Ryosei Nishimura

The landscape of somatic mutations in Down syndrome–related myeloid disorders

Transient abnormal myelopoiesis (TAM) is a myeloid proliferation resembling acute megakaryoblastic leukemia (AMKL), mostly affecting perinatal infants with Down syndrome. Although self-limiting in a majority of cases, TAM may evolve as non-self-limiting AMKL after spontaneous remission (DS-AMKL). Pathogenesis of these Down syndrome–related myeloid disorders is poorly understood, except for GATA1 mutations found in most cases. Here we report genomic profiling of 41 TAM, 49 DS-AMKL and 19 non-DS-AMKL samples, including whole-genome and/or whole-exome sequencing of 15 TAM and 14 DS-AMKL samples. TAM appears to be caused by a single GATA1 mutation and constitutive trisomy 21. Subsequent AMKL evolves from a pre-existing TAM clone through the acquisition of additional mutations, with major mutational targets including multiple cohesin components (53%), CTCF (20%), and EZH2, KANSL1 and other epigenetic regulators (45%), as well as common signaling pathways, such as the JAK family kinases, MPL, SH2B3 (LNK) and multiple RAS pathway genes (47%).

In vivo trafficking and survival of cytokine-induced killer cells resulting in minimal GVHD with retention of antitumor activity.

Cytokine-induced killer (CIK) cells are ex vivo-expanded T lymphocytes expressing both natural killer (NK)- and T-cell markers. CIK cells are cytotoxic against autologous and allogeneic tumors. We previously showed that adoptive transfer of allogeneic CIK cells in a murine model caused minimal graft-versus-host disease (GVHD). However, the precise mechanism of reduced GVHD is not fully understood. Therefore, we evaluated the trafficking and survival of luciferase-expressing CIK cells in an allogeneic bone marrow transplant model. The initial trafficking patterns of CIK cells were similar to conventional T cells that induced GVHD; however, CIK cells infiltrated GVHD target tissues much less and transiently. CIK cells accumulated and persisted in tumor sites, resulting in tumor eradication. We evaluated different properties of CIK cells compared with conventional T cells, demonstrating a slower division rate of CIK cells, higher susceptibility to apoptosis, persistent increased expression of interferon gamma (IFN-gamma), and reduced acquisition of homing molecules required for entry of cells into inflamed GVHD target organs that lack expression of NKG2D ligands recognized by CIK cells. Due to these properties, allogeneic CIK cells had reduced expansion and caused less tissue damage. We conclude that CIK cells have the potential to separate graft-versus-tumor effects from GVHD.

A functional single-nucleotide polymorphism in the human cytidine deaminase gene contributing to ara-C sensitivity.

To test the hypothesis that analyses of drug targets for polymorphism will help to establish gene-based information for the treatment of cancer patients, we investigated the functional single-nucleotide polymorphisms in the human cytidine deaminase (HDCA) gene. The cDNAs from 52 leukaemia/lymphoma samples and 169 control blood samples were direct-sequenced and analysed for the polymorphisms. Three different polymorphisms (A79C, G208A and T435C) were identified in the coding region of the HDCA gene and displayed allelic frequencies of 20.1%, 4.3% and 70.1%, respectively. No association with susceptibility to disease was observed. A novel polymorphism, G208A produced an alanine to threonine substitution (A70T) within the conserved catalytic domain. By introduction of the polymorphic HCDA genes into the yeast CDA-null mutants, the HCDA-70T showed 40% and 32% activity of prototype for cytidine and ara-C substrates, respectively (P < 0.01). The ara-C IC50 value of the yeast transformants carrying HCDA-70T was 757 +/- 33 micromol and was significantly lower (P < 0.01) than that of prototype (941 +/- 58 micromol). This study demonstrated a population characterized with 208A genotype for, which potentially leads one more sensitive to ara-C treatment than prototype. Accumulation of polymorphisms in the genes responsible for drug metabolism and determination of polymorphism-induced biological variations could provide the additional therapeutic strategies in risk-stratified protocols for the treatment of childhood malignancies.

