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© 2000 American Society for Clinical Oncology Survival in Early Breast Cancer Patients Is Favorably Influenced by a Natural Humoral Immune Response to Polymorphic Epithelial MucinFrom the Departments of Obstetrics and Gynecology, Clinical Chemistry, Pathology, and Surgery, Academic Hospital Vrije Universiteit, Amsterdam; and Department of Surgery, Zuiderziekenhuis, Rotterdam, the Netherlands. Address reprint requests to Silvia von Mensdorff-Pouilly, MD, Department of Obstetrics and Gynecology, Academic Hospital Vrije Universiteit, De Boelelaan 1117, 1081 HV Amsterdam, the Netherlands; email s.vonmensdorff{at}azvu.nl
PURPOSE: Polymorphic epithelial mucin (PEM or MUC1) is being studied as a vaccine substrate for the immunotherapy of patients with adenocarcinoma. The present study analyzes the incidence of naturally occurring MUC1 antibodies in early breast cancer patients and relates the presence of these antibodies in pretreatment serum to outcome of disease. MATERIALS AND METHODS: We measured immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies to MUC1 with an enzyme-linked immunoassay (PEM.CIg), which uses a MUC1 triple-tandem repeat peptide conjugated to bovine serum albumin, in pretreatment serum samples obtained from 154 breast cancer patients (52 with stage I disease and 102 with stage II) and 302 controls. The median disease-specific survival time of breast cancer patients was 74 months (range, 15 to 118 months). A positive test result was defined as MUC1 IgG or IgM antibody levels equal to or greater than the corresponding rounded-up median results obtained in the total breast cancer population. RESULTS: A positive test result for both MUC1 IgG and IgM antibodies in pretreatment serum was associated with a significant benefit in disease-specific survival in stage I and II (P = .0116) breast cancer patients. Positive IgG and IgM MUC1 antibody levels had significant additional prognostic value to stage (P = .0437) in multivariate analysis. Disease-free survival probability did not differ significantly. However, stage II patients who tested positive for MUC1 IgG and IgM antibody and who relapsed had predominantly local recurrences or contralateral disease, as opposed to recurrences at distant sites in the patients with a negative humoral response (P = .026). CONCLUSION: Early breast cancer patients with a natural humoral response to MUC1 have a higher probability of freedom from distant failure and a better disease-specific survival. MUC1 antibodies may control hematogenic tumor dissemination and outgrowth by aiding the destruction of circulating or seeded MUC1-expressing tumor cells. Vaccination of breast cancer patients with MUC1-derived (glyco)peptides in an adjuvant setting may favorably influence the outcome of disease.
POLYMORPHIC EPITHELIAL mucin (PEM or MUC1),1-4 which is expressed by most epithelial cancers,5 is attracting increasing interest as a target for the immunotherapy of adenocarcinomas,6 and several MUC1-based vaccines have already been tested in phase I clinical trials.7-10 As MUC1 is a cancer-associated circulating antigen, MUC1 serum assays, such as CA 15.3,11,12 are used in breast cancer patients to monitor therapy and, during follow-up, for early detection of recurrence.13 The MUC1 gene is located on chromosome 1q21-24.14-16 The mucin encoded by it is a high-molecular-weight (> 400 kd) transmembrane glycoprotein that is expressed at the apical cell surface of normal glandular epithelia and overexpressed in epithelial cancers (see review in Patton et al17). MUC1 expression reduces intercellular adhesion18 and participates in epithelial sheet differentiation and lumen formation during organogenesis19; it has lubricating properties and is thought to shield the mucosa from pathogens.18 The extracellular domain of the molecule consists largely of an extended highly glycosylated protein backbone that towers 200 to 500 nm above the plasma membrane18 and all other cell-surface molecules. The backbone is formed mainly of numerous tandemly bound peptide repeats with a highly conserved sequence of 20 amino acids.20-23 Each repeat has five sites, which are O-linked glycosylated in the mature MUC1 mucin molecule.24 In cancer cells, MUC1 not only loses apical distribution, becoming expressed on the whole cell surface, but is also less and aberrantly glycosylated.23-26 Truncated carbohydrate side chains, which are in themselves tumor antigens,27 result in the exposure of numerous repetitive cryptic peptide epitopes on the core protein of the molecule.23 In cancer patients, this altered molecule is shed into the circulation, comes into contact with the immune system, and originates cellular28-30 and humoral31-37 immune responses. Cytotoxic T cells that recognize MUC1 core peptides and mediate lysis of tumor targets in vitro have been obtained from patients