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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.