Droplet digital PCR for detection of BRAF V600E mutation in formalin-fixed, paraffin-embedded melanoma tissues: a comparison with Cobas® 4800, Sanger sequencing, and allele-specific PCR

American Journal of Translational Research

Marian Grendar¹  Eva Minarikova²  Barbora Vanova³  Tatiana Burjanivova⁴  Martina Bobrovska⁵  Eva Jezkova⁶  Bibiana Malicherova⁷  Karin Jasek⁸ 

[1] Bioinformatic Unit, Biomedical Center Martin JFM CU, Jessenius Faculty of Medicine in Martin, Commenius University in Bratislava, Mala Hora 4C, 036 01 Martin;Clinic of Dermatovenerology, Jessenius Faculty of Medicine and University Hospital in Martin, Kollarova 2, 036 01 Martin;Department of Histology and Embryology, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, 036 01 Martin;Department of Molecular Biology, Slovakia JFM CU, Biomedical Center Martin JFM CU, Jessenius Faculty of Medicine in Martin (JFM CU), Commenius University in Bratislava, Mala Hora 4C, 036 01 Martin;Department of Pathological Anatomy, Jessenius Faculty of Medicine and University Hospital in Martin, Kollarova 2, 036 01 Martin;Department of Plastic Surgery, Jessenius Faculty of Medicine and University Hospital in Martin, Kollarova 2, 036 01 Martin;Division of Oncology, Slovakia JFM CU, Biomedical Center Martin JFM CU, Jessenius Faculty of Medicine in Martin (JFM CU), Commenius University in Bratislava, Mala Hora 4C, 036 01 Martin;Division of Respirology JFM CU, Mala Hora 4C, 036 01 Martin

关键词: Melanoma;    ddPCR;    BRAF V600E;    methods;    diagnostic biomarker;    treatment;   

DOI  :  

学科分类：医学（综合）

来源: e-Century Publishing Corporation

【 摘 要 】

Cutaneous melanoma has the worst prognosis of all skin cancers. Although emerging targeted therapies, such as B-Raf kinase inhibitor vemurafenib, improve prognosis they require an accurate and sensitive means of detecting the pathogenic BRAF V600E mutation. We compared the sensitivity of four BRAF V600E detection methods in formalin-fixed, paraffin-embedded melanoma biopsies from 87 consecutive melanoma patients with Breslow stage I-V disease (staging based on the depth of tumor of invasion). The methods assessed were the widely used Cobas® 4800 system based on real-time PCR amplification, Sanger sequencing, allele-specific PCR (AS-PCR), and droplet digital PCR (ddPCR). The BRAF V600E mutation was found in 8 (9.2%), 23 (26.4%), 23 (26.4%) and 31 (35.6%) biopsies, respectively. The limit of detection (LoD) was determined by three different methods: Poisson confidence limits, calibration regression and Tzonev’s method. Pair-wise agreement between the methods was as follows: Cobas vs. Sanger, P = 0.33; Cobas® 4800 vs. AS-PCR, P = 0.33; Cobas® 4800 vs. ddPCR, P = 0.65; Sanger vs. AS-PCR, P = 1; Sanger vs. ddPCR, P = 0.08; AS-PCR vs. ddPCR, P = 0.06. Multinomial logistic regression was used for predictive modeling of the Breslow-Clark score; ddPCR emerged as the best predictor, the other predictors were mitotic activity, type of malignant melanoma and patient’s age. Our results demonstrate that ddPCR is the most sensitive method of detecting the BRAF V600E mutation.

【 授权许可】

CC BY-NC