ALK Rearrangements in NSCLC Detection and Issues in Modern Pathology. 3rd ITOCD Dresden September 13, 2012

Transcription

1 ALK Rearrangements in NSCLC Detection and Issues in Modern Pathology 3rd ITOCD Dresden September 13, 2012

2 Agenda The Importance of Targeted Therapies in NSCLC (Prof. Kenneth O Byrne, Thoracic Oncology Research Group, St James Hospital and Trinity College, Dublin, Ireland). Targeted therapies in NSCLC: Issues in NSCLC pathology (Prof. Keith M Kerr, Aberdeen University Medical School, UK). Detection of ALK Rearrangements in NSCLC (Prof. Patrick Pauwels, University Hospital Antwerpen, Unversity of Antwerpen, Belgium)

3 Housekeeping notes Please turn cell phones/pagers/blackberrys/pdas to silent or off Electronics may interfere with the amplification system Please save questions for faculty for panel discussion at end of presentations Thank you!

4 The Importance of Targeted Therapies in NSCLC Prof Kenneth O Byrne Thoracic Oncology Research Group, St James Hospital and Trinity College, Dublin, Ireland

5 Lung Cancer: Selected Comparative 5 year Survival: males 14 % 5 Year Survival USA Slovakia Iceland France Finland EUROPE Italy England Scotland Denmark Estonia

6 Overall survival % 1 st -line platinum-based CT: Efficacy plateau Overall survival Overall survival Pacli + carbo (PCb) Cis + vin (CV) Pacli + cis (PC) Gem + cis (GC) Doc + cis (DC) Pacli + carbo (PCb) Pacli + carbo (PCb) Gem + cis (GC) Cis + vin (CV) Months Months Months 30 Study arm OS (mo) 1 year (%) Study arm OS (mo) 1 year (%) Study arm OS (mo) 1 year (%) PCb CV OS, overall survival PC GC DC PCb PCb GC CV Kelly et al. JCO 2001; Schiller et al. NEJM2002; Scagliotti et al. JCO 2002

7 1 SQUAMOUS 2 ADENOCA 4 LARGE CELL 3 SMALL CELL

8 Survival Probability Survival Probability Cisplatin/Pemetrexed vs Cisplatin/ Gemcitabine in Advanced NSCLC: Nonsquamous Median Survival 11.8 mos 10.4 mos CP vs CG Adjusted HR 0.81;0.70, Results Squamous Median Survival 9.4;8.4, mos 10.8;9.5, mos CP vs CG Adjusted HR ;1.00, 1.51 Survival Time (months) in Patients With Nonsquamous Histology Survival Time (months) in Patients With SCC Scagliotti, G. J Clin Oncol. 2008

9 Survival Probability Pemetrexed Maintenance: Overall Survival by Histology Non-squamous (n=481) Squamous (n=182) 1.0 HR=0.70 (95% CI: ) P = HR=1.07 (95% CI: ) P = Pemetrexed 15.5 mos 0.5 Pemetrexed 9.9 mos Placebo 10.3 mos Placebo 10.8 mos Time (months) Time (months)

10 RFLP/SNP DNA GENE Polymorphism Deletion Amplification RNA CGH/FISH Transcriptome Signatures Expression array mirs methylation Protein Loss or gain of expression Phenotype IHC Clinical outcome

11 Survival probability (%) BR.21 demonstrated significant improvement in OS versus placebo 100 Tarceva (n=488) Placebo (n=243) Median survival (months) HR=0.73 ( ), p=0.001* 27% reduction in risk of death with Tarceva 42.5% increase in median survival with Tarceva Survival time (months) *HR and p (log-rank test) adjusted for stratification factors at randomisation and EGFR status Shepherd, et al. NEJM 2005 Tarceva Summary of Product Characteristics

12 Prognostic versus predictive markers Prognostic marker Influences clinical outcomes regardless of the therapy received Predictive marker Influences clinical outcomes with a specific therapy A single biomarker can have both predictive and prognostic value Only placebo-controlled studies can determine the prognostic contribution of a biomarker

13 How Does This Enable Personalized Medicine? Right Target Right Drug (or Combinations) Right Patient Genetic validation; Rare phenotypes Selective design and delivery; Combinations for complex diseases Phenotyping and genotyping

