MUC1 Antibodies Research Articles

MUC1 selectively targets human pancreatic cancer in orthotopic nude mouse models.

The goal of this study was to determine whether MUC1 antibody conjugated with a fluorophore could be used to visualize pancreatic cancer. Anti-MUC1 (CT2) antibody was conjugated with 550 nm or 650 nm fluorophores. Nude mouse were used to make subcutaneous and orthotopic models of pancreatic cancer. Western blot and flow cytometric analysis confirmed the expression of MUC1 in human pancreatic cancer cell lines including BxPC-3 and Panc-1. Immunocytochemistry with fluorophore conjugated anti-MUC1 antibody demonstrated fluorescent areas on the membrane of Panc-1 cancer cells. After injecting the conjugated anti-MUC1 antibodies via the tail vein, subcutaneously transplanted Panc-1 and BxPC-3 tumors emitted strong fluorescent signals. In the subcutaneous tumor models, the fluorescent signal from the conjugated anti-MUC1 antibody was noted around the margin of the tumor and space between the cells. The conjugated anti-MUC1 antibody bound the tumor in orthotopically-transplanted Panc-1 and BxPC-3 models enabling the tumors to be imaged. This study showed that fluorophore conjugated anti-MUC1 antibodies could visualize pancreatic tumors in vitro and in vivo and may help to improve the diagnosis and treatment of pancreatic cancer.

MUC1 Promoter-Driven DTA as a Targeted Therapeutic Strategy against Pancreatic Cancer.

Mucin1 (MUC1) is overexpressed in pancreatic ductal adenocarcinoma (PDA) and is associated with tumor aggressiveness, suggesting that MUC1 is a promising therapeutic target for promoter-driven diphtheria toxin A (DTA). Endogenous MUC1 transcript levels were analyzed by quantitative PCR (qPCR) in multiple PDA cells (Capan1, HPAFII, Su.86.86, Capan2, Hs766T, MiaPaCa2, and Panc1). Expression levels were correlated with luciferase activity and cell death after transfection with MUC1 promoter-driven luciferase and DTA constructs. MUC1-positive (+) cells had significantly elevated MUC1 mRNA expression compared with MUC1-negative (-) cells. Luciferase activity was significantly higher in MUC1(+) cells when transfected with MUC1 promoter-driven luciferase and MUC1(+) cells underwent enhanced cell death after transfection with a single dose of MUC1 promoter-driven DTA. IFNγ pretreatment enhanced MUC1 expression in MUC1(-) cells and induced sensitivity to MUC1-DTA therapy. Matched primary and metastatic tumor lesions from clinical specimens revealed similar MUC1 IHC labeling patterns, and a tissue microarray of human PDA biopsies revealed increased immunolabeling with a combination of MUC1 and mesothelin (MSLN) antibodies, compared with either antibody alone. Combining MUC1 with MSLN-targeted DTA enhanced drug efficacy in an in vitro model of heterogeneous PDA. These data demonstrate that MUC1 promoter-driven DTA preferentially kills MUC1-expressing PDA cells and drugs that enhance MUC1 expression sensitize PDA cells with low MUC1 expression. MUC1 expression in primary and metastatic lesions provides a rationale for the development of a systemic MUC1 promoter-driven DTA therapy that may be further enhanced by combination with other promoter-driven DTA constructs.

Usefulness of KL-6 in the subtyping of intraductal papillary mucinous neoplasia of the pancreas, including carcinoma, dysplasia, and hyperplasia.

KL-6 is known as a useful serum biomarker of the disease activity in interstitial pneumonias. We investigated its usefulness as a biomarker for subtyping intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. IPMNs are generally divided into 4 subtypes, namely pancreatobiliary (PB), intestinal (INT), gastric (GS), and oncocytic (ONC). Aside from the KL-6 antibody, the MUC1, MUC2, MUC5AC, MUC6, and MIB-1 antibodies were also examined. Eighteen IPMN cases were examined, including 12 cases of intraductal papillary mucinous carcinomas (IPMCs) simultaneously associated with dysplasia (IPMDs) and hyperplasia (IPMHs) and 6 IPMD cases with IPMH. KL-6 antibody was positive in the 8 IPMC cases, corresponding to a MUC2-negative PB subtype, but negative in 4 IPMC cases, corresponding to the INT subtype, which is positive for MUC2. IPMD of moderate-to-severe degree positively stained for the KL-6 antibody in the IPMC cases of the PB subtype but not in those of the INT subtype. The IPMH cases were mostly negative for KL-6, similar to the mild IPMD cases. In the 6 cases of mild IPMD and/or IPMH, KL-6 and MUC2 expressions were mostly negative. In conclusion, the KL-6 antibody is immunohistochemically a good biomarker of the PB subtype of IPMC, but not the INT subtype. Identifying IPMN subtypes based on KL-6 stainability would be useful. Clinicopathological studies with more IPMC cases might be needed for further progress in this field of study.

