Trisomy 21 in Transient Myeloproliferative Disorder M. J. W. Faed, J. Robertson, A. S. Todd, M. Sivakumaran, and W. O. Tarnow-Mordi
ABSTRACT: Transient leukemia in phenotypically normal children is rare. A n e w b o r n child in whom fever and tachypnea developed'at age 2 d a y s had a white blood cell c o u n t of 20.1 x 109/L and m a n y abnormal blast cells. Chromosome analysis of spontaneously dividing cells f r o m the blood showed these to have trisomy 21, and 80% of cells in the m a r r o w were also trisomic. No trisomic cells were p r e s e n t in skin fibroblast cultures. At age 6 m o n t h s , at which time the blood film a p p e a r e d normal, trisomic cells were no longer present.
INTRODUCTION Down syndrome infants have an increased risk of congenital leukemia; Rosner and Lee [1] showed that in 21 of 56 such newborn infants the leukemia went into spontaneous remission, although in 25% of cases it subsequently recurred. Transient leukemia in Down syndrome infants is apparently megakaryoblastic [2, 3]. About 20% of all cases of leukemia in Down syndrome have been suggested to be of an acute megakaryoblastic type and may yield a history of early transitory leukemia [2]. Transient congenital leukemia has been reported only rarely in infants with a normal karyotype. Van Eys and Flexner [4] reported a patient who had a spontaneous remission of 8 months, and one of the five patients with congenital leukemia reported by Chu et al. [5] remained in remission. More recently, Lampkin et al. [6] reported spontaneous remission in an infant with acute nonlymphoblastic leukemia (ANLL). Most phenotypically normal children with a transient leukemia have been reported to have trisomy 21 mosaicism; three such children evidenced mosaicism in both blood cells and skin fibroblasts [7-9], but in others the trisomic cells were observed only in the marrow or the peripheral blood and only normal cells were present in fibroblast cultures [9-13]. We report sequential studies on another example of apparent transient congenital leukemia in a phenotypically normal infant first investigated because of possible pneumonia. In this case nearly all the spontaneously dividing cells in the peripheral blood during the neonatal period showed trisomy 21, but only normal cells were found in skin fibroblast cultures. These rare observations may be instructive regarding the etiology of leukemia.
From the Human Genetics Laboratory, Departments of Pathology, Haematology, and Child Health, University of Dundee, Scotland.
Address r e p r i n t requests to: M. Faed, Ph.D., Human Genetics Laboratory, Department of Pathology, Ninewells Hospital and Medical School, Dundee DD1 9SY, Scotland. Received February 2, 1990; accepted March 12, 1990.
259 © 1990 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
Cancer Genet Cytogenet 48:259-264 (1990) 0165-4608/90/$3.50
260
M . J . w . Faed et al.
CASE REPORT A n infant was born at 39 weeks of gestation by spontaneous vertex delivery to Scottish parents. Her mother, aged 24 years and a primigravida, had an uneventful pregnancy. Her father was aged 26 years. Her birth weight was 4.36 kg. At age 2 days she was a d m i t t e d to the special care nursery because of fever and tachypnea. Her spleen and liver were both p a l p a b l e at 3 cm below the costal margin but there was no bruising or bleeding. E x a m i n a t i o n was otherwise normal. She received penicillin and gentamicin for 48 hours until blood and surface cultures proved sterile, w h e n antibiotics were stopped. Her h e m o g l o b i n was 17.0 g/dl, her platelet count was 107 x 109/L, and her white blood cell (WBC) count was 17 x 109/L with 24% blast cells. There was no evidence of congenital infection with rubella, toxoplasmosis, syphilis, or Herpes, but her urine grew cytomegalovirus. A bone marrow examination on the fifth day of age confirmed an excess of similar blast cells. Because of the rapid decrease in blast cell numbers, she was discharged from the nursery at age 13 days and made good progress until age 6 weeks w h e n she was admitted to hospital because of an upper respiratory infection and cough treated with erythromycin. Her liver and spleen were then palpable at 2 cm below the costal margin and a chest roentgenogram showed opacities at the left base consistent with pneumonia. Her hemoglobin was 10.9 g/dl, her WBC count was 6.5 x 109/L with no blast cells, and her platelet count was 61 × 109/L. She recovered r a p i d l y and was discharged home after 2 days. On subsequent clinic visits she r e m a i n e d well and her blood and platelet counts were normal. At age 3 months her spleen was no longer palpable and her liver was only 1 cm below the costal margin. At age 18 months she remained healthy with normal d e v e l o p m e n t and no abnormal physical findings. Examination of both parents showed no caf~ au lait spots or other evidence of von Recklinghausen's disease.
Hematological Investigation The blood counts are shown in Table 1. In Romanowsky-stained preparations, the blasts varied in size from 13 to 18/~m in diameter and had the general appearance of myeloblasts with m u l t i p l e nucleoli (up to 6%). The staining reactions were S u d a n black negative, chloracetate esterase negative; nonspecific esterase positive, and periodic acid-Schiff negative. Platelets showed anisocytosis with megathrombocytes up to 13~m in diameter in the blood films.
