Kentaro Yoshinaga

Lack of nucleophosmin mutation in patients with myelodysplastic syndrome and acute myeloid leukemia with chromosome 5 abnormalities

Nucleophosmin (NPM1) gene exon 12 mutations are frequently present in patients with acute myeloid leukemia (AML) with normal karyotype. The NPM1 gene is located on chromosome 5q35, which is often affected in myeloid malignancies including myelodysplastic syndrome (MDS). This suggests that the NPM1 gene is a one of the target genes affected by chromosome 5 abnormalities and play a role in the development of MDS. It has not been clarified whether MPM1 mutations are present in patients with MDS and AML with chromosome 5 abnormalities. Therefore, we carried out a mutational analysis on the NPM1 gene exon 12. NPM1 mutations were not detected in the 28 patients with MDS and AML with chromosome 5 abnormalities.

Effect of interleukin 11 on normal and pathological thrombopoiesis.

Abstract Interleukin 11 (IL-11) is a stromal cell-derived cytokine that has multiple effects on hematopoietic and nonhematopoietic systems. In vitro, it enhances the growth of early progenitors and promotes megakaryocytopoiesis and erythropoiesis. In healthy animals, IL-11 administration stimulates megakaryocyte maturation and increases peripheral platelet counts. IL-11 accelerates the recovery of peripheral neutrophil, erythrocyte, and platelet counts in mice that have undergone cytoablative treatment. Therefore, IL-11 may be useful clinically as an agent promoting recovery from hematopoiesis. However, its clinical use in patients with hematological malignancies may be restricted because IL-11 has been reported to stimulate some leukemia and myeloma cells. In the United States, phase I trials have shown that IL-11 accelerates recovery from chemotherapy-induced or bone-marrow transplantation (BMT)-induced thrombocytopenia. In Japan, phase II trials studying the thrombopoietic effect of IL-11 in patients with solid tumors postchemotherapy, in patients undergoing BMT, and in patients with aplastic or refractory anemia are now under way. Recently, thrombopoietin (TPO) has been cloned, and its thrombopoietic effect and accelerating effect on platelet count recovery in thrombopoietic states have been demonstrated in animal models. The physiological effect of TPO is restricted to hematopoiesis; therefore, it may have fewer side effects than IL-11. However, in addition to its hematopoietic effect, IL-11 administration to mice that have undergone cytoablative therapy significantly decreases morbidity and mortality due to chemotherapy-related endogenous infections caused by gut microorganisms. Therefore, IL-11 can be used in patients postchemotherapy and post-BMT not only to promote platelet recovery but also to prevent life-threatening infections. The use of in-vitro-expanded hematopoietic stem cells for BMT or as target cells for gene therapy is one of the most exciting areas in the field of medicine. Since IL-11 can expand hematopoietic progenitor-cell populations when used in combination with other cytokines, it may be useful as an ex vivo hematopoietic progenitor-cell-amplifying agent

Mechanism of action of antithymocyte globulin in the treatment of aplastic anaemia: in vitro evidence for the presence of immunosuppressive mechanism.

Antithymocyte globulin (ATG) is one of the effective drugs used in the treatment of aplastic anaemia (AA). Although it has been speculated that the mechanism of action of ATG is mediated by its immunosuppressive effect on lymphocytes which might have an inhibitory effect on haemopoietic stem and progenitor cells, no definite evidence of the presence of such a mechanism has been demonstrated. In this study we investigated whether such a mechanism is truly operating in ATG therapy for AA. In five patients who responded to ATG, bone marrow cells were obtained after haematological recovery and CD34‐positive cells were separated by immunobeads. Autologous CD34‐positive cells were mixed with autologous peripheral CD4‐ or CD8‐positive cells obtained before ATG therapy and after haematological recovery, liquid‐cultured for 12 h, and then cultured in methylcellulose for 14 d in the presence of haemopoietic growth factors. In all five cases studied, only the CD8 cells obtained before ATG therapy suppressed the colony forming unit‐granulocyte‐macrophage (CFU‐GM)‐ and burst forming unit‐erythroid (BFU‐E)‐derived colony formation. This result is definite evidence that one of the mechanisms of action of ATG in AA is an inhibitory effect on CD8‐positive cells which have suppressive activity for the growth of haemopoietic progenitor cells.

