Extracellular matrix-induced transforming growth factor-beta receptor signaling dynamics.

Matrix remodeling, degradation, inflammation and invasion liberate peptide fragments that can subsequently interact with cells in an attachment-independent manner. Such 'soluble' matrix components, including collagens, fibronectin and laminin, induced Smad activation (termed crosstalk signaling), which follows a similar chronological sequence and R-Smad specificity as induced by transforming growth factor (TGF)-beta1. Smad4 nuclear translocation occurred in response to collagen binding, indicating downstream signal propagation. TGF-beta scavenging antibody affected only TGF-beta1, but not crosstalk-induced responses. TGF-beta type II receptor mutation (DR26Delta25), which is deficient in TGF-beta type I receptor recruitment to the ligand, induced a heterotetramer signaling complex, and propagated Smad2 activation only through collagen induction and not TGF-beta signaling. Consequentially, TGF-beta ligand participation is not required for crosstalk signaling. This signaling requires a functional integrin beta1 receptor as showed by RNA interference. Co-immunoprecipitation (co-IP) and fluorescent microscopy indicate the involvement of focal adhesion kinase (FAK) and Src activity in collagen-induced signal propagation, and suggest a membrane signaling complex formation that includes both TGF-beta receptors and integrins. The related gene expressional responses are distinct from that evoked by TGF-beta1, supporting its separate function. This signaling mechanism expands and partially explains TGF-beta receptor dynamics and consequential signaling diversity-related gene expressional plasticity.

Decrease in particle-induced osteolysis in obese (ob/ob) mice.

There may be variability in the susceptibility of different individuals to osteolysis from wear debris, and it is not clear whether some individuals may have a genetic predisposition for a more marked osteolytic response. The purpose of this study in mice was to determine whether genetically determined obesity can alter the response to particulate debris. Polyethylene particles were implanted onto the calvaria of seven wild-type mice and seven obese mice (ob/ob). Calvaria from unimplanted wild-type and obese mice served as controls. Calvaria were harvested after 7 days, stained with toluidine blue and for tartrate-specific alkaline phosphatase, and analyzed by histomorphometry. The osteoclast number per mm total bone perimeter was 8.000+/-3.464 in wild-type animals with particles and 2.857+/-1.676 in ob/ob animals with particles (p=0.002; Fisher's PLSD). Bone resorption was 1.895+/-0.713 mm/mm(2) in wild-type animals with particles and 1.265+/-0.494 mm/mm(2) in ob/ob animals with particles (p=0.0438; Fisher's PLSD). Particles induced a diminished osteolytic response in genetically determined obese mice, suggesting that obesity may have a protective role against particle-induced bone resorption-similar to obesity and osteoporosis. These important new findings may help to stimulate clinical studies which may define criteria to better identify patients at risk to develop particle-induced osteolysis.

Calcific myonecrosis: a unique presentation in the upper extremity.

Calcific myonecrosis is a rare and latent condition characterized by a dystrophic calcified lesion that can present 10-64 years following initial trauma. Of the 25 cases documented in English world literature, all have occurred in the lower extremity exclusively. We report a case of a 60-year-old man with a painless enlarging left forearm mass that was subsequently diagnosed as calcific myonecrosis. Awareness of this lesion arising outside of the lower extremity is important to avoid unnecessary surgical intervention and patient reassurance.

Adjuvant chemotherapy for osteosarcoma may not increase survival after neoadjuvant chemotherapy and surgical resection.

