<p>Summary of somatic SVs</p>

Summary of demographic and clinical data for 15 sequenced cancer cases.

<p>IVA disease analysis report</p>

Summary of the WGS mapping data

<p>Summary of agreement between SNP5 and CGI data</p>

<p>Primers used for validating somatic SNVs</p>

<p>IVA disease analysis report</p>

It has legends for the supplementary tables.

<p>Primers used for SV validation experiments</p>

PDF - 194K, Supplementary Figure S1. Immunohistochemistry for PLCE1 protein expression with areas of annotation selected for image quantification (20x.)Supplementary Table S1. PLCE1 mRNA expression in GCA, GNCA and ESCC tumor tissues and tumor-normal expression fold change by characteristics of the subjects. Supplementary Table S2. PLCE1 protein expression in GCA, GNCA and ESCC tumor tissues and tumor-normal expression fold change in ESCC by characteristics of the participants. Supplementary Table S3. The probe-specific association between PLCE1 mRNA expression in tumors and tumor-normal fold change and mortality of ESCC, GCA, and GNCAs. Supplementary Table S4. Spearman correlation coefficients (P values) between PLCE1 mRNA expression in normal tissues of ESCC, GCA, and GNCA and selected PLCE1 SNPs.

<p>Somatic mutations validated by Sanger sequencing</p>

Helicobacter pylori (Hp) is the primary cause of gastric cancer but we know little of its relative abundance and other microbes in the stomach, especially at the time of gastric cancer diagnosis. Here we characterized the taxonomic and derived functional profiles of gastric microbiota in two different sets of gastric cancer patients, and compared them with microbial profiles in other body sites. Paired nonmalignant and tumor tissues were sampled from 160 gastric cancer patients with 80 from China and 80 from Mexico. The 16S rRNA gene V3-V4 region was sequenced using MiSeq platform for taxonomic profiles. PICRUSt was used to predict functional profiles. Human Microbiome Project was used for comparison. We showed that Hp is the most abundant member of gastric microbiota in both Chinese and Mexican samples (51% and 24% respectively), followed by oral-associated bacteria. Taxonomic (phylum-level) profiles of stomach microbiota resembled oral microbiota, especially when the Helicobacter reads were removed. The functional profiles of stomach microbiota, however, were distinct from those found in other body sites and had higher inter-subject dissimilarity. Gastric microbiota composition did not differ by Hp colonization status or stomach anatomic sites, but did differ between paired non-malignant and tumor tissues in either Chinese or Mexican samples. Our study showed that Hp is the dominant member of the non-malignant gastric tissue microbiota in many gastric cancer patients. Our results provide insights on the gastric microbiota composition and function in gastric cancer patients, which may have important clinical implications.

Based on our initial genome-wide association study (GWAS) on esophageal squamous cell carcinoma (ESCC) in Han Chinese, we conducted a follow-up study to examine the single nucleotide polymorphisms (SNPs) associated with family history (FH) of upper gastrointestinal cancer (UGI) cancer in cases with ESCC. We evaluated the association between SNPs and FH of UGI cancer among ESCC cases in a stage-1 case-only analysis of the National Cancer Institute (NCI, 541 cases with FH and 1399 without FH) and Henan GWAS (493 cases with FH and 869 without FH) data (discovery phase). The top SNPs (or their surrogates) from discovery were advanced to a stage-2 evaluation in additional Henan subjects (2801 cases with FH and 3136 without FH, replication phase). A total of 19 SNPs were associated with FH of UGI cancer in ESCC cases with P < 10 −5 in the stage-1 meta-analysis of NCI and Henan GWAS data. In stage-2, the association for rs79747906 (located at 18p11.31, P = 5.79 × 10 −6 in discovery) was replicated ( P = 0.006), with a pooled-OR of 1.59 (95%CI: 1.11-2.28). We identified potential genetic variants associated with FH of UGI cancer. Our findings may provide important insights into new low-penetrance susceptibility regions involved in the susceptibility of families with multiple UGI cancer cases.

