Clinical and molecular characteristics of MEF2D fusion-positive B-cell precursor acute lymphoblastic leukemia in childhood, including a novel translocation resulting in MEF2D-HNRNPH1 gene fusion.

Fusion genes involving MEF2D have recently been identified in precursor B-cell acute lymphoblastic leukemia, mutually exclusive of the common risk stratifying genetic abnormalities, although their true incidence and associated clinical characteristics remain unknown. We identified 16 cases of acute lymphoblastic leukemia and 1 of lymphoma harboring MEF2D fusions, including MEF2D-BCL9 (n=10), MEF2D-HNRNPUL1 (n=6), and one novel MEF2D-HNRNPH1 fusion. The incidence of MEF2D fusions overall was 2.4% among consecutive precursor B-cell acute lymphoblastic leukemia patients enrolled onto a single clinical trial. They frequently showed a cytoplasmic µ chain-positive pre-B immunophenotype, and often expressed an aberrant CD5 antigen. Besides up- and down-regulation of HDAC9 and MEF2C, elevated GATA3 expression was also a characteristic feature of MEF2D fusion-positive patients. Mutations of PHF6, recurrent in T-cell acute lymphoblastic leukemia, also showed an unexpectedly high frequency (50%) in these patients. MEF2D fusion-positive patients were older (median age 9 years) with elevated WBC counts (median: 27,300/ml) at presentation and, as a result, were mostly classified as NCI high risk. Although they responded well to steroid treatment, MEF2D fusion-positive patients showed a significantly worse outcome, with 53.3% relapse and subsequent death. Stem cell transplantation was ineffective as salvage therapy. Interestingly, relapse was frequently associated with the presence of CDKN2A/CDKN2B gene deletions. Our observations indicate that MEF2D fusions comprise a distinct subgroup of precursor B-cell acute lymphoblastic leukemia with a characteristic immunophenotype and gene expression signature, associated with distinct clinical features.

Intraventricular Rituximab in Pediatric CD20-positive Refractory Primary Central Nervous System Lymphoma.

Primary central nervous system lymphoma (PCNSL) is a rare and aggressive type of extranodal non-Hodgkin lymphoma that carries an unsatisfactory prognosis. Treating refractory PCNSL is challenging because of resistance to conventional cytotoxic and intrathecal chemotherapies. Therefore, novel therapeutic approaches are needed. Here, we report a 12-year-old boy with CD20-positive PCNSL, which was refractory to combination chemotherapy and intravenous rituximab. However, the patient achieved complete remission after repeated intraventricular rituximab administration. The results of this case indicate that intraventricular rituximab is an effective option to treat refractory PCNSL in children.

ZNF384-related fusion genes define a subgroup of childhood B-cell precursor acute lymphoblastic leukemia with a characteristic immunotype.

Fusion genes involving ZNF384 have recently been identified in B-cell precursor acute lymphoblastic leukemia, and 7 fusion partners have been reported. We further characterized this type of fusion gene by whole transcriptome sequencing and/or polymerase chain reaction. In addition to previously reported genes, we identified BMP2K as a novel fusion partner for ZNF384 Including the EP300-ZNF384 that we reported recently, the total frequency of ZNF384-related fusion genes was 4.1% in 291 B-cell precursor acute lymphoblastic leukemia patients enrolled in a single clinical trial, and TCF3-ZNF384 was the most recurrent, with a frequency of 2.4%. The characteristic immunophenotype of weak CD10 and aberrant CD13 and/or CD33 expression was revealed to be a common feature of the leukemic cells harboring ZNF384-related fusion genes. The signature gene expression profile in TCF3-ZNF384-positive patients was enriched in hematopoietic stem cell features and related to that of EP300-ZNF384-positive patients, but was significantly distinct from that of TCF3-PBX1-positive and ZNF384-fusion-negative patients. However, clinical features of TCF3-ZNF384-positive patients are markedly different from those of EP300-ZNF384-positive patients, exhibiting higher cell counts and a younger age at presentation. TCF3-ZNF384-positive patients revealed a significantly poorer steroid response and a higher frequency of relapse, and the additional activating mutations in RAS signaling pathway genes were detected by whole exome analysis in some of the cases. Our observations indicate that ZNF384-related fusion genes consist of a distinct subgroup of B-cell precursor acute lymphoblastic leukemia with a characteristic immunophenotype, while the clinical features depend on the functional properties of individual fusion partners.

