Scholarly article on topic 'Comparison of outcome of allogeneic bone marrow transplantation with and without granulocyte colony-stimulating factor (lenograstim) donor-marrow priming in patients with chronic myelogenous leukemia'

Comparison of outcome of allogeneic bone marrow transplantation with and without granulocyte colony-stimulating factor (lenograstim) donor-marrow priming in patients with chronic myelogenous leukemia Academic research paper on "Clinical medicine"

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Abstract of research paper on Clinical medicine, author of scientific article — Shu-Quan Ji, Hui-Ren Chen, Hang-Xiang Wang, Hong-Ming Yan, Shi-Ping Pan, et al.

Abstract To investigate the effect of granulocyte colony-stimulating factor (G-CSF) donor-marrow priming on hematopoietic recovery and clinical outcome after allogeneic hematopoietic stem cell transplantation, we compared HILA-matched related marrow transplantation with and without G-CSF donor priming in a prospective randomized study for a homogeneous group of chronic myelogenous leukemia (CML) patients. Fifty patients (aged 12-41 years) with CML were enrolled in the study. Thirty-two patients (study group) received the marrow grafts primed with G-CSF at 3 to 4 micro/kg per day for 7 days prior to the marrow harvest, and 18 patients (control group) received the marrow grafts without G-CSF priming. All patients received the same graft-versus-host disease (GVHD) prophylaxis (cyclosporine A and methotrexate) and postgraft G-CSF treatment, 3 to 4 micro/kg daily until the absolute neutrophil counts (ANCs) were >10(9)/L. The primary end points were engraftment and incidence of acute GVHD. The secondary end points were the incidence of chronic GVHD, relapse, and overall disease-free survival. The study and control groups were comparable for age, sex, donor selections, conditioning regimens, and disease status. The median times to both neutrophil and platelet engraftment (ANC > 0.5 x 10(9)/L; platelets > 20 x 10(9)/L) were significantly faster in the study group than in the control group, at 15 versus 21 days (P < .001) and 17.5 versus 24 days (P < .001), respectively. G-CSF donor printing yielded significantly higher numbers of total nuclear cells in the marrow grafts compared to the numbers in the control grafts (7.2 versus 2.9 x 10(8)/kg, P < .001). Similar results were seen for CD34+ (6.1versus 2.7 x 10(6)/kg, P < .001) and colony-forming unit-granulocyte/macrophage (CFU-GM) cells (68 versus 16 x 10(4)/kg, P < .001). The incidence of grades II to IV acute GVHD was surprisingly low in the study group: only 2 (6.3%) of 32 transplantation patients in the study group developed grade II acute GVHD, limited to the skin, whereas 5 (27.8%) of 18 patients in the control group developed grades II to IV acute GVHD (P = .032). G-CSF priming did not change the total numbers of CD3+ cells in the marrow grafts but lowered CD4+ cells and increased CD8+ cells, resulting in a significant reduction of CD4:CD8 ratio (P = .018). Six patients in the study group developed chronic GVHD either during or after cyclosporine taper. There were no significant differences in chronic GVHD (24% versus 33.3%), relapse rates (12.5% versus 11.1%), and overall survival rates (78.1% versus 66.7%, P = .32) between the study and control groups during a median follow-up period of 24 months (range, 6-50 months). There was, however, a trend in favor of improved chronic GVHD and disease-free survival in the study group. We conclude that G-CSF donor-marrow priming accelerates both neutrophil and platelet engraftment and is associated with a very low incidence of grades II to IV acute GVHD in CML patients after HLA-matched sibling marrow transplantation. Biol Blood Marrow Transplant 2002;8(5):261-7.

