The pathogenetic significance of exhausted T cells in a mouse model of mature B cell neoplasms.

Dysfunctional anti-tumor immunity has been implicated in the pathogenesis of mature B cell neoplasms, such as multiple myeloma and B cell lymphoma; however, the impact of exhausted T cells on disease development remains unclear. Therefore, the present study investigated the features and pathogenetic significance of exhausted T cells using a mouse model of de novo mature B cell neoplasms, which is likely to show immune escape similar to human patients. The results revealed a significant increase in PD-1+ Tim-3- and PD-1+ Tim-3+ T cells in sick mice. Furthermore, PD-1+ Tim-3+ T cells exhibited direct cytotoxicity with a short lifespan, showing transcriptional similarities to terminally exhausted T cells. On the other hand, PD-1+ Tim-3- T cells not only exhibited immunological responsiveness but also retained stem-like transcriptional features, suggesting that they play a role in the long-term maintenance of anti-tumor immunity. In PD-1+ Tim-3- and PD-1+ Tim-3+ T cells, the transcription factors Tox and Nr4a2, which reportedly contribute to the progression of T cell exhaustion, were up-regulated in vivo. These transcription factors were down-regulated by IMiDs in our in vitro T cell exhaustion analyses. The prevention of excessive T cell exhaustion may maintain effective anti-tumor immunity to cure mature B cell neoplasms.

Genetic subtype classification using a simplified algorithm and mutational characteristics of diffuse large B-cell lymphoma in a Japanese cohort.

Recent large-scale genetic studies have proposed a new genetic classification of diffuse large B-cell lymphoma (DLBCL), which is clinically and biologically heterogeneous. However, the classification methods were complicated to be introduced into clinical practice. Here we retrospectively evaluated the mutational status and copy number changes of 144 genes in 177 Japanese patients with DLBCL, using targeted DNA sequencing. We developed a simplified algorithm for classifying four genetic subtypes-MYD88, NOTCH2, BCL2, and SGK1-by assessing alterations in 18 representative genes and BCL2 and BCL6 rearrangement status, integrating the significant genes from previous studies. In our cohort and another validation cohort from published data, the classification results in our algorithm showed close agreement with the other established algorithm. A differential prognosis among the four groups was observed. The NOTCH2 group showed a particularly poorer outcome than similar groups in previous reports. Furthermore, our study revealed unreported genetic features in the DLBCL subtypes that are mainly reported in Japanese patients, such as CD5-positive DLBCL and methotrexate-associated lymphoproliferative disorders. These results indicate the utility of our simplified method for DLBCL genetic subtype classification, which can facilitate the optimisation of treatment strategies. In addition, our study highlights the genetic features of Japanese patients with DLBCL.

Efficacy of the novel tubulin polymerization inhibitor PTC-028 for myelodysplastic syndrome.

Monomer tubulin polymerize into microtubules, which are highly dynamic and play a critical role in mitosis. Therefore, microtubule dynamics are an important target for anticancer drugs. The inhibition of tubulin polymerization or depolymerization was previously targeted and exhibited efficacy against solid tumors. The novel small molecule PTC596 directly binds tubulin, inhibits microtubule polymerization, downregulates MCL-1, and induces p53-independent apoptosis in acute myeloid leukemia cells. We herein investigated the efficacy of PTC-028, a structural analog of PTC596, for myelodysplastic syndrome (MDS). PTC-028 suppressed growth and induced apoptosis in MDS cell lines. The efficacy of PTC028 in primary MDS samples was confirmed using cell proliferation assays. PTC-028 synergized with hypomethylating agents, such as decitabine and azacitidine, to inhibit growth and induce apoptosis in MDS cells. Mechanistically, a treatment with PTC-028 induced G2/M arrest followed by apoptotic cell death. We also assessed the efficacy of PTC-028 in a xenograft mouse model of MDS using the MDS cell line, MDS-L, and the AkaBLI bioluminescence imaging system, which is composed of AkaLumine-HCl and Akaluc. PTC-028 prolonged the survival of mice in xenograft models. The present results suggest a chemotherapeutic strategy for MDS through the disruption of microtubule dynamics in combination with DNA hypomethylating agents.

Low incidence of thromboembolism in multiple myeloma patients receiving immunomodulatory drugs; a retrospective single-institution analysis.