BACKGROUND AND OBJECTIVES: Osteosarcoma is a primary malignancy of bone. Current therapy includes neoadjuvant chemotherapy, surgery, and postoperative (adjuvant) chemotherapy. Prolonged treatment with chemotherapeutic agents may place patients at increased risk for complications including secondary malignancy. The authors have had promising results with neoadjuvant therapy and surgery alone in the treatment of osteosarcoma. This study retrospectively examines neoadjuvant therapy and surgery alone for the treatment of primary osteosarcoma of bone with no evidence of distant metastases. METHODS: Fifty-four patients, with localized osteosarcoma of bone received neoadjuvant therapy followed by definitive surgical resection. Thirty-five patients received chemotherapy after surgery (adjuvant group) and nineteen patients were followed without postoperative chemotherapy (no adjuvant group). RESULTS: Tumor necrosis was predictive of survival. Kaplan-Meier analysis revealed the use of postoperative chemotherapy was not a predictor of improved outcome. Four patients in the adjuvant therapy group died of secondary malignancy compared with none of the no adjuvant therapy group. Patient age, sex, race, and tumor location were not predictive of survival. CONCLUSIONS: The use of adjuvant chemotherapy in the treatment of localized osteosarcoma of bone did not increase survival after neoadjuvant therapy and definitive surgical therapy. Instead, there was an increased incidence of secondary malignancy after its use.

The clinical significance of tenascin-C splice variant expression in chondrosarcoma.

OBJECTIVES: Tenascin-C (TNC) is an oligomeric glycoprotein of the extracellular matrix that is prominently expressed in malignant tumors. The purpose of this study was: (1) to determine the in vitro TNC splicing pattern in cultured human chondrocytes and chondrosarcoma cells, (2) to determine the in vivo TNC splicing pattern in clinical chondrosarcoma specimens, and (3) to perform survival analysis based on the TNC splicing pattern of the tumor specimens. METHODS: Human articular chondrocytes and chondrosarcoma cells (cell line JJ012) were grown in a three-dimensional alginate bead system and harvested at two time points. Semiquantitative reverse transcription polymerase chain reaction (RT-PCR) was used to determine the in vitro TNC splicing pattern for the two cell types. Clinical chondrosarcoma specimens were obtained intra-operatively and underwent RT-PCR to determine the in vivo TNC splicing pattern. Specific immunohistochemical staining for the large TNC splice variant was performed on the clinical specimens. Survival analysis was used to determine the association between the specific TNC splicing pattern and survival. RESULTS: The in vitro mRNA expression pattern of TNC in normal human articular chondrocytes was characterized by a high ratio of the small to the large splice variant (TNC(small):TNC(large)), whereas the in vitro mRNA expression pattern for cultured chondrosarcoma cells was characterized by a low TNC(small):TNC(large) ratio. Clinical chondrosarcoma specimens with a lower TNC(small):TNC(large) ratio showed a trend towards decreased survival. The TNC splicing pattern of these specimens was verified through specific immunohistochemical staining for the large TNC isoform. CONCLUSIONS: The specific TNC splicing pattern may have clinical significance in chondrosarcoma. TNC expression may therefore play a future role in objective tumor grading and novel therapeutic approaches to this malignancy.

Chondrosarcoma of bone: analysis of 108 cases and evaluation for predictors of outcome.

Chondrosarcoma is the second most common malignant bone tumor and is relatively unresponsive to chemotherapy and radiation regimens. In addition, the clinical course of chondrosarcoma is difficult to predict. The purpose of this study was to review the authors' experience with chondrosarcoma and ascertain any factors related to prognosis and clinical outcome. The medical records of 108 patients followed up for a minimum of 2 years were retrospectively reviewed. There were 31 low-grade and 77 high-grade chondrosarcomas. One hundred one patients underwent surgical resection. There was a statistically significant association between positive margins and local recurrence, metastasis, and death. Tumor grade was not predictive of outcome. Proliferation indices (MIB-1 expression determination through immunohistochemistry) were quantitated in 39 patients. A significant association was seen between MIB-1 expression and recurrence and death. Thus, objective quantitation of tumor proliferation was more predictive than was histologic grade of outcome in chondrosarcoma. Although histologic grade continues to be the standard grading system for chondrosarcoma, the current study contributes to ongoing research and validation of alternative techniques that may be more reliable in guiding prognosis and treatment of chondrosarcoma.

The role of the extracellular matrix in articular chondrocyte regulation.

