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Somatic single-nucleotide variants (SNVs) occur every time a cell divides, appearing even in healthy tissues at low frequencies. These mutations may accumulate as neutral variants during aging, or eventually, promote the development of neoplasia. Here, we present the SP-ddPCR, a droplet digital PCR (ddPCR) based approach that utilizes customized SuperSelective primers aiming at quantifying the proportion of rare SNVs. For that purpose, we selected five potentially pathogenic variants identified by whole-exome sequencing (WES) occurring at low variant allele frequency (VAF) in at-risk colon healthy mucosa of patients diagnosed with colorectal cancer or advanced adenoma. Additionally, two APC SNVs detected in two cancer lesions were added to the study for WES-VAF validation. SuperSelective primers were designed to quantify SNVs at low VAFs both in silico and in clinical samples. In addition to the two APC SNVs in colonic lesions, SP-ddPCR confirmed the presence of three out of five selected SNVs in the normal colonic mucosa with allelic frequencies ≤ 5%. Moreover, SP-ddPCR showed the presence of two potentially pathogenic variants in the distal normal mucosa of patients with colorectal carcinoma. In summary, SP-ddPCR offers a rapid and feasible methodology to validate next-generation sequencing data and accurately quantify rare SNVs, thus providing a potential tool for diagnosis and stratification of at-risk patients based on their mutational profiling., (© 2023. The Author(s).)

Serrated polyposis syndrome (SPS) is associated with a high risk for colorectal cancer. Intense promoter hypermethylation is a frequent molecular finding in the serrated pathway and may be present in normal mucosa, predisposing to the formation of serrated lesions. To identify novel biomarkers for SPS, fresh-frozen samples of normal mucosa from 50 patients with SPS and 19 healthy individuals were analyzed by using the 850K BeadChip Technology (Infinium). Aberrant methylation levels were correlated with gene expression using a next-generation transcriptome profiling tool. Two validation steps were performed on independent cohorts: first, on formalin-fixed, paraffin-embedded tissue of the normal mucosa; and second, on 24 serrated lesions. The most frequently hypermethylated genes were HLA-F, SLFN12, HLA-DMA, and RARRES3; and the most frequently hypomethylated genes were PIWIL1 and ANK3 (Δβ = 10%; P < 0.05). Expression levels of HLA-F, SLFN12, and HLA-DMA were significantly different between SPS patients and healthy individuals and correlated well with the methylation status of the corresponding differentially methylated region (fold change, >20%; r > 0.55; P < 0.001). Significant hypermethylation of CpGs in the gene body of HLA-F was also found in serrated lesions (Δβ = 23%; false discovery rate = 0.01). Epigenome-wide methylation profiling has revealed numerous differentially methylated CpGs in normal mucosa from SPS patients. Significant hypermethylation of HLA-F is a novel biomarker candidate for SPS., (Copyright © 2022 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Optimal selection of high-risk patients with stage II colon cancer is crucial to ensure clinical benefit of adjuvant chemotherapy. Here, we investigated the prognostic value of genomic intratumor heterogeneity and aneuploidy for disease recurrence. We combined targeted sequencing, SNP arrays, fluorescence in situ hybridization, and immunohistochemistry on a retrospective cohort of 84 untreated stage II colon cancer patients. We assessed the clonality of copy-number alterations (CNAs) and mutations, CD8 + lymphocyte infiltration, and their association with time to recurrence. Prognostic factors were included in machine learning analysis to evaluate their ability to predict individual relapse risk. Tumors from recurrent patients displayed a greater proportion of CNAs compared with non-recurrent (mean 31.3% versus 23%, respectively; p = 0.014). Furthermore, patients with elevated tumor CNA load exhibited a higher risk of recurrence compared with those with low levels [p = 0.038; hazard ratio (HR) 2.46], which was confirmed in an independent cohort (p = 0.004; HR 3.82). Candidate chromosome-specific aberrations frequently observed in recurrent cases included gain of the chromosome arm 13q (p = 0.02; HR 2.67) and loss of heterozygosity at 17q22-q24.3 (p = 0.05; HR 2.69). CNA load positively correlated with intratumor heterogeneity (R = 0.52; p < 0.0001). Consistently, incremental subclonal CNAs were associated with an elevated risk of relapse (p = 0.028; HR 2.20), which we did not observe for subclonal single-nucleotide variants and small insertions and deletions. The clinico-genomic model rated an area under the curve of 0.83, achieving a 10% incremental gain compared with clinicopathological markers (p = 0.047). In conclusion, tumor aneuploidy and copy-number intratumor heterogeneity were predictive of poor outcome and improved discriminative performance in early-stage colon cancer. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

