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- The bigger the better? Evaluation of the value of large multi-gene panels in Portuguese cardiomyopathy genetic testingPublication . Baixia, Márcia; Sousa, Sónia; Pina, Maria J.; Silva, Raquel M; Cirnes, Luís; Canedo, Paulo; Castedo, Sérgio; Machado, José C,Introduction: Genetic testing of cardiomyopathies went through major changes in the last few years, from sequential Sanger sequencing of the most likely gene candidates, to multigene panels by NGS, with an ever increasing number of genes analyzed.
- Targeted Gene Next-Generation Sequencing Panel in Patients with Advanced Lung Adenocarcinoma: Paving the Way for Clinical ImplementationPublication . Fernandes, Maria Gabriela O.; Jacob, Maria; Martins, Natália; Moura, Conceição Souto; Guimarães, Susana; Pereira Reis, Joana; Justino, Ana; Pina, Maria João; Cirnes, Luís; Sousa, Catarina; Pinto, Josué; Marques, José Agostinho; Machado, José Carlos; Hespanhol, Venceslau; Costa, José LuisIdentification of targetable molecular changes is essential for selecting appropriate treatment in patients with advanced lung adenocarcinoma. Methods: In this study, a Sanger sequencing plus Fluorescence In Situ Hybridization (FISH) sequential approach was compared with a Next-Generation Sequencing (NGS)-based approach for the detection of actionable genomic mutations in an experimental cohort (EC) of 117 patients with advanced lung adenocarcinoma. Its applicability was assessed in small biopsies and cytology specimens previously tested for epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) mutational status, comparing the molecular changes identified and the impact on clinical outcomes. Subsequently, an NGS-based approach was applied and tested in an implementation cohort (IC) in clinical practice. Using Sanger and FISH, patients were classified as EGFR-mutated (n = 22, 18.8%), ALK-mutated (n = 9, 7.7%), and unclassifiable (UC) (n = 86, 73.5%). Retesting the EC with NGS led to the identification of at least one gene variant in 56 (47.9%) patients, totaling 68 variants among all samples. Still, in the EC, combining NGS plus FISH for ALK, patients were classified as 23 (19.7%) EGFR; 20 (17.1%) KRAS; five (4.3%) B-Raf proto-oncogene (BRAF); one (0.9%) Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2); one (0.9%) STK11; one (0.9%) TP53, and nine (7.7%) ALK mutated. Only 57 (48.7%) remained genomically UC, reducing the UC rate by 24.8%. Fourteen (12.0%) patients presented synchronous alterations. Concordance between NGS and Sanger for EGFR status was very high (κ = 0.972; 99.1%). In the IC, a combined DNA and RNA NGS panel was used in 123 patients. Genomic variants were found in 79 (64.2%). In addition, eight (6.3%) EML4-ALK, four (3.1%), KIF5B-RET, four (3.1%) CD74-ROS1, one (0.8%) TPM3-NTRK translocations and three (2.4%) exon 14 skipping MET Proto-Oncogene (MET) mutations were detected, and 36% were treatable alterations. Conclusions: This study supports the use of NGS as the first-line test for genomic profiling of patients with advanced lung adenocarcinoma.
- How the COVID-19 Pandemic Impacted Oncological Molecular Diagnosis: A Picture from a National Reference Center for Molecular PathologyPublication . Pinto, Daniel; Cirnes, Luís; Pinto, Regina; Pina, Maria João; Giancarlo, Troncone; Schimitt, FernandoThe Portuguese healthcare system had to adapt at short notice to the COVID-19 pandemic. We implemented workflow changes to our molecular pathology laboratory, a national reference center, to maximize safety and productivity. We assess the impact this situation had on our caseload and what conclusions can be drawn about the wider impact of the pandemic in oncological therapy in Portugal. Material and Methods. We reviewed our database for all oncological molecular tests requested between March and April of 2019 and 2020.
- Impact of different storage conditions of formamide in the quality of sequencesPublication . Moura, Daniela; Pina, Maria João; Pereira, Cátia; Baixia, Márcia; Leal, Rafaela; Sousa, Sónia; Vieira, Filipa Quintela; Silva, Regina; Cirnes, LuísObtaining high quality sequences is essential to proper reading and interpretation. A crucial step for this goal is the denaturation of sequencing products which is usually performed using a denaturing agent such as formamide. It has been described that the reduction of formamide quality may cause irregular cytosine and guanine peaks in an electropherogram.According to manufacturers recomendations, formamide should be stored at -20ºC. Under other conditions such as exposition to light and air and temperature changes for a certain period of time, quality may decrease. Objectives: The purpose of this study was to understand which formamide storage-related variables can be responsible for decreasing sequence quality.
