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Erythropoietin Addition to Granulocyte Colony-Stimulating Factor Abrogates Life-Threatening Neutropenia and Increases Peripheral-Blood Progenitor-Cell Mobilization After Epirubicin, Paclitaxel, and Cisplatin Combination Chemotherapy: Results of a Randomized Comparison

From the Cattedra di Ematologia, Istituto di Ostetricia e Ginecologia, Università Cattolica del Sacro Cuore, Rome, Italy.

Address reprint requests to Luca Pierelli, MD, Servizio di Ematologia ed Emotrasfusione, Università Cattolica del Sacro Cuore, Largo A. Gemelli 8, 00168 Roma, Italy; email lpierelli{at}nexus.it

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE AND METHODS: The ability of granulocyte colony-stimulating factor (G-CSF) plus erythropoietin (EPO) treatment was compared in a randomized fashion with that of G-CSF treatment alone in promoting hematologic recovery and peripheral-blood progenitor-cell (PBPC) mobilization in previously untreated patients with advanced ovarian cancer who underwent their first course of epirubicin, paclitaxel, and cisplatin (ETP) chemotherapy during a phase II study of intensive outpatient ETP chemotherapy followed by high-dose carboplatin, etoposide, and melphalan (CEM) late intensification with PBPC support.

RESULTS: Comparative analysis of hematologic recovery of 50 randomized patients, after ETP chemotherapy, showed that life-threatening neutropenia occurred in 88% of the patients treated with G-CSF alone, whereas it occurred in only 4% of patients treated with G-CSF + EPO. Significantly different WBC and polymorphonuclear leukocyte (PMN) counts were observed in the two distinct arms on the day of WBC nadir (P < .0001 and P < .0001, respectively). Moreover, the addition of EPO to G-CSF increased PBPC mobilization and collection as compared with that in G-CSF–treated patients (P = .0009 and P = .0026, respectively), who required a significantly higher number of leukaphereses than G-CSF + EPO–treated patients (P = .0076) to obtain the planned minimum dose of PBPCs. Qualitative analysis by cloning assay of PBPCs collected in both arms revealed that G-CSF– and G-CSF + EPO–mobilized PBPCs have comparable in vitro functional properties.

CONCLUSION: This randomized comparison revealed that EPO significantly increases most of the hematologic effect produced by G-CSF administration after chemotherapy. This biologic property of EPO translated in vivo into a global improvement of patients' hematologic status.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
RECENTLY, THE INTRODUCTION of hematopoietic growth factors into clinical practice has allowed for drug dose intensification in patients with high-risk neoplasms, reducing chemotherapy-associated neutropenia and mobilizing into peripheral blood large quantities of hematopoietic precursors capable of rapidly restoring stable hematopoiesis after chemotherapy/chemoradiotherapy intensification.^1,2 Our group has optimized the procedure for peripheral-blood progenitor-cell (PBPC) mobilization and collection for autotransplantation into patients with high-risk cancer, using the mobilization capacity of disease-oriented intensive chemotherapy and the hematopoietic growth factor granulocyte colony-stimulating factor (G-CSF), which concomitantly reduces chemotherapy-induced neutropenia.³ PBPCs collected using these strategies have shown in vitro comparable or even better functional properties than bone marrow progenitor cells in terms of cloning ability and capacity to sustain long-term hematopoiesis (at the present time, this represents the best stem-cell assay in humans) and have produced accelerated and sustained recovery after high-dose chemotherapy/chemoradiotherapy.^4-6

