Carcinoma-associated Antigen Research Articles

Internal image anti‐idiotype antibodies related to renal‐cell carcinoma‐associated antigen G250

The potential usefulness of internal-image anti-idiotype antibodies (Ab2s) in modulating hosts' immune responses to tumor-associated antigen (TAA) have stimulated considerable interest in the development and characterization of Ab2s. Six different mouse monoclonal Ab2s (NUH31, 44, 51, 71, 82 and 91) were generated against murine monoclonal antibody G250 (MAbG250) which recognizes a human renal-cell carcinoma-associated antigen. All 6 Ab2s showed specificity for the MAbG250 paratope in Western-blot analysis. In inhibition assays, all Ab2s were able to compete with the nominal antigen, albeit with differing efficiency. Based on cross-blocking studies for idiotope mapping, the Ab2s could be divided into 2 groups (group I; NUH31, 51, 71, group 2; NUH44, 82, 91). However, cross-reactivity between these 2 groups was observed, indicating that they recognized partly overlapping epitopes on the paratope of MAbG250. Sera from rabbits immunized with Ab2s showed reactivity with G250 antigen-positive cell lysates, but not with antigen-negative cell lysates. Additional studies revealed that all Ab2s were able to induce anti-anti-idiotype antibodies resembling MAbG250 (Ab1'). These findings suggest that the Ab2s functionally mimic the original G250 antigen and may be of use in the immunotherapy of human renal-cell carcinoma.

Relation between ovarian carcinoma-associated antigens in tumor tissue and detached cyst fluid cells of patients with ovarian neoplasms.

The expression and potential diagnostic value of ovarian carcinoma-associated antigens were estimated in different types of epithelial ovarian neoplasms. The comparison of antigenic expression was performed on solid tumor tissues and loose cyst fluid cells in individual cases of malignant and benign ovarian neoplasms. All studies were performed using monoclonal antibodies (mAbs) against ovarian carcinoma-associated antigens (OC125, OV-TL3, OV632, 10B, 8C) by 3-step peroxidase-antiperoxidase test. All ovarian carcinoma-associated antigens were detected in most serous and endometrioid carcinomas. In mucinous carcinomas as well as in benign ovarian neoplasms these antigens were present only in some cases. Significant inter- and intratumoral immunological heterogeneity was evident; however, the antigens detectable in tissue sections were also found in detached cyst fluid cells. Our results show that mAb show OV-TL3 is the best marker for endometrioid carcinomas and confirmed that mAbs OV632, OC125 and OV-TL3 could be good complementary markers for differentiating malignant and benign lesions in the ovary. The percentage content of all ovarian carcinoma-associated antigens in solid tumors and respective cyst fluid cells was comparable.

An Expression Profile of Active Genes in Human Colonic Mucosa

An expression profile of genes active in the human colonic mucosa was obtained by collecting 959 partial sequences from a 3'-directed cDNA library. Seven genes were found to produce mRNA each of which comprised more than 1% of total mRNA. Four of these genes are novel, and are likely to be uniquely expressed in the colonic mucosa, and the other three have been identified as genes for fatty acid binding protein, immunoglobulin lambda chain, and carcinoma-associated antigen GA733-2. In the remaining 952 clones, 310 were composed of 118 species occurred recurrently but less than 1%, and 533 clones appeared only once. Because the 3'-directed cDNA library faithfully represents the mRNA population in the source tissue, these numbers represent the relative activities of the gene expression. Altogether 156 gene species were identified in GenBank, and a significant portion of these genes encode proteins found in Golgi apparatus and lysosomes, chromosome-encoded mitochondrial proteins, cell surface proteins, and components in the protein synthesis machinery. The types and proportions of genes identified is consistent with the known major activities of the colonic mucosa such as mucous protein production, energy-dependent water absorption, and rapid cell proliferation and turnover.

