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Evidence indicates a complex link between microbiota, tumor characteristics, and host immunity in the tumor microenvironment. In experimental studies, bifidobacteria appear to modulate intestinal epithelial cell differentiation. Accumulating evidence suggests that bifidobacteria may enhance the antitumor immunity and efficacy of immunotherapy. We hypothesized that the amount of bifidobacteria in colorectal carcinoma tissue might be associated with tumor differentiation and higher immune response to colorectal cancer. Using a molecular pathologic epidemiology database of 1313 rectal and colon cancers, we measured the amount of BifidobacteriumDNA in carcinoma tissue by a quantitative PCR assay. The multivariable regression model was used to adjust for potential confounders, including microsatellite instability status, CpG island methylator phenotype, long-interspersed nucleotide element-1 methylation, and KRAS, BRAF, and PIK3CAmutations. Intratumor bifidobacteria were detected in 393 cases (30%). The amount of bifidobacteria was associated with the extent of signet ring cells (P = 0.002). Compared with Bifidobacterium-negative cases, multivariable odd ratios for the extent of signet ring cells were 1.29 (95% CI, 0.74–2.24) for Bifidobacterium-low cases and 1.87 (95% CI, 1.16–3.02) for Bifidobacterium-high cases (Ptrend = 0.01). The association between intratumor bifidobacteria and signet ring cells suggests a possible role of bifidobacteria in determining distinct tumor characteristics or as an indicator of dysfunctional mucosal barrier in colorectal cancer.

Conventional adenomas are initiated by APCgene mutation that activates the WNT signal. Serrated neoplasia is commonly initiated by BRAFor KRASmutation. WNT pathway activation may also occur, however, to what extent this is owing to APCmutation is unknown. We examined aberrant nuclear β-catenin immunolocalization as a surrogate for WNT pathway activation and analyzed the entire APCgene coding sequence in serrated and conventional pathway polyps and cancers. WNT pathway activation was a common event in conventional pathway lesions with aberrant nuclear immunolocalization of β-catenin and truncating APCmutations in 90% and 89% of conventional adenomas and 82% and 70% of BRAFwild-type cancers, respectively. WNT pathway activation was seen to a lesser extent in serrated pathway lesions. It occurred at the transition to dysplasia in serrated polyps with a significant increase in nuclear β-catenin labeling from sessile serrated adenomas (10%) to sessile serrated adenomas with dysplasia (55%) and traditional serrated adenomas (9%) to traditional serrated adenomas with dysplasia (39%) (P=0.0001). However, unlike the conventional pathway, truncating APCmutations were rare in the serrated pathway lesions especially sessile serrated adenomas even when dysplastic (15%) and in the BRAFmutant cancers with microsatellite instability that arise from them (8%). In contrast, APCmissense mutations that were rare in conventional pathway adenomas and cancers (3% in BRAFwild-type cancers) were more frequent in BRAFmutant cancers with microsatellite instability (32%). We conclude that increased WNT signaling is important in the transition to malignancy in the serrated pathway but that APCmutation is less common and the spectrum of mutations is different than in conventional colorectal carcinogenesis. Moderate impact APCmutations and non-APC-related causes of increased WNT signaling may have a more important role in serrated neoplasia than the truncating APCmutations common in conventional adenomas.

ABSTRACTColorectal cancer is a major cause of cancer death and approximately 20% arises within serrated polyps, which are under-recognized and poorly understood. Human serrated colorectal polyps frequently exhibit both oncogenic BRAFmutation and widespread DNA methylation changes, which are important in silencing genes restraining neoplastic progression. Here, we investigated whether in vivoinduction of mutant Brafis sufficient to result in coordinated promoter methylation changes for multiple cancer-related genes. The BrafV637Emutation was induced in murine intestine on an FVB;C57BL/6J background and assessed for morphological and DNA methylation changes at multiple time points from 10 days to 14 months. Extensive intestinal hyperplasia developed by 10 days post-induction of the mutation. By 8 months, most mice had murine serrated adenomas with dysplasia and invasive cancer developed in 40% of mice by 14 months. From 5 months onwards, Brafmutant mice showed extensive, gene-specific increases in DNA methylation even in hyperplastic mucosa without lesions. This demonstrates that persistent oncogenic Brafsignaling is sufficient to induce widespread DNA methylation changes. This occurs over an extended period of time, mimicking the long latency followed by rapid progression of human serrated neoplasia. This study establishes for the first time that DNA methylation arises slowly in direct response to prolonged oncogenic Brafsignaling in serrated polyps; this finding has implications both for chemoprevention and for understanding the origin of DNA hypermethylation in cancer generally.

Given previous biologic evidence of immunomodulatory effects of coffee, we hypothesized that the association between coffee intake of colorectal cancer patients and survival differs by immune responses. Using a molecular pathologic epidemiology database of 4465 incident colorectal cancer cases, including 1262 cases with molecular data, in the Nurses’ Health Study and the Health Professionals Follow-up Study, we examined the association between coffee intake of colorectal cancer patients and survival in strata of levels of histopathologic lymphocytic reaction and T-cell infiltrates in tumor tissue. We did not observe a significant association of coffee intake with colorectal cancer–specific mortality (multivariable-adjusted hazard ratio [HR] for 1-cup increase of coffee intake per day, 0.93; 95% CI, 0.84 to 1.03). Although statistical significance was not reached at the stringent level (α=.005), the association of coffee intake with colorectal cancer–specific mortality differed by Crohn disease–like lymphoid reaction (Pinteraction=.007). Coffee intake was associated with lower colorectal cancer–specific mortality in patients with high Crohn disease–like reaction (multivariable HR for 1-cup increase of coffee intake per day, 0.55; 95% CI, 0.37 to 0.81; Ptrend=.002) but not in patients with intermediate Crohn disease–like reaction (the corresponding HR, 1.02; 95% CI, 0.72 to 1.44) or negative/low Crohn disease–like reaction (the corresponding HR, 0.95; 95% CI, 0.83 to 1.07). The associations of coffee intake with colorectal cancer–specific mortality did not significantly differ by levels of other lymphocytic reaction or any T-cell subset (Pinteraction>.18). There is suggestive evidence for differential prognostic effects of coffee intake by Crohn disease–like lymphoid reaction in colorectal cancer.