CD34+, CD41+ acute megakaryoblastic leukemia in a dog

A clinically normal, 5-year-old intact female German Shepherd dog was presented to the local veterinarian to be spayed. Results of a preoperative CBC included mild nonregenerative anemia, severe thrombocytopenia, and 17% unclassified cells. On cytologic examination of aspirates from the dog's enlarged spleen and peripheral lymph nodes, a population of primitive round cells that occasionally resembled megakaryocytes was observed. A bone marrow aspirate specimen was markedly hypercellular with approximately 65% of marrow cells comprising a homogeneous population of immature hematopoietic cells similar to those found in the spleen, lymph nodes, and peripheral blood. Using immunocytochemical stains with canine-specific antibodies, all neoplastic cells strongly expressed cytoplasmic CD41 and 20-70% of the neoplastic cells expressed CD34 weakly to moderately. Rare (<0.5%) neoplastic cells weakly expressed vWF. The cells were negative for all other markers. Based on these results and the morphology of the neoplastic cells, a diagnosis of acute megakaryoblastic leukemia (AMegL) was made. In spite of treatment, results of a CBC performed 1 week later indicated progressive anemia and thrombocytopenia, and the dog was euthanized. To our knowledge, this report documents the first case of canine AMegL diagnosed with both anti-canine CD34 and CD41 antibodies.     

Disseminated histiocytic sarcoma with peripheral blood involvement in a Bernese Mountain dog

Abstract: A 6‐year‐old Bernese Mountain dog was presented with a history of lethargy and weight loss of 2 weeks duration. On physical examination the dog had pale mucous membranes and tachypnea. Ultrasound examination revealed hepatomegaly, splenomegaly, and mesenteric lymphadenomegaly. Results of a CBC included marked normocytic normochromic nonregenerative anemia, marked thrombocytopenia, and moderate leukocytosis with mild neutrophilia and a large population of unclassified round cells (6.2 × 10³/μL). The unclassified cells occasionally were bi‐ or multinucleated and had variably abundant pale basophilic cytoplasm that contained multiple irregular clear vacuoles and occasionally erythrocytes. Fine needle aspirate specimens of the mesenteric lymph nodes and spleen were composed of a population of round pleomorphic cells with the same features as the circulating cells. On flow cytometric analysis of peripheral blood, the unclassified cells expressed CD18, CD45, CD11c, CD1c, and CD14; immunocytochemical analysis of blood smears also indicated the cells were positive for CD1c, CD1a, and CD11c. The dog died a few hours after referral. The histologic interpretation of samples collected from spleen, liver, and lymph nodes was malignant neoplasia of histiocytic origin. Immunohistochemical staining yielded negative results for CD11d, a marker of red‐pulp macrophages, ruling out hemophagocytic histiocytic sarcoma. Based on clinical and pathologic findings, the final diagnosis was disseminated histiocytic sarcoma (DHS) with peripheral blood involvement. To our knowledge, DHS in a dog with evidence and immunophenotyping of neoplastic cells in peripheral blood has been reported only rarely.     

Spontaneous hemangiosarcoma in the spleen and liver of a young rat

Spontaneous hemangiosarcoma in young rats is rare. In this report, we describe a case of a spontaneous hemangiosarcoma in the spleen and liver of young rats. At necropsy, multiple pale red masses were observed in the spleen. Histopathologically, solid growth and haphazardly arranged neoplastic cells were observed, although no characteristic growth pattern was observed. In contrast, irregularly sized small slit-shaped spaces containing erythrocytes were found among the neoplastic cells. Reticular fibers incompletely surrounding the neoplastic cells were observed by silver staining. Immunohistochemistry revealed that the neoplastic cells were positive for vWF and CD34. Electron microscopic examination revealed that the neoplastic cells had erythrocytes in the lumen and Weibel-Palade bodies in the cytoplasm and were arranged along a discontinuous basal lamina. These features indicate that the tumor originated from vascular endothelial cells. Based on these results, the tumor was diagnosed as a hemangiosarcoma in the spleen and liver.     

