Because of the broad clinical interest which tissue polypeptide antigen (TPA) has attracted as a tumor marker, human cell lines and human tissues have been analyzed for TPA expression using immunofluorescence microscopy. Epithelial cell lines including HeLa, MCF‐7, and A‐431 are recognized by TPA antibodies whereas human lines of non‐epithelial origin are not. The positive staining patterns coincide with keratin‐type intermediate filaments of the cytoskeleton. On tissue sections a subset of epithelial cells including uterine epithelium, bile duct cells in liver and tumor cells in breast carcinoma are strongly positive; cells of the squamous epithelia of skin and tongue as well as cells of non‐epithelial origin are negative. In immunoblots of human epidermis, human tongue mucosa, human hair follicles, Detroit 562 cells, HeLa cells, MCF‐7 and RT‐4 cells, only keratins 8, 18 and 19 show TPA antigenicity. Conversely a TPA preparation is recognized by various antibodies known to react with keratins, including alpha‐IFA, KG 8.13.2 and two antibodies which recognize keratins 18 (CK2) and 19, respectively. Our results thus relate TPA to human keratins 8, 18 and 19 which are known cytoskeletal components in both normal and malignant epithelial cells of simple and non‐squamous origin. We speculate that the elevated levels of circulating TPA antigenicity present in the sera of patients with carcinoma, which are often used to monitor tumor progression, correspond to soluble proteolytic fragments originating from this particular keratin subgroup.
The serological results from apparently healthy individuals and prostate cancer patients were evaluated with a new assay called TPAcyk ELISA. This assay has a biochemical specificity for fragments of cytokeratins 8 and 18, and exhibits a low within- and between-assay imprecision. The data indicate a significant difference between the results of males and females, but no significant age-dependent relation was found. The cut-off value (95% specificity) for healthy individuals was estimated to be 1.27 ng/mL (n = 190) for males and 0.95 ng/mL (n = 81) for females. When using a cut-off value of 1.27 ng/mL, we found a sensitivity for prostate cancer patients with T2-3 N0M0 of about 20%. For patients with metastatic disease, a sensitivity of 75% was found. A higher sensitivity was obtained with patient sera analyzed with PSA than with TPAcyk, particularly in patients with early stages of the disease. We conclude that the results from this new TPAcyk assay were significantly elevated in patients initially diagnosed with poorly differentiated tumors, that patients with localized tumors exhibited low concentrations, and that patients with metastatic disease showed, on average, 8 times higher concentrations than patients with localized disease. The combination of the TPAcyk and PSA results increased the sensitivity for prostate cancer, particularly in patients with metastatic disease.
In the first report of the TD5 workshop (TD5-1), the epitope specificities of 30 different monoclonal antibodies against cytokeratins 8, 18 and 19 were determined. This second report presents the immunohistochemical profiles of these antibodies using human appendix and normal skin for evaluation. Each antibody was tested by one or two different laboratories recruited from the Dutch Working Group on Immunohistochemistry and Cytochemistry. Eight different laboratories participated. The histological specimens were pretreated by the participants in three different ways for immunohistochemistry: microwave antigen retrieval in citrate buffer, enzymatic digestion to restore epitope exposure, no specific treatment (untreated paraffin-embedded samples), and tested blindly without knowledge of cytokeratin or epitope specificity of the antibodies at three different concentrations of 50, 10 and 1 μg/ml. Most of the tested antibodies (29/30) were useful in at least one pretreatment method, with microwave antigen retrieval being the most sensitive approach. For some antibodies, very high backgrounds were observed. Furthermore, it can be concluded that 11 MAbs performed well using all three staining protocols, including untreated paraffin-embedded sections. Interestingly, all the antibodies with documented selected specificity towards cytokeratin 8 (i.e. 178, 191, 199, 202 and 206) are reactive with an immunodominant region corresponding to amino acids 340–365 on cytokeratin 8, which evidently is well-suited as target for immunohistochemical interactions. Similarly, three antibodies with the same capacity to react with untreated samples had specificity against cytokeratin 19 (i.e. 179, 197 and 204) in the corresponding region in this filament, i.e. amino acids 311–335, or the KS 19.1 epitope. None of the six antibodies against the other major cytokeratin 19 epitope (BM 19.21) were found useful for immunohistochemistry on untreated samples. The overall conclusions from the present investigation are that all cytokeratin-8-specific antibodies with defined epitope specificities were very useful. Only one of the major two epitopes on cytokeratin 19 seems to be available for efficient immunohistochemistry. Cytokeratin 18 exposes some epitopes outside the immunodominant region reactive with the antibodies 190, 203 and 205 which can be used for untreated samples. The implications of these findings are of significance both for diagnostic histopathology and for the biology of tumor marker epitope expression in tissues.