Objective Genome-wide association studies (GWAS) of gastric cancer have reported differences in single-nucleotide polymorphism (SNP) associations for tumour subtypes, particularly when divided by location into the gastric cardia versus the non-cardia. Design Here we present results for a GWAS using 2350 East Asian gastric cancer cases divided as 1189 gastric cardia and 1027 gastric non-cardia cases and 2708 controls. We also included up to 3042 cardia cases, 4359 non-cardia cases and 7548 controls for replication from two Chinese studies and one Korean study. From the GWAS, we selected 12 top SNPs for each gastric cancer subtype, 4 top SNPs for total gastric cancer and 1 SNP in MUC1 for replication testing. Results We observed genome-wide significant associations for rs10074991 in PRKAA1 at 5p13.1 for cardia (p=7.36×10−12) and non-cardia cancers (p=2.42×10−23) with per allele OR (95% CI) for the combined endpoint of 0.80 (0.77 to 0.83). At 6p21.1, rs2294693 near UNC5CL was significantly associated with gastric non-cardia cancer risk (p=2.50×10−8), with OR (95% CI) of 1.18 (1.12 to 1.26), but there was only a nominal association for cardia cancer (p=1.47×10−2). We also confirmed a previously reported association for rs4072037 in MUC1 with p=6.59×10−8 for total gastric cancer and similar estimates for cardia and non-cardia cancers. Three SNPs in PSCA previously reported to be associated with gastric non-cardia cancer showed no apparent association for cardia cancer. Conclusions Our results suggest that associations for SNPs with gastric cancer show some different results by tumour location in the stomach.

Abstract Gastric cancer and esophageal cancer are the second and sixth leading causes of cancer-related death worldwide. Multiple genomic alterations underlying gastric cancer and esophageal squamous cell carcinoma (ESCC) have been identified, but the full spectrum of genomic structural variations and mutations have yet to be uncovered. Here, we report the results of whole-genome sequencing of 30 samples comprising tumor and blood from 15 patients, four of whom presented with ESCC, seven with gastric cardia adenocarcinoma (GCA), and four with gastric noncardia adenocarcinoma. Analyses revealed that an A>C mutation was common in GCA, and in addition to the preferential nucleotide sequence of A located 5 prime to the mutation as noted in previous studies, we found enrichment of T in the 5 prime base. The A>C mutations in GCA suggested that oxidation of guanine may be a potential mechanism underlying cancer mutagenesis. Furthermore, we identified genes with mutations in gastric cancer and ESCC, including well-known cancer genes, TP53, JAK3, BRCA2, FGF2, FBXW7, MSH3, PTCH, NF1, ERBB2, and CHEK2, and potentially novel cancer-associated genes, KISS1R, AMH, MNX1, WNK2, and PRKRIR. Finally, we identified recurrent chromosome alterations in at least 30% of tumors in genes, including MACROD2, FHIT, and PARK2 that were often intragenic deletions. These structural alterations were validated using the The Cancer Genome Atlas dataset. Our studies provide new insights into understanding the genomic landscape, genome instability, and mutation profile underlying gastric cancer and ESCC development. Cancer Res; 76(7); 1714–23. ©2016 AACR.

To investigate large structural clonal mosaicism of chromosome X, we analysed the SNP microarray intensity data of 38,303 women from cancer genome-wide association studies (20,878 cases and 17,425 controls) and detected 124 mosaic X events >2 Mb in 97 (0.25%) women. Here we show rates for X-chromosome mosaicism are four times higher than mean autosomal rates; X mosaic events more often include the entire chromosome and participants with X events more likely harbour autosomal mosaic events. X mosaicism frequency increases with age (0.11% in 50-year olds; 0.45% in 75-year olds), as reported for Y and autosomes. Methylation array analyses of 33 women with X mosaicism indicate events preferentially involve the inactive X chromosome. Our results provide further evidence that the sex chromosomes undergo mosaic events more frequently than autosomes, which could have implications for understanding the underlying mechanisms of mosaic events and their possible contribution to risk for chronic diseases.

BACKGROUND Oesophageal cancer is the fourth leading cause of cancer death in China where essentially all cases are histologically oesophageal squamous cell carcinoma (ESCC). Agnostic pathway-based analyses of genome-wide association study (GWAS) data combined with tissue-specific expression quantitative trait loci (eQTL) analysis and publicly available functional data can identify biological pathways and/or genes enriched with functionally-relevant disease-associated variants. METHOD We used the adaptive multilocus joint test to analyse 1827 pathways containing 6060 genes using GWAS data from 1942 ESCC cases and 2111 controls with Chinese ancestry. We examined the function of risk alleles using in silico and eQTL analyses in oesophageal tissues. RESULTS Associations with ESCC risk were observed for 36 pathways predominantly involved in apoptosis, cell cycle regulation and DNA repair and containing known GWAS-associated genes. After excluding genes with previous GWAS signals, candidate pathways (and genes) for ESCC risk included taste transduction (KEGG_hsa04742; TAS2R13, TAS2R42, TAS2R14, TAS2R46,TAS2R50), long-patch base excision repair (Reactome_pid; POLD2) and the metabolics pathway (KEGG_hsa01100; MTAP, GAPDH, DCTD, POLD2, AMDHD1). We identified and validated CASP8 rs13016963 and IDH2 rs11630814 as eQTLs, and CASP8 rs3769823 and IDH2 rs4561444 as the potential functional variants in high-linkage disequilibrium with these single nucleotide polymorphisms (SNPs), respectively. Further, IDH2 mRNA levels were down-regulated in ESCC (tumour:normal-fold change = 0.69, P =  .75E-14). CONCLUSION Agnostic pathway-based analyses and integration of multiple types of functional data provide new evidence for the contribution of genes in taste transduction and metabolism to ESCC susceptibility, and for the functionality of both established and new ESCC risk-related SNPs.

