Shwachman-Diamond syndromes: clinical, genetic, and biochemical insights from the rare variants.

Shwachman-Diamond syndrome is a rare inherited bone marrow failure syndrome characterized by neutropenia, exocrine pancreatic insufficiency, and skeletal abnormalities. In 10-30% of cases, transformation to a myeloid neoplasm occurs. Approximately 90% of patients have biallelic pathogenic variants in the SBDS gene located on human chromosome 7q11. Over the past several years, pathogenic variants in three other genes have been identified to cause similar phenotypes; these are DNAJC21, EFL1, and SRP54. Clinical manifestations involve multiple organ systems and those classically associated with the Shwachman-Diamond syndrome (bone, blood, and pancreas). Neurocognitive, dermatologic, and retinal changes may also be found. There are specific gene-phenotype differences. To date, SBDS, DNAJC21, and SRP54 variants have been associated with myeloid neoplasia. Common to SBDS, EFL1, DNAJC21, and SRP54 is their involvement in ribosome biogenesis or early protein synthesis. These four genes constitute a common biochemical pathway conserved from yeast to humans that involve early stages of protein synthesis and demonstrate the importance of this synthetic pathway in myelopoiesis.

Successful treatment of a novel type I interferonopathy due to a de novo PSMB9 gene mutation with a Janus kinase inhibitor.

BACKGROUND: Type I interferonopathies are a recently established subgroup of autoinflammatory diseases caused by mutations in genes associated with proteasome degradation or cytoplasmic RNA- and DNA-sensing pathways. OBJECTIVE: This study aimed to unveil the molecular pathogenesis of a patient with novel type I interferonopathy, for which no known genetic mutations have been identified. METHODS: We performed the whole-exome sequencing of a 1-month-old boy with novel type I interferonopathy. We also investigated proteasome activities using patient-derived B lymphoblastoid cell lines (LCLs) and normal LCLs transduced with the mutant gene. RESULTS: Whole-exome sequencing identified a de novo proteasome 20S subunit beta 9 (PSMB9) p.G156D mutation in the patient who developed fever, a chilblain-like skin rash, myositis, and severe pulmonary hypertension due to the hyperactivation of IFN-α. Patient-derived LCLs revealed reduced proteasome activities, and exogenous transduction of mutant PSMB9 p.G156D into normal LCLs significantly suppressed proteasome activities, and the endogenous PSMB9 protein was lost along with the reduction of other immunoproteasome subunits, PSMB8 and PSMB10 proteins. He responded to the administration of a Janus kinase inhibitor, tofacitinib, and he was successfully withdrawn from venoarterial extracorporeal membranous oxygenation. At age 7 months, he received an unrelated cord blood transplantation. At 2 years posttransplantation, he no longer required tofacitinib and experienced no disease recurrence. CONCLUSIONS: We present the case of a patient with a novel type I interferonopathy caused by a de novo PSMB9 p.G156D mutation that suppressed the wild-type PSMB9 protein expression. Janus kinase inhibitor and stem cell transplantation could be curative therapies in patients with severe interferonopathies.

Relationship between plasma rabbit anti-thymocyte globulin concentration and immunosuppressive therapy response in patients with severe aplastic anemia.

OBJECTIVES: Patients with acquired aplastic anemia (AA) without HLA-matched sibling donors or aged >40 years receive immunosuppressive therapy (IST) with anti-thymocyte globulin (ATG). We investigated the relationship between plasma rabbit ATG (r-ATG) concentration and IST response. METHODS: From May 2012 to October 2017, 81 patients with severe AA who required initial IST were included. A 1:1 block randomization was employed for 2.5 and 3.5 mg/kg doses of r-ATG. RESULTS: No significant difference in response rates was observed between the 2.5 and 3.5 mg/kg groups (63% vs. 58%, P = .894). Median r-ATG concentrations on days 14 and 28 after IST were 15.2 (0.0-97.7) and 1.8 (0.0-74.9 µg/mL), respectively. According to r-ATG concentration, response rates were significantly higher in the group with higher r-ATG concentration than in those with lower r-ATG concentration (day 14, 88% vs. 52%; P = .006 and day 28, 79% vs. 46%; P = .005). In multivariate analysis, higher r-ATG concentrations at day 28 were independent predictors of favorable response to IST at 6 months (odds ratio, 0.29; 95% confidence interval, 0.09-0.93; P = .037). CONCLUSIONS: The present data indicate that higher r-ATG concentration at day 28 resulted in improved IST response.

