The Comprehensive Cancer Center of Drum Tower Hospital, Jangsu Key Laboratory for Molecular Medicine, Medical School of Nanjing University & Clinical Cancer Institute of Nanjing University, Nanjing 210008, China.
Hum Pathol. 2011 Sep;42(9):1312-8. doi: 10.1016/j.humpath.2010.06.018. Epub 2011 Mar 15.
Conventional polymerase chain reaction-based Sanger sequencing is the standard assay for the detection of K-Ras mutations. However, this method is deficient in identifying small numbers of mutation-bearing cells, and tumor-cell enrichment methods such as microdissection or macrodissection are labor intensive and not always achievable. We applied the recently described coamplification at lower denaturation temperature polymerase chain reaction, which amplifies minority alleles selectively, to detect K-Ras mutations directly in 29 formalin-fixed, paraffin-embedded pancreatic specimens and compared the results with those of conventional polymerase chain reaction. To avoid a false-negative result from the coamplification at lower denaturation temperature polymerase chain reaction assay, we applied a more sensitive peptide nucleic acid polymerase chain reaction method as the gold standard. Dilution experiments indicated an approximately 5-fold improvement in sensitivity with coamplification at lower denaturation temperature polymerase chain reaction-based Sanger sequencing. Conventional polymerase chain reaction detected K-Ras mutations in 11 formalin-fixed, paraffin-embedded pancreatic specimens (37.9%), whereas coamplification at lower denaturation temperature polymerase chain reaction could identify all of those mutations as well as mutations in 10 additional samples, for a total of 21 (72.4%, P = .002) of 29. Unlike peptide nucleic acid polymerase chain reaction, coamplification at lower denaturation temperature polymerase chain reaction identified all K-Ras mutations in specimens in which tumor cells accounted for at least 20% of the total. Adoption of coamplification at lower denaturation temperature polymerase chain reaction is straightforward and requires no additional reagents or instruments. The technique is a good strategy to detect K-Ras mutations selectively in formalin-fixed, paraffin-embedded tissues without tumor-cell enrichment.
传统的聚合酶链反应（PCR）-Sanger 测序是检测 K-Ras 突变的标准检测方法。然而，这种方法在识别少数带有突变的细胞方面存在缺陷，而微切割或宏切割等肿瘤细胞富集方法则劳动强度大，并非总是可行。我们应用了最近描述的在较低变性温度下共扩增 PCR 方法，该方法选择性地扩增少数等位基因，直接在 29 例福尔马林固定、石蜡包埋的胰腺标本中检测 K-Ras 突变，并将结果与传统 PCR 进行比较。为避免在较低变性温度下共扩增 PCR 检测的假阴性结果，我们应用了更敏感的肽核酸 PCR 作为金标准。稀释实验表明，在较低变性温度下共扩增 PCR-Sanger 测序的敏感性提高了约 5 倍。传统 PCR 在 11 例福尔马林固定、石蜡包埋的胰腺标本中检测到 K-Ras 突变（37.9%），而在较低变性温度下的共扩增 PCR 可以识别所有这些突变以及另外 10 个样本中的突变，总共 29 个样本中的 21 个（72.4%，P=0.002）。与肽核酸 PCR 不同，在肿瘤细胞至少占总细胞的 20%的标本中，共扩增 PCR 可以识别所有的 K-Ras 突变。采用在较低变性温度下共扩增 PCR 是一种简单的策略，可以选择性地在未经肿瘤细胞富集的福尔马林固定、石蜡包埋组织中检测 K-Ras 突变。
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