Assessment of postoperative adjuvant treatment using toceranib phosphate against adenocarcinoma in dogs.

Abstract Background Toceranib phosphate (TOC) could be made widely available for treating tumors in dogs if evidence shows that TOC inhibits recurrence after surgery. Objectives To investigate how postoperative adjuvant treatment with TOC modulates the tumor microenvironment (TME), by assessing effects on angiogenic activity, tumor‐infiltrating regulatory T cells (Tregs), and intratumoral hypoxia. Animals Ninety‐two client‐owned dogs were included: 28 with apocrine gland anal sac adenocarcinoma, 24 with small intestinal adenocarcinoma, 22 with lung adenocarcinoma, and 18 with renal cell carcinoma. Methods Retrospective, multicenter study comparing time to progression (TTP) between 42 dogs treated by surgery and TOC and 50 dogs treated by surgery alone. Differences were analyzed in the expression of vascular endothelial growth factor receptor‐2 (VEGFR2) and the number of Foxp3+ Tregs and hypoxia‐inducible factor (HIF)‐1α+ cells in tumor tissues sampled at the first and second (recurrence) surgeries. Results Median TTP for dogs treated by surgery and TOC (360 days) was higher than that for dogs treated by surgery alone (298 days; hazard ratio, 0.82; 95% confidence interval [CI], 0.65‐0.96; P = .02). In dogs treated by surgery and TOC, VEGFR2 expression and the number of Tregs and HIF‐1α+ cells were significantly lower in tissues sampled at the second surgery than in those sampled after the first surgery. In dogs treated by surgery alone, significant differences were found between samples from the 2 surgeries. Conclusions and Clinical Importance Toceranib phosphate could prove to be a useful postoperative adjuvant treatment because of its modulation of the TME.


| INTRODUCTION
Toceranib phosphate (TOC) is a multitarget tyrosine kinase inhibitor (TKI), that exerts antiangiogenic and antitumor effects by targeting molecules such as platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor-2 (VEGFR2), and KIT protooncogene receptor tyrosine kinase (KIT). 1,2 It has shown clinical utility as a single agent in dogs with mast cell tumors, gastrointestinal stromal tumors, and some carcinomas. 3,4 In 1 study, inhibition of PDGFR, VEGFR2, and KIT by TOC-induced antitumor activity resulted in clinical benefits in 63 of 85 dogs with solid tumors (74%). 3 However, the mechanism by which TOC serves as postoperative adjuvant treatment for dogs with adenocarcinoma is unclear. If TOC can support improvement in long-term time to progression (TTP) after surgery in dogs with adenocarcinoma, it could be made widely available for use in this setting.
The presence of microscopic residual disease after surgical removal of tumor tissue is a very important determinant of prognosis in cancer patients. Importantly, recurrence can occur at the primary tumor site even after tumor resection with histopathologically confirmed complete margins. 5 As previously reported, microscopic tumors can survive and resume growth if the tumor microenvironment (TME) is suitably modulated by angiogenic activity, intratumoral hypoxia, and recruitment of tumor-infiltrating regulatory T-cells (Tregs). [6][7][8] Therefore, therapeutically targeting the TME could prove an effective strategy to prevent relapse or delay tumor growth after surgery.
Sunitinib and sorafenib are 2 multitarget TKIs that have similar therapeutic targets to TOC and are commonly used in human cancer patients. Recent studies have reported that sunitinib and sorafenib could suppress expression of VEGFR2 and hypoxia-inducible factor (HIF)-1α as well as decrease the number of tumor-infiltrating Tregs in several carcinomas of humans, suggesting that these TKIs might target the TME. [9][10][11][12][13] Consequently, we hypothesized that TOC also could therapeutically target the TME in dogs with adenocarcinoma, and could help improve long-term TTP after surgery.
Our aim was to investigate the effects of postoperative adjuvant treatment using TOC. Therefore, we retrospectively compared the TTP of 2 subgroups of dogs diagnosed with various types of adenocarcinomas: those treated by surgery alone and those treated by surgery followed by adjuvant treatment with TOC. We also evaluated how TOC affected the TME in terms of VEGFR2 expression and the number of Foxp3 + Tregs and HIF-1α + cells in these tumors. We hypothesized that TOC would improve clinical outcome by modulating the TME.

