Osteosarcoma is a rare form of cancer that account for less than 0.2% of all cancers diagnosed in the United States. Unfortunately, the cause is unknown, but there have been several risks factors accused of causing the disease such as genetics, Paget disease, exposure to radiation or chemicals, and bone marrow transplants. Immunotherapy is a cancer treatment alternative to chemotherapy and radiation therapy. Its goal is to use a patient’s own immune system to fight the tumor. The patient’s immune system is stimulated by exposing synthetic immune molecules into their system. In this paper, we discuss the potential causes of osteosarcomas, the pathophysiology of the disease, and potential ways to cure the disease using different immunotherapy techniques.
Bone cancer is a rare cancer, which occurs due to the unwanted growth of cells in the bone tissues. In 2014, the American Cancer Society estimated that there were 3,020 (1,680 men and 1,340 in women) new cases of bone cancer and 1,460 (830 men and 630 among women) deaths caused by bone cancer in the United States. In adults, over 40% of primary bone cancers are osteosarcoma, which is less than 0.2% of all types of cancer. In children and teenagers (those younger than 20 years), osteosarcoma (56%) and Ewing Sarcoma (34%) are much more common than chondrosarcoma (6%).
The exact cause of most of the bone cancers is not known. Several risk factors have been found that increase the risk of developing bone cancer. These include genetic disorders, Paget disease, radiation, bone marrow transplant, exposure to chemicals, and hormone disorders.
Pathophysiology and molecular basis of Osteosarcoma: The molecular pathophysiology of osteosarcoma consists of chromosomal abnormalities, tumor suppressor gene dysfunction, transcription factors, growth factors, osteosarcoma cell proliferation, apoptosis, anchorage-independent growth, tumor angiogenesis, tumor invasion and osteoclast function, which are explained below. Potential therapeutic targets are mentioned in figure 1.
A.Chromosomal abnormalities:Osteosarcoma has been reported in patients with Bloom syndrome, Rothmund-Thompson syndrome, Werner syndrome, Li-Fraumeni syndrome, and hereditary retinoblastoma [2-4]. Amplifications of chromosomes 6p21, 8q24, and 12q14, as well as loss of heterozygosity of 10q21.1 have been reported in a study of diagnostic biopsy specimens . Numerical chromosomal abnormalities include loss of chromosomes 9, 10, 13, and 17, as well as gain of chromosome 1 .
B. Tumour suppressor Gene dysfunction: In osteosarcoma, there are mutations in both the p53 (22%) and Retinoblastoma (Rb) gene . The p53 expression was higher in low Rosen grade osteosarcoma. Rosen-grade 1: <50% necrosis; grade 2: 50%–90% necrosis; grade 3: >90% necrosis; grade 4: 100% necrosis; grade 1 + 2 = low-grade; grade 3 + 4 = high grade . The p53 mutation has also been shown to be more common in high-grade conventional osteosarcoma versus low-grade central osteosarcoma . The inherited mutation of the Rb gene causes retinoblastoma syndrome, a condition that predisposes a patient to multiple malignancies, including osteosarcoma . Both germ-line and somatic mutations of Rb may confer an increased risk of osteosarcoma .
C. Transcription factors: The activator protein 1 complex (AP1) is a regulator of transcription and consists of Fos and Jun proteins. Both of these proteins are found to be upregulated in high-grade osteosarcoma [7,8]. Overexpression of Myc in bone marrow stromal cells leads to osteosarcoma development . Myc is amplified in U2OS osteosarcoma cell line variants with the highest resistance to doxorubicin. Gain of Myc was found in SaOS-2 methotrexate-resistant variants .
D. Growth factors: High-grade osteosarcoma is found to express TGF-β1 in significantly higher amounts than low-grade osteosarcoma .Insulin-like growth factor (IGF)-I and IGF-II are also overexpressed by osteosarcoma . Connective tissue growth factor (CTGF) is related to a number of proteins in the CCN family (CTGF/Cyr61/Cef10/NOVH) and acts through integrin signaling pathways .
E. Osteoclast function: Osteosarcoma invasion relies on interactions between the bone matrix, osteosarcoma cells, osteoblasts, and osteoclasts.
Figure 1. Potential therapeutic targets in osteosarcoma .
