Predictive biomarkers in diffuse gliomas and non-small cell lung cancer
Year: 2015
Type: dissertation
Study design: Observational Study
Abstract: The presence of certain cancer-related genetic and epigenetic alterations in the tumor affect patients´ response to specific cancer therapies. The accurate screening of these predictive biomarkers in molecular diagnostics is important since it enables the tailoring of an optimal treatment based on molecular characteristics of the tumor. Depending on the type of gene alteration, a wide variety of methods could be applied in biomarker testing. Among the novel methods is next-generation sequencing (NGS) technology, enabling simultaneous detection of multiple alterations. The aim of this thesis was to analyze predictive or potentially predictive genetic and epigenetic alterations of diffuse gliomas and non-small cell lung cancer (NSCLC), and to evaluate the feasibility of pyrosequencing and targeted NGS in the detection of these alterations in formalin-fixed paraffin-embedded (FFPE) tumor tissue specimens. In Study I, we assessed the genetic and epigenetic profile of diffuse gliomas by applying methylation-specific pyrosequencing to detect MGMT promoter hypermethylation, array comparative genomic hybridization to detect chromosomal copy number alterations, and immunohistochemistry (IHC) to detect IDH1 mutation status. MGMT hypermethylation, IDH1 mutations, and losses of chromosome arms 1p and 19q were typical changes in oligodendroglial tumors (grades II-III), whereas losses of 9p and 10q were frequently seen in glioblastomas (grade IV). Furthermore, we detected significant associations of 1) MGMT hypermethylation with IDH1 mutations and loss of 19q, 2) unmethylated MGMT with losses of 9p and 10q and gain of 7p, 3) IDH1 mutations with MGMT hypermethylation, 1p loss, and combined loss of 1p/19q, and 4) non-mutated IDH1 with losses of 10q. Pyrosequencing proved to be a feasible method for determination of MGMT methylation status in FFPE sample material. In Studies II and III, we compared targeted NGS with fluorescence in situ hybridization, IHC, and real-time reverse-transcription PCR in the detection of ALK fusion (Study II), and with real-time PCR in the detection of EGFR, KRAS, and BRAF mutations (Study III). All analyses were successfully performed on all FFPE samples. A good concordance was observed between the results obtained by different methods, and targeted NGS also proved to be advantageous in the identification of novel and rare variants with a potential predictive value. In Study IV, we determined the frequency of ALK fusion in 469 Finnish NSCLC patients, and the association of ALK fusion with clinicopathological characteristics and with the presence of mutations in 22 other driver genes. We detected ALK fusion at a frequency of 2.3%, suggesting that it is a relatively rare alteration in Finnish NSCLC patients. The presence of ALK fusion was significantly linked to younger age and never-/ex-light smoking history. Although most of the ALK-positive tumors had adenocarcinoma histology, also ALK-positive large cell carcinomas were detected. Characterization of ALK-positive cases by targeted NGS showed a coexistence of ALK fusion with mutations in MET, TP53, CTNNB1, and PIK3CA, but the value of these co-occurrences requires further examination. In conclusion, our studies indicate that certain genetic and epigenetic alterations occur together, and the simultaneous screening of multiple alterations may thus allow one to obtain a more comprehensive picture of the molecular background of the tumor, which could facilitate prediction of tumor behavior, prognosis, and treatment response. Our results show the feasibility of pyrosequencing and targeted NGS in FFPE tumor tissue material and also the advantages of targeted NGS over other commonly used methods in the detection of gene rearrangements and mutations, particularly the ability to simultaneously identify multiple alterations.
Source: Työväentutkimus Vuosikirja
Author: Katja Merkkiniemi
Language: English
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W1547921297