Juvenile Hemochromatosis With Non-transfused Hemolytic Anemia Caused by a De Novo PIEZO1 Gene Mutation.

Differential diagnosis of juvenile hemochromatosis along with hemolytic anemia is often difficult. We report a 23-year-old woman with macrocytic hemolytic anemia with iron overload. The patient showed high serum ferritin and transferrin saturation and low serum transferrin and ceruloplasmin. We also noticed stomatocytes in her blood smear, which was confirmed by scanning electron microscopy. Target gene sequencing identified a mutation in PIEZO1 (heterozygous c.6008C>A: p.A2003D). This mutation was reported previously in a family with dehydrated hereditary stomatocytosis (DHS1, [OMIM 194380]), but in the current case, it was identified to be a de novo mutation. We underscore DHS1 in the differential diagnosis of iron overload associated with non-transfused hemolytic anemia in children and young adults.

VLX1570 induces apoptosis through the generation of ROS and induction of ER stress on leukemia cell lines.

A novel proteasome deubiquitinase inhibitor, VLX1570, has been highlighted as a promising therapeutic agent mainly for lymphoid neoplasms and solid tumors. We examined in vitro effects of VLX1570 on eight myeloid and three lymphoid leukemia cell lines. From cell culture studies, 10 out of 11 cell lines except K562 were found to be susceptible to VLX1570 treatment and it inhibited cell growth mainly by apoptosis. Next, to identify the signaling pathways associated with apoptosis, we performed gene expression profiling using HL-60 with or without 50 nmol/L of VLX1570 for 3 hours and demonstrated that VLX1570 induced the genetic pathway involved in "heat shock transcription factor 1 (HSF1) activation", "HSF1 dependent transactivation", and "Regulation of HSF1 mediated heat shock response". VLX1570 increased the amount of high molecular weight polyubiquitinated proteins and the expression of HSP70 as the result of the suppression of ubiquitin proteasome system, the expression of heme oxygenase-1, and the amount of phosphorylation in JNK and p38 associated with the generation of reactive oxygen species (ROS) induced apoptosis and the amount of phosphorylation in eIF2α, inducing the expression of ATF4 and endoplasmic reticulum (ER) stress dependent apoptosis protein, CHOP, and the amount of phosphorylation slightly in IRE1α, leading to increased expression of XBP-1s in leukemia cell lines. In the present study, we demonstrate that VLX1570 induces apoptosis and exerts a potential anti-leukemic effect through the generation of ROS and induction of ER stress in leukemia cell lines.

[Red cell membrane disorders and thalassemia].

Congenital hemolytic anemias are classified into three major categories: red cell membrane disorders, hemoglobinopathies, and red cell enzyme disorders. The membrane disorders are caused by abnormalities in erythrocyte membrane proteins and are often associated with disease-specific deformations of red blood cells. Historically, membrane disorders have been classified according to morphology. In recent years, however, comprehensive genetic analysis with next-generation sequencing has been performed in patients with hemolytic anemia for whom making an accurate diagnosis is difficult. These studies have led to the identification of new causative genes, but there have been inconsistent associations in some cases between the diagnosed disease and the patient's clinical manifestations. Thalassemia is a hemoglobinopathy caused by a quantitative abnormality of one or the other of the globin chains in hemoglobin. Most Japanese patients with thalassemia have mild forms of the disease, which is different from reports in other countries. However, with globalization, the proportion of Japanese patients with intermediate or severe anemia is increasing. Therefore, it is incumbent on hematologists in Japan to be knowledgeable regarding prenatal diagnosis of and gene therapy for thalassemia.

Binimetinib, a novel MEK1/2 inhibitor, exerts anti-leukemic effects under inactive status of PI3Kinase/Akt pathway.

