Volume 13, Issue 12 , Pages 1515-1524, December 2007
CD3+ Cell Dose and Disease Status Are Important Factors Determining Clinical Outcomes in Patients Undergoing Unmanipulated Haploidentical Blood and Marrow Transplantation after Conditioning Including Antithymocyte Globulin
Article Outline
Abstract
Haploidentical transplantation is a feasible alternative for patients with life-threatening hematologic diseases who lack a matched donor. Factors affecting the clinical outcomes of haploidentical transplantation remain under investigation. We analyzed 157 consecutive patients with leukemia who underwent transplantation with nonmanipulated granulocyte colony-stimulating factor (G-CSF)-mobilized marrow and peripheral blood cells (G-BMPBs) from haploidentical donors after receiving myeloablative chemotherapy (Ara-C + BuCy + antithymocyte globulin). Follow up observations after transplantation were made from 48 days to 1191 days (median, 448 days). Multivariate analysis indicated that the cohort given higher doses of CD3+ cells (≥ 177×106 /kg) in allograft transplantation had a significantly lower treatment-related mortality (TRM) (relative risk [RR] = 0.35; 95% CI = 0.16-0.77; P = .0090), better leukemia-free survival (LFS) (RR = 0.46; 95% CI = 0.26-0.84; P = .0106), and better overall survival (OS) (RR = 0.42; 95% CI = 0.23-0.78; P = .0058). Inversely, advanced-stage disease was a strong predictor of greater posttransplantation relapse (RR = 3.48; 95% CI = 1.26- 9.60; P = .0159), worse LFS (RR = 2.56; 95% CI = 1.33-4.95; P = .0050), and worse OS (RR = 2.77; 95% CI = 1.39-5.53; P = .0038). A high number of CD3+ cells (> 177 × 106/kg) given to patients resulted in statistically less TRM and more intensive graft versus leukemia effect without producing more severe grades of GVHD, all resulting in a significantly better overall clinical outcome from haploidentical transplantation.
Key Words: Allogeneic transplantation, CD3+ cell dose, HLA-mismatched/haploidentical, Leukemia
Introduction
Since 1956, when a graft-versus-tumor (GVT) effect was first observed in irradiated mice receiving allogeneic but not syngeneic marrow transplants [1], the concept of allogeneic hematopoietic cell transplantation (HCT) as a form of immunotherapy has been well accepted [2]. The dramatic effect of allogeneic hematopoietic stem cell transplantation (HSCT) demonstrates the ability of donor lymphohematopoietic cells to successfully repopulate treated recipient marrow, making the eradication of certain malignancies possible. Maintaining a stable hematopoietic/immunologic reconstitution in recipients of HCT and also inducing a considerable GVT effect requires relatively large numbers of donor lymphohematopoietic cells. Previous studies have shown that transplantation of a high dose of marrow cells is associated with reduced treatment-related mortality (TRM) and improved survival and results in improved short-term and long-term graft function 3, 4. The doses of CD3+ [5], CD8+ [6], and CD34+ cells 7, 8, 9 may be critical for successful engraftment in a selective CD34+ cell transplantation setting. To date, however, a clear association of those T cell subsets with the clinical outcomes remains uncertain; some results are even controversial 6, 7, 8, 9, 10. Currently, no data are available on the proper or optimal cell dose of nonmanipulated granulocyte colony-stimulating factor (G-CSF)-mobilized marrow and peripheral blood cells (G-BMPBs) in transplantation after a conditioning regimen that includes antithymocyte globulin (ATG). Such studies of graft constituents are drawing increasing attention, not only because these infused cells play a crucial role in hematopoietic/immune reconstitution after transplantation, but also because determination of cell dose is one of the few manageable (or controllable) pretransplantation factors 11, 12. Family HLA-mismatched/haploidentical transplantation offers a feasible therapeutic approach to patients with lethal malignant hematologic diseases without an HLA-matched donor, a situation that is becoming more common as family size shrinks. An earlier study from our center showed that patients with advanced-stage disease and no family or unrelated HLA-matched donor might achieve nearly comparable therapeutic effects from a family mismatched/haploidentical transplantation [13]. Since then, more patients, including those with early-stage disease with high-risk prognostic features, have been enrolled. The cell subpopulations in the donor grafts and the clinical pretransplantation factors of the patients in this study were all carefully evaluated.
