Liver stiffness measured by acoustic radiation force impulse elastography predicted prognoses of hepatocellular carcinoma after radiofrequency ablation

The prognostic factors of patients who undergo radiofrequency ablation (RFA) for hepatocellular carcinoma (HCC) is not fully elucidated. We aimed to investigate the role of liver stiffness (LS) and spleen stiffness (SS) measured by acoustic radiation force impulse (ARFI) elastography in determining the prognoses of patients with HCC after RFA. We prospectively enrolled 173 patients with HCC who underwent ARFI elastography for measurement of LS and SS on the same day of RFA. Overall survival (OS), recurrence-free survival (RFS) after adjusting for competing mortality, and presence of hepatic decompensation were investigated. Patients with LS > 1.5 m/s had significantly shorter OS and RFS than their counterparts. Anti-viral treatment (hazard ratio [HR]: 0.396, p = 0.015) and LS > 1.5 m/s (HR 4.105, p = 0.028) correlated with OS by a multivariate analysis. Besides, serum alpha fetoprotein >10 ng/mL and LS > 1.5 m/s independently predicted poorer RFS. On the other hand, anti-viral treatment (HR: 0.315, p = 0.010), creatinine > 1.5 mg/dL (HR: 9.447, p = 0.006), and SS > 2.7 m/s (HR: 2.869, p = 0.044) predicted a higher risk of hepatic decompensation. In conclusion, LS but not SS measured by ARFI elastography predicted tumor recurrence and OS in RFA-treated HCC; whereas, SS predicted development of hepatic decompensation in these patients.


Development of hepatic decompensation in patients with
In the multivariate analysis that including dimensional ARFI values of LS and SS (model 1), antiviral treatment, serum levels of creatinine and ALT, and ARFI velocity value for SS were independent predictors to hepatic decompensation. In the model 2 which included dichotomous ARFI values, anti-viral treatment, serum creatinine > 1.5 mg/dL, and SS > 2.7 m/s independently predict hepatic decompensation (Table 5).