Integrated molecular profiling of juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML), a rare and aggressive myelodysplastic/myeloproliferative neoplasm that occurs in infants and during early childhood, is characterized by excessive myelomonocytic cell proliferation. More than 80% of patients harbor germ line and somatic mutations in RAS pathway genes (eg, PTPN11, NF1, NRAS, KRAS, and CBL), and previous studies have identified several biomarkers associated with poor prognosis. However, the molecular pathogenesis of 10% to 20% of patients and the relationships among these biomarkers have not been well defined. To address these issues, we performed an integrated molecular analysis of samples from 150 JMML patients. RNA-sequencing identified ALK/ROS1 tyrosine kinase fusions (DCTN1-ALK, RANBP2-ALK, and TBL1XR1-ROS1) in 3 of 16 patients (18%) who lacked canonical RAS pathway mutations. Crizotinib, an ALK/ROS1 inhibitor, markedly suppressed ALK/ROS1 fusion-positive JMML cell proliferation in vitro. Therefore, we administered crizotinib to a chemotherapy-resistant patient with the RANBP2-ALK fusion who subsequently achieved complete molecular remission. In addition, crizotinib also suppressed proliferation of JMML cells with canonical RAS pathway mutations. Genome-wide methylation analysis identified a hypermethylation profile resembling that of acute myeloid leukemia (AML), which correlated significantly with genetic markers with poor outcomes such as PTPN11/NF1 gene mutations, 2 or more genetic mutations, an AML-type expression profile, and LIN28B expression. In summary, we identified recurrent activated ALK/ROS1 fusions in JMML patients without canonical RAS pathway gene mutations and revealed the relationships among biomarkers for JMML. Crizotinib is a promising candidate drug for the treatment of JMML, particularly in patients with ALK/ROS1 fusions.

Risk factors for secondary poor graft function after bone marrow transplantation in children with acquired aplastic anemia.

In patients with acquired AA, PGF is a major cause of cytopenia after hematopoietic stem cell transplantation. An increased incidence of PGF, especially sPGF, has been noted after the introduction of the FLU/CY regimen in children with acquired AA. To clarify the risk factors for sPGF, the clinical data of 49 patients (median age, 11 years; range, 1-19 years) with AA who received allogeneic BMT at Nagoya University Hospital from 1997 to 2016 were analyzed. Out of the 49 patients, 7 developed sPGF, and the 5-year CI was 0.15 (95% CI, 0.04-0.25). Five received the FLU/CY regimen, and the 5-year CI of sPGF was significantly higher in patients who received the regimen (0.36; 95% CI, 0.12-0.62) than in those who were conditioned with the non-FLU/CY regimen (0.06; 95% CI, 0.01-0.17; P = .01). The multivariate analysis confirmed that the FLU/CY regimen (hazard ratio, 6.12; 95% CI, 1.16-32.4; P = .03) was a significant risk factor for sPGF. sPGF improved spontaneously without stem cell boost infusions in 5 patients, ranging from 460 to 3539 days after BMT. The 10-year CI of the spontaneous trilineage recovery was 0.83 (95% CI, 0.00-0.97), and all 7 patients are alive. The FLU/CY regimen was identified as a risk factor for the sPGF development in patients with AA. The establishment of the optimal conditioning regimens for children with AA is warranted.

Danaparoid reduces transplant-related mortality in stem cell transplantation for children.

