1. 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

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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

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

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

11. 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

12. 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

13. 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