Study of biomarkers of prognosis and response to therapy in gastrointestinal stromal tumors (GISTs)

Abstract

Gastrointestinal Stromal Tumors (GISTs) are the most common mesenchymal tumors that arise in the gastrointestinal tract. The majority of these tumors have mutations which activate oncogenes KIT or PDGFRA, encoding tyrosine kinase receptors, inducing a constitutive activation of proliferation, cell survival, and consequently tumor growth. The prognosis of patients with GIST is based on criteria established by the National Institutes of Health (NIH) and the Armed Forces Institutes of Pathology (AFIP), including tumor size, mitotic index and location, and they can be classified as low, intermediate and high risk. Despite this classification, tumor evolution is often unclear, considering the current clinical criteria. Thus, the study of new biomarkers can be crucial for better determining the prognosis of patients with GIST. The microsatellite instability (MSI) occurs in repeat regions of the gnomic DNA due to the failure of the DNA repair system and has been found in several tumor types such as colorectal cancer, where the presence of MSI is an important prognostic factor. Another molecule with prognostic impact in multiple tumors is the Raf kinase inhibitor protein (RKIP). The gene encoding RKIP is considered a metastasis suppressor gene and the loss of RKIP expression is associated with metastasis, prognosis prior and therapy resistance in various neoplasms. In 2013, was discovered the presence of oncogenic mutations in telomerase reverse transcriptase gene (TERT) in a wide range of tumors. These mutations are often associated with poor prognosis of patients with various tumors. In GIST, the evaluation of these three molecules is virtually nonexistent. In this sense, the objective of this study is to elucidate the biological and therapeutic role of these three potential biomarkers, MSI, RKIP and TERT in GISTs. We will evaluate the molecular and immunohistochemical changes of three potential biomarkers in a series of 110 GISTs samples. The presence of the MSI will be assessed by pentaplex PCR with the BAT- 25, BAT- 26, NR21, NR24 and - NR 27 markers followed by fragment analysis in the ABI Prism 3500 Genetic Analyzer system and GeneMaper 4.1 software. We will evaluate mutations in MSI target genes using the same methodology described above and the protein expression of DNA repair (mismatch repair - MMR) by immunohistochemistry. The RKIP expression will be evaluate by the same technique previously mentioned. For the TERT gene will be evaluate the mutations in the promoter region in Ch5: 1,295,228 (herein called C228T) and Ch5: 1295250 (C250T) by direct sequencing. Still, we assess the expression of TERT protein by immunohistochemistry. Then will be evaluate the biological and therapeutic role of potential biomarkers RKIP and TERT by in vitro experiments. RKIP gene silencing will be carried out in an immortalized line GIST882 to assess the viability, apoptosis, proliferation, cell migration and invasion by functional assays. At the same time, the drug response to imatinib will be evaluated in RKIP muted and non- muted cell line. For the TERT gene, will be built two established cell lines (derived from GIST882) expressing the recombinant minimal promoters controlled by the TERT gene (containing the mutation C228T, C250T or wild). Then all the above mentioned functional assays as well as the response to the drug imatinib will be evaluated in TERT wild type and mutated cells. Finally, using the modified clones for RKIP and TERT, the above mentioned, will be conducted in vivo tests through the CAM (chorioallantoic membrane) model for assessment of tumor size and angiogenesis. Statistical analyzes will be performed using SPSS software version 19.0. With this work we intend to contribute to the identification of new biomarkers that may help to better establish the prognosis of patients with GIST. (AU)