Investigating the role of the intestinal microbiota in colorectal cancer development : epidemiological approaches

Le microbiome intestinal désigne l'ensemble des micro-organismes présents dans le tractus gastro-intestinal. Une symbiose dynamique entre l'hôte et le microbiome existe dans l'intestin et des interactions spécifiques entre le microbiome et le métabolisme de l'hôte, et son système immunitaire, sont essentielles pour façonner la physiologie de l'hôte. L'hypothèse que la perturbation de cette symbiose joue un rôle dans le développement de diverses maladies chroniques, y compris du cancer colorectal, a été émise. En effet, il existe de plus en plus de preuves expérimentales selon lesquelles le microbiome intestinal influence potentiellement le développement de tumeurs par le biais d'une dérégulation du métabolisme de l'hôte et de sa fonction immunitaire. Cependant, les données épidémiologiques reliant le microbiome intestinal à la carcinogenèse colorectale restent limitées car très peu de cohortes existantes ont collecté des échantillons fécaux. Les programmes de dépistage du cancer colorectal, dans lesquels des millions d'échantillons de selles sont prélevés chaque année, pourraient fournir de riches opportunités pour établir des cohortes basées sur la population avec des échantillons répétés et prospectivement collectés. La première partie de la thèse résume de manière systématique la littérature épidémiologique actuelle qui a été publiée au cours de la dernière décennie sur l'association entre le microbiome et le cancer. Nos résultats ont souligné que pour la plupart des indicateurs du microbiome, les preuves étaient encore trop faibles pour tirer des conclusions définitives concernant leur rôle dans le cancer. La deuxième partie de la thèse étudie l'association entre la résistance à l'insuline et l'inflammation - des facteurs de risque reconnus du cancer colorectal - et le microbiome intestinal, dans deux cohortes populationnelles : The Northern Finland Birth Cohort et TwinsUK. Notre étude a indiqué que des niveaux plus élevés de résistance à l'insuline et d'autres marqueurs de dysfonctionnement métabolique étaient associés à une diversité amoindrie du microbiome dans les deux cohortes, même après contrôle de l'obésité et d'autres facteurs. Enfin, la troisième partie de la thèse évalue la stabilité et la concordance du microbiome dans les échantillons fécaux prélevés à l'aide de différentes méthodes utilisées dans les programmes de dépistage du cancer colorectal en cours. Nos résultats suggèrent que les collections d'échantillons fécaux couramment utilisées, telles que les tests immunochimiques fécaux et les cartes de prélèvement sur papier, sont en général des supports appropriés pour mesurer la diversité microbiome, bien que des facteurs de stockage, tels que la température ambiante, puissent avoir un impact sur la stabilité de certaines méthodes. En outre, la collecte opportuniste d'échantillons fécaux dans des tubes à tests immunochimiques fécaux après le dépistage du cancer colorectal est probablement une méthode viable pour établir des cohortes avec des échantillons fécaux pré-diagnostics. Dans l'ensemble, cette thèse présente l'état de l'art sur les preuves épidémiologiques du rôle du microbiome dans la tumorigenèse. Elle fournit de nouvelles informations sur l'association des facteurs de risque métaboliques du cancer colorectal avec le microbiome intestinal dans les études populationnelles. Enfin, elle a généré d'importantes données méthodologiques sur l'impact des outils de collecte d'échantillons fécaux sur les mesures microbiennes nécessaires pour les futures recherches épidémiologiques sur le microbiome et le cancer colorectal, ainsi que sur d'autres maladies chroniquesThe gut microbiome is the ensemble of microorganisms that inhabit the gastrointestinal tract. A dynamic host-microbiome symbiosis exists in the intestine and specific interactions between the microbiota and host metabolism and immune system are critical for shaping host physiology. Disturbance of this symbiosis has been hypothesized to play a role in the development of various chronic diseases, including colorectal cancer. Indeed, there is growing experimental evidence that the gut microbiome potentially influences tumor development through dysregulation of host metabolism and immune function. However, epidemiological data linking the gut microbiome with colorectal carcinogenesis remains limited as very few existing cohorts have collected fecal samples. Colorectal cancer screening programs, in which millions of stool samples are collected each year, might provide rich opportunities to establish population-based cohorts with repeated, prospectively collected samples. The first part of the thesis systematically summarizes the current epidemiological literature that has been published in the past decade on the association of the human microbiome with cancer. Our findings emphasised that for most microbiome indicators, the evidence was still too weak to draw firm conclusions in relation to their role in cancer. The second part of the thesis investigates the association between insulin resistance and inflammation - recognised colorectal cancer risk factors - and the gut microbiome in two population-based cohorts – the Northern Finland Birth Cohort and TwinsUK. Our study indicated that higher levels of insulin resistance and other markers of metabolic dysfunction were associated with lower microbiome diversity in both cohorts, even after control for obesity and other factors. Finally, the third part of the thesis evaluates microbiome stability and accuracy in fecal samples collected using different methods employed in ongoing colorectal cancer screening programs. Our findings suggest that commonly used fecal sample collections such as fecal immunotests and paper-based collection cards are, in general, suitable media for microbiome measurements though storage factors such as ambient temperature can impact on stability for some methods. In addition, the opportunistic collection of fecal samples in fecal immunochemical test tubes after colorectal cancer screening is likely a viable method for establishing cohorts with prediagnostic fecal specimens. Overall, this thesis presents the state-of-the-art on epidemiological evidence for the role of the microbiome in tumorigenesis, provides novel insights on the association of metabolic risk factors for colorectal cancer with the gut microbiome in population-based studies, and finally has generated important methodological data on the impact of fecal sample collection tools on microbial measurements that is needed for future epidemiological research on the microbiome and colorectal cancer, as well as other chronic disease

