Definition of the expression of the human carcinoembryonic antigen and non-specific cross-reacting antigen in human breast and lung carcinomas

Mouse cell lines transfected with carcinoembryonic antigen (CEA) and with 2 other members of the human CEA gene family, non-specific cross-reacting antigen (NCA) and biliary glycoprotein (BGP), were used to analyze the specificity of several monoclonal antibodies (MAbs). MAbs COL-1 and COL-6 were shown to react with the transfected CEA gene product but not with NCA, confirming previous results. Cells expressing the transfected BGP gene product also failed to react with COL-1 and COL-6. The MAb B6.2 reacted with cells expressing the NCA gene product but not with those expressing CEA or BGP. The MAb B1.1 reacted strongly with the transfected CEA and BGP gene products but only weakly with the NCA gene product. These antibodies were then utilized in the histochemical analysis of a number of primary and secondary breast and lung tumors. The results indicate that a majority of breast and lung tumors express CEA, and nearly all breast and lung tumors express NCA. Fairly homogeneous expression of CEA and NCA was seen in the majority of both breast and lung tumors. Our results indicate that CEA may be an important target for immunotherapy in a large number of patients with breast and lung tumors.     

Nonspecific crossreacting antigen (NCA) is a major member of the carcinoembryonic antigen (CEA)-related gene family expressed in lung cancer.

Carcinoembryonic antigen (CEA) is one of the most important tumour markers in the management of human carcinoma, including lung cancer. So far, however, because of the nonspecificity of anti-CEA antibodies, it remains unclear whether the experimental measurements of CEA expression really reflect genuine CEA. In normal lung, nonspecific cross reacting antigen (NCA) has been described as a major component of CEA-related antigens. Recently isolated CEA and NCA cDNA clones enabled us to analyse CEA and NCA expression of in vivo tumour specimens and tumour cell lines at mRNA levels. NCA-specific mRNA (but not CEA-specific mRNA) was detected in all normal lung tissues examined. Of 21 lung cancer tissue specimens, nine expressed both NCA and CEA and five expressed only NCA. Of 16 tumour cell lines, two expressed only NCA and one expressed both NCA and CEA, although its level of CEA mRNA was weaker than that of NCA mRNA. Therefore, CEA-related mRNA expression was always accompanied by NCA mRNA expression; there were no cases of CEA mRNA expression alone. These findings suggest that NCA is a major member of the CEA-related gene family expressed in lung cancer.     

Expression of carcinoembryonic antigen (CEA) and nonspecific crossreacting antigen (NCA) in gastrointestinal cancer; the correlation with degree of differentiation.

In spite of its reputation as a tumour marker, little is known about the function of carcinoembryonic antigen (CEA). We examined the mRNA expression of CEA and NCA in 26 gastric and 14 colorectal cancers together with adjacent morphologically normal mucosae. There was no significant difference between the CEA mRNA levels of colorectal cancer and adjacent mucosa, whereas the CEA mRNA levels were significantly elevated in gastric cancer compared with normal gastric mucosa. The expression of NCA, on the other hand, was more cancer-specific and was significantly up-regulated in both gastric and colorectal cancers compared with the corresponding normal mucosae. No correlation was found between the mRNA level and plasma CEA value. No significant up-regulation was recognised in the node positive cancer, cancer with distant metastasis, or the metastatic tissues themselves. Marked diversity in the degree of differentiation in gastric cancer tissues, however, resulted in varied expression of the CEA gene family, compared with the constantly high expression found in colorectal cancer. Further analysis revealed significant up-regulation of NCA in well and moderately differentiated gastric cancers over poorly differentiated cancers, suggesting that NCA might have roles in differentiation.     

Expression of neuroendocrine cell markers L-dopa decarboxylase, chromogranin A, and dense core granules in human tumors of endocrine and nonendocrine origin

