Mitsuoki Eguchi

Establishment of a human leukaemic cell line (CMK) with megakaryocytic characteristics from a Down's syndrome patient with acute megakaryoblastic leukaemia

Summary. A new megakaryoblastic cell line (CMK), which also exhibits erythroid and myeloid markers, was established from a Downs syndrome patient suffering from acute megakaryoblastic leukaemia. The CMK cells were found to be positive in reactions with anti‐platelet antibodies (anti‐glycoproteins IIb/IIIa and Ib, and Plt‐1). Platelet peroxidase (PPO) reactivity was found to be associated with the nuclear envelope and the endoplasmic reticulum but not with the Golgi apparatus. Some cells possessed cytoplasmic granules with the characteristics of α‐granules and demarcation membranes. Karyotyping revealed near‐tetraploidy (modal chromosome number of 95; ranging 87–98) and a translocation der(17)t(11;17), also found in the original leukaemic cells, confirming that the cells were derived from the patients malignant blasts. The CMK cells were also found to be positive in reaction with anti‐glycophorin A antibody, as well as with anti‐myeloid antibodies (MY4, MY7 and MY9). Treatment of CMK cells with phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) greatly enhanced the reactivity with anti‐platelet antibodies, increased the number of cells in which cytoplasm was dissociated into numerous segments and suppressed the reactivity with anti‐glycophorin A. The proliferation of CMK cells was stimulated by interleukin‐3 (IL‐3) and granulocyte‐macrophage colony stimulation factor (GM‐CSF). This cell line should be a useful tool for analysing the basis of the afferent association between megakaryoblastic leukaemia and Downs syndrome, as well as for further study of megakaryocytic differentiation.

Megakaryocytopoiesis in vitro of patients with essential thrombocythaemia: effect of plasma and serum on megakaryocytic colony formation

Summary. TO clarify the mechanism of increased numbers of megakaryocytes in patients with essential thrombocythaemia (ET), we studied in vitro megakaryocytopoiesis in ET and other myeloproliferative disorders, using a megakaryocyte colony assay in methylcellulose containing plasma or serum and medium conditioned by phytohaemagglutinin (PHA) stimulated leucocytes (PHA‐LCM). Megakaryocytic colony formation was supported well by heparinized or citrated plasma and citrated serum which was harvested after clot formation of citrated plasma. Whole serum was inhibitory for megakaryocytic colony growth. The addition of platelet releasates and partially purified platelet derived growth factor (PDGF) resulted in a decrease in the number of megakaryocytic colonies. These findings suggested that platelet‐derived factor(s) in serum was inhibitory to megakaryocytic colony formation.

Ultrastructural and ultracytochemical differences between transient myeloproliferative disorder and megakaryoblastic leukaemia in Down's syndrome

Ultrastructural and ultracytochemical studies were performed on blast cells from 12 Downs syndrome neonates with transient myeloproliferative disorder (TMD) and 13 Downs syndrome patients with megakaryoblastic leukaemia (MKL), in order to clarify the cytological characteristics of these cells. Average platelet peroxidase‐positivity in blast cells of TMD patients was similar to that found in cases of MKL. Blast cells from subjects with TMD contained a number of different granules, namely, alpha granules, those that were myeloperoxidase (MPO)‐positive, electron‐lucent or basophil‐like, and those containing membrane components or ferritin particles. On the other hand, granules found in the blast cells of MKL patients with Downs syndrome included the electron‐lucent variety, those with membrane components and a few that were basophil‐like, but not alpha and MPO‐positive granules nor those containing ferritin particles. A demarcation membrane system was observed in blasts from the TMD group, but not in the MKL group.

