The risk of developing breast and ovarian cancer is higher in families that carry mutations in BRCA1 or BRCA2 genes, and timely mutation detection is critical.  To identify the presence of mutations in the Colombian population and evaluate two testing strategies.  From a total universe of 853 individual blood samples referred for BRCA1 and BRCA2 typing, 256 cases were analyzed by complete direct sequencing of both genes in Myriad Genetics, and the remaining 597 cases were studied by partial sequencing based on founder mutations in a PCR test designed by ourselves ("Profile Colombia").  We found 107 patients carrying deleterious mutations in this group of patients, 69 (64.5%) located in BRCA1, and 38 (35.5%) in BRCA2. Overall, we detected 39 previously unreported mutations in Colombia (22 in BRCA1 and 17 in BRCA2) and only 4 out of the 6 previously reported founder mutations. Sixty four out of 597 patients (10.7%) studied by "Profile Colombia" showed mutations in BRCA1 or BRCA2, and 41/256 patients (16%) showed mutations by complete BRCA1-BRCA2 sequencing.  The spectrum of 44 different mutations in Colombia as detected in our study is broader than the one previously reported for this country. "Profile Colombia" is a useful screening test to establish both founder and new mutations (detection rate of 10.7%) in cases with family history of breast cancer. Complete sequencing shows a detection rate of 16.0%, and should complement the study of the genetic basis of this disease. BRCA1 protein helps prevent the cells from growing in an uncontrolled way by repairing nuclear DNA with proteins encoded by RAD51 and BARD1, and is associated with RNA polymerases in their C-terminal domain allowing its interaction with histone deacetylase complexes participating in cell proliferation, developmental processes and transcription regulation. BRCA2 protein also controls cell growing, and interacts with RAD51 in DNA repair and homologous recombination 7. The development of malignancy (carcinogenesis) is a multi-step process, characterized by a progression of genetic alterations in a single cell line, were cells respond inappropriately to normal regulatory mechanisms ,In some cases genetic mutations in BRCA1 and BRCA2 are inherited, which may generate uncontrolled proliferation of a single cell and contribute to the development of cancer-BRCA1 gene, located in chromosome 17 (locus: 17q12-q21), was identified in 1990 after studying 23 families with a total of 143 cases of breast and ovarian cancer Given the high relation of these mutations with the development of breast cancer, their incidence in Colombia has been studied for many years. In 2007, six deleterious founder mutations were reported, 50% of them where present in families with multiple cases of breast cancer and 33% in families who had breast cancer and/or ovarian cancer, for a total of 53 families included in the study In a second report conducted in 780 patients with sporadic breast cancer (no criteria for family breast cancer), the six founder mutations previously described were studied and it was found that mutations A1708E and 3450delCAAG in BRCA1 were positive in 2.9% and mutation 3034delACAA in BRCA2 was positive in 1.3% of the patients et al., as well as new mutations in the current population in this country. In mutation carrier patients, prevention strategies can be implemented to lower the risk by up to 98% ,An unpublished diagnostic PCR test called "Profile Colombia" was developed by our group as a screening strategy in Colombia by sequencing upstream and downstream framing fragments around the reported founder mutations et al., developed an investigation involving nearly 244 patients with breast cancer from Medellín, where only three mutations, two in BRCA1 -one with family history and one sporadic- and one in BRCA2, a sporadic case, were found in 1.2% of the patients. Of the three mutations reported in Medellín, the first two were included in "Profile Colombia" and the third, 5844del5, had not been previously reported in individuals with Colombian descent The majority of previous studies have been performed in the population of Bogotá, but Hernández The main objective of this study was to determine the mutational spectrum in BRCA1 and BRCA2 genes in Colombia, and compare two diagnostic strategies to detect patients at high risk of developing cancer and establish prevention programs. BRCA1 and BRCA2 genes, including requests for "Perfil Colombia" and requests for "Complete sequence of the BRCA1 and BRCA2 genes". Applications from laboratories outside Colombia (mainly Ecuador and Panama) were excluded. This protocol was performed by convenience as a descriptive analytical study.Eight hundred and fifty three female patients with breast cancer diagnosed at any age in a 6 year period -between 2009 and 2014- were selected for this study from different regions of Colombia, with the following criterium: All samples sent to a reference laboratory in Colombia for analysis of mutations in the Participating centers approved the analytical protocol with the endorsement of the research committee of Instituto de Referencia Andino (IRA), and every patient who participated in the study gave written informed consent. After signing the informed consent, a blood sample was obtained from each patient, and referenced to IRA in Bogotá.® Laboratories, and the remaining 597 cases (70%) were studied by partial sequencing based on founder mutations in a test designed by IRA Laboratories in Bogotá ("Profile Colombia"). The second procedure was carried out based on primers designed along selected BRCA1 and BRCA2 sequences in order to include the six most common mutations in Colombia  as reported by Torres et al., as well as upstream and downstream sequences in short (100-120 bp) framing fragments around the reported founder mutations. The partial sequences obtained were analyzed in a 4.8 Sequencher® program. BRCA1 and BRCA2 complete sequencing was analyzed by Myriad Genetics®. The analytical report and results were both referred to patients with a recommendation for a genetic counseling session. In a global universe of 853 individual blood samples referred for BRCA1 and BRCA2 typing, DNA extraction was performed from all blood samples. Of these, 256 cases (30%) were analyzed by complete direct sequencing of both genes in Myriad Genetics® tables and mutation frequencies were subsequently defined. This protocol was performed by convenience as a descriptive analytical study.Results of molecular analysis of the BRCA1 and BRCA2 gene sequences were registered in Excel,,BRCA1 and BRCA2 genetic analyses from 853 patients were performed, of which 256 (30.0%) were analyzed by total direct sequence test and the remaining 597 (70.0%) were studied by partial sequence-based on founder mutations in the PCR analysis called "Profile Colombia". This study detected 107 patients carrying mutations of which 69 (64.5%) were located in BRCA1 and 38 (35.5%) in BRCA2. Additionally, among patients analyzed with "Profile Colombia", 209 (35.0%) showed a G5337A polymorphism in BRCA1 and 54 (9.0%) patients a A3199G polymorphism in BRCA2, the latter registered as a "class 1" mutation in NIH-BIC database. Overall, 39 new mutations were detected (22 in BRCA1 and 17 in BRCA2) which had not been reported in the previous studies of founder mutations in Colombia Most patients with BRCA1 and BRCA2 mutations among 107 positive individuals came from Bogotá , followed by Barranquilla and Atlántico , Medellín in Antioquia , Bucaramanga in Santander , Cartagena in Bolívar , Ibagué in Tolima , Cúcuta in North of Santander , Cali in Valle del Cauca , Villavicencio in Meta  and Montería in Córdoba . Two patients were received from Pereira in Risaralda, who showed no specific BRCA1/BRCA2 mutations. ,,Among 107 affected patients, 69 (64.5%) were found to carry mutations in BRCA1, of which 40 (58.0%) were either 3450delCAAG or A1708E, previously reported as founder mutations ,,The global deleterious mutational spectrum included 24 different locations in BRCA1 and 22 in BRCA2, of which 22 are new for BRCA1 and 17 are new for BRCA2 in Colombia , as relaWe identified the presence of a simultaneous deleterious mutation and a polymorphism in BRCA1 in the same patient and of simultaneous polymorphisms in both BRCA1 and BRCA2; simultaneous gene mutations and polymorphisms in the same patient were not observed in BRCA2. Both G5337A and A3199G polymorphisms present in BRCA1 and BRCA2 genes respectively, have not yet been reported as deleterious mutations (i.e. mutations directly involved in the development of breast cancer), and in consequence they should be considered as benign polymorphisms.The present study estimated the prevalence of BRCA1 and BRCA2 gene mutations in a broad Colombian population of 853 breast cancer patients. For some patients (30.0%), complete sequencing of BRCA1 and BRCA2 genes, and for the rest (70.0%) an extended profile of six previously reported funder mutations were performed in order to identify eventual immediate upstream and downstream gene mutations previously unreported in Colombia.We identified 43 deleterious mutations in BRCA1 and BRCA2 (39 new and 4 previously reported founder mutations), all of them involved in the development of breast cancer. Among these, 24 were identified in BRCA1, in addition to the G5337A polymorphism, which, given its high prevalence, requires further study to determine its clinical implication. On the other hand, 19 mutations were reported in BRCA2, in addition to the A3199G polymorphism which was also found in a high proportion in this population.Although the clinicians that referred the patients would not report specific clinical criteria taken into account as the basis for their request of genetic testing in a total of 853 patients included in this study, more than a third of the sample (39.3%) had a mutation or polymorphism and, among these, 49 cases (5.7%) showed founder mutations reported by the 2007 study The most frequent mutations in BRCA1 were A1708E (27 patients) and 3450delCAAG (13 patients), followed by four patients with 1793delA, C5141T (3 patients), C39R (234T>C) and V1145F (2 patients each). As for BRCA2, the most frequent mutations besides the previously reported founder mutation 3034delACAA (8 patients), were C5972T (11 patients) and 6328C>T (3 patients), all of which could be considered founder mutations in Colombia  and 2.Although the largest proportion of scientific literature