Carcinoembryonic antigen immunocytochemistry in primary breast cancer

We studied the immunoreactivity by immunohistology of two carcinoembryonic antigens (CEA) with specific and two CEA antibodies with nonspecific cross-reacting antigen (NCA) cross reactivity (CEA/NCA) in 180 primary breast carcinomas. Positive tissue staining was found in more than 90% of the specimens with CEA/NCA antibodies, compared with less than 30% for both CEA-specific antibodies. There was no correlation between the positivity of CEA immunocytochemistry for any of the four antibodies and histologic grade, lymph node stage, locoregional recurrence, disease-free interval (DFI), or patient survival. This large study with a long follow-up period for patients has shown that CEA and CEA/NCA immunocytochemistry have no relation to prognosis in breast cancer. An extensive review of the literature that confirms these findings should end the controversy over the place of CEA and CEA/NCA immunocytochemistry in breast cancer.     

Immunological reactivity of mucinous and serous ovarian adenocarcinomas

Comparison of immunological reactivity of glycoprotein antigens extracted from individual cases of mucinous and serous ovarian adenocarcinomas was performed taking into account the immunological relationship with carcinoembryonic antigen (CEA), nonspecific cross-reacting antigen (NCA), alpha-1-antichymotrypsin, and alpha-1-acid glycoprotein. In all immunological tests, the specific immune sera against perchloric acid extracts of ovarian mucinous and serous cystadenocarcinomas and antisera against the reference antigens mentioned above were used. It was established that: 1) ovarian mucinous and serous adenocarcinomas are immunologically different and possess various tumor-associated antigens, 2) ovarian mucinous adenocarcinomas contain considerable amounts of CEA and NCA, whereas serous type neoplasms show negligible amounts or lack of these antigens; and 3) in both types of tumors, alpha-1-antichymotrypsin and alpha-1-acid glycoprotein activities are found. Immunological data indicate that ovarian mucinous and serous adenocarcinomas derive from separate lineages of epithelium.     

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.     

Immunocytochemical evaluation of human esophageal neoplasms and preneoplastic lesions for beta-chorionic gonadotropin, placental lactogen, alpha-fetoprotein, carcinoembryonic antigen, and nonspecific cross-reacting antigen

Human esophageal neoplasms were studied in comparison to normal, uninvolved, and preneoplastic human esophageal epithelium for the presence of human chorionic gonadotropin (HCG), human placental lactogen (HPL), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and nonspecific cross-reacting antigen (NCA) using the unlabeled antibody peroxidase-antiperoxidase technique. HCG immunoreactivity was identified in 10 of 33 squamous cell carcinomas (33%), in 1 of 6 adenocarcinomas (17%), and 1 of 6 preneoplastic esophageal lesions (17%); while 9 of 33 squamous cell carcinomas (33%) and 1 of 6 adenocarcinomas (17%) contained immunoreactive AFP. Immunoreactive HPL was detected in 6 of 33 squamous cell carcinomas (20%), but in none of the adenocarcinomas. Neither AFP nor HPL immunoreactivity was identified in the 6 hyperplastic lesions which were studied. When stained with an antiserum that was able to detect both CEA and NCA, 27 of 33 squamous cell tumors (82%) and 6 of 6 adenocarcinomas (100%) showed positive immunostaining reactions. Of these, 8 squamous cell carcinomas and 1 adenocarcinoma were subsequently shown to contain only NCA immunoreactivity, while 19 squamous cell carcinomas and 5 adenocarcinomas contained both NCA and CEA immunoreactivity. NCA immunoreactivity alone was identified in 3 of 6 preneoplastic lesions and NCA and CEA immunoreactivity in 1 of 6 preneoplastic lesions. None of the markers was detected in 8 specimens of normal esophageal epithelium which were studied as controls, nor in 6 specimens of uninvolved esophageal epithelium obtained from patients with esophageal cancer. Most tumors expressed 2 or 3 markers, and some tumors were identified which expressed up to 4 of the 5 markers investigated. Only 3 tumors failed to express any of the markers studied. No association was found between the degree of tumor differentiation and presence or absence of HCG immunoreactivity. However, HPL immunoreactivity was more common in poorly differentiated squamous cell carcinomas. In contrast, immunoreactive AFP was more common in well-differentiated squamous cell carcinomas than in other tumor types. Similarly, both CEA and NCA were more frequently expressed in well-differentiated squamous cell carcinomas, adenosquamous carcinomas, and adenocarcinomas than in less differentiated tumors. Our results suggest that HCG, HPL, AFP, CEA, and NCA are tumor-associated antigens in esophageal cancer. Therefore, they could be of value in screening tests for esophageal neoplasms and could be useful in subclassification of esophageal neoplasms.     