Myeloperoxidase-positive acute megakaryoblastic leukemia in a dog

A 16-month-old female spayed Labrador Retriever was referred to the University of Edinburgh for exercise intolerance, inappetence, and severe anemia. A CBC showed severe nonregenerative anemia and moderate numbers of atypical cells with morphologic features most consistent with megakaryoblastic origin. Similar cells were identified in a bone marrow aspirate and accounted for 23% of all nucleated cells. Atypical promegakaryocytes and megakaryocytes were also noted. Myelodysplastic syndrome affecting the megakaryocytic lineage was suspected. Cytologic examination of a fine-needle aspirate of the spleen revealed rare megakaryoblasts similar to those in blood and bone marrow. At necropsy, the bone marrow consisted of atypical megakaryoblasts and megakaryocytes that were also infiltrating spleen, liver, lymph nodes, renal perihilar tissue, and visceral adipose tissue, consistent with acute megakaryoblastic leukemia. Immunohistochemical analysis of splenic sections confirmed megakaryoblastic origin (immunoreactive for CD61 and von Willebrand factor). Some leukemic cells were also immunoreactive for myeloperoxidase (MPO). This aberrant immunophenotype suggested both megakaryocytic and granulocytic/monocytic differentiation of the leukemic cells. To our knowledge, this is the first report of MPO-positive acute megakaryoblastic leukemia in a dog.     

Diagnosis and treatment of chronic T-lymphocytic leukemia in a spotted hyena (Crocuta crocuta).

Physical examination of an asymptomatic 20-yr-old intact female spotted hyena (Crocuta crocuta) revealed a midabdominal mass. A complete blood count (CBC) revealed peripheral lymphocytosis. Abdominal ultrasonography and laparoscopy confirmed severe splenomegaly. Cytologic examination of a bone-marrow core and histologic examination of spleen and liver biopsy samples revealed neoplastic small lymphocytes. Immunohistochemical staining of liver and spleen samples with the use of leukocyte-specific monoclonal antibodies showed that the neoplastic lymphocytes were immunoreactive to T-lymphocyte CD3 receptor and immunonegative to B-lymphocyte CD79a receptor. The morphology and distribution of neoplastic T-lymphocytes within the spleen, liver, peripheral blood, and bone marrow was most consistent with chronic T-lymphocytic leukemia. Treatment with chlorambucil and prednisone effectively decreased the lymphocyte count, but was associated with thrombocytopenia, which resolved after chlorambucil treatment was temporarily discontinued. Chemotherapy was resumed with a single dose of L-asparaginase, followed by a lower dosage of chlorambucil and continued prednisone. Two years after initial diagnosis, the hyena developed a hemoabdomen and was euthanized. Neoplastic T-lymphocytes were present in spleen, liver, visceral and peripheral lymph nodes, lungs, heart, kidney, adrenal glands, mesentery, intestines, pancreas, brain, and bone marrow.     

Spontaneous Malignant T Cell Lymphoma in a Young Male Common Marmoset (Callithrix jacchus)

We histopathologically and immunohistochemically investigated a case of malignant lymphoma that spontaneously developed in a male common marmoset at two years of age. Beginning at two years four months of age, the animal had an enlargement of the submandibular and inguinal lymph nodes, small subcutaneous nodules near the right breast and an approximately fivefold increase in peripheral lymphocyte count compared with the previous examination value. The postmortem findings at two years eight months of age showed lymphadenopathy with enlargement of the thymus and spleen. Small- to intermediate-sized neoplastic lymphocytes had diffusely proliferated in the enlarged nodes. The neoplastic cells were pleomorphic and had irregularly shaped nuclei. The nuclear chromatin staining revealed hyperchromatism in the small-sized cells, and the intermediate-sized cells exhibited vesicular staining. An immunohistochemical examination indicated that the neoplastic lymphocytes were positive for CD3 and negative for CD20, thus suggesting that they had originated from T cells. In addition, the proliferation of high endothelial venules and reactive epithelioid histiocytes was observed. Scattered tingible body-laden macrophages were infrequently detected. Neoplastic lymphocytes were also observed in the thymus, spleen, heart, lungs, liver, kidneys, adrenal glands and femoral and sternal bone marrow. This malignant lymphoma in a young male common marmoset was considered to fit the category of “peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS)” according to the new WHO system of classification.     

