Universidade de Lisboa Faculdade de Farmácia Application and Verification of Next-Generation Sequencing (NGS) for the Molecular Diagnostics of Brain Tumours Maria de Azevedo Pinto Mestrado Integrado em Ciências Farmacêuticas 2017 Universidade de Lisboa Faculdade de Farmácia Application and Verification of Next-Generation Sequencing (NGS) for the Molecular Diagnostics of Brain Tumours Maria de Azevedo Pinto Trabalho de Campo de Mestrado Integrado em Ciências Farmacêuticas apresentada à Universidade de Lisboa através da Faculdade de Farmácia Orientador: Doutor Andreas Waha, Professor Assistente Coorientador: Doutor Rui Eduardo Castro, Professor Assistente 2017 The work that led to the production of this dissertation was carried at the Hirntumor-Referenzzentrum of the Institut für Neuropathologie of the Universität Bonn, under Erasmus+ Programme. Supervisor: PD Dr. Andreas Waha To my father Abstract Primary brain tumours are a critical cause of morbidity and mortality in both adults and children, representing around 1-2% of all newly diagnosed tumours and accounting for about 2% of all cancer-related deaths. Gliomas are the most prevalent primary malignant brain tumour representing 80% of these. Over the past years, distinctive genetic profiles have been identified in several glioma types which have led to the WHO 2016 new classification of CNS tumours that incorporates molecular parameters into the tumour classification criteria, breaking with the previous approach based entirely on histological features. This refines tumour diagnostics and can also provide important prognostic and therapeutic response information. The aim of this study was to apply and verify if Next- Generation Sequencing (NGS) can effectively replace the classical methods – pyrosequencing and Sanger sequencing - in establishing the molecular diagnostics of gliomas. Thus, a glioma-tailored gene panel covering 518 amplicons of 19 genes frequently aberrant in gliomas was designed and applied to assess 30 glioma samples. This targeted NGS approach was carried out by Illumina® TruSeq® technology in Illumina® MiSeq System. DNA libraries preparation showed a success rate of 90%. Data analysis was performed using several bioinformatical software and filtering parameters of variants were optimized to reduce sequencing artefacts in the NGS run. Better DNA quality libraries presented more reliable results showing less DNA sequence changes. The sensitivity and specificity of the 19-gene panel for detection of DNA sequence variants were verified by single-gene analyses which showed to be substantial concerning hotspot mutations but not so trustworthy concerning new-mutations detected by NGS. The presented findings showed that, even though NGS application in routine glioma molecular diagnostics can’t be yet implemented, further investigation of this technology is promising since NGS showed to be a resourceful tool for glioma genetic profiling, displaying its potential as diagnostic method which would facilitate the integrated histological and molecular glioma classification. Key-words: brain tumours, glioma, molecular diagnostics, mutation, Next-Generation Sequencing (NGS) 4 Resumo Os tumores cerebrais primários são uma causa crítica de morbilidade e mortalidade em adultos e crianças, representando cerca de 1-2% de todos os tumores recém- diagnosticados e cerca de 2% de todas as mortes relacionadas com o cancro. Os gliomas são o tipo de tumor cerebral maligno primário mais prevalente representando 80% destes. Ao longo dos últimos anos, foram identificados perfis genéticos distintivos em vários tipos de glioma o que levou à nova classificação de tumores do SNC de 2016, pela OMS, que incorpora os parâmetros moleculares nos critérios de classificação do tumor, quebrando com a abordagem anterior inteiramente baseada nas características histológicas. Esta nova abordagem veio aperfeiçoar o diagnóstico dos tumores e a capacidade de fornecer, também, informações importantes quanto ao prognóstico e resposta à terapêutica. O objetivo deste estudo foi aplicar e verificar se a Sequenciação de Nova Geração (NGS) pode efetivamente substituir os métodos clássicos - pirosequenciação e sequenciação de Sanger - no estabelecimento do diagnóstico molecular dos gliomas. Assim, foi desenhado e aplicado um painel genético adaptado a gliomas que cobriu 518 amplicões de 19 genes frequentemente aberrantes nestes, para analisar 30 amostras de gliomas. Esta abordagem direcionada da NGS foi realizada pela tecnologia TruSeq da Illumina®, no seu sistema MiSeq. A preparação das bibliotecas de DNA mostrou uma taxa de sucesso de 90%. A análise dos dados foi realizada recorrendo a vários softwares bioinformáticos e os parâmetros de filtração das variantes obtidas foram otimizados para reduzir