Multiple epitopes on a human breast-carcinoma-associated antigen

The monoclonal antibody (MAb) NCRC-11 identifies an epitope expressed variably in human breast cancer. The degree of expression of this epitope in primary operable tumours is closely related to the subsequent clinical course of the disease (Ellis et al., 1985). The target antigen for NCRC-11 was isolated from subcellular membranes of breast carcinomas and purified by immunoadsorbent chromatography. NCRC-11 epitopes were expressed upon a large glycoprotein of more than 400 kd. This material was susceptible to degradation by pronase and papain and contained N-acetylglucosamine, as indicated by its binding to wheat-germ agglutinin. The NCRC-11-defined antigen expressed epitopes for the anti-human milk-fat globule membrane antibodies HMFG-1 and HMFG-2, and other antibodies against epithelial membrane antigens (EMA, LICR-LON-M8). The reactivity of these antibodies with tumour membranes was also similar, but not identical, to that of the NCRC-11 antibody. In competitive binding-inhibition assays, these antibodies partially inhibited the binding of 125I-NCRC-11 antibody to antigen, suggesting that the epitopes involved are topographically closely associated. Sandwich immunoassays demonstrated that NCRC-11 epitopes are likely to represent repeated structures of the NCRC-11 antigen. The findings presented are interpreted as indicating that the NCRC-11 antigen expresses a variety of epitopes which are associated with normal differentiation and malignant change.     

C595--a monoclonal antibody against the protein core of human urinary epithelial mucin commonly expressed in breast carcinomas.

Urinary mucins which express determinants for the anti-breast carcinoma monoclonal antibody, NCRC-11 (IgM), closely resemble the mammary mucins found in milk fat globules and carcinomas. An IgG3 monoclonal antibody, C595, was prepared against urinary mucins isolated on a NCRC-11 antibody affinity column, and this 'second generation' antibody was shown to have a very similar pattern of reactivity to the original NCRC-11 antibody. By immunohistology, the profile of reactivity of both antibodies with tumour and normal tissue specimens was virtually identical. Both antibodies reacted with epithelial mucins isolated from breast tumours or normal urine using an NCRC-11 antibody affinity column, although the antibodies were unreactive with other antigen preparations. Heterologous immunoradiometric assays ('sandwich' tests) confirmed that NCRC-11 and C595 epitopes were co-expressed on the same molecule. C595 antibodies inhibited the binding of radiolabelled NCRC-11 antibodies to antigen, suggesting that the two epitopes were in close topographical proximity. The protein core of the mammary mucins has recently been shown to consist predominantly of a repeated 20 amino acid sequence (Gendler et al., 1988). Peptides with this complete sequence and small fragments were synthesised, and the C595 antibody was found to recognise an epitope within this repeat. The ability to identify and synthesise monoclonal antibody-defined determinants, as well as those in the adjacent or overlapping sequences within the protein core of epithelial mucins, is viewed as a strategy for facilitating the production of antibodies of new and novel specificity to complement the panels of existing anti-breast cancer reagents.     

Epitope analysis of monoclonal antibody NCRC-11 defined antigen isolated from human ovarian and breast carcinomas

NCRC-11 is an IgM monoclonal antibody which defines an antigen found in most epithelial malignancies. The antigen has previously been shown to be a high mol. wt. glycoprotein (greater than 400,000) and in this study, antigen preparations were isolated by immunoadsorbent chromatography from ovarian mucinous and ovarian serous cyst adenocarcinoma and from breast carcinoma. Other monoclonal antibodies, against products in normal human milk, and antibodies of the Ca series (Bramwell et al., 1985) reacted with all three antigen preparations. Tests involving epitope mapping were performed to probe the relationships of the various epitopes to that defined by the NCRC-11 antibody, and, of note, the three antigen preparations from different tumour sources were remarkably similar with respect to their relative levels of epitope expression and to their topographical distribution of epitopes. The major differences in epitope expression could be attributed to the degree of sialylation in the three antigens. The antigens from ovarian tumours expressed I(Ma) blood group determinants (defined by the antibody LICR-LON-M18) which were partially masked by sialic acid. With NCRC-11 defined antigen from breast carcinoma, this determinant was totally masked by sialic acid although neuraminidase treatment clearly exposed epitopes reactive with M18 antibodies.     

