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Background: Annually, colorectal cancer (CRC) is diagnosed in >1.4 million subjects worldwide and incidence is increasing. Much effort has therefore been focused on screening, which has proven to reduce cancer-related mortality. The Sept9 DNA-methylation assay is among the most well studied blood-based screening markers. However, earlier reported performances may be misleading: the Sept9 test was recently examined in two screening based cohorts and yielded performances lower than expected. We hypothesize that comorbidities and/or demographic characteristics affect the results of the Sept9 test.Methods: Using a retrospective nested case-control study design, we studied plasma from 150 cancer and 150 controls selected from a well-characterized cohort of 4698 subjects referred for diagnostic colonoscopy due to CRC-related symptoms. The cases and controls were matched on age and gender, and moreover cases were stratified on tumor-site and tumor-stage. The selected cohort included a wide range of comorbidities. Plasma Sept9 levels were assessed using a commercially available PCR based assay (Epi-proColon).Results: Clinical sensitivity for CRC stages I-IV was 37 %, 91 %, 77 %, and 89 %, and the overall sensitivity 73 % (95 % CI, 64-80 %) and specificity 82 % (95 % CI, 75-88 %), respectively. Age >65 was associated with both increased false positive and false negative results (p < 0.05). Arthritis was associated with a higher false negative rate (p = 0.005) whereas Arteriosclerosis was associated with a higher false positive rate (p = 0.007). Diabetes was associated with Sept9 positivity with an OR of 5.2 (95 % CI 1.4-19.1). When the performance of Sept9 was adjusted for these parameters in a final multivariate regression model, the OR for a positive Sept9 test to be associated with CRC increased from 8.25 (95 % CI 4.83-14.09) to 29.46 (95 % CI 12.58-69.02).Conclusions: The results indicate that the performance of the Sept9 assay is negatively affected by several factors commonly associated with CRC screening populations: early-stage disease, age > 65 years, diabetes, arthritis, and arteriosclerosis. This should be taken into account if the Sept9 assay is used as a single marker for CRC screening, but may also have a wider impact, as it is likely that such factors may affect other blood based DNA markers as well. [ABSTRACT FROM AUTHOR]

The cis-regulatory effects responsible for cancer development have not been as extensively studied as the perturbations of the protein coding genome in tumorigenesis. To better characterize colorectal cancer (CRC) development we conducted an RNA-sequencing experiment of 103 matched tumour and normal colon mucosa samples from Danish CRC patients, 90 of which were germline-genotyped. By investigating allele-specific expression (ASE) we show that the germline genotypes remain important determinants of allelic gene expression in tumours. Using the changes in ASE in matched pairs of samples we discover 71 genes with excess of somatic cis-regulatory effects in CRC, suggesting a cancer driver role. We correlate genotypes and gene expression to identify expression quantitative trait loci (eQTLs) and find 1,693 and 948 eQTLs in normal samples and tumours, respectively. We estimate that 36% of the tumour eQTLs are exclusive to CRC and show that this specificity is partially driven by increased expression of specific transcription factors and changes in methylation patterns. We show that tumour-specific eQTLs are more enriched for low CRC genome-wide association study (GWAS) P values than shared eQTLs, which suggests that some of the GWAS variants are tumour specific regulatory variants. Importantly, tumour-specific eQTL genes also accumulate more somatic mutations when compared to the shared eQTL genes, raising the possibility that they constitute germline-derived cancer regulatory drivers. Collectively the integration of genome and the transcriptome reveals a substantial number of putative somatic and germline cis-regulatory cancer changes that may have a role in tumorigenesis. [ABSTRACT FROM AUTHOR]

Background: Patterns of genome-wide methylation vary between tissue types. For example, cancer tissue shows markedly different patterns from those of normal tissue. In this paper we propose a beta-mixture model to describe genome-wide methylation patterns based on probe data from methylation microarrays. The model takes dependencies between neighbour probe pairs into account and assumes three broad categories of methylation, low, medium and high. The model is described by 37 parameters, which reduces the dimensionality of a typical methylation microarray significantly. We used methylation microarray data from 42 colon cancer samples to assess the model. Results: Based on data from colon cancer samples we show that our model captures genome-wide characteristics of methylation patterns. We estimate the parameters of the model and show that they vary between different tissue types. Further, for each methylation probe the posterior probability of a methylation state (low, medium or high) is calculated and the probability that the state is correctly predicted is assessed. We demonstrate that the model can be applied to classify cancer tissue types accurately and that the model provides accessible and easily interpretable data summaries. Conclusions: We have developed a beta-mixture model for methylation microarray data. The model substantially reduces the dimensionality of the data. It can be used for further analysis, such as sample classification or to detect changes in methylation status between different samples and tissues. [ABSTRACT FROM AUTHOR]

