Short-Term Administration of Single-Agent Toceranib in Six Cases of Inoperable Massive Canine Hepatocellular Carcinoma
ABSTRACT
Six dogs with massive hepatocellular carcinoma that was not amenable to surgery were treated by oral administration of single-agent toceranib at a dose of 2.0–3.0 mg/kg every other day for a minimum of 60 days. Partial response was achieved in three dogs, stable disease was achieved in one dog, and progressive disease occurred in two dogs, according to the canine Response Evaluation Criteria in Solid Tumors v1.0. Observed adverse events were mild to moderate in severity and reported in accordance with the Veterinary Cooperative Oncology Group’s common terminology criteria for adverse events v1.1. Activities of alanine aminotransferase and alkaline phosphatase decreased in the cases that were sensitive to treatment with toceranib, whereas the activities remained high in resistant cases. Additionally, the level of phospho-vascular endothelial growth factor receptor 2 was found to be increased in a resistant case. Single-agent toceranib might prove to be an effective treatment for canine hepatocellular carcinoma pending further validation.
Introduction
Hepatocellular carcinoma (HCC) is the most common primary liver tumor in dogs, accounting for 50% of all hepatic masses.1 The prognosis for canine HCC depends mainly on their morphologic types and whether surgical excision is available for the tumors. Massive HCCs, large, solitary masses confined to a single liver lobe, ideally undergo surgical resection, which result in a favorable prognosis with a 1460 day median survival time.2 However, surgery may not be pursued for a variety of reasons. For example, surgery is not recommended for nodular, diffuse, and metastatic HCC because of the presence of disseminated disease. Surgical excision may not be possible in cases of massive HCC involving large vessels such as the vena cava. Owners of dogs who would require multiple procedures might elect not to pursue surgical treatment. These inoperable HCCs have a poor prognosis, and an effective chemotherapeutic protocol has not yet been established.2 Therefore, there is a great deal of interest in the development of novel chemotherapeutic options for the treatment of canine HCC.
Human and canine HCCs possess clear similarities in their histopathological features; however, they differ markedly in their etiologies. Both human and canine HCCs have a rich blood flow resulting from abundance of vascular structures such as hepatic arteries, veins, capillaries, and sinusoids. Tumor cells derived from hepatocytes require intratumoral vessels because of their high metabolic activity. Therefore, targeting these vascular structures might be effective for the treatment of HCCs.
Signals originating from vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) accelerate angiogenesis and contribute to growth and metastasis in various human cancers.3 The VEGF pathway is dysregulated in cases of human HCC, and antiangiogenic agents such as sorafeniba that target VEGF/VEGFR2 have been effective in the treatment of human HCC both in vitro and in vivo.4,5 Toceranibb is a multitargeting antiangiogenic agent used in veterinary medicine. It targets several receptor tyrosine kinases, including VEGFR2, as does sorafenib.6 Toceranib is currently approved only for the treatment of advanced canine mast cell tumors; however, it has the potential to treat a wide variety of canine solid tumors.7 Given the histopathological similarities between human and canine HCC and its abundant vasculature, it is possible that toceranib would be an effective chemotherapeutic agent for the treatment of canine HCC. However, its clinical effectiveness, toxicities, and influence on VEGF activity when administered to dogs with HCC has yet to be explored.
As a first step to developing toceranib-based chemotherapy for canine HCC, we evaluated the short-term clinical outcomes and toxicities in six cases of canine HCC receiving single-agent toceranib. Also, we evaluated the levels of total and phosphorylated VEGFR2 in a toceranib-sensitive and a toceranib-resistant case.
Materials and Methods
Case Selection
We retrospectively collected clinical information including treatment outcomes and toxicities from canine HCC cases treated with single-agent toceranib. In these cases, either the masses were judged to be clinically unresectable or the owners declined our proposal for surgical treatment. An initial contrast-enhanced computed tomography (CT) was performed, and the maximum diameter in each case was determined on each occasion based on 3D-reconstructed CT images. The slice thickness of the CT images was 2 mm. The diagnostic biopsy was performed by using an 18 gauge core biopsy instrument, and all cases were histopathologically diagnosed as HCC or well-differentiated HCC by at least one Japanese College of Veterinary Pathology board-certified pathologist according to the World Health Organization diagnostic criteria of HCC.8
Treatment Protocol
A dose of 2.0–3.0 mg/kg of toceranibb was orally administered every other day (EOD) to each participant for a minimum of 60 days. The dosage was determined according to a previous report.9 In cases 1–4 and case 6, toceranib was the only treatment administered aside from symptomatic treatments for gastrointestinal disorders such as nausea and diarrhea. Treatment for gastrointestinal disorders included standard antiemetic agents such as metoclopramide dihydrochloridec and maropitant citrated.
