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Background:In prospective cohorts, biological samples are generally stored over long periods before an adequate number of cases have accrued. We investigated the impact of sample storage at −80°C for 2 years on the stability of the V4 region of the 16S rRNA gene across seven different collection methods (i.e., no additive, 95% ethanol, RNAlater stabilization solution, fecal occult blood test cards, and fecal immunochemical test tubes for feces; OMNIgene ORAL tubes and Scope mouthwash for saliva) among 51 healthy volunteers.Methods:Intraclass correlation coefficients (ICC) were calculated for the relative abundance of the top three phyla, the 20 most abundant genera, three alpha-diversity metrics, and the first principal coordinates of three beta-diversity matrices.Results:The subject variability was much higher than the variability introduced by the sample collection type, and storage time. For fecal samples, microbial stability over 2 years was high across collection methods (range, ICCs = 0.70–0.99), except for the samples collected with no additive (range, ICCs = 0.23–0.83). For oral samples, most microbiome diversity measures were stable over time with ICCs above 0.74; however, ICCs for the samples collected with Scope mouthwash were lower for two alpha-diversity measures, Faith's phylogenetic diversity (0.23) and the observed number of operational taxonomic units (0.23).Conclusions:Fecal and oral samples in most used collection methods are stable for microbiome analyses after 2 years at −80°C, except for fecal samples with no additive.Impact:This study provides evidence that samples stored for an extended period from prospective studies are useful for microbiome analyses.

Purpose: Chordoma is a rare bone tumor with a high recurrence rate and limited treatment options. The aim of this study was to identify molecular subtypes of chordoma that may improve clinical management. Experimental Design: We conducted RNA sequencing in 48 tumors from patients with Chinese skull-base chordoma and identified two major molecular subtypes. We then replicated the classification using a NanoString panel in 48 patients with chordoma from North America. Results: Tumors in one subtype were more likely to have somatic mutations and reduced expression in chromatin remodeling genes, such as PBRM1 and SETD2, whereas the other subtype was characterized by the upregulation of genes in epithelial–mesenchymal transition and Sonic Hedgehog pathways. IHC staining of top differentially expressed genes between the two subtypes in 312 patients with Chinese chordoma with long-term follow-up data showed that the expression of some markers such as PTCH1 was significantly associated with survival outcomes. Conclusions: Our findings may improve the understanding of subtype-specific tumorigenesis of chordoma and inform clinical prognostication and targeted options.

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Figure S1. A. PBRM1 and SETD2 expression between CC1 and CC2; B. PBRM1 and SETD2 expression between NCC1 and NCC2.

Figure S2. A. Contributions of small indel (ID) signatures (A and E) between CC1 and CC2; B. Number of mutations in small indel (ID) signatures (A and E) between CC1 and CC2.

Figure S5. SNAI1, SNAI3, and TWIST expression in CC1 and CC2 tumors.

Supplementary Legend

Figure S3. Gene expression profiling based on the NanoString panel in 23 skull-base (top) and 25 non-skull-base (bottom) chordoma patients from North America.

Table S1. Genes included in the targeted DNA panel sequencing. Table S2. Differences in clinical characteristics by molecular subtype. Table S3. Distributions of mutation status in PBRM1 and SETD2 by molecular subtype in RNASeq and Nanostring datasets. Table S4. Differentially expressed genes among molecular subtypes. Table S5. GSEA results for up- and down-regulated gene sets comparing CC2 to CC1.

Purpose:Chordoma is a rare bone tumor with a high recurrence rate and limited treatment options. The aim of this study was to identify molecular subtypes of chordoma that may improve clinical management.Experimental Design:We conducted RNA sequencing in 48 tumors from patients with Chinese skull-base chordoma and identified two major molecular subtypes. We then replicated the classification using a NanoString panel in 48 patients with chordoma from North America.Results:Tumors in one subtype were more likely to have somatic mutations and reduced expression in chromatin remodeling genes, such as PBRM1 and SETD2, whereas the other subtype was characterized by the upregulation of genes in epithelial–mesenchymal transition and Sonic Hedgehog pathways. IHC staining of top differentially expressed genes between the two subtypes in 312 patients with Chinese chordoma with long-term follow-up data showed that the expression of some markers such as PTCH1 was significantly associated with survival outcomes.Conclusions:Our findings may improve the understanding of subtype-specific tumorigenesis of chordoma and inform clinical prognostication and targeted options.

Figure S4. Expression of selected markers in relation to overall survival (left) and recurrence-free survival (right) in Chinese chordoma patients with long-term follow-up data. A. CLDN11; B. GLI1; C. EPHA3; D. LEF1.

Supplementary Figure 2: Evaluation of accuracy. Intraclass correlation coefficients (all samples at time zero compared to sample with no additive) for microbiome metrics including the abundance of three phyla, two alpha-diversity metrics (number of observed operational taxonomic units and Shannon index) and four beta-diversity metrics (top PCoA component for unweighted Unifrac, generalized Unifrac, weighted UniFrac, and Bray-Curtis distance) in the Knight laboratory (A) and the Mayo Microbiome laboratory (B).

Odds ratio and 95% confidence intervals for the associations between presence of individual strain/species and Esophageal Squamous Dysplasia (ESD) status

Early-onset breast cancer (EOBC) causes substantial loss of life and productivity, creating a major burden among women worldwide. We analyzed 1,265,548 Hapmap3 single-nucleotide polymorphisms (SNP) among a discovery set of 3,523 EOBC incident cases and 2,702 population control women ages ≤ 51 years. The SNPs with smallest P values were examined in a replication set of 3,470 EOBC cases and 5,475 control women. We also tested EOBC association with 19,684 genes by annotating each gene with putative functional SNPs, and then combining their P values to obtain a gene-based P value. We examined the gene with smallest P value for replication in 1,145 breast cancer cases and 1,142 control women. The combined discovery and replication sets identified 72 new SNPs associated with EOBC (P < 4 × 10−8) located in six genomic regions previously reported to contain SNPs associated largely with later-onset breast cancer (LOBC). SNP rs2229882 and 10 other SNPs on chromosome 5q11.2 remained associated (P < 6 × 10−4) after adjustment for the strongest published SNPs in the region. Thirty-two of the 82 currently known LOBC SNPs were associated with EOBC (P < 0.05). Low power is likely responsible for the remaining 50 unassociated known LOBC SNPs. The gene-based analysis identified an association between breast cancer and the phosphofructokinase-muscle (PFKM) gene on chromosome 12q13.11 that met the genome-wide gene-based threshold of 2.5 × 10−6. In conclusion, EOBC and LOBC seem to have similar genetic etiologies; the 5q11.2 region may contain multiple distinct breast cancer loci; and the PFKM gene region is worthy of further investigation. These findings should enhance our understanding of the etiology of breast cancer. Cancer Epidemiol Biomarkers Prev; 23(4); 658–69. ©2014 AACR.

Supplementary Figure 1: Evaluation of technical reproducibility over time. Intraclass correlation coefficients for microbiome metrics including the abundance of three phyla, two alpha-diversity metrics (number of observed OTUs and Shannon index) and four beta-diversity metrics (top PCoA component for unweighted Unifrac, generalized Unifrac, weighted UniFrac, and Bray-Curtis distance) analyzed at days 1 (A, B) and 4 (C, D) in the Knight laboratory (A, C) and the Mayo Microbiome laboratory (B, D).

Supplementary Table 1: Study design.

Registry-based epidemiologic studies suggest associations between chronic inflammatory intestinal diseases and pancreatic ductal adenocarcinoma (PDAC). As genetic susceptibility contributes to a large proportion of chronic inflammatory intestinal diseases, we hypothesize that the genomic regions surrounding established genome-wide associated variants for these chronic inflammatory diseases are associated with PDAC. We examined the association between PDAC and genomic regions (±500 kb) surrounding established common susceptibility variants for ulcerative colitis, Crohn's disease, inflammatory bowel disease, celiac disease, chronic pancreatitis, and primary sclerosing cholangitis. We analyzed summary statistics from genome-wide association studies data for 8,384 cases and 11,955 controls of European descent from two large consortium studies using the summary data-based adaptive rank truncated product method to examine the overall association of combined genomic regions for each inflammatory disease group. Combined genomic susceptibility regions for ulcerative colitis, Crohn disease, inflammatory bowel disease, and chronic pancreatitis were associated with PDAC at P values < 0.05 (0.0040, 0.0057, 0.011, and 3.4 × 10−6, respectively). After excluding the 20 PDAC susceptibility regions (±500 kb) previously identified by GWAS, the genomic regions for ulcerative colitis, Crohn disease, and inflammatory bowel disease remained associated with PDAC (P = 0.0029, 0.0057, and 0.0098, respectively). Genomic regions for celiac disease (P = 0.22) and primary sclerosing cholangitis (P = 0.078) were not associated with PDAC. Our results support the hypothesis that genomic regions surrounding variants associated with inflammatory intestinal diseases, particularly, ulcerative colitis, Crohn disease, inflammatory bowel disease, and chronic pancreatitis are associated with PDAC.Significance:The joint effects of common variants in genomic regions containing susceptibility loci for inflammatory bowel disease and chronic pancreatitis are associated with PDAC and may provide insights to understanding pancreatic cancer etiology.

