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Granulocyte-Macrophage Colony-Stimulating Factor Treatment Before Doxorubicin and Cyclophosphamide Chemotherapy Priming in Women With Early-Stage Breast Cancer

From MeritCare Roger Maris Cancer Center, Fargo, and Departments of Pediatrics and Internal Medicine, University of North Dakota School of Medicine, Grand Forks, ND; and the Department of Community Health Sciences, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada.

Address reprint requests to Diane E. Sjolander, CCRA, Roger Maris Cancer Center, 820 4th St North, Fargo, ND 58122; email dianesjolander{at}meritcare.coa

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: To determine if inhibition of stem-cell activity induced by granulocyte-macrophage colony-stimulating factor ([GM-CSF]; Sargramostim; Immunex Corporation, Seattle, WA) withdrawal or priming protects hematopoietic stem cells from the cytotoxic effects of adjuvant chemotherapy for early-stage breast cancer.

PATIENTS AND METHODS: Serial blood counts were performed in 20 women with early-stage breast cancer receiving four courses of cyclophosphamide and doxorubicin chemotherapy. By a double-blind, placebo-controlled, balanced randomization, subjects received GM-CSF priming on days 5 to 1 for courses 1 and 3 or courses 2 and 4.

RESULTS: Compared with before priming, after priming the times to neutrophil nadir (12.8 ± 2.5 days v 14.8 ± 1.5 days, respectively; P = .0001) and platelet nadir (mean ± SD, 10.1 ± 1.9 days v 11.1 ± 2.2 days, P < .05) were shorter, indicating a shift of cytotoxicity to later progenitors. The neutrophil nadir was similar with and without priming (mean ± SD, 490 ± 310/µL v 550 ± 350/µL, respectively; P = .2); however, on day 16 the mean neutrophil count was higher (mean ± SD, 1030 ± 580/µL v 690 ± 370/µL, P = .004), and the proportion of patients with a neutrophil count less than 500/µL was lower after priming than before (six of 35 or 17.1% v 12 of 34 or 35.3%, respectively; P = .04). The platelet nadir was higher (mean ± SD, 166,000 ± 51,000/µL after priming v 151,000 ± 45,000/µL before priming, P = .007), and the duration of thrombocytopenia, ie, a platelet count less than 150,000/µL, was shorter (1.5 ± 2.1 days v 2.8 ± 2.9 days, P = .0025) after priming. Episodes of fever and neutropenia were not observed.

CONCLUSIONS: GM-CSF priming from days 5 to 1 before doxorubicin and cyclophosphamide chemotherapy was associated with an earlier neutrophil and platelet nadir. On day 16, a higher mean neutrophil count and a lower proportion of patients with severe (< 500/µL) neutropenia were observed. Beneficial effects on the severity and duration of thrombocytopenia were also noted. These observations support the hypothesis that GM-CSF priming protects hematopoietic progenitors from the cytotoxic effects of chemotherapy.

	INTRODUCTION

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PANCYTOPENIA IS A MAJOR cause of morbidity and mortality after chemotherapy treatment.¹ Outside of the setting of hematopoietic stem-cell reinfusion, pancytopenia limits the intensity of chemotherapy treatment, by making it necessary to either decrease the dose delivered per cycle or prolong the interval between cycles. There is considerable evidence that the efficacy of therapy depends on dosage intensity.^2-4 Therefore, strategies aimed at abrogating hematopoietic toxicity may decrease morbidity and improve the efficacy of chemotherapy.

Granulocyte-macrophage (GM) colony-stimulating factor (CSF) has been shown to be effective in shortening the duration of neutropenia when given prophylactically and therapeutically after chemotherapy.^5-8 The efficacy of GM-CSF administered after chemotherapy is, however, limited by the fact that the cytokine targets an already damaged stem-cell pool.

GM-CSF withdrawal (ie, priming) has been shown to induce a state of hematopoietic stem-cell arrest.^9-11 Based on this observation, GM-CSF administered before chemotherapy may protect hematopoietic stem cells from chemotherapy-induced cytotoxicity and, thereby, decrease the severity and duration of pancytopenia after treatment.

This report describes the results of a randomized trial of GM-CSF versus placebo administered before chemotherapy for early-stage breast cancer. Beneficial effects of priming on neutrophil and platelet recovery were observed.

	PATIENTS AND METHODS

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Twenty women (mean age, 47.7 ± 11.9 years; range, 31 to 69 years), with histologically confirmed early-stage (I and II) breast cancer, scheduled to receive four cycles of cyclophosphamide and doxorubicin chemotherapy were studied. One subject withdrew from the study after the first cycle of therapy. Another subject was inassessable because of the open-labelled use of GM-CSF (cycle 1) and granulocyte (G)-CSF (cycle 2, 3, and 4) without fever after each course of chemotherapy by the treating physician.

