Early and accurate detection of cholangiocarcinoma in patients with primary sclerosing cholangitis by methylation markers in bile

Hege Marie Vedeld, Marit M Grimsrud, Kim Andresen, Heidi D Pharo, Erik von Seth, Tom H Karlsen, Hilde Honne, Vemund Paulsen, Martti A Färkkilä, Annika Bergquist, Marine Jeanmougin, Lars Aabakken, Kirsten M Boberg, Trine Folseraas, Guro E Lind, Hege Marie Vedeld, Marit M Grimsrud, Kim Andresen, Heidi D Pharo, Erik von Seth, Tom H Karlsen, Hilde Honne, Vemund Paulsen, Martti A Färkkilä, Annika Bergquist, Marine Jeanmougin, Lars Aabakken, Kirsten M Boberg, Trine Folseraas, Guro E Lind

Abstract

Background and aims: Primary sclerosing cholangitis (PSC) is associated with increased risk of cholangiocarcinoma (CCA). Early and accurate CCA detection represents an unmet clinical need as the majority of patients with PSC are diagnosed at an advanced stage of malignancy. In the present study, we aimed at establishing robust DNA methylation biomarkers in bile for early and accurate diagnosis of CCA in PSC.

Approach and results: Droplet digital PCR (ddPCR) was used to analyze 344 bile samples from 273 patients with sporadic and PSC-associated CCA, PSC, and other nonmalignant liver diseases for promoter methylation of cysteine dioxygenase type 1, cannabinoid receptor interacting protein 1, septin 9, and vimentin. Receiver operating characteristic (ROC) curve analyses revealed high AUCs for all four markers (0.77-0.87) for CCA detection among patients with PSC. Including only samples from patients with PSC diagnosed with CCA ≤ 12 months following bile collection increased the accuracy for cancer detection, with a combined sensitivity of 100% (28/28) and a specificity of 90% (20/203). The specificity increased to 93% when only including patients with PSC with longtime follow-up (> 36 months) as controls, and remained high (83%) when only including patients with PSC and dysplasia as controls (n = 23). Importantly, the bile samples from the CCA-PSC ≤ 12 patients, all positive for the biomarkers, included both early-stage and late-stage CCA, different tumor growth patterns, anatomical locations, and carbohydrate antigen 19-9 levels.

Conclusions: Using highly sensitive ddPCR to analyze robust epigenetic biomarkers, CCA in PSC was accurately detected in bile, irrespective of clinical and molecular features, up to 12 months before CCA diagnosis. The findings suggest a potential for these biomarkers to complement current detection and screening methods for CCA in patients with PSC.

Conflict of interest statement

Dr. Bergquist received grants from Gilead.

© 2021 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.

Figures

FIGURE 1
FIGURE 1
Flowchart of bile samples included in the study. Number of patients, followed by number of samples in parentheses. Samples are split based on main disease category, including (1) patients with underlying PSC (left panel) and (2) patients with non‐PSC‐related diseases (right panel). PSC samples (left panel) are divided into the following groups: (1) CCA‐PSC (n = 38, 42 samples), including CCA‐PSC ≤ 12 (n = 28, 31 samples; ≤ 12 months from bile sampling to CCA diagnosis) and CCA‐PSC > 12 (n = 10, 11 samples; > 12 months from bile sampling to CCA diagnosis), and (2) all PSC samples (without CCA, n = 205, 272 samples), including PSC‐dysplasia (n = 23, 24 samples; patients with PSC with evidence of biliary dysplasia based on assessment of biliary brush cytology specimens or histological assessment of explant liver ±2 months from bile collection), PSC‐control > 36 (n = 170, 226 samples; patients with PSC with no evidence of CCA or biliary dysplasia based on histological assessment of explanted liver at or after bile collection or > 36 months of follow‐up without established CCA or biliary dysplasia for nontransplanted patients), and PSC‐control < 36 (n = 12, 22 samples; patients with PSC with no evidence of CCA or biliary dysplasia after bile collection but only including nontransplanted patients with < 36 months of follow‐up). Non‐PSC samples (right panel) are divided into (1) CCA (n = 6, 6 samples, of which five were collected at the same time as a confirmed CCA diagnosis and one was collected 3 months prior to diagnosis) and (2) NM liver disease (n = 24, 24 samples; hereditary, idiopathic nonalcoholic fatty liver, biliary stone, and autoimmune liver disease other than PSC)
FIGURE 2
FIGURE 2
ROC curves, calculated AUCs, and sensitivity and specificity values for the four individual DNA methylation biomarkers in bile. (A) Samples from patients with CCA‐PSC (n = 38) versus all PSC (n = 205), (B) samples from patients with PSC diagnosed with CCA ≤ 12 months after bile sampling (CCA‐PSC ≤ 12, n = 28) versus PSC all (n = 205), (C) CCA‐PSC ≤ 12 (n = 28) versus PSC‐control > 36 (n = 170, including samples from patients with PSC showing no evidence of biliary dysplasia or CCA in explanted liver or with > 36 months of follow‐up), and (D) CCA‐PSC ≤ 12 (n = 28) versus PSC‐dysplasia (n = 23, including patients with PSC with evidence of biliary dysplasia based on assessment of biliary brush cytology specimens or histological assessment of explant liver ±2 months from bile collection)
FIGURE 3
FIGURE 3
Comparison of DNA methylation in bile with CA 19‐9 levels and biliary brush cytology. Red circle, sample scored as positive for methylation/cytology/CA 19‐9; white circle, sample scored as negative for methylation/cytology/CA 19‐9. The methylation panel was considered positive if one or more of the four biomarkers were methylated. Biliary brush cytology was scored positive if low/moderate‐grade or high‐grade dysplasia was identified and negative if only normal cells were identified. If more than one brush sample was taken, the case was scored based on the highest grade of dysplasia found. In cases where the sample was scored between two grades of dysplasia, the highest grade was used. For CA 19‐9 levels, 100 U/ml was used as the threshold: a CA 19‐9 level > 100 U/ml was considered positive, while a CA 19‐9 level

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Source: PubMed

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