Christopher M. Reilly

Mitotic Index Is Predictive for Survival for Canine Cutaneous Mast Cell Tumors

Mitotic index (MI) is an indirect measure of cell proliferation that has been demonstrated to be a strong predictor of outcome for several human and canine cancers. The purpose of this study was to evaluate the utility of MI as a predictor of biologic behavior and survival in dogs with cutaneous mast cell tumors (MCTs). Medical records from 148 dogs with histologically confirmed MCTs were reviewed. Information regarding tumor grade, local recurrence, metastatic disease, date of death/last follow-up, and outcome was obtained. The region of the tumor with the highest overall mitotic activity was chosen for evaluation, and the MI value was defined as the number of mitotic figures/10 high-power fields (400X, 2.7 mm2). A Cox proportional hazards regression model was used to compare MI with survival data. A Mann-Whitney test was used to compare MI on the basis of the development of local recurrence and metastatic disease. The MI correlated directly with tumor grade (P < .0001). The median survival time for dogs with an MI ≤5 was significantly longer (70 months) than for those with an MI >5 (2 months), regardless of grade (P < .001). For grade II tumors with an MI ≤5, the median survival time (MST) was 70 months, compared with 5 months for those with an MI >5 (P < .001). For grade III tumors with an MI ≤5, the MST was not reached, compared with <2 months for those with an MI >5 (P < .001). In conclusion, MI is a strong predictor of overall survival for dogs with cutaneous MCTs and should be included as a prognostic indicator when determining therapeutic options.

Elastic modulus and collagen organization of the rabbit cornea: Epithelium to endothelium

The rabbit is commonly used to evaluate new corneal prosthetics and study corneal wound healing. Knowledge of the stiffness of the rabbit cornea would better inform the design and fabrication of keratoprosthetics and substrates with relevant mechanical properties for in vitro investigations of corneal cellular behavior. This study determined the elastic modulus of the rabbit corneal epithelium, anterior basement membrane (ABM), anterior and posterior stroma, Descemets membrane (DM) and endothelium using atomic force microscopy (AFM). In addition, three-dimensional collagen fiber organization of the rabbit cornea was determined using nonlinear optical high-resolution macroscopy. The elastic modulus as determined by AFM for each corneal layer was: epithelium, 0.57 ± 0.29 kPa (mean ± SD); ABM, 4.5 ± 1.2 kPa, anterior stroma, 1.1 ± 0.6 kPa; posterior stroma, 0.38 ± 0.22 kPa; DM, 11.7 ± 7.4 kPa; and endothelium, 4.1 ± 1.7 kPa. The biophysical properties, including the elastic modulus, are unique for each layer of the rabbit cornea and are dramatically softer in comparison to the corresponding regions of the human cornea. Collagen fiber organization is also dramatically different between the two species, with markedly less intertwining observed in the rabbit vs. human cornea. Given that the substratum stiffness considerably alters the corneal cell behavior, keratoprosthetics that incorporate mechanical properties simulating the native human cornea may not elicit optimal cellular performance in rabbit corneas that have dramatically different elastic moduli. These data should allow for the design of substrates that better mimic the biomechanical properties of the corneal cellular environment.

The effect of biophysical attributes of the ocular trabecular meshwork associated with glaucoma on the cell response to therapeutic agents

Glaucoma is a devastating neurodegenerative disease, which can lead to vision loss and is associated with irreversible damage to retinal ganglion cells. Although the mechanism of disease onset remains unknown, we have recently demonstrated that the stiffness of the ocular trabecular meshwork (HTM) increases dramatically in human donor eyes with a history of glaucoma. Here we report that polyacrylamide hydrogels, which mimic the compliant conditions of normal and glaucomatous HTM, profoundly modulate cytoskeletal dynamics and the elastic modulus of the overlying HTM cells. Substratum compliance also modulates HTM cell response to Latrunculin-B, a cytoskeletal disrupting agent currently in human clinical trials for the treatment of glaucoma. Additionally, we observed a compliance-dependent rebound effect of Latrunculin-B with an unexpected increase in HTM cell elastic modulus being observed upon withdrawal of the drug. The results predict that cytoskeletal disrupting drugs may be more potent in advanced stages of glaucoma.

