Ventana s Approach to Lymphoma Biomarkers

Transcription

1 Ventana s Approach to Lymphoma Biomarkers Thomas Grogan, M.D Professor Emeritus, University of Arizona Founder, Ventana Medical Systems. Spanish Society of Pathology Meeting Madrid, Spain 7 February

2 Objectives Demonstrate the new tools to aid lymphoma diagnosis and treatment. Demonstrate new assays and instruments. 2

3 The Ventana Company Founded in 1989 to build new tools to aid the practice of anatomic pathology. Medical driven (14 pathologists) with aid of engineers, chemists and scientists (309 persons). Today 1,500 employees, serving 7,00 hospitals and labs in 63 countries with > 35 million assays per annum. R&D budget of greater than 100m /year. 3

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5 Ventana Product Portfolio Automated solutions spanning the laboratory Pre-Analytical Primary Staining Advanced Staining Tissue Preparation H&E Stain SS IHC ISH Imaging/ Reporting Image Analysis Work Cell Description Tracking Grossing Fixation Processing Vantage Binary Diagnosis Tissue Morphology Symphony Detection of molecular target Diagnosis Predictive/Prognostic NexES Special Stainer BenchMark ULTRA Quantitative analyses Report generation Tele-pathology Archiving VIAS RTD Platforms Workflow and Connectivity Vantage SS= Special Stains, IHC = immunohistochemistry, ISH= in situ hybridization

6 Utility of Slide-Based Tissue Chemistry Simultaneous analysis of morphology, gene and protein status Morphology Gene status Protein status H&E: Hematoxylin&Eosin (Dye) ISH: in situ Hybridization) (DNA/RNA Probe) IHC: Immunohistochemistry (Antibody) 6

7 Next Generation Lymphoma Tools Rabbit monoclonals Super sensitive detection systems Chromogenic single-gene in situ hybridization Combined gene/protein assays Multiplexed assays 7

8 Why Rabbit? 8

9 What can Rb MAb offer? More diverse epitope recognition Better immune response to small epitope and thus higher specificity Phosphorylation site Neo end caused by cleavage Point mutation Higher affinity Possibility to cross react to mouse counterparts average 85% similarity between human and mouse Good immune response to rodent sequences 9

10 Improved Recognition of Small Epitope --- phosphorylation site Anti-S6 ribosomal protein (phospho Ser240/244) Non PPI treated Breast ca. PPI treated Breast ca. Anti- c-met (phospho Tyr1003) Blocked by non phospho peptide Breast ca. Blocked by phospho peptide Breast ca. 10

11 Immunohistochemical Detection of CD3 in T-cell Lymphomas Matter et al. J of Histotechnology, Vol 35, No 4, 2012

12 BCL2 protein expression in follicular lymphomas with t(14;18) chromosomal translocations. Masir et al, Brit J Haematol 144: , 2008 BCL2 pseudonegative follicular lymphoma cases (7/33, 21%) with negative immunostaining by mouse Mab BCL 2/124 but positive with rabbit monoclonal E17. 12

13 BCL2 Protein Expression in Follicular Lymhomas with t(14:18) Chromosomal Translocations British Journal of Hematology, 144,

14 J Clinical Oncology, Vol 30, No 28, Oct 2012.

15 Next Generation Lymphoma Tools Rabbit monoclonals Super sensitive detection systems Chromogenic single-gene in situ hybridization Combined gene/protein assays Multiplexed assays 15

16 Comparison of ALK IHC Detection Schemes Mouse Anti-HQ HRP Multimer Mouse Anti-HQ HRP Multimer Biotinylated Goat Anti-Rabbit Antibody Streptavidin-HRP Complex Goat Anti-Rabbit HQ Multimer HQ-Tyramide Amplified Mouse Anti-HQ HRP Multimer Rabbit Monoclonal Anti-ALK Antibody (Clone D5F3) iview DAB Detection OptiView DAB Detection OptiView DAB Detection & OptiView Amplification

17 ALK- NSCLC ALK+ NSCLC Comparison of IHC Staining iview DAB Detection OptiView DAB Detection OptiView DAB Detection & OptiView Amplification Extended CC1 Clone D5F3 1:70 in dispenser, 16 minute incubation Objective 20X

18 New Tools Aiding the Diagnosis of Lymphoma Rabbit monoclonal antibodies Next generation detection Multiplexing Next generation chromogenic in situ hybridization (CISH) Gene copy number Chromosomal alterations Translocations mrna assays Gene plus protein assays 18

