INCREASED SURVIVIN TRANSCRIPT LEVELS: AN INDEPENDENT NEGATIVE PREDICTOR OF SURVIVAL IN SOFT TISSUE SARCOMA PATIENTS Matthias KAPPLER 1 , Thomas K¨ OHLER 2,6 , Christiane KAMPF 3 , Petra DIESTELK ¨ OTTER 3 , Peter W¨ URL 4 , Marc SCHMITZ 3 , Frank BARTEL 1 , Christine LAUTENSCHL ¨ AGER 5 , Ernst Peter RIEBER 3 , Hannelore SCHMIDT 1 , Matthias BACHE 1 , Helge TAUBERT 1 * and Axel MEYE 1,7 1 Institute of Pathology, University of Halle-Wittenberg, Halle, Germany 2 Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University of Leipzig, Leipzig, Germany 3 Institute of Immunology, Technical University Dresden, Dresden, Germany 4 Surgical Clinic I, Faculty of Medicine, University of Leipzig, Leipzig, Germany 5 Institute of Medical Biometry and Informatics, University of Halle-Wittenberg, Halle, Germany 6 Roboscreen Gesellschaft fu ¨r molekulare Biotechnologie mbH, Leipzig, Germany 7 Clinic of Urology, Technical University Dresden, Dresden, Germany Survivin, a recently identified inhibitor of apoptosis protein (IAP), is expressed in diverse embryonic tissues and in vari- ous human cancers. We have investigated the quantitative expression of survivin mRNA by a sensitive TaqMan™-based RT-PCR assay in tissue samples from 94 patients with soft tissue sarcomas (STS). Survivin transcript levels were mea- sured and normalized to GAPDH transcripts. By using a multivariate Cox regression analysis, we found an inverse correlation between the level of survivin mRNA (ratio >2 zmol survivin/amol GAPDH) and the rate of overall survival (p  0.009, RR  2.7). Survivin transcript variants as detected by qualitative RT-PCR analysis were revealed in 36 of 56 STS patients (64%). Only survivin Ex3 and/or full-length survivin variants but not survivin 2B were identified. Our results suggest that a higher level of survivin mRNA is an indepen- dent predictor of survival for STS patients. © 2001 Wiley-Liss, Inc. Key words: survivin; quantitative RT-PCR; soft tissue sarcoma; mul- tivariate analysis; TaqMan™ Survivin, a recently identified member of the inhibitor of apo- ptosis protein (IAP) family, is supposed to promote cell prolifer- ation by interference with the apoptotic pathway. It was found to be expressed mainly in embryonic tissue and in the majority of tumor cells but was almost undetectable in normal adult tissues. 1–3 Three different survivin transcripts have been identified so far (survivin, survivin 2B and survivin Ex3). It has been shown that there is an elevated survivin expression in patients with pancreatic and prostate carcinoma, 1 neuroblastoma, 4,5 gastric carcinoma, 6 colorectal carcinoma, 1,7,8 lung cancer, 1,9 hepatocellular carci- noma, 10 breast carcinomas, 1,11 bladder cancer, 12 melanomas, 13 B- cell lymphoma 14 and in esophageal cancer. 15 In our study, we investigated the survivin mRNA expression in STS entities by means of quantitative and qualitative RT-PCR techniques. We found that the total survivin mRNA level is in- versely correlated with overall survival of STS patients. In line with the finding in neuroblastoma, 4,5 our study also provides evidence that the survivin mRNA level is, in addition to the well-known molecular genetic alterations of the tumor suppressor p53 and the murine-double minute gene 2, 16 –18 of general impor- tance as an independent negative prognostic factor for STS pa- tients. MATERIAL AND METHODS Tissue samples and histopathologic data We examined 98 frozen tumor samples available from 94 adult STS patients (Institute of Pathology, University of Halle-Witten- berg, Halle, Germany, and Surgical Clinic 1, University of Leipzig, Leipzig, Germany) by quantitative RT-PCR analysis (Ta- ble I). The patients ages ranged from 14 – 84 years (median age 54). Forty-six patients (43%) died from the tumor after an average of 25 months (range 2–201 months), whereas 48 patients (57%) are still alive after an average observation period (i.e., after primary tumor resection) of 35 months (range 6 –145 months). Further- more, we analyzed mRNA expression in nontumor tissue in 22 of the 94 patients, in lymphocytes of 3 none-tumor patients and finally in 4 sarcoma cell lines (LMS6-93, US8-93, A-204, Saos-2). In 56 of the 94 STS patients studied, we investigated qualitatively the occurrence of survivin transcript variants (Table I). RNA preparation, cDNA synthesis and semiautomated transcript analysis by quantitative fluorescence PCR Total RNA was isolated from frozen tissue samples by using the RNeasy Mini Kit according to the manufacturer’s instructions (QIAGEN, Hilden, Germany). For each cDNA synthesis, 1 g RNA per sample, 200 U Superscript™ II RNase H Reverse Tran- scriptase (RT) and 3 g random hexamer primers (GibcoBRL, Karlsruhe, Germany) were mixed. The RT reactions were run for 75 min at 42°C. Survivin mRNA subsequence (77 bp), which does not overlap with the EPR-1 mRNA 19 and which is specific for all 3 survivin transcripts, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript (63 bp) were amplified from cDNA in dupli- cate experiments by ready-to-use PCR assays (Roboscreen, Ge- sellschaft fu ¨r molekulare Biotechnologie mbH, Leipzig, Germany) as previously described. 16 Qualitative RT-PCR In the qualitative RT-PCR for the detection of transcript variants, the RT reaction contained 1 g of total RNA, 1 RT buffer (Pro- mega, Mannheim, Germany), 25 M of each dNTP, 10 pmol of sequence-specific RT primer (5’-TTCCTCCCTCACTTCTCACC- 3’), and 12 U M-MLV RT RNase (Promega, Mannheim, Germany) in a final volume of 20 l. The RT reaction was incubated at 37°C for 1 hr. PCR amplification was performed on a PTC-100 cycler (MJ Research/Biozym Diagnostic, Hess Oldendorf, Germany). Two mi- croliters of cDNA were subjected to amplification in a 25 l mixture Abbreviations: amol, attomoles [10 -18 moles]; FS, fibrosarcoma; GAPDH, glycerine-aldehyde-3-phosphate dehydrogenase; IAP, inhibitor of apoptosis; LMS, leiomyosarcoma; LS, liposarcoma; MFH, malignant fibrous histiocytoma; MNT, malignant neural tumors; RMS, rhabdomyo- sarcoma; RR; relative risk; RT, reverse transcriptase; STS, soft tissue sarcoma; SyS, synovial sarcoma; zmol, zeptomoles [10 -21 moles] Grant sponsor: Wilhelm Sander Stiftung; Grant number: 99.009.1. *Correspondence to: Institute of Pathology, University of Halle-Witten- berg, Magdeburger Strasse 14, D-06097 Halle, Germany. Fax: +49-345-557-1295. E-mail: helge.taubert@medizin.uni-halle.de Received 30 April 2001; Revised 12 June 2001; Accepted 22 June 2001 Published online 00 Month 2001 Int. J. Cancer (Pred. Oncol.): 95, 360 –363 (2001) © 2001 Wiley-Liss, Inc. Publication of the International Union Against Cancer