Monoclonal Antibody DF3 Research Articles

Serum CA153 as biomarker for cancer and noncancer diseases.

CA153 was originally discovered as a tumor antigen recognized by two monoclonal antibodies DF3 and 115D8 simultaneously. Subsequent studies showed that DF3 recognizes the core protein of mucin1 (MUC1 or CD227) whereas 115D8 recognizes part of the glycan chains on MUC1. MUC1 is a highly glycosylated transmembrane protein expressed on the mucosal surfaces of epithelial cells in lung, breast, stomach, gallbladder, lymph node, colon, rectum, and pancreas. The increased serum levels of CA153 have been established as a biomarker for breast cancer diagnosis since 1980s. However, it is unknown if elevated serum CA153 levels were also associated with other cancers and noncancer diseases. In current study, a total of 19,789 clinical lab test results of serum CA153 levels from healthy individuals and patients with 30 different types of diseases during the past 5 years were retrieved and analyzed. According to the mean (SD), median, and p (-Log10p) values calculated, we found that patients suffering lung, breast, ovarian cancers, nephrotic syndrome, type 2 diabetes, endometrial cancer, coronary heart disease, cervical cancer, uremia, and other 12 diseases plus healthy controls >65 years old had significantly (p<0.05, -Log10p>1.30) increased median serum CA153 levels compared to that of healthy controls. Moreover, patients with lymphoma had the highest mean and the biggest SD value for serum CA153. Based on these data and the documented evidence, we proposed that the increased serum CA153 levels might be associated with pathological leakage of the epithelial cell product into the blood circulation in addition to the decreased CA153 clearance rate.

Increased expression of MUC1 in advanced pancreatic cancer.

MUC1 is associated with tumor invasion and metastasis, and is expressed in pancreatic cancer with a high frequency. This study explored whether MUC1 expression affected the survival of patients with pancreatic cancer. Tissue specimens obtained from 70 patients with invasive ductal carcinoma of the pancreas, in pTNM stage III or IV, were immunostained with the anti-MUC1 monoclonal antibody DF3. The results of immunostaining were determined to be positive when more than 50% of the total cancer cells were positively stained. Association of the expression of the DF3 epitope with clinicopathological parameters or patients' survival was statistically evaluated. The incidence of positivity of MUC1 expression was 55.7% (39/70) and this incidence was significantly higher in pTNM stage IV than in stage III (odds ratio [OR], 4.015; 95% confidence interval [CI], 1.459-11.0541; P = 0.0076). As there was a clear difference in overall survival between pTNM stages III and IV ( P = 0.0016), the effect of MUC1 expression on survival was separately evaluated in each stage. It was shown that the expression of MUC1 was associated with unfavorable overall survival in stage IV ( P = 0.0197). Our data suggest that the expression of MUC1 may be related to the progression of pancreatic cancer.

Expression of Different Glycoforms of Membrane Mucin(MUC1) and Secretory Mucin (MUC2, MUC5AC and MUC6) in Pancreatic Neoplasms

Our previous studies of pancreatic tumors have demonstrated that invasive ductal carcinoma (IDC) usually showed expression of MUC1 (membrane bound mucin) detected by monoclonal antibody DF3, whereas intraductal papillary-mucinous neoplasm (IPMN) showed no expression of MUC1. In the present study, we examined 50 IDCs, and 63 IPMNs which were morphologically classified into two histological subtypes, dark cell type (IPMN-D, 27 cases) and clear cell type (IPMN-C, 36 cases). Patients with either of IPMN showed significantly better survival than those with IDC. To clarify the relationship of the expression patterns of mucins with their biological behavior, we examined the expression profiles of various glycoforms of membrane mucin (MUC1) and secretory mucin (MUC2, MUC5AC and MUC6) in the neoplasms using immunohistochemistry. IDCs showed high expression of all the glycoforms of MUC1 (66%-98%). In contrast, IPMNs-D showed no or low expression of all the glycoforms of MUC1 (0%-4%), while IPMNs-C showed low expression of poorly glycosylated MUC1 (3%-6%), but expression of sialylated MUC1 (41%) and fully glycosylated MUC1 (69%). Expression of MUC2 was negative (0%) in IDC, high (96%) in IPMN-D and low (3%) in IPMN-C. MUC5AC was highly expressed in all types. MUC6 expression was higher in IPMNs-C (92%) than in IDCs (56%) and IPMNs-D (37%). In conclusion, the present study demonstrated that IDCs showed high expression of all the glycoforms of MUC1, and also that two types of IPMNs showed different expression patterns of glycosylated MUC1 as well as MUC2 and MUC6. These different expression patterns of mucins may be related with the malignancy potential of pancreatic neoplasms.