Polymorphic epithelial mucin (MUC-1)-containing circulating immune complexes in carcinoma patients.

Circulating immune complexes (CICs) containing polymorphic epithelial mucin (PEM/MUC-1) were found in sera of 24.5% of 151 primary breast carcinoma patients and 18-21.4% of patients with advanced ovarian (n = 56) and breast carcinomas (n = 61), 37% of patients with benign breast tumours, but in only 2.1% of 96 healthy individuals. The incorporation of PEM into CICs affects the detection of circulating PEM in commercial immunoassays such as the CA 15-3 assay, as suggested by a negative correlation between levels of PEM-containing immune complexes (PEM-CICs) and CA 15-3 values, and confirmed by isolation of PEM from CA 15-3-negative sera containing high levels of PEM-CICs. The amounts of PEM masked by human antibodies correspond to significant values of the CA 15-3 assay when monitoring patients for carcinoma. Most antibodies in PEM-CICs were of IgG class, suggesting their specific nature to the PEM epitopes.     

The prognostic value of immunoperoxidase staining with monoclonal antibodies NCRC-11 and 3E1.2 in breast cancer

The variation in survival of women with clinically similar breast cancers may lead to difficulty in clinical management so it is important to recognise factors which indicate the prognosis. Immunoperoxidase staining patterns of primary breast tumours using monoclonal antibody NCRC-11 have been shown to relate to overall survival (Ellis et al., 1985) but the results have not been reproducible in other centres. In this study paraffin sections of 483 primary breast cancers were stained with NCRC-11 and 3E1.2 using an immunoperoxidase system. The tumour staining patterns were compared with overall survival using life tables and tested for relative prognostic significance by Cox's multivariate analysis. NCRC-11 related to survival in all 483 cases (chi 2 5.8, P = 0.02) but both antibodies achieved maximum prognostic significance in lymph node negative patients (chi 2 9.4, P less than 0.002 and chi 2 10.7, P less than 0.001) in whom no other factor was more significant. Immunoperoxidase staining patterns produced by monoclonal antibodies NCRC-11 and 3E1.2 are important prognostic factors in breast cancer.     

Flow cytometric screening of monoclonal antibodies for drug or toxin targeting to human cancer

Monoclonal antibodies (MAbs) that are candidates for antibody-directed therapy were evaluated by a flow cytometric method. This method accurately quantitates the intensity of staining and the percentage of cells from freshly derived primary tumors expressing the relevant cell surface antigens. This method was applied to human colorectal, gastric, and ovarian carcinomas. It allowed calculations of the number of drug molecules that potentially could be delivered by each MAb as well as selection of the optimal combinations of antibodies for treatment of each type of cancer. The binding of all the MAbs varied among the tumors, although combinations of antibodies reduced this problem. A combination of MAbs C14 and NCRC-23 recognized 97% of colorectal tumors. A combination of C14, NCRC-23, and 791T/36 recognized 95% of gastric tumors. Combinations of either 791T/36 and C14 or 791T/36 and NCRC-11 recognized 80% of ovarian tumors. The number of cells binding with a single MAb varied within the tumor. The optimal anti-colorectal tumor antibody was NCRC-23 (anti-carcinoembryonic antigen), which recognized a mean of 65% of the large cells within a tumor at a mean antigen density of 4.9 X 10(5) sites/cell. The optimal anti-gastric tumor antibody was C14 (anti-Y hapten), which recognized a mean of 66% of the large cells within a tumor at a mean antigen density of 4.4 X 10(5) sites/cell. The optimal anti-ovarian antibody was 115/D8, which recognized 54% of the large cells at a mean antigen density of 4.2 X 10(5) sites/cell. These antigen densities were similar to those calculated for HLA/ABC antigens in colorectal and ovarian cancers. However, the gastric tumors expressed elevated levels of major histocompatibility complex class I antigens, with a mean density of 7.3 X 10(5) sites/cell. Combinations of antibodies that recognize a high proportion of tumor cells are likely to be necessary for MAb-drug targeting to prevent tumor recurrence and/or metastases.     

