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Mutationer og blod Peter Meldgaard Aarhus Universitetshospital Evolution of Lung Cancer Evolution of Lung Cancer Evolution of Lung Cancer Evolution of Lung Cancer Genomiske Ændringer ved Lunge Adenocarcinomer No known genotype 1984 - 2003 2009 No known genotype 2004 2013 EGFR mutants in lung adenocarcinoma • Somatic EGFR mutations found in ~30 to 60% of East Asian lung adenocarcinomas, ~10-15% of U.S. and European patients • Clustered in four areas – Nucleotide binding loop (P loop), G719 – Exon 19 deletions (ELREA) – Exon 20 insertions (C-terminal to alpha helix) – Activation loop mutations (L858) N lobe C lobe Associated with response to erlotinib/gefitinib/afatinib (except for exon 20 insertion mutants) Promising results for AZD9291/rocelitinib for T790M resistance mutations • EGFR mutations (M+) are amongst the commonest druggable alterations in NSCLC Waterfall plot depicting best response to erlotinib (EURTAC)1 PFS: 10-14 months RR: 50-80% Primary Resistance Acquired Resistance 1Rosell et al. Lancet Oncology 2012 Personalized medicine aims at selecting the right therapy Cancer Patient Tumour type Oncologist selects therapy based on experience, histology and tumour site Therapy Wrong match The right drug for the tumour type Wrong match Adapted from D Weaver Targeted Therapies Structural DNA/RNA changes Chemosensitivity Immune contexture PDL1 staining Mutational load IHC panels +/- FISH 13 Lung Cancer Patient in the near future Lung nodule/metastases Tumor Biopsy/ blood GLOBAL MOLECULAR PORTRAIT Diagnostic Prognosis Targeted therapy Immunotherapy sensitivity sensitivity PD1/PDL1 ERCC1 OX40 RRM1 Other Pathways … Which treatment and when ? Chemotherapy sensitivity Cancer? Yes/No Need for treatment ? Genome comparison adenocarcinoma vs. squamous cell carcinoma High mutation rates in both lung adenocarcinoma and lung squamous cell carcinoma Lawrence et al., Nature, 2013 Different genes are mutated in lung adenocarcinoma and lung squamous cell carcinoma RTK/Ras/Raf pathway in lung adenocarcinoma: KRAS, EGFR, NF1, BRAF, MET, ERBB2, RIT1, RAF1 Common squamous cell cancer genes including NFE2L2, FAT1, NOTCH1 in lung SqCC Campbell et al., submitted Common and different genes are amplified in lung adenocarcinoma and lung squamous cell carcinoma Lineage oncogenes: SOX2 in squamous; NKX2-1 in adenocarcinoma RTK amplifications mostly in adenocarcinoma but EGFR, PDGFRA, FGFR1 are common TERT and MYC in both Campbell et al., submitted Lung adenocarcinoma driver mutations: current status Multiple known RTK/Ras/Raf drivers known EGFR and ALK already actionable BRAF, ERBB2, MET should be actionable soon New alterations still being discovered: RIT1, ARAF, RAF1, SOS1, RASA1, MAPK1 Many more yet to be discovered—problem of statistical power and functional understanding 18 SPECTA-lung participating sites Ireland Stephen Finn - St James Institute Belgium Thierry Berghmans - Institute Jules Bordet Johan Vansteenkiste - Institute KU Leuven Jan Van Meerbeeck – UZA Antwerp UK Sanjay Popat – Royal Marsden Netherlands Egbert Smit - VU University Medical Center France Benjamin Besse – Gustave Roussy Julien Mazieres - Hôpital Larrey Denmark P. Meldgaard - Aarhus University Hospital Spain Enriqueta Felip – Vall d'Hebron University Hospital Luis Paz-Ares – Hospital Universitario Doce de Octubre Germany Martin Reck - Center of Pneumology and Thoracic Surgery of Grosshansdorf Poland Rafal Dziadziuszko - University of Gdansk Italy Silvia Novello – University of Turin Andrea Ardizzoni – S. Orsola-Malpighi Switzerland Rolf Stahel – University Hospital of Zurich Solange Peters – University of Lausanne Slovenia Tanja Cufer – University Clinic Golnik Central Biobank Gustave Roussy Cancer Campus Next Generation Sequencing (NGS) by 14 MG Resistens Til målrettet behandling Kinase Target Kinase Inhibitor Tumor Response Kinase Target Target Reactivation Activating “downstream” effectors • Secondary genetic alterations • Activation of upstream effectors Pathway Reactivation Histologic Transformation Failure to undergo apoptosis Disease Progression Garraway and Jänne Cancer Discovery 