Desenvolvimento de métodos eficientes para análise de biomarcadores preditivos em biópsias líquidas para aplicação em pacientes com carcinoma do pulmão de não pequenas células
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2021-04-27
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O cancro de pulmão de não pequenas células (CPNPC) é o tipo mais comum de cancro do pulmão, sendo responsável pela maioria casos de cancro. Este encontra-se, geralmente, num estadio avançado quando diagnosticado, diminuindo a taxa de sobrevida. A aplicação de terapias dirigidas (como afitinib, crizotinib) depende da deteção de alterações em genes específicos (como EGFR e ALK), sendo estes considerados biomarcadores preditivos. Atualmente, são utilizadas técnicas de PCR e/ou sequenciação de Sanger para determinar alterações moleculares em biópsias tumorais (que normalmente estão fixadas em formalina e embebidas em parafina – FFPE). Porém, as limitações na dificuldade de obtenção de amostra com qualidade têm levado à necessidade de encontrar alternativas viáveis para determinar a evolução da doença ou até a detetar precocemente. Uma alternativa viável são as biópsias líquidas, consideradas um método confiável e minimamente invasivo para a deteção de mutações genéticas específicas. Este trabalho tem como objetivo desenvolver um teste simples, certificado e com maior sensibilidade analítica para a deteção de mutações associadas a diferentes respostas terapêuticas em biópsias líquidas de doentes com CPNPC. Para tal, foi necessário: definir o método de acondicionamento e extração de cfDNA e desenhar e otimizar ensaios para deteção de mutações no gene EGFR. Relativamente ao acondicionamento e extração de cfDNA a partir de biópsias liquidas, o uso de esferas magnéticas para extração de cfDNA (especificamente o kit GenElute UltraMag Cell-DNA) e os tubos para colheita Cell-Free DNA Collection Tube (CE-IVD) (Roche) mostraram ser adequados e eficientes para obtenção de cfDNA até 6 dias após a de colheita de sangue. Para o desenho dos ensaios de diagnóstico foi feito um estudo das mutações do gene EGFR relacionadas com resposta terapêutica e os “primers” foram desenhados de forma a englobarem todas as alterações relevantes. Após otimização do multiplex PCR foi possível amplificar os exões 18, 19, 20 e 21 do gene EGFR a partir de amostras de cfDNA e sequenciar por NGS. Este método mostrou-se eficiente para a deteção de mutações no gene EGFR, tendo sido detetada uma deleção específica no exão 19 –p.E746_A750delELREA. Desenvolvemos também ensaios de HRM (“High Resolution Melting Temperature”) como uma alternativa de deteção mais rápida e sensível. Os resultados foram promissores, uma vez que conseguimos uma sensibilidade de 0.25% (fração de alelo mutante) para detetar alterações no exão 19 e foi possível também identificar a mutação previamente analisada por NGS (Exão 19- p.E746_A750delELREA) na mesma amostra de cfDNA. O desenho dos ensaios para deteção de alterações no gene ALK encontra-se em desenvolvimento, tendo sido feita a análise “in silico” das mutações/fusões no gene ALK relacionadas com diferentes respostas terapêuticas e o desenho dos “primers” para deteção das mutações em amostras de cfDNA de doentes com CPNPC. Com este trabalho verificamos o potencial que a utilização das biopsias líquidas tem como uma alternativa válida e confiável para a deteção de mutações no EGFR. Esta abordagem permitirá o seguimento (não invasivo) dos doentes com CPNPC durante a terapia, representando uma grande vantagem, pois frequentemente adquirem mutações específicas que provocam resistência aos tratamentos.
Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. This is usually in an advanced stage when diagnosed, decreasing the survival rate. The application of targeted therapies (such as afitinib, crizotinib) depends on the detection of alterations in specific genes (such as EGFR and ALK), which are considered predictive biomarkers. Currently, PCR and / or Sanger sequencing techniques are used to determine molecular changes in tumor biopsies (which are normally formalin fixed and paraffin embedded - FFPE). However, the limitations in obtaining a quality sample have led to the need to find viable alternatives to determine the evolution of the disease or even to its earlier detection. A viable alternative are liquid biopsies, considered a reliable and minimally invasive method for detecting specific genetic mutations. This work aims to develop a simple, certified test with greater analytical sensitivity for detecting mutations associated with different therapeutic responses in liquid biopsies of patients with NSCLC. For that, it was necessary to: define the method of conditioning and extraction of cfDNA and design and optimize assays for detecting mutations in the EGFR gene. Regarding the conditioning and extraction of cfDNA from liquid biopsies, the use of magnetic spheres for extraction of cfDNA (specifically the GenElute UltraMag Cell-DNA kit) and the Cell-Free DNA Collection Tube (CE-IVD) collection tubes (Roche) showed to be adequate and efficient to obtain cfDNA up to 6 days after blood collection. For the design of the diagnostic tests, a study of the EGFR gene mutations related to the therapeutic response was made and the primers were designed to encompass all relevant changes. After optimizing the multiplex PCR, it was possible to amplify exons 18, 19, 20 and 21 of the EGFR gene from cfDNA samples and sequence by NGS. This method proved to be efficient for detecting mutations in the EGFR gene, with a specific deletion being detected in exon 19 –p.E746_A750delELREA. We also developed HRM (High Resolution Melting) tests as a faster and more sensitive detection alternative. The results were promising, since we achieved a sensitivity of 0.25% (fraction of mutant allele) to detect changes in exon 19 and it was also possible to identify the mutation previously analyzed by NGS (Exon 19, p.E746_A750delELREA) in the same sample of cfDNA . The design of assays for detecting alterations in the ALK gene is under development, with an in silico analysis of mutations / fusions in the ALK gene related to different therapeutic responses, and the design of primers to detect mutations in samples of cfDNA of patients with NSCLC. With this work we verify the potential that the use of liquid biopsies has as a valid and reliable alternative for the detection of mutations in EGFR. This approach will allow (noninvasive) follow-up of patients with NSCLC during therapy, representing a great advantage, as they often acquire specific mutations that cause resistance to treatments.
Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. This is usually in an advanced stage when diagnosed, decreasing the survival rate. The application of targeted therapies (such as afitinib, crizotinib) depends on the detection of alterations in specific genes (such as EGFR and ALK), which are considered predictive biomarkers. Currently, PCR and / or Sanger sequencing techniques are used to determine molecular changes in tumor biopsies (which are normally formalin fixed and paraffin embedded - FFPE). However, the limitations in obtaining a quality sample have led to the need to find viable alternatives to determine the evolution of the disease or even to its earlier detection. A viable alternative are liquid biopsies, considered a reliable and minimally invasive method for detecting specific genetic mutations. This work aims to develop a simple, certified test with greater analytical sensitivity for detecting mutations associated with different therapeutic responses in liquid biopsies of patients with NSCLC. For that, it was necessary to: define the method of conditioning and extraction of cfDNA and design and optimize assays for detecting mutations in the EGFR gene. Regarding the conditioning and extraction of cfDNA from liquid biopsies, the use of magnetic spheres for extraction of cfDNA (specifically the GenElute UltraMag Cell-DNA kit) and the Cell-Free DNA Collection Tube (CE-IVD) collection tubes (Roche) showed to be adequate and efficient to obtain cfDNA up to 6 days after blood collection. For the design of the diagnostic tests, a study of the EGFR gene mutations related to the therapeutic response was made and the primers were designed to encompass all relevant changes. After optimizing the multiplex PCR, it was possible to amplify exons 18, 19, 20 and 21 of the EGFR gene from cfDNA samples and sequence by NGS. This method proved to be efficient for detecting mutations in the EGFR gene, with a specific deletion being detected in exon 19 –p.E746_A750delELREA. We also developed HRM (High Resolution Melting) tests as a faster and more sensitive detection alternative. The results were promising, since we achieved a sensitivity of 0.25% (fraction of mutant allele) to detect changes in exon 19 and it was also possible to identify the mutation previously analyzed by NGS (Exon 19, p.E746_A750delELREA) in the same sample of cfDNA . The design of assays for detecting alterations in the ALK gene is under development, with an in silico analysis of mutations / fusions in the ALK gene related to different therapeutic responses, and the design of primers to detect mutations in samples of cfDNA of patients with NSCLC. With this work we verify the potential that the use of liquid biopsies has as a valid and reliable alternative for the detection of mutations in EGFR. This approach will allow (noninvasive) follow-up of patients with NSCLC during therapy, representing a great advantage, as they often acquire specific mutations that cause resistance to treatments.
Descrição
Dissertação de Mestrado apresentada à Universidade de Trás-os-Montes e Alto Douro para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Biotecnologia para as Ciências da Saúde
Palavras-chave
Cancro de pulmão de não pequenas células (CPNPC) , cfDNA