Implementación de la técnica de micromatrices de tejidos para la investigación oncológica en Cuba / Implementation of tissue microarrays technique for cancer research in Cuba

La técnica de micromatrices de tejidos (TMA), basada en la toma de ponches cilíndricos de bloques de parafina donantes y su transferencia a un solo bloque receptor, revolucionó el campo de la patología, por la posibilidad de evaluar múltiples muestras en una sola lámina. En Cuba no existen antecedentes del tema, por lo que el objetivo de este trabajo fue implementar la técnica de TMA. Para ello, se evaluó la concordancia de los resultados que se obtienen por sección completa y por TMA, en la evaluación de los receptores de estrógeno (RE), progesterona (RP) y del factor de crecimiento epidérmico tipo 2 (HER2) en muestras de cáncer de mama. Se estudiaron 45 muestras parafinadas de mujeres con esta enfermedad que se atendieron en el Instituto de Oncología en el año 2012. Se construyeron dos bloques de TMA y posteriormente se determinó, mediante inmunohistoquímica, la expresión de los marcadores RE, RP y HER2, en la sección completa de tejido y en el TMA. Para el análisis de concordancia se empleó el índice Kappa. Se obtuvo una buena concordancia para los tres marcadores (RE k=0,8272; RP k=0,793 y HER2 k=0,716). Este trabajo constituye el primer reporte sobre la técnica de TMA en Cuba y demuestra que es una herramienta valiosa, por lo que se sugiere su potencial uso en la investigación traslacional y en los ensayos clínicos de vacunas(AU)

The tissue microarray (TMA) technique is based on making cylindrical cores from paraffin donor blocks and transfer to a single recipient block. The TMA has revolutionized the field of pathology for the possibility to evaluate multiple samples in one slide. There is no precedent of this subject in Cuba, so the objective of this research was to implement the TMA technique. The concordance of the results obtained by complete section and the TMA were evaluated for this purpose, in the evaluation of the estrogen receptors (ER), progesterone (PR) and epidermal growth factor type 2 (HER2) in samples of breast cancer. Forty-five paraffin-embedded samples from women diagnosed with breast cancer at the Institute of Oncology in 2012 were studied. Two TMA blocks were constructed, and subsequently the expression of markers ER, PR and HER2 was determined by immunohistochemistry, in the complete section of tissue and in the TMA. Kappa index was used for concordance analysis. A good concordance was obtained for all three markers (ER k=0.8272; PR k=0.793 and HER2 k=0.716). This study constitutes the first report on the TMA technique in Cuba and shows that it is a valuable tool, suggesting its potential use in translational research and clinical trials on vaccines(AU)

Normalización de un método inmunoquímico para detectar Papilomavirus humano tipo 16 en lesiones cérvico-uterinas / Standardization of immunochemical method for detection of type 16 human papilomavirus in cervix uterine lesions

Introducción: la infección por Papilomavirus Humano (PVH) es la condición necesaria para la aparición y desarrollo del cáncer cérvico-uterino. Los genotipos de alto riesgo oncogénico son los causantes de este tipo de neoplasia y dentro de ellos el más frecuente es el PVH 16, que se encuentra aproximadamente en el 60 % de los casos. Los métodos de diagnóstico comerciales resultan costosos para países con escasos recursos económicos, lo que sugiere la búsqueda de alternativas empleando protocolos sencillos y baratos. Objetivos: normalizar un método inmunoquímico para la detección del antígeno L1 de PVH tipo 16 en muestras cérvico-uterinas de pacientes con lesiones intraepiteliales escamosas y determinar la coincidencia entre el método normalizado y la Reacción en Cadena de la Polimerasa en Tiempo Real (RCP-TR), como técnica de referencia, para estimar la utilidad de dicho método en el diagnóstico de la infección por este genotipo viral. Métodos: se compararon tres procedimientos de inmunotinción (Indirecto de inmunoperoxidasa en dos pasos, Estreptavidina-Biotina y Amplificación por polímero) respecto a sensibilidad analítica, tinción inespecífica de fondo y tiempo de terminación, para la detección de la proteína L1 de PVH 16 en líneas celulares derivadas de carcinomas cervicales humanos y en muestras cérvico-uterinas utilizadas como controles. El protocolo normalizado se aplicó a muestras cérvico-uterinas de mujeres entre 30 y 59 años, 82 con lesiones intraepiteliales cervicales y 10 sin antecedentes de alteraciones citológicas, a las que además se les determinó PVH 16 mediante RCP-TR. Resultados: el procedimiento de Estreptavidina-Biotina resultó el más sensible y específico. La coincidencia entre el método inmunoquímico y la RCP-TR fue de un 98,6 por ciento, la sensibilidad fue de un 98,57 por ciento y la especificidad de un 91,67 por ciento, con valores predictivos negativo y positivo por encima del 90 por ciento. Conclusiones: se demostró la validez del método inmunoquímico como prueba confirmatoria de la infección por PVH 16. Dicho método probó ser sensible, sencillo y no requiere de una compleja infraestructura para detectar PVH 16 en muestras cervicales. Además, esta técnica permite obtener información rápidamente y evita el uso de métodos invasivos(AU)

