Definitive Characterization of CA 19-9 in Resectable Pancreatic Cancer Using a Reference Set of Serum and Plasma Specimens

Abstract

The validation of candidate biomarkers often is hampered by the lack of a reliable means of assessing and comparing performance. We present here a reference set of serum and plasma samples to facilitate the validation of biomarkers for resectable pancreatic cancer. The reference set includes a large cohort of stage I-II pancreatic cancer patients, recruited from 5 different institutions, and relevant control groups. We characterized the performance of the current best serological biomarker for pancreatic cancer, CA 19-9, using plasma samples from the reference set to provide a benchmark for future biomarker studies and to further our knowledge of CA 19-9 in early-stage pancreatic cancer and the control groups. CA 19-9 distinguished pancreatic cancers from the healthy and chronic pancreatitis groups with an average sensitivity and specificity of 70-74%, similar to previous studies using all stages of pancreatic cancer. Chronic pancreatitis patients did not show CA 19-9 elevations, but patients with benign biliary obstruction had elevations nearly as high as the cancer patients. We gained additional information about the biomarker by comparing two distinct assays. The two CA 9-9 assays agreed well in overall performance but diverged in measurements of individual samples, potentially due to subtle differences in antibody specificity as revealed by glycan array analysis. Thus, the reference set promises be a valuable resource for biomarker validation and comparison, and the CA 19-9 data presented here will be useful for benchmarking and for exploring relationships to CA 19-9.

Fig 1. Differentiating pancreatic cancer from control subjects by two different CA 19–9 assays.

A) CA 19–9 levels in each group. We present the log-transformed values to better visualize all ranges of the values. The boxes indicate the quartiles of the distributions, the horizontal lines in the boxes indicate the medians, and the dashed lines give the ranges, with individual outliers indicated by the circles. A1, Assay 1; A2, Assay 2. B-D) Receiver-operator-characteristic (ROC) curves comparing all pancreatic cancer patients to the indicated control groups. The legends specify the area-under-the-curves (AUCs) for each assay, with the ranges of the 95% confidence intervals.

Fig 2. Correlation between Assay 1 and Assay 2.

The log-transformed CA 19–9 values of all subjects from Assay 1 and Assay 2 were plotted with respect to each other. Each circle indicates an individual patient sample. The trendline is the linear-least squares best fit, and the Spearman’s rank correlation coefficient is 0.74.

Fig 3. Glycan array analysis of three CA 19–9 antibodies.

Each antibody was incubated at 2 μg/mL on a glycan array containing 610 distinct glycans (glycan array version 5.1 from the Consortium for Functional Glycomics). The graph includes the glycans showing the highest levels of binding by any of the antibodies. For each antibody, we normalized the raw fluorescence values to set the highest value to 1. The list specifies the glycans included in the plot, and the labels above the columns indicate the primary motif in each glycan. DRG is the Assay 1 antibody, 9L426 is the Assay 2 antibody, and M081221 is another anti-sialyl Lewis A antibody included for comparison.

Fig 4. Glycan motifs recognized by three CA 19–9 antibodies.

For each antibody, the binding intensity to the indicated glycans, in relative fluorescence units, is given at three antibody concentrations (in μg/mL). Green indicates strong binding, yellow is moderate binding, and pink is weak binding.