Putative Signatures Predict High-Risk Oral SCC: Feasibility of Gene Discovery Using Tumors with Limited Formalin Fixation and the DASL Platform
O Loudig, R Kim, J Segall, A Negassa, M Prystowsky, M Brandwein-Gensler. MMC/Einstein, Bronx, NY; Einstein, Bronx, NY
Background: Our Risk Model is predictive of time to disease-progression for patients with HNSCC. Patients are classified as either low-, intermediate-, or high-risk after examining resection specimens for specific variables; a current limitation is that risk-score cannot be assigned on biopsies as their small size limits full evaluation of these variables. Identifying high-risk on biopsy could assist in planning more aggressive therapy and proper patient counseling. Our goal is to develop an RT-PCR test for formalin fixed paraffin embedded (FFPE) biopsies that is predictive of high-risk and poor-outcome. Our gene discovery approach uses tumor-enriched oral SCC samples subjected to limited FFPE. Array analysis is performed using the Illumina DASL (cDNA-mediated annealing, selection, extension and ligation) 24,000 probe assay, which is designed to detect partially degraded mRNAs as small as 50 nucleotides.
Design: Banked frozen specimens from 17 oral SCC (9 high-, 4 intermediate-, 4 low-risk) had limited FFPE (24 hours). Morphologically-guided tumor-enriched 1 mm cores were procured and mRNA extracted. The DASL platform was used for expression analysis. Raw data was analyzed without background subtraction after robust spline normalization. Standard T-test detected significant fold-change in gene expression between low- and high-risk tumors.
Results: RNA integrity index was ≥ 2.4, confirming suitability for study. Repetition of paired FFPE samples confirmed perfect reproducibility (R = 1.00). Standard T-test analysis revealed 157 genes differentially expressed in high-risk tumors with a 10% false discovery rate (FDR).
Some of these genes are involved in mitotic and signal transduction pathways; others genes regulate cell shape, polarity and migration.
Conclusions: We have demonstrated feasibility of using FFPE tumors for gene discovery and identified 157 genes (FDR 10%) that are differentially expressed in high- and low-risk oral SCC. This approach will facilitate the development of predictive RT-PCR test for routine biopsy samples.
Category: Pan-genomic/Pan-proteomic Approaches to Diseases
Monday, March 22, 2010 1:00 PM
Poster Session II # 229, Monday Afternoon