VEGF and MMP‐9: biomarkers for canine lymphoma

Vascular endothelial growth factor (VEGF) and metalloproteinase (MMP) 2 and 9 are useful biomarkers in human lymphoma. During cancerogenesis, transforming growth factor beta (TGF‐β) stimulates VEGF and MMPs production. VEGF and TGF‐β plasma levels were tested by ELISA, MMP‐2 and MMP‐9 by gelatine zymography in 37 dogs with lymphoma, 13 of which were also monitored during chemotherapy. Ten healthy dogs served as control. Lymphoma dogs showed higher act‐MMP‐9 (P < 0.01) and VEGF (P < 0.05), and lower TGF‐β than controls, and a positive correlation between act‐MMP‐9 and VEGF (P < 0.001). Act‐MMP‐9 and VEGF were significantly higher in T‐cell lymphomas, and in stage V compared with stages III–IV disease, regardless of immunophenotype. VEGF was higher in high‐grade compared with low‐grade T‐cell lymphomas. No correlation was found between cytokines levels at presentation and outcome. During chemotherapy, act‐MMP‐9 and VEGF decreased in B‐cell lymphomas (P < 0.01), suggesting a possible predictive role in this group of dogs.     

Immunohistochemical study of matrix metalloproteinase‐3 (MMP‐3) at the articular cartilage in osteochondrotic pigs

In order to understand the mechanism of osteochondrosis in the pig, articular cartilage was taken from the distal femoral condyles of Duroc pigs exhibiting leg weakness and then examined immunohistochemically for the localization of matrix metalloproteinases‐3 (MMP‐3), one of the enzymes involved in the resolution of cartilage matrix. The articular cartilage had the typical characteristics of osteochondrotic lesions, such as abnormal calcification, clefts of cartilage, disappearance of proteoglycan, and necrotic chondrocytes. The immunoreaction of MMP‐3 was observed in chondrocytes at the boundary between normal cartilage and proteoglycan‐deficient area. Moreover, chondrocytes expressing MMP‐3 showed normal morphology, but the surrounding cartilage matrix did not stain with toluidine blue, which indicated a lack of glycosaminoglycans. These results suggest that MMP‐3 is highly involved in the appearance and expansion of osteochondrotic lesions.     

Collagen,glycosaminoglycans and matrix metalloproteinase‐2 and metalloproteinase‐9 in the cervix of the ewe during prepubertal development

The tortuous nature of the ovine cervix restricts the transcervical passage of the cannula, and many studies have aimed to understand the endocrine mechanism of the remodelling of cervical tissue in adult ewe. However, little is known about the remodelling of the cervical tissue during the prepubertal development of the lambs. To obtain histochemical and biochemical evidence about the nature of the prepubertal development of the cervix of the ewe, cervices of Corriedale lambs obtained at 0, 1, 2, 4, 6 and 8 months of age (n = 5 to 6 in each) were processed. Neutral and acidic glycosaminoglycans (by PAS‐Alcian stain) were weakly in the cervical stroma and not shown change during the development, whereas the percentage volume of fibrillar collagen (by van Gieson stain) increases throughout the experimental period in the superficial fold stroma and deep wall stroma (p < 0.05). The relative cervical weight (g/kg of body weight) and the collagen concentration (by spectrophotometry, mg/mg wet tissue) showed an early decreasing phase from months 0 to 4 and a later increasing phase from months 4 to 8 (p < 0.05). The latent form of matrix metalloproteinase‐2 (MMP‐2) detected by gelatin zymography (ng/mg protein) decreased from months 0 to 2 and increased from months 4 to 8, whereas the activated form decreased from months 0 to 2, remained low until month 6 and then recovered on month 8 (p < 0.0001). Data suggest that the relative cervical weight biphasic pattern during the development is related to MMP‐2‐dependent changes in the collagen content.     