Background: Tobacco-induced lung cancer is characterized by a deregulated inflammatory microenvironment. Variants in multiple genes in inflammation pathways may contribute to risk of lung cancer.Methods: We therefore conducted a three-stage comprehensive pathway analysis (discovery, replication, and meta-analysis) of inflammation gene variants in ever-smoking lung cancer cases and controls. A discovery set (1,096 cases and 727 controls) and an independent and nonoverlapping internal replication set (1,154 cases and 1,137 controls) were derived from an ongoing case–control study. For discovery, we used an iSelect BeadChip to interrogate a comprehensive panel of 11,737 inflammation pathway single-nucleotide polymorphisms (SNP) and selected nominally significant (P < 0.05) SNPs for internal replication.Results: There were six SNPs that achieved statistical significance (P < 0.05) in the internal replication data set with concordant risk estimates for former smokers and five concordant and replicated SNPs in current smokers. Replicated hits were further tested in a subsequent meta-analysis using external data derived from two published genome-wide association studies (GWAS) and a case–control study. Two of these variants (a BCL2L14 SNP in former smokers and an SNP in IL2RB in current smokers) were further validated. In risk score analyses, there was a 26% increase in risk with each additional adverse allele when we combined the genotyped SNP and the most significant imputed SNP in IL2RB in current smokers and a 36% similar increase in risk for former smokers associated with genotyped and imputed BCL2L14 SNPs.Conclusions/Impact: Before they can be applied for risk prediction efforts, these SNPs should be subject to further external replication and more extensive fine mapping studies. Cancer Epidemiol Biomarkers Prev; 21(7); 1213–21. ©2012 AACR.

PDF file - 54K, Summary of Inflammation subpathways, genes and SNP's in Illumina chip

Subjects grouped into clusters (designated as A, B, C) by the UniFrac distance matrix according to the UPGMA method. Note that the figure was constructed from 659 subjects. We included 333 subjects for this study because we did not have serum pepsinogen I and II measurements available for all 659 subjects

Odds ratio and 95% confidence intervals for the associations between presence/absence of individual strain/species and PGI/II ratio

Supplementary Table 2: Change in OTUs. The list of all OTUs showing a change of more than 8 fold between day 4 and day 0 for No Additive and FOBT-Pre treatment. Also included are 2 summary tables showing the overlap between the Mayo and Knight lab sequencing results. Green rows indicate and OTU showing growth in both labs, Yellow rows indicate OTUs showing growth in only one center. NaNs indicate greengene IDs not found in the respective sequencing center (since both centers use different sequencing regions, the assignment may differ between them).

PDF file - 197K, Table S1. Discovery set study populations. Table S2. Distribution of discovery set subjects by site, case-control status and chip. Table S3. Discovery set quality control summary. Table S4. SNPs with score-based P-values < 4 X 10-8 among 58016 SNPs for which Plink aborted the logistic regressions. Table S5. Replication set study populations. Table S6. Distribution of replication set subjects by site, case-control status and

Background: The human upper digestive tract microbial community (microbiota) is not well characterized and few studies have explored how it relates to human health. We examined the relationship between upper digestive tract microbiota and two cancer-predisposing states, serum pepsinogen I/pepsinogen II ratio (PGI/II; predictor of gastric cancer risk) and esophageal squamous dysplasia (ESD; the precursor lesion of esophageal squamous cell carcinoma; ESCC) in a cross-sectional design.Methods: The Human Oral Microbe Identification Microarray was used to test for the presence of 272 bacterial species in 333 upper digestive tract samples from a Chinese cancer screening cohort. Serum PGI and PGII were determined by ELISA. ESD was determined by chromoendoscopy with biopsy.Results: Lower microbial richness (number of bacterial genera per sample) was significantly associated with lower PGI/II ratio (P = 0.034) and the presence of ESD (P = 0.018). We conducted principal component (PC) analysis on a β-diversity matrix (pairwise difference in microbiota), and observed significant correlations between PC1, PC3, and PGI/II (P = 0.004 and 0.009, respectively), and between PC1 and ESD (P = 0.003).Conclusions: Lower microbial richness in upper digestive tract was independently associated with both cancer-predisposing states in the esophagus and stomach (presence of ESD and lower PGI/II).Impact: These novel findings suggest that the upper digestive tract microbiota may play a role in the etiology of chronic atrophic gastritis and ESD, and therefore in the development of gastric and esophageal cancers. Cancer Epidemiol Biomarkers Prev; 23(5); 735–41. ©2014 AACR.

Telomere length is a biomarker reflective of aging and genomic instability. We previously found associations between longer measured leukocyte telomere length (LTL) and increased lung cancer risk in a pooled nested case-control study with 847 cases. Additionally, we reported that longer genetically-predicted telomere length (gTL) using a polygenic risk score (PRS) with 7 single nucleotide polymorphisms (SNPs) was associated with increased lung cancer risk among never-smoking Asian women. To investigate further, we conducted prospective cohort analyses of pre-diagnostic LTL and gTL in relation to risk of overall lung cancer and its subtypes in the UK Biobank. With over triple the number of cases, we had expanded statistical power to investigate lung cancer, adenocarcinoma (LUAD), and squamous cell carcinoma (SCC) among subgroups defined by smoking status and sex in this predominantly European study population. We analyzed 371,890 participants at baseline. Over the 12.36±1.64SD year follow-up, 2829 incident lung cancer cases were diagnosed, including 1078 LUAD and 487 SCC. LTL was measured using qPCR. Using European data, we calculated a weighted PRS for gTL. The PRS was derived from 130 linkage disequilibrium pruned SNPs (r2 > 0.01) associated with longer LTL in the UK Biobank. Multivariable Cox regression was used to estimate the hazard ratios (HR) and 95% confidence intervals (CI) of lung cancer in relation to log-transformed continuous and quartiles (Q) of LTL and gTL separately. Associations were estimated in the overall analytic sample and among smoking-sex subgroups. We conducted Mendelian Randomization (MR) analyses using MR-PRESSO with 130 genetic instruments to assess causal associations between LTL and lung cancer among Europeans.We found a strong dose-response relationship between longer LTL and increased lung cancer risk (p-trend= 2.6E-05), with the highest quartile being statistically significant (HRQ4vs.Q1=1.27, 95%CI: 1.15-1.42). The association was apparent for LUAD (p-trend=6.6E-10; HRQ4vs.Q1=1.78, 95%CI: 1.50-2.12), but not SCC. The lung cancer associations were prominent among never-smoking women (p-trend=4.0E-05) and never-smoking men (p-trend=2.0E-03). We also found dose-response relationships between longer gTL and risk of lung cancer (p-trend=4.2E-06) and LUAD (p-trend=2.1E-08). Additionally, we found evidence for causal associations between longer LTL and risk of lung cancer (HRper 1 SD gTL=1.87, 95%CI: 1.49-2.36, p=4.0E-07) and LUAD (HRper 1 SD gTL=2.45, 95%CI: 1.69-3.57, p=6.5E-06). Longer leukocyte telomere length potentially reflects biological processes relevant to the pathogenesis of lung cancer, particularly LUAD. Citation Format: Jason Yat-Yang Wong, Batel Blechter, Aubrey K. Hubbard, Jianxin Shi, Wei Hu, Mohammad Rahman, Shahinaz Gadalla, Mitchell Machiela, Nathaniel Rothman, Qing Lan. Measured and genetically-predicted leukocyte telomere length and lung cancer risk in the prospective UK Biobank [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3009.

Introduction: There is a critical need for complex microbiome quality control standards representing population-based samples for microbial community profiling and analysis in large scale epidemiologic studies. Methods: We developed standard quality control samples from five volunteers with different phenotypes, comprising one obese female, one healthy male, one male on a low-carb diet, one infant, and one male with Crohn’s Disease, and evaluated their microbial metagenomic profiles within three laboratories at two different timepoints. To quantify the percentage of microbiome variability explained by donors, laboratory and sequencing run, a distance-based coefficient of determination R2 was estimated using a permutational multivariate analysis of variance. In addition, we calculated the intraclass correlation coefficients (ICC) for the relative abundance of the most abundant species, two alpha diversity metrics (i.e., observed number of species and Shannon index) and the first principal coordinates of three beta diversity matrices (i.e., Bray-Curtis, Jaccard and Aitchison) to estimate the accuracy of fecal microbial profiles between the three different laboratories as well as within the laboratories. Results: The variability introduced by the phenotype of the donors explained 82.7% to 95.3% of the overall variability, which was higher than the variability introduced by the laboratories (1.8% to 3.1%) and the sequencing runs (0.6% to 1.7%) - the residual percent variance explained varied between 2.2% and 12.4%. Observations based on principal coordinates analysis showed that samples clustered by donor and not by laboratory or sequencing runs. The five donor clusters were well separated and very distinct. Based on the comparison of species relative abundances, each donor displayed very different microbial profiles; and the microbial profiles of each donor were comparable between the three different laboratories and the two sequencing runs in each laboratory. The reproducibility within and between the laboratories was good to excellent for most diversity metrics (ICCs higher than 0.97) and species relative abundances (range, ICCs=0.70-0.99); however, the reproducibility of the observed number of species was moderate (ICC=0.64 for the first laboratory, ICC=0.78 for the second laboratory, ICC=0.81 for the third laboratory, and ICC=0.42 between the laboratories). Conclusions: These standard quality control samples can be used as a reference in future population-based epidemiologic studies to pool or meta-analyze microbiome data. Citation Format: Semi Zouiouich, Smriti Karwa, Yunhu Wan, Andrew Chan, Joseph Petrosino, Emma Allen-Vercoe, Rob Knight, Jianxin Shi, Mitchell Gail, Christian Abnet, Emily Vogtmann, Rashmi Sinha. Quality control samples for future population-based microbiome studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3050.

