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Background: Hepatocellular carcinoma (HCC) occurs in a well-defined high-risk patient population, but better screening tests are needed to improve sensitivity and efficacy. Therefore, we investigated the use of urine circulating tumour DNA (ctDNA) as a screening test., Methods: Candidate markers in urine were selected from HCC and controls. We then enrolled 609 patients from five medical centres to test the selected urine panel. A two-stage model was developed to combine AFP and urine panel as a screening test., Results: Mutated TP53, and methylated RASSF1a, and GSTP1 were selected as the urine panel markers. Serum AFP outperformed the urine panel among all cases of HCC, but the urine panel identified 49% of HCC cases with low AFP < 20 ng/ml. Using the two-stage model, the combined AFP and urine panel identified 148 of the 186 HCC cases (79.6% sensitivity at 90% specificity), which was 30% more than the cases detected with serum AFP alone. It also increased early-stage HCC detection from 62% to 92% (BCLC stage 0), and 40% to 77% (BCLC stage A)., Conclusion: Urine ctDNA has promising diagnostic utility in patients in HCC, especially in those with low AFP and can be used as a potential non-invasive HCC screening test., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Hepatocellular carcinoma (HCC) is the world's second leading cause of cancer death; 82.4% of patients die within 5 years. This grim prognosis is the consequence of a lack of effective early detection tools, limited treatment options, and the high frequency of HCC recurrence. Advances in the field of liquid biopsy hold great promise in improving early detection of HCC, advancing patient prognosis, and ultimately increasing the survival rate. In an effort to address the current challenges of HCC screening and management, several studies have identified and evaluated liver-cancer-associated molecular signatures such as genetic alterations, methylation, and noncoding RNA expression in the form of circulating biomarkers in body fluids and circulating tumor cells of HCC patients. In this review, we summarize the recent progress in HCC liquid biopsy, organized by the intended clinical application of the reported study., (Copyright © 2018. Published by Elsevier Inc.)

Hepatocellular carcinoma is one of the fastest growing cancers in the US and has a low survival rate, partly due to difficulties in early detection. Because of HCC's high heterogeneity, it has been suggested that multiple biomarkers would be needed to develop a sensitive HCC screening test. This study applied random forest (RF), a machine learning technique, and proposed two novel models, fixed sequential (FS) and two-step (TS), for comparison with two commonly used statistical techniques, logistic regression (LR) and classification and regression trees (CART), in combining multiple urine DNA biomarkers for HCC screening using biomarker values obtained from 137 HCC and 431 non-HCC (224 hepatitis and 207 cirrhosis) subjects. The sensitivity, specificity, area under the receiver operating curve, and variability were estimated through repeated 10-fold cross-validation to compare the models' performances in accuracy and robustness. We show that RF and TS have higher accuracy and stability; specifically, they reach 90% specificity and 86%/87% sensitivity respectively along with 15% higher sensitivity and 10% higher specificity than LR in cross-validation. The potential of RF and TS to develop a panel of multiple biomarkers and the possibility for self-training, cloud-based models for HCC screening are discussed.

Hepatocellular carcinoma (HCC) is the one of the leading causes of cancer mortality in the world, mainly due to the difficulty of early detection and limited therapeutic options. The implementation of HCC surveillance programs in well-defined, high-risk populations were only able to detect about 40-50% of HCC at curative stages (Barcelona Clinic Liver Cancer stages 0 & 1) due to the low sensitivities of the current screening methods. The advance of sequencing technologies has identified numerous modifications as potential candidate DNA markers for diagnosis/surveillance. Here we aim to provide an overview of the DNA alterations that result in activation of cancer pathways known to potentially drive HCC carcinogenesis and to summarize performance characteristics of each DNA marker in the periphery (blood or urine) for HCC screening.

Hypermethylation of the glutathione S-transferase π 1 (GSTP1) gene promoter region has been reported to be a potential biomarker to distinguish hepatocellular carcinoma (HCC) from other liver diseases. However, reports regarding how specific a marker it is have ranged from 100% to 0%. We hypothesized that, to a large extent, the variation of specificity depends on the location of the CpG sites analyzed. To test this hypothesis, we compared the methylation status of the GSTP1 promoter region of the DNA isolated from HCC, cirrhosis, hepatitis, and normal liver tissues by bisulfite-PCR sequencing. We found that the 5' region of the position -48 nt from the transcription start site of the GSTP1 gene is selectively methylated in HCC, whereas the 3' region is methylated in all liver tissues examined, including normal liver and the HCC tissue. Interestingly, when DNA derived from fetal liver and 11 nonhepatic normal tissue was also examined by bisulfite-PCR sequencing, we found that methylation of the 3' region of the promoter appeared to be liver-specific. A methylation-specific PCR assay targeting the 5' region of the promoter was developed and used to quantify the methylated GSTP1 gene in various diseased liver tissues including HCC. When we used an assay targeting the 3' region, we found that the methylation of the 5'-end of the GSTP1 promoter was significantly more specific than that of the 3'-end (97.1% vs. 60%, p<0.0001 by Fisher's exact test) for distinguishing HCC (n = 120) from hepatitis (n = 35) and cirrhosis (n = 35). Encouragingly, 33.8% of the AFP-negative HCC contained the methylated GSTP1 gene. This study clearly demonstrates the importance of the location of CpG site methylation for HCC specificity and how liver-specific DNA methylation should be considered when an epigenetic DNA marker is studied for detection of HCC.

Hypermethylation of the promoter of the tumor suppressor gene, adenomatous polyposis coli (APC), occurs in various malignancies, including hepatocellular carcinoma (HCC). However, reports on the specificity of the methylation of the APC gene for HCC have varied. To gain insight into how these variations occur, bisulfite PCR sequencing was performed to analyze the methylation status of both sense and antisense strands of the APC gene in samples of HCC tissue, matched adjacent non-HCC liver tissue, hepatitis, cirrhosis, and normal liver tissues. DNA derived from fetal liver and 12 nonhepatic normal tissue was also examined. These experiments revealed liver-specific, antisense strand-biased CpG methylation of the APC gene and suggested that, although methylation of the antisense strand of the APC gene exists in normal liver and other non-HCC disease liver tissue, methylation of the sense strand of the APC gene occurs predominantly in HCC. To determine the effect of the DNA strand on the specificity of the methylated APC gene as a biomarker for HCC detection, quantitative methylation-specific PCR assays for sense and antisense strand DNA were developed and performed on DNA isolated from HCC (n = 58), matched adjacent non-HCC (n = 58), cirrhosis (n = 41), and hepatitis (n = 39). Receiver operating characteristic curves were constructed. With the cutoff value set at the limit of detection, the specificity of sense and antisense strand methylation was 84% and 43%, respectively, and sensitivity was 67.2% and 72.4%, respectively. This result demonstrated that the identity of the methylated DNA strand impacted the specificity of APC for HCC detection. Interestingly, methylation of the sense strand of APC occurred in 40% of HCCs from patients with serum AFP levels less than 20 ng/mL, suggesting a potential role for APC as a biomarker to complement AFP in HCC screening.