ultrastructural and ultracytochemical differences between megakaryoblastic leukemia in children and...

451

Ultrastructural and Ultracytochemical Differences Between Megakaryoblastic Leukemia in Children and Adults Analysis of 49 Patients

Mitsuoki Eguchi, M.D., Takebumi Ozawa, M.D., Hitoshi Sakakibara, M.D., Kenichi Sugifa, M.D., Yoshihiko Iwama, M.D., and Toshiharu Furukawa, M.D.

Background. Acute megakaryoblastic leukemia (AMKL) has two peaks in distribution of incidence (in adults and children 1 to 2 years of age) and is frequently seen in children with Down syndrome. The current study was undertaken to disclose whether there were any differences between these groups.

Methods. Electron microscopic and ultrastructural cytochemical features of 49 children and adults with a AMKL or chronic myelogenous leukemia (CML) in megakaryoblastic crisis were compared.

Blast cells from children with AMKL, including those with and without Down syndrome, had immature features lacking typical alpha granules and a demarcation membrane system (DMS). However, blast cells from patients with AMKL with Down syndrome had more theta, electron-lucent, and basophil-like granules, suggesting that the blast cells had more potential to differentiate into other cell lines than megakaryocytes. The AMKL blast cells of adult patients showed a higher percentage of platelet peroxidase (PPO) positivity than other subgroups, and they occasionally contained typical alpha granules and DMS. This indicated that the blast cells of adults with AMKL were more mature than those of children and CML in megakaryoblastic crisis.

By electron microscopic analysis, leukemic megakaryoblasts differed between children with AMKL with and without Down syndrome, adults with AMKL, and patients with CML in megakaryoblastic crisis. Cancer 1992; 70:451-458.

Results.

Conclusions.

Key words: leukemia, peroxidases, electron microscopy.

Down syndrome, acute megakaryoblastic

From the Second Department of Pediatrics, Dokkyo University School of Medicine, Tochigi-ken, Japan.

Supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture, and a grant-in-aid for cancer research from the Ministry of Health and Welfare, Japan.

The authors thank Dr. S. Kojima, Dr. T. Sato, and other physi- cians who provided samples.

Address for reprints: Mitsuoki Eguchi, M.D., The Second Depart- ment of Pediatrics, Dokkyo University School of Medicine, Mibu- machi, Tochigi-ken 321-02, Japan.

Accepted for publication October 15, 1991.

Megakaryoblastic leukemia (AMKL) is a rare type of leukemia characterized by the proliferation of atypical megakaryoblasts in the peripheral blood and bone marrow. It accounts for 3%-4% of all instances of leukemia,”* but it is not so rare in children younger than 3 years, especially those with Down syndrome. This type of leukemia has two peaks in the distribution of its incidence; one is in adults accompanied by myelofibrosis, and the other is in young children of 1-2 years of age. The disease usually is believed to be drug-resistant and to have poor prognosis, but in many children, the disease has been reported to achieve a long remission. There are several reports on subtypes of the disease identified by using monoclonal ant ib~dies .~ AMKL may not be a homogeneous disease and may be divided into several subgroups. In the current study, we studied 49 patients with AMKL, including chronic myelogenous leukemia (CML) in megakaryoblastic crisis, by electron microscopic examination, and detected differences between children with AMKL with or without Down syndrome, adult patients with AMKL, and patients with CML in megakaryoblastic crisis.

Patients

Specimens from 16 children with AMKL without Down syndrome, 20 children with AMKL with Down syndrome, 7 adults with AMKL, and 6 patients with CML in megakaryoblastic crisis were examined by electron microscopic examination. According to the criteria of the French-American-British (FAB) cooperative group,4 the diseases were diagnosed by platelet peroxidase (PPO) reaction and flow cytometry. Specific monoclonal antibodies against platelet glycoproteins Ib or IIb/IIIa were used for flow cytometry, and the patients who were PPO positive and those who were glycoprotein positive (30% or greater) were included in this study. The average age at the time of diagnosis was 1 year, 11 months (range, 3 months to 6 years, 1 month) for children with AMKL without Down syndrome; 1

452 CANCER July 15, 1992, Volume 70, No. 2

year, 5 months (range, 2 months to 3 years) for children with AMKL and Down syndrome; 56 years (range, 19 years to 89 years) for adults with AMKL, and 34 years (range, 10 years to 73 years) for patients with CML in megakaryoblastic crisis. The onset of AMKL in children occurred at younger than 3 years of age in 33 of 36 patients.

