Severe Aplastic Anemia Children Research Articles

Association between Haploidentical Hematopoietic Stem Cell Transplantation Combined with an Umbilical Cord Blood Unit and Graft-Versus-Host Disease in Pediatric Patients with Acquired Severe Aplastic Anemia

Introduction: Haploidentical hematopoietic stem cell transplantation (haplo HSCT) based on granulocyte colony-stimulating factor plus anti-thymocyte regimens ("Beijing Protocol") provides a salvage treatment for patients of acquired severe aplastic anemia (SAA) in China. However, graft-versus-host disease (GVHD) is a major impediment of haplo HSCT due to HLA disparity. Human umbilical cord blood (UCB) is characterized by lower numbers of mature or primed T-cells. Additionally, UCB has been reported to be a rich and rapidly accessible source of regulatory T cells (Tregs) and mesenchymal stromal cells (MSCs). These characteristics of UCB make it a potential prophylactic method for GVHD. Recently, haplo HSCT combined with umbilical cord blood (UCB) (haplo-cord HSCT) is performed in clinical trials to potentially reduce the risk of severe GVHD. Nevertheless, studies comparing GVHD in pediatric patients receiving haplo and haplo-cord HSCT for SAA are limited. Herein, we aim to investigate the impact of UCB co-infusion on GVHD in pediatric patients receiving haplo HSCT for SAA. Methods: Data from 91 consecutive children with SAA treated with "Beijing Protocol"-based haploidentical transplantation with or without co-infusion of UCB between April 2015 and July 2021 in the Children's Hospital of Soochow University were retrospectively analyzed. The cohort included 35 patients in the haplo group and 56 patients in the haplo-cord group. All patients received uniform non-myeloablative conditioning, GVHD prophylaxis. We compared baseline characteristics and transplant outcomes between the haplo and haplo-cord recipients. Log-rank test and Cox proportional regression model were used for univariate and multivariate analysis, respectively. A significance level of 0.05 was used for all analyses. Results: No significant differences in the baseline information were observed between haplo and haplo-cord groups. All 91 patients achieved hematopoietic recovery from haploidentical donors, with a higher 30-day cumulative incidence of platelet engraftment observed with haplo group as compared to haplo-cord group (98.2% versus 97.1%, p = 0.028). Cox regression analysis revealed that the haplo or haplo-cord transplant type was not associated with the time to platelet engraftment, while a low CD34+ cell dose in haploidentical grafts was correlated with delayed platelet recovery (hazard ratio (HR), 2.495; 95% confidence interval (CI), 1.570-3.964; p < 0.001). The haplo-cord group showed a higher incidence of peri-engraftment syndrome compared to the haplo group (75.0% versus 48.6%, p = 0.029), and Cox regression analysis identified haplo-cord HSCT as the only significant factor associated with a higher incidence of peri-ES (HR, 1.767; 95% CI, 1.002-3.115; p = 0.049). Of note, the haplo-cord group showed a lower incidence of II-IV acute GVHD (aGVHD) than the haplo group (16.1% versus 42.9%, p = 0.002). Observed incidences of chronic GVHD (cGVHD) and moderate to severe cGVHD in the haplo-cord group were also lower than that in the haplo group (25.6% versus 51.3%, p = 0.019; 16.2% versus 41.3%, p = 0.016, respectively). Haplo-cord HSCT was identified as the only factor associated with a lower incidence of II-IV aGVHD (HR, 0.335; 95% CI, 0.143-0.784; p = 0.012) and cGVHD (HR, 0.429; 95% CI, 0.203-0.907; p = 0.027) in Cox regression analysis. No differences were observed between the two groups in CMV, EBV infection, overall survival, failure-free survival and GVHD, failure-free survival. Conclusions: Our study provided compelling results of descriptive analysis suggesting that co-infusion of a third-party UCB unit with haplo HSCT might be associated with a lower risk of II-IV aGVHD and cGVHD. Although this co-infusion protocol may come with a higher risk of peri-ES, the survival outcomes may not be negatively affected. Despite the limited sample size, this study is the first real-world comparative study to investigate the impact of haplo-cord HSCT on GVHD in SAA children, which may stimulate further research of haplo-cord HSCT in pediatric patients with SAA and hematologic malignancies, as well as the potential association with GVHD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Significance of paroxysmal nocturnal hemoglobinuria clone in immunosuppressive therapy for children with severe aplastic anemia.

To study the association between paroxysmal nocturnal hemoglobinuria (PNH) clone and immunosuppressive therapy (IST) in children with severe aplastic anemia (SAA). A retrospective analysis was performed on the medical data of 151 children with SAA who were admitted and received IST from January 2012 to May 2020. According to the status of PNH clone, these children were divided into a negative PNH clone group (n=135) and a positive PNH clone group (n=16). Propensity score matching was used to balance the confounding factors, and the impact of PNH clone on the therapeutic effect of IST was analyzed. The children with positive PNH clone accounted for 10.6% (16/151), and the median granulocyte clone size was 1.8%. The children with positive PNH clone had an older age and a higher reticulocyte count at diagnosis (P<0.05). After propensity score matching, there were no significant differences in baseline features between the negative PNH clone and positive PNH clone groups (P>0.05). The positive PNH clone group had a significantly lower overall response rate than the negative PNH clone group at 6, 12, and 24 months after IST (P<0.05). The evolution of PNH clone was heterogeneous after IST, and the children with PNH clone showed an increase in the 3-year cumulative incidence rate of aplastic anemia-PNH syndrome (P<0.05). SAA children with positive PNH clone at diagnosis tend to have poor response to IST and are more likely to develop aplastic anemia-PNH syndrome.