suggests that when cancer is highly related to genetic factors and is attributable to specific founder mutations in a given population, it is possible to perform an inexpensive screening -as compared to the cost of the complete sequencing of each gene Although there were limitations associated with the pre-analytical phase of this study, as data from clinical records were scarce and no follow-up was undertaken directly by us in patients who tested positive for a mutation associated in the development of breast cancer, and even if the genetic sequencing of restricted fragments of BRCA1 and BRCA2 will necessarily leave out an indeterminate proportion of possible mutations in these genes, the record of 39 new deleterious mutations by two complementary molecular testing protocols, which have not been previously reported in Colombia, suggests that Colombians should not be considered as restricted as other populations such as the Finnish or the Ashkenazi Jews, as could have been deduced from the previous report of a small number of founder mutations Finally, the large proportion of patients clinically diagnosed with breast cancer in whom no mutations were found, especially among those with complete sequence of the BRCA1 and BRCA2 genes, supports the fact that the development of breast and ovarian cancer not only depends on a reduced spectrum of genetic factors associated with mutations in these particular genes, and that other genetic and non-genetic factors must be considered. Due to the worldwide high breast cancer incidence and the diversity of mutations identified in a Colombian population, early detection of carriers by profiling and subsequent gene sequencing should allow for monitoring and prevention strategies long before the development of disease. "Profile Colombia" (which includes upstream and downstream neighbor sequencing of previously identified founder mutations) has proved to be a low cost screening PCR analysis useful for local detection of supplementary deleterious and founder mutations.  El cáncer de mama es el segundo cáncer más común en el mundo y el más común entre las mujeres, con una incidencia de 1.7 millones de casos diagnosticados en el 2012 (25% de todos los cánceres) ,et al, la edad estándar de incidencia de carcinoma de ovario en Colombia se estimó como 10.1 casos por cada 100,000 individuos por año, y aproximadamente 11.5% de todos los carcinomas de ovario se hallaron en pacientes que portan una sola mutación de BRCA1, conocida como una mutación fundadora en Colombia ,,En América del Norte y en Europa Occidental, 1 de 8-9 mujeres desarrollarán cáncer de mama durante su vida et al, se han encontrado mutaciones en los genes BRCA1 y BRCA2 afectando entre el 5 y 8% de los pacientes con cáncer de mama en Colombia, y en 10% de los pacientes con carcinoma de ovario ,,-De acuerdo con los resultados obtenidos por Torres Las funciones normales de los genes BRCA1 y BRCA2 son la reparación, transcripción y recombinación del ADN, las cuales previenen el desarrollo del carcinoma como parte del grupo de genes supresores de tumores. La proteína BRCA1 ayuda a prevenir el crecimiento incontrolado de las células reparando el ADN nuclear con proteínas codificadas por los genes RAD51 y BARD1, y está asociada con las polimerasas ARN en el dominio C-terminal, permitiendo su interacción con complejos de histona deacetilasas, participando en la proliferación celular, procesos de desarrollo y regulación de transcripción. La proteína BRCA2 también controla el crecimiento celular e interactúa con el RAD51 en reparación del ADN y recombinación homóloga ,En algunos casos, mutaciones genéticas en BRCA1 y BRCA2 son heredadas, lo cual podría generar proliferación descontrolada de una sola célula y contribuir al desarrollo del cáncer -El gen BRCA1, localizado en el cromosoma 17 (locus: 17q12-q21), fue identificado en 1990 después de estudiar 23 familias con un total de 143 casos de carcinoma de mama y ovario Dada la relación de estas mutaciones con el cáncer de mama, su incidencia en Colombia ha sido estudiada durante muchos años. En 2007 se reportaron seis mutaciones deletéreas fundadoras, 50% de ellas presentes en familias con múltiples casos de cáncer de mama y 33% en familias que tenían cáncer de mama y/o de ovario, para un total de 53 familias incluidas en el estudio En un segundo reporte conducido en 780 pacientes con cáncer de mama esporádico (sin criterio para cáncer de mama familiar), las seis mutaciones fundadoras previamente descritas fueron estudiadas, y se encontró que las mutaciones A1708E y 3450delCAAG en BRCA1 fueron positivas en el 2.9% de los casos, y la mutación 3034delACAA en BRCA2 fue positiva en 1.3% de los pacientes et al., además de encontrar nuevas mutaciones en la población actual en este país. En pacientes portadores de mutación, la detección temprana puede ser implementada para disminuir el riesgo hasta un 98% ,Una prueba de PCR diagnóstica original denominada "Perfil Colombia" fue desarrollada por nuestro grupo como una estrategia de tamizaje en Colombia secuenciando corriente arriba y corriente bajo los fragmentos enmarcados alrededor de las mutaciones fundadoras reportadas previamente et al., desarrollaron una investigación que involucró alrededor de 244 pacientes con cáncer de mama de Medellín, donde solo tres mutaciones, dos en BRCA1 -una con historia familiar y una esporádica- y una en BRCA2, caso esporádico, fueron encontrados en el 1.2% de los pacientes. De las tres mutaciones reportadas en Medellín, las primeras dos fueron incluidas en el "Perfil Colombia" y la tercera, 5844del5, no se había reportado previamente en individuos de ascendencia colombiana La mayoría de los estudios previos fueron realizados en la ciudad de Bogotá, pero Hernández El objetivo principal de este estudio fue determinar el espectro mutacional en los genes BRCA1 y BRCA2 en Colombia, y evaluar dos estrategias diagnosticas para proponer una metodología de detección de pacientes de alto riesgo de desarrollo de cáncer y establecer programas de prevención.Ochocientos cincuenta y tres pacientes del género femenino con cáncer de mama diagnosticados a cualquier edad en un periodo de seis años (entre 2009-2014) fueron seleccionadas para este estudio de diferentes regiones de Colombia bajo el siguiente criterio: Todas las muestras enviadas a un laboratorio de referencia en Colombia para análisis de mutaciones en los genes BRCA1 y BRCA2, incluyendo órdenes para "Perfil Colombia" y órdenes para "Secuenciación completa de genes BRCA1 y BRCA2", excluyendo aplicaciones de laboratorios fuera de Colombia, principalmente de Ecuador y Panamá. Este protocolo fue realizado por conveniencia, como un estudio analítico descriptivo.Los centros participantes aprobaron el protocolo analítico con respaldo del comité del Instituto de Referencia Andino (IRA), y cada paciente participante diligenció el consentimiento informado por escrito. Después de firmar el consentimiento informado, se obtuvo una muestra de sangre de cada paciente y esta fue referenciada al IRA en Bogotá.® (USA), los 597 casos restantes (70%) fueron estudiados por secuenciación parcial basada en las mutaciones fundadoras reportadas previamente en la población, en una prueba diseñada por laboratorios IRA en Bogotá ("Perfil Colombia"). El segundo procedimiento fue llevado a cabo con primers diseñados sobre secuencias seleccionadas de BRCA1 y BRCA2 para incluir las seis mutaciones más comunes en Colombia , como fue reportado por Torres et al. Adicionalmente, se realizaron secuencias cortas (100-120 pares de bases) en el marco de los fragmentos alrededor de las mutaciones fundadoras mencionadas. Las secuencias parciales obtenidas fueron analizadas en un el programa Sequencher® 4.8. La secuenciación completa para BRCA1 y BRCA2 fue llevada a cabo por Myriad Genetics®. El reporte analítico y sus resultados fueron entregados a los pacientes con recomendación de una sesión de consejería genética. En un universo global de 853 muestras de sangre individuales referidas para tipificación de BRCA1 y BRCA2se realizó la extracción de ADN de todas las muestras. De estas, 256 casos (30%) fueron analizados por secuenciación directa para ambos genes en Laboratorios Myriad Genetics® y las frecuencias de mutación fueron subsecuentemente definidas. Este protocolo fue realizado por conveniencia en el marco de un estudio descriptivo analíticoLos resultados de los análisis moleculares de los genes BRCA1 y BRCA2 fueron registrados en tablas Excel,,®.Se realizaron análisis genéticos en los genes BRCA1 y BRCA2 de 853 pacientes colombianos, de los cuales 256 (30.0%), fueron analizados por secuenciación directa total y los 507 restantes (70.0%) fueron estudiados por secuenciación parcial basada en las mutaciones fundadoras en el análisis de PCR denominado "Perfil Colombia". Este estudio detectó 107 pacientes portando mutaciones en estos genes, de las cuales 69 (64.5%) fueron localizadas en BRCA1, y 38 (35.5%) en BRCA2. Adicionalmente, entre los pacientes analizados con "Perfil Colombia", 209 (35.0%) mostraron el polimorfismo G5337A en BRCA1, y 54 (9.0%) de los pacientes el polimorfismo A3199G en BRCA2, este último registrado como mutación "clase 1" en la base de datos del NIH-BIC. En total fueron detectadas 39 nuevas mutaciones (22 en BRCA1 y 17 en BRCA2), las cuales no habían sido reportadas en estudios previos de mutaciones fundadoras en Colombia ,,,,La mayoría de los pacientes con mutaciones BRCA1 y BRCA2 (107 individuos positivos) enviaron su muestra desde Bogotá , seguidos por quienes la enviaron desde Barranquilla y Atlántico , desde Medellín en Antioquia , desde Bucaramanga en Santander , desde Cartagena en Bolívar , desde Ibagué en Tolima , desde Cúcuta en Norte de Santander , desde Cali en Valle del Cauca , desde Villavicencio en Meta . Dos pacientes fueron recibidos de Pereira en Risaralda, y no mostraron mutaciones específicas en BRCA1/BRCA2. La Identificamos también la presencia simultánea de mutaciones deletéreas y polimorfismos en el mismo paciente en BRCA1, y, por otra parte, de polimorfismos simultáneos en ambos genes, BRCA1 y BRCA2; no se observaron mutaciones y polimorfismos simultáneos en el mismo paciente en BRCA2. Estos polimorfismos, G5337A y A3199G, presentes en genes BRCA1 y BRCA2 respectivamente, aún no han sido reportados como mutaciones deletéreas , y en consecuencia deben ser considerados por lo pronto como polimorfismos benignos.El presente estudio estimó la prevalencia de mutaciones en los genes