PMC5402055_01
Bone marrow cytological and histopathological features at diagnosis. a Leishman's (A1), toluidine blue (A2) and chloroacetate esterase (A3) staining of bone marrow (BM) smears from the patient at diagnosis, revealing atypical mast cells (MCs) with elongated cytoplasmic extensions and abnormal granulation (*). A normal MC is shown for comparison (A1, **). b Hematoxilin-eosin (B1, 40x; B2, 400x), Giemsa staining (B3, 400x) and CD117 immunostaining (B4, 400x) of a BM trephine biopsy from the patient at diagnosis, showing normocellular marrow with osteosclerosis (B1, *) and scattered elongated and degranulated abnormal (B2 and B3, *), CD117 positive (B4) MCs
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Fig1
The BM aspirate was normocellular with increased numbers of MC (1.7%), most of which had an atypical (type I) morphology (spindle-shaped MC with irregular distribution and focal accumulation of the cytoplasmic granules, and elongated cytoplasmic extensions); the myeloid/erythroid ratio was normal and there was no myelodysplasia (Fig. 1, panel A). The BM trephine (stained with hematoxylin-eosin and Giemsa), showed a normocellular BM without dysplastic features or increased blast cells, but a few scattered morphologically abnormal MCs, difficult to recognize (Fig. 1, panel B).
Immunophenotypic and genetic features of bone marrow (BM) mast cells (MCs). a Illustrating bivariate dot-plots of the BM cells from the patient (panel A1), and after selecting specifically for BM mast cells (panels A2-A4); as illustrated, bone marrow MCs showed a mature (CD117+high, FcepsilonRI+high) (panel A2), aberrant (CD2+, CD25+) (panel A3) immunophenotype, with expression of activation-associated markers (CD63, CD69) in a large fraction of the cells (panel A4). Corresponding dot plots from normal bone marrow MCs (blue dots) are show for comparison. b Sequences of polymerase chain reaction (PCR) products illustrating the presence of the KIT V560G mutation in bone marrow MCs from the patient, at diagnosis (panel B2); the wild-type (WT) sequence is shown for comparison
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Fig2
Flow cytometry confirmed the presence of BM mast cells with abnormally high light scatter values (SSC and FSC), and a mature (CD117+high, FcepsilonRI+high), but aberrant (CD2+, CD25+) and activated (CD63+high, CD69+high) phenotype (Fig. 2, panel A). Molecular analysis showed the absence of the KIT Asp-816(r)Val mutation; in contrast, the KIT Val-560(r)Gly activating mutation was detected in FACS-purified BM mast cells (Fig. 2, panel B), but not in other BM myeloid and lymphoid cells, including BM precursors.