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Although Castleman disease was first described in 1956, this disease includes various conditions, including unicentric Castleman disease with hyaline vascular histology, human herpesvirus-8 (HHV-8) related multicentric Castleman disease, idiopathic multicentric Castleman disease, and mimics of Castleman disease associated with other conditions. To date, Castleman disease remains incompletely understood due to its rareness and difficulties in clinical and pathological diagnosis. TAFRO syndrome was reported in Japan in 2010. Because lymph node histology is similar in patients with TAFRO syndrome and Castleman disease, TAFRO syndrome is described as a related disorder of Castleman disease. Clinically, however, these conditions differ markedly. Although elevated interleukin-6 (IL-6) expression is characteristic of Castleman disease, increased expression of IL-6 may occur in patients with other diseases, making elevated IL-6 unsuitable for differential diagnosis. Further understanding of these disorders requires the identification of novel disease-specific biomarkers. This review article therefore outlines the characteristics of Castleman disease and TAFRO syndrome.

TAFRO syndrome is a systemic inflammatory disorder of unknown etiology characterized by thrombocytopenia, anasarca, fever, reticulin myelofibrosis, renal dysfunction, and organomegaly. Mortality in patients with this syndrome is high; however, an optimal treatment strategy has not been established. To explore the strategy, we retrospectively analyzed 81 patients with TAFRO syndrome registered in the Multicenter Collaborative Retrospective Study for Establishing the Concept of TAFRO Syndrome in Japan by December 2019. Sixty-eight patients received corticosteroid therapy as the first-line treatment, and as the second-line treatment, 21 received tocilizumab (Toc), 14 received cyclosporine A (CsA), and 8 received rituximab (Rit) in addition to corticosteroids. We compared these second-line treatment groups by setting the primary endpoint as time to next treatment or death (TTNT). Kaplan–Meier analysis showed that the median TTNT in the Toc, CsA, and Rit groups were 2.8 months, 9.2 months, and not reached, respectively. The TTNT of the Rit group was significantly longer than that of the Toc group. In contrast, there were no significant differences in overall survival between groups, indicating that subsequent salvage therapies rescued a large proportion of patients who failed the second-line treatments. Further studies are warranted to establish the optimal treatment strategies for this syndrome.

Various systems for predicting the prognosis of patients with myelodysplastic syndromes (MDS) have been developed. However, associations between performance status (PS) and prognosis of MDS require further investigation. To objectively assess the impact of PS on survival, we examined laboratory findings associated with PS, including serum levels of C-reactive protein (CRP), albumin (ALB), and total cholesterol (CHOL). Patients (n = 123; male 86, female 37; median age 74 yrs.) diagnosed with MDS or myelodysplastic/myeloproliferative neoplasms at Kanazawa Medical University Hospital between 2010 and 2020 were enrolled and grouped by cutoff values determined by receiver operating characteristic analysis: 0.44 mg/dL for CRP, 4.0 g/dL for ALB, and 120 mg/dL for CHOL. The median follow-up period was 17.6 months. Kaplan-Meier analysis revealed that overall survival (OS) in the high CRP, low ALB, and low CHOL groups was significantly shorter than in the low CRP, high ALB, and high CHOL groups, respectively. Multivariable analysis revealed that elevated serum CRP was an independent prognostic risk factor independent of gender, bone marrow blast percentage, and cytogenetics.

X-linked sideroblastic anemia (XLSA) is associated with mutations in the erythroid-specific δ-aminolevulinic acid synthase (ALAS2) gene. Treatment for XLSA is mainly supportive, except in pyridoxine-responsive patients. Female XLSA often represents a late onset of severe anemia, mostly due to the acquired skewing of X-chromosome inactivation. Here, we successfully generated active wild-type and mutant ALAS2 induced pluripotent stem cell (iPSC) lines from the peripheral blood cells of an affected mother and two daughters in a family with pyridoxine-resistant XLSA due to a heterozygous ALAS2 missense mutation (R227C). The erythroid differentiation potential was severely impaired in active mutant iPSC lines compared to that in active wild-type iPSC lines. Most of the active mutant iPSC-derived erythroblasts revealed an immature morphological phenotype, and some showed dysplasia and perinuclear iron deposits. Additionally, globin and HO-1 expression and heme biosynthesis in active mutant erythroblasts were severely impaired compared to that in active wild-type erythroblasts. Furthermore, genes associated with erythroblast maturation and karyopyknosis showed significantly reduced expression in active mutant erythroblasts, recapitulating the maturation defects. Notably, the erythroid differentiation ability and hemoglobin expression of active mutant iPSC-derived hematopoietic progenitor cells (HPCs) were improved by the administration of δ-aminolevulinic acid, verifying the suitability of the cells for drug testing. Administration of a DNA demethylating agent, azacitidine, reactivated the silent wild-type ALAS2 allele in active mutant HPCs and ameliorated erythroid differentiation defects, suggesting that azacitidine is a potential novel therapeutic drug for female XLSA. Our patient-specific iPSC platform provides novel biological and therapeutic insights for XLSA., 女性のX連鎖性鉄芽球性貧血患者さん由来のiPS細胞を使った病態モデルの作製と治療薬候補の発見. 京都大学プレスリリース. 2021-12-01.

Granulocyte transfusion (GTX) is a therapeutic option for severe bacterial or fungal infection in patients with sustained neutropenia after chemotherapy or stem cell transplantation. However, high molecular weight hydroxyethyl starch (HES), which has been used for selective sedimentation of red blood cells during apheresis, is not easily available in many countries including Japan. In this study, we evaluated the efficiency of granulocyte collection using medium molecular weight HES (130 kDa) in combination with the Spectra Optia apheresis system. Apheresis was performed for 2 consecutive days from seven donors and the mean total neutrophil yield from the first and second apheresis was 5.27 ± 3.10 × 1010 and 2.91 ± 2.92 × 1010, respectively. Infusion of concentrates from the first apheresis resulted in a significant neutrophil count increase and concentrates from the second apheresis were enough for maintenance of the neutrophil counts in all the recipients. Although the number of cases is limited, our results clearly show that sufficient number of granulocytes can be harvested by using medium molecular weight HES and this strategy is a safe and effective clinical practice in countries where high molecular weight HES is not available.

Serum ferritin, a marker of systemic iron status, is considered a prognostic factor for patients with myelodysplastic syndromes (MDS), despite the lack of supporting evidence. We investigated the association between serum ferritin levels at diagnosis and the prognoses of Japanese MDS patients with bone marrow blasts

The development of effective therapies has enabled long-term survival for many patients with multiple myeloma (MM). However, the administration of antibody drugs, such as daratumumab, which bind to plasma cell (PC) surface proteins, may prevent PC detection by flow cytometry. We propose VS38 as an alternative antibody for CD38. VS38 recognizes cytoskeleton-linking membrane protein 63 (CLIMP-63) on the rough endoplasmic reticulum, and this protein may be expressed in secretory cells. We investigated VS38 staining in normal hematopoietic cells from five control samples, as well as PCs from 21 patients with plasma cell disorder (PCD). In normal hematopoietic cells, although VS38-stained monocytes, myeloid cells, and a subpopulation of B cells, PCs were significantly and brightly stained by VS38. There was no significant difference in VS38 staining between normal and abnormal PCs obtained from five patients with monoclonal gammopathy of undetermined significance. Furthermore, PCs in 21 PCD cases were clearly identified by VS38 in all cases, in contrast to CD38, even in daratumumab-administered patients whose CD38 epitopes on PCs were masked. These results suggest that the use of the VS38 antibody in flow cytometry contributes to PC detection, independent of therapeutic treatment.

