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Brief report

Erythropoietin-dependent transformation of myelodysplastic syndrome

to acute monoblastic leukemia
Udomsak Bunworasate, Hilal Arnouk, Hans Minderman, Kieran L. OLoughlin, Sheila N. J. Sait, Maurice Barcos,
Carleton C. Stewart, and Maria R. Baer

Acute monoblastic leukemia (acute myeloid leukemia [AML], French-AmericanBritish type M5a) with leukemia cutis developed in a patient 6 weeks after the
initiation of erythropoietin (EPO) therapy
for refractory anemia with ringed sideroblasts. AML disappeared from both marrow and skin after the discontinuation of
EPO. Multiparameter flow cytometric analysis of bone marrow cells demonstrated

coexpression of the EPO receptor with

CD45 and CD13 on the surface of blasts.
The incubation of marrow cells with EPO,
compared to without, resulted in 1.3- and
1.6-fold increases, respectively, in tritiated thymidine incorporation and bromodeoxyuridine incorporation into CD13
cells. Clinical and laboratory findings were
consistent with the EPO-dependent transformation of myelodysplastic syndrome

(MDS) to AML. It is concluded that leukemic transformation in patients with MDS

treated with EPO may be EPO-dependent
and that management should consist of
the discontinuation of EPO followed by
observation, if clinically feasible. (Blood.
2001;98:3492-3494)

2001 by The American Society of Hematology

Introduction

A 66-year-old man was evaluated for mild anemia; BM morphology was

diagnostic of MDS; the French-American-British (FAB) type was refractory anemia with ringed sideroblasts (RARS). Two years later stage IIA
Hodgkin disease was diagnosed. Hemoglobin level was 9.7 g/dL, white

blood cell (WBC) count was 7.6 109/L with a normal differential count,
and platelet count 145 109/L. BM aspirate and biopsy showed dysplastic
erythroid hyperplasia with 90% ringed sideroblasts and 1.6% myeloblasts,
again consistent with RARS. A B-cell clone expressing CD45, HLADr,
CD5, CD19, and light chain was detected by multiparameter flow
cytometry (MFC), consistent with early chronic lymphocytic leukemia.14
Karyotype was normal. Six courses of Adriamycin, bleomycin, vinblastine,
and dacarbazine combination chemotherapy were administered, with
clinical and radiologic resolution of Hodgkin disease. Anemia worsened 3
months later, necessitating monthly red blood cell transfusions. WBC and
platelet counts remained normal, and BM again showed RARS with a
normal karyotype and a B-cell clone by MFC. Serum EPO level was 9.6
mIU/mL. EPO therapy was initiated at a dose of 10 000 IU subcutaneously
3 times per week. Anemia improved, and transfusions were no
longer required.
Six weeks after the initiation of EPO therapy, numerous violaceous
nodules, each measuring 0.5 to 1 cm, appeared across the patients back and
abdomen. Biopsy specimens showed dermal infiltration by monoblasts
staining immunohistochemically for lysozyme (Figure 1A), myeloperoxidase, and CD15. Hemoglobin level was 9 g/dL, WBC count was 26.8 109/
L with 41% neutrophils, 32% lymphocytes, 25% monocytes, 2% basophils,
and 0% blasts, and platelet count was 203 109/L. The BM was
hypercellular, with 45% monoblasts (Figure 1B) staining with -naphthyl
butyrate esterase and expressing CD45, HLADr, CD2, CD4, CD11b,
CD11c, CD13, CD14, CD15, and CD33 by MFC with persistence of the
B-cell clone. Karyotype was again normal, and MLL gene rearrangement
was not detected in 500 interphase cells by fluorescence in situ hybridization with the LSI MLL dual-color rearrangement probe (catalog number
32-190083; Vysis, Downers Grove, IL). A diagnosis of acute monoblastic
leukemia (French-American-British type M5a AML) with leukemia cutis
was made, but chemotherapy was not initiated because of comorbid
conditions. EPO therapy was discontinued. Skin lesions regressed and
disappeared completely by 7 weeks. A BM sample 3 weeks after the

From the Departments of Medicine and Pathology, Leukemia Section, Clinical

Cytogenetics Laboratory and Laboratory of Flow Cytometry, Roswell Park
Cancer Institute, Buffalo, NY

Reprints: Maria R. Baer, Department of Medicine, Leukemia Section, Roswell

Park Cancer Institute, Elm and Carlton Sts, Buffalo, NY 14263; e-mail:
maria.baer@roswellpark.org.