We evaluated the usefulness of L-dopa decarboxylase (DDC) as a tumor marker of neuroendocrine (NE) cell differentiation by measuring its expression in 432 human tumors of diverse types and origins. A subset of these tumors and cell lines derived from them also were studied for expression of two other general NE cell markers, chromogranin A (CgA) and dense core granules (DCG). High concentrations of DDC were present in 96 of 117 (82%) tumors recognized to be of NE or neural origin. As expected, endocrine tumors not recognized to be of NE cell origin, as well as leukemias, lymphomas, sarcomas, melanomas, and germ cell tumors, lacked DDC expression. Of interest, modest concentrations of DDC were present in 46 of 220 (21%) nonendocrine carcinomas, especially non-small cell lung and colorectal carcinomas. We studied concordant expression of the three NE cell markers in lung and colorectal tumors and cell lines. In both tumor types there was nearly 100% concordance between CgA and DCG expression. There was an excellent correlation between DDC and CgA expression in lung cancers, both small cell and non-small cell, but DDC positive colorectal carcinomas usually lacked CgA expression. We conclude: (a) DDC is an excellent cellular marker for tumors of the NE cell system; (b) about 20% of carcinomas not of NE cell origin, especially non-small cell lung and colorectal carcinomas, express DDC, suggesting a common endodermal origin of all of the respiratory and gastrointestinal mucosal cells; and (c) CgA and DCG are expressed concordantly, indicating that CgA expression may be used as a substitute for ultrastructural examination of tumors for DCG expression.     

Production kinetics and immunochemical properties of carcinoembryonic antigen and nonspecific cross-reacting antigen synthesized by various human tumor cell lines

The production kinetics and immunochemical properties of carcinoembryonic antigen (CEA) and nonspecific cross-reacting antigen (NCA) in various human tumor cell lines were studied. By radioimmunoassay (RIA), five CEA-producing tumor cell lines tested--2 derived from colonic (M7609 and CCK-81), one from pancreatic (QGP-1) and 2 from lung (HLC-1 and KNS-62) carcinomas--were found to produce NCA simultaneously. The cellular contents of CEA and NCA and the amounts of both antigens released into the culture medium were highly variable among the cell lines. It was a distinct contrast that one cell line (CCK-81) released very large amounts of CEA and NCA into the medium while having the smallest amounts of both antigens in the cells, whereas the others contained much larger amounts of the antigens in the cells as compared with the amounts released into the medium. For most of the cell lines, the production of both CEA and NCA increased in the stationary phase of growth as compared with the exponential phase. The production kinetics of both CEA and NCA appeared to be parallel with each other in all the cell lines, though the amount ratio of CEA to NCA produced was variable. By means of a double immunodiffusion test with polyclonal antibodies, antigenic uniformity with no unique organ-specificity was confirmed for all the CEA preparations from spent media of the cell lines, though some differences in the sugar moiety of CEA were detected by RIA using monoclonal antibodies. No antigenic differences among NCA preparations were observed. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), molecular heterogeneity was observed among CEA or NCA preparations isolated from cell lysates.     

Decreased expression of biliary glycoprotein in hepatocellular carcinomas

Biliary glycoprotein (BGP) is an adhesion and anti-cell-growth molecule of the carcinoembryonic antigen family. We have earlier demonstrated that BGP mRNA is expressed in hepatocellular carcinomas (HCCs) and the adjacent non-cancerous regions, neither of which express CEA and NCA mRNA. To define an expression level and pattern of BGP at the protein level in HCCs, TS135, a monoclonal antibody (MAb) against BGP, was prepared. This MAb clearly reacted with BGP with a molecular weight of 110 kDa and 85 kDa (BGP-110/85). It cross-reacted weakly with NCA-90 from NCA transfectants, but not at all with CEA-200 from the serum of a colon-cancer patient. The BGP transfectants of cultured hepatocellular carcinoma cHc-4 cells showed Ca²⁻-dependent cell aggregation, which was partially inhibited by modulating BGP on the cell surface with MAb TS135. Immunostaining of non-cancerous liver tissues with MAb TS135 indicated that BGP could be expressed in the bile canalicular domain of hepatocytes. In HCCs, the expression of BGP was predominantly found in the well-differentiated type, where the bile canaliculi and the apical portion of pseudoglands were positively stained, although their staining intensity and stained area were lower and more limited, respectively, than those of non-cancerous regions. The percentage of faintly positive and negative cases (n = 22) from the total (n = 30) was 73%. This suggests that the expression level of BGP decreased in HCCs as compared with adjacent non-cancerous regions. Int. J. Cancer 74:15–19. © 1997 Wiley-Liss, Inc.     

Antigenic heterogeneity of human lung cancers

Antigenic reactivity of 35 perchloric acid (PCA) extracts of different histologic human lung cancer tissues was studied--in comparison with the reactivity of the carcinoembryonic antigen (CEA), the nonspecific cross-reacting antigen (NCA), and alpha-1-antichymotrypsin--with the use of specific immune sera against PCA extracts of lung squamous cell carcinoma, and anti-CEA, anti-NCA, and anti-alpha-1-antichymotrypsin sera. The following antigenic systems were found in lung cancers: a) antigens specific for most squamous cell cancers and adenocarcinomas, which are undetectable in small cell cancers; b) NCA-type antigens; c) CEA-like antigens; and d) the antigen responsible for alpha-1-antichymotrypsin reactivity. A considerable antigenic heterogeneity among lung cancers indicates the necessity for precise histopathologic verification of individual lung cancer cases before commencement of immunologic studies and purification of antigens specific for lung cancers.     

