Hans Guenter Drexler

Occurrence of particular isoenzymes in fresh and cultured leukemia-lymphoma cells. I: Tartrate-resistant acid phosphatase isoenzyme

The cells from 87 leukemia–lymphoma cell lines, 14 B‐lymphoblastoid cell lines, 459 cases of leukemia–lymphoma, normal specimens, 22 leukemia–lymphoma cell lines treated with 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) and 14 cases of chronic lymphocytic leukemia (CLL) and chronic myelocytic leukemia (CML) treated with TPA were analyzed for the expression of tartrate‐resistant acid phosphatase (TracP) isoenzyme separated by isoelectric focusing. The TracP isoenzyme was seen in the following leukemia–lymphoma cell lines: 4 of 30 T‐cell, 2 of 35 B‐cell, 1 of 6 non‐T/non‐B‐cell, 1 of 8 myelomonocytic, 3 of 4 erythroleukemia, and 3 of 4 Hodgkins disease‐derived cell lines. The expression of the TracP band could be induced by treatment with TPA in 3 myelomonocytic leukemia cell lines. Among the different types of leukemia–lymphoma cells freshly obtained from patients, the TracP isoenzyme was detected at a high incidence in cases of B‐CLL, hairy cell leukemia (HCL), and B‐lymphoma. Of the myeloid leukemias, 10% to 20% displayed the TracP isoenzyme. TracP positivity was detected in the peripheral blood, tonsil, bone marrow, spleen, and liver obtained from healthy donors, but not in the thymus. The expression of the TracP band could be newly induced by TPA in cases of CLL and in cases of CML. It is concluded that TracP activity is not specific for HCL, but is found at high incidences in cases of HCL, B‐CLL and B‐lymphoma. The TracP isoenzyme is not expressed by very immature lymphoid leukemia cells, but by cells arrested at later stages of differentiation of the T‐ or B‐cell lineage, and by some myeloid cells.

Occurrence of particular isoenzymes in fresh and cultured leukemia‐lymphoma cells. III. Esterase isoenzyme in monocytes

The expression of a particular α‐naphthyl acetate esterase isoenzyme which is specific for monocytes was examined in a panel of cultured leukemia‐lymphoma cell lines (n = 88), freshly obtained leukemia‐lymphoma cells (n = 527), and in fresh (n = 10) and cultured (n = 22) leukemia cells treated with the phorbol ester 12‐O‐tetradecanoylphorbol 13‐acetate (TPA). The sodium fluoride‐sensitive isoenzyme was separated by isoelectric focusing on horizontal thin‐layer polyacrylamide gels. The esterase isoenzyme was not detected in untreated or TPA‐treated lymphoid, erythroid, or Hodgkins disease‐derived cell lines, but was seen in leukemia cell lines of monocytic origin. TPA induced the new expression of this marker isoenzyme in two leukemia cell lines of promyelocytic and erythroid origin that are known to differentiate along the monocytic‐macrophage cell lineage; TPA stimulation increased the staining intensity of the band in monocytoid cell lines. This esterase isoenzyme was found in 92% of the cases classified morphologically as acute myelomonocytic or monocytic leukemia, but only in 3% of the non‐monocytic acute myeloid leukemias. All lymphoid or erythroid leukemias or lymphomas were negative. Treatment with TPA of AML and CML cells, which commonly differentiate to monocyte/macrophage‐like cells, showed de novo the monocyte‐specific isoenzyme. It is concluded that this isoenzyme is a characteristic marker for monocytic leukemia cells and will be a useful tool for the discriminatory identification of the monocytic element in normal and leukemic cells. Cancer 59:77–82, 1987.

Expression of a monocyte-specific esterase isoenzyme in cases of acute myeloid leukemias.

The carboxylic esterase (E.C. 3.1.1.1) isoenzymes from cases of acute myeloid leukemias were separated by analytical isoelectric focusing on horizontal thin-layer gels. One isoenzyme consisting of one or two components (bands) could be completely and selectively inhibited by addition of 40 mM sodium fluoride (NaF) to the staining bath. The 105 cases were classified into the groups M1-M6 according to the FAB proposals. The NaF-sensitive isoenzyme was not detected in cases of FAB groups M1/2 (acute myeloblastic leukemia without or with maturation), group M3 (acute promyelocytic leukemia) or group M6 (erythroleukemia). Thirty-one out of 33 cases in the FAB group M4 (acute myelomonocytic leukemia) and 9/9 cases in FAB group M5 (acute monocytic leukemia) expressed the NaF-sensitive isoenzyme. The NaF-sensitive isoenzyme was found at different staining intensities; all M5 cases showed the isoenzyme at strong or very strong intensity, whereas most of the M4 cases displayed the isoenzyme at weak, medium or strong staining intensity. The data presented are further evidence that the presence of the NaF-sensitive esterase isoenzyme indicates monocytic involvement or differentiation in cases of myeloid leukemias. The easy and fast to perform method of isoelectric focusing can be used to distinguish the monocytic variants among the acute myeloid leukemias and can supplement the morphological analysis of these cases.