BRCA1 y BRCA2 en una vasta población colombiana de 853 pacientes con cáncer de mama. Para algunos pacientes (30%), se procedió con la secuenciación completa de genes BRCA1 y BRCA2, y para el resto (70%), se realizó un perfil extendido ("Perfil Colombia") de 6 de las mutaciones fundadoras previamente reportadas en orden de identificar mutaciones inmediatas corriente arriba y corriente abajo, no reportadas previamente en Colombia.Identificamos en total 43 mutaciones deletéreas en BRCA1 y BRCA2 (39 nuevas además de las 4 mutaciones fundadoras previamente reportadas), eventualmente involucradas en el desarrollo de cáncer de mama. Sobre estas, 24 fueron identificadas en BRCA1, en adición al polimorfismo G5337A, el cual, dada su alta prevalencia, requiere estudios adicionales para determinar su implicación clínica. Por otro lado, se reportaron 19 mutaciones en BRCA2, en adición al polimorfismo 3199G, el cual también fue hallado en una alta proporción de la población. Aunque los médicos que refirieron los pacientes no reportan los criterios clínicos que los llevaron a solicitar los análisis genéticos más allá del cáncer de mama en sus pacientes, en un total de 853 individuos incluidos en el estudio, más de un tercio de la muestra (39.3%) presentó una mutación o polimorfismo y, entre estos, 49 casos (5.7%) presentaron mutaciones fundadoras reportadas por el estudio de 2007 Las mutaciones más frecuentes en BRCA1 fueron A1708E (27 pacientes) y 3450delCAAG (13 pacientes), seguidas de cuatro pacientes con 1793delA, 3 pacientes con C5141T y 2 pacientes para C39R (234T>C) y para V1145F. En cuanto al BRCA2, las mutaciones más frecuentes además de la mutación fundadora 3034delACAA en 8 pacientes, se encontraron: C5972T (11 pacientes) y 6328C>T (3 pacientes). Todas estas podrían ser consideradas mutaciones fundadoras en Colombia en razón al número de pacientes portadoras  y 2.Aunque la mayor proporción de literatura científica sugiere que el cáncer está altamente relacionado con factores genéticos y es atribuible a mutaciones fundadoras específicas en una población dada, es posible desarrollar un tamizaje de bajo costo, en comparación a la secuenciación completa de cada gen Aunque hubo limitaciones asociadas con la fase pre-analítica de este estudio, ya que la información de las historias clínicas fue escasa y no hubo seguimiento realizado directamente por nosotros a los pacientes que obtuvieron resultados positivos para una mutación asociada con el desarrollo de cáncer, e incluso si la secuenciación genética de fragmentos restringidos de BRCA1 y BRCA2 necesariamente deja por fuera una proporción indeterminada de posibles mutaciones en estos genes, el registro de 39 nuevas mutaciones deletéreas identificadas a través de dos protocolos complementarios de análisis molecular, las cuales no habían sido previamente reportadas en Colombia, sugiere que la población colombiana no debería ser considerada tan restringida genéticamente como otras poblaciones como los finlandeses o los judíos ashkenazi como podría haberse deducido de los reportes previos de un pequeño número de mutaciones fundadoras para esta población de la esquina norte de Suramérica Finalmente, la mayoría de los pacientes diagnosticados clínicamente con cáncer de mama en quienes no se hallaron mutaciones, específicamente entre los analizados con secuenciación completa de genes BRCA1 y BRCA2, soportan el hecho de que el desarrollo de cáncer de mama y de ovario, no solo depende en un espectro reducido de factores genéticos asociados con mutaciones en estos genes en particular, y que otros factores genéticos y no genéticos deben ser considerados en la evaluación diagnóstica de cada población.Debido a la alta incidencia de cáncer de mama a nivel mundial, y a la alta diversidad de mutaciones identificadas en una población colombiana, la detección temprana de portadores realizada por secuenciación de genes y la aplicación de un perfil genético determinado (como el "Perfil Colombia") deben permitir estrategias de prevención y monitoreo mucho antes del desarrollo de esta enfermedad. El "Perfil Colombia" , ha probado ser un análisis de PCR útil como tamizaje, y de bajo costo, para la detección local de mutaciones deletéreas y fundadoras conocidas y suplementarias.


Scientific Reports 10.1038/s41598-017-12819-0, published online 11 October 2017Correction to: In the original version of this Article, the affiliation for Lorena Rojas-Vega and Gerardo Gamba was incomplete. The correct affiliation for these authors appears below.Molecular Physiology Unit, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México and Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico.This error has now been corrected in the PDF and HTML versions of the Article.


Os eventos naturais representados por dados contínuos assumem diferentes distribuições de frequência, entre elas uma distribuição em forma de sino, chamada curva normal ou de Gauss . A curvat de Student, ANOVA, coeficiente de Pearson, regressão linear (resíduos) e análise discriminanteOs principais testes estatísticos empregados na análise de dados clínicos e experimentais são baseados em modelos teóricos que pressupõem a distribuição normal, como teste ,Muitos dados biomédicos apresentam distribuição não normal, especialmente em eventos de grande variabilidade, com desvio padrão maior que a metade do valor médio , contrai,O primeiro passo para a avaliação da normalidade de um conjunto de dados deve ser a visualização de seu histograma, a fim de identificar grandes assimetrias, descontinuidades de dados e picos multimodais. É importante salientar que, na análise de subgrupos ou em comparações múltiplas, todas as categorias ou subamostras sujeitas à análise devem ser submetidas à avaliação da normalidade, e não apenas a amostra globalA Desde que o histograma não apresente inconsistências com a distribuição normal, é recomendável a avaliação dos estimadores de simetria e curtose, que representam aspectos ligados à forma do histograma: desviado para a esquerda/direita (simetria) ou apiculado/achatado (curtose); ambas as medidas se aproximam do zero quando os dados são normais. Como esses estimadores sofrem efeito do tamanho amostral e de valores extremos, é prudente calcular a razão de seus valores pelo erro padrão de suas estimativas. De forma geral, o valor do coeficiente dividido pelo seu erro padrão deve estar entre -1,96 e +1,96 nas distribuições normaisA ,Diagramas quantil-quantil (diagramas Q-Q) são representações gráficas das proporções dos dados da amostra original em comparação com os quantis esperados para uma distribuição normal 2. Nesse,-Há uma dezena de testes estatísticos que verificam o ajuste dos dados à distribuição normal a partir de diferentes pressupostos e algoritmos. Todos os testes pressupõem a hipótese de normalidade dos dados (H0), retornando um p-valor > 0,05 se resultarem na aderência aos parâmetros de normalidade. Diversas simulações demonstram um melhor desempenho para os testes de Shapiro-Wilk e Shapiro-Francia,,Os testes de normalidade sofrem influência do tamanho amostral quanto à sua eficiência. Em amostras pequenas (entre 4 e 30 unidades), há inflação do erro tipo I, sendo preferidos os testes de Shapiro-Wilk e Shapiro-Francia (maior especificidade). À medida que aumentam as amostras, especialmente acima de 500 unidades, todos os testes apresentam melhores desempenhos; entretanto, é prudente adotar o nível de significância de p < 0,01, em função do inflacionamento do erro tipo II causado pelo aumento amostral (perda de sensibilidade),,O teste de D'Agostino-Pearson foi desenvolvido para lidar com amostras mais numerosas (n > 100), apresentando, nesses casos, desempenho próximo ao do Shapiro-Wilk. O teste de Jarque-Bera apresenta bom desempenho na avaliação de normalidade em amostras maiores que 50 unidades, assim como o teste de Anderson-DarlingO teste de Kolmogorov-Smirnov deve ser dedicado apenas à verificação de aderência da amostra a distribuições com outros parâmetros, visto que é superado pelos outros aqui descritos para testar a normalidade dos dados. Por outro lado, o emprego da correção de Lilliefors oferece uma boa opção para analisar normalidade quando a distribuição contiver muitos dados extremos e a amostra for maior que 30 unidades2).Dados que não se revelem aderentes à distribuição normal pelos métodos descritos anteriormente devem ser tratados com cautela pelos pesquisadores. Primeiramente, a descrição da amostra deve ser representada pelos quartis , já que a média e o desvio padrão podem não reproduzir a tendência central e dispersão dos dados. Na ranks) de acordo com a escala de dados. De forma geral, esses testes apresentam maior erro tipo II, especialmente quando as amostras forem de menor dimensão (n < 30), além de tornarem menos generalizáveis as medidas de efeito,Há uma grande variedade de técnicas estatísticas destinadas a examinar amostras independentemente do formato de sua distribuição, que são chamadas estatísticas não paramétricas e englobam testes populares como Mann-Whitney, Wilcoxon, Kruskal-Wallis, Jonckheere-Terpstra, Friedman e coeficiente de Spearman. Essas técnicas substituem os dados originais por postos ordenados , as transformações dos dados alteram a escala entre as medidas, influenciando a interpretação direta e a generalização das medidas de efeitoA transformação dos dados a fim de sua normalização é uma alternativa bastante usual em amostras com distribuição dos dados inclinada para a direita ou esquerda. Raiz quadrada, transformações logarítmicas, exponenciais, angulares (arcsen) e hiperbólicas (1/x) são as mais empregadas. Entretanto, deve-se ter em mente que, da mesma forma que as técnicas que empregam postos ordenados  na escala original; porém, pela maior complexidade dos processos analíticos, recomenda-se o auxílio de um profissional estatístico experiente,Em certas técnicas analíticas multivariadas  ou em análise de medidas repetidas, há a necessidade de comprovação da normalidade multidimensional (esfericidade dos dados). Todavia, esse tópico transcende o escopo do textoFinalmente, as estratégias de avaliação dos dados quanto ao ajuste à distribuição normal devem ser adequadamente descritas na metodologia, sendo essenciais ao sucesso do processo investigativo, além de refletir o cuidado do pesquisador com a análise dos dados, o que gera maior credibilidade aos resultados. When continuous data are used to represent natural events they can take a variety of different frequency