Prognostic value of mutations in c-KIT,PDGFRA, KRAS, NRAS and BRAF genes in Slovene metastatic gastrointestinal stromal tumor patients

Pri približno 85% bolnikov je glavni vzrok za razvoj GIST aktivacijska mutacija (patogena/verjetno patogena različica – PR/VPR, mutacija – PR/VPR) v enem izmed genov c-KIT ali PDGFRA, medtem-ko pri 10-15% bolnikov z GIST omenjena gena nista mutirana (KIT/PDGFRA divji tip GIST). Pri njih se aktivacijske mutacije – PR/VPR lahko pojavijo v genih, ki kodirajo mitohondrijski encimski kompleks sukcinat dehidrogenazo, redkeje pa v genih KRAS, NRAS ali BRAF. Poznavanje mutacijskega statusa vseh naštetih genov, ki so vključeni v patogenezo GIST, je izredno pomembno pri načrtovanju zdravljenja bolnikov z GIST z zaviralci tirozinskih kinaz (TKI), saj je klinični učinek imatiniba, odvisen od genotipa tumorja. Na Oddelku za molekularno diagnostiko Onkološkega inštituta Ljubljana smo v sklopu doktorske raziskave izvedli molekularno karakterizacijo primarnih tumorjeva bolnikov z razsejanimi GIST. V raziskavo smo vključili bolnike, ki so bili po podatkih iz Registra raka RS, med januarjem 2002 in decembrom 2011 zdravljeni s TKI na Onkološkem inštitutu Ljubljana. Vsem bolnikom iz naše raziskave smo z metodo direktnega sekvenciranja po Sangerju določili frekvence, mesta in vrste mutacij – PR/VPR v genu c-KIT (eksoni 9, 11, 13, in 17) in v genu PDGFRA (eksoni 12, 14 in 18). Pri bolnikih z določenim KIT/PDGFRA divjim tipom GIST pa smo z metodo kvantitativnega PCR določili tudi najpogostejše (»hot spot«) mutacije – PR/VPR v genih KRAS, NRAS, BRAF, PIK3CA in AKT1 ter imunohistokemično izražanje SDH kompleksa. Nadalje smo ugotavljali tudi vpliv mesta in vrste dokazanih mutacij – PR/VPR na izhod zdravljenja (mediani čas brez napredovanja bolezni in mediani čas celokupnega preživetja) naših bolnikov z imatinibom. Z doktorsko raziskavo smo ugotovili, da se frekvence mutacij – PR/VPR v preiskovanih genih pri naših bolnikih ujemajo s podatki iz literature. Skupno smo v naši raziskavi dokazali 49 različnih mutacij – PR/VPR v genih c-KIT in PDGFRA. V genu c-KIT smo odkrili 8 novih, v literaturi še nikoli opisanih mutacij – VPRpri enem bolniku pa smo v genu PDGFRA odkrili dve primarni mutaciji – PR/VPR v eksonu 14, kar je v literaturi izjemno redko opisano. V skupini bolnikov s KIT/PDGFRA divjim tipom GIST mutacij – PR/VPR v genih KRAS, NRAS, BRAF, PIK3CA in AKT1 nismo dokazali pri nobenem bolniku, SDH kompleksa pa nista izražala dva od teh bolnikov. Pri ugotavljanju vpliva mesta in vrste dokazanih mutacij – PR/VPR na izhod zdravljenja pa smo ugotovili, da mesto mutacije – PR/VPR v eksonu 11 gena c-KIT klinično pomembno napoveduje ugoden potek zdravljenja z imatinibom tudi pri slovenskih bolnikih z razsejanim GIST ter da vrsta mutacije – PR/VPR ni pomembna za izhod njihovega zdravljenja. Z doktorsko raziskavo potrjujemo, da je molekularna analiza tumorjev bolnikov z GIST nujna in predstavlja pomembno diagnostično orodje pri načrtovanju zdravljenja teh bolnikov z zaviralci tirozinskih kinaz.In total, ~85% of GIST patients harbour activating mutations in one of the genes, c-KIT or PDGFRA, while 10-15% of all GIST patients have no mutations in these two genes (KIT/PDGFRA wild-type GIST). Wild-type GISTs could have alterations in genes of the succinate dehydrogenase (SDH) complex, or rarely in KRAS, NRAS or BRAF genes. The clinical benefit of tyrosine kinase inhibitors (TKIs), such as imatinib, depends on the GIST genotype, therefore molecular characterization of GIST has a crucial role in overall management of these patients. This doctoral study was performed at the Department of molecular diagnostic, Institute of oncology Ljubljana, to molecularly characterize primary tumour samples of patients with metastatic GISTs from the Slovenian Cancer Registry (National Cancer Registry) treated with tyrosine kinase inhibitors (TKIs) between January 2002 and December 2011 at the Institute of Oncology Ljubljana. Direct Sanger sequencing was performed in tumour samples of all patients included in our study to determine mutation spectrum of c-KIT gene (exons 9, 11, 13 and 17) and PDGFRA gene (exons 12, 14 and 18). Moreover, all KIT/PDGFRA wild-type GISTs were tested for the presence of mutations in hot spot regions of KRAS, NRAS, BRAF, PIK3CA and AKT1 genes with quantitative PCR and their expression of SDH complex was assessed by immunohistochemistry. Additionally, we wanted to find out if the location and type of detected mutations have a significant impact on the clinical outcome (progression free and overall survival) of our patients treated with imatinib. Our study concluded that mutation frequencies of c-KIT and PDGFRA genes observed in Slovenian patients are comparable with published series. In our study 49 different mutations were detected in c-KIT and PDGFRA genes. In c-KIT, 8 novel variants that have never been described in the literature before were identified. Moreover, one patient with double mutant GIST, who had two different mutations in PDGFRA exon 14 was identified, which is reported to be very rare. In KIT/PDGFRA wild-type GIST no mutations were found in KRAS, NRAS, BRAF, PIK3CA and AKT1, where SDH-deficiency was found in two patients. When analysing how the location and type of detected mutations effect patients’ clinical outcome, our study concluded that location of the mutation in c-KIT exon 11 is associated with a favourable clinical outcome of the Slovene GIST patients, while the type of detected mutation is not significantly important for their clinical outcome. Our study has confirmed that molecular characterization of GISTs is of great diagnostic value for patients with GIST and their appropriate management and treatment with TKIs