Identification and characterization of new antigenic fragments related to carcinoembryonic antigen in adult feces.

Antigens in human adult feces related to carcinoembryonic antigen (CEA) were analyzed with respect to their molecular masses, CEA domain compositions, and N-terminal amino acid sequences. By avoiding perchloric acid treatment, new fecal antigens related to CEA were identified. The fecal antigens revealed by Western blot were M(r) 78,000, 70,000, 60,000, 50,000, 44,000, 36,000, 33,000, and 25,000 and a species M(r) less than or equal to 14,000. Unlike native CEA, all of the fecal antigens were very poorly soluble in perchloric acid and did not bind to concanavalin A, suggesting that they had undergone significant deglycosylation in the digestive tract. The major fecal antigens were purified by immunoaffinity chromatography and their N-terminal amino acid sequences determined. FA78, FA60, FA33, and the M(r) less than or equal to 14,000 antigen had the N-terminal amino acid sequence of the CEA N-domain, and FA44 and FA25, the sequence of the CEA A2 domain. The CEA domain compositions of the fecal antigens were investigated by probing them with anti-CEA monoclonal antibodies of known domain specificities. The N-terminal amino acid sequences, immunoreactivities with anti-CEA monoclonal antibodies, and apparent molecular masses of the fecal antigens allowed the following domain assignments (based on CEA as N-A1B1-A2B2-A3B3): FA78, N-A1B1-A2B2-A3B3; FA60, N-A1B1-A2B2; FA44, A2B2-A3B3; FA33, N-A1B1; and FA25, A2B2. The M(r) less than or equal to 14,000 antigen was shown to be the N-domain of CEA or nonspecific cross-reacting antigen. FA36 was assigned the N-AB domain structure of nonspecific cross-reacting antigen. The results suggested that FA78, FA60, FA44, FA33, and FA25 were degradation products (including deglycosylation and proteolysis) of CEA and that FA36 was a degradation product of nonspecific cross-reacting antigen.

Immunocytochemical evaluation of neoplastic and non-neoplastic breast diseases with Mab A-80.

Five hundred breast tissue samples from 404 cases were immunostained with A-80, a murine IgM Mab that recognizes a mucinous glycoprotein associated with exocrine differentiation. Samples included 196 primary breast carcinomas, 30 breast carcinoma metastases, 118 fibrocystic disease (FCD), and a further group of 84 samples of FCD from cases known to have breast carcinoma. These samples represented a broad spectrum of common and rare variants of carcinoma and FCD. Samples of fibroadenomas, lactating adenomas, cystosarcoma phylloides, gynecomastia, and normal breasts were similarly studied. The vast majority of carcinomas, 203/212 (95.7%) were immunoreactive; staining varied in extent and intensity, and was virtually unrelated to histologic type and to the presence or absence of recognizable glands. In samples including in-situ and infiltrating ductal or lobular carcinoma, reactivity was frequently stronger in the infiltrating components. No significant difference in reactivity between primary and metastatic carcinomas was noted. Of the group of 118 FCD, 27 were negative whereas 91 showed focal and weak staining. Seventy-two/84 FCD with associated carcinoma were immunostained; in 13 of those 72, staining was strong and extensive. Fibroadenomas, lactating adenomas, gynecomastia, and normal "resting" and lactating breast samples stained focally or not at all. Our findings indicate that Mab A-80 is an excellent immunohistochemical marker for the overwhelming majority of breast carcinomas whereas it marks weakly or not at all the majority of benign neoplasms and normal breast. Moreover, Mab A-80 recognizes a subset of FCD that includes proliferative variants associated with an increased incidence of carcinoma, and FCD in association with carcinoma. Questions regarding rare breast carcinomas that do not react with Mab A-80 remain unclear; yet, we believe that Mab A-80 is a highly promising marker of malignant and dysplastic breast epithelium.

Mucin glycoproteins as tumor markers.