The in vivo role of the extracellular matrix and the manner in which it interfaces with soluble regulators remains largely unknown. The current study reports the extracellular Type II collagen modulation of transforming growth factor-beta 1-stimulated proliferation, proteoglycan synthesis, messenger ribonucleic acid expression for transforming growth factor-beta 1, and integrin messenger ribonucleic acid expression in articular chondrocytes from adults. This study shows that this cytokine modulation occurs through a mechanism initiated by the attachment of Type II collagen to the beta1-integrin. Transforming growth factor-beta 1 stimulated deoxyribonucleic acid and proteoglycan synthesis in a bimodal fashion. Extracellular Type II collagen increased transforming growth factor-beta 1-stimulated deoxyribonucleic acid and proteoglycan synthesis, aggrecan gene expression as much as 400%, and alpha1(II) procollagen gene expression as much as 180% in a dose-dependent fashion. Heat inactivation of the Type II collagen abrogated the observed effects on deoxyribonucleic acid and proteoglycan synthesis. In contrast to Type II collagen, heat-denatured collagen and bovine serum albumin showed none of the observed effects. The presence of Type II collagen in the alginate bead cultures was found to diminish the messenger ribonucleic acid expression for alpha2 integrin and alter the cellular distribution pattern of the beta1 integrin receptors. Blocking of the beta1-integrin with cyclic-peptides containing the Arg-Gly-Asp sequences and antibodies reduced chondrocyte attachment to Type II collagen by 93%. The physiologic effects shown by the chondrocyte as a result of blocking this attachment to Type II collagen were a significant reduction in transforming growth factor-beta 1-stimulated deoxyribonucleic acid and proteoglycan synthesis. The conclusions elucidate the role played by the extracellular matrix in cytokine-specific regulation of the articular chondrocyte. The authors have shown that extracellular Type II collagen acts through a beta1-integrin mediated mechanism to modulate the chondrocyte response to transforming growth factor-beta 1.

Tenascin-C splice variant adhesive/anti-adhesive effects on chondrosarcoma cell attachment to fibronectin.

Tenascin-C is an oligomeric glycoprotein of the extracellular matrix that has been found to have both adhesive and anti-adhesive properties for cells. Recent elucidation of the two major TNC splice variants (320 kDa and 220 kDa) has shed light on the possibility of varying functions of the molecule based on its splicing pattern. Tenascin-C is prominently expressed in embryogenesis and in pathologic conditions such as tumorogenesis and wound healing. Fibronectin is a prominent adhesive molecule of the extracellular matrix that is often co-localized with tenascin-C in these processes. We studied the chondrosarcoma cell line JJ012 with enzyme-linked immunoabsorbance assays, cell attachment assays and antibody-blocking assays to determine the adhesive/anti-adhesive properties of the two major tenascin-C splice variants with respect to fibronectin and their effect on chondrosarcoma cell attachment. We found that the small tenascin-C splice variant (220 kDa) binds to fibronectin, whereas the large tenascin-C splice variant (320 kDa) does not. In addition, the small tenascin-C splice variant was found to decrease adhesion for cells when bound to fibronectin, but contributed to adhesion when bound to plastic in fibronectin-coated wells. Antibody blocking experiments confirmed that both the small tenascin-C splice variant and fibronectin contribute to cell adhesion when bound to plastic. The large tenascin-C splice variant did not promote specific cell attachment. We hypothesize that the biologic activity of tenascin-C is dependent on the tissue-specific splicing pattern. The smaller tenascin-C isoform likely plays a structural and adhesive role, whereas the larger isoform, preferentially expressed in malignant tissue, likely plays a role in cell egress and metastasis.

A phase II trial testing the thermal dose parameter CEM43 degrees T90 as a predictor of response in soft tissue sarcomas treated with pre-operative thermoradiotherapy.