Anti-platelet agents or anticoagulants are administered for patients with multiple myeloma (MM) receiving immunomodulatory drugs (IMiDs) to prevent thrombotic events (TEs). However, there is a discrepancy between current guidelines and clinical practice in thromboprophylaxis and the varied incidence of TEs depending on patient cohort. Therefore, a consensus on the optimal thromboprophylactic strategy is needed. To determine an appropriate strategy for the prevention of TEs in MM patients receiving IMiDs, we performed a retrospective single-institution analysis. In total, 95 MM patients (62% male, median age 65 years, range 30-85 years) from November 2008 to January 2018 were recruited, and 140 cases were analyzed in the medical-record-based study. Thromboprophylactic drugs were given to 69% of patients, anti-platelet agents to 66%, and anticoagulants to 3.0%. Seven TEs (5.0%) and six bleeding events (4.3%) were observed, but no patients died from thrombohemorrhage. The median follow-up period was 184 days (range 21-2224), and the cumulative TE incidence was 1.7% at 3 months, 7.0% at 1 year, and 12.5% at 3 years. Multivariate analysis determined that age > 70 years (p = 0.012) and BMI < 18.5 kg/m2 (p = 0.042) were the significant risk factors of TE. A low incidence of TEs was observed despite the low adherence to guideline recommendations for anticoagulant administration. These results suggest that anti-platelet agents are sufficient for thromboprophylaxis. A high-risk group of TEs in MM patients receiving IMiDs was identified, and a larger study is needed to confirm these findings.

[MALT lymphoma accompanied by elevated serum IgM levels mimicking Waldenström's macroglobulinemia].

A 60-year-old man with chronic hepatitis C was referred to our hospital with significantly elevated total protein and serum IgM (9,500 mg/dl) levels identified via a routine checkup. Blood examination revealed increased serum IgM-monoclonal protein and serum-soluble IL-2 receptor (sIL2R) levels. Computed tomography and fluorodeoxyglucose positron emission tomography revealed pulmonary masses, abnormal soft tissue masses surrounding the bilateral kidneys, and thickened mucous membrane of the bladder with high fluorodeoxyglucose uptake. Pathological examination of the pulmonary mass revealed infiltration of medium-sized lymphocytes and plasma cells. Immunohistochemical analysis revealed tumor cells positive for CD138 and IgM, with a low positive rate of Ki-67 expression. Notably, the tumor cell-surrounding lymphocytes were positive for CD20. Although the patient was initially regarded as having Waldenström's macroglobulinemia owing to the significantly increased serum IgM levels, based on positive IgH-MALT1 translocation and negative MYD88 L265P mutation findings, he was further diagnosed with extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma). Complete remission was achieved following six cycles of rituximab + CHOP therapy. This study data suggest that analysis of the MYD88 L265P mutation in tumor cells is suitable for accurately diagnosing hematopoietic malignancies with increased IgM monoclonal protein.

Detection of clonal plasma cells in POEMS syndrome using multiparameter flow cytometry.

POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal protein [M-protein], and skin changes) is a rare systemic disorder characterized by various symptoms caused by underlying plasma cell (PC) dyscrasia. Detection of monoclonal PCs is mandatory for the diagnosis of POEMS syndrome; however, the usefulness of EuroFlow-based next-generation flow cytometry (EuroFlow-NGF) in POEMS syndrome for detecting monoclonal PCs in bone marrow (BM) and the gating strategy suitable for flow cytometry study of POEMS syndrome remain unknown. We employed EuroFlow-NGF-based single-tube eight-color multiparameter flow cytometry (MM-flow) and established a new gating strategy (POEMS-flow) to detect the monoclonal PCs in POEMS syndrome, gating CD38 broadly from dim to bright and CD45 narrowly from negative to dim compared to MM-flow. MM-flow detected monoclonal PCs in 9/25 (36.0%) cases, including 2/2 immunofixation electrophoresis (IFE)-negative cases (100%). However, POEMS-flow detected monoclonal PCs in 18/25 cases (72.0%), including 2/2 IFE-negative cases (100%). POEMS-flow detected monoclonal PCs with immunophenotypes of CD19- in 17/18 (94.4%). In six cases where post-treatment samples were available, the size of the clones was significantly reduced after the treatment (P = 0.031). POEMS-flow can enhance the identification rate of monoclonal PCs in POEMS syndrome and become a valuable tool for the diagnosis of POEMS syndrome.

UTX inactivation in germinal center B cells promotes the development of multiple myeloma with extramedullary disease.

