466 The number of commercially available PSSAs has steadily grown, as has the number of publications utilizing these reagents in immunohistochemical applications (Fig. 1). Despite this growth, there are still many unan- swered questions about the robustness and reproducibility of immunohistochemical assays of protein phosphorylation, which will be discussed. The focus will be on immunohis- tochemical studies of human solid tumors over the past 5 years. Studies reviewed include novel morphological observations on cellular signaling pathway activation, as well as the potential for PSSAs to provide prognostic infor- mation, prediction of response to targeted therapies, and post-treatment assessment of response to therapy. Due to space limitations only a sampling of the large body of work is discussed here. The dream In the realm of solid tumor assessment, the dream oVered by PSSAs is the ability to provide robust multiparameter measurements of intracellular signaling pathway status, in a way that provides prognostic, predictive, and/or therapeutic information to the clinician. An example of a multipara- meter panel of PSSAs applied to a human glioblastoma is shown in Fig. 2 (unpublished data). In this case, overex- pression of total EGFR was present (compared to no signal in adjacent nonneoplastic brain). In addition, strong tyro- sine phosphorylation of the receptor was found at Tyr1173 but only focal weak phosphorylation at Tyr1068. Down- stream kinases ERK and mTOR show strong cytoplasmic Fig. 1 Continuous growth of publications with immunohistochemical applications of PSSAs. A Google Scholar Advanced search (full text search) using the Boolean search: {(“phosphospeciWc” OR “phospho- speciWc” OR “phosphorylation-speciWc” OR “phosphorylation state- speciWc”) AND “immunohistochemistry”} revealed a continuous increase in the annual number of publications including these key phrases in the full text content. The apparent slight decline in 2007 is likely due to the restricted full text access of many journals in the Wrst year after publication 123 Histochem Cell Biol (2008) 130:465–471 phosphorylation in a subset of tumor cells, whereas the transcription factor CREB is strongly phosphorylated in the majority of tumor cells. Phospho-Histone (H3) is an indica- tor of mitotic activity. Phosphorylation of the actin-associ- ated proteins Ezrin/Radixin/Moesin, potential mediators of tumor invasiveness, is strong in all cells. Finally, a generic anti-phospho-tyrosine antibody is used to assess the general preservation of tyrosine phosphorylation in this tumor sam- ple. In the dream, we can provide quantitative measures of protein phosphorylation, both in terms of percent of protein phosphorylated, as well as in terms of percent cells positive (above a stated threshold). Additionally, the analysis will include information about subcellular localization of the phosphoepitopes under study. All this information is then entered into an algorithm that classiWes the tumor among others of its general type, providing the oncologist with robust and clinically useful information about the activation proWle of signaling networks in this patient’s tumor. But now we must awaken. The reality PSSAs as prognostic markers A central question in the Weld is whether knowledge about the phosphorylation (activation) state of a protein, in addi- tion to its general level of expression, provides nonredun- dant information. Several studies on human solid tumors, most focusing on EGFR family members, have begun to address this question. Because immunohistochemistry for total EGFR failed to demonstrate prognostic importance in non-small cell lung carcinoma (NSCLC), it was hypothe- sized that information about the activation (phosphoryla- tion) of EGFR could reveal prognostic signiWcance. To address this, immunohistochemistry for total and phospho- EGFR as well as phospho-STAT3 was performed on archi- val NSCLC cases. Of the EGFR-expressing tumors, approximately 1/3 showed pEGFR immunoreactivity, but the presence of pEGFR did not provide statistically signiW- cant prognostic information (Cortas et al. 2007). In a similar study on locally advanced breast cancer, total EGFR expression correlated with negative hormone receptor status, and was associated with signiWcantly worse relapse-free survival. However, the authors found no asso- ciation of phospho-EGFR with outcome (Nieto et al. 2007). A large body of evidence indicates that mutated BRAF and NRAS contribute to the genesis and growth of melanomas, presumably by activation of the extracellular signal- regulated kinase (ERK) pathway. The presence of phos- phorylated ERK1/2 was analyzed in 170 melanomas with established NRAS/BRAF mutational status. The authors found notable heterogeneity of phospho-ERK staining with