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Pilot Trial of Interleukin-2 With Granulocyte Colony-Stimulating Factor for the Mobilization of Progenitor Cells in Advanced Breast Cancer Patients Undergoing High-Dose Chemotherapy: Expansion of Immune Effectors Within the Stem-Cell Graft and Post–Stem-Cell Infusion

From the Section of Hematology/Oncology and Department of Ophthalmology-Biostatistics, University of Illinois at Chicago College of Medicine, Chicago; Section of Hematology/Oncology, Cardinal Bernadin Cancer Center, Loyola University Medical Center, Maywood, IL; and Amgen Clinical Research, Thousand Oaks, CA.

Address reprint requests to Jeffrey A. Sosman, MD, University of Illinois at Chicago, Section of Hematology/Oncology, 840 S Wood St (M/C 787) Chicago, IL 60612; email: jasosman{at}tigger.cc.uic.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: To evaluate whether administration of interleukin-2 (IL-2) with granulocyte colony-stimulating factor (G-CSF) improves mobilization of immune effector cells into the stem-cell graft of patients undergoing high-dose chemotherapy and autografting.

PATIENTS AND METHODS: We performed a trial of stem-cell mobilization with IL-2 and G-CSF in advanced breast cancer patients receiving high-dose chemotherapy with cyclophosphamide, thiotepa, and carboplatin and stem cells followed by IL-2. The trial defined immune, hematologic, and clinical effects of IL-2 in this setting.

RESULTS: Of 32 patients enrolled, nine received G-CSF alone for mobilization. Twenty-one of 23 patients mobilized with IL-2 plus G-CSF had stem cells collected with more mononuclear cells than those receiving G-CSF (19.3 v 10.4 x 10⁸/kg; P = .006), but fewer CD34⁺ progenitor cells (6.9 v 22.0 x 10⁶/kg; P = .049). The IL-2 plus G-CSF–mobilized patients had greater numbers of activated T (CD3⁺/CD25⁺) cells (P = .009), natural killer (NK; CD56⁺) cells (P = .007), and activated NK (CD56 bright⁺) cells (P = .039) than those patients mobilized with G-CSF. NK (P = .042) and lymphokine-activated killer (LAK) (P = .016) activity was increased in those mobilized with IL-2 + G-CSF, whereas G-CSF–mobilized patients had a decline in cytolytic activity. In the third week posttransplantation, immune reconstitution was superior in those mobilized with IL-2 plus G-CSF based on greater numbers of activated T cells (P = .003), activated NK cells (P = .04), and greater LAK activity (P = .003). The 16 of 21 IL-2 + G-CSF–mobilized patients with adequate numbers of stem cells (> 1.5 x 10⁶ CD34⁺ cells/kg) collected engrafted rapidly posttransplantation.

CONCLUSION: The results demonstrate that G-CSF + IL-2 can enhance the number and function of antitumor effector cells in a mobilized autograft without impairing the hematologic engraftment, provided that CD34 cell counts are more than 1.5 x 10⁶ cells/kg. Mobilization of CD34⁺ stem cells does seem to be adversely affected. In those mobilized with IL-2 and G-CSF, post–stem-cell immune reconstitution of antitumor immune effector cells was enhanced.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
THE USE OF HIGH-DOSE chemotherapy and autologous stem-cell transplantation for the treatment of advanced regional and metastatic breast cancer has been the focus of significant clinical research efforts over the past 10 to 15 years.^1-4 Although phase II stem-cell transplantation trials have provided promising leads, phase III studies remain inconclusive and trials show that the great majority of patients with metastatic breast cancer are not cured by this approach.^2,3,5-8