Recent studies on the treatment of advanced ovarian cancer suggest that the response to chemotherapy can be improved in patients by combining doxorubicin and paclitaxel with cisplatin during intensive nonmyeloablative treatments.^7,8 In addition, recent phase II studies carried out in our institution suggest that a late intensification with a regimen containing high doses of carboplatin and etoposide with or without melphalan followed by PBPC transplantation confers to advanced ovarian cancer patients a survival advantage, as compared with historical controls.^6,9 In light of these results, a new phase II trial was undertaken, consisting of an outpatient induction program with epirubicin, paclitaxel, and cisplatin (ETP) combination chemotherapy followed by G-CSF administration and PBPC collection, with subsequent PBPC reinfusion after the previously described carboplatin, etoposide, and melphalan (CEM) high-dose regimen. However, owing to recent observations on the potentiation of G-CSF effects by erythropoietin (EPO) both in vitro and in vivo,¹⁰ we decided to compare, in a randomized fashion, the effects of administration of G-CSF or G-CSF + EPO on hematopoietic recovery and PBPC mobilization in ovarian cancer patients receiving their first course of ETP chemotherapy. In vitro functional properties of G-CSF– and G-CSF + EPO–mobilized PBPCs as well as their capacity to recover hematopoiesis after CEM chemotherapy were also compared.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patient Eligibility and Treatment Plan
Fifty patients with stage IIIB, IIIC, or IV ovarian carcinoma with a residual tumor less than 1 cm in size after cytoreductive surgery, ranging in age from 26 to 63 years (median, 51 years), were enrolled onto a phase II study, which includes the present randomized comparison of the hematologic effects of administration of G-CSF or G-CSF + EPO after the first course of ETP chemotherapy (phase A of the study; Fig 1). All patients were previously untreated with chemotherapy or radiotherapy. Eligibility criteria included a performance status of 0 to 2 (World Health Organization scale); adequate pulmonary, cardiac, hepatic, and renal function; absence of underlying infections; a polymorphonuclear leukocyte (PMN) count of more than 2 x 10⁹/L; and a platelet (PLT) count of more than 100 x 10⁹/L. The study was approved by the hospital human investigation review board, and written informed consent was obtained from all patients.

View larger version (29K): [in this window] [in a new window]   Fig 1. Study design of intensive ETP polychemotherapy followed by late intensification with CEM high-dose chemotherapy and PBPC transplantation. Phase A represents the randomized comparison of the effect of G-CSF to that produced by G-CSF + EPO administration on hematologic recovery and PBPC mobilization/collection in patients after their first course of ETP chemotherapy.

Patients were randomly assigned to the alternative growth factor treatments according to a table of random numbers, and they were stratified at randomization for age. Patient characteristics at the time of enrollment are listed in Table 1. The outpatient chemotherapy induction program consisted of three courses of epirubicin 110 mg/m², paclitaxel 175 mg/m² (given over a 3-hour infusion), and cisplatin 100 mg/m², administered the same day (day 1) and repeated every 3 weeks. After the first course of ETP chemotherapy, patients were randomized to receive recombinant human (rh) G-CSF (Neupogen; Dompè Biotec, Milan, Italy) 5 µg/kg/d subcutaneously from day 2 to day 13 or rhG-CSF 5 µg/kg/d and rhEPO (Eprex; Cilag, Milan, Italy) 150 IU/kg every other day from day 2 to day 13.

After the second and third course of ETP, rhG-CSF treatment was started the day after chemotherapy administration, given daily at a dose of 5 µg/kg/d subcutaneously, and discontinued on day 10, in the presence of a neutrophil count of more than 1,000/µL, in all patients; rhEPO was administered at a dose of 150 IU/kg every other day subcutaneously only when hematocrit (Ht) decreased below 30% and was continued until an Ht of 35% was achieved in all patients (phase B of the study; Fig 1). Oral iron therapy was given when an iron deficiency was documented or in the presence of iron functional deficiency (hypochromic RBCs > 10%).

