Fusobacterium species are distinctly associated with patients with Lynch syndrome colorectal cancer.

Accumulating evidence demonstrates clear correlation between the gut microbiota and sporadic colorectal cancer (CRC). Despite this, there is limited understanding of the association between the gut microbiota and CRC in Lynch Syndrome (LS), a hereditary type of CRC. Here, we analyzed fecal shotgun metagenomic and targeted metabolomic of 71 Japanese LS subjects. A previously published Japanese sporadic CRC cohort, which includes non-LS controls, was utilized as a non-LS cohort (n = 437). LS subjects exhibited reduced microbial diversity and low-Faecalibacterium enterotypes compared to non-LS. Patients with LS-CRC had higher levels of Fusobacterium nucleatum and fap2. Differential fecal metabolites and functional genes suggest heightened degradation of lysine and arginine in LS-CRC. A comparison between LS and non-LS subjects prior to adenoma formation revealed distinct fecal metabolites of LS subjects. These findings suggest that the gut microbiota plays a more responsive role in CRC tumorigenesis in patients with LS than those without LS.

Intestinal Bacteria Fluctuating in Early-Stage Colorectal Cancer Carcinogenesis are Associated with Diet in Healthy Adults.

This hospital-based, cross-sectional study aimed to explore the association between diet and fluctuating intestinal bacteria in early-stage colorectal cancer (CRC) (Atopobium parvulum, Actinomyces odontolyticus, Solobacterium moorei, and Bifidobacterium longum). Healthy participants (n = 212) who underwent total colonoscopy at National Cancer Center Hospital (Tokyo, Japan) were divided into two groups according to the relative abundance of bacteria in their feces: those in the top 25% of relative bacterial abundance as cases and the bottom 25% as controls. The participants were divided into three groups (low, medium, and high) according to their intake of food groups associated with CRC. Multivariable logistic regression analysis was conducted to estimate the association between dietary intake and higher relative abundance of bacteria. Dairy products were inversely associated with a higher relative abundance of A. parvulum, A. odontolyticus, and S. moorei, with odds ratios (high vs. low) and 95% confidence interval as follows: 0.16 (0.06-0.44), 0.25 (0.08-0.82), and 0.29 (0.11-0.78), respectively. Additionally, dietary fiber was inversely associated with a higher relative abundance of S.moorei (0.29 [0.11-0.78]). No association was observed between diet and B.longum. In conclusion, healthy adults with a higher intake of dairy products and fiber had lower odds of having a higher relative abundance of CRC-associated microbiota.

Efficient and accurate KRAS genotyping using digital PCR combined with melting curve analysis for ctDNA from pancreatic cancer patients.

A highly sensitive and highly multiplexed quantification technique for nucleic acids is necessary to predict and evaluate cancer treatment by liquid biopsy. Digital PCR (dPCR) is a highly sensitive quantification technique, but conventional dPCR discriminates multiple targets by the color of the fluorescent dye of the probe, which limits multiplexing beyond the number of colors of fluorescent dyes. We previously developed a highly multiplexed dPCR technique combined with melting curve analysis. Herein, we improved the detection efficiency and accuracy of multiplexed dPCR with melting curve analysis to detect KRAS mutations in circulating tumor DNA (ctDNA) prepared from clinical samples. The mutation detection efficiency was increased from 25.9% of the input DNA to 45.2% by shortening the amplicon size. The limit of detection of mutation was improved from 0.41 to 0.06% by changing the mutation type determination algorithm for G12A, resulting in a limit of detection of less than 0.2% for all the target mutations. Then, ctDNA in plasma from pancreatic cancer patients was measured and genotyped. The measured mutation frequencies correlated well with those measured by conventional dPCR, which can measure only the total frequency of KRAS mutants. KRAS mutations were detected in 82.3% of patients with liver or lung metastasis, which was consistent with other reports. Accordingly, this study demonstrated the clinical utility of multiplex dPCR with melting curve analysis to detect and genotype ctDNA from plasma with sufficient sensitivity.

Leveraging explainable AI for gut microbiome-based colorectal cancer classification.

Studies have shown a link between colorectal cancer (CRC) and gut microbiome compositions. In these studies, machine learning is used to infer CRC biomarkers using global explanation methods. While these methods allow the identification of bacteria generally correlated with CRC, they fail to recognize species that are only influential for some individuals. In this study, we investigate the potential of Shapley Additive Explanations (SHAP) for a more personalized CRC biomarker identification. Analyses of five independent datasets show that this method can even separate CRC subjects into subgroups with distinct CRC probabilities and bacterial biomarkers.

