Activated mesenchymal stem/stromal cells promote myeloid cell differentiation via CCL2/CCR2 signaling.

Myeloid cells, which originate from hematopoietic stem/progenitor cells (HSPCs), play a crucial role in mitigating infections. This study aimed to explore the impact of mesenchymal stem/stromal cells (MSCs) on the differentiation of HSPCs and progenitors through the C-C motif chemokine CCL2/CCR2 signaling pathway. Murine MSCs, identified as PDGFRα+Sca-1+ cells (PαS cells), were found to secrete CCL2, particularly in response to lipopolysaccharide stimulation. MSC-secreted CCL2 promoted the differentiation of granulocyte/macrophage progenitors into the myeloid lineage. MSC-derived CCL2 plays an important role in the early phase of myeloid cell differentiation in vivo. Single-cell RNA sequencing analysis confirmed that CCL2-mediated cell fate determination was also observed in human bone marrow cells. These findings provide valuable insights for investigating the in vivo effects of MSC transplantation.

[Durable remission of T-cell prolymphocytic leukemia with CLEC16A::IL2 after allogeneic hematopoietic stem cell transplantation].

A 64-year-old woman presented with fine motor impairment in both hands. MRI revealed a contrast-enhanced lesion in the medulla oblongata. Lymphoid cells with abnormal blebs were observed and a CD4+/CD8+ double positive (DP) T cell population was detected by flow cytometry (FCM) in the bone marrow (BM) and the peripheral blood (PB). CLEC16A::IL2 fusion gene was identified by whole exome sequencing with DNA prepared from DP T cells. Clonal rearrangement of the T-cell receptor gene and expression of TCL1A protein were detected. This led to a diagnosis of T-cell prolymphocytic leukemia (T-PLL) with central nervous system (CNS) infiltration. Abnormal cells in BM and PB became undetectable on microscopy and FCM, and the CNS lesion disappeared on MRI after second-line therapy with alemtuzumab. Meanwhile, the CLEC16A::IL2 fusion mRNA remained detectable in PB. Allogeneic hematopoietic stem-cell transplantation was performed, and the fusion mRNA has now been undetectable for more than 5 years since transplantation. This is the first report of a T-PLL case with a CLEC16A::IL2 fusion gene.

Impact of CD34 positive cell dose in donor graft on the outcomes after haploidentical peripheral blood stem cell transplantation with post-transplant cyclophosphamide - A retrospective single-center study with a Japanese cohort.

BACKGROUND: Haploidentical peripheral blood stem cell transplantation (haplo-PBSCT) with post-transplant cyclophosphamide (PTCy) is an important therapeutic option for patients lacking an HLA-matched donor. However, the significance of CD34+ cell dose in grafts has not been fully elucidated. OBJECTIVE: We aimed to explore the impact of CD34+ cell dose on outcomes after haplo-PBSCT with PTCy. STUDY DESIGN: We retrospectively investigated 111 consecutive patients who underwent haplo-PBSCT with PTCy or HLA-matched PBSCT from related donors. RESULTS: There were no statistically significant differences in 3-year overall survival (p = 0.559) or progression-free survival (p = 0.974) between haplo-PBSCT and matched PBSCT. Delayed neutrophil engraftment and a lower incidence of graft-versus-host disease were observed in haplo-PBSCT. The median dose of CD34+ cells was 4.9 × 106 /kg in 57 haplo-PBSCT and 4.5 × 106 /kg in 54 matched PBSCTs. Importantly, patients who underwent haplo-PBSCT with the administration of CD34+ cell at a dose of ≥4.0 × 106 /kg significantly had improved OS (p = 0.015) and decreased incidence of disease relapse (p = 0.001) without increasing incidence of GVHD. CONCLUSION: Our data suggest that a higher dose of CD34+ cells in haplo-PBSCT with PTCy positively impacts the outcomes without an increase of GVHD.

Purging myeloma cell contaminants and simultaneous expansion of peripheral blood-mobilized stem cells.

