New targets and approaches in osteosarcoma

JPT-06496; No of Pages 11 Pharmacology & Therapeutics xxx (2012) xxx–xxx

Contents lists available at SciVerse ScienceDirect

Pharmacology & Therapeutics journal homepage: www.elsevier.com/locate/pharmthera

Associate editor: B. Teicher

New targets and approaches in osteosarcoma Jonathan Gill a, c, Manpreet K. Ahluwalia a, c, David Geller b, c, Richard Gorlick a, c,⁎ a b c

Department of Pediatrics, Montefiore Medical Center and The Children's Hospital at Montefiore, Bronx, NY, United States Department of Othopedic Surgery, Montefiore Medical Center and The Children's Hospital at Montefiore, Bronx, NY, United States The Albert Einstein College of Medicine, Bronx, NY, United States

a r t i c l e

i n f o

a b s t r a c t Osteosarcoma is the most common primary tumor of bone. Approximately 2/3 of patients who present with localized osteosarcoma can be expected to be cured of their disease with surgery and routine chemotherapy. Only 1/3 of patients with metastases detectable at presentation will be cured. These survival trends have stagnated over the past 20 years using conventional chemotherapy. New agents need to be rationally investigated to strive for improvement in the survival of patients diagnosed with osteosarcoma. This manuscript will review the rationale for conventional chemotherapy used in the treatment of osteosarcoma, as well as agents in varying stages of development that may have promise for treatment in the future. © 2012 Elsevier Inc. All rights reserved.

Keywords: Osteosarcoma Chemotherapy Novel agents Review

Contents 1. Introduction . . . . . . . 2. Conventional chemotherapy 3. Investigational agents . . . 4. Conclusion . . . . . . . . References . . . . . . . . . .

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1. Introduction Osteosarcoma (OS) is the most common primary malignant tumor arising from bone in children and young adults. It arises from mesenchymal cells and is pathologically characterized by spindle cells and aberrant osteoid formation. The incidence of OS has a bimodal distribution with a peak in adolescence and a second peak occurring in the seventh and eighth decade of life. The latter peak, in the era prior to the advent of bisphosphonates, likely represents a consequence of long-standing Paget's disease of bone (Mirabello et al., 2009). In

Abbreviations: IGF, insulin-like growth factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; HER2, human epidermal growth factor receptor 2; MAP, methotrexate, doxorubicin, cisplatin; mTOR, mammalian target of rapamycin; MTPPE, muramyl tripeptide-phosphatidyl ethanolamine; OS, osteosarcoma; PDGF, platelet-derived growth factor; PPTP, Pediatric Preclinical Testing Program; RANK, receptor activator of nuclear factor κB; 153SM-EDTMP, Samarium-153 ethylene diamine tetramethylene phosphonate; VEGF, vascular endothelial growth factor. ⁎ Corresponding author at: Department of Pediatrics, The Children's Hospital at Montefior, 3415 Bainbridge Avenue, Rosenthal 3rd Floor, Bronx, NY, United States. Tel.: +1 718 741 2342; fax: +1 718 920 6506. E-mail address: rgorlick@montefiore.org (R. Gorlick).

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children and young adults, approximately 400 new cases of OS are diagnosed in the United States each year, making it the eighth most common malignancy of childhood (Ottaviani & Jaffe, 2009). High-grade OS can occur in any bone. Most commonly, OS originates juxtaposed to the knee joint involving the distal femur (43%) or proximal tibia (23%). The proximal humerus, the next most common site of disease, represents approximately 10% of cases (Bielack et al., 2002). These bones experience rapid cell division to facilitate the increased growth velocity during adolescence. Within the bone, OS typically occurs in the metaphysis in proximity to the growth plate. The stimulatory signals associated with pubertal growth may represent an inciting event in the oncogenesis of OS. Most patients with newly diagnosed OS present with localized disease. Approximately 15–20% of patients have metastases detectable at presentation. The lung is the most common site of metastasis, comprising more than 85% of metastatic disease, with bone being the second most common site of distant disease (Bielack et al., 2002). However, these data are limited by the technology available to detect metastatic disease. Prior to the era of modern chemotherapy, greater than 80% of patients with localized disease treated by amputation alone developed metastases, most occurring within one year of diagnosis

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Please cite this article as: Gill, J., et al., New targets and approaches in osteosarcoma, Pharmacology & Therapeutics (2012), http://dx.doi.org/ 10.1016/j.pharmthera.2012.09.003

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(Marcove et al., 1970). Given this experience, it is considered that most patients with localized OS have micrometastases at presentation. The curative approach to OS entails both surgery and chemotherapy. The benefit of chemotherapy has been validated by MultiInstitutional Osteosarcoma Study (MIOS) conducted in the 1980's, in which patients were randomized to surgery alone versus adjuvant chemotherapy following amputation for localized OS. The patients randomized to receive chemotherapy had a significant improvement in their 2-year relapse-free survival: 66 versus 17% (Link et al., 1986). This significant difference persisted at follow-up greater than 5-years, validating that the addition of chemotherapy improves overall survival and does not just delay time to progression. Chemotherapy for osteosarcoma is administered in both the neoadjuvant and adjuvant setting. The rationale for preoperative chemotherapy originated in the late 1970's in centers, accustomed to using chemotherapy for osteosarcoma, looking to avoid amputation. Preoperative chemotherapy provided a necessary bridge until a custom-fitted prosthesis could be constructed, which in the initial protocols required several months (Rosen et al., 1979). Despite improvements in manufacturing, this legacy has persisted, and most protocols today mandate 10–12 weeks of preoperative chemotherapy before proceeding to definitive resection. A randomized trial comparing neoadjuvant chemotherapy to surgery followed by adjuvant chemotherapy found that immediate surgery provided no benefit over delayed surgery (Goorin et al., 2003).

