Aplastic Anemia In Children Research Articles

Genetic Background of Idiopathic Bone Marrow Failure Syndromes in Children

IntroductionPancytopenia with a severe decrease in bone marrow (BM) cellularity in children may be caused by a broad variety of underlying disorders. The appropriate classification of bone marrow failure syndromes in children is challenging, particularly with respect to the histological distinction between aplastic anemia (AA), refractory cytopenia of childhood (RCC), and refractory cytopenia with multilineage dysplasia (RCMD). Inherited bone marrow failure syndromes (IBMFS) further defy accurate diagnosis. Clonal hematopoiesis in AA is indicated by the presence of paroxysmal nocturnal hemoglobinuria (PNH) cells and by the identification of uniparental disomies involving chromosome 6p. In addition, "clonal transformation," as defined by the development of myelodysplastic syndromes (MDS) or acute myelogenous leukemia has been noted in about 15% of AA patients. In adult patients with AA, somatic mutations were frequently detected in myeloid malignancy-related genes such as DNMT3A, BCOR, and ASXL1. We aimed to characterize the genetic background of childhood AA/RCC/RCMD.Patients and MethodsWe studied 168 patients with idiopathic AA/RCC/RCMD in children. Diagnosis with AA, RCC, and RCMD was made on the basis of the 2008 World Health Organization classification criteria. Blood, bone marrow, and buccal samples were obtained from the patients after written informed consent was received according to protocols approved by the ethics committee of Nagoya University Graduate School of Medicine. Target sequencing (n = 168) was performed for 88 IBMFS-associated genes and 96 myeloid malignancy-related genes. Furthermore, whole-exome sequencing (WES, n = 25) was performed with matched tumor/normal samples. The mean depth for targeted sequencing was 451x, and the mean depth for WES was 103x. Somatic mutations were detected with the use of a frequency threshold of 0.07 (WES) or 0.02 (targeted sequencing) for variant allele frequency and were individually validated with the use of deep sequencing of polymerase-chain-reaction-amplified targets.ResultsOnly one germline mutation that was diagnostic of IBMFS was detected in our cohort (0.6%). It was a RTEL1 mutation, which supported the diagnosis of dyskeratosis congenita. Telomere length of the patient with a RTEL1 mutation was shorter compared with that of age-matched healthy individuals (−3.2 Standard Deviation). WES, performed in 25 patients, detected only three somatic mutations, all of which affected BCOR. In target sequencing, 20 somatic mutations were detected in 19 patients (11.3%). BCOR (n = 9) and PIGA (n = 4) were recurrently mutated. The mutational frequency of DNMT3A and ASXL1 was very low (0.6%) in our cohort and was clearly different from that of an adult cohort. The majority of somatic mutations carried low variant allele frequency. In case of BCOR mutations, the variant allele frequency tended to be low, suggesting subclonal composition. In case of U2AF1 mutations, the variant allele frequency tended to be high, which suggests that the U2AF1 -mutated clone was dominant in the bone marrow. The difference in the frequency of somatic mutations in AA, RCC, and RCMD was not statistically significant (p = 0.49). However, with regard to the mutated genes, two patients with RCMD carried U2AF1 plus SETBP1 and TP53 mutations, respectively, which are well-known predictors of poor prognosis in adult MDS. The patient who carried U2AF1 plus SETBP1 developed MDS later and underwent bone marrow transplantation. Of the 19 patients with mutated genes, 15 patients were treated with immunosuppressive therapy (IST). The response rate to IST at 6 months was 60% in the patients with somatic mutations, which was equivalent to those without mutations.ConclusionsIn our cohort of children who were clinically diagnosed with AA/RCC/RCMD, the frequency of cryptic IBMFS was considered low. Furthermore, the frequency of detectable somatic mutations in childhood AA was low compared with that in adult AA. No novel mutational target was identified with WES. Idiopathic bone marrow failure syndromes in children were characterized by a paucity of gene mutations irrespective of the histopathological classification. Mutations in adult MDS-related genes suggest molecular pathogenesis is different between RCMD and AA/RCC. In conclusion, our study clarified the yet unrevealed genetic background of idiopathic bone marrow failure syndromes in children. DisclosuresNo relevant conflicts of interest to declare.

