Treatment of Acute Leukemia with Unmanipulated HLA-Mismatched/Haploidentical Blood and Bone Marrow Transplantation Academic research paper on "Clinical medicine"

ASBMI

American Society for Blood and Marrow Transplantation

Treatment of Acute Leukemia with Unmanipulated HLA-Mismatched/Haploidentical Blood and Bone Marrow Transplantation

Xiao-Jun Huang, Dai-Hong Liu, Kai-Yan Liu, Lan-Ping Xu, Huan Chen, Wei Han, Yu-Hong Chen, Xiao-Hui Zhang, Dao-Pei Lu

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains one of the best therapeutic options to cure acute leukemia (AL). However, many patients have no human leukocyte antigen (HLA)-matched donor. Recently, we developed a new method for HLA-mismatched/haploidentical transplantation without in vitro T cell depletion (TCD). This method combined granulotyce-colony stimulating factor (G-CSF)-primed bone marrow and peripheral blood with intensive immunosuppression. We analyzed the outcome of 250 consecutive patients with AL who underwent HLA-mismatched/haploidentical transplantation with 1-3 mismatched loci of HLA-A, B, and DR from family donors via our new transplant protocol. Two hundred forty-nine patients achieved sustained, full donor chimerism. The incidence of grade 2-4 acute graft-versus-host disease (aGVHD) was 45.8%, and that of grades 3 and 4 was 13.4%, which was not associated with the extent of HLA disparity. The cumulative incidence of total chronic GVHD (cGVHD) was 53.9% and that of extensive cGVHD was 22.6% in 217 evaluable patients. One hundred forty-one of the 250 patients survived free of disease recurrence at a median of 1092 days (range: 442-2437 days) of follow-up. Seventeen patients received DLI as a treatment for relapse after transplantation and 7 patients achieved leukemia-free survival (LFS). The 3-year probability of LFS for acute myelogenous leukemia (AML) was 70.7% and 55.9%, and for acute lymphoblastic leukemia (ALL) it was 59.7% and 24.8% in standard-risk and high-risk groups, respectively. Lower LFS were associated with diagnosis of acute leukemia in the high-risk group (P = .001, relative risk [RR], 95% confidence interval [CI]: 2.94[1.535-5.631]) and the occurrence of aGVHD of grades 3 and 4 (P = .004). HLA-mismatched/haploidentical HSCTwas feasible with unmanipulated blood and bone marrow harvest. Biol Blood Marrow Transplant 15: 257-265 (2009) © 2009 American Society for Blood and Marrow Transplantation

KEYWORDS: HLA-mismatched, Haploidentical, Blood and marrow transplantation, Acute leukemia

INTRODUCTION

Acute leukemia (AL) is a common malignant colonal disease of hematopoietic stem cells. Despite recent promising advances in its therapy, allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains one ofthe best therapeutic options to cure AL [1]. Ahuman leukocyte antigen (HLA)-matched related donor is the first choice for allo-HSCT; however, only 25% to 30% of eligible patients have a related donor with suitable or closely matched HLA type. For patients without

From the Institute of Hematology, Peking University, Beijing, People's Republic of China. Financial disclosure: See Acknowledgments on page 264. Correspondence and reprint requests: Xiao-Jun Huang, MD, Institute of Hematology, Peking University, 11 Xizhimen South Street, Beijing 10044, P.R. China (e-mail: xjhrm@medmail. com.cn).

Received August 25, 2008; accepted November 14, 2008

1083-8791/09/152-0001$36.00/0

doi:10.1016/j.bbmt.2008.11.025

HLA identical sibling donors, HLA-matched unrelated bone marrow or cord blood have often been used as the source of stem cells for transplantation [2,3]. Unfortunately, in the case of an unsuccessful donor search or insufficient cord blood cells, this approach may not be viable. The alternative is to obtain hematopoietic stem cells from an HLA-mismatched/haploidential family donor. However, this approach, particularly prior to the end of the last decade, was associated with poor en-graftment, a high risk of early death, and severe graft-versus-host disease (GVHD) [4,5]. Although T cell depletion (TCD) or CD341 cell selection in vitro overcame the HLA barrier, it was challenged because of delayed immune reconstitution, that is, high rate of infections, relapse of leukemia, and consequently, poor survival [6,7].

Recently, we developed a new method for HLA-mismatched/haploidentical transplantation without in vitro TCD [8]. The method involves sequential, in vivo modulation of T cell functions in the recipient and donor, and adjustment of the dose of donor hema-topoietic stem cells. The 4 elements in the GIAC

protocol stand for the following: G, donor treatment with recombinant granulocyte colony-stimulating factor (rhG-CSF); I, intensified immunologic suppression; A, antihuman thymocyte immunoglobulin (ATG) for the prevention of GVHD; C, combination of peripheral blood stem cell transplantation (PBSCT), and bone marrow transplantation (BMT). Using this protocol, promising results for HLA-mis-matched allo-HSCT without in vitro TCD have been achieved at our institute.