2012 BYPASS of (onco)protein effector Mechanisms of drug resistance to EGFR and ALK targeted therapies EGFR mutant Junko Tanizaki ALK rearranged Efficacy of mutant selective EGFR inhibitors in EGFR inhibitor resistant EGFR T790M NSCLC Jänne et al. NEJM 2015; Sequist et al. NEJM 2015 Serial ddPCR profiling of cfDNA reveals 3 molecular subtypes of acquired resistance to AZD9291 EGFR Activating Mutation EGFR T790M EGFR C797S Thress et al, Nature Medicine, 2015 EGFR Activating Mutation EGFR T790M EGFR Activating Mutation Loss of T790M Evolution of resistance mechanisms in EGFR mutant lung cancer following EGFR TKI therapy T790M+ AZD9291 T790M+ plus C797S Erlotinib T790M+ plus unknown resistance T790M+ EGFR activating mutation T790M- No response T790M - Increased Heterogeneity Liquid biopsies Også kaldet blodprøver Tumor diagnostik • Timing • Metastisk sygdom • Beskadigelse af DNA Plasma DNA for tumor diagnostic Fleischhacker & Schmidt Nature Medicine 2008 Blodprøve --- Tumor DNA Bibliotek. Schwarzenbach H, Hoon DS, Pantel K.; Nat Rev Cancer. 2011,Jun;11(6):426-37 What is cell-free DNA? • • • • 166 bp average length Due to apoptotic/necrotic/lytic cell turnover Mostly of hematopoietic origin (disease-free) <2hr half-life Courtesy A Bardelli Cell-free DNA Sequencing as a “Summary” of Inter-Tumor Heterogeneity Plasma DNA---kræft behandling Plasma DNA forudsiger response på erlotinib. Step 1 Step 2 Step 3 Step 4 Blod prøve Oprensning PCR Setup Resultat cobas® 4800 v2.0 2 mL Plasma P<0.05 EGFR M+ EGFR M- Weber et al. 2014, BMC Cancer 10000 Mutated DNA (Copy/ml) Mutated DNA (Copy/ml) Erlotinib --- T790M resistens 5000 100 100 50 0 0 100 200 time (days) Blå = EGFR del19 Rød = EGFR T790M 300 400 10000 5000 100 100 50 0 0 100 200 time (days) 300 400 250 250 00 0 0 0 100 200 300 opsporing 400 50 100 150 => ny behandling Tidligere af0 resistens time (days) time (days) D. 300 400400 600 800 10001200 ! Blood based monitoring of erlotinib in stage 4 EGFRM+ NSCLC Eva Boysen Hansen Peter Meldgaard Boe Sandahl Soerensen Mut ated DNA (Copy / ml ) Background 1 0 Pat ient 3 5 0 1 0 1 0 5 0 0 0 1 0 2 0 3 0 ti me (days) 4 0 A. Mutated DNA (Copy/ml) Mutated DNA (Copy/ml) 800 10000 5000 500 500 250 Patient 2 600 400 200 0 0 0 100 200 300 400 100 200 300 500 150 200 50000 1000 1000 500 250 50 50 400 1000 1000 500 200 300 400 500 Patient 9 10000 5000 500 500 250 0 0 600 100 time (days) Mutated DNA (Copy/ml) Mutated DNA (Copy/ml) Mutated DNA (Copy/ml) 100 0 500000 0 100 time (days) B. Patient 6 150 150 1000000 0 200 100 Patient 8 1500000 500 400 200 time (days) 1000 300 0 0 Patient 7 0 200 250 100000 time (days) 1500 100 time (days) 0 100 50 0 Mutated DNA (Copy/ml) 1000 1000 50 100 100 400 Patient 5 150000 Patient 4 10000 0 Patient 3 5000 time (days) Mutated DNA (Copy/ml) Mutated DNA (Copy/ml) 20000 10000 0 0 time (days) Mutated DNA (Copy/ml) Patient 1 15000 200 300 400 0 500 50 100 150 time (days) time (days) D. C. 500 20000 400 10000 2000 2000 Mutated DNA (Copy/ml) Tissue samples Mutated DNA (Copy/ml) 300 200 100 50 50 40 30 20 300 300 200 100 10 0 0 0 10 20 time (days) Blood samples 1000 30 0 20 40 60 80 100 100 200 300 400400 600 800 10001200 time (days) Soerensen BS et al. ”Monitoring of epidermal growth factor receptor tyrosine kinase inhibitor-sensitizing ! and resistance mutations in the plasma DNA of patients with advanced non-small cell lung cancer during treatment with erlotinib” Cancer. 2014 Aug. Methods • Multicenter: 4 oncology centres in DK (Aalborg, Herning, Odense, Aarhus) • 200 patients in 2 years: Stage 4, EGFRM+, NSCLC (app 80 recruited) • Standard treatment supplemented with blood based monitoring Methods • Examining resistance mechanisms • Ca. 50% are expected to be T790M driven • The rest: ? Exome sequencing of tissue samples at time of diagnosis vs. PD Aims 1. Closer monitoring of erlotinib treatment identification and treatment of oligoprogression. 2. Applicability of blood sample monitoring in a clinically meaningful setting. 