In SCT, death from transplant-related complications is the major obstacle hindering improvement of transplant outcomes, and proper supportive care is essential to reduce TRM. The transplant outcomes of 210 pediatric patients with malignant and non-malignant disorders who consecutively underwent SCT in our institution from 2000 to 2013 were analyzed. The transplant years were divided into three periods: A (2000-2004), B (2005-2008), and C (2009-2013), and an improvement in 5-year OS and a decrease in 5-year TRM were observed over these time periods; that is, OS was 61.5%, 60.3%, and 79.5% (P = .062), and TRM was 19.9%, 7.9%, and 0.0% (P < .001) in periods A, B, and C, respectively. On multivariate analysis, the prognostic factor for TRM for all patients was administration of danaparoid (HR = 0.109, 95% CI = 0.033-0.363, P < .001), and for patients with hematological malignancies in allogeneic SCT, the prognostic factors were danaparoid (HR = 0.046, 95% CI = 0.006-0.326, P = .002) and advanced disease at SCT (HR = 4.802, 95% CI = 1.734-13.30, P = .003). A reduction in TRM after SCT was observed over the time periods, and supportive care with danaparoid was found to be significantly effective in reducing TRM in SCT for children.

[Infected Thoracic Aortic Aneurysm Secondary to the Purulent Pericarditis;Report of a Case].

A 69-year-old man was hospitalized urgently to the department of cardiology, with the progressive general malaise. On admission, his blood pressure was 80/42 mmHg, his white cell count 13,700/µl, and C-reactive protein 25.55 mg/dl suggesting existence of aggressive infection with impaired circulation. Massive pericardial effusion was detected in echocardiography. Pericardial drainage was undergone promptly. There was drainage of 700 ml and the property was purulent. Pneumococcus was detected by the culture test of the pericardial fluid. Antibiotic administration was started by a diagnosis of the purulent pericarditis. His general condition was improved. However, a rapidly expanding saccular aneurysm was found in a descending thoracic aorta by computed tomography( CT). As an infected thoracic aortic aneurysm secondary to the purulent pericarditis, we performed thoracic endovascular aneurysm repair (TEVAR). The intravenous administration of antibiotics was continued for 2 weeks after TEVAR, which was followed by oral antibiotic administration for 1 year. The aneurysm completely disappeared by CT, 10 months after TEVAR. In case with an infected thoracic aortic aneurysm, TEVAR can be a 1st choice of treatment, depending on a causative organism and the morphology of the aneurysm.

Clinical utility of next-generation sequencing-based minimal residual disease in paediatric B-cell acute lymphoblastic leukaemia.

We assessed the clinical utility of next-generation sequencing (NGS)-based monitoring of minimal residual disease (MRD) in a uniformly treated cohort of 79 patients with paediatric B-cell acute lymphoblastic leukaemia. Bone marrow samples were collected at the time of diagnosis, days 33 and 80, pre- (4-5 months) and post- (24 months) maintenance therapy time points, and at relapse. We identified leukaemia-specific CDR3 sequences in 72 of 79 patients (91%) and detected MRD in 59 of 232 samples. Although MRD was detected in 28 of 55 samples (51%) on day 33, the frequencies of MRD detection decreased to 25% (16/65) at day 80, 19% (11/58) at 4-5 months and 7·4% (4/54) at 24 months. In a univariate analysis, positive MRD results on day 80 [relative risk (RR) 95% confidence interval (CI) = 7·438 (2·561-21·6), P < 0·001], at 4-5 months [RR (95% CI) = 10·24 (3·374-31·06), P < 0·001], and at 24 months [RR (95% CI) = 19·26 (4·974-74·59), P < 0·001] exhibited statistically significant associations with inferior leukaemia-free survival; this was confirmed using a Cox proportional hazard model. Our study suggests the promising potential of NGS-MRD for patients with B-cell ALL.

Development of clinical paroxysmal nocturnal haemoglobinuria in children with aplastic anaemia.

