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. 2020 Nov 12:10:579762.
doi: 10.3389/fonc.2020.579762. eCollection 2020.

Multiplexed Droplet Digital PCR Assays for the Simultaneous Screening of Major Genetic Alterations in Tumors of the Central Nervous System

Romain Appay  1   2 , Frederic Fina  1   3 , Doriane Barets  1 , Catherine Gallardo  1 , Isabelle Nanni-Metellus  4 , Didier Scavarda  5 , Daniel Henaff  3 , Juline Vincent  3 , Lise Grewis  3 , Philippe Pourquier  3 , Carole Colin  2 , Dominique Figarella-Branger  1   2
Affiliations

Multiplexed Droplet Digital PCR Assays for the Simultaneous Screening of Major Genetic Alterations in Tumors of the Central Nervous System

Romain Appay et al. Front Oncol. .

Abstract

The increased integration of molecular alterations to define tumor type or grade in central nervous system (CNS) tumor classification brings new challenges for the pathologist to make the best use of a precious limited tissue specimen for molecular studies. Within the different methods available to identify gene alterations, the droplet digital PCR (dPCR) constitutes a rapid, cost-effective, and very sensitive tool. In this study, we describe the development and validation of five multiplexed dPCR assays to detect major CNS biomarkers by using only small amounts of DNA extracted from formalin-fixed paraffin-embedded specimens. When compared to HRM-sequencing, NGS-sequencing, RNA-sequencing, or simplex digital PCR assays used as "gold standard" methods, these multiplexed dPCR assays displayed 100% specificity and sensitivity for the simultaneous detection of: 1/BRAF V600E mutation and KIAA1549:BRAF fusion; 2/FGFR1 N546K and K656E mutations and FGFR1 duplication; 3/H3F3A K27M and G34R/V mutations; 4/IDH1 R132X and IDH2 R172X mutations; and 5/TERT promoter mutations C228T and C250T. In light of the increased integration of molecular alteration, we believe that such strategies might help laboratories to optimize their screening strategies for routine diagnosis of pediatric and adult CNS tumors.

Keywords: biomarkers; formaldehyde-fixed sample tissue; glial and glioneuronal tumors; molecular screening test; multiplexed droplet digital PCR assay; tumors of the central nervous system.

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Figures

Figure 1
Figure 1
Droplet digital PCR results. Two dimensional representations of the BRAF (A), FGFR1 (B), H3F3A (C), IDH1/2 (D) and pTERT (E) multiplexed droplet digital assays. Fluorescence amplitude at channels FAM and HEX are plotted at Y- and at X-axis, respectively. Each dot on the figure represents one droplet, and the corresponding targets are designated for each cluster. Bottom left clusters represent negative droplets.
See this image and copyright information in PMC

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