Expression of microRNA 210 associates with poor survival and age of tumor onset of soft-tissue sarcoma patients.

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.

Co-detection of members of the urokinase plasminogen activator system in tumour tissue and serum correlates with a poor prognosis 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.

Interferon-gamma release assay in the ascites: Early hint for diagnosis of abdominal tuberculosis.

We report on a 20-year-old woman with abdominal tuberculosis.Standard microbiological examination of ascites showed no acid-fast bacilli (AFB), and analysis for the Mycobacterium tuberculosis (MTB)-complex DNA by PCR was negative. However,the interferon-gamma release assay (IGRA) of the ascites was positive after specific stimulation with mycobacterial antigens(ESAT-6/CFP-10/TB7.7[p4]), indicating an infection with MTB.The diagnosis of tuberculosis was later confirmed by histology, MTB culture, and PCR analysis of MTB-complex DNA in tissue samples taken during laparoscopy. Thus, the IGRA of ascites may guide the decision to start active treatment for tuberculosis.

Expression of the stem cell self-renewal gene Hiwi and risk of tumour-related death in patients with soft-tissue sarcoma.

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.

Stem cell-associated genes are extremely poor prognostic factors for soft-tissue sarcoma patients.

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.

The stem cell-associated Hiwi gene in human adenocarcinoma of the pancreas: expression and risk of tumour-related death.

Piwi proteins and their interaction with piRNAs have rapidly emerged as important contributors to gene regulation, indicating their crucial function in germline and stem cell development. However, data on the Hiwi 1 (Hiwi) gene, one of the four human Piwi homologues, are still scarce. Therefore, we investigated the Hiwi mRNA expression in microdissected PDAC tissues from patients with ductal adenocarcinoma of the pancreas (PDAC) by quantitative real-time PCR and the protein expression by immunohistochemistry. Elevated levels of Hiwi mRNA transcripts were measured in 40 out of 56 tissues and a positive immunostaining of Hiwi was detected in tumours of 21 out of 78 patients. There was no general impact of elevated Hiwi mRNA transcript levels or protein expression on survival, as tested by multivariate Cox regression and Kaplan-Meier analysis. However, men showed a significantly increased risk for tumour-related death in case of down- or upregulated expression of Hiwi mRNA (relative risk (RR)=2.78; P=0.034). In summary, we report the first analysis of Hiwi expression in PDAC and its impact on prognosis. We suggest that alterations in mRNA expression of Hiwi can increase the risk of tumour-related death in male PDAC patients.

Histopathological outcome of 597 isolated soft tissue tumors suspected of soft tissue sarcoma: a single-center 12-year experience.

BACKGROUND: The aim of this present report was to analyze the patients referred to us with the presumptive diagnosis of soft tissue sarcoma (STS). METHODS: We reviewed all patients referred to us with suspected soft tissue sarcoma (STS) of the extremities or trunk over a 12-year period. RESULTS: We treated 597 patients with soft tissue tumors. Open biopsy revealed soft tissue sarcoma in 318 cases, benign mesenchymal tumor in 124 cases and isolated metastases (ISTM) from carcinomas in 98 patients; other pathologies were found in 57 patients. The primary carcinomas were lung cancer in 26 patients, breast cancer in 19 patients, renal carcinoma in 16 patients, carcinoma of the esophagus in 12 patients, colonic carcinoma in 5 patients, thyroid gland cancer in 6 patients, and in 14 patients carcinoma of unknown primary was diagnosed. CONCLUSIONS: In our collective with soft tissue tumor, 50% of the patients had the diagnosis of soft tissue sarcoma, 20% presented with a metastasis of carcinoma and 20% had a benign tumor. Referring to our results, in patients with the presumptive diagnosis of soft tissue sarcomas, soft tissue metastasis of a primary carcinoma was unexpectedly common, indicating that greater consideration should be given to this differential diagnosis.

