Michael S. Kent

Blocking Indolamine-2,3-Dioxygenase Rebound Immune Suppression Boosts Antitumor Effects of Radio-Immunotherapy in Murine Models and Spontaneous Canine Malignancies

Purpose: Previous studies demonstrate that intratumoral CpG immunotherapy in combination with radiotherapy acts as an in-situ vaccine inducing antitumor immune responses capable of eradicating systemic disease. Unfortunately, most patients fail to respond. We hypothesized that immunotherapy can paradoxically upregulate immunosuppressive pathways, a phenomenon we term “rebound immune suppression,” limiting clinical responses. We further hypothesized that the immunosuppressive enzyme indolamine-2,3-dioxygenase (IDO) is a mechanism of rebound immune suppression and that IDO blockade would improve immunotherapy efficacy. Experimental Design: We examined the efficacy and immunologic effects of a novel triple therapy consisting of local radiotherapy, intratumoral CpG, and systemic IDO blockade in murine models and a pilot canine clinical trial. Results: In murine models, we observed marked increase in intratumoral IDO expression after treatment with radiotherapy, CpG, or other immunotherapies. The addition of IDO blockade to radiotherapy + CpG decreased IDO activity, reduced tumor growth, and reduced immunosuppressive factors, such as regulatory T cells in the tumor microenvironment. This triple combination induced systemic antitumor effects, decreasing metastases, and improving survival in a CD8+ T-cell–dependent manner. We evaluated this novel triple therapy in a canine clinical trial, because spontaneous canine malignancies closely reflect human cancer. Mirroring our mouse studies, the therapy was well tolerated, reduced intratumoral immunosuppression, and induced robust systemic antitumor effects. Conclusions: These results suggest that IDO maintains immune suppression in the tumor after therapy, and IDO blockade promotes a local antitumor immune response with systemic consequences. The efficacy and limited toxicity of this strategy are attractive for clinical translation. Clin Cancer Res; 22(17); 4328–40. ©2016 AACR.

Canine cancer immunotherapy studies: linking mouse and human

Despite recent major clinical breakthroughs in human cancer immunotherapy including the use of checkpoint inhibitors and engineered T cells, important challenges remain, including determining the sub-populations of patients who will respond and who will experience at times significant toxicities. Although advances in cancer immunotherapy depend on preclinical testing, the majority of in-vivo testing currently relies on genetically identical inbred mouse models which, while offering critical insights regarding efficacy and mechanism of action, also vastly underrepresent the heterogeneity and complex interplay of human immune cells and cancers. Additionally, laboratory mice uncommonly develop spontaneous tumors, are housed under specific-pathogen free conditions which markedly impacts immune development, and incompletely model key aspects of the tumor/immune microenvironment. The canine model represents a powerful tool in cancer immunotherapy research as an important link between murine models and human clinical studies. Dogs represent an attractive outbred combination of companion animals that experience spontaneous cancer development in the setting of an intact immune system. This allows for study of complex immune interactions during the course of treatment while also directly addressing long-term efficacy and toxicity of cancer immunotherapies. However, immune dissection requires access to robust and validated immune assays and reagents as well as appropriate numbers for statistical evaluation. Canine studies will need further optimization of these important mechanistic tools for this model to fulfill its promise as a model for immunotherapy. This review aims to discuss the canine model in the context of existing preclinical cancer immunotherapy models to evaluate both its advantages and limitations, as well as highlighting its growth as a powerful tool in the burgeoning field of both human and veterinary immunotherapy.

Disseminated Lymphoma of Presumptive T-cell Origin in a Great Horned Owl (Bubo virginianus)

ABSTRACT A geriatric male great horned owl (Bubo virginianus) that was a resident at a raptor center was presented for examination because of stridor and weight loss. Results of physical examination, diagnostic imaging, and biopsy were consistent with disseminated lymphoma involving the oropharynx, neck region (including thyroid and parathyroid glands), keel, spleen, and liver. Attempts to treat the owl with chlorambucil failed, and the owl was euthanatized 5 months later. Neoplastic cells from this owl were immunoreactive to CD-3 antibody, suggesting the lymphoma was of T-cell origin.