Comparison of immune manifestations between refractory cytopenia of childhood and aplastic anemia in children: A single-center retrospective study

This retrospective single-center study assessed the incidence and clinical features of immune manifestations of refractory cytopenia of childhood (RCC) and childhood aplastic anemia (AA). We evaluated 72 children with RCC and 123 with AA between February 2008 and March 2013. RCC was associated with autoimmune disease in 4 children, including 1 case each with autoimmune hemolytic anemia, rheumatoid arthritis, systemic lupus erythematosus, and anaphylactoid purpura. No children with AA were diagnosed with autoimmune diseases. Immune abnormalities were common in both RCC and AA; the most significant reductions were in the relative numbers of CD3−CD56+ subsets found in RCC. Despite the many similar immunologic abnormalities in AA and RCC, the rate of autoimmune disease was significantly lower in childhood AA than RCC (p=0.008, χ2=6.976). The relative numbers of natural killer cells were significantly lower in RCC patients than AA patients. By month 6, there was no significant difference in autoimmune manifestations between RCC and AA in relation to the response to immunosuppressive therapy (p=0.907, χ2=0.014). The large overlap of analogous immunologic abnormalities indicates that RCC and childhood AA may share the same pathogenesis.

Diagnosis and management of childhood aplastic anaemia

Abstract Aplastic anaemia is a rare disorder in children. ∼20% of cases are inherited, whilst the rest are considered as acquired or idiopathic where no cause can be found. Diagnosis of the inherited causes of aplastic anaemia can be challenging but it is important for appropriate management and counselling. For idiopathic severe aplastic anaemia, a matched sibling donor haematopoietic stem cell transplant is the treatment of choice. If a matched sibling donor is not available, the options include immunosuppressive therapy with horse ATG and ciclosporin or unrelated donor haematopoietic stem cell transplant. Immunosuppressive therapy with horse anti-thymocyte globulin (ATG) is superior to rabbit ATG. Outcomes following matched unrelated donor haematopoietic stem cell transplantation are now similar to that of matched sibling donor haematopoietic stem cell transplant. The decision to proceed with immunosuppressive with ATG or unrelated donor stem cell transplant will depend on the likelihood of finding a matched unrelated donor and the differing risks and benefits that each therapy provides.

Model-Based Determination of Effective Blood Concentrations of Cyclosporine for Neutrophil Response in the Treatment of Severe Aplastic Anemia in Children.

Optimal immunosuppressive therapy in acquired severe aplastic anemia (SAA) remains to be refined, especially cyclosporine (CsA) use. Current recommendations state that CsA trough blood concentrations (TBC) should be maintained between 200 and 400 ng/mL despite the lack of supporting data. This study aimed at quantifying relationships between CsA exposure and neutrophil response and determining an effective range for CsA TBC. Twenty-three SAA patients treated with CsA were retrospectively analyzed. Nonlinear mixed effect modeling approaches were used to develop a pharmacokinetic-pharmacodynamic model. The pharmacokinetic model described the relationships between CsA doses and TBC. The pharmacodynamic model allowed to estimate boundaries for optimal CsA effects, neutrophils being used as biomarker of response. A time-to-event model linked effective concentration to time-to-therapeutic success. CsA TBC were adequately described by a two-compartment model with first-order absorption, a lag time, and a linear elimination. The efficient range of CsA TBC was estimated between 87 and 120 ng/mL. Model-based simulations and external validation in three additional patients confirmed these results. This original modeling approach was successful in describing the relationship between CsA TBC and neutrophil response in SAA patients. Although further evaluation of the model is necessary, this work suggests that an optimal CsA TBC target of 100 ng/mL would be associated with a better neutrophil response in children with SAA.

Fanconi’s Anemia—Rare Aplastic Anemia at Ten Year-Old Boy in Mogadishu-Somalia: Case Report

Fanconi’s anemia (FA) alson called Fanconi Pancytopenia is a rare, potentially life-threatening failure of haemopoiesis characterized by aplastic anemia that is associated with a variety of congenital abnormalities (Cafe-au-lait spots, abnormalities of fingers, hyperpigmentation of the skin, short stature, microcephaly, deformities of the ear, hypogenitalism, renal anomalies, etc.) and a high risk of developing of malignancy and chromosomal instability. FA is the first described in 1927 by Guido Funconi reported 3 brothers with pancytopenia and physical anomalies. The diagnosis is based on morphological abnormalities, hematologic abnormalities and genetic tests. The present case report describes a 10 years old Somali boy was diagnosed with a Fanconi anemia after recurrent blood transfusion. Though aplastic anaemia in children is an important haematological disorder, there is no study having been undertaken in Somalia and this is the first reported by the patient with Fanconi’s anemia in Somalia. We report this case to create awareness among clinicians the presence of this disease and have a consideration when it comes differentiatal diagnosis of recurrent blood transfusion patients with pancytopenia because it’s a rare genetic disease in Mogadishu and around the world.

