Histological characteristics of 21 Papua New Guinean children with high-grade B-cell lymphoma, which is frequently associated with EBV infection.

The aim of the present study was to confirm the histopathological features of aggressive B-cell lymphoma in Papua New Guinea (PNG)-an EBV endemic region. The immunophenotypic features and expression of EBV-encoded proteins and RNA in B-cell lymphomas were analyzed in 21 PNG children, and compared to the corresponding features of 17 Japanese children with Burkitt lymphoma (BL). Histological diagnosis of the lymphomas from the PNG children was BL in nine patients; atypical Burkitt/Burkitt-like variant of BL (BLL) in three; diffuse large B-cell lymphoma (DLBCL) in four; and B-lymphoblastic lymphoma (B-LBL) in five. The lymphomas from the PNG children had a high positive rate on EBV-RNA in situ hybridization (EBV-ISH; 66.7%). With regard to the histological typing, 10 of 12 patients (83%) with BL/BLL, one of four (25%) with DLBCL, and three of five (60%) with B-LBL were positive for EBV-ISH. The findings of EBV-positive B-LBL were surprising because it is commonly considered that lymphoblastic lymphoma is not associated with EBV. EBV positivity was not detected in the 12 Japanese patients who were available for the EBV-ISH evaluation. It is concluded that it is possible that a proportion of DLBCL and B-LBL besides BL/BLL are associated with EBV in endemic region.

[Graves' disease with splenomegaly and pancytopenia, mimicking B-cell lymphoproliferative disease].

A 48-year-old woman was referred to our hospital because of fever and general fatigue. Peripheral blood analysis showed a hemoglobin level of 82 g/l, a white blood cell count of 1.95 x 10(9)/l and a platelet count of 80 x 10(9)/l. There were 9% CD5-positive B-cells in peripheral blood and 35% CD10-positive B-cells in bone marrow. The patient had a high serum soluble interleukin-2 receptor (SIL-2R) level of 5,185 U/ml and splenomegaly. Lymphoproliferative disease was suspected, however monoclonal rearranged band of immunoglobulin heavy chain was not detected. She also showed hyperthyroidism, Graves' disease and then treatment with thiamazole started. However, the treatment was stopped because of agranulocytosis and she received subtotal thyroidectomy. After treatment for hyperthyroidism, serum SIL-2R level decreased to 504 U/ml and pancytopenia gradually improved. Fifteen months postoperatively, the percentage of CD5-positive B-cells in peripheral blood and CD10-positive B-cells in bone marrow decreased to 8% and 2%, respectively. This clinical course suggests that polyclonal B-cell proliferation was caused by hyperthyroidism.

Inhibition of alpha-globin gene expression by RNAi.

RNA interference (RNAi), a process by which target messenger RNA (mRNA) is cleaved by small interfering complementary RNA (siRNA), is widely used for investigations of regulation of gene expression in various cells. In this study, siRNA complementary to 5' region of exon II of alpha-globin mRNA was examined for its role in erythroid colony forming cells (ECFCs) isolated from normal peripheral blood donor. On day 6 of cell culture, 1x10(6) ECFCs were transfected with lipofectamine-containing alpha-globin specific siRNA. After 48h of transfection, alpha-globin specific siRNA produced significantly reduction of alpha-globin mRNA level in a dose-dependent manner, but it did not affect the level of beta-globin mRNA. Significantly, decreased numbers of hemoglobinized erythroid cells relative to the control were observed supporting the inhibitory effect of this alpha-globin mRNA specific siRNA.

Insulin-like growth factor-II: a novel autocrine growth factor modulating the apoptosis and maturation of umbilical cord blood erythroid progenitors.