We prospectively evaluated whether delivering a thermal dose of > 10 cumulative equivalent minutes at 43 degrees C to >90% of the tumour sites monitored (CEM43 degrees T90) would produce a pathologic complete response (pCR) in > 75% of high-grade soft tissue sarcomas treated pre-operatively with thermoradiotherapy. The impact of thermal dose on local failure (LF), distant metastasis (DM), and toxicity was also assessed. Thirty-five patients > or = 18 years old with grade 2 or 3 soft tissue sarcomas accessible for invasive thermometry were enrolled on the protocol. All patients received megavoltage external beam radiotherapy (RT) in daily fractions of 1.8-2.0 Gy, five times a week, to a median total dose of 50 Gy and an initial hyperthermia treatment (HT) of I h duration utilizing the BSD 2000 with Sigma 60 or MAPA applicators at frequencies of 60-140 MHz. Further HT was given for patients with CEM43 degrees T90 > 0.5 after initial HT ('heatable' patients), twice a week to a maximum of 10 HT or CEM43 degrees T90 > 100. Of the 35 patients entered, 30 had heatable tumours, one of which was inevaluable for pCR or LF as the patient died of DM prior to surgery, leaving 29 evaluable patients. Of these 29 patients, 15 (52%) had a pCR (95% CI: 37-73%), significantly less than the projected rate of > or = 75% (p = 0.02). Of the 25 heatable tumours that achieved CEM43 degrees T90 > or = 10, 14 (56%) had a pCR (95% CI: 39-78%) significantly less than the projected rate (p = 0.06). Three of the 29 patients (10%) with heatable tumours had a LF, versus 1/5 unheatable tumours (p = 0.48). Fourteen of the 30 patients (47%) with heatable tumours developed DM, versus 2/5 unheatable tumours (p = 1.00). Ten of the 30 patients (33%) with heatable tumours developed treatment-induced toxicity. Thus, no correlation of thermal dose with histologic response was observed. Prospective control of CEM43 degrees T90 failed to achieve the projected pCR rate following pre-operative thermoradiotherapy for high-grade soft tissue sarcomas, despite excellent local control. Possible explanations for this outcome are discussed.

Solitary renal cell carcinoma metastasis to the hand: the need for wide excision or amputation.

Metastatic renal cell carcinoma responds poorly to chemotherapy or radiation therapy and is associated with a dismal survival rate. In cases of a solitary acrometastasis, the literature supports complete resection of the lesion in an effort to prolong survival. We report a patient who presented with a solitary metachronous renal cell metastasis to the middle phalanx of the index finger. The lesion was correctly identified as a renal cell metastasis and aggressive surgical management was performed with curative intent.

Knee arthrodesis with circular external fixation.

Knee arthrodesis can enable limb salvage in patients with disability secondary to trauma, infected total knee arthroplasty, pyarthrosis, and other complications. Historically, intramedullary nailing has resulted in the highest overall knee fusion rates. However, intramedullary nailing is relatively contraindicated in the presence of active infection. Nineteen patients who underwent knee arthrodesis with circular external fixation were studied retrospectively. Postoperative radiographs were evaluated for evidence of bony fusion, which was defined as trabecular bridging between the femur and tibia. Patients were interviewed and graded using the functional assessment portion of the Knee Society clinical rating system. Fusion was successful in 13 of 19 (68%) patients. Overall, patients spent an average of 4 months 8 days wearing the circular external fixator. Average time to radiographic and clinical evidence of arthrodesis (defined as lack of motion across the fusion site) was 4 months 18 days. No patient with successful fusion considered himself or herself housebound. All but one of these patients require some form of assistive device for ambulation. Complications occurred in 16 of 19 (84%) patients overall. Superficial pin tract infection (55%) and nonunion (32%) were the most common. Circular external fixation is an effective method for obtaining knee arthrodesis in patients who are not good candidates for intramedullary nailing.

Gene therapy: clinical considerations.

Gene therapy can be defined as the introduction of nucleic acid into cells to ameliorate a disease process. To date there have been more than 313 trials with more than 2000 patients enrolled. The majority of these trials are Phase I or Phase II and have target diseases of either cancer or acquired immunodeficiency syndrome using retroviral and retroviral vectors. The choice of molecular target and the means of delivery have varied and chosen on the basis of the specific indication. Until recently the risks associated with treatment had been under appreciated. The first fatality associated with gene therapy occurred in September 1999 in which an adenoviral vector was used in the treatment of a patient with ornithine transcarbamylase deficiency. Subsequent to this report, other reports have surfaced suggesting that reporting of previous non-fatal reactions may have been minimized. Safety must be considered in relation to the disease process and to alternative treatments available. It may be easier to rationalize placing patients at risk who are facing a fatal disease process without effective alternative therapies. The ultimate goal of gene therapy will be the injection of a vector that has a specific target cell and that will be regulated by physiologic signals. Such a goal will require major improvements in the currently available delivery systems or the development of novel vectors.

