cancer at stage 1A to 3A because these patients are generally eligible for thoracic surgery with curative intent [1726]. Even among these surgical candidates however the number of surgeries performed was significantly lower in the COPD group (64.1%; 59/92 cases) than in the non-COPD group (81.7%; 76/93 cases) (). Furthermore our data showing that COPD-related systemic comorbidities might not be independent factors for proposing thoracic surgery with curative intent was supported by previous data as described above [14]. Thus these data indicate that the decision-making process for the therapeutic management of Japanese lung cancer patients might be affected by the prevalence and severity of COPD. Finally we evaluated whether or not the severity of COPD classified by GOLD grade might be an independent factor affecting the proposal of thoracic surgery with curative intent. Multivariate analysis indicated that severity of COPD was a critical and independent factor for proposing thoracic surgery with curative intent to Japanese patients with lung cancer who underwent bronchoscopy. This finding might be supported by our previous study showing that in comparing patients undergoing thoracic surgery COPD patients with an FEV1/FVC below 0.70 had an average postoperative stay that was 61% higher and a 100% greater need of prolonged oxygen therapy (POT) than patients without COPD [14]. Some limitations of the present study deserve mention. First the reversibility test was performed in only 62.2% of patients (168/270 cases) although COPD was defined as a postpronchodilator FEV1/FVC below 0.7 [16]. This limitation is present in other studies that have evaluated the prevalence of COPD [91027]. The other explanation might be the preoperative pulmonary assessment based on the clinical guidelines in which the need to perform a reversibility test for assessment of airflow obstruction is not mentioned [825]. Although a recent study suggests that some COPD patients show relatively high reversibility for a short-acting beta2-agonist [28] only 1.2% of 168 cases showed significant reversibility in the present study indicating that Japanese patients with both lung cancer and COPD might have different characteristics from that population [27]. Second the present study retrospectively analyzed 270 out of a total of 320 cases with lung cancer in a single institution and therefore might be subject to selection bias. However analyzing the data from 84.4% of all patients in a single institution who were sequentially registered and underwent bronchoscopy from 2010 to 2012 might minimize the possible contribution of the selection bias for patients with lung cancer. Although many studies suggest that COPD remains underdiagnosed in the patients with lung cancer [1314] Zang et al. suggest that awareness of COPD might contribute the conformity to GOLD treatment guideline for stable condition and acute exacerbation of COPD in lung cancer patients during hospitalization [13]. When spirometry was performed at bronchoscopy the median time from the date of spirometry to thoracic surgery was 50 days in the present study. Therefore comprehensive assessment of COPD at bronchoscopy might allow us to implement the optimum management for lung cancer patients [2930]. Conclusions In the present study the high prevalence of COPD among Japanese patients with newly diagnosed lung cancer was shown. Although further investigation into the validity of the assessment of COPD at bronchoscopy from studies of patients with lung cancer from other institutions is warranted we conclude that appropriate risk stratification and comprehensive management of patients with lung cancer and COPD might be made by assessment of the coexistence and severity of COPD at the time of bronchoscopy. Competing interests The authors have declared that no conflict of interest exists. Authors™ contributions NH AM and YH had full access to all of the data in the study and are responsible for the integrity of the data and the accuracy of the data analysis"

Lung_Cancer

".0091811.g002 Heterogeneity of lymphatic vessel density (LVD) microvessel density (MVD) and amount of cancer-associated fibroblasts (CAFs) with respect to tumor location. The LVD (A) MVD (B) and CAF area (C) was significantly different according to each tumor location. .0091811.g003 LVD MVD and CAF area at different distant metastasis sites. The characteristics of cancer-associated stroma differed with respect to the metastatic site. LVD (A) and MVD (B) were greater in the metastatic tumor samples collected from the lung than in samples collected from other metastatic sites (p<0.001). However the amount of CAFs was not significant different between metastatic sites (C). Despite the heterogeneity of stromal characteristics CRC cases with higher LVD MVD and CAFs in center of the primary cancers had a tendency of higher LVD MVD and CAFs in periphery (p<0.05; Table S1). However LVD in center and periphery of primary cancer were not correlated with LVD in related distant metastasis (Table S1). In addition the amount of microvasculature was significantly correlated with the amount of CAFs (Table S2). 2. Clinical significance of cancer-associated stroma in advanced CRCs The MVD LVD and amount of CAFs present at each tumor location were compared according to their clinicopathologic features (). High grade CRCs were associated with lower CAFs in samples taken from the central cancer site (p?=?0.041). When compared with synchronous metastases the patients with metachronous metastases had higher LVD in center and periphery of the primary cancer and had higher MVD in lymph node metastases. Most patients with metachronous metastases were treated by adjuvant chemotherapy before metastasectomy. LVD and MVD in the distant metastases were significantly higher in the patients who had received chemotherapy before metastasectomy than those who did not (p?=?0.011 and 0.048 respectively). .0091811.t002 Clinicopathologic factor and LVD MVD and CAFs. Center (median) Periphery (median) LN metastasis (median) Distant metastasis (median) LVD MVD CAFs LVD MVD CAFs LVD MVD CAFs LVD MVD CAFs Total 39 717 1.13 5 740 1.22 3 888 1.42 3 648 0.91 Histologic grade Low grade 40 717 1.15* 5 741 1.23 3 895 1.43 3 665 0.92 High grade 34 683.5 0.94* 6 643.5 1.18 2 656 1.32 6 498 0.82 pT stage pT2 34 758 1.15 16 870 1.48 6 772 0.73 pT3 47 737 1.19 5 803 1.22 2.5 884 1.43 3 724 0.92 pT4 33 639 1.09 4 630 1.22 3 895 1.41 3 520 0.93 LN metastasis Absent 49 602 1.15 8 712 1.42 4 772 0.94 Present 39 737.5 1.12 4 740 1.21 3 884 1.41 3 617 0.91 Perineural invasion Absent 41 738 1.12 6 772 1.32 5.5 931.5 1.42 4 687 0.94 Present 39 672 1.13 4 702 1.2 2 796 1.39 3 548.5 0.86 Metastasis Synchronous 34* 717.5 1.11 3.0* 741 1.21 3 797* 1.39 3 617 0.93 Metachronous 55* 716 1.21 8.0* 712 1.23 2 1117* 1.63 5 698 0.91 Chemotherapy  Not done 2.0* 597.5* 0.93 Done 10.0* 684* 0.91 * p<0.05; ** p<0.01;   chemotherapy prior to metastatectomy of distant metastasis. 3. Expression loss of PTEN in CAFs PTEN was expressed in cytoplasm and sometimes the nucleus of both cancer and non-neoplastic cells when examined using immunohistochemistry. Expression of PTEN was lost in 8 cases in the center 2 cases in the periphery 4 cases in lymph node metastases and 11 cases in distant metastases (Table S3). In all 11 distant metastases with PTEN loss PTEN expression was intact in both the center and periphery of primary cancer (data not shown). PTEN loss in distant metastasis was correlated with synchronous metastasis (p?=?0.018). 4. Cancer-associated stroma and patient prognosis By using the obtained cut-offs lower LVD MVD and CAFs in the center LVD and CAFs in the periphery and MVD and CAFs in distant metastases were all significantly correlated with lower survival (p<0.05; Fig. S1). Among other clinicopathologic features synchronous metastasis old age larger size high histologic grade advanced pT and pN stage and presence of perineural invasion were associated with a worse prognosis (). By multivariate Cox regression analysis the hazard ratio of synchronous versus metachronous was the highest (4.029) with the lowest p value (p<0.001). CAFs in distant metastasis LVD and MVD in the center LVD in the periphery age and perineural invasion also independently predicted patient survival. In addition loss of PTEN expression in CAFs in distant metastases was associated with a worse prognosis (p?=?0.042; Fig S2) but not in primary cancer or lymph node metastasis. .0091811.t003 Univariate and multivariate survival analysis according to clinicopathologic features. Univariate survival analysis Multivariate survival analysis Factors HR (95% CI) P value HR (95% CI) P value Synchronous vs. Metachronous 4.617 (2.472“8.624) <0.001 3.762 (1.838“7.701) <0.001 Age 1.023 (1.004“1.044) 0.020 1.033 (1.011“1.056) 0.003 Sex (female vs. male) 1.428 (0.920“2.218) 0.113 ” ” Location (left vs. right) 0.503 (0.314“0.806) 0.004 0.700 (0.413“1.188) NS (0.186) Size 1.073 (1.005“1.146) 0.036 1.040 (0.903“1.198) NS (0.584) Histologic grade (high vs. low) 1.862 (1.061“3.269) 0.030 1.491 (0.763“2.912) NS (0.243) pT stage (pT4 vs. pT2/3) 2.341 (1.503“3.645) <0.001 1.137 (0.674“1.921) NS (0.630) pN stage (pN1/2 vs. pN0) 3.848 (1.760“8.411) 0.001 1.773 (0.758“4.146) NS (0.186) Perineural invasion 2.628 (1.640“4.211) <0.001 2.108 (1.265“3.513) 0.004 Venous invasion 1.217 (0.757“1.956) 0.418 ” Center LVD (high vs. low) 0.364 (0.158“0.836) 0.017 0.298 (0.118“0.753) 0.010 Center MVD (high vs. low) 0.391 (0.233“0.655) <0.001 0.437 (0.238“0.801) 0.007 Center CAFs (high vs. low) 0.579 (0.352“0.954) 0.032 1.038 (0.607“1.773) NS (0.892) Periphery LVD (high vs. low) 0.235 (0.086“0.644) 0.005 0.279 (0.096“0.809) 0.019 Periphery MVD (high vs. low) 1.456 (0.911“2.327) 0.117 ” Periphery CAFs (high vs. low) 0.524 (0.336“0.817) 0.004 0.813 (0.499“1.326) NS (0.406) LN LVD (high vs. low) 1.646 (0.874“3.100) 0.123 ” LN MVD (high vs. low) 0.597 (0.294“1.213) 0.154 ” LN CAFs (high vs. low) 0.717 (0.423“1.217) 0.218 ” Metastasis LVD (high vs. low) 0.569 (0.314“1.032) 0.063 ” Metastasis MVD (high vs. low) 0.579 (0.364“0.921) 0.021 1.262 (0.720“2.211) NS (0.417) Metastasis CAFs (high vs. low) 0.492 (0.271“0.894) 0.020 0.290 (0.144“0.582) 0.001 Metastasis PTEN (intact vs. loss) 0.454 (0.208“0.993) 0.048 0.575 (0.239“1.383) NS (0.217) Discussion Carcinoma cells in different tissue areas have distinct characteristics [32]. In central areas of the tumor carcinoma cells maintain an epithelial cell phenotype but carcinoma cells in the invasive front acquire a more malignant and mesenchymal phenotype and are thought to have an increased migratory capacity and contribute to metastatic diseases. These metastatic cells may restore the epithelial phenotype at metastatic sites [33]. In addition to carcinoma cells themselves microenvironment is suggested to be uneven within a given tumor because tumor formation and progression involve the co-evolution of cancer cells and microenvironments [34]. The present study demonstrated that the cancer-associated microenvironment also had distinct characteristics in different areas. Of the sites examined LVD was highest in the center of the primary cancer. MVD was slightly higher in center than at the periphery of the primary cancer but this difference was not statistically significant. Interestingly the amount of CAFs in distant metastases was significantly lower than in center and periphery of the primary cancer. We show that the stromal microenvironment has regional heterogeneity both within the primary tumor and between the primary site and its related metastases. Furthermore our data suggests that the stromal heterogeneity might be attributable to tumor heterogeneity. Therefore it would be beneficial to consider both stromal and tumor cell heterogeneity in order to manage CRC patients better. We evaluated the MVD LVD and amount of CAFs in metastatic tissues of various ans including the liver lung peritoneal seeding distant lymph nodes and ovary. Of the metastatic ans we examined both LVD and MVD were the highest in lung. In our previous study the KRAS discordance rate was also significantly higher in matched lung metastases than in other matched metastatic ans [35]. The underlying mechanism is not known. It could be that primary CRCs with high LVD and MVD have a tendency to produce lung metastases; however our results indicated that LVD and MVD in the center and at the periphery of the primary cancers were lower in the patients with lung metastases (data not shown). Alternatively it may be due to the physiological characteristics of metastatic ans interactions between cancer cells and microenvironment within the metastatic an or the characteristics of the cancer cell clones prone to lung metastasis. However technical or sampling errors also may be possible thus further large-scale studies are required. Although numerous studies have attempted to demonstrate an association between tumor microenvironment characteristics and survival the prognostic impacts of MVD and LVD are still controversial. Some studies have been presented that active angiogenesis and lymphangiogenesis represented by high MVD and LVD are associated with poor prognosis and aggressive clinicopathologic factors [36] [37]. Recent meta-analysis has demonstrated that LVD was significantly associated with disease-free survival but not overall survival [38]. Other studies have reported no statistical significance of MVD and LVD on survival [39]. Prall et al. has reported that high MVD and LVD are related with better survival in a consecutive series and liver metastases [40]. Our results were based on patients with advanced disease with distant metastasis and we showed that high MVD and LVD were related with improved survival. This might be because all the patients in this study had confirmed to have distant metastasis and microvasculatures could influence even delivery of the chemotherapeutic drug into the tumor. However our study had some limitations in terms of the survival analysis. We enrolled the CRC patients with available surgically resected cancer tissues from both primary tumors and corresponding metastatic tumors. Not all advanced CRC patients with metastatic diseases were included and far advanced cases were not enrolled because of their inoperability. Therefore unrecognized biases might have influenced our survival results. Some studies have demonstrated an anti-tumorigenic effect of fibroblasts [20] [21]. However it has become clear that CAFs contribute to the progression of cancer and their prognostic significance in various cancers also has been raised [41] and furthermore several studies have observed genetic alterations in CAFs [26] [27]. PTEN loss of CAFs has been observed in breast cancer and prognostic association of it has been suggested [27] [28]. We observed PTEN loss of CAFs in CRC patients and it was more frequently observed in the corresponding distant metastases. It is suggested that CAFs not only cancer cells have altered gene expression. Moreover loss of PTEN expression of CAFs in distant metastases was significantly correlated with the survival of patients. To our knowledge these are the first results showing PTEN loss in CAFs in CRC patients. Although more research is required we expect that it might be a prognostic factor in CRC patients. In our large cohort of advanced CRC patients with synchronous and metachronous distant metastasis we demonstrated the regional heterogeneity of stromal microenvironment factors according to the tumor location. "

Lung_Cancer

" MTBHsp70 at both flanks. Histopathology Abdominal walls and intestines from mice were fixed for at least 24 h in PBS-buffered 10% formalin. Tissues were routinely embedded in paraffin. 5 ?m thick sections were stained routinely with H&E. For staining tumor-infiltrating T cells mice were perfused with 4% paraformaldehyde (PFA) in PBS and tumor nodules were fixed in 4% PFA/PBS for additional 2 hours washed and infiltrated with 30% sucrose/PBS at 4°C. 6 ?m thick frozen sections were stained with rat anti-mouse CD8 (BD Biosciences 1:100 dilution) or rat anti-mouse Foxp3 (eBioscience 1:12 dilution) followed by polyclonal rabbit anti-rat immunoglobulin/HRP (Dako 1:750 dilution). Signal was developed with diaminobenzidine (DAB Dako). Images were acquired on a Zeiss Axio A1 microscope. All histopathological and immunohistochemical samples were reviewed and the quantitation of the cellular infiltrate was performed in a blinded manner to the observer. Statistical analysis Statistical differences between three or more experimental groups were analyzed using One-Way ANOVA followed by Turkey™s multiple comparison tests when mean of each group is compared with that of every other group or followed by Dunnett™s multiple comparison tests when mean of each group is compared with that of a control group. Statistical differences between two experimental groups were analyzed using Student™s t-test. Survival was analyzed with the Log-rank test. Prism 6.0 software (GraphPad Software) was used for all the statistical analysis. Abbreviations DC: Dendritic cell; scFv: Single-chain antibody variable fragment; MSLN: Mesothelin; MTB: Mycobacterium tuberculosis; Hsp: Heat shock protein; i.p.: Intraperitoneal; i.d.: Intradermal; BMDCs: Bone marrow-derived dendritic cells; APCs: Antigen-presenting cells; PBMCs: Peripheral blood mononuclear cells; PBLs: Peripheral blood leukocytes; LPS: Lipopolysaccharide; H&E: Haematoxylin and eosin; PFA: Paraformaldehyde; DAB: Diaminobenzidine; mAb: monoclonal antibody. Competing interests The authors declare that they have no competing interests. Authors™ contributions JY played a role in the design of the experiments acquisition analysis and interpretation of the data and writing the manuscript. PR JN YY NHA MN GJ-M XT SK HC PU BF TC and PL participated in the performance of experiments. SK and TB were involved in design of the experiments. RB was involved in data analysis. ER was involved in setting up murine ovarian cancer model. SO provided the murine ovarian cancer model. NS provided the plasmid that encodes an scFv fragment specific to MSLN and the recombinant P4 scFv protein. GD NS and SO gave constructive input on experimental design and data analysis. JG played a role in conception and design of the fusion protein. MP and JG were involved in the conceptualization and design of the study analysis and interpretation of datasets and in writing the manuscript. All authors read and approved the final manuscript. Supplementary Material Additional file 1: Figure S1 scFvMTBHsp70 binds to 40L mesothelioma cells. 40L cells were stained with scFvMTBHsp70 or MTBHsp70 followed by mouse anti-MTBHsp70 and Donkey anti-mouse Alexa Fluor 594. Cells were observed using a Nikon Eclipse TiE fluorescence microscope. A Representative pictures from three independent experiments. Scale bar 10 ?m. B Images were analyzed using the NIS-Elements AR Microscope Imaging Software. Mean Fluorescence Intensity was analyzed using ImageJ. P values were determined using One-Way ANOVA followed by Turkey™s multiple comparison tests. ****p?<?0.0001. Click here for file Additional file 2: Figure S2 scFvMTBHsp70 or MTBHsp70 plus P4 scFv treatment does not lead to infiltration of inflammatory cells into abdominal or intestinal mesothelial tissues. Samples of abdominal wall and intestine were prepared from C57BL/6 mice that had previously received multiple i.p. injections of scFvMTBHsp70 MTBHsp70 plus P4 scFv or saline as described in the Methods section. Sections of these tissues were stained with H&E and images were acquired on a Zeiss Axio A1 microscope. Representative images from 3 animals per treatment group are shown. No detectable level of mononuclear cell or granulocyte infiltrate within mesothelial tissues was seen in any sampled tissues. Scale bar 20 ?m. Click here for file Additional file 3: Figure S3 scFvMTBHsp70 treatment does not affect numbers of tumor-infiltrating CD8+ or Foxp3+ T cells. (A) Representative images of intratumoral CD8+ and Foxp3+ T cells from saline (n?