Objectives: Most patients with multiple colonic polyps do not have a known genetic or hereditary origin. Our aim was to analyze the presence of inflammatory cytokines and levels of glucose, insulin, and C-reactive protein (CRP) in patients with multiple colonic polyps., Methods: Eighty-three patients with 10 or more adenomatous or serrated polyps and 53 control people with normal colonoscopy were included. Smoking habits were registered, and glucose, CRP, and basal insulin in the serum/blood were measured. Quantification of IL-2, IL-4, IL-6, IL-10, IL-11, IL-17A, and IL-23 cytokine levels in the serum was performed by a high-sensitivity enzyme-linked immunosorbent assay., Results: Smoking and diabetes were more prevalent in those with colonic polyps than in the control people (67% vs 16%, P = 0.001; 11% vs 2%, P = 0.048). In addition, the cytokine serum levels were higher, i.e., IL-2 (P = 0.001), IL-4 (P = 0.001), IL-6 (P = 0.001), IL-17A (P = 0.001), IL-23 (P = 0.014), and CRP (P = 0.003). Adjusting for sex, smoking, and diabetes in a multivariate analysis, IL-2, IL-4, IL-6, IL-17A, and IL-23 remained independently elevated in cases with multiple polyps., Discussion: These results indicate that immune responses mediated by Th17 cells may be involved in the pathogenesis of multiple colonic polyps.

Colorectal cancer (CRC), a leading cause of cancer-related death worldwide, evolves as a result of the stepwise accumulation of a series of genetic and epigenetic alterations in the normal colonic epithelium, leading to the development of colorectal adenomas and invasive adenocarcinomas. Although genetic alterations have a major role in a subset of CRCs, the pathophysiological contribution of epigenetic aberrations in this malignancy has attracted considerable attention. Data from the past couple of decades has unequivocally illustrated that epigenetic marks are important molecular hallmarks of cancer, as they occur very early in disease pathogenesis, involve virtually all key cancer-associated pathways and, most importantly, can be exploited as clinically relevant disease biomarkers for diagnosis, prognostication and prediction of treatment response. In this Review, we summarize the current knowledge on the best-studied epigenetic modifications in CRC, including DNA methylation and histone modifications, as well as the role of non-coding RNAs as epigenetic regulators. We focus on the emerging potential for the bench-to-bedside translation of some of these epigenetic alterations into clinical practice and discuss the burgeoning evidence supporting the potential of emerging epigenetic therapies in CRC as we usher in the era of precision medicine.

Somatic copy number alterations (CNAs) are a hallmark of cancer, but their role in tumorigenesis and clinical relevance remain largely unclear. Here, we developed CNApp, a web-based tool that allows a comprehensive exploration of CNAs by using purity-corrected segmented data from multiple genomic platforms. CNApp generates genome-wide profiles, computes CNA scores for broad, focal and global CNA burdens, and uses machine learning-based predictions to classify samples. We applied CNApp to the TCGA pan-cancer dataset of 10,635 genomes showing that CNAs classify cancer types according to their tissue-of-origin, and that each cancer type shows specific ranges of broad and focal CNA scores. Moreover, CNApp reproduces recurrent CNAs in hepatocellular carcinoma and predicts colon cancer molecular subtypes and microsatellite instability based on broad CNA scores and discrete genomic imbalances. In summary, CNApp facilitates CNA-driven research by providing a unique framework to identify relevant clinical implications. CNApp is hosted at https://tools.idibaps.org/CNApp/., Competing Interests: SF, LB, MV, EH, RE, MD, JL, AC, SC, JC No competing interests declared, JL is receiving research support from Bayer HealthCare Pharmaceuticals, Eisai Inc, Bristol-Myers Squibb and Ipsen, and consulting fees from Eli Lilly, Bayer HealthCare Pharmaceuticals, Bristol-Myers Squibb, EISAI Inc, Celsion Corporation, Exelixis, Merck, Ipsen, Glycotest, Navigant, Leerink Swann LLC, Midatech Ltd, and Nucleix, (© 2020, Franch-Expósito et al.)

