Recent and current clinical trials in canine appendicular osteosarcoma

Abstract

Osteosarcoma (OSA) is an aggressive primary bone tumor in the domestic dog that most often occurs within the appendicular skeleton. Despite the use of adjuvant chemotherapy, most dogs succumb to metastatic disease within 1 year of diagnosis. To improve this outcome, substantial research is currently focused on investigating novel therapies. Herein, we review emerging treatments and clinical trials that, if proven efficacious, could revolutionize the standard of care for canine appendicular OSA. This article includes a critical perspective on the safety, efficacy, and limitations of select immunotherapy, virotherapy, radiotherapy, targeted therapy, and personalized medicine trials, all of which reflect similar investigations taking place in human oncology. These clinical trials represent a major evolution in the overall approach to therapy for dogs with appendicular OSA that could have significant implications for improving survival.

Introduction

Canine osteosarcoma (OSA) is an aggressive malignancy that accounts for up to 85% of all bone tumors in dogs. Incidence and risk factors are impacted by signalment. In a large population survey of 400 000 insured dogs in Sweden, the overall incidence reported was 5.5 cases per 10 000 dog-years at risk (1). Most canine OSA tumors originate in the metaphyseal region of bones in the appendicular skeleton (2). Reported risk factors for OSA development include sex, breed, history of trauma, and spay or neuter status. Female dogs may be at lower risk of developing OSA, with a reported hazard ratio of 0.71 based on a population study by Egenvall et al (1). Large and giant breed dogs (> 25 kg) are at highest risk, with breeds such as the Irish wolfhound and Leonberger having 126 and 72 cases reported per 10 000 dog-years at risk, respectively, in a Norwegian population study (3).

Affected dogs are typically presented with lameness and swelling that can be localized to the affected site. Due to tumor-related bone destruction, dogs with OSA are at risk for pathologic fracture, with 57.1% of these fractures reported to affect the femur, according to a study by Rubin et al (4). While radiographically detectable pulmonary nodules are present at diagnosis in less than 15% of cases, most dogs (90%) will have undetectable micrometastatic disease. Because of the high overall metastatic rate, the prognosis in canine OSA is poor. With treatment consisting of amputation and follow-up (adjuvant) chemotherapy, disease-free interval and overall survival times are typically what was reported by Selmic et al (5), namely 291 d and 284 d, respectively. With current standard therapies, long-term outcome for localized or disseminated disease have remained static over the past few decades. Thus, novel advances in the treatment of canine OSA are desperately needed.

Current efforts in the diagnosis, treatment, and prognosis of canine appendicular osteosarcoma

In the pre-operative setting, histological confirmation of OSA by bone biopsy may be necessary for definitive diagnosis; however, the biopsy procedure carries a risk of pathologic fracture. As a result, fine-needle aspiration cytology of the bone lesion may be used instead to identify sarcoma cells before histologic confirmation from the surgical specimen obtained at amputation (6). Upon diagnosis, 3-view thoracic radiographs are the minimum required imaging database to screen for evidence of pulmonary metastasis.

Standard of care

Amputation of the affected limb, followed by systemic chemotherapy is the current standard of care for appendicular OSA (5). In select cases, such as small tumors with minimal soft tissue involvement, limb-salvage procedures are considered (7). During a limb-salvage procedure, dogs require reconstruction of the bony defect through an allograft or prosthesis, then arthrodesis of the adjacent joint (7). Distal radial location is most favorable for limb-sparing due to good postoperative limb function (7). External beam radiation therapy is used for patient palliation and is indicated in dogs that do not undergo amputation or limb-sparing surgery (8). The overarching goal is to manage pain with minimal side effects. According to a study of 95 dogs with appendicular OSA, palliative radiation therapy leads to pain relief in 74% of dogs, with a median duration of response of 73 d (8). Bisphosphonates are also commonly prescribed in the palliative setting to reduce bone density loss, and have been combined in some settings with radiation therapy (9).

