Yasunori Ota

Gain‐of‐function mutations and copy number increases of Notch2 in diffuse large B‐cell lymphoma

Signaling through the Notch1 receptor has a pivotal role in early thymocyte development. Gain of Notch1 function results in the development of T‐cell acute lymphoblastic leukemia in a number of mouse experimental models, and activating Notch1 mutations deregulate Notch1 signaling in the majority of human T‐cell acute lymphoblastic leukemias. Notch2, another member of the Notch gene family, is preferentially expressed in mature B cells and is essential for marginal zone B‐cell generation. Here, we report that 5 of 63 (~8%) diffuse large B‐cell lymphomas, a subtype of mature B‐cell lymphomas, have Notch2 mutations. These mutations lead to partial or complete deletion of the proline‐, glutamic acid‐, serine‐ and threonine‐rich (PEST) domain, or a single amino acid substitution at the C‐terminus of Notch2 protein. Furthermore, high‐density oligonucleotide microarray analysis revealed that some diffuse large B‐cell lymphoma cases also have increased copies of the mutated Notch2 allele. In the Notch activation‐sensitive luciferase reporter assay in vitro, mutant Notch2 receptors show increased activity compared with wild‐type Notch2. These findings implicate Notch2 gain‐of‐function mutations in the pathogenesis of a subset of B‐cell lymphomas, and suggest broader roles for Notch gene mutations in human cancers. (Cancer Sci 2009; 100: 920–926)

Identification of a novel fusion, SQSTM1-ALK, in ALK-positive large B-cell lymphoma

ALK-positive large B-cell lymphoma is a rare subtype of lymphoma, and most cases follow an aggressive clinical course with a poor prognosis. We examined an ALK-positive large B-cell lymphoma case showing an anti-ALK immunohistochemistry pattern distinct from those of 2 known ALK fusions, CLTC-ALK and NPM-ALK, for the presence of a novel ALK fusion; this led to the identification of SQSTM1-ALK. SQSTM1 is an ubiquitin binding protein that is associated with oxidative stress, cell signaling, and autophagy. We showed transforming activities of SQSTM1-ALK with a focus formation assay and an in vivo tumorigenicity assay using 3T3 fibroblasts infected with a recombinant retrovirus encoding SQSTM1-ALK. ALK-inhibitor therapies are promising for treating ALK-positive large B-cell lymphoma, especially for refractory cases. SQSTM1-ALK may be a rare fusion, but our data provide novel biological insights and serve as a key for the accurate diagnosis of this rare lymphoma.

High incidence of haemophagocytic syndrome following umbilical cord blood transplantation for adults

Umbilical cord blood transplantation (CBT) is widely accepted, but one critical issue for adult patients is a low engraftment rate, of which one cause is haemophagocytic syndrome (HPS). We aimed to identify the contribution of HPS to engraftment failure after CBT, following preparative regimens containing fludarabine phosphate, in 119 patients (median age, 55 years; range; 17–69 years) with haematological diseases. Graft‐versus‐host disease prophylaxis comprised continuous infusion of a calcineurin inhibitor with or without mycophenolate mofetil. Of the 119 patients, 20 developed HPS within a median of 15 d (cumulative incidence; 16·8%) and 17 of them did so before engraftment. Donor‐dominant chimaerism was confirmed in 16 of 18 evaluable patients with HPS. Despite aggressive interventions including corticosteroid, ciclosporin, high‐dose immunoglobulin and/or etoposide, engraftment failed in 14 of 18 patients. Of these 14 patients, four received second rescue transplantation and all resulted in successful engraftment. Overall survival rates significantly differed between patients with and without HPS (15·0% vs. 35·4%; P < 0·01). Univariate and multivariate analysis identified having fewer infused CD34+ cells as a significant risk factor for the development of HPS (P = 0·01 and 0·006, respectively). We concluded that engraftment failure closely correlated with HPS in our cohort, which negatively impacted overall survival after CBT.

Interspecies organogenesis generates autologous functional islets

Islet transplantation is an established therapy for diabetes. We have previously shown that rat pancreata can be created from rat pluripotent stem cells (PSCs) in mice through interspecies blastocyst complementation. Although they were functional and composed of rat-derived cells, the resulting pancreata were of mouse size, rendering them insufficient for isolating the numbers of islets required to treat diabetes in a rat model. Here, by performing the reverse experiment, injecting mouse PSCs into Pdx-1-deficient rat blastocysts, we generated rat-sized pancreata composed of mouse-PSC-derived cells. Islets subsequently prepared from these mouse–rat chimaeric pancreata were transplanted into mice with streptozotocin-induced diabetes. The transplanted islets successfully normalized and maintained host blood glucose levels for over 370 days in the absence of immunosuppression (excluding the first 5 days after transplant). These data provide proof-of-principle evidence for the therapeutic potential of PSC-derived islets generated by blastocyst complementation in a xenogeneic host.