Association between haploidentical hematopoietic stem cell transplantation combined with an umbilical cord blood unit and graft-versus-host disease in pediatric patients with acquired severe aplastic anemia.

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) based on granulocyte colony-stimulating factor plus anti-thymocyte regimens ('Beijing Protocol') provides a salvage treatment for patients of acquired severe aplastic anemia (SAA) in China. However, graft-versus-host disease (GVHD) is a major impediment of haplo-HSCT due to human leukocyte antigen disparity. Recently, haplo-HSCT combined with umbilical cord blood (UCB) (haplo-cord HSCT) is performed in clinical trials to potentially reduce the risk of severe GVHD. Nevertheless, studies comparing GVHD in pediatric patients receiving haplo and haplo-cord HSCT for SAA are limited. The objective of this study was to investigate the impact of UCB co-infusion on GVHD in pediatric patients receiving haplo-HSCT for SAA. We conducted a retrospective study of 91 consecutive SAA children undergoing haploidentical transplantation based on the 'Beijing Protocol' with or without co-infusion of UCB in our center. All patients received uniform non-myeloablative conditioning and GVHD prophylaxis. We compared baseline characteristics and transplant outcomes between the haplo (n = 35) and haplo-cord (n = 56) recipients. All 91 patients achieved hematopoietic recovery from haploidentical donors, with a higher incidence of peri-engraftment syndrome observed with the haplo-cord group as compared with the haplo group (75.0% versus 48.6%, p = 0.029). Notably, the haplo-cord group showed a lower incidence of II-IV acute GVHD (aGVHD) than the haplo group (16.1% versus 42.9%, p = 0.002). Observed incidences of chronic GVHD (cGVHD) and moderate to severe cGVHD in the haplo-cord group were also lower than that in the haplo group (25.6% versus 51.3%, p = 0.019; 16.2% versus 41.3%, p = 0.016, respectively). Haplo-cord HSCT was identified as the only factor associated with a lower incidence of II-IV aGVHD and cGVHD in multivariate analysis. However, no differences were observed between the two groups for infections and survival outcomes. Our data indicated that co-infusion of UCB in 'Beijing Protocol'-based haplo-HSCT may be effective for reducing the risk of severe GVHD in SAA children.

Clinical Characteristics and Prognosis Analysis of Pediatric Severe Aplastic Anemia

To analyze the clinical characteristics and factors affecting prognosis in children with severe aplastic anemia (SAA). Two hundred and five children with SAA treated in our department from January 2008 to April 2018 were selected, and the clinical characteristics and factors affecting prognosis were retrospectively analyzed. Among 205 SAA children, the effective rate (CR+PR) at 3, 6 and 12 months after immunosuppressive therapy (IST) treatment was 50.9%, 59.0% and 73.9%, respectively, and 5-year overall survival rate was 93.1%±2.0%. Univariate analysis showed that 5-year overall survival rate of SAA children of spontaneous delivery was higher than that of cesarean section (P=0.039), while multivariate analysis showed that birth way had no significant influence on 5-year overall survival rate (P>0.05). The response rate at 3 months after IST of children with a recent history of decoration before SAA onset was higher than those without history of decoration (P<0.05). Most of the SAA children can achieve high response rate and overall survival rate. Patients with recent history of home/school decoration may be the factor affecting hematological response after 3 months of IST, but have no influence on long-term overall survival.

Study of the outcome of pediatric patients with aplastic anemia treated with immunosuppressive therapy-Single center experience

Background: Immunosuppressive therapy is the Initial therapy for children with severe Aplastic anemia (SAA) and lacking a matched related donor. Cyclosporine A (Cy A) alone has been tried as single agent in resource poor Countries. Patients and Methods: This retrospective study was done to assess the outcome of immunosuppressive therapy (IST) in SAA children. It included 23 patients treated at Ain Shams University Pediatrics Hospitals between 2011- 2019 with IST, they received rabbit anti-thymocyte globulin (r ATG) plus (Cy A) (Group I, 8 patients) or Cy A alone (Group II, 15 patients). The median follow- up duration was 59 months. Results: one patient (4.3%) developed AML clone. another patient (4.3%) developed PNH clone. There was no significant difference in response between the two groups, Complete response was 34.7%, 43.5%, and 47.8% and partial response was 34.7%, 26.1%, 21.7% at 3, 6, and 12 months, respectively in the whole cohort. NR was present in 30.4% at 3, 6, and 12 months. Hypertension in 62.5% and higher levels of ALT, total and direct bilirubin were observed in group I as compared to group II. HAAA was clinically evident in 7 patients; 4 of them were hepatitis A, 1 was EBV, and 2 were seronegative for all viruses. At presentation they had significantly higher level of ALT, total bilirubin, and direct bilirubin. Conclusion: IST is an effective therapy in patients with SAA with overall response of 70%. Cy A alone can be an effective therapy in developing countries.