Methods
Data Collection and Patient Selection
A total of 157 consecutive patients who underwent haploidentical transplantation between January 25, 2002, and March 31, 2005, at Peking University Institute of Hematology and Dao-Pei Hospital were enrolled. Only patients with a diagnosis of acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), or myelodysplastic syndrome (MDS) and no HLA-matched related or unrelated donors were eligible for the study. The following criteria for disease stage were used:
The complete immune phenotype was determined for cell populations in each graft. The age range for patients was 6–50 years (median, 25 years), and that for donors was 13–66 years (median, 40 years) (Table 1). A total of 78 patients with early-stage disease were enrolled because most of them had unfavorable prognostic features. The protocol [13] was reviewed and approved by the Institutional Review Board (IRB) at Peking University Institute of Hematology. All patients or their guardians were required to sign consent forms approved by the IRB. Detailed patient and donor characteristics are given in Table 1.
Table 1. Clinical data
| Number of patients | 157 |
| Patient age, median (range), years | 25 (6-50) |
| Donor age, median (range), years | 40 (13-66) |
| Donor–patient sex match, n (%) | |
 Female to male | 26 (17%) |
| Others | 131 (83%) |
| Donor–patient relationship, n (%) | |
| Sibling | 48 (31%) |
| Mother to child | 65 (41%) |
| Others | 44 (28%) |
| Disease type, n (%) | |
| CML | 52 (33%) |
| AML | 41 (26%) |
| ALL | 54 (35%) |
| MDS | 10 (6%) |
| Disease state, n (%) | |
| Early | 78 (50%) |
| Intermediate | 37 (23%) |
| Advanced | 42 (27%) |
| Number of HLA-antigen mismatches, n (%) | |
| 1 Ag | 27 (17%) |
| 2 Ag | 71 (45%) |
| 3 Ag | 59 (38%) |
| Graft type, n (%) | |
| BM + PB | 156 (99.4%) |
| PB alone | 1 (0.6%) |
| MNCs, median (range), ×108/kg | 7.3 (4.2-16.3) |
| CD34+ count, median (range), ×106/kg | 2.3 (0.5-9.7) |
| CD3+ count, median (range), ×106/kg | 177 (15-991) |
| CD4+ count, median (range), ×106/kg | 100 (13-527) |
| CD8+ count, median (range), ×106/kg | 74 (15-467) |
| Follow-up time among living patients, median (range), days | 448 (48-1191) |
Donor Source and HLA Disparity
Family members were tested for HLA compatibility using serology and intermediate-resolution DNA typing for HLA-A, -B, and -C antigens and high-resolution DNA typing for HLA-DRB1, -DQB1, and -DPB1. Donors were patients' mothers in 65 cases (41.4%), fathers in 25 cases (16%), cousins in 4 cases (2.5%), siblings in 48 cases (30.6%), and children in 15 cases (9.5%), with various degrees of HLA disparity in HLA-A, -B, and -DRB1 antigen levels (Table 1, Table 2).
