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Myelodysplasia and acute megakaryoblastic leukemia in down's syndrome
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Leukemia Research Vol. 18. No, 3, pp 163-171. 1994 Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 1tl45-2126/04 $6.(X~ + 0,(X)
0145-2126(93)E0016-O
MYELODYSPLASIA AND ACUTE MEGAKARYOBLASTIC LEUKEMIA IN DOWN'S SYNDROME A. ZIPURSKY,* P. THORNER,* E. DE HARVEN,~ H. CHRISTENSEN~ and J. DOYLE* *Division of Hematology/Oncology, Department of Pediatrics, +Department of Pathology, The Hospital for Sick Children; and ~:Department of Pathology, University of Toronto, Canada
(Received 19 August 1993. Revision accepted 18 November 1993) Abstract--In this report we describe the clinical and hematologic features of 23 cases of myelodysplasia (MDS) or acute megakaryoblastic leukemia (AMKL) occurring in Down's syndrome. MDS was characterized by thrombocytopenia, abnormal megakaryocytopoiesis, megakaryoblasts (<30%) in the marrow and abnormal karyotype, the most common of which was trisomy 8, found in 7/15 patients with MDS. Three of five patients achieved a complete remission with low dose cytosine arabinoside, vincristine and retinyl palmitate. The high cure rate and the distinctive features of the leukemic process in these cases suggest that this type of MDS and AMKL are unique to patients with Down's syndrome.
Key words: Megakaryoblasts,
megakaryoblastic
leukemia,
myelodysplasia,
Down's
syndrome.
CD 13, CD33; platelet antigen GpIIb/IIIa, CD41; and common ALL antigen CD10 (Coulter Immunology, Hialeah, FL). Bone marrow biopsies were fixed in B5, decalcified and embedded in paraffin. Sections were cut at 3 btm and stained with hematoxylin and eosin or Gordon and Sweet's reticulum stain. All sections were reviewed by a pediatric pathologist (P.T). Immunohistochemistry to identify megakaryocytic cells was performed on paraffin sections using a polyclonal antibody to Factor 8-related antigen (Dakopatts, Denmark) by the peroxidase-antiperoxidase method and using the monoclonal antibody MB2 (Biotest, Germany) by the indirect peroxidase method, as previously described [3]. In addition to staining B-lymphocytes as indicated by the manufacturer, in our laboratory this monodonal antibody stains megakaryocytes and their precursors. Electron microscopy specimens were prepared by fixation in glutaraldebyde. Subsequently the cells were treated with monoclonal antibody to CD61 (Gpllla) and then treated with gold-labelled anti-mouse antibody as described previously [4].
Introduction ACUTE MEGAKARYOBLASTIC LEUKEMIA ( A M K L ) is the most common form of leukemia in Down's syndrome [11. It has been estimated that the incidence is 500 times greater in children with Down's syndrome than in normal children [2]. Frequently the disease is preceded by a pre-leukemic phase characterized by abnormal marrow function, a myelodysplastic process. In the present study, we describe the clinical and hematologic features of myelodysplasia in Down's syndrome with particular reference to the abnormal megakaryopoiesis which precedes the development of A M K L . Methods Cases of myelodysplasia (MDS) and AMKL were either studied at The Hospital for Sick Children (cases 6, 7, 13, 14, 18 and 20) or reported to the Canadian Down's syndrome Leukemia Registry. In all cases, blood and bone marrow films were reviewed by a pediatric hematologist (A. Z). Standard hematologic techniques were used for peripheral blood studies and for cytochemistry. Leukocyte membrane antigens were determined using a Coulter Epics Profile Analyzer (Coulter Electronics) and Coulter monoclonal antibodies to T-cell antigens CD2, CD3, CD7; B-cell antigens CD19, CD20, CD22; myeloid/monocytic antigens
Results
Clinical Twenty-three patients with acute megakaryoblastic leukemia and/or myelodysplasia were included in the study. Diagnostic features of MDS included cytopenia, abnormal hematopoiesis and less than 3 0 ~ blasts in the bone marrow. If the marrow contained more than 30% blasts, the patient was considered to have leukemia in keeping with the definition of MDS in other conditions [5]. The typical
Correspondence to: Dr A. Zipursky, Division of Hematology/Oncology, University of Toronto. Toronto, Canada. 163
A. ZIPURSKYet al.
164
TABLE 1. CLINICAL F1NDINGS IN A M K L / M D S IN DOWN'S SYNDROME
Age (months)
Sex
1
39
F
2 3 4 5 6 7 8 9 10
18 21 24 8 37 20 24 21 38
F M F F F M F M M
45+ 3 dead 35 + 18 dead 15+ 8 dead 15+ 4 dead 29+ 16+
Case
Survivalt (months)
Platelets ( x 109/1)
Hemoglobin (g/l)
Blasts (PB)$ (x 199/1)
Blasts$ (BM) %
12 79 40 27 4{1 90 4 200 36
2.1 I) 0 0.144 {1 0.14 0.175 0.312 0.315 0.26 0.07 0.1 0.18 0 0.12 0
22 16 20 15 18 11 23 21 21 6
6.270 0.18 42.4 16.7 11.6
60 80 91 67 58
0.36 0
---
MDS
11
19
M
32 +
12 13 14 15 16
21 24 8 37 37
M M M M F
9 dead 12+ 20+ 28+ 1 dead
90 61 59 17 32 77
102 99 123 92 114 123 68 1(14 86 90 77 110 117 116 88 63
13 26 25 27 14
M M M M F
6 dead 80+ 82+ 15 + 36+
14 24 28 20 15
96 45 50 106 77
M M
0 dead* 13 dead
32 17
8
12 12 8 29 --
AMKL 17 18 19 20 21
AMKL/MDS 22 23
13 26
4.2 4.9
* No treatment given. t Survival from initiation of therapy or from diagnosis (if no therapy given); - - = data not available; MDS = myelodysplasia; A M K L = acute megakaryoblastic leukemia; A M K L / M D S = bone marrow aspiration inadequate to determine percentage of blasts. $ PB = peripheral blood; BM = bone marrow.
