Immunotherapies targeting checkpoint molecule programmed cell death 1 (PD‐1) protein were shown to be effective for treatment of non‐Hodgkin lymphoma in people, but little is known about the ...expression of PD‐1 or its ligand PD‐L1 by canine lymphoma. Therefore, flow cytometry was used to analyse expression of PD‐1 and PD‐L1 in canine lymphoma, using fine‐needle aspirates of lymph nodes from 34 dogs with B cell lymphoma (BCL), 6 dogs with T cell lymphoma (TCL) and 11 dogs that had relapsed. Furthermore, fine‐needle aspirates were obtained from 17 healthy dogs for comparison. Lastly, the impact of chemotherapy resistance on expression of PD‐1 and PD‐L1 was assessed in vitro. These studies revealed increased expression of PD‐L1 by malignant B cells compared to normal B cells. In the case of TCL, tumour cells and normal T cells both showed low to negative expression of PD‐1 and PD‐L1. In addition, tumour infiltrating lymphocytes from both BCL and TCL had increased expression of both PD‐1 and PD‐L1 expression compared to B and T cells from lymph nodes of healthy animals. In vitro, chemotherapy‐resistant BCL and TCL cell lines exhibited increases in both PD‐1 and PD‐L1 expression, compared to non‐chemotherapy selected tumour cells. These findings indicate that canine lymphomas exhibit upregulated checkpoint molecule expression, though the impact of checkpoint molecule expression on tumour biological behaviour remains unclear.
Background: Continuous administration of low doses of cyclophosphamide and standard doses of cyclooxygenase‐inhibiting drugs has been shown to suppress tumor angiogenesis, reverse immunosuppression, ...and deplete regulatory T cells in cancer models.
Hypothesis: We hypothesized that continuous treatment with low‐dose cyclophosphamide and full‐dose piroxicam would delay tumor recurrence in dogs with soft tissue sarcomas (STS).
Animals: Eighty‐five dogs with incompletely resected STS, 30 treated dogs, and 55 contemporary control dogs.
Methods: Treatment outcomes in 85 dogs with incompletely resected STS were evaluated in a retrospective study. Dogs in the treatment group received continuously administered low‐dose cyclophosphamide (10 mg/m2) and standard dose piroxicam (0.3 mg/kg) therapy. Time to local tumor recurrence (disease‐free interval; DFI) was compared between the 30 treated dogs and 55 untreated control dogs matched for age and tumor site and grade.
Results: DFI was significantly (P < .0001) prolonged for STS of all sites (trunk and extremity) in treated dogs compared with untreated control dogs. The DFI also was significantly longer in treated dogs when tumor site (trunk and extremity) was compared. Twelve treated dogs (40%) experienced mild toxicity (grade 1 and 2) at some point during treatment and 1 dog developed grade 4 cystitis. Every other day dosing was tolerated better than daily dosing.
Conclusions: Metronomic therapy with cyclophosphamide and piroxicam was very effective in preventing tumor recurrence in dogs with incompletely resected STS. These findings suggest that further evaluation of this approach is warranted as adjuvant therapy in dogs with highly metastatic tumors such as osteosarcoma and melanoma.
Background
KIT inhibitors, such as toceranib (TOC), and vinblastine (VBL) have not been prospectively compared in the treatment of macroscopic mast cell tumors (MCTs). Also, it is unknown whether VBL ...or TOC is superior for treating MCT without c‐kit mutations.
Hypothesis/Objectives
To determine the value of KIT genotyping and localization in treatment decisions for dogs with macroscopic MCT. We hypothesized that c‐kit mutated MCT would have a better response to TOC than VBL.
Animals
Eighty‐eight client‐owned dogs with macroscopic MCT.
Methods
Prospective, randomized trial. Dogs were randomized to TOC (2.75 mg/kg EOD) or VBL (2.5 mg/m2 weekly × 4 then EOW) by KIT localization and c‐kit mutation status using an adaptive randomization scheme.
Results
Sixty dogs were allocated to TOC and 28 to VBL. Of the dogs receiving TOC, 20% had c‐kit mutations, compared to 30% receiving VBL (P = 0.74). Overall response rates were 46% (TOC) and 30% (VBL) (odds ratio = 1.56 0.62–3.92; P = 0.28). Median progression‐free survival (PFS) for dogs receiving VBL was 78 days (7–1,521) and for TOC 95.5 (14–990); hazard ratio (HR) = 1.34 0.72–2.50; P = 0.36. Median overall survival (OS) was 241.5 days (10–1,521) for the VBL group and 159 (20–990) for the TOC group; HR = 0.80 (0.45–1.41; P = 0.44).
Conclusions and Clinical Importance
Neither PFS nor OS was significantly different between treatment groups. As the proportion of dogs with c‐kit mutations was not different between treatment groups in this population of dogs, c‐kit mutation status did not predict treatment response.
