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The tumor suppressor gene adenomatous polyposis coli (APC) is the initiating mutation in approximately 80% of all colorectal cancers (CRC), underscoring the importance of aberrant regulation of intracellular WNT signaling in CRC development. Recent studies have found that early-onset CRC exhibits an increased proportion of tumors lacking an APC mutation. We set out to identify mechanisms underlying APC mutation-negative (APCmut–) CRCs. We analyzed data from The Cancer Genome Atlas to compare clinical phenotypes, somatic mutations, copy number variations, gene fusions, RNA expression, and DNA methylation profiles between APCmut– and APC mutation-positive (APCmut+) microsatellite stable CRCs. Transcriptionally, APCmut– CRCs clustered into two approximately equal groups. Cluster One was associated with enhanced mitochondrial activation. Cluster Two was strikingly associated with genetic inactivation or decreased RNA expression of the WNT antagonist RNF43, increased expression of the WNT agonist RSPO3, activating mutation of BRAF, or increased methylation and decreased expression of AXIN2. APCmut– CRCs exhibited evidence of increased immune cell infiltration, with significant correlation between M2 macrophages and RSPO3. APCmut– CRCs comprise two groups of tumors characterized by enhanced mitochondrial activation or increased sensitivity to extracellular WNT, suggesting that they could be respectively susceptible to inhibition of these pathways.

Despite improvements over the past 20 years, African Americans continue to have the highest incidence and mortality rates of colorectal cancer (CRC) in the United States. While previous studies have found that copy number variations (CNVs) occur at similar frequency in African American and White CRCs, copy-neutral loss of heterozygosity (cnLOH) has not been investigated. In the present study, we used publicly available data from The Cancer Genome Atlas (TCGA) as well as data from an African American CRC cohort, the Chicago Colorectal Cancer Consortium (CCCC), to compare frequencies of CNVs and cnLOH events in CRCs in the two racial groups. Using genotype microarray data, we analyzed large-scale CNV and cnLOH events from 166 microsatellite stable CRCs-31 and 39 African American CRCs from TCGA and the CCCC, respectively, and 96 White CRCs from TCGA. As reported previously, the frequencies of CNVs were similar between African American and White CRCs; however, there was a significantly lower frequency of cnLOH events in African American CRCs compared to White CRCs, even after adjusting for demographic and clinical covariates. Although larger differences for chromosome 18 were observed, a lower frequency of cnLOH events in African American CRCs was observed for nearly all chromosomes. These results suggest that mechanistic differences, including differences in the frequency of cnLOH, could contribute to clinicopathological disparities between African Americans and Whites. Additionally, we observed a previously uncharacterized phenomenon we refer to as small interstitial cnLOH, in which segments of chromosomes from 1 to 5 Mb long were affected by cnLOH.

BACKGROUND: Mismatch repair (MMR) deficiency is the hallmark of tumours from Lynch syndrome (LS), sporadic MLH1 hypermethylated and Lynch-like syndrome (LLS), but there is a lack of understanding of the variability in their mutational profiles based on clinical phenotypes. The aim of this study was to perform a molecular characterisation to identify novel features that can impact tumour behaviour and clinical management. METHODS: We tested 105 MMR-deficient colorectal cancer tumours (25 LS, 35 LLS and 45 sporadic) for global exome microsatellite instability, cancer mutational signatures, mutational spectrum and neoepitope load. RESULTS: Fifty-three percent of tumours showed high contribution of MMR-deficient mutational signatures, high level of global exome microsatellite instability, loss of MLH1/PMS2 protein expression and included sporadic tumours. Thirty-one percent of tumours showed weaker features of MMR deficiency, 62% lost MSH2/MSH6 expression and included 60% of LS and 44% of LLS tumours. Remarkably, 9% of all tumours lacked global exome microsatellite instability. Lastly, HLA-B07:02 could be triggering the neoantigen presentation in tumours that show the strongest contribution of MMR-deficient tumours. CONCLUSIONS: Next-generation sequencing approaches allow for a granular molecular characterisation of MMR-deficient tumours, which can be essential to properly diagnose and treat patients with these tumours in the setting of personalised medicine.

Many colorectal cancers (CRCs) that exhibit microsatellite instability (MSI) are not explained by MLH1 promoter methylation or germline mutations in mismatch repair (MMR) genes, which cause Lynch syndrome (LS). Instead, these Lynch-like syndrome (LLS) patients have somatic mutations in MMR genes. However, many of these patients are young and have relatives with cancer, suggesting a hereditary entity. We performed germline sequence analysis in LLS patients and determined their tumor's mutational profiles using FFPE DNA. Six hundred fifty-four consecutive CRC patients were screened for suspected Lynch syndrome using MSI and absence of MLH1 methylation. Suspected LS cases were exome sequenced to identify germline and somatic mutations. Single nucleotide variants were used to characterize mutational signatures. We identified 23 suspected LS cases. Germline sequence analysis of 16 available samples identified 5 cases with LS mutations and 11 cases without LS mutations, LLS. Most LLS tumors had a combination of somatic MMR gene mutation and loss of heterozygosity. LLS patients were relatively young and had excess first-degree relatives with cancer. Four of the 11 LLS patients had rare likely pathogenic variants in genes that maintain genome integrity. Moreover, tumors from this group had a distinct mutational signature compared to tumors from LLS patients lacking germline mutations in these genes. In summary, more than a third of the LLS patients studied had germline mutations in genes that maintain genome integrity and their tumors had a distinct mutational signature. The possibility of hereditary factors in LLS warrants further studies so counseling can be properly informed.

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African Americans (AAs) have higher incidence and mortality rates of colorectal cancer (CRC) compared with other US populations. They present with more right-sided, microsatellite stable disease and are diagnosed at earlier ages compared with non-Hispanic Whites (NHWs). To gain insight into these trends, we conducted exome sequencing (n = 45), copy number (n = 33) and methylation analysis (n = 11) of microsatellite stable AA CRCs. Results were compared with data from The Cancer Genome Atlas (TCGA). Two of the 45 tumors contained POLE mutations. In the remaining 43 tumors, only 27 (63%) contained loss-of-function mutations in APC compared with 80% of TCGA NHW CRCs. APC-mutation-negative CRCs were associated with an earlier onset of CRC (P = 0.01). They were also associated with lower overall mutation burden, fewer copy number variants and a DNA methylation signature that was distinct from the CpG island methylator phenotype characterized in microsatellite unstable disease. Three of the APC-mutation-negative CRCs had loss-of-function mutations in BCL9L. Mutations in driver genes identified by TCGA exome analysis were less frequent in AA CRC cases than TCGA NHWs. Genes that regulate the WNT signaling pathway, including SOX9, GATA6, TET1, GLIS1 and FAT1, were differentially hypermethylated in APC-mutation-negative CRCs, suggesting a novel mechanism for cancer development in these tumors. In summary, we have identified a subtype of CRC that is associated with younger age of diagnosis, lack of APC mutation, microsatellite and chromosome stability, lower mutation burden and distinctive methylation changes.

