E. Thiel

Identification and quantitation of human T-cell antigen by antisera purified from antibodies crossreacting with hemopoietic progenitors and other blood cells

Anti-T cell globulin (ATCG) prepared from antihuman thymocyte serum by absorption with kidney, cells from patients with chronic lymphatic leukemias, and several lymphoblastoid cell lines was shown to react specifically with human thymus-derived lymphocytes. While high activity against thymocytes and a T-lymphoblastoid cell line could be demonstrated, ATCG remained negative against several chronic lymphatic leukemias and B-lymphoblastoid cell lines. The ATCG was used in the cytotoxic test, electronmicroscopy, and immunoautoradiography for identification of T cells in thymus, tonsils, spleen, blood, bone marrow, lymphatic leukemias, and lymphoblastoid cell lines. A comparison of these results with the ability to form spontaneous SRBC-rosettes revealed remarkable deviations between both markers in leukemias. Absorption with human brain failed to remove specific activity of ATCG. Labeling experiments by immunoautoradiography and investigations by complement fixation permitted quantitation of relative T-cell antigen concentration on different cell populations. As further evidence for specificity it could be shown that ATCG was no longer toxic for hemopoietic progenitors, whereas unabsorbed globulin reduced the number of colonyforming cells considerably.

High levels of 5′-nucleotidase activity in blastic chronic myelogenous leukemia with common all-antigen

Abstract The activity of 5′-nucleotidase (5′-N) on the surface of leukemia cells was determined in 44 patients with blast crisis of chronic myelogenous leukemia (CML) and in 52 patients with acute myeloblastic (AML) or acute lymphoblastic (ALL) leukemia. The phenotype of blast cells was classified according to immunological surface markers, including the common ALL-antigen (cALLA). High 5′-N activities were found in the presence of cALLA, whereas low to undetectable levels were characteristic for cALLA-negative leukemias, the difference being highly significant (p ≦ 0.0001). While there was a certain overlap of 5′-N levels between cALLA-positive ALL and cALLA-negative AML, no overlap of 5′-N was observed between cALLA-positive lymphoid and cALLA-negative myeloid blast crisis of CML. Thus, 5′-N affords an additional easily-testable biochemical marker that may have its diagnostic and prognostic value especially in the case of blastic chronic myelogenous leukemia.

Classification of normal and malignant lymphatic cells using acid phosphatase and acid esterase.

ZusammenfassungEs wurde geprüft, ob zytochemische Reaktionen (saure Phosphatase, saure Esterase) aufwendigere Membranmarkeruntersuchungen für die Unterteilung normaler und maligner lymphatischer Zellen ersetzen oder ergänzen können. Untersucht wurden normale Lymphozyten, subfraktioniert durch Rosettierung und Differentialzentrifugation sowie bei M. Hodgkin und CLL; maligne lymphatische Zellen bei unterschiedlichen Formen lymphatischer Leukämien; lymphoblastoide Zell-Linien. Im menschlichen Blut sind T-Lymphozyten durch eine deutliche, umschriebene Esterase-Reaktion gekennzeichnet, die bei der zahlenmäßig kleinen (11%) Untergruppe Fc-IgG-positiver T-Lymphozyten etwas geringer ausgeprägt ist; B-Lymphozyten sind negativ oder schwach feingranulär positiv. Lymphoblastoide Zell-Linien vom B- und vom T-Typ sind unterschiedslos überwiegend Esterase- und Phosphatase-positiv. Bei einigen lymphatischen Systemerkrankungen unterstützt ein typisches Muster der Esterase- oder Phosphatase-Aktivität die Diagnose. Sehr hoch ist die Bedeutung der Esterase-Zytochemie einzuschätzen, um den Anteil normaler T-Lymphozyten bei lymphatischen Leukämien, immunologischen Erkrankungen und unter zytostatischer oder immunsuppressiver Behandlung zu bestimmen.SummaryThe usefulness of cytochemical tests (APh and ANAE) to replace or to supplement membrane markers in subclassification of normal and malignant lymphatic cells was investigated. Material: normal lymphocytes subfractionated by rosetting and centrifugation, and in M. Hodgkin and CLL; lymphoblastoid cell lines; malignant lymphatic cells in different types of lymphatic leukemias. In normal human blood, T-lymphocytes are marked by a distinct “dot-like” ANAE-reactivity which is somewhat less pronounced in the small (11%) subgroup of Fc-IgG-receptor positive T-lymphocytes; B-lymphocytes are negative or finely granular positive. Lymphoblastoid cell lines of B- and of T-type are ANAE- and APh-positive. In some lymphatic malignancies, a characteristic pattern of activity of APh or of ANAE may support the diagnosis. The value of ANAE-cytochemistry is highly estimated for the quantitative determination of the percentage of normal T-lymphocytes in lymphatic leukemias, immunological disorders, and during immunosuppressive therapy.

