Immunohistochemical study of the carcinoembryonic antigen (CEA) in human tumors using the CEA-specific oncoprecipitin crustacin

58 human tumors have been studied immunohistologically with the aid of CEA-specific precipitin--crustacin (Cr). The results were in general similar to those which were achieved with anti-CEA-antibodies. But there were some differences. The reaction with Cr in the cytoplasm was mostly diffuse, while the reaction with Ab was localized usually in the peripheral parts of the cytoplasm. The reaction with Cr has a more restricted character and is more expressed in ovarian tumors and less expressed in gastric and colonic tumors than the reaction with Ab.     

Localisation of carcinoembryonic antigen in embryonic and fetal human tissues

Carcinoembryonic antigen (CEA) was localized in various embryonic and fetal human tissues between 8 and 16 weeks of gestation as well as in the colorectal mucosa of older fetuses, newborns and adults. Among the embryonic tissues, CEA was always present in the esophagus, the gastric antrum, the duodenum and the rectum. CEA positive staining of bile canaliculi of the liver was inconstant. All other embryonic tissues were CEA negative. During early fetal development, CEA positive staining of the esophagus, antrum and duodenum was inconstant. However, the whole colon became intensively stained. An inconstant CEA specific staining was found in parts of the midgut and in the bile canaliculi of the liver. The other organs remained CEA negative. Between the 17th week of gestation and birth, CEA staining pattern of the colorectal mucosa did not change. The staining intensity of late fetal colonic mucosa was similar to that of adult colonic mucosa.     

Localisation of carcinoembryonic antigen in embryonic and fetal human tissues

Summary Carcinoembryonic antigen (CEA) was localized in various embryonic and fetal human tissues between 8 and 16 weeks of gestation as well as in the colorectal mucosa of older fetuses, newborns and adults. Among the embryonic tissues, CEA was always present in the esophagus, the gastric antrum, the duodenum and the rectum. CEA positive staining of bile cannaliculi of the liver was inconstant. All other embryonic tissues were CEA negative. During early fetal development CEA positive staining of the esophagus, antrum and duodenum was inconstant. However, the whole colon became intensively stained. An inconstant CEA specific staining was found in parts of the midgut and in the bile cannaliculi of the liver. The other organs remained CEA negative. Between the 17th week of gestation and birth, CEA staining pattern of the colorectal mucosa did not change. The staining intensity of late fetal colonic mucosa was similar to that of adult colonic mucosa.Deceased 20th August 1982     

Immunohistochemical detection of carcinoembryonic antigen (CEA) in non-cancerous and cancerous gastric mucosa

Carcinoembryonic antigen (CEA) was stained by the PAP immunoperoxidase method in cancerous and non-cancerous gastric mucosa of 40 patients (25 non-cancerous dyspeptic patients and 15 patients with gastric carcinoma). The pattern of CEA localization was apical or membranous-cytoplasmic and immuno-reactivity was mild (+), moderate (++) or intensive ( ). No CEA immunoreactivity was detected in normal gastric mucosa whereas it was marked in gastric mucosa of non-cancerous dyspeptic patients with chronic atrophic gastritis and dysplasia (intense). In patients with superficial gastritis and epithelial hyperplasia it was mild or absent. The CEA localization pattern was also apical in non-cancerous dyspeptic patients with microscopic changes, e.g. superficial or chronic atrophic gastritis, epithelial hyperplasia and dysplasia, and in non-cancerous mucosa and cancerous tissue of patients with well (G1) and moderately (G2) differentiated adenocarcinoma.     

Keratin and carcinoembryonic antigen (CEA) in human melanoma cells.