Fanconi anemia is a rare recessive disease characterized by multiple congenital abnormalities, progressive bone marrow failure, and a predisposition to malignancies. It results from mutations in one of the 22 known FANC genes. The number of Japanese Fanconi anemia patients with a defined genetic diagnosis was relatively limited. In this study, we reveal the genetic subtyping and the characteristics of mutated FANC genes in Japan and clarify the genotype-phenotype correlations. We studied 117 Japanese patients and successfully subtyped 97% of the cases. FANCA and FANCG pathogenic variants accounted for the disease in 58% and 25% of Fanconi anemia patients, respectively. We identified one FANCA and two FANCG hot spot mutations, which are found at low percentages (0.04-0.1%) in the whole-genome reference panel of 3,554 Japanese individuals (Tohoku Medical Megabank). FANCB was the third most common complementation group and only one FANCC case was identified in our series. Based on the data from the Tohoku Medical Megabank, we estimate that approximately 2.6% of Japanese are carriers of disease-causing FANC gene variants, excluding missense mutations. This is the largest series of subtyped Japanese Fanconi anemia patients to date and the results will be useful for future clinical management.

The diagnosis of myelodysplastic syndromes (MDS) is based on morphology and cytogenetics. However, limited information is currently available on the interobserver concordance of the assessment of dysplastic lineages (10% or ≥10% in bone marrow (BM)). The revised International Prognostic Scoring System (IPSS-R) described a new threshold (2%) for BM blasts. However, the interobserver concordance of the categories (0-≤2% and2-5%) has limited data. The purpose of the present study was to investigate the assessment of dysplastic lineages and IPSS-R reproducibility. Our study was divided into two Steps. In each Step, the microscopic examinations were performed separately by two morphologists. Regarding the category of BM blasts ≤2% and2-5%, interobserver agreement was more than 'moderate' in all pairs (kappa test: 0.43-0.90). Regarding dysgranulopoiesis (dysG) and dyserythropoiesis (dysE) in BM, interobserver agreement was more than 'moderate' in all pairs (kappa test, dysG: 0.45-0.96, dysE: 0.45-0.81). Regarding the category of dysmegakaryopoiesis (dysMgk) in BM, interobserver agreement was more than moderate in 4 out of 5 pairs (kappa test: 0.58-1.00), and was fair for one pair (kappa test: 0.37). We consider that high interobserver concordance may be possible for the BM blast cell count (≤2% or2-5%) and dysplasia (10% or ≥10%) of each lineage.

To identify prognostic factors for TAFRO syndrome, a rare inflammatory disorder of unknown etiology characterized by thrombocytopenia, anasarca, fever, reticulin myelofibrosis, renal dysfunction, and organomegaly. Data of patients with TAFRO syndrome were extracted from a Japanese patient registry. Patients were divided into groups according to the clinical and laboratory parameters at initial presentation. Cut-off values for the laboratory parameters were determined using receiver operating characteristic curve analysis and by clinical relevance. Patient survival was analyzed by the Kaplan–Meier method. Univariable analysis was performed using log-rank tests. Multivariable analyses were performed with the logistic regression model and the Cox-proportional hazards model. We extracted the data of 83 patients with TAFRO syndrome from the registry. Univariable analysis identified several potential prognostic factors. Of these factors, age ≥ 60 years and D-dimer ≥ 18 μg/dL remained significant predictors of poor overall survival in the multivariable Cox-proportional hazards model. Based on these results, we developed a simple prognostic scoring system for TAFRO syndrome (TS-PSS). Patients in our cohort were stratified into low, intermediate, and high-risk groups by the TS-PSS. This system should be verified with independent patient cohorts in future studies. Supplementary Information The online version contains supplementary material available at 10.1007/s12185-021-03159-x.

Myelodysplastic syndrome (MDS) is a group of clonal stem cell disorders characterized by hematopoietic insufficiency. The accurate risk stratification of patients with MDS is essential for selection of appropriate therapies. We herein conducted a retrospective cohort study to examine the prognostic value of periodic acid-Schiff (PAS) reaction-positive erythroblasts in MDS patients. We examined the PAS positivity of the bone marrow erythroblasts of 144 patients newly diagnosed with MDS; 26 (18.1%) of them had PAS-positive erythroblasts, whereas 118 (81.9%) did not. The PAS-positive group showed significantly poorer karyotypes as defined in the revised International Prognostic Scoring System (IPSS-R) and higher scores in age-adjusted IPSS-R (IPSS-RA) than the PAS-negative group. Overall survival (OS) and leukemia-free survival (LFS) were also significantly shorter in the PAS-positive group than in the PAS-negative group. Similar results were obtained when only high- and very high risk groups were analyzed using IPSS-RA. This retrospective study suggested that the PAS positivity of erythroblasts is an additional prognostic factor combined with other risk scores for OS and LFS in MDS, and our results may contribute to improved clinical decision-making and rapid risk stratification.

Hereditary hemochromatosis (HH) is a group of genetic iron overload disorders that manifest with various symptoms, including hepatic dysfunction, diabetes, and cardiomyopathy. Classic HH type 1, which is common in Caucasians, is caused by bi-allelic mutations of HFE. Severe types of HH are caused by either bi-allelic mutations of HFE2 that encodes hemojuvelin (type 2A) or HAMP that encodes hepcidin (type 2B). HH type 3, which is of intermediate severity, is caused by bi-allelic mutations of TFR2 that encodes transferrin receptor 2. Mutations of SLC40A1 that encodes ferroportin, the only cellular iron exporter, causes either HH type 4A (loss-of-function mutations) or HH type 4B (gain-of-function mutations). Studies on these gene products uncovered a part of the mechanisms of the systemic iron regulation; HFE, hemojuvelin, and TFR2 are involved in iron sensing and stimulating hepcidin expression, and hepcidin downregulates the expression of ferroportin of the target cells. Phlebotomy is the standard treatment for HH, and early initiation of the treatment is essential for preventing irreversible organ damage. However, because of the rarity and difficulty in making the genetic diagnosis, a large proportion of patients with non-HFE HH might have been undiagnosed; therefore, awareness of this disorder is important.

