Adiposity, metabolites, and colorectal cancer risk: Mendelian randomization study

Background: Higher adiposity increases the risk of colorectal cancer (CRC), but whether this relationship varies by anatomical sub-site or by sex is unclear. Further, the metabolic alterations mediating the effects of adiposity on CRC are not fully understood. Methods: We examined sex- and site-specific associations of adiposity with CRC risk and whether adiposity-associated metabolites explain the associations of adiposity with CRC. Genetic variants from genome-wide association studies of body mass index (BMI) and waist-to-hip ratio (WHR, unadjusted for BMI; N = 806,810), and 123 metabolites from targeted nuclear magnetic resonance metabolomics (N = 24,925), were used as instruments. Sex-combined and sex-specific Mendelian randomization (MR) was conducted for BMI and WHR with CRC risk (58,221 cases and 67,694 controls in the Genetics and Epidemiology of Colorectal Cancer Consortium, Colorectal Cancer Transdisciplinary Study, and Colon Cancer Family Registry). Sex-combined MR was conducted for BMI and WHR with metabolites, for metabolites with CRC, and for BMI and WHR with CRC adjusted for metabolite classes in multivariable models. Results: In sex-specific MR analyses, higher BMI (per 4.2 kg/m2) was associated with 1.23 (95% confidence interval (CI) = 1.08, 1.38) times higher CRC odds among men (inverse-variance-weighted (IVW) model); among women, higher BMI (per 5.2 kg/m2) was associated with 1.09 (95% CI = 0.97, 1.22) times higher CRC odds. WHR (per 0.07 higher) was more strongly associated with CRC risk among women (IVW OR = 1.25, 95% CI = 1.08, 1.43) than men (IVW OR = 1.05, 95% CI = 0.81, 1.36). BMI or WHR was associated with 104/123 metabolites at false discovery rate-corrected P ≤ 0.05; several metabolites were associated with CRC, but not in directions that were consistent with the mediation of positive adiposity-CRC relations. In multivariable MR analyses, associations of BMI and WHR with CRC were not attenuated following adjustment for representative metabolite classes, e.g., the univariable IVW OR for BMI with CRC was 1.12 (95% CI = 1.00, 1.26), and this became 1.11 (95% CI = 0.99, 1.26) when adjusting for cholesterol in low-density lipoprotein particles. Conclusions: Our results suggest that higher BMI more greatly raises CRC risk among men, whereas higher WHR more greatly raises CRC risk among women. Adiposity was associated with numerous metabolic alterations, but none of these explained associations between adiposity and CRC. More detailed metabolomic measures are likely needed to clarify the mechanistic pathways

Microbiome Profiling from Fecal Immunochemical Test Reveals Microbial Signatures with Potential for Colorectal Cancer Screening

Simple Summary Colorectal cancer (CRC) is a global healthcare challenge that involves both genetic and environmental factors. Several pieces of evidence suggest that alterations of the gut microbiome can influence CRC development. In the present study we analyzed 16S rRNA sequencing data from fecal immunochemical test (FIT) samples from a large cohort, observing a predictive potential of the microbiome, revealing changes along the path from healthy tissue to carcinoma. Our work has implications in the understanding of the roles of microbes on the adenoma to carcinoma progression and opens the door to an improvement of the current CRC screening programmes. Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer deaths worldwide. Early diagnosis of CRC, which saves lives and enables better outcomes, is generally implemented through a two-step population screening approach based on the use of Fecal Immunochemical Test (FIT) followed by colonoscopy if the test is positive. However, the FIT step has a high false positive rate, and there is a need for new predictive biomarkers to better prioritize cases for colonoscopy. Here we used 16S rRNA metabarcoding from FIT positive samples to uncover microbial taxa, taxon co-occurrence and metabolic features significantly associated with different colonoscopy outcomes, underscoring a predictive potential and revealing changes along the path from healthy tissue to carcinoma. Finally, we used machine learning to develop a two-phase classifier which reduces the current false positive rate while maximizing the inclusion of CRC and clinically relevant samples

Approaches to functionally validate candidate genetic variants involved in colorectal cancer predisposition