Subjects were required to have an anticipated life expectancy of at least 8 months, an Eastern Cooperative Oncology Group functional score 2, a serum bilirubin 2.0 mg/dL, and a serum creatinine 2.0 mg/dL. Subjects with a past history of radiation therapy and/or chemotherapy, with an active hematologic disorder, and/or on bone marrow suppressive medications were excluded from the study.

Approval for the study was obtained from the Investigational Review Board of Meritcare Medical Center. All subjects gave written informed consent after the experimental nature of the investigations to be performed was discussed.

Subjects were randomly assigned by sealed envelope selection to receive GM-CSF or placebo at a dose of 250 µg/m² by subcutaneous injection on day 5, 4, 3, 2, and 1 before each of four courses of chemotherapy. Subjects were instructed to receive the injections at approximately the same time each day. Chemotherapy was initiated not less than 24 hours after the last injection.

GM-CSF was administered before courses 1 and 3 and placebo administered before courses 2 and 4 for half the subjects. For the remaining patients, placebo was administered before courses 1 and 3 and GM-CSF before courses 2 and 4 (ie, a balanced, crossover design). Yeast-derived recombinant GM-CSF (Sargramostim) and identically marked placebo were supplied by Immunex Corporation, Seattle, WA. Neither subjects nor investigators were aware of the randomization assignment. Subjects were asked to record local and systemic side effects experienced after each injection (GM-CSF or placebo). A complete blood count with differential was performed at the time of study entry (ie, baseline) and on days 0, 10, 12, 14, 16, and 21. If the absolute neutrophil count (ANC) on day 16 was less than 500/µL, a complete blood cell count was performed daily until this criterion was met.

Subjects received four courses of cyclophosphamide (600 mg/m²) and doxorubicin (60 mg/m²) at 21-day intervals. Subjects began GM-CSF/placebo injections for their next cycle of chemotherapy on day 16. If the ANC on day 16 was less than 500/µL, daily blood counts were performed and GM-CSF/placebo injections were delayed until an ANC of 500/µL or greater was achieved. The next cycle of chemotherapy was then administered 24 hours after the last injection of GM-CSF/placebo. Open-labelled GM-CSF was not permitted after chemotherapy unless fever with neutropenia was documented. Prophylactic antimicrobials were not used.

Statistical Analysis
Mean values for hemoglobin, ANC, and platelet count at nadir and at days 14 and 16 were compared by repeated measures analysis of variance for subjects receiving placebo and GM-CSF. To evaluate potential effects of treatment order, subjects receiving GM-CSF were compared with subjects receiving placebo for courses 1 and 2, for courses 3 and 4 and for all four courses together. Similar comparisons for time-to-nadir hemoglobin, ANC, and platelet count and for the duration of neutropenia and thrombocytopenia were also performed. Reported side effects in the placebo and GM-CSF groups were compared for all four courses combined. The proportion of patients with severe neutropenia (ANC < 500/µL) in placebo and GM-CSF groups were compared using a one-tailed McNemar test for correlated proportions.

	RESULTS

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Comparability of Treatment Groups
Baseline hematologic parameters in the two study groups were compared. The baseline WBC (mean ± SD, 7,370 ± 1,060/µL v 6,190 ± 1,170/µL, P = .03) and ANC (mean ± SD, 4,750 ± 9,430/µL v 3,720 ± 8,340/µL, P = .02) were higher in the group receiving GM-CSF priming for course 1. These differences, however, are controlled by the randomized crossover study design, ie, both groups received two courses of therapy with placebo priming and two courses of therapy with GM-CSF priming. Differences in the baseline hemoglobins and platelet counts were not observed.

Effects of GM-CSF Priming on Neutrophil Nadir and Recovery
The time to neutrophil nadir (mean ± SD, 2.8 ± 2.5 days for GM-CSF group v 14.8 ± 1.5 days for placebo group, P = .0001) was shorter after priming (Table 1). The neutrophil nadir was similar in both the GM-CSF and placebo groups (mean ± SD, 490 ± 310/µL v 550 ± 350/µL, respectively; P = .2); however, because of the small numbers of subjects in each group, a true difference in the neutrophil nadir could have been missed. On day 16, the mean neutrophil count was higher for the GM-CSF and placebo groups (mean ± SD, 1,030 ± 580/µL v 690 ± 370/µL, respectively; P = .004); this effect was particularly evident for the first two courses (1,060/µL v 650/µL, respectively; P = .013). A trend was also evident for the last two courses (950/µL for GM-CSF v 710/µL for placebo), although the difference was not statistically significant (Table 2). Also on day 16, the proportion of patients with an ANC count less than 500/µL was lower after priming compared with before priming (six of 35 or 17.1% of patients v 12 of 34 or 35.3% of patients, respectively; P = .04).

