Nobuhiro Nishio

Mutations of an E3 ubiquitin ligase c-Cbl but not TET2 mutations are pathogenic in juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) is a rare pediatric myeloid neoplasm characterized by excessive proliferation of myelomonocytic cells. When we investigated the presence of recurrent molecular lesions in a cohort of 49 children with JMML, neurofibromatosis phenotype (and thereby NF1 mutation) was present in 2 patients (4%), whereas previously described PTPN11, NRAS, and KRAS mutations were found in 53%, 4%, and 2% of cases, respectively. Consequently, a significant proportion of JMML patients without identifiable pathogenesis prompted our search for other molecular defects. When we applied single nucleotide polymorphism arrays to JMML patients, somatic uniparental disomy 11q was detected in 4 of 49 patients; all of these cases harbored RING finger domain c-Cbl mutations. In total, c-Cbl mutations were detected in 5 (10%) of 49 patients. No mutations were identified in Cbl-b and TET2. c-Cbl and RAS pathway mutations were mutually exclusive. Comparison of clinical phenotypes showed earlier presentation and lower hemoglobin F levels in patients with c-Cbl mutations. Our results indicate that mutations in c-Cbl may represent key molecular lesions in JMML patients without RAS/PTPN11 lesions, suggesting analogous pathogenesis to those observed in chronic myelomonocytic leukemia (CMML) patients.

Armed Oncolytic Virus Enhances Immune Functions of Chimeric Antigen Receptor–Modified T Cells in Solid Tumors

The clinical efficacy of chimeric antigen receptor (CAR)-redirected T cells remains marginal in solid tumors compared with leukemias. Failures have been attributed to insufficient T-cell migration and to the highly immunosuppressive milieu of solid tumors. To overcome these obstacles, we have combined CAR-T cells with an oncolytic virus armed with the chemokine RANTES and the cytokine IL15, reasoning that the modified oncolytic virus will both have a direct lytic effect on infected malignant cells and facilitate migration and survival of CAR-T cells. Using neuroblastoma as a tumor model, we found that the adenovirus Ad5Δ24 exerted a potent, dose-dependent, cytotoxic effect on tumor cells, whereas CAR-T cells specific for the tumor antigen GD2 (GD2.CAR-T cells) were not damaged. When used in combination, Ad5Δ24 directly accelerated the caspase pathways in tumor cells exposed to CAR-T cells, whereas the intratumoral release of both RANTES and IL15 attracted CAR-T cells and promoted their local survival, respectively, increasing the overall survival of tumor-bearing mice. These preclinical data support the use of this innovative biologic platform of immunotherapy for solid tumors. Cancer Res; 74(18); 5195-205. ©2014 AACR.

Relapse of leukemia with loss of mismatched HLA resulting from uniparental disomy after haploidentical hematopoietic stem cell transplantation.

We investigated human leukocyte antigen (HLA) expression on leukemic cells derived from patients at diagnosis and relapse after hematopoietic stem cell transplantation (HSCT) using flow cytometry with locus-specific antibodies. Two of 3 patients who relapsed after HLA-haploidentical HSCT demonstrated loss of HLA alleles in leukemic cells at relapse; on the other hand, no loss of HLA alleles was seen in 6 patients who relapsed after HLA-identical HSCT. Single-nucleotide polymorphism array analyses of sorted leukemic cells further revealed the copy number-neutral loss of heterozygosity, namely, acquired uniparental disomy on the short arm of chromosome 6, resulting in the total loss of the mismatched HLA haplotype. These results suggest that the escape from immunosurveillance by the loss of mismatched HLA alleles may be a crucial mechanism of relapse after HLA-haploidentical HSCT. Accordingly, the status of mismatched HLA on relapsed leukemic cells should be checked before donor lymphocyte infusion.

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.

Spectrum of molecular defects in juvenile myelomonocytic leukaemia includes ASXL1 mutations

Mutations in NF1, PTPN11, NRAS, KRAS and CBL have been reported to play a pathogenetic role in juvenile myelomonocytic leukaemia (JMML), a rare myelodyplastic/myeloproliferative neoplasm occurring in children. Recently, mutations in ASXL1 were identified in chronic myelomonocytic leukaemia and other myeloid malignancies. We sequenced exon 12 of ASLX1 in 49 JMML patients, and found 2 novel heterozygous (nonsense and frameshift) mutations, one occurring as a sole lesion, the other was in conjunction with a PTPN11 mutation. ASXL1 cooperates with KDM1A in transcriptional repression and thereby ASXL1 mutations may synergize with or mimic other JMML‐related mutations.

