Currently, limited information is available regarding the effects of early lymphocyte recovery on transplant outcomes in pediatric patients with hematological malignancies after unmanipulated haploidentical transplantation. In this study, we evaluated the association of Day 30 absolute lymphocyte count (ALC-30) with transplant outcomes in 60 consecutive pediatric paients with hematological malignancies receiving T-cell-repleted transplantation from an haploidentical related donors. After median follow-up of 36 months (range, 1.4–75 months), higher relapse rate was observed in patients with an ALC-30 < 300 cells/μL compared with patients with an ALC-30 ≥ 300 cells/μL (35.5% vs. 13.8%, P = 0.049). More patients died of infections in those with an ALC-30 < 300 cells/μL compared with patients with an ALC-30 ≥ 300 cells/μL (25.8% vs. 3.4%, P = 0.015). The ALC-30 above the cutoff value 300 cells/μL was associated with improved overall-survival (HR 0.301, 95% CI 0.117–0.771; P = 0.012), leukemia free survival (HR 0.195, 95% CI 0.078–0.498; P = 0.002), less relapse (HR 0.224 95% CI 0.070–0.717; P = 0.012), and less transplant-related mortality (HR = 0.166; 95%CI 0.037–0.750; P = 0.020). Our results suggest that a higher ALC-30 (≥300 cells/μL) could be a useful and simple tool to predict pediatric patients with a superior outcome after unmanipulated haploidentical transplantation. Haploidentical hematopoietic stem cell transplantation (HSCT) is now a viable option for children with hematological malignancies who do not have an human leukocyte antigen (HLA) compatible sibling or fully matched unrelated donor [1-5]. However, relapse and infections are the main complications, which lead to a high mortality and morbidity after HLA-matched or haploidentical HSCT. T cells with cytotoxicity function reconstituted following transplantation can prevent infections and eradicate residual tumor cells and often recover early in the patients with better outcomes after HLA-identical allogeneic HSCT or autologous HSCT [6-15]. Currently, limited information is available regarding the association of early lymphocyte recovery with transplant outcomes after this transplant protocols [2, 3], especially in pediatric patients. In this study, we hypothesize that higher Day 30 absolute lymphocyte count (ALC-30) is associated with superior clinical outcomes in pediatric patients with hematological malignancies following umanipulated haploidentical transplantation [2-4]. Patient characteristics are showed in Table I (please see Supporting Information, available on-line). A total of 60 consecutive pediatric patients with hematological malignancies received unmanipulated HLA-mismatched/haploidentical blood and marrow transplantation during the study period. Two groups were identified based on an ALC of ≥ 300 cells/μL and <300 cells/μL post HSCT. The cutoff of ALC-30 = 300 cells/μL was chosen as a value based on previous report [6, 9, 16, 17]. Moreover, no patients received donor lymphocyte infusion (DLI) [18] prior to Days 30, which excludes the effect of DLI on Days 30 lymphocyte recovery. There were no statistical differences in patient's characteristics between the two groups at Days 30 taking into account a P-value of 0.05, except that more patients with an ALC <300 cells/μL received DLI compared with those with an ALC ≥300 cells/μL (12.9% vs. 0%, P = 0.045). Four of all 60 patients with an ALC-30 < 300 cells/μL received DLI, including acute myeloid leukemia (one patient), acute lymphoblastic leukemia (two patients), and chronic myeloid leukemia (one patient). All of these patients received therapeutic DLI. DLIs were delivered at days 71, 79, 330, and 395, respectively. Two of the four patients are still alive free of leukemia till the end of follow-up. All patients (100%) achieved neutrophil engraftment. One patient (1.67%) failed to platelet engraftment. The median time to neutrophil and platelet recovery was 13 days (range, 10–49 days) and 15.5 days (range, 7–176 days), respectively. There were no significant correlations between ALC-30 and time to neutrophil (r = −0.045, P = 0.733) and platelet engraftment (r = 0.086, P = 0.515). The time for neutophil and platelet engraftment in patients with an ALC-30 ≥ 300 cells/μL was comparable with those with an ALC-30 < 300 cells/μL (P = 0.799, and 0.436, respectively) (Table I). Thirty-two patients developed Grades II–IV acute graft-versus-host disease (aGVHD) with a cumulative incidence of 52.4% ± 8.00%. 