Academic research paper on topic "Comparison of outcome of allogeneic bone marrow transplantation with and without granulocyte colony-stimulating factor (lenograstim) donor-marrow priming in patients with chronic myelogenous leukemia"

Biology of Blood and Marrow Transplantation 8:261-267 (2002) © 2002 American Society for Blood and Marrow Transplantation

Comparison of Outcome of Allogeneic Bone Marrow Transplantation with and without Granulocyte Colony-Stimulating Factor (Lenograstim) Donor-Marrow Priming in Patients with Chronic Myelogenous Leukemia

Shu-Quan Ji,1 Hui-Ren Chen,1 Hang-Xiang Wang,1 Hong-Ming Yan,1 Shi-Ping Pan,1 Chang-Qing Xun2

'Research Center for Hematology, Air Force General Hospital, PLA, Beijing, People's Republic of China; 2Division of Hematology/Oncology and Blood/Marrow Transplant Program, Department of Medicine and Markey Cancer Center, University of Kentucky and VA Medical Center, Lexington, Kentucky, USA.

Correspondence and reprint requests: Shu-Quan Ji, MD and Professor, Research Center for Hematology, Air Force General Hospital, PLA, 30 Fu-Chen Road, Beijing 100036, P. R. China (e-mail: jishuquan@263.net); Chang Q. Xun, MD, Division of Hematology/Oncology and Blood/Marrow Transplant Program, University of Kentucky Medical Center and VA Medical Center, CC301 Markey Cancer Center, 800 Rose St, Lexington, KY 40536 (e-mail: cxun2@uky.edu).

Received August 17, 2001; accepted February 19, 2002

ABSTRACT

To investigate the effect of granulocyte colony-stimulating factor (G-CSF) donor-marrow priming on hematopoietic recovery and clinical outcome after allogeneic hematopoietic stem cell transplantation, we compared HLA-matched related marrow transplantation with and without G-CSF donor priming in a prospective randomized study for a homogeneous group of chronic myelogenous leukemia (CML) patients. Fifty patients (aged 12-41 years) with CML were enrolled in the study. Thirty-two patients (study group) received the marrow grafts primed with G-CSF at 3 to 4 |lg/kg per day for 7 days prior to the marrow harvest, and 18 patients (control group) received the marrow grafts without G-CSF priming. All patients received the same graft-versus-host disease (GVHD) prophylaxis (cyclosporine A and methotrexate) and postgraft G-CSF treatment, 3 to 4 |lg/kg daily until the absolute neutrophil counts (ANCs) were >109/L. The primary end points were engraftment and incidence of acute GVHD. The secondary end points were the incidence of chronic GVHD, relapse, and overall disease-free survival. The study and control groups were comparable for age, sex, donor selections, conditioning regimens, and disease status. The median times to both neutrophil and platelet engraftment (ANC > 0.5 X 109/L; platelets > 20 X 109/L) were significantly faster in the study group than in the control group, at 15 versus 21 days (P < .001) and 17.5 versus 24 days (P < .001), respectively. G-CSF donor priming yielded significantly higher numbers of total nuclear cells in the marrow grafts compared to the numbers in the control grafts (7.2 versus 2.9 X 108/kg, P < .001). Similar results were seen for CD34+ (6.1 versus 2.7 X 106/kg, P < .001) and colony-forming unit-granulocyte/macrophage (CFU-GM) cells (68 versus 16 X 104/kg, P < .001). The incidence of grades II to IV acute GVHD was surprisingly low in the study group: only 2 (6.3%) of 32 transplantation patients in the study group developed grade II acute GVHD, limited to the skin, whereas 5 (27.8%) of 18 patients in the control group developed grades II to IV acute GVHD (P = .032). G-CSF priming did not change the total numbers of CD3+ cells in the marrow grafts but lowered CD4+ cells and increased CD8+ cells, resulting in a significant reduction of CD4:CD8 ratio (P = .018). Six patients in the study group developed chronic GVHD either during or after cyclosporine taper. There were no significant differences in chronic GVHD (24% versus 33.3%), relapse rates (12.5% versus 11.1%), and overall survival rates (78.1% versus 66.7%, P = .32) between the study and control groups during a median follow-up period of 24 months (range, 6-50 months). There was, however, a trend in favor of improved chronic GVHD and disease-free survival in the study group. We conclude that G-CSF donor-marrow priming accelerates both neutrophil and platelet engraftment and is associated with a very low incidence of grades II to IV acute GVHD in CML patients after HLA-matched sibling marrow transplantation.

