Giving Clinical Significance To Molecular Targets<br />
TriStararoundtheworld<br />Tristar Technology Group<br />9700 Great Seneca Highway<br />Rockville, MD 20850<br />U.S.A<br ...
Network of Repositories<br />HematologicalMalignancies<br />Washington DC, USA<br />Headquarters<br />Hamburg, Germany<br ...
General Information<br />Cancer<br />120 Tumor Types<br />Over 2.5 millionsamples<br />Over 60,000 microarrayedsamples<br ...
Biological Samples<br />Tissue micro arrays with matched large sections<br />Tissue blocks with clinical database<br />Pri...
EthicalConsiderations<br />Informed Donor Consent<br />IRB Approval<br />Fully Anonymized<br />Compliant with Current Inte...
Prospective Collection Projects<br />Parameters defined by Customer <br />Protocol Established <br />Timeline & Number of ...
Quality Control<br />Samples are fixed/frozen within 2 – 10 minutes of Excision<br />OCT embedded sample<br />Snap frozen ...
Finding an Attractive Drug Target<br />MolecularEpidemiology<br />Howfrequentisexpression in human cancer?<br />Specificca...
Currentsituation<br />Typicalearlysteps<br />Latersteps<br />usuallynotdone!<br />FunctionalAnalysis<br />MolecularEpidemi...
Finding an Attractive Drug Target<br />Currentsituation<br />Typicalearlysteps<br />Latersteps<br />usuallynotdone!<br />F...
Finding an Attractive Drug Target<br />Optimal situation<br />Latersteps<br />Earlysteps<br />FunctionalAnalysis<br />Mole...
Tissue Micro Array (TMA) Technology<br />A tool for the High-Throughput Analysis of Thousands of Tissue samples <br />Kono...
Frozen OCT Embedded<br />Morphology<br />Formalin Fixed <br />Paraffin Embedded<br />RNA/protein<br />TissueMicroarrays<br...
Tristar: A New Dimension in Human Tissue Analysis<br />
Typical IHC-Based Project<br />ObtainAntibodiesAgainst<br />Target Protein<br />120 Human Cancers (3500 samples)<br />76 N...
Colon		1,500
Lung		1,500
Prostate	3,000
Bladder, Pancreatic, Ovarian etc.</li></ul>Cancer-Specific Analyisis<br />(Prognosis / Progression)<br />5,000 - 10,000 Da...
TriStar Antibody Protocol Development<br />Test Various Normal and Cancer Tissues<br />Test Different Tissue Pre-treatment...
TriStar Antibody Protocol Development<br />Controls:<br />Pre-absorption Control (Test for Target Specificity)<br />Isotyp...
List of Tissues for Cross-Reactivity Study<br />Adrenal * Lung * Spinal Cord Blood Cells * Lymph Node *  Spleen Blood vess...
TriStar Multi-Tumor TissueMicroarray<br />10-50 Samples Each Of 120 Human Cancers<br />Skin: Squamous Cell Carcinoma,  Bas...
TriStar Normal Human TissueMicroarray<br />76 tissue types, 532 cell types, 8 donors each <br />Mesenchymal tissues: aorta...
Validation CaseStudy<br />EstrogenReceptor (ESR1) Gene Amplification<br />Target Identified using aCGH<br />Isitreallytrue...
Validation Platform<br />TriStarBreastCancerPrognosis Array<br />pTstage<br />pNstage<br />Number of nodesexamined<br />Nu...
BreastCancerPrognosis TMA Analysis<br />ESR1 amplification (FISH)<br />ESR1 Amplification* in 358/1739 (21%) of Breast Can...
Does ESR1 Amplification cause ER Overexpression?<br />???<br />ER IHC datafromourdatabase<br />Isthe ESR1 AmplificationFun...
ER expression level<br />(Allred score)<br />ESR1 Amplification Are Linked To ER Protein Overexpression<br />
ESR1 Amplificationarelinked to EarlyStage and Low Grade BreastCancers<br />p=0.7295<br />p&lt;0.0001<br />Holst, Simon et ...
1.0<br />ESR1 amplification (n=43)<br />0.9<br />0.8<br />0.7<br />0.6<br />0.5<br />0.4<br />0.3<br />0.2<br />0.1<br />0...
