Pediatric Aplastic Anemia Patients Research Articles

Potential role of B- and NK-cells in the pathogenesis of pediatric aplastic anemia through deep phenotyping.

Pediatric patients with unexplained bone marrow failure (BMF) are often categorized as aplastic anemia (AA). Based on the accepted hypothesis of an auto-immune mechanism underlying AA, immune suppressive therapy (IST) might be effective. However, due to the lack of diagnostic tools to identify immune AA and prognostic markers to predict IST response together with the unequaled curative potential of hematopoietic stem cell transplantation (HSCT), most pediatric severe AA patients are momentarily treated by HSCT if available. Although several studies indicate oligoclonal T-cells with cytotoxic activities towards the hematopoietic stem cells, increasing evidence points towards defective inhibitory mechanisms failing to inhibit auto-reactive T-cells. We aimed to investigate the role of NK- and B-cells in seven pediatric AA patients through a comprehensive analysis of paired bone marrow and peripheral blood samples with spectral flow cytometry in comparison to healthy age-matched bone marrow donors. We observed a reduced absolute number of NK-cells in peripheral blood of AA patients with a skewed distribution towards CD56bright NK-cells in a subgroup of patients. The enriched CD56bright NK-cells had a lower expression of CD45RA and TIGIT and a higher expression of CD16, compared to healthy donors. Functional analysis revealed no differences in degranulation. However, IFN-γ production and perforin expression of NK-cells were reduced in the CD56bright-enriched patient group. The diminished NK-cell function in this subgroup might underly the auto-immunity. Importantly, NK-function of AA patients with reduced CD56bright NK-cells was comparable to healthy donors. Also, B-cell counts were lower in AA patients. Subset analysis revealed a trend towards reduction of transitional B-cells in both absolute and relative numbers compared to healthy controls. As these cells were previously hypothesized as regulatory cells in AA, decreased numbers might be involved in defective inhibition of auto-reactive T-cells. Interestingly, even in patients with normal distribution of precursor B-cells, the transitional compartment was reduced, indicating partial differentiation failure from immature to transitional B-cells or a selective loss. Our findings provide a base for future studies to unravel the role of transitional B-cells and CD56bright NK-cells in larger cohorts of pediatric AA patients as diagnostic markers for immune AA and targets for therapeutic interventions.

Hypermethylation-mediated downregulation of long non-coding RNA MEG3 inhibits osteogenic differentiation of bone marrow mesenchymal stem cells and promotes pediatric aplastic anemia

BackgroundThe reduced osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is the typical characteristics of pediatric aplastic anemia (AA) pathogenesis. Long non-coding RNA MEG3 is reported to promote osteogenic differentiation of BMSCs via inducing BMP4 expression. ObjectiveThis study aims to investigate the mechanism of DNMT1/MEG3/BMP4 pathway in osteogenic differentiation of BMSCs in pediatric AA. MethodsBMSCs were isolated and purified from bone marrows of pediatric AA patients (n = 5) and non-AA patients (n = 5). The expression of DNMT1, MEG3, and BMP4 in isolated BMSCs was detected using quantitative real-time PCR and western blot analysis. Osteogenic differentiation was determined using Alizarin red staining. The methylation of MEG3 promoter and the interaction between DNMT1 and MEG3 promoter were detected using methylation-specific PCR and chromatin immunoprecipitation assay, respectively. ResultsLowly expressed MEG3 and BMP4 and highly expressed DNMT1 were observed in BMSCs of pediatric AA patients. The overexpression of MEG3 promoted osteogenic differentiation of BMSCs. Luciferase reporter assay showed that MEG3 overexpression increased transcriptional activity of BMP4. The inhibitor of methylation, 5-azacytidine, suppressed DNMT1 expression and reduced methylation of MEG3 promoter. Overexpression of DNMT1 increased the binding between DNMT1 and MEG3 promoter. The simultaneous overexpression of DNMT1 and MEG3 restored the inhibition of osteogenic differentiation caused by DNMT1 overexpression alone. ConclusionsOur findings indicated that DNMT1 mediated the hypermethylation of MEG3 promoter in BMSCs, and DNMT1/MEG3/BMP4 pathway modulated osteogenic differentiation of BMSCs in pediatric AA.