Transplant Outcome of Pediatric and Young Adult Patients with Aplastic Anemia: St Jude Children’s Research Hospital Experience

Background: Bone marrow transplant (BMT) remains the preferred frontline curative modality for patients with aplastic anemia in the presence of a matched sibling donor (MSD). In the absence of MSD the general consensus is frontline immunosuppressive therapy (IST) followed by BMT from a matched unrelated donor (MUD) in case of failure or relapse. No randomized controlled studies have been performed to compare earlier MUD transplant to 2nd cycle IST in case of failure of response to 1st IST cycle. In the pediatric population a higher percentage of congenital marrow failure syndromes is encountered than in adult world; the diagnosis of many of those is challenging clinically and from the molecular and genetic standpoints.Objectives: Review the outcome of 25 patients younger than 20 years diagnosed with aplastic anemia and recently transplanted at our institution. Compare outcomes of MSD versus MUD versus umbilical cord (UC) BMT in this patient population.Methods: A retrospective analysis was performed on 25 patients diagnosed with aplastic anemia who underwent BMT at St Jude Children’s Research Hospital between 9/1/2009 through 8/31/2013. Initial diagnosis and treatment, patients’ characteristics, transplant data, overall survival, and complications were analyzed.Results: Of the 25 patients reviewed, 15 patients were diagnosed with acquired aplastic anemia and 10 with congenital marrow failure, including 7 with dyskeratosis congenita (DKC), 1 with Shwachman Diamond syndrome (SDS), and 2 with congenital marrow failure not otherwise specified (negative genetic testing, but age, family history, and presentation consistent with congenital marrow failure). All 7 DKC patients had telomere lengths less than first percentile, and 2 had documented genetic mutations; of the 7 patients 2 received MSD, 3 received MUD and 2 received UC transplants.Most donors were MUD (13/25), 2 were UC donors as there were no other available donors, and 10 were MSD. All MSD recipients are alive, engrafted and disease free. There were 3 deaths. One MUD recipient with acquired aplastic anemia died of uncontrolled sepsis. Both UC recipients died; one had failure of engraftment and later died of acute myeloid leukemia that had developed on day +224 of transplant while on Danazol and G-CSF, and the other died of pulmonary and hepatic failure. The latter was the only patient who received TBI (2 Gy) as part of his conditioning regimen, engrafted on day +27 and died on day +33. There was no statistical difference in overall survival (OS) between MSD and MUD transplant (100% vs 92.3% at 3 years, respectively, p value = 0.38).Acute graft versus host disease (aGVHD) developed in 5 patients; 3 had received MUD transplant and 2 had received MSD. Two of these 5 patients also developed chronic graft versus host disease (cGVHD), which was limited in both cases. There was no statistically significant difference in engraftment or GVHD occurrence between the MSD and MUD patients.Conclusions: In our recent experience aplastic anemia in children,outcomes from MUD BMT were comparable to MSD BMT, especially with improved supportive care and GVHD prevention and treatment. Cord blood as an alternative donor source remains very challenging in this patient population from the engraftment and toxicity standpoints especially in certain congenital marrow failure syndromes. These data suggest that BMT should be considered earlier when there is MUD availability for patients with aplastic anemia. [Display omitted] P unrelated vs cord = 0.0007P sibling vs cord = 0.0002P unrelated vs sibling = 0.38 DisclosuresReiss:Alexion Pharm: Research Funding.

Whole-Exome Sequencing Reveals a Paucity of Somatic Gene Mutations in Aplastic Anemia and Refractory Cytopenia of Childhood