In this paper, we report the outcome of250 consecutive patients with AL who underwent HLA-mismatched/ haploidentical transplantation via our new transplant protocol.

MATERIALS AND METHODS

Study Setting and Participants

Between November 16,2001 and May 9,2007,250 consecutive patients with AL underwent HLA-mis-matched/haploidentical allo-HSCT at the Institute of Hematology, Peking University. The outcome of 117 patients was reported in 2006 [8]; these patients were followed up again in this study. We enrolled patients with AL suitable for allo-HSCT who did not have HLA-identical related or unrelated donors or a source of stem cells from cord blood. This study was approved by the institutional review board of Peking University, and written informed consent was obtained from all patients and their donors. Patients with any uncontrolled infections or severe liver, renal, lung, or heart diseases were not eligible for HSCT. The characteristics of the patients and donors are shown in Table 1.

Stem Cell Harvest

Family donors were ranked on the basis of HLA match, age (younger preferred), gender (same preferred), and health status (better preferred). To determine HLA-A and B status, low-resolution DNA techniques were used. High-resolution techniques were used for the evaluation of HLA-DRB1, DQB1, DP, and C loci. Maternal and noninherited maternal antigens (NIMA) donors were preferred in the initial stage of study. However, there was no difference between those factors affecting the outcome of transplantation. Hence, there was no preference in the subsequent study.

Donors were treated with rhG-CSF (Filgrastim, Kirin, Japan; 5 mg-kg"1-day"1) injected subcutane-ously (s.c.) for 5-6 consecutive days from day -3. On the 4th day (day 01) of mobilization, bone marrow cells were harvested for the target volume of 10-12 mL-kg"1 of donor weight or the target mononuclear cell (MNC) count of 2 x 108 to 4 x 108 - kg"1 of recipient weight. The cell counts were measured in the operating

Table 1. Characteristics of Patients with Acute Leukemia Receiving HLA-Mismatched/Haploidentical HSCT

Age (years) 25.1(2-56)

Gender

male 154

female 96

HLA-mismatched loci of A, B, and DR Case

1 40(16%)

2 105 (42%)

3 105 (42%) AML 108 (l00%)

Standard risk 74 (68.5%)

CRl 67

High risk 34(31.5%)

CR3/>CR3 11

Ph+(CR+NR) 2

ALL 142(100%)

Standard risk 87 (61.3%)

CRl 82

High risk 55 (38.7%)

CR3/>CR3 17

Ph+(CR+NR) l9 Transplantation yearly

2001-2002 22

2003 42

2004 55

2005 57

2006 47 2007:01-04 27

Donor/recipient gender

female-male 85

female-female 63

male-male 72

male-female 30 Donor

Mother l08 (43.2%)

Father 47 (l8.8%)

Child 15 (6.0%)

Sibling 77 (30.8%)

Uncle or aunt 3 (1.2%)

CRl indicates the first complete remission; NR, nonremission; Ph+, Philadelphia chromosome positive.

room. On the fifth (day 02) of mobilization, PBSC were collected with a COBE Blood Cell Separator (Spectra LRS; COBE BCT Inc., Lakewood, CO) at a rate of 80 mL-min"1 from a total blood volume of 10-12 L. The target MNC from bone marrow and peripheral blood was 4 x 108 to 6 x 108 - kg"1 of recipient weight. Another collection of PBSCs was needed on day 03 if cells collected on the previous 2 days were insufficient. The fresh and unmanipulated bone marrow and PBSCs were infused into the recipient on the day of their collection. In instances of major ABO blood group incompatibility, red blood cells were removed from bone marrow cells by density gradient sedimentation with Hespan (B. Braun Medical Inc., Irvine, CA), according to the manufacturer's instructions. The surface markers of the graft cells were determined by multicolor staining using monoclonal antibodies specific for CD341, CD31, CD41, and CD81 cells, essentially as described by Liu et al. [9].

Conditioning Regimen

As described previously [8], all patients were treated with regimen A, which consisted of the following: cytosine arabinoside (4 g-m"2-day_1, intravenously) on days -10 and -9; busulfan (12 mg-kg"1 p.o. in 12 doses) on days -8, -7, and -6; cyclophosphamide (1.8 g-m"2-day"\ intravenously) on days-5 and -4; semustine (250 mg-m"2, orally) on day -3, and Thymoglobulin (rabbit ATG, Sangstat-Genzyme, 2.5 mg - kg"1 - day"1 intravenously or porcine ATG, Wuhan, China, 20 mg-kg"1 - day"1) through days-5 to-2.

