1. Sample Acquisition With
Annotated Clinical Information
A Critical Success Factor In Biomarker Validation
TriStar
Technology Group
9700 Great Seneca Highway, suite 401
Rockville, MD 20850
(E) info@tristargroup.us
(P) 301-792-633
(W) www.tristargroup.us

2. 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

3. 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

4. Rockville, MD Hamburg, Germany
TMA Repository
Array Manufacturing
Contract Research
Madrid, Spain Rome & Catania, Italy
TMA Repository TMA Repository & Contract Research
Cancer Stem Cell Research

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. 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

7. product groups
Archived 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. our 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. quality control
Samples are fixed/frozen within 2 – 10 minutes of
Excision
OCT embedded sample
Snap frozen sample
Formalin fixed sample

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. primary tumors
with 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. 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 5000
Breast, 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. tissue microarrays
Morphology
Formalin Fixed
Paraffin Embedded
RNA/Protein/DNA
Frozen OCT Embedded DNA

14. tissue microarrays to study tumor
heterogeneity
The 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. tissue microarrays to study tumor
heterogeneity
An optimal way to measure intratumoral heterogeneity
Allows for an overview of the whole tumor
Matched Nodal Total number of
Tumor Type Primary tumors Blocks per tumor Blocks per met
Mets 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. EGFR amplification is often
heterogeneous in lung cancer
Heterogeneity found in 7/13 (54%) EGFR amplified NSCLC
Different areas Different matched
of the primary cancer lymph node metastases
Case
#1
EGFR FISH Result
#2
#3 amplification
#4 polysomy
#5
normal
#6
#7 n.a.

17. heterogeneity TMA: co-analysis of ERG and
PTEN in prostate cancer
35 ERG+PTEN
10 PTEN only
4 ERG only
 PTEN linked to ERG
31 tumors PTEN+ERG
21 ERG precedes PTEN
0 PTEN precedes
PTEN deletions are late events developing  ERG earlier
preferentially in ERG positive prostate
cancers PTEN + ERG
PTEN only
ERG only
P<p<0.0001

18. prostate cancer progression &
prognosis analysis
Frequency of PTEN deletion is strongly linked to prostate cancer
progression (n >2200 donor samples)
50.0 p<0.0001
45.0
40.0
fraction of tumors (%)
35.0 PTEN homozygous
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. 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

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. TriStar: a new dimension in
tissue 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. multi tumor analysis
including 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,
All tumors & sub-types are stained. Customer can select
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,
and pay for data on specific tumors of interest
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.

23. HER2 Expression and Amplification
in Human Cancers
Tapia et al., Modern Pathology, 20(2), 192–198 (2007)
IHC FISH
Urinary bladder cancer
Pancreatic cancer
Endometrial cancer
Gall bladder cancer
Ovarian cancer
0.0 5.0 10.0 15.0 20.0
Fraction of HER2-amplified samples (%)

24. normal tissue analysis
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?

25. multi tumor cell line array
formalin 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 TK 10 A 498
172
IGR-OV1 U 251 ACHN MEL HO (P4) COS-1
K-562 UACC-257 BT-549 COLO-849 HS-766-T
LOX-IMVI UACC-62 CAKI 1 ECV-304 HUT 12
MCF-7 A 549 CCRF-CEM CAKI-2 HUVEC
COLO-205 RT-112 IMR 90
MDA-MB-231 MDA-MB-435 (S) EKVX 293
MOLT 4 A 375 UI-38 Mb(/U-138)
NCI(/L)-H226 NCI-H23 HCC(/L)-2998
NCI-H460 NCI-H322 (M) HOP 62 MBC-5/MRC-5 U-87 MB(/U 87 MG)
PC-3 NCI-H522 HOP 92 SM WS-1
RPMI-8226 OVCAR-3 HS-578T
HS-68
RXF 393 OVCAR-4 KM 12 BT-474(/BT-747) MCF-10A
SF 268 OVCAR-5 M-14 EAL 29
SK-MEL-2 OVCAR-8 MALME-3M
RT 112(/RT II2 D2I)
SK-MEL-28 SF 295 KRIB SJCRH-30WCB MDA-HER-2
SK-MEL-5 SF 539 T-98-G IM 9 MDANEO
SK-OV-3 SNB 75 U-343-MG VM-CUB 1
Partial list CAL-62
SN 12C HELA DBTRG
HACAT HBL-100(WBC)
KU-19-19

26. cancer stem cell(csc) line array
formalin fixed
Cytospins from 33 CSC Lines
Tissue cores from 11 matched & 2 unmatched xenografts
Core diameter: 1.0mm
Cores per donor block: 2
Thyroid Type Donors Cores
GBM 8 16
Breast 1 2
Thyroid 5 10
Melanoma
Colon 7 14
Lung 5 10
Melanoma 7 14
Matched
Lung xenografts:
Colon 4 8
Lung 3 6
Colon Breast 1 2
Melanoma 3 6
Unmatched
xenografts
Glioblastoma Breast 2 4
Total cores 92

27. breast cancer prognosis array
pT stage
pN stage
Number of nodes examined 2,200 Breast Cancers with
Number of positive nodes 5 yr. follow-up information
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, MYC
IHC: ERA, PR, p53, Cytokeratins, EGFR, HER2, CD117, others

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. ESR1 amplification and anti ER
treatment
175 Patients Treated With Tamoxifen Monotherapy
1.0
0.9 ESR1 amplification (n=43)
0.8
0.7
ER IHC positive (n=109)
Surviving
0.6
0.5
0.4 ER IHC negative(n=23)
0.3
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
ESR1 amplification may predict response to Tamoxifen

30. study: TPD52 mRNA expression analysis of 1,000
tumor samples & normal tissues
ABI7900 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. 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
Non-Seminoma
100 data 80
UKE 60 40 20 0 20 40 60 80 100
% of samples showing TPD52 overexpression / downregulation

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. study: sequencing of all 10 PTEN exons in
100 prostate cancer samples
a) c.1067_1070del c) c.1623G>T e) c.352C>T
G C AG AAAC AAAAG G GATGTTTGAAACTAT GCTTTGTCAAGATCA
b) c.2007G>A d) c.1981_1984del
- 5% Mutations
GCAACATGATTGTCA TAAAGTAAGTACTAG
- Mutation Unrelated To Deletion
ABI3100, 16 capillaries tumor type data approx. # p value
Eppendorf pipetting robot PTEN not deleted 95.4% 4.6% 0.3096*
Laser capture micro dissection
(if necessary) PTEN hemizygous
deleted 17 11.8%
UKE data FISH not analyzable 18 0.0%

34. sample data fields
Breast Cancer with Herceptin Treatment & Response Information
LOCALISATION REF# ORGAN UNIQUE ID AGE DATE OF SURGERY DIAGNOSIS
METS POST SURGERY
GRADE T N M (TIME 0) STAGE HER2 ER (%) PR (%) SITE OF MET. METS DIAGNOSIS BY
(MONTHS)
PREV. START TREAT 1 END START TREAT 2 FOLLOW UP
SETTING END TREAT 2 FOLLOW UP 1 FOLLOW UP 2 SURVIVAL
CHEMOTH. TREAT 1 DETAILS TREAT 1 TREAT 2 DETAILS 3

35. sample prospective
collection 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. 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. contact us
TriStar Technology Group LLC
9700 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