The CRCbiome study: a large prospective cohort study examining the role of lifestyle and the gut microbiome in colorectal cancer screening participants.

BACKGROUND: Colorectal cancer (CRC) screening reduces CRC incidence and mortality. However, current screening methods are either hampered by invasiveness or suboptimal performance, limiting their effectiveness as primary screening methods. To aid in the development of a non-invasive screening test with improved sensitivity and specificity, we have initiated a prospective biomarker study (CRCbiome), nested within a large randomized CRC screening trial in Norway. We aim to develop a microbiome-based classification algorithm to identify advanced colorectal lesions in screening participants testing positive for an immunochemical fecal occult blood test (FIT). We will also examine interactions with host factors, diet, lifestyle and prescription drugs. The prospective nature of the study also enables the analysis of changes in the gut microbiome following the removal of precancerous lesions. METHODS: The CRCbiome study recruits participants enrolled in the Bowel Cancer Screening in Norway (BCSN) study, a randomized trial initiated in 2012 comparing once-only sigmoidoscopy to repeated biennial FIT, where women and men aged 50-74 years at study entry are invited to participate. Since 2017, participants randomized to FIT screening with a positive test result have been invited to join the CRCbiome study. Self-reported diet, lifestyle and demographic data are collected prior to colonoscopy after the positive FIT-test (baseline). Screening data, including colonoscopy findings are obtained from the BCSN database. Fecal samples for gut microbiome analyses are collected both before and 2 and 12 months after colonoscopy. Samples are analyzed using metagenome sequencing, with taxonomy profiles, and gene and pathway content as primary measures. CRCbiome data will also be linked to national registries to obtain information on prescription histories and cancer relevant outcomes occurring during the 10 year follow-up period. DISCUSSION: The CRCbiome study will increase our understanding of how the gut microbiome, in combination with lifestyle and environmental factors, influences the early stages of colorectal carcinogenesis. This knowledge will be crucial to develop microbiome-based screening tools for CRC. By evaluating biomarker performance in a screening setting, using samples from the target population, the generalizability of the findings to future screening cohorts is likely to be high. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01538550 .

A comprehensive profile of circulating RNAs in human serum.

Non-coding RNA (ncRNA) molecules have fundamental roles in cells and many are also stable in body fluids as extracellular RNAs. In this study, we used RNA sequencing (RNA-seq) to investigate the profile of small non-coding RNA (sncRNA) in human serum. We analyzed 10 billion Illumina reads from 477 serum samples, included in the Norwegian population-based Janus Serum Bank (JSB). We found that the core serum RNA repertoire includes 258 micro RNAs (miRNA), 441 piwi-interacting RNAs (piRNA), 411 transfer RNAs (tRNA), 24 small nucleolar RNAs (snoRNA), 125 small nuclear RNAs (snRNA) and 123 miscellaneous RNAs (misc-RNA). We also investigated biological and technical variation in expression, and the results suggest that many RNA molecules identified in serum contain signs of biological variation. They are therefore unlikely to be random degradation by-products. In addition, the presence of specific fragments of tRNA, snoRNA, Vault RNA and Y_RNA indicates protection from degradation. Our results suggest that many circulating RNAs in serum can be potential biomarkers.

Species-level verification of Phascolarctobacterium association with colorectal cancer.