Identification of an H antigen-like blood group antigen in sera of cancer patients using a novel monoclonal antibody raised against endometrial carcinoma

An IgM class monoclonal antibody (MAb) was derived by immunizing BALB/c mice with a human endometrial carcinoma cell line. This MAb, termed C12, exhibited strong reactivity against endometrial carcinoma, but lesser reactivity against normal endometria. The antigen recognized by MAb C12 (C12 antigen) was detected by radiometric assay in sera from patients with various carcinomas, but not in sera from patients without carcinomas or in sera from normal individuals. MAb C12 was found to agglutinate blood type O erythrocytes, but not A, B, or AB erythrocytes. To clarify the specificity of MAb C12, tissue staining experiments were performed in parallel using MAb C12, Ulex europaeus lectin I (anti-H), and a monoclonal anti-H antibody. In endometrial carcinoma tissues, both H and C12 antigens increased, but the C12 antigen showed a prominent increase, in contrast to the H antigen. Further, the C12 antigen was not found in endothelial cells of blood type O patients. In sera, the level of the H antigen varied according to the host's blood type. The sera from blood type O individuals possessed higher levels of the H antigen than those with blood type A or B. Thus, the H antigen showed no value as a tumor-associated serum marker. In contrast, the presence of the C12 antigen in sera was not determined by ABO blood group status. Thus, MAb C12 was demonstrated to be a unique MAb that reacts with an H-like antigen occurring in the sera of patients with carcinomas irrespective of ABO blood group status. MAb C12 may prove to be a useful marker for cancer patient serum.     

Cellular effects of tamoxifen in primary breast cancer.

We have investigated the effect of tamoxifen on the biological characteristics of the primary tumour in 33 patients with breast cancer. All patients had pre-treatment biopsy of the primary breast cancer and subsequent trucut biopsy of the primary tumour after 1-4 months on tamoxifen therapy. The staining patterns in the primary tumour of each of the tumour antigens 115D8, DF3, NCRC-11, and carcinoembryonic antigen (CEA) were unaffected by tamoxifen therapy: there was 16%-31% change before vs. during tamoxifen therapy, but this did not reach significance for any of the four antigens. Comparison of the pattern of staining between the four antigens showed 115D8, DF3, and NCRC-11 to be similar to each other both before and during tamoxifen therapy. All three antigens were significantly different from the staining pattern shown by CEA before and during therapy. Tumour antigen expression pre-treatment did not correlate with therapeutic response for any of the four antigens. However, NCRC-11 staining during tamoxifen therapy did correlate with response (p = 0.004). There was no significant difference in oestrogen receptor status before and during tamoxifen therapy, both of which correlated with response. DNA ploidy of the tumour before tamoxifen did not correlate with response to therapy, but there was a weak correlation between response and DNA ploidy measured during tamoxifen therapy. In only a minority of tumours (up to 30%) did tamoxifen exert an effect on the biological characteristics studied. Changes in these biological markers were mainly in tumours which responded to tamoxifen, and the changes seen were a tendency to greater expression of differentiation antigens.(ABSTRACT TRUNCATED AT 250 WORDS)     

A radioimmunoassay for the detection of a human tumor-associated glycoprotein (TAG-72) using monoclonal antibody B72.3

Monoclonal antibody (MAb) B72.3 was generated against a carcinoma metastasis and has been shown to bind with a high degree of selectivity to a tumor-associated glycoprotein (TAG-72) found in human colon and breast carcinomas, while showing minimal reactivity to any normal adult tissues. Competition radioimmunoassays have been developed for the detection of TAG-72 in tumors and sera from both athymic mice bearing human carcinoma xenografts and patients with colon, breast and other carcinomas. The distribution of TAG-72 in human tumor xenografts was restricted to tumors originating from the LS-174T human colon carcinoma, with no significant reactivity being detected in human tumor xenografts from a different colon carcinoma, a human breast carcinoma, or a human melanoma. The levels of TAG-72 in clinical material obtained from surgery were examined; high levels were found in colon carcinomas and to a lesser extent in breast carcinomas, while no detectable levels were found in normal tissues. Sera from apparently normal patients contained a mean level of 2.2 units per ml of TAG-72. A level of 3 standard deviations above the mean level of TAG-72 found in normals was employed as a cut-off value to indicate a positive test result in subsequent studies. No patient with inflammatory disease or other benign colon disease exhibited elevated levels of TAG-72. Seven out of 20 patients with other carcinomas had elevated serum levels of TAG-72. The serum TAG-72 levels were compared with the serum levels of antigens recognized by the MAbs currently used to screen sera of carcinoma patients (CEA, GICA, and OC125). It was clearly demonstrated that TAG-72 is different from these antigens and can be found in some sera in which no antigen is detected by otherwise available MAb RIAs.     

