Novel urine cell-free DNA methylation markers for hepatocellular carcinoma.

An optimized hepatocellular carcinoma (HCC)-targeted methylation next generation sequencing assay was developed to discover HCC-associated methylation markers directly from urine for HCC screening. Urine cell-free DNA (ucfDNA) isolated from a discovery cohort of 31 non-HCC and 30 HCC was used for biomarker discovery, identifying 29 genes with differentially methylated regions (DMRs). Methylation-specific qPCR (MSqPCR) assays were developed to verify the selected DMRs corresponding to 8 genes (GRASP, CCND2, HOXA9, BMP4, VIM, EMX1, SFRP1, and ECE). Using archived ucfDNA, methylation of GRASP, HOXA9, BMP4, and ECE1, were found to be significantly different (p < 0.05) between HCC and non-HCC patients. The four markers together with previously reported GSTP1 and RASSF1A markers were assessed as a 6-marker panel in an independent training cohort of 87 non-HCC and 78 HCC using logistic regression modeling. AUROC of 0.908 (95% CI, 0.8656-0.9252) was identified for the 6-marker panel with AFP, which was significantly higher than AFP-alone (AUROC 0.841 (95% CI, 0.778-0.904), p = 0.0026). Applying backward selection method, a 4-marker panel was found to exhibit similar performance to the 6-marker panel with AFP having 80% sensitivity compared to 29.5% by AFP-alone at a specificity of 85%. This study supports the potential use of methylated transrenal ucfDNA for HCC screening.

Variability in biopsy quality informs translational research applications in hepatocellular carcinoma.

In the era of precision medicine, biopsies are playing an increasingly central role in cancer research and treatment paradigms; however, patient outcomes and analyses of biopsy quality, as well as impact on downstream clinical and research applications, remain underreported. Herein, we report biopsy safety and quality outcomes for percutaneous core biopsies of hepatocellular carcinoma (HCC) performed as part of a prospective clinical trial. Patients with a clinical diagnosis of HCC were enrolled in a prospective cohort study for the genetic, proteomic, and metabolomic profiling of HCC at two academic medical centers from April 2016 to July 2020. Under image guidance, 18G core biopsies were obtained using coaxial technique at the time of locoregional therapy. The primary outcome was biopsy quality, defined as tumor fraction in the core biopsy. 56 HCC lesions from 50 patients underwent 60 biopsy events with a median of 8 core biopsies per procedure (interquartile range, IQR, 7-10). Malignancy was identified in 45/56 (80.4%, 4 without pathology) biopsy events, including HCC (40/56, 71.4%) and cholangiocarcinoma (CCA) or combined HCC-CCA (5/56, 8.9%). Biopsy quality was highly variable with a median of 40% tumor in each biopsy core (IQR 10-75). Only 43/56 (76.8%) and 23/56 (41.1%) samples met quality thresholds for genomic or metabolomic/proteomic profiling, respectively, requiring expansion of the clinical trial. Overall and major complication rates were 5/60 (8.3%) and 3/60 (5.0%), respectively. Despite uniform biopsy protocol, biopsy quality varied widely with up to 59% of samples to be inadequate for intended purpose. This finding has important consequences for clinical trial design and highlights the need for quality control prior to applications in which the presence of benign cell types may substantially alter findings.