Prognosis is correlated with p53 mutation type for soft tissue sarcoma patients.

We investigated the prognostic value of p53 mutation type for 145 soft tissue sarcoma patients. In a PCR-SSCP-sequencing analysis, 15 mutations were identified: 10 non-frameshift (non-fs) and 5 frameshift (fs) mutations. Patients possessing non-fs mutations had a significantly poorer prognosis than patients without p53 mutations (P = 0.014), according to Cox's multivariate analysis. In contrast, the survival of five patients with fs mutations was not affected by their mutation type. Furthermore, occurrence of lymph node metastases and recurrences was correlated with the mutation type; i.e., 4 of 10 and 5 of 10 patients with non-fs mutations showed lymph node metastases and recurrences, respectively, whereas none of the patients with fs mutations showed lymph node metastases and only one a recurrence. Therefore, for the evaluation of prognosis, we suggest applying not the p53 mutational status in general, but the specific type of mutation.

High prognostic significance of Mdm2/p53 co-overexpression in soft tissue sarcomas of the extremities.

Soft tissue sarcomas are a heterogeneous group of neoplasms with various histological subtypes. Up to now, no individual causal molecular markers for prognosis and therapeutic success have been identified. A tumorigenic connection between the oncogene product Mdm2 and tumor suppressor p53 is generally accepted, but their possible clinical relevance has not yet been investigated sufficiently in soft tissue sarcoma. In 86 primary soft tissue sarcoma of the extremities (RO-resected, T1/2 N0 M0), Mdm2 and p53 overexpression were investigated by immunohistochemistry. The results were adjusted to clinico-pathological characteristics and evaluated for their prognostic relevance by multivariate analysis. In Cox's multivariate analysis with stratification of Mdm2 to p53 results, we determined four groups which had different prognostic values for relapse-free and overall survival (Mdm2-/p53- < Mdm2-/p53+ < Mdm2+/p53- < Mdm2+/p53+). The most striking finding was a relative risk (rr) for overall survival of 18.77 (P=0.006) for patients with Mdm2/p53 co-overexpression (n=40). It is noticeably higher than the additive risk from both factors. Coincident Mdm2/p53 overexpression is an independent molecular marker with the highest prognostic relevance described for soft tissue sarcoma. Thus, a high risk sarcoma group has been defined which we believe requires alternative therapeutic approaches.

Low detection rate of p53 antibodies in sera of soft tissue sarcoma patients.

Accumulation of the p53 gene product can lead to its immunogenic appearance and the generation of p53 serum antibodies (p53ab). In different cancer types the occurrence of detectable p53ab has an independent prognostic impact. In spite of the known p53 protein overexpression rate in soft tissue sarcomas (STS), up to 70%, there have been no investigations done on p53ab in serum in STS patients. In this prospective study of 50 STS patients, we investigated the presence of serum p53ab using an enzyme-linked immunosorbent assay system and the presence of p53 overexpression in the appropriate tissue specimen immunohistochemically. Using Kruskal-Wallis chi(2) and Kaplan-Meier tests the results were then correlated to histopathological and clinical data. Six of the 50 patients (12%) showed p53ab detectable in the serum, and 56% (28/50) of the tumors were p53 immunohistochemically positive. Four of the six p53ab positives (66%) had immunohistochemically p53 positive and two (33%) had negative tumors. Altogether four of the 50 patients (8%) were positive for p53ab in serum as well as for p53 immunohistochemistry in tumor tissue specimens. Twenty patients (40%) were negative for both. All of the p53ab positive patients had stage I or II tumors. Excluding tumor stage there was no p53ab correlation to histopathological, clinical or prognostic parameters. We conclude that in STS patients, p53ab also occurs but in contrast to other tumor types at a relatively low frequency. According to our results, the clinical value of p53ab seems to be limited in STS patients.

Gains in chromosomes 7, 8q, 15q and 17q are characteristic changes in malignant but not in benign peripheral nerve sheath tumors from patients with Recklinghausen's disease.