Cytogenetic Studies Blood cultures were established for chromosome preparations in m e d i u m 199 with 5% n e w b o r n calf serum both with and without p h y t o h e m a g g l u t i n i n (PHA). They were harvested at 48 and 72 hours, with colchicine present for the final 2 hours. Marrow cultures were harvested after 1, 18, and 48 hours without mitogens and after 48 hours with PHA. Fibro[llast cultures were established from a small skin b i o p s y and grown in H a m ' s FIO m e d i u m with 15% fetal calf serum for between 22 and 36 days before harvest on coverslips. The results are s u m m a r i z e d in Table 2. The initial observation, made on chromosome preparations of cells derived from the buffy coat and cultured without mitogens for 24 hours, s h o w e d 62 of 64 cells to have trisomy 21. The marrow at 5 days showed about 80% of cells to be trisomic, and a blood sample taken at 6 days s h o w e d nearly all the cells cultured without PHA to have an extra chromosome 21 whereas all PHAstimulated cells had a normal karyotype. Fibroblast cultures from a skin b i o p s y taken at 17 days s h o w e d only normal female cells. A blood sample taken at this time still s h o w e d reasonable numbers of spontaneously dividing cells, all of w h i c h were
Trisomy
261
21 i n T M D
Table 1
Age Days 3 4 5 6 8 9 11 14 17 18 24 Weeks 5 6 7 11 Months 21
B l o o d c o u n t s i n 21 m o n t h s Hb (g/dl)
WBC ( × 109/L}
Blasts ( × 109/L)
Platelets ( × 109/L}
17.7 18.5 18.1 17.5 17.5 16.9 16.0 16.1 16.1 14.5 13.0
17 23.2 20.1 25.8 20.1 25.1 17.1 18.9 15.98 12.4 9.8
4.08 6.03 3.82 3.35 3.02 3.26 2.57 1.89 0.95 0.62 0.30
107 97 80 103 96 98 75 130 165 120 103
10.6 10.9 10.9 11.8
7.1 6.5 8.1 8.5
0 0 0 0
61 61 154 254
11.8
8.7
0
210
Abbreviations: Hb, hemoglobin; WBC, white blood cell.
trisomic; some cells among the PHA-stimulated cultures were also trisomic. Subseq u e n t b l o o d c u l t u r e s at 1 m o n t h a n d 6 m o n t h s s h o w e d a q u i t e d i f f e r e n t p a t t e r n . A t 1 month there were still significant numbers of spontaneously dividing cells, but n e a r l y a l l w e r e c h r o m o s o m a l l y n o r m a l . T h r e e of f o u r c e l l s f o u n d to h a v e a d d i t i o n a l c h r o m o s o m e s 21 w e r e p o l y p l o i d . A t a g e 6 m o n t h s , a f e w m i t o s e s w e r e p r e s e n t i n u n s t i m u l a t e d c u l t u r e s ; t h e s e a l l h a d a n o r m a l k a r y o t y p e , a s d i d all c e l l s i n t h e P H A stimulated cultures.
Table 2 Age (days)
Proportion age shown
o f t r i s o m i c c e l l s p r e s e n t i n c u l t u r e s f r o m s a m p l e s t a k e n at
Tissue
Mitogen
Mitotic index
--
--
64
96.9
5
Buffy coat Marrow
--
6
Blood
17 18
Skin Blood
38
Blood
174
Blood
-PHA -PHA --PHA -PHA -PHA
74 39 91 30 99 25 50 44 101 26 45
79.7 71.8 96.7 0 O 100 8.0 6.8 a 1.0 a 0 0
4
Abbreviation: PHA, phytohemagglutinin.
aThree of 4 cells polyploid.
0.7 1.9 -0.2 2.75 0.5 4.6 0.05 4.15
Mitoses scored
T r i s o m y 21 (%)
262
M . J . W . Faed et al.
