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.

Clinical features and biological implications of acute mixed lineage (hybrid) leukemias.

The composite phenotype of a population of leukemic blast cells is derived through analysis of morphology, cytochemistry, cytogenetics, surface antigens, and gene structure. When analyzed in such a fashion, approximately 10-25% of childhood acute leukemias will show markers of more than one lineage; these may be coexpressed on individual cells (biphenotypic) or appear on two distinct blast populations (bilineal or biclonal). Occasionally, there is conversion from one leukemic phenotype at diagnosis to another phenotype at relapse (lineage shift). Mixed lineage features appear to have biologic and prognostic significance. Some specific mixed lineage leukemia syndromes have been identified; among them are acute nonlymphoid leukemia with T-lymphoid features, CD7+, CD4-, CD8- acute leukemia, CD2+/CD19+ acute lymphoid leukemia, and acute leukemias associated with specific cytogenetic markers, e.g., t(4;11) and t(9;22). In general, these forms of acute leukemia along with others with mixed lineage markers have a poor prognosis, and new therapeutic approaches appear to be indicated.

Changes in clonal growth, immunophenotype, and morphology during a follow-up study of an acute lymphoblastic leukemia.

Cells of a 21-year-old patient with acute lymphatic leukemia were analyzed for morphology and immunophenotype and for genotype consecutively during the course of disease. Initial therapy with the BMFT-ALL protocol (Bundesministerium für Forschung und Technologie) reduced leukemic cells only marginally. The following high-dose Ara-C, mitoxantrone (HAM) chemotherapy led to a cell reduction of 75% and to a drastic change in cell morphology from initially 90% blasts to mainly small lymphoid cells. Immunophenotype, which showed 90% CD7-positive cells in the beginning with a prevalence of helper (60%) over suppressor cells (15%) remained fairly constant until the onset of HAM chemotherapy, which led to a sharp fall and a subsequent slow increase in all T-cell markers. In contrast to pretherapeutic findings, CD7 was now only expressed on the small cells and not on blast cells. Southern blot analysis of the T-cell receptor configuration revealed an initially monoclonal population with rearranged T beta gene. A new band appearing during the clinically ineffective therapy was indicative for development of a second small population which did, however, not emerge in immunophenotype analysis. This second population was eliminated by the HAM chemotherapy, leaving back the initial clone responsible for the final fatal outcome. No activity of the multidrug resistance gene could be detected by Northern blotting.

Expression of c-myb in embryonal carcinoma cells and embryonal stem cells.

Mouse c-myb has been implicated in the regulation of differentiation and proliferation of haematopoietic cells. Analysis of the chromatin structure of the promoter region of c-myb in embryonal carcinoma (EC) cells and embryonal stem (ES) cells reveals a DNAse I-hypersensitive site coincident with a site found in c-myb-expressing haematopoietic cells, but absent in murine fibroblasts (which do not express c-myb). EC and ES cells were found to express c-myb mRNA, albeit at a level lower than found in haematopoietic cells. Differentiation of ES cells into embryoid bodies resulted in an elevated level of c-myb expression.

T-cell receptor delta/alpha rearrangements in lymphoid neoplasms.

Rearrangements within the T-cell receptor (TCR)delta/alpha locus were analyzed in a wide variety of lymphoid neoplasms by eight DNA probes specific for TCR J delta, J alpha and C alpha segments. In all 11 T-cell malignancies, rearrangement and/or deletion of TCR delta was detected irrespective of the stage of maturation of the tumor. The organization of TCR delta correlated with the phenotype of the tumor: In "prethymic" T-cell acute lymphocytic leukemia (ALL), TCR delta was the only TCR gene to be rearranged. More mature T cell malignancies expressing CD4 together with CD3 showed deletion of both alleles of TCR delta, suggestive of TCR V alpha-J alpha rearrangement. All 43 B-cell tumors expressing surface immunoglobulin (sIg), including two cases of adult B-cell ALL, had germline configuration of TCR delta/alpha. In contrast, all 17 B-cell precursor ALLs (null, common, and pre-B-cell ALLs) had rearrangement and/or deletion of TCR delta/alpha. A single case of "histiocytic" lymphoma also showed biallelic deletion of TCR delta. Oligoclonal rearrangements of Ig and TCR genes were observed in two cases of B-cell precursor ALL and in one case of T-cell lymphoblastic lymphoma. Patterns of such "aberrant" TCR rearrangement were similar to those observed in T-lineage malignancies. In particular, seven of eight cases of B-cell precursor ALL and the histiocytic lymphoma which demonstrated biallelic TCR delta deletion, (suggestive of a V alpha-J alpha rearrangement) had clonal TCR beta rearrangement. These data support the hypothesis that supposedly aberrant rearrangements of the TCR genes may follow the same developmental controls as found in T-cell differentiation, despite the lack of evidence for further commitment to the T-cell lineage. TCR delta rearrangement is a useful marker of clonality of immature T-cell tumors which may have only this gene rearranged but is not specific to the T-cell lineage.