Evaluation of 5′-nucleotidase as biochemical marker in leukemias and lymphomas

Summary5′-Nucleotidase (5′-N) is known as a cellular ectoenzyme of wide tissue distribution. The varying expression of the enzyme in leukocytes is thought to be linked to cell differentiation. We, therefore, studied 5′-N activities of bone marrow or peripheral blood mononuclear cells in subgroups of leukemias and lymphomas as defined by morphology and immunological marker expression. In acute leukemias, elevated or very high enzyme activities were correlated with the expression of the common ALL antigen (cALLA). A similar correlation with cALLA was also observed for lymphoid blast crisis in CML. Therefore, 5′-N may be used as an additional diagnostic marker to discriminate cALLA positive leukemic cells from those not expressing cALLA. The 5′-N of leukemia cells of all patients tested gave complete crossreactivity with an antiserum directed against the normal human placental enzyme. Serum-5′-N was not well correlated with cellular enzyme activity in the same patient and therefore appears less suitable as diagnostic marker enzyme. However, elevated serum-5′-N was frequently observed in common ALL.Low enzyme activities in peripheral mononuclear cells were characteristic for some forms of lymphoma like CLL of B-type and hairy cell leukemia. In other lymphoma types, single patients with elevated or even extremely high 5′-N were found, for example in T-CLL, B-PLL and cc-lymphoma. With the limited data available it is too early to decide whether 5′-N could also become an additional useful biochemical marker for the classification of lymphoma subgroups.Though the investigations clearly indicate that 5′-N is not a specific marker for a leukemia or lymphoma subtype, the biochemically measurable variance of its expression appears to be a good indicator for the maturation degree of lymphatic cells.

Interferon-alpha 2b (IFNα) for early-phase chronic lymphocytic leukaemia with high risk for disease progression : results of a randomized multicentre study

The efficacy of interferon‐alpha 2b (IFNα) to prolong progression‐free (PFS) and/or overall survival (OS) in early B‐CLL (Binet stage A) was examined in a risk‐adapted phase III study. 99 previously untreated B‐CLL patients were recruited. 44 patients with expected high risk for disease progression, defined by non‐nodular bone marrow infiltration and lymphocyte doubling time ≤12 months or serum thymidine kinase levels ≥5 U/l, were randomized to either receive IFNα (group 1, n = 21) or not (group 2, n = 23). 55 low‐risk patients were observed to evaluate this risk stratification (group 3). During a median observation time of 36 months, four patients in the IFNα group achieved a partial remission (PR), no patient had stable disease (SD), and 17 patients experienced progressive disease (PD). The four responders had less extensive disease at study entry and tended to exhibit a rise in serum IgG levels. In group 2, no PR, seven SD and 16 PD, whereas in group 3, no PR, 37 SD and 18 PD occurred. PFS in group 1 (6.7 months) was not different from group 2 (13.3 months, P = 0.22), but PFS of groups 1 and 2 differed from group 3 (37 months, P ≤ 0.001). OS was 44.9 months (group 1), 43.1 months (group 2) and 57.9 months (group 3). OS was not significantly different for group 1 v 2, but was significant between groups 1 and 3 (P = 0.023). The higher percentage of PD in group 2 compared to group 3 (70% v 29%) shows that the selected risk factors allow the definition of CLL stage A patients at risk for disease progression within about a year. In conclusion, our data indicate that IFNα does not prolong PFS or OS in stage A CLL patients with high risk for disease progression.