After limb amputation, adjuvant chemotherapy prolongs survival time to an average of 8 to 12 mo, compared to 3 to 5 mo without chemotherapy (10). Common drug protocols include the platinum agent carboplatin, or the anthracycline doxorubicin. For platinum agents, carboplatin is considered the drug of choice due to a lower risk of adverse events, such as nephrotoxicity, nausea, and gastrointestinal toxicity compared to cisplatin (11). When alternating the administration of carboplatin and doxorubicin in 50 dogs in the microscopic disease setting, no improvement in survival time was found compared to single-agent protocols (12). Moreover, in 35 dogs treated with cisplatin and doxorubicin combination therapy, 17 dogs (49%) experienced substantial toxicity that necessitated discontinuation of the treatment (13). In a recently completed phase III trial, the administration of carboplatin alone in 50 dogs led to a higher disease-free interval of 425 versus 135 d compared to alternating carboplatin with doxorubicin (14). Overall, a superior adjuvant chemotherapy protocol has yet to be defined.

Prognostic factors

Several prognostic factors for canine OSA have been reported, including elevated serum alkaline phosphatase (ALP), location of the affected bone, age, and weight. A meta-analysis of these factors concluded that elevated serum ALP and proximal humerus location were significant negative prognosticators (15). Although age is often reported as a risk factor, increasing age did not have significant correlation with disease-free interval and survival time (16). Dogs with an elevated serum ALP at diagnosis had shorter survival compared to dogs with serum ALP within the reference range, with a hazard ratio of 1.62. In a separate prognostic study of 65 appendicular OSA cases, pre-operative proteinuria was clinically associated with poor outcomes (17). Furthermore, dogs with OSA confined to the distal radius had the best prognosis (17). When investigating weight in 54 cases of OSA, underweight dogs had significantly shorter survival times than ideal or overweight dogs, while obesity was not specifically associated with adverse outcomes (18).

Novel clinical trials for canine appendicular osteosarcoma

Canine OSA benefits from extensive research efforts and the ongoing work of many clinical trials. Previous clinical and basic research into canine cancer and immunology have paved the way for a new generation of trials. This emerging forefront of novel therapies may directly impact the future management of canine appendicular OSA. The following categories reflect the current landscape of next-generation therapies being investigated in human oncology. Along with their novelty, potential limitations, and challenges of each treatment are discussed. In addition, a summary of publicized clinical trials for canine OSA obtained from the American Veterinary Medical Association Animal Health Studies Database at the time of manuscript submission (https://ebusiness.avma.org/aahsd) is presented in Table 1.

Table 1.

Summary of selected clinical trials for canine osteosarcoma treatment, based on studies submitted to AAHSD (AVMA Animal Health Studies Database) or the Comparative Oncology Trials Consortium (COTC).

Trial	Type	Target	Phase	Title	Primary institution	Trial ID
Palladia	Targeted therapy	KIT	NA	Toceranib phosphate (Palladia) and carboplatin combination chemotherapy in dogs with naturally occurring cancer.	Kansas State University	AAHSD000179
Rapamycin	Targeted therapy	mTOR	I	Evaluation of orally administered mTOR inhibitor rapamycin in dogs in the adjuvant setting with osteosarcoma.	Multi-institutional (United States, Canada)	COTC021
GD2, GD3	Targeted therapy	GD2/3	I	A ganglioside targeted cancer vaccine for canine osteosarcoma: A phase 1 trial.	University of Florida	AAHSD000140
ADXS31-64	Vaccine/Immunotherapy	HER2/Neu	I	Evaluation of an ADXS31-164 (a recombinant, attenuated Listeria monocytogenes expressing a chimeric human HER2/neu protein) in dogs in the adjuvant setting with osteosarcoma.	Multi-institutional (United States and Canada)	COTC026
VSV-IFNβ-NIS	Oncolytic virotherapy	IFNβ, NIS	I	Defining pharmacokinetics and biological activity of systemic oncolytic VSV within a dose-schedule optimization study.	Multi-institutional (United States)	COTC024
NV-01	Vaccine/Targeted therapy	NGF	II	Open-label, phase II trial of NV-01 for pain palliation in dogs with osteosarcoma.	University of Illinois, Colorado State University	AAHSD000018, AAHSD000063
COXEN	Genomics	NA	NA	Predictive models of drug response in canine osteosarcoma: A prospective clinical trial testing the COXEN approach.	Colorado State University	AAHSD000089
SRT	Radiotherapy	NA	NA	Stereotactic radiation therapy for pain relief and immune modification in dogs with limb osteosarcoma.	University of Wisconsin	AAHSD000072
SRT	Radiotherapy	NA	NA	The analgesic and systemic immune response to stereotactic radiation therapy in canine osteosarcoma.	University of Wisconsin	AAHSD000263

NA — not applicable.

