Jun Minowada

Hodgkin's disease derived cell lines HDLM-2 and L-428: Comparison of morphology, immunological and isoenzyme profiles

The cell lines HDLM-2 and L-428 were established from the pleural effusions of two patients with Hodgkins disease. We studied and compared phenotypic characteristics of HDLM-2 and L-428 cells before and during induction of differentiation with 12-O-tetradecanoylphorbol 13-acetate (TPA) using a number of parameters. TPA-treated HDLM-2 and L-428 cultures did not show adhesion to plastic surface or aggregation of cells; the cells did not develop pseudopodia and were not phagocytic. Only a slight increase in the percentage of NBT-positive cells was observed for L-428 cells. TPA led to a cessation of cell proliferation and to a dose-dependent decrease in the number of viable cells in both cell lines. In HDLM-2 and L-428, treatment with TPA induced distinct morphological changes indicative of a partial differentiation along the myeloid cell lineage. In addition, the production and expellation of benzidine-positive, unnucleated particles were observed in HDLM-2 and L-428 cells. The induced isoenzyme profiles of carboxylic esterase and acid phosphatase resembled those found in myelomonocytic leukemia cell lines. Both cell lines were negative for immunological markers of the T- and B-cell lineages, but reacted with several markers associated with the myelomonocytic cell lineages. HDLM-2 cells produced a factor which could induce differentiation in 12 leukemia cell lines. The overall results suggest that Hodgkin and Sternberg-Reed cells constitute a unique cell type and might be derived from cells of the myelomonocytic cell lineage.

Isoenzyme studies in human leukemia-lymphoma cell lines — I. Carboxylic esterase

The isoenzyme patterns of carboxylic esterase (E.C. 3.1.1.1) were studied in 74 proven human leukemia-lymphoma and 12 normal B-lymphoblastoid cell lines. These cell lines have been extensively phenotyped using poly- and monoclonal antibodies. Esterase isoenzymes were separated by isoelectric focusing and visualized by histo-cytochemical techniques. No leukemia-specific or (except for monocytes) blood cell type-specific isoenzyme or isoenzyme pattern could be detected. The monocytic element in some cell lines was characterized by a strong isoenzyme band which could be selectively and completely inhibited by sodium fluoride. The enzyme phenotypes were stably expressed in all subcultures of a given cell line and did not appear to have any cell cycle dependency. The leukemia-lymphoma cell lines have been subclassified into four major groups according to immunological parameters: T-cell, B-cell, myelomonocytic and non-T, non-B-cell. On the basis of immunological data the T-cell lines were assigned to five stages of differentiation. The number and staining intensity of the isoenzymes increased with differentiation of the T-cells paralleling the expression of immunological markers. The B-cell leukemia-lymphoma cell lines were divided into pre B-, B-, Burkitt lymphoma, multiple myeloma and hairy cell leukemia cell lines. Substantial variability among the isoenzyme patterns was detected ranging from immature profiles of pre B-cell lines to complete isoenzyme repertoires of multiple myeloma cell lines. No significant difference was seen between the isoenzymes of mature B-cell lines and normal B-lymphoblastoid cell lines. The most prominent feature seen in myelomonocytic cell lines was the monocytic band indicating a monocytic origin and separating the monocytoid from the pure myeloid cell lines. Considerable heterogeneity in the isoenzyme patterns was observed in the non-T, non-B cell groups which comprised erythroleukemia cell lines and cell lines arrested at a very early stage of lymphoid differentiation. These latter cell lines together with some T- and B-cell lines shared the common characteristics of positivity for cALLA, TdT and Ia antigens and an immature, incomplete isoenzyme profile. The results support the notions of maturation arrest and normal gene expression in leukemic cell populations. Furthermore, the importance of biochemical studies as part of the multiple marker analysis could be demonstrated.

