Discussion
This large, single-centre study was designed to explore a possible relationship between p53 autoantibody status and breast cancer activity. One thousand and six patients with breast cancer attending a single referral center were included in the study, irrespective of age or disease characteristics at the time of primary diagnosis. The median patient follow up was 4 years. An average of eight serial plasma samples per patient had been cryopreserved, and corresponding clinical information at the time of each sample's collection had been entered into the database. This fully documented library of over 8000 samples was used to look for correlates between tumour behaviour and autoantibodies to p53 during the clinical follow-up period.

Anti-p53 levels were independent of changes in tumour status
As a working hypothesis we proposed that levels of autoantibodies to p53 would reflect tumour behaviour. Thus, for those patients who were positive for p53 autoantibodies at diagnosis, we reasoned that surgical removal of primary tumour might result in reduced p53 autoantibody levels. Should these levels then show a secondary increase associated with relapsed disease, then increasing levels of p53 autoantibodies might act as a biochemical marker of tumour progression. For those patients who were negative for p53 autoantibodies, then, development of recurrent disease may be associated with changes in p53 expression within the metastatic tumour, leading to a switch to an autoantibody-positive status.
We found that the presence or absence of p53 autoantibodies was not predictive for presence or absence of recurrent disease. There was an equivalent incidence of active disease at the time of sampling in the autoantibody-negative and autoantibody-positive groups, these being 25.2% and 28.7%, respectively.
We found that humoral immune activity against p53 appeared to be relatively restricted to a subgroup of patients in whom, once an autoantibody response had been generated, antibody was likely to persist regardless of tumour behaviour. Thus, antibody-positive patients without clinical recurrence remained antibody positive throughout the follow-up period. Conversely, where no detectable p53 autoantibody was present at the time of primary diagnosis, these patients remained similarly negative for antibody irrespective of subsequent disease activity (Table 3).

Immune regulation and potential responsiveness to breast cancer
In contrast to shed markers that correlate with tumour mass, such as CA15.3 for cancer of the breast, any tumour-related immune response will be subject to complex regulation. Autoantibody responses to p53 will require appropriate primary immunization. Initial low-dose antigen exposure may induce immune tolerance and lack of response. Higher antigen doses may activate either antibody-mediated immunity, or cellular immunity.
In breast cancer patients, the present results suggest that, once an active humoral response against p53 is established, this response remains active. This persistent humoral reaction may be driven by persistent antigenic stimulation by p53 protein derived from overexpression of p53 at distant metastatic sites. Alternatively, irradiated normal tissue may be a source of continued antigenic stimulation, because a long-term side effect of radiation therapy is an increased expression of p53 in normal breast tissue, which persists for several years [12]. Since the great majority of our total patient cohort had received radiotherapy, humoral immunity to p53 associated with primary disease might persist, even in those patients who enter remission, due to tumour-independent antigenic stimulation.
Loss of p53 function is known to correlate with loss of efficacy of cancer therapy in vivo [13,14]. This raised the possibility that autoantibodies to p53 that develop during follow up might indicate those patients whose tumor has become resistant to therapy. However, the present results show that if no immunity has been generated at the time of primary diagnosis, then later immunity is unlikely to occur. This corresponds to the finding that expression of p53 antigen in biopsies of locally advanced breast cancer did not correlate with drug resistance [15,16]. Overall, our observations show that screening for p53 autoantibody status is not informative on residual tumour activity, nor on therapeutic responsiveness. We conclude that the potential value of p53 autoantibody screening in patients with breast cancer is limited to the prognostic information obtained at diagnosis.
Table 1  Incidence of autoantibodies to p53 compared with disease status at last clinic attendance  Pearson χ2: P = 0.606. *Information on disease status at last sample time was available for 960 out of 1006 patients
Table 2  Incidence of autoantibodies to p53 compared with menopausal status at diagnosis  Pearson χ2: P = 0.788.
Table 3  Anti-p53 negative patients do not become positive with recurrent disease  Sixty patients who were negative for p53 autoantibodies had also had a plasma sample taken within 30 days of their primary diagnosis of breast cancer. To determine whether antibody status at diagnosis might have been predictive of later disease behaviour (ie independent of the current negative status), we compared two patient subgroups: patients with current nonactive disease and patients with current active disease. With one exception, all patients were antibody negative within 30 days of initial diagnosis. This showed that recurrent disease is highly unlikely to induce a humoral anti-p53 response in those patients who were initially antibody negative.