Occurrence of particular isoenzymes in fresh and cultured leukemia-lymphoma cells. II. Hexosaminidase I isoenzyme.

The isoenzyme profiles of hexosamihidase (N‐acetyI‐β‐D‐glucosaminidase) were analyzed by isoelectric focusing on horizontal polyacrylamide thin‐layer gel with special emphasis on the intermediate isoenzyme (Hex I). The expression of Hex I was examined in 87 leukernia‐lymphoma cell lines, in 14 B‐lymphoblastoid cell lines, in 441 cases of leukemia‐lymphoma (specimens containing 80% or more tumor cells), in 22 leukemia cell lines and in 14 cases of leukemia that had been treated with phorbolesters (TPA) for induction of differentiation, and in the mononuclear cell preparations separated from peripheral blood, lymph node, thymus, bone marrow, tonsil, liver, and spleen specimens from normal donors. Hex I was detected in the leukemia cell lines arrested at early, immature or at late, mature stages of B‐ and T‐cell differentiation, but not in cell lines blocked at intermediate stages of maturation. Most myelomonocytic leukemia cell lines and the erythroleukemia cell lines showed Hex I, whereas the B‐lymphoblastoid cell lines were negative for this marker. During induction of differentiation, the expression of Hex I was lost in 13 of 15 leukemia cell lines that were originally Hex I‐positive. Among the panel of the “fresh” leukemia‐lymphoma cells, Hex I was found predominantly in cases of acute lymphoblastic leukemia and acute myeloblastic/monoblastic leukemia, but rarely or not at all in the mature T‐, B‐ or myeloid malignancies. However, two out of two cases of multiple myeloma were Hex I‐positive, and the Hex I expression could be induced by TPA in three of six B‐cell chronic lymphocytic leukemia cases. Chronic myelocytic leukemia cells remained Hex I‐negative during induction of differentiation. Hex I‐positivity was not detected in the cell preparations from normal tissues, and peripheral blood indicating that the normal cellular counterpart of the Hex I‐positive tumor cells are present at only low percentages within the respective cell populations. It is suggested that Hex I is a marker of early lymphoid and myeloid hematopoiesis that is no longer expressed in intermediate stages of lymphoid differentiation and in later or terminal stages of myeloid differentiation, but that is again detectable in terminally differentiated B‐cells. Further studies will focus on identification and isolation of normal Hex I‐positive cells. Cancer 58:245–251, 1986.

Phenotyping of malignant hematopoietic cells

Summary1255 cases of leukemia-lymphoma were tested between 1972 and 1984 by multiple marker analysis. Routine leukemia phenotyping was performed using standard morphological and cytochemical techniques in combination with clinical and histo-pathological information; the main emphasis was put on immunological surface marker analysis using erythrocyte rosette assays, TdT and a large panel of poly- and monoclonal antibody tests. The 1255 cases were divided into these major types and subtypes: 349 cases of ALL and related immature T- and Burkitt-lymphomas (cALL, pre B-ALL, B-ALL and Burkitt-lymphomas, T-ALL and immature, mostly leukemic T-lymphomas, Null-ALL), 454 cases of mature T- and B-cell malignancies (T-CLL, mycosis fungoides, Sezary-syndrome, T-lymphomas, B-CLL, hairy cell leukemia, multiple myeloma, B-lymphomas), 263 cases of acute myeloid leukemias (AML, AMMoL/AMoL), 182 cases of chronic myeloid leukemias (CML in chronic phase, CMoL, CML in blast crisis), 6 cases of erythroleukemia and 1 case of megakaryoblastic leukemia. A simplified classification scheme which has been used in our laboratories is presented. Phenotyping is of diagnostic, prognostic and therapeutic relevance, most evidently for patients with ALL. Routine leukemia phenotyping should be performed with highly standardized techniques and reagents and by combining information from several fields in the multiple marker analysis. New areas of leukemia research might become very useful for the routine procedure of phenotyping.

Study on Human T Leukemia-Lymphoma Cell Lines by the Second International Workshop Monoclonal Antibodies of the T Cell Protocol

The First International Workshop and Conference on Human Leukocyte Differentiation Antigens was held in November, 1982 in Paris, France; this conference provided means and emphasized the need for a worldwide collaboration in the study of human leukocyte antigens (1). Among numerous impacts of the Workshop, uses of murine monoclonal hybridoma antibodies (mAbs) and of leukemia-lymphoma cell lines as consistent cells were amply recognized (1,2). Limitations and difficulties associated with the use of both mAbs and leukemia-lymphoma cell lines have already been documented (3). Large numbers of growth factor-independent human leukemia-lymphoma cell lines of diverse cell lineages have continued to play significant roles in the research of leukocyte differentiation antigens (4). During the First Workshop 165 coded mAbs were tested on 30 leukemia-lymphoma cell lines (10 T, 10 B, and 10 myelomonocytic or non-T/non-B cell lines) (5).