A porcine lymphoma-derived cell line co-expressing IgM,IgG and IgA

A cell line (PL38PB) was established from blood samples of a 6-month-old pig that was diagnosed with lymphoma with CD5 expression. Histopathological examination revealed neoplastic lesions in the spleen, liver and lymph nodes. Tumor cells were immunohistochemically positive for CD20 and immunoglobulin heavy chains (μ, γ and α). Membranous CD5 and cytoplasmic Immunoglobulin M (IgM), ​Immunoglobulin G (IgG) and ​Immunoglobulin A (IgA) were detected in PL38PB cells by flow cytometry. In addition, the cytoplasm of PL38PB cells were positive for IgM, IgG and IgA by immunofluorescent. However, no Ig secretion was detected in culture supernatant by Ouchterlony gel diffusion method. Results suggest that PL38PB cells express three Ig isotypes that are produced but not secreted.     

Chronic eosinophilic leukemia in a cat: cytochemical and immunophenotypical features

A 3-year-old, male, domestic shorthaired cat was presented with a 3-day history of anorexia and depression. The cat was moderately dehydrated, had pale, slightly icteric, mucous membranes, oral ulcerations, and mild hepatosplenomegaly. A feline leukemia virus (FeLV) antigen test was positive. CBC results obtained at initial presentation included severe normocytic, normochromic, nonregenerative anemia, severe thrombocytopenia, and marked leukocytosis (>100,000/microL) with 77% eosinophils. After 15 days of treatment with prednisone and doxycycline, the cat had persistent severe nonregenerative anemia (HCT 3.4%), thrombocytopenia (28,000/microL), and extreme eosinophilia (total eosinophils, 123.1 x 10(3)/microL; segmented 103.0 x 10(3)/microL; immature 20.1 X 10(3)/microL). Cytologic examination of aspirates from bone marrow, liver, lymph nodes, and spleen revealed a predominance of mature and immature eosinophils, many with dysplastic changes. The M:E ratio was 96.4. On histopathologic examination, multiple organs were infiltrated by eosinophilic granulocytes. Neoplastic cells in blood and bone marrow stained positive for alkaline phosphatase and were negative for myeloperoxidase, chloroacetate esterase, and alpha-naphthyl acetate esterase. On flow cytometric analysis of peripheral blood, the neoplastic cells were positive for CD11b and CD14. These findings were consistent with chronic eosinophilic leukemia. To our knowledge, this is the first report of chronic eosinophilic leukemia in a cat associated with naturally acquired FeLV infection, in which flow cytometry was used to characterize the neoplastic cells.     

Acute myeloid leukemia with multilineage dysplasia in an alpaca

An 18-year-old female alpaca was presented to the Colorado State University Veterinary Teaching Hospital for chronic ill thrift over a 1-year period. Six weeks previously, an infected left mandibular cheek tooth was removed by oral extraction. On physical examination the patient was cachectic, lethargic, and weak. Abnormalities on the CBC included neutropenia, thrombocytosis, and severe nonregenerative, macrocytic, hypochromic anemia. Dysplastic nucleated erythrocytes and micromegakaryocytes were observed on the peripheral blood smear. Neutrophils, bands, and metamyelocytes appeared markedly toxic. Numerous blasts containing variable numbers of fine azurophilic granules were also observed. Based on their morphology, the cells were interpreted to be progranulocytes and myeloblasts, and a presumptive diagnosis of acute myeloid leukemia (AML) was made. The blast cells accounted for 60% of the nucleated cell population on bone marrow aspirates, further supporting a diagnosis of AML with multilineage dysplasia. Post mortem examination showed infiltration of the neoplastic cells into spleen, liver, kidney, and lymph nodes. Based on histologic findings, the morphologic diagnoses were disseminated myeloid neoplasia, chronic regionally extensive tooth root abscess, and membranous glomerulonephritis. The neoplastic cells were CD172a-positive on flow cytometry, chloroacetate esterase-positive by cytochemistry, and myeloperoxidase-positive by immunohistochemistry, confirming myeloid origin. To our knowledge, this is the first case of AML with multilineage dysplasia in an alpaca, with only one other case of myelodysplasia described previously in this species.     