- Resistance profile of osimertinib in pre-treated patients with EGFR T790M-mutated non-small cell lung cancerPublication . Fernandes, Maria Gabriela O.; Sousa, Catarina; Jacob, Maria; Almeida, Leonor; Santos, Vanessa; Araújo, David; Bastos, Hélder Novais; Magalhães, Adriana; Cirnes, Luís; Moura, Conceição Souto; Queiroga, Henrique; Cruz-Martins, Natália; Hespanhol, VenceslauOsimertinib efficacy in pre-treated patients with epidermal growth factor receptor (EGFR) T790M-mutated non-small cell lung cancer (NSCLC) has been demonstrated in clinical trials, but real-world data, particularly regarding resistance profile, remains limited. This study aims to analyze the resistance mechanisms acquired after treatment with Osimertinib. Clinical outcomes and molecular results from re-biopsies at the time of osimertinib progression of EGFR T790M-mutated NSCLC patient were analyzed. Twenty-one patients with stage IV adenocarcinoma were included [median 69 years; 57.1% female; 85.7% never-smokers; 23.8% ECOG performance status (PS) ≥2]. Median PFS and OS were 13.4 (95% CI: 8.0–18.9) and 26.4 (95% IC: 8.9–43.8) months, respectively. At the time of analysis, 10 patients had tumor progression (47.6%). T790M loss occurred in 50%, being associated with earlier progression (median PFS 8.1 vs. 21.4 months, p = 0.011). Diverse molecular alterations were identified, including C797S mutation (n = 1), PIK3CA mutation (n = 2), MET amplification (n = 1), CTNNB1 mutation (n = 1), and DCTN1-ALK fusion (n = 1). Histological transformation into small cell carcinoma occurred in one patient. This real-world life study highlights the relevance of re-biopsy at the time of disease progression, contributing to understand resistance mechanisms and to guide treatment strategies.
- Análise de variantes no gene STK11 e sua influência na resposta à imunoterapiaPublication . Bessa, Ana; Pina, Maria; Domingues, Patrícia; Dias, Margarida; Pinto, Carla; Cirnes, Luís; Silva, Regina A.A imunoterapia é uma das ferramentas terapêuticas de sucesso usadas no tratamento do cancro de pulmão, contudo alguns doentes apresentam progressão da doença ou morte. O gene Serine/ threonineKinase 11 (STK11) está descrito como tendo um papel na fisiologia do sistema imune(SI) e que alterações genéticas no mesmo podem influenciar negativamente a resposta dos pacientes à imunoterapia. Estudar alterações genéticas no gene STK11 em pacientes com cancro de pulmão sujeitos a imunoterapia e a sua possível associação com a resposta do doente à terapêutica. Após extraçãodo DNA de amostras de cancro pulmonar foi realizado um PCR multiplex para amplificação de 9 exões do geneSTK11, os quais foram sequenciados para pesquisa de alterações genéticas. Os pacientes foram divididos em dois grupos: “apresentaram boa resposta à terapia” e “apresentaram má resposta à terapia”, para efetuar curvas de sobrevivência que relacionam resposta à terapia com alterações em STK11. Foram detetadas e classificadas como patogénicas ou provavelmente patogénicas 13 alterações no gene STK11, não tendo sido identificada uma zona do gene ou exão com mais propensão a alterações. Pacientes com alterações no gene STK11 apresentaram pior sobrevida global e pior sobrevida livre de progressão de doença. Estes resultados sugerem que o gene STK11 deve ser estudado na sua totalidade aquando da decisão terapêutica e que deve ser incluído em painéis de Sequenciação de Nova Geração (NGS), uma vez que foram detetadas alterações neste gene com valor preditivo negativo na resposta à imunoterapia.
- Pesquisa de mutação T790M sequencial e alteração da abordagem terapêutica após 2 linhas de quimioterapia – caso clínicoPublication . Estevinho, Fernanda; Silva, Ana Catarina; Cirnes, Luís; Amaro, TeresinaA mutação do EGFR é identificada em 10 -50% dos doentes com cancro do pulmão de não pequenas células (CPNPC). Após terapêutica de primeira linha com inibidores da tirosinacinase (TKI) de 1.ª ou 2.ª linha, em doentes com CPNPC e mutação sensibilizadora do EGFR, o mecanismo de resistência mais frequente é a aquisição de mutação T790M, em 50 -70%. Apresenta -se o caso clínico de uma mulher com CPNPC, cT2bN1M1b, estadio IV, com metastização cerebral e suprarrenal ao diagnóstico e mutação do EGFR. Realizou radioterapia holocraniana e tratamento com erlotinib. Deteção de mutação T790M após 2 esquemas de quimioterapia, com pesquisas prévias em biópsia líquida e histológica negativas para a pesquisa de mutação T790M. Oito anos após o diagnóstico, está funcionalmente autónoma, assintomática, sob terapêutica com osimertinib. Este caso ilustra a importância do conhecimento do mecanismo de resistência nos doentes com CPNPC e mutações do EGFR, e o papel de biopsias sequenciais
- Effects of different storage conditions of sequencing products with formamide in the quality of sequences.Publication . Moura, Daniela; Pina, Maria João; Pereira, Cátia; Baixia, Márcia; Leal, Rafaela; Sousa, Sónia; Vieira, Filipa Quintela; Silva, Regina; Cirnes, LuísDNA sequencing is widely used in molecular diagnosis and good sequence quality is crucial to a correct interpretation. It has been described that formamide quality decrease and sequencing reactions exposure to light, heat and/or oxygen can cause irregular peaks of cytosine and guanine in electropherograms. In a previous study, we concluded that despite the presence of this artifact when formamide is stored under non-ideal conditions, it does not significantly reduce the quality of the sequences.