TriStar Presentation August 2009

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  • 1. Giving Clinical Significance To Molecular Targets
  • 2. TriStararoundtheworld
    Tristar Technology Group
    9700 Great Seneca Highway
    Rockville, MD 20850
    U.S.A
    www.tristargroup.us
    Rockville, MD
    Headquarters
    Hamburg, Germany
    Main Repository
    Array Manufacturing
    Contract Research
    Catania, Italy
    Tissue Repository
  • 3. Network of Repositories
    HematologicalMalignancies
    Washington DC, USA
    Headquarters
    Hamburg, Germany
    Main Repository
    Array Manufacturing
    Contract Research
    Solid Tumors
    InflammatoryDiseases
    Hepatic & RenalDiseases
    CNS
    Solid Tumors
    HematologicalMalignancies
    ProspectiveCollection Projects
    Catania, Italy
    Tissue Repository
  • 4. General Information
    Cancer
    120 Tumor Types
    Over 2.5 millionsamples
    Over 60,000 microarrayedsamples
    Formalin fixed & frozen
    Detailedclinicalinformation
    CNS
    Over 100,000 samples
    Formalin fix & frozen
    Clinicalinformation
    Alzheimer‘sDisease, Parkinson‘sDisease, Stroke, MS,
    VascularDementia, PrionDisease etc.
    Inflammatory Disease
    Over 300,000 samples
    Over 3,000 microarrayedsamples
    Formalin fixed
    Rheumatoid Arthritis, InflammatoryBowelDisease,
    Psoriasis Arthritis etc.
  • 5. Biological Samples
    Tissue micro arrays with matched large sections
    Tissue blocks with clinical database
    Primary cells, RNA & DNA
    Matched FFPE & Frozen samples
    Primary Tumors
    Primary Tumors with Matched Normal Tissues
    Primary Tumors with Matched Metastases
    Nodal & Distant Metasteses
    Samples with Molecular & Clinical Data
  • 6. EthicalConsiderations
    Informed Donor Consent
    IRB Approval
    Fully Anonymized
    Compliant with Current International & EU Regulations
    Blocks That Are in Excess of Diagnostic Sample Only
    Team of Pathologists & Oncologists for Clinical Data Review
  • 7. Prospective Collection Projects
    Parameters defined by Customer
    Protocol Established
    Timeline & Number of Samples
    IRB Approval
    Projects Include Collection of Snap-Frozen Samples with
    Matched Serum/Plasma, CSF as applicable
  • 8. Quality Control
    Samples are fixed/frozen within 2 – 10 minutes of Excision
    OCT embedded sample
    Snap frozen sample
    Formalin fixed sample
    10% Buffered formalin, 10-12 hrs fixation time
    Morphology (H&E) & IHC Markers for immunogenicity
    RNA & DNA Quality (Agilent 2100 Bioanalyzer)
    RIN can be checked & provided upon request
  • 9. Finding an Attractive Drug Target
    MolecularEpidemiology
    Howfrequentisexpression in human cancer?
    Specificcancersubtypesorbiologicalproperties?
    -prognosticrelevance
    What normal tissues do express target?
    Option 2:Performownstudies
    Option 1:Reviewtheliterature
  • 10. Currentsituation
    Typicalearlysteps
    Latersteps
    usuallynotdone!
    FunctionalAnalysis
    MolecularEpidemiology
    Druggability
  • 11. Finding an Attractive Drug Target
    Currentsituation
    Typicalearlysteps
    Latersteps
    usuallynotdone!
    FunctionalAnalysis
    MolecularEpidemiology
    Druggability
    Large sets of
    Human tissues with clinical
    Follow-up required.
    Difficult to get.
  • 12. Finding an Attractive Drug Target
    Optimal situation
    Latersteps
    Earlysteps
    FunctionalAnalysis
    MolecularEpidemiology
    Druggability
    Identifyclinically relevant targets
    beforeincurringsignificantcosts
  • 13. Tissue Micro Array (TMA) Technology
    A tool for the High-Throughput Analysis of Thousands of Tissue samples
    Kononen, Sauter et al. Nat Med. 1998 Jul;4(7):844-7
  • 14. Frozen OCT Embedded
    Morphology
    Formalin Fixed
    Paraffin Embedded
    RNA/protein
    TissueMicroarrays
    2.
    14.
    200.