=?3) scFvMTBHsp70 (n?=?3) or MTBHsp70 plus P4 scFv (n?=?3) -treated mice. Mouse spleen sections were used as positive controls: CD8+ and Foxp3+ T cells are clearly evident in the sections. Scale bar 20 ?m. (B) Numbers of CD8+ and Foxp3+ cells were quantified from 3“5 randomized fields. Click here for file Additional file 4: Figure S4 Validation of in vivo depletion of CD8+ cells in FVB/NJ mice. Mice were injected i.p. with 200 ?g of anti-CD8 mAb or an isotype-matched irrelevant rat IgG2a as described in Methods. All the mice were bled from the tail vein and the depletion of CD8+ cells was examined by flow cytometry analysis of peripheral blood cells stained with fluorophore-conjugated anti-CD8 on days 7 and 28 after tumor inoculation. (A) Representative results of flow analyses on 10 mice per group and reported as the percentage of CD8+ cells in lymphocytes. (B) CD8+ cells in the mice treated with isotype IgG2a or anti-CD8 mAb were compared. ***p< 0.001. Click here for file Acknowledgments This manuscript is dedicated to the memory of Janet Gelfand a victim of ovarian cancer. The authors gratefully acknowledge the continuing support for this work from the Edmund C. Lynch Jr. Cancer Fund Arthur Luxenberg Esq. Perry Weitz Esq. and the VIC Mesothelioma Research and Resource Program at MGH and the Friends of VIC Fund. PU and NHA were supported by the Prof. Dulcie V. Coleman Studentship at Imperial College London. We thank Oliver Mitchell John Cao Lujia Zhou Rumbidzai Mushavi and Sayinthen Vivekanantham for their technical assistances Dr. Yuhui Huang for his useful comments Michael Waring Dr. Michael Santuosuosso and Dr. Ravi Mylvaganam for their technical advice Dr. Musie Ghebremichael for his advice in statistical analysis and Mahnoor Valibhoy for her assistance with the schematic figure. 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10.1111/j.1349-7006.2012.02233.x 22320267 Li H Yu Y Sun L Wang H Zhang P Wei H Wang L Wan M Cao Z Wang Y Chen Y Dong B Wang L Vaccination with B16 tumor cell lysate plus recombinant Mycobacterium tuberculosis Hsp70 induces antimelanoma effect in mice Cancer biotherapy & radiopharmaceuticals 2010 25 185 191 10.1089/cbr.2009.0716 20423232 Cheng WF Hung CF Chai CY Hsu KF He L Rice CM Ling M Wu TC Enhancement of Sindbis virus self-replicating RNA vaccine potency by linkage of Mycobacterium tuberculosis heat shock protein 70 gene to an antigen gene J Immunol 2001 166 6218 6226 11342644 Uto T Tsujimura K Uchijima M Seto S Nagata T Suda T Chida K Nakamura H Koide Y A novel vaccine strategy to induce mycobacterial antigen-specific Th1 responses by utilizing the C-terminal domain of heat shock protein 70 FEMS immunology and medical microbiology 2011 61 189 196 10.1111/j.1574-695X.2010.00762.x 21204994 Rasoli M Omar AR Aini I Jalilian B Syed Hassan SH Mohamed M Fusion of HSP70 gene of 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N Y Acad Sci 2009 1174 6 17 10.1111/j.1749-6632.2009.04933.x 19769731 Banerjee D Matthews P Matayeva E Kaufman JL Steinman RM Dhodapkar KM Enhanced T-Cell responses to glioma cells coated with the anti-EGF receptor antibody and targeted to activating Fc[gamma]Rs on human dendritic cells Journal of Immunotherapy 2008 31 113 120 110.1097/CJI.1090b1013e31815a35892 10.1097/CJI.0b013e31815a5892 18481381 Wang Y Kelly CG Singh M McGowan EG Carrara AS Bergmeier LA Lehner T Stimulation of Th1-polarizing cytokines C-C chemokines maturation of dendritic cells and adjuvant function by the peptide binding fragment of heat shock protein 70 J Immunol 2002 169 2422 2429 12193710 Xing D Orsulic S A mouse model for the molecular characterization of brca1-associated ovarian carcinoma Cancer research 2006 66 8949 8953 10.1158/0008-5472.CAN-06-1495 16982732 Miselis NR Lau BW Wu Z Kane AB Kinetics of host cell recruitment during dissemination of diffuse malignant peritoneal mesothelioma Cancer microenvironment: official journal of the International Cancer Microenvironment Society 2010 4 39 50 21505561 Singh R Paterson Y Vaccination strategy determines the emergence and dominance of CD8+ T-cell epitopes in a FVB/N rat HER-2/neu mouse model of breast cancer Cancer research 2006 66 7748 7757 10.1158/0008-5472.CAN-05-4469 16885378 Huang Y Yuan J Righi E Kamoun WS Ancukiewicz M Nezivar J Santosuosso M Martin JD Martin MR Vianello F Leblanc P Munn LL Huang P Duda DG Fukumura D Jain RK Poznansky MC Vascular normalizing doses of antiangiogenic treatment reprogram the immunosuppressive tumor microenvironment and enhance immunotherapy Proc Natl Acad Sci USA 2012 109 17561 17566 10.1073/pnas.1215397109 23045683 Fields RC Shimizu K Mule JJ Murine dendritic cells pulsed with whole tumor lysates mediate potent antitumor immune responses in vitro and in vivo Proc Natl Acad Sci USA 1998 95 9482 9487 10.1073/pnas.95.16.9482 9689106 Millar DG Garza KM Odermatt B Elford AR Ono N Li Z Ohashi PS Hsp70 promotes antigen-presenting cell function and converts T-cell tolerance to autoimmunity in vivo Nature medicine 2003 9 1469 1476 10.1038/nm962 14625545 Delamarre L Pack M Chang H Mellman I Trombetta ES Differential lysosomal proteolysis in antigen-presenting cells determines antigen fate Science 2005 307 1630 1634 10.1126/science.1108003 15761154 Platt CD Ma JK Chalouni C Ebersold M Bou-Reslan H Carano RA Mellman I Delamarre L Mature dendritic cells use endocytic receptors to capture and present antigens Proc N"

Lung_Cancer

"However the production and administration of these tailor-made DC vaccines are costly and labor-intensive [5]. As a next-step in the development of DC vaccines we designed a recombinant protein that contains a Mycobacterium tuberculosis heat shock protein 70 (MTBHsp70) fused to a single chain variable fragment (scFv) derived from human B cells that targets mesothelin. Mesothelin (MSLN) is a validated immunotherapy target that is highly overexpressed on the surface of common epithelial cancers including ovarian cancers epithelial malignant mesotheliomas ductal pancreatic adenocarcinomas and lung adenocarcinomas while expressed at relatively low levels only in mesothelial cells lining the pleura pericardium and peritoneum in healthy individuals [6-9]. Several therapeutic agents targeting MSLN are evaluated in preclinical and clinical studies such as the recombinant immunotoxin SS1P [9-11]. In our fusion protein the anti-MSLN scFv moiety was originally isolated from a yeast-display human scFv library [12] and demonstrated the ability to recognize both membrane-bound and soluble MSLNs and inhibit CA125/MSLN-dependent cell adhesion [13-15]. The recombinant MTBHsp70 protein provides immunostimulatory functions including the activation of monocytes and DCs to produce CC-chemokines that attract antigen processing and presenting DCs macrophages and effector T and B cells enhanced DC aggregation and maturation [1617] induction of the cytotoxic activity of natural killer cells [18] and improved cross-priming of T cells which is dependent on DCs [19]. The capabilities of MTBHsp70 as a potent immune adjuvant have been well characterized in cancer models including murine models of melanoma and lymphoma [1820-24]. While in these studies proteins or peptides fused with Hsp70 used for immunizations in mice were shown to generate humoral or cellular immune responses we expect that fusion of anti-MSLN scFv and MTBHsp70 takes advantage of the immune-activating action of MTBHsp70 and the tumor-targeting activity of the scFv which will yield anti-tumor responses against the broadest profile of tumor antigens. We evaluated the therapeutic efficacy of this MSLN-targeted fusion protein in syngeneic mouse models of ovarian cancer and mesothelioma and examined its mechanism of action in in vitro and in vivo cross-presentation assay systems. These studies demonstrate that this bifunctional fusion protein significantly enhances survival and slows tumor growth through the augmentation of tumor-specific cell-mediated immune responses. Results Expression of scFvMTBHsp70 fusion protein and MTBHsp70 The structure of scFvMTBHsp70 is shown in Figure 1A. VH and VL from anti-MSLN P4 scFv [13] are linked using a (G4S)3 linker and fused to full length MTBHsp70 with a (G4S)3 linker in between. As shown in Figure 1B only one protein band was observed with a molecular weight of approximately 100 kDa for scFvMTBHsp70 and one protein band with a molecular weight of 70 kDa for MTBHsp70 which match the expected molecular weights of these specific proteins. Endotoxin contamination levels in scFvMTBHsp70 and MTBHsp70 were found to be very low at less than 50 EU per mg of protein. Structure and analysis of scFvMTBHsp70 fusion protein. A anti-MSLN VH and VL are linked with a (G4S)3 linker and fused to full length MTBHsp70 with a (G4S)3 linker. B RAPIDstain based on Coomassie dye following purification and hIgG-Fc tag removal of MTBHsp70 and scFvMTBHsp70. C BR5FVB1 ovarian cancer cells and 40L mesothelioma cells were incubated with 40 ?g/ml scFvMTBHsp70 or 26 ?g/ml MTBHsp70 (blue line) or without either protein (solid) followed by anti-MTBHsp70 (IgG2a) biotinylated anti-IgG2a and Streptavidin-APC and then analyzed by flow cytometry. To confirm that the scFv portion of the fusion protein binds to MSLN on the surface of tumor cells scFvMTBHsp70 or MTBHsp70 was preincubated with 12 ?g/ml recombinant human MSLN for 30 min (red line) before being added to the cells. Data are representative of three independent experiments in duplicate tubes. D Median fluorescence intensity (MFI) values of cells stained with scFvMTBHsp70 or MTBHsp70 normalized to cells stained without either protein. Data are expressed as means?±?SEM in arbitrary units. P values were determined using One-Way ANOVA followed by Turkey™s multiple comparison tests. *p?<?0.05; **p?