Keiki Kawakami

Granulocyte colony-stimulating factor and its receptor in acute promyelocytic leukemia

Expression of granulocyte colony‐stimulating factor (G‐CSF) receptor (G‐CSFR) and in vitro proliferative response to G‐CSF were investigated by quantitative immunofluorescence and [3H] thymidine uptake, respectively, in a series of acute myeloid leukemias (AML). The results indicated that G‐CSFR was detected at high levels in acute promyelocytic leukemia (APL) cells, in comparison with other types of AML. Moreover, APL cells were also seen to predominantly proliferate in response to G‐CSF. Based on these observations, we administered recombinant human G‐CSF to a patient with APL in the third relapse that was resistant to both cytotoxic agents and all trans retinoic acid, in an attempt to sensitize the leukemic cells to cell‐cycle‐dependent agents. Complete remission was achieved. The finding that APL cells are exquisitely responsive to G‐CSF supports the view that G‐CSF is useful for augmentation of their vulnerability to cell‐cycle specific agents. Am. J. Hematol. 58:31–35, 1998.

Hemostatic Abnormalities Following Bone Marrow Transplantation

Hemostatic abnormalities in 26 patients following bone marrow transplantation (BMT) were examined. In the event-free survival group, the plasma levels of antithrombin (AT) and protein C (PC) were significantly decreased 1 and 2 weeks after BMT, and the plasma levels of thrombomodulin (TM) and tissue plasminogen activator-plasminogen activator inhibitor-1 complex (tPA-PAI-I complex) were significantly increased from 4 weeks to 13 weeks after BMT. Excepting AT, there was no significant difference in hemostatic parameters before BMT among the event-free survival, 6-month survival, and death within 6 months groups. On day 0 following BMT, only plasma AT levels were significantly lower in the 6-month survival group than in the death within 6 months group. From 1 to 3 weeks after BMT, plasma levels of AT or PC were significantly lower in the death within 6 months group than in the 6-month survival group. From 1 to 5 weeks after BMT, the plasma levels of TM and tissue type plasminogen activator-plasminogen activator inhibitor-I complex (tPA-PAI-I complex) were significantly higher in the 6-month survival group than in the death within 6 months group. From 1 to 13 weeks after BMT, the plasma levels of D-dimer or soluble fibrin monomer (SFM) were significantly higher in the death within 6 months group than in the 6-month survival group. There was no remarkable difference in plasma levels of thrombin-antithrombin comlex or plasmin-plasmin inhibitor complex following BMT between these groups of patients. These findings suggest that the decrease in the plasma AT or PC level reflects early occurrence of complications of prognostic significance and that the increase in vascular endothelial cell markers such as plasma levels of TM or tPA-PAI-I complex reflects occurrence of complications during the middle course of BMT. Plasma levels of D-dimer and SFM may be useful markers for predicting complications associated with poor prognosis after BMT.

A case of acute myeloblastic leukemia with a novel variant of t(8;21)(q22;q22)

We encountered a case of acute myeloblastic leukemia (AML), with extramedullary leukemia (EML) and a masked type of the variant translocation t(8;21)(q22;q22). Morphologically, the AML M2 subtype according to the French–American–British (FAB) classification was present. Phenotypically, leukemic cells were negative for CD19 and positive for CD56. Clinically, the case showed chemo-refractoriness and a poor outcome. The initial karyotypic interpretation was t(8;9)(q22;q34) on G-banding. Multiplex-fluorescence in situ hybridization (multiplex-FISH) analysis revealed a three-way translocation involving chromosomes 8, 9, and 21, and identified a masked type of variant t(8;21)q22;q22) translocation. The karyotype was finally determined as 45,X,-Y,der(8)t(8;21)(q22;q22), der(9)(8;9)(q22;q34), and der(21)t(9;21)(q34;q22). Results of FISH using the AML1/ETO probe and detection of the AML1/ETO fusion transcripts by reverse transcriptase-polymerase chain reaction (RT-PCR) support the karyotype as well as the sequence of the PCR product. Additionally, C-KIT mutation was detected.

