Dudley Jonathan C, Gurda Grzegorz T, Tseng Li-Hui, Anderson Derek A, Chen Guoli, Taube Janis M, Gocke Christopher D, Eshleman James R, Lin Ming-Tseh
Department of Pathology, Johns Hopkins University School of Medicine, Johns Hopkins Hospital, Park SB202, 600 North Wolfe St., Baltimore, MD, 21287, USA.
Mol Diagn Ther. 2014 Aug;18(4):409-18. doi: 10.1007/s40291-014-0091-6.
Detection of BRAF mutations is an established standard of care to predict small-molecule inhibitor (vemurafenib) response in metastatic melanoma. Molecular assays should be designed to detect not only the most common p.V600E mutation, but also p.V600K and other non-p.V600E mutations.
The purpose of this study was to assess if tumor cellularity can function as a quality assurance (QA) measure in molecular diagnostics. Potential causes of discrepancy between the observed and predicted mutant allele percentage were also explored.
We correlated pathologist-generated estimates of tumor cellularity versus mutant allele percentage via pyrosequencing as a QA measure for BRAF mutation detection in formalin-fixed, paraffin-embedded melanoma specimens.
BRAF mutations were seen in 27/62 (44 %) specimens, with 93 % p.V600E and 7 % non-p.V600E. Correlation between p.V600E mutant percentage and tumor cellularity was poor-moderate (r = -0.02; p = 0.8), primarily because six samples showed a low p.V600E signal despite high tumor cellularity. A QA investigation revealed that our initial pyrosequencing assay showed a false positive, weak p.V600E signal in specimens with a p.V600K mutation. A redesigned assay detected BRAF mutations in 50/131 (38 %) specimens, including 30 % non-p.V600E. This revised assay showed strong correlation between p.V600E BRAF mutant percentage and tumor cellularity (r = 0.76; p ≤ 0.01). Re-evaluation of the previously discordant samples by the revised assay confirmed a high level of p.V600K mutation in five specimens.
Pathologists play important roles in molecular diagnostics, beyond identification of correct cells for testing. Accurate evaluation of tumor cellularity not only ensures sufficient material for required analytic sensitivity, but also provides an independent QA measure of the molecular assays.
检测BRAF突变是预测转移性黑色素瘤对小分子抑制剂（维莫非尼）反应的既定标准治疗方法。分子检测不仅应设计用于检测最常见的p.V600E突变，还应检测p.V600K和其他非p.V600E突变。
本研究的目的是评估肿瘤细胞含量是否可作为分子诊断中的质量保证（QA）指标。还探讨了观察到的和预测的突变等位基因百分比之间差异的潜在原因。
我们通过焦磷酸测序将病理学家生成的肿瘤细胞含量估计值与突变等位基因百分比相关联，作为福尔马林固定、石蜡包埋黑色素瘤标本中BRAF突变检测的QA指标。
在27/62（44%）的标本中发现BRAF突变，其中93%为p.V600E，7%为非p.V600E。p.V600E突变百分比与肿瘤细胞含量之间的相关性为中低水平（r = -0.02；p = 0.8），主要是因为六个样本尽管肿瘤细胞含量高，但p.V600E信号较低。一项QA调查显示，我们最初的焦磷酸测序检测在具有p.V600K突变的标本中显示出假阳性、微弱的p.V600E信号。重新设计的检测在50/131（38%）的标本中检测到BRAF突变，包括30%的非p.V600E。这种修订后的检测显示p.V600E BRAF突变百分比与肿瘤细胞含量之间具有强相关性（r = 0.76；p≤0.01）。通过修订后的检测对先前不一致的样本进行重新评估，证实五个标本中存在高水平的p.V600K突变。
病理学家在分子诊断中发挥着重要作用，不仅仅是识别用于检测的正确细胞。准确评估肿瘤细胞含量不仅可确保有足够的材料用于所需的分析灵敏度，还可为分子检测提供独立的QA指标。
