Breast Reference Set Application: Karen Anderson-ASU (2014)

Abbreviated Name
Breast Ref Set App: Anderson (2014)
Lead Investigator
Anderson, KarenArizona State University
Coordinating Investigator
Feng, Ziding Fred Hutchinson Cancer Center
Involved Investigators


In order to increase the predictive value of tumor-specific antibodies for use as immunodiagnostics, our EDRN BDL has developed a novel protein microarray technology, termed Nucleic Acid Protein Programmable Array (NAPPA), which circumvents many of the limitations of traditional protein microarrays. NAPPA arrays are generated by printing full-length cDNAs encoding the target proteins at each feature of the array. The proteins are then transcribed and translated by a cell-free system and immobilized in situ using epitope tags fused to the proteins. Sera are added, and bound IgG is detected by standard secondary reagents. Using a sequential screening strategy to select AAb from 4,988 candidate tumor antigens, we have identified 28 potential AAb biomarkers for the early detection of breast cancer, and here we propose to evaluate these biomarkers using the EDRN Breast Cancer Reference Set.


The aims of the proposal are to evaluate this panel of 28 AAb biomarkers using the EDRN reference set. We propose to use RAPID ELISA containing each of these 28 AAb in addition to controls and screen the EDRN Reference Set 1 (30 subjects with invasive cancer; 30 subjects with benign disease without atypia) to further evaluate our AAb panel. These data will further inform us of systemic differences in screening and diagnostic populations and determine whether any single or combination of markers has any discriminatory ability when appropriate controls are used. These data will be made publicly available and become part of the background information on the samples that comprise the Reference Set. If the biomarkers demonstrate discrimination between cases and controls in Set 1, we will then progress to evaluating EDRN Reference Set 2 (125 subjects with invasive cancer; 129 benign without atypia; 52 benign with atypia; 40 carcinomas in situ; 125 samples collected at time of mammograms).

Analytic Method

As stipulated, the data will be supplied back to the DMCC within 120 days of the receipt of the samples for analysis. Performance of individual markers will be evaluated: Each array will be normalized at ASU by first removing the background signal estimated by the first quartile of the non-spots and then log-transforming the median-scaled raw intensities to bring the data to the same scale and stabilize the variance across the range of signals. The panel of 28 antigens will then evaluated using a random forest classifier at ASU. ASU will provide to the DMCC, the one-number representing the combination for each sample. We recommend that the performance of the panel be assessed by constructing a receiver operating characteristic (ROC) curve based upon cross validation or its out-of-bag predictions, and summarizing the performance using AUC. We also recommend the DMCC analyze the performance of the panel based on this data using either the partial area under the receiver operating characteristic curve (pAUC) or sensitivity at high specificity. A global assessment of each protein’s and the panel’s ability to distinguish cases and controls should also be conducted using the full area under the receiver operating characteristic curve (AUC).


  • No publications available at this time for this protocol.


Data Collections

  • No data collections available at this time for this protocol.
 Team Project
Finish Date
May 6 2019
Protocol ID
Protocol Type
Reference Set
Field of Research
Collaborative Group
Breast and Gynecologic Cancers Research Group
Cancer Types
  • Malignant neoplasm of breast
Phased Status

Associated Forms