clinical f e a t u r e s of D o w n ' s s y n d r o m e w e r e p r e s e n t in 22 out of 23 cases. C a s e N o . 13 was p h e n o t y p i c a l l y n o r m a l but was p r o v e n to be a m o s a i c with a m i n o r p o p u l a t i o n of t r i s o m y 21 cells. This case is d e s c r i b e d in detail e l s e w h e r e [6]. A s u m m a r y of t h e clinical findings in t h e s e cases is shown in T a b l e 1. This t a b l e is d i v i d e d into cases of m y e l o d y s p l a s i a ( M D S ) in which the m a r r o w blast c o u n t was less t h a n 3 0 % , a c u t e m e g a k a r y o b l a s t i c l e u k e m i a ( A M K L ) in which t h e m a r r o w blast count was g r e a t e r t h a n 30% a n d two cases in which b o n e m a r r o w a s p i r a t i o n was u n s a t i s f a c t o r y . A l l cases in T a b l e 1 w e r e less t h a n 40 m o n t h s of age. S e v e n of the 16 cases of M D S a n d o n e of the five cases of A M K L w e r e f e m a l e . A l l p a t i e n t s with M D S w e r e well at t h e t i m e of diagnosis. T h e r e was no e v i d e n c e of h e p a t o s p l e n o m e g a l y or l y m p h a d e n o p a t h y in 10 of the 11 cases for w h i c h t h e r e w e r e d a t a . A history of n e o n a t a l t r a n s i e n t l e u k e m i a was p r e s e n t in t h r e e of the M D S cases, o n e o f t h e A M K L a n d o n e of the u n d e f i n e d cases. T h u s 5 o u t o f 23 cases of M D S
a n d / o r A M K L h a d a h i s t o r y of t r a n s i e n t l e u k e m i a . In 10 of the 11 M D S cases, t h e d i s e a s e m a n i f e s t e d as i s o l a t e d t h r o m b o c y t o p e n i a ; o n e case m a n i f e s t e d as unexplained anemia. In m a n y cases t h e r a p y was s t a r t e d b e f o r e l e u k e m i a d e v e l o p e d a n d , t h e r e f o r e , t h e d u r a t i o n of m y e l o d y s p l a s i a was u n k n o w n . A l s o in t h o s e cases in which the blast c o u n t was > 30% w h e n first s e e n , it was not known whether there had been a pre-leukemic, m y e l o d y s p l a s t i c p h a s e since, as n o t e d a b o v e , m y e l o d y s p l a s i a in m o s t cases was a s y m p t o m a t i c . In 11 cases t h e p a t i e n t was o b s e r v e d d u r i n g the p e r i o d of m y e l o d y s p l a s i a and the d u r a t i o n v a r i e d f r o m 1-18 m o n t h s with a m e a n v a l u e of 6,3 m o n t h s . P r o g r e s s i o n of M D S to A M K L was d o c u m e n t e d in two cases in which initial m a r r o w blast c o u n t s of 20 a n d 18% w e r e f o l l o w e d 6 a n d 10 m o n t h s l a t e r by an i n c r e a s e in t h e p e r c e n t a g e o f blasts to 41 a n d 9 0 % , respectively. T h e h e m a t o l o g i c f e a t u r e s o f M D S a n d A M K L at the t i m e o f d i a g n o s i s a r e s h o w n in T a b l e 1. T h r o m b o c y t o p e n i a was p r e s e n t in all b u t
Myelodysplasia in Down's syndrome 6007
/ ~
500 400 % 300
% × 200
~ ~ffr!"~¢'~.:::~
jW LU
CYTOSINE • ARABINOSIDE
....
•
•
•
/
loo
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0
1990
JAN FEB 1991
r
OCT
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NOV
--
DEC 199!
,
7
JAN
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FIG. 1. Effect of chemotherapy on platelet count in patient 14. In December 1991, the bone marrow biopsy showed no evidence of myelofibrosis and cytogenetics were normal. For dosage of chemotherapy, see text. Patient continues in remission to date.
one MDS case with platelet counts significantly lower in the A M K L group. Neutropenia (<1000 x 1091) occurred in 4 out of 15 MDS cases and in none of the A M K L or A M K L / M D S cases. In MDS a mild anemia (<100 g/l) was found in 7 of 15 cases whereas
165
it was more common and severe in the A M K L and A M K L / M D S cases. Megakaryoblasts in low numbers (0.1-2.1 x 109/1) were found in 11 of 16 cases of MDS and 1 of 2 cases of A M K L / M D S . In A M K L megakaryoblasts were found in higher numbers in 4 of 5 cases (6.3, 11.6, 16.7 and 42.4 x 109/1). Thrombocytopenia in the MDS group appeared to be due to a failure of production since platelet destruction was not increased as evidenced by apparently normal platelet survival following platelet transfusions in all eight patients so treated. Twenty-one cases received anti-leukemic therapy. Fifteen cases received intensive A N L L treatment which included cytosine arabinoside and anthracycline chemotherapy. Eleven are in continuing clinical remission with a mean follow-up of 31 months (range = 15-73 months). Five cases (cases 1, 5, 6, 12 and 14) received a low dose chemotherapy program which included vincristine 1.0 mg/M 2 and cytosine arabinoside 10 mg/ M 2 every 12 h for 7 days; these drugs were given at 2-week intervals. Also these patients received retinyl palmitate (Aquapalm-Hoffman La Roche) 50,000units/M 2 by mouth daily. Three patients achieved complete remission (cases 1, 12 and 14); in
FIG. 2. Bone marrow biopsy of a Down's syndrome patient with myelodysplasia. The marrow is hypercellular with increased numbers of megakaryocytes, many of which are abnormal in appearance (dysmorphic) with small nuclei or unusual nuclear shapes (hematoxylin and eosin × 800).
166
A. ZIPURSKYet al.
~ii
!~ii~i
¸
•. . . .
FIG. 3. Bone marrow aspirate of a Down's syndrome patient with myelodysplasia (x 1000). A dysplastic megakaryocyte is seen. two this was confirmed by bone marrow examination. Two cases remain in remission, case 1 for 42 months (therapy was stopped after 36 months) and case 14 who is in remission and still on treatment 18 months since starting therapy. Case 12 achieved a hematologic remission but therapy was stopped by the parents after 2 months and the child relapsed shortly thereafter. Cases 5 and 6 did not achieve a remission and were then treated with high dose chemotherapy. Case 5 did not achieve a remission and died whereas Case 6 did and continues in remission 7 months since completion of therapy. The response to low dose therapy in one of our cases is shown in Fig. 1 and demonstrates the rapidity of response to that therapy. Thus, platelet counts began to rise within 2 weeks without any evidence of bone marrow suppression which usually characterizes the hematologic response to high dose A N L L chemotherapy. Case 4 did not recewe chemotherapy and remained well for 18 months, although still thrombocytopenic. She subsequently developed AMKL, did not receive treatment and died shortly thereafter. Case 22 received no therapy and died shortly after diagnosis. Case 8 received oral thioguanine for 5 months, then the low dose protocol and subsequently high dose
therapy. She did not achieve a remission and died 1 year after diagnosis. Laboratory studies
In all cases in our series the blasts were similar on Wright-stained, air-dried smears, varying in size from 15-20 ~m in diameter, with 1-2 nucleoli, a small amount of deeply basophilic cytoplasm and usually distinct cytoplasmic blebbing. Bone marrow biopsies indicated that megakaryocytes were increased in number in the marrow but were small and abnormal in appearance with either small single nuclei or unusual nuclear structure (Fig. 2). This was present in 10 of 11 MDS cases studied. Immunohistochemicai staining of slides showed that these large cells stained positively for Factor 8 and MB2 antigens consistent with a megakaryocytic lineage. Megakaryocytes in bone marrow aspirates also were small and very abnormal in appearance, often containing vacuolated cytoplasm and mononuclear nuclei (Fig. 3). In four cases (three MDS and one AMKL) marrow cells were studied by electron microscopy. Cells in the megakaryocytic lineage were recognized by the presence of the surface antigen CD61 (GpIIIa) as determined by immunogold labelling (Fig. 4). The
Myelodysplasiain Down'ssyndrome
167
FIG. 4. Electron microscopy of a megakaryoblast showing immunogold labelling of surface antigen Gp IIIa (× 40,000), prominent blebs and demarcation vesicles.
abnormal megakaryocytes varied from cells which were blastic in appearance to those with evidence of megakaryocyte differentiation including demarcation membranes and alpha-granules (Fig. 5). In most cases, however, the cells showed little evidence of differentiation. Myelofibrosis was observed in 7 of 11 cases of MDS studied and both cases of AMKL. In 7 of 15 MDS cases dyserythropoiesis was noted in the marrow. Leukocyte surface antigens were studied in six of our patients, information on seven other patients was sent to us. Platelet antigen GpIIb/IIIa was present in 7/10 cases studied by flow cytometry. Two of the negative cases were also studied by electron microscopy and immunogold labelling using monoclonal antibody to platelet antigen GpIIIa; both were positive. The myeloid antigens CD33 (My9) and CD13 (My7) were positive in 6 of 6 and 3 of 4 cases, respectively. Antigens expressed on T- and B-cells were usually negative although the cells of 4 of 12 patients expressed CD3 and/or CD7 and in 1 of 12 cases (case 13) the cells expressed CD10 (CALLA). Cytochemical staining of the leukemic cells for Sudan black, non-specific esterase and PAS were usually negative. Only the acid phosphatase stain was consistently positive.
