Transient myeloproliferative disorder and acute nonlymphoblastic leukemia in Down syndrome

Transient myeloproliferative disorder and acute nonlymphoblastic leukemia in Down syndrome

Transient myeloproliferative disorder and acute nonlymphoblastic leukemia in Down syndrome K. Y. W o n g , MD, Marilyn M. Jones, Ashok K. Srivastava, ...

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Transient myeloproliferative disorder and acute nonlymphoblastic leukemia in Down syndrome K. Y. W o n g , MD, Marilyn M. Jones, Ashok K. Srivastava, MD, a n d Ralph A. G r u p p o , MD From the Children's Hospita! Medical Center and Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center

TWO infants with Down syndrome had transient myeloproliferative disorder (TMD) during the neonatal period and subsequently acute nonlymphoblastic leukemia (ANLL). Histochemically, the blast cells in TMD were indistinguishable from those in ANLL Only the constitutional chromosome (trisomy 21) was found in TMD, whereas new cytogenetic abnormalities emerged in ANLL. A mixed growth pattern in stem cell cultures during TMD suggested the existence of an abnormal clone that migh! be responsible for the evolution into ANLL at a later date. Serial cytogenetic studies and culture studies of peripheral blood cells may help to understand the pathophysiology and risk of ANLL in patients with TMD. (J PEDIATR1988;112:18-22)

Transient myeloproliferative disorder or leukemoid reaction has been described in infants with Down syndrome during the neonatal period. ~'2 This usually resolves gradually over several months without antileukemic treatment. Although there is an increased incidence of leukemia among patients with Down syndrome, 3 the association of TMD and the subsequent development of acute leukemia has been infrequently described.4s We studied two pat!ents with Down syndrome and compared the clinical and laboratory features during the phases of TMD and acute nonlymphoblastic leukemia. An abnormal stem cell growth pattern in TMD suggested the existence of a clone of cells that subsequently evolved into ANLL. This was confirmed with new eytogenetic abnormalities when A N L L was clinically obvious.

Presented in part at the Twenty-First Congress of the International Society of Haematology, Sydney, Australia. SupPorted bY Clinical Cancer Education Grant CA25557-06 from the National Cancer Institute. Submitted for publication July 1, 1987; accepted Aug. 20, 1987. Reprint requests: K. Y. Wong, MD, Division of Hematology: Oncology, Children's Hospital Medical Center, Cincinnati, OH 75229.



Bone marrow specimens were processed with Wright stain for routine morphologic studies. Cytochemical stains to differentiate myeloid from lymphoid cell lines included periodic acid-Schiff, peroxidase, Sudan black, c~-naphthyl esterase, and chloroacetate esterase. Cytogenetic studies were done by direct processing and after 24-hour unstimulated cultures of bone marrow cells.9 TMD ANLL CFU-GM PHA

Transient myeloproliferative disorder Acute nonlymphoblastic leukemia Colony-forming unit--granuloyte/monocyte Phytohemagglutinin

Constitutional karyotypes of the patients were determined from phytohemagglutinin-stimulated blood lymphocyte culture. At least 25 Giemsa banded metaphases were critically analyzed with each study, and four karyotypes were made. Bone marrow cells were cultured in vitro using two methods. Normal C F U - G M cultures were prepared with a modification of the method of Pike and Robinson. 1~ In brief, bone marrow mononuclear cells were separated on Ficoll-Hypaque and resuspended in McCoy 5A enriched

Myeloproliferative disorder and A N L L in Down syndrome

Volume 112 Number I


Table I. Hematologic and m a r r o w findings in t r a n s i e n t myeloproliferative disorder a n d acute nonlymphoblastic leukemia Patient 2

Patient I TMD Hemogram Hemoglobin (g/dL) WBC (x 103//~L) Blast cells (%) Platelet (x103/#L) Bone marrow Cellularity Blast cells (%) Periodic acid-Schiff Peroxidase Sudan black ~-Naphthyl Chloroacetate (est) Karyotype