Implication of therapeutic outcomes associated with molecular characterization of paediatric aplastic anaemia

ObjectivesSevere aplastic anemia is characterized by a hypocellular bone marrow and peripheral cytopenia. Mesenchymal stem cells (MSCs) play a crucial role in haematopoietic stem cells (HSCs) development and the development of microenvironment suitable for hematopoiesis. Molecular characterization of telomere maintenance pathway and gene expression profiling of MSCs can be important for the therapeutic interventions among paediatric aplastic anaemia patients. MethodsThe study involved paediatric aplastic anaemia patients (n = 10) and age matched paediatric healthy donors (n = 8). Peripheral blood samples were collected from the individuals. Average leucocyte telomere length and gene expression of the telomere maintenance genes were determined by quantitative real time PCR. Microarray based gene expression profiles (GSE33812) of MSCs for five paediatric aplastic anaemia patients were analyzed compared to five healthy controls and the data was downloaded from the GEO database. ResultsThe telomere length was significantly shorter among paediatric AA patients compared to age matched healthy donors. Interestingly, one subgroup (n = 2) of paediatric AA patients has moderate telomere length comparable to age matched healthy donors. Based on the gene expression analysis of telomere maintenance pathway, TERF2 was significantly downregulated among paediatric patients with shorter telomere length but not among paediatric patients with moderate telomere length. Gene expression profiling of MSCs revealed three differentially expressed genes (GAS2L3, MK167 and TMSB15A) among the patients and was associated with therapeutic outcome. ConclusionTelomere length estimation and gene expression patterns of the MSCs and telomere length maintenance pathway may serve as a potential biomarker and could be associated with therapeutic choice of paediatric aplastic anaemia patients.

Clinicopathological profile of paroxysmal nocturnal haemoglobinuria clone‐positive aplastic anaemia paediatric patients—A single centre study from North India

There is a paucity of literature related to the prevalence of Paroxysmal Nocturnal haemoglobinuria (PNH) clones in paediatric aplastic anaemia (AA) patients. We performed a retrospective analysis over a period of 42months to study the prevalence of PNH clones in paediatric (age less than 18years) AA cases, using Fluorescein-labelled proaerolysin-based flow cytometric screening and analysed their clinico-pathological features. PNH clone was identified in 100 (33.2%) of the 301 patients screened. These were comprised of 51 cases of non-severe AA, 33 cases of severe AA and 16 cases of very severe AA. The median age was 13years with an M:F ratio of 2.5:1. The median clone size (taken as the proportion of PNH-positive neutrophils) was 2.15% (range: 0.05%-93.1%). Although a majority of patients (n=77) had a clone size of less than 10%, a significant proportion (n=23) did harbour a clone size of more than 10%. Evidence of haemolysis was observed in 3 patients, all of them having a clone size of more than 10%. Interestingly, 1 patient with dural sinus thrombosis harboured a clone size of 1.25% only. Chromosomal breakage analysis was performed in 61 patients, none of which was positive. Complete and partial response to immunosuppressive therapy was found in 55.1% patients (16/29). There is a high prevalence of PNH clones in paediatric AA patients, which in a majority of cases are of small clone sizes. The use of immunosuppressive therapy does not show a better outcome as compared to PNH-negative cases.

