Abstract 2086: Comprehensive microbial and immunological screening of gastroesophageal biopsies: A multi-parametric analysis to study carcinogenesis
Authors: Zhang Chao , Thakkar Prashant V. , Schnoll-Sussman Felice , McClure Bridget , Sonnenberg Greg , Betel Doron , Shah Manish A. 
Published: August 2018

The development of gastric cancer in the context of chronic H. pylori infection is multifactorial, encompassing both bacterial factors and the altered immune microenvironment. 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. Endoscopic biopsies 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 and 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. In our analysis population, in addition to the robust H. pylori signal, 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.

To characterize the immune infiltration in the mucosal biopsy samples, we define a 176-gene immune panel. The result of unsupervised clustering of gene expression 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) 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 (eg. greater the bacterial diversity, the less robust the pro-inflammatory immune signature).

In summary, this study established a methodology for microbiome and immune profiling of gastric biopsy samples. We have identified a pro-inflammatory 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 pro-inflammatory immune signature persisted, suggesting a potential long-term impact of H. pylori infection on mucosal immunity, that might contribute to gastric tumorigenesis.

Citation Format: Chao Zhang, Prashant V. Thakkar, Felice Schnoll-Sussman, Bridget McClure, Greg Sonnenberg, Doron Betel, Manish A. Shah. Comprehensive microbial and immunological screening of gastroesophageal biopsies: A multi-parametric analysis to study carcinogenesis [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 2086.