Abstract 2889: Distinct microbiome in colorectal cancers with the CpG Island Methylator Phenotype
Authors: Sun Ang , Cesaroni Matteo , Jobin Christian , Barrero Carlos , Jelinek Jaroslav , Sautter Jacqueline D. , Rams Thomas E. , Merali Salim , Issa Jean-Pierre 
Published: August 2015

Introduction: Colorectal carcinoma (CRC) can be categorized by aberrant CpG island methylation into CpG Island Methylator Phenotype (CIMP) positive or negative disease. CIMP is also present in many cancers. In glioblastoma, CIMP is associated with IDH mutations and malfunction of ten eleven translocation (TET) enzymes which mediate DNA demethylation. However, IDH and TET mutations are rare in CRC and the mechanism behind CIMP in CRC remains elusive. Recently, our lab reported that Fusobacterium was enriched in CIMP+ CRC cases. In the current study, we explore if there are other CIMP associated microorganisms (CAM) in CRC, and if CIMP is caused by microbial metabolites. Methods: Whole genome sequencing was performed on 25 human CRC (16 CIMP+ and 9 CIMP-) and 19 adjacent normal tissues. The reads that did not map to the human genome were aligned to a microbiome sequence database downloaded from Human Microbiome Project. Bioinformatic analyses were performed to look for CAM. To confirm CAM, qPCR was performed on additional samples. In addition, a multiple reaction monitoring (MRM) method was developed to detect 2-Hydroxygluterate (2HG), an inhibitor of TET enzymes, in candidate bacteria. Results: 1) Unsupervised clustering and Spearman correlation of microbiome data generated two distinct sample groups. One contained 13 cases, all CIMP+ CRC (CIMP+ enriched group, G1). The other had 6 CIMP- CRC and 17 adjacent normal tissues (CIMP- and adjacent normal tissue enriched group, G2). 2) Volcano plot and statistical analysis revealed that 656 and 1051 species/strains were present in G1 and G2, respectively. 3) There was greater (p&lt;0.05) microbial diversity in G2 (&gt;90 species, including bacteria, viruses, and eukaryotes) than in G1 (16 species, all bacteria, including Klebsiella pneumoniae. Most of them belong to Family Enterobacteriaceae). 4) qPCR showed that for CIMP1 CRC, 11/44 (25%) were positive for Klebsiella sp. including 7 (15.9%) that were positive for K. pneumoniae, while only 1/27 (3.7%) CIMP2, 2/45 (4.4%) CIMP0 CRC, and 2/49 (4%) adjacent normal tissues were positive for Klebsiella sp.. 5) We used qPCR to test for pks+ (enterotoxin associated with E. coli and K. pneumoniae) bacteria. Pks was found in 16/83 (19.3%) CRC, including 13/51 (25.5%) CIMP+ CRC, 3/32 (9.4%) CIMP- CRC and 6/66 (9.1%) adjacent normal tissues. 6) MRM detected 2HG production in Fusobacterium nucleatum cultures but not in K. pneumoniae or E.coli. Conclusions: CIMP+ CRC are associated with a distinct microbiome including pks containing bacteria (K. pneumoniae and E. coli) and 2HG producing bacteria (Fusobacterium), raising the possibility that the methylation defect could be contributed to by the microbiome.

Citation Format: Ang Sun, Matteo Cesaroni, Christian Jobin, Carlos Barrero, Jaroslav Jelinek, Jacqueline D. Sautter, Thomas E. Rams, Salim Merali, Jean-Pierre Issa. Distinct microbiome in colorectal cancers with the CpG Island Methylator Phenotype. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2889. doi:10.1158/1538-7445.AM2015-2889