Introduction: Myelodysplastic syndromes (MDS) is thought to develop and progress as a result of accumulation of genetic mutations. This multicenter, open-label, phase II study examined impact of gene mutations on the effect of darbepoetin alfa (DA) for anemia. Death within 1 year and progression to acute myeloid leukemia (AML) were also examined. Methods: DA ≤240 μg was administered once weekly for 16 weeks to DA-naive, low-risk MDS (low or Intermediate-1 [Int-1] risk defined by the International Prognostic Scoring System [IPSS] risk classification) patients with anemia. DNA was extracted from the peripheral blood of patients, and presence of previously reported high-frequency gene mutations in MDS (SF3B1, TET2, SFRS2, ASXL1, DNMT3A, etc.) was analyzed by next-generation sequencing. Primary endpoint was association between frequently observed mutated genes and therapeutic effect of DA (IWG criteria 2006 (HIE)) at 16 weeks. A secondary endpoint was survival analysis results for death and progression to AML within one year after 16 weeks of treatment, with the date of first treatment as the starting date. For AML, progression to AML and subsequent death were stated as events, and the patients without confirmed progression to AML were censored at the date of last known survival. For overall survival, death from any cause was considered an event, and for the survival cases, the study was terminated at the date of last known survival. For the primary endpoint, relative risk and 95% confidence intervals (CI) were calculated using Wilson's score method; statistical significance was assessed using Pearson's chi-square test. Multivariate analysis was performed by adding baseline erythropoietin (EPO) levels (low: Results: Of the 85 patients enrolled between August 2016 and May 2019, 4 patients who were judged ineligible and 2 patients who discontinued the study prior to the start of treatment were excluded, and 79 subjects were included in the analysis (full analysis set). Of 79 subjects, 52 (65.8%) were male (median age 77.0 [29-90] years). IPSS risk was low in 27 (36.7%) and Int-1 in 50 (63.3%) subjects. The frequently mutated genes (³10%) were SF3B1 (24, 30.4%), TET2 (20, 25.3%), SRSF2 (10 cases, 12.7%), ASXL1 (9, 11.4%), and DNMT3A (8, 10.1%). Overall response rate was 70.9%. Univariate logistic regression analaysis did not show any significant association between these mutations and therapeutic efficacy of DA. The same results were obtained when the analysis was limited to red blood cell transfusion-dependent subjects (n=15). After adjusting against baseline EPO values, mutations of ASXL1 gene were associated with significantly worse response (odds ratio 0.180 [0.035-0.928], p = 0.040). Six (7.6%) confirmed cases of AML and 17 (21.5%) deaths (death before confirmed AML) were observed. Overall, 21 deaths were observed including 4 deaths after progression to AML. The median survival time (95% confidence interval) from the start of treatment to confirmed AML or death was 41.3 months (30.6 months - Not reached). Conclusions: The results of this exploratory study suggest that the presence of ASXL1 gene mutations may result in poor response to the anemic treatment with DA in low-risk MDS. The current analysis of progression to AML and death included subjects whose observation period did not reach the prespecified number of days. For such cases, the outcome research will be conducted around October 2020 and updated results will be presented at the ASH 2020 conference. Disclosures Ichikawa: Novartis, Takeda: Honoraria. Shibayama:Taiho: Research Funding; Shionogi: Research Funding; Ono: Honoraria, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Research Funding; Takeda: Honoraria, Research Funding; Merck Sharp & Dohme: Research Funding; Sumitomo Dainippon: Honoraria, Research Funding; Nippon Shinyaku: Honoraria, Research Funding; Daiichi Sankyo: Honoraria; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Chugai: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kyowa Kirin: Honoraria; Otsuka: Honoraria; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Fujimoto: Honoraria; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria; Teijin: Research Funding; Mundi Pharma: Honoraria. Maeda:Kyowa Kirin Co.Ltd.: Honoraria, Research Funding. Kawabata:Celgene Corporation: Consultancy; Celgene Corporation: Honoraria; Sanofi K. K: Honoraria; Novartis Pharma K. K.: Honoraria; Chugai Pharmaceutical Co., Ltd: Honoraria. Matsumura:Shionogi & Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Kyowa Kirin Co., Ltd.: Research Funding; ONO PHARMACEUTICAL CO., LTD.: Research Funding; Eisai Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Janssen Pharmaceutical K.K: Speakers Bureau; Amgen K.K.: Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Speakers Bureau; Astellas Pharma Inc.: Speakers Bureau; DAIICHI SANKYO COMPANY, LIMITED.: Speakers Bureau; Pfizer Japan Inc.: Speakers Bureau; Novartis Pharma KK: Speakers Bureau; Bristol-Myers Squibb Company: Speakers Bureau. Ogawa:Eisai Co., Ltd.: Research Funding; KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding. Mitani:CHUGAI: Research Funding; Takeda: Research Funding; KYOWA KIRIN: Consultancy, Research Funding.

The Japanese National Research Group on Idiopathic Bone Marrow Failure Syndromes has been conducting prospective registration, central review, and follow-up study for patients with aplastic anemia and myelodysplastic syndrome (MDS) since 2006. Using this database, we retrospectively analyzed the prognosis of patients with MDS. As of May 2016, 351 cases were registered in this database, 186 of which were eligible for the present study. Kaplan–Meier analysis showed that overall survival (OS) curves of the five risk categories stipulated by the revised international prognostic scoring system (IPSS-R) were reasonably separated. 2-year OS rates for the very low-, low-, intermediate-, high-, and very high-risk categories were 95, 89, 79, 35, and 12%, respectively. In the same categories, incidence of leukemic transformation at 2 years was 0, 10, 8, 56, and 40%, respectively. Multivariate analysis revealed that male sex, low platelet counts, increased blast percentage (>2%), and high-risk karyotype abnormalities were independent risk factors for poor OS. Based on these data, we classified Japanese MDS patients who were classified as intermediate-risk in IPSS-R, into the lower risk MDS category, highlighting the need for careful assessment of treatments within low- and high-risk treatment protocols.

Castleman disease (CD) is a rare lymphoproliferative disorder that can be unicentric or multicentric. Multicentric CD (MCD) is further subdivided into human herpesvirus type-8-associated, POEMS syndrome-associated, and idiopathic (iMCD). TAFRO syndrome is a newly identified disorder of unknown etiology characterized by thrombocytopenia, anasarca, fever, reticulin myelofibrosis, renal dysfunction, and organomegaly. The TAFRO syndrome is sometimes regarded as a subtype of iMCD (TAFRO-iMCD), whereas iMCD without TAFRO syndrome is considered "not otherwise specified" (iMCD-NOS). However, a proportion of patients with TAFRO syndrome have been diagnosed without lymph node biopsies (TAFRO syndrome without proven iMCD; TAFRO-w/op-iMCD). To clarify the clinical features of iMCD-NOS, TAFRO-iMCD, and TAFRO-w/op-iMCD, we retrospectively analyzed 220 patients extracted from the database of the Multicenter Collaborative Retrospective Study for Establishing the Concept of TAFRO Syndrome. The patients included 87 with iMCD-NOS, 63 with TAFRO-iMCD, and 19 with TAFRO-w/op-iMCD. Patients in all three groups exhibited anemia, hypoalbuminemia, and elevated serum C-reactive protein and interleukin-6 levels. No significant differences in clinical, laboratory, and prognostic features were noted between the TAFRO-iMCD, and TAFRO-w/op-iMCD groups. However, the iMCD-NOS group exhibited polyclonal hyper-γ-globulinemia. The five-year survival rates of patients in the iMCD-NOS and TAFRO-involved groups were 100% and 66.5%, respectively (dropping markedly during the first few months in the latter). The iMCD-NOS and the TAFRO-iMCD samples typically showed plasma cell and mixed-type histologies, respectively. Thus, iMCD can be classified into two distinct subtypes, iMCD-NOS and TAFRO-iMCD. As such, TAFRO-iMCD and TAFRO-w/op-iMCD may be considered the same entity, requiring prompt diagnosis and intensive care.

Late-onset neutropenia after rituximab (RTX) therapy (R-LON) has been widely reported, but clinical studies on a large number of cases are limited. In this study, we aimed to investigate the incidence and risk factors of R-LON.In this study, we retrospectively analyzed data of 213 enrolled B-cell lymphoma patients (male 114; female 99) treated with RTX at a single institution. R-LON was defined as otherwise unexplained grade III-IV neutropenia after RTX. The median age of the patients was 62 years, and 129 of them were initially diagnosed at advanced stages (stage III-IV).R-LON occurred in 19 patients within a median of 121 (range, 49-474) days after the last RTX administration. The 1-year cumulative incidence was 9.0%. On univariate analysis, older age (60 years), advanced stage, and purine analog or methotrexate administration were significant or borderline significant risk factors for R-LON, whereas sex, disease type, bone marrow invasion, combination with cytotoxic chemotherapeutic drugs, intensified therapy (compared with R-CHOP), prior autologous transplantation, and repeated RTX administration were not. On multivariate analysis, older age (hazard ratio (HR), 2.95) and advanced stage (HR, 3.56) were significant risk factors. Treatment with granulocyte colony-stimulating factor was feasible in grade IV R-LON patients with high risk of infection.Careful follow-up is therefore necessary after B-cell lymphoma treatment, especially in high-risk patients with advanced disease or of older age.

We report a series of 14 patients with myelodysplastic syndrome (MDS) accompanied by a monoclonal gammopathy unrelated to therapy. Twelve of these had monoclonal gammopathy of undermined significance (MGUS) and two had smoldering multiple myeloma. These cases represent 10.2 % of all MDS cases seen at our institution over a 14-year period (January 2000 to December 2013). The incidence of MGUS was determined to be significantly higher in MDS than in age-matched concurrent controls by χ(2) test. Absence of prior chemotherapy and simultaneous presentation of MDS and MGUS in most cases suggest true co-occurrence of the two disorders. MGUS was found in all WHO subtypes of MDS with a wide range of risk factors. However, 11 out of the 12 MDS cases accompanied with MGUS had relatively low karyotypic risks. In addition, serum M protein levels remained largely unchanged in 4 cases of MGUS for which serial determinations were performed. These findings indicate that MGUS may not affect the prognosis of MDS.

