Bile-Based Detection of Extrahepatic Cholangiocarcinoma with Quantitative DNA Methylation Markers and Its High Sensitivity

Abstract

Extrahepatic cholangiocarcinoma (EHC) is usually difficult to diagnose by bile cytology because of cellular disintegration. However, DNA samples from bile fluid can provide sufficient materials to screen for the presence of EHC. We developed DNA methylation marker panels that can be used for MethyLight assay-based detection of EHC in bile fluid specimens. The methylation status of 59 DNA methylation markers was investigated in 20 EHC and 20 non-neoplastic gallbladder tissue samples with MethyLight assay to determine cancer-specific DNA methylation markers. Through assaying cancer-specific DNA methylation markers in a training set (n = 40) and validation set (n = 45) of bile fluid specimens from patients with EHC or those without cancer, we selected suitable marker panels that were assessed for their performance in a third set (test set; n = 40). Four marker panels showed a sensitivity of 60% or more and a specificity of 100% in both the training and validation sets, whereas bile cytology displayed a sensitivity of 40% to 46% and a specificity of 100%. In an independent test set of bile fluid samples, a five-gene panel (CCND2, CDH13, GRIN2B, RUNX3, and TWIST1) detected EHC at a sensitivity of 83%, which was far higher than that of bile cytology (46%, P = 0.004). Using bile fluids, a methylation assay consisting of a five-gene panel may be useful for detecting EHC and in helping to increase the sensitivity of preoperative diagnoses.

Methylated Bone Morphogenetic Protein 3 (BMP3) Gene: Evaluation of Tumor Suppressor Function and Biomarker Potential in Biliary Cancer

“…The sensitivity was comparable to that found in other tissue sample studies in which single markers were 14-73% sensitive [16,20] and multi-marker panels were 74-91% sensitive [19-21]. In a multi-marker panel used for screening for colorectal cancer in stools, the high specificity of BMP3 complements other more sensitive methylated and mutant genes [38].…”

“…Of the genes in the panel, three novel genes ( CDO1, DCLK1 , and ZSCAN18 ) were found to be frequently methylated in CC in addition to SFRP1 , which has been previously reported [20]. Additional methylation markers have also been identified in tissues ( CCND2, CDH13, GRIN2B, RUNX3 , and TWIST1 ) and tested in bile samples of ECC patients and controls, the 5-gene panel showed a sensitivity of 83% [21]. While new DNA methylation profiling tools such as methyl-sequencing and methyl-DNA immunoprecipitation arrays [22] are expected to identify many new markers, the mechanistic understanding of the role of methylation of these genes in biliary carcinogenesis remains poor.…”

Promoter hypermethylation of SHOX2 and SEPT9 is a potential biomarker for minimally invasive diagnosis in adenocarcinomas of the biliary tract

“…Interestingly, aberrant DNA methylation patterns have been observed in bile fluid as well as in biliary brush cytology specimens and were described to be capable of differentiating between BTC and benign controls [16, 25–28]. SEPT9 has recently been reported to achieve a sensitivity of 57% with 100% specificity in biliary brush samples from CC patients, while respective tissue samples were methylated in less than 30% [16].…”

Aberrant DNA Methylation as a Biomarker and a Therapeutic Target of Cholangiocarcinoma

“…In order to overcome this problem, Shin et al developed a useful method for the detection of cholangiocarcinoma cells using bile fluid [23]. This method involving DNA methylation assay consisting of a five-gene panel ( CCND2 , CDH13 , GRIN2B , RUNX3 and TWIST1 ) is able to detect cholangiocarcinoma cells with a sensitivity of 83% and a specificity of 100%.…”