Table 2. Patient and donor characteristics according to CD3+ cell dose
| Variable | CD3+ < 177×106/kg | CD3+ ≥ 177×106/kg | P |
|---|---|---|---|
| Number of patients | 78 | 79 | |
| Recipient age, median (range), years | 24 (6-50) | 25 (7-50) | .95 |
| < 20 | 26 (33%) | 28 (35%) | .98 |
| 20-35 | 30 (38%) | 31 (39%) | |
| 35-50 | 21 (27%) | 19 (24%) | |
| ≥ 50 | 1 (1%) | 1 (1%) | |
| Donor age, median (range), years | 40 (14-58) | 40 (13-66) | .29 |
| < 20 | 6 (8%) | 8 (10%) | .51 |
| 20-35 | 11 (14%) | 14 (18%) | |
| 35-50 | 42 (54%) | 45 (57%) | |
| ≥ 50 | 19 (24%) | 12 (15%) | |
| Donor–patient sex matching | .08 | ||
| M to M | 20 (26%) | 25 (32%) | |
| M to F | 35 (45%) | 21 (27%) | |
| F to F | 9 (12%) | 17 (21%) | |
| F to M | 14 (18%) | 16 (20%) | |
| Donor–patient relationship | .04 | ||
| Mother to child | 38 (49%) | 27 (34%) | |
| Father to child | 8 (10%) | 17 (22%) | |
| Cousin | 4 (5%) | 0 | |
| Sibling | 21 (27%) | 27 (34%) | |
| Child to parent | 7 (9%) | 8 (10%) | |
| Blood match | .15 | ||
| Matched | 43 (55%) | 38 (48%) | |
| Minor ABO incompatibility | 18 (23%) | 13 (17%) | |
| Major ABO incompatibility | 17 (22%) | 28 (35%) | |
| HLA-antigen class mismatches | .52 | ||
| Class I (HLA-A and/or HLA-B) | 15 (19%) | 16 (20%) | |
| Class II (HLA-DRB1) | 5 (7%) | 9 (12%) | |
| Class I and II | 58 (74%) | 54 (68%) | |
| Number of HLA-antigen mismatches | .41 | ||
| 1 Ag | 11 (14%) | 16 (20%) | |
| 2 Ag | 39 (50%) | 32 (41%) | |
| 3 Ag | 28 (36%) | 31 (39%) | |
| Disease | .09 | ||
| CML | 30 (38%) | 22 (28%) | |
| AML | 24 (31%) | 17 (21%) | |
| ALL | 20 (26%) | 34 (43%) | |
| MDS | 4 (5%) | 6 (8%) | |
| Disease status | .94 | ||
| Early | 39 (50%) | 39 (49%) | |
| Intermediate | 19 (24%) | 18 (23%) | |
| Advanced | 20 (26%) | 22 (28%) | |
| MNCs, median (range), 108/kg | 6.8 (4.2-12.1) | 7.8 (4.2-16.3) | < .0001 |
| < 7.5 | 55 (71%) | 31 (39%) | < .0001 |
| ≥ 7.5 | 23 (29%) | 48 (61%) | |
| CD34+, median (range), 106/kg | 1.9 (0.5-7.1) | 2.3 (0.5-9.7) | .17 |
| < 2 | 41 (53%) | 28 (35%) | .03 |
| ≥ 2 | 37 (47%) | 51 (65%) | |
| CD4+, median (range), 106/kg | 69 (17-373) | 131 (13-527) | < .0001 |
| < 100 | 65 (83%) | 13 (16%) | < .0001 |
| ≥ 100 | 13 (17%) | 66 (84%) | |
| CD8+, median (range), 106/kg | 49 (15-312) | 103 (25-467) | < .0001 |
| < 75 | 61 (78%) | 19 (24%) | < .0001 |
| ≥ 75 | 17 (22%) | 60 (76%) | |
| Follow-up among survival patients, median (range), days | n = 50 | n = 63 | |
| 414 (48-1150) | 514 (113-1191) | .03 |
Graft Collection and Cell Composition Analysis
Donor BM and/or PB cells were collected using modified mobilization protocols; subcutaneous G-CSF (filgrastim) 5 μg/kg/day was given from day −3 to day 0. BM was harvested at day 0, after 4 days of G-CSF administration, with a target collection dose of 15 mL/kg (recipient weight), and PB, harvested on day 1, 5 days after the first G-CSF injection. The grafts were analyzed for the total number of mononuclear cells (MNCs), and, using a standardized Multi-Set kit (Becton-Dickinson, San Jose, CA), the content of CD34+ cells [14] and subsets of lymphoid cell populations (CD3+, CD4+, and CD8+ cells) was determined (Table 1, Table 2).
Transplantation Protocol
All patients enrolled in this study were treated under a uniform protocol with the conditioning regimen (Ara-C + Bu/Cy +ATG) [13] and GVHD prophylaxis (cyclosporine [CsA] + short-term methotrexate [MTX] 15, 16 + mycophenolate mofetil [MMF]). Pretransplantation conditioning chemotherapy included cytarabine 4 g/m2/day intravenously on days −10 to –9, Bu 4 mg/kg/d orally on days −8 to –6, Cy 1.8 g/m2/day intravenously on days −5 and −4, Me-CCNU 250 mg/m2 orally once on day −3, and ATG (thymoglobuline; Sang Stat, Lyon, France, now marketed by Genzyme) 2.5 mg/kg/day intravenously on days −5 to −2. First-line therapy for ≥ grade II GVHD was 1-2 mg/kg/day of prednisolone equivalents and resumption of full-dose cyclosporine (CSP). Patients who developed steroid-refractory acute GVHD (aGVHD) were given tacrolimus (FK506), MMF, CD25 monoclonal antibody (Simulect, Novartis), or MTX as second-line immunosuppressive therapy.
