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"The dust diseases silicosis and asbestosis were the first occupational diseases to have widespread impact on workers. Knowledge that asbestos and silica were hazardous to health became public several decades after the industry knew of the health concerns. This delay was largely influenced by the interests of Metropolitan Life Insurance Company (MetLife) and other asbestos mining and product manufacturing companies. Objectives: To understand the ongoing corporate influence on the science and politics of asbestos and silica exposure including litigation defense strategies related to historical manipulation of science. Methods: We examined previously secret corporate documents depositions and trial testimony produced in litigation; as well as published literature. Results: Our analysis indicates that companies that used and produced asbestos have continued and intensified their efforts to alter the asbestos“cancer literature and utilize dust-exposure standards to avoid liability and regulation. anizations of asbestos product manufacturers delayed the reduction of permissible asbestos exposures by covering up the link between asbestos and cancer. Once the decline of the asbestos industry in the US became inevitable the companies and their lawyers designed the state of the art (SOA) defense to protect themselves in litigation and to maintain sales to developing countries. Conclusions: Asbestos product companies would like the public to believe that there was a legitimate debate surrounding the dangers of asbestos during the twentieth century particularly regarding the link to cancer which delayed adequate regulation. The asbestos“cancer link was not a legitimate contestation of science; rather the companies directly manipulated the scientific literature. There is evidence that industry manipulation of scientific literature remains a continuing problem today resulting in inadequate regulation and compensation and perpetuating otherwise preventable worker and consumer injuries and deaths. asbestos mesothelioma state-of-the-art corporate corruption MetLife industry knowledge 9421547 4136 Hum Pathol Hum. Pathol. Human pathology 0046-8177 1532-8392 24746212 4271837 10.1016/j.humpath.2014.01.003 NIHMS648391 Y-chromosome status identification suggests a recipient origin of posttransplant non“small cell lung carcinomas: chromogenic in situ hybridization analysis??? Chen Wei MD PhD a Brodsky Sergey V. MD PhD a Zhao Weiqiang MD PhD a Otterson Gregory A. MD b Villalona-Calero Miguel MD b Satoskar Anjali A. MD a Hasan Ayesha MD b Pelletier Ronald MD c Ivanov Iouri MD PhD a Ross Patrick MD PhD c Nadasdy Tibor MD a Shilo Konstantin MD a * aDepartment of Pathology The Ohio State University Wexner Medical Center Columbus OH 43210 bDepartment of Medicine The Ohio State University Wexner Medical Center Columbus OH 43210 cDepartment of Surgery The Ohio State University Wexner Medical Center Columbus OH 43210 *Corresponding author. Department of Pathology The Ohio State University Wexner Medical Center E412 Doan Hall 450 West 10th Ave Columbus OH 43210. Konstantin.ShiloOSUMC.edu (K. Shilo) 13 12 2014 23 1 2014 5 2014 19 12 2014 45 5 1065 1070 2014 Elsevier Inc. All rights reserved. 2014 Summary Owing to the need of lifelong immunosuppression solid-an transplant recipients are known to have an increased risk of posttransplant malignancies including lung cancer. Posttransplant neoplastic transformation of donor-derived cells giving rise to hematopoietic malignancies Kaposi sarcoma and basal cell carcinoma in nongraft tissues has been reported. The goal of this study was to assess the cell origin (donor versus recipient derived) of posttransplant non“small cell lung carcinomas (NSCLCs) in kidney and heart transplant recipients. An institutional database search identified 2557 kidney and heart transplant recipients in 8 consecutive years. Among this cohort 20 (0.8%) renal and 18 (0.7%) heart transplant recipients developed NSCLC. The study cohort comprised 6 of 38 NSCLCs arising in donor-recipient sex-mismatched transplant patients. The tumor cell origin was evaluated by chromogenic in situ hybridization with Y-chromosome probe on formalin-fixed paraffin-embedded tissues. Y-chromosome was identified in 97% ± 1% (range from 92% to 99%) of all types of nucleated cells in male control tissues. In all 5 NSCLCs from male recipients of female donor an Y-chromosome was identified in 97% ± 2% (range from 92% to 100%) of tumor cells statistically equivalent to normal control (P < .001). No Y-chromosome was identified in NSCLC cells from a female recipient of male kidney. These findings suggest a recipient derivation of NSCLC arising in kidney and heart transplant recipients. A combination of histologic evaluation and chromogenic in situ hybridization with Y-chromosome analysis allows reliable determination of tissue origin in sex-mismatched solid-an transplant recipients and may aid in management of posttransplant malignancy in such cases. Post“solid-an transplantation lung cancer Chromogenic in situ hybridization for Y-chromosome 15030100R 648 Ann Thorac Surg Ann. Thorac. Surg. The Annals of thoracic surgery 0003-4975 1552-6259 24576597 4008142 10.1016/j.athoracsur.2013.12.043 NIHMS571118 Accuracy of FDG-PET within the clinical practice of the ACOSOG Z4031 trial to diagnose clinical stage I NSCLC Grogan Eric L. MD MPH a b c Deppen Stephen A. MA MS PhD b c * Ballman Karla V. f Andrade Gabriela M. b Verdial Francys C. b Aldrich Melinda C. b d Chen Chiu L. e Decker Paul A. f Harpole David H. MD g Cerfolio Rrobert J. MD h Keenan Robert J. MD i Jones David R. MD j D™Amico Thomas A. MD g Shrager Joseph B. MD k Meyers Bryan F. MD l Putnam Joe B. Jr. MD a b aVeterans Affairs Medical Center Nashville TN bDepartment of Thoracic Surgery; Department of Medicine Vanderbilt University Medical Center Nashville TN cInstitute for Medicine and Public Health Vanderbilt University Medical Center Nashville TN dDivision of Epidemiology Vanderbilt University Medical Center Nashville TN eCenter for Quantitative Sciences Vanderbilt University Medical Center Nashville TN fDivision of Biomedical Statistics and Informatics Mayo Clinic Rochester MN gDepartment of Surgery Duke University Durham NC hDepartment of Surgery University of Alabama Birmingham AL iDepartment of Surgery Allegheny General Hospital Pittsburgh PA jDepartment of Surgery University of Virginia Charlottesville VA kDepartment of Surgery Stanford University Stanford CA lDepartment of Surgery Washington University St. Louis MO Corresponding Author/Request for Reprints: Eric Grogan M.D. M.P.H. Department of Thoracic Surgery 609 Oxford House 1313 21st Ave. South Nashville TN 37232 Phone: 615-322-0064 Fax: 615-343-9194 eric.groganvanderbilt.edu * Equal shared co-first author 23 4 2014 25 2 2014 4 2014 01 4 2015 97 4 1142 1148 2014 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved. 2014 Background Fluoro-deoxyglucose positron emission tomography (FDG-PET) is recommended for diagnosis and staging of NSCLC. Meta-analyses of FDG-PET diagnostic accuracy demonstrated sensitivity and specificity of 96% and 78% respectively but were performed in select centers introducing potential bias. This study evaluates the accuracy of FDG-PET to diagnose NSCLC and examines differences across enrolling sites in the national ACOSOG Z4031 trial. Methods 959 eligible patients with clinical stage I (cT1-2N0M0) known or suspected NSCLC were enrolled between 2004 and 2006 in the Z4031 trial and 682 had a baseline FDG-PET. Final diagnosis was determined by pathological examination. FDG-PET avidity was categorized into four levels based on radiologist description or reported maximum standard uptake value (SUV). FDG-PET diagnostic accuracy was calculated for the entire cohort. Accuracy differences based on preoperative size and by enrolling site were examined. Results Preoperative FDG-PET results were available for 682 participants enrolled at 51 sites in 39 cities. Lung cancer prevalence was 83%. FDG-PET sensitivity was 82% (95% CI: 79“85) and specificity was 31% (95% CI: 23%“40%). Positive and negative predictive values were 85% and 26% respectively. Accuracy improved with lesion size. Of 80 false positive scans 69% were granulomas. False negative scans occurred in 101 patients with adenocarcinoma being the most frequent (64%) and eleven were ?10mm. The sensitivity varied from 68% to 91% (p=0.03) and the specificity ranged from 15% to 44% (p=0.72) across cities with > 25 participants. Conclusions In a national surgical population with clinical stage I NSCLC FDG-PET to diagnose lung cancer performed poorly compared to published studies. Tumour Biol Tumour Biol Tumour Biology 1010-4283 1423-0380 Springer Netherlands Dordrecht 24510347 4053595 1674 10.1007/s13277-014-1674-x Research The diagnostic and prognostic value of serum human kallikrein-related peptidases 11 in non-small cell lung cancer Xu Chun-Hua Zhang Yu Yu Li-Ke +86-25-8372-8558 +86-25-83728558 yulike_doctor163.com Department of Respiratory Medicine Nanjing Chest Hospital 215 Guangzhou Road Nanjing 210029 China Nanjing Clinical Center of Respiratory Diseases Nanjing China 9 2 2014 9 2 2014 6 2014 35 6 5199 5203 6 12 2013 22 1 2014 The Author(s) 2014 Open Access This is distributed under the terms of the Creative Commons Attribution License which permits any use distribution and reproduction in any medium provided the original author(s) and the source are credited. The aim of this study was to explore the diagnostic and prognostic value of serum human kallikrein-related peptidases 11 (KLK11) level in non-small cell lung cancer (NSCLC). Serum specimens from 138 patients with NSCLC and 40 healthy controls were collected. The concentration of KLK11 was measured by enzyme-linked immunosorbent assay (ELISA). The concentration of KLK11 in NSCLC was significantly higher compared to that in the controls (P?<?0.01). The serum KLK11 levels decreased with stage presence of lymph node and distant metastases regardless of histology age and sex. With a cutoff point of 1.05 ng/ml KLK11 showed a good diagnostic performance for NSCLC. Univariate analysis revealed that NSCLC patients with serum high KLK11 had a longer overall survival (OS) and progression-free survival (PFS) than those with low KLK11 (HR of 0.36 P?=?0.002; HR of 0.46 P?=?0.009). Cox multivariate analysis indicated that KLK11 was an independent prognostic indicator of PFS and OS (HR of 0.53 P?=?0.042; HR of 0.48 P?=?0.037). Kaplan“Meier survival curves further confirmed that patients with high KLK11 have longer PFS and OS (P?=?0.003 and P?=?0.018 respectively). In conclusion the measurement of KLK11 might be a useful diagnostic and prognostic test for NSCLC patients. Keywords Kallikrein-related peptidases 11 Non-small cell lung cancer Diagnosis Prognosis issue-copyright-statement International Society of Oncology and BioMarkers (ISOBM) 2014 Introduction Lung cancer is the leading cause of cancer-related death worldwide with more than 1.2 million deaths each year [1]. Non-small cell lung cancer (NSCLC) accounts for 80“85 % of total lung malignancies [2]. Although advances in noninvasive methods have improved our ability to detect lung cancer more than 75 % of lung cancer patients present an advanced stage of disease [3] and they have little prospect of effective and curative treatment with 5-year survival rates of less than 15 % [4]. 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 Table 1. 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.Table 1Clinical 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 Table 2. 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).Table 2The 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) (Table 3).Table 3Univariate 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 conclusion 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 Boño 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 Tropà 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. Boño 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 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 mwoduke.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 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 lkwradradiol.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 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. "

Lung_Cancer

"a non-taxane microtubule dynamics inhibitor has shown trends towards greater overall survival (OS) compared with progression-free survival in late-stage metastatic breast cancer patients in the clinic. This finding suggests that eribulin may have additional previously unrecognised antitumour mechanisms beyond its established antimitotic activity. To investigate this possibility eribulin's effects on the balance between epithelial“mesenchymal transition (EMT) and mesenchymal“epithelial transition (MET) in human breast cancer cells were investigated. Methods: Triple negative breast cancer (TNBC) cells which are oestrogen receptor (ER?)/progesterone receptor (PR?)/human epithelial growth receptor 2 (HER2?) and have a mesenchymal phenotype were treated with eribulin for 7 days followed by measurement of EMT-related gene and protein expression changes in the surviving cells by quantitative real-time PCR (qPCR) and immunoblot respectively. In addition proliferation migration and invasion assays were also conducted in eribulin-treated cells. To investigate the effects of eribulin on TGF-?/Smad signalling the phosphorylation status of Smad proteins was analysed. In vivo the EMT/MET status of TNBC xenografts in mice treated with eribulin was examined by qPCR immunoblot and immunohistochemical analysis. Finally an experimental lung metastasis model was utilised to gauge the metastatic activity of eribulin-treated TNBC in the in vivo setting. Results: Treatment of TNBC cells with eribulin in vitro led to morphological changes consistent with transition from a mesenchymal to an epithelial phenotype. Expression analyses of EMT markers showed that eribulin treatment led to decreased expression of several mesenchymal marker genes together with increased expression of several epithelial markers. In the TGF-? induced EMT model eribulin treatment reversed EMT coincident with inhibition of Smad2 and Smad3 phosphorylation. Consistent with these changes TNBC cells treated with eribulin for 7 days showed decreased capacity for in vitro migration and invasiveness. In in vivo xenograft models eribulin treatment reversed EMT and induced MET as assessed by qPCR immunoblot and immunohistochemical analyses of epithelial and mesenchymal marker proteins. Finally surviving TNBC cells pretreated in vitro with eribulin for 7 days led to decreased numbers of lung metastasis when assessed in an in vivo experimental metastasis model. Conclusions: Eribulin exerted significant effects on EMT/MET-related pathway components in human breast cancer cells in vitro and in vivo consistent with a phenotypic switch from mesenchymal to epithelial states and corresponding to observed decreases in migration and invasiveness in vitro as well as experimental metastasis in vivo. These preclinical findings may provide a plausible scientific basis for clinical observations of prolonged OS by suppression of further spread of metastasis in breast cancer patients treated with eribulin. triple negative breast cancer eribulin mesilate EMT/MET metastasis Breast cancers can be classified into oestrogen receptors (ERs)+human epithelial growth receptor 2 (HER2)+ and so-called ˜triple negative' breast cancer (TNBC; negative for ER HER2 and progesterone receptors) subtypes. Both ER+ and HER2+ tumours have luminal epithelial characteristics whereas a large fraction of triple negative tumours have stem cell/basal-like properties (Perou et al 2000; Nielsen et al 2004; Neve et al 2006; Shipitsin et al 2007). In the last decade considerable progress has been made in the treatment of ER+ and HER2+ tumours with multiple types of therapies now available including hormonal and monoclonal antibody-based therapies and small molecule inhibitors of tyrosine kinases for both localised and metastatic disease (Arteaga et al 2012). Consequently survival rates of breast cancer patients with luminal subtypes have improved significantly especially for early-stage disease. In contrast TNBC which comprises 10“15% of all breast cancers still lacks effective targeted therapies which when combined with its typically heterogeneous histology and propensity for early metastasis results in poor 5-year survival rates for this breast cancer subtype (Carey et al 2007; Lehmann et al 2011; Shah et al 2012). Given this situation preventing metastatic spread and treatment of existing metastatic disease in TNBC remain among the top challenges in breast cancer treatment today. Eribulin (Halaven) is a non-taxane inhibitor of microtubule dynamics that exerts its primary pharmacologic effects by preventing normal mitotic spindle formation leading to irreversible mitotic blockage and subsequent cell death by apoptosis (Towle et al 2001; Kuznetsov et al 2004; Okouneva et al 2008; Towle et al 2012). Eribulin binds to the plus ends of microtubules (Smith et al 2010) resulting in a pattern of microtubule dynamics inhibition that is distinct from those of other clinically used tubulin agents including epothilones vinca alkaloids and taxanes. Eribulin is currently approved for clinical use in many countries worldwide including United States Japan and EU countries for treatment of certain patients with advanced breast cancer. For instance in the United States and EU eribulin is approved for patients with locally invasive or metastatic breast cancer who have previously received at least two chemotherapeutic regimens for metastatic disease including an anthracycline and a taxane (Cortes et al 2011). Interestingly two different phase 3 clinical trials in metastatic breast cancer patients have suggested that eribulin has more pronounced effects on overall survival (OS) compared with progression-free survival (PFS) (Cortes et al 2011). One possible explanation for this clinical observation is that eribulin may suppress the incidence of new metastasis thus providing an increased survival benefit to patients even under conditions in which the primary tumour and preexisting metastatic tumour may progress. The preclinical studies described herein were designed to assess whether eribulin might have such anti-metastatic properties based on reversion of the metastasis-promoting process termed as epithelial“mesenchymal transition (EMT). Epithelial“mesenchymal transition and its reverse process mesenchymal“epithelial transition (MET) were originally identified as playing central roles during early embryonic development (Yang and Weinberg 2008). More recently EMT has been shown to be a key metastasis-promoting step in many cancers (Gavert and Ben-Ze'ev 2008). In tumours gain of mesenchymal characteristics and loss of epithelial characteristics via EMT correlate well with tumour progression maintenance drug resistance and metastasis (Polyak and Weinberg 2009; Gunasinghe et al 2012). Epithelial“mesenchymal transition progression is characterised by loss of the epithelial marker E-cadherin together with increased expression of mesenchymal markers such as N-cadherin and vimentin. During metastatic progression EMT drives primary epithelial-like tumour cells to acquire invasive mesenchymal phenotypes with increased motility and invasiveness triggering dissemination from the tumour and infiltration into the tumour vascular. These EMT-driven cells then circulate in the blood flow and subsequently redifferentiate via MET during colonisation and growth at distant metastatic sites (Bonnomet et al 2012; Yu et al 2013). Thus considering EMT's role at the onset of the metastatic process controlling EMT in tumours is now considered to be a promising strategy to inhibit metastasis and improve survival of cancer patients. In the present study we investigated potential relationships between EMT/MET balance altered with eribulin treatment as measured in preclinical TNBC models and eribulin's apparent ability to prolong OS in TNBC patients without corresponding increases in PFS. In aggregate these results suggest that eribulin triggers a phenotypic shift in balance from the more aggressive EMT state to a less aggressive MET state the latter being associated with decreased potential for metastasis and invasion. Materials and methods Compounds Eribulin mesilate was chemically synthesised at Eisai Co. Ltd (Tsukuba Japan). 5-Fluorouracil (5-FU) was obtained from Sigma-Aldrich (St Louis MO USA). Cell culture MX-1 was obtained from the United States National Cancer Institute. Hs578T MDA-MB-157 and MCF10A were purchased from American Type Cell Collection. MX-1 was maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum. Hs578T was maintained in DMEM supplemented with 10% fetal bovine serum and 10??g?ml?1 insulin. MDA-MB-157 was maintained in McCoy's 5A medium supplemented with 10% fetal bovine serum. MCF10A was cultured in DMEM/F12 medium supplemented with 5% horse serum 10??g?ml?1 insulin 20?ng?ml?1 EGF 500?ng?ml?1 hydrocortisone and 100?ng?ml?1 cholera toxin. Recombinant TGF-? was purchased from R&D (Minneapolis MN USA). Proliferation assay Cells were plated in 96-well plates and cultured with indicated concentration of compounds for 3 days followed by cell number and viability determinations as measured by CellTiter-Glo Luminescent Cell Viability Assay (Promega Madison WI USA). Gene expression analysis Total RNA from cultured cells treated with eribulin for 7 days was isolated using the RNeasy Mini kit (Qiagen Valencia CA USA). Reverse transcription was carried out using the High Capacity cDNA Reverse Transcription kit (Life Technologies Grand Island NY USA). Synthesised cDNA was amplified with Taqman probes and quantities of DNA were measured by ABI7900 (Applied Biosystems Life Technologies). Assessment of changes in gene expression was conducted by comparison of delta delta Ct values for each sample. TaqMan probes used in this study are summarised in Supplementary Table 1. Western blotting analysis The cultured cells were lysed with Pierce RIPA Buffer (Thermo Scientific Waltham MA USA) with Halt protease inhibitor Cocktail (Thermo Scientific). Lysates mixed with sample buffer were electrophoretically separated and transferred onto membranes. Membranes were blocked with 5% skim milk followed by incubations with anti-human E-cadherin antibody (24E10; Cell Signaling Technology Danvers MA USA) anti-human N-cadherin antibody (#610920; BD Biosciences San Jose CA USA) anti-human vimentin antibody (D21H3; Cell Signaling Technology) anti-human Smad2/3 (#07-408; Millipore Billerica MA USA) anti-human phospho-Smad2 (Ser465/467) (#AB3849; Millipore) anti-human phospho-Smad3 (Ser423/425) (#07-1389; Millipore) and anti-human ?-actin antibody (#4963; Cell Signaling Technology). After washing with TBS-0.05% Tween membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit IgG. After washing with TBS-0.05% Tween membranes were incubated with ECL Prime Western Blotting Detection Reagent (GE Healthcare Little Chalfont UK). Signals were detected and analysed using a Luminescent Image Analyzer LAS-4000 (Fuji Film Tokyo Japan). Signals were detected and analysed using a Luminescent Image Analyzer LAS-4000 (Fuji Film). In vivo tumour xenograft growth assay and sample preparation for in vivo immunohistochemical analyses For in vivo xenograft specimens 10 × 106 MX-1 cells in a Matrigel suspension were subcutaneously injected into the right flank of athymic mice (CAnN.Cg-Foxn1nu/CrlCrlj; Charles River Laboratories Japan Yokohama Japan). Nine to eleven days after inoculation eribulin (0.31 and 3?mg?kg?1) or vehicle was intravenously administrated; this was defined as day 1. On days 4 or 8 mice were killed and tumours were isolated and measured. Collected tumours were fixed in 10% neutral buffered formalin solution (Wako Pure Chemical Industries Osaka Japan) for 24?h followed by embedding in Tissue-Tek VIP 5 Jr. (Sakura Torrance CA USA). Immunohistochemical analysis Sections (5??m) of formalin-fixed paraffin-embedded tumours prepared as above were made using a rotary microtome (Leica Biosystems Wetzlar Germany RM2255). Sections were placed on charged slides and dried on a warm plate at 35“37?°C overnight. Immunohistochemistry (IHC) staining using anti-E cadherin (#3195; Cell Signaling Technology) anti-ZEB1 (#NBP1-05987; Novus Biologicals Littleton CO USA) and anti-N-cadherin (#ab18203; Abcam) primary antibodies was performed on either a Leica BOND-MAX Autostainer (Leica Biosystems) or a Leica ST5020 Multistainer (Leica Biosystems). Stained mounted slides were digitised using a ScanScope XTTM (Aperio Technologies Vista CA USA) whole slide automated scanning system. Image analysis and quantification of IHC staining was performed using Aperio ImageScope software version 11.1.2.760 (Aperio Technologies). Regions of interest (ROIs) for quantification were defined as the entire tumour cross-sectional areas of each sample (one ROI per sample). The following Aperio image analysis algorithms were used to quantify IHC staining within ROIs: IHC nuclear (quantification of ZEB1) IHC membrane (quantification of E-cadherin) and colocalisation (quantification of N-cadherin). In vitro migration and invasion assay BioCoat Matrigel invasion chambers 24-well plates (BD Biosciences) were utilised. For the cell migration assay and the invasion assay 5 × 103 and 2.5 × 103 cells respectively were seeded onto the porous membranes in upper chambers of BioCoat control cell-culture inserts (8.0??m pore size; BD Biosciences) after treatment for 7 days with eribulin or 5-FU. Top chambers (culture inserts) were filled with serum-free medium and bottom chambers were filled with medium containing 20% FBS as a chemoattractant. After 16?h incubation for migration or 26?h incubation for invasion the number of cells that had migrated to bottom surfaces of the membranes was counted after staining with Geimsa solution. Numbers of cells in wells were calculated using means from five randomly selected fields. Lung metastasis model MX-1 cells were pretreated with 1?nM eribulin 3??M 5-FU or DMSO as a control for 7 days. CB17-SCID mice (Charles River Laboratories Japan) were pretreated with cyclophosphamide to eliminate residual immune cells by intraperitoneal injection of 150?mg?kg?1 per day for 2 days (Shankar et al 2007)"

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

"This analysis provided 491 interactions in (21) and 3139 interactions in (31). Owing to the different experimental setups most of these GIs are not directly comparable.Calculating differential correlation scoresGiven a training set containing gene expression profiles of subjects we used the statistical method of (24) to compute for each gene pair its consistent correlation (CC) and DC scores. First DC scores are computed using the real labels of the samples. Then the scores are transformed to log-likelihood ratio (LLR) scores by comparing the original DC scores to scores calculated on the same data with randomly shuffled labels. Thus positive LLR scores mark gene pairs with significant change in DC. The prior probability of real DC changes was set so that only correlation changes of at least 0.4 will have a positive LLR score. This approach guarantees a similar yet slightly more stringent acceptance threshold compared with (24). See Supplementary Text for additional information.GO and microRNA enrichment analysisWe used TANGO (32) for Gene Ontology molecular function and biological process enrichment analysis of modules and FAME (33) for microRNA enrichment analysis. Both tools are available as part of the EXPANDER software (34). When a set of modules was analyzed we corrected for multiple testing using false discovery rate (FDR) with q = 0.05. The background set for the enrichment analysis was defined as the set of genes in the networks and not all genes in the anism. This filtering step reduces bias in case of overrepresentation of GO terms in the networks.Network visualizationNetwork visualization was done using Cytoscape (35).AvailabilityA command line tool for running ModMap is freely available for academic use at http://acgt.cs.tau.ac.il/modmap/.RESULTSSimulationsWe first tested the different algorithms on synthetic graphs H and G. Starting from a perfect module map we first added cliques in H and bicliques in G to represent additional structures that are not part of the map and then introduced random noise to the edges. To generate both sparse and denser graphs we tested a wide range of the noise parameters ? and p in the weighted and the unweighted simulations respectively (see ˜Materials and Methods™ section). The results presented here are for graphs with 500 nodes and six modules per map. We also tested larger graphs with similar results (see Supplementary Figures S3 and S4).We tested 10 combinations of initiator and improver on 10 random data sets for each value of P and ?. We measured the quality of produced solutions using Jaccard coefficient between the reported modules and the known modules. The results of the unweighted and weighted models are shown in A and B respectively. Only the four algorithms that performed best on average in each simulation are shown. Supplementary Table S2 contains the results for all combinations. The local improvement algorithms did not reach perfect scores even on noiseless data. In contrast MBC-DICER and DICER5 followed by global improver reached perfect Jaccard scores when there was no statistical noise. The high performance of MBC-DICER remained robust even when noise levels were as high as P = 0.15 in the unweighted model and ? = 1.2 in the weighted model. A comparison of all algorithms on unweighted graphs with 1000 nodes and 10 modules for noise level P = 0.15 is shown in C. Performance remains high although the graphs are much larger. Using the improvers was beneficial compared with using only the initiator solutions especially for the DICER variants. MBC-DICER with the global improver reached highest performance (0.87). Interestingly the local improver was better than the global improver for all other algorithms (e.g. 0.71 versus 0.59 for DICER5). This is probably because the MBC-DICER initiator detects robust fully connected modules which are a better starting point to the global improver at high noise levels. Tests with different values of k for the DICERk algorithm led us to choosing k = 5 (Supplementary Figure S4). In addition we compared the performance of the global improver with the hypergeometric test and with the Wilcoxon rank-sum test which was used in previous studies. Our results show that using the hypergeometric test reaches similar quality of results but is much faster (see Supplementary Text). Overall the results indicate that MBC-DICER followed by the global improver achieved the best performance on both unweighted and weighted data. We call the resulting algorithm ModMap and will use it as the algorithm of choice from now on.Yeast protein-protein interaction and negative genetic interaction dataWe used PPIs and negative GIs from BIOGRID (30) to find epistatic relations among protein complexes. Only genes that had both types of interactions were used. Overall the networks contained 3979 genes 45 456 PPIs and 76 237 negative GIs (the interactions are listed in Supplementary Table S1). This number of genes and edges is larger than in previous studies. For example (22) covered 1460 genes and (17) covered 743 genes. Therefore our networks have the potential to provide a broader overview of the yeast interactome and allow for a comprehensive performance testing of the different algorithms.As done in previous studies we evaluated solutions by their statistics and the functional characterization of the modules (1722). The calculated solution statistics included the number of modules the number of genes covered and the maximal module size. We used TANGO (34) to measure module functional enrichment and reported the number of discovered GO terms the percent of enriched modules and the percent of module map links for which both modules are enriched (with the same or with different functions) which we call ˜enriched links™. Enriched links represent dense GIs among known biological terms.The solution statistics of all algorithms are shown in Supplementary Table S3. One can observe clear superiority of global over local improvers. In contrast to global improvers which reported at least 100 modules and covered 800“1000 genes the local improvers found 2“28 modules covering only 15“192 genes. Except for DICER the results of all solutions were similar and of high quality. ModMap was the best in terms of the percent of enriched modules (87%) and percent of enriched links (80%). Taken together the map of ModMap was best in combining functional comprehensiveness and quality. We also compared ModMap with other weighted approaches for GI data analysis (2236) on the data of Collins et al. (37). "

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). She J Yang P Hong Q Bai C Lung cancer in china: challenges and interventions Chest 2013 143 4 1117 1126 23546484 Lu Q Lu S Huang L Wang T Wan Y Zhou C Zhang C Zhang Z Li X The expression of V-ATPase is associated with drug resistance and pathology of non-small-cell lung cancer Diagn Pathol 2013 8 145 23984887 Pfannschmidt J Muley T Bülzebruck H Hoffmann H Dienemann H Prognostic assessment after surgical resection for non-small cell lung cancer: experiences in 2083 patients Lung Cancer 2007 55 3 371 17123661 Goya T Asamura H Yoshimura H Kato H Shimokata K Tsuchiya R Sohara Y Miya T Miyaoka E Prognosis of 6644 resected non-small cell lung cancers in Japan: a Japanese lung cancer registry study Lung Cancer 2005 50 2 227 234 16061304 Christiansen A Detmar M Lymphangiogenesis and cancer Genes Cancer 2011 2 12 1146 1158 22866206 El-Gohary YM Metwally G Saad RS Robinson MJ Mesko T Poppiti RJ Prognostic significance of intratumoral and peritumoral lymphatic density and blood vessel density in invasive breast carcinomas Am J Clin Pathol 2008 129 4 578 586 18343785 Karaday? 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

"For mutations with therapeutic significance highly sensitive and specific assays are essential for informing patient therapy and for clinical trials investigating new agents. Relative to published mutations derived from DNA-WES alone the UNCeqRMETA mutations derived from patient-matched DNA-WES and RNA-seq increased the numbers of patients with mutations in genes that are targets for several drugs in clinical trials such as PIK3CA and ERBB2 and for drugs with correlative evidence such as FGFR2 (51). Clinical trials such as NCT01670877 which involve ERBB2 sequencing (52) may be influenced to include RNA-seq due the large mutation rate increase reported here. Although the relative increase in PIK3CA mutations was modest compared to other genes in breast cancer this improved sensitivity is vital for affected patients and could lead to positive clinical trial outcomes. For example some novel canonical mutations in PIK3CA had many mutant reads in RNA-seq but only a few mutant reads in DNA-WES such as the example Luminal A tumor with a single DNA mutant read in the PIK3CA hotspot. This study's results support that RNA sequencing could be beneficial when added to DNA sequencing in clinical settings. Future studies could explore alternative ways to integrate DNA and RNA sequencing beyond UNCeqRMETA which is the first method of this kind. UNCeqRMETA applied the same quality filters for DNA and RNA and potentially different filters could be beneficial. UNCeqRMETA includes a basic indel realignment and integrated DNA and RNA reassembly could potentially be beneficial. Different statistical modeling could further advance the performance displayed by UNCeqRMETA over DNA only based methods. Balancing sensitivity and specificity is important in applying and developing mutation detectors. Receiver operating characteristic curve analysis such as that presented in this study enables assessment of sensitivity and specificity tradeoffs between alternate models. Integrated RNA-seq and DNA-WES mutation detection is important because it boosts sensitivity in low purity tumors in therapeutically-relevant genes and in driver genes relative to DNA-only detection. Integrated mutation detection could also enable more inclusive cohort profiling studies that censor tumors based on purity and could lead to more comprehensive "

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

" 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). 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"

Lung_Cancer

"peptide vaccine CTL clone lung cancer Introduction Lung cancer is one of the main causes of cancer-related mortality. Approximately 85% of lung cancers are diagnosed as non-small cell lung cancer (NSCLC) and the overall survival (OS) rate for advanced NSCLC is poor. The 5-year survival rate is 5% for stage IIIb NSCLC and <1% for stage IV NSCLC (1). Treatment for NCSLC is determined by the patient™s clinical and tumor characteristics performance status (PS) the histological subtype and tumor genotype/phenotype. Recently there have been many studies concerning agents that target molecular changes such as mutations in the epidermal growth factor receptor (EGFR) and the fusion oncogene EML4-ALK in which the echinoderm microtubule-associated protein-like 4 (EML4) is fused with the intracellular domain of anaplastic kinase (ALK) (2“4). Although significant advances have been made in the treatment of NSCLC using molecular targeted therapies such as erlotinib and crizotinib the median OS for patients with advanced NSCLC remains low (56) and acquired resistance to target agents is a major clinical problem. Therefore the development of novel therapies is needed (7). Immunotherapy manipulates the immune system to control and eradicate cancer. Many recent studies provide evidence suggesting that immunotherapeutic manipulations are viable in many tumor types including lung cancer. Numerous trials of peptide vaccines autologous cellular therapy T cell-directed antibody therapy and monoclonal antibody therapy for lung cancer have been carried out around the world (8“10) and some of them have shown favorable results (11“13). The EML4-ALK fusion gene was identified in NSCLC patients by a team led by Professor H. Mano. This fusion gene was formed as the result of a small inversion within the short arm of chromosome 2 that joins differing portions of the EML4 gene with a portion of the ALK gene (1415). As a result of this fusion constant dimerization of the kinase domain of ALK is induced and its catalytic activity increases consequently. The EML4-ALK fusion gene is mainly identified in young never/former light smokers with NSCLC (16). It is estimated that approximately 5% of all NSCLC cases have this fusion gene. A few reports have also identified EML4-ALK in other cancers namely breast cancer and colorectal cancer (1718). For the most part the EML4-ALK fusion gene and other mutations such as those in EGFR and KRAS are mutually exclusive (19). The chromosomal inversion does not always occur in the same location and multiple EML4-ALK variants have been identified (19). At least 11 variants have been reported. The most common variants are E13;A20 (variant 1) and E6a/b;A20 (variant 3a/b) which have been detected in 33% and 29% of NSCLC patients respectively (14). PF-02341066 (crizotinib) is an ALK inhibitor currently under clinical development. Kwak et al conducted an open-label multi-center two-part phase I trial and found a remarkable 57% overall response rate and a 72% 6-month progression-free survival rate (20). In spite of the marked antitumor activity of crizotinib ALK-positive cancers invariably gain resistance to crizotinib. In the case of ALK-positive cancers as well as EGFR-mutant lung cancer resistance develops on average within the first 2 years of therapy (21). The main resistance mutations are L1196M a gatekeeper mutation and C1156M. In addition to ALK mutations other known mechanisms for acquired resistance include ALK amplification (2122) and EGFR activation (2324). To overcome resistance new ALK inhibitors are currently in early phase studies (25). Novel combinatorial strategies to overcome crizotinib resistance and further improve the clinical outcome are needed. We focused on this new fusion array as a novel target of immunotherapy. There are several methods to detect EML4-ALK NSCLC including polymerase chain reaction (PCR) immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) (19). These methods detect high-level EML4-ALK fusion gene expression. Passoni et al identified two HLA-A*02:01-restricted ALK-derived peptides that induce peptide-specific CTL lines (26). We focused on the EML4 array as a novel epitope of immunotherapy. We identified a candidate 9- or 10-amino acid array of novel epitopes using the Bioinformatics and Molecular Analysis Section (BIMAS) software and analyzed its potential as a new immunotherapy epitope with respect to its ability to induce anticancer activity. We then induced and generated a peptide-specific CTL clone from peripheral blood lymphocytes of HLA-A*02:01-positive healthy donors. We report here that an EML4-ALK-derived peptide-specific human CTL clone recognized peptide-pulsed T2 cells and HLA-A*02:01-positive and EML4-ALK-positive tumor cells pretreated with IFN-?. Furthermore we showed that immunotherapy with this novel epitope peptide has potential for treatment of EML4-ALK-positive NSCLC. Materials and methods Peptides Human EML4-ALK-derived peptides carrying binding motifs for HLA-A*02:01-/HLA-A*24:02-encoded molecules were identified by HLA-peptide binding predictions using the BIMAS program (http://bimas.dcrt.nih.gov/molbio/hla_bind/index.html). We purchased a total of seven EML4-ALK-derived peptides carrying HLA-A*02:01 binding motifs and two peptides carrying HLA-A*24:02 binding motifs from Geneworld (Tokyo Japan). Cell lines The H2228 human lung adenocarcinoma cell line and EML4-ALK fusion protein variant 3 (E6; A20) were kindly provided by Professor S. Yano (Kanazawa University). T2 is a lymphoblastoid cell line that lacks TAP function and has HLA-A*02:01 molecules that can easily be loaded with exogenous peptides. T2A24 is the same cell line but with HLA-A*24:02 instead. T2 and T2A24 cells were cultured in RPMI medium supplemented with 10% heat-inactivated FBS. HLA-A*02:01/HLA-A*24:02 binding assay In order to determine the binding ability of the predicted peptides to HLA-A*02:01/HLA-A*24:02 molecules an in vitro cellular binding assay was performed as reported previously (27). Briefly after incubation of the T2/T2A24 cells in culture medium at 26°C for 18 h cells were washed with PBS and suspended in 1 ml Opti-MEM (Invitrogen Carlsbad CA USA) with or without 100 ?g peptide and then incubated at 26°C for 3 h and at 37°C for 3 h. After washing with PBS HLA-A*02:01/HLA-A*24:02 expression was measured by flow cytometry using a FITC-conjugated and HLA-A*02:01-/HLA-A*24:02-specific monoclonal antibody (mAb) and the mean fluorescence intensity was recorded. Generation of dendritic cells CD14+ cells were isolated from human peripheral blood mononuclear cells (PBMCs) using human CD14 microbeads (Miltenyi Biotec Bergisch Gladbach Germany). Immature dendritic cells (DCs) were generated from CD14+ cells using interleukin (IL)-4 (10 ng/ml; PeproTech Inc. Rocky Hill NJ USA) and granulocyte-macrophage colony-stimulating factor (GM-CSF; 10 ng/ml; PeproTech) in RPMI-1640 medium supplemented with 10% FBS. Maturation of DCs was induced by prostaglandin E2 (PGE2; 1 ?g/ml; Sigma St. Louis MO USA) and tumor necrosis factor (TNF-)-? (10 ng/ml; PeproTech). Induction of EML4-ALK-derived peptide-specific CTLs from PBMCs CD8+ cells were isolated from PBMCs using human CD8 microbeads (Miltenyi Biotec Bergisch Gladbach Germany). CD8+ cells (2—106) were stimulated by peptide-pulsed irradiated autologous mature DCs (1—105). Autologous DCs were prepared from a limited supply; artificial antigen presenting cells (aAPCs) (K562/A2 or A24/CD80/CD83) were alternatively used for further examination. After 1 week these cells were stimulated twice per week by peptide-pulsed irradiated artificial APC-A2 or artificial APC-A24 cells (1—105). Supplementation with 10 IU/ml IL-2 (Proleukin; Novartis Pharmaceuticals Basel Switzerland) and 10 ng/ml IL-15 (PeproTech) was performed every 3 to 4 days between stimulations (28). IFN-? ELISPOT assay Specific secretion of IFN-? from human CTLs in response to stimulator cells was assayed using the IFN-? ELISPOT kit (BD Biosciences) according to the manufacturer™s instructions. Stimulator cells were pulsed with peptide for 2 h at room temperature and then washed. Responder cells were incubated with stimulator cells for 20 h. The resulting spots were counted using an ELIPHOTO counter (Minerva Tech Tokyo Japan). HIV-gag (77“85) (SLYNTYATL) was used as an irrelevant peptide in the CTL assay. Generation of CTL clones Cultured cells were incubated with peptide-pulsed T2/T2A24 cells at a ratio of 2:1 for 3.5 h at 37°C. CD107a-specific antibodies (BioLegend San Diego CA USA) were included in the mixture during the incubation period. CD8+CD107a+ cells were sorted using a FACSAria II cell sorter (BD Biosciences). Sorted CTLs were stimulated and the CTL clones were established as described previously (29). Flow cytometry H2228 cells with or without pretreatment with 100 U/ml IFN-? (PeproTech) for 48 h were harvested and stained with anti-HLA-A2 Ab-FITC (MBL Japan) and analyzed using a FACSCanto II flow cytometer (BD Biosciences). Flow cytometry data were analyzed using FlowJo software. Cytotoxicity assay The cytotoxic capacity was analyzed using the Terascan VPC system (Minerva Tech Tokyo). The CTL clone was used as the effector cell type. Target cells treated with 100 U/ml IFN-? (PeproTech) 42 h previously were labeled through incubation in calcein-AM solution for 30 min at 37°C. The labeled cells (1—104) were then co-cultured with the effector cells for 4“6 h. Fluorescence intensity was measured before and after the culture period and specific cytotoxic activity was calculated as described previously (29). HLA-A*02:01 blocking of T-cell activity was tested by pre-incubating the target cells with anti-HLA-A -B -C mAb (W6/32) or an isotype control mAb (mIgG2a?; BioLegend San Diego CA USA). Results Identification of HLA-A*02:01-/HLA-A*24:02-restricted EML4-ALK-derived peptides As candidate EML4-ALK- derived and HLA-A*02:01-/HLA-A*24:02-restricted CTL epitopes we selected nine peptides with highly predicted scores for HLA-A*02:01/HLA-A*24:02 binding calculated using BIMAS software (Tables I and II) and evaluated their ability to bind to HLA-A*02:01/HLA-A*24:02 molecules. All nine peptides were able to bind HLA-A*02:01/HLA-A*24:02 molecules (Fig. 1). Generation of an EML4-ALK-derived peptide-specific CTL clone from human PBMCs We next assessed the capacity of EML4-ALK-derived peptides to generate peptide-specific CTLs in vitro from human PBMCs of HLA-A*02:01/HLA-A*24:02 healthy donors. CTLs were induced by three stimulations with DCs or artificial APCs loaded with the EML4-ALK-derived peptides. CTLs were tested for specificity for each peptide using the IFN-? ELISPOT assay. Peptides A B and C could induce peptide-specific CTLs that were able to specifically recognize T2 cells pulsed with each peptide but not T2 cells without peptides (Fig. 2). Peptides B and C were able to induce CTLs from only one donor (healthy donor 3 for peptide B and healthy donor 4 for peptide C) but peptide A was able to induce CTLs in three of four donors (healthy donors 2 3 and 4). Based on this result we used peptide A for further examinations. Next we obtained one CTL clone from peptide A-specific CTLs that was able to specifically recognize T2 cells pulsed with peptide A but not T2 cells pulsed with an irrelevant HIV-gag peptide using single cell sorting with a CD107a antibody. The population of CD8+CD107a+ cells represented 0.984% of all stimulated cells (Fig. 3A). These cells were sorted as single cells in each well of a 96-well plate. Twenty-one days after cell sorting peptide specificity was assessed using the IFN-? ELISPOT assay (Fig. 3B). The established clone reacted to the T2 cells pulsed with peptide A but not to T2 cells pulsed with the irrelevant HIV-gag peptide. These results indicate that a peptide A-specific CTL clone was successfully established from PBMCs from a healthy donor. The EML4-ALK-specific CTL clone recognizes HLA-A*02:01+ lung carcinoma cells with the EML4-ALK variant 3a/b incubated with IFN-? We next evaluated the ability of the EML4-ALK-specific CTL clone to recognize the cancer cell line H2228 which expresses HLA-A*02:01 and EML4-ALK using the IFN-? ELISPOT assay. Even though the EML4-ALK-specific CTL clone failed to recognize H2228 cells it did recognize those pretreated with 100 U/ml IFN-? 48 h prior to examination (Fig. 4A). We examined the effect of IFN-? on H2228 cells. Incubating target cells with IFN-? for 48 h increased the expression of MHC class I molecules on the cell surface (Fig. 4B). This result indicates that the peptide A-specific CTL clone was able to recognize H2228 cells because of increased expression of MHC-class I on the H2228 cell surface. Specific IFN-? production by the peptide A-specific CTL clone was detectable in H2228 cells treated with IFN-?. The specificity was abolished by an anti-HLA-class I mAb but not by an isotype control suggesting that the observed production was HLA-A2 restricted (Fig. 4C). A cytotoxicity assay was also performed. The peptide A-specific CTL clone was able to specifically lyse H2228 cells pretreated with IFN-? 48 h prior to examination. This specific lysis was blocked by the anti-HLA-class I mAb but not by the isotype control. These results indicate that the peptide A-specific CTL clone showed cytotoxicity and the ability to produce IFN-? against HLA-A*02:01+ EML4-ALK+ NSCLC cell lines (Fig. 5). Discussion In the present study we identified a new tumor-associated CTL epitope (peptide A) derived from EML4-ALK which binds to HLA-A*02:01 molecules and we were able to establish a peptide-specific CTL clone from human PBMCs that specifically recognized cognate peptide-pulsed T2 cells and HLA-A*02:01 tumor cells expressing EML4-ALK that had been pretreated with IFN-?. EML4-ALK-positive lung cancers are highly sensitive to ALK inhibition. However as with trastuzumab or gefitinib (3031) patients typically gain resistance within 1 to 2 years of starting therapy (23). We aimed to overcome these difficulties with immunotherapy. We identified a glypican-3 (GPC3)-derived peptide and showed that GPC3-specific CTL frequency after vaccination correlated with OS. OS was significantly longer in patients with high GPC3-specific CTL frequencies than in those with low frequencies (32). This indicates that the ability to induce a peptide-specific CTL clone is important for effective immunotherapy. We also revealed that GPC3 is an ideal target for anticancer immunotherapy since it is specifically overexpressed in hepatocellular carcinoma (HCC) (33“35). In the present study we chose a peptide array from EML4-ALK from which we were able to induce a peptide-specific CTL clone. EML4-ALK is a strong oncogene overexpressed in cancer cells of NSCLC breast cancer kidney cancer and colon cancer (17). We performed RT-PCR and assayed the EML4 DNA levels of certain lung cancer cell lines. H2228 cells express EML4 moderately but at higher levels than other lung cancer cell lines. EML4 expression has been reported as highly expressed in CD8+ T cells. RT-PCR showed that EML4 DNA levels were high in PBMCs and CD8+ T cells. Because of a lack of suitable antibodies we could not perform western blotting. However our success at inducing a peptide A-specific CTL clone from CD8+ T cells indicated that the CTL clone had no cytotoxicity against CD8+ T cells. This CTL clone could not recognize cancer cell lines without the ability to increase the amount of HLA class I presented on cell surfaces. Further examination is needed to achieve higher tumor reactivity. Combination chemotherapy or radiation therapy plus immunotherapy was recently reported to have a synergistic effect (36). Moreover some mechanisms of synergy between radiation therapy chemotherapy and immunotherapy have been revealed (37). In one of the mechanisms these therapies upregulated tumor antigens and MHC moieties. These results suggest that combination therapy could be used to make tumor cell lines more susceptible to this peptide A-specific CTL clone-mediated cytolysis (38“41). In addition this treatment may be able to overcome resistance to ALK inhibition. Some resistance mechanisms for targeting drugs have been examined. The most commonly identified causes of resistance are point mutations such as L1196M (42“44) G1269A (22) and S1206Y (21). These point mutations occur in the tyrosine kinase domain which plays an important role in oncogenesis. Our peptide array was selected from EML4 which has no correlation with these point mutations. It is possible that this treatment is effective for tumor cells resistant to ALK inhibitors. In this study we identified a new epitope peptide derived from the EML4-ALK fusion gene. We successfully induced an HLA-A*02:01-restricted peptide-specific CTL clone that demonstrated cytotoxicity for EML4-ALK-positive tumor cells. This is a new epitope-based vaccine therapy design for EML4-ALK-positive cancer cells. In order to obtain a stronger effect further analysis is needed. Acknowledgements We thank Professor S. Yano for providing the H2228 cell line which possesses the EML4-ALK fusion gene Professor H. Mano for providing the EML4-ALK fusion DNA and Professor N. Hirano for providing artificial APCs. This study was supported in part by Health and Labor Science Research Grants for Clinical Research and Third Term Comprehensive Control Research for Cancer from the Ministry of Health Labor and Welfare Japan and the National Cancer Center Research and Development Fund (25-A-7). References 1 Silvestri GA Tanoue LT Margolis ML The noninvasive staging of non-small cell lung cancer: the guidelines Chest 123 147S 156S 2003 12527574 2 Reck M What future opportunities may immuno-oncology provide for improving the treatment of patients with lung cancer? Ann Oncol 23 Suppl 8 viii28 viii34 2012 22918925 3 Chang SC Chang CY Shih JY The role of epidermal growth factor receptor mutations and epidermal growth factor receptor-tyrosine kinase inhibitors in the treatment of lung cancer Cancers 3 2667 2678 2011 24212826 4 Gridelli C Peters S Sgambato A Casaluce F Adjei AA Ciardiello F ALK inhibitors in the treatment of advanced NSCLC Cancer Treat Rev 40 300 306 2014 23931927 5 Hall RD Gray JE Chiappori AA Beyond the standard of care: a review of novel immunotherapy trials for the treatment of lung cancer Cancer Control 20 22 31 2013 23302904 6 Jackman DM Miller VA Cioffredi LA Impact of epidermal growth factor receptor and KRAS mutations on clinical outcomes in previously untreated non-small cell lung cancer patients: results of an online tumor registry of clinical trials Clin Cancer Res 15 5267 5273 2009 19671843 7 West H Oxnard GR Doebele RC Acquired resistance to targeted therapies in advanced non-small cell lung cancer: new strategies and new agents Am Soc Clin Oncol Educ Book 2013 10.1200/EdBook_AM.2013.33.e272 http://meetinglibrary.asco.org/content/198-132 8 Wu YL Park K Soo RA INSPIRE: a phase III study of the BLP25 liposome vaccine (L-BLP25) in Asian patients with unresectable stage III non-small cell lung cancer BMC Cancer 11 430 2011 21982342 9 Tyagi P Mirakhur B MAGRIT: the largest-ever phase III lung cancer trial aims to establish a novel tumor-specific approach to therapy Clin Lung Cancer 10 371 374 2009 19808198 10 Quoix E Ramlau R Westeel V Therapeutic vaccination with TG4010 and first-line chemotherapy in advanced non-small-cell lung cancer: a controlled phase 2B trial Lancet Oncol 12 1125 1133 2011 22019520 11 Brahmer JR Tykodi SS Chow LQ Safety and activity of anti-PD-L1 antibody in patients with advanced cancer N Engl J Med 366 2455 2465 2012 22658128 12 Lynch TJ Bondarenko I Luft A Ipilimumab in combination with paclitaxel and carboplatin as first-line treatment in stage IIIB/IV non-small-cell lung cancer: results from a randomized double-blind multicenter phase II study J Clin Oncol 30 2046 2054 2012 22547592 13 Topalian SL Hodi FS Brahmer JR Safety activity and immune correlates of anti-PD-1 antibody in cancer N Engl J Med 366 2443 2454 2012 22658127 14 Soda M Choi YL Mano H Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer Nature 448 561 566 2007 17625570 15 Bonanno L Favaretto A Rugge M Role of genotyping in non-small cell lung cancer treatment: current status Drugs 71 2231 2246 2011 22085382 16 Fukui T Yatabe Y Mitsudomi T Clinicoradiologic characteristics of patients with lung adenocarcinoma harboring EML4-ALK fusion oncogene Lung Cancer 77 319 325 2012 22483782 17 Lin E Li L Guan Y Exon array profiling detects EML4-ALK fusion in breast colorectal and non-small cell lung cancers Mol Cancer Res 7 1466 1476 2009 19737969 18 Robertson FM Petricoin EF III Cristofanilli M Presence of anaplastic lymphoma kinase in inflammatory breast cancer Springerplus 2 497 2013 24102046 19 Sasaki T Rodig SJ J¤nne PA The biology and treatment of EML4-ALK non-small cell lung cancer Eur J Cancer 46 1773 1780 2010 20418096 20 Kwak EL Bang YJ Iafrate AJ Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer New Engl J Med 363 1693 1703 2010 20979469 21 Katayama R Shaw AT Engelman JA Mechanisms of acquired crizotinib resistance in ALK-rearranged lung cancers Sci Transl Med 4 120ra17 2012 22 Doebele RC Pilling AB Camidge DR Mechanisms of resistance to crizotinib in patients with ALK gene rearranged non-small cell lung cancer Clin Cancer Res 18 1472 1482 2012 22235099 23 Shaw AT Engelman JA ALK in lung cancer: past present and future J Clin Oncol 31 1105 1111 2013 23401436 24 Sasaki T Koivunen J J¤nne PA A novel ALK secondary mutation and EGFR signaling cause resistance to ALK kinase inhibitors Cancer Res 71 6051 6060 2011 21791641 25 Latif M Saeed A Kim SH Journey of the ALK-inhibitor CH5424802 to phase II clinical trial Arch Pharm Res 36 1051 1054 2013 23700294 26 Passoni L Scardino A Gambacorti-Passerini C ALK as a novel lymphoma-associated tumor antigen: identification of 2 HLA-A2.1-restricted CD8+T-cell epitopes Blood 99 2100 2106 2002 11877285 27 Hirohashi Y Torigoe T Maeda A An HLA-A24-restricted cytotoxic T lymphocyte epitope of a tumor-associated protein survivin Clin Cancer Res 8 1731 1739 2002 12060610 28 Hirano N Butler MO Xia Z Engagement of CD83 ligand induces prolonged expansion of CD8+T cells and preferential enrichment for antigen specificity Blood 107 1528 1536 2006 16239433 29 Yoshikawa T Nakatsugawa M Sakemura N HLA-A2- restricted glypican-3 peptide-specific CTL clones induced by peptide vaccine show high avidity and antigen-specific killing activity against tumor cells Cancer Sci 102 918 925 2011 21281401 30 Robinson KW Sandler AB EGFR tyrosine kinase inhibitors: difference in efficacy and resistance Curr Oncol Rep 15 396 404 2013 23674236 31 Lesniak D Sabri S Abdulkarim B Spontaneous epithelial- mesenchymal transition and resistance to HER-2-targeted therapies in HER-2-positive luminal breast cancer PLoS One 8 e71987 2013 23991019 32 Sawada Y Yoshikawa T Nakatsura T Phase I trial of a glypican-3-derived peptide vaccine for advanced hepatocellular carcinoma: immunologic evidence and potential for improving overall survival Clin Cancer Res 18 3686 3696 2012 22577059 33 Nakatsura T Yoshitake Y Nishimura Y Glypican-3 overexpressed specifically in human hepatocellular carcinoma is a novel tumor marker Biochem Biophys Res Commun 306 16 25 2003 12788060 34 Okabe H Satoh S Nakamura Y Genome-wide analysis of gene expression in human hepatocellular carcinomas using cDNA microarray: identification of genes involved in viral carcinogenesis and tumor progression Cancer Res 61 2129 2137 2001 11280777 35 Saito-Hisaminato A Katagiri T Nakamura Y Genome-wide profiling of gene expression in 29 normal human tissues with a cDNA microarray DNA Res 9 35 45 2002 12056413 36 Weir GM Liwski RS Mansour M Immune modulation by chemotherapy or immunotherapy to enhance cancer vaccines Cancers 3 3114 3142 2011 24212948 37 Hodge JW Ardiani A Gameiro SR The tipping point for combination therapy: cancer vaccines with radiation chemotherapy or targeted small molecule inhibitors Semin Oncol 39 323 339 2012 22595055 38 Garnett CT Palena C Hodge JW Sublethal irradiation of human tumor cells modulates phenotype resulting in enhanced killing by cytotoxic T lymphocytes Cancer Res 64 7985 7994 2004 15520206 39 Gelbard A Garnett CT Hodge JW Combination chemotherapy and radiation of human squamous cell carcinoma of the head and neck augments CTL-mediated lysis Clin Cancer Res 12 1897 1905 2006 16551875 40 Kaneno R Shurin GV Shurin MR Chemotherapeutic agents in low noncytotoxic concentrations increase immunogenicity of human colon cancer cells Cell Oncol 34 97 106 2011 41 Ramakrishnan R Assudani D Gabrilovich DI Chemotherapy enhances tumor cell susceptibility to CTL-mediated killing during cancer immunotherapy in mice J Clin Invest 120 1111 1124 2010 20234093 42 Choi YL Soda M Yamashita Y EML4-ALK mutations in lung cancer that confer resistance to ALK inhibitors N Engl J Med 363 1734 1739 2010 20979473 43 Katayama R Khan TM Benes C Therapeutic strategies to overcome crizotinib resistance in non-small cell lung cancers harboring the fusion oncogene EML4-ALK Proc Natl Acad Sci USA 108 7535 7540 2011 21502504 44 Lovly CM Pao W Escaping ALK inhibition: mechanisms of and strategies to overcome resistance Sci Transl Med 4 120ps2 2012 Figure 1 EML4-ALK-derived peptides bound to HLA-A2 or HLA-A24 molecules. In vitro cellular peptide binding assays for HLA-A*02:01 (A) or HLA-A*24:02 (B) were performed using a FACS system. Figure 2 IFN-? release by in vitro-induced anti-EML4-ALK CTLs. CD8+ T cells from four healthy donors were stimulated with EML4-ALK-derived peptide-pulsed autologous DCs and aAPCs. CTLs induced by EML4-ALK-derived peptides (1—105) were stimulated with T2 cells pulsed with or without 1 ?M EML4-ALK-derived peptides. IFN-?-producing CTLs were detected by IFN-? ELISPOT assay. DCs dendritic cells; aAPCs artificial antigen presenting cells; CTLs cytotoxic T cells. Figure 3 Peptide A-specific CTL clone established from anti-EML4-ALK CTL. (A) Peptide A-specific CTL clones established using CD107a single cell sorting. Peptide A-specific CTLs (1—105) were incubated with peptide-pulsed T2 cells (5—104) with CD107a-specific antibodies for 3.5 h at 37°C. CD8+CD107a+ cells were sorted using a FACSAria II cell sorter. Square CD8+CD107a+ cells that are peptide A-specific CTL clones. (B) Recognition of peptide-pulsed T2 cells by peptide A-specific CTL clones. A peptide A-specific CTL clone (1—104 cells) was incubated with stimulator cells that had been pulsed with 1 ?M peptide A or HIV-gag peptide. IFN-?-producing CTLs were detected by IFN-? ELISPOT assay. CTLs cytotoxic T cells. Figure 4 Recognition of lung carcinoma cells expressing HLA-A*02:01 and the EML4-ALK fusion gene by the peptide A-specific CTL clone. The peptide A-specific CTL clone recognized H2228 cells pretreated with IFN-? 48 h prior to the assay. (A) The peptide A-specific CTL clone (1—104 cells) was incubated with H2228 cells with or without IFN-?. IFN-? production was detected by IFN-? ELISPOT assay. (B) IFN-? increased expression of HLA-A2 presented on H2228 cells. Incubation of H2228 cells with 100 U/ml IFN-? for 48 h increased HLA-A2 presentation on the cells. Dotted line HLA-A2 on H2228 cells without IFN-?. Black line HLA-A2 on H2228 cells incubated with IFN-? (higher than on H2228 cells without IFN-?). Dashed line and shaded region: no staining of H2228 cells with/without IFN-?. (C) Inhibition of IFN-? production by an anti-HLA-class I mAb. Blocking experiments were performed using an HLA-A -B -C-specific mAb (W6/32) or an isotype control mAb (mIgG2a?). The peptide A-specific CTL clone was incubated with H2228 cells (HLA-A*02:01+/EML4-ALK+) pretreated with IFN-? 48 h prior to examination. IFN-?-producing CTL clones were detected by IFN-? ELISPOT assay. The bar graph shows the percentage of inhibition. CTL cytotoxic T cell. Figure 5 Cytotoxic activity of the peptide A-specific CTL clone against H2228 cells. The peptide A-specific CTL clone was incubated with H2228 cells pretreated with IFN-? 48 h prior to the assay at various E/T ratios and specific lysis was assessed. Blocking experiments were performed using the HLA-A -B -C-specific mAb (W6/32) or the isotype control mAb (mIgG2a?). CTL cytotoxic T cell. Table I HLA-A2 peptide binding predictions of the BIMAS program. Peptide name Peptide sequence Binding scorea A RLSALESRV 69.552 B AISEDHVASV 90.183 C TVLKAALADV 51.79 D KLIPKVTKT 59.989 E YLLPTGEIV 237.82 F MLIWSKTTV 118.238 G VMLIWSKTTV 315.95 a Binding scores were estimated using BIMAS software (http://www-bimas.cit.nih.gov/mobio/hla_bind/). Table II HLA-A24 peptide binding predictions of the BIMAS program. Peptide name Peptide sequence Binding scorea H NYDDIRTEL 369.6 I VYFIASVVVL 200 a Binding scores were estimated using BIMAS software (http://www-bimas.cit.nih.gov/mobio/hla_bind/). Diagn Pathol Diagn Pathol Diagnostic Pathology 1746-1596 BioMed Central 24972450 4085714 1746-1596-9-128 10.1186/1746-1596-9-128 Research Overexpression of both platelet-derived growth factor-BB and vascular endothelial growth factor-C and its association with lymphangiogenesis in primary human non-small cell lung cancer Liu Jiannan 1 2 zdpxf2317@aliyun.com Liu Chuanyong 1 cyl0936@sina.com Qiu Liyun 3 qiuliyun7777@163.com Li Juan 1 lijuanjinan@163.com Zhang Pei 1 sehodzhang@126.com Sun Yuping 1 13370582181@163.com 1Department of Oncology Jinan Central Hospital Affiliated to Shandong University No. 105.Jiefang Road Jinan Shandong 250013 P.R. China 2Department of Oncology Yuhuangding Hospital Yantai Shandong 264000 P.R. China 3Department of Pharmacology Jinan Central Hospital Affiliated to Shandong University Jinan Shandong 250013 P.R. China 2014 27 6 2014 9 128 128 1 4 2014 13 6 2014 Copyright © 2014 Liu et al.; licensee BioMed Central Ltd. 2014 Liu 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 "

