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

Survey of Spanish general practitioners' attitudes toward management of sore throat: an internet-based questionnaire study

BACKGROUND: The management of sore throat varies widely in Europe. The objective of this study was to gain insight into clinicians' perceptions on the current management of sore throat in Spain. METHODS: Cross-sectional, internet-based questionnaire study answered from July to September 2013. General practitioners (GPs) affiliated with the two largest scientific societies of primary care were invited to participate in the study. Questions were asked about physician knowledge, the use of current national guidelines for sore throat management, and management in two clinical scenarios, depicting a young adult with sore throat and: 1. cough, coriza with or without fever, and 2. fever without cough and coriza. RESULTS: The questionnaire was completed by 1476 GPs (5%) and 12.7% declared using rapid antigen detection tests. Antibiotics were considered by 18.8% of the GPs in the first scenario and by 32% in the second scenario (p < 0.001). The antibiotics most commonly mentioned by GPs were amoxicillin and amoxicillin + clavulanate (52.7 and 31.2%, respectively) whereas penicillin V was only prescribed in 11.9% of the cases. The drugs most commonly considered in both scenarios were analgesics and anti-inflammatory drugs. Antitussives, decongestants and expectorants were more commonly prescribed in cases of suspected viral infection (p < 0.001). CONCLUSIONS: GPs have misconceptions as to the indications for using rapid antigen detection tests and prescribing drugs in the management of sore throat. These results suggest that guidelines are seldom followed since one in five GPs declared giving antibiotics for patients with a suspected viral infection and the use of second-choice antibiotics seems considerable

Conjugate Addition to Phenylglycinol-Derived Unsaturated ä-Lactams. Enantioselective Synthesos of Uleine Alkaloids

The stereochemical outcome of the conjugate addition of a variety of stabilized nucleophiles (2indoleaceticenolatesandsulfur-stabilizedanions)tothephenylglycinol-derivedunsaturatedlactams trans-2, cis-2, and its 8-ethyl-substituted analogue 10 is studied. The factors governing the exo or endo facial stereoselectivity are discussed. This methodology provides short synthetic routes to either cis- ortrans-3,4-disubstituted enantiopure piperidines as well as efficient routes for the enantioselective construction of the tetracyclic ring system of uleine alkaloids, both in the normal and 20-epi series. The formal total synthesis of several alkaloids of this group is reported

Guía clínica para el manejo de la faringoamigdalitis aguda del adulto

La faringoamigdalitis aguda (FAA) en el adulto es una de las enfermedades infecciosas más comunes en la consulta del médico de familia. La etiología más frecuente es viral. Dentro de la etiología bacteriana, el principal agente responsable es Streptococcus pyogenes o estreptococo β-hemolítico del grupo A (EBHGA), causante del 5-30% de los casos. En el manejo diagnóstico las escalas de valoración clínica, para predecir la posible etiología bacteriana, son una buena ayuda para seleccionar a qué pacientes se deben practicar las técnicas de detección rápida de antígeno estreptocócico. Es conocido que, en general, sin estas técnicas, se tiende al sobrediagnóstico de FAA estreptocócica, con la consiguiente prescripción innecesaria de antibióticos, muchas veces de amplio espectro. Así, con el manejo de las escalas y la técnica de diagnóstico rápido, elaboramos los algoritmos de manejo de la FAA. Los objetivos del tratamiento son acelerar la resolución de los síntomas, reducir el tiempo de contagio y prevenir las complicaciones supurativas locales y no supurativas. Los antibióticos de elección para el tratamiento de la FAA estreptocócica son penicilina y amoxicilina. La asociación de amoxicilina y clavulánico no está indicada en el tratamiento inicial en la infección aguda. Los macrólidos tampoco son un tratamiento de primera elección; su uso debe reservarse para pacientes con alergia a la penicilina. Es importante en nuestro país adecuar tanto el diagnóstico de la FAA bacteriana y la prescripción de antibióticos a la evidencia científica disponible. La implantación de protocolos de actuación en las farmacias comunitarias puede ser de utilidad para identificar y cribar los casos que no requieran tratamiento antibiótico

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

A colorectal cancer genome-wide association study in a Spanish cohort identifies two variants associated with colorectal cancer risk at 1p33 and 8p12

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 <10-5. We therefore evaluated the 24 loci in another Spanish replication cohort (1481 cases and 1850 controls). Two of these SNPs, rs12080929 at 1p33 (Preplication=0.042; Ppooled=5.523x10-03; OR (CI95%)=0.866(0.782-0.959)) and rs11987193 at 8p12 (Preplication=0.039; Ppooled=6.985x10-5; OR (CI95%)=0.786(0.705-0.878)) were replicated in the second Phase, although they did not reach genome-wide statistical significance. Conclusions: We have performed the first CRC GWAS in a Southern European population and by these means we were able to identify two new susceptibility variants at 1p33 and 8p12 loci. These two SNPs are located near the SLC5A9 and DUSP4 loci, respectively, which could be good functional candidates for the association signals. We therefore believe that these two markers constitute good candidates for CRC susceptibility loci and should be further evaluated in other larger datasets. Moreover, we highlight that were these two SNPs true susceptibility variants, they would constitute a decrease in the CRC missing heritability fraction

