{'fraction_non_alphanumeric': 0.03336823734729494, 'fraction_numerical': 0.01849912739965096, 'mean_word_length': 5.319364799294221, 'pattern_counts': {'":': 0, '<': 2, '<?xml version=': 0, '>': 1, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Prognostic value of postoperative change in liver stiffness in patients with HBV-related hepatocellular carcinoma

Abstract Objective To investigate the prognostic value of change in liver stiffness following surgery, in patients with hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). Methods Patients with HBV-related HCC were included. Preoperative (baseline) liver stiffness and postoperative dynamic change in liver stiffness was evaluated. Results Out of 158 patients in total, postoperative liver stiffness was increased in 98 patients and decreased in 60 patients compared with baseline values. Kaplan-Meier analysis revealed that patients with elevated liver stiffness had significantly worse overall survival outcomes than those with decreased liver stiffness. Similar trends were observed for diseases-free survival and recurrence outcomes. Multivariate analyses showed that Child–Turcotte–Pugh score (hazard ratio [HR] 1.209) and liver stiffness changes (HR 1.891) were independent factors associated with overall survival. Liver stiffness changes (HR 1.521) and α-fetoprotein level (HR 1.210) were found to be independent factors for disease-free survival in patients with HCC. Conclusion Increased postoperative liver stiffness may be an independent risk factor of HCC prognosis. Patients with increased liver stiffness following surgery should undergo additional examinations during follow-up.


Introduction
Due to the prevalence of hepatitis B virus (HBV) and alcohol consumption, hepatocellular carcinoma (HCC) has become one of the most common cancers, with an increasing incidence worldwide, particularly in China. 1,2 Ablation and liver transplantation are effective in treating HCC, however, surgical resection remains the most important therapy, and is conducted in most patients with early stage HCC. [3][4][5] Despite the effectiveness of these treatments, recurrence and metastasis are still major problems for longterm survival in patients with HCC. [6][7][8] Chronic HBV infection is well-known to be associated with the development of cirrhosis and HCC, resulting in hepatic disease-related deaths. [9][10][11] The incidence of HCC is reported to be 0.5% in patients infected with HBV, and ranges between 2.5% and 6% in patients with cirrhosis. 6,[12][13][14] In Asia, over 60% of patients with HCC have an aetiology of chronic HBV infection. [15][16][17] Liver stiffness, measured by transient elastography, has proven to be an effective non-invasive procedure for evaluating liver fibrosis and assessing portal vein hypertension. [18][19][20][21] Liver stiffness can be measured using a FibroScan TM device (Echosens, Paris, France) that detects the propagation speed of an elastic sheer wave triggered by a transducer, which is then converted to a measurement of liver stiffness. 22 High levels of liver stiffness have been reported to be associated with the risk of HCC occurrence. 23,24 To the best of the authors' knowledge, there is no published study to date that evaluates the prognostic effectiveness of liver stiffness measurements in patients with HCC, and thus, the prognostic value of liver stiffness in patients with HCC remains a clinical issue that requires exploration. The aim of the present study was to assess the prognostic value of change in liver stiffness from preoperative values, within one year following hepatic resection in patients with HBV-related HCC.

Transient elastography
All patients underwent liver stiffness measurements by transient elastography on at least two occasions: one prior to surgery (baseline) and the other within 1 year following liver resection surgery. Transient elastography was conducted by Fibroscan TM (Echosens) as previously described. 25 Results were expressed as kPa and the median value of 10 successful measurements in each patient was used as the liver stiffness value.