Background: Mutations in the mismatch repair genes hMLH1 and hMSH2 predispose to hereditary non-polyposis colorectal cancer (HNPCC). Genetic screening of more than 350 Danish patients with colorectal cancer (CRC) has led to the identification of several new genetic variants (e.g. missense, silent and non-coding) in hMLH1 and hMSH2. The aim of the present study was to investigate the frequency of these variants in hMLH1 and hMSH2 in Danish patients with sporadic colorectal cancer and in the healthy background population. The purpose was to reveal if any of the common variants lead to increased susceptibility to colorectal cancer. Methods: Associations between genetic variants in hMLH1 and hMSH2 and sporadic colorectal cancer were evaluated using a case-cohort design. The genotyping was performed on DNA isolated from blood from the 380 cases with sporadic colorectal cancer and a sub-cohort of 770 individuals. The DNA samples were analyzed using Single Base Extension (SBE) Tag-arrays. A Bonferroni corrected Fisher exact test was used to test for association between the genotypes of each variant and colorectal cancer. Linkage disequilibrium (LD) was investigated using HaploView (v3.31). Results: Heterozygous and homozygous changes were detected in 13 of 35 analyzed variants. Two variants showed a borderline association with colorectal cancer, whereas the remaining variants demonstrated no association. Furthermore, the genomic regions covering hMLH1 and hMSH2 displayed high linkage disequilibrium in the Danish population. Twenty-two variants were neither detected in the cases with sporadic colorectal cancer nor in the sub-cohort. Some of these rare variants have been classified either as pathogenic mutations or as neutral variants in other populations and some are unclassified Danish variants. Conclusion: None of the variants in hMLH1 and hMSH2 analyzed in the present study were highly associated with colorectal cancer in the Danish population. High linkage disequilibrium in the genomic regions covering hMLH1 and hMSH2, indicate that common genetic variants in the two genes in general are not involved in the development of sporadic colorectal cancer. Nevertheless, some of the rare unclassified variants in hMLH1 and hMSH2 might be involved in the development of colorectal cancer in the families where they were originally identified. [ABSTRACT FROM AUTHOR]

• The impact of bowel preparation solutions on blood-based, cancer-associated biomarkers have been poorly investigated. • 125 subjects scheduled for a colonoscopy investigating potential colorectal cancer contributed with two blood samples. The collection was before and after usage of bowel preparation solution. • The study demonstrated a systematic, statistical significant difference between pre- and post bowel preparation in three independent biomarkers (BAG4, CyFra21-1 and cell-free DNA). Blood-based, cancer-associated biomarkers are susceptible to a variety of well-known preanalytical factors. The influence of bowel preparation before a diagnostic colonoscopy on biomarker levels is, however, poorly investigated. The present study assessed the influence of bowel preparation on colorectal cancer-associated biomarkers. In addition, the effect of single versus double centrifugation of plasma biomarkers was assessed. Blood samples were collected pre- and post-bowel preparation from 125 subjects scheduled for first time diagnostic colonoscopy due to symptoms attributable to CRC. The samples were separated into serum and EDTA plasma, and analyzed by four independent collaborators for: 1) the proteins AFP, CA19-9, CEA, hs-CRP, CyFra21-1, Ferritin, Galectin-3 and TIMP-1, 2) the proteins BAG4, IL6ST, vWF, CD44 and EGFR, 3) the glycoprotein Galectin-3 ligand, and 4) cell-free DNA (cfDNA). Statistical analysis of biomarker data has been performed using mixed modelling, including repeated measures. The biomarkers generally showed negligible variation between pre- and post-bowel preparation except for CyFra21-1, Ferritin, BAG4 and cfDNA. CyFra21-1 levels were systematically reduced with 29% (95% CI 21–36%) by bowel preparation (p ≤ 0.0001). Ferritin was not significantly different between pre- and post-bowel preparation (p = 0.07), however the estimated difference (increase) was 18%. BAG4 was systematically reduced by 12% (95% CI 1–22%, p = 0.04), while cfDNA showed a significant increase of 28% (95% CI 17–39%, p < 0.0001). Double centrifugation compared to single centrifugation showed reduced vWF (ratio 0.86, p ≤ 0.0001) and CD44 (ratio 0.85, p = 0.016), but increased IL6ST levels (ratio 1.18, p = 0.014). Results of the present study demonstrated systematic, statistically significant differences between pre-bowel and post-bowel preparation levels for three independent blood-based biomarkers (BAG4, CyFra21-1, cfDNA), illustrating the importance of timing of sample collection for biomarker analyses. [ABSTRACT FROM AUTHOR]