Scholarly article on topic 'Effects of Priming with Recombinant Human Granulocyte Colony–Stimulating Factor on Conditioning Regimen for High-Risk Acute Myeloid Leukemia Patients Undergoing Human Leukocyte Antigen–Haploidentical Hematopoietic Stem Cell Transplantation: A Multicenter Randomized Controlled Study in Southwest China'

Effects of Priming with Recombinant Human Granulocyte Colony–Stimulating Factor on Conditioning Regimen for High-Risk Acute Myeloid Leukemia Patients Undergoing Human Leukocyte Antigen–Haploidentical Hematopoietic Stem Cell Transplantation: A Multicenter Randomized Controlled Study in Southwest China Academic research paper on "Clinical medicine"

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Abstract of research paper on Clinical medicine, author of scientific article — Lei Gao, Qin Wen, Xinghua Chen, Yao Liu, Cheng Zhang, et al.

Abstract HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is an effective and immediate treatment for high-risk acute myeloid leukemia (HR-AML) patients lacking matched donors. Relapse remains the leading cause of death for HR-AML patients after haplo-HSCT. Accordingly, the prevention of relapse remains a challenge in the treatment of HR-AML. In a multicenter randomized controlled trial in southwestern China, 178 HR-AML patients received haplo-HSCT with conditioning regimens involving recombinant human granulocyte colony–stimulating factor (rhG-CSF) or non–rhG-CSF. The cumulative incidences of relapse and graft-versus-host disease (GVHD), 2-year leukemia-free survival (LFS), and overall survival (OS) were evaluated. HR-AML patients who underwent the priming conditioning regimen with rhG-CSF had a lower relapse rate than those who were treated with non-rhG-CSF (38.2%; 95% confidence interval [CI], 28.1% to 48.3% versus 60.7%, 95% CI, 50.5% to 70.8%; P < .01). The cumulative incidences of acute GVHD, chronic GVHD, transplantation-related toxicity, and infectious complications appeared to be equivalent. In total, 53 patients in the rhG-CSF–priming group and 31 patients in the non-rhG-CSF–priming group were still alive at the median follow-up time of 42 months (range, 24 to 80 months). The 2-year probabilities of LFS and OS in the rhG-CSF–priming and non-rhG-CSF–priming groups were 55.1% (95% CI, 44.7% to 65.4%) versus 32.6% (95% CI, 22.8% to 42.3%) (P < .01) and 59.6% (95% CI, 49.4% to 69.7%) versus 34.8% (95% CI, 24.9% to 44.7%) (P < .01), respectively. Multivariate analyses indicated that the 2-year probability of LFS of patients who achieved complete remission (CR) before transplantation was better than that of patients who did not achieve CR. The 2-year probability of LFS of patients with no M4/M5/M6 subtype was better than that of patients with the M4/M5/M6 subtype in the G-CSF–priming group (67.4%; 95% CI, 53.8% to 80.9% versus 41.9%; 95% CI, 27.1% to 56.6%; P < .05). This study suggests that the rhG-CSF–priming conditioning regimen is an acceptable choice for HR-AML patients, especially for the patients with no M4/M5/M6 subtype who achieved CR before transplantation.

Academic research paper on topic "Effects of Priming with Recombinant Human Granulocyte Colony–Stimulating Factor on Conditioning Regimen for High-Risk Acute Myeloid Leukemia Patients Undergoing Human Leukocyte Antigen–Haploidentical Hematopoietic Stem Cell Transplantation: A Multicenter Randomized Controlled Study in Southwest China"

AS BMI

American Society for Blood and Marrow Transplantation

Biology of Blood and Marrow Transplantation

journal homepage: www.bbmt.org

Effects of Priming with Recombinant Human Granulocyte Colony-Stimulating Factor on Conditioning Regimen for High-Risk Acute Myeloid Leukemia Patients Undergoing Human Leukocyte Antigen-Haploidentical Hematopoietic Stem Cell Transplantation: A Multicenter Randomized Controlled Study in Southwest China

Lei Gao1, Qin Wen1, Xinghua Chen1, Yao Liu1, Cheng Zhang1, Li Gao1, Peiyan Kong1, Yanqi Zhang2, Yunlong Li1, Jia Liu1, Qingyu Wang1, Yi Su3, Chunsen Wang4, Sanbin Wang5, Yun Zeng6, Aihua Sun1, Xin Du1, Dongfeng Zeng1, Hong Liu1, Xiangui Peng1, Xi Zhang1, *

1 Department of Hematology, Xinqiao Hospital, Third Military Medical University, Chongqing, China

2 Department of Health Statistics, College of Military Preventive Medicine, Third Military Medical University, Chongqing, China