Definition of Endpoints
The study endpoints were engraftment, grade II-IV or III-IV aGVHD, TRM, relapse, leukemia-free survival (LFS), and overall survival (OS).
Neutrophil EngraftmentNeutrophil engraftment after transplantation was defined as a recovery of the blood absolute neutrophil count (ANC) to ≥ 0.5 × 109/L on 3 consecutive days. Platelet recovery was defined as the time after transplantation needed to maintain a blood platelet count ≥ 20 ×109/L without transfusion support for 7 consecutive days.
GVHDaGHVD and chronic GVHD (cGVHD) were defined according to Fred Hutchinson Cancer Research Center criteria 17, 18, 19. Patients who survived for > 100 days after transplantation were evaluated for cGVHD.
RelapseTime to relapse was defined from the date of transplantation to the date of disease recurrence, evaluated by morphological evidence of leukemia in either the BM or any extramedullary site.
TRMTRM was defined as all causes of death other than those related directly to malignant disease itself, occurring at any time after transplantation.
OS and LFSOS was defined as days from transplantation to death from any cause. LFS was defined as days from transplantation to disease progression after transplantation.
Statistical Analysis
The characteristics of patient-related, disease-related, and transplantation-related factors of the low and high CD3+ cell dose groups were compared using the χ2 test for categorical variables and the Mann-Whitney test for continuous variables. Univariate probabilities of achieving ANC and platelet engraftment, developing aGVHD and cGVHD, TRM, and relapse were calculated using cumulative incidence curves to accommodate corresponding competing risks. The Kaplan-Meier estimator was used to evaluate LFS and OS. Comparing the outcomes among CD3+ cell dose groups required adjustment for differences in baseline patient characteristics (Table 2). A Cox proportional hazards model was used to adjust for potential imbalance in baseline characteristics between CD3+ cell dose groups. Outcome events considered in multivariate analyses were TRM, relapse, treatment failure (opposite of LFS), and overall mortality (opposite of OS). A forward stepwise method was used to identify all significant risk factors for each outcome event. The effect of the dose of CD3+ cells transplanted (the main interest in the present study) was included in all steps of model building. The proportionality assumptions were tested by adding a time-dependent covariate; the tests indicated that the proportionality assumptions held for all risk factors. The potential interactions between main treatment groups and significant risk factors were tested, and no interactions were found (Table 3) Adjusted probabilities of LFS and OS were generated from the final Cox models stratified on treatment; weighted averages of covariate values were computed using the sample proportion as the weight function. These adjusted probabilities estimated the likelihood of outcomes in populations with similar prognostic factors. To examine the aGVHD effect, a time-dependent covariate was used in the final Cox regression models (Table 4). Finally, the cell dose effects of MNCs, CD34+, CD4+, and CD8+ were evaluated using the Cox regression method (Table 5).
