Surface markers in acute non-lymphoid leukemia: analysis with a panel of 36 monoclonal antibodies

The reactivity of a panel of monoclonal antibodies was studied in fifty-four cases of acute myeloid (AML) or undifferentiated (AUL) leukemias. Thirty-six antibodies from the Myeloid section of the Second Workshop on Human Leukocyte Differentiation Antigens were used in an indirect immunofluorescence assay. The antibodies could be classified into three groups recognizing respectively granulocytic, monocytic or granulomonocytic leukemias. Most antibodies stained erythroblastic and megakaryoblastic leukemias. In each group, it was possible to define antibodies staining either the less differentiated forms (FAB M 1 and M 5 a) or the more differentiated forms (M 2, M 3, M 4 and M 5 b). Six out of eight AUL were stained by some of the antibodies (mainly from the monocytic group). However, a heterogeneity of stainings in a same blast population was observed.     

Expression of immunological markers on leukemic cells before and after cryopreservation and thawing

We have studied the effects of cryopreservation on the viability and on the expression of surface antigens of acute leukemia cells. Marrow samples were obtained at initial diagnosis from 89 patients with acute myeloid leukemia (AML), acute undifferentiated leukemia (AUL), and acute lymphoid leukemia (ALL). In AML, the mean viability was greater than 90% in the types M1, M4, and M5 of the French-American-British classification, 79% in M2, and 3% in M3 types. The viability was 74% in AUL. In ALL, the viability was 95% for pre-B leukemias, but only 2% in T-cell leukemias. The expression of myeloid antigens was studied before and after freezing and thawing using three monoclonal antibodies (NHL30.5, against poorly differentiated granulocytic leukemias, VIMC6 against differentiated granulocytic leukemias and granulocytes; and UCHM1 or CRIS-6, against monocytic leukemias and monocytes). The percentage of cells stained by NHL30.5 and UCHM1 or CRIS-6 was very similar before and after cryopreservation. For VIMC6, the mean staining after cryopreservation was 60% of the initial one. In pre-B ALL, the stainings by anti common ALL antigen before and after cryopreservation were also very similar. We conclude that leukemic cryopreserved cells are suitable for immunologic studies. The recovery is, however, very low in promyelocytic AML and T-cell ALL.     

Analysis of neoplasms induced by Cas-Br-M MuLV tumor extracts

Cas-Br-M is an ecotropic murine leukemia virus isolated from wild mice that induces a wide spectrum of hematopoietic neoplasms, including T and B cell lymphomas, myelogenous leukemias, and erythroleukemias. The purpose of this study was to determine if the induction of neoplasms belonging to multiple lineages was due to the ecotropic virus itself or to the generation of cell lineage-specific recombinant viruses. The results demonstrate that in some instances (two of 12 tumor extracts tested), recombinant viruses can be recovered from primary Cas-Br-M-induced tumors that will induce lymphomas of single lineages in mice inoculated as newborns. One of these viruses is a recombinant mink cell focus-forming virus that induces T cell lymphomas, and the other is a replication-defective, fibroblast-transforming virus that induces early B lineage lymphomas in mice. Histologic and flow microfluorometric cell surface antigen analyses of primary and in vitro adapted tumors are presented in support of a modified scheme of hematopoietic cell development.     

The "typical" immunophenotype of acute promyelocytic leukemia (APL-M3): does it prove true for the M3-variant?

The immunophenotypes of 12 acute promyelocytic leukemias (APL-M3; eight hypergranular, four microgranular) with documented PML-RAR-alpha fusion gene are presented. Bone marrow mononuclear cells were immunophenotyped using a panel of 20 monoclonal antibodies. The hypergranular APLs exhibited a mature myeloid phenotype as it has been described to be typical for M3. No lineage infidelity was detectable in classic M3 cases. In contrast, among the four cases of M3 variant, all leukemias showed marked expression of CD34 and two of four cases expressed the HLA-DR antigen. The CD2 antigen was expressed in three of four cases. Furthermore, one case showed expression of the CD56 antigen, and one case was positive for the blood group H antigen. The data suggest that microgranular APL is a heterogeneous entity with regard to the immunologic phenotype.     