Selectively induced high MRP gene expression in multidrug-resistant human HL60 leukemia cells

A subclone HL60/DOX was selected from a human leukemic HL60 cell line for resistance to doxorubicin (DOX) by exposure to stepwise increasing concentrations of the drug and coexposure to a potential P-glycoprotein (P-gp) inhibitor, cepharanthine (a biscoclaurine alkaloid). Compared with the parent HL60 cells, the HL60/DOX cells were 13.0-fold more resistant to DOX and showed multidrug-resistant (MDR) phenotype characterized by 4.6-fold, 2.3-fold, and 5.7-fold cross-resistance to vincristine, pirarubicin, and etoposide, respectively, but no cross-resistance to alkylating agent, cisplatin. Immunocytochemical analyses using the specific monoclonal antibody, MRPr1, and quantitative analyses using a competitive reverse transcription-polymerase chain reaction (CRT-PCR) confirmed overexpression of MRP gene products (about 8-fold determined by CRT-PCR) in this resistant clone. The P-gp expression was not detectable by the monoclonal antibody, C219, in the HL60/DOX cells, and that was consistent with extremely low levels of mdr1 mRNA expression determined by CRT-PCR in this clone. Drug accumulation and efflux studies demonstrated the significantly increased efflux rate of DOX compared to the parent HL60 cells. This enhancement of DOX efflux was reversed by the addition of 10 microM verapamil. To investigate the additional underlying mechanisms contributing to MDR phenotype in the HL60/DOX cells, the levels of DNA topoisomerases (Topo) including Topo I, Topo IIalpha, and Topo IIbeta, and gamma-glutamylcystein synthetase (y-GCS) expression were determined using CRT-PCR techniques. Normal expression of each enzyme at the transcriptional level was demonstrated in this resistant clone. Southern blot analysis of the gene organization in the HL60/DOX cells revealed the amplification of MRP gene. These results indicate that alteration of the drug accumulation from enhanced efflux appears to be a major mechanism(s) of MDR phenotype and attributable to high levels of MRP expression in the HL60/DOX cells. Overexpression of MRP in this clone is regulated by the genomic amplification of DNA and increased levels of the MRP mRNA, independently with the normal expression of Topo I, Topo IIalpha, Topo IIbeta, or gamma-GCS.

Reduced-intensity conditioning in unrelated donor cord blood transplantation for familial hemophagocytic lymphohistiocytosis†

Familial hemophagocytic lymphohistiocytosis (FHL) is a disorder of immune homeostasis characterized by fever, cytopenias, hepatosplenomegaly, and coagulopathy. We studied the outcomes of 13 FHL patients who underwent the first unrelated cord blood transplantation (UCBT) after non‐myeloablative conditionings. The major regimen consisted of fludarabine (FLU; n = 12)+melphalan (MEL; n = 11)±low‐dose total body irradiation (TBI 2‐4 Gy; n = 6). The median age at presentation and period to UCBT were 6 and 5 months, respectively. Central nervous system (CNS) disease developed in one infant at diagnosis, and in two others until UCBT. HLH activity was controlled in all but one at the time of UCBT. Ten patients had early engraftment on median day 21 with no grade >2 treatment‐related toxicity and two controllable grade >2 acute GVHD. Two patients with early rejection successfully underwent subsequent UCBT after myeloablative conditioning. Two others had late graft failure following mixed donor chimerism. Two deaths occurred from HLH; early liver failure and late CNS disease. Of 11 FLU+MEL‐conditioned patients, the frequency of disease‐free complete engraftment was higher for MEL (≥120 mg/m2)+TBI, or high‐dose MEL (180 mg/m2) than for others (83% vs. 25%, p = 0.036). The FLU+MEL‐based non‐myeloablative regimen was acceptable for FHL infants undergoing UCBT, although further studies will be needed for confirmation. Am. J. Hematol. 87:637–639, 2012.

