PMC5835284_01
Peripheral blood smears showing different types of morphologically abnormal mast cells. Leishman's stain (a, 40x; b, 100x). Circulating mast cells (MC) had variable morphological features, from metachromatic blasts of medium to large cell size, round to oval shape, fine/immature nuclear chromatin, high nuclear : cytoplasmic ratio, and cytoplasmic metachromatic granules, to atypical MC type 2 with variable, sometimes spindle, cell shape, bilobated nucleus with fine to moderately condensed chromatin, variable nuclear : cytoplasmic ratio, and hypogranulated cytoplasm. Please note that in spite of these heterogeneous morphological features, all the MC present in the peripheral blood had an aberrant immature phenotype, corresponding to abnormal MC precursors (Figure 4).
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fig1
Bone marrow smears showing myeloperoxidase positive blast cells and promyelocytes, and morphologically abnormal toluidine blue positive mast cells. (a) Leishman. (b) Myeloperoxidase. (c) Toluidine blue staining.
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fig2
Bone marrow smears showed 24% myeloperoxidase (MPO) positive BC, 17% promyelocytes, 4% myelocytes, 3% metamyelocytes + neutrophils (AML-M2 classification by cytomorphology), 30% erythroid lineage, and 16% morphologically abnormal toluidine blue positive MC (Figure 2).
Bone marrow dot plots obtained by flow cytometry revealing 12% CD117+ CD34+ myeloid precursor cells (black dots), 5% CD117+CD34-mast cell precursors (orange dots), 13% of abnormal CD2- CD25+ mast cells (red dots), and 49% granulocytic cells with a maturation blockage at the promyelocyte level (purple dots). Myeloid precursor cells (black): CD117+, CD34+, CD2-, CD25-/+, CD13+, CD15-, CD16-, CD30-, CD33+, CD63+, CD65+low, CD69+low, CD123+, HLA-DR+high, and FcepsilonRI/IgE-. Mast cell precursors (orange): CD117+, CD34-, CD2-, CD25+, CD13+, CD15-, CD16-, CD30+, CD33+, CD63+, CD65+low, CD69-, CD123+ high, HLA-DR-/+low, and FcepsilonRI/IgE+. Mast cells (red): CD117+ high, CD34-, CD2-, CD25+ high, CD13+, CD15-, CD16-, CD30+, CD33+, CD63+, CD65+low, CD69+, CD123+ high, HLA-DR+high, and FcepsilonRI/IgE+low.
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fig3
CD45+ (low), CD117+, CD34+ myeloid precursor cells (MPC) also expressing CD123, HLA-DR (high), CD13, CD33, CD65 (low), and CD25 (low, in part of the cells) but lacking CD10, CD15, CD16, CD2, CD30, and FcepsilonRI/IgE; 5% of CD45+ (low), CD117+, CD34- MC precursors (MCP), also being positive for CD123 (high), CD13, CD33, CD65 (low), CD25, CD30, FcepsilonRI/IgE, and HLA-DR (high), and lacking CD10, CD15, CD16, and CD2; 34% promyelocytes with an aberrant phenotype (CD45+, CD34-, CD117+, CD13+low, CD33+, CD65+; CD15+, MPO+, and CD2, CD10, CD11b, CD16, CD25, CD30, FcepsilonRI/IgE, and HLA-DR negative); a maturation blockage at the promyelocyte level, as revealed by an abnormal CD11b/CD13/CD16 maturation pattern, with <1% of CD16+CD10+ mature neutrophils; and 13% of abnormal CD45+, CD34-, CD117+high MC with a relatively immature (CD123+high, FcepsilonRI/IgE+ low, and HLA-DR+high), activated (CD63+, CD69+), and aberrant (CD2-, CD25+, and CD30+) immunophenotype (Figure 3). Cytoplasmic carboxypeptidase and surface CD203 were also positive (data not shown).
Peripheral blood dot plots obtained by flow cytometry showing 3% of CD117+ CD34+ myeloid precursor cells (black dots) and 49% of CD117+ CD34-mast cells precursors (orange dots). Please note that circulating myeloid precursor cells (black dots) and mast cells precursor (orange dots) had an immunophenotype like that observed in bone marrow (BM) myeloid precursor cells and BM mast cell precursors, respectively (please cf. Figure 3, same color code). Please also note that the relatively mature mast cells observed in BM (red dots, Figure 3) were not present in the peripheral blood.
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fig4
In addition, FCM performed in PB, showed 3% CD45+low, CD117+, CD34+ MPC, and 21% of CD45+low, CD117+, CD34- MCP, which were phenotypically similar to the correspondent BM cell populations, at least for the cell surface markers tested, but not CD45+, CD34-, and CD117+ high MC (Figure 4).
Bone marrow trephine biopsy showing a hypercellular marrow (a) with grade 2 fibrosis (b), and a preponderance of myeloid precursors with a diffuse proliferation of myeloperoxidase+ blast cells (c and d) and CD117+ mast cells (e and f). (a) Hematoxylin and eosin (H&E) stain, original amplification x40. (b) Reticulin stain, original magnification x100. (c) H&E stain, original amplification x100. (d) MPO stain, original amplification x100. (e) H&E stain, original amplification x100. (f) CD117 stain, original amplification x100.
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fig5
Bone marrow trephine biopsy revealed a hypercellular marrow with increased proportions of immature MPO+ myeloid cells and morphologically atypical CD117+ fusiform MC forming perivascular dense aggregates, and grade 2 fibrosis (Figure 5).
Serum tryptase levels from diagnosis of MCL-AML to date. Serum tryptase levels were markedly increased at the diagnosis (184 mug/L, October 2015) and decreased transiently to 41 mug/L after two cycles of cladribine (5 days, with one month of interval: December 2015; January 2016). However, one month later (February 2016), the serum tryptase levels increased again to 123 mug/L, and the BM aspirate showed 47% myeloblasts. At that time, the patient received two cycles of combined therapy with idarubicin and cytosine arabinoside (3 + 7 days) (February 2016; April 2016) followed by two cycles of high-dose cytosine arabinoside (June 2016; July 2016). At day  +32 of reinduction chemotherapy (May 2016), the patient achieved complete hematological remission, and the serum tryptase levels decreased to 8 mg/L, remaining normal to date. At the end of August 2016, the patient received allogeneic hematopoietic stem cell transplantation (HSCT) from her sister. By the time of this report (November 2017, 24 months after the diagnosis, 15 months after HSCT), she maintains a complete hematological remission and a complete chimerism in peripheral blood.
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fig6
Then, she received two cycles of cladribine (0.14 mg/kg/day, administered over a 2-hour infusion for 5 days) with one month of interval, and the serum tryptase levels transiently decreased to 41 mug/L (Figure 6). AML consisting of two cycles of idarubicin (12 mg/m2/day, intravenous, for 3 days) and cytosine arabinoside (AraC) (100 mg/m2/day, intravenous, for 7 days), achieving hematological remission and normal tryptase levels after the second induction course (Figure 6). At that time, the By the time of this report (24 months after the diagnosis, 15 months after HSCT), she maintains normal serum tryptase levels, complete hematological remission, and complete chimerism in PB (Figure 6).