3 Department of Hematology, General Hospital of Chengdu Military Region of PLA, Chengdu, China

4 Department of Hematology, Sichuan Provincial Peoples Hospital, Chengdu, China

5 Department of Hematology, General Hospital of Kunming Military Region of PLA, Kunming, China

6 Department of Hematology, Affiliated Hospital of Kunming Medical College, Kunming, China

CrossMark

Article history: Received 19 February 2014 Accepted 3 August 2014

Key Words:

Acute myeloid leukemia High risk Haploidentical Hematopoietic stem cell transplantation Granulocyte

colony—stimulating factor Priming

ABSTRACT

HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is an effective and immediate treatment for high-risk acute myeloid leukemia (HR-AML) patients lacking matched donors. Relapse remains the leading cause of death for HR-AML patients after haplo-HSCT. Accordingly, the prevention of relapse remains a challenge in the treatment of HR-AML. In a multicenter randomized controlled trial in southwestern China, 178 HR-AML patients received haplo-HSCT with conditioning regimens involving recombinant human granulocyte colony—stimulating factor (rhG-CSF) or non—rhG-CSF. The cumulative incidences of relapse and graft-versus-host disease (GVHD), 2-year leukemia-free survival (LFS), and overall survival (OS) were evaluated. HR-AML patients who underwent the priming conditioning regimen with rhG-CSF had a lower relapse rate than those who were treated with non-rhG-CSF (38.2%; 95% confidence interval [CI], 28.1% to 48.3% versus 60.7%, 95% CI, 50.5% to 70.8%; P < .01). The cumulative incidences of acute GVHD, chronic GVHD, transplantation-related toxicity, and infectious complications appeared to be equivalent. In total, 53 patients in the rhG-CSF—priming group and 31 patients in the non-rhG-CSF—priming group were still alive at the median follow-up time of 42 months (range, 24 to 80 months). The 2-year probabilities of LFS and OS in the rhG-CSF—priming and non-rhG-CSF—priming groups were 55.1% (95% CI, 44.7% to 65.4%) versus 32.6% (95% CI, 22.8% to 42.3%) (P < .01) and 59.6% (95% CI, 49.4% to 69.7%) versus 34.8% (95% CI, 24.9% to 44.7%) (P < .01), respectively. Multivariate analyses indicated that the 2-year probability of LFS of patients who achieved complete remission (CR) before transplantation was better than that of patients who did not achieve CR. The 2-year probability of LFS of patients with no M4/M5/M6 subtype was better than that of patients with the M4/M5/M6 subtype in the G-CSF—priming group (67.4%; 95% CI, 53.8% to 80.9% versus 41.9%; 95% CI, 27.1% to 56.6%; P < .05). This study suggests that the rhG-CSF—priming conditioning regimen is an acceptable choice for HR-AML patients, especially for the patients with no M4/M5/M6 subtype who achieved CR before transplantation.

© 2014 Published by Elsevier Inc. on behalf of American Society for Blood and Marrow Transplantation.

Financial disclosure: See Acknowledgments on page 1938.

* Correspondence and reprint requests: Xi Zhang, Department of Hematology, Xinqiao Hospital, Third Military Medical University, Chongqing, China 400037.

E-mail address: zhangxxi@sina.com (X. Zhang).

INTRODUCTION

High-risk acute myeloid leukemia (HR-AML) has not been treated with the same success for favorable or intermediate acute myeloid leukemia [1]. Although the majority of patients with HR-AML achieve complete remission (CR) after intensive induction therapy, most of them will die from

http://dx.doi.org/10.1016/j.bbmt.2014.08.001

1083-8791/® 2014 Published by Elsevier Inc. on behalf of American Society for Blood and Marrow Transplantation.

disease relapse [2]. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an established treatment for patients with HR-AML [3], and the first choice of a donor for a patient with allo-HSCT is an HLA-identical sibling. However, such a donor is not possible for patients with no siblings. The alternatives are an unrelated HLA-matched donor, umbilical cord blood, or a family member who serves as an HLA-haploidentical donor. An unsuccessful or time-consuming donor search process can limit the application of unrelated donors for HSCT. Additionally, the outcome of cord blood transplantation has been limited by incomplete hematopoi-etic and immune reconstruction [4]. As such, donor availability is among the major obstacles to the success of allo-HSCT for the treatment of HR-AML.

The majority of patients have family members who are identical for 1 HLA haplotype and fully mismatched for the other, and who can serve as immediate HSCT donors. Thus, HSCT from an HLA-haploidentical relative offers the option for treatment with an immediate transplantation for virtually all HR-AML patients lacking matched donors [5]. Basic and clinical research on HLA-haploidentical HSCT (haplo-HSCT) has been ongoing for more than 20 years. The best survival rate for AML in adults who underwent transplantation after achieving CR is approximately 55%. However, relapse remains the leading cause of death in patients after haplo-HSCT, especially those with HR-AML [6]. Accordingly, the prevention of relapse remains a challenge in the treatment of HR-AML after haplo-HSCT.

