Eugenie S. Kleinerman

Osteosarcoma: A Randomized, Prospective Trial of the Addition of Ifosfamide and/or Muramyl Tripeptide to Cisplatin, Doxorubicin, and High-Dose Methotrexate

PURPOSE To determine whether the addition of ifosfamide and/or muramyl tripeptide (MTP) encapsulated in liposomes to cisplatin, doxorubicin, and high-dose methotrexate (HDMTX) could improve the probability for event-free survival (EFS) in newly diagnosed patients with osteosarcoma (OS). PATIENTS AND METHODS Six hundred seventy-seven patients with OS without clinically detectable metastatic disease were treated with one of four prospectively randomized treatments. All patients received identical cumulative doses of cisplatin, doxorubicin, and HDMTX and underwent definitive surgical resection of the primary tumor. Patients were randomly assigned to receive or not to receive ifosfamide and/or MTP in a 2 double dagger 2 factorial design. The primary end point for analysis was EFS. RESULTS Patients treated with the standard arm of therapy had a 3-year EFS of 71%. We could not analyze the results by factorial design because we observed an interaction between the addition of ifosfamide and the addition of MTP. The addition of MTP to standard chemotherapy achieved a 3-year EFS rate of 68%. The addition of ifosfamide to standard chemotherapy achieved a 3-year EFS rate of 61%. The addition of both ifosfamide and MTP resulted in a 3-year EFS rate of 78%. CONCLUSION The addition of ifosfamide in this dose schedule to standard chemotherapy did not enhance EFS. The addition of MTP to chemotherapy might improve EFS, but additional clinical and laboratory investigation will be necessary to explain the interaction between ifosfamide and MTP.

Osteosarcoma: The Addition of Muramyl Tripeptide to Chemotherapy Improves Overall Survival—A Report From the Children's Oncology Group

PURPOSE To compare three-drug chemotherapy with cisplatin, doxorubicin, and methotrexate with four-drug chemotherapy with cisplatin, doxorubicin, methotrexate, and ifosfamide for the treatment of osteosarcoma. To determine whether the addition of muramyl tripeptide (MTP) to chemotherapy enhances event-free survival (EFS) and overall survival in newly diagnosed patients with osteosarcoma. PATIENTS AND METHODS Six hundred sixty-two patients with osteosarcoma without clinically detectable metastatic disease and whose disease was considered resectable received one of four prospectively randomized treatments. All patients received identical cumulative doses of cisplatin, doxorubicin, and methotrexate and underwent definitive surgical resection of primary tumor. Patients were randomly assigned to receive or not to receive ifosfamide and/or MTP in a 2 x 2 factorial design. The primary end points for analysis were EFS and overall survival. RESULTS In the current analysis, there was no evidence of interaction, and we were able to examine each intervention separately. The chemotherapy regimens resulted in similar EFS and overall survival. There was a trend toward better EFS with the addition of MTP (P = .08). The addition of MTP to chemotherapy improved 6-year overall survival from 70% to 78% (P = .03). The hazard ratio for overall survival with the addition of MTP was 0.71 (95% CI, 0.52 to 0.96). CONCLUSION The addition of ifosfamide to cisplatin, doxorubicin, and methotrexate did not enhance EFS or overall survival for patients with osteosarcoma. The addition of MTP to chemotherapy resulted in a statistically significant improvement in overall survival and a trend toward better EFS.

Addition of Muramyl Tripeptide to Chemotherapy for Patients with Newly Diagnosed Metastatic Osteosarcoma: a Report from the Children's Oncology Group

The addition of liposomal muramyl tripeptide phosphatidylethanolamine (MTP‐PE) to chemotherapy has been shown to improve overall survival in patients with nonmetastatic osteosarcoma (OS). The authors report the results of addition of liposomal MTP‐PE to chemotherapy for patients with metastatic OS.

miR-20a Encoded by the miR-17–92 Cluster Increases the Metastatic Potential of Osteosarcoma Cells by Regulating Fas Expression