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

Systemic and tumor level iron regulation in men with colorectal cancer: a case control study

BACKGROUND: Increased cellular iron exposure is associated with colorectal cancer (CRC) risk. Hepcidin, a liver peptide hormone, acts as the primary regulator of systemic iron status by blocking iron release from enterocytes into plasma. Concentrations are decreased during low iron status and increased during inflammation. The role of hepcidin and the factors influencing its regulation in CRC remains largely unknown. This study explored systemic and tumor level iron regulation in men with CRC. METHODS: The participants were 20 CRC cases and 20 healthy control subjects. Colonic tissue (adenocarcinoma [cases] healthy mucosa [controls]) was subjected to quantitative PCR (hepcidin, iron transporters and IL-6) and Perls’ iron staining. Serum was analyzed using ELISA for hepcidin, iron status (sTfR) and inflammatory markers (CRP, IL-6, TNF-α). Anthropometrics, dietary iron intake and medical history were obtained. RESULTS: Cases and controls were similar in demographics, medication use and dietary iron intake. Systemically, cases compared to controls had lower iron status (sTfR: 21.6 vs 11.8 nmol/L, p < 0.05) and higher marker of inflammation (CRP: 8.3 vs 3.4 μg/mL, p < 0.05). Serum hepcidin was mildly decreased in cases compared to controls; however, it was within the normal range for both groups. Within colonic tissue, 30% of cases (6/20) presented iron accumulation compared to 5% of controls (1/20) (χ(2) = 5.0; p < 0.05) and higher marker of inflammation (IL-6: 9.4-fold higher compared to controls, p < 0.05). Presence of adenocarcinoma iron accumulation was associated with higher serum hepcidin (iron accumulation group 80.8 vs iron absence group 22.0 ng/mL, p < 0.05). CONCLUSIONS: While CRC subjects had serum hepcidin concentrations in the normal range, it was higher given their degree of iron restriction. Inappropriately elevated serum hepcidin may reduce duodenal iron absorption and further increase colonic adenocarcinoma iron exposure. Future clinical studies need to assess the appropriateness of dietary iron intake or iron supplementation in patients with CRC

Exome sequencing of early-onset patients supports genetic heterogeneity in colorectal cancer

Colorectal cancer (CRC) is a complex disease that can be caused by a spectrum of genetic variants ranging from low to high penetrance changes, that interact with the environment to determine which individuals will develop the disease. In this study, we sequenced 20 early-onset CRC patients to discover novel genetic variants that could be linked to the prompt disease development. Eight genes, CHAD, CHD1L, ERCC6, IGTB7, PTPN13, SPATA20, TDG and TGS1, were selected and re-sequenced in a further 304 early onset CRC patients to search for rare, high-impact variants. Although we found a recurring truncating variant in the TDG gene shared by two independent patients, the results obtained did not help consolidate any of the candidates as promising CRC predisposing genes. However, we found that potential risk alleles in our extended list of candidate variants have a tendency to appear at higher numbers in younger cases. This supports the idea that CRC onset may be oligogenic in nature and may show molecular heterogeneity. Further, larger and robust studies are thus needed to unravel the genetics behind early-onset CRC development, coupled with novel functional analyses and omic approaches that may offer complementary insight

A high degree of LINE-1 hypomethylation is a unique feature of early-onset colorectal cancer

Objective Early-onset colorectal cancer (CRC) represents a clinically distinct form of CRC that is often associated with a poor prognosis. Methylation levels of genomic repeats such as LINE-1 elements have been recognized as independent factors for increased cancer-related mortality. The methylation status of LINE-1 elements in early-onset CRC has not been analyzed previously. Design We analyzed 343 CRC tissues and 32 normal colonic mucosa samples, including 2 independent cohorts of CRC diagnosed ≤50 years old (n = 188), a group of sporadic CRC >50 years (MSS n = 89; MSI n = 46), and a group of Lynch syndrome CRCs (n = 20). Tumor mismatch repair protein expression, microsatellite instability status, LINE-1 and MLH1 methylation, somatic BRAF V600E mutation, and germline MUTYH mutations were evaluated. Results Mean LINE-1 methylation levels (±SD) in the five study groups were early-onset CRC, 56.6% (8.6); sporadic MSI, 67.1% (5.5); sporadic MSS, 65.1% (6.3); Lynch syndrome, 66.3% (4.5) and normal mucosa, 76.5% (1.5). Early-onset CRC had significantly lower LINE-1 methylation than any other group (p<0.0001). Compared to patients with <65% LINE-1 methylation in tumors, those with ≥65% LINE-1 methylation had significantly better overall survival (p = 0.026, log rank test). Conclusions LINE-1 hypomethylation constitutes a potentially important feature of early-onset CRC, and suggests a distinct molecular subtype. Further studies are needed to assess the potential of LINE-1 methylation status as a prognostic biomarker for young people with CRC