Abstract P4-08-04: Assessment of Oncogenic Pathway Activation in Human Breast Cancer Tissue by Multiplex Immunohistochemistry
Authors: Bonnas C , Specht K , Huss R , Arbogast S , Feuerhake F. 
Published: April 2011

Abstract
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 &lt;10 minutes processing time (cold ischemia), in which AKT and MAPK phosphorylation could be accurately detected, where the pMAPK assay was more susceptible to time-dependent decrease in signal than the pAKT assay. In line with this, breast cancer samples obtained from immediately fixed fine needle punch biopsies (n=19) consistently showed strong staining for pMAPK and pAKT, while signals clearly decreased in the corresponding routinely processed surgical resection specimens. Knowing the appropriate processing conditions for multiplexed assessment, we studied pathway activation patterns in a series of invasive breast cancer (n=37) of commercially acquired samples, for which the provider (Indivumed, Hamburg, Germany) guarantees a cold ischemia time &lt;10 minutes and highly standardized, tightly controlled embedding process. 1/37 cases clearly showed predominant AKT activation, 2/37 cases had predominant MAPK activation, and 8/37 cases exhibited concomitant activation of both pathways throughout the tumor, indicating constitutive pathway activity. The remaining 26 cases showed a striking heterogeneity of pathway activation patterns, some with marked pMAPK signals at the interface of tumor to stromal/inflammatory cells, suggesting pathway activation in response to microenvironment interaction. Consistently, activated macrophages showed a strong pMAPK signal. The predominantly pAKT activated case was negative for PTEN by IHC and qRT-PCR. The pMAPK activated cases were ER/PR positive and HER2 negative.
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.