3. Identification of new resistance drivers Urinprøver Dynamic Changes in EGFR Mutation Circulating Tumor DNA in Urine on Anti-EGFR Therapy Hatim Husain MD Center of Personalized Cancer Therapy Liquid Biopsy Program University of California, San Diego Moores Cancer Center September 9, 2015 IASLC World Conference on Lung Cancer 44 Clinical Questions 1. Can EGFR T790M resistance be Detected by ctDNA in urine to inform decisions by about tumor progression in EGFR mutant lung cancer? Yu et al Clin Can Res 2013 2. Can early daily dynamics of ctDNA in urine within the first week on second line anti-EGFR tyrosine kinase therapy provide insight to tumor biology and inform response? Response within 3 weeks of treatment initiation 45 Blocking Wild-Type DNA Amplification Enriches for Mutant Sequences in Urine Daily collection Mutants Wild Type Blocker 10x DNA yield in urine vs plasma due to higher urine volumes collected Blocking technology facilitates PCR enrichment followed by NGS detection (MiSeq) 46 HRPP 130794 Project Schema Specific Aim 1: Test the hypothesis that T790M could be identified prior to radiographic progression • 34 patients receiving treatment with erlotinib monitored longitudinally for acquisition of T790M mutation • Collection frequency q3-6 weeks Specific Aim 2: Test the hypothesis that T790M mutational load in ctDNA within the first week may predict radiographic response to therapy with second line anti-EGFR tyrosine kinase inhibitors • 14 patients positive for T790M by tissue biopsy received treatment with second line anti-EGFR TKIs • Urine specimens were collected from patients prior to treatment, daily for 1 week, then weekly for 3 weeks, then monthly 47 Results: Correlation Data Between Urine and Tissue Biopsy for EGFR T790M T790M Tissue Total Patients (N=65) Positive T790M Tissue Negative T790M Tissue not yet tested Total T790M Urine Positive 14 3 12 29 T790M Urine Negative 1 1 7 9 Total 15 4 19 38 48 Results: Urinary T790M Detection Compared with Date of Radiographic Progression PD 300 T790M detected 200 100 0 Early T790M Detection (Days to Radiographic Progression) T790M detected PD Patient 1 111 Patient 3 52 Patient 10 56 Patient 20 29 28-10-2014 0 400 08-10-2014 10 Patient 3 49 Urine T790M Copies/100K GE 20 500 29-08-2014 18-09-2014 09-08-2014 30-06-2014 21-05-2014 0 30 PD 08-10-2014 20 T790M detected 29-08-2014 40 40 20-07-2014 60 PD Patient 20 50 10-06-2014 T790M detected Urine T790M Copies/100K GE 80 11-04-2014 Urine T790M Copies/100K GE Patient 10 18-09-2014 Patient 1 100 Can the early dynamics of ctDNA within the first week on second line antiEGFR TKI therapy provide insight to tumor biology and inform response? 50 51 Week 1 on drug Week 1 on drug Week 5 Week 4 Week 3 Week 2 Day 7 Day 6 Day 5 0 Day 4 50 20000 Day 3 Ex19 del Day 2 150 Day 1 T790M 4 hr on Drug 200 Urine EGFR Copies/100K GE 250 10/13/2014 Day 7 Day 6 Day 5 Day 4 100 Day 3 Day 2 Day 1 Day 1 (4 hrs) Baseline Urine EGFR Copies/100K GE Results: Daily Urinary ctDNA Detection within the First Week of Therapy 25000 L858R 15000 10000 T790M 5000 0 52 Week 1 on drug 6 wks CT scan 0 0 12 wks CT scan Week 1 on drug 6 wks CT scan 1/19/2015 1/12/2015 1/5/2015 50 12/29/2014 5000 12/22/2014 100 12/15/2014 T790M 100 12/8/2014 10000 150 150 12/1/2014 200 11/24/2014 Size on CT scan 11/17/2014 250 11/10/2014 20000 11/3/2014 300 Urine EGFR Copies/100K GE 250 200 70 Size on CT scan 60 T790M 50 40 Ex19 del 30 50 20 10 0 0 12 wks CT scan Sum of Longest Diameters (mm) 350 10/27/2014 L858R Sum of Longest Diameters (mm) 25000 10/20/2014 1/14/2015 1/7/2015 12/31/2014 12/24/2014 12/17/2014 12/10/2014 12/3/2014 11/26/2014 11/19/2014 11/12/2014 11/5/2014 15000 10/29/2014 10/22/2014 10/15/2014 10/8/2014 Urine EGFR Copies/100K GE Results: Urinary ctDNA Kinetics Compared to Radiographic Change 80 Results: Re-treatment Kinetics in ctDNA EGFR Ex19del and T790M 3.9x3.7 3.7x3.1 3.2x3.0 Change in the Target Lesion on CT (cm) 2.1x1.9 1.7x1.5 200 1st gen TKI 150 3rd gen TKI 100 Held for Toxicity 3rd gen TKI (dose change) 50 T790M 25 E x19del 7 /12/2 015 6 /22/2 015 6 /8 /2 0 1 5 4 /23/2 015 4 /3 /2 0 1 5 0 3 /14/2 015 53 250 2 /22/2 015 U r in e E G F R C o p ie s / 1 0 0 K G E 300 Working Hypothesis: Early Pharmacodynamics of Urine ctDNA as a Putative Biomarker of Response 54 Så: Fremtiden er flydende Både blod og urin