Background
Standard of care treatment for multicentric lymphoma in dogs remains doxorubicin (DOX)‐based combination chemotherapy, but owners may hesitate to commit the time and financial resources to ...complete such a protocol, typically requiring 12–16 visits. Rabacfosadine (RAB), a double prodrug of the nucleotide analog 9‐(2‐phosphonylmethoxyethyl) guanine, has substantial single‐agent activity in dogs with lymphoma, and a different mechanism of action than DOX.
Hypothesis/Objectives
Our objective was to evaluate the efficacy and adverse effect (AE) profile of alternating doses of RAB and DOX in dogs with naïve multicentric lymphoma.
Animals
Fifty‐four dogs with previously untreated lymphoma.
Methods
Open‐label, multicenter prospective clinical trial. Dogs received alternating RAB (1.0 mg/kg IV weeks 0, 6, 12) and DOX (30 mg/m2 IV weeks 3, 9, 15). Dogs that achieved complete response (CR) were followed by monthly evaluations. Complete clinicopathological evaluation and assessment of remission and AEs were performed every 21 days.
Results
The overall response rate was 84% (68%; CR; 16%; partial response PR). The overall median progression‐free interval (PFI) was 194 days (216 for CR and 63 for PR). Most AEs were mild and self‐limiting: gastrointestinal and hematologic AEs were most common. Thirteen dogs experienced dermatologic AEs, and 2 dogs developed grade 5 pulmonary fibrosis.
Conclusions and Clinical Importance
Alternating RAB/DOX generally was well tolerated and resulted in PFIs comparable to standard DOX‐based multi‐agent protocols, with fewer treatment visits. Most adverse events were mild or moderate and self‐limiting. Further studies are warranted to explore long‐term outcome and other RAB chemotherapy combinations.
BACKGROUND: Canine T‐cell lymphoma (TCL) is conventionally considered an aggressive disease, but some forms are histologically and clinically indolent. CD4 TCL is reported to be the most common ...subtype of TCL. We assessed flow cytometric characteristics, histologic features when available, and clinical outcomes of CD4+ TCL to determine if flow cytometry can be used to subclassify this group of lymphomas. OBJECTIVE: To test the hypothesis that canine CD4+ T‐cell lymphoma (TCL) is a homogeneous group of lymphomas with an aggressive clinical course. ANIMALS: Sixty‐seven dogs diagnosed with CD4+ TCL by flow cytometry and treated at 1 of 3 oncology referral clinics. METHODS: Retrospective multivariable analysis of outcome in canine CD4+ TCL including patient characteristics, treatment, and flow cytometric features. RESULTS: The majority of CD4+ TCL were CD45+, expressed low class II MHC, and exhibited an aggressive clinical course independent of treatment regimen (median survival, 159 days). Histologically, CD4+ TCL were classified as lymphoblastic or peripheral T cell. Size of the neoplastic lymphocytes had a modest effect on both PFI and survival in this group. A small number of CD4+ TCL were CD45− and class II MHC high, and exhibited an apparently more indolent clinical course (median survival not yet reached). CONCLUSIONS AND CLINICAL IMPORTANCE: Although the majority of CD4+ TCL in dogs had uniform clinical and flow cytometric features and an aggressive clinical course, a subset had a unique immunophenotype that predicts significantly longer survival. This finding strengthens the utility of flow cytometry to aid in the stratification of canine lymphoma.
Regulatory T cells (Treg) are a distinct group of T lymphocytes with immunosuppressive properties that serve normally to prevent harmful autoimmune responses. However, Tregs can also interfere with ...beneficial immune responses such as anti-tumor and anti-viral immunity in humans and rodents. Given the overall importance of Tregs, it is likely that they play an important role in diseases of dogs as well. However, at present reagents required for identification of Tregs in dogs are not available. Therefore, we investigated whether expression of FoxP3, a transcription factor that is highly expressed in Tregs in humans and rodents could also be used to identify Tregs in dogs. We found that a cross-reactive FoxP3 antibody identified a subset of CD4+ T cells in blood and lymph nodes of dogs. By flow cytometry the mean percentage of FoxP3+CD4+ T cells in normal dogs was 4.3% in blood and 9.8% in the lymph nodes. In dogs with cancer, there was a significant increase in numbers of Treg in blood (7.5%) and tumor-draining lymph nodes (17.1%) compared to age-matched healthy control dogs. We also found that FoxP3+CD4+ T cells in dogs could be significantly expanded in vitro by TCR activation together with addition of TGF-β and IL-2. Treated cells also significantly increased expression of TGF-β and IL-10mRNA. We conclude from these studies that a cross-reactive FoxP3 antibody can be used to identify Tregs in dogs and that this reagent may serve as a useful tool for investigating the role of Treg in a variety of diseases of dogs.
Background: Regulatory T cells (Treg) have been shown to suppress antitumor immunity and often are increased in humans and rodents with cancer. However, Tregs have not been well studied in dogs with ...cancer and it is not known if certain tumor types are associated with increased Tregs.