Development of Paroxysmal Nocturnal Hemoglobinuria in Children with Aplastic Anemia

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a nonmalignant clonal disease of hematopoietic stem cells resulting from a somatic mutation in the PIGA gene. PNH frequently manifests in association with aplastic anemia (AA), in which PIGA mutations are believed to enable escape from the immune-mediated destruction by pathogenic T cells. Recent studies using next-generation sequencing have revealed that frequent somatic PIGA mutationsin AA patients are associated with a better response to IST and prognosis (Yoshizato et al N Engl J Med. 2015; 373: 35-47). However, clinical PNH is a progressive and life-threatening disease driven by chronic hemolysis that leads to thrombosis, renal impairment, poor quality of life, and death. Large studies in adults have reported that clinical PNH developed in 10%-25% of AA patients; however; the frequency of clinical PNH in children with AA has rarely been described. Here we aimed to elucidate the pathological link between PNH and AA in children.Methods: In total, 57 children (35 boys and 22 girls) diagnosed with acquired AA at our hospital between 1992 and 2010 were retrospectively studied. Patients who underwent hematopoietic stem cell transplantation as first-line treatment within 1 year after AA diagnosis and those with clinical PNH at AA diagnosis were excluded. Flow cytometry (FCM) was used to detect PNH CD13+/CD55−/CD59− granulocytes and PNH glycophorin A+/CD55−/CD59− red blood cells (RBCs). Clinical PNH was defined as the presence of intravascular hemolysis and ≥5% PNH granulocytes or PNH RBCs. Minor PNH clones were defined as those with >0.005% PNH granulocytes or >0.010% PNH RBCs. We performed targeted sequencing of bone marrow samples from patients with clinical PNH that were obtained at 2 time points: at AA diagnosis and after PNH development. The panel of 184 genes for targeted sequencing included most of the genes known to be mutated in inherited bone marrow failure syndromes and myeloid cancers, as well as PIGA.Results: The median patient age at AA diagnosis was 9.3 (1.2-17.8) years, and the median follow-up period was 123 (2-228) months. A total of 43 patients were screened for PNH clones by FCM after AA diagnosis, and 21 of these with minor PNH clones were identified. The median percentages of PNH granulocytes and PNH RBCs were 0.001% (0.000%-4.785%) and 0.000% (0.000%-3.829%), respectively. During follow-up, 5 patients developed clinical PNH after adolescence (15-22 years of age). The median time between AA diagnosis and PNH development was 4.9 (3.3-7.9) years. All clinical PNH patients were treated with IST for AA, and complete and partial response after 6 months were achieved in 1 and 4 patients, respectively. Gross hemoglobinuria was present in all clinical PNH patients, but thrombosis was not observed. The size of PNH clones varied greatly among patients: PNH granulocytes and PNH RBCs were 42.96% (10.04%-59.50%) and 48.87% (15.02%-90.80%), respectively. Oral cyclosporine A and intravenous eculizumab were administered to 3 and 1 patients, respectively; all patients showed sustained response as indicated by improvement in gross hemoglobinuria and normal blood counts after treatment. The remaining 1 patient underwent bone marrow transplantation from the HLA-identical mother and was alive without any complications. Overall, the 10-year probability of developing clinical PNH was 10.2% (95%CI, 3.6-20.7). Among 43 patients screened for PNH clones at AA diagnosis, the 10-year cumulative clinical PNH incidence was significantly higher in patients with minor PNH clones than in those without minor PNH clones at AA diagnosis [29% (95% CI, 10%-51%) vs. 0% (95% CI, 0%-0%); p = 0.015]. Among all clinical PNH patients, a total of 8 somatic PIGA mutations were detected (missense, 2; splice site, 2; and frameshift, 4). However, PIGA mutations were not detected at AA diagnosis even in patients who subsequently developed clinical PNH.Conclusion: In our cohort, the percentage of patients who eventually developed clinical PNH was comparable to that reported in adults in a previous study. Furthermore, the current study showed that the presence of minor PNH clones at AA diagnosis was a risk factor for the subsequent development of clinical PNH, although the clones were not detected by targeted sequencing. Thus, pediatric AA patients with PNH clones at AA diagnosis should undergo long-term periodic monitoring for potential clinical PNH development. DisclosuresKojima:SANOFI: Honoraria, Research Funding.

