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1.  ΔNp63α enhances the oncogenic phenotype of osteosarcoma cells by inducing the expression of GLI2 
BMC Cancer  2014;14(1):559.
Background
ΔNp63, a splice variant of p63, is overexpressed and exhibits oncogenic activity in many cancers including pancreatic and breast cancer and promotes cell survival by inhibiting apoptosis. Despite its role in tumorigenesis, mechanistic activity of ΔNp63 mediated oncogenic function in osteosarcoma is poorly understood.
Methods
The expression levels of p63 isoforms in osteosarcoma cell lines were identified using quantitative techniques. Expression profiling using microarray, siRNA mediated loss-of-function, and chromatin immunoprecipitation assays were employed to identify novel ΔNp63α targets in p63-null osteosarcoma SaOS-2 cells that were engineered to express ΔNp63α. The phenotype of SaOS-2-ΔNp63α cells was assessed using wound-healing, colony formation, and proliferation assays.
Results
The comparative expression analyses identified ΔNp63α as the predominant p63 isoform expressed by invasive OS cell lines. Phenotypic analyses of SaOS-2-ΔNp63α cells in vitro indicate that ΔNp63α imparted tumorigenic attributes upon tumor cells. Further, we show that in osteosarcoma cells ΔNp63α directly regulated the transcription factor GLI2, which is a component of the hedgehog signaling pathway, and that functional interactions between ΔNp63α and GLI2 confer oncogenic properties upon OS cells.
Conclusions
Here, we report that GLI2 is the novel target gene of ΔNp63α and that ΔNp63α-GLI2 crosstalk in osteosarcoma cells is a necessary event in osteosarcoma progression. Defining the exact mechanisms involved in this interaction that mediate the pathogenesis of osteosarcoma promises to identify targets for drug therapy.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-559) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2407-14-559
PMCID: PMC4125704  PMID: 25085524
Osteosarcoma; p63; ΔNp63α; GLI2
2.  Extensor Function After Medial Gastrocnemius Flap Reconstruction of the Proximal Tibia 
Background
Reconstruction of the extensor mechanism after resection of the proximal tibia is challenging, and several methods are available. A medial gastrocnemius flap commonly is used, although it may be associated with an extensor lag. This problem also is encountered, although perhaps to a lesser extent, with other techniques for reconstruction of the extensor apparatus. It is not known how such lag develops with time and how it correlates with functional outcome.
Questions/purposes
We therefore (1) assessed patellar height with time, (2) correlated patellar height with function using the Musculoskeletal Tumor Society (MSTS) score, and (3) correlated patellar height with range of motion (ROM) after medial gastrocnemius flap reconstruction.
Methods
Sixteen patients underwent tumor endoprosthesis implantation and extensor apparatus reconstruction between 1997 and 2009 using a medial gastrocnemius flap after sarcoma resection of the proximal tibia. These patients represented 100% of the population for whom we performed extensor mechanism reconstructions during that time. The minimum followup was 2 years (mean, 5 years; range, 2–11 years). Fourteen patients were alive at the time of this study. We used the Blackburne-Peel Index to follow patellar height radiographically with time. Functional outcomes were assessed retrospectively using the MSTS, and ROM was evaluated through active extensor lag and flexion.
Results
Eleven patients had patella alta develop, whereby the maximal patellar height was reached after a mean of 2 years and then stabilized. More normal patellar height was associated with better functional scores, a smaller extensor lag, but less flexion; the mean extensor lag (and flexion) of patients with patella alta was 17° (and 94°) compared with only 4° (and 77°) without.
Conclusions
In our patients patella alta evolved during the first 2 postoperative years. Patella alta is associated with extensor lag, greater flexion, and worse MSTS scores. Surgical fixation of the patellar tendon more distally to its anatomic position or strict postoperative bracing may be advisable.
