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

Technology examples chosen for illustrative purposes only and are not endorsed by the NCI.

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

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

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

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

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

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.

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

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.

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

Department of Pathology Archives Many cases Limited resources

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

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

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

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

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.

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

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

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. 2003 Genome Res 13: 294-307

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

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

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.

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.

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

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

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.

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

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.

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

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

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.