<?0.01;ns non-significant. E scFvMTBHsp70 binds with peritoneal mesothelial cells at a low level compared to ovarian cancer and mesothelioma cells. Binding of the fusion protein is at very low or undetectable levels on PBLs and splenocytes. Thick line with incubation of scFvMTBHsp70; solid without incubation of scFvMTBHsp70. Data are representative of three independent experiments. scFvMTBHsp70 binds to BR5FVB1 ovarian cancer cells and 40L mesothelioma cells through the interaction of scFv with MSLN on the surface of tumor cells Binding of scFvMTBHsp70 or MTBHsp70 to BR5FVB1 ovarian cancer cells or 40L mesothelioma cells as determined by flow cytometry is shown in Figure 1C and D. Binding of scFvMTBHsp70 to MSLN-expressing tumor cells was almost completely inhibited by preincubation of scFvMTBHsp70 with recombinant human MSLN. Although MTBHsp70 also binds to these MSLN-expressing tumor cells the level of binding is not significantly different from background (p?=?0.187 for BR5FVB1 cells and p?=?0.086 for 40L cells). Furthermore the binding of MTBHsp70 to cancer cells cannot be blocked by recombinant MSLN. These data support the view that binding of scFvMTBHsp70 to these tumor cells occurred via the interaction of the scFv portion of the fusion protein with MSLN on the surface of tumor cells. Binding of these proteins with 40L mesothelioma cells was further compared using fluorescence microscopy. scFvMTBHsp70 shows significantly stronger binding intensity as compared to MTBHsp70 (Additional file 1: Figure S1A and B). In order to determine if scFvMTBHsp70 also binds to normal tissue in addition to tumor cells we incubated the fusion protein with peripheral blood leukocytes (PBLs) splenocytes or peritoneal mesothelial cells from healthy FVB/NJ mice and stained the cells using the same method as was used for staining tumor cells. As shown in Figure 1E scFvMTBHsp70 binds with peritoneal mesothelial cells at a low level compared to ovarian cancer and mesothelioma cells. Binding of the fusion protein is at very low or undetectable levels on PBLs and splenocytes. Since scFvMTBHsp70 may potentially target peritoneal mesothelial cells we also explored whether it could induce inflammation in peritoneal mesothelial tissues. We injected na¯ve mice with saline scFvMTBHsp70 or MTBHsp70 plus P4 scFv at the same doses as those used for tumor therapy described in Method sacrificed the mice 7 days post final treatments and examined haematoxylin and eosin (H&E) stained sections prepared from abdominal and intestinal peritoneum. Light microscopic examination revealed no evidence of inflammation and no infiltration of inflammatory cells such as macrophages or granulocytic cells around the mesothelial cells lining the abdominal and intestinal peritoneum of the actively treated or control animals. Representative microscopic images are shown in Additional file 2: Figure S2. scFvMTBHsp70 significantly prolongs ascites-free survival and overall survival in ovarian cancer- or mesothelioma-bearing mice To determine whether scFvMTBHsp70 can prolong survival in tumor-bearing mice we first evaluated the protein in a syngeneic mouse model of papillary ovarian cancer using immune-competent FVB/NJ mice. As shown in Figure 2A scFvMTBHsp70 prolonged both ascites-free and overall survival time compared with saline or the equimolar mixture of MTBHsp70 plus P4 scFv. To further support the efficacy of this fusion protein in prolonging survival in MSLN-expressing tumor-bearing mice we evaluated this protein in a second syngeneic mouse model of mesothelioma using immune-competent C57BL/6 mice. Animals treated with scFvMTBHsp70 showed significantly prolonged ascites-free and overall survival time compared with saline- or MTBHsp70 plus P4 scFv- treated mice (Figure 2B). A and B Kaplan-Meier survival curves of tumor-bearing mice following treatment with scFvMTBHsp70 control proteins or normal saline. A In a syngeneic mouse model of papillary ovarian cancer in immune-competent FVB/NJ mice scFvMTBHsp70 prolonged ascites-free survival time compared with saline (n?=?10 per group representative of two independent experiments; median survival (Med. sur.)?=?47 days vs. 37.5 days) or the mixture of MTBHsp70 plus P4 scFv (Med. sur. = 39 days). scFvMTBHsp70 also prolonged overall survival time in the mice compared with saline (Med. sur. = 51.5 days vs. 43 days) or the mixture of MTBHsp70 plus P4 scFv (Med. sur. = 43 days). B In a syngeneic mouse model of mesothelioma in immune-competent C57BL/6 mice the fusion protein prolonged ascites-free survival time compared with saline-treated mice (n?=?20 per group pooled from two independent experiments; Med. sur. = 28 days vs. 26 days) or the mixture of MTBHsp70 plus P4 scFv (Med. sur. = 27 days). The fusion protein also prolonged overall survival time compared with saline (Med. sur. = 36 days vs. 31 days). P values were determined using the log-rank test. *p?<?0.05; **p?<?0.01; ***p?<?0.001. scFvMTBHsp70 enhances anti-tumor CD8+ T-cell responses in ovarian tumor-bearing mice To investigate whether the anti-tumor effects of scFvMTBHsp70 was associated with anti-tumor effector CD8+ T-cell responses we re-stimulated splenocytes from ovarian tumor-bearing FVB mice that received different treatments with the CD8+ T-cell Her2/neu epitope or MSLN Ld1 as a negative control ex vivo and analyzed the cells for production of IFN? and Granzyme B using flow cytometry. We previously showed that Her2/neu is expressed by BR5FVB1 cells [25]. Ld1 is an in-house designed H2d-restricted MSLN peptide that did not induce ovarian cancer specific T-cell response in H-2q FVB mice. We demonstrated significantly greater anti-Her2/neu CD8+ T-cell responses in splenocytes from scFvMTBHsp70-treated mice compared to mice treated with saline or a simple mixture of MTBHsp70 plus P4 scFv as measured by IFN? and Granzyme B production by CD8+ T cells (Figure 3A and B). This indicates that scFvMTBHsp70 enhances anti-tumor specific CD8+ T-cell responses in ovarian tumor-bearing mice. However no significant difference was seen in the number of tumor-infiltrating CD8+ T cells and no tumor-infiltrating Foxp3+ T cells were seen in tumors from mice in different treatment groups indicating that scFvMTBHsp70 may improve effector cell function rather than the number of intratumoral CD8+ T cells (Additional file 3: Figure S3A and B). Figure 3 Anti-tumor specific CD8+ T-cell functions in tumor-bearing mice following different treatments. A Splenocytes harvested from mice treated with scFvMTBHsp70 fusion protein equimolar mixture of MTBHsp70 plus P4 scFv or saline (n = 10 per group) were re-stimulated with Her2/neu peptide or MSLN Ld1 peptide. Results are reported as the difference between nonstimulated (media alone) and stimulated cells and expressed as the frequency of parent CD3+CD8+ cells. P values were determined using One-Way ANOVA followed by Dunnett™s multiple comparison tests. B Representative flow data are presented. C In vivo CD8+ T-cell depletion study. FVB/NJ mice were injected i.p. with anti-CD8 mAb or an isotype-matched irrelevant rat IgG2a and were treated with scFvMTBHsp70 or saline as described in the methods. CD8+ T-cell depletion significantly and negatively impacted ascites-free survival in the scFvMTBHsp70 treated BR5FVB1 tumor-bearing animals compared to non depleted actively treated (n = 10 per group representative of two independent experiments; Med. sur. = 32.5 days vs. 48 days) animals. After CD8+ T cells depletion scFvMTBHsp70 treatment did not delay onset of disease (clinically evident ascites) compared with saline (Med. sur. = 32.5 days vs. 31.5 days; p = 0.5938). P values were determined using log-rank test. *p< 0.05; **p < 0.01 ***p < 0.001. scFvMTBHsp70 is able to prime an adaptive tumor-specific immune response that has an absolute requirement for tumor-specific CD8+ T cells To determine whether CD8+ T cells play a major role in the protective anti-tumor effects observed in mice treated with scFvMTBHsp70 we conducted in vivo CD8+ T-cell depletion experiments using monoclonal antibodies. The absence of circulating CD8+ cells in peripheral blood following depletion was confirmed by flow cytometry (Additional file 4: Figure S4A and B). As shown in Figure 3C CD8+ T-cell depletion significantly and negatively impacted ascites-free survival in the scFvMTBHsp70-treated BR5FVB1 tumor-bearing animals compared to non-depleted actively-treated animals. Following CD8+ T-cell depletion scFvMTBHsp70 treatment did not delay onset of disease (clinically evident ascites) compared to saline treatment. Therefore our data suggest that the priming of an adaptive tumor-specific immune response by scFvMTBHsp70 treatment is chiefly mediated by tumor-specific CD8+ T cells. scFvMTBHsp70 stimulates maturation of murine bone marrow-derived dendritic cells In order to investigate immunological mechanisms involved in the scFvMTBHsp70-enhanced anti-tumor immune response we first examined if the scFvMTBHsp70 or MTBHsp70 proteins used in our study could stimulate maturation of bone marrow-derived dendritic cells (BMDCs) as shown in previous studies [1617]. We stimulated CD11c+ BMDCs with 2 ?g/ml of scFvMTBHsp70 or an equimolar amount of MTBHsp70 (1.3 ?g/ml). 1 ?g/ml lipopolysaccharide (LPS) was used as positive control. To determine whether the BMDC maturation was attributable to LPS contamination of the recombinant proteins used in this study we also incubated BMDCs with 0.1 ng/ml LPS which was the equivalent amount of endotoxin found in 2 ?g/ml scFvMTBHsp70. After a 24 h-incubation both scFvMTBHsp70 and MTBHsp70 induced DC maturation indicated by an increase in the expression of CD40 CD80 CD86 and MHC class II molecules in comparison to the control cultures in medium. The increased expression of these DC maturation markers were comparable to those on cells stimulated with 1 ?g/ml LPS. The contamination control showed that addition of 0.1 ng/ml LPS did not replicate the effects of scFvMTBHsp70 or MTBHsp70 allowing us to discriminate the scFvMTBHsp70- or MTBHsp70-specific effects from effects of LPS (Figure 4A and B). Figure 4 scFvMTBHsp70 induces DC maturation and promotes antigen presentation and cross-presentation. A CD11c+ BMDCs isolated form FVB/NJ mice were incubated for 24 h with 2 ?g/ml scFvMTBHsp70 1.3 ?g/ml MTBHsp70 1 ?g/ml LPS as positive control or 0.1 ng/ml LPS as contamination control (thick lines) or medium only (solid) stained for CD11c CD40 CD80 CD86 and MHC II and analyzed by flow cytometry. Histograms were gated on CD11c+ DCs. Data are representative of three independent experiments in duplicate wells. B Median fluorescence intensity (MFI) of LPS- or protein-stimulated BMDCs normalized to MFI of BMDCs maintained in medium. Data are expressed as means?