High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples: KRAS codon 12 and 13 mutations in non-small cell lung cancer.
High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples: KRAS codon 12 and 13 mutations in non-small cell lung cancer.
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DOI：
10.1186/1471-2407-6-295
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发表时间：
2006-12-21
期刊：
影响因子：
3.8
通讯作者：
Dobrovic A
中科院分区：
文献类型：
--
作者：
Krypuy M;Newnham GM;Thomas DM;Conron M;Dobrovic A
靶向治疗的发展为癌症的快速和强有力的分子特征创造了迫切的临床需求。我们在这里描述了高分辨率熔融分析(HRM)在临床癌症样本中筛选KRAS突变的应用。在非小细胞肺癌中，KRAS突变已被证明可以识别一组对EGFR靶向治疗无效的患者，因此识别这些突变具有重要的临床意义。我们建立了一种使用嵌合染料Syto 9检测KRAS基因外显子2，特别是密码子12和13的体细胞突变的高分辨熔融(HRM)方法。我们测试了3个已知有KRAS突变的不同细胞株，然后用横跨密码子12和13的189和92个碱基的扩增片段检测突变的敏感性。然后，我们筛选了30例非小细胞肺癌活检组织中的KRAS突变，这些非小细胞肺癌组织之前已经对EGFR外显子18-21的突变进行了测序。细胞系(A549、HCT116和RPMI8226)中已知的KRAS突变很容易用HRM检测到。较短的92bp扩增子在检测突变方面比189bp扩增子更敏感，并且能够可靠地检测到在正常DNA中稀释的每个细胞系DNA的5%-6%。HRM分析发现，30例非小细胞肺癌活检组织中有9例存在KRAS突变。测序结果经标准测序证实。KRAS和EGFR的突变是相互排斥的。HRM是一种敏感的试管内方法，可用于筛选临床样本中的突变。人力资源管理将实现对基因突变的高通量筛选，以便为患者提供适当的治疗选择，并加快旨在识别人类癌症新突变的研究。
The development of targeted therapies has created a pressing clinical need for the rapid and robust molecular characterisation of cancers. We describe here the application of high-resolution melting analysis (HRM) to screen for KRAS mutations in clinical cancer samples. In non-small cell lung cancer, KRAS mutations have been shown to identify a group of patients that do not respond to EGFR targeted therapies and the identification of these mutations is thus clinically important. We developed a high-resolution melting (HRM) assay to detect somatic mutations in exon 2, notably codons 12 and 13 of the KRAS gene using the intercalating dye SYTO 9. We tested 3 different cell lines with known KRAS mutations and then examined the sensitivity of mutation detection with the cell lines using 189 bp and 92 bp amplicons spanning codons 12 and 13. We then screened for KRAS mutations in 30 non-small cell lung cancer biopsies that had been previously sequenced for mutations in EGFR exons 18–21. Known KRAS mutations in cell lines (A549, HCT116 and RPMI8226) were readily detectable using HRM. The shorter 92 bp amplicon was more sensitive in detecting mutations than the 189 bp amplicon and was able to reliably detect as little as 5–6% of each cell line DNA diluted in normal DNA. Nine of the 30 non-small cell lung cancer biopsies had KRAS mutations detected by HRM analysis. The results were confirmed by standard sequencing. Mutations in KRAS and EGFR were mutually exclusive. HRM is a sensitive in-tube methodology to screen for mutations in clinical samples. HRM will enable high-throughput screening of gene mutations to allow appropriate therapeutic choices for patients and accelerate research aimed at identifying novel mutations in human cancer.