19 Ig mrna Status Normal Tonsil Kappa Ig mrna = Black Lambda Ig mrna = Red

20 Gastric DLBCL Ig mrna Status

21 B cell Lymphoma Ig mrna Copy Number DLBCL (37%) Follicular (29%) CLL (12%) MALT (9%) MZL (7%) KAPPA/LAMBDA mrna copy number Current VMSI Assay Flow Cytometry New VMSI Two-color Brightfield Assay

22 Follicular Lymphoma

23 Follicular Lymphoma Ig mrna Status Kappa Ig mrna = Black Lambda Ig mrna = Red

24 Kappa mrna in SLLa 24

25 New Tools Aiding the Diagnosis of Lymphoma Rabbit monoclonal antibodies Next generation detection Multiplexing Next generation chromogenic in situ hybridization (CISH) Gene copy number Chromosomal alterations Translocations mrna assays Gene plus protein assays 25

26 Primary Genomic Aberrations Associated with CLL Aberration Occurrence Prognosis OS Treatments 13q- ~30% Best yrs. Monitor Trisomy 12 ~20% Intermedia te 9-10 yrs. CD20 targeted agents 11q- ~15% Poor 3-6 yrs. 17p- ~7% Very Poor 1-2 yrs. Rituximab, with alkylating agents Novel agents/+/or allogeneic stem cell transplant

27 Chronic lymphocytic leukemia (CLL) Ventana Probe Panel utilizes both Ventana Repeat-free DNA probes (Black) with chromosomal markers (red). Note: Detection of 13q- uses 2 repeat-free DNA probes. TP53 Cen 17 Cen 11 ATM Cen 12 13q12.11 DLEU

28 Bone Marrow Involvement with CLL

29 Bone Marrow Involvement with CLL 17P- (TP53-)

30 Bone Marrow Involvement with CLL 17P- (TP53-)

31 Peripheral Blood CLL TP53 Status

32 New Tools Aiding the Diagnosis of Lymphoma Rabbit monoclonal antibodies Next generation detection Multiplexing Next generation chromogenic in situ hybridization (CISH) Gene copy number Chromosomal alterations Translocations mrna assays Gene plus protein assays 32

33 ALK Break-apart Probe Design Chromosome 2 (p23.2-p23.1) ALK p23 region Telomere 3 ALK probe Probe Hapten: DNP Probe length: 154 kb Probe Coverage: 683 kb 3 5 ALK gene (728 kb) 5 ALK probe Centromere Probe Hapten: DIG Probe length: 113 kb Probe Coverage: 770 kb 33

34 Color Scheme for Brightfield Break-apart In Situ Hybridization + = Blue ISH Signal Red ISH Signal Purple ISH Signal 34

35 ALK Gene Rearrangement Fused 5 &3 ALK 3 ALK 5 ALK 5 ALK 3 ALK 35

36 Repeat-Free Genomic Probes Repetitive DNA Unique DNA 36

37 BCL-2 Breakapart Assay t(14;18) 37

38 New Tools Aiding the Diagnosis of Lymphoma Rabbit monoclonal antibodies Next generation detection Multiplexing Next generation chromogenic in situ hybridization (CISH) Gene copy number Chromosomal alterations Translocations mrna assays Gene plus protein assays 38

39 DLBCL: MYC 10% have translocations, up to 30% have amplifications Over-expression associated with poor outcome New colorimetric ISH and rabbit mab IHC stains may help in identifying cases Normal tonsil amplified Savage, Blood 2009; Stasik, Haematologica 2010; & unpublished 39

40 Increased c-myc copy number in DLBCL case c-myc CEN8 40

41 Cumulative survival DLBCL OS according to normal vs abnormal MYC locus (FISH or CISH) GCB type DLBCL ABC type DLBCL 1.0 Normal MYC locus (n=19) Normal MYC locus (n=23).6 Abnormal MYC locus (n=27).6 Abnormal MYC locus (n=21) P= P= Time (years) Nathlie Johnson (BCCA),

42 Next Generation Lymphoma Tools Rabbit monoclonals Super sensitive detection systems Chromogenic single-gene in situ hybridization Combined gene/protein assays Multiplexed assays 42

43 Brightfield Multiplexed Detection: MYC Gene and myc Protein 60X 10X 60X Tonsil MYC Gene myc Protein 43

44 MYC Gene-Protein Detection in DLBCL MYC Gene Copies and Protein Expression Objective 60X 44

45 Next Generation Lymphoma Tools Rabbit monoclonals Super sensitive detection systems Chromogenic single-gene in situ hybridization Combined gene/protein assays Multiplexed assays 45