Electron Microscopic Examination

Ultrastructural studies were performed on blast cells obtained from the peripheral blood or bone marrow of the patients. Both peripheral blood and bone marrow cells were analyzed in four patients with AMKL to com- pare the ultrastructure of the blast cells.

The cells were centrifuged for 5 minutes at 400 g, and the buffy coat was fixed with 2.5% glutaraldehyde in 0.05 M cacodylate (pH 7.2), washed in cacodylate buffer with 7% sucrose, and postfixed with 170 osmium tetroxide. Specimens were dehydrated and embedded in epoxy resin for transmission electron microscopic examination.

Electron Microscopic Cytochemistry

Platelet peroxidase (PPO). Buffy coat cells were fixed in a mixture containing 0.5% glutaraldehyde, 2% paraformaldehyde, and 1% tannic acid in 0.1 M phosphate-buffered saline for 30 minutes at 4°C. After be- ing washed in phosphate-buff ered saline, the cells were incubated in 2 mg/ml diaminobenzidine medium for 1 hour,5 postfixed in osmium tetroxide, dehydrated, and embedded. As the cytochemical control, hydrogen peroxide was omitted from the diaminobenzidine medium.

Myeloperoxidase (MPO). Fixation of the buffy coat cells was performed for 1 hour at 4°C using 2.5% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.2). The cells were incubated in 0.25 mg/ml diaminobenzidine medium for 1 hour and postfixed in osmium tetroxide.

Acid phosphatase. The glutaraldehyde-fixed specimens were incubated in a mixture of beta-glycerophos- phate and lead nitrate,6 followed by postfixing by osmium tetroxide. As the cytochemical control, specimens were incubated in the medium without beta glycero- phosphate. All of the electron microscopic preparations were analyzed with a JEOL 100-8 electron microscope (Japan Electron Optic Laboratory, Tokyo).

Evaluation of cells. Approximately 100 blast cells from each patient were analyzed ultrastructurally. AMKL cells contain several unusual types of granules that are not observed in normal megakaryocytes, and the frequency of five different types of granules was analyzed in 100 cells from each subject. These were

small granules, alpha granules, electron-lucent granules, theta granules, and basophil-like granules. Be- cause typical alpha granules with a central core are difficult to find in patients with AMKL, especially children, small granules lacking a core and the same size as alpha granules were included in this class as atypical or immature alpha granules. Small granules clustered around the Golgi complex area were excluded because these probably were progranules of lysosomal granules or other Golgi-related large granules.

For evaluating the maturation of blast cells as megakaryocytes, the cells were classified into five types:

1. blast cells that had no alpha or small granules and

2. blast cells that had alpha or small granules but did

3. blast cells with DMS but no small granules; 4. blast cells or small megakaryocytes with small

granules and a DMS; 5. mature megakaryocytes.

no demarcation membrane system (DMS);

not have a DMS;

Differences of these ultrastructural features and PPO positivity between the groups of patients were evaluated by Student’s t test.

Results

Electron Microscopic Morphology

The ultrastructural morphology of the blasts was almost identical in the peripheral blood and bone marrow in each patient. The most prominent nuclear form was round in all of the subgroups of AMKL. Irregular nuclei, as observed in normal mature megakaryocytes, were rare in the AMKL blasts.

