Yasuhiro Enomoto

Myeloproliferative disorders terminating in acute megakaryoblastic leukemia with chromosome 3q26 abnormality

Two cases of myeloproliferative disorders terminating in acute megakaryoblastic leukemia are reported. One case began as primary myelofibrosis and the other as chronic myelogenous leukemia. Blast cells in the acute leukemic phase were identified as megakaryoblasts by the presence of platelet peroxidase. The clinical course is described, and the morphology, immunologic studies, and ultrastructure studies of the blast cells are reported. On cytogenetic analysis both cases had a translocation involving the No. 3 chromosome locus q26.2. The present data suggest that 3q26 may be associated with transformation of the megakaryocytic lineage.

Cytogenetic findings in adult acute leukemia and myeloproliferative disorders with an involvement of megakaryocyte lineage

Cytogenetic analyses were performed on 12 adult patients with abnormal megakaryoblastic proliferation which was detected by ultrastructural cytochemical study (platelet peroxidase) and platelet‐megakaryocytesspecific monoclonal antibodies (TP‐80, Plt1, AN51, and KOR‐77). The patients consisted of two patients with myelodysplastic syndromes (MDS), three with acute megakaryoblastic leukemia (AMKL), six with megakaryoblastic transformation in Philadelphia‐positive chronic myelogenous leukemia (CML‐meg‐BC), and one case of chronic myeloproliferative disorder (CMPD). Among them, an inversion of the long arm of chromosome 3 [inv(3)(q21q26)] was found in one AMKL patient with a normal platelet count. Chromosome change at band 3q26 was also found in one MDS patient without thrombocythemia. Furthermore, the long arm of chromosome 13, where rearrangements in myelofibrosis are clustered (13q12→q22) was seen in one MDS patient. Trisomy of chromosome 19 was found in one AMKL patient and three CML‐meg‐BC patients. These findings indicate that cytogenetic abnormalities involving 3q26, 13q, and trisomy 19 are associated with hematologic neoplasia with megakaryocytic lineage in adult patients, although these abnormalities were not related to the survival of the patients. During the period of this study, two acute myelogenous leukemia patients (AML‐M2 and AML‐M5b) with chromosome rearrangements at band 3q21 and thrombocythemia were found, indicating that chromosome abnormality at band 3q21 is related to quantitative platelet dysfunction, whereas that at 3q26 is related to hematologic malignancies with a proliferation of megakaryocytic lineage.

A novel human leukaemic cell line, CTS, has a t(6;11) chromosomal translocation and characteristics of pluripotent stem cells

A novel human leukaemic cell line, designated CTS, was established from the peripheral blood of a 13‐year‐old girl suffering from acute myeloblastic leukaemia (AML) in relapse. CTS cells expressed CD7, CD13, CD33, CD34 and HLA‐DR antigens, and showed ultrastructural myeloperoxidase activity. In addition, CTS cells showed DNA rearrangements of the immunoglobulin heavy chain gene and the light κ chain gene, and deletions of the T‐cell receptor δ1 gene. Cytogenetic analysis revealed a human female diploid karyotype with a t(6;11)(q27;q23) chromosomal translocation. Molecular studies demonstrated a DNA rearrangement of the MLL gene, the expression of a truncated 11.0 kb MLL mRNA and the detection of the MLL/AF‐6 fusion transcript in CTS cells. To our knowledge, this cell line is the first report of a human leukaemic cell line with a t(6;11) chromosomal translocation. CTS cells showed no significant proliferative response to the cytokines, IL‐2, IL‐3, IL‐6, IL‐11, GM‐CSF, G‐CSF, EPO, SCF, but were induced to differentiate to the T‐cell, B‐cell, erythroid or megakaryocytic lineage in the presence of particular cytokines. This CTS cell line may provide a useful tool in the study of the oncogenesis of mixed lineage leukaemia with 11q23 abnormalities and for the analysis of growth and differentiation of pluripotent stem cells.