Association Between Diet and Fusobacterium nucleatum in the Feces of Healthy Adults: A Hospital-based Cross-sectional Study.

Fusobacterium nucleatum is involved in the development and progression of colorectal cancer. Although the gut microbiota is influenced by diet, studies on the association between diet and F. nucleatum are limited. We aimed to evaluate the association between various dietary factors and fecal F. nucleatum in healthy adults without a history of colorectal cancer or precancerous lesions. This was a cross-sectional study. Subjects who underwent total colonoscopy at the National Cancer Center Hospital (Tokyo, Japan) were included. Healthy subjects (n = 212) were divided into two groups according to the presence or absence of F. nucleatum in their feces which was calculated from data of whole-genome shotgun sequencing, with the group with F. nucleatum serving as cases and the group without F. nucleatum serving as controls. Multivariable logistic regression analysis adjusted potential confounders was conducted to estimate the associations between dietary intake and nutrients estimated by a validated food frequency questionnaire and the presence of F. nucleatum in the feces. There was a significant inverse association between dairy products and the presence of fecal F. nucleatum [high vs. low; OR, 0.41; 95% confidence interval, 0.17-0.95; Ptrend, 0.039]. These results may have important implications for colorectal cancer prevention through nutritional intervention. PREVENTION RELEVANCE: F. nucleatum is well known as a colorectal cancer-associated bacterium. Dietary habits alter the composition and function of the intestinal microbiota. A high intake of dairy products in healthy adults may reduce F. nucleatum and prevent colorectal cancer.

Mediation effect of intestinal microbiota on the relationship between fiber intake and colorectal cancer.

Higher fiber intake has been associated with a lower risk of colorectal cancer (CRC) and has been shown to protect against CRC based on probable evidence. Recent studies revealed a possible mechanism whereby the interaction between intestinal microbiota and fiber intake mediates CRC risk. However, the specific intestinal bacteria and the amount of these bacteria involved in this mechanism are not fully known. Therefore, this single-center study aimed to determine whether specific intestinal bacteria mediated the relationship between fiber intake and CRC risk. We enrolled patients who received colonoscopy at National Cancer Center Hospital. This cross-sectional study included 180 patients with clinically diagnosed CRC and 242 controls. We conducted a causal mediation analysis to assess the natural indirect effect and natural direct effect of specific intestinal bacteria on association between fiber intake and CRC risk. The median age was 64 (interquartile range, 54-70) years, and 58% of the participants were males. We used metagenomics for profiling gut microbiomes. The relative abundance of each species in each sample was calculated. Among the candidate, Fusobacterium nucleatum and Gemella morbillorum had a significant natural indirect effect based on their highest fiber intake compared to the lowest fiber intake, with a risk difference (95% confidence interval, proportion of mediation effect) of -0.06 [-0.09 to -0.03, 23%] and -0.03 [-0.06 to -0.01, 10.5%], respectively. Other bacteria did not display natural indirect effects. In conclusion, Fusobacterium nucleatum and Gemella morbillorum were found to mediate the relationship between fiber intake and CRC risk.

Association between Diet and Fusobacterium nucleatum in the Feces of Healthy Adults: A hospital-based cross-sectional study.

Fusobacterium nucleatum is involved in the development and progression of colorectal cancer. Although the gut microbiota is influenced by diet, studies on the association between diet and F. nucleatum are limited. We aimed to evaluate the association between various dietary factors and fecal F. nucleatum in healthy adults without a history of colorectal cancer or precancerous lesions. This was a cross-sectional study. Subjects who underwent total colonoscopy at the National Cancer Center Hospital (Tokyo, Japan) were included. Healthy subjects (n = 212) were divided into two groups according to the presence or absence of F. nucleatum in their feces which was calculated from data of whole-genome shotgun sequencing, with the group with F. nucleatum serving as cases and the group without F. nucleatum serving as controls. Multivariable logistic regression analysis adjusted potential confounders was conducted to estimate the associations between dietary intake and nutrients estimated by a validated food frequency questionnaire and the presence of F. nucleatum in the feces. There was a significant inverse association between dairy products and the presence of fecal F. nucleatum (High vs. Low, OR 0.41, 95% CI 0.17-0.95, P for trend 0.039). These results may have important implications for colorectal cancer prevention through nutritional intervention.