The use of monoclonal antibodies for the identification and classification of acute myeloid leukemias

We reviewed a library of monoclonal antibodies (MoAbs) detecting antigens on myelomonocytic cells and analysed their reactivity patterns as reported in the literature. On the basis of the frequency of positivity with the myelocytic variants (FAB M1-3) or monocytic variants (FAB M4/5) of acute myeloid leukemias, the MoAbs were assigned to one of four groups. MoAbs of Group I identified most cases of both the myelocytic and the monocytic cell lineages (pan-myelomonocytic reactivity) and can be used to identify acute myeloid leukemias regardless of the subtype. Group II comprised MoAbs which reacted with the majority of FAB M1-3 cases, but showed a preference in reactivity with AMMoL/AMoL cases (reactivity: myelocytic partly, monocytic predominantly). MoAbs of Group III stained most cases with monocytic phenotypes, but labelled only a small percentage of non-monocytic cases. These MoAbs are valuable tools for the detection of cases with monocytic features. Group IV MoAbs reacted with a small to intermediate percentage of myelocytic and/or monocytic cases. Besides their diagnostic application MoAbs might be used in new therapeutic approaches such as in-vivo serotherapy with MoAbs and purging of autologous bone marrow for transplantation. None of the described MoAbs appear to be leukemia-specific. Many MoAbs have been produced against non-myelomonocytic cells and were reactive with cells outside the myelomonocytic cell lineages and the hematopoietic system. Other MoAbs with apparent cell lineage-restricted reactivity regarding normal cells stained leukemic cells of other cell lineages. This phenomenon of translineage reactivity of leukemic cells with mutually exclusive markers indicating a biphenotypic marker profile might be the result of abnormal, disregulated gene expression. New classification systems of acute myeloid leukemias based on immunological marker profiles have been proposed. The analysis of reactivity of normal and malignant myelomonocytic cells with MoAbs has led to refined differentiation schemes of the normal hematopoiesis.

Isoenzyme studies in human leukemia-lymphoma cells lines--II. Acid phosphatase.

This report describes the qualitative acid phosphatase (acP) isoenzyme profiles detected in permanent human hematopoietic cell lines. The acP activity was separated into its isoenzymes by isoelectric focusing on horizontal thin-layer polyacrylamide gels. The pattern of acP isoenzyme was investigated in a total of 86 cell lines. These cell lines were classified into five groups on the basis of their phenotypes characterized in the multiple marker analysis: 74 leukemia-lymphoma cell lines (26 T-, 34 B-, 6 myelomonocytic, 8 Non-T, Non-B cell lines) and 12 so-called normal Epstein-Barr virus transformed B-lymphoblastoid cell lines. Their immunological features had been analysed in detail by use of a large panel of poly- and monoclonal antibodies which led to a further subclassification into stages of differentiation. A progressive increase in number and staining intensity of the isoenzymes which paralleled the expression of surface markers at different stages of differentiation along their developmental pathway was seen in the T- and B-leukemia-lymphoma cell lines. Some cell lines whose isoenzyme profiles did not correspond to the stage of differentiation as evidenced by surface antigen analysis might represent good examples of deranged gene expression in otherwise normally programmed malignant cells, i.e. in our study a mismatch between the isoenzymatic and immunological phenotypes. The tartrate-resistant isoenzyme was detected in 9 out of 74 leukemia-lymphoma cell lines (4 T-, 2 B-, 1 myelomonocytic, 2 Non-T, Non-B cell lines) and in 10 out of 12 normal B-lymphoblastoid cell lines; the only one studied hairy cell leukemia cell line did not express this isoenzyme. The relative specificity of the tartrate-resistant acP is discussed in detail. No leukemia-lymphoma specific isoenzyme or an additional isoenzyme which was not seen in normal hematopoietic cells could be observed. Nor did we find an isoenzyme or isoenzyme pattern characteristic for a certain cell lineage. This underlines the necessity of a combined analysis using markers from different disciplines in the multiple marker analysis in order to accurately characterize normal and malignant blood cells. Furthermore, our results support the concept of maturation arrest at particular stages of differentiation together with the theory of normal gene expression in leukemic cells equivalent to that in their normal counterparts.