GVHD Prophylaxis and Treatment

All transplant recipients received cyclosporine A (CsA), mycophenolate mofetil (MMF), and short-term methotrexate (MTX). The dosage of CsA was 2.5 mg-kg"1-day"1 intravenously from day -9 until bowel function returned to normal. At that point, patients were switched to oral CsA. Every 12 hours, 0.5 g MMF was administered orally from day -9 to day + 30 after transplantation; following this, the dose was tapered to half until day +60 and discontinued thereafter. Following transplantation, a dose of 15 mg-m"2 of mehotrexate was administered intravenously on day +1 and 10 mg-m"2 on days +3, +6, and +11. Whole-blood CsA concentration was monitored weekly using the fluorescence polarization immunoassay technique, and the dosage was adjusted in order to maintain a minimum blood concentration of 150-250 ng-mL"1. In cases where there was no evidence of GVHD by day +40 to approximately +50, CsA dosage was reduced by 1/8 to approximately 1/ 10 dose each month and was discontinued at around 9-10 months. In cases where GVHD occurred in any organ, CsA was continued and adjusted to the blood concentration mentioned above.

Acute GVHD (aGVHD) was treated with methyl-prednisolone (0.5-1 mg-kg"1 -day"1). GVHD manifestation only in the skin was treated with a combination of methylprednisolone and MTX or with MTX alone [10]. MTX was given intravenously on days 1, 3, and 7, then subsequently at intervals of 7 days in the case of complete or partial response for a total of 6-8 doses. The dosage was 5 mg if white blood cells were <2 x 109/Lorplatetelets <50 x 109/L; otherwise the dosage was 10 mg. When there was inadequate or no response to the primary therapy of GVHD, 1 mg-kg"1 of anti-Tac monoclonal antibody (Daclizumab; Roche, Basel, Switzerland) was administered intravenously on days 1, 4, and 8, and subsequently at intervals of 7 days for a total of 3 to 6 doses.

Evaluation and Monitoring of Engraftment, Chimerism, and Minimal Residual Disease

Myeloid recovery was defined as maintenance of an absolute neutrophil count above 0.5 x 109-L"1 for 3

consecutive days after the neutrophil nadir. Chimerism was determined by at least 2 of the following 3 methods: DNA-based HLA typing (for mismatched loci), poly-merase chain reaction (PCR) DNA fingerprinting of short tandem repeats, and chromosomal fluorescence in situ hybridization (FISH) (for Y chromosome). Full donor chimerism was defined as no donor hemato-poietic or lymphoid cells detected with either of the above methods. MRD was monitored with flow cytometry (FCM) or quantitative PCR for BCR/abl, the Wilms tumor gene (WT1), or AML/ETO according to the molecular genetics ofleukemia before transplantation. On days +30, +60, +90, +180, and year +1, + 3, and +5, chimerism and minimal residual disease (MRD) were detected.

Modified Donor Lymphocyte Infusion (DLI)

Modified DLI consists of 2 components: infusion of G-CSF-primed PBSCs instead ofnonprimed donor lymphocytes and short-term immunosuppressive agents to prevent GVHD after DLI. Therapeutic modified DLI was performed in 7 acute myelogenous leukemia (AML) and 10 acute lymphoblastic leukemia (ALL) patients as a treatment for hematologic disease recurrence after transplantation. Cells were from cryo-preserved PBSCs collected on day 02 or day 03 from 11 donors and from newly mobilized PBSCs from the other 6 donors. The median number of CD3 cells infused was 2.09 (0.84-5.6) x 108/kg. Except for the 5 patients in the initial stage of study, 12 patients received CsA or a low dose of MTX for 2-4 weeks after DLI to prevent GVHD [11]. The prophylactic DLI was administered to 13 high-risk patients when they showed evidence of partial recipient chimerism or MRD following withdrawal of immunosuppressants and the absence of acute GVHD [12]. Six of these patients were diagnosed as high risk for AML and 7 were diagnosed as high risk for ALL. The G-CSF-primed PBSCs used for DLI were cryopreserved on day 02 or 03. The median number of CD3+ cells infused was 0.49 (0.2-1.4) x 108/kg. All patients were treated with short-term immunosuppressive agents for 2-4 weeks.

Infection Prevention and Supportive Care

All patients were hospitalized in rooms with high-efficiency particle-arresting (HEPA) air filters for 4-5 weeks from day -10 to the time of neutrophil recovery. They received antibiotic prophylaxis with oral Tri-methoprim-sulfamethoxazole to prevent infection by Pneumocystis carinii from day -10 to +90; Fluconazole for Candida albicans from day -10 to +30; acyclovir for herpes simplex virus and varicella zoster virus from day 11 to the time of CsA discontinuation; Ci-proflaxacin for intestinal decontamination. Ganciclo-vir (5 mg-kg"1) was administered intravenously twice daily from days -10 to -2 for prophylaxis of

