Olaparibe para tratamento de manutenção do câncer de ovário (incluindo trompas de Falópio ou peritoneal primário), seroso ou endometrioide, recém diagnosticado, de alto grau, avançado (estágio FIGO III ou IV), com mutação nos genes BRCA, sensível à quimioterapia de primeira linha, à base de platina / Olaparib for maintenance treatment of newly diagnosed, high-grade, advanced (FIGO stage III or IV) ovarian (including fallopian tube or primary peritoneal), serous or endometrioid cancer with BRCA mutations, sensitive to first-line platinum-based chemotherapy

INTRODUÇÃO: o câncer de ovário é o nono tipo mais incidente entre as mulheres no mundo, correspondendo a 3,6% de todos os cânceres femininos em 2020. No Brasil, o número estimado de novos casos de câncer de ovário, para cada ano do triênio de 2023 a 2025, é de 7.310 casos, correspondendo a um risco estimado de 6,62 casos novos a cada 100 mil mulheres. Os genes BRCA1 e BRCA2 estão entre os genes mais frequentemente mutados no carcinoma seroso ovariano de alto grau, responsável pela grande maioria das mortes por câncer de ovário. Aproximadamente 25% das mulheres diagnosticadas com carcinoma seroso de alto grau apresentam mutações germinativas deletérias em BRCA1 ou BRCA2. Os inibidores da enzima poli-ADP-ribose polimerase (iPARP) têm sido estudados pela sua atividade em pacientes com câncer de ovário, principalmente naquelas com mutação em BRCA1 e 2. No Brasil, apenas olaparibe e niraparibe possuem registro na Agência Nacional de Vigilância Sanitária (Anvisa). Até a chegada destes medicamentos, o tratamento das pacientes se baseava em quimioterapia com derivados de platina, seguido por vigilância ativa. PERGUNTA: olaparibe é seguro e eficaz para o tratamento de pacientes adultas com carcinoma de ovário (incluindo trompa de

Risk of endometrial cancer after RRSO in BRCA 1/2 carriers: a multicentre cohort study.

OBJECTIVE: To know the risk of endometrial cancer (EC) in a population of women with BRCA 1/2 pathogenic or likely pathogenic variants after risk-reducing salpingo-oophorectomy (RRSO). METHODS: The study cohort included data from 857 women with BRCA mutations who underwent RRSO visited four hospitals in Catalonia, Spain, from January 1, 1999 to April 30, 2019. Standardized incidence ratio (SIR) of EC was calculated in these patients using data from a regional population-based cancer registry. RESULTS: After RRSO, eight cases of EC were identified. Four in BRCA 1 carriers and four in BRCA2 carriers. The expected number of cases of EC was 3.67 cases, with a SIR of 2.18 and a 95% CI (0.93-3.95). CONCLUSIONS: In our cohort, the risk of EC in BRCA1/2 carriers after RRSO is not greater than expected. Hysterectomy is not routinely recommended for these patients.

Characterization of Patients with Breast Cancer and National Comprehensive Cancer Network Criteria for Performing BRCA1 and BRCA2 Genetic Test / Caracterização de Pacientes com Câncer de Mama e Critérios da National Comprehensive Cancer Network para Realização do Teste Genético BRCA1 e BRCA22 / Caracterización de Pacientes con Cáncer de Mama y Criterios de la National Comprehensive Cancer Network para Realizar Prueba Genética de BRCA1 y BRCA

Introduction: Approximately 10% of breast cancer cases are attibutable to germinative mutations in susceptibility genes, including BRCA1, BRCA2 and others. The National Comprehensive Cancer Network (NCCN) recommends screening women with breast cancer for mutations in BRCA1/2 in defined scenarios. However, these genetic tests are unavailable at the Brazilian Public Health System (SUS). Objective: This study aimed to characterize women with breast cancer and define the criteria for performing BRCA1/2 test. Method: Quantitative, descriptive, analytic, and retrospective study. Medical records of women diagnosed by SUS with breast cancer between January 2016 and December 2018 were analyzed through the software JAMOVI (version 2.3 - 2022). Results: A total of 245 women were diagnosed. According to NCCN guidelines, 97 women met the criteria for performing BRCA1/2 test, with mean age of 47 years old, predominantly white (90,7%), with comorbidities (55.6%), premenopausal (59.8%), diagnosed in early stages 0 - IIb (68.2%) and 48.4% had familial history of breast cancer. Most frequent histology and molecular subtype was invasive ductal carcinoma (87.2%) and luminal type (59.8%). Conclusion: A significant number of women diagnosed by SUS had indication for BRCA1/2 test. These women are younger, had fewer comorbidities, not menopausal, and differ in terms of the molecular subtype when compared with those without indication for performing the test

Introdução: Aproximadamente 10% dos casos de câncer de mama são atribuíveis a mutações germinativas em genes de suscetibilidade, incluindo BRCA1 e BRCA2. A National Comprehensive Cancer Network (NCCN) recomenda a triagem de mulheres com câncer de mama para mutações em BRCA1/2 em cenários definidos. No entanto, esses testes genéticos não estão disponíveis no Sistema Único de Saúde (SUS). Objetivo: Caracterizar as mulheres com câncer de mama e definir os critérios para realização do teste BRCA1/2. Método: Estudo quantitativo, descritivo, analítico e retrospectivo. Foram analisadosprontuários de mulheres com diagnóstico de câncer de mama pelo SUS entre janeiro de 2016 e dezembro de 2018, por meio do software JAMOVI (versão 2.3 - 2022). Resultados: Foram diagnosticadas 245 mulheres. De acordo com as diretrizes da NCCN, 97 mulheres atenderam aos critérios para realizar o teste BRCA1/2, com idade média de 47 anos, predominantemente brancas (90,7%), com comorbidades (55,6%), na pré-menopausa (59,8%), diagnosticadas nos estágios iniciais 0 - IIb (68, 2%), e 48,4% tinham histórico familiar de câncer de mama. A histologia e o subtipo molecular mais frequentes foram carcinoma ductal invasivo (87,2%) e tipo luminal (59,8%). Conclusão: Considerando os critérios da NCCN, um número significativo de mulheres diagnosticadas pelo SUS teve indicação para realização do teste BRCA1/2. Essas mulheres são mais jovens, têm menos comorbidades, estão em período pré-menopausa mais frequentemente e diferem quanto ao subtipo molecular quando comparadas àquelas sem indicação de realização do exame. Palavras-chave: neoplasias da mama; neoplasias ovarianas; síndrome hereditária de câncer de mama e ovário; genes, BRCA1

