An indirect immunofluorescence technique was used to study the tissue distribution of the epitope recognized by the monoclonal antibody 140.240 which identifies a p97-like melanoma-associated oncofetal antigen. Cryostat sections of various normal and neoplastic human tissues were examined. The presence of antigenic activity was demonstrated in 20 of 39 (51%) primary skin melanomas, in 21 of 52 (40%) metastatic melanomas, and in 20 of 44 (45%) nevi. The reactive nevi were restricted to intradermal, junctional, compound and spindle cell types. Of the 110 samples of 12 major tumor types other than melanoma tested, only 1 of 6 epidermoid tumors, 1 of 4 benign breast tumors and 1 of 5 prostatic tumors gave weak staining. This antibody also reacted with sweat glands and fetal small intestine tissue, but not with other adult or fetal normal tissues. Intrapatient as well as interpatient heterogeneity in the epitope expression was present in primary as well as metastatic tumor lesions surgically removed from patients with melanoma. Evaluation of the immunohistologic data and the clinical outcome of patients with melanoma reveals that the expression of the epitope recognized by this antibody is associated with a more favorable prognosis.

The monocolnal antibody 140.240, which reacts with a melanoma‐associated oncofetal antigen, identifies an epitope present on an 87 kd molecule present on the surface of melanoma cells. This molecule is a singlechain monomer, which by tunicamycin treatment and two‐dimensional tryptic mapping has been shown to arise from a 77 kd polypeptide precursor (p77). This precursor is rapidly glycosylated to an 83 kd intermediate (gp83) which in turn is rapidly further glycosylated into the mature 87 kd glycopolypeptie (gp87). Neuraminidase treatment of the glycopeptides indicates that only gp87 contains accessible sialic acid moieties.

By fusion of mouse NS1 myeloma cells with splenocytes from a BALB/c mouse immunized with human melanoma cells, an IgG1 monoclonal antibody, designated as 140.72, was produced. By the mixed hemadsorption antibody binding assay, 140.72 was shown to react with 17 of 20 melanoma cell lines and with 5 of 14 carcinoma cell lines. This antibody also reacted with 3 of 3 normal melanocyte cultures in much lower titers. It did not react with any of 35 other normal and malignant lines, including neuroblastoma, glioblastoma, sarcoma, teratoma, fibroblast, and lymphoid cell lines. Absorption with fresh melanoma and carcinoma homogenates confirmed the results of direct tests. Fetal reactivity of antibody 140.72 was determined by positive absorption with 10 of 11 tissue homogenates derived from different fetuses of 10-16 weeks' gestation. The reactivity of this antibody was completely removed by absorption with a highly purified preparation of carcinoembryonic antigen (CEA) derived from a colon carcinoma. The antigenic activity was detected in the culture medium of reactive cell lines. Immunoprecipitation analyses of melanoma and carcinoma cells indicated that the antigenic determinant recognized by antibody 140.72 is on a glycoprotein with an apparent molecular weight of 95,000-150,000 common to both serologically reactive cell types. Additionally, a 200,000-molecular-weight glycoprotein corresponding to the CEA molecule was detected only on the reactive carcinoma cells. These data confirmed previous findings obtained with polyclonal anti-CEA antisera for the existence of shared CEA-related antigenic determinants on human carcinomas and melanomas and provided additional molecular characterization of these glycoproteins. Further characterization of the molecules bearing the antigenic determinant recognized by antibody 140.72 should be performed with a view to exploring its potential in the immunodiagnosis and immunotherapy of patients with melanoma.

The HCT-8R clone of the HCT-8 human colon tumor line, which expresses increased quantities of carcinoembryonic antigen (CEA) on its surface, was discovered to have an enhanced susceptibility to lysis by natural killer (NK) cells in human peripheral blood. This increase in susceptibility to lysis by peripheral blood mononuclear cells was not explained by stimulation of interferon release by HCT-8R cells but rather was found to be attributable to an increased susceptibility of HCT-8R cells to lysis by those NK cells that bind to sheep erythrocytes (E-RFC). Cold target competition experiments and single-cell assay for cytotoxic cells suggested that the presence of surface CEA did not increase lysis of HCT-8R by facilitating "recognition" by E-RFC-type cytotoxic cells but by rendering HCT-8R cells more susceptible to the lytic mechanism of NK cells. The magnitude of expression of surface CEA by a variety of human carcinoma cell lines with a few exceptions and subclones of HCT-8 also correlated with increased susceptibility to lysis by blood mononuclear cells. The possible clinical significance of these findings was discussed.

