To minimize the selection bias, we compared the clinicopathologic characteristic between patients who were
selected for this study (n = 200) with those who were not selected (n = 903), and no statistically significant difference was found between the two groups. All patients had previously consented for use of their tissues and clinicopathologic data for research. Five-micron serial sections were cut from FFPE BCa tissue blocks. At least one section was stained with hematoxylin and eosin, assessed by a pathologist, and compared to original report. The study was approved by the Ethical Review Ganetespib clinical trial Committee of the Aga Khan University (2390-RO-ERC-12). Survival analysis was performed
on a subgroup of patients with BCa who had follow-up of at least 5 years or more (n = 82). Patients diagnosed during 2002 to 2008 and followed up until December 2013 or death were included for survival analysis. Overall survival (OS) was calculated from the date of diagnosis to the date of last follow-up or death due to any cause. Immunohistochemistry was performed on FFPE sections to assess the expression of AR, pAkt, and pPTEN as described previously with some modifications [32], [33] and [34]. Dako REAL EnVision Detection System, Peroxidase/DAB +, Rb/Mo (Dako, Glostrup, Denmark) CDK phosphorylation was used for immunohistochemical staining. Briefly, 5-μm serial sections were cut from FFPE tissue onto Superfrost slides (Thermo Scientific, Braunschweig, Germany). Sections were deparaffinized in xylene (BDH, Poole, UK) and rehydrated in a graded series of ethanol (Merck, Darmstadt, Germany). Heat-induced antigen retrieval was performed in 10 mM citrate buffer (pH 6.0) for AR (1 hour), pAkt, and pPTEN (30 minutes) in a boiling water bath (Grant Instruments
Ltd., Cambridge, UK). Endogenous peroxidase activity was blocked by immersing slides in 0.3% find more vol/vol H2O2 at room temperature (RT; 25°C) for 10 minutes. Next, anti-human AR antibody (mouse monoclonal IgG, clone AR441; Dako, diluted 1:50) was applied for 4 hours at RT, and anti-human Ser473 pAkt1/2/3 (rabbit polyclonal IgG; Santa Cruz Biotechnology, diluted 1:50) and Ser380/Thr382/383 pPTEN (rabbit polyclonal IgG; Santa Cruz Biotechnology (Santa Cruz, CA), diluted 1:50) were applied for overnight at 4°C onto serial tissue sections from each case. After three washes for 5 minutes each in phosphate-buffered saline (pH7.4) (Gibco, Carlsbad, CA), HRP-labeled secondary antibody was applied for 1 hour at RT. After washing, substrate was added, and DAB was used for visualization. Hematoxylin (BDH) was used for counterstaining, and images were obtained using microscope (Olympus BX41, Tokyo, Japan, DP70 camera).