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Introduction: Cripto is a founding member of the EGF-CFC family, and plays an important role in tumourigenesis, tumour cell proliferation and migration. We aimed to determine the significance of Cripto expression on the survival of patients with breast cancer., Methods: Immunohistochemical detection of Cripto was performed by using mAb C13 on 120 formalin-fixed paraffin-embedded breast tumour specimens in tissue microarrays. This cohort comprises a series of 120 patients with primary operable breast cancer diagnosed between 1989 and 1995, retrieved from the Concord Repatriation General Hospital breast carcinoma database., Results: Using a cutoff value of 80%, Cripto overexpressed in 57 of the 120 (47.5%) patients. We found significant associations between overexpression of Cripto and the Nottingham Prognostic Index (NPI, p<0.01), histological grade (p<0.01), pathological tumour type (p=0.04), PR (p=0.02) as well as Ki-67 (p=0.02). Univariate analysis reveals that there is a significant correlation between overexpression of Cripto and survival (p=0.0003). Cox regression analysis indicates that the overexpression of Cripto is an independent prognostic factor in breast cancer (HR 2.79, 95%CI 1.20-6.50)., Conclusion: The unique epitope recognized by mAb C13 is overexpressed on breast tumour tissues. In this series of invasive breast cancers, overexpression of Cripto was more often found in high grade and poor prognosis tumours compared to low grade and good prognosis breast cancers. Moreover, overexpression of Cripto was significantly associated with decreased patient survival.

Mucin 1 (MUC1) is highly expressed in breast cancer, has an ubiquitous distribution and, due to altered glycosylation, peptides within the VNTR are exposed. These peptides are the target for anti-MUC1 antibodies, which give a differential reaction on cancer compared with normal tissue. The amino acids, APDTR or adjacent amino acids, are highly immunogenic in mice for antibody production (after immunisation with either breast cancer cells, human milk fat globule (HMFG) or the VNTR peptide). In addition, human studies show that this region of the MUC1 VNTR functions as target epitopes for cytotoxic T cells. We have performed preclinical and clinical studies to examine the immune responses to MUC1 in mice and humans: (a) MUC1+ 3T3 or P815+ 3T3 cells in syngeneic mice are rejected, with the generation of both cytotoxic T lymphocyte (CTL) and DTH responses and a weak antibody response and a weak antibody responses; this type of immunity gives rise to total resistance to re-challenge with high doses of these tumors; (b) immunisation with peptides (VNTR x 2), a fusion protein (VNTR x 5), or HMFG leads to no CTLs, DTH, good antibody production and weak tumour protection (to 10(6) cells, but not 5 x 10(6) cells) (possibly a TH2 type response); (c) immunisation with mannan-fusion protein (MFP) gives rise to good protection (resistance to 50 x 10(6) cells), CTL and DTH responses and weak antibody responses (possibly a TH1 type response, similar in magnitude to that obtained after tumor rejection); (d) established tumors can be rapidly rejected by delayed treatment of MFP; (e) the CTL responses are MHC restricted (in contrast to the human studies); (f) APDTR appears not to be the T cell reactive epitope in mice. On the basis of these findings, two clinical trials are in progress: (a) VNTR x 2 (diphtheria toxoid) which gives rise to some T cell proliferation, DTH and antibody responses in some patients and (b) an MFP trial. The ability to alter the immune response towards cellular immunity with mannan or to humoral immunity with peptides, allows the immune response to be selectively manipulated.

