Diagnostic with alternative sample types (liquid biopsy)

Transcription

1 MOLECULAR DIAGNOSTICS OF EGFR AND T790M MUTATIONS CHALLENGES AND SOLUTIONS Diagnostic with alternative sample types (liquid biopsy) James CH Yang, MD, PhD Director, Professor, Graduate Institute of Oncology National Taiwan University, College of Medicine Director, Department of Oncology, National Taiwan University Hospital

2 Conflict of Interest Statement James Chih-Hsin Yang received honorarium for speech or participated in compensated advisory board of Boehringer Ingelheim, Eli Lilly, Bayer, Roche/Genentech/Chugai, Astellas, MSD, Merck Serono, Pfizer, Novartis, Clovis Oncology, Celgene, Merrimack, Yuhan Pharmaceuticals, BMS and Ono pharmaceutical and uncompensated advisory board of Astrazeneca

3 Which substances can we detect in blood? Blood cells : WBC, RBC, Platelet, etc. Proteins and their fragments: albumin, globulin, et. Metabolites Lipid Tumor cells : circulating tumor cells Cell free DNA (cfdna) : From disintegrated cells Micro RNA Exosomes (DNA and cell contents from alive cells)

4 Crowly et al. Nature Reviews Clinical Oncology 2013

5 Advantage of plasma (cell free) cfdna tests DNA is relatively stable in the circulation and in the plasma after separation Easy for sample acquisition and sample processing Rapid turn around time, eliminate scheduling waiting time for biopsy Possibility for serial monitoring during treatment and follow up Good for genomic mutation detection Possibility to analyze genomic amplification and deletion Summation of tumor heterogeneity

6 Disadvantage of plasma (cell free) cfdna tests cfdna is present in small amount, from both tumor cells and from normal cells, mutant DNA mostly present in very small % PCR amplification is necessary, thus, cross-contamination or artificial introduction of mutant sequence in the procedures is possible Microdissection to increase tumor content is not possible Exosomes may contain high amount of tumor DNA, but separation of exosomes may not be easy Cannot analyze spatial relationship in microenvironment Cannot analyze spatial tumor heterogeneity To analyze genomic deletion is a difficult task even with NGS

7 Technologies to enrich tumor DNA in the circulation for analysis Use circulating tumor cells : technically difficult and expensive Plasma or serum : plasma is a much better source of cfdna From exosomes : technically time consuming, complex and not standardized yet Allelic-specific PCR amplification Use probes to capture sequence of interest Synthesize amplified fragments sequences after capture using next generation sequence (NGS) techniques

8 Current methods to detect cfdna QuantitativePCR Digital PCR Next-Generation Sequencing Real-Time PCR ARMS/Scorpion Mutant allele-specific BEAMing Digital Droplet PCR (ddpcr) Microfluidic digital PCR Hybrid capturebased CAPP-seq Tam-seq Kittable: Therascreen COBAS Highly sensitive, limited foci Highly sensitive, flexible range of coverage Adapted from Qin et al. Chinese Journal of Cancer 2016

9 COBAS for EGFR mutation test

10 Digital droplet PCR (ddpcr)

11 BEAMing techniques

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13 Plasma vs. Serum cfdna to detect EGFR mutations in LL3, LL6 studies Detected by SCORPION/ARMS (Therascreen) Mutation type LUX-Lung 3 Tumour tissue/cfdna cfdna no Sensitivity Specificity +/+ +/ / /+ result (%) (%) Del19 c L858R c Exon20 c G719X c L861Q c S768I c T790M c Overall concordance d % d 28.60% LUX-Lung 6 SERUM Del19 c L858R c Exon20 c PLASMA G719X c L861Q c S768I c T790M c American Society for Clinical Pathology Annual Meeting, 8 10 October 2014

14 Presence of EGFRm in cfdna Predicted PFS (common EGFRm) 13.7 / 8.3M 6.9 / 3.3M Serum DNA - / / 9.7M 5.81 / 4.6M Plasma DNA - / +

15 Presence of EGFRm in cfdna Predicted overall survival Serum DNA + Serum DNA / 14.7M 33.6 / 28.6M Afatinib / CisPem Plasma DNA + Plasma DNA / 17.8M 35.6 / 27.0M Afatinib / CisGem American Society for Clinical Pathology Annual Meeting, 8 10 October 2014

16 Digital droplet PCR for EGFR cfdna

17 Digital Droplet PCR to detect EGFRm cfdna follow up treatment effect

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20 BEAMing for plasma genotyping Sensitivity was 82-86% for sensitizing mutations and 70% for T790M Sensitivity for T790M was highly associated with detection of a sensitizing mutation in cfdna False positive rate was 3-4% for sensitizing mutations but higher (31%) for T790M, perhaps due to heterogeneous presence of a resistance mutation missed in the reference tumour biopsy Oxnard et al. ELCC 2016

21 RR: 26% RR: 46%

22 Plasma T790M(-) Plasma T790M(+)

23 In tissue T790M (-) patients T790M/ActM AF ratio is higher In Tumor T790M(+) than T790M(-) pts Depth of response seems to Correlate to T790M AF ratio >10%

24 PLASMA ANALYSES IN AURA TRIALS Across the AURA trials (NCT , NCT ), plasma was collected for analysis Treatment / dosing AURA Phase I Osimertinib dose escalation and dose expansion cohorts (20 240mg QD) Phase II studies: AURA extension and AURA2 Osimertinib 80 mg QD T790M status T790M positive and negative Only T790M positive Analysis Exploratory post-hoc analysis Intention to treatfor regulatory submission Plasma assay BEAMing cobas Method of comparison ddpcr or cobas NGS ELCC presentation Oxnard G. et al; JCO Jenkins S. et al; 134O [Yang J. presenting] The cobas EGFR Mutation Test for T790M is not available for use with plasma samples in U.S BEAMing, Beads, Emulsification, Amplification and Magnetics; ddpcr, droplet digital PCR; NGS, next generation sequencing; QD, once daily