CELL SURFACE MARKERS IN LEUKEMIA: BIOLOGICAL AND CLINICAL CORRELATIONS*

Recent advances in analysis of leukemic cell phenotypes using cell surface markers have provided important insights into leukocyte differentiation and the cellular origin of leukemia. In addition to the traditional cell surface markers, i.e., surface membrane immunoglobulin and receptors for sheep erythrocytes that define B and T lymphocytes, highly specific monoclonal antibodies have been developed that discriminate various stages of human lymphocyte and granulocyte differentiation. Explorations of the detailed phenotypes of leukemic cells in relation to normal hemopoietic differentiation reveal that consistent, composite phenotypes of different subclasses of lymphoid malignancies closely mimic those of corresponding normal cells at equivalent levels of maturation. This is exemplified in lymphoma cells (chronic lymphocytic leukemia of B or T type, Sezary Syndrome, immunocytoma) that resemble mature and immunocompetent T and B cells, in T cell acute lymphoblastic leukemia (T-ALL) (equivalent to thymus cells) and in non-T ALL (corresponding to lymphoid progenitor cells in the bone marrow). The major phenotypes documented in different leukemias represent the level of maturation arrest imposed on the dominant subclone; this is determined by, but not necessarily synonymous with, the target cell and associated clonogenic cell population in the leukemia. The clinical significance of immunodiagnosis of leukemia cell types becomes best evidenced in acute leukemias. Besides the improvement of diagnosis by using objective criteria, clinically useful subclassifications became evident: five major subtypes of ALL are now recognized, including unclassified or null ALL, common ALL, pre-B-ALL, B-ALL and pre-T/T-ALL. In addition to disclosing that ALL is an heterogeneous disease, such classifications have proved to be prognostically significant. This is exemplified in 248 children and 145 adults with ALL which were analysed for cell type and clinical data. In addition to their utility in leukemia classification, monoclonal antibodies that identify leukemia associated antigens are becoming used therapeutically, e.g., to lyse residual leukemia cells from remission bone marrows removed from leukemia patients before reinfusion. New approaches to the treatment of leukemia in which the objective is to encourage maturation of leukemia cells rather than to achieve leukemia eradication, can be monitored by phenotyping the alterations of the cell surface, and cell markers may hopefully be useful in identifying cell types that can be induced to differentiate.

[Use of specific anti-T-lymphocyte globulin (sATG) for the diagnosis of lymphoproliferative diseases (author's transl)].