Introducción: Aproximadamente el 10% de los casos de cáncer de mama son atribuibles a mutaciones germinales en genes de susceptibilidad, incluidos BRCA1 y BRCA2. La National Comprehensive Cancer Network (NCCN) recomienda la detección de mutaciones BRCA1/2 en mujeres con cáncer de mama en entornos definidos. Sin embargo, estas pruebas genéticas no están disponibles en el Sistema Único de Salud (SUS). Objetivo: Caracterizar mujeres con cáncer de mama y definir los criterios para la realización de la prueba BRCA1/2. Método: Estudio cuantitativo, descriptivo, analítico y retrospectivo. Las historias clínicas de las mujeres diagnosticadas con cáncer de mama entre enero de 2016 y diciembre de 2018, usuarias del SUS, fueron analizadas mediante el software JAMOVI (versión 2.3 - 2022). Resultados: 245 mujeres fueron diagnosticadas. Según las pautas de NCCN, 97 mujeres cumplieron con los criterios para someterse a la prueba BRCA1/2. Las mujeres con indicación para la prueba tenían un promedio de edad de 47 años, eran predominantemente blancas (90,7%), con comorbilidades (55,6%), premenopáusicas (59,8%), diagnosticadas en estadios tempranos 0 - IIb (68,2%) y 48,4% tenía antecedentes familiares de cáncer de mama. Los subtipos histológicos y moleculares más frecuentes fueron el carcinoma ductal invasivo (87,2%) y el tipo luminal (59,8%). Conclusión: Considerando los criterios de la NCCN, un número significativo de mujeres, usuarias del SUS, fueron designadas para hacer la prueba BRCA1/2. Estas mujeres son más jóvenes, tienen menos comorbilidades, están en el periodo de la premenopausia con mayor frecuencia y difieren en el subtipo molecular en comparación con aquellas sin orden de realizarse la prueba

Tamizaje para cáncer de mama con resonancia magnética en mujeres portadoras de mutaciones BRCA y no-BRCA / Magnetic resonance imaging breast cancer screening in women who are carriers of BRCA and non-BRCA gene mutations / Rastreamento de câncer de mama com ressonância magnética em mulheres com mutações BRCA e não BRCA

Introducción: la predisposición hereditaria causada por mutaciones patogénicas de la línea germinal (MPG) explica hasta el 10% de los cánceres de mama. Para reducir su impacto en mujeres mutadas se han propuesto diferentes estrategias, tales como las cirugías reductoras de riesgo o el screening con resonancia magnética (RM) de mamas. Métodos: en este estudio observacional retrospectivo se analizaron los registros de mujeres portadoras de MPG para evaluar las diferentes acciones tomadas luego del test genético. A las pacientes no mastectomizadas se les recomendó ingresar a un programa anual de cribado con RM y se evaluó el porcentaje de adherencia al plan, el número de biopsias efectuadas y el número de cánceres de mama detectados. Resultados: se incluyeron 134 mujeres con MPG, con una distribución en tercios iguales de los genes BRCA1, BRCA2 y genes no-BRCA. Entre las mutadas con indicación de seguimiento, 64% ingresaron al programa de cribado con RM. Las razones que llevaron a las mujeres a no ingresar al programa de seguimiento fueron: la oposición del médico tratante (53%), oposición de la paciente (38%), y falta de recursos (9%). Se realizaron seis biopsias por hallazgos en la RM entre las cuales se detectó un cáncer de mama. La incidencia de cáncer fue de 11 cada 1.000 mujeres-años de riesgo. Conclusiones: nuestro programa de seguimiento con RM de pacientes mutadas logró captar un porcentaje alto de candidatas. Una proporción significativa de las mujeres no ingresó debido la desaprobación del médico tratante o de la propia paciente. La evidencia obtenida revela una necesidad imperiosa de reforzar los programas educativos que destaquen la importancia del seguimiento con RM de las pacientes de alto riesgo en nuestro país.

Summary: Introduction: genetic propensity caused by germline pathogenic mutations explain up to 10% of breast cancer cases. Different strategies have been proposed to reduce its impact on women who are carriers of mutations, such as risk-reducing surgeries or breast magnetic resonance screening. Method: observational, retrospective study analyzing the medical records of women who are carriers of germline pathogenic mutations to assess the different measures taken after the genetic test. Non-mastectomized patients were advised to join an annual MRI screening program and the percentage of adherence to plan was evaluated, along with biopsies performed and the number of breast cancer cases detected. Results: 134 women carriers of germline pathogenic mutations were included in the study, with equal distributions in thirds for BRCA1, BRCA2 and non-BRCA genes. 64% of carriers of mutations who were subject to follow-up checkups joined the RMI screening program. The reasons why women failed to join the follow-up program were: the treating physician objected to the program (53%), the patients opposed to program (38%) and lack of resources (9%). Six biopsies were performed as a consequence of findings in the RMI, and one case of breast cancer was detected. Cancer incidence was 11 out of 1000 women - risk years. Conclusions: our RMI follow-up program for women who are carriers of mutations managed to attract a high percentage of candidates. A significant amount of women failed to join the program because of their treating physician's or their own disapproval. Evidence obtained reveals the dramatic need to reinforce educational programs that emphasize on the importance of RMI follow-up of high risk patients in our country.

Introdução: a predisposição hereditária causada por mutações germinativas patogênicas (GMP) explica até 10% dos cânceres de mama. Para reduzir seu impacto em mulheres com mutações, diferentes estratégias têm sido propostas, como cirurgias de redução de risco ou ressonância magnética (RM) das mamas. Métodos: neste estudo observacional retrospectivo, os registros de mulheres portadoras de MPG foram analisados para avaliar as diferentes ações tomadas após o teste genético. Pacientes não mastectomizadas foram recomendadas a entrar em um programa anual de triagem por ressonância magnética e foram avaliados o percentual de adesão ao plano, o número de biópsias realizadas e o número de cânceres de mama detectados. Resultados: foram incluídas 134 mulheres com MPG, com uma distribuição de terços iguais dos genes BRCA1, BRCA2 e não-BRCA. Entre as mulheres com mutações com indicação de acompanhamento, 64% entraram no programa de triagem por ressonância magnética. Os motivos que levaram as mulheres a não ingressarem ao programa de acompanhamento foram: oposição do médico assistente (53%), oposição da paciente (38%) e falta de recursos (9%). Seis biópsias foram realizadas devido a achados de ressonância magnética, entre os quais foi detectado um câncer de mama. A incidência de câncer foi de 11 por 1.000 mulheres-ano de risco. Conclusões: nosso programa de acompanhamento de ressonância magnética para pacientes com mutação conseguiu capturar uma alta porcentagem de candidatas. Uma proporção significativa de mulheres não entrou devido à falta de aprovação do médico assistente ou da própria paciente. As evidências obtidas revelam a necessidade urgente de reforçar programas educacionais que destaquem a importância do acompanhamento por RM de pacientes de alto risco no Uruguai.