Expression and the molecular forms of neutrophil gelatinase‐associated lipocalin and matrix metalloproteinase 9 in canine mammary tumours

Neutrophil gelatinase‐associated lipocalin (NGAL) is a new biomarker for renal injury. It is also involved in tumorigenesis of different human cancer types. The oncogenic role of NGAL is related to its molecular forms, and heterodimer formation with matrix metalloproteinase 9 (MMP9) promotes human breast cancer (HBC) invasion and metastasis. To date, the levels of NGAL and NGAL/MMP9 complex have not yet been explored in canine mammary tumours (CMTs). Hence, this study aimed to investigate whether NGAL and its molecular forms could be the biomarker for CMT diagnosis. To this end, expression profile of NGAL and MMP9 in mammary epithelial cells as well as in urine samples were detected. By immunohistochemistry staining, NGAL was expressed at variable levels. Unlike HBC, a significant reduction in NGAL expression was demonstrated in benign and malignant CMTs as compared with normal controls. Additionally, NGAL expression was significantly reduced in dogs with metastatic CMTs. By contrast, the mean score of MMP9 expression in ascending order was normal groups, benign, and malignant CMTs. Interestingly, analysis of the molecular form revealed the NGAL/MMP9 complex presents in most mammary tissues and urine of dogs with benign or malignant CMTs, whereas the complex was absent in samples from dogs without CMTs. In conclusion, NGAL and MMP9 are ubiquitously expressed in canine mammary epithelial cells in normal and cancerous status. However, the NGAL/MMP9 complex exclusively presents in mammary tissues and urine of dogs with tumours.     

Increased expression of tissue inhibitor of metalloproteinase‐1 correlates with improved outcome in canine cutaneous mast cell tumours

Canine mast cell tumour (MCT) is a biologically heterogeneous disease. The extracellular matrix degradation promoted by matrix metalloproteinases (MMPs) has been studied in an attempt to elucidate the mechanisms involved in the biological behaviour of tumours. The aim of this study was to characterize the expression of MMP‐2 and ‐9 and tissue inhibitors of metalloproteinase (TIMP)‐1 and ‐2 in canine cutaneous MCTs and to evaluate their prognostic values. Immunohistochemical staining for MMP‐2, MMP‐9, TIMP‐2 and TIMP‐1 was performed in 46 canine cases of MCTs. TIMP‐1 expression showed an independent prognostic value for post‐surgical survival and disease‐related mortality. Dogs with MCTs showing less than 22.9% mast cell TIMP‐1 positivity were more prone to die because of the disease and had a shorter post‐surgical survival. This article suggests the involvement of TIMP‐1 in MCT progression, by contributing to a good outcome in patients with MCTs.     

Large anaplastic spinal B‐cell lymphoma in a cat

Abstract: A 5‐year‐old female spayed domestic shorthair cat was presented for evaluation of tetraparesis. The neurologic lesion was localized to the cervical spinal segment (C1–C6). A left axillary mass was identified, and the results of fine needle aspiration cytology indicated malignant round cell neoplasia of possible histiocytic origin. The cells were large, had marked anisocytosis and anisokaryosis, occasional bi‐ and multinucleation, and cytoplasmic vacuolation. Euthanasia was performed due to the poor prognosis associated with severe, progressive neurologic signs and a malignant neoplasm. Postmortem examination revealed spinal cord compression and an extradural mass at the C1–C2 spinal segment, with neoplastic cells in the adjacent vertebral bodies, surrounding skeletal muscle, left axillary lymph node, and bone marrow from the right femur. The initial histologic diagnosis was anaplastic sarcoma, but immunohistochemical results indicated the cells were CD20+ and CD45R+ and CD3?, compatible with a diagnosis of B‐cell lymphoma. CD79a staining was nonspecific and uninterpretable. Weak to moderate CD18 positivity and E‐cadherin positivity were also observed. Clonality of the B‐cell population could not be demonstrated using PCR testing for antigen receptor gene rearrangement. To the authors' knowledge, this is the first reported case of a feline spinal anaplastic B‐cell lymphoma exhibiting bi‐ and multinucleated cells. The prognostic significance of this cell morphology and immunophenotype is unknown.     

Efficacy of chemotherapy and palliative hypofractionated radiotherapy for cats with nasal lymphoma

Nasal lymphoma (NL) is the most common nasal tumor in cats, and radiotherapy, chemotherapy, or a combination of these treatments have been described as the treatment for this disease. However, the previous studies included various machines and protocols of radiotherapy. Therefore, we aimed to retrospectively compare the prognosis among cases treated with palliative hypofractionated radiotherapy, chemotherapy, and a combination of them with united machine and protocol of radiotherapy. When compared overall survival and progression free survival, there was no significant difference among these three groups. The data of this study suggested that similar efficacy could be achieved by palliative hypofractionated radiotherapy, chemotherapy, or a combination of them.     

