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CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
Sustained response to recombinant human erythropoietin and intermittent all-trans
retinoic acid in patients with myelodysplastic syndromes
Roberto Stasi, Maurizio Brunetti, Edmondo Terzoli, and Sergio Amadori
In vitro studies suggest that all-trans retinoic acid (ATRA) synergizes with erythropoietin (EPO) for the stimulation of hematopoiesis in patients with myelodysplastic
syndrome (MDS). A clinical trial was performed to evaluate whether a combination of these agents was effective in relieving the cytopenias associated with MDS.
Twenty-seven patients with low- or intermediate-risk MDS were enrolled in a 12week study. ATRA was administered orally
at the dose of 80 mg/m2 per day in 2
divided doses for 7 consecutive days
every other week. Recombinant human
EPO was given subcutaneously 3 times a
week. The EPO dose was initiated at 150
U/kg and was increased to 300 U/kg if
after 6 weeks there was no or there was
suboptimal erythroid response. Patients who
responded to therapy were continued on
ATRA and EPO at the same doses for 6
additional months (extension phase). Further treatment was given to patients with a
continued response. Clinically significant
erythroid responses with increases of hemoglobin levels of at least 1 g/dL or reduction
of transfusion needs were seen in 13 (48%)
patients, with 4 showing improved responses after dose escalation of EPO. Ten
(37%) patients displayed continued responses during 6 months of extended treatment, and 7 (26%) are still responsive after a
follow-up period of 13 months. Neutrophil
responses were observed in 5 of 12 patients
with neutropenia, and platelet responses
were observed in 6 of 9 patients with thrombocytopenia. Three patients displayed trilineage responses that were sustained during
continuation therapy. Side effects were observed in all patients but were of mild entity
and did not require discontinuation of
therapy. It is concluded that the combination ATRA ⴙ EPO is an effective and welltolerated treatment for patients with lowand intermediate-risk MDS. The optimal
ATRA and EPO schedule and the role of
maintenance treatment remain to be determined and warrant further investigation.
(Blood. 2002;99:1578-1584)
© 2002 by The American Society of Hematology
Introduction
Myelodysplastic syndrome (MDS) is a heterogeneous group of
clonal hematopoietic stem cell disorders characterized by peripheral cytopenias, functional defects, and a variable propensity for
leukemic transformation. No current treatment has been shown to
be consistently effective in producing sustained improvement in
hematopoiesis in these patients (with the possible exception of
allogeneic bone marrow transplantation in selected younger patients), and supportive care with blood products and administration
of antibiotics remains the mainstay of therapy.
In recent years, experimental therapeutic approaches have been
focused on the use of hematopoietic growth factors and biologic
response modifiers that may act by decreasing the apoptosis rate and
enhancing the differentiation of preleukemic progenitor cells or by
stimulating the growth of residual normal hematopoietic clones. Several
trials have been designed to evaluate the efficacy of recombinant human
erythropoietin (EPO) in the enhancement of erythropoiesis in MDS,
demonstrating an overall response rate of approximately 20%.1 Numerous studies have also explored the use of retinoids, but their results are
difficult to interpret because of the differences in agents, doses, and
schedules and the use of additional agents.2
All-trans retinoic acid (ATRA) has been reported to have poor
activity when used as a single agent,3-5 but in vitro studies support a role
for its use in combination with EPO to reduce apoptosis in patients with
MDS.6,7 It is noteworthy that the declining efficacy of ATRA during
long-term treatment has been associated with a reduction of ATRA
plasma levels.8 Although the mechanisms responsible for this phenomenon are unknown, those hypothesized include malabsorption, induction of cytochrome P450 activity by ATRA, or elevation of cellular
ATRA-binding protein levels, resulting in increased plasma clearance
into nontarget tissues.8 Potential methods to overcome this phenomenon
include blocking oxidation by administration of a P450 inhibitor or
using an intermittent dosing schedule.9 An intermittent schedule of
ATRA administration has a potential advantage over a continuous one.