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.     

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.     

Monkey antisera with increased specificity to carcino-embryonic antigen (CEA).

Three Macaca irus monkeys were immunized with purified human CEA. One received unmodified CEA and two were immunized with haptenated (4-hydroxy-5-iodo-3-nitrophenyl acetyl) CEA. All three monkeys formed precipitating antibodies to CEA. The antisera did not precipitate the CEA-related normal glycoprotein antigen (NCA). In contrast, CEA-immunized rabbits, sheep and goats invariably form antibodies against NCA. Radio-immunoassays showed the monkey antisera to contain trace amounts of antibodies to NCA but the titers were about 10-1,000 times lower than those in rabbit and sheep sera with comparable anti-CEA titers. These results show that monkeys produce antibodies against CEA, but respond weakly to a normal, CEA-related antigen. The weak response to NCA suggests that monkeys may possess an NCA-related antigen. Antisera lacking reactivity to normal CEA-related antigens may be helpful in establishing a more specific diagnostic test for CEA.     

Natural antibodies against the carcinoembryonic antigen (CEA) and a related antigen, NCA, in human sera

Natural antibodies directed against CEA and a related antigen, NCA, have been demonstrated in all normal and pathological sera using an EIA, although they have never been detected by RIA. These antibodies are not anti-blood group antibodies, as their titer was decreased only slightly by absorption with blood group substances. The study of their reactivity with deglycosylated antigens demonstrated that they were directed against peptidic epitopes. Antibodies against NCA or CEA, purified using specific immunosorbents, cross-reacted with all the antigens of the "CEA family" but not or only weakly with unrelated antigens.     

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.     

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.     

Production and characterization of xenogeneic antisera to tumor-associated antigen(s)

Tumor-associated antigen(s) from a specimen of human malignant melanoma and from spent culture medium of a melanoma cell line were extracted and purified. The incidence of antibody activity to these antigens, in sera from cancer patients with neoplasms of various histologic types, was higher (65--83%) than normal donors' (19--25%) by the complement fixation assay. These purified antigens were then used to raise antisera in rabbits and sheep. After absorption with various human normal tissues, these antisera reacted against melanoma, sarcoma, and carcinoma extracts, but not against human normal liver, skin, or muscle extracts. However, the antisera showed reactivity against a human fetal homogenate. Results indicate that the xenogeneic antisera contained antibodies to fetal antigens and to tumor-associated antigen(s). Xenogeneic antisera could be used to purify tumor-associated antigens and oncofetal antigen(s) from crude extracts by affinity chromatography.     

Distribution of tumor-associated antigen cea and cross-reacting NCA in fetal organs

The organ distribution of the tumor-associated car-cinoembryonic antigen (CEA), and that of the normal tissue component NCA (non-specific cross-reacting antigen) have been investigated in the fetus. Organ extracts from five fetuses between 14 and 21 weeks of age were analysed by radioimmunoassay using specific antisera. CEA was detected in large amounts (800-1,650 ng/g) in fetal colon and in barely detectable amounts in lung and placental tissue. This differed from NCA, which could be detected in almost all organ extracts analysed. The highest concentration of NCA was measured in fetal colon and the content increased with the gestational age of the fetus. High amounts of NCA were also found in the liver, spleen and placenta! tissue. The gel elution profiles of CEA and NCA from an amniotic fluid pool and a pool of colonic extracts were also determined. CEA eluted similarly to the marker ¹²⁵I-CEA purified from liver metastasis of colonic carcinoma. The NCA-reactive material was found in three distinct peaks.     

