Clonal evolution process from essential thrombocythemia to acute myeloid leukemia in the original patient from whom the CALR-mutated Marimo cell line was established.

We previously reported the Marimo cell line, which was established from the bone marrow cells of a patient with essential thrombocythemia (ET) at the last stage after transformation to acute myeloid leukemia (AML). This cell line is widely used for the biological analysis of ET because it harbors CALR mutation. However, genetic processes during disease progression in the original patient were not analyzed. We sequentially analyzed the genetic status in the original patient samples during disease progression. The ET clone had already acquired CALR and MPL mutations, and TP53 and NRAS mutations affected the disease progression from ET to AML in this patient. Particularly, the variant allele frequency of the NRAS mutation increased along with the disease progression after transformation, and the NRAS-mutated clone selectively proliferated in vitro, resulting in the establishment of the Marimo cell line. Although CALR and MPL mutations co-existed, MPL was not expressed in Marimo cells or any clinical samples. Furthermore, mitogen-activated protein kinase (MAPK) but not the JAK2-STAT pathway was activated. These results collectively indicate that MAPK activation is mainly associated with the proliferation ability of Marimo cells.

[Cellular kinetics and outcome of tisagenlecleucel for diffuse large B-cell lymphoma].

CD19-targeted chimeric antigen receptor T-cell (CAR-T) therapy has shown promise as treatment of relapsed or refractory B-cell malignancies. However, the clinical utility of early CAR-T monitoring within 1 month after infusion has not been elucidated. In this study, we quantitatively measured CAR-T kinetics in peripheral blood on days 2, 4, 7, 9, 11, 14, 21, and 28 post-infusion using flow cytometry and quantitative polymerase chain reaction in 13 patients with relapsed refractory diffuse large B-cell lymphoma (DLBCL) treated with tisagenlecleucel (tisa-cel). No relationships were identified between bulk CAR-T kinetics and treatment outcomes. Interestingly, the magnitude of CD4+ CAR-T expansion was higher in responders than in nonresponders, while CD8+ CAR-T expansion was minimal in responders. Additionally, CAR-T proliferation was more pronounced in patients with cytokine release syndrome. Our results suggest that CD4+ CAR-T cellular kinetics within 1 month after CAR-T infusion may predict its efficacy after tisa-cel therapy in adult patients with DLBCL.

Comparison of clonal architecture between primary and immunodeficient mouse-engrafted acute myeloid leukemia cells.

Patient-derived xenografts (PDX) are widely used as human cancer models. Previous studies demonstrated clonal discordance between PDX and primary cells. However, in acute myeloid leukemia (AML)-PDX models, the significance of the clonal dynamics occurring in PDX remains unclear. By evaluating changes in the variant allele frequencies (VAF) of somatic mutations in serial samples of paired primary AML and their PDX bone marrow cells, we identify the skewing engraftment of relapsed or refractory (R/R) AML clones in 57% of PDX models generated from multiclonal AML cells at diagnosis, even if R/R clones are minor at <5% of VAF in patients. The event-free survival rate of patients whose AML cells successfully engraft in PDX models is consistently lower than that of patients with engraftment failure. We herein demonstrate that primary AML cells including potentially chemotherapy-resistant clones dominantly engraft in AML-PDX models and they enrich pre-existing treatment-resistant subclones.

[A message to hematology residents in diverse career pathways].

In the present decade, the number of female hematologists and their ratio to the total number of hematologists have increased. This increase in the number of female physicians with various work styles have made physicians aware of diverse career paths. To attain an uninterrupted career, physicians have to overcome obstacles such as severe working environment or intolerance of diversity. The Committee on Studies of Career Education for Female Physicians proposed five learning objectives for all physicians to attain an uninterrupted career: professional awareness of the missions of being a physician, ability to make career plans, flexibility to embrace diverse values of the profession, appropriate attitudes toward supports, and recognition of social gender differences. Learning objectives corresponding to the learning period were also proposed. In order to make career plans, residents need to collect the information on specialty training, perceive a variety of individuals as potential role models, and create, review, and change their own career plan. Residents should become familiar with the process for becoming board-certified hematologists through understanding the new training program for hematology residency.