Treatment of disease-negative but mucin-like carcinoma-associated antigen-positive breast cancer patients with tamoxifen: preliminary results of a prospective controlled randomized trial

Increasing levels of tumor markers such as carcinoembryonic antigen, mucin-like carcinoma-associated antigen (MCA), CA 15.3, and monoclonal antibody H23 in breast cancer patients following the treatment of the primary disease and adjuvant radiation and chemotherapy reflect subclinical development of metastatic disease. Overt metastatic disease is usually incurable and prolongation of life at this stage is impossible, and the treatment is only palliative. The efficacy of tamoxifen, a least-toxic agent, in the treatment of early and minimal metastatic disease detected only by increasing serum levels of MCA was studied prospectively in a randomized study. Our preliminary, albeit encouraging, results showed that the rate of relapse within a median follow-up period of 11 months was 24.1% in the control arm as compared with 0% in the tamoxifen arm (Fisher's exact test, P = 0.012). None of the patients with a relapse had positive progesterone receptors (PR). We may carefully conclude that early treatment may be warranted in young patients with negative PR and continuously increasing serum levels of the marker.

Expression of squamous cell carcinoma-associated antigen in grade 3 pT1 transitional cell carcinoma of the bladder and prediction of its progression and intravesical recurrence

Among superficial transitional cell carcinomas (TCC) of the bladder, Grade 3 pT1 disease is associated with a higher risk of progression and intravesical recurrence. The authors determined whether or not squamous cell carcinoma-associated antigen (SCC antigen) could predict clinical behavior of Grade 3 pT1 disease. SCC antigen was immunohistochemically stained in Grade 3 pT1 TCC of the bladder that had been fixed in formaldehyde solution and embedded in paraffin. Patients were followed up, and disease progression and recurrence were identified. Disease progression was defined as histologically verified muscle invasion or clinically detectable distant metastasis, including pelvic lymph node metastasis. SCC antigen in the cytoplasm was positively identified with immunohistochemical staining in 21 of 55 patients with the disease. Of 21 patients with positive SCC antigen in the cytoplasm, 8 experienced disease progression, whereas there was progression in 5 of 34 patients with negative antigen. The nonprogression rate of Grade 3 pT1 carcinomas with positive antigen in the cytoplasm was significantly lower than that for those with negative antigen. The disease-free rate also was clearly lower in the positive carcinoma than in the negative. Multivariate analysis confirmed that SCC antigen in the cytoplasm was the only significant variable independently affecting progression and intravesical recurrence of the disease. The results suggested that immunohistochemical expression of SCC antigen in the cytoplasm is closely linked with a higher risk for progression and intravesical recurrence of Grade 3 pT1 disease. Detection of the antigen can help to more accurately predict the clinical course of the disease.

Clinical utility and validation of emerging biochemical markers for mammary adenocarcinoma

The clinical utilities of the emerging biochemical markers for mammary adenocarcinoma CA 15-3, CA 549, CA M26, CA M29, and MCA (mucin-like carcinoma-associated antigen) were assessed by a formal rating according to six desirable marker characteristics. All five indicators similarly have good specificities but limited sensitivities. As a consequence, these markers mainly meet just two desirable criteria: their frequency and degree of expression reflect tumor burden and prognosis, and they may correlate with therapeutic results. The validation of these assay properties by clinical trials was evaluated by another rating system, designed to assess proband sample selection, restrictions on allowable observations, and choice of statistical descriptors. By these benchmarks, the estimates of the prior probabilities of test outcome (sensitivity and specificity) are reasonably definitive, but conclusive judgments about the posterior probabilities of test outcome ("predictive values") and about values and costs associated with testing are not possible. Three approaches to enhance the limited clinical utility of biochemical breast cancer markers are considered: shifts of the diagnostic decision threshold, marker panels, and sequential testing. However, none of these strategies improves the described performance characteristics.

Production of a human monoclonal antibody to normal basal and squamous cell carcinoma-associated antigen.