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TriStar Presentation August 2009

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TriStar Presentation August 2009

  1. 1. Giving Clinical Significance To Molecular Targets<br />
  2. 2. TriStararoundtheworld<br />Tristar Technology Group<br />9700 Great Seneca Highway<br />Rockville, MD 20850<br />U.S.A<br />www.tristargroup.us<br />Rockville, MD<br />Headquarters<br />Hamburg, Germany<br />Main Repository<br />Array Manufacturing<br />Contract Research<br />Catania, Italy<br />Tissue Repository<br />
  3. 3. Network of Repositories<br />HematologicalMalignancies<br />Washington DC, USA<br />Headquarters<br />Hamburg, Germany<br />Main Repository<br />Array Manufacturing<br />Contract Research<br />Solid Tumors<br />InflammatoryDiseases<br />Hepatic & RenalDiseases<br />CNS<br />Solid Tumors <br />HematologicalMalignancies<br />ProspectiveCollection Projects<br />Catania, Italy<br />Tissue Repository<br />
  4. 4. General Information<br />Cancer<br />120 Tumor Types<br />Over 2.5 millionsamples<br />Over 60,000 microarrayedsamples<br />Formalin fixed & frozen<br />Detailedclinicalinformation<br />CNS<br />Over 100,000 samples<br />Formalin fix & frozen<br />Clinicalinformation<br />Alzheimer‘sDisease, Parkinson‘sDisease, Stroke, MS,<br />VascularDementia, PrionDisease etc.<br />Inflammatory Disease<br />Over 300,000 samples<br />Over 3,000 microarrayedsamples<br />Formalin fixed<br />Rheumatoid Arthritis, InflammatoryBowelDisease,<br />Psoriasis Arthritis etc.<br />
  5. 5. Biological Samples<br />Tissue micro arrays with matched large sections<br />Tissue blocks with clinical database<br />Primary cells, RNA & DNA<br />Matched FFPE & Frozen samples<br /> Primary Tumors<br /> Primary Tumors with Matched Normal Tissues<br /> Primary Tumors with Matched Metastases<br /> Nodal & Distant Metasteses<br /> Samples with Molecular & Clinical Data<br />
  6. 6. EthicalConsiderations<br />Informed Donor Consent<br />IRB Approval<br />Fully Anonymized<br />Compliant with Current International & EU Regulations<br />Blocks That Are in Excess of Diagnostic Sample Only<br />Team of Pathologists & Oncologists for Clinical Data Review<br />
  7. 7. Prospective Collection Projects<br />Parameters defined by Customer <br />Protocol Established <br />Timeline & Number of Samples <br />IRB Approval <br />Projects Include Collection of Snap-Frozen Samples with <br />Matched Serum/Plasma, CSF as applicable<br />
  8. 8. Quality Control<br />Samples are fixed/frozen within 2 – 10 minutes of Excision<br />OCT embedded sample<br />Snap frozen sample<br />Formalin fixed sample<br />10% Buffered formalin, 10-12 hrs fixation time<br />Morphology (H&E) & IHC Markers for immunogenicity<br />RNA & DNA Quality (Agilent 2100 Bioanalyzer)<br />RIN can be checked & provided upon request<br />
  9. 9. Finding an Attractive Drug Target<br />MolecularEpidemiology<br />Howfrequentisexpression in human cancer?<br />Specificcancersubtypesorbiologicalproperties?<br />-prognosticrelevance<br />What normal tissues do express target?<br />Option 2:Performownstudies<br />Option 1:Reviewtheliterature<br />
  10. 10. Currentsituation<br />Typicalearlysteps<br />Latersteps<br />usuallynotdone!<br />FunctionalAnalysis<br />MolecularEpidemiology<br />Druggability<br />
  11. 11. Finding an Attractive Drug Target<br />Currentsituation<br />Typicalearlysteps<br />Latersteps<br />usuallynotdone!<br />FunctionalAnalysis<br />MolecularEpidemiology<br />Druggability<br />Large sets of<br />Human tissues with clinical<br />Follow-up required.<br />Difficult to get.<br />
  12. 12. Finding an Attractive Drug Target<br />Optimal situation<br />Latersteps<br />Earlysteps<br />FunctionalAnalysis<br />MolecularEpidemiology<br />Druggability<br />Identifyclinically relevant targets<br />beforeincurringsignificantcosts<br />
  13. 13. Tissue Micro Array (TMA) Technology<br />A tool for the High-Throughput Analysis of Thousands of Tissue samples <br />Kononen, Sauter et al. Nat Med. 1998 Jul;4(7):844-7<br />
  14. 14. Frozen OCT Embedded<br />Morphology<br />Formalin Fixed <br />Paraffin Embedded<br />RNA/protein<br />TissueMicroarrays<br />2.<br />14.<br />200.<br />DNA<br />
  15. 15. Tristar: A New Dimension in Human Tissue Analysis<br />
  16. 16. Typical IHC-Based Project<br />ObtainAntibodiesAgainst<br />Target Protein<br />120 Human Cancers (3500 samples)<br />76 Normal Tissues (608 samples)<br />Expression ScreeningAcross<br />Multiple Cancer & Normal Tissues<br />Prognosis Arrays (5 yrfollow up)<br /><ul><li>Breast 2,500 donors
  17. 17. Colon 1,500
  18. 18. Lung 1,500
  19. 19. Prostate 3,000
