C-kit,flt-3, PDGFR-β, and VEGFR2 expression in canine adrenal tumors and correlation with outcome following adrenalectomy

The objective of this retrospective study was to evaluate the expression of receptor tyrosine kinases (RTKs) in canine adrenal tumors and correlate this expression with features of tumor aggressiveness and survival in dogs undergoing adrenalectomy.Forty-three canine adrenal tumors were evaluated for expression of c-kit, fms-like tyrosine kinase 3 (flt-3), platelet-derived growth factor receptor-β (PDGFR-β), and vascular endothelial growth factor receptor 2 (VEGFR2) using immunohistochemistry. Tumor RTK staining characteristics were compared to normal adrenals. Medical records were reviewed for data regarding patient outcome and tumor characteristics. Expression of c-kit, flt-3, PDGFR-β, and VEGFR2 was detected in 26.9%, 92.3%, 96.2%, and 61.5% of cortical tumors and 0%, 63.2%, 47.4%, and 15.8% of pheochromocytomas, respectively. Expression of RTKs was not significantly increased when compared to normal adrenals and did not correlate with survival after adrenalectomy. Receptor tyrosine kinases are not overexpressed in canine adrenal tumors compared to normal adrenal tissue. Therapeutic inhibition of these receptors may still represent an effective approach in cases where receptor activation is present.     

Expression of receptor tyrosine kinases VEGFR-1 (FLT-1), VEGFR-2 (KDR), EGFR-1, PDGFRa and c-Met in canine primary brain tumours

Inhibition of tumour growth and angiogenesis by targeting key growth factor receptors is a promising therapeutic strategy for central nervous system tumours. Characterization of these growth factor receptors in canine primary brain tumours has not been done. Using quantitative real‐time TaqMan polymerase chain reaction (PCR), we evaluated the expression of messenger RNA (mRNA) for five tyrosine kinase growth factor receptors (vascular endothelial growth factor receptor [VEGFR]‐1, VEGFR‐2, endothelial growth factor receptor [EGFR]‐1, platelet‐derived growth factor receptor a [PDGFRa], and c‐Met) relative to normal cerebral cortex in 66 spontaneous canine primary brain tumours. Increased expression of VEGFR‐1 and VEGFR‐2 mRNA was greatest in grade IV astrocytomas (glioblastoma multiforme) and grade III (anaplastic) oligodendrogliomas. EGFR‐1 mRNA expression was more consistently increased than the other receptors in all tumour types, while increased PDGFRa mRNA expression was mostly restricted to oligodendrogliomas. The similarities in increased expression of these tyrosine kinase growth factor receptors in these canine tumours, as compared to data from their human counterparts, suggest that common molecular mechanisms may be present.     

Cullen frontal sinus valved glaucoma shunt: preliminary findings in dogs with primary glaucoma

PURPOSE: To evaluate the efficacy of a novel, professionally manufactured, frontal sinus valved glaucoma shunt in maintaining normal intraocular pressure (IOP) and vision in dogs with primary glaucoma. METHODS: Three eyes of three dogs diagnosed with primary glaucoma were included in this prospective clinical study. A Cullen frontal sinus valved glaucoma shunt was implanted into each glaucomatous globe. Dogs were treated postoperatively with topical neomycin/polymyxin B/0.1% dexamethasone and 0.03% flurbiprofen every 6 h tapered over 8-12 weeks, and meloxicam at 0.1 mg/kg orally every 24 h for 7-10 days. IOP, intracameral shunt position and apparent patency, and vision were assessed twice daily for up to 4 (n= 3 eyes) and 10 (n= 2 eyes) days postoperatively, and then at re-examination periods of up to 36 weeks (n= 1 eye). Postoperative complications were recorded and documented photographically. RESULTS: Normal IOP was maintained in all shunted globes (range 10-29 mmHg; mean = 16.7 mmHg at 24 h; IOP = 23 mmHg at 36 weeks) postoperatively for 2 days (3/3 eyes), 8 weeks (2/2 eyes), and 36 weeks (1/1 eye) without additional antiglaucoma therapies. Photopic vision and shunt position and patency were maintained in all shunted globes for all follow-up periods. Postoperative complications included mild aqueous flare and fibrin (n= 3 eyes for 3-10 days postoperatively); intracameral shunt occlusion with fibrin (n= 1 eye at days 2 and 4); partial anterior chamber tube extrusion (n= 1 eye at day 4), and focal corneal edema (n= 1 eye at 18 weeks). Tissue plasminogen activator injected intracamerally through the silicone tube near the frontal sinus effectively resolved the fibrinous shunt occlusion. CONCLUSIONS: The Cullen frontal sinus valved glaucoma shunt shows promise for the management of canine primary glaucoma.     