Human melanomas are known to contain vimentin intermediate filaments but there has been some dispute about their expression of cytokeratins. The cytoplasm of human M21 melanoma cells maintained in culture reacted with a rabbit anti-keratin antibody and two monoclonal anti-keratin antibodies AE1 and AE2. Cells derived directly from subcutaneous xenografts of M21 melanoma in nude mice, however, failed to express cytokeratins. The presence of keratin filaments in cultured M21 cells was confirmed by electronmicroscopic and immuno-electronmicroscopic examinations of cell extracts. Polyacrylamide gel electrophoresis (PAGE), revealed 46 KD keratin proteins in cultured M21 cells. Small amounts of these low molecular weight keratins were detected by PAGE in M21 melanoma xenografts even though immunofluorescence and immunoperoxidase assays failed to demonstrate keratin at the light microscopic level. Immunofluorescence revealed keratin and carcinoembryonic antigen (hitherto undetected in human melanomas) first on the 9th day of culture of xenograft-derived M21 cells. The appearance of keratin and CEA in M21 melanoma cells in vitro was not affected by inhibition of cellular proliferation or as a result of exposure to methotrexate or adriamycin. However, adriamycin altered the cytoplasmic distribution of keratin.     

Multiple Smith-degradations of carcinoembryonic antigen (CEA) and of asialo CEA

Carcinoembryonic antigen (CEA) and asialo CEA were subjected to multiple Smith-degradation (i.e., for each degradation, application in sequence of periodate oxidation, borohydride reduction, and mild hydrolysis with acid; borohydride-t was substituted for unlabelled borohydride). High yields of modified glycoproteins were obtained at each stage. After three complete degradations and a further periodate-borohydride-t treatment, the carbohydrate content of CEA and of asialo CEA had decreased from 45–50% to 11–12% (i.e., 90% removal of carbohydrate). Glycerol was always one of the products obtained after each degradation, but threitol and erythritol were not detected. The second degradation caused a substantial loss of 2-acetamido-2-deoxyglucose, which is consistent with the location of some of this monosaccharide towards the terminal (non-reducing) end of the oligosaccharides. The “core” region of the oligosaccharides is composed of galactose, mannose, and 2-acetamido-2-deoxyglucose. After the fourth oxidation, 2-acetamido-2-deoxyglucose was 50–60% of the total content of residual carbohydrate. After the first degradation, there was a progressive loss in antigenic activity, but this was associated with a small amount of hydrolysis of the protein moiety of CEA.     

Distribution of tissue polypeptide antigen (TPA) in normal human tissues: Immunohistochemical study on unfixed, methanol-, ethanol-, and formalin-fixed tissues

The distribution of tissue polypeptide antigen (TPA) was studied in unfixed, methanol-, 95% ethanol-1% acetic acid (EA)-, and formalin-fixed paraffin-embedded sections of all adult human tissues using an indirect immunoperoxidase method. The specific staining patterns were virtually identical in unfixed and alcohol-fixed tissues, but in formalin-fixed tissues this similarity was found only after fixation for up to 24 hr and pretreatment with protease for 15 min. Although prolongation of formalin fixation beyond 48 hr increasingly diminished the TPA reactivity, TPA could still be demonstrated in tissues fixed in formalin for up to 6 months. TPA was found to be a cytoplasmic constituent of almost all adult human duct and cavity lining, simple, and stratified epithelia. TPA was not demonstrated in epidermis, renal proximal convoluted and testicular tubules, basket-like myoepithelial cells, nor in most glandular acini, including hepatocytes and pancreatic acinar cells. The TPA staining was also negative in all non-epithelial tissues, including lymph nodes and bone marrow. The well-defined epithelial distribution and the comparable demonstrability in differently preserved tissues make TPA a useful tool for the identification of cells of epithelial character.     

Immunological characteristics of monoclonal antibodies against human carcinoembryonic antigen (CEA)

Four hybridomas secreting monoclonal antibodies (MAbs) of the IgG1 subclass against human carcinoembryonic antigen (CEA) were obtained from fusion of P3-NS1/1-Ag4 myeloma cells with splenic cells from mice immunized with purified CEA. None of the MAbs showed cross-reactivity to perchloric acid extractable antigens from the normal human colon by an inhibition radioimmunoassay. However, MAb C27 showed the highest affinity to CEA. The intensity of immunofluorescence staining of human colorectal cancer cells with MAb C27 correlates well to the cellular CEA content of cancer cells. LS174T showed the highest intensity of fluorescence (95%) while COLO320DM and COLO320HRS were the lowest (0.5%). None of the normal human organs - colon, lungs, liver, spleen or kidneys-showed positive staining by immunoperoxidase anti-peroxidase (PA) techniques, while tissues from colorectal carcinoma (CRC), gastric carcinoma, hepatoma and lung cancer gave a positive rate of 100% (30/30), 96.6% (28/29), 32.1% (9/28) and 82.1% (69/84) respectively. Results suggest that MAb C27 can be used in immunodetection and radiolocalization of colorectal carcinoma.     