14 < 10% PR

15 Mutations identified in EGFR gene EGFR transcript Exons 1 16 Exon 17 Exons Exons Confer sensitivity/resistance to EGFR TKIs G719A/S Deletions D761Y D770_N771 insnpg T790M L858R L861X Unclear effect on sensitivity to EGFR TKIs V689M P694X V700D E709X I715S L730F P733L E746K A763V N765A S768I T783A L792P L798F G810S N826S L838V T847I I853T A859T E866K L688P L718P S720X G735S V738F V742A T751I S752Y D761N L833V H835L H850N V851X G863D A864T Riely, et al. Clin Cancer Res 2006

16 Probability of PFS IPASS: EGFR mutation status defined population benefiting from treatment 1.0 Gefitinib EGFR Mut+ (n=132) Gefitinib EGFR Mut- (n=91) Carboplatin/paclitaxel EGFR Mut+ (n=129) Carboplatin/paclitaxel EGFR Mut- (n=85) Mut +ve Gefitinib vs CT HR=0.48 p< Mut ve Gefitinib vs CT HR=2.85 p< Unknown 6.6 vs 5.8 mos HR = 0.68 p< Time from randomization (months) PFS treatment by EGFR mutation status interaction test: p< Mok et al. NEJM 2009; Fukuoka et al. JCO 2009

17 ALK rearrangement as a Target

18 Clinical profile of ALK-positive NSCLC Gene fusions involving ALK within chromosome 2 have been identified in % of NSCLC tumours 1,2 Incidence rates are influenced by sample size, screening methods, definition of ALK-positivity, and enrichment prior to pre-selection ALK is associated with adenocarcinoma histology Incidence of % 3 5 In squamous cell patients incidence of ALK <1% 1 Trend for ALK patients to be non/light-smokers 2 4 ALK rearrangements, for the most part, appear to occur in the absence of EGFR/KRAS mutations 2,3,6,7 1 Boland JM, et al. Human Pathol. 2009;40:1152 8; 2 Zhang X, et al. Mol Cancer. 2010;9:188; 3 Rodig SJ, et al. Clin Cancer Res. 2009;15: ; 4 Wong DW, et al. Cancer. 2009;115: ; 5 Takahashi T, et al. Ann Surg Oncol. 2010;17:889 97; 6 Horn L and Pao W. J Clin Oncol. 2009;27:4232 5; 7 Shaw AT, et al. J Clin Oncol. 2009;27:

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20 43 yo Male Non-Smoker with NSCLC positive for ALK Pre-Treatment (FLT-PET) After 4 weeks of PF

21 Decrease or increase from baseline (%) First-in-human trial: Tumour responses to PF by patient* n=116** ORR (95% CI) 61% (52, 70) Median response duration 48 weeks Median time to response 8 weeks Disease control rate at 8, 16 weeks 79%, 67% Progressive disease (PD) Stable disease (SD) Partial response (PR) Complete response (CR) *Excludes patients with early death and indeterminate response (n=106) **Includes patients with early death and indeterminate response (n=116) ORR, overall response rate CI, confidence interval Camidge DR, et al. Presented at ASCO 2011; Abstract 2501

22 Oncogene Driver Revolution

23 Lung Cancer Mutation Consortium Incidence of Mutations Detected Mutation found in 54% (280/516) of tumours completely tested (CI 50-59%) Kris et al. ASCO 2011, Abstract 7506

24 Characterising Lung Tumors ETOP Lungscape INCa LCMC CASTLE Cancer Research UK

25 What the future holds... We will require increasing, multidisciplinary collaboration between Clinicians, Pathlogists and Biologists Tissue processing and molecular testing will be paramount

26 Targeted therapies in NSCLC: Issues in NSCLC pathology Prof Keith M Kerr, Aberdeen University Medical School, UK

27 Selective toxicity in NSCLC Therapy 1951 Targetable characteristics of a tumour Mitotic activity Other metabolic functions More fundamental molecular characteristic Implications for diagnosis

