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Background: The overexpression/aberrant activation of the epidermal growth factor receptor and its downstream signaling pathways are common factors associated with the progression of many human tumors. While western blot (WB), RIA, or ELISA are typically used to evaluate relative levels of expression/activation, immunohistochemistry (IHC) provides the ability to quantify and localize intracellular molecules in individual cells and/or within regions of the malignancy and the use of archival pathological specimens. We have validated an approach of IHC staining combined with automated digital microscopy (ADM) analysis against WB using a series of cell culture model systems. This has provided a system by which routine IHC can be optimized to maintain reproducible, precise, and objective quantification. Methods: PhosphoEGFR (pEGFR) was evaluated in A431 cells following stimulation with EGF. Similarly, phosphoMAPK (pMAPK) and phosphoAkt (pAKT) were assessed in Jurkat cells stimulated with PMA or LY294002, respectively. EGFR and HER2 were evaluated in A431, MDA-231, MDA-175, HeLa-S3, and SKBR-3 cell lines. Cell suspensions were split immediately following treatment for WB analysis and formalin fixation/paraffin embedding and IHC with ADM analysis. Immunoblots were digitally scanned and quantified by image analysis. Further refinement of the IHC staining procedure was performed, included varying antigen retrieval, antibody, and substrate conditions, to develop optimal quantitative conditions. Results: Average measurement of pEGFR, pAkt and pMAPK in cell pellets by ADM revealed 5- to 20-fold changes in signal with varying treatments, correlating well with WB analysis (r = 0.960, p < 0.005). Interestingly, in cultured A431 cells there was a 9-fold difference in pEGFR expression in proliferating edges compared to confluent zones. Measurement of erbB receptors in a range of cell lines compared favorably with published values for number of receptors per cell. Refining of IHC staining conditions resulted not only in good correlation with WB, but also specific conditions by which a range of expression differences, even relatively similar ones, can be reproduced. Discussion: These studies suggest that ADM analysis can accurately quantify IHC staining. ADM provides a rigorous system by which routine clinical IHC assays can be optimized to semiquantitatively correlate measured intensity with antigen content. Since a number of receptor-based therapies have been (and continue to be) developed, there is an increasing necessity for a reproducible method by which IHC must provide accurate prognostic and/or diagnostic values. The flexibility and accuracy of ADM analysis affords the possibility of a more objective clinical scoring system that may be used across a variety of histopathological specimens.

[Proc Amer Assoc Cancer Res, Volume 45, 2004]