DNA Copy-number Loss on 1p36.1 Harboring RUNX3 with Promoter Hypermethylation and Associated Loss of RUNX3 Expression in Liver Fluke-associated Intrahepatic Cholangiocarcinoma

Runt-related transcription factor 3 (RUNX3) is a candidate tumor suppressor gene, localized on 1p36, involvedin TGF-ß-Smads signaling. To assess its role in liver fluke-associated intrahepatic cholangiocarcinoma (ICC),the promoter methylation status was investigated in 53 ICCs by methylation-specific PCR, with determinationof loss of 1p36.1 by microarray comparative genomic hybridization and RUNX3 protein expression byimmunohistochemistry. Loss at 1p36.1 was found 41.5% of ICCs (22/53). In addition, DNA hypermethylation ofthe RUNX3 promoter was found in 49.1% (26/53) of cancers and 57.1% (4/7) of ICC cell lines. The protein washighly expressed in normal bile ducts but mostly decreased in ICCs, 67.9% (n=36) being negative forimmunohistochemical staining. Promoter hypermethylation of RUNX3 was associated with reversible decreaseor absence of RUNX3 protein expression (p<0.001), but this was not found to differ with the ICC subtype. Incontrast, loss of 1p36.1 demonstrated a significant link (p=0.020). In conclusion, RUNX3 promoterhypermethylation and loss of 1p36.1 are causal mechanisms for loss of RUNX3 function in liver fluke-associatedICC carcinogenesis.