Quantitative Immunohistochemistry Methods Development for ER Detection in Breast Cancer
KA Vanpatten, M Loftus, J Pettay, L Wang, S Estrada, N Haubein, GS Lett, P Miller, R Tubbs, GA Pestano. Ventana Medical Systens (a Member of the Roche Group), Tucson, AZ; Lerner College of Medicine, Cleveland, OH; The BioAnalytics Group, Jamesburg, NJ
Background: The assessment of cancer biomarker status in tissues represents a critical component in the development of therapeutics as well as in diagnostics. However, tissue-based diagnostics are often subjectively assessed. In addition, manual methods for slide processing promote inconsistency, are labor demanding and time-consuming. Automated IHC and ISH in formalin-fixed paraffin-embedded (FFPE) tissues allows standardized, rapid evaluation of gene and protein biomarkers in a high-throughput format. Further, advances in detection, including multiplexed Quantum dots (Qdots), and spectral imaging, provide a growing set of tools that could complement biomarker studies.
Design: We have applied automated IHC methods with Qdot detection and image analysis to monitor markers in FFPE tissues that may be therapeutic targets, prognostic of disease, or surrogates of drug response. Software algorithms were developed in this study to aid in the deconvolution of hyperspectral data, and to identify cells and cellular compartments. In the study of breast cancer specimens, samples were analyzed for the simultaneous detection of QD 605 CK (cytokeratin), QD 655 ER (estrogen receptor), and the nuclear marker, DAPI. Dual stained CK/ER Qdot IHC results were assessed for concordance with pathology scores, single-stained chromogenic image analysis assays, as well as by qRT/PCR for detection of the estrogen receptor.
Results: The Spearman Rank Correlation test was used to compare chromogenic pathology scores with Qdot image analysis results. Specifically, Qdot detection of ER expression correlated with a statistically significant p value of 0.0003 (rank correlation = 0.927) to the pathology scores. We also noted that significantly enhanced dynamic range of antigen detection was achievable with Qdot detection in FFPE.
Conclusions: In this study we have demonstrated technical feasibility of using multiplexed Qdot IHC coupled with quantitative image analysis. The automated image analysis methods correctly identified ER potein levels, and were consistent with pathology review in breast cancer specimens. These results indicate that advanced analytical methods may be applied to breast cancer biomarker assessment in routinely collected FFPE tissues, but only when using highly standardized techniques.
Wednesday, March 11, 2009 1:00 PM
Poster Session VI # 248, Wednesday Afternoon