KEY WORDS

G-CSF • GVHD • Hematopoietic reconstitution • Allo-BMT

INTRODUCTION

Granulocyte colony-stimulating factor (G-CSF) can promote cell cycling of multipotential hematopoietic progenitors and increase hematopoietic progenitor cell yields in both marrow and peripheral blood [1,2]. Many studies have shown that G-CSF-mobilized peripheral blood progenitor cells (PBPCs) accelerate engraftment and shorten the neutropenic period compared to non-G-CSF-primed steady-state marrow graft [3-6]. G-CSF-mobilized peripheral blood stem cells (PBSCs) spared the donors from the marrow harvest procedure. However, the numbers of T-cells in PBSC collection are 10 to 15 times higher than the numbers in the steady-state marrow graft [7,8]. It is known that donor T-cells in the graft are closely associated with the incidence of graft-versus-host disease (GVHD), especially the mature T-cells collected from the peripheral blood mononuclear fraction [9]. The potential advantage of G-CSF-mobilized PBPCs still needs to be balanced against the currently undefined risk of GVHD that may be associated with the infusion of much higher donor T-lymphocyte numbers in unmanipulated allo-geneic PBSCs than in marrow graft [10,11].

Several pilot studies compared the effects of G-CSF-mobilized PBSCs versus G-CSF-primed marrow on hema-tological recovery and GVHD after allogeneic transplantation [12,13]. The neutrophil recoveries were equivalent in both groups, and the results were very consistent among those studies, although the platelet recoveries were somewhat inconsistent. Patients receiving G-CSF-primed marrow appeared to have a lower incidence of chronic GVHD than did those receiving G-CSF-mobilized PBSCs. It is unclear how G-CSF priming affects relapse rates and overall survival rates because of these studies' mixed patient populations, small patient numbers, and short follow-up periods. This prospective randomized study compared the outcomes of using G-CSF-primed marrow versus steady-state marrow as the stem cell source for HLA-matched sibling transplantation in chronic myelogenous leukemia (CML) patients, with a medium follow-up of 24 months (range, 6-50 months).

PATIENTS AND METHODS

Patients

Patients aged 12 to 50 years with a confirmed diagnosis of CML and an HLA-identical sibling donor were eligible to participate in the study. The CML diagnosis and classification were confirmed by morphologic and cytogenetic analyses of the bone marrow immediately before transplantation. Patients were classified as having chronic phase or accelerated phase according to Hammersmith criteria [14]. The donor and recipient matching was based on the HLA-genotypical typing (A, B, DR) and negative mixed lymphocyte reactions between the donor and recipient. All patients were enrolled from the Air Force General Hospital (AFGH) after the discussion of potential risks and benefits of the study. The study protocol was approved by the Institutional Review Board (IRB) at the AFGH. The patients were prospectively allocated into the donor G-CSF-priming group or the nonpriming group using monthly cycle randomization, ie, first month to non-G-CSF-priming group (control group), second month to G-CSF-priming group (study group). The cycle repeated every 2 months. The pri-

mary end points were engraftment and incidence of acute GVHD. The secondary end points were relapse rate, incidence of chronic GVHD, and overall survival rate. Within 6 months after the first 15 patients were enrolled, a trend strongly suggested fast engraftment without the development of acute GVHD in the donor G-CSF-priming group. After discussions with the IRB and statistician, we decided, because of the benefit to patients, to increase the number of patients in the study group. The patient assignment was then changed to first month to the non-G-CSF-priming control group and the second and third month to the G-CSF-priming study group. The cycle was repeated every 3 months. Patient accrual was initiated in January 1997 and terminated in September 2000 after we were notified that both primary end points achieved significant difference between the study and control group.