Thymic lymphosarcoma of T cell lineage in a koala (Phascolarctos cinereus)

Objective To diagnose and characterise thymic lymphosarcoma in a koala.
Design A pathological case.  

Animal

Seven-year-old female koala.
Procedure The neoplastic process was investigated macroscopically, haematologically, histologically and immunohistologically.
Results The koala had difficulty swallowing because of a medial swelling in the lower neck. Biopsy of this mass and blood examination revealed lymphosarcoma with a leukaemic manifestation; necropsy and histopathological examination showed the mass to be thymus. Palatine tonsils, cervical, axillary and mesenteric lymph nodes, spleen, liver, gut, bronchi, genitalia and bone marrow were infiltrated by neoplastic cells. Immunohistological staining of the thymic mass, cervical and mesenteric lymph nodes, bone marrow, spleen and gut revealed the neoplastic cells to be of T lymphocyte origin (positive for both anti-human CD3 and CD5).  

Conclusions

It is speculated that the neoplastic process originated in the thymus and was disseminated by bloodborne neoplastic cells: This first report of thymic lymphosarcoma in a marsupial confirms that antibodies raised originally to investigate human lymphoid neoplasia can cross-react with neoplastic lymphocytes in koalas.     

Monitoring of large B-cell lymphoma and T-zone lymphoma in a dog via flow cytometry

A 12-y-old, castrated male Pomeranian dog was presented because of mandibular lymph node (LN) enlargement. Physical examination and a complete blood count revealed generalized lymphadenopathy and moderate lymphocytosis. Fine-needle aspirate cytology revealed expansion of medium lymphocytes in the right mandibular LN and expansion of large lymphocytes in the left popliteal LN. Flow cytometry identified 2 aberrant lymphocyte populations in both LNs, namely a CD5+CD45− T-cell population, and a large CD21+ B-cell population. Flow cytometry of the peripheral blood revealed an identical population of aberrant CD45− T cells. The patient was diagnosed with concurrent T-zone lymphoma and leukemia, and B-cell lymphoma. Multi-agent chemotherapy was instituted, and serial clinical and flow cytometric analysis revealed complete remission of the neoplastic B cells, but persistence of the neoplastic T cells and persistent lymphadenopathy. This case affirms the diagnostic value of flow cytometry and reveals a unique limitation of the RECIST criteria.     

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.     

Carcinosarcoma mimicking a feline injection‐site sarcoma in a cat

A 15‐year‐old spayed female domestic short‐haired cat with cutaneous/subcutaneous well‐circumscribed, alopecic mass approximately 25 × 30 mm in diameter, localized to the left shoulder region was brought to the veterinary surgery department. Despite the suggestive location and macroscopic appearance, feline injection‐site sarcoma was not suspected based on the cytologic examination of fine‐needle aspirates. The tumor was surgically resected, and tissue sections were evaluated microscopically. The tumor was found to be nonencapsulated with a distinct border between the neoplastic parenchyma and surrounding connective tissue. The neoplastic tissue consisted of 2 cell populations: elongated to spindle‐shaped cells arranged in bands and cords and malignant epithelial‐like cells. Both populations showed microscopic features of malignancy. Multinucleate giant cells with irregular cytoplasm were scattered among the neoplastic cells. The spindle‐shaped cells strongly expressed vimentin but did not express α‐smooth muscle actin (α‐SMA) or cytokeratin. Desmin was strongly expressed in about 0‐5% of cells. Epithelial‐like cells expressed cytokeratin, but not vimentin, desmin, or α‐SMA. Multinucleate giant cells expressed vimentin, but did not α‐SMA, desmin, or cytokeratin. Based on microscopic observations and IHC results, the final diagnosis was carcinosarcoma with histologic features compatible with feline injection‐site sarcoma, but without the clinical aggressiveness of this tumor.     