DISCUSSION
Transient neonatal leukemia is believed to be rare in p h e n o t y p i c a l l y normal infants. This child was investigated because of possible p n e u m o n i a in the neonatal period, and cytogenetic studies were undertaken as a consequence of finding blast cells in the blood smears. Ascertainment of cases reported by other investigators has varied from routine examination of cord blood [8] to overt pallor [13]. The hematological evidence of leukemia had disappeared within i month. Cytogenetic observations during the leukemic phase showed nearly all the s p o n t a n e o u s l y d i v i d i n g cells from the blood and a high proportion of the cells of the marrow to be trisomic. As the leukemic phase passed, the trisomic cells disappeared; on the last occasion on w h i c h trisomic cells were recognized, three of the four were polyploid. The possibility that these may represent maturing megakaryoblasts is intriguing. It is unfortunate that this possibility could not be explored retrospectively on the material remaining. The relationship of trisomy 21 to leukemia is interesting. Trisomy 21 is the most c o m m o n a d d i t i o n a l finding in leukemic cells in patients with acute lymphoblastic leukemia (ALL) and is also c o m m o n l y found in ANLL. There is an increased risk of l e u k e m i a in Down s y n d r o m e [14, 15]. In particular, there is a proportional increase in megakaryoblastic l e u k e m i a in persons with Down syndrome as c o m p a r e d with the normal population, and the bulk of such cases presenting as transient neonatal l e u k e m i a may be of this type [2]. Of the few cases of transient neonatal leukemia reported in p h e n o t y p i c a l l y normal infants (Table 3), the reactive, p r e s u m a b l y leukemic cells have been shown to be trisomic, although a substantial proportion of these cells in the case reported by Weinberg et al. [10] had a normal karyotype. In a d d i t i o n to those with trisomy 21 shown in Table 3, a case was reported by Van den Berghe et al. [16] in w h i c h the s p o n t a n e o u s l y dividing cells showed pentasomy for chromosome 21. These cells were also present in the marrow, but no abnormality was detected in fibroblasts or in blood or marrow when remission occurred. In our patient, normal cells were found among the s p o n t a n e o u s l y dividing cells, but they constituted a very small p r o p o r t i o n during the leukemic phase. Some spontaneous mitoses are not u n u s u a l in neonates. In this child, small numbers of mitoses persisted even to age 6 months; the child remains well without any clinical or hematological evidence of leukemia after 21 months. Fibroblast cultures have not been examined in all reported cases of p h e n o t y p i c a l l y normal infants. Of the 10 previous cases (Table 3) in which fibroblast cultures were studied, only three have shown trisomic cells and then only in low number. Blast cells from the blood of these infants may proliferate in fibroblast cultures, however, thus giving a false i m p r e s s i o n of constitutional mosaicism. The question of whether the m o s a i c i s m is constitutional or limited to one tissue is not merely academic; if m o s a i c i s m is constitutional, a continuing s u p p l y of trisomic cells in w h i c h malignant change can occur can be presumed. If the trisomy is limited to one t i s s u e - - t h e m a r r o w - - t h e abnormal cell line may be from the committed cell compartment and thus be a t e m p o r a r y population. Indeed, a feature of the p h e n o t y p i c a l l y normal infants is the d i s a p p e a r a n c e or massive reduction of the trisomic cell line. This w o u l d argue against constitutional mosaicism and suggest that in these cases nondisjunction has occurred in a c o m m i t t e d colony-forming cell that ultimately disappears as its proliferative potential is exhausted. The observation of ~n increased frequency of leukemia and in particular of megakaryoblastic l e u k e m i a in Down syndrome, and the suggestion that neonatal transient leukemia, w h i c h m a y also be megakaryoblastic, is invariably a c c o m p a n i e d by cells with trisomy 21, calls for explanation. Neonatal transient leukemia has been described as "ineffective regulation of granulopoiesis w h i c h masquerades as congenital leuke-
263
Trisomy 21 in TMD Table 3
Chromosome findings in cultures on earliest analysis of reported cases of phenotypically normal infants with TLR No. of cells
Reference [71
Age 3 days 3 days 8 days
[81
[10]
11 days 12 days 12mo
[11] Case 1 Case 2
2 days 7 days 3 mo
[12]
Tissue Blood ( P H A + , - ) Bone marrow Skin Blood (unstim) Blood (PHA) Bone marrow Skin Blood (unstim) Blood (PHA) Skin Blood (PHA) Blood (unstim) Blood (PHA) Bone marrow Skin Blood (PHA) Bone marrow Skin
Normal
Trisomic
0 0 96 0" 14 5 38 19 45 85 16 0 65 21 32 14 31 100
50 22 4 36 6 2 3 3 5 0 16 32 0 0 0 3 4 0
[19] Case 1 Case 2 [13l Case 1
Case 2
3 days 3 days
Blood (PHA) Blood (PHA)
6 5 2 5 2
Blood (unstim) Blood (PHA) Bone marrow Skin Blood (unstim) Blood (PHA) Bone marrow Skin
0 38 0 210 0 24 0 60
24 7 55 0 25 1 25 0
Bone marrow Blood (PHA) Skin Blood (unstim) Blood (PHA) Marrow Skin Marrow Blood (unstim) Skin Blood (unstim) Blood (PHA) Bone marrow
0 93 250 3 247 244 200 1 5 99 2 30 15
20 7 0 9 3 6 0 53 20 5 62 0 59
wk days days days wk
7 days 2.5 yr
79% 46%
21% 54%
[9] Case 7
During TLR After TLR
Case 8
During TLR After TLR After TLR Not given During TLR During TLR Not given 4 days 6 days 5 days
Case 9
Present study
Abbreviations: TLR, transient leukemic reaction; PHA, phytohemagglutinin; unstim, unstimulated.
264
M.J.w.