Lung_Cancer

"National Institutes of Health R01 DK082779 9005373 1697 Eur J Cancer Eur. J. Cancer European journal of cancer (Oxford England : 1990) 0959-8049 1879-0852 24780874 4155498 10.1016/j.ejca.2014.03.008 NIHMS621748 Genetic variants of the LIN28B gene predict severe radiation pneumonitis in patients with non-small cell lung cancer treated with definitive radiation therapy Wen Juyi a b 1 Liu Hongliang c 1 Wang Qiming b Liu Zhensheng c Li Yangkai c Xiong Huihua b Xu Ting d Li Peng b Wang Li-E b Liao Zhongxing d * Wei Qingyi c * aDepartment of Radiation Oncology Navy General Hospital Beijing China bDepartment of Epidemiology The University of Texas M.D. Anderson Cancer Center Houston TX 77030 USA cDuke Cancer Institute Duke University Medical Center Durham NC 27710 USA dDepartment of Radiation Oncology The University of Texas M.D. Anderson Cancer Center Houston TX 77030 USA *Corresponding authors: Tel. : +1 919 660 0562; fax: +1 919 681 7385; qingyi.weiduke.edu (Q. Wei) and Tel. :+1 713 563 2300; zliaomdanderson. (Z. Liao) 1 These authors contributed equally to this work. 19 8 2014 26 4 2014 7 2014 05 9 2014 50 10 1706 1716 Background LIN28 is an RNA-binding protein that not only plays key roles in multiple cellular developmental processes and tumorigenesis but also is involved in tissue inflammatory response. However no published study has investigated associations between genetic variants in LIN28 and radiation-induced pneumonitis (RP) in patients with non-small cell lung cancer (NSCLC) treated with definitive radiation therapy. Methods We genotyped eight potentially functional single nucleotide polymorphisms (SNPs) of LIN28A (rs11247946 T>C rs3811464 C>T rs11581746 T>C and rs12728900 G>A) and LIN28B (rs314280 A>G rs12194974 G>A rs17065417 A>C and rs314276 C>A) in 362 patients with NSCLC who received definitive radio (chemo) therapy. The associations between RP risk and genotypes were assessed by hazards ratio (HR) in Cox proportional hazards regression analysis with time to event considered with and without adjustment for potential confounders. Results Multivariate analyses found that patients carrying LIN28B rs314280 AG and AA/AG or rs314276 AC and AA/AC genotypes had a higher risk of grade ?3 RP (for rs314280 AG and AA/AG versus GG adjusted HR= 2.97 and 2.23 95% CI 1.32“6.72 and 1.01“4.94 P = 0.009 and 0.048 respectively; for rs314276 AC and AA/AC versus CC adjusted HR = 2.30 and 2.00 95% CI 1.24“4.28 and 1.11“3.62 and P = 0.008 and 0.022 respectively). Further stratified analyses showed a more profound risk in the subgroups of the age <65 years subjects males stage III/IV ever smokers and MLD ?19.0 Gy. Conclusion Genetic variants of LIN28B but not LIN28A may be biomarkers for susceptibility to severe RP in NSCLC patients. Large prospective studies are needed to confirm our findings. LIN28 polymorphisms inflammation Hazards ratio Non“small cell lung cancer radiation pneumonitis 2984705R 2786 Cancer Res Cancer Res. Cancer research 0008-5472 1538-7445 25339352 4268230 10.1158/0008-5472.CAN-14-1439 NIHMS638086 Oncogenic KRAS confers chemoresistance by upregulating NRF2 Tao Shasha 1 Wang Shue 2 Moghaddam Seyed Javad 3 Ooi Aikseng 1 Chapman Eli 1 Wong Pak K. 2 Zhang Donna D. * 1 1Department of Pharmacology and Toxicology College of Pharmacy The University of Arizona Tucson Arizona 2Department of Aerospace and Mechanical Engineering The University of Arizona Tucson Arizona 3Department of Pulmonary Medicine the University of Texas M. D. Anderson Caner Center Houston Texas *To whom correspondence should be addressed: Donna D. Zhang Department of Pharmacology and Toxicology College of Pharmacy University of Arizona 1703 E. Mable St. Tucson AZ USA Tel: (520) 626-9918; Fax: (520) 626-2466; dzhangpharmacy.arizona.edu 28 10 2014 22 10 2014 15 12 2014 15 12 2015 74 24 7430 7441 Oncogenic KRAS mutations found in 20“30% of all non-small cell lung cancers (NSCLC) are associated with chemoresistance and poor prognosis. Here we demonstrate that activation of the cell protective stress response gene NRF2 by KRAS is responsible for its ability to promote drug resistance. RNAi-mediated silencing of NRF2 was sufficient to reverse resistance to cisplatin elicited by ectopic expression of oncogenic KRAS in NSCLC cells. Mechanistically KRAS increased NRF2 gene transcription through a TPA response element (TRE) located in the NRF2 promoter. In a mouse model of mutant KrasG12D-induced lung cancer we found that suppressing the NRF2 pathway with the chemical inhibitor brusatol enhanced the antitumor efficacy of cisplatin. Co-treatment reduced tumor burden and improved survival. Our findings illuminate the mechanistic details of KRAS-mediated drug resistance and provide a preclinical rationale to improve the management of lung tumors harboring KRAS mutations with NRF2 pathway inhibitors. KRAS NRF2 Brusatol chemoresistance TPA Response Element Chest Chest chest Chest Chest 0012-3692 1931-3543 American College of Chest Physicians 24577678 4137590 chest.13-2281 10.1378/chest.13-2281 Original Research Lung Cancer Assessing the Usefulness of 18F-fluorodeoxyglucose PET-CT Scan After Stereotactic Body Radiotherapy for Early-Stage Non-small Cell Lung Cancer Usefulness of 18F-fluorodeoxyglucose PET-CT Scan Pastis Nicholas J. Jr MD FCCP Greer Travis J. MD Tanner Nichole T. MD FCCP Wahlquist Amy E. MS Gordon Leonie L. MBChB Sharma Anand K. MBBS Koch Nicholas C. PhD Silvestri Gerard A. MD FCCP From the Division of Pulmonary/Critical Care (Drs Pastis Greer Tanner and Silvestri) Public Health Services (Ms Wahlquist) the Department of Radiology (Dr Gordon) and the Department of Radiation Oncology (Drs Sharma and Koch) Medical University of South Carolina Charleston SC. CORRESPONDENCE TO: Nicholas J. Pastis Jr MD FCCP Division of Pulmonary/Critical Care Medical University of South Carolina 96 Jonathan Lucas St Charleston SC 29425; e-mail: pastisnmusc.edu 8 2014 27 2 2014 1 8 2015 146 2 406 411 25 9 2013 1 2 2014 2014 AMERICAN COLLEGE OF CHEST PHYSICIANS 2014 BACKGROUND: Although stereotactic body radiation therapy (SBRT) is an established treatment option for early-stage lung cancer there are no guidelines for reassessing patients for local treatment failure or intrathoracic recurrence after treatment. This study reports the sensitivity specificity and positive and negative predictive values for 18F-fluorodeoxyglucose (FDG) PET-CT scanning when used to evaluate patients after SBRT. METHODS: Charts were reviewed of all patients who received SBRT and a subsequent FDG PET-CT scan at a university hospital over a 5-year period. Pretreatment and 3-month posttreatment tumor characteristics on PET-CT scan and outcome data (adverse events from SBRT need for repeat biopsy rate of local treatment failure and recurrent disease and all-cause mortality) were recorded. RESULTS: Eighty-eight patients were included in the study. Fourteen percent of patients (12 of 88) had positive 3-month PET scans. Of the positive results 67% (eight of 12) were true positives. Eighty-six percent (76 of 88 patients) had negative 3-month FDG PET-CT scans with 89% (68 of 76) true negatives. FDG PET-CT scan performed 3 months after SBRT for non-small cell lung cancer (NSCLC) had a sensitivity of 50% (95% CI 0.26-0.75) a specificity of 94% (95% CI 0.89-1.0) a positive predictive value of 67% (95% CI 0.4-0.93) and a negative predictive value of 89% (95% CI 0.83- 0.96). S: FDG PET-CT scan 3 months after treatment of NSCLC with SBRT was a specific but insensitive test for the detection of recurrence or treatment failure. "

Lung_Cancer

"For mutations with therapeutic significance highly sensitive and specific assays are essential for informing patient therapy and for clinical trials investigating new agents. Relative to published mutations derived from DNA-WES alone the UNCeqRMETA mutations derived from patient-matched DNA-WES and RNA-seq increased the numbers of patients with mutations in genes that are targets for several drugs in clinical trials such as PIK3CA and ERBB2 and for drugs with correlative evidence such as FGFR2 (51). Clinical trials such as NCT01670877 which involve ERBB2 sequencing (52) may be influenced to include RNA-seq due the large mutation rate increase reported here. Although the relative increase in PIK3CA mutations was modest compared to other genes in breast cancer this improved sensitivity is vital for affected patients and could lead to positive clinical trial outcomes. For example some novel canonical mutations in PIK3CA had many mutant reads in RNA-seq but only a few mutant reads in DNA-WES such as the example Luminal A tumor with a single DNA mutant read in the PIK3CA hotspot. This study's results support that RNA sequencing could be beneficial when added to DNA sequencing in clinical settings. Future studies could explore alternative ways to integrate DNA and RNA sequencing beyond UNCeqRMETA which is the first method of this kind. UNCeqRMETA applied the same quality filters for DNA and RNA and potentially different filters could be beneficial. UNCeqRMETA includes a basic indel realignment and integrated DNA and RNA reassembly could potentially be beneficial. Different statistical modeling could further advance the performance displayed by UNCeqRMETA over DNA only based methods. Balancing sensitivity and specificity is important in applying and developing mutation detectors. Receiver operating characteristic curve analysis such as that presented in this study enables assessment of sensitivity and specificity tradeoffs between alternate models. Integrated RNA-seq and DNA-WES mutation detection is important because it boosts sensitivity in low purity tumors in therapeutically-relevant genes and in driver genes relative to DNA-only detection. Integrated mutation detection could also enable more inclusive cohort profiling studies that censor tumors based on purity and could lead to more comprehensive "

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 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 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 (a). 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 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 . 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) (). "

Lung_Cancer

"Sulindac is a ligand of the aryl hydrocarbon receptor (AhR) an xenobiotic-sensing nuclear receptor that can be activated by chemical structures containing planar aromatic hydrocarbons and thus evokes a cellular response that to detoxify xenobiotics. AhR activation leads to transcriptional upregulation of the NQO1 gene [62] [63]. In previous studies [30] [64] [65] sulindac and its two metabolites have been used to treat cancer cells at concentrations of 200 µM “1 mM i.e. the concentrations used in our present study. In addition to reducing the growth of polyps all three increase NQO1 activity and expression in colon cancer cells [28] and might therefore be good candidates to increase the cytotoxic effect of ?-lapachone against lung cancer cells. When two cancer cell lines CL1-1 and CL1-5 with low NQO1 expression and activity were co-incubated with sulindac or its metabolites and ?-lapachone much higher cell death was seen with the CL1-5 cells than the CL1-1 cells ( and 7). These results demonstrated that the effect of sulindac and its metabolites in upregulating NQO1 was greater in CL1-5 cells which has lower NQO1 level and activity than CL1-1 cells showing that sulindac and its metabolites can be used to increase the ?-lapachone sensitivity of cells with lower NQO1 levels. Many other compounds such as toxifolin [32] and resveratrol [66] can increase NQO1 expression or activity but are not FDA-approved. A search is underway for other compounds that can increase the activity or expression of NQO1 using high-throughput library screening and two compounds DMEBP and TRES were recently found to be potent NQO1 inducers with low toxicity [3]. These compounds may also be valuable in increasing ?-lapachone cytotoxicity for cancer cells with low NQO1 expression or activity. The NQO1 Inhibitor Dicoumarol or Transfection with NQO1 siRNA Inhibits the Effect of Sulindac on ?-lapachone Toxicity for Lung Cancer Cells Dicoumarol is widely used as a specific pharmacologic inhibitor of NQO1 and has been shown to inhibit both enzyme activity and expression [45] [67] [68]. NQO1 siRNA designed to specifically target NQO1 mRNA can lower the expression of NQO1 mRNA and protein. In our study both agents blocked the synergistic effect of sulindac or its metabolites and ?-lapachone on decreasing the survival of CL1-1 or CL1-5 cells. Although ?-lapachone is very toxic for many cancer cells cells with lower NQO1 levels are less sensitive. However from the present study we can conclude that sulindac and its metabolites increase NQO1 expression and enzyme activity and thus are potential synergistic drugs that might be used in combination with ?-lapachone to treat cancer cells with high resistance to ?-lapachone cytotoxicity. Supporting Information Figure S1 ?-lapachone causes cell death of CL1-1 and CL1-5 cells by decreasing the mitochondrial membrane potential. (A) Cells were left untreated or were incubated with 5 µM ?-lapachone for the indicated time and then the cell cycle distribution was analyzed using propidium iodide staining and flow cytometry. (B) Cells were incubated with 5 µM ?-lapachone for the indicated time then pro-caspase 3 and caspase 3 levels were analyzed by Western blotting. (C) Cells were incubated with 5 µM ?-lapachone for the indicated time then the mitochondrial membrane potential (MMP) was measured using the dye JC1 (Life Technology) and flow cytometry. (D) Cells were incubated with 5 µM ?-lapachone for the indicated time and then intracellular H2O2 levels were measured. (TIF) Click here for additional data file. Figure S2 zVAD-FMK ALLM and ALLN do not block the cytotoxicity of ?-lapachone. CL1-1 cells (A) or CL1-5 cells (B) were left untreated or were incubated for 1 h with the indicated concentration of the pan caspase inhibitor zVAD (left panels) or the calpain inhibitor ALLM (center panels) or ALLN (right panels) then 5 µM ?-lapachone was added for 12 or 24 h and cell viability measured using the MTT assay and expressed as percentage survival compared to the untreated cells. (TIF) Click here for additional data file. Figure S3 Dicoumarol an NQO1 inhibitor inhibits NQO1 activity and blocks the increase in intracellular calcium levels induced by ?-lapachone. (A) CL1-1 cells (left) or CL1-5 cells (right) were left untreated (CTL) or were incubated with 10 µM dicoumarol for 6 h then NQO1 activity was measured. (B) CL1-1 cells (top panel) or CL1-5 cells (bottom panel) were left untreated or were incubated with 10 µM dicoumarol and/or 5 µM ?-lapachone for 1 h then were stained with Fluo-4 and the intensity of the Fluo-4 fluorescence measured by flow cytometry. (TIF) Click here for additional data file. Figure S4 Sulindac and its metabolites do not affect survival of lung cancer cells. CL1-1 CL1-5 or A549 cells were left untreated or were incubated for 54 h with 100 or 250 µM sulindac (left panel) or sulindac sulfone (center panel) or for 12 h with 100 or 250 µM sulindac sulfide (right panel) then cell survival was measured by the MTT assay and expressed as percentage survival compared to the untreated cells. (TIF) Click here for additional data file. Figure S5 The cytotoxic effect of ?-lapachone on A549 cells is enhanced by sulindac and its metabolites. Two sets of cells were left untreated or were incubated for 6 h with the indicated concentration of sulindac sulindac sulfone or sulindac sulfide then 2 µM ?-lapachone was added to one set and incubation continued for 12 h when cell survival was measured using crystal violet staining and expressed as percentage survival compared to the untreated cells. (TIF) Click here for additional data file. Figure S6 NQO1 siRNA has no effect on cell morphology or cell growth. CL1-1 cells (top) and CL1-5 (bottom) were transfected with negative siRNA or NQO1 siRNA for 1 to 3 days then pictures were taken using a digital camera and phase contrast microscopy. The scale bar represents 50 µm. (TIF) Click here for additional data file. Figure S7 NQO1 RNA levels are decreased by siRNA targeting NQO1. A549 CL1-1 or CL1-5 cells were transfected for 48 h with siRNA targeting NQO1 (siNQO1) or control siRNA (siNeg) and then NQO1 mRNA levels were measured by realtime PCR and expressed as a fold change compared to the value for CL1-5 cells transfected with siNeg. * : p<0.05 compared to the result for the corresponding siNeg-transfected cells. (TIF) Click here for additional data file. Table S1 Primers used in the realtime PCR for actin and NQO1. (TIF) Click here for additional data file. Materials and Methods S1 (DOCX) Click here for additional data file. This work was supported by grants (NSC 101-2320-B-002-020-MY3 NSC 98-2320-B-715-001-MY3 (YPC) and NSC 101-2320-B-002-008) from the National Science Council Taiwan. References 1 PardeeAB LiYZ LiCJ (2002) Cancer therapy with beta-lapachone. Curr Cancer Drug Targets2: 227“24212188909 2 TagliarinoC PinkJJ ReinickeKE SimmersSM Wuerzberger-DavisSM et al (2003) Mu-calpain activation in beta-lapachone-mediated apoptosis. Cancer Biol Ther2: 141“15212750552 3 TanXL MarquardtG MassimiAB ShiM HanW et al (2012) High-throughput library screening identifies two novel NQO1 inducers in human lung cells. Am J Respir Cell Mol Biol46: 365“37122021338 4 MinamiT AdachiM KawamuraR ZhangY ShinomuraY et al (2005) Sulindac enhances the proteasome inhibitor bortezomib-mediated oxidative stress and anticancer activity. Clin Cancer Res11: 5248“525616033843 5 TeraiK DongGZ OhET ParkMT GuY et al (2009) Cisplatin enhances the anticancer effect of beta-lapachone by upregulating NQO1. "

Lung_Cancer

"the pharmacological manipulation of Hsp70 levels in cancer cells may be an effective means of preventing the progression of tumours. We found that the downregulation of Hsp70 by ibuprofen in vitro enhances the antitumoural activity of cisplatin in lung cancer. Ibuprofen prominently suppressed the expression of Hsp70 in A549 cells derived from lung adenocarcinoma and sensitized them to cisplatin in association with an increase in the mitochondrial apoptotic cascade whereas ibuprofen alone did not induce cell death. The cisplatin-dependent events occurring up- and downstream of mitochondrial disruption were accelerated by treatment with ibuprofen. The increase in cisplatin-induced apoptosis caused by the depletion of Hsp70 by RNA interference is evidence that the increased apoptosis by ibuprofen is mediated by its effect on Hsp70. Our observations indicate that the suppression of Hsp70 by ibuprofen mediates the sensitivity to cisplatin by enhancing apoptosis at several stages of the mitochondrial cascade. Ibuprofen therefore is a potential therapeutic agent that might allow lowering the doses of cisplatin and limiting the many challenge associated with its toxicity and development of drug resistance. Hsp70 apoptosis ibuprofen The human Hsp70 family includes ?8 highly homologous members that differ from each other by their intracellular localization and expression patterns.1 Among them the major stress-inducible Hsp70 (also called Hsp72) has an essential role in cell survival under stressful conditions. Compared with its normal counterpart Hsp70 is often overexpressed in various cancer cells and is suspected to contribute to the development of tumours.2 3 Indeed the expression of Hsp70 in certain cancer types has been correlated with poor prognosis and resistance to chemotherapy.45 6 Tumour cells often express several proteins that when abnormally elevated render the tumour resistant to apoptosis.7 Previous studies have confirmed not only that Hsp70 is cytoprotective but also that it interferes effectively with cell death induced by a wide variety of stimuli including several cancer-related stresses. Hsp70 is a potent inhibitor of the stress-activated kinase pathway and apparently blocks apoptotic signals via interactions with JNK Ask1 and SEK1.8910 11 Hsp70 is also a negative regulator of the mitochondrial pathway of apoptosis. Much of the focus on the antiapoptotic function of Hsp70 has been on events that occur after the disruption of the mitochondria. Hsp70 prevents the recruitment of procaspase-9 to the apoptosome and its functional complex formation by direct interaction with apoptotic protease-activating factor 1 (Apaf-1).12 13 Furthermore Hsp70 inhibits the activation of caspase-3 and the cleavage of caspase-3 targets such as ICAD and GATA-1.14 15 On the other hand recent studies have reported that Hsp70 can prevent apoptosis upstream of the mitochondria by inhibiting events which ultimately permeabilize the mitochondrial outer membrane such as the activation of Bax.16 17 As a result of the inhibition by Hsp70 of the apoptosis induced by several anticancer drugs as well as by other stimuli we hypothesized that cancer cells would be sensitized to the induction of apoptosis by the neutralization of Hsp70. Hsp70 has been indeed targeted with pharmaceuticals such as triptolide quercetin and KNK437 which downregulate its expression.1819 20 Although they have prevented the progression of various cancer cells in vitro and in vivo21 22 the optimal clinical use of these small Hsp70 inhibitors singly or combined with other chemotherapeutics remains a challenge. Our overall objective was to pharmacologically control the levels of Hsp70 and increase the effectiveness of anticancer drugs. Several experimental and epidemiologic studies and clinical trials have observed a powerful chemopreventive activity exerted by nonsteroidal anti-inflammatory drugs (NSAIDs).23 24 The anti-carcinogenic properties of NSAID have been attributed to their inhibition of cyclooxygenase (COX) enzymes. However much higher doses of NSAID are needed to obtain an antitumoural effect than to inhibit COX25 suggesting that they also act via COX-independent mechanisms. On the other hand NSAIDs such as aspirin salicylate and sulindac sulphide inhibit the proliferation of cells and induce apoptosis in various cancer cell lines which is considered an important component of their antitumoural activity and increased sensitization of cancer cells to anticancer drugs.262728 29 There is currently interest in the ability of NSAID to directly lower the levels of antiapoptotic molecules such as the Bcl-2 family30 and 14-3-3 protein31 which inhibits the intrinsic mitochondria-dependent apoptosis in various cancer cells. Therefore the NSAID-induced dysfunction of antiapoptotic proteins prompted us to examine whether other antiapoptotic molecules including Hsp70 might also be targets in the prevention of tumour progression by NSAID. In this study we show that ibuprofen is a potent inhibitor of Hsp70 which significantly suppresses its expression by depleting heat shock factor 1 (HSF1) in lung adenocarcinoma-derived A549 cells. The downregulation of Hsp70 by ibuprofen sensitized the cells to cisplatin which was associated with the enhancement of cisplatin-induced apoptotic signalling. Ibuprofen did not only facilitate postmitochondrial events including the activation of cisplatin-induced caspase-9 but also the activation of Bax causing the release of cytochrome c. Besides the demonstration of a similar increase in the sensitivity of A549 cells to cisplatin conferred by Hsp70 knockdown and ibuprofen these observations indicate that ibuprofen accelerates cisplatin-mediated apoptosis at multiple steps of the mitochondrial apoptotic pathway via the inhibition of Hsp70. We conclude that ibuprofen is a potential chemotherapeutic agent which might enable (a) the use of lower less toxic does of cisplatin and (b) the design of a new combination treatment of lung cancer. Results Ibuprofen suppresses the expression of Hsp70 in lung adenocarcinoma cells To define the role of Hsp70 in promoting the formation of tumours we first examined its expression in human lung cancer cell lines. Compared with BEAS-2B a human non-malignant bronchial epithelial cell line the expression levels of Hsp70 in lung cancer cells such as A549 and H358 adenocarcinoma were notably higher (a). As in previous studies which showed an increased expression of Hsp70 in various types of human cancers including breast pancreas and colon we found that Hsp70 is also dysregulated in lung cancer cells. In this study we screened conventional NSAID in search of a new pharmacologic inhibitor which neutralizes Hsp70 as they induce apoptosis in cancer cells by selectively downregulating antiapoptotic proteins. The expression of Hsp70 after the exposure of A549 cells to various NSAID in non-toxic concentrations was analyzed by immunoblot. Ibuprofen in a 400-?M concentration decreased the expression of Hsp70 by 23% in comparison with untreated cells whereas other NSAID had no effect (). b shows the decrease in Hsp70 protein and mRNA levels in A549 cells after treatment with various concentrations of ibuprofen versus no apparent decreases in Hsc70 and Actin. Ibuprofen also decreased the expression of Hsp70 in H358 a human lung adenocarcinoma cell line in a dose-dependent manner (c). These results suggest that ibuprofen decreases the expression of Hsp70 in various lung cancer cell lines. Ibuprofen enhances the apoptosis induced by cisplatin by suppressing Hsp70 As ibuprofen prominently inhibited the expression of Hsp70 we next examined its effect on the proliferation of cancer cells. We observed no significant change in the viability of A549 and H358 cells after the exposure to ?800??M concentrations of ibuprofen alone which downregulates Hsp70 (a) while the exposure to 1.0?mM concentration of ibuprofen caused cell death. Combined these observations indicate that the downregulation of stress-inducible Hsp70 was insufficient to cause the death of A549 and H358 cells. There is evidence that the inhibition of anti-apoptotic molecules such as Hsp70 increases the sensitivity of tumour cells to anticancer drugs thus improving the outcomes of chemotherapy. To study the therapeutic potential of ibuprofen we examined whether its antitumoural effects are synergistic with those of cisplatin widely used in the treatment of lung adenocarcinoma. When we measured the survival of A549 (top of b) and H358 (bottom of b) cells exposed to increasing concentrations of cisplatin incubated in presence versus absence of ibuprofen the latter prominently magnified the apoptosis induced by cisplatin a synergistic effect confirmed by terminal deoxynucleotidyl transferase-mediated dUTP nick and labelling (TUNEL) staining (c). To ascertain the effects conferred by the expression of Hsp70 on cell death while excluding all effects of ibuprofen unrelated to Hsp70 we weakened the expression of Hsp70 by RNA interference (RNAi) (d) and measured its effects on the apoptosis induced by cisplatin. The inhibition of Hsp70 decreased the viability of cisplatin-treated cells by approximately 20% (e). Transfections with scrambled siRNA serving as a control showed no increase in cell death mediated by cisplatin. Cisplatin had no effect on the expression of Hsp70 (g). We quantified the number of apoptotic cells in ibuprofen- and/or cisplatin-treated cultures using the CF488A-annexin V methods. Although cisplatin alone induced apoptosis in 10.2% of A549 cells the co-treatment with ibuprofen increased the percentage of apoptotic cells to 34.0% (f). These observations suggest that ibuprofen sensitizes A549 cells to cisplatin by decreasing the expression of Hsp70. Ibuprofen decreases the expression of Hsp70 via transcriptional inactivation The reverse transcriptase-polymerase chain reaction (RT-PCR) analysis described earlier revealed a decrease in RNA level following treatment with ibuprofen suggesting that the expression of Hsp70 can be downregulated at the transcriptional level. After the recently discovered inhibition by its antagonists of the transcription of Hsp70 in cancer cells by blockade of the activation of HSF118 20 (which is often upregulated and constitutively activated in tumour formation) we studied the effects of ibuprofen on HSF1 in A549 cells. We first performed a ChIP assay to explore whether the inhibitory effect of ibuprofen is at the level of HSF1 DNA binding. As expected we found an unequivocal association between HSF1 and the Hsp70 gene promoter containing the HSE site in ibuprofen-untreated cells (Figure 3a). It is noteworthy that ibuprofen eliminated this binding (Figure 3a) suggesting that it inhibits the expression of Hsp70 via the action of HSF1. This also suggests that ibuprofen blocks the binding of HSF1 chromatin or the steps which precede in several processes needed to activate HSF1. Therefore we broadened our analysis to examine the effect of ibuprofen on the expression of HSF1. Compared with unexposed control cells the HSF1 mRNA level was significantly lower in cells exposed to ibuprofen (bottom of Figure 3b). Consistent with its effect on the expression of mRNA ibuprofen also decreased the expression of HSF1 protein in a dose-dependent fashion (top of Figure 3b). To confirm the inhibition of HSF1-mediated Hsp70 by ibuprofen we lowered the amounts of HSF1 present in A549 cells by RNAi and studied its effect on the expression of Hsp70. The treatment of cells with HSF1 dsRNA decreased the Hsp70 level compared with that measured in cells untreated with dsRNA (Figure 3c). Ibuprofen decreased the expression of HSF1 by 16% in comparison with untreated cells whereas other NSAID had no effect (Table 2). Overall these observations indicate that ibuprofen inhibited the expression of Hsp70 by depleting the HSF1 in A549 cells. Ibuprofen accelerates the mitochondrial apoptotic process induced by cisplatin Several studies have found that mitochondria might be a direct and important target of cisplatin in sensitive cells.32 33 We studied the effects of ibuprofen on the depolarization of mitochondrial membranes and the cytochrome c release induced by cisplatin. A549 cells with or without cisplatin were incubated in absence or presence of ibuprofen and stained with JC-1. Treatment with cisplatin and ibuprofen lowered the mitochondrial membrane potential manifest by an attenuated red and an enhanced green mitochondrial fluorescence (Figure 4a lower right panel) compared with that observed with cisplatin alone (Figure 4a upper right panel) while control (Figure 4a upper left panel) or ibuprofen alone (Figure 4a lower left panel) produced the red-dotted staining pattern of polarized mitochondria. The intensity of green mitochondrial fluorescence in cisplatin-treated cells is significantly increased (36.56 to 55.56%) by the co-treatment with ibuprofen. Ibuprofen also promoted the release of cytochrome c from the mitochondria induced by cisplatin (Figure 4b). These findings unequivocally indicated that in A549 cells ibuprofen enhanced the mitochondria-dependent apoptosis caused by cisplatin. Ibuprofen increases the activation of Bax induced by cisplatin The translocation of the pro-apoptotic protein Bax to the mitochondria is closely associated with the apoptosis induced by cisplatin. To explore the mechanisms by which ibuprofen promotes the apoptosis mediated by mitochondria in response to cisplatin we examined whether it was due to its ability to stimulate the translocation of Bax by cisplatin. We first monitored conformational changes in Bax as indicators of its activation. Western blot analysis of the immunoprecipitates with a conformation specific anti-Bax (6A7) antibody which only recognizes the active form revealed the presence of active Bax in A549 cells treated with cisplatin (Figure 5a lane 4) although not in untreated cells (Figure 5a lanes 1 and 2). Further exposure of the cisplatin-treated cells to ibuprofen caused a 1.5-fold increase in active Bax compared with incubation with cisplatin alone (Figure 5a lane 3)."

Lung_Cancer

"non-small cell lung cancers (NSCLC) and the underlying molecular mechanism. A549 cells were transfected with anti-miR-21 or the negative control oligonucleotides and real-time PCR was applied to detect miR-21 expression level. After ionizing radiation (IR) the survival fractions proliferation apoptosis and expression of phosphorylated-Akt of A549 cells were determined by clonogenic survival analysis MTT assay flow cytometry and Western blotting. Downregulation of miR-21 in radioresistant NSCLC A549 cells inhibited the colony-forming ability and proliferation of A549 cells after IR. Moreover silencing miR-21 enhanced apoptosis of A549 cells induced by IR accompanied by decreased phosphorylated-Akt protein level. However PI3K activator IGF-1 reversed suppression of phosphorylated-Akt protein level and promotion of apoptosis of A549 cells after IR caused by miR-21 knockdown. Silencing miR-21 in radioresistant NSCLC A549 cells sensitized them to IR by inhibiting cell proliferation and enhancing cell apoptosis through inhibition of PI3K/Akt signaling pathway. This might help in sensitization of NSCLC to radiotherapy. 1. Introduction Lung cancer is the leading cause of cancer-related deaths worldwide [1] whereas non-small cell lung cancer (NSCLC) represents the most frequent type of lung cancer [2]. NSCLC accounts for approximately 80% of all lung cancer cases and has a 5-year overall survival rate of less than 15% [3 4]. Approximately 40% of patients diagnosed with NSCLC have unresectable stage III disease or medically inoperable disease [5]. Radiation therapy has been regarded as the main treatment strategy for NSCLC for a long time. However radioresistance is the key issue limiting the effects of radiotherapy [2 6]. It is possibly due to tumor heterogeneity in terms of cell of origin pathology etiology and molecular/genetic pathogenesis [7]. NSCLC cells are often resistant to radiotherapy [8] which in turn induces the local recurrence of NSCLC [9 10]. 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 (Figure 2). 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 Figure 2 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 (Figure 3). 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 Figure 3). 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 Figure 4 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. Authors' Contribution Yongfu Ma and Hui Xia contributed equally to this work. 1 Jemal A Siegel R Ward E Cancer statistics 2008 CA: Cancer Journal for Clinicians 2008 58 2 71 96 2-s2.0-41349099104 2 Siegel R Naishadham D Jemal A Cancer statistics 2012 CA: Cancer Journal for Clinicians 2012 62 1 10 29 2-s2.0-84855792427 3 Fidias P Novello S Strategies for prolonged therapy in patients with advanced non-small-cell lung cancer Journal of Clinical Oncology 2010 28 34 5116 5123 2-s2.0-78650491373 21041704 4 Fuld AD Dragnev KH Rigas JR Pemetrexed in advanced non-small-cell lung cancer Expert Opinion on Pharmacotherapy 2010 11 8 1387 1402 2-s2.0-77952119805 20446853 5 Whitehurst AW Bodemann BO Cardenas J Synthetic lethal screen identification of chemosensitizer loci in cancer cells Nature 2007 446 7137 815 819 2-s2.0-34147198467 17429401 6 le P©choux C Role of postoperative radiotherapy in resected non-small cell lung cancer: a reassessment based on new data Oncologist 2011 16 5 672 681 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. Figure 2 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. Figure 3 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). Figure 4 Apoptosis of NSCLC A549 cells after ionizing radiation (IR). Apoptosis in anti-miR-21- (or anti-miR-NC-) transfected A549 cells combined with (or without) IR (8.0?Gy) was detected through annexin V-FITC/PI staining by flow cytometric analysis. The data represent the means ± SD of three separate experiments. Student's t-test was used to analyze the statistics (*P < 0.05). Figure 5 Suppression of ionizing radiation-induced phosphorylated-Akt (p-Akt) upregulation by anti-miR-21 in NSCLC A549 cells. The expression level of the phosphorylated or total Akt in A549 cells transfected with anti-miR-21 or anti-miR-NC for 48?h followed by ionizing radiation (8?Gy) with or without 10?ng/mL PI3K constituent activator IGF-1 was measured by Western blot. Representative of three independent experiments was shown. Figure 6 Knockdown of miR-21 promoted NSCLC A549 cells apoptosis via inactivation of PI3K-Akt pathway. Apoptosis induced by 8?Gy ionizing radiation in anti-miR-21- (or anti-miR-NC-) transfected A549 cells combined with (or without) PI3K activator IGF-1 treatment (10?ng/mL) was detected through annexin V-FITC/PI staining by flow cytometric analysis. The data represent the means ± SD of three separate experiments. Student's t-test was used to analyze the statistics (*P < 0.05). 1306016 3069 Clin Radiol Clin Radiol Clinical radiology 0009-9260 1365-229X 24857677 4105980 10.1016/j.crad.2014.03.020 NIHMS587373 Article Revisiting the relationship between tumour volume and diameter in advanced NSCLC patients: An exercise to maximize the utility of each measure to assess response to therapy Nishino M. a * Jackman D.M. b DiPiro P.J. a Hatabu H. a J¤nne P.A. b Johnson B.E. b aDepartment of Radiology Dana-Farber Cancer Institute and Brigham and Women™s Hospital 450 Brookline Ave. 75 Francis St. Boston MA 02215 USA bDepartment of Medical Oncology and Department of Medicine Dana-Farber Cancer Institute and Brigham and Women™s Hospital 450 Brookline Ave. Boston MA 02215 USA *Guarantor and correspondent: M. Nishino Department of Radiology Dana-Farber Cancer Institute and Brigham and Women™s Hospital 450 Brookline Ave. Boston MA 02215 USA. Tel.: +1 617 582 7163; fax: +1 617 582 8574. Mizuki_Nishino@dfci.harvard.edu (M. Nishino) 30 5 2014 22 5 2014 8 2014 01 8 2015 69 8 841 848 © 2014 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved. 2014 AIM To revisit the presumed relationship between tumour diameter and volume in advanced non-small-cell lung cancer (NSCLC) patients and determine whether the measured volume using volume-analysis software and its proportional changes during therapy matches with the calculated volume obtained from the presumed relationship and results in concordant response assessment"

Lung_Cancer

"To understand the functional consequences of GWAS loci is challenging and multiple principles for post-GWAS™ functional characterization of genetic loci have been proposed including the exploration of epigenetic mechanisms46. In our study the top GWAS lung cancer loci were strongly associated with methylation levels of CpG sites in nearby gene bodies through cis-regulation and adjusting for smoking status or intensity did not change the results. Furthermore SNPs affecting the DNA methylation of gene bodies (which are typically methylated) were also collectively associated with risk for squamous cell carcinoma after excluding the established GWAS loci and were enriched for genes in cancer pathways. In contrast no enrichment was observed for SNPs affecting the methylation of gene promoters or CGI regions which are typically not methylated in normal tissues. This suggests a potential novel mechanism for genetic effects on cancer risk. In fact gene body-enriched cis-meQTLs outside CGI regions may increase the risk for germline and somatic mutations due to their increased propensity to become mutated1112. Upon spontaneous hydrolytic deamination methylated cytosine residues turn into thymine which are less likely to be efficiently repaired than the uracils that result from deamination of unmethylated cytosine residues. For example about 25% of mutations in TP53 in cancers are thought to be due to epigenetic effects47. Indeed analyses of comprehensive human catalogues of lung tumors have identified frequent G>T mutations enriched for CpG dinucleotides outside CGI regions suggesting a role for methylated cytosine since CGI as we confirmed are usually unmethylated48. A similar signature was recently observed in other tumors14. Thus inherited genetic variation may have a profound impact on carcinogenesis by regulating the human methylome. We observed a high similarity of genetic control on DNA methylation across tissues. Since tissue of origin determines cancer-associated CpG island promoter hypermethylation patterns49 a natural question is whether the genetic regulation of methylation is tissue specific. While the tissue-specificity of eQTLs has been investigated for a few tissues50 for cis-meQTL only a recent investigation was conducted6 showing that 35.7% of 88751 cis meQTLs detected in 662 adipose samples were replicated in ~200 whole blood samples. We found that a large proportion of meQTLs in EAGLE lung samples particularly those with large effect sizes were robustly replicated in breast and kidney tissue samples from TCGA suggesting a high similarity of genetic regulation of methylation across these tissues and related impact on somatic mutation rates1448. The lower replication rate of adipose meQTLs in whole-blood samples6 might be explained by the heterogeneity of different cell types in whole blood and by their more liberal P-value threshold (8.6—10?4) which led to the identification of a large number of weak cis-meQTLs. Compared with cis-regulation trans-eQTL regulation is typically considered to be more complex has smaller effect sizes and is more difficult to be replicated even in the same tissue. However in our study the lung trans-meQTLs are highly reproducible in TCGA lung breast and kidney tissues. Notably this similarity allows mapping meQTLs with substantially improved power by borrowing strength across tissues51. meQTL SNPs are strongly associated with multiple epigenetic marks. Chromatin regulators play a role in maintaining genomic integrity and anization52. We found that meQTL SNPs were strongly enriched for DNase hypersensitive sites and sequences bound by CTCF or modified histones. SNPs could affect these epigenetic marks by several mechanisms such as by affecting the core recognition sequences (exemplified for rs2816057 on chromosome 1 for CTCF) causing loss or gain of a CpG within a binding region which when methylated could affect binding27 or altering the binding sequence for interacting factors53. CTCF could cause changes in epigenetic marks through its multiple key roles including genome anization through mediating intra- and inter-chromosomal contacts5455 the regulation of transcription by binding between enhancers and promoters5456 and the regulation of splicing which may impact tissue specificity during tissue development39. These changes can impact regulation of distant genes and not the genes proximal to the SNPs that would be typically investigated in eQTL studies. This may be one reason for the previously observed lack of correlation between eQTLs and meQTLs347. Future large studies integrating SNP profiles the DNA methylome and transcriptome data through tissue developmental stages will hopefully shed light on this possibility. There may be a myriad of other DNA-binding factors whose binding is directly or indirectly affected by SNPs. For example among the histone marks the strongest enrichment of meQTLs in our study was for H3K4me3 in both SAEC and hAEC cell types. As H3K4me3 is the chromatin mark primarily associated with regulatory elements at promoters and enhancers this suggests a strong influence of meQTLs on regulating gene activity. Unfortunately transcription factor binding data in SAEC or hAEC are not available so we could not test whether SNPs in their core sequence could affect the deposition of epigenetic marks e.g. by recruiting DNA methyltransferases57. It will be important to obtain ChIP data from relevant primary cells for numerous DNA-binding regulatory factors to further elucidate the mechanisms whereby meQTLs and other SNP-affected epigenetic marks arise. In we show here that genetic variation has a profound impact on the DNA methylome with implications for cancer risk tissue specificity and chromatin structure and anization. The meQTL data (Supplementary Data) attached to this manuscript provides an important resource for studying genetic-DNA methylation interactions in lung tissue. Methods Sample collection We assayed 244 fresh frozen paired tumor and non-involved lung tissue samples from Stage I to IIIA non-small cell lung cancer (NSCLC) cases from the Environment And Genetics in Lung cancer Etiology (EAGLE) study18. EAGLE includes 2100 incident lung cancer cases and 2120 population controls enrolled in 2002“2005 within 216 municipalities of the Lombardy region of Italy. Cases were newly diagnosed primary cancers of lung trachea and bronchus verified by tissue pathology (67.0%) cytology (28.0%) or review of clinical records (5.0%). They were 35?79 years of age at diagnosis and were recruited from 13 hospitals which cover over 80% of the lung cancer cases from the study area. The study was approved by local and NCI Institutional Review Boards and all participants signed an informed consent form. Lung tissue samples were snap-frozen in liquid nitrogen within 20 minutes of surgical resection. Surgeons and pathologists were together in the surgery room at the time of resection and sample collection to ensure correct sampling of tissue from the tumor the area adjacent to the tumor and an additional area distant from the tumor (1“5 cm). The precise site of tissue sampling was indicated on a lung drawing and the pathologists classified the samples as tumor adjacent lung tissue and distant non-involved lung tissue. For the current study we used lung tissue sampled from an area distant from the tumor to reduce the potential effects of field cancerization. DNA methylation profiling and data quality control Fresh frozen lung tissue samples remained frozen while approximately 30 mg was subsampled for DNA extraction into pre-chilled 2.0 ml microcentrifuge tubes. Lysates for DNA extraction were generated by incubating 30 mg of tissue in 1 ml of 0.2 mg/ml Proteinase K (Ambion) in DNA Lysis Buffer (10 mM Tris-Cl (pH 8.0) 0.1 M EDTA (pH 8.0) and 0.5% (w/v) SDS) for 24 hrs at 56°C with shaking at 850 rpm in Thermomixer R (Eppendorf). DNA was extracted from the generated lysate using the QIAamp DNA Blood Maxi Kit (Qiagen) according to the manufacturer™s protocol. Bisulfite treatment and Illumina Infinium HumanMethylation450 BeadChip assays were performed by the Southern California Genotyping Consortium at the University of California Los Angeles (UCLA) following Illumina™s protocols. This assay generates DNA methylation data for 485512 cytosine targets (482421 CpG and 3091 CpH) and 65 SNP probes for the purpose of data quality control. Raw methylated and unmethylated intensities were background corrected and dye-bias equalized to correct for technical variation in signal between arrays. For background correction we applied a normal-exponential convolution using the intensity of the Infinium I probes in the channel opposite their design to measure non-specific signal58. Dye-bias equalization used a global scaling factor computed from the ratio of the average red and green fluorescing normalization control probes. Both methods were conducted using the methylumi package in Bioconductor version 2.11. For each probe DNA methylation level is summarized as a ?-value estimated as the fraction of signal intensity obtained from the methylated beads over the total signal intensity. Probes with detection P-values of >0.05 were considered not significantly different from background noise and were labeled as missing. Methylation probes were excluded from meQTL analysis if any of the following criteria was met: on X/Y chromosome annotated in repetitive genomic regions annotated to harbor SNPs missing rate>5%. Because the ?-values for the 65 SNP probes are expected to be similar in matched pair of normal and tumor tissues we performed principal component analysis (PCA) using these 65 SNP probes to confirm the labeled pairs. We then performed PCA using the 5000 most variable methylation probes with var>0.02 and found that the normal tissues were clustered together and well separated from the tumor tissues. We further excluded 5 normal tissues that were relatively close to the tumor cluster. From the remaining 239 normal tissue samples we analyzed 210 with genotype data from a previous GWAS of lung cancer20. Genotype data and genetic association analysis The blood samples were genotyped using the Illumina HumanHap550K SNP arrays in EAGLE GWAS20. The SNPs with call rate >99% minor allele frequency (MAF) >3% and Hardy-Weinberg Equilibrium (HWE) P-value >10?5 were included for analysis. Prior to meQTL analysis each methylation trait was regressed against sex age batches and PCA scores based on methylation profiles. The regression residues were then quantile-normalized to the standard normal distribution N(01) as traits. The genetic association testing was performed using PLINK and R adjusted for the top three PCA scores based on GWAS SNPs to control for potential population stratification. Identification of cis-meQTLs For each CpG methylation probe the cis region was defined as being less than 500kb upstream and downstream from the target CpG-site (1Mb total). A methylation trait was detected to harbor a cis-meQTL if any SNP in the cis region had a SNP-CpG nominal association P-value less than P0 where P0 was chosen to control FDR at 5% by permutations. Here we describe a permutation procedure to choose P0 to control FDR at 5%. For a given P0 let N(P0) be the total number of CpG probes with detected cis-meQTLs and N0(P0) the expected number of CpG probes falsely determined to have cis-meQTLs. FDR is defined as N0(P0)/N(P0). The key is to estimate N0(P0) under the global null hypothesis that no CpG probe has cis-meQTLs. We randomly permuted the genotypes across subjects for 100 times keeping the correlation structure of the 338456 methylation traits in each permutation. Then N0(P0) was estimated as the average number of methylation traits that were detected to harbor cis-meQTL SNPs with nominal P<P0. Control FDR at 5% requires P0=4.0—10?5. The same procedure was applied to detect secondary independently associated cis-meQTL SNPs. With our sample size h2>0.12 is required to detect cis-meQTLs with power greater than 0.8. We note that although we excluded all CpG probes annotated with SNPs there is still the possibility that rare not annotated variants could be associated with the cis-meQTL SNPs. However since common variants and rare variants are known to be poorly correlated and rare variants are uncommon by definition we do not expect this event to be frequent. Identification of trans-meQTLs For each CpG probe the trans region was defined as being more than 500kb from the target CpG-site in the same chromosome or on different chromosomes. For the kth methylation trait with m SNPs in the trans region let (qk1?qkm) be the P-values for testing the marginal association between the trait and the m SNPs. Let pk=min(qk1?qkm) be the minimum P-value for m SNPs and converted pk into genome-wide P-value Pk by performing one million permutations for SNPs in the trans region. Because a cis region is very short (~1M) compared to the whole genome (~3000M) Pk computed based on SNPs in trans regions is very close to that based on permutations using genome-wide SNPs. Thus we use the genome-wide p-value computed based on all SNPs to approximate Pk. Furthermore all quantile-normalized traits follow the same standard normal distribution N(01); thus the permutation-based null distributions are the same for all traits. We then applied the Benjamini-Hochberg procedure to (P1?PN) to identify trans-meQTLs by controlling FDR at 5%. With our sample size h2 >0.24 is required to detect trans-meQTLs with power greater than 0.8. Replication of meQTLs in TCGA samples The replication was performed in TCGA histologically normal tissue samples that had genome-wide genotype (Affymetrix Genome-Wide Human SNP Array 6.0) and methylation profiling (Illumina Infinium HumanMethylation450 BeadChip). We downloaded genotype (level 2) and methylation data (level 3) from the TCGA website22. We also downloaded methylation data for tumor tissue samples and performed PCA analysis to confirm that normal tissue samples were separated from tumor tissue samples. Autosomal SNPs with MAF >3% calling rate >0.99 and HWE P-value > 10?5 were included for imputation using IMPUTE259 and reference haplotypes in the 1000 Genome Project60 (version 2012/03). We only included samples of European ancestry based on EIGENSTRAT analysis. The replication set had 65 lung 87 breast and 142 kidney histologically normal tissue samples after QC. Again each methylation trait was regressed against sex age batches and PCA scores based on methylation profiles. The regression residues were then quantile-normalized to the standard normal distribution N(01) as traits for meQTL analysis. The associations were tested between the quantile-normalized methylation traits and imputed genotypic dosages adjusting for sex age and PCA scores based on SNPs. A genetic association detected in EAGLE lung data was considered replicated if the association had the same direction and FDR<0.05 based on single-sided P-values. Testing genetic associations with methylation and gene expression traits We downloaded gene expression data (level 3) from RNA-seq analysis of 59 histologically normal tissue samples from NSCL patients from TCGA. All samples also had genome-wide genotype data and 28 samples had additional methylation data from Illumina Infinium HumanMethylation450 BeadChips. Regression analysis was performed to test the association of gene expression with methylation levels in the CHRNA5 gene and with methylation levels in PABPC4 STARD3 and SLC35A3 genes. We tested the association between lung cancer GWAS risk SNPs and gene expression using regression analysis under an additive model adjusting for age sex and PCA scores based on genome-wide SNPs. Testing for enrichment of cis-meQTLs in lung cancer GWAS We tested for enrichment in NCI lung cancer GWAS of European ancestry which included three main histologic subtypes of lung cancer (adenocarcinoma (AD) squamous cell carcinoma (SQ) small cell carcinoma (SC)) and a small number of other lung cancer subtypes. We investigated whether the identified cis-meQTL SNPs were collectively associated with lung cancer risk which was tested by examining whether the GWAS P-values for these SNPs deviated from the uniform distribution (i.e. no enrichment). Because the high linkage disequilibrium (LD) in SNPs increased variability of the enrichment statistic and caused a loss of power we first performed LD-pruning using PLINK so that no pair of remaining SNPs had a r2 ?0.8. The enrichment significance was evaluated by 10000 random permutations. The genomic control ?-values61 based on genome-wide SNPs were 1.010.995 0.977 and 1.00 for overall lung cancer AD SC and SQ respectively. Thus the type-I error rates of our enrichment tests were not inflated. The detailed procedure for testing a set of cis-meQTL SNPs is described as follows: Firstly we performed LD-pruning using PLINK so that no pair of remaining SNPs had an r2 ?0.8. Secondly we tested the association for the LD-pruned SNPs (assuming K SNPs left) in a GWAS and computed the P-values (p1?pK). We then tested whether (p1?pK) followed a uniform distribution i.e. no enrichment. Thirdly we transformed P-values into ?12 quantitles qk = F?1(1 ? pk) with F(·) being the cumulative distribution function (CDF) of ?12. We defined a statistic for testing enrichment as Q=?k=1Klog(1?f+fexp(qk/2))3562 where f is a pre-specified constant reflecting the expected proportion of SNPs associated with the disease. Because only a small proportion of SNPs may be associated with the disease we set f=0.05 for this paper. The statistical power was insensitive to the choice of f in the range of [0.01 0.1]62. Finally the significance of the test Q was evaluated by 10000 random permutations. meQTL mediation analysis We investigated whether trans associations were mediated by the methylation levels of CpG probes nearby the trans-acting SNPs. Note that this analysis was only for trans associations with cis effects i.e. the SNP was associated with at least one proximal CpG probes with p<4—10?5. See Fig. 2c. Suppose a SNP G cis-regulates K proximal (<500kb) CpG sites A1?AK with P<4—10?5 and trans-regulates a distal CpG site B. We performed a linear regression: B~?+?G +?kAk. We also computed marginal correlation coefficient cor(GB) and partial correlation coefficient cor(GB|Ak) using an R package œppcor63. A full mediation was detected if G and B were not significantly correlated after conditioning on Ak or equivalently G was not significant (p>0.01) in regression analysis B~?+?G +?kAk for any k. A partial mediation was detected if any Ak had a P<0.05/K (Bonferroni correction) in the regression analysis and |cor(GB)|?| cor(GB|A) |>0.1. An independent effect model (i.e. no mediation) was detected otherwise. Testing enrichment of meQTL SNPs in regulatory regions We obtained peak data for CTCF DNaseI H3K27me3 H3K4me3 and H3K36me of small airway epithelial cells (SAEC) from the ENCODE project and for H3K27me3 H3K4me3 and H3K9-14Ac from human alveolar epithelial cells (hAEC) from our own laboratory. A SNP is determined to be functionally related to a given mark or CTCF binding site if the SNP or any of its LD SNPs (r2 ?0.8 with LD computed using the genotype data of European population in The 1000 Genome Project) resided in any of the mark regions or CTCF binding sites. We explain our enrichment testing using CTCF as an example. We classified genome-wide SNPs into four categories: SNPs not associated with CpG probes in trans or cis (defined as control SNP set) SNPs only associated with proximal CpG probes via cis-regulation (cis-only21119 SNPs) SNPs only associated with distal CpG probes via trans-regulation (trans-only 192 SNPs) and SNPs detected with both trans and cis effects (cis+trans 277 SNPs). For SNPs in the category of cis-only trans-only and cis+trans we computed the proportion of SNPs functionally related to CTCF. To compute the enrichment of cis-meQTLs in CTCF binding sites we defined a control set of SNPs that are not associated with CpG probes via cis- or trans regulation. The selection of the control set was further complicated by the following two observations. (1) cis-meQTL SNPs tended to be more common (data now shown). (2) The probability of a SNP detected as a cis-meQTL SNP positively depended on the density of the CpG probes in the nearby region. Choosing a control set while ignoring these two factors could underestimate the proportion of functionally related SNPs in the control set and thus overestimate the enrichment for cis-meQTLs. Therefore we created 1000 sets of control SNPs with CpG probe density (measured as the number of CpG probes in the cis region of each SNP) and MAF matched with the meQTL SNP set and then averaged the proportions on the 1000 sets. The enrichment was calculated as the fold change with the proportion in the control SNP set as baseline. Next we investigated whether the enrichment was stronger for SNPs more significantly associated with CpG sites. Because we detected only a few hundred trans-meQTLs we focused this analysis on the set of cis-meQTLs. We classified cis-meQTL SNPs into five categories according to the cis-association P-values: P >10?7 (the weakest) 10?10< P ?10?7 10?15 < P ? 10?10 10?20 < P ? 10?15 and P ? 10?20 (the strongest). For each category we computed the proportion of SNPs functionally related to CTCF binding sites. meQTL SNPs affect CTCF binding We found that meQTL SNPs are strongly enriched in CTCF consensus sequences. We used SAEC data from ENCODE to test whether meQTL heterozygous SNPs directly affect CTCF binding by disrupting the CTCF recognition sites. P-values were calculated based on a binomial distribution Binom(N 0.5). Here N is the total number reads covering the SNPs. Raw sequencing data (.fasstq format) from SAEC cells were generated at the University of Washington as part of the ENCODE project and downloaded from the UCSC genome browser. Raw data was aligned to the hg19 genome using CLC genomics workbench (v 5.5.1) parsing out data with less than 80% contiguous alignment to the genome and duplicate reads in excess of 10 copies. We used the CTCFBSDB 2.0 program64 to predict whether the meQTL SNPs or their LD SNPs (r2 ? 0.8) were within CTCF peaks and then examined in SAEC whether CTCF exhibited allele-specific binding. Because common SNPs are more likely to be heterozygous we only looked for SNPs with MAF ?0.4. Here we present two such examples. Systematic investigation of all meQTL SNPs that are heterozygous in SAEC is warranted once more samples with genotypic data are available. Supplementary Material 1 Acknowledgements This study utilized the high-performance computational capabilities of the Biowulf Linux cluster at the NIH Bethesda MD (http://biowulf.nih.gov). We are grateful to the EAGLE participants and the large number of EAGLE collaborators (listed in http://dceg.cancer.gov/eagle) The Cancer Genome Atlas project for the genotype and methylation data and the ENCODE project for the regulatory region data. This work was supported by the Intramural Research Program of NIH NCI Division of Cancer Epidemiology and Genetics and in part by the Norris Comprehensive Cancer Center core grant (P30CA014089) from NCI the Trandisciplinary Research in Cancer of the Lung (TRICL) and the Genetic Associations and Mechanisms in Oncology (GAME-ON) Network (U19CA148127). AW ZW WZ and AH were also funded by the NCI NIH (HSN261200800001E). IALO and ZB were also funded by NIH grants (1 R01 HL114094 1 P30 H101258 and R37HL062569-13) Whittier Foundation and Hastings Foundation. ZB was also funded by the Ralph Edgington Chair in Medicine. CNM was funded by ACS/Canary postdoctoral fellowship (FTED-10-207-01-SIED). Author contributions M.T.L conceived the study. I.A.L.O. supervised DNA methylome analysis. J.S. performed EAGLE TCGA and ENCODE genetic analyses. C.M. performed allele-specific binding analyses. J.D. contributed to genetic analyses and performed GO analyses. J.S. P.L. I.A.L.O. and M.T.L. performed quality control analyses. A.C.P D.C. P.A.B A.W.B N.E.C. and M.T.L. conducted the EAGLE study and provided tissue samples. AW and AH prepared the tissue samples for the analyses. BZ and ZB isolated and cultured alveolar epithelial cells. T.T. and K.D.S. performed methylation normalization. Z.W. and W.W. performed LD analyses. J.S. C.M. J.D. P.L.H. M.C. D.S.L J.H. P-H.C B.S.I.C.W.Z. L.A. M.F. B.P.B. N.C M.A.T. S.J. C. I.A.L.O. M.T.L. contributed to the data interpretation. J.S. and M.T.L. wrote the manuscript. All authors participated in the discussion and reviewed the manuscript."