Humans with breast cancer have T-cells in their lymph nodes which recognize a peptide sequence within the variable number of tandem repeats of the mammary mucin, MUC1, which is overexpressed in breast cancer. To find means of making this recognition event into a potent immune response to breast cancer, we used a murine tumor model and have examined the parameters of the immune response to human mucin (MUC1) expressed in murine BALB/c 3T3 cells. We then sought to boost this response with MUC1-containing synthetic peptides, fusion proteins, and natural mucin (HMFG). MUC1+3T3 cells were found to be rejected by BALB/c mice by day 15 due to a cellular [CD3+, Ly2+ (CD8+)] response. The cellular rejection response was accompanied by the generation of CD8+ cytotoxic T-cells, CD4+ delayed-type hypersensitivity, and little anti-MUC1 antibody. This immune response is presumably of the TH1 type (which occurs in CD8 as well as CD4 cells) of CD8+ cytotoxic cells. By contrast, mice immunized with the MUC1 synthetic peptide, a fusion protein, or HMFG have good antibody responses, a delayed-type hypersensitivity reaction, but no cytotoxic T-cells and less tumor protection, possibly a TH2 type response. We conclude that CD8+ cytotoxic anti-mucin cells can produce significant antitumor responses in vivo to a human "tumor" antigen expressed in murine cells; immunization with soluble synthetic or native materials leads to the "humoral" (TH2) type of immunity, and efforts need to be made to convert this to a TH1-type response.

CD46 (membrane cofactor protein; MCP) is ubiquitously expressed on nucleated human cells; it has a protective function, binding C3b and C4b, which are then cleaved by serum factor I. CD46 molecules (55,000-65,000 MW) have four short consensus repeats (SCR): the function of SCR-1 and -2 is unknown; SCR-3 and -4 bind C3b and C4b. These are succeeded by the STP region, which can contain three separate regions (STP-A, -B, -C) rich in serine, threonine and proline and which are heavily glycosylated, succeeded by transmembrane and cytoplasmic tail regions (of which there are several). Multiple isoforms exist due to the different splicing of exons: STP-A and -B can thus be present or absent. So far these products can only be detected separately by polymerase chain reaction (PCR) and RNA studies; we now describe their detection by anti-peptide antibodies. Peptides whose sequences corresponded with those of STP-A and STP-B were synthesized and used for the immunization of mice; although they differ in only seven of 21 amino acids, monoclonal antibodies (mAb) that reacted specifically with STP-A but not with STP-B, and mAb that reacted specifically with STP-B but not with STP-A, were produced; these reacted specifically with native CD46 on human tissues and cell lines. STP-A mAb reacted with tissues in which STP-A RNA had been found, some leukaemias and cell lines; in normal tissue expression was mainly found in the intestine (large and small) and salivary gland. Anti-STP-B reacted with most tissues and cell lines. The antibodies should be of use in defining the expression and function of CD46 in different tissues.

Breast cancer is considered as the major cause of mortality by cancer for women. Even if chemotherapy, radiotherapy and surgery have improved the life expectancy of patients bearing tumours, breast cancer is responsible for the death of 42,000 women per year in USA and 25,000 women in France. In this context, cancer vaccines may add an attractive alternative therapeutic strategy to the current existing treatments. We describe here the construction of recombinant vaccinia viruses co-expressing a tumour associated antigen (MUC 1) and an "adjuvant" cytokine, which have potential applications in the active immunotherapy of breast cancer. Indeed, recombinant vaccinia viruses have been extensively used during the past decade to induce a protective response against a whole variety of pathogens, and has proven to be of great value in the elicitation of a cellular immune response leading to the rejection of tumour grafts in mouse models.

The expression of mucin carbohydrates [Tn, sialosyl-Tn(STn), and T antigens] and core proteins [MUCI-apomucin-related antigen (ARA) and MUC2-ARA] was examined immunohistochemically in tissues from 40 patients with hepatolithiasis and 26 patients with intrahepatic bile-duct carcinoma. Tn and STn antigens were expressed in most of the carcinomas, and were also often expressed in the atypical bile-duct epithelium of the patients with hepatolithiasis or carcinoma, whereas they were rarely or never expressed in the normal bile duct, suggesting that they are effective tumor markers. T antigen was less useful as a marker for intrahepatic bile-duct carcinoma or the atypical epithelium, because it was expressed in normal bile-duct of some cases. Regarding the expression of ARAs in the carcinomas, non-invasive bile-duct cyst adenocarcinomas with favorable prognosis either expressed no MUCI-ARA with [DF3(-), MUSEII(-) and 139H2(-)] staining pattern or expressed MUCI-ARA with [DF3(-), MUSEII(+) and 139H2(+)] staining pattern. However these tumors often expressed MUC2-ARA with [anti-MRP(+) and CCP58(+)] staining pattern. In contrast, most invasive non-papillary cholangiocarcinomas with poor prognosis expressed MUCI-ARA with [DF3(+), MUSEII(+) and 139H2(+)] staining pattern, but expressed no MUC2-ARA with [anti-MRP(-) and CCP58(-)] staining pattern. These results suggests that different apomucins are produced by bile-duct cystadenocarcinomas and cholangiocarcinomas with differing prognosis. Furthermore, expression of Tn and STn antigens is a useful indicator of malignancy in the intrahepatic duct.

