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OBJECTIVE:The aim of this study was to validate a molecular classification of colorectal cancer (CRC) based on microsatellite instability (MSI), CpG island methylator phenotype (CIMP) status, BRAF, and KRAS and investigate each subtype's response to chemotherapy. DESIGN:This retrospective observational study included a population-based cohort of 878 CRC patients. We classified tumours into five different subtypes based on BRAF and KRAS mutation, CIMP status, and MSI. Patients with advanced stage II (T4N0M0) and stage III tumours received 5-fluoruracil (5-FU)-based chemotherapy or no adjuvant treatment based on clinical criteria. The main outcome was disease-free survival (DFS). RESULTS:Patients with the combination of microsatellite stable (MSS) tumours, BRAF mutation and CIMP positive exhibited the worst prognosis in univariate (log rank P

BACKGROUND:Common low-penetrance genetic variants have been consistently associated with colorectal cancer risk. AIM:To determine if these genetic variants are associated also with adenoma susceptibility and may improve selection of patients with increased risk for advanced adenomas and/or multiplicity (≥ 3 adenomas). METHODS:We selected 1,326 patients with increased risk for advanced adenomas and/or multiplicity and 1,252 controls with normal colonoscopy from population-based colorectal cancer screening programs. We conducted a case-control association study analyzing 30 colorectal cancer susceptibility variants in order to investigate the contribution of these variants to the development of subsequent advanced neoplasia and/or multiplicity. RESULTS:We found that 14 of the analyzed genetic variants showed a statistically significant association with advanced adenomas and/or multiplicity: the probability of developing these lesions increased with the number of risk alleles reaching a 2.3-fold risk increment in individuals with ≥ 17 risk alleles. CONCLUSIONS:Nearly half of the genetic variants associated with colorectal cancer risk are also related to advanced adenoma and/or multiplicity predisposition. Assessing the number of risk alleles in individuals within colorectal cancer screening programs may help to identify better a subgroup with increased risk for advanced neoplasia and/or multiplicity in the general population.

To estimate the long-term need for colonoscopies after a positive fecal immunochemical test (FIT) and post-polypectomy surveillance in the context of a population-based colorectal cancer (CRC) screening program.A discrete-event simulation model was built to reproduce the process of CRC screening and post-polypectomy surveillance following European guidelines in a population of 100,000 men and women aged 50-69 years over a 20-year period. Screening consisted of biennial FIT and colonoscopy in participants with positive results. The model was mainly fed using data from the first and second rounds of a Spanish program (2010-2013). Data on post-polypectomy surveillance results were obtained from the literature. A probabilistic multivariate sensitivity analysis was performed on the effect of participation, FIT positivity, and adherence to surveillance colonoscopies. The main outcome variables were the number of colonoscopies after a positive FIT, surveillance colonoscopies, and the overall number of colonoscopies.An average yearly number of 1,200 colonoscopies after a positive FIT were predicted per 100,000 inhabitants with a slight increase to 1,400 at the end of the 20-year period. Surveillance colonoscopies increased to an average of 1,000 per 100,000 inhabitants in the long-term, showing certain stabilization in the last years of the 20-year simulation horizon. The results were highly sensitive to FIT positivity.Implementing a population-based CRC screening program will increase the demand for colonoscopies, which is expected to double in 20 years, mainly due to an increase in surveillance colonoscopies.

Elevated Microsatellite Alterations at Selected Tetranucleotide repeats (EMAST) is a genetic signature found in up to 60% of colorectal cancers (CRCs) that is caused by somatic dysfunction of the DNA mismatch repair (MMR) protein hMSH3. We have previously shown in vitro that recognition of 5-fluorouracil (5-FU) within DNA and subsequent cytotoxicity was most effective when both hMutSα (hMSH2-hMSH6 heterodimer) and hMutSβ (hMSH2-hMSH3 heterodimer) MMR complexes were present, compared to hMutSα > hMutSβ alone. We tested if patients with EMAST CRCs (hMutSβ defective) had diminished response to adjuvant 5-FU chemotherapy, paralleling in vitro findings. We analyzed 230 patients with stage II/III sporadic colorectal cancers for which we had 5-FU treatment and survival data. Archival DNA was analyzed for EMAST (>2 of 5 markers mutated among UT5037, D8S321, D9S242, D20S82, D20S85 tetranucleotide loci). Kaplan-Meier survival curves were generated and multivariate analysis was used to determine contribution to risk. We identified 102 (44%) EMAST cancers. Ninety-four patients (41%) received adjuvant 5-FU chemotherapy, and median follow-up for all patients was 51 months. Patients with EMAST CRCs demonstrated improved survival with adjuvant 5FU to the same extent as patients with non-EMAST CRCs (P

