DNA Methylation Markers for Detection of Cholangiocarcinoma: Discovery, Validation, and Clinical Testing in Biliary Brushings and Plasma

Abstract

Cholangiocarcinoma (CCA) has poor prognosis due to late-stage, symptomatic presentation. Altered DNA methylation markers may improve diagnosis of CCA. Reduced-representation bisulfite sequencing was performed on DNA extracted from frozen CCA tissues and matched to adjacent benign biliary epithelia or liver parenchyma. Methylated DNA markers (MDMs) identified from sequenced differentially methylated regions were selected for biological validation on DNA from independent formalin-fixed, paraffin-embedded CCA tumors and adjacent hepatobiliary control tissues using methylation-specific polymerase chain reaction. Selected MDMs were then blindly assayed on DNA extracted from independent archival biliary brushing specimens, including 12 perihilar cholangiocarcinoma, 4 distal cholangiocarcinoma cases, and 18 controls. Next, MDMs were blindly assayed on plasma DNA from patients with extrahepatic CCA (eCCA), including 54 perihilar CCA and 5 distal CCA cases and 95 healthy and 22 primary sclerosing cholangitis controls, balanced for age and sex. From more than 3,600 MDMs discovered in frozen tissues, 39 were tested in independent samples. In the clinical pilot of 16 MDMs on cytology brushings, methylated EMX1 (empty spiracles homeobox 1) had an area under the curve (AUC) of 0.98 (95% confidence interval [CI], 0.95-1.0). In the clinical pilot on plasma, a cross-validated recursive partitioning tree prediction model from nine MDMs was accurate for de novo eCCA (AUC, 0.88 [0.81-0.95]) but not for primary sclerosing cholangitis-associated eCCA (AUC, 0.54 [0.35-0.73]). Conclusion: Next-generation DNA sequencing yielded highly discriminant methylation markers for CCA. Confirmation of these findings in independent tissues, cytology brushings, and plasma supports further development of DNA methylation to augment diagnosis of CCA.

FIG. 1

Study flow diagram. From reduced representation bisulfite sequencing discovery, MDMs were advanced to biological validation in independent samples on the basis of broad representation (coverage), high variation between cases and controls, and high statistical significance. Due to high DNA requirements for targeted assays in biological validation (methylation‐specific PCR) it was possible to test only 41 MDMs in independent specimens, which are enumerated in Supporting Table S1. With the exception of BMP3, markers brought forward from biological validation to plasma testing in the clinical pilot met three criteria: AUC values >0.84; fold change >6; and pre‐existing TELQAS assay designs. Markers brought forward to the biliary brushings pilot had either high AUC values, high fold change, or were complementary to those that did in the biological validation (data not shown).

FIG. 2

MDM intensity in biliary brushing sample clinical pilot. A heat matrix shows MDM intensity in DNA extracted from biliary brushing cytology samples from benign biliary stricture controls without PSC, PSC strictures, and dCCA or pCCA cases. Specificity was set to 90% in the normal group. Each row is an MDM, and each column is a unique patient sample. Increasing color intensity from yellow to red indicates deciles of ACTB‐corrected MSP product above the 95th percentile value in the controls without PSC. Values below that threshold are dark gray. Abbreviations: CYP26C1, cytochrome P450 family 26 subfamily C member 1; KCNA1, potassium voltage‐gated channel subfamily A member 1; KLF12, Krüeppel‐like factor 12; PTGDR, prostaglandin D2 receptor; RYR2, ryanodine receptor 2; S1PR1, sphingosine‐1‐phosphate receptor 1; SALL1, spalt like transcription factor 1.

FIG. 3

MDM accuracy in plasma clinical pilot. AUCs with 95% CIs for the cross‐validated recursive partitioning model of a panel of nine MDMs in eCCA cases and controls overall or stratified by presence or absence (+/−) of comorbid PSC.