Table 3. Multivariate analysis of association with TRM, relapse, LFS, and OS
| Outcome | RR (95% CI) | P |
|---|---|---|
| TRM | ||
| CD3+, median (range),106/kg | ||
| < 177 | 1.00 | |
| ≥ 177 | 0.35 (0.16-0.77) | .0090 |
| Relapse | ||
| CD3+: < 177 | 1.00 | |
| ≥ 177 | 0.72 (0.29-1.79) | .4778 |
| Other covariates | ||
| Disease status: Early | 1.00 | .0263∗ |
| Intermediate | 1.02 (0.25-4.06) | .9835 |
| Advanced | 3.48 (1.26-9.60) | .0159 |
| Treatment failure (Opposite of LFS) | ||
| CD3+: < 177 | 1.00 | |
| ≥ 177 | 0.46 (0.26-0.84) | .0106 |
| Other covariates | ||
| Disease status: Early | 1.00 | .0175∗ |
| Intermediate | 1.40 (0.67-2.93) | .3760 |
| Advanced | 2.56 (1.33-4.95) | .0050 |
| Overall mortality (Opposite of OS) | ||
| CD3+: < 177 | 1.00 | |
| ≥ 177 | 0.42 (0.23-0.78) | .0058 |
| Other covariates | ||
| Disease status: Early | 1.00 | .0136∗ |
| Intermediate | 1.46 (0.67-3.18) | .3398 |
| Advanced | 2.77 (1.39-5.53) | .0038 |
∗2 degree-of-freedom test. |
Table 4. Effect of aGVHD on clinical endpoints
| Outcome | RR (95% CI) | P |
|---|---|---|
| TRM | ||
| aGVHD: III-IV versus 0-II | 3.16 (1.43-6.99) | .0046 |
| Relapse | ||
| aGVHD: III-IV versus 0-II | 1.34 (0.38-4.73) | .6524 |
| Treatment failure (Opposite of LFS) | ||
| aGVHD: III-IV versus 0-II | 2.32 (1.18-4.57) | .0150 |
| Overall mortality (Opposite of OS) | ||
| aGVHD: III-IV versus 0-II | 2.64 (1.29-5.43) | .0082 |
Table 5. Effect of MNC, CD34+, CD4+, and CD8+ levels on clinical endpoints
| Outcome | RR (95% CI) | P |
|---|---|---|
| TRM | ||
| MNCs: ≥ 7.5 vs < 7.5×108/kg | 0.70 (0.33-1.49) | .3586 |
| CD34+: ≥ 2 vs < 2 ×106/kg | 0.67 (0.33-1.40) | .2874 |
| CD4+: ≥ 100 vs < 100×106/kg | 0.62 (0.30-1.31) | .2099 |
| CD8+: ≥ 75 vs < 75 ×106/kg | 0.73 (0.35-1.53) | .4090 |
| Relapse | ||
| MNCs: ≥ 7.5 vs < 7.5 ×108/kg | 2.52 (0.95-4.68) | .0632 |
| CD34+: ≥ 2 vs < 2 ×106/kg | 2.58 (0.85-7.80) | .0941 |
| CD4+: ≥ 100 vs < 100×106/kg | 1.33 (0.53-3.33) | .5384 |
| CD8+: ≥ 75 vs < 75×106/kg | 1.40 (0.56-3.51) | .4694 |
| Treatment failure (Opposite of LFS) | ||
| MNCs: ≥ 7.5 vs < 7.5 ×108/kg | 1.11 (0.62-1.96) | .7316 |
| CD34+: ≥ 2 vs < 2 ×106/kg | 1.04 (0.58-1.86) | .8956 |
| CD4+: ≥ 100 vs < 100 ×106/kg | 0.83 (0.47-1.46) | .5151 |
| CD8+: ≥ 75 vs < 75 ×106/kg | 0.97 (0.54-1.73) | .9161 |
| Overall mortality (Opposite of OS) | ||
| MNCs: ≥ 7.5 vs < 7.5×108/kg | 0.97 (0.53-1.76) | .9065 |
| CD34+: ≥ 2 vs < 2 ×106/kg | 0.96 (0.52-1.75) | .8816 |
| CD4+: ≥ 100 vs < 100 ×106/kg | 0.80 (0.44-1.45) | .4537 |
| CD8+: ≥ 75 vs < 75 ×106/kg | 1.05 (0.58-1.91) | .8762 |
Results
Characteristics of Patients Receiving a Low or High CD3+ Cell Dose
The median CD3+ cell number in the transplant inoculums was 177 ×106/kg (range, 15-991 ×106/kg). Patients were stratified into 2 cohorts; 1 cohort was given a lower than median dose of CD3+ cells (< 177 × 106/kg [n = 78]), and the other was given a greater than median or median dose of CD3+ cells (≥ 177 × 106/kg [n = 79]). The differences in clinical characteristics between the 2 cohorts were not statistically significant; the patients in the high-dose CD3+ cell cohort were infused with significantly higher numbers of MNCs, CD34+, CD4+, and CD8+ cells than those in the low-dose CD3+ cell cohort (Table 2).