distributions, one of which is a bell-shaped distribution that is known as the normal or Gaussian curve . Normal t test, ANOVA, Pearson’s coefficient, linear regression , and discriminant analysis.The main statistical tests used for analysis of clinical and experimental data are based on theoretical models that assume a normal distribution, such as Student’s ,Many biomedical data have non-normal distributions, especially those representing events with great variability, with a standard deviation greater than half of the mean value ; which c,The first step in evaluating the normality of a dataset should be to examine its histogram to identify major asymmetries, discontinuity of data, and multimodal peaks. It is also important to stress that when analyzing subsets or conducting multiple comparisons, all of the categories or subsamples being analyzed must be tested for normality, and not just the overall sample.Assuming that the histogram does not reveal elements that are not consistent with the normal distribution, it is then recommended that estimators of symmetry and kurtosis should be calculated. These represent elements related to the shape of the histogram, dislocation to the left/right (symmetry) or peaked/flattened shapes (kurtosis), and both these measures approach zero when data are normal. Since these estimators are affected by sample size and outliers, it is prudent to calculate the ratio of their values to the standard error of their estimates. In general, the result of dividing the value of the coefficient by its standard error should fall in the range -1.96 to +1.96 for normal distributions.,Quantile-quantile plots (Q-Q plots) are graphical illustrations of the proportions of the data from the original sample compared against the quantiles expected for a normal distribution  and 2. Ip-value > 0.05 if the result shows that data do fit the parameters for normality. Several simulations have demonstrated that the Shapiro-Wilk and Shapiro-Francia tests offer better performance.,-There are dozens of statistical tests for verifying the fit of data to a normal distribution, based on different assumptions and using different algorithms. All of them test the null hypothesis (H0) that the data are normal, and so they return ,,The efficacy of normality tests suffers influence from sample size. With small samples (from 4 to 30 units), type I error is inflated and the Shapiro-Wilk and Shapiro-Francia tests are preferable (for better specificity). As sample sizes increase, especially over 500 units, all of the tests offer better performance; however, it is prudent to adopt a significance level of p < 0.01, because of the inflation of type II error caused by larger samples (reducing sensitivity).,,The D´Agostino-Pearson test was developed to deal with larger samples (n > 100), in which case it offers similar performance to the Shapiro-Wilk test. The Jarque-Bera test offers good performance for evaluating normality in samples larger than 50 units, as does the Anderson-Darling test.The Kolmogorov-Smirnov test should be reserved for testing the fit of a sample to distributions with other parameters, since it is outperformed by the other tests mentioned here for testing the normality of data. On the other hand, using the Lilliefors correction is a good option for analyzing normality when the distribution contains many extreme data and the sample is larger than 30 units.2).Data that are proven not to fit the normal distribution using the methods described above should be treated with care by researchers. Initially, the sample should be described using quartiles , since the mean and standard deviation may not reflect the central tendency and dispersion of the data. In ,There is a large number of statistical techniques for analyzing samples that are not dependent on their distribution. These are known as nonparametric statistical techniques and they include popular tests such as the Mann-Whitney, Wilcoxon, Kruskal-Wallis, Jonckheere-Terpstra, Friedman, and also Spearman coefficients. These techniques rely on substituting the original data with their ordered ranks, according to the scale of the data. In general, these tests are subject to greater type II error, especially when the samples are smaller n < 30) and their measures of effect are less generalizable.0 and theOne option that is widely used for samples with distributions shifted to the right or to the left is to perform a mathematic transformation to normalize them. Square roots, logarithmic, exponential, angular (arcsin), and hyperbolic (1/x) transformations are the most usually employed. However, it should be remembered that, in common with techniques that use rank ordering, data transformations alter the scale between measures, influencing interpretation and generalization of measures of effect.-It is also possible to opt for strategies for analysis of data for specific distributions, such as gamma, uniform, log-normal, beta, Tweedie, Poisson, negative binomial, Weibull, and others, which are known as generalized linear models. These analyses offer the advantage of working with the values (and the dimension of the effect) in the original scale; but because of the greater complexity of the analytical processes involved, it is recommended that help is sought from an experienced professional statistician.,For certain multivariate analytical techniques  or in analyses of repeated measures, it is necessary to demonstrate multidimensional normality (sphericity of data). Nevertheless, this is beyond the scope of this editorial.Finally, strategies for assessment of the fit of data to the normal distribution must be adequately described in the methodology, since they are essential to the success of the investigative process, in addition to demonstrating the care researchers have taken with analysis of the data, conferring greater credibility on the results.


Define-se trombose induzida pelo calor endovenoso como a propagação do trombo a partir de uma veia superficial em direção a uma veia mais profunda. Em geral, é considerada clinicamente insignificante quando não há propagação do trombo para o sistema venoso profundo. Essa condição pode ser tratada com terapia anticoagulante, embora a observação pareça ser suficiente, principalmente para graus menores. Neste estudo, relatamos dois casos de trombose induzida pelo calor endovenoso que teriam indicação de heparina de baixo peso molecular até a resolução do quadro. Porém, optou-se pelo uso da rivaroxabana (15 mg de 12 em 12h), com resolução completa do trombo em 4 semanas (caso 1) e em 7 dias (caso 2). A rivaroxabana pode ser uma alternativa promissora no tratamento da trombose induzida pelo calor endovenoso avançada, pela simplicidade da posologia, sem comprometimento da eficácia ou da segurança. São necessários estudos prospectivos, randomizados e controlados que possibilitem melhor entendimento da condição e o desenvolvimento de recomendações mais definitivas sobre opções de prevenção e tratamento. Em geral, a EHIT é considerada clinicamente insignificante quando não há propagação do trombo para o sistema venoso profundo. Como tal, ela quase não é relatada na literatura, e a observação parece ser suficiente, principalmente para graus menoresA trombose induzida pelo calor endovenoso Clinical-Etiology-Anatomy-Physiopathology (CEAP) C5. Foi submetida a raquianestesia e colocada em decúbito dorsal. Posteriormente, foram realizadas punção ecoguiada e passagem de fibra nua (bare fiber) até 2 cm da junção safeno-femoral (JSF)  de 31,2 e quadro clínico de varizes unilaterais de membro inferior esquerdo com classificação al (JSF) 2.linear endovenous energy density – LEED) de 38,5 J/cm, com bom controle ecográfico imediato da JSF  de controle, que evidenciou a presença de EHIT com acometimento menor que 50% da luz da veia femoral comum esquerda; optou-se, então, pelo tratamento anticoagulante ambulatorial da paciente com rivaroxabana 15 mg de 12 em 12h. Repetido o exame em 4 semanas, foram evidenciadas a regressão do trombo e a resolução do quadro, momento em que o anticoagulante foi suspenso.bare fiber) com LEED de 36 J/cm na safena magna direita e de 42 J/cm na safena magna esquerda a partir de 2 cm da JSF, guiado por EDC, sem a realização de tumescência bilateral, sempre com paciente em posição de Trendelemburg. Associada ao procedimento, foi realizada a ressecção de varizes tributárias e perfurantes insuficientes nas pernas.Paciente do sexo feminino, 42 anos, com quadro de insuficiência venosa dos membros inferiores, classificação de CEAP C3, sem outras comorbidades e com história familiar (irmã) de TVP. Foi submetida a raquianestesia, punção de veia safena magna ao nível do joelho e EVLT 1470 nm, com utilização de fibra nua  e Xa também têm sido utilizados para fins de anticoagulação, tornando-se uma opção simplificada para o tratamento da TVPWerth et al.Embora dados de apoio ainda sejam necessários, estamos de acordo com as diretrizes do Fórum Venoso AmericanoNo presente estudo, relatamos dois casos de EHIT classe II de Kabnick e nível 5 de Lawrence em que a indicação seria a utilização de heparina de baixo peso molecular (HBPM) até a resolução do quadro. Mesmo sabendo que o tratamento da EHIT em graus menores é mais controverso e deixado a critério do operador, queríamos tratar esses doentes com pelo menos um curso curto de anticoagulação. Devido ao mecanismo de ação semelhante, à facilidade do tratamento, à possibilidade de administração por via oral e à manutenção do tratamento em regime ambulatorial, optou-se pela utilização de rivaroxabana 15 mg de 12 em 12h, com resolução completa do trombo em 4 semanas no caso 1 e em 7 dias no caso 2.A rivaroxabana e os demais novos anticoagulantes orais podem ser alternativas promissoras para o tratamento dos casos avançados de EHIT, pela simplificação posológica. Com o aumento do número de procedimentos, a disseminação das técnicas de termoablação endovenosa e o aprofundamento do estudo das suas principais complicações, espera-se um maior número de documentações dos casos de EHIT e, consequentemente, a possibilidade de realização de estudos prospectivos, randomizados e controlados que possibilitem um maior entendimento e recomendações mais definitivas das opções de prevenção e tratamento. The mechanism of action of endovenous laser treatment (EVLT) for ablation of varicose veins in the lower limbs is based on generating heat that causes endothelial damage, resulting in thickening and fibrosis and