BIOMARKERS ASSOCIATED WITH HORMONE REFRACTORY PROSTATE CANCER

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Introduction Hormone refractory prostate cancer (HRPC) is the most aggressive and lethal form of prostate cancer with the median survival time of 18 - 24 months [1]. Using genetic mRNA microarray analysis, a panel of 10 genes have been identified as over expressed in hormone refractory prostate cancer (HRPC) tissue specimens, identifying them as potential biomarkers of aggressive disease [2]. The expression of Cell division cycle 2 (CDC2), Ribonucleotidereductase M2 polypeptide (RRM2), and Androgen receptor (AR) were explored in prostate cancer tissue specimens at the protein level using immunohistochemistry (IHC). • Aim • To analyse the expression of CDC2, RRM2 and AR in paired hormone naive and hormone refractory prostate cancer tissue specimens to validate and determine the association of these genes in aggressive prostate cancer at the protein level. • To determine the effect on expression of these proteins in the presence of androgen deprivation. Since androgen receptor is a DNA binding transcription factor, the effect of androgen deprivation itself on the expression of these proteins could represent a confounding factor in either down or up-regulation. Conclusions Biomarkers have been the focus of recent research in many cancers such as breast, colon and prostate [3 - 5]. The pattern of expression observed in the “before” and “after” specimens have validated the results observed in the genetic micro array assay. Included within this panel was androgen receptor, the expression of which is known to be up regulated in advanced prostate cancer [6]. The next phase in this study is to examine the remaining proteins and extend study to include patients on active surveillance to determine the value of these proteins as predictive biomarkers. Results • Methods • GENE MICROARRAY ANALYSIS • Gene array technology (Affymetrics, Santa Clara, California) was used to analyse the RNA expression of micro-dissected epithelial purified (i) organ confined (OCPC) hormone naïve prostate cancer (n=5 individual patients who have undergone radical prostatectomy), (ii) HRPC (n=5 specimens from 3 individual patients with castrate levels of androgen but locally advanced or metastatic disease requiring transurethral prostatectomy to alleviate urinary retention), and (iii) normal prostate tissue derived from organ donors (n=5 individual subjects died from traffic accidents). • IMMUNOHISTOCHEMICAL ANALYSIS Paired tissue samples from with prostate cancer managed with watchful waiting were obtained before and after their prostate cancer had reached a hormone refractory status (n=8). Paraffin embedded blocks were cut using a microtome into 5 μm sections and immunohistochemistry (IHC) staining performed. After non specific protein blocking with horse serum, sections were incubated overnight at 4 °C with a primary monoclonal antibody. A biotinylated secondary antibody (IgG - Anti-mouse/Anti-Goat Envision Kit DAKO™) was incubated for 1 hour at room temperature in a moist chamber. Slides were rinsed with distilled water followed by signal amplification with the addition of avidin-biotin-peroxidase (ABC Vectastain™) followed by 3-3’-Diaminobenzadine chromogen (DAB) (DAKO™) for visualisation. A method control was included in all IHC experiments. Experiments were repeated twice for each primary antibody. No. 027 BIOMARKERS ASSOCIATED WITH HORMONE REFRACTORY PROSTATE CANCER Smith I1,2,6, Sved P2,3, Chan C4, Watson G5, Salomon R 2, Dong Q2 1. Department of Urology, Westmead Hospital. 2. Department of Endocrinology, University of Sydney. 3. Department of Urology, Royal Prince Alfred Hospital. 4. Department of Anatomical Pathology, Concord Hospital. 5. Department of Anatomical Pathology, Royal Prince Alfred Hospital. 6. Department of Urology, Sydney Adventist Hospital. Table 1: Ten genes over expressed in HRPC compared to OCPC and normal prostate tissue Table 1 demonstrates the fold increase expressed as a ratio. Figure 1. Immunostaining for CDC2, RRM2 and AR. Paired specimens from the same patient at hormone naïve (Left) and hormone refractory stages (Right). From figure 1 it can be seen that there was an overall increased expression of CDC2, RRM2, and, AR in the hormone refractory specimens. Poster presentation sponsor