Level of Evidence
Level IV, clinical cohort study. See the Guidelines for Authors for a complete description of levels of evidence.
doi:10.1007/s11999-013-2851-8
PMCID: PMC3676620  PMID: 23423620
3.  Response of an aggressive periosteal aneurysmal bone cyst (ABC) of the radius to denosumab therapy 
Aneurysmal bone cyst (ABC), once considered a reactive lesion, has been proven to be a neoplasia characterized by rearrangements of the USP6-gene. Aggressive local growth and recurrences are common and therapeutic options may be limited due to the vicinity of crucial structures. We describe a case of a locally aggressive, multinucleated giant cell-containing lesion of the forearm of a 21-year old woman, treated with denosumab for recurrent, surgically uncontrollable disease. Under the influence of this RANKL inhibitor, the tumor showed a marked reduction of the content of the osteoclastic giant cells and an extensive metaplastic osteoid production leading to the bony containment, mostly located intracortically in the proximal radius. The diagnosis of a periosteal ABC was confirmed by FISH demonstrating USP6 gene rearrangement on the initial biopsy. Function conserving surgery could be performed, enabling reconstruction of the affected bone. Inhibition of RANKL with denosumab may offer therapeutic option for patients not only with giant cell tumors but also with ABCs.
doi:10.1186/1477-7819-12-17
PMCID: PMC3901337  PMID: 24438319
Aneurysmal bone cyst; FISH; USP6; Denosumab
4.  CXCR4 antibody treatment suppresses metastatic spread to the lung of intratibial human osteosarcoma xenografts in mice 
Current combined surgical and neo-adjuvant chemotherapy of primary metastatic osteosarcoma (OS) is ineffective, reflected by a 5-year survival rate of affected patients of less than 20 %. Studies in experimental OS metastasis models pointed to the CXCR4/CXCL12 homing axis as a novel target for OS metastasis-suppressive treatment. The present study investigated for the first time the CXCR4-blocking principle in a spontaneously metastasizing human 143B OS cell line-derived orthotopic xenograft mouse model. The highly metastatic 143B cells, unlike the parental non-metastatic HOS cells, express functional CXCR4 receptors at the cell surface, as revealed in this study by RT/PCR of gene transcripts, by FACS analysis with the monoclonal anti CXCR4 antibody 12G5 (mAb 12G5) and by CXCL12 time- and dose-dependent stimulation of AKT and ERK phosphorylation. A significantly (p < 0.05) higher CXCL12 dose-dependent chemotactic response of 143B compared to HOS cells in a Boyden chamber trans-well migration assay suggested a crucial role of the CXCL12/CXCR4 homing axis in 143B cell lung metastasis. Repetitive treatment of mice with 143B cell-derived intratibial tumors given intravenous bolus injections of mAb12G5 indeed inhibited significantly (p < 0.01) the number of X-gal-stainable lung micrometastases of lacZ-transduced 143B cells. Antibody treatment had also a mild inhibitory effect on primary tumor growth associated with remarkably less osteolysis, but it did not affect the number of developing lung macrometastases. In conclusion, these results demonstrate considerable potential of high-affinity CXCR4-blocking agents for OS tumor cell homing suppressive treatment in metastasizing OS complementary to current (neo)-adjuvant chemotherapy.
doi:10.1007/s10585-013-9632-3
PMCID: PMC3915086  PMID: 24390633
CXCR4-CXCL12 axis; Lung metastases; lacZ transduction; Osteosarcoma mouse model
5.  Reduced Latency in the Metastatic Niche Contributes to the More Aggressive Phenotype of LM8 Compared to Dunn Osteosarcoma Cells 
Sarcoma  2013;2013:404962.
Metastasis is the major cause of death of osteosarcoma patients and its diagnosis remains difficult. In preclinical studies, however, forced expression of reporter genes in osteosarcoma cells has remarkably improved the detection of micrometastases and, consequently, the quality of the studies. We recently showed that Dunn cells equipped with a lacZ reporter gene disseminated from subcutaneous primary tumors as frequently as their highly metastatic subline LM8, but only LM8 cells grew to macrometastases. In the present time-course study, tail-vein-injected Dunn and LM8 cells settled within 24 h at the same frequency in the lung, liver, and kidney of mice. Furthermore, Dunn cells also grew to macrometastases, but, compared to LM8, with a delay of two weeks in lung and one week in liver and kidney tissue, consistent with prolonged survival of the mice. Dunn- and LM8-cell-derived ovary and spine metastases occurred less frequently. In vitro, Dunn cells showed less invasiveness and stronger contact inhibition and intercellular adhesion than LM8 cells and several cancer- and dormancy-related genes were differentially expressed. In conclusion, Dunn cells, compared to LM8, have a similar capability but a longer latency to form macrometastases and provide an interesting new experimental system to study tumor cell dormancy.
