468 Akt and ERK phosphorylation was investigated by immunohistochemistry in patients with with lymph node- negative breast cancer. P-Akt, but not p-ERK correlated with HER-2/neu overexpression and was related to reduced tumor apoptosis. No association was found between pAkt or pERK with cell proliferation assessed by Ki67 and mitotic count. P-Akt status proved to be a statistically sig- niWcant independent prognostic factor (Schmitz et al. 2004). Similarly, a study on head and neck squamous cell carcinomas (HNSCC) found that Akt phosphorylation (Ser 473) correlates with poor prognosis (Massarelli et al. 2005). Numerous studies have documented the overexpression of the p53 tumor supressor protein in human tumors. P53 function is regulated not only by protein levels, but also by phosphorylation state, which determines stabilization and protection against mdm-2. With this in mind, a study inves- tigated phosphorylation at Ser15 and Ser392 in squamous carcinoma and precursor lesions. Although not tested for prognostic value, the Wndings revealed diVerential staining patterns with the two PSSAs, suggesting that high levels of Ser392 phosphorylation is an early change in the pathogen- esis of SCC (Matsumoto et al. 2004). However, a study on gastroesophageal adenocarcinomas showed no correlation of p53 phosphorylation (Ser15) with p53 mutation, and no correlation with survival. In this setting, analysis of p53 phosphorylation did not appear to have obvious clinical utility. (Puhringer-Oppermann et al. 2006). A study on prostate cancers examined the phosphoryla- tion Akt and relevant downstream substrates glycogen syn- thase kinase 3, mTOR, and the forkhead transcription factor-like 1 (FKHRL1). Although most tumors showed elevated phosphorylation of PKB/Akt in the malignant tis- sue compared with the surrounding benign tissue, only increased phosphorylated-FKHRL1 levels correlated with clinical progression (Jendrossek et al. 2008). Immunohisto- chemical analysis of renal cell carcinomas pAkt and pS6 was found to be independent prognostic factors (Pantuck et al. 2007). Because activation of Akt was documented in hormone- refractory prostate cancer (HRPC) and can result in phos- phorylation of the androgen receptor (AR), IHC analysis of the phosphorylation status of Akt and AR was tested in a series of prostate cancer tumors. In the hormone-refractory cases, phosphorylated AR (pAR) was associated with worse prognosis (McCall et al. 2008). Endocrine therapy is an important treatment option for women with estrogen receptor (ER)-positive breast cancer. Information about the activation state of ER could in theory provide more clinically useful information than mere protein expression. A study of ERalpha serine (Ser) 118 and ERalpha Ser167 revealed that low phosphorylation of ERalpha Ser118 and high phosphorylation of ERalpha Ser167 were associated with signiWcantly improved 123 Histochem Cell Biol (2008) 130:465–471 disease-free and overall survival. (Yamashita et al. 2008)This is a good example of another overlooked aspect, namely careful assessment of dephosphorylation of speciWc epitopes. Overexpression or activation of the transcription factor c-Jun has been implicated in the pathogenesis of several types of cancer. Phospho-c-Jun staining was correlated with signiWcantly shorter overall survival (Kuo et al. 2006). PSSAs as predictive markers A large number of studies have examined the role of immu- nohistochemical tests forErbB-2 (Her2/Neu) as predictive markers for therapeutic responses of breast cancer to anti- Her2 antibodies, culminating in an FDA-approved test (Thor 2001). A logical hypothesis was that assessment of the activation (tyrosine phosphorylation) of Her2 would provide added predictive information. However, immuno- histochemistry with the anti-phospho-ErbB-2 antibody had no added beneWt to identify those patients most likely to beneWt from increased doses of adjuvant chemotherapy (DiGiovanna et al. 2008). However, a similar study but in a diVerent subgroup of breast cancer patients did Wnd clinical utility of PSSAs. The phosphorylation status of Her-2 and EGFR was assessed in Her-2-overexpressing tumor sam- ples from trastuzumab-treated metastatic breast cancers. The presence of ptyr-1248 Her-2 and ptyr-845 or ptyr-1173 EGFR was a strong predictor response of both to anti-Her2 treatment. ptyr-845 EGFR and ptyr-1248 Her-2 were both independent predictors of progression-free survival (Hudel- ist et al. 2006). The rapid growth of clinical trials of tar- geted chemotherapeutics will make the development and validation of reliable PSSA IHC testing a high priority for the next decade. PSSAs as indicators of therapeutic eVect An exciting area application for PSSAs is the assessment of activity in post-treatment tumor samples. As a Wrst step towards clinical application, animal tests were performed using JNJ-10198409, an inhibitor of PDGFR in a nude mouse xenograft model of human colon cancer. The phosphorylation status of phospholipase Cgamma1 (PLCgamma1) was used as a marker of activation of the PDGFR signaling cascade. By using a pair of antibodies in serial sections, one for total (phosphorylated and unphos- phorylated forms) and the other for the phosphorylated form of PLCgamma1 (ph-PLCgamma1), an immunohisto- chemical ratio assay was achieved. Impressively, the authors showed statistically signiWcant, dose-dependent diVerences in the phospho/total PLCgamma1 ratio among the four treatment groups (vehicle, 25, 50, and 100 mg/kg