Another legacy of neoadjuvant chemotherapy is the ability to measure the chemoresponsiveness of a patient's tumor by evaluating the degree of tumor necrosis. This was initially codified into the Huvos grading system, ranging from grade I (little or no effect identified) to grade IV (no histologic evidence of viable tumor identified within the entire specimen) (Rosen et al., 1983). This has been further modified into the tumor necrosis grading which reports the percent necrosis within the resected tumor. The degree of tumor necrosis following neoadjuvant chemotherapy has been shown to be predictive of overall survival. Patients with Huvos grade III or IV, corresponding to greater than 90% necrosis, are generally considered good responders. Patients with a good response to chemotherapy have a disease-free survival greater than 65%. Some studies have demonstrated that patients with a grade IV response to chemotherapy have markedly better outcomes than patients with a grade III response. In comparison, the poorresponders' disease-free survival approaches 50% (Bacci et al., 2005). The tumor necrosis grade can thus be used as a surrogate marker for the chemoresponsiveness of the individual's tumor to neoadjuvant chemotherapy. Investigators attempting to identify new agents or combination regimens have used this tool to extrapolate differences in tumor necrosis to effects on survival. To that effect, it has been demonstrated that increasing the number of agents in the neoadjuavant period increases the percentage of patients with higher grades of necrosis (Bacci et al., 2003). Likewise, it has been shown that increasing the

Table 1 Trials leading to present day use of conventional chemotherapy.

Investigator

Patients

Regimen

Outcome (follow-up)

(year) Marcove (1970)

145 nonmetastatic

Cortes (1972)

13 metastatic

Event-free survival (5-year) 17.4% Doxorubicin

Response rate (5-12 months) 31%

Jaffe (1974)

20 nonmetastatic

Vincristine

Event-free survival (2-23 months)

Methotrexate

80%

Cortes (1974)

21 nonmetastatic

Doxorubicin

Event-free survival (18 months)

Ochs (1978)

8 metastatic

Cisplatin

Response rate

Marti (1985)

18 recurrent

Ifosfamide

Response rate

Link (1986)

156 nonmetastatic

45% 63% 33% 36 randomized

Event-free survival (2 years) No chemotherapy

77 declined

Randomized 17% Non-randomized 9%

randomization

Winkler (1990)

Goorin (2003)

(59 chemotherapy,

Bleomycin, cyclophosphamide, dactinomycin,

18 obervation)

methotrexate, doxorubicin, cisplatin

109 metastatic and

Randomized 66% Non-randomized 67% >90% tumor necrosis

nonmetastatic

Doxorubicin, methotrexate, ifosfamide, IA cisplatin

68%

Doxorubicin, methotrexate, ifosfamide, IV cisplatin

69%

100 nonmetastatic

Methotrexate, doxorubicin, cisplatin, bleomycin,

Event-free survival (2-years)

cyclphosphamide, dactinomycin

Meyers (2008)

Neoadjuvant

61%

Adjuvant

69%

662 nonmetastatic

Event-free survival (4 years) Methotrexate, doxorubicin, cisplatin

65%

Methotrexate, doxorubicin, cisplatin, ifosfamide

66%

Factorial (MTP-PE)

Please cite this article as: Gill, J., et al., New targets and approaches in osteosarcoma, Pharmacology & Therapeutics (2012), http://dx.doi.org/ 10.1016/j.pharmthera.2012.09.003

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duration of preoperative chemotherapy increases the percentage of patients with higher grades of necrosis. However, the predictive value of tumor necrosis grade on overall survival is blunted by the intensification of preoperative chemotherapy (Meyers et al., 1998). Hence, the use of the preoperative window period to test the antitumor activity of new agents has to be done with caution as those results may not reflect an anti-cancer effect leading to improvement in survival, but may be more reflective of the innate biology of the individual's tumor. Likewise, some biomarkers have been shown to correlate with survival but not with histologic response. A case in point is the biomarker, p-glycoprotein, the protein product of the multidrug resistance gene MDR1¸which functions as an ATP dependent efflux pump. In a meta-analysis, p-glycoprotein expression nearly doubles the risk of progression at 2 years, but had a sensitivity and specificity approaching 50% for histologic response (Pakos & Ioannidis, 2003). This discordance highlights the difficulty of using the grade of tumor necrosis as a surrogate marker for overall survival. To date, no molecular markers have been prospectively validated to predict response to chemotherapy and overall survival. For patients with poor response to neoadjuvant chemotherapy, no markers have been validated to help delineate patients who are likely to be cured with the current chemotherapeutic regimens or those who may benefit from investigational or escalation of therapy. 2. Conventional chemotherapy Historically, many agents have been tried by clinicians attempting to improve the outcome of patients with OS. Since the 1990's many protocols have contained a similar three-drug backbone consisting of methotrexate, doxorubicin, and cisplatin (MAP), which are considered active agents in OS, with the possible inclusion of ifosfamide and etoposide (Table 1). Methotrexate has been used to treat osteosarcoma since the early 1970's (Jaffe, 1972). Methotrexate is an analog of folic acid. Its intracellular target is dihydrofolate reductase (DHFR) which converts dihydrofolate to tetrahydrofolate. The inhibition of DHFR leads to impaired purine synthesis. Single agent methotrexate administered every 3 weeks in a case series of 20 patients with a brief follow-up of 2–23 months led to an event-free survival greater than 70% (Jaffe et al., 1974). In the current treatment of OS, methotrexate is required at high-doses of 12 g/m 2, with some protocols suggesting a maximum dose of 20 g. This is typically administered as a bolus infusion over 4 h. After 24 h from the initiation of the infusion, leucovorin the reduced version of folic acid, which bypasses DHFR, is administered as a “rescue”. Doxorubicin, like methotrexate, was first used in OS in the early 1970's (Cortes et al., 1972). Historically, multiple different mechanisms of action have been attributed to doxorubicin including free radical formation, direct DNA damage, as well as interacalation. Currently data suggest that doxorubicin acts through topoisomerase II by stabilizing the intermediary “cleavage DNA” product of topoisomerase II and inhibiting religation. Single-agent doxorubicin (30 mg/m2 ×3 days) in a case series of 21 patients demonstrated activity in osteosarcoma with a disease-free survival of 45% with a follow-up of 1–32 months (median 9 months) (Cortes et al., 1974). Doxorubicin is typically administered in doses approximating 75 mg/m2 per cycle, divided over 2 to 3 days, either as a continuous infusion or bolus injection. Dexrazoxane has been used for cardioprotection in patients receiving bolus injection. Cisplatin was introduced in the treatment of osteosarcoma in the late 1970's (Ochs et al., 1978). Cisplatin's mechanism of action involves directly binding to DNA forming adducts. The most common of which are crosslinks between two adjacent bases. However, distal intrastrand and interstand links have also been observed. In the initial report by Ochs, they reported responses in 5 of 8 patients, all of whom had progressed on prior therapy with methotrexate or doxorubicin. Multiple case series had subsequently been published demonstrating

