Introduction
Outcomes of breast cancer treatment have improved, mostly as a result of earlier diagnosis. Although newer modes of therapy are being applied, traditional therapy involving surgery, radiotherapy and chemotherapy, followed by long-term antioestrogen therapy continues to be used. Immunotherapy could be a useful adjunct to conventional therapy, particularly in an adjuvant setting and (as shown here) in patients with early disease and no metastasis. New therapeutic procedures in breast cancer are usually tried in patients with advanced disease, who may be appropriate candidates for cytotoxic drugs. However, such patients may be unable to respond appropriately to immunotherapy, given that an intact and competent immune system is required to induce a therapeutic immune response.
Treatment of cancer with immunotherapy has been the goal of many researchers since the advent of effective immunization against infectious diseases. Previously, tumour antigens were not easily identified, but currently identified antigens include glycoproteins and glycolipids (for example, gangliosides), developmental and over-expressed antigens (for example, CEA (carcinoembryonic antigen), gp75, MAGE, tyrosinase, melan-A, mucin [MUC]1), and mutated oncogenes (for example, p53, HER-2/neu, ras) [
1]. Our laboratory has focused on MUC1 as a target for tumour immunotherapy. Mucins (such as MUC1) are high-molecular-weight glycoproteins that are secreted by many epithelial cells such as breast, ovary, colon and pancreatic carcinomas. MUC1 is of interest and a potential target for tumour immunotherapy for the following reasons: there is an up to 100-fold increase in the amount of mucin present on cancer cells compared with normal cells; MUC1 has a ubiquitous rather than focal cellular distribution; and MUC1 has altered glycosylation, revealing peptide epitopes not easily identified in normal mucins.
Cloning of the cDNA for MUC1 and definition of the structure revealed that the molecule is transmembranous, with a relatively large extracellular domain and a cytoplasmic tail. It was discovered that most of the immunogenicity (in terms of antibody production) resided in a repeated (variable number of tandem repeats [VNTR]) 20-amino-acid peptide (PDTRPAPGSTAPPAHGVTSA) domain in the extracellular portion of the molecule [
2‐
4]. Such studies of immunogenicity in mice would not be relevant to humans other than the findings that, in humans, MUC1 can stimulate T cells in breast, pancreatic and ovarian cancers [
5‐
7]. Restricted cytotoxic T lymphocytes in humans with breast cancer and in pregnancy, as well as in mice, have also been detected [
8‐
10]. In addition, T cell immunity and B cell immune responses to selected epitopes of MUC1 from ovarian, breast, pancreatic and colon cancer patients have been demonstrated [
11‐
13], as have circulating immune complexes to MUC1 in the serum of breast and ovarian carcinoma patients [
14]. In mice, we demonstrated that a 20-mer MUC1 VNTR (made as a fusion protein comprising five VNTR repeats), when coupled to oxidized mannan (that is, oxidized mannan-MUC1 fusion protein [M-FP]), generates H2 restricted cytotoxic T lymphocytes, which protect mice against challenge with MUC1
+ mouse tumours [
9,
15‐
22].
Over the past 12 years, we have injected many patients with different MUC1 formulations in an effort to induce protective and therapeutic immunity, aiming to reproduce the same effect as seen in mice [
1,
9,
16‐
20,
23‐
35]. In more than 250 patients with advanced cancer, moderate cellular immune responses and substantial antibody responses were noted [
36‐
39]. However, clinical responses were not apparent in these patients, possibly because of the advanced and immunocompromised state of their disease. Therefore, in the present study we aimed to evaluate the effect of M-FP in patients with early disease. Specifically, these patients had stage II disease with fewer than four lymph nodes involved, all of which had been removed, and had no evidence of disease and were entirely healthy at the start of the trial. Although a small number of patients were recruited into this randomized, double-blind pilot study, the early results reported here are promising and justify the performance of a larger study.
Discussion
In this long-term, double-blind study of 31 individuals with stage II breast cancer and no evidence of disease (16 injected with M-FP and 15 with placebo), patients receiving M-FP vaccination appeared to benefit in terms of protection against relapse; none of 16 had a recurrence, but four out of 15 placebo patients had recurrent disease after 7 years and 10 months (December 1997 to October 2005). Although the number of patients in this study is small, the results are statistically significant (P = 0.0292). Most of the treated patients exhibited immunity to MUC1 VNTR, whereas none of the placebo patients had such immune responses. Thus, M-FP appears to confer the survival/disease-free interval advantage in patients with early breast cancer. Importantly, and in contrast to other studies, the findings presented here for vaccination of patients with early disease provide justification for performance of a larger study. Indeed, a strategy of using a small number of patients to obtain an indication of response would be worth considering for immunotherapy trials, in which virtually all patients should exhibit an immune response if the therapy is working, and a high proportion of those should exhibit an anti-tumor response; this contrasts with cancer chemotherapy, in which fewer patients are likely to respond.