os artefactos da sequenciação resultantes da execução da NGS. As bibliotecas de DNA de melhor qualidade apresentaram resultados mais confiáveis exibindo menos alterações nas sequências de DNA. A sensibilidade e a especificidade do painel de 19 genes para a deteção de mutações foram verificadas por análise individual dos genes, indicando ser substanciais em relação às mutações hotspot, mas não tão confiáveis em relação às novas mutações detetadas pela NGS. Os resultados apresentados mostraram que, apesar de não ser possível implementar para já a NGS na rotina do diagnóstico molecular de gliomas, é promissora uma investigação adicional nesta tecnologia, uma vez que a NGS mostrou ser uma ferramenta rica em recursos para delinear o perfil genético dos gliomas, ilustrando o seu potencial como método de diagnóstico que facilitaria a classificação histológica e molecular integrada dos gliomas. Palavras-chave: tumores cerebrais, glioma, diagnóstico molecular, mutação, Sequenciação de Nova Geração (NGS) 5 Acknowledgements I would like to grant my deepest thanks to Prof. Dr. Andreas Waha for giving me the opportunity of working on this research project, at the Hirntumor-Referenzzentrum of Germany. I am deeply grateful for your trust, advice and constant motivation and companionship. It was an honor to form the “team NGS” with such a great scientist and person and, above all, it was an honor to have your guidance. To Prof. Dr. Rui Eduardo Castro for the fundamental final counselling and support. I am very thankful for your prompt availability, help and guidance. I want to particularly thank to Verena Dreschmann and Evelyn Doerner for the technical support and advice. I am very grateful for your patience, availability and hospitality. I am also very thankful to Prof. Dr. Bernd Evert for the tireless motivation and the entertaining “knowledge exchange” sessions in the pauses of the lab work. To my mother, Marta Pinto, for your love and ability for encouraging me in your singular way. I am deeply grateful for having you always by my side. I am profoundly grateful to my uncles, Maria Inês Iglésias and João Tiago Iglésias, for all financial and emotional support. I am very thankful for your constant encouragement and help. I am very thankful to my grandparents, Maria Fernanda Mendoça e José Eduardo Mendoça, for the ongoing support. Specially to my grandmother, thank you for always having the right word at the right time. To my cousin, Mariana Freitas, for being always present, especially when I need it most. I am very grateful for your comforting understanding and constant motivation. To my brother, António Pinto, for the backing and incessant optimism. Finally, I would like to thank to all my friends who supported me during this important phase of my life, particularly to: Sandra Silva, David Pereira, Mariana Alexandre, Alexandra Ângelo, Leonardo Lourenço, André Marques, Jéssica Bronze, Márcio Santos, Jorge Honda, Joana Fernandes, Milene Fortunato, Pedro Maçãs, Cibele Maçãs, Renato Guerreiro, João Matias and Ruben Mendes – because… If I could do this without you? I could, but it wouldn’t be the same! 6 Abbreviations ⁰C degree Celsius µg microgram µl microliter 5-mC 5-methylcytosine 2800M control DNA 2800M A adenine A5XX index i5 adapters A7XX index i7 adapters ACD1 amplicon control DNA ACP3 amplicon control oligo pool 3 ACVR1 activin A receptor type 1 APS adenosine 5’ phosphosulfate ATP adenosine triphosphate ATRX alpha thalassemia/mental retardation syndrome X-linked bp base pair BR broad range BRAF B-Raf proto-oncogene, serine/threonine kinase C cytosine CAT custom amplicon oligo tube CDK4 cyclin-dependent kinase 4 CDK6 cyclin-dependent kinase 6 CDKN2B cyclin-dependent kinase inhibitor 2B Chr chromosome CIC capicua transcriptional repressor CLP clean-up plate CNS central nervous system COSMIC catalogue of somatic mutations in cancer DAL diluted amplicon library dATP deoxyadenosine triphosphate dCTP deoxycytosine triphosphate 7 ddNTP dideoxyribonucleotide triphosphate dGTP deoxyguanosine triphosphate DIPG diffuse intrinsic pontine glioma DNA deoxyribonucleic acid dNTP deoxyribonucleotide triphosphate dsDNA double-stranded DNA dTTP deoxythymidine triphosphate e.g. for example EDP enhanced DNA polymerase EDTA ethylenediaminetetraacetic acid EGFR epidermal growth factor receptor ELB extension-ligation buffer ELE extension-ligation enzyme EMM enhanced master mix ERBB2 Erb-B2 receptor tyrosine kinase 2 EtOH ethanol FFPE formalin-fixed, paraffin-embedded FGFR1 fibroblast growth factor receptor 1 FISH fluorescence in situ hybridisation FUBP1 far upstream element binding protein 1 fw forward primer g gram G guanine Gb gigabase pairs GBM glioblastoma multiforme GcGBM giant-cell glioblastoma multiforme gDNA genomic DNA h hour H3F3A