The ability of osteosarcoma cells to form lung metastases has been inversely correlated to cell surface Fas expression. Downregulation of Fas allows osteosarcoma cells to circumvent FasL-mediated apoptosis upon entrance into the FasL(+) lung microenvironment. However, the mechanism of Fas regulation remains unclear. Here, we show that miRNA plays a role in the downregulation of Fas expression in osteosarcoma. Expression levels of several members of the miR-17-92 cluster including miR-20a and miR-19a were found to be higher in metastatic low-Fas-expressing LM7 cells than in the parental nonmetastatic high-Fas-expressing SAOS-2 cells. We also found an inverse correlation between Fas and miR-20a expression in all 8 cell lines derived from patient samples. Overexpression of miR-20a consistently resulted in the downregulation of Fas expression in SAOS-2 cells and thus in decreased sensitivity to FasL. Conversely, inhibiting miR-20a in LM7 cells increased Fas expression and their sensitivity to FasL. Mice injected with LM7 stably transfected with anti-miR-20a had fewer metastases than those with control plasmids. Taken together, our findings suggest that miR-20a, encoded by miR-17-92, downregulates Fas expression in osteosarcoma, thus contributing to the metastatic potential of osteosarcoma cells by altering the phenotype and allowing survival in the FasL(+) lung microenvironment.

A small interfering RNA targeting vascular endothelial growth factor inhibits Ewing's sarcoma growth in a xenograft mouse model.

Angiogenesis plays an essential role in tumor growth and metastasis and is a promising therapeutic target for cancer. Vascular endothelial growth factor (VEGF) is a key regulator in vasculogenesis as well as in angiogenesis. TC71 human Ewings sarcoma cells overexpress VEGF, with a shift in isoform production from membrane-bound VEGF189 to the more soluble VEGF165. Transfection of TC71 cells with a vector-based VEGF targeted small interfering RNA expression system (VEGFsi) inhibited VEGF165 expression by 80% and VEGF165 protein production by 98%, with no alteration in VEGF189 expression. Human microvascular endothelial cell proliferation and migration induced by conditioned medium from VEGFsi-transfected TC71 cells was significantly less than that induced by conditioned medium from TC71 cells and control vector-transfected TC71 cells. Furthermore, after s.c. injection into athymic nu/nu mice, the tumor growth of VEGFsi-expressing TC71 cells was significantly less than that of parental or control vector-transfected cells. Vessel density as assessed by CD31 immunohistochemical analysis and VEGF165 expression as assessed by Northern blotting were also decreased. Intratumor gene therapy with polyethylenimine/VEGFsi also resulted in tumor growth suppression. When inoculated into the tibias of nude mice, VEGFsi-expressing TC71 cells induced osteolytic bone lesions that were less severe than those induced by control groups. These data suggest that targeting VEGF165 may provide a therapeutic option for Ewings sarcoma.

Phase II study of liposomal muramyl tripeptide in osteosarcoma: The cytokine cascade and monocyte activation following administration

PURPOSE A phase II trial that uses liposome-encapsulated muramyl tripeptide phosphatidylethanolamine (L-MTP-PE) in patients with relapsed osteosarcoma is underway. To determine if in vivo cytokine induction plays a role in the mechanism of action of L-MTP-PE, we investigated the circulating cytokine levels of 16 patients who were undergoing therapy. PATIENTS AND METHODS Patients had histologically proven osteosarcoma and pulmonary metastases that developed either during adjuvant chemotherapy or that were present at diagnosis and persisted despite chemotherapy. Patients were rendered disease-free by surgery. The major goal of the study was to improve the disease-free interval in this high-risk group. L-MTP-PE 2 mg/m2 was infused during a 1-hour period twice a week for 12 weeks, then once a week for 12 weeks. Serial blood samples were collected after L-MTP-PE administration and were assayed for cytokine levels (tumor necrosis factor-alpha [TNF alpha] interleukin-1 alpha [IL-1 alpha], IL-1 beta, IL-6, interferon-gamma [IFN-gamma], neopterin, C-reactive protein). RESULTS After the infusion of L-MTP-PE, there was rapid induction of circulating TNF alpha and IL-6. TNF alpha levels peaked 1 to 2 hours after infusion in 10 of 16 patients, whereas peak IL-6 levels were detected at 2 to 3 hours in all patients. Induction of circulating TNF alpha and IL-6 was evident only after the first dose of L-MTP-PE. Neither IL-1 alpha nor IL-1 beta was detected in the plasma. Neopterin levels increased at 24 hours postinfusion, which indicated macrophage activation, and were not related to the induction of circulating IFN-gamma. C-reactive protein was elevated in all patients at 24 hours and decreased by 72 hours. Unlike circulating TNF alpha and IL-6, elevations in C-reactive protein and neopterin could be detected throughout the treatment course. CONCLUSION It is concluded that L-MTP-PE has specific biologic effects in patients with osteosarcoma that may be important to the drugs immunostimulatory capacity and its effectiveness as an antitumor agent.