Prophylactic antiviral therapy for hepatitis B virus surface antigen-positive patients with diffuse large B-cell lymphoma treated with rituximab-containing chemotherapy.

We conducted a nationwide retrospective analysis of 116 hepatitis B virus (HBV) surface antigen (HBsAg)-positive patients with diffuse large B-cell lymphoma (DLBCL) and 278 HBsAg-negative patients with DLBCL, as a control cohort, who received rituximab-containing regimens as an induction chemotherapy at 30 Japanese medical centers between January 2004 and December 2014. Hepatitis was defined as an absolute serum alanine aminotransferase (ALT) level of ≥100 U/L. HBV reactivation-related hepatitis was defined as hepatitis with an absolute serum HBV DNA level of ≥3.3 log IU/mL or an absolute increase of ≥2 log compared with the baseline value. HBsAg-positive patients were divided into three groups based on anti-HBV prophylactic therapy: no nucleos(t)ide analogue (non-NA, n = 9), lamivudine (LAM, n = 20), and entecavir (ETV, n = 87). The 4-year cumulative incidence (CI) of hepatitis in HBsAg-positive and HBsAg-negative patients was 21.1% and 14.6% (P = .081), respectively. The 4-year CI of HBV reactivation-related hepatitis was higher in HBsAg-positive patients than in HBsAg-negative patients (8.0% vs 0.4%; P < .001). Among HBsAg-positive patients, the 4-year CI of HBV reactivation-related hepatitis was the highest in the non-NA group (33.3%), followed by the LAM (15.0%) and ETV (3.8%) groups (P < .001). Of note, 3 non-NA patients (33%) and 1 LAM patient (5%) (but no ETV patients) died due to HBV hepatitis. Based on Cox multivariate analysis, HBsAg positivity was not associated with poor overall survival. Prophylactic use of ETV would reduce the occurrence of HBV reactivation-related hepatitis and mortality in HBsAg-positive DLBCL patients receiving rituximab-containing chemotherapy.

Phase I clinical trial of intra-bone marrow cotransplantation of mesenchymal stem cells in cord blood transplantation.

Mesenchymal stem cells (MSCs) have immunomodulatory properties and support hematopoiesis in the bone marrow (BM). To develop a new strategy to not only prevent graft-vs-host disease (GVHD) but also to enhance engraftment, a phase I trial of cord blood transplantation (CBT) combined with intra-BM injection of MSCs (MSC-CBT) was designed. Third-party BM-derived MSCs were injected intra-BM on the day of CBT. The conditioning regimen varied according to patient characteristics. GVHD prophylaxis was tacrolimus and methotrexate. The primary endpoint was toxicity related to intra-BM injection of MSCs. Clinical outcomes were compared with those of six controls who received CBT alone. Five adult patients received MSC-CBT, and no adverse events related to intra-BM injection of MSCs were observed. All patients achieved neutrophil, reticulocyte, and platelet recoveries, with median times to recoveries of 21, 35, and 38 days, respectively, comparable with controls. Grade II-IV acute GVHD developed in three controls but not in MSC-CBT patients. No patients developed chronic GVHD in both groups. At 1 year after transplantation, all MSC-CBT patients survived without relapse. This study shows the safety of MSC-CBT, and the findings also suggest that cotransplantation of MSCs may prevent GVHD with no inhibition of engraftment. This trial was registered at the University Hospital Medical Information Network Clinical Trials Registry as number 000024291.

Multi-Lineage BCR-ABL Expression in Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia Is Associated With Improved Prognosis but No Specific Molecular Features.