±?SEM in arbitrary units. P values were determined using One-Way ANOVA followed by Dunnett™s multiple comparison tests. C BMDCs cultured from FVB/NJ mice were pulsed with BR5FVB1 cells alone (Column a) or BR5FVB1 cells pre-complexed with MTBHsp70 (Column b) or scFvMTBHsp70 (Column c) and then incubated with BR5FVB1 tumor cell-primed T cells. Intracellular granzyme B and IFN? expressions in CD3+CD4+ and CD3+CD8+ T cells were analyzed by flow cytometry. Data from three independent experiments in duplicate wells are pooled and analyzed using One-Way ANOVA followed by Turkey™s multiple comparison tests. Data are presented as mean?±?SEM. D Representative flow data are presented. E scFvMTBHsp70 enhanced tumor cell immunogenicity in vivo. Results are reported as the difference between nonstimulated (media alone) and stimulated cells and expressed as the frequency of parent CD3+CD4+ or CD3+CD8+ cells. P values were determined using One-Way ANOVA followed by Turkey™s multiple comparison tests. *p?<?0.05; **p?<?0.01; ***p?<?0.001; ****p?<?0.0001. The scFvMTBHsp70 fusion protein increases tumor antigen presentation and cross-presentation by DC in vitro In the current study we demonstrated that splenic CD8+ T cells from scFvMTBHsp70-treated tumor-bearing mice could produce cytokines upon specific tumor antigen stimulation ex vivo which was associated with their antitumor therapeutic efficacy in vivo. To determine whether scFvMTBHsp70 promotes tumor specific T-cell responses by enhancing antigen presentation and cross-presentation by antigen presenting cells we co-cultured BR5FVB1 tumor cell-primed T cells with DCs that had been pulsed with BR5FVB1 tumor cells in the presence of scFv-MTBHsp70 MTBHsp70 or PBS. The scFvMTBHsp70/tumor cell-pulsed DCs induced significantly higher production of IFN-? and Granzyme B from both CD4+ and CD8+ tumor cell-primed T cells as compared with MTBHsp70 or PBS indicating that scFvMTBHsp70 enhances tumor antigen presentation and cross-presentation by DCs (Figure 4C and D). scFvMTBHsp70 enhances tumor cell immunogenicity in vivo Having demonstrated in vitro that scFvMTBHsp70 enhances tumor antigen presentation and cross-presentation by DCs we next explored whether scFvMTBHsp70 enhances tumor antigen presentation and cross-presentation by DCs and consequently enhances tumor cell immunogenicity in vivo. It has been demonstrated that the high density of DCs at dermal sites facilitates the capture of tumor antigens and that local inflammation induces DC maturation and migration into draining lymph nodes where they present antigens to na¯ve T cells generating a tumor specific immune response [26]. We primed FVB mice with an intradermal (i.d.) injection of mitomycin C-treated BR5FVB1 tumor cells followed by a booster i.d. injection of BR5FVB1 tumor cells with or without scFvMTBHsp70 or MTBhsp70. After 20 days we dissociated skin-draining lymph nodes and re-stimulated lymph node lymphocytes with Her2/neu peptides mitomycin C-treated BR5FVB1 tumor cells or BR5FVB1 tumor cell lysate and performed flow cytometric analysis for the presence of Granzyme B-generating CD4+ and CD8+ T cells. As shown in Figure 4E we demonstrated that Granzyme B-generating CD4+ and CD8+ T cells were significantly enhanced in mice that were immunized with scFv-MTBHsp70-bound tumor cells as compared to those in the mice immunized with tumor cells alone MTBHsp70-bound tumor cells or saline. Discussion We have developed a novel protein-based immunotherapy consisting of a fusion of an anti-MSLN scFv of human origin and recombinant mycobacterial heat shock protein 70 that has the ability to adjuvant significant T-cell responses against specific tumor antigens. P4 scFv directed against MSLN a surface antigen overexpressed on several types of tumor cells is used as a means of targeting the immunotherapeutic agent. We have demonstrated that this bifunctional fusion protein effectively binds BR5FVB1 ovarian cancer cells or 40L mesothelioma cells through the interaction of scFv with MSLN on the surface of tumor cells. We found that the fusion protein significantly prolonged survival time in syngeneic mouse models of papillary ovarian cancer and malignant mesothelioma. Treatment with the fusion protein induced significant tumor-specific CD8+ T-cell immune responses in the splenocytes of ovarian tumor-bearing mice. Furthermore in vivo CD8+ T-cell depletion studies demonstrated that this protective antitumor effect is mainly mediated by tumor-specific CD8+ T cells. Treatment using a mixture of MTBHsp70 plus P4 scFv for ovarian tumor or malignant mesothelioma-bearing mice did not increase survival or enhance tumor-specific immune responses suggesting that only through fusion of the two elements is the immune system effectively activated. We also demonstrated that this approach does not induce inflammation in the abdominal or intestinal mesothelial tissues as a result of a bystander interaction with MSLN on normal mesothelial cells. Several properties of MTBHsp70 appear in this study to contribute to the generation of tumor-specific CD4+ and CD8+ T-cell immune responses. First it induces maturation of DCs. Although several previous studies suggested that MTBHsp70 had pro-inflammatory properties only when contaminated with LPS [2728] other studies have decisively demonstrated that MTBHsp70 alone while not LPS promotes DC maturation and innate immune responses [161729]. In our study we used a fusion protein generated from a mammalian cell expression system ensuring a minimal amount of LPS contamination. We also incubated DCs with the same amount of LPS as that found in the fusion protein and failed to replicate the effects observed with the fusion protein supporting the view that maturation of DCs can be attributed to the fusion protein rather than LPS. Secondly MTBHsp70 is capable of delivering epitopes for enhanced processing and MHC-I presentation by DCs to na¯ve CD8+ T cells a process known as cross-presentation [30]. Mycobacterial Hsp70 fusion proteins have been shown to elicit both CD4+ and CD8+ T-cell responses although priming of CD8+ T cells does not appear to require CD4+ T cells [3132]. We demonstrated in this study that the MSLN-targeted fusion protein elicited significant tumor-specific CD8+ T-cell immune responses in ovarian cancer-bearing mice and this adaptive antitumor response has an absolute requirement for tumor-specific CD8+ T cells. Although at the dosing schedule used in these studies tumor-specific T-cell responses did not eventually lead to rejection of the established tumors they significantly prolonged survival time in tumor-bearing mice. DCs are believed to play a pivotal role in the initiation and programming of tumor-specific T-cell responses and are becoming an essential target in efforts to generate therapeutic immunity against cancer [33]. Two main approaches are currently under consideration for providing DCs with tumor-specific antigens. One approach is to culture patient-derived DCs ex vivo with an adjuvant that induces DC maturation in the presence of tumor specific antigens followed by adoptive transfer into the patient [33]. This approach is fraught with technical and practical difficulties such as selection of a suitable antigenic target inappropriate maturation state of selected DCs and the difficulty of generating a sufficient number of DCs ex vivo. In addition a number of investigators have recently reported that ex vivo-derived DC vaccines have an insignificant role in the direct priming of T cells in vivo[33-35]. An alternative approach to generate tumor-specific antigen bearing DCs is to induce them to take up tumor-specific antigens in vivo. It has been shown that in vivo specific targeting of tumor antigens to DCs improves the induction of antigen-specific CD4+ and CD8+ T-cell immunity. In these studies an agonistic anti-CD40 monoclonal antibody was used to mature DCs and eliminate antigen-specific tolerance [36-39]. MTBHsp70 has also been shown to stimulate inflammation and DC maturation via an interaction with CD40 receptors on both DCs and monocytes thus acting as an alternative ligand to CD40L [2940]. In our study we showed the fusion protein up-regulates surface expression of phenotypic markers of DC maturation. Interestingly in addition to CD80 CD86 and MHC class II molecules the expression of CD40 is also enhanced indicating a possible positive feedback loop involving CD40 signaling components. Beyond promoting DC maturation the scFvMTBHsp70 fusion protein also targets tumor cells towards the matured DCs. We propose that binding of the fusion protein with both tumor cells and DCs improves phagocytosis of parts of tumor cells by DCs and therefore any tumor antigen can be processed and loaded on both MHC class II and MHC class I molecules and presented to CD4+ and CD8+ T cells. This could explain the observed augmentation of tumor antigen presentation and cross-presentation brought about by the fusion protein in vitro. This may also explain the observed increased anti-Her2/neu CD8+ T-cell responses in the scFvMTBHsp70-treated ovarian tumor bearing mice although Her2/neu is not directly targeted. We recapitulated these in vitro findings in an in vivo tumor cell immunogenicity study. We used the fusion protein to activate and mature DCs in the skin such as Langerhans cells. These DCs then captured