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Aberrant DNA methylation detected in liquid biopsies is a promising approach for colorectal cancer (CRC) detection, including premalignant advanced adenomas (AA). We evaluated the diagnostic capability of serum NEUROG1 methylation for the detection of AA and CRC. A CpG island in NEUROG1 promoter was assessed by bisulfite pyrosequencing in a case-control cohort to select optimal CpGs. Selected sites were evaluated through a nested methylation-specific qPCR custom assay in a screening cohort of 504 asymptomatic family-risk individuals. Individuals with no colorectal findings and benign pathologies showed low serum NEUROG1 methylation, similar to non-advanced adenomas. Contrarily, individuals bearing AA or CRC (advanced neoplasia-AN), exhibited increased NEUROG1 methylation. Using >1.3518% as NEUROG1 cut-off (90.60% specificity), 33.33% of AN and 32.08% of AA were identified, detecting 50% CRC cases. Nonetheless, the combination of NEUROG1 with fecal immunochemical test (FIT), together with age and gender through a multivariate logistic regression resulted in an AUC = 0.810 for AN, and 0.796 for AA, detecting all cancer cases and 35-47% AA (specificity 98-95%). The combination of NEUROG1 methylation with FIT, age and gender demonstrated a convenient performance for the detection of CRC and AA, providing a valuable tool for CRC screening programs in asymptomatic individuals.

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Introducción: El diagnóstico de la afectación metastásica ganglionar en el cáncer de pulmón constituye un problema, a pesar de los avances en la estadificación. La determinación del estado de metilación en ganglios podría mejorar la capacidad de las técnicas citohistológicas para detectar afectación metastásica. Nuestro objetivo fue demostrar la viabilidad de realizar estudios de metilación en muestras ganglionares citológicas. Métodos: Estudio prospectivo que incluyó 88 pacientes con diagnóstico o alta sospecha de cáncer de pulmón no microcítico, en los que se realizó una punción citológica por ecobroncoscopia de adenopatías mediastínicas y/o hiliares. Se extrajo ADN a partir de muestras citológicas ganglionares y se realizó el tratamiento con bisulfito de sodio. Los estudios de metilación se realizaron por qPCR-MS y pirosecuenciación en los genes p16/INK4a y SHOX2. Resultados: La metodología empleada permitió obtener ADN de características óptimas/buenas en el 90% de los casos. No se observaron diferencias en la concentración de ADN respecto a la estación ganglionar ni al diagnóstico final. Los análisis por qPCR-MS y pirosecuenciación no fueron posibles en un reducido número de muestras debido a baja concentración de ADN, además de la inadecuada pureza, fragmentación y/o degradación debido al tratamiento con bisulfito de sodio. Conclusión: La cuantificación de la metilación por técnicas como qPCR-MS o pirosecuenciación en muestras ganglionares obtenidas por ecobroncoscopia resulta viable siempre y cuando se logre obtener una concentración adecuada de ADN, contribuyendo a la búsqueda de biomarcadores epigenéticos que mejoren la toma de decisiones en el cáncer de pulmón potencialmente curable en beneficio del paciente

Introduction: The diagnosis of microscopic lymph node metastasis in lung cancer is challenging despite the constant advances in tumor staging. The analysis of the methylation status of certain genes in lymph node samples could improve the diagnostic capability of conventional cyto-histological methods. The aim of this study was to demonstrate the feasibility of methylation studies using cytological lymph node samples. Methods: A prospective study including 88 patients with a diagnosis or strong suspicion of non-small cell lung cancer, in which an echobronchoscopy was performed on mediastinal or hilar lymph nodes for diagnosis and/or staging purposes. DNA was extracted from cytological lymph node samples and sodium bisulfite modification was performed. Methylation studies for p16/INK4a and SHOX2 were accomplished by MS-qPCR and pyrosequencing. Results: The methodology used in our study yielded optimal/good DNA quality in 90% of the cases. No differences in DNA concentration were observed with respect to the lymph node biopsied and final diagnosis. Methylation analyses using MS-qPCR and pyrosequencing were not possible in a small number of samples mainly due to low DNA concentration, inadequate purity, fragmentation and/or degradation as a consequence of bisulfite conversion. Conclusion: Methylation quantification using MS-qPCR and pyrosequencing of cytological lymph node samples obtained using echobronchoscopy is feasible if an appropriate DNA concentration is obtained, notably contributing to the identification of epigenetic biomarkers capable of improving decision making for the benefit of potentially curable lung cancer patients

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Colorectal cancer is characterized by a low survival rate even though the basis for colon cancer development, which involves the evolution of adenomas to carcinoma, is known. Moreover, the mortality rates continue to rise in economically transitioning countries although there is the opportunity to intervene in the natural history of the adenoma-cancer sequence through risk factors, screening, and treatment. Screening in particular accounted for most of the decline in colorectal cancer mortality achieved in the USA during the period 1975-2000. Patients show a better prognosis when the neoplasm is diagnosed early. Among the variety of screening strategies, the methods range from invasive and costly procedures such as colonoscopy to more low-cost and non-invasive tests such as the fecal occult blood test (guaiac and immunochemical). As a non-invasive biological serum marker would be of great benefit because of the performance of the test, several biomarkers, including cytologic assays, DNA and mRNA, and soluble proteins, have been studied. We found that the soluble CD26 (sCD26) concentration is diminished in serum of colorectal cancer patients compared to healthy donors, suggesting the potential utility of a sCD26 immunochemical detection test for early diagnosis. sCD26 originates from plasma membrane CD26 lacking its transmembrane and cytoplasmic domains. Some 90%-95% of sCD26 has been associated with serum dipeptidyl peptidase IV (DPP-IV) activity. DPP-IV, assigned to the CD26 cluster, is a pleiotropic enzyme expressed mainly on epithelial cells and lymphocytes. Our studies intended to validate this test for population screening to detect colorectal cancer and advanced adenomas are reviewed here.