  20. 20. Bladder, Pancreatic, Ovarian etc.</li></ul>Cancer-Specific Analyisis<br />(Prognosis / Progression)<br />5,000 - 10,000 Datapoints<br />Generated Per Antibody<br />Database Generation<br />Genotype vs Phenotype<br />
  21. 21. TriStar Antibody Protocol Development<br />Test Various Normal and Cancer Tissues<br />Test Different Tissue Pre-treatment Conditions:<br />Temperature, Pressure, Enzymatic<br />Antibody Dilution Series for Optimal Background: Signal Ratio<br />
  22. 22. TriStar Antibody Protocol Development<br />Controls:<br />Pre-absorption Control (Test for Target Specificity)<br />Isotype Control Antibody<br />Test for Unspecific (Fc-mediated) Binding<br />Omit Primary Antibody<br />Test for Unspecific Staining Induced by the Detection System<br />
  23. 23. List of Tissues for Cross-Reactivity Study<br />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<br />
  24. 24. TriStar Multi-Tumor TissueMicroarray<br />10-50 Samples Each Of 120 Human Cancers<br />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.<br />In which cancers<br />is the target expressed?<br />
  25. 25. TriStar Normal Human TissueMicroarray<br />76 tissue types, 532 cell types, 8 donors each <br />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), <br />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.<br />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)<br />In which normal tissues<br />is the target expressed?<br />
  26. 26. Validation CaseStudy<br />EstrogenReceptor (ESR1) Gene Amplification<br />Target Identified using aCGH<br />Isitreallytrue?<br />Howfrequent?<br />Histo-pathologicalsubtypes?<br />Isitclinically relevant?<br />Commercial value?<br />ER: Target forhormonetherapy in breastcancer<br />
  27. 27. Validation Platform<br />TriStarBreastCancerPrognosis Array<br />pTstage<br />pNstage<br />Number of nodesexamined<br />Number of positive nodes<br />Tumor diameter<br />BRE grade<br />Polymorphy<br />Tubulusformation<br />Mitoses<br />2,200 Breast Cancers with <br />5 yr.follow-up information<br />Survivalmonths (99%)<br />Survivaltumorspecific (40%)<br />Sometherapyinformation (40%)<br />Molecularparameters: FISH: HER2, EGFR, MDM2, CCND1, MYC<br />IHC: ER, PR, p53, Cytokeratins, EGFR, HER2, CD117, others<br />
  28. 28. BreastCancerPrognosis TMA Analysis<br />ESR1 amplification (FISH)<br />ESR1 Amplification* in 358/1739 (21%) of Breast Cancers<br />Holst, Simon et al, Nat Gen (39), 655-660, 2007<br />
  29. 29. Does ESR1 Amplification cause ER Overexpression?<br />???<br />ER IHC datafromourdatabase<br />Isthe ESR1 AmplificationFunctionallyRelevant ?<br />
  30. 30. ER expression level<br />(Allred score)<br />ESR1 Amplification Are Linked To ER Protein Overexpression<br />
  31. 31. ESR1 Amplificationarelinked to EarlyStage and Low Grade BreastCancers<br />p=0.7295<br />p&lt;0.0001<br />Holst, Simon et al, Nat Gen (39), 655-660, 2007<br />
  32. 32. 1.0<br />ESR1 amplification (n=43)<br />0.9<br />0.8<br />0.7<br />0.6<br />0.5<br />0.4<br />0.3<br />0.2<br />0.1<br />0.0<br />0<br />20<br />40<br />60<br />80<br />100<br />ESR1 amplification and anti ER treatment<br />175 Patients Treated With Tamoxifen<br />No Adjuvant Therapy<br />ER IHC positive (n=109)<br />Surviving<br />ER IHC negative(n=23)<br />p&lt;0.0001<br />months surv<br />Holst, Simon et al, Nat Gen (39), 655-660, 2007<br />ESR1 amplificationpredictsresponse to tamoxifen<br />
  33. 33. ESR1 Validation StudyusingPrognosis TMA: Timeline<br />Affymetrix experiment,<br />ESR1 amp identification<br />March 2006<br />Low density TMA validation<br />April 14th, 2006<br />High density TMA studies<br />April-May, 2006<br />Patent filed<br />July 15th, 2006<br />Paper accepted,<br />Holst et al, Nature Genetics<br />February 2nd, 2007<br />Start to Finish: 11 months<br />
  34. 34. Tristar: A New Dimension in Human Tissue Analysis<br />
  35. 35. Products & Services<br />Product Groups<br /> Archived Human Tissue Repository<br /> High-Density Tissue Micro Arrays (&gt;60,000 donor samples)<br /> Large sections, Blocks, RNA, DNA & Primary Cells<br /> Cancer Stem Cells Arrays<br /> Clinical Data – Prognosis, Progression, Treatment & Response<br /> Molecular Database<br />Services<br /> Compound Screening using Cancer Stem Cells<br /> Antibody Protocol Development (IHC) & Characterization<br /> FISH analysis<br /> Large-Scale Multi-Tumor & Normal Tissue Array Analysis<br /> Large-Scale Analysis of Prognosis<br /> Cross-Reactivity Screening in Normal Tissue (GLP)<br />
  36. 36. Thanks!<br />Tristar Technology Group<br />9700 Great Seneca Highway<br />Rockville, MD 20850<br />U.S.A<br />www.tristargroup.us<br />Rockville, MD<br />Headquarters<br />Hamburg, Germany<br />Main Repository<br />Array Manufacturing<br />Contract Research<br />Catania, Italy<br />Tissue Repository<br />

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