28 Diagnostic Steps Receive, Prepare, Dissect, Sample and Process the specimen in NSCLC Gross examination Morphological Histologic Examination Identify malignancy Levels of Diagnosis in NSCLC Morphology IHC Molecular Therapy Choice Characterize malignancy Lung cancer Not SCLC Subtype NSCLC Molecular Characteristics Protein Gene copy number Translational changes Sequence alterations (mutation, rearrangement) Immunohisto- Chemistry (IHC) Diagnostic IHC In-Situ Hybridization mrna quantitation DNA mutations Prognostic / Predictive

29 Sample of tumour tissue taken Whole tumour / organ Fragment(s) of the tumour Cells from the tumour Section of tumour tissue on microscope slide

30 Pre-analyticals, Processing and Diagnostic Test Outcomes Preparation and Processing to make a diagnosis Alteration of tissue chemistry Morphology and Molecular Is the test feasible and meaningful?

31 How long between Removal (anoxia) and Start of Fixation? Does the sample arrive in fixative?

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38 Small tissue biopsies Fine needle aspirates Fluids

39 Fixation Dehydration Alcohol infusion Acetone Paraffin wax

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41 4 um thick sections

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44 Semi-automated production of Haematoxylin & Eosin stained slides

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48 Diagnostic Steps Receive, Prepare, Dissect, Sample and Process the specimen in NSCLC Cytology Samples Diagnostic Biopsies Resection Specimens Gross examination Morphological Histologic Examination Identify malignancy Characterize malignancy Lung cancer Not SCLC Subtype NSCLC Molecular Characteristics Protein Gene copy number Translational changes Sequence alterations (mutation, rearrangement) Immunohisto- Chemistry (IHC) Diagnostic IHC In-Situ Hybridization mrna quantitation DNA mutations Prognostic / Predictive

49 Technical issues with IHC Fixation too long non-specific overstaining too short poor fixation, lose antigenicity Storage time Antigen retrieval for IHC Heat-induced over or understained Protease loss of staining Endogenous enzyme activity Variable primary antibodies Detection system chemistry Assessment / scoring systems loss of antigenicity (especially cut sections) Bussolati G, Leonardo E, J Clin Pathol 2008; 61,

50 Guidelines for biomarker (EGFR mutation) testing recommend 10% neutral buffered formalin and controlled fixation times Not less than 6 hours and not more than 48 hours fixation time Pirker R, Herth FJF, Kerr KM et al. J Thorac Oncol. 2010;5: Thunnissen E, Kerr KM, Herth FJF et al. Lung Cancer 2012;76:1-18 Lindeman NI, Ladanyi M, Cagle P et al. Joint CAP/IASLC/AMP guidelines in preparation/submitted Yatabe Y, personal communication

51 Pre-analytical issues and (F)ISH Similar to issues with IHC Prolonged fixation - more pre-treatment De-waxing Protein digestion Denaturing DNA Non-specific staining or Hybridization failure Fluorescence issues Loss of morphology Tissue autofluorescence Technology issues

52 Pre-analytical issues and nucleic acids Does preparation facilitate mutation testing? DNA cross-linking during fixation Extended protease treatment PCR products mrna very labile Fresh to Fixed - implications

53 Tumour Heterogeneity The rule, not the exception Biomarker dependent Addictive driver mutations may be different? Minor clones and Resistance Sampling error Does the Test Result adequately reflect the status of the patient s tumour burden? Is the biomarker unique to the malignant cell population?

54 TS protein immunohistochemistry

55 ALK rearrangement L858R mutation exon21 EGFR EGFR protein IHC & H-scoring Alk protein IHC MET protein IHC

56 Conclusions For Molecular testing to be successfull we require: Adequate tissue Quality tissue The menu for molecular testing is increasing Not all tests are equal We must determine the best tests for the right applications to bring the optimal treatment to patients

57 Detection of ALK Rearrangements in NSCLC Prof. Dr. P. Pauwels (UZA, UA )

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66 ALK fusions in NSCLC (2007 present) EML4 ALK Variant 1 2 3a 3b 4 5a 5b V4 V a 8b E17;ins68A20 E20;ins18A20 E6;A19 Coiled-coil domains ALK tyrosine kinase domain KIF5b TFG KLC1 Sanders H, et al. Can Gen. 2011;204;45 52; Togashi Y, et al. PLoS One. 2012;7:e31323; Wong D, et al. Cancer. 2011;117: ; Penzel R, et al. J Thorac Oncol. 2012;7:1198 9; Sasaki T, et al. Euro J Can. 2010;46:

