TriStar Presentation August 2009
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Transcript of TriStar Presentation August 2009
Giving Clinical Significance To Molecular Targets
Tristar Technology Group9700 Great Seneca Highway
Rockville, MD 20850U.S.A
www.tristargroup.us
TRISTAR AROUND THE WORLD
Rockville, MDHeadquarters
Catania, ItalyTissue Repository
Hamburg, GermanyMain Repository
Array ManufacturingContract Research
Hematological Malignancies
NETWORK OF REPOSITORIES
Washington DC, USAHeadquarters
Catania, ItalyTissue Repository
Hamburg, GermanyMain Repository
Array ManufacturingContract Research
Solid TumorsInflammatory Diseases
Hepatic & Renal DiseasesCNS
Solid Tumors Hematological Malignancies
Prospective Collection Projects
GENERAL INFORMATION
Cancer120 Tumor Types
Over 2.5 million samplesOver 60,000 micro arrayed samples
Formalin fixed & frozenDetailed clinical information
CNSOver 100,000 samplesFormalin fix & frozenClinical information
Alzheimer‘s Disease, Parkinson‘s Disease, Stroke, MS,Vascular Dementia, Prion Disease etc.
Inflammatory DiseaseOver 300,000 samples
Over 3,000 micro arrayed samplesFormalin fixed
Rheumatoid Arthritis, Inflammatory Bowel Disease,Psoriasis Arthritis etc.
BIOLOGICAL SAMPLES
Tissue micro arrays with matched large sections
Tissue blocks with clinical databasePrimary 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
ETHICAL CONSIDERATIONS
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
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
QUALITY CONTROL
Samples are fixed/frozen within 2 – 10 minutes of Excision
OCT embedded sampleSnap frozen sample
Formalin fixed sample
10% Buffered formalin, 10-12 hrs fixation timeMorphology (H&E) & IHC Markers for immunogenicity
RNA & DNA Quality (Agilent 2100 Bioanalyzer)RIN can be checked & provided upon request
Finding an Attractive Drug Target
Molecular Epidemiology
What normal tissues do express target?
How frequent is expression in human cancer?
Option 1:Review the
literature
Specific cancer subtypes or biological properties?-prognostic relevance
Option 2:Perform
own studies
Functional Analysis
Druggability
Molecular Epidemiology
Typical early steps
Current situation
Later stepsusually not done!
Large sets ofHuman tissues with clinical
Follow-up required.Difficult to get.
Finding an Attractive Drug Target
Current situation
Functional Analysis
Druggability
Molecular Epidemiology
Typical early steps Later stepsusually not done!
Early steps
Later steps
Optimal situation
Identify clinically relevant targets before incurring significant costs
Finding an Attractive Drug Target
Functional Analysis
Druggability
Molecular Epidemiology
A tool for the High-Throughput Analysis of Thousands of Tissue samples
Kononen, Sauter et al. Nat Med. 1998 Jul;4(7):844-7
Tissue Micro Array (TMA) Technology
DNA
14.
200.
Morphology
RNA/protein
Formalin Fixed Paraffin Embedded
Frozen OCT Embedded
2.
Tissue Microarrays
Tristar: A New Dimension in Human Tissue Analysis
Expression Screening AcrossMultiple Cancer & Normal
Tissues
Obtain Antibodies Against
Target Protein
Database GenerationGenotype vs Phenotype
Cancer-Specific Analyisis
(Prognosis / Progression)
Prognosis Arrays (5 yr follow up)
- Breast 2,500 donors- Colon 1,500- Lung 1,500- Prostate 3,000- Bladder, Pancreatic, Ovarian etc.
5,000 - 10,000 Datapoints
Generated Per Antibody
Typical IHC-Based Project
120 Human Cancers (3500
samples)
76 Normal Tissues (608 samples)
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
TRISTAR ANTIBODY PROTOCOL DEVELOPMENT
Controls:
Pre-absorption Control (Test for Target Specificity)
Isotype Control AntibodyTest for Unspecific (Fc-mediated) Binding
Omit Primary AntibodyTest for Unspecific Staining Induced by the Detection
System
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
10-50 Samples Each Of 120 Human Cancers
In which cancersis the target expressed?
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.
TriStar Multi-Tumor Tissue Microarray
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?
TriStar Normal Human Tissue Microarray
Is it really true?
How frequent?
Histo-pathological subtypes?
Is it clinically relevant?
Commercial value?
Target Identified using aCGH
ER: Target for hormone therapy in breast cancer
Validation Case StudyEstrogen Receptor (ESR1) Gene Amplification
2,200 Breast Cancers with 5 yr.follow-up information
pT stagepN stageNumber of nodes examinedNumber of positive nodesTumor diameterBRE gradePolymorphyTubulus formationMitoses
Survival months (99%)Survival tumor specific (40%)
Some therapy information (40%)
Molecular parameters: FISH: HER2, EGFR, MDM2, CCND1, MYCIHC: ER, PR, p53, Cytokeratins, EGFR, HER2, CD117, others
Validation PlatformTriStar Breast Cancer Prognosis Array
ESR1 Amplification* in 358/1739 (21%) of Breast Cancers
Holst, Simon et al, Nat Gen (39), 655-660, 2007
Breast Cancer Prognosis TMA AnalysisESR1 amplification (FISH)
???
ER IHC data from our database
Is the ESR1 Amplification Functionally Relevant ?
Does ESR1 Amplification cause ER Overexpression?
ER expression level(Allred score)
ESR1 Amplification Are Linked To ER Protein Overexpression
0
5
10
15
20
25
30
35
40
45
pT1(820)
pT2(1023)
pT3(124)
pT4(242)
G1 (545) G2 (844) G3 (655)
% of samples
GAINAMP
p=0.7295 p<0.0001
Holst, Simon et al, Nat Gen (39), 655-660, 2007
ESR1 Amplification are linked to Early Stage and Low Grade Breast Cancers
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Su
rviv
ing
0 20 40 60 80 100
months surv
ER IHC positive (n=109)
p<0.0001
ESR1 amplification (n=43)
ER IHC negative(n=23)
175 Patients Treated With TamoxifenNo Adjuvant Therapy
Holst, Simon et al, Nat Gen (39), 655-660, 2007
ESR1 amplification predicts response to tamoxifen
ESR1 amplification and anti ER treatment
Affymetrix experiment,ESR1 amp identification
Low density TMA validation
High density TMA studies
Patent filed
Paper accepted,Holst et al, Nature Genetics
March 2006
April 14th, 2006
April-May, 2006
July 15th, 2006
February 2nd, 2007
Start to Finish: 11 months
ESR1 Validation Study using Prognosis TMA: Timeline
Tristar: A New Dimension in Human Tissue Analysis
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)
Tristar Technology Group9700 Great Seneca Highway
Rockville, MD 20850U.S.A
www.tristargroup.us
THANKS!
Rockville, MDHeadquarters
Catania, ItalyTissue Repository
Hamburg, GermanyMain Repository
Array ManufacturingContract Research