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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.

This study tested the claim that digital PCR (dPCR) can offer highly reproducible quantitative measurements in disparate laboratories. Twenty-one laboratories measured four blinded samples containing different quantities of a KRAS fragment encoding G12D, an important genetic marker for guiding therapy of certain cancers. This marker is challenging to quantify reproducibly using quantitative PCR (qPCR) or next generation sequencing (NGS) due to the presence of competing wild type sequences and the need for calibration. Using dPCR, 18 laboratories were able to quantify the G12D marker within 12% of each other in all samples. Three laboratories appeared to measure consistently outlying results; however, proper application of a follow-up analysis recommendation rectified their data. Our findings show that dPCR has demonstrable reproducibility across a large number of laboratories without calibration. This could enable the reproducible application of molecular stratification to guide therapy and, potentially, for molecular diagnostics.