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BACKGROUND & AIMS: European guidelines recommend different surveillance intervals of non-dysplastic Barrett’s esophagus (NDBE) based on segment length, as opposed to guidelines in the United States, which do recommend surveillance intervals based on BE length. We studied rates of progression of NDBE to high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC) in patients with short-segment BE using the definition of BE in the latest guidelines (length ≥1 cm). METHODS: We collected demographic, clinical, endoscopy, and histopathology data from 1883 patients with endoscopic evidence of NDBE (mean age, 57.3 years; 83.5% male; 88.1% Caucasians) seen at 7 tertiary referral centers. Patients were followed for a median 6.4 years. Cases of dysplasia or EAC detected within 1 year of index endoscopy were considered prevalent and were excluded. Unadjusted rates of progression to HGD or EAC were compared between patients with short (≥1 and

Summary Background and Aims: As obtaining adequate tissue on biopsy is critical for the detection of residual and recurrent intestinal metaplasia/dysplasia in Barrett’s esophagus (BE) patients undergone Barrett’s endoscopic eradication therapy (BET), we decided to compare the adequacy of biopsy specimens using jumbo versus standard biopsy forceps. Methods: This is a two-center study of patients’ post-radiofrequency ablation of dysplastic BE. After BET, jumbo (Boston Scientific©, Radial Jaw 4, opening diameter 2.8 mm) or standard (Boston Scientific©, Radial Jaw 4, opening diameter 2.2 mm) biopsy forceps were utilized to obtain surveillance biopsies from the neo-squamous epithelium. Presence of lamina propria and proportion of squamous epithelium with partial or full thickness lamina propria was recorded by two experienced gastrointestinal pathologists who were blinded. Squamous epithelial biopsies that contained at least two-thirds of lamina propria were considered ‘adequate’. Results: In a total of 211 biopsies from 55 BE patients, 145 biopsies (29 patients, 18 males, mean age 61 years, interquartile range [IQR] 33–83) were obtained using jumbo forceps and 66 biopsies (26 patients, all males, mean age 65 years, IQR 56–76) using standard forceps biopsies. Comparing jumbo versus standard forceps, the proportion of specimens with any subepithelial lamina propria was 51.7% versus 53%, P = 0.860 and the presence of adequate subepithelial lamina propria was 17.9% versus 9.1%, P = 0.096 respectively. Conclusions: Use of jumbo forceps does not appear to have added advantage over standard forceps to obtain adequate biopsy specimens from the neo-squamous mucosa post-ablation.

AIM To evaluate annual incidence of low grade dysplasia (LGD) progression to high grade dysplasia (HGD) and/or esophageal adenocarcinoma (EAC) when diagnosis was made by two or more expert pathologists. METHODS Studies evaluating the progression of LGD to HGD or EAC were included. The diagnosis of LGD must be made by consensus of two or more expert gastrointestinal pathologists. Articles were searched in Medline, Pubmed, and Embase. Pooled proportions were calculated using fixed and random effects model. Heterogeneity among studies was assessed using the I2 statistic. RESULTS Initial search identified 721 reference articles, of which 53 were selected and reviewed. Twelve studies (n = 971) that met the inclusion criteria were included in this analysis. Among the total original LGD diagnoses in the included studies, only 37.49% reached the consensus LGD diagnosis after review by two or more expert pathologists. Total follow up period was 1532 patient-years. In the pooled consensus LGD patients, the annual incidence rate (AIR) of progression to HGD and or EAC was 10.35% (95%CI: 7.56-13.13) and progression to EAC was 5.18% (95%CI: 3.43-6.92). Among the patients down staged from original LGD diagnosis to No-dysplasia Barrett’s esophagus, the AIR of progression to HGD and EAC was 0.65% (95%CI: 0.49-0.80). Among the patients down staged to Indefinite for dysplasia, the AIR of progression to HGD and EAC was 1.42% (95%CI: 1.19-1.65). In patients with consensus HGD diagnosis, the AIR of progression to EAC was 28.63% (95%CI: 13.98-43.27). CONCLUSION When LGD is diagnosed by consensus agreement of two or more expert pathologists, its progression towards malignancy seems to be at least three times the current estimates, however it could be up to 20 times the current estimates. Biopsies of all Barrett’s esophagus patients with LGD should be reviewed by two expert gastroenterology pathologists. Follow-up strict surveillance programs should be in place for these patients.

Identification of the human microbiome has proven to be of utmost importance with the emerging role of bacteria in various physiological and pathological processes. High throughput sequencing strategies have evolved to assess the composition of the microbiome. To identify possible bias that may exist in the processing of tissue for whole genome sequencing (WGS), it is important to evaluate the extraction method on the overall microbial content and composition. Here we compare two different methods of extraction, homogenization vs. enzymatic lysis, on gastric, esophageal and colorectal biopsies and survey the microbial content and composition using WGS and quantitative PCR (qPCR). We examined total bacterial content using universal 16S rDNA qPCR as well as the abundance of three phyla (Actinobacter, Firmicutes, Bacteroidetes) and one genus (Fusobacterium). We found minimal differences between the two extraction methods in the overall community structure. Furthermore, based on our qPCR analysis, neither method demonstrated preferential extraction of any particular clade of bacteria, nor significantly altered the detection of Gram-positive or Gram-negative organisms. However, although the overall microbial composition remained very similar and the most prevalent bacteria could be detected effectively using either method, the precise community structure and microbial abundances between the two methods were different, primarily due to variations in detection of low abundance genus. We also demonstrate that the homogenization extraction method provides higher microbial DNA content and higher read counts from human tissue biopsy samples of the gastrointestinal tract.

Many patients with a1 cm segment of columnar metaplasia in the distal esophagus, also called an irregular Z line, are encountered. These patients, often referred to as patients with Barrett's esophagus (BE), are enrolled in surveillance programs. However, little is known about their risk of high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC). We aimed to determine the incidence of HGD and EAC in patients with irregular Z line with intestinal metaplasia.We performed a prospective, multicenter cohort study of patients who underwent endoscopic examination for BE at tertiary care referral centers in the United States and Europe. We analyzed data from 1791 patients (mean age, 56 ± 17 years) found to have non-dysplastic BE at the index endoscopy and after 1 year or more of follow-up. Patients were followed for a median of 5.9 years (interquartile range, 3.1-8.3 years). We calculated rates of progression to HGD or EAC between groups of patients with irregular Z line (n = 167) and those with BE of ≥ 1 cm (n = 1624).A higher proportion of patients in the irregular Z-line group were female (26.3%) than in the BE group (14.8% female BE) (P.001). A lower proportion of patients in the irregular Z-line group were smokers (33.5%) than in the BE group (52.6% smokers). None of the patients with irregular Z line developed HGD or EAC during a median follow-up period of 4.8 years (interquartile range, 3.2-8.3 years). All 71 incident cases of HGD or EAC developed in patients with BE of ≥1 cm in length. On multivariate analysis, patients with irregular Z line and patients with BE of ≥ 1 cm did not differ significantly in age, race, or duration of follow-up.In a prospective, multicenter cohort study, we found that patients with irregular Z line do not develop HGD or esophageal cancer within 5 years after index endoscopy.

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.