In order to investigate typical genomic alterations in patients with Recklinghausen's disease (NF1) we studied one from each of the six patients with NF1 several benign and/or malignant tumors. By means of comparative genomic hybridization (CGH) gained results from six benign neurofibromas and 14 malignant peripheral nerve sheath tumors (MPNSTs) were compared with four benign peripheral nerve sheath tumors (BPNSTs) from patients without NF1. In all 14 MPNSTs DNA sequence copy number changes were detected with a mean value of 13.5 imbalances per sample. The most frequent gains were in 8q, 17q (12 tumors each), 7p, 15q (ten tumors each), and 7q (nine tumors). We found ten high-level amplifications in nine of the 14 samples. In two cases, the high-level amplification involved 7p14-pter and 17q24-qter as well. The most frequent loss was in 17p (seven tumors). The benign neurofibromas from NF1-patients and the sporadic BPNSTs revealed only partially DNA sequence copy number changes without any distinct pattern. The gains of #7, 8q, 15q, and 17q were found exclusively in MPNSTs but not in neurofibromas and are supposed to be associated with malignant tumor progression. In comparison of the results of the 14 MPNSTs from NF1-patients with the results of previously published 20 sporadic MPNSTs, we found that the gain of 8q occurs most frequently in both tumor groups. Of course additionally in the sporadic MPNSTs there were more frequent gains of 5p, #6, and statistically significant gains of 20q. On the other hand in the MPNSTs from NF1-patients the most frequent gains were found in #7, and statistically significant in 15q, and 17q.

How is the mutational status for tumor suppressors p53 and p16(INK4A) in MFH of the bone?

Both tumor suppressor genes p53 and p16(INK4A) play a crucial role in the control of cell cycle and tumor development. In this study 19 malignant fibrous histiocytomas of the bone (MFH-b), a very rare sarcoma entity, were investigated for mutations in p53 and p16 genes by a PCR-SSCP-sequencing analysis. In the tumor samples two p53 mutations and two polymorphisms (one in the p53 gene and one in the p16 gene) were found. The occurrence rate for p53 mutations and the absence of p16 mutations in MFH-b are comparable to the findings for MFH of soft tissues (MFH-st) and osteosarcomas, suggesting that p53 rather than p16 may play a role in tumorigenesis of MFH-b.

Clinical relevance of pRb and p53 co-overexpression in soft tissue sarcomas.

The goal of this study was to examine the relationship between immunohistochemical pRb detectability and p53 overexpression in 198 soft tissue sarcomas (STS) with regard to its clinical relevance. Distinct pRb detectability multivariately shows a correlation to survival rate (relative risk (RR)=1.59, P=0.037). p53 positivity was also multivariately correlated to poor prognosis (RR=2.17, P=0.0014). Stratification of pRb staining to p53 results shows a prognostical graduation. Patients with negativity for both proteins have the most favorable prognosis (projected 5-year survival rate (psr)=54.5%). In contrast to this, positivity for both antibodies has the highest risk (RR=2.48, P=0.02) and the poorest prognosis (psr=17.4%). To conclude, these results explain that the clinical relevance of immunohistochemical pRb positivity in STS is connected with p53 in the form of having an increasing effect on the known prognostic relevance of p53 overexpression.

Colony formation of soft tissue sarcoma cells is inhibited by lipid-mediated antisense oligodeoxynucleotides targeting the human mdm2 oncogene.