Abstract B297: Clinical assessment of MUC1 protein expression in FFPE tissue: Developent and validation of an immunohistochemistry assay as a predictive assay for response to MUC1 vaccines.

Abstract Background: Mucin-1 (MUC1, epithelial membrane antigen, EMA) is a transmembrane glycoporotein that is expressed on the apical membrane of epithelial cells of many tissues including breast, prostate, lung, pancreas, stomach, ovaries, intestines, and kidneys. In tumor cells, MUC1 is often overexpressed and aberrantly glycosylated, revealing new epitopes that can trigger a cytotoxic T-cell response. Several approaches are currently being pursued for targeting MUC1 in cancer therapy, mainly focused on vaccines targeting MUC1 antigens. We have developed an immunohistochemistry (IHC) assay to measure MUC1 expression in formalin fixed paraffin-embedded (FFPE) tissues as a potential biomarker assay for MUC1-targeted therapies. Methods: The IHC assays were developed using the Ventana Benchmark™. The MUC1 antibody was obtained from Ventana. The assay was validated using the recommended protocol supplied by the vendor. The assay was optimized using cell pellets generated from cell lines overexpressing MUC1 cDNA as well as an FFPE breast tumor tissue array which consists of 16 cases of breast cancer paired with adjacent normal tissues. In addition, tissue specimens obtained from remnant material from surgery or autopsy were used for the assay optimization and validation. Results: Specificity of the staining was demonstrated by positive staining with T47D breast cancer cell line as well as a B16 cell line overexpressing MUC1 and no staining with HMEC human mammary epithelial cells and the control B16 cell line. Thirty-six FFPE tumor tissues from lung, ovary, breast, colon, kidney, liver, thyroid, and prostate were evaluated for MUC1 expression. Tumor positive specimens were scored based on expression in at least 25% of tumor cells. In addition, staining intensities as well as patterns i.e. apical or membrane polarization and cytoplasmic patterns were noted. 29/36 specimens had positive MUC1 staining; negative staining was reported in thyroid, liver, colon, and prostate tumor tissues. In addition, evaluation of the array of paired normal/tumor breast tissues showed specific staining of tumor tissues in 14/16 specimens with no or little staining in the matched normal tissue. Interestingly, one of the tumor tissues exhibited very distinct apical staining pattern that was unique in the set of 16 breast cancer tissues. Conclusions: We have developed a robust IHC assay for MUC1 that can clearly distinguish MUC1 expression in normal versus tumor tissue as well as demonstrated variable MUC1 expression in a variety of tumor tissues. We are currently exploring the utility of the assay as a biomarker for ONT-10, a novel liposomal cancer vaccine that is currently in clinical trials. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B297. Citation Format: Sabita Sankar, Jennifer Wright, Kevin Klucher, Scott Peterson, Chad Galderisi. Clinical assessment of MUC1 protein expression in FFPE tissue: Developent and validation of an immunohistochemistry assay as a predictive assay for response to MUC1 vaccines. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B297.

Carbohydrate Determinants in Ferret Conjunctiva are Affected by Infection with Influenza H1N1 Virus