A human monoclonal antibody, BM2, was produced by a hybridoma line generated by fusion of lymph node cells from a patient with squamous cell carcinoma (SCC) of the tongue with human B-lymphoblastoid cell line HO-323. BM2, an IgM class antibody, was reactive with all of the SCC cell lines tested. Frozen sections of normal and malignant tumor specimens were investigated to examine the reactivity of BM2 towards them. All 35 oral SCC specimens reacted with BM2. Normal stratified squamous epithelium showed positive staining in basal cells, but no staining was seen in other layers of the stratified epithelium, simple epithelium, and tissues of nonepithelial origin. Ductal basal cells of normal salivary gland also showed positive staining. Western blotting and immunoprecipitation analysis revealed that BM2 recognized 52 kDa membrane-associated protein. BM2 may therefore be a useful tool for biological and clinical studies of SCC.

Induction of delayed-type hypersensitivity responses by monoclonal anti-idiotypic antibodies to tumor cells expressing carcinoembryonic antigen and tumor-associated glycoprotein-72.

The use of anti-idiotypic antibodies as immunogens represents one potential approach to active specific immunotherapy of cancer. Two panels of syngeneic monoclonal anti-idiotypic antibodies were generated. One panel was directed against mAb CC49 and the other to mAb COL-1. mAb CC49 recognizes the pancarcinoma antigen (Ag), tumor-associated glycoprotein-72 (TAG-72), and mAb COL-1 recognizes carcinoembryonic antigen (CEA). Seven anti-idiotypic (AI) antibodies (Ab2) designated AI49-1-7 were generated that recognize the variable region of mAb CC49. These mAb were shown to inhibit the interaction of mAb CC49 (Ab1) with TAG-72 (Ag). Five anti-idiotypic antibodies designated CAI-1-5 were also generated to the anti-CEA mAb, COL-1 (Ab1). These Ab2 were shown to inhibit the interaction between COL-1 (Ab1) and CEA (Ag). Immunization of mice, rats, and rabbits with Ab2 directed against CC49 or COL-1 could not elicit specific Ab3 humoral immune responses, i.e., antibody selectively reactive with their respective target antigens. However, immunization of mice with the CC49 anti-idiotypic antibody (Ab2), designated AI49-3, could induce a delayed-type hypersensitivity response (DTH) specific for tumor cells that express TAG-72. Similarly, immunization of mice with an anti-idiotypic antibody directed against COL-1, designated CAI-1, could induce specific DTH cell-mediated immune responses to murine tumor cells that express human CEA on their surface. These results thus demonstrate that while some anti-idiotype mAb may not be potent immunogens in eliciting Ab3 humoral responses, they are capable of eliciting specific cellular immune responses against human carcinoma-associated antigens. This type of mAb may ultimately be useful in active immunotherapy protocols for human carcinoma.

Expression of Colorectal Carcinoma-Associated Antigens in Colonic Polyps

Immunohistologic techniques were used to study the expression of colorectal carcinoma-associated antigens in colonic polyps and to compare this with expression in the normal colonic epithelium. Forty-nine polyps were studied using monoclonal antibodies to 16 different blood group and differentiation antigens and carcinoemhryonic antigen epitopes. With the Lewis a antigen and the two epitopes of CEA recognized by 3D6 and COL-4 expression in polyp tissue was the same as that in the normal colon. Five types of alteration of antigen expression in polyps were seen. The blood group antigens A, B, and Lewis b, which are expressed only on the right side of the normal adult colon, were detected in both neoplastic and nonneoplastic polyps from the distal colon. The Lewis x antigen and the antigen epitopes detected by the antibodies COL-12, CA19-9, ME491, and GA73,3 showed an increased frequency of expression in all types of polyps in comparison with the normal colonic epithelium, while H-type 2, ND4, and the antigen epitope detected by C029.11 showed a slightly decreased frequency of expression in polyp tissue. The X-like antigen which was expressed in only 7% of normal colon specimens showed increased frequency of expression in polyp tissue with significantly greater expression in neoplastic than hyperplastic lesions ( P = 0.003). The TAG-72 antigen was detected only in adenomas with severe dysplasia ( P = 0.01), correlating well with premalignant histology. These findings have helped us clarify the variation of antigen expression in colonic polyps and allowed us to define which antigens are worthy of further investigation as markers of possible malignant transformation.