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enhanced activity of cisplatin when administered intra-arterially into the tumor bed. Ultimately a randomized trial compared intra-arterial versus intravenous cisplatin as part of a 4 drug regimen. This trial showed no benefit to the intra-arterial administration of cisplatin (Winkler et al., 1990). In most protocols, cisplatin is now administered intravenously at a dose of 120 mg/m 2 over 4 h, either in one day or divided over 2 days. With the backbone of MAP therapy, the long-term survival of patients with localized disease is approximately 65% (Meyers et al., 2008). For patients with metastatic disease detectable at presentation, the long-term survival is nearer to 30–35% (Chou et al., 2009). These results have not improved for the past two decades (Smith et al., 2010). Two other chemotherapeutic agents, used in combination, considered to have activity in OS are ifosfamide and etoposide. Ifosfamide has been used in OS since the 1980's. Ifosfamide is an analog of cyclophosphamide with a similar mechanism of action as an alkylating agent. Single-agent ifosfamide, administered at a dose of 9 g/m 2 divided over 5 days, demonstrated a response rate of 33% in 18 patients with previously treated recurrent osteosarcoma (Marti et al., 1985). When the dose of single-agent ifosfamide is increased to 12 g/m 2 divided over 5 days, the response rate is 27% in patients with previously untreated metastatic OS and 10% in patients with relapsed disease (Harris et al., 1995). The addition of ifosfamide (6 g/m2) to standard MAP therapy increases the percentage of patients with good necrosis. However, it does not improve event-free or overall survival from historical controls (Bacci et al., 2001). Again highlighting that intensification of preoperative chemotherapy blunts the ability of histologic response to predict overall outcomes. Increasing the dose of ifosfamide to 15 g/m2 when used in conjunction with MAP also does not improve survival from historical controls (Ferrari et al., 2005). A randomized trial, using a factorial design comparing the addition of ifosfamide and MTP-PE (to be discussed further in this manuscript) to MAP demonstrated that the addition of ifosfamide (9 g/m2) does not provide benefit over MAP alone (Meyers et al., 2005). Nevertheless, ifosfamide continues to be used in protocols around the world attempting to define its role in the treatment of OS. A recently published trial from the Scandinavian Sarcoma Group adds ifosfamide (10 g/m2) to patients who demonstrated a poor histologic response to MAP. They report that the addition of ifosfamide did not improve the metastasis-free survival of poor responders (48%) to be comparable with good responders (89%) (Smeland et al., 2011). In another strategy to incorporate ifosfamide, a recent single-arm trial published from St. Jude's replaced methotrexate with ifosfamide (8 g/m2) and demonstrated survival similar to historical controls (Daw et al., 2011). Etoposide is frequently paired with ifosfamide in the treatment of sarcomas, even though etoposide has not been demonstrated to have single agent activity. Etoposide is a podophyllotoxin which once again stabilizes topoisomerase II by forming a complex binding DNA and topoisomerase II. A phase II trial using ifosfamide and etoposide in patients with relapsed or refractory disease showed that that the combination had activity in OS with a response rate of 48% (Gentet et al., 1997). When this combination is administered to patients with a newly diagnosed metastatic OS the response rate is 59% (Goorin et al., 2002). In patients with non-metastatic OS, in a single-arm study, the addition of ifosfamide and etoposide to patients with a poor response to neoadjuvant MAP leads to an event-free survival of 56%, compared to good responders' event-free survival of 68% (Bacci et al., 2000). A pilot study, P9754, conducted by an intergroup collaboration between POG and CCG, in one arm added ifosfamide and etoposide to patients with less than 98% necrosis following neoadjuvant chemotherapy with methotrexate, doxorubicin, and cisplatin. They found that the addition of ifosfamide and etoposide was clinically feasible (Schwartz et al., 2004). Ifosfamide and etoposide have also been used to replace doxorubicin in the treatment of patients with newly diagnosed OS in a randomized cross-over trial based on

Please cite this article as: Gill, J., et al., New targets and approaches in osteosarcoma, Pharmacology & Therapeutics (2012), http://dx.doi.org/ 10.1016/j.pharmthera.2012.09.003

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R1507 SCH 717454 IMC-A12

Trastuzumab

Pemetrexed Pralatrexate

IGF

MET

HER2

Crizotinib

PDGF VEGF

Akt Imatinib

TK

mTOR

Sorafenib Pazopanib

DHFR

Rapamycin Ridaforolimus

Trimetrexate Pralatrexate Pemetrexed

Dasatinib Saracatinib

TK TK

src

TK

Aurora Thymidylate synthase

Kinase MLN8237

Pemetrexed Tyrosine Kinase (TK) Serine-threonine Kinase Folate-dependent Enzyme Fig. 1. Schematic of investigational agents and targets.