The pathogenesis and significance of pre-iridal fibrovascular membrane in domestic animals

Histologic examination was made of 1,419 globes from domestic animals (964 dogs, 374 cats, 41 horses, and 40 cattle) with ocular disease; pre-iridal membranes (rubeosis iridis) were found in 98. The membranes originated as endothelial budding from the anterior iridal stroma and seemed to mature into fibrous or fibrovascular membranes that were often followed by hyphema or, occasionally, glaucoma. Pre-existent disease in the 98 affected globes included chronic endophthalmitis (27/98), chronic glaucoma (24/98), anterior uveal melanoma (15/98), ciliary body adenoma (14/98), neoplasms metastatic to the eye (8/98), and chronic retinal detachment (6/98). In terms of likelihood of occurrence, pre-iridal membranes seen in 21% (6/21) of globes with retinal detachment, 19% (14/75) of those with ciliary body adenomas, 14% (24/167) of those with chronic glaucoma, and 10% (15/158) of those with anterior uveal melanoma. They were detected with greatest relative frequency in horses (9/41) followed by dogs (83/964), cats (5/374) and cattle (1/40). These membranes, which are rarely detected by clinical examination, probably form in response to angiogenic factors released by ischemic retina, by neoplasms, or by leukocytes involved in ocular inflammation.     

Mast cell numbers in normal and glaucomatous canine eyes

Numbers of mast cells in the cornea, sclera, choroid, ciliary body, iris, and retina of sections of globes from 35 clinically normal dogs and 34 dogs with secondary glaucoma was determined. Fixed globes were trimmed along a vertical midsagittal plane and embedded in paraffin. Tissue sections, approximately 6 microns thick, were stained with toluidine blue for identification of mast cells. In normal globes, most of the mast cells were observed in the anterior portion of the uvea, and fewer mast cells were seen in the choroid and sclera. Mast cells were not observed in the retina and were seldom observed in the cornea of dogs with or without glaucoma. In sections of glaucomatous globes, mast cells were distributed evenly in the uvea and sclera, and fewer mast cells were present than in normal globes, regardless of the cause of glaucoma.     

Expression of VEGFR and PDGFR‐α/‐β in 187 canine nasal carcinomas

Radiotherapy represents the standard of care for intranasal carcinomas. Responses to tyrosine kinase inhibitors (TKIs) have been reported but data on expression of target receptor tyrosine kinases (rTKs) is limited. This study characterizes the expression of vascular endothelial growth factor receptor (VEGFR), platelet‐derived growth factor receptor (PDGFR)‐α and PDGFR‐β in canine intranasal carcinomas. Histological samples from 187 dogs were retrieved. Immunohistochemistry was performed using commercially available antibodies. Expression of rTKs was classified into weak, moderate or intense and additionally recorded as cytoplasmic, membranous, cytoplasmic‐membranous, nuclear or stromal. VEGFR was expressed in 158 dogs with predominantly moderate expression (36.9%) and a cytoplasmic‐membranous expression pattern (70.9%). PDGFR‐α was detected in 133 with predominantly weak expression (57.9%) and cytoplasmic pattern (87.9%). PDGFR‐β was identified in 74 patients with a predominantly moderate expression (17.6%) and cytoplasmic expression pattern (63.5%). Co‐expression of rTKs was common. These results confirm expression of VEGFR, PDGFR‐α and PDGFR‐β in canine intranasal carcinomas and support the utility of TKIs.     

The immunohistochemical detection of peroxiredoxin 1 and 2 in canine spontaneous vascular endothelial tumors