23.3 25.5 30 Decreased

3.4 8.8 31 7

1+ 13.8 Weak +


+ Negative + Negative 47,XY,+21


59.4 Weak + Negative ND + Negative 48,XY,+7,- 17,+21,+21


12.1 53.5 25 220

8.0 3.6 5 28

8.6 Weak +

3+ 39.5 Weak +

+ + + + 47,XY,+21

Negative Negative + Negative 47,XY,+21/47,XY,+21,t(7;14)(pl 3;ql 2)

ND, not done; est, esterase medium containing 15% fetal bovine s e r u m a n d 0.25% agarose (Sea Plaque, F M C C o r p . / B i o p r o d u c t s Division, Rockland, Maine). These cells were seeded onto previously prepared feeder plates at a final concentration of 2 • 105/ mL. Triplicate 35 m m plates were i n c u b a t e d for 14 days at 37 ~ C in 7.5% CO2, and colonies of more than 50 cells a n d clusters containing fewer t h a n 50 cells were e n u m e r a t e d . A b n o r m a l m y e l o i d / m o n o c y t i c or very early m a r r o w progenitor cells were cultured using a m e t h o d described by Dicke et al. 11 In this assay, 2.5 • 106/mL bone m a r r o w mononuclear cells were suspended in 5 m L Dulbecco modified Eagle m e d i u m containing 10% pooled h u m a n serum and 10% fetal bovine serum. Cells were i n c u b a t e d overnight at 37 ~ C in 7.5% COs with 5% P H A (HA-15, Wellcome Diagnostics, Research Triangle Park, N.C.). Cells were then washed to remove the P H A , resuspended at 2 • 105/mL in Dulbecco complete m e d i u m including 0.25% agarose, and seeded onto feeder plates containing only medium, serum, a n d 0.4% agarose. These plates were incubated for 7 days at 37 ~ C in 7.5% COs, and colonies were scored. CASE R E P O R T S Patient 1. This white newborn infant was referred to Children's Hospital Medical Center because of abdominal distention and failure to pass meconium at 36 hours of age. He had stigmata of Down syndrome, moderate hepatosplenomegaly, and no evidence of congenital heart disease. Hemoglobin was 23.3 g/dL, white blood cell count 25,500/uL with 30% blast cells, and platelets were decreased. A partial exchange transfusion was performed, and suction rectal biopsy tissue examination confirmed the diagnosis of Hirschsprung disease. A bone marrow examination showed normal maturation of the myeloid series but with 13.8%

myeloblasts (Table I). Surgical correction of Hirschsprung disease was carried out with no complication. Follow-up examination at 289 months of age showed resolution of hepatosplenomegaly. Hemoglobin was 12.7 g/dL, WBC 11,200/#L with no blast cells, and platelets 200,000/~zL. He remained in good health until 2~2 years of age, when he was found to have severe anemia during an upper respiratory tract infection. Liver and spleen were not enlarged. Hemoglobin was 3.4 g/dL, WBC 8800/~L with 31% blast cells, and platelets 7000//~L. The bone marrow was hypercellular with 60% blast cells (Table I). Cytosine arabinoside infusion, daunorubicin, VP 16-213, thioguanine, and dexamethasone therapy was administered. Complete remission was achieved in 4 weeks. Bone marrow transplantation from his HLA-matched sister was performed 189 months into remission, with no complication. He has been in continuous complete remission for more than 3 years. Patient ~. This white infant with Down syndrome was delivered by cesarean section because of breech presentation and meconiurn-stained amniotic fluid. He was dusky and mildly tachypneic, with Apgar scores of 5 and 6 at i and 5 minutes, respectively. The liver was 3 cm below the costal margin, and the spleen tip was palpable. Hemoglobin was 12.1 g/dL, WBC 53,500//~L with 25% blast cells, and platelet count 220,000/~L. Eosinophilia and basophilia were present. There was oozing from needle puncture sites, probably secondary to nonfunctioning, large, vacuolated platelets. Thrombocytopenia, hypofibrinogenemia, and microangiopathic blood findings ensued. Bone marrow examination showed 6.8% blast cells, with increase in eosinophilic and basophilic precursor cells. Despite transfusion therapy, massive gastrointestinal bleeding developed on the third day of life. Radionuclide examination did not show the presence of a Meckel diverticulum. Exchange transfusion and vigorous replacement with blood products led to resolution of the bleeding by I week of age. The blood findings normalized at 289 months of age, although both the liver ans spleen were still enlarged to 2 cm below the costal margin.