histologic response at the time resection. The two regimens demonstrated similar outcomes with no statistically significant difference. The reported 3-year event-free survival was 82 and 49% in good and poor responders, respectively, in the doxorubicin arm and 77 and 60% for good and poor responders in the ifosfamide/etoposide arm (Le Deley et al., 2007). This led to EURAMOS I, a multi-national phase III trial of patients with newly diagnosed OS, randomizing patients with poor response to MAP to either continue with MAP or receive MAP with the addition of ifosfamide and etoposide. The study recently completed its planned accrual. The results of which are greatly anticipated. 3. Investigational agents In addition to traditional chemotherapy, there are multiple investigational agents being studied which target pathways that are believed to be active in OS (Fig. 1, Table 2). 3.1. Immunomodulation Muramyl tripeptide-phosphatidyl ethanolamine (MTP-PE) is an analog of muramyl dipeptide. Muramyl dipeptide is a cell wall component of Bacille Calmette–Guerin which activates monocytes and macrophages. In the mouse xenograft of melanoma cell lines, muramyl dipeptide activates alveolar macrophages to eradicate preexisting pulmonary and lymph node metastases (Fidler et al., 1981). In dogs with spontaneous OS, which similarly develop pulmonary metastases following amputation, the administration of MTP-PE led to an improvement of

survival from 77 days for dogs receiving sham injections to 222 days (MacEwen et al., 1989). A phase II trial, conducted in patients with relapsed OS following complete surgical resection of all detectable disease, demonstrated that 24 weeks of treatment with MTP-PE significantly increased time to relapse from 4.5 months for historical controls to 9 months. The patients treated with 12 weeks of MTP-PE did not receive clinical benefit (Kleinerman et al., 1995). A phase III, multi-institutional trial, conducted in patients with newly diagnosed OS, used a factorial design to compare the addition of MTP-PE and/or ifosfamide to standard MAP therapy. This factorial design, when the initial results were released, revealed an unanticipated statistically significant interaction between ifosfamide and MTP-PE which complicated the interpretability of the benefit of MTP-PE (Meyers et al., 2005). However, upon evaluation at a later date, this interaction was no longer statistically significant, and the second analysis demonstrated that the addition of MTP-PE improved overall survival from 70 to 78% (p=.03) (Meyers et al., 2008). In patients with metastatic disease at presentation, MTP-PE improved survival from 40 to 53% (p=.027) (Chou et al., 2009). However, given the unanticipated consequences of the trial design, the benefit of the addition of MTP-PE remains unclear, and further investigation is warranted given these promising findings. The European Medical Agency has approved the use of MTP-PE for patients with osteosarcoma, the Food and Drug Administration has not approved this agent for use in the United States. Another immunomodulator being studied in osteosarcoma is interferon α-2b. Interferon inhibits growth of osteosarcoma cell lines derived from patient tumors grown in culture (Strander & Einhorn, 1977). This effect has been recapitulated in xenograft models of

Please cite this article as: Gill, J., et al., New targets and approaches in osteosarcoma, Pharmacology & Therapeutics (2012), http://dx.doi.org/ 10.1016/j.pharmthera.2012.09.003

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patient derived osteosarcoma cell lines grown in nude mice, with evidence that interferon may lead to tumor differentiation in some samples (Brosjo et al., 1987). In a pilot study of patients with nonmetastatic OS, treated with single-agent adjuvant interferon-α for 3–5 years, the authors report that 63% remained free of disease at 5 years (Strander et al., 1995). Interferon-α has been incorporated into the Euramos I trial. Patients with a good response to neoadjuvant chemotherapy were randomized to interferon maintenance versus observation after completion of adjuvant MAP chemotherapy. As noted previously, this trial has completed accrual, but the results are not available at this time.

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Granulocyte-macrophage colony-stimulating factor (GM-CSF) is another mode of immunomodulation which has been tried in osteosarcoma. The immunomodulatory effects of GM-CSF were demonstrated in mice inoculated with irradiated melanoma cell lines transduced with GM-CSF. Vaccination with these cells was found to be protective from tumor formation when subsequently challenged with nontransduced cells (Dranoff et al., 1993). In a phase I study of aerosolized GM-CSF, of the 7 patients enrolled, one patient with melanoma demonstrated a partial response and one patient with Ewing sarcoma developed a complete response (Anderson et al., 1999). This led to a phase II trial of patients with osteosarcoma with lung metastases in first recurrence. While the

Table 2 Clinical trials of investigational agents in patients with osteosarcoma.

Investigator

Patients

Regimen

Outcome (follow−up)

MTP-PE

Event-free survival

662 nonmetastatic

Randomized factorial design

(4-years/5-years)

91 metastatic

(MAP/I)

70%

78%

40%

53%

(year) Meyers (2008)

Chou (2009)

Strander (1995)

Arndt (2010)

19 nonmetastatic

43 lung metastases

Interferon α-2b

Disease-free survival (5 years)

Single-agent adjuvant

63%

Aersolized GM-CSF

Event-free survival (2 years)

(recurrence) Meyers (2011) 29 nonmetastatic

12.9% Pamidronate

Event-free survival (5 years)

MAP backbone

72%

11 metastatic Berger (2012)

22 relapsed

45% 153

Samarium (

SM-EDTMP)

metastatic (bone)

Progression-free survival (60 days) 45%

Grignani (2012)

35 relapsed

Sorafenib

Unresectable

Response rate /disease control rate 14%/49%

Bond (2008)

10 relapsed/refractory

Imatinib

Response rate 0%

Ebb (2012)

96 metastatic

Trastuzumab for HER2+ only

Event-free survival (30

41 HER2 positive

MAPIE backbone

months)

55 HER2 negative

32% 32%

Bagatell (2011)

Chawla (2012)

3 relapsed/refractory

54 metastatic/unresectable

IGF-1R (R1507) phase 1.

