Jenifer Muñoz

Aberrant gene promoter methylation associated with sporadic multiple colorectal cancer.

Background Colorectal 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 Findings We 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. Conclusions These 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.

MSH6 and MUTYH Deficiency Is a Frequent Event in Early-Onset Colorectal Cancer

Purpose: Early-onset colorectal cancer (CRC) is suggestive of a hereditary predisposition. Lynch syndrome is the most frequent CRC hereditary cause. The MUTYH gene has also been related to hereditary CRC. A systematic characterization of these two diseases has not been reported previously in this population. Experimental Design: We studied a retrospectively collected series of 140 patients ≤50 years old diagnosed with nonpolyposis CRC. Demographic, clinical, and familial features were obtained. Mismatch repair (MMR) deficiency was determined by microsatellite instability (MSI) analysis, and immunostaining for MLH1, MSH2, MSH6, and PMS2 proteins. Germline MMR mutations were evaluated in all MMR-deficient cases. Tumor samples with loss of MLH1 or MSH2 protein expression were analyzed for somatic methylation. Germline MUTYH mutations were evaluated in all cases. BRAF V600E and KRAS somatic mutational status was also determined. Results: Fifteen tumors (11.4%) were MSI, and 20 (14.3%) showed loss of protein expression (7 for MLH1/PMS2, 2 for isolated MLH1, 3 for MSH2/MSH6, 7 for isolated MSH6, and 1 for MSH6/PMS2). We identified 11 (7.8%) germline MMR mutations, 4 in MLH1, 1 in MSH2, and 6 in MSH6. Methylation analysis revealed one case with somatic MLH1 methylation. Biallelic MUTYH mutations were detected in four (2.8%) cases. KRAS and BRAF V600E mutations were present in 39 (27.9%) and 5 (3.6%) cases, respectively. Conclusions: Loss of MSH6 expression is the predominant cause of MMR deficiency in early-onset CRC. Our findings prompt the inclusion of MSH6 and MUTYH screening as part of the genetic counseling of these patients and their relatives. Clin Cancer Res; 16(22); 5402–13. ©2010 AACR.

Susceptibility Genetic Variants Associated With Colorectal Cancer Risk Correlate With Cancer Phenotype

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 x 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 x 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 x 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.

Molecular analysis of the APC and MUTYH genes in Galician and Catalonian FAP families: a different spectrum of mutations?

BackgroundFamilial adenomatous polyposis (FAP) is an autosomal dominant-inherited colorectal cancer syndrome, caused by germline mutations in the APC gene. Recently, biallelic mutations in MUTYH have also been identified in patients with multiple colorectal adenomas and in APC-negative patients with FAP. The aim of this work is therefore to determine the frequency of APC and MUTYH mutations among FAP families from two Spanish populations.MethodsEighty-two unrelated patients with classical or attenuated FAP were screened for APC germline mutations. MUTYH analysis was then conducted in those APC-negative families and in 9 additional patients from a previous study. Direct sequencing, SSCP analysis and TaqMan genotyping were used to identify point and frameshift mutations, meanwhile large rearrangements in the APC gene were screened by multiplex ligation-dependent probe amplification (MLPA).ResultsAPC germline mutations were found in 39% of the patients and, despite the great number of genetic variants described so far in this gene, seven new mutations were identified. The two hotspots at codons 1061 and 1309 of the APC gene accounted for 9,4% of the APC-positive families, although they were underrepresented in Galician samples. The deletion at codon 1061 was not found in 19 APC-positive Galician patients but represented 23% of the Catalonian positive families (p = 0,058). The same trend was observed at codon 1309, even though statistical analysis showed no significance between populations. Twenty-four percent of the APC-negative patients carried biallelic MUTYH germline mutations, and showed an attenuated polyposis phenotype generally without extracolonic manifestations. New genetic variants were found, as well as the two hotspots already reported (p.Tyr165Cys and p.Gly382Asp).ConclusionThe results we present indicate that in Galician patients the frequency of the hotspot at codon 1061 in APC differs significantly from the Catalonian and also other Caucasian populations. Similar results had already been obtained in a previous study and could be due to the genetic isolation of the Galician population. MUTYH analysis is also recommended for all APC-negative families, even if a recessive inheritance is not confirmed.

Somatic POLE proofreading domain mutation, immune response, and prognosis in colorectal cancer: a retrospective, pooled biomarker study