Lung_Cancer

"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 "

Lung_Cancer

"The posttherapy ADC value was higher than pretherapy ADC value (P < 0.001) (). When all tumors were divided into the PR and SD groups according to the mentioned criterion it was noticed that the changes of tumor diameters on T2 images had significant difference between the SD and PR groups (P = 0.007 for longest diameter; P = 0.045 for shortest diameter) (). The pretherapy ADC value had no significant difference between the PR group and SD group (P = 0.517) (). The change of ADC value was statistically significantly higher in PR group compared with that in SD group ( ). The logistic regression model and receiver operator characteristic curve analysis showed that combination of the change of longest diameter and ADC value had a higher area under curve than any other parameter alone for evaluating treatment response in lung cancer (P < 0.01 ). When we used the change of ADC value for differentiating the PR lesion from the SD lesion the best cutoff value was 0.41 — 10?3?mm2/s the overall sensitivity specificity positive predictive value and negative predictive value were 100%0.64710.5714 and 100% respectively and the area under the receiver operator characteristic curve was 0.827. 4. Discussion The chemotherapy response was usually observed after 2 cycles of chemotherapy according to tumor size change by radiographies CT or standard MR rarely by functional imaging dynamic or diffusion weighted MR imaging or PET-CT for example [3621 24]. This study investigated whether the change of ADC value and diameters after chemotherapy could be used to evaluate early treatment response in lung cancer. The ADC value had significant increase after 1 cycle of chemotherapy compared with baseline especially in the PR group (). This result was in agreement with the results of previous studies of both lung cancer and other cancers [17 24“27]. However the current result contradicted with the result of rectal cancer research on the decrease trend of ADC values 2“4 weeks after chemotherapy. Chemotherapy-induced fibrosis might be a contributor to decrease of ADC value [28]. The difference was probably caused by disparity in fibrosis appearance and progression. The increase of ADC value was related to necrosis and reduced cell density histologically [29] while the decrease of ADC value was relevant to cytotoxic edema and fibrosis on histology [28]. From the current results the changes of ADC value were significant between the PR and SD groups () which was in agreement with the previous DWI study of lung cancer treatment response evaluation after chemotherapy [24 26] even when a different b value was used in the previous study [27]. Therefore the noninvasive DWI could be potentially used to early predict and monitor lung cancer response to chemotherapy (). This study further demonstrated that the combination of longest diameter and ADC value change had a higher diagnostic ability than any other parameter alone for evaluating treatment response in lung cancer (P < 0.01 ). The receiver operator characteristic curve showed that the combination of longest diameter change and ADC value change adds additional value for a single parameter alone to predict treatment response. The cutoff value of ADC change could predict response to chemotherapy in lung cancer with 100% sensitivity 64.71% specificity 57.14% positive predictive value 100% negative predictive value and 82.7% accuracy. Comparing with CT the MR imaging had two benefits to evaluate tumor response: first the DWI had the potential to evaluate early treatment response from the tumor inner structure change before the morphological change; second by combining the change of ADC value and tumor diameter the treatment response could be predicted with a high sensitivity and moderate specificity by using MR imaging only. For the small cell lung cancer the tumor diameter change may be significant even in the early stage of chemotherapy. But for the less sensitive non-small cell lung cancer especially on target therapy there may not be apparent anatomical changes initially even if the chemotherapeutic regime was appropriate [29]. Therefore tumor size evaluation alone had a smaller area under the curve than the combination of functional and anatomical assessment. The change of ADC value might have the potential to monitor and early predict lung cancer treatment response to chemotherapy. Furthermore the diagnostic ability increased when combined with the change of ADC value and longest diameter. In our study MR images at baseline and the end of 1 cycle of chemotherapy not only showed the tumor volume change on T2-weighted images but also provided the change of ADC value on DWI. Even through MR imaging could not be regarded as the most appropriate and a œone stop examination method to predict treatment response; it indeed provided precious information to CT. There were several limitations of this study. Firstly the sample number in our study was relatively small especially the pathological subtypes. Secondly the tumor volume pathologic types and chemotherapy regimens were nonuniform which may affect the treatment response of tumor. At last the interval between the start of chemotherapy and treatment response evaluation by DWI was relatively long. One week interval may make the advantage of ADC value change prominent. 5. Conclusions Our data suggested that 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. Acknowledgments The authors thank Rongchao Zhou for collecting part of patients. They appreciate the statistical suggestion from Ningnannan Zhang. This study was supported by Tianjin Medical University Graduate Innovation Fund. Conflict of Interests The authors declared that there was no conflict of interests regarding the publication of this paper. 1 Patz EF Jr. Pinsky P Gatsonis C Overdiagnosis in low-dose computed tomography screening for lung cancer JAMA Internal Medicine 2013 2013 6 pages 12738 2 Smith SM Campbell NC MacLeod U Factors contributing to the time taken to consult with symptoms of lung cancer: a cross-sectional study Thorax 2009 64 6 523 531 2-s2.0-67249109028 19052045 3 Gazala S Pelletier JS Storie D Johnson JA Kutsogiannis DJ Bedard EL A systematic review and meta-analysis to assess patient-peported putcomes after lung cancer surgery Scientific World Journal 2013 2013 789625 4 Sokucu SN Kocaturk C Urer N Evaluation of six patients with pulmonary carcinosarcoma with a literature review Scientific World Journal 2012 2012 167317 5 Pignon J-P Tribodet H Scagliotti GV Lung adjuvant cisplatin evaluation: a pooled analysis by the LACE collaborative group Journal of Clinical Oncology"

Lung_Cancer

"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 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). Apoptosis of NSCLC A549 cells after ionizing radiation (IR). Apoptosis in anti-miR-21- (or anti-miR-NC-) transfected A549 cells combined with (or without) IR (8.0?Gy) was detected through annexin V-FITC/PI staining by flow cytometric analysis. The data represent the means ± SD of three separate experiments. Student's t-test was used to analyze the statistics (*P < 0.05). Suppression of ionizing radiation-induced phosphorylated-Akt (p-Akt) upregulation by anti-miR-21 in NSCLC A549 cells. The expression level of the phosphorylated or total Akt in A549 cells transfected with anti-miR-21 or anti-miR-NC for 48?h followed by ionizing radiation (8?Gy) with or without 10?ng/mL PI3K constituent activator IGF-1 was measured by Western blot. Representative of three independent experiments was shown. Knockdown of miR-21 promoted NSCLC A549 cells apoptosis via inactivation of PI3K-Akt pathway. Apoptosis induced by 8?Gy ionizing radiation in anti-miR-21- (or anti-miR-NC-) transfected A549 cells combined with (or without) PI3K activator IGF-1 treatment (10?ng/mL) was detected through annexin V-FITC/PI staining by flow cytometric analysis. The data represent the means ± SD of three separate experiments. Student's t-test was used to analyze the statistics (*P < 0.05). 1306016 3069 Clin Radiol Clin Radiol Clinical radiology 0009-9260 1365-229X 24857677 4105980 10.1016/j.crad.2014.03.020 NIHMS587373 Revisiting the relationship between tumour volume and diameter in advanced NSCLC patients: An exercise to maximize the utility of each measure to assess response to therapy Nishino M. a * Jackman D.M. b DiPiro P.J. a Hatabu H. a Jnne P.A. b Johnson B.E. b aDepartment of Radiology Dana-Farber Cancer Institute and Brigham and Women™s Hospital 450 Brookline Ave. 75 Francis St. Boston MA 02215 USA bDepartment of Medical Oncology and Department of Medicine Dana-Farber Cancer Institute and Brigham and Women™s Hospital 450 Brookline Ave. Boston MA 02215 USA *Guarantor and correspondent: M. Nishino Department of Radiology Dana-Farber Cancer Institute and Brigham and Women™s Hospital 450 Brookline Ave. Boston MA 02215 USA. Tel.: +1 617 582 7163; fax: +1 617 582 8574. Mizuki_Nishinodfci.harvard.edu (M. Nishino) 30 5 2014 22 5 2014 8 2014 01 8 2015 69 8 841 848 2014 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved. 2014 AIM To revisit the presumed relationship between tumour diameter and volume in advanced non-small-cell lung cancer (NSCLC) patients and determine whether the measured volume using volume-analysis software and its proportional changes during therapy matches with the calculated volume obtained from the presumed relationship and results in concordant response assessment. MATERIALS AND METHODS Twenty-three patients with stage IIIB/IV NSCLC with a total of 53 measurable lung lesions treated in a phase II trial of erlotinib were studied with institutional review board approval. Tumour volume and diameter were measured at baseline and at the first follow-up computed tomography (CT) examination using volume-analysis software. Using the measured diameter (2r) and the equation calculated volume was obtained as (4/3) ?r3 at baseline and at the follow-up. Percent volume change was obtained by comparing to baseline for measured and calculated volumes and response assessment was assigned. RESULTS The measured volume was significantly smaller than the calculated volume at baseline (median 11488.9 mm3 versus 17148.6 mm3; p < 0.0001) with a concordance correlation coefficient (CCC) of 0.7022. At follow-up the measured volume was once again significantly smaller than the calculated volume (median 6573.5 mm3 versus 9198.1 mm3; p = 0.0022) with a CCC of 0.7408. Response assessment by calculated versus measured volume changes had only moderate agreement (weighted ? = 0.545) with discordant assessment results in 20% (8/40) of lesions. Calculated volume based on the presumed relationship significantly differed from the measured volume in advanced NSCLC patients with only moderate concordance in response assessment indicating the limitations of presumed relationship. CMAJ CMAJ 9711805 CMAJ : Canadian Medical Association Journal 0820-3946 1488-2329 Canadian Medical Association 24638027 4016095 10.1503/cmaj.131385 186e296 Practice Five Things to Know About... Screening for lung cancer Kennedy Sean Baerlocher Mark Otto MD School of Medicine (Kennedy) McMaster University Hamilton Ont.; Department of Radiology (Baerlocher) Royal Victoria Hospital Barrie Ont. Correspondence to: Sean Kennedy sean.kennedymedportal.ca 13 5 2014 13 5 2015 186 8 E296 E296 1995-2014 Canadian Medical Association 2014 0042124 4284 Int J Cancer Int. J. Cancer International journal of cancer. Journal international du cancer 0020-7136 1097-0215 24806617 4200482 10.1002/ijc.28958 NIHMS594253 p38 MAPK inhibits breast cancer metastasis through regulation of stromal expansion Hong Bangxing 1 Li Haiyan 1 Zhang Mingjun 1 Xu Jingda 2 Lu Yong 1 Zheng Yuhuan 1 Qian Jianfei 1 Chang Jeffrey T 3 Yang Jing 2 Yi Qing 1 1Department of Cancer Biology Lerner Research Institute Cleveland Clinic Cleveland OH USA 2Department of Lymphoma and Myeloma University of Texas MD Anderson Cancer Center Houston TX USA 3Department of Integrative Biology & Pharmacology University of Texas at Houston Houston TX USA Corresponding author: Qing Yi MD PhD Department of Cancer Biology Cleveland Clinic Lerner Research Institute 9500 Euclid Avenue/NB40 Cleveland OH 44195 USA. Tel: 216-636-7532; Fax: 216-444-3164; yiqccf. 31 5 2014 16 5 2014 1 1 2015 01 1 2016 136 1 34 43 p38 MAPK signaling controls cell growth proliferation and the cell cycle under stress conditions. However the function of p38 activation in tumor metastasis is still not well understood. We report that p38 activation in breast cancer cells inhibits tumor metastasis but does not substantially modulate primary tumor growth. Stable p38 knockdown in breast cancer cells suppressed NF-?B p65 activation inhibiting miR-365 expression and resulting in increased IL-6 secretion. The inhibitory effect of p38 signaling on metastasis was mediated by suppression of mesenchymal stem cell (MSC) migration to the primary tumor and sites of metastasis where MSCs can differentiate into cancer-associated fibroblasts to promote tumor metastasis. The migration of MSCs to these sites relies on CXCR4-SDF1 signaling in the tumor microenvironment. Analysis of human primary and metastatic breast cancer tumors showed that p38 activation was inversely associated with IL-6 and vimentin expression. This study suggests that combination analysis of p38 MAPK and IL-6 signaling in patients with breast cancer may improve prognosis and treatment of metastatic breast cancer. p38 MAPK Breast cancer Metastasis microRNA IL-6 J Mol Diagn J Mol Diagn The Journal of Molecular Diagnostics : JMD 1525-1578 1943-7811 American Society for Investigative Pathology 24813172 4078366 S1525-1578(14)00074-9 10.1016/j.jmoldx.2014.03.006 Regular Detection of Gene Rearrangements in Targeted Clinical Next-Generation Sequencing Abel Haley J. ? Al-Kateb Hussam   Cottrell Catherine E.   Bredemeyer Andrew J.   Pritchard Colin C. ¡ Grossmann Allie H. § Wallander Michelle L. ¶ Pfeifer John D.   Lockwood Christina M.   Duncavage Eric J. eduncavagepath.wustl.edu   ? ?Department of Genetics Washington University St. Louis Missouri  Department of Pathology and Immunology Washington University St. Louis Missouri ¡Department of Laboratory Medicine University of Washington Seattle Washington §Department of Pathology University of Utah and ARUP Laboratories Salt Lake City Utah ¶ARUP Institute for Clinical and Experimental Pathology Salt Lake City Utah ?Address correspondence to Eric J. Duncavage M.D. Department of Pathology and Immunology"

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). She J Yang P Hong Q Bai C Lung cancer in china: challenges and interventions Chest 2013 143 4 1117 1126 23546484 Lu Q Lu S Huang L Wang T Wan Y Zhou C Zhang C Zhang Z Li X The expression of V-ATPase is associated with drug resistance and pathology of non-small-cell lung cancer Diagn Pathol 2013 8 145 23984887 Pfannschmidt J Muley T Bülzebruck H Hoffmann H Dienemann H Prognostic assessment after surgical resection for non-small cell lung cancer: experiences in 2083 patients Lung Cancer 2007 55 3 371 17123661 Goya T Asamura H Yoshimura H Kato H Shimokata K Tsuchiya R Sohara Y Miya T Miyaoka E Prognosis of 6644 resected non-small cell lung cancers in Japan: a Japanese lung cancer registry study Lung Cancer 2005 50 2 227 234 16061304 Christiansen A Detmar M Lymphangiogenesis and cancer Genes Cancer 2011 2 12 1146 1158 22866206 El-Gohary YM Metwally G Saad RS Robinson MJ Mesko T Poppiti RJ Prognostic significance of intratumoral and peritumoral lymphatic density and blood vessel density in invasive breast carcinomas Am J Clin Pathol 2008 129 4 578 586 18343785 Karaday? 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

"630±60?nm band pass filter) and green (?excitation: 470±40?nm band pass filter ?detection: 535±50?nm band pass filter) fluorescence channels. Flow cytometric analysis was assayed with the JC-1 Mitochondrial Membrane Potential Kit (AAT Bioquest Sunnyvale CA USA) according to the manufacturer's directions using a FACSCalibur and the results were analyzed by CellQuest software. In vitro casapse-9 activity determination Caspase-9 activity was measured by a fluorometric assay in whole-cell lysates using Ac-Leu-Glu-His-Asp-MCA substrate (Peptide International Inc. Louisville KY USA). A549 cell extracts were mixed with Ac-LEHD-MCA in ICE standard buffer (100?mM HEPES pH 7.5 10% sucrose 0.1% CHAPS 10?mM DTT 1?mM PMSF) and cleavage of the fluorogenic peptide substrate was monitored at 37?°C for 30?min by a SPECTRA max GEMINI EM (Molecular Device Sunnyvale CA USA) fluorometer with excitation at 370?nm and emission at 460?nm. This work was supported in part by a Grant-in-Aid (23591477) from the Ministry of Education Culture Sports Science and Technology of Japan. Apaf-1 apoptotic protease-activating factor 1 COX cyclooxygenase HSF-1 heat shock factor 1 MTT 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide NSAID nonsteroidal anti-inflammatory drug NSCLC non-small cell lung cancer PCR polymerase chain reaction RNAi RNA interference RT reverse transcriptase TUNEL terminal deoxynucleotidyl transferase-mediated dUTP nick and labelling Edited by G Raschell  The authors declare no conflict of interest. Tavaria M Gabriele T Kola I Anderson RL A hitchhiker's guide to the human Hsp70 family Cell Stress Chaperones 1996 1 23 28 9222585 Jaattela M Escaping cell death: survival proteins in cancer Exp Cell Res 1999 248 30 43 10094811 Aghdassi A Phillips P Dudeja V Dhaulakhandi D Sharif R Dawra R Heat shock protein 70 increases tumorigenicity and inhibits apoptosis in pancreas adenocarcinoma Cancer Res 2007 67 616 625 17234771 Ciocca DR Clark GM Tandon AK Fuqua SA Welch WJ McGuire WL Heat shock protein hsp70 in patients with axillary lymph node-negative breast cancer J Natl Cancer Inst 1993 85 570 574 8455204 Cornford PA Dodson AR Parsons KF Desmond AD Woolfenden A Fordham M Heat shock protein expression independently predicts clinical outcome in prostate cancer Cancer Res 2000 60 7099 7105 11156417 Vargas-Roig LM Gago FE Tello O Aznar JC Ciocca DR Heat shock protein expression and drug resistance in breast cancer patients treated with induction chemotherapy Int J Cancer 1998 79 468 475 9761114 Igney FH Krammer PH Death and anti-death: tumor resistance to apoptosis"

Lung_Cancer

"The presence of 3R/3R polymorphism seemed to predict a higher ORR (100%) compared to the rest of the genotypes with a trend toward statistical significance (p =?0.055). In the subgroup analysis a significantly higher ORR to pemetrexed for wild-type EGFR patients showing a 3R/3R genotype (100%) compared to the 2R/2R (77.8%) 2R/3R (33.3%) and 3R/4R (0%) was observed (p =?0.017). Overall response rate to the treatment and polymorphisms observed Global distribution of polymorphisms (Pol) Response N (%) Stabilization or progression N (%) p value VNTR 2R/2R 7 (77.8) 2 (22.2) 0.055 3R/3R 7 (100) 0 (0) 2R/3R 4 (50) 4 (50) 3R/4R 0 (0) 1 (100) Pol VNTR (Subanalysis by EGFR status; group of native EGFR-patients) 2R/2R 7 (77.8) 2 (22.2) 0.017 3R/3R 7 (100) 0 (0) 2R/3R 2 (33.3) 4 (66.7) 3R/4R 0 (0) 1 (100) Global distribution of SNP Absence 6 (85.7) 1 (14.3) 0.626 Presence 12 (66.7) 6 (33.3) Global distribution of polymorphisms in 3?-UTR +6/+6 10 (83.3) 2 (16.7) 0.234 +6/-6 6 (54.5) 5 (45.5) -6/-6 2 (100) 0 (0) Pol 3?-UTR (Subanalysis by smoking habit stratification; group of active and former smokers) +6/+6 8 (100) 0 (0) 0.085 +6/-6 4 (50) 4 (50) -6/-6 2 (100) 0 (0) No statistically significant differences were observed comparing the presence and the absence of a SNP G >?C as shown in . Overall a non-significant correlation between the different 3?-UTR polymorphisms and the ORR was observed. However the genotype +6/+6 seemed to predict a higher ORR among active/former smokers (A/FS) compared to +6/-6 (100% vs. 50%; p =?0.085). Correlation between PFS and polymorphisms Regarding TSER polymorphisms we found a trend toward statistical significance (p =?0.089) in the differences in PFS observed among the different genotypes in favor of the 3R/3R genotype (A). Kaplan-Meier curves for progression-free survival (PFS) in months (mo) associated with the different TS polymorphisms. A: TSER genotypes. B: Presence or absence of SNP. C: 3´UTR genotypes. In the case of the absence or presence of a SNP at the third repetition (3R allele) we observed a non-significant increased PFS in the subgroup of patients showing an absence of SNP (B). Finally no significant correlations regarding the 3?UTR genotypes and PFS were observed (C). Correlation between OS and polymorphisms In this cohort we found a significant correlation between TSER polymorphisms and OS (A). The median OS was not reached for 3R/3R genotype patients whereas 2R/3R genotype subjects showed a 70 m OS followed by 3R/4R and 2R/2R genotypes with a median OS of 15 m and 13 m respectively (p =?0.019) (A). Kaplan-Meier curves for overall survival (OS) in months (mo) associated with the different TS polymorphisms. A: TSER genotypes. B: Presence or absence of SNP. C: 3´UTR genotypes. No significant differences in OS were observed with regards to the presence/absence of SNP (B) or regarding the 3?-UTR polymorphisms (C). Correlation between toxicity and polymorphisms The most frequent toxicity was grade (G)1 anemia (28%) and nausea (20%) and G2 leucopenia (40%). The most commom G3-4 toxicities were leucopenia (16%) asthenia (8%) anemia (4%) neutropenia (4%) and dyspnea (4%). Overall we found no significant correlations between the toxicity profiles experienced by the patients and the different TS genotypes (). Correlation between grades of toxicity and different genotypes Global distribution polymorphisms (Pol) No toxicity Grade 1-2 Grade 3-4 p value VNTR polymorphisms 2R/2R 2 (22.2) 4 (44.2) 3 (33.4) 0.545 3R/3R 2 (25) 5 (75) 0 (0) 2R/3R 2 (25) 4 (50) 2 (25) 3R/4R 1 (100) 0 (0) 0 (0) SNP polymorphisms Absence 3 (42.9) 4 (57.1) 0 (0) 0.3 Presence 4 (22.2) 9 (50) 5 (27.8) 3?-UTR polymorphisms +6/+6 3 (25) 6 (50) 3 (25) > 0.05 +6/-6 3 (27.3) 6 (54.5) 2 (18.2) -6/-6 1 (50) 1 (50) 0 (0) Discussion Pemetrexed a multitargeted antifolate drug is essential for the first and second-line as well as maintenance treatment of NSCLC patients with non-squamous histology [6]. TS is the main biological target of pemetrexed. Some studies have suggested that TS expression could be a predictive factor of response in NSCLC [19]. Moreover some VNTR genotypes have been associated with TS expression and activity in other tumor types such as colorectal cancer [17]. In NSCLC patients a correlation between different genotypes and the TS protein expression has been shown [20]. Shintani et al. [20] also confirmed that the TS mRNA levels were significantly higher in lung cancer tissues with the 3R/3R genotype as compared to those with the 2R/2R genotype. Nonetheless definitive studies addressing the correlation of the different genotypes of TS in circulating genomic DNA with response to the treatment PFS or OS in pemetrexed-treated NSCLC European patients are lacking. The potential influence of the EGFR status on those polymorphisms and their correlation with clinical outcome after pemetrexed-based treatment is also unexplored. A recent study by Hu et al. [21] investigated the different TS polymorphisms in genomic DNA of 90 Asian NSCLC patients. In contrast with our findings no specific genotype regarding the TSER or 3?-UTR polymorphisms studied seemed to correlate with a significant difference in ORR PFS or OS. This could be explained by substantial clinical differences between both populations. Our cohort was constituted by Caucasian patients compared to the Asian population studied by Hu et al. In addition our patients were mostly current or former smokers (72%) compared to the Asian population that showed 62% of never smokers. Also in our cohort the subjects mainly received pemetrexed-based chemotherapy as a first line regimen (92%) whereas the cohort studied by Hu et al. [21] was treated with pemetrexed as a second or further line in 62.2% of the cases. These remarkable differences in basic clinical characteristics and in particular the ethnicity between both cohorts are probably also explaining the differences observed in the 3?-UTR genotype frequency between our population and the one studied by Hu et al. In our cohort +6 bp/+6 bp +6 bp/-6 bp and -6 bp/-6 bp genotypes were found in 48% 44% and 8% of the cases respectively. In contrast0.078 47.8% and 44.4% were respectively found in the population studied by Hu et al. [21]. In a previous analysis performed on another Caucasian NSCLC population evaluated at the M.D. Anderson Cancer Center [22] a similar proportion of 3?-UTR genotypes according to our findings was observed (49.2% of +6 bp/+6 bp 42.4% of +6 bp/-6 bp and 8.4% of -6 bp/-6 bp). Additionally the low prevalence of the +6 bp/+6 bp genotype in an Asian population compared to our cohort may be confirmed by a recent study in which from 106 Asian NSCLC patients investigated none of them showed a +6 bp/+6 bp genotype in genomic circulating DNA [23]. Nontheless in this latter study [23] a significantly higher ORR was observed among patients showing a -6 bp/-6 bp 3?-UTR genotype compared to the ORR reported for patients presenting a -6 bp/+6 bp polymorphism (32.2% vs 12.7%; p =?0.008). Accordingly in our cohort a higher ORR in patients showing a -6 bp/-6 bp genotype compared to those presenting a -6 bp/+6 bp polymorphism was also observed (100% vs. 54.5%). However the statistical significance was not reached probably due to the relatively low number of patients included in our analysis. Interestingly enough in the subgroup analysis of our data the +6 bp/+6 bp genotype seemed to predict a higher ORR only among active/former smokers compared to +6 bp/-6 bp (100% vs. 50%; p =?0.085). This novel observation if validated in future studies could be relevant for selecting specific drugs for each patient in a second or third line setting. With regards to the TSER polymorphisms the presence of a 3R/3R polymorphism seemed to predict a higher ORR with a clear trend toward statistical significance (p =?0.055). Moreover that difference was even greater and statistically significant benefiting the subpopulation of wild-type EGFR patients. To our knowledge this is the first time that such observation has been made. An interesting preclinical study by Giovannetti et al. [24] investigated the activity profile of a combination therapy against NSCLC cell lines with different genotypes with erlotinib and pemetrexed. Remarkably pemetrexed increased EGFR phosphorylation and reduced Akt phosphorylation. Additionally erlotinib significantly reduced TS expression and activity. Thus when erlotinib and pemetrexed were combined a strong synergism in all NSCLC cells regardless of their genetic signature was observed. This potential crosstalk between the EGFR signaling pathway and the TS expression and activity could in part explain our novel findings showing a significantly higher ORR to pemetrexed in those wild-type EGFR patients harboring a 3R/3R polymorphism."

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

"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

"Twenty-one days after cell sorting peptide specificity was assessed using the IFN-? ELISPOT assay (Fig. 3B). The established clone reacted to the T2 cells pulsed with peptide A but not to T2 cells pulsed with the irrelevant HIV-gag peptide. These results indicate that a peptide A-specific CTL clone was successfully established from PBMCs from a healthy donor. The EML4-ALK-specific CTL clone recognizes HLA-A*02:01+ lung carcinoma cells with the EML4-ALK variant 3a/b incubated with IFN-? We next evaluated the ability of the EML4-ALK-specific CTL clone to recognize the cancer cell line H2228 which expresses HLA-A*02:01 and EML4-ALK using the IFN-? ELISPOT assay. Even though the EML4-ALK-specific CTL clone failed to recognize H2228 cells it did recognize those pretreated with 100 U/ml IFN-? 48 h prior to examination (Fig. 4A). We examined the effect of IFN-? on H2228 cells. Incubating target cells with IFN-? for 48 h increased the expression of MHC class I molecules on the cell surface (Fig. 4B). This result indicates that the peptide A-specific CTL clone was able to recognize H2228 cells because of increased expression of MHC-class I on the H2228 cell surface. Specific IFN-? production by the peptide A-specific CTL clone was detectable in H2228 cells treated with IFN-?. The specificity was abolished by an anti-HLA-class I mAb but not by an isotype control suggesting that the observed production was HLA-A2 restricted (Fig. 4C). A cytotoxicity assay was also performed. The peptide A-specific CTL clone was able to specifically lyse H2228 cells pretreated with IFN-? 48 h prior to examination. This specific lysis was blocked by the anti-HLA-class I mAb but not by the isotype control. These results indicate that the peptide A-specific CTL clone showed cytotoxicity and the ability to produce IFN-? against HLA-A*02:01+ EML4-ALK+ NSCLC cell lines (Fig. 5). Discussion In the present study we identified a new tumor-associated CTL epitope (peptide A) derived from EML4-ALK which binds to HLA-A*02:01 molecules and we were able to establish a peptide-specific CTL clone from human PBMCs that specifically recognized cognate peptide-pulsed T2 cells and HLA-A*02:01 tumor cells expressing EML4-ALK that had been pretreated with IFN-?. EML4-ALK-positive lung cancers are highly sensitive to ALK inhibition. However as with trastuzumab or gefitinib (3031) patients typically gain resistance within 1 to 2 years of starting therapy (23). We aimed to overcome these difficulties with immunotherapy. We identified a glypican-3 (GPC3)-derived peptide and showed that GPC3-specific CTL frequency after vaccination correlated with OS. OS was significantly longer in patients with high GPC3-specific CTL frequencies than in those with low frequencies (32). This indicates that the ability to induce a peptide-specific CTL clone is important for effective immunotherapy. We also revealed that GPC3 is an ideal target for anticancer immunotherapy since it is specifically overexpressed in hepatocellular carcinoma (HCC) (33“35). In the present study we chose a peptide array from EML4-ALK from which we were able to induce a peptide-specific CTL clone. EML4-ALK is a strong oncogene overexpressed in cancer cells of NSCLC breast cancer kidney cancer and colon cancer (17). We performed RT-PCR and assayed the EML4 DNA levels of certain lung cancer cell lines. H2228 cells express EML4 moderately but at higher levels than other lung cancer cell lines. EML4 expression has been reported as highly expressed in CD8+ T cells. RT-PCR showed that EML4 DNA levels were high in PBMCs and CD8+ T cells. Because of a lack of suitable antibodies we could not perform western blotting. However our success at inducing a peptide A-specific CTL clone from CD8+ T cells indicated that the CTL clone had no cytotoxicity against CD8+ T cells. This CTL clone could not recognize cancer cell lines without the ability to increase the amount of HLA class I presented on cell surfaces. Further examination is needed to achieve higher tumor reactivity. Combination chemotherapy or radiation therapy plus immunotherapy was recently reported to have a synergistic effect (36). Moreover some mechanisms of synergy between radiation therapy chemotherapy and immunotherapy have been revealed (37). In one of the mechanisms these therapies upregulated tumor antigens and MHC moieties. These results suggest that combination therapy could be used to make tumor cell lines more susceptible to this peptide A-specific CTL clone-mediated cytolysis (38“41). In addition this treatment may be able to overcome resistance to ALK inhibition. Some resistance mechanisms for targeting drugs have been examined. The most commonly identified causes of resistance are point mutations such as L1196M (42“44) G1269A (22) and S1206Y (21). "

Lung_Cancer

"detection and quantification of cancer DNA molecules from peripheral blood. Using this technique we identified mutant PIK3CA DNA in circulating plasma tumor DNA (ptDNA) from a patient with concurrent early stage breast cancer and non-small cell lung cancer. The patient underwent successful resection of both her breast and lung cancers and using standard Sanger sequencing the breast cancer was shown to harbor the identical PIK3CA mutation identified in peripheral blood. This case report highlights potential applications and concerns that can arise with the use of ptDNA in clinical oncology practice. plasma tumor DNA breast cancer lung cancer PIK3CA digital PCR Br J Cancer Br. J. Cancer British Journal of Cancer 0007-0920 1532-1827 Nature Publishing Group 24983368 4102953 bjc2014353 10.1038/bjc.2014.353 Genetics and Genomics Assessing standardization of molecular testing for non-small-cell lung cancer: results of a worldwide external quality assessment (EQA) scheme for EGFR mutation testing Worldwide external quality assessment for EGFR gene mutation testing Patton S 1 * Normanno N 2 Blackhall F 3 Murray S 4 Kerr K M 5 Dietel M 6 Filipits M 7 Benlloch S 8 Popat S 9 Stahel R 10 Thunnissen E 11 1EMQN Manchester Centre for Genomic Medicine St Mary's Hospital Manchester M13 9WL UK 2Cell Biology and Biotherapy Unit Istituto Nazionale per lo Studio e la Cura dei Tumori ˜Fondazione Giovanni Pascale'”IRCCS 80131 Naples Italy 3Christie Hospital Manchester M20 4BX UK 4Biomarker Solutions Ltd London EC1V 2NX UK 5Department of Pathology Aberdeen Royal Infirmary Aberdeen AB25 2ZN UK 6Charit Humboldt-Universitt zu Berlin Berlin 10117 Germany 7Medical University of Vienna 1010 Vienna Austria 8Pangaea Biotech USP Dexeus University Institute Barcelona 08028 Spain 9Royal Marsden Hospital London SW3 6JJ UK 10University Hospital Z¼rich CH-8091 Z¼rich Switzerland 11Department of Pathology VU University Medical Center Amsterdam 1081 HZ The Netherlands *E-mail: simon.pattoncmft.nhs.uk 15 07 2014 01 07 2014 15 7 2014 111 2 413 420 22 01 2014 19 05 2014 20 05 2014 Copyright 2014 Cancer Research UK 2014 Cancer Research UK 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: The external quality assurance (EQA) process aims at establishing laboratory performance levels. Leading European groups in the fields of EQA Pathology and Medical and Thoracic Oncology collaborated in a pilot EQA scheme for somatic epidermal growth factor receptor (EGFR) gene mutational analysis in non-small-cell lung cancer (NSCLC). Methods: EQA samples generated from cell lines mimicking clinical samples were provided to participating laboratories each with a mock clinical case. Participating laboratories performed the analysis using their usual method(s). Anonymous results were assessed and made available to all participants. Two subsequent EQA rounds followed the pilot scheme. Results: One hundred and seventeen labs from 30 countries registered and 91 returned results. Sanger sequencing and a commercial kit were the main methodologies used. The standard of genotyping was suboptimal with a significant number of genotyping errors made. Only 72 out of 91 (72%) participants passed the EQA. False-negative and -positive results were the main sources of error. The quality of reports submitted was acceptable; most were clear concise and easy to read. However some participants reported the genotyping result in the absence of any interpretation and many obscured the interpretation required for clinical care. Conclusions: Even in clinical laboratories the technical performance of genotyping in EGFR mutation testing for NSCLC can be improved evident from a high level of diagnostic errors. Robust EQA can contribute to global optimisation of EGFR testing for NSCLC patients. non-small-cell lung carcinoma EGFR gene mutations quality assessment Assessment of epidermal growth factor receptor (EGFR) mutations has become mandatory to choose the most active first-line treatment for patients with advanced non-small-cell lung cancer (NSCLC). Indeed randomized phase III clinical trials have demonstrated that first-line administration of an EGFR TKI results in a prolonged progression-free survival as compared with chemotherapy in patients carrying EGFR mutations (Mok et al 2009; Maemondo et al 2010; Mitsudomi et al 2010; Fukuoka et al 2011; Zhou et al 2011; Rosell et al 2012). These studies have also confirmed that EGFR mutations are a reliable marker that predicts sensitivity to EGFR TKIs (Mok et al 2009). Activating mutations occur in exons 18 through 21 of the TK domain of the EGFR gene and either point mutations or in-frame small deletions or insertions (Sharma et al 2007; De Luca and Normanno 2010). Although more than 250 mutations of the EGFR gene have been described to date two mutations a single point mutation in exon 21 the L858R and a series of small in-frame deletions in exon 19 account for ?90% of all EGFR mutations (Sharma et al 2007; Linardou et al 2008). EGFR mutations are strongly associated with defined clinical and pathological features: they are far more frequent in female patients as compared with male; in adenocarcinoma as compared with other histological types; in non-smokers as compared with current smokers or former smokers; and in East-Asian NSCLC patients as compared with Non-East-Asian patients (Normanno et al 2006). External quality assessment (EQA) is a system of objectively checking laboratory results by an independent external agency (van Krieken et al 2013). The main objective of an EQA programme is to establish inter-laboratory comparability. In this respect the EQA process can identify latent systematic errors in methodology that may not be revealed by a laboratory's own internal QA processes. Representatives from ETOP ESMO ESP EMQN and other leading European groups met in July 2010 to discuss a pan-European approach to EQA for EGFR mutation testing in NSCLC. In this paper we present the results of this pilot EQA scheme for EGFR testing that was completed in 2013. Materials and Methods anisation of the scheme A meeting was anised in July 2010 by ETOP and EMQN to bring together a group of professionals representing EMQN ESP ETOP ESMO and other leading European groups involved in NSCLC testing (see Supplementary Information). From this group a steering group of five individuals was formed who planned designed and assessed the results of the pilot EQA scheme. The scheme was coordinated and administered by the EMQN and three rounds were anised within a period of 18 months. The workflow of the scheme process is shown in . 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 (Table 1). 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 (Table 2). 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 (Table 2; 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 (Table 3). 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 (Table 2; 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%) (Figure 2). 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. As this was designed to ascertain proof of principle we did not apply a measure of successful laboratory performance. The pilot established that the scheme design and methods used were acceptable for use in a larger scheme. Second round One hundred and seventeen laboratories from 30 countries registered and 101 participated in the second round (due to customs issues we were not able to get samples to 16 labs) run in the second quarter of 2012. Ninety-one laboratories submitted results within the 8-week time frame “ the remaining 10 labs gave no reason why they did not submit results. A different set of samples from those used in the pilot first round were sent to the laboratories with the emphasis being on the inclusion of mutations at allelic frequencies that would challenge the analytical sensitivity of all the commonly used technologies (Table 2; samples B1“B10). A code number different from the one assigned in the first round was given to the samples. In addition to the genotype results all participating laboratories were also required to submit for assessment copies of their clinical reports for three samples (B1 B4 and B9). The main methodology used by the participants was PCR/sequencing (n=35 laboratories; 39%) and real-time PCR (n=17; 18.6% Figure 2). It was common for labs to use a combination of different methodologies in their testing process (Table 4). A variety of different errors were detected by the second scheme round including 74 (8.1%) genotype errors (false-positive (n=13; 1.5%) false-negative (n=61; 82.4%) and a combination of false-negative and -positive results (n=1; 1.4%)) as well as analytical test failures (n=31; 3.4%) mispositioning of the genotype (n=7; 0.8%) and significant errors in the mutation nomenclature (n=36; 3.9%). Two samples (B2 and B8) gave a disproportionately high error rate compared with the other samples used in this round (Table 5) with 94.1% of errors for B2 made by labs using PCR/sequencing vs 40.7% of errors for sample B8 made by labs using a version of the Therascreen EGFR kit (Qiagen). Laboratories did not lose marks if the declared limitations of their assay meant that they would not detect a particular mutation at the given frequency used in the EQA materials. Eighteen laboratories (19.8%) from 13 countries with a total score below 18 did not pass the second round and were thus classified as poor performers “ 72.2% of these labs used PCR/Sequencing as their main diagnostic test for EGFR mutation status. The interpretation of the test result relative to the clinical referral was reviewed with laboratories receiving comments on their performance but no marks assigned. Overall 46 (50.5%) of the laboratories had a score ?18 in the second round and passed the EQA. All laboratories received a certificate of participation that displayed their performance in the scheme. Third round The 18 laboratories that did not pass the second round were given the opportunity to participate in a third round. One laboratory was unable to participate due to problems with customs sample import permissions. The same set of samples used in the "

Lung_Cancer

"The DNA repair hub module is linked to a module associated with response to DNA damage. It contains five genes: CTF4 ESC4 MMS1 MMS22 and Rt101. The last four genes are part of the cullin-RING ubiquitin ligase complex (GO:0031461). The last three genes were shown to form a complex that stabilizes the replisome during replication stress (4647). The CTF4 gene is related to DNA repair and DNA replication initiation according to its GO annotations. The link suggests that this complex might work together with the DNA repair module for coping with damaged replication forks. Interestingly the two MMS genes were originally detected in MMS sensitivity tests but are not expected to be required for double-stranded repair (47). The RAD52 module (RAD51 RAD52 and RAD59) is related to double-stranded DNA damage repair (48) and is linked both to the DNA damage repair module and to the DNA damage response module suggesting these modules work together in the same pathway as a result of DNA damage to cope both with damaged replication forks and with double-stranded DNA breaks. The fourth linked module contains three genes of the SuperKiller (SKI) complex (SKI2 SKI5 and SKI7). These genes are involved in 3“5 RNA degradation in the cytoplasmatic exosome (4950). Our analysis suggests that this complex might also be involved in response to DNA damage. Previous studies have shown that RNA degradation cytoplasmatic genes might play a role in DNA damage response separately from their cytoplasmatic activity (5152). The suggested roles of RNA degradation genes in DNA damage response include DNA stability and telomere stability related functionality (51) mediating the assembly of multiprotein complexes in double-stranded breaks (52) and specific mRNA degradation on DNA damage (53). Hence our findings match prior studies and strengthen the role of the SKI complex in the response to DNA damage.Analysis of human co-expression and differential correlation networksWe applied ModMap on case-control gene expression data of NSCLC to reveal DC among highly correlated gene modules. The contribution of this part is two fold. First we show that DC among gene modules is reproducible in cross-validation tests. Second we analyze the map of DC patterns between gene modules discovered by ModMap.Given a data set of gene expression profiles from cases and controls we used the method of (24) to compute two scores for each gene pair: the CC score which is positive if the gene pair is consistently correlated across phenotypes and the DC score which is positive if the correlation difference between the cases and controls is higher than expected by chance. These scores were then used as edge weights in networks H and G respectively on which a module map was learned. The methodology was evaluated using cross-validation: given a module map constructed on a set of profiles (the ˜training set™) and a disjoint set of samples (the ˜test set™) the quality of the predicted map was evaluated on the test set by comparing the DC of links and of non-links using Wilcoxon rank-sum test where the null hypothesis is that there is no difference in DC between links and non-links. This measure is parameter-free and reflects all DC changes.We tested several variants of the algorithm using 2-fold cross-validation. The maps produced by the local improver received low P-values but suffered from low coverage. For example for the MBC-DICER initiator the local improver achieved a P-value of 4.43E-4 but the map covered only 197 genes. In contrast when applying ModMap (i.e. MBC-DICER with the global improver) the map covered 1289 genes with P-value of 1.54E-10. Supplementary Text contains further results of testing different parameters of the global improver and tests on Alzheimer™s disease (54) which got similar cross-validation results. The full results are shown in Supplementary Table S11 for lung cancer and in Supplementary Table S12 for Alzehimer's disease. Taken together ModMap produces large maps that are robust when tested on independent data sets.Next we analyzed the module map obtained by running ModMap on all samples of the NSCLC data. The map covered 1921 genes in 76 modules connected by 405 links (see Supplementary Tables S13 and S14 for details). To focus on strong changes in correlation between modules we compared the DC of each link in the map to the DC calculated between random gene sets of the same sizes in 200 repeats and calculated the fold-change between the real link and the best random link as proposed in (24). The link fold-change scores are given in Supplementary Table S14. In all 150 links had fold-change 1.5 with the top five links exceeding 2.3. This indicates that the DC of the linked modules is far stronger than expected by chance. We also analyzed the modules of the top links using pathway enrichment analysis and microRNA enrichment analysis (see Supplementary Table S15 for details). One of the links connected two modules related to immune response activation. The linked modules are shown in . In A we observe many high co-expression edges between the modules (gene pairs with r > 0.4) in the control class. Module 11 is enriched with B-cell receptor signaling pathway genes (6 genes P = 3.1E-8). Module 12 is enriched with T-cell receptor signaling pathway genes (4 genes P = 1.37E-4). B shows GeneMANIA analysis of these 10 genes (755) which confirms that they are connected by several types of interactions. C shows the co-expression of the same modules in the NSCLC class. Within each of the modules a strong level of co-expression is preserved but the co-expression between the modules is abolished suggesting that co-regulation of the different immune responses is lost in NSCLC. Finally module 11 is highly enriched with targets of microRNA 34-a b c family (red nodes in A) whose members are annotated as causal to NSCLC according to the mir-2-disease database (56). Taken together these results show the ability of our analysis to detect NSCLC-related functional modules without using any prior knowledge. .A pair of immune activation-related modules differentially correlated in NSCLC. (A) Two-linked modules which are a part of the constructed module map. Nodes are genes and edges represent correlation >0.4 between the genes in the expression patterns of control class. Edges here correspond to high co-expression between two genes and do not reflect the weights in the CC or DC networks. We observe strong co-expression both within and between the modules. Nodes with black frames are related to immune activation response (six T-cell activation genes in module 11 and four B-cell activation genes in module 12). Red nodes in module 11 are targets of mir-34 family. (B) GeneMANIA analysis of the T-cell and B-cell signaling pathway genes shows that the genes of both modules are expected to interact in healthy controls. (C) The same two modules and their co-expression network in the NSCLC class. As in A the genes within each module are highly co-expressed. In contrast to A co-expression between the modules is completely diminished.DISCUSSIONIn this we presented a methodology for joint analysis of two gene networks each representing a different type of omic relation between genes. The method identifies gene sets as modules and the complex structure of relations among them and summarizes the analysis in a module map. Modules correspond to interacting gene sets in the first network and links in the module map correspond to interacting modules in the second."

Lung_Cancer

"However uncertainty exists in proton-based treatment plans including range uncertainties large sensitivity to position uncertainty and calculation of dose deposition in heterogeneous areas. This study investigated the feasibility of proton transmission beams i.e. without the Bragg peak to treat lung tumors with stereotactic ablative radiotherapy. We compared three representative treatment plans using proton transmission beams versus conformal static-gantry photon beams. It was found that proton treatment plans using transmission beams passing through the patient were feasible and demonstrated lower dose to normal structures and markedly reduced treatment times than photon plans. This is the first study to demonstrate the feasibility of proton-based stereotactic ablative radiotherapy planning for lung tumors using proton transmission beams alone. Further research using this novel approach for proton-based planning is warranted. The authors have no support or funding to report. Introduction Stereotactic ablative radiotherapy (SABR) plays an essential role in the treatment of patients with medically inoperable early stage lung cancer and oligometastasis. The use of protons for lung SABR is emerging as an appealing treatment option because of its potential to deliver higher doses of conformal radiotherapy and spare normal tissues better than traditional photons [1] [2] [3] [4]. This can be achieved because of the natural characteristics of proton beams that deposit its dose at depth with no exit dose referred to as a Bragg peak. However conventional dosimetric models fail to accurately model how protons scatter and deposit dose in highly heterogeneous areas which leads to uncertainties in proton treatment plans [5]. In addition the uncertainties in the stopping power of the various tissues in the body and the interplay effect between spot scanning proton therapy and the target motion leads to large uncertainties in the treatment of lung tumors [5] [6]. In this study we report on the feasibility of proton transmission-beam SABR (PT-SABR) for lung tumors which uses the transmission portion of a spot scanning proton beam i.e. without the Bragg peak. This technique eliminates the major uncertainties of proton therapy mentioned above by having the proton beams pass through the patient. In addition the use of the transmission beam allows an entire field to be treated in one breath hold. This quick treatment and decreased uncertainties lead to smaller planning volumes. To the best of the authors™ knowledge this is the first report on the use of this novel approach to plan SABR with protons without using the Bragg peak which may have dosimetric advantages over photon treatments. Materials and Methods Ethics Statement Written informed consent was obtained from all patients registered in the SABR database. This study including the consent procedure was approved by the Mayo Clinic institutional review board. Patient Cohort Patients were identified from a prospectively collected institutional database of patients treated with SABR. Patients with lung tumors less than one centimetre in maximum dimension were included. The radiation treatment plans of three patients were extracted from the treatment planning system. All patients were treated using three-dimensional conformal multiple static-gantry photon beams. Plans were normalized so that 95% of the planning target volume (PTV) received at least 95% of the prescription dose. The prescription doses for these plans were adjusted to 34 Gy in one fraction based on the recently reported results of Radiation Therapy Oncology Group (RTOG) 0915 which established this dose fractionation regimen as a possible standard dose to be used in future trials [7]. Dose calculations for photon plans used the anisotropic analytical algorithm. Proton Treatment Planning A machine was commissioned in Eclipse v.10 (Varian Medical Systems Palo Alto CA) which allowed for planning and calculating transmission dose plans. The spot size (sigma) of the transmission beam which had an energy of 229 MeV was 2.2 mm. A proton plan that only used the transmission portion of the beam was created for each patient. Proton beam arrangements were selected so that no beams entered through the heart or spinal cord and allowed up to two non-coplanar beams. Four to five beams were used to keep the skin dose comparable to photon plans. The energy of the protons for each spot of a field was 229 MeV; this ensured the Bragg peak was not located within the patient. Dose calculations for the transmission portion of the proton beam were verified with Monte Carlo (Geant4). The proton plans were normalized so that the internal target volume (ITV) receives at least 95% of prescription dose including when range and position errors were included (3.5% and 2 mm) which is standard for spot scanning proton therapy. ITVs were created based on motion of the gross tumor volume in three dimensions using four-dimensional computed tomography image data. The dose constraints from RTOG 0915 were compared for the photon and proton plans as well as the total time that would be required to deliver the treatment. The radiotherapy delivery time per beam was estimated at 1 nC per second for proton therapy which is readily achievable by most spot scanning proton centers and 600 MU per minute for the photon plans. Differences in dosimetric and treatment planning parameters between photon and proton plans were analyzed with two-sided paired t-tests using SAS version 9.2 (SAS Institute Inc. Cary NC). Results The ITVs of the three tumors measured 0.220.42 and 0.99 cubic centimeters. All three proton plans had excellent coverage of the ITV. For all ITVs over 99.4% of the volume received at least 95% of the prescription dose including when uncertainties were examined. This was comparable with the photon plans where 100% of the ITVs received at least 95% of the prescription dose. For most normal tissues lower doses to these ans were achieved with the proton plans compared to the photon plans (). In fact (near) complete sparing of the spinal cord heart and esophagus was possible with protons through careful selection of beam angles (). .0098621.g001 Dose-volume histogram comparison of ans at risk. .0098621.t001 Dosimetric comparison of photon and proton plans. Parameter Photon Proton P-value Mean Range Mean Range Internal target volume (cc) 0.54 0.22“0.99 0.54 0.22“0.99 N/A Spinal cord Maximum dose (Gy) 5.66 2.39“8.07 1.97 0.00“3.06 0.04 Lungs (bilateral) Mean lung dose (Gy) 1.35 0.95“1.92 0.69 0.03“1.36 0.12 V20 (%) 0.66 0.39“1.20 0.49 0.16“1.01 0.06 V5 (%) 7.32 5.4“11.30 6.65 2.96“11.70 0.56 Heart Mean dose (Gy) 8.36 6.27“12.51 0.00 0.00“0.00 0.13 Skin Maximum dose (Gy) 11.75 9.86“13.28 11.40 7.37“16.23 0.89 Esophagus Maximum dose (Gy) 6.49 2.98“9.43 3.40 0.00“7.51 0.05 Homogeneity Index 1.25 1.21“1.29 1.07 1.03“1.11 0.06 Conformity Index 17.14 8.23“30.05 3.47 2.17“4.64 0.15 Proton plans used four to five non-coplanar beams compared to nine to ten beams for photon plans (). The average number of monitor units per field was 818 (range 758“871) with photons and only 38 (range 31“59) with protons. This would translate to an average beam-on time per field of 82 seconds versus 6 seconds for photon and proton plans respectively. These differences in monitor units and beam-on time were statistically significant with P<0.01(). .0098621.g002 Comparison of isodose distributions. Proton (left) and photon (right) treatment plans. .0098621.t002 Comparison of treatment time between photon and proton plans. Parameter Photon Proton P-value Mean Range Mean Range Total monitor units (MU) 7929 6820“8713 178 122“235 <0.01 Fields 9.7 9“10 4.7 4“5 N/A Average MU/field 818 758“871 38 30.5“46.9 <0.01 Beam on time per field (seconds) 81.8 75.6“87.1 5.8 4.7“7.2 <0.01 Discussion Exploiting the transmission beam in proton therapy planning has significant potentials for dose escalation and re-irradiation in lung tumors and eliminates the concern over the uncertainty of the stopping power and its impact on the Bragg peak location. PT-SABR planning requires fewer beams than photons and careful selection of optimal beam angles allows for minimal dose to adjacent normal tissues and tumor dose escalation which may translate to improved local control rates. RTOG 0915 showed that 34 Gy in a single fraction was comparable to 48 Gy in four fractions [7] and the dosimetric constraints from the protocol were easily achieved using both proton and photon plans for patients in this study. Further optimization with proton therapy can allow even higher doses to be delivered while still respecting established dosimetric constraints for normal tissues. This may translate to better tumor control but requires more investigation in a clinical setting. Patients planned with PT-SABR required fewer beams (5 vs. 10) which reduce the total treatment time and the low dose outside the tumor. The average monitor units per field for PT-SABR plans 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 Articles 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; beplerg@karmanos. 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 article 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). The rates of assignment of patients to observation (22%) and chemotherapy (78%) were as expected. CONCLUSIONS 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. To assess DFS the disease status was monitored every 2 months for the first 6 months and subsequently every 3 months by computed tomography after enrollment and according to good medical practice. Toxicities related to the administration of chemotherapy were assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events (version 3.0; ctep.cancer.gov). DFS was defined as the time from the date of enrollment to disease recurrence or death due to any cause and estimated according to the Kaplan-Meier method. A Cox regression model was fit with the time from surgery to enrollment as a covariate to evaluate its effect on DFS. A natural log transformation was applied to the raw protein measurement data and the Pearson correlation coefficient was used to test associations. Bivariate comparison of baseline characteristics between the assigned treatment groups was performed using the Fisher exact test for categorical variables or the Student t test or Wilcoxon rank sum test for continuous variables. A multivariable logistic model to evaluate baseline factors and treatment assignment was fit using backwards selection. Median ERCC1 and RRM1 expression levels were compared with historical medians using the 1-sample Wilcoxon signed rank test. The percentage of patients with both ERCC1 ??65 and RRM1 ??40 was compared with the historical rate using a chi-square test. All statistical analyses and graphics were performed using SAS statistical software (version 9.2; SAS Institute Inc Cary NC). A significance level of 5% was used for all analyses. RESULTS Patient and Trial Characteristics To ensure an adequate sample size of eligible patients and biomarker-specific subgroups a total of 85 patients was registered between April 2 2009 and April 1 2011 from 27 participating sites. Four patients were ineligible; 3 had inadequate lymph node sampling and 1 did not have a tumor measuring ??2 cm. provides the characteristics of the 81 eligible patients. Patient Demographics and Disease Characteristics Variablesa All Patients Assigned to Chemotherapy Assigned to Observation P Refused Assignment Accepted Assignment P N = 81 N = 63 N = 18 N = 20 N = 61 Age y .37 .39 ?Median 64 63.3 68.8 67.2 63.3 ?Mean 63.5 62.9 65.5 65.2 62.9 ?Range 41.6“84.2 41.6“84.2 41.6“81.7 44.2“82.9 41.6“84.2 Sex .18 .61 ?Female 44 (54%) 37 (59%) 7 (39%) 12 (60%) 32 (52%) ?Male 37 (46%) 26 (41%) 11 (61%) 8 (40%) 29 (48%) Ethnicity .65 .18 ?Unknown 7 (8%) 5 (8%) 2 (11%) 0 (0%) 7 (11%) ?Non-Hispanic 74 (91%) 58 (92%) 16 (89%) 20 (100%) 54 (89%) Race .73b .75b ?African American 8 (10%) 8 (13%) 0 (0%) 2 (10%) 6 (10%) ?Asian 3 (4%) 2 (3%) 1 (6%) 0 (0%) 3 (5%) ?Pacific Islander 2 (2%) 1 (2%) 1 (6%) 0 (0%) 2 (3%) ?White 66 (81%) 52 (83%) 14 (78%) 17 (85%) 49 (80%) ?Unspecified 2 (2%) 0 (0%) 2 (11%) 1 (5%) 1 (2%) Histology .06c .60c ?Adeno 52 (64%) 44 (70%) 8 (44%) 14 (70%) 38 (62%) ?Squamous 25 (31%) 17 (27%) 8 (44%) 6 (30%) 19 (31%) ?Large 1 (1%) 1 (2%) 0 (0%) 0 (0%) 1 (2%) ?Bronchioloalveolar 1 (1%) 0 (0%) 1 (6%) 0 (0%) 1 (2%) ?Other 2 (2%) 1 (2%) 1 (6%) 0 (0%) 2 (3%) Stage of disease .16 .27 ?IA (<3 cm) 25 (31%) 22 (35%) 3 (17%) 4 (20%) 21 (34%) ?IB (?3 cm) 56 (69%) 41 (65%) 15 (83%) 16 (80%) 40 (66%) Zubrod performance status .11 1.00 ?0 44 (54%) 31 (49%) 13 (72%) 11 (55%) 33 (54%) ?1 37 (46%) 32 (51%) 5 (28%) 9 (45%) 28 (46%) Weight loss (6 mo) 1.00d .31d ?<5% 64 (79%) 49 (78%) 15 (83%) 14 (70%) 50 (82%) ?5-<10% 9 (11%) 7 (11%) 2 (11%) 3 (15%) 6 (10%) ?10“20% 4 (5%) 3 (5%) 1 (6%) 2 (10%) 2 (3%) ?>20% 1 (1%) 1 (2%) 0 (0%) 0 (0%) 1 (2%) ?Unknown 3 (4%) 3 (5%) 0 (0%) 1 (5%) 2 (3%) Smoking status ?Current 33 (41%) 26 (41%) 7 (39%) 8 (40%) 25 (41%) ?"

Lung_Cancer

"To investigate the efficacy and safety of percutaneous microwave ablation. Methods: Twenty-six rabbits with lung VX2 tumor were randomly divided into experimental and control group. In the experimental group microwave ablation guided by ultrasound or CT was performed based on location of the tumor. Enhanced CT scan was carried out immediately before and after the ablation for all animals. Two animals from each group were sacrificed immediately or 1 week after the ablation respectively and the others were followed for the rest of their lives. Results: CT scan revealed that the tumor was greatly reduced or ablated after ablation. Pathological examination immediately after ablation also confirmed the tumor reduction or ablation. The survival time of the animals in the experimental group was significantly longer than that in the control group. Conclusions: Microwave ablation is a safe and effective method for treating lung cancer in rabbits showing potential clinical applicability. Microwave ablation VX2 tumor lung cancer 9421547 4136 Hum Pathol Hum. Pathol. Human pathology 0046-8177 1532-8392 24444464 3965626 10.1016/j.humpath.2013.10.016 NIHMS537247 A PIK3CA mutation detected in plasma from a patient with synchronous primary breast and lung cancers Jelovac Danijela MD 1 * Beaver Julia A. MD 1 * Balukrishna Sasidharan MD 2 Wong Hong Yuen BS 1 Toro Patricia Valda BS 1 Cimino-Mathews Ashley MD 1 Argani Pedram MD 1 Stearns Vered MD 1 Jacobs Lisa MD 1 VanDenBerg Dustin BS 1 Kessler Jill BS 1 Jeter Stacie BS 1 Park Ben H. MD PhD 1 Wolff Antonio C. MD 1 1The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins 1650 Orleans Street Baltimore MD 21287 2Christian Medical College Vellore Tamil Nadu India 632004 * These authors contributed equally to this work 18 12 2013 31 10 2013 4 2014 01 4 2015 45 4 880 883 2013 Elsevier Inc. All rights reserved. 2013 Digital PCR is a new technology that enables detection and quantification of cancer DNA molecules from peripheral blood. Using this technique we identified mutant PIK3CA DNA in circulating plasma tumor DNA (ptDNA) from a patient with concurrent early stage breast cancer and non-small cell lung cancer. The patient underwent successful resection of both her breast and lung cancers and using standard Sanger sequencing the breast cancer was shown to harbor the identical PIK3CA mutation identified in peripheral blood. This case report highlights potential applications and concerns that can arise with the use of ptDNA in clinical oncology practice. plasma tumor DNA breast cancer lung cancer PIK3CA digital PCR Br J Cancer Br. J. Cancer British Journal of Cancer 0007-0920 1532-1827 Nature Publishing Group 24983368 4102953 bjc2014353 10.1038/bjc.2014.353 Genetics and Genomics Assessing standardization of molecular testing for non-small-cell lung cancer: results of a worldwide external quality assessment (EQA) scheme for EGFR mutation testing Worldwide external quality assessment for EGFR gene mutation testing Patton S 1 * Normanno N 2 Blackhall F 3 Murray S 4 Kerr K M 5 Dietel M 6 Filipits M 7 Benlloch S 8 Popat S 9 Stahel R 10 Thunnissen E 11 1EMQN Manchester Centre for Genomic Medicine St Mary's Hospital Manchester M13 9WL UK 2Cell Biology and Biotherapy Unit Istituto Nazionale per lo Studio e la Cura dei Tumori ˜Fondazione Giovanni Pascale'”IRCCS 80131 Naples Italy 3Christie Hospital Manchester M20 4BX UK 4Biomarker Solutions Ltd London EC1V 2NX UK 5Department of Pathology Aberdeen Royal Infirmary Aberdeen AB25 2ZN UK 6Charit Humboldt-Universitt zu Berlin Berlin 10117 Germany 7Medical University of Vienna 1010 Vienna Austria 8Pangaea Biotech USP Dexeus University Institute Barcelona 08028 Spain 9Royal Marsden Hospital London SW3 6JJ UK 10University Hospital Z¼rich CH-8091 Z¼rich Switzerland 11Department of Pathology VU University Medical Center Amsterdam 1081 HZ The Netherlands *E-mail: simon.pattoncmft.nhs.uk 15 07 2014 01 07 2014 15 7 2014 111 2 413 420 22 01 2014 19 05 2014 20 05 2014 Copyright 2014 Cancer Research UK 2014 Cancer Research UK 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: The external quality assurance (EQA) process aims at establishing laboratory performance levels. Leading European groups in the fields of EQA Pathology and Medical and Thoracic Oncology collaborated in a pilot EQA scheme for somatic epidermal growth factor receptor (EGFR) gene mutational analysis in non-small-cell lung cancer (NSCLC). Methods: EQA samples generated from cell lines mimicking clinical samples were provided to participating laboratories each with a mock clinical case. Participating laboratories performed the analysis using their usual method(s). Anonymous results were assessed and made available to all participants. Two subsequent EQA rounds followed the pilot scheme. Results: One hundred and seventeen labs from 30 countries registered and 91 returned results. Sanger sequencing and a commercial kit were the main methodologies used. The standard of genotyping was suboptimal with a significant number of genotyping errors made. Only 72 out of 91 (72%) participants passed the EQA. False-negative and -positive results were the main sources of error. The quality of reports submitted was acceptable; most were clear concise and easy to read. However some participants reported the genotyping result in the absence of any interpretation and many obscured the interpretation required for clinical care. Conclusions: Even in clinical laboratories the technical performance of genotyping in EGFR mutation testing for NSCLC can be improved evident from a high level of diagnostic errors. Robust EQA can contribute to global optimisation of EGFR testing for NSCLC patients. non-small-cell lung carcinoma EGFR gene mutations quality assessment Assessment of epidermal growth factor receptor (EGFR) mutations has become mandatory to choose the most active first-line treatment for patients with advanced non-small-cell lung cancer (NSCLC). Indeed randomized phase III clinical trials have demonstrated that first-line administration of an EGFR TKI results in a prolonged progression-free survival as compared with chemotherapy in patients carrying EGFR mutations (Mok et al 2009; Maemondo et al 2010; Mitsudomi et al 2010; Fukuoka et al 2011; Zhou et al 2011; Rosell et al 2012). These studies have also confirmed that EGFR mutations are a reliable marker that predicts sensitivity to EGFR TKIs (Mok et al 2009). Activating mutations occur in exons 18 through 21 of the TK domain of the EGFR gene and either point mutations or in-frame small deletions or insertions (Sharma et al 2007; De Luca and Normanno 2010). Although more than 250 mutations of the EGFR gene have been described to date two mutations a single point mutation in exon 21 the L858R and a series of small in-frame deletions in exon 19 account for ?90% of all EGFR mutations (Sharma et al 2007; Linardou et al 2008). EGFR mutations are strongly associated with defined clinical and pathological features: they are far more frequent in female patients as compared with male; in adenocarcinoma as compared with other histological types; in non-smokers as compared with current smokers or former smokers; and in East-Asian NSCLC patients as compared with Non-East-Asian patients (Normanno et al 2006). External quality assessment (EQA) is a system of objectively checking laboratory results by an independent external agency (van Krieken et al 2013). The main objective of an EQA programme is to establish inter-laboratory comparability. In this respect the EQA process can identify latent systematic errors in methodology that may not be revealed by a laboratory's own internal QA processes. Representatives from ETOP ESMO ESP EMQN and other leading European groups met in July 2010 to discuss a pan-European approach to EQA for EGFR mutation testing in NSCLC. In this paper we present the results of this pilot EQA scheme for EGFR testing that was completed in 2013. Materials and Methods anisation of the scheme A meeting was anised in July 2010 by ETOP and EMQN to bring together a group of professionals representing EMQN ESP ETOP ESMO and other leading European groups involved in NSCLC testing (see Supplementary Information). From this group a steering group of five individuals was formed who planned designed and assessed the results of the pilot EQA scheme. The scheme was coordinated and administered by the EMQN and three rounds were anised within a period of 18 months. The workflow of the scheme process is shown in Figure 1."