doi:10.1155/2013/404962
PMCID: PMC3867932  PMID: 24369449
6.  Expression of the Chemokine Receptor CXCR7 in CXCR4-Expressing Human 143B Osteosarcoma Cells Enhances Lung Metastasis of Intratibial Xenografts in SCID Mice 
PLoS ONE  2013;8(9):e74045.
More effective treatment of metastasizing osteosarcoma with a current mean 5-year survival rate of less than 20% requires more detailed knowledge on mechanisms and key regulatory molecules of the complex metastatic process. CXCR4, the receptor of the chemokine CXCL12, has been reported to promote tumor progression and metastasis in osteosarcoma. CXCR7 is a recently deorphanized CXCL12-scavenging receptor with so far not well-defined functions in tumor biology. The present study focused on a potential malignancy enhancing function of CXCR7 in interaction with CXCR4 in osteosarcoma, which was investigated in an intratibial osteosarcoma model in SCID mice, making use of the human 143B osteosarcoma cell line that spontaneously metastasizes to the lung and expresses endogenous CXCR4. 143B osteosarcoma cells stably expressing LacZ (143B-LacZ cells) were retrovirally transduced with a gene encoding HA-tagged CXCR7 (143B-LacZ-X7-HA cells). 143B-LacZ-X7-HA cells co-expressing CXCR7 and CXCR4 exhibited CXCL12 scavenging and enhanced adhesion to IL-1β-activated HUVEC cells compared to 143B-LacZ cells expressing CXCR4 alone. SCID mice intratibially injected with 143B-LacZ-X7-HA cells had significantly (p<0.05) smaller primary tumors, but significantly (p<0.05) higher numbers of lung metastases than mice injected with 143B-LacZ cells. Unexpectedly, 143B-LacZ-X7-HA cells, unlike 143B-LacZ cells, also metastasized with high incidence to the auriculum cordis. In conclusion, expression of the CXCL12 scavenging receptor CXCR7 in the CXCR4-expressing human 143B osteosarcoma cell line enhances its metastatic activity in intratibial primary tumors in SCID mice that predominantly metastasize to the lung and thereby closely mimic the human disease. These findings point to CXCR7 as a target, complementary to previously proposed CXCR4, for more effective metastasis-suppressive treatment in osteosarcoma.
doi:10.1371/journal.pone.0074045
PMCID: PMC3769403  PMID: 24040160
7.  Giant Cell Angioblastoma in an Adult: a Unique Presentation 
Rare Tumors  2013;5(3):e27.
Giant cell angioblastoma is a very rare, locally destructive vascular tumor of intermediate malignancy without metastatic potential. There are only a few cases reported in the literature exclusively in the soft tissue of children. For the first time, we report on an adult patient with a giant cell angioblastoma in the popliteal fossa. The therapy included tumor resection with favorable clinical, oncological and functional outcome. Due to its locally destructive nature, surgery remains the mainstay of treatment. Histologically, giant cell angioblastoma is comprised of nodular aggregates of histiocytoid cells arranged around bland angiomatous spaces. Because of insufficient available data in regard to the definition of the entity, diagnostic criteria and its biological potential, it is not included in the new World Health Organization classification of tumors of soft tissue and bone. The differential diagnosis includes plexiform fibrohistiocytic tumor, myofibroma and giant cell fibroblastoma.
doi:10.4081/rt.2013.e27
PMCID: PMC3804802  PMID: 24179639
giant cell; angioblastoma; vascular tumor; soft-tissue tumor; intermediate malignancy
8.  Physeal Distraction for Joint Preservation in Malignant Metaphyseal Bone Tumors in Children 
Background
Physeal distraction facilitates metaphyseal bone tumor resection in children and preserves the adjacent joint. The technique was first described by Cañadell. Tumor resection procedures allowing limb-sparing reconstruction have been used increasingly in recent years without compromising oncologic principles.
