A new classification for CNS tumors released by the WHO in 2016 considers histopathology and molecular information to establish an integrated diagnostic that should lead a better characterization of CNS tumors. This diagnostic approach for CNS play a crucial role in determining the subsequent treatment plan, so must be carry-out with the most accurate techniques. Regarding techniques used for this molecular characterization, many of them require high amount of DNA and RNA, need highly purified DNA, parameters that could not always be achieve using FFPE sample. In addition, most techniques cannot answer to all questions, and force laboratories to use various approaches. However, digital PCR (dPCR) is getting more interest in oncology, as it is highly sensitive, works in presence of inhibitors, allows copy number variation and gene rearrangement detection using only DNA sample. We decide to develop a dPCR multiplex panel for the molecular characterization of pediatric CNS tumors by detecting mutations on BRAF; FGFR1 and PIK3CA along with KIAA-BRAF fusion; FGFR1 duplication by using only DNA.
DNA sample were isolated from FFPE sample of various CNS tumors prior to be tested by classical approaches (NGS; FISH; QPCR) then by dPCR. A total of 60 samples were tested for the detection of mutation. The dPCR panel for the mutations listed above is a 3-well assay with a sensitivity down to 0.1% fractional abundance.
All sample were well characterized by dPCR, with 100% specificity and sensitivity. Concordance for each molecular alteration was 100%. Sample with low concentration gave expected results, as the biopsie was enriched in cancer cells. The assay efficiently detect KIAA-BRAF fusion and FGFR1 duplication using a CNV approach using DNA as sample.
The use of multiplexed dPCR panel allows to get molecular characterization of brain tumors in 1 day, with high sensitivity and specificity. The assay can be performed by using DNA with no need of RNA for fusion transcript detection. dPCR allow laboratories get access to affordable diagnostic tools. We believe that multiplex dPCR approach will allow every patient to get access to high quality diagnostics. Other studies will be performed on cell-free DNA.
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Legal entity responsible for the study
Assistance Publique – Hopitaux de Marseille.
F. Fina: Consultant: ID-Solutions. D. Henaff: R&D Manager: ID-Solutions. A. Bresson, V. Juline: Training: ID-Solutions. All other authors have declared no conflicts of interest.