g Z o n get al.

The Journal of Pediatrics January 1988

Table II. In vitro growth pattern of marrow cells in transient myeloproliferative disorder and acute nonlymphoblastic leukemia Patient I

CFU-GM culture Colonies Clusters PHA-conditioned culture Colonies Clusters

Patient 2







7 34

24* 36*

42 12

14 52

220 178

171 90

51 85

30* 57*


6 2

0 9

41 109

*Culture of peripheral blood mononuclearcells.

Bone marrow was normal at 4Vz months and demonstrated a normal in vitro growth pattern (Table II). At 2 years of age, he was found to be pale with hepatosplenomegaly. Hemoglobin was 4.8 g/dL, WBC 26,000/~L with no blast cells, and platelet count 29,000/~zL. Bone marrow showed 2.4% blast cells, which we were unable to classify. A marrow biopsy specimen revealed hypercellularity with myelofibrosis and increase in number of megakaryocytes. The in vitro marrow culture results are shown in Table II. He had various degrees of pancytopenia for 9 months; bone marrow study at that time revealed an increase in the number of blast cells, and a translocation between chromosomes 7 and 14 was found (Table I). He was then given intensive chemotherapy for 9 months and has been in remission for over 30 months with no maintenance chemotheraPy.

below the normal range in our laboratory. On the other hand, colonies of immature blast cells were formed in the PHA-conditioned culture. Similar results were observed using peripheral blood mononuclear cells. Studies were repeated when clinical and hematologic findings were normal. Patient 1 had 24 colonies with the C F U - G M culture and 30 with the PHA-conditioned culture from blood mononuclear cells. Patient 2 had normal bone marrow growth pattern, with 220 colonies in the C F U - G M culture and none in the PHA-conditioned culture. The in vitro growth pattern was normal in patient 1 at the time of diagnosis of ANLL. Patient 2 had increased colony counts during the cytopenic phase before diagnosis of ANLL: 171 colonies with C F U - G M cultures and 41 colonies with PHA-conditioned culture.

RESULTS The hematologic and bone marrow findings are summarized in Table I. Both patients had high blast cell counts in the blood (average 104/#L) during T M D when the bone marrow contained only 7% to 14% blast cells. In contrast, pancytopenia was the common feature at diagnosis of A N L L in these children; the bone marrow, however, had 40% to 60% blast cells, which were morphologically myelomonoblastic. Increase in reticulin was demonstrated in the biopsy specimens. Both children had trisomy 21 as the only cytogenetic abnormality in the neonatal period, determined from PHA-stimulated blood lymphocytes as well as from bone marrow cells. New cytogenetic abnormalities were found in the marrow cells of both patients at the time of diagnosis of A N L L. Patient 1 had the karyotype 48,XY,+7,-17, +21,+21 in all marrow cells. Patient 2 had the karyotype 47,XY,+21 in all marrow cells in direct preparation; in the 24-hour unstimulated culture, two of nine cells showed 47,XYIt(7;14)(p13;ql 2),+21. The in vitro growth patterns of marrow cells are summarized in Table 2. At the time of TMD, both patients had few colonies in the C F U - G M marrow culture, much