cytomegalovirus (CMV) infection. Patients were monitored weekly for CMV DNA (real-time PCR) or CMV pp65 antigenemia tests. Patients with CMV antigene-mia were treated with either Ganciclovir or Foscarnet. CMV-related interstitial pneumonia (IP) was defined according to reported criteria [13]. Surveillance for bacterial, fungal, and viral infections were based on clinical requirements. All blood products were irradiated with 2500 cGy before infusion. Patients received red blood cell transfusions if their hemoglobin levels were below 70 g-L"1 or platelet transfusions if their platelet levels dropped below 20 x 109-L_1. G-CSF (5 mg-kg_1day_1, s.c.) was administered to all recipients from day 6 after transplantation until myeloid recovery. Human immunoglobulin (400 mg-kg"1) was given intravenously on days +1, +11, +21, and +31.

Definition and Statistics

Patients with malignancies were categorized as "standard risk'' if they were in the first or second complete remission (CR1 or CR2) of AL without Ph chromosome. Patients were stratified as "high risk'' if they were in more than CR2, in nonremission, and in CR1 or CR2 with t(9;22). The incidence and severity of GVHD was defined based mainly on a consensus conference on GVHD grading, except for manifestation in the gastrointestinal tract, which was scored according to the International Bone Marrow Transplant Registry (IBMTR) system [14,15]. Biopsies of involved organs are routinely performed in the gastrointestinal tract for differential diagnosis. In some cases of suspected chronic GVHD (cGVHD) of the skin, a biopsy was used to differentiate skin allergies or fungal or parasitic infections. Time to GVHD was defined as the time from allo-HSCT to the onset of any grade of GVHD. Patients were evaluated for cGVHD if they survived for at least 100 days after HSCT. Hematologic relapse of leukemia after transplantation was defined as the recurrence of signs and syndromes of leukemia. The first day of stem cell infusion was defined as "day 01,'' and the second day of infusion was "day 02,'' and so on. Days before transplant was preceded by ''-'', and days after the last stem cell infusion was preceded by " + .'' Values were adjusted for engraftment at time of death if the patient had not undergone engraftment; for GVHD, the values were adjusted for survival and relapse at last follow-up. For all surviving patients, the end point of the last follow-up was August 16, 2008. The factors analyzed in univariate analysis included the age and gender of patients and donors, time from diagnosis to transplant, year of transplantation, risk group, extent of HLA disparity, the count of MNC, CD3+, and CD34+ cells infused. Univariate probabilities of developing aGVHD and cGVHD, treatment-related mortality (TRM), and relapse were calculated using cumulative incidence curves to ac-

commodate corresponding competing risk. Distributions for time to overall survival (OS), and leukaemia-free survival (LFS) were evaluated using Kaplan-Meier analysis. Outcomes events considered in multivariate analyses were TRM, relapse, and LFS. First, the test indicated that the proportionality assumptions hold. The final multivariate models were built using a forward stepwise model selection approach. Each model contained all significant risk factors in univariate analysis. Statistical software packages (SPSS 13.0 and SAS) were used for all analyses.

RESULTS

Engraftment

All donors tolerated the procedures, and no severe side effects occurred during either bone marrow harvest or leukapheresis. In the initial stage of study, 16 patients were administered with porcine ATG. Two hundred thirty-four patients were administered with rabbit ATG thereafter. There was no difference related to outcome of transplantation between these 2 groups. The actual ratio of MNC of G-BM to PBSC harvested was from 2:1 to 1:2 in the majority of patients (Table 2). Myeloid, platelet recovery and sustained, full donor chimerism were achieved in 249 patients after transplantation. The median time for myeloid recovery was 12 days (range: 9-26 days) and 15 days (range: 8-151 days) for platelet recovery. There was no statistically significant association between the extent of HLA disparity and the time of engraftment.

Acute GVHD

Of the 250 patients, 86 (34.4%) had no aGVHD, 49 (19.6%) had grade 1, 89 (35.6%) had grade 2, 11 (4.4%) had grade 3, and 15 (6.0%) had grade 4. At 100 days after transplantation, the cumulative incidence of aGVHD grades 2-4 was 45.8%, and that of grades 3 and 4 was 13.4% (Figure 1). No risk factors that might influence the incidence of GVHD grades 3 and 4 were found with univariate analyses. Acute GVHD occurred in 23 (57.5%) of 40 patients with HLA mismatch at 1 locus, 70 (66.7%) of 105 patients with mismatch at 2 loci, and 71 (67.6%) of 105 patients

Table 2. Subtypes of Cells from G-CSF-Primed Bone Marrow and PBSC Infused for Patients with Acute Leukemia Receiving HLA-Mismatched/Haploidentical HSCT

Cell Type

MNC (x 108/kg) CD34+ (x 106/kg) CD3+ (x 108/kg) CD4+ (x 108/kg) CD8+ (x 108/kg) CFU-GM (x 105/kg)

3.7 (0.31-12.2) 1.1 (0.09-8.2) 0.2 (0.02-3.l) 0.1 (0.2-0.6) 0.1 (0.009-0.4) 2.9 (l.2-9.7)

4.9 (0.4-12.2) 1.3 (0.1-5.9) 1.6 (0.2-6.5) 0.9 (0.1-3.4) 0.8(0.1-3.9) 3.7(1.9-10.0)

7.9 (3.1-14.8) 2.1 (0.3-14.2) 2.4 (0.1-10.7) l.l (0.07-4.2) 0.7 (0.1-3.1) 5.6 (2.6-21.4)

CFU-GM indicates colony-forming unit granulomonocyte.

