Quantitative Immunofluorescent Assay for Identification of Biomarkers in Formalin-Fixed Mantle Cell Lymphoma Biopsies
DT Yang, BS Kahl, KH Young, JC Eickhoff, AM Petrich, W Huang, CP Leith. University of Wisconsin, Madison, WI
Background: Identification of prognostic biomarkers is vital in facilitating comparisons across clinical trials and in improving treatment risk stratification. Gene expression profiling (GEP) of mantle cell lymphoma (MCL) has shown over-expression of cell proliferation genes can discriminate between patients with a 6 year median survival difference. However, GEP is currently impractical for clinical use and while immunohistochemistry on formalin-fixed paraffin-embedded (FFPE) tissues has also identified prognostic markers in MCL, limitations in reproducibility and lack of continuous quantification have restricted their clinical application. We assessed the performance of a high throughput assay using automated quantitative analysis (AQUA) of proliferation markers on FFPE on an MCL tissue microarray (TMA).
Design: 20 cases of MCL, diagnosed between 1995 and 2008, were used to construct a TMA with duplicate 1mm cores. Slides were simultaneously stained with DAPI (nuclear mask), anti-CD20 (tumor mask) and 1 of 4 antibodies against proliferation proteins, cdc2, cyclinD1, Ki-67, and mcm2 (targets). Automated quantitative analysis of the target was performed on the AQUA system (HistoRx, New Haven, CT).
Results: AQUA detected a wide range of target expression, particularly of cdc2 and cyclinD1. Results were highly reproducible (>0.70 by intraclass correlation) between duplicate cores. The 25th percentile of the overall survival distribution was 2.7 years. When divided into a poor prognosis group (<3 year survival) and good prognosis group (>3 year survival), two-sample t-test showed a significant difference in cdc2 expression, but not in cyclinD1, Ki-67, or mcm2.
Assessment of proliferation markers by AQUA
|Marker||AQUA range||Correlation coefficient||Clinical p-value|
Conclusions: On a pilot scale TMA of MCL cases, AQUA demonstrated reproducible continuous quantitation of potential biomarkers and identified an association between cdc2 expression level and patient outcome. Application of TMA/AQUA on a full scale likely has the statistical power and high throughput capabilities to identify prognostic biomarkers in MCL.
Wednesday, March 11, 2009 1:00 PM
Poster Session VI # 241, Wednesday Afternoon