Objective: Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome associated with multiple congenital abnormalities and predisposition to malignancies, resulting from mutations in one of the 22 known FA genes (FANCA to W). The proteins encoded by these genes participate in DNA repair pathway (the FA pathway) for endogenous aldehyde damage. Compared to the situation in the US or Europe, the number of Japanese FA patients with genetic diagnosis was relatively limited. In this study, we reveal the genetic subtyping and the characteristics of mutated FA genes in Japanese population and clarify the genotype-phenotype correlations. Results: We studied 117 Japanese FA patients from 103 families (1996 to 2018). The diagnosis of FA was confirmed on the basis of chromosomal breakage tests and clinical features. Molecular diagnosis was obtained in 107 (91.5%) of the 117 patients through direct sequencing of FANCA and FANCG, MLPA analysis for FANCA, targeted exome sequencing (targeted-seq), and whole exome sequencing (WES) analysis (Figure 1). To provide genetic subtyping for the 10 unclassified cases, we tried to apply various technologies. Array CGH revealed large deletions in two FA-B and one FA-T cases. Whole genome sequencing and RNA-sequencing analysis identified splicing site or aberrant splicing mutations among three cases (one FA-B, one FA-C, and one FA-N). Collectively, 113 (97%) of Japanese 117 FA patients were successfully subtyped and a total of 219 mutated alleles were identified. FA-A and FA-G accounted for the disease in 58% and 25% of FA patients, respectively, whereas each of the other complementation groups accounted for less than 5% of FA cases. FANCB was the third most common complementation group (n=4) and only one FA-C case was identified in Japanese FA patients. In the 68 FA-A patients, we identified 130 mutant alleles that included 55 different FANCA variants (17 nucleotide substitutions, 16 small deletions/insertions, 12 large deletions, 1 large duplication and 9 splice site mutation). FANCA c.2546delC was the most prevalent (41/130 alleles; 32%). In the 29 FA-G patients, 57 mutant alleles were identified and seven different FANCG variants were detected. FANCG c.307+1G>C and 1066C>T accounted for most of FANCG mutant alleles (49/57; 88%) in the Japanese FA-G patients. The three hotspot mutations (FANCA c.2546delC, FANCG c.307+1G>C and c.1066C>T) existed at low prevalence (0.04-0.1%) in the whole-genome reference panel of 3554 Japanese individuals (3.5KJPN, Tohoku Megabank). Consistent with the paucity of the FA-C patients as opposed to the previous report (Blood 2000), the FANCC IVS4+4A mutation was absent in the 3.5KJPN database. We were able to examine the hematological outcomes in a subset of our cases (52 FA-A and 23 FA-G). Interestingly, the FA-G patients developed bone marrow failure (BMF) at a significantly younger age than FA-A patients (median age at onset of BMF: 3.1 years vs 5 years). Furthermore, the patients with the FANCA c.2546delC mutation had an increased risk of developing myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), compared to FA-A patients without the mutation. In the rare complementation groups of FA, two FA-B cases with complete loss of FANCB gene and one FA-I patient with N-terminal premature termination codons revealed severe somatic abnormalities, consistent with VACTERL-H association. Two FANCD1 (BRCA2) patients and one FANCN (PALB2) patients did not experience bone marrow failure but developed early-onset malignancies (immature teratoma, T-lymphoblastic lymphoma, adenosquamous lung carcinoma, Wilms tumor). Conclusion: This is the largest series of subtyped Japanese FA patients to date and the results would be useful for future clinical management. To provide molecular diagnosis for FA in Japan, we suggest to start with PCR-direct sequencing of the three common mutations (FANCA c.2546delC, FANCG c.307+1G>C and FANCG c.1066C>T) along with MLPA assay for FANCA. These analyses would enable the identification of about 50% of the mutant alleles. For the rest of the cases, WES or targeted-seq analysis should be useful, however, large deletions and aberrant splicing need to be kept in mind. Disclosures Takaori-Kondo: Pfizer: Honoraria; Novartis: Honoraria; Celgene: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Janssen Pharmaceuticals: Honoraria.

Hepcidin is the central regulator of body iron homeostasis, and dysregulation of hepcidin expression causes various clinical disorders, such as anemia and hemochromatosis. Various stimuli, including iron load and interleukin-6, are involved in the regulation of hepcidin expression. We previously reported that serum hepcidin levels were high in patients with end-stage renal disease, compared with healthy subjects. Since metabolic acidosis is commonly observed in these patients, we hypothesized that acidic milieu might augment hepcidin expression. In this study, we investigated the effect of changes in the pH of the microenvironment on hepcidin expression in human hepatoma and leukemia cell lines. We found that hepcidin expression in these cells was augmented by the acidic milieu created with lactic acid, hydrochloric acid and excess carbon dioxide. Acidic milieu did not clearly enhance hepcidin promoter activity, but rather stabilized hepcidin transcript in the hepatoma cells. We speculate that metabolic acidosis may contribute in part to the elevation of serum hepcidin levels in patients with end-stage chronic kidney disease. Further studies are needed to elucidate the association between acidosis and hepcidin expression in various clinical settings.

Graft failure (GF) in allogeneic hematopoietic stem cell transplantation (SCT) is a life-threatening complication because patients are at high risk of severe infection during prolonged neutropenia and also experience organ toxicity due to previous transplantation. Graft rejection is a major cause of graft failure, and is caused by the immune response of the recipient against the immunohematopoietic cells of the donor.1, 2 Cord blood is an important source for salvage transplantation because of its prompt availability and less stringent HLA compatibility. However, cord blood transplantation (CBT) has a risk of prolonged neutropenia and recurrence of GF. Previous studies reported that the rate of neutrophil engraftment of salvage CBT is 58% and the overall survival is 28–33%; the major cause of death was treatment-related mortality (TRM) in 61% cases, especially due to infection (65% of the cases of TRM).3, 4 In cases of immune-associated graft rejection, it is thought that patients should receive reconditioning to efficiently suppress recipient-derived immunity. Considering that fludarabine, melphalan and low-dose TBI conditioning efficiently suppress alloreactive host immunity and ensure engraftment,5 we have performed salvage CBT following this regimen for patients with graft rejection. We carefully assessed infectious complications before salvage CBT because they may be strongly associated with TRM as carryover infections, leading to death after salvage CBT.

TAFRO syndrome is a systemic inflammatory disorder characterized by thrombocytopenia, anasarca including pleural effusion and ascites, fever, renal insufficiency, and organomegaly including hepatosplenomegaly and lymphadenopathy. Its onset may be acute or sub-acute, but its etiology is undetermined. Although several clinical and pathological characteristics of TAFRO syndrome resemble those of multicentric Castleman disease (MCD), other specific features can differentiate between them. Some TAFRO syndrome patients have been successfully treated with glucocorticoids and/or immunosuppressants, including cyclosporin A, tocilizumab and rituximab, whereas others are refractory to treatment, and eventually succumb to the disease. Early and reliable diagnoses and early treatments with appropriate agents are essential to enhancing patient survival. The present article reports the 2015 updated diagnostic criteria, disease severity classification and treatment strategy for TAFRO syndrome, as formulated by Japanese research teams. These criteria and classification have been applied and retrospectively validated on clinicopathologic data of 28 patients with this and similar conditions (e.g. MCD with serositis and thrombocytopenia).

X-linked sideroblastic anemia (XLSA) is a rare hereditary disorder that typically manifests in males as microcytic anemia. Here, we report a family with XLSA that affects females and manifests as macrocytic anemia. The proband was a Japanese woman harboring a heterozygous mutation c.679C>T in the ALAS2 gene. This mutation causes the amino acid substitution R227C, which disrupts the enzymatic activity of erythroid-specific δ-aminolevulinic acid synthase. The mutation was not detected in the ALAS2 complementary DNA from peripheral blood red blood cells of the proband, indicating that the cells were mostly derived from erythroblasts expressing wild-type ALAS2. The proband's mother, who had been diagnosed with myelodysplastic syndrome, also had XLSA with the same mutation. Clinicians should be aware that XLSA can occur not only in males but also in females, in whom it manifests as macrocytic anemia.