SummaryDifficulties in the production of specific antisera against T-lymphocytes could be overcome by a stepwise absorption and purification procedure of anti-human thymocyte serum. Specific anti-T lymphocyte globulin (sATG) reacted with thymocytes, thymus-derived lymphocytes and a lymphoblastoid cell line of T-cell type whereas no activity was found against lymphoblastoid cell lines of B-cell type. Five chronic and three acute lymphatic leukemias were characterized using sATG in the cytotoxic test, electron microscopy, complement fixation test and quantitative immunoautoradiography, and compared with lymphocyte populations of normal individuals. Three chronic lymphatic leukemias with low numbers of spontaneous rosettes and high percentages of membrane-Ig-positive lymphocytes showed only few T-cell-antigen-positive lymphocytes and were therefore classified as B-cell leukemias. The cells of two chronic lymphatic leukemias with high numbers of spontaneous rosettes carried T-cell-antigen. The T-cell-antigen concentration, however, was lower than that of normal peripheral blood T-lymphocytes. The T-cell nature of two acute lymphatic leukemias with high numbers of spontaneous rosettes was confirmed by a positive reaction of the cells with sATG. In one case of acute lymphatic leukemia most leukemic cells carried T-cell-antigen although these cells did not form spontaneous rosettes. In the first two cases the T-cell-antigen concentration on the cell surface exceeded that of normal blood-T-lymphocytes, in the latter case it was slightly below that. The advantages of the characterization of leukemias with sATG in comparison with the spontaneous rosette formation and the relevance for prognosis are discussed.ZusammenfassungBisherige Schwierigkeiten in der Herstellung spezifischer Antiseren gegen T-Lymphocyten lassen sich durch eine stufenweise Absorptions-und Reinigungsprozedur von Anti-Human-Thymocytenserum überwinden. Spezifisches Anti-Human-Thymocyten-Globulin (sATG) reagierte mit Thymocyten, thymus-abhängigen Lymphocyten und einer lymphoblastoiden Zell-Linie vom T-Zell-Typ, während keine Aktivität gegen lymphoblastoide Zell-Linien vom B-Zell-Type nachzuweisen war. Mit Hilfe von sATG, eingesetzt im Cytotoxizitätstest, in der Elektronenmikroskopie, dem Komplementfixationstest und der quantitativen Immunoautoradiographie, wurden fünf chronische und drei akute lymphatische Leukämien charakterisiert und mit Lymphocytenpopulationen von Normalpersonen verglichen. Drei chronische lymphatische Leukämien mit niedrigen Spontanrosettenzahlen und hohen Prozentzahlen Membran-Ig-positiver Lymphocyten zeigten nur wenige T-Zell-Antigen-positive Lymphocyten und wurden daher als B-Zell-Leukämien klassifiziert. Die Zellen zweier chronischer lymphatischer Leukämien mit hohen Spontanrosettenzahlen trugen das T-Zell-Antigen, die Antigen-Konzentration jedoch lag niedriger als die von normalen peripheren Blut-T-Lymphocyten. Die T-Zell-Natur zweier akuter lymphatischer Leukämien mit hohen Spontanrosettenzahlen wurde durch eine positive Reaktion der Zellen mit sATG bestätigt. In einem Fall von akuter lymphatischer Leukämie wurden trotz fehlender Spontanrosettenbildung überwiegend T-Zell-Antigen-positive Zellen gefunden. In den beiden ersten Fällen lag die T-Zell-Antigen-Konzentration höher als auf normalen Blut-T-Lymphocyten, im letzteren Fall geringfügig darunter. Die Vorteile der Charakterisierung von Leukämien mit sATG im Vergleich mit der Spontanrosettenbildung und die Bedeutung für die Prognostik werden diskutiert.Difficulties in the production of specific antisera against T-lymphocytes could be overcome by a stepwise absorption and purification procedure of anti-human thymocyte serum. Specific anti-T lymphocyte globulin (sATG) reacted with thymocytes, thymus-derived lymphocytes and a lymphoblastoid cell line of T-cell type whereas no activity was found against lymphoblastoid cell lines of B-cell type. Five chronic and three acute lymphatic leukemias were characterized using sATG in the cytotoxic test, electron microscopy, complement fixation test and quantitative immunoautoradiography, and compared with lymphocyte populations of normal individuals. Three chronic lymphatic leukemias with low numbers of spontaneous rosettes and high percentages of membrane-Ig-positive lymphocytes showed only few T-cell-antigen-positive lymphocytes and were therefore classified as B-cell leukemias. The cells of two chronic lymphatic leukemias with high numbers of spontaneous rosettes carried T-cell-antigen. The T-cell-antigen concentration, however, was lower than that of normal peripheral blood T-lymphocytes. The T-cell nature of two acute lymphatic leukemias with high numbers of spontaneous rosettes was confirmed by a positive reaction of the cells with sATG. In one case of acute lymphatic leukemia most leukemic cells carried T-cell-antigen although these cells did not form spontaneous rosettes. In the first two cases the T-cell-antigen concentration on the cell surface exceeded that of normal blood-T-lymphocytes, in the latter case it was slightly below that. The advantages of the characterization of leukemias with sATG in comparison with the spontaneous rosette formation and the relevance for prognosis are discussed.