WASP is involved in proliferation and differentiation of human haemopoietic progenitors in vitro

The Wiskott‐Aldrich syndrome (WAS) is an X‐linked recessive disorder characterized by thrombocytopenia, immunodeficiency and eczema. X‐linked thrombocytopenia (XLT) is a mild form of WAS with isolated thrombocytopenia. Both phenotypes are caused by mutation of the Wiskott‐Aldrich syndrome protein (WASP) gene. In this study we investigated the role of WASP in the differentiation of CD34‐positive (CD34+) cells isolated from the bone marrow of patients with WAS (n = 5) or with XLT (n = 4). Megakaryocyte colony formation was significantly decreased in patients with WAS when compared with normal controls. The formation of granulocyte‐macrophage colonies and erythroid bursts were also decreased in WAS patinets. In contrast, in XLT patients, formation of all these colonies was normal. However, in vitro proplatelet formation of megakaryocytes induced by thrombopoietin was markedly decreased in both XLT and WAS. Electron microscopic examination revealed that megakaryocytes obtained from WAS or XLT patients grown in vitro had abnormal morphologic features, which seemed to be caused by defective actin cytoskeletal organization, including labyrinth‐like structures of the demarcation membrane system and deviated distribution of the α‐granules and demarcation membrane system. These observations indicate that WASP is involved in the proliferation and differentiation of CD34+ haemopoietic progenitor cells probably by its participation in signal transduction and in the regulation of the cytoskeleton.

Fusion of an AF4-related gene, LAF4, to MLL in childhood acute lymphoblastic leukemia with t (2 ; 11) (q11 ; q23)

We showed that the LAF4 gene on 2q11.2–12 was fused to the MLL gene on 11q23 in a pediatric patient with CD10 positive acute lymphoblastic leukemia (ALL) having t(2;11)(q11;q23). The LAF4 gene, which encodes a lymphoid nuclear protein of 1227 amino acids with transactivation potential, is thought to have a role in early lymphoid development. The LAF4 protein was homologous to AF4 and AF5q31 proteins that are fused to MLL in infant early pre-B ALL and the breakpoint of LAF4 was located within the region homologous to the transactivation domain of AF4 and AF5q31. Expression of the 8.5-kb LAF4 transcript was detected in the adult heart, brain, and placenta and in the fetal brain. LAF4 expression was found to be higher in ALL cell lines than in AML and Epstein–Barr virus-transformed B-lymphocyte cell lines. These findings suggest that LAF4, AF4 and AF5q31 might define a new family particularly involved in the pathogenesis of 11q23-associated ALL.

Platelet peroxidase‐positive blast cells in transient myeloproliferative disorder with Down's syndrome

Transient myeloproliferative disorder accompanied by Downs syndrome has been characterized as exhibiting self‐limiting haematological abnormalities. We studied six patients suffering from this disorder in order to clarify the biological nature of their blast cells. Metaphases of leucocytes stimulated with phytohaemagglutinin (PHA) showed trisomy 21 in all patients except one. The exception was constitutionally trisomy 21 mosaic (46, XY = 89/ 47,XY. + 21 = 11). However, metaphases from the peripheral blood cells (blast cells: 70%) without PHA stimulation showed exclusively trisomy 21. Simultaneous examination for morphology and chromosomal analysis on single colonies revealed that granulocyte‐macrophage (GM) colonies and an erythroid colony contained only cells with the trisomy 21 karyotype.

X-linked thrombocytopenia in a girl

Summary. We report X‐linked thrombocytopenia (XLT) in a 6‐year‐old girl with petechiae and thrombocytopenia from the age of 3 months. Her 2‐year‐old brother was also diagnosed with XLT. The Wiskott–Aldrich syndrome protein (WASP) gene was detected as a replacement of +5th G to Aon intron 6 using sequence analysis, and the WASP expression levels in this patient were one‐third those of a healthy control. The X‐inactivation analysis of the patients lymphocytes showed a random pattern of X‐chromosome inactivation. To our knowledge, this is the first confirmed report of XLT in a female.

Human herpesvirus 6 infection associated with hemophagocytic syndrome.

