The Role of Pathology/Molecular Diagnostic in Personalized Medicine

Transcription

1 The Role of Pathology/Molecular Diagnostic in Personalized Medicine Ignacio I. Wistuba, M.D. Jay and Lori Eissenberg Professor in Lung Cancer Director of the Thoracic Molecular Pathology Lab Departments of Pathology and Thoracic/Head & Neck Medical Oncology M. D. Anderson Cancer Center

2 Conflict of Interest Honoraria: Genentech, Glaxo Smith Kline, Boehringer-Ingelheim, Medscape, and AstraZeneca. Research Agreements: Genentech, Pfizer, Astra Zeneca, Myriad, Eli-Lilly, and Merck.

3 3 Consideration for Lung Cancer Molecular Testing In advanced tumors, tissue availability is limited For testing, different types of tumor samples are available: biopsy vs. cytology Molecular testing is required for patients treatment Algorithm for small tissue samples utilization has been developed Tissue sample must represent the setting of the disease

4 Types of Tumor Specimens In Lung Cancer Surgical Resection Advanced Tumor Endobronchial Ultrasound (EBUS) or Pleural Fluid Alcohol-fixed Histology Core Needle Biopsy (CNB) Fine Needle Aspiration (FNA) Alcohol-fixed Cell Block Formalin-fixed and Paraffin-embedded (FFPE) Alcohol-fixed

5 Molecular Testing for NSCLC Traditional Adenocarcinoma ALK RET ROS1 Unknown AKT BRAF VEGFR HER2 EPHA/B PDGFR FGFR INSR EGFR PI3K Squamous Adenocarcinoma KRAS MAPK Large Cell Squamous Cell Ca FGFR1 Amp Adapted from W. Pao and N Girard, Lancet Oncol, 2011 Unknown EGFRvIII PI3KCA EGFR TK DDR2

6 Lung Cancer Targeted Therapy Landscape Change 2012 Adenocarcinoma Frequency Available TKIs - EGFR mutation 15% Erlotinib/Gefitinib - ALK-EML4 fusion 3% Crizotinib - MET amplification 5% Met inhibitors - KIF5B-RET fusion 1% Ret inhibitors - ROS1-FIG fusion 2% Crizotinib - PI3KCA mutation 5% PI3K inhibitors - HER2 mutation 2% Her2 inhibitors Squamous Cell Carcinoma - FGFR1 amplification 22% FGFR TKIs - EGFRvIII mutation 5% EGFR TKIs - PI3KCA mutation 5% PI3KCA inhibitors - DDR2 mutation 3% Dasatinib & Nilotinib

7 Tissue is the Emperor - For diagnosis, the pathologist needs some! Advanced Tumor Core Needle Biopsy (CNB) Fine Needle Aspiration (FNA) What s the problem? I gave you at least 10 cells!

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

9 EGFR Mutations in Lung Cancer Extracellular domain ATP binding cleft Regulatory domain N-lobe C-lobe TK Domain P- loop C- helix A- loop Deletions - 46% Duplications/ Insertions - 9% L858R - 39% Sanger Sequencing (sensitivity: ~20%mutant allele) Exon 19 15bp Deletion Biopsy: - FFPE - Frozen Cytology: - Smears - Cell blocks (FFPE) Deletion 746E-750A CTG858CGG Wild-Type Exon 21 L858R Mutation Wild-Type

10 EML4-ALK Fusion in NSCLC FISH Test: Break-apart Probe 2p23 region Positive Case: >15% Cells Positive ( cells) ALK 29.3 EML variants described Positive Cell: Two signals separation ALK Immunohistochemistry (Clone D5F3) Biopsy: - FFPE EML4-ALK Fusion (+) EML4-ALK Fusion (-) Courtesy of Dr. Y. Yatabe Cytology: - Cell blocks (FFPE)

