Utilization of FFPE in Molecular Utilization of FFPE in Molecular Oncology StudiesOncology Studies

Kishor Bhatia, Ph.D. MRCPath.Kishor Bhatia, Ph.D. MRCPath.

Director, Office of AIDS Malignancy Program, NCIDirector, Office of AIDS Malignancy Program, NCI

Technology examples chosen for Technology examples chosen for illustrative purposes only and are illustrative purposes only and are not endorsed by the NCInot endorsed by the NCI..

Tissue resources; Tissue resources; Responding to changing Responding to changing

scientific needsscientific needs 1960-70’s1960-70’s Serum BanksSerum Banks 1970-80’s1970-80’s Tissue procurement.Tissue procurement. 1980-90’s1980-90’s “BLOT” era. Frozen “BLOT” era. Frozen

samples with limited samples with limited clinical information.clinical information.

1990-20001990-2000 PCR allowed use of PCR allowed use of small small volume samples volume samples

BL

OT

Single geneSingle Protein analysis

IHC

Ch

rom

oso

me

aber

rati

on

s

Xen

og

rafts

PC

R

CH

IP

Mu

ltianalytes

Availability of excision tissue biopsy

Ph

ase III trials

1970 1980

TM

A

2005

OMICs era and Cancer OMICs era and Cancer Research Research

PathwayPathway Harness revolutionary molecular technologies Harness revolutionary molecular technologies

and informatics platforms to translate genomic and informatics platforms to translate genomic and proteomic information from human tissues.and proteomic information from human tissues.

Typing cancers using pattern of gene, protein Typing cancers using pattern of gene, protein expression.expression.

Promise of the Genomic eraPromise of the Genomic era Development of innovative approaches to Development of innovative approaches to

prevention therapy and diagnosis. prevention therapy and diagnosis. Example:Example: Targeted TherapiesTargeted Therapies

Diagnostic elements may include target identificationDiagnostic elements may include target identification

OMICS ERAOMICS ERA• GenomicsGenomics

Gene ExpressionGene Expression Discovery and Discovery and ClinicalClinical

Mutation analysisMutation analysis Discovery and Discovery and ClinicalClinical

SNP analysisSNP analysis Comparative Genomic Hybridization (CGH)Comparative Genomic Hybridization (CGH)

• ProteomicsProteomics Mass Spectrometry TechniquesMass Spectrometry Techniques Protein arraysProtein arrays Affinity arraysAffinity arrays

• Other “omics”Other “omics”• MetabolomicsMetabolomics• GlycomicsGlycomics

Tissue Challenges in Tissue Challenges in Omics eraOmics era

• Conflicting TrendsConflicting Trends Desire for more molecular Desire for more molecular

informationinformation Diminishing size of samples Diminishing size of samples

availableavailable• Accessing the Required Number Accessing the Required Number

of Specimens of Specimens • Requirement for Specimen Requirement for Specimen

AnnotationAnnotation Prospective vs. retrospectiveProspective vs. retrospective

Reliance on Frozen Reliance on Frozen tissuestissues

Frozen samples –golden standard. Frozen samples –golden standard. Molecules in unfixed frozen tissue Molecules in unfixed frozen tissue

remain intactremain intact Validation studies that require large Validation studies that require large

collections of fresh frozen specimen collections of fresh frozen specimen with patient outcome and drug with patient outcome and drug response history will involve years of response history will involve years of monitoring.monitoring.

Volume of sample Volume of sample requirementsrequirements

Reliance on specimens that can be Reliance on specimens that can be acquired as large volume tissue acquired as large volume tissue samples samples Microarray technology requires 10-50 Microarray technology requires 10-50

microgram of RNA.microgram of RNA. Studies conveniently possible on Studies conveniently possible on

disease stages where surgical disease stages where surgical resection is the treatment of choice; resection is the treatment of choice; example early stage NSCLC.example early stage NSCLC.

Need to explore the utilization of low Need to explore the utilization of low volume samples such as guided FNAsvolume samples such as guided FNAs

Departments of Pathology Departments of Pathology Archives : Rich resource of Archives : Rich resource of

tissuestissues Formalin fixed paraffin embedded Formalin fixed paraffin embedded

tissues are widely available and have the tissues are widely available and have the advantage of wealth of information advantage of wealth of information associated with themassociated with them

Routine histological assessment – tissue Routine histological assessment – tissue fixation, usually formaldehyde based fixation, usually formaldehyde based fixatives; buffered formalinfixatives; buffered formalin

Formalin cross linkingFormalin cross linking Analytes derived from FFPEs are poor Analytes derived from FFPEs are poor

quality.quality.

