A longitudinal analysis of paroxysmal nocturnal haemoglobinuria-type cells in patients with bone marrow failure: Results of a prospective multi-centre study in Japan.

To determine the prevalence and clinical relevance of glycosylphosphatidylinositol-anchored protein-deficient (GPI[-]) cell populations (paroxysmal nocturnal haemoglobinuria [PNH]-type cells) in patients with acquired aplastic anaemia (AA) or myelodysplastic syndrome (MDS), we prospectively studied peripheral blood samples of 2402 patients (1075 with AA, 900 with MDS, 144 with PNH, and 283 with other anaemia) using a high-sensitivity flow cytometry assay in a nationwide multi-centre observational study. PNH-type cells were detected in 52.6% of AA and 13.7% of MDS patients. None of the 35 patients with refractory anaemia (RA) with ringed sideroblasts or the 86 patients with RA with excess of blasts carried PNH-type cells. Among the 317 patients possessing PNH-type granulocytes, the percentage of PNH-type granulocytes increased by ≥10% in 47 patients (14.8%), remained unchanged in 240 patients (75.7%), and decreased by ≥10% in 30 patients (9.5%) during 3 years of follow-up. PNH-type granulocyte expansion occurred more frequently (27.1%) in the 144 patients who originally carried PNH-type granulocytes ≥1% than in the 173 patients with PNH-type granulocytes <1% (4.6%). This study confirmed that PNH-type cells are undetectable in authentic clonal MDS patients, and the presence of ≥1% PNH-type granulocytes predicts a higher likelihood of PNH-type cell expansion than with <1% PNH-type granulocytes.

Efficacy and safety of romiplostim in refractory aplastic anaemia: a Phase II/III, multicentre, open-label study.

A previous dose-finding study has suggested that romiplostim is effective in patients with refractory aplastic anaemia (AA) and 10 µg/kg once weekly was recommended as a starting dose. In this Phase II/III, multicentre, open-label study, romiplostim was administered subcutaneously at a fixed dose of 10 µg/kg once weekly for 4 weeks (weeks 1-4) followed by weekly doses (5, 10, 15 and 20 µg/kg) titrated by platelet response for up to 52 weeks (weeks 5-52). A total of 31 patients with AA who were refractory to immunosuppressive therapy (IST) and thrombocytopenia (platelet count of ≤30 × 109 /l) were enrolled. The primary efficacy endpoint of the proportion of patients achieving any haematological (platelet, neutrophil and erythrocyte) response at week 27 was 84% [95% confidence interval (CI) 66-95%]. Trilineage response was 39% (95% CI 22-58%) at week 53. The most common treatment-related adverse events (AEs) were headache and muscle spasms (each 13%). All AEs were mild or moderate except for three patients with Grade 3 hepatic AEs; no AEs necessitated romiplostim discontinuation. Two patients developed cytogenetic abnormalities, of whom one returned to normal karyotype at last follow-up. High-dose romiplostim is effective and well tolerated in the treatment of patients with AA refractory to IST.

The clinical significance of PNH-phenotype cells accounting for < 0.01% of total granulocytes detected by the Clinical and Laboratory Standards Institute methods in patients with bone marrow failure.

Small populations of glycosylphosphatidylinositol-anchored protein-deficient (GPI[-]) cells accounting for up to 0.01% of total granulocytes can be accurately detected by a high-sensitivity flow cytometry (FCM) assay established by the Clinical and Laboratory Standards Institute (CLSI method) and have a prognostic value in bone marrow failure (BMF); however, the significance of GPI(-) granulocytes accounting for 0.001-0.009% of granulocytes remains unclear. To clarify this issue, we examined the peripheral blood of 21 BMF patients in whom minor (around 0.01%) populations of GPI(-) granulocytes had been previously detected by a different high-resolution FCM method (OPTIMA method, which defines ≥ 0.003% GPI(-) granulocytes as an abnormal increase) using both the CLSI and OPTIMA methods simultaneously. These two methods detected an "abnormal increase" in GPI(-) granulocytes in 10 patients (48%) and 17 patients (81%), respectively. CLSI detected 0.002-0.005% (median, 0.004%) GPI(-) granulocytes in 7 patients who were deemed positive for PNH-type cells according to the OPTIMA method, which detected 0.003-0.012% (median 0.006%) GPI(-) granulocytes. The clone sizes of GPI(-) cells detected by each assay were positively correlated (r = 0.994, p < 0.001). Of the seven patients who were judged positive for PNH-type cells by OPTIMA alone, five received immunosuppressive therapy, and all of them achieved a partial or complete response. GPI(-) granulocytes detected in BMF patients by the CLSI method should thus be considered significant, even at percentages of < 0.01%.

