Multisite Analytic Performance Studies of a Real-Time Polymerase Chain Reaction Assay for the Detection of BRAF V600E Mutations in Formalin-Fixed, Paraffin-Embedded Tissue Specimens of Malignant Melanoma
Multisite Analytic Performance Studies of a Real-Time Polymerase Chain Reaction Assay for the Detection of BRAF V600E Mutations in Formalin-Fixed, Paraffin-Embedded Tissue Specimens of Malignant Melanoma
复制标题
DOI：
10.5858/arpa.2011-0505-oa
复制
发表时间：
2012-11-01
影响因子：
4.6
通讯作者：
Dugan, Michael C.
中科院分区：
文献类型：
--
作者：
Anderson, Steven;Bloom, Kenneth J.;Dugan, Michael C.
背景：-基于聚合酶链式反应的伴随诊断(COBAS 4800 BRAF V600突变测试)最近被美国食品和药物管理局批准，以选择BRAF突变的转移性黑色素瘤患者接受BRAF抑制剂Vemurafenib的治疗。目的：(1)通过使用来自Vemurafenib的II期和III期试验的筛选样本来比较COBAS测试和Sanger测序的分析性能，以及(2)评估COBAS测试在不同测试地点的重复性。设计-使用来自477名患者的样本来确定COBAS测试和Sanger测序之间检测V600E(1799T>；A；)突变的阳性和阴性百分比一致性。样本用大规模平行焦磷酸测序方法(454)进行评估，以解决聚合酶链式反应和桑格结果之间的不一致。通过使用3批试剂和8人黑色素瘤样本小组，在3个地点评估了眼镜蛇试验的重复性。结果：所有符合条件的患者都获得了有效的眼镜蛇试验结果。Sanger测序失败率为9.2%(44/477)。其余433例标本，阳性符合率为96.4%(215/223)，阴性符合率为80%(168/210)。在42例眼镜蛇突变阳性/Sanger V600E阴性的标本中，截至454年，17例V600E阳性，24例V600K阳性。眼镜蛇试验检测到70%的V600K突变。在重复性研究中，100%的野生型样本和大于5%的突变等位基因的样本被正确解释；90%的突变等位基因低于5%的样本被检测到V600E突变。结论：眼镜蛇试验(1)检测失败率低于Sanger，(2)对V600E突变的检测更敏感，(3)检测到大多数V600K突变，(4)具有很高的重复性。(ARCH Pathol Lab Med.2012年；第136：1385-1391；DOI：10.5858/arpa.2011-0505-OA)
Context.-A polymerase chain reaction-based companion diagnostic (cobas 4800 BRAF V600 Mutation Test) was recently approved by the US Food and Drug Administration to select patients with BRAF-mutant metastatic melanoma for treatment with the BRAF inhibitor vemurafenib.Objectives.-(1) To compare the analytic performance of the cobas test to Sanger sequencing by using screening specimens from phase II and phase III trials of vemurafenib, and (2) to assess the reproducibility of the cobas test at different testing sites.Design.-Specimens from 477 patients were used to determine positive and negative percent agreements between the cobas test and Sanger sequencing for detecting V600E (1799T>A) mutations. Specimens were evaluated with a massively parallel pyrosequencing method (454) to resolve discordances between polymerase chain reaction and Sanger results. Reproducibility of the cobas test was assessed at 3 sites by using 3 reagent lots and an 8-member panel of melanoma samples.Results.-A valid cobas result was obtained for all eligible patients. Sanger sequencing had a failure rate of 9.2% (44 of 477). For the remaining 433 specimens, positive percent agreement was 96.4% (215 of 223) and negative percent agreement, 80% (168 of 210). Among 42 cobas mutation-positive/Sanger V600E-negative specimens, 17 were V600E positive and 24 were V600K positive by 454. The cobas test detected 70% of V600K mutations. In the reproducibility study, a correct interpretation was made for 100% of wild-type specimens and specimens with greater than 5% mutant alleles; V600E mutations were detected in 90% of specimens with less than 5% mutant alleles.Conclusions.-The cobas test (1) had a lower assay failure rate than that of Sanger, (2) was more sensitive in detecting V600E mutations, (3) detected most V600K mutations, and (4) was highly reproducible. (Arch Pathol Lab Med. 2012;136:1385-1391; doi: 10.5858/arpa.2011-0505-OA)