Once-weekly dosing of recombinant human erythropoietin alpha in patients with myelodysplastic syndromes unresponsive to conventional dosing

R. Stasi1,*, M. Brunetti1, E. Terzoli2, E. Abruzzese3 and S. Amadori3

1 Department of Medical Sciences, Regina Apostolorum Hospital, Albano Laziale; 2 Department of Medical Oncology, I.F.O., Rome; 3 Chair of Hematology, University of Rome ‘Tor Vergata’, Rome, Italy

* Correspondence to: Dr R. Stasi, Department of Medical Sciences, Regina Apostolorum Hospital, Via S. Francesco 50, 00041 Albano Laziale, Italy. Tel: +39-06-932989; Fax: +39-06-233231809; Email: roberto.stasi{at}uniroma2.it


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background: Once-weekly dosing of recombinant human erythropoietin (rhEPO) in patients with myelodysplastic syndromes (MDS) has not been investigated thoroughly. We performed a clinical trial to evaluate the effects of this new dosing regimen in patients with MDS who were unresponsive to the conventional three-times-weekly schedule.

Patients and methods: Forty-eight patients with low- or intermediate-risk MDS were enrolled in a 12-week study. rhEPO alpha (rhEPO{alpha}) was administered once-weekly by subcutaneous injection with a starting dose of 40 000 U fixed dose. The drug dosage was increased to 60 000 U fixed dose if after 6 weeks there was no or suboptimal erythroid response.

Results: Clinically significant responses were seen in 13 (27%) patients, with 11 improving their response after dose escalation of rhEPO{alpha}. Only one patient (case 23) maintains a response after a follow-up period of 14 months. All other patients had responses lasting between 10 and 43 weeks, with a median time to relapse of 20 weeks. Treatment was well tolerated, with no relevant adverse events. Response to therapy was associated with significantly higher concentrations of circulating erythroid blast-forming units and a decrease of the bone marrow fraction of apoptic CD34+ cells.

Conclusions: Once-weekly rhEPO{alpha} therapy results in an improvement of erythropoiesis in a subset of MDS patients who are unresponsive to conventional dosing, and may act by inhibiting apoptosis of erythroid precursors. These results warrant further investigation of this dosing regimen either alone or in combination with other agents.

Key words: apoptosis, erythropoietin, myelodysplastic syndrome, schedule


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Results of several trials have demonstrated clearly that recombinant human erythropoietin (rhEPO) can increase hemoglobin concentration and reduce red blood cell transfusion requirements in selected patients with low-risk myelodysplastic syndromes (MDS), particularly those with a diagnosis of refractory anemia (RA) and a basal serum EPO level of <200 U/l [1Go]. Although rhEPO has been used in a variety of doses and schedules, in most recent series the drug was given subcutaneously daily or three-times a week with a starting dose of 150 U/kg, or 10 000 U fixed dose. The dose was usually doubled if no response was observed after the first 6 weeks of treatment. This ‘conventional’ schedule of rhEPO, adopted by current clinical practice guidelines [2Go–4Go], has been challenged recently. Pharmacodynamic investigations performed in healthy subjects [5Go], as well as clinical trials in both solid and hematological tumors [6Go, 7Go], suggest comparable efficacy between once-weekly and three-times-weekly dosing. The effects of once-weekly rhEPO therapy are being explored in MDS. Recently, once-weekly rhEPO alpha (rhEPO{alpha}) was used in patients with previously untreated MDS either as initial therapy [8Go] or as maintenance treatment of initial responders [9Go], suggesting that it may be at least as active as rhEPO{alpha} given more frequently. However, whether high peak concentrations of rhEPO{alpha} maintained for a short time are more effective than low concentrations maintained for a long period remains undetermined, and clinical data about the efficacy of a schedule switch are lacking. On these grounds, we designed this study to test the effects of once-weekly rhEPO{alpha} doses in MDS patients who were refractory to rhEPO{alpha} given with the conventional schedule.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
Forty-eight patients with low- and intermediate-risk MDS according to the International Prognostic Scoring System [10Go] were included in this study after they had signed an institutional review board-approved informed consent. Patients' clinical and laboratory characteristics at study entry are reported in Table 1. All patients had not responded to a prior treatment with three-times-weekly rhEPO{alpha} given subcutaneously at 10 000 U fixed dose for 6 weeks, and 20 000 U fixed dose given for a further 6 weeks. At least 12 weeks had passed between the last administration of three-times-weekly rhEPO{alpha} and the first administration of once-weekly rhEPO{alpha}. Apart from rhEPO{alpha}, patients had received no other pharmacological treatment for MDS.