A great proportion of the heritability of colorectal cancer (CRC) still remains unexplained, and rare variants, as well as copy number changes, have been proposed as potential candidates to explain the so-called 'missing heritability'. We aimed to identify rare high-to-moderately penetrant copy number variants (CNVs) in patients suspected of having hereditary CRC due to an early onset. We have selected for genome-wide copy number analysis, 27 MMR-proficient early onset CRC patients (< 50 years) without identifiable germline mutations in Mendelian genes related to this phenotype. Rare CNVs were selected by removing all CNVs detected at MAF > 1% in the in-house control CNV database (n = 629 healthy controls). Copy number assignment was checked by duplex real-time quantitative PCR or multiplex ligation probe amplification. Somatic mutation analysis in candidate genes included: loss of heterozygosity studies, point mutation screening, and methylation status of the promoter. We have identified two rare germline deletions involving the AK3 and SLIT2 genes in two patients. The search for a second somatic mutational event in the corresponding CRC tumors showed loss of heterozygosity in AK3, and promoter hypermethylation in SLIT2. Both genes have been previously related to colorectal carcinogenesis. These findings suggest that AK3 and SLIT2 may be potential candidates involved in genetic susceptibility to CRC.

Objective Colorectal cancer (CRC) incidence is higher in African Americans (AAs) compared with non-Hispanic whites (NHWs). A diet high in animal protein and fat is an environmental risk factor for CRC development. The intestinal microbiota is postulated to modulate the effects of diet in promoting or preventing CRC. Hydrogen sulfide, produced by autochthonous sulfidogenic bacteria, triggers proinflammatory pathways and hyperproliferation, and is genotoxic. We hypothesised that sulfidogenic bacterial abundance in colonic mucosa may be an environmental CRC risk factor that distinguishes AA and NHW. Design Colonic biopsies from uninvolved or healthy mucosa from CRC cases and tumour-free controls were collected prospectively from five medical centres in Chicago for association studies. Sulfidogenic bacterial abundance in uninvolved colonic mucosa of AA and NHW CRC cases was compared with normal mucosa of AA and NHW controls. In addition, 16S rDNA sequencing was performed in AA cases and controls. Correlations were examined among bacterial targets, race, disease status and dietary intake. Results AAs harboured a greater abundance of sulfidogenic bacteria compared with NHWs regardless of disease status. Bilophila wadsworthia -specific dsrA was more abundant in AA cases than controls. Linear discriminant analysis of 16S rRNA gene sequences revealed five sulfidogenic genera that were more abundant in AA cases. Fat and protein intake and daily servings of meat were significantly higher in AAs compared with NHWs, and multiple dietary components correlated with a higher abundance of sulfidogenic bacteria. Conclusions These results implicate sulfidogenic bacteria as a potential environmental risk factor contributing to CRC development in AAs.

The development of genotyping technologies has allowed for wider screening for inherited causes of variable outcomes following drug administration. We have performed a genome-wide association study (GWAS) on 221 colorectal cancer (CRC) patients that had been treated with 5-fluorouracil (5-FU), either alone or in combination with oxaliplatin (FOLFOX). A validation set of 791 patients was also studied. Seven SNPs (rs16857540, rs2465403, rs10876844, rs10784749, rs17626122, rs7325568 and rs4243761) showed evidence of association (pooled P-values 0.020, 9.426E-03, 0.010, 0.017, 0.042, 2.302E-04, 2.803E-03) with adverse drug reactions (ADRs). This is the first study to explore the genetic basis of inter-individual variation in toxicity responses to the administration of 5-FU or FOLFOX in CRC patients on a genome-wide scale. © 2013 Macmillan Publishers Limited. All rights reserved 1470-269X/13.

We describe an association between TGFBR1 polymorphism rs868 and mismatch repair proficient hereditary colorectal cancers. This polymorphism results in more binding of TGFBR1 to let-7b-5p miRNA, which would result in lower expression of TGFBR1 and thus higher colorectal cancer risk.The purpose of this study was to identify novel colorectal cancer (CRC)-causing alleles in unexplained familial CRC cases. In order to do so, coding regions in five candidate genes (MGMT, AXIN2, CTNNB1, TGFBR1 and TGFBR2) were sequenced in 11 unrelated microsatellite-stable hereditary non-polyposis CRC (MSS HNPCC) cases. Selected genetic variants were genotyped in a discovery set of 27 MSS HNPCC cases and 85 controls. One genetic variant, rs67687202, in TGFBR1 emerged as significant (P = 0.002), and it was genotyped in a replication set of 87 additional MSS HNPCC-like cases and 338 controls where it was also significantly associated with MSS HNPCC cases (P = 0.041). In the combined genotype data, rs67687202 was associated with a moderate increase in CRC risk (OR = 1.68; 95% CI = 1.13-2.50; P = 0.010). We tested a highly correlated SNP rs868 in 723 non-familial CRC cases compared with 629 controls, and it was not significantly associated with CRC risk (P = 0.370). rs868 is contained in a let-7 miRNA binding site in the 3'UTR of TGFBR1, which might provide a functional basis for the association in MSS HNPCC. In luciferase assays, the risk-associated allele for rs868 was associated with half the luciferase expression in the presence of miRNA let-7b-5p compared with protective allele, suggesting more binding of let-7b-5p and less TGFBR1 expression. Thus, rs868 potentially is a CRC risk-causing allele. Our results support the concept that rs868 is associated with lower TGFBR1 expression thereby increasing CRC risk.

The EPICOLON consortium was initiated in 1999 by the Gastrointestinal Oncology Group of the Spanish Gastroenterology Association. It recruited consecutive, unselected, population-based colorectal cancer (CRC) cases and control subjects matched by age and gender without personal or familial history of cancer all over Spain with the main goal of gaining knowledge in Lynch syndrome and familial CRC. This epidemiological, prospective and multicentre study collected extensive clinical data and biological samples from ∼2000 CRC cases and 2000 controls in Phases 1 and 2 involving 25 and 14 participating hospitals, respectively. Genetic susceptibility projects in EPICOLON have included candidate-gene approaches evaluating single-nucleotide polymorphisms/genes from the historical category (linked to CRC risk by previous studies), from human syntenic CRC susceptibility regions identified in mouse, from the CRC carcinogenesis-related pathways Wnt and BMP, from regions 9q22 and 3q22 with positive linkage in CRC families, and from the mucin gene family. This consortium has also participated actively in the identification 5 of the 16 common, low-penetrance CRC genetic variants identified so far by genome-wide association studies. Finishing their own pangenomic study and performing whole-exome sequencing in selected CRC samples are among EPICOLON future research prospects. © 2012 The Author.

Several studies have suggested that the partially fermentable fibre Plantago ovata husk (PO) may have a protective effect on colorectal cancer (CRC). We studied the potentially pro-apoptotic effect of PO and the implicated mechanisms in CRC cells with different molecular phenotypes (Caco-2, HCT116, LoVo, HT-29, SW480) after PO anaerobic fermentation with colonic bacteria as it occurs in the human colon. The fermentation products of PO induced apoptosis in all primary tumour and metastatic cell lines, independent of p53, adenomatous polyposis coli, β-catenin or cyclo-oxygenase-2 status. Apoptosis was caspase-dependent and both intrinsic and extrinsic pathways were implicated. The intrinsic pathway was activated through a shift in the balance towards a pro-apoptotic environment with an up-regulation of B-cell lymphoma protein 2 homologous antagonist killer (BAK) and a down-regulation of B-cell lymphoma-extra large (Bcl-xL) seen in HCT116 and LoVo cells. This resulted in mitochondrial membrane depolarisation, increased expression of caspase activators second mitochondria-derived activator of caspases (Smac)/Diablo, death effector apoptosis-inducing factor, apoptosome member apoptotic protease activating factor 1 and down-regulation of inhibitors of apoptosis Survivin and X-linked inhibitor of apoptosis in most cells. The extrinsic pathway was activated presumably through the up-regulation of death receptor (DR5). Some important differences were seen between primary tumour and metastatic CRC cells. Thus, metastatic PO-treated LoVo cells had a remarkable up-regulation of TNF-α ligand along with death-inducing signalling complex components receptor interacting protein and TNF-α receptor 1-associated death domain protein. The extrinsic pathway modulator FCICE-inhibitory protein (FLIP), an inhibitor of both spontaneous death ligand-independent and death receptor-mediated apoptosis, was significantly down-regulated after PO treatment in all primary tumour cells, but not in metastatic LoVo. These findings suggest that PO could potentially be a useful chemotherapy adjuvant.