Introduction: The appropriate classification of bone marrow (BM) failure syndromes in children is challenging, particularly in relation to histological distinction between aplastic anemia (AA), refractory cytopenia of childhood (RCC), and refractory cytopenia with multilineage dysplasia (RCMD). The goal of this study is to characterize the molecular pathogenesis of these conditions by identifying the full spectrum of gene mutations in 29 children with three diseases using whole-exome sequencing.Patients and Methods: Wediagnosed AA, RCC, or RCMD on the basis of morphology and histological findings of bone marrow (BM) according to the 2008 World Health Organization (WHO) classification criteria. Patients with AA exhibited hypocellular BM and no morphologically dysplastic changes in any of three hematopoietic cell lineages, while patients with RCC had <10% dysplastic changes in two or more cell lineages or >10% in one cell lineage. Patients with RCMD exhibited >10% dysplastic changes in two or more cell lineages. We obtained peripheral blood and BM samples from 29 children (16 boys and 13 girls) with AA (n = 8), RCC (n = 11), or RCMD (n = 10). The median age at diagnosis was 11 years (range, 2–15 years). We performed exome capture from paired DNA (non-T cells/CD3+ lymphocytes) using SureSelect® Human All Exon V4 kit (Agilent Technologies, Santa Clara, CA), which covered all part of the coding exons, followed by massively-parallel sequencing using HiSeq 2000 (Illumina, San Diego, CA) according to the manufacturer’s protocol. Candidate somatic mutations and germline variants were detected through our pipeline for whole-exome sequencing (Genomon-exome). All candidate somatic nucleotide changes were validated by Sanger sequencing. The ethics committee of Nagoya University Graduate School of Medicine approved this study.Results: Whole-exome sequencing pipeline identified a total of 14 non-synonymous somatic (one nonsense, 11 missense, and two frameshift) changes among the 29 patients, which resulted in only 0.48 mutations per patient. The average numbers of somatic mutations per sample were not significantly different among these groups (0.50 in AA, 0.36 in RCC, and 0.60 in RCMD). As a whole, childhood AA, RCC, and RCMD were characterized by a paucity of somatic mutations compared with adult myelodysplastic syndromes (MDS) in which 10 or more mutations per exome were detected on average. Among the mutated genes, BCOR-inactivating mutations in two patients (p.S158fs in AA and p.E1286X in RCMD) were considered significant genetic events based on previous reports that it is a driver gene in MDS. With regard to germline events, we did not detect any germline mutations of inherited BM failure syndromes. Moreover, we did not identifiy significantly frequent germline events in the entire cohort or any genetic hallmarks to be able to discriminate between these three diseases. When comparing the clinical outcomes of patients with somatic mutations (n = 7) versus those without somatic mutations (n = 22), response rate to immunosuppressive therapy at 6 months (50% vs. 50%), 5-year clonal evolution rate (95% confidential interval) [0% (0%) vs. 6% (0%–26%)], and the 5-year overall survival rate (95% confidential interval) [100% (100%–100%) vs. 95% (70%–99%)] were not significantly different.Conclusion: We usedwhole-exome sequencing analysis for gene mutational profiling of children with AA, RCC, and RCMD. Idiopathic bone marrow failure syndromes in children are characterized by a paucity of somatic gene mutations, irrespective of histological diagnosis. These findings suggest that histological diagnosis based on the WHO classification system does not discriminate the mutational profile of idiopathic BM failure syndromes in children. DisclosuresNo relevant conflicts of interest to declare.

Outcomes of Stem Cell Transplantation with Fludarabine and Melphalan Conditioning for Children with Acquired Bone Marrow Failure: A Nationwide Retrospective Study

Survival after stem cell transplantation (SCT) in children with acquired bone marrow failure (aBMF) has improved over decades. However, we have experienced a certain number of patients who presented with bone marrow aplasia with full donor chimerism after SCT, especially in the last decade. We named the complication “donor-type aplasia”, and now, this is one of the main causes of treatment failure after SCT in children with aBMF. Because fludarabine (FLU)-based conditioning regimen has been often used for Japanese children with aBMF since the 2000s, use of FLU was suspected to associate with donor-type aplasia. On the other hand, when FLU was introduced in the regimen for children with aBMF, the dose of cyclophosphamide (CY) was reduced by half, to reduce the toxicity. Given these, we previously investigated the risk factors for donor-type aplasia, and reported that the dose reduction of CY rather than the use of FLU in itself was relevant to the recent increase of donor-type aplasia (Yoshida N, et al. ASH 2012). To reduce the risk for donor-type aplasia, the conditioning regimen for children with aBMF needs to be reconsidered. The purpose of the present study is to evaluate the outcomes of SCT using FLU/melphalan (MEL)-based conditioning instead of the standard FLU/CY-based regimen in children with aBMF.We retrospectively reviewed the clinical data of 488 patients (<16 years) with aBMF (aplastic anemia and refractory cytopenia of childhood) who received the first SCT from 2000 to 2012 and whose records were available in the Japan Society for Hematopoietic Cell Transplantation Registry. Totally, 28 patients received the FLU/MEL-based regimen, while 233 received the FLU/CY-based regimen. Of the 28 patients, 11 received SCT from a related donor, whereas 17 received it from an unrelated donor. The stem cell source was bone marrow in 19 patients, peripheral blood in 1, or cord blood in 8. The conditioning regimen was based on FLU (100-180 mg/m2) and MEL (70-180 mg/m2), and ATG was included in the regimen in 17 patients, while low dose irradiation was used in 23 patients.The 5-year overall survival and event free survival of all patients who received the FLU/MEL-based regimen was 88%. Engraftment was achieved in 27 out of 28 patients (96%) and secondary graft failure including donor-type aplasia was not observed. Regarding the MEL dose, the favorable survival in patients who received 120 mg/m2 or more was observed (100% vs. 62%; P=0.006). Notably, all patients who received bone marrow transplantation (BMT) were alive without any complication. We then compared the outcomes in the setting of BMT with the FLU/MEL-based regimen (n=19) to those with the FLU/CY-based regimen (n=219). The event free survival was inferior in patients treated with the FLU/CY-based regimen (85% vs. 100%), although this difference was not statistically significant. With the FLU/CY-based regimen, engraftment was achieved in 214 patients (98%), whereas secondary graft failure including donor-type aplasia was seen in 18 patients.In conclusion, the FLU/MEL-based conditioning regimen provided excellent outcomes especially in the setting of BMT. To validate whether this regimen can reduce the risk of donor-type aplasia, a larger study is necessary. Therefore, a prospective study on SCT with this conditioning for aBMF children with high risk of donor-type aplasia is now planned by the Japan Childhood Aplastic Anemia Study Group. DisclosuresNo relevant conflicts of interest to declare.

