Genotyping of the Kirsten Ras (KRAS) proto-oncogene, particularly within its wild-type (WT) codons G12 and G13, is used clinically to define courses of therapy for colorectal cancer (CRC). Stratification of CRC can be improved by genotyping BRAF in conjunction with KRAS, as missense (nonsynonymous) mutations (MTs) in BRAF V600 are frequently observed in MLH1-deficient CRCs, and BRAF V600E-positive tumours correlate with poor CRC patient outcomes. While next-generation sequencing can be used to co-genotype KRAS and BRAF, clinics could benefit from a faster, more cost-effective assay of missense mutations in both KRAS and BRAF that unequivocally discriminates them from all known synonymous WT alleles. We previously reported a novel diagnostic technology that leverages the unique capabilities of droplet digital PCR (ddPCR) to achieve sensitive and quantitative detection of all known missense mutations in BRAF V600. Here, we report a ddPCR-based method to co-genotype KRAS G12/13 missense mutations and unequivocally differentiate them from all WT KRAS alleles. When combined with our test of BRAF status, the resulting inexpensive and rapid multiplex ddPCR assay accurately detects all known missense mutations in both KRAS G12/G13 and BRAF V600. The assay is applied to formalin-fixed paraffin-embedded tumour specimens from a cohort of 87 MLH1-deficient CRC patients, and is shown to correctly identify BRAF V600 and KRAS G12/G13 missense mutations in each case. Two KRAS G12/13 mutation positive patients within the cohort are found to also carry a V600E missense mutation in BRAF. Pathological implications of this rarely observed co-mutation are discussed.

中文翻译：

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使用经济的数字 PCR 检测分析结直肠癌中的 KRAS G12/G13，明确区分错义和同义等位基因

Kirsten Ras ( KRAS ) 原癌基因的基因分型，特别是在其野生型 (WT) 密码子 G12 和 G13 中，在临床上用于定义结肠直肠癌 (CRC) 的治疗过程。通过结合KRAS对BRAF进行基因分型，可以改善 CRC 的分层，因为在 MLH1 缺陷型 CRC 中经常观察到BRAF V600 中的错义（非同义）突变（MT），并且BRAF V600E 阳性肿瘤与较差的 CRC 患者预后相关。虽然新一代测序可用于对KRAS和BRAF进行共基因分型，但临床可以从更快、更具成本效益的KRAS和BRAF明确将它们与所有已知的同义 WT 等位基因区分开来。我们之前报道了一种新型诊断技术，该技术利用液滴数字 PCR (ddPCR) 的独特功能来实现对BRAF V600中所有已知错义突变的灵敏和定量检测。在这里，我们报告了一种基于 ddPCR 的方法来对KRAS G12/13 错义突变进行共基因分型，并将它们与所有 WT KRAS等位基因明确区分开来。结合我们的BRAF状态测试，由此产生的廉价且快速的多重 ddPCR 检测可准确检测KRAS G12/G13 和BRAF 中的所有已知错义突变V600。该检测应用于福尔马林固定石蜡包埋的 87 名 MLH1 缺陷 CRC 患者队列的肿瘤标本，并显示在每种情况下都能正确识别BRAF V600 和KRAS G12/G13 错义突变。发现队列中的两名KRAS G12/13 突变阳性患者在BRAF 中也携带 V600E 错义突变。讨论了这种很少观察到的共突变的病理学意义。