Étude du rôle du microbiote intestinal dans la carcinogenèse colorectale : approches épidémiologiques

The gut microbiome is the ensemble of microorganisms that inhabit the gastrointestinal tract. A dynamic host-microbiome symbiosis exists in the intestine and specific interactions between the microbiota and host metabolism and immune system are critical for shaping host physiology. Disturbance of this symbiosis has been hypothesized to play a role in the development of various chronic diseases, including colorectal cancer. Indeed, there is growing experimental evidence that the gut microbiome potentially influences tumor development through dysregulation of host metabolism and immune function. However, epidemiological data linking the gut microbiome with colorectal carcinogenesis remains limited as very few existing cohorts have collected fecal samples. Colorectal cancer screening programs, in which millions of stool samples are collected each year, might provide rich opportunities to establish population-based cohorts with repeated, prospectively collected samples. The first part of the thesis systematically summarizes the current epidemiological literature that has been published in the past decade on the association of the human microbiome with cancer. Our findings emphasised that for most microbiome indicators, the evidence was still too weak to draw firm conclusions in relation to their role in cancer. The second part of the thesis investigates the association between insulin resistance and inflammation - recognised colorectal cancer risk factors - and the gut microbiome in two population-based cohorts – the Northern Finland Birth Cohort and TwinsUK. Our study indicated that higher levels of insulin resistance and other markers of metabolic dysfunction were associated with lower microbiome diversity in both cohorts, even after control for obesity and other factors. Finally, the third part of the thesis evaluates microbiome stability and accuracy in fecal samples collected using different methods employed in ongoing colorectal cancer screening programs. Our findings suggest that commonly used fecal sample collections such as fecal immunotests and paper-based collection cards are, in general, suitable media for microbiome measurements though storage factors such as ambient temperature can impact on stability for some methods. In addition, the opportunistic collection of fecal samples in fecal immunochemical test tubes after colorectal cancer screening is likely a viable method for establishing cohorts with prediagnostic fecal specimens. Overall, this thesis presents the state-of-the-art on epidemiological evidence for the role of the microbiome in tumorigenesis, provides novel insights on the association of metabolic risk factors for colorectal cancer with the gut microbiome in population-based studies, and finally has generated important methodological data on the impact of fecal sample collection tools on microbial measurements that is needed for future epidemiological research on the microbiome and colorectal cancer, as well as other chronic diseasesLe microbiome intestinal désigne l'ensemble des micro-organismes présents dans le tractus gastro-intestinal. Une symbiose dynamique entre l'hôte et le microbiome existe dans l'intestin et des interactions spécifiques entre le microbiome et le métabolisme de l'hôte, et son système immunitaire, sont essentielles pour façonner la physiologie de l'hôte. L'hypothèse que la perturbation de cette symbiose joue un rôle dans le développement de diverses maladies chroniques, y compris du cancer colorectal, a été émise. En effet, il existe de plus en plus de preuves expérimentales selon lesquelles le microbiome intestinal influence potentiellement le développement de tumeurs par le biais d'une dérégulation du métabolisme de l'hôte et de sa fonction immunitaire. Cependant, les données épidémiologiques reliant le microbiome intestinal à la carcinogenèse colorectale restent limitées car très peu de cohortes existantes ont collecté des échantillons fécaux. Les programmes de dépistage du cancer colorectal, dans lesquels des millions d'échantillons de selles sont prélevés chaque année, pourraient fournir de riches opportunités pour établir des cohortes basées sur la population avec des échantillons répétés et prospectivement collectés. La première partie