Role of Th17/Treg imbalance in the immune pathogenesis and treatment of children with aplastic anemia

To study the role of Th17/Treg imbalance in the immune pathogenesis and therapeutic significance in childhood aplastic anemia (AA). We analyzed data from 43 children (male: female = 14: 29) with AA, all the cases were at the age of 2 to 14 years at diagnosis, and were hospitalized at our department of pediatrics between January 2012 and October 2013 in the Second Hospital of Anhui Medical University. All these patients were divided into 2 groups, severe AA (SAA) group (n = 25, male: female = 8: 17, 2-14 years old) and non-severe AA (NSAA) group (n = 18, male: female = 6: 12, 2-14 years old), depending on the severity at first diagnosis. As to the treatment, we analyzed data at 3 phases of treatment, diagnosis (n = 43, male: female = 14: 29, 2-14 years old), transfusion-indenpendence (n = 8, male: female = 5: 3, 2-11 years old), complete response (n = 6, male: female = 3: 3, 2-11 years old); at the same time, AA children who did not respond to the treatments were considered as failed treatment control (transfusion-indenpendence with failed treatment group, n = 5, male: female = 1: 4, 3-8 years old; complete response failed treatment group, n = 4, male: female = 2: 2, 4-11 years old). The ratio of Treg and Th17 cells in CD4(+) T cells were tested by flow cytometry. The levels of IL-6 and IL-17 in plasma were determined by ELISA. During the same period, 25 age-matched healthy children (male: female = 12: 13, 3-14 years old) were recruited as normal control, 9 cases (male: female = 5: 3, 2-11 years old) of AA children induced by chemotherapy as diagnosis control group. Differences in variables were analyzed using ANOVA and t-tests or the Kruskal-Wallis and Mann-Whitney U-tests, as appropriate. Correlation analysis was evaluated by the Spearman rank correlation test. (1) The ratio of Th17 cells in newly diagnosed AA patients were higher than that of normal group or diagnosis control group [1.63% (1.27%, 2.48%) vs. 0.4% (0.35%, 0.51%) or 0.50% (0.45%, 0.75%), both P < 0.01] while the ratio of Treg cells was lower [4.24% (3.10%, 5.29%) vs. 7.03% (6.56%, 7.48%) or 7.50% (6.60%, 8.30%), both P < 0.01] and the proportion of Th17/Treg were significantly higher [0.53(0.34, 0.69) vs. 0.06 (0.05, 0.07) or 0.09 (0.08,0.11), both P < 0.01]. (2) The levels of IL-6 and IL-17 in newly diagnosed AA patients were higher than in normal group [ (223 ± 92) vs. (116 ± 18) ng/L, (26.2 ± 12.0) ng/L vs. (10.6 ± 2.1) ng/L, P both < 0.01]. There was a positive correlation between Th17 cells and some Th17 cells related cytokines such as IL-17 and IL-6 (r = 0.62, 0.64, P both < 0.01). (3) The ratio of Th17, Th17/Treg, and the levels of IL-6 and IL-17 in children with SAA were also higher than in normal group [1.80% (1.25%, 2.61%) vs. 0.40% (0.35%, 0.51%), 0.57% (5.10%,0.82%) vs. 0.06% (0.05%, 0.07%), (225 ± 108) vs. (116 ± 18) ng/L, (25.9 ± 12.6) vs. (10.6 ± 2.1)ng/L, all P < 0.01]. NSAA also higher than normal group. The ratio of Treg in children with SAA and NSAA was less than that in normal group (P all < 0.01). However, the ratio of Th17, Treg, Th17/Treg, and the levels of IL-6 and IL-17 had no significant difference between SAA and NSAA (all P > 0.05). (4) In different stages of treatment, such as diagnosis, transfusion-indenpendence, complete response, there were significant differences in the ratio of Th17 and Th17/Treg (both P < 0.05) but not in Treg (P > 0.05). The imbalance of Th17/Treg cells and abnormally increased cytokines related to Th17 cells exist in peripheral blood of AA children, but did not significantly affect the severity of AA in preliminary diagnosis. After treatment with immunosuppression, AA was gradually relieved as the imbalance of Th17/Treg was corrected.