| Treatment procedures
All dogs underwent excision of the primary tumor, and also the draining lymph node when lymphadenopathy was detected on imaging or as an intraoperative finding. In all dogs diagnosed with solitary lung tumors, lobectomy was performed. For dogs receiving postoperative adjuvant treatment, TOC (Palladia, Zoetis, Florham Park, New Jersey) was administered PO for 21 days after surgery. The dose was calculated to fall within a dosage range of 2.4-2.9 mg/kg, and was administered every Monday, Wednesday, and Friday. 14,15 Certain additional medications were allowed to be administered concurrently, including gastroprotectants (H 2 receptor antagonists or proton pump inhibitors), F I G U R E 1 Flow diagram for inclusion and exclusion criteria of the studies antiemetics, and antidiarrheals as required. Assessment of AEs was performed every 4 weeks during administration of TOC, in accordance with the veterinary cooperative oncology group-common terminology criteria for AEs. 16 Adverse events were noted on a graded scale of 1 (mild) to 5 (severe) in the medical records. Treatment with TOC was continued until either disease progression was identified, unacceptable toxicity was reached, or the owner requested that the treatment be discontinued. Dogs were excluded from the study if treatment failure occurred <28 days after first administration, because it is difficult to evaluate the effect of TOC in this context, as previously reported. 17 The responses and clinical outcomes documented in the medical records were used to determine TTP. This information included records of regular follow-up communications with the owners and referring veterinarians. Time to progression was defined as the period of time between tumor excision and the point at which local recurrence or metastatic disease was documented.

| Western blot analysis
When tumor recurrence after surgery occurred at approximately the same site as the primary location, the dogs underwent a second operation conducted by the same surgeon with the owner's approval. These tumors were histopathologically confirmed as recurrence of the primary tumor.
Each pair of tumor specimens collected at the first and second excisions were used for molecular biological analysis as described below. Healthcare, Princeton, New Jersey). Bands were quantified using quantification software (ImageJ, US National Institutes of Health, version 1.451 http://imagej.nih.gov/ij/, Bethesda, Maryland) and were presented as relative intensities normalized to that of β-actin.

| Immunohistochemistry
Expression of HIF-1α and Foxp3 proteins in the tumor samples was evaluated using immunohistochemistry (IHC). Sections of the collected Comparison of demographics of the population of dogs with adenocarcinoma classified into two groups: group that received surgery alone and group that received both surgery and toceranib phosphate (TOC) hematoxylin. The HIF-1α + cells were classified as tumor cells in which the nucleus, cytoplasm or both was distinctively stained for HIF-1α. 19,20 Regulatory T-cells were defined as cells with typical lymphocyte morphology and Foxp3 staining in the nucleus but not the cytoplasm. 21 Regulatory T-cells were quantified in 2 different compartments: the intra-tumoral area (defined as the area within nests composed of >5  F I G U R E 3 Expression of VEGFR2 was assessed by western blotting analysis in the tumor specimens collected at first and second surgery (Figure 2 showed small bowel adenocarcinoma). In dogs receiving surgery and toceranib phosphate, the relative intensities of the immunoreactive bands was significantly decreased in the second group of specimens (follow-up) when compared to the first group of specimens (baseline) (P = .03; A). In dogs receiving surgery alone, there was no significant difference between baseline and follow-up (P = .05; B)

