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, 6 (12), e29180

Enhanced Discrimination of Malignant From Benign Pancreatic Disease by Measuring the CA 19-9 Antigen on Specific Protein Carriers

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Enhanced Discrimination of Malignant From Benign Pancreatic Disease by Measuring the CA 19-9 Antigen on Specific Protein Carriers

Tingting Yue et al. PLoS One.

Abstract

The CA 19-9 assay detects a carbohydrate antigen on multiple protein carriers, some of which may be preferential carriers of the antigen in cancer. We tested the hypothesis that the measurement of the CA 19-9 antigen on individual proteins could improve performance over the standard CA 19-9 assay. We used antibody arrays to measure the levels of the CA 19-9 antigen on multiple proteins in serum or plasma samples from patients with pancreatic adenocarcinoma or pancreatitis. Sample sets from three different institutions were examined, comprising 531 individual samples. The measurement of the CA 19-9 antigen on any individual protein did not improve upon the performance of the standard CA 19-9 assay (82% sensitivity at 75% specificity for early-stage cancer), owing to diversity among patients in their CA 19-9 protein carriers. However, a subset of cancer patients with no elevation in the standard CA 19-9 assay showed elevations of the CA 19-9 antigen specifically on the proteins MUC5AC or MUC16 in all sample sets. By combining measurements of the standard CA 19-9 assay with detection of CA 19-9 on MUC5AC and MUC16, the sensitivity of cancer detection was improved relative to CA 19-9 alone in each sample set, achieving 67-80% sensitivity at 98% specificity. This finding demonstrates the value of measuring glycans on specific proteins for improving biomarker performance. Diagnostic tests with improved sensitivity for detecting pancreatic cancer could have important applications for improving the treatment and management of patients suffering from this disease.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Detection of total CA19-9 and CA 19-9 on individual proteins using antibody arrays.
a) High-throughput sample processing and array-based sandwich assays for CA19-9 detection. Forty-eight identical arrays are printed on one microscopic slide, segregated by hydrophobic wax boundaries (left). A set of serum or plasma samples are incubated on the arrays in random order, and the arrays for the entire sample set are probed with the CA 19-9 detection antibody (right). b) Molecular detail. Total CA19-9 is measured at the CA19-9 capture antibody (left), and CA19-9 on specific proteins is measured at the individual antibodies against those proteins (right). b) Representative raw image data from each of the sample groups. Triplicates of each antibody were randomly positioned on the array, as indicated for selected antibodies.
Figure 2
Figure 2. Total CA 19-9 levels and CA 19-9 on specific proteins.
The fluorescence values for the total CA 19-9 (top), CA19-9 on MUC1 (second row), CA 19-9 on MUC16 (third row), and CA 19-9 on MUC5AC (fourth row) are shown for each sample group. The left column compares samples from pancreatitis patients to samples from early-stage pancreatic cancer patients, and the right column compares pancreatitis to late-stage cancer. The sensitivity and specificity at the threshold indicated by the dash line are given.
Figure 3
Figure 3. Diversity in CA 19-9 levels on individual proteins.
Raw antibody images are shown for patient samples representing diverse marker patterns. Data from sample set 3 (replicate 1) are presented. A cancer sample (labeled ‘True positive’) and pancreatitis sample (labeled ‘False positive’) that were high in total CA 19-9 (above a 75% specificity threshold) are in the top left, and pancreatitis samples that were low in total CA 19-9 (‘True negatives’) are in the bottom left. Cancer samples that were low in total CA 19-9 are grouped by relatively high or low signal at one of the mucins in the top right and bottom right, respectively. The sample identifier is given within each array. In the subgroup picked up by the panel (top-right), the antibody showing elevation in a given sample is listed adjacent to each array. The corresponding antibody spots are underlined in white. Two arrays for sample LC3607 are shown, one detected with BPL (rightmost column, row 2), and the other detected with CA19-9 (rightmost column, row 3). All other arrays were detected with CA19-9. The bottom panels show maps of antibodies targeting MUC16 (left), MUC5AC (middle), and MUC1 (right).
Figure 4
Figure 4. Correlations and complementarity between total CA 19-9 and CA 19-9 on MUC16 and MUC5AC.
Each scatter plot compares the values for total CA 19-9 (x axis) to the values for CA 19-9 on MUC16 or MUC5AC. Each point is an individual sample. Samples from Set 1 are presented at top, and samples from Set 2 are presented in the bottom panels. The dashed lines indicate representative thresholds for each marker. The sensitivity and specificity given in each graph represents the performance at those thresholds if a sample exceeding either threshold is called a “case.” The red arrows indicate the samples that are not elevated in total CA 19-9 but are elevated in CA 19-9 on an individual protein. Each ROC curve shows the performance of CA 19-9 alone and the combination of CA 19-9 with the indicated marker. If a sample was elevated in either marker, it was called a “case.” The asterisk indicates the performance at the thresholds in the scatter plots.
Figure 5
Figure 5. Improved classification over CA 19-9 using a three-marker panel.
Each column represents data from a patient sample and each row represents a marker, with the bottom row indicating the patient classification. A threshold was set for each marker, and a yellow square indicates the sample was above the threshold for that marker, and black indicates below the threshold. In the final row, a yellow square indicates the sample was elevated in any of the three markers and classified as a “case.” The true positive (TP) cancer cases that were elevated in CA 19-9 are indicated by ‘TP, CA 19-9’, and the true positive cases elevated only in the other markers are indicated by ‘TP, Panel.’ The false negative (FN) cancer cases are indicated by ‘FN,’ the false positive (FP) control cases that were elevated in a marker are indicated by ‘FP,’ and the true negative (TN) control cases that were low in all markers are indicated by ‘TN.’ Data from Sample Set 1 is presented at top, and data from Sample Set 2 is below.

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