Charles Decraene1,2, Amanda B Silveira1, François-Clément Bidard1,3, Audrey Vallée4, Marc Michel1, Samia Melaabi5, Anne Vincent-Salomon5,6, Adrien Saliou1, Alexandre Houy1,7, Maud Milder1,8, Olivier Lantz1,8,9, Marc Ychou10, Marc G Denis4, Jean-Yves Pierga1,3,11, Marc-Henri Stern1,7, Charlotte Proudhon12. 1. Circulating Tumor Biomarkers Laboratory, SiRIC, Translational Research Department, Institut Curie, PSL Research University, Paris, France. 2. CNRS UMR144, Institut Curie, PSL Research University, Paris, France. 3. Department of Medical Oncology, Institut Curie, PSL Research University, Paris, France. 4. Department of Biochemistry and INSERM U1232, Nantes University Hospital, Nantes, France. 5. Department of Biopathology, Institut Curie, PSL Research University, Paris, France. 6. Inserm U934, Institut Curie, PSL Research University, Paris, France. 7. Inserm U830, Institut Curie, PSL Research University, Paris, France. 8. Inserm CIC BT 1418, Institut Curie, PSL Research University, Paris, France. 9. Inserm U932, Institut Curie, PSL Research University, Paris, France. 10. Department of Medical Oncology, Montpellier Cancer Institute, Montpellier, France. 11. University Paris Descartes, Paris, France. 12. Circulating Tumor Biomarkers Laboratory, SiRIC, Translational Research Department, Institut Curie, PSL Research University, Paris, France; Charlotte.Proudhon@curie.fr.
Abstract
BACKGROUND: Progress in the liquid biopsy field, combined with the development of droplet digital PCR (ddPCR), has enabled noninvasive monitoring of mutations with high detection accuracy. However, current assays detect a restricted number of mutations per reaction. ddPCR is a recognized method for detecting alterations previously characterized in tumor tissues, but its use as a discovery tool when the mutation is unknown a priori remains limited. METHODS: We established 2 ddPCR assays detecting all genomic alterations within KRAS exon 2 and EGFR exon 19 mutation hotspots, which are of clinical importance in colorectal and lung cancer, with use of a unique pair of TaqMan® oligoprobes. The KRAS assay scanned for the 7 most common mutations in codons 12/13 but also all other mutations found in that region. The EGFR assay screened for all in-frame deletions of exon 19, which are frequent EGFR-activating events. RESULTS: The KRAS and EGFR assays were highly specific and both reached a limit of detection of <0.1% in mutant allele frequency. We further validated their performance on multiple plasma and formalin-fixed and paraffin-embedded tumor samples harboring a panel of different KRAS or EGFR mutations. CONCLUSIONS: This method presents the advantage of detecting a higher number of mutations with single-reaction ddPCRs while consuming a minimum of patient sample. This is particularly useful in the context of liquid biopsy because the amount of circulating tumor DNA is often low. This method should be useful as a discovery tool when the tumor tissue is unavailable or to monitor disease during therapy.
BACKGROUND: Progress in the liquid biopsy field, combined with the development of droplet digital PCR (ddPCR), has enabled noninvasive monitoring of mutations with high detection accuracy. However, current assays detect a restricted number of mutations per reaction. ddPCR is a recognized method for detecting alterations previously characterized in tumor tissues, but its use as a discovery tool when the mutation is unknown a priori remains limited. METHODS: We established 2 ddPCR assays detecting all genomic alterations within KRAS exon 2 and EGFR exon 19 mutation hotspots, which are of clinical importance in colorectal and lung cancer, with use of a unique pair of TaqMan® oligoprobes. The KRAS assay scanned for the 7 most common mutations in codons 12/13 but also all other mutations found in that region. The EGFR assay screened for all in-frame deletions of exon 19, which are frequent EGFR-activating events. RESULTS: The KRAS and EGFR assays were highly specific and both reached a limit of detection of <0.1% in mutant allele frequency. We further validated their performance on multiple plasma and formalin-fixed and paraffin-embedded tumor samples harboring a panel of different KRAS or EGFR mutations. CONCLUSIONS: This method presents the advantage of detecting a higher number of mutations with single-reaction ddPCRs while consuming a minimum of patient sample. This is particularly useful in the context of liquid biopsy because the amount of circulating tumor DNA is often low. This method should be useful as a discovery tool when the tumor tissue is unavailable or to monitor disease during therapy.
Authors: Panagiota M Kalligosfyri; Sofia Nikou; Sofia Karteri; Haralabos P Kalofonos; Vasiliki Bravou; Despina P Kalogianni Journal: Biosensors (Basel) Date: 2022-02-04