Growth factors, such as TGF-β are released from the degraded bone matrix during the initial stages of osteosarcoma invasion and act on osteosarcoma cells, stimulating the release of PTHrP, interleukin-6 (IL-6) and interleukin-11 (IL-11) [15,16]. RANK expression is under the control of cytokines IL-1, IL-6, IL-8, tumor necrosis factor-α (TNF-α), PTHrP, and TGF-α . Receptor-ligand binding initiates a cascade of events through binding of TRAF-6. This leads to the activation of both NFκB and MAPK pathways, with the resulting increase in nuclear factor of activated T-cells (NFATc1) activity. RANK/RANKL also activates the c-Fos component of AP-1, resulting in additional NFATc1 upregulation. NFATc1 is an end-point in osteoclast activity and maturation.Crosstalk between cells of bone lineage and immune cells is common as in figure 2.
Figure 2. Crosstalk between cells of bone lineage and immune cells .
Immunotherapy for osteosarcoma:
A. Monoclonal antibodies:
a. Non FDA approved monoclonal antibodies:
1.Bevacizumab: Bevacizumab is a recombinant humanized monoclonal antibody. Its target is vascular endothelial growth factor (VEGF), a pro-angiogenic cytokine. Bevacizumab binds to VEGF and inhibits VEGF receptor binding, thereby preventing the growth and maintenance of tumor blood vessels . Bevacizumab has shown to be clinically beneficial in both preclinical studies and animal models. However, its safety in pediatric groups of patients has been a cause for concern .
2. Cixutumumab: A fully human IgG-1 monoclonal, antineoplastic antibody directed against the human insulin-like growth factor-1 receptor (IGF-1R). Cixutumumab selectively binds to membrane-bound IGF-1R, thereby preventing the binding of the natural ligand IGF-1 and the subsequent activation of PI3K/AKT signaling pathway .Preclinical studies with cixutumumab in osteosarcoma have shown promising results, however, phase II clinical study with the combination of cixutumumab and temsirolimus did not result in objective responses in pediatric and young adults with recurrent or refractory sarcoma .
3. Trastuzumab: A recombinant humanized monoclonal antibody directed against the human epidermal growth factor receptor 2 (HER2) . A phase II clinical trial on Trastuzumab, plus chemotherapy in cases with metastatic osteosarcoma, failed to demonstrate clinical benefit in those cases with tumors overexpressing Her-2-neu. Besides, both tumors with and without Her-2/neu over expression have shown similar poor results .
4. Dinutuximab: Dinutuximab is a chimeric monoclonal antibody that binds to ganglioside GD2, which is commonly overexpressed in malignant melanoma, neuroblastoma, osteosarcoma, and small cell lung cancer . Dinutuximab, in combination with Sargramostim in treating patients with recurrent osteosarcoma, is in a phase II clinical trial.
5.Glembatumumab Vedotin: Targeting glycoproteinnon-metastatic b (GPNMB) with the antibody-drug conjugate, Glembatumumab vedotin, has been suggested based on the preclinical studies evaluating the presence of GPNMB protein expression by immunohistochemistry in human osteosarcoma tumor samples and by enzyme-linked immunosorbent assay (ELISA) in osteosarcoma cell lines. In a single study, GPNMB was expressed in 92.5% (62/67) of osteosarcoma samples. All primary osteosarcoma samples expressed high levels of GPNMB mRNA. Glembatumumab induced cytotoxic effects in 74% (14/19) of osteosarcoma cell lines, and GPNMB protein levels correlated with glembatumumab in vitro cytotoxicity (r = -0.46, P = 0.04) .A phase II clinical study is currently active in treating patients with recurrent or refractory osteosarcomas.
6. Denosumab: Bisphosphonates, which inhibit osteoclast-mediated bone resorption, have been shown to suppress pulmonary metastasis and improve overall survival in an osteosarcoma model, in vivo .Several different types of bisphosphonates have been studied in vitro and in osteosarcoma models in vivo. Denosumab, which inhibits bone resorption by osteoclasts, is a humanized monoclonal antibody to RANKL for the treatment of osteoporosis and bone metastasis. A recent study showed that denosumab was able to reduce tumor size by more than 90% in all patients with RANK-positive giant cell tumors .A phase II clinical trial of Denosumab in relapsed refractory cases of osteosarcoma is currently ongoing.