Figure 1. Incidence of total aGVHD and severe aGVHD after HLA-mismatched/haploidentical HSCT.

Figure 2. Incidence of cGVHD and extensive cGVHD after HLA/mis-matched/haploidentical HSCT.

with mismatch at 3 loci. Grades 3 and 4 of aGVHD were diagnosed in 5 (12.5%) patients with HLA mismatch at 1 locus, 7 (6.7%) patients with mismatch at 2 loci, and 14 (13.3%) patients with mismatch at 3 loci.

Of the 15 patients who developed aGVHD grade 4,8 died of GVHD, 4 patients died of infection at a median of 335 (110-553) days after transplantation after controlling for severe GVHD. The other 3 patients recovered and remained free of GVHD until their last follow-up at 217, 941, and 958 days after transplantation. Of the 11 patients who suffered from aGVHD grade 3,4 died: 1 died of GVHD; 1 died ofveno-occlu-sive disease (VOD), and the remaining 2 died of infection at 132 and 157 days after transplantation after controlling for GVHD. The other 7 recovered and survived until the end of study for a median of 1808 (527-1866) days.

Chronic GVHD

Of the 217 patients who survived for over 100 days after transplantation, 92 (42.4%) developed cGVHD. Sixty-one (28.1%) of these showed limited cGVHD, while 31 (14.3%) showed extensive cGVHD. At 3 years after transplantation, the cumulative incidence of total cGVHD was 53.9% and that of extensive cGVHD was 22.6% (Figure 2). Chronic GVHD was diagnosed in 21 (52.3%) of 38 patients with HLA mismatch at 1 locus, 36 (40.4%) of 89 patients with mismatch at 2 loci, and 35 (38.9%) of 90 patients with mismatch at 3 loci. The development of cGVHD and extensive cGVHD was not associated with the factors analyzed in univariate analyses. The patients who developed aGVHD grade 2 to 4 had a higher risk of developing extensive cGVHD than those without aGVHD grade 2 to 4 (52.8% versus 26.6%, P = .001).

Infection, Transplantation-Related Toxicity and Immune Reconstitution

A total of 120 occurrences of opportunistic infections were recorded in 106 patients during the duration of follow-up. The median time for an opportunistic infection to develop was 280 days (range: 5-1120 days) after transplantation. The infected loci included lungs (74 occurrences), skin (28 occurrences), gastrointestinal tract (24 occurrences), and central nervous system (6 occurrences). The skin infections were caused by the varicella-zoster virus (16 cases) and herpes simplex virus (12 cases). The pathogens found in pneumonia were bacteria in 13 cases, Aspergillus in 18 cases, Candida albicans in 1 case, Pneumocystis carinii in 5 cases, and CMV in 16 cases. The other 21 cases of pneumonia were negative for pathogens, and of these, 9 cases responded to antibiotics. At 3 years after transplantation, the cumulative incidence of opportunistic infections was 49.1%, without risk factors found in statistical analyses.

The reconstitution of natural killer cells returned to normal within 4 weeks after transplantation, as recently reported [16]. At 3 months after HSCT, the peripheral blood counts of CD4+ T cells were 129.00 (24.88-510.82-mL"1. The median of CD8 + cell counts at +60 days was 526.50 (4.54-2742.73)-mL"1 and returned to normal (median: 656.3-mL"1; range: 50.92-mL"1-3708.04-mL"1) within 90 days after transplantation.

Relapse

Of the 250 patients, 45 (AML, 13; ALL, 32) relapsed after transplantation; of these, 22 (AML, 6; ALL, 16) were from the high-risk group. The 3-year probability of relapse in the standard-risk group was 11.9% and 24.3% for AML and ALL, respectively, and that in high-risk group was 20.2% and 48.5%

for AML and ALL, respectively. In multivariate analysis, diagnosis of acute leukemia in the high-risk group was found to be an independent risk factor for relapse; P = .002, relative risk [RR] (95% confidence interval [CI]): 3.018 (1.494-6.097); Figure 3a and 3b. Of the 45 patients, 17 received therapeutic modified DLI; 7 of these patients achieved complete remission and donor chimerism and survived at a median of 520 (300-1810) days after DLI. These 7 patients included 3 standard-risk group patients with ALL, 1 high-risk group patient with ALL, and 3 standard-risk group patients with AML.

