Division of Molecular Pathology, Department of Pathology, Hong Kong Sanatorium and Hospital, Happy Valley, Hong Kong, China.
J Clin Pathol. 2009 Oct;62(10):886-91. doi: 10.1136/jcp.2008.063677.
Mutation of the KRAS gene predicts the clinical response to the monoclonal antibody cetuximab in patients with advanced colorectal cancer (CRC). This study aimed to perform KRAS mutation detection on formalin-fixed paraffin-embedded (FFPE) tumour tissue by two different methods for comparison.
The FFPE sample was microdissected to enrich for tumour cells. KRAS exon 2 mutations were performed on 100 Chinese patients with CRC by direct nucleotide sequencing and high-resolution melting (HRM) analysis.
KRAS exon 2 mutations were detected in a total of 62 patients with the two methods combined, comprising 11 different mutant alleles. Three common mutations p.Gly12Asp, p.Gly12Val and p.Gly13Asp accounted for approximately 70% of all cases. The concordant rate between the two methods was 95%. Four mutations not initially detected by direct sequencing were identified by HRM and confirmed by sequencing of the HRM amplicons. One mutation detected by direct sequencing was inadvertently grouped as a wild-type allele by HRM software, but this was readily rectified through manual review.
HRM analysis is a sensitive method of detecting KRAS mutation on FFPE tumour tissue to guide cetuximab treatment and is applicable to routine molecular diagnostic service. Utilisation of HRM to screen for mutations upfront economises the resource used in the sequencing reaction.
KRAS 基因突变可预测晚期结直肠癌（CRC）患者对单克隆抗体西妥昔单抗的临床反应。本研究旨在通过两种不同方法对福尔马林固定石蜡包埋（FFPE）肿瘤组织进行 KRAS 基因突变检测，以进行比较。
对 100 例中国 CRC 患者的 FFPE 样本进行微切割以富集肿瘤细胞。通过直接核苷酸测序和高分辨率熔解（HRM）分析对 KRAS 外显子 2 突变进行检测。
两种方法联合检测到 62 例患者的 KRAS 外显子 2 突变，共检测到 11 种不同的突变等位基因。三种常见的突变 p.Gly12Asp、p.Gly12Val 和 p.Gly13Asp 约占所有病例的 70%。两种方法的一致性率为 95%。HRM 鉴定出直接测序未最初检测到的 4 种突变，并通过 HRM 扩增子的测序进行了确认。直接测序检测到的 1 种突变被 HRM 软件错误地归类为野生型等位基因，但通过手动审查很容易纠正。
HRM 分析是一种检测 FFPE 肿瘤组织中 KRAS 突变的敏感方法，可指导西妥昔单抗治疗，并适用于常规分子诊断服务。利用 HRM 进行突变筛选可节省测序反应中使用的资源。