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Table 1. Summary of patients' pre-treatment characteristics

 
Eligibility criteria were as follows: primary MDS with <10% blasts on bone marrow examination; Eastern Cooperative Oncology Group performance status of 2 [11Go]; hemoglobin levels <10 g/dl; normal renal and hepatic function; and normal iron, vitamin B12 and folate levels. Exclusion criteria included 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.

Treatment plan
Therapy consisted of a 12-week schedule of rhEPO{alpha} (epoetin alfa, Eprex®; Janssen-Cilag, Milan, Italy) administered subcutaneously once a week. The rhEPO{alpha} dose was initiated at 40 000 U fixed dose and was increased to 60 000 U fixed dose if after 6 weeks there was no or suboptimal erythroid response. Further treatment was given to patients with a continued response.

Response criteria
Responses were categorized according to the criteria developed by Cheson et al. [12Go]. In particular, a major response (MaR) for the erythroid lineage was considered to be a rise in untransfused hemoglobin concentrations of at least 2 g/dl or a 100% decrease in red blood cell (RBC) transfusion requirements during the treatment period. A minor response (MiR) was defined as an increase in untransfused hemoglobin values of 1–2 g/dl or a ≥50% decrease in RBC transfusion requirements. No response was defined as a response less than a MiR.

Study parameters and monitoring of patients
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, USA). Bone marrow aspirates and biopsy specimens were taken at enrollment and at the end of the study (aspirates), or when clinically required. Karyotyping was carried out with standard techniques at study entry and, in responders, at the end of treatment. Erythroid progenitor cell assay was performed at baseline and at 12 weeks. Erythroid blast-forming units (BFU-E) were assayed in viscous medium, as previously described [13Go].

Measurement of apoptosis
Apoptosis was measured by flow cytometry with a FACScan instrument (Becton Dickinson, Mountain View, CA, USA). Mononuclear cell fractions of bone marrow samples were separated after Ficoll–Hypaque gradient centrifugation and washed twice with phosphate-buffered saline. Cells (1 x 106) were then incubated with phycoerythrin-conjugated anti-CD34 mAb (anti-HPCA-2, IgG1; Becton Dickinson) for 10 min at room temperature in the dark and were washed twice with phosphate-buffered saline. Pelleted cells were resuspended in 100 µl binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2; Bender Medsystems, Boehringer Ingelheim, Ridgefield, CT, USA), and were incubated with 2 ml fluorescein isothiocyanate-conjugated annexin V (Bender Medsystems; Boehringer Ingelheim) for 10 min at room temperature in the dark. Afterwards, cells were resuspended in 400 µl 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
The Mann–Whitney U-test was used to compare continuous variables between responders and non-responders. The Wilcoxon matched-pairs test was used to compare repeated measurements in the same patients. Fisher's exact test was used to evaluate the relationship between two dichotomous variables. Correlations of variables with other variables were calculated by Spearman rank correlation coefficient. P <0.05 was designated as statistically significant; all P values were two-tailed.


    Results
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 Materials and methods
 Results
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Response to treatment
All patients completed the 12-week study and were evaluated for toxicity and response. During the study period no patient suffered from infections, underwent general anesthesia or experienced other conditions that might affect the response to rhEPO treatment. Thirteen patients achieved a response after week 12 of treatment, for an overall response rate of 27% (95% confidence interval 0.14–0.40). Changes in blood cell counts, transfusion requirements and laboratory parameters before and after treatment in responders are reported in Table 2.


