Fetal antigens on the surface of human lymphoid cells.

Human B-lymphoblastoid cells established in long-term culture from healthy adults carry surface components that are normally found in human fetal tissues at about 10 wk of age. These antigens are strongly expressed on neoplastic B lymphocytes but not on thymocytes or a cultured T-cell line. They are carried by a small subpopulation of normal adult peripheral blood lymphocytes as well.

ME491 melanoma-associated glycoprotein family: antigenic identity of ME491, NKI/C-3, neuroglandular antigen (NGA), and CD63 proteins.

BACKGROUND: Numerous monoclonal antibodies (MAbs) have been produced to antigens found in human melanomas. Three of the best characterized melanoma antigens include the melanoma-associated glycoproteins (MAGs) defined by two reagent families--the ME491 family (including ME491, 8-1H, and 8-2A) and the NKI/C-3 family (including NKI/C-3 and NKI/black-13)--as well as the neuroglandular antigen (NGA) defined by MAbs LS59, LS62, and LS140. These three antigens have significant similarities in tissue distribution, biosynthesis, and structure. The ME491 MAG has been cloned, mapped, and sequenced. Numerous non-melanoma-associated proteins (Sm23, CO-029, R2, TAPA-1, CD9, CD37, CD53, and CD63) have recently been shown to have significant homology to this sequence. PURPOSE: We conducted this study to investigate the similarity between the two MAG antigens and NGA. METHODS: Several reagents defining the three different melanoma antigens were compared, using competition immunoprecipitation, immunoassay, and inhibition radioimmunoassay techniques. RESULTS: Immunoassay experiments show that MAbs defining the three melanoma antigens bind to affinity-purified ME491 antigen and inhibit each other from binding in an inhibition radioimmunoassay. Competition immunoprecipitation experiments demonstrate that the ME491 and NKI/C-3 antibodies bind to NGA. Rabbit anti-ME491 idiotype serum recognizes determinants shared by NKI/C-3 and the anti-NGA MAbs. A competition immunoprecipitation experiment also confirms the identity of CD63, as defined by MAb RUU-SP 2.28, with the three melanoma antigens. CONCLUSION: These data indicate that the MAGs defined by ME491 and NKI/C-3 as well as the anti-NGA antibodies are epitopes of the same molecule, which is identical to CD63 by both immunochemical and molecular genetic investigations. IMPLICATIONS: Our results indicate that the data obtained in studies of these three melanoma antigens may be pooled, and we propose that the molecule recognized by these reagents be classified as CD63.

Phenol-soluble nonhistone chromatin proteins in chronic lymphocytic leukemia.

The altered gene expression seen in cancer could relate to differences in nonhistone chromatin proteins between normal and malignant tumor cells. Phenol-soluble nonhistone chromatin proteins were isolated from human normal and leukemic (chronic lymphocytic leukemia) B-cells, as well as long-term cultured human B-lymphocyte cell lines. High-resolution two-dimensional electrophoretic maps identified a group of three nuclear proteins with a molecular weight of 45,000 to 50,000 and an isoelectric range of 4.5 to 4.7, which were associated only with the human leukemic B-cells. Leukemic B-cells and cultured B-cell lines also expressed a variant form of nuclear actin and tubulin.

Biosynthesis, glycosylation and intracellular processing of the neuroglandular antigen, a human melanoma-associated antigen.

Neuroglandular antigen (NGA) was identified as a human melanoma-associated antigen by a panel of murine monoclonal antibodies of both IgG2a (LS62, LS76, LS159) and IgG1 (LS113, LS140, LS152) subclasses, developed in this laboratory (L. Sikora, A. Pinto, D. Demetrick, W. Dixon, S. Urbanski, and L. M. Jerry, Int. J. Cancer, 39: 138-145, 1987). Monoclonal antibody LS62 was used to immunoprecipitate NGA from radiolabeled cultured melanoma cells, and it behaved as a heterogeneous glycoprotein "smear" on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (Mr 29,000-70,000). Radioactive pulse-chase time course experiments using human melanoma cells cultured in the presence or absence of inhibitors of protein glycosylation showed that the antigen consisted of a core protein with a molecular weight of 22,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This molecule was modified by the addition of at least three N-linked oligosaccharide side chains (as revealed by limited N-glycanase digestion) to give a precursor form with a molecular weight of approximately 34,000. Subsequent processing steps yielded a heterogeneous family of glycoproteins with varying amounts of covalently attached carbohydrate. Much of this heterogeneity in both molecular weight and pI (as revealed by two-dimensional electrophoresis) could be removed by treatment of the antigen with neuraminidase, suggesting heavy sialylation of the glycoprotein. NGA could be detected on the surface of melanoma cells by fluorescence-activated cell sorter analysis, surface radioiodination, and, as previously shown, immunoperoxidase staining. However, there was a larger intracellular pool of the molecule and the antigen was rapidly released into the culture supernatant. The function of NGA remains unknown but its elevated expression in transformed melanocytes have prompted this characterization to understand its biochemical nature and relation to other melanoma-associated antigens.