Correlation of Clinical Features With the Mutational Status of GM-CSF Signaling Pathway-Related Genes in Juvenile Myelomonocytic Leukemia

Mutations in RAS, neurofibromatosis type 1 (NF1), and PTPN11, constituents of the granulocyte-macrophage colony-stimulating factor signaling pathway, have been recognized in patients with juvenile myelomonocytic leukemia (JMML). We assessed 71 children with JMML for NRAS, KRAS, and PTPN11 mutations and evaluated their clinical significance. Of the 71 patients, three had been clinically diagnosed with neurofibromatosis type 1, and PTPN11 and NRAS/KRAS mutations were found in 32 (45%) and 13 (18%) patients, respectively. No simultaneous aberrations were found. Compared with patients with RAS mutation or without any aberrations, patients with PTPN11 mutation were significantly older at diagnosis and had higher fetal Hb levels, both of which have been recognized as poor prognostic factors. As was expected, overall survival was lower for patients with the PTPN11 mutation than for those without (25 versus 64%; p = 0.0029). In an analysis of 48 patients who received hematopoietic stem cell transplantation, PTPN11 mutations were also associated with poor prognosis for survival. Mutation in PTPN11 was the only unfavorable factor for relapse after hematopoietic stem cell transplantation (p = 0.001). All patients who died after relapse had PTPN11 mutation. These results suggest that JMML with PTPN11 mutation might be a distinct subgroup with specific clinical characteristics and poor outcome.

Relationship between coffee consumption and prevalence of metabolic syndrome among Japanese civil servants.

Background Metabolic syndrome has become a major worldwide public health problem. We examined the relationship between coffee consumption and the prevalence of metabolic syndrome among Japanese civil servants. Methods The study participants were 3284 employees (2335 men and 948 women) aged 20 to 65 years. Using data from their 2008 health checkup records, we analyzed the relationship between coffee consumption and the prevalence of metabolic syndrome. Metabolic syndrome was defined according to the Japanese criteria. Results Metabolic syndrome was diagnosed in 374 of the 2335 men (16.0%) and 32 of the 948 women (3.4%). In univariate and multiple logistic regression analyses, the odds ratios (ORs) among men for the presence of metabolic syndrome were 0.79 (95% CI: 0.56–1.03) and 0.61 (0.39–0.95), respectively, among moderate (≥4 cups of coffee per day) coffee drinkers as compared with non-coffee drinkers. Among all components of metabolic syndrome, high blood pressure and high triglyceride level were inversely associated with moderate coffee consumption in men, after adjusting for age, body mass index, smoking status, drinking status, and exercise. However, in women, moderate coffee consumption was not significantly associated with the prevalence of metabolic syndrome or its components. Conclusions Moderate coffee consumption was significantly associated with lower prevalence of metabolic syndrome in Japanese male civil servants.

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.

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.

Zoledronate sensitizes neuroblastoma-derived tumor-initiating cells to cytolysis mediated by human γδ T cells.

Neuroblastoma is the most common extracranial solid tumor in children that is refractory to intensive multimodal therapy. In particular, tumor-initiating cells (TICs) derived from neuroblastoma are believed responsible for tumor formation and resistance to the conventional therapy; an optimal strategy therefore should target this population. Technically, TICs can be enriched from neuroblastoma-derived spheres when the tumor cells are cultured in a serum-free medium supplemented with certain growth factors. Recently, a line of evidence has suggested antitumor potential of V&ggr;9V&dgr;2 T cells (&ggr;&dgr; T cells), a T-cell population that recognizes and kills target cells independent of surface HLA expressions. Furthermore, a mevalonate pathway inhibitor, zoledronate, has been reported to enhance cytolytic activity of &ggr;&dgr; T cells. On the basis of these findings, we hypothesized that zoledronate would sensitize neuroblastoma TICs to &ggr;&dgr; T-cell–mediated cytolysis and promote therapeutic efficacy against neuroblastoma. In the current study, we show that zoledronate efficiently sensitizes both neuroblastoma-derived adherent cells and sphere-forming cells to &ggr;&dgr; T-cell–mediated cytolysis. Subsequently, in vitro colony formation inhibition assay and in vivo animal studies reveal that the presence of &ggr;&dgr; T cells decelerates outgrowth of neuroblastoma TICs. We finally show that addition of interleukin-15 and/or interleukin-18 in culture enhances the cytolytic activity of &ggr;&dgr; T cells. On the basis of these data, we conclude that ex vivo expanded &ggr;&dgr; T cells are a promising tool for antineuroblastoma immunotherapy with options for further improvement.