TriStar Corporate Presentation


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  • TriStar Corporate Presentation

    1. 1. Sample Acquisition With Annotated Clinical Information : A Critical Success Factor In Biomarker ValidationTriStarTechnology Group9700 Great Seneca Highway, suite 401Rockville, MD 20850(E) 301-792-633(W)
    2. 2. the need for targeted therapeuticswith companion diagnosticsDevelopment of targeted therapeutics requires testing in targeted populationsmatched to a drug’s mechanism of actionEvaluation of Trastuzamab in “all comer” breast cancer patients (25%HER+, 75% HER2-) would not have shown significant benefit in clinical trialsEarly proof of concept in the right patient population is crucialPotentially shorter time to market
    3. 3. An emerging unmet need in oncology drugdevelopment today is service providersthat offer both access to well-annotatedspecimens and sophisticated molecularanalytical capabilities
    4. 4. Rockville, MD Hamburg, Germany TMA Repository Array Manufacturing Contract Research Madrid, Spain Rome & Catania, ItalyTMA Repository TMA Repository & Contract Research Cancer Stem Cell Research
    5. 5. tristar provides Access to 2.5 million archived samples & clinical data Access to patients (prospective collection projects) Fit-for-purpose analytical platforms & services (IHC, FISH, qRT- PCR etc.) Collaboration for Solid tissue biomarker development
    6. 6. 4ethical considerations Informed Donor Consent IRB/EC Approval Fully Anonymized Compliant with Current International & EU Regulations Blocks That Are in Excess of Diagnostic Sample Only Team of 17 Pathologists & 5 Oncologists for Clinical Data Review
    7. 7. 4product groupsArchived Human Tissue Repository >2.5 million samples (FFPE & Frozen). 70% Oncology, 30% CNS, GI etc. High-Density Tissue Micro Arrays>100,000 donor samples with outcome data Outcome Data Treatment, Response rates, disease –free survival (DFS), overall survival (OS) Molecular Data ER/PR/HER2, p53, BRAF, KRAS, EGFR, PIK3CA etc. Blocks & Large sections With matching RNA, DNA Cancer Stem Cell Arrays Lysates & RNA
    8. 8. 4our services Protein Expression IHC (Antibody protocol development, automated or manual staining, reading & interpretation) Large-Scale Analysis of Prognostic markers (500-3500 donor samples) (500-3500 donor samples) Gene Expression RT-PCR Gene copy number FISH/CISH Gene sequencing DNA sequencing Cross-Reactivity Screening in Normal Tissue(GLP)
    9. 9. quality control Samples are fixed/frozen within 2 – 10 minutes of Excision OCT embedded sample Snap frozen sample Formalin fixed sample
    10. 10. quality control 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
    11. 11. primary tumorswith matched mets Primary Tumors Matched Mets Approximate number Nodal 2000 Breast Distant 20 Bone 200 CRC Nodal 2000 Liver 150 Prostate Nodal 500 Bone 300 Lung (NSCLC) Nodal 300 Bone 100 Pancreatic Nodal 100 Head & Neck Nodal/Soft tissue 100 Gastric Nodal, liver etc 200 Melanoma Nodal 50
    12. 12. samples with outcome data tumor type data approximate number 5 yr survival 5000 Breast 10 yr. survival 300 Herceptin 400 (responders & non-responders) 3-5 yr survival 4000 CRC Bevacizumab, Cetuximab 500 (responders & non-responders) Prostate, 10 yr survival 5000Breast, CRC, Ovarian SOC Chemotherapy 1500 (responders & non-responders) 3-5 yr survival 2000 Lung (NSCLC) Docetaxel, Gemcitabine 400 Pancreatic Survival 350 Head & Neck Treatment/survival 200 Gastric Survival 250 NHL Survival 200 Ovarian 3-5 yr. survival 300 Bladder Survival 500
    13. 13. tissue microarrays Morphology Formalin Fixed Paraffin Embedded RNA/protein/DNA Frozen OCT Embedded
    14. 14. tissue microarrays to study tumorheterogeneityThe whole tumor is sectioned Cores are taken from each constituent tumor into 8-10 constituent blocks block and transferred to a TMA.The exact localization of each block is recorded
    15. 15. tissue microarrays to study tumorheterogeneity• An optimal way to measure intratumoral heterogeneity• Allows for an overview of the whole tumor Primary Blocks per Matched Nodal Blocks per Total number ofTumor Type tumors tumor Mets met TMA Cores NSCLC 146 8 66 4 1432 Breast 147 8 32 4 1304 CRC 140 8 42 4 1288 Prostate 190 10 - - 1900 Bladder 147 8 - - 1176
    16. 16. EGFR amplification is oftenheterogeneous in lung cancer Heterogeneity found in 7/13 (54%) EGFR amplified NSCLC Different areas Different matched of the primary cancer lymph node metastasesCase#1 EGFR FISH Result#2 amplification#3#4 polysomy#5 normal#6 n.a.#7