LFS and TRM

Up to August 16,2008,148 patients (AML, 73; and ALL, 75) were alive; 141 achieved LFS throughout the follow-up of 1112 days (range: 442-2437 days).

However, the other 7 patients relapsed after transplantation and received DLI; thereafter, all achieved LFS. The 148 patients comprised 114 (AML, 54; ALL, 60) standard-risk patients and 34 (AML, 19; ALL, 15) high-risk patients. The 3-year probability of LFS for AML was 70.7% and 55.9% and for ALL was 59.7% and 24.8% in the standard-risk and high-risk groups, respectively (Figrue 4a and 4b). Because 7 patients relapsed after transplantation and achieved LFS following therapeutic DLI, the OS was higher than LFS in most groups; those were 72.5%, 55.9% for AML, 65.0%, and 26.5% for ALL in the standard-risk and high-risk groups, respectively (Figure 5aand 5b).Thir-teen patients received prophylactic DLI because of their high risk of disease recurrence and 6 of them survived free of leukemia at a median of 1458 (532-1767) days after transplantation. In term of predictors with multivariate analysis, lower LFS was associated with

Figure 3. (a) Relapse of patients with AML after HLA-mismatched/hap-loidentical HSCT. (b) Relapse of patients with ALL after HLA-mis-matched/haploidentical HSCT.

Figure 4. (a) LFS of patients with AML after HLA-mismatched/haploi-dentical HSCT. (b) LFS of patients with ALL after HLA-mismatched/hap-loidentical HSCT.

Figure 5. (a) OS of patients with AML after HLA-mismatched/haploi-dentical HSCT. (b) OS of patients with ALL after HLA-mismatched/hap-loidentical HSCT.

diagnosis of acute leukemia in high-risk group; P = .001, RR (95% CI): 2.94 (1.535-5.631). The significance of influence of aGVHD grades 3 and 4 on LFS was found in univariate analysis (P = .004), not in multivariate analysis.

Totally, 102 (AML, 35; ALL, 67) out of the 250 patients died: 38 (37.3%) (AML, 11; ALL, 27) from recurrent leukemia and 64 (62.7%) (AML, 22; ALL, 42) from transplant-related complications. The causes of transplantation-related deaths included GVHD in 10 cases (aGVHD, 9; and extensive cGVHD, 1), infection in 42 cases (lung, 29; central nervous system [CNS], 4; and intestinal, 9), organ failure in 3 cases, suicide in 1 case, discontinuation of treatment because of inability to meet expenses in 4 cases, graft failure in 1 case, and posttransplantation lymphoproliferative disorder (PTLD) in 3

cases. The TRM on day 100 after transplantation in the standard- and high-risk groups was 6.8% and 5.9% for AML and 6.9% and 25.9% for ALL, respectively. At 3 years, the TRM in standard-risk and high-risk groups was 19.4% and 29.4% for AML and 21.2% and 50.8% for ALL, respectively. In term of predictors with multivariate analysis, higher TRM was associated with diagnosis of ALL in the high-risk group; P = .049, RR(95% CI):2.422 (1.005-5.835).Thesignif-icance of influence of aGVHD grades 3 and 4 on TRM was found in univariate analysis (P = 0.000), not in multivariate analysis.

DISCUSSION

Our study confirmed and extended our previous finding that transplantation of G-CSF-primed bone marrow (G-BM) and PBSCs from HLA-mismatched/ haploidentical related donors resulted in a high rate of engraftment and a similar incidence of GVHD as that observed in HLA-identical allo-HSCT [8,17]. Usually, it is believed that extensive TCD is essential for the prevention of aGVHD in HLA-mismatched/hap-loidentical HSCT. However, in this study, the T cell number infused was 2.4(0.1-10.7) x 108$kg"1.Moreover, the cumulative incidence of aGVHD of grades 3 and 4 at 100 days was only 13.4% (this was lower than that observed in a previous report). The cumulative incidence of cGVHD and extensive cGVHD at 2 years after transplantation was only 53.9% and 22.6%, respectively, which was not higher than that in HLA-identical sibling donor transplantation [4,17,18]. There was no difference in the cumulative incidence of aGVHD with HLA disparity; this was consistent with our previous results. The rationale for using G-CSF-primed bone marrow (G-BM) combined with PBSC as a graft is 3-fold: (1) G-CSF primed PBSC grafts do not cause higher aGVHD than marrow grafts [19]. Compared with PBSCT, the use of G-BM resulted in comparable engraftment, reduced severity of aGVHD, and less subsequent cGVHD [20]. (2) The experimental data showed that the counts of CD3 +, CD41, and CD81 T cells were different between the mixture grafts and PBSC, steady-state BM, or the G-BM [21]; (3) our recent study demonstrated that the combination use of G-BM and PBSC in proportions from 2:1 to 1:2 of MNC counts maintained T cell hyporesponsiveness and polarization of T cells from Th1 to Th2, which would improve their immune tolerance ability in a HLA-mismatched/haploidentical transplant setting [22]. Our series of experimental and clinical data confirmed the safety and feasibility of the novel regimen, which overcame the HLA barrier without in vitro TCD for HLA-mismatched/haploidenti-cal HSCT. Compared with the data reported by Beatty and Szydlo [23,24], the transplantation