Two murine Mabs VA1(IgG1) and VA2(IgG1) were produced against a bacterial fusion protein comprising glutathione S-transferase and five tandem repeats of the MUC1 protein. Using the immunoperoxidase staining technique, VA1 detected 46/53 and VA2 detected 48/53 breast cancers and both also reacted with a range of other human epithelial carcinomas. In addition VA1 gave weak reactions with normal breast tissues whereas VA2 was non-reactive and could be a relatively tumour specific antibody for breast cancer. The antibodies were also tested by ELISA-VA1 reacted weakly with glycosylated HMFG but strongly with deglycosylated HMFG, whereas VA2 reacted strongly with both forms of HMFG. The reactivities of the two Mabs with synthetic peptides of the MUC1 tandem repeat were used to map the epitopes recognised by VA1 (amino acids RPAPGS) and VA2 (amino acids DTRPA). The use of fusion proteins provides another means of immunisation to produce anti-tumour antibodies.

The secretion and nature of mucins produced from a panel of recently available new gastric and colon carcinoma cell lines (LIM1839, LIM1215, LIM1863, LIM1899, LIM2099, LIM2405, LIM2408, LIM2412, LIM2463), as well as other colon (LS174T, HT29, HT29-SB, COLO533, COLO206), breast (T47D, MCF-7, BT20, ZR75-1) and ovarian (COLO316) tumor cell lines, was investigated. ELISA and Western blotting of the culture supernatants with novel anti-MUC1 and anti-MUC2 monoclonal antibodies (MAbs) specific for mucin core proteins showed their secretion by most of these cell lines. In addition, mucins produced by these cell lines expressed the tumor-associated carbohydrate detected by MAb 3E1.2 (glycolylsialyl-Tn, mammary serum antigen or MSA) and the Tn or T antigens reactive with lectin SSA-M. SSA-M detected MUC1 or MUC2 captured by MAbs BC2 or CCP58, while 3E1.2 only detected MUC1-associated carbohydrate, indicating that the MAb may react with a conformationally dependent epitope, or that the sialyl/glycolyl-transferases involved in MSA production may be sequence specific. In addition, the BC2/SSA-M and CCP58/SSA-M assays detected mucins in some samples which were not detected by BC2/BC2 or CCP58/CCP58 dual determinant assays, indicating that this format may be more appropriate for the detection of tumor-associated mucins in body fluids. These new cell lines and assays should be of use in the investigation of mucin core proteins, particularly LIM2463 and LIM1839 which express significant quantities of both MUC1 and MUC2.

Monoclonal anti-mucine antibodies BC1, BC2 and BC3 produced using human milk fat globule membrane react with a synthetic peptide p1-24 (PDTRPAPGSTAPPAHGVTSAPDTR) representing the repeating amino acid sequence of the mucin core protein. The minimum epitope recognized by these three monoclonal antibodies (mAb) in p1-24 was contained in the five amino acids APDTR. To analyse the variation of position of the epitope, various modifications of the APDTR sequence were made by synthesizing peptides and testing by direct binding and inhibition enzyme-linked immunosorbent assays. Firstly, peptides p13-32 and C-p13-32, in which the epitope APDTR was placed in the middle instead of the C-terminal as in p1-24, were examined. These peptides had a greater reaction with mAb BC1, BC2 and BC3 compared with the reaction with p1-24. Secondly, A-p1-24 and TSA-p1-24 were made wherein two APDTR epitopes were present--these peptides were shown to bind two IgG antibody molecules. Finally, the contribution of each amino acid in the APDTR epitope was studied using the pepscan polyethylene rods, making all 20 of the amino acid substitutions in each position for SAPDTR (the minimum epitope APDTR with an adjacent amino acid S). In the 120 peptides examined there were some 'permissible' substitutions in A, D and T but not in P or R for BC1 and BC2; there were more 'permissible' substitutions for BC3; different substitution patterns were found with each antibody and some substitutions gave an increased reaction compared with the native peptide SAPDTR. The studies are of value in analysing the reaction of antibodies with epitopes expressed in breast cancer and in determining the antigenicity of synthetic peptides.