Cellular effects of tamoxifen in primary breast cancer

We have investigated the effect of tamoxifen on the biological characteristics of the primary tumour in 33 patients with breast cancer. All patients had pre-treatment biopsy of the primary breast cancer and subsequent trucut biopsy of the primary tumour after 1–4 months on tamoxifen therapy.The staining patterns in the primary tumour of each of the tumour antigens 115D8, DF3, NCRC-11, and carcinoembryonic antigen (CEA) were unaffected by tamoxifen therapy: there was 16%–31% change before vs. during tamoxifen therapy, but this did not reach significance for any of the four antigens. Comparison of the pattern of staining between the four antigens showed 115D8, DF3, and NCRC-11 to be similar to each other both before and during tamoxifen therapy. All three antigens were significantly different from the staining pattern shown by CEA before and during therapy.Tumour antigen expression pre-treatment did not correlate with therapeutic response for any of the four antigens. However, NCRC-11 staining during tamoxifen therapy did correlate with response (p=0.004).There was no significant difference in oestrogen receptor status before and during tamoxifen therapy, both of which correlated with response. DNA ploidy of the tumour before tamoxifen did not correlate with response to therapy, but there was a weak correlation between response and DNA ploidy measured during tamoxifen therapy.In only a minority of tumours (up to 30%) did tamoxifen exert an effect on the biological characteristics studied. Changes in these biological markers were mainly in tumours which responded to tamoxifen, and the changes seen were a tendency to greater expression of differentiation antigens. It is uncertain whether these changes are a primary or a secondary effect of tamoxifen with respect to inducing tumour response.     

Identification of the cancer/testis antigens AKAP3 and CTp11 by SEREX in hepatocellular carcinoma

Cancer/testis (CT) antigens are considered promising target molecules for immunotherapy. To efficiently identify potential CT antigens, a testis cDNA library was immunoscreened with sera from hepatocellular carcinoma (HCC) patients. We isolated 3 different antigens, AKAP3, CTp11, and UBQLN3. Although AKAP3 and CTp11 have been previously reported as CT antigens, this is the first time that these 2 antigens have been isolated from HCC patients by SEREX. Conventional RT-PCR analysis showed that AKAP3 was frequently present in HCC cell lines (5/7) and HCC tissues (5/10), and the gene was broadly expressed in several cancer types, including breast cancer cell lines (3/6), breast cancer tissues (6/9), colon cancer cell lines (3/10), colon cancer tissues (5/6), ovary cancer cell lines (6/8), ovary cancer tissues (11/16), lung cancer cell lines (4/7) and lung cancer tissues (6/13). By phage plaque analysis, anti-AKAP3 antibody was detected in sera from 15 of 27 HCC patients and 8 of 27 healthy donors. These data suggest that AKAP3 may be useful for diagnosis and immunotherapy in HCC patients.     

Monoclonal antibody 83D4 immunoreactivity in human tissues: cellular distribution and microcytophotometric analysis of immunoprecipitates on tissue sections.

Immunocytochemical assays were performed on cell cultures as well as on a wide range of human tissues using a monoclonal antibody, MAb 83D4, produced by a murine hybridoma generated by immunization with a cell suspension from a paraffin block of human breast carcinoma tissue. Frozen breast tissue samples (n = 49) were compared to fixed and paraffin-embedded samples (n = 62). Paraffin sections (n = 194) from a variety of human tissues were compared to breast immunoreactivity. Immunoprecipitates, resulting from positive reactions between 83D4 and Avidin Biotin Peroxidase, were evaluated by computer-assisted microcytophotometry (SAMBA). In some frozen breast samples (n = 27), 83D4 antigen distribution was correlated with tumor cell DNA index, ploidy balance, growth fraction (Ki67), hormone receptor (ER, PR) antigenic sites, NORsAg and oncoprotein pHER-2/neu cell content. MAb 83D4 reacted with 3 breast cancer cells lines (MCF7, T47D and H466B) but not with normal epithelial breast cells in culture. The immunostaining in frozen paraffin sections from breast were similar. Like most normal tissues, normal breast did not react with 83D4. Cellular MAb 83D4 antigen concentration increases with the degree of malignancy but is independent of DNA nuclear content, ER, PR, growth fraction and pHER-2neu in cancers. These results suggested that routine immunohistochemical procedures using MAb 83D4 could facilitate the grading of breast cancer, in particular by allowing detection of microvascular invasions in the breast as well as at a distance and of blood-born micrometastases, especially in bone marrow.     