Monoclonal antibodies to the antigen of myeloid cells

ICO-G-2 hybridoma clone was obtained after fusion of spleen cells from BALB/c mice immunized with cells from a patient with acute myelomonoblastic leukemia (AMML) and P3 X 63 Ag8.653 cells, using 50% polyethyleneglycol, molecular weight 1500 KD. The antigen with a molecular weight of 100 KD was present only on polymorphonuclear neutrophils and eosinophils of the peripheral blood. The antigen expression was also found on the majority of myeloid precursors and some nuclear erythroid cells. CFU-GM did not express the antigen. Monoclonal antibodies ICO-G-2 reacted with blast cells of some patients with AML, AMML and CML. The antibodies did not react with cells from patients with AMonL, CMonL, ALL, CLL and LSA. Such pattern of reactivity makes these monoclonal antibodies useful for the differential diagnosis of acute nonlymphocytic leukemias and CML in blast crisis.     

Terminal deoxynucleotidyl transferase, TdT, as a marker for leukemia and lymphoma cells

Terminal deoxynucleotidyl transferase, TdT, was assayed in the mononucleate cells of blood and bone marrow from 121 patients with leukemias at the onset of disease and from 95 subjects with malignant lymphomas at diagnosis. This intracellular marker was also investigated by cytoimmunofluorescent tests in 17 other cases of initial leukemias and in 3 diagnosed lymphoblastic lymphomas. Generally, the TdT levels were significantly enhanced in the blasts of the following: acute undifferentiated leukemias; the more immature types of acute lymphoblastic leukemias i.e., the null, non-T non-B, common, early T and pre-B subgroups; a fraction of blastic crises in chronic myelogenous leukemias; and many lymphoblastic lymphomas. TdT might also be slightly increased in the mononucleate blood cells obtained from the most immature forms of acute myelogenous leukemias. Relapses with changes in cell phenotypes were occasionally observed in previously TdT-positive leukemias as a result of clonal evolution of the disease. The leukemias with blasts containing high levels of TdT were usually responsive to treatment with corticosteroids and vincristine. TdT is an oligoclonal marker characterizing several populations of undifferentiated or poorly differentiated blasts that tend to develop towards or along the lymphoid pathway. Together with specific immunological markers, this enzyme is useful to define the particular type of leukemic cells. It also serves to identify the quasi-lymphoblastic nature of the malignant clone, a helpful indication for the choice of therapy.     

Harvey murine sarcoma virus: influences of coding and noncoding sequences on cell transformation in vitro and oncogenicity in vivo.

The rat-derived Harvey murine sarcoma virus (Ha-MuSV) contains a transduced ras oncogene activated by two missense mutations and flanked by rat retroviruslike VL30 sequences. Ha-MuSV induces focal transformation of mouse NIH 3T3 cells in vitro and tumors (fibrosarcomas and splenic erythroleukemias) in newborn mice. We have used these two assays to study the contribution of coding and noncoding viral sequences to the biological activity of Ha-MuSV. A good correlation was found between the in vitro and in vivo assays. In several different isogenic Ha-MuSV variants, those with a rasH gene that had one or both of the Ha-MuSV missense mutations were much more active biologically than the corresponding proto-oncogene. A Ha-MuSV variant that encoded the proto-oncogene protein induced lymphoid leukemias (with thymomas), with a relatively long latent period, rather than the fibrosarcomas and erythroleukemias characteristic of Ha-MuSV with one or both missense mutations. A VL30-derived segment with enhancer activity was identified downstream from v-rasH. A mutant Ha-MuSV from which this 3' noncoding segment was deleted expressed lower levels of the wild-type viral protein, displayed impaired transforming activity in vitro, and induced lymphoid leukemias (with thymomas). 5' noncoding rat c-rasH sequences were found to increase the biological activity of the virus when substituted for the corresponding segment of v-rasH. We conclude that (i) the biological activity of Ha-MuSV can be influence significantly by noncoding sequences located outside the long terminal repeat as well as by coding sequences, (ii) VL30 sequences positively regulate the expression of v-rasH, (iii) relatively low biological levels of ras, whether resulting from low-level expression of wild type v-rasH or high-levels of ras proto-oncogene protein, induce a type of tumor that differs from tumors induced by high biological levels of ras, and (iv) the in vivo pathogenicity of the Ha-MuSV variants correlated with their transforming activity on NIH 3T3 cells.     

