Genomics and epidemiology for gastric adenocarcinomas (GE4GAC): a Brazilian initiative to study gastric cancer

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.

The global distribution of gastric cancer (GC) is heterogeneous, a consequence of worldwide socio-economic disparities as well as of the human-diversity in terms of ancestry and cultural habits, including diet. This heterogeneity is not limited to global incidence but also embraces treatment response, clinical and pathological presentation, and the mutational landscape. This leads to frail translation of the still limited findings from US/Europe/Asia to other countries, including highly admixed and least-studied populations. In an attempt to reduce the GC knowledge-gap, the GE4GAC project, supported by FAPESP (14/26897-0), was implemented in 2016 at the A.C.Camargo Cancer Center (ACCC C) in São Paulo, Brazil. Collaborations are underway with the National Cancer Institute (NIH, USA); Instituto Nacional de Enfermedades Neoplásicas (Peru) and both the VOGAS (www.vogas.eu) and the StoP (www.stop-project. org) consortia. Here, we present our cohort/sample accrual (May 2019), results and aims.
Complete pathologic-response is seen in~12% of patients. Median follow-up so far was 41 months, and the median survival since neoadjuvant chemotherapy was 73 months. Intention-to-treat analysis showed a 5-year overall survival of 65%. Clinical research includes the identification of surgical prognostic factors [1,2] and the factors that impact outcome [3][4][5]. Patients are also invited to join the GE4GAC to investigate different aspects, such as the factors that contribute to GC development in Brazil, genes relevant for familial GC, predictors of neoadjuvant chemotherapy response and molecular variants that may indicate prognosis. Controls from the endoscopy-sector and the cancerprevention campaign also participate (no-cancer) [6].

Epidemiology
We investigate associations between GC and demographic characteristics, dietary habits, and lifestyle primarily in subjects living in three geographic areas of Brazil: i) São Pãulo-SP (Southeast), ii) Belém-PA (North) and iii) Fortaleza-CE (Northeast). A total of 1121 subjects (Feb-2016 to May-2019), aged 35-74 years were interviewed, including GC-cases (N = 412; C16/ICD-O3) and two control groups including subjects recruited during the cancer-prevention campaigns at ACCCC (N = 153), or from the Endoscopy Department (no-cancer; N = 556).

Basic and translational GC research
Collected samples (N = 2856) include whole-blood, gastricfluids, biopsies, saliva, and leukocytes, derived from > 400 cases+controls. These are used to investigate: i) Gastric Microbiota: We investigate possible links between microbiota and GC, including tumor subtypes/location and neoadjuvant treatment/ response. 16SrRNA sequencing, chosen due to elevated human:eubacteria ratios found in our samples, has been performed for~460 samples, indicating~50 bacteria present in biopsies and 2Xmore for gastric-fluids. Reduced microbiota richness/ diversity in cases was observed compared to controls. Shotgun-metagenomics and metatranscriptomics will be performed for selected samples, together with bacterial cultivation, to determine dead vs. alive bacteria. Virome and fungal populations will also be evaluated. qPCR EBV-analysis (N = 400) showed 12.7% EBV+ in GC-cases and 2.9% in controls. We recently showed a possible new mechanism of EBV-carcinogenesis where EBV-infection triggers APOBEC3 over-expression and an APOBECmutation signature [7]. ii) Whole exome sequencing (WES) and RNA-Seq: These will give comprehensive views of genomewide alterations pertinent to our population and clinical interests, including immune infiltrates. We aim to have WES for~400 patients and RNA-Seq mainly to support treatment-response studies. iii) Genomic ancestry: Using a set of~130,000 Ancestry Informative Markers [8] we found that the average cohort participant has a predominance of Mediterranean (50%), Northern-European (13.5%) and Southwest-Asia (12.5%) ancestries, followed by Sub-Saharan African (4.3%) and Native-American (3.8%) (unpublished). Genomic ancestry will be determined for all subjects and correlated against biological and demographic aspects, such as microbiota, diet, mutation profiles and treatment outcome. iv) Liquid-biopsies: We evaluated the presence of cell-free tumor-derived mutations in gastric washes (GW) collected during endoscopy, comparing mutation levels with biopsies and plasma. Deep sequencing showed enhanced detection when plasma and GW are evaluated together. GW has been shown to carry mutations not found in biopsies, suggesting tumor heterogeneity/cancerizationfield while providing a tool for monitoring treatmentresponse and disease-recurrence [9].
Other liquid-biopsies approaches investigated are Circulating Tumor Cells (CTCs) and Extracellular Vesicles (EVs). EVs-studies have been initiated in-vitro, as a means to study chemotherapy-resistance. Hundreds of patientderived samples have been collected for further studies, including EV-RNA-Seq [10][11][12] and proteomics [13]. The prognostic-value of CTCs in GC was studied in 88 samples, collected from 55 GC-patients (before/after neoadjuvantchemotherapy) [14]. A clinical trial of trastuzumab given to metastatic GC patients with HER2-negative tumor tissue but HER2 FISH-positive CTCs will start soon.