Extensive biochemical studies have shown that mucin tumor antigens have a range of molecular sizes from 200 to greater than 1000 kDa. The molecular size of mucin antigens can be dramatically affected by the source and method of purification. Mucin antigens vary from 24 to 80% in carbohydrate content and their density is usually greater than 1.40 g/ml. Galactose and N-acetyl glucosamine are the predominant sugar residues in many mucins, whereas mannose is usually present in low levels or absent. The amino acids serine, threonine, alanine, glycine, and proline are abundant in mucins. An O-glycosidic linkage between the carbohydrate and protein of mucins is the most common linkage encountered. The gene encoding the core peptide for at least one mucin tumor marker, HMFG, has been identified, sequenced, and expressed. These findings may lead to a better understanding of the multiepitope nature of mucin tumor markers. The advent of hybridoma technology has yielded several monoclonal antibodies that have been used to identify the presence of tumor-associated mucins in the sera of cancer patients. Elevated levels of mucin antigens have been found in the serum of most patients with advanced adenocarcinomas. Many studies have shown that tumor-associated markers are useful in monitoring patients following cancer treatment. Clinically useful immunoassays have been developed for monitoring patients with ovarian, breast, and pancreatic adenocarcinomas. Although individual mucin tumor markers show limited utility in detecting early adenocarcinoma, recent studies using multiple mucin markers have suggested that early detection, at sensitivities greater than 50%, can be achieved.

Expression of a new mucin-type glycoprotein in select epithelial dysplasias and neoplasms detected immunocytochemically with Mab A-80.

We studied by immunocytochemistry 573 tissue and 106 cytologic samples of human tumors, non-neoplastic proliferative lesions and normal tissues with the monoclonal antibody (Mab) A-80 that recognizes a mucinous glycoprotein from the colon carcinoma cell line LS-174T. The spectrum of benign and malignant breast lesions was studied as were epithelial tumors of the colon, stomach, pancreas, lung, salivary glands, thyroid, prostate, kidney, endometrium, skin and mesothelium; non-epithelial tumors included lymphomas, melanomas, gliomas, meningiomas, and sarcomas of soft tissue and bone. With a single exception, breast carcinomas regardless of histologic type were reactive while few fibroadenomas stained weakly and focally. In fibrocystic disease, the presence and intensity of the reactivity paralleled the severity of the epithelial proliferation, e.g. staining was strong in foci of severe or atypical hyperplasia, borderline lesions and carcinomas in situ; apocrine metaplasia stained often but less strongly. Barrett's mucosa, colonic polyps and most gastric and colonic carcinomas stained regardless of glandular features while small cell neuroendocrine carcinomas did not. Adenocarcinomas of the pancreas and lung, and a subset of large cell lung carcinomas reacted whereas neuroendocrine carcinomas of those sites did not. Carcinomas of endometrium, ovary and prostate reacted variably whereas thyroid and renal carcinomas and mesotheliomas were either negative or weakly reactive despite the presence of glands. Lymphomas, skin adnexal tumors, nevi, schwannomas, melanomas, gliomas and sarcomas generally did not react but occasional A-80-positive cells were seen in rare sarcomas and meningiomas. Immunostaining patterns in cytologic specimens were similar to the aforementioned. We conclude that Mab A-80 is an excellent marker for breast carcinomas, and for certain proliferative forms of fibrocystic disease that may precede or be associated with carcinomatous transformation. In colonic, pulmonary and gastric carcinomas, staining with Mab A-80 revealed exocrine features regardless of the absence of glands whereas in renal and thyroid carcinomas and in mesotheliomas staining was focal and weak or absent irrespective of glandular features. We suggest that Mab A-80 is a very promising immunolabel for select exocrine carcinomas, and for some of the dysplasias that may precede their development; its ease of application on tissue sections and cytologic specimens should broaden its usefulness.

Molecular characterization of the epitope in prostate and breast tumor-associated PR92 antigen.

In our previous report, monoclonal antibody PR92 has defined prostate- and breast tumor-associated PR92 antigen. The molecular nature of PR92 antigen, especially the epitope involved in specific interaction with PR92 monoclonal antibody, is described. PR92 antigen was purified from the cell extract or tissue culture medium of prostate cancer cell line DU145 by means of monoclonal antibody-coupled Sepharose 4B affinity chromatography, followed by a Sephacryl S-500 chromatography. Physical and chemical characterization, coupled with high-performance liquid chromatography, determined that PR92 antigen is a glycoprotein with a molecular weight of about 470,000, comprising repeating subunits of about 44,000. Sialic acid was found to form a critical part, while D-galactose and N-acetylgalactosamine were also involved, in the epitope structure. PR92 antigen is rich in serine, threonine, proline, glycine, and alanine and poor in aromatic amino acid residues. The carbohydrate moieties may be predominantly O-linked to polypeptide chains which contribute directly or indirectly to maintain the integrity of the epitope. Elucidation of the molecular nature of PR92 antigen may help understand the mechanism of shedding into the body fluids during tumor progression.