Discussion
This study investigated the role of LS and SS measured by ARFI velocity in predicting the patients' outcomes after RFA. It showed that higher LS values measured by ARFI velocity could be a significant predictor of both HCC recurrence and OS in these patients, but no significant role of SS could be identified in the evaluation of post-RFA outcomes. As patients who receive RFA for HCC usually have more advanced chronic liver disease, or  www.nature.com/scientificreports www.nature.com/scientificreports/ more severe portal hypertension than others who undergo surgical resection. By using more reliable tools for stiffness measurement, such as ARFI elastography, our findings could be applied in clinical practice to optimize the follow-up program for patients with higher risks of recurrence or mortality after RFA treatment.
For patients with early-stage HCC, RFA could provide acceptable long-term OS rates that are comparable or only slightly inferior to that of surgical resection, but the recurrence rates after RFA are still high 23,24 . Our previous study showed that the cumulative 10-year OS and RFS rates after RFA were 48.7% and 12.4%, respectively 23 . To improve the outcomes of patients, it is crucial to elucidate the mechanism and identify the risk factors of tumor recurrence after RFA. Identified predictors of HCC recurrence after curative therapies include tumor factors  www.nature.com/scientificreports www.nature.com/scientificreports/ (including tumor size, number, tumor cell differentiation, vascular invasion, extra-hepatic metastasis, and serum AFP level), liver functional reserve (such as serum albumin level, platelet count, and portal hypertension), and field factors in the background liver (including the grade of hepatic inflammation and steatosis and the stage of liver fibrosis) 12,18,[25][26][27][28][29] .
As the tumor factors might be less apparent in determining the outcomes of patients with early-stage HCC, field factors may play a more important role in tumor recurrence after curative treatments for such patients. To date, only one study from Korea has proposed that the ARFI velocity value of LS assessed at the time of RFA can independently predict the risk of HCC recurrence after treatment 12 . However, that study recruited a relatively small number of patients (n = 120) and could not find a predictor of survival benefit.
In this study, we enrolled 173 HCC patients and confirmed the ability of ARFI elastography to predict not only tumor recurrence but also OS after RFA. The optimal cut-off value of ARFI velocity to predict HCC recurrence was 1.6 m/s in the Korean population 12 . In our cohort, the optimal cut-off value was 1.5 m/s, which could be used to predict both tumor recurrence and OS after RFA effectively. This corresponds to a previous study, in which the cut-off value of ARFI velocity to predict cirrhosis was 1.52 m/s 30 . Thus, the optimal cut-off value in our study seems reasonable because liver cirrhosis is confirmed as an important risk factor for HCC recurrence and mortality after treatment 8 .
In our cohort, patients with significant liver fibrosis measured by ARFI velocity (>1.5 m/s) had poorer liver functional reserve and lower rates of OS and RFS compared to those with non-significant liver fibrosis. The differences of survival benefit and tumor recurrence were still prominent according to LS even after matching for tumor factors and liver reserve. For such patients, improving the prognosis might require following-up patients more closely, arranging salvage liver transplantation in cases of tumor recurrence or liver decompensation, or prescribing adjuvant therapy (such as molecular target therapy or immune check point inhibitors) after RFA. More prospective studies are needed to validate this concept.
Patients who had non-significant liver fibrosis had 5-year RFS rates of only 35.6%, but the long-term outcome was excellent with a 5-year OS rate of 89.5%. This might be due to the patients undergoing a strict surveillance program to detect recurrence after RFA. They could still undergo curative treatment modalities when HCC recurs due to the well-preserved liver function and early tumor stage. This indicates that liver functional reserve and surveillance programs for tumor recurrence are crucial in determining the long-term outcomes of HCC patients who have undergone RFA.
During the long-term follow-up after RFA for HCC, significantly negative effects on OS were found for age, prothrombin activity, advanced CTP grade, tumor size, tumor number, serum level of AFP, and the presence of porto-systemic collateral vessels 23,24,[31][32][33] . LS measured by two-dimensional shear-wave elastography has been reported as a significant predictive factor for OS after RFA for HCC 29 . However, in other studies in Korea, LS measured by TE or ARFI elastography did not have effective prediction performance for OS in HCC patients who received RFA 12,18 . In our study, we identified that an ARFI velocity cut-off value of 1.5 m/s for LS could significantly predict the OS of patients who underwent RFA for HCC. Moreover, patients with significant liver fibrosis measured by ARFI velocity had a relatively poorer liver function as well as higher aminotransferases level and blood neutrophil counts compared to their counterparts. These findings suggested that LS may not only indicate hepatic fibrosis but also correlated with hepatic and systemic inflammation that were closely associated with tumor outcomes 34,35 . On the other hand, antiviral therapy was associated with a lower risk of mortality after RFA, which is consistent with previous studies reporting that ongoing viral replication and antiviral therapy could be used to determine the prognoses of patients with early-stage HCC who underwent RFA 36,37 .
SS has been investigated as a non-invasive marker of portal hypertension. It has a close correlation with the hepatic venous pressure gradient and has been identified as a predictor of cirrhotic complications [19][20][21][22] . According to a previous study, cirrhotic patients with an ARFI velocity value for SS less than 3.25 m/s had a 98.8% probability of not developing hepatic decompensation, and patients with a value greater than 3.43 m/s had a 75.8% probability of mortality 38 . On the other hand, SS measured by TE was suggested to be a predictor of late HCC recurrence at 24 months after liver resection in a recent Italian study 10 . However, the role of SS measured by ARFI velocity in determining the outcomes of HCC after RFA has not been well investigated before. In this study, we identified that SS > 2.7 m/s was associated with a significantly higher risk of hepatic decompensation in HCC patients after RFA treatment even with good liver reserves (Child-Pugh A). However, the status of SS failed to predict survival benefit or tumor recurrence after RFA treatment for HCC even in patients with clinically significant portal hypertension.
To the best of our knowledge, this study is the largest one to compare LS and SS measured by ARFI in determining the prognosis of HCC patients after RFA. Although there was a modest positive correlation between LS and SS measured by ARFI velocity, we could not identify a role of SS in predicting the risk of recurrence or mortality after RFA, even when judged by different cut-off values with increments of 0.1 from 3.0 to 3.5 m/s. Early tumor staging, well-preserved liver function and less significant portal hypertension in our patients may account for such findings. Liver stiffness is a composite of hepatic fibrosis, inflammation, portal pressure, and other factors, while spleen stiffness might more directly reflect portal pressure only. Since portal hypertension is not the only factors associated with hepatic decompensation and mortality, liver stiffness may harbor incremental prognostic information as compared to spleen stiffness 35,39 .
There are some limitations to this study. First, the number of patients was relatively small, and the impact of LS and SS on the recurrence patterns (early or late recurrence) could not be assessed. Even though this is the largest cohort study to investigate the role of LS or SS measured by ARFI elastography in HCC patients who received RFA, further studies with a larger sample size are still needed. Second, the value of ARFI velocity might change during the follow-up period in accordance with the degree of fibrosis, which could progress as part of the natural disease course or could be affected by the anti-viral treatment 40 . Even though our data suggested both anti-viral treatment and advanced LS were significant predictors of OS, longitudinal studies with serial measurements (2020) 10:2006 | https://doi.org/10.1038/s41598-020-58988-3 www.nature.com/scientificreports www.nature.com/scientificreports/ of ARFI velocity for LS should be performed to address this question. Third, stiffness of responding tumor was recently reported to provide a useful tool for early prediction of HCC response to local ablative therapy 41 . However, this information was limited in our study. Fourth, our study only enrolled patients for whom ARFI velocity was measurable at the time of RFA, which might have led to selection bias. Moreover, our report cannot be extended to patients who have experienced other anti-HCC treatments, and further studies are required. Fifth, the predominant underlying liver disease in our HCC cohort was chronic viral hepatitis, which is different from Western populations. It is still undetermined whether ARFI velocity could predict the risks in HCC patients arising from steatohepatitis or alcoholic liver disease. Considering less influence by steatosis 42,43 , ARFI could still be used to determine LS in patients with hepatic steatosis and might have a role in predicting HCC risks.
In conclusion, LS measured by ARFI elastography could predict both HCC recurrence and OS in patients who underwent RFA.

