Bioactive Glass Inhibits Tumor Development from Giant Cell Tumor of Bone-Derived Neoplastic Stromal Cells in a Chicken Chorioallantoic Membrane Assay.

Tumor recurrence is a major problem during the treatment of giant cell tumors of bone (GCTB). We recently identified tumor cell-specific cytotoxic effects of bioactive glasses (BGs) toward neoplastic stromal cells derived from GCTB tissue (GCTSCs) in vitro. Since these data indicated a promising role of BGs in the adjuvant treatment of GCTBs, we aimed to investigate the transferability of the in vitro data into the more complex in vivo situation in the current study. We first analyzed the cytotoxicity of three different BGs in vitro by WST-1 assay after co-cultivation with primary GCTSC cell lines. The effects of BGs on tumor engraftment and growth were analyzed by chicken chorioallantoic membrane (CAM) assays and subsequent quantification of tumor take rates and tumor volumes. In vitro, all tested BGs displayed a cytotoxic effect on GCTSCs that was dependent on BG composition, concentration, and particle size. Comparable effects could be observed within the in vivo environment resulting in reduced tumor take rates and tumor volumes in BG-treated samples. These data indicate a possible clinical application of BGs in the context of GCTB therapy, mediating a reduction of recurrence rates with the simultaneous promotion of bone regeneration.

The Warburg Trap: A Novel Therapeutic Approach for Targeting Osteosarcoma.

Although urgently needed, no significant improvements in osteosarcoma (OS) therapy have been achieved within the last decades. Here, we present a new therapeutic approach based on drug combinations consisting of mitochondrial complex I (MCI) inhibitors and ionophores that induce cancer cell-specific cell death based on a modulation of cellular energy metabolism and intracellular pH (pHi) named the Warburg Trap (WT). The effects of several drug combinations on intracellular pH, cell viability, colony-forming capacity and expression of WNT-target genes were analysed using OS cell lines and primary human osteoblasts (HOB). Tumour take rates and tumour volumes were analysed in vivo using a chicken chorioallantoic membrane assay (CAM). Several WT drug combinations induced the intracellular acidification and apoptotic cell death in OS cells, whereas HOBs tolerated the treatment. A significant inhibition of the colony-forming ability of OS cells and downregulation of WNT-target genes suggest that cancer stem cells (CSCs) are also targeted by the WT approach. In vivo, we observed a significant reduction in the tumour take rates in response to WT drug treatment. Our data suggest that the Warburg Trap is a promising approach for the development of a novel and effective OS therapy to replace or supplement the current OS chemotherapy.

Bioactive glass selectively promotes cytotoxicity towards giant cell tumor of bone derived neoplastic stromal cells and induces MAPK signalling dependent autophagy.

Giant cell tumors of bone (GCTB) are associated with massive bone destructions and high recurrence rates. In a previous study, we observed cytotoxic effects of three different compositions of bioactive glasses (BGs) towards GCTSC but not bone marrow derived stromal cells (BMSC) indicating that BGs represent promising candidates for the development of new therapeutic approaches. In the current study we aimed to investigate the molecular mechanisms that are involved in BG induced cytotoxicity. We observed, that BG treatment was not associated with any signs of apoptosis, but rather led to a strong induction of mitogen activated protein kinases (MAPK) and, as a consequence, upregulation of several transcription factors specifically in GCTSC. Genome wide gene expression profiling further revealed a set of fifteen genes that were exclusively induced in GCTSC or induced significantly stronger in GCTSC compared to BMSC. BG treatment further induced autophagy that was significantly more pronounced in GCTSC compared to BMSC and could be inhibited by MAPK inhibitors. Together with the known osteogenic properties of BGs our findings support the suitability of BGs as therapeutic agents for the treatment of GCTB. However, these data have to be verified under in vivo conditions.

Combined Fluorescence-Based in Vitro Assay for the Simultaneous Detection of Cell Viability and Alkaline Phosphatase Activity during Osteogenic Differentiation of Osteoblast Precursor Cells.