Background: Carbohydrates often accomplish as cell-surface receptors for microorganisms and influenza virus preferentially binds to sialic acid through the viral haemagglutinin. The virus may attach not only to the epithelium in the airways, but also to the surface ocular epithelium.Purpose: To decide if ferrets can be used to study virus induced conjunctivitis and to evaluate changes in the conjunctival glycosylation pattern during an influenza attack.Methods: Ferrets were infected with H1N1 influenza virus via nasal inoculation. The in situ carbohydrate expressions in eyelid sections from ferrets 0 to 10 days after infection was examined using lectin- and immunohistochemistry.Results: The conjunctival cells became hypertrophic with appearance of both PAS positive and PAS + Alcian Blue stained cells 5–6 days after inoculation. The binding of three sialic acid detecting lectins were investigated: WGA, MAA2 and SNA1. While none of them stained conjunctival epithelial cells in the non-infected ferrets to any extent, there was a positive conjunctival reaction in the infected ferret after incubation with all three lectins. Binding of a MUC1 antibody that seems to detect sialylated determinants in the mucin molecule indicates that MUC1 is de novo expressed in most of the squamous conjunctival cells at the start of the influenza infection. MUC5AC positive epithelial cells, probably goblet cells, proliferate in the diseased conjunctiva.Conclusion: Nasal inoculation of H1N1 virus to ferrets has an effect on the conjunctival cells and change their expression of glycans. Synthesized glycans are an integral part of the tear film and the present study contributes to reveal the changes that occur in the surface epithelium in the eyelid and thereby to elucidate the pathophysiology of the virus mediated conjunctivitis. Ferrets are suitable animal models to study human conjunctivitis mediated by human influenza virus.

Synthesis and immunological evaluation of a MUC1 glycopeptide incorporated into l-rhamnose displaying liposomes.

MUC1 variable number tandem repeats (VNTRs) conjugated to tumor-associated carbohydrate antigens (TACAs) have been shown to break self-tolerance in humanized MUC1 transgenic mice. Therefore, we hypothesize that a MUC1 VNTR TACA-conjugate can be successfully formulated into a liposome-based anticancer vaccine. The immunogenicity of the vaccine should be further augmented by incorporating surface-displayed l-rhamnose (Rha) epitopes onto the liposomes to take advantage of a natural antibody-dependent antigen uptake mechanism. To validate our hypothesis, we synthesized a 20-amino-acid MUC1 glycopeptide containing a GalNAc-O-Thr (Tn) TACA by SPPS and conjugated it to a functionalized Toll-like receptor ligand (TLRL). An l-Rha-cholesterol conjugate was prepared using tetra(ethylene glycol) (TEG) as a linker. The liposome-based anticancer vaccine was formulated by the extrusion method using TLRL-MUC1-Tn conjugate, Rha-TEG-cholesterol, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in a total lipid concentration of 30 mM. The stability, homogeneity, and size characterization of the liposomes was evaluated by SEM and DLS measurements. The formulated liposomes demonstrated positive binding with both anti-Rha and mouse anti-human MUC1 antibodies. Groups of female BALB/c mice were immunized and boosted with a rhamnose-Ficoll (Rha-Ficoll) conjugate formulated with alum as adjuvant to generate the appropriate concentration of anti-Rha antibodies in the mice. Anti-Rha antibody titers were >25-fold higher in the groups of mice immunized with the Rha-Ficoll conjugate than the nonimmunized control groups. The mice were then immunized with the TLRL-MUC1-Tn liposomal vaccine formulated either with or without the surface displaying Rha epitopes. Sera collected from the groups of mice initially immunized with Rha-Ficoll and later vaccinated with the Rha-displaying TLRL-MUC1-Tn liposomes showed a >8-fold increase in both anti-MUC1-Tn and anti-Tn antibody titers in comparison to the groups of mice that did not receive Rha-Ficoll. T-cells from BALB/c mice primed with a MUC1-Tn peptide demonstrated increased proliferation to the Rha-liposomal vaccine in the presence of antibodies isolated from Rha-Ficoll immunized mice compared to nonimmune mice, supporting the proposed effect on antigen presentation. The anti-MUC1-Tn antibodies in the vaccinated mice serum recognized MUC1 on human leukemia U266 cells. Because this vaccine uses separate rhamnose and antigenic epitope components, the vaccine can easily be targeted to different antigens or epitopes by changing the peptide without having to change the other components.

The use of a novel MUC1 antibody to identify cancer stem cells and circulating MUC1 in mice and patients with pancreatic cancer

MUC1 is over-expressed and aberrantly glycosylated in >60% of human pancreatic cancer (PC). Development of novel approaches for detection and/or targeting of MUC1 are critically needed and should be able to detect MUC1 on PC cells (including cancer stem cells) and in serum. The sensitivity and specificity of the anti-MUC1 antibody, TAB 004, was determined. CSCs were assessed for MUC1 expression using TAB 004-FITC on in vitro PC cell lines, and on lineage(-) cells from in vivo tumors and human samples. Serum was assessed for shed MUC1 via the TAB 004 EIA. In vitro and in vivo, TAB 004 detected MUC1 on >95% of CSCs. Approximately, 80% of CSCs in patients displayed MUC1 expression as detected by TAB 004. Shed MUC1 was detected serum in mice with HPAF-II (MUC1(high) ) but not BxPC3 tumors (MUC1(low)). The TAB 004 EIA was able to accurately detect stage progression in PC patients. The TAB 004 antibody may be explored as a therapeutic targeting agent for CSCs in PC. The TAB 004 EIA detected circulating MUC1 in a stage-dependent manner in patients with PC and thus may be explored as a PC stage diagnostic biomarker.