Background: The selection of patients for genetic testing to rule out Lynch syndrome is currently based on fulfilment of at least one of the revised Bethesda criteria followed by mismatch repair (MMR) status analysis. A study was undertaken to compare the present approach with universal MMR study-based strategies to detect Lynch syndrome in a large series of patients with colorectal cancer (CRC). Methods: 2093 patients with CRC from the EPICOLON I and II cohorts were included. Immunohistochemistry for MMR proteins and/or microsatellite instability (MSI) analysis was performed in tumour tissue. Germline MLH1 and MSH2 mutation analysis was performed in patients whose tumours showed loss of MLH1 or MSH2 staining, respectively. MSH6 genetic testing was done in patients whose tumours showed lack of MSH6 expression or a combined lack of MSH2 and MSH6 expression but did not have MSH2 mutations. PMS2 genetic testing was performed in patients showing isolated loss of PMS2 expression. In patients with MSI tumours and normal or not available MMR protein expression, all four MMR genes were studied. Results: A total of 180 patients (8.6%) showed loss of expression of some of the MMR proteins and/or MSI. Four hundred and eighty-six patients (23.2%) met some of the revised Bethesda criteria. Of the 14 (0.7%) patients who had a MMR gene mutation, 12 fulfilled at least one of the revised Bethesda criteria and two (14.3%) did not. Conclusions: Routine molecular screening of patients with CRC for Lynch syndrome using immunohistochemistry or MSI has better sensitivity for detecting mutation carriers than the Bethesda guidelines. Instituto de Salud Carlos III (INT09/208 and PI08/0726). Fundación de la CV para la Investigación en el Hospital General Universitario de Alicante. Grant SAF 07-64873 from the Ministerio de Educación y Ciencia. Funds from the AGAUR (2009 SGR 849) Instituto de Salud Carlos III (FI07/00303) CIBERehd is funded by the Instituto de Salud Carlos III. Grant from Conselleria d’Educació de la Generalitat Valenciana (VALi+d).

Background & Aims 5-Fluorouracil (5-FU)–based adjuvant chemotherapy does not increase survival times of patients with colorectal tumors with microsatellite instability. We determined the response of patients with colorectal tumors with the CpG island methylator phenotype (CIMP) to 5-FU–based therapy. Methods We analyzed a population-based cohort of 302 patients with colorectal cancer (CRC) for a median follow-up time of 50.7 months. CIMP status was determined by analysis of the CACNAG1 , SOCS1 , RUNX3 , NEUROG1 , and MLH1 promoters; tumors were considered to be CIMP positive if at least 3 promoters were methylated. Results Tumors from 29.5% of patients (89/302) were CIMP positive; CIMP status did not influence disease-free survival (DFS; log-rank = 0.3). Of tumors of TNM stages II–III (n = 196), 32.7% were CIMP positive. Among patients with stages II–III CRC who did not receive adjuvant 5-FU chemotherapy, those with CIMP-positive tumors had longest times of DFS (log-rank = 0.04); In patients who received chemotherapy, those with CIMP-positive tumors had shorter times of DFS (log-rank = 0.02). In patients with CIMP-negative tumors, adjuvant 5-FU chemotherapy significantly increased time of DFS (log-rank = 0.00001). However, in patients with CIMP-positive tumors, adjuvant 5-FU chemotherapy did not affect time of DFS (log-rank = 0.7). Multivariate analysis showed a significant, independent interaction between 5-FU treatment and CIMP status (hazard ratio [HR], 0.6; 95% confidence interval [CI], 0.5–0.8). Among patients with CIMP-positive tumors, adjuvant chemotherapy was not an independent predictor of outcome (HR, 0.8; 95% CI, 0.3–2.0). In patients who did not receive adjuvant 5-FU chemotherapy, CIMP status was the only independent predictor of survival (HR, 2.0; 95% CI, 1.1–3.8). Conclusions Patients with CIMP-positive colorectal tumors do not benefit from 5-FU–based adjuvant chemotherapy.

Purpose: Several studies have described molecular differences between microsatellite stable hereditary nonpolyposis colorectal cancer (MSS-HNPCC) and microsatellite unstable Lynch syndrome tumors (MSI-HNPCC). These differences highlight the possibility that other instability forms could explain cancer susceptibility in this group of families. The base excision repair (BER) pathway is the major DNA repair pathway for oxidative DNA damage. A defect in this pathway can result in DNA transversion mutations and a subsequent increased cancer risk. Mutations in MUTYH have been associated with increased colorectal cancer (CRC) risk while no association has been described for OGG1 or NUDT1. Experimental Design: We performed mutational screening of the three genes involved in defense against oxidative DNA damage in a set of 42 MSS-HNPCC families. Results: Eight rare variants and 5 frequent variants were found in MSS-HNPCC patients. All variants were previously described by other authors except variant c.285C>T in OGG1. Segregation studies were done and in silico programs were used to estimate the level of amino acid conservation, protein damage prediction, and possible splicing alterations. Variants OGG1 c.137G>A; MUTYH c.1187G>A were detected in Amsterdam I families and cosegregate with cancer. Analysis of OGG1 c.137G>A transcripts showed an inactivation of the splicing donor of exon 1. Conclusions: Two rare variants (OGG1 c.137G>A; MUTYH c.1187G>A) and one common polymorphism (NUDT1 c.426C>T) were associated with CRC risk. We show that the BER pathway can play a significant role in a number of MSS-HNPCC colorectal cancers. More studies could be of interest in order to gain further understanding of yet unexplained CRC susceptibility cases. Clin Cancer Res; 17(7); 1701–12. ©2011 AACR.

Several studies have suggested that the n-3 fatty acids Docosahexaenoic (DHA) and Eicosapentaenoic (EPA) have an important protective effect on colorectal cancer, and this could be at least partly due to their proapoptotic activity. It is unclear, however, how this phenomenon is triggered and what mechanisms are implicated. Here, we show that both DHA and EPA have an important proapoptotic effect on colorectal cancer cells with different molecular phenotypes but not in noncancerous cells. Apoptosis is caspase dependent, and both intrinsic and extrinsic pathways are implicated. The dimerization of Bax and Bak, the depolarization of the mitochondrial membrane, and the subsequent release of cytochrome c and Smac/Diablo to the cytosol evidence the activation of the intrinsic pathway. The implication of the extrinsic pathway is shown by the activation of caspase-8, along with the down-regulation of FLIP. The timing of caspase-8 activation, and the oligomerization of Bid with Bax, suggest a cross-talk with the intrinsic pathway. None of the death receptors that commonly initiate the extrinsic pathway: FAS, TNF-R1, and TRAIL-R2 are found to be responsible for triggering the apoptosis cascade induced by DHA and EPA. Neither PPARγ nor cyclooxygenase-2, two likely candidates to regulate this process, play a significant role. Our findings suggest that the down-regulation of two key regulatory elements of the extrinsic and intrinsic pathways, FLIP and XIAP, respectively, is determinant in the induction of apoptosis by DHA and EPA. These fatty acids could potentially be useful adjuvant anticancer agents in combination with other chemotherapeutic elements.