Immunohistochemical study of matrix metalloproteinases‐2 and ‐9, macrophage inflammatory protein‐2 and tissue inhibitors of matrix metalloproteinases‐1 and ‐2 in normal,purulonecrotic and fungal infected equine corneas

Objective Determine the effects of matrix metalloproteinases (MMPs)‐2, ‐9, macrophage inflammatory protein‐2 (MIP‐2), tissue inhibitors of matrix metalloproteinase (TIMP)‐1 and ‐2 by immunohistochemical expression in fungal affected and purulonecrotic corneas. Procedure Paraffin‐embedded equine corneal samples; normal (n = 9), fungal affected (FA; n = 26), and purulonecrotic without fungi (PN; n = 41) were evaluated immunohistochemically for MMP‐2, ‐9, MIP‐2, TIMP‐1 and ‐2. The number of immunoreactive inflammatory cells was counted and statistics analyzed. Western blot was performed to detect MMP‐2, MMP‐9, TIMP‐1 and TIMP‐2 proteins. Results Matrix metalloproteinases‐2, ‐9, MIP‐2, TIMP‐1 and ‐2 immunoreactivity was identified in corneal epithelium of normal corneas, and in corneal epithelium, inflammatory cells, keratocytes, and vascular endothelial cells of both FA and PN samples. Inflammatory cell immunoreactivity was significantly higher in FA and PN samples than in the normal corneas. There was positive correlation between MMP‐2 and MIP‐2, MMP‐9 and MIP‐2, and MMP‐9 and TIMP‐1 in inflammatory cell immunoreactivity in FA samples. There was positive correlation between MMP‐9 and MIP‐2, MMP‐9 and TIMP‐2, MIP‐2 and TIMP‐1, and MIP‐2 and TIMP‐2 in inflammatory cell immunoreactivity in PN samples. Western blot confirmed the presence of all four proteins in equine corneal samples. Conclusion Increased immunoreactivity of MMP‐2 and ‐9 in FA and PN samples is indirectly related to MIP‐2 through its role in neutrophil chemo‐attraction. Tissue inhibitors of matrix metalloproteinase‐1 and TIMP‐2 are up‐regulated in equine purulonecrotic and fungal keratitis secondary to MMP‐2 and MMP‐9 expression. The correlation between MMPs ‐2 and ‐9, MIP‐2, TIMPs ‐1 and ‐2 suggests that these proteins play a specific role in the pathogenesis of equine fungal keratitis.     

Intraperitoneal antineoplastic drug delivery: experience with a cyclophosphamide,vincristine and prednisolone protocol in cats with malignant lymphoma

In this retrospective study, the efficacy and safety were examined for an intraperitoneal chemotherapy protocol‐cyclophosphamide, vincristine and prednisolone (IP‐COP) in 26 cats with malignant lymphoma. Certainly in cats fiercely resisting IV administration the IP route is a more practical method, safer for the administrator and less stressful for the cat. Complete remission (CR) rate was 76.9% (n = 20). Median duration of first remission was 421 days. Estimated 1‐ and 2‐year disease free period were 67.1 and 48.0%, respectively. Median duration of survival was 388 days and estimated overall 1‐ and 2‐year survival periods were 54.7 and 46.9% respectively. Young cats had a more favourable prognosis. Reaching CR was essential for long‐term survival. No specific IP‐related adverse events (AE) were seen. AE were generally scored as mild and were not excessively abundant. These results indicate that the IP route is a safe and effective alternative for the administration of COP protocol chemotherapeutics.     