Follow-Up
Response to treatment with toceranib was evaluated by measuring the maximum diameter of the target lesion using contrast-enhanced CT. The maximum diameter in each case was determined on each occasion based on 3D-reconstructed CT images. The slice thickness of the CT images was 2 mm. Response to the treatment was classified as complete response, partial response (PR), stable disease (SD), or progressive disease (PD) according to the Canine Response Evaluation Criteria in Solid Tumors v1.0.10 Dogs were considered to have benefited from treatment if their response was classified as complete response, PR, or SD.
The blood activities of liver enzymes including alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were evaluated on the first day of treatment and on the day of follow-up in all cases. Blood samples were taken from the cephalic, saphenous, or jugular veins. Whole blood samples were heparinized and centrifuged for 5 min at 3000 rpm. The activities of plasma ALT and ALP were evaluated using a dry chemistry methode,f,g.
Toxicity Evaluation
Adverse events of treatment were graded according to the Veterinary Cooperative Oncology Group.11 Categories included grade 1 (mild), grade 2 (moderate), grade 3 (severe), grade 4 (life-threatening), and grade 5 (death).
Immunoblotting
Tissue samples were collected from case 1 and 4 using an 18 gauge core biopsy instrument at 126 and 77 days after the start of toceranib administration, respectively. Owner consent was obtained in each instance. Whole-cell lysates were prepared using a radioimmunoprecipitation assay buffer containing protease inhibitorsh and phosphatase inhibitorsi. Protein concentrations were measured using the Bradford protein assayj. Protein samples (5.0 μg/lane) were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to 0.45 μm polyvinylidene fluoride membranesk. To block nonspecific reactions, the membranes were incubated with either 5% Phospho-protein Blocking Reagentl or 5% nonfat milk in phosphate-buffered saline containing 0.1% Tween 20 for 1 hr. The membranes were then incubated overnight at 4°C with the designated primary antibodies. The primary antibodies used in this study were anti-phospho-VEGFR2 (phospho-Tyr1175)m and anti-VEGFR2n. The membranes were washed and subsequently incubated at room temperature with secondary horseradish peroxidase–linked anti-mouse IgG or anti-rabbit IgG antibodieso diluted 1:1000 and then washed three times with phosphate-buffered saline containing 0.1% Tween 20. Immunoblots were visualized by using a chemiluminescent horseradish peroxidase detection reagentp. The loaded amount was verified by reincubating the same membrane with an anti-β-actin mouse monoclonal antibodyq.
Statistical Analysis
GraphPad Prism 7r was used for the statistical analysis. For single comparison, the two-tailed, unpaired Student t test was used.
Results
Response to Treatment with Single-Agent Toceranib for each Case of Canine HCC
We evaluated the records of six dogs with canine HCC treated with single-agent toceranib (cases 1–6). The age, gender, breed, and weight of each patient are shown in Table 1. All dogs were over 10 yr of age and weighed less than 17 kg. Breeds represented included shih tzu, miniature dachshund, and medium-sized mixed-breed. The target lesion, prior treatment, and reason for avoiding surgery were different in each case (Table 1). The six dogs in this study received toceranib at a dose of 2.0–3.0 mg/kg per os EOD for various reasons, such as treating primary, metastatic, or recurrent lesions (Table 2). Case 5 received a low dose of toceranib (2.0 mg/kg EOD) because of the advanced age and the adverse effect of anorexia. All cases did not have apparent evidence of liver dysfunction or hypoalbuminemia prior to the toceranib therapy. Single follow-up was performed in cases 2, 4, and 5 at 112, 82, and 61 days after initiating administration, respectively. Multiple follow-ups were performed in case 1 (two times, at 70 and 126 days after initiating administration), case 3 (three times, at 35, 63, and 105 days after initiating administration), and case 6 (two times, at 35 and 71 days after initiating administration). Response to treatment and occurrence of adverse events following toceranib administration were assessed after treatment intervals of 61 to 126 days (Tables 2, 3). The response to treatment was found to have varied widely among the different cases (Figure 1). Four of the six cases were found to have benefited clinically from toceranib administration. Three of the six cases (cases 1, 3, and 6) showed PR with 34.0–42.4% regression of the disease, and one case (case 2) showed SD with 5.6% regression on the day of follow-up. Two of the six cases were found to be resistant to treatment with toceranib. These cases showed PD with 26.6–31.2% progression despite the administration of toceranib.