UTX/KDM6A, a histone H3K27 demethylase and a key component of the COMPASS complex, is frequently lost or mutated in cancer; however, its tumor suppressor function remains largely uncharacterized in multiple myeloma (MM). Here, we show that the conditional deletion of the X-linked Utx in germinal center (GC) derived cells collaborates with the activating BrafV600E mutation and promotes induction of lethal GC/post-GC B cell malignancies with MM-like plasma cell neoplasms being the most frequent. Mice that developed MM-like neoplasms showed expansion of clonal plasma cells in the bone marrow and extramedullary organs, serum M proteins, and anemia. Add-back of either wild-type UTX or a series of mutants revealed that cIDR domain, that forms phase-separated liquid condensates, is largely responsible for the catalytic activity-independent tumor suppressor function of UTX in MM cells. Utx loss in concert with BrafV600E only slightly induced MM-like profiles of transcriptome, chromatin accessibility, and H3K27 acetylation, however, it allowed plasma cells to gradually undergo full transformation through activation of transcriptional networks specific to MM that induce high levels of Myc expression. Our results reveal a tumor suppressor function of UTX in MM and implicate its insufficiency in the transcriptional reprogramming of plasma cells in the pathogenesis of MM.

Japanese Encephalitis DNA Vaccines with Epitope Modification Reduce the Induction of Cross-Reactive Antibodies against Dengue Virus and Antibody-Dependent Enhancement of Dengue Virus Infection.

Infection with viruses belonging to the genus Flavivirus, such as Japanese encephalitis virus (JEV) and dengue virus (DENV), is a worldwide health problem. Vaccines against JEV and DENV are currently available. However, the dengue vaccine possibly increases the risk of severe dengue due to antibody-dependent enhancement (ADE). Moreover, the Japanese encephalitis (JE) vaccine reportedly induces cross-reactive ADE-prone antibodies against DENV, potentially leading to symptomatic dengue. Therefore, it is necessary to eliminate the risk of ADE through vaccination. In this study, we attempted to develop a JE vaccine that does not induce ADE of DENV infection using an epitope modification strategy. We found that an ADE-prone monoclonal antibody cross-reactive to DENV and JEV recognizes the 106th amino acid residue of the E protein of JEV (E-106). The JE DNA vaccine with a mutation at E-106 (E-106 vaccine) induced comparable neutralizing antibody titers against JEV to those induced by the wild-type JE DNA vaccine. Meanwhile, the E-106 vaccine induced 64-fold less cross-reactive ADE-prone antibodies against DENV. The mutation did not compromise the protective efficacy of the vaccine in the lethal JEV challenge experiment. Altogether, the modification of a single amino acid residue identified in this study helped in the development of an ADE-free JE vaccine.

Construction and evaluation of a self-replicative RNA vaccine against SARS-CoV-2 using yellow fever virus replicon.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a global threat. To forestall the pandemic, developing safe and effective vaccines is necessary. Because of the rapid production and little effect on the host genome, mRNA vaccines are attractive, but they have a relatively low immune response after a single dose. Replicon RNA (repRNA) is a promising vaccine platform for safety and efficacy. RepRNA vaccine encodes not only antigen genes but also the genes necessary for RNA replication. Thus, repRNA is self-replicative and can play the role of an adjuvant by itself, which elicits robust immunity. This study constructed and evaluated a repRNA vaccine in which the gene encoding the spike (S) protein of SARS-CoV-2 was inserted into a replicon of yellow fever virus 17D strain. Upon electroporation of this repRNA into baby hamster kidney cells, the S protein and yellow fever virus protein were co-expressed. Additionally, the self-replication ability of repRNA vaccine was confirmed using qRT-PCR, demonstrating its potency as a vaccine. Immunization of C57BL/6 mice with 1 µg of the repRNA vaccine induced specific T-cell responses but not antibody responses. Notably, the T-cell response induced by the repRNA vaccine was significantly higher than that induced by the nonreplicative RNA vaccine in our experimental model. In the future, it is of the essence to optimize vaccine administration methods and improve S protein expression, like protection of repRNA by nanoparticles and evasion of innate immunity of the host to enhance the immune-inducing ability of the repRNA vaccine.

Unraveling unique features of plasma cell clones in POEMS syndrome with single-cell analysis.