Immunotherapy trials

The use of immunotherapy to treat human malignancies continues to expand, leading to significant tumor control in advanced lymphoma, melanoma, lung, and bladder cancer. Accelerated interest into further application of immunotherapy partially stems from the success of recent human trials blocking cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein (PD1) (19,20). Yet, to date, few studies have examined the potential clinical use or translation of immunotherapy in dogs.

Autologous activated T-cell therapy

The historical use of autologous T-cell therapy for canine OSA dates to adoptive transfer of a xenogeneic, human cytotoxic T-lymphocyte line (TALL-104) after amputation plus cisplatin therapy in 23 dogs with OSA (21). This set a landmark for the development of other vaccines, monoclonal antibodies, and immunomodulators. Preliminary results from a novel autologous T-cell study in 15 dogs with appendicular OSA were recently reported (22). The technology is based on the collection of cancerous OSA tissue from the patient and preparation of an intradermal pre-vaccination series 14 d before apheresis, using canine-specific settings (e.g., for cell size). The collected lymphocytes are expanded in the presence of interleukin-2 (IL-2), after which the activated cells are infused back into the patient.

Among the 15 dogs enrolled in this clinical trial, 12 completed apheresis for the infusion procedure, and 10 completed the activated T-cell therapy (ACT). For dogs receiving ACT, the disease-free interval was 213 d. At the time of reporting, the median survival time of all dogs in the study (including those that did not receive ACT or IL-2) was 339 days, but MST was not reached for dogs that received ACT.

Based on the Veterinary Co-operative Oncology Group-Common Terminology Criteria for Adverse Events (VCOG-CTCAE) scale, few low-grade toxicities (I or II) were observed, including local erythema, vomiting, and diarrhea. Grade III gastrointestinal toxicities were observed in 1 patient before the administration of pre-medications (i.e., NSAIDs, anti-nausea medications).

HER2/neu targeting Listeria vaccine

A recombinant vaccine (ADXS31-164) targeting human epidermal growth factor receptor 2 (HER2/neu) was developed using an attenuated, recombinant Listeria monocytogenes vector. HER2 is an oncogene that is classically overexpressed in various human cancers (e.g., mammary cancer), and is also widely expressed in canine OSA, particularly in cancer stem cells, where it is suggested to lead to reduced response to chemotherapy and shorter survival times (23). In multiple mouse models of primary cancer, recombinant L. monocytogenes delivery elicits cytotoxic T-lymphocyte activity by infecting mononuclear cells and stimulating potent antitumor immunity.

In a phase I dose escalation clinical trial, 18 client-owned dogs that completed the standard of care were enrolled to receive intravenous (IV) infusions of the vaccine at a dose of either 2 × 10⁸, 5 × 10⁸, 1 × 10⁹, or 3.3 × 10⁹ colony-forming units (CFU) every 3 wk for 3 administrations. Before administration of vaccine ADXS31-164, the dogs also received 1 dose of ondansetron [0.2 mg/kg body weight (BW)] to prevent nausea and vomiting, and 1 dose of diphenhydramine (2 mg/kg BW) to prevent anaphylaxis. Dogs that were free of metastatic disease for 5 mo after treatment were offered additional IV infusions every 4 to 6 mo at a dose of 1 × 10⁹ CFU.

The vaccine broke peripheral tolerance against HER2+ OSA tumors, with strong evidence of an interferon-gamma (IFNγ) specific response. Furthermore, the vaccine led to increased tumor-associated T-lymphocyte infiltration, and subsequently lower incidence of metastases when compared to a historical group treated with amputation and carboplatin alone (23). For the 18 dogs treated with ADXS31-164, the median disease-free interval was 956 d compared to 123 to 257 d for amputation and carboplatin alone. Overall 1- and 2-year survival rates were reported to be 77.8% and 67% for the vaccine group, compared to 35.4% and 10% for amputation plus carboplatin, respectively. No dose-dependent effect of ADXS31-164 on HER2/neu-specific immunity was found. Dogs that were “early responders” developed IFNγ-specific responses within 3 wk of administration, and dogs that were “late responders” developed responses within 2 to 6 mo.