Response rate

31 total patients

2/3 stable disease

Ridaforolimus

Response rate

(Bone sarcomas) Trippett (1999)

7 relapsed/

2 patients PR Trimetrexate

refractory Duffaud (2012)

32 relapsed/ refractory

Response rate 2 patients (1 CR, 1 PR)

Pemetrexed

Response rate 1 PR, 5 SD

Please cite this article as: Gill, J., et al., New targets and approaches in osteosarcoma, Pharmacology & Therapeutics (2012), http://dx.doi.org/ 10.1016/j.pharmthera.2012.09.003

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GM-CSF was well-tolerated, 37 of the 43 evaluable patients relapsed. The majority of the recurrences occurred within one year of enrollment, with a median event-free survival of 4.3 months, and a 2-year event-free survival of 12.9%. Likewise, there was no evidence of immunostimulatory effects of the GM-CSF on the resected nodules (Arndt et al., 2010). 3.2. Bone signaling Bisphosphonates and the RANK-ligand inhibitors inhibit bone resorption. These drugs have been studied in multiple models of OS. In OS cell lines, alendronate inhibited cellular invasion, reduced mRNA and protein levels of metalloproteinase, as well as induced apoptosis (Cheng et al., 2004). Another bisphosphonate, zoledronate, has also been shown to affect OS cell lines through several mechanisms. Zoledronate increases the rate of apoptosis, inhibits cell migration, and decreases expression of vascular endothelial growth factor. In a xenograft model, zoledronate reduced primary tumor growth as well as decreased the development of lung metastases (Dass & Choong, 2007). A single-arm pilot study in patients with newly diagnosed OS administered the bisphosphonate pamidronate monthly for 12 months in conjunction with MAP. The addition of pamidronate did not interfere with the ability to administer all planned courses of chemotherapy; neither did it affect the quality of the surgical reconstruction (Meyers et al., 2011). A phase I/II trial of zoledronate in conjunction with MAP and ifosfamide-etoposide in patients with newly diagnosed metastatic osteosarcoma has recently completed accrual. Preliminary results suggest that zoledronate can be safely added to multi-agent chemotherapy for patients with metastatic OS. Results from the expanded cohort are still pending (Goldsby et al., 2011). In India, a phase II/III trial of the addition of zoledronate to cisplatin, doxorubicin, and ifosfamide is ongoing. In France, a phase III trial of zoledronate in combination with a MAP-ifosfamide-etoposide is also accruing patients. The receptor activator of nuclear factor κB (RANK)-ligand is expressed in tumor samples of patients with osteosarcoma. In a retrospective review, 75% of biopsy specimens had a positive staining for RANK-ligand. Half of those samples demonstrated high levels of expression. For the patients with high RANK-ligand expression, the 5-year event-free survival was poor at 17.8% (Lee et al., 2011). Inhibition of RANK in osteosarcoma cell lines has been demonstrated to have limited effect on inhibition of growth, but decreased cell motility and invasion. Likewise RANK activation provided resistance against anchorage-independent cell death (Beristain et al., 2012). Denosumab is a monoclonal antibody to RANK-ligand that inhibits activation of RANK. It has been shown to provide benefit for patients with breast cancer with bone metastases. In these patients, in a phase III trial, denosumab was superior to zoledronate in reducing markers of bone turnover as well as delaying time until the first skeletal related event. Treatment with denosumab did not provide benefit in terms of overall survival or disease progression (Stopeck et al., 2010). There are ongoing trials of the use of denosumab in the prevention of bone metastases in patients with solid tumors. Denosumab has also demonstrated efficacy in the treatment of giant cell tumor of bone. Unlike OS which is believed to be derived from malignant transformation from mesenchymal stem cell/osteoblast origin, giant cell tumor is a benign tumor with rare malignant conversion thought to be derived from osteoclastic origin. Giant cell tumor like osteosarcoma has osteoid matrix deposition either as reactive at the margin or formed within the tumor. Giant cell tumors have been demonstrated to have levels of RANK-ligand expression. Treatment with denosumab has been shown to improve functional status or reduce pain in patients with giant cell tumor, and all the patients whose tumor was evaluable demonstrated greater than 90% reduction in tumor giant cells (Branstetter et al., 2012). Given these results, and the high expression of RANK-ligand in some patients with OS, further investigation of denosumab may be warranted.

Conjugated radioisotopes provide a mechanism of utilizing pathways of bone metabolism to deliver local high-doses of radiation. Samarium-153 ethylene diamine tetramethylene phosphonate ( 153SM-EDTMP) has high specificity for bone uptake and has been approved by the Food and Drug Administration in 1998 for palliation of pain for patients with bone metastases. The major toxicity of this treatment is myelosuppression with all patients observed to have reversible neutropenia and delayed recovery of thrombocytopenia (Turner & Claringbold, 1991). In patients with OS, 153SM-EDTMP has been used at high doses followed by autologous stem cell rescue. At 60 days following administration, 45% of patients had stable disease and 55% had progression. The median progression-free survival observed was 61 days. Pain relief was minimal because of the early pain flair immediately following the administration of the radioisotope, followed by the rapid rate of disease progression (Berger et al., 2012). Unlike 153SM-EDTMP which emits beta-particles, radium223 chloride (Alpharadin) emits alpha-particles which have less tissue penetration. A phase II trial in men with prostate cancer with metastases to bone, found that treatment with radium-223 led to very few patients having any hematologic toxicity (Nilsson et al., 2007). Improvements in pain were dose-dependent, ranging from 40 to 71% of patients (Nilsson et al., 2012).