Distinct Cytoplasmic Expression of KL-6 Mucin in Chromophobe Renal Cell Carcinoma: A Comparative Immunohistochemical Study with Other Renal Epithelial Cell Tumors

The presence of cytoplasmic sialyl glycoproteins is a conspicuous feature in chromophobe renal cell carcinoma (RCC). We compared the immunohistochemical expression of sialyl glycoproteins in chromophobe RCC with that in other types of renal tumors. Formalin-fixed, paraffin-embedded tissues of surgically resected renal tumors (chromophobe RCC, 14 cases [10 cases of classic type and 4 cases of eosinophilic variant]; oncocytoma, 7 cases; and clear cell RCC, 9 cases) and kidneys from immature infants (4 cases) were immunostained with antibodies against sialyl glycoproteins (anti-KL-6 and anti-sialyl MUC1 antibodies). Cytoplasmic expression of KL-6 and sialyl MUC1 was distinctive in the chromophobe RCC and renal oncocytoma cells, and in the intercalated cells in collecting duct epithelia. Apical-surface staining of these sialyl glycoproteins was predominantly observed in clear RCC, in the epithelia of the distal tubule and collecting duct, and in the neonatal renal proximal tubule, but not in those of the adult renal proximal tubule. The above-mentioned observations provide additional evidence for similar phenotypic profiles of chromophobe RCC and renal oncocytoma, and the intercalated cells in collecting ducts and the oncofetal expression of sialyl glycoproteins in clear cell RCC. KL-6 is a potential tumor marker for renal tumors.

MUC1 and the simple mucin-type antigens: Tn and Sialyl-Tn are differently expressed in salivary gland acini and ducts from the submandibular gland, the vestibular folds, and the soft palate

Objective and design Autopsies of the submandibular gland, the vestibular folds and the soft palate from 65–87 old humans were examined to record the immunohistochemical expression of MUC1 and the simple mucin-type antigens Tn and Sialyl-Tn. Results (1) The serous acini in the submucosal glands from the larynx and the soft palate expressed MUC1-associated glycans that were not detectable in the serous acini from the submandibular gland. (2) Virtually all the submucosal acini at oral site of the soft palate are mucous, and in contrast to mucous acini in the vestibular folds and submandibular gland, the palatinal acini in the submucosa underneath the oral mucosa showed a well-defined cytoplasmic reaction with anti-MUC1 antibodies as wells as with anti-Tn. (3) Both the mucous acini and the ducts at the oral site of the soft palate showed reaction for Sialyl-Tn while in the vestibular folds and in the submandibular gland expression for this carbohydrate was observed only in the acini. (4) The staining obtained after incubation with the Tn antibodies showed no cross localization with the staining obtained after incubation with an anti-A blood group antibody. (5) All the autopsies showed reaction in the glands after incubation with the MUC1 antibodies while some autopsies reacted with the anti-Tn antibodies and/or with the anti-Sialyl-Tn antibodies and others did not. Conclusion The mucin expression in the acini and ducts from the upper human aerodigestive tract strongly depended on the location of the glandular tissue.

A randomized, open-label phase IIb study of maintenence therapy with a MUC-1 dendritic cell vaccine in patients with epithelial ovarian cancer in first or second remission.