Microbiome studies have been rapidly increasing over the last decade, providing valuable insights into the commensal bacterial contribution to human physiology and disease, including human cancer. Many studies have demonstrated the important roles of the microbiome in cancer progression, anti-tumor immunity, resistance to therapies, metastasis formation, and in some rare cases even cancer development through the production of genotoxins. However, relatively few studies have analyzed the lung cancer microbiome, and both its composition and role in cancer progression are largely unknown. To characterize the microbiome of tumor and matched normal lung tissues, we performed whole-genome sequencing (WGS) in 872 never smokers using the Kraken pipeline. At the phylum level, WGS analyses of tumor and normal samples showed predominantly Proteobacteria and Actinobacteria (58% and 32%, respectively). Notably, we found overall similar microbiome composition in tumor and normal lung tissue samples. At the genus level, Cutibacterium, Klebsiella, Pseudomonas, Sphingomonas, Staphylococcus, and Acinetobacter genera were among the most abundant bacteria in tumor and normal tissues, alike, and the genera Prevotella, Corynebacterium, and Streptococcus were more abundant in tumor samples compared to normal lung tissue. Comparison of alpha diversity at the genus level between tumors and normal samples showed no substantial difference. We are currently investigating whether the microbiome composition varies by lung tumor anatomical location, sex, histology, study subjects’ geographical location, and immune microenvironment. We will also investigate whether the tumor microbiome composition varies in relation to important genomic features, like cancer driver genes and mutational signatures. Finally, we have 16s rRNA seq data from 771 tumor and normal lung tissues from never smokers and we will compare bacterial microbiome composition and diversity based on WGS and 16s rRNA seq for the same samples. This study, based on the largest analysis of lung microbiome to date, is poised to provide important insights into the role of commensal microbiota in shaping lung tumor development and progression. Citation Format: John McElderry, Tongwu Zhang, Jianxin Shi, Maria Teresa Landi. Characterization of the lung cancer microbiome using whole genome sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5909.

APOBEC enzymes are part of the innate immunity and are responsible for restricting viruses and retroelements by deaminating cytosine residues. Most solid tumors harbor different levels of somatic mutations attributed to the off-target activities of APOBEC3A (A3A) and/or APOBEC3B (A3B). However, how APOBEC3A/B interact with exogenous mutagenic processes in shaping tumor development is largely unknown. Here, by combining deep whole-genome sequencing with multi-omics profiling of 309 lung cancers from smokers with detailed tobacco smoking information, we identify two subtypes defined by low (LAS) and high (HAS) APOBEC mutagenesis. LAS are enriched for A3B-like mutagenesis and KRAS mutations, whereas HAS for A3A-like mutagenesis and TP53 mutations. Unlike APOBEC3A, APOBEC3B expression is strongly associated with an upregulation of the base excision repair pathway. Hypermutation by unrepaired A3A and tobacco smoking mutagenesis combined with TP53-induced genomic instability can trigger senescence, apoptosis, and cell regeneration, as indicated by telomere shortening, high expression of pulmonary healing signaling pathway and stemness markers in HAS. The expected association of tobacco smoking exposure with genomic/epigenomic changes are not observed in HAS, a plausible consequence of frequent cell senescence or apoptosis. HAS tumors have slower clonal expansion and older age at onset compared to LAS, particularly in heavy smokers, consistent with high proportions of newly generated, unmutated cells in HAS. These findings show how heterogeneity in mutational burden across competing mutational processes and cell types contributes to tumor development, with important clinical implications. Citation Format: Tongwu Zhang, Jian Sang, Phuc H. Hoang, Wei Zhao, Jennifer Rosenbaum, Leszek J. Klimczak, John McElderry, Alyssa Klein, Christopher Wirth, Erik N. Bergstrom, Marcos Díaz-Gay, Raviteja Vangara, Amy Hutchinson, Scott M. Lawrence, Nathan Cole, Bin Zhu, Teresa M. Przytycka, Jianxin Shi, Neil E. Caporaso, Robert Homer, Angela C. Pesatori, Dario Consonni, Stephen J. Chanock, David C. Wedge, Dmitry A. Gordenin, Ludmil B. Alexandrov, Reuben S. Harris, Maria Teresa Landi. APOBEC deaminases compete with tobacco smoking mutagenesis and affect age at onset of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1166.

Mutational signatures, the footprints of somatic mutations, are associated with the causes of cancer and have been well-studied for tumors with whole exome or genome sequencing. However, due to the low number of detected mutations, mutational signatures were insufficiently explored for many tumors sequenced by targeted panels in clinics. It impedes the clinical application of mutational signatures. Here, we present a new method, SALMON (Signature Analyzer for Low Mutation cOuNts), to identify mutational signatures in targeted sequenced tumors based on tumor mutational burden. SALMON adjusts for panel size differences and uses a large number of targeted sequenced tumors rather than a large number of mutations per tumor (as with whole exome or genome sequencing) to overcome the challenges of mutational signature analysis for targeted sequencing. Extensive simulations and pseudo-targeted sequenced data show that SALMON can accurately detect spiky or common signatures. We applied SALMON to investigate the pan-cancer patterns of mutational signatures for 100,477 targeted sequenced tumors in AACR Project GENIE, including 14,428 lung and 11,389 breast tumors. We detected well-established signatures in tumor types that have not previously been associated with these signatures, such as the smoking signature in ovarian tumors. Interestingly, analysis of thousands of tumors per cancer type from diverse populations revealed gender discrepancies, self-described race differences, subtype heterogeneity, and metastatic enrichment of mutational signatures. For instance, most sex-biased signatures are more frequently present in males for non-gender-specific cancers. Thiopurine treatment-induced signature in glioma is enriched in Black patients. And endometrioid ovarian or uterine cancers have a higher prevalence of polymerase epsilon (POLE) deficiency-related signatures than non-endometrioid ovarian or uterine cancers, respectively. Our study demonstrates the feasibility and utility of mutational signature analysis for targeted sequenced tumors, enabling precision applications of mutational signatures in the clinical setting. Citation Format: Donghyuk Lee, Min Hua, Difei Wang, Lei Song, Tongwu Zhang, Kai Yu, Xiaohong R. Yang, Jianxin Shi, Stephen J. Chanock, Maria Teresa Landi, Bin Zhu. The mutational signatures of 100,477 targeted sequenced tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5722.

Background: The etiology of lung cancer among never-smokers has not been adequately elucidated despite that globally15% of lung cancer cases in men and 53% in women are not smoking-related. Epigenetic modifications, including changes in DNA methylation (DNAm), have been suggested as possible underlying mechanisms. However, only a few prospective epigenome-wide association studies (EWAS) of lung cancer incidence have been conducted, all exclusively focused on DNAm in peripheral blood cells and included a minimal number of never-smokers. We aimed to investigate genome-wide DNAm associations and epigenetic age acceleration with future risk of lung cancer among never-smokers using pre-diagnostic oral rinse samples. Methods: We conducted a case-control study of 80 never-smoking incident lung cancer cases and 83 comparable never-smoking controls nested in two large prospective cohorts: the Shanghai Women’s Health Study and Shanghai Men’s Health Study. DNAm was measured using the Illumina EPIC array. The top 50 differentially methylated positions (DMPs) were identified from a discovery sample and tested for replication in a validation sample using robust linear regression models. We also conducted an EWAS in the pooled sample. We examined functional overlap enrichment across chromatin states and histone mark broadPeaks for the top 1000 DMPs using eFORGE and constructed enrichment biological pathways analyses. Results: Across discovery and pooled EWAS, we identified four DMPs associated with lung cancer at the epigenome-wide significance level of P Conclusions: To our knowledge, this is the first prospective EWAS of lung cancer among never-smokers using oral rinse samples. Our results show that DNAm in pre-diagnostic oral rinse samples can provide new insights into lung cancer etiology and risk factors. Citation Format: Mohammad L. Rahman, Charles E. Breeze, Xiao-Ou Shu, Jason YY Wong, Andres Cardenas, Xuting Wang, Bu-Tian Ji, Wei Hu, Batel Blechter, Qiuyin Cai, H Dean Hosgood, Gong Yang, Jianxin Shi, Jirong Long, Yu-Tang Gao, Douglas Bell, Wei Zheng, Qing Lan, Nathaniel Rothman. Epigenome-wide association study of lung cancer among never-smokers in two prospective cohorts in Shanghai [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3483.