Reactivity of cross-reacting monoclonal antibodies with canine leukocytes, platelets and erythrocytes

A panel of 380 commercially available monoclonal antibodies (mAbs) against human CD molecules from various sources was tested during the 8th Human Leukocyte Differentiation Antigen Workshop (HLDA8) for cross-reactivity on canine peripheral blood leukocytes by flow cytometry. In addition, all mAbs were used to label a 50:50 mixture of platelets and erythrocytes of the same dogs. This testing resulted in 51 cross-reacting mAbs. mAbs with specificity for CD9, CD29, CD42a, CD61, and CD41/CD61 showed cross-reactivity with canine platelets in a non-polymorphic and one mAb with the erythrocyte antigen CD235a in a polymorphic reaction pattern. Canine leukocyte-reactive mAbs included those with specificity for CD11a, CD11b, CD14, CD18, CD21, CD22, CD47, CD49d, CD49e, CD56, CD62L, CD91, CD94, and CD172a. In addition, several mAbs resulted in a staining pattern of canine cells which suggest that the canine epitope equivalents have an alternate expression pattern from that expected for humans (CD1a, CD35, CD44, CD45, CD75s, CD81). In summary, this study confirmed the reactivity of previously described cross-reactive mAbs with canine cells and resulted in the characterization of mAbs recognizing so far undetectable canine CD molecules.     

Thrombocytosis and central nervous system involvement in a case of canine acute megakaryoblastic leukemia

This case report presents a 14‐month‐old female Poodle mix with acute megakaryoblastic leukemia based on a marked thrombocytosis, abnormal platelet morphology, circulating dwarf megakaryocytes, and blast cells in the blood. Bone marrow abnormalities included dysmegakaryopoiesis dygranulopoiesis, and an increased number of blast cells was observed in the blood. Extensive leukemic involvement was also found in the liver, spleen, lymph nodes, lungs, kidneys, and brain. The cytopathologic features of the abnormal circulating cells were highly suggestive of being megakaryocytic in origin, which was supported by negative myeloperoxidase staining and positive von Willebrand factor staining on immunocytochemistry (ICC). The neoplastic cells were also CD61 positive and had variable von Willebrand factor expression on ICC. Although there were only 25% blast cells in the bone marrow, which theoretically supported myelodysplastic syndrome, the hypothesis that this case represented acute myeloid leukemia of megakaryoblastic origin was confirmed by the continuous increase in circulating blast cell numbers during follow‐up visits and the extensive leukemic involvement of parenchymal organs.     

Horses with equine recurrent uveitis have an activated CD4+ T‐cell phenotype that can be modulated by mesenchymal stem cells in vitro