As detailed in Fig 1, the phase II study included a late chemotherapy intensification with the CEM regimen and reinfusion of PBPCs collected during phase A (phase C), as previously described.⁶ The first 18 consecutive patients who entered phase C (nine G-CSF–treated patients and nine G-CSF + EPO–treated patients) received reinfusions of a fixed dose of 4 x 10⁶/kg CD34⁺ cells to permit an exact comparison of the in vivo functional properties of collected PBPCs. The subsequent 24 consecutive patients (12 G-CSF–treated patients and 12 G-CSF + EPO–treated patients) were given reinfusions of the entire PBPC dose collected after administration of G-CSF or G-CSF + EPO to evaluate the in vivo hematopoietic potential of the entire graft. The day after PBPC reinfusion, all patients were treated with G-CSF + EPO until the achievement of hematopoietic recovery (Fig 1), as previously described.⁶ During the period of myelosuppression, all patients were given transfusions of RBC concentrates when the Ht value was lower than 23% and single-donor PLT concentrates when the PLT count was lower than 10,000/µL or during bleeding episodes. After PBPC transplantation, all patients were discharged from the hospital when the PMN count was more than 500/µL for 3 consecutive days and the PLT count was more than 50,000/µL, in the absence of fever, documented infections, or relevant nonhematologic toxicities.

Circulating CD34⁺ PBPC Identification and Collection
Circulating CD34⁺ cells were detected in patients' blood from day 12 onward after ETP chemotherapy and in leukapheresis products using flow cytometric analysis, as previously described.¹¹ Briefly, 100 µL of whole blood was mixed with 10 µL of a phycoerythrin-conjugated anti-CD34 monoclonal antibody (CD34-PE; Becton-Dickinson, San Jose, CA) and incubated for 30 minutes in the dark at room temperature. Then, erythrocytes were lysed using an NH₄Cl buffer (NH₄Cl 8.29 g/L, KHCO₃ 1 g/L, 4x Na-EDTA 0.037 g/L, pH 7.4) for 10 minutes at room temperature. After washings, the cell suspension was analyzed on a FACScan flow cytometer (Becton-Dickinson). Forward and side scatter signals were collected in linear mode and helped to exclude unwanted events (ie, debris and cell clumps) from cell counts. Fluorescence signals were collected in log mode. Dead cells were excluded from the analysis on the basis of propidium iodide uptake and light scatter signals. Nonspecific binding was checked using nonspecific fluorochrome-conjugated mouse immunoglobulins (Becton-Dickinson). PBPCs were collected by leukapheresis using the Fresenius AS104 blood cell separator (Fresenius, St. Wendel, Germany), as previously described. Collections were started on day 12 after ETP chemotherapy and performed on consecutive days with the theoretical target of collecting at least 4 x 10⁶/kg CD34⁺ cells per patient. A blood volume of about 9 L was processed for single collection, and peripheral venipunctures were used as vascular access in all patients during the outpatient PBPC collection program.

Isolation of CD34⁺ PBPCs From Aliquots of Leukapheresis Products
CD34⁺ circulating progenitors were isolated from small aliquots of leukapheresis products using a CD34 isolation kit (Miltenyi Biotec, Bergish Gladbach, Germany), in accordance with the manufacturer's instructions. The nucleated cell suspension was layered onto a Ficoll-Paque gradient (1.077 g/mL; Pharmacia LKB, Uppsala, Sweden) and centrifuged at 400 x g for 30 minutes at 21°C. Low-density cells were collected and then washed twice with phosphate-buffered saline (PBS). The low-density cell concentration was adjusted to 10⁸ cells/mL with PBS, mixed with 100 µL of CD34 MultiSort MicroBeads (Miltenyi Biotec), and subsequently incubated at 6° to 12°C for 30 minutes. After incubation, cells were loaded onto a VS+ selection column (Miltenyi Biotec) using the VarioMACS magnetic instrument (Miltenyi Biotec), and purified CD34⁺ cells were eluted after removal of the negative fraction and discontinuation of the applied magnetic field. MicroBeads were subsequently removed by MACS MultiSort release reagent (Miltenyi Biotec) and MACS MultiSort stop reagent (Miltenyi Biotec). Finally, CD34⁺ cells were washed twice with PBS by centrifugation.