Cross‐Sectional Imaging Characteristics of Pituitary Adenomas, Invasive Adenomas and Adenocarcinomas in Dogs: 33 Cases (1988–2006)

BACKGROUND Pituitary tumors in dogs can be adenomas, invasive adenomas, or adenocarcinomas. In people, invasive adenomas and pituitary adenocarcinomas carry a worse prognosis than adenomas. HYPOTHESIS/OBJECTIVE To identify differentiating features on cross-sectional imaging in dogs with pituitary adenomas, invasive adenomas, and adenocarcinomas. ANIMALS Thirty-three dogs that had computed tomography (CT) or magnetic resonance imaging (MRI) performed and a necropsy diagnosis of pituitary adenoma (n = 20), invasive adenoma (n = 11), or adenocarcinoma (n = 2). METHODS Medical records were retrospectively reviewed for signalment, history, and diagnosis. CT and MR images were reviewed for characteristics of pituitary tumors. RESULTS Mean (+/- standard deviation) age for dogs with pituitary adenomas (10.6 +/- 2.9 years) was greater than that of those with invasive adenomas (8.3 +/- 2.7 years, P = .04). Eighteen out of 20 (90%) dogs with adenomas had contrast-enhancing masses. Thirteen out of 20 (65%) had homogeneous enhancement. Mean adenoma height was 1.2 +/- 0.7cm. Eight out of 20 (40%) adenomas were round and 8/20 (40%) compressed surrounding brain. Eleven out of 11 dogs (100%) with invasive adenomas had contrast-enhancing masses. Seven out of 11 (64%) masses were homogeneous. Mean invasive adenoma height was 1.8 +/- 0.7 cm, which was significantly greater than adenomas (P = .03). Mass shape varied from round to oval to irregular. Six out of 11 (55%) masses compressed surrounding brain. Clinical and imaging features were variable for 2 dogs with adenocarcinomas. CONCLUSIONS AND CLINICAL RELEVANCE Invasive adenoma should be suspected if a dog with a pituitary tumor is <7.7 years of age and has a mass > 1.9 cm in vertical height. Adenocarcinomas are uncommon and metastatic lesions were not seen with imaging.

Safety and immunomodulatory effects of allogeneic canine adipose-derived mesenchymal stromal cells transplanted into the region of the lacrimal gland, the gland of the third eyelid and the knee joint.

BACKGROUND AIMS Mesenchymal stromal cells (MSCs) have been extensively studied as a cellular therapeutic for various pathologic conditions. However, there remains a paucity of data regarding regional and systemic safety of MSC transplantations, particularly with multiple deliveries of allogeneic cells. The purpose of this study was to investigate the safety and systemic immunomodulatory effects of repeated local delivery of allogeneic MSCs into the region of the lacrimal gland, the gland of the third eyelid and the knee joint in dogs. METHODS Allogeneic adipose tissue-derived canine MSCs were delivered to the regions of the lacrimal gland and the third eyelid gland as well as in the knee joints of six healthy laboratory beagles as follows: six times with 1-week intervals for delivery to the lacrimal gland and the third eyelid gland regions and three to four times with 1- to 2-week intervals for intra-articular transplantations. Dogs were sequentially evaluated by clinical examination. At the conclusion of the study, dogs were humanely euthanized, and a complete gross and histopathologic examination of all organ systems was performed. Mixed leukocyte reactions were also performed before the first transplantation and after the final transplantation. RESULTS Clinical and pathologic examinations found no severe consequences after repeated MSC transplantations. Results of mixed leukocyte reactions demonstrated suppression of T-cell proliferation after MSC transplantations. CONCLUSIONS This is the first study to demonstrate regional and systemic safety and systemic immunomodulatory effects of repeated local delivery of allogeneic MSCs in vivo.

