Patient plasma samples
A library of plasma samples were collected from all patients attending one general oncology clinic for postoperative follow up of breast cancer; samples were taken without any patient selection and irrespective of clinical stage, menopausal status, histological type or degree of differentiation. The operative management of each patient was according to the tenets of their surgeons at the time of presentation; the breast was conserved wherever possible. The majority of patients were treated with adjuvant radio-therapy. Adjuvant cytotoxic chemotherapy, with or without tamoxifen, was the subject of clinical trial at the time.
The clinical status of each patient at time of sampling had been summarized on a database using Helix Express (version 1.0.1; Helix Technologies, Northbrook, IL, USA). The plasma library had been established over a 15-year period, with an average of eight plasma samples per patient being cryopreserved. It is known that cryopreserved p53 autoantibodies are stable in either sera or plasma, and that several cycles of freezing/thawing do not cause loss of titre (Soussi T, personal communication). Although it is theoretically possible that shed p53 protein might partially block autoantibody in some patients, it has already been shown [
17] that autoantibody to p53 is normally in excess, and is thus detectable by ELISA.
P53 autoantibody detection by enzyme-linked immunosorbent assay
The ELISA assay for p53 autoantibodies was developed in-house, based on the ELISA procedure of Lubin
et al [
3] in which the amount of specific p53 autoantibody recorded by ELISA was confirmed by Western blot and immunoprecipitation analyses. Briefly, 96-well assay plates (Falcon 3912; Becton Dickinson and Co, Oxnard, CA, USA) were precoated with baculoviral-derived, purified, wild-type, full-length, human recombinant p53 (0.1 ng/ml phosphate-buffered saline [PBS]) using 30 μl per well. After overnight incubation at 4°C, the plates were washed three times with PBS-0.1% Tween at room temperature. Wells were then blocked for 2 h with 250 μl PBS-0.5% Tween. After five washes with PBS-0.1% Tween, 30 μl of each plasma sample was incubated on ice for 2 h. The plate was again washed five times as above before addition of horse radish peroxidase-linked goat antiserum to human immunoglobulin (Sigma A8667; Sigma Aldrich Co Ltd, Poole, Dorset, UK) and incubation on ice for 1 h. After a further five washes, bound horse radish peroxidase was detected by conventional methods using
O-phenylenediamine dihydrochloride (30 min), stopping with 3 mol/l H
2SO
4, and reading the optical density (OD) at 492 nm.
An OD reading of less than 0.4 was taken as a negative result. This cutoff was determined by titration of positive and negative samples (1/10, 1/50, 1/100 and 1/500) and gave good discrimination between positive and negative samples at 1/500 dilution. All assay runs included the same internal standards of a sample known to be positive at 1/500 dilution and a sample known to be negative at 1/10 dilution, in order to confirm assay reproducibility throughout the series.
The intial screen of all patients was at a plasma dilution of 1/10 in PBS-0.1% Tween. Samples from all patients with a positive OD reading at the 1/10 dilution were then re-assayed at 1/500 (see Sample processing, below). The positive and negative internal standards ensured that all results were directly comparable. Background controls received no p53 protein. Specificity controls used soluble p53 protein added to the positive plasma sample before assay; this resulted in loss of signal.
In one assay series we compared a commercial ELISA kit for p53 autoantibodies (produced by Dianova GmbH; licenced to CalBiochem-Novabiochem Corp; distributed by Oncogene Research Products, Cambridge, MA, USA; cat no. QIA 16) with our in-house ELISA. A total of 20 patients' samples were tested, representing a range of positive and negative readings. Two samples were scored strongly positive by the in-house assay, but only one of these two scored positive by the Dianova assay. Having established sensitivity, specificity and reproducibility of the in-house assay, we judged that this was superior to the commercial assay both in terms of sensitivity and of cost (£1 per test compared with £23 per test). The in-house assay was thus used throughout the study presented here.
Sample processing
The last sample obtained from each of the 1006 patients was screened for anti-p53 antibodies at a 1/10 dilution. Those patients who proved to be positive for p53 autoantibodies at this low dilution were then tested for autoantibody status using all samples from each patient; here a 1/500 dilution of plasma was used because this was found to give good specificity and sensitivity for known positive and negative controls. Of those patients who were antibody negative in the initial screen, 60 had had a plasma sample obtained around the time of their primary diagnosis of breast cancer; these primary samples were assayed (1/10 dilution) to look for any positive to negative switches in autoantibody levels during clinical follow up.
Clinical fields
The presence or absence of autoantibodies to p53 was compared with the clinical status of each patient, which was classified: (i) primary remission; (ii) secondary remission following a previous relapse; (iii) active relapsed disease; or (iv) continuous active disease since first diagnosis. For those patients who were anti-p53 positive, their clinical history was compared with levels of measured p53 autoantibodies throughout their follow-up period.