First-Line Choice for Severe Aplastic Anemia in Children: Transplantation from a Haplo-Identical Donor Versus Immunosuppressive Therapy

Background: Severe aplastic anemia (SAA) is a fatal disorder in children if treated inappropriately. According to the current algorithm, transplantation from a matched related donor (MRD) is recommended as the first-line option in children with SAA, if a MRD is unavailable, IST with a combination of anti-thymocyte globulin (ATG) and cyclosporine (CsA) represents the first-line choice. However, numerous limitations of immunosuppressive therapy (IST) exists. IST children may have incomplete recovery, and appear to be associated with an increased risk of clonal evolution and subsequent relapse. Additionally, the unavailability of horse-ATG in China also make the response of IST at a discount greatly. At the same time, great progresses in recent years in haploidentical transplantation might also promote changes in the algorithm of SAA treatment.Methods: We retrospectively compared the outcomes of children with SAA who received IST or who underwent hematopoietic stem cell transplantation (HSCT) from a haplo-identical donor (HID) as first-line choice between 2007 and 2016. The conditioning regimen for SCT from a haploidentical donor consisted of intravenous busulfan, cyclophosphamide and rabbit ATG. All of children were administered with granulocyte colonystimulating factor (G-CSF)-primed bone marrow (BM) combined with G-CSF-primed peripheral blood (PB) hematopoietic stem cells.Results: A total of 52 SAA children under the age of 17 years were initially treated with IST (n=24) or haplo-identical HSCT (n=28) as first-line choice. The last follow-up for all surviving patients was June 1, 2017. In IST group, by 6 months, 15 of the 21 subjects (71.4%) improved with first-line IST and achieved a partial response (n=10) or complete response (n=5). In SCT group, 27 patients (96.4%) achieved primary engraftment at a median of 12 (range, 10-21) days except one suffered from primary graft failure. The cumulative incidences (CI) of grade II-IV and of grade III-IV acute graft versus host disease (GVHD) were 48.1±1.0% and 11.1±0.4%, respectively. The 3-year CI of extensive chronic GVHD was 3.7±0.1%. The estimated 10-year overall survival were 73.4±12.6% and 89.3±5.8% in patients treated with IST or HID-HSCT (P=0.806). However, the failure-free survival was significantly inferior in patients receiving IST than in those undergoing transplantation from a HID [52.6±10.5% versus 89.3±5.8, P=0.008]. In univariate analysis, the choice of first-line immunosuppressive therapy was the only adverse predictor for failure-free survival. At the last follow-up, completely normal blood count was observed in 11 of 20 (55.0%) and 24 of 25 (96.0%) alive cases in IST and HID-HSCT cohort (P=0.003).Conclusions: Based on the superior FFS, high rate of engraftment and acceptable incidence of GVHD, the choice of HID HSCT for SAA children without a matched related donor as a first choice option seems reasonable. These suggest that SCT from a haplo-identical donor could be considered as first-line choice in children who lack a matched related donor, especially in experienced transplantation centers. DisclosuresNo relevant conflicts of interest to declare.

Combination of umbilical cord mesenchymal stem cells and standard immunosuppressive regimen for pediatric patients with severe aplastic anemia

BackgroundDefects of bone marrow mesenchymal stem cells (BM-MSCs) in proliferation and differentiation are involved in the pathophysiology of aplastic anemia (AA). Infusion of umbilical cord mesenchymal stem cells (UC-MSCs) may improve the efficacy of immunosuppressive therapy (IST) in childhood severe aplastic anemia (SAA).MethodsWe conducted an investigator-initiated, open-label, and prospective phase IV trial to evaluate the safety and efficacy of combination of allogenic UC-MSCs and standard IST for pediatric patients with newly diagnosed SAA. In mesenchymal stem cells (MSC) group, UC-MSCs were injected intravenously at a dose of 1 × 106/kg per week starting on the 14th day after administration of rabbit antithymocyte globulin (ATG), for a total of 3 weeks. The clinical outcomes and adverse events of patients with UC-MSCs infusion were assessed when compared with a concurrent control group in which patients received standard IST alone.ResultsNine patients with a median age of 4 years were enrolled as the group with MSC, while the data of another 9 childhood SAA were analysed as the controls. Four (44%) patients in MSC group developed anaphylactic reactions which were associated with rabbit ATG. When compared with the controls, neither the improvement of blood cell counts, nor the change of T-lymphocytes after IST reached statistical significance in MSC group (both p > 0.05) and there were one (11%) patient in MSC group and two (22%) patients in the controls achieved partial response (PR) at 90 days after IST. After a median follow-up of 48 months, there was no clone evolution occurring in both groups. The 4-year estimated overall survival (OS) rate in two groups were both 88.9% ± 10.5%, while the 4-year estimated failure-free survival (FFS) rate in MSC group was lower than that in the controls (38.1% ± 17.2% vs. 66.7% ± 15.7%, p = 0.153).ConclusionsConcomitant use of IST and UC-MSCs in SAA children is safe but may not necessarily improve the early response rate and long-term outcomes. This clinical trial was registered at ClinicalTrials.gov, identifier: NCT02218437 (registered October 2013).