Questions/purposes
We report our results with Cañadell’s technique by assessing tumor control, functional outcome, and complications.
Methods
Six consecutive children with primary malignant metaphyseal bone tumors underwent physeal distraction as a part of tumor resection. Tumor location was the distal femur in four patients, the proximal humerus in one patient, and the proximal tibia in one patient. The functional outcome was evaluated after a minimum of 18 months (median, 62 months; range, 18–136 months) using the Musculoskeletal Tumor Society (MSTS) score and the Toronto Extremity Salvage Score (TESS).
Results
At latest followup, five patients were alive and disease-free and one had died from metastatic disease. All tumor resections resulted in local control; there were no local recurrencies. The mean MSTS score was 79% (range, 53%–97%) and corresponding mean TESS was 83% (range, 71%–92%). In one case, postoperative infection required amputation of the proximal lower leg. All physeal distractions were successful except for one patient in whom distraction resulted in rupturing into the tumor. This situation was salvaged by transepiphyseal resection.
Conclusions
We consider Cañadell’s technique a useful tool in the armamentarium to treat children with malignant tumors that are in close proximity to an open physis.
Level of Evidence
Level IV, therapeutic study. See Guidelines for Authors for a complete description of levels of evidence.
doi:10.1007/s11999-011-2224-0
PMCID: PMC3348308  PMID: 22203330
9.  Silencing of CD44 Gene Expression in Human 143-B Osteosarcoma Cells Promotes Metastasis of Intratibial Tumors in SCID Mice 
PLoS ONE  2013;8(4):e60329.
Osteosarcoma (OS) is the most frequent primary malignant bone cancer in children and adolescents with a high propensity for lung metastasis. Therefore, it is of great importance to identify molecular markers leading to increased metastatic potential in order to devise more effective therapeutic strategies that suppress metastasis, the major cause of death in OS. CD44, the principal receptor for the extracellular matrix component hyaluronan (HA), is frequently found overexpressed in tumor cells and has been implicated in metastatic spread in various cancer types. Here, we investigated the effects of stable shRNA-mediated silencing of CD44 gene products on in vitro and in vivo metastatic properties of the highly metastatic human 143-B OS cell line. In vitro, CD44 knockdown resulted in a 73% decrease in the adhesion to HA, a 57% decrease in the migration rate in a trans-filter migration assay, and a 28% decrease in the cells' capacity for anchorage-independent growth in soft agar compared to the control cells, implicating that CD44 expression contributes to the metastatic activity of 143-B cells. However, making use of an orthotopic xenograft OS mouse model, we demonstrated that reduced CD44 expression facilitated primary tumor growth and formation of pulmonary metastases. The enhanced malignant phenotype was associated with decreased adhesion to HA and reduced expression of the tumor suppressor merlin in vivo. In conclusion, our study identified CD44 as a metastasis suppressor in this particular experimental OS model.
doi:10.1371/journal.pone.0060329
PMCID: PMC3614951  PMID: 23565227
10.  Genes Regulated in Metastatic Osteosarcoma: Evaluation by Microarray Analysis in Four Human and Two Mouse Cell Line Systems 
Sarcoma  2012;2012:937506.
Osteosarcoma (OS) is a rare bone neoplasm that affects mainly adolescents. It is associated with poor prognosis in case of metastases formation. The search for metastasis predicting markers is therefore imperative to optimize treatment strategies for patients at risk and important for the search of new drugs for the treatment of this devastating disease. Here, we have analyzed by microarray the differential gene expression in four human and two mouse OS cell line systems consisting of parental cell lines with low metastatic potential and derivatives thereof with increased metastatic potential. Using two osteoblastic cell line systems, the most common OS phenotype, we have identified forty-eight common genes that are differentially expressed in metastatic cell lines compared to parental cells. The identified subset of metastasis relevant genes in osteoblastic OS overlapped only minimally with differentially expressed genes in the other four preosteoblast or nonosteoblastic cell line systems. The results imply an OS phenotype specific expression pattern of metastasis regulating proteins and form a basis for further investigation of gene expression profiles in patients' samples combined with survival analysis with the aim to optimize treatment strategies to develop new drugs and to consequently improve the survival of patients with the most common form of osteoblastic OS.
doi:10.1155/2012/937506
PMCID: PMC3504467  PMID: 23213280
11.  Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells 
Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of micrometastases by radiologic imaging and the success in their therapeutic eradication remain limited.