Tumor-associated antigens in effusions of malignant and benign origin

We determined the concentration and effusion/serum ratio of mucin-like carcinoma-associated antigen (MCA) in comparison to carcinoembryonic antigen, carbohydrate antigen 19-9, cancer antigen 125, and cancer antigen 15-3 in 80 sera and 99 effusions from 64 patients with histologically confirmed malignancies (4 patients out of this group showed various effusions simultaneously, which were analyzed separately) and 31 patients with various nonneoplastic diseases. Tumor cells were detected by cytological examination in 41 effusions (60.3%) from patients with neoplastic diseases, while in another 27 cases this method failed to demonstrate the malignant origin of the effusion. Of the cytological "positive" malignant effusions 90% were also correctly identified by an elevated MCA concentration at a cutoff level of 10 U/ml, whereas only one effusion of benign origin (3%) showed a slightly elevated MCA concentration of 10.5 U/ml. In 33% of cytologically "negative" effusions of patients with neoplastic diseases, the MCA concentration was also elevated, with a maximum of 453 U/ml. Increased MCA levels in cytologically confirmed malignant effusions were not restricted to metastatic breast cancer. All 17 cytologically "positive" "non-breast cancer" effusions were correctly identified by their MCA concentrations. None of the other tumor markers reached this high sensitivity at the same level of specificity. The ratio of effusion/serum concentration of all tumor markers as well as the concentration of cancer antigen 125 in effusions was of little diagnostic value. Our results indicate that the MCA concentration in an effusion correlates very closely with its malignant origin and is superior to all the other antigens tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Mucin-like carcinoma-associated antigen (MCA) in breast cancer: clinical experience at the National Cancer Institute of Milan.

Mucin-like Carcinoma-associated Antigen (MCA) is a glycoprotein belonging to the mucin family; it is defined by the monoclonal antibody b-12. Mucins represent an interesting group of tumor markers and are widely utilized in the clinical monitoring of neoplastic patients. These molecules show a certain degree of tissue specificity and MCA is preferentially associated with breast tissue. Several studies have demonstrated that patients with breast cancer usually have high MCA serum levels. In this paper the experience of the National Cancer Institute of Milan with the clinical use of MCA in breast cancer patients is reported. The observed sensitivity of the MCA test was poor in patients with early-stage disease, while it was acceptable in patients with advanced breast cancer. MCA concentrations appeared to be directly related to disease spread. A clear relationship was seen between MCA levels and lymph-nodal status. The highest MCA plasma levels were observed in patients with metastatic disease. In this group of patients the sensitivity of the test on the basis of a cut-off of 11 U/mL was 52%.

Biochemistry and Molecular Biology of MCA

The mucin-like carcinoma-associated antigen (MCA) is a mucin with a molecular weight of 350-500 kD. It circulates in the serum and its serum content can be determined with the Cobas Core MCA EIA test. Patients with breast cancer show elevated MCA serum levels. The molecule has a polypeptide backbone consisting of three parts: the C-terminus the N-terminus and the transmembrane sequences. The protein is heavily glucosylated with carbohydrate side chains that contain fucose, galactose and N-acetyl galactosamine. The antibody b-12 recognizes a repetitive epitope on the peptide portion of the MCA molecule. The epithelial mucin, which is coded by a unique gene, was cloned using PCR technology. Peptides corresponding to the N- and C-terminus were expressed in E. coli. Analysis of the purified peptides revealed molecular weights of 12 and 18 kD.