Novel bone substitute materials need to be evaluated in terms of their osteogenic differentiation capacity and possible unwanted cytotoxic effects in order to identify promising candidates for the therapy of bone defects. The activity of alkaline phosphatase (ALP) is frequently quantified as an osteogenic marker, while various colorimetric assays, like MTT assay, are used to monitor cell viability. In addition, the DNA or protein content of the samples needs to be quantified for normalization purposes. As this approach is time consuming and often requires the analysis of multiple samples, we aimed to simplify this process and established a protocol for the combined fluorescence-based quantification of ALP activity and cell viability within one single measurement. We demonstrate that the fluorogenic substrate 4-methylumbelliferone-phosphate (4-MUP) and the commonly used para-nitrophenylphosphate (p-NPP) produce comparable and highly correlating results. We further show that fluorescein-diacetate (FDA) can be used to quantify both cell viability and cell number without interfering with the quantification of ALP activity. The measurement of additional normalization parameters is, therefore, unnecessary. Therefore, the presented assay allows for a time-efficient, simple and reliable analysis of both ALP activity and cell viability from one sample and might facilitate experiments evaluating the osteogenic differentiation of osteoblast precursor cells.

Human Platelet Lysate Can Replace Fetal Calf Serum as a Protein Source to Promote Expansion and Osteogenic Differentiation of Human Bone-Marrow-Derived Mesenchymal Stromal Cells.

Fetal calf serum (FCS) is frequently used as a growth factor and protein source in bone-marrow-derived mesenchymal stromal cell (BMSC) culture media, although it is a xenogenic product presenting multiple disadvantages including but not limited to ethical concerns. A promising alternative for FCS is human platelet lysate (hPL), which is produced out of human platelet concentrates and happens to be a stable and reliable protein source. In this study, we investigated the influence of hPL in an expansion medium (ESM) and an osteogenic differentiation medium (ODM) on the proliferation and osteogenic differentiation capacity of human BMSC. Therefore, we assessed population doublings during cell expansion, performed alizarin red staining to evaluate the calcium content in the extracellular matrix and determined the activity of alkaline phosphatase (ALP) as osteogenic differentiation correlates. The proliferation rate of BMSC cultured in ESM supplemented with hPL exceeded the proliferation rate of BMSC cultured in the presence of FCS. Furthermore, the calcium content and ALP activity was significantly higher in samples incubated in hPL-supplemented ODM, especially in the early phases of differentiation. Our results show that hPL can replace FCS as a protein supplier in cell culture media and does not negatively affect the osteogenic differentiation capacity of BMSC.

Tumor Suppressor Function of miR-127-3p and miR-376a-3p in Osteosarcoma Cells.

Since the introduction of high-dose chemotherapy about 35 years ago, survival rates of osteosarcoma patients have not been significantly improved. New therapeutic strategies replacing or complementing conventional chemotherapy are therefore urgently required. MicroRNAs represent promising targets for such new therapies, as they are involved in the pathology of multiple types of cancer, and aberrant expression of several miRNAs has already been shown in osteosarcoma. In this study, we identified silencing of miR-127-3p and miR-376a-3p in osteosarcoma cell lines and tissues and investigated their role as potential tumor suppressors in vitro and in vivo. Transfection of osteosarcoma cells (n = 6) with miR-127-3p and miR-376a-3p mimics significantly inhibited proliferation and reduced the colony formation capacity of these cells. In contrast, we could not detect any influence of miRNA restoration on cell cycle and apoptosis induction. The effects of candidate miRNA restoration on tumor engraftment and growth in vivo were analyzed using a chicken chorioallantoic membrane (CAM) assay. Cells transfected with mir-127-3p and miR-376a-3p showed reduced tumor take rates and tumor volumes and a significant decrease of the cumulative tumor volumes to 41% and 54% compared to wildtype cells. The observed tumor suppressor function of both analyzed miRNAs indicates these miRNAs as potentially valuable targets for the development of new therapeutic strategies for the treatment of osteosarcoma.

MicroRNA-365a-3p inhibits c-Rel-mediated NF-κB signaling and the progression of pancreatic cancer.