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.

Expression and significance of mTOR signaling pathway in T cells in childhood aplastic anemia

Objective To sudy the changes in mTOR signaling pathway in childhood aplastic anemia(AA) by detecting the expression levels of the molecules of mTOR signaling pathway in T cells, and to explore immunologoical pathogenesis of AA in children from T cell intracellular signal transduction pathway. Methods Peripheral blood samples were collected from 16 newly diagnosed severe AA(SAA) patients and 8 patiens treated with effective immunosuppressive therapy, and the findings were compared with those of 17 healthy children (normal controls) and CEM cells(positive controls). The expressions of p-Akt, p-TSC2, p-mTORC1, p-4EBP1, p-p70S6K in CD3+ T cells in peripheral blood were detected by flow cytometry(FCM). Results 1.The expressions of p-Akt, p-TSC2, p-mTORC1, p-4EBP1, p-p70S6K of the newly diagnosed SAA group were higher than those of the normal control group(P 0.05), but the expressions of p-4EBP1 were higher(P<0.05). The MFI was followed by 3.28±1.27, 16.50±10.91, 3.54±1.66, 74.89±49.69 and 4.21±1.69. Conclusions 1.The expressions of p-Akt, p-TSC2, p-mTORC1, p-4EBP1, p-p70S6K were increased in the newly diagnosed SAA patients, the mTOR signaling pathway was activated in SAA patients.2.The expressions of p-Akt, p-TSC2, p-mTORC1, p-4EBP1, p-p70S6K were lower than those of the newly diagnosed SAA patients.The degree of activation of mTOR signaling pathway was associated with disease status.The signaling pathways may be involved in the T cells of AA of the immune abnormalities. Key words: Aplastic anemia; T cell; Mammalian target of rapamycin; Signaling pathway

Peripheral blood lymphocyte telomere length as a predictor of response to immunosuppressive therapy in childhood aplastic anemia.

Predicting the response to immunosuppressive therapy could provide useful information to help the clinician define treatment strategies for patients with aplastic anemia. In our current study, we evaluated the relationship between telomere length of lymphocytes at diagnosis and the response to immunosuppressive therapy in 64 children with aplastic anemia, using flow fluorescence in situ hybridization. Median age of patients was ten years (range 1.5-16.2 years). Severity of the disease was classified as very severe in 23, severe in 21, and moderate in 20 patients. All patients were enrolled in multicenter studies using antithymocyte globulin and cyclosporine. The response rate to immunosuppressive therapy at six months was 52% (33 of 64). The probability of 5-year failure-free survival and overall survival were 56% (95% confidence interval (CI): 41-69%) and 97% (95%CI: 87-99%), respectively. Median telomere length in responders was -0.4 standard deviation (SD) (-2.7 to +3.0 SD) and -1.5 SD (-4.0 to +1.6 (SD)) in non-responders (P<0.001). Multivariate analysis showed that telomere length shorter than -1.0 SD (hazard ratio (HR): 22.0; 95%CI: 4.19-115; P<0.001), platelet count at diagnosis less than 25×10(9)/L (HR: 13.9; 95%CI: 2.00-96.1; P=0.008), and interval from diagnosis to immunosuppressive therapy longer than 25 days (HR: 4.81; 95%CI: 1.15-20.1; P=0.031) were the significant variables for poor response to immunosuppressive therapy. Conversely to what has been found in adult patients, measurement of the telomere length of lymphocytes at diagnosis is a promising assay in predicting the response to immunosuppressive therapy in children with aplastic anemia.

Comparison of rabbit antithymocyte globulin and Jurkat cell-reactive anti-t lymphocyte globulin as a first-line treatment for children with aplastic anemia

The purpose of this study was to investigate the effects of rabbit antihuman thymocyte globulin (R-ATG) and Jurkat cell-reactive anti-T lymphocyte globulin (ATG-F) in the treatment of childhood aplastic anemia (AA) and compare their efficacy and side effects. A total of 53 children with AA were analyzed in the present study, including 32 cases of severe AA, 10 cases of very severe AA and 11 cases of transfusion-dependent nonsevere AA. While receiving immunosuppressive therapy (IST), 29 and 24 patients, all of whom received long-term oral supplement with cyclosporin A (CSA), androgen, and traditional Chinese medicines, were treated with R-ATG and ATG-F, respectively. If necessary, the patients were also given supportive care such as component transfusion and/or infection control. Absolute counts of peripheral blood lymphocyte at various time points were dynamically measured after ATG therapy. According to the International AA Treatment and Effect standards, we found that there were no statistically significant differences in the response rate (70.83% vs. 68.97%, p > 0.05) and the overall survive rate (83.33% vs. 82.76%, p > 0.05) between the ATG-F and R-ATG groups. In addition, no obvious differences were observed between these two groups in the response time, efficacy in severe AA and very severe AA, or the incidence rates of ATG-related adverse reactions. After ATG treatment, the extent of peripheral blood lymphocyte reduction and duration in peripheral blood were similar between the ATG-F and R-ATG groups. The results of this study showed that ATG-F and R-ATG had similar efficacy and adverse reactions in the first-line treatment of childhood AA, despite being derived from different immunogens.

