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Utilization of FFPE in Molecular Oncology Studies Kishor Bhatia, Ph.D. MRCPath. Director, Office of AIDS Malignancy Program, NCI.

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Presentation on theme: "Utilization of FFPE in Molecular Oncology Studies Kishor Bhatia, Ph.D. MRCPath. Director, Office of AIDS Malignancy Program, NCI."— 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 sSerum Banks sSerum Banks sTissue procurement sTissue procurement sBLOT era. Frozen samples with limited clinical information sBLOT era. Frozen samples with limited clinical information PCR allowed use of small volume samples PCR allowed use of small volume samples

4 BLOT Single gene Single Protein analysis IHC Chromosome aberrations Xenografts PCR CHIP Multianalytes Availability of excision tissue biopsy Phase III trials TMA 2005

5 OMICs era and Cancer Research Pathway Pathway Harness revolutionary molecular technologies and informatics platforms to translate genomic and proteomic information from human tissues. Harness revolutionary molecular technologies and informatics platforms to translate genomic and proteomic information from human tissues. Typing cancers using pattern of gene, protein expression. Typing cancers using pattern of gene, protein expression. Promise of the Genomic era Promise of the Genomic era Development of innovative approaches to prevention therapy and diagnosis. Development of innovative approaches to prevention therapy and diagnosis. Example:Targeted Therapies Example:Targeted Therapies Diagnostic elements may include target identification Diagnostic elements may include target identification

6 OMICS ERA Genomics Genomics Gene ExpressionDiscovery and Clinical Gene ExpressionDiscovery and Clinical Mutation analysisDiscovery and Clinical Mutation analysisDiscovery and Clinical SNP analysis SNP analysis Comparative Genomic Hybridization (CGH) Comparative Genomic Hybridization (CGH) Proteomics Proteomics Mass Spectrometry Techniques Mass Spectrometry Techniques Protein arrays Protein arrays Affinity arrays Affinity arrays Other omics Other omics Metabolomics Metabolomics Glycomics Glycomics

7 Tissue Challenges in Omics era Conflicting Trends Conflicting Trends Desire for more molecular information Desire for more molecular information Diminishing size of samples available Diminishing size of samples available Accessing the Required Number of Specimens Accessing the Required Number of Specimens Requirement for Specimen Annotation Requirement for Specimen Annotation Prospective vs. retrospective Prospective vs. retrospective

8 Reliance on Frozen tissues Frozen samples –golden standard. Frozen samples –golden standard. Molecules in unfixed frozen tissue remain intact Molecules in unfixed frozen tissue remain intact Validation studies that require large collections of fresh frozen specimen with patient outcome and drug response history will involve years of monitoring. Validation 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 samples Reliance on specimens that can be acquired as large volume tissue samples Microarray technology requires microgram of RNA. Microarray technology requires microgram of RNA. Studies conveniently possible on disease stages where surgical resection is the treatment of choice; example early stage NSCLC. 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 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 them Formalin fixed paraffin embedded tissues are widely available and have the advantage of wealth of information associated with them Routine histological assessment – tissue fixation, usually formaldehyde based fixatives; buffered formalin Routine histological assessment – tissue fixation, usually formaldehyde based fixatives; buffered formalin Formalin cross linking Formalin cross linking Analytes derived from FFPEs are poor quality. Analytes derived from FFPEs are poor quality.

11 Shifts in tissue usability Changes in technology have enhanced the value of FFPE tissues Changes in technology have enhanced the value of FFPE tissues

12 Department of Pathology Archives Many cases Many cases Limited resources Limited resources

13 Technology tools to recover information from available tissues Challenges Challenges Ability to conduct multiple analysis from limited volume tissues. Ability to conduct multiple analysis from limited volume tissues. Technologies to interrogate paraffin embedded samples. Technologies to interrogate paraffin embedded samples.

14 Genomics DNA analysis. DNA analysis. Mutation detection Mutation detection Sensitivity, Heterogeneity, Rapid analysis for target identification. Sensitivity, Heterogeneity, Rapid analysis for target identification. SNP, Clinical data, Epidemiologic data. SNP, Clinical data, Epidemiologic data. Genotyping Genotyping Large Cancer Epidemiology studies Large Cancer Epidemiology studies Several Genotyping platforms Several Genotyping platforms Multiple DNA isolation methods Multiple DNA isolation methods

15 Genomics Challenge Challenge DNA amount available from samples not sufficient to complete multiple studies. DNA amount available from samples not sufficient to complete multiple studies. Solution Solution Replicate genetic information Replicate genetic information

16 Technology Requirement Accuracy Accuracy Representation of the amplified DNA such that there is minimal loci and allele bias Representation of the amplified DNA such that there is minimal loci and allele bias Stability and usability of amplified DNA Stability and usability of amplified DNA Methods must be easily adaptable robust and scaleable Methods must be easily adaptable robust and scaleable Whole genome amplification Whole genome amplification

17 Whole Genome Amplification Unlimited quantity of Genomic DNA for unlimited analysis Unlimited quantity of Genomic DNA for unlimited analysis Amplification of 100, ,000 fold Amplification of 100, ,000 fold Input of 10ng of un-degraded DNA sufficient. Input of 10ng of un-degraded DNA sufficient. Direct amplification from a wide variety of samples Direct amplification from a wide variety of samples Genomic DNA, blood, FNAs, buccal washes etc. Genomic DNA, blood, FNAs, buccal washes etc.

