Tissue Microarrays: New Tools And New Demands on Biorepositories

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Tissue MicroarraysNew Tools And New Demands on
Biorepositories

• Stephen M. Hewitt, M.D., Ph.D.
• Tissue Array Research Program,
• National Cancer Institute, Bethesda MD

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Biomedicine Has Become A High Throughput Endeavor
Integrated -omic Analysis
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Crossroads Of Biomedical Research
Individual Proteins
Empiric
Biochemistry Molecular Biology
-Omic HighThroughput Biology
Knowledge
Qualitative
Quantitative
Biologic Systems
Hypothesis
Number Of Proteins
Modified From Gavin MacBeath, Nat Gen 32 Sup 526
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Tissue In Biomedical Research
Information That Can Not Be Reduced To A Single
Measurement
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The Essence Of Biomarkers

• If You Dont Take A Temperature, You Cant
  Find A Fever.
• Samuel Shem, House of God

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Biomarkers

• Any Parameter That Can Be Measured About A
  Patient That Relates To A Specified Outcome
• Must Add To Diagnostic Utility
• Must Demonstrate Benefit Over Current Grading and
  Staging Schema
• Better, Faster, Cheaper

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Uses Of Biomarkers

• Diagnostic
• Determining The Presence Of Disease
• Prognostic
• Forecasting The Outcome (Survival) Of The Disease
• Predictive
• Predicting Response To Therapy
• Often Prognostic Markers Are Predictive

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Framing The Question
Early Diagnosis, Prognostic
Drug Effect Surrogate Endpoint
Progression, Regression
Intervention
Disease Threshold
Time
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Improved Discrimination
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Developing A Useful Biomarker

• Requires Input From Users
• Basic Scientist
• Clinicians
• Business Standpoint
• Often Not The First Answer Considered
• Not X From Y, But Given X, Separate Q and R
• Advance From Current Standard Of Care
• Not As Good As, But Better, Faster, Cheaper

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Discovery Vs Clinical Tool

• Biomarker Discovery
• Research Tool
• To Identify Proteins/Patterns
• Final Assay Will Be Reformulated
• Clinical Tool
• Reproducible
• Hardened Assay
• FDA

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Biomarkers Meets The Clinic

• Practical Questions
• Introduction Into The Current Care
  Environment
• Intellectual Property
• Will Patients Tolerate The Test?
• Cost

• The Hazing Test
• Would You Use This Test On Your Mother?
• Would You Perform The Assay Day In Day Out?

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Validation

• Available Specimens
• Yours
• Collaborators
• Retrospective
• Prospective
• Single Institution Trials
• Multi-center Trials
• Community Use - Cohorts
• Population Based Assessment

Assay Hardening
Commercial Formulation Of Assay
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Tissue Is The Issue

• Drug Development
• Outcome Of Patient Is Primary Issue
• Correlation With Biology Is Secondary
• Assays Often Performed In Model Systems
• Biomarker Development
• Measurement Of The Patient Is Primary
• Correlation With Biology Is Desired
• Assays Performed On Patient Samples

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Tissue Biomarkers

• BM Measured In Tissue
• Requires A Priori Identification Of Presence Of
  Disease
• Typical
• IHC, ISH, FISH
• Forefront
• Expression Profile, CGH, Mass Spec
• Integration With Current Diagnostic Environment
  Is Essential

Pharm Dx
Critical Path
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The Challenges Of Tissue

• Finding Tissue
• Permission To Obtain Tissue
• Obtaining Tissue
• Preserving Tissue
• Storing Tissue
• Reducing Tissue To A Reagent
• Performing Assays On Tissue
• Interpretation Of Results

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Quality Is EverythingQuality Remains Subjective

• Tissue Quality
• Histology
• Proteins
• Nucleic Acids

• Clinical Data
• Complete
• Detailed

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Measuring Quality In Paraffin Embedded Tissue

• Histology
• Primary
• Diagnosis Based On Fund Of Knowledge
• Protein
• Immunohistochemistry
• Primary Molecular Use
• Optimization/Recovery of Damage
• Extracted Proteins

• Nucleic Acids
• DNA
• Length
• RNA
• Length
• Quantity
• Most Delicate

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What Is A Tissue Array?

• A Block Of Samples From Hundreds Of Blocks
• Multiple Samples
• Paraffin Embedded Or Frozen Tissue
• Arranged In An Organized Fashion
• Platform For High- Throughput Pathology

NCI TARP LAB TARP2 C-052
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What Is A Tissue Array?