Lung_Cancer

"Lung cancer is the most common malignancy and the leading cause of cancer deaths worldwide. While smoking is by far the leading cause of lung cancer other environmental and genetic factors influence the development and progression of the cancer. Since unique mutations patterns have been observed in individual cancer samples identification and characterization of the distinctive lung cancer molecular profile is essential for developing more effective tailored therapies. Until recently personalized DNA sequencing to identify genetic mutations in cancer was impractical and expensive. The recent technological advancements in next-generation DNA sequencing such as the semiconductor-based Ion Torrent sequencing platform has made DNA sequencing cost and time effective with more reliable results. Using the Ion Torrent Ampliseq Cancer Panel we sequenced 737 loci from 45 cancer-related genes to identify genetic mutations in 76 human lung cancer samples. The sequencing analysis revealed missense mutations in KRAS EGFR and TP53 genes in the breast cancer samples of various histologic types. Thus this study demonstrates the necessity of sequencing individual human cancers in order to develop personalized drugs or combination therapies to effectively target individual breast cancer-specific mutations. This research was supported by the grants from National Natural Science Foundation of China the Wu Jieping Foundation and the National Institute of Health (R01 CA90427 & R01 AI084811 to SY Chen). 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 most common malignancy worldwide and also the leading cause of cancer related deaths. In 2008 an estimated 1.61 million new cases were reported globally accounting for 12.7% of all new cancers [1]. Additionally roughly 1.38 million deaths (18.2% of total cancer deaths) were reported around the world [2]. In China lung cancer has the highest incidence of all new cancer cases in both men and women (21.7% in 2008) with more than a 24.9% mortality rate [2]. Women in China reported only a slightly higher incidence of lung cancer over breast cancer this same year; however the mortality rate of lung cancer is more than 3 times higher than that of breast cancer (20.2% vs. 6.1% respectively) [2]. Lung cancer often exhibits non-specific symptoms and diagnosis often occurs at an advanced stage or after metastasis has already occurred [3]. While efforts continue to improve early diagnosis and treatment of lung cancer the staggering incidence poor prognosis and considerable mortality rate prevails. The leading cause of lung cancer is cigarette smoking and increased exposure is directly correlated with an increased risk of developing lung cancer [4]. 85“90% of lung cancer deaths are associated with smoking and current smokers are 15 times more likely to die from lung cancer than never-smokers [5]. There are two major forms of lung cancer: non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC). NSCLC which accounts for roughly 85% of all lung cancers can be further divided into three major histologic subtypes: squamous-cell carcinoma (SCC) adenocarcinoma and large-cell lung cancer. While smoking can be attributed to all forms of lung cancer it is most commonly linked to SCLC and SCC. Never-smokers on the other hand are most commonly diagnosed with adenocarcinoma [3] [5]. Interestingly only 10“24% of smokers develop lung cancer indicating the importance of other environmental and individual genetic factors [6] [7]. Aside from tobacco smoke other etiologic agents and risk factors have been identified including occupation exposure to second-hand smoke asbestos radon gas and air pollution in addition to genetic factors [8]“[10]. Roughly 10“15% of lung cancers arise in patients that report never having smoked and these cancers do so spontaneously with an accumulation of genetic and epigenetic changes [5]. Despite ongoing efforts to improve screening and treatment of lung cancers the prognosis of patients with most forms of lung cancer remains poor [3]. Because the genetic and environmental factors causing lung cancer vary widely each tumor has the potential to exhibit a unique gene mutation profile. As such accumulating evidence suggests that individualized tailored therapies are essential for effective treatment against lung cancers. This can be accomplished by profiling an individual's cancer genome in order to dissect the oncogenic mechanisms that regulate the progression of the cancer. Recently a new technology based on semiconductor sequencing called Ion Torrent sequencing [11] is tackling many of the issues associated with other sequencing methods namely the cost time and overall practicality of individualized genome sequencing. In this study we have used Ion Torrent sequencing to analyze 76 clinical lung cancer samples to identify the genetic mutations in 737 loci of 45 known cancer-related genes. Results Mutation analysis of human lung cancer tumors with Ion Ampliseq Cancer Panel A total of 76 Lung cancer samples () was analyzed using Ion Torrent Ampliseq Cancer Panel to identify mutations in 737 loci of 45 oncogenes and tumor suppressor genes in human lung cancers. These Lung cancer samples were all from Chinese patients ranging from 28“80 years old represented by 40 men with a mean age of 62 years and 36 women with a mean age of 59 years. .0095228.t001 Detected mutations (including Missense point mutations/deletion/insertion) in 45 genes (737 loci) of 76 human lung cancer samples."

Lung_Cancer

"adenocarcinoma in Chinese female non-smokers has not been well addressed. ATM rs189037 was a common polymorphism in the promoter of ATM gene. Studies have shown that this site possibly may regulate ATM protein activity due to regulation function of promoter as shown in most genes. And specific genotypes or haplotypes of ATM may play an important role in carcinogenesis through expression regulation or alternative splicing of the ATM gene [31]. We searched through NCBI (National Center for Biotechnology Information) dbSNP database to get the allele frequency of this polymorphism. The data indicated that the frequency of wild-type allele G was 61.1% and the frequency of variant allele A was 38.9% in Chinese Han population (http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?rs=rs189037). In this study our results was in accordance with the data from NCBI. In 2006 Kim et al.[25] evaluated the role of ATM rs189037 in lung cancer development In Korean population for the first time. No significant association was found between this polymorphism and lung cancer risk (P>0.05). They recruited 616 lung cancer patients in which 78.4% were male and 79.6% were cigarette smokers. As cigarette smoking might modulate the risk of lung cancer in turn it could be a confounder in the association between ATM rs189037 and lung cancer risk. Besides there was no gene-environment interactions be considered in their research. In 2010 Lo et al.[23] suggested that ATM rs189037 was associated with lung cancer risk among never smokers (AA vs GG: OR?=?1.61 95%CI 1.10“2.35) and this association might be modified by passive smoking. Although they have eliminated the cofounding effect of cigarette smoking by conducting their study in non-smokers the risk of lung cancer among different histological types still needed to be clarified. Recently Hsia et al.[24] put their attention on the association of ATM rs189037 with lung cancer susceptibility among ever smokers. No genotype frequency difference was found between lung cancer cases and controls among ever smokers (P>0.05). After summing up the omissions of their studies and combining with the current situation that Chinese non-smoking female lung adenocarcinoma incidence and fatality rate was increasingly rising up we performed this case-control study to elucidate the association between ATM rs189037 and lung adenocarcinoma risk. To the best of our knowledge this is the first study that has investigated whether ATM rs189037 was associated with lung adenocarcinoma risk in non-smoking Han-Chinese females. Our results have shown that individuals with exposure to cooking oil fume had a 1.63-fold increased risk of developing lung adenocarcinoma (P?=?0.011). Similar significant associations were observed in our previous studies of Chinese non-smoking females. Experimental studies have presented that fumes from cooking oils could be genotoxic because of the potential carcinogenic components such as polycyclic aromatic hydrocarbons (PAHs) and benzo[a]pyrene 78-diol 910-epoxide (BPDE) which involved in inducing DNA adducts and thus made a predisposition to lung adenocarcinoma [32]“[34]. Besides the method of cooking and throat or eyes irritation the interviewers also asked each woman the information on cooking oil fumes exposure such as the types of cooking oils she used the frequency she used stir frying or deep frying to prepare food ventilation conditions and the use of a fume extractor. Increasing epidemiological studies have reported cooking method and types of cooking oils on lung cancer susceptibility among Chinese females. Seow et al.[35]found that women who reported that they stir fried daily had a significantly increased risk of lung cancer (OR?=?2.0 95%CI 1.0“3.8) and risk was enhanced for those who stir fried meat daily (OR?=?2.7 95%CI 1.3“5.5). The elevated lung cancer risk might be attributed to heterocyclic amines generated during frying of meats. In addition the frequency of stir frying seemed to be related with lung cancer susceptibility. Gao et al. [36]investigated the association between the frequency of stir frying and lung cancer risk in Chinese females they observed that stir frying more than 30 dishes per week was associated with high risk of lung cancer (OR?=?2.6 95%CI 1.3“5.0). In a case-control study in northeast China Wu-Williams et al.[37] found that women who deep fried twice per month had a 2.1-fold increased risk of developing lung cancer than those who never used deep frying method. And there was a significant trend in risk with increasing number of meals cooked by deep frying. Also this kind of correlation was found in both non-smokers and lung adenocarcinoma population. For types of cooking oils Zhong et al.[38] reported that soybean oil was most commonly used in Shanghai and the use of rapeseed oil was associated with a higher risk of lung cancer (OR?=?1.84 95%CI 1.12“3.03). In this study we observed that ATM rs189037 AA genotype carriers were more susceptible to lung adenocarcinoma than GA or GG genotype carriers in a recessive model. This might not give direct support for AA genotype as a risk factor for lung adenocarcinoma. But the results reflected that G allele might be a protective factor for lung adenocarcinoma. So we compared AA genotype with GA genotype and our data showed that women who were AA genotype carriers had an elevated risk of lung adenocarcinoma (OR?=?1.74 95%CI 1.10“2.74 P?=?0.018). In other words GA genotype might be protective for developing lung adenocarcinoma. In the stratified analysis of cooking oil fumes exposure we also found that AA genotype carriers had a predisposition to lung adenocarcinoma in women who had no exposure of cooking oil fumes (OR?=?1.89 95%CI 1.03“3.49). Considering that G allele might be a protective factor for lung adenocarcinoma we then compared AA genotype with GA genotype to further validate our previous results. And it turned out that in the non-exposed group women who were AA genotype carriers had a higher risk of lung adenocarcinoma than those GA genotype carriers (OR?=?1.98 95%CI 1.15“3.40 P?=?0.014) which was in accordance with our previous data that G allele might be a protective factor for lung adenocarcinoma. But in the combined analysis of interaction of cooking oil fumes exposure and rs189037 polymorphism no significant association was found. We have described the distribution of any possible factors such as age passive smoking status fuel smoke exposure family history of cancer between cooking oil fumes exposed group and non-exposed group that might affect the association but none of these seemed to be different between exposed group and non-exposed group (). As tumor is a multifactorial disease we could infer that there might be other risk factors playing a role in the development of lung adenocarcinoma. We tended to believe that there might be other host genetic susceptibility or unknown risk factors caused the results. .0096911.t005 Comparisons of distribution of risk factors between cooking oil fumes exposed group and non-exposed group. Variable Exposed(%) Non-exposed(%) P value Mean age (±S.D.) 56.3±11.7 56.1±11.1 0.871a Fuel smoke exposure 44(28.2%) 98(27.0%) 0.777b Passive smoking exposure 96(61.5%) 203(55.9%) 0.235b Family history of cancer 19(12.2%) 37(10.2%) 0.504b a Student's t-test was used to compare the frequency distribution of demographic variables between the exposed group and non-exposed group. b Peason's chi square was used to compare the frequency distribution of demographic variables fuel smoke exposure family history of cancer passive smoking between the exposed group and non-exposed group. There are several limitations in the current study. First hospital-based studies are likely to include some controls with non-malignant lung diseases especially those associated with chronic inflammatory processes are suspected to have predisposing factors for lung cancer."

Lung_Cancer

"of erythrocytes in systemic lupus erythematosus: analysis of the stability of the defect and of a restriction fragment length polymorphism of the CR1 gene J Immunol 1987 138 2708 2710 2881967 Xiang L Rundles JR Hamilton DR Wilson JG Quantitative alleles of CR1: coding sequence analysis and comparison of haplotypes in two ethnic groups J Immunol 1999 163 4939 4945 10528197 Birmingham DJ Chen W Liang G Schmitt HC Gavit K Nagaraja HN A CR1 polymorphism associated with constitutive erythrocyte CR1 levels affects binding to C4b but not C3b Immunology 2003 108 531 538 10.1046/j.1365-2567.2003.01579.x 12667215 Holers VM Chaplin DD Leykam JF Gruner BA Kumar V Atkinson JP Human complement C3b/C4b receptor (CR1) mRNA polymorphism that correlates with the CR1 allelic molecular weight polymorphism Proc Natl Acad Sci U S A 1987 84 2459 2463 10.1073/pnas.84.8.2459 3031685 Zhang Q Yu JT Zhu QX Zhang W Wu ZC Miao D Tan L Complement receptor 1 polymorphisms and risk of late-onset Alzheimer™s disease Brain Res 2010 1348 216 221 20558149 He JR Xi J Ren ZF Qin H Zhang Y Zeng YX Mo HY Jia WH Complement receptor 1 expression in peripheral blood mononuclear cells and the association with clinicopathological features and prognosis of nasopharyngeal carcinoma Asian Pac J Cancer Prev 2012 13 6527 6531 10.7314/APJCP.2012.13.12.6527 23464487 Srivastava A Mittal B Complement receptor 1 (A3650G RsaI and intron 27 HindIII) polymorphisms and risk of gallbladder cancer in north Indian population Scand J Immunol 2009 70 614 620 10.1111/j.1365-3083.2009.02329.x 19906204 Ferreira CG Lung cancer in developing countries Am Soc Clin Oncol Educ Book 2013 327 331 PMID:23714537 23714537 Amos CI Wu X Broderick P Gorlov IP Gu J Eisen T Dong Q Zhang Q Gu X Vijayakrishnan J Sullivan K Matakidou A Wang Y Mills G Doheny K Tsai YY Chen WV Shete S Spitz MR Houlston RS Genome-wide association scan of tag SNPs identifies a susceptibility locus for lung cancer at 15q25.1 Nat Genet 2008 40 616 622 10.1038/ng.109 18385676 Hung RJ McKay JD Gaborieau V Boffetta P Hashibe M Zaridze D Mukeria A Szeszenia-Dabrowska N Lissowska J Rudnai P Fabianova E Mates D Bencko V Foretova L Janout V Chen C Goodman G Field JK Liloglou T Xinarianos G Cassidy A McLaughlin J Liu G Narod S Krokan HE Skorpen F Elvestad MB Hveem K Vatten L Linseisen J A susceptibility locus for lung cancer maps to nicotinic acetylcholine receptor subunit genes on 15q25 Nature 2008 452 633 637 10.1038/nature06885 18385738 Wang Y Broderick P Webb E Wu X Vijayakrishnan J Matakidou A Qureshi M Dong Q Gu X Chen WV Spitz MR Eisen T Amos CI Houlston RS Common 5p15.33 and 6p21.33 variants influence lung cancer risk Nat Genet 2008 40 1407 1409 10.1038/ng.273 18978787 McKay JD Hung RJ Gaborieau V Boffetta P Chabrier A Byrnes G Zaridze D Mukeria A Szeszenia-Dabrowska N Lissowska J Rudnai P Fabianova E Mates D Bencko V Foretova L Janout V McLaughlin J Shepherd F Montpetit A Narod S Krokan HE Skorpen F Elvestad MB Vatten L Nj¸lstad I Axelsson T Chen C Goodman G Barnett M Loomis MM Lung cancer susceptibility locus at 5p15.33 Nat Genet 2008 40 1404 1406 10.1038/ng.254 18978790 Markiewski MM Lambris JD The role of complement in inflammatory diseases from behind the scenes into the spotlight Am J Pathol 2007 171 715 727 10.2353/ajpath.2007.070166 17640961 Ricklin D Lambris JD Complement-targeted therapeutics Nat Biotechnol 2007 25 1265 1275 10.1038/nbt1342 17989689 Hugli TE Biochemistry and biology of anaphylatoxins Complement 1986 3 111 127 3542363 Ostrand-Rosenberg S Cancer and complement Nat Biotechnol 2008 26 1348 1349 10.1038/nbt1208-1348 19060872 Markiewski MM DeAngelis RA Benencia F Ricklin-Lichtsteiner SK Koutoulaki A Gerard C Coukos G Lambris JD Modulation of the antitumor immune response by complement Nat Immunol 2008 9 1225 1235 10.1038/ni.1655 18820683 Markiewski MM Lambris JD Is complement good or bad for cancer patients? A new perspective on an old dilemma Trends Immunol 2009 30 286 292 10.1016/j.it.2009.04.002 19428302 Fan Q He JF Wang QR Cai HB Sun XG Zhou XX Qin HD Shugart YY Jia WH Functional polymorphism in the 5?-UTR of CR2 is associated with susceptibility to nasopharyngeal carcinoma Oncol Rep 2013 30 11 16 23612877 Cerhan JR Novak AJ Fredericksen ZS Wang AH Liebow M Call TG Dogan A Witzig TE Ansell SM Habermann TM Kay NE Slager SL Risk of non-Hodgkin lymphoma in association with germline variation in complement genes Br J Haematol 2009 145 614 623 10.1111/j.1365-2141.2009.07675.x 19344414 Zhang Z Yu D Yuan J Guo Y Wang H Zhang X Cigarette smoking strongly modifies the association of complement factor H variant and the risk of lung cancer Cancer Epidemiol 2012 36 e111 e115 10.1016/j.canep.2011.11.004 22197220 Shen M Vermeulen R Rajaraman P Menashe I He X Chapman RS Yeager M Thomas G Burdett L Hutchinson A Yuenger J Chanock S Lan Q Polymorphisms in innate immunity genes and lung cancer risk in Xuanwei China Environ Mol Mutagen 2009 50 285 290 10.1002/em.20452 19170196 Fearon DT Identification of the membrane glycoprotein that is the C3b receptor of the human erythrocyte polymorphonuclear leukocyte B lymphocyte and monocyte J Exp Med 1980 152 20 30 10.1084/jem.152.1.20 6967510 Besaratinia A Pfeifer GP Second-hand smoke and human lung cancer Lancet Oncol 2008 9 657 666 10.1016/S1470-2045(08)70172-4 18598930 Coyle YM Minahjuddin AT Hynan LS Minna JD An ecological study of the association of metal air pollutants with lung cancer incidence in Texas J Thorac Oncol 2006 1 654 661 10.1097/01243894-200609000-00009 17409932 Garshick E Laden F Hart JE Rosner B Smith TJ Dockery DW Speizer FE Lung cancer in railroad workers exposed to diesel exhaust Environ Health Perspect 2004 112 1539 1543 10.1289/ehp.7195 15531439 Brennan P Buffler PA Reynolds P Wu AH Wichmann HE Agudo A Pershagen G Jockel KH Benhamou S Greenberg RS Merletti F Winck C Fontham ET Kreuzer M Darby SC Forastiere F Simonato L Boffetta P Secondhand smoke exposure in adulthood and risk of lung cancer among never smokers: a pooled analysis of two large studies Int J Cancer 2004 109 125 131 10.1002/ijc.11682 14735478 Lee G Walser TC Dubinett SM Chronic inflammation chronic obstructive pulmonary disease and lung cancer Curr Opin Pulm Med 2009 15 303 307 10.1097/MCP.0b013e32832c975a 19417670 Engels EA Inflammation in the development of lung cancer: epidemiological evidence Expert Rev Anticancer Ther 2008 8 605 615 10.1586/14737140.8.4.605 18402527 Kullo IJ Ding K Shameer K McCarty CA Jarvik GP Denny JC Ritchie MD Ye Z Crosslin DR Chisholm RL Manolio TA Chute CG Complement receptor 1 gene variants are associated with erythrocyte sedimentation rate Am J Hum Genet 2011 89 131 138 10.1016/j.ajhg.2011.05.019 21700265 Teeranaipong P Ohashi J Patarapotikul J Kimura R Nuchnoi P Hananantachai H Naka I Putaporntip C Jongwutiwes S Tokunaga K A functional single-nucleotide polymorphism in the CR1 promoter region contributes to protection against cerebral malaria J Infect Dis 2008 198 1880 1891 10.1086/593338 18954261 Chen GB Xu Y Xu HM Li MD Zhu J Lou XY Practical and theoretical considerations in study design for detecting gene-gene interactions using MDR and GMDR approaches PLoS One 2011 6 e16981 .0016981 21386969 Lou XY Chen GB Yan L Ma JZ Zhu J Elston RC Li MD A generalized combinatorial approach for detecting gene-by-gene and gene-by-environment interactions with application to nicotine dependence Am J Hum Genet 2007 80 1125 1137 10.1086/518312 17503330 PLoS One one 1932-6203 Public Library of Science San Francisco USA 24416392 3887046 PONE-D-13-33822 .0085329 Research Mathematics Statistics Biostatistics Statistical Methods Medicine Clinical Research Design Meta-Analyses Oncology Basic Cancer Research Metastasis Cancers and Neoplasms Lung and Intrathoracic Tumors Pulmonology Surgery Thoracic Surgery Evaluation of Video-Assisted Thoracoscopic Surgery for Pulmonary Metastases: A Meta-Analysis VATS for Pulmonary Metastases Dong Siyuan Zhang Lin * Li Wenya Du Jiang Liu Xiangli Chen Xitao Department of Thoracic Surgery First Hospital of China Medical University Shenyang Liaoning Province People's Republic of China Arnold Paul Editor University of Kansas United States of America * E-mail: zhanglincmu@163.com Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: SYD LZ. Performed the experiments: SYD LZ WYL JD XLL XTC. Analyzed the data: SYD LZ WYL JD XLL XTC. Contributed reagents/materials/analysis tools: SYD LZ WYL JD XLL XTC. Wrote the paper: SYD LZ. 2014 9 1 2014 9 1 e85329 16 8 2013 25 11 2013 2014 Dong 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 To evaluate the evidence comparing video-assisted thoracic surgery (VATS) and open thoracotomy in the treatment of metastatic lung cancer using meta-analytical techniques. Methods A literature search was undertaken until July 2013 to identify the comparative studies evaluating disease-free survival rates and survival rates. The pooled odds ratios (OR) and the 95% confidence intervals (95% CI) were calculated with the fixed or random effect models. Results Six retrospective studies were included in our meta-analysis. These studies included a total of 546 patients: 235 patients were treated with VATS and 311 patients were treated with open thoracotomy. The VATS and the thoracotomy did not demonstrate a significant difference in the 1-3-5-year survival rates and the 1-year disease-free survival rate. There were significant statistical differences between the 3-year disease free survival rate (p?=?0.04) which favored open thoracotomy. Conclusions The VATS approach is a safe and feasible treatment in terms of the survival rate for metastatic lung cancer compared with the thoracotomy. The 3-year disease-free survival rate in the VATS group is inferior to that of open thoracotomy. The VATS approach could not completely replace open thoracotomy. The authors have no support or funding to report. Introduction Metastasectomy is considered a beneficial treatment for a patient with metastatic lung cancer whose primary tumor has been well controlled[1].After surgery 5-year survival rates of 30% to 50% could be achieved depending on the underlying primary cancer[2]“[4].In practice the surgical approaches to pulmonary metastases are variable. Video-assisted thoracoscopic surgery (VATS) is an emerging technique; many procedures that had previously required a thoracotomy have been performed with the minimally invasive VATS. VATS has been used for the treatment of pulmonary metastases. The routine use of VATS for the treatment of respectable metastatic lung cancer remains controversial. Critics of the VATS approach have argued that it might not be an equivalent oncological operation[5] [6]. A prospective study by Cerfolio[7]found that 22% of the nodules that could be detected by thoracotomy were missing by VATS.Whether the VATS approach can provide a satisfactory outcome is unknown. An evidenced-based investigation of the VATS approach is needed we undertook this meta-analysis to achieve a more objective assessment of the published studies and to provide a more accurate comparison between VATS and thoracotomy for metastatic lung cancer. Methods Search Strategy Electronic searches were of the MEDLINECochrane Controlled Trial Register (CENTRAL) Ovid MEDILINE PubMed and Embase databases were performed until July 2013.The following MeSH search headings were used: œmetastatic lung cancer œpulmonary metastases œvideo-assisted thoracic surgery œthoracotomy and œcomparative study.We searched the reference lists of relevant studies reviews editorials lettersand meeting abstracts. We used the Science Citation Index to cross-reference for further studies that met our criteria. Study Selection The studies included in this meta-analysis were based on our predetermined criteria as follows: (1) clinical trials that include the full text of the paper published in peer-reviewed English journals or reports of presentations at major thoracic surgery meetings; (2) comparison of the efficacy of VATS to that of thoracotomy in patients with metastatic lung cancer; and (3) similarity in the patients' baseline characteristics. Data extraction and quality assessment Two independent reviewers (Siyuan and Wenya) assessed the quality and the risk of bias of the included trials as follows: (1) the studies that did not include a comparative group with surgery as a form of intervention were excluded; (2) the trials focusing on patients undergoing surgery for primary lung cancer were excluded; (3) the studies on robotic video-assisted thoracic surgery were excluded; (4) if there was an overlap between authors centers or patient cohorts evaluated in the published literature only the most recent report was included; (5) studies published more than 20 years ago were excluded because of the significant technological changes that has occurred. The s were evaluated with the Downs and Black quality assessment method[8]. Discrepancies between the two investigators were resolved by discussion and consensus with a senior investigator. The final results were reviewed by two senior investigators (Lin and Jiang).The disease-free survival was defined as the date of the initial metastasectomy until the date of a recurrence. Statistical and sensitivity analyses The meta-analysis was performed using the RevMan 5.1.0. software package. The odds ratio (OR) or the mean difference with 95% confidence intervals (95% CI) was calculated for the dichotomous outcomes and the continuous outcomes respectively. A P value<0.05 was considered a significant difference in the value between the two groups. We used the I2 statistic to investigate the heterogeneity among the studies.The heterogeneity was explored by X2 and I2; I2<25% and I2>50% reflect a small and large inconsistency respectively. P<0.05 was considered significant. If there were a statistical difference in terms of the heterogeneity (P?0.05) a random-effect model was selected to pool the data. Otherwise a fixed-effect model was used. Taking into account the presence of different sample sizes of the included studies a sensitivity analysis was performed to compare the of 1-year survival rate and the 3-year disease free survival rate between VATS and open thoracotomy. Publication bias A funnel plot was used to explore bias. Asymmetry in the funnel plot of trial size against treatment effect was used to assess the risk of bias. Results Description of the studies Six retrospective cohort studies the met our criteria were included in this meta-analysis. A total of 546 patients were included in the six studies;235 patients were allocated to the VATS group whereas 311 were allocated to the open thoracotomy group to evaluate their survival rate.The search algorithm results of the search strategies and selection criteria are shown in Fig 1. The patient characteristics and evaluation index are shown in Table 1. .0085329.g001 Figure 1 Identification of studies for inclusion. .0085329.t001 Table 1 Study Design Country NO(V/O) Gender (M/F) Mean age (years) Assessment score Nakajima2001[28] OC Japan 45/55 V59/41 O34/21 V55±15 O55±14 13 Mutsaerts2002[29] OC Netherlands 8/12 NR NR 19 Nakas2009[30] OC UK 25/27 V16/9 O 19/8 V69 O66 16 Carballo2009[31] OC USA 36/135 V18/18 O82/53 V58.5 O49 15 Gossot2009[32] OC France 31/29 V21/10 O13/16 V43 O40 18 Chao2012[33] OC Taiwan 90/53 V49/41 O35/18 NR 13 V VATS; O Open thoracotomy; NR Not reported; OC observational cohort. Assessment of Recurrence and Survival Six studies documented the 1-year survival rateand there was no significant heterogeneity among the six studies (x2?=?3.79 P?=?0.58I2?=?0%).A fixed effect model was used.The combined result is shown in Fig 2(OR?=?1.15; 95%CI 0.72“1.84; p?=?0.58). Because of the heterogeneity in sample size the sensitivity analyses were conducted using larger sample sizes. There was no difference between the two surgical methods with an OR of 1.00(95%CI 0.55“1.79) and with heterogeneity(?2?=?3.23P?=?0.07 I2?=?69%). Five studies reported the 3-year survival rate and heterogeneity was identified through the five studies (x2?=?11.32P?=?0.02I2?=?65%); and a random effect model was adopted (OR?=?1.07; 95%CI 0.50“2.27; p?=?0.86) (Fig 3). Three studies compared the 5-year survival rate (OR?=?0.96; 95%CI 0.34“2.71; p?=?0.93) with certain heterogeneity(x2?=?8.86P?=?0.01I2?=?77%) (Fig 4). .0085329.g002 Figure 2 1-year survival rate. Forest plot of the Odds Ratio(OR) of the 1-year survival rate following VATS versus open thoracotomy for metastatic lung cancer.The estimate of the OR of each individual trial corresponds to the middle of the squares and horizontal line gives the 95% CI.On each linethe numbers of events as a fraction of the total number randomized are shown for both treatment groups.For each subgroupthe sum of the statistics along with the summary OR is represented by the middle of the solid diamonds.A test of heterogeneity between the trials within a subgroup is given below the summary statistics. .0085329.g003 Figure 3 3-year survival rate. Forest plot of the Odds Ratio(OR) of the 3-year survival rate following VATS versus open thoracotomy for metastatic lung 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

"64Cu-AcD10 (33%) and 64Cu-(M10)2 (38%)) at 4 h. As expected the multivalent conjugates (60-73% at 1h 33-38% at 4 h) showed higher serum stability than the monovalent conjugate (55% and 20% at 1 and 4 h). It is important to note that all the divalent conjugates showed similar stability irrespective of their design. Surprisingly acetylation of the amino terminus of the peptide did not increase the serum stability of the peptide according to this assay. However rat serum has notorious endopeptidase activity which would not be blocked by acetylation 35. MicroPET Imaging The feasibility of ?v?6-integrin targeted NSCLC tumor detection in vivo was evaluated in SCID mice bearing H2009 (?v?6+) xenografts. Effect of multivalency on imaging signal amplification was evaluated by comparing the tumor uptake of divalent probe (64Cu-(M10)2) with the monovalent one (64Cu-M10). As summarized in 64Cu-M10 showed moderate H2009 tumor uptake (0.55 ± 0.17 %ID/g) at 1 h p.i. but the tumor became barely visible at 4 h (0.33 ± 0.11 %ID/g) and 24 h p.i. (0.28 ± 0.11%ID/g). By contrast the scaffold based divalent probe 64Cu-(M10)2 showed an approximate 3-fold tumor uptake increase (p < 0.01) at all three time points (1.6 ± 0.20 %ID/g 0.85 ± 0.19 %ID/g and 0.79 ± 0.03 %ID/g at 1 h 4 h and 24 h p.i. respectively) ( ) supporting the premise of signal enhancement through dimerization of the H2009.1-10mer peptide. It is noteworthy that although the peptide based (64Cu-D10) and scaffold based (64Cu-(M10)2) divalent probes are considerably different in their peptide's spatial orientations they displayed a similar level of tumor signal enhancement at all three time points ( ). This observation is consistent with their binding affinity measurements. It must be pointed out that the higher tumor uptake of 64Cu-(M10)2 and 64Cu-D10 was accompanied by their higher uptake in other ans particularly kidneys ( and 2). Being the major clearance an for all the probes kidneys showed the highest uptake with respect to other ans at every time point. Among the three probes (64Cu-M10 64Cu-(M10)2 and64Cu-D10) the monovalent 64Cu-M10 showed the lowest kidney uptake (6.83 ± 0.35 %ID/g 6.23 ± 0.32 %ID/g and 3.50 ± 0.36 %ID/g at 1 h 4 h and 24 h p.i. respectively) while an approximate 6-fold increase of kidney uptake was observed for both divalent probes (64Cu-(M10)2: 53.6 ± 8.68 %ID/g 46.25 ± 10.39 %ID/g and 27.33 ± 7.3 %ID/g at 1 h 4 h and 24 h p.i. respectively; 64Cu-D10: 43.46 ± 1.8 %ID/g 39.3 ± 5.66 %ID/g and 21.36 ± 0.57 %ID/g at 1 h 4 h and 24 h p.i. respectively) ( and ). Peptide Acetylation Acetylation of the N-terminus of the H2009.1-10mer peptide was employed to protect the peptide from serum peptidases 36. This is particularly important as the critical RGD lies directly at the N-terminus of the peptide. Although acetylation did not affect serum stability in rat serum it may protect the peptide in vivo. Additionally acetylation reduces the charge of each peptide branch by +1 which may reduce nonspecific binding of the peptide and improve the in vivo biodistribution characteristics. The acetylated divalent probe 64Cu-AcD10 was produced by capping the N-terminus of H2009.1-10mer peptide. Tumor accumulation of the acetylated probe 64Cu-AcD10 was almost identical to the non-acetylated dimeric probes (64Cu-(M10)2 and 64Cu-D10) (). Impressively 64Cu-AcD10 showed a drastic reduced kidney uptake level as compared to the non-acetylated 64Cu-D10 and 64Cu-(M10)2 (). Remarkably it also displayed a much more favorable biodistribution profile than the non-acetylated dimers (). Of particular importance the low background uptake of 64Cu-AcD10 in lung (1.33 ± 0.23 %ID/g 0.65 ± 0.03 %ID/g and 0.34 ± 0.08 %ID/g at 1 h 4 h and 24 h p.i. respectively) and liver (1.08 ± 0.11 %ID/g 0.72 ± 0.09 %ID/g and 0.55 ± 0.10 %ID/g at 1 h 4 h and 24 h p.i. respectively) is the highly desirable feature for NSCLC imaging. Not only was liver uptake of 64Cu-AcD10 significantly lower than the liver accumulation of the non-acetylated dimers (p < 0.01) in fact it was also at the same level as or less than that was observed for monomeric 64Cu-(M10)2 (). The 64Cu-AcD10 showed highest tumor-to-lung contrast among all the probes (). In sum 64Cu-AcD10 exhibits superior biodistribution properties compared to the other probes. Integrin Specificity To assure that the probes are specific for ?v?6 the four probes were tested in animals bearing xenograft tumors which do not express ?v?6 8. The large cell lung cancer cell line H460 was employed as this cell line does not express ?v?6 as determined by western immunoblotting flow cytometry and immunocytochemistry (). In addition no specific staining of ?v?6 was observed in a H460 tumor harvested from the xenograft model. By comparison strong staining is observed on a similarly treated H2009 tumor. Consistent with these observations the fluorescently labeled dimeric H2009.1-10mer peptide binds H2009 cells but not H460 cells as determined by flow cytometry over a concentration range of 0.10-200 nM. Binding was undetectable on H460 cells even at concentrations as high as 200 nM (A). These data support the use of H460 NSCLC tumors an appropriate ?v?6-negative control. Consistent with the H2009.1-10mer binding ?v?6 the radiotracers showed significantly lower (p < 0.01) uptake in H460 tumor at all three time points compared to the H2009 tumors ( ). Furthermore the ratio of H460 tumor-to-lung and tumor-to-heart ratios are ? 1 at 24 hours for all probes. Most importantly individual probes showed similar biodistribution profile in H2009 and H460 tumor bearing mice (Tables 1 and 2). These data demonstrate the ?v?6-integrin specificity of the probes 64Cu-M10 64Cu-(M10)2 64Cu-D10 and 64Cu-AcD10. Probe uptake is not a general phenomenon of all tumors but requires expression of ?v?6. One concern is the potential for our RGD containing peptide to bind the more common ?v?3 integrin which is a marker for neovasculature and is found on virtually all tumor vasculature. While ?v?6 is expressed specifically in H2009 tumors ?v?3 is expressed in both H2009 and H460 tumors (A) and tumor vasculature. Although our previous data suggests that this peptide is specific for ?v?6 we determined the binding affinity of the H2009.1-10mer peptide on purified ?v?6 ?v?3 and ?v?5 (). The dimeric H2009.1-10mer peptide has a Kd of 0.65 ± 0.26 nM for ?v?6 100 ± 8.5 nM for ?v?3 and 133 nM for ?v?5. "

Lung_Cancer

"contributed to collection of the data. NI: contributed to interpretation of the study data. SI: contributed to interpretation of the radiological data. KW: contributed to the development of the analytic concept data analyses. KI: contributed to interpretation of the study data. KY: contributed to critical revision of the manuscript. 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/1471-2466/14/14/prepub Acknowledgements This work is partially supported by Aichi Health Promotion Foundation and Grant-in-Aid. We thank an experienced medical editor in NAI Inc. for English check and revision. Mathers CD Loncar D Projections of global mortality and burden of disease from 2002 to 2030 PLoS Med 2006 3 11 e442 17132052 Mannino DM Buist AS Global burden of COPD: risk factors prevalence and future trends Lancet 2007 370 9589 765 773 17765526 Sekine Y Behnia M Fujisawa T Impact of COPD on pulmonary complications and on long-term survival of patients undergoing surgery for NSCLC Lung Cancer 2002 37 1 95 101 12057873 Lopez-Encuentra A Astudillo J Cerezal J Gonzalez-Aragoneses F Novoa N Sanchez-Palencia A Prognostic value of chronic obstructive pulmonary disease in 2994 cases of lung cancer Eur J Cardiothorac Surg 2005 27 1 8 13 15736303 Turner MC Chen Y Krewski D Calle EE Thun MJ Chronic obstructive pulmonary disease is associated with lung cancer mortality in a prospective study of never smokers Am J Respir Crit Care Med 2007 176 3 285 290 17478615 Raviv S Hawkins KA DeCamp MM Jr Kalhan R Lung cancer in chronic obstructive pulmonary disease: enhancing surgical options and outcomes Am J Respir Crit Care Med 2011 183 9 1138 1146 21177883 Fabbri LM Luppi F Beghe B Rabe KF Complex chronic comorbidities of COPD Eur Respir J 2008 31 1 204 212 18166598 Brunelli A Charloux A Bolliger CT Rocco G Sculier JP Varela G Licker M Ferguson MK Faivre-Finn C Huber RM ERS/ESTS clinical guidelines on fitness for radical therapy in lung cancer patients (surgery and chemo-radiotherapy) Eur Respir J 2009 34 1 17 41 19567600 Loganathan RS Stover DE Shi W Venkatraman E Prevalence of COPD in women compared to men around the time of diagnosis of primary lung cancer Chest 2006 129 5 1305 1312 16685023 Young RP Hopkins RJ Christmas T Black PN Metcalf P Gamble GD COPD prevalence is increased in lung cancer independent of age sex and smoking history Eur Respir J 2009 34 2 380 386 19196816 Mitsudomi T Kosaka T Endoh H Horio Y Hida T Mori S Hatooka S Shinoda M Takahashi T Yatabe Y Mutations of the epidermal growth factor receptor gene predict prolonged survival after gefitinib treatment in patients with non-small-cell lung cancer with postoperative recurrence J Clin Oncol 2005 23 11 2513 2520 15738541 Matsuo K Ito H Yatabe Y Hiraki A Hirose K Wakai K Kosaka T Suzuki T Tajima K Mitsudomi T Risk factors differ for non-small-cell lung cancers with and without EGFR mutation: assessment of smoking and sex by a case-control study in Japanese Cancer Sci 2007 98 1 96 101 17054433 Zhang J Zhou JB Lin XF Wang Q Bai CX Hong QY Prevalence of undiagnosed and undertreated chronic obstructive pulmonary disease in lung cancer population Respirology 2013 18 2 297 302 23051099 Matsuo M Hashimoto N Usami N Imaizumi K Wakai K Kawabe T Yokoi K Hasegawa Y Inspiratory capacity as a preoperative assessment of patients undergoing thoracic surgery Interact Cardiovasc Thorac Surg 2012 14 5 560 564 22307392 Pellegrino R Viegi G Brusasco V Crapo RO Burgos F Casaburi R Coates A van der Grinten CP Gustafsson P Hankinson J Interpretative strategies for lung function tests Eur Respir J 2005 26 5 948 968 16264058 Rabe KF Hurd S Anzueto A Barnes PJ Buist SA Calverley P Fukuchi Y Jenkins C Rodriguez-Roisin R Van Weel C Global strategy for the diagnosis management and prevention of chronic obstructive pulmonary disease: GOLD executive summary Am J Respir Crit Care Med 2007 176 6 532 555 17507545 Detterbeck FC Boffa DJ Tanoue LT The new lung cancer staging system Chest 2009 136 1 260 271 19584208 Maeda R Yoshida J Ishii G Hishida T Nishimura M Nagai K The prognostic impact of cigarette smoking on patients with non-small cell lung cancer J Thorac Oncol 2011 6 4 735 742 21258254 Rudin CM Avila-Tang E Harris CC Herman JG Hirsch FR Pao W Schwartz AG Vahakangas KH Samet JM Lung cancer in never smokers: molecular profiles and therapeutic implications Clin Cancer Res 2009 15 18 5646 5661 19755392 Matsuda A Matsuda T Shibata A Katanoda K Sobue T Nishimoto H Cancer incidence and incidence rates in Japan in 2007: a study of 21 population-based cancer registries for the monitoring of cancer incidence in Japan (MCIJ) project Jpn J Clin Oncol 2013 43 3 328 336 23296772 Society AC Cancer facts and figures 2013 2013 http://www.cancer./Research/CancerFactsFigures/CancerFactsFigures/2013-cancer-facts-and-figures.pdf Haiman CA Stram DO Wilkens LR Pike MC Kolonel LN Henderson BE Le Marchand L Ethnic and racial differences in the smoking-related risk of lung cancer N Engl J Med 2006 354 4 333 342 16436765 de Torres JP Marin JM Casanova C Cote C Carrizo S Cordoba-Lanus E Baz-Davila R Zulueta JJ Aguirre-Jaime A Saetta M Lung cancer in patients with chronic obstructive pulmonary disease“ incidence and predicting factors Am J Respir Crit Care Med 2011 184 8 913 919 21799072 Okada M Nishio W Sakamoto T Harada H Uchino K Tsubota N Long-term survival and prognostic factors of five-year survivors with complete resection of non-small cell lung carcinoma J Thorac Cardiovasc Surg 2003 126 2 558 562 12928658 Colice GL Shafazand S Griffin JP Keenan R Bolliger CT Physiologic evaluation of the patient with lung cancer being considered for resectional surgery: ACCP evidenced-based clinical practice guidelines (2nd edition) Chest 2007 132 3 Suppl 161S 177S 17873167 Baldwin DR White B Schmidt-Hansen M Champion AR Melder AM Diagnosis and treatment of lung cancer: summary of updated NICE guidance Bmj 2011 342 d2110 21525094 Fukuchi Y Nishimura M Ichinose M Adachi M Nagai A Kuriyama T Takahashi K Nishimura K Ishioka S Aizawa H COPD in Japan: the Nippon COPD epidemiology study Respirology 2004 9 4 458 465 15612956 Tashkin DP Celli B Decramer M Liu D Burkhart D Cassino C Kesten S Bronchodilator responsiveness in patients with COPD Eur Respir J 2008 31 4 742 750 18256071 Kobayashi S Suzuki S Niikawa H Sugawara T Yanai M Preoperative use of inhaled tiotropium in lung cancer patients with untreated COPD Respirology 2009 14 5 675 679 19476597 Bolukbas S Eberlein M Eckhoff J Schirren J Short-term effects of inhalative tiotropium/formoterol/budenoside versus tiotropium/formoterol in patients with newly diagnosed chronic obstructive pulmonary disease requiring surgery for lung cancer: a prospective randomized trial Eur J Cardiothorac Surg 2011 39 6 995 1000 20970351 Radiat Oncol Radiat Oncol Radiation Oncology (London England) 1748-717X BioMed Central 24456714 3946177 1748-717X-9-32 10.1186/1748-717X-9-32 Research Clinical outcome of postoperative highly conformal versus 3D conformal radiotherapy in patients with malignant pleural mesothelioma Krayenbuehl Jr´me 1 Jerome.krayenbuehlusz.ch Dimmerling Peter 1 peter.dimmerlingusz.ch Ciernik I Frank 2 3 ilja.ciernikklinikum-dessau.de Riesterer Oliver 1 oliver.riestererusz.ch 1Department of Radiation Oncology Zurich University Hospital Rmistrasse 100 8091 Zurich Switzerland 2Center for Clinical Research University of Zurich Zurich Switzerland 3Department of Radiotherapy and Radiation Oncology Dessau Municipal Hospital Dessau Germany 2014 24 1 2014 9 32 32 6 9 2013 3 1 2014 Copyright 2014 Krayenbuehl et al.; licensee BioMed Central Ltd. 2014 Krayenbuehl 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. 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 Radiotherapy (RT) is currently under investigation as part of a trimodality treatment of malignant pleural mesothelioma (MPM). The introduction of highly conformal radiotherapy (HCRT) technique improved dose delivery and target coverage in comparison to 3-dimensional conformal radiotherapy (3DCRT). The following study was undertaken to investigate the clinical outcome of both radiation techniques. Methods Thirty-nine MPM patients were treated with neoadjuvant chemotherapy extrapleural pneumonectomy (EPP) and adjuvant RT. Twenty-five patients were treated with 3DCRT and 14 with HCRT (Intensity modulated radiotherapy or volumetric modulated arc therapy). Overall survival disease free survival locoregional recurrence and pattern of recurrence were assessed. A matched pair analysis was performed including 11 patients of each group. Results After matching for gender age histology tumor stage and resection status HCRT seemed superior to 3DCRT with a local relapse rate of 27.3% compared to 72.7% after 3DCRT (p = 0.06). The median time to local relapse was increased by 49% with HCRT in comparison to 3DCRT from 10.9 ± 5.4 months to 16.2 ± 3.1 months (p = 0.06). The median overall survival was 22.3 ± 15.3 months for HCRT and 21.2 ± 9.2 months for 3DCRT (p = 0.57). Recurrence analysis showed that in-field local relapses occurred in previously underdosed regions of the tumor bed in 16% of patients treated with 3DCRT and in 0% of HCRT patients. Conclusions The use of HCRT increases the probability of local control as compared to 3DCRT by improving target volume coverage. HCRT did not improve overall survival in this patient series due to the high rate of distant recurrences. Mesothelioma Radiation therapy Extrapleural pneumonectomy Volumetric modulated arc therapy Intensity modulated radiotherapy Multimodal therapy Introduction Malignant pleural mesothelioma (MPM) is a rare and aggressive malignancy associated with poor prognosis. Although MPM is often initially confined to the hemithorax it has a high potential for metastatic spread in the course of disease [1]. The mainstay of treatment is surgery consisting of either pleurectomy/decortication (PD) or radical extrapleural pneumonectomy (EPP) in combination with cisplatin/pemetrexed and in selected cases postoperative radiotherapy [2-5]. The rationale to apply postoperative radiotherapy after EPP has been the high rate of local recurrence after EPP alone of about 40% [6]. The pattern of pleural dissemination infiltrative growth and the manipulations within the chest cavity during surgery place the entire ipsilateral chest wall at high risk for post-surgical relapse especially at the diaphragm insertion the pericardium mediastinum and bronchial stump. Technically hemithoracic radiotherapy is challenging due to various reasons. Firstly the size of the volume to be treated is large and may cover up to six liters. Secondly the target lies in close proximity to various ans at risk (OAR) such as the heart ipsilateral kidney liver remaining lung esophagus and/or spinal cord. Thirdly the thoracic cavity has a complex shape with its costodiaphragmatic recess extending around the liver and the kidney. Previous publications showed that highly conformal radiotherapy (HCRT) such as intensity modulated radiotherapy (IMRT) or volumetric modulated arc therapy (VMAT) can improve the dose distribution in respect to target coverage and dose to OAR [78]. However to our knowledge there is no clinical study published that investigated and compared clinical outcome after both radiation techniques. In order to verify if the technical improvements introduced with IMRT or VMAT have translated into a clinical benefit we evaluated the clinical outcome of MPM patients treated with chemotherapy surgery and 3DCRT or HCRT at our institution. Material and methods We reviewed the clinical outcome of 39 consecutive patients treated either with 3DCRT (25 patients) or HCRT (11 IMRT patients and 3 VMAT patients). Patient staging was established using FDG-PET/CT and/or conventional thoraco-abdominal CT. The patients with clinical stage T1-T3 N0-2 M0 R0-2 were treated with 3 cycles of preoperative chemotherapy (pemetrexed and cisplatin) followed by EPP and RT [7]. All histological subtypes were accepted for RT. Patients were not selected for this review if they had metastatic disease or a local relapse before the start of RT. The study was approved by the local ethics committee of the University Hospital of Zurich. Radiation techniques The 3DCRT group was treated between 1999 and 2005. These patients were treated with 25 — 1.8 Gy?=?45 Gy to the hemithorax and subsequently in a second series a boost of 7 — 1.8 Gy?=?12.6 Gy was given to the incompletely resected area (total dose 57.6 Gy). Dose calculation was performed on Pinnacle planning system (Philips Medical Systems) for a linear accelerator (Clinac 2100C Varian Medical Systems). Details of the treatment technique have previously been published [7]. HCRT has been used at our institution since 2005 for the treatment of MPM patients. Of the 14 patients treated with HCRT 11 were treated with conventional static field IMRT and 3 patients with rotational IMRT (volumetric arc radiotherapy i.e. Rapid Arc® in the present series). IMRT and VMAT plans achieved similar dose distributions [910]."

