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In various tumors, the hypoxia inducible factor-1α ( HIF-1α ) and the epidermal growth factor-receptor ( EGFR ) have an impact on survival. Nevertheless, the prognostic impact of both markers for soft tissue sarcoma (STS) is not well studied. We examined 114 frozen tumor samples from adult soft tissue sarcoma patients and 19 frozen normal tissue samples. The mRNA levels of HIF-1α, EGFR, and the reference gene hypoxanthine phosphoribosyltransferase (HPRT) were quantified using a multiplex qPCR technique. In addition, levels of EGFR or HIF-1α protein were determined from 74 corresponding protein samples using ELISA techniques. Our analysis showed that a low level of HIF-1α or EGFR mRNA (respectively, relative risk (RR) = 2.8; p = 0.001 and RR = 1.9; p = 0.04; multivariate Cox´s regression analysis) is significantly associated with a poor prognosis in STS patients. The combination of both mRNAs in a multivariate Cox's regression analysis resulted in an increased risk of early tumor-specific death of patients (RR = 3.1, p = 0.003) when both mRNA levels in the tumors were low. The EGFR protein level had no association with the survival of the patient's cohort studied, and a higher level of HIF-1α protein associated only with a trend to significance (multivariate Cox's regression analysis) to a poor prognosis in STS patients (RR = 1.9, p = 0.09). However, patients with low levels of HIF-1α protein and a high content of EGFR protein in the tumor had a three-fold better survival compared to patients without such constellation regarding the protein level of HIF-1α and EGFR. In a bivariate two-sided Spearman's rank correlation, a significant correlation between the expression of HIF-1α mRNA and expression of EGFR mRNA ( p < 0.001) or EGFR protein ( p = 0.001) was found, additionally, EGFR mRNA correlated with EGFR protein level ( p < 0.001). Our results show that low levels of HIF-1α mRNA or EGFR mRNA are negative independent prognostic markers for STS patients, especially after combination of both parameters. The protein levels showed a different effect on the prognosis. In addition, our analysis suggests a possible association between HIF-1α and EGFR expression in STS.

The capillary morphogenesis gene 2 (CMG2), also known as the anthrax toxin receptor 2 (ANTXR2), is a transmembrane protein putatively involved in extracellular matrix (ECM) adhesion and tissue remodeling. CMG2 promotes endothelial cell proliferation and exhibits angiogenic properties. Its downregulation is associated with a worsened survival of breast carcinoma patients. Aim of this study was to analyze the CMG2 mRNA and protein expression in soft tissue sarcoma and their association with patient outcome. CMG2 mRNA was measured in 121 tumor samples of soft tissue sarcoma patients using quantitative real-time PCR. CMG2 protein was evaluated in 52 tumor samples by ELISA. CMG2 mRNA was significantly correlated with the corresponding CMG2 protein expression (r s = 0.31; p = 0.027). CMG2 mRNA expression was associated with the mRNA expressions of several ECM and tissue remodeling enzymes, among them CD26 and components of the uPA system. Low CMG2 mRNA expression was correlated with a worsened patients' disease-specific survival in Kaplan-Meier analyses (mean patient survival was 25 vs. 96 months; p = 0.013), especially in high-stage tumors. A decreased CMG2 expression is a negative prognostic factor for soft tissue sarcoma patients. CMG2 may be an interesting candidate gene for the further exploration of soft tissue sarcoma genesis and progression., Competing Interests: The authors declare no conflict of interest.

Chemokines are involved in both the negative and positive regulation of inflammatory processes, angiogenesis and cancer/cancer stem cell proliferation as well as the chemoattraction of tumor cells to metastatic sites. The aim of this study was to measure the mRNA expression levels of three chemokines, CCL2, CCL7 and CX3CL1, in soft tissue sarcomas (STSs) and to assess the correlations between these levels as well as their correlations with clinicopathological data and the disease-specific survival of STS patients. The mRNA levels of CCL2, CCL7 and CX3CL1 were analyzed in tumor tissues from 126 STS patients using qPCR. Low mRNA expression of CCL2 and CX3CL1 was significantly correlated with a worse prognosis (RR = 1.98; p = 0.019 and RR = 2.10; p = 0.014; multivariate Cox's regression analysis). A combined low expression of CCL2 and CX3CL1 was associated with a significantly increased risk of tumor-related death as compared to patients with high expression levels of both chemokines (RR = 3.08; p = 0.003). A gender-specific multivariate analysis revealed that female STS patients with low CX3CL1 mRNA expression had a 3.46-fold increased risk of death (p = 0.004). Low expression of both CCL2 and CX3CL1 mRNAs resulted in an additive 5.37-fold increased risk of tumor-related death (p = 0.003) as compared to those with high expression of both parameters in female patients. In conclusion, this is the first study to show a significant correlation between combined low expression of CCL2 and CX3CL1 and a poor prognosis for STS patients, particularly in female patients., (© 2014 UICC.)