Assessment of small in-frame indels and C-terminal nonsense variants of BRCA1 using a validated functional assay.

BRCA1 (Breast Cancer 1, early onset) is linked to breast and ovarian cancer predisposition. Still, the risks conferred by a significant portion of BRCA1 variants identified in the population remains unknown. Most of these variants of uncertain significance are missense alterations. However, the functional implications of small in-frame deletions and/or insertions (indels) are also difficult to predict. Our group has previously evaluated the functional impact of 347 missense variants using an extensively validated transcriptional activity assay. Here we show a systematic assessment of 30 naturally occurring in-frame indels located at the C-terminal region of BRCA1. We identified positions sensitive and tolerant to alterations, expanding the knowledge of structural determinants of BRCA1 function. We further designed and assessed the impact of four single codon deletions in the tBRCT linker region and six nonsense variants at the C-terminus end of BRCA1. Amino acid substitutions, deletions or insertions in the disordered region do not significantly impact activity and are not likely to constitute pathogenic alleles. On the other hand, a sizeable fraction of in-frame indels at the BRCT domain significantly impact function. We then use a Bayesian integrative statistical model to derive the probability of pathogenicity for each variant. Our data highlights the importance of assessing the impact of small in-frame indels in BRCA1 to improve risk assessment and clinical decisions for carriers.

Implicações éticas dos testes genéticos de suscetibilidade ao câncer de mama / Ethical implications of genetic testing of susceptibility to breast cancer / Implicaciones éticas de las pruebas genéticas de susceptibilidad al cáncer de mama

Resumo O câncer de mama representa um problema de saúde pública por ser a neoplasia maligna de maior incidência em mulheres no mundo. A forma hereditária corresponde a cerca de 5% a 10% de todos os casos e está diretamente relacionada à herança de mutações genéticas, sendo as principais nos genes supressores de tumor BRCA1 e BRCA2. A identificação dessas mutações é de extrema importância pelo elevado risco de desenvolvimento de câncer de mama nessa população, permitindo estratégias de rastreamento diferenciado e adoção de medidas de redução de risco. Entretanto, é importante e necessário refletir sobre os aspectos éticos relacionados ao uso indiscriminado de testes genéticos. O objetivo deste trabalho foi avaliar o conhecimento e a opinião de médicos de um centro de referência oncológico sobre a indicação dos testes genéticos de suscetibilidade ao câncer de mama mediante dilemas éticos aos quais são submetidos na prática médica.

Abstract Breast cancer is a public health problem because it is the malignant neoplasm with the highest incidence in women worldwide. The hereditary form corresponds to about 5% to 10% of all cases and is directly related to the inheritance of genetic mutations. The main ones occur in the BRCA1 and BRCA2 tumor suppressor genes. The identification of these mutations is extremely important because of the high risk of breast cancer development in this population, allowing differentiated screening strategies and the adoption of risk reduction measures. However, reflections on the ethical aspects related to the indiscriminate use of genetic testing are important and necessary. The objective of this study was to evaluate the knowledge and opinion of physicians of an oncology reference center on the indication of genetic tests for susceptibility to breast cancer given the ethical dilemmas to which they are submitted in medical practice.

Resumen El cáncer de mama representa un problema de salud pública, ya que es la neoplasia maligna con mayor incidencia en las mujeres de todo el mundo. La forma hereditaria corresponde a entre el 5% y el 10% de todos los casos y está directamente relacionada con la herencia de mutaciones genéticas, y las principales se dan en los genes supresores de tumores BRCA1 y BRCA2. La identificación de estas mutaciones es extremadamente importante debido al elevado riesgo de esta población de desarrollar cáncer de mama, además de permitir estrategias de rastreo diferenciadas y la adopción de medidas de reducción del riesgo. Sin embargo, es importante y necesario reflexionar sobre los aspectos éticos relacionados con el uso indiscriminado de las pruebas genéticas. El objetivo de este estudio fue evaluar el conocimiento y la opinión de los médicos de un centro oncológico de referencia sobre la indicación de las pruebas genéticas de susceptibilidad al cáncer de mama mediante los dilemas éticos a los que se ven sometidos en la práctica médica.

Population-based screening of Uruguayan Ashkenazi Jews for recurrent BRCA1 and BRCA2 pathogenic sequence variants.

In Ashkenazi Jews (AJ) three recurring pathogenic sequence variants (PSVs) are detected in ~2.5% of the general population in the BRCA1 (c.68_69del = 185delAG, c.5266dup = 5382insC), and BRCA2 (c.5946del = 6174delT). Population-based screening for these PSVs in AJ women is part of the health basket in Israel. To assess the feasibility and outcome of BRCA genotyping in the Jewish population of Uruguay, AJ in the greater Montevideo area were recruited using ethically approved protocol and without pretest counseling were genotyped for the three predominant AJ PSVs in the BRCA genes. Independently confirmed PSV carriers were counseled, and genetic testing was offered to additional family members. Overall, 327 participants were enrolled: 312 (95%) female, 261 (80%) had all four grandparents AJ, and 14 (4%) women were breast cancer survivors with a mean age ± standard deviation (SD) 50 ± 11.5 years. The BRCA1 c.68_69del PSV was detected in three cancer free participants (0.92%, CI 95% 0.31-2.6), all with a suggestive family history. No carriers of the other two recurrent PSVs were detected. Online oncogenetic counseling was provided for all carriers. In conclusion, the rate of the BRCA1 c.68_69del PSV was similar with the rate in other AJ communities. AJ population BRCA genotyping screens in Uruguay seem feasible and should be promoted.