NF-κB contributes to the aggressiveness of pancreatic ductal adenocarcinoma (PDA), which is counteracted by the bioactive agent sulforaphane. We investigated sulforaphane-induced microRNA signaling and its influence on progression features. Using established cell lines, microRNA and gene arrays, we predicted miR-365a as the top candidate for the sulforaphane-induced inhibition of the NF-κB subunit c-Rel. The lipofection of miR-365a-3p mimics inhibited the luciferase activity of a c-Rel 3'-UTR construct, as well as c-Rel expression, NF-κB activity, and tumor viability, migration, and clonogenicity, whereas apoptosis was induced. In vivo, miR-365a-3p reduced the volume of tumor xenografts and the expression of progression markers. In a tissue array, the expression of miR-365a-3p was absent in almost all 91 malignant tissues but not in 5 normal tissues, thus confirming the previous results. Our observations suggest that sulforaphane-induced miR-365a-3p expression inhibits NF-κB activity by downregulating c-Rel, which prevents the progression of PDA.

Outcome of conservative and surgical treatment of enchondromas and atypical cartilaginous tumors of the long bones: retrospective analysis of 228 patients.

BACKGROUND: Sufficient data on outcome of patients with clinically and radiologically aggressive enchondromas and atypical cartilaginous tumors (ACT) is lacking. We therefore analyzed both conservatively and surgically treated patients with lesions, which were not distinguishable between benign enchondroma and low-grade malignant ACT based upon clinical and radiologic appearance. METHODS: The series included 228 consecutive cases with a follow-up > 24 months to assess radiological, histological, and clinical outcome including recurrences and complications. Pain, satisfaction, functional limitations, and the musculoskeletal tumor society (MSTS) score were evaluated to judge both function and emotional acceptance at final follow-up. RESULTS: Follow-up took place at a mean of 82 (median 75) months. The 228 patients all had comparable clinical and radiological findings. Of these, 153 patients were treated conservatively, while the other 75 patients underwent intralesional curettage. Besides clinical and radiological aggressiveness, most lesions were histologically judged as benign enchondromas. 9 cases were determined to be ACT, while the remaining 7 cases had indeterminate histology. After surgery, three patients developed a recurrence, and a further seven had complications of which six were related to osteosynthesis. Both groups had excellent and almost equal MSTS scores of 96 and 97%, respectively, but significantly less functional limitations were found in the non-surgery group. Further sub-analyses were performed to reduce selection bias. Sub-analysis of histologically diagnosed enchondromas in the surgery group found more pain, less function, and worse MSTS score compared to the non-surgery group. Sub-analysis of smaller lesions (< 4.4 cm) did not show significant differences. In contrast, larger lesions displayed significantly worse results after surgery compared to conservative treatment (enchondromas > 4.4 cm: MSTS score: 94.0% versus 97.3%, p = 0.007; pain 2.3 versus 0.8, p = 0.001). The majority of lesions treated surgically was filled with polymethylmethacrylate bone-cement, while the remainder was filled with cancellous-bone, without significant difference in clinical outcome. CONCLUSION: Feasibility of intralesional curettage strategies for symptomatic benign to low-grade malignant chondrogenic tumors was supported. Surgery, however, did not prove superior compared to conservative clinical and radiological observation. Due to the low risk of transformation into higher-grade tumors and better functional results, more lesions might just be observed if continuous follow-up is assured.

Enchondromas and atypical cartilaginous tumors at the proximal humerus treated with intralesional resection and bone cement filling with or without osteosynthesis: retrospective analysis of 42 cases with 6 years mean follow-up.

