Giving Clinical Significance To Molecular Targets

NETWORK OF REPOSITORIES

Washington DC, USA

TMA Repository

Catania, ItalyTMA Repository

Cancer Stem Cell Research

Hamburg, Germany

TMA RepositoryArray ManufacturingContract Research

Madrid, SpainTMA Repository

GENERAL INFORMATION

Cancer120 Tumor Types

Over 2.5 million samplesOver 60,000 micro arrayed samples

Formalin fixed & frozenDetailed clinical information

CNSOver 100,000 samplesFormalin fixed & frozen

Clinical informationAlzheimer‘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

• Tumor Heterogeneity Arrays• Primary Tumors with Matched Nodal & Distant Metastases• Samples with Molecular & Clinical Data

UNIQUE CAPABILITIES

Extensive, Oncology-focused Archived Human Tissue Repository

Over 120 Types of Cancers, including less prevalent/rare

Largest Commercially Available TMA repository

High Density TMAs (>3,000 donor samples) enabling large scale studies

Detailed Clinical Info: Prognosis, Progression, Treatment & Response

Extensive Molecular Database

UNIQUE CAPABILITIES

Research Focused: TMAs constructed accordingly

QC capabilities: 17 pathologists, 2 Oncologists

Follow up data from donors eligible for and who received current monoclonal antibody therapies

Strong Scientific Background – Contract Research Studies (IHC/FISH)

Large-Scale Analysis of Prognosis

Cancer Stem Cell Arrays, In-Vitro & In-Vivo Research

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 17 Pathologists & 2 Oncologists for Clinical Data Review

PROJECT EXAMPLES (ARCHIVED SAMPLES)

Diffused Large B Cell Lymphoma (DLBCL)50 donor samples with Treatment & Response Information

10 serial sections per donor

Diagnosis CD20 CD10 BCL2 AgePerformance

StatusDate of

diagnosisSITE*

Symptoms StageBone

marrowBulky Epo

International Prognostic

Index

Extranodal Location

GCSF

Treatment start

Treatment schedule

Therapy reduction

Rituximab Cycles RadiotherapyResponse to

therapy

Maintainance therapy with Rituximab ?

Follow up Date of relapse

Second treatment

Response to therapy

D.O.D. Due to

lymphomaNote

PROJECT EXAMPLES (ARCHIVED SAMPLES)

Ovarian Cancer100 donor samples with Treatment & Response

Information10 serial sections per donor

ID # AGE RACE DIAGNOSISDATE of

SURGERYT N M (T0)

GRADE STAGE FIGOPREV.

CHEMOTH.SETTING

START TREATMENT

DETAILSEND

TREATMENTPERFORMANCE STATUS (ECOG)

PROGRESSION DISEASE DATE TREATMENT FREE INTERVAL D.O.D NOTES

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

Typical early steps

Current situation

Later stepsusually not done!

Large sets ofLarge sets ofHuman tissues with clinicalHuman tissues with clinical

Follow-up required.Follow-up required.Difficult to get.Difficult to get.

Large sets ofLarge sets ofHuman tissues with clinicalHuman tissues with clinical

Follow-up required.Follow-up required.Difficult to get.Difficult to get.

Finding an Attractive Drug Target

Current situation

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

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

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 FROZEN TISSUES FOR CROSS-REACTIVITY STUDY

FDA Recommended List of Frozen Normal Human TissuesGLP or Non-GLP Study

3 unrelated donors each

Adrenal * Lung * Spinal Cord * Blood Cells * Lymph Node * Spleen Blood vessels (endothelium) * Ovary * Striated

(skeletal) Muscle Bone Marrow * Fallopian Tube (oviduct) * Testis * Brain * Pancreas * Thymus * Parathyroid * Thyroid * Breast (mammary gland) * Peripheral Nerve * Tonsil * Eye *

Pituitary * Ureter * Gastrointestinal Tract * Placenta * Urinary Bladder * Heart * Prostate * Uterus (endometrium)

Kidney (glomerulus, tubule) * Salivary Gland * Uterus – cervix * Liver * Skin

10-50 Samples Each Of 120 Human CancersSkin: 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)

TriStar Normal Human Tissue Microarray

Is it really true?

How frequent?

Histo-pathological subtypes?

Is it clinically relevant?

Commercial value?

Target Identified using aCGH

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

Holst, Simon et al, Nat Gen (39), 655-660, 2007

ESR1 Amplification are linked to Early Stage and Low Grade Breast Cancers

ER IHC positive (n=109)

ER IHC negative(n=23)

175 Patients Treated With TamoxifenNo Adjuvant Therapy

Holst, Simon et al, Nat Gen (39), 655-660, 2007

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

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 Cell Arrays

Clinical Data – Prognosis, Progression, Treatment & Response

Molecular Database

PRODUCTS & SERVICES

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 Group, LLC9700 Great Seneca Highway

Rockville, MD 20850U.S.A

www.tristargroup.us

THANK YOU

Washington DC, USATMA Repository

Catania, ItalyTMA Repository

Cancer Stem Cell Research

Hamburg, GermanyTMA Repository

Array ManufacturingContract Research

Madrid, SpainTMA Repository