TriStarTechnology Group

9700 Great Seneca Highway, suite 401Rockville, MD 20850(E) info@tristargroup.us(P) 301-792-633 (W) www.tristargroup.us

Sample Acquisition With Annotated Clinical Information

A Critical Success Factor In Biomarker Validation

the need for targeted therapeutics with companion diagnostics

Development of targeted therapeutics requires testing in targeted populations matched to a drug’s mechanism of action

Evaluation of Trastuzamab in “all comer” breast cancer patients (25% HER+, 75% HER2-) would not have shown significant benefit in clinical trials

Early proof of concept in the right patient population is crucial

Potentially shorter time to market

An emerging unmet need in oncology drug development today is service providers that offer both access to well-annotated specimens and sophisticated molecular analytical capabilities

Rockville, MD

Rome & Catania, Italy

Hamburg, Germany

Madrid, Spain

TMA Repository

TMA Repository TMA Repository & Contract Research

Array ManufacturingContract Research

Cancer Stem Cell Research

tristar providesAccess 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

ethical 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

product 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 DataER/PR/HER2, p53, BRAF, KRAS, EGFR, PIK3CA etc.

Cancer Stem Cell ArraysLysates & RNA

Blocks & Large sectionsWith matching RNA, DNA

Protein ExpressionIHC (Antibody protocol development, automated or manual staining, reading & interpretation)

Gene ExpressionRT-PCR

Gene copy numberFISH/CISH

Cross-Reactivity Screening in Normal Tissue(GLP)

Large-Scale Analysis of Prognostic markers (500-3500 donor samples)(500-3500 donor samples)

Gene sequencingDNA sequencing

our services

quality controlSamples are fixed/frozen within 2 – 10 minutes of Excision

OCT embedded sample

Snap frozen sample

Formalin fixed sample

quality control10% 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

Primary Tumors Matched Mets Approximate number

BreastNodal

DistantBone

200020

200

CRC NodalLiver

2000150

Prostate NodalBone

500300

Lung (NSCLC) NodalBone

300100

Pancreatic Nodal 100

Head & Neck Nodal/Soft tissue 100

Gastric Nodal, liver etc 200

Melanoma Nodal 50

primary tumors with matched mets

tumor type data approximate number

Breast5 yr survival

10 yr. survivalHerceptin

5000300

400 (responders & non-responders)

CRC3-5 yr survival

Bevacizumab, Cetuximab4000

500 (responders & non-responders)

Prostate,Breast, CRC, Ovarian

10 yr survivalSOC Chemotherapy

50001500 (responders & non-responders)

Lung (NSCLC)3-5 yr survival

Docetaxel, Gemcitabine2000400

Pancreatic Survival 350

Head & Neck Treatment/survival 200

Gastric Survival 250

NHL Survival 200

 Ovarian 3-5 yr. survival 300

Bladder Survival 500

samples with outcome data

Formalin Fixed Paraffin Embedded

Frozen OCT Embedded

tissue microarrays

Morphology

RNA/Protein/DNA

DNA

tissue microarrays to study tumor heterogeneity

The whole tumor is sectioned into 8-10

constituent blocks The exact localization of each

block is recorded

Cores are taken from each constituent tumor block and transferred to a TMA.

Tumor Type Primary tumors

Blocks per tumor

Matched Nodal Mets

Blocks per met

Total number of 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

tissue microarrays to study tumor heterogeneity

Allows for an overview of the whole tumorAn optimal way to measure intratumoral heterogeneity

Heterogeneity found in 7/13 (54%) EGFR amplified NSCLC

EGFR FISH Result

amplification

normal

polysomy

n.a.

