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
BLOT
IHC
CHIP
PCR
TMA
Multianalytes
Xenografts
Chromosome aberrations
Phase III trials
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 Proteomics Other “omics”
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 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 Genotyping
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 Solution
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 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. Methods of WGA Methods PCR approaches Non PCR approaches
Degenerate oligonucleotide primed PCR
Primer extension preamplification
Non PCR approaches
T7 based Linear amplification
F 29 DNA polymerase strand displacement amplification

19. High quality Genomic DNA
Method
Technical
Template Input
Applications
DOD-PCR
I-PEP
Easy
Low quantity
Poor-quality
Microsatellite
Sequencing
MDA/SDA
High quality Genomic DNA
Array CGH
RQ-PCR
SNP
S.Blotting
T7-Linear
Amplification
Cumbersome
Poor quality

20. Strand-displacement Amplification Reaction
Hexamer Primers
No common primer sequence
Isothermal reaction (30oC)
ng of DNA
Uniform yeild
Phi29 DNA polymerase
Strand displacement
Synthesis rate of nt/s
Processive (70kb)
Thermolabile
Proof reading (error < 106)
Lage et al Genome Res 13:

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

22. Strand Displacement Amplification
Additional applications
CGH.
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
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.