Citations

Abstract Lung cancer is the leading cancer diagnosis worldwide and the number one cause of cancer deaths. Exposure to cigarette smoke, the primary risk factor in lung cancer, reduces epithelial barrier function and increases susceptibility to infections. Herein, we hypothesized that somatic mutations together with cigarette smoke create a dysbiotic microbiota that is associated with lung carcinogenesis. Using lung tissue from controls (n=33) or cancer cases (n=143), we conducted 16S rRNA gene sequencing (MiSeq), with RNA-seq data from lung cancer cases in The Cancer Genome Atlas (n=1112) serving as the validation cohort. We demonstrate a lower alpha diversity in normal lung as compared to non-tumor adjacent or tumor tissue, indicating a shift in the overall microbial community in lung cancer patients as compared to those without cancer. Lung cancer cases were classified by the relative abundance of two taxa, Variovorax and Streptococcus, with an increase in Variovorax abundance in tumors as compared to non-tumor adjacent paired lung tissue (FDR corrected P=0.013). The species of Variovorax was identified histologically, and also by two additional 16S rRNA strategies (Resphera Insight analysis and PacBio sequencing). A group of taxa were associated with squamous cell carcinoma (SCC), of which Acidovorax were enriched in smokers (P =0.0013). Further, these taxa, including Acidovorax, exhibited higher abundance among the subset of SCC cases with TP53 mutations, an association not seen in adenocarcinomas (AD). Therefore, we observed a microbiome-gene and a microbiome-exposure interaction in SCC lung cancer tissue. Together, these results open the door for future biomarker research that could be used to improve screening and direct mechanistic studies of lung cancer therapy. Citation Format: Leigh Greathouse, James White, Valery Bliskovsky, Ashley Vargas, Eric Polley, Elise Bowman, Mohammed Khan, Ana Robles, Brid Ryan, Amiran Dzutsev, Giorgio Trinchieri, Marbin Pineda, Sven Bilke, Paul Meltzer, Marina Walther-Antonio, Garth Ehrlich, Joshua Mell, Joshua Earl, Sergey Balashov, Archana Bhat, Alex Valm, Clayton Deming, Sean Conlan, Julia Oh, Julie Segre, Curtis Harris. Microbiome-TP53 gene interaction in human lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4925. doi:10.1158/1538-7445.AM2017-4925

Abstract Purpose: Lung cancer is the leading cause of cancer deaths worldwide. Most present symptomatically at late stage with high lethality. Early detection reduces mortality but accurate and readily accessible tools for population screening are limited. By whole methylome sequencing, we have identified novel methylated DNA markers (MDMs) for lung cancer in tissue (Giakoumopoulos et al. ASCO 2016). Using top candidate MDMs in the present study, we now explore their clinical accuracy for lung cancer detection when assayed from plasma. Experimental Procedures: Archival plasmas from two independent study groups were tested in blinded fashion. Lung cancer cases and controls (apparently healthy smokers) for each group were balanced on age and sex (Group 1: 64 cases, 231 controls; Group 2: 23 cases, 80 controls). Using multiplex PCR followed by QuARTS (Quantitative Allele-Specific Real-time Target and Signal amplification) assay, a post-bisulfite quantification of MDMs on DNA extracted from plasma was performed. We selected 31 MDM candidates for initial evaluation in Group 1 (1 ml plasma/patient); top individual MDMs were subsequently tested in Group 2 to identify optimal MDM panels for lung cancer detection (2 ml/patient). Results: From Group 1 analyses, 13 high performance MDMs were selected for further testing (CYP26C1, SOBP, SUCLG2, SHOX2, ZDHHC1, NFIX, FLJ45983, HOXA9, B3GALT6, ZNF781, SP9, BARX1, EMX1) with individual areas under the receiver operator curve (AUCs) ranging from 0.593 to 0.939. Discrimination by individual MDMs was corroborated in Group 2 in which data was analyzed using two methods: a logistic regression fit and a regression partition tree approach. The logistic fit model identified a 4-marker panel (ZNF781, BARX1, EMX1, and SOBP) with an AUC of 0.96 and an overall sensitivity of 91% and 90% specificity. Analysis of the data using a regression partition tree approach identified 4 markers (ZNF781, BARX1, EMX1, and HOXA9) with AUC of 0.93 and an overall sensitivity of 96% and specificity of 94%. For both approaches, B3GALT6 was used as a standardizing marker of total DNA input. Conclusion: A panel of MDMs assayed in plasma achieved high sensitivity and specificity for all types of lung cancer. Further clinical evaluation and validation of this promising panel in larger patient groups are clearly indicated. Citation Format: Hatim T. Allawi, Maria Giakoumopoulos, Evan Flietner, Austin Oliphant, Carla Volkmann, Brian Aizenstein, Tamara Sander, Drew Eckmayer, Ashley Poenitzsch Strong, Melissa Gray, Barry Berger, Tracy Yab, William Taylor, Douglas Mahoney, John B. Kisiel, David E. Midthun, David A. Ahlquist, Graham P. Lidgard. Detection of lung cancer by assay of novel methylated DNA markers in plasma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 712. doi:10.1158/1538-7445.AM2017-712