Follow-up study comparing necropsy rates and discrepancies between clinical and pathologic diagnoses at a veterinary teaching hospital: 2009 versus 1989 and 1999

OBJECTIVES A follow-up, retrospective study to determine whether the proportion of discrepancies between clinical and pathological diagnoses made during 2009, 1999 and 1989 and the proportion of dogs necropsied have changed. METHODS Medical records of 148 hospitalised dogs that died or were euthanased in a veterinary medical teaching hospital during 2009 were reviewed. Clinical and pathological diagnoses were recorded, categorised and compared to historical controls using a data set of 623 dogs from a previous study. RESULTS The proportion of discrepancies was significantly (P<0·001) lower in 2009 (14·9%), compared to both 1999 (37%) and 1989 (39·8%). There was also a significant (P<0·001) decrease in the number of necropsies performed during 2009 (21·4%) compared to both 1999 (48·4%) and 1989 (58·9%). CLINICAL SIGNIFICANCE There was a marked improvement in the ante-mortem diagnosis of patients in 2009 compared with both 1989 and 1999 as evidenced by the decrease in the proportion of discrepancies between the clinical and pathological diagnoses. Necropsies should still be regarded as a vital tool for teaching, determining the pathological basis of disease, identification of new and emerging diseases, and for an individual animal determining the cause of death.

Can a commercially available EPID dosimetry system detect small daily patient setup errors for cranial IMRT/SRS?

PURPOSE The purpose of this study was to determine if the Sun Nuclear PerFRACTION electronic portal imager device dosimetry software would be able to detect setup errors in a clinical setting and would be able to correctly identify the direction in which the setup error was introduced. METHODS AND MATERIALS A 7-field intensity modulated radiation therapy (IMRT) treatment plan for a centrally located tumor was developed for 1 phantom and 5 canine cadaver heads. Systematic setup errors were introduced by manually moving the treatment couch by 1, 3, and 5 mm in each translational direction to assess stereotactic radiation surgery (SRS), IMRT, and 3-dimensional (3D) treatment tolerances after the initial alignment was performed. An angular setup error of 5° yaw was also assessed. The delivered treatment fluence was automatically imported in the PerFRACTION software and compared with the baseline fluence. RESULTS In the canine phantom, a 5-mm shift was undetected by gamma analysis, and up to a 2-cm shift had to be introduced for the gamma pass rate of 3%/3 mm to fall below a 95% pass rate criterion. The same 5-mm shift using 3% difference caused the pass rates for 2 fields to drop below the 95% tolerance. For each respective translational shift, the affected beam angles were consistent across the cadaver heads and correlated with the direction of translational shift. The best field pass rate, worst field pass rate, and average pass rate across all 7 fields was analyzed to develop clinical guidance on parameter settings for SRS, IMRT, and 3D tolerances. CONCLUSIONS PerFRACTION 2-dimensional mode successfully detected setup errors outside the systematic error tolerance for SRS, IMRT, and 3D when an appropriate analysis metric and pass/fail criteria was implemented. Our data confirm that percent difference may be more sensitive in detecting plan failure than gamma analysis.

Evaluation of optimal water fluoridation on the incidence and skeletal distribution of naturally arising osteosarcoma in pet dogs.

Experimental toxicological studies in laboratory animals and epidemiological human studies have reported a possible association between water fluoridation and osteosarcoma (OSA). To further explore this possibility, a case-control study of individual dogs evaluated by the UC Davis Veterinary Medical Teaching Hospital was conducted using ecologic data on water fluoridation based on the owners residence. The case group included 161 dogs with OSA diagnosed between 2008-2012. Two cancer control groups included dogs diagnosed with lymphoma (LSA) or hemangiosarcoma (HSA) during the same period (n = 134 and n = 145, respectively). Dogs with OSA were not significantly more likely to live in an area with optimized fluoride in the water than dogs with LSA or HSA. Additional analyses within OSA patients also revealed no significant differences in age, or skeletal distribution of OSA cases relative to fluoride status. Taken together, these analyses do not support the hypothesis that optimal fluoridation of drinking water contributes to naturally occurring OSA in dogs.

Principles and Applications of Radiation Therapy in Exotic Animals

Radiation therapy is a treatment modality for cancer that is widely used in veterinary medicine, although its use in exotic animal practice has remained limited. However, there are case reports and case series of treating birds, small mammals, and reptiles for a variety of cancers with radiotherapy with varied outcomes. In this article the basic principles of radiation therapy are reviewed and the literature regarding its use in exotic animal practice is summarized. Side effects of radiotherapy are also discussed.