Studies performed in patients with acute promyelocytic leukemia9 and
chronic myelogenous leukemia10 indicate that a period of time without
drug administration allows a return to baseline plasma clearance levels
and a down-regulation of cellular ATRA-binding protein, which results in
higher ATRA plasma concentrations and possibly less cytoplasmic
binding of the drug. In this report we detail the results of a phase 2 clinical
trial in which we evaluated the biologic effects, tolerance, and safety of the
combination EPO ⫹ intermittent ATRA in patients with MDS.
From the Department of Medical Sciences, Regina Apostolorum Hospital,
Albano Laziale; the Department of Complementary Oncology, Regina Elena
Institute, Rome; and the Department of Hematology, University of Rome Tor
Vergata, S. Eugenio Hospital, Italy.
Apostolorum Hospital, Via S. Francesco 50, 00041 Albano Laziale, Italy;
e-mail: [email protected].
Patients and methods
Patients
Twenty-seven patients with low- or intermediate-risk MDS according to the
International Prognostic Scoring System criteria11 were entered into the
Submitted July 18, 2001; accepted October 26, 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.
Reprints: Roberto Stasi, Department of Medical Sciences, Regina
© 2002 by The American Society of Hematology
1578
BLOOD, 1 MARCH 2002 䡠 VOLUME 99, NUMBER 5
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BLOOD, 1 MARCH 2002 䡠 VOLUME 99, NUMBER 5
EPO PLUS ATRA IN MDS
study after informed consent had been obtained. These were consecutive
eligible patients from a series of 41 patients with low- or intermediate-risk
MDS. All patients had histologically confirmed MDS; bone marrow
aspirates were classified according to the French-American-British (FAB)
criteria.12 Pretreatment clinical and hematologic characteristics of the
patients are shown in Table 1. Median time from diagnosis to initiation of
growth factor therapy was 23 months (range, 8-56 months). At enrollment,
21 patients were transfusion dependent; the median was 2.5 U (range, 2-4
U) packed red blood cell (RBC) transfusions per month for 3 months before
the study. Transfusions were usually given when the hemoglobin concentration was less than 8 g/dL, though occasionally we adopted different
thresholds in individual patients in accordance with their compliance with
low hemoglobin levels. In addition, sometimes transfusions were delayed
because of a shortage of blood products, which occurred frequently during
the holidays. None of the patients had previously undergone cytotoxic
therapy for MDS.
Eligibility criteria were as follows: primary MDS with less than 10%
blasts on bone marrow examination; performance status of 2 according to
the Eastern Cooperative Oncology Group (ECOG) scale13; hemoglobin
levels lower than 10 g/dL; normal renal and hepatic function; normal iron,
vitamin B12, and folate levels, clinically significant heart and central
nervous system disease, uncontrolled hypertension, florid infections, or
other malignancies. On study entry, patients gave signed, institutional
review board–approved informed consent.
Table 1. Summary of pretreatment characteristics
No. patients
27
Men
13
Women
14
Age (y)
Median
68
Range
52-78
19
RARS
3
RAEB
5
12
Intermediate
6
Poor
1
IPSS†
Low risk
5
Intermediate-1 risk
13
Intermediate-2 risk
1
Not applicable
8
Hemoglobin levels
Median
8.1 g/dL
Range
6.1-9.5 g/dL
Absolute neutrophil count
Median
Range
Therapy consisted of a 12-week schedule of ATRA and EPO. ATRA
(Vesanoid; Roche, Milan, Italy) was administered orally at a dose of 80
mg/m2 per day in 2 divided doses for 7 consecutive days every other week.
Recombinant human EPO (Eprex; Janssen-Cilag, Milan, Italy) was given
subcutaneously 3 times a week. The EPO dose was initiated at 150 U/kg and
was increased to 300 U/kg if after 6 weeks there was no or there was
suboptimal erythroid response. Patients who responded to therapy were
continued on ATRA and EPO at the same doses for 6 more months
(extension phase). Further treatment was given to patients who had
continued responses. Patients were questioned weekly concerning possible
adverse events. Treatment was discontinued at the patient’s request if severe
side effects or transformation to AML occurred.