    DNA
  • 15. Tristar: A New Dimension in Human Tissue Analysis
  • 16. Typical IHC-Based Project
    ObtainAntibodiesAgainst
    Target Protein
    120 Human Cancers (3500 samples)
    76 Normal Tissues (608 samples)
    Expression ScreeningAcross
    Multiple Cancer & Normal Tissues
    Prognosis Arrays (5 yrfollow up)
    • Breast 2,500 donors
    • 17. Colon 1,500
    • 18. Lung 1,500
    • 19. Prostate 3,000
    • 20. Bladder, Pancreatic, Ovarian etc.
    Cancer-Specific Analyisis
    (Prognosis / Progression)
    5,000 - 10,000 Datapoints
    Generated Per Antibody
    Database Generation
    Genotype vs Phenotype
  • 21. TriStar Antibody Protocol Development
    Test Various Normal and Cancer Tissues
    Test Different Tissue Pre-treatment Conditions:
    Temperature, Pressure, Enzymatic
    Antibody Dilution Series for Optimal Background: Signal Ratio
  • 22. TriStar Antibody Protocol Development
    Controls:
    Pre-absorption Control (Test for Target Specificity)
    Isotype Control Antibody
    Test for Unspecific (Fc-mediated) Binding
    Omit Primary Antibody
    Test for Unspecific Staining Induced by the Detection System
  • 23. List of Tissues for Cross-Reactivity Study
    Adrenal * Lung * Spinal Cord Blood Cells * Lymph Node * Spleen Blood vessels (endothelium) * Ovary * Striated (skeletal) Muscle Bone Marrow * Fallopian Tube (oviduct) * Testis Brain – cerebrum (cortex) * Pancreas * Thymus Brain – cerebellum * Parathyroid * Thyroid Breast (mammary gland) * Peripheral Nerve * Tonsil Eye * Pituitary * Ureter Gastrointestinal Tract2 * Placenta * Urinary Bladder Heart * Prostate * Uterus- body (endometrium) Kidney (glomerulus, tubule) * Salivary Gland * Uterus – cervix Liver * Skin
  • 24. TriStar Multi-Tumor TissueMicroarray
    10-50 Samples Each Of 120 Human Cancers
    Skin: Squamous Cell Carcinoma, Basal Cell Carcinoma, Merkel Cell Carcinoma. Uterine Corpus: Endometrioid Adenocarcinoma, Serous. Parathyroid Gland: Adenoma, Carcinoma. Mammary Gland: Intraductal Carcinoma, Lobular Carcinoma In Situ, Invasive Ductal Carcinoma, Invasiv Lobular Carcinoma, Mucinous Carcinoma, Papillary Carcinoma, Tubular Carcinoma. Kidney: Clear Cell Type, Papillary Type, Chromophobe Cell Type. Urinary Bladder: Non-Invasive Papillary Tumor (Pta), Transitional Cell Carcinoma, Squamous Cell Carcinoma, Adenocarcinoma, Small Cell Carcinoma. Salivary Glands: Mixed Tumor, Adenolymphoma, Adenoma, Mucoepidermoid Carcinoma, Acinic Cell Carcinoma, Adenocarcinoma, Adenoid Cystic Carcinoma. Esophagus: Squamous Cell Carcinoma, Adenocarcinoma. Stomach: Adenocarcinoma Diffuse Type, : Adenocarcinoma Intestinal Type. Adrenal Gland: Adrenal Cortical Adenoma, Adrenal Cortical Carcinoma, Pheochromocytoma. Pancreas: Adenocarcinoma, Adenoma. Mediastinum: Thymoma. Small Intestine: Adenocarcinoma, Carcinoid. Large Intestine: Adenoma, Adenocarcinoma. Appendix: Adenocarcinoma, Carcinoid. Anal: Small Cell Carcinoma. Prostate: Prostatic Adenocarcinoma Untreated, Hormone Refractory Adenocarcinoma Adenocarcinoma, Clear Cell Adenocarcinoma, Atypical Hyperplasia. Cervix: Squamous Cell Carcinoma, Adenocarcinoma. Vagina: Squamous Cell Carcinoma, Adenocarcinoma. Vulva: Squamous Cell Carcinoma. Thyroid Gland: Follicular Carcinoma, Papillary Carcinoma, Anaplastic Carcinoma, Medullary Carcinoma, Adenoma. Lung: Squamous Cell Carcinoma, Adenocarcinoma, Undifferentiated Large Cell Carcinoma, Small Cell Carcinoma, Carcinoid. Testis: Seminoma, Teratoma, Embryonal Carcinoma, Choriocarcinoma, Yolk-Sac-Tumor, Teratocarcinoma. Ovary: Serous Carcinoma, Mucinous Carcinoma, Endometrioid Carcinoma, Brenner Tumor, Germ Cell Tumors. Liver: Hepatocellular Carcinoma, Cholangiocarcinoma. Fibrohistiocytic: Fibrosarcoma, Benign Histiocytoma, Dermatofibrosarcoma Protuberans, Atypical Fibroxanthoma, Malignant Fibrous Hiostiocytoma Lipomatous: Lipoma, Lioposarcoma. Smooth Muscle: Leiomyoma, Leiomyosarcoma, Leiomyoblastoma. Skletal Muscle: Rhabdomyoma, Rhabdomyosarcoma. Blood And Lymph Vessels: Angioma, Epitheloid Hemangioma, Hemangioendothelioma, Angiosarcoma, Kaposi Sarcoma. Perivascular: Glomus Tumor, Hemangiopericytoma. Synovial: Benign Giant Cell Tumor Of Tendon Sheath, Synovial Sarcoma. Mesothelial: Solitary Fibrous Tumor Of Pleura And Peritoneum, Adenomatoidtumor, Malignes Mesothelioma. Neural: Neurofibroma, Neurinoma. Granular Cell Tumor, Malignant Peripheral Nerve Sheath Tumor. Clear Cell Sarcoma. Paraganglioma, Ganglioneuroma. Pnet: Ganglioneuroblastoma, Neuroblastoma, Neuoepithelioma, Extraskelettal Ewings-Sarcoma. Malignant Mesenchymoma. Alveolar Soft Part Sarcoma. Epitheloid Sarcoma. Osseous: Osteoidosteoma, Osteoblastoma, Osteosarcoma. Chondrous: Chondroblastom, Chondrom, Chondrosarcoma, Chordomas. Ewing Sarcoma. Giant Cell Tumor Of The Bone. Brain: Astrocytoma, Glioblastoma Multiforme, Oligodendroglioma, Ependymoma, Medulloblastoma, Medulloepithelioma, Craniopharyngeoma, Esthesioneuroblastoma, Retinoblastoma. Nevus Naevocellularis, Malignant Melanoma, Gastrointestinal Stromatumor, Endometrioid Stromal Sarcoma, Mixed Malignent Mesodermal Tumor, Aml, Cml, Cll, Immunocytic Lymphoma, Plasmocytoma, Centrocytic Lymphoma, Centroblastic Centrocytic Lymphoma, Centroblastic Lymphoma, Immunoblastic Lymphoma, Burkitt Lymphoma, T-Cell Lymphoma Low Grade, T-Cell Lymphoma High Grade, M Hodgkin Lymphocytic Depletion, M Hodgkin Mixed Cell Type, M Hodgkin Nodular Sclerosing etc.
    In which cancers
    is the target expressed?
  • 25. TriStar Normal Human TissueMicroarray
    76 tissue types, 532 cell types, 8 donors each
    Mesenchymal tissues: aorta/intima, aorta/media, heart (left ventricle), sceletal muscle, sceletal muscle/tongue, myometrium, appendix (muscular wall), esophagus (muscular wall), stomach (muscular wall), ileum (muscular wall), colon descendens (muscular wall), kidney pelvis (muscular wall), urinary bladder (muscular wall), penis (glans/corpus spongiosum), ovary (stroma), fat tissue (white),
    Surfaces: skin (surface), skin (hairs, sebaceous glands), lip (epithelium), oral cavity, tonsil (surface epithelium), anal canal (skin), anal canal (transition epithelium), exocervix, esophagus, kidney pelvis, urinary bladder, amnion/chorion, stomach (antrum), stomach (fundus and corpus), small intestine, duodenum, small intestine, ileum, appendix, colon descendens, rectum, gallbladder, bronchus, paranasal sinus.
