Identification of S100 calcium binding protein A9 as a prognostic biomarker in gallbladder cancer

Abstract

BackgroundGallbladder cancer (GBC) is the most lethal orphan malignancy of biliary tract, with poor prognosis and varied global incidences, predominately reported from, Northern India. Moreover, early diagnostic biomarker(s) of GBC is still undetermined. Earlier studies have demonstrated that S100A9 is associated with the prognosis of various malignancies. S100A9 (calgranulin B or Myeloid Related Protein-14) is a calcium binding protein which has wide range of possible intracellular as well as extracellular functions. Previous reports showed that S100A9 stimulates malignant development and cancer progression, and is also involved in other inflammatory diseases. So far, no study has been made on the expression and epigenetic regulation of S100A9 in GBC. Therefore, the present study aimed to investigate the expression and promoter methylation status of S100A9 in GBC to evaluate its utility as a diagnostic biomarker for GBC. Materials and methodMethylation specific PCR (MS-PCR) was done to determine the methylation status of S100A9 gene promoter. Semi quantitative and quantitative RT-PCR were performed to assess its expression level in tumor and non tumor tissues. Immunohistochemistry (IHC) was carried out to understand its cellular distribution. Statistical analysis was performed using student’s t-test for paired case and control tissue samples, and one way ANOVA for multiple comparison procedures. ResultsMS-PCR of S100A9 showed methylation (p =0.022) in the promoter region of about i.e.,75% (9/12) in adjacent non tumor (ANT) tissues and about 25% (3/12) in GBC cases. RT-PCR analyses revealed upregulation of S100A9 in GBC at transcriptional level. IHC analysis further supported the change in the transcriptional level, scored positive for S100A9 (62%; 36/58), in GBC and about (40%; 12/30) in non tumor (p <0.048). ConclusionThe study shows that elevated expression and hypomethylation in the S100A9 gene promoter suggesting that DNA methylation plays a crucial role in the regulation of S100A9 expression in GBC. However, further studies are warranted to confirm this in larger pool of samples before concluding its usefulness as a biomarker of GBC.