A MEK1/2 inhibitor, binimetinib is promising as a therapeutic agent for malignant melanoma with N-RAS mutation. We examined in vitro effects of binimetinib on 10 human myeloid/lymphoid leukemia cell lines, and found that three of five cell lines with N-RAS mutation and one of five without N-RAS mutation were responsive to treatment with binimetinib. Binimetinib inhibited cell growth mainly by inducing G1 arrest and this action mechanism was assisted by gene set enrichment analysis. To identify signaling pathways associated with binimetinib response, we examined the status of MAP kinase/ERK and PI3Kinase/Akt pathways. The basal levels of phosphorylated ERK and Akt varied between the cell lines, and the amounts of phosphorylated ERK and Akt appeared to be reciprocal of each other. Interestingly, most of the binimetinib-resistant cell lines revealed strong Akt phosphorylation compared with binimetinib-sensitive ones. The effect of binimetinib may not be predicted by the presence/absence of N-RAS mutation, but rather by Akt phosphorylation status. Moreover, combination of binimetinib with a PI3K/Akt inhibitor showed additive growth-suppressive effects. These results suggest that binimetinib shows potential anti-leukemic effects and the basal level of phosphorylated Akt might serve as a biomarker predictive of therapeutic effect.

Utility of a Fluorescence Microscopy Imaging System for Analyzing the DNA Ploidy of Pathological Megakaryocytes Including 5q- Syndrome.

To investigate megakaryocyte (MK) DNA ploidy in various hematological diseases, fluorescence microscopy imaging system (FMI) can be used to analyze DNA ploidy with cell morphology at the single-cell level by using specialized image-processing software. Here we compared DNA ploidy obtained by FMI measured with that obtained flow cytometry (FCM). With FMI, we could evaluate the DNA ploidy in long-term preserved bone marrow smear samples after staining. We next analyzed the MK DNA ploidy in 42 bone marrow smear samples including 26 myeloid neoplasm cases, and we compared the DNA ploidy and platelet counts in the patients' peripheral blood; the production of platelets was significantly high compared to DNA ploidy in the myeloproliferative neoplasms group. The FMI method revealed that the patients with 5q- syndrome exhibited relatively low DNA ploidy despite high platelet counts, and this result suggested that increased DNA ploidy is not indispensable to abundant platelet production. The FMI method for DNA ploidy will be a useful tool to clarify the relationship between DNA ploidy and platelet production by MKs.

Low neutrophil alkaline phosphatase score is a new aspect of calreticulin-mutated myeloproliferative neoplasms.

Calreticulin (CALR) and JAK2-V617F gene mutations, which are major genetic mutations in patients with primary myelofibrosis (PMF) and essential thrombocythemia (ET), exert different effects on the clinical features and outcomes of these diseases. We analyzed 88 and 9 patients with ET and PMF, respectively, and determined the differences in the clinical characteristics of ET patients with JAK2-V617F compared with CALR mutations. The frequency of the JAK2-V617F and CALR mutations were 64 and 22 %, respectively. Patients with CALR mutations were younger, had a lower white blood cell count, and had a lower rate of thrombotic events than patients with the JAK2 mutation. The neutrophil alkaline phosphatase (NAP) score of 16 patients with CALR mutations was significantly lower than the normal controls, which was mainly due to the high proportion of NAP-negative neutrophils. This is the first report to show an association between CALR mutations in patients with myeloproliferative neoplasms (MPN) and the NAP score. Although the mechanism is unclear, the NAP score could be a useful and reliable biochemical marker to discriminate the mutational status of MPN patients. Further investigation is warranted to determine whether these characteristics contribute to the pathogenesis of MPN and the NAP score.

Withaferin A suppresses the growth of myelodysplasia and leukemia cell lines by inhibiting cell cycle progression.

Treatment outcomes for acute myeloid leukemia and myelodysplastic syndromes (MDS) remain unsatisfactory despite progress in various types of chemotherapy and hematopoietic stem cell transplantation. Therefore, there is a need for the development of new treatment options. We investigated the growth-suppressive effects of withaferin A (WA), a natural plant steroidal lactone, on myelodysplasia and leukemia cell lines. WA exhibited growth-suppressive effects on the cell lines, MDS-L, HL-60, THP-1, Jurkat and Ramos, and induction of cell cycle arrest at G2/M phase at relatively low doses. Evaluation by annexin V/PI also confirmed the induction of partial apoptosis. Gene expression profiling and subsequent gene set enrichment analysis revealed increased expression of heme oxygenase-1 (HMOX1). HMOX1 is known to induce autophagy during anticancer chemotherapy and is considered to be involved in the treatment resistance. Our study indicated increased HMOX1 protein levels and simultaneous increases in the autophagy-related protein LC3A/B in MDS-L cells treated with WA, suggesting increased autophagy. Combined use of WA with chloroquine, an autophagy inhibitor, enhanced early apoptosis and growth suppression. Together with the knowledge that WA had no apparent suppressive effect on the growth of human normal bone marrow CD34-positive cells in the short-term culture, this drug may have a potential for a novel therapeutic approach to the treatment of leukemia or MDS.