BACKGROUND: Recently, various blood cell lineages expressing the BCR-ABL fusion gene in Philadelphia chromosome (Ph)-positive acute lymphoblastic leukemia (ALL) have been reported. However, the biological and clinical significance of these BCR-ABL lineages has not been established; therefore, we aimed to clarify the impacts of these different BCR-ABL-expressing lineages. PATIENTS: Multi-lineage BCR-ABL expression (multi-Ph) was defined as BCR-ABL expression outside of the B-lineage compartment, as determined by fluorescence in situ hybridization (FISH) in peripheral blood neutrophils and bone marrow clots, and flow cytometry-sorted polymerase chain reaction (PCR). We analyzed IKZF1 deletion patterns by PCR, examined gene expression profiles using RNA sequencing, and compared treatment outcomes across different BCR-ABL-expressing lineages. RESULTS: Among the 21 multi-Ph patients in our 59-patient cohort (36%), BCR-ABL expression was detected at the multipotential progenitor level. However, no IKZF1 deletion patterns or gene expression profiles were identified that were specific for multi-Ph. However, multi-Ph patients were found to have better survival rates than patients with uni-lineage BCR-ABL expression [event-free survival (EFS): 74 vs. 33%, P = 0.01; overall survival (OS): 79 vs. 44% at 4 years, P = 0.01]. In multivariate analyses, multi-Ph was identified as a good prognostic factor for both EFS and OS. CONCLUSION: We confirmed that more than one-third of Ph+ALL patients could be classified as mutli-Ph. Although no specific molecular characteristics were identified for multi-Ph, this phenotype was associated with better treatment outcomes.

Identification of the novel deletion-type PML-RARA mutation associated with the retinoic acid resistance in acute promyelocytic leukemia.

All-trans retinoic acid (ATRA) and arsenic trioxide (ATO) are essential for acute promyelocytic leukemia (APL) treatment. It has been reported that mutations in PML-RARA confer resistance to ATRA and ATO, and are associated with poor prognosis. Although most PML-RARA mutations were point mutations, we identified a novel seven amino acid deletion mutation (p.K227_T233del) in the RARA region of PML-RARA in a refractory APL patient. Here, we analyzed the evolution of the mutated clone and demonstrated the resistance of the mutated clone to retinoic acid (RA). Mutation analysis of PML-RARA was performed using samples from a chemotherapy- and ATRA-resistant APL patient, and the frequencies of mutated PML-RARA transcript were analyzed by targeted deep sequencing. To clarify the biological significance of the identified PML-RARA mutations, we analyzed the ATRA-induced differentiation and PML nuclear body formation in mutant PML-RARA-transduced HL-60 cells. At molecular relapse, the p.K227_T233del deletion and the p.R217S point-mutation in the RARA region of PML-RARA were identified, and their frequencies increased after re-induction therapy with another type of retinoiec acid (RA), tamibarotene. In deletion PML-RARA-transduced cells, the CD11b expression levels and NBT reducing ability were significantly decreased compared with control cells and the formation of PML nuclear bodies was rarely observed after RA treatment. These results indicate that this deletion mutation was closely associated with the disease progression during RA treatment.

Mutation analysis of therapy-related myeloid neoplasms.

We analyzed the genetic mutation status of 13 patients with therapy-related myeloid neoplasms (t-MN). Consistent with previous reports, t-MN cells preferentially acquired mutations in TP53 and epigenetic modifying genes, instead of mutations in tyrosine kinase and spliceosome genes. Furthermore, we compared the mutation status of three t-MN cells with each of the initial lymphoid malignant cells, and identified common mutations among t-MN and the initial malignant cells in two patients. In a patient who developed chronic myelomonocytic leukemia (CMML) after follicular lymphoma (FL), TET2 mutation was identified in both CMML and FL cells. Notably, the TET2 mutation was also identified in peripheral blood cells in the disease-free period with the same allelic frequency as CMML and FL cells, but not in a germ-line control, indicating that the TET2 mutation occurred somatically in the initiating clone for both malignant cells. On the other hand, a germ-line MYB mutation was identified in a patient who developed myelodysplastic syndromes (MDS) after FL. These results suggest that germ-line deposition and clonal hematopoiesis are closely associated with t-MN susceptibility; however, further analysis is necessary to clarify the mechanism required to provide the initiating clone with lineage commitment and clonal expansion.

Phase I study of cord blood transplantation with intrabone marrow injection of mesenchymal stem cells: A clinical study protocol.