Although fluoroquinolones or other antibiotics are commonly used to prevent bacterial infections after hematopoietic cell transplantation (HCT), because of the growing presence of multidrug-resistant microorganisms, it is important to identify patients who are more likely to benefit from antibacterial prophylaxis. To evaluate risk factors for early bacterial infection after allogeneic HCT, we retrospectively analyzed clinical data for 112 consecutive adult patients with hematological malignancies who received transplants without any antibacterial prophylaxis. The cumulative incidence of bacterial infection at 30 days after transplantation was 16%. Among various pre-transplant factors, only high serum ferritin (>700 ng/mL, 47 patients) and high C-reactive protein (CRP) (>0.3 mg/dL, 28 patients) levels were significantly associated with the development of bacterial infection in a multivariate analysis (hazard ratio (95% confidence interval): ferritin, 4.00 (1.32–12.17); CRP, 3.64 (1.44–9.20)). In addition, septic shock and sepsis with organ failure were exclusively observed in patients who had high ferritin and/or high CRP levels. These results suggest that pretransplant serum ferritin and CRP levels can be useful markers for predicting the risk of early bacterial infection after allogeneic HCT. It may be prudent to limit antibacterial prophylaxis to patients with predefined risk factors to ensure the safety of HCT with the use of fewer antibiotics.

Dysregulated production of hepcidin is implicated in anemia of inflammation, whereas interleukin-6 (IL-6) is a major inducer of hepcidin production. Overproduction of IL-6 is responsible for pathogenesis of multicentric Castleman disease (MCD), a rare lymphoproliferative disorder accompanied by systemic inflammatory responses and anemia. In this study, we investigated the roles of hepcidin and IL-6 in anemia of inflammation and the long-term effects of anti–IL-6 receptor antibody (tocilizumab) treatment on serum hepcidin and iron-related parameters in MCD patients. We found that tocilizumab treatment resulted in a rapid reduction of serum hepcidin-25 in 5 of 6 MCD patients. Long-term reductions, accompanied by progressive normalization of iron-related parameters and symptom improvement, were observed in 9 of 9 cases 1.5, 3, 6, and 12 months after the start of tocilizumab treatment. In in vitro experiments, IL-6–induced up-regulation of hepcidin mRNA in hepatoma cell lines was completely inhibited by tocilizumab but increased in the presence of patients' sera. Our results suggest that, although multiple factors affect serum hepcidin levels, IL-6 plays an essential role in the induction of hepcidin in MCD. This accounts for the long-term ameliorative effect of IL-6 blockage with tocilizumab on anemia by inhibiting hepcidin production in MCD patients.

Sideroblastic anemias consist of a heterogeneous group of inherited and acquired disorders. The most common hereditary type is X-linked sideroblastic anemia (XLSA), which is associated with mutations in the erythroid-specific δ-aminolevulinic acid synthase (ALAS2) gene. Heme synthesis starts with the polymerization of glycine and succinyl CoA polymerization and synthesis of δ-aminolevulinic acid (ALA) in the mitochondria. ALAS2 encodes the enzyme that catalyzes these first steps in the heme synthetic pathway in erythroid cells, steps that require pyridoxal 5'-phosphate (PLP) as a cofactor. It has been found that treatment with PLP is effective for a small fraction of XLSA patients, but there are no effective treatments for the other fraction. The aim of this study is to explore the molecular mechanisms of XLSA and to develop new effective therapies. We used episomal methods to generate induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMCs) of three late-onset XLSA female patients in one family. The cells harbored the heterozygous mutation (R227C) in the ALAS2 gene. Because ALAS2 is located in the X-chromosome, either wild-type or mutant ALAS2 gene is inactivated in the erythroid cells of female heterozygotes. All three patients showed severe anemia and their PBMCs showed skewed X-chromosome inactivation with preferential inactivation of the X chromosome carrying wild-type ALAS2, indicating a condition associated with unbalanced lyonization. From each patient, we successfully established iPSC lines with the active mutant ALAS2 allele and with the active wild-type ALAS2 allele. We assessed the hematopoietic differentiation potential of these two types of iPSC lines derived from the same patient. Differentiation into hematopoietic progenitor cells (HPCs) using embryoid body formation was comparable in the two groups. However, further differentiation in erythroid culture was significantly impaired in iPSC lines harboring the active mutant ALAS2 allele compared with those harboring the active wild-type ALAS2 allele (CD235a+ cells: 59.20±12.16% with the active wild-type ALAS2 allele vs. 3.95±4.71% with the active mutant ALAS2 allele, p We examined the effect of ALA on the erythroid differentiation of the HPCs. The CD235a-positive erythroid cell ratio of HPCs with the active wild-type ALAS2 allele did not increase following administration of ALA. By contrast, the ratio reached normal levels for HPCs with the active mutant ALAS2 allele (CD235a+ cells: 6.10± 5.61% vs. 85.34± 11.05%, p Our data suggest that our iPSC-based system could be useful for studying the precise molecular mechanisms of XLSA and drug testing. Figure Figure. Disclosures Morimoto: Grant-in-Aid for JSPS Research Fellow: Research Funding. Takaori-Kondo: celgene: Honoraria, Research Funding; Bristol-Myers Squibb, Novartis, Janssen pharma, Pfizer: Honoraria.

Overproduction of hepcidin by interleukin-6 (IL-6) is considered to be the main factor responsible for the development of anemia in inflammatory conditions. Since oncostatin M (OSM), a member of the IL-6 family, plays an important role in immune and inflammatory responses, we assessed the effect of OSM on hepcidin expression, as well as that of leukemia inhibitory factor (LIF), another member of the IL-6 family. We found that hepcidin expression was markedly induced by OSM and LIF in a time- and dose-dependent manner in hepatoma cell lines, and this expression was induced independent of IL-6/IL-6 receptor signaling. Luciferase assay revealed that OSM and LIF stimulated a -1.3-kb hepcidin promoter. This effect was markedly reduced when the signal transducer and activator of transcription (STAT) site of the promoter was mutated, and was almost completely abolished in the presence of AG-490, a Janus kinase (JAK) inhibitor. Hence, the JAK/STAT pathway plays a major role in OSM- and LIF-induced activation of the hepcidin promoter. In conclusion, we demonstrated that OSM and LIF can induce hepcidin expression mainly through the JAK/STAT pathways. Further studies are warranted to evaluate the clinical significance of OSM and LIF in the development of anemia in various inflammatory diseases.

The association of iron overload with complications of allogeneic hematopoietic stem cell transplantation (HSCT) has been suggested in previous studies. Because hepcidin plays a central role in the regulation of iron homeostasis, we analyzed the association between pretransplant serum hepcidin-25 levels and early infectious complications after allogeneic HSCT. We studied 55 consecutive adult patients with a median age of 47 years (range: 20–64 years) who underwent allogeneic HSCT for hematologic malignancies at our institution. Thirty-two patients had myelogenous malignancies; the remaining 23 had lymphogenous malignancies. The median pretransplant serum hepcidin level of patients in the study was 21.6 ng/mL (range: 1.4–371 ng/mL), which was comparable to that of healthy volunteers (median: 19.1 ng/mL [range: 2.3–37 ng/mL]; n=17). When cumulative incidences of documented bacterial and cytomegalovirus (CMV) infections at day 100 were compared according to pretransplant hepcidin-25 levels, the incidence of bacterial, but not CMV, infection, was significantly higher in the high-hepcidin group (≥50 ng/mL; n=17) than in the low-hepcidin group (

The relationship between serum hepcidin, a key regulator of body iron homeostasis, and erythropoiesis was investigated before and after stem cell transplantation in 31 patients with hematopoietic malignancies. Serum hepcidin-25 was monitored using a liquid chromatography-tandem mass spectrometry-based assay system. Other iron- and erythropoiesis-related parameters and known hepcidin regulators, such as interleukin-6 and growth differentiation factor-15, were also monitored. The serum hepcidin level peaked one week after stem cell transplantation, followed by a gradual decrease with a parallel change in interleukin-6 and a reciprocal change in reticulocyte count. Multivariate regression analysis demonstrated that the serum hepcidin level at four weeks after stem cell transplantation showed significant inverse correlations with erythropoietic activity markers, such as the soluble transferrin receptor, but not with growth differentiation factor-15. These results indicate the existence of an unknown functional erythropoiesis-associated circulating factor, other than growth differentiation factor-15, that negatively regulates hepcidin production in stem cell transplantation settings.

