Abstract
Background and aim
An increasing number of clinical data suggests the importance of the assessment of serum levels of the squamous cell carcinoma antigen (SCCA)-lgM immune complex for the diagnosis of hepatocellular carcinoma (HCC). In addition, monitoring of SCCA-IgM immune complexes has been described as a useful prognostic approach in patients with cirrhosis since the progressive increase of SCCA-IgM over time is associated with a higher risk of HCC development. Because other assays in patients with cirrhosis have been affected by interfering IgMs, the aim of the present study was to assess the specificity of SCCA-IgM reactivity in cirrhotic patients by evaluating SCCA-IgM detection dependence on different capturing phases and by measuring the recovery of SCCA-IgM reactivity after serum fractionation.
Patients and methods
Serum samples from 82 patients with cirrhosis (M/F ratio 3/1; mean age ± SD: 56 ± 9 years) were collected at the Liver Unit of the Department of Clinical and Experimental Medicine, University of Padua, according to the approved institutional procedures. Serum levels of SCCA-IgM were measured using two different ELISA tests: the reference assay based on a rabbit oligoclonal anti-human SCCA antibody (Hepa IC, Xeptagen, Italy) and a dedicated ELISA with a mouse monoclonal anti-SCCA as capture antibody.
Results
Serum levels of SCCA-IgM measured with the reference assay (median value 87 AU/mL) were higher than levels measured with the mouse monoclonal test (median value 78 AU/mL), but the differences were not statistically significant (Mann-Whitney U test, p>0.05). When SCCA-IgM levels measured with both tests were compared, a linear correlation was found (r = 0.77, p < 0.001). An in silico analysis using available structural data of SCCA showed the presence of up to three putative antigenic sites localized on the SCCA surface, thus providing evidence that the test with the mouse monoclonal anti-SCCA antibody could underestimate the total circulating immune complexes due to the steric hindrance of the enormous mass of IgM (900 kDa) that could mask on the SCCA (45 kDa) surface the binding site recognized by the monoclonal antibody. To show that the SCCA-IgM assay was not affected by interfering IgMs, we measured the recovery of SCCA-IgM reactivity after serum fractionation of ten of the most reactive samples in both assays. Total recovery of SCCA-IgM reactivity was obtained with both assays in the fractions corresponding to components with higher molecular weight than IgM and SCCA (>2000 kDa).
Conclusions
The results of this study indicate that the reactivity measured in cirrhotic patients is related only to SCCA-IgM immune complexes and is not affected by other serum components, supporting the analytical specificity of the SCCA-IgM assay and validating the importance of SCCA-IgM as a risk biomarker for HCC development.