Probabilistic application of plasma carcinoembryonic antigen assay in cancer patients.

Plasma carcinoembryonic antigen (C.E.A.) levels in inpatients proved at necropsy to be cancer free were used to assess the ability of the C.E.A. assay to distinguish benign and malignant disease. The patients had a mean C.E.A. level significantly greater than that for young healthy people. In view of the considerable overlap of the ranges of plasma C.E.A. concentration in cancer patients and patients with non-malignant disease a probabilistic interpretation is advocated rather than the use of a simple cut-off between positive and negative. On the basis of the cancer-free control group, 19 out of 64 untreated patients with various solid tumours had plasma C.E.A. levels considered to correspond to a greater than 95% probability of cancer.     

Prostate-specific antigen levels among cirrhotic patients

PURPOSE: The aim of this study was to determine serum prostate-specific antigen (PSA) levels in patients with liver cirrhosis. PATIENTS AND METHODS: Between January 1995 and August 2001, 216 men with cirrhosis were evaluated. The extent of their liver disease was classified according to the Child-Pugh classification. Serum PSA levels were measured with the Hybritech Tandem-R RIA method and matched with age-related reference PSA levels. Digital rectal examination (DRE) was performed in all patients. Patients with elevated PSA levels and/or abnormal DRE were recommended to undergo further assessment including transrectal ultrasonography (TRUS) and biopsy performed by an urologist. RESULTS: Two hundred and sixteen men (mean age 54.09 +/- 9.09 years, range 25-76) with cirrhosis were examined. Their mean PSA value was 0.57 +/- 0.84 ng/mL and tended to be lower than in the normal population. The degree of PSA decrease was found to parallel the severity of the liver disease (p=0.002). The mean serum PSA level increased with each age decade in a statistically significant manner (p<0.001). Four patients (three with elevated PSA values) underwent prostate biopsy. Three biopsies were positive for prostate cancer, the other showed evidence of benign prostatic hyperplasia (BPH). CONCLUSION: Serum PSA is influenced by the severity of liver disease and its levels tend to be lower in cirrhotic patients than in the normal population. However, serum PSA can still be considered a reliable marker in the clinical management of prostatic disease in patients with cirrhosis.     

Biosynthesis and glycosylation of the carcinoembryonic antigen.

Human gastric adenocarcinoma MKN-45 cells were found to synthesize actively carcinoembryonic antigen (CEA). The biosynthesis and carbohydrate processing of CEA were studied in these cells by means of metabolic labelling followed by immunoadsorption with a specific polyclonal-antibody preparation and gel electrophoresis. Pulse-chase studies with [14C]leucine and [3H]mannose (shortest pulse 3 min) showed that N-linked oligosaccharide side chains are added to the protein co-translationally, producing a high-mannose immature CEA; the average molecular mass of this form is 145 kDa. The protein is later translocated to the Golgi apparatus and here undergoes additional processing; these modifications are visible in our system as a broadening of the CEA band and require about 4 h. The upper limit of mature CEA band reaches 200 kDa, but radioactivity is maximally incorporated at 168 kDa. The extent of co-translational glycosylation was measured by treating the cells with tunicamycin; in the presence of this inhibitor, a 74 kDa aglyco-CEA was produced and was still recognized by the antibody. Monensin, an ionophore which interferes with glycoprotein maturation and terminal sugar addition, blocked broadening of the CEA band, producing a sharp 141 kDa peak. In conclusion, CEA appears to be synthesized as a 145 kDa high-mannose immature form, the protein core accounting for about half of its molecular mass. Full maturation results in a broad band at 168 kDa.     

Visual detection of carcinoembryonic antigen on surfaces.

An attempt is made to detect carcinoembryonic antigen (CEA) at clinically interesting concentrations by using a simple immunologic surface test. Antibodies to CEA are detected in a direct test at concentrations below 1 ng/ml. The sensitivity of this assay is mainly limited by diffusion to the reacting surface. CEA is detected in an inhibition test at concentrations down to 20 ng/ml. The sensitivity of this inhibition test is limited by the average equilibrium constant in solution of the antibody-antigen reaction.     

Carcinoembryonic antigen gene and carcinoembryonic antigen expression in the liver metastasis of colorectal carcinoma.

The presence of the carcinoembryonic antigen (CEA) gene and CEA expression in the liver was tested to identify their possible roles in the liver metastasis of colorectal carcinoma. The CEA gene in the liver was identified by amplifying the CEA-specific N-terminal domain exon with digoxigenin-dUTP labeling in 16 colorectal carcinomas with liver metastases. Next, CEA expression was tested by immunostaining using the anti-CEA monoclonal antibody (T84.66, ATCC). Liver tissues from 13 stomach cancer patients and 12 colorectal cancer patients without liver metastasis were also tested as control groups. Three grades (<25%, 25-50%, and 50%< or =) were given according to the proportion of positive cells. The CEA gene was amplified in the metastatic tumor cells of the liver (2.6 +/- 0.2, mean grade +/- SEM) and their surrounding hepatocytes (1.5 +/- 0.2) in all cases. CEA expression was found in all metastatic tumor cells and 14 cases of the surrounding hepatocytes. Among the control groups, the CEA gene of the hepatocytes was found in 9 cases each of the colorectal and the stomach cancers that did not exhibit CEA expression. The level of serum CEA was related with the numbers and volume of liver metastases, but not with CEA expression in tumor cells and surrounding hepatocytes. The CEA gene in the metastatic tumor cells, not in the hepatocytes, was closely associated with CEA expression in the surrounding hepatocytes (p<0.01). Although the precise mechanism of CEA gene regulation in hepatocytes remains to be proven, the CEA gene in the metastatic tumor of the liver seems to affect CEA expression in the surrounding hepatocytes facilitating liver metastasis in colorectal carcinoma.     