Immunohistochemical analysis of colon carcinomas applying exocrine and neuroendocrine markers.

Eighty colon carcinomas reflecting the histologic spectrum were studied immunohistochemically; their epithelial characteristics had been established by demonstrating cytokeratin polypeptides. Paraffin sections were immunostained with monoclonal antibody (Mab) A-80 that recognizes a mucin-like glycoprotein related to exocrine differentiation. Sequential sections were immunostained with neuroendocrine (NE) differentiation antibodies: NSE, human chromogranin A, serotonin, somatostatin, substance P and VIP. Twenty-one/80 carcinomas immunoreacted exclusively with Mab A-80; these included adenocarcinomas with variably defined glands, colloid, "solid", and linitits plastica carcinomas. Eleven/80 carcinomas immunoreacted only with antibodies to NE markers. Twenty-nine/80 carcinomas of histologically variable patterns expressed both exocrine and NE antigens. A notable group of 19 adenocarcinomas immunostaining with Mab A-870 included a minority NE cell subpopulation. We tentatively conclude that given a limited battery of immunoprobes, colon carcinomas comprise 4 groups: 1) pure exocrine carcinomas, 2) pure NE carcinomas, 3) mixed exocrine and NE carcinomas, and 4) exocrine carcinomas with occasional NE cells. Thus, phenotypically mixed exocrine and NE carcinomas comprise the largest group while the second largest group exhibited exclusively features of exocrine phenotype. Preliminary clinical correlative data indicate that pure NE colon carcinomas behave more aggressively than their exocrine counterparts; moreover, colon carcinomas containing a NE subpopulation, even if small, also seem to behave worse than their counterparts without an NE subpopulation.

Monoclonal antibody PR92 with restricted specificity for tumor-associated antigen of prostate and breast carcinoma.

A unique mouse hybridoma, PR92, was obtained using human prostate adenocarcinoma cell line DU145. The monoclonal antibody produced by the PR92 clone was reactive with DU145, MCF-7 (breast adenocarcinoma), and CHAGO (lung carcinoma), but not with normal cell lines and 16 other cell lines of cancerous origin. A homologous solid-phase sandwich radioimmunoassay (PR92-RIA) was developed utilizing PR92 monoclonal antibody. The PR92-RIA recognized unique antigen present in DU145 cell extract but did not detect 16 other known tumor-associated markers. In preliminary studies, the PR92-RIA measured greater than 5 units/ml level of PR92 monoclonal antibody-binding antigen in 23 of 31 (74%) serum specimens of active prostate cancer and 27 of 31 (87%) active breast carcinoma patients. Only 1 of 79 (1%) sex-matched normal donors and 1 of 57 (2%) benign disease control patients showed the serum antigen level greater than 5 units/ml. A high degree of correlation was observed between the PR92 antigen activity and the clinical status of four prostate and four breast cancer patients during therapeutic treatment. Thus, the PR92-RIA detects new tumor-associated antigen which may be useful in detection and monitoring of prostate and breast carcinoma patients.

Comparison of T-antigen expression in normal, premalignant, and malignant human colonic tissue using lectin and antibody immunohistochemistry.

The Thomsen-Friedenreich antigen has been implicated as a cancer-associated antigen in some human organs including the colon. Most previous studies of Thomsen-Friedenreich antigen expression in the colon used peanut agglutinin (PNA) to identify the immunodeterminant in tissues. However, evidence from other organs suggests that anti-T antibodies have specificities which differ from those of peanut lectin. To elucidate the nature of the T-immunodeterminant in colonic mucosa, we compared staining by PNA to that of a polyclonal (PAb) and monoclonal (MAb) anti-T antibody. PNA demonstrated the best sensitivity (91%) in cancer tissues but the lowest specificity (68%) in normal mucosa. Staining with MAb was only 76% sensitive but 100% specific. Sensitivity and specificity of PAb were intermediate between PNA and MAb. MAb stained fewer adenomatous polyps than either PNA or PAb, but staining appeared to correlate with premalignant features of the polyps. PNA-binding sites were more prevalent than either PAb or MAb in hyperplastic polyps. Cell cytoplasm was stained by both antibodies more often than by PNA. The majority of fetal colonic specimens stained with all three reagents suggesting that Thomsen-Friedenreich antigen may be an oncodevelopmental antigen in human colon. Differences in staining patterns in some tissues may be due to different antigenic specificities among PNA, PAb, and MAb.