Population pharmacokinetics of thrombomodulin alfa in pediatric patients with hematological malignancy and disseminated intravascular coagulation.

BACKGROUND: Thrombomodulin alfa (TM-α) is a new class of anticoagulant drug for patients with disseminated intravascular coagulation (DIC). This study aimed to determine the pharmacokinetics of TM-α and determine the optimal dose in pediatric patients with hematological malignancy and DIC. PROCEDURE: Pediatric patients with hematological malignancy and DIC were administered TM-α at a dose of 0.06 mg/kg (380 U/kg) over 30 min every 24 hr. Blood samples were taken at steady state before the start, immediately after the end, and 24 hr after the start of the sixth administration. Population pharmacokinetic analysis was performed using sparse samples with the nonlinear mixed-effect modeling program NONMEM® , version 7.3. RESULTS: The actual and predicted plasma concentrations of TM-α based on the final population pharmacokinetic model showed a good linear correlation. Clearance and volume of distribution of TM-α were affected by body weight. The clearance of TM-α in pediatric patients with hematological malignancy and DIC was higher than that in adults as previously reported. Six of eight patients did not achieve the target trough concentration at steady state. Furthermore, the pharmacokinetic simulation based on the estimated pharmacokinetic parameters from the final model demonstrated that TM-α administered at a dose of 0.06 mg/kg every 24 hr also failed to achieve the target trough concentration at steady state in the majority of pediatric patients. CONCLUSIONS: Our study shows that further dose adjustment of TM-α is necessary considering the higher clearance per body weight in pediatric patients with hematological malignancy and DIC.

Flow cytometric analysis as an additional predictive tool of treatment response in children with chronic-phase chronic myeloid leukemia treated with imatinib.

Bone marrow samples of newly diagnosed children with chronic-phase chronic myeloid leukemia (CML) were obtained at diagnosis and after imatinib initiation and stained with anti-human CD34, CD38, CD123, CD45RA, cMpl, and lineage antibodies. Flow cytometric analysis revealed that granulocyte macrophage progenitor predominance in CML progenitors at diagnosis and elevated cMpl expression in bone marrow progenitors at 3 months may predict poor outcome in children with chronic-phase CML treated with imatinib. We recommend flow cytometric analysis of bone marrow in the early phase of treatment, as it is a convenient tool that may predict treatment response and guide CML management.

The Potential and Limits of Hematopoietic Stem Cell Transplantation for the Treatment of Autosomal Dominant Hyper-IgE Syndrome.

PURPOSE: Autosomal dominant hyper-IgE syndrome (AD-HIES) is included among primary immunodeficiencies, and results from heterozygous mutations in the signal transduction and activator of transcription 3 (STAT3) gene. AD-HIES leads to impaired Th17 cell differentiation and IL-17 production, and is associated with increased susceptibility to bacteria and fungi. It was reported that several patients with AD-HIES were treated with hematopoietic stem cell transplantation (HSCT). The efficacy of HSCT in treating AD-HIES is variable. This study aims to evaluate the long-term clinical and immunological efficacy of HSCT for AD-HIES. METHODS: We have followed for more than 8 years two patients with AD-HIES who were treated with HSCT. Their ability of IL-17 production was evaluated by flow cytometry. RESULTS: Both patients indicated the normal ability of IL-17 production and their serum IgE levels decreased after HSCT. On the other hand, they suffered from pulmonary complications of AD-HIES such as pneumatoceles and bronchiectasis even after HSCT; however, the frequency of infections was decreased. CONCLUSIONS: Although the dysfunction of STAT3 in non-hematological tissues such as the lungs could not be corrected by HSCT, AD-HIES patients with risk factors for pulmonary complications may benefit from immunological correction by HSCT before severe pulmonary complications occur. Future studies should investigate risk factors for pulmonary complications in AD-HIES patients.