outcomes, such as survival and mortality, were better in the present study. The reason for the improvement might be because of the use of G-BM combined with PBSC as a graft, use of ATG and other immunosup-pressants, as well as better supportive care and training of clinical staff in the past decade. However, the rate of engraftment and incidence of aGVHD grade 3-4 were better than those in previous reports, which might suggest the advantage of this protocol in inducing immune tolerance.

In the standard-risk group, the 3-year probability of relapse for AML and ALL was similar, that is,, 14.8% and 15.4% respectively. Surprisingly, in AML, the relapse rate is similar between standard- and high-risk groups. This may be partially explained by the application of prophylactic DLI, which might be helpful in decreasing the relapse rates in high-risk AML patients following transplantation [12]. Although the prophylactic DLI resulted in a 20.7% incidence of severe GVHD, death from aGVHD was very low because of the strategy of preemptive evaluation and the prevention of and therapy for GVHD. Despite giving prophylactic DLI, disease recurrence in the high-risk ALL patients was 56.6%. The inclusion of patients with advanced stage of disease in the present study may have contributed to the high rate of relapse in high-risk ALL. One-third of our patients (55 of 142) presented with highly advanced refractory ALL, including Philadelphia chromosome-positive (Ph+) ALL. Further, 34.5% (19 of 55) were in the nonremission state, 30.9% (17 of 55) >CR2 and only 7 patients received prophylactic DLI. Moreover, this indicated that even in HLA-mismatched HSCT, ALL such as Ph+ ALL may be insensitive to DLI; thus, the risk of leukemia relapse was still present, particularly in patients with high-risk ALL compared to those with AML. In our study, the LFS in the standard-risk group patients was 70.7% for AML and 55.9% for ALL, while the corresponding values reported by Ruggeri et al. [18] were 60% for AML and 40% for ALL, respectively. Moreover, we obtained better results for patients in the nonremission stage (59.7% for AML and 24.8% for ALL) than previously reported (10% and 13%, respectively) [18]. However, given the different patient populations, and the nonrandomized comparison, the results should be cautiously interpreted and compared.

Because of high levels of T cell infusion, the reconstitution of T cells was much faster in our study than the slow rate of T cell recovery previously reported in T cell depleted settings; this may have contributed to the relatively lower percentage of nonleukemic deaths [25,26]. Although CMV infection was higher (68%), IP occurred in only 6.4% patients (unpublished data). A previous study on HSCT with ATG conditioning reported complications with a high rate of lymphoproli-ferative disorders (LPD) related to the Epstein-Barr virus [26]. In this study, the incidence of PTLD was

only 0.12% (3 cases), probably because of a more rapid immune reconstitution.

In conclusion, the overall results of our study in terms of TRM and disease-free survival (DFS) showed favorable outcomes compared with those expected for patients at a similar stage of disease following haploi-dentical transplantation with in vitro TCD [5]. The high rate of engraftment, relatively lower incidence of GVHD, and higher OS demonstrated that HLA-mismatched/haploidentical HSCT was feasible with unmanipulated blood and bone marrow harvest. In the future, further reduction of relapse and other complications will lead to improved clinical outcomes.

ACKNOWLEDGMENTS

We are grateful for the critical reading and careful revision of English diction and grammar by the native-speakers in Enpapers.

Financial disclosure: The authors have nothing to disclose.

REFERENCES

1. Horowitz MM, Gale RP, Sondel PM, et al. Graft-versus-leuke-mia reactions after bone marrow transplantation. Blood. 1990;75: 555-562.

2. Ottinger HD, Ferencik S, Beelen DW, et al. Hematopoietic stem cell transplantation: contrasting the outcome of transplantations from HLA-identical siblings, partially HLA-mismatched related donors, and HLA-matched unrelated donors. Blood. 2003;102:1131-1137.

3. Benito AI, Diaz MA, Gonzalez-Vicent M, et al. Hematopoietic stem cell transplantation using umbilical cord blood progenitors: review of current clinical results. Bone Marrow Transplant. 2004;33:675-690.

4. Aversa F, Tabilio A, Velardi A, et al. Treatment of high-risk acute leukemia with T-cell-depleted stem cells from related donors with 1 fully mismatched HLA haplotype. N Engl J Med. 1998;339:1186-1193.