While animal models have proven to be invaluable tools for cancer research1, the monitoring/visualization of micrometastases remains a challenge and inaccurate evaluation of metastatic spread in preclinical studies potentially leads to disappointing results in clinical trials2. Consequently, there is great interest in refining the methods to finally allow reproducible and reliable detection of metastases down to the single cell level in normal tissue. The main focus therefore is on techniques, which allow the detection of tumor cells in vivo, like micro-computer tomography (micro-CT), positron emission tomography (PET), bioluminescence or fluorescence imaging3,4. We are currently optimizing these techniques for in vivo monitoring of primary tumor growth and metastasis in different osteosarcoma models. Some of these techniques can also be used for ex vivo analysis of metastasis beside classical methods like qPCR5, FACS6 or different types of histological staining. As a benchmark, we have established in the present study the stable transfection or transduction of tumor cells with the lacZ gene encoding the bacterial enzyme β-galactosidase that metabolizes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to an insoluble indigo blue dye7 and allows highly sensitive and selective histochemical blue staining of tumor cells in mouse tissue ex vivo down to the single cell level as shown here. This is a low-cost and not equipment-intensive tool, which allows precise validation of metastasis8 in studies assessing new anticancer therapies9-11. A limiting factor of X-gal staining is the low contrast to e.g. blood-related red staining of well vascularized tissues. In lung tissue this problem can be solved by in-situ lung perfusion, a technique that was recently established by Borsig et al.12 who perfused the lungs of mice under anesthesia to clear them from blood and to fix and embed them in-situ under inflation through the trachea. This method prevents also the collapse of the lung and thereby maintains the morphology of functional lung alveoli, which improves the quality of the tissue for histological analysis. In the present study, we describe a new protocol, which takes advantage of a combination of X-gal staining of lacZ-expressing tumor cells and in-situ perfusion and fixation of lung tissue. This refined protocol allows high-sensitivity detection of single metastatic cells in the lung and enabled us in a recent study to detect "dormant" lung micrometastases in a mouse model13, which was originally described to be non-metastatic14.
doi:10.3791/4162
PMCID: PMC3487289  PMID: 22929213
Cancer Biology; Issue 66; Medicine; Molecular Biology; Cellular Biology; lung metastasis; lacZ-tagging; 5-Bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal) staining;  in-situ lung perfusion; metastases; imaging
12.  Extraarticular Knee Resection for Sarcomas with Preservation of the Extensor Mechanism: Surgical Technique and Review of Cases 
Background
Sarcomas in or contaminating the knee are rare but extremely challenging to treat. Complete resection of the joint is necessary, and often the entire extensor mechanism is removed as well. Reconstruction of the knee is challenging, and the resulting function may be compromised.
Description of technique
We describe a surgical technique of extraarticular resection of the knee while preserving the extensor mechanism combined with prosthetic reconstruction. The medial and lateral retinaculum is prepared such that it allows extraarticular placement of K-wires that are driven through the patella and the proximal tibia, serving as in situ guides for the osteotomies.
Patients and Methods
We retrospectively reviewed 11 patients with sarcomas contaminating the knee. The minimum followup was 14 months (mean, 38 months; range, 14–80 months).
Results
At last followup patients had a mean flexion of 88° (range, 65°–120°). We observed no complications related to the extensor mechanism, and there was one local recurrence.
Conclusions
We believe extraarticular resection of the knee with preservation of the extensor mechanism is a reasonable treatment option for intraarticular sarcomas with functional scores comparable to those for patients having intraarticular resections.