de la thèse résume de manière systématique la littérature épidémiologique actuelle qui a été publiée au cours de la dernière décennie sur l'association entre le microbiome et le cancer. Nos résultats ont souligné que pour la plupart des indicateurs du microbiome, les preuves étaient encore trop faibles pour tirer des conclusions définitives concernant leur rôle dans le cancer. La deuxième partie de la thèse étudie l'association entre la résistance à l'insuline et l'inflammation - des facteurs de risque reconnus du cancer colorectal - et le microbiome intestinal, dans deux cohortes populationnelles : The Northern Finland Birth Cohort et TwinsUK. Notre étude a indiqué que des niveaux plus élevés de résistance à l'insuline et d'autres marqueurs de dysfonctionnement métabolique étaient associés à une diversité amoindrie du microbiome dans les deux cohortes, même après contrôle de l'obésité et d'autres facteurs. Enfin, la troisième partie de la thèse évalue la stabilité et la concordance du microbiome dans les échantillons fécaux prélevés à l'aide de différentes méthodes utilisées dans les programmes de dépistage du cancer colorectal en cours. Nos résultats suggèrent que les collections d'échantillons fécaux couramment utilisées, telles que les tests immunochimiques fécaux et les cartes de prélèvement sur papier, sont en général des supports appropriés pour mesurer la diversité microbiome, bien que des facteurs de stockage, tels que la température ambiante, puissent avoir un impact sur la stabilité de certaines méthodes. En outre, la collecte opportuniste d'échantillons fécaux dans des tubes à tests immunochimiques fécaux après le dépistage du cancer colorectal est probablement une méthode viable pour établir des cohortes avec des échantillons fécaux pré-diagnostics. Dans l'ensemble, cette thèse présente l'état de l'art sur les preuves épidémiologiques du rôle du microbiome dans la tumorigenèse. Elle fournit de nouvelles informations sur l'association des facteurs de risque métaboliques du cancer colorectal avec le microbiome intestinal dans les études populationnelles. Enfin, elle a généré d'importantes données méthodologiques sur l'impact des outils de collecte d'échantillons fécaux sur les mesures microbiennes nécessaires pour les futures recherches épidémiologiques sur le microbiome et le cancer colorectal, ainsi que sur d'autres maladies chronique

Human microbiome and metabolic health : an overview of systematic reviews

To summarize the microbiome's role in metabolic disorders (insulin resistance, hyperglycemia, type 2 diabetes, obesity, hyperlipidemia, hypertension, nonalcoholic fatty liver disease [NAFLD], and metabolic syndrome), systematic reviews on observational or interventional studies (prebiotics/probiotics/synbiotics/transplant) were searched in MEDLINE and Embase until September 2020. The 87 selected systematic reviews included 57 meta-analyses. Methodological quality (AMSTAR2) was moderate in 62%, 12% low, and 26% critically low. Observational studies on obesity (10 reviews) reported less gut bacterial diversity with higher Fusobacterium, Lactobacillus reuteri, Bacteroides fragilis, and Staphylococcus aureus, whereas lower Methanobrevibacter, Lactobacillus plantarum, Akkermansia muciniphila, and Bifidobacterium animalis compared with nonobese. For diabetes (n = 1), the same was found for Fusobacterium and A. muciniphila, whereas higher Ruminococcus and lower Faecalibacterium, Roseburia, Bacteroides vulgatus, and several Bifidobacterium spp. For NAFLD (n = 2), lower Firmicutes, Rikenellaceae, Ruminococcaceae, whereas higher Escherichia and Lactobacillus were detected. Discriminating bacteria overlapped between metabolic disorders, those with high abundance being often involved in inflammation, whereas those with low abundance being used as probiotics. Meta-analyses (n = 54) on interventional studies reported 522 associations: 54% was statistically significant with intermediate effect size and moderate between-study heterogeneity. Meta-evidence was highest for probiotics and lowest for fecal transplant. Future avenues include better methodological quality/comparability, testing functional differences, new intervention strategies, and considerating other body habitats and kingdoms

Stability of the Fecal and Oral Microbiome over 2 Years at -80°C for Multiple Collection Methods.

