Use of DNA image cytometry in addition to flow cytometry for the study of patients with advanced ovarian cancer.

Forty-five patients with advanced ovarian cancer were studied with both DNA flow cytometry (FCM) and automatic DNA image cytometry carried out with the Leiden Television Analysis System (Leytas). There was a significant difference in survival between the diploid and nondiploid cases as determined by FCM. Furthermore, the presence of nuclei with a high DNA content (defined as a DNA content higher than 5C) as determined by Leytas indicated a poor prognosis. When the combined results of FCM and Leytas were taken into account, three different groups of patients could be distinguished. The group of patients with a diploid malignancy (n = 12) had a median survival of more than 60 months. The group of patients (n = 11) with a nondiploid tumor having fewer than 100 nuclei with a high DNA content per 1600 microscope fields formed an intermediate group (median survival, 42 months), whereas the median survival of the remaining patients (n = 22), who had a nondiploid malignancy combined with more than 100 of these nuclei per 1600 microscope fields, was only 15 months. In addition, comparison of the clinical parameters by means of a multivariate analysis (Cox regression model) showed that the combined results of FCM and DNA image cytometry had the largest influence on survival. It is concluded that DNA image cytometry appears to be supplementary to FCM for the study of DNA ploidy abnormalities and that the combined results of these methods have a major influence on the clinical outcome.

Appearance of high affinity receptors for type beta transforming growth factor during differentiation of murine embryonal carcinoma cells.

Recent studies have demonstrated that type beta transforming growth factor (TGF-beta) not only influences cell growth but also affects cell differentiation. In the present study, two different embryonal carcinoma (EC) cell lines and their differentiated cells were examined for the presence of TGF-beta receptors and for their responses to this factor. F9 and PC-13 EC cells bind little, if any, TGF-beta and do not appear to respond to TGF-beta in monolayer or soft agar cultures. Treatment of both EC cell lines with retinoic acid leads to the appearance of irreversibly differentiated cells that begin to exhibit receptors for TGF-beta by 48 h. After an additional 3-5 days, the differentiated cells express approximately 6000 high affinity receptors for TGF-beta, with an apparent dissociation constant of 45 PM. In contrast to the results observed with the parental EC cells, TGF-beta influences the growth of the differentiated cells cultured in both serum-free and serum-containing media. However, TGF-beta, alone or in combination with other growth factors, does not induce the differentiated cells to form colonies in soft agar. The possible relationship of these results to the roles of growth factors during early mammalian development is discussed.

Nuclear and cytoplasmic distribution of cellular myb protein in human haematopoietic cells evidenced by monoclonal antibody.

A monoclonal antibody was used for analysing the expression of the cellular myb (c-myb) protein in a variety of established human tumor cell lines and its decrease after induction of differentiation. Differentiated resting human T-cells and B-cells do not express detectable amounts of c-myb protein. However, upon mitogenic stimulation in vitro T-cells exhibit strong expression of the c-myb protein as demonstrated by immunocytochemical staining and indirect immunoprecipitation. In contrast to the transformed T-lymphoblastic cell line Molt-4, where c-myb protein is a nuclear antigen, it was found in proliferating normal T-cells almost exclusively distributed in the cytoplasm. Screening of a total of 70 fresh human malignant lymphomas by immunohistochemical staining indicates the presence of the c-myb protein primarily in non-Hodgkin's lymphomas with a large growth fraction, i.e. precursor cell-derived lymphoblastic lymphomas of B-cell type and T-cell type (9/10, 3/4, respectively) and anaplastic large cell Ki-1 lymphomas (5/9), which originate from activated lymphoid cells. The c-myb protein was located predominantly in the nucleus and in some cases additionally in the cytoplasm. The different subcellular locations suggest a dual functional role. While nuclear localisation is exhibited by transformed haematopoietic cells, cytoplasmic localisation appears to be characteristic for proliferating normal T-cells and points to a second property of the c-myb protein other than interaction with DNA.

Flow cytometry, cellular DNA content, and prognosis in human malignancy.