Registry-based epidemiologic studies suggest associations between chronic inflammatory intestinal diseases and pancreatic ductal adenocarcinoma (PDAC). As genetic susceptibility contributes to a large proportion of chronic inflammatory intestinal diseases, we hypothesize that the genomic regions surrounding established genome-wide associated variants for these chronic inflammatory diseases are associated with PDAC. We examined the association between PDAC and genomic regions (±500 kb) surrounding established common susceptibility variants for ulcerative colitis, Crohn's disease, inflammatory bowel disease, celiac disease, chronic pancreatitis, and primary sclerosing cholangitis. We analyzed summary statistics from genome-wide association studies data for 8,384 cases and 11,955 controls of European descent from two large consortium studies using the summary data-based adaptive rank truncated product method to examine the overall association of combined genomic regions for each inflammatory disease group. Combined genomic susceptibility regions for ulcerative colitis, Crohn disease, inflammatory bowel disease, and chronic pancreatitis were associated with PDAC at P values < 0.05 (0.0040, 0.0057, 0.011, and 3.4 × 10−6, respectively). After excluding the 20 PDAC susceptibility regions (±500 kb) previously identified by GWAS, the genomic regions for ulcerative colitis, Crohn disease, and inflammatory bowel disease remained associated with PDAC (P = 0.0029, 0.0057, and 0.0098, respectively). Genomic regions for celiac disease (P = 0.22) and primary sclerosing cholangitis (P = 0.078) were not associated with PDAC. Our results support the hypothesis that genomic regions surrounding variants associated with inflammatory intestinal diseases, particularly, ulcerative colitis, Crohn disease, inflammatory bowel disease, and chronic pancreatitis are associated with PDAC. Significance: The joint effects of common variants in genomic regions containing susceptibility loci for inflammatory bowel disease and chronic pancreatitis are associated with PDAC and may provide insights to understanding pancreatic cancer etiology.

Background: A number of cohort studies have collected Scope mouthwash samples by mail, which are being used for microbiota measurements. We evaluated the stability of Scope mouthwash samples at ambient temperature and determined the comparability of Scope mouthwash with saliva collection using the OMNIgene ORAL Kit. Methods: Fifty-three healthy volunteers from Mayo Clinic and 50 cohort members from Bangladesh provided oral samples. One aliquot of the OMNIgene ORAL and Scope mouthwash were frozen immediately and one aliquot of the Scope mouthwash remained at ambient temperature for 4 days and was then frozen. DNA was extracted and the V4 region of the 16S rRNA gene was PCR amplified and sequenced using the HiSeq. Intraclass correlation coefficients (ICC) were calculated. Results: The overall stability of the Scope mouthwash samples was relatively high for alpha and beta diversity. For example, the meta-analyzed ICC for the Shannon index was 0.86 (95% confidence interval, 0.76–0.96). Similarly, the ICCs for the relative abundance of the top 25 genera were generally high. The comparability of the two sample types was relatively low when measured using ICCs, but were increased by using a Spearman correlation coefficient (SCC) to compare the rank order of individuals. Conclusions: Overall, the Scope mouthwash samples appear to be stable at ambient temperature, which suggests that oral rinse samples received by the mail can be used for microbial analyses. However, Scope mouthwash samples were distinct compared with OMNIgene ORAL samples. Impact: Studies should try to compare oral microbial metrics within one sample collection type.

Objective: This nested case-control study in the NIH-AARP Diet and Health Study was carried out to prospectively investigate the relationship of oral microbiome with head and neck cancer. Methods: Among 34,262 participants with oral wash samples, 60 incident head and neck cancer cases were identified during a mean of 6.25±1.28 years of follow-up. Two controls were matched to each case by age at sample collection, sex, and baseline smoking history, and available controls were not diagnosed with head and neck cancer over follow-up. Whole genome shotgun metagenomic sequencing was used to measure the overall oral microbiome, and ITS2 gene qPCR was used to measure the presence of fungi in pre-diagnostic oral wash samples. ITS2 gene sequencing was performed on ITS2 qPCR positive samples. Taxonomic and functional alpha-diversity and beta-diversity metrics were computed. Presence/absence and relative abundance of groups of red- and/or orange-complex periodontal pathogens were also calculated. Conditional logistic regression models and MiRKAT were used to investigate the case-control associations. Results: Participants with higher taxonomic microbial alpha-diversity had a decreased risk of head and neck cancer with odds ratios per every 10 observed species of 0.87 (95% CI=0.75-1.004, P=0.057), and per one unit of Faith’s PD and the Shannon index of 0.84 (95% CI=0.72-0.99, P=0.032) and 0.52 (95% CI=0.29-0.94, P=0.032), respectively. No case-control differences were found for beta diversity (all p>0.05). The presence of red-complex periodontal pathogens was non-significantly associated with reduced risk of head and neck cancer (OR=0.41, 95% CI=0.17-1.01, P=0.052). Greater relative abundance of red-complex (OR=0.29, 95% CI=0.09-0.94, P=0.040), orange-complex (OR=0.45, 95% CI=0.22-0.89, P=0.022), and both complexes combined (OR=0.46, 95% CI=0.25-0.82, P=0.009), were associated with reduced risk of head and neck cancer. The presence of oral fungi was also strongly associated with reduced risk of head and neck cancer compared with controls (OR=0.39, 95% CI=0.17-0.87, P=0.022). Conclusions: Greater taxonomic alpha-diversity and the presence or relative abundance of multiple bacterial or fungal species, including the red- and orange-complex periodontal pathogens, were associated with reduced risk of head and neck cancer. Citation Format: Zeni Wu, Yongli Han, Yunhu Wan, Xing Hua, Casey L. Dagnall, Kristine Jones, Amy Hutchinson, Belynda D. Hicks, Weiyin Zhou, Linda Liao, Heather Hallen-Adams, Jianxin Shi, Christian C. Abnet, Rashmi Sinha, Anil Chaturvedi, Emily Vogtmann. The oral microbiome and the risk of head and neck cancer: A nested case-control study in the NIH-AARP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 687.

Lung cancer in never smokers (LCINS) is a common cause of cancer mortality, but is poorly characterized. Here we show, through high coverage whole genome sequencing of 232 tumors/blood pairs, that LCINS can be separated into three subtypes, defined by somatic copy number aberrations. While the dominant subtype (‘piano'), rarely found in lung cancer from smokers, is characterized by quiet copy number profiles, the other subtypes are associated with specific arm-level amplifications and EGFR mutations (‘mezzo-forte'), and whole genome doubling (‘forte'). Piano tumors feature somatic UBA1 mutations, germline AR variants, and stem cell-like properties, including low mutational burden, infrequent TP53 alterations, high intra-tumor heterogeneity, long telomeres, and slow growth as suggested by the occurrence of cancer driver genes decades prior to tumor diagnosis. In contrast, driver mutations in mezzo-forte and forte tumors are generally late clonal events acquired close to tumor diagnosis, thus potentially facilitating target identification with a single biopsy. The mutational signature associated with direct exposure to tobacco smoking (SBS4) was not observed in our study, even in cases with reported exposure to second-hand tobacco smoke. No significant differences of mutational spectra were found between passive and non-passive smokers. While genes within the RTK-RAS pathway have distinct impacts on survival, mutations in TP53, CHEK2, EGFR, or loss of 15q or 22q independently doubled mortality. These genomic alterations in forte and mezzo-forte subtypes, and stem cell-like features in piano tumors create avenues for personalized therapeutic strategies in LCINS. Citation Format: Tongwu Zhang, Ludmil B. Alexandrov, Wei Zhao, Jian Sang, Jianxin Shi, Bin Zhu, Dmitry A. Gordenin, Montserrat Garcia-Closas, Qing Lan, Nathaniel Rothman, Stephen J. Chanock, David C. Wedge, Maria Teresa Landi. Genomic and evolutionary classification of lung cancer in never smokers from the Sherlock-Lung study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 865.

Genome wide association studies (GWAS) in 9,013 pancreatic cancer patients and 12,452 controls of European ancestry have discovered over 20 risk loci in the human genome. Here, we fine mapped one such locus on chromosome 16q23.1 in the vicinity of two Chymotrypsinogen precursor genes, CTRB1 and CTRB2. We fine-mapped this locus to rs72802365 (P=2.51x10-17, OR=1.36, 95% CI=1.31-1.40) and identified colocalization (PP=87%) with aberrant exon 5→7 CTRB2 splicing in pancreatic tissues. Imputation of a 584 bp insertion/deletion variant overlapping exon 6 of CTRB2 into the GWAS datasets resulted in a highly significant association with pancreatic cancer risk (P=2.83x10-16, OR=1.36, 95% CI=1.31-1.42), indicating that it may underlie this signal. Exon skipping attributable to the deletion (risk) allele introduces a premature stop codon in exon 7 of CTRB2, yielding a truncated chymotrypsin B2 protein that lacks chymotrypsin activity, is poorly secreted, and accumulates intracellularly in the endoplasmic reticulum (ER). The intracellular accumulation of a nonfunctional chymotrypsinogen B2 protein may lead to ER stress and pancreatic inflammation, which in turn, may explain the increased pancreatic cancer risk in carriers of CTRB2 exon 6 deletion alleles. Citation Format: Ashley Jermusyk, Jun Zhong, Naomi Gordon, Katelyn Connelly, Sumeth Perera, Ehssan Abdolalizadeh, Tongwu Zhang, Aidan O'Brien, Jason Hoskins, Irene Collins, Daina Eiser, Chen Yuan, Harvey Risch, Eric J. Jacobs, Donghui Li, Mengmeng Du, Rachael Stolzenberg-Solomon, Alison P. Klein, Jill P. Smith, Brian M. Wolpin, Stephen J. Chanock, Jianxin Shi, Gloria M. Petersen, Christopher Westlake, Laufey T. Amundadottir. An exception to the rule: A coding functional variant at a pancreatic cancer GWAS locus [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr IA-11.

