Tissue or Liquid Biopsy? ~For Diagnosis, Monitoring and Early detection of Resistance~

16 th Dec. 2016. ESMO Preceptorship Program Non-Small-Cell Lung Cancer @Singapore Tissue or Liquid Biopsy? ~For Diagnosis, Monitoring and Early detection of Resistance~ Research Institute for Disease of the Chest, Kyushu University, Fukuoka, Japan Eiji Iwama

Contents What is Liquid Biopsy? How to detect mutations Diagnosis and monitoring of EGFR activating mutations in cfdna Detection of resistance mutations in cfdna

Liquid biopsy Haber DA, et al. Cancer Discovery, 2014 Jun 4(6);650-61.

Liquid biopsy Circulating free DNA (cfdna) Circulating tumor DNA (ctdna) Haber DA, et al. Cancer Discovery, 2014 Jun 4(6);650-61.

Current proceedings against NSCLC Imaging <Chest X-ray> Invasive method <Bronchoscopy> Pathological diagnosis <Chest CT> <CT-guided biopsy> Genetic testing Recurrence Treatment

Introduction of Liquid Biopsy to clinical practice Imaging <Chest X-ray> Liquid Biopsy Advantage: Monitoring of patients during treatment Potential to provide a genetic signature of the patient s entire tumor (information including heterogeneity) <Chest CT> Genetic testing Recurrence Treatment

Contents What is Liquid Biopsy? How to detect mutations Diagnosis and monitoring of EGFR activating mutations in cfdna Detection of resistance mutations in cfdna

Sanger sequencing <T790M (C to T transition)> Sanger sequencing needs >10% target frequency to detect a target mutation. (Detection limit = 10%) Kobayashi S, N Engl J Med. 2005 It is difficult to detect mutations in cfdna.

Allele specific PCR-based method (Cobas, ARMS ) Cut-off is determined with the Ct value. Ct = Ct (Target allele) - Ct (Control allele) (Detection limit = 1%)

Digital PCR FluidigmCorporation s BioMarkHD System for digital PCR and qpcr Detection limit; 0.1% 0.01% 5 mutant 1,000 wild = 0.5% (Allele Specific-PCR cannot detect) 1 mutant 40 wild = 2.5% (Allele Specific-PCR can detect!!)

Droplet digital PCR (ddpcr) Oxnard GR, Clin Cancer Res., 2014 Bio-Rad s QX100 droplet digital PCR System RainDropSource and RainDropSense machines for droplet digital PCR Detection limit; 0.005% 0.01% Detection limit; 0.001%

Next Generation Sequencing (NGS) (http://www.slideshare.net/ueb52/introduction-to-next-generation-sequencing-v2)

Contents What is Liquid Biopsy? How to detect mutations Diagnosis and monitoring of EGFR activating mutations in cfdna Detection of resistance mutations in cfdna

Biomarker study of Afatinib (Afatinib IIS) Iwama E. et al. Ann Oncol. 2016, published online Advanced adenocarcinoma EGFR mutation (Ex19 del, L858R) (+) EGFR-TKIs-naïve PS0-1 Tumor samples available 35 patients were enrolled Pre-treatment (Baseline) Afatinib (2 nd gen. EGFR-TKI) 40mg/day (Enrollment: May 2014 to Nov 2014) 4 w 24 w Post-treatment (After PD) Tumor samples Blood samples dpcr dpcr dpcr dpcr NGS NGS NGS NGS

Biomarker study of Afatinib (Afatinib IIS) Iwama E. et al. Ann Oncol. 2016, published online Advanced adenocarcinoma EGFR mutation (Ex19 del, L858R) (+) EGFR-TKIs-naïve PS0-1 Tumor samples available 35 patients were enrolled Pre-treatment (Baseline) Afatinib (2 nd gen. EGFR-TKI) 40mg/day (Enrollment: May 2014 to Nov 2014) 4 w 24 w Post-treatment (After PD) 100% 80% 60% 81% 72% Detection frequency of EGFR activating mutations in cfdna 100% 60% dpcr NGS 40% 20% 0% 13% 13% 0% 0% Pre treatment 4w 24w After PD