The use of flow cytometry to analyze the cellular DNA content of human malignancies has become increasingly commonplace. The relationship between abnormalities in DNA content or proliferative characteristics and prognosis is becoming clear for a variety of malignancies in part through new techniques that permit analysis of archival material. High- and low-risk groups of patients with early breast and bladder carcinomas, non-small-cell lung cancer, and colorectal, ovarian, and cervical carcinoma can be distinguished on the basis of abnormal stemline DNA content. In several hematologic and common pediatric malignancies, the prognostic relevance of DNA content flow cytometry has been similarly established. Though the interpretation of tumor cell cycle analyses is less certain, this characteristic may also be prognostically important. However, generalizations cannot be made when applying flow cytometric DNA analysis to clinical decision making. The prognostic importance of an abnormal DNA histogram for an individual patient must be assessed on the basis of the relevant data base for that particular tumor type. The current extent of this data base for various malignancies is reviewed.

Expression of oncogenes and cell cycle related genes in acute and chronic leukemias.

The authors have assayed the level of expression of several cell-cycle related genes in several populations of circulating myeloid leukemic blast cells. The genes explored included oncogenes such as c-myc, c-myb, p53, and cell-cycle-related genes such as vimentin, calcyclin, ornithine decarboxylase (ODC) and histone H3. Particular attention was given to analysis of the relationship existing between the mRNA levels of the histone H3 gene, which is expressed specifically in the S phase of the cell cycle, and the levels of other genes that are expressed in different stages of the G1 phase. Remarkable differences were observed among the different cases indicating that a differential expression of cell-cycle-related genes characterizes many acute leukemias. This differential expression is reflected in an altered ratio among G1-related genes and the H3 histone gene. The large fraction of leukemic cells which does not express histone H3 and therefore is functionally noncycling, shows a heterogeneous pattern of G1-related gene expression. This reflects the inability of most leukemic cells to progress through the G1 phase into the S phase of the cell cycle. This inability represents an abnormality of the cell cycle. It is concluded that the study of the expression of cell-cycle genes and protooncogenes in in understanding how leukemic cells enter a state of proliferation arrest, which appears to occur in a large fraction of leukemic cells.

Heterogeneity in protein patterns of cells from terminal blast crisis in chronic granulocytic leukemia: discrimination between lymphatic and myeloid lineages.

Terminal blast crisis cells of chronic granulocytic leukemia are biochemically distinct. Triton-X-114 detergent phase lysates revealed that myeloid types express predominantly proteins of a 24 kd apparent molecular weight range, whereas lymphatic types do not express these molecules but a 55 kd protein band. These biochemical differences, observed in the poorly differentiated blast crisis cells, are also found in well differentiated hemopoietic malignancies such as chronic lymphatic leukemia, or in mature granulocytes isolated from healthy individuals. The results support the concept of the different lineages of blast crisis cells in chronic granulocytic leukemia but question the role of these proteins in cell differentiation. In addition, the presence or absence of these proteins provide a helpful tool for classifying blast crises of chronic granulocytic leukemia.

The 5q-- deletion: correlation of breakpoints with the immunophenotype of leukemic blast cells.

Cytogenetic and immunologic features of 13 patients with the 5q- deletion with the diagnosis of acute leukemia, either as the blastic transformation of a typical 5q- syndrome or myeloproliferative disorder or as a de novo presentation, were studied. Variable 5q- breakpoints were identified: The common interstitial deletion at q13q33 was found in nine cases, and a terminal deletion at q13, q22, q22, q31, respectively, was found in four cases, two of which had unbalanced translocations. A comparison of 5q- breakpoints with the blast cell immunophenotype indicated an association of myeloid and TdT+/myeloid leukemias with interstitial deletions and of monoblastic phenotypes with terminal deletions.

A mouse pluripotent embryonal stem cell line stage-specifically regulates expression of homeo-box containing DNA sequences during differentiation in vitro.

