Using custom protein microarrays to identify autoantibody biomarkers for the early detection of breast cancer.

Abstract

#2003 Background: Cancer patients make antibodies to tumor-derived proteins that are potential biomarkers for early detection. To detect autoantibodies to tumor antigens in patient sera, we have adapted novel high-density custom protein microarrays (NAPPA) expressing 6,500 candidate tumor antigens for biomarker detection. These arrays are probed with sera from patients with early stage breast cancer and healthy women. Using this approach, we identified antibodies in the sera of breast cancer patients.
 Methods: 6,500 full-length human antigens were expressed using mammalian reticulocyte lysate and captured onto NAPPA protein microarrays. Protein expression (>90%) was confirmed with anti-GST antibodies. Patient sera were added, and bound IgG detected with secondary antibodies. Serum samples were obtained from 103 patients with stages I-III breast cancer, and 103 age-matched control women, all undergoing routine mammography.
 Results: Using high-density protein microarrays, sera from breast cancer patients (n=53) and healthy donor sera (n=53) were screened for autoantibodies to 6,500 protein antigens. Antigens were selected for further analysis if the 95th percentile of signal of cases and controls were significantly different (p<0.05) and if the number of cases with signals above the 95th percentile of controls was larger than the number expected due to random chance (p<0.05). Known tumor antigens, such as p53, were detected, as well as novel antigens such as DCC1, Rab7L and USP44. In total, 768 unique antigens were selected for further analysis with an independent set of breast cancer patient (n=50) and healthy donor sera (n=50).
 Conclusions: Custom in-situ protein microarrays can be used to detect serum tumor antigen-specific antibodies and enables the rapid, simultaneous detection of immunogenic tumor antigens from patient sera. These autoantibodies are being evaluated as potential biomarkers for the early diagnosis of breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2003.

p53 Autoantibodies as Potential Detection and Prognostic Biomarkers in Serous Ovarian Cancer

“…Serum was used at a dilution of 1:300 for all subsequent ELISA analyses. Inter-assay coefficients of variation of RAPID ELISA is 3.9%(14). Both p21-GST and p53-GST are readily expressed and detected by immunoblotting with anti-GST Mab (Supplemental Figure 1B, left), but only p53-GST is detected with the ovarian cancer patient serum (Supplemental Figure 1B, right).…”

“…Detection of p53 antibodies in patient sera using Rapid Antigenic Protein In situ Display (RAPID) ELISA was performed as described(14). Briefly, 96-well detection plates coated with anti-GST antibody (GE Healthcare, Piscataway, NJ) were blocked overnight at 4°C with blocking buffer (5% milk/0.2% Tween20 in phosphate buffered saline (PBS-T)).…”

“…We have developed a custom ELISA assay, termed Rapid Antigenic Protein In situ Display (RAPID), for the detection of antibodies to tumor antigens in patient sera(14). RAPID ELISA eliminates the need for a priori protein purification and minimizes the risk of immunogenic co-purified bacterial antigens as cross-reacting serologic targets in ELISA.…”

Protein Microarray Signature of Autoantibody Biomarkers for the Early Detection of Breast Cancer

“…Arrays were stored in an air-tight container at room temperature, protected from light. The printed DNA was transcribed and translated in situ using previously published protocols 25, 28 . Protein expression was detected using anti-GST MAb (Cell Signaling, Danvers, MA) diluted at 1:200.…”

Autoantibody biomarkers for the detection of serous ovarian cancer

“…Tumor antigen-associated autoantibodies (TAAb) represent a well-documented source of potential early diagnostic biomarkers for ovarian cancer [6–10]. TAAbs are generated in response to protein overexpression or mutations in cancer patients.…”