Only low-grade (I or II) transient toxicities were observed within the dose range investigated, the most common of which were hypertension (19/23) and thrombocytopenia (13/23). Since the immune targeting of HER2 has been reported to cause cardiotoxicity in human cancers, cardiac status was evaluated by echocardiography and serum cardiac troponin I levels. No significant or sustained changes in cardiac parameters were identified.

Recombinant Salmonella expressing IL-2 vaccine

Orally administered, attenuated and genetically engineered Salmonella enterica serovar Typhimurium encoding IL-2 (SalpIL2) was developed and tested in combination with amputation and adjuvant doxorubicin (24). The innate biodistribution of Salmonella spp. in hypoxic or anaerobic environments makes this an attractive vector for the delivery of intratumoral immunotherapy. IL-2 is a pleiotropic cytokine released early during an immune response. The mechanism of action of SalpIL2 involves immunemediated cytotoxicity, leading to the immunogenic killing of S. typhimurium-infected tumor cells.

SalpIL2 was administered to 19 client-owned dogs (3 × 10⁸ CFU/kg BW) in a phase I clinical trial at the University of Minnesota. The dose was given at day 0, after which amputation was performed at day 10, and doxorubicin chemotherapy started 2 wk after. Safety and efficacy were the primary and secondary measures, respectively.

The therapy led to disease-free intervals ranging from 69 to 880 d with a median of 199 d (24). Three of the 19 dogs did not develop metastases during the study period. Dogs treated with a lower dose of SalpIL2 had longer disease-free intervals than dogs treated using the highest SalpIL2 dose. Although toxicities were observed, they were not attributed to the administration of SalpIL2.

Limitations and challenges

Success in immunotherapy is challenged by poor immunogenicity of cancers and difficulty in overcoming tumor-induced immune suppression. Developing immunotherapy regimens that are consistently effective for each patient and identifying biomarkers of response to immunotherapy are often difficult. For autologous T-cell therapy, factors such as availability of apheresis centers and turnaround times must be considered. Administration of pre-medicants, such as non-steroidal anti-inflammatory drugs (NSAIDs) or anti-nausea medications for ACT, may further reduce the incidence of adverse effects in dogs. Live, attenuated cancer vaccines have the potential to invoke strong immune responses by enhancing antigen delivery through microbial delivery systems, yet this approach could pose risks for immunocompromised patients. Safety and quality control of the treatment should be considered a priority for dogs receiving live attenuated vaccines.

Virotherapy trials

Virotherapy is a strategy that directs the use of viruses for primary or metastatic tumor cell destruction. Viruses can be exploited as vectors for the delivery of exogenous agents or to mediate tumor cell destruction (oncolytic virotherapy). When used to illicit an immune response, virotherapy is considered a form of immunotherapy.

Adenoviral gene therapy

In canine OSA, neoadjuvant gene therapy with delivery of a replication-deficient adenovirus vector (Ad-FasL) was tested for intratumoral activation of FasL (25). FasL is a type II transmembrane secreted protein member of the tumor necrosis factor family that, upon engaging with the Fas “death receptor” (CD95 or APO-1), mediates apoptosis. In previous studies involving anti-Fas antibodies, there was evidence of apoptosis in Fas⁺ tumors; however, this treatment led to high lethality in mice due to engagement of Fas antigen in the liver, leading to fulminant hepatitis (26).

A phase I trial involving Ad-FasL delivery in 56 dogs with appendicular OSA was completed. Administration of Ad-FasL was followed by the standard of care after a 10-day delay. This period of delay was based on preclinical data showing statistically significant immunologic protection in animals treated after 10 d with FasL gene transfer. Ad-FasL delivery demonstrated significant survival improvement (98 wk versus 37 wk in historical controls) in dogs with high inflammation or high lymphocyte infiltration scores (> 1), especially in tumors that expressed low levels of FasL. This result suggests that Ad-FasL may be most effective when OSA tumors fail to express, or express low levels of FasL. Inflammation, apoptosis, or necrosis following FasL activation resulted in better outcomes (25). However, survival in dogs with low inflammation scores (< 1) was not different from the current standard of care.