DISCUSSION A myelocytic leukemoid picture may be found in newborn infants with Down syndrome and may be confused with congenital leukemia. ~,12-15 Characteristically, the abnormal hematologic picture resolves without treatment over weeks or months. If the infant dies of other medical causes, there is no evidence of leukemia at autopsy. The spontaneous resolution of clinical and hematologic findings at 2 months of age in our patients certainly conforms with the diagnosis of TMD. High blast cell count was seen in the circulation during TMD when the bone marrow was minimally infiltrated with blast cells. In contrast, pancytopenia was present in both patients when the bone marrow was diagnostic of ANLL. The discrepancy in blast cell count may suggest a difference in the growth rate, maturation potential, or the regulatory mechanism of the abnormal clone of cells in TMD and in ANLL. Clonal chromosome abnormalities are common in A N L L and have diagnostic and prognostic importance. ~6,~7 Cytogenetic studies of TMD in Down syndrome have been infrequent, especially with banding techniques. One patient had 57 chromosomes, which disappeared when

Volume 112 Number 1

Myeloproliferative disorder and A N L L in Down syndrome

TMD re solved.12 One patient had constriction abnormalities in chromosomes 1 and 2.5 One patient had X;8 translocation and several double minutes, which never recurred. 18One patient in the neonatal period had a minor clone of marrow cells with 48 chromosomes (+C,+21), which persisted during spontaneous remission and emerged as the major abnormality during exacerbation of the leukemia. 4 This last case probably was truly congenital leukemia with a persistent clone of chromosomally abnormal cells. The incidence of abnormal cytogenetic findings with TMD could not be ascertained, as most of the patients did not undergo cytogenetic studies. Both of our patients had trisomy 21 as the only abnormal finding. In patients who have developed ANLL after TMD, new clonal chromosome abnormalities have occurred. One patient had 59 chromosomes in the leukemic cells, with gains in C, D, E, F, and G groups? Another patient had translocation between chromosomes 1 and 19 in the leukemic cells. 8 Unfortunately, cytogenetic studies were not available in these patients during TMD. Common chromosomal abnormalities in patients with Down syndrome with acute leukemia include missing chromosomes, partial deletions, chromosome rearrangements, and extra chromosomes commonly involving +8, +19, +21, and +22) 9-22Our patient 1 had trisomy 7 and monosomy 17. Trisomy 7 is an uncommon finding, although trisomy 7q was reported as part of the cytogenetic abnormalities in a patient with acute megakaryoblastic leukemia,z2 Monosomy 17 has not been reported in patients with Down syndrome and ANLL. The significance of 7/14 translocation in marrow cells of our patient 2 is not known at this time. This abnormality has been observed infrequently in PHA-stimulated lymphocyte cultures exclusively and not in bone marrow preparations, fibroblast, or amniocyte cultures. It is not known to be associated with any malignancy23.24 The growth pattern of marrow cells or peripheral blood cells in soft agar can be helpful in diagnosing leukemia? ~A maturing pattern has been observed with marrow cells from patients with TMD, 25'26 implying the benign nature and difference from true congenital leukemia. However, a leukemic pattern has also been found in soft agar culture with peripheral blood cells in a patient with TMD who subsequently died of cardiac disease at age 1 year with no evidence of leukemia.27 Both of our patients had a mixed growth pattern in stem cell cultures when studied initially, suggesting the existence of an abnormal clone, which might account for the subsequent development of ANLL. Although the cytogenetic abnormality observed in the ANLL phase was not demonstrated during TMD, it might be related to the size of the abnormal clone. Unfortunately, cytogenetic studies could not be performed with the cells


from the colonies in the culture system to see whether the observed chromosomal abnormalities were already present during TMD. The causal relationship between TMD and ANLL is not understood. We recommend that all Down syndrome patients with TMD should be studied serially with in vitro cultures and cytogenetic analysis of peripheral blood mononuclear cells to understand the pathophysiology and risk of ANLL in TMD.


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The Journal of Pediatrics January 1988

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