A qualitative agar gel immunoprecipitin (IP) test for detection of fecal occult human hemoglobin.

A versatile, qualitative, agarose gel immunoprecipitation (IP) test for the detection of fecal occult hemoglobin (Hb) was developed to provide a more accurate method for the detection of occult blood in stool. This test allowed detection of 0.2 mg human hemoglobin/g of stool after a 2-h incubation. In addition, a specimen application device which can accommodate a variety of specimens was developed for this test. A study of 252 clinical stool specimens revealed a close correlation in the results obtained at three separate laboratories. The IP test identified blood in 11 out of 24 specimens collected from patients with colorectal carcinoma as compared to 4 of 24 specimens positive with a guaiac-based Hemoccult II test. The simplicity of immunoprecipitation coupled with the high sensitivity and specificity of this technique suggests that this new test would be a very useful and effective means with which to screen for occult blood in stool.

Immunoperoxidase localization of estrogen receptors in human brest carcinoma.

Monoclonal antibodies to the estrogen receptor protein were used to evaluate the specificity of the peroxidase antiperoxidase (PAP) method for localization of estrogen receptors (ER) in human breast carcinoma. Twenty-five cases of breast carcinoma were selected, and they were roughly classified as ER-rich and ER-poor tumors based on ER levels as determined by dextran-coated charcoal (DCC) assay. ER-rich tumors showed strongly positive cytoplasmic and, to a lesser degree, nuclear staining. Both the cytoplasmic and nuclear staining were less intense in ER-poor tumors, and the staining intensity varied from one to another cell population in the same tumor. Completely negative cells were also observed. The results of the PAP and DCC methods correlated fairly well in ER-rich tumors but not in ER-poor tumors. The PAP method provided visual localization of ER combined with excellent tissue morphology. Application of both methods (DCC and PAP) may provide better assessment of the estrogen receptor levels in breast carcinomas.

Galactosyltransferase variant in pleural effusion.

In measuring total galactosyltransferase activity in the pleural effusions from patients with benign or malignant diseases, we found no significant difference between the two groups (p greater than 0.05). However, a small amount of a galactosyltransferase variant, GT(l), could be separated from other galactosyltransferase enzymes in malignant pleural effusions by anion-exchange chromatography (DEAE-cellulose) with a buffer of low ionic strength. Other galactosyltransferases were eluted from the column with buffer of higher ionic strength. Using a mini-column method, we detected GT(l) enzyme in 19 of 26 specimens fro cancer patients, as compared with eight of 25 specimens from patients with benign disorders. The appearance of GT(l) enzyme in pleural effusion may be a tumor-associated phenomenon.

Preparation and specificities of antisera to the amino-terminal sequence of the carcinoembryonic antigen.

A tetracosapeptide (peptide-24) corresponding to the amino-terminal sequence of the carcinoembryonic antigen (CEA) was synthesized and characterized. Antisera were produced to the peptide-24, and a radioimmunoassay was developed utilizing peptide-24 with a tyrosine residue on the amino-terminal end (Tyr-peptide-24). Inhibitions of anti-peptide-24-125I-Tyr-peptide-24 complex formation were done with several preparations of CEA and the normal cross-reacting antigen. The extent of cross-reactivities was low, one CEA preparation requiring a 250-fold molar quantity greater than peptide-24 to obtain the same degree of inhibition. Attempts to degrade the CEAs and normal cross-reacting antigens in order to possibly expose the amino-terminal ends for reactivity with antibody did not result in any great increase in inhibitory capacity. It was concluded that either the conformations of the antigenic determinant(s) of the peptide-24 and of the amino-terminal end of CEA were sufficiently different to result in little cross-reacting antigen are blocked for reactivity with antibody by other portions of the molecule.