Ferritin is an iron-storage protein composed of different ratios of 24 light (L) and heavy (H) subunits. The serum level of ferritin is a clinical marker of the body's iron level. Transferrin receptor (TFR)1 is the receptor not only for transferrin but also for H-ferritin, but how it binds two different ligands and the blood cell types that preferentially incorporate H-ferritin remain unknown. To address these questions, we investigated hematopoietic cell-specific ferritin uptake by flow cytometry. Alexa Fluor 488-labeled H-ferritin was preferentially incorporated by erythroid cells among various hematopoietic cell lines examined, and was almost exclusively incorporated by bone marrow erythroblasts among human primary hematopoietic cells of various lineages. H-ferritin uptake by erythroid cells was strongly inhibited by unlabeled H-ferritin but was only partially inhibited by a large excess of holo-transferrin. On the other hand, internalization of labeled holo-transferrin by these cells was not inhibited by H-ferritin. Chinese hamster ovary cells lacking functional endogenous TFR1 but expressing human TFR1 with a mutated RGD sequence, which is required for transferrin binding, efficiently incorporated H-ferritin, indicating that TFR1 has distinct binding sites for H-ferritin and holo-transferrin. H-ferritin uptake by these cells required a threshold level of cell surface TFR1 expression, whereas there was no threshold for holo-transferrin uptake. The requirement for a threshold level of TFR1 expression can explain why among primary human hematopoietic cells, only erythroblasts efficiently take up H-ferritin.

The association of Epstein-Barr virus (EBV) with human immunodeficiency virus-negative T-cell lymphoma was examined in 68 patients using the polymerase chain reaction (PCR) with DNA obtained from formalin-fixed paraffin-embedded tissues and an in situ hybridization technique. EBV-encoded RNA (EBER) was detected in 43 of 68 cases (63%) of peripheral T-cell lymphoma: in 100% (11 of 11 cases) of NK/T-cell lymphomas, 70% (14 of 20 cases) of angioimmunoblastic T-cell lymphomas (AILT) and 49% (18 of 37 cases) of other types of peripheral T-cell lymphoma. A positive band was also detected at high incidence (36 of 65 cases; 55%) in a PCR analysis using primers to detect the Bam HI-W fragment of EBV. In the immunohistochemical analysis using a monoclonal antibody to latent membrane protein 1 (LMP-1) of EBV, one of the EBV-encoded latent gene products, LMP-1, was found to be expressed in 13 of 64 cases (20%), but EBNA-2 was not expressed in all the cases examined (0 of 59 cases; 0%). The 5-yr survival rate was 28% for peripheral T-cell lymphomas overall, 0% for NK/T-cell lymphomas, 38% for AILTs and 28% for other types of peripheral T-cell lymphoma. The difference in the overall survival rate between NK/T-cell lymphoma and non-NK/T-cell lymphoma was significant (P = 0.0498 by Log-rank test). Among peripheral T-cell lymphoma patients overall, the group severely infected with EBV (EBER-ISH ++) had a lower 5-yr survival rate (8%) than the group slightly (EBER-ISH +) or not infected (38%; P = 0.0013).

Bendamustine is a chemotherapeutic drug that has recently come to be used frequently in the treatment of indolent B cell lymphomas. Skin toxicity is recognized as one of its characteristic side effects, but detailed information on such reactions has not yet been obtained. To clarify the clinical features of skin toxicity associated with bendamustine, we retrospectively analyzed skin reactions that developed in patients treated with bendamustine and rituximab (BR). Of 34 patients treated with 3-6 cycles of BR, 11 developed late-onset, persistent skin eruptions. These patients demonstrated increases in CD8(+) T cell number and CD8(+):CD4(+) cell ratio at the end of chemotherapy. In contrast, peripheral eosinophil count was not associated with such adverse events, whereas eosinophil infiltration was frequently observed in the skin. Patients with skin reactions tended to have higher seropositivity of hepatitis Bcore antibody, and multiplex viral screening PCR of the frozen sera demonstrated cytomegalovirus-DNA in some of the eruption-positive patients. It is speculated that inappropriate activation of CD8(+) T cells by latently infected pathogens may be one of the triggers of late-onset skin reactions caused by bendamustine., First online: 02 April 2015

Diffuse large B-cell lymphoma (DLBCL) can be divided into prognostically important categories such as germinal center B (GCB)-like and non-GCB-like groups. The t(14;18)(q32;q21) translocation defines a unique subset of DLBCL cases with a GCB gene expression profile. Two-color fluorescence in situ hybridization (FISH) analysis was applied to detect t(14;18) (q32;q21) in the nuclei of paraffin-embedded tissue sections from 61 patients with de novo DLBCL. Nine (15%) of 61 cases had a positive pattern. Fifty-seven cases were subclassified in an immunohistochemical study with anti-CD10, anti-bcl-6, and anti-MUM1 antibodies. In this classification, 21 cases (37%) were placed in the GCB group, and 36 (63%) were placed in the non-GCB group. There was a discrepancy between t(14;18) occurrence and bcl-2 protein expression. Bcl-2 protein expression was positive in 40 (67%) of 60 cases. The expression of bcl-2 protein in the GCB and non-GCB groups was not significantly different: 15 (71%) of 21 cases in the GCB group and 24 (67%) of 36 cases in the non-GCB group tested positive. We found no difference between the FISH-positive and FISH-negative groups in overall survival time (P = .6019, log-rank test). The overall survival rates of GCB and non-GCB groups did not differ significantly by immunohistochemical classification (P = .5399, log-rank test). Overall survival was significantly longer in the group with a low International Prognostic Index (IPI) score than in the group with a high IPI score (P = .0002, log-rank test). Our results suggest that immunohistochemical study and cytogenetic study with t(14;18) FISH cannot predict the clinical outcomes of DLBCL patients. A study with a larger number of patients may show a difference in clinical outcomes between FISH-positive and FISH-negative groups and between GCB and non-GCB groups.

Transferrin receptor 2 alpha (TfR2 alpha), the major product of the TfR2 gene, is the second receptor for transferrin (Tf), which can mediate cellular iron uptake in vitro. Homozygous mutations of TfR2 cause haemochromatosis, suggesting that TfR2 alpha may not be a simple iron transporter, but a regulator of iron by identifying iron-Tf. In this study, we analysed the ligand specificity of TfR2 alpha using human transferrin receptor 1 (TfR1) and TfR2 alpha-stably transfected and expressing cells and flow-cytometric techniques. We showed that human TfR2 alpha interacted with both human and bovine Tf, whereas human TfR1 interacted only with human Tf. Neither human TfR1 nor TfR2 alpha interacted with either lactoferrin or melanotransferrin. In addition, by creating point mutations in human TfR2 alpha, the RGD sequence in the extracellular domain of TfR2 alpha was shown to be crucial for Tf-binding. Furthermore, we demonstrated that mutated TfR2 alpha (Y250X), which has been reported in patients with hereditary haemochromatosis, also lost its ability to interact with both human and bovine Tf. Although human TfR1 and TfR2 alpha share an essential structure (RGD) for ligand-binding, they have clearly different ligand specificities, which may be related to the differences in their roles in iron metabolism.