Hematopoeitic Stem Cell Transplant in Aplastic Anemia: Is It Time to Revise the Treatment Alogirthm

Background: Aplastic anemia (AA) is a rare bone marrow failure (BMF) syndrome of unknown etiology characterized by hypocellular bone marrow and severe persistent pancytopenia. Pediatric AA is more common in adolescent age group with better outcomes as compared to adults. Current treatment modalities include immunosuppressive therapy (IST) or matched related bone marrow transplant, when possible. There is a high risk of disease relapse with IST and clonal evolution into myelodysplastic syndrome. There is limited pediatric data on outcomes with different treatment options.Methods: Retrospective database analysis of Pediatric Health Information Systems (PHIS) database was performed to evaluate healthcare outcomes of pediatric patients (age less than 21 years) with AA from January 1, 2006 to December 31, 2015. PHIS is an administrative quality-controlled database from 43 not-for-profit children's hospitals. ICD-9 code 284.9 was used to identify patients with acquired AA. Appropriate procedure and pharmacy billing codes were used to obtain information regarding transplants and various drugs used. SAS based statistical software was used to perform analysis.Results: A total of 5127 inpatient admissions were noted for AA during the study period. An overall increasing trend of transplant was observed from 2006-2015 (Figure 1). Almost equal inpatient admission rate was noted in males and females, with a slightly higher rate amongst males who underwent transplant (55% vs 45%). Inpatient admission was more common amongst whites in both transplant (n=407, 8%) and non-transplant groups (n=4717, 92%; Table 1). Overall inpatient mortality was about 2% with a similar mortality in those who underwent transplant (5%). Cyclosporine (n= 1785, 38%) and steroids were most common immunosuppressive drugs used in non-transplant group. In the transplant group (Table 2), graft failure (20%) was the most common complication, while Cyclophosphamide (77%), Fludarabine (53%) and anti-thymocyte globulin (31%) based conditioning was most commonly used. Transplant itself was an independent risk factor for mortality (p=0.04) along with graft failure (p=0.02). Steroids (92%) and Cyclosporine (73%) were the most common immunosuppression used.Conclusion: Hematopoietic stem cell transplants have good outcomes in pediatric AA patients. With an increasing trend of HSCT in these patients, successful outcomes with other transplant options such as match unrelated donor transplants should be considered. Treatment algorithm for Pediatric AA needs to be revised to include other transplant sources. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Frequency of Paroxysmal Nocturnal Hemoglobinuria Clones by Multiparametric Flow Cytometry in Pediatric Aplastic Anemia Patients of Indian Ethnic Origin.

The literature on paroxysmal nocturnal hemoglobinuria (PNH) in aplastic anemia (AA) is largely focused on adults with few studies in children. Moreover, large studies are conspicuously absent from developing countries. Knowledge of the prevalence and utility of their detection is required before widespread use of PNH screening in pediatric AA in resource-limited settings. We performed a retrospective audit over a period of 9 years to study the prevalence of PNH clones by flow cytometry (FCM) in children ≤12 years of age presenting with AA, and analyzed their response to immunosuppressant therapy. Nine (12.9%) out of 70 patients had PNH clones comprising >1% of the target cell population, including five patients (7.14%) with PNH clone size >10%. The clone size in monocytes ranged from 3.7% to 95.2% (median 21.1%) and in neutrophils from 1.6% to 87.6% (median 19.5%). Fluorescent aerolysin (FLAER)-based FCM screening significantly improved the detection of PNH clones compared to non-FLAER based screening techniques (18.4% vs. 6.25%). One child showed chronic intravascular hemolysis and another developed arterial stroke during the course of illness. None of our PNH-positive AA patients tested for chromosome breakage studies (n = 8) showed increased clastrogen-induced breakage. A lower frequency but moderate/large-sized PNH clones were seen in our pediatric AA population, compared to western data. FLAER-based FCM screening significantly improved the detection of PNH clones. We recommend routine FLAER-based screening of PNH in pediatric AA patients.