Prethymic phenotype and genotype of pre-T (CD7+/ER-)-cell leukemia and its clinical significance within adult acute lymphoblastic leukemia.

Pretreatment blast cells from 739 adults with acute lymphoblastic leukemia (ALL) were immunophenotyped as part of a prospective treatment protocol study. Among 192 patients (26%) with T lineage ALL, 47 (6%; 24% of T lineage ALL) had lymphoblasts without sheep erythrocyte rosette formation, but with pan-T antigen CD7 on the membrane and intracellular CD3 proteins mostly in perinuclear accumulation. The T-cell surface antigens CD5 and/or CD2 and focal acid phosphatase were additional markers of this subgroup traditionally called pre-T ALL, whereas thymocyte antigen CD1 as well as CD4 and CD8 antigens were not expressed. Hematopoietic progenitor cell markers, namely terminal deoxynucleotidyl transferase (TdT), and in part common ALL antigen (CD10), HLA-DR antigens, and/or My-10 (CD34), a unique antigen of marrow cells absent in thymus cells, further characterized this immature T-ALL form of putative prothymocytic phenotype (CD7+/intracellular CD3+/TdT+/My-10+/-/HLA-DR+/-/CD10+/-). The prethymic T cell character was supported by germ-line T-cell receptor beta genes found in 21 of 36 patients analyzed. In five cases only T gamma-chain genes were rearranged. Fifteen patients, however, had rearrangements of both T beta and T gamma genes. Immunoglobulin heavy chain genes were rearranged only in two cases. Pre-T ALL differed significantly from E-rosette+ T-ALL in some presenting clinical features, namely mediastinal mass, lymphoadenopathy, and platelet count, and independently of clinical factors in prognosis (P = .02, median remission duration: 15.7 v 33.5 months, and P = .02, median survival time: 24.6 v 50.7 months). We conclude that ALL classification based solely on T- or B-cell lineage affiliation is not sufficient but needs further subdivision according to relevant maturation stages as exemplified here within the T-cell axis. The putative prethymic T cell progenitor phenotype described might help elucidate the sequence of genetic events that commit normal hematopoietic cells to the T-cell lineage.

Comparative biochemical and cytogenetic studies of childhood acute lymphoblastic leukemia with the Philadelphia chromosome and other 22q 11 variants.

We studied the relationship of direct karyotypes, determined at diagnosis and remission, to Abelson-related tyrosine kinase activity and the cytogenetic features of erythroid and myeloid colonies derived from remission marrow of six children with acute lymphoblastic leukemia (ALL). These patients had either the characteristic Philadelphia chromosome (Ph1) [t(9;22)(q34;q11)] or cytogenetically similar variants with a 22q11 breakpoint but no detectable cytogenetic involvement of 9q34. The findings suggested two distinct subtypes of ALL: one defined by t(9;22)(q34;q11) and expression of P185BCR-ABL tyrosine kinase and one with variant karyotypes and no P185BCR-ABL expression. The former comprises cases with Ph1 + marrow cells and Ph1 + erythroid and (or) myeloid colonies in remission marrow and others in which the t(9;22) is undetectable in remission marrow cells. In the latter subgroup, the disease may reflect more extreme mosaicism with a similar stem cell that is cytogenetically undetectable. Variant karyotypes included a del(22)(q11) in one patient and a t(6;22;15;9) (q21;q11;q?22;q21) in another; in both instances, the malignant blast cells lacked P185BCR-ABL expression. Thus ALL with t(9;22)(q34;q11) should be distinguished from ALL with other involvement of the 22q11 breakpoint by molecular studies including protein expression. The diversity of karyotypic findings in cases with involvement of 22q11 suggests at least two mechanisms of leukemogenesis in patients with ALL defined by this breakpoint.