Neutrophil and Platelet Recovery
All patients in the study achieved complete granulocyte engraftment, as reflected by the ANC attained. The median time after transplant to achieve an ANC of ≥ 0.5 × 109/L was 12 days (range, 8-25 days); the median time to achieve a blood platelet count of ≥ 20 × 109/L was 15 days (range, 7-169 days) in all but 5 patients. Three of these 5 patients had died from nonrelapse-related mortality (NRM) at day 36-84 posttransplantation. Two were alive; 1 survived to 399 days, the other to 206 days. The cumulative incidence of platelet engraftment was 97% (95% confidence interval [CI] = 94%-100%) in the low CD3+ cell dose cohort (< 177 × 106/kg) versus 95% (95% CI = 89%-98%) in the high CD3+ cell dose cohort (≥ 177 × 106/kg) (P = .3626). There was no significant difference in the cumulative incidence of ANC and platelet engraftment between the 2 CD3+ cell dose cohorts (Figures 1A and B).
Figure 1
Cumulative incidence of outcomes of haploidentical HCT for patients with leukemia. A, ANC (P = 1.0000 at day 100). B, Platelet engraftment (P = .3626 at day 100). C, aGVHD, grade 2-4 (P = .1165 at day 100). D, aGVHD, grade 3-4 (P = .5449 at day 100). E, Relapse (P = .3962 at 2 years). F, TRM (P = .0181 at 2 years).
GVHD
High CD3+ cell dose (≥ 177 ×106/kg) transplantation correlated with a higher incidence of grade II-IV aGVHD, 51% (95% CI = 40%-61%), compared with low CD3+ cell dose (< 177 × 106/kg) transplantations, 39% (95% CI = 28%-49%), but the difference was not statistically different (P = .1165) (Figure 1C). No significant difference was found in the cumulative incidence of grade III-IV aGVHD between the high CD3+ cell cohort, 19% (95% CI = 11%-28%), and the low CD3+ cell cohort, 15% (95% CI = 8%-24%), (P = 0.5449) (Figure 1D). In the 157 patients evaluated, the cumulative incidence of cGVHD at 2 years was 44% (95% CI = 32%-55%) in the low CD3+ cell dose cohort and 52% (95% CI = 41%-63%), in the high CD3+ cell dose cohort (P = .3100).
Death
RelapsePatients undergoing transplantations containing high CD3+ cell doses (≥ 177 × 106 CD3+ cells/kg) had fewer relapses at 2 years, 12% (95% CI = 6%-21%), than in the cohort receiving low CD3+ cell doses (< 177 × 106 CD3+ cells/kg), 18% (95% CI = 8%-31%), but the difference was not statistically significant (P = .3962) (Figure 1E). Thirteen patients died from disease relapse between 36 and 945 days after transplantation with various coexisting complications: severe infection, 43%; grade III-IV aGVHD, 35.7%; and interstitial pneumonia (IPn), 50%; along with varying grades of aGVHD.
TRMA total of 31 patients died from various complications between 72 and 590 days after transplantation: 27.3% from Ipn, 30.3% from lung infection due to mixed pathogens; 57.6% from nonpulmonary infection, and 24.2% from severe (grade 3-4) aGVHD. In patients whose grafts included high CD3+ cell doses (≥ 177 × 106/kg), the 2-year TRM of 13% (95% CI = 6%-22%) was statistically lower than the 30% (95% CI = 19%-41%) in those whose grafts contained fewer CD3+ cells (< 177 × 106/kg) (P = .0181) (Figure 1F).
