Abstract P4-08-04: Assessment of Oncogenic Pathway Activation in Human Breast Cancer Tissue by Multiplex Immunohistochemistry

A multitude of cancer-associated genomic aberrations converge into activation of a limited repertoire of oncogenic ‘core’ pathways. Assessment of the activation status of such key signaling cascades is necessary to characterize pathway dependency of tumors, to predict response to specific pathway inhibitors, and to track drug effects in pharmacodynamic studies. We established a duplex immunohistochemistry (IHC) tool for the combined assessment of two key pathways involved in breast cancer biology, PI3K/AKT and MAPK. Validation of the duplex IHC assay was performed by tracking activation kinetics of phospho-AKT and phospho-MAPK in Heregulin (HRG-≥1) stimulated cultured MDA-MB453 cells by Western Blotting and immunostaining of immediately paraformaldehyde-fixed cells. In addition, one-sided inhibition of PI3K/AKT and MAPK signaling cascades was performed using the MEK1-inhibitor PD98059 and the PI3K inhibitor LY294002. Xenograft tumors were used to define the acceptable cold ischemia time for complex tissues. Consistently, we found a narrow time window of In conclusion, duplex IHC for concomitant detection of pAKT and pMAPK can be robustly applied to immediately processed breast cancer fine needle biopsies, whereas the use in surgical resection specimens is likely to be inaccurate unless the material has been processed under tightly controlled conditions. The observed staining patterns suggest distinctive activation patterns pointing to genomic aberrations causing constitutive pathway activation. As tumor heterogeneity and inflammatory cells are likely to influence any pathway analysis on homogenized human breast cancer tissue, we recommend to consider morphological assessment to localize pathway activation to relevant cell populations. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-08-04.