with breast, pancreatic, and ovarian carcinomas.28-30 Agrawal et al38 demonstrated the presence of T cells that specifically proliferate in response to MUC1 in multiparous, but not in nulliparous, women. MUC1 peptide–specific major histocompatability complex class I–restricted cytotoxic T lymphocytes have been generated in vitro using T cells from multiparous women stimulated with synthetic MUC1 peptide–loaded, autologous antigen-presenting cells.39 As with the cellular immune responses, humoral immune responses are not restricted to patients with a malignant tumor but are also found in physiologic and benign situations. MUC1 antibodies that are directed to the peptide core of the molecule have been described in ulcerative colitis.31 Circulating MUC1 immunoglobulin M (IgM) antibodies are present in patients with breast, colon, and pancreatic cancer,32 in healthy women, and in patients with benign and malignant ovarian tumors.35 B cells from tumor-draining lymph nodes of ovarian cancer patients produce antibodies that react with the MUC1 protein core.33,34,36 Recently, circulating MUC1 immunoglobulin G (IgG) antibodies have been detected in patients with colorectal cancer.40 Previously, we developed an assay for the measurement of circulating MUC1 immune complexes41 and obtained preliminary results suggesting that circulating immune complexes that contain MUC1 are related to a favorable disease outcome in breast cancer patients.42 Recently, we developed an assay for the measurement of unbound circulating MUC1 IgG and IgM antibodies that are directed to the peptide core of the molecule.43 The objective of the present study was to analyze the incidence of these naturally occurring MUC1 antibodies in healthy subjects and in patients with benign and malignant breast tumors and to relate their presence in pretreatment serum to overall and disease-free survival in breast cancer patients.
The study was performed retrospectively using a total of 456 serum samples from healthy individuals and from patients with benign and malignant breast tumors.
Breast Cancer Patients
Information on patient outcome was available in all but six patients, who were treated as censored observations. The principal events analyzed were recurrence and death from breast cancer. Disease-free survival was defined as the time elapsed between the start of primary treatment and the first reappearance of breast cancer at any site (local, contralateral, or distant) (Table 1). Freedom from distant failure was defined as the time elapsed between primary treatment and the first confirmed distant metastasis. Disease-specific survival was defined as the time elapsed between primary treatment and death from breast cancer (Table 1). Five patients died of unrelated causes, four were free of recurrence, and one showed no evidence of disease after surgical treatment of a local recurrence; these patients were treated as censored observations. Five-year disease-specific survival was analyzed in patients (n = 122) who were followed-up for at least 5 years since primary treatment (n = 106) and in patients who died from breast cancer within 5 years since primary treatment (n = 16).
Control Study Population Serum samples were collected, aliquoted, and stored at -70°C until analyzed.
MUC1 Antibody Assay (PEM.CIg)
MUC1 Serum Levels
Immunohistochemistry
Statistical Methods
MUC1 was overexpressed in all 25 primary tumors stained with MAb 115D8.
Assay Results
Median MUC1 IgG and IgM levels were not substantially different in the total control population and the breast cancer patients. Median MUC1 IgG and IgM levels in the total breast cancer population were 0.646 OD (range, 0.328 to 1.514 OD) and 0.778 OD (range, 0.178 to 1.655 OD), respectively (Table 2). On the basis of these results, MUC1 IgG and IgM antibody levels  0.7 OD and 0.8 OD, respectively, were arbitrarily defined as positive. Individuals with concomitantly positive MUC1 IgG and MUC1 IgM test results (Table 2) were defined as being MUC1 antibody-positive; conversely, those with negative MUC1 IgG or negative MUC1 IgM antibody levels were defined as MUC1 antibody-negative. The number of MUC1 antibody–positive individuals was significantly higher in benign breast tumor patients than in nulligravidae (P = .038) and pregnant women (P = .003) and was significantly higher in healthy women than in pregnant women (P = .013). The number of breast cancer patients with MUC1 antibody–positive test results was significantly higher than that found in the total control population (P = .006), in nulligravidae (P = .015), and in pregnant women (P = .002). The number of breast cancer patients with MUC1 antibody–positive test results did not differ significantly between lobular and ductal carcinomas, between histologic grade 1, 2, and 3, between ER-positive and ER-negative tumors, between MAI less than or 10, nor between stage I and II.
