Giving Clinical Significance To Molecular Targets

NETWORK OF REPOSITORIES

Washington DC, USA Hamburg, Germany

TMA Repository TMA Repository
Array Manufacturing
Contract Research

Madrid, Spain Catania, Italy

TMA Repository TMA Repository
Cancer Stem Cell Research
GENERAL INFORMATION

Cancer
120 Tumor Types
Over 2.5 million samples
Over 60,000 micro arrayed samples
Formalin fixed & frozen
Detailed clinical information

CNS
Over 100,000 samples
Formalin fixed & frozen
Clinical information
Alzheimer‘s Disease, Parkinson‘s Disease, Stroke, MS,
Vascular Dementia, Prion Disease etc.

Inflammatory Disease
Over 300,000 samples
Over 3,000 micro arrayed samples
Formalin fixed
Rheumatoid Arthritis, Inflammatory Bowel Disease,
Psoriasis Arthritis etc.
BIOLOGICAL SAMPLES

Tissue micro arrays with matched large sections

Tissue blocks with clinical database
Primary 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

Performance Date of
Diagnosis CD20 CD10 BCL2 Age SITE*
Status diagnosis
International
Bone Extranodal
Symptoms Stage Bulky Epo Prognostic GCSF
marrow Location
Index
Maintainance
Treatment Treatment Therapy Response to
Rituximab Cycles Radiotherapy therapy with
start schedule reduction therapy
Rituximab ?
Date of Second Response Due to
Follow up D.O.D. Note
relapse treatment to therapy lymphoma
 PROJECT EXAMPLES (ARCHIVED SAMPLES)

Ovarian Cancer
100 donor samples with
Treatment & Response Information
10 serial sections per donor

DATE of
ID # AGE RACE DIAGNOSIS T N M (T0)
SURGERY
PREV. START END PERFORMANCE
GRADE STAGE FIGO SETTING DETAILS
CHEMOTH. TREATMENT TREATMENT 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 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
Finding an Attractive Drug Target

Molecular Epidemiology

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
 Current situation

Typical early steps Later steps

usually not done!
Finding an Attractive Drug Target

Current situation

Typical early steps Later steps

usually not done!

Molecular
Epidemiology

Large sets of
Human tissues with clinical
Follow-up required.
Difficult to get.
 Finding an Attractive Drug Target

Optimal situation

Later steps
Early steps

Identify clinically relevant targets

before incurring significant costs
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
Tissue Microarrays

2.

14. Morphology

Formalin Fixed
Paraffin Embedded
200.
RNA/protein

Frozen OCT Embedded

DNA
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 Antibody

Test for Unspecific (Fc-mediated) Binding

Omit Primary Antibody

Test for Unspecific Staining Induced by the Detection System
LIST OF FROZEN TISSUES FOR CROSS-REACTIVITY STUDY

FDA Recommended List of Frozen Normal Human Tissues

GLP 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
 TriStar Multi-Tumor Tissue Microarray

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.
 TriStar Normal Human 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)
 Validation Case Study
Estrogen Receptor (ESR1) Gene Amplification

Target Identified using aCGH

Is it really true?
How frequent?
Histo-pathological subtypes?
Is it clinically relevant?
Commercial value?
Validation Platform
TriStar Breast Cancer Prognosis Array

pT stage
pN stage 2,200 Breast Cancers with
Number of nodes examined 5 yr.follow-up information
Number of positive nodes
Tumor diameter
BRE grade
Polymorphy
Tubulus formation
Mitoses

Survival months (99%)

Survival tumor specific (40%)
Some therapy information (40%)

Molecular parameters:
FISH: HER2, EGFR, MDM2, CCND1, MYC
IHC: ER, PR, p53, Cytokeratins, EGFR, HER2,
CD117, others
Breast Cancer Prognosis TMA Analysis
ESR1 amplification (FISH)

ESR1 Amplification* in 358/1739 (21%) of Breast Cancers

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

Does ESR1 Amplification cause ER Overexpression?

???

ER IHC data from our database

Is the ESR1 Amplification Functionally Relevant ?

ESR1 Amplification Are Linked To ER Protein Overexpression

ER expression level
(Allred score)
ESR1 Amplification are linked to Early Stage and Low Grade Breast Cancers

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

ESR1 amplification and anti ER treatment

175 Patients Treated With Tamoxifen

No Adjuvant Therapy

ER IHC positive (n=109)

ER IHC negative(n=23)

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

ESR1 Validation Study using Prognosis TMA: Timeline

Affymetrix experiment, March 2006

ESR1 amp identification

Low density TMA validation April 14th, 2006

High density TMA studies April-May, 2006

Patent filed July 15th, 2006

Paper accepted, February 2nd, 2007

Holst et al, Nature Genetics
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, LLC
THANK YOU 9700 Great Seneca Highway
Rockville, MD 20850
U.S.A
Washington DC, USA www.tristargroup.us
TMA Repository

Hamburg, Germany
TMA Repository
Array Manufacturing
Contract Research

Catania, Italy
TMA Repository
Cancer Stem Cell Research

Madrid, Spain
TMA Repository