Makito Tanaka

Acute megakaryoblastic leukaemia (AMKL) in children: a comparison of AMKL with and without Down syndrome

To characterize childhood acute megakaryoblastic leukaemia (AMKL), we reviewed 45 children with AMKL diagnosed between 1986 and 2005 at Nagoya University Hospital and Japanese Red Cross Nagoya First Hospital. Twenty‐four patients (53%) had AMKL associated with Down syndrome (DS‐AMKL) and 21 (47%) had non‐DS‐AMKL. The median age of the DS‐AMKL patients was 21 months (range, 8–38 months) and that of non‐DS‐AMKL patients was 15 months (range, 2–185 months). The morphology of blast cells was categorized into three groups according to the stage of megakaryocyte maturation. The blast cells were more immature in DS‐AMKL than in non‐DS‐AMKL in terms of morphology and immunophenotyping. Cytogenetic abnormalities of leukaemic cells were classified into seven categories: normal karyotype including constitutional trisomy 21 in DS‐AMKL; numerical abnormalities only; t(1;22)(p13;q13); 3q21q26 abnormalities; t(16;21)(p11;q22); −5/del(5q) and/or −7/del(7q); and other structural changes. The outcome of children with either DS‐AMKL or non‐DS‐AMKL is excellent. The 10‐year overall survival estimate was 79% [95% confidence interval (CI): 54–90] for DS‐AMKL and 76% (95% CI: 58–91) for non‐DS‐AMKL (P = 0·81) with a median follow‐up of 78 months (range, 20–243 months). Our study shows the diverse heterogeneity of childhood AMKL and the need for subclassification according to cytogenetic and morphological features.

Late-onset non-infectious pulmonary complications following allogeneic hematopoietic stem cell transplantation in children

Late-onset non-infectious pulmonary complications (LONIPCs) that arise beyond 3 months after allogeneic hematopoietic SCT include bronchiolitis obliterans (BO), bronchiolitis obliterans with organizing pneumonia (BOOP) and idiopathic pneumonia syndrome (IPS). We retrospectively analyzed the incidence and outcome of LONIPCs among pediatric hematopoietic SCT recipients. We included 97 patients who survived for more than 3 months among the 114 who underwent allogeneic hematopoietic SCT between April 1997 and May 2007. Of the 97 enrolled patients, 10 (10.3%) developed LONIPCs at a median of 187 days after hematopoietic SCT (range, 123–826 days). Of the 10 patients with LONIPCs, eight had BO and two had IPS. Multivariate analysis showed that the onset of LONIPCs was associated with high-risk underlying disease and extensive chronic GVHD (hazard ratio, 5.42 (95% confidence interval, 1.36–21.7) and hazard ratio, 11.7 (95% confidence interval, 2.40–57.1), respectively). Only two patients responded to therapy with steroids and six of the 10 patients died. The 5-year OS rate was significantly lower among patients with, than without LONIPCs (28.0 vs 87.2%, P=0.000). Considering that we are lacking optimal therapies for LONIPCs, strategies aimed at the prevention of LONIPCs should be attempted.

Comparison of matched-sibling donor BMT and unrelated donor BMT in children and adolescent with acquired severe aplastic anemia

From January 1991 to March 2007, 61 children and adolescent with acquired severe aplastic anemia received BMT in our institutions. We retrospectively compared the outcome of 30 cases of matched-sibling donor BMT (MSD-BMT) and 31 cases of unrelated donor BMT (URD-BMT). We observed one graft failure among MSD-BMT recipients and three graft failures among URD-BMT recipients, respectively. No patients in the MSD-BMT group developed grades II–IV acute GVHD compared with 11 of 30 patients (37%) in the URD-BMT group (P<0.001). One of 30 MSD-BMT recipients (3%) developed chronic GVHD compared with 8 of 30 URD-BMT recipients (27%) (P=0.013). The incidence of EBV and CMV reactivation was 11 of 20 URD-BMT recipients and 23 of 30, respectively. One patient in the URD-BMT group died of a motor accident 5.5 years after BMT. Ten-year OS was 100% in MSD-BMT recipients and 93.8% (95% CI, 81.9–100%) in URD-BMT recipients, respectively (P=0.252). Ten-year failure-free survival was 96.7% (95% CI, 90.2–100%) in the MSD-BMT group and 84.7% (95% CI, 70.2–99.2%) in the URD-BMT group, respectively (P=0.161).

