CA125 immune complexes in ovarian cancer patients with low CA125 concentrations

Abstract

Background: About 20% of women with ovarian cancer have low concentrations of serum cancer antigen 125 (CA125), and this important tumor marker cannot be used to monitor their disease. The measured concentration for mucin 1 (MUC1), or CA15-3, another tumor marker, can be lowered in breast and ovarian cancer patients when circulating immune complexes (CICs) containing antibodies bound to the free antigen are present. Because CA125 and MUC1 are related members of the mucin family, we sought to determine whether CICs might also exist for CA125 and interfere with its clinical assay.

Methods: We developed an antigen capture-based assay to determine the presence of CICs for CA125. We spotted mouse antibodies to CA125 onto nanoparticle slides, incubated them with patient serum, and added Cy5-tagged goat antihuman IgG antibodies. Fluorescence intensities were read and normalized to the intensities for glutathione S-transferase A1 as a control.

Results: Assay results for 23 ovarian cancer cases with high CA125 concentrations, 43 cases with low CA125 concentrations, and 19 controls (mean CA125 concentrations, 2706, 23, and 11 kilounits/L, respectively) revealed mean fluorescence intensities for CA125 CIC of 2.30, 2.72, and 1.99 intensity units (iu), respectively. A generalized linear model adjusted for batch and age showed higher CA125 CIC fluorescence intensities in low-CA125 cases than in high-CA125 cases (P = 0.03) and controls (P = 0.0005). Four ovarian cancer patients who had recurrent disease and always had low CA125 values had a mean CA125 CIC value of 3.06 iu (95% CI, 2.34-4.01 iu).

Conclusions: These preliminary results suggest the existence of CICs involving CA125, which may help explain some ovarian cancer cases with low CA125 concentrations.

Fig. 1. Illustration of antigen capture–based assays for CA125 free antigen and CA125 in immune complexes

Mouse monoclonal antibodies to CA125 are spotted onto a nanoparticle slide and patient serum is added. After washing, rabbit anti-CA125 antibody is added, followed by Cy5- tagged goat antirabbit antibody (A). When CA125 is bound in an immune complex with human anti-CA125, the rabbit anti-CA125 antibodies may be blocked (B). CA125 immune complexes are measured by adding Cy5-tagged goat antihuman IgG antibodies immediately following incubation with the sera (C). The inserts of panels A-C illustrate the array-based antigen and immune complex reactions in an invasive serous case with a CA125 concentration of 3331 kilounits/L, compared with an invasive serous case with a CA125 concentration of 26.7 kilounits/L. The free-antigen assay is strongly positive for the high-CA125 case and weakly positive for the low-CA125 case. The opposite occurs for the CA125 CIC assay. Green spots are BSA controls as orientation markers. We subsequently introduced an array that included a mouse monoclonal antibody to GSTA1, which served as a negative control and allowed calculation of a "normalized" intensity for the CA125-CIC (D). A pair of cases from Table 1— one with a CA125 concentration of 21 kilounits/L and a normalized fluorescence intensity of 2.9 iu (right) and a second with a CA125 of 2880 kilounits/L and normalized fluorescence intensity of 1.1 iu (left).