Raynaud's phenomenon is a rare condition in the pediatric age group. Because of this, there is little information on the outcome of these patients. We have followed three patients in our clinic for extended period of time, and all remain well clinically despite the presence of im munologic abnormalities in two. This suggests that, while careful follow up is necessary, the presence of Raynaud's phenomenon and immunologic abnormalities in the pediatric age group are not necessarily of bad prognostic significance.

Human and animal melanomas undergo maturation spontaneously in vivo and in vitro, and as a result of experimental manipulation in vitro. To gain a better understanding of this phenomenon, we studied the effects of theophylline on a cultured human malignant melanoma cell line (CaCL 73-36). We observed a dose-dependent inhibition of cell growth with a reduction in plating efficiency of 16, 64, and 99% at concentrations of theophylline of 0.1, 1.0, and 2.0 mM, respectively. Theophylline-treated cells showed morphological changes consistent with a more differentiated state such as increased dendrite formation and contact inhibition. Expression of surface HLA-A,B,C antigens and beta-2-microglobulin was enhanced 15- and fivefold, respectively. Finally, cells treated with theophylline for 96 h showed a five- to eightfold increase in sensitivity to lysis by natural killer cells. These findings have obvious bearing on the potential use of theophylline in the treatment of malignant melanoma.

Two human malignant melanoma cell lines, differing in their patterns of HLA-DR antigen expression, were examined for changes in antigen expression following theophylline treatment. In one line, the basal HLA-DR antigen content of which remained constant during culture, theophylline decreased HLA-DR expression, accompanied by morphological changes indicating increased differentiation. In the second line, the surface HLA-DR antigen expression decreased with time during culture and showed no decrease in antigen expression or morphological changes when cultured in the presence of theophylline.

While patients with melanoma are known to produce antibodies against melanoma cells, the tumor specificity of these reactions has not been well documented. Using the sensitive mixed hemadsorption assay we have identified antibody against one or more of nine different cultured melanoma cell lines in only nine of 48 patients with melanoma. Reactivity against melanoma cell lines was seen only in females, 9/27 versus 0/21 males. The strongest melanoma reactivity was seen in sera which also contained lymphocytotoxic antibody. The reactivity was not melanoma specific because it could be removed by absorption either with pooled platelets, nonmelanoma tumor cells or in two cases, by both. We conclude that the occurrence of specific antimelanoma antibodies against common or shared surface melanoma associated antigens is an uncommon event in melanoma patients not receiving specific active immunotherapy. The clinical significance of the observed reactivity and whether it is directed against cancer associated determinants, fetal antigens, or normal tissue or histocompatibility antigens requires further study.

A microassay is described to determine immunoglobulin Ig class or IgG subclass of mouse monoclonal antibodies by mixed hemadsorption assay. Monoclonal antibody bound to adherent target cells is reacted with serial dilutions of a panel of class or subclass specific rabbit anti-mouse Ig antisera and binding of the latter is traced by anti-rabbit globulin-coated indicator erythrocytes. The class or subclass of the bound monoclonal antibody is revealed by preferential binding of the corresponding rabbit antibody. Unlike gel immunodiffusion analysis, the mixed hemadsorption assay may be performed with unconcentrated hybridoma culture supernatants.

The hybridoma system has been utilized to produce antibodies to characterize the cell surface antigens on human melanoma cells. On initial screening, two antibodies derived by the fusion of mouse myeloma cells (SP2/0‐Ag14) and splenocytes from a mouse immunized with a melanoma cell line (CaCL 78‐1) showed cross‐reactivity with 10 melanoma cell lines and did not react with any of 4 epithelial cancer lines, or 4 normal adult fibroblast lines. However, because of reactivity with 2 neuroblastoma cell lines, additional testing with other neuroectodermal derivatives was carried out and revealed a broad cross‐reactivity among melanomas, neuroblastomas, retinoblastomas and glioblastomas and against antigens shared by fetal but not adult brain. Thus, these results indicate the existence of common neuroectodermal antigens on melanoma cells. Before melanoma specificity can be claimed for, an antibody reactivity with nonmelanoma cells bearing these neuroectodermal antigen should be excluded.

The mixed hemadsorption assay is a sensitive method for the detection of antibodies to cell surface antigens as well as to soluble antigens. Miniaturization of this assay through the use of microplates with 12 μl rather than 400 μl wells allows a 5-fold reduction in volume of test serum and equivalent reduction in target antigen and other reagents used in the indicator system. Using 5 different antigen systems, melanoma-associated antigens, carcinoembryonic antigen, HLA antigen, β-2 microglobulin and fetal calf serum, a 4–8-fold increase in sensitivity is realized. The geometry of the 12 μl wells results in no significant detachment of target cells which may account for the improved sensitivity and quality of the titration curves.