Chromosomal analysis in the MDS group revealed, in addition to trisomy 21, seven cases with trisomy 8, two with monosomy 7 and two with other findings, Trisomy 21 alone was found in six of the MDS cases and in the three AMKL cases studied. Discussion Acute megakaryoblastic leukemia (AMKL) is the commonest form of leukemia occurring in Down's syndrome [1]. It is often preceded by a pre-leukemic hematologic disorder which, in this report, is referred to as myelodysplasia (MDS). We have arbitrarily specified three criteria for the diagnosis of MDS in Down's syndrome: (1) cytopenia (usually thrombocytopenia); (2) dysmegakaryopoiesis; and (3) increased numbers of megakaryoblasts in marrow aspirates but the total percentage should be less than 30% in keeping with the recommendations described by Bennett et al. [5] for other forms of MDS. MDS was documented in 11 out of 23 cases in the present series. It is likely that the true incidence was higher because in those cases in which leukemia was diagnosed when the patient was first seen it was not possible to know whether there had been a preceding pre-leukemic phase since that phase is usually
168
A. ZIPURSKYet al.
FIG. 5. Electron microscopy of a megakaryoblast showing demarcation membranes and alpha granules (× 31,992).
asymptomatic. Hayashi et al. reported that 11 of 13 cases of A M K L in Down's syndrome were preceded by a pre-leukemic syndrome [8] of myelodysplasia. Similarly Kojima et al. [9] described 14 cases of AMKL of whom seven had a pre-leukemic period of myelodysplasia. The pre-leukemic phase was characterized in 10 of 11 of the cases in this series by thrombocytopenia. This was due to failure of production since platelet survival was normal as evidenced by the good response to platelet transfusions in all eight patients so treated. Despite the evidence of production failure, bone marrow biopsies indicated that the numbers of megakaryocytes were increased in the marrow and their appearance was abnormal with many of the cells smaller than normal megakaryocytes and often contained single or bizarre nuclei (Figs 2 and 3). The duration of MDS before becoming A M K L is unknown. In two of our cases MDS progressed to leukemia within 6 and 11 months, respectively. One of our cases was followed for 18 months with no therapy and then developed AMKL. In most of our cases, since therapy was started when the patient was myelodysplastic, the natural history of the disease was difficult to determine. There have been other reports of progression from MDS to AMKL. Broomhead et al. reviewed four
cases associated with trisomy 8 in which the preleukemic phase lasted 8-22 months [10]. Kaneko et al. [11] described two cases in which progression occurred over a period of 8 and 22 months, respectively. There have been reports of other cases in which progression occurred over 4 weeks [121 and 4 months
[131. If it is assumed that all MDS cases will develop A M K L the results of therapy in A M K L / M D S have been excellent (Table 2). In both the Nordic [14] and POG [151 series, the MDS and A M K L cases were lumped together. In our series (Tables 1 and 2) both MDS and AMKL responded to therapy. In the MDS group, three of the five deaths occurred during induction. All relapses occurred in the first 9 months. Five cases were treated with low dose cytosine arabinoside, vincristine and retinyl palmitate and three of these patients achieved a remission [7]. Two of them remain in remission 40 and 18 months since starting therapy while one relapsed shortly after therapy was stopped by the parents. The response to this low dose therapy (Fig. 1) was characterized by a rapid rise in platelet count, usually, without evidence of the bone marrow suppression which is seen following high dose chemotherapy and which usually precedes the appearance of remission. Case 22 received no therapy and died within 1 month.
169
Myelodysplasiain Down's syndrome TABLE 2. TREATMENTOF AMKL/MDS IN DOWN'SSYNDROME
POG [15] Nordic [14]* Present series
No. studied
No. in CCR
%
Duration of follow-up (months)
12 17 22
12 13 16
100 76 64
27-60 5-89 12-80
* A study of the Nordic Society of Pediatric Hematology/Oncology. The survival of this group of AMKL/MDS patients is significantly better than that reported for cases of AMKL in non-Down's syndrome children. A group of 18 such patients reported by Schaison et al. [16] had a median survival of 5 months, with only one patient still alive 12 months after diagnosis. The blast cells in these patients were considered to be megakaryoblasts because of their morphology and the presence of platelet-specific antigens on their surface. In all cases in our series the blasts were similar, varying in size from 15-20 ~tm in diameter, with 1-2 nucleoli, a small amount of deeply basophilic cytoplasm and usually distinct cytoplasmic blebbing. This morphology is consistent with the appearance of megakaryoblasts [17]. When viewed by electron microscopy the blasts were usually undifferentiated although in some there was evidence of demarcation membranes and alphagranules. Eguchi et al. found no evidence of demarcation membranes or alpha-granules in 13 cases of AMKL in Down's syndrome [18]. Four cases were studied and in all the blast cells were labelled with anti-CD61 (GplIIa) as identified by immunogold (Figs 4 and 5). Distinct blebs were noted on all surfaces in all four cases (Figs 4 and 5). Most reported studies of membrane antigens found that the blasts of AMKL/MDS in Down's syndrome contain the platelet antigen GplIb/IIIa. Koller et al. [19] reported that CD61 (anti-GplIIa) was more sensitive than Gp41a (anti-GpIIb/IIIa) and this is consistent with our finding of two cases that were negative with anti-GplIb/IIIa on flow cytometry but positive with anti-GplIIa on electron microscopy. In our series the blast cells frequently contained the antigens My9 and My7, typical of myeloid cells, consistent with the findings of Kaneko et al. [11]. Cases have been reported of pre-leukemia in young children with Down's syndrome in which antigens usually expressed in myeloid and T-cells were positive in the blasts [20] and in one case where CD10 (CALLA) was positive [8]. In our opinion it is likely that these are cases of acute leukemia of the type described in the present study. The leukemic cell in this disorder may express multiple aberrant antigens. As noted above the morphology of the leukemic