Most next generation sequencing (NGS) studies identified candidate genetic variants predisposing to colorectal cancer (CRC) but do not tackle its functional interpretation to unequivocally recognize a new hereditary CRC gene. Besides, germline variants in already established hereditary CRC-predisposing genes or somatic variants share the same need when trying to categorize those with relevant significance. Functional genomics approaches have an important role in identifying the causal links between genetic architecture and phenotypes, in order to decipher cellular function in health and disease. Therefore, functional interpretation of identified genetic var iants by NGS platforms is now essential. Available approaches nowadays include bioinformatics, cell and mo lecular biology and animal models. Recent advances, such as the CRISPR-Cas9, ZFN and TALEN systems, have been already used as a powerful tool with this objective. However, the use of cell lines is of limited value due to the CRC heterogeneity and its close interaction with microenvironment. Access to tridimensional cultures or organoids and xenograft models that mimic the in vivo tissue architecture could revolutionize functional ana lysis. This review will focus on the application of state-of-the-art functional studies to better tackle new genes involved in germline predisposition to this neoplasm

Multiple Sporadic Colorectal Cancers Display a Unique Methylation Phenotype

The members of the Gastrointestinal Oncology Group of the Spanish Gastroenterological Association are: Hospital 12 de Octubre, Madrid: Juan Diego Morillas (local coordinator), Raquel Muñoz, Marisa Manzano, Francisco Colina, Jose Díaz, Carolina Ibarrola, Guadalupe López, Alberto Ibáñez; Hospital Clínic, Barcelona: Antoni Castells (local coordinator), Virgínia Piñol, Sergi Castellví-Bel, Francesc Balaguer, Victoria Gonzalo, Teresa Ocaña, María Dolores Giráldez, Maria Pellisé, Anna Serradesanferm, Leticia Moreira, Miriam Cuatrecasas, Josep M. Piqué; Hospital Clínico Universitario, Zaragoza: Ángel Lanas (local coordinator), Javier Alcedo, Javier Ortego; Hospital Cristal-Piñor, Complexo Hospitalario de Ourense: Joaquin Cubiella (local coordinator), Ma Soledad Díez, Mercedes Salgado, Eloy Sánchez, Mariano Vega; Hospital del Mar, Barcelona: Montserrat Andreu (local coordinator), Anna Abuli, Xavier Bessa, Mar Iglesias, Agustín Seoane, Felipe Bory, Gemma Navarro, Beatriz Bellosillo, Josep Ma Dedeu, Cristina Álvarez, Begoña Gonzalez; Hospital San Eloy, Baracaldo and Hospital Donostia, CIBERehd, University of Country Basque, San Sebastián: Luis Bujanda (local coordinator) Ángel Cosme, Inés Gil, Mikel Larzabal, Carlos Placer, María del Mar Ramírez, Elisabeth Hijona, Jose M. Enríquez-Navascués, Jose L. Elosegui; Hospital General Universitario de Alicante: Artemio Payá (EPICOLON I local coordinator), Rodrigo Jover (EPICOLON II local coordinator), Cristina Alenda, Laura Sempere, Nuria Acame, Estefanía Rojas, Lucía Pérez-Carbonell; Hospital General de Granollers: Joaquim Rigau (local coordinator), Ángel Serrano, Anna Giménez; Hospital General de Vic: Joan Saló (local coordinator), Eduard Batiste-Alentorn, Josefina Autonell, Ramon Barniol; Hospital General Universitario de Guadalajara and Fundación para la Formación e Investigación Sanitarias Murcia: Ana María García (local coordinator), Fernando Carballo, Antonio Bienvenido, Eduardo Sanz, Fernando González, Jaime Sánchez, Akiko Ono; Hospital General Universitario de Valencia: Mercedes Latorre (local coordinator), Enrique Medina, Jaime Cuquerella, Pilar Canelles, Miguel Martorell, José Ángel García, Francisco Quiles, Elisa Orti; CHUVI-Hospital Meixoeiro, Vigo: EPICOLON I: Juan Clofent (local coordinator), Jaime Seoane, Antoni Tardío, Eugenia Sanchez; EPICOLON II: Ma Luisa de Castro (local coordinator), Antoni Tardío, Juan Clofent, Vicent Hernández; Hospital Universitari Germans Trias i Pujol, Badalona and Section of Digestive Diseases and Nutrition, University of Illinois at Chicago, Chicago, IL: Xavier Llor (local coordinator), Rosa M. Xicola, Marta Piñol, Mercè Rosinach, Anna Roca, Elisenda Pons, José M. Hernández, Miquel A. Gassull; Hospital Universitari Mútua de Terrassa: Fernando Fernández-Bañares (local coordinator), Josep M. Viver, Antonio Salas, Jorge Espinós, Montserrat Forné, Maria Esteve; Hospital Universitari Arnau de Vilanova, Lleida: Josep M. Reñé (local coordinator), Carmen Piñol, Juan Buenestado, Joan Viñas; Hospital Universitario de Canarias: Enrique Quintero (local coordinator), David Nicolás, Adolfo Parra, Antonio Martín; Hospital Universitario La Fe, Valencia: Lidia Argüello (local coordinator), Vicente Pons, Virginia Pertejo, Teresa Sala; Hospital Sant Pau, Barcelona: Dolors Gonzalez (local coordinator), Eva Roman, Teresa Ramon, Maria Poca, Ma Mar Concepción, Marta Martin, Lourdes Pétriz; Hospital Xeral Cies, Vigo: Daniel Martinez (local coordinator); Fundacion Publica Galega de Medicina Xenomica (FPGMX), CIBERER, Genomic Medicine Group-University of Santiago de Compostela, Santiago de Compostela, Galicia, Spain: Ángel Carracedo (local coordinator), Clara Ruiz-Ponte, Ceres Fernández-Rozadilla, Ma Magdalena Castro; Hospital Universitario Central de Asturias: Sabino Riestra (local coordinator), Luis Rodrigo; Hospital de Galdácano, Vizcaya: Javier Fernández (local coordinator), Jose Luis Cabriada; Fundación Hospital de Calahorra (La Rioja) La Rioja: Luis Carreño (local coordinator), Susana Oquiñena, Federico Bolado; Hospital Royo Villanova, Zaragoza: Elena Peña (local coordinator), José Manuel Blas, Gloria Ceña, Juan José Sebastián; Hospital Universitario Reina Sofía, Córdoba: Antonio Naranjo (local coordinator).Epigenetics are thought to play a major role in the carcinogenesis of multiple sporadic colorectal cancers (CRC). Previous studies have suggested concordant DNA hypermethylation between tumor pairs. However, only a few methylation markers have been analyzed. This study was aimed at describing the epigenetic signature of multiple CRC using a genome-scale DNA methylation profiling. We analyzed 12 patients with synchronous CRC and 29 age-, sex-, and tumor location-paired patients with solitary tumors from the EPICOLON II cohort. DNA methylation profiling was performed using the Illumina Infinium HM27 DNA methylation assay. The most significant results were validated by Methylight. Tumors samples were also analyzed for the CpG Island Methylator Phenotype (CIMP); KRAS and BRAF mutations and mismatch repair deficiency status. Functional annotation clustering was performed. We identified 102 CpG sites that showed significant DNA hypermethylation in multiple tumors with respect to the solitary counterparts (difference in β value ≥0.1). Methylight assays validated the results for 4 selected genes (p = 0.0002). Eight out of 12(66.6%) multiple tumors were classified as CIMP-high, as compared to 5 out of 29(17.2%) solitary tumors (p = 0.004). Interestingly, 76 out of the 102 (74.5%) hypermethylated CpG sites found in multiple tumors were also seen in CIMP-high tumors. Functional analysis of hypermethylated genes found in multiple tumors showed enrichment of genes involved in different tumorigenic functions. In conclusion, multiple CRC are associated with a distinct methylation phenotype, with a close association between tumor multiplicity and CIMP-high. Our results may be important to unravel the underlying mechanism of tumor multiplicity.This work was supported by grants from the Hospital Clínic of Barcelona (Josep Font grant), Ministerio de Economí­a y Competitividad (SAF 2007-64873 and SAF2010-19273), Fundación Científica de la Asociación Española contra el Cáncer, and Instituto de Salud Carlos III (PI10/00384). “Cofinanciado por el Fondo Europeo de Desarrollo Regional (FEDER). Unión Europea. Una manera de hacer Europa”. CIBEREHD is funded by the Instituto de Salud Carlos III