INTRODUCTION: Delayed hematological recovery, graft failure, and acute graft-versus-host disease (GVHD) still remain major problems in cord blood transplantation (CBT). Mesenchymal stem cells (MSCs) are known to support bone marrow stroma and promote hematopoiesis. Additionally, MSCs possess immunomodulatory properties and are used clinically for the treatment of acute GVHD. Therefore, the use of MSCs to enhance engraftment and prevent GVHD after allogeneic hematopoietic cell transplantation has been explored. Recent clinical trials have shown the feasibility and safety of intravenous cotransplantation of MSCs with cord blood cells in pediatric patients, but not in adult patients, who are at greater risk of graft failure. As for the route of administration of MSCs, direct intrabone marrow injection of MSCs is thought to enhance the engraftment of cord blood cells more than intravenous injection. Based on these background findings, this clinical trial was designed to develop a new strategy to enhance engraftment and prevent GVHD after CBT. METHODS AND ANALYSIS: This is a single-center, phase I, clinical study to evaluate the safety of CBT combined with intrabone marrow injection of ex vivo expanded MSCs from bone marrow of a third-party donor. Adult patients with hematological disorders are eligible for this study. The target sample size is 5, and the registration period is 3 years. The target dose of MSCs infused is 0.5 × 10 cells/kg of patient body weight. On the day of CBT, MSCs are injected into the intrabone marrow of the patient 4 hours before the infusion of a single cord blood unit. The conditioning regimen varies according to patient age and disease. GVHD prophylaxis consists of a combination of tacrolimus and methotrexate. The primary endpoint of this study is infusional toxicity of MSCs within 14 days after transplantation.

Phase II study of intrabone single unit cord blood transplantation for hematological malignancies.

The outcomes of cord blood transplantation with non-irradiated reduced-intensity conditioning for hematological malignancies need to be improved because of graft failure and delayed engraftment. Intrabone infusion of cord blood cells has the potential to resolve the problems. In this phase II study, 21 adult patients with hematological malignancy received intrabone transplantation of serological HLA-A, B, and DR ≥4/6 matched single cord blood with a median number of cryopreserved total nucleated cells of 2.7 × 107 /kg (range, 2.0-4.9 × 107 /kg) following non-irradiated fludarabine-based reduced-intensity conditioning. Short-term methotrexate and tacrolimus were given as graft-versus-host disease prophylaxis, and granulocyte colony-stimulating factor was given after transplantation. No severe adverse events related to intrabone injection were observed. The cumulative incidences of neutrophils ≥0.5 × 109 /L, reticulocytes ≥1%, and platelets ≥20 × 109 /L recoveries were 76.2%, 71.4%, and 76.2%, respectively, with median time to recoveries of 17, 28, and 32 days after transplantation, respectively. The probability of survival with neutrophil engraftment on day 60 was 71.4%, and overall survival at 1 year after transplantation was 52.4%. The incidences of grade II-IV and III-IV acute graft-versus-host disease were 44% and 19%, respectively, with no cases of chronic graft-versus-host disease. The present study showed the safety of direct intrabone infusion of cord blood. Further analysis is required to confirm the efficacy of intrabone single cord blood transplantation with non-irradiated reduced-intensity conditioning for adult patients with hematological malignancy. This study was registered with UMIN-CTR, number 000000865.

Malignant lymphoma with cardiac involvement.

Malignant lymphoma with cardiac involvement is difficult to diagnose and treatment selection decisions can be challenging, because patients usually present with atypical disease involvement and the incidence is low. Herein, we describe the clinical characteristics and courses of three non-Hodgkin lymphoma patients showing cardiac involvement. All three patients were male, ages 32, 74 and 64 years. All three patients had presented with cardiac involvement mainly in the right heart system. We promptly performed needle biopsies for patients 1 and 3, and open-heart biopsy for patient 2, which showed PMBL for patient 1, DLBCL for patients 2 and 3. Since we were concerned regarding possible transient exacerbation of heart failure or the occurrence of fatal arrhythmia, we chose to start with relatively low dose chemotherapeutic interventions or pre-phase steroid therapy. After one course of chemotherapy or pre-phase steroid therapy, symptoms associated with heart failure almost completely subsided, and we further administered full-dose chemotherapy thereafter, resulting in complete responses in 2 cases. This case series demonstrates that malignant lymphoma with cardiac involvement is a treatable disease, despite widespread involvement. Furthermore, rapid and appropriate diagnostic imaging and biopsy are important when this disease is suspected.