The clinical significance of paroxysmal nocturnal haemoglobinuria (PNH) in children with aplastic anaemia (AA) remains unclear. We retrospectively studied 57 children with AA between 1992 and 2010. During the follow-up, five patients developed clinical PNH, in whom somatic PIGA mutations were detected by targeted sequencing. The 10-year probability of clinical PNH development was 10·2% (95% confidence interval, 3·6-20·7%). Furthermore, the detection of minor PNH clones by flow cytometry at AA diagnosis was a risk factor for the subsequent development of clinical PNH. These patients with PNH clones at AA diagnosis should undergo periodic monitoring for potential clinical PNH development.

Clinical utility of next-generation sequencing for inherited bone marrow failure syndromes.

PURPOSE: Precise genetic diagnosis of inherited bone marrow failure syndromes (IBMFS), a heterogeneous group of genetic disorders, is challenging but essential for precise clinical decision making. METHODS: We analyzed 121 IBMFS patients using a targeted sequencing covering 184 associated genes and 250 IBMFS patients using whole-exome sequencing (WES). RESULTS: We achieved successful genetic diagnoses for 53 of 121 patients (44%) using targeted sequencing and for 68 of 250 patients (27%) using WES. In the majority of cases (targeted sequencing: 45/53, 85%; WES: 63/68, 93%), the detected variants were concordant with, and therefore supported, the clinical diagnoses. However, in the remaining 13 cases (8 patients by target sequencing and 5 patients by WES), the clinical diagnoses were incompatible with the detected variants. CONCLUSION: Our approach utilizing targeted sequencing and WES achieved satisfactory diagnostic rates and supported the efficacy of massive parallel sequencing as a diagnostic tool for IBMFS.Genet Med advance online publication 19 January 2017.

A Cytokine-Based Diagnostic Program in Pediatric Aplastic Anemia and Hypocellular Refractory Cytopenia of Childhood.

BACKGROUND: Distinguishing hypocellular refractory cytopenia of childhood (RCC) from aplastic anemia (AA) is challenging. Thus far, no studies have compared the cytokine profiles in patients with AA to those with hypocellular RCC. In the present study, we addressed whether thrombopoietin (TPO) and interleukin 17 (IL-17) plasma levels are useful for differentiating between the two diseases. METHODS: We measured the endogenous plasma concentrations of TPO and IL-17 in 29 patients with AA, 34 patients with hypocellular RCC, and 31 healthy controls using sensitive enzyme-linked immunosorbent assays. RESULTS: The TPO and IL-17 plasma levels were significantly lower in patients with hypocellular RCC than in patients with AA (P < 0.001 and P = 0.007, respectively). The multivariate logistic regression analysis identified moderate disease severity, TPO levels of <1,369.8 pg/ml (TPO-low group, n = 32; odds ratio (OR), 13.40; 95% confidence intervals (CI), 3.001-51.254; P < 0.001), and IL-17 levels of <22.2 pg/ml (IL-17-low group, n = 33; OR, 4.11; 95% CI, 1.033-19.404; P = 0.031) as independent factors discriminating hypocellular RCC from AA. Importantly, 25 (78.1%) of 32 patients in the TPO-low group and 25 (75.8%) of 33 patients in the IL-17-low group were diagnosed as having hypocellular RCC. Moreover, 22 (71%) of 31 patients in the TPO-high group and 21 (70%) of 30 patients in the IL-17-high group were diagnosed as having AA. CONCLUSIONS: TPO and IL-17 levels are useful for differentiating hypocellular RCC from AA. Prospective studies are required to confirm our findings.

Correlation of rabbit antithymocyte globulin serum levels and clinical outcomes in children who received hematopoietic stem cell transplantation from an alternative donor.