Lung_Cancer

"To understand the functional consequences of GWAS loci is challenging and multiple principles for post-GWAS™ functional characterization of genetic loci have been proposed including the exploration of epigenetic mechanisms46. In our study the top GWAS lung cancer loci were strongly associated with methylation levels of CpG sites in nearby gene bodies through cis-regulation and adjusting for smoking status or intensity did not change the results. Furthermore SNPs affecting the DNA methylation of gene bodies (which are typically methylated) were also collectively associated with risk for squamous cell carcinoma after excluding the established GWAS loci and were enriched for genes in cancer pathways. In contrast no enrichment was observed for SNPs affecting the methylation of gene promoters or CGI regions which are typically not methylated in normal tissues. This suggests a potential novel mechanism for genetic effects on cancer risk. In fact gene body-enriched cis-meQTLs outside CGI regions may increase the risk for germline and somatic mutations due to their increased propensity to become mutated1112. Upon spontaneous hydrolytic deamination methylated cytosine residues turn into thymine which are less likely to be efficiently repaired than the uracils that result from deamination of unmethylated cytosine residues. For example about 25% of mutations in TP53 in cancers are thought to be due to epigenetic effects47. Indeed analyses of comprehensive human catalogues of lung tumors have identified frequent G>T mutations enriched for CpG dinucleotides outside CGI regions suggesting a role for methylated cytosine since CGI as we confirmed are usually unmethylated48. A similar signature was recently observed in other tumors14. Thus inherited genetic variation may have a profound impact on carcinogenesis by regulating the human methylome. We observed a high similarity of genetic control on DNA methylation across tissues. Since tissue of origin determines cancer-associated CpG island promoter hypermethylation patterns49 a natural question is whether the genetic regulation of methylation is tissue specific. While the tissue-specificity of eQTLs has been investigated for a few tissues50 for cis-meQTL only a recent investigation was conducted6 showing that 35.7% of 88751 cis meQTLs detected in 662 adipose samples were replicated in ~200 whole blood samples. We found that a large proportion of meQTLs in EAGLE lung samples particularly those with large effect sizes were robustly replicated in breast and kidney tissue samples from TCGA suggesting a high similarity of genetic regulation of methylation across these tissues and related impact on somatic mutation rates1448. The lower replication rate of adipose meQTLs in whole-blood samples6 might be explained by the heterogeneity of different cell types in whole blood and by their more liberal P-value threshold (8.6—10?4) which led to the identification of a large number of weak cis-meQTLs. Compared with cis-regulation trans-eQTL regulation is typically considered to be more complex has smaller effect sizes and is more difficult to be replicated even in the same tissue. However in our study the lung trans-meQTLs are highly reproducible in TCGA lung breast and kidney tissues. Notably this similarity allows mapping meQTLs with substantially improved power by borrowing strength across tissues51. meQTL SNPs are strongly associated with multiple epigenetic marks. Chromatin regulators play a role in maintaining genomic integrity and anization52. We found that meQTL SNPs were strongly enriched for DNase hypersensitive sites and sequences bound by CTCF or modified histones. SNPs could affect these epigenetic marks by several mechanisms such as by affecting the core recognition sequences (exemplified for rs2816057 on chromosome 1 for CTCF) causing loss or gain of a CpG within a binding region which when methylated could affect binding27 or altering the binding sequence for interacting factors53. CTCF could cause changes in epigenetic marks through its multiple key roles including genome anization through mediating intra- and inter-chromosomal contacts5455 the regulation of transcription by binding between enhancers and promoters5456 and the regulation of splicing which may impact tissue specificity during tissue development39. These changes can impact regulation of distant genes and not the genes proximal to the SNPs that would be typically investigated in eQTL studies. This may be one reason for the previously observed lack of correlation between eQTLs and meQTLs347. Future large studies integrating SNP profiles the DNA methylome and transcriptome data through tissue developmental stages will hopefully shed light on this possibility. There may be a myriad of other DNA-binding factors whose binding is directly or indirectly affected by SNPs. For example among the histone marks the strongest enrichment of meQTLs in our study was for H3K4me3 in both SAEC and hAEC cell types. As H3K4me3 is the chromatin mark primarily associated with regulatory elements at promoters and enhancers this suggests a strong influence of meQTLs on regulating gene activity. Unfortunately transcription factor binding data in SAEC or hAEC are not available so we could not test whether SNPs in their core sequence could affect the deposition of epigenetic marks e.g. by recruiting DNA methyltransferases57. It will be important to obtain ChIP data from relevant primary cells for numerous DNA-binding regulatory factors to further elucidate the mechanisms whereby meQTLs and other SNP-affected epigenetic marks arise. In we show here that genetic variation has a profound impact on the DNA methylome with implications for cancer risk tissue specificity and chromatin structure and anization. The meQTL data (Supplementary Data) attached to this manuscript provides an important resource for studying genetic-DNA methylation interactions in lung tissue. Methods Sample collection We assayed 244 fresh frozen paired tumor and non-involved lung tissue samples from Stage I to IIIA non-small cell lung cancer (NSCLC) cases from the Environment And Genetics in Lung cancer Etiology (EAGLE) study18. EAGLE includes 2100 incident lung cancer cases and 2120 population controls enrolled in 2002“2005 within 216 municipalities of the Lombardy region of Italy. Cases were newly diagnosed primary cancers of lung trachea and bronchus verified by tissue pathology (67.0%) cytology (28.0%) or review of clinical records (5.0%). They were 35?79 years of age at diagnosis and were recruited from 13 hospitals which cover over 80% of the lung cancer cases from the study area. The study was approved by local and NCI Institutional Review Boards and all participants signed an informed consent form. Lung tissue samples were snap-frozen in liquid nitrogen within 20 minutes of surgical resection. Surgeons and pathologists were together in the surgery room at the time of resection and sample collection to ensure correct sampling of tissue from the tumor the area adjacent to the tumor and an additional area distant from the tumor (1“5 cm). The precise site of tissue sampling was indicated on a lung drawing and the pathologists classified the samples as tumor adjacent lung tissue and distant non-involved lung tissue. For the current study we used lung tissue sampled from an area distant from the tumor to reduce the potential effects of field cancerization. DNA methylation profiling and data quality control Fresh frozen lung tissue samples remained frozen while approximately 30 mg was subsampled for DNA extraction into pre-chilled 2.0 ml microcentrifuge tubes. Lysates for DNA extraction were generated by incubating 30 mg of tissue in 1 ml of 0.2 mg/ml Proteinase K (Ambion) in DNA Lysis Buffer (10 mM Tris-Cl (pH 8.0) 0.1 M EDTA (pH 8.0) and 0.5% (w/v) SDS) for 24 hrs at 56°C with shaking at 850 rpm in Thermomixer R (Eppendorf). DNA was extracted from the generated lysate using the QIAamp DNA Blood Maxi Kit (Qiagen) according to the manufacturer™s protocol. Bisulfite treatment and Illumina Infinium HumanMethylation450 BeadChip assays were performed by the Southern California Genotyping Consortium at the University of California Los Angeles (UCLA) following Illumina™s protocols. This assay generates DNA methylation data for 485512 cytosine targets (482421 CpG and 3091 CpH) and 65 SNP probes for the purpose of data quality control. Raw methylated and unmethylated intensities were background corrected and dye-bias equalized to correct for technical variation in signal between arrays. For background correction we applied a normal-exponential convolution using the intensity of the Infinium I probes in the channel opposite their design to measure non-specific signal58. Dye-bias equalization used a global scaling factor computed from the ratio of the average red and green fluorescing normalization control probes. Both methods were conducted using the methylumi package in Bioconductor version 2.11. For each probe DNA methylation level is summarized as a ?-value estimated as the fraction of signal intensity obtained from the methylated beads over the total signal intensity. Probes with detection P-values of >0.05 were considered not significantly different from background noise and were labeled as missing. Methylation probes were excluded from meQTL analysis if any of the following criteria was met: on X/Y chromosome annotated in repetitive genomic regions annotated to harbor SNPs missing rate>5%. Because the ?-values for the 65 SNP probes are expected to be similar in matched pair of normal and tumor tissues we performed principal component analysis (PCA) using these 65 SNP probes to confirm the labeled pairs. We then performed PCA using the 5000 most variable methylation probes with var>0.02 and found that the normal tissues were clustered together and well separated from the tumor tissues. We further excluded 5 normal tissues that were relatively close to the tumor cluster. From the remaining 239 normal tissue samples we analyzed 210 with genotype data from a previous GWAS of lung cancer20. Genotype data and genetic association analysis The blood samples were genotyped using the Illumina HumanHap550K SNP arrays in EAGLE GWAS20. The SNPs with call rate >99% minor allele frequency (MAF) >3% and Hardy-Weinberg Equilibrium (HWE) P-value >10?5 were included for analysis. Prior to meQTL analysis each methylation trait was regressed against sex age batches and PCA scores based on methylation profiles. The regression residues were then quantile-normalized to the standard normal distribution N(01) as traits. The genetic association testing was performed using PLINK and R adjusted for the top three PCA scores based on GWAS SNPs to control for potential population stratification. Identification of cis-meQTLs For each CpG methylation probe the cis region was defined as being less than 500kb upstream and downstream from the target CpG-site (1Mb total). A methylation trait was detected to harbor a cis-meQTL if any SNP in the cis region had a SNP-CpG nominal association P-value less than P0 where P0 was chosen to control FDR at 5% by permutations. Here we describe a permutation procedure to choose P0 to control FDR at 5%. For a given P0 let N(P0) be the total number of CpG probes with detected cis-meQTLs and N0(P0) the expected number of CpG probes falsely determined to have cis-meQTLs. FDR is defined as N0(P0)/N(P0). The key is to estimate N0(P0) under the global null hypothesis that no CpG probe has cis-meQTLs. We randomly permuted the genotypes across subjects for 100 times keeping the correlation structure of the 338456 methylation traits in each permutation. Then N0(P0) was estimated as the average number of methylation traits that were detected to harbor cis-meQTL SNPs with nominal P<P0. Control FDR at 5% requires P0=4.0—10?5. The same procedure was applied to detect secondary independently associated cis-meQTL SNPs. With our sample size h2>0.12 is required to detect cis-meQTLs with power greater than 0.8. We note that although we excluded all CpG probes annotated with SNPs there is still the possibility that rare not annotated variants could be associated with the cis-meQTL SNPs. However since common variants and rare variants are known to be poorly correlated and rare variants are uncommon by definition we do not expect this event to be frequent. Identification of trans-meQTLs For each CpG probe the trans region was defined as being more than 500kb from the target CpG-site in the same chromosome or on different chromosomes. For the kth methylation trait with m SNPs in the trans region let (qk1?qkm) be the P-values for testing the marginal association between the trait and the m SNPs. Let pk=min(qk1?qkm) be the minimum P-value for m SNPs and converted pk into genome-wide P-value Pk by performing one million permutations for SNPs in the trans region. Because a cis region is very short (~1M) compared to the whole genome (~3000M) Pk computed based on SNPs in trans regions is very close to that based on permutations using genome-wide SNPs. Thus we use the genome-wide p-value computed based on all SNPs to approximate Pk. Furthermore all quantile-normalized traits follow the same standard normal distribution N(01); thus the permutation-based null distributions are the same for all traits. We then applied the Benjamini-Hochberg procedure to (P1?PN) to identify trans-meQTLs by controlling FDR at 5%. With our sample size h2 >0.24 is required to detect trans-meQTLs with power greater than 0.8. Replication of meQTLs in TCGA samples The replication was performed in TCGA histologically normal tissue samples that had genome-wide genotype (Affymetrix Genome-Wide Human SNP Array 6.0) and methylation profiling (Illumina Infinium HumanMethylation450 BeadChip). We downloaded genotype (level 2) and methylation data (level 3) from the TCGA website22. We also downloaded methylation data for tumor tissue samples and performed PCA analysis to confirm that normal tissue samples were separated from tumor tissue samples. Autosomal SNPs with MAF >3% calling rate >0.99 and HWE P-value > 10?5 were included for imputation using IMPUTE259 and reference haplotypes in the 1000 Genome Project60 (version 2012/03). We only included samples of European ancestry based on EIGENSTRAT analysis. The replication set had 65 lung 87 breast and 142 kidney histologically normal tissue samples after QC. Again each methylation trait was regressed against sex age batches and PCA scores based on methylation profiles. The regression residues were then quantile-normalized to the standard normal distribution N(01) as traits for meQTL analysis. The associations were tested between the quantile-normalized methylation traits and imputed genotypic dosages adjusting for sex age and PCA scores based on SNPs. A genetic association detected in EAGLE lung data was considered replicated if the association had the same direction and FDR<0.05 based on single-sided P-values. Testing genetic associations with methylation and gene expression traits We downloaded gene expression data (level 3) from RNA-seq analysis of 59 histologically normal tissue samples from NSCL patients from TCGA. All samples also had genome-wide genotype data and 28 samples had additional methylation data from Illumina Infinium HumanMethylation450 BeadChips. Regression analysis was performed to test the association of gene expression with methylation levels in the CHRNA5 gene and with methylation levels in PABPC4 STARD3 and SLC35A3 genes. We tested the association between lung cancer GWAS risk SNPs and gene expression using regression analysis under an additive model adjusting for age sex and PCA scores based on genome-wide SNPs. Testing for enrichment of cis-meQTLs in lung cancer GWAS We tested for enrichment in NCI lung cancer GWAS of European ancestry which included three main histologic subtypes of lung cancer (adenocarcinoma (AD) squamous cell carcinoma (SQ) small cell carcinoma (SC)) and a small number of other lung cancer subtypes. We investigated whether the identified cis-meQTL SNPs were collectively associated with lung cancer risk which was tested by examining whether the GWAS P-values for these SNPs deviated from the uniform distribution (i.e. no enrichment). Because the high linkage disequilibrium (LD) in SNPs increased variability of the enrichment statistic and caused a loss of power we first performed LD-pruning using PLINK so that no pair of remaining SNPs had a r2 ?0.8. The enrichment significance was evaluated by 10000 random permutations. The genomic control ?-values61 based on genome-wide SNPs were 1.010.995 0.977 and 1.00 for overall lung cancer AD SC and SQ respectively. Thus the type-I error rates of our enrichment tests were not inflated. The detailed procedure for testing a set of cis-meQTL SNPs is described as follows: Firstly we performed LD-pruning using PLINK so that no pair of remaining SNPs had an r2 ?0.8. Secondly we tested the association for the LD-pruned SNPs (assuming K SNPs left) in a GWAS and computed the P-values (p1?pK). We then tested whether (p1?pK) followed a uniform distribution i.e. no enrichment. Thirdly we transformed P-values into ?12 quantitles qk = F?1(1 ? pk) with F(·) being the cumulative distribution function (CDF) of ?12. We defined a statistic for testing enrichment as Q=?k=1Klog(1?f+fexp(qk/2))3562 where f is a pre-specified constant reflecting the expected proportion of SNPs associated with the disease. Because only a small proportion of SNPs may be associated with the disease we set f=0.05 for this paper. The statistical power was insensitive to the choice of f in the range of [0.01 0.1]62. Finally the significance of the test Q was evaluated by 10000 random permutations. meQTL mediation analysis We investigated whether trans associations were mediated by the methylation levels of CpG probes nearby the trans-acting SNPs. Note that this analysis was only for trans associations with cis effects i.e. the SNP was associated with at least one proximal CpG probes with p<4—10?5. See Fig. 2c. Suppose a SNP G cis-regulates K proximal (<500kb) CpG sites A1?AK with P<4—10?5 and trans-regulates a distal CpG site B. We performed a linear regression: B~?+?G +?kAk. We also computed marginal correlation coefficient cor(GB) and partial correlation coefficient cor(GB|Ak) using an R package œppcor63. A full mediation was detected if G and B were not significantly correlated after conditioning on Ak or equivalently G was not significant (p>0.01) in regression analysis B~?+?G +?kAk for any k. A partial mediation was detected if any Ak had a P<0.05/K (Bonferroni correction) in the regression analysis and |cor(GB)|?| cor(GB|A) |>0.1. An independent effect model (i.e. no mediation) was detected otherwise. Testing enrichment of meQTL SNPs in regulatory regions We obtained peak data for CTCF DNaseI H3K27me3 H3K4me3 and H3K36me of small airway epithelial cells (SAEC) from the ENCODE project and for H3K27me3 H3K4me3 and H3K9-14Ac from human alveolar epithelial cells (hAEC) from our own laboratory. A SNP is determined to be functionally related to a given mark or CTCF binding site if the SNP or any of its LD SNPs (r2 ?0.8 with LD computed using the genotype data of European population in The 1000 Genome Project) resided in any of the mark regions or CTCF binding sites. We explain our enrichment testing using CTCF as an example. We classified genome-wide SNPs into four categories: SNPs not associated with CpG probes in trans or cis (defined as control SNP set) SNPs only associated with proximal CpG probes via cis-regulation (cis-only21119 SNPs) SNPs only associated with distal CpG probes via trans-regulation (trans-only 192 SNPs) and SNPs detected with both trans and cis effects (cis+trans 277 SNPs). For SNPs in the category of cis-only trans-only and cis+trans we computed the proportion of SNPs functionally related to CTCF. To compute the enrichment of cis-meQTLs in CTCF binding sites we defined a control set of SNPs that are not associated with CpG probes via cis- or trans regulation. The selection of the control set was further complicated by the following two observations. (1) cis-meQTL SNPs tended to be more common (data now shown). (2) The probability of a SNP detected as a cis-meQTL SNP positively depended on the density of the CpG probes in the nearby region. Choosing a control set while ignoring these two factors could underestimate the proportion of functionally related SNPs in the control set and thus overestimate the enrichment for cis-meQTLs. Therefore we created 1000 sets of control SNPs with CpG probe density (measured as the number of CpG probes in the cis region of each SNP) and MAF matched with the meQTL SNP set and then averaged the proportions on the 1000 sets. The enrichment was calculated as the fold change with the proportion in the control SNP set as baseline. Next we investigated whether the enrichment was stronger for SNPs more significantly associated with CpG sites. Because we detected only a few hundred trans-meQTLs we focused this analysis on the set of cis-meQTLs. We classified cis-meQTL SNPs into five categories according to the cis-association P-values: P >10?7 (the weakest) 10?10< P ?10?7 10?15 < P ? 10?10 10?20 < P ? 10?15 and P ? 10?20 (the strongest). For each category we computed the proportion of SNPs functionally related to CTCF binding sites. meQTL SNPs affect CTCF binding We found that meQTL SNPs are strongly enriched in CTCF consensus sequences. We used SAEC data from ENCODE to test whether meQTL heterozygous SNPs directly affect CTCF binding by disrupting the CTCF recognition sites. P-values were calculated based on a binomial distribution Binom(N 0.5). Here N is the total number reads covering the SNPs. Raw sequencing data (.fasstq format) from SAEC cells were generated at the University of Washington as part of the ENCODE project and downloaded from the UCSC genome browser. Raw data was aligned to the hg19 genome using CLC genomics workbench (v 5.5.1) parsing out data with less than 80% contiguous alignment to the genome and duplicate reads in excess of 10 copies. We used the CTCFBSDB 2.0 program64 to predict whether the meQTL SNPs or their LD SNPs (r2 ? 0.8) were within CTCF peaks and then examined in SAEC whether CTCF exhibited allele-specific binding. Because common SNPs are more likely to be heterozygous we only looked for SNPs with MAF ?0.4. Here we present two such examples. Systematic investigation of all meQTL SNPs that are heterozygous in SAEC is warranted once more samples with genotypic data are available. Supplementary Material 1 Acknowledgements This study utilized the high-performance computational capabilities of the Biowulf Linux cluster at the NIH Bethesda MD (http://biowulf.nih.gov). We are grateful to the EAGLE participants and the large number of EAGLE collaborators (listed in http://dceg.cancer.gov/eagle) The Cancer Genome Atlas project for the genotype and methylation data and the ENCODE project for the regulatory region data. This work was supported by the Intramural Research Program of NIH NCI Division of Cancer Epidemiology and Genetics and in part by the Norris Comprehensive Cancer Center core grant (P30CA014089) from NCI the Trandisciplinary Research in Cancer of the Lung (TRICL) and the Genetic Associations and Mechanisms in Oncology (GAME-ON) Network (U19CA148127). AW ZW WZ and AH were also funded by the NCI NIH (HSN261200800001E). IALO and ZB were also funded by NIH grants (1 R01 HL114094 1 P30 H101258 and R37HL062569-13) Whittier Foundation and Hastings Foundation. ZB was also funded by the Ralph Edgington Chair in Medicine. CNM was funded by ACS/Canary postdoctoral fellowship (FTED-10-207-01-SIED). Author contributions M.T.L conceived the study. I.A.L.O. supervised DNA methylome analysis. J.S. performed EAGLE TCGA and ENCODE genetic analyses. C.M. performed allele-specific binding analyses. J.D. contributed to genetic analyses and performed GO analyses. J.S. P.L. I.A.L.O. and M.T.L. performed quality control analyses. A.C.P D.C. P.A.B A.W.B N.E.C. and M.T.L. conducted the EAGLE study and provided tissue samples. AW and AH prepared the tissue samples for the analyses. BZ and ZB isolated and cultured alveolar epithelial cells. T.T. and K.D.S. performed methylation normalization. Z.W. and W.W. performed LD analyses. J.S. C.M. J.D. P.L.H. M.C. D.S.L J.H. P-H.C B.S.I.C.W.Z. L.A. M.F. B.P.B. N.C M.A.T. S.J. C. I.A.L.O. M.T.L. contributed to the data interpretation. J.S. and M.T.L. wrote the manuscript. All authors participated in the discussion and reviewed the manuscript."

Lung_Cancer

"In the case of HCRT only one series was applied with 26 — 1.75 Gy?=?45.5 Gy delivered to the hemithorax with a simultaneous integrated boost of 26 — 2.15 Gy?=?55.9 Gy delivered to the R1/R2 region. Planning and dose calculation was performed on the Eclipse planning system (Varian Medical Systems Palo Alto CA) for a linear accelerator (Clinac 6EX or Trilogy Varian Medical Systems). The treatment technique and dose-volume constraints have been previously published [71011]. Follow-Up and recurrence analysis Patients were followed up every three to four months with clinical examinations and CT or PET/CT scans. Local tumor progression or recurrence was defined as an increasing radiographic abnormality within or partially within the irradiation field. Recurrence adjacent to the field border but not in-field was defined as marginal miss recurrence. Regional recurrence was defined as recurrence in close proximity but not within the irradiated field. Tumor recurrence in the contralateral hemithorax or abdominal cavity was classified as a distant recurrence [12]. All in-field recurrences were carefully analyzed by 2 of the authors (JK PD) in order to assess if they occurred in previously underdosed areas by comparing the respective diagnostic image with the radiation therapy treatment plan. Statistics All survival endpoints as well as tumor recurrence were measured from the date of treatment start (neoadjuvant chemotherapy) and were evaluated using the Kaplan Meier Method. In a subset of the cohort a matched pair analysis was performed in order to compare outcome after 3DCRT and HCRT. For this analysis the patients were matched for age preoperative TNM R and histology and sex (except one pair). Results Between 1999 and 2011 39 patients were treated with neoadjuvant chemotherapy and EPP followed by RT. All follow up patients were deceased at the time of this study. Matched pair analysis In the matched pair analysis 11 HCRT and 11 3DCRT patients were matched based on tumor staging resection status tumor histology age and gender (except one pair were the gender was not matched). In each group 3 patients had a tumor stage T1N0M0 with resection R0 and 8 patients tumor stage T2N0M0 with resection R1. Tumor histology was epithelioid for 6 patients and biphasic for 5 patients in each group. The mean age was 59.6 years and 59.8 years for patient™s in the HCRT and 3DCRT group. The median time to local relapse was increased by 49% with HCRT in comparison to 3DCRT from 10.9?±?5.4 months to 16.2?±?3.1 months (p?=?0.06) as displayed in . Three (27.3%) and eight patients (72.7%) had a local relapse after HCRT and 3DCRT respectively. Nine HCRT (81.8%) and nine 3DCRT (81.8%) patients developed metastases within a median time of 18.4?±?10.7 months and 10.9?±?8.6 months (p?=?0.21). The difference in disease free survival between HCRT and 3DCRT was not significant (p?=?0.72). The median overall survivals were 22.3?±?15.3 months for HCRT and 21.2?±?9.2 months for 3DCRT and are displayed in (p?=?0.57). Local control for 11 matched modulated radiotherapy (HCRT) patients and 11 3-dimensional conformal radiotherapy (3DCRT) patients. Overall survival for 11 matched modulated radiotherapy (HCRT) patients and 11 3-dimensional conformal radiotherapy (3DCRT) patients. Outcome of the entire cohort Fourteen HCRT and 25 3DCRT patients were treated and reviewed. Patient™s sex age tumor characteristics and resection are displayed on . Patient demographics and tumor characteristics of 39 patients who underwent neoadjuvant chemotherapy extrapleural pneumonectomy and radiotherapy Characteristic HCRT 3D-CRT n?=?14 n?=?25 Sex ??¢ Male 13 22 ??¢ Female 1 3 Mean age (years) 61 61 Side ??¢ Right 9 15 ??¢ Left 5 10 Tumor histology ??¢ Epithelioid 8 17 ??¢ Biphasic 6 8 Initial Tumor stage ??¢ T1 3 15 ??¢ T2 10 10 ??¢ T3 1 0 Initial Nodal stage ??¢ N0 14 22 ??¢ N1 0 1 ??¢ N2 0 2 Resection ??¢ R0 4 8 ??¢ R1 9 15 ??¢ R2 1 2 Abbreviation: HCRT: Highly conformal modulated radiotherapy 3DCRT: 3-dimensional conformal radiotherapy. The median overall survival was 20.8?±?14.4 months for the HCRT group and 26.9 ± 11.8 months for the 3DCRT group (p?=?0.48). In the HCRT group 10 patients (71%) died of progressive disease and 4 patients (29%) due to intercurrent disease: one patient died of septic shock one of acute myocardial infarction one of progressive biventricular heart failure and another patient who was well and without evidence of disease at two days before his sudden death most likely also died due to a cardiac event. In the 3DCRT group 24 patients (96%) died of progressive disease and one of septic shock (4%). The local control rates were improved after HCRT (p?=?0.30). Four HCRT patients (28.6%) suffered from locoregional relapse in comparison to 15 patients (60%) treated with 3DCRT. Analysis of tumor recurrence For patients treated with HCRT local relapse occurred in-field in 3 patients (21.4%) all within areas that had been treated with doses between 43 Gy to 59 Gy (according to our treatment planning protocol 95% of the prescribed 45 Gy (=43 Gy) should enclose the target volume which in this case is the tumor bed of the hemithorax ) and none in a clearly underdosed region. One patient (7.1%) had a marginal miss recurrence at the field border (13 Gy). In the 3DCRT group twelve patients (48%) had in-field recurrences in regions treated with doses between 30 Gy and 56 Gy (). Notably in 16% of patients treated with 3DCRT (4/25) in-field recurrences occurred in regions that were covered with doses of only 30 to 43 Gy instead of the prescribed ?45 Gy. One patient (4.0%) had a marginal miss recurrence (18 Gy). In one patient with a regional recurrence (4.0%) the delivered dose was not possible to define because no diagnostic CT was available. In one patient (4.0%) the site of recurrence could not be determined because of missing radiographs during follow-up. Locoregional recurrences in patients who underwent highly conformal modulated radiotherapy Localization Age/sex Histology Time to recurrence Dose in recurrence region (Months) (Gray) In-field recurrence 67/m Epithelioid 10 54 In-field recurrence 62/m Biphasic 15 56“59 In-field recurrence 64/m Epithelioid 10 43“50 Marginal miss 65/m Biphasic 17 13 Recurrence Locoregional recurrences in patients who underwent 3D-conformal radiotherapy Localization Age/sex Histology Time to recurrence Dose in recurrence region (Months) (Gray) In-field recurrence 61/m Epithelioid 21 50 In-field recurrence 68/m Biphasic 16 36 In-field recurrence 68/m Epithelioid 13 30 In-field recurrence 65/m Biphasic 19 56 In-field recurrence 65/m Biphasic 16 50 In-field recurrence 50/m Epithelioid 29 48 In-field recurrence 55/m Biphasic 8 50 In-field recurrence 66/m Biphasic 20 36 501 In-field recurrence 68/m Epithelioid 11 32“43 46“501 In-field recurrence 58/m Epithelioid 9 50 In-field recurrence 58/m Epithelioid 11 50 In-field recurrence 65/m Biphasic 19 30 Marginal miss recurrence 55/m Epithelioid 19 18 Regional recurrence 62/m Epithelioid 13 Not available Unknown 56/m Epithelioid 13 Not available 1The recurrence occurred in two separate regions. Distant recurrences occurred in ten patients (71.4%) treated with HCRT and in twenty 3DCRT patients (80%). "

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

" 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

"lung tumors. The use of proton beam therapy can further reduce dose to normal structures. However uncertainty exists in proton-based treatment plans including range uncertainties large sensitivity to position uncertainty and calculation of dose deposition in heterogeneous areas. This study investigated the feasibility of proton transmission beams i.e. without the Bragg peak to treat lung tumors with stereotactic ablative radiotherapy. We compared three representative treatment plans using proton transmission beams versus conformal static-gantry photon beams. It was found that proton treatment plans using transmission beams passing through the patient were feasible and demonstrated lower dose to normal structures and markedly reduced treatment times than photon plans. This is the first study to demonstrate the feasibility of proton-based stereotactic ablative radiotherapy planning for lung tumors using proton transmission beams alone. Further research using this novel approach for proton-based planning is warranted. The authors have no support or funding to report. Introduction Stereotactic ablative radiotherapy (SABR) plays an essential role in the treatment of patients with medically inoperable early stage lung cancer and oligometastasis. The use of protons for lung SABR is emerging as an appealing treatment option because of its potential to deliver higher doses of conformal radiotherapy and spare normal tissues better than traditional photons [1] [2] [3] [4]. This can be achieved because of the natural characteristics of proton beams that deposit its dose at depth with no exit dose referred to as a Bragg peak. However conventional dosimetric models fail to accurately model how protons scatter and deposit dose in highly heterogeneous areas which leads to uncertainties in proton treatment plans [5]. In addition the uncertainties in the stopping power of the various tissues in the body and the interplay effect between spot scanning proton therapy and the target motion leads to large uncertainties in the treatment of lung tumors [5] [6]. In this study we report on the feasibility of proton transmission-beam SABR (PT-SABR) for lung tumors which uses the transmission portion of a spot scanning proton beam i.e. without the Bragg peak. This technique eliminates the major uncertainties of proton therapy mentioned above by having the proton beams pass through the patient. In addition the use of the transmission beam allows an entire field to be treated in one breath hold. This quick treatment and decreased uncertainties lead to smaller planning volumes. To the best of the authors™ knowledge this is the first report on the use of this novel approach to plan SABR with protons without using the Bragg peak which may have dosimetric advantages over photon treatments. Materials and Methods Ethics Statement Written informed consent was obtained from all patients registered in the SABR database. This study including the consent procedure was approved by the Mayo Clinic institutional review board. Patient Cohort Patients were identified from a prospectively collected institutional database of patients treated with SABR. Patients with lung tumors less than one centimetre in maximum dimension were included. The radiation treatment plans of three patients were extracted from the treatment planning system. All patients were treated using three-dimensional conformal multiple static-gantry photon beams. Plans were normalized so that 95% of the planning target volume (PTV) received at least 95% of the prescription dose. The prescription doses for these plans were adjusted to 34 Gy in one fraction based on the recently reported results of Radiation Therapy Oncology Group (RTOG) 0915 which established this dose fractionation regimen as a possible standard dose to be used in future trials [7]. Dose calculations for photon plans used the anisotropic analytical algorithm. Proton Treatment Planning A machine was commissioned in Eclipse v.10 (Varian Medical Systems Palo Alto CA) which allowed for planning and calculating transmission dose plans. The spot size (sigma) of the transmission beam which had an energy of 229 MeV was 2.2 mm. A proton plan that only used the transmission portion of the beam was created for each patient. Proton beam arrangements were selected so that no beams entered through the heart or spinal cord and allowed up to two non-coplanar beams. Four to five beams were used to keep the skin dose comparable to photon plans. The energy of the protons for each spot of a field was 229 MeV; this ensured the Bragg peak was not located within the patient. Dose calculations for the transmission portion of the proton beam were verified with Monte Carlo (Geant4). The proton plans were normalized so that the internal target volume (ITV) receives at least 95% of prescription dose including when range and position errors were included (3.5% and 2 mm) which is standard for spot scanning proton therapy. ITVs were created based on motion of the gross tumor volume in three dimensions using four-dimensional computed tomography image data. The dose constraints from RTOG 0915 were compared for the photon and proton plans as well as the total time that would be required to deliver the treatment. The radiotherapy delivery time per beam was estimated at 1 nC per second for proton therapy which is readily achievable by most spot scanning proton centers and 600 MU per minute for the photon plans. Differences in dosimetric and treatment planning parameters between photon and proton plans were analyzed with two-sided paired t-tests using SAS version 9.2 (SAS Institute Inc. Cary NC). Results The ITVs of the three tumors measured 0.220.42 and 0.99 cubic centimeters. All three proton plans had excellent coverage of the ITV. For all ITVs over 99.4% of the volume received at least 95% of the prescription dose including when uncertainties were examined. This was comparable with the photon plans where 100% of the ITVs received at least 95% of the prescription dose. For most normal tissues lower doses to these ans were achieved with the proton plans compared to the photon plans (). In fact (near) complete sparing of the spinal cord heart and esophagus was possible with protons through careful selection of beam angles (). .0098621.g001 Dose-volume histogram comparison of ans at risk. .0098621.t001 Dosimetric comparison of photon and proton plans. Parameter Photon Proton P-value Mean Range Mean Range Internal target volume (cc) 0.54 0.22“0.99 0.54 0.22“0.99 N/A Spinal cord Maximum dose (Gy) 5.66 2.39“8.07 1.97 0.00“3.06 0.04 Lungs (bilateral) Mean lung dose (Gy) 1.35 0.95“1.92 0.69 0.03“1.36 0.12 V20 (%) 0.66 0.39“1.20 0.49 0.16“1.01 0.06 V5 (%) 7.32 5.4“11.30 6.65 2.96“11.70 0.56 Heart Mean dose (Gy) 8.36 6.27“12.51 0.00 0.00“0.00 0.13 Skin Maximum dose (Gy) 11.75 9.86“13.28 11.40 7.37“16.23 0.89 Esophagus Maximum dose (Gy) 6.49 2.98“9.43 3.40 0.00“7.51 0.05 Homogeneity Index 1.25 1.21“1.29 1.07 1.03“1.11 0.06 Conformity Index 17.14 8.23“30.05 3.47 2.17“4.64 0.15 Proton plans used four to five non-coplanar beams compared to nine to ten beams for photon plans (). The average number of monitor units per field was 818 (range 758“871) with photons and only 38 (range 31“59) with protons. This would translate to an average beam-on time per field of 82 seconds versus 6 seconds for photon and proton plans respectively. These differences in monitor units and beam-on time were statistically significant with P<0.01(). .0098621.g002 Comparison of isodose distributions. Proton (left) and photon (right) treatment plans. .0098621.t002 Comparison of treatment time between photon and proton plans. Parameter Photon Proton P-value Mean Range Mean Range Total monitor units (MU) 7929 6820“8713 178 122“235 <0.01 Fields 9.7 9“10 4.7 4“5 N/A Average MU/field 818 758“871 38 30.5“46.9 <0.01 Beam on time per field (seconds) 81.8 75.6“87.1 5.8 4.7“7.2 <0.01 Discussion Exploiting the transmission beam in proton therapy planning has significant potentials for dose escalation and re-irradiation in lung tumors and eliminates the concern over the uncertainty of the stopping power and its impact on the Bragg peak location. PT-SABR planning requires fewer beams than photons and careful selection of optimal beam angles allows for minimal dose to adjacent normal tissues and tumor dose escalation which may translate to improved local control rates. RTOG 0915 showed that 34 Gy in a single fraction was comparable to 48 Gy in four fractions [7] and the dosimetric constraints from the protocol were easily achieved using both proton and photon plans for patients in this study. Further optimization with proton therapy can allow even higher doses to be delivered while still respecting established dosimetric constraints for normal tissues. This may translate to better tumor control but requires more investigation in a clinical setting. Patients planned with PT-SABR required fewer beams (5 vs. 10) which reduce the total treatment time and the low dose outside the tumor. The average monitor units per field for PT-SABR plans 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 Articles 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; beplerg@karmanos. 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 article 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). The rates of assignment of patients to observation (22%) and chemotherapy (78%) were as expected. CONCLUSIONS 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. To assess DFS the disease status was monitored every 2 months for the first 6 months and subsequently every 3 months by computed tomography after enrollment and according to good medical practice. Toxicities related to the administration of chemotherapy were assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events (version 3.0; ctep.cancer.gov). DFS was defined as the time from the date of enrollment to disease recurrence or death due to any cause and estimated according to the Kaplan-Meier method. A Cox regression model was fit with the time from surgery to enrollment as a covariate to evaluate its effect on DFS. A natural log transformation was applied to the raw protein measurement data and the Pearson correlation coefficient was used to test associations. Bivariate comparison of baseline characteristics between the assigned treatment groups was performed using the Fisher exact test for categorical variables or the Student t test or Wilcoxon rank sum test for continuous variables. A multivariable logistic model to evaluate baseline factors and treatment assignment was fit using backwards selection. Median ERCC1 and RRM1 expression levels were compared with historical medians using the 1-sample Wilcoxon signed rank test. The percentage of patients with both ERCC1 ??65 and RRM1 ??40 was compared with the historical rate using a chi-square test. All statistical analyses and graphics were performed using SAS statistical software (version 9.2; SAS Institute Inc Cary NC). A significance level of 5% was used for all analyses. RESULTS Patient and Trial Characteristics To ensure an adequate sample size of eligible patients and biomarker-specific subgroups a total of 85 patients was registered between April 2 2009 and April 1 2011 from 27 participating sites. Four patients were ineligible; 3 had inadequate lymph node sampling and 1 did not have a tumor measuring ??2 cm. provides the characteristics of the 81 eligible patients. Patient Demographics and Disease Characteristics Variablesa All Patients Assigned to Chemotherapy Assigned to Observation P Refused Assignment Accepted Assignment P N = 81 N = 63 N = 18 N = 20 N = 61 Age y .37 .39 ?Median 64 63.3 68.8 67.2 63.3 ?Mean 63.5 62.9 65.5 65.2 62.9 "

Lung_Cancer

"for use in lung cancer clinical trials Eur J Cancer 1994 30 5 635 642 10.1016/0959-8049(94)90535-5 8080679 Osoba D Zee B Pater J Warr D Kaizer L Latreille J Psychometric properties and responsiveness of the EORTC Quality of Life Questionnaire (QLQ-C30) in patients with breast ovarian and lung cancer Qual Life Res 1994 3 5 353 364 10.1007/BF00451727 7841968 Osoba D Aaronson N Zee B Sprangers M te Velde A Modification of the EORTC QLQ-C30 (version 2.0) based on content validity and reliability testing in large samples of patients with cancer Qual Life Res 1997 6 2 103 108 9161109 Hjermstad MJ Fossa SD Bjordal K Kaasa S Test/retest study of the European anization for Research and Treatment of Cancer Core Quality of Life Questionnaire J Clin Oncol 1995 13 1249 1254 7738629 Bergner M Bobbitt RA Carter WB Gilson BS The Sickness Impact Profile: development and final revision of a health-status measure Med Care 1981 19 8 787 805 10.1097/00005650-198108000-00001 7278416 Pot AM van Dyck R Deeg DJH Ervaren Druk door Informele Z: constructie van een schaal [Self-Perceived Pressure from Informal Care: construction of a scale] Tijdschrift voor Gerontologie en Psychiatrie 1995 26 5 214 219 Given CW Given B Stommel M Collins C King S Franklin S The Caregiver Reaction Assessment (CRA) for caregivers to persons with chronic physical and mental impairments Res Nurs Health 1992 15 4 271 283 10.1002/nur.4770150406 1386680 Nijboer C Triemstra M Tempelaar R Sanderman R van den Bos GAM Measuring both negative and positive reactions to giving care to cancer patients: psychometric qualities of the Caregiver Reaction Assessment (CRA) Soc Sci Med 1999 48 1259 1269 10.1016/S0277-9536(98)00426-2 10220024 Rusbult CE Martz JM Agnew CR The Investment Model Scale: measuring commitment level satisfaction level quality of alternatives and investment size Pers Relat 1998 5 4 357 391 10.1111/j.1475-6811.1998.tb00177.x Lewis FM Fletcher KA Cochrane BB Fann JR Predictors of depressed mood in spouses of women with breast cancer J Clin Oncol 2008 26 8 1289 1295 10.1200/JCO.2007.12.7159 18323552 de Jager-Meezenbroek E van den Berg M Tuytel G Visser A Garssen B Het meten van spiritualiteit als een universeel fenomeen: de ontwikkeling van de Spirituele Attitude en Interesse Lijst (SAIL) [Measuring spirituality as a universal human experience: the development of the Spiritual Attitude and Involvement List (SAIL)] Psychosociale Oncologie 2006 14 3 14 15 Hakkaart- van Roijen L Tan SS Bouwmans CAM Handleiding voor kostenonderzoek: methoden en standaard kostprijzen voor economische evaluaties in de gezondheidsz. [Manual for cost research: methods and unit-prices for economic evaluations in health care] 2010 Diemen: College voor Zverzekeringen [actualized version] Koopmanschap MA Rutten FFH van Ineveld BM van Roijen L The friction cost method for measuring indirect costs of disease J Health Econ 1995 14 2 171 189 10.1016/0167-6296(94)00044-5 10154656 Baer RA Smith GT Lykins E Button D Krietemeyer J Sauer S Walsh E Duggan D Williams JMG Construct validity of the Five Facet Mindfulness Questionnaire in meditating and nonmeditating samples Assessment 2008 15 3 329 342 10.1177/1073191107313003 18310597 Veehof MM ten Klooster PM Taal E Westerhof GJ Bohlmeijer ET Psychometric properties of the Dutch Five Facet Mindfulness Questionnaire (FFMQ) in patients with fibromyalgia Clin Rheumatol 2011 30 8 1045 1054 10.1007/s10067-011-1690-9 21347605 Carmody J Baer RA Relationships between mindfulness practice and levels of mindfulness medical and psychological symptoms and well-being in a Mindfulness-Based Stress Reduction program J Behav Med 2008 31 1 23 33 10.1007/s10865-007-9130-7 17899351 Neff KD The development and validation of a scale to measure self-compassion Self Identity 2003 2 3 223 250 10.1080/15298860309027 Neff KD Vonk R Self-compassion versus global self-esteem: two different ways of relating to oneself J Pers 2009 77 1 23 50 10.1111/j.1467-6494.2008.00537.x 19076996 Kuyken W Watkins E Holden E White K Taylor RS Byford S Evans A Radford S Teasdale JD Dalgleish T How does Mindfulness-Based Cognitive Therapy work? Behav Res Ther 2010 48 11 1105 1112 10.1016/j.brat.2010.08.003 20810101 Treynor W Gonzalez R Nolen-Hoeksema S Rumination reconsidered: a psychometric analysis Cogn Ther Res 2003 27 3 247 259 10.1023/A:1023910315561 Labelle LE Campbell TS Carlson LE Mindfulness-Based Stress Reduction in oncology: evaluating mindfulness and rumination as mediators of change in depressive symptoms Mindfulness 2010 1 28 40 10.1007/s12671-010-0005-6 Horowitz M Wilner N Alvarez W Impact of Event Scale: measure of subjective stress Psychosom Med 1979 41 3 209 218 472086 Brom D Kleber RJ De Schok Verwerkings Lijst [The Impact of Event Scale] Ned Tijdschr Psychol 1985 40 164 168 Sundin EC Horowitz MJ Impact of Event Scale: psychometric properties Br J Psychiatry 2002 180 205 209 10.1192/bjp.180.3.205 11872511 van Aalderen JR Donders ART Giommi F Spinhoven P Barendregt HP Speckens AEM The efficacy of Mindfulness-Based Cognitive Therapy in recurrent depressed patients with and without a current depressive episode: a randomized controlled trial Psychol Med 2012 42 05 989 1001 10.1017/S0033291711002054 22017808 Teasdale JD Segal ZV Williams JMG Ridgeway VA Soulsby JM Lau MA Prevention of relapse/recurrence in major depression by mindfulness-based cognitive therapy J Consult Clin Psychol 2000 68 4 615 623 10965637 Cohen J Statistical power analysis for the behavioral sciences 1988 2 Hillsdale: L. Erlbaum Preacher KJ Hayes AF Asymptotic and resampling strategies for assessing and comparing indirect effects in multiple mediator models Behav Res Methods 2008 40 3 879 891 10.3758/BRM.40.3.879 18697684 W¼rtzen H Dalton SO Andersen KK Elsass P Flyger H Sumbundu A Johansen C Who participates in a randomized trial of Mindfulness-Based Stress Reduction (MBSR) after breast cancer? A study of factors associated with enrollment among Danish breast cancer patients Psychooncology 2012 22 5 1180 1185 22592966 Schofield P Ugalde A Carey M Mileshkin L Duffy M Ball D Aranda SK Lung cancer: challenges and solutions for supportive care intervention research Palliat Support Care 2008 6 281 287 18662422 J Hematol Oncol J Hematol Oncol Journal of Hematology & Oncology 1756-8722 BioMed Central 24565018 3943805 1756-8722-7-15 10.1186/1756-8722-7-15 Research A novel mycobacterial Hsp70-containing fusion protein targeting mesothelin augments antitumor immunity and prolongs survival in murine models of ovarian cancer and mesothelioma Yuan Jianping 1 jyuan2partners. Kashiwagi Satoshi 1 skashiwagipartners. Reeves Patrick 1 pmreevespartners. Nezivar Jean 1 jedone1gmail.com Yang Yuan 1 amyyang1114gmail.com Arrifin Nadiah Hashim 1 Arrifinnadiah.hashim-arrifin09imperial.ac.uk Nguyen Mai 1 mai.nguyencantab.net Jean-Mary Gilberte 1 gilberte.JeanMary001umb.edu Tong Xiaoyun 1 tongxiaoyun2003gmail.com Uppal Paramjit 1 paramjit.uppal10imperial.ac.uk Korochkina Svetlana 1 sv_koroyahoo.com Forbes Ben 1 bforbespartners. Chen Tao 1 tchen5partners. Righi Elda 1 elda.righilibero.it Bronson Roderick 2 roderick_bronsonhms.harvard.edu Chen Huabiao 3 hchen13partners. Orsulic Sandra 4 sandra.Orsuliccshs. Brauns Timothy 1 tBRAUNSPARTNERS. Leblanc Pierre 1 pleblancmclean.harvard.edu Scholler Nathalie 5 nathalie.schollersri.com Dranoff Glenn 6 7 glenn_dranoffdfci.harvard.edu Gelfand Jeffrey 1 jGELFANDPARTNERS. Poznansky Mark C 1 mpoznanskypartners. 1Vaccine and Immunotherapy Center Division of Infectious Diseases Department of Medicine Massachusetts General Hospital 149 13th Street Charlestown Boston MA 02129 USA 2Department of Pathology Harvard Medical School Boston USA 3Ragon Institute of MGH MIT and Harvard University Boston USA 4Women™s Cancer Research Institute Cedars-Sinai Medical Center Los Angeles USA 5Penn Ovarian Cancer Research Center Department of Obstetrics and Gynecology University of Pennsylvania Philadelphia USA 6Department of Medical Oncology and Cancer Vaccine Center Dana-Farber Cancer Institute Boston USA 7Department of Medicine Brigham and Women™s Hospital Harvard Medical School Boston USA 2014 24 2 2014 7 15 15 29 9 2013 2 2 2014 Copyright © 2014 Yuan et al.; licensee BioMed Central Ltd. 2014 Yuan 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 credited. 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 Although dendritic cell (DC) vaccines are considered to be promising treatments for advanced cancer their production and administration is costly and labor-intensive. 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]."

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

"The objective response rate (ORR) for patients with adenocarcinoma (primary endpoint) was 5% (2 partial responses; 1?sided P?=?.372 for null hypothesis [H0]: ORR ? 5%) and 6% (1 partial response) for patients with nonadenocarcinoma. Responders included: 2 of 25 EGFR mutation?positive tumors; 1 of 3 EGFR wild?type with HER2 amplification. Median progression?free survival was 12 weeks overall (n?=?66) and 18 weeks (n?=?26) for patients with EGFR mutation?positive tumors. Common treatment?related adverse events were of grade 1 or 2 severity manageable with standard supportive care and included diarrhea (grade 3 [G3] 12%) acneiform dermatitis (G3 6%) exfoliative rash (G3 3%) dry skin (G3 0%) fatigue (G3 3%) and stomatitis (G3 2%). Six patients (9%) discontinued due to treatment?related adverse events. By patient report NSCLC symptoms of dyspnea cough and pain (chest arm/shoulder) showed improvement first observed after 3 weeks on therapy. S Dacomitinib demonstrated preliminary activity and acceptable tolerability in heavily pretreated patients and may offer benefit in molecularly defined patient subsets. Cancer 2014;120:1145“1154. 2014 The Authors. Cancer published by Wiley Periodicals Inc. on behalf of American Cancer Society. This study investigated the efficacy and safety of dacomitinib in advanced refractory non“small cell lung cancer (NSCLC) selecting for patients with KRAS wild?type tumors to exclude those least likely and simultaneously enrich for those most likely to benefit from therapy. Although the observed response rate was low a number of patients experienced prolonged disease control accompanied by rapid and durable lung cancer symptom relief suggesting that dacomitinib has relevant activity against KRAS wild?type NSCLC. dacomitinib PF?00299804 non“small cell lung cancer erlotinib adenocarcinoma nonadenocarcinoma source-schema-version-number 2.0 component-id cncr28561 cover-date 15 April 2014 details-of-publishers-convertor Converter:WILEY_ML3GV2_TO_NLM version:4.0.5 mode:remove_FC converted:21.05.2014 We thank all of the participating patients and their families as well as the global network of investigators research nurses study coordinators and operations staff. Medical writing support was provided by Christine Arris at ACUMED (Tytherington UK) with funding from Pfizer Inc. Introduction Following failure of chemotherapy and erlotinib treatment options are limited for patients with advanced non“small cell lung cancer (NSCLC). Reversible epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) such as erlotinib and gefitinib selectively target EGFR/HER1 one of the members of the human epidermal growth factor receptor (HER) family and are most effective in cancers harboring EGFR mutations. The remaining members of the HER family comprise HER2 and HER4 tyrosine kinases and the kinase?null HER3.1 HER family members act together via hetero? and homodimerization to enable downstream signaling pathways modulating a range of cellular activities including growth proliferation differentiation and migration.1 In contrast to patients with EGFR?mutation?positive tumors patients with KRAS?mutant NSCLC are unlikely to respond to gefitinib or erlotinib and do not have an improved progression?free survival (PFS) compared with those who have placebo following erlotinib therapy.2 Dacomitinib (PF?00299804) is a potent irreversible oral small?molecule inhibitor of HER1/EGFR HER2 and HER4 tyrosine kinases with antitumor activity in both gefitinib?sensitive and gefitinib?resistant including EGFR T790M preclinical NSCLC models.3 Dacomitinib demonstrated encouraging antitumor activity against NSCLC in Western and Japanese patients in phase 1 studies5 further supported by preliminary data from phase 2 NSCLC studies conducted in Asian patients with KRAS wild?type refractory disease7; unselected patients previously treated with chemotherapy8; and patients with EGFR?mutant disease (first?line treatment).9 This phase 2 trial (ClinicalTrials.gov identifier NCT00548093) assessed the efficacy safety and impact on health?related quality of life (HRQoL) of dacomitinib in patients with KRAS wild?type NSCLC who progressed after 1 or 2 chemotherapy regimens and erlotinib. Materials and Methods Patient Population Main inclusion criteria were age??18 years histologically or cytologically confirmed advanced NSCLC progression on erlotinib and 1 or 2 regimens of chemotherapy confirmation of KRAS wild?type tumor or known EGFR exon 19 deletion or EGFR exon 21 mutation (previously documented EGFR mutation was accepted when insufficient tissue was available for KRAS testing) and Eastern Cooperative Oncology Group performance status (ECOG PS) of 0 to 2. Exclusion criteria included chemotherapy radiotherapy biological or investigational agents or surgery within 4 weeks of study entry; EGFR inhibitors within 2 weeks of study entry; intolerance to erlotinib; prior investigational EGFR?targeted therapy without written agreement of the study sponsor; and uncontrolled or significant cardiovascular disease. Trial Design and Treatment This was a multicenter open?label phase 2 trial. To address differences in the expected response rates between tumors of different histologies10 2 cohorts comprising patients with adenocarcinoma and those without adenocarcinoma (nonadenocarcinoma) were enrolled. Patients received 45 mg of dacomitinib once daily on an empty stomach (2 hours before or after dacomitinib intake) on a continuous basis during a 21?day cycle. Dose interruptions of?<2 weeks without discontinuation from the study were allowed for toxicity; 2 dose attenuation levels of 30 mg and then 20 mg were allowed. Treatment was discontinued for disease progression intolerance (grade 3 or 4 toxicity or intolerable grade 2 toxicity that does not resolve to grade 1 or baseline after 2 weeks' interruption) global deterioration of health?related symptoms protocol noncompliance or patient withdrawal. The primary endpoint was best overall response (BOR) according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.012 for patients with tumors of adenocarcinoma histology. Secondary efficacy endpoints included: BOR in patients with tumors of nonadenocarcinoma histology duration of objective response PFS PFS at 6 months (PFS6M) overall survival (OS) and OS at 6 (OS6M) and 12 (OS12M) months. Other secondary endpoints were safety; patient?reported outcomes (PROs) of HRQoL"

Lung_Cancer

"Therefore using the combined sample from the LSC and PLuSS ROC curves were generated using the 3-gene model the full 11-gene model and covariates-only model in male former smokers (Figure 1). Likelihood ratio tests confirmed that both the 3-gene and 11-gene models are significantly more discriminative than the covariates only model (p?=?0.0002 and p?=?0.002 respectively); however the 3- and 11-gene models were not significantly different from each other (p?=?0.29). Areas under the curve (AUC) were 0.74 and 0.80 for the 3- and 11-gene models respectively while the AUC was 0.55 for the covariates only model. Although sample sizes were small in cohort-stratified analyses of male former smokers these analyses demonstrate that the increased discriminative power of the 3-gene model is observed in two independent cohorts (Additional file 1: Figure S1). ROC curves comparing the sensitivity and specificity of 3- and 11-gene methylation panels for classifying CMH. ROC curves were generated by applying logistic regression models to male former smokers (n?=?139) from the combined PLuSS and LSC. The covariates included age pack years education and COPD. AUC is indicated in parentheses. Discussion This study demonstrates a significant association between CMH and prevalence of promoter methylation in sputum of lung cancer risk genes in two geographically distinct cohorts. This association was especially strong in males and in former smokers and SULF2 was the most consistently associated gene. Importantly the overall association between CMH and methylation and the specific effects of sex and smoking status were observed independently in both cohorts. Combining the two cohorts strengthened the statistical significance of these associations. The central finding of our study is that male former smokers with unresolved CMH may be at an increased risk of lung cancer. Given that 50% of persons diagnosed with lung cancer are former smokers prospective studies evaluating the methylation status of former smokers with CMH who subsequently develop lung cancer are needed [26]. The eleven genes examined in this study were selected based on prior evidence that they are associated with lung cancer risk [1718]. Therefore increased prevalence of methylation of these genes may predict lung cancer among subjects with CMH. These gene promoters have all been shown to be methylated in tumors [2728] and are proposed to represent an expanding field of precancerous epigenetic changes in the aerodigestive tract of smokers [1729]. This hypothesis is supported by the observation that the methylation prevalence of these gene promoters increases as the time to lung cancer diagnosis decreases [17]. Mounting evidence indicates that these changes are causal for tumor initiation [30-33]. The association between methylation and CMH was markedly stronger in males than in females (Table 4). Univariate analysis of males and females in both cohorts (Additional file 1: Tables S3 and S4) reveals that females with CMH are significantly younger than female controls in the LSC; however this was not true in the PLuSS. Additionally age was a covariate in all adjusted analyses and thus is unlikely to account for the lack of association between methylation and CMH in females. This apparent protective mechanism in females warrants further study. The association between methylation and CMH was also stronger in former than in current smokers (Table 5). The increase in effect size in former smokers may be due to several reasons: (1) the CMH phenotype in former smokers may not be confounded by cough and phlegm caused by irritation due to current smoking; (2) in susceptible smokers CMH that persists in spite of smoking cessation may represent a phenotype with a more distinct molecular pathology; (3) The association between CMH and gene promoter methylation may be stronger with age. In the LSC and PLuSS cohorts former smokers were significantly older than current smokers (mean age difference 4.2 years data not shown). This age difference between former and current smokers also likely explains the puzzling observation that current smokers have lower overall methylation compared to former smokers (Additional file 1: Table S2); current smokers are younger and younger age is associated with less total methylation in these lung cancer risk genes. Numerous studies have demonstrated that prior CMH significantly increases the risk for later development of lung cancer (reviewed in [1516]). Assessment of the latency period between diagnosis of CMH and diagnosis of lung cancer has shown that this risk increases with time since diagnosis of CMH [34]. In one study [34] the odds ratio nearly quadrupled at latency >15 years compared to latency 1“5 years. Importantly this suggests that CMH may serve as a precursor to lung carcinogenesis [34]. We hypothesize that the increased prevalence for methylation of the lung cancer risk genes seen in this study may help explain the epidemiological link between CMH and lung cancer. Further studies are needed to establish a direct link between gene methylation and lung cancer. Interestingly while SULF2 p16 JPH3 and PCDH20 all demonstrate evidence for association with CMH in the current study a previous study determined that GATA4 promoter methylation was associated with airflow obstruction [35]. These findings suggest that major differences exist in the genes affected by aberrant promoter methylation in distinct COPD sub-phenotypes. This is consistent with the major pathophysiological differences that underlie emphysema and chronic mucous hypersecretion [36] and suggests the role basal cell hyperplasia may play in development of lung cancer [37]. Of the 11 genes analyzed SULF2 demonstrated the strongest association with CMH. SULF-2 is an extracellular enzyme that catalyzes the hydrolysis of 6-O-sulfo groups from heparan sulfate polysaccharides [38-40]. Heparan sulfate proteoglycans (HSPGs) are widely distributed on cell membranes and the extracellular matrix and serve as coreceptors for many growth factors and cytokines [41] and the position of 6-O sulfates is of particular importance for ligand binding [38-40]. Epigenetic inactivation of SULF2 either by siRNA treatment or promoter methylation activates numerous type I interferon (IFN)-inducible genes [42]. It was proposed that silencing of SULF2 prevents the removal of sulfate groups from IFN-binding sites which may preserve either the binding affinity or bioavailability of interferons leading to increased transcription of multiple IFN-inducible genes [42]. It is plausible that CMH caused by metaplastic mucous cells that are sustained due to dysregulated cell death mechanisms that involve IFN signaling [43-45] creates an inflammatory milieu which causes methylation of SULF2. In turn the type 1 interferon response induced by methylation of SULF2 may help to perpetuate the inflammation associated with CMH. This is the first report of epigenetic changes in the airways of individuals with CMH. Strengths of the study include the use of the large well-characterized LSC for the initial phase of study and excellent replication of all main findings in the geographically distinct PLuSS. We chose the standard definition for chronic bronchitis in the LSC and a definition that most closely captured the standard clinical definition of chronic bronchitis in the PLuSS. While the differences in questionnaires used to define CMH could be considered a limitation in the study the definition for CMH was applied to PLuSS subjects prior to any data analysis and was not subsequently modified. We propose that this approach improves the rigor of our validation. "

Lung_Cancer

"This is a requirement for all labs that are accredited to the ISO 15189:2012 standard (International anisation for Standardization (ISO) 2010). A fundamental aspect of all diagnostic testing is the accurate reporting of the results. The quality of reports submitted was acceptable with a large proportion being comprehensive stand-alone documents containing most of the basic core elements. However the report is meaningless if the referring clinician cannot easily extract the relevant information. Therefore it is essential that that the report is clear concise and easy to read. Many of the reports obscured the take home message and there was often a lack of clarity and balance between the test information and the clinical context. Standardisation of the reporting and naming of mutations is also important and we assessed labs against the nomenclature guidelines from the Human Genome Variation Society (HGVS) (Human Genome Variation Society (HGVS) 2014). For example we considered that it was not acceptable to report the amino acid change only as redundancy in the genetic code means that different changes at the nucleotide level can result in the same change at the amino acid level. In the results of this EQA scheme suggest that the technical quality of EGFR mutational analysis could be improved as evidenced from a high level of diagnostic errors. Overall the standard of reporting was acceptable. These findings also underline the importance of EQA as a mechanism to reveal errors in methodology and to ensure an adequate quality of molecular testing. Regular participation in EQA should be seen as a routine part of the diagnostic testing process for all labs helping to improve and standardise their processes. We have established a model for a robust and scalable EQA that can contribute to global optimisation and improvements in the overall quality of EGFR testing for patients with NSCLC. "

Lung_Cancer

"Then 1—106 freshly prepared cells were suspended in 100 µl PBS and stained with combination of fluorochrome-coupled antibodies to CD11b and Gr1. Cells were collected by FCM. Data were analyzed with FlowJo software. Western Blot Analysis The western blot analysis was performed as described previously with some modifications [33]. Briefly 4T1 cells were treated with niclosamide in designed concentration for 24 hours then cells were washed with cold PBS twice and lysed in RIPA buffer. Protein concentrations were measured using the Lowry method and equalized before loading. Equal amounts of total protein from each sample was applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Amersham Bioscience Piscataway NJ). After electrophoresis the membranes were blocked for 2 h at 37°C and incubated with specific primary antibodies overnight at 4°C followed by the secondary antibody conjugated to horseradish peroxidase. The reactive bands were detected using a commercially available enhanced chemiluminescence kit (Amersham Piscataway NJ). Boyden Chamber Migration and Invasion Assay Boyden chamber (8 µm pore size) migration assay was performed as previously described with some modification [34]. Briefly 5—104 4T1 cells or MDA-MB-231 cells in 100 µl serum-free medium were added in the top chamber then 600 µl of medium with 10% FBS was added to the bottom chamber. Different concentrations of niclosamide were added in both chambers. Cells were allowed to migrate for 20 hours. Non-migrated cells in the top chamber were removed. The migrated cells were fixed in 4% paraformaldehyde and stained with 0.5% crystal violet. Migrated cells were counted and photographed under a light microscope. Invasion assay was conducted according to previous study [34]. Briefly the upper surface of the transwell plate was coated with 60 µl Matrigel (BD Biosciences). After Matrigel polymerization the bottom chambers were filled with 500 µl medium containing 10% FBS. 5—104 4T1 cells or MDA-MB-231 cells in 100 µl serum-free medium were added in the upper part of each transwell and treated with different concentrations of niclosamide. After incubation for 20 hours non-migrated cells on the top side of the filter were removed and migrated cells were fixed with 100% methanol and stained with 0.5% crystal violet then migrated cells were counted and photographed under a light microscope. Percentage of migrated cells inhibited by niclosamide was quantified. Mice and Tumor Model All animal experiments were approved and conducted by the Institutional Animal Care and Treatment Committee of Sichuan University in China (Permit Number: 20121101). Female BALB/c mice (Six- to eight-week-old) used in this study were obtained from Beijing HFK bioscience CO. Ltd Beijing China. Briefly 100 µL 4T1 tumor cell suspension containing 1.0—106 cells were injected subcutaneously in the right flank of BALB/c mice. About seven days after inoculation tumor cells the tumor-bearing mice were randomized into three groups (8 mice per group) and received intraperitoneally injection (i.p.) of niclosamide 20 mg/kg 10 mg/kg or vehicle respectively once daily for 21 days. Tumor volumes and body weight were assessed every three days. The tumor size was calculated according to the formula: Tumor volume (mm3)?=?0.52—L—W2 where L is the length and W is the width. Moreover when all animals were euthanized by cervical dislocation the lungs were harvested total number of lung metastases was counted. Immunohistochemistry Immunohistochemistry staining of tumor sections were described previously [18]. One part of paraffin tumor sections was stained with hematoxylin and eosin (H&E). The other part was stained with Ki67 cleaved casepase-3 VEGF antibodies using immunohistochemistry staining to investigate tumor cell proliferation and apoptosis respectively. In addition paraffin-embeded tumor sections were stained with an anti-CD31 antibody to examine blood vessel density. Images were taken with Leica microscope (Leica DM4000B). Toxicity Evaluation To test potential side effects or toxicity on mice during the treatment all the animals were observed continuously for relevant indexes such as body weight anorexia diarrhea and other clinical symptoms. At the 28th day all animals were euthanized by cervical dislocation after taking blood from eyeball. Blood was obtained for blood routine analysis by Nihon Kohden MEK-5216K Automatic Hematology Analyzer. The tissues of heart lung spleen liver and kidney were stained with H&E for histopathologic examination. Statistical Analysis Data represented as means±SD of three independent experiments. The statistical comparisons were made by Student™s T test and statistically significant p values were labeled as follows: *P<0.05; **P<0.01; ***P<0.001. Results The Anti-proliferation Effects of Niclosamide against Breast Cancer Cells In order to investigate whether niclosamide has direct effects on breast cancer cells we tested the proliferation inhibition caused by niclosamide treatment on different breast cancer cell lines by MTT. After exposure to niclosamide for 72 h the IC50 of MDA-MB-231 MCF-7 MDA-MB-468 were 0.95 µM 1.05 µM and 1.88 µM respectively (A). Exposure of 4T1 cells to niclosamide for 24 h 48 h and 72 h respectively resulted in decrease of the cell proliferation (B). Therefore these results demonstrated that niclosamide inhibited breast cancer cells proliferation in a time- and concentration-dependent manner. Thus we chose 4T1 and MDA-MB-231 cell lines for further experiments. .0085887.g001 The effect of niclosamide on breast cancer cells viability. (A) Proliferation of MDA-MB-231 MCF-7 MDA-MB-468 cells treated with various concentrations (0“10 µM) of niclosamide for 72 hours respectively. Cell viability was detected by MTT assay. The data are expressed as the means ± SD from three independent experiments. (B) MTT assays showed niclosamide inhibited 4T1 breast cancer cells proliferation concentration- and time-dependently. Values represented means ± SD from three experiments (*p<0.05; **p<0.01; ***p<0.001). (C) The effects of niclosamide (a-f:0“1.25 µM) on colony formation in 4T1 cells 12 days the statistic results of colony formation assays presented as surviving colonies. Data are expressed as means ± SD from three experiments (*p<0.05; **p<0.01). (D) The fluorescence microscopic appearance of Hoechst 33342 staining nuclei of 4T1 cells with various concentration niclosamide for 24 h (40—). Data are the representative from three parallel experiments. To further determine whether niclosamide could inhibit the proliferation of 4T1 we conducted colony formation assay after niclosamide treatment. As shown in C clonogenic assay clearly showed that clone formation of 4T1 cells was reduced in a concentration-dependent manner after exposure to niclosamide. Furthermore the size of the colonies treated with niclosamide was significantly smaller than the control. Induction of Apoptosis by Niclosamide As D indicates the 4T1 cells exhibited features of apoptosis as showed by Hoechst 33342 staining such as bright-blue fluorescent condensed nuclei nuclear fragmentation and reduction of cell volume. To further confirm the induction of apoptosis in 4T1 cells with niclosamide treatment we also investigated the levels of apoptosis using the AnnexinV-FITC/PI dual-labeling technique by FCM. As shown in A and B after niclosamide treatment for 24 h the apoptosis induction effect was apparently observed. When the 4T1 cells were treated with 1.25 µM niclosamide the apoptosis rate was 13.7% whereas the apoptosis cells increased to 19.0% 25.5% and 31.3% when cells were treated with 2.5 µM 5 µM and 10 µM niclosamide respectively. Moreover we examined Bcl-2 Mcl-1 Survivin and cleaved caspase-3 expression levels in 4T1 cells after niclosamide-treated for 24 h by western blotting analysis. The expression of Bcl-2 Mcl-1 and Survivin significantly decreased while that of cleaved caspase-3 increased in a concentration-dependent manner (C) which was coincident with the results of Hoechst 33342 staining and FCM assays. .0085887.g002 Niclosamid induces 4T1 breast cancer cells apoptosis. (A) 4T1 cells were treated with niclosamide at indicated doses for 24 hours and the level of apoptosis was evaluated using the Annexin V/PI dual-labeling technique as determined by FCM. Data shown are representative of three independent experiments. (B) Statistic results of apoptosis assays 4T1 cells positive for both Annexin V and PI were considered apoptotic. Data are expressed as means ± SD from three independent experiments (*p<0.05; **p<0.01). (C) Western blot analyses of 4T1 cells treated (24 h) with different concentrations of niclosamide to evaluate protein expression of Bcl-2 Mcl-1 Cleaved caspase-3 Survivin and ?-actin was employed as a standard. Niclosamide Suppresses Breast Cancer Cell Migration and Invasion Breast cancer metastasis poses a predominant threat to cancer related mortality. Moreover one of the key steps in successful cancer metastasis is tumor cell migration and invasion [34] [35]. Therefore in order to examine whether niclosamide could inhibit breast cancer cell migration and invasion we performed transwell migration and invasion assays on 4T1 and MDA-MB-231 cell lines. As shown in Figure 3A niclosamide-treated groups showed reduced migrated cell numbers on 4T1 cells similar results was obtained in invasion assay (Figure 3B). Meanwhile niclosamide obviously inhibited MDA-MB-231 migration and invasion were also observed (Figure 3C). Moreover we also investigated whether Stat3 Focal Adhesion Kinase (FAK) and Src which are considered to be related with cell migration and invasion are involved in niclosamide-mediated migration and invasion [34]. As Figure 3D indicates niclosamide treatment decreased the expression of phosphorylated-STAT3 (Tyr705) phosphorylated-FAK (Tyr925) and phosphorylated-Src (Tyr416) without affecting their total expression level. Taken together these results suggested that niclosamide could suppress breast cancer cell migration and invasion in a concentration-dependent manner. .0085887.g003 Figure 3 Niclosamide inhibits breast cancer cell 4T1 and MDA-MB-231 migration and invasion and inhibits FAK-involved pathway. (A) A total of 5—104 4T1 cells were seeded in the top chamber of transwell with serum-free medium and treated with different concentration of niclosamide. After 20 hours migrated cells were stained photographed (10—) and quantified. (B) A total of 5—104 4T1 cells were treated with various concentration of niclosamide and allowed to invade through Matrigel and Transwell membrance Invaded cell number was stained photographed (20—) and counted. (C) Niclosamide inhibited MDA-MB-231 migation and invasion. The number of migrated cells and invaded cells was counted respectively. Data represent means ±SD. (n?=?3 in triplicate; *p<0.05; **p<0.01; ***p<0.001). (D) 4T1 cells were treated with different concentration of niclosamide (0“5 µM). After 24 hours cell lysates were blotted with spectfic antibodies (anti-phospho-STAT3 anti-phospho-FAK anti-phospho-Src STAT3 FAK and Src) ?-actin was the loading control. Anti-tumor Efficacy of Niclosamide in 4T1 Mouse Mammary Tumor Model To study the antitumor activity of niclosamide in vivo 4T1 tumor-bearing mice were treated with niclosamide at the dose of 10 mg/kg and 20 mg/kg. From the results (Figure 4A) it was found that the tumor growth of the niclosamide groups become slowed 7 days after treatment. After 21 days treatment niclosamide substantially suppressed tumor growth in a dose-dependent manner compared with the control. Moreover after treatment with niclosamide for 21 days body weight of the mice were statisticed and no significant differences in body weight were found among the three groups (Figure 4B). Furthermore previous studies have showed that 4T1 mouse breast cancer have a high metastatic potential and spontaneously metastasize to secondary foci from the primary sites and one of the fatal metastatic organs is lung as early as 2 weeks after inoculation [36] [37]. In the present study we seek to evaluate whether treatment of niclosamide could reduce the occurrence of lung metastasis. The data in Figure 4C and D showed that niclosamide-treated at 20 mg/kg resulted in significant reduction in the number of lung metastases compared with other groups. In addition histological analyses proved that the number of micrometastatic nodules per field in the niclosamide-treated at 20 mg/kg group was also significant fewer than other groups (Figure 4C). These results further indicated that high dose of niclosamide could inhibit tumor metastasis in breast cancer. .0085887.g004 Figure 4 Effect of niclosamide treatment on primary tumor growth and pulmonary metastasis. (A) 4T1 tumor-bearing female BALB/c mice were treated as described with vehicle niclosamide at 10 and 20 mg/kg the mean tumor volumes ± SD of six mice per every group. (B) After 28 days of tumor cell inoculation the body weight of the niclosamide treatment and vehicle groups were statisticed and there were no significant difference among the groups. (C) Lung metastatic nodules were visualized to show the inhibitory effect of niclosamide on 4T1 tumor 21 days after treatment. Arrow indicated metastatic nodules (up) The H&E staining of lungs from each group (10—). (D) The mean lung metastasis nodules of each group the treatment with niclosamide at 20 mg/kg resulted in significant inhibition of lung metastasis versus vehicle control. "