Treatment of aplastic anemia in children

Treatment of aplastic anemia in children

Immunosuppressive therapy with horse anti-thymocyte globulin and cyclosporine as treatment for fulminant aplastic anemia in children

Patients with severe aplastic anemia (SAA) and an absolute neutrophil count (ANC) of 0 typically have fatal outcomes. We defined fulminant AA (FAA) as ANC = 0 for at least 2weeks prior to and after immunosuppressive therapy (IST). We analyzed the outcomes of 35 children with FAA among 288 children who enrolled in a prospective study for AA (AA-97 study). AA was classified as FAA (n = 35), very SAA (vSAA; n = 129), or SAA (n = 124). All of the children received the IST with horse anti-thymocyte globulin (ATG) and cyclosporine (CsA). A significantly lower response rate at 6months was seen in children with FAA when compared to those with vSAA or SAA (40.0, 63.6, and 63.7%, respectively; p = 0.027). Of 20 nonresponder patients in the FAA group, 11 were rescued by alternative donor transplantation, and 5 patients showed a late response after 6months. Consequently, no significant difference was noted in overall survival when comparing the FAA, vSAA, and SAA groups (88.5, 95.8, and 96.8%). These findings indicate that IST with ATG and CsA is justified as a first-line treatment for children with FAA who lack a human leukocyte antigen-matched sibling donor.

Hematopoietic Stem Cell Transplantation in Children and Young Adults with Secondary Myelodysplastic Syndrome and Acute Myelogenous Leukemia after Aplastic Anemia

Secondary myelodysplastic syndrome and acute myelogenous leukemia (sMDS/sAML) are the most serious secondary events occurring after immunosuppressive therapy in patients with aplastic anemia. Here we evaluate the outcome of hematopoietic stem cell transplantation (HSCT) in 17 children and young adults with sMDS/sAML after childhood aplastic anemia. The median interval between the diagnosis of aplastic anemia and the development of sMDS/sAML was 2.9 years (range, 1.2 to 13.0 years). At a median age of 13.1 years (range, 4.4 to 26.7 years), patients underwent HSCT with bone marrow (n = 6) or peripheral blood stem cell (n = 11) grafts from HLA-matched sibling donors (n = 2), mismatched family donors (n = 2), or unrelated donors (n = 13). Monosomy 7 was detected in 13 patients. The preparative regimen consisted of busulfan, cyclophosphamide, and melphalan in 11 patients and other agents in 6 patients. All patients achieved neutrophil engraftment. The cumulative incidence of grade II-IV acute graft-versus-host disease (GVHD) was 47%, and that of chronic GVHD was 70%. Relapse occurred in 1 patient. The major cause of death was transplant-related complication (n = 9). Overall survival and event-free survival at 5 years after HSCT were both 41%. In summary, this study indicates that HSCT is a curative therapy for some patients with sMDS/sAML after aplastic anemia. Future efforts should focus on reducing transplantation-related mortality.

Low Expression of Basic Fibroblastic Growth Factor in Mesenchymal Stem Cells and Bone Marrow of Children with Aplastic Anemia

Background: Our previous experiments with gene chip suggested that basic fibroblastic growth factor (FGF2) levels were lower in mesenchymal stem cell (MSC) from aplastic anemia patients. The purpose of this study was to determine the expression of FGF2 in MSC and in bone marrow of children with aplastic anemia to better understand the role of low FGF2 expression in the pathogenesis of aplastic anemia. Procedure: MSCs from the bone marrow of aplastic anemia children and control group were cultured in vitro. Growth curves of primary and passage MSC were plotted. FGF2 gene expression in MSCs was detected using quantitative real-time polymerase chain reaction (RT-PCR). FGF2 protein expression in mononuclear cells and FGF2 protein level in extracellular fluid of bone marrow were also investigated. Result: Decreased growth of MSCs from aplastic anemia children was observed after passage 8 in serial subcultivation, and FGF2 gene expression was downregulated. Within the patients’ bone marrow, low FGF2 expression was validated both in mononuclear cells and in the extracellular fluid. Conclusion: Low FGF2 gene expression in MSCs and low FGF2 protein level in bone marrow of aplastic anemia may involve to pathogenesis of aplastic anemia.

