Evaluation of the "Burgenland PREvention trial of colorectal cancer Disease with ImmunologiCal Testing" (B-PREDICT)-a population-based colorectal cancer screening program.

BACKGROUND: The colorectal cancer (CRC) screening program B-PREDICT is a population based invited two stage screening project using a faecal immunochemical test (FIT) for initial screening followed by a colonoscopy for those with a positive FIT. B-PREDICT was compared with the opportunistic screening colonoscopy (OPP-COL), performed in course of the nationwide screening program. METHODS: Within B-PREDICT all residents of the Austrian federal state Burgenland, aged between 40 and 80 are annually invited to FIT testing. All individuals who underwent initial colonoscopy in Burgenland between 01/2003 and 12/2014, were included in this study. Individuals from the FIT-triggered invited screening program B-PREDICT were compared with those from the non-FIT triggered OPP-COL. RESULTS: 15 133 individuals from B-PREDICT were compared to 10 045 individuals with OPP-COL. CRC detection rates were 1.34% (CI-95%, [1.15; 1.52]) in B-PREDICT compared to 0.54% in OPP-COL (95%-CI, [0.39; 0.68] p < 0.001). The decrease in the age standardized incidence rates of CRC was more pronounced in the population screened with FIT than in the general population screened with colonoscopy. Changes in incidence rates per year were -4.4% (95%-CI, [-5.1; -3.7]) vs. -1.8% (95%-CI, [-1.9; -1.6] p < 0.001). CONCLUSIONS: B-PREDICT shows a two-fold higher detection rate of CRC as well as HRA compared to OPP-COL.

Using fecal immmunochemical cartridges for gut microbiome analysis within a colorectal cancer screening program.

The colorectal cancer (CRC) screening program B-PREDICT is an invited two-stage screening project using a fecal immunochemical test (FIT) for initial screening followed by a colonoscopy for those with a positive FIT. Since the gut microbiome likely plays a role in the etiology of CRC, microbiome-based biomarkers in combination with FIT could be a promising tool for optimizing CRC screening. Therefore, we evaluated the usability of FIT cartridges for microbiome analysis and compared it to Stool Collection and Preservation Tubes. Corresponding FIT cartridges as well as Stool Collection and Preservation Tubes were collected from participants of the B-PREDICT screening program to perform 16S rRNA gene sequencing. We calculated intraclass correlation coefficients (ICCs) based on center log ratio transformed abundances and used ALDEx2 to test for significantly differential abundant taxa between the two sample types. Additionally, FIT and Stool Collection and Preservation Tube triplicate samples were obtained from volunteers to estimate variance components of microbial abundances. FIT and Preservation Tube samples produce highly similar microbiome profiles which cluster according to subject. Significant differences between the two sample types can be found for abundances of some bacterial taxa (e.g. 33 genera) but are minor compared to the differences between the subjects. Analysis of triplicate samples revealed slightly worse repeatability of results for FIT than for Preservation Tube samples. Our findings indicate that FIT cartridges are appropriate for gut microbiome analysis nested within CRC screening programs.

Untargeted Metabolomics Reveals Major Differences in the Plasma Metabolome between Colorectal Cancer and Colorectal Adenomas.

Sporadic colorectal cancer is characterized by a multistep progression from normal epithelium to precancerous low-risk and high-risk adenomas to invasive cancer. Yet, the underlying molecular mechanisms of colorectal carcinogenesis are not completely understood. Within the "Metabolomic profiles throughout the continuum of colorectal cancer" (MetaboCCC) consortium we analyzed data generated by untargeted, mass spectrometry-based metabolomics using plasma from 88 colorectal cancer patients, 200 patients with high-risk adenomas and 200 patients with low-risk adenomas recruited within the "Colorectal Cancer Study of Austria" (CORSA). Univariate logistic regression models comparing colorectal cancer to adenomas resulted in 442 statistically significant molecular features. Metabolites discriminating colorectal cancer patients from those with adenomas in our dataset included acylcarnitines, caffeine, amino acids, glycerophospholipids, fatty acids, bilirubin, bile acids and bacterial metabolites of tryptophan. The data obtained discovers metabolite profiles reflecting metabolic differences between colorectal cancer and colorectal adenomas and delineates a potentially underlying biological interpretation.