The study is of interest for several reasons. First, we highlight the characteristics of the patients evaluated in the present study; these patients had early disease, they presented with a primary lesion, they had lymph nodes surgically removed, and at the time of commencement of the trial they had no evidence of disease. We consider these characteristics to be crucial if immunotherapeutic studies are to yield useful findings. In another trial [
43], a potentially therapeutic monoclonal antibody (CO17-1A) was used in 83 patients with minimal residual colorectal cancer (Dukes C) after surgery, and some patients remained tumour free for many years. Furthermore, in a placebo-controlled clinical trial using NY-ESO-1 protein (cancer testis protein) with ISCOMATRIX adjuvant in early-stage melanoma patients [
44], immunized patients exhibiting antibody, delayed-type hypersensitivity, and CD4
+ and CD8
+ T cell responses appeared to have superior clinical outcomes to those treated with placebo or protein alone. Unfortunately, most cancer trials involve patients with advanced disease, selected on the basis of traditional therapy involving toxic chemotherapeutic agents. However, such patients are likely to have poor immune responses, significant tumour bulk, limited time for the induction and effector phases of specific immunity, and are the least likely to have therapeutic benefit from immunotherapy regimens. We do not advocate abandonment of the traditional approach of phase I studies, in which the primary end-point is assessment of toxicity, but we do recommend that these pilot studies of immunotherapeutic agents be quickly followed by larger studies in patients with early disease. In our experience to date, more than 250 other patients have received M-FP by direct injection but all had advanced disease; no toxic effects were detected but – importantly – none had objective clinical responses [
36‐
39]. We hope that the present study will encourage use of immunotherapy in patients with early disease, but we note the long follow-up time required (and the large number of patients) if meaningful results are to be to obtained.
A second reason why this study is of importance is that some of the patients had a cellular immune response (of the samples that could be tested), and most had a significant antibody response. In contrast to patients from other clinical trials of M-FP [
36‐
39,
41], we did not detect IFN-γ, cytotoxic T lymphocytes, or proliferation of T cells to pVNTR in pretreatment samples. Interestingly, pre-existing peptide-reactive T cell responses (IFN-γ) to MUC1, as measured using quantitative polymerase chain reaction, have been detected in normal donors and in patients with primary breast cancers [
45]. However, the responses were against different MUC1 antigens, namely short peptide epitopes MUC1
950–958 (STAPPVHNV) and MUC1
12–20 (LLLLTVLTV), which are outside the MUC1 VNTR region and were not incorporated in our M-FP vaccine. We did not detect anti-MUC1 antibody levels in pretreatment samples in other M-FP clinical trials either [
36‐
39,
41]. Although our finding is contradictory to those of previous studies [
11,
12,
46], this is possibly due to differences in ELISA methodology or the antigen used to coat the ELISA plates (recombinant pVNTR in our studies versus peptide in other studies), which would mean that the conformation of the antigen is different [
11]. We also noted that no immunized patients exhibited strong antibody responses after the limited course of subcutaneous injections (that is, titres were <1/40 to 1/80 only), and so the results shown for 1/40 serum dilutions are specific and represent essentially maximum reactivity.
It is difficult to conclude firmly that antibody and/or T cell responses led to nonrecurrence of tumour, but it is possible that the persistent presence of antibody would bind to small metastatic deposits, leading to their eradication, whereas with large deposits there is the difficulty of penetration of the antibody into the tumour cell mass; similar comments apply to T cells. We note a study [
47] in which it was demonstrated that the occurrence of MUC1 antibodies without immunization in early breast cancer patients (stages I and II) were associated with significant benefit in terms of disease-specific survival. Patients immunized in our trial and who had developed IgM and IgG antibodies appeared to have benefit, with significantly delayed recurrence. We await the results of a larger study (in excess of 360 patients, which is in progress (EOF ethics approval, 30 December 2004; no. 64055)) before we draw firm conclusions on the role of anti-MUC1 immunity and lack of recurrence.
Finally, although antibody production and cellular immunity occurred only in the immunized group, there was no control group with oxidized mannan only; it is theoretically possible that the mannan, by cross-linking mannose receptors on macrophages and dendritic cells, could lead to their activation. An endogenous immune response could eradicate tumour deposits or activate macrophages, which could eradicate tumour cells because macrophages are known to be cytocidal to tumour cells in the absence of any immune response. However, in many experimental studies mannan mixed with MUC1 FP failed to induce the necessary immune responses and tumour protection to have any impact on tumour cell growth in mice [
19,
20]. Therefore, this mechanism is unlikely to account for the results presented here.
Acknowledgements
This work was supported by the New Idea Breast Cancer Funds, Hellenic Funds and Prolipsis Medical Center Funds. VA is an NH and MRC R. Douglas Wright Fellow 223316 and MP is an NHMRC Senior Research Fellow. All authors were also supported by the Burnet Institute at Austin (VA, GP, MP, DP, BL, SP and IM), Prolipsis Medical Center (SV, AT, AT and HD) and The National Hellenic Research Foundation (MNA). tamoxifen used in the study was provided by A Dervos – G. Dimitrakopoulos & Co OE, Athens Greece. The authors thank Ms Violeta Bogdanovska and Mr Wenjun Li for preparation of GLP grade M-FP and placebo, Mr Brendan Toohey for preparation of pVNTR, Ms Vicky Psichou for PBMC isolation and Ms Carla Osinski, Mr Wenjun Li, Ms Jodie Lodding and Mr Harry Aletras for technical assistance in analysis of immunological samples. We also thank Dr Dishan Gunawardana for preparation of ethics applications and randomization of vaccine/placebo samples.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
VA, GAP, IFM and SV participated in the design of the study. VA and GAP supervised production of Good Lab Practice (GLP) grade vaccine/placebo. SV collected blood samples and MNA isolated and stored sera and PBMCs. VA, GAP, BEL, SJP, MP and DSP all assessed and performed immunological analysis of blood samples. SV performed the clinical trial at Prolipsis Medical Center, and HD, A Tsibanis and A Tsikkinis were involved in the clinical trial management, patient recruitment, injection and planning. VA and GAP also performed the statistical analysis. All authors read and approved the final manuscript.