Available online at www.sciencedirect.com

ScienceDirect Recent advances in osteosarcoma Sander M Botter1, Dario Neri2 and Bruno Fuchs1 Although osteosarcoma (OS) is a rare malignancy, it is ranked among the leading causes of cancer-related death in the pediatric age group. The cancer’s low prevalence and its large tumor heterogeneity make it difficult to obtain meaningful progress in patient survival. In this review we present an overview of current clinical trials which largely focus on stimulation of the immune system or rely on the inhibition of kinases such as Src and mTOR. The potential efficacy of tumortargeted TNFalpha is discussed, as well as the importance of preclinical validation of new targets. To improve the success of future clinical trials, clinicians and basic researchers need to intensify their exchange. Finally, a case is made for individualized treatment of OS patients, based on interdisciplinary cooperation in dedicated Sarcoma Centers. Addresses 1 Sarcoma Center & Laboratory for Orthopedic Research, Department of Orthopedics, Balgrist University Hospital, University of Zurich, Forchstrasse 340, 8008 Zurich, Switzerland 2 Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zurich Wolfgang-Pauli-Str. 10, 8093 Zurich, Switzerland Corresponding author: Fuchs, Bruno ([email protected])

Current Opinion in Pharmacology 2014, 16:15–23 This review comes from a themed issue on Musculoskeletal Edited by Alison Gartland and Lynne J Hocking

1471-4892/$ – see front matter, Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.coph.2014.02.002

approximately half of the bone tumor patients eventually succumb to the disease. Although conventional chemotherapy remains the golden standard for treatment of OS, the survival plateau of OS patients forces us to look for new therapeutic agents. This review will focus on advances in currently employed targeted therapies, as well as addressing novel targets that may be used in future clinical trials. Next to the low disease prevalence, the high amount of tumor heterogeneity is the main cause of slow progress in testing these new agents in clinical trials. Therefore, in vitro and in vivo preclinical screening is a vital element in OS drug studies.

Tumor heterogeneity in OS and its consequences There exists a large amount of inter-patient, as well as inter-tumor and intra-tumor heterogeneity in OS. This heterogeneity can be divided into three layers. First, there is a number of different OS subtypes, many of them having distinct histological or radiological features [7]. Second, the genomic organization of OS is extremely complex [8]: aberrant karyotypes are a hallmark of OS, and modern genomic screening have recently identified chromothripsis, a single catastrophic genomic instability event where hundreds of genomic rearrangements cause the remodeling of an entire chromosome, as a common phenomenon in bone cancers and OS [9,10]. As a result, hundreds of newly generated fusion products can be found (S Lorenz et al., abstract 17, 18th annual meeting of Connective Tissue Oncology Society, New York; November 2013). Finally, the third, more general source of heterogeneity is formed by the tumor environment, such as the stroma, the availability of a vascular network, and the hosts’ immune system [11].

Introduction Primary bone tumors are rare malignancies; approximately 10 patients per million people are diagnosed, with about 2–5 patients per million people, particularly in the pediatric age group, suffer from osteosarcoma (OS), the most prevalent primary bone tumor [1]. Because these tumors have a high propensity to metastasize, they are ranked among the most frequent causes of cancer-related death, despite the fact that this cancer type only accounts for 5–6% of all childhood tumors [2–4]. With the introduction of chemotherapy in the 1970s, the 5-year survival rate has increased from about 20% to 65–70% [5]. However, during the last two decades no further improvements have been made in terms of survival, and even remain at 20–30% for almost one quarter of all patients with clinically detectable metastatic disease at the time of initial diagnosis [6], similar to historical controls. Overall, www.sciencedirect.com

The large tumor heterogeneity introduces several problems, such as finding a reliable biomarker that can be used for targeting strategies, to perform molecular profiling in order to identify recurrent genetic changes, or even to answer the basic question which cell type is the cause of OS [12,13]. It also conveys resistance of the tumor to drugs, giving rise to relapse and metastasis. One theory that may explain these last processes involves the presence of a multipotent cancer stem cell (CSC), which is capable of both self-renewal and maintaining the tumor cell mass, yet is relatively resistant to chemotherapeutic drugs (see Figure 1). If it were possible to target CSCs, this would improve the survival for all OS patients, in particular those where the primary tumor has metastasized. There are a discrete number of techniques available to isolate and characterize the presumed sarcoma Current Opinion in Pharmacology 2014, 16:15–23