Safety and tolerability of ibrutinib monotherapy in Japanese patients with relapsed/refractory B cell malignancies.

UNLABELLED: In this phase I dose-escalation study we evaluated the safety, tolerability, pharmacokinetics, and antitumor activity of ibrutinib, an oral covalent inhibitor of Bruton's tyrosine kinase (BTK, in Japanese patients with relapsed/refractory B cell malignancies (RRBCM). Fifteen patients aged 42-78 years were enrolled to one of three cohorts. Cohort 1 (n = 3) consisted of two phases, a single-dose (140 and 280 mg) phase and a multiple-dose (420 mg) phase of ibrutinib; cohort 2 (n = 6) included multiple doses of ibrutinib 560 mg; and cohort 3 (n = 6) included only patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) dosed at ibrutinib 420 mg. One patient (CLL/SLL cohort) experienced grade 3 pneumonia and sepsis, which were considered dose-limiting toxicities. No deaths were reported. The most common (≥ 20% patients) adverse events were neutropenia, anemia, nasopharyngitis, increased bilirubin, and rash. Dose-dependent increase in maximum plasma concentration and area under the concentration from 0 to the last quantifiable time was observed, while time to reach maximum plasma concentration and elimination half-life was similar between doses. The overall response rate was 73.3% (11/15) for all cohorts combined. Overall, ibrutinib (420 and 560 mg) was tolerable with acceptable safety profiles and effective for Japanese patients with RRBCM including CLL/SLL. CLINICAL TRIAL REGISTRATION: NCT01704963.

Generation and Characterization of UL21-Null Herpes Simplex Virus Type 1.

UL21 of herpes simplex virus type 1 (HSV-1) is an accessory gene that encodes a component of the tegument. Homologs of this protein have been identified in the alpha, beta, and gamma herpesvirus subfamilies, although their functions are unclear. To clarify the functions of UL21, we generated a UL21-null HSV-1 mutant. Growth analysis showed that the synthesis of infectious UL21-null HSV-1 in glial cells was delayed and that the overall yield was low. The plaque sizes of the UL21-null mutant were smaller than those of wild-type HSV-1. We identified several candidate UL21-interacting proteins, including intermediate filaments, by yeast two-hybrid screening. The distribution of glial fibrillary acidic protein (GFAP), which is the main component of intermediate filaments, was altered in UL21-null mutant-infected glial cells compared to wild-type virus-infected cells. These results will help clarify the function of UL21 and broaden our understanding of the life cycle of HSV.

Bortezomib induces apoptosis in T lymphoma cells and natural killer lymphoma cells independent of Epstein-Barr virus infection.

Epstein-Barr virus (EBV), which infects not only B cells, but also T cells and natural killer (NK) cells, is associated with multiple lymphoid malignancies. Recently, the proteasome inhibitor bortezomib was reported to induce apoptosis of EBV-transformed B cells. We evaluated the killing effect of this proteasome inhibitor on EBV-associated T lymphoma cells and NK lymphoma cells. First, we found that bortezomib treatment decreased the viability of multiple T and NK cell lines. No significant difference was observed between EBV-positive and EBV-negative cell lines. The decreased viability in response to bortezomib treatment was abrogated by a pan-caspase inhibitor. The induction of apoptosis was confirmed by flow cytometric assessment of annexin V staining. Additionally, cleavage of caspases and polyadenosine diphosphate-ribose polymerase, increased expression of phosphorylated IκB, and decreased expression of inhibitor of apoptotic proteins were detected by immunoblotting in bortezomib-treated cell lines. We found that bortezomib induced lytic infection in EBV-positive T cell lines, although the existence of EBV did not modulate the killing effect of bortezomib. Finally, we administered bortezomib to peripheral blood mononuclear cells from five patients with EBV-associated lymphoproliferative diseases. Bortezomib had a greater killing effect on EBV-infected cells. These results indicate that bortezomib killed T or NK lymphoma cells by inducing apoptosis, regardless of the presence or absence of EBV.