BACKGROUND: Enchondromas and atypical cartilaginous tumors (ACT) are often located at the proximal humerus. Most lesions can be followed conservatively, but surgical resection may alleviate pain, avoid pathological fractures, and prevent transformation into higher grade chondrosarcomas. Rigorous intralesional resection and filling with polymethylmethacrylate bone cement has been proposed for enchondromas but also for ACT, as an alternative for extralesional resection. We intended to analyze radiological, clinical, and functional outcome of this strategy and compare bone cement without osteosynthesis to bone cement compound osteosynthesis, which has not been analyzed so far. METHODS: We retrospectively analyzed 42 consecutive patients (mean follow-up 73 months; range 8-224) after curettage and bone cement filling with or without osteosynthesis. Exclusion criteria were Ollier's disease and cancellous bone filling. Twenty-five patients only received bone cement. Seventeen patients received additional proximal humerus plate for compound osteosynthesis to increase stability after curettage. Demographics and radiological and clinical outcome were analyzed including surgery time, blood loss, hospitalization, recurrences, and complications. An additional telephone interview at the final follow-up assessed postoperative satisfaction, pain, and function in the quick disabilities of the arm, shoulder, and hand (DASH) score and the Musculoskeletal Tumor Society (MSTS) score. Statistics included the Student T tests, Mann-Whitney U tests, and chi-square tests. RESULTS: No osteosynthesis compared to compound osteosynthesis showed smaller tumors (4.2 (± 1.5) cm versus 6.6 (± 3.0) cm; p = 0.005) and smaller bone cement fillings after curettage (5.7 (± 2.1) cm versus 9.6 (± 3.2) cm; p = 0.0001). A score evaluating preoperative scalloping and soft-tissue extension did not significantly differ (1.9 (± 0.9) versus 2.0 (± 1.0); rating scale 0-4; p = 0.7). Both groups showed high satisfaction (9.2 (± 1.5) versus 9.2 (± 0.9); p = 0.5) and low pain (1.0(±1.7) versus 1.9(±1.8); p = 0.1) in a rating scale from 0 to 10. Clinical and functional outcome was excellent for both groups in the DASH score (6.0 (± 11.8) versus 11.0 (± 13.2); rating scale 0-100; p = 0.2) and the MSTS score (29.0 (± 1.7) versus 28.7 (± 1.1); rating scale 0-30; p = 0.3). One enchondroma recurrence was found in the group without osteosynthesis. Complications (one fracture and one intra-articular screw) were only detected after osteosynthesis. Osteosynthesis had longer surgery time (70 (± 21) min versus 127 (± 22) min; p < 0.0001), more blood loss (220 (± 130) ml versus 460 (± 210) ml; p < 0.0001), and longer stay in the hospital (6 (± 2) days versus 8 (± 2) days; p = 0.004). CONCLUSIONS: Intralesional tumor resection was oncologically safe and clinically successful with or without osteosynthesis. Osteosynthesis did not reduce the risk for fracture but was more invasive.

microRNA-210 overexpression inhibits tumor growth and potentially reverses gemcitabine resistance in pancreatic cancer.

Resistance to first-line chemotherapies like gemcitabine contributes to high disease lethality in pancreatic cancer. By microarray and qRT-PCR, we observed significant downregulation of microRNA-210 in gemcitabine-resistant cells. The overexpression of microRNA-210 was toxic to gemcitabine-resistant cells and enhanced gemcitabine sensitivity. MicroRNA-210 overexpression induced caspase-3-mediated apoptosis, and inhibited colony formation. Computationally, ABCC5, a highly expressed gene in our array data, was identified as a potential target of microRNA-210 and the overexpression of ABCC5 in gemcitabine-resistant cells was confirmed by qRT-PCR. MicroRNA-210 overexpression reduced ABCC5 mRNA levels and inhibited a luciferase reporter expressing the ABCC5 3' UTR. The expression pattern of microRNA-210 and ABCC5 was mirrored in all of 5 pancreatic cancer cell lines used. Likewise, microRNA-210 transfection nearly totally inhibited tumor xenograft growth, proliferation and metastasis without obvious side effects in vivo. Also, an absence or low expression of microRNA-210 correlated to high ABCC5 expression in the majority of malignant patient tissues from a total of 101 patient tissues examined. Our observations provide at first glance, an important function for microRNA-210 in regulation of gemcitabine responsiveness by it's target gene ABCC5.

CD8+/FOXP3+-ratio in osteosarcoma microenvironment separates survivors from non-survivors: a multicenter validated retrospective study.

