Cancer/Testis antigens as potential predictors of biochemical recurrence of prostate cancer following radical prostatectomy

Prostate tissue specimens from 72 clinically localized PCa cases with follow-up data (29 without recurrence and 43 with recurrence) and 17 clinically localized PCa without follow-up data were collected and frozen at the time of radical prostatectomy (RP), from 1993 to 2007, at the Johns Hopkins Hospital. Twenty histologically normal samples of prostate tissues were collected from cancer-free regions of surgical specimens obtained at the time of RP. The tissue specimens were processed as described previously before RNA extraction [13]. OCT-embedded frozen tissue blocks were manually trimmed to enrich the content of target tissue lesions (e.g., normal or cancer) prior to sectioning and RNA extraction. For tumor samples, tumor cells made up more than 70% of the tissue content (calculated by averaging the % tumor content in the first and last sections) in all cases [14]. Metastatic PCa tissues (n = 21) were collected from soft tissue metastasis of patients who died from PCa, as part of the Johns Hopkins Autopsy Study of lethal PCa [15]. Autopsy RNA specimens had high quality as assessed by RNA index number (RIN), and were prepared and processed as previously described [16, 17]. Twenty normal prostate tissues, 17 localized PCa without follow-up data and 21 metastatic PCa samples were used to validate the microarray data obtained from database (Additional file 1). Seventy two localized PCa samples were used to investigate whether selected CTAs were associated with disease recurrence following RP. The use of surgical and autopsy specimens for molecular analysis was approved by the Johns Hopkins Medicine Institutional Review Boards. Biochemical recurrence was defined as a postoperative elevation of serum PSA (0.2 ng/ml or greater) after RP [18, 19]. The endpoint of the follow-up in this study was the time to biochemical recurrence.

First strand cDNA was made from 0.5 μg RNA using iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., Hercules, CA) following the manufacturer's protocol in a total volume of 20 μl. All primers and probes (Additional file 2) were obtained from Integrated DNA Technologies (Coralville, IA). Quantitative singleplex and multiplex real-time PCR were carried out with the CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.). The singleplex PCR reactions were performed with 0.2 μl of cDNA template in 25 μl of reaction mixture containing 12.5 μl of iQ SYBR Green Supermix (Bio-Rad Laboratories, Inc.) and 0.25 μmol/L each primer. PCR reaction was subjected to hot start at 95°C for 3 minutes followed by 45 cycles of denaturation at 95°C for 10 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 1 minute. Quantitative multiplex real-time PCR reaction was carried out with 0.2 μl of cDNA template in 25 μl of reaction mixture containing 12.5 μl of iQ Multiplex Powermix (Bio-Rad Laboratories, Inc.), 0.3 μmol/L each primer and 0.2 μmol/L probe. The reaction was subjected to hot start at 95°C for 3 minutes followed by 45 cycles of denaturation at 95°C for 10 seconds, and annealing and extension at 60°C for 60 seconds. Amplification of β-actin was used as a control for RNA integrity for all samples. We confirmed that almost identical results were obtained when using TATA binding protein as a reference gene instead of β-actin (data not shown). A reference RNA from pooled cells and prostate tissue were included on each plate to provide standardization across PCR plates. Analysis and fold-change differences were determined using the comparative CT method. Fold change was calculated from the ΔΔCT values with the formula 2 ^-ΔΔCT.

CTA expression was normalized by dividing each individual value by their respective β-actin value. Distributions of normalized CTAs were compared among categories of prognostic factors using the Wilcoxon test (for binary factors) or Kruskal-Wallis test (for factors with more than 2 categories). Correlations between the normalized CTAs and other continuous variables were evaluated by linear regression and the Pearson correlation coefficient. Normalized CTAs were dichotomized at the median of the distribution among prostate cancer patients who did not have a recurrence. Risk of recurrence associated with CTAs was evaluated in univariate and multivariable proportional hazards models. All statistical analyses were performed with SAS v9.2 (SAS Institute, Cary, NC).