Faed et al.
m i a " [17]. D e s p i t e the w i d e l y r e c o g n i z e d difficulty of d i s t i n g u i s h i n g b e t w e e n a transient m y e l o p r o l i f e r a t i v e d i s o r d e r and c o n g e n i t a l l e u k e m i a , the p r e s e n c e of cells w i t h t r i s o m y 21 w i t h o u t a d d i t i o n a l clonal a b n o r m a l i t y is itself probably b e n i g n [3]. T h e t r a n s i e n t n a t u r e of the p h e n o m e n o n suggests, rather, that there is a greater r e l a t i v e e x p a n s i o n of t r i s o m i c cells in the p r o l i f e r a t i v e phase in the p r o g r e s s i o n f r o m c o l o n y f o r m i n g cell to m a t u r e m e g a k a r y o c y t e . In vitro studies on c o l o n y g r o w t h m a y h e l p to r e s o l v e this q u e s t i o n . A n e x p e c t e d effect of a d d i t i o n a l p r o l i f e r a t i o n w o u l d be to i n c r e a s e the risk of a s e c o n d m u t a t i o n a l e v e n t w h i c h m i g h t result in l e u k e m i a . This agrees w i t h the K n u d s o n " t w o - h i t " h y p o t h e s i s of c a n c e r initiation [18] and offers an e x p l a n a t i o n for the i n c r e a s e d risk of l e u k e m i a in D o w n s y n d r o m e .
REFERENCES 1. Rosner E, Lee SL (1972): Down's syndrome and acute leukemia'. Myelogenous or lymphoblastic? Reports of forty-three cases and a review of the literature. Am J Med 53:203-218. 2. Zipursky A, Peeters M, Poon A (1987): Megakaryoblastic leukemia and Down's syndrome--a review. In: Oncology and Immunology of Down Syndrome. Alan R. Liss, New York, pp. 33-56. 3. Hayashi Y, Eguchi M, Sugita K, Nakazawa S, Sato T, Kojima S, Bessho F, Knoishi 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 72:15-23. 4. Van Eys J, Flexner JM (1969): Transient spontaneous remission in a case of untreated congenital leukemia. Am J Dis Child 118:507. 5. Chu J-Y, O'Connor DM, Gale GB (1982): Five cases of congenital leukemia. Pediatr Res 16:201A. 6. Lampkin BC, Peipon JJ, Price JK, Bove KE, Srivastava AK, Jones MM (1985): Spontaneous remission of presumed conenital acute non lymphoblastic leukemia (ANLL) in a karyotypically normal neonate. Am J Pediatr Hematol Oncol 7:346-351. 7. Brodeur GM, Dahl GV, William DL, Tipton RE, Kalwinsky DK (1980): Transient leukemoid reaction and trisomy 21 mosaicism in a phenotypically normal newborn. Blood 55:691-693. 8. Heaton DC, Fitzgerald PH, Fraser GJ, Abbott GD (1981): Transient leukemoid proliferation of the cytogenetically unbalanced + 21 cell line of a constitutional mosaic boy. Blood 57:883-887. 9. Abe K, Kajii T, Niikawa N (1989): Disomic homozygosity in 21-trisomic cells: A mechanism responsible for transient myeloproliferative syndrome. Human Genet 82:313-316. 10. Weinberg AG, Schiller G, Windmiller J (1982): Neonatal leukemoid reaction. An isolated manifestation of mosaic trisomy 21. Am J Dis Child 136:310-311. 11. Seibel NL, Sommer A, Miser J (1984): Transient neonatal leukemoid reactions in mosaic trisomy 21. J Pediatr 104:251-254. 12. Hanna MD, Melvin SL, Dow LW (1985): Transient myeloproliferative syndrome in a phenotypically normal infant. Am J Pediatr Hematol Oncol 7:79-81. 13. Jones GR, Weaver M, Laug WE (1987): Transient blastemia in phenotypically normal newborns. Am J Pediatr Hematol Oncol 9:153-157. 14. Wald N, Borges WL, Li CC, Turner JH, Harnois MC (1961): Leukemia associated with mongolism. Lancet 1:228. 15. Stewart A, Webb l, Hewitt D (1958): A survey of childhood malignancies. Br Med J 1:1495-1508. 16. Van den Berghe H, Vermaelen K, Broeckhaer-Van Orshoven A, Delbeke M-J, Benoit Y, Orye E, Van Eygen M, Logghe N (1983): Pentasomy 21 characterizing spontaneously regressing congenital acute leukemia. Cancer Genet Cytogenet 9:19-24. 17. Ross JD, Moloney WC, Desforges JF (1963). Ineffective regulation of granulopoiesis masquerading as congenital leukemia in a monogoloid child. ] Pediatr 63:1-10. 18. Knudson AG (1971): Mutation and cancer: Statistical study of retinoblastoma. Proc Natl Acad Sci USA 68:820-823. 19. Laing D-C, Shen E-Y, Chyou S-C (1986): To early distinguish neonatal transient leukemoid proliferation from congenital leukemia by in vitro cell growth. Blut 53:101-106.