cells in AMKL/MDS suggests that these cells are megakaryoblasts. It should be pointed out, however, that in 6 of 16 MDS cases there was evidence of dyserythropoiesis (including PAS positivity of normoblasts [21]). Some of our cases had been diagnosed as erythroleukemia. Also there have been reports of an increased incidence of erythroleukemia in patients with Down's syndrome [22]. In some of these cases, the leukemic cells had properties of both erythroid and megakaryocytic lineage [23]. We have postulated previously [1] that in AMKL/MDS or erythroleukemia in Down's syndrome the leukemic cell is a biphenotypic cell with both megakaryocytic and erythroid features. Such biphenotypic hematopoietic progenitor cells have been isolated from normal bone marrow [24]. The megakaryocytic nature of the leukemic cells in myelodysplasia is supported also by the presence of myelofibrosis in 7 of 11 MDS cases studied by us. Myelofibrosis is closely associated with acute megakaryoblastic leukemia in adults [17[, and in nonDown's syndrome children [25]. Chromosomal abnormalities were found frequently in our cases of myelodysplasia. Of interest, 7 of 14 cases of MDS had trisomy 8. Three cases of AMKL were studied and only trisomy 21 was found. It would appear that trisomy 8 is closely related to the development of myelodysplasia in Down's syndrome. Hecht et al. in 1986 [26] reviewed all published chromosomal studies of leukemia in Down's syndrome. In that series there were 17 cases under age 4 with ANLL. Trisomy 8 was found in 6 out of 17 cases, one with an associated trisomy 19. Hayashi et al. studied 13 cases of AMKL in Down's syndrome and found two with trisomy 8 [8]. Wang et al. [27] described four cases of acute leukemia in children younger than 4 years of age of whom three had trisomy 8. Broomhead et al. described one such case [10] and postulated that 'the triad of pre-leukemic (thrombocytopenia), AMKL and trisomy 8 in children with Down's syndrome represents a specific type of acute l e u k e m i a . . . ' . Trisomy 8 was found only in 1 of 10 cases described by Cairney et al. [25] and in none of 14 cases described by Schaison et al. [16]. Combining our series of 17 cases studied with
170
A. Z|PURSKYet al. TABLE 3. LEUKEMIA IN DOWN'S SYNDROME: THE HOSPITAL FOR SICK CHILDREN (1985-
1991)
ALL ANLL AMKL* Other Total
Non-Down's syndrome
Down's syndrome
O/Et
325 (85%)
6 (50%)
=13 x
7 (2%) 51 383 (13%)
6 (50%) 0 12
=600 x =22 x
* AMKL includes cases diagnosed as either acute megakaryoblastic leukemia or erythroleukemia. t O/E = Observed over expected number of cases assuming that the prevalence of Down's syndrome is 1 in 700 children. the cases studied by others [8, 26, 27] trisomy 8 has been found in 18 of 51 (35%) of patients with A M K L / MDS in Down's syndrome. It would appear, therefore, that trisomy 8 is frequently associated with the acute megakaryoblastic leukemia that occurs in Down's syndrome. It is apparent that acute megakaryoblastic leukemia including the associated myelodysplastic syndrome is relatively common in Down's syndrome. We estimated previously that its incidence is approximately 500 times more common in Down's syndrome than in normal children [2]. To analyze this further, we reviewed all cases of leukemia admitted to The Hospital for Sick Children from 1985 to 1991 to determine the types of leukemia seen in Down's syndrome and in normal children (Table 3). The prevalence of Down's syndrome is thought to be 1 in 700 of the population. Accordingly the experience at The Hospital for Sick Children (Table 3) indicates that the total incidence of leukemia in Down's syndrome children was 22 times greater than in normal children; however, the incidence of A M K L was 600 times greater than expected. A L L was only 13 times greater. There may be a referral bias in these data, nevertheless all children with leukemia in our region are referred to this hospital and, therefore, our experience probably reflects the true incidence of the types of leukemia occurring in children with Down's syndrome. Accordingly it would appear that the incidences of both A L L and A M K L are increased in Down's syndrome, but the incidence of the latter is remarkably high. Why is there such a high incidence of A M K L / M D S in Down's syndrome? Is A M K L / M D S in Down's syndrome a different type of megakaryoblastic leukemia than that found in normal children or adults? There is considerable evidence to suggest that the A M K L / M D S of Down's syndrome is a unique disease that differs from A M K L found in normal subjects. The first difference is that none of the reported series of A M K L in non-Down's syndrome
children or adults have described a high incidence of myelodysplasia and the unique bone marrow findings in the myelodysplastic phase of leukemia in Down's syndrome. Secondly, hepatosplenomegaly is not a common feature of MDS or A M K L in Down's syndrome, whereas it is common in A M K L in normal children [24] and in infants suffering from A M K L in association with a 1:22 translocation [28]. However, the major difference between the A M K L / M D S of Down's syndrome and that in normal individuals is the high cure rate of this disease in Down's syndrome (Table 3). Schaison et al. [15] reported a series of 18 normal children with A M K L in which the median survival with therapy was 5 months with only one child, a recipient of a bone marrow transplant, alive after 1 year. Infants with A M K L and a 1:22 translocation [28] as well as adults [29] with A M K L have a very poor response to therapy. Lu et al. [30] recently reported a review of cytogenetic changes found in acute megakaryoblastic leukemia and concluded that there are at least three distinct types of AMKL. One is that found in Down's syndrome, one is the infant form associated with a 4 : 11 translocation and the third refers to other cases, most of which occur in adults. All the above evidence suggests that acute megakaryoblastic leukemia in Down's syndrome is a unique disease characterized by a long pre-leukemic, myelodysplastic phase, unusual chromosomal findings and a high cure rate. Acknowledgements--We gratefully acknowledge the support of the Leukemia Research Fund of Canada.
References 1. Zipursky A., Poon A. & Doyle J. (1992) Leukemia in Down's syndrome. A review. Pediat. Hemat. Oncol. 9, 139. 2. Zipursky A., Peeters M. & P o o n A. (1987) Megakaryoblastic leukemia and Down's syndrome. Pediat. Hemat. Oncol. 4, 211.
Myelodysplasia in Down's syndrome 3. Kahn H. J. & Thorner P. S. (1989) Immunohistochemical staining of pediatric tumors with the monoclonal antibody MB2: a potential marker for Ewing's sarcoma and peripheral neuroectodermaltumor. Pediat. Path. 9, 153. 4. deHarven E., Soligo D. & Christensen H. (1990) Double labelling of cell surface antigens with colloidal gold markers. Histochem. J. 22, 18. 5. Bennett J. M., Catovsky D., Daniel M. T., Flandrin G., Galton D. A. G,, Gralnick H. R. & Sultan C. (1989) Proposals for the classification of the myelodysplastic syndromes. Br. J. Haemat. 51, 189. 6. Doyle J., Thorner P. & Zipursky A. (in preparation). 7. Zipursky A., Rivard G. & Freedman M. H. (1992) Differentiation of leukemic megakaryoblasts in Down's syndrome. Blood 80, 32a. 8. Hayashi Y., Eguchi M., Sugita K., Nakazawa S., Sato T., Kojima S., Bessho F., Konishi S., Inaba T., Hanada R. & Yamamoto K. (1988) Cytogenetic findings and clinical features in acute leukemia and transient myeloproliferative disorder in Down's syndrome. Blood 72, 15. 9. Kojima S., Matsuyama T., Sato T., Horibe K., Konishi S., Tsuchida M., Hayashi Y., Kigasawa H., Akiyama Y., Okamura J., Nakahata T., Bessho F., Eguchi M., Nakazawa S. & Ueda R. (1990) Down's syndrome and acute leukemia in children: an analysis of phenotype by use of monoclonal antibodies and electron microscopic platelet peroxidase reaction. Blood 76, 2348. 10. Broomhead A. F., Kwan Y. L., Zell N. A., Lam-PoTan P. R. & O'Gorman H. D. (1985) Acute leukemia characterized by trisomy 8 in Down's syndrome. Pathology 17, 111. 1l. Kaneko Y., Rowley J. D., Variakojis D. et al. (1981) Chromosome abnormalities in Down's syndrome patients with acute leukemia. Blood 58, 459. 12. Suarez C. R., LeBeau M., Silberman S., Fresco R. & Rowley J. D. (1985) Acute megakaryoblastic leukemia in Down's syndrome: report of a case and review of cytogenetic findings. Med. Ped, Oncol. 13, 225. 13. Slavc I., Urban C., Haas O. A., Kroisel P. M. & K611er U, (1991) Acute megakaryocytic leukemia in children. Cancer 68, 2266. 14. Lie S. O. (personal communication). 15. Ravindranath Y., Abella E., Krischer J., Civin C. I., Harris M., Chauvenet A., Alvarado C., Dubowy R., Ritchey A. K. & Steuber (2. P. (1990) Acute myeloid leukemia (AML) in Down's syndrome (DS): a highly curable disease. Blood 76, 311a. 16. Schaison G., Daniel M. T., Berger R. & Baruchel A. (1990) De novo acute megakaryoblastic leukemia (AMKL) in children. A study of 22 cases. Blood 76, 316a. 17. Bevan D., Rose M. & Greaves M. (1982) Leukemia of platelet precursors: diverse features in four cases. Br. J. Haernat. 51, 147. 18. Eguchi M., Sakakibara H., Suda J., Ozawa J., Hayashi
