Acute Megakaryoblastic Leukemia in Dogs: A Report of Three Cases and Review of the Literature
Three dogs of different breeds, ages, and genders were presented with pale mucous membranes, depression, anorexia, and splenomegaly. Observed were severe normocytic, nor-mochromic, nonregenerative anemia, thrombocytopenia, and leukopenia. Blood smears contained large, atypical cells with blue vacuolated cytoplasm, cytoplasmic blebs, round to oval central nuclei, and elevated numbers of cytoplasmic fragment resembling macroplatelets. Bi- and multinucleated atypical cells were found mainly in spleen, lymph nodes, and bone marrow. A final diagnosis of acute megakaryoblastic leukemia (AMegL) was made based on morphology and positivity to the megakaryocyte-derived cell-specific markers von Willebrand factor and CD61. In case nos. 1 and 2, no treatment was initiated, and the dogs died on days 4 and 3, respectively. Case no. 3 received supportive therapy with prednisone, and after a brief improvement the dog died spontaneously 35 days after initial presentation. Only 11 cases of AMegL have been reported in dogs, and the specific diagnostic criteria have not been well established. The presence of vacuolization, cytoplasmic blebs, central round nuclei, cytoplasmic fragments, and multinucleated cells in these three cases were considered useful to differentiate AMegL from other hematopoietic neoplasms.
Introduction
Acute megakaryoblastic leukemia (AMegL or AML7) is a subtype of acute myeloid leukemia (AML), which has previously been described in only 11 dogs.1–10 Diagnostic criteria have not been well established. According to the French-American-British cooperative group, definitive diagnosis requires identification of >30% (20% according to World Health Organization guidelines) of the total nucleated cells in bone marrow as blasts. These blasts are identified as megakaryocytic precursors by positivity to megakaryocyte-derived cell-specific markers11 including CD61, von Willebrand factor (vWF), and (in human medicine) Factor XIII, CD41, CD36, and CD42. Canine-specific markers recognizing most of the above-mentioned molecules are not currently available. Additionally, most of these markers are not commonly included in antibody panels utilized for flow cytometric or immunocytochemical phenotyping of hematopoietic malignancies, even though inclusion of CD41 into panels has been widely recommended by the American College of Veterinary Pathologists’ (ACVP) Oncology Committee for immunophe-notyping of animal leukemia.12
In humans, the diagnosis of AMegL is commonly associated with an extremely poor prognosis,13,14 particularly when associated with chromosomal abnormalities such as Down syndrome.15,16 In dogs, survival time is very short, despite different chemotherapeutic approaches.1,3,5,7,10
This manuscript describes clinical and hematological features of three dogs with AMegL (previously unreported) and critically reviews cases reported in the literature. The major features identified in AMegL are described in order to identify parameters useful in differential diagnosis.
Case Reports
Case No. 1
An 8-month-old, female Bernese mountain dog was presented with clinical signs of asthenia and weight loss of approximately 1 month’s duration. Three days prior to presentation, the dog became anorexic and had a single episode of vomiting. Physical examination revealed muscular atrophy and pale mucous membranes. Hepato- and splenomegaly were evident, and prescapular and popliteal lymph nodes were moderately enlarged. Blood samples were submitted for hematology and clinical biochemical analysis together with submandibular and prescapular lymph node and splenic fine-needle aspiration biopsies (FNB). Flow-cytometric immunophenotyping of atypical cells was performed on blood and bone marrow using the following antibodies:17 CD45 (clone YKIX716.13,a all leukocytes), CD18 (clone CA1.4E9,a all leukocytes), CD3-FITC (clone CA17.2A12,a T-lymphocytes), CD4-FITC (clone YKIX302.9,a neutrophils and T-helper lymphocytes), CD8-PE (clone YCATE55.9,a T-cytotoxic), CD21 (clone CA21D6,a B-lymphocytes), CD14-PE (clone TUK4,a monocytes/macrophages), CD11b (clone CA163E10,a granulocytes and monocytes), CD11c (clone Ca11.6A1,a granulocytes and monocytes), and CD34-PE (clone 1H6,b precursor cells). Blood, lymph node, and bone marrow smears were also submitted for immunocytochemical evaluation18 using a mouse monoclonal anti-CD61 antibody (antihuman gpIIIa, clone Y2/51c) and a rabbit polyclonal anti-vWF (anti-Factor VIII-related antigenc).