[Emerging infectious disease and transfusion-transmitted infection].

Hepatitis B, hepatitis C and HIV infections are well-known infectious diseases caused by blood products, but recently there have been almost no reports. In 2011, hepatitis E virus (HEV) antibody test was covered by insurance in Japan, and certain numbers of transfusion-transmitted HEV were reported, and five cases were recognized in 2019. As the Japanese Red Cross has started to examine individual NAT of HEV for all blood donors since August 2020, and the number of transfusion-transmitted HEV will decrease. In addition, Trypanosoma cruzi antibody test for selective blood donors has been examined since August 2016, and the risk of Chagas disease infection by blood transfusion will be decreased. In the future, it is important for protecting against the transfusion-transmitted infection to inspect bacterial tests and know the information of emerging infectious disease occurring overseas. When the emerging infectious diseases such as DENV (dengue virus), WNV (West Nile virus), Zika (Zika virus) and CHIKV (chikungunya virus) occur in Japan, it is necessary to stimulate countermeasures and prepare what kind of tests should be performed for blood donors or criteria for blood donation.

Soluble NKG2D Ligands Are Potential Biomarkers and Sentinels of Immune-Mediated Bone Marrow Injury in Bone Marrow Failure Syndromes.

Immune-mediated processes are considered important in the pathogenesis of bone marrow failure syndromes (BFS). We previously reported that natural killer group 2D (NKG2D) ligands were expressed on pathological blood cells of patients with BFS and that NKG2D immunity may be involved in bone marrow failure. In addition to membranous NKG2D ligands on the cell surface, soluble NKG2D ligands can exist in plasma. We therefore examined the relationship between soluble NKG2D ligands and blood cell counts in 86 patients with BFS, including aplastic anemia, myelodysplastic syndrome with single lineage dysplasia, and paroxysmal nocturnal hemoglobinuria. Approximately half of the BFS patients were positive for soluble NKG2D ligands in the plasma by enzyme-linked immunosorbent assay, and soluble NKG2D ligand-positive BFS patients exhibited severe cytopenia regardless of membranous NKG2D ligand expression. In vitroanalyses demonstrated that soluble ULBP1, an NKG2D ligand, down-regulated NKG2D receptors on CD2-positive cells in peripheral blood. Moreover, soluble ULBP1 attenuated the cytotoxic effects of peripheral blood mononuclear cells on K562, which express membranous ULBP1. Our results suggest that soluble NKG2D ligands can be easy-to-measure biomarkers for the prediction of activity of immune-meditated bone marrow injury in BFS and that soluble NKG2D ligands suppress redundant immune-mediated bone marrow injury.

Reduction in adverse transfusion reactions with increased use of washed platelet concentrates in Japan-A retrospective multicenter study.

Plasma removal by washing platelet concentrates (PCs) is effective in preventing adverse reactions to PC transfusions. The Japanese Red Cross Society (JRCS) started releasing washed PCs (WPCs) as a commercially approved blood product in September 2016. This retrospective multicenter study investigated the change in the number of transfused WPCs and the impact on the incidence of adverse reactions to PCs before and after the release. The numbers and types of transfused PCs and the adverse reactions to the PCs for a year before the start of the WPC release and for a year after the release were reported by 27 medical institutes in Japan. Transfusion information for approximately 8% of the amount of PCs supplied in Japan was analyzed during the study period. After the start of WPC release by the JRCS, the number of transfused WPCs doubled. The rate of adverse reactions to PCs decreased significantly (p = 0.0223), from 4.30% before the release to 4.05% after the release. The rates of adverse reactions to unwashed and WPCs were 4.13% and 0.84%, respectively. Allergic adverse reactions were significantly decreased after the release (3.60% before versus 3.37% after). No severe allergic reactions to WPCs were reported. The release of WPCs by the JRCS significantly reduced transfusion-related adverse reactions to PCs in Japan.

Establishment of a flow cytometry assay for detecting paroxysmal nocturnal hemoglobinuria-type cells specific to patients with bone marrow failure.