Plasma metabolites associated with colorectal cancer stage: Findings from an international consortium.

Colorectal cancer is the second most common cause of cancer-related death globally, with marked differences in prognosis by disease stage at diagnosis. We studied circulating metabolites in relation to disease stage to improve the understanding of metabolic pathways related to colorectal cancer progression. We investigated plasma concentrations of 130 metabolites among 744 Stages I-IV colorectal cancer patients from ongoing cohort studies. Plasma samples, collected at diagnosis, were analyzed with liquid chromatography-mass spectrometry using the Biocrates AbsoluteIDQ™ p180 kit. We assessed associations between metabolite concentrations and stage using multinomial and multivariable logistic regression models. Analyses were adjusted for potential confounders as well as multiple testing using false discovery rate (FDR) correction. Patients presented with 23, 28, 39 and 10% of Stages I-IV disease, respectively. Concentrations of sphingomyelin C26:0 were lower in Stage III patients compared to Stage I patients (pFDR < 0.05). Concentrations of sphingomyelin C18:0 and phosphatidylcholine (diacyl) C32:0 were statistically significantly higher, while citrulline, histidine, phosphatidylcholine (diacyl) C34:4, phosphatidylcholine (acyl-alkyl) C40:1 and lysophosphatidylcholines (acyl) C16:0 and C17:0 concentrations were lower in Stage IV compared to Stage I patients (pFDR < 0.05). Our results suggest that metabolic pathways involving among others citrulline and histidine, implicated previously in colorectal cancer development, may also be linked to colorectal cancer progression.

Genome-wide association analysis of diverticular disease points towards neuromuscular, connective tissue and epithelial pathomechanisms.

OBJECTIVE: Diverticular disease is a common complex disorder characterised by mucosal outpouchings of the colonic wall that manifests through complications such as diverticulitis, perforation and bleeding. We report the to date largest genome-wide association study (GWAS) to identify genetic risk factors for diverticular disease. DESIGN: Discovery GWAS analysis was performed on UK Biobank imputed genotypes using 31 964 cases and 419 135 controls of European descent. Associations were replicated in a European sample of 3893 cases and 2829 diverticula-free controls and evaluated for risk contribution to diverticulitis and uncomplicated diverticulosis. Transcripts at top 20 replicating loci were analysed by real-time quatitative PCR in preparations of the mucosal, submucosal and muscular layer of colon. The localisation of expressed protein at selected loci was investigated by immunohistochemistry. RESULTS: We discovered 48 risk loci, of which 12 are novel, with genome-wide significance and consistent OR in the replication sample. Nominal replication (p<0.05) was observed for 27 loci, and additional 8 in meta-analysis with a population-based cohort. The most significant novel risk variant rs9960286 is located near CTAGE1 with a p value of 2.3×10-10 and 0.002 (ORallelic=1.14 (95% CI 1.05 to 1.24)) in the replication analysis. Four loci showed stronger effects for diverticulitis, PHGR1 (OR 1.32, 95% CI 1.12 to 1.56), FAM155A-2 (OR 1.21, 95% CI 1.04 to 1.42), CALCB (OR 1.17, 95% CI 1.03 to 1.33) and S100A10 (OR 1.17, 95% CI 1.03 to 1.33). CONCLUSION: In silico analyses point to diverticulosis primarily as a disorder of intestinal neuromuscular function and of impaired connective fibre support, while an additional diverticulitis risk might be conferred by epithelial dysfunction.

Plasma metabolites associated with colorectal cancer: A discovery-replication strategy.