| Clinical outcomes
In all dogs, histopathologically complete excision was achieved at the initial surgery for the primary tumor. In 35 of the 50 dogs treated by surgery alone, the regional lymph nodes were removed, and metastasis was detected in 10 of these dogs. In 30 of the 42 dogs treated by surgery and TOC, the regional lymph nodes were removed, and metastasis was detected in 9 of these dogs (Table 3) Figure 2 and Table 3). Comparisons of other variables are shown in Table 3. Among SBA, LA, and RCC cases, the TTP for dogs treated by surgery and TOC was significantly longer than for those treated by surgery alone, but no significant difference in TTP was found between the 2 treatment groups for AGASA cases (Table 3). Among dogs with an invasive type of tumor (ie, >20/10 HPF mitotic index and lymph node metastases), the TTP for dogs treated by surgery and TOC was significantly longer than for those treated by surgery alone (Table 3). In the 42 dogs that received surgery and TOC, no significant differences were found when comparing the incidence of AEs (Table 4). However, a significantly longer TTP was associated with the following variables during TOC treatment: systolic blood pressure > 136 mm Hg (HR, 0.76; 95% CI, 0.59-0.93; P = .04), neutrophil count ≤4200/μL (HR, 0.81; 95% CI, 0.63-0.97; P = .02), total dose >112 mg/kg (HR, 0.72; 95% CI, 0.45-0.89; P = .0082), and administration period >124 days (HR, 0.78; 95% CI, 0.62-0.91; P = .02; Table 4).

| Molecular biological assessment
Expression of VEGFR2 protein was comparatively evaluated between specimens collected at the first and second surgeries in 16 of the 18 cases that underwent repeat surgery (Figure 3). Expression of HIF-1α and Foxp3 proteins was comparatively evaluated between specimens taken at the 2 surgeries in all 18 cases (Figures 4 and 5).
In dogs that were treated by surgery and TOC, the relative intensities of the immunoreactive bands of VEGFR2 were significantly lower in the second group of specimens (follow-up) than in the first group of specimens (baseline; P = .03; Figure 3A). In dogs that were treated by surgery alone, no significant difference was found in VEGFR2 expression between baseline and follow-up (P = .05; Figure 3B). respectively, which represents no significant difference between these results (P = .16; Figure 5B).

| DISCUSSION
Ours is the first study to investigate the efficacy of a single agent, TOC, as a postoperative adjuvant treatment for dogs with adenocarcinoma. We compared the clinical outcomes of 2 subgroups with similar demographic characteristics. In veterinary oncology, survival time often is used as the primary end point because it is easily defined.
However, it can be affected by owner-driven factors such as the delay of initiation of additional treatments or euthanasia. We therefore used TTP, because it is a more reliable method for assessing response to treatment. In our study, adjuvant treatment with TOC was associated with an overall improvement of TTP in dogs with adenocarcinoma.
Intriguingly, TOC significantly increased the TTP for dogs suffering from SBA, LA, and RCC, and some advanced types of adenocarcinomas that had higher mitotic index, invasive characteristics, and lymph node metastases. Several studies have reported that postoperative adjuvant treatment with sunitinib and sorafenib was efficacious in human patients with advanced RCC. [22][23][24] Reports in the veterinary literature also have indicated that postoperative adjuvant treatment may improve survival time in dogs with SBA. 25 Our data suggest that the effects of TOC might depend on the type of adenocarcinoma in dogs that it is used to treat.
Dogs treated with TOC in general tolerated it well, but these cases had a higher rate of AEs as compared to those treated by surgery alone. These incidence rates are consistent with a previously published study. 26 [19][20][21]28,29 the relationship between the modulation of expression of these proteins and the mechanism of recurrence remains unclear. One investigator suggested that tumor recurrence might be independent of angiogenesis. 30 In some dogs with adenocarcinomas, tumor recurrence might not be influenced by Foxp3 + Tregs, VEGFR2 or HIF-1α, and may effectively take advantage of other critical microenvironmental factors. To establish the therapeutic approach of using TOC to target the TME, further prospective research on the relationship between TME modulation and the process of recurrence is required.
In conclusion, we determined that postoperative adjuvant treatment with TOC was associated with a significantly longer TTP for dogs with adenocarcinomas compared to treatment by surgery alone, and that this inhibition of recurrence possibly is caused by the longterm inhibition of Tregs, VEGFR2, and HIF-1α associated with TOC treatment. Our data should contribute to developing a prospective therapeutic approach for using TOC to target the TME. Further research is required to establish clinical applications in small animals.