OBJECTIVE: To search a novel function of erythroid progenitor cells circulating as the major nucleated cell population in umbilical cord blood (CB) cells. MATERIALS AND METHODS: Human CB-derived CD36(+) erythroid progenitors were subjected to cDNA microarray. Gene expression and biological property of CB-erythroid progenitors and adult peripheral blood (PB)-erythroid progenitors were compared by using real-time polymerase chain reaction (PCR) and serum-free culture system with erythropoietin (EPO). RESULTS: The microarray revealed 124-fold higher levels of insulin-like growth factor-II (IGF-II) gene expression in CB-CD36(+) erythroid progenitors than in stimulated lymphocytes of adult PB. Real-time PCR verified that IGF-II mRNA levels were highest in CB-CD36(+) erythroid progenitors compared to other CB- or adult PB-fractionated cells. When CB-CD36(+) erythroid progenitors were cultured with EPO in serum-free medium, anti-IGF-II-antibody (Ab) reduced the number of erythroid colonies. When CB- and adult PB-derived erythroid colony-forming cells (ECFCs) were cultured with interleukin-3, stem cell factor, and EPO, mRNA levels per cells of IGF-II peaked on day 12, but those of type 1 and type 2 receptors did not increase with ECFCs maturation. The maturation rate by IGF-II was higher in CB-ECFCs than in adult PB-ECFCs. The majority of CB-ECFCs expressed IGF-II protein. Anti-IGF-II-Ab, but not anti-IGF-I-Ab, reduced the number of CB-ECFCs in liquid culture with EPO. Anti-IGF-II-Ab accelerated apoptosis of ECFCs, assessed by dimethylthiazole tetrazolium bromide, bromodeoxyuridine, and flow cytometric analyses. ECFCs failed to attain full maturity in the presence of anti-IGF-II-Ab. CONCLUSIONS: These results suggest that IGF-II is produced by erythroid progenitors themselves, and has a crucial role in fetal erythropoiesis by modulating apoptosis and maturation in an autocrine fashion.

Frequent microsatellite instability in non-Hodgkin lymphomas irresponsive to chemotherapy.

Microsatellite instability (MSI) in haematopoietic malignancies has been controversial. Particularly in non-Hodgkin lymphoma, the data published to date lack unity. Using a unique fluorescent technique, we found MSI in eight (14%) tumours in a panel of 59 carefully selected non-Hodgkin lymphoma patients. Our fluorescent technique also reveals two qualitatively distinct modes of MSI, i.e. Type A and Type B. Based on our previous studies using DNA mismatch repair (MMR) gene-knock out animals, we have concluded that Type A MSI is a direct consequence of defective MMR. MSI observed in non-Hodgkin lymphomas was uniformly Type A, which implies that MMR deficiency occurs in this malignancy. Intriguingly, in non-Hodgkin lymphoma patients treated by CHOP/VEPA-based therapies, response to chemotherapy was significantly worse in those with microsatellite-unstable tumours (p=0.027). As a consequence, the patient outcomes at 1 year after treatment were significantly less favourable in this population (p=0.046), although the survival difference was not statistically confirmed in a longer term. These findings suggest that in some non-Hodgkin lymphomas MMR deficiency may lead to drug resistance in tumour cells and, consequently, to poor patient outcomes. In non-Hodgkin lymphoma, MSI may be a potential biomarker that predicts the tumour response against chemotherapy.

Expression patterns of microRNAs 155 and 451 during normal human erythropoiesis.

To clarify the roles of microRNAs (miRNAs) in erythropoiesis, the expression of miR-155, miR-221, miR-223, and miR-451 were analyzed during the differentiation of purified normal human erythroid progenitors in a liquid culture system. Cells increased almost 500-fold in a number, and differentiated to benzidine-positive mature erythroblasts. Analyses of miRNA expression using the quantitative real-time polymerase chain reaction showed that the expression level of miR-155 decreased about 200-fold, and that the expression of miR-451 increased about 270-fold during 12 days of cultures. A moderate down-regulation of miR-221 and miR-223 was observed. MiR-451 was expressed in red blood cells about 10(4)-fold more than in granulocytes, obtained from normal human peripheral blood. These observations suggest that miR-155 and miR-451 are key molecules for normal erythroid differentiation, and that quantitative assays of the two miRNAs may be a relevant method for analyzing pathological erythropoiesis.

