Hepatitis B virus pre-S2 deletion (nucleotide 1 to 54) in plasma predicts recurrence of hepatocellular carcinoma after curative surgical resection

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. Despite curative surgical resection, high recurrence of HCC after surgery results in poor patient survival. To develop prognostic markers is therefore important for better prevention and therapy of recurrent HCC to improve patient outcomes. Deletion mutations over the pre-S1 and pre-S2 gene segments of hepatitis B virus (HBV) have been closely associated with recurrence of HCC after curative surgical resection. In this study, we applied a next-generation sequencing-based approach to further evaluate the association of pre-S deletion regions with HCC recurrence. We demonstrated that the pre-S2 deletion (nucleotide 1 to 54) was the most predominant deletion regions of pre-S gene in plasma of HBV-related HCC patients. Moreover, patients with the pre-S2 deletion (nucleotide 1 to 54) exhibited a significantly higher risk of HCC recurrence after curative surgical resection than those without. The pre-S2 deletion (nucleotide 1 to 54) in plasma represented a prognostic factor that independently predicted HCC recurrence with greater performance than other clinicopathological and viral factors. Our data suggest that detection of the pre-S2 deletion (nucleotide 1 to 54) in plasma may be a promising noninvasive strategy for identifying patients at high risk for HCC recurrence after curative surgical resection.


Introduction
Among the most common and deadly human cancers worldwide, hepatocellular carcinoma (HCC) accounts for at least 700,000 deaths annually [1][2][3]. Although curative surgical resection of HCC is available [4][5][6], the 5-year recurrence rate of HCC after surgery remains up to 80%, resulting in poor patient survival [7][8][9]. Therefore, development of biomarkers for predicting HCC recurrence risk after surgical resection is important in allowing for early prevention and timely treatment of the recurrent HCC to ameliorate patient outcomes.
Chronic infection with hepatitis B virus (HBV) is intimately associated with the development of HCC, responsible for as high as 50% of HCC cases in the world [10][11][12]. In chronic HBV infection, ground glass hepatocytes (GGHs) in liver tissues represent preneoplastic lesions of HCC [13]. Two types of GGHs (designated type I and II) are identified to consistently express the pre-S1 and pre-S2 mutant proteins, respectively, which contain deletion mutations in the pre-S1 and pre-S2 gene segments of HBV large surface proteins [14,15]. Both types of pre-S mutants have been well demonstrated to dysregulate various oncogenic signaling pathways, leading to tumorigenesis of hepatocytes and eventually HCC development [13,[16][17][18]. Chronic HBV-infected patients and HBV-related HCC patients who harbor pre-S mutants exhibit significantly higher incidences of HCC development [19][20][21] and recurrence after curative surgical resection [22][23][24][25][26][27], respectively. As a result, the presence of pre-S mutants is proposed as a valuable biomarker for the prognosis of HBV-related HCC.
Several approaches have been applied to qualitatively and semi-quantitatively detect pre-S mutants in blood and liver tissue specimens of HBV-related patients, including the approaches utilizing immunohistochemistry staining [15,22], polymerase chain reaction (PCR) [19,20,28], and the Pre-S Gene Chip [21,24]. Recently, we have developed a new approach based on next-generation sequencing (NGS) for quantitative detection of pre-S mutants from patient plasma with superior sensitivity, efficiency, and fidelity [29,30]. In this study, we further evaluated the association between deletion regions of pre-S mutants and HCC recurrence in a cohort of 75 HBV-related HCC patients receiving curative surgical resection.