16 Musculoskeletal

Figure 1

High stem cell marker expression/multipotent

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Late accumulation of genetic mutations (p53, Rb, Cdkn2, chromothripsis)

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Relapse & metastasis Current Opinion in Pharmacology

A potential role for mesenchymal stem cell (MSC)-like OS cells that help explain relapse and metastasis following chemotherapy. Mutations, either aquired stochastically or radically, can accumulate during each phase of the mesenchymal osteoblastic differentiation process, giving rise to cells that possess MSC-like properties in different degrees. These different cell populations are reflected in the heterogeneous tumor mass, where the tumor stroma/micro-environment plays a role in maintaining the MSC-like population, and in which differentiated OS cells may switch to a more dedifferentiated, MSC-like state, or even recruit stromal MSCs. This heterogeneous cell population may directly resist chemotherapeutic treatment, which has been shown to enrich for MSC-like cells, or part of the cells may enter a state of dormany, after which they can contribute to tumor relapse and metastasis formation.

CSCs [14,15], and although these techniques identified subpopulations of cells that have more stem-cell like properties, an enhanced tumorigenic capacity and/or drug resistance, none of them are really specific nor do they enable the isolation of a pure CSC population. Many of these techniques have been derived from studies that addressed CSCs in more common epithelial cancers, of which extrapolation to OS may be misleading. Finally, the finding that differentiated cancer cells, similar to normal differentiated cells [16,17], can return back to a stem-like state [18], has cast more and more doubts on the reality of CSC existence in OS [19]. Thus, it is as of yet unclear whether these cells represent a completely distinct population of cells, or simply represent a property or Current Opinion in Pharmacology 2014, 16:15–23

phenotype which differentiated cancer cells acquire in response to their environment.

Advances in osteosarcoma therapy The number of clinical trials testing substances or treatment methods against OS has increased over the last two decades [20]. At present, there are 70 active clinical trials listed that include osteosarcoma patients, of which 21 trials are specifically aimed at targeting OS (see Supplementary Tables 1 and 2). The largest and most important ongoing OS trial is the EURAMOS-1 (EURopean and AMerican Osteosarcoma Studies, ClinicalTrials.gov identifier NCT00134030) trial, an inter-continental collaboration of 17 countries which started in 2005, with 2260 www.sciencedirect.com

Recent advances in osteosarcoma Botter, Neri and Fuchs 17

registered OS patients as of June 2011 (estimated trial end date for the primary outcome measure, 3-year event-free survival: January 2015). Below we will give an overview of some other ongoing trials, which can be roughly divided into four different groups: chemotherapeutic drugs and combinations thereof, immunomodulation, specific target inhibition using small molecules, and trials involving bisphosphonates. A graphical overview of the presented molecular targets is given in Figure 2. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.coph.2014.02.002.

Chemotherapeutic drugs and combinations thereof

The gold standard of OS treatment includes cytotoxic drugs such as methotrexate (M), cisplatin (A), doxorubicin (P), ifosfamide (I), and etoposide (E). Combinations of these classic drugs, mostly in the form of neoadjuvant as well as adjuvant MAP, are currently the most efficacious way to reduce OS tumor growth and to increase the survival changes of patients [21], and different combinations or administration schemes of these drugs are still under investigation. In the EURAMOS-1 trial, one treatment arm is testing the addition of IE to standard adjuvant MAP treatment; ifosfamide given as neoadjuvant drug did not seem to confer additional benefit [22]. One ongoing Phase II trial (NCT01650090, estimated completion date: December 2014) is testing the local administration of inhaled cisplatin, to treat recurrent lung metastases. Trials testing other types of drugs, such as pemetrexed or the combination gemcitabine/docetaxel proved to be unsuccessful [23–25]. A Phase I trial evaluating the effect of trabectedin yielded stable disease in two out of a total of three OS patients [26], although a pediatric-sarcoma Phase II trial did not turn out to be as successful [27]. Interestingly however, successful treatment with this drug was reported in two OS patients with a wild-type genotype of the DNA repair gene ERCC5 [28], which may be explained by the drugs’ known and rather unique dependence of the cell’s DNA repair status [29], and may necessitate a more detailed molecular characterization of the patients’ tumor before drug allocation. This concept is also tested in another combinational trial which is currently recruiting patients (NCT01459484, estimated completion date: January 2020), where the efficacy of MAP versus MAPI and muramyl tripeptide, an already approved immunostimulant, will be tested by first assessing the expression of ABCB1, a protein that confers multidrug resistance to the tumor cell. Immunomodulation