Shifts in tissue usabilityShifts in tissue usability

Changes in technology have Changes in technology have enhanced the value of FFPE tissuesenhanced the value of FFPE tissues

Department of Pathology Department of Pathology ArchivesArchives

Many casesMany cases Limited resourcesLimited resources

Technology tools to Technology tools to recover information from recover information from

available tissuesavailable tissues• ChallengesChallenges• Ability to conduct multiple Ability to conduct multiple

analysis from limited volume analysis from limited volume tissues. tissues.

• Technologies to interrogate Technologies to interrogate paraffin embedded samples.paraffin embedded samples.

GenomicsGenomics

DNA analysis.DNA analysis. Mutation detectionMutation detection

Sensitivity, Heterogeneity, Rapid Sensitivity, Heterogeneity, Rapid analysis for target identification.analysis for target identification.

SNP, Clinical data, Epidemiologic data.SNP, Clinical data, Epidemiologic data. GenotypingGenotyping

Large Cancer Epidemiology studiesLarge Cancer Epidemiology studies Several Genotyping platformsSeveral Genotyping platforms Multiple DNA isolation methodsMultiple DNA isolation methods

GenomicsGenomics

Challenge Challenge DNA amount available from samples DNA amount available from samples

not sufficient to complete multiple not sufficient to complete multiple studies.studies.

SolutionSolution Replicate genetic informationReplicate genetic information

Technology RequirementTechnology Requirement AccuracyAccuracy

Representation of the amplified DNA Representation of the amplified DNA such that there is minimal loci and such that there is minimal loci and allele biasallele bias

Stability and usability of amplified DNAStability and usability of amplified DNA Methods must be easily adaptable robust Methods must be easily adaptable robust

and scaleableand scaleable Whole genome amplificationWhole genome amplification

Whole Genome Whole Genome AmplificationAmplification

Unlimited quantity of Genomic Unlimited quantity of Genomic DNA for unlimited analysisDNA for unlimited analysis Amplification of 100,000 -1000,000 Amplification of 100,000 -1000,000

foldfold Input of 10ng of un-degraded DNA Input of 10ng of un-degraded DNA

sufficient.sufficient. Direct amplification from a wide Direct amplification from a wide

variety of samplesvariety of samples Genomic DNA, blood, FNAs, buccal Genomic DNA, blood, FNAs, buccal

washes etc.washes etc.

MethodsMethods of WGAof WGA

MethodsMethods PCR approachesPCR approaches

Degenerate oligonucleotide Degenerate oligonucleotide primed PCRprimed PCR

Primer extension preamplificationPrimer extension preamplification Non PCR approaches Non PCR approaches

T7 based Linear amplificationT7 based Linear amplification 29 DNA polymerase strand 29 DNA polymerase strand

displacement amplificationdisplacement amplification

MethodMethod TechnicalTechnical TemplatTemplate Inpute Input

ApplicatioApplicationsns

DOD-PCRDOD-PCR

I-PEPI-PEP

EasyEasy Low Low quantityquantity

Poor-Poor-qualityquality

MicrosateMicrosatellitellite

SequenciSequencingng

MDA/SDAMDA/SDA EasyEasy Low Low quantityquantity

High High quality quality Genomic Genomic DNADNA

Array Array CGHCGH

RQ-PCRRQ-PCR SNPSNP S.BlottingS.Blotting

T7-LinearT7-Linear

AmplificatiAmplificationon

CumbersoCumbersomeme

Low Low quantityquantity

Poor Poor qualityquality

Lage et al. Lage et al. 20032003 Genome Res 13: 294-307Genome Res 13: 294-307

Strand-displacement Strand-displacement AmplificationAmplification Reaction Reaction

•Hexamer Primers•No common primer sequence

•Isothermal reaction (30oC)•10-100 ng of DNA

•Uniform yeild•Phi29 DNA polymerase

•Strand displacement•Synthesis rate of 50-200nt/s•Processive (70kb)•Thermolabile•Proof reading (error < 106)

WGA DNA ApplicationsWGA DNA Applications

Luthra R and Medeiros J. Journal of Mol Diag: 5, 236-242, 2004

Strand Displacement Strand Displacement AmplificationAmplification

Additional applicationsAdditional applications CGH.CGH. Microarray based Genome-wide Microarray based Genome-wide

scalable SNP genotypingscalable SNP genotyping (Gunderson et al; Nature Genetics, 17, (Gunderson et al; Nature Genetics, 17,

549-554, 2005)549-554, 2005)

AdvantageAdvantage small sample size small sample size usableusable

Gene Expression ProfilingGene Expression Profiling

Analytical technique to measure the Analytical technique to measure the expression of a large number of genes in tissue expression of a large number of genes in tissue specimens simultaneously. specimens simultaneously.