    17. 17. heterogeneity TMA: co-analysis of ERG andPTEN in prostate cancer 35 ERG+PTEN 10 PTEN only 4 ERG only  PTEN linked to ERG P<p<0.0001 31 tumors PTEN+ERG 21 ERG precedes PTEN 0 PTEN precedes ERG  ERG earlier PTEN deletions are late events developing preferentially in ERG positive prostate PTEN + ERG cancers PTEN only ERG only
    18. 18. prostate cancer progression &prognosis analysisFrequency of PTEN deletion is strongly linked to prostate cancer progression (n >2200 donor samples) 50.0 p<0.0001 45.0 40.0 35.0 PTEN homozygous fraction of tumors (%) 30.0 25.0 PTEN hemizygous 20.0 15.0 10.0 5.0 0.0 PIN (n=29) BPH (n=20) pT2 pT3a pT3b pT4 (n=24) HR (n=54) (n=1085) (n=360) (n=227)
    19. 19. tissue micro arrays to studytumor heterogeneity The level of heterogeneity of therapy target genes may be relevant for diagnosis and response HER2 is homogenous in breast cancer but heterogeneous in colon cancer Tumor heterogeneity is clinically important and can be optimally addressed by heterogeneity TMAs
    20. 20. molecular epidemiology Most oncology drugs in development are expected to be active only in sub-sets of patients How frequent is expression in human cancer? Specific cancer subtypes or biological properties? -prognostic relevance What normal tissues do express target? Option 1: Option 2: Review the Perform literature own studies
    21. 21. TriStar: a new dimension intissue biomarker analysis Prognosis TMA-Based Target Evaluation Strategy (IHC) Multi-Tumor Tissue Array Normal Tissue Array Tumor Cell Line Array 3,500 donor samples 600 donor samples 140 Cell Lines All Cancer Types 532 Cell Types Including NCI 60 √ Expression in cancer types (including niche cancers) √ Complete normal tissue expression information √ Cell lines identified for functional studies/drug screening Cancer – Specific Prognosis TMA Analaysis Prostate Cancer Breast Cancer Lung Cancer Bladder Cancer (3,000 donors) (2,000 donors) (1,400 donors) (1,100 donors) Colon Cancer Pancreatic Cancer Ovarian Cancer NHL (1,400 donors) (300 donors) (200 donors) (200 donors)Relationship of Molecular Target to Prognosis, Histological Sub-type, Response to Treatment etc
    22. 22. multi tumor analysisincluding less prevalent tumor types 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, All tumors & sub-types are stained. Customer can select Endometrioid Carcinoma, Brenner Tumor, Germ Cell Tumors. Liver: Hepatocellular Carcinoma, Cholangiocarcinoma. Fibrohistiocytic: Fibrosarcoma, Benign Histiocytoma, Dermatofibrosarcoma Protuberans, Atypical Fibroxanthoma, Malignant Fibrous Hiostiocytoma and pay for data on specific tumors of interest 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.
    23. 23. HER2 Expression and Amplification in Human CancersTapia et al., Modern Pathology, 20(2), 192–198 (2007) IHC FISH Breast cancer Urinary bladder cancer Stomach cancer Pancreatic cancer Esophageal cancer Endometrial cancer Vulva cancer Gall bladder cancer Lung cancer Ovarian cancer 0.0 5.0 10.0 15.0 20.0 Fraction of HER2-amplified samples (%)
    24. 24. normal tissue analysis 76 tissue types, 532 cell types, 8 donors eachMesenchymal tissues: aorta/intima, aorta/media, heart (left ventricle), sceletal muscle, sceletalmuscle/tongue, myometrium, appendix (muscular wall), esophagus (muscular wall), stomach(muscular wall), ileum (muscular wall), colon descendens (muscular wall), kidney pelvis (muscularwall), 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 (surfaceepithelium), anal canal (skin), anal canal (transition epithelium), exocervix, esophagus, kidneypelvis, urinary bladder, amnion/chorion, stomach (antrum), stomach (fundus and corpus), smallintestine, duodenum, small intestine, ileum, appendix, colondescendens, rectum, gallbladder, bronchus, paranasal sinus.Solid organs: lymph node, spleen, thymus, tonsil, liver, pancreas, parotid gland, submandibullarygland, sublingual gland, lip (small salivary gland), duodenum (Brunner gland), kidneycortex, 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 (corpusluteum), ovary (follicular cyst), placenta (first trimenon), placenta (mature), adrenalgland, parathyroid gland, thyroid, cerebellum, cerebrum, pituitary gland (posterior lobe), pituitarygland (anterior lobe) In which normal tissues is the target expressed?