Osteosarcoma is the most common primary bone tumor characterized by juvenile onset, tumor heterogeneity, and early pulmonary metastasis. Therapeutic improvement stagnates since more than two decades. Unlike major malignancies, biomarkers as prognostic factors at time of diagnosis are missing. Disease rareness hampers study recruitment of patient numbers sufficient to outweigh tumor heterogeneity. Here, we analyzed in a multicenter cohort the osteosarcoma microenvironment to reduce effects of tumor cell heterogeneity. We hypothesized that quantitative ratios of intratumoral CD8+T-cells to FOXP3+T-cells (CD8+/FOXP3+-ratios) provide strong prognostic information when analyzed by whole-slide imaging in diagnostic biopsies. We followed recommendations-for-tumor-marker-prognostic-studies (REMARK). From 150 included cases, patients with complete treatment were identified and assigned to the discovery (diagnosis before 2004) or the validation cohort (diagnosis 2004-2012). Highly standardized immunohistochemistry of CD8+ and FOXP3+, which was validated by methylation-specific gene analysis, was performed followed by whole-slide analysis and clinical outcome correlations. We observed improved estimated survival in patients with CD8+/FOXP3+-ratios above the median (3.08) compared to patients with lower CD8+/FOXP3+-ratios (p = 0.000001). No patients with a CD8+/FOXP3+-ratio above the third quartile died within the observation period (median follow-up 69 mo). Multivariate analysis demonstrated independence from current prognostic factors including metastasis and response to neoadjuvant chemotherapy. Data from an independent validation cohort confirmed improved survival (p = 0.001) in patients with CD8+/FOXP3+-ratios above 3.08. Multivariate analysis proofed that this observation was also independent from prognostic factors at diagnosis within the validation cohort. Intratumoral CD8+/FOXP3+-ratio in pretreatment biopsies separates patients with prolonged survival from non-survivors in osteosarcoma.

Improved survival in osteosarcoma patients with atypical low vascularization.

BACKGROUND: Osteosarcoma is considered a highly vascularized bone tumor with early metastatic dissemination through intratumoral blood vessels mostly into the lung. Novel targets for therapy such as tumor vascularization are highly warranted since little progress has been achieved in the last 30 years. However, proof of relevance for vascularization as a major prognostic parameter has been hampered by tumor heterogeneity, difficulty in detecting microvessels by immunohistochemistry, and small study cohorts. Most recently, we demonstrated that highly standardized whole-slide imaging could overcome these limitations (Kunz et al., PloS One 9(3):e90727, 2014). In this study, we applied this method to a multicenter cohort of 131 osteosarcoma patients to test osteosarcoma vascularization as a prognostic determinant. METHODS: Computer-assisted whole-slide analysis, together with enzymatic epitope retrieval, was used for CD31-based microvessel quantification in 131 pretreatment formalin-fixed and paraffin-embedded biopsies from three bone tumor centers. Kaplan-Meier-estimated survival and chemoresponse were determined and multivariate analysis was performed. Conventional hot-spot-based microvessel density (MVD) determination was compared with whole-slide imaging. RESULTS: We detected high estimated overall (p ≤ 0.008) and relapse-free (p ≤ 0.004) survival in 25 % of osteosarcoma patients with low osteosarcoma vascularization in contrast to other patient groups. Furthermore, all patients with low osteosarcoma vascularization showed a good response to neoadjuvant chemotherapy. Comparison of conventional MVD determination with whole-slide imaging suggests false high quantification or even exclusion of samples with low osteosarcoma vascularization due to difficult CD31 detection in previous studies. CONCLUSION: Low intratumoral vascularization at the time of diagnosis is a strong predictor for prolonged survival and good response to neoadjuvant chemotherapy in osteosarcoma.

Epigenetic silencing of genes and microRNAs within the imprinted Dlk1-Dio3 region at human chromosome 14.32 in giant cell tumor of bone.

BACKGROUND: Growing evidence exists that the neoplastic stromal cell population (GCTSC) within giant cell tumors (GCT) originates from mesenchymal stem cells (MSC). In a previous study we identified a microRNA signature that differentiates between these cell types. Five differentially expressed microRNAs are located within the Dlk1-Dio3 region on chromosome 14. Aberrant regulation within this region is known to influence cell growth, differentiation and the development of cancer. The aim of this study was to elucidate the involvement of deregulations within the Dlk1-Dio3 region in GCT pathogenesis. METHODS: Quantitative gene and microRNA expression analyses were performed on GCTSCs and MSCs with or without treatment with epigenetic modifiers. Methylation analysis of differentially methylated regions was performed by bisulfite sequencing. RESULTS: In addition to microRNA silencing we detected a significant downregulation of Dlk1, Meg3 and Meg8 in GCTSCs compared to MSCs. DNA methylation analyses of the Meg3-DMR and IG-DMR revealed a frequent hypermethylation within the IG-DMR in GCTs. Epigenetic modification could restore expression of some but not all analyzed genes and microRNAs suggesting further regulatory mechanisms. CONCLUSION: Epigenetic silencing of genes and microRNAs within the Dlk1-Dio3 region is a common event in GCTSCs, in part mediated by hypermethylation within the IG-DMR. The identified genes, micro RNAs and microRNA target genes might be valuable targets for the development of improved strategies for GCT diagnosis and therapy.