B‐cell intestinal lymphoma with Mott cell differentiation in a 1‐year‐old miniature Dachshund

Abstract: A 1‐year‐old intact female miniature Dachshund was presented with hematochezia, vomiting, and diarrhea of more than 1‐week duration. An abdominal mass was palpated, which at exploratory surgery was found to be a 7‐cm‐long thickened section of ileum. The thickened ileum was resected. Impression smears revealed numerous small‐ to medium‐sized lymphocytes, with a smaller number of cells resembling Mott cells. The Mott‐like cells contained multiple pale vacuoles that were positive for periodic acid‐Schiff (PAS) in wet‐fixed smears, consistent with Russell bodies. Histologic evaluation of the surgically excised ileum revealed 2 populations of neoplastic lymphoid cells. The majority were uniform medium‐sized lymphocytes with hyperchromatic oval or round nuclei and inconspicuous nucleoli. The remaining cells resembled Mott cells, which contained several PAS‐positive eosinophilic globules in the cytoplasm, occasionally compressing the nucleus. The majority of neoplastic cells stained positively for vimentin, CD20, CD79a, and Pax‐5, but were negative for CD3 and lysozyme; 43.5% of cells stained positively for Ki‐67. The Mott cells were strongly positive for immunoglobulin but were negative for Pax‐5. Using electron microscopy, a homogenous substance of intermediate electron density was observed frequently in the cisternae of rough endoplasmic reticulum in the cytoplasm of the Mott cells, and rarely in the perinuclear cisternae of the lymphoid cells, corresponding to the site of immunoglobulin staining. Monoclonal rearrangement of immunoglobulin heavy‐chain (IgH) gene was observed by PCR testing for lymphocyte–antigen receptor rearrangement. The morphologic features, immunophenotype, and IgH gene rearrangement verified the lymphoid cells were neoplastic (mature cell type) and had a B‐cell phenotype, with evidence of immunoglobulin production and differentiation into Mott cells. This case was unusual because of the age of the dog and because most intestinal lymphomas are T‐cell phenotype. The Mott cell morphology also differed from typical mature B‐cell lymphoma types and may be a unique B‐cell lymphoma variant.     

Cutaneous T‐cell lymphoma – Sézary syndrome in a Boxer

A 7‐year‐old male Boxer with a 3.5‐year history of atopy and food hypersensitivity was presented with multiple poorly circumscribed nodules and maculae of the skin and tongue, and jaundiced mucosal membranes. Cytologic and histopathologic examination of the skin lesions revealed cutaneous epitheliotropic lymphoma. Cells were CD3⁺ and CD8⁺ in flow cytometry. The CBC showed a moderate leukocytosis with 16% atypical lymphocytes with irregularly cleaved nuclei. Flow cytometric phenotyping of peripheral blood showed an elevated proportion of the CD8⁺ T‐lymphocyte subpopulation, indicating a malignant population of T‐cell origin, and the electropherogram of the PCR antigen receptor rearrangement produced a monoclonal peak for TCRγ. Liver enzyme activities were markedly increased and abdominal ultrasound examination showed increased echogenicity of the liver and enlarged abdominal lymph nodes. Fine‐needle aspirates of the liver confirmed infiltration with lymphocytes exhibiting the same morphology as the cells detected in skin and peripheral blood. Treatment was induced with L‐asparaginase, lomustine, and prednisone. Partial clinical remission of the skin and tongue lesions was achieved within 10 days, and hematologic abnormalities resolved. Despite further treatment with L‐asparaginase and lomustine, the dog relapsed within one month and was euthanized. Presence of malignant lymphocytes in skin, peripheral blood, and liver indicate a rare variant of leukemic cutaneous T‐cell lymphoma, equivalent of Sézary syndrome in a dog. This case report describes the use of flow cytometry as a complementary tool for lymphocyte characterization of skin lesions for the first time.     

Biphenotypic B‐cell lymphoma in 2 cats

The clinical and pathologic features of biphenotypic B‐cell lymphoma in 2 cats are reported. Clinical presentation varied from multiple cutaneous masses identified on the thigh in one cat to signs of lethargy from acute hemorrhage due to neoplastic infiltration of one kidney in the other. Cytology and histopathology confirmed round cell neoplasia in both cats and immunochemical staining demonstrated expression of both B‐ and T‐lymphocyte markers by the neoplastic cells in both animals. In PCR analysis of antigen receptor gene rearrangement, clonal rearrangement of B‐cell receptor genes and polyclonal T‐cell receptor gene rearrangement were demonstrated in both lymphomas. These findings were consistent with a diagnosis of B‐cell lymphoma with aberrant CD3 expression in both cases. Clinical progression of disease post diagnosis was rapid in both cats, suggesting a poor prognosis for this lymphoma type. Although bigenotypic receptor rearrangement of lymphoma cells appears relatively common, this is the first known report of actual biphenotypic lymphoma in cats.     

