Eighteen mouse monoclonal antibodies were selected for reactivity with cell surface antigens of the immunizing human melanoma cell line SK-MEL-28. Six distinct antigenic systems were defined by direct serological assays and absorption tests with a panel of 41 cell lines derived from normal and malignant human tissues. Biochemical analysis indicated that two of the antigens are glycoproteins with molecular sizes of 95,000 and 150,000 daltons (gp95 and gplS0). Two other antigenic systems (05 and the R24 group) are associated with heat-stable molecules having the characteristics of glycolipids.The remaining two antigens (M19 and R%) are heat labile, but molecular characterization has not been possible. Each of the antigenic systems has a distinctive pattern of distribution on various cell types, varying from a broad representation to a more restricted occurrence. Os appears to be a species antigen, being present on virtually every human cell type tested. gp95, gplS0, M19, and R8 are found on a characteristic proportion of melanomas, astrocytomas, and epithelial cancers and on normal kidney cells. The antigen defined by the R24 antibody has the most restricted distribution of all. Reactivity is found with melanomas and astrocytomas, whereas epithelial cell types, fibroblasts, and cells of hematopoietic origin lack R24. Although occurrence of gp95, gpl50, M19, and B8 distinguishes a small subset of melanomas not expressing these antigens, R24 is found on all melanoma cells. Cloning of Hybridomas and Passage in Mice. After cell colonies 1-2 mm in diameter formed, samples of supernatants were harvested and assayed for antibody by using PA-MHA tests against an initial screening panel of 10 human cell lines-5 melanoma cell lines (SK-MEL-13, -27, -28, -31, -37) and 5 nonmelanoma cell lines (SK-RC-6 and -7, ME-180, EBV-B cells, and sldn fibroblasts). Cells from wells containing antibody were passaged at low cell density (two to five cells per well) and, if antibody continued to be detected, the cells were cloned (one cell per well) two consecutive times. Cultures of cloned hybridomas were injected subcutaneously into nu/nu mice (Swiss background) and also were stored in liquid nitrogen. Sera from mice with progressively growing tumors were collected, stored at either -20'C or -70'C, and used for serological and biochemical analysis. Sera were obtained from mice prior to tumor inoculation, and only mice with low levels of preexisting natural antibody against human cells were used.Immunoprecipitation Procedures. SK-MEL-28 cells were labeled as follows: (i) surface labeling with 125I by the lactoperoxidase procedure (4), (ii) metabolic incorporation of[a5S]methionine (Amersham, 1000 Ci/mmol; 1 Ci = 3.7 X 101" becquerels) by using 250 MCi in 10 ml of methionine-free minimal essential medium containing 1% fetal bovine serum for 16 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely...
We have previously described (1) a mouse IgG3 monoclonal antibody (AbR24) with a high degree of serological specificity for cultured human melanoma cells. All melanoma cell lines and two astrocytomas examined were positive for the heat-stable cell surface antigen detected by this antibody. Although choroidal melanocytes and brain had low levels of the antigen, a wide variety of other cells and tissues were unreactive. Three other monoclonal antibodies (Abs C5, I24, and K9), having a similar restricted specificity, were derived from the same fusion. These antibodies showed the same strong reactivity with melanomas and lack of reactivity with epithelial cells, but had a slightly wider specificity range in that they also reacted weakly with MOLT-4 (a T cell line), leukocytes, and some fetal tissues.In this communication, we identify the antigen detected by AbR24 as GD3, a previously characterized disialoganglioside.
Defects of DNA mismatch repair (MMR) cause the high level microsatellite instability (MSI-H) phenotype. MSI-H cancers may develop either sporadically or in the context of the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome that is caused by germline mutations of MMR genes. In colorectal cancer (CRC), MSI-H is characterized by a dense lymphocytic infiltration, reflecting a high immunogenicity of these cancers. As a consequence of immunoselection, MSI-H CRCs frequently display a loss of human leukocyte antigen (HLA) class I antigen presentation caused by mutations of the b2-microglobulin (b2m) gene. To examine the implications of b2m mutations during MSI-H colorectal tumor development, we analyzed the prevalence of b2m mutations in MSI-H colorectal adenomas (n 5 38) and carcinomas (n 5 104) of different stages. Mutations were observed in 6/38 (15.8%) MSI-H adenomas and 29/104 (27.9%) MSI-H CRCs. A higher frequency of b2m mutations was observed in MSI-H CRC patients with germline mutations of MMR genes MLH1 or MSH2 (36.4%) compared with patients without germline mutations (15.4%). The high frequency of b2m mutations in HNPCC-associated MSI-H CRCs is in line with the hypothesis that immunoselection may be particularly pronounced in HNPCC patients with inherited predisposition to develop MSI-H cancers. b2m mutations were positively related to stage in tumors without distant metastases (UICC I-III), suggesting that loss of b2m expression may promote local progression of colorectal MSI-H tumors. However, no b2m mutations were observed in metastasized CRCs (UICC stage IV, p 5 0.04). These results suggest that functional b2m may be necessary for distant metastasis formation in CRC patients. ' 2007 Wiley-Liss, Inc.