Reactivity pattern of ‘myeloid monoclonal antibodies’ with emphases on MCS-2

The reactivity pattern of the murine monoclonal antibody (MoAb) MCS-2 was tested on a panel of 724 cases of leukemia-lymphoma. MCS-2 was positive in 178/185 (96%) cases of AML (FAB M1-3), 10/10 cases of AMMol/AMoL (FAB M4/5), 42/45 (93%) cases of CML, 1/1 case of CMoL, 37/38 (97%) cases of CML-myeloid blast crisis, 0/9 cases of CML-lymphoid blast crisis. No positive staining was seen in 238 cases of T-CLL, mycosis fungoides, Sèzary-syndrome, B-CLL, hairy cell leukemia, multiple myeloma and T- and B-lymphoma nor in 32 cases of B-ALL, Burkitt-lymphoma, Null-ALL and immature T-lymphoma. A positive expression was found in 8/110 cases of cALL, 1/6 cases of pre B-ALL and 1/35 cases of T-ALL. Fifteen other MoAbs (MCS-1, OKM1, My-1, Leu-M1, Leu-M3, CA-2-38, MY4, MY7, MY8, MY9, VIM-D2, VIM-D5, Mol, Mo2, 63D3) which are associated with the myelomonocytic cell lineages were tested by indirect immunofluorescence on 60 or more patients (62-149 cases). A wide variability in the frequency of positivity was seen for the panel of cases studied and for the blast cell populations per individual samples: 21-96% of the AML cases (FAB M1-3) and 31-100% of the AMMoL/AMoL cases (FAB M4/5) were positive for the various MoAbs. None of the analysed MoAbs stained only myelocytic or only monocytic leukemias, but a certain degree of preference for the monocytic variants was noted for Leu-M3, CA-2-38, MY4, VIM-D2, Mo2 and 63D3. The detection of MCS-2 on immature ALL blast cells might indicate a coexpression of lymphoid and myeloid markers on very immature cells, or an abnormal gene expression by malignant cells, or the identification of a so far undetected subclass of acute leukemias.

Burkitt cell acute lymphoblastic leukemia with partial expression of T-cell markers and subclonal chromosome abnormalities in a man with acquired immunodeficiency syndrome

A 45-year-old white male, bisexual, with a 2-year history of acquired immunodeficiency syndrome (AIDS) prodrome, developed a Burkitt cell-like acute lymphoblastic leukemia (ALL). Marker studies of marrow blasts show an unusual and possibly unique pattern, in that an unequivocal monoclonal B cell leukemia, having K-IgM with HLA-DR and B cell subset antigen (BA-1) expression, was superimposed with a mature suppressor T cell marker profile (pan-T, mature T, and suppressor/cytotoxic T antigens). The leukemic blasts were totally negative for terminal deoxynucleotidyl transferase (TdT), human T cell leukemia-lymphoma virus (HTLV) p19 antigen, and other immunoglobulin isotypes. Chromosome analysis of marrow cells disclosed that 70% of the cells had 47,XY, + 12,t(8;14)(q24;q32) chromosome complement, and 30% of the cells had a 47,XY, + 12,dup1q + (q22-31),t(8;14)(q24;q32). The consistent finding of the specific chromosome rearrangement (8/14 translocation) in all abnormal cells suggests that the cells were derived from a common precursor. With regard to the partial T cell marker expression, the significance of these markers in B cell leukemia is unclear, as is their relation to the additional chromosome abnormalities that apparently developed in the process of clonal evolution.

Isoenzyme studies in human leukemia-lymphoma cell lines. IV: Lactate dehydrogenase

Isoelectric focusing (IEF) in horizontal polyacrylamide gels has been used to separate lactate dehydrogenase (LDH) isoenzymes in 97 human permanent hematopoietic cell lines (85 leukemia-lymphoma cell lines and 12 normal B-lymphoblastoid cell lines). Maximally 8 LDH bands were seen; the electrophoretically detectable bands 4 and 5 could be separated by IEF into 2 and 3 isoenzymes, respectively. The LDH patterns have been found to vary both in number of isoenzymes and in relative intensity in different cell lines depending upon the stage at which arrest of differentiation occurred. These differences can be used to analyse and distinguish different cell lines. The method should provide a valuable supplement to the enzymatic phenotyping and complete characterization of fresh and cultured leukemias and for the monitoring of phenotypic changes occurring during induction of differentiation.