46 Five-Color Multiplexed Cell Labeling Antigen Pseudo Color Secondary Ab Primary Ab Nuclear antigen Cyan 655 goat anti-human Human anti-nuclear Ki-67 nuclear protein Magenta 605 goat anti-rabbit Rabbit anti-ki-67 Mitochondria Orange 525 goat anti-mouse Mouse anti-mito Microtubules Green 565 goat anti-rat Rat anti-tubulin Actin Red 705 streptavidin Biotin-phalloidin 46

47 Spectral Imaging 47

48 T-Cell Profile in B-Cell Lymphoma CD20 CD3 CD4 20x 20x 20x CD20 CD8 FOXP3 40x 20x 20x CONFIDENTIAL

49 4-Plex Composite on Lymphoma CD3 QD565 CD4 QD525 CD8 QD605 FP3 QD CD3/CD4/FOXP3 CD3/CD4 CD3/CD CD3/CD4/FOXP3 CD3/CD CONFIDENTIAL

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51 Neutralizing Tumor-Promoting Chronic Inflammation: A Magic Bullet? Coussens et al. Science 339, 286 (2013)

52 Targets of Therapy High through-put technologies Targets, diagnostics, and agents for: Cell of origin: ABC-DLBCL vs. GCB-DLBCL Survival strategies (ABC): BCR signalling, NF-kB Epigenetics (GCB): DNA methyltransferases Oncogenes: BCL2, MYC Microenvironment Enhancing immune surveillance Inhibiting angiogenesis 52

53 Anti-CD20 Therapy (Rituxamab) in DLBCL Two prognostic risk groups: Germinal center B cell like (GCB): 80% at 5 yr, median PFS not reached Activated B cell like (ABC): 40% at 5 yr, median PFS 1.5 yr Lenz, et al, NEJM

54 ABC-DLBCL: B-cell receptor signalling Mutations in: CARD11 CD79A/B MYD88 (gain of function) A20 Lead to activation of: NF-kB (MYD88 or CARD11) JAK kinase activation of STAT3 (MYD88) Increases autocrine IL6, IL10, and beta-ifn PI3K/AKT/mTOR pathway ABC DLBCL (n=155) MYD88 (23%) L265P (29%) 2 CARD (8%) CD79A/B 8 A20 (23%) MYD88 other(8%) 51 Lenz et al, Science 2008; Compagno Nature 2009; Ngo et al, Nature 2011; Shipp et al, Nat Med 2002; and others) 54

55 Next Gen Cell Signaling Assays PI3k February 10,

56 The Challenges Deliver more results relevant to patient prognosis and therapy. Deliver more molecular-based assays. Deliver more automated, standardized results. Communicate results better. 56

57 Prescription for Success Know our strengths: Knowledge of tissue: the tissue is the issue. Integration abilities: molecular morphology our field. Better tools: Better assays: Rabbit monoclonal Chromogen single-gene in situ hybridization Combined gene and protein assays Multiplexed assays Better platforms: Medical cockpit Pre-Analytic Platform 57

58 Medical Cockpit 58

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60 Computer Assisted Detection of Gene Status in Dual ISH Assay Slides 60

61 Quad-plexed Quantum Dot Analyses Discovery XT Fluorescence Analysis - VMSI x Image Cube: Composite Image: Normalize Image Brightness Image cube base name Unmixed Image Name Blur Image before processing Browse Wavelength 585 nm Primary Localization Nucleus Smd4 Label 655 nm Nucleus Ki nm Membrane 565 nm Nucleus Analyze Configure Automated quantitative analyses Help CD-20 DAPI Exit Interactivity between plots and image allow intuitive exploration of the data Interactivity between plots and image allow intuitive exploration of the data. 505 nm 565 nm Pre-process individual frequency slices and display results on composite image 625 nm Composite 61

62 Analog Capture (2) Channel Select (3) SDI (3), sync/trig (2) Pre-Analytic Platform US Measurement Channel, 3-5 sensors, sequentially pulsed Programmable Sine Burst Generator AD5930 Eval Amplifier 7 Vpp out Multiplexer 1:5 Target Attenuator Phase/Ratio (2) Phase/Ratio Comparator AD8302 Eval Multiplexer 5:1 Automated Tissue Arduino Microprocessor Using internal Sampling Clock, timed mode acquisition, spread spectrum US frequency Analog in, Digital I/O bit, asynchronous In Reference position (1) Ready/In position (1) Start/Stop (1) Stepper Controller, 1 Axis - 11 position indexing, - repeat home, single run - pulse out when in position Preservation pakt 62

63 The Ideal Future Multiparameter, multiplex results with morphology, phenotype, and genotype with full definition of all therapeutic targets communicated in a same day patient report integrating pathology, radiology, surgery and oncology findings. Results communicated by a multitasking pathologist who serves as diagnostician, lab director and information integrator. 63

64 We Innovate Healthcare 64