Case of B-Cell Lymphoma with Rearrangement of the BCL1, BCL2, BCL6 , and c-MYC Genes

We managed a peculiar case of lymphoma showing immunohistochemical overexpression of cyclin D1. At initial examination the patient had meningeal lymphomatosis and general lymphadenopathy. Histologic examination of biopsy specimens of inguinal lymph nodes showed tumor cells and vague nodular growth resembling lymphoblasts. The results of flow cytometric analysis were positive for CD10, CD20, CD103, and immunoglobulin G (IgG) and Igκ and were negative for CD5, CD23, and terminal deoxynucleotidyl transferase activity. Results of immunohistochemical analysis of paraffin-embedded specimens were positive for cyclin D1 and Bc12 in the tumor cells. Sixty percent of tumor cells had positive results for MIB1/Ki67. Cytogenetic and molecular studies revealed tumor cells simultaneously had t(14;18)(q32;q21), t(11;22)(q13;q11), t(8;14)(q24;q32), and t(3;14)(q27;q32) with the rearrangement of BCL1, BCL2, BCL6, and c-MYC genes. Lymphadenopathy showed a quick and complete response to doxorubicin-containing systemic chemotherapy with rituximab, but the central nervous system disease progressed and killed the patient.

Molecular analysis of PDGFRα/β genes in core binding factor leukemia with eosinophilia

Abstract:  Eosinophilia sometimes occurs in acute myeloid leukemia (AML), especially in core binding factor (CBF) leukemia. However, the pathogenesis of the differentiation from leukemic progenitors to eosinophils is not well understood in this type of leukemia. Recent reports showed that a novel fusion tyrosine kinase, Fip1‐like1 (FIP1L1) platelet‐derived growth factor receptor alpha (PDGFRα), is found in idiopathic hypereosinophilic syndrome. The involvement of another chimeric gene, PDGFRβ, was also reported in myeloproliferative disorder with eosinophilia. These chimeric genes cause constitutive activation of PDGFR tyrosine kinases. On the other hand, a two‐hit model for the pathogenesis of AML, which seems to be caused by inactivating mutations in transcription factors and genetic lesions in tyrosine kinase resulting in constitutive activation, has been proposed. On the basis of these findings, we screened for the expression of the FIP1L1‐PDGFRα fusion gene and for mutations in the juxtamembrane and tyrosine kinase domains of PDGFRα/β genes in 22 cases of CBF leukemia with eosinophilia. Among these cases, no FIP1L1‐PDGFRα fusion gene was found. Although cDNA sequencing also detected three types of single‐nucleotide alterations at kinase domains in PDGFRα/β genes, all of them were silent changes and polymorphisms. Therefore, PDGFRα/β genes do not appear to play a significant pathogenetic role in eosinophilia or leukemogenesis of CBF leukemia.

Molecular cloning of IGλ rearrangements using long‐distance inverse PCR (LDI‐PCR)

Malignant cells of mature B‐cell origin show tumor‐specific clonal immunoglobulin gene (IG) rearrangements, including V(D)J recombinations, nucleotide mutations, or translocations. Rapid molecular cloning of the breakpoint sequence by long‐distance inverse PCR (LDI‐PCR) has so far been applied to rearrangements targeted to IGH joining, IGH switch, and IGκ regions. We tended to apply LDI‐PCR method for cloning of IGλ rearrangements.

