The sTRA Plasma Biomarker: Blinded Validation of Improved Accuracy Over CA19-9 in Pancreatic Cancer Diagnosis

Abstract

Purpose: The CA19-9 biomarker is elevated in a substantial group of patients with pancreatic ductal adenocarcinoma (PDAC), but not enough to be reliable for the detection or diagnosis of the disease. We hypothesized that a glycan called sTRA (sialylated tumor-related antigen) is a biomarker for PDAC that improves upon CA19-9.

Experimental design: We examined sTRA and CA19-9 expression and secretion in panels of cell lines, patient-derived xenografts, and primary tumors. We developed candidate biomarkers from sTRA and CA19-9 in a training set of 147 plasma samples and used the panels to make case-control calls, based on predetermined thresholds, in a 50-sample validation set and a blinded, 147-sample test set.

Results: The sTRA glycan was produced and secreted by pancreatic tumors and models that did not produce and secrete CA19-9. Two biomarker panels improved upon CA19-9 in the training set, one optimized for specificity, which included CA19-9 and 2 versions of the sTRA assay, and another optimized for sensitivity, which included 2 sTRA assays. Both panels achieved statistical improvement (P < 0.001) over CA19-9 in the validation set, and the specificity-optimized panel achieved statistical improvement (P < 0.001) in the blinded set: 95% specificity and 54% sensitivity (75% accuracy), compared with 97%/30% (65% accuracy). Unblinding produced further improvements and revealed independent, complementary contributions from each marker.

Conclusions: sTRA is a validated serological biomarker of PDAC that yields improved performance over CA19-9. The new panels may enable surveillance for PDAC among people with elevated risk, or improved differential diagnosis among patients with suspected pancreatic cancer.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest

A.D. Singhi is a consultant/advisory board member for Foundation Medicine. No potential conflicts of interest were disclosed by the other authors.

©2019 American Association for Cancer Research.

Figures

Figure 1.

The CA19–9 and sTRA assays. A, The epitopes detected by the CA19–9 and TRA-1–60 antibodies. B, Potential secretion of carriers of single or dual antigens. C, In the CA19–9 assay, both the capture and detection antibodies detect the glycan epitope of the CA19–9 antibody. In the sTRA assay, the capture antibodies target either the CA19–9 antigen or a protein carrier of sTRA. After sample incubation, the captured material is treated with sialidase and then probed with the TRA antibody.

Figure 2.

Complementary elevations of CA19–9 and sTRA in model systems. A, Immunofluorescence staining of mouse xenografts of cell lines showed variable expression of the 2 markers. B, Quantification of the cell surface and secreted levels showed the certain cell lines produced primarily one or the other glycans. C, Immunofluorescence staining of PDX tissue also showed variable expression of the 2 markers. D, Quantification of the levels in the mouse tissue and sera showed complementary patterns of expression.

Figure 3.

Complementary elevations in primary tumors and plasma. A, Immunofluorescence staining showed expression of one, both, or neither of the markers. B, The quantification of tissue and plasma levels revealed low correspondence between the 2 markers. A substantial group of patients was elevated in only sTRA, based on thresholds set to the highest control samples (dashed lines), but the high correlation (0.74) was caused by one outlier value (arrowhead).

Figure 4.

Biomarker panel development. A, The CA19–9 and sTRA assays were quantified in 72 case and 75 control plasma specimens. As a single marker, the CA19–9:sTRA assay performed similarly to CA19–9. B, The correlations between the sTRA markers and CA19–9 were very low, with samples elevated in one, both, or neither of the markers. C, A threshold was applied to each marker in the panel or to CA19–9 alone, and samples with an elevation in any marker were called as cases. In the panel optimized for specificity shown here, the panel identified more of the cases than CA19–9. D, The performance of both panels was better than CA19–9 in the training set and in the application of the predetermined thresholds to the 50-sample validation set. For both panels, the difference in the average of sensitivity and specificity was significant (P < 0.001). The difference is the average over 1,000-fold bootstrapping analysis, and the error bars are the 95% confidence intervals. E, The breakdown of marker contributions and the improvement in final performance were similar between the training and validation sets.

Figure 5.

Application to blinded samples. The 2 biomarker panels were applied to a blinded set of 147 samples, using predetermined marker thresholds and classification rules. A, Both panels improved upon CA19–9. The difference in the average of sensitivity and specificity was significant (P < 0.001) for the specificity panel, based on 1,000-fold bootstrapping analysis. B, The individual marker performances matched the training set. C, The sTRA and CA19–9 markers showed complementary elevations. The higher correlation (0.68) was caused by a sample that was very high in both (arrowhead). The dashed lines show the predetermined thresholds for the specificity panel. D, The improvements in either sensitivity or specificity were consistent between the training and test sets. E, The independent contributions of each panel member and the improvements of the panels over CA19–9 were consistent between the training and test sets.