Fetal antigens in nonneoplastic conditions.

During studies that showed the presence of fetal antigens on the surface of human malignant melanoma tumor cells, polyvalent antisera specific for human fetal tissues of varying ages were developed. These reagents demonstrated varying patterns of expression of fetal antigens at different ages in various tissues of the human fetus. The possibility that nonneoplastic adult cells showing either maturation arrest or excessive proliferation also might express fetal antigens led to studies of human bone marrow. Although normal bone marrow cells expressed low levels of fetal antigens, large amounts were seen on bone marrow cells of patients with anemias due to iron, B12, or folic acid deficiencies, as well as on those with leukemia. Moreover, normal adult tissues adapted to long-term culture also expressed fetal antigens. After 3 weeks in organ culture adult human skin showed morphological changes similar to those seen in fetal periderm and strongly expressed fetal antigens. In addition, lymphoblasts in long-term cultured human lymphoid cell lines established from normal donors also carried surface fetal antigens. These latter antigens were shared with neoplastic B-cells (chronic lymphocytic leukemia) but not with T-cells. Their expression varied with the cell cycle. The reexpression of fetal antigens on malignant cells is thought to signal a basic derangement in the control of differentiation which is considered to be peculiar to neoplasia. However, these studies indicate that normal adult cells also may reexpress fetal antigens under circumstances unrelated to neoplasia but associated with either maturation arrest or rapid and excessive proliferation.

Production and characterization of tumor infiltrating lymphocyte clones derived from B16-F10 murine melanoma.

The adoptive transfer of tumor infiltrating lymphocytes (TIL) in conjunction with recombinant interleukin-2 (rIL-2) for the treatment of advanced cancer has recently been under intense investigation. Despite extensive research, the precise surface phenotype of TIL remains to be fully defined. To elucidate this unsolved problem, we established 11 TIL clones derived from rIL-2 expanded TIL obtained from B16-F10 murine melanoma tumors. These clones could be divided phenotypically into four groups: CD8 (+) T-cell clones, natural killer (NK)-cell clones, NK-like CD8 (+) T-cell clones, and double negative T-cell clones. Functionally, CD8 (+) T-cell clones demonstrated specific cytotoxic activity against B16-F10 melanoma cells, whereas NK-cell clones and double negative T-cell clones demonstrated only non-specific cytotoxic activity against NK-sensitive YAC-1 cells. NK-like CD8 (+) T-cell clones showed dual cytotoxic activity. Clones T1 [a CD8 (+) T-cell clone] and T2 [an NK-like CD8 (+) T-cell clone] which had cytotoxic activity against B16-F10 melanoma cells, demonstrated a proliferative response against immunoblotted B16-F10 melanoma antigens, whereas clones T7 (an NK-cell clone) and T10 (a double negative T-cell clone), which had no cytotoxic activity against B16-F10 cells, demonstrated no proliferative response against them. Winn assays revealed that only the CD8 (+) T-cell clone (T1) had an antitumor effect in vivo, whereas the double negative T-cell clone (T10) and NK-like CD8 (+) T-cell clone (T2) stimulated tumor growth in vivo. Adoptive immunotherapy using tumor-specific, highly cytotoxic TIL clones may represent a useful future immunotherapeutic option for the treatment of human tumors.

Characterization of a human melanosome-associated antigen recognized by monoclonal antibody, HMSA-2.