Hematopoietic growth factors stimulate AML cells in culture, activating metabolic processes and the cell cycle. In vitro, the simultaneous exposure of leukemic cells to chemotherapy and growth factors, such as granulocyte colony—stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor, and interleukin-3, referred to as growth-factor priming, increased the susceptibilityofcells to killing by chemotherapy, especially by the cell cycle—specific agent cytosine arabinoside (Ara-C) [7-9]. A number of studies have addressed this concept in the remission induction of AML patients [10-12]. We previously reported a randomized trial involving 114 refractory and relapsed AML patients who were treated with recombinant human G-CSF (rhG-CSF), in addition to Ara-C, etoposide, and aclarubicin, and we found a favorable effect of rhG-CSF—priming chemotherapy on the refractory and relapse AML [13].

Furthermore, rhG-CSF-priming conditioning regimens have been applied sporadically in the setting of refractory and relapsed AML patients before allo-HSCT [14]. However, there have not been any studies on rhG-CSF priming in myeloablative haplo-HSCT. We conducted a multicenter randomized controlled study to determine whether treatment with rhG-CSF during the conditioning regimen before haplo-HSCT improved leukemia-free survival (LFS) in patients with HR-AML by reducing the rate of relapse.

PATIENTS AND METHODS Patients and Donors

HR-AML patients (n = 178) who required allo-HSCT but lacked HLA-matched donors were enrolled in this study. The patients were hospitalized at 5 transplantation centers in southwest China. The patients were included if they fulfilled at least 1 of the following criteria defining HR-AML: no response to induction chemotherapy, relapse within 6 months after induction or consolidation therapy, relapse with 6 months after induction therapy that could not be relieved using the original induction therapy, >2 relapses or relapse after autologous HSCT, or unfavorable cytogenetics [15]. Patients with serious liver and kidney dysfunction, those who were considered to have allergic manifestations for rhG-CSF, and those who had additional tumors in need of treatment were excluded. This study enrolled

patients from October 29, 2007, through June 27, 2011. It was approved by the ethics committees of the participating institutions and was conducted in accordance with the Declaration of Helsinki. All of the participants provided their written informed consent.

Study Design and Conditioning Regimen

The patients were enrolled and randomly assigned to receive the rhG-CSF—priming regimen or non-rhG-CSF—priming regimen before haplo-HSCT. The non-rhG-CSF—priming regimen consisted of chlorethyl cyclo-hexyl nitrosourea, Ara-C, busulfan, cyclophosphamide, and antithymocyte globulin (ATG). The doses used were as follows: chlorethyl cyclohexyl nitrosourea 200 mg/m2 orally once on day -9, Ara-C 4 g/m2 once daily intravenously on days -8 to -7, busulfan 3.2 mg/kg once daily intravenously on days -6 to -4, cyclophosphamide 1.8 g/m2 once daily intravenously on days -3 to -2, and ATG (Thymoglobulin, Genzyme, Cambridge, MA) 2.5 mg/ kg once daily intravenously for 4 consecutive days on days -5 to -2. For the rhG-CSF—priming regimen, rhG-CSF (rhG-CSF, Filgrastim, Kirin, Gunma, Japan; 5 mg/kg once daily subcutaneously on days -10 to -7) was added to the above conditioning regimen.

Donor Selection and Stem Cell Graft Harvesting

HLA-A, HLA-B, and HLA-DR typing were performed using a highresolution DNA technique [16]. For the 178 haplo-HSCT patients, 114 donors were full haplotype mismatches, 56 donors were haploidentical with 2 mismatched loci, and 8 donors were haploidentical with 1 mismatched locus. All of the patients received peripheral blood (PB) combined with bone marrow (BM) HSCT. The donor PB and BM cells were collected using standard mobilization protocols [16]. Generally, >2.0 $ 108/kg peripheral blood monocytes, >4.0 x 108/kg BM nucleated cells, and >6.0 x 106/kg CD34+ cells were obtained.

Evaluation of Engraftment

Neutrophil engraftment was defined as an absolute neutrophil count (ANC) > .5 x 109/L for 3 consecutive days. Platelet engraftment was defined as a platelet count >20 x 109/L for 3 consecutive days without transfusion. Hematopoietic chimerism was evaluated by fluorescence in situ hybridization for sex-mismatched patient-donor pairs and by PCR amplification of short tandem repeats using peripheral blood samples from the donor and the recipient for sex-matched pairs.