Background & Aims: Mismatch repair (MMR) deficiencies are the hallmark of tumors arising in Lynch syndrome, however, in approximately 15% of sporadic colorectal cancers (CRC) these deficiencies most often are associated with somatic methylation of the MMR gene MLH1. Recently, an oncogenic mutation in the BRAF gene has been involved in sporadic CRC showing MMR deficiencies as a result of MLH1 promoter methylation. The aim of this study was to evaluate the contribution of BRAF V600E mutation analysis in the identification of MSH2/MLH1 gene mutation carriers in newly diagnosed CRC patients. Methods:BRAF V600E mutation was analyzed in CRC patients with MMR deficiencies (microsatellite instability and/or lack of MLH1/MSH2 protein expression) in the EPICOLON population-based study. The effectiveness and efficiency of different strategies were evaluated with respect to the presence of MSH2/MLH1 germline mutations. Results: MMR deficiencies were detected in 119 of the 1222 CRC patients with tumors showing either microsatellite instability (n = 111) or loss of protein expression (n = 81). BRAF mutation was detected in 22 (18.5%) of the patients. None of the patients with unambiguous germline mutation had BRAF mutation. Regardless of the strategy used to identify MSH2/MLH1 gene carriers, the introduction of BRAF analysis in these patients slightly improves their effectiveness. The introduction of BRAF mutation analysis as a step before germline genetic testing in patients with MMR deficiencies achieved a significant reduction in costs per mutation detected. Conclusions: Detection of BRAF V600E mutation could simplify and improve the cost effectiveness of genetic testing for hereditary nonpolyposis colorectal cancer, especially in patients whose family history is incomplete or unknown.

Colonoscopy is one of the methods of choice for screening relatives of patients with colorectal cancer.To evaluate the rate of adherence to colonoscopy in first-degree relatives of patients with colorectal cancer and describe the lesions found.A prospective, cross-sectional, multicentre, nationwide study was conducted. The study population was composed of first-degree relatives of patients with colorectal cancer selected randomly from the EPICOLON study. Seventy-four index patients were included. These had 342 living first-degree relatives (parents, siblings and children), of whom 281 were interviewed.The adherence rate was 38% (107/281). Adherence was greater in families with a higher degree of familial aggregation for colorectal cancer (88.9% for Amsterdam vs 33.3% for Bethesda and sporadic cancer; p0.05), an index patient aged under 65 years (60% for patients65 years vs 32.9% for patientsor=65 years; p0.05) and an index patient who was female (46.2% for women vs 31% for men; p = 0.28). Adherence was also greater in relatives under 65 years (54% in patients65 years vs 18% in patientsor=65 years; p = 0.05), in female relatives (49% in female relatives vs 27.3% in male relatives; p0.05) and in siblings and children (40% in siblings and children vs 13% in parents; p0.05). Lesions were found in 26% (28/107) of the study population. Nine (8.4%) individuals had a total of 18 advanced lesions.These results indicate that adherence to colonoscopy in our population of first-degree relatives was low. The adherence was more frequently associated with a higher degree of familial aggregation, a relative age of under 65 years, a sibling or offspring relationship, and female sex.

ARLTS1 was recently identified in chromosome 13q14 as a tumor suppressor gene of the ADP-ribosylation factor family with pro-apoptotic characteristics. Additionally, one of its genetic variants (W149X) was hypothesized to be a polymorphism associated with familial cancer. We performed a large case-control association study within the EPICOLON project aimed at evaluating the sporadic and familial colorectal cancer (CRC) risk associated with ARLTS1 genetic variants. Whereas P131L and W149X did not seem to affect CRC risk, C148R did show, for the first time in CRC, statistically significant differences between cases and controls [odds ratio (OR) = 1.45, 95% confidence interval (95% CI) = 1.13-1.86, P = 0.003], sporadic cases and controls (OR = 1.59, 95% CI = 1.13-2.23, P = 0.007) and familial cases and controls (OR = 1.55, 95% CI = 1.10-2.19, P = 0.01) in agreement with a hypothetical moderate increase of the cancer risk linked to the C148R ARLTS1 variant, both in sporadic and familial CRC cases.

Background & Aims: Whereas it has conclusively been demonstrated that biallelic MutY human homolog (MYH) mutations confer a significant risk for colorectal cancer (CRC), the influence of monoallelic mutations remains controversial. Characterization of MYH-associated CRC is critical to identify individuals who might benefit from preventive strategies. This prospective, multicenter, case-control, population-based study was aimed at (1) establishing the CRC risk associated with specific germline MYH mutations and (2) devising a set of clinical criteria to identify MYH carriers among newly diagnosed CRC. Methods: Genotyping for Y165C and G382D was performed by TaqMan technology. Single-stranded conformation polymorphism analysis was performed in heterozygotes to screen for mutations in the entire gene. All individuals were re-screened for any additional pathogenic variant. Results: Biallelic and monoallelic MYH mutations were found in 8 (0.7%) and 19 (1.7%) of 1116 CRC patients, respectively. None of the 934 control subjects carried biallelic mutations, whereas 22 (2.3%) of them were monoallelic carriers. In a meta-analysis including all previous case-control studies, monoallelic MYH carriers were not at increased risk for CRC (odds ratio, 1.11; 95% confidence interval, 0.90–1.37), although a significant association was found with the Y165C mutation in either homozygotes or heterozygotes (odds ratio, 1.67; 95% confidence interval, 1.17–2.40). Furthermore, presence of more than 15 synchronous colorectal adenomas or CRC diagnosed before the age of 50 years was the most effective set of criteria for the identification of biallelic MYH mutation carriers. Conclusions: This study proposes the first set of clinical criteria designed to identify CRC patients with biallelic MYH mutations, and it argues against an increased risk for monoallelic carriers.

Background Colorectal tumors caused by failure of the DNA mismatch repair system commonly show microsatellite instability. Our goals were to compare the performance of two panels of markers (a panel previously recommended by the National Cancer Institute [NCI] and a pentaplex of mononucleotide repeats) and to devise the simplest diagnostic strategy for identification of patients with colorectal cancer characterized by defects in mismatch repair. Methods We recruited 1058 patients who were newly diagnosed with colorectal cancer. DNA from fresh-frozen and paraffin-embedded tumors was tested for microsatellite instability, using the NCI-recommended panel of microsatellite markers and the pentaplex panel of mononucleotide repeats , respectively, as templates for polymerase chain reactions (PCRs). Microsatellite instability in fresh-frozen tumors was also assessed using the pentaplex panel of mononucleotides in a crossover analysis. The expression of mismatch repair proteins (MLH1, MSH2, MSH6, and PMS2) in the tumors was determined immunohistochemically. The sensitivity and specificity with which the marker panels identified tumors with deficiencies in the expression of mismatch repair proteins were calculated. All statistical tests were two-sided. Results The sensitivity and positive predictive value of the NCI panel were 76.5% (95% confidence interval [CI] = 61% to 92%) and 65.0% (95% CI = 49% to 81%), respectively; corresponding values for the mononucleotide pentaplex panel were 95.8% (95% CI = 89% to 103%) and 88.5% (95% CI = 79% to 98%), respectively. A panel consisting of the mononucleotide repeat markers BAT26 and NR24 alone had the same predictive value as the pentaplex panel of mononucleotide repeats. Conclusions The pentaplex panel of mononucleotide repeats performs better than the NCI panel for the detection of mismatch repair – deficient tumors. Simultaneous assessment of the instability of BAT26 and NR24 is as effective as use of the pentaplex panel for diagnosing mismatch repair deficiency. J Natl Cancer Inst 2007;99: 244 – 52