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<0.0001) and multivariate analyses (hazard ratio 1.75, 95% CI 1.05-2.93, P = 0.03) after adjusting for age, sex, chemotherapy, and TNM stage. Treatment with 5-FU-based regimens improved prognosis in patients with the combination of MSS tumours, KRAS mutation and CIMP negative (log rank P = 0.003) as well as in patients with MSS tumours plus BRAF and KRAS wild-type and CIMP negative (log-rank P<0.001). After adjusting for age, sex, and TNM stage in the multivariate analysis, only patients with the latter molecular combination had independently improved prognosis after adjuvant chemotherapy (hazard ratio 2.06, 95% CI 1.24-3.44, P = 0.005)., Conclusion: We confirmed the prognostic value of stratifying CRC according to molecular subtypes using MSI, CIMP status, and somatic KRAS and BRAF mutation. Patients with traditional chromosomally unstable tumours obtained the best benefit from adjuvant 5-FU-based chemotherapy., Competing Interests: The authors have declared that no competing interests exist.

Purpose: A recent study reported that 5-fluorouracil (5-FU)-based chemotherapy is less effective in treating patients with advanced colorectal cancer demonstrating hypermethylation of the TFAP2E gene. The aim of our study was to confirm and validate these findings in large, uniformly treated, well-characterized patient cohorts. Experimental Design: Two cohorts of 783 patients with colorectal cancer: 532 from a population-based, multicenter cohort (EPICOLON I) and 251 patients from a clinic-based trial were used to study the effectiveness of TFAP2E methylation and expression as a predictor of response of colorectal cancer patients to 5-FU-based chemotherapy. DNA methylation status of the TFAP2E gene in patients with colorectal cancer was assessed by quantitative bisulfite pyrosequencing analysis. IHC analysis of the TFAP2E protein expression was also performed. Results: Correlation between TFAP2E methylation status and IHC staining was performed in 607 colorectal cancer samples. Among 357 hypermethylated tumors, only 141 (39.6%) exhibited loss of protein expression. Survival was not affected by TFAP2E hypermethylation in stage IV patients [HR, 1.21; 95% confidence interval (CI), 0.79-1.87; log-rank P = 0.6]. In stage II-III cases, disease-free survival was not influenced by TFAP2E hypermethylation status in 5-FU-treated (HR, 0.91; 95% CI, 0.52-1.59; log-rank P = 0.9) as well as in nontreated patients (HR, 0.88; 95% CI, 0.5-1.54; log-rank P = 0.7). Conclusions: TFAP2E hypermethylation does not correlate with loss of its protein expression. Our large, systematic, and comprehensive study indicates that TFAP2E methylation and expression may not play a major role in predicting response to 5-FU-based chemotherapy in patients with colorectal cancer. Clin Cancer Res; 24(12); 2820-7. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Background: Mutational signatures have been proved as a valuable pattern in somatic genomics, mainly regarding cancer, with a potential application as a biomarker in clinical practice. Up to now, several bioinformatic packages to address this topic have been developed in different languages/platforms. MutationalPatterns has arisen as the most efficient tool for the comparison with the signatures currently reported in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. However, the analysis of mutational signatures is nowadays restricted to a small community of bioinformatic experts., Results: In this work we present Mutational Signatures in Cancer (MuSiCa), a new web tool based on MutationalPatterns and built using the Shiny framework in R language. By means of a simple interface suited to non-specialized researchers, it provides a comprehensive analysis of the somatic mutational status of the supplied cancer samples. It permits characterizing the profile and burden of mutations, as well as quantifying COSMIC-reported mutational signatures. It also allows classifying samples according to the above signature contributions., Conclusions: MuSiCa is a helpful web application to characterize mutational signatures in cancer samples. It is accessible online at http://bioinfo.ciberehd.org/GPtoCRC/en/tools.html and source code is freely available at https://github.com/marcos-diazg/musica .

