Bache et al. BMC Cancer 2010, 10:132 http://www.biomedcentral.com/1471-2407/10/132 Page 3 of 8 Table 2 Primer sequences and primer locations applied for qRT-PCR analysis of survival. A p-value of less than 0.05 was con- sidered to be statistically significant. gene HPRT OPN primer sense antisense sense antisense sequence 5’ ! 3’ 5’-TTGCTGACCTGCTGGATTAC-3’ 5’-CTTGCGACCTTGACCATCTT-3’ 5’-TGGCCGAGGTGATAGTGTG-3’ 5’-CGGGGATGGCCTTGTATG-3’ Localization 309-328 551-570 555-573 686-703 Karlsruhe, Germany). The cDNA synthesis was per- formed as described previously [28]. The mRNA levels of OPN and the housekeeping gene, hypoxanthin phos- phoribosyl-transferase (HPRT), were quantified using Quantitect SYBRGreen PCR kit (Qiagen, Hilden, Ger- many) in a Rotorgene 3000 thermal cycler (LTF Labor- technik, Wasserburg, Germany). The OPN and HPRT transcripts were amplified with primer pairs of Table 2. The PCR cycling conditions included incubation for 15 min at 95°C, followed by 40 cycles of 30 sec at 95°C, 30 sec at 60°C and 30 sec at 72°C. A melting curve analysis in a range from 65°C to 95°C (5°C/sec) was appended. OPN mRNA was quantified as molecules of OPN mRNA/pg HPRT mRNA. OPN-ELISA measurements Tumor tissue extracts were prepared from frozen tissues using a standard extraction protocol. Briefly, 20 slices of 10 μm thickness were cut. The total protein was extracted by solubilizing the tissues in 1 ml Giordano buffer using sonication on ice. The cell lysates were clar- ified by centrifugation at 15000 rpm for 15 min. The protein content was determined using the Bradford assay. Fresh blood samples collected before surgical treatment were coagulated for 20 min at room tempera- ture and centrifuged at 3000 rpm for 10 min (Sarstedt monovette, Nümbrecht, Germany). The serum and tumor tissue extracts were aliquoted and stored at -80°C until analysis. Aliquots of each sample were analyzed in duplicate using a commercial ELISA system for OPN (IBL International, Hamburg, Germany) according to the manufacturer’s instructions [3]. Statistical methods All statistical analyses were performed with the SPSS software package 16.0 for Windows (SPSS Inc., Chicago, IL). The differences in the numerical data between two groups were evaluated using the Pearson’s test or Fish- er’s exact test. For survival analyses the overall survival of STS patients was used as follow-up end point. The survival curves were generated using Kaplan-Meier ana- lysis and the log rank test was used to test for differ- ences. For univariate and multivariate analyses (adjusted to tumor stage) the Cox’s proportional hazard regression model was used to calculate the hazard ratio in the Results Osteopontin levels in serum and tumor tissue In patients with soft tissue sarcoma (STS) the median protein concentration of OPN in the serum and tumor tissue extracts was 704 ng/ml (184-2660 ng/ml) and 51.9 ng/mg (3.2-868 ng/mg), respectively (Fig. 1). The median OPN/HPRT transcript level ratio was 12.8 mole- cules OPN/pg HPRT (with a range of 0-110 molecules OPN/pg HPRT, Fig. 1). In particular, there was no cor- relation between OPN mRNA and OPN protein levels of tumors (n = 66; p = 0.4), serum and tumor OPN pro- tein levels (n = 73; p = 0.9) or tumor OPN mRNA and serum OPN protein levels (n = 62; p = 0.7). Osteopontin levels and survival analyses In subgroups split at the median serum or tumor OPN protein level, there was no significant difference in over- all survival. However, when specimens were divided into four groups according to their OPN protein levels, the serum and tumor OPN protein levels did associate with different clinical parameters and overall survival. The group with the lowest tumor (≤ 34.34 ng/mg) or serum (≤ 516.54 ng/ml) OPN protein levels, i. e., the lowest quartile, had a favorable outcome compared to the other three groups (Table 3). Kaplan-Meier analyses showed a significant decrease of overall survival for the three groups with high OPN pro- tein levels compared to the group with the lowest tumor (p = 0.007; log-rank test) and serum (p = 0.04; log-rank test) OPN protein levels. The mean survival time of the patient group with the lowest OPN protein level was 103 months (serum) and 68 months (tumor) whereas patients of the three remaining groups (high OPN protein levels) survived on average 60 months and 58 months (for serum and tumor tissue OPN protein levels, respectively). In addition, patients with both high serum and high tumor OPN protein levels had the worst prognosis (data not shown). Using a univariate Cox regression hazard model for STS patients with high OPN protein level in the serum or in tumor tissue extracts, the risk of tumor- related death was a 2.2 (95% confidence interval (CI) 1.01-5.1; p < 0.05) and 3.7 (95% confidence interval (CI) 1.3-10.4; p = 0.01), respectively (Fig. 2, Table 3). How- ever, in a multivariate Cox’s regression hazard model adjusted to tumor stage OPN protein level was not signif- icantly correlated with prognosis. Bivariate analysis of osteopontin levels with clinical parameters Bivariate analysis of OPN expression did show an asso- ciation of serum and tumor OPN protein levels with