culminating in non-thrombotic occlusion of the incompetent veins.The intensity of vein wall contraction appears to be important, because the residual vein lumen after laser treatment is subject to occlusion by clot formation.This paper describes two cases of EHIT from a retrospective analysis of 278 great and small saphenous veins treated with the EVLT technique at our service over a 5-year period. These two cases were treated using rivaroxaban for anticoagulation because it simplifies posology, without affecting efficacy or safety, making it an attractive option for prevention and treatment of deep venous thrombosis (DVT).The patient was a 52-year-old female with diabetes and hypertension, a body mass index (BMI) of 31.2 and a clinical diagnosis of unilateral varicose veins of the left lower limb with a Clinical-Etiology-Anatomy-Physiopathology (CEAP) classification of C5. She was given spinal anesthesia and placed in decubitus dorsal. Next, ultrasound-guided puncture was performed and a bare optical fiber advanced up to 2 cm from the saphenopopliteal junction (SPJ)  and 2.Tumescence was induced with saline solution at room temperature and then EVLT was conducted with a 1470 nm laser and linear intravenous energy density (LEED) of 38.5 J/cm, with a good control ultrasound of the SPJ immediately postoperatively .During the same procedure, tributary varicose veins and incompetent perforating-communicating veins were removed. The patient was given a single 40 mg dose of enoxaparin 6 hours after the end of surgery and was encouraged to walk as soon as she had recovered from the anesthetic. She was discharged 10 hours after the operation, with a prescription for anti-inflammatories and analgesics.Five days after the procedure, at the first follow-up appointment, the patient was examined with color Doppler ultrasonography (CDU), which detected an EHIT compromising less than 50% of the lumen and a decision was taken to choose outpatient anticoagulant treatment with 15 mg of rivaroxaban every 12h. The examination was repeated 4 weeks later and it was found that the thrombus had undergone regression and the clinical picture had normalized, which is when the anticoagulant was withdrawn.The patient was a 42-year-old female with venous insufficiency of the lower limbs and a CEAP classification of C3, free from other comorbidities, but with a family history (sister) of DVT. Spinal anesthesia was given and then the great saphenous vein was punctured at the level of the knee and EVLT was conducted with a bare fiber and a 1470 nm laser using a LEED of 36 J/cm for the right great saphenous vein and a LEED of 42 J/cm for the left great saphenous vein, starting 2 cm from the SPJ and guided by CDU, without inducing tumescence in either limb and with the patient in the Trendelenburg position throughout. During the same procedure, incompetent perforating veins and tributary varicose veins in the legs were resected.The patient was discharged the same day on anti-inflammatories and a single 40 mg dose of enoxaparin 6 hours after the end of surgery and encouraged to walk every day. Despite having been instructed to return to the clinic 7 days later, the patient only returned 28 days after the operation, when she underwent a control CDU, which showed EHIT with a thrombus that appeared to be floating compromising less than 50% of the lumen of the right common femoral vein . She wasEndothermal heat-induced thrombosis is a finding that is expected during follow-up after EVLT of the great saphenous vein. However, what is not entirely clear in the literature is the clinical progression of patients who exhibit EHIT close to or extending to the SPJ.Although there is no evidence of a correlation between the distance from the SPJ at which thermoablation is started and the likelihood of EHIT, it is possible that the fiber can migrate after tumescence is induced, with minimal displacement of the fiber by external compression.-Several systems for EHIT classification have been developed and they can differ in terms of the need for and type of treatment and anticoagulation dose and duration.Kabnick et al.Lawrence et al.,Harlander-Locke et al.There are no convincing data on which routine use of anticoagulants for EVLT at prophylactic doses could be based. Selected patients with a history of superficial venous thrombosis, DVT or obesity are candidates for prophylaxis.Sufian et al. conducted a prospective study to evaluate the incidence of EHIT and its progression and concluded that the risk factors include: size of the vein, age and concomitant phlebectomies.Thankfully, PE is rare after EVLT procedures. Rosales-Velderrain et al. reported on three cases of patients who developed PE after radio frequency ablation of the great saphenous vein and mini-phlebectomies of primary symptomatic varicose veins of the lower limbs.Nowadays, drugs that work by direct inhibition of factor II (thrombin) and factor Xa are also being used for anticoagulation, offering a simplified treatment option for DVT.Werth et al.Although supporting data are needed, we are in agreement with the American Venous Forum guidelinesIn this paper we have described two cases of EHIT, Kabnick class II and Lawrence level 5, in which the standard indication would be low molecular weight heparin until resolution of the condition. Despite being aware that treatment of less severe EHIT is more controversial and left to the surgeon’s judgment, we wanted to treat these patients with at least one short course of anticoagulation. In view of the similar mechanism of action, the ease of treatment, the possibility of oral administration and of maintenance of treatment in an ambulatory setting, it was decided to use 15 mg of rivaroxaban every 12h, with complete resolution of the thrombus in 4 weeks in case 1 and in 7 days in case 2.Rivaroxaban and other new oral anticoagulants may be promising options for treatment of advanced cases of EHIT because of the posological simplification they offer. As the number of procedures increases, the techniques for endovenous thermoablation become more widespread and their principal complications are better studied, it is expected that more cases of EHIT will be documented, making it possible to conduct prospective, randomized and controlled studies that offer better understanding and lead to more definitive recommendations on the options for prevention and treatment.


Herpes simplex virus (HSV)-mediated delivery of the HSV thymidine kinase (TK) gene to tumor cells in combination with ganciclovir (GCV) may provide an effective suicide gene therapy for destruction of glioblastomas, prostate tumors and other neoplasias by recruiting tumor-infiltrating lymphocytes into the tumor. The development of new treatment strategies or combination of available innovative therapies to improve cell cytotoxic T lymphocytes trafficking into the tumor mass and the production of inhibitory molecules blocking tumor tissue immune-tolerance are crucial to improve the efficacy of cancer therapy. The immune system interacts closely with tumors during the disease development and progression to metastasis. The complex communication between the immune system and the tumor cells can prevent or promote tumor growth. New therapeutic approaches harnessing protective immunological mechanisms have recently shown very promising results. This is performed by blocking inhibitory signals or by activating immunological effector cells directly. Immune checkpoint blockade with monoclonal antibodies directed against the inhibitory immune receptors CTLA-4 and PD-1 has emerged as a successful treatment approach for patients with advanced melanoma. Ipilimumab is an anti-CTLA-4 antibody which demonstrated good results when administered to patients with melanoma. Gene therapy has also shown promising results in clinical trials. Particularly,  Cancer progression is accompanied by a strong suppression of the immune system (IS), which interferes with effective antitumor response and diminishes tumor eradication  A better knowledge of the interaction between the tumor and the IS has allowed the development of specific therapies designed to improve patient´s immune response. Tumor immunotherapy has two strategies: attack the tumor directly or activate the IS by the use of cell therapies, like stimulatory agonists or the immune-checkpoint blockade  This article reviews the use of immune-checkpoint blockade and suicide gene therapy as different alternatives for cancer therapy and analyzes the possible synergic effects that can be reach with the combination of this both therapies.The IS interacts intimately with the tumors during the process of disease development and its progression to metastasis   Tumor cells change their surface markers recurrently.. For example, they express tumor-associated antigens (TAA) ,In the adaptive response, the processed TAA are presented by the MHC class I and II molecules from APCs to the specific receptors of T CD8+ and CD4+ cells respectively for their activation Tumors can also deregulate the IS by altering a complex balance between activating and inhibitory signals (checkpoints) in different pathways that regulate the function of T cells ,Treg cells are relevant to the maintenance of the immunological homeostasis: they preserve the tolerance to self-antigens, prevent the autoimmune diseases, modulate the development of an immune response and favor the escape of tumor cells from immune control ,,Because Treg suppresses the immune response against self-antigens The most frequents TAA are own-antigens subexpressed in normal cells but highly expressed in tumor cells ,--,,The Treg lymphocytes have four main mechanisms of action to regulate the immune response In the case of T cells, the amplitude and quality of the response after recognition of an antigen is regulated by a balance between costimulatory and inhibitory signals (immune-checkpoint) ,,As previously mentioned, the activation and inhibition of different receptors regulate the balance between immune response and immunotolerance It has been demonstrated that tumors use some immunological control pathways as a mechanism of immune resistance Antitumor antibodies that block immune-checkpoints are directed against lymphocyte receptors or their ligands ,,,The CTLA-4 receptor is the first immune-checkpoint used as clinical target CTLA-4 is crucial in T-cell activation. This is demonstrated by the lethal phenotype of the hyperactivated immune system in CTLA-4 