Graft-versus-Host Disease Prophylaxis and Management

In addition to basic treatment with ATG in the conditioning regimen, all of the transplant recipients received cyclosporine (CsA), mycophenolate mofetil (MMF), and short-term methotrexate (MTX) for graft-versus-host disease (GVHD) prophylaxis. On day +1, MTX (15 mg/m2) was administered intravenously, and 10 mg/m2 was given on days +3, +6, and +11 after transplantation. Continuous intravenous CsA (1.5 mg/kg once daily) was started on day -7, increased to 2.5 mg/kg once daily on day -1, and was continued until the patients were able to tolerate oral medication. CsA (2.5 mg/kg twice a day) was given orally with trough levels targeted between 200 and 400 ng/mL until 180 days; it was then tapered and taken for an additional 180 days until fully discontinued. MMF (7.5 mg/kg twice per day) was started on day +1 and was discontinued on day +90. Acute GVHD (aGVHD) and chronic GVHD (cGVHD) were treated as previously described by Gao Let al. [16,17].

Infection Prevention and Supportive Care

All patients were cared for in a laminar air flow room and received prophylactic antibiotics when their ANC was less than .5 x 109/L. Nor-floxacin, trimethoprim sulfamethoxazole, and ganciclovir were routinely administered as previously reported [18]. All blood products were irradiated and filtered. Red cell and platelet transfusions were administered to maintain hemoglobin levels of >80 g/L and platelet counts >20 x 109/L. All patients received rhG-CSF beginning from ANC < .5 x 109/L after hemato-poietic stem cell infusion until donor engraftment was achieved.

Statistical Analyses

The date of the last follow-up for all of the surviving patients was June 27, 2013. The primary objective of the study was to determine the effects of adding rhG-CSF to the conditioning regimen on the cumulative incidence of relapse and on LFS after haplo-HSCT. A secondary objective was to assess the incidences of engraftment, GVHD, infection and transplantation-related toxicity (TRT), and overall survival (OS). We had planned to enroll 178 patients over a close to 4-year period, with an additional follow-up of 2 years after the enrollment of the last patient. This number of patients would have given the study a power of 86% to show an absolute increase of 20% in the rate of 2-year LFS (from 50% to 70%) with the use of rhG-CSF. The Mann-

Table 1

Patient, Donor, and Graft Characteristics

Variable G-CSF Non-rhG-CSF Statistics P Value

No. of patients 89 (100.0) 89 (100.0)

Age at transplantation, yr -.139 .889*

<18 22 (24.7) 20 (22.5)

18-40 53 (59.6) 56 (62.9)

>40 14 (15.7) 13 (14.6)

Patient sex .563 .453y

Male 40 (44.9) 45 (50.6)

Female 49 (55.1) 44 (49.4)

Diagnosis of FAB .362 .548y

M4/M5/M6 43 (48.3) 40 (44.9)

No- M4/M5/M6 46 (51.7) 49 (55.1)

Disease status before HSCT .882 .663y

CR1 38 (42.7) 44 (49.5)

>CR2 22 (24.7) 19(21.3)

PR/NR/RE 29 (32.6) 26 (29.2)

HLA compatibility - .795z

1 loci-mismatched 4 (4.5) 3 (3.4)

2 loci-mismatched A, B 28 (31.5) 32 (36.0)

3-loci-mismatched A, DRB1 57 (64.0) 54 (60.6)

Donor-recipient sex match .630 .889y

Female-female 19 (21.4) 18 (20.2)

Female-male 18 (20.2) 21 (23.6)

Male-male 22 (24.7) 24 (27.0)

Male-female 30 (33.7) 26 (29.2)

Donor-recipient relationship .251 .882y

Mother-child 16 (18.0) 15 (16.9)

Father-child 29 (32.6) 33 (37.1)

Siblings 44 (49.3) 41 (46.1)

Prognostic risk category -.532 .595*

Favorable 8 (9.0) 8 (9.0)

Intermediate 47 (52.8) 51 (57.3)

Unfavorable 34 (38.2) 30 (33.7)

PR indicates partial remission; NR, nonremission; RE, relapse. Data presented are n (%). * Mann-Whitney test. y Chi-square test. z Fisher exact test.

Whitney test, chi-square test, and Fisher exact test were used to analyze the differences in the baseline characteristics, therapeutic effects and adverse effects between the 2 groups. The Kaplan-Meier analysis and log-rank test were used to analyze the 2-year LFS and OS of each group. In these analyses, the patients were recorded as either deceased or censored at the end of the study.

RESULTS

Patient and Donor Characteristics and Engraftment

Between October 2007 and June 2013, 89 HR-AML patients were assigned to the conditioning regimen combined with rhG-CSF (group A) and 89 HR-AML patients were assigned to the conditioning regimen without rhG-CSF (group B). The characteristics of the patients and donors before transplantation are shown in Table 1. The 2 treatment groups did not differ in their clinical, hematological, cyto-genetic, or therapeutic features.