TPS171 Background: MUC-1 is a highly immunogenic antigen that is widely expressed in epithelial ovarian carcinoma (EOC) making it an ideal target for vaccine therapy. Prior phase I and II studies conducted in Australia of the MUC-1 dendritic cell vaccine (CVac) in heavily pretreated, advanced disease patients showed minimal toxicities and prolonged disease stabilization. The purpose of this trial is to determine the safety and efficacy of a MUC-1 dendritic cell vaccine (CVac) in ovarian cancer patients who are in clinical remission after first or second-line therapy. Methods: Women with stage III or IV ovarian, fallopian tube, or primary peritoneal carcinoma who have had a complete response (CR) based on clinical and radiologic studies after first or second-line chemotherapy will be eligible for this study. Planned sample size is 60 patients. The first 6 patients will be treated with CVac open-label to ensure consistency of manufacturing. The next 54 patients will be randomized 1:1 to CVac or standard of care (observation). Patients receiving vaccine will undergo leukopheresis followed by in vitro loading of dendritic cells with MUC-1. They will receive a total of 10 intradermal vaccine administrations over 52 weeks (every 4 weeks × 7, then every 8 weeks × 3). Clinical assessments will be every 4 weeks and radiologic assessments will be every 12 weeks. Primary endpoints are the safety of CVac in this population and effect on progression-free survival (PFS). Secondary endpoints are effect on overall survival (OS) and immunologic parameters, including MUC-1 antibodies and intracelluar cytokine assays. No significant financial relationships to disclose.

Abstract 5696: Down-regulation of MUC1 by the herbal formulation, triphala in colon cancer cells

Abstract Natural products from medicinal plants and traditional medicine have been a fertile source for identification of novel molecules with human health-protective effects. Triphala is an Ayurvedic medicine used as a dietary supplement to prevent and cure ailments of the gastro-intestinal tract in India from ancient times. It is a formulation made from dry powder of three fruits, Terminalia chebula, Emblica officinalis and Terminalia bellerica and has been used to treat microbial infections and ulcers of stomach and colon. In order to identify bioactive principles and their molecular targets, colon cancer cells HCT-116 (5×103 cells) were seeded in 96 well plates and treated with the methanolic extract of triphala at varying concentrations for 24, 48 and 72 hours. Cell proliferation assay demonstrated a dose-dependent inhibition of cell growth and the IC50 was determined to be ∼100 μg/ml. RNA isolated from colon cancer cells treated for 6 hours with the methanolic extract at 100 and 200 μg/ml concentrations revealed a dose-dependent down-regulation of MUC1 gene expression as detected by qRT-PCR. Also, MUC1 antibody immunohistochemistry showed down-regulation of MUC1 protein expression, when cells grown in chamber slides were treated with the methanolic extract. In addition, qRT-PCR revealed down-regulation of expression of the inflammatory molecules IL-6, IFNγ, COX-2, NF-κB and the cell cycle-regulatory molecule, cyclin D1. These results suggest that triphala extract exerts its anti-tumorigenic effects by influencing the expression of mucins via modulation of inflammatory molecules that are up-stream of mucin expression, in colon cancer cells. FTICR-MS (Fourier-transform Ion Cyclotron Resonance MS) analysis of the methanolic extract of triphala readily confirmed the presence of previously reported compounds such as gallic acid, ellagic acid, several mucic acid gallates, chebulic acid, ascorbic acid, citric acid, and several tannins. Sub-fractionation of the methanolic extract with ethyl acetate, when tested on colon cancer cells, showed higher efficacy (IC50 = 50 µg/ml) suggesting enrichment of select bioactive principles. FTICR-MS analysis of the ethyl acetate extract revealed enrichment of gallic acid, ellagic acid, and several other tannins, while mucic acid and their gallates were not. HPLC fingerprinting of the methanolic and ethyl acetate extracts confirmed the enrichment of phenolics into ethyl acetate fraction. The fractionation results suggest that enrichment of distinct set of active principles of triphala into different fractions is a viable strategy to help identify their specific molecular targets. More extensive fractionation of the methanolic extract of triphala is currently underway (Supported from Agnes Brown Duggan Endowment). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5696.

MUC1 Limits Helicobacter pylori Infection both by Steric Hindrance and by Acting as a Releasable Decoy

The bacterium Helicobacter pylori can cause peptic ulcer disease, gastric adenocarcinoma and MALT lymphoma. The cell-surface mucin MUC1 is a large glycoprotein which is highly expressed on the mucosal surface and limits the density of H. pylori in a murine infection model. We now demonstrate that by using the BabA and SabA adhesins, H. pylori bind MUC1 isolated from human gastric cells and MUC1 shed into gastric juice. Both H. pylori carrying these adhesins, and beads coated with MUC1 antibodies, induced shedding of MUC1 from MKN7 human gastric epithelial cells, and shed MUC1 was found bound to H. pylori. Shedding of MUC1 from non-infected cells was not mediated by the known MUC1 sheddases ADAM17 and MMP-14. However, knockdown of MMP-14 partially affected MUC1 release early in infection, whereas ADAM17 had no effect. Thus, it is likely that shedding is mediated both by proteases and by disassociation of the non-covalent interaction between the α- and β-subunits. H. pylori bound more readily to MUC1 depleted cells even when the bacteria lacked the BabA and SabA adhesins, showing that MUC1 inhibits attachment even when bacteria cannot bind to the mucin. Bacteria lacking both the BabA and SabA adhesins caused less apoptosis in MKN7 cells than wild-type bacteria, having a greater effect than deletion of the CagA pathogenicity gene. Deficiency of MUC1/Muc1 resulted in increased epithelial cell apoptosis, both in MKN7 cells in vitro, and in H. pylori infected mice. Thus, MUC1 protects the epithelium from non-MUC1 binding bacteria by inhibiting adhesion to the cell surface by steric hindrance, and from MUC1-binding bacteria by acting as a releasable decoy.