Long-term outcome after immunosuppressive therapy with horse or rabbit antithymocyte globulin and cyclosporine for severe aplastic anemia in children

Some prospective studies showed that rabbit antithymocyte globulin was inferior to horse antithymocyte globulin as first-line therapy for patients with severe aplastic anemia. We retrospectively analyzed the clinical outcome of 455 children with severe aplastic anemia who received horse antithymocyte globulin (n=297) or rabbit antithymocyte globulin (n=158) combined with cyclosporine as first-line therapy between 1992 and 2010. The response rates were comparable between the horse and rabbit antithymocyte globulin groups at 3 months [46% (136/294) versus 42% (66/153), P=0.55] and 6 months [60% (178/292) versus 55% (87/143), P=1.0]. Using multivariate analysis, differences in antithymocyte globulin preparations were not associated with response rates. However, 2-year and 10-year overall survival rates in the horse antithymocyte globulin group were significantly better than those in the rabbit antithymocyte globulin group (2-year overall survival: 96% versus 87%, 10-year overall survival: 92% versus 84%, P=0.004). On the basis of multivariate analysis, use of rabbit antithymocyte globulin was a significant adverse factor for overall survival (hazard ratio = 3.56, 95% confidence interval, 1.53 - 8.28, P=0.003). Rabbit antithymocyte globulin caused more profound immunosuppression, which might be responsible for the higher incidence of severe infections. Considering that there are no studies showing the superiority of rabbit antithymocyte globulin over horse antithymocyte globulin, horse antithymocyte globulin should be recommended as a first-line therapy. However, our results justify the use of rabbit antithymocyte globulin as first-line therapy if horse antithymocyte globulin is not available.

Epidemiological Features of Aplastic Anemia in Indian Children.

To study the socio-demographic profile and clinico-hematological features of aplastic anemia in children presenting at a single institution over 5y. Patients below the age of 15y presenting with features of aplastic anemia were included in the study. Epidemiology, clinico-hematological features and treatment received were recorded in all the cases. Serology for hepatitis A, B, C, E, EBV, parvovirus and HIV was carried out. Cytogenetic studies were available in approximately half of the patients. One hundred eighty five patients were diagnosed with aplastic anemia. Ten patients with inherited bone marrow failure syndrome (IBMFS) were excluded. Median age was 8y (range 4-14y) with a male to female ratio of 2.4:1. Pallor (100%) followed by bleeding manifestations (83.8%) and fever (73.5%) were most common presenting symptoms. One hundred twenty patients (70%) were classified as severe, 36 (21%) very severe and 17 (9%) non-severe aplastic anemia. Viral markers for parvovirus, Epstein barr virus and hepatitis were positive in 25.8%, 20% and 6.7% patients respectively. Six patients had history of varicella infection in recent past (within 6mo). Very few patients (30) could afford immunosuppressive therapy (IST) and had suboptimal response (29.7%). Majority of patients had idiopathic aplastic anemia. Very severe aplastic anemia and severe aplastic anemia were frequent. Few patients received IST and had suboptimal response. There is need to establish a national registry for aplastic anemia.

Pharmacokinetics of antithymocyte globulin in the treatment of childhood aplastic anemia

Objective To investigate the pharmacokinetics of antithymocyte globulin(ATG)in the treatment of childhood aplastic anemia(AA). Methods Six cases of of childhood AA treated with rabbit ATG(R-ATG, Genzyme company, USA and Fresenius company, Germany)were in the study.Enzyme-linked immunosorbent assay(ELISA) was used to detect the concentrations of ATG in plasma during the treatment period of the first day to the 5th day, and during the 7th, 21th, 35th, 60th and 90th after the treatment finished respectively(all 12 time nodes). The pharmacokinetic data was detect with the Practical Pharmacokinetics-program(3P97). Results The peak levels of ATG concentrations in plasma(30-32 mg/L)were reached at the third to forth day during the treatment period, and then gradually descended to about half of the peak level in the 21st day(16-18 mg/L), and the microcomponent of ATG in plasma could be detected in the 90th day after the therapy.There were not evident difference of plasma concentrations in every time nodes, the peak levels, and pharmacokinetic data including the drug half-life, area under the plasma concentration-time curve(AUC)between the 2 forms of R-ATG(Genzyme company, USA and Fresenius company, Germany). The therapy effective rate in the 6 cases AA of this group was 66.7% in the followed-up for mean 12 months. Conclusions Successfully set up the techniques to detect the pharmacokinetics of ATG in the treatment of childhood AA, will help to research the relationship between the pharmacokinetics with the side effects and outcomes of ATG in the treatment of AA. Key words: Pharmacokinetics; Antithymocyte globulin; Immunosuppressive therapy; Aplastic anemia