POEMS syndrome is a rare monoclonal plasma cell disorder, with unique symptoms distinct from those of other plasma cell neoplasms, including high serum VEGF levels. Because the prospective isolation of POEMS clones has not yet been successful, their real nature remains unclear. Herein, we performed single-cell RNA-Seq of BM plasma cells from patients with POEMS syndrome and identified POEMS clones that had Ig λ light chain (IGL) sequences (IGLV1-36, -40, -44, and -47) with amino acid changes specific to POEMS syndrome. The proportions of POEMS clones in plasma cells were markedly smaller than in patients with multiple myeloma (MM) and patients with monoclonal gammopathy of undetermined significance (MGUS). Single-cell transcriptomes revealed that POEMS clones were CD19+, CD138+, and MHC class IIlo, which allowed for their prospective isolation. POEMS clones expressed significantly lower levels of c-MYC and CCND1 than MM clones, accounting for their small size. VEGF mRNA was not upregulated in POEMS clones, directly indicating that VEGF is not produced by POEMS clones. These results reveal unique features of POEMS clones and enhance our understanding of the pathogenesis of POEMS syndrome.

Outcomes of poor peripheral blood stem cell mobilizers with multiple myeloma at the first mobilization: A multicenter retrospective study in Japan.

Autologous stem cell transplantation (ASCT) remains an important therapeutic strategy for multiple myeloma; however, a proportion of patients fail to mobilize a sufficient number of peripheral blood stem cells (PBSCs) to proceed to ASCT. In the present study, we aimed to clarify the characteristics and outcomes of poor mobilizers. Clinical data on poorly mobilized patients who underwent PBSC harvest for almost 10 years were retrospectively collected from 44 institutions in the Japanese Society of Myeloma (JSM). Poor mobilizers were defined as patients with less than 2 × 106/kg of CD34+ cells harvested at the first mobilization. The proportion of poor mobilization was 15.1%. A sufficient dataset including overall survival (OS) was evaluable in 258 poor mobilizers. Overall, 92 out of 258 (35.7%) poor mobilizers did not subsequently undergo ASCT, mainly due to an insufficient number of PBSCs. Median OS from apheresis was longer for poor mobilizers who underwent ASCT than for those who did not (86.0 vs. 61.9 mon., p = 0.02). OS from the diagnosis of poor mobilizers who underwent ASCT in our cohort was similar to those who underwent ASCT in the JSM database (3y OS rate, 86.8% vs. 85.9%). In this cohort, one-third of poor mobilizers who did not undergo ASCT had relatively poor survival. In contrast, the OS improved in poor mobilizers who underwent ASCT. However, the OS of extremely poor mobilizers was short irrespective of ASCT.

Successful allogeneic bone marrow transplantation after massive gastrointestinal bleeding in a patient with myelodysplastic syndrome associated with intestinal Behçet-like disease.

A 45-year-old woman was diagnosed with myelodysplastic syndrome (MDS) with trisomy 8 and Behçet-like disease (BLD) with multiple colorectal ulcers. Nonspecific inflammatory cells were infiltrated in the intestinal mucosa, whereas fluorescence in situ hybridization (FISH) analysis revealed only sporadic trisomy 8-positive cells. She presented massive lower gastrointestinal bleeding early after bone marrow transplantation but achieved long-term remission of both MDS and BLD. This is the first report of massive gastrointestinal bleeding after transplantation for MDS with BLD. Based on FISH analysis, dysregulation of systemic inflammation may be involved in BLD rather than direct invasion by trisomy 8-positive MDS clones.

The combination of the tubulin binding small molecule PTC596 and proteasome inhibitors suppresses the growth of myeloma cells.

The novel small molecule PTC596 inhibits microtubule polymerization and its clinical development has been initiated for some solid cancers. We herein investigated the preclinical efficacy of PTC596 alone and in combination with proteasome inhibitors in the treatment of multiple myeloma (MM). PTC596 inhibited the proliferation of MM cell lines as well as primary MM samples in vitro, and this was confirmed with MM cell lines in vivo. PTC596 synergized with bortezomib or carfilzomib to inhibit the growth of MM cells in vitro. The combination treatment of PTC596 with bortezomib exerted synergistic effects in a xenograft model of human MM cell lines in immunodeficient mice and exhibited acceptable tolerability. Mechanistically, treatment with PTC596 induced cell cycle arrest at G2/M phase followed by apoptotic cell death, associated with the inhibition of microtubule polymerization. RNA sequence analysis also revealed that PTC596 and the combination with bortezomib affected the cell cycle and apoptosis in MM cells. Importantly, endoplasmic reticulum stress induced by bortezomib was enhanced by PTC596, providing an underlying mechanism of action of the combination therapy. Our results indicate that PTC596 alone and in combination with proteasome inhibition are potential novel therapeutic options to improve outcomes in patients with MM.