Human hematopoietic stem cells (HSCs) are widely used as a cellular source for hematopoietic stem cell transplantation (HSCT) in the clinical treatment of hematological malignancies. After transplantation therapy, delays in hematopoietic recovery due to insufficient donor-derived HSCs can lead to increased risks of life-threatening infections and bleeding. Our previous studies developed an efficient ex vivo expansion culture medium (3a medium) for umbilical cord blood-derived HSCs (CBSCs), offering a potential solution to this problem. Nevertheless, the broader applicability of our culture method to alternative cell sources and, of greater significance, its efficacy in eliminating potentially disease-associated contaminated tumor cells, especially in autologous transplantation, raise critical clinical questions. In this study, we modified the 3a medium by incorporating UM729 to replace UM171, adding FMS-like tyrosine kinase 3 (Flt3) ligand, and adjusting the concentrations of butyzamide, 740Y-P, polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (PCL-PVAc-PEG, Soluplus) to create the modified-3a medium. This sophistication allowed the efficient expansion of not only CBSCs but also peripheral blood-mobilized HSCs (PBSCs). Additionally, we successfully removed contaminated myeloma cells by adding bortezomib and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) at appropriate concentrations, although we maintained HSCs through the addition of lenalidomide. Our research findings present the potential for widespread clinical application of the modified-3a medium and suggest a safe ex vivo culture technique for expanding human HSCs within peripheral blood-derived donor grafts used for autologous HSCT.

Safety of romiplostim administered immediately after cord-blood transplantation: a phase 1 trial.

Graft failure and delayed hematopoietic recovery are the major limitations of cord-blood transplantation (CBT). Romiplostim, a thrombopoietin-receptor agonist, promotes megakaryopoiesis and multilineage hematopoiesis in aplastic anemia. The decreased number of hematopoietic stem cells in the early phase after CBT and aplastic anemia share certain characteristics. Therefore, we hypothesized that romiplostim administration immediately after CBT may promote multilineage hematopoietic recovery. We investigated the safety and preliminary efficacy of administering romiplostim a day after CBT. This phase 1 dose-escalation study included six adults with hematologic malignancies in remission. Romiplostim was administered subcutaneously within 7 days after single-unit CBT, initially at doses of 5 µg/kg or 10 µg/kg in three patients, then once a week for 14 weeks or until platelet recovery. The maximum dose was 20 µg/kg. The median number of romiplostim administrations was 6 (range, 3-15). Romiplostim-related adverse events included bone pain (3/6) and injection site reaction (1/6). Non-hematological grade ≥ 3 toxicities were observed in four patients; febrile neutropenia was the most common (4/6). All patients achieved neutrophil engraftment and the median time was 14 days (range, 12-32). Platelet counts ≥ 50 × 109 /L were recorded in all patients except for one who died on day 48; the median time was 34 days (range, 29-98). No relapse, thrombosis, or bone marrow fibrosis was observed during a median follow-up of 34 months. Romiplostim may be safely administered in the early phase of CBT. Further phase 2 trial is warranted for its efficacy evaluation. Trial registration number: UMIN000033799, August 18, 2018.

Repeated immunosuppressive rabbit antithymocyte globulin therapy for adult patients with relapsed or refractory aplastic anemia.

OBJECTIVES: Immunosuppressive therapy (IST) with antithymocyte globulin (ATG) and cyclosporin A is the standard treatment for aplastic anemia (AA). However, the efficacy of repeated IST with rabbit ATG (rATG) as salvage therapy remains unclear in patients with relapsed or refractory AA. METHODS: We retrospectively evaluated the efficacy and safety of IST2 with rATG (IST2-rATG) in 19 consecutive patients with relapsed or refractory AA who received first-line IST with rATG in two centers between 2009 and 2020. RESULTS: The overall 6-month response rate of the patients was 58%. The response rates were similar between patients with relapsed and refractory AA. The presence of glycophosphatidylinositol-deficient blood cells was associated with a better response to IST2-rATG. Despite retreatment with the same rATG, serum disease and severe allergic reactions were not observed. CONCLUSION: IST2-rATG is effective and safe for the treatment of adult patients with relapsed and refractory AA after receiving first-line IST with rATG.

Risk factors for CAR-T cell manufacturing failure among DLBCL patients: A nationwide survey in Japan.