Depleting dietary valine permits nonmyeloablative mouse hematopoietic stem cell transplantation

Hematopoietic stem cell maintenance requires the amino acid valine. How to maintain hematopoietic stem cells Hematopoiesis provides the body with a continuous supply of blood cells (see the Perspective by Sommerkamp and Trumpp). Taya et al. report that amino acid content is important for hematopoietic stem cell (HSC) maintenance in vitro and in vivo. Dietary valine restriction seems to “empty” the mouse bone marrow niche. Ito et al. used single-cell approaches and cell transplantation to identify a subset of HSCs at the top of the HSC hierarchy. Self-renewal relied on the induction of mitophagy, a quality-control process linked to a cells metabolic state. Both studies may be helpful in improving clinical bone marrow transplantation. Science, this issue p. 1103, p. 1152; see also p. 1156 A specialized bone marrow microenvironment (niche) regulates hematopoietic stem cell (HSC) self-renewal and commitment. For successful donor-HSC engraftment, the niche must be emptied via myeloablative irradiation or chemotherapy. However, myeloablation can cause severe complications and even mortality. Here we report that the essential amino acid valine is indispensable for the proliferation and maintenance of HSCs. Both mouse and human HSCs failed to proliferate when cultured in valine-depleted conditions. In mice fed a valine-restricted diet, HSC frequency fell dramatically within 1 week. Furthermore, dietary valine restriction emptied the mouse bone marrow niche and afforded donor-HSC engraftment without chemoirradiative myeloablation. These findings indicate a critical role for valine in HSC maintenance and suggest that dietary valine restriction may reduce iatrogenic complications in HSC transplantation.

Hunner-Type (Classic) Interstitial Cystitis: A Distinct Inflammatory Disorder Characterized by Pancystitis, with Frequent Expansion of Clonal B-Cells and Epithelial Denudation

Interstitial cystitis (IC) is a chronic bladder disease with urinary frequency, bladder discomfort or bladder pain of unknown etiology. Based on cystoscopic findings, patients with IC are classified as either Hunner-type/classic IC (HIC), presenting with a specific Hunner lesion, or non-Hunner-type IC (NHIC), presenting with no Hunner lesion, but post-hydrodistension mucosal bleeding. Inflammatory cell infiltration, composed predominantly of lymphocytes, plasma cells and epithelial denudation, has in the past been documented as a major pathological IC finding. However, the significance of the pathological evaluation of IC, especially with regard to the difference between HIC and NHIC, has been downplayed in recent years. In this study, we performed immunohistochemical quantification of infiltrating T-lymphocytes, B-lymphocytes and plasma cells, and measured the amount of residual epithelium in urinary bladder biopsy specimens taken from patients with HIC and NHIC, and those with no IC, using image analysis software. In addition, in situ hybridization of the light chains was performed to examine clonal B-cell expansion. Lymphoplasmacytic infiltration was significantly more severe in HIC specimens than in NHIC specimens (P <0.0001). Substantial lymphoplasmacytic inflammation (≥200 cells/mm2) was observed in 93% of HIC specimens, whereas only 8% of NHIC specimens were inflamed. Plasmacytic infiltration was more prominent in HIC specimens compared with NHIC and non-IC cystitis specimens (P <0.005). Furthermore, expansion of light-chain-restricted B-cells was observed in 31% of cases of HIC. The amount of residual epithelium was decreased in HIC specimens compared with NHIC specimens and non-IC cystitis specimens (P <0.0001). These results suggest that NHIC and HIC are distinct pathological entities, with the latter characterized by pancystitis, frequent clonal B-cell expansion and epithelial denudation. An abnormality in the B-cell population may be involved in the pathogenesis of HIC.

Successful engraftment after reduced-intensity umbilical cord blood transplantation for myelofibrosis

Although allogeneic hematopoietic stem cell transplantation has recently been applied to patients with myelofibrosis with reproducible engraftment and resolution of marrow fibrosis, no data describe the outcomes of umbilical cord blood transplantation. We describe 14 patients with primary (n = 1) and secondary myelofibrosis (n = 13) who underwent reduced-intensity umbilical cord blood transplantation. Conditioning regimens included fludarabine and graft-versus-host disease prophylaxis composed cyclosporine/tacrolimus alone (n = 6) or a combination of tacrolimus and mycophenolate mofetil (n = 8). Thirteen patients achieved neutrophil engraftment at a median of 23 days. The cumulative incidence of neutrophil and platelet engraftment was 92.9% at day 60 and 42.9% at day 100, respectively. Posttransplantation chimerism analysis showed full donor type in all patients at a median of 14 days. The use of umbilical cord blood could be feasible even for patients with severe marrow fibrosis, from the viewpoint of donor cell engraftment.

