Osteosarcoma of the tibiotarsus with possible pulmonary metastasis in a ring-necked dove (Streptopelia risoria).

An unknown-age, adult female ring-necked dove (Streptopelia risoria) was presented with an ulcerated mass on the medial side of the right tibiotarsus. Radiographs revealed severe boney lysis with proliferative periosteal reaction. Surgical amputation was performed at the level of the mid femur and histopathologic examination of the mass identified an osteosarcoma. At the 6-month recheck, the bird was in good condition with no evidence of tumor regrowth or metastasis; however, at 8 months, the dove was found dead. On necropsy, a large mass was present in the coelomic cavity invading the left pulmonary parenchyma. Histopathologic examination indicated a spindle cell sarcoma. Immunohistochemical staining for osteocalcin and osteonectin was performed on the confirmed osteosarcoma in the tibiotarsus and the spindle cell sarcoma mass. Results indicated positive intracytoplasmic immunoreactivity for osteocalcin and osteonectin in the confirmed osteosarcoma neoplasm. Very rare positive cytoplasmic immunoreactivity occurred in the spindle cell sarcoma.

Gene expression profiling identifies inflammation and angiogenesis as distinguishing features of canine hemangiosarcoma.

BACKGROUND: The etiology of hemangiosarcoma remains incompletely understood. Its common occurrence in dogs suggests predisposing factors favor its development in this species. These factors could represent a constellation of heritable characteristics that promote transformation events and/or facilitate the establishment of a microenvironment that is conducive for survival of malignant blood vessel-forming cells. The hypothesis for this study was that characteristic molecular features distinguish hemangiosarcoma from non-malignant endothelial cells, and that such features are informative for the etiology of this disease. METHODS: We first investigated mutations of VHL and Ras family genes that might drive hemangiosarcoma by sequencing tumor DNA and mRNA (cDNA). Protein expression was examined using immunostaining. Next, we evaluated genome-wide gene expression profiling using the Affymetrix Canine 2.0 platform as a global approach to test the hypothesis. Data were evaluated using routine bioinformatics and validation was done using quantitative real time RT-PCR. RESULTS: Each of 10 tumor and four non-tumor samples analyzed had wild type sequences for these genes. At the genome wide level, hemangiosarcoma cells clustered separately from non-malignant endothelial cells based on a robust signature that included genes involved in inflammation, angiogenesis, adhesion, invasion, metabolism, cell cycle, signaling, and patterning. This signature did not simply reflect a cancer-associated angiogenic phenotype, as it also distinguished hemangiosarcoma from non-endothelial, moderately to highly angiogenic bone marrow-derived tumors (lymphoma, leukemia, osteosarcoma). CONCLUSIONS: The data show that inflammation and angiogenesis are important processes in the pathogenesis of vascular tumors, but a definitive ontogeny of the cells that give rise to these tumors remains to be established. The data do not yet distinguish whether functional or ontogenetic plasticity creates this phenotype, although they suggest that cells which give rise to hemangiosarcoma modulate their microenvironment to promote tumor growth and survival. We propose that the frequent occurrence of canine hemangiosarcoma in defined dog breeds, as well as its similarity to homologous tumors in humans, offers unique models to solve the dilemma of stem cell plasticity and whether angiogenic endothelial cells and hematopoietic cells originate from a single cell or from distinct progenitor cells.

High incidence of lymphoid neoplasia in a colony of Egyptian spiny-tailed lizards (Uromastyx aegyptius).