Clinical impact of HLA-DR15, a minor population of paroxysmal nocturnal haemoglobinuria-type cells, and an aplastic anaemia-associated autoantibody in children with acquired aplastic anaemia

Aplastic anaemia (AA) is defined as a pancytopenia caused by bone marrow failure, and its pathogenesis is thought to involve autoimmune processes. Several predictive markers of the response to immunosuppressive therapy (IST) have been proposed, which appear to reflect the immune pathophysiology. We prospectively investigated the presence of human leucocyte antigen (HLA)‐DR15, a minor population of paroxysmal nocturnal haemoglobinuria (PNH)‐type cells, and antibodies to the recently identified autoantigen postmeiotic segregation increased 1 (PMS1) in 103 children with AA enrolled in a multicentre study. In contrast to adults, children with AA did not show an increased frequency of HLA‐DR15. In addition, a sensitive flow cytometric assay revealed that children with AA have a much lower prevalence of PNH‐type cells (21·4%) than reported for adults with this disease. An immunoblotting assay detected anti‐PMS1 antibody in 15 of 103 (14·6%) of the children. Finally, the response rate to IST was not significantly different between patients with and without DR15 (45·5% vs. 54·0%), PNH‐type cells (68·2% vs. 53·1%) or anti‐PMS1 antibody (40·0% vs. 59·1%). The current study did not confirm a correlation between these markers and the response to IST, suggesting that there is a difference in the pathophysiologies of adult and paediatric AA.

Downregulation of GATA-2 and overexpression of adipogenic gene-PPARγ in mesenchymal stem cells from patients with aplastic anemia

Aplastic anemia (AA) is characterized by a reduced number of hematopoietic stem cells and fatty replacement in the bone marrow. Transcriptional factor GATA-2 plays several important roles in both hematopoiesis and adipogenesis. Decreased levels of GATA-2 compromise the proliferation and survival of hematopoietic stem cells. GATA-2 suppresses adipocyte differentiation through direct inhibition of adipogenic factors, including peroxisome proliferator-activated receptor-gamma (PPARgamma). Previous studies have shown that expression of GATA-2 is decreased in marrow CD34-positive cells in AA. To elucidate the mechanisms of fatty marrow replacement, we evaluated the mRNA expression for GATA-2 and PPARgamma in mesenchymal stem cells (MSCs) from patients with AA by quantitative real-time polymerase chain reaction. GATA-2 expression by MSCs from AA patients was significantly lower than in normal subjects. Conversely, expression of PPARgamma was significantly higher in AA patients. Western blot analysis demonstrated that protein levels of GATA-2 were lower in AA patients than those in normal subjects. Moreover, incubation with interferon-gamma induced downregulation of GATA-2 levels in MSCs from normal subjects. These findings indicate that fatty marrow replacement in AA patients can be explained by downregulation of GATA-2 and overexpression of PPARgamma in MSCs. Decreased expression of GATA-2 might be responsible for the pathogenesis and development of the clinical features of the disease.

Correlation of cyp2c19 phenotype with voriconazole plasma concentration in children

Background: Voriconazole is a triazole antifungal agent with potent activity against a broad spectrum of pathogens, including Aspergillus and Candida species. In human adults, allelic polymorphisms of CYP2C19 are known to correlate with significant variation in voriconazole plasma concentration. Here, we report an analysis of CYP2C19 phenotype and voriconazole plasma concentrations in children. Methods: This retrospective study included 37 children who had voriconazole plasma concentrations measured from May 2006 to June 2011. All had single-nucleotide polymorphisms that define the 3 major CYP2C19 alleles. Patients were classified as follows: normal metabolizers, intermediate metabolizers, poor metabolizers, or hypermetabolizers. Results: The frequencies of the 3 CYP2C19 genetic polymorphisms were similar to those previously reported for Japanese adults. Trough plasma concentrations of voriconazole were significantly higher in the poor metabolizer and intermediate metabolizer groups compared with the normal metabolizer and hypermetabolizer groups (P=0.004). Two patients with high plasma concentrations experienced voriconazole-related severe adverse events (syndrome of inappropriate antidiuretic hormone secretion and cardiac toxicities). Conclusions: The current study suggests that a significant association exists in children between the voriconazole plasma concentration and the CYP2C19 phenotype. Dose adjustment based on CYP2C19 phenotype may be useful during voriconazole therapy, especially for Japanese children, who as a group have a higher incidence of the poor metabolizer and intermediate metabolizer phenotypes.