Characterization of a variant of carcinoembryonic antigen (CEA) using monoclonal and polyclonal anti-CEA antibodies

A variant of the carcinoembryonic antigen (CEA) with lower molecular weight than a CEA reference preparation has been separated from CEA. Using a polyclonal, spleen absorbed anti-CEA antiserum, the variant crossreacts with reference CEA in immunodiffusion. The CEA-activity of the variant has been demonstrated using an enzyme-immunoassay with monoclonal CEA specific antibodies. There is sufficient immunological evidence that this variant is a distinct antigen different from the crossreactive antigens described so far. The reactivity of the polyclonal anti-CEA antiserum with the CEA variant was abolished by absorption against the immobilized variant.     

Primary structure of human carcinoembryonic antigen (CEA) deduced from cDNA sequence

The cDNAs corresponding to the mRNA encoding a polypeptide which is immunoreactive with the antisera specific to carcinoembryonic antigen (CEA) (1) are cloned. The amino acid sequences deduced from the nucleotide sequences of the cDNAs show that it is synthesized as a precursor with a signal peptide followed by 668 amino acids of the putative mature CEA peptide, whose N-terminal 24 amino acids and amino acids 286 to 295 exactly coincide with those known for N-terminal sequences of CEA (2) and NFA-1 (3), respectively. The first 108 N-terminal residues are followed by three very homologous repetitive domains of 178 residues each and then by 26 mostly hydrophobic residues which probably comprise a membrane anchor. Each repetitive domains contains 4 cysteines at precisely the same positions and as many as 28 possible N-glycosylation sites are found in the CEA peptide region agreeing with high carbohydrate content of purified CEA.     

DNA aptamers selection for carcinoembryonic antigen (CEA)

Carcinoembryonic antigen (CEA) is a glycoprotein antigen generally used for diagnosis, prognosis and treatment monitoring of several types of tumors, including colorectal cancer. Nucleic acid aptamers are DNA or RNA oligonucleotides capable of binding with high specificity and affinity to a molecular target. The aim of this study was to obtain aptamers specific to CEA for use as radiopharmaceuticals in colorectal cancer diagnosis. Five aptamers were selected through the Systematic Evolution of Ligands by EXponencial Enrichment (SELEX) and tested using T84 (CEA+) and Hela (CEA−) cells. Apta 3 and Apta 5 showed the best results presenting high specificity and affinity for T84 cells, with dissociation constants (K_d) of 60.4 ± 5.7 nM and 37.8 ± 5.8 nM, respectively. These results indicate that Apta 3 and Apta 5 are promising candidates for identifying tumor cells that overexpress CEA.     

Escherichia coli of human origin binds to carcinoembryonic antigen (CEA) and non-specific crossreacting antigen (NCA)

Immobilized carcinoembryonic antigen (CEA) and non-specific crossreacting antigen (NCA) bound 3 strains of E. coli of human origin. The binding was dose dependent, saturable, and of high avidity. Binding of the bacteria to CEA and NCA was completely abolished in the presence of 10 mM alpha-methyl D-mannopyranoside. Bacteria did not bind to concanavalin A. In addition, binding to deglycosylated CEA was either absent or significantly reduced. These findings indicate that the E. coli strains bind to D-mannosyl residues in CEA and NCA. Considering the tissue distribution of CEA (brush border of colonic epithelium) and NCA (granulocytes), these glycoproteins may be involved in the recognition of bacteria.     