Biomarker study of Afatinib (Afatinib IIS) Iwama E. et al. Ann Oncol. 2016, published online PFS according to the detection of EGFR activating mutations in cfdna by dpcr <Baseline> <4 weeks after the initiation of afatinib treatment> Not detected mpfs: 14.4 months (N = 6) (N = 6) Not detected mpfs: 14.3 months (N = 26) Detected mpfs: 12.8 months (N = 26) Detected mpfs: 10.0 months (N = 4) (months) (months)

Detection of EGFR activating mutations in cfdna 100% 80% Sensitivity and Specificity for detection of EGFR activating mutations in cfdna Highly dpcr, sensitive BEAMing method (Highly(dPCR, sensitive BEAMing) method) Cobas (Allele specific PCR) (Compared with tissue DNA using an allele specific PCR) 80% 77% 95% 98% 60% 40% 20% Cobas EGFR Mutation Test v2 for plasma sample was approved for companion diagnostics of TARCEVA (Erlotinib) by FDA (June, 2016) 0% Sensitivity Specificity (Yung TKF, CCR, 2009; Karlovich C, CCR, 2016, http://blog.aacr.org/fda-approval-liquid-biopsy-test-lung-cancer/)

Contents What is Liquid Biopsy? How to detect mutations Diagnosis and monitoring of EGFR activating mutations in cfdna Detection of resistance mutations in cfdna

Detection of T790M in tissue samples Mechanisms of acquired resistance to 1 st gen. EGFR-TKIs Yu HA, Clin Cancer Res., 2013 Using PCR based method (ARMS, cobas...), T790M has been detected in approximately 60% of the patients with acquired resistance to 1 st gen. EGFR-TKIs.

Detection of EGFR T790M mutation in cfdna Sensitivity and Specificity for detection of T790M in cfdna (Compared with tissue DNA using an allele specific PCR) 100% Highly dpcr, sensitive BEAMing (Highly method sensitive (BEAMing) method) Cobas (Allele specific PCR) 80% 60% 70% 69% 59% 80% 40% 20% 0% Sensitivity Specificity (Oxnard GR, JCO, 2016) ( cobas EGFR Mutation Test v2 information)

Detection of EGFR T790M mutation in cfdna What causes low sensitivity and specificity in the detection of T790M in cfdna? Alleles with T790M are fewer than that with activating mutations. ( low sensitivity) cfdna potentially has the information including primary and metastatic tumors (inter-tumor heterogeneity) ( low specificity) Highly sensitive method may detect the T790M mutation which is not responsible for the resistance to EGFR-TKIs. ( low specificity)

Efficacy of Osimertinib (AZD9291, TAGRISSO TM ) according to T790M status in the patients with acquired resitance to 1 st gen. EGFR-TKI <Tissue assessed by Cobas > <cfdna assessed by BEAMing> <T790M negative in cfdna assessed by BEAMing> (Oxnard GR, JCO, 2016)

Combination use of liquid and tissue biopsy for detection of T790M Strategy 1 Acquired resistance to 1 st or 2 nd gen. EGFR-TKI Strategy 2 Acquired resistance to 1 st or 2 nd gen. EGFR-TKI T790M assessment with cfdna Positive Negative T790M assessment with Tissue Positive Negative T790M assessment with Tissue Positive Unknown T790M assessment with cfdna Positive Negative Negative 3 rd gen. TKI Chemotherapy 3 rd gen. TKI Chemotherapy (Oxnard GR, JCO, 2016) (Tan DSW, JTO, 2016) Liquid biopsy reduces the occasion of tissue biopsy (invasion, cost, time) Liquid biopsy may salvage the patients whose tissue samples are not obtained or insufficient Cobas EGFR Mutation Test v2 for plasma sample was approved for companion diagnostics of TAGRISSO TM (Osimertinib) by FDA (Sep, 2016)

Exploration of resistance mechanisms to EGFR-TKIs using deep sequencing (NGS) Putative resistance mechanisms to Rociletinib (3 rd generation TKI) in cfdna MET copy-number gain was the most frequent mechanism Significant fraction of patients develop multiple resistance mechanisms, reflecting the clinical importance of tumor heterogeneity (Chabon JJ, Nature communications. 2016)

Conclusions Technological advancement has made it possible to analyze cfdna. Liquid biopsy cannot replace the tissue biopsy, but can complement it. Development of best strategy for combination use of liquid biopsy and tissue biopsy in the detection of T790M is warranted.