Hematopoietic malignancies are the most commonly reported neoplasms in lizards, occurring sporadically as in other reptiles. An unusually high incidence of lymphoid neoplasia occurred in a collection of Egyptian spiny-tailed lizards (Uromastyx aegyptius) from 1993-2001. Eight of 15 lizards necropsied at the Louisville Zoological Garden (53%) had multicentric lymphoma. Immunohistochemistry was not useful in characterizing the lineage of normal or neoplastic lymphocytes. By light and electron microscopy (EM), the neoplasms had plasmacytoid morphologic features suggesting B-cell origin, although some tumors also had a primitive lymphoblast component. A concurrent leukemic blood profile was identified in seven of the cases (88%). All were adult animals and no sex predilection was observed. No exposure to exogenous carcinogens was observed. Some of the lizards were unrelated, so hereditary factors were unlikely. Although examination by EM and viral isolation performed on archived tissues and plasma failed to detect viruses, an infectious etiology still warrants consideration.

Cellular immunolocalization of gastric and pancreatic lipase in various tissues obtained from dogs.

OBJECTIVE: To determine cellular immunolocalization of canine gastric lipase (cGL) and canine pancreatic lipase (cPL) in various tissues obtained from clinically healthy dogs. SAMPLE POPULATION: Samples of 38 tissues collected from 2 climically healthy dogs. PROCEDURES: The cGL and cPL were purified from gastric and pancreatic tissue, respectively, obtained from dogs. Antisera against both proteins were developed, using rabbits, and polyclonal antibodies were purified by use of affinity chromatography. Various tissues were collected from 2 healthy dogs. Primary antibodies were used to evaluate histologic specificity. Replicate sections from the collected tissues were immunolabeled for cGL and cPL and examined by use of light microscopy. RESULTS: Mucous neck cells and mucous pit cells of gastric glands had positive labeling for cGL, whereas other tissues did not immunoreact with cGL. Pancreatic acinar cells had positive labeling for cPL, whereas other tissues did not immunoreact with cPL. CONCLUSIONS AND CLINICAL RELEVANCE: We concluded that cGL and cPL are exclusively expressed in gastric glands and pancreatic acinar cells, respectively. Also, evidence for cross-immunoreactivity with other lipases or related proteins expressed by other tissues was not found for either protein. Analysis of these data suggests that gastric lipase is a specific marker for gastric glands and that pancreatic lipase is a specific marker for pancreatic acinar cells. These markers may have clinical use in the diagnosis of gastric and exocrine pancreatic disorders, respectively.

Use of the Cell-Dyn 3500 to predict leukemic cell lineage in peripheral blood of dogs and cats.

BACKGROUND: Morphology and cytochemistry are the foundation for classification of leukemias in dogs and cats. Advances in automated hematology instrumentation, immunophenotyping, cytogenetics, and molecular biology are significantly improving our ability to recognize and classify spontaneous myeloproliferative and lymphoproliferative disorders. OBJECTIVE: The purpose of this study was to assess the utility of flow cytometry-based light scatter patterns provided by the Cell-Dyn 3500 (CD3500) automated hematology analyzer to predict the lineage of leukemic cells in peripheral blood of dogs and cats. METHODS: Leukemic cells from 15 dogs and 6 cats were provisionally classified using an algorithm based on the CD3500 CBC output data and were subsequently phenotyped by enzyme cytochemistry, immunocytochemistry, indirect flow cytometry, and analysis of antigen receptor gene rearrangement. RESULTS: The algorithm led to correct predictions regarding the ontogeny of the leukemic cells (erythroid/megakaryocytic potential, myeloid leukemia, monocytic leukemia, chronic granulocytic leukemia, lymphoid leukemia) in 19/21 animals. Mismatches in the WBC impedance count and the WBC optical count in conjunction with microscopic assessment of blasts in the blood were useful for predicting myeloproliferative disorders with erythroid or megakaryocytic potential. The leukocyte light scatter patterns enabled distinction among myeloid leukemias (represented by acute myelomonocytic leukemia, acute monocytic leukemia, chronic granulocytic leukemia) and lymphocytic leukemias (including acute and chronic lymphocytic leukemias). One case of acute lymphocytic leukemia was misidentified as chronic lymphocytic leukemia. CONCLUSIONS: Algorithmic analyses can be applied to data generated by the CD3500 to predict the ontogeny of leukemic cells in the peripheral blood of dogs and cats. This rapid and quantitative technique may be used to improve diagnostic decisions, expand therapeutic choices, and increase prognostic accuracy.

