TFAP2E Methylation and Expression Status Does Not Predict Response to 5-FU-based Chemotherapy in Colorectal Cancer

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, wellcharacterized 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 P¼ 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 ¼ 0.9) as well as in nontreated patients (HR, 0.88; 95% CI, 0.5–1.54; log-rank P ¼ 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

Copy-number intratumor heterogeneity increases the risk of relapse in chemotherapy-naive stage colon cancer

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

CNApp, a tool for the quantification of copy number alterations and integrative analysis revealing clinical implications.

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/

DNA Methylation Profiling of SPS

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 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

Increased Th17-Related Cytokine Serum Levels in Patients With Multiple Polyps of Unexplained Origin

OBJECTIVES: Most patientswith 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 weremeasured. 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 diabetesweremore 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 amultivariate 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

Characterization of a novel POLD1 missense founder mutation in a Spanish population

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.This work was supported by the Institute for Health and Biomedical Research of Alicante (ISABIAL, UGP‐16‐146). RFA is recipient of a Fellowship from the Consellería Educación of the Valencian Community. ACA is funded by the Acción Juvenil from the Spanish Ministry of Economy and Competitiveness. VDO is recipient of a Fellowship from the Spanish Association Against Cancer (AECC). AC and MIC are funded by Health and Biomedical Research Foundation from the Valencian Region (FISABIO). EHI is recipient of a fellowship from the Fondo Investigación Sanitaria ISCIII (FI12/00233)

Profundizando en el riesgo (epi)genético a cáncer colorrectal: Nuevos genes responsables y marcadores moleculares para el cribado

El cáncer colorrectal (CCR) constituye en la actualidad la neoplasia más frecuente en España. Hasta un 30% de los casos de CCR se incluyen dentro del CCR familiar y alrededor de un 3-5% se deben a mutaciones en genes de elevada penetrancia que siguen una herencia mendeliana Éstos últimos incluyen al CCR hereditario no polipósico: principalmente el Síndrome de Lynch (SL), debido a mutaciones germinales en los genes de sistema reparador de errores (MMR) MLH1, MSH2, MSH6 y PMS2; y varios síndromes polipósicos, como la poliposis adenomatosa familiar, asociada al gen APC y la poliposis asociada al gen MUTYH. En aproximadamente un 70% de los casos analizados de pacientes con sospecha de SL no se encuentran mutaciones germinales y en torno a un 10% de estos casos, con pérdida de expresión de MLH1 e hipermetilación del promotor de dicho gen en su tumor, se explican por epimutaciones constitucionales en MLH1. No obstante, la prevalencia de este fenómeno en la población general es desconocida. También existen casos de CCR con elevada carga familiar, pero sin alteración del sistema MMR, de los que se desconoce su origen genético (CCR familiar tipo X). Lo mismo ocurre para algunos casos de poliposis adenomatosa con agregación familiar, que no se explican por mutaciones en los genes APC o MUTYH. Recientemente se han descrito dos mutaciones recurrentes en los genes POLE y POLD1 (p.Leu424Val y p.Ser478Asn respectivamente) como responsables de un pequeño porcentaje de estos casos de CCR y poliposis no clasificados pero, debido a la escasez de casos descritos hasta la fecha, fenotipo clínico exacto no se ha definido. Así, los objetivos de este trabajo han sido: en primer lugar, determinar la prevalencia de las epimutaciones constitucionales en MLH1 como una causa de SL en una serie no seleccionada de CCR y compararla con la prevalencia de este fenómeno en una serie seleccionada; y en segundo lugar, analizar la prevalencia de las mutaciones recurrentes en los genes POLE y POLD1 en casos de CCR y poliposis familiar de aparición temprana, sin mutación en los genes responsables de los síndromes de sospecha, y así contribuir a definir el fenotipo clínico asociado a las mutaciones en dichos genes. Se evaluó la prevalencia de epimutaciones constitucionales de MLH1 en una cohorte de CCR no seleccionado, y se comparó con una cohorte de CCR familiar seleccionado. Así, no se detectaron epimutaciones constitucionales en MLH1 en la población no seleccionada. No obstante, el 15,6% de la serie seleccionada fueron positivos para epimutaciones en MLH1. Por otro lado, se realizó la búsqueda de mutaciones recurrentes en POLE y POLD1 en casos de CCR y/o poliposis familiar mediante ensayos de genotipado KASPar y/o secuenciación Sanger. Se identificó la mutación POLE_p.Leu424Val como mutación de novo, ningún caso presentó la mutación POLD1_p.Ser478Asn, pero se identificó una nueva mutación en POLD1: p.Leu474Pro, con patogenicidad sustentada mediante diversas aproximaciones, en una familia de CCR familiar tipo X con también casos de cáncer de endometrio. Así, estos resultados sugieren una prevalencia insignificante de epimutaciones constitucionales en MLH1 en cohortes no seleccionadas de CCR. Así, el análisis de epimutación constitucional en MLH1 debería dirigirse exclusivamente a pacientes que cumplen los criterios revisados de Bethesda y cuyo tumor presente pérdida de expresión y metilación de MLH1. Por otro lado, las mutaciones en POLE y POLD1 explican una pequeña proporción de casos de CCR y poliposis familiar. La secuenciación de al menos estas mutaciones recurrentes debería considerarse en el diagnóstico genético rutinario de los casos con sospecha de CCR o poliposis hereditarios, sin mutaciones en los genes de sospecha clásicos

CNApp, a tool for the quantification of copy number alterations and integrative analysis revealing clinical implications

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 . In most cases, human cells contain two copies of each of their genes, yet sometimes this can change, an effect called copy number alteration (CNA). Cancer is a genetic disease and thus, studying the DNA from tumor samples is crucial to improving diagnosis and choosing the right treatment. Most tumors contain cells with CNAs; however, the impact of CNAs in cancer progression is poorly understood. CNAs can be studied by examining the genome of tumor cells and finding which regions display an unusual number of copies. It may also be possible to gather information about different cancer types by analyzing the CNAs in a tumor, but this approach requires the analysis of large amounts of data. To aid the analysis of CNAs in cancer cells, Franch-Expósito, Bassaganyas et al. have created an online tool called CNApp, which is able to identify and count CNAs in genomic data and link them to features associated with different cancers. The hope is that a better understanding of the effect of CNAs in cancer could help better diagnose cancers, and improve outcomes for patients. Potentially, this could also predict what type of treatment would work better for a specific tumor. Besides, by using a machine-learning approach, the tool can also make predictions about specific cancer subtypes in order to facilitate clinical decisions. Franch-Expósito, Bassaganyas et al. tested CNApp using previously existing cancer data from 33 different cancer types to show how CNApp can help the interpretation of CNAs in cancer. Moreover, CNApp can also use CNAs to identify different types of bowel (colorectal) cancer in a way that could help doctors to make decisions about treatment. Together these findings show that CNApp provides an adaptable and accessible research tool for the study of cancer genomics, which could provide opportunities to inform medical procedures

Additional file 1: of Mutational Signatures in Cancer (MuSiCa): a web application to implement mutational signatures analysis in cancer samples

Figure S1. Somatic mutational prevalence in MuSiCa web app. Figure S2. Mutational profile representation in MuSiCa web app. Figure S3. Reconstruction of mutational profile in MuSiCa web app. Figure S4. Comparison with cancer signatures in MuSiCa web app. Figure S5. Principal component analysis in MuSiCa web app. (PDF 3039 kb