A tagged helper-free Friend virus causes clonal erythroblast immortality by specific proviral integration in the cellular genome.

A colinear molecular clone of the Lilly-Steeves polycythemia strain of Friend spleen focus-forming virus (SFFV) was modified by inserting a 215-base-pair tag of simian virus 40 DNA into its nonfunctional pol gene region. The DNA was then transfected into psi-2 packaging cells, and helper-free tagged SFFV was recovered in the culture medium. Injection of this helper-free virus into NIH/Swiss mice caused transient mild splenomegaly and formation of spleen foci at 9 to 10 days. Although the vast majority of infected erythroblast clones then differentiated and died out, rare cell clones that were present in only 20 to 30% of the mice grew extensively by 26 to 33 days to form transplantable leukemias. The clonality of these leukemias was established by Southern blot analysis of their DNAs by using several restriction endonucleases and the simian virus 40 tag as a hybridization probe. All transplantable leukemias lacked helper virus contamination and contained a single tagged SFFV provirus that expressed the mitogenic env gene product gp55. The SFFV proviruses in these leukemias also appeared to be integrated into a few tightly clustered sites in the cellular genome. Although the tagged SFFV caused polycythemia during the polyclonal early stage of erythroblastosis, growth of the helper-free clonal erythroleukemias caused severe anemia. These results suggest that a single SFFV can cause mitosis of erythroblasts, and that cell immortalization also occurs when the provirus integrates into a critical site in the host genome. We propose that mice with clonal-stage leukemia become anemic because the immortalizing proviral integrations interfere with the cellular commitment to differentiate.     

ICO-13 monoclonal antibodies to the differentiation antigen of human hemopoietic cells

Monoclonal antibodies (Mab) ICO-13 of IgM isotype reacted in indirect surface immunofluorescence test with 88.5 +/- 3.4% of thymocytes and 7.1 +/- 2.4% of T cells, but failed to react with other peripheral blood cells from healthy donors. The antigen disappeared in cells stored at 4 degrees C overnight or at -196 degrees C in liquid nitrogen. The antigen detected by Mab ICO-13 was expressed on blast cells of acute lymphoblast leukemias and lymphomas. The antigen was absent in chronic lymphoblast leukemia and blast crisis of chronic myeloid leukemia.     

BK virus-transformed inbred hamster brain cells. I. Status of the viral DNA and the association of BK virus early antigens with purified plasma membranes

Inbred LSH hamster brain cells were transformed in vitro by the GS strain of BK virus (BKV), and transplantable tumors classified as undifferentiated glioblastomas were induced in the syngeneic host. The viral status in the transformed cells, designated LSH-BR-BK, was established. About 46 genome equivalents per cell of viral DNA was detected, with the majority of sequences in a free form. The transformed cells expressed large quantities of tumor (T) antigen as well as surface (S) antigen as demonstrated by indirect immunofluorescence. Sixty-three percent of tumor-bearing hamsters produced high-titer antibodies against T, whereas 3 of 14 (21%) hamsters also produced antibodies against the BKV-specific S antigen. Furthermore, the relatedness of BKV early gene products, including T, S, and tumor-specific transplantation antigen, was established by the production of a rabbit antiserum against highly purified plasma membranes of LSH-BR-BK cells and by the induction of a BKV-specific tumor-specific transplantation antigen response by these plasma membranes in the syngeneic host.     