Gelatinase and inhibitor expression in soft tissue sarcomas: lack of correlation with distant metastasis.

The predominant mode of death for most patients with soft tissue sarcomas (STS) remains distant metastasis (DM). Current clinical predictors of DM are unreliable. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) correlate with biologic aggression in other tumors. The gene expression of the gelatinase, MMP-2 and MMP-9, and their respective inhibitors, TIMP-1 and TIMP-2, in STS was evaluated. Twelve fresh-frozen surgical specimens from patients with large (>5 cm) STS were analyzed. Six patients developed DM while 6 survived disease-free (DFS) at a minimum follow-up of 13 months. Following mRNA isolation, reverse transcription-polymerase chain reaction was performed using primers for MMP-2, MMP-9, TIMP-1, and TIMP-2. Gene expression was determined by band densitometry. Ratios of MMP-9/TIMP-1 and MMP-2/ TIMP-2 gene expression as well as MMP-2 protein activation ratio (active/inactive enzyme determined by gelatin zymography) were analyzed for correlation with DM and DFS. MMP-2 gene was expressed in 12 specimens, while MMP-9 was detectable in 9. Relative levels of MMP-2 and MMP-9, MMP2/TIMP-2 ratio, and MMP-9/TIMP-1 ratio were not significantly correlated with DM. Poor DFS was significantly correlated with high MMP-9/TIMP-1 ratio (p = 0.02). Active MMP-2 protein was detected in 12 specimens, while active MMP-9 protein was detected in 2. No association was found between MMP-2 protein activation ratio and DM or DFS. While MMP-2 gene expression and protein activity occurred in these 12 specimens, gelatinase/inhibitor ratios (for both MMP-2 and MMP-9) appear to be poor predictors of DM in STS.

Marshall Urist Award. Interstitial collagenase gene expression correlates with in vitro invasion in human chondrosarcoma.

Matrix metalloproteinases contribute to the processes of local invasion and metastasis by providing cells with the ability to traverse tissue boundaries. The levels of gene expression were quantitated for matrix metalloproteinases-1 and tissue inhibitors of metalloproteinases-1 in human chondrosarcoma cell lines, and the results were correlated with cell differentiation, collagenase activity, and in vitro invasion. Three well characterized human cell lines were used in this study, with the level of chondrocytic differentiation confirmed to be JJ012, FS090, and 105KC in increasing order on the basis of aggrecan and collagen gene expression. The matrix metalloproteinases-1/tissue inhibitors of metalloproteinases-1 ratio correlated with the level of differentiation in an inverse fashion. Collagenase activity paralleled matrix metalloproteinases-1/tissue inhibitors of metalloproteinases-1 gene expression and was associated with a more invasive phenotype in an in vitro assay. In this report, matrix metalloproteinase-1 and tissue inhibitors of metalloproteinases-1 expression in human chondrosarcoma tumor cell lines were quantitated, and it was shown that interstitial collagenase gene expression correlates inversely with chondrocytic differentiation. Differences in collagenase activity and in vitro invasion correlate inversely with the level of differentiation. These findings are consistent with the hypothesis that collagenase activity is associated with a poorer prognosis in chondrosarcoma by facilitating cell egress from the tumor matrix.

Extracellular collagen regulates expression of transforming growth factor-beta1 gene.

