Applications of molecular barcode sequencing for the detection of low‐frequency variants in circulating tumour DNA from hepatocellular carcinoma

Abstract

Purpose Liquid biopsy has emerged as a promising tool for minimally invasive and accurate detection of various malignancies. We aimed to apply molecular barcode sequencing to circulating tumour DNA (ctDNA) from liquid biopsies of hepatocellular carcinoma (HCC). Study Design Patients with HCC or benign liver disease were enrolled between 2017 and 2018. Matched tissue and serum samples were obtained from these patients. Plasma cell‐free DNA was extracted and subjected to targeted sequencing with ultra‐high coverage and molecular barcoding. Results The study included 143 patients: 102 with HCC, 7 with benign liver tumours and 34 with chronic liver disease. No tier 1/2 or oncogenic mutations were detected in patients with benign liver disease. Among the HCC patients, 49 (48%) had tier 1/2 mutations in at least one gene; detection rates were higher in advanced stages (75%) than in early stages (26%–33%). TERT was the most frequently mutated gene (30%), followed by TP53 (16%), CTNNB1 (14%), ARID2 (5%), ARID1A (4%), NFE2L2 (4%), AXIN1 (3%) and KRAS (1%). Survival among patients with TP53 mutations was significantly worse (p = 0.007) than among patients without these mutations, whereas CTNNB1 and TERT mutations did not affect survival. ctDNA testing combined with α‐fetoprotein and prothrombin induced by vitamin K absence‐II analyses improved HCC detection, even in early stages. Conclusions ctDNA detection using molecular barcoding technology offers dynamic and personalized information concerning tumour biology, such information can guide clinical diagnosis and management. This detection also has the potential as a minimally invasive approach for prognostic stratification and post‐therapeutic monitoring.

HCC biomarkers – state of the old and outlook to future promising biomarkers and their potential in everyday clinical practice

“…The number of patients and the control groups are also large enough in most studies, and international studies are available in addition to many Asian studies. Most studies with ctDNA are in biomarker development phase 1 or 2 ( 57 , 84 , 94 , 95 ). The limitations are that it is difficult to distinguish between ctDNA and circulating free DNA (released from non-malignant cells) and the low concentration of ctDNA in blood.…”

Liquid Liver Biopsy for Disease Diagnosis and Prognosis

“…Furthermore, targeted sequencing of ctDNA can detect tumor-associated mutations with high sensitivity, in cases without prior knowledge of their presence in the tumor tissue, indicating strong application for cancer diagnostics and patient stratification for targeted therapy. 70,71,103 Positive ctDNA detection and gene analysis in HCC patients prior to surgery was also associated with an increased risk of early recurrence and extrahepatic metastasis. 72 Furthermore, continuous assessment of ctDNA could inform on therapy response and disease progression.…”