In the second treatment arm of the EURAMOS-1 trial, adjuvant treatment of pegylated interferon-alpha (IFNalpha) is tested in patients that responded well to neoadwww.sciencedirect.com

juvant MAP treatment, compared to MAP alone. Results from this treatment arm have recently been presented, and unfortunately show that the addition of IFNalpha to MAP did not lead to improved event free survival (S Bielack et al., J Clin Oncol 31, 2013, Suppl; abstract LBA10504). Although disappointing, this outcome may not be surprising, given that the amount of (pre-)clinical evidence that demonstrates efficacy of IFNalpha against OS dates back to the 1970s and is rather limited [30]. Another immunomodulatory approach that is currently tested is the viral delivery of granulocyte macrophage colony-stimulating factor (GM-CSF, NCT01169584, primary completion date: March 2012, estimated study completion date: June 2014), which showed efficacy against solid tumors [31], albeit not against pulmonary OS metastases [32]. Aerosolic delivery of the cytokine IL2 (NCT01590069, primary completion date: June 2016), and administration of antibodies directed against GD2 (NCT00743496, primary completion date: June 2015), a widely expressed tumor marker, is also under trial. Specific target inhibition using small molecules

These trials encompass small-molecule kinase inhibitors that inhibit serine/threonine kinases such as mTOR or Aurora A, or thyrosine kinases such as Src, IGF-1R, VEGFR, PDGFR, or c-Kit. Rapamycin (serolimus), studied in relation to cancer since the 1990s, or analogous substances are potent inhibitors of mTOR, a serine/ threonine protein kinase that promotes cell growth and cell survival. The inhibition of aurora A, a serine/thereonine kinase that plays a role in the mitotic spindle pole formation, thereby promote cell cycle progression, is currently undergoing Phase II testing (NCT01154816, primary completion date: March 2016). Four trials testing inhibitors of the thyrosine kinase Src are currently underway, despite conflicting in vivo evidence [33,34]. Finally, the multikinase inhibitors, which may limit the chance of tumor cell resistance, are a relatively new subclass of drugs under review. Examples include OSI-930, which has not yet been tested in OS but shows promising results in other solid tumors [35], and pazopanib, for which a trial is about to start (NCT01759303, primary completion date: June 2015). Bisphosphonates

Besides inhibiting bone loss in osteoporotic patients, for which bisphosphonates were originally designed, researchers have shown that these compounds also exhibit direct anti-tumor properties [36,37]. A study where pamidronate, a second generation bisphosphonate, was added to MAP chemotherapy showed a favorable safety profile in adolescent OS patients [38] and the same was found for the more potent zoledronic acid [39]. Results from other clinical trials, albeit not planned at this moment, are eagerly awaited.

Advances in osteosarcoma research Current Opinion in Pharmacology 2014, 16:15–23

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Figure 2

Bevacizumab Cixutumumab

VEGF PDGFR

VEGFR

IGF-1R

Integrins

Sorafenib Pazopanib Dasatinib Saracatinib

Src

Rapamycin Temsirolimus Everolimus CC-115 FKBP

PI3K

Ras

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c-Raf

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MAP Cyclophosphamide Decitabine Irinotecan Dacarbazine Thiotepa Radiotherapy (Ra-223, heavy ion, protons)

elF4E

Cell cycle progression & cell survival Current Opinion in Pharmacology

Schematic overview of targeted molecular pathways in osteosarcoma.

Immunological activation using TNFalpha

Since the introduction of ifosfamide over two decades ago, the only new drug that has been approved for the treatment of OS is muramyl tripeptide (MTP), a synthetic derivative of a naturally occurring immune stimulatory component of cell walls from Mycobacterium species [40]. This principle had already been recognized in the late 19th century, when intratumoral injections Current Opinion in Pharmacology 2014, 16:15–23

with ‘Coley’s toxin’, a heat-inactivated mixture of streptococci, proved to be successful against OS [41]. In general it is known that infection, and the concomitant activation of the immune system, confers protection to OS progression [42,43,44], and for these reasons a growing portion of the latest clinical OS trials investigates ways to stimulate the immune system [45]. www.sciencedirect.com

