Performance comparison of two qPCR platforms for the detection of BRAF V600E mutation

CARDOSO N,; COLLI S; MANGONE F; ROZAS D; DE MATTEO E; M V PRECIADO; LORENZETTI M

Congreso; Reunión Anual Sociedades de Biociencia. SAIC, SAI &FAIC, SAFIS; 2022

V600 mutation in BRAF modulates Ras-MEK signaling pathway in a ligand-dependent fashion and was described as a driver mutation in multiple tumor types, including central nervous system (CNS). A main drawback for gold-standard Sanger sequencing detection of V600 mutation in small, formalin-fixed paraffin-embedded (FFPE) CNS tumor biopsies is low DNA yield and integrity. Here, qPCR is a suitable alternative to detect the mutation in these cases.Our aim was to compare two qPCR commercial kits for the detection of BRAF V600E mutation in FFPE CNS tumor biopsies. The performance of the BRAF V600 Mutations Detection kit (AmoyDX) and LightMix BRAF V600E/K kit (Roche) was assessed and compared. DNA extracted from 18 FFPE Glioma biopsies, previously characterized by Sanger sequencing, 11 with V600E variant, were re-tested.AmoyDX kit correctly identified all mutated (11/11) and WT (7/7) samples. Roche kit failed to detect 3 mutated samples, while correctly identifying WT cases, thus rendering 3 false-negative results. Both kits showed 100% specificity; however, AmoyDX kit had a significantly higher sensitivity over Roche, 100% vs 73%, respectively (χ2 test, p=0.031). Both kits had positive likelihood ratios (LR+) above 10 (15.3 for AmoyDX and 11.3 for Roche), but their LR+ difference was not significant. Similarly, and although not statistically significant, the LR- was lower for the AmoyDx kit (0.04 for AmoyDX vs 0.31 for Roche). The positive predicted value (PPV) was 100% for both kits, while the negative predictive value (NPV) was 1 for AmoyDX vs 0.7 for Roche.The kit from AmoyDX represents a better diagnostic tool for the detection of BRAF V600 variant since it presented higher sensitivity and a moderately higher LR+ and NPV; being an open-platform kit represents an additional advantage. Since qPCR detects small DNA targets, assessing BRAF V600 by qPCR is of paramount importance when dealing with scarce FFPE CNS biopsies, where DNA is usually fragmented during fixation.