Analiza učestalosti i tipova mutacija KRAS i BRAF gena u karcinomima kolorektuma u populaciji Srbije

Abstract

Uvod: Kolorektalni kancer (CRC) se po svojoj učestalosti i smrtnosti nalazi u samom vrhu svih kancera kod oba pola, kako u svetu, tako i u Srbiji. Za ranu kancerogenezu ovog maligniteta karakteristične su somatske genske promene KRAS i BRAF protoonkogena. Mutacije ovih gena predstavljaju molekularne biomarkere odgovora na ciljanu terapiju, dok su rezultati o njihovoj ulozi kao prognostičkih i prediktivnih faktora kontradiktorni. Podaci o učestalosti i tipu pojedinačnih KRAS i BRAF mutacija kod CRC-a u našoj zemlji su malobrojni, a njihova veza sa kliničko-histopatološkim karakteristikama tumora, karakteristikama bolesnika i tokom bolesti nije dovoljno ispitana. Cilj: Utvrđivanje prisustva, učestalosti i tipa KRAS mutacija i specifične BRAF p.V600E mutacije kod bolesnika sa CRC-om u populaciji Srbije i povezanost prisustva i tipa ovih mutacija sa kliničko-histopatološkim karakteristikama tumora i karakteristikama bolesnika. Takođe, ispitivanje navedenih mutacija kao prediktivnih biomarkera adjuvantne i EGFR-ciljane terapije. Materijal i metode: U radu je analizirano 188 parafinskih uzoraka karcinoma kolorektuma. DNK je izolovana komercijalnim kitom za izolaciju genomske DNK iz parafinskih tkiva (QIAamp® DNA FFPE Tissue kit, QIAGEN). Detekcija prisustva i tipa mutacija kodona 12 i 13 KRAS gena urađena je pomoću dva dijagnostički validirana testa bazirana na eseju lančane reakcije polimeraze (PCR) u realnom vremenu (DxS TheraScreen K-RAS PCR kit, QIAGEN) i PCR reakciji praćenoj reverznom hibridizacijom (KRAS StripAssayTM, ViennaLab Diagnostics). Detekcija p.V600E mutacije u BRAF genu urađena je analizom krive topljenja (HRM) PCR produkta i metodom direktnog sekvenciranja. Za statističku obradu podataka korišćeni su Fišerov egzaktni, χ2 i Log-Rank testovi...

Introduction: Colorectal cancer (CRC) incidence and mortality rates are among the highest in both sexes worldwide, as well as in Serbia. The early carcinogenesis of this malignancy is characterized by somatic gene alterations of KRAS and BRAF protooncogenes. Mutations of these genes represent molecular biomarkers of response to targeted therapy, while their roles as prognostic and predictive factors are still contradictory. There are few data about the frequency and types of single KRAS and BRAF mutations in CRC in our country, and their correlation with clinicohistopathological characteristics of tumor, characteristics of patients and the course of disease is still to be established. Aim: Determination of presence, frequency and types of KRAS mutations and specific BRAF p.V600E mutation in CRC patients in Serbian population and correlation of presence and types of these mutations with tumor clinicohistopathological characteristics and patient characteristics. Also, the examination of specified mutations as potential predictive biomarkers of adjuvant and EGFR-targeted therapy. Material and methods: In this study 188 CRC paraffin samples were analyzed. DNA was extracted using the commercial kit for genomic DNA isolation from paraffin tissues (QIAamp® DNA FFPE Tissue kit, QIAGEN). Detection of presence and types of KRAS codon 12 and 13 mutations was performed with two validated diagnostic tests based on real-time polymerase chain reaction (PCR) assay (DxS TheraScreen K-RAS PCR kit, QIAGEN) and PCR reaction followed by reverse hybridization (KRAS StripAssayTM, ViennaLab Diagnostics). BRAF p.V600E mutation was assessed by high resolution melting (HRM) analysis of PCR products and automatic DNA sequencing. Fisher exact, χ2 and Log-Rank tests were used for statistical analysis...