Application of protein microarrays for multiplexed detection of antibodies to tumor antigens in breast cancer

Abstract

There is strong preclinical evidence that cancer, including breast cancer, undergoes immune surveillance. This continual monitoring, by both the innate and the adaptive immune systems, recognizes changes in protein expression, mutation, folding, glycosylation, and degradation. Local immune responses to tumor antigens are amplified in draining lymph nodes, and then enter the systemic circulation. The antibody response to tumor antigens, such as p53 protein, are robust, stable, and easily detected in serum; may exist in greater concentrations than their cognate antigens; and are potential highly specific biomarkers for cancer. However, antibodies have limited sensitivities as single analytes, and differences in protein purification and assay characteristics have limited their clinical application. For example, p53 autoantibodies in the sera are highly specific for cancer patients, but are only detected in the sera of 10-20% of patients with breast cancer. Detection of p53 autoantibodies is dependent on tumor burden, p53 mutation, rapidly decreases with effective therapy, but is relatively independent of breast cancer subtype. Although antibodies to hundreds of other tumor antigens have been identified in the sera of breast cancer patients, very little is known about the specificity and clinical impact of the antibody immune repertoire to breast cancer. Recent advances in proteomic technologies have the potential for rapid identification of immune response signatures for breast cancer diagnosis and monitoring. We have adapted programmable protein microarrays for the specific detection of autoantibodies in breast cancer. Here, we present the first demonstration of the application of programmable protein microarray ELISAs for the rapid identification of breast cancer autoantibodies.

Figure 1

Detection of p53-specific antibodies in early-stage breast cancer. A. Sera derived from breast cancer patients and healthy female donors were tested for p53-specific antibodies (left) and EBNA-1 specific antibodies (right) by protein ELISA. The sera used consisted of normals (n=46); Stage I (n=29); Stage II (n=70); Stage III (n=47) and Stage IV breast cancer (n=40). The median level is shown with a bar. P53 antibody detection correlates with presence of breast cancer (p<0.0001). In contrast, EBNA-1 antibodies are detected at comparable intensity in normals (n=64) and patients (n=132), independent of stage. B. Rapid and durable loss of p53 antibodies with neoadjuvant chemotherapy. Two patients with early-stage Her2+ breast cancer who had detectable antibodies to p53, underwent neoadjuvant chemotherapy treatment with trastuzumab and chemotherapy over a 14-week period. P53 antibodies were detected in serial serum samples by ELISA, and show a rapid loss with the onset of treatment. C. Autoantibodies in breast cancer can be subtype-specific. i) Detection of p53-specific antibodies in breast cancer subtypes. Sera from Figure 1A were divided by tumor subtype. Subgroup analysis showed borderline significant preferential detection of anti-p53 antibodies in patients with ER- (n=26) compared with ER+(n=93) cancer, but no preference between HER2+ (n=22) vs. HER2- (n=73) breast cancer. ii) Selective detection of survivin-specific antibodies in HER2- breast cancer. Sera from the breast cancer patients in (A) were tested for anti-survivin specific antibodies using recombinant GST-tagged survivin. Subgroup analysis showed selective detection of anti-survivin antibodies in patients with HER2- breast cancer, independent of estrogen receptor (ER) status.

Figure 2

Development of protein microarrays for detection of autoantibodies. A. Detection of p53 antigen with standard ELISA, using reference p53-antibody positive sera and negative sera. B and C. 4 antigens (S100A7, p21, ML-IAP, and p53) are expressed in microarray format, and p53 antigen is specifically detected with the reference sera. All four proteins are strongly expressed, as measured by detection of GST tag using anti-GST antibody. D. Sensitivity of detection. P53 positive serum was serially diluted and tested by standard ELISA and NAPPA. Standard deviation for ELISA was based on two samples and for NAPPA was based on four features on the array.

Figure 3

Detection of multiple distinct epitopes of p53 antigen expressed by NAPPA. 7 different monoclonal antibodies to p53 antigen (D01, D02, D07,D013,D014,240, and 243) were used to detect determinants on the p53 molecule expressed by NAPPA. As shown with the anti-GST control which detects the c-terminal GST fusion protein, all p53 antigenic determinants are specifically detected by NAPPA fluorescence, 96-well ELISA, and by western blotting of expressed recombinant protein. The negative control (no antigen) and the control antigen p21-GST are not detected.

Figure 4

Comparable specificities of NAPPA microarray and protein ELISA for the detection of p53 antibodies in metastatic breast cancer patient sera. 15 sera from patients with metastatic breast cancer were diluted 1:100 and tested for p53-specific antibodies by protein ELISA and by NAPPA microarray. Two sera were positive by protein ELISA and were also positive by NAPPA microarray. The images of the quadruplicate-spotted antigens detected are shown in red on the bottom.

Figure 5

Development of custom tumor antigen high-density programmable protein microarrays for the detection of autoantibodies in patient sera. A) Over 1,700 candidate tumor antigens were expressed and captured in a microarray format, and protein expression was detected using anti-GST antibody. B. The arrays were probed with sera from a healthy individual, and patients with melanoma, breast and ovarian cancer. All arrays were incubated with 1:300 diluted serum and developed using a HRP conjugated anti-human IgG and TSA. (i) Shows EBNA response from healthy serum (red circle, CV=40% across 4 slides) (ii) Response to ML-IAP from melanoma serum (CV not tested) (iii) Response to p53 from breast cancer sera (CV=11% across 4 slides) (iv) Response to p53 from ovarian cancer sera (CV=49% across 2 slides).