Cloning Assays of Isolated CD34⁺ Cells
Colony-forming cells (colony-forming unit granulocyte-macrophage [CFU-GM] and burst-forming unit–erythroid [BFU-E]) were evaluated in CD34⁺ cells by a modification of a previously described method.¹⁰ Briefly, 1,000 cells were seeded in Iscove's modified Dulbecco's medium (Gibco, Grand Island, NY) supplemented with 25% fetal calf serum (Gibco) and 0.3% soft agar, in the presence of 10 ng/mL stem-cell factor (Amgen, Thousand Oaks, CA), 20 ng/mL interleukin-3 (Genzyme, Cambridge, MA), 20 ng/mL granulocyte-macrophage-CSF (GM-CSF) (Schering-Plough, Milan, Italy), 20 ng/mL G-CSF (Sigma Chemical Co, Milan, Italy), 4 IU/mL EPO (R&D System, Oxon, United Kingdom), and 10 ng/mL Flt3 ligand (Genzyme) in 35-mm Petri dishes. Cultures were incubated at 37°C with 5% CO₂-95% air in a fully humidified atmosphere for 14 days. On day 14, aggregates with more than 40 cells were scored as colonies by examination using an inverted microscope.

Statistical Analysis
Comparisons between groups of patients were performed by Mann-Whitney U nonparametric tests. A P value of less than .05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Effect of G-CSF and G-CSF + EPO on Hematologic Recovery and PBPC Mobilization
At the present time, 50 patients have completed phase A of our study, which includes the randomized comparison of the effects of administration of G-CSF and G-CSF + EPO after the first course of ETP chemotherapy on hematologic toxicity and PBPC mobilization (Fig 1). Table 1 shows that all patients enrolled in the G-CSF and G-CSF + EPO arms were comparable in terms of age, previous treatments, and hematologic characteristics. All patients had undergone only cytoreductive surgery as antineoplastic treatment and had no previous chemoradiotherapy.

Table 2 lists the blood cell counts of patients in the two study arms on the day of WBC nadir, which occurred in both arms after an average time interval of 7 days after ETP chemotherapy. With regard to hematologic toxicity, the addition of EPO to G-CSF abrogated life-threatening neutropenia in 96% of the patients, whereas neutropenia occurred in 88% of the patients treated with G-CSF alone after ETP chemotherapy. Significantly higher WBC and PMN counts were observed in G-CSF + EPO–treated patients, and an average difference of about 1 log was observed between the PMN counts of G-CSF– and G-CSF + EPO–treated patients on the day of WBC nadir. Thus, G-CSF–treated patients had median WBC and PMN counts of 1,150/µL and 230/µL, respectively, whereas G-CSF + EPO–treated patients had counts of 3,000/µL (P < .0001) and 1,950/µL (P < .0001), respectively, on the day of WBC nadir. Consequently, life-threatening neutropenia occurred in 22 of 25 G-CSF–treated patients and lasted a median period of 2 days in these patients, whereas only one of 25 patients treated with G-CSF + EPO experienced 2 days of life-threatening neutropenia.

A slightly, although significantly, higher Ht average value was observed in EPO + G-CSF–treated patients as compared with G-CSF–treated patients on the day of WBC nadir. At that time, a median Ht of 33% was observed in G-CSF + EPO–treated patients as compared with the median value of 29.4 (P = .0052) observed in G-CSF–treated patients.

During the period of myelosuppression, none of the patients in either study arm required systemic antibiotic treatment, although nine patients in the G-CSF arm experienced a fever episode with a temperature above 38°C. In both arms, none of the patients experienced a clinically or microbiologically documented infection during myelosuppression.