The importance of MUC1 and cyclin B1 antibodies in nipple aspiration fluid (NAF): preliminary results

MUC1 glycoprotein is produced by normal breast epithelial cells and cyclin B1 is involved in the transition from G2-to-M phase of the normal cell cycle. In cancer cells, including breast cancer, MUC1 and cyclin B1 are overexpressed and aberrantly expressed leading to their recognition by the immune system and production of specific antibodies detected in the serum of cancer patients. Because MUC1 and cyclin B1 are expected to be deregulated early in disease, we hypothesized that monitoring these antibody responses may be of diagnostic or prognostic value in breast cancer (BC). Furthermore, we wanted to test if NAF can be an alternative source of these antibodies.

KL-6 is another useful marker in assessing a micropapillary pattern in carcinomas of the breast and urinary bladder, but not the colon

To evaluate the peculiar "inside-out" pattern in micropapillary (MP) carcinoma, we investigated the usefulness of KL-6 antibody in the assessment of the MP pattern of cancers, in comparison with antibodies to epithelial membrane antigen (EMA), MUC1 (CD227), and CD 10. Immunohistochemical investigation was performed on specimens exhibiting an MP pattern obtained from 12 persons with cancer: 4 with breast carcinoma, 3 with carcinoma of the urinary bladder, and 5 with colonic carcinoma. Immunohistochemical study with KL-6, EMA, and MUC1 antibodies revealed similar continuous linear positive patterns restricted to the surface of the MP pattern in both breast and urinary bladder cancers, revealing the peculiar "inside-out" morphology. However, EMA also gave cytoplasmic positivity in most of the cases tested, and MUC1 was also present in the cytoplasm of some cases. In sharp contrast, immune reactions of colon carcinomas with these antibodies were negative, except for focal positivity for KL-6 and MUC1 antibodies in some cases. CD10 was only focally positive in an MP pattern in 4 of the 5 cases of colon carcinoma and in 1 case with carcinoma of the urinary bladder. These findings suggest that KL-6 is a useful marker to assess the MP character of breast and urinary bladder carcinomas; that MUC1 was similarly positive, with the addition of cytoplasmic positivity in some cases; and that the MP pattern of colon cancer, positive for CD 10, was different in character from both breast and urinary bladder carcinomas, although all these cancers seemingly exhibit similar MP patterns on histopathology. This heterogeneity of the MP pattern in various cancers needs to be investigated when more cases have been accumulated.

Labeled anti-mucin antibody detectable by infrared-fluorescence endoscopy

The goal of our study was to develop a method for molecular imaging of the gastrointestinal tract using an infrared fluorescence endoscope (IRFE) and antibodies labeled with an indocyanine green (ICG) derivative to detect cancerous tissue. The IRFE comprised an infrared endoscope equipped with excitation (710-790 nm) and barrier (810-920 nm) filters. We developed ICG-N-hydroxysulfosuccinimide ester (ICG-sulfo-OSu) and 3-ICG-acyl-1,3-thiazolidine-2-thione (ICG-ATT) as infrared fluorescent-labeling reagents, and anti-human carcinoembryonic antigen (CEA) antibody and MUC1 antibody were labeled with the ICG-derivatives. Freshly resected specimens of gastric cancer were observed by IRFE after reaction with ICG-derivative-labeled antibodies. Positive fluorescence was observed at the tumor location by IRFE, and the immunofluorescent images correlated well with the tumor sites. The immunofluorescence studies suggested that the intensity of the infrared fluorescence of the ICG-ATT-labeled MUC1 antibody is stronger than the ICG-sulfo-OSu-labeled MUC1 antibody. We concluded that specific antibodies for gastrointestinal cancer labeled with an ICG-derivative accompanied by a reinforcing agent and an optimal electronic device can generate a strong enough fluorescent signal to visualize cancer proteins.