knockout mice It has been proposed that CTLA-4 expression attenuates the activation of T cells by a cascade of inhibitory signals CTLA-4 blockade can affect the intratumoral immune response by inactivating Treg tumor-infiltrating lymphocytes ,PD-1 is also a key protein in immune regulation After binding to its ligand, PD-1 suppresses T cell activation by recruiting SHP-2, which dephosphorylates and inactivates Zap70, an important component in the TCR signaling pathway. As result, PD-1 inhibits T-cell proliferation and its effector functions, such as the production of IFN-γ ,,,Although CTLA-4 and PD-1 negatively regulate the activation of T cells by blocking the CD3/CD28 pathway, these receptors have different roles There are preclinical studies that propose a combined therapy using antibodies for the blockade of both pathways simultaneously (anti-CTLA-4 plus anti-PD-1) Ipilimumab (anti-CTLA-4) is a recombinant human monoclonal antibody (IgG1 kappa immunoglobulin) approved by the Food and Drug Administration (FDA) in 2011 for the treatment of metastatic melanoma. The human monoclonal antibody nivolumab (anti-PD-1) is an IgG4 kappa immunoglobulin authorized in Japan in 2014 for the treatment of unresectable melanoma. The FDA approved the humanized monoclonal antibody pembrolizumab (IgG4 kappa immunoglobulin) against PD-1 in September 2014 and the nivolumab in December 2014, both for the treatment of advanced melanoma. In March 2015 the FDA approved nivolumab for the treatment of lung cancer ,There are phase II studies proving increased survival in patients with metastatic melanoma who received ipilimumab In 2015, the safety and efficacy of nivolumab and ipilimumab were assessed separately and compared vs nivolumab+ipilimimab as a novel combined therapy . They observed a survival of 11.4 months for the combined treatment versus 6.9 months for treatment with nivolumab alone and 2.9 months for ipilimumab alone The use of ipilimumab and tremelimumab has been associated with adverse events affecting the skin , intestine (diarrhea and colitis), liver (hepatitis and elevated liver enzymes) and endocrine glands  The selectivity of the antineoplastic agents is limited because cancer cells are resistant to apoptosis, cell cycle arrest, and senescence. Besides that, some resistant cells subpopulations may emerge in response to the neoplastic agent The herpes simplex virus-thymidine kinase/ganciclovir (HSV-TK/GCV) system is selective for tumor cells because it affects the active replication of DNA, which is one characteristic of tumor cells. This activity is decreased in the surrounding stromal cells, many of which are in the quiescent state ,The enzymes used in suicide gene therapy are divided into two groups. The first one includes enzymes of non-mammalian origin (e.g. HSV-TK). The second comprises enzymes of human origin that are absent or subexpressed in tumor cells ,,The HSV-TK/GCV system uses ganciclovir and its analogs as prodrugs. These are analogs of purine nucleosides ,,-,,,,,The HSV-TK/GCV system has been tested in pre-clinical ,,,,It has been shown that HSV-TK/GCV therapy induces anti-tumor immunity ,,,During the HSV-TK/GCV tumor treatment, the levels of some cytokines that stimulate APCs and T cells, such as IL-2, IL-12, IFNγ, TNFα and GM-CSF, are increased; whereas inhibitory cytokines like IL- 4, IL-6 and IL-10 are not stimulated ,The viral TK protein also functions as a superantigen, stimulating a highly immunogenic tumor microenvironment ,,The viral vector-mediated HSV-TK/GCV therapy has shown effective progression to phase III in some clinical trials when it was used alone or in combination with chemotherapy or radiation for prostate cancer, hepatocellular carcinoma, or glioblastoma multiforme Despite the promising results, there are some disadvantages. The replication-deficient adenoviral vectors trigger a strong humoral and cellular immune response that limits its effectiveness to a period of two to three weeks. Regardless these vectors may be useful for therapeutic applications where a very high level of transient expression of the therapeutic gene is desirable, as would occur in cancer gene therapy ,,Phase I and II studies have shown some side effects, such as mild fever, neutropenia, headache, thrombocytopenia, and impaired hepatic enzymes, among others. Fortunately, these events are transient and easy to tolerate The combined treatment of gene therapy and immunotherapy is an attractive option that recent advances in cancer therapeutics have made possible. The use of a suicide gene therapy system would lead to the sudden and massive presentation of TAA over a sustained period of weeks or months. It is reasonable to think that this therapy can be synergistically enhanced by its combination with a systemically administered immune-checkpoint inhibitor drug such as those described in this review. To explain this idea in a better way, it could be assumed that a tumor treated with HSV-TK/GCV will generate sudden and massive exposure of TAA to the immune system, which in other conditions wouldn't generate an effective immunoreactivity due to the decrease of MCH I and costimulating molecules and by the induction of Treg. This event would trigger the attraction and activation of APC and some TAA could be expected to induce an increase in the number of intratumoral Treg lymphocytes (even before the intervention with gene therapy), leading to an immunosuppressive environment. In order to prevent this immunological phenomenon and to enhance the antitumor response, subsequent administration of an immune-checkpoint inhibitor, for example an anti-PD1 antibody, would affect the activity of intratumoral Treg cells. This would lead to an increase in the proliferation of the effector T cells capable of fighting the tumor and would reinforce an immune memory response that would potentially have a long-term protective effect Recently performed tests with adenoviral vectors that carry the HSV-TK gene and the PD-1 extracellular domain sequence fused to the Fc portion of mouse IgG2a was recently assayed to produce the soluble PD-1 (sPD1-Ig) segment. This segment inhibits activity of the complete ligand competitively, and consequently, inhibits the apoptotic effect of T cells mediated by the immunosuppressive interaction of the whole ligand with its receptor. This vector was administered in a murine model of colon carcinoma and demonstrated a synergy between HSV/TK therapy and competitive blockade of PD-1/PD-L1 binding. There was a significant decrease in tumor volume in the group of mice treated with HSV-TK/sPD1 compared to the control groups, including the group of mice treated with the simple scheme of HSV-TK / GCV  It is possible that in the near future, preclinical and clinical trials will continue to test hypotheses similar to the one proposed in this section and will certainly have very effective clinical results, and above all, with a high level of therapeutic selectivity, which will favor even more the development of the precision medicine in the area of oncology.The increase of the immune response against tumors could be a key strategy to fight against cancer. The TK/GCV system induces the massive presentation of TAA effectively. On the other hand, the expression of the TK super-antigen facilitates antitumor cellular immunity. The use of monoclonal antibodies against the immune-checkpoints, such as CTLA-4 and PD-1, can decrease the tumor immunosuppression. Until now both strategies are found in clinical trials and have shown promising results. It would be expected that the combination of these two types of therapies would be synergistic, more selective and effective and would have a long-term protective effect. La progresión de muchos tipos de cáncer se acompaña de una fuerte supresión del sistema inmune (SI), lo que interfiere con la respuesta antitumoral y la erradicación eficaz del tumor La mejor comprensión del sistema inmunológico del tumor ha permitido el desarrollo de terapias específicas diseñadas para mejorar la respuesta inmune de un paciente. La inmunoterapia tumoral tiene dos estrategias: atacar al tumor directamente o activar el SI usando terapias celulares, como la estimulación con agonistas o el bloqueo de puntos de control inmunológicos En este artículo se hace una revisión del uso de terapia génica suicida y el bloqueo de los puntos de control inmunológicos como alternativas para el tratamiento del cáncer y se analizan los posibles efectos sinérgicos que podrían lograrse con la combinación de estas dos terapias. El SI interactúa íntimamente con los tumores durante el proceso del desarrollo de la enfermedad y su progresión a metástasis  Las células tumorales presentan cambios en sus marcadores de superficie. Por ejemplo, expresan antígenos asociados a tumor (TAA) ,En la respuesta adaptativa, los TAA procesados son presentados por moléculas MHC clase I y II de las APC a los receptores específicos para la activación de las células T CD8+ o CD4+, respectivamente Los tumores también pueden desregular el SI alterando un complejo balance entre señales inhibitorias (puntos de control) y activadoras en diferentes vías que regulan la acción de las células T ,Los linfocitos Treg son relevantes para el mantenimiento de la homeostasis inmunológica: mantienen la tolerancia a antígenos propios, evitan la aparición de enfermedades autoinmunes, controlan el desarrollo de una respuesta inmune y favorecen el escape de las células tumorales del control inmunológico,,Debido a que los Treg suprimen la respuesta inmune contra antígenos propios 17. El uso de estas células como blanco puede impactar las estrategias terapéuticas contra el cáncer Los TAA más frecuentes son antígenos propios poco expresados en células normales, pero altamente expresados en las tumorales ,-,,Los linfocitos Treg tienen cuatro mecanismos principales de acción para regular la respuesta inmune En el caso de las células T, la amplitud y calidad de la respuesta después del reconocimiento de un antígeno, está regulada por un balance entre señales coestimuladoras e inhibitorias (puntos de control inmunológico) ,,Como se comentó previamente, la activación e inhibición de diferentes receptores regula el balance entre la respuesta y la tolerancia inmune Los anticuerpos antitumorales que bloquean los puntos de control inmunológico están dirigidos contra receptores de linfocitos o sus ligandos ,,,El receptor CTLA-4 es el primer punto de control inmunológico usado como blanco