tumor cells or tumor cell fragments through the connection established by the fusion protein and migrated to the draining lymphoid organs where they presented tumor antigens to na¯ve T cells. T cells recovered from the draining lymph node showed significantly enhanced responses to stimulation with a range of tumor antigens. Conclusion Our study provides preclinical evidence that supports a protein-based immunotherapy that induces anti-tumor immune responses which normally require dendritic cell-based approaches. The MSLN-targeted MTBHsp70 fusion protein binds MSLN on tumor cells recruits and activates APCs including DCs loads DCs in vivo with the broadest profile of naturally processed tumor antigens promotes tumor antigen presentation and cross-presentation and enhances tumor specific CD4+ and CD8+ T-cell responses (Figure 5). Our study supports the continued exploration of this novel fusion protein alone or in combination with immune checkpoint inhibitors following conventional surgical reduction and chemotherapy for MSLN-expressing cancers. This new approach could significantly increase time to recurrence and survival in humans with ovarian cancer and mesothelioma where effective second line treatment options are very limited. Figure 5 A schematic model showing that the scFvMTBHsp70 fusion protein binds with MSLN on tumor cells and activates antigen presenting cells (APCs) thus promoting uptake of tumor cells or tumor cell fragments and promoting tumor antigen presentation and cross-presentation as well as adjuvanting tumor specific CD4 + and CD8 + T-cell responses. Methods Production of proteins The plasmid pQE30-MTBhsp70 that encodes full length MTBHsp70 was a generous gift from Dr. Peter Sveshnikov (Moscow Medical Academy Russia). The plasmid pTOR2-scFv that encodes an scFv fragment specific to MSLN and the recombinant P4 scFv protein [13] generated and purified from yeast were generous gifts from Dr. Nathalie Scholler (Penn Ovarian Cancer Research Center University of Pennsylvania). The DNA fragment corresponding to a 15 amino acid linker (GGGGSGGGGSGGGGS) was connected to the scFv at its C-terminal using an overlap PCR approach. The PCR product scFv-linker was subcloned into pQE30-MTBhsp70 at the N-terminal of MTBhsp70. The DNA fragment for scFvMTBhsp70 was PCR amplified and cloned into pPMY5 (Promab) downstream of a human IgG1 Fc domain and separated from the Fc region by the signal cleavage sequence for Tobacco Etch Virus protease (TEV enzyme). scFvMTBHsp70 the MSLN-targeted fusion protein was generated from HEK293 cells and purified using Protein G resin (Pierce). The Fc region of the Protein G eluted protein was then cleaved from the fusion protein by TEV enzyme (Promab) digestion. MTBHsp70 was generated using the same expression system. The production and purification of these two proteins was accomplished by Promab Biotechnologies Inc. at Richmond CA. After purification and hIgG-Fc tag removal the integrity of scFv-MTBHsp70 and MTBHsp70 were determined by SDS-PAGE followed by staining with RAPIDstain (G-Bioscience). Endotoxin contamination levels in scFvMTBHsp70 and MTBHsp70 were determined by Limulus Amebocyte Lysate Assay (LAL-assay Cambrex). Cells The BR5FVB1 ovarian cancer cells a kind gift from Dr. Orsulic in Women™s Cancer Research Institute at Cedars-Sinai Medical Center [41] or 40L mesothelioma cells a kind gift from Dr. Kane in Department of Pathology and Laboratory Medicine at Brown University [42] were maintained at 37°C in DMEM with 2 mmol/L L-glutamine 10 units/ml penicillin 10 ?g/ml streptomycin and 10% fetal bovine serum in humidified atmosphere with 5% CO2. Cells were cultured until 80% confluent and harvested with enzyme-free cell-dissociation buffer (Gibco) for in vitro tumor cell binding assays and cross-presentation studies or harvested with Trypsin EDTA (Mediatech) for animal injections. Mouse PBLs were obtained from FVB mice via tail vein bleeds after lysis of erythrocytes using M-lyse buffer (R&D systems). Small pieces of parietal peritoneal membrane were taken from the mice and digested in enzyme-free cell-dissociation buffer to obtain mouse peritoneal mesothelial cells. To test whether scFvMTBHsp70 or MTBHsp70 binds to the MSLN-expressing tumor cells or non-cancerous cells we incubated BR5FVB1 ovarian tumor cells 40L mesothelioma cells or normal cells from FVB mice including PBLs splenocytes and peritoneal mesothelial cells with 40 ?g/ml scFvMTBHsp70 or 26 ?g/ml MTBHsp70 followed by anti-MTBHsp70 (IgG2a) (Biodesign International) biotinylated anti-IgG2a (BD Bioscience) and Streptavidin-APC (BioLegend) and then analyzed the tumor cells by flow cytometry. As controls cells were incubated with the reagents described above except scFvMTBHsp70 or MTBHsp70. To confirm that scFv portion of the fusion protein binds to MSLN on the surface of tumor cells scFvMTBHsp70 or MTBHsp70 was preincubated with 12 ?g/ml of recombinant human MSLN (R&D Systems) for 30 min before adding to the cells. For fluorescence microscopy cells were cultured on coverslips until 50% confluent stained with 10 ?g/ml scFvMTBHsp70 or 6.5 ?g/ml MTBHsp70 followed by mouse anti-MTBHsp70 (1:500 dilution) and Donkey anti-mouse Alexa Fluor 594 (Invitrogen 1:500 dilution). Cells were observed using a Nikon Eclipse TiE fluorescence microscope. In some experiments tumor cells were treated with 20 ?g/ml mitomycin C at a concentration of 5 — 106/ml for 1 h in a 37°C water bath and washed with complete medium at least 3 times before use. Animal models and tumor treatment Ovarian cancer was established by i.p. injection of syngeneic cancer cells BR5FVB1 (107 cells per mouse) into 6-week-old female FVB/NJ mice as previously described [25]. All mice were purchased from Jackson laboratories. Intraperitoneal mesotheliomas were established by i.p. injection of syngeneic 40L cells (2?—?106 per mouse) into 6-week-old male C57BL/6 mice as previously described [42]. Mice with ovarian tumors were treated 7 days after BR5FVB1 tumor cell inoculation with i.p. injections of scFvMTBHsp70 (2 ?g per mouse) normal saline or an equimolar mixture of MTBHsp70 plus P4 scFv. This was followed by "

Lung_Cancer

"Methods Lung flooding was performed in four human lung lobes which were resected from non-small cell lung cancers. B-mode imaging and temperature measurements were simultaneously obtained during high-intensity focused ultrasonography of centrally located lung cancers. The tumour was removed immediately following insonation and processed for nicotinamide adenine dinucleotide phosphate-diaphorase and H&E staining. In addition the left lungs of three pigs were flooded. Purified BSA in glutaraldehyde was injected centrally into the left lower lung lobe to simulate a lung tumour. The ultrasound was focused transthoracically through the flooded lung into the simulated tumour with the guidance of sonography. The temperature of the tumour was simultaneously measured. The vital signs of the animal were monitored during the procedure. Results A well-demarcated lesion of coagulation necrosis was produced in four of four human lung tumours. There did not appear to be any damage to the surrounding lung parenchyma. After high-intensity focused ultrasound insonation the mean temperature increase was 7.5-fold higher in the ex vivo human tumour than in the flooded lung tissue (52.1 K?±?8.77 K versus 7.1 K?±?2.5 K). The transthoracic high-intensity focused ultrasound of simulated tumours in the in vivo model resulted in a mean peak temperature increase up to 53.7°C (±4.5). All of the animals survived the procedure without haemodynamic complications. Conclusions High-intensity focused ultrasound with lung flooding produced a thermal effect in an ex vivo human lung carcinoma and in vivo simulated lung tumours in a porcine model. High-intensity focused ultrasound is a potential new strategy for treating lung cancer. High-intensity focused ultrasound Lung cancer ablation Lung flooding Background Lung cancer remains the leading cause of cancer-related death in the western population [12]. In addition to primary tumours the lung is the most common site for metastatic spread of other malignancies. Curative treatment of lung tumours or metastases requires surgical resection which results in the loss of functional lung parenchyma. Unfortunately fewer than half of patients are eligible for curative surgery due to limited lung function [3]. Radiation or chemotherapy alone rarely cures patients. Minimally invasive local ablation (for example percutaneous laser or radiofrequency) results in major complications in 6% of patients and procedure-specific 30-day mortality rates of 2.6% [4]. Furthermore the local progression rate is 35% [5]. Therefore these procedures are palliative strategies. High-intensity focused ultrasound (HIFU) is a non-invasive highly precise procedure to locally destroy tissue through ablation. Current clinical trials are evaluating the effectiveness of HIFU for the treatment of cancers of the brain breast liver bone and prostate [6-9]. However lung cancers have never been treated with this approach because the ventilated lung is a total acoustic absorber and reflector [10]. This problem was solved by lung flooding. Lung flooding enables efficient lung sonography and tumour imaging in ex vivo human and in vivo porcine lung cancer models [11]. The conditions for applying HIFU to lung tumours are provided. The aim of this study was to evaluate the effectiveness of sonography-guided HIFU with lung flooding for lung tumour ablation in ex vivo human and in vivo animal models. Methods Ex vivo examinations Preparation of resected human lung lobes with carcinoma Four patients with non-small cell lung cancer (NSCLC) received therapeutic lobectomies of the right lower lobe (n?=?2) right upper lobe (n?=?1) or left lower lobe (n?