Background: Human epidermal growth factor receptor 2 (ErbB2) is overexpressed in a variety of human malignancies. Moreover, ErbB2 has been reported to influence cancer patient survival and progression of different tumor entities. However, information regarding the prognostic impact of ErbB2 in soft tissue sarcoma (STS) patients is limited and conflicting., Material and Methods: ErbB2 mRNA and protein levels were defined by quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA), and the prognostic impact of ErbB2 mRNA and protein levels in tumor tissue of 124 soft tissue sarcoma patients were investigated., Results: The median ErbB2 mRNA expression level in tumor tissue was decreased 3.9-fold compared to non-neoplastic surrounding tissue (p = 0.001). Furthermore, an increased ErbB2 mRNA expression level was associated with an improved tumor-specific survival (p = 0.01, log rank test). Multivariate Cox's proportional hazard regression analyses revealed an increased ErbB2 mRNA expression level as an independent favorable prognostic factor for tumor-specific survival of STS patients (n = 124; RR = 3.0; 95 % CI = 1.6-5.7; p < 0.001). In addition, multivariate Cox's proportional hazard regression analyses showed that an increased ErbB2 protein expression level correlated with poorer recurrence-free survival of STS patients (n = 47; RR = 9.9; 95 % CI = 1.7-59.7; p = 0.012), in particular for STS patients who received postoperative radiotherapy (n = 27; RR = 17.9; 95 % CI = 1.3-247.7; p = 0.031)., Conclusion: This study suggests an inverse prognostic value of ErbB2 mRNA and protein expression level.

Background: Argonaute genes are essential for RNA interference, stem cell maintenance and differentiation. The Piwi-like genes, a subclass of the Argonaute genes, are expressed mainly in the germline. These genes may be re-expressed in tumors, and expression of the Piwi-like genes is associated with prognosis in several types of tumors., Methods: We measured the expression of Piwi-like mRNAs (Piwi-like 2-4) in 125 soft tissue sarcoma (STS) samples by qPCRs. Statistical tests were applied to study the correlation of expression levels with tumor-specific survival for STS patients., Results: In multivariate Cox's regression analyses, we showed that low Piwi-like 2 and Piwi-like 4 mRNA expression were significantly associated with a worse prognosis (RR = 1.87; p = 0.032 and RR = 1.82; p = 0.039). Low expression of both genes was associated with a 2.58-fold increased risk of tumor-related death (p = 0.01). Piwi-like 4 and combined Piwi-like 2 and 4 mRNA levels correlated significantly with prognosis (RR = 3.53; p = 0.002 and RR = 5.23; p = 0.004) only for female but not for male patients. However, combined low Piwi-like 2 and 3 transcript levels were associated with worse survival (RR = 5.90; p = 0.02) for male patients., Conclusions: In this study, we identified a significant association between the expression of Piwi-like 2 and 4 mRNAs and the tumor-specific survival of soft tissue sarcoma patients. Furthermore, a connection between sex and the impact of Piwi-like mRNA expressions on STS patients' prognosis was shown for the first time.

Background: It is well known that osteopontin (OPN) plays an important role in tumor progression and that a high OPN expression level in several tumor entities correlates with poor prognosis in cancer patients. However, little is known about the prognostic relevance of the OPN mRNA splice variants., Methods: We analyzed the mRNA expression levels of different OPN splice variants in tumor tissue of 124 soft tissue sarcoma (STS) patients. Quantitative real-time PCR (qRT-PCR) was used to analyze the mRNA expression level of three OPN splice variants (OPN-a, -b and -c)., Results: The multivariate Cox's proportional hazard regression model revealed that high mRNA expression levels of OPN splice variants are significantly associated with poor prognosis in STS patients (n = 124). Women (n = 68) with high mRNA expression levels of OPN-a and OPN-b have an especially elevated risk of tumor-related death (OPN-a: RR = 3.0, P = 0.01, CI = 1.3-6.8; OPN-b: RR = 3.4, P = 0.01, CI = 1.4-8.2). In particular, we found that high mRNA expression levels of OPN-b and OPN-c correlated with a high risk of tumor-related death in STS patients that received radiotherapy (n = 52; OPN-b: RR = 10.3, P < 0.01, CI = 2.0-53.7; OPN-c: RR = 11.4, P < 0.01, CI = 2.2-59.3)., Conclusion: Our study shows that elevated mRNA expression levels of OPN splice variants are negative prognostic and predictive markers for STS patients. Further studies are needed to clarify the impact of the OPN splice variants on prognosis.