Non‐immunosuppressive FTY720‐derivative OSU‐2S mediates reactive oxygen species‐mediated cytotoxicity in canine B‐cell lymphoma

OSU‐2S is a FTY720 (Fingolimod) derivative that lacks immunosuppressive properties but exhibits strong anti‐tumour activity in several haematological and solid tumour models. We have recently shown OSU‐2S to mediate potent cytotoxicity in human mantle cell lymphoma cell lines and primary cells. We report here the pre‐clinical activity of OSU‐2S in spontaneous B‐cell lymphoma of dogs which shares many characteristics of human lymphoma. OSU‐2S mediated apoptosis in canine B‐cell lines and primary B‐cell lymphoma cells obtained from spontaneous lymphoma bearing dogs. OSU‐2S induced reactive oxygen species (ROS) in canine lymphoma cells and inhibition of ROS partially rescued OSU‐2S‐mediated cell death. These studies provide a rational basis for the use of spontaneous lymphoma in pet dogs as a preclinical large animal model for the development of OSU‐2S as small molecule for treating people and dogs with lymphoma.     

Expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its expected roles in the bovine endometrium during gestation

Extracellular matrix metalloproteinase inducer (EMMPRIN) and its induced matrix metalloproteinases (MMPs) play a crucial role in tissue remodeling during the peri-implantation period. However, the role of EMMPRIN in the bovine placenta is still unclear. We have postulated that EMMPRIN might play a regulatory role in trophoblastic cell functions during gestation by itself or through the regulation of MMP expression. In this study, EMMPRIN mRNA was detected in the bovine placentome and interplacentome throughout gestation, and its expression was significantly higher in the cotyledon during late gestation. In situ hybridization showed that EMMPRIN mRNA was expressed in the caruncular epithelium and the cotyledonary epithelium, including binucleate cells. Western blot analysis detected a band representing a protein of approximately 65 kDa in the caruncular and cotyledonary tissues, and the intensity of its expression was increased in both of these tissues during late gestation. The expression levels of MMP-2 and MMP-14 in the bovine placenta were higher during late gestation, as was observed for EMMPRIN. Therefore, EMMPRIN might regulate trophoblastic cell functions, especially those of binucleate cells, through MMP expression in the bovine placenta.     

Prognostic value of pretreatment plasma D‐dimer level in dogs with intermediate to high‐grade non‐Hodgkin lymphoma

Pretreatment D‐dimer levels have been reported to predict survival in several types of malignancies in human patients. The objective of this study was to evaluate the prognostic value of pretreatment D‐dimer level in dogs with intermediate to high‐grade non‐Hodgkin lymphoma (NHL). In a prospective, randomized, double‐blind study of F14512 vs etoposide phosphate, we assessed the prognostic value of pretreatment plasma D‐dimer level in 48 client‐owned dogs diagnosed with intermediate to high‐grade NHL. The correlation between pretreatment plasma D‐dimer level and various clinical features, progression‐free survival (PFS) and overall survival (OS) was analysed. The median value of pretreatment plasma D‐dimer level was 0.4 μg/mL (range: 0.1‐14.3 μg/mL). High pretreatment plasma D‐dimer level (>0.5 μg/mL) was detected in 44% (21/48) of dogs. High D‐dimer levels were not correlated with naive vs relapsed lymphoma, clinical stage, substage, immunophenotype or treatment group. D‐dimer levels >0.5 μg/mL were significantly associated with inferior median PFS (54 vs 104 days, P = .011) and OS (93 vs 169 days, P = .003). In the multivariate analysis, high D‐dimer levels remained an independent predictor for worse PFS (HR: 3.21, 95% CI: 1.57‐6.56, P = .001) and OS (HR: 3.87, 95% CI: 1.88‐7.98; P < .001). This study suggests that pretreatment plasma D‐dimer level can serve as a predictor of prognosis in dogs with intermediate to high‐grade NHL. Further studies are warranted to confirm these findings.