3.3. Membrane tyrosine kinase receptor inhibitors OS expresses in varying degrees cell surface transmembrane receptors. These include vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), human epidermal growth factor receptor 2 (HER2), and MET. These all share common features of being transmembrane receptors with tyrosine kinase activity. Intracellularly they activate different signaling cascades which have been implicated in oncogenesis. VEGF receptors comprise a family of receptors of which VEGFR-3 has been demonstrated to be overexpressed in OS cell lines (Hassan et al., 2012). The expression of VEGFR-3, but not its homologues, has been correlated with worse event-free and overall survival (Abdeen et al., 2009). Inhibitors of VEGF receptors studied in OS target the tyrosine kinase pocket of the receptor. The Pediatric Preclinical Testing Program (PPTP) serves as a multi-institutional NCI contract funded consortium for investigating new agents in multiple primary tumor cell lines and xenograft models. This group has tested several tyrosine kinase inhibitors including sorafenib, sunitinib, cediranib, and pazopanib. Each of these agents has the ability to inhibit multiple different tyrosine kinases with varying degrees among the agents. All of these agents, when tested by the PPTP, demonstrate growth inhibition in the OS models. Only sorafenib has been studied specifically in patients with OS. Singleagent sorafenib was administered, in a single-arm phase II trial in patients with relapsed unresectable OS. The median progression-free survival observed was 4 months, with an overall response rate of 14% and the disease control rate (which includes patients with stable disease) was 49% (Grignani et al., 2012). Pazopanib, a very non-specific tyrosine kinase inhibitor with multiple intracellular targets, has been studied in soft tissue sarcomas, most recently with the publication of the results of the PALETTE trial. This was a multi-national placebo controlled phase III trial in patients with metastatic soft tissue sarcoma who had failed prior therapy. Pazopanib increased the median progression time from 1.6 months for the placebo arm to 4.6 months (van der Graaf et al., 2012). Although patients with OS were not included in this report, this single-agent activity in patients with chemoresistant sarcomas does suggest that this class of agents may have a role in treating other sarcomas as well. A major hurdle for the adoption of these tyrosine kinase inhibitors is the legacy of bevacizumab, a targeted antibody to the VEGF receptor. Despite bevacizumab's promising preclinical results, clinical trials rarely report remission. Regulatory agencies may be reluctant to support trials with the use of these agents.

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Unlike VEGF, the data for the inhibition of PDGF receptors is less promising. Tumor samples taken from initial biopsies of patients with OS have varying expression of PDGF-A. There are conflicting reports about whether PDGF-A expression correlate with prognosis (Kubo et al., 2008; Sulzbacher et al., 2010). Imatinib is a tyrosine kinase inhibitor which has been described to have inhibitory activity on PDGF-A. In cell culture, OS cell lines' resistance to imatinib is thought to be secondary to one of the down-stream targets, mitogen-activated protein kinase, being constitutively active (Kubo et al., 2008). In a phase II study of imatinib in children with refractory or relapsed solid tumors, 10 patients with osteosarcoma were evaluable, none of whom demonstrated response to imatinib (Bond et al., 2008). HER2, like PDGFR, has some controversy regarding its clinical significance in osteosarcoma. Approximately 40% of osteosarcoma patient samples demonstrate high levels of HER2 expression. Increased expression of HER2 was found to occur more frequently in patients who presented with metastases at the time of diagnosis and at relapse. Expression of HER2 also correlated with worse tumor necrosis grade at the time of resection and with a worse event-free survival (Gorlick et al., 1999; Onda et al., 1996). However, other investigators found minimal HER2 expression on the cell membrane in osteosarcoma samples. For patients with cytoplasmic HER2 expression, they found no associate with response to preoperative chemotherapy or prognosis (Kilpatrick et al., 2001; Thomas et al., 2002). Given the initial findings, in 2001 a phase II trial was initiated in patients with metastatic osteosarcoma whose tumors overexpress HER2. Trastuzumab, a monoclonal antibody to HER2, was administered weekly in conjunction with standard MAP chemotherapy. Highlighting the difficulty of conducting clinical trials of targeted therapy in rare diseases several years were required to complete enrollment. The recently published results of this trial were difficult to interpret, demonstrating no statistical difference in event-free and overall survival between the HER2-positive group treated with trastuzumab and the HER-2 negative group treated with cytotoxic chemotherapy alone (Ebb et al., 2012). The IGF receptor is another membrane tyrosine kinase receptor which has been studied in OS. IGF-R is a similar in structure to the insulin receptor, but has disparate downstream targets and has an increased affinity for IGF. In lower organisms, growth and glucose metabolism are regulated by the same pathway. In higher organisms the pathways have diverged but remain structurally related. There exist two known subtypes of the IGF-R: IGF-1R and IGF-2R. In osteosarcoma cell lines, variable expression of IGF-1R was observed. However, a high level of expression of IGF-2R was noted across all cell lines (Hassan et al., 2012). In tumor samples both IGF-1R and IGF-2R were found to be expressed in approximately half of the samples (45 and 56%, respectively) (Abdeen et al., 2009). In osteosarcoma, there is some evidence that increased IGF-1R expression is correlated with the presence of metastases at diagnosis and overall survival (Wang et al., 2012). The role of IGF-2R is much less clearly elucidated. IGF-2R has been implicated as a suppressor of growth by sequestering IGF-2 and limiting its ability to stimulate growth signaling (Rezgui et al., 2009). Germ-line allelic variations with unknown significance on protein function in the IGF-2R have been demonstrated to increase the propensity for developing osteosarcoma (Savage et al., 2007). Unlike in Ewing sarcoma, in which inhibitory antibodies to the IGF-1R have demonstrate clinical benefit, limited clinical data is available on the role of IGF-1R inhibition in osteosarcoma. However, in a phase I study of R1507, 2 patients with osteosarcoma had stable disease for greater than 1 year (Bagatell et al., 2011). SCH 717454, another antibody to the IGF-1 receptor, has been studied in a phase II trial in patient with relapsed osteosarcoma and Ewing sarcoma. The study has completed accrual, with the preliminary results indicating stable disease in some patients with OS (Anderson et al., 2008). The major concern with these agents has been their availability.

