Leukemia Research Vol. 15, No. 10, pp. 887-897, 1991.

0145-2126/91 $3.00 + .fl~ Pergamon Press plc

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M O R P H O L O G I C A N D FLOW CYTOMETRIC ANALYSIS OF C I R C U L A T I N G M E G A K A R Y O B L A S T S IN C H R O N I C M Y E L O I D LEUKAEMIA ANDRAS MATOLCSY, ENDRE KALMAN, L,~SZL0 PAJOR, TIBOR KONYA and EDIT WEBER Department of Pathology, University Medical School of P6cs, Hungary (Received 13 March 1991. Accepted 13 April 1991) Abstract--The immunophenotype (a), ultrastructural features (b) and cell kinetics (c) of circulating megakaryoblasts have been studied in two cases of pure megakaryoblastic and one case of mixed (myeloblastic, megakaryoblastic) cell proliferation in chronic myeloid leukaemia (CML). (a) The blast cells showed early megakaryocyte differentiation antigen (HLA-DR), platelet specific GplIIa (CD61) and Gplfb-IIIa (CD41) antigens in different percentages. (b) The megakaryoblasts were recognized by the presence of platelet GplIIa (CD61) demonstrated by an immunoelectron microscopic method. The labelled cells were "lymphocyte-like" megakaryoblasts and cells with features of cytoplasmic maturation (demarcation membranes, alpha granules and vacuoles). (c) Cellular DNA content of the megakaryoblasts was measured by propidium iodide (PI) staining of cells expressing platelet Gplfla (CD61). Flow cytometric (FC) DNA analysis revealed no aneuploidy and high ploidy (>4N) cell population. In the two cases of pure megakaryoblastic proliferation a high percentage of the megakaryoblasts were in the S-phase, while the non-megakaryoblastic cell fraction showed no elevated S-phase compartment. It is concluded that in CML the circulating megakaryoblasts (1) have a nuclear maturation arrest and accumulation at the level of tetraploid DNA content, (2) surface antigen expression and cytoplasmic organelles show a tendency to mature and (3) in pure megakaryoblastic proliferation the myeloid cells are not in the cell compartment showing high proliferation. Key words: Cell cycle distribution, chronic myeloid leukaemia, DNA content, flow cytometry, immunoelectron microscopy, megakaryoblast.

INTRODUCTION

Hibbin et al. [12] have shown that the number of circulating megakaryocyte progenitors (CFU-Mk) are increased in patients with CML compared with normal individuals, and that they have a remarkable tendency to unstimulated colony formation [13-15]. Cultures of megakaryocytes from patients undergoing megakaryoblastic transformation of CML showed a tendency to mature; however, dysplastic features of the cytoplasm are detected [16]. Although a large number of cytometric studies have been cartied out on CML [17-22], the cell kinetics of circulating megakaryoblasts has not been studied. The present study was designed to analyse the cell cycle distribution of circulating leukaemic megakaryoblasts in CML and to compare their electron microscopic features and antigen expression.

ABNORMAL proliferation of the megakaryocytic cell line has been demonstrated in the bone marrow and peripheral blood from patients with chronic myeloid leukaemia (CML) [1-3]. Such an expansion of the megakaryocytic compartment is characterized by the presence of cells with a number of different histologic features including dwarf megakaryocytes, promegakaryocytes, micromegakaryocytes and megakaryoblasts [4--6]. The recently developed monoclonal antibodies (MoAb) specific for megakaryocyte and platelet antigens enable recognition of the circulating megakaryoblasts and analysis of their immunophenotypes and morphological aberrations [7-11]. Abbreviations: ANAE, alpha-naphthyl acetate esterase; AP, acid phosphatase; CD, cluster designation; CML, chronic myeloid leukaemia; FC, flow cytometry; FITC, fluorescein isothiocyanate; MoAb, monoclonal antibody; MPO, myeloperoxidase; PAS, periodic acid-Schiff; PBS, phosphate-buffered saline; PI, propidium iodide. Correspondence to: Dr A. Matolcsy, Department of Pathology, University Medical School, Szigeti ut 12, H7643 P6cs, Hungary.