As planned, on day 12 after ETP chemotherapy, all patients were evaluated for PBPC mobilization by analyzing circulating CD34⁺ cells, and those who showed a CD34⁺ cell count of more than 20/µL underwent leukapheresis for the collection of a minimum dose of 4 x 10⁶/kg CD34⁺ cells. On day 12, G-CSF + EPO–treated patients had twice as many WBCs and PMNs as G-CSF–treated patients (Table 3). WBC and PMN median counts were 10,000/µL and 8,000/µL, respectively, in G-CSF–treated patients, as compared with the values of 22,000/µL (P = .0035) and 19,000/µL (P = .0051), respectively, observed in the G-CSF + EPO arm. Furthermore, the CD34⁺ cell count was significantly increased in G-CSF + EPO–treated patients: the median count of CD34⁺ cells was 75/µL in the G-CSF arm, whereas G-CSF + EPO–treated patients had a median CD34⁺ cell count of 118/µL (P = .0009). No significant differences were observed in PLT and Ht counts on day 12. All patients of both arms achieved a CD34⁺ cell count of more than 20/µL on day 12, and all patients underwent leukapheresis. A significantly lower number of leukaphereses made possible the collection of a significantly higher number of CD34⁺ cells per kilogram in G-CSF + EPO–treated patients, as compared with patients in the G-CSF–treated arm (Table 4). An average of 1.04 leukaphereses enabled the collection of a median of 8.7 x 10⁶/kg CD34⁺ cells in G-CSF + EPO–treated patients, whereas 1.48 leukaphereses (P = .0076) harvested a median of 5.5 x 10⁶/kg CD34⁺ cells (P = .0026) in G-CSF–treated patients. A double safe minimum dose of 4 x 10⁶/kg CD34⁺ cells was obtained in 72% of G-CSF + EPO–treated patients, as compared with 20% in the G-CSF–treated arm.

None of the patients in either arm experienced a toxic effect attributable to growth factor treatment, and in none of them was discontinuation of treatment required. Relevant nonhematologic toxicities consisted of grade 2 nausea/vomiting, grade 3 alopecia, and grade 2 mucositis in most patients, which were comparable in both arms.

At the present time, 46 patients have completed phase B of the study, 24 in the G-CSF + EPO arm and 22 in the G-CSF arm. No significant differences were found during this phase in terms of days of G-CSF administration, EPO administration, or antibiotic treatment. One patient in the G-CSF + EPO arm and two patients in the G-CSF arm required one transfusion of RBC concentrates during phase B, and none of the patients in either arm required PLT transfusions.

In Vitro Functional Assay of Collected CD34⁺ PBPCs and Their Ability to Reconstitute Hematopoiesis After High-Dose Treatments
Cloning assays in the presence of hematopoietic cytokines were performed to evaluate the cloning ability of collected PBPCs in both arms. The frequency of both CFU-GM and BFU-E was statistically comparable in both G-CSF– and G-CSF + EPO–mobilized CD34⁺ cells (Table 5). Immunomagnetically isolated CD34⁺ cells from leukapheresis products collected in G-CSF + EPO–treated patients generated a median of 60/20 CFU-GM/BFU-E per 1,000 seeded cells; these figures were statistically comparable to those observed in G-CSF–treated patients, in whom we found a cloning ability of 55/25 CFU-GM/BFU-E per 1,000 CD34⁺ (P = .7075 and P = .7950, respectively).

At present, 42 patients have completed phase C of our phase II study, and 21 patients from each arm of phase A have received transplants. The first 18 consecutive patients, who were randomized in phase A, treated in phase B, and subsequently given transplants in phase C, were given reinfusions, after CEM high-dose chemotherapy, of a fixed dose of 4 x 10⁶/kg CD34⁺ cells (a fixed dose of 4 x 10⁶/kg CD34⁺ cells was frozen separately, and the remaining cells were frozen in additional bags as a reserve or as an additional source of PBPCs for further hematopoietic rescue) to avoid interference of progenitor dose in the comparison of hematopoietic recovery and clinical management after transplantation (Table 6). The results obtained in terms of hematopoietic recovery in patients given transplants of G-CSF– or G-CSF + EPO–mobilized PBPCs indicate that an identical number of these progenitors mediate identical hematopoietic recovery after CEM high-dose chemotherapy, requiring a statistically comparable number of days to achieve 500 PMNs/µL, 1,000 WBCs/µL, 50,000 PLTs/µL, and 20,000 reticulocytes/µL (Table 6). In summary, the data revealed that patients in both arms required a median of 10 days to achieve a PMN count of more than 500/µL and 11 days for untransfused PLTs to exceed 50,000/µL.