Analysis of Hereditary Elliptocytosis with Decreased Binding of Eosin-5-maleimide to Red Blood Cells.

Flow cytometric test for analyzing the eosin-5-maleimide (EMA) binding to red blood cells has been believed to be a specific method for diagnosing hereditary spherocytosis (HS). However, it has been reported that diseases other than HS, such as hereditary pyropoikilocytosis (HPP) and Southeast Asian ovalocytosis (SAO), which are forms in the category of hereditary elliptocytosis (HE), show decreased EMA binding to red blood cells. We analyzed EMA binding to red blood cells in 101 healthy control subjects and 42 HS patients and obtained a mean channel fluorescence (MCF) cut-off value of 36.4 (sensitivity 0.97, specificity 0.95). Using this method, we also analyzed 12 HE patients. Among them, four HE patients showed the MCF at or below the cut-off value. It indicates that some HE patients have decreased EMA binding to red blood cells. Two of these four HE patients were classified as common HE, and two were spherocytic HE with reduced spectrin. This study demonstrates that, in addition to patients with HPP or SAO, some HE patients have decreased EMA binding to red blood cells.

Five-aza-2'-deoxycytidine-induced hypomethylation of cholesterol 25-hydroxylase gene is responsible for cell death of myelodysplasia/leukemia cells.

DNA methyltransferase inhibitors (DNMT inhibitors) are administered for high-risk MDS, but their action mechanisms are not fully understood. Hence, we performed a genome-wide DNA methylation assay and focused on cholesterol 25-hydroxylase (CH25H) among the genes whose expression was up-regulated and whose promoter region was hypomethylated after decitabine (DAC) treatment in vitro. CH25H catalyzes hydroxylation of cholesterol and produces 25-hydroxycholesterol (25-OHC). Although CH25H mRNA expression level was originally low in MDS/leukemia cell lines, exposure to DNMT inhibitors enhanced CH25H mRNA expression. The promoter region of CH25H was originally hypermethylated in HL-60 and MDS-L cells, but DAC treatment induced their hypomethylation together with increased CH25H mRNA expression, activation of CH25H-oxysterol pathway, 25-OHC production and apoptotic cell death. We further confirmed that normal CD34-positive cells revealed hypomethylated status of the promoter region of CH25H gene. CH25H-knockdown by transfection of shRNA lentiviral vector into the cell lines partially protected the cells from DAC-induced cell death. Exogenous addition of 25-OHC suppressed leukemic cell growth. The present study raises a possibility that DNMT inhibitors activate CH25H-oxysterol pathway by their hypomethylating mechanism and induce leukemic cell death. Further investigations of the promoter analysis of CH25H gene and therapeutic effects of DNMT inhibitors on MDS/leukemia will be warranted.

[Hereditary red cell membrane disorders in Japan: comparison with other countries].

Red cell membrane disorders are the most common type of inherited hemolytic disorders in the Japanese population. In hereditary spherocytosis (HS), the primary presentation is a loss of membrane surface area, leading to reduced deformability because of defects in the membrane proteins ankyrin, band 3, ß-spectrin, α spectrin, or protein 4.2 (P4.2). Complete P4.2 deficiencies, which are inherited in an autosomal recessive manner, comprise a unique HS subgroup and are common in Japanese, but rare in other populations. In contrast, the principle presentation in hereditary elliptocytosis (HE) is mechanical weakness of the erythrocyte membrane skeleton due to defects in α-spectrin, ß-spectrin, or protein 4.1. Although α-spectrin mutations are the most frequent cause of HE in Caucasian, African, and Mediterranean populations, these mutations are rare in the Japanese population, in which P4.1 deficiencies are instead most common. Furthermore, hereditary stomatocytoses (HSt) are disorders of monovalent cation permeability in the red cell membrane.

[Band 3 deficiency as a cause of hereditary spherocytosis].