Isoenzyme studies in human leukemia-lymphoma cell lines — III. β-hexosaminidase (E.C. 3.2.1.30)☆

The hexosaminidase (beta-N-acetylglucosaminidase) isoenzyme profiles of 86 human hematopoietic cell lines grown actively in suspension culture were analysed by isoelectric focusing and by conventional disc electrophoresis on horizontal thin-layer polyacrylamide gels. A maximum of three hexosaminidase (Hex) isoenzymes (A = anodic, I = intermediate, B = basic) could be demonstrated. The immunological phenotyping of 74 leukemia-lymphoma derived cell lines had led to a categorization into four groups with a subclassification of the T- and B-cell lines into several stages of differentiation: 26 T-cell, 34 B-cell, 6 myelomonocytic and 8 Non-T, Non-B cell leukemia-lymphoma cell lines. Twelve so-called normal B-lymphoblastoid cell lines were also available. Distinct isoenzyme profiles were seen in the different stages of differentiation in the T- and B-leukemia-lymphoma cell lines. Among the 12 normal B-lymphoblastoid cell lines heterogeneity in the isoenzymatic phenotypes was detected. Hex isoenzyme expression in normal and neoplastic lymphoid cell lines represents hypothetically sequential stages of T- and B-cell differentiation. Myelomonocytic cell lines displayed strongly stained bands of all three isoenzymes. Heterogeneity was seen in the group of Non-T, Non-B cell lines. Four out of 5 pre B-cell lines and 4 out of 4 Non-T, Non-B cell lines which are comparable to cases of pre B- and common ALL revealed a high Hex I/Hex A ratio in terms of intensity of the isoenzyme bands. The analysis of Hex isoenzymes is useful for characterizing lymphoid and myeloid populations (both normal and malignant, cultured or fresh), particularly with regard to their stage of differentiation. But this enzyme should be part of a multiple enzyme study where the information obtained is complementary. In turn, enzyme marker analysis should be included in the multiple marker analysis for an optimized characterization of leukemic cells.

Catalogue of Epstein-Barr virus (EBV) receptors on human malignant and non-malignant hematopoietic cell lines☆

Epstein-Barr virus (EBV) can induce a broad spectrum of hematological diseases, especially in immune deficient patients. We assayed for receptor for EBV (EBVR) using fluoresceinated viral particles on 44 human hematopoietic cell lines derived from patients with T, B, and non-T, non-B acute lymphocytic leukemia (ALL), non-lymphoid leukemia, Burkitt lymphoma, myeloma and several unique lines we and others have recently developed. All 31 EBV nuclear-associated antigen (EBNA) negative cell lines were of neoplastic origin. Seven of 13 EBNA-positive cell lines were of normal cell origin. Four of 25 non-B (surface immunoglobulin negative) EBNA-negative neoplastic cell lines were EBVR-positive. Three of six EBNA-negative B-cell (surface immunoglobulin positive) lines were EBVR-positive. Nine of 13 EBNA-positive Burkitt and non-Burkitt cell lines strongly expressed EBVR. Four EBNA-positive Burkitt lymphoma cell lines exhibited EBVR only to a limited degree. Studies of the cell lines for EBVR, complement receptors (CR) and surface immunoglobulin (SIg) revealed that presence of SIg does not obligate the presence of EBVR. Functional EBVR accompanied SIg among EBNA-negative cell lines. SIg-negative cell lines can possess EBVR. Fourteen of 16 EBVR-positive lines were also positive for CR. The EBVR assay is a useful tool for assessing the potential role of EBV in the induction of hematopoietic disorders.

Heterogeneity of marker expression in B-cell leukemias and its diagnostic significance

Lymphoproliferative disorders of B-cell origin are identified by surface immunoglobulin markers. Clinically, these include several types of leukemias and lymphomas. We have attempted to further characterize B-cell leukemias immunologically using monoclonal antibodies Leu-1 and FMC-7. While cases of classical B-CLL have been shown to react with Leu-1 monoclonal, most other B-cell leukemias often do not react with Leu-1. Our results show that use of an additional monoclonal FMC-7 does not contribute towards diagnostic reliability.