Survival
Follow-up of surviving patients from 48 to 1191 days posttransplantation (median, 448 days) revealed a 53% (95% CI = 39%-66%) probability of 2-year LFS for patients with grafts containing fewer CD3+ cells (< 177 × 106/kg) and a 75% (95% CI = 64%-84%) probability of 2–year LFS in those with grafts containing more CD3+ cells (≥ 177 × 106/kg) (P = .0115) (Figure 2A). OS after haploidentical transplantation in the cohort was 70.2%. Univariate analysis demonstrated that only disease status and CD3+ cell dose in the graft were important predictors of survival. The 2-year probability of survival was 77% in those who underwent transplantation in the early stage of disease, 57% in those who did so in the intermediate stage, and 48% in those who did so in an advanced stage. There was no statistical difference in survival probability between those with intermediate-stage disease and advanced disease (log-rank test; P = .3246); however, a significant difference in survival probability was seen between patients who underwent transplantation in early-stage disease (77%; 95% CI = 66%-86%) and those who did so in intermediate-stage or advanced disease (56%; 95% CI = 43%-68%) (P = .0052). In addition, the probability of 2-year survival in the cohort undergoing transplantation with a low CD3+ cell dose (< 177 × 106/kg), 52% (95% CI = 37%-67%), was lower than that seen in the cohort who underwent transplantation with a high CD3+ cell dose (≥ 177 × 106/kg), 77% (95% CI = 66%-86%) (P = .0075) (Figure 2B).
Figure 2
Adjusted probabilities of LFS and OS after haploidentical HCT for patients with leukemia (adjusted for early, intermediate, and advanced disease status). A, Adjusted probability of LFS (P = .0115 at 2 years). B, Adjusted probability of OS (P = .0075 at 2 years).
Multivariate Analysis
Variables considered in the multivariate analyses were patient age group (< 20, 20-34, ≥ 35 years), donor–patient sex match, donor type (sibling vs others), number of HLA antigen mismatches, disease type, disease stage, graft type, CMV, MNCs (≥ 7.5 vs < 7.5 × 108/kg), CD34+ (≥ 2 vs < 2 × 106/kg), CD3+ (≥ 177 vs < 177 × 106/kg), CD4+ (≥ 100 vs < 100 × 106/kg), and CD8+ (≥ 75 vs < 75 × 106/kg). Multivariate analysis (Table 3) showed that advanced-stage disease at the time of transplantation was associated with a worse LFS (2.56; 95% CI = 1.33-4.95; P = .0050). Inversely, a high dose of CD3+ cells in the graft was associated with better LFS (0.46; 95% CI = 0.26-0.84; P = .0106) (Table 3). The disease status before transplantation and CD3+ cell dose given also had a significant influence on OS. Advanced-stage disease predicted a worse outcome (relative risk [RR] = 2.77; 95% CI = 1.39-5.53; P = .0038). However, patients had a significantly better survival if the transplant contained a high CD3+ cell dose, ≥ 177 × 106/kg (RR = 0.42; 95% CI = 0.23-0.78; P = .0058) (Table 3). Donor–recipient pair relationships showed no significant effect on outcomes.
Grades III-IV aGVHD posttransplantation had a significant effect on the various endpoint outcomes measured (Table 4). However, cGVHD had no obvious impact on these endpoints—a finding that could change with a longer follow-up time. Univariate and multivariate analysis showed that the numbers of MNCs, CD34+, CD4+, or CD8+ cells in the transplants had no significant influence on these clinical endpoints (Table 5).
Discussion
The present study is the largest study to date evaluating the predictors of developing GVHD, TRM, relapse, and survival in patients who, after receiving an identical conditioning regimen, (including ATG), underwent transplantation with unmanipulated nucleated cells from haploidentical blood and marrow donors. Stable engraftment and long-term survival after family mismatched/haploidentical HCT likely involves multiple mechanisms such as the conditioning regimen 11, 12, inherited maternal/paternal antigen (NIMA/NIPA) mismatch existing in donor-recipient pairs 20, 21, disease status 22, 23, donor versus recipient natural killer cell alloreactivity 23, 24, and the number of CD34+ cells infused 8, 11, 23. Previous reports were based mainly on the results of CD34+ cell selected transplantation, which involved marked T-cell depletion in vitro. Thus, the CD3+ cell doses given in those studies were very low, ranging from 103/kg to 105/kg of recipients' body weight 8, 11, 25, 26. Including ATG in the pretransplantation conditioning regimen provides an effective means of depleting T cells in vivo and preventing severe GVHD. But intensive T cell depletion in vivo by ATG also can delay immune reconstitution, resulting in a high mortality from viral and fungal infections 27, 28. Studies of ATG clearance in HSCT recipients, using ELISA for its detection, showed that significant levels of ATG can remain in the blood for up to 5 weeks posttransplantation 29, 30. Using different commercial preparations and conditioning regimens, as well as different timings and doses of ATG, may contribute to the discrepancies in the reports from different centers.
