Hiroshi Kawabata

The synthetic furanonaphthoquinone induces growth arrest, apoptosis and differentiation in a variety of leukaemias and multiple myeloma cells.

2‐Methyl‐naphtho[2,3‐b]furan‐4,9‐dione (FNQ3), a synthetic analogue of the quinone kigelinone, has demonstrated a real potential for use in the treatment of a variety of solid tumours. Unlike other quinones, such as mitomycin‐C and adriamycin, the cytotoxicity of FNQ3 is often 10‐ to 14‐fold more potent towards the tumour cells than their normal counterparts. We report, for the first time, that the drug had activity against a broad spectrum of leukaemias and multiple myeloma cells. It decreased the growth of acute myeloid leukaemia (AML) and multiple myeloma cell lines in a dose‐dependent fashion (50% inhibitory concentration ≈1·25u2003μg/ml against most of the leukaemia cell lines). This dose apparently initiated mitochondrial collapse as measured by depolarisation of the mitochondrial membrane. FNQ3 potentiated the differentiation of HL‐60 myeloid cells in the presence of either 1α, 25(OH)2 dihydroxyvitamin D3 [1α,25(OH)2D3] or all‐trans‐retinoic acid (ATRA). FNQ3 inhibited the proliferation of primary AML cells while inducing apoptosis. Eleven of 14 (79%) AML marrow samples had a prominent decrease in their clonogenic growth when cultured in the presence of the drug. In summary, this drug has growth inhibitory, apoptotic and differentiative effects against myeloid leukaemias and multiple myeloma cells. FNQ3 may represent a new therapeutic approach to these malignancies.

Association of Epstein-Barr virus with human immunodeficiency virus-negative peripheral T-cell lymphomas in Japan.

Abstract:u2002 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 (Pu2003=u20030.0498 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%; Pu2003=u20030.0013).

Incidence of Diffuse Large B-Cell Lymphoma of Germinal Center B-Cell Origin in Whole Diffuse Large B-Cell Lymphoma : Tissue Fluorescence In Situ Hybridization Using t(14 ; 18) Compared with Immunohistochemistry

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.Astudy 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.

Clinical Significance of Serum Hepcidin Levels on Early Infectious Complications in Allogeneic Hematopoietic Stem Cell Transplantation

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 (> or = 50 ng/mL; n = 17) than in the low-hepcidin group (<50 ng/mL; n = 38) (65% [95% confidence interval, 38%-82%] versus 11% [3%-23%]; P < .001). This finding was confirmed by multivariate Cox analysis adjusted for confounders, including pretransplant ferritin and C-reactive protein (CRP) levels. No fungal infection was documented in either group. These results suggest that the pretransplant serum hepcidin-25 level may be a useful marker for predicting the risk of early bacterial complications after allogeneic HSCT. Larger prospective studies are, however, warranted to confirm our findings.

Epstein-Barr Virus-Associated B-Cell Type Non-Hodgkin’s Lymphoma with Concurrent p53 Protein Expression in a Rheumatoid Arthritis Patient Treated with Methotrexate

A Japanese male patient received various medications for his long-standing rheumatoid arthritis (stage IV, class II). He developed a mass on the right anterior chest wall after being treated with methotrexate (MTX) for 4 months. A biopsy of the mass showed it to be Epstein Barr virus (EBV)-associated lymphoma of B-cell phenotype stage IE (bulky mass), with positive EBV-encoded small RNAs (EBERs) in situ hybridization, EBV latent membrane protein-1 (LMP-1) negative, EB nuclear antigen-2 (ERNA-2) negative, CD20/L26 (+), CD45RO/UCHL-1 (-). A single band of the joined termini of the EBV genome was demonstrated in DNA extracted from the mass, suggesting a clonal disorder of the mass. Immunostaining of the mass with p53 antibody was also positive.With discontinuation of MTX and administration of chemotherapy, the tumor disappeared but recurred after 3 months. This case suggests that concordant p53 expression and latent EBV infection may play an important role in the pathogenesis of lymphomas arising in patients with rheumatoid arthritis who are immunosuppressed with MTX.

Double Ph‐positive megakaryoblastic transformation of chronic myeloid leukemia

Abstract: 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.