Case

#1

#2

#3

#4

#5

#6

#7

Different areasof the primary cancer

Different matched lymph node metastases

EGFR amplification is often heterogeneous in lung cancer

PTEN + ERG

PTEN only

ERG only

PTEN deletions are late events developing preferentially in ERG

positive prostate cancers

heterogeneity TMA: co-analysis of ERG and PTEN in prostate cancer

35 ERG+PTEN10 PTEN only4 ERG only PTEN linked to ERG

P<p<0.0001

31 tumors PTEN+ERG21 ERG precedes PTEN0 PTEN precedes ERG earlier

Frequency of PTEN deletion is strongly linked to prostate cancer progression (n >2200 donor samples)

p<0.0001

PIN (n=29)

BPH (n=20)

pT2 (n=1085)

pT3a (n=360)

pT3b (n=227)

pT4 (n=24)

HR (n=54)

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5.0

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25.0

30.0

35.0

40.0

45.0

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PTEN homozygous

PTEN hemizygous

fracti

on o

f tu

mors

(%

)

prostate cancer progression & prognosis analysis

tissue micro arrays to study tumor 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

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

Most oncology drugs in development are expected to be active only in sub-sets of patients

molecular epidemiology

Prognosis TMA-Based Target Evaluation Strategy (IHC)

Multi-Tumor Tissue Array3,500 donor samples

All Cancer Types

Normal Tissue Array600 donor samples

532 Cell Types

Tumor Cell Line Array140 Cell Lines

Including NCI 60

Cancer – Specific Prognosis TMA Analaysis

Breast Cancer(2,000 donors)

Prostate Cancer(3,000 donors)

Lung Cancer(1,400 donors)

Bladder Cancer(1,100 donors)

Pancreatic Cancer(300 donors)

Colon Cancer(1,400 donors)

Ovarian Cancer(200 donors)

NHL(200 donors)

Relationship of Molecular Target to Prognosis, Histological Sub-type, Response to Treatment etc

TriStar: a new dimension in tissue biomarker analysis

√ Expression in cancer types (including niche cancers)√ Complete normal tissue expression information√ Cell lines identified for functional studies/drug screening

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.

multi tumor analysis including less prevalent tumor types

All tumors & sub-types are stained. Customer can selectand pay for data on specific tumors of interest

Ovarian cancer

Gall bladder cancer

Endometrial cancer

Pancreatic cancer

Urinary bladder cancer

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0

Fraction of HER2-amplified samples (%)

Tapia et al., Modern Pathology, 20(2), 192–198 (2007)

IHC FISH

HER2 Expression and Amplification in Human Cancers

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?

normal tissue analysis

140 Human Cell Lines including NCI 60

To identify tumor cell lines for functional studies/drug screening HCT-116

HCT-15HEP-G-2HT29IGR-OV1K-562LOX-IMVIMCF-7

MDA-MB-231

NCI(/L)-H226NCI-H460PC-3RPMI-8226RXF 393SF 268SK-MEL-2SK-MEL-28SK-MEL-5SK-OV-3SN 12C

SNB 19SW-620T 47 DTK 10U 251UACC-257UACC-62A 549

MDA-MB-435 (S)MOLT 4NCI-H23NCI-H322 (M)NCI-H522OVCAR-3OVCAR-4OVCAR-5OVCAR-8SF 295SF 539SNB 75

SRUO-31786-OA 498ACHNBT-549CAKI 1CCRF-CEMCOLO-205EKVX

HCC(/L)-2998HOP 62HOP 92HS-578TKM 12M-14MALME-3MKRIBT-98-GU-343-MG

LN-401LN-229BS 149

MEL HO (P4)COLO-849ECV-304CAKI-2RT-112293A 375

MBC-5/MRC-5SMBT-474(/BT-747)EAL 29

SJCRH-30WCBIM 9VM-CUB 1HELAHACATKU-19-19

GAMG p6IGR-1(/IGR 1)CRL-7930172COS-1HS-766-THUT 12HUVECIMR 90

UI-38 Mb(/U-138)

U-87 MB(/U 87 MG)WS-1HS-68MCF-10A

RT 112(/RT II2 D2I)MDA-HER-2MDANEOCAL-62DBTRGHBL-100(WBC)

Partial list

multi tumor cell line array formalin fixed

Cytospins from 33 CSC Lines

Tissue cores from 11 matched & 2 unmatched xenograftsCore diameter: 1.0mmCores per donor block: 2