Assessment of four monoclonal antibodies as serum markers in breast cancer

Four monoclonal-antibody-defined serum markers (CA15-3, HMFG1, HMFG2 and NCRC-11) were examined in five groups of subjects: controls, benign breast disease and stage I/II, stage III and metastatic breast cancer. None of the markers were significantly elevated in primary breast cancer (i.e. stage I/II or stage III) compared with controls or patients with benign breast disease. These markers therefore have no role in screening or in the diagnosis of primary breast cancer. CA15-3, HMFG2 and NCRC-11 were significantly increased in the patients with metastatic breast cancer (P < 0.001), indicating a potential use in the diagnosis of symptomatic metastases. In patients with metastases, sequential changes in CA15-3 correlated significantly with clinical response to therapy. Thus CA15-3 is a powerful marker of response and in combination with other markers, may provide an objective measurement of response to therapy in patients with advanced breast cancer.     

Reactivity of monoclonal antibody DF3 with a high molecular weight antigen expressed in human ovarian carcinomas

We have previously described the monoclonal antibody (MAb) DF3, prepared against a human breast carcinoma. MAb DF3 reacts with a high molecular weight glycoprotein detectable in human breast carcinomas and in human milk. Previous studies have demonstrated that DF3 antigen levels are elevated in the plasma of patients with breast and ovarian cancer. The present study has further examined the reactivity of MAb DF3 with human ovarian carcinomas. Immunoperoxidase staining demonstrated reactivity of MAb DF3 with 95% of benign, borderline, and malignant tumors (serous, mucinous, and endometrioid) of the ovary. Furthermore, malignant tumors contained cytoplasmic DF3 antigen while benign tumors expressed the antigen only on apical surfaces. Western blot analyses demonstrated that the MAb DF3 reactive ovarian antigen (DF3-O) was a glycoprotein with a heterogenous molecular weight ranging between 300,000 and 450,000. This antigen was detectable by immunofluorescence on the cell surface of five of six cultured human ovarian carcinoma cell lines. The extent of cell surface reactivity with MAb DF3 was equivalent to or greater than that obtained with MAb OC125, an antibody generated against coelomic epithelium and developmental amnion. Furthermore, uptake of 125I-labeled MAb DF3 by human ovarian carcinoma xenografts in athymic mice was 5.4- and 6.2-fold higher than the respective uptake noted in liver and control tumor (P = 0.031). These findings suggest that DF3-O antigen is similar if not identical to the antigen detected in human breast carcinomas by MAb DF3. Thus, MAb DF3 may be a useful reagent in immunodiagnostic evaluation of patients with ovarian cancer.     

Inhibition of human tumor xenograft growth in nude mice by a novel monoclonal anti-HSPG isolated from human liver

Heparan sulfate proteoglycans (HSPGs) were isolated from normal human liver and α monoclonal antibody (MAb) was raised against them. Preliminary studies showed that MAb clone 1E4-1C2 was able to react with many cell lines tested, including hematopoietic cells and solid tumors. MAb1E4-1C2 was used to study whether HSPG was involved in growth and proliferation of human liver cancer using hepatocellular carcinoma (HCC) cell line (HepG2) as a model. Inhibition by MAb1E4-1C2 of HepG2 cell proliferation was studied in vitro by MTT assay. For in vivo assay, xenograft induction in athymic mice was performed. The results showed that MAb1E4-1C2 inhibited proliferation of HepG2 cells significantly, compared to isotype and medium control. MAb1E4-1C2 also suppressed the growth of tumor, resulting in smaller tumor size and weight. The investigation also showed that MAb1E4-1C2 inhibited proliferation and restricted tumor growth through the induction of apoptosis. The results suggest that HSPG might be involved in liver cancer cell proliferation. Therefore, a specific MAb that was raised against liver HSPG might be an alternative therapeutic agent for the treatment of human liver cancer.     