MATERIALS AND METHODS Patients Three patients with CML were diagnosed according to the currently accepted clinical, haematological, cytogenetical and histological criteria [1, 2]. They were selected 887

888

A. MATOLCSYet

al.

TABLE 1. HAEMATOLOGICALDATA OF THE 3 CML PATIENTSWITH MEGAKARYOBLASTICCELL PROLIFERATION No.

Age/sex (yr)

WBC (x 109/1)

1 2 3

64/M 55/M 60/M

15.2 22.0 19.2

Platelets HTC (× 109/1) (%) 30 600 220

22.0 25.0 34.0

Blasts (%)

Splenomegaly (cm)

Duration (months)

Treatment

MF

Phl

20 28 21

16 22 --

4 30 36

Regular transfusion Regular transfusion Splenectomy

++ +++ +++

+ + +

Duration, duration of the disease; WBC, white blood cell count; HTC, haematocrit; MF, myelofibrosis (+ - mild, + + - moderate, + + + - severe). from patients exhibiting a variable percentage of megakaryoblasts in the peripheral blood and bone marrow. Their clinical data are presented in Table 1.

Cytochemistry May-Griinwald-Giemsa, myeloperoxidase (MPO), periodic acid-Schiff (PAS), acid phosphatase (AP) and alphanaphthyl acetate esterase (ANAE) reactions were performed on blood smears, using standard methods [23].

Antibodies The MoAbs employed in this study and detailed in Table 2 were obtained through the courtesy of Prof. Dr W. Knapp, Institute of Immunology, University of Vienna.

lmmunoelectron microscopy [33] Washed mononuclear cells (5 x 106 cell/ml) were fixed for 5 min in PBS, pH 7.2, containing 1.0% glutaraldehyde. The fixed cells were washed three times in PBS, pH 7.2, and treated for 1 h at 4°C with 100 p.l of VI-PL2 (CD61) MoAb diluted 1 : 10 with PBS. After three washes in PBS the cells were incubated for 1 h with 100 lxl peroxidaseconjugated goat anti-mouse immunoglobulin (DAKO) diluted 1 : 50 with PBS. After three washes with the same buffer, the cells were fixed in PBS containing 1.0% glutaraldehyde. Following a further washing in PBS, peroxidase activity was revealed in a 2 mg/ml DAB medium [34]. The cells were then postfixed in osmium tetroxide, dehydrated and embedded in Epon. Ultrathin sections were examined in a Jeol 100 electron microscope.

Cell preparation

Simultaneous immunofluorescence and DNA staining [35]

Peripheral venous blood from the three patients with CML was drawn into syringes containing preservative-free heparin. All samples were subjected to a red blood cell sedimentation. After 1 h, the buffy-coat was removed and centrifuged at low speed (150g, 10min) to remove the platelets. The leukocyte pellet was washed twice in 1.0 M phosphate-buffered saline (PBS).

1 × 106 cells were incubated with 5 ~tl VI-PL2 MoAb at 4°C for 30 min. Following two washes in cold (4°C) PBS, the cells were incubated for an additional 30 rain at 4°C in 100 ~tl of fluorescein isothiocyanate (FITC)-conjugated F(ab)2 rabbit anti-mouse IgG (DAKO). After fluorescence staining with VI-PL2 MoAb, the cells were washed twice in PBS, then fixed on ice in 70% ethanol for 30 min and subsequently treated with 1 mg/ml of RNAase (Sigma) at 37°C for 30 min. Propidium iodide (PI, Calbiochem) was added to a final concentration of 5 ~tg/ml.

Immunophenotyping of blast cells Immunocytochemical studies were performed on routinely prepared, air-dried blood smears and cytocentrifuge preparations, using the alkaline phosphatase anti-alkaline phosphatase method [32].