Leukostasis in Children and Adolescents with Chronic Myeloid Leukemia: Japanese Pediatric Leukemia/Lymphoma Study Group.

BACKGROUND: The details of leukostasis in children and adolescents with chronic myeloid leukemia (CML) are unknown. This study determined the characteristics of leukostasis in children and adolescents with CML. PROCEDURE: A total of 256 cases from a retrospective study of patients with CML conducted by the Japanese Pediatric Leukemia/Lymphoma Study Group from 1996 to 2011 were analyzed, and of these, 238 cases were evaluated in this study. RESULTS: Leukostasis was diagnosed in 23 patients (9.7%). The median leukocyte count and spleen size below the left costal margin in cases with leukostasis were significantly higher and larger when compared to those in cases without leukostasis (458.5 × 10(9) /l vs. 151.8 × 10(9) /l (P < 0.01), and 13 vs. 5 cm (P < 0.01), respectively). Leukostasis occurred with ocular symptoms in 14 cases, priapism in four cases, and dyspnea, syncope, headache, knee pain, difficulty hearing, and aseptic necrosis of the femoral head in one case each. One case had two leukostasis symptoms simultaneously. Three cases were diagnosed before imatinib became available. Five cases received special treatment, and in the remaining 15 cases, all of these symptoms resolved after treatment with imatinib. CONCLUSIONS: This retrospective study represents the largest series of children and adolescents in which leukostasis of CML has been reported. Our data provide useful insight into the characteristics of leukostasis in recent cases of children and adolescents with CML.

Role of Second Transplantation for Children With Acute Myeloid Leukemia Following Posttransplantation Relapse.

BACKGROUND: In children with acute myeloid leukemia (AML), hematopoietic stem cell transplantation (HSCT) in first remission is indicated for patients with a relatively high risk of relapse. Second HSCT is a curative option; however, few reports have been published about a second HSCT in children for AML with posttransplantation relapse. PROCEDURE: Using the database provided by the Japanese Society of Hematopoietic Cell Transplantation, we analyzed 46 children with AML who underwent a second allogeneic HSCT after achieving a second remission. RESULTS: The median duration from the first to second HSCT was 20 months, and the source of the second HSCT was related bone marrow (BM) in 22, related peripheral blood in 6, unrelated BM in 14, and unrelated cord blood in 4 patients. Twenty-five children eventually died of the following causes: progressive disease in 14 and transplant-related toxicities in 9. The 5-year overall survival rate was 41.7 ± 7.7%. An interval of less than 24 months between the first and second HSCT was a significant poor prognostic factor. CONCLUSIONS: Children with AML who experience a relapse after HSCT in first remission have a good chance of survival with a second HSCT if a second remission is achieved.

Comparison of second transplantation and donor lymphocyte infusion for donor mixed chimerism after allogeneic stem cell transplantation for nonmalignant diseases.

BACKGROUND: Donor mixed chimerism (MC) is an increasing problem after hematopoietic stem cell transplantation (HSCT) for nonmalignant diseases. PROCEDURE: In this study, a self-administered questionnaire was used to retrospectively compare efficacy and safety in 49 patients undergoing second HSCT (n = 13) or donor lymphocyte infusion (DLI; n = 36) as treatment for MC. RESULTS: The response rate to DLI of patients with secondary graft failure (GF) (25.0%) was significantly lower than that of patients without secondary GF (81.3%; P = 0.041). Among patients undergoing DLI, the rates of successful response were significantly higher in patients having at least 30% donor chimerism (94.1%) than in patients having less than 30% donor chimerism (61.1%; P = 0.041). Furthermore, the rates of successful response were significantly higher in patients receiving larger first or maximum doses of DLI. Sixteen (50.0%) of 32 patients without secondary GF attained complete chimerism after DLI. The cumulative incidence of grade II-IV acute graft-versus-host disease and cytopenia was 37.6 and 26.1%, respectively. CONCLUSIONS: DLI yields promising response rates in most patients with higher donor chimerism levels, whereas second HSCT is more likely to benefit patients with lower donor chimerism levels.