Based upon the hypothesis that the Based upon the hypothesis that the constellation of multiple genes will be more constellation of multiple genes will be more predictive of clinical outcome than any single predictive of clinical outcome than any single gene alone. gene alone. Gene expression signatures have been shown to Gene expression signatures have been shown to

predict prognosis of several cancers as well as predict prognosis of several cancers as well as response to particular chemotherapy regimens. response to particular chemotherapy regimens.

Continued progress and ultimate routine Continued progress and ultimate routine clinical use, is limited by requirements for clinical use, is limited by requirements for fresh tumor tissue. fresh tumor tissue.

Strategies for Gene Strategies for Gene Expression signatures from Expression signatures from

Paraffin embedded Paraffin embedded tissues/FNAtissues/FNA

Discovery Discovery Amplification of RNAAmplification of RNA

Validation and clinical application Validation and clinical application Multi gene expression using Real Time Multi gene expression using Real Time

Quantitative PCR. Quantitative PCR.

Analyte Amplification - Analyte Amplification - RNARNA

• ChallengesChallenges RNA present over large RNA present over large

concentration rangeconcentration range RNA amplification while RNA amplification while

maintaining sequence maintaining sequence representationrepresentation

• MethodsMethods Poly A or random primer PCR Poly A or random primer PCR T7 RNA polymerase amplificationT7 RNA polymerase amplification Combination of PCR/T7 Combination of PCR/T7

amplificationamplification

Use of Paraffin Use of Paraffin Embedded SpecimensEmbedded Specimens

• Improved TechnologiesImproved Technologies Illumina DASLIllumina DASLTM TM assayassay Affymetrix X3P microarraysAffymetrix X3P microarrays

Validation Validation Multi-gene expression Multi-gene expression

using Real time RT-PCRusing Real time RT-PCR Panel of genes identified from frozen Panel of genes identified from frozen

tissue analysistissue analysis Gene specific primers to measure Gene specific primers to measure

short RNA fragmentsshort RNA fragments Sufficient RNA can be isolated from Sufficient RNA can be isolated from

few 10 micron slide mounted few 10 micron slide mounted sections to quantitate up to 30 sections to quantitate up to 30 genes.genes.

RNA/DNA Isolation

RQ PCR

Data Analysis

RNA

FFPE tumor micro-dissectionDNA

Sequence

0

2

4

6

8

10

12

0 10 20 30 40

Cycle Number

Rel

ativ

e F

luo

resc

ence

Validation : Real time PCR analysis of Gene Expression

RTArray

Measuring Multi-gene Measuring Multi-gene expression in fixed tissuesexpression in fixed tissues

Develop methodology Develop methodology for robust multi gene for robust multi gene measurements in RNA measurements in RNA from archival samples.from archival samples. Cronin M et al. Am J. Cronin M et al. Am J.

Pathol. 164, 35-42, Pathol. 164, 35-42, 2004.2004.

Primers designed such Primers designed such that Amplicon sizes that Amplicon sizes limited to 100 bases in limited to 100 bases in length.length.

Example: Oncotype Dx Example: Oncotype Dx AssayAssay

Panel of 21 Genes selected. Panel of 21 Genes selected. Based upon assessment of 250 Based upon assessment of 250

candidate genes previously identified candidate genes previously identified using fresh frozen tissues. using fresh frozen tissues.

668 paraffin blocks from tamoxifen 668 paraffin blocks from tamoxifen treated node negative breast cancers.treated node negative breast cancers.

Score based upon expression levels Score based upon expression levels obtained from paraffin embedded obtained from paraffin embedded tissues allowed identification of patients tissues allowed identification of patients with low- high risk of recurrence. with low- high risk of recurrence.

Paik et al. New England Journal of Medicine 351 (27): 2817, 2004

Interface of Technologies and Interface of Technologies and Specimen for the Development Specimen for the Development

of Biomarkersof Biomarkers• What is the clinical question/need?What is the clinical question/need?

• Interface organization of archival material Interface organization of archival material with specific projectswith specific projects

• Selection of appropriate specimens to Selection of appropriate specimens to address the clinical questionaddress the clinical question• Paraffin embedded tissues with clinical Paraffin embedded tissues with clinical

informationinformation

• Develop appropriate study designDevelop appropriate study design• Tissue micro arrays.Tissue micro arrays.

• Develop core collaborative centers to Develop core collaborative centers to allow access to expertiseallow access to expertise

SummarySummary

• Technological solutions continue to Technological solutions continue to evolve to allow use of a wide variety of evolve to allow use of a wide variety of samples samples • Use of small volume specimens is possible Use of small volume specimens is possible

in omics erain omics era• Clinical annotation enhances the value Clinical annotation enhances the value

of paraffin embedded specimens.of paraffin embedded specimens.• Large clinical sets of archival samples Large clinical sets of archival samples

in departments of pathology can be in departments of pathology can be significant tools in translational significant tools in translational cancer research.cancer research.