A 56-year-old man was diagnosed with immunoglobulin (Ig) A-κ multiple myeloma in July 2007. After three courses of vincristine, adriamycin, and dexamethasone (VAD) chemotherapy, autologous peripheral blood stem cell transplantation was performed and achieved a very good partial response. In February 2010, an increase in the M-protein concentration and plasmacytoma in the L3/4 lumbar vertebrae were observed, and radiation treatment was performed. This was followed by administrations of bortezomib, lenalidomide, and thalidomide, none of which achieved a good response. In November 2011, the patient presented with obstructive jaundice, and imaging tests revealed tumorous lesions in the lower bile duct region and bilateral kidneys. Plasmacytoma was diagnosed from biopsy of the right renal mass. Radiotherapy to the common bile duct tumor resulted in jaundice amelioration, but the patient died despite subsequent treatment efforts. Autopsy revealed multiple extramedullary lesions in the abdominal cavity and in the region around the common bile duct. CD138 shedding was observed in the myeloma cells. We include a discussion of the literature on CD138 shedding and 38 reports of obstructive jaundice associated with extramedullary disease.

Twenty-nine transplant eligible newly diagnosed multiple myeloma (NDMM) patients have received Cyclophosphamide-Bortezomib-Dexamethasone (CyBorD) as induction treatment in our institute since November 2011. CyBorD is composed of CPA 300 mg/m2 p.o., Bor 1.3 mg/m2 i.v. or s.c., and Dex 40 mg/body p.o. on days 1, 8, 15, and 22. The median number of CyBorD cycles was 4 (range 2-6), except in one patient who progressed during the first cycle. Grade 4 neutropenia was observed in 2 patients, but none experienced grade 2 thrombocytopenia. Grade 3 non-hematologic adverse events were observed in two patients with varicella-zoster virus reactivation. Responses after CyBorD were ≥PR in 72%, ≥VGPR in 52%, ≥CR in 21%, and sCR in 21%. Autologous stem cells were harvested in 27 patients. Seventeen of these 27 patients received high-dose melphalan and autologous stem cell transplantation (ASCT) within 12 months after diagnosis. Patients with ≥CR increased to 59% after ASCT. Our data suggest the efficacy and the feasibility of administering CyBorD to transplant eligible NDMM patients.

Mutations in isocitrate dehydrogenase (IDH) 1 and 2 are frequently observed in acute myeloid leukemia (AML), glioma, and many other cancers. While wild-type IDHs mediate exchanges between isocitrate and α-ketoglutarate (α-KG), mutant IDHs convert α-KG to oncometabolite 2-hydroxyglutarate (2-HG), which causes dysregulation of a set of α-KG-dependent dioxygenases such as TET, histone demethylase and others. Because mutant IDH has no necessary functions in normal cells, inhibitors directed against mutant IDH are not expected to have the side effects as anti-cancer agents. To determine whether mutant IDH enzymes are valid targets for cancer therapy, we created a mouse model of mutant IDH2-dependent AML. By using a combination of AML model mice with cre-loxp, we conditionally deleted mutant IDH2 from AML mice, which resulted in the loss of leukemia stem cells and significantly delayed the progression of AML. These results indicate that mutant IDHs are promising targets for anticancer therapy.

Intravascular large B-cell lymphoma (IVLBCL) is a rare distinct disease entity of the extranodal large B-cell lymphoma type, characterized by the selective growth of tumor cells in the lumina of small vessels of various organs. Lack of remarkable lymphadenopathy and non-specific clinical abnormalities including fever of unknown origin and lactate dehydrogenase elevation generally make timely and accurate diagnosis difficult. Recent diagnostic advances in the detection of this disease using FDG-PET/CT and random skin biopsies are expected to increase the diagnostic yield. Regarding the therapeutic aspects of this disease, improvement of clinical outcomes by the application of anti-CD20 monoclonal antibody, rituximab, and a high risk of CNS recurrence have been indicated. Thus, a prospective phase II trial of immunochemotherapy combined with CNS prophylaxis is now ongoing. Previously, the difficulty of obtaining sufficient tumor samples hampered biological investigations but the novel technique developing xenograft models is opening the door to uncovering the underlying mechanisms by focusing on the fundamental biological question: "Why do tumor cells become lodged in the lumina of vessels?" This review describes the current understanding of research on IVLBCL and discusses the future prospects for this disease entity.