Somatic mosaicism for oncogenic NRAS mutations in juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) is a rare pediatric myeloid neoplasm characterized by excessive proliferation of myelomonocytic cells. Somatic mutations in genes involved in GM-CSF signal transduction, such as NRAS, KRAS, PTPN11, NF1, and CBL, have been identified in more than 70% of children with JMML. In the present study, we report 2 patients with somatic mosaicism for oncogenic NRAS mutations (G12D and G12S) associated with the development of JMML. The mutated allele frequencies quantified by pyrosequencing were various and ranged from 3%-50% in BM and other somatic cells (ie, buccal smear cells, hair bulbs, or nails). Both patients experienced spontaneous improvement of clinical symptoms and leukocytosis due to JMML without hematopoietic stem cell transplantation. These patients are the first reported to have somatic mosaicism for oncogenic NRAS mutations. The clinical course of these patients suggests that NRAS mosaicism may be associated with a mild disease phenotype in JMML.

Engraftment syndrome following allogeneic hematopoietic stem cell transplantation in children

Abstract:  ES is a complication that occurs immediately before or at the timing of neutrophil engraftment following autologous or allogeneic SCT. It is characterized by fever, skin rash, and non‐cardiac pulmonary infiltrates. We evaluated the incidence, risk factors, and outcomes of ES following allogeneic SCT in children. Of 100 pediatric patients, 20 (20%) developed ES occurring at a median of 14 days (range 8–27 days) post‐transplant. Patients presented with fever (100%), skin rash (100%), diffuse pulmonary infiltration (25%), and body weight gain (85%). On multivariate analysis, significant risk factors for ES included younger age (<8 yr old) and human leukocyte antigen disparity between donors and recipients. Univariate analysis showed that patients with ES had a higher incidence of developing chronic graft‐versus‐host disease and ES was not associated with other complications. Event‐free survival did not significantly differ between patients with and without ES regardless of the presence of malignant or non‐malignant diseases.

Feasibility and results of bone marrow transplantation from an HLA-mismatched unrelated donor for children and young adults with acquired severe aplastic anemia.

Treating patients with severe aplastic anemia (SAA) who fail to respond to immunosuppressive therapy (IST) and do not have an HLA-matched donor is challenging. We report favorable outcomes in 11 patients who underwent bone marrow transplantation (BMT) from an HLA-mismatched unrelated donor. The median age was 11 years (range, 3–20 years). The conditioning regimen consisted of cyclophosphamide (200 mg/kg), antithymocyte globulin (10 mg/kg), and total body irradiation (5 Gy). Patients received tacrolimus and methotrexate for prophylaxis against graft-versus-host disease (GVHD). Donor-recipient pairs were mismatched for the HLA-DR antigen in 8 patients by serologic typing. HLA-A and HLA-B antigens were mismatched in 1 and 2 patients, respectively. Ten patients achieved engraftment. One patient who failed to engraft was rescued by a second transplantation from her mother, who was mismatched at 2 HLA antigens. Acute GVHD of grades II to IV occurred in 2 patients. Three patients developed limited chronic GVHD, and 1 patient developed extensive chronic GVHD of the lung. All patients are alive at 9 to 56 months after transplantation (median, 33 months). Considering our encouraging results, HLA-mismatched unrelated-donor BMT for SAA is feasible as a salvage therapy for nonresponders to IST.

De novo childhood myelodysplastic/myeloproliferative disease with unique molecular characteristics.

Myelodysplastic/myeloproliferative uclassifiable (MDS/MPN‐U) is a rare myeloid neoplasm characterized by myelodysplasia and myeloproliferation at the time of initial presentation, which is usually a diagnosis of exclusion. The molecular pathogenesis of MDS/MPN‐U patients remains to be elucidated. Among five patients diagnosed with MDS/MPN‐U, three patients harboured RUNX1 (AML1) mutations; one carried somatic mosaicism of RUNX1 mutation with JAK2V617F mutation and one had dual RUNX1 and FLT3‐internal tandem duplication mutations with progression to acute myeloid leukaemia (AML). Germline mutation of TP53 was detected as a sole genetic lesion in one patient. JAK2V617F and somatic mosaicism of KRAS and TET2 mutations co‐existed in one patient. Otherwise, no alterations were detected in PTPN11, NRAS, CBL and ASXL1 genes. ETV6‐PDGFRB fusion transcript was not detected in all patients. Four patients recieved haematopoietic stem cell transplantation (HSCT); three patients relapsed and one achieved complete remission after three donor lymphocyte infusions. Our findings suggest that the mutational spectrum observed in childhood MDS/MPN‐U is quite different from that seen in juvenile myelomonocytic leukaemia and, to some extent, resemble chronic myelomonocytic leukaemia. Moreover, two patients had constitutional alterations of genes frequently found in AML. Further investigations are required to define the roles of these genetic alterations in the pathogenesis of childhood MDS/MPN‐U.