Pre-existing anti-HLA antibodies negatively impact survival of pediatric aplastic anemia patients undergoing HSCT.

Graft failure and survival are the major problems for patients with aplastic anemia undergoing hematopoietic stem cell transplantation (HSCT). Previous studies showed that anti-HLA antibodies negatively impact engraftment in HSCT. This retrospective study of 51 pediatric patients with acquired aplastic anemia who underwent allogeneic HSCT at a single institution between 2006 and 2012 investigated the influence of anti-HLA antibodies on the outcome of HSCT. Serum samples collected before HSCT were tested for the presence of anti-HLA antibodies. Pre-existing anti-HLA antibodies were detected in 54.9% (28/51) of patients, among whom 39.2% (20/51) had anti-HLA class I antibodies. Anti-HLA antibodies were associated with worse five-yr survival (78.6% vs. 100%, p=0.021) and higher treatment-related mortality (21.4% vs. 0%, p=0.028) compared with antibody-negative patients. Anti-HLA class I antibody-positive patients had poorer five-yr survival (75.0%) than anti-HLA class I&II antibody-positive and antibody-negative patients (87.5% and 100.0%, respectively, p=0.039). Presence of anti-HLA class I antibodies (p=0.024) and older age (10yr or more; p=0.027) significantly increased the risk of post-HSCT mortality. Pre-existing anti-HLA antibodies negatively affect the outcome of HSCT in pediatric patients with aplastic anemia. Routine testing for anti-HLA antibodies concurrent with efficient treatment should be conducted prior to HSCT.

Paroxysmal Nocturnal Hemoglobinuria Clones in Children with Acquired Aplastic Anemia: A Multicentric Study