// Hassan Ashktorab 1 , Sadhna Ahuja 2 , Lakshmi Kannan 1 , Xavier Llor 3 , Nathan A. Ellis 4 , Rosa M. Xicola 3 , Adeyinka O. Laiyemo 1 , John M. Carethers 5 , Hassan Brim 2 , Mehdi Nouraie 1 1 Department of Medicine and Cancer Center, Howard University College of Medicine, Washington DC, USA 2 Department of Pathology, Howard University College of Medicine, Washington DC, USA 3 Department of Medicine and Cancer Center, Yale University, New Haven, CT, USA 4 Cancer Biology Research Program, The University of Arizona Cancer Center, Tucson, AZ, USA 5 Division of Gastroenterology, Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA Correspondence to: Hassan Ashktorab, email: hashktorab@howard.edu Keywords: MSI, colorectal cancer, African Americans, Hispanics, Caucasians Received: December 14, 2015 Accepted: March 28, 2016 Published: April 23, 2016 ABSTRACT PURPOSE: African Americans (AA) are at a higher risk of colorectal cancer (CRC) and some studies report a higher frequency of microsatellite instability (MSI) in this population while others report lower frequency compared to Caucasians. AIM: To determine and evaluate the association of race and clinical factors with MSI frequency through meta- analysis. METHODS: Twenty-two studies out of 15,105 (1997-2015) were evaluated after a search in different literature databases, using keywords “colorectal cancer, microsatellite instability, African Americans, Caucasians and Hispanics”. We used random effect meta-analysis to calculate the MSI frequency in all studies as well as in African American and Caucasian samples. Meta-regression analysis was used to assess the univariate effect of race, gender, age, tumor location and stage on MSI frequency. RESULTS: The overall MSI frequency among CRCs was 17% (95%CI: 15%-19%, I²=91%). In studies with available race data, The MSI rate among AAs, Hispanics and Caucasians were 12%, 12% and 14% respectively and was not significantly different. Sub-group analysis of studies with racial information indicates MSI OR of 0.78 for AAs compared to Caucasians. CONCLUSION: CRCs demonstrate an overall MSI frequency of 17%. MSI frequency differences between AAs and Caucasians were not pronounced, suggesting that other factors contribute to the racial disparity. The methodological approaches and biological sources of the variation seen in MSI frequency between different studies need to be further investigated.

Disruption of the gene encoding Protein Tyrosine Kinase 6 ( Ptk6 ) delayed differentiation and increased growth in the mouse intestine. However, Ptk6 -null mice were also resistant to azoxymethane-induced colon tumorigenesis. To further explore functions of PTK6 in colon cancer, expression of epithelial and mesenchymal markers, as well as proliferation, migration, and xenograft tumor growth, was examined in human colon tumor cell lines with knockdown or overexpression of PTK6. PTK6 protein, transcript, and activation were also examined in a human colon tumor tissue array, using immunohistochemistry and qRT-PCR. Knockdown of PTK6 led to the epithelial–mesenchymal transition (EMT) in SW480 and HCT116 cells, whereas overexpression of PTK6 in SW620 cells restored an epithelial phenotype in a kinase-independent manner. PTK6 knockdown also increased xenograft tumor growth of SW480 cells, suggesting tumor suppressor functions. In clinical specimens, PTK6 expression was highest in normal differentiated epithelial cells and reduced in tumors. In contrast, overexpression of constitutively active PTK6 promoted STAT3 and ERK5 activation in colon cancer cells, and endogenous PTK6 promoted cell survival and oncogenic signaling in response to DNA-damaging treatments. These data indicate that PTK6 has complex, context-specific functions in colon cancer; PTK6 promotes the epithelial phenotype to antagonize the EMT in a kinase-independent manner, whereas activation of PTK6 promotes oncogenic signaling. Implications: Understanding context-specific functions of PTK6 is important, because although it promotes cell survival and oncogenic signaling after DNA damage, expression of PTK6 in established tumors may maintain the epithelial phenotype, preventing tumor progression. Mol Cancer Res; 14(6); 563–73. ©2016 AACR . This article is featured in Highlights of This Issue, [p. 505][1] [1]: /lookup/volpage/14/505?iss=6

Identification of individuals who should undergo hereditary nonpolyposis colorectal cancer (HNPCC) genetic testing is a critical and difficult issue. For this purpose, the National Cancer Institute outlined a set of recommendations, the Bethesda guidelines, which have recently been revised.To compare the clinical performance of original and revised Bethesda guidelines for the detection of MSH2/MLH1 gene carriers in patients with colorectal cancer.A total of 1,222 patients with newly diagnosed colorectal cancer were included in the EPICOLON study, a prospective, multicenter, nationwide epidemiology survey aimed at establishing the incidence of HNPCC in Spain (JAMA 2005; 293:1986-1994). Performance characteristics of the original and revised Bethesda guidelines were assessed with respect to the presence of MSH2/MLH1 germline mutations. Logistic regression analysis was performed to establish the most effective strategy.Original or revised Bethesda guidelines were equivalent strategies in terms of sensitivity (100%vs 100%; ns), specificity (98.1%vs 97.9%; ns), and overall accuracy (98.1%vs 97.9%; ns), as well as positive (25.8%vs 24.2%) and negative predictive values (100%vs 100%). The most discriminating individual variables were criteria number 1 (i.e., fulfillment of the Amsterdam criteria; RR = 34.14; 95% CI = 6.85-170.16; p0.001) and number 2 (i.e., individuals with two HNPCC-related neoplasms; RR = 35.63; 95% CI = 4.83-262.6; p0.001) of the original guidelines, and criterion number 1 of the revised guidelines (i.e., colorectal cancer diagnosed under 50 yr of age; RR = 29.34; 95% CI = 3.81-225.96; p= 0.001). The aggregation of these three criteria was equivalent to both Bethesda guidelines in terms of sensitivity (100%) and negative predictive value (100%), but superior to the revised criteria regarding specificity (98.5%; p0.05), overall accuracy (98.5%; p0.05), and positive predictive value (30.8%).Original and revised Bethesda guidelines are equivalent, highly effective criteria for the identification of MSH2/MLH1 gene mutation carriers in patients with newly diagnosed colorectal cancer. A new set of recommendations, based on a combination of some of their individual criteria, may provide additional advantages in terms of effectiveness.