All patients received 9.84 (group A: 9.36; group B: 10.03) x 108/kg mononuclear cells. The median doses of infused CD34+ and CD3+ cells were 7.21 (group A: 7.81; group B: 6.92) x 106/kg and 2.14 (group A: 1.88; group B: 2.47) x 108/kg, respectively. All patients achieved full donor chimerism by day 28 after HSCT. ANC exceeded .5 x 109/L within 13 days (range, 10 to 18) in group A and 12 days (range, 10 to 16) in group B. All patients in both groups achieved platelet engraftment after a median of 16 days (range, 12 to 23 days in group A; 13 to 26 days in group B). Secondary granulocytopenia and thrombocytopenia occurred in 6 patients (2 in group A and 4 in group B). Among these patients, 3 relapsed on days +106, +142, and +177,

respectively, whereas the other patients recovered after treatment with rhG-CSF and platelet transfusions.

GVHD Incidence and Severity

In group A, 37 (41.6%) patients had grade II aGVHD, and 9 (10.1%) patients had grade III and IV aGVHD. In group B, 34 (38.2%) patients had II aGVHD, and 13 (14.6%) patients had grade III and IV aGVHD. The 100-day cumulative incidences of grade II to IV aGVHD and grade III and IV aGVHD were 42.4% (95% confidence interval [CI], 32.0% to 52.9%) versus 39.3% (95% CI, 28.9% to 49.7%) (P > .05) and 10.2 (95% CI, 3.9% to 16.5%) versus 15.7 (95% CI, 7.8% to 23.6%) (P > .05), respectively (Table 2, Figure 1). The clinical manifestations of grade III to IV aGVHD included severe skin rash, diarrhea, hepatic dysfunction, and hematuria. Methylprednisolone (2 mg/kg once daily) was administered intravenously and was tapered as scheduled or based on the therapeutic response. The 5 patients with grade III aGVHD who were resistant to methylprednisolone received daclizumab, and CsA was changed to tacrolimus to control aGVHD. Among these patients, 3 (1 in group A and 2 in group B) showed recurrent episodes of serious aGVHD, and they died from infections on days +115, +149, and +212, respectively.

All patients who survived longer than 100 days after HSCT were evaluated for incidence of cGVHD. Chronic GVHD developed in 47 (52.8%) patients in group A and in 35 (39.3%) patients in group B. In total, 34 of47 (72.3%) patients in group A and 25 of 35 (71.4%) patients in group B showed limited cGVHD when CsA was tapered at the scheduled time (as

Table 2

Clinical Outcomes of rhG-CSF and Non-rhG-CSF- -Priming Haplo-HSCT

Outcomes rhG-CSF Non-rhG-CSF Statistics P Value

Engraftment 89(100) 89(100)

Acute GVHD

Grade II 37 (41.6) 34 (38.2) .211 .646*

Grade III-IV 9(10.1) 13 (14.6) .83 .362*

Chronic GVHD

Total 47 (52.8) 35 (39.3) 3.256 .071*

Limited 34 (38.2) 25 (28.1) 2.054 .152*

Extensive 13 (14.6) 10(11.2) .449 .503*

Infection within 100 d 4.608 .466*

No infection 10(11.2) 8 (9.0)

Severe septicemia 16(18.0) 9(10.1)

CMV antigenemia 18 (20.2) 24 (27.0)

Zoster 5 (5.6) 3 (3.4)

IFI 7 (7.9) 11 (12.4)

Mixed infection 11 (12.4) 14(15.7)

Fever 27 (30.3) 23 (25.8)

Gastrointestinal tract 89(100) 89(100) - -

Oral mucositis 60 (67.4) 68 (76.4) 1.780 .182*

Transaminase increase 67 (75.3) 62 (69.7) .704 .401 *

Serum creatinine level increase 4 (4.5) 3 (3.4) - 1.000y

Causes of death

aGVHD 2 (2.2) 1(1.1) - 1.000y

Infection 4 (4.5) 5 (5.6) - 1.000y

Relapse 30 (33.7) 52 (58.4) 10.944 .001 *

Two-year OS 59.6 (49.4-69.7) 34.8 (24.9-44.7) 7.825 .005z

Two-year LFS 55.1 (44.7-65.4) 32.6 (22.8-42.3) 8.119 .004z

Data presented are n (%). * Chi-square test. y Fisher exact test.

z Kaplan-Meier analysis and log-rank test.

shown in Table 2). The 2-year cumulative incidence of cGVHD for group A and group B was 53.3 (95% CI, 42.8% to 63.8%) and 39.3 (95% CI, 29.2% to 49.5%), respectively (P > .05) (Figure 2). Patients with cGVHD remained on CsA/ tacrolimus and steroids (long-term taper).

