Evolución Clonal de los Tumores y Biopsia Líquida en Cáncer de Pulmón

Transcription

1 Evolución Clonal de los Tumores y Biopsia Líquida en Cáncer de Pulmón Ignacio I. Wistuba, M.D. Professor and Chair Department of Translational Molecular Pathology The University of Texas M. D. Anderson Cancer Center Bouston, Texas

2 2 Disclosures Bonoraria: Genentech, Roche, Bristol-Myers Squibb, Boehringer Ingelheim, Medscape, Astra Zeneca, Pfizer, Ariad, BTG Molecular, Asuragen, Merck. Research support: Genentech, Oncoplex, BTG Molecular, DepArray, Merck, Medimmune.

3 Molecular Testing for NSCLC Traditional Adenocarcinoma AKT BRAF VEGFR ALK HER2 RET EPHA/B RdS1 PDGFR Unknown FGFR INSR EGFR PI3K Squamous Adenocarcinoma KRAS MAPK Large Cell Squamous Cell Ca FGFR1 Amp Adapted from W. Pao and N Girard, Lancet dncol, 2011 Unknown EGFRvIII PI3KCA EGFR TK DDR2

4 Diagnostic Algorithm for Small Biopsy and Cytology Specimens Tumor Positive Biopsy Cytology SCLC LCNEC Squamous Adenoca NSCLC-NOS Morphology Morphology IBC NE (+) Morphology IBC p63/p40 (+) Morphology IBC TTF1 (+) Morphology IBC (-) Molecular Testing: EGFR mutation, ALK and RdS1 Fusion; MET ex14

5 echanisms of Resistance to EGFR and ALK TKIs in Lung Adenocarcinoma EGFR ALK Unknown (18%) Alternate Oncogene (EGFR, KRAS) (36%) Unknown (30%) SCLC Features (14%) EGFR T790M Mutation (49%) PI3KCA Mut (2%) MET Ampl (2%) EMT Change (14%) ALK Resistance Mutations (~36%) ALK Copy Number Gain (CNG) (~18%) odified from Sequist L V et al. Sci Transl Med 2011; Doebele RC et al, Clin Cancer Res, 2012

6 Mechanisms of Resistance to EGFR TKI Adenocarcinoma SCLC B&E Synaptophysin B&E Synaptophysin Sequist L V et al. Sci Transl Med 2011;3:75ra26-75ra26

7 Non-Small Cell Lung Carcinomas (NSCLC) Show Bigh Number of Somatic Mutations Lawrence et al., Nature, 2013 TCGA, Squamous Cell Carcinoma TCGA, Adenocarcinoma PS Hammerman et al., (TCGA) Nature, 2012 EA Collisson et al., (TCGA), Nature, 2014

8 Next Generation Sequencing Platforms MDACC Clinical Laboratory Ion Torrent (Life Technologies) Semiconductor based detection of ph change Illumina Flow cell based, 4-color optical imaging of fluorescent labeled nucleotides PGM Proton MiSeq BiSeq Small Gene Panels ( Gbases/run) Large gene panels and Whole Exome Sequencing (10 Gbases/run) Small Gene Panels ( Gbases/run) For Whole Exome and Genome Sequencing (600 Gbases/run) Buman Genome: 3 Gb Whole Exome: 30 Mb Courtesy of Dr. Kenneth Aldape, MD Anderson Cancer Center, Houston, USA

9 NGS for Mutation Analysis Samples Using FFPE Tumor Tissues J. Izzo et al, 2015

10 Next-Generation Sequencing (NGS) Panel - Major Benefits Provide information in multiple targetable gene abnormalities. Data on mutation, copy number variations, indels and translocations Can be performed in routine small FFPE tissue samples and liquid biopsy (cfdna, CTCs, exosome DNA). Turn around time acceptable for clinical management and costs being significantly reduced. Clinically, it offers to patients more options to get off-label treatment and enter in genomic-based clinical trials.