Expression of microRNAs can affect age of tumor onset and prognosis of cancer patients. However, nothing is known about the effects of microRNAs on altered age of cancer onset and disease-specific survival of soft-tissue sarcoma (STS) patients. The levels of miR-210, also known as hypoxia-regulated microRNA, were analyzed by quantitative real-time (RT)-PCR in the tumors of 78 STS patients. The patients were stratified according to their microRNA levels with low, intermediate and high expression levels and the association of microRNA expression and patients' survival was analyzed using multivariate Cox's regression hazard analyses. A significant correlation between an intermediate miR-210 expression and disease-specific death of STS patients [relative risk (RR) = 3.19; p = 0.018] was observed compared with patients with high expression levels in their tumors. Interestingly, the association between an intermediate expression of miR-210 and a poor prognosis was only significant in female STS patients (RR = 11.28; p = 0.010), but not observed in male individuals. Furthermore, the expression of miR-210 showed a significant association with the age of tumor onset in a gender-specific manner. Specifically, male patients with an intermediate expression of miR-210 associated with a 9.6-year later age of tumor onset (p = 0.017) compared with males with a low expression of miR-210 in their tumors. However, no significant differences in the female patients were observed. This study provides the first evidence of a correlation of expression levels of a single microRNA (miR-210) with the prognosis and age of tumor onset in a gender-specific manner in STS patients., (Copyright © 2011 UICC.)

Background: The human leucine-rich, repeat-containing G protein-coupled receptor (LGR) 5, also called GPR49, is a marker of stem cells in adult intestinal epithelium, stomach and hair follicles. LGR5/GPR49 is overexpressed in tumors of the colon, ovary and liver and in basal cell carcinomas. Moreover, an expression in skeletal muscle tissues was also detected. However, there has been no investigation regarding the expression and function of LGR5/GPR49 in soft-tissue sarcomas (STS) yet., Methods: Seventy-seven frozen tumor samples from adult STS patients were studied using quantitative real-time TaqMan™ PCR analysis. The mRNA levels of wild type LGR5/GPR49 and a newly identified splice variant of LGR5/GPR49 lacking exon 5 (that we called GPR49Δ5) were quantified., Results: A low mRNA expression level of GPR49Δ5, but not wild type LGR5/GPR49, was significantly correlated with a poor prognosis for the disease-associated survival of STS patients (RR = 2.6; P = 0.026; multivariate Cox's regression hazard analysis). Furthermore, a low mRNA expression level of GPR49Δ5 was associated with a shorter recurrence-free survival (P = 0.043). However, tumor onset in patients with a lower expression level of GPR49Δ5 mRNA occurred 7.5 years later (P = 0.04) than in patients with a higher tumor level of GPR49Δ5 mRNA., Conclusion: An attenuated mRNA level of the newly identified transcript variant GPR49Δ5 is a negative prognostic marker for disease-associated and recurrence-free survival in STS patients. Additionally, a lower GPR49Δ5 mRNA level is associated with a later age of tumor onset. A putative role of GPR49Δ5 expression in tumorigenesis and tumor progression of soft tissue sarcomas is suggested.

Background: Members of the urokinase-type plasminogen activator (uPA) system are up-regulated in various solid malignant tumors. High antigen levels of uPA, its inhibitor PAI-1 and its receptor uPAR have recently been shown to be associated with poor prognosis in soft-tissue sarcoma (STS) patients. However, the mRNA expression of uPA system components has not yet been comprehensively investigated in STS patients., Methods: The mRNA expression level of uPA, PAI-1, uPAR and an uPAR splice variant, uPAR-del4/5, was analyzed in tumor tissue from 78 STS patients by quantitative PCR., Results: Elevated mRNA expression levels of PAI-1 and uPAR-del4/5 were significantly associated with clinical parameters such as histological subtype (P = 0.037 and P < 0.001, respectively) and higher tumor grade (P = 0.017 and P = 0.003, respectively). In addition, high uPAR-del4/5 mRNA values were significantly related to higher tumor stage of STS patients (P = 0.031). On the other hand, mRNA expression of uPA system components was not significantly associated with patients' survival. However, in STS patients with complete tumor resection (R0), high PAI-1 and uPAR-del4/5 mRNA levels were associated with a distinctly increased risk of tumor-related death (RR = 6.55, P = 0.054 and RR = 6.00, P = 0.088, respectively). Strikingly, R0 patients with both high PAI-1 and uPAR-del4/5 mRNA expression levels showed a significant, 19-fold increased risk of tumor-related death (P = 0.044) compared to the low expression group., Conclusion: Our results suggest that PAI-1 and uPAR-del4/5 mRNA levels may add prognostic information in STS patients with R0 status and distinguish a subgroup of R0 patients with low PAI-1 and/or low uPAR-del4/5 values who have a better outcome compared to patients with high marker levels.