Study of the antigenic cross reactivity between carcinoembryonic antigen and "nonspecific cross reacting antigens" (NCA and NCA 2).

The immunochemical relationship between CEA, NCA and NCA 2 was studied in guinea-pigs. Strong cross reactions were found between these antigens, either in delayed or anaphylactic reactions. Some specific determinants for each antigen could still be demonstrated. Delayed hypersensitivity is likely to be due to the protein moiety of the molecules while anaphylactic reactivity could probably be related to their glucidic parts. Consequently, CEA and NCA have common antigenic determinants on their glucidic and peptidic moieties, perhaps more on the latter ones.     

Chromosomal localization of the carcinoembryonic antigen gene family and differential expression in various tumors

Carcinoembryonic antigen (CEA) is a glycoprotein which is important as a tumor marker for a number of human cancers. It is a member of a gene family comprising about 10 closely related genes. In order to characterize mRNAs transcribed from individual genes we have identified by DNA and RNA hybridization experiments, gene-specific sequences from the 3' noncoding regions of CEA, and of nonspecific cross-reacting antigen (NCA) mRNAs, which have been recently cloned. With these probes, CEA mRNAs with lengths of 3.5 and 3.0 kilobases and an NCA mRNA species of 2.5 kilobases were identified in various human tumors. A 2.2-kilobase mRNA species, however, could only be detected in leukocytes of patients with chronic myeloid leukemia by hybridization with a probe from the immunoglobulin-like repeat domain of CEA. This region is known to be very similar among the various members of the CEA gene family, and indeed the probe hybridizes with all four mRNA species. In situ hybridization with a cross-hybridizing probe from the NCA gene localized the members of the CEA gene family to the short and to the long arm of chromosome 19. In addition, a CEA cDNA probe was found to hybridize to the long arm of chromosome 19 only.     

Carcinoembryonic antigen gene family and its clinical application

Carcinoembryonic antigen (CEA) gene was cloned in 1987. Thereafter, the structures of non-specific cross-reacting antigen (NCA) and biliary glycoprotein I (BGPI) have also been clarified. These three antigens contain immunoglobulin-like domains in their basic structures and it could be possible that CEA gene was originated from this basic structure by internal gene multiplication. Each CEA and NCA has hydrophobic domain in the C-terminus consisting of 26 amino acids which is eliminated when it binds with membrane and is reconstituted by combining with phosphatidylinositol glycan, whereas BGPI contains transmembrane and cytoplasmic domains. It is of interest that CEA and NCA have been found to function as adhesion molecules. The structure and possible function of another CEA gene family, PS beta G, were also introduced in this short review.     

Coexpression of ganglioside antigen Fuc-GM1, neural-cell adhesion molecule, carcinoembryonic antigen, and carbohydrate tumor-associated antigen CA 50 in lung cancer.

With the aid of specific monoclonal antibodies, tumor tissues from 68 patients with lung cancer were examined for their expression of two small cell lung carcinoma (SCLC) antigens, Fuc-GM1 (fucosyl GM1; IV2FucII3NeuAc GgOse4) and neural-cell adhesion molecule (NCAM), and two broader tumor antigens, carcinoembryonic antigen (CEA) and carbohydrate cancer-associated antigen CA 50. Expression of Fuc-GM1 was seen in 75% and NCAM in 78% of the SCLC specimens, but also in 12 and 20% of non-SCLC. Either or both of these antigens were expressed in more than 90% of SCLC and in 25% of non-SCLC. CEA was found in more than 80% of SCLC and non-SCLC. Expression of CA 50 was seen in 65-68% of non-SCLC and SCLC, showing preference for SCLC and lung adenocarcinoma. In SCLC, cellular expression of Fuc-GM1 was generally seen together with NCAM and CA 50, but rarely with CEA. There was considerable inter- and intratumor heterogeneity in the expression of all four antigens. The results suggest that CEA is the antigen of choice for the detection of lung cancer regardless of histotype. In combined analysis of CEA, CA 50, Fuc-GM1 and NCAM, two patterns of antigen expression were recognized that appear to discriminate between SCLC and non-SCLC tumors, respectively. A considerable fraction of SCLC and non-SCLC tumors, however, exhibited similar patterns of antigen expression. The biological and clinical significance of these observations remains to be investigated.     