Efficacy of interferon-alpha in essential thrombocythemia during pregnancy

Dear Editor, Essential thrombocythemia (ET) is a chronic myeloproliferative neoplasm associated with an increase in platelet counts and risk of vascular complications. When young women with ET become pregnant, an adverse outcome of pregnancy caused by thromboembolic complications is a matter of concern [1]. Reduction of platelet counts by conventional chemotherapeutic agents is not advisable due to their potential fetal toxicities. Two observational studies involving retrospective multicenter cases suggested a role of interferon-alpha in this clinical setting [1, 2]. Herein, we report four consecutive pregnancies in three ET patients (aged 22 to 32 years) successfully treated with interferon-alpha in our institution between 2001 and 2016. To our knowledge, this represents the largest single-center series. No patients had a prior history of pregnancy or thrombosis. The time from diagnosis of ET to pregnancy ranged from 19 to 77 months. Patient 1 became pregnant twice at the age of 22 and 27 years. All patients were prophylactically given acetylsalicylic acid (ASA) shortly after the diagnosis of ET. Patients 1 and 3 had calreticulin mutations but not Jak 2 mutation, while genetic data were not available for patient 2. Platelet counts ranged from 1095 to 1741 × 10/L at the time of diagnosis (Table 1) and decreased slightly when patients became pregnant. After obtaining informed consent and IRB approval, interferon-alpha was started from 26 to 29 weeks of gestation in patients 1 and 2, respectively. Because of the highly elevated platelet counts, interferon was started at 12 weeks of gestation in patient 3. The interferon dose was three million units given subcutaneously twice or three times per week. In all patients, platelet counts showed a gradual decrease during the treatment, which was well tolerated with no noteworthy adverse events except for a transient liver dysfunction in patient 3. Full-term delivery was obtained in all but patient 3, in whom a cesarean section was performed because of breech presentation. All the babies had normal birth weight and platelet counts (Table 1). Interferon-alpha is known to have anti-proliferative effect on pluripotent as well as lineage-committed progenitor cells. In addition, interferon-alpha induction of SOCS-1 impairs thrombopoietin signaling, leading to direct suppression of megakaryocyte growth [3]. Platelet count before conception may not correlate or predict the risk of pregnancy complication [4, 5]. However, reduction of platelet counts with interferon-alpha has yielded uneventful delivery in most cases. Indeed, an Italian registry [1] reported live births in 19 out of 20 pregnant ET patients given interferon, as did an UK multicenter study [2] in 9 out of 10 pregnant ET patients treated with pegylated interferon-alpha-2a. A systematic survey indicates that interferon-alpha does not significantly increase the fetal risks above that in general population rates [6]. In conclusion, our study supports that interferon-alpha is the treatment of choice in the management of ET patients in pregnancy [7]. * Yataro Yoshida yoshida@takedahp.or.jp

Epstein-Barr virus-associated anaplastic large cell variant of diffuse large B-cell-type non-Hodgkin's lymphoma with concurrent p53 protein expression.

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-, CDl0-, 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.

Tentative diagnostic criteria and disease severity classification for Castleman disease: A report of the research group on Castleman disease in Japan

Abstract Objectives: To determine the tentative diagnostic criteria and disease severity classification for Castleman disease (CD) and describe the clinical and pathologic features among human herpesvirus 8 (HHV-8) negative idiopathic multicentric CD (iMCD) in the Japanese population. Methods: We established the working groups for the research of CD in Japan and had meetings to discuss and define the tentative diagnostic criteria and disease severity classification for CD. We subsequently analyzed 142 patients classified into iMCD by using the nationwide Japanese patient registry. Results: We proposed the preliminary diagnostic criteria and disease severity classification for CD based on our discussion. In addition, we made a proposal for the disease activity score. We identified clinical and pathological features of patients with iMCD diagnosed by these diagnostic criteria. In the disease severity classification, 37, 33 and 30% patients were categorized into mild, moderate and severe diseases, respectively. Conclusion: This is the first proposal for diagnosis and classification of CD by the Japanese group. Further studies are required to validate whether they can distinguish CD from other inflammatory diseases and to determine their sensitivity and specificity.

Transferrin and transferrin receptors update

ABSTRACT In vertebrates, transferrin (Tf) safely delivers iron through circulation to cells. Tf‐bound iron is incorporated through Tf receptor (TfR) 1‐mediated endocytosis. TfR1 can mediate cellular uptake of both Tf and H‐ferritin, an iron storage protein. New World arenaviruses, which cause hemorrhagic fever, and Plasmodium vivax use TfR1 for entry into host cells. Human TfR2, another receptor for Tf, is predominantly expressed in hepatocytes and erythroid precursors, and holo‐Tf dramatically upregulates its expression. TfR2 forms a complex with hemochromatosis protein, HFE, and serves as a component of the iron sensing machinery in hepatocytes. Defects in TfR2 cause systemic iron overload, hemochromatosis, through down‐regulation of hepcidin. In erythroid cells, TfR2 forms a complex with the erythropoietin receptor and regulates erythropoiesis. TfR2 facilitates iron transport from lysosomes to mitochondria in erythroblasts and dopaminergic neurons. Administration of apo‐Tf, which scavenges free iron, has been explored for various clinical conditions including atransferrinemia, iron overload, and tissue ischemia. Apo‐Tf has also been shown to ameliorate anemia in animal models of &bgr;‐thalassemia. In this review, I provide an update and summary on our knowledge of mammalian Tf and its receptors. Graphical abstract Figure. No Caption available. HighlightsTransferrin receptor 1 (TfR1) mediates cellular uptake of holo‐Tf and H‐ferritin.New World arenaviruses and Plasmodium vivax use TfR1 for entry into host cells.In hepatocytes, TfR2 forms a complex with HFE and serves as an iron sensor.In erythroblasts, TfR2 forms a complex with EPO receptor and regulates erythropoiesis.TfR2 facilitates iron transport from the lysosomes to mitochondria in erythroblasts.