Esophageal cancer is the eighth most common and sixth most frequent fatal human cancer in the world, and the fourth most common incident cancer in China. Shanxi Province, a region in north central China, has among the highest esophageal cancer rates in China, and nearly all of these cases are esophageal squamous cell carcinoma (ESCC). Understanding the molecular mechanisms underlying esophageal carcinogenesis and its molecular pathology will facilitate the development of biomarkers for early detection to reduce ESCC mortality. Genome-wide association studies (GWAS) have found many loci associated with the development of ESCC, however, most of the significant SNPs identified by GWAS are noncoding variants with unknown functions located in intergenic regions far from genes and are difficult to study functionally. As an initial effort to identify causal variants for ESCC, we focused here on loci near genes with known biological functions. Our analysis consists of three steps. We identified some functional variants for ESCC in the second and third steps of the analysis. First, from the GWAS analysis we selected those loci in or around the genes in three important pathways - inflammation, immunity, and DNA repair - using information from KEGG, Biocarta, and GO databases. We selected loci which had their 95% confidence intervals of the odds ratios not including OR = 1 based on GWAS analysis. Second, we integrated the expression and genotype data from ESCC somatic and germline samples and computed eQTLs using the normal tissues for n = 100 cases which had both gene expression data and GWAS genotype data. We found some interesting results from the second step analysis. The top 20 most significant genes included XPC, TP73, CASP8, DAPK1, IGF1R and LEPR, which were implicated in different cancers (Cancer Genetics Web: http://www.cancer-genetics.org). CASP8 was known to be involved in apoptosis processes. It was significantly associated with early onset of esophageal adenocarcinoma (Wu et al, Neoplasia. 2011 Apr;13(4):386-92). It had been shown that IGF1R had a key role in malignant progression of esophageal cancer (Kalinina et al, Int J Cancer. 2010 Oct 15;127(8):1931-40; Doyle et al, Am J Gastroenterol. 2012 Feb;107(2):196-204). Finally, we further intersected these significant loci in the eQTL analysis with the loci that had significant association between genotype and the somatic DNA copy number alterations. One of the significant genes was ST6GAL1. It had been reported this gene may promote malignant progression by promoting the TGF-beta induced EMT through down-regulation of E-cadherin-mediated cell adhesion and up-regulation of integrin-mediated cell migration (J. Lu et al, J Biol Chem. 2014 Dec 12;289(50):34627-41). We also found some novel genes in the analyses and their relation to ESCC will be investigated in future. Citation Format: Howard H. Yang, Hua Su, Huaitian Liu, Nan Hu, Chaoyu Wang, Lemin Wang, Ti Ding, Carol Giffen, Alisa M. Goldstein, Philip R. Taylor, Maxwell P. Lee. Integrated analysis of ESCC tumor/normal tissues and patient blood samples using expression, genotypes and DNA somatic change data. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2561.

Tumor:normal fold change for expression of 396 miRNAs in 30 ESCC cases. (XLS 283 kb)

Analyses of genome-wide association study (GWAS) data have revealed that detectable genetic mosaicism involving large (>2 Mb) structural autosomal alterations occurs in a fraction of individuals. We present results for a set of 24,849 genotyped individuals (total GWAS set II [TGSII]) in whom 341 large autosomal abnormalities were observed in 168 (0.68%) individuals. Merging data from the new TGSII set with data from two prior reports (the Gene-Environment Association Studies and the total GWAS set I) generated a large dataset of 127,179 individuals; we then conducted a meta-analysis to investigate the patterns of detectable autosomal mosaicism (n = 1,315 events in 925 [0.73%] individuals). Restricting to events >2 Mb in size, we observed an increase in event frequency as event size decreased. The combined results underscore that the rate of detectable mosaicism increases with age (p value = 5.5 × 10(-31)) and is higher in men (p value = 0.002) but lower in participants of African ancestry (p value = 0.003). In a subset of 47 individuals from whom serial samples were collected up to 6 years apart, complex changes were noted over time and showed an overall increase in the proportion of mosaic cells as age increased. Our large combined sample allowed for a unique ability to characterize detectable genetic mosaicism involving large structural events and strengthens the emerging evidence of non-random erosion of the genome in the aging population.