171
Y., Sato T., Fojima S. & Furukawa T. (1989) Ultrastructural and ultracytochemical differences between transient myeloproliferative disorder and megakaryoblastic leukemia in Down's syndrome. Br. J. Haemat. 73, 315. 19. Koller U., Haas O., Ludwig W. G., Harbott J., Creutzig U.. Bettelheim P., Krieger O., Gadner W. & Knapp W. (1991) Acute megakaryoblastic leukemia: a comparison of phenotypic, genotypic and clinical features in children and adults. Proc. Am. Ass. Cancer Res. 32, 179. 2/). Penchansky L., Kaplan S. S., Stoic V. & Krause J. R. (1991) Down's syndrome and mixed acute leukemia in infants. Cancer 68, 414. 21. Quaglino D. & Hayhoe F. G. H. (1960) Periodic acidSchiff positivity in erythroblasts with spinal reference to Di Guglielmo's syndrome. Br. J. Haemat. 6, 26. 22. Malkin D. & Freedman M. (1989) Childhood erythroleukcmia: review of clinical and biological features. Am. J. Pediat. Hemat. Oncol. il, 348. 23. Debili N., Kieffer N., Mitjavila M. T., Villeval J. L., Guichard J., Teillet F., Henri A.. Clemetson K. J., Vainchenker W. & Breton-Gorius J. (1989) Expression of platelet glycoprotein by erythroid blasts in four cases of trisomy 21. Leukemia 3, 669. 24. Nishi T.. Nakahata T., Koike K., Takagi M., Naganurea K. & Akabane T. (1990) Induction of mixed crythroid-megakaryocyte colonies and bipotential blast cell colonies by recombinant human erythropoietin in serum-free culture. Blood 76, 1330. 25. Cairney E. A.. McKenna R., Arthur D. C., Nesbit M. V. Jr & Woods W. J. (1986) Acute megakaryoblastic leukemia in children. Br. J. Haemat. 63, 541. 26. Hecht F., tlecht B. K. & Morgan R. (1986) Chromosome clues to acute leukemia in Down's syndrome. Cancer Genet. Cytogenet. 21, 93. 27. Wang N., Leung J., Warrier R. P., Schorin M., Kirpatrick D., Nowak M. J. & Strand R. (1987) Nonrandom chromosomal aberrations and clonal chromosomal evolution in acute leukemia associated with Down's syndrome. Cancer Genet, Cvtogenet. 28, 155. 28. Carroll A., Civin C., Schneider N., Dahl G., Pappo A., Bowman P., Emami A., Gross S., Alvarado C., Phillips C., Krischer J., Crist W., Head D., Gresik M., Ravindranath Y. & Weinstein H. (1991) The t (1;22) (p13;q13) is nonrandom and restricted to infants with acute megakaryoblastic leukemia. A pediatric oncology group study, Blood 78, 748. 29. San Miguel J. F., Gonzalez M., Canizo M. C., Ojeda E., Orfao A., Caballero M. D., Moro M. J., Fisac P. & Lopez Borrasca A. L. (1988) Leukemias with megakaryoblastic involvement: clinical, hematologic, and immunologic characteristics. Blood 72, 402. 30. Lu G., Altman A. J. & Benn P, A. (1993). Review of the cytogenetic changes in .acute megakaryoblastic leukemia: one disease or several'? Cancer Genet. Cytogenet. 67, 81.
Pergamon
Leukemia Research Vol. 18. No, 3, pp 163-171. 1994 Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 1tl45-2126/04 $6.(X~ + 0,(X)
0145-2126(93)E0016-O
MYELODYSPLASIA AND ACUTE MEGAKARYOBLASTIC LEUKEMIA IN DOWN'S SYNDROME A. ZIPURSKY,* P. THORNER,* E. DE HARVEN,~ H. CHRISTENSEN~ and J. DOYLE* *Division of Hematology/Oncology, Department of Pediatrics, +Department of Pathology, The Hospital for Sick Children; and ~:Department of Pathology, University of Toronto, Canada
(Received 19 August 1993. Revision accepted 18 November 1993) Abstract--In this report we describe the clinical and hematologic features of 23 cases of myelodysplasia (MDS) or acute megakaryoblastic leukemia (AMKL) occurring in Down's syndrome. MDS was characterized by thrombocytopenia, abnormal megakaryocytopoiesis, megakaryoblasts (<30%) in the marrow and abnormal karyotype, the most common of which was trisomy 8, found in 7/15 patients with MDS. Three of five patients achieved a complete remission with low dose cytosine arabinoside, vincristine and retinyl palmitate. The high cure rate and the distinctive features of the leukemic process in these cases suggest that this type of MDS and AMKL are unique to patients with Down's syndrome.
Key words: Megakaryoblasts,
megakaryoblastic
leukemia,
myelodysplasia,
Down's
syndrome.
CD 13, CD33; platelet antigen GpIIb/IIIa, CD41; and common ALL antigen CD10 (Coulter Immunology, Hialeah, FL). Bone marrow biopsies were fixed in B5, decalcified and embedded in paraffin. Sections were cut at 3 btm and stained with hematoxylin and eosin or Gordon and Sweet's reticulum stain. All sections were reviewed by a pediatric pathologist (P.T). Immunohistochemistry to identify megakaryocytic cells was performed on paraffin sections using a polyclonal antibody to Factor 8-related antigen (Dakopatts, Denmark) by the peroxidase-antiperoxidase method and using the monoclonal antibody MB2 (Biotest, Germany) by the indirect peroxidase method, as previously described [3]. In addition to staining B-lymphocytes as indicated by the manufacturer, in our laboratory this monodonal antibody stains megakaryocytes and their precursors. Electron microscopy specimens were prepared by fixation in glutaraldebyde. Subsequently the cells were treated with monoclonal antibody to CD61 (Gpllla) and then treated with gold-labelled anti-mouse antibody as described previously [4].
Introduction ACUTE MEGAKARYOBLASTIC LEUKEMIA ( A M K L ) is the most common form of leukemia in Down's syndrome [11. It has been estimated that the incidence is 500 times greater in children with Down's syndrome than in normal children [2]. Frequently the disease is preceded by a pre-leukemic phase characterized by abnormal marrow function, a myelodysplastic process. In the present study, we describe the clinical and hematologic features of myelodysplasia in Down's syndrome with particular reference to the abnormal megakaryopoiesis which precedes the development of A M K L . Methods Cases of myelodysplasia (MDS) and AMKL were either studied at The Hospital for Sick Children (cases 6, 7, 13, 14, 18 and 20) or reported to the Canadian Down's syndrome Leukemia Registry. In all cases, blood and bone marrow films were reviewed by a pediatric hematologist (A. Z). Standard hematologic techniques were used for peripheral blood studies and for cytochemistry. Leukocyte membrane antigens were determined using a Coulter Epics Profile Analyzer (Coulter Electronics) and Coulter monoclonal antibodies to T-cell antigens CD2, CD3, CD7; B-cell antigens CD19, CD20, CD22; myeloid/monocytic antigens
Results
Clinical Twenty-three patients with acute megakaryoblastic leukemia and/or myelodysplasia were included in the study. Diagnostic features of MDS included cytopenia, abnormal hematopoiesis and less than 3 0 ~ blasts in the bone marrow. If the marrow contained more than 30% blasts, the patient was considered to have leukemia in keeping with the definition of MDS in other conditions [5]. The typical
Correspondence to: Dr A. Zipursky, Division of Hematology/Oncology, University of Toronto. Toronto, Canada. 163