Detection of mRNAs of Carcinoembryonic Antigen and Nonspecific Cross-reacting Antigen Genes in Colorectal Adenomas and Carcinomas by in situ Hybridization

Expression of mRNAs of carcinoembryonic antigen (CEA) and nonspecific cross-reacting antigen (NCA) genes in colorectal carcinomas and adenomas was investigated by in situ hybridization (ISH) with specific biotinylated probes. Hybridization was clearly detected throughout the cytoplasm of 7 out of 15 adenomas and 13 out of 15 carcinomas with the CEA cDNA probe, and in 6 out of 15 adenomas and 10 out of 15 carcinomas with the NCA cDNA probe. The intensity of signal appeared to be stronger in carcinomas than that in adenomas, and the CEA and NCA mRNAs were expressed together in most of the positive tissue specimens. On the other hand, noninvaded tissues adjacent to the carcinoma did not show any signal except for 4 cases faintly stained with the NCA probe. This finding was partly confirmed by Northern blot analysis which indicated that the specific bands for the CEA and NCA mRNAs were more intense in RNAs from carcinoma tissues than in those from adjacent noninvaded tissues. These data suggest that the ISH technique with biotinylated probes could be of use for analyzing expression and localization of CEA and related genes on tissue sections.     

Detection of mRNAs of carcinoembryonic antigen and nonspecific cross-reacting antigen genes in colorectal adenomas and carcinomas by in situ hybridization

Expression of mRNAs of carcinoembryonic antigen (CEA) and nonspecific cross-reacting antigen (NCA) genes in colorectal carcinomas and adenomas was investigated by in situ hybridization (ISH) with specific biotinylated probes. Hybridization was clearly detected throughout the cytoplasm of 7 out of 15 adenomas and 13 out of 15 carcinomas with the CEA cDNA probe, and in 6 out of 15 adenomas and 10 out of 15 carcinomas with the NCA cDNA probe. The intensity of signal appeared to be stronger in carcinomas than that in adenomas, and the CEA and NCA mRNAs were expressed together in most of the positive tissue specimens. On the other hand, noninvaded tissues adjacent to the carcinoma did not show any signal except for 4 cases faintly stained with the NCA probe. This finding was partly confirmed by Northern blot analysis which indicated that the specific bands for the CEA and NCA mRNAs were more intense in RNAs from carcinoma tissues than in those from adjacent noninvaded tissues. These data suggest that the ISH technique with biotinylated probes could be of use for analyzing expression and localization of CEA and related genes on tissue sections.     

Immunophenotypic analysis of colorectal carcinomas with monoclonal antibodies 47D10 and anti-carcinoembryonic antigen

A series of 80 colorectal adenocarcinomas were analyzed immunohistochemically for the antigen recognized by a new monoclonal antibody (MCA) 47D10. These antigens are part of a complex family of substances similar to, yet distinct from carcinoembryonic antigen (CEA), and are termed nonspecific cross-reacting antigens (NCA). Formalin-fixed paraffin-embedded sections of colorectal adenocarcinomas were evaluated for the expression of these antigens and compared to the expression of CEA. Our study shows that 83.8% of the cases were positively stained for NCA while 91.3% were positive for CEA. Both antigens were coexpressed in 80% of the cases. No correlation was found between MCA 47D10 immunoreactivity and tumor grade, stage, size or location within the colon. In 25 cases, the benign colonic mucosa adjacent to the carcinoma stained positively with MCA 47D10. Normal colon does not express NCA as recognized by MCA 47D10, except in rare cells. Forty-eight of these cases had serum available for study. Both NCA and CEA were determined in these serum samples. Forty-two of these sera demonstrated elevated CEA levels, whereas only 8 showed increased levels of the 47D10 antigen(s). These findings suggest that the gene product(s) recognized by MCA 47D10 can be independently expressed or, more commonly, coexpressed with CEA in these tissues.     

