Whereas cancer patients have benefited from liquid biopsies, the scenario for gastric adenocarcinoma (GAC) is still dismal. We used next‐generation deep sequencing of TP53—a highly mutated and informative gene in GAC—to assess mutations in tumor biopsies, plasma (PL) and stomach fluids (gastric wash—GW). We evaluated their potential to reveal tumor‐derived mutations, useful for monitoring mutational dynamics at diagnosis, progression and treatment. Exon‐capture libraries were constructed from 46 patients including tumor biopsies, GW and PL pre and post‐treatment (196 samples), with high vertical coverage >8,000×. At diagnosis, we detected TP53 mutations in 15/46 biopsies (32.6%), 7/46 GW‐ (15.2%) and 6/46 PL‐samples (13%). Biopsies and GW were concordant in 38/46 cases (82.6%) for the presence/absence of mutations and, furthermore, four GW‐exclusive mutations were identified, suggesting tumor heterogeneity. Considering the combined analysis of GW and PL, TP53 mutations found in biopsies were also identified in 9/15 (60%) of cases, the highest detection level reported for GAC. Our study indicates that GW could be useful to track DNA alterations, especially if anchored to a comprehensive gene‐panel designed for this malignancy.

Gastric cancer (GC) is the fifth most common type of cancer worldwide with high incidences in Asia, Central, and South American countries. This patchy distribution means that GC studies are neglected by large research centers from developed countries. The need for further understanding of this complex disease, including the local importance of epidemiological factors and the rich ancestral admixture found in Brazil, stimulated the implementation of the GE4GAC project. GE4GAC aims to embrace epidemiological, clinical, molecular and microbiological data from Brazilian controls and patients with malignant and pre-malignant gastric disease. In this letter, we summarize the main goals of the project, including subject and sample accrual and current findings.

Whereas targeted and shotgun sequencing approaches are both powerful in allowing the study of tissue-associated microbiota, the human: microorganism abundance ratios in tissues of interest will ultimately determine the most suitable sequencing approach. In addition, it is possible that the knowledge of the relative abundance of bacteria and fungi during a treatment course or in pathological conditions can be relevant in many medical conditions. Here, we present a qPCR-targeted approach to determine the absolute and relative amounts of bacteria and fungi and demonstrate their relative DNA abundance in nine different human tissue types for a total of 87 samples. In these tissues, fungi genomes are more abundant in stool and skin samples but have much lower levels in other tissues. Bacteria genomes prevail in stool, skin, oral swabs, saliva, and gastric fluids. These findings were confirmed by shotgun sequencing for stool and gastric fluids. This approach may contribute to a more comprehensive view of the human microbiota in targeted studies for assessing the abundance levels of microorganisms during disease treatment/progression and to indicate the most informative methods for studying microbial composition (shotgun versus targeted sequencing) for various samples types.

Objective: To investigate the diagnostic and prognostic role of gastric fluid DNA (gfDNA) in gasric cancer (GC) patients and controls submitted to upper digestive endoscopy. Design: The concentration of gfDNA was evaluated in 941 samples, including subjects with normal gastric mucosa (n = 10), peptic diseases (n = 596), pre-neoplastic conditions (n = 99), and cancer (n = 236). gfDNA levels were evaluated according to age, gender, BMI, gastric fluids pH, use of proton-pump inhibitors, GC tumor subtypes, histological grades, clinical stages, and disease progression/outcome. Results: In the non-cancer group, we observed that gfDNA levels are increased in women as compared to men (p=7.44e-4). Remarkably, gfDNA levels are increased in GC patients as compared to non-GC (normal + peptic diseases, p=5.67e-13) and in GC versus pre-neoplastic disease (p=1.53e-6). Similar differences were also seen when more advanced tumors (T3) were compared to early stages (T2 and below) (p=5.97-4). Moreover, our results suggest the prognostic value of gfDNA as GC-patients with higher gfDNA concentrations (<1.28ng/microliter) had increased infiltration of immune cells in the tumor (p=1.06e-3), which parallels with better disease-free survival (p= 0.014). Conclusion: These findings highlight the significance of collecting and studying stomach fluids from gastric cancer patients and reveals the potential impact of this approach as well as its diagnostic and prognostic value for disease management.

Although numerous studies have shown the relevance of the gut microbiome in several diseases, underlying questions remain concerning the stomach microbiome and the establishment of a causal link between the microbiota and the development of gastric diseases, much beyond Helicobacter pylori and Epstein Barr virus. In this study, we aimed to characterize the bacterial composition of the stomach of subjects undergoing upper endoscopy, including gastric cancer (GC) individuals, aiming to identify fluctuations in bacterial populations that might be associated with stomach health. During endoscopic examination at A.C. Camargo Cancer Center (Sao Paulo, Brazil), gastric fluids (GF) were recovered from either GC patients (113) or individuals with gastric-related complaints, such as superficial gastritis (SG; 79), atrophic gastritis (AG; 12), and intestinal metaplasia (IM; 33). For eubacteria identification, the V3-V4 region of the 16S rRNA gene was amplified and paired-end sequenced (Illumina MiSeq). Analyses were carried out using Qiime2 and phyloseq packages. On average, we identified between 14 and 104 OTUs per subject, evidencing the potential of GFs for determining the stomach microbial composition and the interindividual variation. Testing of sample richness between GC and controls showed significant differences between the number of OTUs observed in each group (an average of 44 and 52 OTUs, respectively—Mann-Whitney test, p&lt;0.05) and SG patients had a significantly increased alpha diversity (Shannon, p&lt;0.05) as compared to AG, IM, and GC patients (both intestinal and diffuse subtypes), indicating dysbiosis already in early carcinogenesis steps. Additionally, the prolonged use of proton pump inhibitors or the presence of H. pylori (except for SG) did not seem to interfere with bacterial diversity. Specific genera are enriched in the sample subsets, including a lower presence of Corynebacterium and increased Streptococcus (Linear discriminant analysis Effect Size, LDA score &gt;2) in AG samples as compared to SG patients, which are also increased in the IM, and GC. These results indicate these bacteria to be potentially associated with the stomach dysbiosis that may lead to the carcinogenesis cascade. Besides the regular turnover of the gastric epithelial tissue and pouring of cells onto the gastric cavity, GF certainly contains a high proportion of transient oral-derived bacteria, while stomach-resident microbiota is expected to be in close contact with the gastric epithelia. Preliminary data analyzing the bacterial content of the saliva x GF in a subset of patients showed no significant beta-diversity differences, but both fluids differ significantly from their biopsies’ composition. We are currently performing culturomics and transcriptomics to identify bacteria that are alive in the stomach. As GC is a complex malignancy with limited treatment options, these results may contribute to developing new interventions to treat and to better understand this disease. Citation Format: Thais F. Bartelli, Gabriela E. Albuquerque, Alexandre Defelicibus, Marianna S. Serpa, Lais L.S. Abrantes, Rodrigo Borges, Felipe Coimbra, Adriane G. Pelosof, Israel T. Silva, Diana N. Nunes, Emmanuel Dias-Neto. Unravelling the gastric microbiome in health and disease: Gastric cancer beyond Helicobacter pylori in a Brazilian cohort [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B09.