Citation: Journal of the American Animal Hospital Association 55, 1; 10.5326/JAAHA-MS-6788
Association Between Responses to Toceranib Administration and Plasma Liver Enzyme Activities
Liver enzymes such as ALT and ALP, which are used to evaluate hepatocellular injury or bile duct obstruction, are typically increased in the blood of canine HCC patients; therefore, we speculated that these enzymes might be biomarkers for monitoring response to treatment in canine HCC. After we had confirmed that ALT and ALP activities were increased in all cases on the first day of treatment, we evaluated their activities on the day of follow-up. Case 5 was excluded from the evaluation of liver enzyme activities because this case received prednisolone, which may affect plasma liver enzyme activities, at a dose of 0.3 mg/kg EOD for 58 days to treat comorbidities including perianal fistula and intervertebral disk displacement. We found that ALT and ALP activities decreased in the toceranib-sensitive cases, whereas the activities remained high in the toceranib-resistant cases. Although we acknowledged the limited statistical power in the small numbers of cases, we performed the statistical analysis as reference information. ALP showed a significant difference between the sensitive and resistant cases (P = .031, two-tailed unpaired Student t test), whereas ALT did not (P = .054, two-tailed unpaired Student t test).
Clinical Toxicities of Single-Agent Toceranib Administration in Cases of Canine HCCs
Adverse events observed in this study were graded minimal to moderate (Table 3) and were well controlled with symptomatic therapy. One of the six cases experienced no adverse events. Four of the six cases experienced mild toxic side effects including grade 1 diarrhea, anorexia, neutropenia, and lethargy. Only a single case experienced moderate toxic side effects, which included grade 2 diarrhea in addition to grade 1 anorexia and neutropenia. In this case, toceranib was discontinued at 20 days after the follow-up exam because of the adverse effects. The neutrophil counts for this case on days 0, 70, 126, and 146 were 5000, 7000, 2500, and 4100 per µL, respectively.
Increased Phosphorylation of VEGFR2 in Tumor Tissue from a Toceranib-Resistant HCC Case
We evaluated total and phosphorylated levels of VEGFR2 in a toceranib-sensitive (case 1) and a toceranib-resistant HCC case (case 4). We found that the level of phospho-VEGFR2 in the toceranib-resistant case (PD; case 4) was remarkably higher in a sample taken from tumor tissue than it was in the sample taken from normal tissue. Conversely, the level of phospho-VEGFR2 was equivalent in both tissues in the toceranib-sensitive case (PR; case 1). The tumor tissue samples expressed total VEGFR2 at much lower levels than did the normal tissue samples in both toceranib-sensitive and toceranib-resistant cases (Figure 2).
Citation: Journal of the American Animal Hospital Association 55, 1; 10.5326/JAAHA-MS-6788
Discussion
In this study, single-agent administration of toceranib benefitted four of the six canine HCC cases, although the remaining two cases showed no response to treatment. We found that the ALT and ALP activities decreased in the toceranib-sensitive cases but not in the toceranib-resistant cases. In addition, an increased level of phosphorylated VEGFR2 was found in a tumor tissue sample from the toceranib-resistant case.
Four of the six patients with canine HCC treated with single-agent toceranib were judged to have benefited clinically based on the Canine Response Evaluation Criteria in Solid Tumors v1.0 criteria. The target lesions of the patients were classified as PR or SD after minimum of 60 days of treatment. Moreover, the dosage of 2.0–3.0 mg/kg per os EOD was well tolerated by these dogs. The adverse events observed were graded as minimum to moderate. Furthermore, symptomatic therapy was effective in controlling the clinical signs well in all cases. The results of this study agreed with a previous study that reported on the ability of antiangiogenic agents such as sorafenib to successfully treat human HCC.5 The grades of adverse events seen in this study were also consistent with a previous report evaluating other types of canine tumors.9 These results suggest that single-agent toceranib might be a new chemotherapeutic option for the treatment of canine HCC that is not amendable to surgery, although further validation is required.
