Bache et al. BMC Cancer 2010, 10:132 http://www.biomedcentral.com/1471-2407/10/132 Page 2 of 8 serum levels of 93 histological verified adult STS patients. Moreover, to characterize the role of OPN in soft tissue sarcoma, we correlated OPN levels with clini- cal parameters and prognosis. Methods Patients and tumor material Tumor tissue and serum samples of 93 adult patients with histologically verified primary STS were analyzed (Table 1). In detail, we have analyzed OPN protein levels in serum (86 patients) and tumor tissue (80 patients) and OPN mRNA level in tumor tissue (68 patients). Patients and tissue samples have been partially described previously [24-26]. The study was carried out in compliance with the Helsinki Declaration and it was approved by the Ethics Committee of the Medical Faculty of the University Halle. All patients gave written informed consent (Institute of Pathology, University of Halle, Germany and Department of Surgery 1, Univer- sity of Leipzig, Germany). Patients’ mean age was 56 years (19 to 87 years). The median follow-up time was 44 months (2 to 146 months). The overall survival according to stage was 86% for stage I (n = 15), 59% for stage II (n = 39), 37% for stage III (n = 19) and 0% for stage IVa (n = 9). Tumor tissue was collected following surgical resection and snap-frozen in liquid nitrogen. Tumors were classified according to the van Unnik grading system [27]. Reverse transcription-polymerase chain reaction and real- time quantitative PCR Total RNA was isolated using the RNeasy Mini Kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany). Subsequently, 1000 ng of RNA was reverse transcribed with random hexamer primers and Superscript II reverse transcriptase (Invitrogen, Table 1 Histopathological and clinical data in soft tissue sarcoma patients Serum OPN protein Tumor OPN protein Tumor OPN mRNA Total Tumor stage Tumor type Irradiation Chemotherapy Tumor size LS FS and MFH NS RMS and LMS Other STS I II III IV Yes No Yes No T1 T2 Yes No Tumor resection Radical (R0) Not Radical (R1) Distant metastases M0 M1 Relapse 86 14 37 26 9 21 24 8 22 11 31 55 8 78 12 74 57 29 79 7 55 31 Abbreviations: OPN: osteopontin, LS: liposarcoma, MFH: malignant fibrous histiocytoma; FS: fibrosarcoma; NS: neurogenic sarcoma; LMS: leiomyosarcoma; RMS: rhabdomyosarcoma; STS: soft tissue sarcoma; 80 13 32 26 9 21 20 7 22 10 32 48 7 73 11 69 54 26 73 7 51 29 68 12 26 22 8 18 17 3 20 10 29 39 5 63 10 58 49 19 62 6 47 21