Pancreatic islet fibrosis in rock hyrax (Procavia capensis), Part 2: Pathology, immunohistochemistry, and electron microscopy.

Pancreatic islet fibrosis with varying degrees of islet cell hyperplasia or islet effacement was diagnosed histologically in 19 rock hyraxes (Procavia capensis) from seven zoological parks. Some, but not all, affected hyraxes were from a common lineage. The condition was associated with apparent hyperglycemia in seven and diabetes mellitus in two. Immunohistochemistry revealed hyperplasia of beta, alpha, and delta cells proportional to the degree of the fibrosis. Electron microscopy revealed collagen deposition and fibroplasia within and around the islets. Special stains and electron microscopy were negative for the presence of amyloid. Beta cell depletion was never identified. The condition has morphologic features that resemble islet fibrosis of human infants born to diabetic mothers.

The use of cytochemistry, immunophenotyping, flow cytometry, and in vitro differentiation to determine the ontogeny of a canine monoblastic leukemia.

We evaluated the utility of cytochemistry, immunophenotyping, flow cytometry, and in vitro culture with forced differentiation of leukemic cells as diagnostic aids to identify the malignant cell ontogeny in a dog with leukemia. A tentative diagnosis of monoblastic leukemia was established by microscopic examination of Romanowsky-stained blood smears and bone marrow aspirate smears. This diagnosis also was supported by the light scatter signature that identified the blast cells as large, non-granular monocytic cells using a CellDyn 3500 automated hematology analyzer; as well as by the detection of N-butyrate esterase and the lack of choloroacetate esterase or leukocyte peroxidase by cytochemical staining. Subsequently, leukemic cells were isolated from the dog's peripheral blood and placed into tissue culture or cryopreserved. The leukemic cells grew in suspension cultures and proliferated spontaneously for up to 4 days. By day 7, proliferation was negligible. Upon culture with conditioned supernatant using mitogen-stimulated human T cells as a source of cytokines, an increased proportion of cells entered S phase by day 2 of culture; however, proliferation declined markedly by day 4, at which time the cells had apparently differentiated to adherent, vacuolated macrophages. The cytokine-stimulated leukemic cells were positive for the monocyte/macrophage specific markers alpha-1-antitrypsin, alpha-1-antichymotrypsin, lysozyme, CD14, MHC class II, and calprotectin, an antigen found in differentiated macrophages and granulocytes. Despite the strong tendency of the leukemic cells towards monocytic differentiation, our results suggested that they retained some features of a myelomonocytic precursor. These data show that cytochemistry, immunophenotyping, flow cytometry, and in vitro differentiation of canine leukemia cells are useful tools for confirming the lineage of malignant hematopoietic cells.

Canine malignant hemangiosarcoma as a model of primitive angiogenic endothelium.

Hemangiosarcoma (HSA) is a common untreatable cancer of dogs that resembles human angiosarcoma. Detailed studies of these diseases have been historically hindered by the paucity of suitable reagents. Here, we show that expression of CD117 (c-Kit) can distinguish primitive (malignant) from mature (benign) proliferative endothelial lesions, and we describe eight independent cell lines derived from canine HSA explants. Endothelial origin was confirmed by sustained expression of surface CD105 (endoglin), CD146 (MUC18), and CD51/CD61 (alpha(v)beta(3) integrin). The cell lines showed anchorage-independent growth and were motile and invasive when cultured on a basement membrane matrix. They required endothelial growth factors for growth and survival, and they could be induced to form tubular structures resembling blood vessels when cultured under low calcium conditions. The formation of vessel-like structures was blocked by nicotine, and restored by FK506, suggesting that 'nuclear factor of activated T cells' activity prevents differentiation of these cells. In summary, these cell lines represent a unique and novel resource to improve our understanding of endothelial cell biology in general and canine HSA in particular.