• A Protein Array With Retained
  Histomorphology
• Cell Type Localization
• Cell Type Quantification
• Subcellular Localization
• Platform For High- Throughput Proteomics

NCI TARP LAB TARP2 C-052
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Uses Of Tissue Microarrays

• Immunohistochemistry 95
• In Situ Hybridization 4
• Other 1

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Immunohistochemistry

• Strengths
• Histomorpholoy
• Cytomorphology
• Simple
• Robust
• In The Clinic
• No Specialized Equipment
• Relatively Cheap

• Weaknesses
• Quantitation
• Standards
• FFPE Tissue
• Solutions
• Standards
• Tissue Collection
• Assay Performance
• Image Analysis

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Multifaceted Role Of TMAs In Translational
Research

• TMAs
• Multiple Samples
• Different Levels Of Annotation
• XMAs
• Xenografts
• Pediatric Cancers
• NCI60
• CMAs
• Cell Lines
• NCI60

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Pathways To TMAs In Biomarker Development
In Vitro Screening Methods
Clinical Trials
Epidemiology Based Studies
Multi-tumor Systems
Other Methods
Micro-Arrays
Tissue Microarrays
Validation In Real Patient Samples
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From Tissue To Arrays

• Develop Hypothesis
• Identify Tissue
• Prepare Tissue
• Array Tissue
• Section Arrays
• Perform Experiment
• Analyze Data
• Publish
• Repeat

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Arraying The Correct Tissue

• A Fresh Section Is Cut And HE Made
• The Section Is Reviewed, And The Target Tissue Is
  Outlined
• Smallest Reasonable Target 1.5 mm
• When Arraying, The Slide Is Overlaid With The
  Block

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Tissue Arrayers

• Manual
• Simple To Use
• Allows Special Techniques

• Automated (ATA-27)
• 4-5 X Efficiency In Tissue Utilization
• Video-Overlap Essential

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Arraying
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Arraying
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Arraying
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Arraying
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Arraying
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Arraying
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Challenges In The Construction Of A TMA

• Identifying Adequate/Appropriate Material
• Negotiating MTA/IRB Approval
• Specimens Present Special Challenges
• Biopsy Small Specimens Are Hard To Array
• Prior Use Of Material For Research
• Diversity Of Specimen Sources
• Regional Differences In Diagnosis Treatment
• Differences In Specimen Handling
• Informatics
• Managing Expectations
• Pathologist Vs Scientist

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Proteomic Profiling Of Tumors

• Goals
• Describe Signaling Pathways
• Describe Differences In Signaling Between Tumor
  Types
• Provide Basis For Clinical Test To Predict
  Response

• Challenges
• Phospho-Specific Antibodies
• Image Analysis
• New Models To Explain Interactions
• Correlation With Outcome

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Akt Pathway
GSK-3b 4EBP-1 P-PDK-1 p70 mTOR
P-S473 P-T308
PDK-1
Other Signals
Akt
mTOR
p70
GSK-3
4EBP-1
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P-4EBP-1
P-p70S6K
P-mTOR
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Traditional Data
0, 1, 2, 3, 4
, -
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AKT Pathway Activation With Semi-Quantitative
Scoring
Akt
Upstream
Downstream
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Analysis With Quantitative Image Analysis

• Seven Phospho-specific Antibodies On A 500 Core
  Multitumor Array
• Image Analysis
• Scored Intensity Of Staining
• Automatic Threshold/Background Correction
• Pair-wise Correlation
• Correlation Coefficient
• T-test Calculated P Value
• P lt0.0025 Considered Significant

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Akt Pathway Propagation
Colon Cancer
Breast Cancer
Prostate Cancer
Lung Cancer
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Appropriate Therapeutic Targets Of The Akt
Pathway
PDK-1
After Looking At 5 Additional Tumor Types
Other Signals
Akt
mTOR
p70
GSK-3
4EBP-1
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Beyond Immunohistochemistry

• TMAs Are A Protein Array Platform To Present
  Tissue For Biochemical/Biophysical Analysis
  Discovery Platform
• Tissue Immunoblotting
• Spectroscopic Analysis
• FTIR
• Raman
• X-ray
• Reduction But Retention
  Of Histomorphology

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Tissue Immunoblotting

• Goals
• FFPE Tissue
• Multiplex
• Quantitative
• Utility
• Pathway Analysis
• Total/Phospho
• Histo-geography

• Restrictions
• Archival Material
• Material On A Glass
• Surface
• Exploring
• Cytokines
• Abs That Do Not Perform In FFPE

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Tissue Immunoblotting
Sealed Envelopoe
Block (1 Kg)
Glass slide
Absorbent pads
FITC (for specific Ag )
Nitrocellulose membrane
Biotinylation
3MM filter paper
Enzyme treatment
P-FILM membrane stack
Biotin
Spacer
Protein on membrane
Tissue slide
Cy 5 (for total protein)
Absorbent pads
Glass slide
Heat source
Routine Immunodetection
Heat-facilitated Capillary Transfer
Detection With A Microarray Scanner
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Transfer Kinetics
Intra-Transfer Reproducibility
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Application To Whole Tissue
Pan CK
CK-14
CK-4
N
S
D
T
T
SPARC
Annexin I
p53
COX 2
Squamous Cell Carcinoma Of The Esophagus
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Biomarker Profiles
3
Pan-CK
2.5
2
Relative expression intensity
1.5