Venous thromboembolism incidence in cancer patients with germline BRCA mutations.

BACKGROUND: Germline BRCA (gBRCA) mutations predispose to an increased risk of breast and ovarian cancer among other neoplasms. Recently, several genomic alterations such as ALK and ROS-1 rearrangements have been described as molecular drivers of venous thromboembolism (VTE). The association of gBRCA mutations and VTE is unknown. MATERIALS AND METHODS: We performed an observational, retrospective, single-center study to determine the VTE incidence in consecutive patients with gBRCA mutations and cancer diagnosis attended in the multidisciplinary heredofamiliar cancer unit (HFCU) of Hospital General Universitario Gregorio Marañón, Spain, from 2010 to 2019. RESULTS: One-hundred and forty-one patients were included in the analysis. The overall VTE incidence was 12.8%. The highest incidence was reported in ovarian cancer patients (20.0%), followed by patients with both ovarian and breast cancers (16.6%) and the lowest was found in breast cancer (4.9%). No difference in the type of gBRCA mutation (1 or 2) in terms of VTE rate was observed. Sixty one percent of the patients were receiving anti-cancer therapy at the time of VTE diagnosis and the majority of the events (83.3%) were diagnosed in ambulatory setting. Khorana score was of limited value to detect high-risk patients. CONCLUSIONS: The VTE incidence observed in our study is consistent with prior data described in general population of breast and ovarian cancer. The risk of VTE in these patients seems to be driven by the type of cancer. We have not observed any significant interaction of gBRCA mutation status and cancer-associated thrombosis.

Correlation between the number of false positive variants and the quality of results using Ion Torrent PGM™ sequencing to screen BRCA genes. / Correlación entre el número de variantes de falsos positivos y la calidad de los resultados en la secuenciación con Ion Torrent PGM™ para seleccionar genes BRCA

INTRODUCTION: Next Generation Sequencing (NGS) is cost-effective and a faster method to study genes, but its protocol is challenging. OBJECTIVE: To analyze different adjustments to the protocol for screening the BRCA genes using Ion Torrent PGM sequencing and correlate the results with the number of false positive (FP) variants. MATERIAL AND METHODS: We conducted a library preparation process and analyzed the number of FP InDels, the library concentration, the number of cycles in the target amplification step, the purity of the nucleic acid, the input, and the number of samples/Ion 314 chips in association with the results obtained by NGS. RESULTS: We carried out 51 reactions and nine adjustments of protocols and observed eight FP InDels in homopolymer regions. No FP Single-Nucleotide Polymorphism variant was observed; 67.5% of protocol variables were jointly associated with the quality of the results obtained (p<0.05). The number of FP InDels decreased when the quality of results increased. CONCLUSION: The Ion AmpliSeq BRCA1/BRCA2 Community Panel had a better performance using four samples per Ion-314 chip instead of eight and the optimum number of cycles in the amplification step, even when using high-quality DNA, was 23. We observed better results with the manual equalization process and not using the Ion Library Equalizer kit. These adjustments provided a higher coverage of the variants and fewer artifacts (6.7-fold). Laboratories must perform internal validation because FP InDel variants can vary according to the quality of results while the NGS assay should be validated with Sanger.

Introducción. La secuenciación de nueva generación es un método rentable y rápido para el estudio de los genes, pero su protocolo entraña desafíos. Objetivo. Investigar diferentes ajustes del protocolo de selección de los genes BRCA mediante secuenciación de Ion Torrent PGM™ y correlacionar los resultados con el número de variantes de falso positivo. Materiales y métodos. El proceso de preparación de la biblioteca, el número de falsos positivos InDels, la concentración de la biblioteca, el número de ciclos en el paso de amplificación de objetivos, la pureza del ácido nucleico, la entrada y el número de muestras por chip del Ion-314 se analizaron en asociación con los resultados obtenidos por secuenciación de nueva generación secuenciación de nueva generación. Resultados. Se hicieron 51 reacciones y nueve ajustes de los protocolos, y se observaron ocho falsos positivos InDels en las regiones de homopolímeros. No se observó ninguna variante de polimorfismo de nucleótido simple falso positivo. En 67,5 % de los casos, las variables de protocolo en su conjunto se asociaron con la calidad de los resultados obtenidos (p<0,05). El número de falsos positivos InDels disminuyó al aumentar la calidad de los resultados. Conclusiones. El panel comunitario Ion AmpliSeq BRCA1/BRCA2 tuvo un mejor rendimiento, con cuatro muestras por chip Ion-314 en lugar de ocho, y el número de ciclos en el paso de amplificación, incluso con ADN de alta calidad, fue mejor con 23. Se observaron mejores resultados con el proceso de ecualización manual y sin el uso del kit Ion Library Equalizer. Estos ajustes proporcionaron una mayor cobertura de las variantes y menos artefactos. Los laboratorios deben realizar la validación interna porque las variantes de falsos positivos InDel pueden variar según la calidad de los resultados. La secuenciación de próxima generación debe validarse con Sanger.

Presentación de Caso Clínico: Carcinoma de mama luego de mastectomía de reducción de riesgo en paciente con mutación BRCA1 / Case report: Breast carcinoma after risk reducing mastectomy in a BRCA+ patient

Las pacientes sanas portadoras de mutaciones patogénicas del gen BRCA1 tiene un riesgo muy elevado de desarrollar cáncer de mama y ovario, con cifras reportadas que ascienden a 84% y 59% respec- tivamente.¹ Esto motivó a implementar distintas medidas de segui- miento y de reducción de riesgo, como la mastectomía bilateral de reducción de riesgo (MRR) y la salpingo-ooforectomía de reducción de riesgo (SORR). Pero aún así, distintas experiencias publicadas re- fieren que en pacientes portadoras de mutacion BRCA1 sometidas a MRR, el riesgo subsiguiente de desarrollar un cáncer primario de mama puede llegar al 10%.2 Por otro lado, las técnicas de mastectomías en general han experi- mentado modificaciones en menos, conservando las estructuras te- gumentarias y el tejido adiposo que la recubre. Últimamente se ha expuesto que, con la guía de las imágenes, se puede resecar menos tejido aun, fundamentalmente en los contornos mamarios donde se observa tejido adiposo y nada (o muy escaso) tejido glandular.3 Esta recomendación hoy es empírica, ya que no hay evidencia de la segu- ridad oncológica de conservar estos tejidos