Clinical significance of the new tumor marker MCA in the follow-up of patients with mammary carcinoma.

MCA is a mucin-like carcinoma-associated antigen which has been found in all breast cancer independent of histological type and degree of differentiation. A two-step solid-phase EIA was developed and the serum concentration of MCA was measured in 176 breast cancer patients after surgery. Using a cutoff of 11.0 U/ml the test showed a sensitivity of 77% and a specificity of 82% when compared to clinical status. The predictive value of this tumor marker was 72% for a positive diagnosis and 93% for a negative diagnosis. Based on these observations it is recommended that determination of MCA values take place 2 to 3 times in the first postoperative week in all women with surgically treated mammary carcinoma. These MCA values should then serve as reference for further determinations, which should be performed at each check-up. A subsequent increase in the MCA value should be considered as a first sign of metastasis.

Retroposition in a family of carcinoma-associated antigen genes.

The gene encoding the carcinoma-associated antigen defined by the monoclonal antibody GA733 is a member of a family of at least two type I membrane proteins. This study describes the mechanism of evolution of the GA733-1 and GA733-2 genes. A full-length cDNA clone for GA733-1 was obtained by screening a human placental library with a genomic DNA probe. Comparative analysis of the cDNA sequence with the previously determined genomic sequence confirmed that GA733-1 is an intronless gene. The GA733-2 gene encoding the monoclonal antibody-defined antigen was molecularly cloned with a cDNA probe and partially sequenced. Comparison of GA733-2 gene sequences with the previously established cDNA sequence revealed that this gene consists of nine exons. The putative promoter regions of the GA733-1 and GA733-2 genes are unrelated. These findings suggest that the GA733-1 gene was formed by the retroposition of the GA733-2 gene via an mRNA intermediate. Prior to retroposition, the GA733-2 gene had been affected by exon shuffling. Analysis of GA733-2 exons revealed that many delineate structural motifs. The GA733-1 retroposon was localized either to chromosome region 1p32-1p31 or to 1p13-1q12, and the GA733-2 founder gene was localized to chromosome 4q.

Retroposition in a Family of Carcinoma-Associated Antigen Genes

The gene encoding the carcinoma-associated antigen defined by the monoclonal antibody GA733 is a member of a family of at least two type I membrane proteins. This study describes the mechanism of evolution of the GA733-1 and GA733-2 genes. A full-length cDNA clone for GA733-1 was obtained by screening a human placental library with a genomic DNA probe. Comparative analysis of the cDNA sequence with the previously determined genomic sequence confirmed that GA733-1 is an intronless gene. The GA733-2 gene encoding the monoclonal antibody-defined antigen was molecularly cloned with a cDNA probe and partially sequenced. Comparison of GA733-2 gene sequences with the previously established cDNA sequence revealed that this gene consists of nine exons. The putative promoter regions of the GA733-1 and GA733-2 genes are unrelated. These findings suggest that the GA733-1 gene was formed by the retroposition of the GA733-2 gene via an mRNA intermediate. Prior to retroposition, the GA733-2 gene had been affected by exon shuffling. Analysis of GA733-2 exons revealed that many delineate structural motifs. The GA733-1 retroposon was localized either to chromosome region 1p32-1p31 or to 1p13-1q12, and the GA733-2 founder gene was localized to chromosome 4q.

Characterization of cis-acting elements regulating transcription of the human DF3 breast carcinoma-associated antigen (MUC1) gene.

The present studies have examined the sequences responsible for regulating transcription of the human DF3 breast carcinoma-associated antigen (MUC1) gene. A region 1656 base pairs upstream to the DF3 transcription initiation site was fused to the chloramphenicol acetyltransferase gene. Transient expression assays using a series of deleted constructs demonstrated that the region from position -618 contains the regulatory sequences necessary for DF3 transcription in human MCF-7 breast cancer cells. Further analysis with internal deletion vectors and heterologous promoter constructs indicated the involvement of cis-acting elements in the fragment extending from positions -598 to -485. By gel retardation and DNA footprinting, we have identified a protein in MCF-7 cells that recognizes sequences between positions -505 and -485. The results of Southwestern studies demonstrate that this protein has an apparent molecular mass of 45 kDa. Taken together, these results suggest that DF3 gene transcription is regulated by a previously undescribed transacting factor.