Purpose: African Americans (AA) have the highest incidence of colorectal cancer compared with other U.S. populations and more proximal colorectal cancers. The objective is to elucidate the basis of these cancer disparities. Experimental design: Of note, 566 AA and 328 non-Hispanic White (NHW) colorectal cancers were ascertained in five Chicago hospitals. Clinical and exposure data were collected. Microsatellite instability (MSI) and BRAF (V600E) and KRAS mutations were tested. Statistical significance of categorical variables was tested by the Fisher exact test or logistic regression and age by the Mann–Whitney U test. Results: Over a 10-year period, the median age at diagnosis significantly decreased for both AAs (68–61; P < 0.01) and NHWs (64.5– 62; P = 0.04); more AA patients were diagnosed before age 50 than NHWs (22% vs. 15%; P = 0.01). AAs had more proximal colorectal cancer than NHWs (49.5% vs. 33.7%; P < 0.01), but overall frequencies of MSI, BRAF and KRAS mutations were not different nor were they different by location in the colon. Proximal colorectal cancers often presented with lymphocytic infiltrate (P < 0.01) and were diagnosed at older ages (P = 0.02). Smoking, drinking, and obesity were less common in this group, but results were not statistically significant. Conclusions: Patients with colorectal cancer have gotten progressively younger. The excess of colorectal cancer in AAs predominantly consists of more proximal, microsatellite stable tumors, commonly presenting lymphocytic infiltrate and less often associated with toxic exposures or a higher BMI. Younger AAs had more distal colorectal cancers than older ones. These data suggest two different mechanisms driving younger age and proximal location of colorectal cancers in AAs. Clin Cancer Res; 20(18); 4962–70. ©2014 AACR.

Purpose Disparities in both colorectal cancer (CRC) incidence and survival impact African Americans (AAs) more than other US ethnic groups. Because vitamin D is thought to protect against CRC and AAs have lower serum vitamin D levels, genetic variants that modulate the levels of active hormone in the tissues could explain some of the cancer health disparity. Consequently, we hypothesized that genetic variants in vitamin D-related genes are associated with CRC risk. Methods To test this hypothesis, we studied 39 potentially functional single-nucleotide polymorphisms (SNPs) in eight genes (CYP2R1, CYP3A4, CYP24A1, CYP27A1, CYP27B1, GC, DHCR7, and VDR) in 961 AA CRC cases and 838 healthy AA controls from Chicago and North Carolina. We tested whether SNPs are associated with CRC incidence using logistic regression models to calculate p values, odds ratios, and 95 % confidence intervals. In the logistic regression, we used a log-additive genetic model and used age, gender, and percent West African ancestry, which we estimated with the program STRUCTURE, as covariates in the models. Results A nominally significant association was detected between CRC and the SNP rs12794714 in the vitamin D 25-hydroxylase gene CYP2R1 (p = 0.019), a SNP that has previously been associated with serum vitamin D levels. Two SNPs, rs16847024 in the GC gene and rs6022990 in the CYP24A1 gene, were nominally associated with left-sided CRC (p = 0.015 and p = 0.018, respectively). Conclusions Our results strongly suggest that genetic variation in vitamin D-related genes could affect CRC susceptibility in AAs. Electronic supplementary material The online version of this article (doi:10.1007/s10552-014-0361-y) contains supplementary material, which is available to authorized users.

Colorectal cancer (CRC) is the second leading cause of cancer-related death among men and women in Western countries. Once a tumour develops, a differentiated prognosis could be determined by lifestyle habits or inherited and somatic genetic factors. Finding such prognostic factors will be helpful in order to identify cases with a shorter survival or at a higher risk of recurrence that may benefit from more intensive treatment and follow-up surveillance. Sixteen CRC genetic susceptibility variants were directly genotyped in a cohort of 1235 CRC patients recruited by the EPICOLON Spanish consortium. Univariate Cox and multivariate regression analyses were performed taking as primary outcomes overall survival (OS), disease-free survival and recurrence-free interval. Genetic variants rs9929218 at 16q22.1 and rs10795668 at 10p14 may have an effect on OS. The G allele of rs9929218 was linked with a better OS [GG genotype, genotypic model: hazard ratio (HR) = 0.65, 95% confidence interval (CI) 0.45-0.93, P = 0.0179; GG/GA genotypes, dominant model: HR = 0.66, 95% CI 0.47-0.94, P = 0.0202]. Likewise, the G allele of rs10795668 was associated with better clinical outcome (GG genotype, genotypic model: HR = 0.73, 95% CI 0.53-1.01, P = 0.0570; GA genotype, genotypic model: HR = 0.66, 95% CI 0.47-0.92, P = 0.0137; GG/GA genotypes, dominant model: HR = 0.68, 95% CI 0.50-0.94, P = 0.0194). In conclusion, CRC susceptibility variants rs9929218 and rs10795668 may exert some influence in modulating patient's survival and they deserve to be further tested in additional CRC cohorts in order to confirm their potential as prognosis or predictive biomarkers.

Background: Colorectal cancer (CRC) is a disease of complex aetiology, with much of the expected inherited risk being due to several common low risk variants. Genome-Wide Association Studies (GWAS) have identified 20 CRC risk variants. Nevertheless, these have only been able to explain part of the missing heritability. Moreover, these signals have only been inspected in populations of Northern European origin. Results: Thus, we followed the same approach in a Spanish cohort of 881 cases and 667 controls. Sixty-four variants at 24 loci were found to be associated with CRC at p-values

Genome-wide association studies have successfully identified 20 colorectal cancer susceptibility loci. Amongst these, four of the signals are defined by tagging single nucleotide polymorphisms (SNPs) on regions 14q22.2 (rs4444235 and rs1957636) and 20p12.3 (rs961253 and rs4813802). These markers are located close to two of the genes involved in bone morphogenetic protein (BMP) signaling (BMP4 and BMP2, respectively). By investigating these four SNPs in an initial cohort of Spanish origin, we found substantial evidence that minor allele frequencies (MAFs) may be different in northern and southern European populations. Therefore, we genotyped three additional southern European cohorts comprising a total of 2028 cases and 4273 controls. The meta-analysis results show that only one of the association signals (rs961253) is effectively replicated in the southern European populations, despite adequate power to detect all four. The other three SNPs (rs4444235, rs1957636 and rs4813802) presented discordant results in MAFs and linkage disequilibrium patterns between northern and southern European cohorts. We hypothesize that this lack of replication could be the result of differential tagging of the functional variant in both sets of populations. Were this true, it would have complex consequences in both our ability to understand the nature of the real causative variants, as well as for further study designs.

Background & Aims: Colorectal cancers (CRCs) with microsatellite instability (MSI) and a mismatch repair (MMR) immunohistochemical deficit without hypermethylation of the MLH1 promoter are likely to be caused by Lynch syndrome. Some patients with these cancers have not been found to have pathogenic germline mutations and are considered to have Lynch-like syndrome (LLS). The aim of this study was to determine the risk of cancer in families of patients with LLS. Methods: We studied a population-based cohort of 1705 consecutive patients, performing MSI tests and immunohistochemical analyses of MMR proteins. Patients were diagnosed with Lynch syndrome when they were found to have pathogenic germline mutations. Patients with MSI and loss of MSH2 and/or MSH6 expression, isolated loss of PMS2 or loss of MLH1 without MLH1 promoter hypermethylation, and no pathogenic mutation were considered to have LLS. The clinical characteristics of patients and the age- and sex-adjusted standardized incidence ratios (SIRs) of cancer in families were compared between groups. Results: The incidence of CRC was significantly lower in families of patients with LLS than in families with confirmed cases of Lynch syndrome (SIR for Lynch syndrome, 6.04; 95% confidence interval [CI], 3.58–9.54; SIR for LLS, 2.12; 95% CI, 1.16–3.56; P < .001). However, the incidence of CRC was higher in families of patients with LLS than in families with sporadic CRC (SIR for sporadic CRC, 0.48; 95% CI, 0.27–0.79; P < .001). Conclusions: The risk of cancer in families with LLS is lower that of families with Lynch syndrome but higher than that of families with sporadic CRC. These results confirm the need for special screening and surveillance strategies for these patients and their relatives. This work was supported by grants from Instituto de Salud Carlos III (PI-080726, INT-09/208, and PI11/026030), the Fondo de Investigación Sanitaria/FEDER (PS09/02368, 10/00384, 10/00918, 11/00219, and 11/00681), Fundació Olga Torres (CRP) and FP7 CHIBCHA Consortium (SCB and ACar), the Ministerio de Economía y Competitividad (SAF2010-19273), and Agència de Gestió d’Ajuts Universitaris i de Recerca (2009 SGR 849). SCB is supported by a contract from the Fondo de Investigación Sanitaria (CP03-0070). CIBERER and CIBERehd are funded by the Instituto de Salud Carlos III.