18 Methods of WGA Methods PCR approaches PCR approaches Degenerate oligonucleotide primed PCR Degenerate oligonucleotide primed PCR Primer extension preamplification Primer extension preamplification Non PCR approaches Non PCR approaches T7 based Linear amplification T7 based Linear amplification 29 DNA polymerase strand displacement amplification 29 DNA polymerase strand displacement amplification

19 MethodTechnical Template Input Applications DOD-PCRI-PEPEasy Low quantity Poor-quality Microsatellite Microsatellite Sequencing Sequencing MDA/SDAEasy Low quantity High quality Genomic DNA Array CGH Array CGH RQ-PCR RQ-PCR SNP SNP S.Blotting S.Blotting T7-LinearAmplificationCumbersome Low quantity Poor quality

20 Lage et al Genome Res 13: Strand-displacement Amplification Reaction Hexamer Primers No common primer sequence Isothermal reaction (30 o C) ng of DNA Uniform yeild Phi29 DNA polymerase Strand displacement Synthesis rate of nt/s Processive (70kb) Thermolabile Proof reading (error < 10 6 )

21 WGA DNA Applications Luthra R and Medeiros J. Journal of Mol Diag: 5, , 2004

22 Strand Displacement Amplification Additional applications Additional applications CGH. CGH. Microarray based Genome-wide scalable SNP genotyping Microarray based Genome-wide scalable SNP genotyping (Gunderson et al; Nature Genetics, 17, , 2005) (Gunderson et al; Nature Genetics, 17, , 2005) Advantagesmall sample size usable Advantagesmall sample size usable

23 Gene Expression Profiling Analytical technique to measure the expression of a large number of genes in tissue specimens simultaneously. 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. 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. 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. 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 Discovery Discovery Amplification of RNA Amplification of RNA Validation and clinical application Validation and clinical application Multi gene expression using Real Time Quantitative PCR. Multi gene expression using Real Time Quantitative PCR.

25 Analyte Amplification - RNA Challenges Challenges RNA present over large concentration range RNA present over large concentration range RNA amplification while maintaining sequence representation RNA amplification while maintaining sequence representation Methods Methods Poly A or random primer PCR Poly A or random primer PCR T7 RNA polymerase amplification T7 RNA polymerase amplification Combination of PCR/T7 amplification Combination of PCR/T7 amplification

26 Use of Paraffin Embedded Specimens Improved Technologies Improved Technologies Illumina DASL TM assay Illumina DASL TM assay Affymetrix X3P microarrays Affymetrix X3P microarrays

27 Validation Multi-gene expression using Real time RT-PCR Validation Multi-gene expression using Real time RT-PCR Panel of genes identified from frozen tissue analysis Panel of genes identified from frozen tissue analysis Gene specific primers to measure short RNA fragments Gene specific primers to measure short RNA fragments Sufficient RNA can be isolated from few 10 micron slide mounted sections to quantitate up to 30 genes. Sufficient RNA can be isolated from few 10 micron slide mounted sections to quantitate up to 30 genes.

28 RNA/DNA Isolation RQ PCR Data Analysis RNA FFPE tumor micro-dissection DNA Sequence Validation : Real time PCR analysis of Gene Expression RT Array

29 Measuring Multi-gene expression in fixed tissues Develop methodology for robust multi gene measurements in RNA from archival samples. Develop methodology for robust multi gene measurements in RNA from archival samples. Cronin M et al. Am J. Pathol. 164, 35-42, Cronin M et al. Am J. Pathol. 164, 35-42, Primers designed such that Amplicon sizes limited to 100 bases in length. Primers designed such that Amplicon sizes limited to 100 bases in length.

30 Example: Oncotype Dx Assay Panel of 21 Genes selected. Panel of 21 Genes selected. Based upon assessment of 250 candidate genes previously identified using fresh frozen tissues. Based upon assessment of 250 candidate genes previously identified using fresh frozen tissues. 668 paraffin blocks from tamoxifen treated node negative breast cancers. 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. 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? What is the clinical question/need? Interface organization of archival material with specific projects Interface organization of archival material with specific projects Selection of appropriate specimens to address the clinical question Selection of appropriate specimens to address the clinical question Paraffin embedded tissues with clinical information Paraffin embedded tissues with clinical information Develop appropriate study design Develop appropriate study design Tissue micro arrays. Tissue micro arrays. Develop core collaborative centers to allow access to expertise Develop core collaborative centers to allow access to expertise

32 Summary Technological solutions continue to evolve to allow use of a wide variety of samples Technological solutions continue to evolve to allow use of a wide variety of samples Use of small volume specimens is possible in omics era Use of small volume specimens is possible in omics era Clinical annotation enhances the value of paraffin embedded specimens. Clinical 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. Large clinical sets of archival samples in departments of pathology can be significant tools in translational cancer research.


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