DHODH inhibition synergizes with DNA-demethylating agents in the treatment of myelodysplastic syndromes.

Dihydroorotate dehydrogenase (DHODH) catalyzes a rate-limiting step in de novo pyrimidine nucleotide synthesis. DHODH inhibition has recently been recognized as a potential new approach for treating acute myeloid leukemia (AML) by inducing differentiation. We investigated the efficacy of PTC299, a novel DHODH inhibitor, for myelodysplastic syndrome (MDS). PTC299 inhibited the proliferation of MDS cell lines, and this was rescued by exogenous uridine, which bypasses de novo pyrimidine synthesis. In contrast to AML cells, PTC299 was inefficient at inhibiting growth and inducing the differentiation of MDS cells, but synergized with hypomethylating agents, such as decitabine, to inhibit the growth of MDS cells. This synergistic effect was confirmed in primary MDS samples. As a single agent, PTC299 prolonged the survival of mice in xenograft models using MDS cell lines, and was more potent in combination with decitabine. Mechanistically, a treatment with PTC299 induced intra-S-phase arrest followed by apoptotic cell death. Of interest, PTC299 enhanced the incorporation of decitabine, an analog of cytidine, into DNA by inhibiting pyrimidine production, thereby enhancing the cytotoxic effects of decitabine. RNA-seq data revealed the marked downregulation of MYC target gene sets with PTC299 exposure. Transfection of MDS cell lines with MYC largely attenuated the growth inhibitory effects of PTC299, suggesting MYC as one of the major targets of PTC299. Our results indicate that the DHODH inhibitor PTC299 suppresses the growth of MDS cells and acts in a synergistic manner with decitabine. This combination therapy may be a new therapeutic option for the treatment of MDS.

Acquired von Willebrand Syndrome due to Aortic Valve Stenosis in a Case with Antiphospholipid Antibody.

Acquired von Willebrand syndrome (AVWS) is a bleeding disorder caused by an acquired deficiency of von Willebrand factor (vWF). Some patients with AVWS show a low bleeding tendency and are diagnosed by the presence of a mild prolongation of activated partial thromboplastin time (APTT) preoperatively. Another cause of APTT prolongation is the presence of antiphospholipid antibody (aPL). We experienced a case of AVWS due to aortic valve stenosis in a patient with aPL in whom aortic valve replacement surgery was successful with vWF replacement. In patients with AVWS-associated disorders who are identified based on APTT prolongation at the preoperative examination, both vWF and aPL screening tests must be performed.

Fulminant Bacillus cereus septicaemia with multiple organ ischaemic/haemorrhagic complications in a patient undergoing chemotherapy for acute myelogenous leukaemia.

Bacillus cereus is a Gram-positive spore-forming rod widely found in the environment and is thought to be a frequent source of contamination. This micro-organism is reportedly a significant pathogenic agent among immunocompromised individuals. Furthermore, multiple cases of fulminant septicaemia have been reported among individuals receiving chemotherapy for acute myelogenous leukaemia. In some cases, B. cereus septicaemia was associated with multiple haemorrhages. We, herein, describe a patient with an extremely acute course of B. cereus septicaemia characterised by haemorrhage and infarction of multiple organs, which led to his death. Our findings suggest that delayed treatment of B. cereus in patients with haematologic malignancies undergoing chemotherapy may result in extremely poor outcomes; thus, immediate empirical treatment with vancomycin should be considered.

Immune pancytopenia after chemotherapy in a patient with diffuse large B-cell lymphoma.

During treatment for malignant lymphoma, cytopenia can develop for several reasons. In the treatment of cytopenia, various possibilities should be considered because inadequate treatment causes exacerbation of cytopenia and can lead to fatal conditions, such as infection and bleeding. Herein, we describe immune pancytopenia 3 months after the last exposure to chemotherapy in a patient with diffuse large B-cell lymphoma (DLBCL). She suffered from severe pancytopenia after two courses of rituximab and bendamustine therapy for a second relapse of DLBCL. Immune pancytopenia was diagnosed with bone marrow tests and the presence of autoantibodies; it promptly resolved after initiation of prednisolone therapy. Clinicians should be aware of immune cytopenia and monitor for it carefully, even if patients have already finished chemotherapy treatment.