Submitted May 22, 2001; accepted July 31, 2001.

The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked advertisement in accordance with 18 U.S.C. section 1734.

The myelodysplastic syndromes (MDS) are heterogeneous clonal

bone marrow (BM) disorders characterized by ineffective hematopoiesis.1 Anemia develops in 90% of patients. Erythropoietin
(EPO) therapy stimulates erythropoiesis in approximately 25% of
patients.2,3 Decreased sensitivity of erythroid precursors to EPO at
the initial level of signal transduction may contribute to anemia in
MDS4; EPO therapy may overcome this decreased sensitivity.
The risk for transformation of MDS to acute myeloid leukemia
(AML) correlates with the percentage of BM myeloblasts, cytogenetic abnormalities, and number of cytopenias.5 Marrow blasts may
increase, albeit infrequently, in MDS patients receiving granulocytemonocyte or granulocyte colony-stimulating factor.6-9 Leukemic
transformation of MDS is rare during EPO therapy, is seen
primarily in patients with refractory anemia with excess blasts or
refractory anemia with excess blasts in transformation,3,10,11 and is
thought to reflect natural history rather than EPO effect. AML has
also developed in patients with end-stage renal failure treated with
EPO12,13; the role of EPO in this setting is unknown. We report a
patient with clinical and laboratory findings consistent with EPOdependent transformation of MDS to AML.

Study design
Clinical course

Supported in part by shared resources of the Roswell Park Cancer Center

Support Grant (P30 CA16056) and by the Leonard S. LoVullo Memorial Fund
for Leukemia Research.

3492

2001 by The American Society of Hematology

BLOOD, 1 DECEMBER 2001 VOLUME 98, NUMBER 12

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BLOOD, 1 DECEMBER 2001 VOLUME 98, NUMBER 12

EPO-DEPENDENT TRANSFORMATION OF MDS TO AML

3493

Figure 1. Erythropoietin-dependent acute monoblastic leukemia. Histopathology. Skin biopsy showing infiltration by monoblasts staining with lysozyme (panel A, 50
magnification) and bone marrow aspirate smear showing
acute monoblastic leukemia (panel B, 200 magnification), both during EPO therapy. Bone marrow aspirate
smear showing the absence of leukemia 3 weeks after
the discontinuation of EPO therapy (panel C, 50
magnification).

discontinuation of EPO showed reversion to MDS, without increased blasts

(Figure 1C) and without abnormal myeloid cells by MFC; the B-cell clone
persisted. Anemia worsened again, necessitating transfusions. The patient
had a fatal myocardial infarction 4 months later; leukemia had not recurred.
Laboratory studies
Samples. BM mononuclear cells obtained by Ficoll-Hypaque density
centrifugation at presentation of AML, 3 months earlier, and 6 weeks later
were cryopreserved in RPMI 1640 medium (Gibco, Grand Island, NY) with
20% fetal calf serum and 20% dimethyl sulfoxide using standard procedures.
EPO receptor expression. EPO receptor expression was studied by
MFC with the rabbit polyclonal antibody EpoR (C-20) (Santa Cruz
Biotechnology, Santa Cruz, CA), detected with fluorescein isothiocyanateconjugated goat antirabbit immunoglobulin G (Caltag, Burlingame, CA).
The primary antibody was titered using the UT-7/EPO cell line15 provided
by Dr Kamatsu (Tochigi, Japan). The EPO receptor was coexpressed with
CD45 and CD13 on the surface of blasts, defined by forward versus side
scatter characteristics, but not on lymphocytes, defined by forward versus
side scatter and absence of CD13 expression. Cells coexpressing the EPO
receptor, CD45, and CD13 were detected in samples from all 3 time points,
but were most numerous at the presentation of AML (Figure 2A).
In vitro EPO response. BM cells cryopreserved before the initiation of
EPO therapy were incubated for 4 hours in RPMI 1640 medium supplemented with 10% fetal calf serum, with and without 1 U/mL recombinant
EPO (Amgen, Thousand Oaks, CA). DNA synthesis was measured by
radiolabeled thymidine ([methyl-3H], 1 Ci [3.7 104 Bq], 10 minutes)