Immunohistochemical analysis of pulmonary and pleural neoplasms using a monoclonal antibody (47D10) which reacts with nonspecific cross-reacting antigen

A series of 251 human pulmonary carcinomas were analyzed immunohistochemically for the antigens recognized by a new monoclonal antibody (MAb) 47D10. These antigens are part of a complex family of substances similar to, yet distinct from carcinoembryonic antigen (CEA), and are termed 'nonspecific cross-reacting antigens' (NCAs). The NCA epitope recognized by the MAb 47D10 is expressed on the cell surface and has previously been shown to be distinct from epitopes detected by several anti-CEA MAbs, as well as by MAbs 19-9 and Du-PAN-2. The NCA epitope recognized by MAb 47D10 is well preserved in formalin-fixed and paraffin-embedded tissues. Using immunohistochemistry, this epitope has been shown to have a limited biodistribution in normal tissues, and to be expressed by adenocarcinomas arising in the pancreas, colon, breast, ovary, prostate and lung. The frequency and pattern of NCA expression in human pulmonary neoplasms was found to correlate with the known distribution of CEA: and was often present in the non-small-cell carcinomas. In addition, the expression of CEA relative to NCA was evaluated in a select group of non-small-cell carcinoma cases using several anti-CEA MAbs, to directly compare the expression of CEA to NCA. In general, the NCA reaction pattern is more intense and expressed on more cells within the tumors than that of CEA expression.     

Carcinoembryonic antigen as differentiation marker for small cell lung cancer in vitro and its clinical relevance

Fifteen small cell lung cancer (SCLC) cell lines and 249 histopathological specimens from patients with SCLC were investigated for carcinoembryonic antigen (CEA) expression. Quantitative determinations of CEA in cell line homogenates correlated significantly (p less than 0.001) with L-DOPA decarboxylase (DDC) activities and population doubling times (PDT). Using monoclonal antibodies specific for CEA (BW 431/31) and crossreactive with non-specific crossreacting antigens (NCA) 55 and 95 (BW 250/183 and BW 374/14), the CEA-reactive moiety in SCLC was immunologically identified as CEA. In vivo studies demonstrated CEA by immunohistochemistry in 57% (141/249) of newly diagnosed SCLC. The presence of CEA and the degree of immunostaining did not correlate with clinical parameters.     

Adhesion to carcinoembryonic antigen by human colorectal carcinoma cells involves at least two epitopes

Carcinoembryonic antigen (CEA) may be involved in both cell-cell and cell-substrate adhesion. Our purpose was to determine whether epitopes involved in the homophilic binding of human colorectal carcinoma cells to CEA participated in adhesion to basement membrane proteins. Three human colorectal adenocarcinoma cell lines and one CHO cell line transfected with CEA cDNA were tested in a solid-phase adhesion assay. The 2 CEA-expressing carcinoma cell lines (KM-12c and CCL 188) and the transfectant, but not the parental CHO line, bound to CEA. The CEA-non-producing carcinoma line (Clone A) did not bind to CEA. All colorectal carcinoma cell lines, the transfectant and the parental CHO line bound to laminin, while the colorectal carcinoma lines bound to type-IV collagen. MAbs to epitopes on CEA that cross-react with non-specific cross-reacting antigen (NCA) inhibited adhesion of CEA-expressing cells to CEA. MAbs to non-cross-reactive epitopes of CEA did not block adhesion to CEA. When the inhibitory anti-CEA antibodies were compared in a competitive radioimmunoassay, 2 distinct epitopes were identified. Epitope I is in the N-terminal domain and defined by MAbs MN3, T84.1 and C110, whereas epitope II is located in the repeating loop domains and is recognized by antibodies MN15, PR3B10 and NPI. None of the antibodies to epitope I or II blocked adhesion by KM-12c or CCL 188 cells to laminin or type-IV collagen. Thus, at least 2 different regions on CEA participate in adhesion to CEA but not to collagen or laminin by CEA-expressing human colorectal carcinoma cells.     

Carcinoembryonic antigen-like substances of human bile. Isolation and partial characterization.

Three species of carcinoembryonic antigen (CEA)-like macromolecules, called the biliary glycoprotein I, II and III (BGP I, II and III) were identified in human bile. BGP I was found in normal gall-bladder bile and hepatic bile but not in bile subjected to inflammation ("white bile"). It was immunologically related to CEA and NGP (the "normal CEA-like glycoprotein". Synonyms: NCA, CCEA-2, etc.). BGP I differed immunologically from CEA in that it lacked the tumor-associated determinants of CEA. It was different from NGP (and CEA) in that it contained BGP I specific determinants as revealed by anti-BGP I antibodies. In bile from gallbladders with obstructed outlet and subjected to cholecystitis, a non-malignant inflammatory process, BGP I was replaced by BGP II and BGP III. Immunologically and physicochemically, BGP II and BGP III appeared to be closely similar to NGP and CEA, respectively.