Overall, the phase I data concluded that gene therapy with viral vectors can be safely administered due to the replication-deficient nature of adenoviruses. Transient increases in aspartate transaminase and creatine phosphokinase were observed, but with no attributable clinical symptoms. Of the 54 dogs that were evaluated, 22 had no reportable toxicity, 26 exhibited grade I or grade II toxicity, 4 exhibited grade III toxicity, and 2 had grade IV toxicity. Both cases of grade IV toxicity involved hypotension and azotemia that were not attributed to the administration of Ad-FasL.

Vesicular stomatitis virus

VSV-hIFN-NIS is a recombinant vesicular stomatitis virus (VSV) engineered to express interferon beta (IFNβ) and the sodium-iodide symporter (NIS). In a preliminary syngeneic model of murine myeloma, single shot systemic therapy with VSV-hIFN-NIS resulted in tumorspecific uptake and viral replication, leading to tumor remission (27). IFNβ enhances specificity of VSV and activates innate immunity to initiate antiviral responses. Specific replication of the virus is monitored by single-photon emission computerized tomography (SPECT)/CT imaging, using a NIS-specific radiotracer.

A pre-clinical dose-escalation study was first performed in purpose-bred, healthy beagles to define a safe systemic dose range (28). Systemic VSV therapy at a tissue-culture infective dose (TCID) of 1 × 10¹⁰ TCID₅₀/0.5 m² was well-tolerated, and 10-fold dose-limiting toxicities included hepatotoxicity and coagulopathy. Eight client-owned dogs with a variety of spontaneous cancers were then recruited, and this dose-feasibility trial defined the pharmacokinetics (PK) and biological activity of systemic oncolytic VSV (29). Within this study, 2 intravenous doses of VSV-hIFN-NIS were delivered to 1 client-owned dog with metastatic maxillary OSA, showing tumor-specific virus replication and delayed viral decay. The dog exhibited stable disease for 6 mo before progression of the primary lesion. No shedding of the virus was observed in urine or buccal swab samples of all 8 dogs, and correlative PK studies demonstrated elevated levels of VSV RNA in the blood. Transient hepatotoxicity resolved in 2/8 dogs following systemic VSV-hIFN-NIS treatment.

Limitations and challenges

Efficacy and specificity of the virus are often considered key limitations in the use of virotherapy. Many oncolytic viruses have favorable safety and toxicity profiles and are potent anti-cancer agents in vitro, but demonstrate limited clinical efficacy as a single agent (30). Accurate delivery of the virus to the target tumor tissue remains a challenge for virotherapy. Multi-site injections when compared to IV injections have shown promise in enhancing immune responses to virotherapy. Due to the replication-deficient nature of the virus, specificity is also of concern, and viral modifications may be necessary to increase specificity. Establishment of alternate regimens, such as the evaluation of combined therapies may improve responses to virotherapy.

Radiation therapy trials

Radiation therapy (RT) is often the treatment of choice in the palliative setting to alleviate local pain, but newer technologies may be offered with curative intent. The killing of tumor cells or the inhibition of osteoclast-mediated osteolysis are factors that may contribute to pain reduction. Radiation treatment typically involves 2 to 4 relatively large doses given once per week, after which pain relief begins in 70% of dogs after 11 to 15 d, and lasts for 60 to 120 d (8). Combined chemotherapy and RT have also been shown to be more effective than radiation therapy alone (9).

Bisphosphonates and radiation therapy

With the introduction of aminobisphosphonate therapy in dogs with appendicular OSA, more studies are aimed at determining their effects as single agents and in combination with radiation therapy. Aminobisphosphonate reduces malignant osteolysis by inducing osteoclast apoptosis. An early study by Fan et al (31) showed that single agent pamidronate treatment in 43 dogs with appendicular OSA provided durable pain relief in 12/43 dogs, lasting a median of 231 d.