Immature blast cells of AMKL, which did not have granule formation, were characterized by round nuclei with sparse nuclear chromatin and a large nucleolus. The cytoplasm was not so extensive and contained numerous dark mitochondria (Fig. 1). The electron microscopic morphology of the immature blast cells without granules was similar among the AMKL and CML in megakaryoblastic crisis subgroups. The mean of blast cells without granules was 63% (standard deviation [SD], *14%) for children with AMKL without Down syndrome; 57% (SD, +-13%) for children with AMKL and Down syndrome; 53% (SD, +18%) for adults with AMKL; and 58% (SD, *14%) for patients with CML in megakaryoblastic crisis. Statistically, there were no differences among these ratios (Fig. 2).

Blast cells that contained some granules, suggesting more advanced maturation, were identified in all subgroups. The granules observed were of five types; alpha

Megakaryoblastic LeukemialEguchi e t al . 453

Figure 1. Immature blast cell of acute megakaryoblastic leukemia Numerous dark mitochondoria are observed in the cytoplasm (X9000).

granules, small granules, electron-lucent granules, theta granules, and basophil-like granules (Fig. 3). Blast cells with typical alpha granules, suggesting more advanced megakaryocytic maturation, were observed in three adults with AMKL but were not observed in any of the children with AMKL. Small granules of the same size as the alpha granules but lacking a central core were observed occasionally in children with AMKL. Blast cells with small granules were significantly less common in children with AMKL with Down syndrome (P < 0.05) and were most frequently observed in blast cells from adults with AMKL (P < 0.05) (Fig. 2). Blast cells containing electron-lucent granules were most frequently observed in children with AMKL and Down syndrome (P < 0.05) (Fig. 2).

Granules containing a membranous component, which have been designated as theta granules because of their resemblance to the Greek letter theta, were observed in 12% (SD, *8%) of the blast cells from patients with Down syndrome, 1% (SD, +2%) of the blast cells of adult patients, and 1% (SD, +2%) of the blast cells of children without Down syndrome. Such granules were not seen in the patients with CML. Theta granules were significantly more common in the children with Down syndrome (P < 0.05) (Fig. 2).

Basophil-like granules were observed in children with AMKL and Down syndrome, and even in this group they were not common. The granules were almost absent in the other subgroups (Fig. 2).

For evaluating the maturation of blast cells as megakaryocytes, the cells were classified (as reported) after electron microscopic examination (Fig. 4). More than 60% of the blasts in all subgroups were cells without alpha or small granules and DMS. The percentage of cells without the granules and DMS was significantly higher in children with AMKL and Down syndrome (93% t 7%; P < 0.05) than in the other subgroups, whereas cells with small granules but no DMS were the least common in Down syndrome (6% k 6%; P < 0.05) (Fig. 4). The numbers of more matured megakaryoblasts (blast cells with DMS but no small granules; blast cells or small megakaryocytes with small granules and a DMS; or mature megakaryocytes) were almost negligi- ble, except for blast cells with small granules and DMS in adult patients (6% * 6%, Fig. 5).

Electron Microscopic Cytochemistry Platelet peroxidase (PPO). The PPO reaction was

positive in the nuclear envelope and rough endoplasmic reticulum and negative in the Golgi apparatus and the alpha, small, electron-lucent, and theta granules

10 20 30 40 %

' D ( - )

Blasts with any types of granules ADT

CML

D ( + )

' D ( - )

D ( + ) Blasts with small or a granules ADT

~ CML

D ( - )

CML

CML

basophil

Figure 2. Percentage of blast cells with granules. D(-): children with AMKL without Down syndrome; D(+): children with AMKL and Down syndrome; ADL: adults with AMKL: CML: patients with CML in megakaryoblastic crisis.

454 CANCER July 25, 1992, Volume 70, No. 2

(Fig. 6). Basophil-like granules were faintly positive in several cells. The mean of positive blast cells in children with AMKL without Down syndrome was 58% (SD, +21%); in children with AMKL and Down syndrome 57% (SD, +17%); in adults with AMKL, 74% (SD, ?lo%); and in patients with CML in megakaryocytic crisis 51% (SD, -t19'/0). Positivity was significantly higher in adults with AMKL than in children with AMKL with or without Down syndrome. Cells incubated in the diaminobenzidine medium without hydrogen peroxide as the cytochemical control showed no reactivity.