Background and objective: Breast cancer burden is rising in Africa; however, breast cancer risk factors remain largely understudied in this population. The gut microbiome may play a role in the etiology of breast cancer through the regulation of estrogen homeostasis and other metabolic or immunologic pathways. We investigated the association of fecal bacteria with breast neoplasms in a case-control study conducted in Accra and Kumasi, Ghana, comprising 102 non-malignant breast disease cases, 379 invasive breast cancer cases, and 414 population-based controls nested within the Ghana Breast Health Study. Methods: We used the MO-BIO PowerMag kit for DNA extraction and sequenced the V4 region of 16S rRNA gene to characterize bacteria in fecal samples. We used DADA2 to process sequencing data. We calculated estimates of alpha diversity (observed sequence variants, Shannon index, and Faith's phylogenetic diversity), beta diversity (Bray Curtis and unweighted/weighted UniFrac), and presence/absence and relative abundance of select taxa. We then estimated the associations of the microbial metrics with non-malignant breast disease and malignant breast cancer using multivariable unconditional logistic regression. Results: Odds of malignant breast cancer were statistically significantly lower with increasing alpha diversity (all Ptrends 0.1) but was statistically significantly different for both case groups compared controls (all p's Conclusions: Our findings suggest lower fecal alpha diversity, differences in overall microbial composition, and prevalence of certain taxa involved in estrogen metabolism may be associated with both non-malignant breast disease and invasive breast cancer. Given that the gut bacteria are potentially modifiable and targetable, our findings warrant further investigation into the role of the gut microbiota in hormone regulation and etiology of breast cancer in integrative, prospective studies. Citation Format: Doratha Byrd, Emily Vogtmann, Zeni Wu, Autumn Hullings, Xing Hua, Thomas Ahearn, James J. Goedert, Jianxin Shi, Jonine Figueroa, Louise Brinton, Montserrat Garcia-Closas, Rashmi Sinha. Associations of fecal microbial profiles with non-malignant breast disease and breast cancer in the Ghana Breast Health Study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4638.

Background: The oral microbiota may be associated with lung cancer risk through direct mechanisms, including infection, immune responses, and periodontal disease, and through indirect mechanisms such as the modification of the oral microbiota by tobacco. We conducted a case-cohort study nested within three US prospective cohort studies to evaluate the association between oral microbiota ascertained years before a cancer diagnosis and risk of lung cancer. Methods: Incident lung cancer cases within the Agricultural Health Study (AHS; N=244), NIH-AARP Diet and Health Study (N=376), and the Prostate, Lung, Colorectum, and Ovarian Cancer Screening Trial (PLCO; N=700) who provided an oral wash sample were identified. The median time between oral sample collection and diagnosis was approximately 6.6 years, 3.4 years, and 4.5 years for AHS, NIH-AARP, and PLCO, respectively. A referent subcohort was randomly selected by strata of age, sex, and cigarette smoking history. We extracted DNA using the DSP DNA Virus Pathogen kit, and the V4 region of the 16S rRNA gene was PCR amplified and sequenced using the MiSeq. The sequencing data were processed using QIIME2 with the DADA2 plugin and we generated alpha and beta diversity metrics. Cox proportional hazards models were used to evaluate the hazard ratios (HR) and 95% confidence intervals (CI) for the association between the oral microbial measures and the risk of lung cancer with adjustment for known lung cancer risk factors, and estimates from the three cohorts were meta-analyzed. Results: Increased alpha diversity was associated with decreased lung cancer risk, although only the association with the Shannon Index reached statistical significance (HR for 5th quintile versus 1st quintile 0.74; 95% CI 0.60, 0.92) with no evidence of between-study heterogeneity (p = 0.5968). Specific principal coordinate vectors from the beta diversity matrices were also significantly associated with lung cancer risk, suggesting differing bacterial communities between future lung cancer cases. When stratified by histologic subtypes, the inverse association with alpha diversity was restricted to squamous cell carcinoma, with all alpha diversity metrics reaching statistical significance (e.g., Faith’s phylogenetic diversity HR for 5th quintile versus 1st quintile 0.57; 95% CI: 0.37, 0.87). Similarly, when stratified by smoking history, the inverse association with alpha diversity was restricted to former smokers (e.g., observed species HR for 5th quintile versus 1st quintile 0.63; 95% CI: 0.44, 0.89). Conclusions: In oral wash samples collected years before diagnosis, we found significant associations between both alpha and beta diversity metrics of the oral microbial communities and risk of lung cancer. Additional work is required to understand the associations by histologic subtype and smoking history. Citation Format: Emily Vogtmann, Xing Hua, Guoqin Yu, Autumn Hullings, Yunhu Wan, Casey L Dagnall, Kristine Jones, Belynda D. Hicks, Amy Hutchinson, Shalabh Suman, Bin Zhu, Barry Graubard, Mitchell H. Gail, J. Gregory Caporaso, William Wheeler, Dale Sandler, Laura E. Beane Freeman, Linda Liao, Neal D. Freedman, Neil Caporaso, Rashmi Sinha, Jianxin Shi, Christian C Abnet. The human oral microbiota and risk of lung cancer: An analysis of three prospective cohort studies [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr A39.

Globally, about 2 million people are diagnosed with lung cancer annually, making it the most common type of cancer in the world. In addition, lung cancer is the leading cause of cancer death among both men and women, accounting for ~25% of all cancer deaths in the United States and ~19% of cancer deaths worldwide. Although tobacco smoking is the most common cause of lung cancer, it is estimated that 10-25% of all patients diagnosed with lung cancer worldwide are never smokers, with higher proportions in Asians and women. Compared to former and current smokers with lung cancer, lung cancers in never smokers tend to present at an earlier age and more frequently have adenocarcinoma histology. Although a few risk factors are known to contribute to the etiology of lung cancer in never smokers, a large fraction of cancer cases cannot be explained by established environmental and genetic risk factors, highlighting the need for research in this area. One promising approach to identify the etiological factors involved in lung tumorigenesis in never smokers is based on the study of the “mutational signatures” that the exogenous and endogenous processes leave on the tumor tissue and surrounding areas. Somatic mutations accumulate throughout individuals’ lifetime, as a result of the balance between DNA damage and DNA repair. Mutational sources can change over time and mutational signatures can provide information on the tumor evolutionary trajectory, tracking activities of mutational processes. We designed “Sherlock-Lung” to identify mutational signatures in lung cancers from never smokers and order them based on their clonal and subclonal localization to have an insight on the factors involved in the initiation of the tumors. Prevention and treatment strategies are likely to be more effective when targeting the tumor initiating factors rather than those responsible for the tumor progression. Here, we present the Sherlock-Lung study design, which includes a combination of lung cancer cases from populations highly exposed to lung cancer risk factors and populations with unknown exposures. We also present preliminary data from this study and what we have learned from whole genome sequencing analysis of the mutational landscape of lung cancers from smokers and nonsmokers and from different histological subtypes. For example, although the mutational signature associated with tobacco smoking is largely clonal in all lung cancers from smokers, consistent with an initiating role of tobacco smoking in lung tumorigenesis, the mutational signatures distribution varies across histological subtypes. Moreover, the frequency of mutations in major cancer driver genes varies dramatically between smokers and never smokers, and mutational signatures distribution appears to differ between tumors with and without specific driver genes. In never smokers, without the dominant mutational source of tobacco smoking, other signatures emerged, e.g., that associated with reactive oxygen species. Although this approach has already revealed important information on the etiological processes involved in tumorigenesis of lung cancer and other cancer types, there is a growing list of open questions and challenges that need to be addressed. In population studies, careful exposure assessment to identify and validate new mutational signatures and the inclusion of study subjects from different geographical locations and ethnicities are critical. Citation Format: Maria Teresa Landi, Tongwu Zhang, Montserrat Garcia-Closas, Yohan Bossé, Jianxin Shi, Bin Zhu, Qing Lan, Nathaniel Rothman, Jiyeon Choi, Neil E. Caporaso, Wei Zhao, Chongyi Chen, Eytan Ruppin, Jennifer Rosenbaum, Dmitry Gordenin, Sai Yendamuri, Chris Amos, Paul Brennan, David C. Christiani, Geoff Liu, Bonnie Gould Rothberg, Matthew Schabath, Han Liang, Ludmil Alexandrov, David Wedge, Stephen Chanock. Sherlock-Lung: Tracing lung cancer mutational processes in never smokers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr SY26-02.