Background: Osteopontin (OPN) overexpression is correlated with a poor prognosis for tumor patients. However, only a few studies investigated the prognostic impact of expression of OPN in soft tissue sarcomas (STS) yet., Methods: This study is based on tumor and serum samples from 93 adult STS patients. We investigated OPN protein levels in serum (n = 86) and tumor tissue (n = 80) by ELISA and OPN mRNA levels in tumor tissue (n = 68) by quantitative real-time PCR., Results: No correlation was found between OPN levels in serum and tumor tissue. Moreover, an elevated OPN protein level in the serum was significantly associated with clinical parameters such as higher stage (p = 0.004), higher grade (p = 0.003), subtype (p = 0.002) and larger tumor size (p = 0.03). OPN protein levels in the tumor tissue were associated with higher stage (p = 0.06), higher grade (p = 0.003), subtype (p = 0.07) and an increased rate of relapse (p = 0.02). In addition, using a Cox's proportional hazards regression model, we found that an elevated OPN protein level in the serum and tumor tissue extracts is a significant negative prognostic factor for patients with STS. The relative risks of tumor-related death were 2.2 (p < 0.05) and 3.7 (p = 0.01), respectively., Conclusion: Our data suggest OPN protein in serum as well as in tumor tissue extracts is an important prognostic factor for soft tissue sarcoma patients.

Background: The urokinase plasminogen activator (uPA) system is one of the best-investigated protease systems, both under physiological and pathological conditions, including various types of cancer. However, effects of co-expression of members of the uPA system in soft-tissue sarcoma (STS) patients at the protein level in both tumour tissue and serum have not been investigated yet., Methods: We examined 82 STS patients for protein levels of uPA, PAI-1and uPAR in tumour tissue and serum by ELISA., Results: A significant correlation between high antigen levels of uPA, PAI-1 or uPAR in tumour tissue, and of uPAR in serum, with poor outcome of STS patients was found for the first time. Most strikingly, we observed an additive effect of combined uPA, PAI-1 or uPAR levels in tumour tissue extracts with uPAR levels in serum on patients' prognosis. High uPA/uPAR, PAI-1/uPAR and uPAR/uPAR antigen levels in tumour tissue/serum were associated with a 5.9-fold, 5.8-fold and 6.2-fold increased risk of tumour-related death (P=0.003, 0.001 and 0.002, respectively) compared with those patients who displayed low levels of the respective marker combination., Conclusion: As expression of members of the uPA system in tumour tissue and serum is additively correlated with prognosis of STS patients, our results suggest that combinations of these biomarkers can identify STS patients with a higher risk of tumour-related death.

The p53 stress response is crucial for the prevention of tumor formation. The oncogene HDM2 is one of the key negative regulators of p53 and is a central node in the p53 pathway. P53 and HDM2 form an oscillating feedback loop. HDM2 expression is regulated by different promoters. To evaluate its clinical relevance, we determined the levels of HDM2 transcripts originating from the constitutive P1 and p53-sensitive P2 promoter in 133 soft tissue sarcomas and correlated the results with the age of diagnosis and the patients' outcome. We show that only high levels of the HDM2-P1 transcript but not the P2 transcript are associated with an 11-year earlier age of onset (50.5 years) compared with low P1 levels (61.5 years; P < 0.0001, t test). In addition, low P1 and P2 mRNA expression levels were independent predictors of poor outcome for patients with soft tissue sarcomas (low P1: relative risk, 3.7; P < 0.0001; low P2: relative risk, 2.5; P = 0.001). A change in the expression levels of the HDM2 transcripts originating from the two HDM2 promoters could disrupt the oscillating P53-HDM2 feedback loop in a way that elevated levels of HDM2-P1 transcript are associated with an earlier age of tumor onset and that reduced levels of HDM2-P1 or HDM2-P2 transcripts are correlated with poor prognosis of patients with soft tissue sarcomas.

The inhibitor of apoptosis wild-type survivin is a multifunctional protein that suppresses apoptosis and regulates cell cycle progression. An association between wild-type survivin expression and radiosensitivity has been described in different tumor cells. The effects of siRNA-induced knockdown of wild-type survivin and survivin-splice variants survivin-2B and survivin-Delta3 were investigated under normoxic and hypoxic conditions in the human sarcoma cell line US 8-93 (mutant p53). Inhibition of the survivin isoforms by siRNA resulted in a decrease of target mRNA down to 14-70% compared to cells treated with control siRNA independent of the oxygen level. The mRNA expression of survivin isoforms was decreased by the factor of 1-12 when the cells were cultivated under hypoxic conditions. Moreover, the knockdown of wild-type survivin reduced colony formation independent of oxygen concentration down to 70% and induced formation of polyploid cells. Less reduction of plating efficiency was observed after specific knockdown of survivin-2B and survivin-Delta3 under hypoxic or normoxic conditions. A knockdown of wild-type survivin, survivin-Delta3 and survivin-2B isoforms in combination with irradiation caused no radiosensitization in cell line US 8-93, neither under hypoxic nor under normoxic conditions tested in the colony-forming assay. However, knockdown of wild-type survivin caused radiosensitization in the megacolony assay.