11 Types of Tumor Beterogeneity Mariam Jamal-Hanjani et al. Clin Cancer Res 2015;21:

12 Intra-tumor Beterogeneity (ITB) Whole-genome (WGS) or whole-exome sequencing (WES) studies have shown: Branched evolution Spatial heterogeneity, intra-tumor heterogeneity: spatial ITB Genomic patterns varying overtime: temporal ITB Genomic changes (ITB) varying in response to therapy and may be associated to resistance to targeted therapy

13 Intratumor Tumor Beterogeneity (ITB) Trunk and Branch Model Swanton, Cancer Research, 2012

14 Clonal Evolution and Phylogenetic Analyses The clonality of somatic mutations can be estimated by bioinformatic analysis using tumor purity, allelic copy number and mutation variant allele frequency. Mariam Jamal-Hanjani et al. Clin Cancer Res 2015;21:

15 Intratumor Beterogeneity (ITB) and Clonal Evolution Mariam Jamal-Hanjani et al. Clin Cancer Res 2015;21:

16 Clinical Implications of ITB Tumor development Tumor progression and metastasis Tumor aggressiveness and poor outcome Immune response Resistance or sensitivity to therapy Chemotherapy Targeted therapy Immunotherapy

17 ITB in Localized Lung Adenocarcinomas 76% of all mutations were trunk mutations 13/14 known cancer gene mutations were trunk mutations J. Zhang et al, Science, vol 346, 2014

18 ITB Defined by Exome Sequencing vs. Deep Sequencing A. Exome sequencing: average sequencing depth of 277x B. Deep sequencing: average sequencing depth of 863x A KRAS p.g12c mutation was detected in only 1 of 4 tumor regions at exome depth but detected in all four tumor regions by deep sequencing J. Zhang et al, Science, vol 346, 2014

19 Bigh Concordance of Genomic Changes Between Matched Primary and Metastasis NSCLCs 15 primary (P) and mets (M) tumor pairs. Targeted NGS on 3,230 exons of 182 cancerrelated genes and 37 introns of 24 often rearranged genes. 311 genomic alterations detected: 63 recurrent and 248 non-recurrent (passengers) Concordance between matched P and M was: 93% recurrent 63% non-recurrent. Stéphane Vignot et al. JCd 2013;31:

20 WES of Matched Primary and Brain Metastasis NSCLCs (n=6 Cases) 1T 1M KRAS G12V LRP1B stop gain ARID1A Stop gain PIK3CA E545K TP53 stop gain TP53 E285K STK-11 stop gain KEAP1 G423V EGFR A755D EGFR Exon 19 deletion TP53 S241A EPBA3 Stop gain SYNE1 stop gain ARID1A stop gain On average, 51% of all mutations (24% to 93%), and all 14 canonical cancer gene mutations were shared between primary and metastasis tumors. Metastases (average 852) had larger mutation burdens than paired primary tumors (average 596). Mutation mechanism are preserved during cancer evolution even at the metastatic sites. J. Zhang, D. Gomez et al, MD Anderson Cancer Center, AACR 2016

21 Practical Implications of Lung Cancer ITB Taking multiple tumor samples from advanced metastatic tumors is not practical Approaches to understand the evolutionary rules book of tumors: Novel computational approaches to asses spatial ITB Longitudinal collection of tumors to assess temporal ITB Deep sequencing (~800x) may be useful to identify most driver and targetable genomic changes Liquid biopsy assessing circulating free (cf)dna, circulating tumor cells (CTCs) and exosome-derived (exo)dna

22 Battle of Clones Role of Liquid Biopsy Intra-tumor and Inter-tumor Beterogeneity R. Burrell and C. Swanton, Molecular dncology, Volume 8, 2014, Mutational monitoring though Liquid Biopsy Circulating Free (cf) DNA Exosomes Circulating Tumor Cells (CTCs)

23 TEM with immunogold Atomic Force Microscopy Raposo et al, doi: /jcb Source of Tumor DNA in the Bloodstream of Patients Circulating free (tumor) DNA (cfdna) Circulating Tumor Cell (CTC) Exosomes Endocytic origin intracellular nm Cargo loading seems to be (at least, partially) regulated event Crowley, E Nat Rev Clin dnc 10,

24 Liquid Biopsy in Lung Cancer Non-invasive approach that detects diagnostic, prognostic and predictive biomarkers in cancer patients Currently, it is being tested in cancer patients with metastatic disease to deliver targeted therapy Applications: Can be easily repeated to control treatment efficiency and/or the detection of genomic changes resulting from resistance to therapy (e.g., EGFR T790M) It is an alternative to patients with solid tumors when biopsies are inaccessible or after more than one attempt the yield was unsatisfactory It may be able to address the issue of tumor heterogeneity

25 Circulating Cell-free Tumor DNA 167 bp Fragments of Nuclear DNA, a Nucleosome The linker DNA between nucleosomes is cleaved leaving 167 bp cell-free DNA fragments (145 bp plus a ~20 bp segment wrapping histone H1). Originally described by Wyllie in Chandrananda et al BMC Medical Genomics Wyllie 1980 Nature.; Slide from Rick Lanman