Germline variants in cancer-predisposing genes in pancreatic cancer patients with a family history of cancer.

BACKGROUND: Our phase II trial (FABRIC study) failed to verify the efficacy of gemcitabine plus oxaliplatin (GEMOX) in patients with pancreatic ductal adenocarcinoma (PDAC) with a familial or personal history of pancreatic, breast, ovarian or prostate cancer, which suggested that a family and personal history may be insufficient to determine response to platinum-based chemotherapy. METHODS: This ancillary analysis aimed to investigate the prevalence of germline variants of homologous recombination repair (HRR)-related genes and clarify the association of germline variants with the efficacy of GEMOX and patient outcome in PDAC patients. Of 45 patients enrolled in FABRIC study, 27 patients were registered in this ancillary analysis. RESULTS: Of the identified variants in HRR-related genes, one variant was considered pathogenic and eight variants in six patients (22%) were variants of unknown significance (VUS). Objective response to GEMOX was achieved by 43% of the seven patients and tended to be higher than that of patients without such variants (25%). Pathogenic/VUS variant in HRR-related genes was an independent favorable factor for progression-free survival (hazard ratio, 0.322; P = 0.047) and overall survival (hazard ratio, 0.195; P = 0.023) in multivariable analysis. CONCLUSIONS: The prevalence of germline variants in PDAC patients was very low even among patients with a familial/personal history of pancreatic, breast, ovarian or prostate cancer. Patients with one or more germline variants in HRR-related genes classified as pathogenic or VUS may have the potential to obtain better response to GEMOX and have better outcomes.

Safety and efficacy of S-IROX (S-1, irinotecan and oxaliplatin combination therapy) in patients with advanced pancreatic cancer: A multicenter phase 1b dose-escalation and dose-expansion clinical trial.

BACKGROUND: This phase 1b trial evaluated the toxicity and efficacy of S-1, irinotecan, and oxaliplatin combination therapy (S-IROX) as first-line chemotherapy in patients with advanced pancreatic cancer (APC). METHODS: Patients aged 20-75 years with APC and an Eastern Cooperative Oncology Group performance status of 0 or 1 were eligible to receive escalating doses of S-1 (60 or 80 mg m2·day) on days 1-7, fixed doses of oxaliplatin (85 mg/m2) biweekly, and escalating doses of irinotecan (150, 165, or 180 mg/m2) once every 2 weeks. In the dose-escalation cohort, a 3 + 3 design was used to determine the maximum-tolerated dose (MTD) and explore the recommended dose (RD). A dose-expansion cohort was added to further evaluate the safety and efficacy of the combination. This trial was registered at UMIN-CTR (UMIN000012054). RESULTS: Approximately 47 patients were enrolled, of whom 45 were eligible for the analysis. The MTD was not determined, but the RD was determined to be dose level 1 based on a review of data from each level. Among the 45 patients, the ORR was 51.1% [95% confidence interval (CI), 35.8-66.3%]. The median progression-free survival and median overall survival was 6.9 months (95% CI, 5.1-8.8 months) and 15.8 months (95% CI, 9.8-20.8 months), respectively. Common adverse events included neutropenia, elevated liver enzyme levels, diarrhoea, and nausea. CONCLUSIONS: The S-IROX regimen showed promising efficacy with manageable toxicities in Japanese patients with APC. A randomised phase 2/3 trial comparing S-IROX, mFOLFIRINOX, and gemcitabine plus nab-paclitaxel is currently ongoing (jRCTs031190009).

Surgical Treatment for Colorectal Cancer Partially Restores Gut Microbiome and Metabolome Traits.