Lung_Cancer

"In the liver lymphatic vessels run parallel to the interlobular vessels and bile duct. Lymphatic vessels can be classified as deep or superficial lymphatic collecting ducts. Superficial lymphatic collecting ducts are often found in the connective tissues of the liver capsule and the lymph is transferred into the parasternal paracardial and abdominal lymph nodes. The deep lymphatic collecting ducts connect to one another to form upstream and downstream trunks which transport lymph into the phrenic lymph nodes around the terminal segment of the inferior vena cava and hepatic and left gastric lymph nodes. Thus lymphatic metastasis of liver carcinoma may be found in the hepatic hilar upper abdominal and retroperitoneal lymph nodes because of the parallel distribution between lymphatic vessels and major abdominal blood vessels. In addition skip metastasis may be found in several groups of lymph nodes. Overall PLC is rare in liver carcinoma. To date to the best of our knowledge PLC has not been identified in patients who have undergone liver transplantation due to liver carcinoma. PLC patients usually develop progressive dyspnea cough weight loss fatigue and other symptoms accompanied by hypoxemia restrictive ventilatory dysfunction and diffusion dysfunction [5]. The history of cancer or surgery and characteristic features identified on lung CT scans can be used to diagnose PLC after exclusion of interstitial pneumonia pulmonary fibrosis sarcoidosis pulmonary embolism heart failure and hematogenous disseminated pulmonary tuberculosis. Biopsy and subsequent pathologic examination are not required for the diagnosis of PLC [69]. In the early stages lung CT shows interstitial lesions linear and reticular shadows and interlobar fissure thickening. Approximately one-third of PLC patients present with pleural effusion (unilateral or bilateral). Once patients develop dyspnea other findings may be present including irregular thickening of the tracheal vascular bundles and interlobular septa as well as multiple beaded small nodules of varying sizes (usually smaller than 3 mm in diameter) distributed along the interlobular septa and pleura. In our present case report the patient was suspected to have interstitial pneumonia due to Pneumocystis carinii after transplantation. Thus management was geared toward removing the edema and treating the pulmonary infection (SMZ and caspofungin); however the response to treatment was poor. On the basis of examination of cancer cells in the pleural effusion and the PET-CT scan PLC was subsequently diagnosed. The positive rate of cancer cells in pleural effusion is 40% to 50%. This diagnostic accuracy rate may increase if the sediment from the pleural effusion is used for examination after being kept for 24 hours. Although biopsy via bronchoscopy pleurocentesis lung puncture or thoracoscopy and subsequent pathologic examination may confirm the diagnosis of PLC biopsy increases the risk of pneumothorax. Sputum collection is relatively easy but examination of exfoliated cells in the sputum is associated with a low positive rate [3]. Lung CT and PET-CT findings and cytology from the pleural effusion can confirm the diagnosis of PLC. Although a false-negative diagnosis of primary liver carcinoma is possible with the use of PET-CT (40% to 50%) PET-CT has favorable sensitivity in the detection of extrahepatic metastasis of liver carcinoma. Acikgoz et al. [10] reported that the detection rate of extrahepatic metastatic foci ?1 cm in diameter was as high as 92.9% in liver carcinoma patients after liver transplantation. There is evidence that the specificity of PET-CT for PLC is 100% and that the sensitivity is 86%. The mean SUV in the region of PLC (1.37?±?0.64) was significantly greater than that in the normal lung (0.5?±?0.29) (P?<?0.0001) [11]. Thus combined examinations have an elevated detection rate compared to a single examination. Examinations selected according to the disease condition may significantly increase the detection rate. To date no effective strategies have been developed for the treatment of PLC. Currently antitumor therapy and antispasmodic therapy of the airway with theophylline or ?2-adrenergic receptor agonists are used. However these treatments usually have poor efficacy and PLC is associated with a poor prognosis. Patients usually develop progressive dyspnea and die as a result of respiratory failure and/or heart failure. Approximately 50% to 85% of PLC patients have a survival time between 3 and 6 months [1213]. In our patient PLC progressed rapidly because of immunosuppression after liver transplantation. Although immunosuppressive therapy was discontinued promptly the severity of the patient™s symptoms increased rapidly and he died as a result of respiratory failure within 1 month. In 1975 Kane et al. [14] reported the autopsy findings from 7524 patients with solid cancers that originated from the prostate breast stomach pancreas and liver. Involvement of the pulmonary lymphatic system by cancer cells was noted in 1085 patients (only 1% of these patients died as a result of respiratory failure). Although PLC is rarely reported in liver carcinoma the incidence of liver carcinoma“induced PLC might be far higher than previously reported. In addition liver carcinoma is highly malignant and progresses rapidly."

Lung_Cancer

"s with the gatekeeper T790M mutation [34] monotherapy with a mutant-selective EGFR-TKI may not be sufficient to inhibit the growth of tumors with acquired resistance to gefitinib. Our findings suggest that the combination of a MEK-TKI and a PIK3/mTOR-TKI may be effective in controlling these resistant tumors. Many KRAS-mutant cancer cells have been shown to be sensitive to MEK inhibitors [35] and KRAS mutations can be detected in up to 30% of lung cancers dependent upon histology and ethnicity [36-38] suggesting that a subset of lung cancers would likely be highly sensitive to AZD6244. Our finding that AZD6244 was effective in one distinct KRAS mutant human lung cancer NCI-H460 models supports and validates this hypothesis. Although monotherapy with AZD6244 resulted in antitumor and some antiangiogenic effects in all of our lung cancer models the antitumor effects were more apparent in the NCI-H1993 lung adenocarcinoma model. The increased antitumor efficacy observed in this model is associated with differences in the inhibitory effect of p-AKT signaling pathway in NCI-H1975 and NCI-H460 lung tumors. However additional studies are needed to elucidate this phenomenon. In this study we evaluated therapy directed against MEK and PI3K/mTOR in distinct gefitinib-resistant NSCLC xenograft models. MEK or PI3K/mTOR inhibition resulted in antitumor effects for these gefitinib-resistance NSCLC models by blocking key intracellular pathways controlling cell proliferation and survival as demonstrated both in vitro and in vivo. Surprisingly PI3K/mTOR inhibition by BEZ235 also suppressed lung tumor angiogenesis and targeted both MEK and PI3K/mTOR activation in lung tumors resulting in substantial antiangiogenic effects which may due to the significantly reduced expression of MMP-9 in tumors. We concluded that the combination of a selective MEK inhibitor and a PI3K/mTOR inhibitor was effective in suppressing the growth of gefitinib-resistant tumors caused by EGFR T790M mutation MET amplification and KRAS/PIK3CA mutation. These findings represent a promising strategy for the treatment of gefitinib-resistant NSCLC and provide a strong basis for the design of clinical trials for this purpose. Competing interests The authors declare that they have no competing interests. Authors™ contributions YQQ conceived and designed the experiments. XXW YHY YY and HL performed the experiments. XXW YHY and DDM analyzed the data. XXW YHY and HL wrote the paper. YQQ supervised the whole experimental work and revised the manuscript. All authors read and approved the manuscript. She J Yang P Hong Q Bai C Lung cancer in China: challenges and interventions Chest 2013 143 1117 1126 23546484 Weir HK Thun MJ Hankey BF Ries LA Howe HL Wingo PA Jemal A Ward E Anderson RN Edwards BK Annual report to the nation on the status of cancer 1975“2000 featuring the uses of surveillance data for cancer prevention and control J Natl Cancer Inst 2003 95 1276 1299 12953083 Schiller JH Harrington D Belani CP Langer C Sandler A Krook J Zhu JM Johnson DH Comparison of four chemotherapy regimens for advanced nonsmall-cell lung cancer N Engl J Med 2002 346 92 98 11784875 Pao W Chmielecki J Rational biologically based treatment of EGFR mutant non-small-cell lung cancer Nat Rev Cancer 2010 10 760 774 20966921 Bean J Brennan C Shih JY Riely G Viale A Wang L Chitale D Motoi N Szoke J Broderick S Balak M Chang WC Yu CJ Gazdar A Pass H Rusch V Gerald W Huang SF Yang PC Miller V Ladanyi M Yang CH Pao W Met amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib Proc Natl Acad Sci U S A 2007 104 20932 20937 18093943 Engelman JA Zejnullahu K Mitsudomi T Song Y Hyland C Park JO Lindeman N Gale CM Zhao X Christensen J Kosaka T Holmes AJ Rogers AM Cappuzzo F Mok T Lee C Johnson BE Cantley LC Jnne PA MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling Science 2007 316 1039 1043 17463250 Sequist LV Waltman BA Dias-Santagata D Digumarthy S Turke AB Fidias P Bergethon K Shaw AT Gettinger S Cosper AK Akhavanfard S Heist RS Temel J Christensen JG Wain JC Lynch TJ Vernovsky K Mark EJ Lanuti M Iafrate AJ Mino-Kenudson M Engelman JA Genotypic and histological evolution of lung cancers acquiring resistance to EGFR inhibitors Sci Transl Med 2011 3 75ra26 Pao W Wang TY Riely GJ Miller VA Pan Q Ladanyi M KRAS mutations and primary resistance of lung adenocarcinomas to gefitinib or erlotinib PLoS Med 2005 2 e17 15696205 Huynh H Soo KC Chow PK Tran E Targeted inhibition of the extracellular signal-regulated kinase kinase pathway with AZD6244 (ARRY-142886) in the treatment of hepatocellular carcinoma Mol Cancer Ther 2007 6 138 146 17237274 Yeh TC Marsh V Bernat BA Ballard J Colwell H Evans RJ Parry J Smith D Brandhuber BJ Gross S Marlow A Hurley B Lyssikatos J Lee PA Winkler JD Koch K Wallace E Biological characterisation of ARRY-142886 (AZD6244) a potent highly selective mitogen-activated protein kinase kinase 1/2 inhibitor Clin Cancer Res 2007 13 1576 1583 17332304 Huynh H Chow PK Soo KC AZD6244 and doxorubicin induce growth suppression and apoptosis in mouse models of hepatocellular carcinoma Mol Cancer Ther 2007 6 2468 2476 17876044 Sos ML Fischer S Ullrich R Peifer M Heuckmann JM Koker M Heynck S St¼ckrath I Weiss J Fischer F Michel K Goel A Regales L Politi KA Perera S Getlik M Heukamp LC Ansn S Zander T Beroukhim R Kashkar H Shokat KM Sellers WR Rauh D Orr C Hoeflich KP Friedman L Wong KK Pao W Thomas RK Identifying genotype-dependent efficacy of single and combined PI3K- and MAPK-pathway inhibition in cancer Proc Natl Acad Sci U S A 2009 106 18351 18356 19805051 Hoeflich KP O™Brien C Boyd Z Cavet G Guerrero S Jung K Januario T Savage H Punnoose E Truong T Zhou W Berry L Murray L Amler L Belvin M Friedman LS Lackner MR In vivo antitumor activity of MEK and phosphatidylinositol 3-kinase inhibitors in basal-like breast cancer models Clin Cancer Res 2009 15 4649 4664 19567590 Engelman JA Chen L Tan X Crosby K Guimaraes AR Upadhyay R Maira M McNamara K Perera SA Song Y Chirieac LR Kaur R Lightbown A Simendinger J Li T Padera RF Garc­a-Echeverr­a C Weissleder R Mahmood U Cantley LC Wong KK Effective use of PI3K and MEK inhibitors to treat mutant Kras G12D and PIK3CA H1047R murine lung cancers Nat Med 2008 14 1351 1356 19029981 Liu P Cheng H Roberts TM Zhao JJ Targeting the phosphoinositide 3-kinase pathway in cancer Nat Rev Drug Discov 2009 8 627 644 19644473 Mallon R Feldberg LR Lucas J Chaudhary I Dehnhardt C Santos ED Chen Z dos Santos O Ayral-Kaloustian S Venkatesan A Hollander I Anti-tumor effect of PKI-587 a highly potent dual PI3K/mTOR kinase inhibitor Clin Cancer Res 2011 17 3193 3203 21325073 Sunaga N Shames DS Girard L Peyton M Larsen JE Imai H Soh J Sato M Yanagitani N Kaira K Xie Y Gazdar AF Mori M Minna JD Knockdown of oncogenic KRAS in non-small cell lung cancers suppresses tumor growth and sensitizes tumor cells to targeted therapy Mol Cancer Ther 2011 10 336 346 21306997 Bhardwaj V Zhan Y Cortez MA Ang KK Molkentine D Munshi A Raju U Komaki R Heymach JV Welsh J C-Met inhibitor MK-8003 radiosensitizes c-Met-expressing non-small-cell lung cancer cells with radiation-induced c-Met-expression J Thorac Oncol 2012 7 1211 1217 22617250 Davies BR Logie A McKay JS Martin P Steele S Jenkins R Cockerill M Cartlidge S Smith PD AZD6244 (ARRY-142886) a potent inhibitor of mitogen-activated protein kinase/extracellularsignal-regulated kinase kinase 1/2 kinases: mechanism ofaction in vivo pharmacokinetic/pharmacodynamicmodels relationship and potential for combination in preclinical Mol Cancer Ther 2007 6 2209 2219 17699718 Sano T Takeuchi S Nakagawa T Ishikawa D Nanjo S Yamada T Matsumoto K Yano S The novel phosphoinositide 3-kinase“mammalian target of rapamycin inhibitor BEZ235 circumvents erlotinib resistance of epidermal growth factor receptor mutant lung cancer cells triggered by hepatocyte growth factor Int J Cancer 2013 133 505 514 23319394 Chou TC Talalay P Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors Adv Enzyme Regul 1984 22 27 55 6382953 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 Friday BB Adjei AA Advances in targeting the Ras/Raf/MEK/Erk mitogenactivated protein kinase cascade with MEK inhibitors for cancer therapy Clin Cancer Res 2008 14 342 346 18223206 Robinson KW Sandler AB The role of MET receptor tyrosine kinase in non-small cell lung cancer and clinical development of targeted anti-MET agents Oncologist 2013 18 115 122 23345546 Regales L Gong Y Shen R de Stanchina E Vivanco I Goel A Koutcher JA Spassova M Ouerfelli O Mellinghoff IK Zakowski MF Politi KA Pao W Dual targeting of EGFR can overcome a major drug resistance mutation in mouse models of EGFR mutant lung cancer J Clin Invest 2009 119 3000 3010 19759520 Fremin C Meloche S From basic research to clinical development of MEK1/2 inhibitors for cancer therapy J Hematol Oncol 2010 3 8 14 20149254 Adjei AA Cohen RB Franklin W Morris C Wilson D Molina JR Hanson LJ Gore L Chow L Leong S Maloney L Gordon G Simmons H Marlow A Litwiler K Brown S Poch G Kane K Haney J Eckhardt SG Phase I pharmacokinetic and pharmacodynamic study of the oral small-molecule mitogen-activated protein kinase kinase 1/2 inhibitor AZD6244 (ARRY-142886) in patients with advanced cancers J Clin Oncol 2008 26 2139 2146 18390968 Watson AL Anderson LK Greeley AD Keng VW Rahrmann EP Halfond AL Powell NM Collins MH Rizvi T Moertel CL Ratner N Largaespada DA Co-targeting the MAPK and PI3K/AKT/mTOR pathways in two genetically engineered mouse models of schwann cell tumors reduces tumor grade and multiplicity Oncotarget 2014 5 1502 1514 24681606 Wee S Jagani Z Xiang KX Loo A Dorsch M Yao YM Sellers WR Lengauer C Stegmeier F PI3K pathway activation mediates resistance to MEK inhibitors in KRAS mutant cancers Cancer Res 2009 69 4286 4293 19401449 Shimizu T Tolcher AW Papadopoulos KP Beeram M Rasco DW Smith LS Gunn S Smetzer L Mays TA Kaiser B Wick MJ Alvarez C Cavazos A Mangold GL Patnaik A The clinical effect of the dual-targeting strategy involving PI3K/AKT/mTOR and RAS/MEK/ERK pathways in patients with advanced cancer Clin Cancer Res 2012 18 2316 2325 22261800 Pao W Miller VA Politi KA Riely GJ Somwar R Zakowski MF Kris MG Varmus H Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain PLoS Med 2005 2 e73 15737014 Li D Ambrogio L Shimamura T Kubo S Takahashi M Chirieac LR Padera RF Shapiro GI Baum A Himmelsbach F Rettig WJ Meyerson M Solca F Greulich H Wong KK BIBW2992 an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models Oncogene 2008 27 4702 4711 18408761 Wong KK Fracasso PM Bukowski RM Lynch TJ Munster PN Shapiro GI Jnne PA Eder JP Naughton MJ Ellis MJ Jones SF Mekhail T Zacharchuk C Vermette J Abbas R Quinn S Powell C Burris HA A phase I study with neratinib (HKI-272) an irreversible pan ErbB receptor tyrosine kinase inhibitor in patients with solid tumors Clin Cancer Res 2009 15 2552 2558 19318484 Uramoto H Shimokawa H Hanagiri T Kuwano M Ono M Expression of selected gene for acquired drug resistance to EGFR-TKI in lung adenocarcinoma Lung Cancer 2011 73 361 365 21315472 Garon EB Finn RS Hosmer W Dering J Ginther C Adhami S Kamranpour N Pitts S Desai A Elashoff D French T Smith P Slamon DJ Identification of common predictive markers of in vitro response to the Mek inhibitor selumetinib (AZD6244; ARRY-142886) in human breast cancer and non-small cell lung cancer cell lines Mol Cancer Ther 2010 9 1985 1994 20587667 Mitsudomi T Steinberg SM Oie HK Mulshine JL Phelps R Viallet J Pass H Minna JD Gazdar AF Ras gene mutations in non-small cell lung cancers are associated with shortened survival irrespective of treatment intent Cancer Res 1991 51 4999 5002 1654209 Mitsudomi T Viallet J Mulshine JL Linnoila RI Minna JD Gazdar AF Mutations of ras genes distinguish a subset of non-small-cell lung cancer cell lines from small-cell lung cancer cell lines Oncogene 1991 6 1353 1362 1679529 Murray S Karavasilis V Bobos M Razis E Papadopoulos S Christodoulou C Kosmidis P Fountzilas G Molecular predictors of response to tyrosine kinase inhibitors in patients with Non-Small-Cell Lung Cancer J Exp Clin Cancer Res 2012 31 77 22992338 PLoS One PLoS ONE plos plosone PLoS ONE 1932-6203 Public Library of Science San Francisco USA 24887068 4041776 PONE-D-14-02596 10.1371/journal.pone.0098621 Research Article Medicine and Health Sciences Oncology Cancer Treatment Radiation Therapy Feasibility of Proton Transmission-Beam Stereotactic Ablative Radiotherapy versus Photon Stereotactic Ablative Radiotherapy for Lung Tumors: A Dosimetric and Feasibility Study Lung SABR Using Transmission Proton Beams Mou Benjamin Beltran Chris J. * Park Sean S. Olivier Kenneth R. Furutani Keith M. Department of Radiation Oncology Mayo Clinic Rochester Minnesota United States of America Deutsch Eric Editor Institut Gustave Roussy France * E-mail: Beltran.Chris@mayo.edu Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: BM KMF SSP KRO CJB. Analyzed the data: BM KMF SSP KRO CJB. Contributed reagents/materials/analysis tools: BM KMF SSP KRO CJB. Wrote the paper: BM KMF SSP KRO CJB. 2014 2 6 2014 9 6 e98621 21 1 2014 6 5 2014 2014 Mou 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. Stereotactic ablative radiotherapy is being increasingly adopted in the treatment of lung tumors. The use of proton beam therapy can further reduce dose to normal structures. However uncertainty exists in proton-based treatment plans including range uncertainties large sensitivity to position uncertainty and calculation of dose deposition in heterogeneous areas. This study investigated the feasibility of proton transmission beams i.e. without the Bragg peak to treat lung tumors with stereotactic ablative radiotherapy. We compared three representative treatment plans using proton transmission beams versus conformal static-gantry photon beams. It was found that proton treatment plans using transmission beams passing through the patient were feasible and demonstrated lower dose to normal structures and markedly reduced treatment times than photon plans. This is the first study to demonstrate the feasibility of proton-based stereotactic ablative radiotherapy planning for lung tumors using proton transmission beams alone. Further research using this novel approach for proton-based planning is warranted. The authors have no support or funding to report. Introduction Stereotactic ablative radiotherapy (SABR) plays an essential role in the treatment of patients with medically inoperable early stage lung cancer and oligometastasis. The use of protons for lung SABR is emerging as an appealing treatment option because of its potential to deliver higher doses of conformal radiotherapy and spare normal tissues better than traditional photons [1] [2] [3] [4]. This can be achieved because of the natural characteristics of proton beams that deposit its dose at depth with no exit dose referred to as a Bragg peak. However conventional dosimetric models fail to accurately model how protons scatter and deposit dose in highly heterogeneous areas which leads to uncertainties in proton treatment plans [5]. In addition the uncertainties in the stopping power of the various tissues in the body and the interplay effect between spot scanning proton therapy and the target motion leads to large uncertainties in the treatment of lung tumors [5] [6]. In this study we report on the feasibility of proton transmission-beam SABR (PT-SABR) for lung tumors which uses the transmission portion of a spot scanning proton beam i.e. without the Bragg peak. This technique eliminates the major uncertainties of proton therapy mentioned above by having the proton beams pass through the patient. In addition the use of the transmission beam allows an entire field to be treated in one breath hold. This quick treatment and decreased uncertainties lead to smaller planning volumes. To the best of the authors™ knowledge this is the first report on the use of this novel approach to plan SABR with protons without using the Bragg peak which may have dosimetric advantages over photon treatments. Materials and Methods Ethics Statement Written informed consent was obtained from all patients registered in the SABR database. This study including the consent procedure was approved by the Mayo Clinic institutional review board. Patient Cohort Patients were identified from a prospectively collected institutional database of patients treated with SABR. Patients with lung tumors less than one centimetre in maximum dimension were included. The radiation treatment plans of three patients were extracted from the treatment planning system. All patients were treated using three-dimensional conformal multiple static-gantry photon beams. Plans were normalized so that 95% of the planning target volume (PTV) received at least 95% of the prescription dose. The prescription doses for these plans were adjusted to 34 Gy in one fraction based on the recently reported results of Radiation Therapy Oncology Group (RTOG) 0915 which established this dose fractionation regimen as a possible standard dose to be used in future trials [7]. Dose calculations for photon plans used the anisotropic analytical algorithm. Proton Treatment Planning A machine was commissioned in Eclipse v.10 (Varian Medical Systems Palo Alto CA) which allowed for planning and calculating transmission dose plans. The spot size (sigma) of the transmission beam which had an energy of 229 MeV was 2.2 mm. A proton plan that only used the transmission portion of the beam was created for each patient. Proton beam arrangements were selected so that no beams entered through the heart or spinal cord and allowed up to two non-coplanar beams. Four to five beams were used to keep the skin dose comparable to photon plans. The energy of the protons for each spot of a field was 229 MeV; this ensured the Bragg peak was not located within the patient. Dose calculations for the transmission portion of the proton beam were verified with Monte Carlo (Geant4). The proton plans were normalized so that the internal target volume (ITV) receives at least 95% of prescription dose including when range and position errors were included (3.5% and 2 mm) which is standard for spot scanning proton therapy. ITVs were created based on motion of the gross tumor volume in three dimensions using four-dimensional computed tomography image data. The dose constraints from RTOG 0915 were compared for the photon and proton plans as well as the total time that would be required to deliver the treatment. The radiotherapy delivery time per beam was estimated at 1 nC per second for proton therapy which is readily achievable by most spot scanning proton centers and 600 MU per minute for the photon plans. Differences in dosimetric and treatment planning parameters between photon and proton plans were analyzed with two-sided paired t-tests using SAS version 9.2 (SAS Institute Inc. Cary NC). Results The ITVs of the three tumors measured 0.220.42 and 0.99 cubic centimeters. All three proton plans had excellent coverage of the ITV. For all ITVs over 99.4% of the volume received at least 95% of the prescription dose including when uncertainties were examined. This was comparable with the photon plans where 100% of the ITVs received at least 95% of the prescription dose. For most normal tissues lower doses to these organs were achieved with the proton plans compared to the photon plans (Table 1). In fact (near) complete sparing of the spinal cord heart and esophagus was possible with protons through careful selection of beam angles (Figure 1). 10.1371/journal.pone.0098621.g001 Figure 1 Dose-volume histogram comparison of organs at risk. 10.1371/journal.pone.0098621.t001 Table 1 Dosimetric comparison of photon and proton plans. Parameter Photon Proton P-value Mean Range Mean Range Internal target volume (cc) 0.54 0.22“0.99 0.54 0.22“0.99 N/A Spinal cord Maximum dose (Gy) 5.66 2.39“8.07 1.97 0.00“3.06 0.04 Lungs (bilateral) Mean lung dose (Gy) 1.35 0.95“1.92 0.69 0.03“1.36 0.12 V20 (%) 0.66 0.39“1.20 0.49 0.16“1.01 0.06 V5 (%) 7.32 5.4“11.30 6.65 2.96“11.70 0.56 Heart Mean dose (Gy) 8.36 6.27“12.51 0.00 0.00“0.00 0.13 Skin Maximum dose (Gy) 11.75 9.86“13.28 11.40 7.37“16.23 0.89 Esophagus Maximum dose (Gy) 6.49 2.98“9.43 3.40 0.00“7.51 0.05 Homogeneity Index 1.25 1.21“1.29 1.07 1.03“1.11 0.06 Conformity Index 17.14 8.23“30.05 3.47 2.17“4.64 0.15 Proton plans used four to five non-coplanar beams compared to nine to ten beams for photon plans (Figure 2). The average number of monitor units per field was 818 (range 758“871) with photons and only 38 (range 31“59) with protons. This would translate to an average beam-on time per field of 82 seconds versus 6 seconds for photon and proton plans respectively. These differences in monitor units and beam-on time were statistically significant with P<0.01(Table 2). 10.1371/journal.pone.0098621.g002 Figure 2 Comparison of isodose distributions. Proton (left) and photon (right) treatment plans. 10.1371/journal.pone.0098621.t002 Table 2 Comparison of treatment time between photon and proton plans. Parameter Photon Proton P-value Mean Range Mean Range Total monitor units (MU) 7929 6820“8713 178 122“235 <0.01 Fields 9.7 9“10 4.7 4“5 N/A Average MU/field 818 758“871 38 30.5“46.9 <0.01 Beam on time per field (seconds) 81.8 75.6“87.1 5.8 4.7“7.2 <0.01 Discussion Exploiting the transmission beam in proton therapy planning has significant potentials for dose escalation and re-irradiation in lung tumors and eliminates the concern over the uncertainty of the stopping power and its impact on the Bragg peak location. PT-SABR planning requires fewer beams than photons and careful selection of optimal beam angles allows for minimal dose to adjacent normal tissues and tumor dose escalation which may translate to improved local control rates. RTOG 0915 showed that 34 Gy in a single fraction was comparable to 48 Gy in four fractions [7] and the dosimetric constraints from the protocol were easily achieved using both proton and photon plans for patients in this study. Further optimization with proton therapy can allow even higher doses to be delivered while still respecting established dosimetric constraints for normal tissues. This may translate to better tumor control but requires more investigation in a clinical setting. Patients planned with PT-SABR required fewer beams (5 vs. 10) which reduce the total treatment time and the low dose outside the tumor. The average monitor units per field for PT-SABR plans were a fraction of those needed for the photon plans (Table 2). 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 photo"

Lung_Cancer

"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 - 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 Organisation 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."

Lung_Cancer

"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 - 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). 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"

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

" The median time to distant metastases was 18.4 ± 10.7 months in HCRT group and 16.7 ± 7.7 months in 3DCRT group (p?=?0.7). In the HCRT group distant metastases involved only the contralateral chest in three patients (30%) and only the abdominal cavity in three patients (30%). Both sites were affected by distant metastases in four further patients (40%). Discussion We demonstrate in a retrospective analysis of patients with MPM and treated at our institution with trimodality therapy that the use of postoperative highly conformal radiation techniques (HCRT) reduces local recurrence in comparison to 3DCRT. A recurrence analysis showed that in the case of 3DCRT 4 of 25 patients (16%) had a local recurrence in regions that were clearly underdosed according to current radiation protocols (doses???45 Gy are recommended e.g. SAKK 17/04) in contrast to 0% of patients treated with HCRT. This supports the hypothesis that HCRT should improve local control in comparison to 3DCRT by improving target volume coverage. In our study patients treated with HCRT showed a tendency for improved progression free survival and local relapse free survival but did not benefit in terms of overall survival due to the high rates of distant relapses. Local control is important in patients with MPM for symptom control but also because some patients might benefit in terms of improved overall survival. Better local control after HCRT did not translate into improved overall survival in our patient series. Remarkably the rate of death due to intercurrent disease most often cardiac events was higher after HCRT (29%) in comparison to 3DCRT (4%). Since cardiac sparing is rather improved with HCRT the most likely explanation for this difference is patient selection. The urgent research question if postoperative radiotherapy impacts on overall survival after EPP is addressed by a randomized study currently conducted in Switzerland SAKK 1704. Patient accrual for this study was terminated in 2012 and the results are awaited. Even after trimodality treatment local recurrence remains high in some patient series. In a retrospective series of 49 patients treated with 3D-conformal RT after EPP and chemotherapy 67% of all recurrences included the ipsilateral hemithorax and 25% of all recurrences were local only [12]. Therefore improvement of radiotherapy is mandatory. In recent years radiotherapy has made enormous technical advances. More sophisticated highly conformal radiation techniques (HCRT) such as IMRT or rotational RT (VMAT) have become available and substituted for the older 3DCRT technique. The use of HCRT enables improvement in the dose distribution and target volume coverage. This is because with HCRT even complex target volumes such as the tumor bed of the costodiaphragmatic recess or the pericardium can be treated without or with little dose compromise and at the same time with optimal sparing of the normal tissue due to a steeper dose fall-off. Thus the use of HCRT should intuitively improve treatment outcome in terms of local tumor control. Our data suggest indeed that the use of HCRT bears considerable potential to improve on hemi-thoracic tumor control rates most likely due to improved target volume coverage. The poor local control rates and high rates of in-field recurrences following 3DCRT in our cohort may be due to suboptimal dose coverage or the restriction of the target volume to avoid critical ans both limitations inherent to the technique. After 3DCRT 4/24 (16.6%) in-field recurrences occurred in regions covered with only 30“43 Gy. In the case of 3DCRT mixed beams of photons and electrons were used to optimize dose coverage. The match of these beams often causes cold and hot spots of dose coverage. Poor matching during daily treatment can result in >20% dose inhomogeneity in the junction area [7]. In addition as the spinal cord is blocked when the tolerance dose of 45 Gy is reached insufficient dose delivery to parts of the mediastinum has been observed resulting in underdosage to the tumor bed [7]. Favorable tumor control after IMRT as part of a trimodality therapy has previously been reported by Rice et al. [13]. The median overall survival of their 61 patients treated was 14.2 months with a locoregional recurrence rate of 13% and only 5% local in-field recurrences reported. The median dose prescribed was only 45 Gy and half of all patients received doses even less than 45 Gy. The reason for the comparatively higher local control rate reported by Rice et al. in comparison to our study remains unclear. It may be explained by patient selection and the comparatively short median overall survival of 14.2 months in comparison to 20.8 months in the present series and by the retrospective study design. The shorter median overall survival reported by Rice et al. could be caused by more advanced tumor stages (40 T3 8 T4 26 N2) more aggressive subtypes (14 biphasic 4 sarcomatoid) and the fact that neoadjuvant chemotherapy was not routinely administered. With regard to toxicity the major dose limiting an for postoperative radiotherapy of MPM is the contralateral lung. Lung complications such as radiation pneumonitis are likely to be higher with multi-field techniques such as IMRT or VMAT in comparison to 3DCRT where opposed beams from 0 and 180 degrees are usually used thereby optimally sparing the contralateral lung. With regard to dose escalation and lung sparing surgery protons might prove superior to IMRT/VMAT. Severe complications of the lung with grade 4 and 5 pneumonitis after IMRT have been reported [712]. Since then special attention to the contralateral lung dose has been given during the treatment planning process and pneumonitis rates should be lower today. Intuitively the use of HCRT should reduce toxicity and complication probabilities of esophagus heart liver and kidney however no data with regard to these toxicity endpoints comparing both treatment techniques are available. In recent years the need for extensive surgery has been questioned and less radical surgery has been advocated such as pleurectomy/decortication. In the context of reduced surgery the anatomical situation makes it difficult for RT to be applied to the entire pleural space however it can still be considered as a targeted local postoperative option in case of incomplete resection. Future clinical studies are required to define the role of radiotherapy in combination with lung sparing surgery. Conclusions In summary the use of HCRT for treatment of patients with MPM after EPP is likely to improve local control rates. The local control improvement did not improve the overall survival due to the high rates of distant relapses in this series. Further improvement of trimodal or systemic therapy is required to tackle the high risk of distant recurrences. "

Lung_Cancer

"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 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). 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) (). 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 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 (). 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 Roentg"

Lung_Cancer

"These findings may have important therapeutic implications. Pancreatic cancer is one of the deadliest human malignancies. A striking feature of pancreatic cancer is that activating Kras mutations are found in ?90% of cases. However apart from a restricted population of cells expressing pancreatic and duodenal homeobox 1 (PDX1) most pancreatic cells are refractory to Kras-driven transformation. In the present study we sought to determine which subsets of PDX1+ cells may be responsible for tumor growth. Using the Lox-Stop-Lox“KrasG12D genetic mouse model of pancreatic carcinogenesis we isolated a population of KrasG12D-expressing PDX1+ cells with an inherent capacity to metastasize. This population of cells bears the surface phenotype of EpCAM+CD24+CD44+CD133“SCA1? and is closer in its properties to stem-like cells than to more mature cell types. We further demonstrate that the tumorigenic capacity of PDX1+ cells is limited becoming progressively lost as the cells acquire a mature phenotype. These data are consistent with the hypothesis that the adult pancreas harbors a dormant progenitor cell population that is capable of initiating tumor growth under conditions of oncogenic stimulation. We present evidence that constitutive activation of the mitogen-activated protein kinase (MAPK/ERK) signaling and stabilization of the MYC protein are the two main driving forces behind the development of pancreatic cancer cells with stem-cell“like properties and high metastatic potential. Our results suggest that pancreatic cells bearing Kras mutation can be induced to differentiate into quasi-normal cells with suppressed tumorigenicity by selective inhibition of the MAPK/ERK/MYC signaling cascade. pancreatic ductal adenocarcinoma cell of origin Chest Chest chest Chest Chest 0012-3692 1931-3543 American College of Chest Physicians 25117058 4188148 chest.14-0477 10.1378/chest.14-0477 Original Research Critical Care Aggressiveness of Intensive Care Use Among Patients With Lung Cancer in the Surveillance Epidemiology and End Results-Medicare Registry ICU Use Among Elderly Patients With Lung Cancer Cooke Colin R. MD Feemster Laura C. MD Wiener Renda Soylemez MD O™Neil Maya E. PhD Slatore Christopher G. MD From the Division of Pulmonary and Critical Care Medicine (Dr Cooke) Center for Healthcare Outcomes and Policy Institute for Healthcare Innovation and Policy Michigan Center for Integrative Research in Critical Care University of Michigan Ann Arbor MI; the Division of Pulmonary and Critical Care Medicine (Dr Feemster) VA Puget Sound Healthcare System and University of Washington School of Medicine Seattle WA; Boston University School of Medicine (Dr Wiener) Boston MA; Edith Nourse Rogers Memorial VA Hospital (Dr Wiener) Bedford MA; Health Services Research and Development (Drs O™Neil and Slatore) and Section of Pulmonary and Critical Care Medicine (Dr Slatore) Portland VA Medical Center; and the Division of Pulmonary and Critical Care Medicine (Dr Slatore) Department of Medicine Oregon Health and Science University Portland OR. CORRESPONDENCE TO: Colin R. Cooke MD University of Michigan Center for Healthcare Outcomes and Policy 2800 Plymouth Rd Bldg 16 Room 127W Ann Arbor MI 48109; e-mail: cookecrumich.edu 10 2014 19 6 2014 1 10 2015 146 4 916 923 25 2 2014 14 5 2014 2014 AMERICAN COLLEGE OF CHEST PHYSICIANS 2014 BACKGROUND: Approximately 65% of elderly patients with lung cancer who are admitted to the ICU will die within 6 months. Efforts to improve end-of-life care for this population must first understand the patient factors that underlie admission to the ICU. METHODS: We performed a retrospective cohort study examining all fee-for-service inpatient claims in the Surveillance Epidemiology and End Results (SEER)-Medicare registry for elderly patients (aged > 65 years) who had received a diagnosis of lung cancer between 1992 and 2005 and who were hospitalized for reasons other than resection of their lung cancer. We calculated yearly rates of ICU admission per 1000 hospitalizations via room and board codes or International Classification of Diseases Ninth Revision Clinical Modification and diagnosis-related group codes for mechanical ventilation stratified the rates by receipt of mechanical ventilation and ICU type (medical/surgical/cardiac vs intermediate) and compared these rates over time. RESULTS: A total of 175756 patients with lung cancer in SEER were hospitalized for a reason other than surgical resection of their tumor during the study period49373 (28%) of whom had at least one ICU stay. The rate of ICU admissions per 1000 hospitalizations increased over the study period from 140.7 in 1992 to 201.7 in 2005 (P < .001). The majority of the increase in ICU admissions (per 1000 hospitalizations) between 1992 and 2005 occurred among patients who were not mechanically ventilated (118.2 to 173.3 P < .001) and among those who were in intermediate ICUs (20.0 to 61.9 P < .001) but increased only moderately in medical/surgical/cardiac units (120.7 to 139.9 P < .001). S: ICU admission for patients with lung cancer increased over time mostly among patients without mechanical ventilation who were largely cared for in intermediate ICUs. Cell Death Dis Cell Death Dis Cell Death & Disease 2041-4889 Nature Publishing Group 24481441 4040650 cddis2013550 10.1038/cddis.2013.550 Original Ibuprofen enhances the anticancer activity of cisplatin in lung cancer cells by inhibiting the heat shock protein 70 Ibuprofen and cisplatin-mediated apoptosis Endo H 1 2 Yano M 1 2 * Okumura Y 1 Kido H 1 1Division of Enzyme Chemistry Institute for Enzyme Research The University of Tokushima Tokushima Japan 2Department of Nutrition School of Human Cultures The University of Shiga Prefecture Shiga Japan *Department of Nutrition School of Human Cultures The University of Shiga Prefecture Hikone Shiga 522-8533 Japan. Tel: +81 749 28 8441; E-mail: yano.mshc.usp.ac.jp 01 2014 30 01 2014 1 1 2014 5 1 e1027 25 06 2013 27 11 2013 10 12 2013 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/ Hsp70 is often overexpressed in cancer cells and the selective cellular survival advantage that it confers may contribute to the process of tumour formation. Thus the pharmacological manipulation of Hsp70 levels in cancer cells may be an effective means of preventing the progression of tumours. We found that the downregulation of Hsp70 by ibuprofen in vitro enhances the antitumoural activity of cisplatin in lung cancer. Ibuprofen prominently suppressed the expression of Hsp70 in A549 cells derived from lung adenocarcinoma and sensitized them to cisplatin in association with an increase in the mitochondrial apoptotic cascade whereas ibuprofen alone did not induce cell death. The cisplatin-dependent events occurring up- and downstream of mitochondrial disruption were accelerated by treatment with ibuprofen. The increase in cisplatin-induced apoptosis caused by the depletion of Hsp70 by RNA interference is evidence that the increased apoptosis by ibuprofen is mediated by its effect on Hsp70. Our observations indicate that the suppression of Hsp70 by ibuprofen mediates the sensitivity to cisplatin by enhancing apoptosis at several stages of the mitochondrial cascade. Ibuprofen therefore is a potential therapeutic agent that might allow lowering the doses of cisplatin and limiting the many challenge associated with its toxicity and development of drug resistance. Hsp70 apoptosis ibuprofen The human Hsp70 family includes ?8 highly homologous members that differ from each other by their intracellular localization and expression patterns.1 Among them the major stress-inducible Hsp70 (also called Hsp72) has an essential role in cell survival under stressful conditions. Compared with its normal counterpart Hsp70 is often overexpressed in various cancer cells and is suspected to contribute to the development of tumours.2 3 Indeed the expression of Hsp70 in certain cancer types has been correlated with poor prognosis and resistance to chemotherapy.45 6 Tumour cells often express several proteins that when abnormally elevated render the tumour resistant to apoptosis.7 Previous studies have confirmed not only that Hsp70 is cytoprotective but also that it interferes effectively with cell death induced by a wide variety of stimuli including several cancer-related stresses. Hsp70 is a potent inhibitor of the stress-activated kinase pathway and apparently blocks apoptotic signals via interactions with JNK Ask1 and SEK1.8910 11 Hsp70 is also a negative regulator of the mitochondrial pathway of apoptosis. Much of the focus on the antiapoptotic function of Hsp70 has been on events that occur after the disruption of the mitochondria. Hsp70 prevents the recruitment of procaspase-9 to the apoptosome and its functional complex formation by direct interaction with apoptotic protease-activating factor 1 (Apaf-1).12 13 Furthermore Hsp70 inhibits the activation of caspase-3 and the cleavage of caspase-3 targets such as ICAD and GATA-1.14 15 On the other hand recent studies have reported that Hsp70 can prevent apoptosis upstream of the mitochondria by inhibiting events which ultimately permeabilize the mitochondrial outer membrane such as the activation of Bax.16 17 As a result of the inhibition by Hsp70 of the apoptosis induced by several anticancer drugs as well as by other stimuli we hypothesized that cancer cells would be sensitized to the induction of apoptosis by the neutralization of Hsp70. Hsp70 has been indeed targeted with pharmaceuticals such as triptolide quercetin and KNK437 which downregulate its expression.1819 20 Although they have prevented the progression of various cancer cells in vitro and in vivo21 22 the optimal clinical use of these small Hsp70 inhibitors singly or combined with other chemotherapeutics remains a challenge. Our overall objective was to pharmacologically control the levels of Hsp70 and increase the effectiveness of anticancer drugs. Several experimental and epidemiologic studies and clinical trials have observed a powerful chemopreventive activity exerted by nonsteroidal anti-inflammatory drugs (NSAIDs).23 24 The anti-carcinogenic properties of NSAID have been attributed to their inhibition of cyclooxygenase (COX) enzymes. However much higher doses of NSAID are needed to obtain an antitumoural effect than to inhibit COX25 suggesting that they also act via COX-independent mechanisms. On the other hand NSAIDs such as aspirin salicylate and sulindac sulphide inhibit the proliferation of cells and induce apoptosis in various cancer cell lines which is considered an important component of their antitumoural activity and increased sensitization of cancer cells to anticancer drugs.262728 29 There is currently interest in the ability of NSAID to directly lower the levels of antiapoptotic molecules such as the Bcl-2 family30 and 14-3-3 protein31 which inhibits the intrinsic mitochondria-dependent apoptosis in various cancer cells. Therefore the NSAID-induced dysfunction of antiapoptotic proteins prompted us to examine whether other antiapoptotic molecules including Hsp70 might also be targets in the prevention of tumour progression by NSAID. In this study we show that ibuprofen is a potent inhibitor of Hsp70 which significantly suppresses its expression by depleting heat shock factor 1 (HSF1) in lung adenocarcinoma-derived A549 cells. The downregulation of Hsp70 by ibuprofen sensitized the cells to cisplatin which was associated with the enhancement of cisplatin-induced apoptotic signalling. Ibuprofen did not only facilitate postmitochondrial events including the activation of cisplatin-induced caspase-9 but also the activation of Bax causing the release of cytochrome c. Besides the demonstration of a similar increase in the sensitivity of A549 cells to cisplatin conferred by Hsp70 knockdown and ibuprofen these observations indicate that ibuprofen accelerates cisplatin-mediated apoptosis at multiple steps of the mitochondrial apoptotic pathway via the inhibition of Hsp70. We conclude that ibuprofen is a potential chemotherapeutic agent which might enable (a) the use of lower less toxic does of cisplatin and (b) the design of a new combination treatment of lung cancer. Results Ibuprofen suppresses the expression of Hsp70 in lung adenocarcinoma cells To define the role of Hsp70 in promoting the formation of tumours we first examined its expression in human lung cancer cell lines. Compared with BEAS-2B a human non-malignant bronchial epithelial cell line the expression levels of Hsp70 in lung cancer cells such as A549 and H358 adenocarcinoma were notably higher (Figure 1a). As in previous studies which showed an increased expression of Hsp70 in various types of human cancers including breast pancreas and colon we found that Hsp70 is also dysregulated in lung cancer cells. In this study we screened conventional NSAID in search of a new pharmacologic inhibitor which neutralizes Hsp70 as they induce apoptosis in cancer cells by selectively downregulating antiapoptotic proteins. The expression of Hsp70 after the exposure of A549 cells to various NSAID in non-toxic concentrations was analyzed by immunoblot. Ibuprofen in a 400-?M concentration decreased the expression of Hsp70 by 23% in comparison with untreated cells whereas other NSAID had no effect (Table 1). Figure 1b shows the decrease in Hsp70 protein and mRNA levels in A549 cells after treatment with various concentrations of ibuprofen versus no apparent decreases in Hsc70 and Actin. Ibuprofen also decreased the expression of Hsp70 in H358 a human lung adenocarcinoma cell line in a dose-dependent manner (Figure 1c). These results suggest that ibuprofen decreases the expression of Hsp70 in various lung cancer cell lines. Ibuprofen enhances the apoptosis induced by cisplatin by suppressing Hsp70 As ibuprofen prominently inhibited the expression of Hsp70 we next examined its effect on the proliferation of cancer cells. We observed no significant change in the viability of A549 and H358 cells after the exposure to ?800??M concentrations of ibuprofen alone which downregulates Hsp70 (Figure 2a) while the exposure to 1.0?mM concentration of ibuprofen caused cell death. Combined these observations indicate that the downregulation of stress-inducible Hsp70 was insufficient to cause the death of A549 and H358 cells. There is evidence that the inhibition of anti-apoptotic molecules such as Hsp70 increases the sensitivity of tumour cells to anticancer drugs thus improving the outcomes of chemotherapy. To study the therapeutic potential of ibuprofen we examined whether its antitumoural effects are synergistic with those of cisplatin widely used in the treatment of lung adenocarcinoma. When we measured the survival of A549 (top of Figure 2b) and H358 (bottom of Figure 2b) cells exposed to increasing concentrations of cisplatin incubated in presence versus absence of ibuprofen the latter prominently magnified the apoptosis induced by cisplatin a synergistic effect confirmed by terminal deoxynucleotidyl transferase-mediated dUTP nick and labelling (TUNEL) staining (Figure 2c). To ascertain the effects conferred by the expression of Hsp70 on cell death while excluding all effects of ibuprofen unrelated to Hsp70 we weakened the expression of Hsp70 by RNA interference (RNAi) (Figure 2d) and measured its effects on the apoptosis induced by cisplatin. The inhibition of Hsp70 decreased the viability of cisplatin-treated cells by approximately 20% (Figure 2e). Transfections with scrambled siRNA serving as a control showed no increase in cell death mediated by cisplatin. Cisplatin had no effect on the expression of Hsp70 (Figure 2g). We quantified the number of apoptotic cells in ibuprofen- and/or cisplatin-treated cultures using the CF488A-annexin V methods. Although cisplatin alone induced apoptosis in 10.2% of A549 cells the co-treatment with ibuprofen increased the percentage of apoptotic cells to 34.0% (Figure 2f). These observations suggest that ibuprofen sensitizes A549 cells to cisplatin by decreasing the expression of Hsp70. Ibuprofen decreases the expression of Hsp70 via transcriptional inactivation The reverse transcriptase-polymerase chain reaction (RT-PCR) analysis described earlier revealed a decrease in RNA level following treatment with ibuprofen suggesting that the expression of Hsp70 can be downregulated at the transcriptional level. After the recently discovered inhibition by its antagonists of the transcription of Hsp70 in cancer cells by blockade of the activation of HSF118 20 (which is often upregulated and constitutively activated in tumour formation) we studied the effects of ibuprofen on HSF1 in A549 cells. We first performed a ChIP assay to explore whether the inhibitory effect of ibuprofen is at the level of HSF1 DNA binding. As expected we found an unequivocal association between HSF1 and the Hsp70 gene promoter containing the HSE site in ibuprofen-untreated cells (Figure 3a). It is noteworthy that ibuprofen eliminated this binding (Figure 3a) suggesting that it inhibits the expression of Hsp70 via the action of HSF1. This also suggests that ibuprofen blocks the binding of HSF1 chromatin or the steps which precede in several processes needed to activate HSF1. Therefore we broadened our analysis to examine the effect of ibuprofen on the expression of HSF1. Compared with unexposed control cells the HSF1 mRNA level was significantly lower in cells exposed to ibuprofen (bottom of Figure 3b). Consistent with its effect on the expression of mRNA ibuprofen also decreased the expression of HSF1 protein in a dose-dependent fashion (top of Figure 3b). To confirm the inhibition of HSF1-mediated Hsp70 by ibuprofen we lowered the amounts of HSF1 present in A549 cells by RNAi and studied its effect on the expression of Hsp70. The treatment of cells with HSF1 dsRNA decreased the Hsp70 level compared with that measured in cells untreated with dsRNA (Figure 3c). Ibuprofen decreased the expression of HSF1 by 16% in comparison with untreated cells whereas other NSAID had no effect (Table 2). Overall these observations indicate that ibuprofen inhibited the expression of Hsp70 by depleting the HSF1 in A549 cells. Ibuprofen accelerates the mitochondrial apoptotic process induced by cisplatin Several studies have found that mitochondria might be a direct and important target of cisplatin in sensitive cells.32 33 We studied the effects of ibuprofen on the depolarization of mitochondrial membranes and the cytochrome c release induced by cisplatin. A549 cells with or without cisplatin were incubated in absence or presence of ibuprofen and stained with JC-1. Treatment with cisplatin and ibuprofen lowered the mitochondrial membrane potential manifest by an attenuated red and an enhanced green mitochondrial fluorescence (Figure 4a lower right panel) compared with that observed with cisplatin alone (Figure 4a upper right panel) while control (Figure 4a upper left panel) or ibuprofen alone (Figure 4a lower left panel) produced the red-dotted staining pattern of polarized mitochondria. The intensity of green mitochondrial fluorescence in cisplatin-treated cells is significantly increased (36.56 to 55.56%) by the co-treatment with ibuprofen. Ibuprofen also promoted the release of cytochrome c from the mitochondria induced by cisplatin (Figure 4b). These findings unequivocally indicated that in A549 cells ibuprofen enhanced the mitochondria-dependent apoptosis caused by cisplatin. Ibuprofen increases the activation of Bax induced by cisplatin The translocation of the pro-apoptotic protein Bax to the mitochondria is closely associated with the apoptosis induced by cisplatin. To explore the mechanisms by which ibuprofen promotes the apoptosis mediated by mitochondria in response to cisplatin we examined whether it was due to its ability to stimulate the translocation of Bax by cisplatin. We first monitored conformational changes in Bax as indicators of its activation. Western blot analysis of the immunoprecipitates with a conformation specific anti-Bax (6A7) antibody which only recognizes the active form revealed the presence of active Bax in A549 cells treated with cisplatin (Figure 5a lane 4) although not in untreated cells (Figure 5a lanes 1 and 2). Further exposure of the cisplatin-treated cells to ibuprofen caused a 1.5-fold increase in active Bax compared with incubation with cisplatin alone (Figure 5a lane 3). When we analyzed the effects conferred by ibuprofen on the translocation of Bax to mitochondria in cisplatin-treated cells we observed an approximately 1.3-fold increase in the amount of translocated Bax (Figure 5b). To exclude an effect of ibuprofen unrelated to the inhibition of Hsp70 we performed RNAi for a selective knock-down of Hsp70 and we studied its effects on the activation of Bax. Consistent with the earlier data presented for ibuprofen the depletion of Hsp70 increased the activation of Bax in cisplatin-treated cells although its extent was greater with Hsp70 RNAi than with ibuprofen (Figure 5c). These observations confirmed that (a) ibuprofen promotes the activation of Bax dependent on cisplatin and its translocation to the mitochondria in A549 cells and (b) its mechanism of action is mediated by the inhibition of Hsp70. Ibuprofen facilitates events occurring upstream and downstream of mitochondrial disruption in cisplatin-mediated apoptosis Previous studies have shown that Hsp70 can inhibit apoptosis by acting downstream of the mitochondria.121314 15 Hsp70 interacts directly with Apaf-1 to prevent the formation of cytochrome c-mediated apoptosome and subsequent activation of caspase-9. To examine whether ibuprofen also influences the downstream mitochondrial events we measured its effects on the cleavage of procaspase-9 in the apoptosis mediated by cisplatin. With an anti-active caspase-9 antibody fully processed caspase-9 was predominantly identified in cisplatin-treated A549 cells (Figure 6a lane 3) over untreated cells (Figure 6a lanes 1 and 2). It is noteworthy that treatment with ibuprofen increased >4-fold the amount of active caspase-9 in cells treated with cisplatin compared with cells incubated with cisplatin alone (Figure 6a lane 4). As as reported earlier the highest increases in the activation of Bax and release of cytochrome c by ibuprofen were <2-fold these observations suggest that ibuprofen also facilitates the post mitochondrial process taking place between the release of cytochrome c and the activation of caspase-9. To verify that this is a specific effect we studied the effect of Hsp70 knock-down on the activation of caspase-9 mediated by cisplatin. The caspase-9 activity in cells depleted of Hsp70 with cisplatin was fourfold greater than in control (scrambled) siRNA-treated cells (Figure 6b). We obtained similar results when we measured the activity of caspase-9 in cells treated with ibuprofen (Figure 6c) or siRNA against Hsp70 (Figure 6d) by a fluorometric assay using a synthetic substrate. Overall these observations confirmed unambiguously that ibuprofen intensified the apoptosis induced by cisplatin by its effects on the events occurring downstream of the mitochondria by inhibiting Hsp70 although whether it stimulated the formation of apoptosome (essential for the recruitment of procaspase-9) remains to be determined. We conclude that ibuprofen promotes the apoptosis induced by cisplatin at multiple stages of the mitochondrial cascade by attenuating the expression of Hsp70 in A549 cells. Discussion We found that compared with non-malignant bronchial epithelial cells human lung cancer cells overexpressed Hsp70. This is an important observation as targeting the expression or function of Hsp70 has been suggested as an effective treatment strategy in several cancers based on the hypothesis that higher levels of Hsp70 protect against cell death and increase the survival rate against modalities used in chemotherapy.11 15 In fact it is well documented that the expression of Hsp70 is significantly increased in cancer tissues and/or serums obtained from patients with non-small cell lung cancer (NSCLC)34353637 38 and its overexpression correlates with poor prognosis in NSCLC.36 Several reports have indicated that functionally related small molecules that inhibit Hsp70 decrease the viability of colo-rectal or pancreatic cancer cells by promoting apoptosis via the downregulation of Hsp70 and may be a promising new class of cancer chemotherapeutics.1921 22 We showed that ibuprofen a relatively non-toxic and widely used NSAID significantly decreased the expression of Hsp70 in lung adenocarcinoma cell lines. We also clearly demonstrated that the inhibitory mechanisms of ibuprofen on Hsp70 are due to a decrease in HSF1 expression. Although the fundamental mechanism behind the reduction in HSF-1 expression is unknown a previous study has indicated that the nuclear factor 1 family member NFIX which codes for site-specific DNA-binding proteins known to have multiple roles in replication signal transduction and transcription exerts a transcriptional repressive effect on the expression of HSF1 in cancer cells.39 Whether NFIX is indeed involved in the inhibition of HSF1 expression evoked by ibuprofen is applicable in further studies. To the best of our knowledge this is the first study of the inhibitory effects of NSAID on the cellular expression of Hsp70. In addition we showed that ibuprofen does not influence the cell viability without additional stimuli unlike its maximal effect on the expression of Hsp70. The lack of inhibitory efficacy of ibuprofen against tumours is consistent with a previous study which showed that low-dose ibuprofen did not induce apoptosis in mouse and human colorectal cancer cell lines.29 Similar observations were made following RNAi of Hsp70 suggesting that the attenuation of Hsp70 per se is insufficient to cause the death of A549 and perhaps other cells. It has been shown that the knockdown of Hsp70 has no effect on the viability of several cancer cell lines although sensitized them to anticancer drugs.40 41 Therefore the therapeutic potential of ibuprofen combined with chemotherapeutic agents needs to be explored. Cisplatin is one of most effective chemotherapeutic drugs against NSCLCs.42 It is noteworthy that damage to DNA caused by cisplatin enables apoptosis involving mitochondrial pathways which is negatively regulated by Hsp70. As ibuprofen prominently suppressed the expression of Hsp70 in A549 and H358 cells we examined the possible synergistic activity of ibuprofen and cisplatin against cancer. As expected ibuprofen potentiated synergistically the anti-proliferative effect of cisplatin in A549 and H358 cells. Despite its potent antitumoural properties the therapeutic use of cisplatin in oncology is seriously limited by dose-dependent adverse effects and frequent development of drug resistance.43 Therefore our findings may make useful contributions toward the development of new and less toxic chemotherapy against NSCLCs. We also examined the molecular mechanisms of these synergistic properties of ibuprofen. Hsp70 protects cells against mitochondria-dependent apoptosis at different levels although the precise mechanism remains hypothetical because of regular contradictory descriptions of Hsp70 function. Earlier reports have shown a protective effect of Hsp70 against cellular apoptosis by inhibition of the apoptosome function a protein complex comprising Apaf-1 and cytochrome c.12 13 However recent reports have questioned this repression of apoptosis downstream of the mitochondrial membrane permeabilization."