Correlation between the number of false positive variants and the quality of results using Ion Torrent PGM™ sequencing to screen BRCA genes / Correlación entre el número de variantes de falsos positivos y la calidad de los resultados en la secuenciación con Ion Torrent PGM™ para seleccionar genes BRCA

Abstract | Introduction: Next Generation Sequencing (NGS) is cost-effective and a faster method to study genes, but its protocol is challenging. Objective: To analyze different adjustments to the protocol for screening the BRCA genes using Ion Torrent PGM sequencing and correlate the results with the number of false positive (FP) variants. Materials and methods: We conducted a library preparation process and analyzed the number of FP InDels, the library concentration, the number of cycles in the target amplification step, the purity of the nucleic acid, the input, and the number of samples/Ion 314 chips in association with the results obtained by NGS. Results: We carried out 51 reactions and nine adjustments of protocols and observed eight FP InDels in homopolymer regions. No FP Single-Nucleotide Polymorphism variant was observed; 67.5% of protocol variables were jointly associated with the quality of the results obtained (p<0.05). The number of FP InDels decreased when the quality of results increased. Conclusion: The Ion AmpliSeq BRCA1/BRCA2 Community Panel had a better performance using four samples per Ion-314 chip instead of eight and the optimum number of cycles in the amplification step, even when using high-quality DNA, was 23. We observed better results with the manual equalization process and not using the Ion Library Equalizer kit. These adjustments provided a higher coverage of the variants and fewer artifacts (6.7-fold). Laboratories must perform internal validation because FP InDel variants can vary according to the quality of results while the NGS assay should be validated with Sanger.

Resumen | Introducción. La secuenciación de nueva generación es un método rentable y rápido para el estudio de los genes, pero su protocolo entraña desafíos. Objetivo. Investigar diferentes ajustes del protocolo de selección de los genes BRCAmediante secuenciación de Ion Torrent PGM™ y correlacionar los resultados con el número de variantes de falso positivo. Materiales y métodos. El proceso de preparación de la biblioteca, el número de falsos positivos InDels, la concentración de la biblioteca, el número de ciclos en el paso de amplificación de objetivos, la pureza del ácido nucleico, la entrada y el número de muestras por chip del Ion-314 se analizaron en asociación con los resultados obtenidos por secuenciación de nueva generación secuenciación de nueva generación. Resultados. Se hicieron 51 reacciones y nueve ajustes de los protocolos, y se observaron ocho falsos positivos InDels en las regiones de homopolímeros. No se observó ninguna variante de polimorfismo de nucleótido simple falso positivo. En 67,5 % de los casos, las variables de protocolo en su conjunto se asociaron con la calidad de los resultados obtenidos (p<0,05). El número de falsos positivos InDels disminuyó al aumentar la calidad de los resultados. Conclusiones. El panel comunitario Ion AmpliSeq BRCA1/BRCA2 tuvo un mejor rendimiento, con cuatro muestras por chip Ion-314 en lugar de ocho, y el número de ciclos en el paso de amplificación, incluso con ADN de alta calidad, fue mejor con 23. Se observaron mejores resultados con el proceso de ecualización manual y sin el uso del kit Ion Library Equalizer. Estos ajustes proporcionaron una mayor cobertura de las variantes y menos artefactos. Los laboratorios deben realizar la validación interna porque las variantes de falsos positivos InDel pueden variar según la calidad de los resultados. La secuenciación de próxima generación debe validarse con Sanger.

Development and Pilot Implementation of the Genomic Risk Assessment for Cancer Implementation and Sustainment (GRACIAS) Intervention in Mexico.

PURPOSE: Genomic cancer risk assessment (GCRA) is standard-of-care practice that uses genomic tools to identify individuals with increased cancer risk, enabling screening for early detection and cancer prevention interventions. GCRA is not available in most of Mexico, where breast cancer (BC) is the leading cause of cancer death and ovarian cancer has a high mortality rate. METHODS: Guided by an implementation science framework, we piloted the Genomic Risk Assessment for Cancer Implementation and Sustainment (GRACIAS) intervention, combining GCRA training, practice support, and low-cost BRCA1/2 (BRCA) gene testing at four centers in Mexico. The RE-AIM model was adapted to evaluate GRACIAS intervention outcomes, including reach, the proportion of new patients meeting adapted National Comprehensive Cancer Network criteria who participated in GCRA. Barriers to GCRA were identified through roundtable sessions and semistructured interviews. RESULTS: Eleven clinicians were trained across four sites. Mean pre-post knowledge score increased from 60% to 67.2% (range 53%-86%). GCRA self-efficacy scores increased by 31% (95% CI, 6.47 to 55.54; P = .02). Participant feedback recommended Spanish content to improve learning. GRACIAS promoted reach at all sites: 77% in Universidad de Guadalajara, 86% in Instituto Nacional de Cancerología, 90% in Tecnológico de Monterrey, and 77% in Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán. Overall, a pathogenic BRCA variant was identified in 15.6% (195 of 1,253) of patients. All trainees continue to provide GCRA and address barriers to care. CONCLUSION: We describe the first project to use implementation science methods to develop and deliver an innovative multicomponent implementation intervention, combining low-cost BRCA testing, comprehensive GCRA training, and practice support in Mexico. Scale-up of the GRACIAS intervention will promote risk-appropriate care, cancer prevention, and reduction in related mortality.

SEOM clinical guidelines for the treatment of advanced prostate cancer (2020).

The treatment of advanced prostate cancer has evolved due to recent advances in molecular research and new drug development. Dynamic aberrations in the androgen receptor, DNA repair genes, PTEN-PI3K, and other pathways drive the behavior of advanced prostate cancer allowing a better selection of therapies in each patient. Tumor testing for BRCA1 and BRCA2 is recommended for patients with metastatic prostate cancer, also considering a broad panel to guide decisions and genetic counseling. In symptomatic metastatic patients, castration should be stared to palliate symptoms and prolong survival. In high-risk or high-volume metastatic hormone-naïve patients, castration should be combined with docetaxel, abiraterone, enzalutamide or apalutamide. Radiotherapy to the primary tumor combined with systemic therapy is recommended in low-volume mHNPC patients. In patients with non-metastatic castration-resistant tumors, risk stratification can define the frequency of imaging. Adding enzalutamide, darolutamide or apalutamide to these patients prolongs metastasis-free and overall survival, but potential adverse events need to be taken into consideration. The choice of docetaxel, abiraterone or enzalutamide for treating metastatic castration-resistant patients depends on previous therapies, with cabazitaxel being also recommended after docetaxel. Olaparib is recommended in BRCA1/BRCA2 mutated castration-resistant patients after progression on at least one new hormonal therapy. Aggressive variants of prostate cancer respond to platinum-based chemotherapy. To optimize treatment efficiency, oncologists should incorporate all of these advances into an overall therapeutic strategy.