Epigenetics are thought to play a major role in the carcinogenesis of multiple sporadic colorectal cancers (CRC). Previous studies have suggested concordant DNA hypermethylation between tumor pairs. However, only a few methylation markers have been analyzed. This study was aimed at describing the epigenetic signature of multiple CRC using a genome-scale DNA methylation profiling. We analyzed 12 patients with synchronous CRC and 29 age-, sex-, and tumor location-paired patients with solitary tumors from the EPICOLON II cohort. DNA methylation profiling was performed using the Illumina Infinium HM27 DNA methylation assay. The most significant results were validated by Methylight. Tumors samples were also analyzed for the CpG Island Methylator Phenotype (CIMP); KRAS and BRAF mutations and mismatch repair deficiency status. Functional annotation clustering was performed. We identified 102 CpG sites that showed significant DNA hypermethylation in multiple tumors with respect to the solitary counterparts (difference in β value ≥0.1). Methylight assays validated the results for 4 selected genes (p = 0.0002). Eight out of 12(66.6%) multiple tumors were classified as CIMP-high, as compared to 5 out of 29(17.2%) solitary tumors (p = 0.004). Interestingly, 76 out of the 102 (74.5%) hypermethylated CpG sites found in multiple tumors were also seen in CIMP-high tumors. Functional analysis of hypermethylated genes found in multiple tumors showed enrichment of genes involved in different tumorigenic functions. In conclusion, multiple CRC are associated with a distinct methylation phenotype, with a close association between tumor multiplicity and CIMP-high. Our results may be important to unravel the underlying mechanism of tumor multiplicity.

Aberrant hypermethylation of cancer-related genes has emerged as a promising strategy for the development of diagnostic, prognostic and predictive biomarkers in human cancer, including colorectal cancer (CRC). The aim of this study was to perform a systematic and comprehensive analysis of a panel of CRC-specific genes as potential diagnostic, prognostic and predictive biomarkers in a large, population-based CRC cohort.Methylation status of the SEPT9, TWIST1, IGFBP3, GAS7, ALX4 and miR137 genes was studied by quantitative bisulfite pyrosequencing in a population-based cohort of 425 CRC patients.Methylation levels of all genes analyzed were significantly higher in tumor tissues compared to normal mucosa (p

Colorectal cancer is one of the most frequent neoplasms and an important cause of mortality in the developed world. Mendelian syndromes account for about 5% of the total burden of CRC, being Lynch syndrome and familial adenomatous polyposis the most common forms. Lynch syndrome tumors develop mainly as a consequence of defective DNA mismatch repair associated with germline mutations in MLH1, MSH2, MSH6 and PMS2. A significant proportion of variants identified by screening these genes correspond to missense or noncoding changes without a clear pathogenic consequence, and they are designated as "variants of uncertain significance", being the c.1852_1853delinsGC (p.K618A) variant in the MLH1 gene a clear example. The implication of this variant as a low-penetrance risk variant for CRC was assessed in the present study by performing a case-control study within a large cohort from the COGENT consortium-COST Action BM1206 including 18,723 individuals (8,055 colorectal cancer cases and 10,668 controls) and a case-only genotype-phenotype correlation with several clinical and pathological characteristics restricted to the Epicolon cohort. Our results showed no involvement of this variant as a low-penetrance variant for colorectal cancer genetic susceptibility and no association with any clinical and pathological characteristics including family history for this neoplasm or Lynch syndrome.

