Case of the month #11
by Giulia Mangiagalli (DipECVCP)
The smear has good cellularity and preservation, with high numbers of erythrocytes on a finely pink granular background. Main population of intermediate to large round cells is noted. These cells have scant to moderate amount of dark blue cytoplasm occasionally with perinuclear clear halo or with few clear vacuoles, mostly with well-defined borders and cytoplasmic protrusions (blebs). The nucleus is 2-4 RBC in diameter, mostly round, paracentrally to centrally located, with smooth chromatin and inconspicuous nucleoli; bi-multinucleation and mitosis are often noted. Few foamy macrophages occasionally in cyto-phagocytosis are also seen.
Cytological findings are consistent with malignant round cell tumor. Main differential diagnoses are acute leukemia and intermediate to large cell lymphoma.
Looking at cyto-morphological aspects, acute megakaryoblastic leukemia (AML-M7) should be suspected.
Immunophenotyping by flow cytometry of the thoracic effusion previously collected in EDTA tube was performed. This exam identified a main population of intermediate to large immature cells positive to CD45/CD18/CD34/CD61, and negative to the other lymphoid and myeloid markers.
This phenotype pattern was consistent with acute megakaryoblastic leukemia (AML-M7).
The owner did not accede to perform additional tests (e.g. bone marrow, and spleen cytological sampling), and any chemotherapy was rejected. Immunosuppressive therapy based on glucocorticoids (1 mg/kg twice per day) was established as therapeutic support. After 3 weeks, humane euthanasia was elected given the worsening of clinical conditions.
Acute megakaryoblastic leukemia (AML-M7) is a rare acute neoplastic hematological disorder which involves the megakaryocytic lineage. Only 19 spontaneous cases have been reported in the literature, and in several canine breeds: German shepherd seems to be over-represented.
Clinical and clinicopathological findings
Clinical alterations are not-specific such as pale mucous membrane, depression, anorexia, splenomegaly and lymphadenomegaly: metastasis to the spleen and lymph nodes are commonly seen. Bleeding disorders may be also reported.
Severe nonregenerative normocytic normochromic anemia is the main hematological alteration reported in affected dogs, as in all acute leukemia usually occurs; rubricytosis (normoblastemia) with dysplastic features is also seen suggesting dyserythropoiesis. Leukocytosis occurs rarely, while leukopenia is more commonly seen. Severe thrombocytopenia is a common hematological alteration as result of maturation arrest due to neoplastic involvement of the lineage, but a minority of patients may present thrombocytosis as well.
Diagnosis
A definitive diagnosis of AML-M7 is usually based on electron microscopy for the ultrastructural detection of membrane-bound vacuoles, a dilated canalicular system, and cytoplasmic “bull’s eye” alpha-granules or positivity to platelet-specific antigens. Electron microscopy is not useful for diagnostic purposes because it is time-consuming and expensive, while immunophenotyping by different techniques is considered more reliable to assess the lineage of neoplastic cells.
However, identification of specific cytomorphological aspects is useful to achieve a preliminary diagnosis of this disease and to rapidly determine which antigenic positivity must be investigated.
The neoplastic cells may show 5 characteristic morphological aspects: bi-multinucleation, central round nucleus, multiple discrete cytoplasmic vacuoles, cytoplasmic projections (called blebs) or pseudopodia, and cytoplasmic fragments resembling macroplatelets on the background. Finally, purple granules may be also noted. The presence of at least 4 of the above-mentioned features is highly suggestive of this neoplastic disorder.
By flow cytometry, these neoplastic cells are identified as megakaryoblasts positive to CD61 (member of beta-3 integrin also called glycoprotein IIIa, expressed early in megakariopoiesis and involved in platelet adhesion and interaction with fibrinogen, von Willebrand factor, fibronectin, thrombospondin, and vitronectin). Neoplastic cells are negative to lymphoid and myeloid antigens, while common leukocytes markers CD45 and CD18 bear usually negative or with low expression: this aspect is common to all acute leukemias due to cells immaturity. Unfortunately, progenitor antigen CD34 is positive in a small percentage of cases (around 15%).
Moreover, megarkaryoblasts are generally positive to immunohistochemistry or immunocytochemistry for vonWillebrand factor, CD41 (glycoprotein IIb), and CD62P (P selectin). Blasts may express ANBE but are negative for other myeloid histo or cytochemical stains (SBB, MPx, CAE).
The bone marrow is usually largely replaced by neoplastic cells which may be up to four times as large as erythroid precursors and show cytomorphological aspects described before. AML-M7 is diagnosed when megakaryoblasts account for 20% or more of nucleated cells in bone marrow or consists of ≥50% cells of megakaryocyte lineage. Sings of dysplasia in megakaryocytes (and other lineages), and fibrosis may be also present. Cytology of spleen and lymph nodes which are often involved may be of value for the complete staging of the neoplasia.
Differential diagnoses
Possible differential diagnoses of AML-M7 include several acute leukemias (e.g. AML without maturation or with minimal differentiation, acute monoblastic leukemia, acute erythroid leukemia) and large cell lymphoid neoplasms. Disseminated histiocytic sarcomas should be considered since it shares the presence of cytoplasmic vacuolization, bi-multinucleated cells, and occasionally cytoplasmic fragments.
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