1

0.5

0
S
D
T
21
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Biomarker Profiles
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Distribution Analysis Of Phospho-Proteins
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RNA From FFPEThe Ultimate Quest

• History of RNA Isolation From FFPE Is L ong
  Poorly Documented
• No Comprehensive Studies
• Ad Hoc Methods
• Modified Approaches From Frozen Tissue
• Proteinase K
• Guanidinium
• No Studies Referenced To Frozen Tissue Adequately

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Optimization Of RNA Recovery From FFPE Impact Of
Temperature and Additional Time
Days Of Lysis In GT
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RNA Quantity and Quality
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RNA Recovery From Tissue
RNA In FFPE -Anticipated
RNA In Frozen Tissue
RNA In FFPE -Revised
Results Of RT-PCR
?
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Quantitative Amplification Based On RNA Source
Primer Location
Same Quantity of Starting RNA Random Prime cDNA
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Standard Protocol Formalin Fixation Paraffin
Embedding

• Misnomer
• Every Institution Is Different
• Different Protocols For Different Specimen Types
• Biopsies
• Excisional Specimens
• Drift Of Protocols With Time

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Basic Design
60
45
CaCl2
0
36
12
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Fixation Time Hrs
24 hrs 30 min Phosphate
Fixative Buffer
15
Tris
Processor Time Min Per Step
0
None
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Effects Of Formalin Buffer On Histology
CaCl2
Phosphate
No Buffer
Tris
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Effect Of Formalin Buffers On RNA Quality
A
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Effect Of Fixation Time On RNA Quality
B
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Effect Of Processor Time On RNA Quality
C
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Tissue Preservation Processing

• Warm Ischemia Time
• Poor Fixation
• Inadequate Volume
• Inadequate Time
• Poor Processing
• Inadequate Dehydration
• Paraffin Quality
• Hard Paraffins
• Contaminants

• Metabolic Impact
• Impact Of Buffers
• What Is The Buffer?
• Alternative Processing
• Microwave
• Reagent Substitutions
• Storage
• Blocks
• Slides

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Assay DevelopmentHardening The Assay

• Inadequate Sample
• Inappropriate/Damaged Sample
• Measures Of Assay Performance
• Irreproducibility
• Throughput
• Data Interpretation

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Tissue Handling Impacts Results

• Immunohistochemistry Is As Robust As Any Other
  Means Of Measuring A Protein
• Tissue Handling Processing Is What Limits The
  Assays
• Frozen Tissue Is Impractical
• Formalin Fixation Is Poorly Standardized
• Tissue Processing Is Poorly Standardized
• Efforts In the US Europe To Standardize Tissue
  Collection, Handling Storage
• NCI, BIG, CLSI, FDA, Tubafrost.

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Prospective Collection Of Tissue

• Standardized Collection Processing
• Alternative Fixatives
• Collection Of Pre-specified Tissues
• Allows Systematic Study Of Material That Can Not
  Be Adequately Obtained In Retrospective Studies
• Excess Material From Non-enrolling /Ineligible
  Patients
• What Is State Of The Art Today, Is Not State Of
  The Art Tomorrow

New FDA Guidelines
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Specimen HandlingWhat Does NOT Impact Results?

• Factors That Are Known To Impact Quality
• Warm Ischemia Time
• Fixation Time
• Fixative Buffer
• Processor Time
• Paraffin Type
• Slide Storage
• Factors That Remain To Be Studied
• Alternative Reagents
• Block Storage
• Everything Else
• Factors That Do Not Impact Quality
• None Documented

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The Essence Of Biomarkers

• If You Dont Take A Temperature, You Cant
  Find A Fever.
• Samuel Shem, House of God

If You Dont Specify An Analyte, You Cant
Perform An Assay. SMH
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Acknowledgements

• TARP Lab
• Kimberly Tuttle
• Joon-Yong Chung
• Mikiko Takikita
• Langston Lim
• Yvonne Gathright
• Till Braunschweig
• Phil Dennis
• Jen Jin
• Javed Khan
• Chand Khanna
• Phil Taylor

• Division Of Cancer Therapeutics Control
• Division Of Cancer Epidemiology Genetics
• SEER Program
• Developmental Therapeutics Program
• Cancer Therapeutic Evaluation Program
• Comparative Oncology Program
• Michael Emmert-Buck
• Ira Levin
• Robert Star
• Max Robinowitz

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Tissue Array Research Program
www.cancer.gov/tarp
genejock_at_helix.nih.gov TARP Lab Advanced
Technology Center MSC 4605, Bethesda, MD
20892-4605
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