ABSTRACT: Transient leukemia in phenotypically normal children is rare. A n e w b o r n child in whom fever and tachypnea developed'at age 2 d a y s had a white blood cell c o u n t of 20.1 x 109/L and m a n y abnormal blast cells. Chromosome analysis of spontaneously dividing cells f r o m the blood showed these to have trisomy 21, and 80% of cells in the m a r r o w were also trisomic. No trisomic cells were p r e s e n t in skin fibroblast cultures. At age 6 m o n t h s , at which time the blood film a p p e a r e d normal, trisomic cells were no longer present.
INTRODUCTION Down syndrome infants have an increased risk of congenital leukemia; Rosner and Lee [1] showed that in 21 of 56 such newborn infants the leukemia went into spontaneous remission, although in 25% of cases it subsequently recurred. Transient leukemia in Down syndrome infants is apparently megakaryoblastic [2, 3]. About 20% of all cases of leukemia in Down syndrome have been suggested to be of an acute megakaryoblastic type and may yield a history of early transitory leukemia [2]. Transient congenital leukemia has been reported only rarely in infants with a normal karyotype. Van Eys and Flexner [4] reported a patient who had a spontaneous remission of 8 months, and one of the five patients with congenital leukemia reported by Chu et al. [5] remained in remission. More recently, Lampkin et al. [6] reported spontaneous remission in an infant with acute nonlymphoblastic leukemia (ANLL). Most phenotypically normal children with a transient leukemia have been reported to have trisomy 21 mosaicism; three such children evidenced mosaicism in both blood cells and skin fibroblasts [7-9], but in others the trisomic cells were observed only in the marrow or the peripheral blood and only normal cells were present in fibroblast cultures [9-13]. We report sequential studies on another example of apparent transient congenital leukemia in a phenotypically normal infant first investigated because of possible pneumonia. In this case nearly all the spontaneously dividing cells in the peripheral blood during the neonatal period showed trisomy 21, but only normal cells were found in skin fibroblast cultures. These rare observations may be instructive regarding the etiology of leukemia.
From the Human Genetics Laboratory, Departments of Pathology, Haematology, and Child Health, University of Dundee, Scotland.
Address r e p r i n t requests to: M. Faed, Ph.D., Human Genetics Laboratory, Department of Pathology, Ninewells Hospital and Medical School, Dundee DD1 9SY, Scotland. Received February 2, 1990; accepted March 12, 1990.
259 © 1990 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
Cancer Genet Cytogenet 48:259-264 (1990) 0165-4608/90/$3.50
260
M . J . w . Faed et al.
CASE REPORT A n infant was born at 39 weeks of gestation by spontaneous vertex delivery to Scottish parents. Her mother, aged 24 years and a primigravida, had an uneventful pregnancy. Her father was aged 26 years. Her birth weight was 4.36 kg. At age 2 days she was a d m i t t e d to the special care nursery because of fever and tachypnea. Her spleen and liver were both p a l p a b l e at 3 cm below the costal margin but there was no bruising or bleeding. E x a m i n a t i o n was otherwise normal. She received penicillin and gentamicin for 48 hours until blood and surface cultures proved sterile, w h e n antibiotics were stopped. Her h e m o g l o b i n was 17.0 g/dl, her platelet count was 107 x 109/L, and her white blood cell (WBC) count was 17 x 109/L with 24% blast cells. There was no evidence of congenital infection with rubella, toxoplasmosis, syphilis, or Herpes, but her urine grew cytomegalovirus. A bone marrow examination on the fifth day of age confirmed an excess of similar blast cells. Because of the rapid decrease in blast cell numbers, she was discharged from the nursery at age 13 days and made good progress until age 6 weeks w h e n she was admitted to hospital because of an upper respiratory infection and cough treated with erythromycin. Her liver and spleen were then palpable at 2 cm below the costal margin and a chest roentgenogram showed opacities at the left base consistent with pneumonia. Her hemoglobin was 10.9 g/dl, her WBC count was 6.5 x 109/L with no blast cells, and her platelet count was 61 × 109/L. She recovered r a p i d l y and was discharged home after 2 days. On subsequent clinic visits she r e m a i n e d well and her blood and platelet counts were normal. At age 3 months her spleen was no longer palpable and her liver was only 1 cm below the costal margin. At age 18 months she remained healthy with normal d e v e l o p m e n t and no abnormal physical findings. Examination of both parents showed no caf~ au lait spots or other evidence of von Recklinghausen's disease.
Hematological Investigation The blood counts are shown in Table 1. In Romanowsky-stained preparations, the blasts varied in size from 13 to 18/~m in diameter and had the general appearance of myeloblasts with m u l t i p l e nucleoli (up to 6%). The staining reactions were S u d a n black negative, chloracetate esterase negative; nonspecific esterase positive, and periodic acid-Schiff negative. Platelets showed anisocytosis with megathrombocytes up to 13~m in diameter in the blood films.
