Predictive biomarkers in diffuse gliomas and non-small cell lung cancer

11 TIIVISTELMÄ . 13 INTRODUCTION . 15 REVIEW OF THE LITERATURE . 16 Genetic and epigenetic alterations in cancer . 16 1.1 Genetic alterations in cancer . 16 1.1.1 Small DNA sequence alterations . 17 1.1.2 Structural and numerical chromosome alteratio ns . 17 1.2 Epigenetic alterations in cancer . 18 1.2.1 DNA methylation . 19 1.2.2 Histone modifications . 19 1.2.3 Small non-coding miRNAs . 20 1.3 Predictive biomarkers for targeted therapies. 21 Gliomas . 23 2.1 Epidemiology . 23 2.2 Histopathology and grading of gliomas . 23 2.3 Predictive biomarkers in adult diffuse gliomas . 24 2.3.1 Codeletion of chromosomes 1p and 19q . 24 2.3.2 MGMT promoter hypermethylation . 25 2.3.3 IDH1 mutations . 26 2.3.4 Other potential therapeutic molecular targets in gliomas . 2 7 2.4 Treatment of gliomas . 28 Non-small cell lung cancer. 29 3.1 Epidemiology . 29 3.2 Histopathology and staging of NSCLC tumors . 29 3.3 Predictive biomarkers in non-small cell lung ca ncer . 30 3.3.1 EGFR mutations . 30 3.3.1.1. Resistance to EGFR tyrosine kinase inhibit or therapy . 31 3.3.2 ALK fusions . 32 3.3.2.1. Resistance to ALK tyrosine kinase inhibito r therapy . 3 3 3.3.3 Other potential therapeutic molecular targets in NSCLC . 33 3.4 Treatment of NSCLC . 34 Methods in the analysis of predictive biomarkers i n diffuse gliomas and non-small cell lung cancer . 36 4.1 Testing of MGMT gene promoter hypermethylation in diffuse gliomas . 36 4.1.1 Pyrosequencing as a method for MGMT promoter methylation testing . 37 4.2 Detection of ALK fusions and EGFR mutations in NSCLC . 38 4.2.1 Targeted next-generation sequencing as a meth od for detection of genetic alterations . 39 AIMS OF THE STUDY . 41 MATERIALS AND METHODS. 42 Study samples . 42 5.1 Glioma patient samples (I) . 42 5.2 Non-small cell lung cancer patient samples (II, III, IV) . 42 Analysis methods . 43 6.1 Nucleic acid extraction (I, II, III, IV) . 43 6.2 Pyrosequencing (I) . 43 6.3 Array comparative genomic hybridization (I) . 44 6.4 Immunohistochemistry (I, II, IV) . 44 6.5 Fluorescence in situ hybridization (II, IV) . 45 6.6 Real-time RT-PCR (II) . 45 6.7 Real-time PCR (III) . 45 6.8 Targeted next-generation sequencing (II, III, I V) . 46 6.8.1 Targeted next-generation sequencing with Illu mina HiSeq2000 system (II, III) . 46 6.8.2 Targeted next-generation sequencing with Ion Torrent PGM system (IV) . 47 6.9 Sanger sequencing (III) . 47 6.10 Statistical analyses (I, IV) . 47 Ethical permissions . 48 RESULTS AND DISCUSSION. 49 Genetic and epigenetic profiling of diffuse glioma s (I) . 49 8.1 Pyrosequencing in detection of MGMT promoter hypermethylation. 49 8.2 Chromosomal copy number aberrations, IDH1 mutation and MGMT promoter hypermethylation in glioma subtypes . 50 8.3 Associations between genetic and epigenetic alt erations . 51 Next-generation sequencing in detection of genetic alterations in non-small cell lung cancer (II, III) . 54 9.1 Correlation of targeted NGS with FISH, IHC, rea l-time RT-PCR, and real-time PCR in detection of genetic alterations (II, III) . 54 9.2 ALK fusion and mutations in EGFR, KRAS, and BRAF in adenocarcinoma-enriched NSCLC patient cohorts (II, III) . 57 9.3 Detection of rare and novel mutations by target ed NGS (III) . 58 Clinicopathological and molecular characterization of non-small cell lung cancer patients harboring ALK fusion (IV). 60 10.1 ALK fusion frequency in Finnish NSCLC patients . 60 10.2 Clinicopathological characteristics of patient s harboring ALK fusion . 61 10.3 Presence of other driver gene mutations in NSC LC patients harboring ALK fusion . 61 CONCLUSIONS . 64 ACKNOWLEDGMENTS . 66 WEB-BASED RESOURCES . 68 REFERENCES . 69 ORIGINAL PUBLICATIONS . 89