By mining publicly available microarray data from the Gene Expression Omnibus http://​www.​ncbi.​nlm.​nih.​gov/​geo in conjunction with our own data [12], we first selected candidate CT-X and non-X CTAs for investigation. As described in Additional file 1, the CT-X antigens (SSX2, CSAG2, MAGEA2, and MAGEA12) were highly up-regulated only in metastatic PCa but were not expressed in benign and primary PCa. The highest expression of non-X CT antigens (CEP55, NUF2, PBK and TKK) was seen in metastatic PCa similar to the CT-X antigens; however the non-X CT antigens were also up-regulated in primary PCa compared to benign prostate disease. Interestingly, a member of the CT-X antigen family, PAGE4, was highly expressed in benign and primary PCa but not in metastatic PCa.

To validate the microarray data, we performed quantitative real-time PCR using samples from normal prostate (n = 20), clinically localized PCa (n = 17) and metastatic PCa (n = 21) (Figure 1). Consistent with the microarray data, all of the CTAs with the exception of PAGE4 were highly expressed in metastatic disease. Furthermore, the expression of four non-X CT antigens (CEP55, NUF2, PBK and TKK) was significantly different between normal prostate and clinically localized PCa. In contrast, there were no significant differences between normal prostate and clinically localized PCa for the four CT-X antigens namely, SSX2, CSAG2, MAGEA2, and MAGEA12. However, PAGE4 showed the highest expression in normal prostate but was significantly down-regulated in metastatic samples.

The aim of this study was to determine if CTAs are associated with disease recurrence after radical prostatectomy (RP). Since the following five CTAs, CEP55, NUF2, PBK, TKK and PAGE4 showed differential expression in both clinically localized PCa and metastatic PCa compared to normal prostate, we selected these five CTAs for developing a multiplex PCR assay that has the potential to stratify patients with and without PCa recurrence after RP.

In order to characterize the selected CTAs, we analyzed 72 samples from PCa patients who underwent radical prostatectomies for their disease (29 patients without recurrence and 43 patients with recurrence). Characteristics of the prostatectomy patients are described in Table 1. In this cohort, 36% of the patients had Gleason 8-10 and a fairly large fraction (60%) experienced biochemical recurrence within a median of 1 year follow-up (range, 1 to 8 years), indicating that this cohort was a very high risk cohort. As shown in Figure 2A-D, CEP55 (p < 0.0001), NUF2 (p < 0.0001), PBK (p = 0.0045) and TTK (p = 0.0386) were significantly higher in prostate samples from patients with recurrent disease. In contrast, PAGE4 (p = 0.0019) was significantly lower in the same patients (Figure 2E). However, there was no significant difference in the CT-X antigens (SSX2, CSAG2, MAGEA2, and MAGEA12) between patients with or without recurrence (data not shown).

We next compared CTA expression patterns with prognostic variables such as the age of patients, preoperative PSA, Gleason grade, and tumor stage. All of the CTAs with the exception of TTK, were significantly correlated with the prostatectomy Gleason score (CEP55; p = 0.0057, NUF2; p = 0.0051, PBK; p = 0.0295, PAGE4; p = 0.0236); however none were correlated with age, preoperative PSA and tumor stage.

Kaplan-Meier analyses were performed with dichotomized CTA expression (according to the median) and the time parameter of biochemical-free survival. Higher expression of CEP55 and NUF2 was significantly correlated with shorter biochemical recurrence-free time (Figure 3A, B). In contrast, higher expression of PAGE4 was significantly correlated with longer biochemical recurrence-free time (Figure 3C). In univariate proportional hazards models, CEP55 (HR = 3.59, 95% CI: 1.50-8.60), p = 0.004; NUF2 (HR = 2.28, 95% CI: 1.11-4.67), p = 0.024; and PAGE4 (HR = 0.44, 95% CI: 0.21-0.93), p = 0.031 were significantly associated with the risk of PCa recurrence (Table 2). Multivariable proportional hazards models evaluated the effect of established prognostic factors including age, PSA, pathologic stage, and prostatectomy Gleason score; only the latter was statistically significant in the multivariable model. Each CTA was tested individually in a multivariable model that also adjusted for prostatectomy Gleason score (Table 3). None of the CTAs retained statistical significance after adjustment for prostatectomy Gleason score, indicating that at least in this cohort the CTAs are not independent predictors of biochemical recurrence.