* This work was supported by a Grant-in-Aid from the Ministry of
Health and Welfare as part of a comprehensive 10-year Strategy for
Cancer Control; by a Grant for Pediatric Research 63-06 from the
Ministry of Health and Welfare; by a Grant-in-Aid for Cancer Research;
and by the Japanese Foundation for Multidiciplinary Treatment of
1 Department of Virology and
2 Department of Pathology, the National Children's Medical Research
Center, Tokyo, Japan.
3 Department of Hematology /Oncology, Saitama Children's Hospital,
4 Department of Pediatrics, Dokkyo University Medical School, Tochigi,
Transient myeloproliferative disorder is a hematological condition
observed during the neonatal period in patients with Down syndrome
[ 1] .This disorder mimics congenital leukemia and the blast cells
were often reported to show characteristics compatible with those
of megakaryoblasts  or pluripotent stem cells . Although this
disorder usually resolves gradually without antileukemic treatment,
in some patients, leukemia develops after periods of spontaneous
remission , raising questions about the benign origin of this
disorder . It is therefore unclear if transient myeloproliferative
disorder is aleukemia, preleukemia, or transient dysplasia of myelopoiesis.
This important issue might be resolved by analysis of the clonality
of the blast cells of this disorder. We therefore studied the clonality
of blast cells in transient myeloproliferative disorder using antigen
receptor genes as well as X chromosome inactivation patterns.
Materials and Methods
Patient Samples. The patients studied included three newborn infants,
all of whom had Down syndrome with standard trisomy 21 (Table 1
). They all developed transient myeloproliferative disorder during
the neonatal period with more blast cells in the peripheral blood
than in the bone marrow, and were followed by spontaneous resolution
without antileukemic treatment. The follow-up period of these patients
was from 72 days to 5 months. One out of three patients was alive
at the time of this study (patient 3). The remaining two patients
died while there was no evidence of leukemia. The clinical course
of patient 2 was reported previously .
Cell Separation and Immunological Analysis.
Heparinized peripheral blood was separated on a density gradient
of FicollMetrizoate (Lymphoprep, Nyegaard, Oslo, Norway). The interface
of mononuclear cells was subjected to immunological and DNA analysis.
Reactivity of the blast cells with monoclonal antibodies against
lineage-specific antibodies (Table 2) was assayed by an indirect
immunofluorescence technique using a fluorescence-activated cell
shorter as described before .
Southern Blot Analysis.
High-molecular weight DNA was extracted from the mononuclear cells
according to the method described previously . For the antigen
receptor gene analysis, Ig1H, Cß, 1y, Cdelta, and 1delta2 probes
were employed, with 6 µg of BamHI-digested DNA samples. Clonal analysis
with the X-linked phosphoglycerate kinase (PGK) probe  was performed
as described by Vogelstein et al. .
Table1. Clinical data of three patiens
with transient abnormal myelopoiesis
Immunological Analysis of Blast Cells.
All of the samples of blast cells that we studied were positive
for platelet associated antigens (CD41, CD42b, or KOR-P 77 ).
Two out of three samples also showed positivity for CD 7 antigen.
N one of three samples expressed lymphoid lineage-associated antigens
such as CD 3, CD 10 or CD 19 (Table 2).
Antigen Receptor Gene Configuration of Blast Cells.
N one of the three samples of blast cells showed any rearrangements
of the IgH or TCR loci using 1H, TCRß, y, and delta probes (data
X Chromosome Inactivation Analysis of Blast Cells.
94% , 99% , and 82% of the peripheral mononuclear cells were blast
cells after the density gradient cell separation in patients 1,
2, and 3 respectively. After an additional digestion by restriction
enzyme Hpall, DNA of the blast cells of the three patients showed
complete loss of either the 1.05 kb or the 0.9 kb band, suggesting
monoclonal expansion of the blast cells (Fig. 1, lanes 2, 6, and
8). In contrast, the mononuclear cells of patient 1 after spontaneous
remission showed the retention of both of the alleles (Fig. 1, lane
Our initial study into the lineage and clonality of blast cells
using the antigen receptor genes Ig1H and TCRß, y, and delta showed
no rearrangements of these loci in the blasts of any of the three
Table 2. Peripheral blood immunophenotyping
As all the patients demonstrate more than 82% of blast cells in the
mononuclear cell fraction, our negative findings for clonal rearrangement
cannot be attributed to the low sensitivity of this assay. Furthermore,
as almost all of the leukemias with lymphoid characteristics show
antigen receptor gene rearrangement , our results suggest that
the proliferating blast cells are of nonlymphoid origin. In order
to study the clonality of blast cells, we employed the X chromosome
linked polymorphic gene, PGK. In patient 1, mononuclear cells were
compared during blastic phase and remission phase, which was achieved
without therapy, for sensitivity to HpalI restriction enzyme digestion.
As the results show, the absence at blastic phase, but the presence
at remission phase of the allele identified at 1.05 kb indicates that
mononuclear cells at blastic phase are of single clonal origin.