Equine recurrent uveitis (ERU) is an immune‐mediated disease causing repeated or persistent inflammatory episodes which can lead to blindness. Currently, there is no cure for horses with this disease. Mesenchymal stem cells (MSCs) are effective at reducing immune cell activation in vitro in many species, making them a potential therapeutic option for ERU. The objectives of this study were to define the lymphocyte phenotype of horses with ERU and to determine how MSCs alter T‐cell phenotype in vitro. Whole blood was taken from 7 horses with ERU and 10 healthy horses and peripheral blood mononuclear cells were isolated. The markers CD21, CD3, CD4, and CD8 were used to identify lymphocyte subsets while CD25, CD62L, Foxp3, IFNγ, and IL10 were used to identify T‐cell phenotype. Adipose‐derived MSCs were expanded, irradiated (to control proliferation), and incubated with CD4⁺ T‐cells from healthy horses, after which lymphocytes were collected and analyzed via flow cytometry. The percentages of T‐cells and B‐cells in horses with ERU were similar to normal horses. However, CD4⁺ T‐cells from horses with ERU expressed higher amounts of IFNγ indicating a pro‐inflammatory Th1 phenotype. When co‐incubated with MSCs, activated CD4⁺ T‐cells reduced expression of CD25, CD62L, Foxp3, and IFNγ. MSCs had a lesser ability to decrease activation when cell‐cell contact or prostaglandin signaling was blocked. MSCs continue to show promise as a treatment for ERU as they decreased the CD4⁺ T‐cell activation phenotype through a combination of cell‐cell contact and prostaglandin signaling.     

Progression of cutaneous plasmacytoma to plasma cell leukemia in a dog

A 5‐year‐old male neutered Bernese Mountain Dog was presented for cutaneous plasmacytoma, which was treated by surgical excision. Four months later, the dog developed multiple skin masses, hyphema, pericardial and mild bicavitary effusions, myocardial masses, and marked plasmacytosis in the peripheral blood. Circulating plasma cells expressed CD34 and MHC class II by flow cytometry. Immunocytochemistry demonstrated that these cells were strongly positive for multiple myeloma oncogene 1/interferon regulatory factor 4 (MUM‐1) and weakly to moderately positive for Pax5. The dog was hypoglobulinemic but had a monoclonal IgA gammopathy detected by serum immunofixation electrophoresis. The PCR analysis of antigen receptor gene rearrangements (PARR) by fragment analysis using GeneScan methodology revealed that plasmacytoid cells in the original cutaneous plasmacytoma and peripheral blood had an identical immunoglobulin heavy chain gene (IgH) rearrangement, indicating that both populations were derived from the same neoplastic clone. Canine cutaneous plasmacytoma rarely progresses to a malignant form and plasma cell leukemia is rarely diagnosed in the dog. This report describes a case of cutaneous plasmacytoma progressing to plasma cell leukemia with a rapid and aggressive clinical course. This report also highlights the utility of flow cytometry, immunocytochemistry, immunofixation electrophoresis, and PARR by fragment analysis using GeneScan methodology in the diagnosis of this hematopoietic neoplasm.     

Bone marrow necrosis and myelophthisis: manifestations of T‐cell lymphoma in a horse

Abstract: A 14‐year‐old spayed American Paint mare was evaluated for mild colic, anorexia, pyrexia, and pancytopenia. Physical examination revealed mild tachycardia, tachypnea, and pale mucous membranes. Serial laboratory analyses revealed progressive pancytopenia, hyperfibrinogenemia, and hyperglobulinemia. A few large atypical cells were observed in peripheral blood smears. Results of tests for equine infectious anemia and antipenicillin antibody were negative. Serum protein electrophoresis indicated a polyclonal gammopathy. Smears of bone marrow aspirates contained hypercellular particles, but cell lines could not be identified because the cells were karyolytic, with pale basophilic smudged nuclei and lack of cellular detail. A diagnosis of bone marrow necrosis was made. Treatment consisted of antimicrobials, nonsteroidal anti‐inflammatory drugs, and corticosteroids. The pyrexia resolved; however, the pancytopenia progressively worsened and petechiation and epistaxis developed. The horse was humanely euthanized. Postmortem examination revealed a diffuse round cell neoplasm infiltrating the kidneys, spleen, lymph nodes, lungs, and bone marrow. Immunophenotyping results (CD3+, CD79α−) indicated the neoplastic cells were of T‐cell lineage. Infiltration of lymphoma cells into the bone marrow appeared to have resulted in severe myelophthisis and bone marrow necrosis. Bone marrow necrosis has been associated previously with lymphoma in humans and dogs. To our knowledge, this is the first reported case of lymphoma resulting in bone marrow necrosis in a horse.