Cancer stem cells can play an important role in tumorigenesis and tumor progression. However, it is still difficult to detect and isolate cancer stem cells. An alternative approach is to analyse stem cell-associated gene expression. We investigated the coexpression of three stem cell-associated genes, Hiwi, hTERT and survivin, by quantitative real-time-PCR in 104 primary soft-tissue sarcomas (STS). Multivariate Cox's proportional hazards regression analyses allowed correlating gene expression with overall survival for STS patients. Coexpression of all three stem cell-associated genes resulted in a significantly increased risk of tumor-related death. Importantly, tumors of patients with the poorest prognosis were of all four tumor stages, suggesting that their risk is based upon coexpression of stem cell-associated genes rather than on tumor stage.

Self-renewal is considered as a common property of stem cells. Dysregulation of stem cell self-renewal is likely a requirement for the development of cancer. Hiwi, the human Piwi gene, encodes a protein responsible for stem cell self-renewal. In this study, we investigated the expression of Hiwi at the RNA level by real-time quantitative PCR in 65 primary soft-tissue sarcomas (STS) and ascertained its impact on prognosis for STS patients. In a multivariate Cox's proportional hazards regression model, we found that an increased expression of Hiwi mRNA is a significant negative prognostic factor for patients with STS (P=0.017; relative risk 4.6, 95% confidence interval (CI) 1.3-16.1) compared to medium expression of Hiwi transcript. However, a low expression of Hiwi transcript is correlated with a 2.4-fold (CI 0.7-8.0) increased risk, but this effect was not significant (P=0.17). Altogether, high-level expression of Hiwi mRNA identifies STS patients at high risk of tumour-related death. This is the first report showing a correlation between expression of a gene involved in stem cell self-renewal and prognosis of cancer patients.

The product of the HDMX (or MDM4) gene is structurally related to the MDM2 oncoprotein and is also capable of interacting with the tumor suppressor protein p53. The aim of our study was to determine the amplification status of the HDMX gene and the expression of the HDMX mRNA (particularly that of the HDMX-S splice variant) in soft-tissue sarcomas (STS). Patients with STS were evaluated for the status of HDMX gene amplification (n = 66) and HDMX-S mRNA expression (n = 57) within their tumors. DNA, total RNA and protein were isolated from frozen tumor tissue. We determined that the HDMX-S splice variant transcript was predominant in a subset (14%) of tumor samples and that its expression was correlated with decreased patient survival (15 vs. 53 months, p < 0.0001, log-rank test) and with a 17-fold increased risk of a tumor-related death (p < 0.0001, multivariate Cox's regression model). The tumors from these patients also expressed elevated levels of HDMX-S protein. The HDMX gene was amplified in 17% of STSs, and the gene amplification was associated with poor prognosis (RR = 6.5, p < 0.0001). There was no correlation between the HDMX gene amplification and overexpression of the HDMX-S splice variant. In summary, our data indicate that both the overexpression of the HDMX-S transcript as well as HDMX gene amplification are important prognostic markers for STS., ((c) 2005 Wiley-Liss, Inc.)

The aim of this study was to investigate the level and the prognostic value of the expression of different survivin transcript variants--survivin, survivin-DeltaEx3 and survivin-2B--in tumours of 76 soft tissue sarcoma (STS) patients. The expression of survivin transcript variants in STS tissue samples and in 12 nonmalignant control tissues was analysed by quantitative RT-PCRs. Expression levels of all survivin transcript variants were strongly elevated in STS compared to normal tissues. A positive correlation between expression of splice variants and tumour stage was found (P=0.02; chi2 test). The multivariate Cox's proportional hazards regression model revealed a 7.3-fold increased risk of tumour-related death for patients with survivin-DeltaEx3 overexpressing tumours (P=0.007). The effect of surivivin (wildtype variant) and survivin-2B was less pronounced but still significant (2.2- and 1.9-fold, resp., P<0.05 each). Our results show for the first time that mRNA expression of survivin-variants is significantly correlated to a poor prognosis for STS patients, and we suggest expression of survivin splice variants together with tumour stage as independent predictor of survival.