This study elucidates the nature of antigens recognized by monoclonal antibody (MoAb) HMSA-2, which was developed against human melanosome-associated antigen (HMSA) of malignant melanoma (Maeda and Jimbow, Cancer 59:415-423, 1987). Through flow cytometry analyses, indirect immunoprecipitation of antigen biosynthetically labeled with 35S-methionine, enzyme-linked immunosorbent assays and immunoelectron microscopy, we found that a) the antigens recognized by MoAb HMSA-2 were melanosomal matrix glycoproteins; b) these antigens were expressed mainly in the cytoplasm, although they could also be detected on the cell surface; c) the cytoplasmic expression of MoAb HMSA-2 was cell-cycle dependent; d) large amounts of these antigens were released into culture supernatants; e) MoAb HMSA-2 immunoprecipitated two major glycoproteins with molecular weights of 94 and 53 kDa from culture supernatants, and f) both components have complex N-linked oligosaccharide chains with sialic acid, suggesting that these melanosomal proteins are derived from the trans-cisternae of the Golgi. These human melanosome-associated antigens may prove useful not only for studying the immunobiology of melanogenesis, but also for the immunodiagnosis of melanocytic disorders.

Melanocytic differentiation of human neuroblastoma: expression of a human melanosome-associated antigen.

Five human neuroblastoma cell lines were examined for expression of a human melanosome-associated antigen (HMSA). Only cell line SK-N-SH reacted with a monoclonal antibody, HMSA-2, shown to recognize melanosomal glycoproteins. To further characterize the melanocytic lineages of SK-N-SH, three morphologically distinct clones designated SK-N-SH-N (neuroblast type), SK-N-SH-F (fibroblast type), and SK-N-SH-EP (epithelial type) were established by colony formation cloning. By fluorescence-activated cell sorter analysis and tyrosinase assay, we found that only SK-N-SH-EP and SK-N-SH-F reacted with HMSA-2 and had tyrosinase activity. These results suggest that epithelial-type and fibroblast-type cells appear to possess the melanocytic potential, but not neuroblast-type cells. Furthermore, SK-N-SH-EP was found to spontaneously convert to neuroblast-type or fibroblast-type cells, whereas SK-N-SH-N and SK-N-SH-F clones have remained morphologically stable. Our results suggest that at least one neuroblastoma cell line, SK-N-SH, may be an excellent model for investigating clonal maturation and the melanocytic differentiation of neuroblastoma.

Induced morphological changes in human small cell lung carcinoma cells.

Several theories suggest that lung carcinomas are not totally separate entities, but are derived from a common precursor, probably of endodermal origin. The histological classification of lung cancers is complex, with much overlap between groups broadly designated as small cell (SCLC), squamous cell, adenocarcinoma and all others simply termed non-small cell. It is shown here that in vitro exposure of classic, non-adherent SCLC lines to 10 microM 5' bromodeoxyuridine (BrdU) results in a rapid cell-line dependent change to a morphology consistent with an adherent, non-small cell phenotype. Accompanying this morphological shift is a decreased expression of the amplified N-myc protooncogene. These induced changes underline the morphological relatedness of lung carcinoma cell lines.

Spindle-cell epithelial thymoma. Fine-structural and tumor lymphocyte observations.

A fine-structural study of a spindle-cell epithelial thymoma in a patient with pemphigus and autoimmune hemolytic anemia is presented and compared with the few previously described. Because light-microscopic features suggested hemangiopericytoma, critical fine-structural comparisons between spindle-cell epithelial thymoma and hemangiopericytoma are detailed. Based upon groups of tonofilaments with desmosomal insertions, abundant well-formed desmosomes, negligible numbers of pinocytotic vesicles, and an absence of myofilaments and dense bodies, an epithelial origin for this tumor is proposed. Langerhans' cell granules, a new observation in thymoma, were found in cells of probably histiocytic origin. Tumor lymphocyte studies revealed that more than 95% of cells formed E rosettes, 36% formed EAC rosettes, yet none contained surface immunoglobulin. The significance of these observations is discussed.

Malignant melanoma (stage 1): a clinical trial of adjuvant BCG immunotherapy.

A prospectively controlled randomized clinical trial of adjuvant BCG immunotherapy in patients with stage 1B malignant melanoma (Clark's level 3--5) is described. The combination of intradermal and oral BCG allows approximation of the bacilli to any microscopic foci of residual disease. The trial was activated in May 1975 and to date 107 patients have been admitted to the trial, 49 patients being randomil entered patients, there have been five relapses of 49 patients in the treatment group and ten relapses of 58 patients in the control group. This encouraging trend is not yet statsitically significant at the 5% level (P = 0.17). If evaluable patients with Clark's level 3 and 4 lesions are assessed separately, there is a significant trend in favour of the immunotherapy group (P less than 0.05) with three relapses in the treatment group and ten relapses in the control group.