For successful chimeric antigen receptor T (CAR-T) cell therapy, CAR-T cells must be manufactured without failure caused by suboptimal expansion. In order to determine risk factors for CAR-T cell manufacturing failure, we performed a nationwide cohort study in Japan and analysed patients with diffuse large B-cell lymphoma (DLBCL) who underwent tisagenlecleucel production. We compared clinical factors between 30 cases that failed (7.4%) with those that succeeded (n = 378). Among the failures, the proportion of patients previously treated with bendamustine (43.3% vs. 14.8%; p < 0.001) was significantly higher, and their platelet counts (12.0 vs. 17.0 × 104 /µL; p = 0.01) and CD4/CD8 T-cell ratio (0.30 vs. 0.56; p < 0.01) in peripheral blood at apheresis were significantly lower than in the successful group. Multivariate analysis revealed that repeated bendamustine use with short washout periods prior to apheresis (odds ratio [OR], 5.52; p = 0.013 for ≥6 cycles with washout period of 3-24 months; OR, 57.09; p = 0.005 for ≥3 cycles with washout period of <3 months), low platelet counts (OR, 0.495 per 105 /µL; p = 0.022) or low CD4/CD8 ratios (

[Waldenström macroglobulinemia complicated by peripheral neuropathy due to neural infiltration].

A 51-year-old man with the chief complaint of glove- and stocking-type dysesthesia for >3 years was diagnosed with Waldenström's macroglobulinemia (WM) based on IgM-type M-proteinemia, bone marrow infiltration of plasmacytoid B cells, multiple lymphadenopathies, and splenomegaly. A nerve conduction examination suggested demyelinating neuropathy. Serum anti-myelin-associated glycoprotein antibody was negative. Sural nerve biopsy showed myelin thinning, suggesting demyelination. Axonal damage and tumor cell infiltration in the intrafascicular epineurium were also observed. After chemotherapies with rituximab and bendamustine, M-proteinemia and lymphadenopathies disappeared. However, abnormalities in the nerve conduction examination and dysesthesia were only slightly alleviated. As articles describing patients with WM with peripheral nerve infiltration are limited, we report this case with a literature review.

[Hematopoietic recovery by ASXL1-mutated clones after immune suppressive therapy in a patient with severe aplastic anemia].

Sequencing technology has identified aplastic anemia (AA) not only as an autoimmune bone marrow failure syndrome, but also as a clonal hematopoietic disease. Here, we present a case in which an ASXL1-mutated clone was predominantly expanded during the treatment of AA. A 58-year-old man with chronic glomerulonephritis on maintenance hemodialysis presented with pancytopenia. The findings of bone marrow biopsy indicated a hypoplastic bone marrow. Magnetic resonant imaging showed fatty changes in the bone marrow. The patient was eventually diagnosed with severe AA. He was treated with anti-human thymocyte globulin, cyclosporine, granulocyte colony-stimulating factor, and the thrombopoietin receptor agonist (TPO-RA) eltrombopag. After switching to another TPO-RA, romiplostim, the neutrophil, reticulocyte, and platelet counts gradually improved, and blood transfusion was not needed 1 year after treatment. Mutational analyses revealed that reconstituted hematopoietic cells originated from the ASXL1-mutated clone. Nevertheless, the patient's blood cell counts remained normal 2 years after treatment.

[Lymphoplasmacytic lymphoma accompanied by severe myelofibrosis].

A 61-year-old female was referred to our hospital because of pancytopenia and febrile neutropenia. On admission, computed tomography showed mild hepatosplenomegaly and intra-abdominal abscess formation in the right pelvic region; however, no lymphadenopathy was found. Bone marrow (BM) examination showed severe fibrosis by silver staining. Several small- to medium-sized lymphocytes with a constriction in the nuclei were observed, exhibiting CD3 (-), CD10 (-), CD20 (+), BCL-2 (+-), and CD138 (+-). Genetic testing revealed that BM cells were positive for MYD88 mutation and positive for IgH rearrangement, whereas neither JAK2 nor CALR mutation was positive. A diagnosis of BM infiltration of lymphoplasmacytic lymphoma (LPL) was made. Rituximab monotherapy was administered once a week for four times. BM examination 4 weeks after the end of treatment showed that lymphoma cells had disappeared and that myelofibrosis had been almost gone. The MYD88 mutation of BM turned out to be negative at that moment.

Chemically defined cytokine-free expansion of human haematopoietic stem cells.