A. ZIPURSKYet al.
164
TABLE 1. CLINICAL F1NDINGS IN A M K L / M D S IN DOWN'S SYNDROME
Age (months)
Sex
1
39
F
2 3 4 5 6 7 8 9 10
18 21 24 8 37 20 24 21 38
F M F F F M F M M
45+ 3 dead 35 + 18 dead 15+ 8 dead 15+ 4 dead 29+ 16+
Case
Survivalt (months)
Platelets ( x 109/1)
Hemoglobin (g/l)
Blasts (PB)$ (x 199/1)
Blasts$ (BM) %
12 79 40 27 4{1 90 4 200 36
2.1 I) 0 0.144 {1 0.14 0.175 0.312 0.315 0.26 0.07 0.1 0.18 0 0.12 0
22 16 20 15 18 11 23 21 21 6
6.270 0.18 42.4 16.7 11.6
60 80 91 67 58
0.36 0
---
MDS
11
19
M
32 +
12 13 14 15 16
21 24 8 37 37
M M M M F
9 dead 12+ 20+ 28+ 1 dead
90 61 59 17 32 77
102 99 123 92 114 123 68 1(14 86 90 77 110 117 116 88 63
13 26 25 27 14
M M M M F
6 dead 80+ 82+ 15 + 36+
14 24 28 20 15
96 45 50 106 77
M M
0 dead* 13 dead
32 17
8
12 12 8 29 --
AMKL 17 18 19 20 21
AMKL/MDS 22 23
13 26
4.2 4.9
* No treatment given. t Survival from initiation of therapy or from diagnosis (if no therapy given); - - = data not available; MDS = myelodysplasia; A M K L = acute megakaryoblastic leukemia; A M K L / M D S = bone marrow aspiration inadequate to determine percentage of blasts. $ PB = peripheral blood; BM = bone marrow.
clinical f e a t u r e s of D o w n ' s s y n d r o m e w e r e p r e s e n t in 22 out of 23 cases. C a s e N o . 13 was p h e n o t y p i c a l l y n o r m a l but was p r o v e n to be a m o s a i c with a m i n o r p o p u l a t i o n of t r i s o m y 21 cells. This case is d e s c r i b e d in detail e l s e w h e r e [6]. A s u m m a r y of t h e clinical findings in t h e s e cases is shown in T a b l e 1. This t a b l e is d i v i d e d into cases of m y e l o d y s p l a s i a ( M D S ) in which the m a r r o w blast c o u n t was less t h a n 3 0 % , a c u t e m e g a k a r y o b l a s t i c l e u k e m i a ( A M K L ) in which t h e m a r r o w blast count was g r e a t e r t h a n 30% a n d two cases in which b o n e m a r r o w a s p i r a t i o n was u n s a t i s f a c t o r y . A l l cases in T a b l e 1 w e r e less t h a n 40 m o n t h s of age. S e v e n of the 16 cases of M D S a n d o n e of the five cases of A M K L w e r e f e m a l e . A l l p a t i e n t s with M D S w e r e well at t h e t i m e of diagnosis. T h e r e was no e v i d e n c e of h e p a t o s p l e n o m e g a l y or l y m p h a d e n o p a t h y in 10 of the 11 cases for w h i c h t h e r e w e r e d a t a . A history of n e o n a t a l t r a n s i e n t l e u k e m i a was p r e s e n t in t h r e e of the M D S cases, o n e o f t h e A M K L a n d o n e of the u n d e f i n e d cases. T h u s 5 o u t o f 23 cases of M D S
a n d / o r A M K L h a d a h i s t o r y of t r a n s i e n t l e u k e m i a . In 10 of the 11 M D S cases, t h e d i s e a s e m a n i f e s t e d as i s o l a t e d t h r o m b o c y t o p e n i a ; o n e case m a n i f e s t e d as unexplained anemia. In m a n y cases t h e r a p y was s t a r t e d b e f o r e l e u k e m i a d e v e l o p e d a n d , t h e r e f o r e , t h e d u r a t i o n of m y e l o d y s p l a s i a was u n k n o w n . A l s o in t h o s e cases in which the blast c o u n t was > 30% w h e n first s e e n , it was not known whether there had been a pre-leukemic, m y e l o d y s p l a s t i c p h a s e since, as n o t e d a b o v e , m y e l o d y s p l a s i a in m o s t cases was a s y m p t o m a t i c . In 11 cases t h e p a t i e n t was o b s e r v e d d u r i n g the p e r i o d of m y e l o d y s p l a s i a and the d u r a t i o n v a r i e d f r o m 1-18 m o n t h s with a m e a n v a l u e of 6,3 m o n t h s . P r o g r e s s i o n of M D S to A M K L was d o c u m e n t e d in two cases in which initial m a r r o w blast c o u n t s of 20 a n d 18% w e r e f o l l o w e d 6 a n d 10 m o n t h s l a t e r by an i n c r e a s e in t h e p e r c e n t a g e o f blasts to 41 a n d 9 0 % , respectively. T h e h e m a t o l o g i c f e a t u r e s o f M D S a n d A M K L at the t i m e o f d i a g n o s i s a r e s h o w n in T a b l e 1. T h r o m b o c y t o p e n i a was p r e s e n t in all b u t
Myelodysplasia in Down's syndrome 6007
/ ~
500 400 % 300
% × 200
~ ~ffr!"~¢'~.:::~
jW LU
CYTOSINE • ARABINOSIDE
....
•
•
•
/
loo
//~
0
1990
JAN FEB 1991
r
OCT
f
NOV
--
DEC 199!
,
7
JAN
.... FEB
FIG. 1. Effect of chemotherapy on platelet count in patient 14. In December 1991, the bone marrow biopsy showed no evidence of myelofibrosis and cytogenetics were normal. For dosage of chemotherapy, see text. Patient continues in remission to date.
one MDS case with platelet counts significantly lower in the A M K L group. Neutropenia (<1000 x 1091) occurred in 4 out of 15 MDS cases and in none of the A M K L or A M K L / M D S cases. In MDS a mild anemia (<100 g/l) was found in 7 of 15 cases whereas
165
it was more common and severe in the A M K L and A M K L / M D S cases. Megakaryoblasts in low numbers (0.1-2.1 x 109/1) were found in 11 of 16 cases of MDS and 1 of 2 cases of A M K L / M D S . In A M K L megakaryoblasts were found in higher numbers in 4 of 5 cases (6.3, 11.6, 16.7 and 42.4 x 109/1). Thrombocytopenia in the MDS group appeared to be due to a failure of production since platelet destruction was not increased as evidenced by apparently normal platelet survival following platelet transfusions in all eight patients so treated. Twenty-one cases received anti-leukemic therapy. Fifteen cases received intensive A N L L treatment which included cytosine arabinoside and anthracycline chemotherapy. Eleven are in continuing clinical remission with a mean follow-up of 31 months (range = 15-73 months). Five cases (cases 1, 5, 6, 12 and 14) received a low dose chemotherapy program which included vincristine 1.0 mg/M 2 and cytosine arabinoside 10 mg/ M 2 every 12 h for 7 days; these drugs were given at 2-week intervals. Also these patients received retinyl palmitate (Aquapalm-Hoffman La Roche) 50,000units/M 2 by mouth daily. Three patients achieved complete remission (cases 1, 12 and 14); in
FIG. 2. Bone marrow biopsy of a Down's syndrome patient with myelodysplasia. The marrow is hypercellular with increased numbers of megakaryocytes, many of which are abnormal in appearance (dysmorphic) with small nuclei or unusual nuclear shapes (hematoxylin and eosin × 800).
166
A. ZIPURSKYet al.
~ii
!~ii~i
¸
•. . . .