Aminobisphosphonate therapy generally has a safe toxicity profile, although a recent case report highlighted osteonecrosis of the jaw as a possible complication of long-term use of the potent NBP zoledronate in the management of OSA complications, which has also been reported in human NBP use and pediatric OSA (32). Additionally, in a retrospective study of 50 canine appendicular OSA cases, addition of pamidronate to RT resulted in a decreased MST of 69 d compared to 309 d for combination chemotherapy and RT (9). Due to the retrospective of the study, it is possible that bias towards combination therapy for cases with more severe signs existed, and prolonged use of NBPs with RT in dogs requires further prospective evaluation.

Stereotactic radiation therapy

For dogs that are poor candidates for amputation or limb-sparing procedures, stereotactic radiation therapy has been evaluated as an advanced treatment technique. Stereotactic radiation therapy involves the administration of high dose fractions of radiation (20 to 30 Gy) to the target site using an external radiation beam, sparing surrounding tissues with submillimeter accuracy (33). Conventionally used to treat brain tumors in human patients (34), select veterinary radiation oncology practices have investigated stereotactic radiation delivery systems for the treatment of appendicular canine OSA. Unfortunately, a multi-institutional retrospective review of 18 dogs treated with stereotactic radiation therapy concluded that stereotactic radiation therapy led to major complications, the most common of which were surgical site infections and pathological fractures, with 9 dogs facing amputation at a median of 152 d (35). The major disadvantage of stereotactic radiation therapy may be that pathological fractures are extremely difficult to repair. Fracture repair using internal fixation could be considered a viable treatment for pathologic fractures in tumors that have been treated with stereotactic radiation therapy, using either an open approach or minimally invasive percutaneous osteosynthesis (MIPO) (36). The high risk for fractures may also justify prophylactic stabilization (35).

Overall, stereotactic radiation therapy when combined with surgery, could be considered a high-risk procedure for the dog. Alternatively, curative-intent methods other than stereotactic radiation therapy could be considered for canine appendicular OSA treatment (35).

Limitations and challenges

Despite the goal of only irradiating the tumor, radiation therapy does not exclusively target tumor cells. Whether radiation therapy is delivered with curative or palliative intent, development of pathologic fracture and radiation side effects should be considered during the protocol. Generally, radiation adverse effects, such as erythema, edema, and desquamation can be self-limiting (37) and are less common with palliative protocols. However, late side effects in tissues with slow turnover (i.e., bone) can lead to tissue fibrosis, necrosis, loss of function, or death, though these effects are less likely in the acute pain palliation setting. Radiation therapy is often contraindicated for sites of pathologic fracture. Therefore, appropriate patient selection, with careful consideration and planning must be performed before beginning a radiation therapy protocol.

Targeted therapy trials

Molecular targeted therapy involves the specific targeting of the cancer’s genes, proteins, or the tumor microenvironment to induce tumor remission. This approach often involves treatment with a monoclonal antibody, leading to specific targeting of proteins, or small molecule inhibitors, which lead to modulation of pathway expression in cancer.

Targeting of receptor tyrosine kinases

Toceranib (Palladia) is an oral receptor tyrosine kinase inhibitor approved by the US Federal Drug Administration for the treatment of Patnaik grade II or III, recurrent, cutaneous mast cell tumors with or without regional lymph node involvement in dogs. This drug is considered one of the first targeted therapies available in North America for canine cancer treatment. A safety evaluation of combination carboplatin and Palladia was performed in dogs with various cancers, including OSA (38). The protocol was well-tolerated overall, with the dose-limiting toxicity for Palladia administration being neutropenia.

The efficacy and clinical benefit of Palladia, which is defined as an objective response or stabilization of macroscopic disease, is reportedly limited with canine OSA treatment (39–41). The concurrent addition of Palladia to metronomic cyclophosphamide did not improve outcomes in microscopic disease, and the use of Palladia may not lead to reliable clinical responses of pulmonary macroscopic metastases (41,42).