31 of 57 evaluable patients surviving more than 100 days developed chronic GVHD (cGVHD: limited 20 [35.09%]; extensive 11 [19.30%]) with a cumulative incidence of 59.7% ± 7.50%. There were no significant differences in the incidence of aGVHD and cGVHD between patients with an ALC-30 ≥ 300 cells/μL and those with an ALC-30 < 300 cells/μL post HSCT (Table I). No effects of age, sex, HLA-mismatch, ABO-mismatch, CD3+ and CD34+ cell dose on the incidence of aGVHD and cGVHD were observed. Fourteen of 60 (23.33%) patients died from nonrelapse causes, with a cumulative incidence of transplant-related mortality (TRM) of 28.5% ± 7.70%. Causes of death were: bacterial/fungal/viral infection [9]; GVHD-related [4]; post-transplant lymphoproliferative disorder (PTLD) [1]. Factors associated with a significantly increased risk of TRM were ALC-30 < 300 cells/μL (P = 0.008); lymphocytes in allograft (P = 0.041); and disease risk (high risk [HR] versus standard risk [SR], P = 0.044). In multivariate analysis, ALC-30 (HR = 0.166; 95%CI 0.037–0.750; P = 0.020) were independently associated with an increased risk of TRM. The causes of death due to infections were significantly higher in patients with ALC-30 < 300 cells/μL (8/31, 25.8%) compared with those with ALC-30 ≥ 300 cells/μL (1/29, 3.4%) (P = 0.015). There was a statistically significant increase in relapse rate for patients with an ALC-30 < 300 cells/μL (HR = 0.224; 95%CI 0.070–0.717; P = 0.012) (Table I) and those in high-risk groups (HR = 4.753; 95% CI 1.653–13.668; P = 0.004) after transplantation. This significant difference in relapse led to a significantly superior three-year leukemia-free survival (LFS) for the groups of ALC-30 ≥ 300 cells/μL compared with those of ALC-30 < 300 cells/μL (79.3% versus 32.3%, P < 0.001) (Fig. 1A and Table I). Leukemia-free survival (A), overall survival (B) curves in pediatric patients with hematological malignancies after unmanipulated HLA-mismatched/haploidentical blood and marrow transplantation. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Currently, 38 of 60 patients are alive and 33 are disease free. In univariate analysis, factors associated with improved overall survival (OS) were standard risk disease (74.4% versus 42.9% for high risk disease; P = 0.016) and ALC-30 ≥ 300 cells/μL (79.3% versus 48.4% for ALC-30 < 300 cells/μL; P = 0.013). There were no impacts of type of age, sex, time from diagnosis to transplantation, CD3+ and CD34+ cell dose on OS. Multivariate Cox regression analysis showed that standard risk (HR 2.521, 95% CI 1.090-5.831; P = 0.031) and ALC-30 ≥ 300 cells/μL (HR 0.301, 95% CI 0.117–0.771; P = 0.012) were associated with improved OS (Fig. 1B and Table II). The association of rapid early lymphocyte recovery with better transplant outcomes has been demonstrated in both HLA-identical allogeneic and autologous transplant settings [6, 7, 9, 11, 13, 14, 19]. We have identified an independent prognostic effect of early lymphocyte recovery in hematological malignant patients receiving unmanipulated haploidentical transplant [17]. To minimize heterogeneity due to ages, we focused our analysis on a select group of pediatric patients after a long follow-up. Similar to the results obtained in general population, [17] pediatric patients with a higher ALC-30 (≥300 cells/μL) also experienced superior OS and LFS (Fig. 1A,B). Impressively, we identified ALC-30 as a strong independent predictor of treatment outcome in this subpopulation. Our previous study demonstrated that NK cells constitute 27% (range, 0.07%–86%) of the early recovered lymphocytes and patients with more CD56bright NK cells in the recovery stage have a better survival [20]. Available data suggest that early recovered NK cells may be the main effector cells in eliminating leukemia cells [8, 21]. In addition to NK cells, the compositions of early reconstituted lymphocyte include T cells, monocytes, and dendritic cells. Therefore, further studies are needed to elucidate the role of above-mentioned immune cells played