Donor Priming Regimen and Bone Marrow Harvesting

Informed consent was obtained from each donor using the forms approved by the IRB at the AFGH. Donors were primed with G-CSF (lenograstim) (Chugai Pharmaceutical, Tokyo, Japan) at 3 to 4 |Jg/kg per day by a single daily subcutaneous injection for 7 consecutive days as previously described [15-17]. On the eighth day the bone marrow cells were harvested from the posterior iliac crests while the patients were under epidural anesthesia, with approximately 10 mL of bone marrow collected per each aspiration. The target volume for each donor marrow collection was 18 to 20 mL/kg recipient body wt. Total nucleated cell (TNC) counts were obtained using an automated counter instrument. The numbers of CD34+ and CD3+, CD4+, and CD8+ T-cells were assessed by immunophenotyping and flow cytometric analysis. The fresh and unmanipulated marrow was infused on the same day after the marrow harvest (day 0). In ABO major blood group incompatibility, the red cells in the marrow were removed by sediment manipulation.

Conditioning Regimen

For patients with CML in chronic phase, the conditioning regimens were either cyclophosphamide (Cy) 120 mg/kg over 2 days and total body irradiation (TBI) with 1000 cGy by 2 fractions at a dose rate of 5 to 6 cGy/min (Cy + TBI); or busulfan (Bu) 16 mg/kg administered orally in 16 doses over 4 days plus Cy 120 mg/kg over 2 days (BuCy). In February 1998, our original study of overall leukemia patients (CML, acute myelogenous leukemia, acute lymphoblastic leukemia) with standard risk showed that BuCy conditioning regimens were superior to Cy + TBI conditioning, with lower rates of transplantation-related complications and significantly lower cost [17]. After February 1998, the conditioning regimen for all standard-risk leukemia patients was changed to BuCy. For all high-risk patients with CML accelerated phase or disease duration >3 years, the regimen consisted of Bu (8 mg/kg in 8 doses over 2 days), Cy (120 mg/kg over 2 days), and TBI 1000 cGy in 2 fractions [17].

Supportive Care

All patients were hospitalized in rooms with high-efficiency particulate air filters and received standard antibiotic

Table 1. Patient and Transplantation Characteristics

Study Group Control Group P

No. of patients 32 18

Age, median (range), y 32.5 (12-42) 29 (16-41) .9

Sex, F/M 10/22 6/12 .91

Disease status at BMT .91

CML, chronic phase 27 15

CML, accelerated phase 5 3

Interval from diagnosis to BMT .95

<1 year 8 6

2-3 years 17 8

>3 years 7 4

Conditioning regimens .99

Cy + TBI 7 4

Bu + Cy + TBI 9 5

Bu + Cy 16 9

Donor age, median (range), y 30 (15-42) 27 (17-48) .9

Donor sex, F/M 14/18 7/11 .92

Donor/recipient sex match .9

M/M 10 6

F/M 12 6

M/F 8 5

F/F 2 1

prophylactic therapy consisting of oral trimethoprim-sulfamethoxazol (TMP-SMZ), fluconazole, and acyclovir. Intravenous immunoglobulins were given at a dosage of 500 mg/kg weekly starting on day 1. G-CSF (lenograstim), 3 to 4 |g/kg per day, was given to all recipients subcuta-neously from the second day of transplantation until the neutrophil counts reached 0.5 X 109/L for 3 consecutive days. Patients received transfusions if hemoglobin or platelet levels were below 8.0 g/dL or 20 X 109/L, respectively. All blood products were irradiated.