Cytogenetic Studies Blood cultures were established for chromosome preparations in m e d i u m 199 with 5% n e w b o r n calf serum both with and without p h y t o h e m a g g l u t i n i n (PHA). They were harvested at 48 and 72 hours, with colchicine present for the final 2 hours. Marrow cultures were harvested after 1, 18, and 48 hours without mitogens and after 48 hours with PHA. Fibro[llast cultures were established from a small skin b i o p s y and grown in H a m ' s FIO m e d i u m with 15% fetal calf serum for between 22 and 36 days before harvest on coverslips. The results are s u m m a r i z e d in Table 2. The initial observation, made on chromosome preparations of cells derived from the buffy coat and cultured without mitogens for 24 hours, s h o w e d 62 of 64 cells to have trisomy 21. The marrow at 5 days showed about 80% of cells to be trisomic, and a blood sample taken at 6 days s h o w e d nearly all the cells cultured without PHA to have an extra chromosome 21 whereas all PHAstimulated cells had a normal karyotype. Fibroblast cultures from a skin b i o p s y taken at 17 days s h o w e d only normal female cells. A blood sample taken at this time still s h o w e d reasonable numbers of spontaneously dividing cells, all of w h i c h were
Trisomy
261
21 i n T M D
Table 1
Age Days 3 4 5 6 8 9 11 14 17 18 24 Weeks 5 6 7 11 Months 21
B l o o d c o u n t s i n 21 m o n t h s Hb (g/dl)
WBC ( × 109/L}
Blasts ( × 109/L)
Platelets ( × 109/L}
17.7 18.5 18.1 17.5 17.5 16.9 16.0 16.1 16.1 14.5 13.0
17 23.2 20.1 25.8 20.1 25.1 17.1 18.9 15.98 12.4 9.8
4.08 6.03 3.82 3.35 3.02 3.26 2.57 1.89 0.95 0.62 0.30
107 97 80 103 96 98 75 130 165 120 103
10.6 10.9 10.9 11.8
7.1 6.5 8.1 8.5
0 0 0 0
61 61 154 254
11.8
8.7
0
210
Abbreviations: Hb, hemoglobin; WBC, white blood cell.
trisomic; some cells among the PHA-stimulated cultures were also trisomic. Subseq u e n t b l o o d c u l t u r e s at 1 m o n t h a n d 6 m o n t h s s h o w e d a q u i t e d i f f e r e n t p a t t e r n . A t 1 month there were still significant numbers of spontaneously dividing cells, but n e a r l y a l l w e r e c h r o m o s o m a l l y n o r m a l . T h r e e of f o u r c e l l s f o u n d to h a v e a d d i t i o n a l c h r o m o s o m e s 21 w e r e p o l y p l o i d . A t a g e 6 m o n t h s , a f e w m i t o s e s w e r e p r e s e n t i n u n s t i m u l a t e d c u l t u r e s ; t h e s e a l l h a d a n o r m a l k a r y o t y p e , a s d i d all c e l l s i n t h e P H A stimulated cultures.
Table 2 Age (days)
Proportion age shown
o f t r i s o m i c c e l l s p r e s e n t i n c u l t u r e s f r o m s a m p l e s t a k e n at
Tissue
Mitogen
Mitotic index
--
--
64
96.9
5
Buffy coat Marrow
--
6
Blood
17 18
Skin Blood
38
Blood
174
Blood
-PHA -PHA --PHA -PHA -PHA
74 39 91 30 99 25 50 44 101 26 45
79.7 71.8 96.7 0 O 100 8.0 6.8 a 1.0 a 0 0
4
Abbreviation: PHA, phytohemagglutinin.
aThree of 4 cells polyploid.
0.7 1.9 -0.2 2.75 0.5 4.6 0.05 4.15
Mitoses scored
T r i s o m y 21 (%)
262
M . J . W . Faed et al.