Immunohistological characterization of human monoclonal antibody against lung cancer

Using immunoperoxidase staining method, a human monoclonal antibody (MAb), termed HB4C5, which was generated by fusion of NAT-30 cells with regional lymph node lymphocytes from a lung cancer patient, was examined for tumor specificity. The determinant recognized by HB4C5 MAb was well preserved in usual formalin-fixed paraffin-embedded tissue sections. Experiments with four major histological types of the lung cancer showed that HB4C5 MAb reacted with adenocarcinoma (19/27), squamous cell carcinoma (2/15), and large cell carcinoma (4/6), but not with small cell carcinoma (0/7). The immunoreaction was seen in the cytoplasm. In addition, this MAb also reacted with some gastric and colon adenocarcinomas. Further study on normal lung and nonpulmonary tissues revealed that HB4C5 MAb bound only type II pneumocytes and proximal tubules of normal kidney.     

Comparison of monoclonal antibodies for the detection of occult breast carcinoma metastases in bone marrow

Summary Twenty percent (n = 6) of Stage III or IV breast cancer patients (n = 30) had bone marrow metastases detected in bilateral bone marrow biopsy/aspiration preparations using standard histologic preparations. Each metastasis was also detected by four separate monoclonal antibodies (MAbs) which recognize breast carcinoma associated antigens (DF3, anti-EMA, HMFG-2, and CAM5.2). These MAbs were then utilized to stain other bone marrow preparations (n = 81) to determine their utility for the detection of micrometastatic breast carcinoma. MAbs HMFG-2, anti-EMA, and DF3 were each strongly reactive with bone marrows containing histologically-evident metastatic breast carcinoma (18/18). These anti-epithelial membrane antigen MAbs, however, were also reactive with rare plasma cells and immature cells (as well as cell clusters) in some of the control bone marrow samples tested, including those from normal patients and patients with hematologic disorders. They also reacted with some of the preparations from patients with leukemia and lymphoma, and with uninvolved marrows from patients with non-epithelial malignancies. The anti-keratin MAb CAM5.2, in contrast, reacted with 83% (15/18) breast cancer metastases and failed to stain any cells in the various categories of control marrow preparations. These data suggested that MAb CAM5.2 might be utilized to immunohistochemically differentiate micrometastatic breast carcinoma from immature myeloid or erythroid elements.Each MAb was then reacted with histologically uninvolved marrow preparations from the remaining 24 of 30 breast cancer patients in an attempt to identify occult breast carcinoma metastases. While MAbs HMFG-2, DF3, and anti-EMA demonstrated reactive cells in some of these marrows, this reactivity was similar to that seen with control preparations. MAb CAM5.2, in contrast, was negative with all specimens. These data suggest that MAb CAM5.2 may be a useful immunologic probe for the detection and confirmation of metastatic breast carcinoma in bone marrow, while more caution must be employed in the interpretation of results obtained using MAbs anti-EMA, DF3, and HMFG-2.     

Antibody reacting with the murine mammary tumor virus in the serum of patients with breast carcinoma: a possible serological detection method for breast carcinoma

Sera from patients with Stages A and B infiltrating ductal carcinoma of the breast, benign breast disease, cancers other than breast carcinoma, and normal female controls were examined by indirect immunoelectron microscopy (IEM) and a viral agglutination test for evidence of antibodies directed against murine mammary tumor virus (MMTV). Sera from 41 (79%) of 52 patients with breast carcinoma and eight (19%) of 42 normal subjects or patients with benign breast disease (noncancer subjects) showed evidence of MMTV labeling by IEM. In the MMTV agglutination test, significant virus agglutination (2+ to 4+) was present in eight (13%) of 61 noncancer sera, 58 (86%) of 68 breast carcinoma sera, and two (11%) of 18 other cancer sera. The results of the more rapid MMTV agglutination test correlated well with IEM. Analysis of reacting antibody by IEM revealed no immunoglobulin A and significant immunoglobulin M and immunoglobulin G antibody. Serum reactivity against MMTV was completely absorbed by MMTV but not by the glycoprotein with a molecular weight of 52,000 of MMTV, Friend murine leukemia virus, avian myeloblastosis virus, or sheep erythrocytes. It is concluded that reactivity of human antibodies to MMTV is strongly associated with, but is not entirely specific for, breast carcinoma. It remains to be determined if normal persons with these antibodies will ultimately develop breast cancer and should therefore be considered at high risk. These tests may have potential usefulness as a diagnostic screen for breast cancer.     