Level of Evidence
Level IV, therapeutic study. See the Guidelines for Authors for a complete description of levels of evidence.
doi:10.1007/s11999-010-1359-8
PMCID: PMC3008904  PMID: 20419485
13.  A Rare Periosteal Diaphyseal Lesion of the Ulna 
Periosteal lesions of the ulna diaphysis are rare, include a wide spectrum of tumors, and may cause considerable diagnostic problems. Surgical treatment may vary widely, based on an accurate diagnosis. We present the case of a periosteal, extraskeletal low grade myxoid chondrosarcoma of the ulna diaphysis. The surgical therapy included an en-bloc resection with allograft reconstruction. The patient showed a favorable outcome. Careful preoperative evaluation and planning are imperative to obtain a satisfactory oncological and functional outcome, especially with uncommon tumor presentations at rare locations.
doi:10.2174/1874325001206010008
PMCID: PMC3282889  PMID: 22371818
Allograft; diaphyseal lesion; periosteal; ulna.
14.  A New Incision Technique to Reduce Tibiofemoral Mismatch in Rotationplasty 
Rotationplasty provides stable and durable biologic reconstruction after tumor resection around the knee and renders reliable results, in young patients. However, after resection of the tumor, there is often a mismatch between the circumference of the proximal (femoral) and the distal (tibial) parts. Because rotationplasty includes an intercalary amputation where the ends are readapted, there is always a mismatch of the proximal and distal circumferences of the soft tissue envelope. To facilitate skin closure without tension and to avoid impaired wound healing and subsequent infections, the type of incision is critical and must be carefully planned. We present a new incision technique for rotationplasty about the knee. Half of the difference of the incision length of the proximal and distal circumferences represents the base of the triangle proximally, medially and laterally of the thigh. After adapting both ends, the peak of this flat triangle is distally adapted via a vertical incision which allows it to match unequal circumferences. This technique was used in eight patients, in all of whom the wounds healed uneventfully.
doi:10.1007/s11999-009-1093-2
PMCID: PMC2853676  PMID: 19756901
15.  Preferential Expression of the Secreted and Membrane forms of Tumor Endothelial Marker 7 transcripts in Osteosarcoma 
Anticancer research  2009;29(11):4317-4322.
Background
High expression of tumor endothelial marker 7 (TEM7) is correlated with osteogenic sarcoma (OS) metastasis and poor survival of patients. The TEM7 gene produces four alternatively spliced transcripts with distinct functional domains; the expression pattern of these transcripts in OS is unknown.
Materials and Methods
mRNA expression was assessed in 5 OS cell lines, 7 normal bone, and 9 OS tumor specimens by reverse transcriptase polymerase chain reaction.
Results
All OS cell lines, 6/9 tumors but none of the bone specimens expressed mRNA of TEM7 secreted forms 1 and 2. A total of 3/5 OS cell lines, 8/9 of tumors and 4/7 of bone specimens expressed mRNA of the TEM7 intracellular form. One out of 5 cell lines, 2/7 tumors and none of the bone specimens expressed mRNA of the TEM7 membrane form. The secreted forms had 20-fold higher expression in metastatic (LM7) compared to non-metatstatic (SAOS-2) cells.
Conclusion
The mRNA of secreted and the membrane forms of TEM7 are preferentially expressed in OS.
PMCID: PMC2800050  PMID: 20032373
TEM7; alternative splicing; osteosarcoma; PCR; metastasis
16.  Novel Measurement Technique of the Tibial Slope on Conventional MRI 
The posterior inclination of the tibial plateau, which is referred to as posterior tibial slope, is determined routinely on lateral radiographs. However, radiographically, it is not always possible to reliably recognize the lateral plateau, making a separate assessment of the medial and lateral plateaus difficult. We propose a technique to measure the plateaus separately by defining a tibial longitudinal axis on a conventional MRI. The medial plateau posterior tibial slope obtained from radiographs was compared with MR images in 100 consecutive patients with knee pain when ligament or meniscal injury was assumed. The posterior tibial slope on MRI correlated with those on radiographs. The mean posterior tibial slope was 3.4° smaller on MRI compared with radiographs (4.8° ± 2.4° versus 8.2° ± 2.8°, respectively). The reproducibility was slightly better on radiographs than MRI (± 0.9° versus ± 1.4°). Twenty-one of the 100 cases had more than a 5° difference (range, −8.7° to 8.9°) between the medial and lateral plateaus. The proposed technique allows measurement of the posterior tibial slope of the medial and lateral plateaus on a standard knee MRI. By using this novel measurement technique, a reliable assessment of the medial and lateral tibial plateaus is possible.