Colorectal cancer is known to arise from multiple tumorigenic pathways; however, the underlying mechanisms remain not completely understood. Metabolomics is becoming an increasingly popular tool in assessing biological processes. Previous metabolomics research focusing on colorectal cancer is limited by sample size and did not replicate findings in independent study populations to verify robustness of reported findings. Here, we performed a ultrahigh performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) screening on EDTA plasma from 268 colorectal cancer patients and 353 controls using independent discovery and replication sets from two European cohorts (ColoCare Study: n = 180 patients/n = 153 controls; the Colorectal Cancer Study of Austria (CORSA) n = 88 patients/n = 200 controls), aiming to identify circulating plasma metabolites associated with colorectal cancer and to improve knowledge regarding colorectal cancer etiology. Multiple logistic regression models were used to test the association between disease state and metabolic features. Statistically significant associated features in the discovery set were taken forward and tested in the replication set to assure robustness of our findings. All models were adjusted for sex, age, BMI and smoking status and corrected for multiple testing using False Discovery Rate. Demographic and clinical data were abstracted from questionnaires and medical records.

The Immunome of Colon Cancer: Functional In Silico Analysis of Antigenic Proteins Deduced from IgG Microarray Profiling.

Characterization of the colon cancer immunome and its autoantibody signature from differentially-reactive antigens (DIRAGs) could provide insights into aberrant cellular mechanisms or enriched networks associated with diseases. The purpose of this study was to characterize the antibody profile of plasma samples from 32 colorectal cancer (CRC) patients and 32 controls using proteins isolated from 15,417 human cDNA expression clones on microarrays. 671 unique DIRAGs were identified and 632 were more highly reactive in CRC samples. Bioinformatics analyses reveal that compared to control samples, the immunoproteomic IgG profiling of CRC samples is mainly associated with cell death, survival, and proliferation pathways, especially proteins involved in EIF2 and mTOR signaling. Ribosomal proteins (e.g., RPL7, RPL22, and RPL27A) and CRC-related genes such as APC, AXIN1, E2F4, MSH2, PMS2, and TP53 were highly enriched. In addition, differential pathways were observed between the CRC and control samples. Furthermore, 103 DIRAGs were reported in the SEREX antigen database, demonstrating our ability to identify known and new reactive antigens. We also found an overlap of 7 antigens with 48 "CRC genes." These data indicate that immunomics profiling on protein microarrays is able to reveal the complexity of immune responses in cancerous diseases and faithfully reflects the underlying pathology.

Leukocyte telomere length throughout the continuum of colorectal carcinogenesis.

Considering the high prevalence of colorectal cancer (CRC) and relatively high mortality there is strong interest in identification of clinically relevant biomarkers. Telomere shortening is supposed to contribute to genomic instability and crucially involved in process of carcinogenesis. Peripheral blood leukocyte (PBL) telomere length was previously investigated in several studies as potential biomarker for CRC but with controversial results. This prompted us to investigate relative PBL telomere length in association with different histological findings throughout the continuum of colorectal carcinogenesis in order to reflect the whole spectrum of putative CRC development in a large study involving 2011 individuals. The study based on the Colorectal Cancer Study of Austria (CORSA), including 384 CRC cases as well as age- and gender-matched 544 high-risk adenomas, 537 low-risk adenoma patients and 546 colonoscopy-negative controls. Relative expression of telomeric repeats and the single copy reference gene, albumin (T/S ratio) was determined using monochrome multiplex quantitative PCR (MMQPCR). Telomeres were found to be significantly longer in CRC patients compared to control subjects (P = 3.61x10-6). Yet, no significant differences in telomere length could be detected for high-risk (P = 0.05956) and low-risk colorectal adenoma patients (P = 0.05224). In addition, results presented in this manuscript highlight the impact of various epidemiological factors on PBL telomere length and its involvement in CRC. However, further large studies also including colorectal adenomas are necessary to confirm these results.

Bayesian and frequentist analysis of an Austrian genome-wide association study of colorectal cancer and advanced adenomas.

