Leukemia-associated antigens in man.

Rabbit antisera raised against acute lymphoblastic leukemia (ALL) cells were used to distinguish ALL from other leukemias, to identify rare leukemia cells in the bone marrow of patients in remission, and to define human leukemia-associated antigens. Antibody binding was studied with the use of immunofluorescence reagents and the analytic capacity of the Fluorescence Activated Cell Sorter-1 (FACS-1). The results indicated that most non-T-cell ALL have three leukemia-associated antigens on their surface which are absent from normal lymphoid cells: 1) an antigen shared with myelocytes, myeloblastic leukemia cells, and fetal liver (hematopoietic) cells; 2) an antigen shared with a subset of intermediate normoblasts in normal bone marrow and fetal liver; and 3) an antigen found thus far only on non-T-cell ALL and in some acute undifferentiated leukemias, which we therefore regard as a strong candidate for a leukemia-specific antigen. These antigens are absent from a subgroup of ALL patients in which the lymphoblasta express T-cell surface markers. Preliminary studies on the bone marrow samples of patients in remission indicated that rare leukemia cells were present in some samples. The implications of these findings with respect to the heterogeneity and cell origin(s) of ALL, its diagnosis, and its potential monitoring during treatment were discussed.

Co-ordinate expression of BI.3C5 and HLA-DR antigens on haemopoietic progenitors from chronic myeloid leukaemia.

Haemopoietic cells isolated from the peripheral blood of patients with chronic myeloid leukaemia (CML), have been extensively purified and enriched using either Percoll density gradients or Percoll density gradients combined with elutriation. The quantitative expression of the BI.3C5 associated antigen and the co-expression of BI.3C5 and HLA-DR antigens on these two populations has been studied using either single or simultaneous two colour FACS sorting, following by in-vitro culture for single and multilineage haemopoietic progenitors thus obtained. The data show that the CFU-GEMM are always found in the most strongly BI.3C5 positive fraction, irrespective of the separation procedure and that the bulk of the CFU-GEMM co-express BI.3C5 and HLA-DR. The cell types initiating these CFU-GEMM are morphologically immature blasts. The more mature cells of the myelomonocytic and erythroid lineages forming single lineage colony types show variable BI.3C5 expression, although most are HLA-DR positive. Such enriched populations of malignant progenitors could provide a useful source of material to study both gene expression and the molecular mechanisms underlying malignant transformation.

Clinical importance of DNA content in rectal cancer measured by flow cytometry.

The DNA content of 369 rectal cancers was measured by flow cytometry. One hundred and four (28%) were diploid, 252 (68%) were aneuploid, and 13 (3.5%) were tetraploid. Diploid cancers were associated with an improved 5 year survival (p less than 0.001) and were more likely to present at an early stage. DNA content, however, did not confer independent prognostic information in a Cox model based on four discrete pathological variables. Patients were classified by a new system of prognostic grouping and those with a very good or a very poor outlook were removed leaving 137 prognostic group III patients. No further substratification of this group by DNA content or by four additional pathological variables could be achieved. As the new prognostic system is not improved by the addition of ploidy, routine adoption of flow cytometry in the assessment of rectal cancer cannot be recommended.

Analysis of human leukaemic cells using cell surface binding probes and the fluorescence activated cell sorter.

Cell surface binding fluorescent ligands have been used to distinguish between different types of leukaemic cells and between leukaemic cells and their presumed normal counterparts or progenitors. Binding of these probes was evaluated using the Fluorescence Activated Cell Sorter (FACS) which provides both rapid, objective and quantitative recording of fluorescent signals from individual cells plus physical separation of cells of particular interest. Binding sites for cholera toxin (monosialoganglioside GM1) were found to be normally expressed in chronic leukaemias but greatly diminished or absent in acute leukaemias irrespective of their morphological type. Antibodies specific for the common form of acute lymphoblastic leukaemia (ALL, non-T, non-B) have been produced in rabbits. After extensive absorption and testing these were shown to define a cell surface antigen of non-T, non-B type ALLs. The antigen is absent from other leukaemias with two interesting exceptions--the majority of acute undifferentiated leukaemias express the antigen as do a proportion of chronic granulocytic leukaemias in blast crisis relapse. The anti-ALL antibodies can therefore be used to distinguish different leukaemias and, more significantly, can identify the existence of relatively rare leukaemic cells in the blood of untreated patients and the marrow of treated patients considered to be in remission.

