Laboratory of Molecular Oncologic and Transplantation Pathology, S. Orsola-Malpighi Hospital, Bologna, Italy.
Onco Targets Ther. 2013 Aug 5;6:1057-64. doi: 10.2147/OTT.S42369. eCollection 2013.
Detection of KRAS mutations in archival pathology samples is critical for therapeutic appropriateness of anti-EGFR monoclonal antibodies in colorectal cancer. We compared the sensitivity, specificity, and accuracy of Sanger sequencing, ARMS-Scorpion (TheraScreen®) real-time polymerase chain reaction (PCR), pyrosequencing, chip array hybridization, and 454 next-generation sequencing to assess KRAS codon 12 and 13 mutations in 60 nonconsecutive selected cases of colorectal cancer. Twenty of the 60 cases were detected as wild-type KRAS by all methods with 100% specificity. Among the 40 mutated cases, 13 were discrepant with at least one method. The sensitivity was 85%, 90%, 93%, and 92%, and the accuracy was 90%, 93%, 95%, and 95% for Sanger sequencing, TheraScreen real-time PCR, pyrosequencing, and chip array hybridization, respectively. The main limitation of Sanger sequencing was its low analytical sensitivity, whereas TheraScreen real-time PCR, pyrosequencing, and chip array hybridization showed higher sensitivity but suffered from the limitations of predesigned assays. Concordance between the methods was k = 0.79 for Sanger sequencing and k > 0.85 for the other techniques. Tumor cell enrichment correlated significantly with the abundance of KRAS-mutated deoxyribonucleic acid (DNA), evaluated as ΔCt for TheraScreen real-time PCR (P = 0.03), percentage of mutation for pyrosequencing (P = 0.001), ratio for chip array hybridization (P = 0.003), and percentage of mutation for 454 next-generation sequencing (P = 0.004). Also, 454 next-generation sequencing showed the best cross correlation for quantification of mutation abundance compared with all the other methods (P < 0.001). Our comparison showed the superiority of next-generation sequencing over the other techniques in terms of sensitivity and specificity. Next-generation sequencing will replace Sanger sequencing as the reference technique for diagnostic detection of KRAS mutation in archival tumor tissues.
检测存档病理学样本中的 KRAS 突变对于结直肠癌中抗 EGFR 单克隆抗体的治疗适用性至关重要。我们比较了 Sanger 测序、ARMS-Scorpion（TheraScreen®）实时聚合酶链反应（PCR）、焦磷酸测序、芯片杂交和 454 下一代测序在 60 例非连续选择的结直肠癌病例中检测 KRAS 密码子 12 和 13 突变的敏感性、特异性和准确性。在 60 例中有 20 例用所有方法检测为 KRAS 野生型，特异性为 100%。在 40 例突变病例中，至少有 1 种方法与 10 例不一致。Sanger 测序、TheraScreen 实时 PCR、焦磷酸测序和芯片杂交的敏感性分别为 85%、90%、93%和 92%，准确性分别为 90%、93%、95%和 95%。Sanger 测序的主要局限性是分析灵敏度低，而 TheraScreen 实时 PCR、焦磷酸测序和芯片杂交的灵敏度较高，但受到预设计检测的限制。Sanger 测序方法之间的一致性为 k = 0.79，其他技术的 k 值大于 0.85。肿瘤细胞富集与 KRAS 突变脱氧核糖核酸（DNA）的丰度显著相关，用 TheraScreen 实时 PCR 的 ΔCt（P = 0.03）、焦磷酸测序的突变百分比（P = 0.001）、芯片杂交的比值（P = 0.003）和 454 下一代测序的突变百分比（P = 0.004）进行评估。此外，与所有其他方法相比，454 下一代测序在突变丰度的定量方面表现出最佳的交叉相关性（P < 0.001）。我们的比较表明，下一代测序在敏感性和特异性方面优于其他技术。下一代测序将取代 Sanger 测序成为存档肿瘤组织中 KRAS 突变诊断检测的参考技术。