Figure 2. EPO receptor expression on CD13 blasts and in vitro proliferation of

CD13 cells in response to EPO. Upper panels (A) demonstrate gating on blasts
defined by forward scatter characteristics (FSC) versus side scatter characteristics
(SSC) (left) and coexpression of the EPO receptor with CD45 (center) and CD13
(right) on cells in the gated region. Lower panels (B) demonstrate the incorporation of
BrdU into CD13 cells in the absence (left) and presence (right) of EPO. Increased
DNA synthesis is seen in CD13 cells in the presence of EPO.

incorporation. In 2 experiments with 5 106 cells each, DNA synthesis

increased 1.27- and 1.33-fold (mean, 1.3-fold) in the presence of EPO.
To identify the population proliferating in response to EPO, cells
incubated for 4 hours with and without EPO were labeled with 10 M
5-bromo-2-deoxyuridine (BrdU) for 30 minutes then were stained with
CD13 antibody and analyzed by MFC.16 Incubation of marrow cells with
versus without EPO resulted in a 1.6-fold increase in BrdU incorporation
(from 3.3% to 5.2%) into myeloid blasts, defined by forward versus side
scatter characteristics and CD13 expression.

Results and discussion

AML developed in our patient during EPO therapy and regressed
after its discontinuation. Transformation to acute leukemia was
unexpected, given the RARS subtype of MDS, the normal karyotype, and the presence of anemia as the sole cytopenia.5 Adriamycin administered to treat Hodgkin disease could have been leukemogenic, but MLL gene rearrangement, characteristic of topoisomerase
II inhibitor-induced AML,17 was not found. The development of
AML during EPO therapy and the disappearance of leukemia from
BM and skin after the discontinuation of EPO were consistent with
EPO-dependent transformation of MDS to AML. In vitro results
also support this hypothesis; myeloid blasts expressed the EPO
receptor and proliferated in response to EPO. Relatively modest
EPO-induced proliferation in vitro is explained by the absence of
leukocyte-conditioned medium or costimulatory cytokines in the
cultures.18,19 Of note, the B-cell clone detected by MFC might
possibly have produced costimulatory factors in vivo.
EPO has been reported to stimulate clonogenic leukemic
erythroid progenitors in erythroleukemia20 and leukemic blast
colony growth in the presence of phytohemagglutinin-stimulated,
leukocyte-conditioned medium in other AML subtypes.18,19 EPO
receptor expression was found on leukemia cells in 81 of 136
(60%) patients with all French-American-British types of AML,
and in vitro proliferation in response to EPO was seen in 16% of
patients, all with expression of the receptor.21 Moreover, compared
with those without EPO receptor expression, remission duration
was shorter in patients whose cells expressed the EPO receptor and
proliferated in response to EPO.21
To our knowledge, this is the first demonstration of EPO-dependent
leukemic transformation of MDS. EPO is used increasingly in patients
with MDS, and close observation for leukemic transformation is
warranted. EPO therapy should be discontinued in patients with MDS
that progresses to AML, and, if clinically feasible, those patients should
be observed for the regression of AML.

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3494

BUNWORASATE et al

BLOOD, 1 DECEMBER 2001 VOLUME 98, NUMBER 12

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2001 98: 3492-3494

doi:10.1182/blood.V98.12.3492

Erythropoietin-dependent transformation of myelodysplastic syndrome to

acute monoblastic leukemia
Udomsak Bunworasate, Hilal Arnouk, Hans Minderman, Kieran L. O'Loughlin, Sheila N. J. Sait, Maurice
Barcos, Carleton C. Stewart and Maria R. Baer

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