Electron microscopy and physical characterization of the carcinoembryonic antigen.

Carcinoembryonic antigen (CEA), a glycoprotein material purified from human tumors, has been visualized by electron microscopy. At neutral pH, it consists largely of relatively homogenous, morphologically distinctive twisted rod or cruller shaped particles, with dimensions 9 x 40 nm. The particle length is considerably diminished at pH 40.0, which correlates with a known diminution of charge. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a molecular weight of 180,000 in the peak region of the CEA band for both 10 and 15% acrylamide. When native CEA was treated with neuraminidase, reduced, and alkylated, a relatively compact random coil was produced, whereas reduction and alkylation without neuraminidase treatment produced a less configuration, as determined by sedimentation studies and by electron microscopy. Electrophoretic migration, however, was apparently unaffected by reduction and alkylation. Thus the characteristic CEA particle appears by several lines of evidence to be substantially folded into a recognizably tertiary structural arrangement.     

Immunocytochemical localization of polyclonal carcinoembryonic antigen in hepatocellular carcinomas.

Hepatocellular carcinomas exhibit a unique canalicular immunocytochemical staining pattern with polyclonal antibodies directed against carcinoembryonic antigen (pCEA). The use of this method to facilitate a definitive diagnosis of hepatocellular carcinoma on fine needle aspirates of the liver was evaluated using aspirates and the corresponding core biopsy samples from nine cases. Immunoperoxidase staining with pCEA produced an identical canalicular staining pattern in 6 (66%) of 9 aspirates and 6 (75%) of 8 biopsy samples. The negative results in three aspirates may be due to their lack of the tissue fragments necessary to show this staining pattern. These findings indicate that the expression of this unique immunocytochemical staining pattern may aid in the diagnosis of primary hepatocellular carcinoma by fine needle aspiration cytology.     

Isolation of carcinoembryonic antigen]

A method employed in the authors' laboratory for isolating the carcinoembryonic antigen (CEA) from tumours of the entodermal digestive tract and their metastases is described. The main step is fractionation of the preparations obtained by perchloric acid extraction using gel filtration on Sephadex G-200, Sepharose 4 B, and Sepharose 6 B at pH 4.5 and 7.0. The peaks with CEA-specific antigeneity were characterized by the distribution coefficient of the CEA between the mobile and the stationary phases (Kav), and by the relative elution volume (ve/vo). The CEA purified by gel filtration could not reliably be enriched through preparative electrophoresis in Sephadex G-25 gel, polyacrylamide gel or Pevikon powder. The isolation procedures used were compared with those reported in the literature, and the results obtained are discussed.     

Structural studies on carcinoembryonic antigen. Periodate oxidation.

Periodate oxidation has been applied to examine the carbohydrate structure of carcinoembryonic antigen (CEA) and the possible role of the carbohydrate residues in its antigenic activity. Sialic acid (N-acetylneuraminic acid) and fucose were completely destroyed, and galactose and mannose were partially destroyed by a single periodate treatment. Serial periodate treatment (Smith degradation) destroyed additional amounts of galactose and mannose as well as significant amounts of N-acetylglucosamine. Prior removal of sialic acid by neuraminidase treatment led to increased destruction of galactose by periodate. Antigenic activity persisted indicating that the residues destroyed played little, if any, part in the antigenicity of CEA. These results yield an initial view of the structural arrangement of the carbohydrate residues in the CEA molecule.     

Phosphohexose isomerase and carcinoembryonic antigen in the sera of patients with primary lung cancer

Phosphohexose isomerase (PHI) and carcinoembryonic antigen (CEA) were measured at the time of diagnosis in 300 patients with lung cancer. Serum levels were high in 75.7% and 53.0% of patients respectively. PHI levels were higher in large cell and small cell carcinomas (p less than 0.001). CEA levels were higher in adenocarcinomas (p less than 0.001). Metastatic carcinomas showed higher levels on PHI and CEA than localized cases. Survival was significantly longer in patients with normal PHI (p less than 0.001) and normal CEA (p less than 0.005) than in cases with elevated markers. The prognostic significance of PHI persisted in the different pathological types and stages, whereas CEA only had prognostic impact in non-small cell cases. Serial PHI determinations were useful for follow-up in 82.4% of cases with initial abnormal values and in 55.4% of cases with a normal value. Serial CEA was useful in 41% of cases with initially high value but in less than 15% of those with baseline normal. We conclude that PHI has prognostic significance independently of pathology and stage, whereas CEA was a prognostic indicator only in non-small cell cases; serial PHI determinations were useful more often than CEA for follow-up.     