|Drug||Clinical trial identifier no.||Phase||Study Design||Target|
|Bevacizumab||NCT00667342||Phase II||Non-Randomized, Safety/Efficacy Study, Open Label||VEGF|
|Cixutumumab||NCT00831844||Phase II||Safety/Efficacy Study, Open Labe||EGFR|
|Dinutuximab||NCT02484443||Phase II||Biomarker/Laboratory analysis, Treatment||Ganglioside GD2|
|Glembatumumab||NCT02487979||Phase II||Biomarker/Laboratory analysis, Treatment||GPNMB|
|Denosumab||NCT02470091||Phase II||Biomarker/Laboratory analysis, Treatment||RANKL|
Table 1. Monoclonal antibody drugs in phase I-III [19-28].
1.DEC-205-NY-ESO-1 fusion protein vaccine: A phase I clinical trial is ongoing of mTOR inhibition with Rapamycin for enhancing intranodal, dendritic cell vaccine induced, anti-tumor immunity in patients with NY-ESO-1 expressing solid tumors.
|Drug||Clinical trial identifier no.||Phase||Study Design||Target|
|DEC-205-NY-ESO1 fusion protein vaccine||NCT01522820||Phase I||Non-Randomized, Safety Study, Open Label||Tumour cells, which express NY-ESO-1|
Table 2. Vaccine in phase I trial .
C. Adoptive cell therapy
1. GD2 bispecific antibody : Ganglioside GD2 is highly expressed on neuroectoderm derived tumors and sarcomas such as osteosarcoma, rhabdomyosarcoma, Ewing’s sarcoma in children and adolescents, liposarcoma, fibrosarcoma, leiomyosarcoma and other soft tissue sarcomas in adults. GD2 expression in normal tissues is restricted to the brain and is inaccessible to circulating antibodies. GD2 expression in peripheral nerves and melanocytes is considered as a suitable target for systemic tumor immunotherapy. Anti-GD2 antibodies tested in clinical trials for neuroblastoma for over the past two decades has proven the safety and efficacy . T cells coated with GD2 bispecific antibody helps the T-cells to recognize neuroblastoma and osteosarcoma cells and kill these cancer cells.
|Drug||Clinical trial identifier no.||Phase||Study Design||Target|
|GD2 bispecific antibody||NCT02173093||Phase II||Safety/Efficacy Study, Open Label||GD2 positive cells|
Table 3. Adoptive cell therapy in clinical trials [30-31].
D. mTOR inhibitors:
1. Sirolimus : A natural macrocyclic lactone produced by the bacterium, Streptomyces hygroscopicus, with immunosuppressant properties. In cells, sirolimus binds to the immunophilin FK Binding Protein-12 (FKBP-12) to generate an immunosuppressive complex that binds to and inhibits the activation of the mammalian target of rapamycin (mTOR), a key regulatory kinase. This results in the inhibition of T lymphocyte activation and proliferation in response to antigenic and cytokine (IL-2, IL-4, and IL-15) stimulation and inhibition of antibody production. It has been investigated in a phase II clinical trial in combination with cyclophosphamide in advanced sarcoma patients. It has been well tolerated with a partial response rate of about 20% as a stable disease lasting for 6 months .
2. Everolimus : A derivative of the natural macrocyclic lactone sirolimus with immunosuppressant and anti-angiogenic properties. In cells, everolimus binds to the immunophilin FK Binding Protein-12 (FKBP-12) to generate an immunosuppressive complex that binds to and inhibits the activation of mTOR. Inhibition of mTOR activation results in the inhibition of T lymphocyte activation and proliferation associated with antigen and cytokine (IL-2, IL-4, and IL-15) stimulation and the inhibition of antibody production. A non-randomized phase II clinical trial using Everolimus in combination with sorafenib has been completed in patients with unresectable high-grade osteosarcoma progressing after standard treatment. Thirty-eight patients have been recruited between June 16, 2011, and June 4, 2013. Seventeen (45%; 95% CI 28–61) of 38 patients were progression free at 6 months. Toxic effects led to dose reductions, or short interruptions, or both in 25 (66%) of 38 patients and permanent discontinuation for 2 (5%) patients. The most common grade 3–4 adverse events were lymphopenia and hypophosphataemia each in 6 (16%) patients, hand and foot syndrome in 5 (13%), thrombocytopenia in 4 (11%), and fatigue, oral mucositis, diarrhoea, and anaemia each in 2 (5%). One patient (3%) had a grade 3 pneumothorax that required trans-thoracic drainage. That had recurred at the time of disease progression. This was reported as a serious adverse event related to the study drugs in both instances. No other serious adverse events were reported during the trial. There were no treatment-related deaths .
