BMC Cancer 2005, 5:111 http://www.biomedcentral.com/1471-2407/5/111 iltpbFAininryvintgoeeaRu)srlcyTtnr(aosl-aeatinPlsentC1ccoerot1Rofon3lgm,tue)rn;snyoioMenla,gWRneinxdβNpw-(Aarmnhcedt(oslirsanleionoycgebniueelo1lnlaed2orvae)deb;wdlpfsareaotoiogis7omfhi4tnpti01v)5(e10lba3acpnsSoaee,tns-iptep1r5nao0tirlso4(SbaL1faaNt1nen)dCrd;nrapaePs5dg1ciac0e-alSl Analysis of gene expression levels of p503S, p504S and p510S by RT-PCR using β-actin yielded a 740 base-pair band as internal control, in whole blood from 11 patients after radical prostatectomy and androgen ablation (lanes 1 to 11); nega- tive control, no RNA (lane 12); positive control (LNCaP cell line) (lane13); MW (molecular weight). and p510S circulating cells by reverse transcriptase- polymerase chain reaction (RT-PCR) assay of peripheral blood (PB) from patients with CaP. The aim of this study was to investigate whether p503S, p504S and p510S genes are diagnostic or prognostic pros- tate-specific markers for monitoring disease progression and recurrence. Extending our previous research investi- gating biomarkers that are clinically important to distin- guish patients with prostate cancer at risk for early relapse from patients in clinical remission [12-14], in this study, we analyzed by RT-PCR assays p503S, p504S and p510S gene markers in PB from 42 patients with treated or untreated CaP and with benign prostatic hyperplasia (BPH) (controls). Methods Patients and control tissue specimens The 42 participants included in this study were consecu- tively selected from patients treated at the Department of Urology "U. Bracci", University of Rome "La Sapienza". We studied three clinical groups. The first group com- prised 11 patients with CaP (mean age at diagnosis 68.9 years; range 45–89) who underwent androgen ablation (AA) followed by retropubic radical prostatectomy (RP) and bilateral lymphadenectomy between January 1997 and December 1999. Tumors were graded using the Gleason system for histologic grading of CaP: two tumors were Gleason score sum 4, 7 Gleason sum 7 and 2 Gleason sum 8. Tumors were then evaluated clinico-path- ologically using the tumor-node metastatic (TNM) staging system 2002: 7 tumors were organ-confined diseases (pT2) and 4 were extra-prostatic diseases (pT3). Follow- up after treatment ranged from 3–84 months (mean, 27 months). At the time of venipuncture for PB, we distin- guished two subgroups: patients who responded to the first 24 months of treatment without clinical progression; and AA resistant patients who received neoadjuvant androgen deprivation therapy because recurrent disease developed within 24 months after RP. We defined recur- rence by serum PSA levels, transrectal ultrasound (TRUS) and computed tomographic (CT) scan. Men were consid- ered disease free after RP if they had serum PSA levels less than 0.1 ng/ml. The second subgroup comprised 11 patients with elevated serum PSA levels (more than 4.0 ng/ml), who had received a diagnosis of CaP based on TRUS-guided-prostate biopsy (1 tumor was Gleason sum 6 and 10 were Gleason sum 7). All 11 patients had organ- confined disease: 4 were pT1 and 7, pT2. In patients treated for CaP, PB samples were obtained 27 to 84 months (mean 55 months) after treatment ended; in patients who had received a diagnosis of CaP based on clinical and biochemical evidence of malignancy, PB sam- ples were obtained before biopsy and before treatment began. The third group (control group) comprised 20 men 50 years of age or older (mean age 58.7 years; range 45–79) with no known malignancy, with a histologic diagnosis of BPH in tissue obtained by fine-needle biopsy of the prostatic glands and on conventional clinical data (serum PSA levels less than 4.0 ng/ml, and digital rectal examination and TRUS negative for CaP). The control group also included a PB sample from a patient who underwent radical cystectomy for bladder cancer (G3T1) and 6 neoplastic and non neoplastic prostatic tissues; 6 tissues from various organs (adrenal gland, heart, myo- metrium and uterine cervix); and 6 prostatic (SV48, LS147D, SCOV 3 and MCF7) and non prostatic cell lines (PC3 and LNCaP). Reverse-transcription polymerase-chain-reaction (RT- PCR) Total RNA was extracted from whole blood cells and cell lines using the technique previously described [12-15]. Total RNA from frozen tissue was extracted using the pro- tocol developed by Ambion, and detailed in the RNA iso- lation Kit (Ambion Inc., U.S.A). cDNA (250 ng) from each sample was amplified by PCR using the following prim- ers: sense, 5'TGCCCTCGTGACGTTCTTCT3' and antisense 5'TCTTTCTTGATGGCAGGCACTAC3' of P503, 136 bp (GeneBank accession number AF065388 (16) using 35 cycles (94°C for 30", 60°C for 30" and 72°C for 60 sec); sense 5'AAATGGTTATCATTAGGGCTTTTGA3' and anti- sense 5'TTCCTTTTTCACTAGAACCCATTCA3' of P504S, 149 bp (GeneBank accession number 4204097 [17] using 35 cycles (94°C for 30", 55°C for 30" and 72°C for 60 sec); sense 5'TTGAACAGCTACTACGGTCAATGTATT3'and antisense 5' GCAGAGAGCAACCGATGTTTT3' of P510S, 96 bp Page 2 of 7 (page number not for citation purposes)