GVHD Prophylaxis

All patients received cyclosporine A (CsA) and methotrex-ate (MTX) for GVHD prophylaxis. The dosage of MTX was 7.5 mg/M2 intravenously on days 1, 3, 6, and 11 posttransplantation. The dosage of CsA was 2.5 mg/kg per day intravenously on day -1 until bowel function was normal and then 5 mg/kg per day orally in 3 divided doses. From day 50, the dosage was reduced by 5% every week and stopped on day 180 if there was no chronic GVHD. If chronic GVHD developed, CsA was continued for a longer time. Whole-blood CsA trough levels were measured weekly using fluorescence polarization immunoassay. The CsA dose was reduced if the trough level of CsA was more than 300 ng/mL or the serum creatinine level exceeded 2 mg/dl. The diagnosis and grading of GVHD was established according to previous definable criteria [18]. Acute GVHD of grade II or higher was treated with methylpred-nisone 1 to 2 mg/kg per day.

Evaluation of Engraftment

Engraftment was defined as absolute neutrophil count (ANC) > 0.5 X 109/L after neutrophil nadir. Bone marrow aspiration, biopsy, and cytogenetic studies were done at 1 month posttransplantation to assess engraftment. Further bone marrow evaluations were done if clinically indicated.

Progenitor Cell and T-Cell Analyses in the Marrow Grafts

Cell surface markers were determined by the dual or 3-color staining method using monoclonal antibodies directly against CD34+, CD3+, CD4+, and CD8+ (Coulter, Fullerton, CA). The marrow cells were incubated with the monoclonal antibodies for 15 minutes at room temperature. The cells were then washed and analyzed by flow cytometer (EPICS XL, Coulter). The numbers of CD34+ and subsets of T-cells were calculated by the numbers of total nuclear cells per microliter, multiplying the percentage of their corresponding immunophenotype. CFU-GM was determined by semisolid agar culture [19].

Statistical Analysis

Patients, disease stages, and transplantation-related characteristics were compared between the study and control groups using the chi-square test. A comparison between classified variables was performed by the Fisher exact test. The Student t test was used to compare the means of continuous variables between the 2 groups. The cumulative probabilities of GVHD, relapse rate, and disease-free survival were calculated using the Kaplan-Meier and log-rank tests. The date of the final analysis was March 15, 2001.

RESULTS

Patient Characteristics

Fifty patients with Philadelphia chromosome-positive CML undergoing allogeneic transplantations from HLA-identical siblings were enrolled in this study. Thirty-two patients, of which 27 were in chronic phases and 5 in accelerated phases, were allocated into the study group with donor G-CSF priming, and 18 patients, of which 15 were in chronic phases and 3 in accelerated phases, were allocated into the control group without donor G-CSF priming. All patients underwent transplantations during the same time period. Both groups of patients were comparable in the most important parameters such as patient age, sex, disease status, donor selections, and conditioning regimens (Table 1).

Marrow Graft Characteristics

Table 2 summarizes the contents of marrow grafts in the study and control groups. The median volumes harvested were 1.10 L (range, 0.60-1.50 L) in the study group and 1.20 L (range, 0.75-1.60 L) in the control group, with the goal being 18 to 20 mL/kg of recipient body wt. The median numbers of TNCs, CD34+ cells, and CFU-GM cells were significantly higher in the study group (P < .001)

Table 2. Graft Characteristics*

Study Group Control Group P

TNC, x l08/kg 7.2 (3.5-ll.5) 2.9 (l.9-4.6) .00l

CD34+, x l06/kg 6.l(3.5-ll.2) 2.7 (l.4-4.2) 00l

CFU-GM, x l04/kg 68 (32-96) l6 (8-27) 00l

CD3+, x l06/kg 35.7 (27-43.0) 34.5(28-42.2) .9

CD4+, x l06/kg l6.0 (l0.2-24.4) 20.2 (l5.8-29.3) .02

CD8+, x l06/kg l6.2 (9.5-25) l2.9 (8.8-20.9) .02

*Values are median (range).

Days to ANC > 0.5 X 107L 15 (10-22) 21 (13-29) .001

Days to platelet count > 20 X 109/L 17.5 (12-28) 24 (17-32) .001

Units of red cell transfusion 2 (0-7) 3 (0-10) .3

Units of platelets transfusion 4 (1-9) 7 (4-15) .01

*Values are median (range).

