Histochem Cell Biol (2008) 130:465–471 467 Fig. 2 An example of a panel of phosphorylation state-speciWc anti- bodies applied to a human glioblastoma multiforme. a Overexpression of total EGFR is apparent [(compared to little or no signal in adjacent nonneoplastic brain, (not shown)]. In addition, strong tyrosine phos- phorylation of the receptor was found at Tyr1173 (b) but only focal weak phosphorylation at Tyr1068 (c). Downstream intracellular kinas- es ERK (d) and mTOR (e) show strong cytoplasmic and phosphoryla- only a minority of tumor cells positive in most cases. More- over, the percentage of phospho-ERK-positive tumor cells did not correlate with prognosis (Houben et al. 2008). A frequently overlooked phenomenon is staining of non- tumor stromal cells and the vasculature with PSSAs. A large body of evidence points at the tumor microenviron- ment as critical to oncogenesis (Roskelley and Bissell 2002). In addition, vascular-targeted therapies are now in common use, making PSSA assessment of stromal cell and vascular cell signaling a possibly ripe area of investigation. Mouse xenograft studies were performed with tumors that produced TGF-alpha, stimulating endothelial cells to express and activate EGFR (Kuwai et al. 2008). EGFR blockade in these tumors lead to endothelial cell apoptosis and subsequent tumor necrosis. Importantly, tumor xeno- grafts in which TGF-alpha was knocked down with shRNA did not stimulate endothelial EGFR activation, and these tumors were resistant to therapy. Based on this and other similar studies, the assessment of human tumor endothelial cell EGFR, PDGFR or VEGFR activation status could pro- vide clinically valuable information, but to date has not been speciWcally analyzed. tion in a subset of tumor cells, whereas the transcription factor CREB (f) is strongly phosphorylated in the nuclei of most tumor cells. Phos- pho-Histone H3 (g) is a marker of all cells in mitosis. Phosphorylation of the actin-associated proteins Ezrin/Radixin/Moesin, potential medi- ators of tumor invasiveness, is strong in all cells (h). Finally, a generic anti-phospho-tyrosine antibody reveals the general preservation of tyrosine phosphorylation in this tumor section Because the activation of mitogen-activated protein kinase (MAPK) and members of the Akt pathway can pro- mote glioblastoma cell proliferation, survival, and resis- tance to radiation, assessment of signaling pathway activation was undertaken in a series of these tumors. Ele- vated p-MAPK was most strongly associated with poor response to radiotherapy. Elevated p-mTOR, p-p70S6K, and p-MAPK were associated with shorter survival. How- ever, only p-MAPK proved to be an independent prognostic factor after factoring for other clinical variables (Pelloski et al. 2006). Another study from the same group found that among tumors lacking EGFRvIII expression (a truncated and mutationally activated form of the receptor), the status of phospho-AKT and phospho-ERK was prognostically sig- niWcant. However, when EGFRvIII was expressed these markers did not provide additional prognostic power (Pelloski et al. 2007). It seems likely that the EGFRvIII- negative tumors represent a more heterogeneous group from the standpoint of molecular pathogenesis and thus beneWt more from subclassiWcation using phospho-ERK and phospho-AKT assessment. 123