26 cfdna Genotyping Analysis TISSUE BLOOD PLASMA ION PGM 50 genes Pre-analytical Issues ION PGM 318V2 Chip (~5 million reads) Ultra deep sequencing 4 sample/run Bio Rad ddpcr Tissue ION PROTO N CCP (409 genes) CBPv2 : Cancer hot spot panel V2; CCP: Comprehensive cancer panel Plasma Amount of blood/plasma Type of tubes Time for processing Variant Call Confirmation ION PROTON Ion PI HiQ Chip (~80 million reads) Ultra deep sequencing 32 samples/run Meenakshi Mehrotra et al, MD Anderson Cancer Center

27 Genotyping Methodologies ctdna Platform Sensitivity (for clinical use) Type of Mutations Quantitative Sanger 20% mut, indel No Pyro-seq 5% mut Yes Sequenom/ ABI SNaPshot Next Generation Sequencing (NGS) Allele Specific detection 10% mut Yes 1%-0.01% (MDACC) mut, indel, translocation, copy number Yes 1%-0.01% Botspot, mut Yes BEAMing >0.01% Botspot, mut Yes Digital PCR, Deep NGS, UltraSeek MassArray (Sequenom) > % Platform dependent Yes

28 Guardant360 Panel 2015 All NCCN Somatic Genomic Targets in a Single Test POPNO MUOAOPONS T Complete* or Critical Exon Coverage in 70 Genes AKT1 ALK APC AR ARAF ARID1A ATM BRAF BRCA1 BRCA2 CCDN1 CCND2 CCNE1 CDH1 CDK4 CDK6 CDKN2 A CDKN2 B CTNNB1 EGFR ERBB2 ESR1 EZH2 FBXW7 FGFR1 FGFR2 FGFR3 GATA3 GNA11 GNAQ GNAS HNF1A HRAS IDH1 IDH2 JAK2 JAK3 KIT KRAS MAP2K1 MAP2K2 MET MLH1 MPL MYC NF1 NFE2L2 NOTCH1 NPM1 NRAS NTRK1 PDGFR A PIK3CA PTEN PTPN11 RAF1 RB1 RET RHEB RHOA RIT1 ROS1 SMAD4 SMO SRC STK11 TERT TP53 TSC1 VHL AMPLPFPCAOPONS AR BRAF CCNE1 CDK4 CDK6 EGFR ERBB2 FGFR1 FGFR2 KIT KRAS MET MYC PDGFRA PIK3CA RAF1 FUSPONS ALK FGFR2 FGFR3 RET ROS1 NTRK1 PNDELS EGFR exons 19/20 ERBB2 exons 19/20 MET exon 14 skipping

29 ctdna and Tissue Concordance Studies

30 Detection of EGFR Mutations in Lung Adenocarcinoma Using cfdna EGFR mutations examined using digital droplet (dd)pcr using cfdna. Analysis of activating EGFR mutations in 34 patients with mutant tumors. L858R was detected in 8/12 and Del- 19 in 9/23 patients with mutant tumors. Four patients treated with EGFR TKI were examined serially: increasing levels of T790M emerged before OR. Geoffrey R. dxnard et al. Clin Cancer Res 2014;20:

31 Tony Mok et al. Clin Cancer Res 2014;21: ctdna EGFR Mutation and Survival in NSCLC Patients Treated w/firstline Intercalated Erlotinib and Chemotherapy Randomization 6 cycles of Gemcitabine/Platinum + sequential Erlotinib or placebo (FASTACT-2, Lancet dncology). Blood 3 time-points (baseline, cycle 3 and progression); matched tdna in 238 cases. cfdna assayed by Cobas, 41 EGFR mutation hotspots. Concordance between cf- tdna 88%; 75% sensitivity, 96% specificity. EGFR + EGFR - EGFR + EGFR - By cycle 3, median OS cfdna EGFR mut months vs for cfdna EGFR mut(-).

32 Response in Patients With Lung Tumors Treated with Osimertinib Based on T790M Status in Tumor and Plasma Best Percentage Change in Target Lesion Patients With T790M+ Plasma/ T790M Tumors Geoffrey R. dxnard et al. JCd doi: /jcd

33 Kaplan-Meier Curves of Progression-free Survival in T790M+ and T790M Subpopulations Treated with Osimertinib T790M Tumor Testing T790M Plasma Testing T790M Plasma Negative T790M Plasma Positive Geoffrey R. dxnard et al. JCd doi: /jcd

34 A Proposed Paradigm for Use of Plasma Genotyping for EGFR T790M Mutation Geoffrey R. dxnard et al. JCd doi: /jcd

35 Liquid Biopsy in Cancer Ilie et al, Ann Transl Med, 2014