(calculated per recipient body weight). There was no difference in the total numbers of CD3+ cells between the study and control marrow grafts. The study group had fewer CD4+ cells (P = .02) but more CD8+ cells (P = .02), resulting in a marked decrease in the CD4+:CD8+ ratio (P = .018).

Engraftment

All patients achieved trilineage engraftment. Patients in the study group had a consistently faster time to engraftment in both neutrophils and platelets (Table 3). The median times to ANC > 0.5 x 109/L were 15 days in the study group versus 21 days in the control group (P = .001). The median times to platelets > 20 x 109/L were 17.5 days in the study group versus 24 days in the control group (P = .001). Patients receiving G-CSF-primed marrow had fewer platelet transfusions before hematopoietic reconstitution (P = .01).

Graft-versus-Host Disease

Eleven (34.4%) of 32 patients in the study group and 8 (44.4%) of 18 patients in the control group developed grade I acute GVHD, which was not a significant difference (P = .8). Two (6.3%) of 32 patients in the study group and 5 (27.8%) of 18 patients in the control group developed grades II to IV acute GVHD (P = .032) (Figure 1). The acute GVHD of the 2 patients in the study group was grade II, limited only to the skin and controlled by a short course of methylprednisone. Of the 5 patients with grades II to IV acute GVHD in the control group, 1 died of acute GVHD of the gastrointestinal tract and 1 died of acute and chronic GVHD of the liver. Four (33.3%) of the 12 patients in the control group who survived more than 6 months had chronic GVHD. Three patients had limited disease, and 1 patient died of extensive disease. Six (24%) of the 25 patients in the study group who survived more than

No. of patients 32 18

Median follow-up (range), mo 24 (6-50) 26 (7-48)

No. of surviving patients 25 12 Causes of death, n

Relapse 4 2

GVHD 0 2

Hepatitis 1 0

Infection 2 2

6 months developed chronic GVHD either during or after CsA was tapered. There were no GVHD-related deaths in the study group. The difference between incidence rates of chronic GVHD in the study and control groups was not significant (24% versus 33.3%, P = .41).

Follow-up

The median follow-up time was 24 months (range,

6-50 months) in the study group and 26 months (range,

7-48 months) in the control group. Four (12.5%) of 32 patients in the study group and 2 (11.1%) of 18 patients in the control group died of disease relapse (P = .87). Of the 4 patients in the study group who died of relapse, 3 were in CML accelerated phase prior to undergoing transplantation. Three patients in the study group (9.4%) and 4 patients in the control group (22.2%) died of transplantation-related causes (Table 4). As of this report, 25 of 32 patients in the study group and 12 of 18 patients in the control group were still alive. The estimated probabilities of disease-free survival at 3 years for the study group and control group were 78.1% and 66.7%, respectively (Figure 2) and were not statistically significant according to the log-rank test (P = .32). However, there was a trend in favor of improved disease-free survival in the study group.

DISCUSSION

This study, performed during 1997-2000 in a single center, compared the outcomes in a homogenous group of patients with CML of HLA-identical marrow transplantations with and without donor G-CSF priming prior to the mar-

Figure 1. Probability of developing acute grade II to IV GVHD.

Figure 2. Disease-free survival rates.

Table 5. Comparison of Engraftment and Incidence Rates of Acute GVHD from Various Studies

Median Time Median Time

to ANC > to Platelets > Postgrafting Acute

Study Author Donor G-CSF Treatment Types of Grafts 500/mm3, d 20,000/mm3, d G-CSF GVHD (II-IV)