Background & Aims: High-risk features of colonic polyps are based on size, number, and pathologic characteristics. Surveillance colonoscopy is often recommended according to these findings. This study aimed to determine whether the molecular characteristics of polyps might provide information about the risk of metachronous advanced neoplasia., Methodology: We retrospectively included 308 patients with colonic polyps. A total of 995 polyps were collected and tested for somatic BRAF and KRAS mutations. Patients were classified into 3 subgroups, based on the polyp mutational profile at baseline, as follows: non-mutated polyps (Wild-type), at least one BRAF-mutated polyp, or at least one KRAS-mutated polyp. At surveillance, advanced adenomas were defined as adenomas ≥ 10 mm and/or with high grade dysplasia or a villous component. In contrast, advanced serrated polyps were defined as serrated polyps ≥ 10 mm in any location, located proximal to the splenic flexure with any size or with dysplasia., Results: At baseline, 289 patients could be classified as wild-type (62.3%), BRAF mutated (14.9%), or KRAS mutated (22.8%). In the univariate analysis, KRAS mutations were associated with the development of metachronous advanced polyps (OR: 2.36, 95% CI: 1.22-4.58; P = 0.011), and specifically, advanced adenomas (OR: 2.42, 95% CI: 1.13-5.21; P = 0.023). The multivariate analysis, adjusted for age and sex, also showed associations with the development of metachronous advanced polyps (OR: 2.27, 95% CI: 1.15-4.46) and advanced adenomas (OR: 2.23, 95% CI: 1.02-4.85)., Conclusions: Our results suggested that somatic KRAS mutations in polyps represent a potential molecular marker for the risk of developing advanced neoplasia.

Background: We identified a new and a recurrent POLD1 mutation associated with predisposition to colorectal cancer (CRC). We characterized the molecular and clinical nature of the potential POLD1 founder mutation in families from Valencia (Spain)., Methods: Clinical and molecular data were collected from four independent families known to have a POLD1 Leu474Pro mutation. To establish its founder effect, haplotype construction was performed using 14 flanking POLD1 polymorphic markers. We calculated penetrance estimates and clinical expressivity, globally and stratified by age and sex., Results: We included 32 individuals from the four families: 20 carriers and 12 noncarriers. A common haplotype was identified in these families in a region comprising 2,995 Mb, confirming L474P as the first founder POLD1 mutation identified. Thirteen tumors diagnosed in 10 POLD1 carriers: eight CRC, three endometrial and two other tumors were considered. The median age of cancer onset for POLD1 mutation carriers was 48 years. The observed penetrance was 50% and the cumulative risk at age of 50 years was 30%., Conclusions: The findings of the present study contribute to a better understanding of CRC genetics in the Spanish population. The clinical phenotype for this mutation is similar to that in Lynch syndrome. Future studies using next generation sequencing with large gene panels for any hereditary cancer condition will offer the possibility of detecting POLE/POLD1 mutations in unsuspected clinical situations, demonstrating a more real and unbiased picture of the associated phenotype., (Copyright © 2017 John Wiley & Sons, Ltd.)

Molecular advances support the existence of an alternative pathway of colorectal carcinogenesis that is based on the hypermethylation of specific DNA regions that silences tumor suppressor genes. This alternative pathway has been called the serrated pathway due to the serrated appearance of tumors in histological analysis. New classifications for colorectal cancer (CRC) were proposed recently based on genetic profiles that show four types of molecular alterations: BRAF gene mutations, KRAS gene mutations, microsatellite instability, and hypermethylation of CpG islands. This review summarizes what is known about the serrated pathway of CRC, including CRC molecular and clinical features, prognosis, and response to chemotherapy.