Phenotypic changes in acute lymphoblastic leukemia cells of the common type in diffusion chambers.

Abstract cALL positive blast cells from peripheral blood of four children as well as cells from the Reh line were cultivated in DC in order to promote cell differentiation. DC were implanted into the peritoneal cavity of pre-irradiated CBA mice. At different intervals during the culture period changes in the membrane marker profile were determined by direct immunofluorescence after labeling the cells with AcALLG, ATCG and polyvalent AIg. In addition, the ability of cells to form rosettes with SRBC, AET-treated sheep erythrocytes and mouse red blood cells was tested. In the course of the culture the majority of cALL positive cells from three patients (T.H., V.M., I.G.) simultaneously developed a positive reaction with polyvalent AIg. Furthermore, a remarkable portion of cells in V.M. turned out to become positive with only AIg. Comparable results together with an increasing percentage of M-RFC were obtained in the fourth patient, C.M., using AIg(Fab) 2 . Besides this development, quite a few cells expressing the T-cell antigen occurred in the latter two patients. In the case of the Reh line, the cells developed T-cell antigen, and in one of two experiments they further acquired an E-receptor. Conversely, in the second experiment a receptor for mouse red blood cells was detected. Our data suggest that malignant cells carrying the cALLA are representative of an early developmental stage of lymphatic ontogeny.

Prolymphocytic leukemia@@@Prolymphozytäre Leukämie

ZusammenfassungBei der prolymphozytären Leukämie handelt es sich um eine wohldefinierte, großzellige Sonderform der CLL, deren hervorstehendste Merkmale Milztumor, hohe Leukozytenzahl, typische Morphologie der leukämischen Zellen und schlechtes Ansprechen auf eine bei CLL meist erfolgreiche Therapie sind. Die PL-Zellen sind durch einen hohen Gehalt an Lysosomen und an saurer Phosphatase gekennzeichnet, das Chromatinmuster liegt zwischen dem bei CLL und bei ALL, die leukämischen Zellen eines Patienten können Merkmale von B- oder von T-Lymphozyten besitzen. Die Bezeichnung “prolymphozytär” entspricht zwar kaum den pathophysiologischen Vorstellungen vom Differenzierungsgrad dieser Zellen; er sollte aber beibehalten werden, da er zur Bezeichnung dieser Sonderform einer lymphatischen Leukämie nützlich und in der Literatur eingebürgert ist.SummaryThe term “prolymphocytic leukemia” designates a lymphoproliferative disorder closely related to CLL. Its most conspicuous features are: massive splenic enlargement, high leukocyte counts, typical morphology of the leukemic cells, and poor response to modes of treatment usually effective in CLL. PL-cells are characterized by a high content of APh and lysosomes, and their pattern of chromatin moves between that observed in CLL and in ALL. Leukemic cells in this disease may be of B- or of T-cell nature. The term “prolymphocytic” does not represent our knowledge on the stage of differentiation of the leukemic cells, but should nevertheless still be applied as it is useful to describe a special type of lymphatic leukemia and is widely used in hematological literature.

Use of Specific Antisera against Leukemia-Associated Antigens in the Diagnosis of Childhood Acute Lymphoblastic Leukemia

A series of 66 children with acute lymphoblastic leukemia (ALL) at diagnosis were investigated (simultaneously) for various surface markers. For this purpose the reaction of specific antisera against ALL antigens and T cell antigens was analysed in every case by several test systems, namely immunofluorescence, microcytotoxicity and complement fixation. A clearly defined classification in 6 subgroups of ALL emerged. The clinical data at presentation and possible correlations with the immunological subgroups were demonstrated.