Translocation t(3;4)(q26;q21) in myelodysplastic syndrome with megakaryoblastic proliferation

A 74-year-old Japanese male with a 4-year history of refractory anemia with excess of blasts is reported here. Chromosome study revealed the bone marrow cells of this patient to contain a t(3;4)(q26;q21). Ultrastructural analysis of platelet peroxidase and immunocytochemical study using monoclonal antibody for platelet antigen revealed a large number of blasts in the bone marrow to be megakaryoblasts. Thus, this case was thought to be one of a myelodysplastic syndrome with excess of blasts including megakaryoblastic proliferation showing chromosome changes at 3q26 and 4q21. The relationship of the anomaly on the long arm of a chromosome #3, especially at band 3q26, to abnormal megakaryoblastic proliferation is discussed.

EXPERIMENTAL PHOSPHOLIPIDOSIS INDUCED BY 4,4′-DIETHYL-AMINOETHOXYHEXESTROL

The effect of a generalized phospholipidosis inducing drug, diethylamino‐ethoxyhexestrol (DH, a coronary vasodilator), was studied using rats. The initial alterations are characterized by the appearance of abnormal cytoplasmic inclusion bodies. At the early stage of DH administraion, they appeared near the Golgi apparatus. Histochemical and ultrastructural evidence showed that the inclusion bodies consisted of polar lipid, mainly of phospholipids. From cytochemical and biochemical observation, the lysosome was regarded as the primary site of the drug‐induced morphological changes. The drug‐induced abnormal cytoplasmic inclusion bodies were of three basic morphological types, i.e., multilamellated, crystalloid and finger‐print‐like bodies. Additionally, many intermediate forms were found showing structural features of those basic types. These drug‐induced cytoplasmic changes, namely storage of phospholipids, were considered to be reversible both morphologically and biochemically after the cessation of DH administration.

An increase in basophils in a case of acute myelomonocytic leukaemia associated with marrow eosinophilia and inversion of chromosome 16

A patient with acute myelomonocytic leukaemia (M4 subtype) with pericentric inversion of chromosome 16, inv(16)(p13q22), and a marked basophilia is described. Results from transmission electron microscopy suggested that the basophils were immature. Clinically, although leukaemic cells had a tendency to infiltrate a number of tissues and organs, such as skin, gingiva, liver and lung, the patient was successfully treated with chemotherapy. The increase in basophils accompanied by marrow eosinophilia may be the result of abnormal differentiation of leukaemic cells that have the capacity to differentiate into both the eosinophilic and basophilic pathways.

Monoclonal Antibody Against Bone Marrow Stromal Cells Its Production and Characterization

Bone marrow stromal cells play an essential role in the proliferation and differentiation of hematopoietic stem cells (1,2). As a means of analyzing of the bone marrow microenvironment immunohistochemically, we attempted to produce a rat monoclonal antibody against the murine preadipocyte line HI derived from long‐term bone marrow culture (LTBMC) of C57BL/6 mice (3,4). A newly established monoclonal antibody, designated R4‐A9, was obtained from a hybridoma prepared by fusion of Y.B2/ 3.0Ag20(YO) rat myeloma cells with spleen cells of LEW rats immunized with HI cells. The immunofluorescence of live HI cells showed that the antigen reacting with this antibody was strongly expressed on the cell surface. The specificity of R4‐A9 was assessed immunohistochemically on frozen sections of various tissues from normal adult mice. R4‐A9 demonstrated specificity for hematopoietic stroma in bone marrow and spleen. No staining was observed in thymus, lymph nodes or other tissues examined, with the exception of Leydig cells in the testis and the endothelium of small arteries in several organs. Detailed immunohistochemical observations at both the light microscopy and electron microscopy level showed that R4‐A9 selectively reacted with the sinusoidal endothelium, peri‐sinusoidal adventitial cells (5) (adventitial reticular cells (6)) and intersinusoidal reticular cells (5) and the reticular cells of the splenic red pulp. These findings indicate that reticular cells and the endothelium of the bone marrow possess the common cell surface molecules recognized by R4 A9. SDS‐PAGE analysis showed that R4‐A9‐immuno‐precipitated proteins had a molecular mass of 100 kDa under reducing conditions. These data show that R4‐A9 is highly specific for reticular cells and the endothelium of murine bone marrow and spleen, providing a new approach for detailed structural analysis of the constituents of bone marrow stroma. Acta Pathol Jpn 41: 499–506, 1991.