B-cell lymphoma during pregnancy associated with hemophagocytic syndrome and placental involvement.

We report a case of B-cell lymphoma during pregnancy associated with hemophagocytic syndrome and placental involvement. A 33-year-old Japanese woman developed pancytopenia, hepatosplenomegaly, and a high-grade fever for 2 weeks at 23 weeks of gestation. The demonstration of hemophagocytes in her bone marrow confirmed the diagnosis of hemophagocytic syndrome. She was referred at 25 weeks of gestation for evaluation of hemophagocytic syndrome. The screening for infection and autoimmune disease was negative. Clinical manifestation suggested malignant lymphoma as the underlying cause of hemophagocytic syndrome, but we could not confirm any lymphoma involvement in the bone marrow aspiration. Glucocorticoid therapy did not arrest the hemophagocytic process. Her general status worsened, and reduction of amniotic fluid was noted. At 28 weeks of gestation, we performed a Cesarean section because of fetal distress. Microscopic examination of placental specimen revealed diffuse infiltration of large, atypical lymphoid cells involving the intervillous space. Using immunohistochemical study, we made the diagnosis of B-cell lymphoma. R-CHOP (rituximab/cyclophosphamide/doxorubicin/vincristine/prednisone) chemotherapy was administered on the eighth postpartum day. After 2 cycles of R-CHOP chemotherapy, hematopoiesis became normal and hepatosplenomegaly almost completely disappeared. After 6 cycles of R-CHOP, the patient received autologous peripheral-blood stem cell transplantation, and she is currently in complete remission 1 year after diagnosis. The infant did well, without clinical or laboratory manifestations of malignant lymphoma. In cases with suspected malignancy associated with hemophagocytic syndrome during pregnancy, it is important to verify placental microscopic examination for evaluating the causative disease of hemophagocytic syndrome.

[Human herpesvirus 6 encephalitis in a patient with adult T-cell leukemia/lymphoma].

Human herpesvirus 6 (HHV-6) reactivates in immunocompromised patients, and HHV-6 encephalitis has often been reported as a complication of transplantation. We describe a 37-year-old woman with the acute type of adult T-cell leukemia/lymphoma who developed HHV-6 encephalitis before chemotherapy. The patient's main symptoms were disorientation and short-term memory loss. Magnetic resonance imaging of the brain showed a bilateral T2 prolongation within the temporal lobes, and HHV-6 DNA was detected in the cerebrospinal fluid (CSF). After treatment with ganciclovir, HHV-6 DNA disappeared from the CSF and the patient's symptoms gradually improved. HHV-6 encephalitis should be listed as a differential diagnosis of encephalopathy developing in immunocompromised patients.

Epithelioid hemangioendothelioma presenting with severe myelofibrosis and a high serum hyaluronan level.

Epithelioid hemangioendothelioma (EHE) is a rare tumor originating from the vascular endothelium; it has an intermediate malignant potential. EHEs affect all age groups and mostly originate from the soft tissues of the extremities, lungs, and liver. Spinal EHEs, especially those occurring in the bone marrow region, are extremely rare. We report a case of EHE with massive involvement of the liver, vertebrae, and cranial bones that caused severe myelofibrosis (MF) in a 67-yr-old-male patient. Hyaluronan deposits were diffusely observed in the tumor tissue biopsies obtained from both the liver and bone marrow. Furthermore, the serum hyaluronan level increased markedly along with rapid progression of the disease. To the best of our knowledge, this is the first report of MF occurring in an EHE; hyaluronan may have played an important role in the pathogenesis of fibrosis in this case.

Mortalin is a novel mediator of erythropoietin signaling.