    25. 25. multi tumor cell line arrayformalin fixed 140 Human Cell Lines including NCI 60 To identify tumor cell lines for functional studies/drug screening HCT-116 SNB 19 SR LN-401 GAMG p6 HCT-15 SW-620 UO-31 LN-229 IGR-1(/IGR 1) HEP-G-2 T 47 D 786-O BS 149 CRL-7930 HT29 A 498 MEL HO (P4) TK 10 172 ACHN COLO-849 IGR-OV1 U 251 COS-1 ECV-304 K-562 UACC-257 BT-549 HS-766-T CAKI-2 LOX-IMVI UACC-62 CAKI 1 HUT 12 RT-112 MCF-7 CCRF-CEM HUVEC A 549 293 COLO-205 IMR 90 MDA-MB- MDA-MB-435 A 375 EKVX UI-38 Mb(/U-138) 231 (S) MBC-5/MRC- U-87 MB(/U 87 MG) NCI(/L)- MOLT 4 HCC(/L)- 5 WS-1 H226 NCI-H23 2998 SM HS-68 NCI-H460 NCI-H322 (M) HOP 62 BT-474(/BT- MCF-10A PC-3 HOP 92 747) NCI-H522 RT 112(/RT II2 D2I) HS-578T EAL 29 RPMI-8226 OVCAR-3 MDA-HER-2 SJCRH- RXF 393 OVCAR-4 KM 12 MDANEO 30WCB SF 268 OVCAR-5 M-14 IM 9 CAL-62 SK-MEL-2 OVCAR-8 MALME-3M DBTRG VM-CUB 1 SK-MEL-28 SF 295 KRIB HBL-100(WBC) HELA SK-MEL-5 SF 539 T-98-G HACAT SK-OV-3 SNB 75 U-343-MG KU-19-19 Partial list SN 12C
    26. 26. cancer stem cell(csc) line arrayformalin fixed Cytospins from 33 CSC Lines Tissue cores from 11 matched & 2 unmatched xenografts Core diameter: 1.0mm Thyroid Cores per donor block: 2 Type Donors Cores GBM 8 16 Breast 1 2 Melanoma Thyroid 5 10 Colon 7 14 Lung 5 10 Melanoma 7 14 Matched Lung xenografts: Colon 4 8 Lung 3 6 Breast 1 2 Melanoma 3 6 Colon Unmatched xenografts Breast 2 4 Glioblastoma Total cores 92
    27. 27. TriStar breast cancer prognosisarray pT stage 2,200 Breast Cancers with pN stage 5 yr. follow-up information Number of nodes examined Number of positive nodes Tumor diameter BRE grade Polymorphy Tubulus formation Mitoses Tumor specific & raw survival Radiotherapy (Y/N) Chemotherapy (Y/N)Molecular data:FISH: HER2, EGFR, MDM2, CCND1, MYCIHC: ER, PR, p53, Cytokeratins, EGFR, HER2,CD117, others
    28. 28. breast cancer prognosis TMA analysis ESR1 Amplification* in 358/1739 (21%) of Breast Cancers Holst, Simon et al, Nat Gen (39), 655-660, 2007
    29. 29. ESR1 amplification and anti ER treatment175 Patients Treated With Tamoxifen Monotherapy 1.0 ESR1 amplification (n=43) 0.9 0.8 0.7 ER IHC positive (n=109) 0.6 Surviving 0.5 ER IHC negative(n=23) 0.4 0.3 ESR1 amplification may predict response to Tamoxifen 0.2 0.1 p<0.0001 0.0 0 20 40 60 80 100 months surv Holst, Simon et al, Nat Gen (39), 655-660, 2007
    30. 30. study: TPD52 mRNA expression analysis of 1,000 tumor samples & normal tissuesABI7900 based qRT-PCR, TPD52 vs GAPDH Skin 2 Pancreas 1 Lymph node 2 Stomach 2 Lung 2 Kidney 2 Oral cavitiy 2 Prostate 2 Normal tissues Breast 1 Testis 3 Endometrium 2 Bladder 2 Ovar 2 Thyroid gland 2 Vulvar 2 Brain 2 Myometrium 2 Skeletal muscle 2 Liver 3 Fat tissue 2 Malignant melanoma 11 Liver cancer 50 Larynx carcinoma 39 Pancreatic cancer 38 Lung cancer 134 Stomach cancer 50 Oral cavity cancer 56 Renal cell cancer 59 Breast cancer 53 Prostate cancer 48 Cancers Endometrial cancer 31 Testis cancer 59 Ovarian cancer 33 Urinary bladder cancer 55 Uterus cervix carcinoma 28 Thyroid gland cancer 40 Vulvar cancer 39 Leiomyosarcoma 42 Colon cancer 50 Liposarcoma 36 Esophageal cancer 48