AbstractAbstract 1269 Introduction.Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic disorder characterized by the clonal expansion of a PIG-A mutated stem cell and consequent defective synthesis of glycosil phosphatidyl-inositol-anchored proteins, complement-mediated hemolysis, increased incidence of thrombosis, bone marrow failure. PNH and acquired aplastic anemia (AA) are closely related and a reciprocal progression is possible. A relative resistance of the PNH stem cell to the immune-mediated damage can explain the PNH clonal expansion in AA. High resolution flow cytometry analysis (FCA) has revealed a high incidence of minor PNH clones in adult AA patients at diagnosis, predictive for some Authors of a favourable response to the immunosuppressive therapy (IST) (Maciejevki et al, 2001; Ishiyama et al, 2003; Sugimori et al, 2006). “Pure” PNH is a very rare disease in children. Only a few studies have so far evaluated longitudinally PNH clones in pediatric AA patients. Materials and Methods.Ninety AA patients diagnosed in 8 AIEOP (Italian Association of Pediatric Hematology-Oncology) Centers (age at diagnosis 1–20 years, median =10.8, 51 severe AA, 30 very severe AA, 9 non severe AA) were studied: forty-one since diagnosis, 25 during IST, 20 off therapy and 4 selected cases after hematopoietic stem cell transplantation (HSCT). Among the patients followed since diagnosis, 8 received an HLA matched sibling donor HSCT as first line therapy, whereas the other 33 patients were treated with IST according to EBMT protocols (anti-lymphocyte globulin/anti-thymocyte, ciclosporin ± granulocyte colony stimulating factor). The study started in 1998. Peripheral blood PNH cells were detected by lack of CD59 expression on granulocytes by a two-color FCA for CD59 (clone p282-FITC Becton-Dickinson) and CD11b (clone D12-PE Becton-Dickinson); at least 105 cells were analyzed, for a total of 1104 tests. The presence of a population CD11b+/CD59- > 0.15% was defined as abnormal; the cut off value was established in 1998 by evaluating 87 normal controls (PNH clones: median = 0.001%, mean+2SD=0.10%). Since 2009 FCA results were confirmed by more sensitive techniques with three or six-color sequential gating analysis for CD45/33/66b or CD45/33/15/24/14/FLAER. Results.A PNH+ clone was observed in 15 patients (36.6%) at diagnosis (clone size 0.17–10.4%), in 10 patients (40%) during IST (clone size 0.16–12.6%) and in 8 patients (40%) off-therapy (clone size 0.16–4.0%). The presence of a PNH+ clone at diagnosis did not predict a favourable response to IST, both in ALG and ATG-treated patients. In 33 patients (16 at diagnosis, 9 in IST, 8 off therapy), the presence of the PNH clone was sporadic or intermittent, whereas in 13 patients (9 at diagnosis, 3 in IST, 1 off therapy) the clone persisted for more than 3 following controls (follow up 6–60 months). Among the 26 PNH- patients at diagnosis, in 10 a PNH clone (clone size 0.16–1.7%) appeared later during IST. Among the 25 patients studied during IST, in one patient PNH clone appearance was associated with the tapering of cyclosporine (figure 1), in two with the relapse when off therapy. In one out of 4 patients treated with HSCT, a PNH clone appeared at time of relapse and disappeared after starting IST with cyclosporine (figure 2). A mild hemolysis was observed in the only 2 patients with a major PNH clone (clone size 12.6 and 10.4% respectively). No thrombotic events were reported. Conclusions.We have observed a significant incidence of minor PNH clones in pediatric AA at diagnosis, as reported in adults. Whereas previous studies in adults correlated the presence of pre-treatment minor PNH clones with a favourable response to IST, we do not confirm those observations both in the present multi-centre as in our previous single-centre study (Timeus et al, 2010), in agreement with Yoshida et al (2008) and Scheinberg et al (2010). The appearance of a PNH clone in a PNH- patient at diagnosis is described as uncommon (Sugimori et al, 2009), however in our series this was observed in 38% of previously PNH- patients. In AA the presence of PNH clones seems related to complex interactions between stem cells, immune-mediated damage and immunosuppressive therapy. A periodic screening for PNH clones in patients with AA is recommended, permitting modulation in IST, early identification of major PNH clones and prompt diagnosis of a frank PNH. [Display omitted] [Display omitted] Disclosures:Timeus:Alexion Pharma Italy s.r.l.: Research Funding. Dufour:Pfizer: Consultancy.

Immunosuppressive activity of mesenchymal stem cells is not decreased in children with aplastic anemia