Lung_Cancer

"Ann Surg233: 189“19411176124 29 YamashitaJI KurusuY FujinoN SaisyojiT OgawaM (2000) Detection of circulating tumor cells in patients with non-small cell lung cancer undergoing lobectomy by video-assisted thoracic surgery: a potential hazard for intraoperative hematogenous tumor cell dissemination. J Thorac Cardiovasc Surg119: 899“90510788810 Int J Clin Exp Pathol Int J Clin Exp Pathol ijcep International Journal of Clinical and Experimental Pathology 1936-2625 e-Century Publishing Corporation 24695377 3971289 Original Interleukin 7 signaling prevents apoptosis by regulating bcl-2 and bax via the p53 pathway in human non-small cell lung cancer cells Liu Zi-Hui 1 Wang Ming-Hui 3 Ren Hong-Jiu 1 Qu Wei 1 Sun Li-Mei 1 Zhang Qing-Fu 1 Qiu Xue-Shan 1 2 Wang En-Hua 1 2 1 Department of Pathology College of Basic Medical Sciences China Medical University Shenyang China 2 Department of Pathology The First Affiliated Hospital of China Medical University Shenyang China 3 Department of Breast Surgery The Affiliated Hospital of Chengde Medical College Chengde China Address correspondence to: Dr. Xueshan Qiu Department of Pathology College of Basic Medical Sciences China Medical University Department of Pathology The First Affiliated Hospital of China Medical University Heping Ward North 2nd Road 92 Shenyang China. Tel: +86 02423261638; +86 13386881964; E-mail: xsqiumail.cmu.edu.cn 2014 15 2 2014 7 3 870 881 22 12 2013 06 1 2014 IJCEP Copyright 2014 2014 Interleukin 7/Interleukin 7 receptor (IL-7/IL-7R) signaling induces the upregulation of cyclin D1 to promote cell proliferation in lung cancer but its role in preventing the apoptosis of non-small cell lung cancer (NSCLC) cell lines remains unknown. To study the role of IL-7 in lung cancer cell apoptosis normal HBE cells as well as A549 and H1299 NSCLC cells were examined using flow cytometry. The results showed that the activation of IL-7R by its specific ligand exogenous interleukin-7 was associated with a significant decline in apoptotic cells. Western blot and real-time PCR assays indicated that the activation of IL-7/IL-7R significantly upregulated anti-apoptotic bcl-2 and downregulated pro-apoptotic bax and p53 at both protein and mRNA levels. The knockdown of IL-7R through small interfering RNAs significantly attenuated these effects of exogenous IL-7. However there was no significant anti-apoptotic effect in H1299 (p53-) cells. Furthermore the inhibition of p53 significantly abolished the effects of IL-7/IL-7R on lung cancer cell apoptosis. These results strongly suggest that IL-7/IL-7R prevents apoptosis by upregulating the expression of bcl-2 and by downregulating the expression of bax potentially via the p53 pathway in A549 and HBE cells. Interleukin 7 interleukin 7 receptor p53 apoptosis NSCLC PLoS One one 1932-6203 Public Library of Science San Francisco USA 24743794 3990524 PONE-D-13-45521 .0094644 Research Medicine and Health Sciences Oncology Cancers and Neoplasms Lung and Intrathoracic Tumors Non-Small Cell Lung Cancer Biology and Life Sciences Computational Biology Genome Analysis Transcriptome Analysis Genome Expression Analysis Gene Regulatory Networks Genetics Human Genetics Genetic Association Studies Cancer Genetics Genomics Systems Biology Identification of Logic Relationships between Genes and Subtypes of Non-Small Cell Lung Cancer Gene-Subtype Logic Relationships of NSCLC Su Yansen * Pan Linqiang * Key Laboratory of Image Information Processing and Intelligent Control School of Automation Huazhong University of Science and Technology Wuhan Hubei China Zhang Yan Editor Harbin Medical University China * E-mail: suyansen1985163.com (YS); lqpanmail.hust.edu.cn (LP) Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: YS LP. Performed the experiments: YS. Analyzed the data: YS. Contributed reagents/materials/analysis tools: YS. Wrote the paper: YS. Revised the paper: YS LP. 2014 17 4 2014 9 4 e94644 20 11 2013 18 3 2014 2014 Su Pan 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. Non-small cell lung cancer (NSCLC) has two major subtypes: adenocarcinoma (AC) and squamous cell carcinoma (SCC). The diagnosis and treatment of NSCLC are hindered by the limited knowledge about the pathogenesis mechanisms of subtypes of NSCLC. It is necessary to research the molecular mechanisms related with AC and SCC. In this work we improved the logic analysis algorithm to mine the sufficient and necessary conditions for the presence states (presence or absence) of phenotypes. We applied our method to AC and SCC specimens and identified lower and higher logic relationships between genes and two subtypes of NSCLC. The discovered relationships were independent of specimens selected and their significance was validated by statistic test. Compared with the two earlier methods (the non-negative matrix factorization method and the relevance analysis method) the current method outperformed these methods in the recall rate and classification accuracy on NSCLC and normal specimens. We obtained biomarkers."

Lung_Cancer

"(a) shows a frame from the 4D acquisition from Patient number 2. A surface rendered tumor-inclusive volume-of-interest in four different respiratory phases is shown in (b). Both the tumor as well as the surrounding anatomy exhibit significant deformation from phase to phase. shows motion trajectories extracted from two time series one from each patient. MRI-based monitoring over multiple respiratory cycles yields some interesting observations. In the case of Patient number 1 there is little cycle-to-cycle variation in the respiratory pattern as evidenced by the motion trajectory of the diaphragm. Furthermore the motion of the tumor centroid is well correlated with the motion of the diaphragm ((a); R2 = 0.99) indicating that in this case diaphragmatic motion is an appropriate surrogate for tumor motion. Finally the motion of individual points on the tumor boundary (i.e pixels comprising the edges of the tumor mass) is well correlated with that of the tumor centroid ((b); R2 = 0.9 to 1.0) indicating the absence of any significant rotation or deformation in the tumor mass. In the case of Patient number 2 while the respiratory pattern is quite regular (as seen from the motion trajectory of the diaphragm) the motion of the tumor centroid is very poorly correlated with diaphragmatic motion ((c); R2 = 0.16) and shows significant cycle-to-cycle variation. This behavior indicates that in this case diaphragmatic motion is a poor surrogate for tumor motion. Furthermore the motion of the tumor centroid is also relatively poorly correlated with that of individual points on the tumor boundary ((d); R2 = 0.56 to 0.94) indicating the occurrence of significant rotation/deformation of the tumor mass. The complex motion observed in Patient number 2 is likely due to the proximity of tumor to the cardiac wall which almost touches the edge of the tumor ((c)) and serves as a second actuator of motion (the first being the diaphragm). These results demonstrate that the current clinical practice of using the motion of the diaphragm (or external or internal surrogates for diaphragmatic motion) has significant limitations when the tumor mass is located in the proximity of other moving structures. The goal of this work was to demonstrate the feasibility and the potential advantages of using rapid MRI as a pretreatment image-guidance tool for lung RT. These early results from rapid MRI of NSCLC patients show that for guidance-quality imaging the inherent contrast presented by the tumor mass and critical structures against the signal-poor lung parenchyma enables us to sacrifice SNR in order to achieve adequate acquisition speed to capture respiratory motion. Furthermore in the case of Patient number 2 we observe that through long-term prospective MR imaging one can capture spatiotemporal effects that are not captured by 4DCT. This is due to the fact that 4DCT projections are sorted using an external surrogate for diaphragmatic motion thereby implicitly assuming that a perfect correlation exists between diaphragmatic motion and tumor motion. The choice of a 1.5?T scanner for this work was motivated by the fact that several lung motion investigations have been performed at this field strength [12 18]. Observer studies comparing 1.5?T and 3?T scanners for lung MRI show that there is no significant difference in overall image quality [19 20] suggesting that the expected benefits of higher SNR at 3?T are somewhat mitigated due to the accompanying increase in susceptibility artifacts. Furthermore at this initial stage we chose to use existing coils and sequences. As seen from the results while this strategy was adequate for cine-2D imaging very large improvements in acquisition speed are required for truly 4D MRI. This is evidenced by the fact that even with the use of parallel acceleration = 4 the acquisition time for the 4D time series shown in was ~1.5?s/volume. Thus there is much room for exploration of other rapid MRI sequences and for developing sequences specifically optimized for RT guidance. In particular we expect the largest improvements in imaging speed to come from strategies based on sparse sampling and reconstruction such as k-t Broad-use Linear Acquisition Speed-up Technique (k-t BLAST) and its parallel imaging version k-t SENSitivity Encoding (k-t SENSE). Beyond the current scope it is expected that the information obtained from rapid MRI (cine-2D or 4D) can be merged with that from 3DCT or 4DCT to create a fused pretreatment 4D image that combines the soft-tissue contrast and temporally dense information from MRI with the spatial accuracy and electron density information from CT. Admittedly this is a nontrivial problem because one has to account for MRI artifacts correct for geometric distortions of the anatomy due to the relatively narrow bore of the magnet and develop robust multimodality image registration tools. Furthermore since this was a feasibility study the patients were not asked to lie in the treatment position for the MRI scan. However for future studies which aim to fuse the MRI with CT patients will be required to do so. However if these challenges are addressed fused 4D images would provide a more realistic picture of the behavior of thoracic anatomy over multiple respiratory cycles. Such guidance would enable the development of novel 4D treatment planning paradigms that explicitly account for effects such as baseline shifts and changes in abdominal versus thoracic breathing. Finally several investigators are working on integrated MRI+linac designs [21“23]. Online prospective 4D MRI would enable such systems to perform real-time monitoring and potentially real-time beam adaptation. 4. Conclusion We have investigated the feasibility of rapid MRI as a modality for image-based guidance in lung radiotherapy. While the acquisition speeds of cine-2D imaging are adequate for capturing most respiratory motion significant further improvements are required to achieve comparable speeds for truly 4D MRI acquisition. Nevertheless these early results indicate that rapid MRI offers a highly attractive noninvasive imaging tool for respiratory motion management. The ability to perform dose-free long-term monitoring over multiple respiratory cycles yields valuable information that is not currently available with 4DCT. We expect that such image-guidance will lay the groundwork for significantly better respiratory motion management in lung radiotherapy. Acknowledgments This work was partially supported by the American Association of Physicists in Medicine (AAPM) Research Seed Funding Grant 2008. Conflict of Interests The authors declare that there is no conflict of interests regarding the publishing of this paper. 1 Keall PJ Mageras GS Balter JM The management of respiratory motion in radiation oncology report of AAPM Task Group 76 Medical Physics 2006 33 10 3874 3900 2-s2.0-33749422038 17089851 2 Mageras GS Pevsner A Yorke ED Measurement of lung tumor motion using respiration-correlated CT International Journal of Radiation Oncology Biology Physics 2004 60 3 933 941 2-s2.0-4744343520 3 Keall P 4-Dimensional Computed Tomography Imaging and Treatment Planning Seminars in Radiation Oncology 2004 14 1 81 90 2-s2.0-0842306300 14752736 4 Wink NM Panknin C Solberg TD Phase versus amplitude sorting of 4D-CT"

Lung_Cancer

"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 (C). Strong ALK expression was identified in 32 cases weak expression in 12 cases and no expression in 253 cases (). 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. 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. FISH analysis was performed on the 297 cases to evaluate ALK gene rearrangement status. Two hundred and eighty-six out of 297 cases were informative for FISH analysis and 33 cases were identified with ALK+?(E). Thirty of the 33 ALK+?cases showed strong ALK expression and the other 3 showed weak ALK expression. Therefore there were 11 cases that showed ALK expression but were ALK-. We re-reviewed the FISH slides of the 11 discordant cases (2 cases with strong and 9 cases with weak ALK expression) and 3 cases (1 with strong and 2 with weak ALK expression) were identified as ALK+?while 8 (1 case with strong and 7 with weak ALK expression) were still ALK- ( F). Regarding the 3 ALK+?cases which were not identified by the original FISH analysis a case-by-case analysis revealed the following: Case 1 The dominant FISH signal pattern in this case was more than one copy of a single green signal without a corresponding orange signal in addition to fused signals (D). According to the ALK signal enumeration guide this indicated a deletion of the orange portion of the ALK probe which targeted the drug targeting area. Therefore we initially considered this case as negative. After re-reviewing the FISH analysis we found there were some areas containing scattered ALK+?cells with one or more copies of single green signals in addition to fused signals and a single red signal. The first 50 cells counted revealed 8 ALK+?cells. The second and third cell count in another 100 cells by different readers revealed 6 and 7 ALK+?cells respectively. If the first and third 50-cell count was considered the average percentage of positive cells reached 15%. Therefore this sample should be considered positive. Case 1 and 3 For these two cases originally constructed on TMA and IHC analysis showed strongly positive staining in one core and weakly positive staining in the other. After re-reviewing the FISH slides we found that there was indeed a small area of each core with a few cells containing subtle break-apart signals. As cell counts were difficult to perform in small areas containing not many cancer cells we cut the tissue sections. The IHC analysis still demonstrated strongly and weakly positive ALK expression respectively. The FISH analysis in the tissue sections showed ALK+. According to the final result of FISH analysis 36 out of the 286 lung adenocarcinoma cases were identified with ALK+. None of IHC negative cases were ALK+ demonstrating 100% sensitivity. Eight IHC-positive cases (1 strongly and 7 weakly positive cases) did not show ALK gene rearrangement resulting in 81.8% specificity. The concordance rate of IHC and FISH is 97.2% (). qRT-PCR and VENTANA ALK IHC analysis of discordant cases To further identify whether eight discordant cases of IHC and FISH carried ALK fusion at the RNA level a qRT-PCR analysis was applied. Positive qRT-PCR results were observed in 5 cases (1 strongly and 4 weakly positive cases) (Table 2). Among the 5 cases 3 (1 strongly and 2 weakly positive cases) were shown to have ALK expression using VENTANA ALK IHC analysis (G). The ALK fusion in these 3 cases was detected at around 14 of 30 qRT-PCR cycles (J). Regarding the other two cases although weak staining in cancer cells could be observed (H) they were considered negative according to the manufacturer™s scoring algorithm (details in Materials and Method section). The ALK fusion in these 2 cases was detected at around 28 of 30 qRT-PCR cycles (K). The remaining 3 of the 8 discordant cases showed neither VENTANA ALK staining nor ALK fusion (Figures 1I and 1L). Table 2 VENTANA IHC and qRT-PCR analysis of all weakly positive and discordant cases detected by CST ALK (D5F3) Sample ID FISH IHC (CST) IHC (VENTANA) qRT-PCR Variant type 9 Positive 1+ Positive EML4-ALK variant 1/2/3a/3b 37 Positive 1+ Positive EML4-ALK variant 1/2/3a/3b 67 Positive 1+ Positive EML4-ALK variant 1/2/3a/3b 94 Positive 1+ Positive EML4-ALK variant 1/2/3a/3b 98 Positive 1+ Positive EML4-ALK variant 1/2/3a/3b 28 Negative 2+ Positive EML4-ALK variant 1/2/3a/3b 171 Negative 1+ Positive EML4-ALK variant 1/2/3a/3b 203 Negative 1+ Positive EML4-ALK variant 1/2/3a/3b 21 Negative 1+ Negative EML4-ALK variant 1/2/3a/3b 36 Negative 1+ Negative EML4-ALK variant 1/2/3a/3b 41 Negative 1+ Negative Negative 39 Negative 1+ Negative Negative 74 Negative 1+ Negative Negative VENTANA ALK IHC and qRT-PCR assays were also applied to the remaining 5 of the 12 ALK weakly expressed cases which were concordant with FISH analysis. These 5 cases were shown to have ALK expression detected by VENTANA ALK IHC and ALK fusion revealed by qRT-PCR analysis (Table 2). Clinicopathological characteristics of patients with ALK+ Using FISH analysis as a standard detection method the clinicopathological characteristics of the ALK+?and ALK- patients were compared and the results are shown in Table 3. As the median ages of the positive and negative groups were 48 and 58 years respectively the ALK+ patients were significantly younger (p <0.001). Patients with ALK+ were more likely to have lymph node metastasis compared to ALK- patients (p = 0.002). No correlation was observed between ALK+ and ALK- cases in terms of sex smoking habit tumor size pT M factors or pathologic TNM stage. Table 3 Clinicopathologic comparisons between EML4“ALK fusion-positive and fusion-negative lung adenocarcinomas Overall EML4“ALK(+) EML4“ALK(?) n?=?287 n?=?37 (%) n?=?250 (%) P value Age Mean?±?SD 48.16?±?11.529 58.17?±?10.03 <0.001 Median 48 58 Range 24-70 25-81 Sex Male 20(13.1) 133(86.9) 0.999 Female 17(13.1) 113(86.9) Nonavailable 0 4 Smoking Never smoker 22(13.8) 138(86.2) 0.239 Ever smoker 9(8.9) 92(91.1) Nonavailable 6 20 Tumor size(mm) Mean?±?SD 38.42?±?24.263 37.59?±?16.837 0.872 Median 32.50 35.00 Range 7-100 10-90 pT status pT1 4(13.8) 25(86.2) 0.350 pT2 14(8.3) 154(91.7) pT3-T4 5(15.6) 27(84.4) Nonavailable 14 44 pN status pN0 2(2.0) 100(98.0) 0.002 pN1 11(16.7) 55(83.3) pN2-3 7(13.5) 45(86.5) Nonavailable 17 50 pTNM stages pStage I 2(2.5) 79(97.5) 0.028 pStage II 9(12.2) 65(87.8) pStage III 9(14.3) 54(85.7) Nonavailable 17 52 Discussion In this study we applied IHC and FISH analyses using CST™s D5F3 antibody in a Chinese lung adenocarcinoma sample cohort. An accurate FISH analysis depends on multiple factors including fine equipment skilled personnel well-preserved FFPE samples enough cancer cells etc. In this study two cores in TMAs were not identified with ALK+?in initial FISH analysis due to a lack of cancer cells. Similarly in biopsies the numbers of cancer cells is often very limited making an accurate FISH analysis difficult. With the IHC analysis in this study almost all of the cancer cells in the two cores showed ALK expression despite the fact that only a few ALK+?cells were revealed by FISH analysis. A <100% rate of cellular positivity in ALK+?tumors has been demonstrated to be due to the technical limitations of FISH analysis [13]. Therefore combining IHC and FISH analyses results in ALK status being more accurately evaluated in biopsies. IHC analysis using CST™s D5F3 antibody has been demonstrated with 100% sensitivity [1214-16] suggesting that IHC analysis is an effective way to prescreen patients for FISH analysis in the clinical diagnosis process [141517]. For IHC negative cases FISH analysis is not necessary. In strongly positive IHC cases FISH analysis also may not be necessary. Although there was one strongly positive IHC case which was shown with ALK- by FISH analysis the VENTANA ALK assay and qRT-PCR analysis revealed ALK expression and ALK fusion respectively. In addition it has been reported that the lung cancer patient with IHC-positive and FISH-negative ALK had a dramatic response to crizotinib [18]. Therefore the patient in our case may benefit from crizotinib. Weakly positive IHC cases must be carefully examined. In this study 7 out of 12 (58.3%) weakly positive cases were discordant with FISH analysis. Using the VENTANA ALK IHC assay three out of the seven weakly positive cases showed ALK expression and could be treated with crizotinib. Using qRT-PCR analysis five out of the seven weakly positive cases showed ALK fusion at the RNA level. Therefore there were two cases in which the qRT-PCR analysis result was discordant with the VENTANA ALK IHC assay. Compared to negatively expressed ALK cases without any staining (I) these two cases were indeed weakly stained in cancer cells using the VENTANA ALK IHC analysis (H). "

Lung_Cancer

"The main complexity of modeling and interpreting such phenomena lies in the additional temporal dimension needed to express the association as the risk depends on both intensity and timing of past exposures. This type of dependency is defined here as exposure“lag“response association. In this contribution I illustrate a general statistical framework for such associations established through the extension of distributed lag non-linear models originally developed in time series analysis. This modeling class is based on the definition of a cross-basis obtained by the combination of two functions to flexibly model linear or nonlinear exposure-responses and the lag structure of the relationship respectively. The methodology is illustrated with an example application to cohort data and validated through a simulation study. This modeling framework generalizes to various study designs and regression models and can be applied to study the health effects of protracted exposures to environmental factors drugs or carcinogenic agents among others. 2013 The Authors. Statistics in Medicine published by John Wiley & Sons Ltd. latency distributed lag models exposure“lag“response delayed effects splines 1. Introduction In biomedical research it is commonly appreciated that an exposure event produces effects lasting well beyond the exposure period with an increase in risk occurring from few hours to many years later depending on the physiological processes linking the exposure and the health outcome. The problem is made even more complicated in the presence of protracted time-varying exposures when the health effect measured at a given time can be described as the result of multiple exposure events of different intensities sustained in the past. This phenomenon common to various research fields has been associated for example with peak 1 or chronic exposures 2 to environmental stressors drug intake 34 or occupational exposures to carcinogenic substances 5. The main complexity of modeling and interpreting such dependencies lies in the additional temporal dimension needed to express the association beyond the usual exposure“response relationship as the risk depends on both intensity and timing of past exposures. Nonetheless the appropriate representation of the temporal pattern of risks may provide further insights on the association of interest in particular regarding the underlying pathophysiological mechanisms and prevent biases in estimates and predictions. Revising previous terminology 6 I define these dependencies as exposure“lag“response associations. In particular this issue has been debated in cancer epidemiology 7“9. Analytical approaches extend simple indices such as cumulative exposure in order to accommodate the temporal variation in risk because of protracted exposures. In particular the pioneering work by Thomas 106 helped develop sophisticated statistical methods on the basis of weighting past exposures through specific functions whose parameters are estimated by the data. Vacek 11 Langholz and colleagues 12 and Richardson 13 provided interesting applications in case-control studies with weights represented through simple parametric functions. The methodology was improved by Hauptmann and colleagues in a series of papers 14“16 by using flexible and smooth spline functions. Sylvestre and Abrahamowicz 17 and Abrahamowicz and colleagues 18 extended the spline methods to the analysis of time-to-event data with a cohort design and presented their applications in pharmaco-epidemiology. The main limitation of the statistical techniques described in these papers is the assumption of a linear exposure“response relationship. Models for nonlinear dependencies introduce further nontrivial complexities from both statistical and interpretational perspectives as the problem becomes inherently bidimensional. Abrahamowicz and Mackenzie 19 proposed a model for analyzing the nonlinear time-dependent effects of fixed exposures while Vacek 11 and Berhane and colleagues 20 extended this scheme to the case of protracted time-varying exposures. However the modeling techniques illustrated in these other papers still face some limitations as they are based on complex estimation routines with convergence issues and problems in producing uncertainty measures such as standard errors and confidence intervals. Interestingly equivalent approaches were previously established in time series analysis on the basis of distributed lag models (DLMs) a methodology originally formulated in econometrics 21 then applied in epidemiological research 22. These models involve the definition of a distributed lag function analogous to the weighting function described before. In particular Armstrong 23 generalized the method to distributed lag non-linear models (DLNMs) a class of models with different options for the functions applied to model nonlinearity and distributed lag effects. The theory of DLMs and DLNMs have been recently re-evaluated 24 offering a well-grounded statistical tool and a comprehensive scheme for interpretation. In this paper I aim to establish a general conceptual and statistical framework for modeling exposure“lag“response associations built upon the paradigm of DLMs and DLNMs. This modeling class extended beyond time series analysis provides a unified methodology applicable in different study designs data structures and regression models including most of the previous methods as specific cases. Also the statistical framework is defined by completely parametric functions and fitted through standard regression methods with measures of uncertainty and fit statistics easily available. The R package dlnm originally developed for time series data 25 is extended in parallel offering a easy-to-use implementation of the modeling approach. The manuscript is structured as follows. The development and algebraic definition of the modeling framework is described in Section 2. As an illustrative example in I apply the method for investigating the relationship between occupational exposure to radon and lung cancer mortality by using the data from the Colorado Plateau miners cohort. The modeling framework is then validated in a simulation study n. A final discussion is provided in. Information on data and software implementation is included in. The R code and data are included in the supporting information together with additional details making the results of the illustrative example and of the simulation study entirely reproducible. 2. Modeling framework The modeling skeleton is derived by extending the class of DLNMs beyond the time series context. This extension provides a neat algebraic representation and a comprehensive statistical definition. The focus is on a function here defined s(xt) which describes the dependency in terms of the exposure history to x evaluated at time t. The function s(xt) is commonly included in regression models in order to estimate the association while controlling for potential confounders. Although the regression model varies depending on the study design and the type of data the definition of s(xt) provided later and the related modeling framework generally apply. 2.1. Models for linear exposure“response relationships Previous studies on the topic have defined the function s(xt) by using slightly different algebraic formulae 1026111417. Assuming a linear exposure“response relationship a general notation can be given by (1a) (1b) (1c) In (1a) the increase in risk at time t is defined as the integral of the instantaneous exposure intensity xu over the period ?t = [t0t1] with t0 and t1 representing the times of the first and last relevant exposures. Here w(t ? u) is the weighting function previously described in which assigns weights to past exposures experienced at time t ? u on the basis of their contribution to the risk at time t. The model can be reparameterized as in (1b) where the risk is now expressed along the lag with ? ? [?0L]. Here L ? ?0 = t1 ? t0 is interpreted as the lag period over which an exposure to x is assumed to affect the risk at time t usually with ?0 = 0. This parameterization offers the advantage that the function w is now directly defined in the new dimension of lag ? and it is independent of the time axis chosen for t which may represent different time scales depending on the study design. The function w(?) termed from here on as the lag“response function models the lag“response curve associated with exposure x. Finally for computational purposes the integral is approximated in (1c) by a sum of terms derived by partitioning the lag interval in equally spaced discrete units and assuming the protracted exposure as a sequence of exposure events xt ? ? at lags ? = ?0 ¦ L. A statistical model for (1) can be defined by expressing the lag“response function w(?) as a linear combination of terms obtained through basis transformation with related parameters. By using matrix notation let the vector qxt of exposure history be defined by (2) Such exposure history changes along time depending on the time t at which the vector qxt is computed. Given (2) the cumulative function s(xt) in (1) can be written using a compact and general matrix notation as (3) The (L ? ?0 + 1) × v? matrix C is obtained from the transformation of the lag vector ? = [?0 ¦ ? ¦ L]T by choosing a specific basis with dimension v? for w(?) which defines the related basis functions. In this parameterization the function s(xt) representing the integral of x · w(?) over the interval [?0L] is defined as a lag“basis function with parameters ?. Interestingly the equation in (3) is almost identical to that defining DLMs 24 Eq. (4). The different indexing in the original version reflects the specific application in time series where the data are perfectly ordered in time and the matrix Q has a structure such that qt? ? qt + 1? + 1. However this is a specific case of the general representation in (2)“(3). The theory and software already developed for DLMs can be therefore extended in parallel. Alternative lag“basis functions for representing s(xt) are derived through different lag“response functions w(?) in (1). In particular the traditional index of unweighted cumulative exposure is a specific case of (3) where reduces to with w(?) equal to a constant c. This is obtained by specifying C as an (L ? ?0 + 1)-dimensional vector of 1's with v? = 1. More sophisticated models with splines or other functions such as those illustrated in publications cited in only require the application of different bases for deriving C but are nevertheless represented by 3). 2.2. Extension to nonlinear exposure“response relationships The extension to the nonlinear case presents further complexities as anticipated earlier. The model in (1) can be extended by defining an additional exposure“response function f(x) to express the potentially nonlinear exposure“response curve along the dimension of the predictor. An intuitive generalization of (1) is: (4) with f(x) as the standard exposure“response function. However the function f(x) · w(?) in 4) previously proposed 1119 is not easily represented as a linear combination of basis variables and generates models that are not linear in their parameters and thus require ad hoc optimization routines. More importantly this representation is based on the strong assumption of independency between f(x) and w(?) namely that the exposure“response shape is the same at each lag ? and vice versa that the lag structure is the same at each value of x. This assumption can be relaxed by expressing s(xt) as a truly bivariate function with the more flexible representation: (5) Here the bidimensional function f · w(x?) is defined as the exposure“lag“response function and models simultaneously the exposure“response curve along x and lag“response curve along ? namely an exposure“lag“response surface. Differently from 4) the exposure“lag“response function in 5) can be expressed as a linear combination of basis variables and related parameters through a special tensor product. As anticipated earlier Armstrong 23 proposed the same approach for time series data within the DLNM framework generalizing this tensor product parameterization through the concept of cross-basis. Specifically two sets of basis functions are independently chosen to represent f(x) and w(?) respectively. The cross-basis is the bidimensional space of functions obtained by the combination of the two sets integrated over the lag dimension and represents the core of DLNMs. The algebraic representation has been previously presented 24 and a revised version is proposed here. Briefly the simpler lag-basis for DLMs in 3) can be extended by choosing an additional basis with dimension vx for representing f(x). The application of the related basis functions to the vector of exposure history qxt obtained by 2) generates a (L ? ?0 + 1) × vx matrix Rxt. Let Axt be: (6) with 1v as a v-dimensional vector of 1's and C defined in 3). The cross-basis function s(xt;?) can be defined as (7) In this case the dimension of the cross-basis is determined by the product of the dimensions of the bases for the two spaces and the association is expressed through vx · v? values W and related parameters ?. The cross-basis function s(xt) represents the integral of f · w(x?) over the interval [?0L] cumulating the contributions of events representing the exposure history. In spite of the relatively complex algebraic form the definition of cross-basis and the specification of DLNMs only amount to the choice of the bases for the functions f(x) and w(?). These can be independently selected between several options such as splines linear threshold or piecewise constant (step) functions. The DLNM modeling class comprises the simpler DLMs from Section 2.1. For example the bidimensional exposure“lag“response function f · w(x?) in 5) reduces to a non-linear function for un-weighted cumulative exposure f(x) · c when w(?) is a constant function c and to the lag“response function x · w(?) in (1) when f(x) is simply an linear function of the untransformed x. The model proposed by Berhane and colleagues 20 can be written in the form of 6)“7) when both f(x) and w(?) are cubic B-splines. 2.3. Estimation and prediction Although the lag-basis and cross-basis functions in (1)“(3) and (5)“(7) involve a nonstandard parameterization in terms of exposure histories DLMs and DLNMs do not require specialized estimation procedures. The association is entirely expressed by the vx × v? parameters ? of the cross-basis values W. The computation of the exposure history in (2) can be extended to all N observations with x measured at time t producing an N × (L ? ?0 + 1) matrix of exposure histories Q. The matrix of transformed variables W in (3) and (7) is consequently derived. This matrix can be included in the design matrix of standard regression models to estimate the parameters ?. In the completely parametric development proposed here the number of coefficients vx × v? represents the degrees of freedom (df) used to model the association. Inference on the parameters ? and interpretation of the estimated association is aided by the prediction of specific risk measures. For simpler DLMs that assume a linear exposure“response relationship this step reduces to the computation of a series of estimated risk contributions at lag ?p with ?0 ? ?p ? L and the associated (co)variance matrix . The series of risk contributions is provided by (8) with Cp obtained from the vector of lag ?p used for prediction by applying the same basis functions for w(?) used for estimation. These estimated risk contributions compose the lag“response curve and can be interpreted using either a forward or backward perspective. Namely represents the risk contribution at time t + ?p in the future from a unit increase in exposure x at time t or the contribution from a unit increase in exposure x occurring at time t ? ?p in the past to a given risk measured at time t. The estimated risk contributions associated with different exposure increases are easily derived. The equations in (8) only apply to DLMs with lag-bases as defined in (3). For DLNMs the association is allowed to vary nonlinearly in the space of x. Moreover the specification in (5)“(7) allows the lag-response curve to change depending on the level of the exposure. The prediction of risk contributions corresponding to a specific exposure intensity xp at lag ?p involves a more complex procedure. First let be the (L ? ?0 + 1)-dimensional vector of exposure history with constant exposure xp. The related matrices and are derived from (6) substituting qxt and C with and Cp by applying the same two sets of basis functions for f · w(x?) chosen for estimation. The exposure-specific risk contributions and associated (co)variance matrix are provided by (9) The estimated risk contributions may be interpreted as a lag-response curve similar to in (8) but this time associated with a specific exposure level xp instead of a unit increase. These measures may be used to define a grid of predicted risk contributions defined within the ranges of the exposure x and the lag ? thus obtaining a bi-dimensional representation of the association. From this grid besides above it is also possible to derive the vector of lag-specific risk contributions expressing the exposure-response curve for lag ?p. As noted in Section 2.2 the truly bivariate definition of (7) allows both the lag-response curve and exposure-response curve defined by and respectively to change depending on the specific exposure and lag values xp and ?p. The grid is interpreted as a risk surface along x and ? representing the exposure“lag“response. In addition predictions in (8)“(9) may be extended to a generic exposure history qh. Substituting it into in (9) provides the vector of lag-specific risk contributions for each exposure that occurred within the lag period. The overall cumulative effect of such exposure history with associated (co)variance matrix may be computed with: (10) The Equation (10) can be used to estimate the predicted cumulative risk for a given pattern of exposure qh. This method can also be applied to investigate how the risk progressively evolves along an exposure profile computing the cumulative risk at each time associated with the time-varying exposure history qh. 2.4. Identifiability and constraints The tensor product structure of the cross-basis defined in (5)“(7) poses some identifiability issues. In particular each of the vx basis variables in R is multiplied by each of the v? basis variables in C. If an intercept is included in f(x) the related matrix of cross-basis variables W is not of full rank and the parameters of the regression model are not identifiable even when a common intercept is not included. Therefore the cross-basis in (7) should always be defined without an intercept in the basis functions for x. Also these basis functions can be centered on a specific exposure value x0 which will represent the reference for the risk summaries computed by (8)“(10). The bidimensional shape of the exposure“lag“response can be constrained to follow a prespecified pattern. In particular a priori assumptions on the lag structure can be imposed through functional constraints on the basis for the space of ?. Left and right constraints on the extremes of the supporting interval ?0“L are particularly meaningful for smooth functions. A left constraint can be imposed by excluding the intercept from the basis. This step will force the lag“response curve to predict a null risk at the beginning of the lag period. A right constraint on a B-splines basis can be produced by excluding specific basis variables as previously described for linear exposure“response relationships 17. The constraint produces a smooth dependency which approaches a null risk at the end of the lag period. Such constraints are particularly useful in the presence of sparse data in order to limit the flexibility of the model under specific assumptions about the lag“response curve. However biases can be introduced if these assumptions are not met. Additional information is provided in Section D1 of the supporting information. The functional constraints discussed in this section can be specified without introducing customized optimization methods for estimating the parameters ? in (3)“(7). More sophisticated methods are required for example to constrain the lag“response curve to be non-negative in the whole lag period L. These approaches have been previously proposed for linear dependencies 141718 and introduce further complexities in the bidimensional context of DLNMs. This development is not pursued here. 2.5. Model selection and inferential procedures The framework described in Sections 2.1“2.2 includes a fairly large number of models defined by different functions for each of the two dimensions and by different choices regarding each function such as number and location of knots in splines. This raises the issue of selecting the optimal model for describing the exposure“lag“response association. Previous studies on temporal dependencies have proposed selection procedures on the basis of profile likelihood 15 AIC 141620 or BIC 17. Simulation studies seems to indicate a better performance of AIC when compared with BIC in this context 18 a result consistent with unpublished simulations performed on time series data for DLNMs. Inference on the models illustrated in the previous sections primarily focuses on the specification of confidence intervals for the risk measures in Section 2.3 and on the definition of tests for a set of null hypotheses. Confidence intervals for lag“response curves exposure“response curves and cumulative risks obtained through and can be easily derived from the diagonal of the related (co)variance matrices in (8)“(10) assuming a multivariate normal distribution of the estimators. Regarding hypothesis testing two null hypotheses are particularly relevant in this framework. The first one postulates a linear exposure“response relationship namely H0 : f(x) = x. The second one assumes a constant risk namely H0 : w(?) = c. Tests on constrained models can be also defined. The assumption of independency is not easily tested as the form in (4) cannot be expressed as a model linear in its parameters. However defining general inferential procedures in this setting is not straightforward. First the null hypotheses H0 : f(x) = x and H0 : w(?) = c are not independent and an incorrect assumption about the association in one dimension may bias the test estimator for the hypothesis related to the other space as previously reported 19. In addition estimates are usually conditional on a posteriori selection of a best-fitting model based on the selection methods discussed before. Under these conditions the estimators for the (co)variance matrices in (8)“(10) are likely to underestimate the true sampling (co)variance and the distribution of the test statistics may be different from that assumed unconditional on the selection procedure. This may generate undercoverage of confidence intervals and inflated type I error for tests 1727. Given these complexities a general framework for hypothesis testing embedded in the model selection procedure is not provided here. An assessment through simulations of the performance of estimators generated by AIC and BIC-selected models will be presented in. Specifically simulations will provide an empirical evaluation of the ability of the information criteria to identify the correct model between those defining the null or alternative hypotheses about linearity and constant effects and measures of performance such as bias coverage and root mean square error. 3. An application The conceptual and statistical framework of DLNMs described in extended beyond time series data is general and applicable in different study designs. As an illustrative example I propose here an application in survival analysis of time-to-event data. This represents one of the most complex settings as the temporal pattern of risk is produced by exposure histories that vary during the follow-up of each subject. Specifically the methodology is used to investigate the association between occupational exposure to radon and mortality for lung cancer. The analysis is based on data from the Colorado Plateau uranium miners cohort already used in previous methodological contributions 121520. Section A of the supporting information provides a list of the main steps to replicate the analysis in other real-life examples. 3.1. Data The cohort data used in this example were collected by the National Institute for Occupational Safety and Health. Detailed information on the cohort is given elsewhere 12. Briefly subjects were eligible to enter the cohort if they worked in mines within the Colorado Plateau area between 1950 and 1960 and provided demographic personal and occupational information during their working period. Vital status and cause of death were ascertained by linkage with different sources. The data used in this example refer to the follow-up of the cohort on December 311982 including 3347 subjects and 258 lung cancer deaths. Exposure data available in the data set include cumulative measures of radon and smoking in 5-year age intervals. The radon exposure history for each subject expressed in working-level months (WLM) was reconstructed by linking employment information with measured or predicted levels in each mine in each year. The smoking history expressed in the number of cigarettes packs × 100 was reported by each subject during his working period and assumed constant after the last reporting age. A summary of the data is provided in Table I. Table I Descriptive statistics of the Colorado Plateau uranium miners cohort. The data included here refer to the follow-up on December 31 1982. Exposure to radon is measured in working level months (WLM) while smoking is reported as packs of cigarettes/100 Full cohort Lung cancer cases N % N % Subjects 3347 100.0 258 7.7 Deaths (%) 1258 37.6 258 100.0 Ever smokers (%) 2656 79.4 238 92.2 Median Min 25th 75th Max Median Min 25th 75th Max Age at entry 34.0 15.8 25.8 44.0 80.0 41.6 18.6 34.3 48.0 63.9 Follow-up time (years) 23.9 0.1 19.6 25.5 32.5 18.3 0.3 12.9 22.0 30.8 Exposure to radon Exposure period (years) 6.7 0.1 2.7 11.8 53.0 12.8 0.1 7.8 17.6 39.5 Total cumulative exposure (WLM/year) 429.0 0.0 153.5 1016.8 10000.0 1231.9 8.0 553.7 2528.6 10000.0 Yearly exposure (WLM/year) All 60.2 0.1 26.7 122.2 3245.3 81.6 1.0 42.3 165.4 1295.7 Lag 0“9 52.4 0.1 23.8 102.5 2994.0 61.4 3.9 31.3 144.7 1110.8 Lag 10“19 53.8 0.1 24.3 112.5 3245.3 78.3 1.0 42.9 164.0 1295.7 Lag 20“29 74.0 0.1 33.0 141.7 3245.3 104.7 4.1 52.2 180.0 1295.7 Lag 30“40 95.7 0.2 48.0 151.6 2994.0 104.7 5.5 60.0 175.3 860.2 Smoking Exposure period (years) 38.0 5.0 31.0 46.0 75.0 40.0 14.0 33.0 48.0 72.0 Total cumulative exposure (packs × 100) 131.6 0.4 94.5 174.5 676.3 147.4 21.8 109.5 188.1 567.2 Yearly exposure (packs × 100) 3.6 0.0 2.5 3.6 24.4 3.6 0.0 3.5 4.2 13.4 3.2. Modeling strategy For this illustrative example the analysis is performed through a Cox proportional-hazard model with time-varying covariates by using age as the time axis. Effect measures are reported as a hazard ratio (HR). The model is represented by the following: (11) where the log-hazard log [h(t)] is expressed as a sum of baseline log-hazard log [h0(t)] and contributions of additional covariates. These comprise cross-basis functions sx(xt) and sz(zt) for radon and smoking respectively as defined in (1)“(7) and a linear term for calendar time u in order to control for secular trends in lung cancer risk not accounted for by the delayed effects of the two exposures. Radon is the exposure of interest and is modeled with different combinations of bases for f(x) and w(?) in the cross-basis sx(xt). Given the limited information on smoking histories in this analysis the cross-basis sz(zt) is a priori defined with a natural cubic B-spline with one knot at the median of 2.5 yearly packs × 100 for the exposure“response and a step function with a single cut-off at lag 20 for the lag structure with lag period 2“40 years. However different cross-basis functions can be applied. The model spends 5 df controlling for confounders and a different amount for modeling the effect of radon depending on the chosen cross-basis sx(xt). Modeling exposure“lag“response associations in time-to-event data assumes the definition of an extended version of continuous time-varying predictors namely the varying exposure history for each subject at the ages he contributes to different risk sets 28. The lag scale is chosen as years with lag 0 identifying the exposure during the last year. The lag period is fixed at 2“40 assuming no effect of exposure after 40 years and in the last 2 years consistently with previous analyses. Multiple exposure histories are computed for each subject at the ages he contributed to each risk set given his exposure profile reconstructed from the 5-year periods. This step produced matrices of exposure histories Qx and Qz for radon and smoking respectively as defined in (2). These matrices are used to specify the lag-bases or cross-bases matrices Wx and Wz from (3)“(7) included in the design matrix of the Cox model."

Lung_Cancer

"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). .0091811.g001 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 . 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). .0091811.t001 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)."

Lung_Cancer

"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). 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. Figure 1 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. To assess DFS the disease status was monitored every 2 months for the first 6 months and subsequently every 3 months by computed tomography after enrollment and according to good medical practice. Toxicities related to the administration of chemotherapy were assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events (version 3.0; ctep.cancer.gov). DFS was defined as the time from the date of enrollment to disease recurrence or death due to any cause and estimated according to the Kaplan-Meier method. A Cox regression model was fit with the time from surgery to enrollment as a covariate to evaluate its effect on DFS. A natural log transformation was applied to the raw protein measurement data and the Pearson correlation coefficient was used to test associations. Bivariate comparison of baseline characteristics between the assigned treatment groups was performed using the Fisher exact test for categorical variables or the Student t test or Wilcoxon rank sum test for continuous variables. A multivariable logistic model to evaluate baseline factors and treatment assignment was fit using backwards selection. Median ERCC1 and RRM1 expression levels were compared with historical medians using the 1-sample Wilcoxon signed rank test. The percentage of patients with both ERCC1 ??65 and RRM1 ??40 was compared with the historical rate using a chi-square test. All statistical analyses and graphics were performed using SAS statistical software (version 9.2; SAS Institute Inc Cary NC). A significance level of 5% was used for all analyses. RESULTS Patient and Trial Characteristics To ensure an adequate sample size of eligible patients and biomarker-specific subgroups a total of 85 patients was registered between April 2 2009 and April 1 2011 from 27 participating sites. Four patients were ineligible; 3 had inadequate lymph node sampling and 1 did not have a tumor measuring ??2 cm. Table 1 provides the characteristics of the 81 eligible patients. Table 1 Patient Demographics and Disease Characteristics Variablesa All Patients Assigned to Chemotherapy Assigned to Observation P Refused Assignment Accepted Assignment P N = 81 N = 63 N = 18 N = 20 N = 61 Age y .37 .39 ?Median 64 63.3 68.8 67.2 63.3 ?Mean 63.5 62.9 65.5 65.2 62.9 ?Range 41.6“84.2 41.6“84.2 41.6“81.7 44.2“82.9 41.6“84.2 Sex .18 .61 ?Female 44 (54%) 37 (59%) 7 (39%) 12 (60%) 32 (52%) ?Male 37 (46%) 26 (41%) 11 (61%) 8 (40%) 29 (48%) Ethnicity .65 .18 ?Unknown 7 (8%) 5 (8%) 2 (11%) 0 (0%) 7 (11%) ?Non-Hispanic 74 (91%) 58 (92%) 16 (89%) 20 (100%) 54 (89%) Race .73b .75b ?African American 8 (10%) 8 (13%) 0 (0%) 2 (10%) 6 (10%) ?Asian 3 (4%) 2 (3%) 1 (6%) 0 (0%) 3 (5%) ?Pacific Islander 2 (2%) 1 (2%) 1 (6%) 0 (0%) 2 (3%) ?White 66 (81%) 52 (83%) 14 (78%) 17 (85%) 49 (80%) ?Unspecified 2 (2%) 0 (0%) 2 (11%) 1 (5%) 1 (2%) Histology .06c .60c ?Adeno 52 (64%) 44 (70%) 8 (44%) 14 (70%) 38 (62%) ?Squamous 25 (31%) 17 (27%) 8 (44%) 6 (30%) 19 (31%) ?Large 1 (1%) 1 (2%) 0 (0%) 0 (0%) 1 (2%) ?Bronchioloalveolar 1 (1%) 0 (0%) 1 (6%) 0 (0%) 1 (2%) ?Other 2 (2%) 1 (2%) 1 (6%) 0 (0%) 2 (3%) Stage of disease .16 .27 ?IA (<3 cm) 25 (31%) 22 (35%) 3 (17%) 4 (20%) 21 (34%) ?IB (?3 cm) 56 (69%) 41 (65%) 15 (83%) 16 (80%) 40 (66%) Zubrod performance status .11 1.00 ?0 44 (54%) 31 (49%) 13 (72%) 11 (55%) 33 (54%) ?1 37 (46%) 32 (51%) 5 (28%) 9 (45%) 28 (46%) Weight loss (6 mo) 1.00d .31d ?<5% 64 (79%) 49 (78%) 15 (83%) 14 (70%) 50 (82%) ?5-<10% 9 (11%) 7 (11%) 2 (11%) 3 (15%) 6 (10%) ?10“20% 4 (5%) 3 (5%) 1 (6%) 2 (10%) 2 (3%) ?>20% 1 (1%) 1 (2%) 0 (0%) 0 (0%) 1 (2%) ?Unknown 3 (4%) 3 (5%) 0 (0%) 1 (5%) 2 (3%) Smoking status ?Current 33 (41%) 26 (41%) 7 (39%) 8 (40%) 25 (41%) ?Former (quit ?1 y) 39 (48%) 30 (48%) 9 (50%) 10 (50%) 29 (48%) ?Never 9 (11%) 7 (11%) 2 (11%) 1.00e 2 (10%) 7 (11%) 1.00e Abbreviation: Adeno adenocarcinoma. a All P values shown are 2-sided. b White versus all other races. c Adenocarcinoma versus all other histologies. d Weight loss <5% versus ?5%. e Derived using the Freeman-Halton exact test. The distribution of assignment to chemotherapy and observation was 63 patients (78%) and 18 patients (22%) respectively which was not significantly different (P?=?.20 Fisher exact test) from the expected rates of 70% (129 patients) and 30% (55 patients) respectively.16 Based on protein levels in these 81 patients the number of those with low ERCC1 and low RRM1 was 31 patients (38%) 22 patients had low ERCC1 and high RRM1 (27%) 10 patients had high ERCC1 and low RRM1 (12%) and 18 patients had high ERCC1 and RRM1 (22%) which is not significantly different from prior results (P?=?.14 Fisher exact test; 54 of 184 29%; 38 of 184 21%; 37 of 184 20%; and 55 of 1840.3 respectively). We investigated whether treatment arm assignment varied by patients' smoking status histology age and sex. In bivariate comparisons no statistically significant associations were found. However the multivariable logistic model found that patients with adenocarcinoma (P?=?.03) and potentially stage IA disease (P?=?.06) were more likely to be assigned to adjuvant chemotherapy (ie they were more likely to have low levels of ERCC1 RRM1 or both). One of the 18 patients assigned to observation and 19 of the 63 patients assigned to chemotherapy rejected this choice and withdrew consent. There was no statistically significant difference in patient characteristics between those who accepted and those who refused their treatment assignment (Table 1). Feasibility The trial achieved its primary feasibility objective with a treatment assignment within the prespecified timeframe in 71 of 81 patients (88%). We successfully determined protein levels in all 85 patients. Ten of the 81 eligible patients did not achieve assignment to treatment versus observation within the 84-day time interval from surgical resection. The time interval from surgery to assignment ranged from 86 days to 105 days in these 10 patients. For 3 patients the specimens were received after the 84-day limit had passed. For the other 7 patients the time interval from receipt to reporting ranged from 7 days to 25 days (median 18 days). For the 71 patients with a successful assignment within the 84-day time interval from surgical resection the time from receipt to reporting ranged from 3 days to 26 days (median 8 days). The reasons for reporting results in excess of 14 days were equipment failure and inadequate expression values in control specimens which required equipment recalibration and a repeat processing of the specimens. Overall the time from receipt of specimens to reporting ranged from 1 day to 27 days (median 11 days; mean 12 days) which is similar to that reported for patients with advanced NSCLC (range 1 day-47 days; median 11 days; mean 12 days).18 Survival and Toxicity Survival analyses were performed on the 61 patients who accepted assignment to treatment (44 patients) or surveillance (17 patients). Patients who rejected their treatment assignment withdrew consent and thus could not be followed for survival. Fourteen patients had DFS events; 2 had died (1 from disease recurrence and the other from cardiac disease without recurrence). The median follow-up among those patients still alive at the time of last follow-up was 27 months (range 3 months-44 months). Six patients had <?24 months of follow-up. The collective 2-year DFS and OS rates were 80% (95% confidence interval [95% CI] 67%-88%) (Fig. 2A) and 96% (95% CI 87%-99%) from the date of registration. The 2-year DFS rate was 83% (95% CI 68%-92%) for patients who received chemotherapy (Fig. 2B) and it was 71% (95% CI 43%-87%) for those observed (Fig. 2C). Table 2 includes 2-year DFS estimates within each of the 3 gene expression categories in the chemotherapy arm. The median time from surgery to enrollment was 41 days (range 11 days-79 days). The time from surgery was added as a covariate to a Cox regression model and was not found to be significantly related to DFS (P?=?.22) or OS (P?=?.36). Table 2 Disease-Free Survival Rates Patient Group No. DFS (95% CI) 1-Year 2-Year Accepted assigned treatment 61 88% (77%-94%) 80% (67%-88%) Received chemotherapy 44 95% (83%-99%) 83% (68%-92%) By protein level category (for those that received chemotherapy) ?Low ERCC1/low RRM1 20 95% (69%-99%) 84% (59%-95%) ?Low ERCC1/high RRM1 18 94% (65%-99%) 82% (55%-94%) ?High ERCC1/low RRM1 6 100% (100%-100%) 100% (100%-100%) Abbreviations: 95% CI 95% confidence interval; DFS disease-free survival; ERCC1 excision repair cross-complementing group 1; RRM1 ribonucleotide reductase M1. Figure 2 Kaplan-Meier survival estimates are shown. (A) Collective disease-free survival is shown for patients who accepted adjuvant chemotherapy or observation based on gene expression analysis. (B) Disease-free survival is shown for patients who received adjuvant chemotherapy. (C) Disease-free survival is shown for patients in the observation group. Conf Int indicates confidence interval. A total of 22 patients discontinued chemotherapy because of treatment-related toxicity (50%). None of the patients died because of treatment-related toxicity. Details are provided in Table 3. Table 3 Number of Patients With Grade 3 and Grade 4 Adverse Events Among the 44 Patients Who Received Chemotherapya Level of Severity Adverse Event Grade 3 Grade 4 No. of patients with events 13 14 Type of events ?Neutropenia 11 6 ?Thrombocytopenia 4 4 ?Nausea 4 0 ?Vomiting 4 0 ?Anemia 2 0 ?Anorexia 2 0 ?Fatigue 2 0 ?Febrile neutropenia 1 1 ?Thromboembolism 1 1 ?Dehydration 1 0 ?Hearing impairment 1 0 ?Mucositis 1 0 ?Pleural effusion 1 0 ?Renal failure 1 0 ?Bradycardia (sinus) 1 0 ?Syncope 1 0 ?ALT elevation 1 0 ?Hypokalemia 1 0 ?Hyponatremia 0 2 Abbreviation: ALT alanine aminotransferase. a Adverse events were assessed according to the Common Terminology Criteria for Adverse Events (version 3.0). In Situ ERCC1 and RRM1 Protein Levels RRM1 levels ranged from 2.4 to 234.3 (median 39.7; mean 48.1) which were not significantly different from the expected values (median 40.5; range 8.3-96.2) (P?=?.87).16 ERCC1 protein levels ranged from 4.3 to 211.2 (median 41.9; mean 58.8) and these values were significantly different from the expected values (median 65.9; range 1.9-178.7) (P?=? 0.02). There was a significant correlation noted between ERCC1 and RRM1 levels (correlation coefficient 0.39; P?=?.0003) (Fig. 3) as previously reported.91618 Figure 3 Distribution of excision repair cross-complementing group 1 (ERCC1) and ribonucleotide reductase M1 (RRM1) levels in eligible patients is shown. The median protein levels of ERCC1 in adenocarcinomas squamous cell carcinomas and the other histologies were 34.257.1 and 121.5 respectively. The corresponding median levels of RRM1 were 38.142.6 and 48.9 respectively. Although the levels were higher in squamous cell carcinomas compared with adenocarcinomas the medians were not statistically significant (ERCC1: P?=?.16; RRM1: P?=?.72). DISCUSSION Disease stage is a predictor of benefit from adjuvant chemotherapy in patients with NSCLC. Patients with stage III disease derive the most benefit and those with stage I are reported to derive the least.12419“23 Although not statistically significant for patients with stage I disease and a tumor diameter >?3 cm a numerical risk reduction of 7% has been reported and for those with tumors measuring ??3 cm a numerical risk increase of 40% has been reported.23 A significant treatment-related toxicity is febrile neutropenia which has been reported in 7% to 24% of patients.242022 Treatment-related deaths occur in 0.5% to 2% of patients.122022 The inclusion of molecular markers predictive of therapeutic efficacy into adjuvant decision algorithms would greatly improve the clinical benefit and reduce toxicity for patients with NSCLC. This approach is particularly attractive for patients with stage I disease in whom the parameters for weighing risks and benefits are to our knowledge the least well defined. Recent advances in molecular diagnostics have resulted in improved outcomes for patients whose tumors harbor mutations in oncogenic signal transduction molecules that can be inactivated by therapeutic agents. Similarly platinum agents target DNA and gemcitabine targets ribonucleotide reductase; both are unequivocally required not only for cellular proliferation but also for other essential cellular functions. Although to our knowledge specific oncogenic mutations have not been identified to date ERCC1 and RRM1 have emerged as promising predictors of efficacy for cisplatin and gemcitabine respectively. We conducted a phase 2 trial of treatment selection based on the levels of protein expression of ERCC1 and RRM1 for patients with completely resected stage I NSCLC and tumor diameters ??2 cm primarily to establish feasibility but also to evaluate preliminary efficacy as assessed by 2-year survival rates. We achieved our primary goal by demonstrating within a cooperative group environment that treatment assignment can be achieved for >?85% of patients within 84 days (12 weeks) the established timeframe for the initiation of adjuvant therapy from surgery in patients with NSCLC.12420“22 At first glance our demonstration of feasibility should not be surprising. However it is important to note that surgical practice has not usually engaged a medical"

Lung_Cancer

"Background Although dendritic cell (DC) vaccines are considered to be promising treatments for advanced cancer their production and administration is costly and labor-intensive. 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. 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]."