clínico 4. A pesar de que CTLA-4 se expresa en células efectoras T CD8+ activadas, su función fisiológica más importante se ejerce a través de distintos efectos en los dos subconjuntos principales de células T CD4+: la disminución de la actividad de las células T cooperadoras (Th1) y el potenciamiento de la actividad inmunosupresora de las células Treg La importancia de CTLA-4 en la activación de células T está demostrada por el fenotipo letal del sistema inmune hiperactivado en ratones knockout para CTLA-4Se ha propuesto que la expresión de CTLA-4 atenúa la activación de las células T por una cascada de señales inhibitorias El bloqueo de CTLA-4 puede afectar la respuesta inmune intratumoral mediante la inactivación de los linfocitos Treg infiltrantes de tumor ,PD-1 también es una proteína clave en la regulación inmune Después de la unión con su ligando, PD-1 suprime la activación de células T reclutando SHP-2, la cual desfosforila e inactiva Zap70, un componente importante en la vía de señalización del TCR. Como resultado, PD-1 inhibe la proliferación de células T y sus funciones efectoras, como la producción de IFN-γ ,,,Aunque CTLA-4 y PD-1 regulan de manera negativa la activación de las células T al bloquear la vía CD3/CD28, estos receptores tienen distintos papeles Existen estudios preclínicos que proponen una terapia combinada utilizando anticuerpos para el bloqueo simultáneo de ambas vías (anti-CTLA-4 más anti-PD-1) Food and Drug Administration (FDA) en el 2011 para el tratamiento de melanoma metastásico. El anticuerpo monoclonal humano nivolumab (anti-PD-1) es una inmunoglobulina IgG4 kappa autorizada en Japón en el 2014 para el tratamiento del melanoma no resecable. La FDA aprobó el anticuerpo monoclonal humanizado pembrolizumab (inmunoglobulina IgG4 kappa) contra PD-1 en septiembre de 2014 y el nivolumab en diciembre de 2014, ambos para el tratamiento de melanomas avanzados. En marzo del 2015 la FDA aprobó el nivolumab para el tratamiento de cáncer de pulmón El ipilimumab (anti-CTLA-4) es un anticuerpo monoclonal recombinante humano (inmunoglobulina IgG1 kappa) aprobado por la ,Existen estudios de fase II que muestran una mayor sobrevida en pacientes con melanoma metastásico a los que se les administró ipilimumab En 2015 se evaluó la seguridad y eficiencia del nivolumab e ipilimumab por separado y en comparación con una terapia combinada nivolumab + ipilimimab , observándose una sobrevida de 11.4 meses para el tratamiento combinado, contra con 6.9 meses para el tratamiento solo con nivolumab y 2.9 meses para el ipilimumab ,El uso de ipilimumab y tremelimumab se ha asociado con eventos adversos que afectan la piel , el intestino (diarrea y colitis), el hígado (hepatitis y enzimas hepáticas elevadas) y las glándulas endócrinas  La selectividad terapéutica de los agentes antineoplásicos es limitada, porque las células cancerosas son resistentes a la apoptosis, al arresto del ciclo celular y a la senescencia, además que pueden emerger subpoblaciones celulares resistentes ,El sistema HSV-TK/GCV es selectivo para las células tumorales debido a que afecta la replicación activa del DNA de manera preferencial en las células tumorales, actividad que es baja en las células estromales circundantes, muchas de las cuales están en estado quiescente Herpes simplex [HSV-TK]. El segundo comprende enzimas de origen humano que están ausentes o se expresan a bajas concentraciones en células tumorales ,Las enzimas utilizadas en terapia génica suicida se dividen en dos grupos. El primero comprende enzimas de origen no mamífero , propiciándose un ambiente inmunosupresor. Para prevenir este fenómeno inmunológico y reforzar la respuesta antitumoral, la subsiguiente administración de un inhibidor de punto de control inmunológico, por ejemplo un anticuerpo anti-PD1, afectaría la actividad de las células Treg intratumorales, lo que conduciría a un incremento en la proliferación de las células T efectoras capaces de combatir al tumor y reforzaría una respuesta de memoria inmunológica que potencialmente tendría un efecto protector de largo plazo HSV-TK y la secuencia del dominio extracelular de PD-1 fusionada a la porción Fc de la IgG2a de ratón, para producir el segmento PD-1 soluble (sPD1-Ig), que inhibe la actividad del ligando completo de manera competitiva, y por consecuencia, inhibe el efecto apoptótico de las células T mediado por la interacción inmunosupresora del ligando íntegro con su receptor. Éste se administró en un modelo murino de carcinoma de colon y demostró una sinergia entre la terapia HSV/TK y el bloqueo competitivo de la unión PD1/PD-L1. Hubo un decremento significativo en el volumen tumoral en el grupo de ratones tratados con HSV-TK/sPD1 en comparación con los grupos controles, incluyendo el grupo de ratones tratados con el esquema simple HSV-TK/GCV Recientemente se ensayó un vector adenoviral portando el gen Es posible que en un futuro cercano se continúen realizando ensayos preclínicos y clínicos que prueben hipótesis similares a la que se plantea en este apartado y que seguramente tendrán resultados clínicos muy efectivos, y sobre todo, con un alto nivel de selectividad terapéutica, lo cual favorecerá aún más el desarrollo de la medicina de precisión en el área de la oncología.La potencialización de la respuesta inmune contra los tumores podría ser una estrategia clave para combatir el cáncer. El sistema TK/GCV induce la presentación masiva de TAAs de manera efectiva. Por otra parte, la expresión del súper-antígeno TK facilita la inmunidad celular antitumoral. El uso de anticuerpos monoclonales contra puntos de control de la respuesta inmune, como CTLA-4 y PD-1, contrarrestan la inmunosupresión ocasionada por el tumor. Hasta ahora ambas estrategias se encuentran en ensayos clínicos y han demostrado resultados prometedores. Se esperaría que la combinación de estos dos tipos de terapias fuera sinérgica, más selectiva y efectiva y que tuviera un efecto protector de largo plazo.


The Brazilian Journal of Cardiovascular Surgery (BJCVS) is grateful for the reviewers,listed below, which collaborated in this edition. Without their work would be impossibleto keep the high scientific standard of our journal.Alexandre HuebDouglas BolzanEduardo K. SaadiFernando PlataniaGabriel LiguoriGibran FeguriJoão Carlos Ferreira LealJoão de Deus e BritoJosé Carlos M. PachonJose Dario Frota FilhoKarlos VilarinhoLindemberg SilveiraLuciano AlbuquerqueLuís DallanMagaly Arrais dos SantosMarcos Aurelio Barboza de OliveiraMichel Pompeu B. de O. SáMilton SantoroPaulo Roberto EvoraPedro SalernoRenato ArnoniRicardo Carvalho LimaRodrigo MilaniStevan Martins


The main objective of this paper was to report the clinical effectiveness of leukocyte- platelet- rich fibrin (L-PRF) in the treatment of a combined endo-periodontal lesion of an upper first premolar.  The tooth had a profound abfraction on the vestibular aspect and presented no mobility but revealed a deep pocket measuring of 11 mm on the mesial vestibular aspect and 14 mm on the mesial palatine aspect. The three dimensional image analysis showed total bone loss in the mesial aspect and an extensively bone loss of the vestibular aspect of the vestibular root.  Endodontic treatment was performed and periodontal access surgery  was done with the application of autologous L-PRF. Three month and 6 months after surgery, the cone beam computed tomography (CBCT) exams showed no bone regeneration in any aspect of the tooth. However, periodontal examination showed a significative improvement in the deepness of surcus. The mesial vestibular aspect had a deep pocket of 3 mm and 5 mm on the mesial palatine aspect showing a reduction in deepness of 8 mm and 9 mm, respectively. The actual treatment for teeth with bad prognosis is the extraction and replacement with implants. Even though implants are capable of restore function and aesthetic, the abuse of this approach have led to the loss of teeth that could be successfully treated with a less invasive technique. The prognosis of teeth with endoperiodontal lesion is poor but could be enhanced with regenerative therapies. Until now there are no clinical trials and just four case report about the treatment of these teeth with platelet rich fibrin. The periodontium and the pulp are closely related, having embryonic, anatomic and functional interrelationship. The main anatomic pathways by which the pulp and periodontal ligament communicate are dentinal tubules, lateral and accessory canals and the apical foramen The treatment of endo-periodontal lesion depends on the diagnosis and differentiating between endodontic and periodontal disease Leukocyte- platelet- rich fibrin (L-PRF) is an autologous concentrate obtained through the centrifugation of blood of the same patient in which it will be used. In this concentrate most of the platelets, leucocytes, growth factors and cytokines are contained in a strong fibrin matrix. The fibrin matrix influences the biology of the material and the cells trapped inside The main objective of this paper was to report the clinical effectiveness of leukocyte- platelet- rich fibrin in the treatment of a combined endo-periodontal lesion.This research was conducted in concordance with Helsinki Declaration and patient signed the informed consent. A 52 year's old male patient was derivate to the Dental Clinic of the Universidad de La Frontera, for the endodontic treatment of the upper left first premolar. Clinically the tooth had a profound abfraction on the vestibular aspect, in close proximity with the root canal. The tooth had no mobility but revealed a deep pocket measuring of 11 mm on the mesial vestibular aspect and 11 mm on the mesial palatine aspect. There were no other periodontal involved teeth in the remaining dentition. The patient was a smoker (6/day) and had no systemic diseases associated.The three dimensional image analyses were realized by Cone Beam Computed Tomography (CBCT). The tooth in question showed total bone loss in the mesial aspect of the vestibular root and an extensively bone loss of the vestibular aspect of the vestibular root. The palatine root had bone loss involvement in the mesial aspect  A-B.At clinical examination, was observed that tooth 2.4 was asymptomatic, without any endodontic treatment started, and a slight discoloration. Vitality tests were negative. The tooth present two root and there was observed no bleeding at the endodontic access. In the treatment stage, initially the two root canals were