Comprehensive study for BRCA1 and BRCA2 entire coding regions in breast cancer.

BACKGROUND: About 5-10% of incidences of breast cancers have been reported as a result of germline mutations of BRCA genes. However, the mutational spectrum of BRCA1 and BRCA2 genes among breast cancer Saudi women patients is inadequate at present. Therefore, the present study aimed to report the specific germinal mutation of BRCA1 and BRCA2 in the entire coding regions, to investigate the prevalence rate of BRCA1 & BRCA2 mutations among Saudi women and the effect of these mutations, both benign and malignant tumors. METHODOLOGY: A total of 270 tissue samples of benign and malignant breast tumors were collected from Saudi women patients, Riyadh, Saudi Arabia. Examination of BRCA1 and BRCA2 germline mutations was performed using heteroduplex DNA analysis (HDA) or single-stranded conformation analysis (SSCA). 177 breast cancer women with malignant tumors and 93 with benign tumors were enrolled in the study. A total of 62 out of 177 breast cancer patients carried a BRCA1 or BRCA2 mutation (54 BRCA1 and 8 BRCA2). The analysis was done using the Sanger sequence assay. RESULTS: Point and frameshift mutations through the entire coding area of the two genes indicated that all the mutations were germline alterations and of early-onset breast cancers. The mean ages of diagnosed breast cancer women for BRCA1 and BRCA2 mutation carriers were 36.3 (± 3.5) and 37.9 (± 3.7) years, whereas that of benign control was 35(± 2.5) years. CONCLUSION: Point and frameshift mutations across the entire coding region of BRCA1 and BRCA2 are responsible for many breast cancers cases.

Influence of germline BRCA genotype on the survival of patients with triple-negative breast cancer.

The presence of BRCA pathogenic variants (PVs) in triple-negative breast cancer (TNBC) is associated with a distinctive genomic profile that makes the tumor particularly susceptible to DNA-damaging treatments. However, patients with BRCA PVs can develop treatment resistance through the appearance of reversion mutations and restored BRCA expression. As copy-number variants (CNV) could be less susceptible to reversion mutations than point mutations, we hypothesize that carriers of BRCA CNVs may have improved survival after treatment compared to carriers of other BRCA PVs or BRCA wild-type. Women diagnosed with stage I-III TNBC at ≤50 years at a cancer center in Mexico City were screened for BRCA PVs using a recurrent PV assay (HISPANEL; 77% sensitivity). The recurrence-free (RFS) and overall survival (OS) were compared according to mutational status. Among 180 women, 17 (9%) were carriers of BRCA1 ex9-12del CNV and 26 (14%) of other BRCA PVs. RFS at ten years for the whole cohort was 79.2% (95% CI 72.3-84.6%), with no significant differences according to mutational status. 10-year OS for the entire cohort was 85.3% (95%CI: 78.7-90.0%), with BRCA CNV carriers demonstrating numerically superior OS rates other PV carriers and non-carriers (100% vs. 78.6% and 84.7%; log-rank p=0.037 and p=0.051, respectively). This study suggests that BRCA1 ex9-12del CNV carriers with TNBC may have a better OS, and supports the hypothesis that the genotype of BRCA PVs may influence survival by limiting treatment resistance mediated by reversion mutations among CNV carriers.

Avaliação do silenciamento de BRCA1 por mecanismos epigenéticos e genéticos em câncer de mama triplo-negativo metastático e sua associação com ancestralidade e prognóstico / Evaluation of BRCA1 silencing by epigenetic and genetic mechanisms in metastatic triple-negative breast cancer and its association with ancestry and prognosis