5. Aversa F, Terenzi A, Felicini R, et al. Haploidentical stem cell transplantation for acute leukemia. Int J Hematol. 2002; 7(Suppl):165-168.

6. Aversa F, Martelli MF. Transplantation of haploidentically mismatched stem cells for the treatment of malignant diseases. Springer Semin Immunopathol. 2004;26:155-168.

7. Kodera Y, Nishida T, Ichinohe T, Saji H. Human leukocyte antigen haploidentical hematopoietic stem cell transplantation; indications and tentative outcomes in Japan. Semin Hematol. 2005; 42:112-118.

8. Huang XJ, Liu DH, Liu KY, et al. Haploidentical hematopoietic stem cell transplantation without in vitro T-cell depletion for the treatment of hematological disease. Bone Marrow Transplant. 2006;38:291-297.

9. Liu Y, Chen S, Yu H. [Standardization and quality control in flow cytometric enumeration of CD34(+) cells]. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2000;8:302-306.

10. Huang XJ, Jiang Q, Chen H, et al. Low-dose methotrexate for the treatment of graft-versus-host disease after allogeneic hema-topoietic stem cell transplantation. Bone Marrow Transplant. 2005;36:343-348.

11. Huang XJ, Liu DH, Liu KY, et al. Donor lymphocyte infusion for the treatment of leukemia relapse after HLA-mismatched/ haploidentical T-cell-replete hematopoietic stem cell transplantation. Haematologica. 2007;92:414-417.

12. Huang XJ, Liu DH, Liu KY, et al. Donor lymphocyte infusion after HLA-mismatched/habloidentical T-cell-replete hemato-poietic stem cell transplantation for prophylaxis of relapse of leukemia in patients with advanced leukemia. J Clin Immunol. 2008;28:276-283.

13. Meyers JD, Flournoy N, Thomas ED. Nonbacterial pneumonia after allogeneic marrow transplantation. A review of ten years' experience. Rev Infect Dis. 1982;4:1119-1132.

14. PrzepiorkaD, Weisdorf D, Martin P, et al. 1994 Consensus conference on acute GVHD grading. Bone Marrow Transplant. 1995; 15:825-828.

15. Rowlings PA, Przepiorka D, Klein JP, et al. IBMTR Severity Index for grading acute graft-versus-host disease: retrospective comparison with Glucksberg grade. Br J Haematol. 1997;97: 855-864.

16. Chang YJ, Zhao XY, Huang XJ. Effects of the NK cell recovery on outcomes of unmanipulated haploidentical blood and marrow transplantation for patients with hematologic malignancies. Biol Blood Marrow Transplant. 2008;14:323-334.

17. Lu DP, Dong LJ, Wu T, et al. Conditioning including antithy-mocyte HLA-mismatched/haploidentical blood and marrow transplantation can achieve comparable outcomes with HLA-identical sibling transplantation. Blood. 2006;107:3065-3073.

18. Ruggeri L, Capanni M, Urbani E, et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science. 2002;295:2097-2100.

19. Brensinger WI, Clift RA, Applebaum FA, et al. Transplantation of allogeneic peripheral stem cells mobilized by recombinant

human granulocyte-colony stimulating factor. Stem Cells. 1996;14:90.

20. Elfenbein GJ, Sackatein R. Primed marrow for autologous and allogeneic transplantation: a review comparing primed marrow to mobilized blood and steady-state marrow. Exp Hematol. 2004;32:327-339.

21. Huang XJ, Chang YJ, Zhao XY. A direct comparison of immunological characteristics of granulocyte-colony stimulating factor (G-CSF)-primed bone marrow grafts and G-CSF-mobilized peripheral blood grafts. Hematologica. 2005; 90:715-716.

22. Huang X-J, Chang Y-J, Zhao X-Y. Mainitaining hyporespon-siveness and polarization potential of T cells after in vitro mixture of G-CSF mobilized peripheral blood grafts and G-CSF primed bone marrow grafts in different proportion. Transplant Immunol. 2007;17:193-197.

23. Beatty P, Clift R, Mickelson E, et al. Marrow transplantation from related donors other than HLA-identical siblings. N Engl J Med. 1985;313:765-771.

24. Szydlo R, Goldman J, Klein J, et al. Results of allogeneic bone marrow transplants for leukemia using donors other than HLA-identical siblings. J Clin Oncol. 1997;15:1767-1777.

25. Rizzieri DA, Koh LP, Long GD, et al. Partially matched, non-myeloablative allogeneic transplantation: clinical outcomes and immune reconstitution. J Clin Oncol. 2007;25:690-697.

26. Waller EK, Langston AA, Lonial S, et al. Pharmacokinetics and pharmacodynamics of anti-thymocyte globulin in recipients of partially HLA-matched blood hematopoietic progenitor cell transplantation. Biol Blood Marrow Transplant. 2003;9:460-471.