DNA methylation changes are important in cancer development and the etiology of the CpG Island Methylator Phenotype (CIMP), the most extreme form of aberrant promoter DNA methylation in cancer, can only partially be explained by genetic changes. An association between CIMP and the gut microbiota has been implicated in colorectal cancer (CRC) pathogenesis but the exact mechanisms underlying the observations are poorly understood. In order to clarify the link between CIMP and the microbiome in CRC, we studied human primary tumor and adjacent mucosal tissues (normal) from CRC patients (n=46). The patient samples were categorized by tumor sites (23 proximal and 23 distal), sex (28 males and 18 females), and age (69 yrs ±11). Bisulfite pyrosequencing and Digital Restriction Enzyme Analysis of Methylation (DREAM) were performed to determine the methylation of CpG sites in the CRC tumors. The tumor samples were selected based on their CIMP status (10 high, 10 low, and 26 negative) and the tumor sites. 16S rRNA gene sequencing using Illumina-Hi-Seq was performed on those samples. Principle Coordinate analysis of the 16S rRNA sequences showed that the microbiota of CIMP-positive (CIMP-high and -low combined) tumors was significantly different from the microbiota of the CIMP-negative tumors (PERMANOVA, P = 0.013). Linear discriminant analysis effect size (LEfSe) showed an enrichment of specific bacterial taxa in the CIMP-positive microbiota. Fusobacterium (LDA score = 4.71, p=0.033) and Erysipelotrichaceae (LDA score = 3.77, p=0.004) were most enriched in the CIMP-positive tumors along with Bacteroides. The microbiota of different tumor sites also showed significant difference with Fusobacterium (LDA score = 4.32, p=0.047) and Erysipelotrichaceae (LDA score = 3.45, p=0.039) being enriched in the proximal tumors compared to the distal tumors. All these bacterial taxa have previous been found to be associated with CRC and/or metabolic disorders. We validated the 16S rRNA data by qPCR analysis using genus-specific probes. In the same samples, Pan-Fusobacterium and Bacteroides fragilis were enriched in the CIMP-positive tumors. The median number of Pan-Fusobacterium per 100 human cells was 12-fold higher in the CIMP-positive tumors (p=0.0015) and 6-fold higher in the case of B. fragilis (p=0.0228). Thus, our data show broad difference in the microbiota of CIMP-positive CRCs compared to CIMP-negative CRCs. The significantly enriched bacterial taxa in CIMP-positive CRC suggest that these specific taxa could play an important role in aberrant DNA methylation modulation in colorectal cancer, and further studies should elucidate the mechanism underlying the CIMP/microbiota link in the context of CRC.

Citation Format: Pyoung Hwa Park, Raad Z. Gharaibeh, Lauren Cole, Ang Sun, Woonbok Chung, Jaroslav Jelinek, Jillian L. Pope, Christian Jobin, Jean-Pierre J. Issa. CIMP is associated with altered microbiota composition in colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-141.