Minor populations of glycosylphosphatidylinositol-anchored protein-deficient (GPI[-]) cells in the peripheral blood may have a prognostic value in bone marrow failure (BMF). Our objective is to establish the optimal flow cytometry (FCM) assay that can discriminate GPI(-) populations specific to BMF from those of healthy individuals. To identify a cut-off that discriminates GPI(-) rare cells from GPI(+) cells, we determined a position of the borderline that separates the GPI(-) from GPI(+) cells on a scattergram by testing more than 30 healthy individuals, such that no GPI(-) dot fell into the upper left quadrant where fluorescein-labeled aerolysin (FLAER)-CD11b+ granulocytes and CD55-CD59- glycophorin A+ erythrocytes were positioned. This method allowed us to define ≥ 0.003% CD11b+FLAER- granulocytes and ≥ 0.005% glycophorin A+CD55-CD59- erythrocytes to be specific to BMF patients. Longitudinal cross-validation studies showed minimal (< 0.02%) inter-laboratory differences in the GPI(-) cell percentage. An analysis of 1210 patients with BMF revealed a GPI(-) cell population in 56.3% of patients with aplastic anemia and 18.5% of patients with myelodysplastic syndrome. The GPI(-) granulocyte percentages was 0.003-0.01% in 3.7% of patients. This FCM assay effectively identified an increase in the percentage of GPI(-) rare cells that are specific to BMF patients and allowed different laboratories to accurately detect 0.003-0.01% of pathological GPI(-) cells.

A nationwide survey of hypoplastic myelodysplastic syndrome (a multicenter retrospective study).

Hypoplastic myelodysplastic syndrome (hMDS) is a distinct entity with bone marrow (BM) hypocellularity and the risk of death from BM failure (BMF). To elucidate the characteristics of hMDS, the data of 129 patients diagnosed between April 2003 and March 2012 were collected from 20 institutions and the central review team of the National Research Group on Idiopathic Bone Marrow Failure Syndromes, and compared with 115 non-hMDS patients. More RA and fewer CMMoL and RAEB-t in French-American-British (FAB) and more RCUD and MDS-U and fewer RCMD in World Health Organization (WHO) classifications were found in hMDS than non-hMDS with significant differences. The overall survival (OS) and AML progression-free survival (AML-PFS) of hMDS were higher than those of non-hMDS, especially in patients at age ≥50 and of lower risk in Revised International Prognostic Scoring System (IPSS-R). In competing risks analysis, hMDS exhibited decreased risk of AML-progression in lower IPSS or IPSS-R risk patients, and higher risk of death from BMF in patients at age ≥50. Poor performance status (PS ≥2) and high karyotype risks in IPSS-R (high and very high) were significant risk factors of death and AML-progression in Cox proportional hazards analysis.

Long-term outcome of patients with acquired chronic pure red cell aplasia (PRCA) following immunosuppressive therapy: a final report of the nationwide cohort study in 2004/2006 by the Japan PRCA collaborative study group.

Immunosuppressive therapy has been employed as the initial treatment for acquired chronic pure red cell aplasia (PRCA), such as idiopathic, thymoma-associated, or large granular lymphocyte (LGL) leukaemia-associated PRCA, which is thought to be immune-mediated. To explore the overall long-term outcome following immunosuppression and to identify the risk factors for death in these disorders, we conducted nationwide surveys in Japan 2004 and 2006, and identified a total of 185 patients with acquired chronic PRCA, including 72 idiopathic, 41 thymoma-associated and 14 LGL leukaemia-associated cases of PRCA for whom data was available. The present study evaluated 127 patients with these three subsets of PRCA. The median overall survival has not yet been reached in idiopathic PRCA. The estimated median overall survival times in patients with thymoma-associated and LGL leukaemia-associated PRCA were 142·1 and 147·8 months, respectively. Twenty-two deaths were reported, and the response to induction therapy and relapse of anaemia were found to be associated with death. The major causes of death were infection in seven patients and organ failure in another seven patients. The results suggest that maintenance therapy and the management of infectious complications are crucial for improving the prognosis of chronic PRCA.

[Regression of primary rectal MALT lymphoma after Helicobacter pylori eradication].

A 74-year-old-man was referred to our hospital to undergo examination and therapy for a rectal polypoid lesion. MALT lymphoma of the rectum (stage IE) was diagnosed by performing a biopsy of the tumor during colonoscopy. The patient received eradication therapy against Helicobacter pylori (H. pylori) because of chronic gastritis due to H. pylori, and a complete remission was documented after this therapy. However, MALT lymphoma recurred 18 months later. Our observations in this case suggest that eradication therapy against H. pylori may be useful not only for gastric MALT lymphoma but also for colorectal MALT lymphoma.

Online reporting system for transfusion-related adverse events to enhance recipient haemovigilance in Japan: a pilot study.