Two-parameter flow cytometry (FC) Analyses of the cells for surface immunofluorescence

TABLE 2. MONOCLONALANTIBODIESUSED IN THIS STUDY CD

Antibody

Specificity

CD3 CD4 CD8 CD10 CD14 CD15 CD24 CD41 CD61 CDw65 ---

Leu-4 Leu-3a Leu-8 VIL-A1 VIM-13 VIM-D5 VIB-E3 VI-PL1 VI-PL2 VIM-2 VID-1 VIE-G4

pan-T lymphocytes T helper T suppressor common ALL antigen myelomonocytic cells granulocytic cells B cells platelet (GpIIb-IIIa) platelet (GpIIIa) myeloid HLA-DR (glycophorin A)

Source and references Becton-Dickinson Becton-Dickinson Becton-Dickinson Knapp, W. [24] Knapp, W. [25] Knapp, W. [26] Knapp, W. [27] Knapp, W. [28] Knapp, W. [28] Knapp, W. [29] Knapp, W. [30] Knapp, W. [31]

CD nomenclature from the Fourth International Workshop on Human Leukocyte Differentiation (Vienna, February 1989).

Circulating megakaryoblastsin CML and DNA content were performed using a Partec PAS II flow cytometer equipped with a high pressure mercury lamp (100 W). Measurements of the cells were made in a band from 470 to 500 nm excitation using an Ex 488 BP filter. The emitted light was split with a TK560 dichroic mirror. The surface fluorescence emission (FITC-CD61) was measured in a band from 515 to 560 nm, and nucleic acid associated fluorescence emission (PI-DNA) in a band above 590 nm. For each sample 50 000 cells were analysed. The proportion of cells in the G0/G1, S, and G2 + M phases of the cell cycle were calculated according to the method described by Baisch et al. [36].

RESULTS

Cytology and cytochemistry The percentage of blast cells in the peripheral blood of the three patients ranged between 20 and 28% meeting the requirements of the accelerated phase of CML. The morphological appearance of the peripheral blast cells varied from case to case: they were generally undifferentiated, either small "lymphocyte-like" forms with cytoplasmic blebs or larger cells with a more abundant basophilic cytoplasm (Fig. 1). The majority of blast cells were positive for ANAE and PAS. All the blast cells in cases 1 and 3 were MPO-negative. In case 2 nearly half the blast cells showed a fine, granular MPO reaction. No APpositive blast cell population was detected. Immunophenotype The reactivity of MoAbs to white blood cells of the three CML patients is detailed in Table 3. MoAb against HLA-DR, platelet GplIIa (CD61) and platelet GplIb-IIIa (CD41) antigens reacted with a different percentage of blast cells. In cases 1 and 3, the cells labelled with myelomonocytic MoAbs (CD15 and CDw65) had the morphological structure of granulocytes, myelocytes and promyelocytes; the blast cells did not show a positive reaction. In case 2, half the cells with a blastic character (13% of all white blood cells) revealed a positive reaction with CD15 and CDw65 MoAbs. Very few small cells were labelled with lymphoid (CD3, CD4, CD8, CD10, CD24) markers, and there was no reaction with MoAb against erythroid cells (VI-EG4). Electron microscopy The megakaryoblasts were surface labelled with anti-platelet GplIIa (CD61), and studied by immunoelectron microscopy. The positive reaction exhibited a distinct linear electron dense pattern at the cell surface membranes (Figs 2 and 3). The blast cells varied in diameter between 10 and 30 ~tm. The most primitive, undifferentiated promegakaryoblasts were reminiscent of "small lymphocytes" with a narrow

889

cytoplasm poor in organelles. The cytoplasm frequently showed pseudopodes composed of organellefree cytoplasm (Fig. 2). The larger cells with surface reaction of CD61 showed some tendency to differentiate. The cytoplasm was wide and rich in organelles except for the periphery where it was organellefree. The specific organelles (alpha-granules, vacuoles and demarcation membranes) showing perinuclear clustering. In the nuclei there was marked chromatin condensation (Fig. 3).