Genetic engineering for dividing and proliferating cells, such as iPS and mesenchymal stem cells, must avoid insertional carcinogenesis. A strategy utilizing the integration machinery of a non-pathogenic adeno-associated virus (AAV) that achieves site-specific insertion of its genome into the AAVS1 locus on chromosome 19 minimizes the risk of insertional mutagenesis. The AAVS1 site is also reportedly one of the safe harbors for transgene insertion. Any DNA linked with the inverted terminal repeat (ITR) or the p5 promoter sequence with co-expression of AAV Rep protein is easily targeted to the AAVS1 site. Large DNA sequences exceeding 100 kb can be inserted at this site. The author introduces the principle of this method and the recent applications for iPS and other cells.

Gene therapy targeting hematopoietic stem cells (HSC) can now be recognized as a curative treatment option for patients with genetic disorders, including primary immunodeficiency (PID) diseases. Despite an increasing number of successfully treated cases, the therapeutic benefits still vary considerably among trials. To further optimize HSC gene therapy, it is hoped that a research model system capable of faithful recapitulation of the disease phenotypes can be established. Recently, a new model system that may meet this goal has become a reality; that is, patient-derived induced pluripotent stem cells (iPSCs). iPSCs are useful for modeling genetic disorders, because of their potential to differentiate into various types of somatic cells while retaining the specific genetic mutations. They are also susceptible to genetic manipulation in vitro, thus enabling pre-clinical assessment of candidate treatment strategies for their performance. This article introduces a proof that patient-derived iPSCs represent an invaluable tool for modelling genetic diseases such as PIDs, and also provide an indispensable research model usable for the development of ideal therapeutic modalities.

Recent studies of hematopoietic stem and progenitor cells (HSPCs) have led to the development of new ways to detect and purify HSPCs and have also revealed several intrinsic and extrinsic factors that control the molecular signals fundamental to self-renewal and differentiation of HSPCs. These findings have provided new approaches for expanding HSPCs ex vivo utilizing protein factors and small-molecule compounds (SMCs) and have also demonstrated promising outcomes in clinical trials. Although further technical innovation is still needed, elucidation of the whole picture of signaling pathways critical to HSPCs and manipulation of such pathways by SMCs could establish efficient and robust methods for ex vivo expansion of HSPCs.

Aberrant activation of the Wnt, Notch and Hedgehog pathways via mutations or ligand overexpression has been implicated in a large number of cancer types where they are involved in functions ranging from tumor initiation to cancer stem cell (CSC) maintenance and angiogenesis. Agents targeting each one of these three pathways have now reached clinical trials, and the first one of these, Vismodegib, a hedgehog pathway inhibitor, was approved in 2012 by US FDA for the treatment of advanced basal cell carcinoma. Development of agents that target critical steps in these pathways as novel signal transduction pathways will be complicated by signaling cross-talk. The role that embryonic signaling pathways play in the function of CSCs, the development of new anti-CSC therapeutic agents, and the complexity of potential CSC signaling cross-talk are being explored coupled with early phase I clinical studies.

Improvement of cell purification and transplantation techniques have contributed to the identification of cell populations known as tumor-initiating cells (TICs). Although it was hypothesized that tumors are organized as hierarchies of tumor cells that are sustained by rare TICs, like normal tissue stem cells, there are several controversies towards such cancer stem cell model, e.g. reversible change of stem cell like population based on epigenetic changes, clonal genetic evolution and problems in xenotransplantation system. Despite complexity in cancer stem cell models, studies in cancer stem cell field have revealed that there are close relationship between cancer malignancy and stem cell properties, called "stemness". Understanding molecular mechanisms for controlling stemness would contribute to establishment of novel diagnostics or therapeutics for cancer.

Although allogeneic stem cell transplantation (allo-SCT) is a potentially curative treatment option for multiple myeloma (MM), it is not recognized as a standard of care because of the high associated incidences of both treatment related mortality and relapse. We administered lenalidomide (Len) as maintenance therapy for patients with MM undergoing allo-SCT who were at high risk of disease relapse. Graft-versus-host disease was induced by Len administration in two patients, but was manageable with dose reduction. Although Len has a direct anti-myeloma effect and can also induce tumor immunity against residual myeloma cells, it is important to identify how to optimize the safety and the effects of Len administration after allo-SCT. Further accumulation of data including those from prospective clinical trials is urgently needed.

