Utilization of FFPE in Molecular Oncology Studies

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 • 1960-70’s Serum Banks • 1970-80’s Tissue procurement. • 1980-90’s “BLOT” era. Frozen samples with limited clinical information. • 1990-2000 PCR allowed use of small volume samples

4. Availability of excision tissue biopsy BLOT IHC CHIP TMA PCR Multianalytes Xenografts Phase III trials Chromosome aberrations Single gene Single Protein analysis 1970 1980 2005

5. OMICs era and Cancer Research • Pathway • Harness 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 era • Development of innovative approaches to prevention therapy and diagnosis. • Example: Targeted Therapies • Diagnostic elements may include target identification

6. OMICS ERA • Genomics • Gene Expression Discovery and Clinical • Mutation analysis Discovery and Clinical • SNP analysis • Comparative Genomic Hybridization (CGH) • Proteomics • Mass Spectrometry Techniques • Protein arrays • Affinity arrays • Other “omics” • Metabolomics • Glycomics

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

8. Reliance on Frozen tissues • Frozen samples –golden standard. • 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.

9. Volume of sample requirements • Reliance on specimens that can be acquired as large volume tissue samples • Microarray technology requires 10-50 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 them • Routine histological assessment – tissue fixation, usually formaldehyde based fixatives; buffered formalin • Formalin cross linking • Analytes 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 cases • Limited resources

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

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

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

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

17. Whole Genome Amplification • Unlimited quantity of Genomic DNA for unlimited analysis • Amplification of 100,000 -1000,000 fold • Input of 10ng of un-degraded DNA sufficient. • Direct amplification from a wide variety of samples • Genomic DNA, blood, FNAs, buccal washes etc.

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

20. Strand-displacement Amplification 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) Lage et al. 2003Genome Res 13: 294-307

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

22. Strand Displacement Amplification • Additional applications • CGH. • Microarray based Genome-wide scalable SNP genotyping • (Gunderson et al; Nature Genetics, 17, 549-554, 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 • Discovery • Amplification of RNA • Validation and clinical application • Multi gene expression using Real Time Quantitative PCR.

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

26. Use of Paraffin Embedded Specimens • Improved Technologies • Illumina DASLTM assay • Affymetrix X3P microarrays

27. Validation Multi-gene expression using Real time RT-PCR • Panel of genes identified from frozen tissue analysis • 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.

28. Validation : Real time PCR analysis of Gene Expression RNA/DNA Isolation RNA DNA FFPE tumor micro-dissection Sequence Array RT RQ PCR Data 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 projects • Selection of appropriate specimens to address the clinical question • Paraffin embedded tissues with clinical information • Develop appropriate study design • Tissue micro arrays. • 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 • Use of small volume specimens is possible in omics era • 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.