    Solid organs: lymph node, spleen, thymus, tonsil, liver, pancreas, parotid gland, submandibullary gland, sublingual gland, lip (small salivary gland), duodenum (Brunner gland), kidney cortex, kidney medulla, prostate, seminal vesicle, epididymis, testis, lung (parenchyma), lung (bronchial glands), breast, endocervix, endometrium (proliferation), endometrium (secretion), fallopian tube, endometrium (early decidua), ovary (stroma), ovary (corpus luteum), ovary (follicular cyst), placenta (first trimenon), placenta (mature), adrenal gland, parathyroid gland, thyroid, cerebellum, cerebrum, pituitary gland (posterior lobe), pituitary gland (anterior lobe)
    In which normal tissues
    is the target expressed?
  • 26. Validation CaseStudy
    EstrogenReceptor (ESR1) Gene Amplification
    Target Identified using aCGH
    Isitreallytrue?
    Howfrequent?
    Histo-pathologicalsubtypes?
    Isitclinically relevant?
    Commercial value?
    ER: Target forhormonetherapy in breastcancer
  • 27. Validation Platform
    TriStarBreastCancerPrognosis Array
    pTstage
    pNstage
    Number of nodesexamined
    Number of positive nodes
    Tumor diameter
    BRE grade
    Polymorphy
    Tubulusformation
    Mitoses
    2,200 Breast Cancers with
    5 yr.follow-up information
    Survivalmonths (99%)
    Survivaltumorspecific (40%)
    Sometherapyinformation (40%)
    Molecularparameters: FISH: HER2, EGFR, MDM2, CCND1, MYC
    IHC: ER, PR, p53, Cytokeratins, EGFR, HER2, CD117, others
  • 28. BreastCancerPrognosis TMA Analysis
    ESR1 amplification (FISH)
    ESR1 Amplification* in 358/1739 (21%) of Breast Cancers
    Holst, Simon et al, Nat Gen (39), 655-660, 2007
  • 29. Does ESR1 Amplification cause ER Overexpression?
    ???
    ER IHC datafromourdatabase
    Isthe ESR1 AmplificationFunctionallyRelevant ?
  • 30. ER expression level
    (Allred score)
    ESR1 Amplification Are Linked To ER Protein Overexpression
  • 31. ESR1 Amplificationarelinked to EarlyStage and Low Grade BreastCancers
    p=0.7295
    p<0.0001
    Holst, Simon et al, Nat Gen (39), 655-660, 2007
  • 32. 1.0
    ESR1 amplification (n=43)
    0.9
    0.8
    0.7
    0.6
    0.5
    0.4
    0.3
    0.2
    0.1
    0.0
    0
    20
    40
    60
    80
    100
    ESR1 amplification and anti ER treatment
    175 Patients Treated With Tamoxifen
    No Adjuvant Therapy
    ER IHC positive (n=109)
    Surviving
    ER IHC negative(n=23)
    p<0.0001
    months surv
    Holst, Simon et al, Nat Gen (39), 655-660, 2007
    ESR1 amplificationpredictsresponse to tamoxifen
  • 33. ESR1 Validation StudyusingPrognosis TMA: Timeline
    Affymetrix experiment,
    ESR1 amp identification
    March 2006
    Low density TMA validation
    April 14th, 2006
    High density TMA studies
    April-May, 2006
    Patent filed
    July 15th, 2006
    Paper accepted,
    Holst et al, Nature Genetics
    February 2nd, 2007
    Start to Finish: 11 months
  • 34. Tristar: A New Dimension in Human Tissue Analysis
  • 35. Products & Services
    Product Groups
    Archived Human Tissue Repository
    High-Density Tissue Micro Arrays (>60,000 donor samples)
    Large sections, Blocks, RNA, DNA & Primary Cells
    Cancer Stem Cells Arrays
    Clinical Data – Prognosis, Progression, Treatment & Response
    Molecular Database
    Services
    Compound Screening using Cancer Stem Cells
    Antibody Protocol Development (IHC) & Characterization
    FISH analysis
    Large-Scale Multi-Tumor & Normal Tissue Array Analysis
    Large-Scale Analysis of Prognosis
    Cross-Reactivity Screening in Normal Tissue (GLP)
  • 36. Thanks!
    Tristar Technology Group
    9700 Great Seneca Highway
    Rockville, MD 20850
    U.S.A
    www.tristargroup.us
    Rockville, MD
    Headquarters
    Hamburg, Germany
    Main Repository
    Array Manufacturing
    Contract Research
    Catania, Italy
    Tissue Repository