Negru Serban, Papadopoulou Eirini, Apessos Angela, Stanculeanu Dana Lucia, Ciuleanu Eliade, Volovat Constantin, Croitoru Adina, Kakolyris Stylianos, Aravantinos Gerasimos, Ziras Nikolaos, Athanasiadis Elias, Touroutoglou Nikolaos, Pavlidis Nikolaos, Kalofonos Haralabos P, Nasioulas George
University of Medicine and Pharmacy of Timisoara, Timisoara, Romania.
Department of Molecular Biology, GENEKOR, Athens, Greece.
BMJ Open. 2014 May 23;4(5):e004652. doi: 10.1136/bmjopen-2013-004652.
Treatment 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.
Diagnostic molecular laboratory located in Athens, Greece.
2425 patients with colorectal cancer participated in the study.
2071 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.
KRAS 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.
The 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.
结直肠癌的治疗决策通常由肿瘤组织分子分析指导。本研究的目的是开发并验证一种高分辨率熔解（HRM）方法，用于检测希腊和罗马尼亚结直肠癌患者中的KRAS、NRAS和BRAF突变，并确定这些突变在各自人群中的频率。
位于希腊雅典的诊断分子实验室。
2425例结直肠癌患者参与了本研究。
对2071例结直肠癌患者（1699例希腊患者和372例罗马尼亚患者）进行KRAS外显子2突变分析。此外，对连续患者的354个肿瘤（196例希腊患者和161例罗马尼亚患者）进行KRAS（外显子2、3和4）、NRAS（外显子2、3和4）和BRAF（外显子15）的全面分析。通过新设计的HRM分析方案进行KRAS、NRAS和BRAF突变检测，随后进行桑格测序。
在分析的1699例希腊结直肠癌患者中，702例（41.3%）检测到KRAS外显子2突变（密码子12/13），在罗马尼亚患者中为39.2%（146/372）。在接受KRAS、NRAS和BRAF全面分析的354例患者中，40.96%有KRAS外显子2突变（密码子12/13）。在KRAS外显子2野生型患者中，15.31%有额外的RAS突变，12.44%有BRAF突变。所使用的新设计的HRM方法与测序方法相比显示出更高的灵敏度。
所开发的HRM方法被证明是一种检测KRAS、NRAS和BRAF突变的可靠方法。此外，希腊和罗马尼亚结直肠癌患者之间在KRAS、NRAS和BRAF的突变频率上未观察到差异。