The carcinoembryonic antigen (CEA) gene family in non-human primates

Carcinoembryonic antigen (CEA) is a tumor marker of wide clinical use though its function remains unknown. The CEA counterpart and some related macromolecules cannot be demonstrated in mice, thus prohibiting studies of CEA function by gene disruption strategies. In an attempt to find a relevant animal model for functional studies of CEA we have investigated the occurrence of CEA subgroup members in baboon and African green monkey at the genomic and mRNA levels. The investigation was focused on the characteristic immunoglobulin-variable region-like (IgV-like) N-terminal domain of the family members. Based on N-domain sequences 3 and 4 different CEA subgroup genes, respectively, were identified. One sequence in each monkey species corresponded to human CEACAM8, while it was not possible to assign an obvious human counterpart for the other N-domain sequences. However, studies of cDNAs from African green monkey COS-1 cells identified one of the sequences as CEACAM1. Expression of CEACAM1 mRNA and protein was upregulated by IFNgamma as has previously been demonstrated for human CEACAM1. Presence of GPI-linked CEA subgroup members in African green monkey was suggested by sequencing. Both monkey species would thus seem suitable for functional studies of selected CEA subgroup members.     

CA 15-3 in human breast cancer. Comparison with tissue polypeptide antigen (TPA) and carcinoembryonic antigen (CEA)

CEA, TPA, CA 15-3 were assayed in 238 patients in follow-up for breast cancer after surgery. CA 15-3 showed the best sensitivity and specificity; the predictive value of a positive CA 15-3 test was three times higher than CEA and TPA. No association was found between marker positivity and the number of organs involved by metastases. CA 15-3 positivity was significantly associated with visceral rather than soft tissue recurrences; no significant similar association was observed for CEA and TPA. CA 15-3 serum levels were early predictors of relapse in four out of nine patients within a 6-12 month follow-up period.     

Mouse models expressing human carcinoembryonic antigen (CEA) as a transgene: evaluation of CEA-based cancer vaccines

In recent years, investigators have carried out several studies designed to evaluate whether human tumor-associated antigens might be exploited as targets for active specific immunotherapy, specifically human cancer vaccines. Not too long ago such an approach would have been met with considerable skepticism because the immune system was believed to be a rigid discriminator between self and non-self which, in turn, protected the host from a variety of pathogens. That viewpoint has been challenged in recent years by a series of studies indicating that antigenic determinants of self have not induced absolute host immune tolerance. Moreover, under specific conditions that evoke danger signals, peptides from self-antigen can be processed by the antigen-presenting cellular machinery, loaded onto the major histocompatibility antigen groove to serve as targets for immune intervention. Those findings provide the rationale to investigate a wide range of tumor-associated antigens, including differentiation antigens, oncogenes, and tumor suppressor genes as possible immune-based targets. One of those tumor-associated antigens is the carcinoembryonic antigen (CEA). Described almost 40 years ago, CEA is a M(r) 180-200,000 oncofetal antigen that is one of the more widely studied human tumor-associated antigens. This review will provide: (i) a brief overview of the CEA gene family, (ii) a summary of early preclinical findings on overcoming immune tolerance to CEA, and (iii) the rationale to develop mouse models which spontaneously develop gastrointestinal tumors and express the CEA transgene. Those models have been used extensively in the study of overcoming host immune tolerance to CEA, a self, tumor-associated antigen, and the experimental findings have served as the rationale for the design of early clinical trials to evaluate CEA-based cancer vaccines.     

The expression of Lewisx on carcinoembryonic antigen (CEA)-related glycoproteins of normal and inflamed oesophageal squamous mucosa