Genomic studies have revealed remarkable intratumoral heterogeneity (ITH) in lung adenocarcinoma (LUAD) and increasing evidence suggests its impact in cancer treatment. However, to what extent epigenomic abnormalities and somatic copy number alterations (SCNAs) are involved in shaping genetic clones in lung tumors and are associated with clinical outcome is under-appreciated. Here we applied Infinium HumanMethylation450K BeadChips, Infinium Global Screening Arrays and ultra-deep (>500X) targeted sequencing of 35 LUAD driver genes to 369 samples (including 77 normal tissues) from 84 LUAD patients to investigate the genetic and epigenetic architecture of subclonal diversification. SCNAs were mostly clonal events. For patients with more than three tumor regions assayed, 66% SCNAs were located on the trunk of the evolutionary trees. Similar evolutionary trajectories were inferred from SCNAs and DNA methylation. The driver genes’ sequencing depth allowed to identify subclonality even in TP53, EGFR, and BRAF mutations, which were largely reported as clonal events in previous studies. Elevated copy number ITH was associated with higher risk of death, while no significant association was found between clinical outcome and DNA methylation ITH or heterogeneous CpG island methylator phenotype (CIMP), in contrast with the observed prognostic capability of DNA methylation ITH in hematological malignancies. These findings expand our understanding of the coevolution of genetic and epigenetic aberrations in tumorigenesis, the subclonal complexity for therapeutic targets and the implication of ITH in cancer prognosis. Citation Format: Wei Zhao, Xing Hua, Tongwu Zhang, Dario Consonni, Angela Cecilia Pesatori, Jianxin Shi, Maria Teresa Landi. Genetic and epigenetic intratumor heterogeneity of lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1601.

Pancreatic Ductal Adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths in United States with a 5-year survival rate of only 8%. Inherited predisposition plays an important role in PDAC risk. Rare, moderately to highly penetrant mutations in hereditary cancer and pancreatitis genes, identified in families with a high incidence of disease, account for a small fraction of cases. At the other end of the spectrum, common risk variants of low penetrance have been discovered through genome-wide association studies (GWAS). Currently, over 20 common PDAC risk signals have been identified through GWAS in populations of European ancestry. However, these loci explain only a small fraction of genetic heritability for PDAC, and the causal genes for the large majority of these are unknown. Most susceptibility alleles discovered through GWAS are located in noncoding regions of the genome and are likely to function through allele specific regulation of gene expression. Transcriptome-wide association studies integrate expression quantitative trait loci (eQTL) with GWAS data to discover genes whose cis-regulated expression is associated with complex traits. To identify novel susceptibility loci and likely causal genes, we applied this approach to PDAC. Firstly, we used two reference datasets generated from histologically normal pancreatic tissue samples from individuals of European ancestry, the DCEG/LTG (n=95) and Genotype-Tissue Expression (GTEx v7, n=174), to build robust predictors for gene expression (n=2,872-5,906; r2>0.01), using 5 different models (Top1, Lasso, Enet, Blup and Bslmm) in FUSION and PrediXcan. Gene expression predictors (n=2,043-21,422) were also generated by Enet in 48 different tissues (n=74-421 samples) from 608 individuals of European ancestry (GTEx). We then applied these predictor models to PDAC GWAS summary statistics from 9,040 PC cases and 12,496 controls. We discovered 24 genes whose genetically predicted expression is significantly associated with PDAC risk (FDR < 0.05), including 13 candidate genes at 10 novel loci (CELA3B, SMC2, SMC2-AS1, RP11-80H5.9, SMUG1, BTBD6, RCCD1, PNMT, CDK12, PGAP3, SUPT4H1, RP11-888D10.3 and PGPEP1) and 11 at 5 known risk loci (TERT, CLPTM1L, ZDHHC11B, INHBA, ABO, PDX1, KLF5, WDR59, CFDP1, BCAR1 and TMEM170A). Pathway enrichment analysis for genes differentially expressed in the top vs. bottom quartile of CELA3B expression highlighted higher expression of inflammatory and immune response genes in both the DCEG/LTG and GTEx pancreatic transcriptome datasets, indicating that low expression of this gene may be associated with an inflammatory state in the pancreas. Our findings showcase the utility of integrating multiple datasets to generate hypotheses and provide novel clues for the genetic mechanisms underlying pancreatic cancer risk. Citation Format: Jun Zhong, Ashley Jermusyk, Lang Wu, Jason W. Hoskins, Irene Collins, Mingfeng Zhang, Song Lei, Charles C. Chung, Tongwu Zhang, Wenming Xiao, Rachael Stolzenberg-Solomon, Alison P. Klein, Brian M. Wolpin, Xiao-Ou Shu, Stephen J. Chanock, Sara Olson, Nilanjan Chatterjee, Jill Smith, Jianxin Shi, Peter Kraft, Gloria M. Petersen, Wei Zheng, Laufey T. Amundadottir. Large-scale transcriptome-wide association study (TWAS) identifies novel candidate susceptibility genes for pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1591.

Background: Accounting for approximately 1.76 million annual deaths worldwide, lung cancer is a significant global health burden. While smoking is the most common cause of lung cancer, up to 25% of all lung cancer patients worldwide are never smokers. Lung cancer is the leading cause of cancer mortality in China, where most women do not smoke, making women in Asia an ideal population to study. Previously conducted genome-wide association studies (GWAS) of lung cancer risk among never-smoking women in Asia identified 10 lung cancer susceptibility loci. Indoor air pollution from coal burned for home cooking and heating is known to contain lung carcinogens and has been found to be causally associated with lung cancer. In the current analysis, we evaluated gene-environment interaction between a polygenic risk score (PRS) and coal use in relation to lung adenocarcinoma. Methods: Three studies (Taiwan, Shanghai, Shenyang) from the Female Lung Cancer Consortium in Asia (FLCCA) were used for the primary analysis (1,419 cases; 1,446 controls). A replication study was conducted using samples from Xuanwei, China (159 cases; 572 controls), where lung cancer rates for never-smokers are among the highest in the world and attributed to widespread coal use. We calculated a PRS as the weighted sum of the risk allele counts across the 10 loci, and modeled PRS as a continuous variable scaled by the standard deviation in controls. Logistic regression was used to estimate the main effects of the PRS and coal use, and a likelihood ratio test was used to evaluate the interaction. Models were adjusted for age ( Results: Coal use was associated with an increased risk of lung adenocarcinoma (OR=1.31, 95% CI: 1.01-1.68). We observed an exposure-response relationship between PRS and lung adenocarcinoma (p-trend= 2x10-16) and found a significant multiplicative interaction between PRS and coal use (p-interaction= 0.005). The association between PRS and lung adenocarcinoma was significantly higher among the never coal users (OR=1.68, 95% CI: 1.52-1.86) compared to ever coal users in the three studies (OR=1.24, 95% CI: 1.03-1.50) (p-interaction=0.005), as well as between never coal users in the three studies and ever coal users in Xuanwei (OR=1.25, 95% CI: 1.04-1.49) (p-interaction=0.004). Conclusion: We observed an antagonistic interaction between PRS and coal use with lung adenocarcinoma, where the genetic effect was attenuated among those exposed to coal combustion in the home. We replicated the finding in Xuanwei. These results suggest that the pathogenesis of lung cancer among never-smoking women in Asia differs by exposure to coal combustion emissions and provides one of the few examples of sub-multiplicative gene-environment interactions in the cancer literature. Citation Format: Batel Blechter, Chao Agnes Hsiung, Zhihua Yin, Xiao-Ou Shu, H. Dean Hosgood, Jason Y.Y Wong, Jianxin Shi, Wei Hu, Bryan Bassig, Wei Jie Seow, Yu -ang Gao, Qiuyin Cai, Yong-Bing Xiang, I-Shou Chang, Baosen Zhou, Wei Zheng, Kyoung-Mu Lee, Stephen Chanock, Nilanjan Chatterjee, Nathaniel Rothman, Qing Lan. Analysis of polygenic risk score interaction with coal use and risk of lung adenocarcinoma among never-smoking women in Asia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2682.

Background: Tobacco-induced lung cancer is characterized by a deregulated inflammatory microenvironment. Variants in multiple genes in inflammation pathways may contribute to risk of lung cancer. Methods: We therefore conducted a three-stage comprehensive pathway analysis (discovery, replication, and meta-analysis) of inflammation gene variants in ever-smoking lung cancer cases and controls. A discovery set (1,096 cases and 727 controls) and an independent and nonoverlapping internal replication set (1,154 cases and 1,137 controls) were derived from an ongoing case–control study. For discovery, we used an iSelect BeadChip to interrogate a comprehensive panel of 11,737 inflammation pathway single-nucleotide polymorphisms (SNP) and selected nominally significant (P < 0.05) SNPs for internal replication. Results: There were six SNPs that achieved statistical significance (P < 0.05) in the internal replication data set with concordant risk estimates for former smokers and five concordant and replicated SNPs in current smokers. Replicated hits were further tested in a subsequent meta-analysis using external data derived from two published genome-wide association studies (GWAS) and a case–control study. Two of these variants (a BCL2L14 SNP in former smokers and an SNP in IL2RB in current smokers) were further validated. In risk score analyses, there was a 26% increase in risk with each additional adverse allele when we combined the genotyped SNP and the most significant imputed SNP in IL2RB in current smokers and a 36% similar increase in risk for former smokers associated with genotyped and imputed BCL2L14 SNPs. Conclusions/Impact: Before they can be applied for risk prediction efforts, these SNPs should be subject to further external replication and more extensive fine mapping studies. Cancer Epidemiol Biomarkers Prev; 21(7); 1213–21. ©2012 AACR.