Response criteria
Responses were categorized according to recently published criteria by an
international working group.14 Regarding the erythroid lineage, a major
response (MaR) was considered a rise in untransfused hemoglobin concentrations of at least 2 g/dL or a 100% decrease in RBC transfusion
requirements during the treatment period. A minor response (MiR) was
defined as an increase in untransfused hemoglobin values of 1 to 2 g/dL or a
50% or greater decrease in RBC transfusion requirements. No response was
defined as a response less than a minor response. Regarding platelets, for
patients with pretreatment platelet counts less than 100 ⫻ 109/L, MaR was
defined as an absolute increase of 30 ⫻ 109/L or more, and MiR was a 50%
increase in platelet count with a net increase greater than 10 ⫻ 109/L but not
exceeding 30 ⫻ 109/L. Regarding neutrophils, for patients with pretreatment absolute neutrophil counts (ANCs) less than 1.5 ⫻ 109/L, MaR was
considered at least a 100% increase or an absolute increase of 0.5 ⫻ 109/L,
whichever was greater; MiR required a 100% or greater increment in ANC,
but absolute increase required less than 0.5 ⫻ 109/L.
Patient evaluation before entry included complete history and physical
examination. All patients underwent chest roentgenography and electrocardiography. Baseline laboratory evaluation included a complete blood cell
count with reticulocytes, serum EPO, serum ferritin, vitamin B12 and folate
levels, routine serum chemistry, coagulation tests, and urinalysis. Vital
signs and complete blood cell counts were monitored once a week. Serum
EPO levels were determined using a commercially available enzyme-linked
immunoassay (Quantikine IVD Erythropoietin; R&D Systems, Minneapolis, MN). Bone marrow aspirates and biopsy specimens were taken at
diagnosis and at the end of the study (aspirates) or when clinically required.
Karyotyping was carried out with standard techniques15 at study entry and,
in responders, at the end of treatment.
Karyotype*
Good
Study design
Study parameters and monitoring of patients
FAB subtype
RA
1579
1.9 ⫻ 109/L
0.5-4.1 ⫻
109/L
Platelet count
Median
145 ⫻ 109/L
Range
32-298 ⫻ 109/L
Serum erythropoietin (n ⫽ 21)
Median
368.5 mIU/mL
Range
80-1482 mIU/mL
Serum ferritin
Median
654 ng/mL
Range
157-1348 ng/mL
MDS duration before rhEPO treatment
Median
23 months
Range
8-56 months
*Good indicates normal (Y, del[5q], del[20q]). Poor indicates complex (ⱖ 3
abnormalities) or chromosome 7 anomalies. Intermediate indicates other
abnormalities.
†International Prognostic Scoring System.11
Erythroid progenitor cell assay
Heparinized blood samples were collected at baseline, at 12 weeks, and, in
responders, during the extension phase. Erythroid blast-forming units
(BFU-E) were assayed in viscous medium using a modification of the
method of Iscove and colleagues,16 as previously described. Briefly,
2 ⫻ 105 peripheral blood mononuclear cells were plated in triplicate in
35-mm Petri dishes with 1-mL aliquots of 0.9% methylcellulose viscous
Iscoves modified Dulbecco medium (Gibco, Grand Island, NY) supplemented with 30% human AB serum, 10% bovine serum albumin (Fraction
V; Sigma, St Louis, MO), 1 ⫻ 10⫺4 M 2-mercaptoethanol (Sigma), and 2 U
EPO (Ortho Diagnostic Systems, Raritan, NJ). After incubation for 14 days
at 37°C in a humidified atmosphere supplemented with 5% CO2, the
cultures were scored for BFU-E (defined as bursts of colonies consisting of
hemoglobinated cells) with an inverted microscope.
