Institut für Pathologie, Charité Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany.
J Mol Diagn. 2010 Jan;12(1):35-42. doi: 10.2353/jmoldx.2010.090079. Epub 2009 Dec 10.
KRAS mutation testing before anti-epidermal growth factor receptor therapy of metastatic colorectal cancer has become mandatory in Europe. However, considerable uncertainty exists as to which methods for detection can be applied in a reproducible and economically sound manner in the routine diagnostic setting. To answer this question, we examined 263 consecutive routine paraffin slide specimens. Genomic DNA was extracted from microdissected tumor tissue. The DNA was analyzed prospectively by Sanger sequencing and array analysis as well as retrospectively by melting curve analysis and pyrosequencing; the results were correlated to tissue characteristics. The methods were then compared regarding the reported results, costs, and working times. Approximately 40% of specimens contained KRAS mutations, and the different methods reported concordant results (kappa values >0.9). Specimens harboring fewer than 10% tumor cells showed lower mutation rates regardless of the method used, and histoanatomical variables had no influence on the frequency of the mutations. Costs per assay were higher for array analysis and melting curve analysis when compared with the direct sequencing methods. However, for sequencing methods equipment costs were much higher. In conclusion, Sanger sequencing, array analysis, melting curve analysis, and pyrosequencing were equally effective for routine diagnostic KRAS mutation analysis; however, interpretation of mutation results in conjunction with histomorphologic tissue review and on slide tumor tissue dissection is required for accurate diagnosis.
KRAS 基因突变检测在转移性结直肠癌的抗表皮生长因子受体治疗之前已在欧洲成为强制性要求。然而，在常规诊断环境中以可重复且经济合理的方式应用哪种检测方法仍然存在相当大的不确定性。为了回答这个问题，我们检测了 263 例连续的常规石蜡切片标本。从微切割的肿瘤组织中提取基因组 DNA。前瞻性地通过 Sanger 测序和阵列分析以及回顾性地通过熔解曲线分析和焦磷酸测序对 DNA 进行分析；将结果与组织特征相关联。然后，根据报告的结果、成本和工作时间比较了这些方法。大约 40%的标本含有 KRAS 突变，并且不同的方法报告了一致的结果（kappa 值>0.9）。无论使用哪种方法，含有少于 10%肿瘤细胞的标本的突变率均较低，组织解剖学变量对突变的频率没有影响。与直接测序方法相比，阵列分析和熔解曲线分析的每个检测的成本更高。然而，对于测序方法，设备成本要高得多。总之，Sanger 测序、阵列分析、熔解曲线分析和焦磷酸测序在常规诊断 KRAS 突变分析中同样有效；然而，为了进行准确的诊断，需要结合组织形态学组织复查和载玻片上肿瘤组织的切割来解释突变结果。