The Outcome of Severe Aplastic Anemia Children Treated with Reduced Dose of Cyclophosphamide Combined Cyclosporine A

To analyze the clinical efficacy and side effects of reduced-dose of cyclophosphamide combined cyclosporine A for severe aplastic anemia(SAA) children. Ten pediatric patients with SAA from January 2008 to May 2012 were enrolled. All the patients were treated with reduced dose of cyclophosphamide combined cyclosporine A. The dose of cyclophosphamide was 30 mg/(kg·d)×4 d, the dose of cyclosporine A gradually increased >15 mg/L accroding to the blood concentration. The median follow-up time of the 10 pediatric patients was 100 months (6-126 months). Among 10 children with SAA, 4 cases achieved complete response(CR), 3 cases obtained partial response (PR) and the overall response rate was 70%, the remaining 3 cases showed no response (NR). One refractory patient treated by cyclophosphamide was progressed to paroxysmal nocturnal hemoglobinuria(PNH) at 25 months and was dead at 42 months after therapy. The results show that reduced-dose cyclophosphamide (30 mg/kg·d for 4 consecutive days) combinated with CsA (initial dose 4 mg/kg·d, and drugvallery concentration >150 ng/ml) can make 7 of 10 children with severe aplastic anemia achieve complete response or partial response, and this regimen may be the second line regimen selected for some SAA children.

Alternative donor HSCT for 109 children with acquired severe aplastic anemia: a single center retrospective analysis

目的了解替代供者（AD）移植一线治疗儿童再生障碍性贫血（AA）的疗效及安全性。方法回顾性分析2010年4月1日至2016年12月31日在上海儿童医学中心一线接受AD移植治疗的AA患儿临床资料，统计分析总生存（OS）率、植入成功率、移植物抗宿主病（GVHD）发生率等指标。结果共纳入109例患者，极重型AA（VSAA）32例，重型AA（SAA）64例，非重型AA（NSAA）伴输血依赖13例，中位年龄6（0.8~18）岁，其中44例患者接受全相合无关供者（MUD）移植，44例接受8–9/10位点不全相合无关供者（MMUD）移植，21例接受不全相合亲缘供者（MMRD）移植，所有患者均接受以外周血干细胞（PBSC）为主的移植，≥3个位点不合的单倍型移植加第三方脐血（UCB）一份。所有患者移植前均未接受过抗胸腺细胞球蛋白（ATG）治疗，并排除活动性感染。106例（97.2%）获造血重建，中性粒细胞中位重建时间为13（9~19）d，血小板中位重建时间为16（10~81）d。死亡13例，5年OS率为88.1%（95%CI 81.1%~91.4%），MUD、MMUD及MMRD三组患者OS率差异无统计学意义（P=0.361）。总体急性GVHD（aGVHD）及Ⅱ~Ⅳ度aGVHD发生率分别为74.3%和39.4%，总体慢性GVHD（cGVHD）和中度cGVHD发生率分别为30.7%和9.9%，无一例患者发生重度cGVHD。结论对于无同胞全相合供者的SAA/VSAA患儿，早期一线接受AD移植可能是一个选择，但需要进一步探索更有效的预防及治疗GVHD的措施。

Predicting response to porcine antilymphocyte globulin plus cyclosporine A in children with acquired severe aplastic anemia.

In severe aplastic anemia (SAA), predictive markers of response to immunosuppressive therapy (IST) of porcine antilymphocyte globulin (pALG) have not been well defined. We investigated whether clinical and laboratory findings before treatment could predict response in a pediatric cohort. In this study, we included 70 newly diagnosed SAA children and treated them with pALG. The response rate was documented during follow-up. The log-rank test compared response rates between the potential predictive factors. The response rate was 57.1% at 24 months follow-up. In log-rank test, mild disease severity was the most significant predictive marker of better response (P < 0.001); SAA patients with higher absolute reticulocyte count (ARC) and platelet level showed a higher response rate (both P < 0.001). Although insignificantly, elderly children and male sex show better response rate after treatment. The response rate worsened when the time interval before IST was more than 60 days. Modified IST with pALG was suitable for SAA children, and favorable response correlates with mild disease severity was identified. ARC and platelet status also appeared to be a reproducible prognostic model for response rate. IST should be started as soon as possible, given that the response rate worsens as the interval between diagnosis and treatment increases.

Modified immunosuppressive therapy with porcine antilymphocyte globulin plus delayed cyclosporine A in children with severe aplastic anemia.

Immunosuppressive therapy (IST) with antithymocyte globulin (ATG) and cyclosporine (CsA) is the standard treatment for children with severe aplastic anemia (SAA) with no human leukocyte antigen-matched siblings. Due to the unavailability of horse ATG in China, porcine antilymphocyte globulin (p-ALG), which is less expensive and more effective than rabbit ATG, is widely used. We sought to evaluate the efficacy and safety profile of modified IST with p-ALG plus delayed CsA at day 21 in 50 SAA children. Eighteen SAA patients who progressed from nonsevere aplastic anemia (NSAA) were classified as SAA-II; the other 32 patients were classified as SAA-I. Overall response (OR) rates at 3, 6 and 12months were 56, 64 and 62%, respectively. The 10-year overall survival (OS) rate and disease-free survival (DFS) rate were 80 and 56%. The OR, OS and DFS rates in the SAA-I group were clearly better than those in the SAA-II group. Death rate from infection within 30days was 4%. Modified IST with p-ALG plus delayed CsA is a reliable and well-tolerated treatment for children with SAA, and reduces early death due to infection. Modified IST is more suitable for children with SAA-I.