Abstract Mutant peptides presented in cancer are superior vaccine candidates than self peptides. The efficacy of mutant K-Ras, P53 and EFGR peptides have been tested as cancer vaccines in pancreatic cancer, colorectal cancer, and lung adenocarcinoma. However, the occurrence of these mutations in cancer is limited. Further more, these mutant peptides can only be presented by certain MHC alleles. Neoantigens expressed by passenger mutations which are not involved in common proliferative molecular pathways are potential candidates for personalized vaccine design. Such passenger mutations have drawn attention by the recent clinical data that colorectal cancer with a large number of somatic mutations due to mismatch-repair defects are more susceptible to immune checkpoint blockade by PD1 antibody therapy. In this study, we analyzed the next generation sequencing data of 147 lung adenocarcinoma patients from The Cancer Genome Atlas and predicted neoantigens presented by MHC Class I and Class II molecules. We found 18175 expressed clonal somatic mutations, with an average of 124 per patient. Based on such somatic mutations, we predicted 8731 neo-peptides, among which 3888 strong-binders and 4843 weak-binders to MHC Class I molecules were found by NetMHCPan. For HLA-A*02:01, 778 strong binding and 822 weak binding neo-peptides were found. In average, 11 neo-antigen peptides of mutations were predicted per individual patient with HLA-A*02:01 allele. Smokers expressed 5.73 fold higher mutant neo-antigen peptides than non-smokers. The 20 most commonly mutated genes with predicted neo-antigens are TTN, RYR2, MUC16, KRAS, ZFHX4, TP53, USH2A, NAV3, KEAP1, ANK2, DNAH9, ADAMTS12, CSMD1, CSMD3, SPTA1, FAT3, ZNF804A, PAPPA2, PCLO, EGFR. As a positive control, the frequency of known proliferation-related mutations (KRAS, P53, and EGFR) were determined in 147 patients, which are consistent with previously next generation sequencing studies of lung adenocarcinoma. Mutation hotspots were found in proliferation-related mutations (KRAS), but not in passenger mutations. 6950 neo-peptides, including 259 strong binders and 6691 weak binders were found for HLA DRB1, an MHC Class II molecule. INDEL mutation type of neo-epitopes were found in 119 of 147 patients, with an average of 4.34 INDEL mutations presented by MHC Class I and 0.86 INDEL mutations presented by MHC Class II molecule HLA DRB1 in every patient. Our results support the feasibility of discovering individualized mutant peptides as candidates for immunodiagnosis and immunotherapy of lung adenocarcinoma. Neo-epitopes which can be presented by both MHC Class I and MHC Class II molecules are attractive targets for designing long-peptide vaccines. Note: This abstract was not presented at the meeting. Citation Format: Dapeng Zhou, Weijing Cai, Deng Pan, Tan Wen Ling, Jiaqian Wang, Caichun Zhou, Yanyan Lou. Neoantigens predicted by clonal mutation analysis in lung adenocarcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 628. doi:10.1158/1538-7445.AM2017-628