Type Donors   CoresGBM 8   16

Breast 1   2Thyroid 5   10Colon 7   14Lung 5   10

Melanoma 7   14Matched

xenografts:      Colon 4   8Lung 3   6

Breast 1   2Melanoma 3   6

Unmatched xenografts      

Breast 2   4       

Total cores     92

Thyroid

Melanoma

Lung

Colon

Glioblastoma

cancer stem cell(csc) line arrayformalin fixed

2,200 Breast Cancers with 5 yr. follow-up information

pT stagepN stageNumber of nodes examinedNumber of positive nodesTumor diameterBRE gradePolymorphyTubulus formationMitoses

Tumor specific & raw survivalRadiotherapy (Y/N)

Chemotherapy (Y/N)

Molecular data: FISH: HER2, EGFR, MDM2, CCND1, MYCIHC: ERA, PR, p53, Cytokeratins, EGFR, HER2, CD117, others

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 analysis

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rviv

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0 20 40 60 80 100months surv

ER IHC positive (n=109)

p<0.0001

ESR1 amplification (n=43)

ER IHC negative(n=23)

175 Patients Treated With Tamoxifen Monotherapy

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

ESR1 amplification and anti ER treatment

ESR1 amplification may predict response to Tamoxifen

ABI7900 based qRT-PCR, TPD52 vs GAPDH

Normal tissues

Skin 2 Pancreas 1Lymph node 2 Stomach 2Lung 2 Kidney 2Oral cavitiy 2 Prostate 2Breast 1 Testis 3Endometrium 2 Bladder 2Ovar 2 Thyroid gland 2Vulvar 2 Brain 2Myometrium 2 Skeletal muscle 2Liver 3 Fat tissue 2

Cancers

Malignant melanoma 11 Liver cancer 50Larynx carcinoma 39 Pancreatic cancer 38Lung cancer 134 Stomach cancer 50Oral cavity cancer 56 Renal cell cancer 59Breast cancer 53 Prostate cancer 48Endometrial cancer 31 Testis cancer 59Ovarian cancer 33 Urinary bladder cancer 55Uterus cervix carcinoma 28 Thyroid gland cancer 40Vulvar cancer 39 Leiomyosarcoma 42Colon cancer 50 Liposarcoma 36Esophageal cancer 48

study: TPD52 mRNA expression analysis of 1,000 tumor samples & normal tissues

Frequency of TPD52 expression

UKE data

study: TPD52 mRNA expression analysis In 1,000 tumor samples & normal tissues

UKE data

TPD52 expression levels

study: TPD52 mRNA expression analysis in 1,000 tumor samples & normal tissues

- 5% Mutations- Mutation Unrelated To Deletion

ABI3100, 16 capillariesEppendorf pipetting robotLaser capture micro dissection (if necessary)

UKE data

tumor type data approx. # p value

PTEN not deleted 95.4% 4.6% 0.3096*

PTEN hemizygous deleted 17 11.8%FISH not

analyzable 18 0.0%

study: sequencing of all 10 PTEN exons in 100 prostate cancer samples

Breast Cancer with Herceptin Treatment & Response Information

LOCALISATION REF# ORGAN UNIQUE ID AGE DATE OF SURGERY DIAGNOSIS

GRADE T N M (TIME 0) STAGE HER2 ER (%) PR (%)METS POST SURGERY

(MONTHS)SITE OF MET. METS DIAGNOSIS BY

PREV. CHEMOTH.

SETTINGSTART

TREAT 1TREAT 1DETAILS

END TREAT 1

START TREAT 2

TREAT 2DETAILS

END TREAT 2

FOLLOW UP 1

FOLLOW UP 2

FOLLOW UP 3

SURVIVAL

sample data fields

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

sample prospective collection projects

overview

Complexity of translational biomarker research supporting drug & diagnostic development increasingly requires knowledge-based services/partnerships that go beyond the traditional fee-for-service modelService 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-effectiveVery large product offering, standardized QC, top-notch scientific capabilities

contact usTriStar Technology Group LLC9700 Great Seneca Highway

Rockville, MD 20852

For more information please visit our website at www.tristargroup.us

p. 1-866-851-STAR

f. 1-509-471-1765

e. info@tristargroup.us