Macrophages and cancer

The uncontrolled growth of metastases resistant to conventional therapeutic modalities is a major cause of death from cancer. Data from our laboratory and others indicate that metastases arise from the nonrandom spread of specialized malignant cells that preexist within a primary neoplasm. These metastases can be clonal in their origin, and different metastases can originate from different progenitor cells. In addition, metastatic cells can exhibit an increased rate of spontaneous mutation compared with benign nonmetastatic cells. These data provide an explanation for the clinical observation that multiple metastases can exhibit different sensitivities to the same therapeutic modalities. These findings suggest that the successful therapy of disseminated metastases will have to circumvent the problems of neoplastic heterogeneity and the development of resistance.Appropriately activated macrophages can fulfill these demanding criteria. Macrophages can be activated to become tumoricidal by interaction with phospholipid vesicles (liposomes) containing immunomodulators. Tumoricidal macrophages can recognize and destroy neoplastic cells in vitro and in vivo, leaving non-neoplastic cells uninjured. Although the exact mechanism(s) by which macrophages discriminate between tumorigenic and normal cells is unknown, it is independent of tumor cell characteristics such as immunogenicity, metastatic potential, and sensitivity to cytotoxic drugs. Moreover, macrophage destruction of tumor cells apparently is not associated with the development of tumor cell resistance.Macrophages are found in association with malignant tumors in a definable pattern, suggesting that the most direct way to achieve macrophage-mediated tumor regression is in situ macrophage activation. Intravenously administered liposomes are cleared from the circulation by phagocytic cells, including macrophages, so when liposomes containing immunomodulators are endocytosed, cytotoxic macrophages are generated in situ. The administration of such liposomes in certain protocols has been shown to bring about eradication of cancer metastases.Macrophage destruction of metastases in vivo is significant, provided that the total tumor burden at the tart of treatment is minimal. For this reason, we have been investigating various methods to achieve maximal cytoreduction in metastases by modalities such as chemotherapy or radiotherapy prior to macrophage-directed therapy. It is important to note that even the destruction of 99.9% of cells in a metastasis measuring 1 cm2 would leave 106 cells to proliferate and kill the host. The ability of tumoricidal macrophages to distinguish neoplastic from bystander nonneoplastic cells presents an attractive possibility for treatment of the few tumor cells which escape destruction by conventional treatments.Macrophage-directed therapy has been studied in several human protocols, yielding important biological information about the use of liposome-encapsulated macrophage activators in cancer patients. Currently, Phase II and Phase JB protocols are being conducted based on the results from Phase I trials.

Phase I trial of liposomal muramyl tripeptide phosphatidylethanolamine in cancer patients.

Twenty-eight evaluable patients with metastatic cancer refractory to standard therapy received escalating doses of muramyl tripeptide phosphatidylethanolamine (MTP-PE) (.05 to 12 mg/m2) in phosphatidylserine (PC):phosphatidylcholine (PS) liposomes (lipid:MTP-PE) ratio 250:1). Liposomal MTP-PE (L-MTP-PE) was infused over 1 hour twice weekly; doses were escalated within individual patients every 3 weeks as tolerated for a total treatment duration of 9 weeks. Routine clinical laboratory parameters, acute phase reactants and various immunologic tests were monitored at various time points during treatment. Toxicity was moderate (less than or equal to grade II) in 24 patients with chief side effects being chills (80% of patients), fever (70%), malaise (60%), and nausea (55%). In four patients L-MTP-PE treatment was deescalated due to severe malaise and recurrent fever higher than 38.8 degrees C. The maximum-tolerated dose (MTD) was 6 mg/m2. Significant (P less than .05) increases in WBC count, absolute granulocyte count, ceruloplasmin, beta 2-microglobulin, c-reactive protein, monocyte tumoricidal activity, and serum IL-1 beta were found. Significant decreases in serum cholesterol were also observed. Clearance of intravenously (iv)-infused technetium-99 (99mTc)-labeled liposomes containing MTP-PE in four patients was biphasic; gamma camera scans revealed uptake of radiolabel in liver, spleen, lung, nasopharynx, thyroid gland, and tumor (two patients). No objective tumor regression was seen. In view of its definite immunobiologic activity and lack of major toxicity, additional phase II and adjuvant trials of L-MTP-PE are warranted.

