Protein microarray signature of autoantibody biomarkers for the early detection of breast cancer

Abstract

Cancer patients spontaneously generate autoantibodies (AAb) to tumor-derived proteins. To detect AAb, we have probed novel high-density custom protein microarrays (NAPPA) expressing 4988 candidate tumor antigens with sera from patients with early stage breast cancer (IBC), and bound IgG was measured. We used a three-phase serial screening approach. First, a prescreen was performed to eliminate uninformative antigens. Sera from stage I-III IBC (n = 53) and healthy women (n = 53) were screened for AAb to all 4988 protein antigens. Antigens were selected 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 significant (p < 0.05). These 761 antigens were screened using an independent set of IBC sera (n = 51) and sera from women with benign breast disease (BBD) (n = 39). From these, 119 antigens had a partial area under the ROC curve (p < 0.05), with sensitivities ranging from 9-40% at >91% specificity. Twenty-eight of these antigens were confirmed using an independent serum cohort (n = 51 cases/38 controls, p < 0.05). Using all 28 AAb, a classifier was identified with a sensitivity of 80.8% and a specificity of 61.6% (AUC = 0.756). These are potential biomarkers for the early detection of breast cancer.

Figure 1. Schematic of the detection of autoantibodies with NAPPA protein microarrays

1) Replicate NAPPA protein microarrays expressing 4,988 candidate tumor antigens are probed with sera from patients with cancer and healthy controls (2). Detection of IgG autoantibodies in patient sera is compared with control sera (3) and patterns of immune responses are identified (4). Antigens that correspond to antibodies detected only in sera from cancer cases are then selected for further confirmation.

Figure 2. Schematic of Serum Screening Strategy

Breast cancer sera were sequentially tested on custom microarrays as shown. Initial screening was performed using arrays expressing 4,988 unique full length cDNAs and case/control sera derived from a screening mammography clinic (Cohort 1). Secondary screening was performed using arrays expressing 761 unique full length cDNAs and case/control sera derived from a diagnostic mammography clinic, to control for benign breast disease (Cohort 2). From these, 119 potential biomarkers were selected. An independent blinded validation set of case/control sera (Cohort 3) were used to validate the top 28 biomarkers.

Figure 3. Detection of autoantibodies with NAPPA protein microarrays

Left, anti-GST stain (red), which binds to a c-terminal tag present on all proteins, confirms total protein display; Right, 3-dimensional renderings of the signal intensities for representative images of one block (in green box on left) probed with four different serum samples (2 cases and 2 controls) and detected with anti-human IgG. Spots (gene SF3A1 arrayed in duplicate) indicated by yellow circles and arrows display differential reactivity between cases and controls. Insets show original scanned images.

Figure 4. Receiver operating characteristic curve

for the random forest classifier with 2000 trees and 3 random features. The curve is calculated using the predicted class probabilities from a leave-one-out cross classification study with five random seeds.

Figure 5. Independent evaluation of ATP6AP1 biomarker

A. ATP6AP1 protein is overexpressed in a number of breast cancer cell lines. Immunoblotting of cell lysates separated by SDS-PAGE for ATP6AP1 protein was performed. ATP6AP1 (arrow) was strongly detected in the cell lines shown. Actin protein is shown in the lower panel. B. ATP6AP1 AAb detection in sera by ELISA. Sera derived from healthy normal controls (n=64) and Stage I-III breast cancer (n=148) were tested for ATP6AP1 IgG AAb using recombinant ATP6AP1-GST and GST proteins (p=0.059). Comparison of IgG responses in these sera to the negative control antigen PCNA and the positive control antigen EBNA-1 are shown.

Figure 5. Independent evaluation of ATP6AP1 biomarker

A. ATP6AP1 protein is overexpressed in a number of breast cancer cell lines. Immunoblotting of cell lysates separated by SDS-PAGE for ATP6AP1 protein was performed. ATP6AP1 (arrow) was strongly detected in the cell lines shown. Actin protein is shown in the lower panel. B. ATP6AP1 AAb detection in sera by ELISA. Sera derived from healthy normal controls (n=64) and Stage I-III breast cancer (n=148) were tested for ATP6AP1 IgG AAb using recombinant ATP6AP1-GST and GST proteins (p=0.059). Comparison of IgG responses in these sera to the negative control antigen PCNA and the positive control antigen EBNA-1 are shown.