Disease-Specific Survival
Survival analysis in relation to stage is shown in Fig 1. Probability of survival was significantly higher for pT1 in comparison to pT2 node-negative patients (P = .0261). None of the patients with lobular adenocarcinomas, and likewise none of the patients with grade 1 tumors died during the observation period. No relation was found between ER status and disease-specific survival. Patients with MAI less than 10 had a higher probability of disease-specific survival (P = .0179) than patients with MAI
A positive MUC1 IgM antibody test in pretreatment serum had, by itself, no impact on disease-specific survival. A benefit in disease-specific survival was found in patients with node-positive disease (P = .0444) and stage II disease (P = .0738) and in patients with stage I and II disease (P = .0508) in addition to a positive MUC1 IgG antibody test. Thirty-seven breast cancer patients (24%) had a MUC1 antibody–positive test result (ie, a concomitant elevation of MUC1 IgG and IgM antibodies in pretreatment serum); none died of breast cancer during the observation period. Results from Kaplan-Meier analyses for disease-specific survival and 5-year disease-specific survival as well as for disease-free survival and freedom from distant failure in relation to MUC1 antibody test results are listed in Table 3. In patients with stage I and II disease, when regarded as one group (Fig 2), probability of survival for MUC1 antibody–negative patients was 80%; at the time of analysis, no deaths from breast cancer had occurred among the MUC1 antibody–positive patients (P = .0116). A significant benefit in disease-specific survival was also observed in MUC1 antibody–positive breast cancer patients with stage II disease (Fig 3), also after adjustment for histologic grade (P = .0233), ER (P = .0205), MAI (P = .05), and nodal involvement (P = .0244). The disease-specific survival benefit that was observed in patients with stage I disease was not significant.
Five-Year Disease-Specific Survival Five-year disease-specific survival analysis could be performed in 122 patients (Table 3). Probability of survival was significantly higher for patients with stage I disease as compared with patients with stage II disease (P = .0207). MUC1 antibody–positive patients had a significantly higher probability of survival (P = .0343) than MUC1 antibody–negative patients. A benefit in survival for MUC1 antibody–positive patients (P = .0542) was also observed in patients with stage II disease (n = 83).
Disease-Free Survival
Freedom From Distant Failure Multivariate analysis showed stage to be the only independent variables (P = .0085) for prediction of freedom from distant failure in the study population. A MUC1 antibody–positive test result was associated with a lower, albeit not significant, RR for distant metastases (P = .1301; RR, 0.44; 95% CI, 0.15 to 1.28).
Kaplan-Meier analysis showed a significantly higher probability of freedom from distant failure in patients with stage I than in stage II disease (P = .0041) and in patients with MAI less than 10 than patients with MAI
Free, natural MUC1 antibodies are present in the circulation of healthy subjects as well as in cancer patients. Pregnancy and lactation, as well as inflammation of glandular epithelial tissues and tissular disarray associated with benign diseases, are physiologic and benign situations that can lead to a transitory escape to the circulation of MUC1 glycoforms and induce immune responses. In our study, nulliparous women had significantly lower absolute levels of MUC1 IgG antibodies than all other groups studied. The low MUC1 IgM levels observed in pregnant women may be due to the relative immunosuppression that is associated with pregnancy. As cellular immune responses to MUC1 have been described in multiparous women38,39 who have necessarily undergone changes in the mammary gland in preparation for lactation, the possible participation of lactation in unleashing this immune response should be investigated. It is attractive to link these observations with those obtained from epidemiologic studies indicating that nulliparity as well as an older age at first delivery increase the lifetime risk of breast cancer, whereas high parity47 and prolonged breast feeding seem to reduce the risk of breast cancer.48 Although it can be explained otherwise,49 the observed association could suggest a role of MUC1 immune responses in the immune surveillance of breast cancer. In this retrospective study, we have found a benefit in disease-specific survival in patients with stage II breast cancer who have above-median levels of circulating MUC1 IgG and IgM antibodies; however, such a benefit was not observed in disease-free survival. This disease-specific survival benefit could result from a control of disease spreading, as manifested in a lower frequency of distant metastases in MUC1 antibody–positive patients. The fact that the benefit in survival that was observed in stage I MUC1 antibody–positive patients did not reach significance may be due to the small number of patients included in the study. Studies have shown that isolated disseminated tumor cells can be found at the time of primary surgery in the bone marrow of 30% of lymph node-negative breast cancer patients50 and that their presence is associated with reduced distant disease-free survival and disease-specific survival.50,51 Antibodies may be effective in eradicating these circulating tumor cells while exerting a limited effect against the primary tumor.52,53 Unlike the primary tumor, tumor cells reach the circulation as a single cell or small clumps and are therefore