DNA methylation is a fundamental epigenetic mechanism in regulating the expression of genes controlling crucial cell functions in cancer development. Methylation defects (both global hypomethylation and hypermethylation of CpG islands) are implicated in colorectal carcinogenesis. Some nutrients have a clear effect on methylation, suggesting that some dietary-associated differences in the incidence of colorectal cancer could be due to the effect of diet on methylation. The presence of methylation defects has clear diagnostic and prognostic implications. Thus, several tests are being used for colorectal cancer screening based on methylated gene analysis, whether in feces or blood. In addition, the reversibility of methylation processes allows the development of chemotherapies that regulate this process through their antineoplastic activity.

Colorectal cancer (CRC) is the second most common cancer in Western countries. Hereditary forms only correspond to 5% of CRC burden. Recently, genome-wide association studies have identified common low-penetrant CRC genetic susceptibility loci. Early-onset CRC (CRC50 years old) is especially suggestive of hereditary predisposition although 85-90% of heritability still remains unidentified. CRC50 patients (n = 191) were compared with a late-onset CRC group (CRC65 years old) (n = 1264). CRC susceptibility variants at 8q23.3 (rs16892766), 8q24.21 (rs6983267), 10p14 (rs10795668), 11q23.1 (rs3802842), 15q13.3 (rs4779584), 18q21 (rs4939827), 14q22.2 (rs4444235), 16q22.1 (rs9929218), 19q13.1 (rs10411210) and 20p12.3 (rs961253) were genotyped in all DNA samples. A genotype-phenotype correlation with clinical and pathological characteristics in both groups was performed. Risk allele carriers for rs3802842 [Odds ratio (OR) = 1.5, 95% confidence interval (CI) 1.1-2.05, P = 0.0096, dominant model) and rs4779584 (OR = 1.39, 95% CI 1.02-1.9, P = 0.0396, dominant model) were more frequent in the CRC50 group, whereas homozygotes for rs10795668 risk allele were also more frequent in the early-onset CRC (P = 0.02, codominant model). Regarding early-onset cases, 14q22 (rs4444235), 11q23 (rs3802842) and 20p12 (rs961253) variants were more associated with family history of CRC or tumors of the Lynch syndrome spectrum excluding CRC. In our entire cohort, sum of risk alleles was significantly higher in patients with a CRC family history (OR = 1.40, 95% CI 1.06-1.85, P = 0.01). In conclusion, variants at 10p14 (rs10795668), 11q23.1 (rs3802842) and 15q13.3 (rs4779584) may have a predominant role in predisposition to early-onset CRC. Association of CRC susceptibility variants with some patient's familiar and personal features could be relevant for screening and surveillance strategies in this high-risk group and it should be explored in further studies.

Thirty-five percent of colorectal cancer (CRC) susceptibility is thought to be attributable to genetics, but only a small proportion of the cases (

Background & Aims Ten common low-penetrant genetic variants have been consistently associated with colorectal cancer (CRC) risk; little is known about the correlation between these variants and CRC phenotype. Characterization of such a correlation would improve CRC management and prevention programs. We assessed the association between these genetic variants and CRC phenotype in patients and modeled pairwise combinations to detect epistasis. Methods The validation population corresponded to a prospective, multicenter, population-based cohort (EPICOLON I) of 1096 patients with newly diagnosed CRC. The replication set was an independent, prospective, multicenter Spanish cohort (EPICOLON II) of 895 patients with newly diagnosed CRC. For individual single nucleotide polymorphism (SNP) association analyses, a multivariate method using logistic regression was applied in EPICOLON I and subsequently prospectively validated in EPICOLON II. Interactions between SNPs were assessed using the likelihood ratio test. Results Validated results confirmed that the C allele on 8q23.3 (rs16892766) was significantly associated with advanced-stage tumors (odds ratio [OR], 1.48; 95% confidence interval [CI], 1.15–1.90; P value=4.9 × 10 −3 ). The G allele on 8q24.21 (rs6983267) was more common in patients with a familial history of CRC (OR, 2.02; 95% CI, 1.35–3.03; P value=3.9 × 10 −4 ). The combination of rs6983267 on 8q24.21 and rs9929218 on 16q22.2 was associated with a history of colorectal adenoma (carriers of GG and AA, respectively; OR, 2.28; 95% CI, 1.32–3.93; P = 5.0 × 10 −4 ). Conclusions CRC susceptibility variants at 8q23.3, 8q24.21, and 16q22.2 appear to be associated with cancer phenotype. These findings might be used to develop screening and surveillance strategies.

The aim of this study is to evaluate if mismatch repair (MMR) defective colorectal cancer has a different response to adjuvant 5-fluorouracil (5-FU) chemotherapy in a cohort of patients prospectively followed during 5 years.The cohort included 754 surgically treated patients with colorectal cancer. MMR status was diagnosed by MLH1 and MSH2 immunohistochemistry and microsatellite instability analysis. Median follow-up was 49.2 months (range 1-73). At inclusion, 505 patients were diagnosed as TNM II or III stage, analysis of the efficacy of adjuvant chemotherapy was made on this population. Adjuvant chemotherapy was applied to 248 patients (98.2% 5-FU based).MMR deficiency was found in 76 patients (10.1%). No differences were found in overall survival (log-rank p=0.3) or disease-free survival (log-rank p=0.3) regarding MMR status. Adjuvant chemotherapy improves survival in patients in the II or III stage, but this improvement is only evident in patients with MMR-competent tumours (log-rank p=0.00001). Survival of patients with MMR-defective tumours does not improve with adjuvant chemotherapy (log-rank p=0.7). A multivariate analysis showed an independent effect of the interaction between MMR status and adjuvant chemotherapy (Hazard ratio 2.04; 95% confidence interval: 1.42-2.93).In a cohort of colorectal cancer patients, those with MMR-deficient tumours seem not to benefit from 5-FU-based chemotherapy.

