• Serban Negru, Eirini Papadopoulou, Angela Apessos, Dana Lucia Stanculeanu, Eliade Ciuleanu, Constantin Volovat, Adina Croitoru, Stylianos Kakolyris, Gerasimos Aravantinos, Nikolaos Ziras, Elias Athanasiadis, Nikolaos Touroutoglou, Nikolaos Pavlidis, Haralabos P Kalofonos, and George Nasioulas.
• University of Medicine and Pharmacy of Timisoara, Timisoara, Romania.
• BMJ Open. 2014 May 23; 4 (5): e004652.

ObjectivesTreatment decision-making in colorectal cancer is often guided by tumour tissue molecular analysis. The aim of this study was the development and validation of a high-resolution melting (HRM) method for the detection of KRAS, NRAS and BRAF mutations in Greek and Romanian patients with colorectal cancer and determination of the frequency of these mutations in the respective populations.SettingDiagnostic molecular laboratory located in Athens, Greece.Participants2425 patients with colorectal cancer participated in the study.Primary And Secondary Outcome Measures2071 patients with colorectal cancer (1699 of Greek and 372 of Romanian origin) were analysed for KRAS exon 2 mutations. In addition, 354 tumours from consecutive patients (196 Greek and 161 Romanian) were subjected to full KRAS (exons 2, 3 and 4), NRAS (exons 2, 3 and 4) and BRAF (exon 15) analysis. KRAS, NRAS and BRAF mutation detection was performed by a newly designed HRM analysis protocol, followed by Sanger sequencing.ResultsKRAS exon 2 mutations (codons 12/13) were detected in 702 of the 1699 Greek patients with colorectal carcinoma analysed (41.3%) and in 39.2% (146/372) of the Romanian patients. Among the 354 patients who were subjected to full KRAS, NRAS and BRAF analysis, 40.96% had KRAS exon 2 mutations (codons 12/13). Among the KRAS exon 2 wild-type patients 15.31% harboured additional RAS mutations and 12.44% BRAF mutations. The newly designed HRM method used showed a higher sensitivity compared with the sequencing method.ConclusionsThe HRM method developed was shown to be a reliable method for KRAS, NRAS and BRAF mutation detection. Furthermore, no difference in the mutation frequency of KRAS, NRAS and BRAF was observed between Greek and Romanian patients with colorectal cancer.Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

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