Transfusion requirements and hospital stay were statistically comparable in both arms. The subsequent consecutive series of 24 patients who completed the study were given reinfusions, after CEM, of the entire dose of CD34⁺ cells per kilogram collected during phase A (Table 7). Thus, these patients received reinfusions of significantly different CD34⁺ cell doses; 8.7 was the median CD34⁺ cell dose infused per kilogram in the G-CSF + EPO arm, and 5.5 was the median dose (P = .0015) in G-CSF–treated patients. These patients experienced a comparable WBC, PMN, and reticulocyte recovery, but G-CSF + EPO–treated patients had a significantly faster PLT recovery than G-CSF patients, with the median number of days for untransfused PLTs to reach more than 50,000/µL 10 and 11 days (P = .0011) for G-CSF + EPO and G-CSF patients, respectively (Table 7). This phenomenon translated into a reduction of PLT transfusion requirements in the G-CSF + EPO patients, who received transfusions of an average of 0.50 single donor units, as compared with the average of 1.4 units (P = .0073) infused in G-CSF patients. Finally, no significant differences between the two arms were found in RBC transfusions and length of hospital stay.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
The usefulness of G-CSF administration in patients with high-risk cancer is now well established.^1,2 The reduction of chemotherapy-induced neutropenia and the enhancement of chemotherapy-elicited PBPC mobilization represent the clinical effects through which this cytokine plays a substantial role in the treatment of cancer patients. EPO exerts its clinical effect by increasing erythroid production in anemic patients in whom the underlying cause of RBC reduction is the inappropriate production of endogenous EPO.^12-16 Cisplatin- or carboplatin-based chemotherapy regimens represent a potential cause of reduced EPO production by renal cells, and exogenous EPO is currently administered with clinical benefit in these patients. Several investigational reports indicate that EPO has additional biologic properties that result in a potentiation of human hematopoiesis by increasing the proliferating state, the frequency, and the recirculation of progenitors of multiple lineages.^17-19 Moreover, EPO, when associated with G-CSF or GM-CSF, has been shown to be capable of potentiating multilineage recovery in aplastic anemia, in myelodisplastic syndromes, and after hematopoietic progenitor transplantation.^20-25

With this background, we planned a randomized comparison to evaluate the effect of the addition of EPO to G-CSF treatment in patients who underwent intensive cisplatin-based chemotherapy for advanced ovarian cancer (epirubicin 110 mg/m², paclitaxel 175 mg/m² for 3 hours, and cisplatin 100 mg/m²). The comparison was carried out after the first course of chemotherapy, when all patients were chemotherapy-naive, and PBPC monitoring and collection were performed. In patients in the G-CSF + EPO arm, EPO was administered subcutaneously every other day at a dose of 150 IU/kg for 12 days, irrespective of the Ht level, to verify the effect of pharmacologic doses of EPO in association with G-CSF in promoting hematologic recovery and PBPC mobilization for subsequent collection. The results observed indicate that EPO significantly enhanced all of the previously described hematologic effects of G-CSF after chemotherapy.