=?1). The average age of the patients was 75.7 years (range 68 to 82 years). The mean tumour diameter was determined by computed tomography and was 4.1 cm (range 3.2 to 5.1 cm). Pre-surgical histological diagnoses of three adenocarcinomas and one large cell neuroendocrine carcinoma were confirmed with percutaneous or transbronchial needle biopsies. The lobes were cooled in a 15°C isotonic liquid bath immediately following resection and prepared for liquid filling (flooding) in tabula. An expanded polytetrafluoroethylene graft was then anastomosed end-to-end with the lobar bronchi. An infusion system was connected to the conduit and the lobes were continuously filled with isotonic saline (15°C) until the functional residual capacity was reached. The lung lobe was examined transpleurally by ultrasound (MicroMaxx„¢ Portable Ultrasound System; SonoSite Inc. Bothell WA USA) in the liquid bath with a linear probe (L38e 10 to 5 MHz; SonoSite Inc. Bothell WA USA) in fundamental B-mode. The lobes were selected for HIFU only in cases with gas-free filling and complete sonographic imaging of the tumour. This study was approved by the ethics committee of the Medical Association of Thuringia. HIFU application The experimental HIFU setup contained the HIFU transducer H102 (Sonic Concepts Inc. Bothell WA USA) a power amplifier (RF Source Athena Greece) a Type K thermocouple (XF339 Labfacility Ltd. Sheffield South Yorkshire UK) and a digital multimeter (34401A Agilent Santa Clara CA USA). The HIFU setup and alignment with the tumour are illustrated in . The thermocouple was guided through the centre hole of the HIFU transducer into the tumour. The alignment of the HIFU focal zone within the tumour and the thermocouple was monitored by ultrasound with a curved imaging probe (C11e SonoSite Inc. Bothell WA USA) which was mounted sideways on the HIFU transducer. Self-heating of the thermocouple was excluded by applying HIFU to the thermocouple in water. An impedance-matching network provided by the manufacturer drives the HIFU probe with a radiofrequency (RF) amplifier. The acoustic power of the focal zone in water was estimated to be 2500 W/cm2 at 1.1 MHz (ISPTP spatial peak temporal peak) based on the manufacturer™s calibration sheet. A duty cycle of 50% and a repetition frequency of 10 KHz were applied. The cancer tissue was exposed to HIFU for ten seconds. The focal zone was immediately moved through the tumour in lateral 2 mm increments. HIFU was initiated again for ten seconds and ultrasound B-mode imaging was then performed. HIFU exposure ended after one slice of the HIFU zone moved through the tumour volume. Subsequently the HIFU focus and thermocouple were placed into flooded lung tissue. The procedure was repeated as described above. All experimental steps were performed within 45 minutes of surgical resection. Schematic setup of ex vivo high-intensity focused ultrasound application. High-intensity focused ultrasound transducer (1) flooded lung (2) cancer (3) attenuator (4) impedance matching network (5) power RF amplifier (6) signal generator (7) thermocouple (8) multimeter (9) thermo-control (10) and sector-array probe (11). Histopathology and enzyme histochemistry The specimen including the tumour was cut at a 90° angle to the treated cross section immediately following the last HIFU exposure. Each half of the tumour was sectioned parallel to the cutting surface to show the transition from ablated to non-ablated tumour. One part was fixed in 4% formaldehyde for H&E and the other was frozen in liquid nitrogen for nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining. An intralobar metastasis from one case served as an untreated tissue control. In vivo examination Animal preparation Animal experiments were performed on three female pigs (Deutsches Landschwein breed; weight range: 33 to 38 kg average: 35.5 kg) with permission from the Veterinary Department of the Thuringian State Authority for Food Protection and Fair Trading in compliance with the National Animal Protection Act. Total intravenous anaesthesia was initiated with propofol (10 mg/kg/h) fentanyl (0.05 to 0.08 ?g/kg/min) and pancuronium bromide (2.5 ?g/kg/min). A left-sided Robertshaw double-lumen tube with an extra-long bronchial lane (size 39 Ch; special product by Mallinckrodt Medical Dublin Ireland) was inserted after tracheotomy. Mechanical ventilation was performed with an ICU respirator (Servo 900 Siemens AG Munich Germany) on a volume-controlled setting (FIO2?=?1.0; tidal volume 10 ml/kg; respiratory rate 16 to 20/min; positive end expiratory pressure?=?6 cm H2O). The electrocardiogram arterial blood pressure capillary oxygen saturation and expiratory CO2 concentration were measured and recorded continuously (Datex AS/3 Compact Multiparameter Patient Monitor; Datex-Ohmeda Corp. Helsinki Finland). Arterial blood gases were analysed every 30 minutes (ABL System 625; Radiometer Medical Copenhagen Denmark). Tumour simulation and HIFU application The HIFU setup for transthoracic application on pigs contained a self-manufactured sample holder. The HIFU transducer (SU102 Sonic Concepts Inc. Bothell WA USA) was arranged with the sideways attached puncture channel to guide the thermocouple and curved imaging probe (C11e MicroMaxx; SonoSite Inc. Bothell WA USA). The three elements were arranged such that the thermocouple tip was in the focus of the HIFU beam. A rib was resected after lung flooding without pleural injuries. The HIFU applicator was placed above the resected rib (Figure 2). A 17-G needle was placed in the centre of the lower left lung lobe with the guidance of a percutaneous transpleural ultrasound. Five millilitres of a fluid composed of purified BSA and glutaraldehyde (Bioglue®; CryoLife Europa Guildford UK) were injected to simulate a lung tumour. A thermocouple was immediately inserted through the needle into the simulated tumour. The alignment of the focal zone with the target lesion and thermocouple was monitored by ultrasound imaging. The surgical HIFU probe with an outer diameter of 35 mm operates at 3.0 MHz. The focal acoustic intensity was estimated to be 2000 W/cm2 MHz (ISPTP? spatial peak temporal peak) based on the manufacturer™s calibration sheet. HIFU was applied according to a ˜one second on one second off™ scheme. The temperature inside the simulated tumour was recorded by the thermocouple during HIFU exposure. Data were transferred and stored on a laptop. Figure 2 In vivo high-intensity focused ultrasound application after rib resection. Thermocouple (1) high-intensity focused ultrasound transducer (2) and sector-array probe (C11e 8 to 5 MHz Micromaxx Sonosite Inc. Bothell WA USA) (3). Results Ex vivo examinations The mean temperature in the tumour tissue increased by 52.1 K?±?8.77 K after ten seconds of HIFU exposure. The same acoustic intensity produced a mean temperature increase of 7.1 K?±?2.5 K in a flooded lung. The temperature increase in the ex vivo human NSCLC was 7.5-fold higher than that in the flooded lung tissue (Figure 3). The sonoablated cancer tissue appeared to be strongly hyperechoic immediately following HIFU application (Figure 4). The strength of echogenicity decreased 30 seconds after HIFU exposure but did not disappear. The ablated cancer tissue appeared to be slightly whitish in all four cases. A demarcation between treated and non-treated areas was observed (Figure 5). Figure 3 Mean temperature increase. The mean temperature increases (± standard deviation) within an adenocarcinoma surrounded with flooded lung tissue (n?=?3) and within flooded lung alone (n?=?3) are shown. Temperatures were measured with a thermocouple ten seconds after ex vivo high-intensity focused ultrasound insonation. Figure 4 Sonomorphology of adenocarcinoma in ex vivo flooded human lung before and after high-intensity focused ultrasound. (A) B-mode ultrasound image of adenocarcinoma in flooded lung before ex vivo high-intensity focused ultrasound insonation. Adenocarcinoma (1) flooded lung (2) thermocouple (3) and focal zone of high-intensity focused ultrasound beam (4)"

Lung_Cancer

"human FTSJ2 and porcine FTSJ2. (A) The protein structure of E. coli RrmJ which was resolved by B¼gl et al. (2000) (PDB ID: 1EIZ) [7]. (B) The protein structure of human FTSJ2 which was resolved by Wu et al. (2009) (PDB ID: 2NYU) [36]. (C) The protein structure of porcine FTSJ2 which was predicted using the SWISS-MODEL website with human FTSJ2 as a template. The ?-helices and ?-strands are shown in green and yellow respectively. The SAM residues and the K-D-K-E catalytic center are shown in the ball and stick representations respectively. (TIF) Click here for additional data file. Figure S2 Porcine Ftsj2 mRNA expression in porcine tissues. (A) Expression of porcine Ftsj2 mRNA as measured by semi-quantitative RT-PCR. Porcine ?-actin mRNA was used as a loading control. (B) Quantification of the porcine Ftsj2 mRNA expression which normalized to the ?-actin mRNA expression. The values are equal to?=?the means of duplicate experiments. (TIF) Click here for additional data file. The authors would like to thank Dr. Jeremy J.W. Chen for providing the CL1-0 and CL1-5 cell lines. We also like to thank our colleagues (Drs. Tung-Chou Tsai Yu-Tang Tung and Zi-Lun Lai) in the Molecular Embryology and DNA Methylation Laboratory for their help with discussions and technical issues. References 1 AngM LiberekK SkowyraD ZyliczM GeopoulosC (1991) Biological role and regulation of the universally conserved heat shock proteins. J Biol Chem266: 24233“242361761528 2 BenjaminIJ McMillanDR (1998) Stress (heat shock) proteins: molecular chaperones in cardiovascular biology and disease. Circ Res83: 117“1329686751 3 RichmondCS GlasnerJD MauR JinH BlattnerFR (1999) Genome-wide expression profiling in Escherichia coli K-12. Nucleic Acids Res27: 3821“383510481021 4 OguraT TomoyasuT YukiT MorimuraS BeggKJ et al (1991) Structure and function of the ftsH gene in Escherichia coli. Res Microbiol142: 279“2821925026 5 CaldasT BinetE BoulocP CostaA DesgresJ et al (2000) The FtsJ/RrmJ heat shock protein of Escherichia coli is a 23 S ribosomal RNA methyltransferase. J Biol Chem275: 16414“1641910748051 6 LapeyreB (2004) Conserved ribosomal RNA modification and their putative roles in ribosome biogenesis and translation. Curr Genet12: 263“284 7 B¼glH FaumanEB StakerBL ZhengF KushnerSR et al (2000) RNA methylation under heat shock control. Mol Cell6: 349“36010983982 8 BlanchardSC PuglisiJD (2001) Solution structure of the A loop of 23S ribosomal RNA. Proc Natl Acad Sci USA98: 3720“372511259644 9 CaldasT BinetE BoulocP RicharmeG (2000) Translational defects of Escherichia coli mutants deficient in the Um(2552) 23S ribosomal RNA methyltransferase RrmJ/FTSJ. Biochem Biophys Res Commun271: 714“71810814528 10 FederM PasJ WyrwiczLS BujnickiJM (2003) Molecular phylogenetics of the RrmJ/fibrillarin superfamily of ribose 2?