Most genome-wide association studies (GWAS) were analyzed using single marker tests in combination with stringent correction procedures for multiple testing. Thus, a substantial proportion of associated single nucleotide polymorphisms (SNPs) remained undetected and may account for missing heritability in complex traits. Model selection procedures present a powerful alternative to identify associated SNPs in high-dimensional settings. In this GWAS including 1060 colorectal cancer cases, 689 cases of advanced colorectal adenomas and 4367 controls we pursued a dual approach to investigate genome-wide associations with disease risk applying both, single marker analysis and model selection based on the modified Bayesian information criterion, mBIC2, implemented in the software package MOSGWA. For different case-control comparisons, we report models including between 1-14 candidate SNPs. A genome-wide significant association of rs17659990 (P=5.43×10-9, DOCK3, chromosome 3p21.2) with colorectal cancer risk was observed. Furthermore, 56 SNPs known to influence susceptibility to colorectal cancer and advanced adenoma were tested in a hypothesis-driven approach and several of them were found to be relevant in our Austrian cohort. After correction for multiple testing (α=8.9×10-4), the most significant associations were observed for SNPs rs10505477 (P=6.08×10-4) and rs6983267 (P=7.35×10-4) of CASC8, rs3802842 (P=8.98×10-5, COLCA1,2), and rs12953717 (P=4.64×10-4, SMAD7). All previously unreported SNPs demand replication in additional samples. Reanalysis of existing GWAS datasets using model selection as tool to detect SNPs associated with a complex trait may present a promising resource to identify further genetic risk variants not only for colorectal cancer.

Association of genetic variants of human telomerase with colorectal polyps and colorectal cancer risk.

Human telomerase reverse transcriptase (TERT) gene encodes the catalytic subunit of telomerase and is located on chromosome 5p15, a genomic region which was found to be associated with multiple cancer types. But no associations with colorectal cancer (CRC) have been reported until recently. Therefore, the purpose of this study was to investigate the influence of seven single-nucleotide polymorphisms (SNPs) of TERT on susceptibility to colorectal polyps and CRC. The study population of our ongoing colorectal cancer study of Austria (CORSA) comprised 3,842 Caucasian participants. A total of 3,264 participants was genotyped including 142 CRC cases, 492 high-risk polyps, 837 low-risk polyps, and 1,793 polyp-free controls verified by colonoscopy. Genotyping was performed by TaqMan assay using genomic DNA. The impact of each SNP was estimated by multiple logistic regression analyses performed with R Version 2.11.1. None of the investigated TERT SNPs (rs2736122, rs2853676, rs2735940, rs2736098, rs2075786, rs2736100, rs4975605) were found to be associated with risk of CRC nor colonic polyps. However, the haplotype CGTATGG was associated with a significantly increased risk of high-risk polyps (OR = 1.48, 95% CI 1.01-2.17, P = 0.043). In accordance with other studies our results suggest no major influence of the investigated TERT SNPs on CRC and colorectal polyp risk. However, relevance of telomerase in tumorigenesis of multiple malignancies demands further investigations of the 5p15 locus concerning CRC susceptibility.

No association of XRCC1 polymorphisms Arg194Trp and Arg399Gln with colorectal cancer risk.