Peroxiredoxin (PRDX) is an antioxidant enzyme family with six isoforms (PRDX1–6). The main function of PRDXs is to decrease cellular oxidative stress by reducing reactive oxygen species, such as hydrogen peroxide, to H₂O. Recently, it has been reported that PRDXs are overexpressed in various malignant tumors in humans, and are involved in the development, proliferation, and metastasis of tumors. However, studies on the expression of PRDXs in tumors of animals are limited. Therefore, in the present study, we immunohistochemically investigated the expression of PRDX1 and 2 in spontaneous canine hemangiosarcoma (HSA) and hemangioma (HA), as well as in selected normal tissue and granulation tissue, including newly formed blood vessels. Although there were some exceptions, immunolocalization of PRDX1 and 2 in normal canine tissues was similar to those in humans, rats, or mice. In granulation tissue, angiogenic endothelial cells were strongly positive for PRDX1 and 2, whereas quiescent endothelial cells in mature vessels were negative. Both PRDX1 and 2 were significantly highly expressed in HSA compared to HA. There were no significant differences in the expression of PRDX1 and 2 among the subtypes and primary sites of HSA. These results suggest that PRDX1 and 2 may be involved in the angiogenic phenotypes of endothelial cells in granulation tissue as well as in the behavior in the malignant endothelial tumors.     

Detection of indoleamine 2,3-dioxygenase 1-expressing cells in canine normal and tumor tissues

Cancer immunotherapy is a novel cancer treatment for canine tumors. Indoleamine 2,3-dioxygenase 1 (IDO1) is overexpressed in some human tumors and inhibits antitumor immunity. In this study, we comprehensively evaluated expression pattern of IDO1 and the nature of IDO1-expressing cells in canine normal and tumor tissues. In normal tissue samples, IDO1 expression was detected only in the lymph nodes, spleen, tonsil tissues, and colon tissues. In contrast, IDO1-positive tumor cells were observed in several tumor tissue types. This is the first study to evaluate IDO1 expression in canine normal and tumor tissues, and the results suggest that IDO1 is a promising target for novel cancer immunotherapy in dogs with tumors.     

Canine pre-iridal fibrovascular membranes: morphologic and immunohistochemical investigations

Objective  Pathologic intraocular neovascularization is a key component of many canine ophthalmic diseases such as uveitis, retinal detachment, intraocular neoplasms, and corneal perforation. The purpose of this study was to evaluate the structure of pre-iridal fibrovascular membranes (PIFMs) associated with several different disease processes and to identify specific factors associated with their development in the canine eye.
Procedure  This study examined 36 enucleated canine eyes with the diagnosis of PIFM and one of the following: lens-induced uveitis, retinal detachment, iridociliary adenoma, corneal perforation, severe hyphema, or vitreal gliovascular membranes (canine ocular gliovascular syndrome, COGS). Three histologic stains and six immunohistochemical stains were performed in all 36 PIFM eyes and four histologically normal eyes, including: hematoxylin and eosin, alcian blue periodic acid schiff (PAS), Masson's trichrome, platelet endothelial cell adhesion molecule-1 (CD31), smooth muscle actin, vimentin, laminin, vascular endothelial growth factor (VEGF), and cyclooxygenase-2 (COX-2).
Results  Pre-iridal fibrovascular membrane extracellular matrix staining was consistent with collagen and mucins in all cases and positive for laminin in most cases. All PIFMs contained CD31-positive vessels and predominantly lymphoplasmacytic inflammation. Both PIFM vessels and spindle cells were positive for laminin, vimentin, smooth muscle actin, VEGF, and COX-2. Secondary intraocular pathology and immunohistochemical staining of other intraocular structures are also reported.
Conclusions  Pre-iridal fibrovascular membrane morphology and immunohistochemical characteristics were similar across six canine disease processes, suggesting analogous pathophysiologic mechanisms. COX-2 and VEGF were identified using immunohistochemistry and may play a role in PIFM development.     

Uveitic glaucoma in the horse

Glaucoma is a serious complication of anterior uveitis that can result in significant ocular discomfort and loss of vision. As glaucoma is insidious and mostly asymptomatic until late in the course of disease, it is often undiagnosed. Equine recurrent uveitis is the most common underlying disorder: it is a disease of high prevalence, characterised by recurrent‐remitting episodes of ocular inflammation. Here, the inflammatory cells and mediators they release alter the normal anatomic structure of the anterior chamber and aqueous drainage pathways of the eye. These changes alter the homeostatic mechanisms of intraocular pressure control. Management of glaucoma secondary to uveitis can be challenging due to the difficulties of managing the underlying disorder and its effects on the eye. This article reviews the pathogenesis of uveitic glaucoma in the horse and medical and surgical management options.     