Influence of level and depth on recurrence rate in thin melanomas.

A population-based study of the biology of the thin-level melanoma according to site, Breslow's thickness, and Clark's level was undertaken. Two hundred fifteen patients were studied with a mean follow-up of 41 months. Overall, 23 patients (10.7%) had recurrences, 8 locally, 9 regionally, and 6 systemically, despite an adequate local excision. A multivariate analysis was done. In the patients with thin lesions (less than 1 mm), increasing level (p < 0.002) and head and neck site (p < 0.04) increased the risk of recurrence. Increasing thickness of melanoma up to 1 mm did not influence the risk. This study identifies a group of high-risk melanoma patients for whom adjuvant therapy to decrease recurrences should be studied.

Zinc, carotene, and retinol in melanoma and non-melanoma skin cancer.

Serum zinc, beta-carotene, retinol, retinol binding protein and prealbumin were measured in 22 cutaneous melanoma patients at diagnosis and in 17 patients with 1 or more basal cell carcinomas of the skin recently removed. The indices measured were found to distinguish melanoma patients from non-melanoma patients with a high level of accuracy. Moreover, the predictability of zinc depends on the level of beta-carotene, and this dependence differs between melanoma and non-melanoma patients.

Childhood melanoma.

In melanoma patients, the prognostic value of tumor depth, Clark's level, the presence of ulceration, and regional involvement have not been clearly documented in the pediatric population. This report correlates these factors in a population-based study of patients under the age of 20 years. Of the initial 35 melanoma patients registered in southern Alberta with the Alberta Cancer Board, 14 were found on review to have a diagnosis other than melanoma. In the remaining 21 cases the diagnosis of melanoma was confirmed. There was a suggestion that patients with deeper lesions had a worse prognosis, but this was statistically confirmed only using Clark's levels. The children were then compared with all melanoma patients diagnosed in southern Alberta over the same time period. There was no difference in tumor depth, Clark's level, ulceration, regional involvement, or survival between these two groups. The natural history in children appears to be similar to that of the adult population, contrary to previous reports suggesting a markedly worse prognosis.

Ocular melanoma: a population-based study.

During the 10-year period ending December 1976 ocular malignant melanoma developed in 99 patients in Alberta. To investigate the natural history of this disease we reviewed certain clinical and epidemiologic features of these cases. Of all the melanomas during that time 16% occurred in the eye, and of all the ocular malignant diseases 70% were malignant melanomas. The more malignant mixed cell tumours were much more frequent in the women than in the men, while the converse was true of the less malignant spindle cell melanomas. Within each cell type the women survived longer than the men. The actuarial 5-year survival rate of the entire group was 62%. Metastases occurred in 29 of the 99 patients; the liver was the only or initial site in 22 (76%). Our study shows that there has been no improvement in the survival rate of patients with ocular melanoma over the past 10 years. Our therapeutic methods must be improved.

The lymphocyte plasma membrane: locus of control in the immune response.

The lymphocyte plasma membrane is the locus of events which control the immune response. T and B lymphocytes, which mediate cellular and humoral immunity respectively, show distinctive plasma membrane morphologies and cell surface receptors. The dynamic state of these plasma components is emphasized by their lateral mobility in the fluid plane of the membrane, as well as variation in their structure or expression as the lymphocyte proliferates and differentiates in response to stimulation by antigen or mitogens. The best understood membrane glycoproteins are surface membrane immunoglobulins that serve as antigen receptors on B cells, and the histocompatability-beta2 microglobulin complex that has an immunoglobulin-like structure. Other less well defined surface structures showing modulation during the cell cycle may affect growth regulation of proliferating lymphocytes. Some of these are shared by fetal and neoplastic cells. Major theories of lymphocyte signaling are discussed, and the early events in lymphocyte activation are reviewed. While a complete model encompassing all these early events is not yet possible, the central issues can be usefully discussed in term of receptor-transducer-effector concepts derived by strong parallels from a knowledge of hormone-membrane interactions.