Equine Degenerative Myeloencephalopathy in Lusitano Horses

BACKGROUND Equine degenerative myeloencephalopathy (EDM) is a neurodegenerative disorder that has been previously associated with low vitamin E concentrations. OBJECTIVE To describe the clinical, electrophysiologic, and pathologic features of EDM in a group of related Lusitano horses. ANIMALS Fifteen Lusitano horses. PROCEDURES Neurologic examinations were conducted, and serum vitamin E concentrations were measured. Three neurologically abnormal horses were further evaluated by ophthalmologic examination, electroretinography, electroencephalography, muscle and nerve biopsies, and post-mortem examination. RESULTS Six horses appeared neurologically normal, 6 were neurologically abnormal, and 3 had equivocal gait abnormalities. Abnormal horses demonstrated ataxia and paresis. An inconsistent menace response was noted in 4 neurologically abnormal horses and in 1 horse with equivocal findings. All horses had low serum vitamin E concentrations (<1.5 ppm). Ophthalmologic examinations, electroretinograms, electroencephalograms, and muscle and peripheral nerve biopsies were unremarkable in 3 neurologically abnormal horses. At necropsy, major neuropathological findings in these horses were bilaterally symmetric, severe, neuro axonal degeneration in the gracilis, cuneatus medialis, cuneatus lateralis, and thoracicus nuclei and bilaterally symmetric axonal loss and demyelination mainly in the dorsolateral and ventromedial tracts of the spinal cord. A diagnosis of EDM was made based on these findings. Pedigree analysis identified 2 sires among the affected horses. CONCLUSIONS AND CLINICAL RELEVANCE Equine degenerative myeloencephalopathy is a neurodegenerative disorder that causes ataxia and, in severe cases, paresis, in young Lusitano horses. The disease appears to have a genetic basis, and although vitamin E deficiency is a common finding, low serum vitamin E concentrations also may occur in apparently unaffected related individuals.

Presumed solitary intraocular or conjunctival lymphoma in dogs and cats: 9 cases (1985–2013)

OBJECTIVE To determine prevalence, reason for evaluation, treatment, and outcome for dogs and cats with presumed solitary ocular lymphoma (PSOL). DESIGN Retrospective case series. ANIMALS 7 dogs and 2 cats with PSOL. PROCEDURES Medical records were reviewed. Progression-free survival time (PFST) and overall survival time (OST) were determined. RESULTS Animals with intraocular (4 dogs and 1 cat) or conjunctival (3 dogs and 1 cat) lymphoma represented 0.1% and 0.08% of patients with lymphoma evaluated at the hospital during the study period, respectively. Animals with intraocular lymphoma represented 0.19% of all patients with uveitis; animals with conjunctival lymphoma represented 0.16% of all patients with conjunctivitis. Tumors included B-cell (2 intraocular and 1 conjunctival), non-B-cell, non-T-cell (1 intraocular), and T-cell (3 conjunctival) neoplasms; immunophenotype of 2 uveal lymphomas was not determined. Treatments included enucleation (4 intraocular) and chemotherapy (3 intraocular and 2 conjunctival). All dogs with intraocular lymphoma developed neurologic signs. Lymph node metastasis was detected in 2 patients with conjunctival lymphoma. Median PFST and OST were 178 days for all animals with PSOL, dogs with PSOL, and animals with intraocular lymphoma. Median PFST and OST for animals with conjunctival lymphoma were 221 and 549 days, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated PSOL was uncommon, but should be considered a differential diagnosis for animals with uveitis or conjunctivitis. Performance of MRI and cytologic analysis of CSF and regional lymph node aspirate samples may be beneficial for such patients. Prognosis seemed to be better for animals with conjunctival lymphoma than it was for those with intraocular lymphoma.

