Elucidating the colorectal cancer gut microbiome

Abstract

Colorectal cancer (CRC) is one of the most prevalent malignancies in Malaysia and usually diagnosed at advanced stages, leading to poor prognoses. It is a multifactorial disease, in which recent research showed that some of these factors might lead to aberration of the gut microbiome and cause CRC. Numerous research has been carried out in profiling the CRC gut microbiome, in which gut bacteria such as Fusobacterium nucleatum has been identified as being over-represented in CRC patients. In this study, gut mucosal tissues from 18 CRC patients and 18 non-CRC controls were collected from the Endoscopy suite, UKM Medical Centre, Kuala Lumpur, from 2014 to 2017. 16S rRNA gene sequencing targeting the V3/V4 regions was then performed with an Illumina MiSeq® sequencer. Raw FASTQ files of the sequencing results were filtered and trimmed by Trimmomatic v0.36, merged using PEAR v0.98 and then converted to FASTA using the FAST-X Toolkit. Operational taxonomic unit (OTU) and taxanomical analyses were performed using QIIME v1.9.1 against the Greengenes database, and also using the One Codex platform against the Targeted Loci database. Metagenome functional prediction was done using PICRUSt v1.1.2, and differential abundance analysis between CRC and non-CRC gut microbiome was carried out. We also attempted to associate specific gut microbiota with CRC demographics of ethnicity, gender and staging. Parvimonas micra, Fusobacterium nucleatum, Peptostrepotococcus stomatis and Akkermansia muciniphila were found to be over represented in our CRC patients compared to non-CRC controls. This finding was further validated using a species-specific quantitative PCR in a validation cohort of 40 CRC and 20 non-CRC tissues samples from the UMBI-PPUKM Biobank, in which the combination of these four bacteria markers distinguished CRC from controls (AUROC = 0.925). Nevertheless, due to small sample size of the discovery cohort, bacteria identified as significantly different in various CRC demographics and specific to certain ethnicity, gender and CRC staging could not be further verified in our validation cohort, even though it was observed that Gamella morbillorum was abundant in Chinese male CRC patients. In summary, P. micra, F. nucleatum, P. stomatis and A. muciniphila were identified to enriched in our CRC patients of the Malaysian nationality. Nevertheless, a larger, multi-centre study on patients from hospitals in Malaysia will be required to test the reproducibility of these results, before these bacteria could be utilised as screening biomarkers for CRC in Malaysians.