TY - JOUR
TI - Direct comparison study between droplet digital PCR and a combination of allele-specific PCR, asymmetric rapid PCR and melting curve analysis for the detection of BRAF V600E mutation in plasma from melanoma patients
AU - Tzanikoua, E.
AU - Haselmanna, V.
AU - Markou, A.
AU - Duda, A.
AU - Utikal, J.
AU - Neumaier, M.
AU - Lianidou, E.S.
JO - Clinical Chemistry and Laboratory Medicine (CCLM)
PY - 2020
VL - 58
TODO - 11
SP - 1799-1807
PB - De Gruyter Open Ltd
SN - null
TODO - 10.1515/cclm-2019-0783
TODO - B Raf kinase;  cell free DNA;  circulating tumor DNA;  DNA;  unclassified drug;  B Raf kinase;  BRAF protein, human;  circulating tumor DNA, adult;  aged;  allele specific polymerase chain reaction;  Article;  blood sampling;  cancer staging;  comparative study;  controlled study;  DNA isolation;  droplet digital polymerase chain reaction;  female;  gene amplification;  gene mutation;  human;  limit of blank;  limit of detection;  limit of quantitation;  major clinical study;  male;  melanoma;  mutational analysis;  priority journal;  Sanger sequencing;  blood;  cohort analysis;  genetics;  liquid biopsy;  melanoma;  middle aged;  mutation;  polymerase chain reaction;  procedures;  specimen handling;  time factor;  very elderly, Adult;  Aged;  Aged, 80 and over;  Circulating Tumor DNA;  Cohort Studies;  Female;  Humans;  Liquid Biopsy;  Male;  Melanoma;  Middle Aged;  Mutation;  Polymerase Chain Reaction;  Proto-Oncogene Proteins B-raf;  Specimen Handling;  Time Factors
TODO - 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/asymmetricrapid 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 preanalytical 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. © 2020 De Gruyter. All rights reserved.
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