Development of Diffuse Large Cell Lymphoma from Follicular Lymphoma with Multiple Immunoglobulin Heavy Chain Gene Rearrangement Occurring in a Patient with Wiskott-Aldrich Syndrome

Patients with Wiskott-Aldrich syndrome (WAS), a congenital immunodeficiency, show a markedly increased risk of developing non-Hodgkin’s lymphoma compared with the general population [1]. Malignant lymphoma in WAS commonly presents as extranodal disease, and its histology almost always shows diffuse lymphoma, especially of large cell or immunoblastic cell type, as in other congenital or acquired immunodeficiencies [2]. We report a case of B-cell lymphoma occurring in a patient with WAS. At presentation the disease was follicular lymphoma with different histological, immunohistochemical, and molecular biological findings node by node, suggesting the presence of more than a single neoplastic clone. Eventually, the patient was killed by the systemic invasion of monoclonal diffuse large cell lymphoma, which appeared to have existed since the onset of the disease. A 21-year-old Japanese man was admitted because of general lymphadenopathy. Because the patient had been suffering since early infancy from recurrent respiratory infections, atopic eczema, and a tendency to bleed due to thrombocytopenia, his condition was diagnosed as WAS. Physical examination and computed tomographic scans revealed generalized lymphadenopathy. The patient showed a marked Development of Diffuse Large Cell Lymphoma from Follicular Lymphoma with Multiple Immunoglobulin Heavy Chain Gene Rearrangement Occurring in a Patient with Wiskott-Aldrich Syndrome Keiki Kawakami,a Motoko Yamaguchi,b Yasuyuki Watanabe,a Tetsuya Muratac

A newly established human acute lymphoblastic leukemia cell line with characteristics of the earliest B-cell maturation

SummaryA new human acute lymphoblastic leukemia (ALL) cell line, designated HBL-3, was established from the bone marrow of a patient with non-T-ALL. The HBL-3 cell line expressed B4 (CD 19), BA-1 (CD 24) and HLA-DR antigens, but not surface immunoglobulin (SIg) or cytoplasmic immunoglobulin (CIg). The cell line lacked the common acute lymphoblastic leukemia antigen (CALLA) and antigenic markers characteristic of T-cell and myeloid cell lineages. The HBL-3 cells had structural rearrangements of both the homologous chromosome 9s, including a translocation with chromosome 1 which has been reported in a patient with common ALL. The cell line had rearranged immunoglobulin heavy chain genes but retained germ-line κ light chain genes and germ-line T-cell receptorβ- and γ-chain genes. The HBL-3 cell line was strongly positive for terminal deoxynucleotidyl transferase (TdT). These findings indicate that the HBL-3 cell line is derived from the earliest B-cell committed to B-cell lineage.

A case of acute myeloid leukemia with t(7;11)(p15;p15) mimicking myeloid crisis of chronic myelogenous leukemia

The chromosome aberration t(7;11)(p15;p15) is uncommon but recurrent in leukemia. We experienced a case of acute leukemia with t(7;11)(p15;p15), the hematological appearance of which mimicked myeloid crisis in chronic myeloid leukemia (CML). This case showed splenomegaly, a decreased neutrophil alkaline phosphatase (NAP) score, increased vitamin B12 value, and cells at all stages of neutrophilic maturation in both bone marrow and peripheral blood. We initially had difficulty differentiating acute myeloid leukemia (AML) M2 with marked myeloid differentiation from myeloid crisis of Philadelphia chromosome (Ph)-negative CML. Immature myeloid cells in the peripheral blood disappeared and cytogenetic analysis indicated that marrow cells changed to the normal karyotype after remission induction therapy. Therefore, this case was thought not to be myeloid crisis but AML M2 subtype. TheNUP98/HOXA9 fusion transcript was detected by reverse transcription-polymerase chain reaction (RT-PCR) at exon A but not exon B ofNUP98.

Genetic heterogeneity in blast crisis of chronic myelocytic leukemia.

Fourteen patients with lymphoid and mixed blast crisis (BC) of chronic myelocytic leukemia were studied by immunophenotyping and genotyping. Rearrangements of immunoglobulin heavy chain (IgH), T-cell receptor (TcR) gamma and TcR beta genes were detected in all 14, in nine and in four patients, respectively. Interestingly, more than two rearranged bands of IgH gene in three lymphoid BC and two rearranged bands with germ line band in 1 biphenotypic BC indicated the genetic heterogeneity of the blasts. Some blastic transformations are thought to occur at a more immature stage of hematopoietic differentiation than that indicated by the phenotype and genotype of BC cells.