Haematopoietic stem cells (HSCs) are a rare cell type that reconstitute the entire blood and immune systems after transplantation and can be used as a curative cell therapy for a variety of haematological diseases1,2. However, the low number of HSCs in the body makes both biological analyses and clinical application difficult, and the limited extent to which human HSCs can be expanded ex vivo remains a substantial barrier to the wider and safer therapeutic use of HSC transplantation3. Although various reagents have been tested in attempts to stimulate the expansion of human HSCs, cytokines have long been thought to be essential for supporting HSCs ex vivo4. Here we report the establishment of a culture system that allows the long-term ex vivo expansion of human HSCs, achieved through the complete replacement of exogenous cytokines and albumin with chemical agonists and a caprolactam-based polymer. A phosphoinositide 3-kinase activator, in combination with a thrombopoietin-receptor agonist and the pyrimidoindole derivative UM171, were sufficient to stimulate the expansion of umbilical cord blood HSCs that are capable of serial engraftment in xenotransplantation assays. Ex vivo HSC expansion was further supported by split-clone transplantation assays and single-cell RNA-sequencing analysis. Our chemically defined expansion culture system will help to advance clinical HSC therapies.

Cardiac Tamponade as a Recurrence of Angioimmunoblastic T-Cell Lymphoma with the Detection of a p.Gly17Val RHOA Mutation in the Pericardial Effusion.

Angioimmunoblastic T-cell lymphoma (AITL) is an intractable type of T-cell lymphoma. We and others have identified that the p.Gly17Val RHOA mutation is specifically identified in AITL. We herein report a patient whose condition deteriorated, resulting from massive pericardial effusion one month after undergoing autologous transplantation for AITL. He was diagnosed with cardiac tamponade caused by AITL recurrence in the presence of the p.Gly17Val RHOA mutation as well as T-lineage cells with an aberrant immune-phenotype in the pericardial effusion. This case suggests that a precision medicine approach by detecting the presence of a p.Gly17Val RHOA mutation is useful for the management of AITL.

Triple-negative Thrombocythemia and Subsequent Acute Lymphoblastic Leukemia with Additional Somatic Mutations.

Triple-negative essential thrombocythemia (ET) is a condition in which mutations in JAK2, CALR and MPL are all negative. Transformation to acute myeloid leukemia may occur during the course of ET, while B-acute lymphoblastic leukemia B-(ALL) is rare. We experienced a case diagnosed as B-ALL during the course of triple-negative ET. Notably, cytoreduction was required for the excessive increase in blood cells during the bone marrow recovery period after chemotherapies. Whole exome sequencing identified 17 somatic mutations: 9 were identified in both ET and B-ALL samples, while 8 were specific to B-ALL, suggesting that these 8 might have caused the transformation.

[Acute myeloid leukemia harboring NUP98::DDX10].

NUP98::DDX10 is a rare fusion gene associated with acute myeloid leukemia (AML), for which the prognosis and indication for allogeneic hematopoietic stem cell transplantation are unknown. A 48-year-old woman was diagnosed with AML harboring NUP98::DDX10. The results of quantitative RT-PCR of the fusion mRNA as a minimal residual disease (MRD) marker guided the treatment. In August 2019, the patient achieved hematological remission following standard remission induction therapy with idarubicin and cytarabine. After four cycles of consolidation therapies, MRD was detected, and she underwent allogeneic stem cell transplantation in May 2020. As MRD persisted in June, the immunosuppressant was stopped and three cycles of azacitidine were administered. Despite this, a hematological relapse occurred in January 2021 that was resistant to high-dose cytarabine and an investigational agent. She died as a result of the disease's progression. Thus, a second thought should be given to the timing of transplantation, the bridging, and the intervention for relapse after transplantation. The cases must be accumulated.

Administration of brentuximab vedotin to a Hodgkin lymphoma patient with liver dysfunction due to vanishing bile duct syndrome resulting in a partial response without any severe adverse events.