Measurement of apoptosis
Apoptosis was measured by flow cytometry with a FACScan instrument
(Becton Dickinson, Mountain View, CA). Mononuclear cell fractions of
bone marrow samples were separated after Ficoll-Hypaque gradient
F
M
64
57
52
62
75
78
77
56
73
71
68
54
72
62
69
76
70
64
77
68
66
61
70
63
72
74
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
RAEB
RA
RA
RA
RA
9
22
8
13
34
19
27
31
18
47
52
16
15
9
21
55
51
33
44
18
23
56
38
15
12
47
25
MDS
duration
(mo)
715
838
ND
273
165
658
ND
175
560
135
ND
181
269
ND
363
1482
80
125
231
1115
938
ND
586
374
281
673
513
Baseline
serum EPO
(mIU/mL)
3
2
3
0
2
2
3
0
3
3
2
0
0
3
2
4
2
0
2
2
3
2
4
0
0
2
3
3
0
2
0
0
1
3
0
2
0
2
0
0
3
1
4
1
0
2
0
2
1
2
0
0
2
1
6.8
7.1
6.9
8.5
8.1
6.7
7.9
9.1
8.1
7.6
8.3
8.6
9.3
7.4
8.5
6.3
8.6
9.5
8.1
7.2
6.1
7.7
7.4
8.6
8.5
8.4
7.9
7.3
8.6
7.3
8.2
9.3
8.0
7.5
10.0
8.6
9.6
9.0
9.1
11.4
8.3
8.1
6.1
8.7
9.3
8.7
8.5
6.4
7.8
8.1
8.3
10.9
7.9
7.3
At 6 wks
Before
Before
After
Hb (g/dL)*
RBC
transfusions
7.5
10.4
8.0
8.7
9.5
7.8
8.1
9.8
8.4
10.9
8.7
9.7
11.6
8.1
8.8
6.6
9.5
10.1
7.8
8.3
6.6
8.6
7.9
8.0
11.3
8.5
8.2
At 12 wks
NR
MaR
NR
NR
MiR
MiR
NR
NR
NR
MaR
NR
MiR
MaR
NR
MiR
NR
MiR
NR
NR
MaR
NR
MiR
MiR
NR
MaR
NR
MiR
Erythroid
response
NA indicates not applicable; ND, not done; Int, intermediate.
*Blood counts before and after treatment were calculated by averaging the results of 3 counts taken in a 2-week period.
M
M
F
F
M
RAEB
RA
RARS
RA
RA
RA
RAEB
RA
RARS
RAEB
RA
RA
RAEB
RA
RA
RARS
RA
RA
RA
RA
RA
RA
FAB
subtype
1.0
1.3
0.9
2.3
1.4
1.1
2.0
4.1
2.3
3.1
1.0
1.3
3.7
2.1
1.9
1.2
3.2
1.4
0.9
2.4
1.9
3.3
0.5
1.3
2.6
1.7
2.5
Before
0.8
1.6
0.7
2.0
3.1
2.7
2.6
4.4
2.1
4.3
1.8
3.5
4.1
3.2
2.4
1.5
3.5
1.5
1.2
2.2
2.1
2.9
1.2
1.2
4.4
1.6
2.7
At 12 wks
ANC (⫻ 109/L)*
NR
NR
NR
NA
MaR
MaR
NA
NA
NA
NA
MaR
MaR
NA
NA
NA
NR
NA
NR
NR
NA
NA
NA
MaR
NR
NA
NA
NA
Neutrophil
response
67
93
131
147
86
158
141
229
126
74
102
32
104
85
201
298
117
136
84
220
151
168
54
131
214
145
93
Before
78
145
126
128
137
179
173
247
295
166
92
53
123
77
152
263
191
163
75
246
191
238
121
115
266
175
158
At 12 wks
Platelets
(⫻ 109/qL)*
NR
MaR
NA
NA
MaR
NA
NA
NA
NA
MaR
NA
MiR
NA
NR
NA
NA
NA
NA
NR
NA
NA
NA
MaR
NA
NA
NA
MaR
Platelet
response
ND
Good
Good
Good
Int
Good
Int
Good
Good
ND
Good
Poor
ND
ND
Int
ND
ND
Int
Int
Good
ND
Int
Good
Good
ND
Good
Good
Karyotype
NA
0.5
0.5
0
1
1
0.5
0
0
NA
0.5
2
NA
NA
1
NA
NA
1.5
1
0
NA
0.5
0.5
0.5
NA
0
0.5
IPSS score
value
NA
Int-1
Int-1
Low
Int-1
Int-1
Int-1
Low
Low
NA
Int-1
Int-2
NA
NA
Int-1
NA
NA
Int-1
Int-1
Low
NA
Int-1
Int-1
Int-1
NA
Low
Int-1
IPSS risk
group
STASI et al
F
F
F
M
F
F
M
F
M
F
F
M
F
M
F
M
M
F
M
M
61
1
Sex
Age
(y)
Patient
no.
