Presentation on theme: "Utilization of FFPE in Molecular Oncology Studies"— Presentation transcript:
1 Utilization of FFPE in Molecular Oncology Studies Kishor Bhatia, Ph.D. MRCPath.Director, Office of AIDS Malignancy Program, NCI
2 Technology examples chosen for illustrative purposes only and are not endorsed by the NCI.
3 Tissue resources; Responding to changing scientific needs ’s Serum Banks’s Tissue procurement.’s “BLOT” era. Frozen samples with limited clinical information.PCR allowed use of small volume samples
4 Availability of excision tissue biopsy BLOTIHCCHIPPCRTMAMultianalytesXenograftsChromosome aberrationsPhase III trialsSingle geneSingle Protein analysis197019802005
5 OMICs era and Cancer Research PathwayHarness revolutionary molecular technologies and informatics platforms to translate genomic and proteomic information from human tissues.Typing cancers using pattern of gene, protein expression.Promise of the Genomic eraDevelopment of innovative approaches to prevention therapy and diagnosis.Example: Targeted TherapiesDiagnostic elements may include target identification
6 OMICS ERA Genomics Proteomics Other “omics” Gene Expression Discovery and ClinicalMutation analysis Discovery and ClinicalSNP analysisComparative Genomic Hybridization (CGH)ProteomicsMass Spectrometry TechniquesProtein arraysAffinity arraysOther “omics”MetabolomicsGlycomics
7 Tissue Challenges in Omics era Conflicting TrendsDesire for more molecular informationDiminishing size of samples availableAccessing the Required Number of SpecimensRequirement for Specimen AnnotationProspective vs. retrospective
8 Reliance on Frozen tissues Frozen samples –golden standard.Molecules in unfixed frozen tissue remain intactValidation studies that require large collections of fresh frozen specimen with patient outcome and drug response history will involve years of monitoring.
9 Volume of sample requirements Reliance on specimens that can be acquired as large volume tissue samplesMicroarray technology requires microgram of RNA.Studies conveniently possible on disease stages where surgical resection is the treatment of choice; example early stage NSCLC.Need to explore the utilization of low volume samples such as guided FNAs
10 Departments of Pathology Archives : Rich resource of tissues Formalin fixed paraffin embedded tissues are widely available and have the advantage of wealth of information associated with themRoutine histological assessment – tissue fixation, usually formaldehyde based fixatives; buffered formalinFormalin cross linkingAnalytes derived from FFPEs are poor quality.
11 Shifts in tissue usability Changes in technology have enhanced the value of FFPE tissues
12 Department of Pathology Archives Many casesLimited resources
13 Technology tools to recover information from available tissues ChallengesAbility to conduct multiple analysis from limited volume tissues.Technologies to interrogate paraffin embedded samples.
14 Genomics DNA analysis. Mutation detection Genotyping Sensitivity, Heterogeneity, Rapid analysis for target identification.SNP, Clinical data, Epidemiologic data.GenotypingLarge Cancer Epidemiology studiesSeveral Genotyping platformsMultiple DNA isolation methods
15 Genomics Challenge Solution DNA amount available from samples not sufficient to complete multiple studies.SolutionReplicate genetic information
16 Technology Requirement AccuracyRepresentation of the amplified DNA such that there is minimal loci and allele biasStability and usability of amplified DNAMethods must be easily adaptable robust and scaleableWhole genome amplification
17 Whole Genome Amplification Unlimited quantity of Genomic DNA for unlimited analysisAmplification of 100, ,000 foldInput of 10ng of un-degraded DNA sufficient.Direct amplification from a wide variety of samplesGenomic DNA, blood, FNAs, buccal washes etc.
18 Methods of WGA Methods PCR approaches Non PCR approaches Degenerate oligonucleotide primed PCRPrimer extension preamplificationNon PCR approachesT7 based Linear amplificationF 29 DNA polymerase strand displacement amplification
19 High quality Genomic DNA MethodTechnicalTemplate InputApplicationsDOD-PCRI-PEPEasyLow quantityPoor-qualityMicrosatelliteSequencingMDA/SDAHigh quality Genomic DNAArray CGHRQ-PCRSNPS.BlottingT7-LinearAmplificationCumbersomePoor quality
20 Strand-displacement Amplification Reaction Hexamer PrimersNo common primer sequenceIsothermal reaction (30oC)ng of DNAUniform yeildPhi29 DNA polymeraseStrand displacementSynthesis rate of nt/sProcessive (70kb)ThermolabileProof reading (error < 106)Lage et al Genome Res 13:
21 WGA DNA ApplicationsLuthra R and Medeiros J. Journal of Mol Diag: 5, , 2004
22 Strand Displacement Amplification Additional applicationsCGH.Microarray based Genome-wide scalable SNP genotyping(Gunderson et al; Nature Genetics, 17, , 2005)Advantage small sample size usable
23 Gene Expression Profiling Analytical technique to measure the expression of a large number of genes in tissue specimens simultaneously.Based upon the hypothesis that the constellation of multiple genes will be more predictive of clinical outcome than any single gene alone.Gene expression signatures have been shown to predict prognosis of several cancers as well as response to particular chemotherapy regimens.Continued progress and ultimate routine clinical use, is limited by requirements for fresh tumor tissue.
24 Strategies for Gene Expression signatures from Paraffin embedded tissues/FNA DiscoveryAmplification of RNAValidation and clinical applicationMulti gene expression using Real Time Quantitative PCR.
25 Analyte Amplification - RNA ChallengesRNA present over large concentration rangeRNA amplification while maintaining sequence representationMethodsPoly A or random primer PCRT7 RNA polymerase amplificationCombination of PCR/T7 amplification
26 Use of Paraffin Embedded Specimens Improved TechnologiesIllumina DASLTM assayAffymetrix X3P microarrays
27 Validation Multi-gene expression using Real time RT-PCR Panel of genes identified from frozen tissue analysisGene specific primers to measure short RNA fragmentsSufficient RNA can be isolated from few 10 micron slide mounted sections to quantitate up to 30 genes.
28 Validation : Real time PCR analysis of Gene Expression RNA/DNA IsolationRNADNAFFPE tumor micro-dissectionSequenceArrayRTRQ PCRData Analysis
29 Measuring Multi-gene expression in fixed tissues Develop methodology for robust multi gene measurements in RNA from archival samples.Cronin M et al. Am J. Pathol. 164, 35-42, 2004.Primers designed such that Amplicon sizes limited to 100 bases in length.
30 Example: Oncotype Dx Assay Panel of 21 Genes selected.Based upon assessment of 250 candidate genes previously identified using fresh frozen tissues.668 paraffin blocks from tamoxifen treated node negative breast cancers.Score based upon expression levels obtained from paraffin embedded tissues allowed identification of patients with low- high risk of recurrence.Paik et al. New England Journal of Medicine 351 (27): 2817, 2004
31 Interface of Technologies and Specimen for the Development of Biomarkers What is the clinical question/need?Interface organization of archival material with specific projectsSelection of appropriate specimens to address the clinical questionParaffin embedded tissues with clinical informationDevelop appropriate study designTissue micro arrays.Develop core collaborative centers to allow access to expertise
32 SummaryTechnological solutions continue to evolve to allow use of a wide variety of samplesUse of small volume specimens is possible in omics eraClinical annotation enhances the value of paraffin embedded specimens.Large clinical sets of archival samples in departments of pathology can be significant tools in translational cancer research.