Erythropoietin (EPO) stimulates erythroid growth by enhancing the proliferation, maturation and survival of late-stage erythroid progenitor cells. However, the entire process of EPO stimulation remains undetermined. To further clarify the intracellular mechanisms by which EPO affects the growth of erythroid progenitor cells, we analyzed proteins obtained from purified human erythroid colony-forming cells (ECFCs) cultured with or without EPO, and one of the proteins apparently related with EPO stimuli was identified as mortalin (mthsp70/PBP74/Grp75/mot-2), which is a member of the heat shock protein 70 family of chaperones. The amount of mortalin mRNA in ECFCs increased in an EPO dose-dependent manner, and ECFC growth was dependent on the amount of mortalin. Furthermore, expression of mortalin in ECFCs was suppressed by a phosphatidylinositol 3-kinase inhibitor. Finally, we analyzed gene expression patterns in ECFCs cultured with or without EPO after treatment with mortalin small interfering RNA (siRNA) using a DNA microarray. When ECFCs treated with mortalin siRNA were cultured with EPO, the expression of several genes overlapped with the profile seen in control ECFCs cultured without EPO. Our data suggest that mortalin is involved in the mediation of EPO signaling and plays an important role in stimulating the growth of erythroid progenitor cells.

[Plasma cell leukemia presenting with cleaved nuclei and a monocytoid appearance].

A 74-year-old man presented with anemia, thrombocytopenia and leukocytosis with 41% abnormal cells having cleaved and monocytoid nuclei. The bone marrow was infiltrated with 43.6% abnormal cells that were negative for peroxidase staining and positive for PAS staining. Surface antigen analysis revealed no expression of CDs 2, 3, 4, 8, 10, 19, 20, 13 or 33. Serum immunoelectrophoresis detected a monoclonal band, which was identifiable as kappa-type IgA protein. Immunostaining also revealed expression of IgA and kappa-type in the abnormal cells. The patient was diagnosed as having plasma cell leukemia but the morphological findings were unusual. Surface antigen analysis revealed expression of CD38 later. This case suggests that immunostaining of abnormal cells is required for the differential diagnosis of plasma cell leukemia in leukocytosis.

Therapeutic angiogenesis by ex vivo expanded erythroid progenitor cells.

Recent reports have demonstrated that erythroid progenitor cells contain and secrete various angiogenic cytokines. Here, the impact of erythroid colony-forming cell (ECFC) implantation on therapeutic angiogenesis was investigated in murine models of hindlimb ischemia. During the in vitro differentiation, vascular endothelial growth factor (VEGF) secretion by ECFCs was observed from day 3 (burst-forming unit erythroid cells) to day 10 (erythroblasts). ECFCs from day 5 to day 7 (colony-forming unit erythroid cells) showed the highest VEGF productivity, and day 6 ECFCs were used for the experiments. ECFCs contained larger amounts of VEGF and fibroblast growth factor-2 (FGF-2) than peripheral blood mononuclear cells (PBMNCs). In tubule formation assays with human umbilical vein endothelial cells, ECFCs stimulated 1.5-fold more capillary growth than PBMNCs, and this effect was suppressed by antibodies against VEGF and FGF-2. Using an immunodeficient hindlimb ischemia model and laser-Doppler imaging, we evaluated the limb salvage rate and blood perfusion after intramuscular implantation of ECFCs. ECFC implantation increased both the salvage rate (38% vs. 0%, P < 0.05) and the blood perfusion (82.8% vs. 65.6%, P < 0.01). In addition, ECFCs implantation also significantly increased capillaries with recruitment of vascular smooth muscle cells and the capillary density was 1.6-fold higher than in the control group. Continuous production of human VEGF from ECFCs in the skeletal muscle was confirmed at least 7 days after the implantation. Implantation of ECFCs promoted angiogenesis in ischemic limbs by supplying angiogenic cytokines (VEGF and FGF-2), suggesting a possible novel strategy for therapeutic angiogenesis.

[Successful cyclosporine treatment for thrombocytopenia after salvage therapy with arsenic trioxide therapy followed by autologous hematopoietic stem cell transplantation in acute promyelocytic leukemia].