    31. 31. study: TPD52 mRNA expression analysis In 1,000 tumor samples & normal tissues Frequency of TPD52 expression ≥2 fold down-regulated ≥2 fold up-regulated Lung, small cell Oral cavity Thyroid gland Renal, clear cell Renal, papillary Leiomyosarcoma Lung, adeno Liposarcoma Pancreas Vulvar Liver Lung, large cell Melanoma Lung, squamous Endometrium Stomach Prostate Cervix* Ovar Larynx* Colon* Esophagus squamous* Mamma, lobular Mamma, ductal Esophagus adeno* Bladder , non-invasive Seminoma Bladder, invasive UKE data Non-Seminoma100 80 60 40 20 0 20 40 60 80 100 % of samples showing TPD52 overexpression / downregulation
    32. 32. study: TPD52 mRNA expression analysis in 1,000 tumor samples & normal tissues TPD52 expression levels Renal, papillary Leiomyosarcoma Renal, clear cell Liposarcoma down-regulated Lung, small cell Lung, adeno Oral cavity Vulvar Pancreas Thyroid gland Liver Lung, large cell Melanoma Endometrium Lung, squamous Stomach Prostate Larynx* Cervix* up-regulated Esophagus squamous* Colon* Esophagus adeno* Bladder , non-invasive Ovar Bladder, invasive Mamma, lobular Mamma, ductal Seminoma UKE data Non-Seminoma-4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 avr. TPD52 expression level (log2)
    33. 33. study: sequencing of all 10 PTEN exons in100 prostate cancer samplesa) c.1067_1070del c) c.1623G>T e) c.352C>TGCAGAAACAAAAGG GATGTTTGAAACTAT GCTTTGTCAAGATCAb) c.2007G>A d) c.1981_1984del - 5% Mutations - Mutation Unrelated To DeletionGCAACATGATTGTCA TAAAGTAAGTACTAGABI3100, 16 capillaries tumor type data approx. # p valueEppendorf pipetting robot PTEN notLaser capture micro dissection deleted 95.4% 4.6% 0.3096*(if necessary) PTEN hemizygous deleted 17 11.8% FISH notUKE data analyzable 18 0.0%
    35. 35. sample prospectivecollection projects Prospective collection of formalin fixed samples of mantle cell lymphoma from lymph node sites only with 5-10ug matching RNA per sample Prospective collection of Frozen OCT & FFPE samples of IBD & Ulcerative Colitis, recently diagnosed, diseased + adjacent normal. Matched Serum & Whole Blood with Clinical Labs Prospective collection of formalin fixed & OCT samples of metastatic NSCLC (adenocarcinoma & SCC) with matched nodal mets, serum & RNA Prospective collection of formalin fixed & OCT samples of esophageal adenocarcinoma, serum & RNA
    36. 36. overview Complexity of translational biomarker research supporting drug & diagnostic development increasingly requires knowledge-based services/partnerships that go beyond the traditional fee-for-service model Service providers must offer a range of services, histology labs, analytical platforms, top academic opinion leaders etc. TriStar’s service platform is sustainable, scalable, flexible & cost-effective Very large product offering, standardized QC, top-notch scientific capabilities
    37. 37. 11contact usTriStar Technology Group LLC9700 Great Seneca HighwayRockville, MD 20852For more information please visit ourwebsite at www.tristargroup.usp. 1-866-851-STARf. 1-509-471-1765e.