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Table 2. Changes in hematological and clinical parameters following once-weekly rhEPO{alpha} therapy in responders

 
The four patients who attained an MaR (patients 16, 23, 37 and 43) exhibited a suboptimal rise of hemoglobin levels at the dose of 40 000 U and required a dose escalation. Two of the nine patients with an MiR (patients 26 and 40) had signs of erythroid improvement after the first 6 weeks of treatment, but did not benefit from the higher rhEPO{alpha} dose; the other seven patients showed a response only after being challenged with rhEPO{alpha} at 60 000 U. Five patients (4, 12, 19, 40 and 47) exhibited a response that was based solely on a decrease in transfusion requirements. The hemoglobin levels at which their transfusions were ordered prior to and after treatment was initiated were, respectively: patient 4, 7.9 and 8.4 g/dl; patient 12, 6.8 and 7.3 g/dl; patient 19, 7.0 and 7.7 g/dl; patient 40, 7.2 and 7.6 g/dl; and patient 47, 7.7 and 8.0 g/dl. No significant changes in neutrophil and platelet counts were observed. In responders, the increase in hemoglobin concentration was associated with a significant increase in reticulocyte counts. Mean (±SD) reticulocyte count was 16 024 ± 6085/µl before treatment versus 27 789 ± 6294/µl at week 12 of treatment (P=0.0014).

Thus far, only one patient (case 23) maintains a response after a follow-up of 14 months. All other patients relapsed between 10 and 43 weeks from the achievement of response, with a median time to relapse of 20 weeks.

Safety
The treatment was well tolerated, with no serious adverse events. Only one patient (case 23) had a mild increase in arterial blood pressure after 6 weeks of treatment that was easily controlled by medical therapy. Five patients complained of pain with or without erythema at the site of rhEPO{alpha} injections, although they did not interrupt rhEPO{alpha} administration.

Laboratory studies
As shown in Figure 1, the number of circulating BFU-E in responders during week 12 of treatment consistently increased compared with baseline (P=0.0014). Analysis of karyotype at the end of the study (available in 10 patients) did not show significant changes. Pretreatment determination of the degree of apoptosis in hematopoietic progenitors by means of the annexin V method did not show significant differences between the French-American-British/World Health Organization (FAB/WHO) subgroups. In RA and RA with ringed sideroblasts (RARS), the median CD34+ cell apoptosis was 57.5% (range 37.8% to 83.5%), whereas in RA with excess blasts (RAEB) it was 48.1% (range 33.7% to 56.2%) (P not significant). The control group had a median CD34+ cell apoptosis of 14.8% (range 5.5% to 27.9%), which was significantly different from that of MDS patients (P < 0.001). At week 12 of treatment, CD34+ cell apoptosis was significantly decreased in responders (median 38.8%; range 21.7% to 51.2%) compared with non-responders (median 55.4%; range 40.5% to 77.2%) (P < 0.001). Figure 2 reports the double-color analyses with antibodies against CD34 (phycoerythrin) and against annexin V (fluorescein isothiocyanate) in patient 35. Double-positive cells (upper right quadrant) dropped from 39.5% before treatment (Figure 2A) to 21.7% at week 12 of treatment (Figure 2B).



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Figure 1. Behavior of circulating erythroid blast-forming units (BFU-E) concentrations before and after 12 weeks of treatment in responders and non-responders.

 


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Figure 2. Flow cytometric evaluation of CD34+ cell apoptosis in a responder. See Results for details. (A) Dot plot histogram before treatment. (B) Dot plot histogram at week 12 of treatment.

 
Prognostic factors
Table 3 shows the clinical and laboratory characteristics of the 13 patients who had erythroid responses (MaR + MiR) to once-weekly rhEPO{alpha} treatment compared with those of the 35 patients who did not respond. No variable was significantly different between the two groups of patients in univariate analysis. Similarly, no combination of these characteristics demonstrated a significant association with response. It should be noted, however, that no patient with RARS and only one patient with RAEB had a response to treatment.


