Tzanikou Eleni, Haselmann Verena, Markou Athina, Duda Angelika, Utikal Jochen, Neumaier Michael, Lianidou Evi S
Analysis of Circulating Tumor Cells Lab, Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Athens, Greece.
Institute for Clinical Chemistry, University Medical Center, Ruprecht-Karls University of Heidelberg, Mannheim, Germany.
Clin Chem Lab Med. 2020 Oct 25;58(11):1799-1807. doi: 10.1515/cclm-2019-0783.
Background In metastatic melanoma, 40%-50% of patients harbor a BRAF V600E mutation and are thereby eligible to receive a combined BRAF/MEK inhibitor therapy. Compared to standard-of-care tissue-based genetic testing, analysis of circulating tumor DNA (ctDNA) from blood enables a comprehensive assessment of tumor mutational status in real-time and can be used for monitoring response to therapy. The aim of our study was to directly compare the performance of two highly sensitive methodologies, droplet digital PCR (ddPCR) and a combination of ARMS/asymmetric-rapid PCR/melting curve analysis, for the detection of BRAF V600E in plasma from melanoma patients. Methods Cell-free DNA (cfDNA) was isolated from 120 plasma samples of stage I to IV melanoma patients. Identical plasma-cfDNA samples were subjected to BRAF V600E mutational analysis using in parallel, ddPCR and the combination of ARMS/asymmetric-rapid PCR/melting curve analysis. Results BRAF V600E mutation was detected in 9/117 (7.7%) ctDNA samples by ddPCR and in 22/117 (18.8%) ctDNA samples by the combination of ARMS/asymmetric- rapid PCR/melting curve analysis. The concordance between these two methodologies was 85.5% (100/117). The comparison of plasma-ctDNA analysis using ddPCR and tissue testing revealed an overall agreement of 79.4% (27/34), while the corresponding agreement using the combination of ARMS/asymmetric-rapid PCR/melting curve analysis was 73.5% (25/34). Moreover, comparing the detection of BRAF-mutant ctDNA with the clinics, overall agreement of 87.2% (48/55) for ddPCR and 79.2% (42/53) was demonstrated. Remarkably, the duration of sample storage was negatively correlated with correctness of genotyping results highlighting the importance of pre-analytical factors. Conclusions Our direct comparison study has shown a high level of concordance between ddPCR and the combination of ARMS/asymmetric-rapid PCR/melting curve analysis for the detection of BRAF V600E mutations in plasma.
背景 在转移性黑色素瘤患者中，40%-50%的患者存在BRAF V600E突变，因此有资格接受BRAF/MEK抑制剂联合治疗。与基于组织的标准护理基因检测相比，对血液中循环肿瘤DNA（ctDNA）的分析能够实时全面评估肿瘤突变状态，并可用于监测治疗反应。我们研究的目的是直接比较两种高灵敏度方法——液滴数字PCR（ddPCR）和ARMS/不对称快速PCR/熔解曲线分析组合——检测黑色素瘤患者血浆中BRAF V600E的性能。方法 从120例I至IV期黑色素瘤患者的血浆样本中分离游离DNA（cfDNA）。将相同的血浆cfDNA样本同时采用ddPCR以及ARMS/不对称快速PCR/熔解曲线分析组合进行BRAF V600E突变分析。结果 通过ddPCR在9/117（7.7%）的ctDNA样本中检测到BRAF V600E突变，通过ARMS/不对称快速PCR/熔解曲线分析组合在22/117（18.8%）的ctDNA样本中检测到该突变。这两种方法之间的一致性为85.5%（100/117）。使用ddPCR进行血浆ctDNA分析与组织检测的总体一致性为79.4%（27/34），而使用ARMS/不对称快速PCR/熔解曲线分析组合时相应的一致性为73.5%（25/34）。此外，将BRAF突变ctDNA的检测结果与临床情况进行比较，ddPCR的总体一致性为87.2%（48/55），ARMS/不对称快速PCR/熔解曲线分析组合的一致性为79.2%（42/53）。值得注意的是，样本储存时间与基因分型结果的正确性呈负相关，突出了分析前因素的重要性。结论 我们的直接比较研究表明，在检测血浆中BRAF V600E突变方面，ddPCR与ARMS/不对称快速PCR/熔解曲线分析组合之间具有高度一致性。
