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Long term in vitro cultures of six human malignant gliomas were established to obtain permanent lines and to assess, under conditions of prolonged culture, changes in morphology and phenotype of neoplastic cells and the extent of these modifications. We analyzed expression of the following markers by immunocytochemistry: glioma-specific antigens (GE2 and CG12), fibronectin, intermediate filaments (GFAP, vimentin, neurofilaments), class I and II histocompatibility antigens (HLA-ABC and HLA-DR), growth factor and receptor (alpha TGF and EGF-receptor), proliferation-associated antigen (Ki-67). Strong and stable staining with the two antiglioma monoclonal antibodies (GE2 and CG12) was seen, with coexpression of GFAP and fibronectin in five of six cell lines (after 20 passages) and presence of vimentin and neurofilaments. HLA-DR expression was heterogeneous, with a peculiar intracellular compartmentation in four of six cell lines. Cells showed clear cytoplasmic positivity for alpha TGF and strong membrane staining for EGF-receptor. In previous studies we showed that these cell lines have increased copies of chromosome 7; therefore we speculate that an autocrine pathway of stimulation may maintain the neoplastic growth. The percentage of Ki-67 positive proliferating cells ranged from 40 to greater than 60%, depending on cell line and passage. A slight decrease in the positivity of some markers (GFAP, vimentin and HLA-DR in 2/6 cell lines) was observed after prolonged in vitro culture (greater than 12 months), but morphophenotypic modifications, established within a few passages after explanation, were maintained with time. A clonogenic assay showed values of plating efficiency (PE) higher than corresponding values of other similar cell lines with a tendency to increase in the late passages. PE and Ki-67 positivity were not associated with tumorigenicity into nude mice (except the Hu 197 cell line). These results indicate that, in culture, all six cell lines acquired stable morphology, a well defined antigenic phenotype and high growth rate. Further studies will be performed on these permanent cell lines to clarify differentiation steps of malignant gliomas.

The sera of 212 patients with malignant and non-malignant diseases have been radioimmunoassayed for the presence of the carcinoembryonic antigen (CEA) using 3 different kits produced of Hoffman La Roche, Switzerland (RCK), BY Sorin-IRE, Italy and Belgium (SCK), and by the Istituto Sieroterapico Milanese, Italy (ICK). In the presence of endodermically-derived system carcinomas, the RCK gave more positive results (72.6%) than did the SCK (63.1%) or ICK (56.2%). With regard to other carcinomas, ICK (50.0%) and SCK (47.1%) gave better results than did RCK (30.6%). The results are discussed in terms of clinical usefulness of the CEA assay and as regards reproducibility, procedural advantages, and economical cost of each kit. It is concluded that the CEA assay cannot be used for the diagnosis of gastrointestinal cancers, although it is useful as a measure of "cancerosity" for prognostic purposes. In this sense the double antibody method employed by SCK and ICK is clinically more advantageous than is the perchloric acid extraction-zirconyl phosphate gel precipitation method of RCK.

A human melanoma cell line (HU 214) with high growth potential was established from a lymph node metastasis of a patient with advanced cutaneous melanoma. The cells of this line were able to grow in monolayer (according to the Rosenblum technique) and in agar (according to the Courtenay-Mills method), and formed tumors when injected in nude mice. The line has been maintained in culture for more than 47 passages. The cell cultures were periodically characterized (every 6-8 passages) by immunohistochemistry using a panel of monoclonal antibodies (MoAbs) including MoAbs against tumor-associated antigens (antimelanoma, antiglioma and anti-LLA), against vimentin, and against major histocompatibility antigens, and including also Ki 67, a MoAb which reacts with a nuclear antigen associated with cell proliferation. The results of this characterization indicate that we have established a human melanoma cell line with a stable antigenic phenotype during subculturing, poorly differentiated cells, and a high growth potential.