Transferrin receptor 1 (TfR1) is a type II membrane protein that mediates cellular iron uptake. Transferrin receptor 2(TfR2), another receptor for transferrin (Tf), has recently been cloned. We examined expression levels of TfR1, TfR2-alpha (membrane form) and TfR2-beta (non-membrane form) transcripts in cells from 67 patients with de novo acute myeloid leukaemia (AML) using reverse transcription-polymerase chain reaction (RT-PCR), and correlated the results with a variety of clinical features and disease outcomes of these patients. Significant correlations were noted between the levels of both TfR1 and TfR2-alpha (r = 0.771, P < 0.001) and TfR1 and TfR2-beta (r = 0.534, P < 0.001). Unexpectedly, initial white blood cell (WBC) counts were inversely correlated with levels of expression of either TfR1(r = -0.357, P = 0.003), TfR2-alpha (r = -0.486, P < 0.0001), or TfR2-beta (r = -0.435, P = 0.0003). Only TfR2 expression was significantly associated with either serum iron (r = -0.270, P = 0.045) or serum ferritin (r = -0.364, P = 0.008). Multivariate analyses using Cox's proportional hazard model showed that elevated TfR2-alpha, but not TfR1 or TfR2-beta mRNA levels significantly contributed to a better prognosis for AML patients. Furthermore, a group with high expression levels of both TfR2-alpha and TfR2-beta survived significantly longer than a group without high expression of both of them (P < 0.01 by log-rank). The present study suggests that (i) TfRs-independent iron uptake might have an important role in in vivo proliferation of AML cells; (ii) expression of TfR2 (especially the alpha form) is a novel prognostic factor for patients with AML.

In the new World Health Organization (WHO) classification of malignant lymphoma, anaplastic large cell lymphoma of B-cell phenotype is classified either as the anaplastic large cell variant of diffuse large B-cell lymphoma or as Hodgkin's lymphoma. A 71-year-old Japanese man developed fever and generalized lymphadenopathy. Biopsy of the right axillary node revealed morphology of malignant lymphoma in which large cells with abundant cytoplasm and pleomorphic nuclei were scattered among small lymphocytes. Immunostaining with various monoclonal antibodies revealed the large cells to be CD79+, CD20/L26+, CD45RO/UCHL-(1-), CD3-, CD10-, CD30+, NPM/ALK-, EMA-, CD15-, and bcl-(2-). Amplification of the J region of the immunoglobulin heavy chain by polymerase chain reaction revealed a single rearranged band. Therefore the diagnosis of anaplastic large cell variant of diffuse large B-cell lymphoma, stage IIIB, was made from the standpoint of the new WHO classification of malignant lymphoma. Biopsy led to findings of Epstein-Barr virus (EBV)-associated lymphoma with positive in situ hybridization results for EBV small RNAs, positive results of immunostaining with EBV latent membrane 1 antibody, and negative results of immunostaining with Epstein-Barr nuclear antigen 2. Results of immunostaining of the mass with p53 antibody also were positive for lymphoma cells. The findings in this case may suggest a close relationship between p53 expression and latent EBV infection.

A 64-yr-old Japanese man presented with mild anemia, leukocytosis, and thrombocytosis in November 1999. A diagnosis of chronic myeloid leukemia was made with a positive Ph chromosome, and interferon α treatment was started, 6 million units a day. Two years later, in October 2001, the patient developed leukocytosis, an increased LDH level, and large blasts with basophilic cytoplasm with cytoplasmic projections appeared in the peripheral blood. Bone marrow aspiration revealed increased blasts (59.6%). These blasts were negative on peroxidase stain, positive on acid phosphatase, and weakly positive on α naphthyl butyrate esterase stain and periodic acid-Schiff stain. Immunohistochemical staining with monoclonal antibodies revealed that these blasts were strongly positive with anti-CD41 (glycoprotein IIb/IIIa), weakly positive with CD7, CD33, and CD34, and negative with other monoclonal antibodies. A diagnosis of megakaryoblastic transformation from chronic myeloid leukemia was therefore made. Two-color fluorescence in situ hybridization (FISH) for portions of the major-bcr and abl genes from bone marrow cells revealed two fused signals in 90.6% and one fused signal in 5.8% of 106 cells. A cytogenetic study revealed that bone marrow cells were 69, XYY, + 6, -7, + 8, -9, t(9;22)(q34;q11), + 11, + 13, - 15, - 16, dic(17;18)(p11;p11), - 18, + 19, + 21, der(22)t(9;22) in six of nine examined cells. These findings confirmed that these megakaryoblasts originated from megakaryocytes of the chronic myeloid leukemia clone.

Summary. Expression of the transferrin receptor (TfR) is controlled essentially by post-transcriptional regulation, which is mediated by the interaction between iron regulatory proteins (IRPs) and iron-responsive elements (IREs) located in the 3′-untranslated region (UTR) of TfR mRNA. We examined whether additional controls of expression might occur by investigating the regulation of TfR in the absence of its' IREs by cloning 3′-UTR truncated TfR cDNA (TfRΔIRE) into pcDNA3·1(+) and stably expressing it in CHO-TRVb (Chinese Hamster Ovary, TfR variant cells, clone b) cells which have no endogenous TfR. Both the surface and the total cell TfR protein levels increased significantly after treating the stably transfected cells with the iron chelator desferrioxamine (DFO) compared with the control cells. In contrast, expression of the transferrin receptor 2 (TfR2), which was also cloned in the same vector and stably expressed in the TRVb-CHO cells, was not regulated by iron chelation. In addition, TfR2 fused to the 3′-UTR of TfR (TfR2-IRE) was cloned into the same vector and stably expressed in the CHO-TRVb cells. A comparison showed that expression of full-length TfR and TfR2-IRE increased by fivefold and twofold, respectively, after addition of DFO. Further experiments showed that TRVb-CHO cells, which were stably transfected with TfRΔIRE and exposed to either normal medium or DFO, resulted in similar levels of TfR mRNA, but the TfR protein biosynthetic rate increased with iron chelation. In summary, expression of TfR in cells placed in an iron-deficient milieu can increase by a mechanism which is independent of the IREs of TfR.

Hepcidin is the central regulator of systemic iron homeostasis; dysregulation of hepcidin expression causes various iron metabolic disorders, including hereditary hemochromatosis and anemia of inflammation. To identify molecules that modulate hepcidin expression, we developed a bioassay system for hepcidin gene (HAMP) promoter activity by stable transfection of Hep3B hepatoma cells with an expression plasmid in which EGFP was linked to a 2.5-kb human HAMP promoter. Interleukin 6, bone morphogenetic protein 6 (BMP-6), and oncostatin M, well-characterized stimulators of the HAMP promoter, strongly enhanced the green fluorescence intensity of these cells. Dorsomorphin, heparin, and cobalt chloride, known inhibitors of hepcidin expression, significantly suppressed green fluorescence intensity, and these inhibitory effects were more prominent when the cells were stimulated with BMP-6. Employing this system, we screened 1,280 biologically active small molecules and found several candidate inhibitors of hepcidin expression. Apomorphine, benzamil, etoposide, CGS-15943, kenpaullone, and rutaecarpine (all at 10 μmol/L) significantly inhibited hepcidin mRNA expression by Hep3B cells without affecting cell viability. CGS-15943 was the strongest suppressor of BMP-6-induced hepcidin-25 secretion in these cells. We conclude that our newly developed hepcidin promoter bioassay system is useful for identifying and evaluating compounds that modulate hepcidin expression.