The potentiation of G-CSF effects also includes a significant increase in PBPC mobilization and collection. This property of EPO was first observed in our institution, was reported in a preliminary study,¹⁰ and subsequently was confirmed by Olivieri et al²⁶ in a nonrandomized setting. The present study represents the first randomized comparison in which this potentiating effect of EPO has been demonstrated. EPO significantly increased both PBPC mobilization and collection as compared with G-CSF alone, and this event translates into the possibility of collecting two doses of 4 x 10⁶/kg CD34⁺ cells in 72% of G-CSF + EPO–treated patients with a single collection procedure. Consequently, EPO administration significantly reduced the number of leukaphereses required to obtain the theoretical target of 4 x 10⁶/kg CD34⁺ cells. The qualitative analysis of mobilized PBPCs by cloning assay and transplantation procedures revealed that G-CSF + EPO–mobilized cells have identical biologic properties as compared with those mobilized by G-CSF alone, mediating an identical hematopoietic recovery with comparable clinical management when reinfused in equivalent amounts into patients treated with high-dose chemotherapy. Importantly, when patients were given reinfusions of the entire dose of PBPCs collected after G-CSF or G-CSF + EPO (as previously detailed, G-CSF + EPO patients had significantly higher PBPC doses), a significantly faster PLT recovery and a significantly lower PLT transfusional requirement were observed in G-CSF + EPO–treated patients, as compared with the G-CSF arm. These results confirm the previously described effects of CD34⁺ cell dose on the rate of PLT recovery after PBPC²⁷ and underline one of the potential advantages in having an increased dose of CD34⁺ cells collected (an additional advantage could be the availability of two well-dosed grafts for multiple intensification).

Collectively, in the 12 patients who were randomized to the G-CSF + EPO arm and who were given reinfusions of the entire PBPC collected dose, we observed a cost reduction of US$1,200 per patient (this estimate of costs included the extra cost of EPO administration) because of the reduced number of leukaphereses/PLT transfusions as compared with those required by G-CSF–treated patients. A novel effect produced by the addition of EPO to G-CSF treatment observed in the present comparison is represented by the abrogation of life-threatening neutropenia in most of the patients of the G-CSF + EPO arm. In fact, 88% of the G-CSF–treated patients had life-threatening neutropenia, with an average length of 2 days after ETP chemotherapy, whereas most of the G-CSF + EPO–treated patients (96%) experienced just a slight decrease in WBC and PMN counts. None of the enrolled patients experienced clinically or microbiologically documented infections during G-CSF or G-CSF + EPO treatments, but in the G-CSF arm, nine patients experienced fever episodes with temperatures above 38°C, which, however, did not require antibiotic therapy. Collectively, the addition of EPO to G-CSF produced a global potentiation of hematopoietic functions that was revealed by the increased amount of multilineage circulating progenitors and by relevant effects on granulocytopoiesis, which is a hematopoietic differentiating pathway in which EPO is not directly involved.

The mechanism through which EPO potentiates the G-CSF effect on both multilineage progenitor mobilization and granulocytopoiesis is unknown. The distribution of detectable levels of EPO receptor is apparently confined to erythroid progenitors, and undifferentiated/unstimulated CD34⁺ progenitors have low levels of EPO receptor expression.²⁸ In harmony with this biologic evidence, EPO administration in the absence of pharmacologic doses of other hematopoietic cytokines does not produce any relevant effect on differentiating pathways other than RBC production. A previous report by Pene et al²⁹ indicates that the granulopoietic effect of GM-CSF can be significantly enhanced by EPO administration during the hematopoietic recovery produced by autologous bone marrow transplantation in patients treated with high-dose chemotherapy. Other clinical reports showed that EPO addition to G-CSF or GM-CSF can improve impaired granulopoiesis, erythropoiesis, and megakaryocytopoiesis in some patients.^20-25 Therefore, previous reports and our present randomized comparison indicate that pharmacologic doses of EPO exert a biologic effect on G-CSF/GM-CSF–exposed multipotent progenitors. A possible explanation of GM-CSF/G-CSF potentiation by EPO might lie in the ability of G-CSF or GM-CSF to recruit hematopoietic progenitors into the proliferative compartment of the cell cycle,^30,31 where low levels of EPO receptor expression might be induced and EPO might mediate a survival advantage³² and/or a mitogenic signaling³³ to proliferating progenitors.

In conclusion, the present study indicates that EPO could be administered to potentiate G-CSF effects after platinum-based intensive polychemotherapy. Nevertheless, the small number of patients enrolled onto the present study suggests caution in the combined use of EPO and G-CSF as systematic treatment after platinum-based chemotherapy until larger studies confirm these data in patients who undergo multiple courses of chemotherapy.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
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Submitted July 6, 1998; accepted November 24, 1998.

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