Comparative Doppler imaging of the ophthalmic vasculature in normal Beagles and Beagles with inherited primary open-angle glaucoma

The objective of this study was to compare orbital and ocular vasculature velocity, measured by Doppler imaging, in normal Beagles and Beagles with inherited primary open-angle glaucoma. Eight normal Beagles and 13 Beagles with different stages of primary open-angle glaucoma were evaluated twice with a 2–4-week period between measurements. Doppler imaging was performed with the dogs anesthetized, and the Doppler transducer applied directly on the corneal surface. The majority of the orbital vasculature (external ethmoidal artery; internal ophthalmic artery and vein; and external ophthalmic artery and vein) and ocular blood vessels (anterior ciliary artery and veins; long posterior ciliary arteries; short posterior ciliary arteries; primary retinal arteries; and the vortex veins) were identified and Doppler blood velocity parameters were determined. The glaucomatous dogs demonstrated significant differences in the Doppler velocity parameters of several orbital vessels (external ethmoidal, external ophthalmic, and internal ophthalmic arteries), and several ocular vessels (anterior ciliary, short posterior ciliary, and long posterior ciliary arteries). These differences included decreased blood velocities, and increased pulsatility and resistive indexes. The Doppler blood flow velocities of the primary retinal arteries were unchanged between the normal and glaucomatous dogs. In the glaucomatous dogs, the Doppler imaging suggests increased vascular resistance downstream in both the orbital and ocular vasculature. These blood velocity parameter changes may be primary or secondary, and may offer therapeutic opportunities to increase perfusion, prolong the retina and optic nerve head function, and maintain vision in the canine glaucomas.     

Localization of smooth muscle actin in the iridocorneal angle of normal and spontaneous glaucomatous beagle dogs

Purpose To date, our knowledge of the canine trabecular meshwork (TM) with regard to contractility is incomplete. It is important to understand the potential contractile capability within the TM and possible changes associated with spontaneous hypertensive glaucoma. To that end we have examined the presence of actin, including smooth muscle (SM) actin, in the normal and glaucomatous canine iridocorneal angle (ICA) morphologically and immunohistochemically. Methods Sections from the ICAs of 12 Beagles with inherited glaucoma (3 months to 6 years old) and age‐matched normal Beagles were treated with target retrieval, protein and power blocked and sequentially incubated with the primary antibody (rat anticanine SM actin) and the secondary antibody (rabbit antirat immunoglobulin), followed by peroxidase labeled streptavidin and incubation with substrate‐chromogen solution (AEC). Smooth muscle fibers that lined an artery within canine heart tissue were used as positive controls. Separate specimens were prepared for ultrastructual observation. Results Ultrastructurally, cells within the inner, posterior region of the corneoscleral TM and outer, posterior region of the uveal TM contained many microfilaments, 6 nm in diameter (i.e. actin). Immunohistochemistry demonstrated that cells within these regions possessed SM actin, having been greatest posteriorly, but extended anteriorly to a lesser extent. In the preglaucomatous affected dog the localization pattern for SM actin was identical to that seen in the normal dogs. With the progression of the disease the pattern disappeared. Conclusions The interior presence of myofibroblastic cells within the canine ICA suggests that these cells and the smooth muscle cells of the ciliary body along the same plane of orientation function to facilitate the removal of aqueous humor and are likely to be influenced by vascular mediators. The contractile apparatus for the ICA in the dog with inherited glaucoma appeared identical to that of the normal dog prior to expression of the disease, but weakened as the disease progressed.     

Canine ocular onchocerciasis in the United States: two new cases and a review of the literature

Since 1991, 53 cases of canine ocular onchocerciasis have been reported in the literature worldwide, 43 of these were from Greece, five from Hungary, and five from the western United States. Information on the histopathologic features of canine ocular onchocerciasis is limited. We describe the histopathologic features of canine ocular onchocerciasis in two dogs from California that presented clinically with firm episcleral nodules and uveitis unilaterally. Pertinent literature and pathogenesis are reviewed; recognizable clinical features and treatment are discussed. The cases presented were diagnosed via histopathology of the enucleated globes and episcleral granulomas at the Comparative Ocular Pathology Laboratory of Wisconsin (COPLOW). Positive identification of adult Onchocerca within episcleral granulomas was made based on light-microscopy features. Histopathologic examination of both globes revealed episcleral parasites surrounded by granulomas containing few to moderate numbers of eosinophils. Other sequelae, in both cases, included lymphoplasmacytic uveitis, preiridal fibrovascular membranes, peripheral anterior synechiae, retinal degeneration, and optic nerve head cupping. Both male and female worms were present, as were in utero microfilariae in both cases. Worms in both cases were tentatively identified as Onchocerca lienalis. Ocular onchocerciasis should be a differential consideration in cases of canine conjunctival nodules or periorbital swelling, particularly in dogs from the western United States.     

