Exome and Tissue-Associated Microbiota as Predictive Markers of Response to Neoadjuvant Treatment in Locally Advanced Rectal Cancer

The clinical and pathological responses to multimodal neoadjuvant therapy in locally advanced rectal cancers (LARCs) remain unpredictable, and robust biomarkers are still lacking. Recent studies have shown that tumors present somatic molecular alterations related to better treatment response, and it is also clear that tumor-associated bacteria are modulators of chemotherapy and immunotherapy efficacy, therefore having implications for long-term survivorship and a good potential as the biomarkers of outcome. Here, we performed whole exome sequencing and 16S ribosomal RNA (rRNA) amplicon sequencing from 44 pre-treatment LARC biopsies from Argentinian and Brazilian patients, treated with neoadjuvant chemoradiotherapy or total neoadjuvant treatment, searching for predictive biomarkers of response (responders, n = 17; non-responders, n = 27). In general, the somatic landscape of LARC was not capable to predict a response; however, a significant enrichment in mutational signature SBS5 was observed in non-responders (p = 0.0021), as well as the co-occurrence of APC and FAT4 mutations (p < 0.05). Microbiota studies revealed a similar alpha and beta diversity of bacteria between response groups. Yet, the linear discriminant analysis (LDA) of effect size indicated an enrichment of Hungatella, Flavonifractor, and Methanosphaera (LDA score ≥3) in the pre-treatment biopsies of responders, while non-responders had a higher abundance of Enhydrobacter, Paraprevotella (LDA score ≥3) and Finegoldia (LDA score ≥4). Altogether, the evaluation of these biomarkers in pre-treatment biopsies could eventually predict a neoadjuvant treatment response, while in post-treatment samples, it could help in guiding non-operative treatment strategies.Fil: Takenaka, Isabella Kuniko T. M.. No especifíca;Fil: Bartelli, Thais F.. No especifíca;Fil: Defelicibus, Alexandre. No especifíca;Fil: Sendoya, Juan Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Golubicki, Mariano. Gobierno de la Ciudad de Buenos Aires. Hospital de Gastroenterología "Dr. Carlos B. Udaondo"; ArgentinaFil: Robbio, Juan. No especifíca;Fil: Serpa, Marianna S.. No especifíca;Fil: Branco, Gabriela P.. No especifíca;Fil: Santos, Luana B. C.. No especifíca;Fil: Claro, Laura C. L.. No especifíca;Fil: Oliveira dos Santos, Gabriel. No especifíca;Fil: Kupper, Bruna E. C.. No especifíca;Fil: da Silva, Israel T.. No especifíca;Fil: Llera, Andrea Sabina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: de Mello, Celso A. L.. No especifíca;Fil: Riechelmann, Rachel P.. No especifíca;Fil: Dias Neto, Emmanuel. Universidade de Sao Paulo; BrasilFil: Iseas, Soledad. Gobierno de la Ciudad de Buenos Aires. Hospital de Gastroenterología "Dr. Carlos B. Udaondo"; ArgentinaFil: Aguiar, Samuel. No especifíca;Fil: Nunes, Diana Noronha. No especifíca

Evaluation of Bacteria and Fungi DNA Abundance in Human Tissues

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

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

Abstract 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 Helicobacter pylori Genome Project : insights into H. pylori population structure from analysis of a worldwide collection of complete genomes

Helicobacter pylori, a dominant member of the gastric microbiota, shares co-evolutionary history with humans. This has led to the development of genetically distinct H. pylori subpopulations associated with the geographic origin of the host and with differential gastric disease risk. Here, we provide insights into H. pylori population structure as a part of the Helicobacter pylori Genome Project (HpGP), a multi-disciplinary initiative aimed at elucidating H. pylori pathogenesis and identifying new therapeutic targets. We collected 1011 well-characterized clinical strains from 50 countries and generated high-quality genome sequences. We analysed core genome diversity and population structure of the HpGP dataset and 255 worldwide reference genomes to outline the ancestral contribution to Eurasian, African, and American populations. We found evidence of substantial contribution of population hpNorthAsia and subpopulation hspUral in Northern European H. pylori. The genomes of H. pylori isolated from northern and southern Indigenous Americans differed in that bacteria isolated in northern Indigenous communities were more similar to North Asian H. pylori while the southern had higher relatedness to hpEastAsia. Notably, we also found a highly clonal yet geographically dispersed North American subpopulation, which is negative for the cag pathogenicity island, and present in 7% of sequenced US genomes. We expect the HpGP dataset and the corresponding strains to become a major asset for H. pylori genomics

Evidence for Mitochondrial Genome Methylation in the Yeast Candida albicans: A Potential Novel Epigenetic Mechanism Affecting Adaptation and Pathogenicity?