L265P Mutation of the MYD88 Gene Is Frequent in Waldenström’s Macroglobulinemia and Its Absence in Myeloma

L265P mutation in the MYD88 gene has recently been reported in Waldenström’s macroglobulinemia; however the incidence has been different according to the methods used. To determine the relevance and compare the incidence by different methods, we analyzed the L265P mutation in bone marrow mononuclear cells from lymphoid neoplasms. We first performed cloning and sequencing in 10 patients: 8 Waldenström’s macroglobulinemia; 1 non-IgM-secreting lymphoplasmacytic lymphoma; and 1 low grade B-cell lymphoma with monoclonal IgG protein. The L265P mutation was detected in only 1/8 Waldenström’s macroglobulinemia patients (2 of 9 clones). To confirm these results, direct sequencing was performed in the 10 patients and an additional 17 Waldenström’s macroglobulinemia patients and 1 lymphoplasmacytic lymphoma patient. Nine of 28 patients (7/25 Waldenström’s macroglobulinemia, 1/2 lymphoplasmacytic lymphoma, and B-cell lymphoma) harbored the mutation. We next tested for the mutation with BSiE1 digestion and allele-specific polymerase chain reaction in the 28 patients and 38 patients with myeloma. Aberrant bands corresponding to the mutation were detected by BSiE1 digestion in 19/25 patients with Waldenström’s macroglobulinemia (76%), 1/2 lymphoplasmacytic lymphoma and B-cell lymphoma, but not in the 38 myeloma patients. The L265P mutation was more frequent in patients with Waldenström’s macroglobulinemia than in those with myeloma (p=1.3x10-10). The mutation was detected by allele-specific polymerase chain reaction in 18/25 Waldenström’s macroglobulinemia patients (72%). In the 25 Waldenström’s macroglobulinemia patients, the L265P was more frequently detected by BSiE1 digestion than by direct sequencing (p=5.3x10-4), and in males (15/16, 94%) than in females (4/9, 44%) (p=1.2x10-2). No siginificant difference was observed in the incidence of the L265P mutation between BSiE1 digestion and allele-specific polymerase chain reaction (p=0.32). These results suggest that the L265P mutation is involved in the majority of Waldenström’s macroglobulinemia. BSiE1 digestion and allele-specific polymerase chain reaction may detect a small fraction of mutated cells in some cases.

Risk factor analysis of vasovagal reaction from blood donation

BACKGROUND Vasovagal reaction (VVR) is the most frequent side effect at blood collection sites. AIMS To protect donors, factors contributing to VVR were analysed. MATERIALS AND METHODS Complications following whole blood and apheresis donations have been recorded and accumulated by the Japanese Red Cross Tokyo Blood Centre. A dataset of 43,948 donors who had no complications was prepared as a control by randomly selecting days in each season in the 2006 and 2007 fiscal years. Factors contributing to 4924 VVR incidents in the 2006 and 2007 fiscal years were analysed by univariate and multivariate logistic regression. RESULTS The age, weight, body mass index (BMI), predonation systolic and diastolic pressure, and circulating blood volume were lower, and the pulse was higher, for the VVR group compared to the control group (p<0.0001). The VVR group had more female donors, less sleep, and more time since a meal than the control. In multivariate analysis, significant risk factors for 400 ml whole blood donors, which are the majority of donors, were an age <50 years, being female, a BMI <25, pulse ≥90/min, sleep duration <8 h, the time after eating ≥4 h, a first time donation and circulating blood volume of <4.3 l. Sleep duration of <6 h was shown to be a VVR risk as much as a first time donation. CONCLUSION From our analysis, the amount of sleep obtained the previous night should be considered at the reception of donors.

Aberrant methylation of the RIZ1 gene in myelodysplastic syndrome and acute myeloid leukemia.