25 PLASMA SAMPLE COLLECTION Matched plasma samples were collected from screened patients in the Phase II AURA studies (AURA extension and AURA2) for retrospective analysis N=873 screened patients pooled AURA Phase II studies (N=401 in AURA extension; N=472 in AURA2) N=710 FFPE tissue tested N=440 T790M positive N=257 T790M negative N=13 Invalid test N=551 (77.6%) Matched plasma N=416 (94.5%) Matched plasma N=127 (49.4%) Matched plasma N=8 Matched plasma Yang JC presented at ELCC 2016

26 T790M CONCORDANCE BETWEEN COBAS AND NGS cobas tissue test - tissue vs tissue AURA2 (N=383) Using MiSeq NGS of tissue as reference PPA / sensitivity NPA / specificity OPA / concordance 88.3% ( ) 97.3% ( ) 91.0% ( ) cobas ctdna test - plasma vs plasma AURA2 plasma samples (N=344) Using MiSeq NGS of plasma as reference PPA / sensitivity NPA / specificity OPA / concordance 91.5% ( ) 91.1% ( ) 91.3% ( ) Using NGS of plasma ctdna as a reference method, the cobasplasma test is highly sensitive and specific for T790M detection. NGS, next generation sequencing; NPA, negative percentage agreement; OPA, overall percentage agreement; PPA; positive percentage agreement Yang JC presented at ELCC 2016

27 COBAS PLASMA TEST VERSUS COBAS TISSUE TEST AS A REFERENCE METHOD Pooled AURA Phase II studies (AURA extension and AURA2) cobas plasma test performance Using cobas tissue test as reference L858R Exon 19 deletion T790M PPA / sensitivity 75.6% 85.1% 61.4% NPA / specificity 98.1% 98.0% 78.6% OPA / concordance 90.9% 90.0% 65.4% Differences in detection of T790M using tissue and plasma are thought to reflect tumour biology and molecular heterogeneity in the resistance setting Yang JC presented at ELCC 2016

28 OBJECTIVE RESPONSE RATE BASED ON TISSUE AND PLASMA MUTATION RESULTS ORR (95% CI) AURA extension AURA2 ctdna T790M positive subset 59.1% (50.0, 67.7) 69.7% (60.2, 78.2) Pooled AURA Phase II studies (AURA extension and AURA2) % (57.5, 70.1) Evaluable for response set (tissue T790M positive) 61.3% (54.2, 68.1) 70.9% (64.0, 77.1) 66.1% (61.2, 70.7) In the AURA Phase II pooled analysis, the ORR for the plasma T790M-positive subset was similar to that of the evaluable for response set (selected using tissue testing) Yang JC presented at ELCC 2016

29 Acquired EGFR C797S mutation Resistance to AZD9291 in NSCLC harbouring EGFR T790M cfdna collected from patients in AURA (Ph-I) whose tumours developed resistance NGS of cfdna from 7 subjects detected an acquired EGFR C797S mutation in one ddpcr was performed on serial cfdna specimens collected from 15 patients All were T790M positive before AZD9291 treatment, but upon developing resistance, three molecular subtypes emerged: Persistence of T790M and acquired the C797S mutation (6 cases) Persistence of T790M but did not acquire C797S (5 cases) Lost of T790M, but underlying EGFR activating mutation (4 cases) Thress KS et al. Nature Med published online 4 May 2015; doi: /nm.3854

30 Acquired EGFR C797S mutation Resistance to AZD9291 in NSCLC harbouring EGFR T790M Maintained T790M and acquired the C797S mutation Maintained T790M but did not acquire C797S Lost T790M, despite presence of underlying EGFR activating mutation Thress KS et al. Nature Med published online 4 May 2015; doi: /nm.3854

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32 CAPP-Seq of cfdna in rociletinib treated patients Chabon et al Nature Communications 2016

33 CAPP-Seq of cfdna before and after in 43 rociletinib treated patients Chabon et al Nature Communications 2016

34 MET copy number gain predicted inferior response and short duration of PFS of Rociletinib treatment Chabon et al Nature Communications 2016

35 trovera Quantitative Next-Generation Sequencing Assays for Urine EGFR Mutation Testing1 Presented By Heather Wakelee at 2016 ASCO Annual Meeting

36 TIGER-X: Sample Types Submitted for Pretreatment <br />EGFR Testing Presented By Heather Wakelee at 2016 ASCO Annual Meeting

37 Plasma Detection Is Sensitive and Can Complement Tissue T790M Testing Presented By Heather Wakelee at 2016 ASCO Annual Meeting

38 Urine Detection Is Equally Sensitive and Can Complement Tissue T790M Testing Presented By Heather Wakelee at 2016 ASCO Annual Meeting

39 What can cfdna help us in the clinic? In EGFR mutation NSCLC Initial molecular diagnosis Sensitivity : 70-95% Specificity: >95% Follow up for drug treatment effect? Prediction of PFS or OS Determine molecular progressiontime Diagnosisof molecular resistance mechanisms and guide for selection of subsequent regimen Discovery ofother and novelresistance mechanisms Presence predict for poor prognosis? Howdoes the result compare to imaging progression T790M(+) vs. T790M(-), T790M(-) and actm(-) are nonshedders, require re-biopsy. T790M(-)but actm(+) are not good candidate for osimertinib C797S, B-RAF,K-rasmutations, cmet, and novel ones etc.