Targeting of mTOR pathway

A systematic genome-wide screen in OSA identified dual phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibition as a conserved therapeutic vulnerability in OSA (43). Clinically, a dose escalation study of the mTOR inhibitor rapamycin determined a safe and active pharmacological oral dose in 22 dogs with appendicular OSA (44). This parenteral formulation resulted in the modulation of the mTOR pathway targets in tumor and peripheral blood mononuclear cells (PBMC), even at low doses of 0.01 mg/kg BW, compared with high doses at 0.08 mg/kg BW (44). AKT is a serine-threonine kinase that is implicated in downstream signaling and activation of mTOR. However, in rapamycin treated tumors, no differences in AKT expression or phosphorylation levels were observed. No serious adverse effects were reported from the study, but self-limiting grade I or II toxicities that could be attributed to rapamycin administration include vomiting, diarrhea, anorexia, and thrombocytopenia. While rapamycin clearance could not be studied due to the long half-life of the drug, plasma accumulation was evident at day 15 (44). Further results of this parenteral formulation are expected during the optimization of drug schedules and clinical trial evaluation. To this end, the Comparative Oncology Trials Consortium (COTC) of the US National Cancer Institute has been prospectively evaluating rapamycin in the adjuvant setting following limb amputation and chemotherapy. Results of this large multi-institutional clinical trial have not yet been released.

Targeting of nerve growth factor

Targeting skeletal pain ligands associated with nociceptors has been suggested to improve pain control, particularly in canine OSA, where local pain is often associated with tissue injury from inflammation (45). Moreover, because not all patients are elected for amputation due to coinciding nervous system or joint diseases, pain management in the palliative setting is often necessary for these patients.

Nociception in nerves of the normal bone is known to be controlled by the nerve growth factor (NGF) (46). An open-label, phase II trial has begun for pain palliation in canine OSA using a fully caninized, monoclonal antibody against NGF (NV-01), based on evidence that canine OSA cells express and secrete NGF ligands (45). In a pilot study involving kaolin injections into the canine footpad to mimic lameness and inflammatory pain, 32 research dogs were recruited for induction of paw inflammation (46). The degree of lameness was scored, then NV-01 was administered IV and compared to meloxicam or phosphate-buffered saline administration. The recovery period ranged from 7 to 14 d, after which the dogs were returned to the colony.

NV-01 was extremely well-tolerated and reduced signs of lameness comparable to meloxicam treatment. No adverse effects were observed over the 2-week monitoring period. Pharmacokinetic analysis revealed a distribution phase half-life of approximately 12 h and an elimination phase half-life of approximately 9 d. For meloxicam, significant improvement in lameness scores (compared to placebo) was observed at 6 h and on days 1, 5, 6, and 7. With NV-01 delivered IV, significance in lameness reduction compared to placebo was observed on days 1, 3, 6, and 7.

Limitations and challenges

Innate and acquired resistance to targeted therapy is a challenge faced by numerous human and veterinary targeted cancer therapy trials. Intrinsic resistance is often characterized by de novo activation of signaling pathways independent of the signaling target initially inhibited (47). Clonal evolution of resistant mutants is an acquired strategy for tumors to develop a diminished response to targeted therapy. This clonal outgrowth can produce unfavorable long-term outcomes for patients receiving monoclonal antibodies or small molecule inhibitors. Off-target side effects of targeted therapies are also of concern, which can lead to unique and dose-limiting toxicities. During clinical development, careful consideration of potential resistance mechanisms may lead to better targeting and drug combination approaches to improve long-term outcomes.

Metronomic chemotherapy

Low dose metronomic chemotherapy is an investigational treatment that may be incorporated concurrent and sequential to standard of care adjuvant chemotherapy or used in the palliative gross disease setting. This treatment approach involves use of a low dose, continuous administration of a chemotherapeutic, such as the alkylating agent cyclophosphamide. Due to toxicities reported at high doses, the metronomic approach for drug dosing may achieve clinical benefit without the same toxicity profile seen at the maximum-tolerated dose. Low dose metronomic chemotherapy has been reported to inhibit both tumor angiogenesis and immune suppressive regulatory T-cells, while conventional chemotherapy at the maximum-tolerated dose, kills rapidly dividing tumor cells.