Allogeneic hematopoietic stem cell transplantation for Chediak-Higashi syndrome.

The clinical outcome of allogeneic HSCT was retrospectively analyzed in eight patients with CHS. In total, six of these eight patients are alive. Four of five patients transplanted with MAC achieved prompt engraftment, and three of the four patients, including two patients with AP before transplant, are alive without disease. In contrast, three patients transplanted with RIC without active AP disease achieved prompt engraftment and survive long term. RIC-HSCT might be an alternative treatment for CHS similar to other types of HLH, at least for patients without active AP disease.

Inhibition of cathepsin S reduces allogeneic T cell priming but not graft-versus-host disease against minor histocompatibility antigens.

Cathepsin (Cathepsin) S, L, and B proteases mediate antigen presentation on major histocompatibility complex (MHC) class II by degrading the invariant chain Ii, which blocks peptide loading. The ability of the Cathepsin S inhibitor LHVS (morpholinurea-leucine-homophenylalanine-vinylsulfone phenyl) to impede antigen presentation has led its development as a therapy for autoimmune diseases. There is substantial evidence that donor T cell recognition of host minor histocompatibility antigens (miHA) and subsequent destruction of host tissue mediates graft-versus-host disease (GVHD). We hypothesized that enzymes involved in antigen presentation may play a role in the development of GVHD. Using the C57BL/6 → BALB.B minor mismatch acute GVHD (aGVHD) model, we found that the cathepsin S activity of spleens from allogenetically transplanted mice were significantly increased 1 week after transplantation compared with syngeneic mice. Although LHVS decreased T cell priming responses against both single OVA antigen and miHA in vitro, LHVS did not reduce the severity of aGVHD. In fact, LHVS exacerbated a CD4(+)-T cell-dependent model of GVHD similar to chronic GVHD. This suggests that cytokines rather than T cells may mediate much of the damage in the aGVHD model and that therapeutics based on inhibition of antigen presentation for GVHD must be approached with caution.

Methylated chrysin reduced cell proliferation, but antagonized cytotoxicity of other anticancer drugs in acute lymphoblastic leukemia.

The efficacy of 5,7-dimethoxyflavone (DMF), a methylated analog of chrysin, as a therapeutic agent to treat acute lymphoblastic leukemia (ALL) was investigated. Using a panel of ALL cell lines, the IC50 (half-maximal inhibitory concentration) of DMF varied between 2.8 and 7.0 µg/ml. DMF induced G0/G1 cell cycle arrest, concomitant with a decreased expression of phosphorylated retinoblastoma-associated protein 1. DMF increased the rate of apoptosis, although it was apparent only after a long period of exposure (96 h). The accumulation of oxidative stress was not involved in the growth-inhibitory effects of DMF. As DMF reduced the intracellular levels of glutathione, the combination effects of DMF with other anticancer drugs were evaluated using the improved Isobologram and the combination index method. In the simultaneous drug combination assay, DMF antagonized the cytotoxicity of 4-hydroperoxy-cyclophosphamide, cytarabine, vincristine, and L-asparaginase in all tested ALL cells. This study demonstrated that DMF, a methylated flavone, was an effective chemotherapy agent that could inhibit cell cycle arrest and induce apoptosis in ALL cell lines. However, combination therapy with DMF and other anticancer drugs is not recommended.