-O-methyltransferases. Gene302: 129“13812527203 11 PintardL LecointeF BujnickiJM BonnerotC GrosjeanH et al (2002) Trm7p catalyses the formation of two 2?-O-methylriboses in yeast tRNA anticodon loop. EMBO J21: 1811“182011927565 12 PintardL BujnickiJM LapeyreB BonnerotC (2002) MRM2 encodes a novel yeast mitochondrial 21S rRNA methyltransferase. EMBO J21: 1139“114711867542 13 BonnerotC PintardL LutfallaG (2003) Functional redundancy of Spb1p and a snR52-dependent mechanism for the 2?-O-ribose methylation of a conserved rRNA position in yeast. Mol Cell12: 1309“131514636587 14 KresslerD RojoM LinderP CruzJ (1999) Spb1p is a putative methyltransferase required for 60S ribosomal subunit biogenesis in Saccharomyces cerevisiae. Nucleic Acids Res27: 4598“460810556316 15 LapeyreB PurushothamanSK (2004) Spb1p-directed formation of Gm2922 in the ribosome catalytic center occurs at a late processing stage. 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" non“small-cell lung cancer Clin Lung Cancer 10 252 256 19632943 Patel JD Hensing TA Rademaker A Hart EM Blum MG Milton DT Bonomi PD 2009 Phase II study of pemetrexed and carboplatin plus bevacizumab with maintenance pemetrexed and bevacizumab as first-line therapy for nonsquamous non“small-cell lung cancer J Clin Oncol 27 3284 3289 19433684 Ramlau R Gorbunova V Ciuleanu TE Novello S Ozguroglu M Goksel T Baldotto C Bennouna J Shepherd FA Le-Guennec S Rey A Miller VA Thatcher N Scagliotti GV 2012 Aflibercept and docetaxel versus docetaxel alone after platinum failure in patients with advanced or metastatic non-small-cell lung cancer: a randomized controlled phase III trial J Clin Oncol 30 3640 3647 22965962 Reck M von Pawel J Zatloukal P Ramlau R Gorbounova V Hirsh V Leighl N Mezger J Archer V Moore N Manegold C 2009 Phase III trial of cisplatin plus gemcitabine with either placebo or bevacizumab as first-line therapy for nonsquamous non-small-cell lung cancer: AVAiL J Clin Oncol 27 1227 1234 19188680 Riess JW Logan AC Krupitskaya Y Padda S Clement-Duchene C Ganjoo K Colevas AD Pedro-Salcedo MS Kuo CJ Wakelee HA 2012 Maintenance bevacizumab is associated with increased hemoglobin in patients with advanced nonsquamous non-small cell lung cancer Cancer Invest 30 231 235 22360362 Sandler AB Gray R Perry MC Brahmer J Schiller JH Dowlati A Lilenbaum R Johnson DH 2006 Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer N Engl J Med 355 2542 2550 17167137 Scagliotti GV Parikh P von Pawel J Biesma B Vansteenkiste J Manegold C Serwatowski P Gatzemeier U Digumarti R Zukin M Lee JS Mellemgaard A Park K Patil S Rolski J Goksel T de Marinis F Simms L Sugarman KP Gandara D 2008 Phase III study comparing cisplatin plus gemcitabine with cisplatin plus pemetrexed in chemotherapy-naive patients with advanced-stage non-small-cell lung cancer J Clin Oncol 26 3543 3551 18506025 Schiller JH Harrington DP Belani CP Langer CJ Sandler AB Krook JE Zhu J Johnson DH the Eastern Cooperative Oncology Group 2002 Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer N Engl J Med 346 92 98 11784875 Sher A Wu S 2011 Anti-vascular endothelial growth factor antibody bevacizumab reduced the risk of anemia associated with chemotherapy“A meta-analysis Acta Oncol 50 997 1005 21554028 Sitohy B Nagy JA Jaminet S-CS Dvorak HF 2011 Tumor-surrogate blood vessel subtypes exhibit differential susceptibility to anti-VEGF therapy Cancer Res 71 7021 7028 21937680 Tam BYY Wei K Rudge JS Hoffman J Holash J Park S Yuan J Hefner C Chartier C Lee J-S Jiang S Nayak NR Kuypers FA Ma L Sundram U Wu G Garcia JA Schrier SL Maher JJ Johnson RS Yancopoulos GD Mulligan RC Kuo CJ 2006 VEGF modulates erythropoiesis through regulation of adult hepatic erythropoietin synthesis Nat Med 12 793 800 16799557 Tew WP Gordon M Murren J Dupont J Pezzulli S Aghajanian C Sabbatini P Mendelson D Schwartz L Gettinger S Psyrri A Cedarbaum JM Spriggs DR 2010 Phase 1 study of aflibercept administered subcutaneously to patients with advanced solid tumors Clin Cancer Res 16 358 366 20028764 Therasse P Arbuck SG Eisenhauer EA Wanders J Kaplan RS Rubinstein L Verweij J Van Glabbeke M van Oosterom AT Christian MC Gwyther SG 2000 New guidelines to evaluate the response to treatment in solid tumors (RECIST Guidelines) J Natl Cancer Inst 92 205 216 10655437 Van Cutsem E Tabernero J Lakomy R Prenen H Prausova J Macarulla T Ruff P van Hazel GA Moiseyenko V Ferry D McKendrick J Polikoff J Tellier A Castan R Allegra C 2012 Addition of aflibercept to fluorouracil leucovorin and irinotecan improves survival in a phase III randomized trial in patients with metastatic colorectal cancer previously treated with an oxaliplatin-based regimen J Clin Oncol 30 3499 3506 22949147 Vaughn C Zhang L Schiff D 2008 Reversible posterior leukoencephalopathy syndrome in cancer Curr Oncol Rep 10 86 91 18366965 (A) The Kaplan“Meier curve of PFS (N=38). Median PFS: 5 months (95% CI 4.3“7.1). (B) Brain MRI of a patient diagnosed with RPLS. Extensive increased T2 signal of a few scattered areas of restricted diffusion and vasogenic oedema in the frontal cortex/subcortical regions near the vertex and the parietal and occipital regions bilaterally (arrows). No focal enhancing lesions to suggest metastatic disease. Baseline demographics (N=42) Age (years) Median 61.5 Range (years) 38“77 Age (years) by category N (%) <65 23 (54.8) ?65 19 (45.2) Gender N (%) Male 23 (54.8) Female 19 (45.2) Race N (%) African American 2 (4.8) Asian American 4 (9.5) Caucasian American 36 (85.7) ECOG performance status N (%) 0 21 (50.0) 1 21 (50.0) Abbreviation: ECOG=Eastern Cooperative Oncology Group. Summary of most common (>10%) TEAEs by NCI CTCAE grade (N=42) All grades N (%) Grade 3/4 N (%) Number of patients with most common TEAE with NCI grade 40 (95.2) 27 (64.3) Nausea 29 (69.0) 0 Fatigue 28 (66.7) 3 (7.1) Hypertension 24 (57.1) 15 (35.7) Constipation 21 (50.0) 0 Diarrhoea 16 (38.1) 1 (2.4) Dysgeusia 15 (35.7) 0 Stomatitis 15 (35.7) 0 Dysphonia 14 (33.3) 0 Headache 14 (33.3) 1 (2.4) Proteinuria 14 (33.3) 1 (2.4) Anorexia 13 (31.0) 0 Dehydration 12 (28.6) 3 (7.1) Dyspepsia 12 (28.6) 0 Epistaxis 11 (26.2) 0 Vomiting 10 (23.8) 0 Weight loss 10 (23.8) 0 Blood creatinine increased 8 (19.0) 0 Cough 8 (19.0) 0 Dyspnoea 7 (16.7) 1 (2.4) Hyponatraemia 7 (16.7) 2 (4.8) Neutropaenia 7 (16.7) 6 (14.3) Thrombocytopaenia 7 (16.7) 3 (7.1) Dizziness 6 (14.3) 0 Hyperglycaemia 6 (14.3) 1 (2.4) Hypokalaemia 6 (14.3) 4 (9.5) Hypomagnesaemia 6 (14.3) 0 Insomnia 6 (14.3) 0 Rhinorrhaea 6 (14.3) 0 Anaemia 5 (11.9) 2 (4.8) Dry mouth 5 (11.9) 0 Hiccups 5 (11.9) 0 Hyperkalaemia 5 (11.9) 2 (4.8) Hypocalcaemia 5 (11.9) 1 (2.4) Musculoskeletal pain 5 (11.9) 0 Oedema peripheral 5 (11.9) 0 Pain in extremity 5 (11.9) 0 Pneumonia 5 (11.9) 2 (4.8) Abbreviations: CTCAE=Common Terminology Criteria for Adverse Events; TEAE=treatment-emergent adverse events. Mean (s.d.) observed noncompartmental pharmacokinetic parameters for free ziv-aflibercept Ziv-aflibercept Parameter N Mean s.d. t 1/2 Day 7 4.62 1.46 CL l per day per kg 7 0.016 0.004 V ss l?kg?1 7 0.081 0.015 C max mg?l?1 21 104 26.2 C last mg?l?1 21 65.2 50.8 t max Day 21 0.0695 0.019 t last Day 21 7.02 9.32 AUCinf Day — mg?l?1 7 402 100 MRTinf Day 7 5.35 1.28 Abbreviations: AUCinf=area under the concentration curve from time zero extrapolated to infinity; CL=clearance; Clast=last positive plasma concentration; Cmax=maximal plasma concentration; MRTinf=mean residence time extrapolated to infinity; tlast=time of the last positive plasma concentration; tmax=time required to reach maximal plasma concentration; t1/2=observed half-life; Vss=volume of distribution at steady state. PLoS One one 1932-6203 Public Library of Science San Francisco USA 24505377 3913724 PONE-D-13-49110 .0088064 Research Biology Molecular Cell Biology Signal Transduction Signaling in Selected Disciplines Oncogenic Signaling Medicine Oncology Basic Cancer Research Immune Evasion Metastasis Oxidative Damage Tumor Physiology Cancer Prevention Cancer Vaccines Cancer Treatment Gene Therapy Cancers and Neoplasms Lung and Intrathoracic Tumors Impact of p120-catenin Isoforms 1A and 3A on Epithelial Mesenchymal Transition of Lung Cancer Cells Expressing E-cadherin in Different Subcellular Locations Effect of p120ctn Isoforms on EMT of Lung Cancer Zhang Yijun 1 Zhao Yue 1 Jiang Guiyang 1 Zhang Xiupeng 1 Zhao Huanyu 1 Wu Junhua 1 Xu Ke 2 Wang Enhua 1 * 1 Department of Pathology First Affiliated Hospital and College of Basic Medical Sciences China Medical University Shenyang China 2 Department of Radiology First Affiliated Hospital of China Medical University Shenyang China Andr Frdric Editor Aix-Marseille University France * E-mail: wangehhotmail.com Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: YJZ GYJ EHW. Performed the experiments: YJZ XPZ JHW. Analyzed the data: YJZ HYZ YZ. Contributed reagents/materials/analysis tools: YJZ YZ KX. Wrote the paper: YJZ KX EHW. 2014 4 2 2014 9 2 e88064 21 11 2013 6 1 2014 2014 Zhang et al This is an open-access distributed under the terms of the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original author and source are credited. The epithelial mesenchymal transition (EMT) is an important process in tumor development."

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