1580
Table 2. Clinical and hematologic characteristics of MDS patients administered EPO plus ATRA
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BLOOD, 1 MARCH 2002 䡠 VOLUME 99, NUMBER 5
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BLOOD, 1 MARCH 2002 䡠 VOLUME 99, NUMBER 5
Figure 1. Response versus time. Kaplan-Meier plot of probability of response
versus time for the 13 patients with erythroid responses.
centrifugation and washed twice with phosphate-buffered saline. Cells
(1 ⫻ 106) were then incubated with phycoerythrin-conjugated anti-CD34
mAb (anti–HPCA-2, IgG1; Becton Dickinson) for 10 minutes at room
temperature in the dark and were washed twice with phosphate-buffered
saline. Pelleted cells were resuspended in 100 ␮L binding buffer (10 mM
HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2; Bender Medsystems, Boehringer Ingelheim, Ridgefield, CT) and were incubated with 2 ␮L
fluorescein isothiocyanate–conjugated annexin V (Bender Medsystems;
Boehringer Ingelheim) for 10 minutes at room temperature in the dark.
Afterward, cells were resuspended in 400 ␮L binding buffer before flow
cytometric analysis. Analysis was based on gating of subpopulations of
CD34⫹ cells by forward scatter versus side scatter and by side scatter versus
fluorescence-2. Negative controls included peripheral blood mononuclear
cells incubated with neither CD34-PE mAb nor annexin V–FITC and cells
incubated with CD34-PE mAb only. Bone marrow from 10 healthy donors
was used as reference.
Statistical analysis
Statistical evaluation was performed with the STATISTICA for Windows
(StatSoft, Tulsa, OK) software package on an IBM-compatible computer.
Mann-Whitney U test was used to compare continuous variables between
responders and nonresponders. Wilcoxon matched-pairs test was used to
compare repeated measurements in the same patients. Fisher exact test was
used to evaluate the relationship between 2 dichotomous variables.
Correlations of variables with other variables were calculated by Spearman
rank correlation coefficient. P ⬍ .05 was designated as statistically significant; all P values were 2-tailed.
Results
EPO PLUS ATRA IN MDS
1581
In responders, the increase in hemoglobin concentration was
associated with a significant increase in reticulocyte counts. Mean
reticulocyte count was 20 531/␮L (95% CI, 13 326-27 736/␮L)
before treatment versus 50 423/␮L (95% CI, 35 459-65 387/␮L)
on week 12 of treatment (P ⫽ .001475). Ten of the 13 patients
administered ATRA ⫹ EPO during the extension phase had
continued response. Although we had initially planned to stop
treatment after 6 months, the good compliance to combination
therapy and the requests from the patients themselves induced us to
prolong treatment. Thus far, with a median follow-up of 15 months,
7 patients are still responsive. Figure 1 illustrates a Kaplan-Meier
plot of probability of response versus time for the 13 patients with
erythroid responses.
Five of the 12 patients with pretreatment neutropenia had
increases in neutrophil count of 0.5 ⫻ 109/L or greater (range of
ANC increase, 0.7 to 2.2 ⫻ 109/L), for a response rate of 42%
(95% CI, 0.09-0.74). Ten other patients who did not have neutropenia before treatment showed an increase in neutrophil count
ranging from 0.2 to 1.8 ⫻ 109/L. The neutrophil response began to
appear after 2 weeks of treatment and was continued throughout
treatment, though we observed that the ANC tended to be higher
when patients were administered ATRA. At the time of this report,
all 5 patients maintain their response.
Five patients with pretreatment thrombocytopenia exhibited
increases in platelet count exceeding 30 ⫻ 109/L (range of platelet
count increment, 33 to 92 ⫻ 109/L), and one patient (patient 16)
had an MiR and an increase in platelet count of 21 ⫻ 109/L. When
only patients with pretreatment platelet counts less than 100 ⫻ 109/
L (n ⫽ 9) were considered, the response rate was 67% (95% CI,
0.28-1.05). In 12 patients who had normal counts before treatment,
we observed platelet increases ranging from 18 to 169 ⫻ 109/L.