Lung_Cancer

"Methods: Twenty-six rabbits with lung VX2 tumor were randomly divided into experimental and control group. In the experimental group microwave ablation guided by ultrasound or CT was performed based on location of the tumor. Enhanced CT scan was carried out immediately before and after the ablation for all animals. Two animals from each group were sacrificed immediately or 1 week after the ablation respectively and the others were followed for the rest of their lives. Results: CT scan revealed that the tumor was greatly reduced or ablated after ablation. Pathological examination immediately after ablation also confirmed the tumor reduction or ablation. The survival time of the animals in the experimental group was significantly longer than that in the control group. Conclusions: Microwave ablation is a safe and effective method for treating lung cancer in rabbits showing potential clinical applicability. Microwave ablation VX2 tumor lung cancer 9421547 4136 Hum Pathol Hum. Pathol. Human pathology 0046-8177 1532-8392 24444464 3965626 10.1016/j.humpath.2013.10.016 NIHMS537247 A PIK3CA mutation detected in plasma from a patient with synchronous primary breast and lung cancers Jelovac Danijela MD 1 * Beaver Julia A. MD 1 * Balukrishna Sasidharan MD 2 Wong Hong Yuen BS 1 Toro Patricia Valda BS 1 Cimino-Mathews Ashley MD 1 Argani Pedram MD 1 Stearns Vered MD 1 Jacobs Lisa MD 1 VanDenBerg Dustin BS 1 Kessler Jill BS 1 Jeter Stacie BS 1 Park Ben H. MD PhD 1 Wolff Antonio C. MD 1 1The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins 1650 Orleans Street Baltimore MD 21287 2Christian Medical College Vellore Tamil Nadu India 632004 * These authors contributed equally to this work 18 12 2013 31 10 2013 4 2014 01 4 2015 45 4 880 883 2013 Elsevier Inc. All rights reserved. 2013 Digital PCR is a new technology that enables detection and quantification of cancer DNA molecules from peripheral blood. Using this technique we identified mutant PIK3CA DNA in circulating plasma tumor DNA (ptDNA) from a patient with concurrent early stage breast cancer and non-small cell lung cancer. The patient underwent successful resection of both her breast and lung cancers and using standard Sanger sequencing the breast cancer was shown to harbor the identical PIK3CA mutation identified in peripheral blood. This case report highlights potential applications and concerns that can arise with the use of ptDNA in clinical oncology practice. plasma tumor DNA breast cancer lung cancer PIK3CA digital PCR Br J Cancer Br. J. Cancer British Journal of Cancer 0007-0920 1532-1827 Nature Publishing Group 24983368 4102953 bjc2014353 10.1038/bjc.2014.353 Genetics and Genomics Assessing standardization of molecular testing for non-small-cell lung cancer: results of a worldwide external quality assessment (EQA) scheme for EGFR mutation testing Worldwide external quality assessment for EGFR gene mutation testing Patton S 1 * Normanno N 2 Blackhall F 3 Murray S 4 Kerr K M 5 Dietel M 6 Filipits M 7 Benlloch S 8 Popat S 9 Stahel R 10 Thunnissen E 11 1EMQN Manchester Centre for Genomic Medicine St Mary's Hospital Manchester M13 9WL UK 2Cell Biology and Biotherapy Unit Istituto Nazionale per lo Studio e la Cura dei Tumori ˜Fondazione Giovanni Pascale'”IRCCS 80131 Naples Italy 3Christie Hospital Manchester M20 4BX UK 4Biomarker Solutions Ltd London EC1V 2NX UK 5Department of Pathology Aberdeen Royal Infirmary Aberdeen AB25 2ZN UK 6Charit Humboldt-Universitt zu Berlin Berlin 10117 Germany 7Medical University of Vienna 1010 Vienna Austria 8Pangaea Biotech USP Dexeus University Institute Barcelona 08028 Spain 9Royal Marsden Hospital London SW3 6JJ UK 10University Hospital Z¼rich CH-8091 Z¼rich Switzerland 11Department of Pathology VU University Medical Center Amsterdam 1081 HZ The Netherlands *E-mail: simon.pattoncmft.nhs.uk 15 07 2014 01 07 2014 15 7 2014 111 2 413 420 22 01 2014 19 05 2014 20 05 2014 Copyright 2014 Cancer Research UK 2014 Cancer Research UK 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: The external quality assurance (EQA) process aims at establishing laboratory performance levels. Leading European groups in the fields of EQA Pathology and Medical and Thoracic Oncology collaborated in a pilot EQA scheme for somatic epidermal growth factor receptor (EGFR) gene mutational analysis in non-small-cell lung cancer (NSCLC). Methods: EQA samples generated from cell lines mimicking clinical samples were provided to participating laboratories each with a mock clinical case. Participating laboratories performed the analysis using their usual method(s). Anonymous results were assessed and made available to all participants. Two subsequent EQA rounds followed the pilot scheme. Results: One hundred and seventeen labs from 30 countries registered and 91 returned results. Sanger sequencing and a commercial kit were the main methodologies used. The standard of genotyping was suboptimal with a significant number of genotyping errors made. Only 72 out of 91 (72%) participants passed the EQA. False-negative and -positive results were the main sources of error. The quality of reports submitted was acceptable; most were clear concise and easy to read. However some participants reported the genotyping result in the absence of any interpretation and many obscured the interpretation required for clinical care. Conclusions: Even in clinical laboratories the technical performance of genotyping in EGFR mutation testing for NSCLC can be improved evident from a high level of diagnostic errors. Robust EQA can contribute to global optimisation of EGFR testing for NSCLC patients. non-small-cell lung carcinoma EGFR gene mutations quality assessment Assessment of epidermal growth factor receptor (EGFR) mutations has become mandatory to choose the most active first-line treatment for patients with advanced non-small-cell lung cancer (NSCLC). Indeed randomized phase III clinical trials have demonstrated that first-line administration of an EGFR TKI results in a prolonged progression-free survival as compared with chemotherapy in patients carrying EGFR mutations (Mok et al 2009; Maemondo et al 2010; Mitsudomi et al 2010; Fukuoka et al 2011; Zhou et al 2011; Rosell et al 2012). These studies have also confirmed that EGFR mutations are a reliable marker that predicts sensitivity to EGFR TKIs (Mok et al 2009). Activating mutations occur in exons 18 through 21 of the TK domain of the EGFR gene and either point mutations or in-frame small deletions or insertions (Sharma et al 2007; De Luca and Normanno 2010). Although more than 250 mutations of the EGFR gene have been described to date two mutations a single point mutation in exon 21 the L858R and a series of small in-frame deletions in exon 19 account for ?90% of all EGFR mutations (Sharma et al 2007; Linardou et al 2008). EGFR mutations are strongly associated with defined clinical and pathological features: they are far more frequent in female patients as compared with male; in adenocarcinoma as compared with other histological types; in non-smokers as compared with current smokers or former smokers; and in East-Asian NSCLC patients as compared with Non-East-Asian patients (Normanno et al 2006). External quality assessment (EQA) is a system of objectively checking laboratory results by an independent external agency (van Krieken et al 2013). The main objective of an EQA programme is to establish inter-laboratory comparability. In this respect the EQA process can identify latent systematic errors in methodology that may not be revealed by a laboratory's own internal QA processes. Representatives from ETOP ESMO ESP EMQN and other leading European groups met in July 2010 to discuss a pan-European approach to EQA for EGFR mutation testing in NSCLC. In this paper we present the results of this pilot EQA scheme for EGFR testing that was completed in 2013. Materials and Methods anisation of the scheme A meeting was anised in July 2010 by ETOP and EMQN to bring together a group of professionals representing EMQN ESP ETOP ESMO and other leading European groups involved in NSCLC testing (see Supplementary Information). From this group a steering group of five individuals was formed who planned designed and assessed the results of the pilot EQA scheme. The scheme was coordinated and administered by the EMQN and three rounds were anised within a period of 18 months. The workflow of the scheme process is shown in Figure 1. 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 (Table 1). 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 (Table 2). 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 (Table 2; 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 (Table 3). 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 (Table 2; 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%) (Figure 2). 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. As this was designed to ascertain proof of principle we did not apply a measure of successful laboratory performance. The pilot established that the scheme design and methods used were acceptable for use in a larger scheme. Second round One hundred and seventeen laboratories from 30 countries registered and 101 participated in the second round (due to customs issues we were not able to get samples to 16 labs) run in the second quarter of 2012. Ninety-one laboratories submitted results within the 8-week time frame “ the remaining 10 labs gave no reason why they did not submit results. A different set of samples from those used in the pilot first round were sent to the laboratories with the emphasis being on the inclusion of mutations at allelic frequencies that would challenge the analytical sensitivity of all the commonly used technologies (Table 2; samples B1“B10). A code number different from the one assigned in the first round was given to the samples. In addition to the genotype results all participating laboratories were also required to submit for assessment copies of their clinical reports for three samples (B1 B4 and B9). The main methodology used by the participants was PCR/sequencing (n=35 laboratories; 39%) and real-time PCR (n=17; 18.6% Figure 2). It was common for labs to use a combination of different methodologies in their testing process (Table 4). A variety of different errors were detected by the second scheme round including 74 (8.1%) genotype errors (false-positive (n=13; 1.5%) false-negative (n=61; 82.4%) and a combination of false-negative and -positive results (n=1; 1.4%)) as well as analytical test failures (n=31; 3.4%) mispositioning of the genotype (n=7; 0.8%) and significant errors in the mutation nomenclature (n=36; 3.9%). Two samples (B2 and B8) gave a disproportionately high error rate compared with the other samples used in this round (Table 5) with 94.1% of errors for B2 made by labs using PCR/sequencing vs 40.7% of errors for sample B8 made by labs using a version of the Therascreen EGFR kit (Qiagen). Laboratories did not lose marks if the declared limitations of their assay meant that they would not detect a particular mutation at the given frequency used in the EQA materials. Eighteen laboratories (19.8%) from 13 countries with a total score below 18 did not pass the second round and were thus classified as poor performers “ 72.2% of these labs used PCR/Sequencing as their main diagnostic test for EGFR mutation status. "

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

"As one of the DNA repair genes ataxia-telangiectasia mutated (ATM) gene which is responsible for the multisystem autoxomal recessive disorder ataxia-telangiectasia (A“T) plays a crucial role in the recognition signaling and repair of DNA damage especially DNA double-strand breaks (DSBs) [4] [5]. The ATM protein is a member of phosphoinositide 3-kinase (PI-3 kinases) and can be activated by DSBs caused by ionizing radiation or reactive oxygen intermediates [6] [7]. Once activated ATM can phosphorylate various downstream substates that function in cell cycle arrest apoptosis and DNA repair such as p53 NBS1 BRCA1 and Chk2 [8] [9]. Therefore genetic variants in ATM gene may lead to the structure and function change of the protein and act as important factors indicating individual susceptibility to cancer. ATM -111G>A (rs189037) resides in the promoter of ATM gene. Increasing studies have shown that variations in the DNA promoter sequence may potentially alter the affinities of multiple regulatory proteins-DNA interactions or the specificity of the transcriptional process [10]“[13]. Although this polymorphism makes no amino acid change the alleles may have different binding affinity to the transcription factor and exhibit different levels of mRNA expression [14] [15]. Zhang et al. [16]declared that ATM rs189037 AA genotype was associated with a lower ATM mRNA levels than GG genotype in lung tissue samples. Their results showed that the G-to-A change might create a transcriptional inhibitor-binding site for ATM rs189037 A allele promoter and subsequently reduce the ATM mRNA expression. Consequently lower expression of ATM might cause elevated sensitivity to ionizing radiation defects in the activation of cell cycle checkpoints a reduced capacity for DNA repair and abnormal apoptosis. All of these features would contribute to increased individual cancer susceptibility. In recent years a number of studies have evaluated the association between this polymorphism and cancer risk such as thyroid carcinoma [17] oral cancer [18] breast cancer [19] leukemia [20] nasopharyngeal carcinoma [21] glioma [22] and lung caner [23]“[25]. Previous studies of ATM rs189037 have included cigarette smokers as cases and controls that made it difficult to judge whether this polymorphism were associated with lung cancer or tobacco use. Considering the facts in China the incidence and death rate of lung cancer in women continues to increase and this phenomenon is frequently occurring in those who have never smoked. In order to have a better control of confounding of gender or smoking we performed a case-control study to identify the association between the polymorphism of ATM rs189037 and the risk of lung cancer in the non-smoking females in Chinese Han population. We also investigated the interaction between genetic polymorphism and environmental exposure in lung cancer. Methods Subjects This hospital-based case-control study included 487 lung cancer patients and 516 cancer-free hospital controls. All subjects were female non-smokers and they were from unrelated ethic Han Chinese. The cases were recruited during January 2002 to November 2012 at Liaoning Cancer Hospital & Institute. All patients were histologically confirmed to have lung cancer before any radiotherapy and chemotherapy. During the same time controls were selected from patients with other lung diseases but free of cancer history and symptom. Controls suffered mainly from bronchitis pneumonias fibrosis sarcoidosis chronic obstructive pulmonary disease and emphysema. Controls were all non-smoking females and frequency-matched to case subjects for age (±5 years). This study was approved by the institutional review board of China Medical University and written informed consent was obtained from each participant or each participant's representatives if direct consent could not be obtained. Data Collection A total of 10 ml of venous blood was collected from each patient. Patients were interviewed to collect information for demographics and environmental exposure at the time they were admitted to hospital. Information concerning demographic characteristics passive smoking cooking oil fume exposure fuel smoke exposure family history of cancer occupational exposure and dietary habit was obtained for each case and control by trained interviewers. An individual was defined as a smoker if she had consumed a total of 100 cigarettes in her lifetime; otherwise she was considered as a non-smoker. About fuel smoke exposure participants who used coal-fuel-burning stoves without chimneys were regarded as fuel smoke exposure. For exposure to cooking oil fumes participants were mainly asked about the method of cooking and eyes or throat irritation. For cooking methods participants were asked whether they cooked food in a stir-frying way and how many times a week; for eyes or throat irritation participants were asked how often they felt eyes or throat irritated by the oily smoke. There were four possible responses ranging from œnever œseldom œsometimes and œfrequently. Subjects were considered as cooking oil fume exposure if they met criteria as follows: (1) have cooked for over 15 years; (2) cooked food in a stir-frying way for more than twice a week; (3) felt eyes or throat irritated by oily smoke. Exposure for cooking oil fume was categorized as an indicator variable equal to 1 if participants reported frequently or sometimes and equal to 0 otherwise. Genotype Analysis Genomic DNA was extracted from peripheral blood samples by the conventional phenol-chloroform extraction method. SNP was genotyped by investigators blinded to case-control status in order to avoid any genotyping bias using TaqMan methodology and read with the Sequence Detection Software on an Applied Biosystems 7500 FAST Real-Time PCR System according to the manufacturer's instructions (Applied Biosystems Foster City CA). Amplification was done under the following conditions: 95°C for 10 min followed by 47 cycles of 92°C for 30 s and 60°C for 1 min. In this study 487 lung cancer patients and 516 controls were all genotyped successfully and 5% duplicated samples were randomly selected to assess the reproducibility for quality control with a concordance rate of 100%. Statistical Analysis The x2 test and t test were applied to estimate differences in demographic variables and distributions of genotypes between cases and controls. The association of genotypes of ATM rs189037 with risk of lung cancer was estimated by computing the odds ratios (ORs) and 95% confidence intervals (CIs) in unconditional logistic regression analysis. The Hardy-Weinberg equilibrium (HWE) was tested using goodness-fit x2 test to compare the genotype frequencies in the control subjects from those expected. A logistic regression model was used to evaluate gene-environment interactions. All data were analyzed with Statistical Product and Service Solutions (SPSS) v13.0 for Windows if not otherwise specified. All statistical analysis were two-sided and the significance level was set at P<0.05. Results Population characteristics A total of 487 lung cancer and 516 age-matched cancer-free controls were enrolled in this study. As shown in the mean ages of cases and controls (mean ±S.D.) were almost identical (56.5±11.7 and 56.3±12.5 respectively). All cases were female non-smoking lung cancer patients. No statistically significant difference was found between cases and controls in terms of age (P?=?0.248) and monthly income (P?=?0.084). Cases included 434 non-small cell lung cancer (NSCLC) patients and 53 small cell carcinoma patients. In the NSCLC cases there were 320 adenocarcinomas 73 squamous cell carcinomas and 41 other tumors with a variety of different pathologies (such as large cell carcinomas mixed cell carcinomas or undifferentiated carcinomas). .0096911.t001 Characteristics of lung cancer cases and controls. Variables Cases(%) Controls(%) P value Female 487 516 Mean age (years) 56.5±11.7 56.3±12.5 0.248a Income (yuan/month) 628.9±419.3 563.5±387.6 0.084a Never smoker 487 516 Histological type NSCLC 434(89.1) Adenocarcinoma 320(65.7) Squamous cell carcinoma 73(15.0) Small cell carcinoma 53(10.9) Other 41(8.4) a Student's t-test was used to compare the frequency distributions of demographic variables between the cases and controls. Association analysis The observed genotype frequencies among the control subjects was in agreement with that expected under the Hardy-Weinberg equilibrium (P?=?0.119). The distribution of ATM rs189037 genotypes among subjects were displayed in Table 2. Using subjects with the ATM rs189037 GG genotype as the reference group we calculated the ORs and 95%CIs for heterozygous carriers of GA genotype and homozygous carriers of AA genotype. No significant difference was observed between lung cancer cases and controls in each test (P>0.05). In order to increase the statistical power we combined the GA genotype with the AA genotype to compare with GG genotype as a dominant model and combined the GA genotype with the GG genotype to compare with AA genotype as a recessive model. The results indicated that individuals with AA genotype had a significantly elevated risk of lung adenocarcinoma compared with those carrying the GG or GA genotype (OR?=?1.44 95%CI 1.02“2.02 P?=?0.039). .0096911.t002 Table 2 Distribution of ATM rs189037 genotypes and ORs for lung cancer cases and controls. Genotype Cases(%) Controls(%) ORc 95%CI P overall (n?=?487) GG 148(30.4) 152(29.5) ref GA 240(49.3) 272(52.7) 0.91 0.68“1.20 0.494 AA 99(20.3) 92(17.8) 1.11 0.77“1.59 0.590 dominant modela 0.96 0.73“1.25 0.742 recessive modelb 1.18 0.86“1.61 0.313 NSCLC (n?=?434) GG 129(29.7) 152(29.5) ref GA 213(49.1) 272(52.7) 0.92 0.68“1.24 0.573 AA 92(21.2) 92(17.8) 1.18 0.81“1.71 0.397 dominant model 0.98 0.74“1.30 0.906 recessive model 1.24 0.90“1.71 0.192 Adenocarcinoma (n?=?320) GG 94(29.4) 152(29.5) ref GA 150(46.9) 272(52.7) 0.89 0.64“1.23 0.485 AA 76(23.7) 92(17.8) 1.33 0.90“1.99 0.156 dominant model 1.00 0.74“1.36 0.987 recessive model 1.44 1.02“2.02 0.039* Squamous cell carcinoma (n?=?73) GG 24(32.9) 152(29.5) ref GA 39(53.4) 272(52.7) 0.90 0.52“1.56 0.706 AA 10(13.7) 92(17.8) 0.69 0.32“1.51 0.355 dominant model 0.85 0.50“1.43 0.537 recessive model 0.74 0.37“1.50 0.400 *P<0.05. a GA+AA vs GG. b AA vs GA+GG. c adjusted for age and data were calculated by unconditional logistic regression. According to the results above we assumed that ATM rs189037 AA genotype might affect lung adenocarcinoma risk among non-smoking Chinese females. To test this hypothesis and explore the gene-environment interaction we adopted all the lung adenocarcinoma patients and cancer-free controls whose information about environmental risk factors were completely obtained such as fuel smoke exposure cooking oil fume exposure passive smoking and family history of cancer. Cases and controls were not included in the association analysis if any item of their environmental risk factors data was incomplete. After screening we had 242 lung adenocarcinoma cases and 277 cancer-free controls that were eligible. Selected demographic variables and environmental risk factors for the cases and controls were listed in Table 3."

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

"However it remains to be elucidated whether NME2-mediated alteration of nucleosomal reanization possesses any unique features of the histone modification language (involving specific enzyme complexes and histone chaperones) that could be essential in mitigating metastasis. SUPPLEMENTARY DATASupplementary Data are available at NAR Online. SUPPLEMENTARY DATA Research Fellowship from CSIR (V.Y. A.B. and A.K.) UGC (R.K.T.) and ICMR (R.H. and A.S.) is acknowledged. The ˜CSIR“Mayo Clinic Collaboration for Innovation and Translational Research™ is acknowledged for helping to initiate the collaboration with Eric Wieben's group. Lung lysates were kindly provided by I. Boldogh from UTMB Galveston TX 77555 USA. We thank Vani Brahmachari (ACBR India) for helping us with mononucleosome preparation and other members of the Chowdhury lab especially Parashar Dhapola for stimulating discussions and suggestions. We also thank Munia Ganguli (CSIR-IGIB) for careful reading and editing of the manuscript.   The authors wish it to be known that in their opinion the first two authors should be regarded as Joint First Authors. FUNDING Council of Scientific and Industrial Research Project MLP1203 and Wellcome Trust/Department of Biotechnology India Alliance (DBT) [500127/Z/09/Z to S.C.]. Funding for open access charge: Wellcome Trust/DBT India Alliance grant no. [500127/Z/09/Z to S.C.]. Senior Research Fellowship from Wellcome Trust/ Department of Biotechnology India Alliance [500127/Z/09/Z to S.C.]; National Institutes of Health [CA129537 and CA154320 to T.K.P.]. Conflict of interest statement. None declared. REFERENCES 1. Hay E.D. The mesenchymal cell its role in the embryo and the remarkable signaling mechanisms that create it Dev. Dyn. 2005 233 706 720 15937929 2. Koerber R.T. Rhee H.S. Jiang C. Pugh B.F. Interaction of transcriptional regulators with specific nucleosomes across the Saccharomyces genome Mol.Cell 2009 35 889 902 19782036 3. Fidler I.J. The pathogenesis of cancer metastasis: the ˜seed and soil™ hypothesis revisited Nat. Rev. Cancer 2003 3 453 458 12778135 4. Hanahan D. Weinberg R.A. Hallmarks of cancer: the next generation Cell 2011 144 646 674 21376230 5. Sethi N. Kang Y. Unravelling the complexity of metastasis”molecular understanding and targeted therapies Nat. Rev. Cancer 2011 11 735 748 21941285 6. Jiang C. Pugh B.F. Nucleosome positioning and gene regulation: advances through genomics Nat. Rev. Genet. 2009 10 161 172 19204718 7. Barski A. Cuddapah S. Cui K. Roh T.Y. Schones D.E. Wang Z. Wei G. Chepelev I. Zhao K. High-resolution profiling of histone methylations in the human genome Cell 2007 129 823 837 17512414 8. Liu C.L. Kaplan T. Kim M. Buratowski S. Schreiber S.L. Friedman N. Rando O.J. Single-nucleosome mapping of histone modifications in S. cerevisiae PLoS Biol. 2005 3 e328 16122352 9. Zhang L. Ma H. Pugh B.F. Stable and dynamic nucleosome states during a meiotic developmental process Genome Res. 2011 21 875 884 21515815 10. Robinson P.J. An W. Routh A. Martino F. Chapman L. Roeder R.G. Rhodes D. 30 nm chromatin fibre decompaction requires both H4-K16 acetylation and linker histone eviction J. Mol. Biol. 2008 381 816 825 18653199 11. Routh A. Sandin S. Rhodes D. Nucleosome repeat length and linker histone stoichiometry determine chromatin fiber structure Proc. Natl. Acad. Sci. U.S.A 2008 105 8872 8877 18583476 12. Segal E. Widom J. What controls nucleosome positions Trends Genet. 2009 25 335 343 19596482 13. Bargaje R. Alam M.P. Patowary A. Sarkar M. Ali T. Gupta S. Garg M. Singh M. Purkanti R. Scaria V. Proximity of H2A.Z containing nucleosome to the transcription start site influences gene expression levels in the mammalian liver and brain Nucleic Acids Res. 2012 40 8965 8978 22821566 14. Hogan G.J. Lee C.K. Lieb J.D. Cell cycle-specified fluctuation of nucleosome occupancy at gene promoters"

Lung_Cancer

"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). 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. Figure 1 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. To assess DFS the disease status was monitored every 2 months for the first 6 months and subsequently every 3 months by computed tomography after enrollment and according to good medical practice. Toxicities related to the administration of chemotherapy were assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events (version 3.0; ctep.cancer.gov). DFS was defined as the time from the date of enrollment to disease recurrence or death due to any cause and estimated according to the Kaplan-Meier method. A Cox regression model was fit with the time from surgery to enrollment as a covariate to evaluate its effect on DFS. A natural log transformation was applied to the raw protein measurement data and the Pearson correlation coefficient was used to test associations. Bivariate comparison of baseline characteristics between the assigned treatment groups was performed using the Fisher exact test for categorical variables or the Student t test or Wilcoxon rank sum test for continuous variables. A multivariable logistic model to evaluate baseline factors and treatment assignment was fit using backwards selection. Median ERCC1 and RRM1 expression levels were compared with historical medians using the 1-sample Wilcoxon signed rank test. The percentage of patients with both ERCC1 ??65 and RRM1 ??40 was compared with the historical rate using a chi-square test. All statistical analyses and graphics were performed using SAS statistical software (version 9.2; SAS Institute Inc Cary NC). A significance level of 5% was used for all analyses. RESULTS Patient and Trial Characteristics To ensure an adequate sample size of eligible patients and biomarker-specific subgroups a total of 85 patients was registered between April 2 2009 and April 1 2011 from 27 participating sites. Four patients were ineligible; 3 had inadequate lymph node sampling and 1 did not have a tumor measuring ??2 cm. Table 1 provides the characteristics of the 81 eligible patients. Table 1 Patient Demographics and Disease Characteristics Variablesa All Patients Assigned to Chemotherapy Assigned to Observation P Refused Assignment Accepted Assignment P N = 81 N = 63 N = 18 N = 20 N = 61 Age y .37 .39 ?Median 64 63.3 68.8 67.2 63.3 ?Mean 63.5 62.9 65.5 65.2 62.9 ?Range 41.6“84.2 41.6“84.2 41.6“81.7 44.2“82.9 41.6“84.2 Sex .18 .61 ?Female 44 (54%) 37 (59%) 7 (39%) 12 (60%) 32 (52%) ?Male 37 (46%) 26 (41%) 11 (61%) 8 (40%) 29 (48%) Ethnicity .65 .18 ?Unknown 7 (8%) 5 (8%) 2 (11%) 0 (0%) 7 (11%) ?Non-Hispanic 74 (91%) 58 (92%) 16 (89%) 20 (100%) 54 (89%) Race .73b .75b ?African American 8 (10%) 8 (13%) 0 (0%) 2 (10%) 6 (10%) ?Asian 3 (4%) 2 (3%) 1 (6%) 0 (0%) 3 (5%) ?Pacific Islander 2 (2%) 1 (2%) 1 (6%) 0 (0%) 2 (3%) ?White 66 (81%) 52 (83%) 14 (78%) 17 (85%) 49 (80%) ?Unspecified 2 (2%) 0 (0%) 2 (11%) 1 (5%) 1 (2%) Histology .06c .60c ?Adeno 52 (64%) 44 (70%) 8 (44%) 14 (70%) 38 (62%) ?Squamous 25 (31%) 17 (27%) 8 (44%) 6 (30%) 19 (31%) ?Large 1 (1%) 1 (2%) 0 (0%) 0 (0%) 1 (2%) ?Bronchioloalveolar 1 (1%) 0 (0%) 1 (6%) 0 (0%) 1 (2%) ?Other 2 (2%) 1 (2%) 1 (6%) 0 (0%) 2 (3%) Stage of disease .16 .27 ?IA (<3 cm) 25 (31%) 22 (35%) 3 (17%) 4 (20%) 21 (34%) ?IB (?3 cm) 56 (69%) 41 (65%) 15 (83%) 16 (80%) 40 (66%) Zubrod performance status .11 1.00 ?0 44 (54%) 31 (49%) 13 (72%) 11 (55%) 33 (54%) ?1 37 (46%) 32 (51%) 5 (28%) 9 (45%) 28 (46%) Weight loss (6 mo) 1.00d .31d ?<5% 64 (79%) 49 (78%) 15 (83%) 14 (70%) 50 (82%) ?5-<10% 9 (11%) 7 (11%) 2 (11%) 3 (15%) 6 (10%) ?10“20% 4 (5%) 3 (5%) 1 (6%) 2 (10%) 2 (3%) ?>20% 1 (1%) 1 (2%) 0 (0%) 0 (0%) 1 (2%) ?Unknown 3 (4%) 3 (5%) 0 (0%) 1 (5%) 2 (3%) Smoking status ?Current 33 (41%) 26 (41%) 7 (39%) 8 (40%) 25 (41%) ?Former (quit ?1 y) 39 (48%) 30 (48%) 9 (50%) 10 (50%) 29 (48%) ?Never 9 (11%) 7 (11%) 2 (11%) 1.00e 2 (10%) 7 (11%) 1.00e Abbreviation: Adeno adenocarcinoma. a All P values shown are 2-sided. b White versus all other races. c Adenocarcinoma versus all other histologies. d Weight loss <5% versus ?5%. e Derived using the Freeman-Halton exact test. The distribution of assignment to chemotherapy and observation was 63 patients (78%) and 18 patients (22%) respectively which was not significantly different (P?=?.20 Fisher exact test) from the expected rates of 70% (129 patients) and 30% (55 patients) respectively.16 Based on protein levels in these 81 patients the number of those with low ERCC1 and low RRM1 was 31 patients (38%) 22 patients had low ERCC1 and high RRM1 (27%) 10 patients had high ERCC1 and low RRM1 (12%) and 18 patients had high ERCC1 and RRM1 (22%) which is not significantly different from prior results (P?=?.14 Fisher exact test; 54 of 184 29%; 38 of 184 21%; 37 of 184 20%; and 55 of 1840.3 respectively). We investigated whether treatment arm assignment varied by patients' smoking status histology age and sex. In bivariate comparisons no statistically significant associations were found. However the multivariable logistic model found that patients with adenocarcinoma (P?=?.03) and potentially stage IA disease (P?=?.06) were more likely to be assigned to adjuvant chemotherapy (ie they were more likely to have low levels of ERCC1 RRM1 or both). One of the 18 patients assigned to observation and 19 of the 63 patients assigned to chemotherapy rejected this choice and withdrew consent. There was no statistically significant difference in patient characteristics between those who accepted and those who refused their treatment assignment (Table 1). Feasibility The trial achieved its primary feasibility objective with a treatment assignment within the prespecified timeframe in 71 of 81 patients (88%). We successfully determined protein levels in all 85 patients. Ten of the 81 eligible patients did not achieve assignment to treatment versus observation within the 84-day time interval from surgical resection. The time interval from surgery to assignment ranged from 86 days to 105 days in these 10 patients. For 3 patients the specimens were received after the 84-day limit had passed. For the other 7 patients the time interval from receipt to reporting ranged from 7 days to 25 days (median 18 days). For the 71 patients with a successful assignment within the 84-day time interval from surgical resection the time from receipt to reporting ranged from 3 days to 26 days (median 8 days). The reasons for reporting results in excess of 14 days were equipment failure and inadequate expression values in control specimens which required equipment recalibration and a repeat processing of the specimens. Overall the time from receipt of specimens to reporting ranged from 1 day to 27 days (median 11 days; mean 12 days) which is similar to that reported for patients with advanced NSCLC (range 1 day-47 days; median 11 days; mean 12 days).18 Survival and Toxicity Survival analyses were performed on the 61 patients who accepted assignment to treatment (44 patients) or surveillance (17 patients). Patients who rejected their treatment assignment withdrew consent and thus could not be followed for survival. Fourteen patients had DFS events; 2 had died (1 from disease recurrence and the other from cardiac disease without recurrence). The median follow-up among those patients still alive at the time of last follow-up was 27 months (range 3 months-44 months). Six patients had <?24 months of follow-up. The collective 2-year DFS and OS rates were 80% (95% confidence interval [95% CI] 67%-88%) (Fig. 2A) and 96% (95% CI 87%-99%) from the date of registration. The 2-year DFS rate was 83% (95% CI 68%-92%) for patients who received chemotherapy (Fig. 2B) and it was 71% (95% CI 43%-87%) for those observed (Fig. 2C). Table 2 includes 2-year DFS estimates within each of the 3 gene expression categories in the chemotherapy arm. The median time from surgery to enrollment was 41 days (range 11 days-79 days). The time from surgery was added as a covariate to a Cox regression model and was not found to be significantly related to DFS (P?=?.22) or OS (P?=?.36). Table 2 Disease-Free Survival Rates Patient Group No. DFS (95% CI) 1-Year 2-Year Accepted assigned treatment 61 88% (77%-94%) 80% (67%-88%) Received chemotherapy 44 95% (83%-99%) 83% (68%-92%) By protein level category (for those that received chemotherapy) ?Low ERCC1/low RRM1 20 95% (69%-99%) 84% (59%-95%) ?Low ERCC1/high RRM1 18 94% (65%-99%) 82% (55%-94%) ?High ERCC1/low RRM1 6 100% (100%-100%) 100% (100%-100%) Abbreviations: 95% CI 95% confidence interval; DFS disease-free survival; ERCC1 excision repair cross-complementing group 1; RRM1 ribonucleotide reductase M1. Figure 2 Kaplan-Meier survival estimates are shown. (A) Collective disease-free survival is shown for patients who accepted adjuvant chemotherapy or observation based on gene expression analysis. (B) Disease-free survival is shown for patients who received adjuvant chemotherapy. (C) Disease-free survival is shown for patients in the observation group. Conf Int indicates confidence interval. A total of 22 patients discontinued chemotherapy because of treatment-related toxicity (50%). None of the patients died because of treatment-related toxicity. Details are provided in Table 3. Table 3 Number of Patients With Grade 3 and Grade 4 Adverse Events Among the 44 Patients Who Received Chemotherapya Level of Severity Adverse Event Grade 3 Grade 4 No. of patients with events 13 14 Type of events ?Neutropenia 11 6 ?Thrombocytopenia 4 4 ?Nausea 4 0 ?Vomiting 4 0 ?Anemia 2 0 ?Anorexia 2 0 ?Fatigue 2 0 ?Febrile neutropenia 1 1 ?Thromboembolism 1 1 ?Dehydration 1 0 ?Hearing impairment 1 0 ?Mucositis 1 0 ?Pleural effusion 1 0 ?Renal failure 1 0 ?Bradycardia (sinus) 1 0 ?Syncope 1 0 ?ALT elevation 1 0 ?Hypokalemia 1 0 ?Hyponatremia 0 2 Abbreviation: ALT alanine aminotransferase. a Adverse events were assessed according to the Common Terminology Criteria for Adverse Events (version 3.0). In Situ ERCC1 and RRM1 Protein Levels RRM1 levels ranged from 2.4 to 234.3 (median 39.7; mean 48.1) which were not significantly different from the expected values (median 40.5; range 8.3-96.2) (P?=?.87).16 ERCC1 protein levels ranged from 4.3 to 211.2 (median 41.9; mean 58.8) and these values were significantly different from the expected values (median 65.9; range 1.9-178.7) (P?=? 0.02). There was a significant correlation noted between ERCC1 and RRM1 levels (correlation coefficient 0.39; P?=?.0003) (Fig. 3) as previously reported.91618 Figure 3 Distribution of excision repair cross-complementing group 1 (ERCC1) and ribonucleotide reductase M1 (RRM1) levels in eligible patients is shown. The median protein levels of ERCC1 in adenocarcinomas squamous cell carcinomas and the other histologies were 34.257.1 and 121.5 respectively. The corresponding median levels of RRM1 were 38.142.6 and 48.9 respectively. Although the levels were higher in squamous cell carcinomas compared with adenocarcinomas the medians were not statistically significant (ERCC1: P?=?.16; RRM1: P?=?.72). DISCUSSION Disease stage is a predictor of benefit from adjuvant chemotherapy in patients with NSCLC. Patients with stage III disease derive the most benefit and those with stage I are reported to derive the least.12419“23 Although not statistically significant for patients with stage I disease and a tumor diameter >?3 cm a numerical risk reduction of 7% has been reported and for those with tumors measuring ??3 cm a numerical risk increase of 40% has been reported.23 A significant treatment-related toxicity is febrile neutropenia which has been reported in 7% to 24% of patients.242022 Treatment-related deaths occur in 0.5% to 2% of patients.122022 The inclusion of molecular markers predictive of therapeutic efficacy into adjuvant decision algorithms would greatly improve the clinical benefit and reduce toxicity for patients with NSCLC. This approach is particularly attractive for patients with stage I disease in whom the parameters for weighing risks and benefits are to our knowledge the least well defined. Recent advances in molecular diagnostics have resulted in improved outcomes for patients whose tumors harbor mutations in oncogenic signal transduction molecules that can be inactivated by therapeutic agents. Similarly platinum agents target DNA and gemcitabine targets ribonucleotide reductase; both are unequivocally required not only for cellular proliferation but also for other essential cellular functions. Although to our knowledge specific oncogenic mutations have not been identified to date ERCC1 and RRM1 have emerged as promising predictors of efficacy for cisplatin and gemcitabine respectively. We conducted a phase 2 trial of treatment selection based on the levels of protein expression of ERCC1 and RRM1 for patients with completely resected stage I NSCLC and tumor diameters ??2 cm primarily to establish feasibility but also to evaluate preliminary efficacy as assessed by 2-year survival rates. We achieved our primary goal by demonstrating within a cooperative group environment that treatment assignment can be achieved for >?85% of patients within 84 days (12 weeks) the established timeframe for the initiation of adjuvant therapy from surgery in patients with NSCLC.12420“22 At first glance our demonstration of feasibility should not be surprising. However it is important to note that surgical practice has not usually engaged a medical"

Lung_Cancer

"These suggest that the three aspects are genetically connected but the cause and effect relationships are still unknown. For example physiologic TF binding studies involve many TFs consequently it is difficult to assign nucleosome reanization to the binding site occupancy of any particular TF. Therefore several aspects remain unclear: does TF binding influence nucleosome (re)anizations locally or impact the chromatin landscape at a more global level; are all or only a fraction of TF binding a result of reanization in nucleosome occupancy and do all TF binding and associated changes in nucleosome occupancy result in altered gene expression? With these in mind following characterization of two states (before and after induction of a single TF of choice) we determined: (i) genomic binding sites of the TF (ii) promoter nucleosome occupancy and (iii) transcriptome profiles. Results demonstrated that promoter-proximal TF binding influenced expression of the target gene when it was coupled to nucleosome repositioning at or close to its binding site in most cases. In contrast only in few cases change in target gene expression was found when TF binding occurred without local nucleosome reanization. cover-date 2 September 2014 INTRODUCTION Although transcription factors (TFs) can recognize cognate binding sites on the nucleosomal surface functional binding is almost always associated with chromatin modification remodeling and finally displacement or compaction of nucleosomes. Specific epigenetic signals determine the site and state of this reanization based on the bound TF leading to cellular differentiation. Modification of the cellular phenotype from a group or ˜community™-like (epithelial) state to a more solitary (mesenchymal) form (epithelial to mesenchymal transition EMT) is a basic developmental feature that has been well studied (reviewed in (12)). Metastasis or spreading of cancer from the site of origin is also manifested with enhanced mesenchymal features in tumor cells (34). In this context though gene expression networks for the EMT are well studied regulation of the nucleosomal or chromatin state is poorly understood (5). Though there are several minor variations the overall chromatin architecture of a given transcription unit may be summarized as: a ˜?1™ nucleosome positioned upstream of the transcription start site (TSS) a nucleosome-free region (NFR) followed by a ˜+1™ nucleosome downstream of TSS in addition to an array of positioned nucleosomes throughout the gene body (6). This basic anization is stabilized by modified histones or histone variants placed at specific regions within genes (78). anizational change in chromatin such as altered positioning of the +1 and/or ?1 nucleosome with resultant change in the NFR (9) altered inter-nucleosomal spacing due to chromatin compaction (10“12) and/or histone variant occupancy near TSS (13) may result in transcriptional response (14“16). Together these can modulate accessibility of genomic DNA in vivo toward binding of TFs (6). This model is supported by high-resolution nucleosome maps generated for human promoters by subjecting chromatin to micrococcal nuclease (MNase) and detecting the undigested DNA with tiling arrays (17) or deep sequencing (MNase-Seq) (15). By employing the yeast system nucleosome dynamics during transcriptional changes has been investigated in detail. It was found that nucleosome reanization as a result of physiological perturbations by means of heat-shock induced large-scale transcriptome changes in yeast. However at the nucleosomal level most changes were limited to one or two nucleosomes per promoter (16). Furthermore genome-wide analysis of nucleosome dynamics during meiotic development in yeast revealed dramatic reanization of chromatin (9). A combined analysis of nucleosome architecture transcriptional states and status of DNA binding factors from publicly available data sets in yeast suggested DNA binding factors may control promoter nucleosome architecture (18). Other studies found that nucleosome repositioning events may facilitate TF binding and gene expression upon androgen treatment (19) during differentiation of hematopoietic stem cells to erythrocytes (20) or interferon-beta activation following virus infection (21). Together these suggest a process where nucleosome remodelers general TFs and the transcriptional elongation machinery together orchestrate the nucleosome-positioning pattern in vivo (22“24). This understanding prompts further interesting questions regarding both the nature of TF/nucleosome interactions and to what extent this influences transcriptional response. For example in earlier studies induced physiological perturbations activated multiple TFs (19“21) it is not clear how unique site-specific TF binding influences nucleosome (re)anizations locally (in close vicinity of their binding sites). Another pertinent question is: do all sites occupied by a TF and associated nucleosome occupancy changes result in altered gene expression? Keeping these in mind we sought to study binding of single TF that would also induce the physiological change. A candidate TF was first identified from analysis of early and advanced lung cancer transcriptome profiles. In vivo binding sites of the TF nucleosome positions and transcriptome profiles of both the metastatic and induced non-metastatic state in lung cancer cells were then determined (see Scheme S1 in Supplementary Information for a summary of the overall design) and using these the correlation between target site occupancy nucleosome reanization and their combined effect on the transcriptome was examined. Results that suggest a model where TF binding coupled with nucleosome reanizations that influence transcription are: (i) almost always associated with nucleosome repositioning that is at close proximity to the TF binding site (TFBS) and (ii) constrained to specific loci and not spread over the whole genome. MATERIALS AND METHODS Chromatin immunoprecipitation and sequencing Chromatin immunoprecipitation (ChIP) assays were performed following the Fast ChIP protocol (25). Briefly two plates were made with 1 — 105 A549 cells in each on achieving 80% confluency the first plate was transfected with the clone pcDNA3-NME2-Myc to induce non-metastatic 2 (NME2). The second plate was left untreated (un-induced condition). After 48 h ChIP was performed using cells from each plate independently and cells were fixed with 1% formaldehyde for 10 min lysed and sheared (?300 bp) using a Misonix 3000 sonicator. Twenty-five per cent of lysate was used to isolate input chromatin using phenol“chloroform and ethanol precipitation. The remaining lysate was pre-cleared using protein-A sepharose beads and further divided into two equal portions: one part was immunoprecipitated using antibody specific to the Myc epitope (clone 9E10 anti-c-Myc monoclonal Sigma) and a negative control of immunoprecipitation was prepared by adding isotypic control (IgG) to the second portion. Immunoprecipitation was done by incubating overnight at 4°C with 2 ?g of antibody. The un-induced experiment was treated in a similar fashion before immunoprecipitation with nm23-h2-L-16 antibody (sc-17587 Santa Cruz Biotechnology Inc.); IgG was used as the respective isotypic control. Immune complexes were collected using herring sperm DNA-saturated protein-A Sepharose washed and eluted using Chelex-100 after proteinase K treatment. Reads were aligned (mapped) to the unmasked human reference genome (NCBI v36 hg18) using the MAQ (Mapping and Assembly with Qualities) (26) after clipping to 24 bp based on quality scores. The sequences were then mapped to the reference human genome (NCBI Build 36 UCSC hg18). Only those that aligned to the reference genome were further considered for peak generation. To accommodate variations relative to the reference genome up to two mismatches were allowed. Commercially available small interfering RNAs (siRNAs) from Dharmacon Inc. USA were used to silence expression of NME2 (transient depletion) in A549 wherever required. Peak generation following ChIP-seq The resulting sequence read distribution was processed with ChIP-Seq peak locator utility CisGenome (26). CisGenome uses a conditional binomial model to identify regions in which the ChIP reads are significantly enriched relative to the control reads. A false discovery rate of 10% was considered while predicting NME2 target regions. In order to filter out low-quality sites we applied two post-processing options boundary refinement and single-strand filtering. Mononucleosome preparation and hybridization to promoter tiling array A549 cells (NME2-induced or -depleted along with respective control cells) were grown in T-75 flasks till 80% confluency in Dulbecco's modified Eagle's medium trypsinized pelleted and washed with ice-cold 1X phosphate buffered saline. Five times the packed pellet volume of 1X hypotonic buffer was added cells homogenized IGEPAL (a non-ionic non-denaturing detergent) was added to final concentration of 0.6% lysate vortexed and centrifuged at 12 000 revolutions per minute. Pellet (nuclei) was resuspended in MNase digestion buffer and incubated on ice. OD (Optical Density) was measured and 1 unit of MNase/OD was added to the suspension. Following incubation at 37ºC for 30 min the reaction was stopped using stop-buffer treated with proteinase K (1 mg/ml) overnight at 37ºC before phenol“chloroform purification and ethanol precipitation to pellet down the mononucleosomal DNA. Precipitated DNA was washed with 70% alcohol and dissolved in water. DNase-treated cells were taken as control. Mixture containing 3 ?g of DNA 50 mMTris-Cl 5 mM MgCl2 and 0.3 ?g/?l of random hexamers was incubated at 95ºC for 5 min and chilled to 4ºC. 5 ?l dNTP (1.2 mM dGTP dCTP dATP and 0.25 mM dUTP) and 50 units of Klenow were incubated first at 22ºC for 10 min then 37ºC for 30 min and finally 95ºC for 5 min and chilled as above. Fifty units of Klenow were added again and the mix first kept at 22ºC for 10 min and then 37ºC for 30 min. Reaction was stopped by phenol“chloroform followed by purification using Qiagen columns. Purified product was fragmented and labeled as per Affymetrix Chromatin Immunoprecipitation Assay protocol before hybridization to Gene Chip Human Promoter Tiling array 1.0R (Affymetrix) as per manufacturer's protocol. The control DNase-treated cells were also subjected to labeling reactions as above before hybridization. Nucleosome positions were identified using iChIP (Bioconductor package). Nucleosome occupancy analysis A549 cells depleted for NME2 were generated using commercially available short hairpin RNAs (from Origene Inc. USA; catalog no. TR311160) and stable cell clones were selected in presence of puromycin. For analysis of nucleosome repositioning we considered a distance of 300 bp (+/? 150 bases) in other words a nucleosome was denoted as repositioned in the NME2-induced condition when detected beyond 300 bp of a nucleosome found in the cells before NME2 induction. In order to avoid arbitrary assignments in cases where a gene belonged to more than one category (while assigning NME2 nucleosome associations during repositioning analysis (Figure 5)) it was considered in all the respective cases. Figure 1. NME2 binding sites before or after inducing NME2. (A) A Circos plot showing ChIP-seq peak distribution of NME2 on all chromosomes (1“22 and X and Y); peaks in red represent NME2 binding sites before induction; peaks in blue represent NME2 binding sites after induction (replicates are shown in both cases). Two projections from chromosomes 9 and 15 are shown. (B) Twelve-mer consensus NME2 binding motif identified using Gibbs sampler (upper panel)”distribution of the 12-mer motif within NME2 peaks constructed from read counts is shown in the lower panel. Figure 2. Nucleosome positions detected in A549 cells before and after inducing NME2. (A) UCSC browser representation of nucleosome positions with corresponding probe intensity at two loci on chromosome 12. (B) Nucleosome occupancy around TSS and expression level of corresponding genes in cells before (left panels) and after NME2 induction (right panels); gene expression was normalized within respective cases by z-transformation with respect to the mean expression level of the data sets. (C) Frequency of occurrence and location of nucleosomes around TSS. ?1123 +4 denote sequential presence of nucleosomes with respect to their occurrence from TSS; percentage of total number of nucleosomes found in respective cases before or after induction of NME2. (D) Left panel: distribution of repositioned nucleosomes around TSSs in NME2-induced cells relative to the status in cells before NME2 was induced; number of repositioned nucleosomes in a window size of 100 bp is shown on top. Right panel: expression of corresponding genes shown in triplicate before and after NME2 induction. Figure 3. Nucleosome depletion and NME2 occupancy. (A) Schematic representation of possible relationship between nucleosome positions before and after inducing NME2. (B) Nucleosome occupancy is depleted on or near NME2 binding sites on inducing NME2 relative to the condition before NME2 induction. Ratio of number of nucleosomes detected after/before NME2 induction in 300 bp windows is shown; x-axis denotes the distance of nucleosomes from the nearest NME2 binding site in NME2-induced cells. (C) Schematic representation of nucleosome shift between two conditions was represented by ?Ndisplacement (left panel). Percentage of shifted nucleosomes plotted for a given ?Ndisplacement is shown in the right panel (x-axis was plotted to indicate: no shift shift in 100 bp windows and shift exceeding 300 bases). Distribution of the nucleosome shift was also found for the NME2-depleted condition minus A549 cells; significance of the difference in distributions was tested using the Wilcoxon rank sum test (P = 0.00016). (D) Position of the nearest nucleosome with respect to NME2 binding sites in cells before (green) or after inducing NME2 (red; left panels); 791 genes where the nearest nucleosome was within 300 bp and shifted in the NME2-induced condition are shown. Expression level of corresponding genes in triplicate before or after NME2 induction is shown in the right panels. Figure 4. Validation by quantitative real-time PCR (Polymerase Chain Reaction) for NME2 binding and nucleosome occupancy. Nucleosome positions and NME2 binding sites are shown before and after NME2 induction at six different loci in the left panel (genes shown in UCSC browser representation); right panel shows validation at the corresponding loci by quantitative real-time PCR for NME2 binding and nucleosome occupancy in cells before and after inducing NME2. Relative fold change is shown on x-axis. Experiments were performed in triplicate; error bars are for standard deviation (* and ** represent P < 0.05 and P < 0.01 respectively). Figure 5. TF binding and nucleosome repositioning models analyzed in this study. Nucleosome shift in the vicinity of TF binding results in repositioning of the ?1 nucleosome (upper panel) whereas co-occupancy (center panel) or target site binding that has no associated nucleosome repositioning in close vicinity either before or after induction of the TF (lower panel) shows no significant change in ?1 or +1 nucleosome positions. Percentage of nucleosomes is based on genome-wide total for respective cases; to avoid arbitrary assignment genes were considered in more than one category where applicable. Quantitative real-time PCR for validating ChIP-nucleosome positioning For validating individual nucleosome positions quantitative real-time PCRs were performed using ChIPped DNA on ABI's (Applied Biosystems Inc.) 7500 fast platform. Equal concentration of ChIP DNA was taken from the test (from NME2-induced cells) and contro˜l (un-induced) samples. The results were analyzed using comparative computed tomography method. Primers were designed against the obtained peak region using ABI's Primer Express software (Supplementary Table S1). Western blotting Western blot for NME2 was done using antibody from Abcam Cambridge MA USA (catalog no. ab60602). "

Lung_Cancer

"are associated with erythrocyte sedimentation rate Am J Hum Genet 2011 89 131 138 10.1016/j.ajhg.2011.05.019 21700265 Teeranaipong P Ohashi J Patarapotikul J Kimura R Nuchnoi P Hananantachai H Naka I Putaporntip C Jongwutiwes S Tokunaga K A functional single-nucleotide polymorphism in the CR1 promoter region contributes to protection against cerebral malaria J Infect Dis 2008 198 1880 1891 10.1086/593338 18954261 Chen GB Xu Y Xu HM Li MD Zhu J Lou XY Practical and theoretical considerations in study design for detecting gene-gene interactions using MDR and GMDR approaches PLoS One 2011 6 e16981 .0016981 21386969 Lou XY Chen GB Yan L Ma JZ Zhu J Elston RC Li MD A generalized combinatorial approach for detecting gene-by-gene and gene-by-environment interactions with application to nicotine dependence Am J Hum Genet 2007 80 1125 1137 10.1086/518312 17503330 PLoS One one 1932-6203 Public Library of Science San Francisco USA 24416392 3887046 PONE-D-13-33822 .0085329 Research Mathematics Statistics Biostatistics Statistical Methods Medicine Clinical Research Design Meta-Analyses Oncology Basic Cancer Research Metastasis Cancers and Neoplasms Lung and Intrathoracic Tumors Pulmonology Surgery Thoracic Surgery Evaluation of Video-Assisted Thoracoscopic Surgery for Pulmonary Metastases: A Meta-Analysis VATS for Pulmonary Metastases Dong Siyuan Zhang Lin * Li Wenya Du Jiang Liu Xiangli Chen Xitao Department of Thoracic Surgery First Hospital of China Medical University Shenyang Liaoning Province People's Republic of China Arnold Paul Editor University of Kansas United States of America * E-mail: zhanglincmu163.com Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: SYD LZ. Performed the experiments: SYD LZ WYL JD XLL XTC. Analyzed the data: SYD LZ WYL JD XLL XTC. Contributed reagents/materials/analysis tools: SYD LZ WYL JD XLL XTC. Wrote the paper: SYD LZ. 2014 9 1 2014 9 1 e85329 16 8 2013 25 11 2013 2014 Dong 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 To evaluate the evidence comparing video-assisted thoracic surgery (VATS) and open thoracotomy in the treatment of metastatic lung cancer using meta-analytical techniques. Methods A literature search was undertaken until July 2013 to identify the comparative studies evaluating disease-free survival rates and survival rates. The pooled odds ratios (OR) and the 95% confidence intervals (95% CI) were calculated with the fixed or random effect models. Results Six retrospective studies were included in our meta-analysis. These studies included a total of 546 patients: 235 patients were treated with VATS and 311 patients were treated with open thoracotomy. The VATS and the thoracotomy did not demonstrate a significant difference in the 1-3-5-year survival rates and the 1-year disease-free survival rate. There were significant statistical differences between the 3-year disease free survival rate (p?=?0.04) which favored open thoracotomy. Conclusions The VATS approach is a safe and feasible treatment in terms of the survival rate for metastatic lung cancer compared with the thoracotomy. The 3-year disease-free survival rate in the VATS group is inferior to that of open thoracotomy. The VATS approach could not completely replace open thoracotomy. The authors have no support or funding to report. Introduction Metastasectomy is considered a beneficial treatment for a patient with metastatic lung cancer whose primary tumor has been well controlled[1].After surgery 5-year survival rates of 30% to 50% could be achieved depending on the underlying primary cancer[2]“[4].In practice the surgical approaches to pulmonary metastases are variable. Video-assisted thoracoscopic surgery (VATS) is an emerging technique; many procedures that had previously required a thoracotomy have been performed with the minimally invasive VATS. VATS has been used for the treatment of pulmonary metastases. The routine use of VATS for the treatment of respectable metastatic lung cancer remains controversial. Critics of the VATS approach have argued that it might not be an equivalent oncological operation[5] [6]. A prospective study by Cerfolio[7]found that 22% of the nodules that could be detected by thoracotomy were missing by VATS.Whether the VATS approach can provide a satisfactory outcome is unknown. An evidenced-based investigation of the VATS approach is needed we undertook this meta-analysis to achieve a more objective assessment of the published studies and to provide a more accurate comparison between VATS and thoracotomy for metastatic lung cancer. Methods Search Strategy Electronic searches were of the MEDLINECochrane Controlled Trial Register (CENTRAL) Ovid MEDILINE PubMed and Embase databases were performed until July 2013.The following MeSH search headings were used: œmetastatic lung cancer œpulmonary metastases œvideo-assisted thoracic surgery œthoracotomy and œcomparative study.We searched the reference lists of relevant studies reviews editorials lettersand meeting s. We used the Science Citation Index to cross-reference for further studies that met our criteria. Study Selection The studies included in this meta-analysis were based on our predetermined criteria as follows: (1) clinical trials that include the full text of the paper published in peer-reviewed English journals or reports of presentations at major thoracic surgery meetings; (2) comparison of the efficacy of VATS to that of thoracotomy in patients with metastatic lung cancer; and (3) similarity in the patients' baseline characteristics. Data extraction and quality assessment Two independent reviewers (Siyuan and Wenya) assessed the quality and the risk of bias of the included trials as follows: (1) the studies that did not include a comparative group with surgery as a form of intervention were excluded; (2) the trials focusing on patients undergoing surgery for primary lung cancer were excluded; (3) the studies on robotic video-assisted thoracic surgery were excluded; (4) if there was an overlap between authors centers or patient cohorts evaluated in the published literature only the most recent report was included; (5) studies published more than 20 years ago were excluded because of the significant technological changes that has occurred. The s were evaluated with the Downs and Black quality assessment method[8]. Discrepancies between the two investigators were resolved by discussion and consensus with a senior investigator. The final results were reviewed by two senior investigators (Lin and Jiang).The disease-free survival was defined as the date of the initial metastasectomy until the date of a recurrence. Statistical and sensitivity analyses The meta-analysis was performed using the RevMan 5.1.0. software package. The odds ratio (OR) or the mean difference with 95% confidence intervals (95% CI) was calculated for the dichotomous outcomes and the continuous outcomes respectively. A P value<0.05 was considered a significant difference in the value between the two groups. We used the I2 statistic to investigate the heterogeneity among the studies.The heterogeneity was explored by X2 and I2; I2<25% and I2>50% reflect a small and large inconsistency respectively. P<0.05 was considered significant. If there were a statistical difference in terms of the heterogeneity (P?0.05) a random-effect model was selected to pool the data. Otherwise a fixed-effect model was used. Taking into account the presence of different sample sizes of the included studies a sensitivity analysis was performed to compare the of 1-year survival rate and the 3-year disease free survival rate between VATS and open thoracotomy. Publication bias A funnel plot was used to explore bias. Asymmetry in the funnel plot of trial size against treatment effect was used to assess the risk of bias. Results Description of the studies Six retrospective cohort studies the met our criteria were included in this meta-analysis. A total of 546 patients were included in the six studies;235 patients were allocated to the VATS group whereas 311 were allocated to the open thoracotomy group to evaluate their survival rate.The search algorithm results of the search strategies and selection criteria are shown in Fig 1. The patient characteristics and evaluation index are shown in Table 1. .0085329.g001 Figure 1 Identification of studies for inclusion. .0085329.t001 Table 1 Study Design Country NO(V/O) Gender (M/F) Mean age (years) Assessment score Nakajima2001[28] OC Japan 45/55 V59/41 O34/21 V55±15 O55±14 13 Mutsaerts2002[29] OC Netherlands 8/12 NR NR 19 Nakas2009[30] OC UK 25/27 V16/9 O 19/8 V69 O66 16 Carballo2009[31] OC USA 36/135 V18/18 O82/53 V58.5 O49 15 Gossot2009[32] OC France 31/29 V21/10 O13/16 V43 O40 18 Chao2012[33] OC Taiwan 90/53 V49/41 O35/18 NR 13 V VATS; O Open thoracotomy; NR Not reported; OC observational cohort. Assessment of Recurrence and Survival Six studies documented the 1-year survival rateand there was no significant heterogeneity among the six studies (x2?=?3.79 P?=?0.58I2?=?0%).A fixed effect model was used.The combined result is shown in Fig 2(OR?=?1.15; 95%CI 0.72“1.84; p?=?0.58). Because of the heterogeneity in sample size the sensitivity analyses were conducted using larger sample sizes. There was no difference between the two surgical methods with an OR of 1.00(95%CI 0.55“1.79) and with heterogeneity(?2?=?3.23P?=?0.07 I2?=?69%). Five studies reported the 3-year survival rate and heterogeneity was identified through the five studies (x2?=?11.32P?=?0.02I2?=?65%); and a random effect model was adopted (OR?=?1.07; 95%CI 0.50“2.27; p?=?0.86) (Fig 3). Three studies compared the 5-year survival rate (OR?=?0.96; 95%CI 0.34“2.71; p?=?0.93) with certain heterogeneity(x2?=?8.86P?=?0.01I2?=?77%) (Fig 4). .0085329.g002 Figure 2 1-year survival rate. Forest plot of the Odds Ratio(OR) of the 1-year survival rate following VATS versus open thoracotomy for metastatic lung cancer.The estimate of the OR of each individual trial corresponds to the middle of the squares and horizontal line gives the 95% CI.On each linethe numbers of events as a fraction of the total number randomized are shown for both treatment groups.For each subgroupthe sum of the statistics along with the summary OR is represented by the middle of the solid diamonds.A test of heterogeneity between the trials within a subgroup is given below the summary statistics. .0085329.g003 Figure 3 3-year survival rate. Forest plot of the Odds Ratio(OR) of the 3-year survival rate following VATS versus open thoracotomy for metastatic lung cancer.The estimate of the OR of each individual trial corresponds to the middle of the squares and horizontal line gives the 95% CI.On each linethe numbers of events as a fraction of the total number randomized are shown for both treatment groups.For each subgroupthe sum of the statistics along with the summary OR is represented by the middle of the solid diamonds.A test of heterogeneity between the trials within a subgroup is given below the summary statistics. .0085329.g004 Figure 4 5-year survival rate. Forest plot of the Odds Ratio(OR) of the 5-year survival rate following VATS versus open thoracotomy for metastatic lung cancer."

Lung_Cancer