Mouse embryonal stem (ES) cells have been shown to provide a new model system suitable for the analysis of different aspects of murine development. This report gives evidence that ES cell lines are also most useful for the study of developmentally regulated gene expression in vitro. Homeo-box containing genes which are suggested to play a key role in the regulation of differentiation steps occurring during embryogenesis are stage-specifically transcribed in differentiating murine ES cells: (i) A mouse embryonal stem cell line (ES-12957) was isolated and characterized with respect to its differentiation potential. When injected subcutaneously into syngeneic mice, ES-12957 cells formed fully differentiated teratomas representing derivatives of all three germ layers. When allowed to grow in suspension cultures in vitro, the cells followed a reproducible developmental pathway forming complex organized 'embryoid bodies' which resembled mouse early postimplantation embryos. (ii) A mouse DNA sequence with homeo-box homology (MH-121) was isolated and structurally analyzed. Transcription of a 1.7 kb RNA species from this DNA sequence was demonstrated in ES-12957 cells which were differentiated in vitro. A second, previously described homeo-box gene (Mo-10) was also shown to be expressed in ES-12957 cells in a stage-specific manner. A 4-kb transcript could be identified exclusively in RNA of cells which were allowed to differentiate for 9 days. These findings support the suggestion that the homeo-box genes of mammals, like those of Drosophila, may have important functions during embryonic development.

Isolation of a teratocarcinoma stem cell lectin implicated in intercellular adhesion.

We have previously identified a cell surface teratocarcinoma stem cell lectin with a fucan/mannan specificity. We now report the purification of the hemagglutinin (lectin) from stem cell conditioned medium by exclusion on a Sepharose 2B column, followed by elution with 0.5M NaCl from DEAE-cellulose, providing an overall purification of about 90-fold. When this material was analyzed, by SDS-polyacrylamide gel electrophoresis, a major band of Mr 56000 was consistently observed. Hemagglutination activity was renatured from the gels and localized exclusively to a region of the gel that, as detected by fluorography, contains only the 56-kDa component. This suggested that this polypeptide comprises the lectin.

In situ staining of bromodeoxyuridine positive cells in normal and neoplastic colony-forming units grown on plasma clots.

Bromodeoxyuridine, an analogue of thymidine, can be detected by means of monoclonal antibodies and utilized as a marker of the S-phase of the cell cycle. In this paper a method for the detection of the labeling index of normal and neoplastic colony-forming units (CFU) growing in plasma clot semisolid medium is described and preliminary results on the cell cycle of 7th and 14th CFU granulocyte-macrophage are discussed.

A simple method for simultaneous detection of Fc receptors and differentiation antigens on normal and neoplastic murine hematopoietic cells.

Cell surface receptors for immunoglobulin Fc regions can be detected by light microscopy of cytocentrifuge preparations of normal or malignant murine hematopoietic cells. The cells are first incubated in a culture supernate containing a monoclonal anti-hapten antibody, then with heat-killed, haptenated bacteria. Using 2 morphologically distinct strains of bacteria, we can detect both Fc receptors and cell surface differentiation antigens.

Dysplastic eosinophils in three patients with acute promyelocytic leukaemia.

Three cases of acute promyelocytic leukaemia (M3) with dysplastic eosinophils are reported. Promyelocytes from these cases showed classical features of hypergranular acute promyelocytic leukaemia. All the cases had minimal organomegaly and disturbances of coagulation were minimal. All the cases were associated with dysplastic eosinophils in the marrow but very few of these eosinophils were found in peripheral smear. One of the three patients presented with a mass in the vertebral column, a feature not uncommonly seen in a variant form of AML-M2 with eosinophilia. Dysplastic eosinophils appear to arise from leukaemic clone itself. These three cases probably represent hitherto undescribed morphological variant of acute promyelocytic leukaemia where leukaemic cells show limited differentiation capability to dysplastic eosinophils.

Rearrangement of T-cell receptor and immunoglobulin heavy chain genes in childhood acute mixed lineage leukaemia.

Rearrangement of the beta and gamma chain genes of the TCR gene complex and of the Ig heavy chain genes were examined in three cases of childhood acute mixed lineage leukaemia. Blast cells, classified morphologically as acute lymphoblastic leukaemia (ALL) in one child and acute non-lymphocytic leukaemia (ANLL) in the other two, all co-expressed markers associated with both T (CD7, TdT) and myeloid (CD33) cells. Cytogenetic analysis detected abnormalities associated with myeloid leukaemia. Immunoglobulin heavy chain genes were not rearranged in two patients but a novel rearrangement was seen in the third. No rearrangement of the beta or gamma chains of the T-cell receptor complex were seen. Acute mixed lineage leukaemia may thus arise from a pluripotent precursor cell capable of both lymphoid and myeloid differentiation.