BACKGROUND Precision cancer medicine depends on defining distinct tumour subgroups using biomarkers that may occur at very modest frequencies. One such subgroup comprises patients with exceptionally mutated (ultramutated) cancers caused by mutations that impair DNA polymerase epsilon (POLE) proofreading. METHODS We examined the association of POLE proofreading domain mutation with clinicopathological variables and immune response in colorectal cancers from clinical trials (VICTOR, QUASAR2, and PETACC-3) and colorectal cancer cohorts (Leiden University Medical Centre 1 and 2, Oslo 1 and 2, Bern, AMC-AJCC-II, and Epicolon-1). We subsequently investigated its association with prognosis in stage II/III colorectal cancer by Cox regression of pooled individual patient data from more than 4500 cases from these studies. FINDINGS Pathogenic somatic POLE mutations were detected in 66 (1·0%) of 6517 colorectal cancers, and were mutually exclusive with mismatch repair deficiency (MMR-D) in the 6277 cases for whom both markers were determined (none of 66 vs 833 [13·4%] of 6211; p<0·0001). Compared with cases with wild-type POLE, cases with POLE mutations were younger at diagnosis (median 54·5 years vs 67·2 years; p<0·0001), were more frequently male (50 [75·8%] of 66 vs 3577 [55·5%] of 6445; p=0·0010), more frequently had right-sided tumour location (44 [68·8%] of 64 vs 2463 [39·8%] of 6193; p<0·0001), and were diagnosed at an earlier disease stage (p=0·006, χ2 test for trend). Compared with mismatch repair proficient (MMR-P) POLE wild-type tumours, POLE-mutant colorectal cancers displayed increased CD8+ lymphocyte infiltration and expression of cytotoxic T-cell markers and effector cytokines, similar in extent to that observed in immunogenic MMR-D cancers. Both POLE mutation and MMR-D were associated with significantly reduced risk of recurrence compared with MMR-P colorectal cancers in multivariable analysis (HR 0·34 [95% CI 0·11-0·76]; p=0·0060 and 0·72 [0·60-0·87]; p=0·00035), although the difference between the groups was not significant. INTERPRETATION POLE proofreading domain mutations identify a subset of immunogenic colorectal cancers with excellent prognosis. This association underscores the importance of rare biomarkers in precision cancer medicine, but also raises important questions about how to identify and implement them in practice. FUNDING Cancer Research UK, Academy of Medical Sciences, Health Foundation, EU, ERC, NIHR, Wellcome Trust, Dutch Cancer Society, Dutch Digestive Foundation.

Whole-exome sequencing identifies rare pathogenic variants in new predisposition genes for familial colorectal cancer.

Purpose:Colorectal cancer is an important cause of mortality in the developed world. Hereditary forms are due to germ-line mutations in APC, MUTYH, and the mismatch repair genes, but many cases present familial aggregation but an unknown inherited cause. The hypothesis of rare high-penetrance mutations in new genes is a likely explanation for the underlying predisposition in some of these familial cases.Methods:Exome sequencing was performed in 43 patients with colorectal cancer from 29 families with strong disease aggregation without mutations in known hereditary colorectal cancer genes. Data analysis selected only very rare variants (0–0.1%), producing a putative loss of function and located in genes with a role compatible with cancer. Variants in genes previously involved in hereditary colorectal cancer or nearby previous colorectal cancer genome-wide association study hits were also chosen.Results:Twenty-eight final candidate variants were selected and validated by Sanger sequencing. Correct family segregation and somatic studies were used to categorize the most interesting variants in CDKN1B, XRCC4, EPHX1, NFKBIZ, SMARCA4, and BARD1.Conclusion:We identified new potential colorectal cancer predisposition variants in genes that have a role in cancer predisposition and are involved in DNA repair and the cell cycle, which supports their putative involvement in germ-line predisposition to this neoplasm.Genet Med 17 2, 131–142.

Susceptibility genetic variants associated with early-onset colorectal cancer.

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 (CRC<50 years old) is especially suggestive of hereditary predisposition although 85-90% of heritability still remains unidentified. CRC<50 patients (n = 191) were compared with a late-onset CRC group (CRC>65 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 CRC<50 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 patients 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.

Prevalence of somatic mutl homolog 1 promoter hypermethylation in Lynch syndrome colorectal cancer.

Colorectal cancers (CRCs) that have microsatellite instability (MSI) and mutL homolog 1 (MLH1) immunoloss are observed in 3 clinical scenarios: Lynch syndrome (LS), sporadic MSI CRC, and Lynch‐like syndrome (LLS). v‐Raf murine sarcoma viral oncogene homolog B1 (BRAF) mutational analysis is used to differentiate LS from sporadic MSI CRC. The role of MLH1 promoter methylation status for the differential diagnosis of these clinical forms is not well established. The objectives of this study were: 1) to analyze MLH1 promoter methylation in MLH1‐deficient CRCs by pyrosequencing, and 2) to assess its role in the differential diagnosis of MLH1‐deficient CRCs.

Case-control study for colorectal cancer genetic susceptibility in EPICOLON: previously identified variants and mucins.

BackgroundColorectal 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.MethodsCRC 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.ResultsNone 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).ConclusionsARL11, 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.

New genes emerging for colorectal cancer predisposition

Colorectal cancer (CRC) is one of the most frequent neoplasms and an important cause of mortality in the developed world. This cancer is caused by both genetic and environmental factors although 35% of the variation in CRC susceptibility involves inherited genetic differences. Mendelian syndromes account for about 5% of the total burden of CRC, with Lynch syndrome and familial adenomatous polyposis the most common forms. Excluding hereditary forms, there is an important fraction of CRC cases that present familial aggregation for the disease with an unknown germline genetic cause. CRC can be also considered as a complex disease taking into account the common disease-commom variant hypothesis with a polygenic model of inheritance where the genetic components of common complex diseases correspond mostly to variants of low/moderate effect. So far, 30 common, low-penetrance susceptibility variants have been identified for CRC. Recently, new sequencing technologies including exome- and whole-genome sequencing have permitted to add a new approach to facilitate the identification of new genes responsible for human disease predisposition. By using whole-genome sequencing, germline mutations in the POLE and POLD1 genes have been found to be responsible for a new form of CRC genetic predisposition called polymerase proofreading-associated polyposis.