Fig.1. PGK analysis of X chromosome inactivation patterns
in three patients. An auto radiograph of a Southern hybridization
experiment is shown, wherein a PGK gene probe was hybridized to
DNA from mononuclear cells from patient 1 (initial presentation
and after remission, lanes 1 and 2 and lanes 3 and 4, respectively)
and mononuclear cells from patients 2 and 3 (initial presentation,
lanes 5 and 6 and lanes 7 and 8, respectively). In lanes 1,3,5,
and 7 the DNA has been digested with restriction enzymes (BstXI,
PstI) that reveal two polymorphic PGK alleles (1.05-kb and 0.9-kb
bands). In lanes 2,4,6, and 8 DNA has been digested with these enzymes
and also with HpaII, an enzyme that distinguishes active from inactive
alleles through methylation differences.
Although we could not examine the samples at remission phase in
patients 2 and 3, the complete absence of one of two alleles indicates
a monoclonal proliferation of blast cells. Altogether our results
strongly suggest that monoclonal expansion of a progenitor cell
with nonlymphoid characteristics occurs in this disorder with Down
syndrome. Megakaryoblastic features characterized by expression
of CD41 (patients 1 and 3), CD42b (patient 1), or KOR-P 77 (patients
1 and 2) are of interest in the light of the recent recognition
that acute leukemia in Down syndrome is much more frequently of
a megakaryoblastic nature than previously reco gnized [ 11] . There
are several possible explanations for blast cell proliferation in
this disorder . Firstly, blast cells might proliferate in response
to an increase in production of growth factors such as IL-3 and/or
IL-6 . Secondly, hemopoietic dysregulation, including that caused
by defective immunological surveillance during the neonatal period,
might be responsible for this disorder. Thirdly, transient myeloproliferative
disorder could be a preleukemic state. Since in the former two interpretations
polyclonal proliferation of blast cells is expected, our findings
strongly support the third possibility. This idea is also supported
by other reports that this is due to the result of a spontaneous
resolution of a malignant clone [4, 13]. Alternatively this condition
might be heterogeneous, some cases being benign reactive conditions
with polyclonal myeloproliferation, while others are actually preleukemic
conditions. Studies on more patients with a longer survival period
will clarify whether there is any heterogeneity in clonality among
patients with this disorder.
We are indebted to Drs. J. Singer-Sam, P. Leder, T. Rabbits, and
T. W. Mak for providing the DNA probes, PGK, IgJH (lamda CH 28-6),
TCR y, and TCR ß and delta respectively. We also thank Dr. S. Nakazawa
for immunopheno typing data from patient 1.
References 1. Nagao T, Lampkin BC, Hug G (1970) A neonate
with Down's syndrome and transient abnormal myelopoiesis: serial
blood and bone marrow studies. Blood 36:443447
2. Koike T, Aoki S, Maruyama S, Narita M, Ishizuka T, Imanaka H,
Adachi T, Maeda H, Shibata A (1987) Cell surface phenotyping of
megakaryoblasts. Blood 69 : 957-962
3. Suda J, Eguchi M, Akiyama Y, Iwama Y, Furukawa T, Sato Y, Miura
Y, Suda T, Saito M (1987) Differentiation of blast cells from a
Down's syndrome patient with transient myeloproliferative disorder
. Blood 69: 508- 513
4. Morgan R, Hecht F, Cleary ML, Sklar J, Link MP (1985) Leukemia
with Down's syndrome: translocation between chromosomes 1 and 19
in acute myelomonocytic leukemia following transient congenital
myeloproliferative syndrome. Blood 66:1466-1471
5. Nakagawa T, Nishida H, Arai T, Yamada T, Fukuda M, Sukamoto S
(1988) Hyperviscosity syndrome with transient abnormal myelopoiesis
in Down syndrome. J Pediatr 112:58-61
6. Nakamura K, Sasaki M, Fujimoto J, Enomoto Y, Kaneko Y, Ozaki
M, Miyashita T, Tsunematsu Y, Hata J, Kobayashi N, Mizutani S (1990)
Molecular diversity of precursor B acute lymphoblastic leukemias
identified by the immunoglobulin heavy chain gene organization.
7. Keith DH, Singer-Sam J, Riggs AD (1986) Active X chromosome DNA
is unmethylated at eight CCGG sites clustered in a guanine-plus-cytosine-rich
island at the 5' end of the gene for phosphoglycerate kinase. Mol
Cell BioI 6:4122-4128
8. Vogelstein B, Fearon ER, Hamilton SR, Feinberg AP (1985) Use
of restriction fragment length polymorphisms to determine the clonal
origin of human tumors. Science 227:642-646
9. Nakazawa S, Sugita K (1987) Establishment and characterization
of a murine anti-platelet monoclonal antibody and its usefulness
for the identification of megakaryocyte-lineage cells. Keio J Med
10. Asou N, Hat tori T, Matsuoka M, Kawano F, Takatsuki K (1989)
Rearrangements of T -cell antigen receptor delta chain gene in hematologic
neoplasms. Blood 74:2707-2712
11. Zipursky A, Peeters M, Poon A (1986) Megakaryoblastic leukemia
and Down's syndrome -a review. In: McCoy EE, Epstein CJ (eds) Oncology
and immunology of Down syndrome. Liss, New York, pp 33-56
12. Ikebuchi K, Wong 00, Clark SC, I hIe JN, Hirai Y, Ogawa M (1987)
Interleukin 6 enhancement of interleukin 3-dependent proliferation
of multipotential hematopoietic progenitors. Proc Natl Acad Sci
13. Lazarus KH, Heerema NA, Palmer CO, Baehner RL (1981) The myeloproliferative
reaction in a child with Down syndrome: cytological and chromosomal
evidence for a transient leukemia. Am J HematoI11:417-422