Band 3 protein accounts for the largest percentage of whole erythrocyte membrane proteins. Abnormalities in this protein are closely associated with pathologies including hereditary spherocytosis (HS), Southeast Asian ovalocytosis and distant renal tubular acidosis. Currently, EMA binding capacity measurement in erythrocytes is the most useful screening test for diagnosing HS. We have also demonstrated reduced EMA binding capacity in patients with HS who have deficiencies of membrane proteins such as ankyrin not directly binding to EMA and who have as yet undetectable abnormalities of membrane proteins. However, even patients with hereditary elliptocytosis, who have a partial spectrin deficiency, were found to show reduced EMA binding capacity. Six of 7 had spherocytic elliptocytosis. Therefore, it is necessary to meticulously diagnose HS by ruling out all other possibilities.

Rigosertib induces cell death of a myelodysplastic syndrome-derived cell line by DNA damage-induced G2/M arrest.

A multi-kinase inhibitor, rigosertib (ON 01910.Na) has recently been highlighted as a novel type of anti-cancer agent for the treatment of the myelodysplastic syndromes (MDS), but its action mechanisms remain to be clarified. We investigated the in vitro effects of rigosertib on an MDS-derived cell line MDS-L and a myeloid leukemia cell line HL-60. Rigosertib suppressed the proliferation of both HL-60 and MDS-L cells and induced apoptosis by inhibition of the PI3 kinase/Akt pathway. As the effects on cell cycle, rigosertib treatment promoted the phosphorylation of histone H2AX and led to the DNA damage-induced G2/M arrest. In addition, an immunofluorescence staining study demonstrated the abnormal localization of aurora A kinase, suggesting that rigosertib causes perturbation of spindle assembly and deregulated mitotic patterns towards cell cycle arrest and apoptosis. We also found that rigosertib exerted growth inhibitory effects on two lymphoid cell lines, Jurkat and Ramos. We further examined the molecular pathways influenced by rigosertib from the gene expression profiling data of MDS-L cells and found a possible involvement of rigosertib treatment in the upregulation of the genes related to microtubule kinetics and the downregulation of the mRNA degradation system. The gene set enrichment analysis showed the suppression of "nonsense-mediated mRNA decay (NMD)" as the most significantly affected gene set. These data provide a new aspect and a potential utility of rigosertib for the treatment of refractory hematopoietic malignancies.

Varied clinical course of aplastic crisis in hereditary spherocytosis.

This study is the first to report a familial case involving differing clinical courses of aplastic crisis triggered by parvovirus B19 in two patients with HS, although similar eosin-5-maleimide-binding test and sodium dodecylsulfate-polyacrylamide gel electrophoresis results had been obtained for both. One patient had short-term mild symptoms, whereas the other patient developed severe anemia that required blood transfusion, experienced fever for 13 days, and did not have any rash. The severity of aplastic crisis is reported to be correlated with the severity of the underlying hemolytic anemia; the present findings show that the severity of infection should also be considered as an important predictive factor of the severity of aplastic crisis.

Rab27a is essential for the formation of neutrophil extracellular traps (NETs) in neutrophil-like differentiated HL60 cells.

Neutrophils play a crucial role in host defence. In response to a variety of inflammatory stimulation, they form neutrophil extracellular traps (NETs). NETs are extracellular structures composed of chromatin fibers decorated with antimicrobial proteins and developing studies indicate that NETs contribute to extracellular microbial killing. While the intracellular signaling pathways that regulate NET formation remain largely unknown, there is growing evidence that generation of reactive oxygen species (ROS) is a key event for NET formation. The Rab family small GTPase Rab27a is an important component of the secretory machinery of azurophilic granules in neutrophils. However, the precise mechanism of NET formation and whether or not Rab27a contributes to this process are unknown. Using neutrophil-like differentiated HL60 cells, we show here that Rab27a plays an essential role in both phorbol myristate acetate (PMA)- and Candida albicans-induced NET formation by regulating ROS production. Rab27a-knockdown inhibited ROS-positive phagosome formation during complement-mediated phagocytosis. To investigate the role of Rab27a in neutrophil function in detail, both primary human neutrophils and neutrophil-like differentiated HL60 cells were treated with PMA, and NET formation process was assessed by measurement of release of histone H3 into the medium, citrullination of the arginine in position 3 of histone H4 and chase of the nuclear change of the living cells in the co-existence of both cell-permeable and -impermeable nuclear indicators. PMA-induced NET formation occured sequentially in both neutrophil-like differentiated HL60 cells and primary neutrophils, and Rab27a-knockdown clearly inhibited NET formation in association with reduced ROS production. We also found that serum-treated Candida albicans triggers NET formation in a ROS-dependent manner, and that Rab27a-knockdown inhibits this process as well. Our findings demonstrate that Rab27a plays an important role in NET formation induced by both Candida albicans infection and PMA treatment by regulating ROS production.