Abstract This study assessed the inter-user reproducibility of Computer-Aided Nodule Assessment and Risk Yield (CANARY), a novel analytical tool that risk stratifies lung adenocarcinomas (ADCs) according to defined computed tomography (CT) structural characteristics. CANARY detects nine distinct voxel signatures in ADCs based on standard CT imaging, thereby defining nodule characteristics correlating with patient outcomes, and corresponding to invasion or lepidic growth histologically. A software user segments the borders of each ADC prior to voxel analysis, introducing potential variability into the assessment. While CANARY is a promising method, it requires validation of the analytical variability between users and prediction of accuracy in an independent cohort. To evaluate the reproducibility of CANARY analysis, three independent users who are not part of the CANARY development team segmented and analyzed 50 biopsy-confirmed primary lung ADCs from Vanderbilt University Medical Center. The CT scans of ADCs were selected retrospectively based on the following criteria: ADC-histology proven on biopsy, diagnosed between 2009-2015, less than 3cm in greatest diameter, and stages IA-IV. Users followed a standard operating procedure established at the Mayo Clinic, and were blinded to clinical characteristics and patient outcomes. Results: To measure inter-user variability of ADC voxel composition, the intraclass correlation coefficient (ICC) was calculated based upon the percentage of each voxel subtype within the whole ADC. An ICC of 1 reflects high reproducibility between users. Amongst all 50 ADCs, the average ICC for all nine voxel types was 0.828 (95% CI 0.76, 0.895). The ICC of the four voxel types associated with invasive features on CT was 0.865 (95% CI 0.805, 0.924). ICCs were also calculated using a logarithmic transformation for data normalization, generating an ICC of 0.745 (95% CI 0.663, 0.826) for all nine voxel types, and an ICC of 0.995 (95% CI 0.993, 0.997) for the four voxel types associated with invasion. Conclusions: (1) CANARY analysis of lung ADC voxel signatures appears to be reproducible amongst users, making it a reliable tool for the evaluation of ADC voxel density. (2) Correlation of invasive signatures associated with more aggressive ADCs was nearly perfect amongst users. Additional validation metrics for CANARY with larger datasets are being evaluated, including the accuracy of tumor prognostic predictions between users and analysis of ADC datasets from other institutions. Citation Format: Erica C. Nakajima, Michael P. Frankland, Tucker Johnson, Sanja L. Antic, Ronald A. Karwoski, Bennett Landman, Heidi Chen, Ronald C. Walker, Brian J. Bartholmai, Tobias Peikert, Srinivasan Rajagopalan, Pierre P. Massion, Fabien Maldonado. Assessing the reproducibility of computer-aided nodule assessment and risk yield (CANARY) method to characterize lung adenocarcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3723. doi:10.1158/1538-7445.AM2017-3723

Abstract Pulmonary fibrosis is a significant complication in cancer patients treated with radiation or various chemotherapeutic agents. One such reagent, bleomycin, is used to treat a variety of cancers including, but not limited to, lymphoma, ovarian cancer, and melanoma. Its efficacy, however, is impacted by the incidence of significant pulmonary fibrosis, which often becomes dose limiting. In studies investigating the role(s) of metabolism in the profibrotic actions of transforming growth factor beta, we previously determined that increased fatty acid synthase (FASN) expression was observed in the fibrotic areas of lungs from idiopathic pulmonary fibrosis (IPF) patients compared to healthy controls. In that FASN levels are significantly increased in numerous human tumors including breast, prostate, colorectal, and ovarian, we wished to integrate these findings by examining whether inhibiting FASN activity could reduce pulmonary fibrosis. To that end, the cerulenin-derived FASN inhibitor C75 was tested in a murine treatment model of bleomycin-induced lung fibrosis. Following addition of C75, there was significant diminution in a number of profibrotic targets including collagen 1α1, fibronectin, connective tissue growth factor, and total lung collagen as assessed by hydroxyproline content. Not only was there a C75 dose-dependent decrease in the aforementioned targets, but lung function as assessed by peripheral blood oxygenation (on room air) was also stabilized/improved. Thus, FASN inhibition might reflect a possible target to maintain normal lung physiology in the context of cancer chemotherapy. This work was supported by Public Health Service grants GM-055816 and GM-054200 from the National Institutes of General Medical Sciences. Citation Format: Mi-Yeon Jung. Fatty acid synthase is a risk factor for bleomycin-induced lung fibrosis in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 346. doi:10.1158/1538-7445.AM2017-346