Anti-HuC and -HuD autoantibodies are differential sero-diagnostic markers for small cell carcinoma from large cell neuroendocrine carcinoma of the lung

Aiming to identify novel sero-diagnostic markers for neuroendocrine carcinomas of the lung, the two-dimensional gel electrophoresis-immunoblot method was used to analyze tumor-associated autoantibodies in patients with small cell lung carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC). Several autoantigens were revealed and anti-HuC autoantibody was detected only in sera of SCLC patients. Since Hu family proteins including HuC are well-known causes of paraneoplastic encephalomyelitis/sensory neuronopathy (PEM/SN), the expression of HuC as well as HuD mRNAs and their proteins was studied in 11 lung cancer cell lines. The expression of HuC and HuD mRNAs and proteins was only detected in SCLC- and LCNEC-derived cells. To validate the existence of anti-HuC and -HuD auto-antibodies, we studied a large number of sera including those from lung cancer patients employing dot blot analysis. Anti-HuC and -HuD autoantibodies were detected only in SCLC cases with or without PEM/SN, and not in the sera of LCNEC patients. The mechanism leading to different anti-HuC and -HuD autoantibody production between SCLC and LCNEC is unclear; however, the results from the present and previous studies suggest that anti-HuC and -HuD auto-antibodies are novel differential sero-diagnostic markers for SCLC from LCNEC.     

Purification and characterisation of a breast-cancer-associated glycoprotein not expressed in normal breast and identified by monoclonal antibody 83D4.

Monoclonal antibody (mAb) 83D4 was generated using formol-fixed paraffin-embedded human breast carcinoma tissue as the immunogen. Previous studies demonstrated that it was reactive with breast carcinoma tissues, but not with normal breast. The antigen identified by mAb 83D4 was detected, using ELISA, in MCF7 breast carcinoma cell line membrane extracts, in primary breast and colon carcinoma tissue extracts and in pleural effusion fluid from patients with metastatic breast cancer. No reactivity with 83D4 was found in either human milk fat globule membranes or skimmed milk. 83D4 reactive antigen was found to be a heterogeneous high molecular weight (MW) protein (apparent Mr:300-400 to over 1000 kDa) by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The antigen was purified from MCF7 cells, breast and colon carcinomas and effusion fluid, by perchloric acid solubilisation followed by immunoaffinity chromatography with 83D4. The immunopurified antigen from MCF7 cells and pleural effusion fluid was further analysed by gel filtration and ion-exchange chromatography, which confirmed the high MW and indicated the charge heterogeneity of the reactive molecules. The 83D4 reactive antigen strongly bound to wheat-germ agglutinin and weakly to peanut lectin. No binding was found with lentil lectin or concanavalin A. Antigenic activity was strongly reduced by trypsin and subtilysin digestion and by treatment with sodium periodate, but it was not affected by neuraminidase. These results imply the glycoprotein nature of the 83D4-defined antigen and the involvement of carbohydrate, but probably not sialic acid, in the epitope. Purified 83D4 antigen did not display reactivity for mAb HMFG-1, directed against a polymorphic epithelial mucin, PEM, using ELISA, but bound mAb CC49 and weakly mAb B72.3, antibodies which define a tumour associated glycoprotein, TAG-72. Moreover CC49 and 83D4 showed similar reactivity pattern in immunoblotting assays. A double determinant radioimmunoassay confirmed that 83D4 antigen carries epitopes for mAb B72.3 and CC49. Competition radioimmunoassays clearly distinguished the 83D4 defined epitope from those recognised by B72.3 and CC49, demonstrating that antibody 83D4 identifies a unique epitope. It is suggested that the antigens identified by mAb 83D4 and by mAb B72.3 and CC49 may form part of the same family of carcinoma associated glycoproteins.