Proliferating cell nuclear antigen (PCNA) and human malignant tumor nucleolar antigens (HMTNA) in nucleoli of human hematological malignancies

Lymphoma (Lymphocytic non-Hodgkin's malignant lymphoma) and leukemic (chronic lymphocytic, acute and chronic myeloid, myelomonocytic leukemia) cells were studied by indirect immunofluorescence to evaluate the presence of proliferating cell nuclear antigen (PCNA) and human malignant tumor nuclear antigen (HMTNA) in their nucleoli. Most cells in lymph node smears of lymphocytic non-Hodgkin's malignant lymphoma (NHML) developed a bright nucleolar fluorescence with HMTNA antibodies. PCNA was detected in nucleoli of a limited number of cells which apparently represent the proliferating cell population in these lymphomas. Similarly, in the bone marrow smears of patients with chronic lymphocytic leukemia most cells possessed a nucleolar fluorescence for HMTNA and PCNA was present in nucleoli of a limited number of cells. In the bone marrow smears of patients with myeloid or myelomonocytic leukemias most blastic or monocytoid cells also developed a bright nucleolar fluorescence with HMTNA antibodies and PCNA was present only in a small percentage of these cells. Leukemic cells with PCNA in their nucleoli like thekhuntigen might represent a proliferating cell population in late G1-early S phase.     

Different modes of mesenteric infiltration displayed by two rat leukemias. A study by scanning and transmission electron microscopy and by microcinematography

Infiltration of the mesentery after intraperitoneal implantation of two transplantable rat leukemias, the undifferentiated L5222 and the myeloid BNML, was studied by means of scanning and transmission electron microscopy, and microcinematography. In animals implanted with L5222 cells, contraction of the mesenteric mesothelium is a conspicuous feature. It occurs within the first 24h after implantation and influences decisively the course of infiltration. In contrast, The presence of BNML cells leads to mesothelial contraction only in the terminal stage and, therefore, exerts no direct effect on infiltration. In addition, the two leukemias differ with regard to their cellular motility. Whereas L5222 cells locomote within the mesentery, only stationary movements are recorded with BNML cells. Based on the different interactions with the mesothelium and cell motilities, two distinct modes of infiltrating the mesentery could be ascertained for the two rat leukemias.     

Cell surface molecules of friend erythroleukemias: Decrease in T200 glycoprotein expression after induction

Monoclonal antibodies against the Thy-1 and T200 glycoproteins were used to study the expression of cell surface molecules on mouse hematopoietic cell lines. Friend erythroleukemias express T200 glycoprotein but do not express significant amounts of Thy-1 glycoprotein on their cell surface. The rate of T200 glycoprotein synthesis in maximally-induced Friend erythroleukemia 745.6 cells is less than 10% that in noninduced cells, although total protein synthesis shows only a twofold decline and induced cells express 2-6-fold less T200 glycoprotein on their surface compared to noninduced cells. T200 glycoprotein expression is reduced in a variant cell line obtained by selection for growth in dimethylsulfoxide, showing that the reduction in T200 glycoprotein synthesis characteristic of induced cells is an event that can be dissociated from commitment and hemoglobin synthesis. Analysis of T200 glycoprotein negative cell lines, isolated by cytotoxic immunoselection against T200 glycoprotein, indicates that the presence of T200 glycoprotein on the cell surface is not necessary for induction of hemoglobin synthesis and terminal differentiation of Friend erythroleukemias.     

Molecular basis of a unique tumor antigen of radiation leukemia virus-induced leukemia B6RV2: its relation to MuLV gp70 of xenotropic class

Hybridomas secreting monoclonal antibodies that reacted with the B6 radiation leukemia virus (RadLV)-induced leukemia B6RV2 were produced by fusion of BALB/c NS-1 myeloma cells with spleen cells from (BALB/c X B6)F1 mice immunized with B6RV2. By direct and absorption analyses with 28 B6 and BALB/c leukemias, the monoclonal antibodies NU7-4 and NU7-99 were shown to react only with B6RV2, indicating that they recognized an individually distinct antigen on B6RV2 that was identified previously with conventional (BALB/c X B6)F1 anti-B6RV2 serum. Another monoclonal antibody, NU1-132, showed relatively restricted reactivity with B6 RadLV leukemias. These three monoclonal antibodies all precipitated material of approximately 80,000 daltons, which is the same size as that precipitated by anti-xenotropic MuLV gp70 serum. Sequential immunoprecipitation analysis revealed that the molecules precipitated by NU7-4 were not removed by pretreatment of NU7-99 or NU1-132 and that the molecules precipitated by NU7-99 were not removed by NU7-4 or NU1-132. The molecules precipitated by NU1-132 were partially removed by pretreatment with NU7-4, but not with NU7-99. The molecules precipitated by these three monoclonal antibodies were removed by pretreatment with anti-xenotropic gp70. These results suggested heterogeneity of the xenotropic MuLV gp70-related molecules expressed on B6RV2 and a possible relation between serologically defined unique tumor antigens and gp70-related molecules.     

