Multiple Tumor-associated Autoantibodies As Diagnostic Biomarkers In The Detection Of Breast Cancer

Breast cancer is a common cancer in women among the world, and it is the leading cause of female deaths. Currently, mammography is a common technique to diagnose breast cancer, but it is not popularized among population for the limitations of ages and breast densities in patients. Tumor-associated autoantibodies(TAAbs), as serological biomarkers, have been studied by many investigators. However, most researchers still have not found the specific TAAbs or TAAb combinations with more diagnostic potential for breast cancer by the general indirect ELISA method. PurposeTo evaluate the immunodiagnosis values of autoantibodies against p53, cyclin B1, cyclin D1, p16, IMP1, p62, koc, c-myc, survivin, 14-3-3ξ, and RalA in the detection of breast cancer, and to establish an autoantibody combination with more diagnostic potentials in breast cancer. Methods1. We used E. coli prokaryotic expression system and pET-28a(+) vector to induce and express p53, p62, 14-3-3ξ and RalA recobination proteins labelled by 6×histidine tags, and purified them by affinity column chromatography.2. Sample sizes were calculated according to the methods of diagnostic test using PASS13.0 software. Then, we collected 415 sera including sera from 197 patients with breast cancer, 109 patients with breast benign disease and 109 normal individuals.3. Indirect enzyme-linked immunosorbent assay(ELISA) was used to examine the presence of eleven TAAbs. During the experiments, a series of diluted Ig G gradients was added to every plate, so as to manufacture standard curves to further standardize test results, and this method was called relative quantitative ELISA. Afterwards, western blot was used to validate ELISA results.4. One-way analysis of variance and SNK method were used to compare the mean levels of autoantibodies among three groups. Besides, Chi-squared test was applied to examine the the frequency of autoantibodies in different comparison groups. Furthermore, in the light of the evaluation method of diagnostic test, we assessed the validity and reliability of single autoantibody and autoantibody combinations in the detection of breast cancer as diagnostic biomarkers. Eventually, we examined the distribution of autoantibodies in different clinical stages of breast cancer using Kruskal-Wallis test. Results1. We purified p53, p62, 14-3-3ξ and RalA recombinant proteins successfully. Their concentrations were 0.24 mg/ml, 0.19mg/ml, 0.76mg/ml, and 0.50mg/ml, respectively. Also, the purity of these autoantibodies was high enough.2. The positive rates of autoantibodies to p53, cyclin B1, p16, p62, c-myc, survivin, and 14-3-3ξ in breast cancer group were significantly higher than those in the groups of breast benign disease(P < 0.05) and normal controls(P < 0.01). Thereinto, the sensitivity of autoantibody to cyclin B1 was the highest(23.9%), followed by autoantibodies against survivin(19.3%) and p53(14.7%).3. When we tested the significant autoantibodies in parallel, with the successive addition of TAAs, the sensitivities of selected autoantibody panels were increased to 62.4% from 23.9% in the diagnosis of breast cancer. Besides, the area under the receiver operating characteristic curve(AUC) was up to 0.78 for the optimal autoantibody panel.4. The autoantibody to p16 was found to be significantly elevated in stageⅠpatients of breast cancer compared with other clinical stages(P < 0.01). Conclusions1. The autoantibodies against p53, cyclin B1, p16, p62, c-myc, survivin, and 14-3-3ξ were the potential biomarkers in the immunodiagnosis of breast cancer, and the p16 autoantibody is proved to be the diagnostic biomarker in the early detection of breast cancer.2. The detection of single TAAb usually offer lower sensitivities, and multiple detection of a set of TAAbs was shown to be more sensitive.3. Combined detection of autoantibodies to p53, cyclin B1, p16, p62, c-myc, survivin, and 14-3-3ξ, can improve the power of immunodiagnosis in breast cancer.