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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
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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.