Combined mRNA expression levels of members of the urokinase plasminogen activator (uPA) system correlate with disease-associated survival of soft-tissue sarcoma patients

For the present study, tumor tissue samples of 78 adult patients with histologically verified soft-tissue sarcomas that have been described in previous studies were used [20, 22]. The study adhered to national regulations on ethical issues and was approved by the Ethics Committee from the Medical Faculty of the Martin-Luther-University Halle-Wittenberg, Germany. All patients gave written informed consent (Institute of Pathology, University of Halle-Wittenberg, Germany and Department of Surgery 1, University of Leipzig, Germany). The median age of the patients at surgery was 59 years (range 22-83 years). The median follow-up time of the patients was 42 months (range 2 to 146 months after primary tumor resection). During that time, 30 patients experienced a locoregional recurrence, seven developed a distant metastasis and 42 of the patients have died. The tumors were staged according to the UICC system. All of the tumor samples were collected before radio- or chemotherapy. Relevant data on the clinical and histomorphological parameters of the STS patients are shown in Table 1 and in Additional file 1 .

Tissue processing, isolation of total RNA, cDNA synthesis, and evaluation of the quantity and quality of the isolated RNA were performed as described [22]. Among the 78 STS patients, the mRNA expression in tumor tissue could be investigated in all cases for uPA and PAI-1 expression, in 77 cases for expression of wild-type uPAR (uPAR-wt) and in 76 cases for expression of the uPAR splice variant uPAR-del4/5.

For quantification of uPA, PAI-1, uPAR-wt and uPAR-del4/5 mRNA, gene-specific hybridization probes and highly sensitive RT-PCR assays (LC FastStart DNA Master Hybridization Probes, Roche Diagnostics, Mannheim, Germany) applying LightCycler technology (Light Cycler 1.0; Software Version 3.5, Roche) were used as previously described [14, 23]. Briefly, the PCR assays were performed using 2 μl of a 1:10 dilution of the respective cDNA products. Primer sequences, concentration of primers and probes as well as PCR conditions for amplification are shown in Additional file 2. All HPLC-purified primers were purchased from TibMolBiol (Berlin, Germany). Five-log-range calibration curves were generated for each PCR run using eight glass capillaries coated with defined numbers of linearized plasmid molecules (10¹ - 10⁶ molecules), carrying either the uPA, the PAI-1 or the uPAR (wild-type or -del4/5) cDNA (Roboscreen, Leipzig, Germany). All measurements were performed using aliquots of the same cDNA dilutions within short time intervals to ensure comparable conditions. The cDNA samples were quantified at least in duplicate in independent PCR runs for the appropriate marker transcripts. The mean values of all measurements were used for further calculations. The levels of specific mRNAs were evaluated relative to the average expression levels of the medium abundance housekeeping gene hypoxanthine guanine phosphoribosyltransferase (HPRT) as described [24]. Relative mRNA expression ratios (amol of appropriate marker per amol HPRT) were used for all further calculations and statistical analyses.

The levels of significance between continuous variables of the biological markers were calculated with Spearman's rank correlation test (r_s). The relationship between biological marker expression levels and clinical and histomorphological parameters was evaluated using non-parametric Mann-Whitney or Kruskal-Wallis tests. For survival analyses the disease-associated survival of STS patients was used as the follow-up end point. The disease-associated survival was defined as the time from the day of primary surgery to tumor-related death of the patients. Statistical analyses of the association between mRNA expression of uPA system components and prognosis were performed by Kaplan-Meier analyses (log-rank tests) and using the Cox proportional hazard regression model. The multivariate Cox regression model was adjusted for the following known clinical prognostic factors in STS patients: tumor stage, histological subtype, type of tumor resection, and tumor localization. All calculations were performed using the SPSS 17.0 program (SPSS-Science, Chicago, IL). P-values < 0.05 were considered statistically significant.

The expression of uPA and PAI-1 mRNA was determined in 78 cases, and the expression of uPAR-wt and uPAR-del4/5 mRNA was determined in 77 and 76 STS samples, respectively (Table 1). The normalized mRNA expression values of uPA, PAI-1, uPAR-wt and uPAR-del4/5 ranged from 0-16.2 (median 0.23; 25th and 75th percentile 0.055 and 1.03, respectively), from 0-181.9 (median 0.73; 25th and 75th percentile 0.10 and 3.08, respectively), from 0-24.1 (median 0.18; 25th and 75th percentile 0.044 and 1.15, respectively), and from 0-0.29 (median 0.004; 25th and 75th percentile 0.0001 and 0.012, respectively), respectively. For statistical analysis, the median value was set as the cut-off point to separate STS patients in groups with low or high tissue mRNA expression.