Flow cytometry Measurement of the single-parameter DNA content of the peripheral white blood cells is presented in Fig. 4. The distribution of the DNA content shows a highly proliferating cell population. There are no high-ploid (>4N) and aneuploid peaks. No signs of cell aggregation are detected. To determine the cell cycle distribution of megakaryoblasts and to compare them with the non-megakaryoblastic cells population, a two-parameter (CD61, DNA) FC analysis was performed. The CD61-positive cell population showed two distinct accumulations: the first represented the G0/G1 phase, the second the G2 + M phase of the cycle; the S-phase of the cell cycle was between the two (Fig. 5). The cell cycle distribution of CD61-positive and CD61-negative cells is presented in Table 4. The percentage of CD61-positive cells in the S-phase (16.2-26.3%) was considerably increased in all three patients. In two of them with pure megakaryoblastic proliferation (cases 1 and 3), the non-megakaryoblastic cell population (CD61-negative cells) did not show accumulation at the S-phase. In case 2 with mixed, myeloblastic-megakaryoblastic proliferation both the CD61-positive (megakaryoblastic) and the CD61-negative (non-megakaryoblastic) cell populations had an increased percentage of cells in the S-phase. The percentage of CD61-positive cells in G2 + M phase is also elevated (4.8-10.6%), while this elevation of the CD61-negative cell population in the G2 + M phase is not observed. DISCUSSION In this study three cases of CML with megakaryoblastic proliferation were immunophenotyped, evaluated ultrastructurally by immunoelectron microscopy and analysed by two-parameter FC. Cytochemistry and immunophenotype of the blast cells revealed pure megakaryoblastic proliferation in two patients (cases 1 and 3) and bilinear (myeloblastic and megakaryoblastic) blast cell proliferation in one patient (case 2). The megakaryoblasts showed

890

A. MATOLCSYet al.

TABLE 3. REACTIVITY OF MONOCLONALANTIBODIES (MoAb) WITHBLOODCELLSUSINGTHE ALKALINEPHOSPHATASEANTI-ALKALINEPHOSPHATASEMETHOD Case MoAb CD3 CD4 CD8 CD10 CD14 CD15 CD24 CD41 CD61 CDw65 VID-1 VIE-G4

1

2

3

-----32% -16% 17% 23% 16% --

10% 5% 5% -ND 32% -10% 15% 35% 5% --

15% 5% 8% --60% -18% 20% 65% ---

ND, not detected.

immune reactions with megakaryocyte-specific anti bodies (CD41, CD61) and H L A - D R in different percentages from case to case, but in all three cases GpIIIa (CD61) antigen expression was the highest. Several investigators, including ourselves, have suggested that a wide variety of antigen expression on megakaryoblasts corresponds to different levels of maturation [8, 32]. Using a wide range of MoAbs, it was revealed that transient, early differentiation antigens (HLA-DR, CD33, CD13, vimentin) and markers which appear later in the process of megakaryocyte development (factor VIII-related antigen, CD41) were present on leukaemic megakaryoblasts at the same time [37, 38]. The ultrastructural evaluation of megakaryoblasts, using immunoelectron microscopy, also revealed heterogeneity of the blast cells. The appearance of demarcation membranes, vacuoles and alpha-granules in megakaryoblasts might be interpreted as an incipient cytoplasmic maturation. The characteristic features of normal megakaryocyte differentiation and development, such as cell surface antigen changes, appearance of specific organelles and alteration of D N A content, may be reflected in leukaemic blasts [11, 39--41[. Cellular D N A content within megakaryoblastic cells was determined by measuring PI staining of the blood cells expressing GpIIIa antigens. Singleparameter D N A analysis showed a high proliferation fraction, but no aneuploidy or high-ploidy (>4N) cells. It was reported that the S-phase compartment was high at the time of CML myeloblastic crisis, but

