EGFR ctdna Testing. Andrew Wallace 21/09/2015 Genomic Diagnostics Laboratory St. Mary s Hospital, Manchester

Transcription

1 EGFR ctdna Testing Andrew Wallace 21/09/2015 Genomic Diagnostics Laboratory St. Mary s Hospital, Manchester

2 ctdna & EGFR Testing in NSCLC EGFR ctdna testing Non-invasive - patients too sick/biopsy or cytology is not possible or inadequate (~25% patients with advanced disease) License for Gefitinib/Iressa since Sep 2014 for ctdna EGFR M+ve where no biopsy possible Mutation status reflects current tumour load - monitor disease progression/response New drugs in clinical trials targeting common EGFR resistance mutation p.t790m Unknowns Concordance with tumour genotype Cost Practicality Choice of Methodology RT-PCR BEAMing NGS Digital PCR

3 Patients All patients Diagnosed with NSCLC Consented to provide samples to help test development Biopsy EGFR result Majority EGFR mutation positive Cohort 1 22 patients newly diagnosed pre-treatment Cohort 2 8 patients whose treatment had commenced

4 Samples Samples collected between EDTA blood 2-4mL Plasma separated within 24hrs of phlebotomy 1mL plasma aliquots stored at -80 o C

5 DNA Extraction QIAamp Circulating Nucleic Acid Kit Extracted DNA used immediately postextraction 0.8mL 2mL plasma

6 EGFR ctdna Testing Method EGFR therascreen RGQ assay Analysed on ABI7900HT Real-Time PCR Normal control 7 Mutant reactions LOD range 7% - 0.5% Custom Mutant Delta Cts Ct 28.8 Delta Ct 6.2 Ct 35.0 Reaction Delta Ct cut off p.g719x 12 p.s768i 12 Exon 19 deletions 12 p.t790m 8 Exon 20 insertions 12 p.l858r 12 p.l861q 12

7 Biopsy EGFR Results Cohort 1 22 untreated patients 18 with biopsy mutation(s) detectable by Qiagen Therascreen 3 with no detectable mutation (biopsy adequate) 1 with rare Exon 19 deletion (not detected by Qiagen Therascreen)

8 Biopsy EGFR Results Cohort 2 8 patients undergoing treatment 4 with biopsy mutation(s) detectable by Qiagen Therascreen 3 with KRAS mutation & no detectable EGFR mutation 1 with rare Exon 19 deletion (not detected by Qiagen Therascreen)

9 Ct Normal vs Plasma Volume Plasma extractions all gave sufficient EGFR DNA for Therascreen assay Kit valid range for input FFPE DNA is Ct Max Ct = 31.1 Min Ct = 23.7

10 Specificity - Mutation Negative delta Cts Biopsy negative tests 97/98 True negative 1/98 False positive Insertions Ct = % specificity Low quality call L861Q S768I Insertions G719X L858R False positive could not be replicated x2 Deletions T790M

11 Sensitivity Mutation Positive delta Cts Biopsy EGFR positive mutations 18/21 detected 85.7% sensitivity Biopsy EGFR positive patients 16/18 detected 88.9% sensitivity

12 False Negative Results Patient Biopsy Result Biopsy Date - Plasma Date c.2573t>g p.(leu858arg) & c.2499g>t p.(leu833phe) p.g719x & p.s768i 20/04/12 27/04/12 19/09/14 14/10/14 Neoplastic content >30% <30% Comment 1 Comment 2 Comment 3 Rare variant c.2499g>t p.(leu833phe) not known whether in cis/trans with c.2573t>g p.(leu858arg) c.2499g>t p.(leu833phe) may interfere with ARMS priming Biopsy DNA c.2573t>g p.(leu858arg) detected but low Ct = 8.0. cf 4/5 plasma EGFR delta Cts <8.0 Common doublet mutation, can assume in cis. Single miss Not visible on Sanger in biopsy Biopsy DNA p.g719x positive Ct = 6.3 & p.s768i positive Ct = 7.9

13 Cohort 2 Patients Undergoing Treatment 4/6 (66.7%) mutations detected 3/4 (75%) mutations in patients detected Patient 3 Biopsy reported 04/03/2015 Sanger detected Exon 19del Palliative radiotherapy 12/03/15 23/03/15 Plasma taken 07/05/2015 Patient 4 Treated for 14 months on EGFR-TKI Biopsy reported 06/07/2012 Sanger detected Exon 19del & p.t790m Cisplatin & Pemetrexed chemotherapy After progression on AZD /04/2013 to 05/05/2013 Plasma taken 05/05/2013 after progression on AZD

14 Reduction in ctdna following Treatment Quantity of detectable mutant ctdna falls during successful treatment/ stable disease Mok et al 2015 Detection and Dynamic Changes of EGFR Mutations from Circulating Tumor DNA as a Predictor of Survival Outcomes in NSCLC Patients Treated with First-line Intercalated Erlotinib and Chemotherapy. Clin Cancer Res; 21:

15 Results Concordance Concordance by Patient Biopsy DNA Concordance by Assay Biopsy DNA M +ve M -ve M +ve M -ve Plasma DNA M +ve 16 0 M -ve 2 4 Plasma DNA M +ve 18 1 M -ve 3 96 Concordance 20/22 (90.1%) Concordance 114/118 (96.6%)

16 Conclusions Amount of ctdna isolated from 0.8mL 2.0mL plasma adequate for Therascreen EGFR analysis Custom/historic delta Ct cut-offs on plasma DNA discriminate well between biopsy M+ve and biopsy M-ve Analytical specificity of assays >98% is good, can be reduced further by repeat/confirmation analysis Analytical sensitivity in untreated patients ~85% is good and compares well with other studies Analytical sensitivity in treated patients appears lower and linked to treatment Clinical sensitivity of assay is reduced due to EGFR mutations undetectable by assay e.g. rare exon 19 deletions (2/10 in study) and rare passenger/doublet mutations

17 Acknowledgements Dr F Blackhall recruitment & selection of patients Lynsey Priest sampling of patients, plasma separation Shuwen Huang & Carolyn Gokhale ctdna testing Cancer Team routine EGFR testing

18 EGFR ctdna Testing Andrew Wallace 21/09/2015 Genomic Diagnostics Laboratory St. Mary s Hospital, Manchester

19 c.2573t>g (p.leu858arg) c.2499g>t p.(leu833phe) c.2573t>g (p.leu858arg)