Dr. Kenichi Sugita, The Second Department of Pediatrics, Dokkyo University School of Medicine, 880 Kitakobayashi, Mibu Tochigi, 321-02 (Japan) Table 1. Serological studies Virus-associated hemophagocytic syndrome (VAHS), first described by Risdall et al. [1], is a potentially self-limited proliferation of cytologically benign histiocytes exhibiting marked hemophagocytosis, in association with a systemic viral infection. Thus far, Epstein-Barr virus, cytomegalovirus, herpes simplex virus and adenovirus have been known as the most common causative agents [2-4]. We describe a girl who had VAHS associated with human herpesvirus 6 (HHV-6) infection. A 15-year-old girl, previously well, complained about common-cold-like symptoms, a sore throat, malaise and had otitis media in April 1993. Several days later, she was referred to our hospital because of fever and for evaluation of leukopenia. Physical examination revealed pharyngitis and slightly enlarged and tender cervical lymph nodes. An enlarged liver extending 1 cm below the right costal margin was palpated but no splenomegaly was found. The blood count revealed: Hb 13.2 g/dl, platelet count 115 × 109/1 and WBC 0.5 × 109/1 (band 14%, segmented neutrophils 35%, monocytes 6%, lymphocytes 45%). The CD4+/CD8+ ratio was 0.68. Bone marrow aspiration from the iliac crest revealed normal cellularity (total nuclear cells, 169 × 109/1) with a normal megakaryocyte count (0.09 × 109/1) and profuse macrophage infiltration (4.5 %) with evidence of hemophagocytosis. The coagulation screen was within normal limits. The serum lactate dehydrogenase (LDH) level was 1,097 WU (normal range: 185-330 WU), ferritin 0.631 g/l (normal range: 0.003-0.078 g/l) and total cholesterol and triglycerides were within the normal range. On serological examination, the titers of anti-HHV-6 IgM were 40 and 10 in the first and the second measurement, respectively and those of IgG were 10 and 10. The HHV-6 antibody was evaluated using the indirect immunofluorescence method as described previously [5]. Serum specimens that showed fluorescence at a 10-fold dilution were considered positive for HHV-6 antibody. During the clinical course, the titer of IgM decreased to less than 10 while that of IgG increased to 80 (table 1). These findings suggested that she had a recent HHV-6 infection. However, HHV-6 DNA could not be detected in the cultures of mononuclear cells (obtained on May 20, 1993) or in the serum (sample from June 12, 1993). No evidence of

Establishment of a novel human myeloid leukaemia cell line (FKH‐1) with t(6;9)(p23;q34) and the expression of dek‐can chimaeric transcript

Translocation t(6;9)(p23;q34), resulting in a dek‐can gene fusion, is a recurrent chromosomal abnormality mainly associated with specific subtypes of acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS). Patients with this type of chromosomal change are usually young and their prognosis is poor. The role of fusion protein generated from dek‐can chimaeric transcript on the leukaemogenesis of t(6;9) AML or MDS is as yet unknown. We have established the first permanent cell line (FKH‐1) with t(6;9), derived from the peripheral blood of a patient with t(6;9) AML transformed from Philadelphia chromosome (Ph1)‐negative chronic myelocytic leukaemia (CML). The FKH‐1 expressed myelomonocytic markers and dek‐can chimaeric transcript. In the presence of 10 ng/ml recombinant human granulocyte colony‐stimulating factor (G‐CSF), the cells doubled every 54 h and showed multilineage myeloid differentiation, resulting in heterogenous morphologies such as macrophages, basophils, eosinophils and neutrophils. Thus, this cell line may be derived from a pluripotent myeloid stem cell and should be a useful tool for biomolecular studies on the pathogenesis of t(6;9) myeloid malignancies which have rarely been investigated because of the lack of continuously proliferating cells.

Ultrastructural and ultracytochemical differences between megakaryoblastic leukemia in children and adults analysis of 49 patients

Background. Acute megakaryoblastic leukemia (AMKL) has two peaks in distribution of incidence (in adults and children 1 to 2 years of age) and is frequently seen in children with Down syndrome. The current study was undertaken to disclose whether there were any differences between these groups.