Improvements in stem-cell transplantation have largely been directed at the tolerability, rapid hematologic engraftment, and cost reduction of this approach.^9-14 These measures are unlikely to lead to improved disease-free survival. On the other hand, high-dose chemotherapy with stem-cell infusion may offer a unique clinical setting in which to apply immunotherapy. The time after the stem-cell transplantation procedure is likely optimal to apply immunotherapy based on the maximally reduced tumor burden. Furthermore, patients recovering from autologous stem-cell transplantation have been shown to quickly recover their non–major histocompatability complex–restricted cytolytic immune cell populations with natural killer (NK) and lymphokine-activated killer (LAK) function.^15,16 In fact, enhanced numbers of NK populations with increased cytolytic activity can be observed in the peripheral blood.¹⁶ Although specific active immunotherapy (vaccination) or specific T-cell adoptive immunotherapy may be viewed as more attractive approaches, the problem of limited antigenic tumor targets and the limited ability to ex vivo expand T cells may hinder these strategies. An even greater obstacle is that posttransplantation T-cell function may not easily allow for effective vaccine approaches.^17,18

Recent data demonstrate that standard granulocyte colony-stimulating factor (G-CSF) mobilization of peripheral-blood progenitor cells can diminish NK/LAK function, with a poorer yield and function of NK cells in the graft.¹⁹ This may impair a potential immune effector function involved in the graft-versus-tumor effect, if it exists in autologous stem-cell transplantation. Therefore, we have explored the use of interleukin-2 (IL-2) in the mobilization and post–stem-cell infusion phases of autologous stem-cell transplantation in a phase I trial in patients with advanced breast cancer. Continuous intravenous (IV) IL-2 was administered for two 4-day courses to better activate immune cells without a significant delay of the transplantation, which more extended IL-2 therapy would require. Additionally, previous data suggest that IL-2 could enhance the hematopoietic potential of the peripheral blood with increased granulocyte-macrophage colony-forming units and induction of cytokines (IL-6, G-CSF, granulocyte-macrophage colony-stimulating factor, and so on) with hematopoietic stimulatory activity in vivo.^20,21 Therefore, we attempted to develop a schedule of IL-2 with a dose that was tolerable and would enhance immune activity of the stem-cell graft and hopefully enhance posttransplantation immune reconstitution. In this report we describe our clinical and biologic findings from this pilot trial.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Treatment Plan
Eligibility. Patients had histologically confirmed advanced breast cancer that was either metastatic or locally advanced (IIIB or IV). Patients were eligible for this trial if they had received no more than two chemotherapy regimens for metastatic disease and had either stable disease or response to the last chemotherapy regimen. Patients with recurrent or metastatic disease that had been completely resected were eligible. Patients with bone marrow involvement were eligible provided that less than 30% of the overall cellularity of the bone marrow core biopsy was represented by breast cancer. Organ function was within normal range, and all patients were required to undergo pulmonary function tests and multigated cardiac scans. Normal cardiac stress testing was required for those patients who were older than 50 years of age or had significant risk factors or symptoms suspicious for coronary vascular disease.

Treatment plan.
The treatment plan is illustrated in Fig 1. Patients received IL-2 as part of the mobilizing regimen for 4 days (96 hours) via continuous IV infusion then rested off treatment for approximately 3 days (72 hours) and were then treated for an additional 4 days of IL-2 in combination with daily subcutaneous injection of G-CSF at 10 µg/kg/d. The patients completed their second course of 96 hours of IL-2 and then began their stem-cell apheresis 24 hours later. G-CSF injections were continued throughout their apheresis for the 3- to 5-day period. Aphereses were performed using Cobe Spectra (Lakewood, CO) Instruments with 10- to 12-L aphereses performed daily for a minimum of 3 days and a maximum of 5 days to achieve a minimum of 1.5 x 10⁶ CD34⁺ cells/kg and a target of 5.0 x 10⁶ CD34⁺ cells/kg. To protect against very poor or failed engraftment, patients who could not mobilize a minimum of 1.5 x 10⁶ CD34⁺ mononuclear cells/kg in three to five aphereses were removed from the protocol and mobilization was considered to have failed. Some of these patients (three of the five) went on to stem-cell transplantation after subsequent mobilization or bone marrow collection.