Effects of GM-CSF Priming on Platelet Nadir and Recovery
Compared with before priming, the time to platelet nadir after priming was shorter (mean ± SD, 10.1 ± 1.9 days v 11.1 ± 2.2 days, respectively; P < .05), and the platelet nadir was higher (mean ± SD, 166,000 ± 51,000/µL v 151,000 ± 45,000/µL, respectively; P = .007) after (Tables 1 and 2). The effect on the platelet nadir was particularly evident for the first two courses (176,300/µL v 150,000/µL, respectively; P = .0009). A trend was also evident for the last two courses (155,700/µL v 151,300/µL, respectively), although the difference was not statistically significant. After priming, the duration of a platelet count less than 150,000/µL was shorter compared with before priming (mean ± SD, 1.5 ± 2.1 days v 2.8 ± 2.9 days, respectively; P = .0025). This effect was particularly evident for the first two courses (0.9 days v 2.9 days, respectively; P = .0005). A trend was also evident for the last two courses (2.1 days v 2.7 days, respectively), although the difference was not statistically different.

Effects of GM-CSF on Hemoglobin Nadir and Recovery
Overall, with and without priming the time-to-hemoglobin nadir (mean ± SD, 11.8 ± 1.9 days v 11.6 ± 1.9 days, respectively; P = .59) and the hemoglobin nadir (10.9 ± 1.1 gm/dL v 10.9 ± 0.8 gm/dL, respectively; P = .81) were similar (Tables 1 and 2). However, when each course was compared individually, a benefit with priming was observed (course 1, P = .0007; course 2, P = .008; course 3, P = .0186; and course 4, P = .0136).

Toxicity of GM-CSF Priming
Reported (grade 1 and 2) toxic effects of GM-CSF are listed in Table 3. Severe (grade 3 and 4) toxic effects were not observed. No subjects withdrew from the study because of side effects related to priming. Headaches, myalgias, and other systemic symptoms including itching, tachycardia, heartburn, and dyspepsia were more commonly reported with GM-CSF than with the placebo. A difference in the incidence and severity of local reactions was not reported. Changes in the biochemical profile with GM-CSF priming were not observed. Episodes of fever and neutropenia were not observed.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
GM-CSF priming from days 5 to 1 before doxorubicin and cyclophosphamide chemotherapy was associated with an earlier neutrophil and platelet nadir, a higher mean ANC, and a lower proportion of severe neutropenia on day 16. A minor protective effect on the hemoglobin nadir was also observed. These findings support the study hypothesis that GM-CSF priming protects stem cells from chemotherapy-related cytotoxicity by inducing a state of stem-cell arrest.

The a priori protection of hematopoietic progenitors by stem-cell arrest offers several theoretical advantages over the post facto stimulation of an already damaged progenitor pool. By decreasing stem-cell damage, GM-CSF priming may decrease progressive bone marrow hypoplasia and peripheral blood pancytopenia, often observed after sequential courses of chemotherapy. Similarly, GM-CSF priming may prevent the development of myelodysplasia and/or secondary acute myeloid leukemia. GM-CSF priming may be substantially less expensive than the prophylactic or therapeutic administration of GM-CSF after chemotherapy (eg, 5 v 7 to 10 days of treatment, respectively). Furthermore, the degree of stem-cell protection provided by GM-CSF priming may be independent of the intensity of the chemotherapy treatment administered.

In the present study, protective effects of GM-CSF were primarily observed during the first two courses of therapy. Tachyphylaxis to GM-CSF priming with the last two courses of therapy is unexplained but may have been because of chemotherapy-induced recruitment of earlier progenitor cells not sensitive to GM-CSF withdrawal. A longer period of cytokine withdrawal before each course of chemotherapy (eg, 48 or 72 hours) may have been more effective and/or prevented tachyphylaxis.

Treatment delays were less frequent after priming compared with before (three of 36 or 8.3% v eight of 36 or 22.2%, respectively; P = .048). This likely reflects an artifact of the experimental design. According to the protocol, GM-CSF/placebo priming was to be started on day 16 of the preceding course if the ANC was greater than 500/µL. For patients with an ANC less than 500/µL on day 16, priming was to be delayed until this criterion was met. The proportion of patients with an ANC less than 500/µL on day 16 was lower, with GM-CSF priming than with placebo (six of 35 or 17.1% v 12 of 34 or 35.3% of patients, respectively; P = .04). Thus, treatment delays were less common in patients receiving GM-CSF priming. In routine clinical practice, however, treatment is typically delayed for an ANC less than 1,500/µL on day 21 rather than for an ANC less than 500/µL on day 16. As a result, the clinical relevance of this observation cannot be determined from this study.