Aplastic anemia (AA) is characterized by a reduced number of hematopoietic stem cells (HSCs), resulting in peripheral pancytopenia. Several pathogeneses for AA have been hypothesized, and among these, the autoimmune-associated hypothesis is considered to be the most likely [1, 2]. Mesenchymal stem cells (MSCs) have been shown to exert immunosuppressive capacity both in vitro and in vivo. Normal bone marrow (BM)-derived MSCs suppress T cell function and may provide an immunoprotected environment for HSCs [3, 4]. Bacigalupo et al. [5] reported that MSCs derived from the BM of adult AA patients are defective in their ability to suppress T cell proliferation and cytokine release. They hypothesized that these defects may result in increased activation/proliferation of BM T cells, which suppress HSCs in AA patients. The aim of the present study was to investigate whether MSCs from pediatric AA patients also exert decreased suppressive activity on T cells when compared with those derived from normal individuals. Bone marrow samples from 34 AA children (16 males, 18 females) and 15 healthy volunteers were obtained following informed consent from either patients or parents. Median patient age was 10 years (range 2–21 years). The timing of sampling was at diagnosis in 15 patients and after immunosuppressive therapy (IST) with anti-thymocyte globulin and cycrosporin A in 19 patients. Eleven of these were IST responders and eight were non-responders. Aplasia was secondary to acute hepatitis in four patients and the cause was unknown in 30 patients. Seven patients had very severe AA, 19 patients had severe AA and eight patients had moderate AA. Bone marrow mononuclear cells (MNCs) were isolated and cultured as reported previously [5]. Briefly, MNCs were plated in a 25-cm tissue culture flask with complete MesenCult Basal Medium (Stem cell technologies, Vancouver, Canada) containing human MSC supplements. Adherent cells were expanded to the third generation. Characterization of MSCs was determined by flow cytometry and their differentiation into adipogenic and osteoblastic lineages. Surface markers of the MSCs were CD29?, CD44?, CD90?, CD105?, CD73?, CD34-, CD45and CD14-. To demonstrate capacity of MSCs to differentiate to adipocytes, cells were further cultured at 2 9 10 cells/flask with MesenCult basal medium supplemented with human adipocytogenic supplements (Stem cell technologies). Adipogenic differentiation was confirmed by the presence of lipid droplets under optical microscopy after 3 weeks. Similarly, for differentiation to osteoblasts, MSCs were cultured with MesenCult basal medium supplemented with an osteogenic differentiation kit (Stem cell technologies) containing dexamethasone (0.1 lmol/l), ascorbic acid (0.05 mmol/l) and glycerophosphate (10 mmol/l). After 5 weeks of culture, cell layers were stained with silver nitrate (Von Kossa method) in order to detect calcium deposition, which indicates osteogenesis. There were no differences in the expression of MSC surface markers and the differentiation capacity between AA patients and normal individuals. Immunosuppressive activity of MSCs from pediatric patients with AA was tested by their inhibitory effects on PHA-induced T cell proliferation. Peripheral blood MNCs from normal adult volunteers (2 9 10) were dispensed Y. Xu Y. Takahashi A. Yoshimi M. Tanaka H. Yagasaki S. Kojima (&) Department of Pediatrics, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan e-mail: kojimas@med.nagoya-u.ac.jp

Predicting Response to Immunosuppressive Therapy in Childhood Aplastic Anemia.

Aplastic anemia (AA) is defined as a pancytopenia caused by bone marrow failure; its pathogenesis is thought to involve autoimmune processes. Immunosuppressive therapy (IST) with antithymocyte globulin (ATG) and cyclosporine (CyA) provides response rates of 50–70% for children with AA, but predictive markers of response to therapy have not been well defined. We previously investigated the clinical relevance of HLA, a minor population of paroxysmal nocturnal hemoglobinuria-type cells, and a specific auto-antibody associated with AA in pediatric patients and reported that there was no correlation between these markers and response to therapy (Yoshida N, et al. Br J Haematol, 2008). In the current study, we prospectively evaluated whether clinical and laboratory findings before treatment, including age, sex, interval between diagnosis and treatment, etiology, severity of disease, white blood cell (WBC) count, neutrophil count, hemoglobin level, reticulocyte count, and platelet count, could predict the IST response at 6 months. Subjects included a large population of pediatric AA patients enrolled in a multicenter AA-97 study conducted by the Japan Childhood Aplastic Anemia Study Group. Between October 1997 and September 2006, 312 children (186 boys and 126 girls) younger than 18 years who were newly diagnosed with AA were enrolled in the study and treated with a combination of ATG and CyA. The median age at diagnosis was 8 years (range, 1–17 years). Of the 312 patients, 261 had idiopathic AA, 44 had hepatitis-associated AA, and 7 had AA from other causes. In terms of severity, 156 patients had very severe disease, 107 had severe disease, and 49 had moderate disease. The median interval between diagnosis and treatment was 15 days (range, 1–180 days). The overall response rate was 56%. In multivariate analyses, lower WBC count, shorter interval between diagnosis and therapy, and male sex were predictive markers of better response. Patients with WBC <2500/μl had a significantly higher response rate than those with WBC ≥2500/μl (61 vs. 48%; P=0.008); this was the strongest predictor of response. Notably, response rate was inversely related to the interval between diagnosis and treatment; response rates of patients with an interval between diagnosis and treatment of <30 and ≥30 days were 60% and 43%, respectively (P=0.02). Boys had a better response compared with girls (62 vs. 48%; P=0.02). In conclusion, pretreatment clinical and laboratory findings influence the response rate to IST. Response is well correlated with WBC count rather than neutrophil count or severity of disease. IST should be started as soon as possible after diagnosis of AA, given that the response rate worsens as the interval between diagnosis and treatment increases.