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MET expression has also been implicated in oncogenesis. Lentiviral transfection leading to the overexpression of MET transforms primary human osteoblasts to become osteosarcomas (Patane et al., 2006). Likewise, MET has been demonstrated to be overexpressed in 60% of osteosarcoma patient samples (Ferracini et al., 1995). Patients with amplification of MET have a poor 5-year event-free survival of 28% (Entz-Werle et al., 2007). The inhibition of Met with an oral Met inhibitor, PF-2341066, also known as crizotinib, limited the growth of osteosarcoma in xenografts (Sampson et al., 2011). Crizotinib, which also has inhibitory effects on Anaplastic Lymphoma Kinase (ALK), has demonstrated very promising results in ALK-driven malignancies including anaplastic large cell lymphoma and neuroblastoma. Preliminary results demonstrate 7/8 complete responses for patients with anaplastic large cell lymphoma and 2 patients with neuroblastoma having partial responses (Mosse et al., 2012). In patients with advanced non-small-cell lung cancer, crizotinib treatment led to a 1-year overall survival rate of 74% (Shaw et al., 2011). The presence of abnormalities in ALK has not been determined in OS. However, given the promising results of crizotinib in ALK-driven tumors, and the possible off-target effects on MET, this agent may have promise in OS as well. Crizotinib is currently being studied in a phase I/II trial of children with relapsed and refractory solid tumors. 3.4. Intracellular signaling inhibitors As well as receptor tyrosine kinases, intracellular tyrosine kinases which serve as intermediaries in the signaling cascade have also been targets of drug development with possible implications for the treatment of osteosarcoma. Src is a nonreceptor tyrosine kinase. Upon activation, it signals STAT, a cytoplasmic transcription factor to move to the nucleus, with the net result of transitioning through the cell cycle (Yu et al., 1995). Src transformed cells lose adhesion, have increased motility, and increased invasiveness (Yeatman, 2004). Dasatinib is a tyrosine kinase inhibitor, which has classically been used for imatinib resistant chronic myelogenous leukemia. It is also known to have inhibitory effects on src. In osteosarcoma cell lines, dasatinib inhibits src's ability to activate downstream targets. However, dasatinib has no effect on osteosarcoma cell line growth in vitro, nor does it have any inhibitory effect on tumor growth or metastasis in vivo (Hingorani et al., 2009). Saracatinib is an oral specific inhibitor of src, which has had disappointing single-agent activity in phase II trials in head and neck squamous cell carcinoma, gastric adenocarcinoma, and breast cancer (Fury et al., 2011; Gucalp et al., 2011; Mackay et al., 2012). There is currently a placebo-controlled phase II trial of saractinib in patients with relapsed osteosarcoma who have been rendered disease-free, which is still in the process of patient accrual. The mammalian target of rapamycin (mTOR) is involved in the regulation of protein synthesis. It is a serine-threonine protein kinase which upon activation stimulates the progression from G1 to the S phase of cell cycle. The inhibition of mTOR by rapamycin, also known as sirolimus, on osteosarcoma cells in vitro leads to G1 arrest. Rapamycin monotherapy tested by the PPTP led to a sustained complete response in one osteosarcoma cell line (Houghton et al., 2008). An analog of rapamycin, ridaforolimus, has been studied in a phase II trial of patients with advanced bone and soft tissue sarcomas. The predominant benefit of single-agent ridaforolimus was disease stabilization with a median progression-fee survival in patients of 15.4 weeks in patients with bone sarcomas. From a total of 54 patients with heavily pretreated bone sarcomas, two patients with osteosarcoma had a partial response (Chawla et al., 2012). Currently there are several ongoing trials of rapamycin analogs in patients with relapsed solid tumors including osteosarcoma. In response to the inhibition of mTOR, Akt, another serinethreonine kinase upstream of mTOR, becomes hyperphosphorylated and activated. Akt activation leads to resistance to apoptosis and