Accumulating evidence indicates that the gut microbiome and metabolites are associated with colorectal cancer (CRC). However, the influence of surgery for CRC treatment on the gut microbiome and metabolites and how it relates to CRC risk in postoperative CRC patients remain partially understood. Here, we collected 170 fecal samples from 85 CRC patients pre- and approximately 1 year postsurgery and performed shotgun metagenomic sequencing and capillary electrophoresis-time of flight mass spectrometry-based metabolomics analyses to characterize alterations between pre- and postsurgery. We determined that the relative abundance of 114 species was altered postsurgery (P < 0.005). CRC-associated species, such as Fusobacterium nucleatum, were decreased postsurgery. On the other hand, Clostridium scindens, carcinogenesis-associated deoxycholate (DCA)-producing species, and its biotransformed genes (bai operon) were increased postsurgery. The concentration of 60 fecal metabolites was also altered postsurgery (P < 0.005). Two bile acids, cholate and DCA, were increased postsurgery. We developed methods to estimate postoperative CRC risk based on the gut microbiome and metabolomic compositions using a random forest machine-learning algorithm that classifies large adenoma or early-stage CRC and healthy controls from publicly available data sets. We applied methods to preoperative samples and then compared the estimated CRC risk between the two groups according to the presence of large adenoma or tumors 5 years postsurgery (P < 0.05). Overall, our results show that the gut microbiome and metabolites dynamically change from pre- to postsurgery. In postoperative CRC patients, potential CRC risk derived from gut microbiome and metabolites still remains, which indicates the importance of follow-up assessments. IMPORTANCE The gut microbiome and metabolites are associated with CRC progression and carcinogenesis. Postoperative CRC patients are reported to be at an increased CRC risk; however, how gut microbiome and metabolites are related to CRC risk in postoperative patients remains only partially understood. In this study, we investigated the influence of surgical CRC treatment on the gut microbiome and metabolites. We found that the CRC-associated species Fusobacterium nucleatum was decreased postsurgery, whereas carcinogenesis-associated DCA and its producing species and genes were increased postsurgery. We developed methods to estimate postoperative CRC risk based on the gut microbiome and metabolomic compositions. We applied methods to compare the estimated CRC risk between two groups according to the presence of large adenoma or tumors after 5 years postsurgery. To our knowledge, this study is the first report on differences between pre- and postsurgery using metagenomics and metabolomics data analysis. Our methods might be used for CRC risk assessment in postoperative patients.

Comprehensive Genomic Profiling of Neuroendocrine Carcinomas of the Gastrointestinal System.

The neuroendocrine carcinoma of the gastrointestinal system (GIS-NEC) is a rare but highly malignant neoplasm. We analyzed 115 cases using whole-genome/exome sequencing, transcriptome sequencing, DNA methylation assays, and/or ATAC-seq and found GIS-NECs to be genetically distinct from neuroendocrine tumors (GIS-NET) in the same location. Clear genomic differences were also evident between pancreatic NECs (Panc-NEC) and nonpancreatic GIS-NECs (Nonpanc-NEC). Panc-NECs could be classified into two subgroups (i.e., "ductal-type" and "acinar-type") based on genomic features. Alterations in TP53 and RB1 proved common in GIS-NECs, and most Nonpanc-NECs with intact RB1 demonstrated mutually exclusive amplification of CCNE1 or MYC. Alterations of the Notch gene family were characteristic of Nonpanc-NECs. Transcription factors for neuroendocrine differentiation, especially the SOX2 gene, appeared overexpressed in most GIS-NECs due to hypermethylation of the promoter region. This first comprehensive study of genomic alterations in GIS-NECs uncovered several key biological processes underlying genesis of this very lethal form of cancer. SIGNIFICANCE: GIS-NECs are genetically distinct from GIS-NETs. GIS-NECs arising in different organs show similar histopathologic features and share some genomic features, but considerable differences exist between Panc-NECs and Nonpanc-NECs. In addition, Panc-NECs could be classified into two subgroups (i.e., "ductal-type" and "acinar-type") based on genomic and epigenomic features. This article is highlighted in the In This Issue feature, p. 587.

Clonal dynamics of circulating tumor DNA during immune checkpoint blockade therapy for melanoma.

Assessment of treatment efficacy of immune checkpoint inhibitors in melanoma patients is difficult as the response to these therapies varies among patients or lesions. The clonal evolution of cancer during immune checkpoint blockade therapy could cause treatment resistance. We investigated the potential of liquid biopsy in monitoring the mutational profiles of metastatic melanoma during immunotherapy. Plasma samples collected from 21 Japanese metastatic melanoma patients before immune checkpoint blockade therapy were subjected to whole-exome sequencing (WES). Furthermore, 14 Japanese patients with melanoma were enrolled for longitudinal analysis of circulating tumor DNA (ctDNA). Plasma samples were collected prospectively before and during therapy and sequenced. WES of the pretreatment plasma from Japanese melanoma patients showed detectable ctDNA levels with wide ranges of variant allele frequencies within a sample, suggesting clonal and subclonal mutations in ctDNA. In targeted sequencing using longitudinal samples, ctDNA levels correlated with increased tumor size, while ctDNA content immediately decreased after a surge in a patient exhibiting pseudo-progression, suggesting the potential of ctDNA analysis in discriminating between pseudo- and true progression. Mutant ctDNA levels showed different patterns within the clinical course of specific patients, suggesting that these mutations were derived from different tumor clones with distinct therapeutic responses. During further investigation, WES of plasma samples from 1 patient showed marked differences in the mutational profiles of ctDNA, including expansive tumor evolution during an acute exacerbation. Immunotherapy may induce characteristic clonal evolutions of tumors; longitudinal analysis of ctDNA has the potential of determining these tumor evolution patterns and therapeutic responses.