A 55-year-old man with acute promyelocytic leukemia in the first relapse was treated with arsenic trioxide as salvage therapy. After obtaining molecular remission, he underwent autologous peripheral blood stem cell transplantation (PBSCT) with busulfan and melphalan conditioning. The transplant dose of CD 34-positive cells was sufficient, and engraftment was prompt. Platelet count increased to 320 x 10(9)/1 on day 21; however, it rapidly decreased to 27 x 10(9)/l on day 37. Despite treatment with corticosteroid, the platelet count decreased to 6 x 10(9)/l on day 55. About one month after cyclosporine administration, thrombocytopenia gradually improved. This clinical course suggests immune-mediated thrombocytopenia following autologous PBSCT.

Pivotal role of Notch signaling in regulation of erythroid maturation and proliferation.

Notch signaling plays an important role in cell fate decisions in developmental systems. To clarify its role in committed hematopoietic progenitor cells, we investigated the effects of Notch signaling in erythroid colony forming cells (ECFCs) generated from peripheral blood. ECFCs express Notch receptors, Notch1 and Notch2, and Notch ligands Delta1, Delta4, and Jagged1. When we assayed the effects of Notch ligands on erythroid maturation by flow cytometry, we found that immobilized Delta1 and immobilized Delta4 in particular inhibited maturation, whereas Jagged1 had no effect. In addition, Delta4 inhibited proliferation without reducing cell viability. Increases in expression levels of the Notch target gene hairy enhancer of split (HES) -1 were evident by real-time PCR after stimulation with immobilized Delta4. The effect of soluble Delta4 on expression of HES-1 was less pronounced than that seen with the immobilized form, indicating that all surface-bound ligands are important for effective signal transduction. When ECFCs were cultured in the presence of soluble Delta4 at a low cell concentration, erythroid maturation was slightly inhibited, but at a high concentration, maturation was promoted via competition of soluble Delta4 with endogenous ligands. These results indicate a pivotal role of Notch signaling in regulating erythroid maturation and proliferation, and further suggest that cell-cell interactions modulate growth of erythroid progenitor cells via Notch system.

Single nucleotide polymorphisms and haplotypes of protein C and protein S genes in the Thai population.

Protein C (PC) and protein S (PS) play key roles in an anticoagulant pathway in order to control the haemostatic system. We identified single nucleotide polymorphisms (SNPs) and/or haplotypes in the promotor and exons of the whole PC and PS genes and in the 3'-untranslated region of the PS gene in 55 Thai individuals. The PC gene revealed 10 haplotypes. One synonymous SNP at 2196 was found in the normal Thai population with a minor allele frequency of 4.90%. One homozygous mutation in exon 7, R147W, co-segregated with the synonymous SNP 2196 (homozygote) of the PC gene, resulting in decreased PC activity and antigenic levels. The PS gene revealed three haplotypes with two frequent dimorphisms in exon 15 and the 3'-untranslated region. The most frequent haplotype in the PS gene was H3 (wild type). There was no correlation between the haplotypes of PC and PS genes with functional and antigenic levels of PC and PS.

Hydroxyurea responses and fetal hemoglobin induction in beta-thalassemia/HbE patients' peripheral blood erythroid cell culture.

