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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Patients and methods: Forty-eight patients with low- or intermediate-risk MDS were enrolled in a 12-week study. rhEPO alpha (rhEPO) 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. 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 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Treatment plan
Therapy consisted of a 12-week schedule of rhEPO (epoetin alfa, Eprex®; Janssen-Cilag, Milan, Italy) administered subcutaneously once a week. The rhEPO
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. [12]. 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 12 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 [13].
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 FicollHypaque 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 MannWhitney 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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 injections, although they did not interrupt rhEPO
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).
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 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
. For instance, in a double-blind, placebo-controlled study, 36.8% of patients with low-risk MDS who were randomized to receive rhEPO
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
was given using a modified schedule of 150300 U/kg on alternate days [19
]. In addition, Terpos et al. [20
] have reported recently that prolonged administration of rhEPO
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
therapies. In fact, at least 12 weeks had passed between the last administration of three-times-weekly rhEPO
and the first administration of once-weekly rhEPO
.
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 [1].
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 [21]. 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 [22, 23
]. 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 [23
]. 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
therapy involve inhibition of apoptosis of the dysplastic clone [24
].
In conclusion, a single, weekly, subcutaneous administration of rhEPO 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
require further investigation, and findings could lead to better understanding of the pathophysiology of MDS.
![]() |
Acknowledgements |
---|
Received for publication February 2, 2004. Revision received May 31, 2004. Accepted for publication June 17, 2004.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Rizzo JD, Lichtin AE, Woolf SH et al. Use of epoetin in patients with cancer: evidence-based clinical practice guidelines of the American Society of Clinical Oncology and the American Society of Hematology. Blood 2002; 100: 23032320.
3. Alessandrino EP, Amadori S, Barosi G et al. Evidence- and consensus-based practice guidelines for the therapy of primary myelodysplastic syndromes. A statement from the Italian Society of Hematology. Haematologica 2002; 87: 12861306.[ISI][Medline]
4. Bowen D, Culligan D, Jowitt S et al. Guidelines for the diagnosis and therapy of adult myelodysplastic syndromes. Br J Haematol 2003; 120: 187200.[CrossRef][ISI][Medline]
5. Cheung W, Minton N, Gunawardena K. Pharmacokinetics and pharmacodynamics of epoetin alfa once weekly and three times weekly. Eur J Clin Pharmacol 2001; 57: 411418.[CrossRef][ISI][Medline]
6. Gabrilove JL, Cleeland CS, Livingston RB et al. Clinical evaluation of once-weekly dosing of epoetin alfa in chemotherapy patients: improvements in hemoglobin and quality of life are similar to three-times-weekly dosing. J Clin Oncol 2001; 19: 28752882.
7. Cazzola M, Beguin Y, Kloczko J et al. Once-weekly epoetin beta is highly effective in treating anaemic patients with lymphoproliferative malignancy and defective endogenous erythropoietin production. Br J Haematol 2003; 122: 386393.[CrossRef][ISI][Medline]
8. Musto P, Falcone A, Sanpaolo G et al. Efficacy of a single, weekly dose of recombinant erythropoietin in myelodysplastic syndromes. Br J Haematol 2003; 122: 269271.[CrossRef][ISI][Medline]
9. Aloe-Spiriti MA. Effects of 40.000 IU bi-weekly induction dose of Epoetin alpha followed by 40.000 IU once weekly in low risk myelodysplastic syndrome patients. Blood 2002; 100: 790a.
10. Greenberg P, Cox C, LeBeau MM et al. International scoring system for evaluating prognosis in myelodysplastic syndromes. Blood 1997; 89: 20792088.
11. Miller AB, Hoogstraten B, Staquet M, Winkler A. Reporting results of cancer treatment. Cancer 1981; 47: 207214.[ISI][Medline]
12. Cheson BD, Bennett JM, Kantarjian H et al. Report of an international working group to standardize response criteria for myelodysplastic syndromes. Blood 2000; 96: 36713674.
13. Stasi R, Brunetti M, Bussa S et al. Response to recombinant human erythropoietin in patients with myelodysplastic syndromes. Clin Cancer Res 1997; 3: 733739.[Abstract]
14. Stein RS, Abels RI, Krantz SB. Pharmacologic doses of recombinant human erythropoietin in the treatment of myelodysplastic syndromes. Blood 1991; 78: 16581663.[Abstract]
15. Goy A, Belanger C, Casadevall N et al. High doses of intravenous recombinant erythropoietin for the treatment of anaemia in myelodysplastic syndrome. Br J Haematol 1993; 84: 232237.[ISI][Medline]
16. Bowen D, Ehmer B, Neubert P et al. The clearance of a single i.v. bolus of recombinant human erythropoietin from the serum of patients with myelodysplastic syndromes and its effects on erythropoiesis. Exp Hematol 1991; 19: 613616.[ISI][Medline]
17. Aloe Spiriti MA, Petti MC, Latagliata R et al. Recombinant human erythropoietin in the treatment of myelodysplastic syndromes. An interim report. Haematologica 1993; 78: 123126.[ISI][Medline]
18. Stone RM, Bernstein SH, Demetri G et al. Therapy with recombinant human erythropoietin in patients with myelodysplastic syndromes. Leuk Res 1994; 18: 769776.[CrossRef][ISI][Medline]
19. A randomized double-blind placebo-controlled study with subcutaneous recombinant human erythropoietin in patients with low-risk myelodysplastic syndromes. Italian Cooperative Study Group for rHuEpo in Myelodysplastic Syndromes. Br J Haematol 1998; 103: 10701074.[CrossRef][ISI][Medline]
20. Terpos E, Mougiou A, Kouraklis A et al. Prolonged administration of erythropoietin increases erythroid response rate in myelodysplastic syndromes: a phase II trial in 281 patients. Br J Haematol 2002; 118: 174180.[CrossRef][ISI][Medline]
21. Merchav S, Nielsen OJ, Rosenbaum H et al. In vitro studies of erythropoietin-dependent regulation of erythropoiesis in myelodysplastic syndromes. Leukemia 1990; 4: 771774.[ISI][Medline]
22. Parker JE, Mufti GJ, Rasool F et al. The role of apoptosis, proliferation, and the Bcl-2-related proteins in the myelodysplastic syndromes and acute myeloid leukemia secondary to MDS. Blood 2000; 96: 39323938.
23. Stasi R, Brunetti M, Terzoli E, Amadori S. Sustained response to recombinant human erythropoietin and intermittent all-trans retinoic acid in patients with myelodysplastic syndromes. Blood 2002; 99: 15781584.
24. Greenberg PL. Apoptosis and its role in the myelodysplastic syndromes: implications for disease natural history and treatment. Leuk Res 1998; 22: 11231136.[CrossRef][ISI][Medline]