MUC1 mucin expression in follicular dendritic cells and lymphoepithelial lesions of gastric mucosa-associated lymphoid tissue lymphoma.

Membrane-associated mucin MUC1 is expressed in various adenocarcinoma cells and active T lymphocytes. We tried to find out whether MUC1 is expressed in gastric mucosa-associated lymphoid tissue (MALT) lymphoma lesion. MUC1 was not expressed in infiltrating T lymphocytes; however, MUC1 was found on the cell surface of follicular dendritic cells (FDC) of germinal centers and in the epithelial cytoplasm of lymphoepithelial lesion (LEL) of the lymphoma, which were immunohistochemically detected by monoclonal antibodies DF3 and MY.1E12. MUC1 was also expressed in the FDC of control cases (gastrectomy specimen containing reactive lymphoid follicles, n = 10, MUC1/ DF3, 100%; MUC1/MY.1E12, 40%), and FDC in MALT lymphomas (n = 59) showed lower MUC1 expression rates (MUC1/ DF3, 32%; MUC1/MY.1E12, 0%) than the control (P < 0.001). Lymphoepithelial lesion in the low-grade MALT lymphomas (n = 23) showed a higher MUC1/DF3 expression rate (30%) than those in the high-grade MALT lymphomas (n = 36; 6%; P < 0.05). T lymphocytes in the surface mucosa were more frequent in MALT lymphoma (91.4 +/- 80.6/unit area) than those in the control (20.0 +/- 23.6) (P < 0.001). S100-positive dendritic cells around LEL were more frequent in the low-grade (19.0 +/- 9.4/unit area) than in the high-grade (11.7 +/- 9.7) (P < 0.005). This study demonstrated MUC1 mucin expression on FDC for the first time. Mucosa-associated lymphoid tissue lymphoma, especially low-grade, shows immunologically active state, where FDC MUC1 expression may be suppressed by some factors released from lymphoma cells. Further study to elucidate the pathogenetic role of MUC1 in MALT lymphoma is necessary.

Abnormal expression of MUC1 apomucin and mature MUC1 mucin in biliary epithelial cells in various cystic liver diseases

MUC1 apomucin is proposed as an "oncofetal" antigen in the biliary system. We surveyed the biliary expression of MUC1 apomucin and mature MUC1 mucin (highly-glycosylated MUC1 apomucin) in hepatic cysts, biliary microhamartomas, and intrahepatic bile ducts in various cystic liver diseases and clarified the relationship between the expression of these mucins and cystogenesis in the liver. The expression of MUC1 apomucin and mature MUC1 mucin were detected immunohistochemically using the monoclonal antibodies DF3 and SM3, and HMFG1, respectively. We surveyed six livers with adult-type polycystic disease (APCD), six with multiple hepatic cysts without polycystic kidney, 11 with solitary hepatic cysts, and 11 "histologically normal" livers. Biliary microhamartomas were frequently found in the former three. MUC1 apomucin was frequently expressed at the luminar surface of the epithelial cells lining cysts and also in biliary microhamartomas, independent of the type of cystic disease. MUC1 apomucin detected by both monoclonal antibodies (MAbs) DF3 and SM3 was also frequently expressed in the intrahepatic bile ducts from polycystic diseases, particularly the adult-type, whereas such expression was rare in the intrahepatic bile ducts in normal livers. Mature MUC1 mucin was expressed on the luminal surface of the epithelial cells lining cysts in approximately half of hepatic cysts studied, but it was infrequently expressed in biliary microhamartomas and almost absent in the intrahepatic bile ducts. The biliary epithelial cells lining hepatic cysts, biliary microhamartomas, the apparently normal intrahepatic bile ducts in APCD, multiple hepatic cysts, and to a lesser degree, solitary hepatic cysts, express abnormal levels MUC1 apomucin. This implies that biliary epithelial cells expressing MUC1 apomucin are related to the whole cystogenetic process in these diseases. In contrast, the expression of mature MUC1 mucin may be related to the late cystogenetic process in these diseases. (Hepatology 1996 Sep;24(3):539-43)