Radiation up-regulates the expression of VEGF in a canine oral melanoma cell line

To evaluate radiosensitivity and the effects of radiation on the expression of vascular endothelial growth factor (VEGF) and VEGF receptors in the canine oral melanoma cell line, TLM 1, cells were irradiated with doses of 0, 2, 4, 6, 8 and 10 Gray (Gy). Survival rates were then determined by a MTT assay, while vascular endothelial growth factor receptor (VEGFR)-1 and -2 expression was measured by flow cytometry and apoptotic cell death rates were investigated using an Annexin assay. Additionally, a commercially available canine VEGF ELISA kit was used to measure VEGF. Radiosensitivity was detected in TLM 1 cells, and mitotic and apoptotic cell death was found to occur in a radiation dose dependent manner. VEGF was secreted constitutively and significant up-regulation was observed in the 8 and 10 Gy irradiated cells. In addition, a minor portion of TLM 1 cells expressed vascular endothelial growth factor receptor (VEGFR)-1 intracellularly. VEGFR-2 was detected in the cytoplasm and was down-regulated following radiation with increasing dosages. In TLM 1 cells, apoptosis plays an important role in radiation induced cell death. It has also been suggested that the significantly higher VEGF production in the 8 and 10 Gy group could lead to tumour resistance.     

Vascular endothelial growth factor expression in canine intracranial meningiomas and association with patient survival

BACKGROUND: Vascular endothelial growth factor (VEGF) is a regulator of angiogenesis and vascular permeability. In human patients with meningiomas, increased VEGF expression is predictive of postsurgical recurrence. The objectives of this study were to evaluate VEGF expression in canine intracranial meningiomas and to determine whether an association between VEGF expression and patient survival existed. METHODOLOGY: Tumor tissue from 17 dogs with histologically confirmed intracranial meningiomas was obtained surgically. All dogs then were treated with radiotherapy. Immunohistochemistry was performed on 5-microm sections of paraffin-embedded tumor tissue with rabbit anti-human VEGF polyclonal antibody. The extent, intensity, and distribution of VEGF staining for each section were assessed with light microscopy by means of a semiquantitative scale. Survival was analyzed by the Kaplan-Meier procedure. Survival rates among groups were compared by log-rank tests with the significance set at P < or = .05. FINDINGS: VEGF expression was detected in all tumors, with >50% of cells staining positively in tissues from 15/17 dogs. Shorter survival times were associated with greater VEGF expression (P = .01). CONCLUSIONS: VEGF expression can be measured in canine intracranial meningiomas and may be associated with poor outcome. SIGNIFICANCE: The extent of VEGF expression in canine intracranial meningiomas may be used as a prognostic marker and suggests a potential future target for therapy.     

Solid intraocular xanthogranuloma in three Miniature Schnauzer dogs

OBJECTIVE: Macrophages that contain abundant intracytoplasmic lipid are called 'foam cells'. In four canine globes submitted to the Comparative Ocular Pathology Laboratory of Wisconsin (COPLOW), foam cells formed a solid intraocular mass. The purpose of this study was to describe the histopathologic findings in these cases. PROCEDURE: The electronic COPLOW database (1993-2006) was searched for the diagnosis of 'foam cell tumor'. Clinical history, gross pathology and histopathology (5-micron sections, hematoxylin and eosin and Alcian blue periodic acid Schiff) were reviewed in all cases. Cases were included if the globe was grossly filled by a solid mass and if all intraocular structures were effaced by lipid-laden foam cell macrophages admixed with birefringent, Alcian blue-positive crystals oriented in stellate patterns. RESULTS: All three patients (four globes) satisfying the selection criteria were Miniature Schnauzers. In all cases the clinical history included diabetes mellitus, hyperlipidemia and chronic bilateral uveitis that was interpreted to be lens-induced. All globes were enucleated because of glaucoma. CONCLUSIONS: The term solid intraocular xanthogranuloma was used to describe these cases because the intraocular contents were effaced by a solid mass of foam cells and birefringent crystals. The cases in this report suggest that diabetic Miniature Schnauzers with hyperlipidemia are at risk for lipid and macrophage-rich uveitis, which may in some cases form a solid inflammatory intraocular mass, precipitate glaucoma, and lead to enucleation.     