Treatment of primary central nervous system lymphoma with induction of complement-dependent cytotoxicity by intraventricular administration of autologous-serum-supplemented rituximab

We describe an immunocompetent 19‐year‐old man with CD20‐positive primary central nervous system (CNS) lymphoma refractory to chemotherapy and irradiation. After intraventricular administration of rituximab, a chimeric anti‐CD20 monoclonal antibody, supplemented with autologous serum, a remarkable response developed to the CNS parenchymal lymphoma. Cytotoxicity assays showed that untreated patients serum with rituximab, but not that of heat‐inactivated patients serum with rituximab or rituximab alone, induced potent rituximab‐mediated cytotoxicity against tumor cells in the patients cerebrospinal fluid, suggesting induction of complement‐dependent cytotoxicity against CNS lymphoma. (Cancer Sci 2006; 97: 80 –83)

Down-regulation of CD5 expression on activated CD8+ T cells in familial hemophagocytic lymphohistiocytosis with perforin gene mutations

Hemophagocytic lymphohistiocytosis (HLH) is characterized by uncontrolled activation of T cells and macrophages with overproduction of cytokines. Familial HLH type 2 (FHL2) is the most common form of primary HLH and is caused by mutations in PRF1. We have recently described a significant increase in the subpopulation of CD8(+) T cells with clonal expansion and CD5 down-regulation in Epstein-Barr virus associated-HLH, which represented a valuable tool for its diagnosis. However, this unusual phenotype of CD8(+) T cells has not been investigated fully in patients with FHL2. We performed immunophenotypic analysis of peripheral blood and measured serum pro-inflammatory cytokines in five patients with FHL2. All patients showed significantly increased subpopulations of activated CD8(+) T cells with down-regulation of CD5, which were negligible among normal controls. Analysis of T-cell receptor Vβ repertoire suggested the reactive and oligoclonal expansion of these cells. The proportion of the subset declined after successful treatment concomitant with reduction in the serum levels of cytokines in all patients except one who continued to have a high proportion of the subset and died. These findings suggest that down-regulation of CD5 on activated CD8(+) T cells may serve as a useful marker of dysregulated T cell activation and proliferation in FHL2.

Stem cell transplantation for paediatric patients with non-anaplastic peripheral T-cell lymphoma in Japan

Reports of non‐anaplastic peripheral T‐cell lymphoma (PTCL) in paediatric patients, especially results of stem cell transplantation (SCT), are relatively rare. We herein report the results of SCT using the Transplant Registry Unified Management Program system of the Japanese Society of Stem Cell Transplantation in paediatric patients with non‐anaplastic PTCL. We analysed 26 patients (13 females and 13 males) aged ≤18 years with non‐anaplastic PTCL who underwent a total of 28 SCT. Median age at transplantation was 13·5 years (range: 0–18 years). PTCL not otherwise specified was diagnosed in 17 patients; extranodal Natural Killer (NK)/T cell lymphoma, nasal type in nine; and subcutaneous panniculitis‐like T‐cell lymphoma in two. Transplantation was with syngeneic donor in one, related donor in 10; unrelated donor in 10; and auto transplantation in 7. Five‐year overall survival rate and event‐free survival rate was 62·96% and 55·56%, respectively. Male gender, chronic graft‐versus‐host disease (GVHD), and reduced intensity conditioning were good prognostic factors in all patients. In 20 patients with refractory or relapsed disease, male gender and chronic GVHD were also good prognostic factors. This study is the first report concerning transplantation in children with non‐anaplastic PTCL, although the number of patients was small. Larger studies are needed to confirm these findings.

Microsatellite analysis of childhood leukemia: Correlation of 9p and 12p chromosome abnormalities with expression of related genes

Abstract Background: Loss of heterozygosity (LOH) and DNA replication error (RER) have been thought to be involved in carcinogenesis, but have not been investigated in childhood leukemia and lymphoma.

Massive hyper-reactive hematopoietic nests in bilateral iliac bones in a patient with mild aplastic anemia

To the Editor: We encountered a patient with aplastic anemia (AA) whose bone marrow (BM) specimens showed hypercellular marrow with slight dysplastic signs, because relatively massive hematopoietic nests were retained in limited areas, especially in BM biopsy sites. In this situation, it seemed to be hard to think of AA in a differential diagnosis. A 14-year-old male was found to have mild pancytopenia. His complete blood count showed a red blood cell count of 3.24 10/L, hemoglobin level of 11.0 g/dL, MCV of 96.9 fL, and a reticulocyte count of 38 10/L. White blood cell count was found to be 2.85 10/L with a differential count of 36% neutrophils, 58% lymphocytes, 1% eosinophils, 1%