Frequent Expression of Muc1 Apomucin on Biliary Epithelial Cells of Damaged Small Bile Ducts in Primary Biliary Cirrhosis and Chronic Viral Hepatitis: An Immunohistochemical Study

MUC1 apomucin is a specific target tumor antigen recognized by cytotoxic T cells in a major histocompatibility complex (MHC) unrestricted fashion in patients with pancreatic and breast cancer. This T–cell–mediated immune mechanism against MUC1 apomucin expressing cells has not been evaluated in nonneoplastic immune–mediated diseases. Therefore, we immunohistochemically surveyed the expression of MUC1 apomucin on biliary epithelial cells of small bile ducts in various hepatobiliary diseases, including primary biliary cirrhosis (PBC). MUC1 apomucin was detected using the monoclonal antibody DF3 and the streptavidin–biotin complex, in livers from 31 patients with PBC, 67 with chronic viral hepatitis (CH) with or without cirrhosis, 31 with extrahepatic biliary obstruction (EBO), 30 with hepatolithiasis, and from 23 normal individuals. MUC1 apomucin was infrequently and focally expressed in the biliary epithelial cells of the small bile ducts in 3 of 23 normal livers. In contrast, MUC1 apomucin was frequently and strongly expressed on the luminal surface of biliary epithelia] cells of small bile duct, in 22 of 31 patients with PBC, and in 50 of 67 patients with CH. In particular, high levels of MUC1 apomucin were expressed in bile ducts showing chronic nonsuppurative destructive cholangitis (CNSDC) in PBC and hepatitic duct injuries in CH. In EBO and hepatolithiasis, MUC1 apomucin was focally and weakly expressed in 29% and 30% of livers examined, respectively. More MUC1 apomucin was expressed in PBC and CH than in EBO, hepatolithiasis, and normal liver ( P &lt; .01, respectively). Frequent and high luminal expression of MUC1 apomucin on biliary epithelial cells in damaged small bile ducts in PBC and CH may be related to T–cell–mediated immunologic mechanisms in these diseases, probably by an MHC–unrestricted recognition process.

Frequent expression of MUC1 apomucin on biliary epithelial cells of damaged small bile ducts in primary biliary cirrhosis and chronic viral hepatitis: An immunohistochemical study

MUC1 apomucin is a specific target tumor antigen recognized by cytotoxic T cells in a major histocompatibility complex (MHC) unrestricted fashion in patients with pancreatic and breast cancer. This T-cell-mediated immune mechanism against MUC1 apomucin expressing cells has not been evaluated in nonneoplastic immune-mediated diseases. Therefore, we immunohistochemically surveyed the expression of MUC1 apomucin on biliary epithelial cells of small bile ducts in various hepatobiliary diseases, including primary biliary cirrhosis (PBC). MUC1 apomucin was detected using the monoclonal antibody DF3 and the streptavidin-biotin complex, in livers from 31 patients with PBC, 67 with chronic viral hepatitis (CH) with or without cirrhosis, 31 with extrahepatic biliary obstruction (EBO), 30 with hepatolithiasis, and from 23 normal individuals. MUC1 apomucin was infrequently and focally expressed in the biliary epithelial cells of the small bile ducts in 3 of 23 normal livers. In contrast, MUC1 apomucin was frequently and strongly expressed on the luminal surface of biliary epithelia] cells of small bile duct, in 22 of 31 patients with PBC, and in 50 of 67 patients with CH. In particular, high levels of MUC1 apomucin were expressed in bile ducts showing chronic nonsuppurative destructive cholangitis (CNSDC) in PBC and hepatitic duct injuries in CH. In EBO and hepatolithiasis, MUC1 apomucin was focally and weakly expressed in 29% and 30% of livers examined, respectively. More MUC1 apomucin was expressed in PBC and CH than in EBO, hepatolithiasis, and normal liver (P < .01, respectively). Frequent and high luminal expression of MUC1 apomucin on biliary epithelial cells in damaged small bile ducts in PBC and CH may be related to T-cell-mediated immunologic mechanisms in these diseases, probably by an MHC-unrestricted recognition process. (Hepatology 1996 Jun;23(6):1313-7)