More than one third of human soft tissue sarcoma (STS) have elevated levels of the MDM2 oncoprotein, resulting either from gene amplification or alternate mechanisms. MDM2 functions as a negative feedback regulator of the tumor suppressor p53. The aim of the present study was to investigate whether mdm2-antisense oligodeoxyribonucleotides (AS-ODNs) can influence the growth characteristics of two MDM2-overexpressing STS cell lines (US8-93, LMS6-93) where both have heterozygous p53 non-missense mutations. Cells were treated with lipofectamine-complexed mdm2 AS-ODNs complementary to a sequence of the mdm2 cDNA initiation site in comparison to sense control ODNs. After seeding and cultivation of a defined cell number the clonogenic survival was performed. The treatment of US8-93 cells with AS-ODNs, but not with sense ODNs, decreased the number of colonies up to > 80%. Western blot analysis demonstrated a significant decreasing of MDM2 protein level in AS-ODN transfected cells indicating an AS-specific inhibition of mdm2 transcription in US8-93 cells. Additionally, an increase of the G2/M population was found. In contrast, in the LMS6-93 cells treated with AS-ODNs only a decrease in clonogenic survival up to 26%, no change in MDM2 protein level and no cell cycle alterations were seen. All these factors taken together into consideration can be suggest that lipid-mediated mdm2 AS-ODNs could be as an effective therapeutic strategy for STS with an abnormal mdm2 overexpression.

Expression of alternatively and aberrantly spliced transcripts of the MDM2 mRNA is not tumor-specific.

The MDM2 proto-oncogene encodes a 90-kDa protein that binds to and inactivates the tumor suppressor p53. Several reports describe the presence of different alternatively, as well as, aberrantly spliced transcripts of the MDM2 mRNA in a variety of human cancers that have lost the ability to bind p53. Due to the transforming ability of at least some of the isoforms it has been suggested that they might contribute to tumorigenesis. Here we show that shorter MDM2 transcripts are also widely expressed in normal tissues, including lung and renal tissue, and in lymphocytes. Alteration in MDM2 RNA transcripts were found in the majority of the samples. Although we cannot exclude that alterations in MDM2 preferentially occur during cancer development, our data rather indicate that in this context the commonly observed transcript variants may also possess a normal physiological function.

Detection of numerical chromosomal changes in 20 malignant fibrous histiocytomas by FISH.

We investigated 20 malignant fibrous histiocytomas (MFHs) with the help of specific centromeric probes for chromosomes 1, 3, 4, 6, 8, 9, 12, 16, 17 and 18. The results show a broad variation in the number of signals per nucleus. However, tumors can be assigned into four groups: i) with mostly disomic clones, ii) with a high percentage of polysomic clones, iii) with a considerable amount of monosomic and nullisomic clones and iv) with a tendency in both directions. A gain of spots per nucleus takes place in 75-100% of the investigated tumors - the highest incidence occurring with respect to chromosome 3. A loss of spots per nucleus occurred in 20-60% of the tumors - predominantly with respect to chromosome 1.

Morphological and molecular characterization of an undifferentiated soft tissue sarcoma cell line and derivative clones.

From an undifferentiated soft tissue sarcoma (STS) a cell line designated US8-93 has been established. At subcloning the cell line US8-93 three different lines (US8-93A, B and C) could be set up. In a subsequent study characteristics for ultrastructure, growth, cell cycle distribution, karyotype, protein overexpression detected by immunohistochemistry (IHC) and p53 mutational status were determined. The cell line US8-93 as well as subclones contain mainly bipolar spindle-shaped cells and additionally some polygonal and multinucleated cells. Cells possess the characteristics of primitive mesenchymal cells based on their positive reactions with anti-vimentin and negative reactions for desmin, cytokeratin, myoglobin, S100, and NSE, implying a classification as an undifferentiated STS. Cytogenetic analysis revealed nearly diploid cells with several structural and numerical aberrations for chromosomes 1, 3, 4, 6, 9, 10, 12, 13, 15 and 18. IHC positivity was found for the tumor suppressor proteins p53 and Rb, the oncogene products Bcl-2, K-ras, N-ras, P-glycoprotein Mdr-1 and MDM-2. In the p53 gene a nonsense mutation in exon 4 was detected, that was confirmed in the original primary tumor and in three derivative clonal lines. The described STS cell line represents a valuable supplementation to the relatively small number of human STS cell lines currently available and may also provide a good in vitro model for studies of STS tumorigenesis in respect to a mutated p53 gene.