Vanishing bile duct syndrome (VBDS) is a rare hepatic disorder which leads to liver failure as a result of progressive destruction of the intrahepatic bile ducts. There are no treatment modalities for VBDS itself and severe hepatic dysfunction restricts the treatment of underlying diseases. We safely treated a case of classic Hodgkin lymphoma (HL) with VBDS using brentuximab vedotin (BV). The patient was treated with 5 cycles of reduced BV and a partial metabolic response was obtained. Moreover, a standard dose of BV for another 5 cycles was accomplished with minimal adverse events. Our experience indicates that BV could be a treatment option for classic HL with VBDS.

Hemolysis induced by SARS-CoV-2 mRNA vaccination in patients with paroxysmal nocturnal hemoglobinuria.

Autoimmune and complement-related hematological side effects have been observed with messenger ribonucleic acid (mRNA) vaccines. Here, we report the incidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine-induced hemolysis in patients with paroxysmal nocturnal hemoglobinuria (PNH). We reviewed the medical records of seventeen patients with PNH visiting the University of Tsukuba Hospital who had received two doses of the SARS-CoV-2 mRNA vaccine between May 2021 and November 2021. Twelve patients were being treated with complement inhibitors. The median age of all patients was 62 years (range 29-89 years).; six were males and eleven were females. Fourteen patients received the BNT162b2 vaccine (Pfizer/BioNTech) and three received the mRNA-1273 vaccine (Moderna). The median percentages of PNH clones in erythrocytes and granulocytes were 37.61% (range 8.11-85.71%) and 59.73% (range 3.76-97.82%), respectively. Of the twelve patients receiving complement inhibitors, only one had a hemolytic reaction after vaccination, but it did not meet the definition of breakthrough hemolysis. By contrast, hemolytic attacks were observed in two of the five untreated patients with PNH, and one of them required a blood transfusion. Appropriate administration of complement inhibitors to patients with PNH may prevent hemolysis induced by SARS-CoV-2 mRNA vaccination.

A single-cell atlas of non-haematopoietic cells in human lymph nodes and lymphoma reveals a landscape of stromal remodelling.

The activities of non-haematopoietic cells (NHCs), including mesenchymal stromal cells and endothelial cells, in lymphomas are reported to underlie lymphomagenesis. However, our understanding of lymphoma NHCs has been hampered by unexplained NHC heterogeneity, even in normal human lymph nodes (LNs). Here we constructed a single-cell transcriptome atlas of more than 100,000 NHCs collected from 27 human samples, including LNs and various nodal lymphomas, and it revealed 30 distinct subclusters, including some that were previously unrecognized. Notably, this atlas was useful for comparative analyses with lymphoma NHCs, which revealed an unanticipated landscape of subcluster-specific changes in gene expression and interaction with malignant cells in follicular lymphoma NHCs. This facilitates our understanding of stromal remodelling in lymphoma and highlights potential clinical biomarkers. Our study largely updates NHC taxonomy in human LNs and analysis of disease status, and provides a rich resource and deeper insights into LN and lymphoma biology to advance lymphoma management and therapy.

Salvage Cord Blood Transplantation Using a Short-term Reduced-intensity Conditioning Regimen for Graft Failure.

Objective Graft failure (GF) is a life-threatening complication of hematopoietic stem cell transplantation (HSCT). A standardized conditioning regimen and an appropriate graft source of salvage HSCT for GF have not yet been established. Some case series have shown good hematopoietic recoveries after salvage HSCT using a short-term reduced-intensity preparative regimen consisting of fludarabine (30-90 mg/m2), cyclophosphamide (2 g/m2), and total-body irradiation (2 Gy). However, the dose of fludarabine has varied in these reports based on the clinical condition of the patients, resulting in very limited experiences with each dose of fludarabine. Methods We retrospectively analyzed 10 patients who developed GF after allogeneic HSCT and underwent salvage cord blood transplantation (CBT) using the above-mentioned conditioning regimen with a fixed dose (90 mg/m2) of fludarabine. Results Eight patients (80.0%) achieved neutrophil engraftment within 30 days from salvage HSCT with a median of 21 (range, 17-23) days. The 1-year overall survival (OS) rate after the salvage HSCT was 50.0%, and the median OS was 281 (range, 23-1,638) days. Cumulative incidences of non-relapse mortality and relapse at 1 year were 50.0% and 10.0%, respectively. Conclusion CBT using this short-term reduced-intensity conditioning regimen may be a promising salvage therapy for GF.