Expression of Mucin Core Protein of Mammary Type in Primary Liver Cancer

Expression of mucin core protein of mammary type (MUC-1 core protein) was investigated in primary hepatic carcinoma (25 cases of cholangiocarcinoma, 15 cases of combined hepatocellular-cholangiocellular carcinoma and 18 cases of hepatocellular carcinoma) with monoclonal antibody DF3 and a standard avidinbiotin complex method. MUC-1 core protein was almost always expressed in cholangiocarcinoma and in the cholangiocarcinoma area of hepatocellular-cholangio-cellular carcinoma to a varied degree, whereas such expression was virtually absent in hepatocellular carcinoma and the hepatocellular carcinoma areas of hepatocellular-cholangiocellular carcinoma. Nonneoplastic intrahepatic biliary epithelium, as well as hepatocytes, was virtually negative for this protein. In well-differentiated cholangiocarcinoma, this protein tended to be expressed on luminal surfaces, whereas poorly differentiated cholangiocarcinoma showed cell membranous or diffuse cytoplasmic staining patterns. Double staining with Alcian blue (pH 2.5) and immunostaining for MUC-1 core protein showed that although some parts of cancerous areas were positive for both stains, most cancerous areas were only positive for one. Alcian blue--positive areas were dominant over MUC-1 core protein--expressing areas in well-differentiated cholangiocarcinoma, whereas the reverse was the case in poorly differentiated cholangiocarcinomas and also in cholangiocarcinoma areas of hepatocellular-cholangiocellular carcinoma. This study is the first report to document that cholangiocarcinoma and cholangiocarcinoma areas of hepatocellular-cholangiocellular carcinoma express MUC-1 core protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of normal breast epithelial cells in primary cultures: differentiation and growth factor receptors studies.

The growth and differentiation of normal human mammary epithelial cells (HMEC) were studied after propagation of serial cultures from breast tissue biopsies from 42 mammoplasty patients. Cells were grown for up to 7 mo. in low calcium medium. HMEC cultures displayed heterogeneous growth patterns, according to the average doubling time of 44 +/- 6 h for 32 generations. Proliferation peaked at Day 30. HMEC maintained a normal karyotype and were organized in ductlike structures when cultured in collagen gel matrix. The cultures retained several phenotype traits of the epithelial lineage, including the expression of cytokeratins 18 and 19, specific mammary gland antigens, as shown by indirect HMEC immunostaining by the monoclonal antibodies DF3, EMA, 7B10, and 1BE12. Estrogen receptors were undetectable, whereas progesterone receptors were present at very low density. High-affinity cell surface receptors for epidermal growth factor (EGF) (Kd = 1.1 x 10(-10) M) were observed at a density of 50,000 to 100,000 sites per cell. Accordingly, [3H]thymidine incorporation in HMEC was optimally stimulated by EGF at concentrations of 10(-11) to 10(-10) M. HMEC were also seen to possess functional VIP receptors linked to the adenylate cyclase system, as we previously observed in seven human breast cancer cell lines. These results show that long-term cultures of HMEC provide useful models for studying the growth and differentiation of the normal human mammary gland, and the role of growth factors and hormones in these functions.