Patient specimen collection
In this study, the HCC patients who had HBV infection and received curative surgical resection were included; the HCC patients who had infection with other types of hepatitis viruses and did not receive resection surgery were excluded. The plasma samples were collected from 75 HBV-related HCC patients who underwent surgery at the China Medical University Hospital (Taichung, Taiwan (Invitrogen, Carlsbad, CA, USA) together with specific primer pairs [first round: forward, 5'-GCGGGTCACCATATTCTTGGG-3' (corresponding to nucleotide (nt) 2818 to 2837 in HBV genome sequence) and reverse, 5'-GAGTCTAGACTCTGCGGTAT-3' (corresponding to nt 236 to 255 in HBV genome sequence); second round: forward, 5'-GCGGGTCACCAT ATTCTTGGG-3' (corresponding to nt 2818 to 2837 in HBV genome sequence) and reverse, 5'-TAACACGAGCAGGGGTCCTA-3' (corresponding to nt 180 to 199 in HBV genome sequence)] following the program [stage 1: 95˚C for 1 minute; stage 2 (35 cycles): 95˚C for 1 minute, 55˚C for 1 minute, and 72˚C for 1 minute; stage 3: 72˚C for 7 minutes]. Next, the PCR products of pre-S gene (approximately ranging from 350 to 600 base pairs in size) were analyzed by NGS on the NextSeq 500 system (Illumina, San Diego, CA, USA) according to the manufacturer's instructions in the Department of Laboratory Medicine at the China Medical University Hospital. All of the sequence reads were compared with the master sequences from the reference sets of HBV genotypes, which were available on the NCBI website (https://www. ncbi.nlm.nih.gov/projects/genotyping/view.cgi?db=2), by using BLAST. The deletion types, regions, and percentages of pre-S gene were determined by using our customized scripts. Finally, the pre-S deletion regions with the highest percentage in each type were identified for statistical analysis. S1 Table summarized the pre-S genotyping results of all patients.

Statistical analysis
The univariate and multivariate analyses of pre-S deletions for overall (OS) and recurrencefree survival (RFS) were conducted by the Cox proportional-hazards regression model. The OS and RFS curves were analyzed by the Kaplan-Meier method and the log-rank test. The receiver operating characteristic (ROC) curves of pre-S deletions were established for discriminating patients with HCC recurrence from those without and the area under the ROC curves (AUCs) were determined and compared by the Hanley-McNeil test. All analyses were performed using SAS version 9.4 (SAS Institute Inc., Cary, NC, USA) [31]. A P value < 0.05 indicated a statistically significant difference.

Patients with the pre-S2 deletion (nt 1 to 54) in plasma as a population at high risk for HCC recurrence after curative surgical resection
The deletion regions of pre-S gene whose percentages in patient plasma ranked among the top in each type were selected for clinicopathological analysis, including the pre-S1 deletion (nt 2854 to 2970), the pre-S1 deletion (nt 2855 to 2872), the pre-S2 deletion (nt 1 to 54), and the  The HBV surface gene is composed of the pre-S1, pre-S2, and S gene segments. The arrow above the diagram indicates the start site (nt 1) of the circular HBV genome that goes clockwise and ends at nt 3221 (not shown). The nt numbers below the diagram indicate the positions of three gene segments in the HBV genome. The grey boxes represent the deletion regions detected in the pre-S1 and pre-S2 gene segments. Here only the deletion regions found in at least 2 of 75 patients were shown and designated in order from the top to bottom as follows: the pre-S1 deletion (nt 2854 to 2970), the pre-S1 deletion (nt 2854 to 3021), the pre-S1 deletion (nt 2855 to 2872), the pre-S1 deletion (nt 2858 to 2986), the pre-S1 deletion (nt 2910 to 3089), the pre-S2 deletion (nt 1 to 54), the pre-S2 deletion (nt 1 to 57), and the pre-S1+pre-S2 deletion (nt 2855 to 2872, 1 to 54). The frequency of these pre-S deletion regions in 75 HCC patients as well as the number of patients with HBV genotype B or C for each type of pre-S deletion region were shown on the right half of the figure. Abbreviations: HCC, hepatocellular carcinoma. pre-S1+pre-S2 deletion (nt 2855 to 2872, 1 to 54). Among these deletion regions, only the pre-S2 deletion (nt 1 to 54) had a significantly positive correlation with HCC recurrence (P value = 0.0080) ( Table 3). Moreover, the pre-S2 deletion (nt 1 to 54), along with the Child-Pugh cirrhosis score and AJCC TNM stage, were significantly and independently associated with poor RFS in patients (pre-S2 deletion (nt 1 to 54), hazard ratio (HR) = 2.392, 95% confidence interval (CI) 1.297 to 4.410, P value = 0.0052; Child-Pugh cirrhosis score, HR = 2.065, 95% CI 1.086 to 3.927, P value = 0.0271; AJCC TNM stage, HR = 3.411, 95% CI 1.710 to 6.804, P value = 0.0005) ( Table 4). Patients with the pre-S2 deletion (nt 1 to 54), Child-Pugh cirrhosis score (B/C), or AJCC TNM stage (IIIA/IIIB/IIIC/IVA/IVB) had a significantly shorter median RFS than those without (pre-S2 deletion (nt 1 to 54), 7.7 vs. 31.7 months, P value = 0.0283; Child-Pugh cirrhosis score, 5.1 vs. 15.1 months, P value = 0.0093; AJCC TNM stage, 5.0 vs.