In myeloid malignancies, mutations have occurred in epigenetic regulator genes, including Ten-Eleven-Translocation 2 (TET2). TET2 is an enzyme that catalyzes the conversion of 5-methylcytosine into 5-hydroxymethylcytosine (5-hmC) which is a key intermediate for oxidative DNA demethylation. We analyzed the in vivo phenotype of TET2 failure using Ayu17-449 (TET2(trap/trap)) mice created by the gene-trap method in which TET2 mRNA levels were decreased to about 20% of the level in wild-type (WT) mice. In TET2(trap/trap) mice the levels of 5-hmC in genomic DNA from bone marrow (BM) cells were decreased in comparison to WT mice. TET2(trap/trap) mice were born at an expected Mendelian frequency but died at a high rate by postnatal day 3, indicating TET2 to be essential for survival. In analysis of the hematopoietic system, transplantation of TET2(trap/trap), but not WT fetal liver cells, led to mild myeloid hyperplasia and splenomegaly in WT recipient mice, but no onsets of lethal hematological malignancies were observed during a follow-up period of 12 months. TET2 knockdown led to an increased serial replating capacity of BM cells in vitro and increased hematopoietic stem cell (HSC) self-renewal in vivo in competitive repopulation and serial transplantation assays. These data indicate that TET2 has a critical role in survival and HSC homeostasis.

TET (Ten Eleven Translocation) family proteins are dioxygenases that convert methylcytosine to hydroxymethylcytosine, and play an important role in the DNA demethylation process. Most notably, TET2 mutations have frequently been identified in myeloid malignancies, such as myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia, and acute myeloid leukemias, as well as angioimmunoblastic T-cell lymphoma and a proportion of peripheral T-cell lymphomas. To date, various types of Tet2 knockout/knockdown mice have been generated. Tet2 mutations induce enhanced self-renewal ability and competitive repopulation capacity in hematopoietic stem cells, and various MPN/MDS-like diseases and T-cell lymphoma consequently develop in model mice. These findings appear to have a strong correlation with the recently identified TET2 mutations in a significant proportion of healthy elderly people, and suggest that TET2 mutations lead to a pre-cancer state in hematopoietic stem/progenitor cells. In conclusion, TET2 might play a major role as a gate keeper for hematopoietic stem/progenitor cells preventing them from developing into various hematologic malignancies by acquiring additional disease-specific gene mutations.

Aggressive natural killer cell leukemia (ANKL) is a malignant disorder of mature NK cells that is relatively common in East Asia. It is associated with Epstein-Barr virus in more than 80% of patients. The median survival is not more than three months after diagnosis. In a recent survey of 34 cases with ANKL, the median patient age was 40 years and the age distribution followed a bimodal pattern. Morphologically, the ANKL cells varied from large granular lymphocytes to atypical cells with pleomorphic-like appearances. Clinical characteristics include fever, liver dysfunction, hemophagocytic syndrome, and a rapidly progressive course. In one third of patients, tumor cells in peripheral blood or the bone marrow are below 20% at initial presentation, which might lead to diagnostic delay. L-asparaginase-based chemotherapy plus allogeneic hematopoietic stem cell transplantation (HSCT) is a potential therapeutic option with curative intent.  As for extranodal NK/T cell lymphoma, nasal type, HSCT could be anticipated to provide survival benefit or a chance of cure for patients in clinical stage II to IV with complete remission at transplantation. However, the optimal stem cell source, timing of transplantation, and preconditioning regimen require further elucidation.

Mutations in JAK2, MPL and CALR are regarded as driver mutations, and are mutually exclusively detected in more than 90% of myeloproliferative neoplasms (MPNs). In addition, mutations in epigenetic regulator genes such as TET2 or DNMT3A are detected in MPNs. Although the roles of mutations in epigenetic regulator genes were clarified in normal hematopoiesis, their roles have remained unclear in malignant hematopoiesis of MPNs. We analyzed three lines of mutant mice: mice with JAK2V617F, a representative of driver gene mutations; mice with loss of TET2, a representative of epigenetic abnormalities; and mice with both. We thereby clarified two roles of loss of TET2 in malignant hematopoiesis of JAK2-mutated MPNs: one is "disease initiator and sustainer" via reinforcing the function of JAK2-mutated hematopoietic stem cells, and the other is "disease accelerator". New strategies in risk assessment or treatment are required, considering not only single but also multiple mutations.

Identification of cellular constituents of the hematopoietic stem cell (HSC) niche has recently been the subject of intensive investigation. To investigate the spatial localization of the HSC niches in bone marrow, we have established a whole-mount immunofluorescence imaging technique in which the 3D spatial relationships between stromal structures and HSCs in the BM can be precisely determined. The imaging assessment combined with computational simulations has uncovered a significant association between HSCs and arterioles, ensheathed exclusively by rare Nestin(bright) NG2⁺ pericytes (Nes(peri) cells), distinct from sinusoid-associated Nestin(dim) Leptin receptor (LepR)⁺ (Nes(retic)) cells which reportedly represent peri-vascular niche cells. Depletion of NG2⁺ cells using NG2-creERTM / inducible diphtheria toxin receptor (iDTR) mice changed HSC localization away from arterioles, induced HSC cycling and reduced long-term repopulation of HSCs in BM, suggesting that periarteriolar NG2⁺ cells form quiescent niches for HSCs. These results form the basis of studies that will allow us to genetically dissect the functions of distinct vascular niches. This vascular niche model, in which arterioles and sinusoids differentially regulate HSC quiescence and proliferation, respectively, have implications for the behavior of healthy HSC and may be useful in the future for evaluating the niches for cancer (leukemic) stem cells.