View larger version (16K): [in this window] [in a new window]   Fig 1. Treatment schedule. Patients received stem-cell mobilization with IL-2 and G-CSF as outpatients. Twenty-four hours after completion of the second weekly course of IL-2, patients underwent leukapheresis. Transplantation patients were hospitalized until they were afebrile with an absolute neutrophil count greater than 500/µL. A number of patients completed their posttransplantation IL-2 as outpatients.

Dose escalation of IL-2.
Patients were enrolled onto the IL-2 dose levels in groups of cohorts that included patients receiving G-CSF alone and/or IL-2 post–stem-cell infusion as controls ( Table 1). Patients who received mobilizing IL-2 at a defined dose level were assigned to receive post–stem-cell IL-2 in the next group only after the initial cohort of patients demonstrated the safety and engraftment without the post–stem-cell IL-2 (at the same mobilizing dose of IL-2). Within a group (Table 1; groups 1, 2, or 3) of patients assignment to a treatment was random to prevent bias. Patients received mobilizing doses of IL-2 at 1.8, 2.7, or 3.6 million IU/m²/d. The post–stem-cell IL-2 was initially set at 0.9 million IU/m²/d for 14 days. This was found to be intolerable and was later revised to 0.45 million IU/m²/d for 12 days, with tolerable toxicity in subsequent patients (see Results). Standard National Cancer Institute common toxicity criteria (Version 2.0) were used, and posttransplantation engraftment was considered present when platelet count was more than 20,000/µL without any further platelet transfusions and the absolute neutrophil count was more than 500/µL and maintained without further administration of G-CSF.

Recombinant methionyl granulocyte colony-stimulating factor (r-metHuG-CSF; G-CSF; filgrastim) is a purified, hydrophobic, nonglycosylated protein composed of 175 amino acids with a molecular weight of 18,800 da. G-CSF was stored at 2 to 8°C and allowed to reach room temperature for a maximum of 24 hours before injection.

Flow Cytometry
The relative numbers of CD56⁺, CD56 bright⁺, CD3/CD25⁺, and CD34⁺ mononuclear cells were analyzed by two- and three-color immunofluorescence and flow cytometry. On collection, samples were immediately processed (Ficoll-Hypaque purified) and cryopreserved. On completion of treatment, individual patient samples were thawed and assayed simultaneously. Antibodies were purchased from Becton Dickinson (San Jose, CA). Analysis of surface phenotype included phycoerythrin-conjugated anti-CD56, cytochrome-conjugated anti-CD3, fluorescence-conjugated anti-CD25, or phycoerythrin-conjugated anti-CD34. CD15 cells were excluded from the analysis to exclude mature myeloid cells when analyzing for expression of lymphoid markers CD56, CD3, and CD25. A minimum of 10,000 CD15- cells were analyzed by flow cytometry using a FACStar Plus flow cytometer (Becton Dickinson, San Jose, CA).

Chromium Release Assays for NK/LAK Cytotoxicity
K562 (NK-sensitive and LAK-sensitive) and Daudi (NK-resistant and LAK-sensitive) tumor cell lines were labeled with 200 µCi of chromium-51 for 1 hour at 37°C and washed three times in media and 10% fetal calf serum. Five thousand labeled target cells (K562 or Daudi) were incubated with effector cells (fresh peripheral-blood mononuclear cells, thawed and assayed simultaneously) at various effector:target ratios for 4 hours at 37°C. The supernatant was collected and assayed for radioactivity by gamma scintillation counting. Data were presented as the percentage of specific chromium-51 release or as lytic units (LU), defined as the number of effector cells per 10⁷ required to lyse 30% of the target cells.²² All samples from a single patient were assayed simultaneously from their cryopreserved samples.