Immunohistochemical analysis of lens epithelial-derived membranes following cataract extraction in the dog

The objective of the study was to characterize the morphologic and immunohistochemical features of lens epithelial-derived proliferative membranes from the anterior segment of canine globes. These features were correlated with those previously identified for diseases resulting from lens epithelial cell (LEC) proliferation including posterior capsular opacification, traumatic subcapsular cataract, and subcapsular plaques associated with hypermature cataracts. Sixteen canine globes were removed as a result of glaucoma or other complications following cataract extraction. Light microscopic and immunohistochemical analysis was performed on sections from formalin-fixed, paraffin-embedded globes. The tissues were stained with a variety of antibodies for cellular markers for LECs, growth factors or other cellular constituents relevant to cellular metaplasia and proliferation. The membranes were composed of monolayers or multilayers of spindle-shaped cells on the external surfaces of the anterior and posterior lens capsule, ciliary processes, iris leaflets, and iridocorneal angle, and they could be seen extending from an obvious monolayer of LEC within the capsular sac. Variably, scattered pigment cells, presumably of uveal origin, were concurrently present. Cellular components of the membranes stained positive for vimentin, transforming growth factor-beta, basic fibroblast growth factor, and smooth muscle actin. An amorphous eosinophilic extracellular matrix consisting predominately of collagen was associated with the membranes. Proliferative anterior segment membranes following cataract surgery were morphologically and immunohistochemically similar to cellular and matrix components of posterior capsular opacification and capsular plaques seen with hypermature cataracts, both of which result from metaplasia and proliferation of LEC. The presence of these LEC-derived membranes in association with secondary glaucoma suggests that exuberant proliferation of LEC outside the confines of the lens capsular sac may cause pathologic alterations in the eye following cataract surgery in the dog.     

Cellular characterization of multidrug resistance P-glycoprotein, alpha fetoprotein, and neovascular endothelium-associated antigens in canine hepatocellular carcinoma and cirrhotic liver

P-glycoprotein (P-gp), which is encoded by the multidrug resistance gene (MDR-1); alpha fetoprotein (AFP); and vascular endothelium-associated antigens are well-known markers for human and canine hepatic diseases. We obtained liver tissues from 5 dogs with hepatocellular carcinoma (HCC) and 12 dogs with cirrhosis, and we performed histopathologic and immunohistochemical evaluations using anti-P-gp, anti-AFP, anti-CD31, and anti-CD34 antibodies. P-gp was expressed at higher levels in HCC than in cirrhotic livers ( P < .01), and was most commonly localized in biliary canaliculi and small ductuli. AFP was localized mainly in the cytoplasm in HCC ( P < .01) and in a few cases of cirrhosis. In both HCC and cirrhosis, the AFP-positive cells were morphologically similar to normal hepatocytes and showed an even cytoplasmic distribution of AFP. The endothelial markers CD31 and CD34 were used to investigate vascular distribution. CD31 was expressed strongly in the portal area and parenchyma in HCC, but it was rarely observed in the parenchyma in cirrhosis. CD34 expression could not be detected in both HCC and cirrhosis. This study constitutes the first comprehensive study of P-gp, AFP, and endothelial markers in canine HCC and cirrhosis. The importance of these markers in HCC and cirrhosis in dogs was demonstrated and provides a more accurate basis for a definitive diagnosis of HCC and cirrhosis in dogs.     

Tarantula cubensis extract alters the degree of apoptosis and mitosis in canine mammary adenocarcinomas

In the present study, 13 clinical cases of canine mammary adenocarcinoma were evaluated in order to understand the effect of Tarantula cubensis extract (TCE) on tumor tissue. Punch biopsies were taken from the tumors before treatment with TCE. Subcutaneous injections of TCE were administered three times at weekly intervals (3 mL per dog). Between days 7 and 10 after the third injection, the tumor masses were extirpated by complete unilateral mastectomy. Pre- and post-treatment tumor tissues were immunohistochemically assessed. The expression of B-cell lymphoma 2 (Bcl-2) was found to be higher in pre-treatment compared to post-treatment tissues (p < 0.01) whereas Ki-67 expression was lower in post-treatment tissues (p < 0.01). No significant differences in fibroblast growth factor or vascular endothelial growth factor expression were observed between pre- and post-treatment tissues (p > 0.05). The apoptotic index was determined to be low before treatment and increased during treatment. These results suggest that TCE may be effective for controlling the local growth of canine mammary adenocarcinoma by regulating apoptosis.