Biochemical results were within reference intervals except for a mild hypoproteinemia (53.5 g/L, reference interval 55.0 to 73.0 g/L), hyperphosphatemia (2.02 mmol/L, reference interval 0.83 to 2.05 mmol/L), and a twofold increase of alkaline phosphatase activity (368 IU/L, reference interval 0 to 150 IU/L). Complete blood count (CBC) results showed severe normocytic, normochromic, nonregenerative anemia; severe thrombocytopenia; moderate neutropenia; lymphopenia; and a prevalent population (about 90%) of atypical cells [Table 1]. Atypical cells were medium sized (12 to 25 μ in diameter) with moderately abundant blue cytoplasm, often with distinct and round vacuoles. Nuclei were central, round, and occasionally had visible nucleoli. Many cells had cytoplasmic projections (blebs), and many large cytoplasmic fragments were identified as atypical platelets [Figures 1A, 1B]. Hypocellular and hemodiluted bone marrow samples contained >70% atypical cells along with a very low percentage of erythroid and myeloid precursors and an absence of stainable iron deposits and particles. Fine-needle aspiration biopsy of the spleen and prescapular lymph node were hypercellular and contained >80% round, variably sized, often bi- to multinu-cleated atypical cells. These cells had prominent anisokaryosis; round to irregularly shaped nuclei; finely to coarsely stippled chromatin; occasionally visible nucleoli; and basophilic, frequently microvacuolated cytoplasm [Figure 2]. Rare, residual, small lymphocytes and occasional plasma cells were also found.
With flow cytometry of blood and bone marrow samples, neoplastic cells were characterized by elevated forward scatter (FSC) and intermediate side scatter (SSC). Results of cell phenotypes are summarized in Table 2. Immunocytochemistry for platelet-specific antigens showed positivity to both CD61 and vWF [Figures 3A, 3B]. Because of the dog’s rapid deterioration and poor prognosis, the owner elected euthanasia 4 days after initial presentation.
Case No. 2
A 5-year-old, male French bulldog with a history of juvenile demodicosis and prior surgical removal of a cutaneous mastocytoma was presented with depression, pale mucous membranes, and prominent splenomegaly. A CBC revealed severe nonregenerative, normocytic, normochromic anemia; severe thrombocytopenia; and leukopenia with 66% atypical nucleated cells (2840 cells/μL) on the blood film. Atypical cells were 15 to 25 μ in diameter, with moderate basophilic cytoplasm; several round vacuoles and cytoplasmic blebs; round, central nuclei with diffuse chromatin; and occasionally one to two visible nucleoli. Many cytoplasmic fragments similar to macroplatelets and occasionally binucleated cells and cells with karyolysis were seen.
Flow cytometry of atypical blood cells was performed for the following antigens: CD45, CD18, CD3, CD4, CD8, CD21, CD14, CD11b, CD34, and NSA (neutrophil-specific antigen, clone CAD048Ad). A prevalent population of cells with high FSC and intermediate SSC properties was found. Results of immunophenotyping have been summarized in Table 2. A final diagnosis of acute megakaryoblastic leukemia was made. The owner refused any further investigation (including bone marrow aspirate) and elected euthanasia within 3 days of initial presentation.
Case No. 3
A 9-year-old, spayed female Corso mastiff was presented with a history of hyperthermia (39.4°C), depression, anorexia, and mild polydipsia. A mild splenomegaly was found on clinical examination, and a splenic FNB was performed. A CBC revealed a moderate normocytic normochromic anemia (packed cell volume [PCV] 22%) with thrombocytopenia. Total leukocyte and neutrophil counts were within the established reference intervals, but the dog was lymphopenic, and 61% of total nucleated cells were atypical medium to large round cells. Similarly abnormal cells (approximately 60%) were also noted on examination of the splenic FNB. A tentative diagnosis of acute leukemia was made.
Six days after initial presentation, the anemia had worsened (PCV 13.9%), and approximately 44% of all circulating nucleated cells were atypical. Atypical cells were 12 to 30 μ in diameter with basophilic cytoplasm, round cytoplasmic vacuoles, and occasional cytoplasmic blebs. Nuclei were round to oval with diffuse chromatin and occasional nucleoli. Cytoplasmic fragments and some erythroid precursors with dysplastic changes (i.e., asynchronous maturation and megaloblasts) were found in the peripheral blood. Bone marrow was hemodiluted with neoplastic cells, which represented >80% of the total nucleated cells. Many multi-nucleated or binucleated cells of megakaryocytic lineage were present. Erythrophagocytic macrophages also were evident in bone marrow aspirates. Flow cytometric immunophenotyping of atypical cells in peripheral blood and bone marrow samples was performed using the following antigens: CD45, CD18, CD3, CD4, CD8, CD21, CD14, CD11b, and CD34. Intracytoplasmic staining with anti-CD79a-PE (clone HM57,e B cells, all stages) was performed after permeabilization of the cells using BD FACS permeabilizing solution 2.f Atypical cells showed high FSC and intermediate SSC properties and were faintly positive only to common markers CD45 and CD18 [Table 2].