Update on genetic predisposition to colorectal cancer and polyposis

The present article summarizes recent developments in the characterization of genetic predisposition to colorectal cancer (CRC). The main themes covered include new hereditary CRC and polyposis syndromes, non-CRC hereditary cancer genes found mutated in CRC patients, strategies used to identify novel causal genes, and review of candidate genes that have been proposed to predispose to CRC and/or colonic polyposis. We provide an overview of newly described genes and syndromes associated with predisposition to CRC and polyposis, including: polymerase proofreading-associated polyposis, NTHL1-associated polyposis, mismatch repair gene biallelic inactivation-related adenomatous polyposis (including MSH3- and MLH3-associated polyposes), GREM1-associated mixed polyposis, RNF43-associated serrated polyposis, and RPS20 mutations as a rare cause of hereditary nonpolyposis CRC. The implementation of next generation sequencing approaches for genetic testing has exposed the presence of pathogenic germline variants in genes associated with hereditary cancer syndromes not traditionally linked to CRC, which may have an impact on genetic testing, counseling and surveillance. The identification of new hereditary CRC and polyposis genes has not deemed an easy endeavor, even though known CRC-related genes explain a small proportion of the estimated familial risk. Whole-genome sequencing may offer a technology for increasing this proportion, particularly if applied on pedigree data allowing linkage type of analysis. The final section critically surveys the large number of candidate genes that have been recently proposed for CRC predisposition