Mycoplasma contamination of established cell lines is a well-known but often poorly controlled artefactual problem in immunological studies of human tumor cell lines. We have evaluated four methods for detecting mycoplasmas in cell lines, namely direct culture, DNA staining, uridine phosphorylase assay, and a fourth technique based on our finding that the supernatant medium of mycoplasma-infected cell cultures inhibits thymidine uptake of mitogen-stimulated peripheral blood lymphocytes. In our hands the simplest, most reliable, and least expensive means of monitoring cell cultures for mycoplasma proved to be DNA staining. The uridine phosphorylase assay was unsuitable for use with melanoma cell lines, as six of eight lines that were negative with the other three techniques were positive with this assay.

Antisera highly specific for carcinoembryonic antigen (CEA) from New Zealand White rabbits and a goat reacted strongly in antibody binding tests with cultured tumor cell lines, irrespective of the ability of the cell lines to produce CEA. The most reactive were colon carcinoma and melanoma cell lines, the former known to produce CEA and the latter not associated with CEA production. The reactivity was not diminished by absorption with perchloric acid extracts of normal lung or spleen, whereas absoprtion with purified CEA preparations abolished the reactivity. Quantitative absorption studies indicated that reactivity against CEA-producing cell lines could be totally removed by absorption with other CEA-producing lines but not with melanoma cell lines. Reactivity against melanoma cell lines could be completely removed by colon carcinoma cells as well as by melanoma cells. Antisera raised against purified CEA, after absorption with extracts of normal lung, still contained two populations of antibodies, one that binds a newly described antigen cross-reacting with CEA which is present on melanoma cells.

The effect of human leukocyte interferon (IFN) on the in vitro growth and expression of melanoma-associated antigens (MAA). beta 2-microglobulin (beta 2m) and HLA-DR antigen on cultured human melanoma cells was studied. Exposure of melanoma cells to IFN for 64 hours resulted in a dose-dependent inhibition of growth with 46% reduction in cell number at 10(3) U IFN/ml and 74% reduction at 10(5) U/ml. Quantitative absorption experiments in the mixed hemadsorption assay determined that the expression of MAA and beta 2m on treated cells was enhanced at 10(2)-10(5) U IFN/ml, twofold to fivefold for MAA and fivefold to twelvefold for beta 2m. No change was seen in HLA-DR antigen expression. The IFN-induced enhancement of MAA and beta 2m could be detected as early as after 16 hours and a maximum expression was reached at 96 hours after IFN exposure. The IFN-induced enhancement of MAA and beta 2m on melanoma cells was reversible. Studies with melanoma cells grown in stationary phase and serum-deprived conditions indicated that IFN-induced augmentation of MAA and beta 2m did not require cell proliferation. The data suggest that the effect of IFN on antigen expression is independent of its effect on cell growth. Further studies are needed to fully elucidate the mechanism.

The presence of immune complexes and carcinoembryonic antigen (CEA) was investigated in 50 patients with bronchogenic carcinoma at the time of and/or following diagnostic or definitive surgery. Immune complexes were measured by the C1q binding test and CEA by the Z gel method and elevations defined as values in excess of 2 S.D. above the normal mean, greater than or equal to 9.2% for C1q binding activity (C1q-BA) and greater than or equal to 5.0 ng/ml for CEA. The overall incidence of elevated values was 30.7% for C1q-BA and 34.2% for CEA. There was a greater incidence of elevated values of C1q-BA among patients with clinically evident disease. The differences with respect to CEA elevation were not significant due to the fact that 6 of 9 samples with elevated CEA values obtained from patients with no evident disease were in fact associated with the presence of clinically undetectable disease in these patients. Elevation of C1q-BA and CEA beyond the immediate postoperative period was predictive of a significantly shorter median survival time. The most significant differences in survival time were seen between patients with normal values for C1q-BA and CEA and those with elevations of one or both parameters, 6.0 vs. 19.5 months (p less than 0.001). Elevation of either parameter during the immediate pre- and postoperative period was not predictive of a poor survival. In terms of clinical application, it appears that CEA estimation had the best predictive value but that the addition of C1q-BA measurement may provide additional prognostic information, particularly in patients who do not have elevated CEA values.