Reactivity patterns of monoclonal antibodies positive on myelomonocytic leukemia cells as defined by esterase isoenzyme analysis

SummaryThe reactivity with monoclonal antibodies (MoAbs) specific for myelomonocytic cells and the expression of a particular esterase isoenzyme were analyzed in 159 cases of acute myeloid leukemias. The incidence of positivity of 16 MoAbs (MCS-2, MCS-1, OKM1, My-1, Leu-M1, Leu-M3, CA-2-38, MY4, MY7, MY8, MY9, VIM-D2, VIM-D5, Mo1, Mo2, 63D3) was studied using the indirect immunofluorescence technique. A carboxylic esterase isoenzyme which can be inhibited completely and selectively by sodium fluoride (NaF) was demonstrated by isoelectric focusing on horizontal polyacrylamide gels. This NaF-sensitive isoenzyme indicated the monocytic origin of the blast cells as it is specific for this cell lineage. Prior to the immunological-isoenzymatic analysis all cases were categorized into two subtypes according to morphological criteria of the FAB classification system: 147 cases of AML (FAB M1-3) and 12 cases of AMMoL/AMoL (FAB M4/5). However, 15 out of 147 cases of AML expressed the NaF-sensitive isoenzyme and were therefore assigned to the group AMMoL/AMoL. Likewise, 1 case, diagnosed morphologically as AMMoL, was negative for this marker isoenzyme and was assigned to the other leukemia subtype. The incidence of reactivity varied widely for the MoAbs tested regarding the overall results on all cases and the positivity on cases of either AML or AMMoL/AMoL. The MoAbs were grouped into four classes depending on the pattern of reactivity with myeloblastic or monoblastic or both subtypes of acute myeloid leukemia. The MoAbs MCS-2, MY7, Leu-M1, and MY9 detected the vast majority of cases with either myelocytic or monocytic involvement (group-I: “pan-myelomonocytic” reactivity). The MoAbs MCS-1, OKM1, VIM-D5, and Mo1 showed a predominance in their staining pattern for monocytic variants, but were also positive on a substantial percentage of nonmonocytic cases (group-II: predominantly reactive with monocytic, but also myelocytic cases). The MoAbs Leu-M3, MY4, VIM-D2, Mo2, and MY8 reacted with the large majority of AMMoL/AMoL cases and with a small number of AML cases (group-III: monocyte-“specific” reactivity). The MoAbs of group-I are useful in differentiating acute lymphoid from acute myeloid leukemias. The MoAbs of group-III, and to a lower extent those of group-II, will be of considerable value in the subtyping of acute myeloid leukemias. The results show that (1) accuracy of leukemia classification might not always be achieved by morphology alone, but that immunological and biochemical aspects should be included as well, and (2) several MoAbs are very useful tools for classification and subtyping of acute myeloid leukemias.

High concordance between marker profiles of 22 human leukemia-lymphoma cell lines tested with the same monoclonal antibodies before and during the second international workshop on human differentiation antigens

SummaryOur laboratory participated in the Second International Workshop and Conference on Human Leucocyte Differentiation Antigens. In this international study the reactivity profiles of monoclonal antibodies were analyzed on normal and malignant hematopoietic cells. The Workshop was divided into three categories: the T-cell, B-cell and myelomonocytic cell studies. We blindly tested 159 coded monoclonal antibodies of the panel for the T-cell study on 22 permanently established leukemia cell lines. The monoclonal antibodies were provided by the Workshop Committee and their reactivity with the target cells was visualized by standardized indirect immunofluorescence. After decoding it was recognized that 11 monoclonal antibodies had been examined on these cell lines prior to the Workshop. The reactivity of these 11 monoclonal antibodies was analyzed and compared with the earlier results. From a total of 217 paired tests done blindly in the Workshop study and prior to the Workshop, 191 tests (88%) did not show significantly different data. The possible reasons for discrepancies include nonspecific Fc-receptor-binding on some cell lines and a relatively nonspecific reactivity of some monoclonal antibodies.This analysis demonstrates the stability of the antigen expression on human leukemia-lymphoma cell lines grown at consistently optimal conditions, for the tests, using the same monoclonal antibodies as in the Workshop, had been performed 0.5–5 years prior to the Workshop study. On the other hand, nonspecific Fc-binding, wide “specificity” of monoclonal antibodies and a shift in antigen expression of the cells (due to poor growth conditions, involuntary induction of differentiation and other factors) must be taken into consideration upon immunological analysis.