We performed methylation specific PCR analysis on the RIZ1 promoter in MDS and AML. Methylation was detected in 17 of 34 MDS (50%) and 22 of 72 AML (31%) (p=0.053). Methylation was detected in eleven of 17 secondary AML from MDS (65%), and eleven of 55 de novo AML (20%) (p=0.0005). Bisulfite sequence revealed methylation at many CpG sites in the promoter. Decreased RIZ1 expression was accompanied by methylation in six of nine samples examined, while it was also observed in seven of 13 without methylation. Treatment of AML cells, that have RIZ1 methylation, with 5-Aza-dC, induced growth suppression with RIZ1 restoration. Our results suggest that the RIZ1 gene is inactivated in MDS and AML in part by methylation, whereas another mechanism should be involved in others.

Development of diffuse large B-cell lymphoma in a patient with Waldenström’s macroglobulinemia/ lymphoplasmacytic lymphoma: clonal identity between two B-cell neoplasms

Waldenströms macroglobulinemia (WM)/ lymphoplasmacytic lymphoma (LPL) is an indolent mature B-cell neoplasm. In rare cases of WM/LPL, diffuse large B-cell lymphoma (DLBCL) develops as a result of histologic transformation. In this report, we present a case of DLBCL developing in a patient with WM/LPL. Combination chemotherapy for DLBCL was effective and complete remission was eventually achieved. We attempted to determine the clonal relatedness between WM/LPL and DLBCL in the patient by analyzing complementarity-determining region 3 (CDR3) in the immunoglobulin heavy chain gene. A common CDR3 sequence was found in tumor cells of DLBCL and those of WM/LPL, indicating that tumor cells of DLBCL are clonally identical to those of WM/LPL. Therefore, in the present case, DLBCL is developed from WM/LPL cells by clonal evolution.

K‐252a‐induced polyploidization and differentiation of a human megakaryocytic cell line, Meg‐J: transient elevation and subsequent suppression of cyclin B1 and cdc2 expression in the process of polyploidization

Megakaryocytes are unique haemopoietic cells which undergo DNA replication, giving rise to polyploid cells. However, little is known about the mechanism of megakaryocytic polyploidization. To address this issue, we used the human megakaryocytic cell line Meg‐J. In the presence of K‐252a (an indolocarbasole derivative), Meg‐J cells stopped proliferation and exhibited additional megakaryocytic features, including morphological changes, polyploidization, and increases in the levels of surface expression of platelet glycoprotein (GP) IIb/IIIa and GPIb. Thrombopoietin (TPO) promoted the K‐252a‐induced polyploidization and megakaryocytic differentiation. In the process of K‐252a‐induced polyploidization, levels of expression of both cdc2 and cyclin B1 were elevated transiently and subsequently decreased. This suggested that the polyploidization process in Meg‐J cells was at least in part associated with a transient elevation and subsequent decrease in the expression of cdc2/cyclin B1 complex, a critical kinase involved in G2/M cell cycle transition.

Anti-lymphoma effect of naproxen and indomethacin in a patient with relapsed diffuse large B-cell lymphoma.

A 77‐year‐old man with relapsed non‐Hodgkins lymphoma, diffuse large B‐cell type, was treated with naproxen, a nonsteroidal anti‐inflammatory drug (NSAID), for paraneoplastic fever.

Long-Term Remission of Primary Bone Marrow Diffuse Large B-Cell Lymphoma Treated with High-Dose Chemotherapy Rescued by In Vivo Rituximab-Purged Autologous Stem Cells

Primary bone marrow diffuse large B-cell lymphoma (DLBCL) is a rare type of extranodal lymphoma with poor prognosis. Here, we report a case of primary bone marrow DLBCL successfully treated with high-dose chemotherapy and rescued by in vivo rituximab-purged autologous stem cells. A 39-year-old woman visited our hospital because of anemia. Bone marrow examination revealed a large B-cell lymphoma invasion. An 18F-fluorodeoxyglucose positron emission tomography scan revealed disseminated bone marrow uptake without evidence of dissemination at other sites. These findings led to a diagnosis of primary bone marrow DLBCL. Our patient underwent R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone) chemotherapy and achieved complete remission. Subsequently, she received high-dose chemotherapy with an in vivo rituximab-purged autologous stem cell transplant. Seven years have passed since the transplantation, and she remains in remission. This suggests that transplantation of an in vivo rituximab-purged autograft is a promising strategy for primary bone marrow DLBCL.