10-Year-Outcome of Acquired Aplastic Anemia Children Treated with Antithymocyte Globulin, Cyclosporine with or without G-CSF.

BACGROUND: Acquired aplastic anemia (AA) is thought to be an immune-mediated disease. Immunosuppressive therapy (IST) has been the treatment of choice for patients who did not have suitable donors. Previously, we had published promising results of IST for children with acquired AA. In the study, overall survival rate (OS) at 4 years was 83% in patients with vSAA and 92% in those with SAA/nonSAA.OBJECTIVES: Here we report follow-up results focusing on pediatric patients with relapse and clonal diseases, MDS/AML and PNH.PATIENTS and TREATMENT: From 1992 to 1997, 119 newly diagnosed children with acquired AA (median age; 9) entered AA-92 study. 50 vSAA patients were treated with ATG+CyA+mPSL+danazole +G-CSF, 36 SAA and 28 nonSAA patients were treated with ATG+CyA+mPSL+danazole +/−G-CSF. Complete remission (CR) was defined as a neutrophil count >1.5x10^9/L, a platelet count >100xx10^9/L, and a hemoblobin level of >11 g/dl. Partial response (PR) was defined as a neutrophil count >0.5x10^9/L, a platelet count >20x10^9/L, and a hemoglobin level of >8.0 g/dl. Relapse was indicated by the return of the PB counts to levels meeting the definition of SAA and/or the requirement for blood transfusion. Response rate was 71% at 6 months in vSAA patients, 65% in SAA/nonSAA patients, respectively. There was no statistical difference in 6-month response rate between G-CSF +/− treatments. No patient responded after 6 months. Therefore, 75 responders and 29 non-responders at 6 months were analyzed their OS, relapse rate (RR), and treatment-failure-free survival (TFFS). The median observation time of surviving patients is 118 months, ranging from 75 to 168 months.RESULTS: Among 119 patients, 38 patients received BMT and 17 patients died during an observation period. The OS was 81.9+−3.8% at 10 years, but has not reached plateau. Of the 75 6mo-responders, 23 patients relapsed and the RR was 33.6+−6.0% at 10 years. TFFS of the 75 was 67.4+−5.5%. Fourteen patients received 2nd ATG therapy and 5 of them responded. Eleven of the 23 patients with relapse received alternative donor BMT and 8 are alive. 10y-OS of these relapsed patients was 73.4+−9.4%. Nineteen of the 29 6mo-non-responders received alternative donor BMT and 15 are alive. There is no statistically significant difference in 10y-OS between the responders and the non-responders (89.1+−3.7% vs. 75.0+−8.2%, p=0.09). New clonal abnormalities appeared in 9 of 119 patients (10.0+−3.2%KM probability): monosomy 7(3 patients), trisomy 8(3 patients), trisomy 9, trisomy11, del (13)(1 patient each). There is no statistically difference in 10y-OS, RR, and incidence of clonal abnormality between randomized G-CSF+/− treatments. Clinical PNH with symptomatic hemolysis developed in 3 of the responder patients, 96, 105 and 138 months after the AA diagnosis, respectively. Among 65 surviving responders, 33(51%) have CR and 26(40%) PR at last follow-up time.CONCLUSIONS: Our data demonstrate that IST is effective for children with acquired AA, but relapse are common. Effective 2nd line treatment should be developed. PNH developed even in pediatric AA patients during long-term observation.