Statistics
Statistical analysis was performed using the nonparametric tests of the Wilcoxon signed ranks test using two-sided P values. Additionally, the Mann-Whitney U test and Kruskal-Wallis one-way analysis of variance were performed for nonparametric analysis between treatment groups. P values are presented based on pooled variances. The limit of significance was fixed at .05 with 95% confidence intervals (CI).

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patient Enrollment and Characteristics
Thirty-two patients were enrolled onto the protocol. Patient characteristics are listed in Table 2. The patients received a median and mean of approximately two prior chemotherapy regimens, with a range of one to three based on the required eligibility criteria (including adjuvant chemotherapy). Of the 32 patients, disseminated stage IV disease was present in 27 and locally advanced disease (IIIB) in five patients. None of the stage IV chemotherapy-treated patients had attained a complete radiologic response before transplantation. The subsequent treatment of the 32 enrolled patients is illustrated in Fig 2.

View larger version (30K): [in this window] [in a new window]   Fig 2. Patient outcome. Boxes represent subsequent treatment pathways for all 32 patients entered onto the protocol. Four patients were lost to follow-up. Only one of these patients underwent stem-cell transplantation after removal from the protocol. Abbreviations: VOD, veno-occlusive disease; f/u, follow-up. ** Denotes patients without long-term follow-up.

Adequacy of Stem-Cell Source to Proceed With Transplantation
All nine patients who were mobilized with G-CSF alone had an adequate yield of CD34⁺ mononuclear cells (1.5 x 10⁶/kg) in three aphereses as mandated by the protocol ( Table 3). On the other hand, mobilization with both G-CSF and IL-2 at 1.8 to 3.6 million IU/m²/d led to successful collections in 16 of 21 patients (failure rate, 0 of nine v five of 21). Although this did not reach statistical significance, it seemed clinically relevant in this cohort. Fourteen of these patients had three aphereses, whereas two patients required four aphereses to reach the required CD34 yield. The patients for whom mobilization was unsuccessful were among those treated at 1.8 million IU (four patients) and 2.7 million IU (one patient) IL-2 dose levels and in subsequent attempts mobilized poorly ( 2.0 x 10⁶/kg CD34⁺ cells) with G-CSF alone or chemotherapy and G-CSF. Comparing the CD34⁺ cell yields among those mobilized with G-CSF versus IL-2 plus G-CSF, there was a significant difference in CD34⁺ cell yield in favor of G-CSF–mobilized patients alone(P = .049). A median of 6.8 x 10⁶/kg CD34⁺ cells were collected from the nine patients receiving G-CSF alone for mobilization, whereas the 21 patients mobilized with IL-2 and G-CSF had a median of 3.5 x 10⁶/kg CD34⁺. However, the total mononuclear cell count was greater in those patients mobilized with IL-2 and G-CSF compared with those receiving G-CSF alone, as listed in Table 3 (P = .006). As shown in Fig 2, five patients did not mobilize adequately with IL-2 plus G-CSF; three patients underwent subsequent mobilization regimens, including even bone marrow collection in two patients and then went on to high-dose therapy and a stem-cell transplantation. Two patients were not mobilized further and received standard chemotherapy without stem-cell transplantation.

Clinical Toxicity of Post–Stem-Cell IL-2 Therapy
The 0.9 million IU/m²/d IL-2 post–stem-cell infusion dose was quite toxic and was accompanied by grade 3 fevers (frequently above 40°C) with shaking chills, diarrhea, and diffuse erythema. Patients did not continue beyond 7 to 8 days. No obvious infections were observed. Only two patients were treated at this dose. In the subsequent eight patients who received the post–stem-cell IL-2 at 0.45 million IU/m²/d, toxicity consisted of some fevers, rash, and diarrhea, which were tolerable (grade 1 or 2) for the planned 12 days.