Introdução e contexto: O câncer de mama do subtipo triplo-negativo (TN) representa cerca de 15% de todos os cânceres de mama e é negativo para expressão dos receptores de estrógeno e progesterona e superexpressão/amplificação de HER2. É mais prevalente em mulheres jovens e negras, apresenta maior risco de desenvolvimento de metástases e pior prognóstico. Parte significativa de tumores TN apresentam deficiência na via de reparo de recombinação homóloga (DRH), resultado de mecanismos genéticos, como variantes P/PP germinativas em BRCA1/2, sendo mutação em BRCA1 a mais frequente, e/ou silenciamento epigenético de BRCA1 e RAD51C no tumor. Não há dados sobre o status de mutação germinativa em BRCA1/2 em relação ao padrão metastático dos TN e nem em relação a mecanismos envolvidos na DRH em mulheres brasileiras. Objetivos: Avaliar o status de mutação germinativa nos genes BRCA1 e BRCA2 em mulheres com tumores TN e sua associação com padrão metastático, mecanismos de DRH e ancestralidade. Metodologia: Estudo retrospectivo unicêntrico. A avaliação de BRCA1/2 foi realizada a partir do DNA de leucócito/tecido não tumoral, e quando não disponíveis, tecido tumoral fresco ou emblocado em parafina. O status de metilação de BRCA1 e RAD51C foi determinado por sequenciamento de próxima geração (NGS) de amplicon usando DNA tumoral tratado com bissulfito de sódio; o critério de classificação foi >40% hipermetilado; entre ≥20% à <40% intermediário. A análise de ancestralidade foi realizada por eletroforese capilar com análise de fragmentos ou por genotipagem usando o array PMDA, utilizando DNA de leucócito. Informações clínicas de toda a casuística foram obtidas dos prontuários eletrônicos disponíveis na Instituição. Resultados: Foram incluídos 400 casos de tumores TN, 132 com diagnóstico de metástase e 268 sem metástase. A caracterização genética de BRCA1/2 foi obtida para 399 casos, sendo que 21,6% (86/399) apresentaram variantes patogênicas (P) ou provavelmente patogênicas (PP), onde 81,4% (70/86) das portadoras de variantes P/PP eram portadoras de variante P/PP em BRCA1, correspondento a 17,5% dos casos avaliados (70/399), e 18,6% (16/86) portadoras de variante P/PP em BRCA2, correspondendo a 4,0% dos casos (16/399). A média de idade ao diagnóstico foi menor para portadoras de variantes P/PP em BRCA1 que apresentaram idade de 41,7 anos, sendo que a idade das portadoras de variantes P/PP em BRCA2 e mulheres sem variantes P/PP ou VUS (do inglês, variant of uncertain significance) foi de 47,1 e 47,9 anos, respectivamente. Dos casos metastáticos, 131 tiveram BRCA1/2 testados, sendo 12,2% (16/131) BRCA1-P/PP e 6,1% (8/131) BRCA2-P/PP. Em relação ao padrão metastático dos tumores TN, os sítios mais acometidos em BRCA1-P/PP e sem variantes P/PP foram pulmão (50% e 37%, respectivamente) e SNC (31% e 23%, respectivamente), enquanto que para BRCA2-P/PP, os sítios mais acometidos foram SNC (62%) e ossos (37%). Em análise com modelos de regressão logística multivariada, observou-se que o risco para desenvolver metástase no grupo BRCA2-P/PP foi significativamente maior, chegando a quase 4 vezes mais para desenvolver em SNC (p=0,035) quando comparado com o grupo sem variantes P/PP e perto da significância (p=0,067) para desenvolver em ossos. O grupo BRCA1-P/PP não apresentou risco aumentado quando comparado com o grupo sem variantes P/PP. A análise de metilação nos genes BRCA1/RAD51C foi realizada no tumor primário para 177 casos, apresentando hipermetilação de 55,4% em um dos dois genes, sendo 43,5% (77/177) hipermetilados em BRCA1; e para RAD51C, 11,9% (21/177) hipermetilados. Já a análise de metilação nos tumores metastáticos foi realizada para 25 casos, sendo 32% hipermetilados em um dos dois genes, sendo 20% (5/25) hipermetilados em BRCA1 e 12% (3/25) em RAD51C. A análise de ancestralidade foi realizada para 216 casos, onde a maioria (68%) foi classificada como ancestralidade predominantemente europeia; foi investigada se havia associação entre as ancestralidades e o evento genético ou epigenético subjacente a DRH, porém não ficou evidente nenhuma associação. Conclusões: BRCA1 confirmou ser o gene que mais frequentemente apresenta variantes P/PP em mulheres com CMTN, principalmente em mulheres diagnosticadas em idade jovem. A maioria dos casos cujo DNA tumoral apresentou hipermetilação em um dos dois genes investigados (BRCA1 e RAD51C) foram em mulheres jovens, sendo o gene BRCA1 mais frequentemente metilado. Observou-se que há um risco elevado de até 4 vezes para desenvolver metástase no SNC no grupo de mulheres com BRCA2-PP. Não foi possível estabelecer associação significativa entre os mecanismos genéticos e epigenéticos subjacentes a DRH com ancestralidades europeias ou africanas na população estudada.

Introduction: Triple-negative (TN) subtype breast cancer accounts for about 15% of all breast cancers and is negative for estrogen and progesterone receptor expression and HER2 overexpression/amplification. It is more prevalent in young, black women, has a higher risk of developing metastases and a worse prognosis. A significant part of TN tumors have a deficiency in the homologous recombination repair (HRD) pathway, resulting from genetic mechanisms, such as germline pathogenic mutations in BRCA1/2, with BRCA1 being the most frequent mutation, and/or epigenetic silencing of BRCA1 and RAD51C in the tumor. There are no data regarding BRCA1/2 germline mutation status in relation to the metastatic pattern of TN tumors or about the mechanisms involved in HRD in Brazilian women. Objectives: To evaluate the germline mutation status in BRCA1 and BRCA2 genes in women with TNBC and its association with metastatic pattern, HRD mechanisms and ancestry. Methodology: Single-center retrospective study. BRCA1/2 evaluation was performed from leukocyte/non-tumor tissue DNA, and when not available, fresh tumor tissue or embedded in paraffin. The methylation status of BRCA1 and RAD51C was determined by next-generation amplicon sequencing (NGS) using tumor DNA treated with sodium bisulfite; the classification criteria was >40% hypermethylated; between 20% to <39% intermediate. Ancestry analysis was performed by capillary electrophoresis with fragment analysis using the PMDA array using leukocyte DNA. Clinical information from the entire sample was obtained from electronic medical records available at the institution. Results: 400 cases of TNBC were included, 132 diagnosed with metastasis and 268 without metastasis. The genetic characterization of BRCA1/2 was obtained for 399 cases, and 21.6% (86/399) presented pathogenic (P) or probably pathogenic (PP) variants, where 81.4% (70/86) of the carriers of P/PP variants carried a pathogenic variant in BRCA1, corresponding to 17.5% of the evaluated cases (70/399), and 18.6% (16/86) carried P/PP variants in BRCA2, corresponding to 4, 0% of cases (16/399). The mean age at diagnosis was lower for carriers of P/PP variants in BRCA1, who were 41.7 years old, and the age of carriers of P/PP variants in BRCA2 and women without P/PP or VUS (Variant of Uncertain Significance) was 47.1 and 47.9 years, respectively. In regarding to metastatic cases, 131 had BRCA1/2 tested, with 12.2% (16/131) BRCA1-P/PP and 6.1% (8/131) BRCA2-P/PP. Regarding the metastatic pattern of TN tumors, the most affected sites in BRCA1-P/PP and without P/PP variants were lung (50% and 37%, respectively) and CNS (31% and 23%, respectively), while for BRCA2-P/PP, the most affected sites were the CNS (62%) and bones (37%). In analysis with multivariate logistic regression models, it was observed that the risk of developing metastasis in the BRCA2-P/PP group was significantly higher, reaching almost 4 times more to develop in the CNS (p=0.035) when compared to the group without P/PP variants and close to significance (p=0.067) to develop in bone. The BRCA1-P/PP group was not at increased risk when compared to the group without P/PP variants. The analysis of methylation in the BRCA1/RAD51C genes was performed in the primary tumor for 177 cases, showing 55.4% hypermethylation in one of the two genes, with 43.5% (77/177) being hypermethylated in BRCA1; and for RAD51C, 11.9% (21/177) hypermethylated. The analysis of methylation in metastatic tumors was performed for 25 cases, 32% of which were hypermethylated in one of the two genes, 20% (5/25) hypermethylated in BRCA1 and 12% (3/25) in RAD51C. Ancestry analysis was performed for 216 cases, where the majority (68%) were classified as predominantly European ancestry; it was investigated whether there was an association between ancestry and the genetic or epigenetic event underlying the HRD, but no association was evident. Conclusions: BRCA1 confirmed to be the gene that most frequently presents P/PP variants in women with TNBC, mainly in women diagnosed at a young age. Most cases whose tumor DNA showed hypermethylation in one of the two genes investigated (BRCA1 and RAD51C) were in young women, with the BRCA1 gene being more frequently methylated. It was observed that there is an up to 4-fold increased risk of developing CNS metastasis in the group of women with BRCA2-PP. It was not possible to establish a significant association between the genetic and epigenetic mechanisms underlying HRD with European or African ancestry in the population studied