3. Ridaforolimus : A small molecule and non-prodrug analogue of the lipophilic macrolide antibiotic rapamycin with potential antitumor activity. Ridaforolimus binds to and inhibits the mTOR, which may result in cell cycle arrest and, consequently, the inhibition of tumor cell growth and proliferation. Upregulated in some tumors, mTOR is a serine/threonine kinase involved in regulating cellular proliferation, motility, and survival that is located downstream of the PI3K/Akt signaling pathway.A phase II clinical trial with ridaforolimus in patients with advanced bone and soft tissue sarcoma has led to PFS results that compare favorably with historical metrics. Adverse events were mostly mild to moderate and mainly consisted of stomatitis, mucosal inflammation, mouth ulceration, rash, and fatigue .
4. Temsirolimus : An ester analog of rapamycin. Temsirolimus binds to and inhibits the mammalian target of rapamycin (mTOR), resulting in decreased expression of mRNAs necessary for cell cycle progression and arresting cells in the G1 phase of the cell cycle. mTOR is a serine/threonine kinase which plays a role in the PI3K/AKT pathway that is upregulated in some tumors. Efficacy of temsirolimus in osteosarcoma models has been evaluated, combined with cisplatin or bevacizumab on the growth of human osteosarcoma xenografts (OS-33 and OS-1) in vivo. This incorporates functional imaging techniques and microscopic analyses to unravel mechanisms of response. In both OS-33 and OS-1 models, the activity of temsirolimus was significantly enhanced by the addition of cisplatin (TC) or bevacizumab (TB) .
|Drug||Clinical trial identifier no.||Phase||Study Design||Target|
|Sirolimus||NCT00743509||Phase II||Non-Randomized, Open Label||mTOR kinase|
|Everolimus||NCT01830153||Phase II||Non-Randomized, Safety/Efficacy Study, Open Label||mTOR kinase|
|Ridaforolimus||NCT00538239||Phase III||Randomized, Double Blind||mTOR kinase|
|Temsirolimus||NCT01016015||Phase II||Efficacy Study, Open Label||mTOR kinase|
|Everolimus||NCT01216826||Phase II||Non-Randomized, Safety/Efficacy Study, Open Label||mTOR kinase|
Table 4. mTOR inhibitors under clinical trials phase I-III [32-40].
E. Kinase inhibitors:
1. Pazopanib  : A small molecule inhibitor of multiple protein tyrosine kinases with potential antineoplastic activity. Pazopanib selectively inhibits vascular endothelial growth factor receptors (VEGFR) -1,-2 and -3, c-kit and platelet derived growth factor receptor (PDGF-R), which may result in the inhibition of angiogenesis in tumors, in which these receptors are upregulated. It has been approved by the FDA for the treatment of specific soft tissue sarcomas. Moreover, case studies in relapsed refractory cases of osteosarcoma showed that pazopanib might be effective in matastatic cases of osteosarcoma. The efficacy and safety of this targeted therapy needs to be validated in large randomized clinical trials .
2. Regorafenib : Regorafenib binds to and inhibits vascular endothelial growth factor receptors (VEGFRs) 2 and 3, and Ret, Kit, PDGFR and Raf kinases, which may result in the inhibition of tumor angiogenesis and tumor cell proliferation. VEGFRs are receptor tyrosine kinase that plays an important role in tumor angiogenesis; the receptor tyrosine kinases RET, KIT, and PDGFR, and the serine/threonine-specific Raf kinase are involved in tumor cell signaling. A phase I clinical study in patients with advanced solid tumors showed its benefit . Phase II clinical trial in patients with osteosarcoma is currently recruiting patients.