Leukemia Relapse

Relapse occurred in 34 of 89 (38.2%) patients in group A and in 54 of 89 (60.7%) patients in group B. The estimated cumulative incidences of relapse were 38.2% (95% CI, 28.1% to 48.3%) and (60.7% [95% CI, 50.5% to 70.8%], respectively P < .01) (Figure 3). Next, we performed multivariate analyses based on the subtype of AML, cytogenetics, and disease status before HSCT. In group A, the relapse rate of patients with no M4/M5/M6 was lower than that of patients with M4/ M5/M6 (28.3% [95% CI, 15.2% to 41.3%] versus 48.8 [95% CI,

33.9% to 63.8%], P < .05). In group B, there was no difference between the 2 subtype groups (58% [95% CI, 44.3% to 71.7%] versus 64.1% [95% CI, 49.0% to 79.2%], P > .05). In both group A and group B, the relapse rate in patients with CR (CR1 or > CR2) was lower than that of patients with partial remission, no remission, or relapse. However, there were no significant differences among the different prognostic risk categories between the 2 groups (Table 3).

The rescue therapy used to treat relapse included donor lymphocyte infusion in 21 patients, chemotherapy in 19 patients, donor lymphocyte infusion and chemotherapy in 27 patients, haplo-HSCT from the same donors in 4 patients, and transplants from other haploidentical donors in 7 patients. Ten patients did not receive any treatment because of rapid, aggressive relapses. Six patients achieved CR again after the second transplantation, whereas 78 patients died (52 of

Figure 1. The cumulative incidence of aGVHD. (A)Shows Group A and (B) shows group B.

Figure 2. The cumulative incidence of cGVHD. (A) Shows group A and (B) shows group B.

leukemia, 23 of serious infection in BM suppression, and 3 of transplantation-related mortality (TRM) after a second transplantation).

TRTs and Infectious Complications

All 178 patients had gastrointestinal (GI) toxicity, including vomiting, anorexia, and diarrhea. 88.8% (158 of 178) patients had grade 1 to 2 GI toxicity, and only 11.2% (20 of 178) patients had grade 3 GI toxicity. Oral mucositis occurred in 128 patients (60 in group A and 68 in group B, P > .05), but none suffered from grade 4 oral mucositis. A slightly elevated transaminase level was commonly observed (67 in group A and 62 in group B, P > .05). Serum creatinine levels increased 2-fold or more during the 100 days after transplantation, compared with that before transplantation, in only 7 patients (4 in group A and 3 in group B, P > .05). No deaths resulted from lethal organ toxicities from the conditioning regimen during the 100 days after HSCT.

The following infectious complications were observed during the 100 days after haplo-HSCT: cytomegalovirus (CMV) antigenemia, 20.2% versus 27.0%; zoster, 5.6% versus 3.4%; severe septicemia, 18.0% versus 10.1%; invasive fungi infections (IFIs), 7.9% versus 12.4%; mixed infection, 12.4% versus 15.7%; and fever with no identifiable infection, 30.3% versus 25.8% for group A and group B, respectively. Eight

0.00 20.00 40.00 60.00 80.00

Time after transplant (months)

Figure 3. The cumulative incidence of relapse in different groups (P = .004)

patients with CMV antigenemia developed CMV pneumonia. These infectious complications, with the exception of 5 cases of IFIs and 4 cases of severe septicemia, were controllable with the use of conventional antimicroorganism therapies.

Survival

As of June 30,2013, 53 patients in group A and 31 patients in group B were still alive at the median follow-up time of 42 months (range, 24 to 80 months). The causes of TRM included leukemia relapse in 82 cases, severe infection in 9 cases, and recurrent episodes of serious aGVHD in 3 cases. The 2-year probabilities of LFS and OS in the group A and group B were 55.1 (95% CI, 44.7% to 65.4%) versus 32.6 (95% CI, 22.8% to 2.3%) and 59.6 (95% CI, 49.4% to 69.7%) versus 34.8 (95% CI, 24.9% to 44.7%) (P < .01) (Table 2, Figure 4). Multivariate analyses indicated that disease status at transplantation and French-American-British (FAB) subtype of AML were the only significant factors (Table 4) (P < .05). In both groups, the 2-year probability of LFS of patients who achieved CR before transplantation was better than that of patients who remained at partial remission, nonremission, or relapse (63.3% [95% CI, 51.1% to 75.5%] versus 37.9% [95% CI, 20.3% to 55.6%] in group A, P < .05; 38.1% [95% CI, 26.1% to 50.1%] versus 19.2% [95% CI, 4.1% to 34.4%] in group B, P < .01). The 2-year probability of LFS of patients with no M4/M5/M6 subtype was better than that of patients with the M4/M5/M6 subtype in group A (67.4% [95% CI, 53.8% to 80.9%] versus 41.9% [95% CI, 27.1% to 56.6%], P < .05). In contrast, no difference was observed in group B (35.9% [95% CI, 20.8% to 51.0%] versus 30.0% [95% CI, 17.3% to 42.7%], P > .05).