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Table 3. Comparison of baseline clinical and laboratory characteristics of MDS patients responding and not responding to once-weekly rhEPO{alpha} treatment

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The results in this large series of patients with low- and intermediate-risk MDS indicate that, along with the dose, the schedule of administration may be relevant in determining response to rhEPO{alpha}. Early trials in patients with MDS employing rhEPO{alpha} intravenously failed to demonstrate significant advantages of very high doses, up to 100 000 U, given twice weekly [14Go, 15Go]. However, pharmacokinetic studies have shown that the half-life of intravenously injected rhEPO{alpha} can be as short as 3 h in patients with MDS [16Go]. Thus, this route of administration is unlikely to result in high response rates. On the other hand, rhEPO{alpha} has been rarely used subcutaneously at doses in excess of 400 U/kg per single administration [17Go, 18Go].

When assessing our results, it is important to underline that because of the variability inherent with transfusion therapy, the rate of MiR may have been overestimated. In fact, a significant clinical benefit was restricted to the four complete responders who became transfusion-independent, only one of whom had an increase in hemoglobin >2 g/dl. Thus, using stringent criteria the clinical impact of once-weekly rhEPO{alpha} appears limited to no more than 10% of patients unresponsive to the conventional three-times-weekly schedule. Furthermore, our findings may not be applicable to all MDS patients. The results of other trials indicate that in some patients more frequent administrations could be necessary to elicit the biological response to rhEPO{alpha}. For instance, in a double-blind, placebo-controlled study, 36.8% of patients with low-risk MDS who were randomized to receive rhEPO{alpha} subcutaneously at the daily dose of 150 U/kg responded to treatment, but the response rate dropped to 16.2% in a following open phase, during which rhEPO{alpha} was given using a modified schedule of 150–300 U/kg on alternate days [19Go]. In addition, Terpos et al. [20Go] have reported recently that prolonged administration of rhEPO{alpha} subcutaneously at a dose of 150 U/kg three-times a week may significantly increase the erythroid response rate in low-risk MDS patients. In this regard, our study was designed to avoid possible delayed effects of previous rhEPO{alpha} therapies. In fact, at least 12 weeks had passed between the last administration of three-times-weekly rhEPO{alpha} and the first administration of once-weekly rhEPO{alpha}.

As expected, low-risk patients showed a tendency towards a higher response rate. This is in line with previously reported trials, and may be explained by the fact that these patients have a higher percentage of apoptotic cells in the bone marrow that may be sensitive to the antiapoptotic effects of erythropoietin. Endogenous erythropoietin levels were not predictive of response, but it should be noted that most of the patients in this series had baseline EPO levels in excess of 200 mIU/ml, which are known to predict an unfavorable response to conventional three-times-weekly treatment [1Go].

The heterogeneity of erythroid precursors of MDS to the anti-apoptotic effects of rhEPO might be the key to the different response patterns observed with different dosing regimens. Previous studies have shown that maximal growth of erythroid colonies in MDS require extremely variable rhEPO concentrations, five- to 20-fold higher than normal controls, with BFU-E sensitivity being a critical factor in determining response to rhEPO [21Go]. As a consequence, the optimal dosing regimen of rhEPO probably needs to be defined according to the individual patient.

The results of laboratory investigations performed in this study are in line with previous reports, and show that CD34+ cell apoptosis in MDS is significantly higher than in normal controls [22Go, 23Go]. Furthermore, we confirm previously reported data indicating that response to treatment is associated with higher concentrations of BFU-E in the peripheral blood and a remarkable decrease of the bone marrow fraction of apoptic CD34+ cells [23Go]. Whether these findings represent a stimulation of residual polyclonal hematopoiesis or an actual reduction in the degree of ineffective hematopoiesis remains undetermined, although it is likely that the mechanisms of the positive effects of rhEPO{alpha} therapy involve inhibition of apoptosis of the dysplastic clone [24Go].

In conclusion, a single, weekly, subcutaneous administration of rhEPO{alpha} at the dose of 40 000 or 60 000 U results in an improvement of erythropoiesis in a subset of MDS patients who are refractory to conventional dosing regimens. The mechanisms of response of once-weekly rhEPO{alpha} require further investigation, and findings could lead to better understanding of the pathophysiology of MDS.


    Acknowledgements
 
We thank Janssen-Cilag (Milan, Italy) for supplying the recombinant human erythropoietin used in this study.

Received for publication February 2, 2004. Revision received May 31, 2004. Accepted for publication June 17, 2004.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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