Abstract
Background
Several studies have shown that the main circulating biomarkers of liver and colorectal cancer can be detected in the bloodstream and are also associated with immunoglobulin M to form stable complexes. These immune complexes show increased capacity of discrimination between cancer patients and healthy controls if combined with the free biomarker form. Within the context of the Project FIRB 2003 - Nanosized Cancer Polymarker Biochip - we wanted to investigate if IgM complexes have importance also in breast cancer. We focused our study on the immune complexes between IgM and CA 15.3 because free CA 15.3 is the most commonly used breast cancer biomarker in clinical practice. However, this biomarker alone lacks satisfactory sensitivity especially in early cancer detection.
Aim
The aim of our study was to assess the occurrence of immune complexes between CA 15.3 and IgM in sera from patients with primary breast cancer and in sera from healthy controls to evaluate its putative diagnostic value compared with the diagnostic value of free CA 15.3.
Methods
A total of 130 serum samples were obtained from 56 healthy women (mean age±SD, 45±8.23 years) and 74 women with stage l and II breast cancer (mean age±SD, 59±13.6 years) before any treatment, either surgical or chemo-therapeutic. Serum samples were collected, aliquoted and stored at-80°C in the centralized biobank of the project according to very stringent standard operating procedures (SOPs) distributed by the coordinating unit.
To evaluate the presence of CA 15.3-lgM immune complexes, we developed and validated a novel enzyme-linked immunosorbent assay (ELISA) with a polyclonal rabbit anti-human CA 15.3 antibody (Abcam) as the catcher antibody. CA 15.3-lgM was detected with peroxidase-conjugated anti-human IgM and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and hydrogen peroxide as substrate (Sigma Aldrich, Italy). The levels of CA 15.3-lgM were expressed in arbitrary units per mL (AU/mL) by interpolation on a calibration curve obtained by serial dilution of a reference calibrator purified by gel filtration chromatography from a pool of serum samples with high levels of CA 15.3-lgM. Serum levels of free CA 15.3 were assessed in parallel on each sample using an automated immunoassay system (ADVIA Centaur-Siemens Diagnostics) and expressed in U/mL.
Results
To discriminate between cancer patients and healthy controls, we used as cutoff values 31.5 U/mL for free CA 15.3 (corresponding to the cutoff used in the clinical routine) and 794 AU/mL for CA 15.3-lgM (representing the 95th percentile of the distribution of serum levels of CA 15.3-lgM in healthy controls). By using these cutoff values, we obtained a sensitivity of 1 0% (7/74 cases) and a specificity of 95% (53/56 controls) for CA 15.3-lgM. The sensitivity and specificity of free CA15.3 were 7% (5/74 cases) and 1 00% (56/56 controls), respectively. Interestingly, the serum levels of the two biomarkers did not overlap, so their combination at 95% specificity identified 12/74 cases (16.2%). When we took a cutoff of 22 U/ mL for free CA15.3 (the 95th percentile of its distribution in healthy controls), we calculated a sensitivity of 26% (1 9/74 cases) and a specificity of 95% (53/56 controls); its combination with CA 15.3-lgM had a sensitivity of 34% (25/74 cases) and a specificity of 90% (50/56 controls).
Conclusions
These results demonstrate for the first time the presence of CA 15.3-lgM in the bloodstream of patients with breast cancer. In addition, our data suggest that CA 15.3-lgM is a complementary serological marker to free CA 15.3 and the combination of these biomarkers could improve the diagnosis of breast cancer.