67 Methods for Detecting ALK Rearrangements Fluorescence in-situ hybridization (FISH) Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Next Generation DNA Sequencing (NGS) Immunohistochemistry (IHC) CC CC Fusion Gene DNA/RNA Sequence Detection Fusion Protein Detection

68 RT-PCR and DNA Sequencing to Detect ALK Neither RT-PCR nor DNA sequencing are standard techniques for the identification of ALK rearrangements, and have not been validated in clinical trials to identify responders to ALK targeted therapies Although there are promising developments in the literature Soda M, et al. Clin Cancer Res Aug 20 [Epub ahead of print]c Peled N, et al. J Thorac Oncol. 2012;7:e14 6

69 1. ALK FISH testing in wet lab Vysis ALK probe DESIGN of Vysis ALK dual color, break apart rearrangement probe Identifies all rearrangements regardless of fusion partner (EML4, TGF, KIF5B etc.)

70 Vysis ALK Break Apart FISH Probe Kit Optimised for lung specimens (FFPET) Ready to use in vitro diagnostic kit Gold standard FISH test used in clinical trials CE marked and FDA approved Vysis ALK Break Apart FISH Probe Kit, Package Insert, /R1

71 2. Enumeration PRINCIPLE

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80 Conclusions FISH is currently the Gold Standard to detect ALK rearrangements in NSCLC IHC is in development along with RT-PCR and NGS, however at this time these techiques are not clinically validated or standardized Some of these techniques do look promising

81 Chairman s Summary Oncogene Driver Revolution

82 Lung Cancer Mutation Consortium Incidence of Mutations Detected Mutation found in 54% (280/516) of tumours completely tested (CI 50-59%) Kris et al. ASCO 2011, Abstract 7506

83 Multiplex Testing in Squamous Cell Lung Cancer: SQ-MAP integrated results Target N Frequency 95% CI FGFR1 amplification PTEN mutation 17% PTEN loss, complete PIK3CA mutation KRAS mutation DDR2 mutation 13/52 25% 15 38% 3/18 17% 5 37% 3/27 11% 3 26% 4/52 8% 2 17% 1/52 2% 1 9% 0/18 0% 0 15% Paik et al. J Clin Oncol 30: 2012 (suppl; abstr 7505)

84 Cancer therapy: progress in the last decade Targeted therapies = a new era shaping cancer therapy Targeting specific pathways that control the biological processes associated with cancer Holding the promise of better efficacy and fewer side-effects

85 But what do we need to be successful? Multidisciplinary collaboration Training for Molecular techniques Quality Assurance schemes

86 European Society of Pathology (ESP): European External Quality Assessment (EQA) program Aim: ensure optimal accuracy and proficiency in mutation testing for patients with NSCLC across all countries Project group: E. Thunnissen, Amsterdam, the Netherlands (scheme organiser) E. Dequeker, Leuven, Belgium (scheme coordinator logistics) L. Bubendorf, Basel, Switzerland K. Kerr, Aberdeen, Scotland P. Pauwels, Antwerp, Belgium E. Schuuring, Groningen, the Netherlands K. Miller, UKNEQAS, London, UK Scheme includes FISH and IHC

87 NEQAS ALK EQA Program Has created cell lines for ALK EQA with FISH Pilot rounds in progress Should be available to rest of Europe by end of 2012 Contact: Keith Miller, UKNEQAS, London, UK There are also various local EQA schemes in development Italy, Germany, France, etc...

88 (coming Jan. 2013) Aim of the Academy is to promote education and training and encourage innovation in molecular testing Stimulate dialogue and foster collaborations between clinicians and pathologists in molecular testing and targeted therapies in oncology In , this programme will primarily focus upon the important area of ALK gene rearrangements in NSCLC 3 Pillars: Faculty of world leading key opinion leaders Website and interactive on-line learning resources Practical workshops to train in molecular techniques Visit the website today to register your interest!