Platelet response was maintained throughout treatment for 4 of the
6 patients, whereas patients 1 and 26 had relapses after 65 and 28
weeks of extended treatment, respectively. It is noteworthy that the
platelet response was slower than the hemoglobin and neutrophil
responses because it began to appear after 4 weeks from the start of
treatment. Three patients (patients 5, 16, and 23) displayed
trilineage hematologic improvement that was sustained during
continuation therapy.
A representative responding patient’s course (patient 18) is
shown in Figure 2. The patient was a 70-year-old woman with a
Response to treatment
All patients completed the 12-week study and were evaluated for
toxicity and response. Changes in blood cell counts and transfusion
requirements before and after treatment are reported in Table 2.
According to defined response criteria, on week 12 of treatment the
erythroid response rate was 48% (95% confidence interval [CI],
0.28-0.68). The response was major in 5 patients, minor in 8, and
absent in 14. Of the 5 patients who attained MaR, 4 exhibited an
optimal response at the dose of 150 IU/kg and were maintained
with that dose, whereas the fifth achieved an MaR only at the
higher dose. Five of the 8 patients with MiR had signs of response
after the first 6 weeks of treatment but did not benefit from the
higher EPO dose; the other 3 patients showed a response only after
challenge with EPO at 300 IU/kg. Five patients (patients 1, 5, 6, 11,
and 13) exhibited responses based solely on a decrease in
transfusion requirements. Hemoglobin levels at which their transfusions were ordered before and after treatment initiation were:
patient 1, 7.5 g/dL and 7.3 g/dL; patient 5, 7.8 g/dL and 7.4 g/dL;
patient 6, 7.3 g/dL and 7.9 g/dL; patient 11, 8.0 g/dL and 8.2 g/dL;
patient 13, 7.6 and 7.7 g/dL.
Figure 2. Response to combination therapy. Clinical course of a patient with MDS
responding to combination therapy with EPO and ATRA. See “Results” for details.
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1582
BLOOD, 1 MARCH 2002 䡠 VOLUME 99, NUMBER 5
STASI et al
4-year history of MDS and a transfusion need of 3 RBC units per
month. Significant improvement of hemogram parameters was
observed after 4 weeks of treatment, at which time her hemoglobin
concentration rose to 8.3 g/dL without transfusions and her platelet
count increased to 93 ⫻ 109/L from a baseline value of 74 ⫻ 109/L.
A constant increase in hemoglobin and platelet values was noted
thereafter, with hemoglobin (11.2 g/dL) and platelet (189 ⫻ 109/L)
peaks recorded in week 11. This patient eventually maintained
hemoglobin concentrations in the 10 to 11 g/dL range and platelet
values above 100 ⫻ 109/L during the extension phase.
Examination of marrow aspirates on conclusion of the study
showed a nonsignificant increase in the percentage of erythroid
cells in 1 of 3 major responders and in 2 of 6 minor responders. As
assessed by clinical parameters and bone marrow morphology,
disease progression was not observed in any patient.
Laboratory studies
As shown in Figure 3, the number of circulating BFU-E in
responders during week 12 of treatment consistently increased
compared with baseline (P ⬍ .001). Analysis of karyotype at the
end of the study (available in 12 patients) did not show remarkable
changes. Pretreatment determination of the degree of apoptosis in
hematopoietic progenitors by means of the annexin V method
indicated no significant differences between the FAB subgroups. In
refractory anemia (RA) and RA with ringed sideroblasts (RARS),
the median CD34⫹ cell apoptosis was 52.3% (range, 24.3%86.4%), whereas in RA with excess blasts (RAEB) it was 49.3%
(range, 38.7%-61.2%; P not significant). Control patients had
median CD34⫹ cell apoptosis of 14.8% (range, 5.5%-27.9%;
P ⬍ .001). In week 12 of treatment, CD34⫹ cell apoptosis was
significantly decreased in responders (median, 35.1%; range,
15.4%-56.4%) compared to nonresponders (median, 57.3%; range,
43.4%-81.2%; P ⬍ .001). Further determinations during the extension phase in 6 patients produced results almost superimposable
with those obtained in week 12. Figure 4 reports the double-color
analyses with antibodies against CD34 (phycoerythrin) and against
Annexin V (fluorescein isothiocyanate) in patient 18. Doublepositive cells (upper right quadrant) dropped from 38.6% before
treatment (Figure 4A) to 15.4% during the extension phase
(Figure 4B).
