TGF-b in prostate cancer Reis ST et al. associated with the conversion from a benign state to a malignant state: 1) a reduction or loss of the sensitivity to the inhibitory effects of TGF-b and 2) an increase in the ability to elevate TGF-b expression levels, which in turn promotes carcinogenesis by stimulating extracellular matrix production, promoting angiogenesis, and inhibiting the host immune system.8 To evaluate whether the expression levels of TGF-b1 in prostate cancer cells are associated with prognosis, as predicted by the TGF-b elevation in carcinogenesis, we investigated a putative correlation between TGF-b gene expression and Gleason score, pathological stage, and PSA serum level. PATIENTS AND METHODS Patients This study was conducted using surgical specimens from 79 patients with clinically localized PCa who underwent radical prostatectomy in our institution between 1993 and 2007; patients with a minimum follow-up of five years were randomly selected from a frozen tumor tissue database (Table 1). Benign tissue samples from an additional 10 patients with PCa who also underwent radical prostatect- omy were included in this group. Patients who had undergone other treatments for PCa were excluded. All of the subjects provided their informed consent to participate in the study and to allow their biological samples to be genetically analyzed. Approval for the study was given by the Institutional Board of Ethics (n˚0453/08). We correlated the expression levels of the TGF-b1 genes in each sample with the patient’s Gleason score, pathologic stage according to the TNM 2002 staging system, and serum PSA level (in ng/ml). For the analysis, the pathologic stage was considered as an organ-confined (pT2) or non-organ- confined (pT3) disease. The Gleason score was classified as low grade (,7) or high grade ($7), and preoperative PSA was used to identify patients at high ($10 ng/ml) or low risk (,10 ng/ml) for disease recurrence. In addition, at a mean follow-up time of 60 months, we analyzed the gene expression levels in patients with biochemical recurrence, which was defined as a PSA level .0.4 ng/ml. The control group consisted of tissue specimens from 11 patients with benign prostate hyperplasia (BPH) who presented with lower urinary tract symptoms and had an interest in undergoing surgery (mean age 64¡6.0 years). Table 1 - Age and clinical characteristics of 79 men who underwent radical prostatectomy to treat prostate cancer. Age (years) Mean Min - Max PSA (ng/ml) Mean Min - Max ,10 n (%) $10 n (%) Stage pT2 n (%) pT3 n (%) Gleason Score ,7 n (%) $7 n (%) 63 41 - 79 10.8 2.0 - 37.0 47 (59.5) 32 (40.5) 38 (48) 41 (52) 32 (41.7) 46 (58.3) 1144 CLINICS 2011;66(7):1143-1147 RNA Isolation and cDNA Synthesis All of the tumor samples were obtained from surgical specimens and immediately frozen in liquid nitrogen at -170˚C. A mirror image slide of the frozen fragment was stained with hematoxylin and eosin to verify that the tumor represented at least 75% of the fragment in the cancer patients and that no tumors were present in the control patients with BPH. Total RNA was isolated using the RNAaqueous Kit (Applied Biosystems, CA, USA) according to the manufac- turer’s instructions. The RNA concentration was deter- mined by the ratio of the absorbance at 260 and 280 nm using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, USA). cDNA was generated using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The reactions were incubated at 25˚C for 10 min, followed by 37˚C for 120 min and 85˚C for 5 min. The cDNA was stored at -20˚C until use. Quantitative Real-Time Polymerase Chain Reaction and Gene Expression The expression level of the TGF-b1 gene was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR) with the ABI 7500 Fast system (Applied Biosystems). The target sequences were amplified in a 10 ml reaction volume containing 5 ml TaqMan Universal PCR Master Mix, 0.5 ml TaqMan Gene Expression Assays (TGF-b Hs00852894_m1 and B2M Hs99999907_m1), 1 ml cDNA, and 3.5 ml DNase-free water. The polymerase chain reaction (PCR) cycling conditions were 2 min at 50˚C and 10 min at 95˚C, followed by 40 cycles of 15 seconds at 95˚C and 1 min at 60˚C. The TaqMan endogenous control assay utilized b-2-microglobulin (B2M). We used the DDCT method to calculate the relative expression levels of the two target genes using the formula DDCT = (CT target gene, PCa sample - CT endogenous control, PCa sample) – (CT target gene, BPH sample - CT endogenous control, BPH sample). The fold change in gene expression was calculated as 2-DDCT. Statistical Analyses Quantitative variables were expressed as median values, interquartile ranges (Q1-Q3), and minimum and maximum values. Qualitative variables were expressed as numbers and percentages. We constructed a box plot for a descriptive analysis of TGF-b expression according to pathologic stage, Gleason score, and PSA level. We used the Mann-Whitney test to compare categories. Statistical analyses were performed using GraphPad Prism 3, and significance was set at p#0.05. RESULTS As shown in Figure 1, an analysis of the 79 patients with PCa showed that TGF-b1 was underexpressed (median 6.07610-1-fold) in malignant prostatic tissue as compared to the BPH samples. The TGF-b1 expression levels in relation to the Gleason scores are shown in Table 2. The median TGF-b1 expression levels were significantly higher (p = 0.0065) among patients with a Gleason score $7 (9.4610-1-fold) compared with those patients with a Gleason score ,7 (4.6610-1-fold). With regard to the pathologic stage, the expression of TGF-b1 was similar (p = 0.2514) between stage pT3 patients (median 4.9610-1-fold) and stage pT2 patients (median 6.6610-1-fold). When the results were evaluated according