Improved Outcome of Unrelated Cord Blood Transplantation in Children with Severe Aplastic Anemia

IntroductionHematopoietic stem cell transplantation (HSCT) from an HLA-matched related donor is a first-line treatment for children with severe aplastic anemia (SAA). HSCT from an HLA-matched unrelated donor (MUD) is indicated as a salvage treatment for non-responders to immunosuppressive therapy (IST).Because unrelated cord blood transplantation (UCBT) has been considered as an experimental therapy, information of UCBT in patients with SAA is scarce, especially in pediatric patients. We analyzed the outcomes of UCBT in children with AA registered in the Transplant Registry Unified Management Program (TRUMP) conducted by the Japanese Society for Hematopoietic Cell Transplantation.Patients and MethodsBetween 1998 and 2013, 27 patients <15 years old who underwent an UCBT as the first HSCT were registered in the TRUMP database. Patients with congenital bone marrow failure syndrome such as Fanconi anemia were excluded from the analysis. Treatment failure were defined as death by all causes, graft failure (GF) and secondary malignancy. Data collected as October 2014 were analyzed.ResultsPatients' characteristicsAmong the 27 patients, 10 patients underwent UCBT during 1998 - 2005 and 17 patients underwent UCBT during 2006 - 2013. The median age at the time of UCBT was 6 years (range, 0 - 14). The male/female ratio was 12/15. The median interval between the diagnosis of AA and CBT was 212 days (range, 39 - 2,982). Sixteen patients received antithymocyte globulin (ATG) as immunosuppressive therapy (IST) before UCBT. The median total nucleated cell number and CD34 positive cell number at cryopreservation were 3.99 x 107/kg (range, 0.09 - 16.11 x 107/kg) and 1.31 x 105/kg (range, 0.39 - 6.17 x 105/kg), respectively. Ten patients received fludarabine (FLU) + cyclophosphamide (CY) + irradiation ± ATG. Six received FLU + CY + ATG. Four received FLU + melphalan (MEL) + irradiation and the remaining 7 received other various conditioning regimens. Cyclosporine was used in 9 patients and tacrolimus was used in 17 patients for graft-versus-host disease (GVHD) prophylaxis.Transplantation outcomesNeutrophil recovery was observed in 19 patients and the median time to engraftment was 20 days. Platelets engraftment was observed in 17 patients and the median time to engraftment was 50 days. One patient experienced secondary GF. Sustained engraftment was observed in 18 patients. Grade II - IV acute GVHD, limited chronic GVHD, and extensive chronic GVHD were observed in 6 (26%), 2 (10%), and 2 patients (10%), respectively.With median follow-up times of 18 months after CBT, 19 of 27 patients were alive. Causes of death included bacterial/fungus infections in 3, veno-occulusive disease (VOD) in 2, EBV-LPD, hematologic malignancy, and secondary GF in each 1 patient.The 5-year overall and failure-free survival (OS, FFS) of all patients were 69.5±9.0% and 59.3±9.5%, respectively. The 5-year OS and FFS of 17 patients who underwent UCBT after 2006 was 93.8±6.1% and 76.0±10.5%, respectively. Primary GF was observed in 8 patients, including 3 who underwent UCBT during 2006 - 2013. Two patients, who underwent UCBT after 2006, received FLU + CY + ATG without irradiation and remaining one patient received CY + ATG + total body irradiation (TBI). We screened anti-HLA antibodies in 8 patients but none of them were positive. The conditioning regimens including ATG exhibited a significantly worse FFS than those without ATG (33.3±12.2 % vs 91.7±8.0 %, P=0.004). On the other hand, the conditioning regimens including irradiation exhibited a better FFS than those without irradiation (75.0±9.7 % vs 14.3±13.2 % , P=0.01). The 5 year FFS of FLU + MEL + irradiation regimen (n=4) and FLU + CY + irradiation regimen (n=10) was 100% and 90%±9.5%, respectively.Univariate analysis resulted in no statistical significant differences in OS and FFS in HLA disparities, total nucleated cell number and CD34 positive cell number at cryopreservation, and methods of GVHD prophylaxis. One patient developed secondary malignancy.DiscussionOur study showed that a reduced conditioning regimen with Flu, MEL/CY and low-dose irradiation may be an optimal regimen for SAA children receiving UCBT. An important finding is that ATG has no benefit in this setting. A prospective study with larger number of patients is warranted to confirm our promising results. DisclosuresKojima:SANOFI: Honoraria, Research Funding.

Comparison between two different dose of r-ATG combined with CsA for treating children with severe aplastic anemia