Differential expression of cell surface binding sites for cholera toxin in acute and chronic leukaemias.

Binding of purified cholera toxin to cell surface receptors has been visualized by an indirect immunofluorescence procedure. Normal nucleated cells from blood, bone marrow and lymphoid tissues, express these receptors with the possible exception of erythroid precursors. Cells from patients with chronic lymphoid or myeloid leukaemias have a normal receptor expression but acute leukaemic cells showed a marked deficiency in cholera toxin binding. Insertion of purified Gm ganglioside into membranes of acute leukaemic cells provided cellular binding sites for the toxin.

Membrane marker analysis of 'lymphoid' and myeloid blast crisis in PH1 positive (chronic myeloid) leukemia.

The membrane phenotype of leukaemic cells was analysed during different stages of chronic myeloid leukaemia by a panel of markers. These included antisera against ALL antigen, p23,30 (Ia-like structure) and other T cell, B cell and myeloid markers 'Lymphoid' blast crisis shares the phenotype of common ALL (of non-T, non-B variety). Both leukemias react with anti-ALL serum and have pre-myeloid, pre-B lymphoid and pre-thymocyte characteristics. Their phenotype may reflect the characteristics of the pluripotential stem cell from which they derive. Nevertheless both leukaemias retain their undifferentiated characteristics and lack overt myeloid, B cell and thymocyte differentiation markers. Myeloid blast crisis and AML are negative with anti-ALL serum but some of the poorly differentiated myeloblasts react with anti-p23,30 serum (and negative for SmIg). The anti-p23,30 serum (used in a double marker assay combined with anti-immunoglobulin) detects some (4-11%) intermediate sized agranular p23,30+/SmIg-cells in peripheral blood during the chronic phase of CML as well as in normal foetal bone marrow. These could be myeloid stem cells (from which in CML the myeloid blast crisis arises). The results demonstrate that surface membrane analysis can aid exact diagnosis in different stages of CML.

Expression of HPCA-1 and HLA-DR antigens on growth factor- and stroma-dependent colony forming cells.

The expression of HLA-DR and HPCA-1 antigens (recognized by the L243 and BI.3C5 antibodies respectively) on adult human bone marrow cells was examined by fluorescence activated cell sorting and colony assays. Nearly all the (day 14) lineage restricted and multipotential colony forming cells analysed in methylcellulose cultures in the presence of added growth factors express HLA-DR and HPCA-1 determinants. Two colour cell sorting reveals that the lineage restricted HLA-DR positive progenitors express variable levels of BI.3C5 positivity whereas most of the multipotential progenitors, the multi-CFC or CFU-GEMM, are highly BI.3C5 positive. The isolated HLA-DR and BI.3C5 positive populations also contain haemopoietic precursors which adhere to and form colonies on pre-formed stromal layers. Thus, haemopoietic progenitors assayed in both types of culture system can be analysed and enriched by simultaneous two-colour sorting using anti-HLA-DR and BI.3C5 monoclonal antibodies. Similarities in the antigenic phenotype of such cells, however, precludes the use of these reagents for segregating growth factor-dependent from stroma-dependent progenitors.

Dysplasia and deoxyribonucleic acid aneuploidy in the assessment of precancerous changes in chronic ulcerative colitis. Observer variation and correlations.

Cancer prevention in patients with long-standing ulcerative colitis depends on the detection of epithelial dysplasia in colorectal biopsy specimens. Deoxyribonucleic acid analysis by flow cytometry has also been used to examine biopsy specimens, and might be a more quantitative method of detecting precancerous change. Histology and flow cytometry were used to analyze 333 paraffin blocks from colectomy specimens of 58 patients with extensive ulcerative colitis; 22 of these patients had developed carcinoma. Interobserver agreement between three experienced pathologists grading the sections was good for high-grade dysplasia and no dysplasia, but poor for low-grade and indefinite dysplasia. Deoxyribonucleic acid aneuploidy was easier to recognize than dysplasia and, as with dysplasia, it was found to be associated with patients who had developed carcinomas. The presence of deoxyribonucleic acid aneuploidy correlated with the presence of dysplasia. We believe that dysplasia is a useful marker of premalignant change and that flow cytometry may be useful as a complement to histologic examination when dysplasia is suspected.