Carcinoembryonic antigen (CEA)-related antigens were detected histologically in normal and inflamed oesophageal squamous mucosa using polyclonal anti-CEA antisera and monoclonal antibodies recognizing CEA or NCAs (non-specific cross-reacting antigens). Expression was limited to the surface of more mature squames. Immunoblotting of detergent extracts of oesophageal mucosa separated on polyacrylamide gels using polyclonal anti-CEA antisera showed a number of CEA-related proteins, of 195, 145, and 80 kDa. CEA-specific monoclonal antibodies recognized only the 195-kDa glycoprotein. The lower molecular weight species were recognized by anti-NCA antibody DD9 and a CD66 antibody. The carboyhydrate antigen Lewis^x (Le^x, CD15), previously shown to be a marker of mature squames, was present predominantly on a subpopulation of the 195-kDa antigen and was demonstrable on the higher molecular weight component of a doublet recognized by the CEA antibodies. Expression of Le^x carbohydrate antigens in inflamed oesophageal squamous mucosa was shown to be significantly reduced relative to the expression seen in normal tissue. A suprabasal layer of CEA-positive, Le^x-negative cells became apparent in inflamed tissue showing altered glycosylation of the CEA under these conditions. It is postulated that CEA plays a role in maintaining the integrity of the squamous mucosa.     

Colorectal hyperplasia and dysplasia due to human carcinoembryonic antigen (CEA) family member expression in transgenic mice

CEA and CEACAM6 are immunoglobulin family intercellular adhesion molecules that are up-regulated without structural mutations in approximately 70% of human cancers. Results in in vitro systems showing tumorigenic effects for these molecules suggest that this correlation could indicate an instrumental role in tumorigenesis. To test whether this applies in vivo, transgenic mice harboring 187 kb of the human genome containing four CEA family member genes including the CEA and CEACAM6 genes were created and their copy numbers increased by mating until colonocyte expression levels reached levels seen in human colorectal carcinomas. The colonocyte surface level of integrin alpha5 and the activation of AKT increased progressively with the expression levels of CEA/CEACAM6. Colonic crypts showed a progressive increase in colonocyte proliferation, an increase in crypt fission, and a strong inhibition of both differentiation and anoikis/apoptosis. All transgenic mice showed massively enlarged colons comprising a continuous mosaic of severe hyperplasia, dysplasia and serrated adenomatous morphology. These results suggest that up-regulated non-mutated adhesion molecules could have a significant instrumental role in human cancer.     

A carcinoembryonic antigen (CEA) specific monoclonal hybridoma antibody that reacts only with high-molecular-weight CEA

Summary A monoclonal antibody secreted by a hybridoma, formed by the fusion of a spleen cell from an immunized mouse and a myeloma cell, has been shown to react only with a form of carcinoembryonic antigen (CEA) with a molecular weight of 180,000 daltons. The monoclonal antibody precipitates a 180,000-dalton form of CEA from colorectal carcinoma cell lysates, purified iodinated CEA and iodinated CEA-S, but does not precipitate the cross-reactive antigen (NCA) from normal colon.     

Long-range chromosomal mapping of the carcinoembryonic antigen (CEA) gene family cluster

A long-range physical map of the carcinoembryonic antigen (CEA) gene family cluster, which is located on the long arm of chromosome 19, has been constructed. This was achieved by hybridization analysis of large DNA fragments separated by pulse-field gel electrophoresis and of DNA from human/rodent somatic cell hybrids, as well as the assembly of ordered sets of cosmids for this gene region into contigs. The different approaches yielded very similar results and indicate that the entire gene family is contained within a region located at position 19q13.1-q13.2 between the CYP2A and the D19S15/D19S8 markers. The physical linkage of nine genes belonging to the CEA subgroup and their location with respect to the pregnancy-specific glycoprotein (PSG) subgroup genes have been determined, and the latter are located closer to the telomere. From large groups of ordered cosmid clones, the identity of all known CEA subgroup genes has been confirmed either by hybridization using gene-specific probes or by DNA sequencing. These studies have identified a new member of the CEA subgroup (CGM8), which probably represents a pseudogene due to the existence of two stop codons, one in the leader and one in the N-terminal domain exons. The gene order and orientation, which were determined by hybridization with probes from the 5' and 3' regions of the genes, are as follows: cen/3'-CGM7-5'/3'-CGM2-5'/5'-CEA-3'/5'-NCA-3'/5'-CGM1- 3'/3'-BGP-5'/3'- CGM9-5'/3'-CGM6-5'/5'-CGM8-3'/PSGcluster/qter.