MPO. The cells had no reaction for myeloperoxidase in the rough endoplasmic reticulum, nuclear envelope, Golgi apparatus, and all types of granules.

Acid phosphatase. A positive reaction for acid phosphatase was observed in electron-lucent and theta granules and in the Golgi apparatus (Fig. 7). Alpha granules and small granules were negative for acid phosphatase. Golgi-endoplasmic reticulum-lysosomes (GERL) were well-developed in children with AMKL with or without Down syndrome; these organelles usually were positive for acid phosphatase (Fig. 8).

Figure 3. Five different types of granules in megakaryoblastic leukemia. (A) Alpha granules (X90,OOO). (B) Small granule (X90,OOO). (C) Electron lucent granule (X50,OOO). (D) Theta granules (X50,OOO). (E) Basophil-like granule (X50,OOO).

Discussion

Megakaryoblasts are small cells that resemble the L1 or L2 blasts of the French-American-British classification and are difficult to differentiate from acute lymphoblas- tic leukemia cells by light microscopic e~amina t ion .~ ,~ A diagnosis of AMKL must be confirmed by immunologic and electron microscopic cytochemical studies8 The PPO reaction of electron microscopic cytochemistry has been found to be more sensitive and to detect more immature cells than monoclonal antibodies directed against platelet glycoprotein IIb-IIIa or Ib.9,'0 However, PPO is not specific to megakaryocytes and platelets3 because the reaction also is seen in immature erythroblasts," mast cell precursors,'* and common ALL cells.13 However, the monoclonal antibodies also react with hemopoietic stem cells.14 Thus, it is necessary to use electron microscopic and immunologic techniques to diagnose AMKL to exclude cells other than megakaryoblasts. However, if patients negative for PPO or the monoclonal antibodies are excluded from the diagnosis, it is possible to exclude immature megakaryoblasts positive for PPO and negative for monoclonal antibodies"

Megakaryoblastic Leukemia/Eguchi et al.

(5)

455

’ D ( -1 D (+)

ADT

, CML

20 40 60 a0 vn

Figure 4. Five types of megakaryoblasts classified by maturation as megakaryocytes. The types are described in the text (see “Electron Microscopic Morphology”).

or PPO-deficient megakary~blasts.’~,’~ Nevertheless, we included only doubly positive patients in this study so as to exclude patients with an uncertain diagnosis.

AMKL often is believed to be resistant to treatment and associated with a poor prognosis. However, in children there are a number of reports that the disease re- sponds well to treatment and is associated with good progn~sis .~,’~,’~ Some studies have shown better prognosis associated with AMKL than with acute nonlym- phocytic leukemia.” In children, AMKL usually de- velops before the child is 3 years old,” and it often accompanies Down In the current series, the average age of onset in the 36 children studied was 1 year, 8 months, and 89% of them had disease develop when they were younger than 3 years of age. Acute AMKL in children often is accompanied by leukocytosis and ~rganomegaly,~ which are not observed in adults with AMKL. Thus, the features of AMKL differ between children and adults, suggesting that there may be several variants of the disease.”

Electron microscopic examination usually shows that AMKL blasts have round nuclei and lack alpha granules and a DMS,4,5,23-25 although a few patients

were noted in blast cellsz6 Breton-Gorius and Vain- chenker3 classified AMKL into four subtypes. Type 4 was the most mature, and the blasts had alpha granules and a DMS.3 In the current study, only a few of the blast cells had either of these structures. Alpha granules with central core resembling a bull’s-eye were observed in blast cells obtained from three adults with AMKL. This finding and the higher percentage of PPO positivity suggest that AMKL blasts in adults are more mature than those in children and patients with CML in megakaryoblastic crisis.