Construction via polymerase chain reaction of a mouse/human chimeric heavy chain gene of a monoclonal antibody toward human colorectal carcinoma-associated antigen

Construction via polymerase chain reaction of a mouse/human chimeric heavy chain gene of a monoclonal antibody toward human colorectal carcinoma-associated antigen

The comparison of the expression of ovarian carcinoma-associated antigens and CEA in tissue sections and respective cyst fluid cells of ovarian neoplasms

The comparison of the expression of ovarian carcinoma-associated antigens and CEA in tissue sections and respective cyst fluid cells of ovarian neoplasms

A rapid and simple method for the purification of tumor cells from ascitic fluid of ovarian carcinoma

Rapid isolation of tumor cells growing in clumps from the ascitic fluid of patients with ovarian carcinoma was achieved by harvesting cells from ascitic fluid and subsequently passing them over a 30-microns nylon mesh filter. Single cells and small cell aggregates passed through the mesh, and large cell clumps that had not passed through the filter were isolated by backwashing. Morphologically, the cells in the clumps consisted almost entirely of tumor cells. The clumps were analyzed by immunocytochemistry and only a small proportion of cells (3.1 +/- 0.5% to 8.3 +/- 0.8%) stained with anti-CD45 monoclonal antibody (a panleukocyte marker for cells of hematopoietic origin). Most of the cells in clumps stained positively (90.6 +/- 1.7% to 97.5 +/- 0.5%) with 2G3 (a monoclonal antibody binding to a high-molecular-weight carcinoma-associated antigen). Unfiltered cell populations, however, contained up to 34.4 +/- 2.1% contaminating CD45+ non-tumor cells. This method should be useful for rapidly and easily obtained highly enriched fresh ovarian carcinoma cells from ascitic fluid in a form in which they grow naturally.

Tumour detection and localization using 99Tcm-labelled OV-TL 3 Fab?? in patients suspected of ovarian cancer

Fab' fragments of the monoclonal antibody OV-TL 3, that recognizes an ovarian carcinoma-associated antigen (OA3), were labelled with 99Tcm using D-glucarate as a ligand. Twenty patients suspected of having primary or recurrent ovarian cancer received intravenously 1 mg of the Fab' labelled with 740 MBq 99Tcm. Both planar and single photon emission computed tomographic (SPECT) scintigraphy were performed up to 30 h after intravenous infusion. In 19 out of 20 patients surgical and histopathological evaluation was performed between 2 and 6 days postinfusion. Imaging results were compared with X-ray computed tomography (CT), ultrasonography (US) and CA 125 serum level. Blood clearance was fast with median t1/2 beta of 9.5 h. Thirty-seven per cent of the injected dose (% ID) was excreted in the urine within the first 24 h, whereas 7% ID was excreted in the 24 h faeces. In one patient with an OA3 negative ovarian carcinoma, radioimmunoscintigraphy (RIS) did not visualize the tumour. In two other patients a benign ovarian cyst was found, also showing no elevated uptake. In 13 out of 17 patients ovarian cancer lesions were detected with RIS, whereas CT and US detected lesions in, respectively, 15 and 12 patients. Of 36 surgically defined tumour deposits larger than 1 cm in diameter, 53% were detected and localized with RIS, whereas CT and US detected 61 and 40%, respectively. Radioimmunoscintigraphy with 99Tcm-OV-TL 3 Fab' is less distressing for the patients but the overall imaging performance is not improved when compared with 111In-OV-TL 3 F(ab')2.