Murine monoclonal anti-idiotype antibody as a potential network antigen for human carcinoembryonic antigen.

Carcinomas of the gastrointestinal tract are not curable by standard therapies. Thus, new therapeutic approaches for this disease are needed. This study proposes the use of anti-Id mAb as Ag substitutes to induce anti-tumor immunity in gastrointestinal cancer patients. Recently, we have generated and characterized one monoclonal anti-Id antibody, designated 3H1 (Ab2), which mimics biologically and antigenically a distinct and specific epitope of the 180,000 m.w. carcinoembryonic antigen (CEA) primarily expressed in high density by human pancreatic and colonic tumor cells. This epitope is unique to CEA and not present on other CEA-related lower m.w. members of the Ag family also found on normal tissues. The antigenic determinant as defined by the mAb 8019 (Ab1) against which the Ab2, 3H1 was raised, is absent on normal adult tissues by immunoperoxidase staining and haematopoietic cells including granulocytes by flow cytometry analysis. Anti-Id (Ab2) 3H1 induced CEA-specific antibodies in mice and rabbits. The immune sera from both mice and rabbits competed with Ab1 for binding to the colon carcinoma cell line LS174T and inhibited the binding of radioiodinated Ab1 to Ab2. This indicates that anti-anti-Id (Ab3) in mice and rabbits share idiotopes with Ab1 (8019). Furthermore, monoclonal Ab3 that bind to CEA have been generated from mice immunized with 3H1. The Ab3 (both polyclonal as well as monoclonal) immunoprecipitated the same 180,000 m.w. CEA as Ab1 (8019) by Western blotting analysis and showed almost identical immuno-staining patterns as Ab1 on colonic adenocarcinoma tissue sections from several patients. Collectively these data suggest that Ab2 3H1 could potentially be used clinically as a network Ag for immunotherapy of patients with CEA positive tumors.     

Bacterial single-chain antibody fragments, specific for carcinoembryonic antigen.

We have produced single-chain antibody (scFv) fragments in bacteria specific for carcinoembryonic antigen (CEA). Polymerase chain reaction (PCR) was used for the cloning and modification of the heavy and light variable regions (VH and VL) of the mouse monoclonal antibody (MAb) CB-CEA.1. A 14-amino acid linker was used in the synthesis of the scFv gene. The VH and VL regions were amplified from cDNA by PCR using 5' end FR1 and 3' end constant region primers, and then sequenced. VH was then amplified by PCR using an exact 5' end FR1 primer, and a phosphorylated (PP) 3' end primer for J2 that also encoded the first 7 amino acids of the linker. VL was amplified with a PP 5' end primer for FR1, also encoding the remaining 7 amino acids of the linker, and a 3' end primer for J5, plus a stop codon and a BglII restriction site. The fragments were ligated and reamplified with the PP VH 5' and VL 3' end primers. The VH-linker-VL structure was blunt-cloned into expression vectors bearing the tryptophan promoter and pelB or ompA signal peptide sequences. Culture supernatant, bacteria pellet and periplasm preparations were assayed in Western blot and a protein of about 27 kDa was identified with rabbit antibodies specific for the Fab of CB-CEA.1. Bacterial supernatant and periplasm preparations also inhibited the recognition of CEA by HRP-labeled CB-CEA.1 in enzyme-linked immunosorbent assay (ELISA). Periplasm preparations were purified by affinity chromatography with specific anti-idiotypic MAbs. The Western blot of the eluates identified a protein of approximately 27 kDa that blocked the recognition of CEA by HRP-labeled CB-CEA.1 in ELISA. The VH-linker-VL structure was cloned into a vector bearing the lacZ promoter and the pelB signal peptide. The recombinant bacterial clones also expressed about 27 kDa scFv, specific for CEA.     

Combined carcinoembryonic antigen and cytopathologic examination in ascites

OBJECTIVE: To investigate use of the combined carcinoembryonic antigen (CEA) test and cytopathologic examination to improve the diagnosis of neoplastic vs. nonneoplastic ascites. STUDY DESIGN: The tests were performed prospectively on 130 patients with ascites whose effusions were submitted for cytologic examination. RESULTS: Sixty-seven patients had epithelial tumors, and the cytologic examination was positive in 39 (58.2%). The CEA level was > or = 11.0 ng/mL in 36 patients (53.73%). CEA was helpful in the diagnosis in 18 cases, increasing to 57 (85.07%) the number of positive diagnoses. Eight samples of nonepithelial tumors had low levels of CEA. In 55 patients with nonneoplasic ascites the cytopathologic examination was negative, but the CEA assay was > 11.0 ng/mL in 3 patients. CONCLUSION: The cytopathologic examination should be performed in all cases, and the CEA assay should be done in suspected cases of epithelial neoplasia in which the cytologic examination was negative, there was uncertainty about the histologic type of neoplasia, or a diagnosis of nonepithelial neoplasia was made. When ascitic leukocytosis or hepatic failure is present, one should be cautious in interpreting the CEA assay because false positivity can occur.