Erythromyeloid lineage fidelity is conserved in erythroleukaemia.

Blast cells from eight patients with erythroleukaemia and one with erythroid blast crisis of chronic myeloid leukaemia were studied for the co-expression of cell surface myeloid and erythroid markers, and the phenotype compared with that of erythroblasts from two patients with megaloblastic anaemia. The technique of dual indirect immunofluorescence was used with a panel of seven mouse monoclonal antibodies against well-defined myeloid antigens (CD11b, 13, 14, 15, 33 and HLA-DR) and a rat antibody, YTH89.1, specific for glycophorin A. No dual fluorescence, emanating from myeloid or erythroid lineage markers, was found to occur in either the neoplastic or non-neoplastic erythroid cells studied. These data support the hypothesis that lineage fidelity is conserved in leukaemia.

Preferential in-vitro growth and expansion of leukemic T lymphoblasts.

An in-vitro culture system was used to selectively grow malignant cells from the bone marrow of a patient with acute T-lymphoblastic leukemia. Molecular analysis of DNA extracted from the bone marrow cells before culture showed the presence of both rearranged and germ line patterns for the T-cell beta receptor (CTB) gene, and chromosomal analysis revealed the presence of a major and a minor abnormal clone. The cells were cultured in RPMI medium supplemented with 20% fetal calf serum, 2% lymphocyte conditioned medium, L-glutamine and antibiotics. The presence of malignant cells in the cultured population was confirmed by morphologic, molecular probing and cytogenetic analysis. After four weeks in culture, DNA extracted from the cultured cells showed only the rearranged pattern for the CTB gene. Chromosomal analysis of the same cultured sample revealed only the presence of the initially predominant abnormal clone. Shortly thereafter, analysis of fresh uncultured bone marrow cells from the patient in relapse revealed that the same chromosomally abnormal clone also predominated in vivo. Thus, our results demonstrate the selective nature of this culture system and its ability to amplify leukemic T-lymphoblasts. This culture system is also useful for detecting occult malignant cells in histologically normal bone marrow.

Detection of minimal residual disease in acute myelogenous leukemia by RNA-in situ hybridization.

Several proto-oncogenes have been reported to be expressed in normal and malignant hematopoietic cells. Since these studies have been done almost exclusively by Northern and dot-blot analyses using mixed populations of cells, any conclusions concerning quantitative changes in gene expression are difficult to document. We have developed a rapid and sensitive RNA-in situ hybridization technique permitting detection of as few as 5 copies of mRNA per individual cell. Using this technique we have studied the expression levels of several oncogenes including MYC, SIS, FMS, p53, FOS and RAF in both normal hematopoietic cells and bone marrow (BM) cells obtained from acute myelogenous leukemia (AML) patients at presentation, at relapse and in complete remission (CR). Two of these oncogenes, MYC and SIS, are expressed at levels at least 2-5-fold higher in hematopoietic cells obtained from leukemia patients than in any normal hematopoietic cell examined, including cells obtained from regenerating bone marrow. The proportion of abnormal cells correlated well with the percentage of blast cells determined by morphological examination. In 7 out of 10 AML patients in morphological remission, a subpopulation of cells is detectable with abnormally high levels of MYC and/or SIS mRNA. These high levels of MYC expression are similar to those found in BM cells obtained from AML patients at presentation or relapse, but the percentage of cells with this abnormality is generally much lower. Continued follow-up of these patients has shown that 5 of them relapsed within 8 months. At this time, none of the 3 patients which were negative for MYC overexpression has relapsed.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of glycoprotein-bound carbohydrates from pluripotent embryonal carcinoma cells by pokeweed agglutinin-agarose.