3. Cabozantinib : Cabozantinib strongly binds to and inhibits several RTKs, which are often overexpressed in a variety of cancer cell types, including hepatocyte growth factor receptor (MET), RET (rearranged during transfection), vascular endothelial growth factor receptor types 1 (VEGFR-1), 2 (VEGFR-2), and 3 (VEGFR-3), mast/stem cell growth factor (KIT), FMS-like tyrosine kinase 3 (FLT-3), TIE-2 (TEK tyrosine kinase, endothelial), tropomyosin-related kinase B (TRKB) and AXL. This may result in an inhibition of both tumor growth and angiogenesis, and eventually, lead to tumor regression.
|Drug||Clinical trial identifier no.||Phase||Study design||Target|
|Pazopanib||NCT01759303||Phase II||Efficacy Study, Open Label||VEGFR-1, -2 and -3, c-kit and platelet derived growth factor receptor (PDGFR)|
|Regorafenib||NCT02048371||Phase II||Randomized, Efficacy Study, Double blind||VEGFRs 2 and 3, and Ret, Kit, PDGFR and Raf kinases|
|Cabozantinib-smalate||NCT02243605||Phase II||Efficacy Study, Open Label||RTK|
Table 5. Kinase inhibitors in clinical trials phase I-III [41-45].
Bone cancer is not a common cancer. Conventional treatment for bone cancer consists of chemotherapy, surgical resection and radiation therapy. Although early tumor detection, novel surgical technics, neoadjuvant chemotherapy with effective high dose protocols and a multidisciplinary approach to osteosarcoma has led to better tumor control and limb salvage tumor removal and even cure in selected patients, the prognosis for locally advanced, metastatic and recurrent cases is still poor and the clinical outcomes provided by these therapeutic modalities have not significantly improved in recent decades. Advanced knowledge of the tumorigenesis and tumor promoting molecular pathways and interaction between immune system and tumor cells in different types of cancer as well as osteosarcoma has led to the concept of targeting these known molecular pathways and augmenting anti-tumor immune response as a potential therapeutic modality either alone or in combination with conventional therapeutic approach for an optimal response and potential less toxic therapeutic modality. Multiple drugs of these categories are under clinical trials for the treatment of osteosarcomas, such as mAbs, adoptive therapy and vaccines as well as targeted therapies addressing mediators in the carcinogenesis pathways such as mTOR inhibitors and kinase inhibitors. The available preclinical data on animal models has shown the feasibility of these therapeutic options, however, many of these treatment modalities are still under clinical investigation in early phase clinical trials and none of them has been approved for the treatment of osteosarcoma yet. Further clinical studies and enrolling patients in clinical trial may turn the light into these novel treatment modalities and their place in the therapeutic approach to osteosarcoma.
1. Soft tissue sarcoma. American cancer society.
2. HT Ta, CR Dass, PFM Choong, DE Dunstan. Osteosarcoma treatment: state of the art. Cancer Metastasis Rev. 2009, 28(1- 2): 247–263.
3. X Hu, AX Yu, BW Qi, Tao Fu, Gang Wu et al. The expression and significance of IDH1 and p53 in Osteosarcoma. J Exp Clin Cancer Res. 2010, 29(1): 43.
4. Park HR, Jung WW, Bertoni F, Bacchini P, Park JH et al. Molecular analysis of p53, MDM2 and H-ras genes in low-grade central Osteosarcoma. Pathol Res Pract. 2004, 200(6): 439–445.
5. B Alberts, A Johnson, J Lewis et al. Molecular biology of the cell. Garland Science. 2008.
6. Longhi A, Benassi MS, Molendini L, Macchiagodena M, Picci P et al. Osteosarcoma in blood relatives. Oncol Rep. 2001, 8(1): 131–136.
7. Wu JX, Carpenter PM, Gresens C, Keh R, Niman H, et al. The proto-oncogene c-fos is over-expressed in the majority of human Osteosarcoma. Oncogene. 1990, 5(7): 989–1000.
8. A Franchi, ACalzolari, G. Zampi. Immunohistochemical detection of c-fos and c-jun expression in osseous and cartilaginous tumors of the skeleton. Virchows Arch. 1998; 432(6): 515–519.
9. Shimizu T, Ishikawa T, Sugihara E, Kuninaka S, Miyamoto T et al. c-MYC overexpression with loss of Ink4a/Arf transforms bone marrow stromal cells into Osteosarcoma accompanied by loss of adipogenesis. Oncogene. 2010, 29(42): 5687–5699.
10. Hattinger CM, Stoico G, Michelacci F, Pasello M, Scionti I et al. Mechanisms of gene amplification and evidence of co amplification in drug-resistant human Osteosarcoma cell lines. Genes Chromosomes Cancer. 2009; 48(4): 289–309.
