BMC Cancer 2005, 5:111 http://www.biomedcentral.com/1471-2407/5/111 Table 2: p503S, p504S and p510S positive cells in peripheral blood of patients with prostate cancer (CaP) in relation to Gleason score and TNM stage. (Continued) Group 2 (untreated CaP) Total (n = 11) Gleason score 6–7 (n = 11) TNM Organ-confined disease (n = 11) T1 (n = 4) T2 (n = 7) 2 (18.18) 0 2 (28.6) 9 (81.81) 3 (75 6 (85.7) 11 (100) 4 (100) 7 (100) (GeneBank accession number AF071202 [18] using 35 cycles (94°C for 30", 57°C for 30" and 72°C for 60 sec) by Platinum Taq DNA polymerase (Gibco BRL), accord- ing to the manufacturer's protocols. The integrity of RNA was checked in a preliminary PCR reaction for a human β- actin 743 base-pair (bp) fragment [19]. Each RT-PCR experiment included a non retrotranscribed sample as a negative control and c-DNA from LNCaP cell lines as a positive control. PCR-amplified P510S sequence specifi- city was checked by direct sequencing of the amplified product by the automated DNA sequencer A.L.F. (Phar- macia, Freiburg, Germany). The sequencing reaction was run on an Abby Applied 377 Sequencer (Applied Biosys- tems). Band identity was verified by comparison with the genomic and cDNA P510S sequences at the GeneBank using the BLAST program. Results RT- PCR analysis amplified p5103S-, p504S- and p510S- mRNA in PB from nearly all patients who underwent AA and RP, who had recurrent disease and p503S and p504S in only 2 (40%) of the 5 patients without recurrence (Fig. 1; Table 1). Comparing the RT-PCR findings with the his- tological Gleason scores we found p503S- and p504S-pos- itive cells in a higher percentage of patients with higher- grade Gleason scores than lower-grade scores. P503S were more often expressed in extraprostatic disease (pT3N1 and pT3N2) and p504S in organ-confined disease (pT2N0). The p510S gene was amplified in all patients regardless of the clinical subgroup (Table 2). Two samples from 11 untreated patients with biopsy- proven CaP contained p503S mRNA. This gene was expressed significantly less frequently than p504S and p510S (18% vs 81 and 100% (P <.01, by chi-square test) (Fig. 2; Table 1). PB from the control patient with non prostate malignancies, who underwent radical cystectomy for transitional-cell carcinoma (G3T1), contained p504S- and p510S-but no p503S-positive cells (Fig. 2, lane 12). Nearly all untreated patients with Gleason score 6–7 bAiacFnfeyintglelaRurlcyTnrabs-aeriPlsocC2icponosRfoyngmtue(rslanoaienol,gefeisnxtβh1p-weartcehbotsoilsna1liedo1ydnb)ielaolrednov(edldedaolnsfarneoe71fm42p0a)51t0ba1ies3anpsSceta,o-tpwpnie5atint0rihrto4sSlbtrwaaannintddhsiatpCsi5oa1nP0alS 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 with CaP after biopsy (lanes 1 to 11) and one patient with transitional cell carcinoma of the bladder (lane 12) as control. Negative control (no RNA) (lane 13); positive control (LNCaP cell line) (lane 14); MW (molecular weight). tumors and organ-confined disease had p504S- and p510S-positive cells, whereas only 2 of the 11 patients had p503S-positive cells. p503S-positive cells were detectable in patients with stage pT2 and undetectable in those with stage pT1 disease. p510S-positive cells were expressed in similar percentages of patients with pT1 and pT2 disease (Table 2). In PB samples from the control group (BPH), PCR ampli- fied more p504S- and p510S- than p503S-positive cells (55 and 75% vs 25%) (Fig. 3). Nearly all the prostatic and non prostatic-tissues and cell lines used as controls expressed p503S-, p504S-, and p510S-mRNA (Table 1) (Fig. 4). DNA sequencing of the amplified 96 bp fragment confirmed the specificity of these primers for the p510S gene (Fig. 5). Page 4 of 7 (page number not for citation purposes)