Presença da mutação do gene BRCA1 em cadelas com tumores mamários malignos / Presence of BRCA1 gene mutation in bitches with malignant mammary tumors

Mammary tumors (MTs) in bitches are similar to breast cancers in women. Thus, they can be used as a model for human breast cancer and findings can be extrapolated for use in human medicine. BRCA1 is a tumor suppressor gene. When the gene has a mutation, it cannot repair damaged DNA, which causes genetic instability and tumorigenesis. Therefore, we aimed to study the frequency of single nucleotide polymorphisms (SNPs) in the BRCA1 gene that are associated with distinct histological types of malignant MT in bitches. The study population consisted of 91 bitches, including a control group of 6 animals with healthy mammary glands and 85 animals with MTs. All animals underwent a presurgery evaluation consisting of a questionnaire administered to the person responsible for the animal, a physical examination, collection of peripheral blood for hematological and serum biochemistry evaluations, an electrocardiogram, and a preanesthesia evaluation. In addition, distant metastasis was studied via chest radiography and abdominal ultrasound. After evaluations were complete, the animals that could undergo surgery were administered general anesthesia and underwent a mastectomy or mammary gland sample collection. Histopathological examination and molecular analysis were performed to identify mutations in the BRCA1 gene. Histopathological examinations found 10 different types of malignant tumors in 36 sick animals. Tumor samples plus samples from the 6 control animals were subjected to DNA extraction, polymerase chain reaction (PCR) analysis, and genetic sequencing. The tumor with the highest incidence (33.33%) was a complex carcinoma, followed by carcinoma in mixed tumor (13.88), tubular carcinoma (13.88) and carcinosarcoma (13.88). Molecular analysis revealed 3 different SNP points in 5 samples (4006G>A, 3619A>G, and 3761C>T). The allelic variant 4006G>A (1/36) resulted in the alteration of the amino acid valine by isoleucine (V1336 I). The mutation 3619A>G (2/36) inserted the amino acid alanine instead of threonine (T1207 A). The mutation 3761C>T (2/36) led to the alteration of the amino acid serine by phenylalanine (S1254 F), a mutation for which there are no published reports. The histological types that showed BRCA1 mutations were complex carcinoma (1/5), carcinoma in mixed tumor (1/5), papillary carcinoma (1/5) and tubular carcinoma (2/5). Software analysis identified the new SNP (nucleotide 3761) in BRCA1 and 2 point mutations in nucleotides 4006 and 3619 and responsible for genetic instability. The development of breast cancer is caused by many endogenous and exogenous factors. The results of our study show that these factors have a greater presence in female, mixed breed, uncastrated, and older dogs, confirming the data in the veterinary literature. In the present study, we found different histological types of malignant breast tumors with mutations in the BRCA1 gene, as other authors have reported. However, we also found the mutation 3761C>T, which, to the best of our knowledge, has not been reported in the literature. This shows the need for studies in veterinary medicine that assess mutations in the BRCA1 gene and the most common histological types. In conclusion, SNPs in the BRCA1 gene cause genetic instability, resulting in additional mutations that lead to the development of breast tumors. They are point mutations that affect transcription, resulting in truncated proteins. These proteins may have a loss of function, leading to carcinogenesis.(AU)

Management of early breast cancer during the COVID-19 pandemic in Brazil.

PURPOSE: The COVID-19 pandemic has impacted early breast cancer (EBC) treatment worldwide. This study analyzed how Brazilian breast specialists are managing EBC. METHODS: An electronic survey was conducted with members of the Brazilian Society of Breast Cancer Specialists (SBM) between April 30 and May 11, 2020. Bivariate analysis was used to describe changes in how specialists managed EBC at the beginning and during the pandemic, according to breast cancer subtype and oncoplastic surgery. RESULTS: The response rate was 34.4% (503/1462 specialists). Most of the respondents (324; 64.4%) lived in a state capital city, were board-certified as breast specialists (395; 78.5%) and either worked in an academic institute or one associated with breast cancer treatment (390; 77.5%). The best response rate was from the southeast of the country (240; 47.7%) followed by the northeast (128; 25.4%). At the beginning of the pandemic, 43% changed their management approach. As the outbreak progressed, this proportion increased to 69.8% (p < 0.001). The southeast of the country (p = 0.005) and the state capital cities (p < 0.001) were associated with changes at the beginning of the pandemic, while being female (p = 0.001) was associated with changes during the pandemic. For hormone receptor-positive tumors with the best prognosis (Ki-67 < 20%), 47.9% and 17.7% of specialists would recommend neoadjuvant endocrine therapy for postmenopausal and premenopausal women, respectively. For tumors with poorer prognosis (Ki-67 > 30%), 34% and 10.9% would recommend it for postmenopausal and premenopausal women, respectively. Menopausal status significantly affected whether the specialists changed their approach (p < 0.00001). For tumors ≥ 1.0 cm, 42.9% of respondents would recommend neoadjuvant systemic therapy for triple-negative tumors and 39.6% for HER2 + tumors. Overall, 63.4% would recommend immediate total breast reconstruction, while only 3.4% would recommend autologous reconstruction. In breast-conserving surgery, 75% would recommend partial breast reconstruction; however, 54.1% would contraindicate mammoplasty. Furthermore, 84.9% of respondents would not recommend prophylactic mastectomy in cases of BRCA mutation. CONCLUSIONS: Important changes occurred in EBC treatment, particularly for hormone receptor-positive tumors, as the outbreak progressed in each region. Systematic monitoring could assure appropriate breast cancer treatment, mitigating the impact of the pandemic.