We studied immunohistochemical stains for TPA and CA125 in patients with benign and malignant gynecologic diseases. The results were as follows: (1) CA125 was not found in ovarian mucinous cystadenocarcinoma but was demonstrated immunohistochemically in 82% of ovarian serous cystadenocarcinomas and 83% of Krukenberg's tumors. (2) TPA was demonstrated in 65% of ovarian serous and 75% of ovarian mucinous cystadenocarcinomas, and in 58% of endometrial carcinomas. (3) TPA was found in all trophoblastic tumors examined, while CA125 was found in none. Eighty-three percent of patients with trophoblastic diseases had raised serum TPA levels. (4) When serum CA125 levels were raised CA125 was demonstrated immunohistochemically in 71% of patients with ovarian serous cystadenocarcinomas, 67% of patients with Krukenberg's tumors and 100% of patients with tubal carcinomas. (5) Despite elevated serum levels, CA125 and TPA were not identified by immunohistochemistry in 64% cases of benign ovarian disease and in 80% of patients with uterine myomata. (6) It would seem that CA125 was more easily released from tumor cells than TPA.
The epitope specificities of 30 monoclonal antibodies (MAbs) against the most common human cytokeratins, i.e., Nos. 8, 18, and 19, in epithelial cells were investigated in the ISOBM TD-5 Workshop. Seven research groups from universities or companies participated independently in the evaluation of the antibody specificities. The complex assembly of cytokeratins in vivo, with obligatory heterologous dimeric combinations of different cytokeratins from each of the two major groups, comprising together more than 20 different individual cytokeratins, made analysis of the antibody reactivity patterns with isolated single cytokeratins necessary. The concordance of the evaluations was striking and independent of the technologies used. As antigens purified individual cytokeratins, chemically degraded purified cytokeratins, recombinant intact and truncated cytokeratins, as well as specific synthesized shorter peptides were used. In order to elucidate the epitope specificity, reactivity patterns in ELISA assays and immunoblots with partial enzymatic degradation of the antigens were performed. Competitive cross-inhibition experiments between antibodies using antigens and antibodies in all possible combinations were performed with radioimmunometric assays, BIAcore, and ELISA technology.All 30 antibodies could convincingly be classified with regard to target cytokeratin. One MAb (192) had to be deleted due to dual specificities in both isotype and epitope specificity against its target. Six antibodies bound selectively to cytokeratin 8, 14 to cytokeratin 18, and 10 to cytokeratin 19, as demonstrated by using native, recombinant, and synthesized antigens. The immunodominant part of the molecule for all three types of cytokeratins was located in the region of amino acid (aa) 270–400. Out of the six MAbs reactive with cytokeratin 8, four MAbs, i.e., 178, 199, 202, and 206, were reactive with a sequence in the interval aa 340–365, and MAb 191 reacted with a closely related epitope. The remaining antibody, 192, presented dual specificities. At least two closely related major immunogenic epitopes could be identified in cytokeratin 8. In cytokeratin 18 four distinct epitopes could be documented, again with the dominating sequence region 270–429 as target for 10 (181, 184, 186, 188, 189, 190, 193, 196, 198, and 200) out of 14 antibodies. Since MAb 193 is known to react with the M3 epitope, aa 322–342 in cytokeratin 18, this entire group is reactive in the region close to the charge shift, in the middle of the rod 2B region, as shown by competitive binding. The remaining four anticytokeratin 18 antibodies (180, 185, 203, and 205) displayed unique, noncompetitive binding to this filament. Cytokeratin 19, reactive with altogether ten antibodies, displayed two major epitopes, all of them also within the large immunodominant region. MAbs 179, 195, 197, and 204 were reactive with the peptides aa 311–335 also known as the KS 19.1 epitope, and MAbs 182, 183, 187, 194, and 201 bound to peptide aa 346–367, known as the BM 19.21 epitope. One ant...
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