DNA mismatch repair deficiency is observed in about 10% to 15% of all colorectal carcinomas and in up to 90% of hereditary nonpolyposis colorectal cancer (HNPCC) patients. Tumors with mismatch repair defects acquire mutations in short repetitive DNA sequences, a phenomenon termed high-level microsatellite instability (MSI-H). The diagnosis of MSI-H in colon cancer is of increasing relevance, because MSI-H is an independent prognostic factor in colorectal cancer, seems to influence the efficacy of adjuvant chemotherapy, and is the most important molecular screening tool to identify HNPCC patients. To make MSI typing feasible for the routine pathology laboratory, highly reproducible and cost effective laboratory tests are required. Here, we describe a novel T 25 mononucleotide marker in the 3Vuntranslated region of the CASP2 gene (CAT25) that displayed a quasimonomorphic repeat pattern in normal tissue of 200 unrelated individuals of Caucasian origin. In addition, CAT25 was monomorphic also in all tested donors of African and Asian origin (n = 102 and n = 79, respectively) and thus differs from the most commonly used markers BAT25 and BAT26. Without the analysis of corresponding normal tissue, CAT25 correctly detected 56 of 57 colorectal cancer specimens classified as MSI-H by using the standard National Cancer Institute/International Collaborative Group-HNPCC marker panel. Combined with the standard markers BAT25 and BAT26 in a multiplex PCR, all MSI-H colorectal cancer samples were typed correctly. No falsepositive results were obtained in 60 non-MSI-H control colorectal cancer specimens. These data suggest that CAT25 should be included into novel marker panels for microsatellite testing thus allowing for a significant reduction of the complexity and costs of MSI typing. Moreover, CAT25 represents a highly promising marker for early detection of colorectal cancer in HNPCC germ line mutation carriers. (Cancer Res 2005; 65(18): 8072-8)
Background:High-level microsatellite instability (MSI-H) has been reported as a prognostic marker in colon cancer. We here analysed the prognostic significance of MSI and mutations of the Beta2-Microglobulin (B2M) gene, which occur in about 30% of MSI-H colon cancer, in the cohort of the prospective FOGT-4 (Forschungsruppe Onkologie Gastrointestinale Tumoren, FOGT) trial.Methods:Microsatellite instability status was determined using standard protocols (NCI/ICG-HNPCC panel and CAT25) in 223 colon cancer lesions. Beta2-Microglobulin mutation status was evaluated by exon-wise sequencing in all MSI-H lesions.Results:Patients with MSI-H (n=34) colon cancer presented with a significantly lower risk of relapse after 12 months of follow-up compared with MSS (n=189) colon cancer patients (5 year time to relapse: MSI-H 0.82 vs MSS 0.66, P=0.03). No significant difference in overall survival was detected. Beta2-Microglobulin mutations were identified in 10 (29.4%) out of 34 MSI-H colon cancers and were associated with a complete absence of disease relapse or tumour-related death events (P=0.09).Conclusion:The risk of late disease relapse was significantly lower in patients with MSI-H compared with MSS colon cancer. Moreover, B2M mutations may contribute to the favourable outcome of MSI-H colon cancer patients and should therefore be evaluated as a potential prognostic marker in future clinical trials.
The expression of intercellular adhesion molecule-I (ICAM-1, CD54)
Proliferation of cytotoxic T‐cells, a prerequisite for the development of antitumor vaccines, was induced by 1, but not by its partial structures A and B. The conjugate 1 containing a tumor‐associated Sialyl–TN–MUC‐1 glycopeptide antigen A and a T‐cell epitope B of tetanus toxin was synthesized by fragment condensation on a solid phase.
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