Influence of CYP3A5 and ABCB1 gene polymorphisms on calcineurin inhibitor-related neurotoxicity after hematopoietic stem cell transplantation.

BACKGROUND: One severe side effect of calcineurin inhibitors (CNIs: such as cyclosporine [CsA] and tacrolimus [FK506]) is neurotoxicity. CNIs are substrates for CYP3A5 and P-glycoprotein (P-gp), encoded by ABCB1 gene. In the present study, we hypothesized that genetic variability in CYP3A5 and ABCB1 genes may be associated with CNI-related neurotoxicity. METHODS: The effects of the polymorphisms, such as CYP3A5 A6986G, ABCB1 C1236T, G2677T/A, and C3435T, associated with CNI-related neurotoxicity were evaluated in 63 patients with hematopoietic stem cell transplantation. RESULTS: Of the 63 cases, 15 cases developed CNI-related neurotoxicity. In the CsA patient group (n = 30), age (p = 0.008), hypertension (p = 0.017), renal dysfunction (p < 0.001), ABCB1 C1236T (p < 0.001), and G2677T/A (p = 0.014) were associated with neurotoxicities. The CC genotype at ABCB1 C1236T was associated with it, but not significantly so (p = 0.07), adjusted for age, hypertension, and renal dysfunction. In the FK506 patient group (n = 33), CYP3A5 A6986G (p < 0.001), and ABCB1 C1236T (p = 0.002) were associated with neurotoxicity. At least one A allele at CYP3A5 A6986G (expressor genotype) was strongly associated with it according to logistic regression analysis (p = 0.01; OR, 8.5; 95% CI, 1.4-51.4). CONCLUSION: The polymorphisms in CYP3A5 and ABCB1 genes were associated with CNI-related neurotoxicity. This outcome is probably because of CYP3A5 or P-gp functions or metabolites of CNIs.

Graft versus leukemia effect against juvenile myelomonocytic leukemia after unrelated cord blood transplantation.

A 13-month-old female underwent unrelated cord blood transplantation (CBT) for juvenile myelomonocytic leukemia (JMML). In spite of progression of the disease after a conditioning regimen with high-dose chemotherapy, a complete remission was induced in concordance with development of acute GVHD after reduction of the immunosupressant. She has been in complete remission for 1 year after transplantation. This case illustrates that CBT can provide a potent graft versus leukemia (GVL) effect against JMML.

[Cutaneous non-tuberculous mycobacterial infection following cord blood stem cell transplantation].

We describe a 4-year-old-girl with familial hemophagocytic lymphohistiocytosis (FHL) who developed disseminated cutaneous nontuberculous mycobacterial (NTM) infection after unrelated cord blood stem cell transplantation (uCBSCT). After transplantation, the patient developed steroid-refractory acute graft-versus-host disease, and was given methylprednisolone, cyclosporin and mycophenolate mofetil. Six months after uCBSCT, cutaneous lesions that looked like insect bites appeared and spread widely over the thighs. NTM infection was diagnosed by skin biopsy although no organism could be identified. Minocycline (MINO) and sulfamethoxazole/trimethoprim (ST) were administered. However, the cutaneous disease followed a course of remissions and exacerbations. One month after the skin biopsy, mycobacterium chelonae was detected by bacteriological culture of abscess drainage. Ten months after uCBSCT, the cutaneous lesions quickly progressed and the inguinal lymph nodes became enlarged and painful. Then the antibiotics were switched from MINO and ST to amikacin and clarithromycin (CAM) based on the results of mycobacterial susceptibility test. The cutaneous lesions gradually improved after continuous administration of CAM. Cutaneous NTM infection is rare, but it may occur in immunocompromised patients after SCT.

Association of isochromosome (7)(q10) in Shwachman-Diamond syndrome with the severity of cytopenia.

