Bolton Louise, Reiman Anne, Lucas Katie, Timms Judith, Cree Ian A
Department of Pathology, Queen Alexandra Hospital, Portsmouth, United Kingdom.
Department of Pathology and Warwick Medical School, University Hospitals Coventry and Warwickshire, Coventry, United Kingdom.
PLoS One. 2015 Jan 8;10(1):e0115672. doi: 10.1371/journal.pone.0115672. eCollection 2015.
KRAS mutation assays are important companion diagnostic tests to guide anti-EGFR antibody treatment of metastatic colorectal cancer. Direct comparison of newer diagnostic methods with existing methods is an important part of validation of any new technique. In this this study, we have compared the Therascreen (Qiagen) ARMS assay with Competitive Allele-Specific TaqMan PCR (castPCR, Life Technologies) to determine equivalence for KRAS mutation analysis.
DNA was extracted by Maxwell (Promega) from 99 colorectal cancers. The ARMS-based Therascreen and a customized castPCR assay were performed according to the manufacturer's instructions. All assays were performed on either an Applied Biosystems 7500 Fast Dx or a ViiA7 real-time PCR machine (both from Life Technologies). The data were collected and discrepant results re-tested with newly extracted DNA from the same blocks in both assay types.
Of the 99 tumors included, Therascreen showed 62 tumors to be wild-type (WT) for KRAS, while 37 had KRAS mutations on initial testing. CastPCR showed 61 tumors to be wild-type (WT) for KRAS, while 38 had KRAS mutations. Thirteen tumors showed BRAF mutation in castPCR and in one of these there was also a KRAS mutation. The custom castPCR plate included several other KRAS mutations and BRAF V600E, not included in Therascreen, explaining the higher number of mutations detected by castPCR. Re-testing of discrepant results was required in three tumors, all of which then achieved concordance for KRAS. CastPCR assay Ct values were on average 2 cycles lower than Therascreen.
There was excellent correlation between the two methods. Although castPCR assay shows lower Ct values than Therascreen, this is unlikely to be clinically significant.
KRAS突变检测是指导转移性结直肠癌抗表皮生长因子受体（EGFR）抗体治疗的重要伴随诊断检测。将更新的诊断方法与现有方法进行直接比较是任何新技术验证的重要组成部分。在本研究中，我们将Therascreen（Qiagen）ARMS检测与竞争性等位基因特异性TaqMan PCR（castPCR，Life Technologies）进行了比较，以确定KRAS突变分析的等效性。
使用Maxwell（Promega）从99例结直肠癌中提取DNA。基于ARMS的Therascreen检测和定制的castPCR检测均按照制造商的说明进行。所有检测均在Applied Biosystems 7500 Fast Dx或ViiA7实时PCR仪（均来自Life Technologies）上进行。收集数据，并对两种检测类型中来自相同组织块的新提取DNA重新检测不一致的结果。
在纳入的99个肿瘤中，Therascreen检测显示62个肿瘤KRAS为野生型（WT），而37个在初次检测时有KRAS突变。CastPCR检测显示61个肿瘤KRAS为野生型（WT），而38个有KRAS突变。13个肿瘤在castPCR检测中显示BRAF突变，其中1个同时有KRAS突变。定制的castPCR板包含了一些Therascreen未涵盖的其他KRAS突变和BRAF V600E，这解释了castPCR检测到的突变数量较多的原因。3个肿瘤需要重新检测不一致的结果，所有这些肿瘤随后在KRAS检测上达成一致。CastPCR检测的Ct值平均比Therascreen低2个循环。
两种方法之间存在极好的相关性。虽然castPCR检测的Ct值低于Therascreen，但这在临床上不太可能具有显著意义。