Mobilization of Immune Cells and their Cytolytic Function
Viable peripheral-blood samples were not available for the initial 10 patients. Adequate pretreatment and posttreatment peripheral-blood samples were available for a total of 17 patients (12 mobilized with IL-2 and G-CSF and five mobilized with G-CSF alone). We examined the blood of these patients for CD56⁺, CD56 bright⁺, and CD3⁺/CD25⁺ lymphoid cells that could potentially mediate antitumor effects in vivo.^23-25 The CD15- mononuclear cells obtained from the peripheral blood at the time of the leukapheresis (each of the three initial aphereses) were evaluated for CD56⁺, CD56 bright⁺, and CD3/CD25⁺ cells via flow cytometry ( Table 4). There were marked differences in the number of CD56⁺ mobilized cells favoring those patients receiving IL-2 and G-CSF, with a mean of 872 x 10⁶/kg CD56⁺ cells (95% CI, 653 to 1,191 x 10⁶/kg CD56⁺ cells) versus 160 x 10⁶/kg CD56⁺ cells (95% CI, 127 to 191 x 10⁶/kg CD56⁺ cells) in those receiving G-CSF alone (P = .007). Even more impressive was the disparity of CD56 bright⁺ lymphocytes, with a mean of 360 x 10⁶/kg (95% CI, 214 to 506 x 10⁶/kg) among those mobilized with IL-2 and G-CSF, compared with a mean of only 21 x 10⁶/kg (95% CI, 12 to 30 x 10⁶/kg) for the five patients mobilized with G-CSF alone (P = .039). Although the numbers of CD3⁺ T lymphocytes were similar among those receiving IL-2 and G-CSF compared with those receiving G-CSF alone (data not shown), the numbers of CD3⁺/CD25⁺ (IL-2Rß) was nearly a log greater in those receiving IL-2 and G-CSF compared with G-CSF alone (196 x 10⁶/kg [95% CI, 136 to 256 x 10⁶/kg] v 24 x 10⁶/kg [95% CI, 5 to 43 x 10⁶/kg] CD3/CD25⁺) (P = .009). Overall, these data show that IL-2 doses of 1.8 to 3.6 million IU/m²/d of Amgen IL-2 induced marked expansion of NK, activated NK, and activated T cells into the mobilized peripheral blood for stem-cell collection.

View larger version (17K): [in this window] [in a new window]   Fig 3. Four patients mobilized with G-CSF alone and 11 mobilized with IL-2 and G-CSF had peripheral-blood mononuclear cells (PBMCs) assayed for NK and LAK activity. PBMCs were collected before and after mobilization on day 2 of the apheresis. (A) NK activity against the K562 target; (B) LAK activity against the Daudi target. NK/LAK assays had SEM less than 15% and spontaneous release of target cells less than 20% of the maximal release.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

In contrast to the immune activation, there was a modest but clinically significant decrease in the numbers of CD34⁺ cells mobilized, and only 16 of 21 of the IL-2 and G-CSF–treated patients mobilized 1.5 x 10⁶ CD34⁺ cells/kg. The five patients did not continue on protocol based on our concern for engraftment failure. All 16 patients who mobilized adequately ( 1.5 x 10⁶ CD34⁺ cells/kg) engrafted well with minimal toxicity if they did not receive IL-2 after transplantation. The deleterious effect of IL-2 on progenitor-cell mobilization was unexpected. Preclinical models suggest that activated NK cells might actually enhance engraftment in bone marrow transplantation models.^21,30,31 Additionally, human clinical trials with IL-2 in solid tumor patients have shown that cytokines that enhance hematopoiesis, such as IL-6, G-CSF, and granulocyte-macrophage colony-stimulating factor, are induced in the blood and granulocyte-macrophage colony-forming units and erythroid burst-forming units can be increased in the blood after administration of IL-2.²⁰ Other studies do show that NK cells suppress hematopoiesis and certainly some of the secondary cytokines induced by IL-2 such as interferon and tumor necrosis factor gammaa are suppressive to bone marrow function.^32,33