Expression of the Clustered NeuAcα2–3Galβ O-Glycan Determines the Cell Differentiation State of the Cells

Human embryonic stem cells (hESCs) are pluripotent stem cells from early embryos, and their self-renewal capacity depends on the sustained expression of hESC-specific molecules and the suppressed expression of differentiation-associated genes. To discover novel molecules expressed on hESCs, we generated a panel of monoclonal antibodies against undifferentiated hESCs and evaluated their ability to mark cancer cells, as well as hESCs. MAb7 recognized undifferentiated hESCs and showed a diffuse band with molecular mass of >239 kDa in the lysates of hESCs. Although some amniotic epithelial cells expressed MAb7 antigen, its expression was barely detected in normal human keratinocytes, fibroblasts, or endothelial cells. The expression of MAb7 antigen was observed only in pancreatic and gastric cancer cells, and its levels were elevated in metastatic and poorly differentiated cancer cell lines. Analyses of MAb7 antigen suggested that the clustered NeuAcα2–3Galβ O-linked oligosaccharides on DMBT1 (deleted in malignant brain tumors 1) were critical for MAb7 binding in cancer cells. Although features of MAb7 epitope were similar with those of TRA-1–60, distribution of MAb7 antigen in cancer cells was different from that of TRA-1–60 antigen. Exposure of a histone deacetylase inhibitor to differentiated gastric cancer MKN74 cells evoked the expression of MAb7 antigen, whereas DMBT1 expression remained unchanged. Cell sorting followed by DNA microarray analyses identified the down-regulated genes responsible for the biosynthesis of MAb7 antigen in MKN74 cells. In addition, treatment of metastatic pancreatic cancer cells with MAb7 significantly abrogated the adhesion to endothelial cells. These results raised the possibility that MAb7 epitope is a novel marker for undifferentiated cells such as hESCs and cancer stem-like cells and plays a possible role in the undifferentiated cells.     

Diagnosis of human childhood rhabdomyosarcoma of antibodies to desmin, the structural protein of muscle specific intermediate filaments

Four embryonal rhabdomyosarcomas, one tumor diagnosed as an undifferentiated sarcoma, probably a rhabdomyosarcoma, and six different non-muscular sarcomas were investigated with antibodies specific for different intermediate filament types. The tumor cells in the rhabdomyosarcomas and the undifferentiated tumor were stained clearly by antibodies to desmin, the intermediate filament type characteristic of muscle. The staining of tumor cell by antibodies to vimentin, the intermediate filament type characteristic of certain cell types of mesenchymal origin including myoblasts, was different in these 5 cases. In one case of embryonal rhabdomyosarcoma nearly all tumor cells were stained, but in the remaining cases few or no tumor cells were positive with the vimentin antibody. In these rhabdomyosarcomas not only the large rhabdomyoblasts, but also the small undifferentiated cells were labeled by antibodies to desmin. In the latter cell type the desmin filaments were arranged typically in coils. In contrast, tumor cells in the non-muscular mesenchymal sarcomas were stained only by antibodies to vimentin but not by antibodies to desmin or prekeratin. The retention of the desmin marker characteristic of normal muscle in cases of rhabdomyosarcoma not only allowed the undifferentiated desmin-positive sarcoma to be classified as rhabdomyosarcoma but also suggests that the use of antibodies to desmin could be very helpful in the future for the diagnosis of undifferentiated rhabdomyosarcomas.     

Stages of B-lymphocyte differentiation in acute lymphoblastic leukemia

Blasts phenotype was determined in 61 children with the acute lymphoblastic leukemia. Non-T-cell acute lymphoblastic leukemia was diagnosed in 51 children. Stages of blasts differentiation were determined with the aid of monoclonal antibodies set using alkaline phosphatase-anti-alkaline phosphatase technique. Blasts in 50 patients belonged to B subpopulation confirmed by the presence of panB CD19 and CD22 antigens. Common antigen was seen in 76.5% of the examined patients with non-T-cell acute lymphoblastic leukemia. Cases of non-T-cell acute lymphoblastic leukemia were divided into 8 subgroups depending on the antigens of B-cells differentiation. An identification of pre-B subgroups of the acute lymphoblastic leukemia indicates heterogenicity of the acute lymphoblastic leukemias in childhood and enables their classification into groups corresponding to the early stages of lymphoblasts maturation.     