Figure 4. Flow cytometric evaluation of CD34ⴙ cell apoptosis in a responder.
See “Results” for details. (A) Dot plot histogram before treatment. (B) Dot plot
histogram during the extension phase.
erythema at the site of recombinant human (rh)EPO injections,
though it did not interrupt rhEPO administration. ATRA-related
side effects were observed in all patients and are detailed in
Table 3. In particular, changes in the skin and mucous membranes developed in 22 (81%) patients and consisted of erythema (particularly in the areas of greater distribution of the
sebaceous glands), hyperkeratosis, cheilitis, conjunctivitis, and
nail dystrophy. All these side effects were grade 1 or 2 according
to the National Cancer Institute toxicity scale. While on the
protocol, approximately 50% of the patients experienced elevations of serum triglycerides; biochemical signs of liver cell
damage or renal toxicity were not noted.
Safety
Prognostic factors
Treatment was well tolerated overall. Patient 15 had a mild
increase in arterial blood pressure after 8 weeks of treatment that
was easily controlled by medical therapy. Patient 3 had painful
Table 4 shows the clinical and laboratory characteristics of the 13
patients who had erythroid responses (MaR⫹MiR) to ATRA plus
EPO treatment compared with those of the 14 patients who did not
respond. No variable was significantly different between the 2
groups of patients in univariate analysis. Similarly, no combination
of these characteristics demonstrated a significant association with
response. We were unable to show the predictive value of these
variables for neutrophil and platelet responses.
Table 3. Side effects associated with therapy
Side effect
Figure 3. Responders versus nonresponders. Comparison of circulating BFU-E
before and after 12 weeks of treatment in responders and nonresponders. Horizontal
lines represent mean values.
No. patients
Dry skin
22
Cheilitis
17
Conjunctivitis
7
Nail dystrophy
3
Nausea
2
Headache
1
Myalgias
1
Hypertension
1
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BLOOD, 1 MARCH 2002 䡠 VOLUME 99, NUMBER 5
EPO PLUS ATRA IN MDS
Table 4. Comparison of clinical and laboratory characteristics of MDS patients
responding and not responding to ATRA plus EPO treatment
Median age, y
M/F
MDS duration, mo
Serum EPO
Responders
(range)
Nonresponders
(range)
66 (54-77)
69 (52-78)
.319 330
6/7
7/7
1.000 000
22 (12-56)
322 (80-1115)
25 (8-55)
P
.644 601
467 (125-1482)
.468 262
Prior RBC transfusion
requirements, U/mo
Hb levels, g/dL
Reticulocytes, ⫻ 109/L
2.5 (0-4)
.507 629
7.9 (6.7-9.3)
2 (0-4)
8.1 (6.1-9.5)
.884 009
22.33 (5.9-80.0)
18.89 (4.86-86.3)
.752 408
ANC, ⫻ 109/L
2.4 (0.5-3.7)
1.55 (0.9-4.1)
.144 909
Platelets, ⫻ 109/L
104 (32-220)
133.5 (67-298)
.528 024
Cytogenetics,
normal/abnormal
5/4
7/3
.649 920
Discussion
In this study, 27 patients with low- or intermediate-risk MDS were
treated with a combination of intermittent oral ATRA and subcutaneous EPO for a 12-week study period. The EPO dose was doubled
if after 6 weeks there was no erythroid response or a suboptimal
erythroid response. Erythroid responses were seen in 13 (48%)
patients (MaR in 5 patients, MiR in 8 patients), with 10 (37%)
patients displaying a continued response during 6 months of
extended treatment and 7 (26%) maintaining their response after a
median follow-up period of 15 months. Neutrophil response was
observed in 5 (42%) and platelet response in 6 (67%) patients,
respectively. Three patients displayed trilineage response that was
sustained during continuation therapy. Side effects were observed
in all patients but were of mild entity and did not require the
discontinuation of treatment.