Survivin, a member of the inhibitor-of-apoptosis family is an essential protein for regular mitosis and is involved in an anti-apoptotic pathway. In some studies, an association between survivin expression and radiosensitivity has been described for tumor cells, but the relationship between p53 and survivin regarding radioresistance remains to be clarified. In order to increase the effect of irradiation on two sarcoma cell lines, A-204 with wt-p53 and US 8-93 with mt-p53, siRNA was applied to knock down survivin expression. The effects of combined treatment of siRNA treatment and irradiation were investigated by clonogenic survival assay, measurement of activity of caspases 3 and 7, Western blot hybridization for survivin and p53, and morphological analysis of apoptosis. Survivin knock down caused radiosensitization in the cell line A-204 (wt-p53) with an enhancement factor of 1.8 at 2 Gy (p=0.05) and 2.5 at 4 Gy (p=0.02), respectively. No radiosensitization was found in the cell line US 8-93 (mt-p53), when clonogenic survival was analyzed. These findings were supported by an increase in activity (up to 5.2-fold) of caspases 3 and 7 in cell line A-204 (wt-p53), but not in cell line US 8-93 (mt-p53) after a combined treatment of siRNA and irradiation. Our findings suggest that the wt-p53-caspase pathway is of importance for the radiosensitization induced by targeting survivin, which may have an impact on future gene therapeutical treatments.

Side effects make it necessary to seek new radiosensitizers with low systemic actions. Sodium peroxodisulfate is a strong oxidizer classified as a safe agent with low systemic effects. We have examined the effect of this oxidizer on the radiosensitivity of the radioresistant human soft tissue sarcoma cell line US8-93 (mt-p53). The effects of peroxodisulfate (0.02-3.0 mM) with or without irradiation were studied by clonogenic survival assay, comet assay and the induction of apoptosis. We found sodium peroxodisulfate to be nontoxic for US8-93 up to a concentration of 0.1 mM. The combination of 0.1 mM sodium peroxodisulfate and irradiation showed a slight radiosensitizing effect with an enhancement factor of up to 1.5. This was coupled with an increase in apoptosis from 12 to 22% and an inhibition of repair of irradiation-induced DNA damage. Incubation with concentrations between 0.1-1.0 mM sodium peroxodisulfate resulted in a strong decrease of clonogenic survival with an IC50 of 0.28 mM. This was correlated with an increase in cross links. Furthermore, a strong additive effect was observed for the combination of 0.3 mM sodium peroxodisulfate and irradiation resulting in an increase in enhancement ratio from 1.3 at 2 Gy to 3.1 at 6 Gy (p

Survivin, a member of the inhibitors-of-apoptosis gene family, is overexpressed in many tumor types. Survivin is a prognostic marker of soft-tissue sarcomas, but the downregulation of survivin expression and the possible dependency of survivin downregulation on p53 in these tumors have not been investigated. Therefore, we applied small interfering RNA (siRNA) to knock down the expression of survivin in five human sarcoma cell lines with wild-type or mutant p53 alleles. Compared with survivin mRNA expression in the nonsense siRNA-treated sarcoma cell lines, expression after treatment with survivin-specific siRNA was reduced by 73-88%; survivin protein expression was reduced by 52-81%. This finding was coupled with a reduction in clonogenic survival ranging from 65-86%. However, less than 10% of cells treated with survivin-specific siRNA underwent apoptosis. Cell-cycle and morphologic analyses showed that after a dramatic increase in the number of treated cells in the G2/M phase, some of the cells became polyploid; this result indicates that mitosis of a substantial number of treated cells was incomplete. Our findings suggest that survivin-specific siRNA could be a selective treatment to kill sarcoma cells regardless of the presence or absence of wild-type p53 alleles.

The aim of this study was to determine the amplification status of the HDMX gene and the expression of the HDMX mRNA particularly that of the HDMX-S splice variant in soft tissue sarcomas (STS). We show that the HDMX gene is amplified in 27% of STSs, which was associated with a worse prognosis (RR = 2.8, p = 0.03). We have also found that the transcript of the HDMX-S variant was predominant in a subset of (14%) of tumor samples, which was correlated with a significantly decreased overall survival time (15 vs. 53 months, p < 0.0001, log-Rank-test) and with a 9-fold-increased risk of tumor-related death (p < 0.0001). There was no correlation between the HMDX gene amplification and the HDMX-S splice variant overexpression. In summary, our data indicate that both the overexpression of the HDMX-S transcript, as well as, the HDMX gene amplification are important prognostic markers for STS.

Few studies have investigated the loss of heterozygosity and microsatellite instability in soft tissue sarcomas. Therefore, we analyzed samples of human soft tissue sarcomas to determine the status of the chromosomal region 12q14-15, which contains the MDM2 gene encoding the well-known counterpart of the tumor suppressor p53. In addition, we determined whether an amplified MDM2 gene was present in the samples. Of the 88 soft tissue sarcoma samples, 24 (27%) showed evidence of loss of heterozygosity of markers representing 12q14-15, and 12 (14%) showed evidence of microsatellite instability. Of the 72 samples analyzed by semiquantitative polymerase chain reaction, 15 (21%) possessed an amplified MDM2 gene. Loss of heterozygosity (P =.008) and microsatellite instability (P =.035) were significantly more common in Stage I tumors than in higher stage tumors. This result indicated that these alterations occur early in soft tissue sarcoma progression and possibly define a subgroup of soft tissue sarcoma. Surprisingly, MDM2 amplification in soft tissue sarcoma patients was associated with a prognosis better than that of patients without the amplification; however, this difference was not statistically significant (P =.6). Furthermore, of the tumors with an MDM2 amplification, 40% (6/15) also experienced loss of heterozygosity at 12q14-15; in contrast, only 16% of tumors without an MDM2 amplification (9/57) underwent a loss of heterozygosity. A concomitant occurrence of deletions and amplifications resulting from deficiencies in the nonhomologous end-joining pathway could in part explain this finding.