in improving transplant outcomes. Studies in animals suggest that post-transplantation interleukin-15 (IL-15) and IL-7 administration significantly increased donor-derived T cells, NK cells, and NKT cells in mice recipients after bone marrow transplantation, which could enhance the graft-versus-leukemia effects [22-24]. Thus, enhancing the immune recovery in pediatric patients with lower ALC-30 (<300 cells/μL) is important to improve the outcomes of these patients who underwent unmanipulated haploidentical transplantation. Infection is one of the fatal complications after allo-HSCT [25]. In our study, the higher ALC-30 (≥300 cells/μL) group showed a significant decreased percentages of infection-related death compared to the low group (25.8% vs. 3.4%, P = 0.015), this may be related to the early recovered lymphocytes, which could contribute to prevent fatal opportunistic infections after transplantation. These data suggest that maximum efforts should be pursued in patients with ALC-30 < 300 cells/μL to prevent and monitor opportunistic infections, including extended antimicrobial, antifungal prophylaxes. Currently, the promising effects of different immunological strategies, including interleukin-2 (IL-2), [26] and DLI, [18] have been showed in our transplant settings. In conclusion, our results suggest that the prevention of infection and relapse by the early recovered lymphocytes may contribute to the improved transplant outcomes of pediatric patients after unmanipulated haploidentical transplantation. Our data also suggest that the status of the immune system post-transplant should be carefully monitored as a potential variable influencing outcome and further immunological therapies for these patients (with ALC-30 < 300 cells/μL) should be considered. Pediatric patients with hematological malignancies suitable for allogeneic HSCT without HLA-identical related or unrelated donors were candidates for the haploidentical HSCT. Sixty patients with hematological malignancies, who underwent unmanipulated haploidentical transplantation between October 2002 and August 2007, were enrolled in this retrospectve study. For the pediatric patients, written informed consent from patients' parents to perform transplantation and to collect blood was obtained, both patients and their donors provided written informed consent, and the Institutional Review Board of Peking University Institute of Hematology approved this study. The characteristics of the 60 patients are shown in Table I (please see supporting information, available on-line), disease status at transplantation was defined according to the data published previously [2, 17]. Follow-up for all patients went through January 10, 2010. All patients received myeloablative regimens including a combination of cytosine arabinoside (Ara-C) (4 g/m2 × 2 d, on Days −10 and −9), busulfan (12 mg/kg administered orally in 12 doses over 3 d, on Days −8, −7, and −6), cyclophosphamide (1.8 g/m2 × 2 d, on days −5 and −4), Simustine (MeCCNU) (250 mg/m2 orally once on Day −3), and rabbit anti-human thymocyte immunoglobulin (Thymoglobulin 2.5 mg/kg per day i.v. of the Sangstat product) on Days −5 through −2 [2, 3]. Donors received recombinant human granulocyte colony-stimulating factor (rhG-CSF, Filgrastim) 5 μg/kg daily for 5 to 6 days. On the 4th day, bone marrow cells were harvested. The fresh and unmanipulated bone marrow and PBPCs were infused into the recipients on the day of collection [2, 3]. Prophylaxis for GVHD included cyclosporine A (CSA) and short-term methotrexate (MTX) with mycophenolate mofetil (MMF). CSA was started intravenously on Day −9, at the dosage of 2.5 mg/kg, and switched to oral formulation as soon as the patient was able to take medication after engraftment. Thedosage was adjusted to blood levels. MMF was administered orally, 0.5 g every 12 hr, from Day 9 before transplantation to Day 30 after transplantation, then 0.25 twice a day for one to two months. The dosage of MTX was 15 mg/m2, administered i.v. on Day 1, and 10 mg/m2 on Days 3, 6, and 11 after transplantation. Filgrastim (rhG-CSF) 5 μg/kg per day was given to all recipients subcutaneously from Day 6 after transplantation until the neutrophil count reached 0.5 × 109 cells/L for three