This study was purposed to compare the efficacy and safety of two different doses of rabbit anti-thymocyte globulin (r-ATG) combined with cyclosporine (CsA) for treating children with severe aplastic anemia (SAA). From January 2005 to July 2010, a total of 95 children with SAA accepted intensive immunosuppressive therapy (IIST) in our department, out of them 55 cases were treated with r-ATG 2.5 mg/(kg·d) for 5 days in combination with CsA (group I) and other 40 cases were treated with r-ATG 3.5 mg/(kg·d) for 5 days in combination with CsA (group II). The responsive rate, adverse reactions, early mortality, relapse and clonal disease were analyzed retrospectively and results between the two groups were compared. Out of 95 patients 43 were boys and 52 were girls, their ages were from 1 to 16 years. The sex, age, severity and course of the disease were comparable between the two groups. The results showed that after treating for 3 and 6 months, the response of patients in group II was higher than that of patients in group I (50% vs 32.1%, P = 0.08 and 65% vs 45.3%, P = 0.059), at 9 and 12 months the response rate of patients in group II and group I did not show significant difference (70.0% vs 71.1%,P = 0.904 and 82.5% vs 80.8%,P = 0.832); at 12 months of treatment, the complete response rate of patients in group II was significantly higher than that of patients in group I (40.0% vs 23.1%,P = 0.08); at 3, 6, 9 months of treatment, the complete response rate of 2 groups showed no obvious difference. The incidence of serum disease, early infection and early mortality did not show statistical difference between two groups. There was no statistical difference in 2 year overall survival rate of two groups. In group I 39 patients were followed-up for more than 2 years, among them 3 patients relapsed, 1 patient died and 1 patient was diagnosed as acute monocytic leukemia (M5). In group II 15 patients were followed up for more than 2 years, there were no relapse, death and clonal disease. It is concluded that the r-ATG combined with CsA is an effective and safe therapeutic regimen for the SAA children. The effect of r-ATG 3.5 mg/(kg·d) is better than the 2.5 mg/(kg·d). The early safety is comparable between the two groups. However, the long-term effect, complications and survival rate need longer follow-up study to evaluate.

Downregulated CXCL12 expression in mesenchymal stem cells associated with severe aplastic anemia in children.

The mechanisms of idiopathic severe aplastic anemia (SAA) in children are not completely understood. Insufficiency of the bone marrow microenvironment, in which mesenchymal stem cells (MSCs) are an important element, can be a potential factor associated with hematopoietic impairment. In the current study, we studied whether aberrant gene expression could be found in MSCs from children with SAA. Using microarray analysis, two different patterns of global gene expression were detected in the SAA MSCs. Fourteen genes (POLE2, HGF, KIF20A, TK1, IL18R1, KITLG, FGF18, RRM2, TTK, CXCL12, DLG7, TOP2A, NUF2, and TYMS), which are related to DNA synthesis, cytokines, or growth factors, were significantly downregulated. Further, knockdown of gene expression was performed using the small hairpin RNA (shRNA)-containing lentivirus method. We found that knockdown of CXCL12, HGF, IL-18R1, FGF18, or RRM2 expression compelled MSCs from the controls to behave like those from the SAA children, with decreased survival and differentiation potential. Among them, inhibition of CXCL12 gene expression had the most profound effects on the behavior of MSCs. Further experiments regarding re-introduction of the CXCL12 gene could largely recover the survival and differentiation potential in MSCs with inhibition of CXCL12 expression. Our findings suggest that MSCs from children with SAA exhibit aberrant gene expression profiles and downregulation of CXCL12 gene may be associated with alterations in the bone marrow microenvironment.

Association of Telomere Length of Peripheral Blood Leukocyte with Hematological Response of Immunosuppressive Therapy in Children with Severe Aplastic Anemia

AbstractAbstract 4403 Introduction:Severe aplastic anemia (SAA) is characterized by life-threatening cytopenias. SAA can be cured by hematopoietic stem cell transplantation, but in children when a sibling donor is unavailable, immunosuppressive therapy with antithymocyte globulin (ATG) plus cyclosporine is effective. Variations in telomere length have been reported in severe aplastic anemia but their clinical significance is not clear,The aim of our study is relationship between telomere length and clinical response to immunosuppressive therapy children with severe aplastic anemia. Material and methods:We measured telomere length in children with SAA at our institution who had received ATG plus cyclosporine between 2009 to 2011and analyzed its relationship with hematologic recovery. Patients 18 (9 girls and 9 boys) aged 7–19 yrs with SAA without sibling donor, have all received rabbit ATG in a dose of 3.75 mg/kg/day for 5 days and CSA in a dose of 5mg/kg/day for 12 months). Remission was assessed on 84th, 112th, 180th and 360th day of treatment. Patients were divided into 3 groups: I group: non responders NR, II group: early remission -ER (complete-CR and partial remission PR on 84th and 112th day), III late remission LR (CR and PR on 180th and 360th day). We analyzed telomere length in 20 parents (11 mothers and 9 fathers aged 39–55yrs) our 11 patients. Control peripheral blood samples were obtained from 12 healthy children (3 girls and 9 boys, aged 3–17 yrs), and 10 healthy adult (aged 28–55yrs). Informed consent was obtained from all adult donors and from parents of participating children.Genomic DNA was extracted using AxyPrep Blood Genomic DNA Miniprep Kit (Axygene). Telomere length was measured using the quantitive PCR (qPCR) method described by Elisa Pavesi et al (1). Pavesi E. et al. Pediatr Blood Cancer. 2009;53(3):411-6. Two 96-well plates were prepared for measurements, first plate was used for amplification of telomere product and second one was used for single copy gene (SEP15) product amplification. Each reaction contained: Power SYBR1 Green PCR Master Mix (Applied Biosystems), DNA and primers (270nM Tel1 and 900nM Tel2 or 500nM each SEP15 primers). Reactions were preformed in Real Time PCR: CFX96 system (Biorad) with thermal cycling conditions: 95C–10 minutes; 95C–15 seconds, 54C 2 minutes (35 cycles).Telomere/single copy gene (T/S) ratios for samples were calculated from Human reference DNA (Applied Biosystems) standard curve and T/S ratios values were expressed in relative to telomere length in controls. Statistica software version 9.0 (StatSoft, Tulsa, OK) was used for statistical analysis. Mann–Whitney U test was employed to compare telomere length among samples groups. p value <0.05 was considered statistically significant. Results:The telomere length in children with SAA were shorter than health children but this not significantly to (p=0, 16)Twelve patients (66, 6%) responded to immunosuppressive therapy. There was correlation between telomere length and the late response. Children with late response were shorter telomere than children with control group (Fig 1). [Display omitted] We not noted in our patient relapse and clonally transformation, but time of observation was only 3 yearParents children with SAA telomere length were shorter than health control adult people, but without significantly (p=0, 15) (Fig 2). [Display omitted] We found correlation between shorter telomere length parents and late response to immunosupression therapy in children with SAA (p= 0,012) (Fig 3). [Display omitted] Conclusions:Our observations indicate that SAA patients with shortened telomeres respond to immunosuppressive therapy better then those with elongated telomeres.Telomere length parents of SAA children with response of IST were shorter than parent's in children with SAA without response.Telomere length may be considered therefore as a predicting factor in these patients.Studies on a larger group of patients should be performed to confirm our observations. Disclosures:No relevant conflicts of interest to declare.