Studies in animal models and humans suggest anti-inflammatory roles on the N-acylethanolamide (NAE)-peroxisome proliferators activated receptor alpha (PPARα) system in inflammatory bowel diseases. However, the presence and function of NAE-PPARα signaling system in the ulcerative colitis (UC) of humans remain unknown as well as its response to active anti-inflammatory therapies such as 5-aminosalicylic acid (5-ASA) and glucocorticoids. Expression of PPARα receptor and PPARα ligands-biosynthetic (NAPE-PLD) and -degrading (FAAH and NAAA) enzymes were analyzed in untreated active and 5-ASA/glucocorticoids/immunomodulators-treated quiescent UC patients compared to healthy human colonic tissue by RT-PCR and immunohistochemical analyses. PPARα, NAAA, NAPE-PLD and FAAH showed differential distributions in the colonic epithelium, lamina propria, smooth muscle and enteric plexus. Gene expression analysis indicated a decrease of PPARα, PPARγ and NAAA, and an increase of FAAH and iNOS in the active colitis mucosa. Immunohistochemical expression in active colitis epithelium confirmed a PPARα decrease, but showed a sharp NAAA increase and a NAPE-PLD decrease, which were partially restored to control levels after treatment. We also characterized the immune cells of the UC mucosa infiltrate. We detected a decreased number of NAAA-positive and an increased number of FAAH-positive immune cells in active UC, which were partially restored to control levels after treatment. NAE-PPARα signaling system is impaired during active UC and 5-ASA/glucocorticoids treatment restored its normal expression. Since 5-ASA actions may work through PPARα and glucocorticoids through NAE-producing/degrading enzymes, the use of PPARα agonists or FAAH/NAAA blockers that increases endogenous PPARα ligands may yield similar therapeutics advantages.

Early-onset colorectal cancer (CRC) represents a clinically distinct form of CRC that is often associated with a poor prognosis. Methylation levels of genomic repeats such as LINE-1 elements have been recognized as independent factors for increased cancer-related mortality. The methylation status of LINE-1 elements in early-onset CRC has not been analyzed previously.We analyzed 343 CRC tissues and 32 normal colonic mucosa samples, including 2 independent cohorts of CRC diagnosed ≤ 50 years old (n=188), a group of sporadic CRC >50 years (MSS n=89; MSI n=46), and a group of Lynch syndrome CRCs (n=20). Tumor mismatch repair protein expression, microsatellite instability status, LINE-1 and MLH1 methylation, somatic BRAF V600E mutation, and germline MUTYH mutations were evaluated.Mean LINE-1 methylation levels (± SD) in the five study groups were early-onset CRC, 56.6% (8.6); sporadic MSI, 67.1% (5.5); sporadic MSS, 65.1% (6.3); Lynch syndrome, 66.3% (4.5) and normal mucosa, 76.5% (1.5). Early-onset CRC had significantly lower LINE-1 methylation than any other group (p

BackgroundColorectal cancer (CRC) multiplicity has been mainly related to polyposis and non-polyposis hereditary syndromes. In sporadic CRC, aberrant gene promoter methylation has been shown to play a key role in carcinogenesis, although little is known about its involvement in multiplicity. To assess the effect of methylation in tumor multiplicity in sporadic CRC, hypermethylation of key tumor suppressor genes was evaluated in patients with both multiple and solitary tumors, as a proof-of-concept of an underlying epigenetic defect.Methodology/principal findingsWe examined a total of 47 synchronous/metachronous primary CRC from 41 patients, and 41 gender, age (5-year intervals) and tumor location-paired patients with solitary tumors. Exclusion criteria were polyposis syndromes, Lynch syndrome and inflammatory bowel disease. DNA methylation at the promoter region of the MGMT, CDKN2A, SFRP1, TMEFF2, HS3ST2 (3OST2), RASSF1A and GATA4 genes was evaluated by quantitative methylation specific PCR in both tumor and corresponding normal appearing colorectal mucosa samples. Overall, patients with multiple lesions exhibited a higher degree of methylation in tumor samples than those with solitary tumors regarding all evaluated genes. After adjusting for age and gender, binomial logistic regression analysis identified methylation of MGMT2 (OR, 1.48; 95% CI, 1.10 to 1.97; p = 0.008) and RASSF1A (OR, 2.04; 95% CI, 1.01 to 4.13; p = 0.047) as variables independently associated with tumor multiplicity, being the risk related to methylation of any of these two genes 4.57 (95% CI, 1.53 to 13.61; p = 0.006). Moreover, in six patients in whom both tumors were available, we found a correlation in the methylation levels of MGMT2 (r = 0.64, p = 0.17), SFRP1 (r = 0.83, 0.06), HPP1 (r = 0.64, p = 0.17), 3OST2 (r = 0.83, p = 0.06) and GATA4 (r = 0.6, p = 0.24). Methylation in normal appearing colorectal mucosa from patients with multiple and solitary CRC showed no relevant difference in any evaluated gene.ConclusionsThese results provide a proof-of-concept that gene promoter methylation is associated with tumor multiplicity. This underlying epigenetic defect may have noteworthy implications in the prevention of patients with sporadic CRC.