FIG. 3. Bone marrow aspirate of a Down's syndrome patient with myelodysplasia (x 1000). A dysplastic megakaryocyte is seen. two this was confirmed by bone marrow examination. Two cases remain in remission, case 1 for 42 months (therapy was stopped after 36 months) and case 14 who is in remission and still on treatment 18 months since starting therapy. Case 12 achieved a hematologic remission but therapy was stopped by the parents after 2 months and the child relapsed shortly thereafter. Cases 5 and 6 did not achieve a remission and were then treated with high dose chemotherapy. Case 5 did not achieve a remission and died whereas Case 6 did and continues in remission 7 months since completion of therapy. The response to low dose therapy in one of our cases is shown in Fig. 1 and demonstrates the rapidity of response to that therapy. Thus, platelet counts began to rise within 2 weeks without any evidence of bone marrow suppression which usually characterizes the hematologic response to high dose A N L L chemotherapy. Case 4 did not recewe chemotherapy and remained well for 18 months, although still thrombocytopenic. She subsequently developed AMKL, did not receive treatment and died shortly thereafter. Case 22 received no therapy and died shortly after diagnosis. Case 8 received oral thioguanine for 5 months, then the low dose protocol and subsequently high dose
therapy. She did not achieve a remission and died 1 year after diagnosis. Laboratory studies
In all cases in our series the blasts were similar on Wright-stained, air-dried smears, varying in size from 15-20 ~m in diameter, with 1-2 nucleoli, a small amount of deeply basophilic cytoplasm and usually distinct cytoplasmic blebbing. Bone marrow biopsies indicated that megakaryocytes were increased in number in the marrow but were small and abnormal in appearance with either small single nuclei or unusual nuclear structure (Fig. 2). This was present in 10 of 11 MDS cases studied. Immunohistochemicai staining of slides showed that these large cells stained positively for Factor 8 and MB2 antigens consistent with a megakaryocytic lineage. Megakaryocytes in bone marrow aspirates also were small and very abnormal in appearance, often containing vacuolated cytoplasm and mononuclear nuclei (Fig. 3). In four cases (three MDS and one AMKL) marrow cells were studied by electron microscopy. Cells in the megakaryocytic lineage were recognized by the presence of the surface antigen CD61 (GpIIIa) as determined by immunogold labelling (Fig. 4). The
Myelodysplasiain Down'ssyndrome
167
FIG. 4. Electron microscopy of a megakaryoblast showing immunogold labelling of surface antigen Gp IIIa (× 40,000), prominent blebs and demarcation vesicles.
abnormal megakaryocytes varied from cells which were blastic in appearance to those with evidence of megakaryocyte differentiation including demarcation membranes and alpha-granules (Fig. 5). In most cases, however, the cells showed little evidence of differentiation. Myelofibrosis was observed in 7 of 11 cases of MDS studied and both cases of AMKL. In 7 of 15 MDS cases dyserythropoiesis was noted in the marrow. Leukocyte surface antigens were studied in six of our patients, information on seven other patients was sent to us. Platelet antigen GpIIb/IIIa was present in 7/10 cases studied by flow cytometry. Two of the negative cases were also studied by electron microscopy and immunogold labelling using monoclonal antibody to platelet antigen GpIIIa; both were positive. The myeloid antigens CD33 (My9) and CD13 (My7) were positive in 6 of 6 and 3 of 4 cases, respectively. Antigens expressed on T- and B-cells were usually negative although the cells of 4 of 12 patients expressed CD3 and/or CD7 and in 1 of 12 cases (case 13) the cells expressed CD10 (CALLA). Cytochemical staining of the leukemic cells for Sudan black, non-specific esterase and PAS were usually negative. Only the acid phosphatase stain was consistently positive.
Chromosomal analysis in the MDS group revealed, in addition to trisomy 21, seven cases with trisomy 8, two with monosomy 7 and two with other findings, Trisomy 21 alone was found in six of the MDS cases and in the three AMKL cases studied. Discussion Acute megakaryoblastic leukemia (AMKL) is the commonest form of leukemia occurring in Down's syndrome [1]. It is often preceded by a pre-leukemic hematologic disorder which, in this report, is referred to as myelodysplasia (MDS). We have arbitrarily specified three criteria for the diagnosis of MDS in Down's syndrome: (1) cytopenia (usually thrombocytopenia); (2) dysmegakaryopoiesis; and (3) increased numbers of megakaryoblasts in marrow aspirates but the total percentage should be less than 30% in keeping with the recommendations described by Bennett et al. [5] for other forms of MDS. MDS was documented in 11 out of 23 cases in the present series. It is likely that the true incidence was higher because in those cases in which leukemia was diagnosed when the patient was first seen it was not possible to know whether there had been a preceding pre-leukemic phase since that phase is usually
168
A. ZIPURSKYet al.
FIG. 5. Electron microscopy of a megakaryoblast showing demarcation membranes and alpha granules (× 31,992).
asymptomatic. Hayashi et al. reported that 11 of 13 cases of A M K L in Down's syndrome were preceded by a pre-leukemic syndrome [8] of myelodysplasia. Similarly Kojima et al. [9] described 14 cases of AMKL of whom seven had a pre-leukemic period of myelodysplasia. The pre-leukemic phase was characterized in 10 of 11 of the cases in this series by thrombocytopenia. This was due to failure of production since platelet survival was normal as evidenced by the good response to platelet transfusions in all eight patients so treated. Despite the evidence of production failure, bone marrow biopsies indicated that the numbers of megakaryocytes were increased in the marrow and their appearance was abnormal with many of the cells smaller than normal megakaryocytes and often contained single or bizarre nuclei (Figs 2 and 3). The duration of MDS before becoming A M K L is unknown. In two of our cases MDS progressed to leukemia within 6 and 11 months, respectively. One of our cases was followed for 18 months with no therapy and then developed AMKL. In most of our cases, since therapy was started when the patient was myelodysplastic, the natural history of the disease was difficult to determine. There have been other reports of progression from MDS to AMKL. Broomhead et al. reviewed four
cases associated with trisomy 8 in which the preleukemic phase lasted 8-22 months [10]. Kaneko et al. [11] described two cases in which progression occurred over a period of 8 and 22 months, respectively. There have been reports of other cases in which progression occurred over 4 weeks [121 and 4 months
[131. If it is assumed that all MDS cases will develop A M K L the results of therapy in A M K L / M D S have been excellent (Table 2). In both the Nordic [14] and POG [151 series, the MDS and A M K L cases were lumped together. In our series (Tables 1 and 2) both MDS and AMKL responded to therapy. In the MDS group, three of the five deaths occurred during induction. All relapses occurred in the first 9 months. Five cases were treated with low dose cytosine arabinoside, vincristine and retinyl palmitate and three of these patients achieved a remission [7]. Two of them remain in remission 40 and 18 months since starting therapy while one relapsed shortly after therapy was stopped by the parents. The response to this low dose therapy (Fig. 1) was characterized by a rapid rise in platelet count, usually, without evidence of the bone marrow suppression which is seen following high dose chemotherapy and which usually precedes the appearance of remission. Case 22 received no therapy and died within 1 month.