Although lung cancer is largely caused by tobacco smoking, inherited genetic factors play a role in its etiology. Genome-wide association studies in Europeans have only robustly demonstrated 3 polymorphic variations that influence the risk of lung cancer. Tumor heterogeneity may have hampered the detection of association signal when all lung cancer subtypes were analyzed together. In a genome-wide association study of 5,355 European ever-smoker lung cancer patients and 4,344 smoking control subjects, we conducted a pathway-based analysis in lung cancer histologic subtypes with 19,082 single-nucleotide polymorphisms mapping to 917 genes in the HuGE-defined “inflammation” pathway. We identified a susceptibility locus for squamous cell lung carcinoma at 12p13.33 (RAD52, rs6489769) and replicated the association in 3 independent studies totaling 3,359 squamous cell lung carcinoma cases and 9,100 controls (OR = 1.20, Pcombined = 2.3 × 10−8). Significance: The combination of pathway-based approaches and information on disease-specific subtypes can improve the identification of cancer susceptibility loci in heterogeneous diseases. Cancer Discovery; 2(2); 131–9. © 2011 AACR. Read the Commentary on this article by Sellers and Chen, p. 110. This article is highlighted in the In This Issue feature, p. 95.

Breast cancer incidence rates, clinical features, and risk factors are known to vary significantly by race/ethnicity. Recently, tumor sequencing and profiling analyses have improved molecular classification and refined existing breast cancer subtypes. However, detailed genomic analyses are limited in Asian populations. The goal of this study was to profile fresh frozen breast tumors and paired normal tissue in breast cancer cases who had surgeries at two hospitals in Hong Kong. Only tumors containing 50% or more tumor cells were included for sequencing. RNA sequencing (RNASeq), whole exome sequencing, and SNP array were conducted in ~100 tumors. In 92 tumors with RNASeq data, the unsupervised clustering analysis based on 2000 most variable genes identified two subgroups in patients with luminal tumors (defined by PAM50). The most significantly differentially expressed genes between the two luminal subgroups were enriched in immune and inflammation pathways. We further conducted the clustering analysis within 72 luminal tumors (luminal A + luminal B) using 700 immune genes, which again classified the patients into two subgroups (high-immune luminal [HILum] and low-immune luminal [LILum]). Three computational algorithms, CIBERSORT, MCP-counter, and ESTIMATE, were used to infer the fraction or abundance of immune cell populations in each tumor using RNASeq data. We found significant differences in the abundance of most immune cell populations between the two luminal subgroups. The HILum group, which consisted of 53% of all luminal tumors, had a higher overall immune score as well as a higher score for each immune cell subpopulation measured by MCP-counter, at levels similar to those in patients with HER2-positive or basal-like tumors. HILum tumors also expressed significantly higher levels of immune checkpoint genes such as PD1, PDL1, and CTLA4 compared with LILum tumors. Interestingly, HILum was also associated with lower ESR1 expression, earlier age onset, higher level of ploidy and loss-of-heterozygosity, and higher proportions of APOBEC signatures (mutation signatures 2 and 13 in COSMIC) and lower occurrence of the defective DNA mismatch repair signature (signature 6) compared with LILum. The HILum subtype is not specific to Hong Kong since similar results were seen in both TCGA white and Asian breast cancer patients. In summary, we identified a group of luminal patients who had higher levels of immune cell infiltration and immune checkpoint gene expression and are associated with distinct genomic features. These patients may potentially benefit from the immune checkpoint inhibitor therapies. Citation Format: Xiaohong (Rose) Yang, Bin Zhu, Difei Wang, Hela Koka, Tongwu Zhang, Feng Wang, Cherry Wu, Koon Ho Tsang, Wing-cheong Chan, Sze Hong Law, Priscilla Lee, Mengjie Li, Wentao Li, Suyang Wu, Zhiguang Liu, Mingyi Wang, Kristine Jones, Amy Hutchinson, Belynda Hicks, Jianxin Shi, Shelly Lap Ah Tse. Identification of a luminal subtype with high immune abundance among breast cancer patients in Hong Kong, China [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-165.

Melanoma susceptibility SNPs can be categorized by their impact on known susceptibility phenotypes (pigmentation, nevus count, telomere length, or other). In an ongoing meta-analysis of melanoma, at this stage involving 17,800 cases but soon expanding to more than 30,000 cases, 26 SNPs have achieved genome-wide significance. UK Biobank is a population-based cohort of 500,000 UK persons aged at recruitment 40-69 years; participants reported pigmentation characteristics including ease of tanning, natural hair color, skin color (in the absence of tan), and number of childhood sunburns. To conduct analyses, we separately analyzed the red hair phenotype while ordering the other hair colors from black to blond. Among the 26 SNPs from the melanoma GWAS, 15 were associated with at least one pigmentation factor assessed in UK Biobank (each at p Supported by Cancer Research UK (C588/A19167) & NIH (CA083115). This research has been conducted using the UK Biobank Resource. Citation Format: David T. Bishop, Mark M. Iles, Julia A. Newton-Bishop, Jennifer H. Barrett, Adam Trouwer, Matthew Law, Stuart MacGregor, David Duffy, Nicholas G. Martin, Nicholas Hayward, Anne Cust, Maria T. Landi, Alisa Goldstein, Jianxin Shi, Mitch Machiela, Florence Demenais, Peter Kanetsky, GenoMEL & MELANOSTRUM. Understanding melanoma susceptibility through GWAS of risk phenotypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 234.

Limited knowledge about the intratumor heterogeneity (ITH) of non-clear-cell renal cell carcinoma (nccRCC) hinders therapeutic efficacy. We performed multi-region whole-genome sequencing, genome-wide methylation profiling and deep targeted sequencing of both primary and paired metastatic tumors in 124 samples from 29 subjects with papillary renal cell carcinoma type 1, papillary renal cell carcinoma type 2 and collecting duct kidney tumors. Samples were taken from the tumors' center to the periphery at ~1.5cm from each other. We conducted integrative ITH analysis of single nucleotide variants, somatic copy number alterations, structural variants, transposon element insertions, telomere length and DNA methylation, and inferred their respective evolutionary history. We also analyzed the concordance between physical and lineage phylogenetic evolution, and inferred the regions from which the metastases likely originated. ITH varied significantly across tumors and histological subtypes. For example, the average number of structural variants per tumor was 1.2 in papillary RCC type 1 vs. 23.6 in papillary RCC type 2. In papillary tumors, ITH was lowest for copy number alterations (~5% subclonal changes across all tumor types), intermediate for single nucleotide variants (~32%), and highest for structural variants (~60%), suggesting a tumor molecular evolution along these steps. Lineage trees commonly showed branching evolutions with a dominant subclone. Focal deletion of the CDKN2A gene characterized the collecting duct tumors. Methylation ITH was highest in the enhancer regions. Metastatic and primary tumors shared similar mutation signatures. Telomere length was generally longer in normal vs. tumor samples and was similar in physically adjacent samples. In summary, our study characterized ITH of nccRCC with unprecedented details for multiple types of somatic alterations, and delineated the branching architecture of clonal evolution, including the metastatic phase. Citation Format: Bin Zhu*, Luana Poeta*, Manuela Costantini*, Tongwu Zhang*, Steno Sentinelli, Jianxin Shi, Kevin Brown, Xing Hua, Meredith Yeager, Mingyi Wang, Belynda Hicks, Stephen Chanock*, Michele Gallucci*, Vito Fazio*, Maria Teresa Landi*. Multiple region whole-genome sequencing reveals intratumor heterogeneity and branching clonal architecture of non-clear-cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2185.