Relevance of ultrastructural immunocytochemistry in the characterization of unclassifiable leukemias: correlation with phenotypic and genic studies.

Nine cases of acute leukemia presenting unusual phenotype were studied by light microscopy (LM) cytochemistry and transmission electron microscopy (TEM) immunocytochemistry with the immunogold staining (IGS) method; in addition, cytogenetic and molecular analyses were performed. The presence of myeloperoxidase (MPO) was studied at TEM in combination with immunophenotype to identify minor populations not characterizable at LM. Four of nine cases had no TEM/MPO reactivity, whereas the remaining five showed variable percentages of positive cells. Of the MPO negative cases, one was a megakaryoblastic leukemia with a positive platelet peroxidase (PPO) reaction, and three were lymphoid. Among the peroxidase positive cases, the percentage of MPO reactive cells was higher at TEM than at LM examination. In case 5 TEM analysis indicated that cells with some MPO reactivity at LM were non neoplastic myeloid cells. With this combined technique in cases 1 and 2 we excluded the presence of the MPO enzyme in CD15 positive lymphoid cells and, in another case, we documented the existence of CD19/MPO positive cells. The value of cytochemistry and immunology at the ultrastructural level for the characterization of blast cells and for the precise diagnosis of leukemia with "unusual" phenotype is illustrated.

Murine embryonal carcinoma cell-surface sialyl LeX is present on a novel glycoprotein and on high-molecular-weight lactosaminoglycan.

Carbohydrate-specific monoclonal antibodies were used to demonstrate the expression of a new membrane glycoprotein on F9 murine embryonal carcinoma cells. Sialyl LeX was detected using monoclonal antibody FH6 in a sensitive, cell monolayer radioimmunoassay. The antigen codistributed in gel filtration of a crude homogenate and in a membrane-enriched fraction with two known lactosaminoglycan markers, i and SSEA-1 (LeX or X hapten). Sialyl LeX was further shown to be carried by a novel glycoprotein, termed small lactosaminoglycan-like glycoprotein (sLAG) which could be purified by immunoaffinity chromatography. In two-dimensional polyacrylamide gel electrophoresis this glycoprotein had an apparent molecular weight of 45 kDa and a pI of about 6.5. The more differentiated cell line PYS-2 also expressed sialyl LeX and i antigens but not LeX, and FH6-reactive sLAG could be extracted from PYS-2 membranes. Sialylation of fucosylated type 2 carbohydrate chains (X haptens) thus may be an early modification of embryonic carbohydrate antigens.

Differential timing of nuclear lamin A/C expression in the various organs of the mouse embryo and the young animal: a developmental study.

In mouse embryos, acquisition of the nuclear lamin polypeptides A/C varies according to developmental stage and tissue type. In order to determine the precise time points and cell types in which lamin A/C are first observed, we have used two monoclonal antibodies in immunofluorescence studies of different tissues of developing mouse embryos and of young mice. One antibody (mAB346) is specific for lamins A and C, while the other (PKB8) detects lamins A, B and C. Dividing uterine development into three phases--germ layer formation, organogenesis and tissue differentiation--our results show that lamin A/C expression in the embryo proper is not observed until the third phase of development. Lamin A/C first appears at embryonic day 12 in muscle cells of the trunk, head and the appendages. Three days later it is also seen in cells of the epidermis where its appearance coincides with the time of stratification. In the simple epithelial of lung, liver, kidney and intestine, as well as in heart and brain, lamins A/C do not appear until well after birth. Embryonal carcinoma (EC) cells express lamin B but not lamin A/C. Lamin A/C expression is noted in some EC cells after they are induced to differentiate and in several differentiated teratocarcinoma cell lines. Our results suggest that commitment of a cell to a particular pathway of differentiation (assayed by cell-type-specific expression of intermediate filament proteins) usually occurs prior to the time that lamin A/C can be detected. Thus lamin A/C expression may serve as a limit on the plasticity of cells for further developmental events.

Hematogones: a multiparameter analysis of bone marrow precursor cells.