Introduction: Human Papillomavirus (HPV) infection is the necessary condition for the occurernce and development of cervical cancer. The high oncogenic risk genotypes are the responsible for this type of neoplasia and the most frequent is HPV 16 that affects roughly 60 percent of cases. Commercial kits for HPV detection are expensive for resource-poor countries, which suggests the search for alternative throguh non-expensive simple protocoles. Objectives: to standardize an immunochemical method for the detection of HPV 16 L1 antigen in cervical samples of patients with squamous intraepithelial lesions and to determine the diagnostic coincidence between the immunochemical method and the real-time polymerase chain reaction to estimate the usefulness of this method for the detection of cervical infection with this viral genotype. Methods: three immunostaining methods (Two-Step Indirect Immunoperoxidase, Labelled Streptavidin-Biotin and Enhanced Polymer) were compared in terms of analytical sensitivity, nonspecific background staining and time of completion, for the detection of protein L1 of HPV-16 in a cell line derived from human cervical carcinoma and clinical samples from uterine cervix. The optimized protocol was applied to 82 cervical samples from women aged 30-59 years with squamous intraepithelial lesions and to 10 samples of sexually active women without previous signals of positive cytology. The presence of type 16 HPV was also detected with the aid of RT-PCR. Results: the Streptavidin-Biotin system was the most sensitive and specific. The diagnostic agreement between the immunochemical method and the real-time polymerase chain reaction reached 98.6 percent, sensitivity was 98.57 percent and specificity was 91.67 %, with positive and negative predictive values above 90 percent. Conclusions: the validity of the immunochemical method as a confirmatory test for infection by HPV-16 has been demonstrated. The normalized immunochemical method proved to be a sensitive, simple, relatively fast method to detect HPV from clinical samples of cervical cells. Furthermore, this method provides information quickly, avoiding the use of invasive methods in patients(AU)

Normalización de un método inmunoquímico para detectar Papilomavirus humano tipo 16 en lesiones cérvico-uterinas / Standardization of immunochemical method for detection of type 16 human papilomavirus in cervix uterine lesions

Introducción: la infección por Papilomavirus Humano (PVH) es la condición necesaria para la aparición y desarrollo del cáncer cérvico-uterino. Los genotipos de alto riesgo oncogénico son los causantes de este tipo de neoplasia y dentro de ellos el más frecuente es el PVH 16, que se encuentra aproximadamente en el 60 por ciento de los casos. Los métodos de diagnóstico comerciales resultan costosos para países con escasos recursos económicos, lo que sugiere la búsqueda de alternativas empleando protocolos sencillos y baratos. Objetivos: normalizar un método inmunoquímico para la detección del antígeno L1 de PVH tipo 16 en muestras cérvico-uterinas de pacientes con lesiones intraepiteliales escamosas y determinar la coincidencia entre el método normalizado y la Reacción en Cadena de la Polimerasa en Tiempo Real (RCP-TR), como técnica de referencia, para estimar la utilidad de dicho método en el diagnóstico de la infección por este genotipo viral. Métodos: se compararon tres procedimientos de inmunotinción (Indirecto de inmunoperoxidasa en dos pasos, Estreptavidina-Biotina y Amplificación por polímero) respecto a sensibilidad analítica, tinción inespecífica de fondo y tiempo de terminación, para la detección de la proteína L1 de PVH 16 en líneas celulares derivadas de carcinomas cervicales humanos y en muestras cérvico-uterinas utilizadas como controles. El protocolo normalizado se aplicó a muestras cérvico-uterinas de mujeres entre 30 y 59 años, 82 con lesiones intraepiteliales cervicales y 10 sin antecedentes de alteraciones citológicas, a las que además se les determinó PVH 16 mediante RCP-TR. Resultados: el procedimiento de Estreptavidina-Biotina resultó el más sensible y específico...