Background: Deregulation of DNA mismatch repair is a common mechanism for the development of hereditary nonpolyposis colon carcinoma and related familiar cancers, but it also plays a role in the tumorigenesis of sporadic cancers. Although the protein expression of the two main components of DNA mismatch repair, hMSH2 and hMLH1, has been described in soft tissue sarcoma (STS) patients, its prognostic impact is yet to be determined., Methods: The authors investigated the expression of the DNA repair proteins hMSH2 and hMLH1 by Western blot analysis in tumor samples of 57 STS patients. The correlation between the expression of hMSH2/hMLH1 and survival was studied in a Cox proportional hazards regression model, which was adjusted for the prognostic effects of staging, tumor entity, and radicality of tumor resection., Results: Nine of 57 STS (16%) showed reduced expression of hMSH2 and reduced expression of hMLH1 was detected in seven STS patients (12%). In a Kaplan-Meier analysis, the median survival for patients with reduced expression of the hMSH2 protein was 18 months, whereas the patients with a normal expression of hMSH2 survived for an average of 68 months. A multivariate Cox proportional hazards regression model revealed a significant correlation between the reduced expression of the hMSH2 protein and poor survival (relative risk = 4.7; 95% confidence interval [CI]: 1.3-17.2; P = 0.019)., Conclusions: Reduced expression of the hMSH2 protein is an independent negative prognostic factor for STS patients., (Copyright 2003 American Cancer Society.DOI 10.1002/cncr.11326)

Purpose: Survivin is a member of the inhibitor-of-apoptosis gene family and is known to be overexpressed in a number of tumor types. The aim of this study was to evaluate the prognostic value of survivin protein expression in tumor tissue extracts in a group of well-characterized soft-tissue sarcoma (STS) patients., Experimental Design: In this investigation, malignant tissue samples from 63 STS patients as well as from a panel of tumor cell lines were investigated, with nonmalignant tissues serving as controls. The survivin protein level was quantified by a novel ELISA and by Western blot analysis. Results obtained by both methods were compared with clinicopathological parameters regarding tumor grade and tumor entity, and they were then correlated to survival in a multivariate Cox regression model., Results: High survivin levels were detected by ELISA and Western blot analysis in tumor tissue extracts and in lysates of tumor cell lines. None or only weak expression of survivin protein was found in nonmalignant cells and tissues. When comparing survivin values obtained by ELISA or Western blot, we found a significant correlation between both methods (P = 0.013, Pearson test). Our findings revealed that, in multivariate Cox regression analyses, survivin levels measured by ELISA and Western blot were significantly associated with tumor-related death in STS patients (P = 0.001, RR = 19.8, and P = 0.004, RR = 5.1, respectively). However, in a direct comparison of both survivin protein detection assays, we found a higher sensitivity and a stronger correlation to prognosis in survivin ELISA as compared with the Western blot assays. Furthermore, a higher tumor grade and more aggressive STS entity showed an elevated survivin protein expression level., Conclusion: Altogether, an elevated survivin content in tumor tissue extracts has a significant and independent negative predictive value on the survival-rate of STS patients. This finding corresponds well to data obtained for the mRNA level of survivin, as shown previously (M. Kappler et al., Int. J. Cancer, 95: 360-363, 2001).