DISCUSSION
Transient neonatal leukemia is believed to be rare in p h e n o t y p i c a l l y normal infants. This child was investigated because of possible p n e u m o n i a in the neonatal period, and cytogenetic studies were undertaken as a consequence of finding blast cells in the blood smears. Ascertainment of cases reported by other investigators has varied from routine examination of cord blood [8] to overt pallor [13]. The hematological evidence of leukemia had disappeared within i month. Cytogenetic observations during the leukemic phase showed nearly all the s p o n t a n e o u s l y d i v i d i n g cells from the blood and a high proportion of the cells of the marrow to be trisomic. As the leukemic phase passed, the trisomic cells disappeared; on the last occasion on w h i c h trisomic cells were recognized, three of the four were polyploid. The possibility that these may represent maturing megakaryoblasts is intriguing. It is unfortunate that this possibility could not be explored retrospectively on the material remaining. The relationship of trisomy 21 to leukemia is interesting. Trisomy 21 is the most c o m m o n a d d i t i o n a l finding in leukemic cells in patients with acute lymphoblastic leukemia (ALL) and is also c o m m o n l y found in ANLL. There is an increased risk of l e u k e m i a in Down s y n d r o m e [14, 15]. In particular, there is a proportional increase in megakaryoblastic l e u k e m i a in persons with Down syndrome as c o m p a r e d with the normal population, and the bulk of such cases presenting as transient neonatal l e u k e m i a may be of this type [2]. Of the few cases of transient neonatal leukemia reported in p h e n o t y p i c a l l y normal infants (Table 3), the reactive, p r e s u m a b l y leukemic cells have been shown to be trisomic, although a substantial proportion of these cells in the case reported by Weinberg et al. [10] had a normal karyotype. In a d d i t i o n to those with trisomy 21 shown in Table 3, a case was reported by Van den Berghe et al. [16] in w h i c h the s p o n t a n e o u s l y dividing cells showed pentasomy for chromosome 21. These cells were also present in the marrow, but no abnormality was detected in fibroblasts or in blood or marrow when remission occurred. In our patient, normal cells were found among the s p o n t a n e o u s l y dividing cells, but they constituted a very small p r o p o r t i o n during the leukemic phase. Some spontaneous mitoses are not u n u s u a l in neonates. In this child, small numbers of mitoses persisted even to age 6 months; the child remains well without any clinical or hematological evidence of leukemia after 21 months. Fibroblast cultures have not been examined in all reported cases of p h e n o t y p i c a l l y normal infants. Of the 10 previous cases (Table 3) in which fibroblast cultures were studied, only three have shown trisomic cells and then only in low number. Blast cells from the blood of these infants may proliferate in fibroblast cultures, however, thus giving a false i m p r e s s i o n of constitutional mosaicism. The question of whether the m o s a i c i s m is constitutional or limited to one tissue is not merely academic; if m o s a i c i s m is constitutional, a continuing s u p p l y of trisomic cells in w h i c h malignant change can occur can be presumed. If the trisomy is limited to one t i s s u e - - t h e m a r r o w - - t h e abnormal cell line may be from the committed cell compartment and thus be a t e m p o r a r y population. Indeed, a feature of the p h e n o t y p i c a l l y normal infants is the d i s a p p e a r a n c e or massive reduction of the trisomic cell line. This w o u l d argue against constitutional mosaicism and suggest that in these cases nondisjunction has occurred in a c o m m i t t e d colony-forming cell that ultimately disappears as its proliferative potential is exhausted. The observation of ~n increased frequency of leukemia and in particular of megakaryoblastic l e u k e m i a in Down syndrome, and the suggestion that neonatal transient leukemia, w h i c h m a y also be megakaryoblastic, is invariably a c c o m p a n i e d by cells with trisomy 21, calls for explanation. Neonatal transient leukemia has been described as "ineffective regulation of granulopoiesis w h i c h masquerades as congenital leuke-
263
Trisomy 21 in TMD Table 3
Chromosome findings in cultures on earliest analysis of reported cases of phenotypically normal infants with TLR No. of cells
Reference [71
Age 3 days 3 days 8 days
[81
[10]
11 days 12 days 12mo
[11] Case 1 Case 2
2 days 7 days 3 mo
[12]
Tissue Blood ( P H A + , - ) Bone marrow Skin Blood (unstim) Blood (PHA) Bone marrow Skin Blood (unstim) Blood (PHA) Skin Blood (PHA) Blood (unstim) Blood (PHA) Bone marrow Skin Blood (PHA) Bone marrow Skin
Normal
Trisomic
0 0 96 0" 14 5 38 19 45 85 16 0 65 21 32 14 31 100
50 22 4 36 6 2 3 3 5 0 16 32 0 0 0 3 4 0
[19] Case 1 Case 2 [13l Case 1
Case 2
3 days 3 days
Blood (PHA) Blood (PHA)
6 5 2 5 2
Blood (unstim) Blood (PHA) Bone marrow Skin Blood (unstim) Blood (PHA) Bone marrow Skin
0 38 0 210 0 24 0 60
24 7 55 0 25 1 25 0
Bone marrow Blood (PHA) Skin Blood (unstim) Blood (PHA) Marrow Skin Marrow Blood (unstim) Skin Blood (unstim) Blood (PHA) Bone marrow
0 93 250 3 247 244 200 1 5 99 2 30 15
20 7 0 9 3 6 0 53 20 5 62 0 59
wk days days days wk
7 days 2.5 yr
79% 46%
21% 54%
[9] Case 7
During TLR After TLR
Case 8
During TLR After TLR After TLR Not given During TLR During TLR Not given 4 days 6 days 5 days
Case 9
Present study
Abbreviations: TLR, transient leukemic reaction; PHA, phytohemagglutinin; unstim, unstimulated.
264
M.J.w.