Morphologically distinct lymphoid cells with homogeneous, condensed chromatin and scant cytoplasm can be observed in large numbers in the bone marrow of children with a variety of hematologic and nonhematologic disorders. In some patients, these cells may account for greater than 50% of the bone marrow cells, creating a picture that can be confused with acute lymphoblastic leukemia (ALL) or metastatic tumor. Although originally called hematogones (HGs), a variety of other names have been proposed for these unique cells. The clinical significance of expanded HGs has not been resolved, and the biologic features of these cells are incompletely described. In this study, we correlate the clinical, morphologic, cytochemical, flow cytometric, molecular, and cytogenetic properties of bone marrow samples from 12 children with substantial numbers of HGs (range 8% to 55% of bone marrow cells). Diagnoses in these patients included anemia, four; neutropenia, one; anemia and neutropenia, one; idiopathic thrombocytopenic purpura, two; retinoblastoma, two; Ewing's sarcoma, one; and germ cell tumor, one. Flow cytometric analyses of bone marrow cells demonstrated a spectrum extending from early B-cell precursors (CD10+, CD19+, TdT+, HLA-Dr+) to mature surface immunoglobulin-bearing B cells in these patients, corroborating our morphologic impression of HGs, intermediate forms, and mature lymphocytes. DNA content was normal, and no clonal abnormality was identified by either cytogenetic or immunoglobulin and T-cell receptor (TCR) gene rearrangement studies. Follow-up ranged from 3 months to 3 years. None of the patients has developed acute leukemia or bone marrow involvement by solid tumor. The possible role of HGs in immune recovery and hematopoiesis is presented.

The protein composition of the nuclear matrix of murine P19 embryonal carcinoma cells is differentiation-stage dependent.

The protein composition of the nuclear matrix of murine P19 embryonal carcinoma (EC) cells was compared with that of clonal derivatives of P19 EC differentiated in vitro, and with that of P19 EC cells induced to differentiate with retinoic acid (RA). Several major differences in nuclear matrix protein composition were found between the cell lines tested. Some polypeptides were found to occur only in EC cells, whereas others proved to be restricted to one or more of the differentiated derivatives. During RA treatment of EC cells a transient expression of some matrix proteins was observed. Several new proteins appeared, and others disappeared. Our data indicate that the protein composition of the nuclear matrix is a sensitive gauge for the differentiation state of cells.

Acute promyelocytic leukemia with T-cell markers and particular response to treatment. Report of a case.

A case of acute leukemia with atypical malignant cells is reported. The clinical picture and coagulation studies were consistent with a disseminated intravascular coagulation syndrome. Morphologically, the leukemic cells from the peripheral blood and bone marrow showed azurophilic granules. More than 80% of cells were hypergranulated, resembling the macrogranular type of promyelocytes. Ultrastructural studies and the pattern of endogenous peroxidase were consistent with the microgranular type of promyelocytes in about 20% of the leukemic cells. Auer bodies were present in both types of atypical promyelocytes. Cytochemically, the whole malignant population exhibited intense peroxidase activity. Studies with monoclonal antibodies showed that about 45% of the proliferating cells expressed T-cell markers T3, T4, T8 and T11, but the cells were not reactive with OKM1 monoclonal antibodies. The chemotherapy for acute promyelocytic leukemia was inefficient, and the prompt disappearance of the blood abnormalities was observed only when chemotherapy for acute lymphoblastic leukemia was started. Therefore, it seems that in some cases of leukemia with hybrid types of malignant cells the morphological features determine the clinical picture, while the patient's response to the therapy is conditioned mainly by the cell surface phenotype.

The use of clone-specific anti-VJ junction oligonucleotides to identify minor clonal lymphoid populations.

The polymerase chain reaction was used to amplify rearrangements of T-cell receptor gamma genes from 2 leukemic DNA samples involving the same variable segment. One V-J junction was sequenced and an anti-junctional oligonucleotide synthesized. This oligonucleotide was used as probe on in vitro amplified DNA. We show that this strategy allows the detection of the corresponding clonal DNA at dilutions of up to 10(6) fold in germline DNA. Moreover we demonstrate that it is possible to differentiate this clone from the second leukemic clone and from polyclonal T-cells.

Immunologic analysis of a thousand cases of acute leukemia. French Groupe d'Etude Immunologique des Leucémies (GEIL).

The French Groupe d'Etude Immunologique des Leucémies (GEIL), now in its fifth year of existence, has collected extensive immunological information on more than 1,000 leukemia patients. The rationale of such multicentric group is to improve immuno-phenotyping and investigate rare phenotypes, with relevance to the clinical outcome of the patients. These aims are described and exemplified through 3 of the first studies performed.