Small granules of the same size as the alpha granules but lacking a central core were observed in all of the subgroups of AMKL in some degree. It is uncertain whether or not these small granules were an immature form of alpha granules. However, photographs in an atlas of blood cells show granules without central core in some of normal megakaryocytes and platelet^.'^ In the current study, these small granules without a central core were observed most often in adult patients with AMKL who showed a higher ratio of PPO positivity and had alpha granules. Thus, the small granules could belong to the same category of granules as alpha granules. The electron-lucent granules noted in this study have been reported previously as small round

Figure 5. Small megakaryocyte from an adult with AMKL. Numerous alpha granules and demarcation membrane are seen in

Lave been reported in whom alpha iranules aAd DMS the cytoplasm (X<OOO).

456 CANCER July 25,2992, Volume 70, No. 2

Figure 7. Acid phosphatase reaction of an AMKL blast. Theta granules show the reactivity (x~o,ooo~,

Figure 6 . Platelet peroxidase reaction of an AMKL blast from a patient with Down syndrome. All electron lucent granules show no reaction (X 10,000).

vacuoles in AMKL cells in ~hildren.’~ We found that the electron-lucent granules were positive for acid phosphatase and negative for MPO and PPO, indicating that the granules were lysosomes but not the same as the primary granules of granulocytes, which are MPO- positive.

Electron-lucent granules with a membranous component (theta granules) were observed previously in blast cells from children with AMKL with Down syndrome.16 Parkin et aLZ8 found theta granules in basophil precursor cells. Breton-Gorius et al.3,11,29 reported that these granules were a marker of immature erythroblasts in erythroleukemia because the cells containing them reacted with FA-152, a monoclonal antibody for immature erythroblasts. However, FA-152 also reacts with monocytes, blast cells, megakaryocytes, and plate- l e t ~ . ~ ’ ~ ~ ’ Thus, theta granules appear to be a marker of all of these kinds of cells and do not seem to restrict one type of cell. In the current study, theta granules were observed most frequently in the blasts of children with AMKL with Down syndrome, indicating that the blasts in children with Down syndrome had more multipotential activity than those in the other subgroups. There is a report that AMKL blasts in patients with Down syndrome transformed into basophils during cult~re,~’

Figure 8. Acid phosphatase reaction of GERL in a AMKL blast (X40,OOO).

Megakaryoblastic LeukemialEguchi et al.

and the current study showed that AMKL blasts in children with Down syndrome had basophil granules. These results could support the observation by Parkin et a1.” that the theta granules were basophil in nature and that AMKL blasts in patients with Down syndrome were more basophilic than those of other subgroups.

Transient myeloproliferative disease (TMD) is a self-limiting megakaryoblastic leukemoid reaction oc- curring in Down syndrome. TMD blasts resemble AMKL blasts and can not be differentiated by light microscopic techniques, although the two have chromosomal and electron microscopic d i f f e ren~es .*~ ,~~ The chromosome in TMD blasts shows few abnormalities except trisomy 21, whereas AMKL blasts may show various other clonal chromosomal abn~rmali t ies .~~

TMD blasts ha\ been shown to have highly heterogeneous characteristics by electron microscopic examination,I6 monoclonal antibody studies,34 and tissue culture s t ~ d i e s , ~ ~ , ~ ~ and TMD is thought to be a disease arising from multipotential stem cells with megakaryocytic differentiati~n.~~ To a lesser extent, the AMKL blasts in children with Down syndrome also had heterogeneous characteristics in our electron microscopic study. Several patients with AMKL developing after re- covery from TMD in the neonatal period have been rep~rted.~~,~’”’ These facts might suggest a relationship between TMD and AMKL.

Megakaryoblastic infiltration is reported to occur in 30% of patients with CML in blastic crisis,39 but it is difficult to differentiate from crisis of other cell lineage without PPO and monoclonal antibody ~ tud ie s .~ ,~ ’ In the current study, blasts from patients with megakaryocytic crisis of CML resembled those of the other subgroups in their fine structure but showed slight differences in maturation.

In conclusion, ultrastructural analysis showed that blast cells from adults with AMKL were more mature than those from children with AMKL or from patients with CML in crisis. Blast cells from children with AMKL and Down syndrome were more heterogeneous than those from the other subgroups that we studied.

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ultrastructural and ultracytochemical differences between megakaryoblastic leukemia in children and...

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