TA-MUC1 epitope in non-small cell lung cancer

MUC1 (CD227), an established tumor marker, is expressed on glandular epithelia and on epithelial tumors. Tumor MUC1 differs from normal MUC1 by modified glycan side chains. Recently, a novel carbohydrate-induced conformational tumor-associated MUC1 epitope (TA-MUC1) was described, whose clinical relevance in lung cancer is not known. Eighty-five paraffin embedded tissue sections of non-small cell lung cancer (NSCLC) patients (73% male; mean age 64+/-9 years) were stained with the monoclonal antibody PankoMab (against TA-MUC1) and compared with the established antibodies E29 and 214D4 regarding prognostic relevance. TA-MUC1 is virtually absent in bronchial epithelium. As shown by multivariate analysis, only staining with PankoMab, but not with E29 or 214D4, was correlated with patients' survival (p=0.029). Moreover, when regarding interactions of MUC1 antibody staining results and clinico-pathological parameters, patients with lymph node metastasis lacking PankoMab staining were attributed the highest risk by far (Hazard ratio=4.6, 95% CI: 2.1-9.7, p=0.000). In summary, the presence of TA-MUC1 is a favorable prognostic factor in this cohort of NSCLC patients, in particular if lymph node metastases are present. This is in contrast to the results for E29 and 214D4, which recognize less or not glycosylation dependent epitopes. As this is the first report on a well-defined MUC1 epitope associated with improved survival in NSCLC, a more differentiated view on MUC1 may be mandatory.

Radioimmunoscintigraphy of pancreatic cancer in tumor-bearing athymic nude mice using 99mtechnetium-labeled anti-KL-6/MUC1 antibody

KL-6 is an extracellular epitope of MUC1, a membrane-penetrating glycoprotein, and its overexpression has been reported in pancreatic cancer. The aim of this study was to examine whether radiolabeled anti-KL-6/MUC1 antibody could be used for molecular imaging of pancreatic cancer in vivo. Anti-KL-6/MUC1 antibody was labeled with 99mTC by the stannous reduction method. Immunoreactivity of the 99mTc-labeled anti-KL-6/MUC1 antibody was evaluated by a whole-cell binding study. In vivo experiments were performed by injecting the 99mTc-labeled anti-KL-6/MUC1 antibody into athymic nude mice bearing the KP-1NL pancreatic cancer cell line. A whole-cell binding study showed that the radiolabeled antibody retained its immunoreactivity. On scintigrams, the density of the tumors remained unchanged during the 16-32 h after injection, whereas that of the kidneys decreased time-dependently. The radioactivity levels of the kidneys and tumors were measured densitometrically, and we found that the intensity in the tumors relative to that in the kidneys increased time-dependently. Radioactivity levels were the highest in the blood 32 h after injection, and those in the liver, kidney, lung, and tumor were also rather high. 99mTc-labeled anti-KL-6/MUC1 antibody appears to be a promising agent as a tumor-specific radiotracer for pancreatic cancer.

The MUC1 HMFG1 Glycoform Is a Precursor to the 214D4 Glycoform in the Human Uterine Epithelial Cell Line, HES1

MUC1, a type I transmembrane glycoprotein expressed on most epithelia and many cancer cells, is involved in embryo implantation and tumor progression. A series of antibodies directed against the MUC1 ectodomain have been used to study MUC1 expression in the female reproductive tract, sometimes with apparently contradictory results. In the current study, we used two monoclonal MUC1 antibodies, 214D4 and HMFG1, to study the relationship between these MUC1 glycoforms in the human uterine epithelial cell line, HES, and human endometrial extracts. In response to tumor necrosis factor stimulation, accumulation of the HMFG1-reactive forms preceded that of the 214D4-reactive forms. Following inhibition of protein synthesis by cycloheximide, HMFG1-reactive species were lost rapidly (metabolic half-life [T(1/2)] = 20 min), while there was no change in the level of the 214D4-reactive forms even after 80 min. HMFG1-reactive forms had smaller oligosaccharide chains than the 214D4-reactive forms, and could not be detected on the cell surface of intact cells or in the shed (media) fraction, although they were readily detected in permeabilized cells. Both 214D4- and HMFG1-reactive species were detected in human endometrial extracts throughout the cycle; however, consistent with the HES cell studies, the HMFG1-reactive species were both smaller and less abundant than the 214D4-reactive species. Consistent with this observation, we found that HMFG1-reactive species were difficult to detect in tissue sections unless predigested with neuraminidase, indicating that these structures are rapidly sialylated during synthesis. In contrast, 214D4-reactive species were robustly detected in both proliferative and secretory stages. Collectively, these studies indicate that the HMFG1-reactive glycoform is a precursor of the 214D4-reactive glycoform in HES cells and normal uterine epithelia. Therefore, discrepancies in patterns of MUC1 expression in other studies may be due to failure to account for these glycoform relationships.