IQGAP1 is an oncogenic target in canine melanoma

Canine oral mucosal melanoma is an aggressive malignant neoplasm and is characterized by local infiltration and a high metastatic potential. The disease progression is similar to that of human oral melanomas. Whereas human cutaneous melanoma is primarily driven by activating mutations in Braf (60%) or Nras (20%), human mucosal melanoma harbors these mutations much less frequently. This makes therapeutic targeting and research modeling of the oral form potentially different from that of the cutaneous form in humans. Similarly, research has found only rare Nras mutations and no activating Braf mutations in canine oral melanomas, but they are still reliant on MAPK signaling. IQGAP1 is a signaling scaffold that regulates oncogenic ERK1/2 MAPK signaling in human Ras- and Raf- driven cancers, including melanomas. To investigate whether IQGAP1 is a potential target in canine melanoma, we examined the expression and localization of IQGAP1 in primary canine melanomas and canine oral melanoma cell lines obtained from the University of California-Davis. Using CRISPR/Cas9 knockout of IQGAP1, we examined effects on downstream ERK1/2 pathway activity and assayed proliferation of cell lines when treated with a peptide that blocks the interaction between IQGAP1 and ERK1/2. We observed that canine IQGAP1 is expressed and localizes to a similar extent in both human and canine melanoma by qPCR, Western blot, and immunofluorescence. Deletion of IQGAP1 reduces MAPK pathway activation in cell lines, similar to effects seen in human BrafV600E cell lines. Additionally, we demonstrated reduced proliferation when these cells are treated with a blocking peptide in vitro.

PROSPECTIVE COMPARISON OF TUMOR STAGING USING COMPUTED TOMOGRAPHY VERSUS MAGNETIC RESONANCE IMAGING FINDINGS IN DOGS WITH NASAL NEOPLASIA: A PILOT STUDY

Identification of nasal neoplasia extension and tumor staging in dogs is most commonly performed using computed tomography (CT), however magnetic resonance imaging (MRI) is routinely used in human medicine. A prospective pilot study enrolling six dogs with nasal neoplasia was performed with CT and MRI studies acquired under the same anesthetic episode. Interobserver comparison and comparison between the two imaging modalities with regard to bidimensional measurements of the nasal tumors, tumor staging using historical schemes, and assignment of an ordinal scale of tumor margin clarity at the tumor-soft tissue interface were performed. The hypotheses included that MRI would have greater tumor measurements, result in higher tumor staging, and more clearly define the tumor soft tissue interface when compared to CT. Evaluation of bone involvement of the nasal cavity and head showed a high level of agreement between CT and MRI. Estimation of tumor volume using bidimensional measurements was higher on MRI imaging in 5/6 dogs, and resulted in a median tumor volume which was 18.4% higher than CT imaging. Disagreement between CT and MRI was noted with meningeal enhancement, in which two dogs were positive for meningeal enhancement on MRI and negative on CT. One of six dogs had a higher tumor stage on MRI compared to CT, while the remaining five agreed. Magnetic resonance imaging resulted in larger bidimensional measurements and tumor volume estimates, along with a higher likelihood of identifying meningeal enhancement when compared to CT imaging. Magnetic resonance imaging may provide integral information for tumor staging, prognosis, and treatment planning.

Factors Affecting Platelet Concentration in Platelet Concentrates from Canine Blood Donors

Background Physiologic factors in dogs that might contribute to enhanced platelet yield in platelet concentrates (PCs) are largely unknown. Objective To determine whether individual differences in weight, age, preprocessing blood chemistry, and CBC variables predict the final platelet concentrations in PCs. Our hypotheses were (1) increased lipemic indices would be positively associated with increased platelet concentrations in PCs and (2) increased preprocessing platelet concentrations would be associated with higher platelet concentrations in the PCs. Animals All blood donation records of dogs from February 2, 2009 through April 1, 2015 at the University of California—Davis Veterinary Blood Bank were examined with 104 cases included in this study. Methods In this retrospective study, data were collected from medical records of canine blood donors. Records were reviewed for internal consistency and accuracy and subjects were included in the study if donor screening and donation occurred on the same day and a viable PC was obtained. Univariate and multivariable regressions were used to test the impact that each variable had on the final platelet concentration in PCs. Results Final platelet concentration in PCs was positively associated with the predonation CBC platelet values (P < .001), lipemic index (P = .01), and phosphorous levels (P = .001). Collectively these 3 variables explained 29% of the variance in platelet concentrations in PCs. Conclusions and clinical importance Future prospective studies are required to determine if canine blood donations from dogs with lipemia yield PCs with higher platelet concentrations without negatively affecting other blood components.