Résumé
Background: Recent progress in the ‘liquid biopsy’ field in combination with the development of the droplet digital PCR (ddPCR) technology now enables non-invasive monitoring of cancer-related genomic alterations with high detection accuracy. Current ddPCR techniques represent the gold standard for the detection of point mutations that have been previously characterized on tumor tissues. However, ddPCR screen for only one mutation (or a few mutations, with multicolor ddPCR) per reaction. This is a clear limitation to use it as a discovery tool, to detect resistance-associated mutations that may appear during therapy (e.g. ESR1 activating mutations in breast cancer) or when tumor tissue is not available (e.g. EGFR activating mutations in lung cancers).
Methods:
We developed the Multiple Hotspot mutations detection by Single droplet digital PCR (MHS-ddPCR) method, a variant of the conventional ddPCR technique, which detects all genomic alterations within a hotspot region, using a unique couple of Taqman oligo-probes. We first established two specific assays covering KRAS and EGFR mutation hotspot regions, which are of clinical importance in the context of colorectal cancer. The assay for KRAS scans for the 7 most common mutations in codons 12 and 13 of the gene as well as all other mutations with lower frequency (<1%). The EGFR assay screens for all in-frame deletions of exon 19, which are frequent activating events in EGFR.
Results:
Sensitivity and specificity of MHS-ddPCR were assessed on decreasing fractions of tumor DNA mutated for KRAS. The KRAS assay reaches a sensitivity of 0.02% and was validated on plasma and tumor samples harboring a panel of different KRAS mutations. Similarly, the EGFR assay can detect numerous exon 19 deletions from patient samples and with limited amounts of tumor DNA.
Conclusions:
The MHS-ddPCR is extremely sensitive, works for numerous hotspot regions with different types of alterations (SNV, deletion) and is cost-effective as it combines multiple assays in one reaction.
Citation Format: Charles Decraene, Francois-Clement Bidard, Samia Belaabi, Etienne Rouleau, Adrien Saliou, Alexandre Houy, Maud Milder, Olivier Lantz, Marc Ychou, Jean-Yves Pierga, Marc-Henri Stern, Charlotte Proudhon. Exhaustive scanning of mutation hotspot regions using a new tool: The MHS-ddPCR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 410.