Serody Filgrastim 10 |g/kg/d X 4 d Marrow (n = 26) I6 I6 No 27%

Filgrastim 10 |g/kg/d X 4 d PBSCs (n = 20) I7 I3 No 60%

Isola Filgrastim 10 |g/kg/d X 2 d Marrow (n = 17) I7 2I No I9%

None Marrow (n = 112) 24 2S No I9%

Ji Lenograstim 3-4 |g/kg/d X 7 d Marrow (n = 32) IS I7.S Yes 6.3%

None Marrow (n = 18) 2I 24 Yes 27.8%

Couban Filgrastim 10-15 |g/kg/d X 4 d Marrow (n = 29) I8 22 No N/A

None Marrow (n = 20) 22 27 No N/A

row harvest. Many groups have consistently shown that G-CSF-mobilized PBSCs engraft earlier than non-G-CSF-stimulated steady-state marrow in matched sibling transplantation settings [3-6]. There were only a few reported studies comparing G-CSF-primed marrow versus G-CSF-mobilized PBSCs [12,13] and G-CSF-primed versus non-primed donor marrow [20,21]. Our results confirmed the findings of the Isola and Couban groups [20,21] that G-CSF donor priming facilitates the marrow engraftment. The median times for neutrophil engraftment after G-CSF-primed marrow transplantations were almost identical among these studies, and there was no difference between G-CSF-mobilized PBSCs versus G-CSF-primed marrow transplantations, as summarized in Table 5.

The median times to platelets > 20 x 109/L after G-CSF-primed marrow transplantation were also significantly faster than those for nonprimed marrow grafts in all 4 studies (Table 5). G-CSF marrow priming facilitated both neu-trophil and platelet engraftment, suggesting that G-CSF increased not only committed myeloid progenitor cells but also early noncommitted stem cells. The prompt engraft-ment observed in G-CSF-primed marrow transplantations was probably caused by the relatively high numbers of CD34+ cells infused. Mavroudis et al. reported 4 incidents of late graft failure after the initial engraftment of 12 patients who received G-CSF-stimulated marrow grafts with CD34+ cell selection [22]. We and other groups saw no incidence of late graft failure in the patients who received G-CSF-primed marrow without further manipulation.

The G-CSF doses used for donor-marrow priming differed in these 4 studies (Table 5). The neutrophil and platelet engraftment were very similar among the G-CSF-primed marrow transplantation studies. We used postgrafting G-CSF treatment until the ANC was > 0.5x109/L, and other study groups did not use postgrafting G-CSF. It appeared that postgrafting G-CSF did not further facilitate engraftment if the donor marrow had already been exposed to G-CSF. The question that remains to be answered is, what is the optimal dose of G-CSF for donor-marrow priming?

Bensinger et al. compared G-CSF-mobilized PBSCs to historical steady-state marrow transplantations and reported a similar incidence of GVHD, although the T-cell numbers in G-CSF-mobilized PBPCs were often 10 times higher than those in the marrow [6]. Serody et al. and Darrant and Morton compared the incidence rates of acute and chronic

GVHD in patients treated with G-CSF-mobilized PBSCs and those treated with G-CSF-primed marrow [12,13]. Both studies found that patients treated with G-CSF- primed marrow grafts had lower incidence rates of both acute and chronic GVHD, especially chronic GVHD. One of the explanations for the lower rates was that the numbers of T-cells in the G-CSF-primed marrow were much lower than the numbers in the G-CSF-mobilized PBSCs. The incidence of acute GVHD in our study was surprisingly low in the patients who received G-CSF-primed marrow grafts and postgrafting G-CSF. Only 2 (6.3%) of 32 patients developed grade II acute GVHD, which was limited to the skin and quickly resolved with a short course of steroid treatment. The incidence rates of grades II to IV acute GVHD were not only significantly lower in our study group than in our control group, but also much lower than the incidence rates for G-CSF-primed marrow transplantations done by other groups, as shown in Table 5. Serody et al. reported a 27% incidence rate of grades II to IV acute GVHD after the matched sibling G-CSF-primed marrow transplantations. Both our study and the study of Serody et al. were done with patients who received matched sibling transplants with similar CsA and MTX as GVHD prophylaxis. The major difference in the studies was that we routinely used postgrafting G-CSF to ANC > 0.5 x 109/L, whereas the other groups did not use the postgrafting G-CSF. We used lenograstim, which is a glycosylated recombinant human G-CSF (Chugai Pharmaceutical, Tokyo, Japan). The others most likely used filgrastim, a nonglycosylated G-CSF (Amgen, Thousand Oaks, CA), considering that the studies from other groups were done within the market territory of Amgen company, although the authors did not specifically mention the G-CSF sources in their report. Our results demonstrated that G-CSF did not change the total numbers of CD3+ T-cells but altered subsets of T-cells and significantly lowered the CD4:CD8 ratio. It is known that cytokines produced by CD4+ and CD8+ T-cells can be characterized into 2 patterns, Th1 (interleukin [IL]-2 and interferon-y) and Th2 responses (IL-4 and IL-10)