A final diagnosis of AMegL was made, but the owner refused chemotherapy. Supportive therapy including corti-costeroids (prednisone 32 mg per os q 24 hours) was administered. According to the owner, clinical status improved for 2 weeks; then the dog worsened rapidly and died spontaneously 35 days after initial presentation.
Discussion
Megakaryoblastic leukemia is an acute neoplastic disorder rarely recognized in dogs. To our knowledge, only 11 cases of spontaneous AMegL have been previously described.1–10 Clinicopathological features of these reported cases have been summarized and compared with the three cases described herein [Table 3]. Morphological descriptions of neoplastic cells were drawn from the text or attached figures of reported papers in order to define some common features useful to diagnose canine AMegL.
Canine AMegL was diagnosed in 12 different breeds, but three cases were in German shepherd dogs. Eight females and six males ranging in age from 8 months to 10 years were affected.
A definitive diagnosis of AMegL is usually based on the ultrastructural detection of membrane-bound vacuoles, a dilated canalicular system, and cytoplasmic “bull’s eye” alpha-granules1–5 or positivity to platelet-specific antigens.17,19–20 Electron microscopy is not useful for diagnostic purposes because of its time constraints and costs. While flow cytometry might be useful, platelet-specific markers are not usually included in most reported panels for flow cytometric diagnosis of acute leukemia.6,8–10 In both humans and dogs, AMegL is often a rapidly progressive disease with a poor prognosis. As a result, specific cytomorphological analysis is needed to achieve a preliminary diagnosis of the disease and to rapidly determine which ancillary tests would be required to help achieve a definitive diagnosis.
A severe, nonregenerative, normocytic normochromic anemia is a feature in all reported canine cases of canine AMegL, but this form of anemia is also found in many other neoplastic and nonneoplastic hematological diseases. Anemia is especially common in acute leukemias—both myeloid and lymphoid leukemias.12–13 Leukocytosis occurred in only three (21%) of 14 reported cases with a moderately severe neutrophilia. Leukopenia was more common, occurring in six (43%) of the 14 cases. The number of circulating neoplastic cells ranged from 0 to 16.0 × 109/L. Rubricytosis was found in seven (50%) cases, sometimes with dysplastic features suggesting dyserythropoiesis. While leukopenia and a limited number of neoplastic cells in peripheral blood are common features in human AMegL,14–15 leukopenia is not usually seen in other AMLs in either humans or dogs. In a retrospective study of human patients with AMegL and AMLs other than AMegL, leukocyte count and percentage of blast cells in peripheral blood were statistically lower in AMegL.14 Vernau and Moore describe leukocytosis as a common feature in most canine AMLs, as >77% of dogs with acute leukemia showed extreme leukocytosis (i.e., a white blood cell count >50 × 109/L).13 No extensive data exist regarding the percentage of circulating blast cells in different AML subtypes; however, in the authors’ experience, the presence of a high percentage of circulating blasts is a frequent feature in most canine AMLs (unpublished data).
Even though thrombocytosis has been found in some dogs with AMegL,4,7,9 severe thrombocytopenia occurred in 10 (77%) of the 13 reported cases. Thrombocytopenia is a common feature in most acute leukemias of myeloid and lymphoid origin.
The bone marrow of dogs with AMegL is frequently extensively replaced by neoplastic cells. These neoplastic cells may be up to four times as large as red cells. The cytoplasm of the abnormal cells can be scarce to moderately abundant; it is usually basophilic and often contains several clear cytoplasmic vacuoles and blebs. Cytoplasmic granules are occasionally recognized. Nuclei are usually round and centrally located. Bi- to multinucleated neoplastic giant cells, closely resembling immature or dysplastic megakaryocytes, are frequently recognized in bone marrow as well as in splenic and lymph node aspirates.