Diagnosis and anti-virus treatment
Fibrosis and HCC were diagnosed by histopathology of tissue specimens acquired during liver surgery. The maximum tumour diameter was calculated with images obtained during preoperative contrast-enhanced computed tomography (CT) using a GE LightSpeed VCT system (GE Healthcare, Tokyo, Japan). HBV infection was diagnosed with a positive serum viral marker and/or elevated serum HBV-DNA level (>1000 copies/ml during two consecutive detections). Briefly, serum HBV DNA was detected using a Daan test (Daan Gene Co., Ltd., Guangdong, China) according to the manufacturer's instructions. DNA was extracted from 100 ll of serum and detected by real-time polymerase chain reaction (PCR) with HBV-DNA specific fluorescence probing, using a Roche LightCycler 480 system (Roche Diagnostics Ltd., Rotkreuz, Switzerland). HCC recurrence during follow-up was screened by contrast-enhanced CT and ultrasonography (GE Volusion TM E10 ultrasound system; GE Healthcare, Shanghai, China). Blood laboratory parameters were obtained for each patient, and Child-Turcotte-Pugh score classification was applied for consideration of prognosis, as previously described. 25,26 All patients received anti-HBV regimens (entecavir) for at least 6 months prior to surgery and after surgery, as previously described. 27,28

Study outcomes
The primary outcome of this study was change in liver stiffness following hepatic resection, from preoperative (baseline) value, as a prognostic marker in patients with HBVrelated HCC. Secondary outcomes included independent factors associated with prognosis following hepatic resection among patients with HBV-related HCC.

Statistical analyses
Data are presented as n (%) prevalence, median (range) or mean AE SD. Student's t-test and Wilcoxon signed-rank test was used to analyse continuous variables with or without normal distribution, respectively. Categorical variables were analysed using v 2 -test and Fisher's exact test. Survival was assessed using Kaplan-Meier estimator and Log-rank test, as appropriate. Multivariate analysis was performed using Cox proportional hazards model. All statistical analyses were conducted using SPSS software, version 16.0 (SPSS Inc., Chicago, IL, USA) and a P value < 0.05 was regarded as statistically significant.

Baseline patient characteristics
Out of 186 patients initially enrolled, a total of 158 patients were included in the final analyses ( Figure 1; Table 1). Male patients were predominant (n ¼ 123 [77.85%]), and median age was 50 years (range, 22-74 years).
More than half of the patients were confirmed by pathology to have fibrosis (67.72%). Most patients were Child-Turcotte-Pugh class A (n ¼ 147 [93.04%]), and all patients received anti-virus therapy prior to hepatic resection. The median serum HBV DNA level was 4.1 log copies/ ml. A total of 102 patients (64.56%) had serum HBV DNA level <3 log copies/ml (the lower limit of detection) prior to surgery, and 56 patients (35.44%) were serum HBV DNA positive. The median maximum tumour diameter was 4.1 (range, 3.0-5.3) cm, and median liver stiffness was 6.7 (range, 4.3-12.5) kPa (Table 1).

Demographic and clinical characteristics in patients with increased or decreased postoperative liver stiffness
All patients received liver stiffness assessments on at least two occasions.
Mean baseline liver stiffness was 8.53 AE 2.41 Kpa and was 9.26 AE 2.29 Kpa after surgery. Compared with baseline values prior to surgery, 98 patients (62.03%) experienced increased liver stiffness (mean change, 3.8 AE 1.3 kPa) and 60 patients (37.97%) had decreased liver stiffness (mean change, 2.4 AE 1.1 kPa) within 12 months following surgery (dynamic changes are shown in Figure 2). In patients categorised according to postoperative increase or decrease in liver stiffness, there was a statistically significant between-group difference in platelet count only (P ¼ 0.04; Table 2).
Prognostic performance of liver stiffness changes in patients with HCC Patient outcomes were analysed using Kaplan-Meier estimator in patients categorised according to postoperative increase or decrease in liver stiffness, in order to investigate the potential difference in prognosis between the two groups. Patients with increased postoperative liver stiffness were revealed to have significantly worse overall survival outcomes than those with decreased postoperative liver stiffness (P ¼ 0.042). Similar trends were observed for diseases-free survival and recurrence outcomes, which showed that patients with increase postoperative liver stiffness had significantly worse outcomes than those with decreased liver stiffness (P ¼ 0.045 for disease-free survival and P ¼ 0.041 for recurrence outcomes; Figure 3).