Multicenter Retrospective Analysis of Chemotherapy for Advanced Pancreatic Acinar Cell Carcinoma: Potential Efficacy of Platinum- and Irinotecan-Containing Regimens.

OBJECTIVES: The aim of this multicenter retrospective study was to identify the optimal chemotherapeutic regimen for advanced pancreatic acinar cell carcinoma (PACC). METHODS: Fifty-eight patients with histopathologically confirmed advanced PACC who had received chemotherapy between 1996 and 2013 were enrolled. The clinical characteristics of the patients and the treatment efficacy data were collected from the medical records at 16 Japanese institutions, using standardized data collection instrument. RESULTS: The most commonly selected treatment regimens were gemcitabine-, fluoropyrimidine-, platinum-, and irinotecan-containing regimens. The overall response rate in the patients who received first-line chemotherapy were 7% and 38%, respectively, and the median overall survival was 13.2 months. When the data for all the treatment lines were aggregated, the response rates to gemcitabine-, fluoropyrimidine-, platinum-, and irinotecan-containing regimens were 7%, 18%, 40%, and 29%, respectively. The overall survival tended to be better in patients who had received a platinum-containing regimen (hazard ratio, 0.50; 95% confidence interval, 0.23-1.11; P = 0.08) or irinotecan-containing regimen (hazard ratio, 0.42; 95% confidence interval, 0.15-1.19; P = 0.09) at least once in the treatment course as compared with those who had not. CONCLUSIONS: Our findings suggested that platinum- and irinotecan-containing regimens exhibited some potential efficacy in patients with advanced PACC.

Feasibility study of nivolumab as neoadjuvant chemotherapy for locally esophageal carcinoma: FRONTiER (JCOG1804E).

One of the standard treatments of resectable esophageal squamous cell carcinoma (ESCC) is neoadjuvant chemotherapy followed by surgery. Nivolumab showed efficacy for metastatic ESCC. However, the safety and efficacy of neoadjuvant nivolumab with chemotherapy for ESCC is unknown. Therefore, we will conduct FRONTiER to evaluate the safety and efficacy of nivolumab adding to neoadjuvant chemotherapy. FRONTiER comprises four experimental cohorts: (A) including nivolumab plus 5-FU+CDDP (cisplatin and 5-fluorouracil [CF]); (B) including one prior administration of nivolumab and the cohort A regimen; (C) including nivolumab plus docetaxel+ CF; (D) including one prior administration of nivolumab and the cohort C regimen; an expanded cohort. The primary end point is the incidence of dose-limiting toxicities from the initial dose to the 30th postoperative day. Clinical Trial Identifier: NCT03914443.

Influence of gastrectomy for gastric cancer treatment on faecal microbiome and metabolome profiles.

OBJECTIVE: Recent evidence points to the gut microbiome's involvement in postoperative outcomes, including after gastrectomy. Here, we investigated the influence of gastrectomy for gastric cancer on the gut microbiome and metabolome, and how it related to postgastrectomy conditions. DESIGN: We performed shotgun metagenomics sequencing and capillary electrophoresis time-of-flight mass spectrometry-based metabolomics analyses on faecal samples collected from participants with a history of gastrectomy for gastric cancer (n=50) and compared them with control participants (n=56). RESULTS: The gut microbiota in the gastrectomy group showed higher species diversity and richness (p<0.05), together with greater abundance of aerobes, facultative anaerobes and oral microbes. Moreover, bile acids such as genotoxic deoxycholic acid and branched-chain amino acids were differentially abundant between the two groups (linear discriminant analysis (LDA) effect size (LEfSe): p<0.05, q<0.1, LDA>2.0), as were also Kyoto Encyclopedia of Genes and Genomes modules involved in nutrient transport and organic compounds biosynthesis (LEfSe: p<0.05, q<0.1, LDA>2.0). CONCLUSION: Our results reveal alterations of gut microbiota after gastrectomy, suggesting its association with postoperative comorbidities. The multi-omic approach applied in this study could complement the follow-up of patients after gastrectomy.