169
Myelodysplasiain Down's syndrome TABLE 2. TREATMENTOF AMKL/MDS IN DOWN'SSYNDROME
POG [15] Nordic [14]* Present series
No. studied
No. in CCR
%
Duration of follow-up (months)
12 17 22
12 13 16
100 76 64
27-60 5-89 12-80
* A study of the Nordic Society of Pediatric Hematology/Oncology. The survival of this group of AMKL/MDS patients is significantly better than that reported for cases of AMKL in non-Down's syndrome children. A group of 18 such patients reported by Schaison et al. [16] had a median survival of 5 months, with only one patient still alive 12 months after diagnosis. The blast cells in these patients were considered to be megakaryoblasts because of their morphology and the presence of platelet-specific antigens on their surface. In all cases in our series the blasts were similar, varying in size from 15-20 ~tm in diameter, with 1-2 nucleoli, a small amount of deeply basophilic cytoplasm and usually distinct cytoplasmic blebbing. This morphology is consistent with the appearance of megakaryoblasts [17]. When viewed by electron microscopy the blasts were usually undifferentiated although in some there was evidence of demarcation membranes and alphagranules. Eguchi et al. found no evidence of demarcation membranes or alpha-granules in 13 cases of AMKL in Down's syndrome [18]. Four cases were studied and in all the blast cells were labelled with anti-CD61 (GplIIa) as identified by immunogold (Figs 4 and 5). Distinct blebs were noted on all surfaces in all four cases (Figs 4 and 5). Most reported studies of membrane antigens found that the blasts of AMKL/MDS in Down's syndrome contain the platelet antigen GplIb/IIIa. Koller et al. [19] reported that CD61 (anti-GplIIa) was more sensitive than Gp41a (anti-GpIIb/IIIa) and this is consistent with our finding of two cases that were negative with anti-GplIb/IIIa on flow cytometry but positive with anti-GplIIa on electron microscopy. In our series the blast cells frequently contained the antigens My9 and My7, typical of myeloid cells, consistent with the findings of Kaneko et al. [11]. Cases have been reported of pre-leukemia in young children with Down's syndrome in which antigens usually expressed in myeloid and T-cells were positive in the blasts [20] and in one case where CD10 (CALLA) was positive [8]. In our opinion it is likely that these are cases of acute leukemia of the type described in the present study. The leukemic cell in this disorder may express multiple aberrant antigens. As noted above the morphology of the leukemic
cells in AMKL/MDS suggests that these cells are megakaryoblasts. It should be pointed out, however, that in 6 of 16 MDS cases there was evidence of dyserythropoiesis (including PAS positivity of normoblasts [21]). Some of our cases had been diagnosed as erythroleukemia. Also there have been reports of an increased incidence of erythroleukemia in patients with Down's syndrome [22]. In some of these cases, the leukemic cells had properties of both erythroid and megakaryocytic lineage [23]. We have postulated previously [1] that in AMKL/MDS or erythroleukemia in Down's syndrome the leukemic cell is a biphenotypic cell with both megakaryocytic and erythroid features. Such biphenotypic hematopoietic progenitor cells have been isolated from normal bone marrow [24]. The megakaryocytic nature of the leukemic cells in myelodysplasia is supported also by the presence of myelofibrosis in 7 of 11 MDS cases studied by us. Myelofibrosis is closely associated with acute megakaryoblastic leukemia in adults [17[, and in nonDown's syndrome children [25]. Chromosomal abnormalities were found frequently in our cases of myelodysplasia. Of interest, 7 of 14 cases of MDS had trisomy 8. Three cases of AMKL were studied and only trisomy 21 was found. It would appear that trisomy 8 is closely related to the development of myelodysplasia in Down's syndrome. Hecht et al. in 1986 [26] reviewed all published chromosomal studies of leukemia in Down's syndrome. In that series there were 17 cases under age 4 with ANLL. Trisomy 8 was found in 6 out of 17 cases, one with an associated trisomy 19. Hayashi et al. studied 13 cases of AMKL in Down's syndrome and found two with trisomy 8 [8]. Wang et al. [27] described four cases of acute leukemia in children younger than 4 years of age of whom three had trisomy 8. Broomhead et al. described one such case [10] and postulated that 'the triad of pre-leukemic (thrombocytopenia), AMKL and trisomy 8 in children with Down's syndrome represents a specific type of acute l e u k e m i a . . . ' . Trisomy 8 was found only in 1 of 10 cases described by Cairney et al. [25] and in none of 14 cases described by Schaison et al. [16]. Combining our series of 17 cases studied with
170
A. Z|PURSKYet al. TABLE 3. LEUKEMIA IN DOWN'S SYNDROME: THE HOSPITAL FOR SICK CHILDREN (1985-
1991)
ALL ANLL AMKL* Other Total
Non-Down's syndrome
Down's syndrome
O/Et
325 (85%)
6 (50%)
=13 x
7 (2%) 51 383 (13%)
6 (50%) 0 12
=600 x =22 x
* AMKL includes cases diagnosed as either acute megakaryoblastic leukemia or erythroleukemia. t O/E = Observed over expected number of cases assuming that the prevalence of Down's syndrome is 1 in 700 children. the cases studied by others [8, 26, 27] trisomy 8 has been found in 18 of 51 (35%) of patients with A M K L / MDS in Down's syndrome. It would appear, therefore, that trisomy 8 is frequently associated with the acute megakaryoblastic leukemia that occurs in Down's syndrome. It is apparent that acute megakaryoblastic leukemia including the associated myelodysplastic syndrome is relatively common in Down's syndrome. We estimated previously that its incidence is approximately 500 times more common in Down's syndrome than in normal children [2]. To analyze this further, we reviewed all cases of leukemia admitted to The Hospital for Sick Children from 1985 to 1991 to determine the types of leukemia seen in Down's syndrome and in normal children (Table 3). The prevalence of Down's syndrome is thought to be 1 in 700 of the population. Accordingly the experience at The Hospital for Sick Children (Table 3) indicates that the total incidence of leukemia in Down's syndrome children was 22 times greater than in normal children; however, the incidence of A M K L was 600 times greater than expected. A L L was only 13 times greater. There may be a referral bias in these data, nevertheless all children with leukemia in our region are referred to this hospital and, therefore, our experience probably reflects the true incidence of the types of leukemia occurring in children with Down's syndrome. Accordingly it would appear that the incidences of both A L L and A M K L are increased in Down's syndrome, but the incidence of the latter is remarkably high. Why is there such a high incidence of A M K L / M D S in Down's syndrome? Is A M K L / M D S in Down's syndrome a different type of megakaryoblastic leukemia than that found in normal children or adults? There is considerable evidence to suggest that the A M K L / M D S of Down's syndrome is a unique disease that differs from A M K L found in normal subjects. The first difference is that none of the reported series of A M K L in non-Down's syndrome
children or adults have described a high incidence of myelodysplasia and the unique bone marrow findings in the myelodysplastic phase of leukemia in Down's syndrome. Secondly, hepatosplenomegaly is not a common feature of MDS or A M K L in Down's syndrome, whereas it is common in A M K L in normal children [24] and in infants suffering from A M K L in association with a 1:22 translocation [28]. However, the major difference between the A M K L / M D S of Down's syndrome and that in normal individuals is the high cure rate of this disease in Down's syndrome (Table 3). Schaison et al. [15] reported a series of 18 normal children with A M K L in which the median survival with therapy was 5 months with only one child, a recipient of a bone marrow transplant, alive after 1 year. Infants with A M K L and a 1:22 translocation [28] as well as adults [29] with A M K L have a very poor response to therapy. Lu et al. [30] recently reported a review of cytogenetic changes found in acute megakaryoblastic leukemia and concluded that there are at least three distinct types of AMKL. One is that found in Down's syndrome, one is the infant form associated with a 4 : 11 translocation and the third refers to other cases, most of which occur in adults. All the above evidence suggests that acute megakaryoblastic leukemia in Down's syndrome is a unique disease characterized by a long pre-leukemic, myelodysplastic phase, unusual chromosomal findings and a high cure rate. Acknowledgements--We gratefully acknowledge the support of the Leukemia Research Fund of Canada.
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