Colon-specific eQTL analysis to inform on functional SNPs

BACKGROUND: Genome-wide association studies on colorectal cancer have identified more than 60 susceptibility loci, but for most of them there is no clear knowledge of functionality or the underlying gene responsible for the risk modification. Expression quantitative trail loci (eQTL) may provide functional information for such single nucleotide polymorphisms (SNPs). METHODS: We have performed detailed eQTL analysis specific for colon tissue on a series of 97 colon tumours, their paired adjacent normal mucosa and 47 colon mucosa samples donated by healthy individuals. R package MatrixEQTL was used to search for genome-wide cis-eQTL and trans-eQTL fitting linear models adjusted for age, gender and tissue type to rank transformed expression data. RESULTS: The cis-eQTL analyses has revealed 29,073 SNP-gene associations with permutation-adjusted P-values < 0.01. These correspond to 363 unique genes. The trans-eQTL analysis identified 10,665 significant SNP-gene associations, most of them in the same chromosome, further than 1 Mb of the gene. We provide a web tool to search for specific SNPs or genes. The tool calculates Pearson or Spearman correlation, and allows to select tissue type for analysis. Data and plots can be exported. CONCLUSIONS: This resource should be useful to prioritise SNPs for further functional studies and to identify relevant genes behind identified loci

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/

Rectal Aberrant Crypt Foci in Humans Are Not Surrogate Markers for Colorectal Cancer Risk

NTRODUCTION: Over the past 20 years, aberrant crypt foci (ACF) have emerged as potential precursors and biomarkers for colorectal cancer (CRC). However, data regarding their molecular pathogenesis, as well as their endoscopic and histological identification, remain inconsistent. METHODS: A wide cohort of ACF from 100 control subjects and 100 case patients, including patients with adenoma and CRC, were characterized for endoscopic, morphologic, and molecular features. RESULTS: We observed that among all the endoscopic features evaluated, only the number of large ACF correlated with CRC risk (P = 0.003), whereas the histological classification, as assessed by 2 different pathologists, was inconsistent and did not differ between control and case patients. Moreover, only a few APC and BRAF mutations and no microsatellite instability were detected in our samples. KRAS mutations were detected in 16.3% of ACF samples, which also exhibited increased MGMT hypermethylation. However, none of those events were found to be predictive of CRC risk. DISCUSSION: Although ACF might be preneoplastic lesions of the colon, they are not suitable biomarkers for assessing CRC progression

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

Using linkage studies combined with whole-exome sequencing to identify novel candidate genes for familial colorectal cancer

Colorectal cancer (CRC) is a complex disorder for which the majority of the underlying germline predisposition factors remain still unidentified. Here, we combined whole‐exome sequencing (WES) and linkage analysis in families with multiple relatives affected by CRC to identify candidate genes harboring rare variants with potential high‐penetrance effects. Forty‐seven affected subjects from 18 extended CRC families underwent WES. Genome‐wide linkage analysis was performed under linear and exponential models. Suggestive linkage peaks were identified on chromosomes 1q22-q24.2 (maxSNP = rs2134095; LODlinear = 2.38, LODexp = 2.196), 7q31.2-q34 (maxSNP = rs6953296; LODlinear = 2.197, LODexp = 2.149) and 10q21.2-q23.1 (maxSNP = rs1904589; LODlinear = 1.445, LODexp = 2.195). These linkage signals were replicated in 10 independent sets of random markers from each of these regions. To assess the contribution of rare variants predicted to be pathogenic, we performed a family‐based segregation test with 89 rare variants predicted to be deleterious from 78 genes under the linkage intervals. This analysis showed significant segregation of rare variants with CRC in 18 genes (weighted p‐value > 0.0028). Protein network analysis and functional evaluation were used to suggest a plausible candidate gene for germline CRC predisposition. Etiologic rare variants implicated in cancer germline predisposition may be identified by combining traditional linkage with WES data. This approach can be used with already available NGS data from families with several sequenced members to further identify candidate genes involved germline predisposition to disease. This approach resulted in one candidate gene associated with increased risk of CRC but needs evidence from further studies