Abstract Introduction: DNA topoisomerase inhibitors (e.g. etoposide, irinotecan, teniposide, topotecan) are commonly used for treating small cell lung cancer (SCLC). Tyrosyl-DNA phosphodiesterase (TDP1) plays a role in repairing DNA damage caused by DNA topoisomerase inhibitors and it is believed to be responsible for resistance to this class of drugs. The purpose of this study was to determine whether common TDP1 polymorphisms are associated with overall survival among SCLC patients. Method: Two common TDP1 SNPs (rs942190 and rs2401863, r2(European)=0.63 and r2(East Asian)=0.26) were analyzed in 898 patients from 10 studies in the International Lung Cancer Consortium, conducted in the U.S., Canada, Japan, Italy, Spain and the United Kingdom. Genotype data were obtained from either the OncoArray Consortium or other existing genome-wide association studies. Cox proportional hazard regression models with robust standard error were used to evaluate genotype associations for overall survival at 36 months post-diagnosis, adjusting for age, sex, race and tumor stage. Results: Patients homozygous for the minor allele (GG) of TDP1 SNP rs942190 were associated with poorer survival compared to those carrying both major alleles (AA), with a hazard ratio (HR) of 1.36 (95% confidence interval (CI): 1.07-1.72, p-value=0.01), but there was no association with survival for patients with the heterozygous genotype (HR=1.02, 95% CI: 0.83-1.27, p-value=0.83). For rs2401863, patients carrying both minor alleles (CC) tended to have better survival than patients carrying AA alleles, but this was within the limits of chance given no significant association (HR=0.79, 95% CI: 0.61-1.02, p-value=0.07). In addition, multi-tissue expression quantitative trait loci (eQTL) analyses from the Genotype-Tissue Expression Project indicated a potential effect of rs942190 on lung tissue, with higher TDP1 gene expression for the GG than AG or AA genotypes. Conclusion: We found that among SCLC patients the TDP1 rs942190 GG genotype was associated with poor overall survival. Further study could determine if this genotype can serve as a predictive marker for the treatment efficacy of DNA topoisomerase inhibitors in SCLC. Citation Format: Pawadee Lohavanichbutr, Lori C. Sakoda, Christopher I. Amos, Susanne M. Arnold, David C. Christiani, Michael P. Davies, John K. Field, Eric B. Haura, Rayjean J. Hung, Takashi Kohno, Maria Teresa Landi, Geoffrey Liu, Yi Liu, Michael W. Marcus, Grainne M. O'Kane, Matthew B. Schabath, Kouya Shiraishi, Stacey A. Slone, Adonina Tardón, Ping Yang, Kazushi Yoshida, Ruyang Zhang, Xuchen Zong, Noel S. Weiss, Chu Chen. Common TDP1 polymorphisms in relation to survival among small cell lung cancer patients in a multicenter study from the International Lung Cancer Consortium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2248. doi:10.1158/1538-7445.AM2017-2248

Abstract The Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO) in collaboration with the Colorectal Cancer Family Registry (CCFR) aims to identify genetic variants and environmental risk factors that impact colorectal cancer (CRC). Over 30 studies from North America, Europe, and Australia participate in GECCO. These studies have collected clinical, epidemiological, and survival data, as well as blood and tumor biospecimens, for over 80,000 CRC cases and controls. The current study aims to conduct targeted deep sequencing of tumors and matching normal DNA to identify recurrent and novel somatic and germline variants in 4,200 CRC cases. To achieve this goal, an AmpliSeq targeted sequencing panel of 1.12 Mbp was constructed to sequence the coding regions of 190 significantly mutated genes identified from whole exome sequencing datasets generated by the Nurses’ Health Study and Health Professional’s Follow-up Study, and The Cancer Genome Atlas. The panel also covers coding regions of 15 genes with germline high penetrance mutations in CRC, 54 regions to detect CRC-related copy number alterations (CNAs), and microsatellite and homopolymer repeat regions to identify defective DNA mismatch repair. Primers were also included to detect Fusobacterium nucleatum DNA in tumor biopsies, as F. nucleatum is thought to promote CRC carcinogenesis. Sequencing of the DNA libraries on Illumina HiSeq 2500 produced a mean coverage of greater than 500X for tumor DNA and 100X for normal DNA, with >85% of the bases covered at the target at 50x. So far, targeted sequencing of >1,500 DNA samples from CRC tumors and normal tissues has identified recurrent and novel somatic mutations, germline genetic variants, and hypermutation status of the tumors due to defective DNA mismatch repair or pathogenic mutations in the POLE gene. Targeted sequencing has also allowed quantification of the F. nucleatum DNA in tumor biopsies; the results were validated by a multiplex QPCR assay. At the AACR annual meeting, we will present targeted sequencing results generated from the first two GECCO-participating studies (n=1,300 cases). These data will be valuable for future association testing of somatic mutations, CNAs, hypermutation status, and F. nucleatum with germline genetic variants, lifestyle, and environmental risk factors and survival. This large study will allow development of better strategies for diagnosis, treatment, and prevention of CRC. Citation Format: Syed H. Zaidi, Wei Sun, Jeroen Huyghe, Catherine S. Grasso, Quang Trinh, Charles Connolly, Amy French, Jasmine Mu, Marios Giannakis, Eve Shinbrot, Ivan Borozan, Michael J. Quist, Hermann Brenner, Daniel Buchanan, Peter Campbell, Andrew Chan, Jenny Chang-Claude, Vincent Ferretti, Charles Fuchs, Andrea Gsur, Marc Gunter, Tabitha Harrison, Michael Hoffmeister, Wen-Yi Huang, Paul Krzyzanowski, Stephen Lee, Mathieu Lemire, Jessica Miller, Danielle Pasternack, Cherie Teney, Elaine Mardis, Polly Newcomb, Lincoln Stein, Lee Timms, David Wheeler, Christina Yung, Niha Zubair, Levi Garraway, Shuji Ogino, Li Hsu, Steven Gallinger, Stephen Thibodeau, Thomas Hudson, Ulrike Peters. Targeted deep sequencing of colorectal tumor tissues to study associations of tumor subtypes with germline genetic, lifestyle, and environmental risk factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1286. doi:10.1158/1538-7445.AM2017-1286