Aerosol gemcitabine inhibits the growth of primary osteosarcoma and osteosarcoma lung metastases.

Osteosacarcoma (OS) lung metastases are often resistant to chemotherapy. Most anticancer drugs are administered systemically. In many cases this is followed by dose‐dependent toxicity, which may not allow the achievement of therapeutic levels in lungs to eradicate metastases. We determined the efficacy of gemcitabine (GCB) by administering it directly to the lungs via aerosol and studied the role of the Fas pathway in response to the therapy. We used 2 osteosarcoma lung metastases animal models: human LM7 cells that form lung metastases in mice following intravenous injection and murine LM8 cells, which grows subcutaneously in mice and spontaneously metastasize to the lung. Treatment was initiated when the presence of lung metastases had been established. Aerosol GCB inhibited the growth of lung metastases in mice. Intraperitoneal GCB administration at similar dosage had no effect on lung metastases. Besides its direct effect on lung metastases, aerosol GCB suppressed the growth of subcutaneous LM8 tumor. Histopathological examination of mice receiving aerosol GCB showed no evidence of toxicity. Lungs are distinguished from other tissues by the constitutive expression of FasL. Since exposure of tumor cells to GCB upregulated Fas expression, we hypothesized that the susceptibility of the tumor cells to ligand‐induced cell death by resident lung cells may be increased. Therefore, the Fas pathway may contribute to the therapeutic effect of aerosol GCB.

Efficacy of liposomal muramyl tripeptide (CGP 19835A) in the treatment of relapsed osteosarcoma

Muramyl tripeptide phosphatidylethanolamine (MTP-PE) is a synthesized lipophilic analogue of muramyl dipeptide. MTP-PE encapsulated in liposomes (L-MTP-PE) allows selective delivery to pulmonary macrophages and circulating monocytes. In vivo administration has resulted in tumor regression in mice with B16 melanoma lung and lymph node metastases and a 40% long-term disease-free survival in dogs with osteosarcoma. Phase I studies have demonstrated that the drug is well tolerated. A Phase II trial using L-MTP-PE was undertaken in relapsed osteosarcoma patients to determine whether L-MTP-PE therapy could improve the progression-free interval in this high-risk group of patients. Patients had histologically proven osteosarcoma and pulmonary metastases that had developed during adjuvant chemotherapy or that were present at diagnosis and had persisted despite chemotherapy. Patients were rendered disease free by surgery. L-MTP-PE, 2 mg/m2, was infused over a 1-hour period twice a week for 12 weeks in 12 patients (Group 1). Sixteen patients (Group 2) received 2 mg/m2 L-MTP-PE twice a week for 12 weeks, then once a week for 12 weeks, for a total of 24 weeks of therapy. Progression-free intervals in each group were calculated from the day of surgery to the day of relapse and compared with the progression-free interval of a historical control group (Group 3) treated postoperatively with chemotherapy at M. D. Anderson Cancer Center between 1980 and 1990. Patients who received 24 weeks of L-MTP-PE therapy had a significant prolongation in time to relapse but those who received 12 weeks of therapy did not. The median time to relapse for group 2 patients was 9.0 months compared with 4.5 months for the control group (Group 3). These data suggest that L-MTP-PE deserves further investigation in a more appropriate adjuvant setting. A nationwide randomized Phase III trial is now underway in newly diagnosed osteosarcoma patients in conjunction with the Childrens Cancer Study Group and the Pediatric Oncology Group.