From a cytomorphological perspective, five major features of AMegL cells can be described from the literature and observed cases. These major features include: 1) central round nucleus, 2) clear cytoplasmic vacuoles, 3) cytoplasmic blebs, 4) bi- or multinucleated cells, and 5) large cytoplasmic fragments/macroplatelets. Despite the limited number of reported cases of canine AMegL, we believe that the presence of at least four of the above-mentioned major morphological criteria is highly suggestive of AMegL. These criteria were found in all of the previously reported cases in which accurate morphological data were available (five of 11 cases) and in all three cases reported herein. Unfortunately, in six of the reported cases,1,5–7,9 descriptions of morphological aspects and images were not adequate to correctly evaluate the presence of all major features described above, but a minimum of two features were found in each of them.
Disseminated histiocytic sarcomas (DHS) should be considered as an important differential diagnosis for AMegL, since both neoplasms share the presence of cytoplasmic microvacuolization, bi- or multinucleated cells, and occasionally cytoplasmic fragments. Disseminated histiocytic sarcoma, however, is rarely leukemic. Moreover, in DHS, cytoplasmic borders of neoplastic cells are often indistinct, and nuclei are usually peripherally placed and oval to indented in shape. In histiocytic disorders, flow cytometry identifies a high expression of CD45 and CD18, and most neoplastic cells are positive to CD14, CD11c, CD1a, and CD1c.21,22 In contrast, the cases of canine AMegL described here showed low expression of common antigens, and CD14 was negative.
To our knowledge, only one previous case report evaluating canine AMegL immunophenotyping by flow cytometry has been published.10 In this report, as in our three cases, neoplastic cells were negative to all lymphoid (CD3, CD4, CD8, CD79a, CD21) and myeloid markers (CD11b, CD11c, CD14), while common markers CD18 and CD45 bear negative to low expression. Acute leukemias of lymphoid and myeloid lineage are often characterized by low expression of leukocyte common markers because of the immaturity of cells.18,23 Precursor antigen CD34 was positive in a small percentage (15.5%) of neoplastic cells in the peripheral blood of one of the three cases described in the current report. In humans, CD34 is reported to be expressed in 40% to 60% of AMegL cases.24
A complete immunophenotyping of leukemic cells has been performed, applying immunocytochemistry or immunohistochemistry in a wide number of reported cases in dogs, and the reports mainly agree on the lack of all lymphoid and myeloid markers in AMegL cells. Park et al reported positivity to CD79a (a B-cell marker).9 In the present report, CD79a was performed only in case no. 3 and was negative—a finding that was consistent with most of the other reported cases as well as in the human literature.9–10
Conclusion
Acute megakaryoblastic leukemia may be an underestimated entity in dogs, mainly because immunophenotyping panels do not often include megakaryocyte/platelet markers. In dogs, AMegL is an aggressive disease with a poor prognosis, involving bone marrow, internal organs, and lymph nodes. Although a final diagnosis requires positivity to platelet-specific markers, the identification of morphological features could be useful to reduce possible differentials and to expedite achieving a tentative diagnosis.
AbD Serotec; Kidlington, United Kingdom OX5 1GE
BD Pharmingen; BD Biosciences, San Josè, CA 95131
Dako; Glostrup, Denmark DK-2600
VMRD Inc., Pullman, WA 99163
Dako; Glostrup, Denmark DK-2600
FACSort; Becton Dickinson, San Josè, CA 95131
Acknowledgments
We thank Drs. Raffaella Capitelli and Marco Trabucco for submitting cases and clinical data. We also thank Dr. Manuela Florenti for performing immunocytochemical analysis.
Citation: Journal of the American Animal Hospital Association 46, 5; 10.5326/0460327
Citation: Journal of the American Animal Hospital Association 46, 5; 10.5326/0460327
Citation: Journal of the American Animal Hospital Association 46, 5; 10.5326/0460327
Blood smear (feathered edge) of the dog described as case no. 1. Cells with central round nuclei and microvacuoles are seen in both images. A circulating binucleated neoplastic cell is evident (A), and cytoplasmic blebs and large cytoplasmic fragments are shown (B). (May Grünwald-Giemsa stain, 1000×; bar=20 μm.)
Fine-needle biopsy from prescapular lymph node of the dog described as case no. 1. Many neoplastic cells with vacuolated cytoplasm and bi- to multinucleated neoplastic megakaryocytes are seen. (May Grünwald-Giemsa stain, 1000×; bar=20 μm.)
Immunocytochemistry of bone marrow from the dog described as case no. 1. Neoplastic cells were both CD61 (A) and vWF (B) positive. (1000×; bar=20 μm.)