Recent advances in osteosarcoma Botter, Neri and Fuchs 19

Today, it is thought that the well-known immunocytokine TNFalpha played an important role in reducing the tumor sizes of the ‘Coley’ toxin’ patients [46], and the fact that MTP stimulates the release of TNFalpha [47,48] may explain its effect on overall survival. Therefore, it is conceivable to further explore the anti-tumor capacity of TNFalpha towards OS. However, although its high tumor-killing capacity makes TNFalpha an appealing therapeutic drug, its substantial toxicity prevents it from being administered systemically. Still, recombinant TNF is nowadays successfully used for local treatment of soft tissue sarcomas, accomplished using a rather complex and invasive surgical procedure [49–51]. To overcome the toxicity problem, an alternative strategy is to couple TNFalpha to an antibody directed against the tumor stroma. In soft tissue sarcomas, these ‘armed antibodies’ led to decreased tumor growth and, combined with doxorubicin, to complete tumor eradication and a lasting antitumor immunity [52], demonstrating synergistic interaction between chemotherapy and immunological compounds in the treatment of sarcomas. Two clinical trials testing this armed antibody in solid tumors showed a favorable safety profile [53,54], clearly demonstrating that the targeting strategy had worked. The fact that promising preclinical results with the use of TNFalpha in OS have been observed [55,56] make this strategy even more interesting to pursue. Bone metabolism

In cancer types where bone metastases are especially prevalent, such as breast and prostate cancer, interplay between tumor cells and bone-resorbing osteoclasts is thought to play an important role in the growth of the tumor tissue. This so-called vicious cycle of bone turnover releases growth factors stored inside the bone matrix, allowing the tumor to expand more rapidly. Targeting this aberrant bone turnover has been presented as a new way of inhibiting tumor growth in OS [57], and a growing body of data points to the involvement of osteoclasts in OS metastasis formation [58]. Recently, the GRM4 locus, implemented to play a role in bone formation and resorption, has been associated with OS susceptibility [59]. Additionally, an imbalance of the RANK/RANKL/OPG axis, associated with bone remodeling and bone metabolism, may also be associated with OS development [60,61]. Role of microRNAs in osteosarcoma

Since the term microRNA (miRNAs, mi-Rs) was first posed by Science in 2001, research in this field has bloomed, resulting in a surge of publications in all fields on cancer, including OS. Over the last two years only, over 60 papers have been published in which new miRNA targets and their relation to OS biology have been described. Using miRNA arrays, researchers have identified hundreds of miRNAs that are differentially regulated when comparing osteosarcoma cells with norwww.sciencedirect.com

mal bone osteoblasts. It is thought that these miRNAs modulate the expression of specific target oncogenes or tumor suppressor genes, thereby affecting tumorigenesis and tumor growth, but also play important roles in processes such as cell migration, invasion and resistance to chemotherapeutics [62]. For instance, it has been shown that miR-17/miR-221 and miR-128 play opposite roles in maintaining PTEN expression [63,64,65]. Ezrin, another well-known target in osteosarcoma biology, is down regulated by miR-183 [66,67], and Cyr61, previously identified to predict poor patient prognosis [68], is regulated by miR-100 [69]. Alternatively, miRs can be used by tumor suppressor genes to exert their function; for instance, expression of the miR-34 family (consisting of miR34a, 34b and 34c), who target cell cycle proteins, can be induced by P53 in response to DNA damage or oncogenic stress. Many more miRs have been described to play a role in OS so far, of which more comprehensive overviews have been presented elsewhere [70,71]. A still open question is how these miRs should be targeted as new therapeutic entity. This can either be done directly, for example, by blocking the expression of oncogenic miRs or by substitution of tumor-suppressor miRs using virus-based constructs, or indirectly, via drugs to modulate miRNA expression by targeting their transcription and/or processing [72]. Both strategies have successfully been used [69,73], demonstrating their potential as novel OS therapy. Preclinical validation of new targets

One reason why clinical OS trials often fail may not be because of the lack of molecular targets [74], but simply because preclinical validation of these targets has not been performed well enough before the start of a new clinical trial. Indeed, because of the low disease incidence and large tumor heterogeneity of OS, rigorous in vitro and in vivo testing is imperative before embarking on any new strategy in a clinical setting. The objective of the Pediatric Preclinical Testing Program (PPTP), a consortium of institutions across the United States and in Australia, is to test novel agents in panels of preclinical mouse xenograft models representing the most common pediatric cancers, among which OS is represented [75]. Since 2007, over 50 candidate drugs have been tested, providing a rationale for further testing in clinical trials [76]. However the Program, being important, does not faithfully recapitulate the human disease: for instance, all OS cell lines are implanted subcutaneously, whereas for many cancers including OS the micro-environment is known to play an important role in disseminating tumor cells throughout the body. For OS, this means that the tumor cells in these induced models should be implanted orthotopically, that is, by intratibial or intrafemural injections [68,77]. Current Opinion in Pharmacology 2014, 16:15–23