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increased cell growth. This effect is abrogated by inhibition of IGF-1R (Wan et al., 2007). Pathways independent of Akt have also been implicated in the mechanism by which IGF-1 signaling can bypass rapamycin-induced apoptosis (Thimmaiah et al., 2010). In xenograft models of osteosarcoma, the anti-tumor effect of rapamycin has been shown to be additive with two different anti-IGF-1R antibodies: IMC-A12 and R1507 (Kolb et al., 2010, 2012). A phase I study of temsirolimus, another homologue of rapamycin, in combination with IMC-A12 in children with relapsed and refractory solid tumors has been recently completed. A phase II trial of IMC-A12 and temsirolimus in patients with metastatic sarcomas is still in the process of completing accrual. Aurora kinases are another family of serine/threonine protein kinases. The aurora kinase family is involved in preparing the chromosomes for cell division. Aurora kinase A specifically has been shown to be important in centrosome maturation, bipolar spindle assembly, and cell cycle regulation by activation Polo-like kinase 1 (Vader & Lens, 2008). MLN8237 is a small molecule inhibitor of Aurora kinase A. In the PPTP evaluation of MLN8237, in vivo testing in osteosarcoma xenografts demonstrated intermediate to high anti-tumor activity (Maris et al., 2010). A single-arm phase II trial of MLN8237 is still accruing patients with relapsed/refractory solid tumors and leukemias. 3.5. Antifolates Osteosarcoma responds poorly to conventional dose of methotrexate. However, high‐dose methotrexate has been one of the mainstays of treatment since its introduction in the 1970's. Methotrexate resistance has been attributed to loss or decreased function of the reduced folate carrier, increased DHFR expression, and decreased retention of methotrexate within the cell by lack of polyglutamylation. Trimetrexate is another antifolate, but it does not require the reduced folate carrier for entry into the cell. It provides an attractive alternate to methotrexate for patients with osteosarcoma, as impairment in methotrexate transport due to abnormalities in the reduced folate carrier has been demonstrated in the majority of osteosarcoma tumor samples (Sowers et al., 2011). A phase II trial of trimetrexate in patients with refractory acute lymphoblastic leukemia and osteosarcoma demonstrated a response rate in 2 of 7 evaluable patients with osteosarcoma (1 complete response, 1 partial response) (Trippett et al., 1999). This has led to a phase I trial combining trimetrexate with high-dose methotrexate in patients with recurrent osteosarcoma. Pemetrexed is another antifolate drug, but it inhibits multiple folate-dependent enzymes needed for cell division, most importantly thymidylate synthase. Like methotrexate, pemetrexed requires the reduced-folate carrier for entry into the cell (Rollins & Lindley, 2005). In osteosarcoma cell lines, pemetrexed demonstrated anti-tumor activity; however to a lesser degree than methotrexate (Bodmer et al., 2008). In a phase II trial of patients with refractory/relapsed osteosarcoma, pemetrexed demonstrated that 1 of 32 patients had a partial response and 5 patients had stable disease. Although median progression-free survival was only 1.4 months, greater than 30% of patients were still alive at 1 year (Duffaud et al., 2012). Pralatrexate is a methotrexate analog with increased transport across the reduced folate carrier and increased intracellular accumulation by polyglutamation than methotrexate, and, like methotrexate, functions as an inhibitor of dihydrofolate reductase (DeGraw et al., 1993). In patients with relapsed or refractory peripheral T-cell lymphomas, pralatrexate demonstrated a response rate of 29% in 109 evaluable patients. (11% complete responses, 18% partial responses) (O'Connor et al., 2011). Pralatrexate administered to patients with previously treated non-small cell lung cancer led to a response rate of 10%, with a 1 year survival rate of 56% (Krug et al., 2003). In patients with mesothelioma, the results of single-agent pralatrexate were more disappointing with no observed complete or partial responses, 3 of the 16 patients enrolled had stable disease for greater

than 9 months (Krug et al., 2007). Pralatrexate has not been studied in patients with OS. 3.6. Targets of genomic instability Most osteosarcoma samples harbor a complex karyotype, with cytogenetic heterogeneity with the cells of the same tumor (Bridge et al., 1997). This may, in part, be due to aberrations in p53 in osteosarcoma. Osteosarcoma cell lines have been demonstrated to have loss of p53, mutations in p53, as well as wild-type p53 (Diller et al., 1990). MDM2, a known suppressor of p53, is significantly amplified in osteosarcoma samples with wild-type p53. However, MDM2 amplification was observed only in 5 of the 32 samples studied (Overholtzer et al., 2003). The Nutlins (1, 2, and 3) have been identified as small molecule inhibitors of the interaction between p53 and MDM2 (Vassilev et al., 2004). In osteosarcoma cell lines that express wildtype p53, Nutlin-3a, an enantiomer of Nutlin-3, suppresses proliferation and promotes apoptosis (Wang et al., 2012). These agents are currently in preclinical testing, but may be promising for a subset of patients with osteosarcoma. The complex karyotype associated with osteosarcoma has recently been suggested to be the product of a sentinel event, termed chromothripsis. From Greek, this translates to chromosomes shattering into pieces. The mechanism behind this process remains unclear (Stephens et al., 2011). However, given the unique nature of the complex karyotype within the osteosarcoma cell, as compared to the surrounding tissues, future therapies may be developed to target this abnormality as we begin to learn more about this process. 4. Conclusion The majority of patients with osteosarcoma are cured with the current treatment strategies. However, approximately 1/3 of patients will relapse of which only a minority will not ultimately succumb from their disease. In the treatment of osteosarcoma, four agents have been demonstrated to have proven activity: methotrexate, doxorubicin, cisplatin, and ifosfamide. Combination regimens using three of these agents have demonstrated similar results. However, several trials have demonstrated that 4-drug regimens do not proved superior outcomes than the standard MAP therapy. A meta-analysis has confirmed these findings that there is no significant difference in event-free and overall survival between three and four-drug regimens in the treatment of osteosarcoma (Anninga et al., 2011). The role of the combination of ifosfamide and etoposide in the treatment of osteosarcoma has yet to be fully elucidated. The results of the international collaboration to answer this question will take several more years for the data to reach maturity, as this trial has only recently reached its accrual target. In addition to traditional chemotherapy, there are a host of targeted agents which have demonstrated activity in vitro and in vivo. Further investigation of many of these agents is warranted. However, several questions remain that will define how we understand the activity of these agents in osteosarcoma. Radiographically, responses are difficult to assess due to limited change in tumor size with treatment because of matrix deposition. Testing these agents in patients with detectable disease may be setting them up for failure. Newly diagnosed patients with osteosarcoma given active traditional chemotherapy have a low probability of cure if part of their detectable disease remains unresected, and so surgical resection of all identified disease is the standard of care. In the relapsed setting, with chemoresistant disease, could an investigational agent be expected to do better? Secondarily, if a patient who relapses is rendered free of disease and then given an experimental agent, what would be the appropriate outcome to measure? The gold-standard of measuring survival compared to placebo will consume a significant number of patients and time, especially given the understanding that ultimately most of these novel agents may have limited

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benefit. Lastly, should these agents be investigated in patients with metastatic disease in an effort to improve their survival? What then of the concern of possibly hampering the ability to deliver active chemotherapy to patients who can reasonably be expected to be cured and engender patients to have a worse cure rate? Due to the great successes of the past, the introduction of new agents into the treatment of osteosarcoma is complicated within our current paradigm of trial design. Given the plateau in survival from osteosarcoma over the past two decades, new agents are clearly necessary, as the benefits from traditional chemotherapy have seemingly been maximized. The challenges ahead lay not only defining which agents need priority for further investigation but how we measure their success.

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