Thank you for your attention Kyushu University Hospital

Back Up Slide

Efficacy of EGFR-Tyrosine Kinase Inhibitors EGFR-Tyrosine Kinase Inhibitors (EGFR-TKIs) for EGFR active mutations positive NSCLC pts. 1 st gen. EGFR-TKI: Gefitinib, Erlotinib 2 nd gen. EGFR-TKI: Afatinib ORR:60~80% MST:2 years ~ Genetic mutations in adeno ca. of lung cancer (Japan) Cytotoxic chemotherapy for NSCLC pts. ORR:30~40% MST:1 year RET 2% BRAF 1% HER2 3% ROS1 3% ALK 5% Others KRAS 10% EGFR 40-55% Kohno et al. Cancer Sci. 2013

Circulating free DNA (cfdna) Heitzer E, et al. Clin Chem., 2015 Jan;61(1):112-23

Detection frequency of T790M in tissue samples obtained before treatment with EGFR-TKIs Technique Detection frequency of T790M Sanger Sequencing 2.2% (11/503) OxnardGR, JTO,2012 Scorpion-ARMS (ARMS) 1.4% (21/1450) ShiY,JTO,2014 MALDI-TOF MS 31.5% (23/73) SuKY,JCO,2012 Colony hybridization 78.9% (30/38) Fujita Y, JTO,2012 Microdissection + PCR-based method 65.3% (62/95) Costa C, CCR. 2014 Digital PCR 79.9% (298/373) WatanabeM,CCR,2015 Digital PCR 100% (13/13) IwamaE,Oncotarget,2015 The detection frequency of T790M is dependent on the sensitivity of techniques.

Model of the emergence of T790M Only highly sensitive methods such as dpcr can detect T790M. Sanger sequencing or allele specific PCR can detect T790M. T790M detected by these methods is responsible for the resistance to EGFR-TKIs!!

Are there any solutions to improve the specificity for detecting T790M in cfdna?? Evaluate the T/A (T790M/Activating mutation) ratio with a highly sensitive and quantitative manner. <Efficacy of rociletinib according to the T/A ratio> Tissue Plasma (Quantification with allele specific PCR or NGS) Piotrowska Z, Cancer Discovery, 2015 (BEAMing) Karlovich C, Clin. Can. Res. 2016

Sensitivity to erlotinib according to the frequency of T790M positive cells Chmielecki J, Science Translational Med., 2011

Highly sensitive and quantitative evaluation of the EGFR T790M mutation by dpcr <Pre-TKI sample> L858R Panel (1) Panel (2) Estimated number of target alleles Panel (1) (2) 188 212 Panel (1) + (2) 400 T/A (%); The ratio of the number of T790M alleles to the number of activating mutation alleles T790M 57 46 103 0.52% Control 389 373 762 <Post-TKI sample (Post PD)> Panel (1) Panel (2) L858R Panel (1) (2) Panel (1) + (2) 22 30 52 T790M 475 539 1014 48.84% Control 689 653 1342 Iwama E et al, Oncotarget 2015. Aug 21;6(24):20466-73

Highly sensitive and quantitative evaluation of the EGFR T790M mutation by dpcr Pre-TKI Post-TKI Iwama E et al, Oncotarget 2015. Aug 21;6(24):20466-73

各検査法の検出限界検出限界 Diaz LA Jr, et al. J Clin Oncol. 2014;32(6):579-86

Detection of EGFR T790M mutation in cfdna ddpcr (Sacher AG, JAMA Oncol., 2016)

Detection of EGFR T790M mutation in cfdna Sensitivity and Specificity for detection of T790M in cfdna (Compared with tissue DNA using an allele specific PCR) 100% Highly sensitive method (BEAMing) Cobas 80% 60% 70% 69% 59% 80% 40% 20% 0% Sensitivity No difference in PFS between the patients with detectable T790M in Tissue specimen and those with detectable T790M in cfdna. Specificity (Oxnard GR, JCO, 2016)

I declare no conflicts of interest.