G-CSF–mobilized bone marrow induces early neutrophil engraftment with a reduced risk of GVHD 31, 32. Ji et al [32] reported that using G-CSF–primed haploidentical marrow grafts along with combined sequential use of CSP, MMF, ATG, and MTX provided an excellent alternative for the treatment of high-risk hematologic malignancy in patients without matched donors. The incidence of aGVHD was surprisingly low, only 6.3% in G-CSF–primed marrow transplants, compared with 33% in steady-state marrow transplants [32]. Recently, the clinical phenomenon has been confirmed by experimental data; in vitro mixtures of G-PB and G-BM in different proportions (G-PB:G-BM = 2:1, 1:1, and 1:2) has successfully introduced T cell hyporesponsiveness and polarization of T cells from Th1 to Th2 [33]. Under the current BM/PB harvest protocol, G-BM was harvested on day 0 after 4 days of G-CSF (target dose, 15 mL/kg recipient weight), and G-PB was harvested in a single leukapheresis on day 1 after 5 days of G-CSF therapy. In general, the number of MNCs obtained from G-BM was 3.87 (1.1∼9.52) ×108/kg, and that obtained from G-PB was 3.795 (1.17∼12.1) ×108/kg, with a BM:PB ratio of 1.006 (0.2∼6.5) (results from analysis of our unpublished data, n = 98).
G-BM also may add BM-derived mesenchymal stromal cells (MSCs) to grafts. In vitro, MSCs inhibit the proliferation of activated T cells and the formation of cytotoxic T cells. In vivo, they appear to have anti-inflammatory effects. Some previous results have shown that MSC therapy can facilitate engraftment [34] and decrease GVHD 35, 36, 37. The affect of MSCs co-transplanted in allo-HSCT on the effect of graft versus leukemia (GVL) response and the pathophysiologic mechanism for decreasing graft versus host (GVH) response remains under investigation 38, 39, 40. Furthermore, the significance of the BM:PB ratio in the graft, the immune characteristics of the T-cell subsets, dendritic cells, monocytes, natural killer cells (NKC), and the cytokine profile introduced by combining G-mobilized BM with G-PB need further investigation.
In our results, overall CD34+ doses below those reported by other centers may result from strict gating for CD34+ cell assay. Moreover, the dose of G-CSF that we used (5 μg/kg/day) was significantly lower than that reported by others (10-15 μg/kg/day) 31, 41, 42. Grafts mobilized by G-CSF have marked tolerogenic properties. The immune characteristics of the G-CSF–mobilized peripheral blood stem cells (PBSCs) and BM may partially explain why the relatively high CD3+ cell dose in the transplant did not increase the cumulative incidence of severe GVHD over 100 days or increase the incidence of cGVHD posttransplantation. Our results correspond somewhat to those of a recent report in which the 2 risk factors traditionally associated with aGVHD—age and the infused doses of CD3+, CD4+, CD8+, or CD34+ cells with PBSC grafts—did not significantly increase the incidence of GVHD [43].
In conclusion, our data demonstrate that with the currently available treatments, the dose of donor T cells used in this protocol is rational and acceptable. Relatively high numbers of MNCs and CD34+, CD4+, and CD8+ cells and a significantly high CD3+ cell dose contribute to better posttransplantation survival and enhanced LFS, resulting in part from a reduction in treatment-related toxicity without an increase in severe GVHD.
Acknowledgments
We are indebted to the patients and their families and express our gratitude for their participation in this study. We also acknowledge grants from the national “211 Project” (92000-242156014) and research financial support from the Peking University EBM group. Special thanks go to all of the staff members at the Division of Hematopoietic Cell Transplantation, the HLA Lab, and the FCM Lab at Peking University and Beijing Dao-Pei Hospital for their diligence and excellent work. We also thank Dr John C. Herion for his critical reading and helpful revisions to the manuscript and Dr Yan-Rong Liu for excellent laboratory support by providing the FACS analysis.
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PII: S1083-8791(07)00455-7
doi:10.1016/j.bbmt.2007.09.007
© 2007 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.
Volume 13, Issue 12 , Pages 1515-1524, December 2007