11 Practical Considerations for Molecular Testing of Lung Cancer Test: EGFR (exons 18-21) mutations and ALK fusion Histology: All tumors w/adenocarcinoma component, and in small samples NSCLC-NOS and other histologies (incomplete sampling) Specimen: Upfront collection of as much tissue as possible at diagnosis Consider re-biopsy: If diagnostic sample is inadequate for molecular testing At time of recurrence, or disease progression on targeted therapy Metastasis vs. primary: Most accessible site (tissue quality is more important) Test metastasis if developed after therapy IASLC/AMP/CAP guidelines in draft and NCCN Clinical Guidelines NSCLC v2 2012

12 Practical Considerations for Molecular Testing of Lung Cancer - Reports Samples availability for testing: In house: less than 24 hours Outside: less than 3 days Quality control by pathologist: At least 500 cells 50% tumor (vs. no-malignant) cells, and gross dissection recommended for enrichment 50 cells/slide Molecular Test: No specific platform is recommended Report: 10 days max Indicate platform Indicate suboptimal fixation in the report IASLC/AMP/CAP guidelines in draft NCCN Clinical Guidelines NSCLC v2 2012

13 Types of Gene Mutation Assays PCR-based Sanger Sequencing PCR-based Pyrosequencing Real-time PCR DxS Test Multiplex and Flexible Tests PCR-based SNaPshot (Applied Biosystem) PCR-based Mass ARRAY SNP Sequenom, Inc Next-Generation of Sequencing (NGS)

14 Multiplexed Mutation Assays Tumor Tissue Multiplex PCR Resected Specimen Core Biopsy SNaPshot (Applied Biosystem) Mass ARRAY SNP - Sequenom, Inc Dias-Santagata, EMBO Mol Med 2:146, 2010 Sensitivity:10% mutant allele / ~20ng DNA/multiplex reaction

15 Use of Cytological Material for Molecular Diagnosis of Lung Cancer EGFR/KRAS mutation and ALK fusions FNA cell blocks, fluids, endobronchial ulstrasound (EBUS), and archival slides, all have been used successfully Touch preps done to ascertain the adequacy of core biopsy material Study Specimen Test N % Suitable Smouse, Can Cyt, 2009 Routine EGFR mut Schuubiers, JTO, 2010 EBUS - FNA EGFR KRAS mut Sakairi, CCR 2010 EBUS - FNA ALK fusion Rekhtman, JTO, 2011 Routine EGFR KRAS mut Navani, AJRCCM, 2012 EBUS - FNA EGFR mut Courtesy of M. Zakowski (modified), New York MSKCC

16 NSCLC Molecular Diagnosis Tumor (CNB) FFPE DNA Extraction Multiplex PCR ~20ng DNA/multiplex reaction Next-Generation of Sequencing (NGS): DNA- & RNA-seq Sequenom (BRAF: G464-G1391) Wild-type Mutant ~10% Sensitivity

17 NGS as a Single Platform to Evaluate Multiple Alterations ( Genes) Tumors Mutation detection DNA copy number detection Translocations/gene fusions RNA-seq: gene expression, alternative splicing Characteristics: High coverage: multiple (~500x) reads of the same sequence to gain confidence in result Critical when ratio of neoplastic to non-neoplastic cells is low Allows signal to be sifted from the noise Examination of reads in both directions to rule out artifacts Confirm or rule out sequence variant using an additional method (e.g. Sanger)

18 Current: Next Generation of Sequencing Illumina HiSeq 2000 Illumina MiSeq Ion Torrent PGM Gigabases 6 11 days 1.5 Gigabases 1 day 1 Gigabase 6 hours Emerging: Illumina HiSeq 2500 Ion Torrent Proton Human Genome in a Day

19 Tissue Availability in Advanced NSCLC Re-biopsy Chemo-naïve Refractory to Chemotherapy Resistance to Targeted Therapy Bone Liver Adrenal Bone Liver Adrenal Bone Liver Adrenal Adapted from Herbst et al, N Engl J Med 359:1367, 2008

