Evaluación de la metilación del DNA en tejidos de cáncer colorrectal fijados en formalina y embebidos en parafina (FFPE) / Evaluation of DNA methylation in formalin-fixed, paraffin embedded colorectal cancer tissues (FFPE)

Resumen Las alteraciones en la metilación de dinucleótidos CpG en regiones promotoras es uno de los mecanismos epigenéticos implicados en cáncer que tiene uso potencial como biomarcador. Su evaluación, a partir de tejidos fijados en formalina y embebidos en parafina (FFPE), representa un gran desafío dadas la degradación parcial, el entrecruzamiento y las bajas cantidades del DNA obtenido. En esta nota técnica, describimos un protocolo para el estudio del estado de metilación del promotor distal del proto-oncogén K-RAS, a partir de varias muestras obtenidas de dos tejidos FFPE de cáncer colorrectal con antigüedad de 11 años. Se empleó un protocolo de conversión con bisulfito alternativo al usual; se usó una DNA polimerasa modificada y una PCR anidada y se optimizó la secuenciación directa del DNA convertido con bisulfito. Este protocolo podría ser aplicado para determinar estados de metilación en otros genes y tipos de cáncer en tejidos FFPE.

Abstract Alterations in the methylation of CpG dinucleotides in promoter regions is one of the epigenetic mechanisms involved in cancer that has potential use as a biomarker. Its evaluation from formalin-fixed and paraffin-embedded (FFPE) tissues represents a great challenge given the partial degradation, crosslinking, and low amounts of the obtained DNA. In this technical note we describe a protocol for the study of the methylation status of the distal promoter of the K-RAS proto-oncogene from several samples obtained from two 11-years old FFPE tissues of colorectal cancer. An alternative bisulfite conversion protocol to the usual one was used; a modified DNA polymerase and a nested PCR were used and the direct sequencing of the converted DNA with bisulfite was optimized. This protocol could be applied to determine methylation states in other genes and types of cancer.

Citología convencional y en base líquida en muestra compartida de tomas cervicouterinas / Conventional and liquid-based cytology in shared cervical samples

Estandarizar la técnica de citología en base líquida mediante el uso del medio fijador (BDSUREPAth) y de citocentrífuga e identificar las posibles ventajas sobre citología convencional en muestra compartida de tomas cervicouterinas. Métodos: se incluyeron 92 muestras de mujeres que asistieron a las campañas del programa de proyección social de la Fundación Universitaria de Ciencias de la Salud, Bogotá DC, Colombia. Resultados: de las 92 muestras, 89 fueron negativas para citología convencional y 75 para base líquida, observándose un mayor número de casos con anormalidades en células escamosas en citología en base líquida. Conclusiones: las células escamosas mantienen su tonalidad citoplasmática siendo basófilas o acidófilas, conservando lo translúcido de los citoplasmas; las células glandulares preservan su patrón en panal de abejas o empalizada. Mediante citología en base liquida se obtienen fondos más limpios, las atipias de las células epiteliales fueron más fáciles de identificar al igual que los microorganismos patógenos...

Objective: to standardize the use of BD SurePath and centrifuged liquid-based cytology and identify possible advantages over the use of conventional cytology by comparing results on shared cervical samples analysis. Methods: we included 92 samples collected from women who attended the social projection campaign conducted by Fundación Universitaria de Ciencias de la Salud, Bogotá DC, Colombia. Results: out of the 92 samples, 89 were negative by conventional cytology and 75 by liquid-based cytology, evidencing a greater number of cases where squamous cell anomalies were detected using liquid-based cytology. Conclusions: squamous cells cytoplasma maintains its tone, being basophilic and acidophilic, conserving translucence; glandular cells preserve their honeycomb or palisade pattern. Liquid-based cytology gives a more clean background and improves identification of epithelial cell atypia as well as pathogenic microorganisms...

Types and variants of human papillomavirus in patients with cervical cancer submitted to radiotherapy

INTRODUCTION: high risk HPV infections and variants presence has been associated to increase the risk of cervical cancer. However, there are few studies that analyze the presence of them in patients with cervical cancer before and alter radiotherapy treatment. OBJECTIVES: to analyse the human papilloma virus presence and E7/HPV16 variants in 60 women with cervical cancer before and after radiotherapy. MATERIALS AND METHODS: HPV detection and typing were based on a GP5+/GP6+ PCR – Enzyme immune assay. E7/HPV16 variants were detected by PCR -Single strand conformation polymorphism (SSCP) and confirmed by direct sequence. RESULTS: before radiotherapy, 50/60 patients (83.3 percent) were HPV positive and HPV16 (53.3 percent) was the most prevalent type. After 3 months of radiotherapy, 55 patients attended to consult; of them, 19 (34.6 percent) were HPV positive, this decrease in the HPV detection was significant (p<0.0005). The E7/HPV16 analysis showed that 20 samples (62.5 percent) amplified before radiotherapy, 18 of them (90 percent) had identical SSCP pattern to the prototype and 2 showed a different SSCP pattern. The sequence of these two samples showed silent mutations at nt. 732 (T-to-C), 789 (T-to-C) and 795 (T-to-G). After radiotherapy, the was not detection of new mutations, 6 patients showed persistent HPV16 infection with the same SSCP pattern to the prototype, and samples that initially showed a different SSCP pattern were negative to E7/ HPV16 after radiotherapy. CONCLUSION: few E7/HPV16 variants were detected before radiotherapy and it seems that the treatment did not cause mutations in this gene