We analyzed the correlation between rabbit ATG (rATG) serum levels and clinical outcomes in 37 children who received rATG at a total dose of 10 or 15 mg/kg during HSCT conditioning from an alternative donor. Fourteen patients had advanced malignant diseases, 13 had severe AA, and 10 had inherited disorders. Complete engraftment was achieved in all patients, and no rejection occurred. The cumulative incidence of grades II-IV acute GVHD and extensive chronic GVHD was 27% (95% CI, 12.5-39.6%) and 8.1% (95% CI, 0-23.1%), respectively. Multivariate analysis identified lower rATG levels at week 4 as an independent risk factor in the development of grades II-IV acute GVHD (p = 0.037). Serious infections were not observed in any patient following HSCT. No correlation was found between EBV reactivation and rATG levels at week 2 and week 4 after HSCT. Furthermore, no correlation was found between relapse and rATG levels two and four wk post-transplantation. The probability of five-yr OS among patients was 70.3% (95% CI, 59.8-79.2%). Our results suggest that targeted rATG administration may protect patients from severe acute GVHD without increasing the risk of EBV reactivation or relapse.

Abnormal urinalysis on day 7 in patients with IgA vasculitis (Henoch-Schönlein purpura).

Rare progression to renal failure imposes a burden on children with IgA vasculitis (Henoch-Schönlein purpura, HSP). An abnormal urinalysis on day 7 (7d-UA) may be a surrogate marker for persistent nephritis, but this has not been established. We retrospectively analyzed the risk factors for persistent nephritis in a cohort of 138 children. Of 35 children with abnormal 7d-UA, 24 (69%) had an abnormal urinalysis 6 months after the diagnosis of HSP, which was significantly more than 6 of 103 children (6%) with normal 7d-UA (P < 0.0001). The negative predictive values for normal urinalysis and negative proteinuria 6 months after diagnosis were 0.94 (95% confidence interval [CI], 0.90-0.97) and 0.98 (95% CI, 0.95-0.99), respectively. When children with abnormal urinalysis 6 months after diagnosis were compared with those without, the following factors were significantly associated: age at diagnosis, abnormal urinalysis at diagnosis, abnormal 7d-UA, complement C3, steroid treatment, and presence of abdominal pain. However, multivariate analysis revealed that abnormal 7d-UA was the only significant risk factor for abnormal urinalysis 6 months after diagnosis (odds ratio 54.3, 95% CI 15.3-275, P = 1.89 × 10-6). Abnormal 7d-UA may be an independent risk factor for persistent nephritis, but this should be confirmed in a prospective study.

Choreito formula for BK virus-associated hemorrhagic cystitis after allogeneic hematopoietic stem cell transplantation.

Therapy for BK virus (BKV)-associated hemorrhagic cystitis (BKV-HC) is limited after hematopoietic stem cell transplantation (HSCT). We examined whether choreito, a formula from Japanese traditional Kampo medicine, is effective for treating BKV-HC. Among children who underwent allogeneic HSCT between October 2006 and March 2014, 14 were diagnosed with BKV-HC (median, 36 days; range, 14 to 330 days) after HSCT, and 6 consecutive children received pharmaceutical-grade choreito extract granules. The hematuria grade before treatment was significantly higher in the choreito group than in the nonchoreito group (P = .018). The duration from therapy to complete resolution was significantly shorter in the choreito group (median, 9 days; range, 4 to 17 days) than in the nonchoreito group (median, 17 days; range, 15 to 66 days; P = .037). In 11 children with macroscopic hematuria, the duration from treatment to resolution of macroscopic hematuria was significantly shorter in the choreito group than in the nonchoreito group (median, 2 days versus 11 days; P = .0043). The BKV load in urine was significantly decreased 1 month after choreito administration. No adverse effects related to choreito administration were observed. Choreito may be a safe and considerably promising therapy for the hemostasis of BKV-HC after HSCT.

Late-Onset Combined Immunodeficiency with a Novel IL2RG Mutation and Probable Revertant Somatic Mosaicism.