Increased Telomere Length Is a Negative a Predicting Factor for IST in Children with Severe Aplastic Anemia?

Abstract 4381Shortening of telomeres is observed in 1/3 of peripheral blond samples, collected from patients with severe aplastic anemia (SAA). Probably, this phenomenon is associated with poor response to immunosuppressive therapy –IST (antithymocyte globulin -ATG and cyklosporin-CSA). The aim of this study was to assess the length of telomeres in peripheral blood of children with SAA treated with antithymocyte globulin (ATG) and cyklosporin (CSA) Materials and Methods:Peripheral blood samples were collected from 13 children with confirmed SAA. Patients (9 girls and 4 boys) aged 7–19 yrs have all received rabbit ATG in a dose of 3.75 mg/kg/day for 5 days and CSA in a dose of 5mg/kg/day for 12–14 months). Remission was assessed on 180th and 360th day of treatment. Control peripheral blood samples were obtained from 12 healthy children (3 girls and 9 boys, aged 3–17 yrs), 12 healthy adultand from 4 SAA children parents. Informed consent was obtained from all adult donors and from parents of participating children.DNA extraction and qPCR Genomic DNA was extracted from whole blood using AxyPrep Blood Genomic DNA Miniprep Kit (Axygene). Telomere length was determined using the quantitive PCR (qPCR) method described by Elisa Pavesi et al (1). Briefly, two qPCR reactions were run for each DNA sample: amplification of telomere product and amplification of a single copy gene (SEP15). Each qPCR reaction contained: Power SYBR1 Green PCR Master Mix (Applied Biosystems), template DNA and primers (270nM Tel1 and 900nM Tel2 or 500nM of each SEP15 primers). Telomere/single copy gene (T/S) ratios for samples were calculated using Human reference DNA (Applied Biosystems) standard curve. Reactions were preformed in Real Time PCR CFX96 system (Biorad). Statistica software version 9.1 (StatSoft) was used for statistical analysis Results:Among 13 SAA patients telomere shortening was found in 9 children while 4 patients had significantly elongated telomeres. All of the latter ones responded poorly to IST treatment: 3 of them did not respond (NR) to IST (neither on day 180 nor on 360) and one patient had a partial remission (PR). 2 patients in the NR group were randomized to unrelated bone marrow transplantation and one had another course of IST (no unrelated donor), after which he reached a PR. All patients with elongated telomeres had normal serum adriostendion level. Telomere elongation was also found in parents compared to the control group of adults (n=12, age range 31–57). Among 9 patients with shortened telomeres, 4 achieved complete remission (CR), 1 - PR and 4 did not respond (NR) to treatment on check times points. Conclusions:Our observations indicate that SAA patients with shortened telomeres respond to immunosuppressive therapy better then those with elongated telomeres. Telomere length may be considered therefore as a predicting factor in this patients. Studies on a larger group of patients should be performed to confirm our observations.Table 1Children telomere length (T/S) comparisonAplastic anemia groupControlp-value*T/S median (mean)0,025984 (0,078277)0,075671 (0,1270218)0,31T/S Min. value0,0006680,000505T/S Max. value0,4342040,585426Quartile controln(%)Control quartile value rangeI6(46)<0,0241II3(23)0,0241≤n<0,0756III2 (15,5)0,0756≤n<0,1516IV2(15,5)0,1516≥n*- U-test comparison between Aplastic anemia group and controls.Short telomere definition –samples, which T/S value is less than the first Quartile of controls. Long telomere - samples, which T/S value is in III quartile of controls. Very long telomere - samples, which T/S value is higher than the IV Quartile of controls. Disclosures:No relevant conflicts of interest to declare.

Allogeneic hematopoietic stem cell transplantation for childhood aplastic anemia: Prospective trial in China

We aim to investigate the efficacy and safety of the treatment with fully matched allogeneic hematopoietic stem cell transplants for children with severe aplastic anemia (SAA) in the first prospective trial in China. Six SAA children received allogeneic hematopoietic stem cell transplantation combined with chemotherapy. Five patients had successful engraftment while the sixth child regained normal peripheral blood counts consistent with spontaneous autologous hematopoiesis. Mean duration of follow-up was 2.75 years, and survival was 83%(5/6). The results indicated that allogeneic hematopoietic stem cell transplantation is a good option for the treatment of children with severe aplastic anemia.