The MDM2 oncogene plays an important role in tumorigenesis and especially in soft tissue sarcomas (STS). Overexpression of the MDM2 protein is associated with a poorer prognosis for STS patients. An MDM2 antisense approach to reduce MDM2 protein levels has been successfully applied on several carcinomas in vitro and used on a few in vivo cases. However, antisense treatment not only resulted in an MDM2 protein reduction but also in a wild-type P53 gene (wt-P53) mediated tumor growth suppression due to its genetic wt-P53 status. In this study, we used a clinically relevant xenotransplanted STS model with a mutated P53 gene (mt-P53) in order to exclude the influence of wt-P53. The human STSs were surgically implanted and one week later osmotic pumps were implanted intraperitoneally into nude rats releasing MDM2-antisense ODNs (AS ODNs) continuously for one week. As controls MDM2-sense ODN (SE ODN) or a 0.9% NaCl solution (saline solution) were administered. After one week animals treated with MDM2-AS ODN (100 or 200 microg) showed a reduction in tumor mass in comparison to animals treated with MDM2-SE ODN. The reduction in tumor mass was significant in animals treated with MDM2-AS ODNs in comparison to the saline solution treated ones (p=0.018 or p=0.007). Furthermore, a significant reduction in macroscopically visible tumor number with MDM2-AS ODN treatments (100 or 200 microg) in comparison to MDM2-SE ODN or saline solution treatment (both p=0.009) was observed for the first time. As expected a reduction in MDM2 protein expression in the MDM2-AS ODN treated tumors when compared to the MDM2-SE ODN or saline solution treated tumors was detected in Western blot analyses and immunohistochemically. In addition, an unexpected reduction in mt-P53 protein expression after AS ODN therapy was also observed. In short, we have demonstrated in vivo for the first time that MDM2-AS ODN treatment of xenotransplanted STS (mt-P53) reduces tumor mass, tumor number, MDM2 protein and mt-P53 protein expression. Our findings support the hypothesis that MDM2-AS ODN treatment may exert a tumor inhibiting effect on all MDM2 expressing tumors regardless of the P53-status, this in turn may be of general importance in gene therapy of cancer.

Survivin, a recently identified inhibitor of apoptosis protein (IAP), is expressed in diverse embryonic tissues and in various 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 measured 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 DeltaEx3 and/or full-length survivin variants but not survivin 2B were identified. Our results suggest that a higher level of survivin mRNA is an independent predictor of survival for STS patients., (Copyright 2001 Wiley-Liss, Inc.)

We analysed the effects of caffeine and taxol on the radiobiological behaviour of two human sarcoma cell lines (RD, SK-LMS-1) each with a p53 missense mutation. Treatment with 2 mM caffeine resulted in an inhibition of the irradiation induced G2/M arrest in both cell lines. This effect was coupled with a radiosensitization in cell line SK-LMS-1 after an irradiation with 6 Gy (enhancement factor of 5.0). However, the effect of radiosensitization was not correlated with an induction of apoptosis. Incubation with 20 nM taxol increased the irradiation induced apoptosis almost 3-fold in cell line SK-LMS-1, but not in cell line RD. However, taxol had no effect on the irradiation induced G2/M arrest or radiosensitivity in either cell line. The results support the hypothesis that the prevention of irradiation induced G2/M arrest but not the induction of apoptosis plays a critical role in determining radiosensitivity in sarcoma cell lines with p53 mutations.

The MDM2 gene encodes a 90-kDa oncoprotein that is overexpressed in several human carcinomas, osteosarcomas, gliomas and soft tissue sarcomas (STSs). This overexpression is the result of several mechanisms, for example, enhanced transcription or translation, gene amplification and alternative splicing. We found that 19 of 67 (28.4%) STS specimens contained an amplified MDM2 gene. The amplification was more likely to be present in grade 1 tumors than in grade 2 or 3 tumors (58% of grade 1 tumors vs. 15% of grade 2 or 3 tumors, p = 0.001, chi(2) test). Furthermore, patients with tumors that contained an amplified MDM2 gene had a survival estimate (87 months) that was longer than that of patients with tumors that lacked an amplified gene (40 months; p = 0.02, log-rank test). Alternatively and aberrantly spliced MDM2 mRNAs were detected in human STSs by a highly sensitive reverse transcription-polymerase chain reaction method. Of 71 tumor samples, 38 (54%) showed evidence of the spliced forms, which included MDM2-A, MDM2-B and several variants exclusively expressed in STSs. A common feature of all forms was the absence of the MDM2 N-terminal region, which includes the TP53-binding region. Furthermore, the presence of the spliced forms was associated with elevated levels of TP53 (p = 0.01, chi(2) test). Although the presence of spliced forms was associated with late-stage tumor phenotypes (p = 0.05, chi(2) test), we observed no relationship between the presence of splice variants and patient outcome., (Copyright 2001 Wiley-Liss, Inc.)

The human MDM2 oncogene, well known as the tumor suppressor gene p53's partner, plays an important role in tumorigenesis whether it is dependent on or independent of TP53. In this study, we investigated in a PCR-sequencing analysis the exon 11 of the human MDM2 gene for gene alterations. A MboII polymorphism occurs in 8% of normal blood donors (8 out of 100 probands) and in 13% of the soft tissue sarcoma patients (11 out of 82 patients). Of note was that two STS patients carried the gene alteration only in the tumor specimens heterozygously but not in normal tissue. In a Kaplan-Meier analysis, patients without the polymorphism, indicated a median survival rate of 57 months, whereas, patients with the polymorphism survived on average only 38 months. We suggest that this polymorphism might be associated with an increased cancer susceptibility.