Immunohistochemical Characteristics of Normal Canine Eyes

Immunohistochemistry is widely utilized in diagnostic laboratories to study neoplastic and nonneoplastic diseases. Knowledge of the immunohistochemical characteristics of normal tissue is essential for interpretation of immunoreactivity in pathologic conditions. In this study, immunohistochemistry was performed with a broad panel of diagnostically relevant antibodies on 4 normal canine globes—namely, vimentin, pan-cytokeratin (AE1/AE3), cytokeratin 7, cytokeratin 8/18, cytokeratin 20, α–smooth muscle actin, muscle specific actin, desmin, Melan-A, microphthalmia transcription factor, S-100, glial fibrillary acidic protein, triple neurofilaments, neuron-specific enolase, chromogranin A, synaptophysin, laminin and CD31. Results include cytokeratin immunoreactivity limited to the conjunctival epithelium, corneal epithelium, and retinal pigment epithelium; distinct patterns of immunopositivity of muscle markers; and widespread immunoreactivity for vimentin and most neural/neuroendocrine markers. These findings in normal eyes provide the basis for interpretation of ocular immunohistochemistry in dogs. Published immunophenotypes of primary ocular neoplasms are also reviewed.

Aggressive local therapy combined with systemic chemotherapy provides long‐term control in grade II stage 2 canine mast cell tumour: 21 cases (1999–2012)

This retrospective case series evaluates the outcome of 21 dogs with grade II stage 2 mast cell tumour (MCT) treated with adequate local therapy and adjuvant systemic chemotherapy (prednisone, vinblastine and CCNU). The median survival for all dogs was 1359 days (range, 188-2340). Median disease-free interval was 2120 days (149-2325 days). Dogs treated with surgery and chemotherapy had shorter survival (median, 1103 days; 188-2010 days) than those that underwent surgery, radiation therapy and chemotherapy as part of their treatment (median, 2056 days; 300-2340 days). Two patients had local recurrence in the radiation field and four patients had de novo MCT. Distant metastasis was not observed in any dogs. The results of this study suggest that, in the presence of loco-regional lymph node metastasis in grade II MCT, the use of prednisone, vinblastine and CCNU after adequate local-regional therapy can provide a median survival in excess of 40 months.

Histologic and immunohistochemical predictors of clinical behavior for feline diffuse iris melanoma

OBJECTIVE To determine histologic and immunohistochemical predictors of metastasis of feline diffuse iris melanoma (FDIM). ANIMALS Globes from 47 client-owned cats enucleated for FDIM between January 1985 and December 2013. PROCEDURES Hematoxylin and eosin-stained sections were evaluated for neoplastic invasiveness and cell morphology, necrosis within the neoplasm, inflammation, and glaucoma. Sections were immunolabeled with antibodies against melan-A, PNL2, E-cadherin, or B-Raf, and label intensity, percentage of labeled cells, and label homogeneity were semi-quantitatively graded. Medical records were evaluated, and referring veterinarians and clients were contacted to determine whether cats developed metastasis following enucleation. The log-rank test or Cox proportional hazards model was used to determine associations between histologic or immunohistochemical parameters and metastasis. RESULTS Metastasis was suspected or confirmed in 9/47 (19%) cats. Extrascleral extension, necrosis within the neoplasm, a mitotic index of >7 mitoses in 10 high-power (×400) fields, choroidal invasion, and increased E-cadherin and melan-A label intensity were each associated with increased rate of metastasis. PNL2 label homogeneity was associated with decreased rate of metastasis. Decreased PNL2 label intensity and an increasing percentage of neoplastic cells labeled for melan-A each approached significance for increased rate of metastasis. CONCLUSIONS We report four histologic and three immunohistochemical parameters helpful in determining cats at risk of metastasis of FDIM. Further studies should determine if B-Raf mutations identified in human malignant melanomas are found in cats with FDIM and assess benefits of adjunctive therapy following enucleation of eyes with FDIM bearing poor prognostic indicators.