Poor potential of proliferation and differentiation in bone marrow mesenchymal stem cells derived from children with severe aplastic anemia

The pathogenesis of severe aplastic anemia (SAA) has not been completely understood, and insufficiency of the hematopoietic microenvironment can be an important factor. Here, we compared the basic properties of mesenchymal stem cells (MSCs), a major component of bone marrow microenvironment, from five SAA children with those of MSCs from five controls. Although MSCs from SAA children and controls were similar in morphology and immunophenotypic profile, SAA MSCs had slower expansion rate and smaller cumulative population doubling (1.83 +/- 1.21 vs 3.36 +/- 0.87; p = 0.046), indicating lower proliferative capacity. After osteogenic induction, SAA MSCs showed lower alkaline phosphatase activity (optical density, 1.46 +/- 0.04 vs 2.27 +/- 0.32; p = 0.013), less intense von Kossa staining, and lower gene expression of core binding factor alpha1 (0.0015 +/- 0.0005 vs 0.0056 +/- 0.0017; p = 0.013). Following adipogenic induction, SAA MSCs showed less intense Oil red O staining (optical density, 0.86 +/- 0.22 vs 1.73 +/- 0.42; p = 0.013) and lower lipoprotein lipase expression (0.0105 +/- 0.0074 vs 0.0527 +/- 0.0254; p = 0.013). These findings provided evidence that defects in bone marrow MSCs of SAA children do exist.

Poor Potential of Proliferation and Differentiation in Bone Marrow Mesenchymal Stem Cells Derived From Children with Severe Aplastic Anemia.

Abstract 5081 Introduction Idiopathic severe aplastic anemia (SAA), characterized by failure of hematopoiesis, is rare and potentially life-threatening to children. However, the pathogenesis has not been completely understood, and insufficiency in the hematopoietic microenvironment can be an important factor. Mesenchymal stem cells (MSCs) play an important role in maintaining bone marrow microenvironment. Therefore, we aimed at the intrinsic defects of bone marrow MSCs derived from SAA children. Materials and Methods Bone marrow MSCs were obtained from 5 SAA children and 5 controls. The morphology, immunophenotyping, proliferative capacity and differentiation potential of MSCs from SAA children were determined and compared with those of MSCs from controls. Results In vitro, MSCs of SAA and control group shared a similar spindle-shaped morphology. Both revealed a consistent immunophenotypic profile which was negative for CD45, CD14 and CD34, and positive for CD105, CD73, and CD44. However, SAA MSCs had slower expansion rate and smaller cumulative population doubling from passage 4 to 6 (1.83± 1.21 vs 3.36± 0.87; p = 0.046), indicating lower proliferative capacity. Besides, only 3 of 5 cultures of SAA group retained the ability to continue expansion till 80%-90% confluent cell layer beyond passage 6, suggesting earlier senescence of SAA MSCs. After osteogenic induction, SAA MSCs showed lower alkaline phosphatase activity (1.46± 0.04 vs 2.27± 0.32; p = 0.013), less intense von Kossa staining and lower gene expression of core binding factora1 (0.0015± 0.0005 vs 0.0056± 0.0017; p = 0.013). Following adipogenic induction, SAA MSCs showed less intense Oil red O staining (0.86± 0.22 vs 1.73± 0.42; p = 0.013) and lower lipoproteinlipase expression (0.0105± 0.0074 vs 0.0527± 0.0254; p = 0.013).The results of real time-PCR analysis for the assessment of lineage-specific genes were consistent with the findings of histochemical stains, and both indicated that SAA MSCs had poor osteogenic and adipogenic potential. Conclusions In this study, we demonstrated that bone marrow MSCs from children with SAA had poor potential of proliferation and differentiation. These alterations in MSCs may contribute to the failure of hematopoiesis, and lead to the development of the disease. Further studies are needed to elucidate the relationship between MSCs and SAA. Disclosures No relevant conflicts of interest to declare.

Endogenous interleukin-11 (IL-11) levels in newly diagnosed children with acquired severe aplastic anemia (SAA)

Background: Impaired hematopoietic growth factor production is a hypothetical contributing factor to the development of acquired severe aplastic anemia (SAA). The serum levels of most hematopoietic cytokines in SAA patients are elevated. Objective: To measure interleukin-11 levels in newly diagnosed SAA children and attempt to correlate levels with disease severity and response to therapy. Design/methods: Following enrollment into a clinical study but prior to treatment, serum samples were obtained from 11 newly diagnosed children with acquired SAA. These samples were collected between May 2000 and September 2002. IL-11 levels were quantified utilizing an ELISA technique. Results: Ten of the 11 patients had low or normal levels of IL-11 (<85 pg/mL) and one had an elevated level of 409 pg/mL (normal range 15–200 pg/mL). Conclusions: The production of IL-11 does not increase in response to thrombocytopenia in most children with SAA. The significance of this laboratory observation is not clear at this time. Further studies are warranted to determine what, if any, role this plays in the development of this disorder and if the administration of recombinant human IL-11 might be beneficial in the treatment of acquired SAA.