ABSTRACT: We assessed differential gene expression in tissue specimens of PCa and BPH patients who underwent radical prostatectomy by customized expression microarrays (Agilent). To detect gene signatures of diagnostic and prognostic value, we assessed fresh frozen radical prostatectomy (RP) samples of PCa and BPH patients by transcriptome-wide sequencing and Agilent custom microarrays. We stratified PCa patients according to seven clinical risk groups based on Gleason Score (GS), the presence of regional lymph node metastases (pN) and the occurrence of death of disease (DoD): (i) very low risk (group V: GS<7, pN0), (ii) low risk (group L: GS=7, pN0), (iii) medium risk (group M: GS<=7, pN1), (iv) high risk survivors without lymph node infiltration (group H-s: GS>7, pN0), (v) high risk non-survivors without lymph node infiltration (group H-d: GS>7, pN0, DoD), (vi) high risk survivors with lymph node infiltration (group H+s: GS>7, pN1), (vii) high risk non-survivors with lymph node infiltration (group H+d: GS>7, pN1, DoD). For high risk groups adjacent tumor-free prostate tissue samples were obtained (groups H+sf, H-sf, H+df, H+df). The control group contained patients with benign prostate hyperplasia (group C). Information on the course of the disease, survival of the patients and the cause of death were obtained from the general practitioners or treating urologists or from records of the regional tumor registry. Clinicopathological parameters were obtained by routine histopathological examination of the surgical specimens. Serum levels of the prostate-specific antigen (PSA) were determined preoperatively (Abbott, Wiesbaden, Germany). Frozen tissue samples were embedded in Tissue-Tek OCT-compound (Sakura Finetek GmbH, Staufen im Breisgau, Germany) and fixed on metal indenters by freezing. Cryo-sections were prepared using a cryo-microtome (Leica Biosystems, Nußloch, Germany) equipped with a microtome blade C35 cooled to -28 °C. For every tissue three stacks of consecutive cryo-sections (10 µm) were generated, each bordered/flanked by HE-stained cryo-sections (4 µm), which were evaluated by a pathologist with regard to tumor and stroma cell content. PCa tissue stacks had to be flanked on either side by sections containing at least 50 % tumor cells to be used for further analyses. Sections of the tissue stacks were immediately transferred to 1 ml Qiazol (Qiagen, Hilden, Germany) and stored at -80 °C until RNA isolation.
