The diagnosis of
cholangiocarcinoma (CCA) is often challenging, leading to poor prognosis. CCA arises via chronic
inflammation which may be associated with
autoantibodies production. This study aims to identify
IgG antibodies directed at self-
proteins and
tumor-associated
antigens.
Proteins derived from immortalized cholangiocyte cell line (MMNK1) and CCA cell lines (M055, M214 and M139) were separated using 2-dimensional electrophoresis and incubated with pooled plasma of patients with CCA and non-neoplastic controls by immunoblotting. Twenty five immunoreactive spots against all cell lines-derived
proteins were observed on stained
gels and studied by LC-MS/MS. Among these,
heat shock protein 70 (HSP70),
enolase 1 (ENO1) and
ribonuclease/angiogenin inhibitor 1 (RNH1) obtained the highest matching scores and were thus selected for further validation. Western blot revealed immunoreactivity against HSP70 and RNH1 in the majority of CCA cases and weakly in healthy individuals. Further, ELISA showed that plasma HSP70
autoantibody level in CCA was significantly capable to discriminate CCA from healthy individuals with an area under the receiver operating characteristic curve of 0.9158 (cut-off 0.2630, 93.55% sensitivity and 73.91% specificity). Plasma levels of
IgG autoantibodies against HSP70 were correlated with progression from healthy individuals to
cholangitis to CCA (r = 0.679, P<0.001). In addition, circulating ENO1 and RNH1
autoantibodies levels were also significantly higher in
cholangitis and CCA compared to healthy controls (P<0.05). Moreover, the combinations of HSP70, ENO1 or RNH1
autoantibodies positivity rates improved specificity to over 78%. In conclusion, plasma
IgG autoantibodies against HSP70, ENO1 and RNH1 may represent new diagnostic markers for CCA.