Discussion
Although curative surgical resection is available for treating HCC patients, high recurrence rate of HCC after surgery is still a big threat, causing poor patient outcomes [32][33][34]. Patients carrying HBV pre-S mutants, which contain deletions over the pre-S1 and pre-S2 regions, have been demonstrated to be at high risk for HCC recurrence after curative surgical resection [22][23][24][25]. In this study, we further evaluated the association of pre-S deletion regions in plasma with HCC recurrence and identified the pre-S2 deletion (nt 1 to 54) as an independent prognostic biomarker for HCC recurrence with greater performance than other clinicopathological and viral factors.
Several reports have examined the deletion incidences and patterns of HBV pre-S gene in patients with different stages of chronic HBV infection-related liver diseases, including chronic hepatitis, liver cirrhosis, and HCC. The incidences of overall pre-S deletions (including the pre-S1 and pre-S2 deletions) are gradually increased from chronic hepatitis patients to liver cirrhosis patients and eventually reach a peak in HCC patients [13,21,28,[35][36][37]. The pre-S1 deletion is most frequently detected in liver cirrhosis patients, while the pre-S2 deletion is most frequently detected in HCC patients [35][36][37]. The distributions of the pre-S1 deletion regions nearly cover the entire pre-S1 gene segment with the highest prevalence in the former region, but the distributions of the pre-S2 deletion regions predominantly fall in the former region of pre-S2 gene segment [28,[35][36][37]. Moreover, patients with pre-S deletions show a significantly higher risk of developing liver cirrhosis and HCC than those without [19][20][21]. Consistent with these findings, in this study the pre-S genotyping results from plasma of HBVrelated HCC patients displayed similar distribution patterns of pre-S deletion regions, among which the pre-S2 deletion (nt 1 to 54) had the highest incidence. Furthermore, we provided evidence supporting that patients with the pre-S2 deletion (nt 1 to 54) represented a population at high risk of HCC recurrence after curative surgical resection. Considering that the pre-S2 region (nt 1 to 54) coincides with the B-and T-cell epitopes of HBV large surface proteins [28,[38][39][40], the pre-S2 mutant proteins harboring the pre-S2 deletion (nt 1 to 54) may emerge as an immune escape mutant that may possibly explain their high incidence in HCC and high association with HCC recurrence after curative surgical resection.
The clinical correlation between HBV pre-S deletions and HCC recurrence after curative surgical resection has been well documented [41]. Both the presence and higher percentage of pre-S deletion mutations in liver tissues and serum/plasma of HCC patients have been independently associated with a higher risk of HCC recurrence after resection surgery [22][23][24][25][26][27]. However, before this study, the association of specific pre-S deletion regions with HCC recurrence remains poorly defined. In this study, we for the first time identified that the presence of the pre-S2 deletion (nt 1 to 54) in plasma of HCC patients was an independent biomarker for prediction of HCC recurrence after curative surgical resection. Our finding may potentially support the development of approaches toward focusing on detection of specific pre-S deletion regions, thus facilitating the clinical application of pre-S deletions as biomarkers in prediction of HCC recurrence. Although a previous report by Jia et al has shown that deletions in the pre-S gene display consistent patterns and incidences between the matched serum and liver tissues in HBV-related HCC patients [37], whether the pre-S2 deletion (nt 1 to 54) may be also detected in liver tissues of HCC patients to predict the recurrence of HCC after resection surgery still needs to be investigated. In addition, although the clinicopathological characteristics of the cohort of 75 HCC patients analyzed in this study coincided with the representative features of a large population of HCC patients in Taiwan [42], a large cohort of patients from different clinical centers are needed to further validate this finding in clinical practice.

Conclusions
Our results demonstrated that patients with the HBV pre-S2 deletion (nt 1-54) in plasma had a higher risk of HCC recurrence than those without after curative surgical resection. Detection of the pre-S2 deletion (nt 1-54) in plasma may be a promising noninvasive strategy for identifying patients at high risk of postoperative HCC recurrence, allowing them for early preventive and timely therapeutic managements for better survival.