A microRNA signature differentiates between giant cell tumor derived neoplastic stromal cells and mesenchymal stem cells.

Giant cell tumor (GCT) derived stromal cells (GCTSCs) have been identified as the neoplastic cell population of GCTs. Within these stromal cells a subpopulation has been identified that shares several features with mesenchymal stem cells (MSCs) indicating that these neoplastic cells develop from MSCs. Although spontaneous transformations of MSC have already been observed in vitro and in vivo the underlying molecular mechanisms are poorly understood. As microRNAs are crucially involved in tumorigenesis and the modulation of stem cell fate and behavior, they represent promising candidates for the regulation of this process. Therefore, the aim of this study was the comparative analysis of the microRNA expression profiles of GCTSCs and MSCs in order to identify differentially expressed microRNAs and their target genes. We could identify a microRNA signature consisting of 26 differentially expressed microRNAs that perfectly separates these two cell types. One of the microRNAs with the most pronounced differences in expression levels was miR-224. We could confirm the already known regulation of the apoptosis inhibitor API5 by miR-224 and could further identify three novel miR-224 target genes (SMAD5, SLMAP, H3.3B). The involvement of these genes in the regulation of apoptosis resistance, proliferation, differentiation and the regulation of gene transcription suggests pivotal roles of these genes in the neoplastic transformation of MSCs during GCT development.

Xenogenic transplantation of human mesenchymal stem cells in a critical size defect of the sheep tibia for bone regeneration.

Mesenchymal stem cells (MSCs) represent an attractive cell population for the regeneration of mesenchymal tissues. Their special immunological characteristics suggest that MSCs could be used in nonautologous applications. In this study, the regenerative capacity of human and ovine MSCs was assessed in an ovine critical size defect model. Human and ovine MSCs from bone marrow were cultured on mineralized collagen and implanted into a 3.0-cm-long sheep tibia bone defect (n = 7). Unloaded mineralized collagen served as control. Bone healing was assessed until euthanasia 26 weeks after surgery by radiology and histologically after euthanasia. The presence of human cells after xenogenic transplantation was analyzed using human-specific in situ hybridization. Both radiology and histology demonstrated significantly better bone formation after transplantation of autologous ovine MSCs on mineralized collagen compared with unloaded matrices and with the xenogenic treatment group. Nevertheless, no local or systemic rejection reactions could be observed after transplantation of human MSCs, although the presence of human MSCs could be demonstrated. It can be concluded that despite successful demonstration of the presence of human MSCs after xenogenic transplantation, the xenogenic transplantation of human MSCs leads to poorer bone regeneration than autologous transplantation of ovine MSCs.

Expression of TRAIL and the death receptors DR4 and DR5 correlates with progression of degeneration in human intervertebral disks.

Intervertebral disks degenerate far earlier than other musculoskeletal tissues and apoptosis has been suggested to have a vital function in promoting the degeneration process that is strongly associated with back pain. However, the molecular mediators of apoptosis in the intervertebral disk are poorly understood. Fas/FasL, TRAIL/DR4, TRAIL/DR5 and TNF-alpha/TNFR1 are ligand/receptor pairs of the tumor necrosis factor/nerve growth factor family, which are able to induce apoptosis by trimerization of the receptor by its corresponding ligand. We investigated which of these molecules are expressed in intervertebral disks and whether their expression correlates to disk degeneration. Intervertebral disks from 28 donors (age 12-70 years) suffering from scoliosis, vertebrae fracture or disk degeneration were scored histologically for degeneration and analyzed for gene expression of FasL/Fas, TRAIL/DR4, TNF-alpha/TNFR1 and caspase 8. Protein expression of FasL and TRAIL was assessed by immunohistology and apoptotic cell death was quantified by poly(ADP-ribose) polymerase (PARP) p85 staining. Isolated disk cells were analyzed by flow cytometry for Fas, FasL, TRAIL, DR4 and DR5 expression. Gene expression of TRAIL (P=0.002) and caspase 8 (P=0.027) significantly correlated with degeneration. TRAIL expression further correlated with cellularity (P=0.04), muccoid matrix changes (P=0.009) and tears and cleft formation (P=0.019). FasL and TRAIL expression was confirmed by immunohistology and PARP cleavage was significantly associated with degeneration (P=0.027). Flow cytometry on isolated disk cells revealed correlations between DR4 and degeneration (P=0.014), DR4/DR5 double-positive cells and degeneration (P=0.019), as well as DR5 and changes in tissue granularity (P=0.03). This is the first study that shows that intervertebral disk cells express TRAIL, DR4 and DR5, which correlate to the degenerative state of the disk. Therefore, disk cells inherit the molecular machinery to induce and undergo cellular apoptosis, and the frequency of cytokine expression suggests that the TRAIL/DR4/DR5 axis is an important molecular mediator of apoptosis induction in disk tissue.