BACKGROUND: A surveillance system for transfusion-related adverse reactions and infectious diseases in Japan was started at a national level in 1993, but current reporting of events in recipients is performed on a voluntary basis. A reporting system which can collect information on all transfusion-related events in recipients is required in Japan. METHODS: We have developed an online reporting system for transfusion-related events and performed a pilot study in 12 hospitals from 2007 to 2010. RESULTS: The overall incidence of adverse events per transfusion bag was 1.47%. Platelet concentrates gave rise to statistically more adverse events (4.16%) than red blood cells (0.66%) and fresh-frozen plasma (0.93%). In addition, we found that the incidence of adverse events varied between hospitals according to their size and patient characteristics. CONCLUSION: This online reporting system is useful for collection and analysis of actual adverse events in recipients of blood transfusions and may contribute to enhancement of the existing surveillance system for recipients in Japan.

Pembrolizumab-related Immune Thrombocytopenia in a Patient with Lung Adenocarcinoma Treated by Radiotherapy: Potential Immune-related Adverse Event Elicited by Radiation Therapy.

The effect of radiotherapy during immunotherapy on immune-related adverse events (irAEs) is not fully understood. We herein report a 74-year-old woman diagnosed with lung adenocarcinoma with programmed death ligand 1 expression ≥50% and treated with pembrolizumab. She developed fatal immune thrombocytopenia associated with pembrolizumab immediately following radiotherapy. A flow cytometry analysis of peripheral blood detected an increased expression of programmed death-1 (PD-1) and Ki-67 in CD4+ and CD8+ T cells after radiotherapy, compared with pre-irradiation measurements. This case suggests that radiotherapy may evoke irAEs during treatment with anti-PD-1 antibodies, which physicians should consider when using radiotherapy in patients treated with these drugs.

Long-term follow-up of patients with paroxysmal nocturnal hemoglobinuria treated with eculizumab: post-marketing surveillance in Japan.

All Japanese patients with paroxysmal nocturnal hemoglobinuria (PNH) treated with eculizumab were enrolled in post-marketing surveillance (PMS) between June 2010 and August 2019 to assess the long-term effectiveness and safety of eculizumab. The reduction in intravascular hemolysis, the change in hemoglobin (Hb) level, and the change in renal function were assessed to determine the effectiveness of eculizumab. The types and frequencies of adverse events (AEs) were assessed to determine its safety. A total of 632 patients were enrolled and the median treatment duration was 3.6 years. Treatment with eculizumab significantly reduced lactate dehydrogenase (LDH) levels and significantly increased Hb levels. These changes were maintained for up to 5 years of treatment. An estimated glomerular filtration rate ≥ 60 ml/min/1.73 m2 and higher LDH level at baseline were associated with increases in Hb levels during eculizumab treatment. The overall incidence of any AE was 69.92/100 patient-years. Hemolysis was the most common AE (6.43/100 patient-years). The incidence of infection-related AEs was 20.57/100 patient-years, and included meningococcal infection in three patients (0.12/100 patient-years). This long-term follow-up of patients with PNH demonstrated the sustained effectiveness of eculizumab and supports its well-established safety profile.

Glycosylated ferritin as an improved marker for post-transfusion iron overload.

Red blood cell (RBC) transfusion is an effective therapy for anemia, but repeated transfusions may cause iron overload-related damage to various organs. Iron chelation therapy, now widely available for patients who have received transfusions, is expected to reduce organ damage even in patients who received many transfusions. Therefore, determining when to start iron chelation therapy is important. In guidelines for iron chelation therapy, the serum ferritin level has been widely accepted as a practical marker for estimating iron overload. However, guidelines recommend multiple measurements of serum ferritin, because levels often fluctuate. Here, we investigated the usefulness of glycosylated ferritin as a marker of iron overload using a cohort consisted of 103 patients who had a total ferritin value over 1000 ng/mL. We found that the volume of RBCs transfused was clearly associated with the glycosylated ferritin level. We also found that acute inflammation, as represented by C-reactive protein values, was associated with increased non-glycosylated ferritin and that patients with hematopoietic diseases had higher glycosylated ferritin levels, possibly because of repeated RBC transfusions. We thus conclude that glycosylated ferritin may be an improved marker for predicting iron overload status.

Results from multinational phase 3 studies of ravulizumab (ALXN1210) versus eculizumab in adults with paroxysmal nocturnal hemoglobinuria: subgroup analysis of Japanese patients.

