PMC4130965_01
Morphology of neoplastic cells. (A) Composite showing examples of pleomorphic circulating leukemic cells in peripheral blood obtained from subsequent peripheral blood smears. (B) Bone marrow aspirate smear showing four blastoid looking cells (arrows) and dysplastic megakaryocyte with multiple widely separated nuclei) (Lower left corner).
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Fig1
The majority of circulating lymphocytes were small and mature with very rarely encountered atypical-primitive looking lymphoid cells (Figure 1A). The dysplastic megakaryocytes were also present in the aspirate smear, however, their percentage cannot be determined due to insufficient aspiration material obtained (Figure 1B). Deletion of the p53 gene was confirmed by FISH in 11% of the nuclei cells analyzed. Interphase nuclei cells showed normal hybridization pattern when MLL gene was analyzed. During the patient's stay in the hospital, his general condition was deteriorated with increase of the tumor burden, aggravation of ascites, hepatosplenomegally, and lymphadenopathy along with progressive pancytopenia and increase in the percentage of the abnormal lymphoid cells in the peripheral smear (Figure 1A).
Bone marrow biopsy findings. (A) Biopsy section (H&E), at low magnification (20x) showing infiltration by abnormal lymphoid cells. (B) CD56 immunostain highlighting marked increase in NK cells (50x). (C) Reticulin stain highlighted an increase in reticulin fibrosis (50x), (D) Trichrome stain highlighting focal bundles of collagen deposition (50x).
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Fig2
Examination of bone marrow biopsy revealed hypercellular marrow with few necrotic areas; that is extensively infiltrated with atypical lymphoid cells with round or irregular nuclei, rather condensed chromatin, multiple small nucleoli residing within a reactive inflammatory back ground composed of small lymphocytes, histiocytes, neutrophils, eosinophils, and polyclonal plasma cells (Figure 2A).
Megakaryocytic atypia and anisocytosis. (A) Biopsy section (reticulin stain) showing megakaryocytic atypia with small clusters, (B) CD61 immunostain showing megakaryocytic clustering (10x), (C) CD61 immunostain showing anisocytosis (50x).
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Fig3
In addition, there was striking megakaryocytic hyperplasia with some clustering, marked atypia in the form of abnormal separation of nuclear lobes with evident anisocytosis and some micromegakaryocytes (Figure 3).
Immunohistochemistry performed on bone marrow biopsy section (20x). The lymphoid infiltrate is positive for CD3 (epsilon) (A), negative for CD5 (B), positive for CD56 (C) positive for EBV (D).
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Fig4
By immunohistochemical stains, the abnormal cells were strongly positive for CD3 epsilon (CD3epsilon polyclonal, Dako, Denmark), CD56 and EBV RNA (using ISH technique) (Figure 4) and negative for CD20, CD5, CD4 & CD8 with partial weak positivity for CD7.
Flow cytometry performed on ascitic fluid. The population of interest expresses CD2, CD7 and CD56, and lacks surface CD3, CD16, CD57 and TCR alpha-beta. Abbreviations ECD, phycoerythrin-Texas Red; FTIC, fluorescein isothiocyanate; PE, phycoerythrin.
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Fig5
Flow cytometry was then performed on ascitic fluid using antibody panel: CD45, CD2, sCD3, CD5, CD7, CD4, CD8, CD56, CD57, CD16, TCR alpha-beta and TCR gamma-delta, it revealed approximately 20% NK-cells expressing CD2, CD7 & CD56, they were negative for CD3 (surface), CD4, CD5, CD8, CD57, TCR alpha-beta and TCR gamma-delta (Figure 5).
Karyotype by conventional cytogenetics. ISCN:44,X,-Y, der(2) t(2;10) (q37;q11.2), der(17) (p?11.2) (Zhang et al.). A minimum of 20 metaphases were karyotyped according to the International System for Human Cytogenetic Nomenclature (Shaffer et al.), image capturing, processing and data were carried out using microscope Zeiss-AX10 scope.A1 with IKAROS imaging system (Metasystems, Germany).
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Fig6
Conventional Cytogenetic analysis using GTG-banding was performed on unstimulated 24 and 48 hours culture of bone marrow aspirate according to standard procedures; it revealed an abnormal clone with complex karyotype (>=3 aberrations) consisting of aneuploidy cells with 44, X, (missing the sex chromosome Y), derivative chromosome 17, derivative chromosome 2 that resulted from reciprocal translocation between the chromosomes 2 and 10 at bands q37 and q11.2, respectively, besides an additional material of unknown origin attached at band p?11.2 in 15 cells (Figure 6).