Introduction: Human Papillomavirus (HPV) infection is the necessary condition for the occurernce and development of cervical cancer. The high oncogenic risk genotypes are the responsible for this type of neoplasia and the most frequent is HPV 16 that affects roughly 60 percent of cases. Commercial kits for HPV detection are expensive for resource-poor countries, which suggests the search for alternative throguh non-expensive simple protocoles. Objectives: to standardize an immunochemical method for the detection of HPV 16 L1 antigen in cervical samples of patients with squamous intraepithelial lesions and to determine the diagnostic coincidence between the immunochemical method and the real-time polymerase chain reaction to estimate the usefulness of this method for the detection of cervical infection with this viral genotype. Methods: three immunostaining methods (Two-Step Indirect Immunoperoxidase, Labelled Streptavidin-Biotin and Enhanced Polymer) were compared in terms of analytical sensitivity, nonspecific background staining and time of completion, for the detection of protein L1 of HPV-16 in a cell line derived from human cervical carcinoma and clinical samples from uterine cervix. The optimized protocol was applied to 82 cervical samples from women aged 30-59 years with squamous intraepithelial lesions and to 10 samples of sexually active women without previous signals of positive cytology. The presence of type 16 HPV was also detected with the aid of RT-PCR. Results: the Streptavidin-Biotin system was the most sensitive and specific...

Hormone receptors and other prognostic factors in breast cancer in Cuba.

Clinical management of breast cancer, making a prognosis and deciding on treatment, currently depend on defining prognostic factors, especially hormone receptors (HR). In addition to confirming the heterogeneity of the disease, these biological parameters are indispensable tools for designing personalized treatment. In this study, 1509 tumors from Cuban women diagnosed with breast cancer were examined. Hormone receptor (HR) expression was determined and correlated with a group of prognostic factors, such as age, tumor size, histological type, nuclear grade, histological grade, number of metastatic axillary lymph nodes, and clinical stage. Estrogen receptor (ER) expression was associated with low nuclear grade and histological grade, and with smaller tumor size (p < 0.05). Analysis of age at the time of diagnosis showed that ER expression was greater in patients in the group aged >50 years (p < 0.05). In general, ER expression was greater in patients in earlier clinical stages (p < 0.05). With regard to HR expression, 53% of tumors in this sample were ER+ and 49% were PR+. In 38% of cases, both receptors were positive and in 28% both receptors proved negative. The ER+/PR- combination was observed in 23% of cases while only 11% exhibited the ER-/PR+ combination. These findings indicate that approximately 72% of the tumors studied expressed some level of hormone dependency. This is the first report of HR expression in Cuba using immunohistochemical techniques and a representative sample of breast tumors diagnosed in different provinces around the country.

Relationship between IL-10 and tumor markers in breast cancer patients.

Tumor markers are expressed due to molecular alterations of the tumor cells, and we can relate them to the immune system to find new associations to improve prognosis. IL-10 inhibits the generation of immune responses at the tumor site. To determine IL-10 expression in the tumor microenvironment and to associate it with certain tumor markers, 27 breast cancer patients were monitored by immunohistochemistry. The results showed that 23 breast cancer samples exhibited a strong expression of IL-10. IL-10 was associated with some poor prognosis tumor makers. A direct association between IL-10, Bcl-2, and Bax was detected. The relationship between IL-10 and Bax was statistically significant (P=0.001). An inverse association of IL-10 with p53 was observed. IL-10 reflects a suppressive tumor microenvironment, and its relationship with apoptosis markers can suggest an increase in the aggressiveness of the tumor even if it still is at an early stage.