Prediction of prognosis in primary breast cancer by detection of a high molecular weight mucin-like antigen using monoclonal antibodies DF3, F36/22, and CU18: a Cancer and Leukemia Group B study.

Three monoclonal antibodies (MAbs) (DF3, F36/22, CU18) were used to monitor expression of distinct epitopes present within a family of mucin-like, breast carcinoma-associated molecules. Primary tumor specimens from more than 190 stage II breast cancer patients were evaluated for expression of the high molecular weight antigens. With a median follow-up of 6 years, patients whose tumors exhibited high immunoperoxidase staining scores (greater than 50% positive cells) with MAb DF3 had a superior disease-free survival ([DFS] 56% +/- 6% v 37% +/- 5% at 6 years; P = .0088) and overall survival ([OS] 72% +/- 5% v 59% +/- 5% at 6 years; P = .025). Staining scores with the other two antibodies did not correlate with improved prognosis. For MAbs DF3 and CU18, patients whose tumors exhibited predominantly apical cellular reactivity patterns had improved DFS, although differences reached conventional levels of statistical significance only with MAb CU18. In multivariate analyses, the prognostic value of MAb DF3 staining was independent of other identified prognostic factors. Furthermore, the concordance between primary and axillary lymph node metastases staining with each MAb was 73%, 80%, and 85% for MAbs DF3, F36/22, and CU18, respectively. These results suggest that staining with MAb DF3 identifies a group of node-positive women with a relatively favorable prognosis. Expression of the DF3 mucin-like glycoprotein is related to better differentiation, and staining with MAb DF3 provides an accurate and objective estimate of clinical outcome independent of histopathologic evaluation.

Recognition of the Polypeptide Core of Mucin by Monoclonal Antibody MUSE11 against an Adenocarcinoma‐associated Antigen

Monoclonal antibody (MAb) MUSE11 detects an adenocarcinoma‐associated antigen and is useful for the serodiagnosis of pancreas cancer. We established a sandwich enzyme immunoassay using MAb MUSE11 and MAb DP3 against a breast cancer‐associated mucin core protein as a catcher and a tracer, respectively. With this assay system, the binding of the tracer MAb DF3 to an antigen in the human kidney tissue lysate was clearly inhibited by MAb MUSE11. In addition, MAb MUSE11 showed a significant binding activity to the synthetic peptide corresponding to the tandem repeat of a human epithelial mucin core protein. These data suggest that MAb MUSE11 could detect the polypeptide core of a mucin, and may be of use for studying mucin as a gene product.

Effect of radioiodination on the binding of monoclonal antibody DF3 to breast carcinoma cells

The murine monoclonal antibody (MAb) designated DF3 is an IgG 1 prepared against a membrane enriched fraction of human breast carcinoma. MAb DF3 reacts with a family of large molecular weight glycoproteins expressed by 78% of breast cancer cells and 95% of epithelial ovarian cancer cells. Binding to the brest cancer cell line, MCF-7, of native MAb DF3 was compared to that of MAb DF3 exposed to different concentrations of Iodogen or Bolton-Hunter reagent. The amount of MAb DF3 required to obtain half-maximal binding ( B 1 2max ) with MCF-7 extract for native MAb DF3 IgG was 180 ng, while the B 1 2max for MAb DF3 IgG exposed to 1 and 10 μg lodogen was 580 ng and 1800 mg, respectively. In contrast, the B 1 2max for MAb DF3 IgG treated with Bolton-Hunter reagent was not different from that for native MAb DF3 IgG. Similar results were obtained with F(ab′) 2. Immunoreactive fractions for the 125I-labeled MAb DF3 were 0.13,0.24 and 0.65 for IgG after exposure to 1 μg Iodogen, 10 μg lodogen and Bolton-Hunter, respectively. Immunoreactive fractions for F(ab′) 2 were 0.08, 0.08, and 0.53 for 1 and 10 μg Iodogen and Bolton-Hunter reagent, respectively. Association constants ( K a ) were significantly higher for MAb DF3 IgG radioiodinated with Bolton-Hunter (3.97 ± 0.38 × 10 8 M −1) than for IgG exposed to 1 μg lodogen (2.78 ± 0.30 × 10 8 M −1) or 10 μg Iodogen (1.03 ± 0.12 × 10 8M −1). Similarly, the K a ratio observed for the F(ab′) 2 radioiodinated with Bolton-Hunter reagent, 1 or 10 μg Iodogen was 22 to 3 to 1. Affinity constants for immunoreactive IgG ( K a (ir)) for the three preparations were similar, ranging from 1.16 ± 0.29 × 10 9M −1 to 1.33 ± 0.2 × 10 9M −1. Likewise, the K a (ir) for the immunoreactive F(ab′) 2 ranged from 3.74 ± 0.97 × 10 8M −1 to 4.74 ± 0.92 × 10 8M −1.