BACKGROUND: X-ray repair cross complementation group 1 (XRCC1) plays a key role in base excision repair. The purpose of this study was to examine the association of two genetic polymorphisms in XRCC1 (rs1799782 and rs25487) with risk of colorectal polyps and colorectal cancer (CRC). METHODS: In the ongoing colorectal cancer study of Austria (CORSA), a total of 3091 Caucasian participants was genotyped using 5'-nuclease TaqMan assays. Multiple logistic regression was applied to compare individuals of the control group against three different case groups namely CRC cases, high-risk and low-risk polyps. RESULTS: The two investigated SNPs in XRCC1 were not found to be associated with neither CRC risk nor polyp risk. Comparing the CRC cases versus the controls the OR was 0.60 (95%CI 0.27-1.31) for the heterozygous polymorphic genotype of SNP rs1799782 and 1.47 (95%CI 0.81-2.65) for the homozygous polymorphic genotype of SNP rs25487. Comparing the high-risk polyp group versus the controls the OR was 2.64 (95%CI 0.61-11.42) for the homozygous polymorphic genotype of SNP rs1799782 and 0.89 (95%CI 0.60-1.33) for SNP rs25487, respectively. In an haplotype analysis also no statistically significant association was found. CONCLUSION: Our finding that none of the two investigated SNPs of XRCC1 were significantly associated with risk of CRC or polyps is consistent with the results of a recently published meta-analysis.

MNS16A tandem repeats minisatellite of human telomerase gene: a risk factor for colorectal cancer.

Telomerase reactivation and expression of human telomerase gene [human telomerase reverse transcriptase (hTERT)] are hallmarks of unlimited proliferation potential of cancer cells. A polymorphic tandem repeats minisatellite of hTERT gene, termed MNS16A was reported to influence hTERT expression. To assess the role of MNS16A as potential biomarker for colorectal cancer (CRC), we investigated for the first time the association of MNS16A genotypes with risk of colorectal polyps and CRC. In the ongoing colorectal cancer study of Austria (CORSA), 3842 Caucasian participants were recruited within a large screening project in the province Burgenland including 90 CRC cases, 308 high-risk polyps, 1022 low-risk polyps and 1822 polyp free controls verified by colonoscopy. MNS16A genotypes were determined by polymerase chain reaction from genomic DNA. Associations of MNS16A genotypes with CRC risk were estimated by logistic regression analysis computing odds ratios (ORs) and 95% confidence intervals (CIs). We identified five different variable number of tandem repeats (VNTRs) of MNS16A including VNTR-364, a newly discovered rare variant. VNTR-274 allele was associated with a 2.7-fold significantly increased risk of CRC compared with the VNTR-302 wild-type (OR = 2.69; 95% CI = 1.11-6.50; P = 0.028). In our CORSA study, the medium length VNTR-274 was identified as risk factor for CRC. Although, this population-based study herewith reports the largest cohort size concerning MNS16A thus far, further large-scale studies in diverse populations are warranted to confirm hTERT MNS16A genotype as potential biomarker for assessment of CRC risk.

Association of IGF1 and IGFBP3 polymorphisms with colorectal polyps and colorectal cancer risk.

PURPOSE: Insulin-like growth factor 1 (IGF1) is a peptide growth factor that promotes cell proliferation and inhibits apoptosis. The bioavailability of IGF1 is regulated by the insulin-like growth factor binding protein 3 (IGFBP3). The purpose of this study was to examine the association of genetic variants in IGF1 (rs6214, rs6220, and rs35767) and IGFBP3 (rs2854744 and rs2854746) with risk of colorectal polyps and colorectal cancer. METHODS: In this ongoing colorectal cancer study of Austria (CORSA), a total of 3,360 Caucasian participants, consisting of 178 colorectal cancer patients, 328 patients with high risk polyps, 1,059 patients with low risk colorectal polyps, and 1,795 colonoscopy-negative controls, were recruited within a large colorectal screening project in the province Burgenland and from three hospitals in Vienna. Multiple logistic regression was applied to compare individuals of the control group against three different risk groups, namely, colorectal cancer group, high risk polyp group, and low risk polyp group. RESULTS: Carriers of the homozygous polymorphic genotype of the SNP rs6214 were associated with an increased colorectal risk (OR = 1.79, 95% CI 1.04-1.90) compared to the colonoscopy-negative controls; this was also found when combining colorectal cancer cases and high risk polyp group (OR = 1.39, 95% CI 1.01-1.90). CONCLUSION: Our results suggest that the SNP rs6214 of IGF1 could have an impact on developing colorectal cancer and colorectal polyps with villous elements.