highly accessible and more vulnerable to destruction. Taking this further than the classical mechanism of action of antibodies (ie, antibody-dependent cell-mediated cytotoxicity and complement-mediated lysis), antibodies to MUC1 could be involved in restoring cell adhesion, uncovering cell surface receptors involved in immune recognition, and neutralizing the immunosuppressive effect of soluble MUC1. Cell adhesion and antiadhesion are required for tumor invasion to occur, and MUC1 seems to play a role in both of these aspects.18,54 The extracellular domain of the molecule extends high above the plasma membrane,18 shielding the cancer cell and towering above other cell-surface molecules, such as those involved in cell-cell adhesive interactions55 and in immune recognition.56 Steric hindrance by MUC1 strongly decreases cell-cell57 and cell-matrix interactions and prevents epithelial cell aggregation by interfering with integrin-mediated adhesion,55 favoring invasion of tumor cells into the underlying stroma, lymph, and blood vessels.58 As is the case with monoclonal antibodies to the MUC1 repeat domain,55 antibodies that are bound to MUC1 could lead to a capping or clustering of MUC1 on the cell surface, restoring cell adhesion and limiting cancer invasion. A redistribution of MUC1 could also be instrumental in unmasking cell surface antigens that are involved in immune recognition processes56 and enabling recognition and destruction of the tumor cell by cellular effectors of the immune system. The benefit in survival observed in patients with stage I and II disease and positive levels of MUC1 IgG antibodies could be signalling the presence of a MUC1 specific T-helper lymphocyte response. This possibility is strengthened by our recent results that indicate a predominance of IgG2 subclass in these responses. On the other hand, the effectors do not necessarily have to be specific T cells, at an early tumor stage, immune surveillance and checking disease spread could be achieved by the broadly reactive properties of natural killer cells.59 MUC1 seems to be a ligand for the intercellular adhesion molecule 1 (ICAM-1), a member of the immunoglobulin superfamily.60-62 Adhesion of cancer cells to E-selectin and ICAM-1 on the cell surface of activated fibroblasts and endothelial cells could favor migration of cancer cells away from the tumor site and into the circulation. Cancer cells in the bloodstream could then, by the same mechanism, adhere to the endothelium, extravasate, and metastasize. The counter-receptor to ICAM-1 is the lymphocyte function-associated antigen (LFA-1), a member of the integrin family that is required for a broad range of leukocyte functions, including T-cell–mediated killing, T-helper responses, and B-lymphocyte responses.63 The finding that soluble MUC1 inhibits adhesion of MUC1-expressing cells to ICAM-160 and to E-selectin64 suggests an immunosuppressive role61,65,66 for the high levels of circulating mucin that are often found in patients with advanced stage disease. Furthermore, recent studies have shown expression of MUC1 on activated T cells67 as well as on normal B cells,68 which suggests that the molecule may be involved in immune modulation. The benefit in survival that was observed in the group of patients with MUC1 IgG and IgM responses could define the subset of patients with an effective primary IgM response.69 It may be that these IgM antibodies that lead to a maturation of the immune response are the ones capable of sufficiently complexing with the mucin to mediate some of the effects mentioned or, by blocking binding sites on the mucin molecule, counteract its immunosuppressive action. More than one epitope is involved in these immune responses (unpublished observations); definition of their significance in restraining tumor progression will help define the best vaccination material. In conclusion, these preliminary results seem to indicate that naturally occurring MUC1 antibodies may check disease spread in patients with breast cancer, possibly by destroying circulating or seeded isolated disseminated tumor cells (micrometastases) that eventually could lead to metastatic disease and death. Vaccination of breast cancer patients with MUC1-derived (glyco)peptides after (or even before) primary surgery could in an adjuvant setting engender immune responses that may favorably influence outcome of disease. The stronger immune response and the immunologic memory that are associated with active immunotherapy would provide a constant surveillance mechanism to protect cancer patients from recurrence of disease. A word of caution is necessary, taking into account the retrospective nature of this study. We are in the process of validating these preliminary results in a prospective study of breast cancer patients that analyzes levels of MUC1 antibodies in pretreatment serum, the detection of isolated disseminated tumor cells in the bone marrow at primary surgery, pretreatment immunologic status, and response to treatment in relation to outcome of disease.
Supported in part by a grant from the Dutch Cancer Society (KWF grant no. VU96-1318), the Biocare Foundation (grant no. 94-23), and a European Communities Concerted Action (contract no. BMH1-CT94-1462). We thank K. van Uffelen and M.J.J. Poort-Keesom for expert technical assistance.
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ABSTRACT
INTRODUCTION
2 test or, when the sample size was small, a Fisher’s exact test. Multivariate analysis of disease-specific survival, disease-free survival, and freedom from distant failure was performed using the Cox proportional hazards regression model.
