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Although Barrett’s esophagus is the primary precursor of esophageal adenocarcinoma, the annual incidence of adenocarcinoma is 0.12% in a Barrett’s esophagus population. We hypothesized that the esophageal microbiome may be associated with mucosal immunity changes that may determine subsequent carcinogenesis. To better understand the association among the local microbiota composition, immune response and progression of esophageal cancer, we performed whole genome sequencing (WGS) and bulk RNASeq in parallel, from endoscopic biopsies (n=23) collected from patients with normal squamous cell (n=9), non-dysplastic Barrett’s esophagus (n=8) and esophageal adenocarcinoma (n = 6). Using a novel customized computational pipeline to identify and characterize bacteria from low microbial content endoscopic samples (Zhang et al, Genome Biology, 2015). WGS analysis revealed a higher microbial diversity in normal squamous cell carcinomas compared to the non-dysplastic Barrett’s esophagus with high abundance of Veilonella Parvula, Prevotella melaninogenica and Streptococcus parasanguinis. Unsupervised clustering analysis of the entire data set revealed high abundance of Fusobacterium nucleatum in adenocarcinoma samples, which has been associated with a shorter survival in esophageal cancer. To characterize the immune infiltration in the mucosal biopsy samples, we defined a 784-gene immune panel. Unsupervised clustering analysis of gene expression revealed a much higher immune infiltration in most adenocarcinoma and some non-dysplastic Barrett’s compared with normal esophageal squamous tissue samples. Especially CD4+ Th1 and Th2 helper cell as well as CD8+ T cell associated immune response are highly enriched in some adenocarcinoma and non-dysplastic Barrett’s esophageal samples. To further investigate the effect of immune tumor microenvironment on efficacy of immunotherapy, we performed single cell sequencing on matching normal and tumor tissues collected at baseline and post-treatment from 6 patients subjected to anti-PD1 therapy. Using this approach, we can identify both innate and adaptive immune cells in both pre and post treatment biopsies. We specifically observe upregulation of MHCII associated cells in post-treatment biopsies. Together, our data suggest that esophageal cancers display distinct microbial patterns associated with chronic inflammation and a tumor-promoting pro-inflammatory microenvironment. Note: This abstract was not presented at the meeting. Citation Format: Chao Zhang, Prashant V. Thakkar, Prateek Sharma, Sreekar Vennelaganti, Doron Betel, Manish A. Shah. Understanding associations among local microbiome, immune response, and efficacy of immunotherapy in esophageal 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 2826.

Gastric and Esophageal cancers remain one of the leading causes of cancer related deaths. Dysbiosis of gut microbiome is a strong risk factor for gastrointestinal cancer, likely due to altered mucosal immunity and pro-inflammatory immune microenvironment. However, molecular interactions between host human cells and microbiome are poorly understood in context of gastric and esophageal cancer. Our main goal is to understand how altered mucosal microbiome alters tumor microenvironment, thus creating conditions conducive to tumorigenesis. Using a novel computational pipeline combined with low-pass (10-30x) whole genome sequencing (WGS), we examined microbiome composition of 15 gastric cancer (GC) and adjacent normal tissue (n=30). We found a higher overall bacterial content in tumor samples compared to that of matching normal tissues. Furthermore, we observed dysbiosis in H. pylori positive samples, consistent with previous findings. Among other microbes, we found specific enrichment of V. Parvula (12/15) and P. melaninogenica (10/15) in tumor tissues, both of which have been implicated in tumorigenicity. A subset of these samples (10 GC and 12 normal) underwent parallel RNAseq analysis. Using gene expression signature panel of 176 genes, we examined association of various immune cell types with tumor versus normal mucosa. We found an overall higher immune response, and higher association of Th1 and Th2 helper cells and macrophages, with tumors as compared to normal mucosa. We further corroborated these findings using single cell RNAseq approach. Using ELISA, we detected significantly increased expression of pro-inflammatory cytokines such as TNF-α, IL-8, GRO, MCP-1 and IL-1a in tumor samples than normal mucosa. Increased TNF-α and IL-8 expression has been directly correlated to tumor invasion and metastasis, and tumor vascularity respectively. Additionally, MCP-1 is known to recruit macrophages and is secreted by gastric epithelial cells in response to pro-inflammatory cytokines upon H. pylori infection. Using a similar approach, we performed WGS and RNAseq, on 9 normal squamous cell carcinoma (NSCC), 8 non-dysplastic Barrett's esophagus (BE) and 6 esophageal adenocarcinoma samples. WGS analysis revealed higher microbial diversity in NSCCs compared to BE samples with high abundance of V. Parvula, P. melaninogenica and S. parasanguinis. Unsupervised clustering analysis revealed high abundance of F. nucleatum in adenocarcinoma, which has been associated with shorter survival in esophageal cancer. Unsupervised clustering analysis of RNAseq data revealed high enrichment of CD4+ Th1 and Th2 helper cell and CD8+ T cell associated immune response in some adenocarcinoma and BE samples. Together our data suggest that both gastric and esophageal cancers display distinct microbial patterns associated with chronic inflammation and tumor-promoting pro-inflammatory microenvironment. Citation Format: Prashant V. Thakkar, Chao Zhang, Prateek Sharma, Sreekar Vennelaganti, Doron Betel, Manish A. Shah. Understanding the association of gut microbiota and tumor microenvironment in gastric and esophageal cancer [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 5128.