Crystal digital PCR for detection and quantification of circulating EGFR mutations in advanced non-small cell lung cancer Cécile Jovelet Postdocoral fellow Translational research lab Gustave Roussy Institute, France

detection of EGFR mutations in cfDNA by dPCR Introduction Lung cancer: third most frequent cancer Non-Small Cell Lung Cancer (NSCLC): 80% cases of lung cancer Sensitivity to EGFR-TKIs for patients harboring EGFR activating mutations 11% of NSCLC Deletion exon 19, L858R, L861Q Development of secondary resistance to EGFR-TKIs 50% of resistance: mutation T790M in exon 20 of the EGFR gene Third-generation EGFR-TKIs: active on tumor cells harboring also EGFR T790M resistant mutations. Their prescription: requirement of detection of resistance mutation in a new tissue biopsy. 12/04/2017 detection of EGFR mutations in cfDNA by dPCR

detection of EGFR mutations in cfDNA by dPCR Limitations for NSCLC rebiopsying: lack of available tissue for molecular profile assessment, the location or size of the tumor, the insufficient quality or quantity of genetic material, and the risk of complications Plasma circulating free DNA (cfDNA) analysis: minimally-invasive alternative to tissue biopsy Potential applications of cfDNA based analysis: molecular abnormalities characterization at diagnosis, patient selection for targeted therapies, monitoring of treatment response, and screening for acquired mutations at the time of resistance 12/04/2017 detection of EGFR mutations in cfDNA by dPCR

detection of EGFR mutations in cfDNA by dPCR Main objectives: Detect EGFR activating and resistance mutations in cfDNA of NSCLC patients by digital PCR. Compare the dPCR results with NGS analysis Monitor the cfDNA in longitudinal samples during follow-up. Methods: Extraction: Blood sample: 10mL, EDTA tubes Double centrifugation, < 4h after sampling Extraction: 3 mL Plasma, QIAamp circulating nucleic acid Kit (Qiagen) Detection of mutation: NGS (PGM, Ion Torrent), Crystal digital PCR (Stilla) 12/04/2017 detection of EGFR mutations in cfDNA by dPCR

Detection of EGFR mutations by CrystalTM Digital PCR Nucleic acid extraction Under pressure, sample is compartmentalized into 20,000 to 30,000 droplets Target amplification by PCR Each droplet is analysed individually (fluorescence quantification) 12/04/2017   detection of EGFR mutations in cfDNA by dPCR 22/09/2018 cfDNA 5

detection of EGFR mutations in cfDNA by dPCR EGFR L858R Wild-type Targets of EGFR assay: EGFR activating mutations: exon 19 deletion, L858R, L861Q EGFR resistance mutation: T790M EGFR T790M High sensibility (AF > 0,05 %) Limit of detection is 2 copies of mutated DNA per chamber 12/04/2017   detection of EGFR mutations in cfDNA by dPCR 22/09/2018 cfDNA 6

Detection of EGFR mutations by NGS Plasma samples were analyzed by PGM (Ion Torrent) 10ng of cfDNA Cancer Hotspot Panel v2 (CHP2) targeting 50 cancer genes (EGFR) Patients Investigation of the EGFR mutational status in cfDNA of 61 patients with advanced stage IV NSCLC and for whom re-biopsy was not feasible. 11 patients: no tumor EGFR mutation in non-synchronous tumor tissue (by NGS) 50 patients: EGFR mutations detected in non-synchronous tumor tissue (by NGS), treated by EGFR TKI therapies Among the 50 patients with EGFR mutation in tumor tissue, 14 patients had at least one follow-up sample 7 patients had at least 3 samples. in order to monitor mutations detected at baseline or to detect new mutations 12/04/2017 detection of EGFR mutations in cfDNA by dPCR

detection of EGFR mutations in cfDNA by dPCR Results 11 patients with no EGFR mutation detected in tumor DNA No EGFR mutation detected in cfDNA (dPCR and NGS) 50 patients harboring EGFR activating mutations in tumor DNA (non-synchronous) In Day 0 samples: 29 EGFR activating mutations 14 EGFR resistance mutations T790M 12/04/2017 detection of EGFR mutations in cfDNA by dPCR

detection of EGFR mutations in cfDNA by dPCR 12/04/2017 detection of EGFR mutations in cfDNA by dPCR

detection of EGFR mutations in cfDNA by dPCR 12/04/2017 detection of EGFR mutations in cfDNA by dPCR

dPCR is a more sensitive technique than NGS Good correlation of mutation allele frequencies detected by NGS and dPCR ddPCR NGS Day 0 samples EGFR activating mutations 29 28 (n=50) EGFR resistance mutation 14 11 Follow-up samples 17 (n=26) 7 6 total 67 56 dPCR is a more sensitive technique than NGS 12/04/2017 detection of EGFR mutations in cfDNA by dPCR

detection of EGFR mutations in cfDNA by dPCR Conclusion: Detection of EGFR mutations in cfDNA Detection of EGFR resistance mutation for 14 patients: change of treatment Better sensitivity of dPCR compared to NGS accurate monitoring of EGFR mutations in follow-up samples. reflect of the evolution of the disease along the treatments dPCR: better suited to the detection of known mutations than NGS sensitivity, cost and time (2h vs 2days) 12/04/2017 detection of EGFR mutations in cfDNA by dPCR

Thanks to Thank you for your attention Dr. Ludovic Lacroix Dr. Benjamin Besse Translationnal research Lab Team Patients Dr. Magali Droniou Dr. Jordan Madic ?? 12/04/2017