Although in this study we used oral ATRA in alternate weeks at
80 mg/m2 per day, the optimal schedule in association with EPO is
unknown because other schedules have not been systematically
investigated. In addition, EPO has been used in MDS in a variety of
doses and schedules. In most reports, we find starting doses of 450
U/kg per week, as in the current study.
When interpreting our results, it seems reasonable to ask what is
the relative contribution of ATRA and EPO to the results and
whether their effects are synergistic or simply additive. Although
these issues were not investigated directly, some considerations can
be inferred from our data and other reported studies. We know that
improvement of hemoglobin levels in response to EPO therapy in
patients with MDS stands, on average, at approximately 20%1 but
that by patient selection (FAB types RA and RAEB, serum EPO
levels less than 200 mIU/mL, no transfusion need) responses to
EPO alone can be in excess of 50%.1 Because in our series only 2
patients displayed the pretreatment characteristics of good responders to EPO, it seems unlikely that the high response rate of this
study could be obtained with EPO alone. Nevertheless, the limited
sample size of this study and the wide confidence interval
(28%-68%) did not allow us to fully support superiority of
combination therapy over EPO alone or to conclude that the use of
1583
ATRA results in a synergistic effect on erythroid precursors.
Regarding myeloid and megakaryocytic lineages, the response rate
was in the magnitude of 25%, far above the expected additive effect
of the single drugs, but again we have to acknowledge that the
relatively small number of patients raises concerns about the
reproducibility of these findings.
A remarkable result of our study is the sustained response to
therapy in 26% of the patients because the clinical usefulness of
any therapy in MDS depends on response duration and on response
rate. Actually, we do not know whether these patients would stay in
prolonged remission only with the combination therapy or with
either agent as single therapy. For example, in the study by Negrin
et al16 in which EPO was combined with granulocyte–colonystimulating factor, approximately half the responders maintained
an erythroid response with EPO alone. Incidentally, in that study
the variables that turned out to be predictive of erythroid response
were serum EPO, reticulocyte count, and cytogenetic pattern,
whereas in our own study no pretreatment characteristic was
significantly associated with response. As stated before, the good
compliance with combination therapy and requests from the
patients themselves induced us to prolong unchanged treatment
until relapse occurred.
Results of laboratory investigations performed in this study
indicate that response to treatment is associated with higher
concentrations of BFU-E in the peripheral blood and with a
remarkable decrease of the bone marrow fraction of apoptotic
CD34⫹ cells. Whether these findings represent a stimulation of
residual polyclonal hematopoiesis or an actual reduction in the
degree of ineffective hematopoiesis remains undetermined. Studies
suggest that one mechanism of the positive effects of combination
therapy with growth factors is in fact the inhibition of apoptosis.17
The current study confirms previous findings that bone marrow of
patients with MDS contains significantly more apoptotic cells than
bone marrow of healthy persons; it also confirms that there is ample
variation between patients.18-20 There was no significant relationship between pretreatment values for apoptosis and treatment
outcome. This suggests that apoptosis in itself is not the only factor
for a clinical response to the combination therapy we used. In
drawing these conclusions, however, we must emphasize that there
are many techniques now available to assess apoptosis, each with a
different sensitivity. Although our results are perfectly in line with
those of other researchers,17,20 this aspect should be carefully
evaluated when comparing results from different studies.
In conclusion, the combination of intermittent oral ATRA and
subcutaneous EPO is an effective and well-tolerated treatment for
patients with low- and intermediate-risk MDS. Several issues such
as optimal ATRA and EPO schedules, role of maintenance treatment, and mechanisms of response remain to be determined and
deserve further investigation.
Acknowledgment
We thank Janssen-Cilag and Roche Companies (Milan, Italy) for
supplying, in part, the recombinant human erythropoietin and the
all-trans retinoic acid used in this study.
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2002 99: 1578-1584
doi:10.1182/blood.V99.5.1578
Sustained response to recombinant human erythropoietin and intermittent alltrans retinoic acid in patients with myelodysplastic syndromes
Roberto Stasi, Maurizio Brunetti, Edmondo Terzoli and Sergio Amadori
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