20 MD Anderson BATTLE Program Stages I-III Resected Stage IV Untreated Stage IV Refractory BATTLE-Prevention (in preparation) PIs: E. Kim S. Swisher BATTLE-FL (=300) (started 6/2011, n=29) PIs: E. Kim J. Heymach BATTLE (n=324) (completed, 11/2009) PIs: E. Kim R. Herbst BATTLE (n=400) (started 6/2011, n=93) PI: V. Papadimitrakopoulou

21 Kim et al (Cancer Discovery 2011) and V. Papadimitrakopoulou (unpublished) BATTLE-1 and -2 Trial Schemas BATTLE-1 Protocol enrollment Biopsy performed BATTLE-2 Protocol enrollment Biopsy performed 11 Molecular Marker Analysis (14 days) Stage 1: (n=97) Equal Randomization Stage 1: (n=158) Adaptive Randomization Stage 1: (n=200) Adaptive Randomization by KRAS Mutation Status EML- ALK Fusion EGFR Μut exclusio n Statistical modeling, biomarker selection Stage 2: (n=200) Refined Adaptive Randomization Best discovery markers/signatures Erlotinib Sorafenib Bexarotene +Erlotinib Vandetanib Erlotinib Sorafenib Erlotinib +AKTi MEKi +AKTi Primary endpoint: 8-week disease control

22 BATTLE-1 and -2 Tissue Collection and Molecular Analysis Sample/Marker BATTLE-1 BATTLE-2 Tissue Cores 2-3/case (1 frozen) 5/case (3 frozen) Cytology (FNA) No Yes Protein (IHC) Yes (n=5) Yes (n=6) Gene Copy # (FISH) Yes (n=2) No Mutation Yes (3 genes) Yes (9 genes) mrna-affy Array Yes (3 signatures developed) Yes (Test BATTLE-1: WEE, EMT, Sorafenib; and develop new) Proteomic (RPPA) Yes Yes MicroRNA Array No Yes Next-gen Sequencing No Yes (RNA-Seq/DNA Targeted Mut) Kim et al (Cancer Discovery 2011) and V. Papadimitrakopoulou (unpublished)

23 Tissue Quality Control for Molecular Testing by Pathologist: Refractory NSCLC Core Needle Biopsy (CNB) Adequacy Biopsies for Molecular Profiling (DNA, RNA and Proteins) in NSCLC Refractory Tumors: CT BATTLE-1 = 270/324, 83% (3 CNBs and no FNA) BATTLE-2 (3/2012) = 74/77, 96% (5 CNBs and FNA) SCC Necrosis Fibrosis

24 Mechanisms Fig. 1 The frequency of of Resistance observed drug resistance to mechanisms. EGFR TKIs in Lung Adenocarcinoma Adenocarcinoma Unknown (30%) SCLC Features (14%) EGFR T790M Mutation (49%) H&E SCLC Synaptophysin PI3KCA Mut (2%) MET Ampl (2%) EMT Change (14%) H&E Synaptophysin Modified from Sequist L V et al. Sci Transl Med 2011;3:75ra26-75ra26

25 Mechanisms Fig. 1 The frequency of of Resistance observed drug resistance to mechanisms. ALK TKIs in Lung Adenocarcinoma Doebele RC et al, Clin Cancer Res2012 Mar 1;18(5): Epub 2012 Jan 10

26 NSCLC: Re-biopsy Diagnosis At Tumor Progression FNA and Cell Block Core Needle Biopsy Histology Diagnosis & Quality Control for Molecular Testing Molecular Testing: Mutation, Copy Number Analysis, Gene Expression, etc

27 Molecular Testing in Lung Cancer How Do We Deal With Pathologists? Advocating for and/or providing enough tissue Being reasonable on the request (enough tissue available for histology and molecular diagnosis) Guiding on the important question: tumor (yes/no), histology type and molecular change, to prioritize tissue Reassure that the material will be returned and the information will be shared Being nice!