Radiation induced G2/M block and apoptosis in two human sarcoma cell lines with different p53 gene status.

We investigated a wild-type (wt) p53 rhabdomyosarcoma (A-204) and a mutated (mt p53) undifferentiated sarcoma cell line (US8-93) for their response to X-rays. The observation period was 0 to 96 h after irradiation. Both cell lines showed a strikingly delayed G2/M arrest and an induction of apoptosis after irradiation. Compared with the cell line A-204 (wt p53), the cell line US8-93 (mt p53) revealed a stronger G2/M arrest. In agreement with this, in terms of viability as well as the rate of apoptosis, A-204 (wt p53) showed a stronger response to irradiation than US8-93 (mt p53). We suggest that the different p53 gene status might be the cause for a different response to irradiation.

Molecular characterization and liposomal transfection of a p53-mutated cell line established from a poorly differentiated leiomyosarcoma.

A human cell line LMS6-93 has been established from a leiomyosarcoma (LMS). Characteristics for ultrastructure, growth characteristics, cell cycle distribution, karyotype, protein expression detected by immunohistochemistry (IHC), p53 mutational status and liposomal transfection behaviour were studied and determined. The primary tumor was clearly positive for á-smooth muscle type actin and desmin in moderately differentiated areas and indicated a loss of myogenic differentiation in other regions and therefore was classified as a poorly differentiated LMS. The cell line LMS6-93 contains mainly polymorphic spindle shaped or polygonal tumor cells which possess the characteristics of primitive mesenchymal cells, based on their morphology and positive reaction with an antibody to vimentin. IHC staining for S100, synaptophysin A, NSE, neurofilament proteins and cytokeratins were negative. Cytogenetic analysis revealed in the cell line diploid karyotypes comparatively close to several structural and numerical aberrations for chromosomes 2, 5, 6, 9, 10, 12, 14, 17, 18, 20, 22, and Y. IHC positivity was found for the tumor suppressor protein Rb and the oncogene product MDM2. In a p53 mutational analysis a 1 bp insertional mutation in exon 6 (G insertion in codon 215) was detected and confirmed in the original primary tumor. The other p53 allele appears to be wild-type as indicated in Western hybridization. Using different cationic lipid formulations complexed with a reporter expression vector (GFP) successful transfection into LMS6-93 cells was observed. The highest transfection rates (20-30% GFP expression in the viable cell population) were obtained with lipofectin. These results suggest that LMS6-93 functions as a good in vitro model for transfection studies on an LMS cell line carrying a heterozygous p53-frameshift mutation.

Expression of cathepsin B, D and L protein in juvenile idiopathic arthritis.

Juvenile idiopathic arthritis (JIA) is the most common childhood autoimmune rheumatic disease and like rheumatoid arthritis (RA), it is characterized by inflammation and the progressive destruction of joints. In RA, cathepsins as proteinases play a major role in destroying synovial tissue and cartilage matrix. So far no data on cathepsin expression in pannus tissue of HA patients exist. The aim of this study was to characterize the expression levels of cathepsins B, D, H, and L in HA and to compare them with those in RA. Synovectomy tissue from 16 HA and 12 RA patients was investigated for cathepsin expression levels by Western blot analysis. Expression of cathepsins B, D and L was on comparable levels in the synovectomy tissue of HA and RA patients. The following graduation of expression was determined: cathepsin D > cathepsin L > cathepsin B. Cathepsin H was neither found to be expressed in HA nor in RA patients. The expression levels of cathepsins in pannus tissue showed no clear difference between patients with systemic JIA and patients with monoarticular JIA. In summary, the comparable expression of cathepsins B, D and L in RA and JIA synovectomy tissue suggests that they may play a similarly important role in destroying synovial tissue and cartilage matrix in the course of HA and RA.