permeabilized with #10 and #15 K-files . The working length was determined with a periapical radiograph and verified with the aid of an electronic apex locator . After this, the canals shaping was performed by a rotatory reciprocating files  at 350 rpm, following the manufacturers indication, with a Crown-down technique. This system used an only single file Primary 025/08 in all canals. In the entire canal shaping stage, was perfused with sodium hypochlorite at 2.5% and permeabilized with #15 K-file to remove the smear layer of the root canals. The canal obturation was realized with WaveOne® points with the same taper of the Primary file, achieving a proper fit in the apex. As a final root canal sealer, TopSeal®  was used. Finally a radiographical and clinical control was done at 7 and 14 days after the treatment, observing no clinical symptoms or radiographical findings of treatment failure.The periodontal surgery was made two week after the endodontic treatment. The patient was informed about the imperative necessity of stop smoking and 1 hour prior the surgery he took Amoxicilin 875 mg  and Ketoprofen 200 mg  and mouth rinse with chlorhexidine 0.12% .The surgical area was anesthetized with lidocaine 2%. Sulcular incision around the involved tooth, followed by a releasing vertical incision on the mesial side, was made with 15 scalpel blade . A full flap was raise, but minimally extended on the distal side  B. AfterOnce the roots and the furcation were clean, fibrin clots were placed in bone defect, one on top of the other and in opposite directions, and gently placed on the vestibular aspect, covering the root D. ClosurThree month and 6 months after surgery, the CBCT exams showed no bone regeneration in any aspect of the tooth . However, periodontal examination shows a significative improvement in the deepness of surcus. The mesial vestibular aspect had a deep pocket of 3 mm and 5 mm on the mesial palatine aspect showing a reduction in deepness of 8 mm and 9 mm, respectively  B-D.The actual treatment for teeth with bad prognosis is the extraction and replacement with implants. Even though implants are capable of restore aesthetic and function, the abuse of this approach have led to the loss of teeth that could be successfully treated with a less invasive technique, and still be functionally useful. In this case report, an upper premolar with a severe endo-periodontal lesion was successfully treated with a conservative treatment.The prognosis of a true combined endoperiodontal lesion is often poor or even hopeless, especially when periodontal lesions, like in this case report, compromises an extensive loss of attachment ,Leukocytes and platelet rich fibrin is a second generation of platelet concentrated in which most of the platelets and leucocytes are contained within a strong fibrin matrix In vitro studies have shown that L-PRF stimulates the proliferation of fibroblasts, periodontal progenitor cells and osteoblasts ,,,-In vivo studies have shown that L-PRF promotes periodontal regeneration and enhances alveolar bone augmentation et al. In this case report was observed a gain in the clinical attachment level and reduction in probing depth. However we could not observe any bone gain at any aspect of the tooth. Karunakar ,,,It is questionable if the reduction in probing deep obtained in this study would be different if there were no use of a platelet concentrate. In despite of the potential positives effects of the L-PRF very few studies had tempted to prove its clinical and radiographic effectiveness in the treatment of endoperiodontal lesions  ). Además, el surco palatogingival, las perforaciones radiculares y las fracturas radiculares verticales se describen como vías comunicativas. Estas vías permiten el intercambio de bacterias y bioproductos inflamatorios entre la pulpa y el ligamento periodontal. Por lo tanto, una enfermedad periodontal primaria puede causar un proceso degenerativo en la pulpa y, de la misma manera, una infección intrapulpar puede degenerar el periodonto .El periodonto y la pulpa están estrechamente relacionados, a través de una interrelación embrionaria, anatómica y funcional. Las principales vías anatómicas por las que se comunican la pulpa y el ligamento periodontal son los túbulos dentinarios, los canales laterales y accesorios y el foramen apical ).El tratamiento de la lesión endoperiodontal depende del diagnóstico y de la diferenciación entre enfermedad endodóntica y periodontal La fibrina rica en plaquetas y leucocitos es un concentrado autólogo obtenido mediante la centrifugación de sangre del mismo paciente en el que se usará. En este concentrado, la mayoría de las plaquetas, leucocitos, factores de crecimiento y citoquinas están contenidas en una matriz de fibrina sólida. La matriz de fibrina influye en la biología del material y en las células contenidas en su interior El objetivo principal de este trabajo fue informar la efectividad clínica de la fibrina rica en plaquetas y leucocitos en el tratamiento de una lesión endoperiodontal combinada.Esta investigación se realizó en base a la Declaración de Helsinki y el paciente en cuestión firmó el consentimiento informado. Un paciente masculino de 52 años fue derivado a la Clínica Dental de la Universidad de La Frontera para el tratamiento endodóntico del primer premolar superior izquierdo. Clínicamente, el diente tenía una profunda abfracción en la superficie vestibular, muy cerca del canal radicular. El diente no tenía movilidad, pero reveló una bolsa profunda que medía 11 mm en la superficie mesio vestibular y 11 mm en la superficie palato mesial. No hubo otros dientes periodontalmente comprometidos en la dentición restante. Paciente fumador (6/día) y no presentaba enfermedades sistémicas asociadas.El análisis de imagen tridimensional se realizó mediante la tomografía computarizada Cone Beam (CBCT). El diente en cuestión mostró una pérdida ósea total en la superficie mesial de la raíz vestibular y una pérdida ósea extensa de la superficie vestibular de la raíz vestibular. La raíz palatina presentaba pérdida ósea en la superficie mesial  A-B.Al examen clínico, se observó que el diente 2.4 no presentaba síntomas, sin que se iniciara ningún tratamiento endodóntico; sin embargo, presentaba una ligera decoloración. Las pruebas de vitalidad fueron negativas. El diente presentó dos raíces y no se observó sangrado al realizar el acceso endodóntico.En la etapa de tratamiento, inicialmente los dos canales radiculares se permeabilizaron con limas K #10 y #15 . La longitud de trabajo se determinó con una radiografía periapical y se verificó con la ayuda de un localizador electrónico apical (Root ZX(r), J. Morita, Japón). Después de esto, la conformación de los canales se realizó mediante limas rotatorias reciprocantes (WaveOne(r), Dentsply Maillefer, Ballaigues, Suiza) a 350 rpm, siguiendo la indicación de los fabricantes, mediante una técnica de Crown-down. Este sistema utilizó sólo la lima primary 025/08 en todos los canales. En toda la etapa de conformación del canal, se irrigó con hipoclorito de sodio al 2,5% y se permeabilizó con un lima K #15 para eliminar la capa de residuos de los canales radiculares. La obturación del canal se realizó con puntas WaveOne® con la misma conicidad de la lima primary, logrando un ajuste adecuado a nivel apical. Como sellador del canal radicular, se utilizó TopSeal(r) . Finalmente, se realizó un control radiográfico y clínico a los 7 y 14 días después del tratamiento, sin observar síntomas clínicos ni hallazgos radiográficos relacionados con algún fracaso del tratamiento.La cirugía periodontal se realizó dos semanas después del tratamiento endodóntico.El paciente fue informado sobre la necesidad de dejar de fumar y 1 hora antes de la cirugía tomó Amoxicilina de 875 mg  y Ketoprofeno 200 mg  y se aplicó enjuague bucal con clorhexidina 0.12% .El área quirúrgica se anestesió con lidocaína al 2%. Se realizó una incisión sulcular alrededor del diente involucrado, seguida de una incisión de descarga vertical en el lado mesial, con una hoja de bisturí tipo 15 . Se levantó un colgajo de espesor total, pero se extendió mínimamente en el lado distal  B. DespuUna vez que las raíces y la furcación estuvieron limpias, se colocaron coágulos de fibrina en el defecto óseo, uno encima del otro y en direcciones opuestas, y se colocaron suavemente en la superficie vestibular, cubriendo la raíz D. El cieTres meses y 6 meses después de la cirugía, los exámenes CBCT no mostraron regeneración ósea en ningún aspecto del diente  C-D. SinEl tratamiento actual para los dientes con mal pronóstico es la extracción y el reemplazo con implantes. A pesar de que los implantes son capaces de restaurar la estética y la función, el abuso de éstos ha llevado a la pérdida de dientes que no presentarían problema con una técnica menos invasiva, permaneciendo funcionalmente útiles. En este caso, un premolar superior con una lesión endoperiodontal severa se trató satisfactoriamente con un tratamiento conservador.El pronóstico de una lesión combinada endoperiodontal verdadera a menudo es pobre, especialmente cuando las lesiones periodontales, como en este caso, comprometen una pérdida extensa de la inserción ,La fibrina rica en plaquetas y leucocitos es una segunda generación de plaquetas concentradas en la que la mayoría de las plaquetas y leucocitos están contenidos dentro de una fuerte matriz de fibrina in vivo han demostrado que L-PRF promueve la regeneración periodontal y mejora el aumento óseo alveolar in vitro demuestran que L-PRF estimula la proliferación de fibroblastos, las células progenitoras periodontales y los osteoblastos . También se informa que L-PRF promueve la diferenciación de los osteoblastos y la producción de proteínas ,,-Los estudios En este caso, se observó una ganancia en el nivel de inserción clínica y una reducción en la profundidad del sondaje. Sin embargo, no se pudo observar ninguna ganancia ósea en ninguna superficie del diente. Karunakar et al. ,,,Es cuestionable si la reducción en la profundidad de sondeo obtenida en este estudio sería diferente si no hubiera uso de un concentrado de plaquetas. A pesar de los posibles efectos positivos de la L-PRF, muy pocos estudios habían tenido la intención de demostrar su efectividad clínica y radiográfica en el tratamiento de las lesiones endoperiodontales


The affiliation of the author Juan Berenguer is incorrect. The correct affiliation is: Hospital Universitario Gregorio Marañón, Madrid, Spain.