Faed et al.
m i a " [17]. D e s p i t e the w i d e l y r e c o g n i z e d difficulty of d i s t i n g u i s h i n g b e t w e e n a transient m y e l o p r o l i f e r a t i v e d i s o r d e r and c o n g e n i t a l l e u k e m i a , the p r e s e n c e of cells w i t h t r i s o m y 21 w i t h o u t a d d i t i o n a l clonal a b n o r m a l i t y is itself probably b e n i g n [3]. T h e t r a n s i e n t n a t u r e of the p h e n o m e n o n suggests, rather, that there is a greater r e l a t i v e e x p a n s i o n of t r i s o m i c cells in the p r o l i f e r a t i v e phase in the p r o g r e s s i o n f r o m c o l o n y f o r m i n g cell to m a t u r e m e g a k a r y o c y t e . In vitro studies on c o l o n y g r o w t h m a y h e l p to r e s o l v e this q u e s t i o n . A n e x p e c t e d effect of a d d i t i o n a l p r o l i f e r a t i o n w o u l d be to i n c r e a s e the risk of a s e c o n d m u t a t i o n a l e v e n t w h i c h m i g h t result in l e u k e m i a . This agrees w i t h the K n u d s o n " t w o - h i t " h y p o t h e s i s of c a n c e r initiation [18] and offers an e x p l a n a t i o n for the i n c r e a s e d risk of l e u k e m i a in D o w n s y n d r o m e .
REFERENCES 1. Rosner E, Lee SL (1972): Down's syndrome and acute leukemia'. Myelogenous or lymphoblastic? Reports of forty-three cases and a review of the literature. Am J Med 53:203-218. 2. Zipursky A, Peeters M, Poon A (1987): Megakaryoblastic leukemia and Down's syndrome--a review. In: Oncology and Immunology of Down Syndrome. Alan R. Liss, New York, pp. 33-56. 3. Hayashi Y, Eguchi M, Sugita K, Nakazawa S, Sato T, Kojima S, Bessho F, Knoishi 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 72:15-23. 4. Van Eys J, Flexner JM (1969): Transient spontaneous remission in a case of untreated congenital leukemia. Am J Dis Child 118:507. 5. Chu J-Y, O'Connor DM, Gale GB (1982): Five cases of congenital leukemia. Pediatr Res 16:201A. 6. Lampkin BC, Peipon JJ, Price JK, Bove KE, Srivastava AK, Jones MM (1985): Spontaneous remission of presumed conenital acute non lymphoblastic leukemia (ANLL) in a karyotypically normal neonate. Am J Pediatr Hematol Oncol 7:346-351. 7. Brodeur GM, Dahl GV, William DL, Tipton RE, Kalwinsky DK (1980): Transient leukemoid reaction and trisomy 21 mosaicism in a phenotypically normal newborn. Blood 55:691-693. 8. Heaton DC, Fitzgerald PH, Fraser GJ, Abbott GD (1981): Transient leukemoid proliferation of the cytogenetically unbalanced + 21 cell line of a constitutional mosaic boy. Blood 57:883-887. 9. Abe K, Kajii T, Niikawa N (1989): Disomic homozygosity in 21-trisomic cells: A mechanism responsible for transient myeloproliferative syndrome. Human Genet 82:313-316. 10. Weinberg AG, Schiller G, Windmiller J (1982): Neonatal leukemoid reaction. An isolated manifestation of mosaic trisomy 21. Am J Dis Child 136:310-311. 11. Seibel NL, Sommer A, Miser J (1984): Transient neonatal leukemoid reactions in mosaic trisomy 21. J Pediatr 104:251-254. 12. Hanna MD, Melvin SL, Dow LW (1985): Transient myeloproliferative syndrome in a phenotypically normal infant. Am J Pediatr Hematol Oncol 7:79-81. 13. Jones GR, Weaver M, Laug WE (1987): Transient blastemia in phenotypically normal newborns. Am J Pediatr Hematol Oncol 9:153-157. 14. Wald N, Borges WL, Li CC, Turner JH, Harnois MC (1961): Leukemia associated with mongolism. Lancet 1:228. 15. Stewart A, Webb l, Hewitt D (1958): A survey of childhood malignancies. Br Med J 1:1495-1508. 16. Van den Berghe H, Vermaelen K, Broeckhaer-Van Orshoven A, Delbeke M-J, Benoit Y, Orye E, Van Eygen M, Logghe N (1983): Pentasomy 21 characterizing spontaneously regressing congenital acute leukemia. Cancer Genet Cytogenet 9:19-24. 17. Ross JD, Moloney WC, Desforges JF (1963). Ineffective regulation of granulopoiesis masquerading as congenital leukemia in a monogoloid child. ] Pediatr 63:1-10. 18. Knudson AG (1971): Mutation and cancer: Statistical study of retinoblastoma. Proc Natl Acad Sci USA 68:820-823. 19. Laing D-C, Shen E-Y, Chyou S-C (1986): To early distinguish neonatal transient leukemoid proliferation from congenital leukemia by in vitro cell growth. Blut 53:101-106.