Embryoglycan is the high-molecular-weight poly-N-acetyllactosamine characteristically and abundantly present in early embryonic cells. Among lectins reacting with poly-N-acetyllactosamines pokeweed agglutinin (PWA) was found to be most useful in analyzing glycoproteins from HM-1 pluripotent embryonal carcinoma (EC) cells, since virtually all glycoproteins carrying embryoglycan in these cells could be isolated by affinity chromatography on PWA-agarose. Furthermore almost all of embryoglycan from HM-1 cells bound to PWA-agarose. Since PWA-agarose used for the present study was confirmed to bind branched but not linear poly-N-acetyllactosamines, the above result indicated that embryoglycan lacking branched poly-N-acetyllactosaminyl chain was scarcely present in these cells. The same approach was used as a mean to show that embryoglycan with Lotus tetragonolobus agglutinin receptor activity also usually has the branched poly-N-acetyllactosamine structure in EC cells. Glycoprotein fractions from PYS-2 parietal endoderm cells and from STO fibroblasts also bound to PWA-agarose. However, the ratio of PWA binding fraction to the total [14C]galactose-labeled glycoproteins was less in these cells as compared to the value in EC cells, and the poly-N-acetyllactosamines from these cells exhibited lower molecular weights than embryoglycan. These results are consistent with our proposal that the complexity and abundance of poly-N-acetyllactosamines distinguishes EC cells from most other cells.

Glycoprotein-bound large carbohydrates of early embryonic cells: structural characteristic of the glycan isolated from F9 embryonal carcinoma cells.

The high-molecular-weight glycopeptides characteristic of early embryonic cells were isolated from F9 embryonal carcinoma cells grown in vitro and also from the cells grown in vivo as subcutaneous tumors. The two preparations had similar carbohydrate compositions. The major components were galactose and N-acetylglucosamine (molar ratio 1:0.86) in the glycan isolated from the cultured cells. In addition, small amounts of fucose, N-acetylgalactosamine and mannose were present. The glycan from the in vitro grown cells was found to have a molecular weight of more than 10,000 by gel filtration after mild alkaline treatment or hydrazinolysis. The structural characteristics of the core portion of the glycan were studied by using the radioactively labeled glycopeptide from the in vitro grown cells. Methylation analysis provided the following informations. 1) The glycan was highly branched at galactosyl residues. 2) Large numbers of galactosyl residues were also present at non-reducing termini. 3) Monosubstitution of galactose occurred at C-3. 4) Glucosamine residues were mainly monosubstituted. That the disaccharide GlcNAc-Gal was the major structural unit of the glycan was suggested by the isolation of the deacetylated disaccharide after alkaline thiophenol cleavage followed by acid hydrolysis. Furthermore, methylation analysis of the glycan from the in vivo grown tumors indicated that monosubstitution of glucosamine occurred at C-4 and that disubstitution of galactose occurred at least mainly at C-3 and C-6. We propose that the basic structural unit of the core portion is 4GlcNAc 1 leads to 3Gal, and that the galactosyl residue serves as a branching point at C-6. Thus, the structural unit of the core portion of the large glycan appears to be the same as that of lactosaminoglycans found in adult cells.

High sensitivity and specificity assay for detection of leukemia/lymphoma cells in human bone marrow.

An assay of high sensitivity and specificity for detection of residual malignant cells in remission bone marrow from patients with acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL) has been developed. The assay combines an immunoselection step with immunoglobulin gene rearrangement analysis by high specific-activity DNA probes. In experimental conditions, less than one contaminating tumor cell out of 1000 bone marrow cells was detected. A bone marrow contamination was detected in morphologically negative bone marrow specimens from two intermediate-grade non-Hodgkin's lymphoma patients. This method can be of value in determining a true remission status in high-risk ALL and NHL patients.

Early somatostatinoma of the papilla of the duct of Santorini.

We studied a patient with a very small somatostatinoma that arose from the prominence of the orifice of the duct of Santorini. The patient presented clinically with epigastric discomfort, marked loss of weight, diarrhea, exertional dyspnea, and chest pain. He flushed intermittently and had occasional tachycardia and hypertension. Levels of serum serotonin and urinary 5-hydroxyindoleacetic acid were normal. A small ampullary tumor was resected and identified by immunohistochemical staining to be a somatostatinoma. The patient had gained 6.75 kg and was essentially free of symptoms 16 months after surgery.