Background: The prevalence of MLH1 constitutional epimutations in the general population is unknown. We sought to analyse the prevalence of MLH1 constitutional epimutations in unselected and selected series of patients with colorectal cancer (CRC)., Methods: Patients with diagnoses of CRC (n=2123) were included in the unselected group. For comparison, a group of 847 selected patients with CRC who fulfilled the revised Bethesda guidelines (rBG) were also included. Somatic and constitutional MLH1 methylation was assayed via methylation-specific multiplex ligation-dependent probe amplification of cases lacking MLH1 expression. Germline alterations in mismatch-repair (MMR) genes were assessed via Sanger sequencing and methylation-specific multiplex ligation-dependent probe amplification., Results: Loss of MLH1 expression occurred in 5.5% of the unselected series and 12.5% of the selected series (p<0.0001). No constitutional epimutations in MLH1 were detected in the unselected population (0/62); five cases from the selected series were positive for MLH1 epimutations (15.6%, 5/32; p=0.004)., Conclusions: Our results suggest a negligible prevalence of MLH1 constitutional epimutations in unselected cases of CRC. Therefore, MLH1 constitutional epimutation analysis should be conducted only for patients who fulfil the rBG and who lack MLH1 expression with methylated MLH1., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Background and Aims: Individuals with tumours showing mismatch repair (MMR) deficiency not linked to germline mutations or somatic methylation of MMR genes have been recently referred as having 'Lynch-like syndrome' (LLS). The genetic basis of these LLS cases is unknown. MUTYH-associated polyposis patients show some phenotypic similarities to Lynch syndrome patients. The aim of this study was to investigate the prevalence of germline MUTYH mutations in a large series of LLS patients., Methods: Two hundred and twenty-five probands fulfilling LLS criteria were included in this study. Screening of MUTYH recurrent mutations, whole coding sequencing and a large rearrangement analysis were undertaken. Age, sex, clinical, pathological and molecular characteristics of tumours including KRAS mutations were assessed., Results: We found a prevalence of 3.1% of MAP syndrome in the whole series of LLS (7/225) and 3.9% when only cases fulfilling clinical criteria were considered (7/178). Patients with MUTYH biallelic mutations had more adenomas than monoallelic (P=0.02) and wildtype patients (P<0.0001). Six out of nine analysed tumours from six biallelic MUTYH carriers harboured KRAS-p.G12C mutation. This mutation was found to be associated with biallelic MUTYH germline mutation when compared with reported series of unselected colorectal cancer cohorts (P<0.0001)., Conclusions: A proportion of unexplained LLS cases is caused by biallelic MUTYH mutations. The obtained results further justify the inclusion of MUTYH in the diagnostic strategy for Lynch syndrome-suspected patients., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Germline mutations in DNA polymerase ɛ (POLE) and δ (POLD1) have been recently identified in families with multiple colorectal adenomas and colorectal cancer (CRC). All reported cases carried POLE c.1270C>G (p.Leu424Val) or POLD1 c.1433G>A (p.Ser478Asn) mutations. Due to the scarcity of cases reported so far, an accurate clinical phenotype has not been defined. We aimed to assess the prevalence of these recurrent mutations in unexplained familial and early-onset CRC and polyposis, and to add additional information to define the clinical characteristics of mutated cases. A total of 858 familial/early onset CRC and polyposis patients were studied: 581 familial and early-onset CRC cases without mismatch repair (MMR) deficiency, 86 cases with MMR deficiency and 191 polyposis cases. Mutation screening was performed by KASPar genotyping assays and/or Sanger sequencing of the involved exons. POLE p.L424V was identified in a 28-year-old polyposis and CRC patient, as a de novo mutation. None of the 858 cases studied carried POLD1 p.S478N. A new mutation, POLD1 c.1421T>C (p.Leu474Pro), was identified in a mismatch repair proficient Amsterdam II family. Its pathogenicity was supported by cosegregation in the family, in silico predictions, and previously published yeast assays. POLE and POLD1 mutations explain a fraction of familial CRC and polyposis. Sequencing the proofreading domains of POLE and POLD1 should be considered in routine genetic diagnostics. Until additional evidence is gathered, POLE and POLD1 genetic testing should not be restricted to polyposis cases, and the presence of de novo mutations, considered., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)