Heritability of cutaneous melanoma is among the highest across cancer types and is mostly determined by common, noncoding genetic variants. Previous genome-wide association studies (GWAS), mostly including subjects from Australia and Northern European countries, had identified twenty genetic loci associated with melanoma risk. To characterize the genetic landscape of cutaneous melanoma across different populations, we conducted a new association study in Southern European countries including 6,043 melanoma cases and 10,383 controls from the MelaNostrum Consortium. Moreover, we largely expanded the analyses of melanoma cases and controls from other populations, for a total of 27,450 histologically confirmed melanoma cases and 49,888 controls from Northern Europe, Southern Europe, USA and Australia. Imputation was done using the Haplotype Reference Consortium as a reference panel. We assessed the association between cutaneous melanoma risk and single-nucleotide polymorphisms (SNPs) adjusting for country and ancestry-informative principal components. We identified 26 new genetic loci associated with melanoma risk achieving genome-wide significance. The pigmentation pathway, with TYRP1 and MITF, and the telomere-related pathway with RTEL1, TERC, and POT1 among the candidate genes, continued to show an important role for melanoma susceptibility. Additional candidate pathways emerged, including signaling (with GPRC5A and DOCK8), cell-cell junction (with CDH1), immune-related functions (with DCST2, involved in antigen processing), transcriptional regulation (with FOXD3, which represses MITF expression), or tumor-suppressor genes (TP53). Adding genotype data from 6,130 self-reported melanoma cases in a sensitivity analysis identified eight additional novel genetic loci involved in the HLA region, signaling transduction and transcription factor functions. A combined analysis of the melanoma GWAS with the GWAS of pigmentation characteristics from the UK Biobank including 500,000 UK persons, and with a GWAS of nevus density including over 52,000 persons, revealed substantial overlap across phenotypes. Although the effect size of the SNPs on melanoma risk was consistent across populations, substantial differences in minor allele frequency (MAF) were observed in pigmentation-related genes between Northern and Southern European populations; for example, MC1R, SLC45A2, and ASIP SNP MAF was 8.2, 3.1 and 14.2 in Northern and 2.6, 8.8, and 5.5 in Southern European subjects, respectively. Additional analyses based on melanoma histologic subtypes and body site distribution are ongoing. These results provide further insight into genetic susceptibility to cutaneous melanoma and provide the opportunity for genetic risk scores in risk-prediction models for early detection and targeted prevention of the disease. Citation Format: Cristina Pellegrini, Matthew Law, Mark Iles, Mitch Machiela, Alex Stratigos, Tongwu Zhang, Kevin Brown, Julia Newton-Bishop, Nick Hayward, Nick Martin, Alisa Goldstein, Rose Yang, Stephen Chanock, Florence Demenais, Susana Puig, Eduardo Nagore, Jianxin Shi, Tim Bishop, Stuart McGregor, Maria Teresa Landi, GenoMEL Consortium, MelaNostrum Consortium. Association analysis across different populations identifies 26 new cutaneous melanoma risk loci [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 228.

Both lung squamous cell carcinoma (LUSC) and lung cancer with neuroendocrine features (LUNE; i.e., small cell carcinoma (SCLC), large cell carcinoma (LCNEC) and other subtypes) are associated with tobacco smoking and have a very poor prognosis. We sought to characterize their clonal architecture and illustrate how the evolutionary patterns differ across these lung cancer subtypes. We performed multi-region whole genome sequencing of 83 samples from 36 LUSC and 67 samples from 23 LUNE patients that had been resected before systemic therapy. Among somatic copy number alterations, we observed genome doubling intra-tumor heterogeneity (ITH) and massive genomic instability in both subtypes. For somatic nucleotide variants (SNVs), the total mutation burden was higher in LUSC (16.7 average number of mutations/Mb) than LUNE (12.7/Mb), but for SNVs ITH was higher in LUNE. Among driver genes, TP53, CDKN2A, CR1, and NRK in LUSC and TP53, KRAS, KMT2D, and RB1 in LUNE were frequently mutated and were always clonal. Evidence of positive selection was found for TP53, PTEN, KMT2D and KEAP1 in LUSC, and for TP53 and KRAS in LUNE. As expected, the dominant mutation signatures in LUSC clones included signatures 4, 1, and 5, while APOBEC signatures (2 and 13) were predominantly found in sub-clones. In contrast, no evidence of APOBEC signatures were found in LUNE. Among structural variants, LUNE had more events (339.25 average number of events/tumor) than LUSC (206.7/tumor), with considerable ITH in both subtypes, particularly in LUNE. In LUNE, the dominant mechanism responsible for SVs included fork stalling and template switching within the microhomology-mediated break-induced repair (MMBIR). We found recurrent fusion events with hotspots including those previously identified near TTC28/CHEK2 (42% LUSC and 35% LUNE) and LRP1B (42% LUSC). Additional hotspots were found in chromosome bands 20p12.1 (22% LUSC), 1p31.1 (19% LUSC) and 12p11.22 (22% LUNE). We found >10-fold higher transposon insertions (LINE1) in LUSC (71.9 average number of events/tumor) than LUNE (6.3/tumor). Our findings improve our evolutionary understanding of these lung cancer subtypes and warrant further investigations on the mechanisms involved in clonal genomic instability. Citation Format: Tongwu Zhang, Joshua Sampson, Pier Alberto Bertazzi, Angela Pesatori, Dario Consonni, Bin Zhu, Belynda Hicks, Xing Hua, Jianxin Shi, Kevin Brown, Stephen Chanock, Maria Teresa Landi. Comparison of intra-tumor heterogeneity and clonal evolution across lung cancer subtypes by multi-region whole genome sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1187.

Objective: To elucidate the genetic architecture of gene expression in pancreatic tissues. Design: We performed expression quantitative trait locus (eQTL) and allele specific expression (ASE) analyses using RNA-sequence data and 1000 Genomes (1000G) imputed GWAS genotypes from 95 fresh frozen histologically normal pancreatic tissue samples. Data from 115 pancreatic tumor-derived tissue samples from The Cancer Genome Atlas (TCGA) was included for comparison. Results: We identified 38,615 cis-eQTLs (corresponding to 484 Genes) in histologically normal tissues and 39,713 cis-eQTL (corresponding to 237 Genes) in tumor tissues (FDR Conclusions: We have identified cis-eQTLs representing potential functional regulatory variants in the pancreas and generated a rich dataset for further studies on gene expression and regulation in pancreatic tissues. Citation Format: Laufey T. Amundadottir, Soren Lykke Andresen, Wenming Xiao, Jason Hoskins, Ashley Jermusyk, Lauren Rost, Irene Collins, Jinping Jia, Michael Mobaraki, Bin Zhu, Robert Kurtz, Hemang Parikh, Lei Song, Meredith Yeager, Torben Jensen, William Bamlet, Nilanjan Chatterjee, Brian Wolpin, Jill Smith, Sara Olson, Gloria Petersen, Jianxin Shi, Mingfeng Zhang. Analysis of cis-eQTLs in normal and tumor-derived pancreatic tissues reveals functional insights, including for the 9q34.1 ABO pancreatic cancer risk locus [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 1442. doi:10.1158/1538-7445.AM2017-1442

Cutaneous malignant melanoma (CMM) is an etiologically heterogeneous disease with genetic, host, environmental factors, and their interactions contributing to its development. CDKN2A and CDK4 are the two established major susceptibility genes for melanoma identified so far. Recent evidence suggests that copy number variations (CNVs) may contribute to disease susceptibility in several inherited diseases including cancer. The goals of this study were 1) to assess whether the frequency of CNVs varied by CMM or CDKN2A/CDK4 mutation status; and 2) to identify rare CNVs that were related to melanoma predisposition in these high-risk families. We used genome-wide tiling CGH arrays (Nimblegen 720K exon-focused) to investigate characteristics of CNVs in 174 CMM cases, 44 high-risk unaffected family members (with dysplastic nevi/large number of moles or germline CDKN2A mutations), and 48 unrelated spouses from 50 American melanoma-prone families (21 CDKN2A+, 2 CDK4+, 27 mutation negative). We used the Nexus Copy Number™ built-in FASST2 algorithm to identify significant CNVs (significant threshold = 0.000001; minimal number of probes per segment = 5; log2 ratio>0.3 for gains and -0.3 for losses). We found that the median number of total CNVs, or gains or losses separately, did not show significant differences in CMM cases, high-risk unaffected family members, and unrelated controls. Among CMM cases, CNV frequencies were not significantly associated with germline CDKN2A/CDK4 mutation status, age at melanoma diagnosis, or number of melanomas. Similar results were obtained when number of genes and lengths of DNA segments affected by CNVs were analyzed. Restriction to large CNVs (>10 kb or >100 kb) or rare CNVs (not reported in the Toronto CNV database) did not change results significantly. On the other hand, we identified several rare large CNVs (>10 kb, not reported in unrelated controls) that either involved known melanoma genes or co-segregated with melanoma (observed in multiple CMM cases) within families. These included a 1.3Mb deletion in PARP1 in a single CMM case, a 10 kb deletion in CDKN2A in 4 of 5 CMM cases and two obligate gene carriers in a large family that was negative for CDKN2A mutations by sequencing, a 175 kb deletion in LINGO2 in 2 of 3 CMM cases in one family, a 10 kb deletion in 8q24 in 3 of 4 CMM cases in one family, a 110 kb deletion in 2q22.1 in all three cases in a family, and a 57 kb duplication in 4q32.2 in all five cases in a CDKN2A mutation positive family. The role of these CNVs, particularly those involving genes that have unknown function related to CMM development, in CMM susceptibility remain to be investigated. Citation Format: Xiaohong (Rose) Yang, Jianxin Shi, Hunter Bennett, Laura Burke, Casey Dagnall, Laurie Burdette, Belynda Hicks, Margaret Tucker, Alisa Goldstein. Germline copy number variations in melanoma families with/without CDKN2A/CDK4 mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2755. doi:10.1158/1538-7445.AM2015-2755