We report two male siblings with SDS. They have the same compound heterozygous mutations. Only one of the siblings acquired cytogenetic abnormality of i(7q) 2 years after diagnosis, became transfusion-dependent, and underwent allogeneic hematopoietic stem cell transplantation. These cases indicate that i(7q) is associated with significant cytopenia in SDS patients.

Treatment outcome of children with acute lymphoblastic leukemia: the Tokyo Children's Cancer Study Group (TCCSG) Study L04-16.

The survival rate of children with acute lymphoblastic leukemia (ALL) has increased to approximately 90% after substantial progress in risk-oriented treatment strategies. Between 2005 and 2013, the Tokyo Children's Cancer Study Group (TCCSG) conducted a risk-oriented, non-randomized study, L04-16. The principal aim of this study was to assemble background characteristics and treatment outcomes, and gather genetic information on leukemic cells under central diagnosis. This report outlines the background characteristics and treatment outcomes of 1033 children with ALL treated according to a TCCSG platform. The 5-year event-free and overall survival (OS) rates for all children were 78.1 ± 1.3 and 89.6 ± 1.0%, respectively. The OS rate was significantly higher in children with B-cell precursor (BCP)-ALL (91.9 ± 1.0%, n = 916) than in those with T-ALL (71.9 ± 4.3%, n = 117, p < 0.001). In univariate analysis for BCP-ALL, children aged 1-6 years (5y-OS: 94.2 ± 1.0%), with an initial white blood cell count of < 20,000/µL (94.0 ± 1.0%), high hyperdiploidy (95.4 ± 1.6%), ETV6-RUNX1 (97.4 ± 1.2%) or TCF3-PBX1 (96.9 ± 2.1%), and "Day8NoBlasts" (96.4 ± 1.1%) had the best outcomes. Genetic investigation revealed two novel fusion genes within this cohort: ETV6-ZNF385A and ZNF362-TCF4. Our study highlighted the clinical aspects of genomic features of ALL in Japanese children. We provide fundamental information for the further molecular investigation of this disease.

Lack of mitochondrial depolarization by oxidative stress is associated with resistance to buthionine sulfoximine in acute lymphoblastic leukemia cells.

Raised intracellular glutathione is one characteristics of high-risk childhood acute lymphoblastic leukemia (ALL). Depletion of glutathione by buthionine sulfoximine (BSO) has been reported to be toxic against some cancer cells. To assess the role of glutathione in ALL, the toxicity of BSO was studied in B-precursor ALL cell lines. BSO increased oxidative stress equally in all cell lines; however mitochondrial depolarization was observed only in BSO-sensitive cells. BSO up-regulated Bcl-2 protein, and antagonized the anti-ALL effect of prednisolone in BSO-resistant cells. A lack of mitochondrial death-signal activation by oxidative stress seemed to be associated with BSO-resistance in ALL.

[Reactivation of familial hemophagocytic lymphohistiocytosis after non-myeloablative cord blood stem cell transplantation resulted in the early graft failure].

A 4-year-old girl with familial hemophagocytic lymphohistiocytosis (FHL) underwent unrelated cord blood stem cell transplantation (uCBSCT) using the fludarabine-based non-myeloablative conditioning regimen. Shortly after the uCBSCT, she experienced the reactivation of the FHL, and subsequent graft failure. Considering that the residual recipient lymphocytes, which survived after non-myeloablative conditioning, had a role in the FHL reactivation and in the graft rejection, myeloablative conditioning combined with horse ATG was used to eradicate the recipient lymphocytes in the second CBSCT. Furthermore, in order to prevent rejection, cord blood, the DNA type of which was completely matched in a host to graft (HVG) direction, was selected. In the second uCBSCT, FHL reactivation was noted at a limited level. Complete chimerism was achieved and the NK-cell activity recovered to the normal range. Our case suggests the necessity of the conditioning regimen to eradicate host lymphocytes in the stem cell transplantation for FHL, and the significance of HLA DNA-typing in uCBSCT for pediatric patients.