We have previously demonstrated an association between increased abundance of Phascolarctobacterium and colorectal cancer (CRC) and adenomas in two independent Norwegian cohorts. Here we seek to verify our previous findings using new cohorts and methods. In addition, we characterize lifestyle and sex specificity, the functional potential of the Phascolarctobacterium species, and their interaction with other microbial species. We analyze Phascolarctobacterium with 16S rRNA sequencing, shotgun metagenome sequencing, and species-specific qPCR, using 2350 samples from three Norwegian cohorts-CRCAhus, NORCCAP, and CRCbiome-and a large publicly available data set, curatedMetagenomicData. Using metagenome-assembled genomes from the CRCbiome study, we explore the genomic characteristics and functional potential of the Phascolarctobacterium pangenome. Three species of Phascolarctobacterium associated with adenoma/CRC were consistently detected by qPCR and sequencing. Positive associations with adenomas/CRC were verified for Phascolarctobacterium succinatutens and negative associations were shown for Phascolarctobacterium faecium and adenoma in curatedMetagenomicData. Men show a higher prevalence of P. succinatutens across cohorts. Co-occurrence among Phascolarctobacterium species was low (<6%). Each of the three species shows distinct microbial composition and forms distinct correlation networks with other bacterial taxa, although Dialister invisus was negatively correlated to all investigated Phascolarctobacterium species. Pangenome analyses showed P. succinatutens to be enriched for genes related to porphyrin metabolism and degradation of complex carbohydrates, whereas glycoside hydrolase enzyme 3 was specific to P. faecium.IMPORTANCEUntil now Phascolarctobacterium has been going under the radar as a CRC-associated genus despite having been noted, but overseen, as such for over a decade. We found not just one, but two species of Phascolarctobacterium to be associated with CRC-Phascolarctobacterium succinatutens was more abundant in adenoma/CRC, while Phascolarctobacterium faecium was less abundant in adenoma. Each of them represents distinct communities, constituted by specific microbial partners and metabolic capacities-and they rarely occur together in the same patients. We have verified that P. succinatutens is increased in adenoma and CRC and this species should be recognized among the most important CRC-associated bacteria.

Long-term follow-up of colorectal cancer screening attendees identifies differences in Phascolarctobacterium spp. using 16S rRNA and metagenome sequencing.

Background: The microbiome has been implicated in the initiation and progression of colorectal cancer (CRC) in cross-sectional studies. However, there is a lack of studies using prospectively collected samples. Methods: From the Norwegian Colorectal Cancer Prevention (NORCCAP) trial, we analyzed 144 archived fecal samples from participants who were diagnosed with CRC or high-risk adenoma (HRA) at screening and from participants who remained cancer-free during 17 years of follow-up. We performed 16S rRNA sequencing of all the samples and metagenome sequencing on a subset of 47 samples. Differences in taxonomy and gene content between outcome groups were assessed for alpha and beta diversity and differential abundance. Results: Diversity and composition analyses showed no significant differences between CRC, HRA, and healthy controls. Phascolarctobacterium succinatutens was more abundant in CRC compared with healthy controls in both the 16S and metagenome data. The abundance of Bifidobacterium and Lachnospiraceae spp. was associated with time to CRC diagnosis. Conclusion: Using a longitudinal study design, we identified three taxa as being potentially associated with CRC. These should be the focus of further studies of microbial changes occurring prior to CRC diagnosis.

Cisplatin treatment of testicular cancer patients introduces long-term changes in the epigenome.

BACKGROUND: Cisplatin-based chemotherapy (CBCT) is part of standard treatment of several cancers. In testicular cancer (TC) survivors, an increased risk of developing metabolic syndrome (MetS) is observed. In this epigenome-wide association study, we investigated if CBCT relates to epigenetic changes (DNA methylation) and if epigenetic changes render individuals susceptible for developing MetS later in life. We analyzed methylation profiles, using the MethylationEPIC BeadChip, in samples collected ~ 16 years after treatment from 279 Norwegian TC survivors with known MetS status. Among the CBCT treated (n = 176) and non-treated (n = 103), 61 and 34 developed MetS, respectively. We used two linear regression models to identify if (i) CBCT results in epigenetic changes and (ii) epigenetic changes play a role in development of MetS. Then we investigated if these changes in (i) and (ii) links to genes, functional networks, and pathways related to MetS symptoms. RESULTS: We identified 35 sites that were differentially methylated when comparing CBCT treated and untreated TC survivors. The PTK6-RAS-MAPk pathway was significantly enriched with these sites and infers a gene network of 13 genes with CACNA1D (involved in insulin release) as a network hub. We found nominal MetS-associations and a functional gene network with ABCG1 and NCF2 as network hubs. CONCLUSION: Our results suggest that CBCT has long-term effects on the epigenome. We could not directly link the CBCT effects to the risk of developing MetS. Nevertheless, since we identified differential methylation occurring in genes associated with conditions pertaining to MetS, we hypothesize that epigenomic changes may also play a role in the development of MetS in TC survivors. Further studies are needed to validate this hypothesis.