Hypothesis: We hypothesized that Treg percentages would be increased in dogs with cancer and that Treg percentages would be higher in dogs with certain types of cancer.
Animals: The percentages and numbers of Tregs and nonregulatory T cells and B cells were assessed in 34 dogs with cancer and 9 age‐matched control dogs. Dogs evaluated included 14 dogs with sarcoma, 7 dogs with carcinoma, 7 dogs with lymphoma, and 6 dogs with mast cell tumor.
Methods: Numbers and percentages of Tregs, CD4+, and CD8+ T cells and B cells were determined using flow cytometry and compared between control dogs and dogs with cancer.
Results: The percentage of Tregs was significantly increased overall in dogs with cancer compared with control dogs. When tumor types were compared, Treg percentages were significantly increased in dogs with carcinoma. The Treg/CD8 T cell ratio was significantly higher in dogs with cancer compared with control dogs and was also significantly increased in 2 dogs with T‐cell lymphoma.
Conclusions: Treg percentages in blood were increased in dogs with cancer, particularly in dogs with carcinoma. The Treg/CD8 ratio also identified tumor‐specific abnormalities in dogs with cancer. These findings indicate that tumor‐specific factors may affect Tregs in dogs.
Increased numbers of tumour‐associated macrophages correlate with rapid tumour growth and metastasis in tumours. Thus, macrophage depletion has potential as a novel cancer therapy and positive ...responses have been reported in rodent tumour models. To investigate the effectiveness of this approach in dogs with cancer, we evaluated the effects of the macrophage‐depleting agent liposomal clodronate (LC) in dogs with soft‐tissue sarcoma (STS). To this end, we conducted a clinical trial of LC therapy in 13 dogs with STS. Repeated LC administration was well tolerated clinically. Preliminary examination of tumour biopsy sets from 5 of the 13 dogs demonstrated that the density of CD11b+ macrophages was significantly decreased after LC treatment. Circulating concentrations of interleukin‐8 were also significantly reduced. These preliminary studies are the first to suggest that LC can be used as a systemic macrophage‐depleting agent in dogs to reduce numbers of tumour‐associated macrophages.
Background
Effective treatments for dogs with advanced stage mast cell tumors (MCT) remain a pressing need. A micellar formulation of paclitaxel (paclitaxel micellar) has shown promise in early‐phase ...studies.
Hypothesis/Objectives
The objective was to demonstrate greater activity for paclitaxel (micellar) compared with lomustine. The null hypothesis was μp = μL (ie, proportion of responders for the paclitaxel micellar and lomustine groups, respectively).
Animals
Two hundred and fifty‐two dogs with advanced stage nonresectable grade 2 or 3 MCT.
Methods
Prospective multicenter randomized double‐blind positive‐controlled clinical trial. The primary endpoint was confirmed overall response rate (CORR) at 14 weeks. A secondary endpoint, biologic observed response rate (BORR), also was calculated. Safety was assessed by the characterization and grading of adverse events (AE).
Results
Overall CORR (7% versus 1%; P = .048) and BORR (23% versus 10%; P = .012) were greater for paclitaxel (micellar) compared with lomustine. Paclitaxel (micellar)‐treated dogs were 6.5 times more likely to have a confirmed response and 3.1 times more likely to experience a biologic observed response. The majority of AE with paclitaxel (micellar) were transient and clinically manageable. Twenty‐seven dogs (33%) receiving lomustine were discontinued because of hepatopathy compared with 3 dogs (2%) receiving paclitaxel (micellar) (P < .0001; odds ratio 26.7).
Conclusions and Clinical Importance
Paclitaxel (micellar)'s activity and safety profile are superior to lomustine. The addition of an active and novel taxane to the veterinary armamentarium could fill a substantial need and, as its mechanism of action and AE profile do not overlap with currently available TKI, its availability could lead to effective combination protocols.
Intravenous gene delivery using liposome-DNA complexes (LDC) has previously been shown to elicit antitumor activity, but only in rodent tumor models. Therefore, we conducted a study to determine in a ...large animal spontaneous tumor model whether intravenous infusions of LDC could target gene expression to cutaneous tumor tissues and whether repeated treatments had an effect on tumor growth or angiogenesis. A total of 13 dogs with cutaneous soft tissue sarcomas were enrolled in the study and were randomized to receive a series of 6 weekly infusions of LDC containing either canine endostatin DNA or DNA encoding an irrelevant gene (luciferase). Serial tumor biopsies were obtained to assess transgene expression, tumor microvessel density (MVD), and intratumoral leukocyte inflammatory responses. We found that intravenous infusion of LDC did not result in detectable gene expression in cutaneous tumor tissues. However, two of 13 treated dogs had objective tumor responses and eight dogs had stable disease during the treatment period. In addition, a significant decrease in tumor MVD was noted in six of 12 treated dogs at the completion of six treatments. These results suggest that intravenous infusions of LDC may elicit nonspecific antitumor activity and inhibit tumor angiogenesis.
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