Combined Staining of TAG-72, MUC1, and CA125 Improves Labeling Sensitivity in Ovarian Cancer

Single antigen-targeted intraperitoneal radioimmunotherapy for ovarian cancer has shown limited success. Due to the heterogeneous expression of tumor antigens on cancer cells, a multi-antigen targeting approach appears logical to augment the therapeutic efficacy of antibody-guided therapy. In the interest of developing this novel approach, ovarian cancer tissue microarray slides containing cancer and benign/non-neoplastic tissue samples (n=92) were processed for single-, double-, and triple-antigen labeling using antibodies for the tumor-associated antigens TAG-72, MUC1, and CA125. Among all ovarian cancer types, 72%, 61%, and 50% of the samples showed immunolabeling for TAG-72, MUC1, and CA125, respectively. Expression level of these antigens was significantly (p<0.005) higher in advanced stage carcinomas compared with early stage. Of the 48 epithelial ovarian cancer samples, individual anti-TAG-72, MUC1, and CA125 antibody probing showed labeling in 89.5%, 87.5%, and 73.0% of the cases, respectively. In the majority of the cancer samples (>70%), a heterogeneous labeling pattern was observed (only 30-40% of the cancer cells within the sample were labeled). However, upon combining the three antigens (triple-antigen labeling), 98% of the epithelial ovarian cancer samples were labeled and >95% of the cancer cells within each sample were labeled. Our data indicate that the heterogeneous expression of cancer antigens appears to be a major obstacle in antibody-guided therapy, and this can be overcome by multiple antigen targeting. Therapeutic efficacy of antibody-guided therapy for ovarian cancer treatment will be enhanced by the combined targeting of TAG-72, MUC1, and CA125.

Humoral immune responses to MUC1 in women with a BRCA1 or BRCA2 mutation

Introduction Breast cancer patients with early disease and a natural humoral response to MUC1 have a favourable prognosis, suggesting a possible role of MUC1 antibodies (ab) in controlling haematogenous tumour dissemination and outgrowth. The aim of the study was to evaluate humoral immune responses to MUC1 in women at hereditary high risk of breast cancer to investigate whether this immune response could play a role in the prevention of disease. Materials and methods CA15.3 (U/mL), and IgG and IgM ab to MUC1 (arbitrary units per mL, Arb-U/mL) were measured in serum samples obtained from 422 women at hereditary high risk of breast/ovarian cancer, of whom 127 BRCA1/2 carriers, attending the Familial Cancer Clinic of the VU University Medical Centre, and from 370 age-matched healthy controls. Serum samples obtained from women who developed breast cancer ( N = 12) or breast cancer recurrence ( N = 17), and from women who underwent prophylactic mastectomy ( N = 12) and had no breast lesions were also tested. Results CA15.3 ranked significantly higher in mutation carriers than in controls ( P = 0.03). MUC1 IgG ab levels ranked significantly lower in BRCA1/2 mutation carriers than in controls ( P = 0.003). MUC1 IgG levels were not significantly different ( P = 0.53) between women who developed primary breast cancer (median 0.72 Arb-U/ml, range 0.52–2.44 Arb-U/ml) and women who underwent prophylactic mastectomy and had no breast lesions (median 1.04 Arb-U/ml, range 0.43–2.88 Arb-U/ml). Conclusion Serum levels of natural IgG ab to MUC1 are lower in BRCA1/2 mutation carriers than in healthy controls. Furthermore, in contrast to previous results in women with sporadic breast cancer, no elevated MUC1 IgG ab were seen in women at hereditary high risk who developed breast cancer. Prophylactic immunotherapy with MUC1 substrates may be a strategy to reduce the risk of breast cancer in BRCA1/2 mutation carriers, strengthening tumour immune surveillance.