Comparison of portal and peripheral blood levels of carcinoembryonic antigen, CA 19-9, and CA 125 tumor-associated antigens in patients with colorectal and pancreatic cancer

Synchronous serum specimens from the systemic and portal circulations of 43 patients with gastrointestinal cancer were assayed for levels of carcinoembryonic antigen, CA 19-9, and CA 125 tumor-associated antigens. The number of patients having a mean ratio of portal to systemic levels greater than 1 and the observed quantity of tumor-associated antigens were significant for carcinoembryonic antigen and CA 125 only in patients with colorectal cancer. No correlations were noted with the surgical stage of disease or with high or low (normal) levels of the three tumor-associated antigens. These findings suggest that peripheral concentrations of these antigens are in equilibrium with shedding from tumors and that hepatic clearance of a single pass does not significantly alter peripheral concentrations.     

Proteolytic release of antigenic fragments corresponding to normal fecal antigen and non-specific cross-reacting antigen from carcinoembryonic antigen.

Three immunogenic parts have so far been identified in the carcinoembryonic antigen (CEA) molecule. These are: determinants cross-reactint with the normal fecal antigen (NFA) (NFA determinant); determinants cross-reacting antigen (NCA) (NCA determinant); and determinants which appear to be more cancer-specific (cancer determinant). The chemical nature of these parts of the CEA molecule was investigated by digestion with proteolytic enzymes together with anti-CEA preparations with which these three immunogenic parts of CEA molecule could be identified. The CEA digest obtained with pepsin did not react in immunodiffusion and radioimmunoassay, indicating that pepsin completely destroyed all the antigenic parts. Digestion by pronase E destroyed only the cancer determinant and liberated two antigenic fragments corresponding to the NFA determinant and the NCA determinant, respectively. These results suggest that the cancer determinant may reside in a protein or a peptide part of the molecule. The chemical nature of the NFA and NCA determinant remains to be clarified.     

Studies on circulating antibody against carcinoembryonic antigen (CEA) and CEA-like antigen in cancer patients

Characteristics of auto-antibodies for carcinoembryonic antigen (CEA) detected in sera from 3 cancer patients (2 colorectal and 1 breast cancer) were examined. The antibodies belonged to polyclonal immunoglobulin G (IgG). The binding of auto-antibodies with the labeled CEA was inhibited by not only the unlabeled CEA but also NCA-2 (feces and meconium). However, no binding with NCA was observed. Among these auto-antibodies the antibody directed against blood group Lewis determinants which are known to be present in many purified CEA preparations was not found. Previously we had suggested that CEA, NCA-2 and NCA may contain immune determinant in common with alpha 1-acid glycoprotein (AG). These auto-antibodies showed significantly enhanced reactivity for the labeled CEA preparation after purification by anti-AG affinity chromatography in spite of no immunological reaction with AG. These results suggest that auto-antibodies are raised against the common antigenic determinants of both CEA and NCA-2 which do not exist in NCA. These antibodies might be directed to common amino acid sequence shared by CEA and NCA-2, though not excluding the carbohydrate moiety. We surveyed about 500,000 cancer patients but could find only 3 patients who showed a difference in the values of CEA by the indirect and direct method. Thus, the existence of this type auto-antibody to CEA in cancer patients is a rare phenomenon.