Objective To compare the effect of tocilizumab (TCZ) with other biologic therapies in improving anemia of rheumatoid arthritis (RA) patients. Methods We compared the change of hemoglobin (Hb) levels in a cohort of 147 consecutive RA patients who were treated with biologics for more than 12 weeks. Twenty eight patients were treated with TCZ, and 119 patients were treated with biologics other than TCZ (87 with TNF inhibitors and 32 with abatacept). The change of Hb levels from baseline to week 12 was compared between the TCZ and the non-TCZ groups. We performed univariate and multivariate analyses with adjustment of potential confounders such as baseline characteristics, concomitant treatment, and the clinical response to treatment. Results Hb levels generally increased after biologic therapies both in the TCZ and the non-TCZ groups. The increase of Hb levels was greater in the TCZ group than in the non-TCZ groups (1.1 g/dL in the TCZ group vs 0.3 g/dL in the non-TCZ group, p = 0.009). Univariate analysis revealed that increase of Hb levels was also significantly associated with lower Hb, higher Low Hemoglobin Density, and higher CRP levels at baseline and greater reduction in the clinical disease activity index. TCZ therapy was significantly associated with the increase of Hb levels even after adjustment for these factors by multivariate analysis (p

Myelodysplastic syndrome (MDS) is a heterogeneous group of hematopoietic stem cell disorders characterized by ineffective hematopoiesis and a high propensity to transform into acute myeloid leukemia (AML). Although hematopoietic stem cell transplantation can cure some MDS patients, most patients are not eligible to undergo the treatment because of advanced age or comorbidities. In order to choose the optimal treatments, predicting prognosis is crucial. Chromosomal karyotype, bone marrow (BM) blast percentage, and cytopenia are well-known prognostic factors for MDS patients. These factors are incorporated into the international prognostic scoring system and its revised version (IPSS-R). However, the impact of morphological abnormalities has not been fully determined. Thus, to elucidate this, we examined the influence of the number of dysplastic lineages and the presence of increased ring sideroblasts (RS) on the survival of MDS patients whose BM blast percentages were Since 2006, the National Research Group on Idiopathic Bone Marrow Failure Syndromes, Japan, has been conducting the Prospective Registration, Central Review and Follow-up Study for Aplastic Anemia and Myelodysplastic Syndrome. Patients with BM blasts of By May 2016, 351 cases had been registered to the depository. After central review, 216 cases were diagnosed as MDS according to definition by the French-American-British Cooperative Group. Cases that were not registered within 1 year after the time of institutional diagnosis and cases with follow-up periods of 15% RS in erythroblasts. The mean BM blast percentage was significantly lower in patients with RCUD than in those with RCMD, consistent with the result of our pervious study that used a different cohort where the BM blast percentages in patients with RA according to the 2008 WHO classification were significantly lower than those in patients with RCMD (Matsuda A, et al. Eur J Haematol. 2013;90:79-80). The 2-year OS of patients with RCUD was significantly higher than that of patients with RCMD (84% vs 76%, P = 0.014; Fig. 1a), which is consistent with the results of a German study (Maassen A, et al. Leuk Res. 2013;37:64-70). The cumulative incidence of progression to AML tended to be higher in RCMD than in RCUD, although the difference was not statistically significant (P = 0.0656, Fig. 1b). Among patients with RCMD, those with increased number of RSs showed significantly lower hemoglobin levels and higher cytogenetic scores according to the IPSS-R definition than those without increased RS (Table 2). The 2-year OS of the former was significantly inferior to that of the latter in our cohort (58% vs 87%, P = 0.009; Fig. 1c). Increased number of RSs is known to be associated with SF3B1 mutations, which in turn have been shown to be associated with favorable prognosis. However, the results of the present study indicate that in RCMD, an increase in the number of RSs may be an unfavorable indicator of patient survival. Disclosures Ozawa: Celgene Japan: Consultancy; Sumitomo Dainippon Pharma Co. Ltd: Research Funding; JCR Pharmaceutical Inc: Consultancy; Takara Bio Inc: Research Funding. Takaori-Kondo:Pfizer: Research Funding; Eisai: Research Funding; Takeda Pharmaceutical: Research Funding; Astellas Pharma: Research Funding; Kyowa Kirin: Research Funding; Cognano: Research Funding; Toyama Chemical: Research Funding; Mochida Pharmaceutical: Research Funding; Shionogi: Research Funding; Alexion Pharmaceuticals: Research Funding; Chugai Pharmaceutical: Research Funding; Janssen Pharmaceuticals: Speakers Bureau; Merck Sharp and Dohme: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau.

Endonucleases capable of producing internucleosomal DNA cleavage are one of the key enzymes in apoptosis. We examined endonuclease activities contained in nuclei of CD34 + and erythroid cells in the bone marrow (BM) from 12 patients with the myelodysplastic syndromes. The levels of Mg 2+ -dependent and acidic endonucleases showed little changes as compared with those from normal BM. By contrast, the level of Ca 2+ Mg 2+ -dependent endonuclease was appreciably higher in MDS erythroid cells than normal counterparts, although the activity varied markedly in CD34 + and erythroid cells. Our results suggested that Ca 2+ Mg 2+ -dependent endonuclease is related to ineffective erythropoiesis in MDS.

Transplantation-associated thrombotic microangiopathy (TA-TMA) is a devastating complication of hematopoietic stem cell transplantation. TA-TMA likely represents the final stage of vascular endothelial injury; however, its pathophysiology is largely unknown, making clinical management difficult. Recently, the association of neutrophil extracellular traps (NETs) with the development of thrombotic thrombocytopenic purpura and hemolytic uremic syndrome has been reported. Thus, we explored whether NETs are also relevant to the occurrence of TA-TMA. We retrospectively analyzed post-transplant trends of serum NET levels in 90 patients, 11 of whom developed TA-TMA. Relative to baseline (before the conditioning regimen), elevated serum NET levels either at 4 weeks after transplantation or as early as the day of transplantation were associated with significantly increased risk of TA-TMA. In contrast, thrombomodulin, a potential marker for TA-TMA, was not helpful to predict the occurrence of TA-TMA in our study. In addition, we directly detected glomerular deposition of NETs in 2 TA-TMA patients. Increased NET levels are a significant risk factor for TA-TMA, suggesting that NET level is a useful biomarker for TA-TMA.

Studies have suggested an association between pretransplant serum levels of ferritin and C-reactive protein (CRP) and complications of allogeneic hematopoietic stem cell transplantation (HSCT). To evaluate the prognostic impact of these biomarkers on the development of acute and chronic graft-versus-host disease (GVHD), we retrospectively studied 211 patients who underwent allogeneic HSCT for hematologic diseases at our institution. The cumulative incidence rate of chronic GVHD at 3 years was 40.7 %. In the multivariate analysis, elevated CRP levels (≥2 mg/L) were significantly associated with a high incidence of chronic GVHD, whereas high ferritin levels (≥880 ng/mL) showed a tendency, though not statistically significant, to association with a low incidence of chronic GVHD. No significant association was observed between the pretransplant serum ferritin or CRP levels and the incidence of acute GVHD. Multivariate analysis indicated that high pretransplant serum ferritin levels were significantly associated with increases in treatment-related mortality and relapse rates. Overall, an elevated pretransplant serum ferritin level, but not an elevated serum CRP level, is a strong risk factor for overall mortality (hazard ratio, 2.16; P = 0.002). Our results also indicate that pretransplant serum CRP levels may be a useful biomarker for predicting the risk of chronic GVHD.

Iron overload is frequently observed in patients with hematologic diseases before and after allogeneic stem-cell transplantation because they usually receive multiple red blood cell transfusions. Elevated pretransplant serum ferritin levels, which are widely used as indicators of body iron status, are significantly associated with a lower overall survival rate and a higher incidence of treatment-related complications; for example, infections and hepatic veno-occlusive disease. As serum ferritin levels are affected, not only by iron loading but also by inflammation, imaging techniques to quantify tissue iron levels have been developed, for example, quantitative MRI using the transverse magnetic relaxation rate, and superconducting quantum interference devices. Iron chelators, such as deferasirox, a new oral iron-chelating agent, reduce iron load in transfusion-dependent patients. Iron-chelating therapy before and/or after transplantation is a promising strategy to improve the clinical outcomes of transplant patients with iron overload. However, further research is needed to prove the direct relationship between iron overload and adverse outcomes, as well as to determine the effects of treatment for iron overload on outcomes of allogeneic stem-cell transplantation.