AbstractBackgroundTranscatheter aortic valve replacement (TAVR) is typically performed under general anesthesia (GA). However, there is increasing data supporting the safety of performing TAVR under local anesthesia/conscious sedation (LA). We performed a meta‐analysis to gain better understanding of the safety and efficacy of LA versus GA in patients with severe aortic stenosis undergoing TAVR.Methods and ResultsWe comprehensively searched EMBASE, PubMed, and Web of Science. Effect sizes were summarized using risk ratios (RRs) difference of the mean (DM), and 95% CIs (confidence intervals) for dichotomous and continuous variables respectively. Twenty‐six studies and 10,572 patients were included in the meta‐analysis. The use of LA for TAVR was associated with lower overall 30‐day mortality (RR, 0.73; 95% CI, 0.57–0.93; P = 0.01), use of inotropic/vasopressor drugs (RR, 0.45; 95% CI, 0.28–0.72; P < 0.001), hospital length of stay (LOS) (DM, −2.09; 95% CI, −3.02 to −1.16; P < 0.001), intensive care unit LOS (DM, −0.18; 95% CI, −0.31 to −0.04; P = 0.01), procedure time (DM, −25.02; 95% CI, −32.70 to −17.35; P < 0.001); and fluoroscopy time (DM, −1.63; 95% CI, −3.02 to −0.24; P = 0.02). No differences were observed between LA and GA for stroke, cardiovascular mortality, myocardial infarction, permanent pacemaker implantation, acute kidney injury, paravalvular leak, vascular complications, major bleeding, procedural success, conduction abnormalities, and annular rupture.ConclusionOur meta‐analysis suggests that use of LA for TAVR is associated with a lower 30‐day mortality, shorter procedure time, fluoroscopy time, ICU LOS, hospital length of stay, and reduced need for inotropic support.

Tumor-initiating cells (TIC) are dynamic cancer cell subsets that display enhanced tumor functions and resilience to treatment but the mechanism of TIC induction or maintenance in lung cancer is not fully understood. In this study, we show the calcium pathway transcription factor NFATc2 is a novel regulator of lung TIC phenotypes, including tumorspheres, cell motility, tumorigenesis, as well as in vitro and in vivo responses to chemotherapy and targeted therapy. In human lung cancers, high NFATc2 expression predicted poor tumor differentiation, adverse recurrence-free and cancer-specific overall survivals. Mechanistic investigations identified NFATc2 response elements in the 3’ enhancer region of SOX2, and NFATc2/SOX2 coupling upregulates ALDH1A1 by binding to its 5’ enhancer. Through this axis, oxidative stress induced by cancer drug treatment is attenuated, leading to increased resistance in a mutation-independent manner. Targeting this axis provides a novel approach for the long-term treatment of lung cancer through TIC elimination.