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.

BackgroundCisplatin-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.ResultsWe 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.ConclusionOur 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.

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.

BackgroundThe 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.MethodsFrom 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.ResultsDiversity 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.ConclusionUsing 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.

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: We analysed 144 archived faecal samples from participants in the NORwegian Colorectal CAncer Prevention (NORCCAP) trial diagnosed with CRC or high-risk adenomas (HRA) at screening, or who remained cancer-free during 17 years of follow-up. We performed 16S rRNA sequencing of all 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 to healthy controls in both the 16S and metagenome data. The abundance of Bifidobacterium and Lachnospiraceae spp. were 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.

BackgroundColorectal 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.MethodsThe 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.DiscussionThe 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 RegistrationClinicalTrials.gov Identifier: NCT01538550

There is justified optimism regarding the use of miRNAs as early detection biomarkers of cancer. They are well characterized and are involved in all the hallmarks of cancer. Less is known about the role of most other non-coding RNA (ncRNAs) classes in normal physiology and tumorigenesis. The JanusRNA dataset consist of circulating RNA profiles of pre-clinical samples from 1631 cancer patients and 673 cancer-free controls. We studied eight cancer types including cancer of the: lung, colon, rectum, prostate, breast, testis, ovaries and gallbladder. JanusRNA has its origin from the large population-based Janus Serum Bank Cohort which consists of 318 628 Norwegians. The dataset combines information from the complete nationwide cancer registry, RNA sequencing profiles from 1631 cancer patients and 673 cancer-free controls, as well as data on lifestyle, anthropometry and biochemical measurements from national health surveys. The Janus Serum Bank is specifically suited for studies of early detection and risk biomarkers of cancer, since samples are collected nationwide over a large time span, pre-clinically and cancer occurs at different points in time after blood draw. We used a nested case-control design, selecting both cases and controls among the Janus cohort members. We restricted our selection to cases with at least one sample collected within 10 years prior to cancer diagnosis. We selected 673 cancer-free Janus participants for comparison of RNA levels with the cancer cases. The controls were frequency matched to the case group on sex, age at blood donation and date of blood donation. The JanusRNA dataset has been used to investigate the natural variation of circulating RNAs in cancer-free individuals. This data resource was also used in a study of variation in RNA expression associated with common traits like age, sex, smoking, BMI and physical activity in cancer-free individuals. RNA dynamics in lung and testicular carcinogenesis throughout a 10-year follow-up has also been studied.

Testicular cancer (TC) survival rates have increased substantially over the last decades, largely due to the introduction of cisplatin (CP) chemotherapy. This treatment is, however, associated with increased risk of developing metabolic syndrome (MetS), defined according to the National Cholesterol Education Program. We aimed to investigate if CP treatment were associated with epigenetic changes, and whether these changes render TC survivors susceptible for developing MetS later in life. We included 279 Norwegian TC survivors with and without CP treatment and MetS, matched on age at blood sampling (Table 1). The TC survivors were re-examined on average 16 years after the orchiectomy, and for some patients, CP treatment. Whole genome DNA methylation profiles were measured with MethylationEPIC BeadChip and analyzed with the R package minfi. We used a linear regression model adjusting for smoking, age and cell type composition to identify CP differentially methylated CpG sites and logistic regression adjusting for smoking and age to identify differentially methylated CpG sites associated with MetS. Gene enrichment analyses were based on Fisher's exact test using KEGG and Reactome pathways. 32 and 15 differentially methylated CpG sites were associated with CP treatment after adjusting for multiple testing with False Discovery Rate (FDR) and Bonferroni correction, respectively. The PTK6-RAS-MAPk pathway was significantly enriched with the FDR significant CpGs (p-value < 0.1). We could not identify FDR significant differentially methylated CpGs associated with MetS with our sample size. In conclusion, our results suggest that CP treatment has long-term effects on the epigenome. Genes involved in double-strand break repair, consistent with the cytotoxicity of CP treatment, are among the differentially methylated CpGs. Our top list of differentially methylated CpGs associated with MetS (lowest unadjusted p-values), should be further explored in a larger sample set of TC survivors. Table 1: Overview of sample characteristicsCP+ MetS+CP- MetS+CP+ MetS-CP- MetS-N613411569Mean age at surgery31312730Mean age at sample collection49484443Time between surgery and sample collection17181714 Citation Format: Trine B. Rounge, Cecilie Bucher-Johannessen, Christian M. Page, Trine B. Haugen, Sophie D. Fosså, Hege S. Haugnes, Tom Grotmol. Cisplatin treatment of testicular cancer introduces long-term changes to the epigenome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4325.