Author Correction: Metagenomic analysis of colorectal cancer datasets identifies cross-cohort microbial diagnostic signatures and a link with choline degradation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Metagenomic and metabolomic analyses reveal distinct stage-specific phenotypes of the gut microbiota in colorectal cancer.

In most cases of sporadic colorectal cancers, tumorigenesis is a multistep process, involving genomic alterations in parallel with morphologic changes. In addition, accumulating evidence suggests that the human gut microbiome is linked to the development of colorectal cancer. Here we performed fecal metagenomic and metabolomic studies on samples from a large cohort of 616 participants who underwent colonoscopy to assess taxonomic and functional characteristics of gut microbiota and metabolites. Microbiome and metabolome shifts were apparent in cases of multiple polypoid adenomas and intramucosal carcinomas, in addition to more advanced lesions. We found two distinct patterns of microbiome elevations. First, the relative abundance of Fusobacterium nucleatum spp. was significantly (P < 0.005) elevated continuously from intramucosal carcinoma to more advanced stages. Second, Atopobium parvulum and Actinomyces odontolyticus, which co-occurred in intramucosal carcinomas, were significantly (P < 0.005) increased only in multiple polypoid adenomas and/or intramucosal carcinomas. Metabolome analyses showed that branched-chain amino acids and phenylalanine were significantly (P < 0.005) increased in intramucosal carcinomas and bile acids, including deoxycholate, were significantly (P < 0.005) elevated in multiple polypoid adenomas and/or intramucosal carcinomas. We identified metagenomic and metabolomic markers to discriminate cases of intramucosal carcinoma from the healthy controls. Our large-cohort multi-omics data indicate that shifts in the microbiome and metabolome occur from the very early stages of the development of colorectal cancer, which is of possible etiological and diagnostic importance.

Meta-analysis of fecal metagenomes reveals global microbial signatures that are specific for colorectal cancer.

Association studies have linked microbiome alterations with many human diseases. However, they have not always reported consistent results, thereby necessitating cross-study comparisons. Here, a meta-analysis of eight geographically and technically diverse fecal shotgun metagenomic studies of colorectal cancer (CRC, n = 768), which was controlled for several confounders, identified a core set of 29 species significantly enriched in CRC metagenomes (false discovery rate (FDR) < 1 × 10-5). CRC signatures derived from single studies maintained their accuracy in other studies. By training on multiple studies, we improved detection accuracy and disease specificity for CRC. Functional analysis of CRC metagenomes revealed enriched protein and mucin catabolism genes and depleted carbohydrate degradation genes. Moreover, we inferred elevated production of secondary bile acids from CRC metagenomes, suggesting a metabolic link between cancer-associated gut microbes and a fat- and meat-rich diet. Through extensive validations, this meta-analysis firmly establishes globally generalizable, predictive taxonomic and functional microbiome CRC signatures as a basis for future diagnostics.

Metagenomic analysis of colorectal cancer datasets identifies cross-cohort microbial diagnostic signatures and a link with choline degradation.

Several studies have investigated links between the gut microbiome and colorectal cancer (CRC), but questions remain about the replicability of biomarkers across cohorts and populations. We performed a meta-analysis of five publicly available datasets and two new cohorts and validated the findings on two additional cohorts, considering in total 969 fecal metagenomes. Unlike microbiome shifts associated with gastrointestinal syndromes, the gut microbiome in CRC showed reproducibly higher richness than controls (P < 0.01), partially due to expansions of species typically derived from the oral cavity. Meta-analysis of the microbiome functional potential identified gluconeogenesis and the putrefaction and fermentation pathways as being associated with CRC, whereas the stachyose and starch degradation pathways were associated with controls. Predictive microbiome signatures for CRC trained on multiple datasets showed consistently high accuracy in datasets not considered for model training and independent validation cohorts (average area under the curve, 0.84). Pooled analysis of raw metagenomes showed that the choline trimethylamine-lyase gene was overabundant in CRC (P = 0.001), identifying a relationship between microbiome choline metabolism and CRC. The combined analysis of heterogeneous CRC cohorts thus identified reproducible microbiome biomarkers and accurate disease-predictive models that can form the basis for clinical prognostic tests and hypothesis-driven mechanistic studies.