Ravulizumab demonstrated noninferior efficacy and comparable safety to eculizumab in two open-label, phase 3 studies in patients with paroxysmal nocturnal hemoglobinuria (PNH) who complement inhibitor-naive (Study 301) or were previously treated with eculizumab (Study 302). This subgroup analysis assessed ravulizumab's efficacy and safety in Japanese patients in Studies 301 and 302, who are known to have different clinicopathologic features from white patients. Patients were randomly assigned (1:1) to eculizumab every-two-weeks or weight-based dosing of ravulizumab every-eight-weeks for 26 weeks. Co-primary endpoints were transfusion avoidance and lactate dehydrogenase (LDH) normalization in Study 301 and percentage change in LDH levels from baseline to day 183 in Study 302. Thirty-three Japanese patients (n = 18 ravulizumab; n = 15 eculizumab) enrolled in Study 301; 12 enrolled in Study 302 (n = 5 ravulizumab; n = 7 eculizumab). In the Study 301 ravulizumab group, 83.3% (15/18) of patients avoided transfusion; the adjusted prevalence of LDH normalization was 52.1%. In the Study 302 ravulizumab group, the least-squares-mean percentage change from baseline in LDH was 8.34%. No deaths or meningococcal infections occurred during the 6-month primary evaluation period in either study. In conclusion, ravulizumab's efficacy and safety were consistent in the Japanese and global patient populations with PNH in the phase 3 studies. Clinical Trial Identifier: NCT02946463; NCT03056040.

NKG2D-mediated immunity underlying paroxysmal nocturnal haemoglobinuria and related bone marrow failure syndromes.

It is considered that a similar immune mechanism acts in the pathogenesis of bone marrow (BM) failure in paroxysmal nocturnal haemoglobinuria (PNH) and its related disorders, such as aplastic anaemia (AA) and myelodysplastic syndromes (MDS). However, the molecular events in immune-mediated marrow injury have not been elucidated. We recently reported an abnormal expression of stress-inducible NKG2D (natural-killer group 2, member D) ligands, such as ULBP (UL16-binding protein) and MICA/B (major histocompatibility complex class I chain-related molecules A/B), on granulocytes in some PNH patients and the granulocyte killing by autologous lymphocytes in vitro. The present study found that the expression of NKG2D ligands was common to both granulocytes and BM cells of patients with PNH, AA, and MDS, indicating their exposure to some incitement to induce the ligands. The haematopoietic colony formation in vitro by the patients' marrow cells significantly improved when their BM cells were pretreated with antibodies against NKG2D receptor, suggesting that the antibodies rescued haematopoietic cells expressing NKG2D ligands from damage by autologous lymphocytes with NKG2D. Clinical courses of patients with PNH and AA showed a close association of the expression of NKG2D ligands with BM failure and a favourable response to immunosuppressive therapy. We therefore propose that NKG2D-mediated immunity may underlie the BM failure in PNH and its-related marrow diseases.

Long-term response and outcome following immunosuppressive therapy in thymoma-associated pure red cell aplasia: a nationwide cohort study in Japan by the PRCA collaborative study group.

BACKGROUND: Thymoma-associated pure red cell aplasia (PRCA) accounts for a significant proportion of cases of secondary PRCA and immunosuppressive therapy has been reported to be useful in this condition. However, because of its rarity, the long-term response and relapse rates after immunosuppressive therapy are largely unknown, and optimal management of this disorder remains unclear. The aim of this study was to collect more information on the outcome of patients with thymoma-associated PRCA. DESIGN AND METHODS: We conducted a nationwide survey in Japan. From a total of 185 patients, comprising 73 with idiopathic and 112 with secondary PRCA, 41 patients with thymoma were evaluated for this report. End-points of this study were the response rate, duration of the response after immunosuppressive therapy and overall survival. RESULTS: Surgical removal of thymoma was reported in 36 patients, 16 of whom developed PRCA at a median of 80 months post-thymectomy. First remission induction therapy was effective in 19 of 20 patients treated with cyclosporine, 6 of 13 patients treated with corticosteroids and 1 of 1 treated with cyclophosphamide. No cyclosporine-responders relapsed within a median observation period of 18 months (range; 1 to 118 months). Relapse of anemia was observed in three corticosteroid-responders who did not receive additional cyclosporine. Only two patients were in remission after stopping therapy for 19 and 67 months. The estimated median overall survival time of all patients was 142 months. CONCLUSIONS: Thymoma-associated PRCA showed an excellent response to cyclosporine and cyclosporine-containing regimens were effective in preventing relapse of anemia. It does, however, remain uncertain whether cyclosporine can induce a maintenance-free hematologic response.