Level of Evidence: Level III, diagnostic study. See the Guidelines for Authors for a complete description of levels of evidence.
doi:10.1007/s11999-009-0711-3
PMCID: PMC2706341  PMID: 19190973
17.  Osteoblastic and Osteolytic Human Osteosarcomas can be Studied with a new Xenograft Mouse Model Producing Spontaneous Metastases 
Cancer investigation  2009;27(4):435-442.
There is no animal model that reflects the histological and radiographical heterogeneity of osteosarcoma. We assessed seven osteosarcoma cell lines for their potential to develop orthotopic tumors and lung metastasis in SCID mice. Whereas radiologically, 143B developed osteolytic tumors, SaOS-LM7 developed osteoblastic primary tumors. The mineralization status was confirmed by assessing the alkaline phosphatase activity and the microarray expression profile. We herein report a xenograft orthotopic osteosarcoma mouse model to assess osteoblastic and osteolytic lesions, which may contribute in the search for new diagnostic and therapeutic approaches.
doi:10.1080/07357900802491477
PMCID: PMC2723944  PMID: 19212826
Osteosarcoma; Animal Model; Xenograft; Orthotopic; Lung metastasis
18.  Osteosarcoma of the Pelvis: Outcome Analysis of Surgical Treatment 
Risk factors to explain the poor survival of patients with osteosarcoma of the pelvis are poorly understood. Therefore, we attempted to identify factors affecting survival and development of local recurrence and metastasis. We retrospectively reviewed 43 patients who had high-grade pelvic tumors and were treated surgically. Twenty lesions were chondroblastic, 10 fibroblastic, 11 osteoblastic, and one each was giant cell-rich and small cell osteosarcomas. At a median of 3.5 years (range, 0.3–21 years) postoperatively, 13 patients were alive with no evidence of disease. The overall and disease-free 5-year survival rates were 38% and 29%, respectively, at 5 years. Anatomic location, tumor size, and margin predicted survival. Fifteen patients (35%) had local recurrence. The 5-year cumulative incidence of recurrence with death as a competing risk factor was 34%. Location in the ilium and size of the tumor predicted local recurrence. Twenty-one (49%) of 43 patients had metastases develop. The cumulative incidence of metastasis with death as a competing risk factor was 48% at 5 years. Six patients who presented with metastasis had a worse survival than patients who had no evidence of metastasis at presentation (2-year survival, 33% versus 76%). If distant metastasis is diagnosed subsequent to primary treatment, aggressive therapy may be justified.
Level of Evidence: Level II, prognostic study. See the Guidelines for Authors for a complete description of levels of evidence.
doi:10.1007/s11999-008-0495-x
PMCID: PMC2628496  PMID: 18855090
19.  Expression of Atrophy mRNA Relates to Tendon Tear Size in Supraspinatus Muscle 
Skeletal muscle atrophy and fatty infiltration develop after tendon tearing. The extent of atrophy serves as one prognostic factor for the outcome of surgical repair of rotator cuff tendon tears. We asked whether mRNA of genes involved in regulation of degradative processes leading to muscle atrophy, ie, FOXOs, MSTN, calpains, cathepsins, and transcripts of the ubiquitin-proteasome pathway, are overexpressed in the supraspinatus muscle in patients with and without rotator cuff tears. We evaluated biopsy specimens collected during surgery of 53 consecutive patients with different sizes of rotator cuff tendon tears and six without tears. The levels of corresponding gene transcripts in total RNA extracts were assessed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Supraspinatus muscle atrophy was assessed by MRI. The area of muscle tissue (or atrophy), decreased (increased) with increasing tendon tear size. The transcripts of CAPN1, UBE2B, and UBE3A were upregulated more than twofold in massive rotator cuff tears as opposed to smaller tears or patients without tears. These atrophy gene products may be involved in cellular processes that impair functional recovery of affected muscles after surgical rotator cuff repair. However, the damaging effects of gene products in their respective proteolytic processes on muscle structures and proteins remains to be investigated.
