Analysis of 16S rRNA sequencing in advanced colorectal cancer tissue samples

163 Background: Changes in the colon’s microbiota composition are contributors to the pathogenesis of colorectal cancer (CRC). Features of this microbiome may have prognostic significance. For this study, we analyzed 16S rRNA sequencing data from tumor tissue samples of advanced CRC patients and determined if there were potential correlations between microbiome composition and clinical outcomes. Methods: One hundred and thirty three advanced CRC patients in St. Vincent’s Hospital in Korea were enrolled. DNA was extracted from collected tissue samples, the V3-V4 regions were amplified, and a 16S rRNA gene amplicon library was prepared using an Illumina protocol. DNA was sequenced on an Illumina MiSeq instrument. We used three different bioinformatic packages to process the sequence data and evaluate the microbiome composition of each tumor. Results: The classification performances of three different analytic pipelines (Kraken2, QIIME2, and DADA2) were compared in a microbiome control sample. Among these, DADA2 and QIIME2 were chosen for use in subsequent analysis, due to their lower Chi-square (χ2) test statistic values on the control data. After excluding samples that retained less than 5% of total reads after merging, 120 samples were analyzed. The median age of the cohort was 63 years, 63.3% were male, and all were Korean. The distribution over primary sites was 27.5% from the right-side colon, 30.8% from the left-side colon, and 41.7% from the rectum. All subjects received the first line of systemic treatment. The median progression-free survival time was 8.9 months. Twenty-nine patients (24.2%) survived more than 24 months. When examining the results from the two bioinformatic packages, pairwise comparisons showed positive correlations in the relative abundances of the top 20 genera. Fusobacterium, a microbe known to relate to pathogenesis and prognosis of CRC, was not detected by QIIME2. Stratifying by primary site, rectal cancers showed higher alpha diversity than left- or right-side colon cancers. Separately, a higher serum CEA level (≥8) at diagnosis, and the presence of lung metastasis were both found to be related to higher alpha-diversity, a global indicator of microbiome composition. When excluding minimally abundant (< 1% per patient) genera, beta-diversity was found to be differentiable by T stage, the presence of lung metastasis, and the presence of liver metastasis. Most notably, beta-diversity differed between patients who survived more than two years and patients who died within 2 years. Using the DADA2 results, we confirmed that the presence of Fusobacterium nucleatum in CRC tissue was found to be a strong predictor of poor overall survival (OS), along with old age and liver metastasis. Conclusions: This study suggests potential associations between microbiome composition and clinical parameters of advanced CRC. Microbial biomarkers may be a valuable prognostic tool in this population.