Histochem Cell Biol (2008) 130:465–471 b.i.d.). This study should serve as a model for future work in animal models as well as in the more complex and hetero- geneous human samples (D’Andrea et al. 2005). A neoadjuvant trial of rapamycin was recently com- pleted in patients with recurrent glioblastoma, whose tumors lacked expression of the tumor suppressor PTEN. Several interesting observations were noted. The magnitude of mTOR inhibition (measured by S6 protein phosphorylation) varied substantially. Also, rapamycin treatment led to Akt activation in seven patients, which was associated with shorter time-to-progression (Cloughesy et al. 2008). Radiation therapy has profound eVects on cell signaling pathways, underlying some aspects of its therapeutic acti- vity. A study of ERK phosphorylation in colorectal cancer revealed that most patients who were phospho-ERK posi- tive before RT converted to ERK negativity after RT (Corn et al. 2008). Although intriguing, further studies are needed to determine if measurement of phospho-ERK can be pre- dictive of response or an indicator of the eVect of to radia- tion therapy. The use of surrogate cells, obtained noninvasively, to assess chemotherapeutic activity would be a boon for oncologists and much preferred by patients. Buccal mucosa cells were tested as a model in which to assess geWtinib activity in patients with advanced non-small cell lung can- cer (Loprevite et al. 2007). Pretreatment and post-treatment cells were tested for expression of p-EGFR, p-ERK and p-AKT. Baseline p-AKT expression, but not that of EGFR, p-EGFR, and p-ERK showed a potentially predictive role, although the study did not reach statistical signiWcance. Further work on buccal cells and other possible surrogate cells will be a fruitful area. New technologies Quantitative image analysis Like any immunohistochemical assay, a frequent bottle- neck is the unbiased scoring of staining intensity, percent positive cells, and subcellular localizations. Application of an immunoXuorescence-based quantitative image analysis system (AQUA) to the study of AKT phosphorylation (Ser473) revealed the marker as a strong negative prognos- tic factor in oropharyngeal squamous cancer(Yu et al. 2007). Future applications of this and other image analysis methods using ratio imaging of phospho- to total protein immunoreactivity, as achieved with cultured cells (Mandell and Banker 1996) could provide a readout of relative (%) phosphorylation, more appropriate for comparisons of mul- tiple samples in which the total amount of the phosphopro- tein could vary. 469 FRET Fluorescence resonance energy transfer (FRET) involves the transfer of energy from an excited donor molecule to a nearby (<7 nm) spectrally overlapping acceptor. Although most widely applied to the study of interacting engineered autoXuorescent proteins, FRET can be used to measure the proximity of two appropriately labeled antibodies. Proof- of-principle of this method was obtained in cancer cell lines using the acceptor photobleaching Xuorescence resonance energy transfer imaging approach to measure the phosphor- ylation state of EGFR (Keese et al. 2005). In a study on head and neck cancers, a generic anti-phospotyrosine anti- body and an anti-EGFR antibody were successfully used to measure EGFR phosphorylation (Kong et al. 2006). FRET eYciency correlated with worsening disease-free survival but not with overall survival. FRET-based methods are much more technically challenging than traditional immu- nohistochemistry but have some potential advantages. One is the measurement of phosphorylation for proteins for which no good PSSAs are available, by means of a pair of a general (protein-speciWc) antibody and a generic anti-phos- photyrosine, phosphothreonine, or phosphoserine antibody. The other is the inherent quantitative nature of the measure- ments. Finally, the method can potentially be used to mea- sure the binding interactions of any pair of molecules in tissue Wxed tissue samples. The hard work ahead: antibody validation and phosphoepitope characterization An obvious need for PSSAs, as well as for the entire Weld of immunohistochemistry, is improved antibody validation. A good example of the multiple approaches necessary to truly validate antibodies for quantitative immunohisto- chemistry was recently published for the case of C-Met (Pozner-Moulis et al. 2007). Much more work will be needed to bring the validation of a large set of PSSAs. Ide- ally, information gleaned from these types of studies can be made available to the research community via public web- sites, such as the Abminer site hosted by the National Can- cer Institute (http://discover.nci.nih.gov/abminer/). Because reversible protein phosphorylation is inherently labile, the use of PSSAs requires an additional set of assur- ances beyond those for general IHC antibodies. For exam- ple, surgically excised tissues are often kept at ambient temperatures for long periods in the operating room or in surgical pathology labs during the busy workday. A largely unaddressed issue is the relative stability/lability of various phosphoepitopes ex vivo. We initiated a pilot investigation by growing xenografts in nude mice, excising the tumors, and either Wxing immediately, or delaying Wxation for four hours (Fig. 3). We were encouraged that some markers, 123