BACKGROUND: In prospective cohorts, biological samples are generally stored over long periods before an adequate number of cases have accrued. We investigated the impact of sample storage at -80°C for 2 years on the stability of the V4 region of the 16S rRNA gene across seven different collection methods (i.e., no additive, 95% ethanol, RNAlater stabilization solution, fecal occult blood test cards, and fecal immunochemical test tubes for feces; OMNIgene ORAL tubes and Scope mouthwash for saliva) among 51 healthy volunteers. METHODS: Intraclass correlation coefficients (ICC) were calculated for the relative abundance of the top three phyla, the 20 most abundant genera, three alpha-diversity metrics, and the first principal coordinates of three beta-diversity matrices. RESULTS: The subject variability was much higher than the variability introduced by the sample collection type, and storage time. For fecal samples, microbial stability over 2 years was high across collection methods (range, ICCs = 0.70-0.99), except for the samples collected with no additive (range, ICCs = 0.23-0.83). For oral samples, most microbiome diversity measures were stable over time with ICCs above 0.74; however, ICCs for the samples collected with Scope mouthwash were lower for two alpha-diversity measures, Faiths phylogenetic diversity (0.23) and the observed number of operational taxonomic units (0.23). CONCLUSIONS: Fecal and oral samples in most used collection methods are stable for microbiome analyses after 2 years at -80°C, except for fecal samples with no additive. IMPACT: This study provides evidence that samples stored for an extended period from prospective studies are useful for microbiome analyses

The human microbiome in relation to cancer risk : a systematic review of epidemiologic studies

The microbiome has been hypothesized to play a role in cancer development. Because of the diversity of published data, an overview of available epidemiologic evidence linking the microbiome with cancer is now needed. We conducted a systematic review using a tailored search strategy in Medline and EMBASE databases to identify and summarize the current epidemiologic literature on the relationship between the microbiome and different cancer outcomes published until December 2019. We identified 124 eligible articles. The large diversity of parameters used to describe microbial composition made it impossible to harmonize the different studies in a way that would allow meta-analysis, therefore only a qualitative description of results could be performed. Fifty studies reported differences in the gut microbiome between patients with colorectal cancer and various control groups. The most consistent findings were for Fusobacterium, Porphyromonas, and Peptostreptococcus being significantly enriched in fecal and mucosal samples from patients with colorectal cancer. For the oral microbiome, significantly increased and decreased abundance was reported for Fusobacterium and Streptococcus, respectively, in patients with oral cancer compared with controls. Overall, although there was a large amount of evidence for some of these alterations, most require validation in high-quality, preferably prospective, epidemiologic studies

Taxonomic Composition and Diversity of the Gut Microbiota in Relation to Habitual Dietary Intake in Korean Adults

We investigated associations of habitual dietary intake with the taxonomic composition and diversity of the human gut microbiota in 222 Koreans aged 18–58 years in a cross-sectional study. Gut microbiota data were obtained by 16S rRNA gene sequencing on DNA extracted from fecal samples. The habitual diet for the previous year was assessed by a food frequency questionnaire. After multivariable adjustment, intake of several food groups including vegetables, fermented legumes, legumes, dairy products, processed meat, and non-alcoholic beverages were associated with major phyla of the gut microbiota. A dietary pattern related to higher α-diversity (HiαDP) derived by reduced rank regression was characterized by higher intakes of fermented legumes, vegetables, seaweeds, and nuts/seeds and lower intakes of non-alcoholic beverages. The HiαDP was positively associated with several genera of Firmicutes such as Lactobacillus, Ruminococcus, and Eubacterium (all p < 0.05). Among enterotypes identified by principal coordinate analysis based on the β-diversity, the Ruminococcus enterotype had higher HiαDP scores and was strongly positively associated with intakes of vegetables, seaweeds, and nuts/seeds, compared to the two other enterotypes. We conclude that a plant- and fermented food-based diet was positively associated with some genera of Firmicutes (e.g., Lactobacillus, Ruminococcus, and Eubacterium) reflecting better gut microbial health