We, however, identified that two of the six cases were toceranib-resistant, as determined by progression of the target lesion despite treatment with toceranib. These results suggest that there is a toceranib-resistant population among canine HCC patients, and that the response to treatment should be frequently reviewed during administration because of the potential population of toceranib-resistant HCC. HCC is a drug-resistant tumor with a high detoxification ability and expression of P-glycoprotein, which excretes cytotoxic drugs from tumor cells.12 These features of HCC might have contributed to the development of drug resistance to toceranib therapy in these cases, although we were not able to elucidate the mechanism of resistance to toceranib in this study. It is noteworthy that all cases receiving toceranib for the treatment of their primary lesion were resistant, whereas all cases that received toceranib for the treatment of metastatic and recurrent lesion were sensitive. This result might suggest that treatment with toceranib for a primary lesion of HCC should be avoided, although such cases were only two of a total six cases, and this result might be by chance. Interestingly, ALT and ALP activities were decreased in all toceranib-sensitive cases, whereas the activities remained at high levels in the toceranib-resistant cases. This finding suggests that ALT and ALP might be used as low-invasive biomarkers to measure the response to toceranib therapy. However, the use of the liver enzymes may be limited for the patients concurrently receiving steroids in addition to toceranib therapy because steroids affect the activities of these enzymes. The low dose of toceranib (2.0 mg/kg EOD) did not show effectiveness in case 5 (PD). Although a low dose of sorafenib was reportedly beneficial for human HCC, it cannot be ruled out that the low plasma concentration of toceranib affected the response in the case.13 However, further study is warranted for determining whether a dose of toceranib lower than 2.4 mg/kg EOD is beneficial, as many cases have no other option but receive a limited dose of toceranib for unavoidable reasons.
The VEGF/VEGFR2 signaling pathway plays essential roles in the sensitivity of human HCC to sorafenib treatment.14 The phosphorylation of VEGFR2 may also be associated with the sensitivity of canine HCC to toceranib treatment because canine and human HCC share many similarities. In this study, the levels of phosphorylated VEGFR2 were increased in a tumor tissue sample from a resistant case (case 4) versus a normal tissue sample from the same patient. Conversely, the levels of phospho-VEGFR2 were equivalent in the tumor tissue and normal tissue samples obtained from the sensitive case (case 1). The result suggests that toceranib failed to sufficiently prevent the receptor phosphorylation of VEGFR2 and the downstream signal transduction in the resistant case. This result is consistent with the study showing that elevated tissue expression of VEGFR2 was associated with poor outcome in human HCC treated with sorafenib.15 The significance of VEGFR2 phosphorylation is still unclear because we evaluated phospho-VEGFR2 levels in only two HCC cases. However, VEGFR2 molecular expression or phosphorylation level might prove to be a key feature used to distinguish toceranib-resistant from toceranib-sensitive cases of canine HCC after further validation in the future.
We acknowledge several limitations to this study. First, we included only six cases with variations in the clinical presentation, dose of medication administered, duration of follow-up, and reason that surgery was avoided. Therefore, our results do not define the clinical usefulness of toceranib in the treatment of canine HCC. However, our results provide preliminary information that could be helpful to develop a toceranib-based therapy for the treatment of canine HCC. Future studies are required to validate the usefulness of toceranib in the treatment of canine HCC. Second, the effect of long-term toceranib administration might be different from that of short-term administration. One concern is that the administration of an antiangiogenic agent might increase the aggressiveness of neoplastic cells through the development of hypoxia in the tumor itself. Although this is merely hypothetical, it could ultimately shorten the patient’s survival time.16 Further studies are required to evaluate the long-term effects of toceranib administration on overall survival time, prognosis, and potential for metastasis in canine HCC patients.
Conclusion
Single-agent toceranib administration showed a clinical benefit in four of the six cases of canine HCC. The remaining two cases were resistant to treatment with toceranib. The activities of ALT and ALP and the levels of phosphorylated VEGFR2 might be key factors to distinguish toceranib-resistant cases from sensitive cases, although further validation is required. A large-scale study is warranted to determine the clinical usefulness of toceranib administration in the case of canine HCC.
Changes in contrast-enhanced CT images of a toceranib-sensitive (case 1) and a toceranib-resistant case (case 4). Single-agent toceranib administration decreased the maximum diameter of the target lesion in a toceranib-sensitive case (case 1). The target lesion in case 4 was progressive, and an example of a toceranib-resistant case. CT, computed tomography.
The levels of total and phosphorylated VEGFR2 in a toceranib-sensitive case (case 1) and a toceranib-resistant case (case 4). The figure displays the results of phospho-VEGFR2 and total VEGFR2 by immunoblot. The level of VEGFR2 in a toceranib-resistant case was over-phosphorylated in the tumor tissue compared with the surrounding normal liver tissue. However, the levels of phosphorylated VEGFR2 were equivalent between the tumor and normal tissue samples in the toceranib-sensitive case. The total amount of VEGFR2 in tumor tissue was lower than that found in the surrounding normal liver tissues in both the sensitive and resistant cases. N, surrounding normal liver tissue; T, tumor tissue; VEGFR2, vascular endothelial growth factor receptor 2.
Contributor Notes