[23]. The Thl-type responses are critical for acute GVHD

[24]. Treatment that induces the Th2 response reduces GVHD [25]. G-CSF polarizes the T-cell differentiation from Th1- to Th2-type cells and induces Th2 responses with the production of IL-4 and IL-10 [26]. Tayebi et al. compared phenotypic and functional properties of lymphocytes from bone marrow or PBSC donors after G-CSF treatment in a

randomized study [27]. These researchers found not only that the lymphocyte counts in the marrow grafts were 10-fold lower than the counts in the G-CSF- mobilized PBCS grafts, but also that the production of Th1 cytokines (EL-2, interferon^, tumor necrosis factor) after G-CSF treatment was also severely impaired [27]. Thus, modulation of cytokine production by G-CSF may help to explain the surprisingly low incidence of clinical acute GVHD observed in our study group with the combination of donor G-CSF marrow priming and postgrafting G-CSF treatment. Whether the glycosy-lation of G-CSF increases T-cell polarization and Th2 cytokine production remains to be answered.

The incidence rates of chronic GVHD and relapse are relatively low in CML patients with HLA-identical sibling transplantations compared to patients with mismatched transplantations and those with advanced/high-risk leukemia/ lymphoma. Our study showed a trend of decreasing of chronic GVHD and improving disease-free survival in the patients who received the G-CSF-primed marrow grafts (78.1% versus 66.7%), but the difference did not reach statistical significance according to the log-rank test. To compare the survival curves using a 2-tailed log-rank test at the 0.05 level of significance with 1 group having 78.1% long-term survival rate and the other a 66.7% survival rate would require us to randomize 243 patients per treatment arm (80% power) or 326 patients per treatment arm (90% power). Enrolling this number of patients would require an 8- to 10-year enrollment period for a single center and, certainly, would need a multicenter collaborative study.

In summary, the present study describes the allogeneic marrow transplantation outcome for 50 patients with CML using HLA-identical sibling marrow grafts with and without G-CSF donor priming prior to the marrow harvest. G-CSF donor priming increased the numbers of CD34+ and CFU-GM cells and facilitated both neutrophil and platelet reconstitution. The median times to neutrophil and platelet engraftment were comparable to those of G-CSF-mobilized PBSCs. Postgrafting G-CSF treatment did not further accelerate the engraftment if the donor marrow had been primed with G-CSF. However, the postgrafting G-CSF treatment administered to the recipients appeared to reduce the incidence of acute GVHD. G-CSF alters the CD4:CD8 ratio and polarizes T-cells from Th1 to Th2 responses. The combination of G-CSF donor-marrow priming and postgrafting G-CSF treatment dramatically reduced the incidence of grades II to IV acute GVHD to a surprisingly low rate, 6.3% in HLA-identical sibling marrow transplantations. There were no significant differences in the incidence rates of chronic GVHD, relapse rates, and overall disease-free survival rates between the G-CSF-primed and nonprimed marrow transplantation patients during a median of 24 months of follow-up. However, there was a trend in favor of improved disease-free survival at 3 years in G-CSF-primed marrow transplantation patients. A multicenter collaborative trial with large patient numbers is warranted.

ACKNOWLEDGMENT

Dr. Xun is supported by the National Marrow Donor Program, Ami Strelzer Manasevit Scholars Program, and NIH grant, CA91795-02.

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