The trial also demonstrated four potentially important effects of IL-2 and G-CSF combination in a mobilization regimen. First, NK and LAK function was diminished in the peripheral blood by G-CSF mobilization. This has been reported previously by Miller et al.¹⁹ The decline of NK/LAK cytotoxicity was not only prevented, but the cytotoxicity was actually enhanced by IL-2 administration. The clinical importance of this finding has not yet been established, but this immune effect may be a factor in the induction of an antitumor effect. Second, patients frequently tolerated the second week (96-hour infusion) of IL-2 better than the initial week. There was less fever, dehydration, nausea/vomiting, and need for fluids. The second week of treatment was accompanied by G-CSF administration. G-CSF may block some of the cytokine storm (systemic inflammatory response syndrome) associated with IL-2 and sepsis.³⁴ This may provide an unexpected approach to diminish IL-2 toxicity. Third, after transplantation (days 14 to 21), patients mobilized with IL-2 and G-CSF demonstrated greater numbers of activated NK and activated T cells as well as enhanced LAK cytotoxicity in their peripheral blood compared with those who received only G-CSF for mobilization. This suggests more rapid and vigorous immune reconstitution of lymphoid cells capable of antitumor effects. Finally, a number of patients (seven of 18) who received IL-2 as part of their mobilization regimen remain progression-free from 16+ to 42+ months from the date of their transplantation. The role that IL-2 played in this favorable outcome is unclear. These patients may also have remained progression-free because of their favorable clinical characteristics before transplantation (single disease site, one prior chemotherapy, younger age, lack of liver metastases).²⁶ Furthermore, the long-term outcome of the six patients mobilized with G-CSF alone and then treated with IL-2 after stem-cell infusion did not seem as promising, with only one (one of six) long-term progression-free survivor. Because the formulation of IL-2 in the trial is not available, we are completing dose escalation with much higher doses (10 to 15 million IU/m²/d) of Chiron IL-2. We have also split the G-CSF administration to a twice-daily schedule in an attempt to improve CD34⁺ cell mobilization.³⁵ We believe that with further IL-2 dose escalation, we can define a safe, tolerable, outpatient IL-2 regimen.

Our approach is not unique; many other investigators, including Fefer et al, have targeted hematologic malignancies with a posttransplantation IL-2 course of treatment.^36-39 Work from the lab of Mazunder et al has led to numerous clinical trials that used ex vivo bone marrow or peripheral-blood progenitor cell activation with very high-dose IL-2 followed by systemic IL-2 after stem-cell infusion.^40-44 Finally, allogeneic bone marrow transplantation may offer real clues to a graft-versus-tumor effect in breast cancer.⁴⁵ This approach, although it faces significant obstacles, is of great interest and has already attracted a number of investigators, especially with the use of nonmyeloablative regimens. As we stated in our introduction, a tumor-specific vaccine or specific adoptive cellular therapy in the transplantation setting is what we aim for in the future.^46-49 This report demonstrates that although IL-2 does not enhance hematopoietic mobilization, it can greatly enhance the number and function of immune effector cells within the graft and can enhance immune reconstitution shortly after posttransplantation engraftment of lymphoid populations important in mediating antitumor effects. To definitively understand the significance of this type of pilot trial, phase II and more importantly phase III trials must be conducted. However, these trials will likely have to wait until the final results of multicenter trials define whether there is any role for high-dose chemotherapy and stem-cell rescue in the management of patients with breast cancer.

	ACKNOWLEDGMENTS

 
Supported by NCI-RO-1 CA727707 (JAS) and a research grant from Amgen Clinical Research Division to University of Illinois at Chicago College of Medicine and Loyola University Medical Center; Cardinal Bernadin Cancer Center.

	NOTES

 
Presented in part at the American Society of Hematology Annual Meeting, December 4-8, 1996, Orlando, FL, and at the International Society of Experimental Hematology Annual Meeting, August 1998, Vancouver, Canada.

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TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
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Submitted October 22, 1999; accepted September 21, 2000.

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