A Next-Generation Sequencing-Based Platform for Quantitative Detection of Hepatitis B Virus Pre-S Mutants in Plasma of Hepatocellular Carcinoma Patients

Abstract

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. Early diagnosis and treatment of HCC remain a key goal for improving patient survival. Chronic hepatitis B virus (HBV) infection is a major risk factor for HCC development. Pre-S mutants harboring deletions in HBV large surface antigen have been well demonstrated as HBV oncoproteins that dysregulate multiple signaling pathways in hepatocytes, leading to HCC formation. The presence of pre-S mutants in plasma represents important predictive and prognostic markers for HCC in patients with chronic HBV infection. However, the method to detect pre-S mutants remains to be optimized. In this study, we developed a platform, based on the next-generation sequencing (NGS) technology, for detection of pre-S mutants in plasma of HBV-related HCC patients. Compared to the current TA cloning-based analysis, the NGS-based analysis could detect pre-S deletion quantitatively, and the detection rate was significantly more sensitive in 49 plasma analyzed (McNemar’s paired proportion test, P value < 0.0001; simple kappa coefficient, κ = 0.29 (95% CI, 0.12 to 0.46)). Our data suggest that the NGS-based platform may hold a promise for improving the clinical application of pre-S mutants in serving as predictive and prognostic markers for HBV-related HCC.

Detection of hepatitis B virus pre-S mutants in plasma by a next-generation sequencing-based platform determines their patterns in liver tissues

“…The presence of pre-S mutants in liver tissues or blood has emerged as a valuable biomarker for HBV-related HCC [19][20][21][22][23]. Recently, we have developed a NGS-based platform for quantitative detection of pre-S mutants in patient plasma with high sensitivity and accuracy using easy and relatively less invasive methods [24]. In this study, we further demonstrated that the NGS-based pre-S genotyping in plasma could also efficiently determine the patterns of pre-S mutants in liver tissues in HBV-related HCC patients.…”

“…However, these two methods provide only qualitative and semi-quantitative results and remain to be optimized in practical operation. The TA cloning-based analysis is mainly dependent on cloning and sequencing of pre-S gene PCR bands that are clearly separated and visualized in agarose gel; the unseparated or invisible PCR bands may be omitted during the procedure, thus leading to lower detection sensitivity [24]. Also, the cloning procedure is somewhat time-consuming.…”

“…The NGS-based detection of pre-S mutants was performed as previously described [24]. Briefly, DNA isolated from plasma specimens was used as template for PCR amplification of the pre-S gene.…”

“…Two methods have been commonly used to detect the presence of pre-S mutants in chronic HBV carriers and HBV-related HCC patients, one based on immunohistochemistry (IHC) staining of HBV surface antigen (HBsAg) for GGH visualization in liver tissues [14,21,22], and another TA cloning following polymerase chain reaction (PCR) amplification of pre-S gene for DNA sequencing in serum/plasma samples [13,19,20]. Unlike these two methods providing only qualitative and semi-quantitative results, we have recently developed a nextgeneration sequencing (NGS)-based platform for quantitative detection of pre-S mutants in patient plasma [24]. In contrast to the TA cloning-based method, the NGS-based platform can detect the presence of pre-S mutants (not only types but also levels) in patient plasma with higher sensitivity and accuracy [24].…”

“…Unlike these two methods providing only qualitative and semi-quantitative results, we have recently developed a nextgeneration sequencing (NGS)-based platform for quantitative detection of pre-S mutants in patient plasma [24]. In contrast to the TA cloning-based method, the NGS-based platform can detect the presence of pre-S mutants (not only types but also levels) in patient plasma with higher sensitivity and accuracy [24]. However, it remains unclear whether the pre-S genotyping results by the NGS-based platform from the plasma of patients may correspond with those by the IHC-based method from the liver tissues.…”