H-2-specific antibodies induced by injection of syngeneic leukemia cells

AKR/J mice immunized with several syngeneic leukemia cells contained antibodies in their sera which reacted with certain AKR leukemia cell lines, depending on their H-2 expression, and precipitated H-2K antigens from lysates of leukemia cells. Precipitation of H-2K was not due to virus-specific antibodies: it could not be blocked by prior absorption with H-2-negative leukemias, but was blocked by certain allogeneic lymphocytes. Tumor-specific H-2K antibodies did not react with H-2K from normal AKR lymphocytes either on the cell surface or after detergent solubilization; however, they did react with H-2K from mitogen-activated AKR and BALB.K lymphoblasts. Since both these latter cells were also lysed by AKR-Gross/MuLV-specific and H-2K^k-restricted cytotoxic T lymphocytes, we consider the possibility that antibodies detecting conformational alterations induced in H-2K^k molecules by viral association may be present in syngeneic AKR antileukemia sera.Abbreviations used in this paper GCSA Gross-virus-induced cell-surface antigen - MCF mink cell focus-forming virus - MuLV murine leukemia virus - Th T helper     

Detection of P-glycoprotein in human leukemias using monoclonal antibodies

Overexpression of a Mr 170,000 membrane glycoprotein (P-glycoprotein) is consistently associated with multidrug resistance in cell lines. Two monoclonal antibodies (Mab) against P-glycoprotein (265/F4 and C 219) were used to examine tumour samples from patients with leukemias for evidence of P-glycoprotein overexpression. High levels of P-glycoprotein (greater than 5% positive cells) were detected with both antibodies in samples from 3 out of 18 patients suggesting that a multidrug resistant phenotype may also occur in human leukemias.     

Clinical relevance of autoantibodies in systemic rheumatic diseases

Autoantibodies directed to intracellular antigens are serological hallmarks of systemic rheumatic diseases. Identification of circulating autoantibodies is helpful in establishing the correct diagnosis, indicating the prognosis and providing a guide to treatment and follow-up. Some autoantibodies are included in diagnostic and classification criteria for diseases such as anti-Sm antigen and anti-double-stranded DNA antibodies in systemic lupus erythematosus, anti-U1 nuclear ribonucleoprotein antibodies in mixed connective tissue disease, and anti-SS-A/Ro and anti-SS-B/La antibodies in Sjögren's syndrome. Over the past 30 years, the identification of new autoantibody systems was advanced by the initiation or adaptation of novel techniques such as double immunodiffusion to detect antibodies to saline-soluble nuclear antigens, extraction-reconstitution and ELISA techniques to detect histone and chromatin antibodies, immunoblotting and immunoprecipitation to detect a wide range of antibodies directed against naturally occurring and recombinant proteins. These techniques have been made possible by advances in cellular and molecular biology and in turn, the sera from index patients have been important reagents to identify novel intracellular macromolecules. This paper will focus on the clinical relevance of several autoantibody systems described by Tan and his colleagues over the past 30 years.Abbreviations ANA antinuclear antibody - CENPs centromere proteins - CTD connective tissue disease - DIA drug-induced autoimmunity - DIL drug-induced lupus - HIV human immunodeficiency virus - IIF indirect immunofluorescence - JCA juvenile chronic arthritis - MCTD mixed connective tissue disease - MSA mitotic spindle apparatus - NOR nucleolar organizer - NuMA nuclear mitosis antigen - PBC primary biliary cirrhosis - PCNA proliferating cell nuclear antigen - PM polymyositis - RA rheumatoid arthritis - RNP ribonucleoprotein - SLE systemic lupus erythematosus - SS Sjögren's syndrome - SSc systemic sclerosis - UCTD undifferentiated connective tissue disease