11. A Franchi, L Arganini, G Baroni, Anna Calzolari, Rodolfo Capanna et al. Expression of transforming growth factor β isoforms in Osteosarcoma variants: association of TGFβ1 with high-grade Osteosarcoma. Journal of Pathology. 1998, 185(3): 284–289.
12. B Rikhof, S De Jong, A J H Suurmeijer, C Meijer, WTA van der Graaf. J Pathol. 2009, 217(4): 469–482.
13. Lau LF, Lam SC. The CCN family of angiogenic regulators: the integrin connection. Exp Cell Res. 1999; 248(1): 44–57.
14. Kansara M, Teng MW, Smyth MJ, Thomas DM. Translational Biology of Osteosarcoma. Nat Rev Cancer. 2014, 14(11): 722- 735.
15. TA Guise, JM Chirgwin. Transforming growth factor-beta in osteolytic breast cancer bone metastases. Clin Orthop Relat Res. 2003, (415Suppl): S32–38.
16. Quinn JM, Itoh K, Udagawa N, Hausler K, Yasuda H et al. Transforming growth factor β affects osteoclast differentiation via direct and indirect actions. J Bone Miner Res. 2001, 16(10): 1787–1794.
17. Hofbauer LC, Heufelder AE. Osteoprotegerin and its cognate ligand: a new paradigm of osteoclastogenesis. Eur J Endocrinol. 1998, 139(2): 152–154.
18. H Takayanagi. The role of NFAT in osteoclast formation. Ann N Y Acad Sci. 2007, 1116: 227–237.
19. St. Jude Children’s Research Hospital; Genentech, Inc. A Study of Bevacizumab in Combination with Chemotherapy for Treatment of Osteosarcoma. In: ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2015 Jan 2.
20. Scharf VF, Farese JP, Coomer AR, Milner RJ, Taylor DP et al. Effect of bevacizumab on angiogenesis and growth of canine osteosarcoma cells xenografted in athymic mice. Am J Vet Res. 2013, 74(5): 771-778.
21. National Cancer Institute (NCI); National Cancer Institute (NCI). Cixutumumab in Treating Patients With Relapsed or Refractory Solid Tumors. In: ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2015 Jan 8. NCT Identifier: NCT00831844.
22. Wagner LM, Fouladi M, Ahmed A, Krailo MD, Weigel B et al. Phase II study of cixutumumab in combination with temsirolimus in pediatric patients and young adults with recurrent or refractory sarcoma: a report from the Children’s Oncology Group. Pediatr Blood Cancer. 2015, 62(3): 440-444.
23. Memorial Sloan Kettering Cancer Center; National Cancer Institute (NCI).Trastuzumab in Treating Patients with Recurrent Osteosarcoma. In: ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2015 Feb 27. NCT Identifier: NCT00005033.
24. Ebb D, Meyers P, Grier H, Bernstein M, Gorlick R et al. Phase II trial of trastuzumab in combination with cytotoxic chemotherapy for treatment of metastatic osteosarcoma with human epidermal growth factor receptor 2 overexpression: a report from the children’s oncology group. J Clin Oncol. 2012, 30(20): 2545-2551.
25. Dhillon S. Dinutuximab: first global approval. Drugs. 2015, 75(8): 923-927.
26. Roth M, Barris DM, Piperdi S, Kuo V, Everts S et al. Targeting Glycoprotein NMB With Antibody-Drug Conjugate, Glembatumumab Vedotin, for the Treatment of Osteosarcoma. Pediatr Blood Cancer. 2015, 63(1): 32-38.
27. Ory B, Heymann MF, Kamijo A, Gouin F, Heymann D. Zoledronic acid suppresses lung metastases and prolongs overall survival of osteosarcoma-bearing mice. Cancer. 2005, 104(11): 2522–2529.
28. Branstetter DG1, Nelson SD, Manivel JC, Blay JY, Chawla S et al. Denosumab induces tumor reduction and bone formation in patients with giant-cell tumor of bone. Clin Cancer Res. 2012, 18(16): 4415-4424.
29. Roswell Park Cancer Institute.; National Cancer Institute (NCI). Vaccine Therapy With or Without Sirolimus in Treating Patients With NY-ESO-1 Expressing Solid Tumors. In: ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2015 Jan 2.