DISCUSSION

For decades, the cell cycle—dependent agent Ara-C has been a cornerstone of the treatment of patients with AML. The combined exposure of AML cells to cytokines, such as G-CSF, granulocyte macrophage colony—stimulating factor, and IL-3, with Ara-C increases the intracellular levels of the active metabolite, cytosine arabinoside triphosphate, elevates the incorporation of Ara-C into cellular DNA, and enhances the killing of leukemic blasts and leukemic progenitor cells by the antimetabolite [19,20]. Previous studies by Pabst [10] and by our group [13] have shown that AML patients benefited from rhG-CSF—priming remission conduction, with improved rates of CR.

In the current multicenter randomized controlled study, we addressed the effects of different priming conditioning regimens for HR-AML patients before haplo-HSCT. The

Table 3

Multivariate Analysis of Relapse in rhG-CSF and Non- rhG-CSF Priming Haplo-HSCT

Risk Factor rhG-CSF (n = 89) Non-rhG-CSF (n = 89)

Relapse, % (95% CI) Chi-Square P Value Relapse, % (95% CI) Chi-Square P Value

Diagnosis of FAB 4.268 .039 .398 .528

M4/M5/M6 48.8 (33.9-63.8) 64.1 (49.0-79.2)

No- M4/M5/M6 28.3 (15.2-41.3) 58 (44.3-71.7)

Disease status before HSCT 8.402 .004 4.998 .025

CR1 and >CR2 28.3 (16.9-39.7) 55.6 (43.3-67.8)

PR/NR/RE 58.6 (40.7-76.5) 73.1 (56.0-90.1)

Prognostic risk category .088 .957 4.058 .131

Favorable 37.5 (4.0-71.0) 37.5 (4.0-71.0)

Intermediate 36.2 (22.4-49.9) 56.9 (43.3-70.5)

Unfavorable 41.2 (24.6-57.7) 73.3 (57.5-89.2)

Kaplan-Meier analysis and log-rank test were used.

concept of the rhG-CSF-priming conditioning regimen was to eradicate micro-residual disease of AML by promoting the entry of AML cells into the cell cycle with rhG-CSF before the initiation of high-dose chemotherapy conditioning regimens for haplo-HSCT. Although all patients in the 2 cohorts experienced CR after transplantation, the relapse rate of the patients in group A was lower than that of the patients in group B. Moreover, the 2-year probabilities of LFS and OS in group A were higher than those in group B. The significant differences in relapse, LFS, and OS resulted from the activation of AML cells mediated by rhG-CSF, indicating the potential of the rhG-CSF-priming conditioning regimen protocol to eradicate leukemia [21,22]. Our data also compare favorably with recent reports of haplo-HSCT for hematological malignancies that did not use rhG-CSF before the conditioning regimen [23,24]. We previously reported the results of haplo-HSCT using a non-rhG-CSF-priming conditioning regimen for treating hematological malignancies [16]. This earlier study showed that the probabilities of 2-year LFS and OS were 68% and 70%, respectively. Although LFS and OS did not improve in the current study, the leukemia risk classification of the patients enrolled in the 2 studies was different. Specifically, the current study only enrolled patients with HR-AML.

After a median follow-up of 42 months, our data showed that there was no significant difference in the incidence of grade II to IV aGVHD between the 2 groups (42.4% [95% CI, 32.0% to 52.9%] versus 39.3% [95% CI, 28.9% to 49.7%], P > .05). However, this result was lower than the incidences of 55% and 78% that were reported by other investigators in studies in

which G-CSF-priming conditioning regimens were not used [24,25] and they were similar to our previous results [18]. Similar to that of aGVHD, the incidence of cGVHD was not significantly different between the 2 groups. The lower incidence of aGVHD in our haplo-HSCT recipients in both group A and group B was associated with GVHD prophylaxis with CsA plus short-term MTX, MMF, and the addition of ATG to the conditioning regimen [16]. Many experimental and clinical studies have reported that the use of rhG-CSF before and/or after transplantation and the infusion of PB stem cells mobilized by rhG-CSF, which modulates the balance between Th1 and Th2 cells, could lead to T cell hyper-responsiveness [26,27]. Morris et al. confirmed that by modifying its pegylation and using it in combination with Flt-3 ligand, G-CSF might lead to the activation and amplification of donor-invariant natural killer cells, a marked increase in post-transplantation cell-mediated CD8+ T cytotoxicity, and the enhancement of graft-versus-leukemia effects [28]. In the current study, rhG-CSF was used before and/or after transplantation, and PB stem cells were mobilized by rhG-CSF. The difference between group A and group B was the application of rhG-CSF on days -10 to -7. This slight difference in treatment regimens might have resulted in the small effects observed on the incidence of GVHD after transplantation.