Herpes simplex virus UL56 interacts with and regulates the Nedd4-family ubiquitin ligase Itch.

BACKGROUND: Herpes simplex virus type 2 (HSV-2) is one of many viruses that exploits and modifies the cellular ubiquitin system. HSV-2 expresses the tegument protein UL56 that has been implicated in cytoplasmic transport and/or release of virions, and is a putative regulatory protein of Nedd4 ubiquitin ligase. In order to elucidate the biological function of UL56, this study examined the interaction of UL56 with the Nedd4-family ubiquitin ligase Itch and its role in the regulation of Itch. Additionally, we assessed the similarity between UL56 and regulatory proteins of Itch and Nedd4, Nedd4-family-interactins proteins (Ndfip). RESULTS: UL56 interacted with Itch, independent of additional viral proteins, and mediated more striking degradation of Itch, compared to Nedd4. Moreover, it was suggested that the lysosome pathway as well as the proteasome pathway was involved in the degradation of Itch. Other HSV-2 proteins with PY motifs, such as VP5 and VP16, did not mediate the degradation of endogenous Itch. Ndfip1 and Ndfip2 were similar in subcellular distribution patterns to UL56 and colocalized with UL56 in co-transfected cells. CONCLUSIONS: We believe that this is the first report demonstrating the interaction of a HSV-specific protein and Itch. Thus, UL56 could function as a regulatory protein of Itch. The mechanism, function and significance of regulating Itch in HSV-2 infection remain unclear and warrant further investigation.

Herpes simplex virus type 2 tegument protein UL56 relocalizes ubiquitin ligase Nedd4 and has a role in transport and/or release of virions.

BACKGROUND: The ubiquitin system functions in a variety of cellular processes including protein turnover, protein sorting and trafficking. Many viruses exploit the cellular ubiquitin system to facilitate viral replication. In fact, herpes simplex virus (HSV) encodes a ubiquitin ligase (E3) and a de-ubiquitinating enzyme to modify the host's ubiquitin system. We have previously reported HSV type 2 (HSV-2) tegument protein UL56 as a putative adaptor protein of neuronal precursor cell-expressed developmentally down-regulated 4 (Nedd4) E3 ligase, which has been shown to be involved in protein sorting and trafficking. RESULTS: In this study, we visualized and characterized the dynamic intracellular localization of UL56 and Nedd4 using live-cell imaging and immunofluorescence analysis. UL56 was distributed to cytoplasmic vesicles, primarily to the trans-Golgi network (TGN), and trafficked actively throughout the cytoplasm. Moreover, UL56 relocalized Nedd4 to the vesicles in cells transiently expressing UL56 and in cells infected with HSV-2. We also investigated whether UL56 influenced the efficiency of viral replication, and found that extracellular infectious viruses were reduced in the absence of UL56. CONCLUSION: These data suggest that UL56 regulates Nedd4 and functions to facilitate the cytoplasmic transport of virions from TGN to the plasma membrane and/or release of virions from the cell surface.

Absence of transovarial transmission of Borrelia duttonii, a tick-borne relapsing fever agent, by the vector tick Ornithodoros moubata.

We examined the vector competence of the tick, Ornithodoros moubata, using laboratory-reared gerbils as hosts. Transmission of the relapsing fever agent Borrelia duttonii occurred efficiently from infected ticks to uninfected gerbils and from infected gerbils to uninfected ticks. Spirochetes were maintained stably in the ticks for at least 3 months, but they disappeared from the bloodstream of infected gerbils after three episodes of spirochetemia. We also examined transovarial transmission of B. duttonii during the gonotrophic cycle and filial generation. No spirochetes could be detected from the offspring generation of the ticks by culture and polymerase chain reaction (PCR) methods, although spirochetes were still found in the female ticks. The results indicate that, because of the rarity of transovarial infection, the role of transovarial passage of B. duttonii to eggs and larval O. moubata ticks is limited in maintaining B. duttonii. Our findings strongly suggest that B. duttonii is maintained through the O. moubata tick-human transmission cycle in tick-borne relapsing fever endemic areas.