Effects of vitamin D receptor gene polymorphisms on low-resistance training using exercise machines: the 'Power Rehabilitation' program.

The enhancement and promotion of health is necessary to maintain the quality of life (QOL) of the aged population in developed nations such as Japan where the number of elderly has been increasing rapidly. For this purpose, low-resistance training using exercise machines ('Power Rehabilitation') has been established as a rehabilitation program. To investigate the individual factors which influence the effects of 'Power Rehabilitation', single nucleotide polymorphisms (SNPs) in the vitamin D receptor (VDR) gene and the ciliary neurotrophic factor (CNTF) gene were analyzed, and the relationship between SNP patterns and the effects of 'Power Rehabilitation' was evaluated. 'Power Rehabilitation' had an effect on the physiological functions involved in the activities of daily life (ADL) rather than muscle strength and size. In addition, certain SNP patterns showed better improvement of parameters associated with the effects of 'Power Rehabilitation' as analyzed by comparison between SNP patterns and factor analysis. Large scale analyses are required to ensure this tendency and to discover individual factors which may help to promote the health and QOL of the aged population.

[CD56-positive peripheral T-cell lymphoma primarily presenting with tonsillar swelling].

A 51-year-old woman was admitted to our hospital with tonsillar swelling. After tonsillectomy was performed, she was diagnosed as having CD56-positive T-cell lymphoma, mainly composed of small and medium-sized atypical cells. An immunohistochemical study showed that the malignant lymphocytes were positive for CD3, CD8, CD56, TIA-1 and granzyme B, while negative for CD20, CD5 and CD10. Flowcytometry demonstrated the lymphocytes were positive for CD56. Southern blot analysis revealed a rearrangement of the T-cell receptor gamma chain. The disease stage by Ann Arbor staging classification was II B. We provided MCEC therapy followed by autologous peripheral blood stem cell transplantation, and complete remission (CR) was achieved. Two months after CR, however, the patient relapsed with peritonitis due to perforation of an ileal tumor, and died of sepsis. It is rare for CD56-positive T-cell lymphoma to occur primarily in the tonsils. Because small bowel ulcers were revealed during the course of induction chemotherapy, we report a valuable case in which suspected CD56-positive enteropathy-type T-cell lymphoma (ETL) occurred primarily in the tonsils.

Multiple myeloma complicated by autoimmune hemolytic anemia.

A 57-year-old man was admitted with severe anemia and hypergamma globulinemia. After a diagnosis of multiple myeloma and autoimmune hemolytic anemia was made, chemotherapy rapidly decreased the M-protein level and improved his anemia with normalization of the direct Coombs test. The immunoglobulin binding to the patient's red cells was immunoglobulin G kappa chain like the myeloma M-protein. However, monoclonal immunoglobulin G derived from short-term culture of the patient's bone marrow mononuclear cells did not bind to a panel of red cells. Therefore, the relationship between the M protein produced by his myeloma cells and hemolysis remained unclear.

[Seven patients with stage I and II primary testicular lymphoma].

Seven patients with stage I and II primary testicular lymphoma (PTL) have been treated since 1990 to the present at Kawasaki Medical School. All patients, whose median age was 56 yrs, had initially complained of swelling of the scrotal contents. The lesions were on the right in two patients, on the left in five, and no patient had bilateral lesions. The histological diagnosis was diffuse large B-cell type in all patients. Five patients were classified as Ann Arbor stage I, and two at stage II. After high-orchiectomy for resection of tumor, two patients received chemotherapy alone, with a combination of chemotherapy and irradiation of the contralateral testis in the remaining five. Complete remission was achieved in all seven patients, but relapse occurred later in one. As it is recognized that, even in localized stage or low risk group PTL patients, the relapse rate in the central nervous system (CNS) and contralateral testis is quite high, chemotherapy, prophylactic CNS treatment and radiation of the contralateral testis after tumor resection should be included in the management of PTL.