consecutive days. Bone marrow aspiration and cytogenetics studies were performed at 1, 2, and 3 months after transplantation to assess engraftment. HLA DNA typing and PCR-DNA fingerprinting (short tandem repeat) were used for donor chimerism detection. For each patient, at least two methods were used to confirm donor chimerism [2, 3]. Neutrophil engraftment after transplantation was defined as an absolute neutrophil count (ANC) exceeding 500/μL for three consecutive days. The day of engraftment was determined to be the first of these three consecutive days. Platelet recovery was defined the time after transplantation needed to maintain a blood platelet count exceeding 20 × 109/L without transfusion support for seven consecutive days. The severity of aGVHD was graded according to the consensus criteria [27]. Chronic GVHD was assessed and graded according to the standard criteria [28, 29]. Time to relapse was defined from the date of transplantation to the date of disease recurrence, evaluated by morphological evidence of leukemia in either the bone marrow or any extramedullary site. TRM was defined as all causes of death other than those related directly to malignant disease itself, occurring at any time after transplantation. OS was defined as days from transplantation to death from any cause. LFS was defined as days from transplantation to disease progression after transplantation. The primary endpoint of the study was to assess the impact on OS based on ALC-30 after transplantation. The secondary endpoint was to determine whether the ALC-30 was an independent prognostic factor for OS in pediatric patients with hematological malignancies. Summary statistics, such as proportions, means, standard deviations, 95% confidence intervals (CIs), medians, and ranges, were used to describe the patient characteristics, pretransplant variables, and post-transplant outcomes. The associations between lymphocyte recovery and transplant outcomes were analyzed by the methods of Kaplan-Meier or calculated using cumulative incidence curves to accommodate competing risks [30]. Log-rank test was used in survival and Gray test was used in cumulative incidence analyses. To confirm outcomes and adjust for potential confounding factors, multivariate Cox proportional hazards models were assessed for the proportional hazards assumption and for testing interaction terms with covariates. Variables included in the models were: age (less than versus greater than or equal median), sex, donor-patient sex match (female to male versus others), ABO match (identical+minor versus major+bidirectional), HLA-match (one locus versus two locus versus three locus), disease risk (HR versus SR), CD34 dose (less than versus greater than or equal to median), CD3 dose (less than versus greater than or equal to median), ALC-30 (less than versus greater than or equal to median). The final multivariate models were built using a forward stepwise model selection approach. Patients' characteristics in “high” versus “low” lymphocyte groups were compared using chi-square statistics for categorical variables and Mann-Whitney U for continuous variables, respectively. The calculations were carried out with “SPSS 13.0” statistical software. R software was used to calculate the cumulative incidence considering the presence of competing risk. Ying-Jun Chang: performed research, analysis, and interpretation of data, and drafting of the article, and gave final approval of the version to be published; Xiang-Yu Zhao: performed research, analysis and interpretation of data, and drafting of the article and gave final approval of the version to be published; Xiao-Jun Huang: involved in conception and design, revising the article critically, and final approval of the version to be published; All others: Study design discussions, treated patients at Peking University Institute of Hematology. Additional Supporting Information may be found in the online version of this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Ying-Jun Chang*, Xiang-Yu Zhao*, Ming-Rui Huo*, Lan-Ping Xu*, Dai-Hong Liu*, Kai-Yan Liu*, Xiao-Jun Huang*, * Peking University People's hospital, Peking University Institute of Hematology, Beijing, China.
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