Univariate and multivariate analyses of prognostic variables in patients with HCC
The potential correlations between clinical parameters and overall survival, diseasefree survival and recurrence were analysed using Cox proportional hazards model in patients with HCC. Univariate analyses showed that age, Child-Turcotte-Pugh score and postoperative change in liver stiffness were all prognostic variables for overall survival. Multivariate analysis revealed that only Child-Turcotte-Pugh score (hazard ratio [HR] 1.209, P ¼ 0.039) and liver stiffness changes (HR 1.891, P ¼ 0.042) were independent prognostic variables that were associated with overall survival (Table 3).
To evaluate whether postoperative change in liver stiffness was an independent risk factor for disease-free survival and recurrence outcomes, both univariate and multivariate analyses were conducted (Table 4 and Table 5). Multivariate analyses showed that a-fetoprotein (AFP) level

Discussion
The present study demonstrated that increased postoperative liver stiffness may be a potential risk factor for poor HCC outcomes in patients with HBV-related HCC.
The study showed that patients with decreased postoperative liver stiffness values had better survival outcomes than patients with elevated liver stiffness values. In addition, Child-Turcotte-Pugh score and liver stiffness changes were found to be independent variables associated with overall survival. AFP level and liver stiffness changes were found to be independent variables associated with HCC disease-free survival while serum HBV DNA viral load, AFP level and liver stiffness changes were independent variables associated with HCC recurrence. As such, these factors may have prognostic value in patients with HBVrelated HCC.
Although liver histology is useful regarding diagnosis of hepatic diseases, such as cirrhosis, the invasive nature of the procedure limits its wider use in clinical practice. 29,30 FibroScan TM (Echosens) has been routinely applied for liver diseases detection worldwide, and allows clinicians to quantitatively evaluate the status of the liver. 31 As a non-invasive procedure, FibroScan TM is also suitable for patients following liver resections.
Previous advances have  indicated that elevated liver stiffness is associated with HCC, but the mechanisms for liver regeneration after surgery are complicated in patients with underlying HBV infection. 32 Postoperative screening with transient elastography provides a reproducible method of quantification to assess the alteration of liver regeneration, as well as potential impact on the prognosis of patients with HCC. 33 The present study found a correlation between dynamic changes of liver stiffness and prognosis in patients with HBV-related HCC, which may be utilized as a dynamic approach to  monitor HCC or evaluate the recovery of patients. Several markers have been identified as effective in evaluating prognosis in patients with HCC. [34][35][36] For example, AFP has been widely reported as a prognostic marker in patients with HCC, [37][38][39] and is a serum HCC marker that is used to diagnosis and monitor HCC progression. However, AFP levels are not only elevated in HCC. Other diseases, such as those involving tumours of the female reproductive system, may also manifest with elevated AFP levels. 40,41 Although high postoperative AFP levels are associated with poorer HCC prognosis, there is a certain proportion of patients with HCC who are AFP negative. 42 For those AFP-negative patients with HCC, AFP is not suitable as a prognostic marker. According to the present study, patients with HCC with abnormal or normal AFP levels may instead be effectively stratified using liver stiffness measured by transient elastography.
The present study results may be limited by several factors. The relatively small sample size may limit the generalizability of the results, and the retrospective study design may have biased the results. In the present study, the antiviral treatment was determined by clinical experts at Yantai Infectious Diseases Hospital, liver stiffness was measured at Jining No.1 People's Hospital, and HCC diagnosis and surgery were performed at Heze Municipal Hospital. In addition, patients included in the present study received various different treatments following surgery, for example, some received no further treatment following radical resection, while some were treated with sorafenib, and these factors were not included in the present analyses. All of these additional variables may have affected the present results, and whether or not postoperative treatment may further improve the prognosis of patients with HCC remains unclear. Data were collected and analysed at a single centre. Larger multicentre prospective studies are required to validate the present results and further investigate the value of liver stiffness as a prognostic marker in patients with HCC.
In conclusion, the present study demonstrated an association between dynamic change in liver stiffness and prognosis in patients with HCC. Liver stiffness measurement with transient elastography may allow the evaluation of outcomes in patients with HCC, and thus, may be used to guide routine follow-up for these patients. In particular, patients with HCC with increased liver stiffness following surgery should undergo more frequent follow-up examinations, even if the most recent exam following liver resection indicates no cause for concern.

Declaration of conflicting interest
The authors declare that there is no conflict of interest.

Funding
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.