First, the correlation between the mRNA expression levels of the biological markers was analyzed using Spearman's rank correlation. In general, all factors displayed a strong correlation with each other with r_s-values of approximately 0.80; uPA mRNA values significantly correlated with PAI-1 (r_s = 0.82, P < 0.001), with uPAR-wt (r_s = 0.81, P < 0.001) and with uPAR-del4/5 (r_s = 0.74, P < 0.001) mRNA levels. In addition, significant correlations were found between uPAR-wt mRNA levels and both PAI-1 and uPAR-del4/5 mRNA values (r_s = 0.85, P < 0.001, and r_s = 0.82, P < 0.001, respectively) as well as between PAI-1 and uPAR-del4/5 mRNA values (r_s = 0.78, P < 0.001).

The relationship between the mRNA expression levels and the clinical and histomorphological parameters of the STS patients is summarized in Table 1. A significant association was observed between mRNA expression levels of PAI-1 (P = 0.037), uPAR-wt (P = 0.044) and uPAR-del4/5 (P < 0.001) and the histological subtype of STS patients. High mRNA expression levels of these three markers (and of uPA) were observed, especially in leiomyosarcomas. Furthermore, mRNA levels of PAI-1 (P = 0.017) and uPAR-del4/5 (P = 0.003) were significantly related to tumor grade, and high uPAR-del4/5 mRNA values were associated with a higher tumor stage in STS patients (P = 0.031). In contrast, mRNA levels of uPA did not differ significantly between tumors in relation to clinical and histomorphological parameters (Table 1).

The association of clinical and histomorphological parameters and tumor biological factors with disease-associated survival is presented in Table 2. In univariate Cox regression hazard analysis, two of the clinical parameters, tumor stage and tumor resection status were significantly related to prognosis, i.e. patients with high stage tumors or with tumors that were not completely resected (R1) had, as expected, a higher risk for tumor-related death (Table 2). No significant association between uPA, PAI-1, uPAR-wt and uPAR-del4/5 mRNA values with disease-associated survival of STS patients was observed in both univariate (Table 2) and multivariate Cox regression analyses (data not shown).

We next performed analyses in the subgroup of STS patients with complete tumor resection (R0) who were at lower risk for tumor-related death compared to the patients whose tumors were not completely resected (R1, Table 2; [25]). We assessed whether some of the biological markers might add prognostic information for patients' survival in the R0 subgroup of STS patients. In R0 patients, survival time is inversely significantly correlated with expression of PAI-1 mRNA (r_s = -0.337, P = 0.014) and of uPAR-del4/5 mRNA (r_s = -0.346, P = 0.013) by bivariate correlation analysis using Spearman's Rho test (Additional file 3: A). Furthermore, using linear regression analysis about a third of patients with lower expression rates for both markers showed the best survival (Additional file 3: B). Therefore it was implicated to apply the 33% percentiles as cut-off points for R0 patients according to their mRNA expression levels, i.e. for PAI-1 (33% percentile: 0.228) and for uPAR-del4/5 mRNA expression (33% percentile: 0.001). Accordingly, R0 patients (n = 52) with mRNA values in the range of the 0% to the 33% percentile were allocated to the low expressing group, and all other patients with mRNA values above the 33% percentile (>33% to 100%) were designated as the high expressing group. We found an association between high PAI-1 and uPAR-del4/5 mRNA expression levels and an increased risk of tumor-related death for R0 patients using multivariate Cox regression analysis with relative risks (RR) of 6.55 (95%CI = 1.0-44.5) and 6.00 (95%CI = 0.8-47.1), respectively, with a trend towards significance (P = 0.054 and P = 0.088, respectively; Table 3). A moderately increased risk of tumor-related death in R0 patients was also observed for high uPA and uPAR-wt mRNA values (RR = 3.53, 95%CI = 0.7-16.8, and RR = 2.66, 95%CI = 0.5-14.4, respectively). However, this risk was not significantly different than for R0 patients expressing low uPA or uPAR-wt mRNA levels (Table 3).

Finally, we assessed whether a combination of PAI-1 and uPAR-del4/5 mRNA values might improve the prognostic impact for patients' survival. Again, the mRNA expression levels of both PAI-1 and uPAR-del4/5 were divided into groups with low (0-33% percentile) versus high (> 33% to 100% percentile) values, respectively. In multivariate Cox regression analysis, we found that patients with low mRNA values of both factors were characterized by a much better disease-associated survival than patients with tumors in which one or both mRNA values were high (Figure 1). R0 patients with high PAI-1/uPAR-del4/5 (n = 30) revealed a significant 19-fold increased risk of tumor-related death (RR = 19.1, 95%CI = 1.1-335.3, P = 0.044) compared to R0 patients who showed low PAI-1/uPAR-del4/5 mRNA expression levels (n = 13). Thus, a pronounced additive effect on prognosis of R0 patients was identified when combined PAI-1/uPAR-del4/5 mRNA expression levels in tumor tissue were used. The risk of tumor-related death for R0 patients with a low/high mRNA expression (high PAI-1/low uPAR-del4/5 and low PAI-1/high uPAR-del4/5) was not significantly different from that for R0 patients with a low PAI-1/uPAR-del4/5 mRNA expression in their tumors. We did not observe any significant correlation for either group with the disease-associated survival of all STS patients or for the R1 patients. The combination of other factors did also not provide any additional prognostic information.