BACKGROUND: Colorectal cancer (CRC) is considered a complex disease, and thus the majority of the genetic susceptibility is thought to lie in the form of low-penetrance variants following a polygenic model of inheritance. Candidate-gene studies have so far been one of the basic approaches taken to identify these susceptibility variants. The consistent involvement of some signaling routes in carcinogenesis provided support for pathway-based studies as a natural strategy to select genes that could potentially harbour new susceptibility loci. METHODOLOGY/PRINCIPAL FINDINGS: We selected two main carcinogenesis-related pathways: Wnt and BMP, in order to screen the implicated genes for new risk variants. We then conducted a case-control association study in 933 CRC cases and 969 controls based on coding and regulatory SNPs. We also included rs4444235 and rs9929218, which did not fulfill our selection criteria but belonged to two genes in the BMP pathway and had consistently been linked to CRC in previous studies. Neither allelic, nor genotypic or haplotypic analyses showed any signs of association between the 37 screened variants and CRC risk. Adjustments for sex and age, and stratified analysis between sporadic and control groups did not yield any positive results either. CONCLUSIONS/SIGNIFICANCE: Despite the relevance of both pathways in the pathogenesis of the disease, and the fact that this is indeed the first study that considers these pathways as a candidate-gene selection approach, our study does not present any evidence of the presence of low-penetrance variants for the selected markers in any of the considered genes in our cohort.

BACKGROUND:Recent studies suggest potential roles of the endocannabinoid system in gastrointestinal inflammation. Although cannabinoid CB(2) receptor expression is increased in inflammatory disorders, the presence and function of the remaining proteins of the endocannabinoid system in the colonic tissue is not well characterized. METHODOLOGY:Cannabinoid CB(1) and CB(2) receptors, the enzymes for endocannabinoid biosynthesis DAGLalpha, DAGLbeta and NAPE-PLD, and the endocannabinoid-degradating enzymes FAAH and MAGL were analysed in both acute untreated active ulcerative pancolitis and treated quiescent patients in comparison with healthy human colonic tissue by immunocytochemistry. Analyses were carried out according to clinical criteria, taking into account the severity at onset and treatment received. PRINCIPAL FINDINGS:Western blot and immunocytochemistry indicated that the endocannabinoid system is present in the colonic tissue, but it shows a differential distribution in epithelium, lamina propria, smooth muscle and enteric plexi. Quantification of epithelial immunoreactivity showed an increase of CB(2) receptor, DAGLalpha and MAGL expression, mainly in mild and moderate pancolitis patients. In contrast, NAPE-PLD expression decreased in moderate and severe pancolitis patients. During quiescent pancolitis, CB(1), CB(2) and DAGLalpha expression dropped, while NAPE-PLD expression rose, mainly in patients treated with 5-ASA or 5-ASA+corticosteroids. The number of immune cells containing MAGL and FAAH in the lamina propria increased in acute pancolitis patients, but dropped after treatment. CONCLUSIONS:Endocannabinoids signaling pathway, through CB(2) receptor, may reduce colitis-associated inflammation suggesting a potential drugable target for the treatment of inflammatory bowel diseases.