TRM was also observed in a significant proportion of patients. The leading causes of reported deaths were infections, GVHD, and TRTs. Perugia et al. [29] reported that 40% of their patients died of nonrelapse causes and that most of the deaths were caused by infections, mainly CMV and asper-gillus. Under the Tuebingen protocol, 7 of 27 adult patients

—i-1-1-1-r-1 i-1-1-1-1—

0.00 20.00 40.00 60.00 80.00 0.00 20.00 40.00 60.00 80.00

Time after transplant (months) Time after transplant (months)

Figure 4. The

2-year probabilities of LFS and OS in different groups. (A) Shows group A (P = .004) and (B) shows group B (P =

.005).

Table 4

Multivariate Analysis of Two-Year LFS in rhG-CSF and Non-rhG-CSF—Priming Haplo-HSCT

rhG-CSF (n = 89) Non—rhG-CSF (n = 89)

LFS, % (95% CI) Chi-Square P Value LFS, % (95% CI) Chi-Square P Value

Diagnosis of FAB 5.927 .015 .259 .611

M4/M5/M6 41.9 (27.1-56.6) 35.9 (20.8-51.0)

No- M4/M5/M6 67.4 (53.8-80.9) 30.0(17.3-42.7)

Disease status before HSCT 5.624 .018 7.030 .008

CR1 and >CR2 63.3 (51.1-75.5) 38.1 (26.1-50.1)

PR/NR/RE 37.9 (20.3-55.6) 19.2 (4.1-34.4)

Prognostic risk category .576 .750 2.230 .328

Favorable 62.5 (29.0-96.0) 50.0 (15.4-84.6)

Intermediate 53.2 (38.9-67.5) 35.3 (22.2-48.4)

Unfavorable 55.9 (39.2-72.6) 23.3 (8.2-38.5)

Kaplan-Meier analysis and log-rank test were used.

and 2 of 21 children died. Of these patients, 6 had infections and 3 had GVHD [30]. The Dana Farber group [31] reported early deaths in 50% of their 24 patients, with the most common causes being bacterial or fungal. Additionally, Peking University reported TRMs of 9.1% and 12.7% at day 100 in standard- and high-risk groups, respectively. The TRMs increased to 19.5% and 31.1% at 2 years, and most of the TRMs were due to opportunistic infections [24]. In our study, GI toxicity and oral mucositis were the most common side effects. Most of the patients had infectious complications after transplantation, with 6 of 89 patients in group A and 6 of 89 patients in group B dying from IFI, GVHD, or severe septicemia.

At the same time, we analyzed the influence of established risk factors on outcomes. Interestingly, only the stage of the disease before transplantation had a significant influence on outcomes, and cytogenetics did not affect the prognosis of the disease. We also found that not all AML subtypes benefited from the rhG-CSF-priming conditioning regimen. The 2-year LFS of FAB-type no-M4/M5/M6 was better than that of FAB-type M4/M5/M6. This difference could be explained by the differences in the expression of G-CSF receptor (G-CSFR) on the different types of blasts. Graf et al. [32] found that the monocytic subtypes (FAB-type M4/ M5) expressed significantly more granulocyte macro-phage-CSFR, whereas the FAB-type M3 subtype mainly expressed G-CSFR. Moreover, Kutlay et al. [33] demonstrated that the G-CSFR expression rate was higher in the M2 and M3 subtypes and lower in the M5 and M6 subtypes. Recently, priming with rhGM-CSF during the first induction course has been reported to have positive results, which also supports our findings [34].

In summary, the results from this multicenter randomized controlled study were encouraging, and they suggest that haplo-HSCT with rhG-CSF-priming conditioning regimens might be feasible for high-risk AML patients without HLA-identical siblings or with unrelated donors. According to the results of multivariate and subtype analyses, the rhG-CSF-priming conditioning regimen is the best choice for HR-AML patients with no M4/M5/M6 subtype and who achieved CR before transplantation.

ACKNOWLEDGMENTS

Authorship statement: X.Z., Lei Gao, and Q.W. contributed to the concept design, data collection and analysis, as well as drafting and critically revising the manuscript. Y.Z. performed statistical analyses of the data. The other authors were involved in the collection and assembly of data and data interpretation. The authors thank Y.L., X.L., T.C., H.L.

for their collection of the data. All authors have agreed to the submission of this manuscript.

Financial disclosure: This research was supported in part by the Research Fund of the Natural Science Foundation of Chongqing (No. 2009BA5056), the Clinical Foundation of TMMU, and the "1130" Foundation of Xinqiao Hospital.

Conflict of interest statement: There are no conflicts of interest to report.

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