A complex interrelationship exists between the extracellular matrix and cytokine signaling in articular chondrocytes. We sought to determine whether the extracellular matrix serves as a regulatory component of transforming growth factor-beta1 expression. Bovine articular chondrocytes were isolated and resuspended in alginate, yielding final extracellular protein concentrations of 0 to 1.5% (wt/vol) for type-II or type-I collagen. Cultures were maintained for 7 days in the presence or absence of transforming growth factor-beta1-supplemented medium (10 ng/ml). The amount of transforming growth factor-beta1 mRNA was examined with quantitative competitive reverse transcription-polymerase chain reaction analysis. The results indicate that exogenous transforming growth factor-beta1 stimulates endogenous transforming growth factor-beta1 mRNA expression approximately 8-fold. This effect depended on the concentration of extracellular type-II collagen. As the concentration of extracellular type-II collagen is increased, the expression of transforming growth factor-beta1 mRNA decreases in both basal and transforming growth factor-beta1-stimulated cultures. Exogenous extracellular type-I collagen also served to negatively modulate transforming growth factor-beta1 gene expression but with a different concentration profile. The results demonstrate that transforming growth factor-beta1 mRNA expression was upregulated by exogenous transforming growth factor-beta1 and was downregulated by extracellular type-I and type-II collagens. The profoundly different effects on transforming growth factor-beta1 expression by the two collagens are consistent with those reported for mammary epithelial cells and likely serve as a negative feedback mechanism to preserve tissue homeostasis.

The treatment of high-grade soft tissue sarcomas with preoperative thermoradiotherapy.

PURPOSE: To explore the use of a novel program of preoperative radiation and hyperthermia in the management of high-grade soft tissue sarcomas (STS). METHODS AND MATERIALS: Eligible patients were adults over 18 with Grade 2 or 3 STS, surgically resectable without a local excision prior to referral to Duke University Medical Center and without distant metastases. Patients were staged generally with CT and/or MR imaging. The diagnosis was established with fine needle aspiration or incisional biopsy. Patients were then treated with 5000 to 5040 cGy, 180-200 cGy per fraction. Chemotherapy was usually not employed. Generally two hyperthermia treatments per week were given with a planned thermal dose of 10-100 CEM 43 degrees T90. Invasive thermometry and thermal mapping were done in all patients. Surgical resection was planned 4-6 weeks after the completion of radiation and hyperthermia. RESULTS: Ninety-seven patients were treated on study between 1984 and 1996. Follow-up ranged from 12 to 155 months (median 32). All tumors were high-grade in nature, 44 greater than 10 cm in size (maximum tumor diameter), 43 5-10 cm in size, 10 less than 5 cm. Seventy-eight of the 97 tumors were located in an extremity. Of the 97 patients, 48 remain alive and continually free of disease following initial therapy. Of the remaining 49 patients, 44 have relapsed (34 dead, 10 living with disease), 3 have died secondary to complications of therapy, and 2 have died of unrelated causes. Ten-year actuarial overall survival, cause-specific survival, and relapse-free survival are 50, 47, and 47% respectively. The predominant pattern of failure has been distant metastases with only 2 patients developing local failure alone. Ten-year actuarial local control for extremity tumors is 94%, 63% for the 19 patients with tumors at sites other than the extremity. Of the 78 patients with extremity lesions, 63 have had limb preservation and remain locally controlled. Overall 38 patients experienced 57 major complications. There were 3 deaths, one due to adriamycin cardiomyopathy and two secondary to wound infections. Four patients required amputation secondary to postoperative wound healing problems. Complications directly attributable to hyperthermia occurred in 15 patients with 11 instances of second- or third-degree burns and two instances of subcutaneous fat necrosis. The hyperthermia complications were generally not severe and either healed readily or were excised at the time of surgical resection of the primary tumor. CONCLUSIONS: For these aggressive high-grade soft tissue sarcomas, this treatment program of preoperative thermoradiotherapy provided excellent local regional control for extremity lesions (95%) and satisfactory local regional control (63%) of nonextremity sarcomas, but did not appear to influence the rate of distant metastases or survival. Complications were frequent but apart from the direct thermal burns, not too different from those reported for preoperative radiotherapy alone. More effective adjuvant systemic therapy is necessary to impact favorably on survival.