Mechanistic/mammalian target protein of rapamycin (mTOR) is an evolutionarily conserved kinase that plays a critical role in sensing and responding to growth factors, cytokines and nutrients, including amino acids and glucose. Although mTOR was originally discovered as a target protein of rapamycin, the inhibitory effects of rapamycin on mTORC1 substrates are complex. Recent studies using genetic approaches have clearly demonstrated the physiological roles of mTOR complexes in regulating the maintenance of hematopoietic and leukemia stem cells. Advances in understanding of how mTOR signaling is involved in mechanisms of normal hematopoiesis and leukemogenesis may lead to novel therapeutic approaches that can successfully eradicate leukemia.

A significant number of reports have described hematopoietic improvement after iron chelation therapy in iron-overloaded (IO) patients. These observations indicate negative impacts of excess iron on hematopoiesis. To investigate how excess iron affects hematopoiesis, we generated IO mice and examined hematopoietic parameters in these mice. IO mice did not show significant defects in the hematopoietic data of peripheral blood. Myeloid progenitor cells in the bone marrow were increased in IO mice, but the number and function of the erythroid progenitors and hematopoietic stem cells were not significantly affected. However, bone marrow transplantation from normal donors to IO recipients showed delayed hematopoietic reconstitution. Quantitative RT-PCR analyses of the bone marrow stromal cells demonstrated remarkably reduced expressions of several important cytokines, e.g. CXCL12, VCAM-1, Kit-ligand and IGF-1, in the IO mice. In addition, erythropoietin and thrombopoietin levels were significantly suppressed, and oxidative stress was significantly increased in the IO bone marrow and liver. Our findings thus indicate that excess iron can damage bone marrow stromal cells and other vital organs that support hematopoiesis, presumably via increased oxidative stress.

A 64-year-old woman underwent reduced-intensity conditioning cord blood transplantation (RIC-CBT) for refractory acute myeloid leukemia (AML). A 6/6 antigen-level HLA-identical cord blood from a male infant was transfused. After successful engraftment with complete donor chimerism, the patient developed mixed chimera (XX 8.8%) on day 82. Tapering of tacrolimus was started on day 96. Bone marrow chimerism analysis showed a decreasing recipient cell population (XX 2.2%) on day 117 and tacrolimus was discontinued with no clinical signs of GVHD on day 123. However, pancytopenia with agranulocytosis was detected on day 138. She was diagnosed as having secondary graft failure associated with Coombs-positive immune hemolytic anemia and immune thrombocytopenia (ITP). At the same time, the percentage of recipient T cell chimerism in peripheral blood was about 50% and the B cell population showed lambda light chain restriction. On day 180, she received a second RIC-CBT due to lack of improvement of agranulocytosis. A single dose of rituximab was administered on day - 11 before the second CBT to eliminate the activated B cells. Prompt neutrophil engraftment was achieved and both hemolytic anemia and ITP also showed resolution. She is currently well (30 months after the second CBT), showing normal blood cell counts and complete second donor chimerism of marrow cells.

ATL is an aggressive T-cell malignancy caused by HTLV-1 virus infection. Tax, which is the most important regulatory protein of HTLV-1, is associated with aggressive proliferation of host cells and is also a major target antigen for CD8⁺ cytotoxic T-cells (CTLs). Recently, allogeneic hematopoietic stem cell transplantation (allo-HSCT) has proven effective for ATL, and donor-derived Tax-specific CTL might contribute to graft-versus-ATL effects in some recipients who maintained complete remission after allo-HSCT. We, for the first time, analyzed the Tax-specific T-cell receptor (TCR) repertoire, phenotypes and functions of Tax-specific CTLs at single-cell levels in HLA-A24⁺ ATL patients who underwent allo-HSCT. We found that 1) a particular amino acid sequence motif (PDR) in the CDR3 region of TCR-β was conserved in different patients and also within the same patient before and after allo-HSCT, and 2) the PDR⁺ Tax-specific CTL clone selectively expanded in ATL long-term survivors as less-differentiated effector memory CTLs. Actually, the PDR⁺ CTL showed not only strong binding activity for the Tax-tetramer but also strong killing activity against patients' HTLV-1-infected T-cells without any reaction against normal cells. We are presently evaluating the killing activities of PDR⁺ TCR-transduced T-cells against Tax in immunodeficient mice, with the aim of developing a new immunotherapy for ATL.