Primary immunodeficiency disease (PID) is caused by mutations of more than two hundred immunity-related genes. In addition to the heterogeneity of the diseases, the atypical presentation of each disease caused by hypomorphic mutations or somatic mosaicism makes genetic diagnosis challenging. Next-generation sequencing tests all genes simultaneously and has proven its innovative efficacy in genomics. We describe a male PID patient without any family history of immunodeficiency. This patient suffered from recurrent infections from 1 year of age. Laboratory analysis showed hypogammaglobulinemia. T, B, and NK cells were present, but the T cell proliferative response decreased. Whole-exome sequencing analysis identified an IL2RG p.P58T missense mutation. CD8(+) and CD56(+) cells showed revertant somatic mosaicism to the wild-type allele. A late-onset and atypical presentation of the X-linked severe combined immunodeficiency (X-SCID) phenotype might be associated with revertant somatic mosaicism in T and NK cells. This patient is the seventh reported case of X-SCID with revertant somatic mosaicism. His classical clinical management did not result in a molecular diagnosis because of the atypical presentation. The coverage that is provided by whole-exome sequencing of most PID genes effectively excluded differential diagnoses other than X-SCID. As next-generation sequencing becomes available in clinical practice, it will enhance our knowledge of PID and rescue currently undiagnosed patients.

Paroxysmal nocturnal hemoglobinuria and telomere length predicts response to immunosuppressive therapy in pediatric aplastic anemia.

Acquired aplastic anemia is an immune-mediated disease characterized by severe defects in stem cell number resulting in hypocellular marrow and peripheral blood cytopenias. Minor paroxysmal nocturnal hemoglobinuria populations and a short telomere length were identified as predictive biomarkers of immunosuppressive therapy responsiveness in aplastic anemia. We enrolled 113 aplastic anemia patients (63 boys and 50 girls) in this study to evaluate their response to immunosuppressive therapy. The paroxysmal nocturnal hemoglobinuria populations and telomere length were detected by flow cytometry. Forty-seven patients (42%) carried a minor paroxysmal nocturnal hemoglobinuria population. The median telomere length of aplastic anemia patients was -0.99 standard deviation (SD) (range -4.01-+3.01 SD). Overall, 60 patients (53%) responded to immunosuppressive therapy after six months. Multivariate logistic regression analysis identified the absence of a paroxysmal nocturnal hemoglobinuria population and a shorter telomere length as independent unfavorable predictors of immunosuppressive therapy response at six months. The cohort was stratified into a group of poor prognosis (paroxysmal nocturnal hemoglobinuria negative and shorter telomere length; 37 patients) and good prognosis (paroxysmal nocturnal hemoglobinuria positive and/or longer telomere length; 76 patients), respectively. The response rates of the poor prognosis and good prognosis groups at six months were 19% and 70%, respectively (P<0.001). The combined absence of a minor paroxysmal nocturnal hemoglobinuria population and a short telomere length is an efficient predictor of poor immunosuppressive therapy response, which should be considered while deciding treatment options: immunosuppressive therapy or first-line hematopoietic stem cell transplantation. The trial was registered in www.umin.ac.jp with number UMIN000017972.

Fulminant adenovirus hepatitis after hematopoietic stem cell transplant: Retrospective real-time PCR analysis for adenovirus DNA in two cases.

BACKGROUND: Viral infection is one of the major causes of mortality in patients undergoing hematopoietic stem cell transplant (HSCT). Systemic infection of adenovirus (AdV) has emerged as a not uncommon viral infection with significant morbidity and mortality as with cytomegalovirus and Epstein-Barr virus infection. Routine surveillance for these viruses has become a clinical practice and subsequent preemptive therapy improves patients' outcomes; however, the effectiveness of preemptive therapy for AdV has not been fully investigated in patients with a lethal form of AdV infection. METHODS: Sequential AdV loads were retrospectively analyzed in children with fulminant AdV hepatitis after HSCT. RESULTS: The AdV DNA became detectable (1 × 10(4) copies/mL) as early as 2 weeks after HSCT. These levels reached >1 × 10(8) copies/mL at the onset of fulminant hepatitis. However, we determined that γ-glutamyltransferase levels were elevated to >100 IU/L at least 2 weeks before the diagnosis of hepatitis. CONCLUSIONS: Our observation raises the possibility that elevated γ-glutamyltransferase could be a sentinel marker for AdV hepatitis, which prompts elaborated monitoring of AdV load and targeted treatment.

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.