Two monoclonal antibodies (MoAbs) were produced against human colorectal cancer and were found to react with many colorectal carcinomas (MoAb DH-1 detected 25 of 32 and MoAb SMA-1 detected 28 of 32) by the immunoperoxidase technique. These two antibodies were found to detect epitopes present on human milk fat globules (HMFG) by enzyme-linked immunosorbent assays (ELISAs). There have been few reports of anti-mucin antibodies being produced to colon cancer and so we examined the reactions of these and 13 other anti-HMFG MoAbs with colonic cancers. Thirty-two colonic tumours were examined and different staining patterns were noted. Staining was particularly marked on the cell membranes and glandular deposits of the tumours rather than in the cytoplasm of the cells. All colonic tumours tested were positive with at least one MoAb, but no single MoAb stained all tumours; this suggested that at least one epitope of HMFG could be found on all colonic tumours but no epitope detected by any one MoAb could be found on all colonic tumours. The 15 MoAbs were also tested on a panel of adult tissues, and with the exception of two, all had unique reaction patterns. Thus, at least 13 different epitopes associated with HMFG could be detected by the panel of MoAbs based on their different tissue distributions. However, no normal tissue expressed all the epitopes detected by all the MoAbs (i.e. none was positive with all the MoAbs) and no 'monomorphic' HMFG epitope could be found. The antibodies were shown to react with carbohydrate or peptide-based epitopes, but this reactivity had no relationship with the pattern of tissue reaction and the significance of the expression of different epitopes is not clear. Preliminary results of serum tests with three different anti-HMFG MoAbs showed that approximately 10% of colorectal cancer patients had elevated HMFG levels but with the cut-off level selected, 10% of normal subjects also had raised levels. From this study, antibodies to HMFG, commonly associated with breast cancer, can also react with carcinoma of the colon and can indeed be useful in detecting this disease histologically (e.g. MoAbs CC3, 4 and 5 which detect greater than 80% of colonic tumours). In addition it is clear that some of the mucins produced in carcinoma of the colon are similar to those produced by breast tumours.

Mammary mucins are increased in amounts in breast cancer patient sera, and most anti-breast cancer antibodies react with such mucins. One such mucin is found in human milk fat globule membrane and consists predominantly of O-linked sugars and a protein core. Partial complementary DNA clones for the protein core have recently been obtained. The nucleotide sequence is of interest as it contains a 60-base pair repeat, giving rise to a repeated 20-amino acid sequence (PDTRPAPGSTAPPAHGVTSA). Peptides with various lengths were synthesized using this sequence and the adjacent 4 amino acids (PDTR). Three anti-human milk fat globule membrane antibodies produced in our laboratory (BC1, BC2, and BC3) were tested to determine their reactivity with these synthetic peptides. Using three different assays (direct enzyme-linked immunosorbent assay test on peptides, direct enzyme-linked immunosorbent assay test on bovine serum albumin-conjugated peptides, and an inhibition test with the peptides in liquid, rather than solid phase), it was shown that APDTR was the minimum amino acid sequence required to form a reactive epitope with all 3 antibodies, although individual differences in the reactivities of the antibodies were noted. The addition of alanine (A) converted a nonreactive PDTR peptide to a reactive one, and the deletion of arginine (R) did the reverse; thus APDTR is the smallest peptide which reacts with these anti-human milk fat globule membrane antibodies.