In a retrospective study of 50 dogs treated with chronic LDM cyclophosphamide, adverse effects were observed that resulted in discontinuation of treatment in 22 dogs (44%), while 16 dogs (32%) developed sterile hemorrhagic cystitis leading to irritating bladder symptoms and hematuria (48). Moreover, metronomic chemotherapy has also been combined with toceranib phosphate (Palladia) and was found to have no improvement on the disease-free interval after amputation and carboplatin chemotherapy (42).

Limitations and challenges

Prospective clinical trial validation for the benefit of LDM chemotherapy in veterinary oncology is currently lacking. The administration of cyclophosphamide with an LDM schedule warrants additional monitoring for cystitis, especially after high cumulative doses.

Despite a generally well-tolerated acute toxicity profile, chronic administration of LDM chemotherapy may be associated with potential complications.

Personalized medicine trials

Personalized medicine is a strategy that separates patients into groups based on predictions about their individualized response to treatment. High throughput research models such as genetic models of disease allow for the robust prediction of potential treatment efficacy. In canine OSA, a gene expression model has been recently developed for doxorubicin and carboplatin treatment by comparing the drug sensitivity data of canine OSA cell lines and tumor datasets (49). Through a bioinformatics approach, predictions in differential gene expression were made in canine OSA tumor samples, and then matched with the clinical outcome of patients after chemotherapy treatment in a retrospective setting. Dogs whose treatment matched the predictions in gene expression had significantly better clinical outcomes, leading to longer disease-free intervals (49). The prediction of drug response may direct future decisions regarding the choice of chemotherapeutic or experimental therapy.

Personalized medicine algorithms (Pmed)

A recent pioneering effort among US veterinary hospitals also supports that personalized medicine is feasible as an approach for OSA treatment. This multi-site study adopted personalized medicine algorithms involving genomic profiling and bioinformatics to look at the feasibility of determining suitable therapies for an individual dog with an average turnaround time of 5 business days (50). After submission, the samples were verified by quality control for RNA quality prior to processing, after which the personalized medicine algorithms report was relayed to the attending veterinarian. Of the 20 patient samples submitted, 13 were successfully profiled, while others were hindered by either pathology or RNA quality control failure (50). Based on the report, opinions that were communicated from attending veterinarians about feasibility of the process were overall positive, with constructive information provided that could potentially guide further clinical trials.

Although Pmed approaches at present are performed with single gene evaluations for Palladia and masitinib in dogs with mast cell tumors, there is potential translational value of conducting personalized medicine for treating future OSA cases. As such, this client-tailored approach may soon be part of a powerful armamentarium for monitoring drug resistance and the decision-making process. Prospective clinical trials are warranted to assess the impact of such approaches on the prognosis of dogs with OSA.

Limitations and challenges

Personalized medicine remains a growing and immature field that relies heavily on technology and human interpretation of patient data. This data generation and interpretation can raise issues with cost, confidentiality, and potential ethical concerns. Despite these challenges, personalized approaches have strong potential to see routine implementation into the clinic as the ability to genetically interrogate tumors becomes faster and easier. In veterinary oncology, and in the specific setting of canine appendicular OSA, policies regulating use and interpretation of this genetic data may be necessary.

Conclusions

Canine OSA remains the most common aggressive primary bone tumor in dogs. The chronology of amputation or RT followed by chemotherapy is standard treatment that many dogs face when diagnosed with this disease. A wealth of past research into canine OSA, cancer biology and immunology may soon begin to pay clinical dividends. With emerging therapeutic strategies, such as immunotherapy and targeted therapy, owners increasingly have access to new treatment options for their companion animals. In addition, even more personalized medicine may soon open additional doors for owners to expand upon currently available therapeutic opportunities. In veterinary oncology, pinpointing realistic molecular targets for canine appendicular OSA has tremendous potential in halting its metastatic progression. It is important to note that, because many of the results of the novel therapy trials reported herein remain inconclusive, more research is warranted to translate such therapies into a new validated standard of care.

While outcomes with conventional surgery and chemotherapy may have plateaued over the last several years, novel therapeutic approaches, currently under investigation based on decades of preclinical investigation, see the OSA field poised to make strides in the future to significantly extend survival of dogs affected by this aggressive disease.