HepaClear, a blood-based panel combining novel methylated CpG sites and protein markers, for the detection of early-stage hepatocellular carcinoma.

BACKGROUND: Early screening and detection of hepatocellular carcinoma (HCC) can efficiently improve patient prognosis. We aimed to identify a series of hypermethylated DNA markers and develop a blood-based HCC diagnosis panel containing DNA methylation sites and protein markers with improved sensitivity for early-stage HCC detection. RESULTS: Overall, 850K methylation arrays were performed using paired tissue DNA samples from 60 HCC patients. Ten candidate hypermethylated CpG sites were selected for further evaluation by quantitative methylation-specific PCR with 60 pairs of tissue samples. Six methylated CpG sites, along with α-fetoprotein (AFP) and des-gamma-carboxyprothrombin (DCP), were assayed in 150 plasma samples. Finally, an HCC diagnosis panel, named HepaClear, was developed in a cohort consisting of 296 plasma samples and validated in an independent cohort consisting of 198 plasma samples. The HepaClear panel, containing 3 hypermethylated CpG sites (cg14263942, cg12701184, and cg14570307) and 2 protein markers (AFP and DCP), yielded a sensitivity of 82.6% and a specificity of 96.2% in the training set and a sensitivity of 84.7% and a specificity of 92.0% in the validation set. The HepaClear panel had higher sensitivity (72.0%) for early-stage HCC than AFP (≥ 20 ng/mL, 48.0%) and DCP (≥ 40 mAU/mL, 62.0%) and detected 67.5% of AFP-negative HCC patients (AFP ≤ 20 ng/mL). CONCLUSIONS: We developed a multimarker HCC detection panel (HepaClear) that shows high sensitivity for early-stage HCC. The HepaClear panel exhibits high potential for HCC screening and diagnosis from an at-risk population.

Epigenetic Signatures Discriminate Patients With Primary Sclerosing Cholangitis and Ulcerative Colitis From Patients With Ulcerative Colitis.

Background: Primary sclerosing cholangitis (PSC) is a chronic inflammatory liver disease affecting the intra- and extrahepatic bile ducts, and is strongly associated with ulcerative colitis (UC). In this study, we explored the peripheral blood DNA methylome and its immune cell composition in patients with PSC-UC, UC, and healthy controls (HC) with the aim to develop a predictive assay in distinguishing patients with PSC-UC from those with UC alone. Methods: The peripheral blood DNA methylome of male patients with PSC and concomitant UC, UC and HCs was profiled using the Illumina HumanMethylation Infinium EPIC BeadChip (850K) array. Differentially methylated CpG position (DMP) and region (DMR) analyses were performed alongside gradient boosting classification analyses to discern PSC-UC from UC patients. As observed differences in the DNA methylome could be the result of differences in cellular populations, we additionally employed mass cytometry (CyTOF) to characterize the immune cell compositions. Results: Genome wide methylation analysis did not reveal large differences between PSC-UC and UC patients nor HCs. Nonetheless, using gradient boosting we were capable of discerning PSC-UC from UC with an area under the receiver operator curve (AUROC) of 0.80. Four CpG sites annotated to the NINJ2 gene were found to strongly contribute to the predictive performance. While CyTOF analyses corroborated the largely similar blood cell composition among patients with PSC-UC, UC and HC, a higher abundance of myeloid cells was observed in UC compared to PSC-UC patients. Conclusion: DNA methylation enables discerning PSC-UC from UC patients, with a potential for biomarker development.