Abstract A04: Microbial and immunologic characterization of gastroesophageal tissue biopsy samples: A multiparametric analysis
Authors: Zhang Chao , Thakkar Prashant Vijay , Schnoll-Sussman Felice , McClure Bridget , Bigg Michelle , Sonnenberg Greg , Betel Doron , Shah Manish 
Published: February 2019

Abstract
Gastric cancer carcinogenesis is associated with chronic inflammation, most commonly the result of Helicobacter pylori chronic infection in the stomach antrum. The development of gastric cancer in the context of chronic H. pylori infection is multifactorial, encompassing both bacterial factors and the altered immune microenvironment. However, a comprehensive analysis of the relation between inflammation and host microbial population in patient tissue samples has not previously been explored. We proposed an unbiased study to evaluate the relationships among microbiome composition, host immune response and genomic characterization from next-generation sequencing of gastric biopsy samples.
Patients undergoing upper endoscopy without chronic inflammatory disease or chronic NSAID use were eligible for participation. Endoscopic biopsies from gastric fundus, body, and antrum were collected from patients with active H. pylori infection (n=21), prior infection (n=22) and no prior infection (n=26), and were sequenced at 10X to 30X coverage. In total, 77 gastric biopsies from 69 patients were freshly frozen for whole genome sequencing (WGS) and transcriptome (RNASeq) analysis.
Detecting the microbiome from human biopsy sequencing data directly is challenging due to the low microbial content. A novel computational pipeline was developed to address this problem specifically (Zhang et al., Genome Biology 2015). A robust H. pylori signal was detected in samples from clinically verified H. pylori infected patients, and the results were further validated by qPCR. In our analysis population, in addition to identification of H. pylori, several bacteria associated with other cancers were also detected in several biopsy samples, such as Prevotella melaninogenica, Veillonella parvula and Fusobacterium nucleatum. H. pylori infection was associated with reduced microbial biodiversity compared to prior infection or control tissue (p=0.02). H. pylori active infection samples have a distinct non-H. pylori microbiome compared to prior infection and control samples. We also identified 5 patients with prior infection and 1 control patient with occult H. pylori infection (e.g., asymptomatic patients).
To characterize the immune infiltration in the mucosal biopsy samples, we developed a 176-gene panel, collected from multiple published studies, to define the immune signatures. The expression profile of this immune gene panel was used evaluate the immune infiltration levels of multiple immune cell types. The result of unsupervised clustering revealed a much higher immune infiltration in H. pylori positive samples compared to uninfected samples, especially for CD8+, Th2 and Th17 cell populations. Two orthogonal experimental essays (ELISA and Flow Cytometry) were performed independently to verify the results. ELISA results confirmed the RNAseq-based expression profiling of inflammatory cytokines such as GRO, IL8, TNFa and SCD40L. Importantly, in 2 patients with prior H. pylori infection, the pro-inflammatory immune signature characteristic of H. pylori persisted. Finally, amongst H. pylori active infection patients, the biodiversity of the other bacteria present was inversely correlated with local immune infiltration (e.g., greater the bacterial diversity, the less robust the proinflammatory immune signature).
In summary, this study established a methodology for microbiome and immune profiling of gastric biopsy samples. We have identified a proinflammatory immune response signature associated with H. pylori infection, confirmed and validated by orthogonal assays. Notably, in several samples from previously eradicated H. pylori infection, the proinflammatory immune signature persisted, suggesting a potential long-term impact of H. pylori infection on mucosal immunity, that might contribute to gastric tumorigenesis. Finally, we demonstrate that the biodiversity of the bacterial species correlates with the character of immune infiltration.
Citation Format: Chao Zhang, Prashant Vijay Thakkar, Felice Schnoll-Sussman, Bridget McClure, Michelle Bigg, Greg Sonnenberg, Doron Betel, Manish Shah. Microbial and immunologic characterization of gastroesophageal tissue biopsy samples: A multiparametric analysis [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A04.