Identification of a family of high molecular weight tumor-associated glycoproteins.

The monoclonal antibody (MAb) designated DF3 was prepared against a human breast carcinoma metastatic to liver. This MAb reacts with a high molecular weight glycoprotein detectable in human breast carcinomas and human milk. In contrast, MAb F36/22 was prepared against the MCF-7 breast carcinoma cell line, MAb 115-D8 against human milk fat globule membrane (HMFGM) and MAb Ca1 against the HEp-2 human laryngeal carcinoma cell line. These MAb have similar patterns of reactivity with normal tissues and tumors based upon immunoperoxidase staining. In the present study we have monitored reactivity of these MAb against DF3 antigen purified from human breast carcinoma cell lines (MCF-7, BT-20) and HMFGM. Solid phase immunoassays and immunoblotting demonstrate that MAb DF3, F36/22, 115-D8, and Ca1 all react with the same purified DF3 antigen. Furthermore, immunoblot analysis indicates that the DF3 antigen reactive with these MAb differs structurally in preparations from breast carcinoma cells and HMFGM. We also demonstrate that MAb F36/22 completely inhibits MAb DF3 binding in competitive blocking assays. In contrast, the results indicate that MAb 115-D8 and Ca1 only partially block MAb DF3 reactivity and the extent of this inhibition varies with DF3 antigen purified from breast carcinoma cells and HMFGM. Taken together, these findings with multiple MAb prepared against a variety of immunogens suggest that existence of a family of related but not identical high molecular weight tumor-associated glycoproteins.

Monoclonal antibody DF3 correlates with tumor differentiation and hormone receptor status in breast cancer patients.

The murine monoclonal antibody (MAb) designated DF3, reacts with a 300-kd human mammary epithelial antigen which is expressed on apical borders of secretory mammary epithelial cells and in the cytosol of less differentiated malignant cells. Human mammary tumors have been evaluated for the level of DF3 antigen as a correlate to clinicopathologic parameters related to degree of tumor differentiation: nuclear grade (NG), histologic grade (HG), and estrogen receptor status (ER). More DF3 antigen was present in breast carcinomas with NG 1 and 2 as compared to tumors with NG 3 (p = .002). Similarly DF3 antigen presence was greater in HG 1 and 2 tumors than in HG 3 (p less than .001). The results also demonstrate that quantitative differences in the presence of the DF3 differentiation antigen correlate with estrogen receptor status. Twenty-two of 23 ER positive tumors were also DF3 positive. Only 6 of 23 ER negative tumors were reactive to MAb DF3 (p less than .001). There was, however, no correlation between DF3 reactivity and absolute levels of estrogen or progesterone receptor. These findings confirm our hypothesis that MAb DF3 reacts to a differentiation antigen present in some human breast carcinomas. The DF3 antigen phenotype can serve as an independent phenotypic marker with correlations to standard indicators of degree of differentiation and estrogen receptor status of infiltrating ductal carcinomas of the breast, and should thus be evaluated as a prognostic indicator in breast cancer patients. The data also suggests that DF3 histochemistry may be a useful alternative in assessing estrogen receptor status of small breast cancers where there is an insufficient amount of tumor present for biochemical assay of hormone receptor levels.