Due to genetic heterogeneity of beta-thalassemia (beta-thal) patients, several efforts have been undertaken to determine the efficacy of hydroxyurea treatment. The aim of this work is to determine the responder and nonresponder for hydroxyurea treatment in beta-thal intermedia based on gamma-globin mRNA and fetal hemoglobin (HbF) induction in human erythroid progenitor cells purified from a patient's peripheral blood. Eighteen beta-thal/hemoglobin E patients [13 beta(E)/codon41/42(-TCTT), 4 beta(E)/codon17, and 1 beta(E)/IVS-654], requiring blood transfusion occasionally, with Hb levels of 5.20-8.50 g/dl were studied. The relative levels of gamma-globin mRNA was measured by real-time reverse-transcription polymerase chain reaction and HbF by high-performance liquid chromatography. The results indicated that erythroid progenitor cells treated with 30 mumol/l hydroxyurea for 96 h preferentially enhanced (G)gamma-and (A)gamma-globin mRNA. The mean values of (G)gamma-globin mRNA fold induction were higher than (A)gamma-globin mRNA (12+/-4 vs 4+/-0.30), the Pearson's correlation of (G)gamma-and (A)gamma-globin mRNA was r=0.80. Induction of (G)gamma/(A)gamma globin mRNA is up to ninefold. A 30% increase in the proportion of HbF out of the total Hb was found in cultures derived from four patients, 20-30% in cultures from nine patients, and less than 20% in cultures from five patients. In cultures from only two patients, increase in the proportion of HbF was less than 3%, and (G)gamma/(A)gamma globin mRNA is less than 0.50.

Sustained molecular remission by non-myeloablative stem cell transplantation after autologous hematopoietic stem cell transplantation in a patient with multiple myeloma.

Multiple myeloma (MM) is refractory to conventional chemotherapy. To achieve a sustained complete remission, we performed planned non-myeloablative allogeneic stem cell transplantation (NST) after autologous hematopoietic stem cell transplantation (HSCT) in a patient with stage III MM. Autologous HSCT was performed using high-dose melphalan after conventional chemotherapy, followed by NST from an HLA-identical sibling using low-dose total body irradiation (200 cGy) for conditioning. Cyclosporine and mycophenolate mofetil were used for graft-vs-host disease (GVHD) prophylaxis. Acute GVHD was transiently seen in the skin and intestine, while, in addition, mild chronic GVHD was seen in the oral mucosa and skin. Complete donor chimerism was achieved and the disappearance of tumor-derived monoclonal B cells was confirmed based on an analysis of immunoglobulin light chain messenger signals on day 156 when chronic GVHD occurred. The clinical course in this case strongly suggested the existence of a graft-vs-myeloma effect.

Genome-wide array-based comparative genomic hybridization of natural killer cell lymphoma/leukemia: different genomic alteration patterns of aggressive NK-cell leukemia and extranodal Nk/T-cell lymphoma, nasal type.

Natural killer (NK) cell lymphomas/leukemias are highly aggressive lymphoid malignancies, but little is known about their genomic alterations, and thus there is an urgent need for identification and analysis of NK cell lymphomas/leukemias. Recently, we developed our own array-based comparative genomic hybridization (array CGH) with an average resolution of 1.3 Mb. We performed an array CGH analysis for 27 NK-cell lymphoma/leukemia cases that were classified into two disease groups based on the World Health Organization Classification (10 aggressive NK-cell leukemia cases and 17 extranodal NK/T-cell [NK/T] lymphomas, nasal type). We identified the differences in the genomic alteration patterns of the two groups. The recurrent regions characteristic of the aggressive NK-cell leukemia group compared with those of the extranodal NK/T lymphoma, nasal-type group, were gain of 1q and loss of 7p15.1-p22.3 and 17p13.1. In particular, gain of 1q23.1-24.2 (P = 0.041) and 1q31.3-q44 (P = 0.003-0.047), and loss of 7p15.1-p22.3 (P = 0.012-0.041) and 17p13.1 (P = 0.012) occurred significantly more frequently in the former than in the latter group. Recurrent regions characteristic of the extranodal NK/T lymphoma, nasal-type group, compared with those of the other group were gain of 2q, and loss of 6q16.1-q27, 11q22.3-q23.3, 5p14.1-p14.3, 5q34-q35.3, 1p36.23-p36.33, 2p16.1-p16.3, 4q12, and 4q31.3-q32.1. Our results can be expected to provide further insights into the genetic basis of lymphomagenesis and the clinicopathologic features of NK-cell lymphomas/leukemias.