30. Barbara Ann Karmanos Cancer Institute Maxim Yankelevich.; National Cancer Institute (NCI).Activated T Cells Armed With GD2 Bispecific Antibody in Children and Young Adults With Neuroblastoma and Osteosarcoma. In: ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2015 Jan 2.
31. Ahmed M, Cheung NK. Engineering anti-GD2 monoclonal antibodies for cancer immunotherapy. FEBS Lett. 2014, 588(2): 288-297.
32. University of Michigan Cancer Center; University of Michigan Cancer Center. A Phase II Study of Oral Cyclophosphamide and Sirolimus (OCR) in Advanced Sarcoma.In: ClinicalTrials. gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2015 Jan 2. NLM Identifier: NCT00743509
33. Schuetze SM, Zhao L, Chugh R, Thomas DG, Lucas DR et al. Results of a phase II study of sirolimus and cyclophosphamide in patients with advanced sarcoma. Eur J Cancer. 2012, 48(9): 1347-1353.
34. Asan Medical Center; Jin-HeeAhn, Asan Medical Center. RAD001 in Advanced Sarcoma.In: ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2015 Jan 2.NLM Identifier: NCT01830153.
35. Grignani G, Palmerini E, Ferraresi V, D’Ambrosio L, Bertulli R et al. Sorafenib and everolimus for patients with unresectable high-grade osteosarcoma progressing after standard treatment: a non-randomised phase 2 clinical trial. Lancet Oncol. 2015, 16(1): 98-107.
36. Merck Sharp & Dohme Corp; Merck Sharp & Dohme Corp. Ridaforolimus in25reatment of Sarcoma-SUCCEED (Sarcoma Multi-Center Clinical Evaluation of the Efficacy of Ridaforolimus)(8669-011 AM6). In: ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2015 Jan 2. NLM Identifier: NCT00538239.
37. Chawla SP, Staddon AP, Baker LH, Schuetze SM, Tolcher AW et al. Phase II study of the mammalian target of rapamycin inhibitor ridaforolimus in patients with advanced bone and soft tissue sarcomas. J Clin Oncol. 2012, 30(1): 78-84.
38. National Cancer Institute (NCI); National Cancer Institute (NCI). Temsirolimus and Cixutumumab in Treating Patients with Locally Advanced, Metastatic, or Recurrent Soft Tissue Sarcoma or Bone Sarcoma. In: ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2015 Jan 8. NCT Identifier: NCT01016015
39. Fleuren ED, Versleijen-Jonkers YM, Roeffen MH, Franssen GM, Flucke UE et al. Temsirolimus combined with cisplatin or bevacizumab is active in osteosarcoma models. Int J Cancer. 2014, 135(12): 2770-2782.
40. Sidnei Epelman; SidneiEpelman, Hospital Santa Marcelina. Phase II Study of Everolimus in Children and Adolescents With Refractory or Relapsed Osteosarcoma. In: ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2015 April 16. NCT Identifier: NCT01216826.
41. Vector Oncology; Vector Oncology. Study of Pazopanib in the Treatment of Osteosarcoma Metastatic to the Lung.In: ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2015 April 16. NCT Identifier: NCT01759303.
42. Safwat A, Boysen A, Lücke A, Rossen P. Pazopanib in metastatic osteosarcoma: significant clinical response in three consecutive patients. Acta Oncol. 2014, 53(10):1451-1454.
43. Sarcoma Alliance for Research through Collaboration; Sarcoma Alliance for Research through Collaboration. A Blanket Protocol to Study Oral Regorafenib in Patients With Refractory Liposarcoma, Osteogenic Sarcoma, and Ewing/Ewing-like Sarcomas. In: ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2015 April 16. NCT Identifier: NCT02048371.
44. Mross K, Frost A, Steinbild S, Hedbom S, Büchert M et al. A phase I dose-escalation study of regorafenib (BAY 73-4506), an inhibitor of oncogenic, angiogenic, and stromal kinases, in patients with advanced solid tumors. Clin Cancer Res. 2012, 18(9): 2658-2667.
45. National Cancer Institute (NCI); National Cancer Institute (NCI). Cabozantinib-s-malate in Treating Patients With Relapsed Osteosarcoma or Ewing Sarcoma. In: ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2015 April 16. NCT Identifier: NCT02243605.