The commensal yeast Candida albicans is an opportunistic pathogen. In order to successfully colonize or infect the human body, the fungus must adapt to the host’s environmental conditions, such as low oxygen tension (hypoxia), temperature (37°C), and the different carbon sources available. Previous studies demonstrated the adaptive importance of C. albicans genetic variability for its pathogenicity, although the contributions of epigenetic and the influence of environmental factors are not fully understood. Mitochondria play important roles in fungal energetic metabolism, regulation of nuclear epigenetic mechanisms and pathogenicity. However, the specific impact of inter-strain mitochondrial genome variability and mitochondrial epigenetics in pathogenicity is unclear. Here, we draw attention to this relevant organelle and its potential role in C. albicans pathogenicity and provide preliminary evidence, for the first time, for methylation of the yeast mitochondrial genome. Our results indicate that environmental conditions, such as continuous exposure for 12 weeks to hypoxia and 37°C, decrease the mitochondrial genome methylation in strains SC5314 and L757. However, the methylation decrease is quantitatively different in specific genome positions when strains SC5314 and L757 are compared. We hypothesize that this phenomenon can be promising for future research to understand how physical factors of the host affect the C. albicans mitochondrial genome and its possible impact on adaptation and pathogenicity

Evidence for Mitochondrial Genome Methylation in the Yeast Candida albicans: A Potential Novel Epigenetic Mechanism Affecting Adaptation and Pathogenicity?

The commensal yeast Candida albicans is an opportunistic pathogen. In order to successfully colonize or infect the human body, the fungus must adapt to the host's environmental conditions, such as low oxygen tension (hypoxia), temperature (37 degrees C), and the different carbon sources available. Previous studies demonstrated the adaptive importance of C. albicans genetic variability for its pathogenicity, although the contributions of epigenetic and the influence of environmental factors are not fully understood. Mitochondria play important roles in fungal energetic metabolism, regulation of nuclear epigenetic mechanisms and pathogenicity. However, the specific impact of inter-strain mitochondrial genome variability and mitochondrial epigenetics in pathogenicity is unclear. Here, we draw attention to this relevant organelle and its potential role in C. albicans pathogenicity and provide preliminary evidence, for the first time, for methylation of the yeast mitochondrial genome. Our results indicate that environmental conditions, such as continuous exposure for 12 weeks to hypoxia and 37 degrees C, decrease the mitochondrial genome methylation in strains SC5314 and L757. However, the methylation decrease is quantitatively different in specific genome positions when strains SC5314 and L757 are compared. We hypothesize that this phenomenon can be promising for future research to understand how physical factors of the host affect the C. albicans mitochondrial genome and its possible impact on adaptation and pathogenicity.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), BrazilConselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), BrazilUniv Fed Sao Paulo, Lab Evolutionary Genom & Biocomplex, Dept Microbiol Immunol & Parasitol, Sao Paulo, BrazilAC Camargo Canc Ctr, Lab Med Genom, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Hlth Informat, Sao Paulo, BrazilUniv Fed Sao Paulo, Lab Evolutionary Genom & Biocomplex, Dept Microbiol Immunol & Parasitol, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Hlth Informat, Sao Paulo, BrazilFAPESP: 2013/08457-0FAPESP: 2013/07838-0CNPq: 303905/2013-1Web of Scienc

Table_2_Bacterial and fungal characterization of pancreatic adenocarcinoma from Endoscopic Ultrasound-guided biopsies.docx

IntroductionThe tumor microbiome (TM) has been linked to pancreatic cancer prognosis. Specific microbes can confer tumor resistance to therapies. Early knowledge of the TM at time of diagnosis would be clinically relevant for precision therapy based on microbial composition. However, it is difficult to define the TM prior to surgical resection.MethodsIn this pilot feasibility study, patients underwent Endoscopic Ultrasound-Fine Needle Aspiration (EUS-FNA) biopsy of pancreatic adenocarcinoma. These samples were analyzed using 16S rRNA and internal transcribed spacer (ITS) sequencing for characterization of the tumor bacteria and fungi.ResultAfter in silico decontamination and comparison to non-matched tumor, we were able to characterize the TM in biopsies, which was comparable to the TM from surgical specimens.DiscussionEUS-FNA biopsy may represent a feasible modality to characterize the pancreatic TM prior to surgical resection with proper decontamination strategies and improvements in matched controls.</p

Table_1_Bacterial and fungal characterization of pancreatic adenocarcinoma from Endoscopic Ultrasound-guided biopsies.docx

IntroductionThe tumor microbiome (TM) has been linked to pancreatic cancer prognosis. Specific microbes can confer tumor resistance to therapies. Early knowledge of the TM at time of diagnosis would be clinically relevant for precision therapy based on microbial composition. However, it is difficult to define the TM prior to surgical resection.MethodsIn this pilot feasibility study, patients underwent Endoscopic Ultrasound-Fine Needle Aspiration (EUS-FNA) biopsy of pancreatic adenocarcinoma. These samples were analyzed using 16S rRNA and internal transcribed spacer (ITS) sequencing for characterization of the tumor bacteria and fungi.ResultAfter in silico decontamination and comparison to non-matched tumor, we were able to characterize the TM in biopsies, which was comparable to the TM from surgical specimens.DiscussionEUS-FNA biopsy may represent a feasible modality to characterize the pancreatic TM prior to surgical resection with proper decontamination strategies and improvements in matched controls.</p

Evaluation of Bacteria and Fungi DNA Abundance in Human Tissues

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