20 Musculoskeletal

To simulate the full heterogeneity of the OS disease process as close as possible, animal models in which OS develops spontaneously should also be considered for preclinical validation. Because mice do not spontaneously develop osteosarcomas, elaborate, but time-consuming gene targeting strategies need to be undertaken [78]. Alternatively, OS development in large breeds of dogs occurs at a 10–30 times higher frequency [79], and shows striking similarities with the human disease [80]. The samples used for basic research purposes are usually derived from spontaneous OS cases arising in domestic pets, sharing the same environmental conditions as humans. Genetic analysis of these samples confirmed that the gene expression signatures of canine and human OS are highly similar, and even parallel human survival analyses [81,82]. Because of these striking similarities, canine osteosarcoma is gaining fast attention as a valid clinical model for the human disease [83,84], and more effort should be put in setting up drug trials with canine patients, as an important clinical surrogate to start human clinical trials.

milestone for the treatment of OS patients [52–54,85]. Yet, not all antibody products efficiently localize to the tumor site and may thus display insufficient activity in vivo. We anticipate that Nuclear Medicine techniques (e.g. Immuno-PET, [86]) may play a crucial role both for the selection of the best therapeutic antibody for clinical development and, potentially, for patient selection. The quest to perform large international trials is evident, however, considering the currently ongoing 70 trials, less likely to happen. Probably all too often, clinical trials are initiated without rigorous preclinical testing. We may need to internationally establish criteria to be fulfilled before starting a clinical OS trial, based on preclinical data obtained from in vitro, induced and spontaneous models. In order for this to happen, it will be imperative that clinicians, biologists and basic researchers move closer together. Only the synthesis of the entire knowledge will allow the optimal conduct of a clinical trial leading to success.

Conflict of interest Conclusions Advances in the outcome of OS patients over the last few decades have plateaued despite enormous efforts. There are numerous and well accepted reasons to explain this, but maybe the time has come to make a stop and think about the future strategy. To start, the high inter- and intratumor heterogeneity has consequences for the way in which patient treatment should be conducted after OS diagnosis. The treatment protocol of each patient needs to be discussed in a multidisciplinary tumor board, providing a pleiotropic expert input. Furthermore, to ensure the highest standard of care, each patient should preferably be referred to a dedicated Sarcoma Center, which concentrates the latest knowledge and care regarding treatment of OS. Because of the intra-tumor and inter-tumor heterogeneity, it is very unlikely that targeting a single genetic event in OS with a favorable clinical response, proven successful for some types of cancer (use of vemurafenib in BRAF V600E-mutated metastatic melanoma, use of imatinib in gastrointestinal stromal tumors), will make the disease disappear. Therefore, we may need to stop performing single agent analyses but instead concentrate on combinatorial approaches, targeting functional processes such as bone remodeling, immunomodulation, and the colonization of metastatic tumor cells. Furthermore, more effort should be undertaken in biodistribution studies to select those compounds that really target the tumor and not affect the surrounding healthy tissues. Recent breakthrough results in the development of immunostimulatory antibodies and armed antibody products (e.g. antibody–drug conjugates, antibody–cytokine fusion proteins) in a variety of solid tumors suggest that immunological approaches may also represent the next Current Opinion in Pharmacology 2014, 16:15–23

Dario Neri is a cofounder and shareholder of Philogen S.p.A. (Siena, Italy), a biotech company which develops armed antibody products. The other authors declare no conflict of interest.

Acknowledgements Our work is supported by the Zurcher Krebsliga (Zurich, Switzerland), the University of Zurich, the Schweizerischer Verein Balgrist (Zurich, Switzerland), the Walter L. & Johanna Wolf Foundation (Zurich, Switzerland), the Highly Specialized Medicine for Musculoskeletal Oncology program of the Canton of Zurich, and the Swiss National Science Foundation SNF Nr.310030_149649. The funders had no role in the preparation of the manuscript.

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