doi:10.1007/s11999-008-0565-0
PMCID: PMC2628494  PMID: 18941855
20.  Sclerosing Epithelioid Fibrosarcoma: Case Presentation and a Systematic Review 
In sclerosing epithelioid fibrosarcoma (SEF), a rare variant of low-grade fibrosarcoma, treatment results and therapeutic options are poorly characterized. We systematically analyzed the data of all 89 patients (43 female, 46 male; mean age, 47 years [range, 14–87 years]) reported in the literature concerning clinical presentation, histopathology, differential diagnosis, treatment, survival rates, and prognosis, and we present an additional case. Information detailing treatment, disease control, and followup was available in 60 (67%), 75 (84%), and 68 patients (76%), respectively. Case history was variable with one-third of patients reporting a painful, enlarging mass. Ten patients (13%) presented with metastases, 23 (31%) had metastases develop after diagnosis, and 28 (37%) had local recurrence. Low cellularity, mild pleomorphy, and sclerotic hyaline matrix of SEF suggest a benign clinical behavior, and cell morphology allows for the wide differential diagnosis of benign, pseudosarcomatous, and malignant proliferations. In addition to surgery, 11 patients (15%) had chemotherapy, 22 (29%) had postoperative radiation therapy, and three (4%) had a combination of both. Twenty-three patients (34%) died from their disease after a mean of 46 months, 24 (35%) were alive with disease, and 20 (31%) were alive without evidence of disease. Patients with SEF of the head and neck had the worst prognosis.
Level of Evidence: Level III, prognostic study. See the Guidelines for Authors for a complete description of levels of evidence.
Electronic supplementary material
The online version of this article (doi:10.1007/s11999-008-0205-8) contains supplementary material, which is available to authorized users.
doi:10.1007/s11999-008-0205-8
PMCID: PMC2384045  PMID: 18340502
21.  High expression of Tumor Endothelial Marker 7 is associated with metastasis and poor survival of patients with osteogenic sarcoma 
Gene  2007;399(2):137-143.
Our objective is to identify genes regulating metastasis of osteogenic sarcoma (OGS) since metastasis is the primary cause of mortality among patients with OGS. To identify such genes, we first created a database of differentially expressed genes between six low-grade and six high-grade OGS tumors, and between a normal immortalized osteoblast cell line (FOB) and four commercially available OGS-derived cell lines. We specifically searched for surface-proteins over-expressed in high-grade OGS, since we hypothesize that tumor-cell specific surface markers are key to metastasis. A gene encoding Tumor Endothelial Marker7 (TEM7) was selected as a candidate for further study. TEM7 expression pattern was assessed by RT-PCR, Western blotting and immunostaining. TEM7 mRNA was abundantly expressed in SAOS cells (derived from high-grade OGS), but not in FOB or MG63 cells (derived from low-grade OGS). Virtually no expression of TEM7 protein was observed in FOB cells but abundant expression was noted in SAOS and TE85 cells. Employing immunostaining of 92 human OGS specimens (50 high grade and 42 low-grade) collected before chemotherapy show 97% (37 of 38) of high-grade OGS specimens with metastasis have high TEM7 staining. Further, we found that elevated expression of TEM7 correlated with poor survival (p<0.04) of affected patients. Inhibiting TEM7 function by siRNA inhibited invasion and migration of OGS cells with metastatic potential. Our results suggest TEM7 expression level closely parallels histology-based prognostication of OGS metastasis and, therefore, it is a therapeutic target. This is the first demonstration of a link between TEM7 and cancer metastasis.
doi:10.1016/j.gene.2007.05.003
PMCID: PMC2066185  PMID: 17560052
TEM7; Osteogenic sarcoma; Metastasis; siRNA; Tumor marker

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