Reprobing the blots with antibodies recognizing actin or heat shock protein confirmed loading of approximately equal amounts of total proteins from all lysates tested. Correlations of biomarker immunostaining data with RFS for stage T prostate cancer patients.The logrank test was used for correlation of immunoscore data with patient survival.RFS curves for patients expressing high levels of markers are denoted in black symbols.White symbols indicate patients whose tumors contain lower protein levels, using the median immunoscore for dichotomizing data.Median immunoscores were for cIAP, for cIAP, for XIAP, and for survivin.Significance was calculated based on a logrank test.The prostate glands of a total of TRAMP male mice and agematched control mice were investigated by HE histology.The numbers of animals developing PIN or invasive cancer are indicated at various ages.PIN and cancer lesions were multifocal in nearly all animals in which transformation of prostate epithelium was found.Gleason score data were available for of these tumors, whereas clinical stage information was known for of tumor specimens.In addition to invasive or metastatic cancer, these microarrays contained separate cases of BPH for comparison with cancers.In addition, roughly of tumor specimens on the arrays contained nonneoplastic prostate epithelium adjacent to tumor tissue, permitting additional comparisons of nontransformed epithelium with invasive cancer.Microarrays were immunostained using the aforementioned antibodies specific for cIAP, cIAP, XIAP, or survivin. The specificity of these immunostaining results was confirmed by control stainings performed using either preimmune serum or immune antisera that had been preabsorbed with the relevant immunogens. Immunostaining results were quantified according to the approximate percentage of immunopositive cells and immunointensity on a scale, and then an immunoscore was calculated from the product of the percentage immunopositivity and immunointensity. To characterize the expression of cIAP, cIAP, XIAP, and survivin in TRAMP mice, paraffin sections from male urogenital system were immunostained.Comparisons of untreated tumors with hormonerefractory specimens on the microarray revealed significant correlations of refractory disease with higher cIAP and with lower cIAP and lower survivin. Further evidence of a correlation of higher cIAP expression with aggressive disease was found by comparing the immunoscores of metastases collected at the autopsies from patients, who had undergone androgen deprivation by orchiectomy and had subsequently died of endstage metastatic prostate cancer, revealing higher levels of cIAP protein compared with primary tumors. To more precisely contrast the levels of IAP family protein expression in tumor versus normal prostate tissue, we analyzed skinnyneedle biopsies from a small cohort of men who were uniformly treated with external beam irradiation.During needle biopsy before radiotherapy, several cores of tissue were obtained from each patient, providing casematched tissue samples containing only normal prostatic epithelium for of the tumor biopsies available.Using the normal and tumor specimens derived from these earlystage patients, we evaluated the expression of the cIAP, cIAP, XIAP, and survivin proteins by immunohistochemistry and scored the results as described above. Whereas immunostaining results varied Norgestimate widely among specimens examined, the overall immunoscores for the cancers displayed clear elevations in immunoreactivity when compared with histologically normal specimens. For example, whereas of normal prostate specimens had cIAP immunoscores of, of, thus suggesting that many prostate cancers develop pathological elevations in the levels of this antiapoptotic protein.

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