A Safeguard System for Induced Pluripotent Stem Cell-Derived Rejuvenated T Cell Therapy

Summary The discovery of induced pluripotent stem cells (iPSCs) has created promising new avenues for therapies in regenerative medicine. However, the tumorigenic potential of undifferentiated iPSCs is a major safety concern for clinical translation. To address this issue, we demonstrated the efficacy of suicide gene therapy by introducing inducible caspase-9 (iC9) into iPSCs. Activation of iC9 with a specific chemical inducer of dimerization (CID) initiates a caspase cascade that eliminates iPSCs and tumors originated from iPSCs. We introduced this iC9/CID safeguard system into a previously reported iPSC-derived, rejuvenated cytotoxic T lymphocyte (rejCTL) therapy model and confirmed that we can generate rejCTLs from iPSCs expressing high levels of iC9 without disturbing antigen-specific killing activity. iC9-expressing rejCTLs exert antitumor effects in vivo. The system efficiently and safely induces apoptosis in these rejCTLs. These results unite to suggest that the iC9/CID safeguard system is a promising tool for future iPSC-mediated approaches to clinical therapy.

Physiological Srsf2 P95H expression causes impaired hematopoietic stem cell functions and aberrant RNA splicing in mice

Splicing factor mutations are characteristic of myelodysplastic syndromes (MDS) and related myeloid neoplasms and implicated in their pathogenesis, but their roles in the development of MDS have not been fully elucidated. In the present study, we investigated the consequence of mutant Srsf2 expression using newly generated Vav1-Cre-mediated conditional knockin mice. Mice carrying a heterozygous Srsf2 P95H mutation showed significantly reduced numbers of hematopoietic stem and progenitor cells (HSPCs) and differentiation defects both in the steady-state condition and transplantation settings. Srsf2-mutated hematopoietic stem cells (HSCs) showed impaired long-term reconstitution compared with control mice in competitive repopulation assays. Although the Srsf2 mutant mice did not develop MDS under the steady-state condition, when their stem cells were transplanted into lethally irradiated mice, the recipients developed anemia, leukopenia, and erythroid dysplasia, which suggests the role of replicative stress in the development of an MDS-like phenotype in Srsf2-mutated mice. RNA sequencing of the Srsf2-mutated HSPCs revealed a number of abnormal splicing events and differentially expressed genes, including several potential targets implicated in the pathogenesis of hematopoietic malignancies, such as Csf3r, Fyn, Gnas, Nsd1, Hnrnpa2b1, and Trp53bp1 Among the mutant Srsf2-associated splicing events, most commonly observed were the enhanced inclusion and/or exclusion of cassette exons, which were caused by the altered consensus motifs for the recognition of exonic splicing enhancers. Our findings suggest that the mutant Srsf2 leads to a compromised HSC function by causing abnormal RNA splicing and expression, contributing to the deregulated hematopoiesis that recapitulates the MDS phenotypes, possibly as a result of additional genetic and/or environmental insults.

Classification of AIDS-related lymphoma cases between 1987 and 2012 in Japan based on the WHO classification of lymphomas, fourth edition

The introduction of combined antiretroviral therapy (ART) has reduced the mortality of patients with human immunodeficiency virus‐1 infection worldwide. However, malignant lymphoma is a severe and frequent complication seen in patients with acquired immunodeficiency syndrome (AIDS). The diagnostic criteria for some categories of AIDS‐related lymphoma were revised in the World Health Organization International Classification of Lymphoma, fourth edition. The purpose of this study was to assess the clinicopathological characteristics of Japanese patients with AIDS‐related lymphoma according to the revised classification. In this retrospective study, 207 AIDS‐related lymphoma cases diagnosed between 1987 and 2012 in Japan were subjected to histological subtyping and clinicopathological analyses. Diffuse large B‐cell lymphoma (DLBCL) was the predominant histological subtype throughout the study period (n = 104, 50%). Among the DLBCL cases, 24% were of the germinal center (GC) type and 76% were of the non‐GC type. Non‐GC‐type cases showed a significantly lower 1‐year survival rate (43%) than the GC‐type cases (82%). Cases of Burkitt lymphoma (n = 57, 28%), plasmablastic lymphoma (n = 16, 8%), primary effusion lymphoma (n = 9, 4%), Hodgkin lymphoma (n = 8, 4%), and large B‐cell lymphoma arising in Kaposi sarcoma‐associated herpesvirus‐associated multicentric Castleman disease (n = 2, 1%) were also observed. Hodgkin lymphoma was more common in patients receiving ART (11.1%) than in ART‐naïve patients (1.4%). Statistical analyses identified CD10 negativity, BCL‐6 negativity, Epstein–Barr virus positivity, and Kaposi sarcoma‐associated herpesvirus positivity as risk factors for poor prognosis. This information will help in the early diagnosis of lymphoma in patients with AIDS.