Human colon adenocarcinoma cells. III. In vitro organoid expression and carcinoembryonic antigen kinetics in hollow fiber culture.

Cell line LS174T was established in our laboratory from a primary human colon adenocarcinoma and was serially cultured for 4 years. Following 1 month of culture in perfused hollow fiber matrices, organoid growth reminiscent of the patient's original tumor was observed. The cellular organization was predominantly glandular with mucin located within gland lumina and cells. Glands had irregular brush borders, well-developed junctional complexes, and intracytoplasmic lumina lined with microvilli, features found in adenocarcinoma tissue. Alternate-day determinations of carcinoembryonic antigen (CEA) revealed biphasic kinetics in the extracapillary fluids of the hollow fiber system but not in identically treated monolayer cultures. The initial rate of CEA release was similar to that of monolayer cultures. A significantly accelerated secondary CEA release phase was observed in 10,000- and 50,000-molecular weight (MW)-exclusion fiber cultures (P less than or equal to 0.001 and P = 0.05, respectively). Totals of 43.8 micrograms and 112.6 micrograms CEA were released into the extracapillary fluids of 10,000- and 50,000-MW-exclusion fiber cultures, respectively, which were onefold and 2.3-fold increases, respectively, over monolayer supernatant yields. Most CEA was released during the final days of the secondary phase. In monolayer cultures, maximum CEA release occurred during the stationary phase of growth.

Extraction of human plasma or sera by heat treatment for a solid-phase radioimmunoassay of carcinoembryonic antigen.

Heat treatment and a solid-phase radioimmunoassay are combined to give a relatively simple and rapid procedure for assay of carcinoembryonic antigen in plasma or serum. The new way we describe to extract this antigen is an alternative to the conventional method of extraction with perchloric acid. Heating plasma or serum samples in acetate buffer (0.16 mol/L, pH 5.0) at 70 degrees C for 15 min precipitates out most of the heat-labile, nonspecific plasma proteins, but leaves most of the antigen in solution, with its immunochemical properties apparently unaffected. Comparison between the heat treatment and the perchloric acid extraction yielded comparable values when tested either by solid-phase radioimmunoassay or by the zirconyl phosphate precipitation method. An added advantage of our method is that it gives the same assay values for both plasma and serum. Results for a group of pathological plasma samples, assayed by both our method and the perchloric acid-zirconyl phosphate precipitation method, gave a correlation coefficient of 0.90.

A simplified solid-phase radioimmunoassay for carcinoembryonic antigen.

A simple and rapid solid-phase radioimmunoassay for the measurement of carcinoembryonic antigen (CEA) levels in sera or plasma is described. The procedure involves perchloric acid (PCA) extraction of the samples, followed by a rapid neutralization of excess acid with alkaline buffer solution. The PCA extracts are assayed uing antibody-coated plastic tubes and radio-labeled anti-CEA antibody as a marker. The assay may be completed in one day. The solid-phase direct-binding assay demonstrated a sensitivity of 0.5 ng CEA/ml plasmin and a marked tolerance to variations in pH and ionic strength of the system. A fair correlation between the CEA levels determined by the solid-phase radioimmunoassay and zirconium phosphate gel method was observed.

Human colon adenocarcinoma cells. II. Tumorigenic and organoid expression in vivo and in vitro.

A human colon epithelial tumor cell line (LS174T) recultured in vitro following passage through hamsters displayed differences in its cell doubling time and synthesis of carcinoembryonic antigen when compared with the cells grown solely in vitro. These animal-passaged cells more closely resembled the parent tumor cell line (LS180) derived from the primary tumor than LS174T, the trypsinized variant of LS180. Analysis of lactate dehydrogenase isoenzymes indicated that the tumor cells recovered from the hamsters were free of xenogeneic host tissue. Furthermore, LS174T grafted to athymic (nude) mice grew as a mucinous adenocarcinoma microscopically resembling the original tumor. The altered growth potential of LS174T was also demonstrated on confluent feeder monolayers of normal cells and by uninhibited multiplication in vitro. These results suggest that, at least in this one case, short-term passage of long-term cultured cells into xenogeneic hosts may effect a phenotypic reversion such that the cells regain properties observed in the primary tumor and the initial in vitro explant.