Background & Aims Approximately half of the families that fulfill Amsterdam criteria for Lynch syndrome or hereditary nonpolyposis colorectal cancer (HNPCC) do not have evidence of the germline mismatch repair gene mutations that define this syndrome and result in microsatellite instability (MSI). The carcinogenic pathways and the best diagnostic approaches to detect microsatellite stable (MSS) HNPCC tumors are unclear. We investigated the contribution of epigenetic alterations to the development of MSS HNPCC tumors. Methods Colorectal cancers were divided into 4 groups: (1) microsatellite stable, Amsterdam-positive (MSS HNPCC) (N = 22); (2) Lynch syndrome cancers (identified mismatch repair mutations) (N = 21); (3) sporadic MSS (N = 92); and (4) sporadic MSI (N = 46). Methylation status was evaluated for CACNAG1 , SOCS1 , RUNX3 , NEUROG1 , MLH1 , and long interspersed nucleotide element-1 ( LINE-1 ). KRAS and BRAF mutation status was analyzed. Results MSS HNPCC tumors displayed a significantly lower degree of LINE-1 methylation, a marker for global methylation, than any other group. Although most MSS HNPCC tumors had some degree of CpG island methylation, none presented a high index of methylation. MSS HNPCC tumors had KRAS mutations exclusively in codon 12, but none harbored V600E BRAF mutations. Conclusions Tumors from Amsterdam-positive patients without mismatch repair deficiency (MSS HNPCC) have certain molecular features, including global hypomethylation, that distinguish them from all other colorectal cancers. These characteristics could have an important impact on tumor behavior or treatment response. Studies are underway to further assess the cause and effects of these features.

ContextThe selection of individuals for hereditary nonpolyposis colorectal cancer (HNPCC) genetic testing is challenging. Recently, the National Cancer Institute outlined a new set of recommendations, the revised Bethesda guidelines, for the identification of individuals with HNPCC who should be tested for microsatellite instability.ObjectiveTo establish the most effective and efficient strategy for the detection of MSH2/MLH1 gene carriers.Design, Setting, and PatientsA prospective, multicenter, nationwide study (the EPICOLON study) in 20 hospitals in the general community in Spain of 1222 patients with newly diagnosed colorectal cancer between November 1, 2000, and October 31, 2001.InterventionsMicrosatellite instability testing and MSH2/MLH1 immunostaining in all patients regardless of age, personal or family history, and tumor characteristics. Patients whose tumors exhibited microsatellite instability and/or lack of protein expression underwent MSH2/MLH1 germline testing.Main Outcome MeasuresEffectiveness and efficiency of both microsatellite instability testing and immunostaining, either directly or previous selection of patients according to the revised Bethesda guidelines, were evaluated with respect to the presence of MSH2/MLH1 germline mutations.ResultsTwo hundred eighty-seven patients (23.5%) fulfilled the revised Bethesda guidelines. Ninety-one patients (7.4%) had a mismatch repair deficiency, with tumors exhibiting either microsatellite instability (n = 83) or loss of protein expression (n = 81). Germline testing identified 11 mutations (0.9%) in either MSH2 (7 cases) or MLH1 (4 cases) genes. Strategies based on either microsatellite instability testing or immunostaining previous selection of patients according to the revised Bethesda guidelines were the most effective (sensitivity, 81.8% and 81.8%; specificity, 98.0% and 98.2%; positive predictive value, 27.3% and 29.0%, respectively) to identify MSH2/MLH1 gene carriers. Logistic regression analysis confirmed the revised Bethesda guidelines as the most discriminating set of clinical parameters (odds ratio, 33.3; 95% confidence interval, 4.3-250; P = .001).ConclusionThe revised Bethesda guidelines constitute a useful approach to identify patients at risk for HNPCC. In patients fulfilling these criteria, both microsatellite instability testing and immunostaining are equivalent and highly effective strategies to further select those patients who should be tested for MSH2/MLH1 germline mutations.

Background Colorectal cancer (CRC) is the second leading cause of cancer death in developed countries. Familial aggregation in CRC is also important outside syndromic forms and, in this case, a polygenic model with several common low-penetrance alleles contributing to CRC genetic predisposition could be hypothesized. Mucins and GALNTs (N-acetylgalactosaminyltransferase) are interesting candidates for CRC genetic susceptibility and have not been previously evaluated. We present results for ten genetic variants linked to CRC risk in previous studies (previously identified category) and 18 selected variants from the mucin gene family in a case-control association study from the Spanish EPICOLON consortium. Methods CRC cases and matched controls were from EPICOLON, a prospective, multicenter, nationwide Spanish initiative, comprised of two independent stages. Stage 1 corresponded to 515 CRC cases and 515 controls, whereas stage 2 consisted of 901 CRC cases and 909 controls. Also, an independent cohort of 549 CRC cases and 599 controls outside EPICOLON was available for additional replication. Genotyping was performed for ten previously identified SNPs in ADH1C, APC, CCDN1, IL6, IL8, IRS1, MTHFR, PPARG, VDR and ARL11, and 18 selected variants in the mucin gene family. Results None of the 28 SNPs analyzed in our study was found to be associated with CRC risk. Although four SNPs were significant with a P-value < 0.05 in EPICOLON stage 1 [rs698 in ADH1C (OR = 1.63, 95% CI = 1.06-2.50, P-value = 0.02, recessive), rs1800795 in IL6 (OR = 1.62, 95% CI = 1.10-2.37, P-value = 0.01, recessive), rs3803185 in ARL11 (OR = 1.58, 95% CI = 1.17-2.15, P-value = 0.007, codominant), and rs2102302 in GALNTL2 (OR = 1.20, 95% CI = 1.00-1.44, P-value = 0.04, log-additive 0, 1, 2 alleles], only rs3803185 achieved statistical significance in EPICOLON stage 2 (OR = 1.34, 95% CI = 1.06-1.69, P-value = 0.01, recessive). In the joint analysis for both stages, results were only significant for rs3803185 (OR = 1.12, 95% CI = 1.00-1.25, P-value = 0.04, log-additive 0, 1, 2 alleles) and borderline significant for rs698 and rs2102302. The rs3803185 variant was not significantly associated with CRC risk in an external cohort (MCC-Spain), but it still showed some borderline significance in the pooled analysis of both cohorts (OR = 1.08, 95% CI = 0.98-1.18, P-value = 0.09, log-additive 0, 1, 2 alleles). Conclusions ARL11, ADH1C, GALNTL2 and IL6 genetic variants may have an effect on CRC risk. Further validation and meta-analyses should be undertaken in larger CRC studies.

Background Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) associated with diseases of the colon including inflammatory bowel diseases (IBD) and colorectal cancer (CRC). However, the functional role of many of these SNPs is largely unknown and tissue-specific resources are lacking. Expression quantitative trait loci (eQTL) mapping identifies target genes of disease-associated SNPs. This study provides a comprehensive eQTL map of distal colonic samples obtained from 40 healthy African Americans and demonstrates their relevance for GWAS of colonic diseases. Results 8.4 million imputed SNPs were tested for their associations with 16,252 expression probes representing 12,363 unique genes. 1,941 significant cis-eQTL, corresponding to 122 independent signals, were identified at a false discovery rate (FDR) of 0.01. Overall, among colon cis-eQTL, there was significant enrichment for GWAS variants for IBD (Crohn’s disease [CD] and ulcerative colitis [UC]) and CRC as well as type 2 diabetes and body mass index. ERAP2, ADCY3, INPP5E, UBA7, SFMBT1, NXPE1 and REXO2 were identified as target genes for IBD-associated variants. The CRC-associated eQTL rs3802842 was associated with the expression of C11orf93 (COLCA2). Enrichment of colon eQTL near transcription start sites and for active histone marks was demonstrated, and eQTL with high population differentiation were identified. Conclusions Through the comprehensive study of eQTL in the human colon, this study identified novel target genes for IBD- and CRC-associated genetic variants. Moreover, bioinformatic characterization of colon eQTL provides a tissue-specific tool to improve understanding of biological differences in diseases between different ethnic groups. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1292-z) contains supplementary material, which is available to authorized users.