The use of GM-CSF priming to prevent chemotherapy-induced cytotoxicity has been previously reported. Results have been inconsistent. Aglietta et al¹² observed higher ANC counts and fewer treatment delays in women with node-positive breast cancer treated with GM-CSF priming from days 6 to 4 compared with women who received chemotherapy alone. Edmonson et al¹³ similarly observed beneficial effects on leukopenia and thrombocytopenia in subjects with advanced cancer treated with 18 days versus 14 days of GM-CSF after chemotherapy. Treatment was administered at 21-day intervals. Thus, subjects who received 18 days of GM-CSF effectively received GM-CSF priming from days 6 to 3.¹³ Bernstein et al¹⁴ administered GM-CSF until a WBC of 100,000/µL was achieved. Chemotherapy was administered 48 hours thereafter. A beneficial effect of priming was not observed. Prolonged exposure to GM-CSF may have resulted in recruitment of earlier progenitors, thereby abrogating any effect of GM-CSF withdrawal on stem-cell arrest.¹⁴ Schwartzberg et al¹⁵ observed no effect on hematopoietic recovery after the administration of GM-CSF on days 7 to 3 before intensive chemotherapy. An effect may have been masked by the use of G-CSF after chemotherapy. A lower peripheral blood stem cell yield was also observed after GM-CSF priming in this study. This effect may have been because of a decrease in chemotherapy induced cytotoxicity resulting from cytokine withdrawal.¹⁵

In the present study, the most commonly observed side effects of GM-CSF priming included headache, myalgias, and other minor (grade 1 and 2) systemic complaints. Severe (grade 3 and 4) toxic effects were not observed. Toxic effects of GM-CSF priming reported by Bernstein et al¹⁴ and by Edmonson et al¹³ were more severe than those observed in the present study, presumably because of the prolonged duration of GM-CSF used by these investigators.^14,15 The side effects of GM-CSF priming observed in the present study were similar to the side effects of GM-CSF used prophylactically or therapeutically.¹⁶

This study has identified interesting and potentially useful biologic effects of GM-CSF priming; however, the findings are clearly limited by the small number of patients evaluated. Furthermore, the adjuvant chemotherapy regimen used was modest in intensity. Blood product support and hospitalization for fever and neutropenia were not required. An impact on these clinical end points could therefore not be evaluated. Therefore, future studies should evaluate GM-CSF priming in a more intensive clinical setting and with larger numbers of patients.

	ACKNOWLEDGMENTS

 
Supported by a grant from the Immunex Corporation, Seattle, WA

We thank Paul Etzell, MD, Jean Garvey PA-C, Louis Geeraerts, MD, Gerald Gross, MD, and John Leitch, MD, for their suggestions concerning trial design and for enrolling subjects onto the study. We also thank the nursing and pharmacy staff of the Roger Maris Cancer Center and the library staff of MeritCare Medical Center for their dedication and assistance with the trial.

	REFERENCES

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8. Bregni M, Siena S, Di Nicola M, et al: Comparative effects of granulocyte-macrophage colony-stimulating factor and granulocyte-colony stimulating factor after high-dose cyclophosphamide cancer therapy. J Clin Oncol14:628-635, 1996[Abstract/Free Full Text]

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10. Vadhan-Raj S, Broxmeyer H, Hittelman W, et al: Abrogating chemotherapy (CT)-induced myelosuppression by GM-CSF: Optimizing the schedule. Proc Am Soc Clin Oncol10:349, 1991 (abstr 1241)

11. Vadhan-Raj S, Broxmeyer H, Hittelman W, et al: Abrogating chemotherapy-induced myelosuppression by recombinant granulocyte-macrophage colony-stimulating factor in patients with sarcoma: Protection at the progenitor cell level. J Clin Oncol10:1266-1277, 1992[Abstract/Free Full Text]

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14. Bernstein S, Christiansen N, Fay J, et al: Granulocyte-macrophage colony-stimulating factor (GM-CSF) priming of high-dose etoposide and cyclophosphamide: A pilot trial. Exp Hematol24:1363-1368, 1996[Medline]

15. Schwartzberg L, West W, Birch R, et al: Randomized prospective trial ± pretreatment with GM-CSF prior to high-dose cyclophosphamide, etoposide and cisplatin plus G-CSF. Proc Am Soc Clin Oncol12:452, 1993 (abstr 1568)

16. Jones S, Schottstaedt M, Duncan L, et al: Randomized double-blind prospective trial to evaluate the effects of Sargramostim verses placebo in a moderate-dose flurouracil, doxorubicin and cyclophosphamide adjuvant chemotherapy program for stage II and III breast cancer. J Clin Oncol14:2976-2983, 1996[Abstract]

Submitted March 31, 1998; accepted July 14, 1999.

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