not in the chronic phase of the disease [18, 20]. Twoparameter cell cycle analysis revealed a high percentage of S-phase cells (16.2-26.3%) among the megakaryoblasts (CD61-positive cells). In the two patients with pure megakaryoblastic proliferation (cases 1 and 3) evaluation of the non-megakaryoblastic cells (CD61-negative cells), which are mostly myeloid cells, revealed that the S-phase compartment was not increased (4.9-7.6%). In the patient with mixed blastic proliferation (case 2) both myeloid and megakaryoblastic cell population comprised high S-phase compartments. D6rmer et al. [17] carried out a complete cell cycle analysis of the myeloblastic crises of CMLs. They did not find one definite morphologic cell compartment to account for the high proliferation. High proliferation was also detected among myeloblasts and promyelocytes. However, since CML is a stern cell disease involving all haemopoietic cell lines, our findings suggest that non-myeloblastic crises, especially pure megakaryoblastic crises, may develop without considerable proliferation of the myeloid compartment. Brach et al. [42] showed that megakaryoblasts from M7 patients were able to produce and secrete interleukin 6 (IL6) and express IL-6 receptors to stimulate their own DNA synthesis by acting in a paracrine fashion. Kinetic studies have lead to the conclusion that normal megakaryocyte development includes proliferative and endoreduplication phases [43]. The

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FIG. 2. Electron micrographs of megakaryoblasts from the peripheral blood of patient 2. Reaction product of CD61 MoAb is present on the cell surface membrane as an electrondense precipitate. Small, "lymphocyte-like" promegakaryoblast without cytoplasmic organelles. Cytoplasmic blebs can be detected. Part of a surface-negative myeloid cell (M) is present. (Without uranyl acetate and lead citrate, x 13.200.)

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FIG, 3. Blood megakaryoblast from panent 3 exhibits surface labelling with CD61 MoAb. There are features of cytoplasmic differentiation: perinuclear clustering of organelles (demarcation membranes, vacuoles, mitochondria) and organelle-free peripheral cytoplasm. (Uranyl acetate and lead citrate. × 13.200.)

893

Circulating megakaryoblasts in CML

895

TABLE 4. PERIPHERAL BLOOD CELL CYCLE DISTRIBUTION OF CD61-POSITIVE AND NEGATIVE CELL POPULATIONS IN 3 CASES OF CML WITH MEGAKARYOBLASTIC PROLIFERATION

No. 1 2 3

CD61-positive cells (%)

CV (%)

CD61-negative cells (%)

CD61-positive cells (%)

G0/G1

S

G2 + M

G0/G1

S

G2 + M

19.9 16.4 22.2

3.3 8.0 5.5

91.0 82.7 92.8

7.6 15.6 4.9

1.4 1.7 2.3

79.0 68.7 71.8

16.2 26.3 17.6

4.8 5.0 10.6

CV, coefficient of variation.

! G2*M

7

!

/

/

/ ! / /

/

indicated by the D N A content, was obvious, the appearance of specific organeUes in the cytoplasm and the heterogeneity of antigen expression were suggestive of a tendency to mature. Roth et al. [47] reported that a phorbol ester stimulated leukaemic megakaryoblast cell line was able to overcome the maturation block, and that the ploidy of the megakaryoblasts increased. Advances in the humoral regulation of megakaryopoiesis showed that thrombopoietin stimulated progenitor cells to polyploidization and cytoplasmic maturation [48]. The effects of humoral factors on the leukaemic megakaryocyte cell cycle and maturation might be the subject of further studies.

//

/ FIG. 5. Computer-drawn histogram demonstrating analysis of CD61 and DNA in peripheral blood cells of patient 2. Cell counts per channel are represented by the vertical axis. G0/G1 stands for the region containing cells in the G1 phase of the cell cycle. G2 + M is the region with twice the PI (DNA) fluorescence intensity of the G0/G1 region and contains cells at G2 + M. S represents theregion between G0/G1 and G2 + M and contains cells undergoing DNA synthesis.

proliferative phase, which comprises mostly immature forms with diploid and tetraploid DNA classes, is a pool of more mature polyploidy megakaryocytes [44]. Megakaryocyte colony-forming units (CFU-M) are composed of megakaryocytes in the proliferative phase showing a high S-phase compartment (8-23%) without polyploidization [45]. The proliferative phase is followed by from three to five waves of an endomitotic cycle resulting in mature polyploidy megakaryocytes [46]. The accumulation of CD61-positive cells in the G2 + M phase and the lack of higher ploidy classes (>4N) suggest that leukaemic megakaryoblasts are incapable of entering the pathway of an endomitotic cell cycle and remain in the proliferative phase. Although the maturation arrest,

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