Comparison of Fecal Sample Collection Methods for Microbial Analysis Embedded within Colorectal Cancer Screening Programs

International audienceBackground: Colorectal cancer screening programs with fecal sample collection may provide a platform for population-based gut microbiome disease research. We investigated sample collection and storage method impact on the accuracy and stability of the V3-V4 region of the 16S rRNA genes and bacterial quantity across seven different collection methods [i.e., no solution, two specimen collection cards, and four types of fecal immunochemical test (FIT) used in four countries] among 19 healthy volunteers. Methods: Intraclass correlation coefficients (ICC) were calculated for the relative abundance of the top three phyla, the most abundant genera, alpha diversity metrics, and the first principal coordinates of the beta diversity matrices to estimate the stability of microbial profiles after storage for 7 days at room temperature, 4ºC or 30ºC, and after screening for the presence of occult blood in the stool. In addition, accuracy was estimated for samples frozen immediately compared to samples with no solution (i.e., the putative gold standard). Results: When compared with the putative gold standard, we observed significant variation for all collection methods. However, interindividual variability was much higher than the variability introduced by the collection method. Stability ICCs were high (≥0.75) for FIT tubes that underwent colorectal cancer screening procedures. The relative abundance of Actinobacteria (0.65) was an exception and was lower for different FIT tubes stored at 30ºC (range, 0.41–0.90) and room temperature (range, 0.06–0.94). Conclusions: Paper-based collection cards and different types of FIT are acceptable tools for microbiome measurements. Impact: Our findings inform on the utility of commonly used fecal sample collection methods for developing microbiome-focused cohorts nested within screening programs

Markers of metabolic health and gut microbiome diversity:findings from two population-based cohort studies

Abstract Aims/hypothesis: The gut microbiome is hypothesised to be related to insulin resistance and other metabolic variables. However, data from population-based studies are limited. We investigated associations between serologic measures of metabolic health and the gut microbiome in the Northern Finland Birth Cohort 1966 (NFBC1966) and the TwinsUK cohort. Methods: Among 506 individuals from the NFBC1966 with available faecal microbiome (16S rRNA gene sequence) data, we estimated associations between gut microbiome diversity metrics and serologic levels of HOMA for insulin resistance (HOMA-IR), HbA1c and C-reactive protein (CRP) using multivariable linear regression models adjusted for sex, smoking status and BMI. Associations between gut microbiome diversity measures and HOMA-IR and CRP were replicated in 1140 adult participants from TwinsUK, with available faecal microbiome (16S rRNA gene sequence) data. For both cohorts, we used general linear models with a quasi-Poisson distribution and Microbiome Regression-based Kernel Association Test (MiRKAT) to estimate associations of metabolic variables with alpha- and beta diversity metrics, respectively, and generalised additive models for location scale and shape (GAMLSS) fitted with the zero-inflated beta distribution to identify taxa associated with the metabolic markers. Results: In NFBC1966, alpha diversity was lower in individuals with higher HOMA-IR with a mean of 74.4 (95% CI 70.7, 78.3) amplicon sequence variants (ASVs) for the first quartile of HOMA-IR and 66.6 (95% CI 62.9, 70.4) for the fourth quartile of HOMA-IR. Alpha diversity was also lower with higher HbA1c (number of ASVs and Shannon’s diversity, p < 0.001 and p = 0.003, respectively) and higher CRP (number of ASVs, p = 0.025), even after adjustment for BMI and other potential confounders. In TwinsUK, alpha diversity measures were also lower among participants with higher measures of HOMA-IR and CRP. When considering beta diversity measures, we found that microbial community profiles were associated with HOMA-IR in NFBC1966 and TwinsUK, using multivariate MiRKAT models, with binomial deviance dissimilarity p values of <0.001. In GAMLSS models, the relative abundances of individual genera Prevotella and Blautia were associated with HOMA-IR in both cohorts. Conclusions/interpretation: Overall, higher levels of HOMA-IR, CRP and HbA1c were associated with lower microbiome diversity in both the NFBC1966 and TwinsUK cohorts, even after adjustment for BMI and other variables. These results from two distinct population-based cohorts provide evidence for an association between metabolic variables and gut microbial diversity. Further experimental and mechanistic insights are now needed to provide understanding of the potential causal mechanisms that may link the gut microbiota with metabolic health