Prognostic significance of drug-regulated genes in high-grade osteosarcoma.

About 25-45% of patients with high-grade osteosarcoma poorly respond to chemotherapy with an increased risk of relapse and the development of metastasis. Therefore, the aim of this study was the evaluation of the prognostic value of eight previously identified drug-regulated candidate genes on osteosarcoma therapy outcome. Gene expression of 8 candidate genes was analyzed in 35 formalin-fixed, paraffin-embedded, laser-microdissected osteosarcoma biopsies. The prognostic value of these genes was evaluated by the correlation of gene expression with therapy outcome, overall survival and event-free survival in univariate and multivariate analysis. Upon univariate analysis, the expression of MALAT-1, IMPDH2, FTL and RHOA significantly correlated with response to chemotherapy. Expression of all four genes was increased in the poor responder group. Upon multivariate analysis, IMPDH2 maintained its independent prognostic value (P=0.025). Concerning the overall survival of the patients, we observed a significant association with the expression of FTL, PHB, ATAD2, ACTN1 and RRM2 as well as lactate dehydrogenase serum levels. In the subgroups of patients with high expression of these genes and those with elevated lactate dehydrogenase levels, the mean overall survival was decreased 1.7-, 1.9-, 2.2-, 2.4-, 1.5- and 4.5-fold, respectively. Except RRM2, all genes and lactate dehydrogenase serum levels remained significant in the multivariate analysis. In addition, the event-free survival was significantly decreased in the subgroups of patients with high FTL, ATAD2 and IMPDH2 expression (1.8-, 6.3- and 2.4-fold, respectively). These data demonstrate that among the identified genes are valuable markers for the prediction of osteosarcoma therapy outcome. Especially IMPDH2 and FTL are promising candidates for the stratification of osteosarcoma patients into low- and high-risk groups. Owing to their involvement in drug action these genes may further be potential targets for the modulation of drug sensitivity.

Evaluation of the predictive value of Her-2/neu gene expression on osteosarcoma therapy in laser-microdissected paraffin-embedded tissue.

Histologic response to chemotherapy is currently the strongest prognostic factor in high-grade osteosarcoma, but it can only be assessed after several weeks of therapy. Thus, detection of chemosensitivity at the time of diagnosis would be of great clinical importance. The expression of the proto-oncogene Her-2/neu has been shown to be of predictive value in breast cancer and has also been considered as prognostic marker for osteosarcomas, but reports of mainly immunohistochemical studies are controversial. Therefore, the aim of this study was to investigate Her-2/neu gene expression in laser-microdissected osteosarcoma cells. Laser microdissection enables the precise isolation of morphological defined cells from archival tissue specimens and is in combination with the highly sensitive real-time RT-PCR technique a valuable tool for cell-specific analysis of gene expression. Through optimization of current protocols, we could show that this technique can be successfully applied on formalin-fixed, paraffin-embedded and decalcified osteosarcoma tissue with high sensitivity and reproducibility. In all 17 osteosarcoma biopsies analyzed, we could detect Her-2/neu gene expression. Expression correlated significantly with the response to preoperative chemotherapy, which was assessed histologically according to the six-grade scale of Salzer-Kuntschik. Risk assessment on the basis of increased Her-2/neu gene expression matched the histologic findings in 16 out of 17 cases (94%). These data demonstrate the reliability of laser microdissection in the analysis of gene expression and suggest a possible role of Her-2/neu as prognostic marker for therapy outcome in osteosarcomas.