Methods
Patients. This study prospectively enrolled 173 patients who had treatment-naive HCC and underwent RFA as a first treatment at Taipei Veterans General Hospital from January 2013 to December 2017. The diagnosis and staging of HCC were performed according to the guidelines of the American Association for the Study of Liver Disease 44 . The indications of RFA were as follows: (A) solitary tumor with size < 5 cm or 2-3 tumors all with sizes < 3 cm; (B) an absence of extra-hepatic metastasis or major vascular invasion; (C) grade A CTP classification of liver functional reserve; (D) no ascites; (E) platelet count > 50,000/mm3; and (F) no other major comorbidities that might complicate the RFA procedure (such as infections, arrhythmias, acute myocardial infarction, uncontrolled congestive heart failure, chronic obstructive pulmonary disease with acute exacerbation, or recent stroke) 23 .
The study was conducted in accordance with the ethical guidelines of the 1975 Declaration of Helsinki and current ethical guidelines. It was also approved by the Institutional Review Board, Taipei Veterans General Hospital. Informed consent was obtained from all patients before the entered the study.
RFA and follow-up. The RFA device, procedure, and follow-up have been described previously 23,32 . Briefly, RFA was performed by experienced hepatologists or interventional radiologists with the confirmation of complete ablation. Regular follow-up with clinical assessment were performed one month after RFA and every 3-6 months later to monitor HCC recurrence. The procedures involved physical examination, laboratory exams including serum AFP levels, and contrast-enhanced image studies by computed tomography scans or magnetic resonance imaging 44,45 . All of the patients were followed until the end of 2018. The primary endpoint of this study was HCC recurrence, and the second endpoint was OS.
Biochemical and serologic markers. Serum biochemistries were measured using a Roche/Hitachi Modular Analytics System (Roche Diagnostics GmbH, Mannheim, Germany). The serum level of AFP was tested using a radio-immunoassay kit (Serono Diagnostic SA, Coinsin/VD, Switzerland). Serum levels of hepatitis B surface antigen (HBsAg) and hepatitis C virus antibody were tested by radio-immunoassay (Abbott Laboratories, North Chicago, IL) and second-generation enzyme immunoassay (Abbott). Previously described methods were used to calculate the ratio (AAR) of AST to ALT, APRI, and ALBI grade 46-48 . Acoustic radiation force impulse elastography measurements. ARFI elastography was performed in fasting status of the patient 49 to assess LS and SS on the same day as RFA with targeted selection of the non-tumor part by an experienced technician who was blinded to the clinical information of the patients. The detailed technique of ARFI elastography has been reported in previous studies, and the results are expressed in meters per second (m/s) 11 . A result is considered to be reliable when 10 validated measurements are within a ratio of IQR to the median value (IQR/M) of less than 0.3 with a success rate of more than 60% 50 .

Statistical analysis.
Data are shown as the median (IQR) or n (%) values as appropriate. Variables were compared using the chi-squared test or Fisher's exact test for categorical values and a student's t-test or the Mann-Whitney test for continuous values. The correlation between LS and SS was investigated using the Spearman correlation test. The optimal cutoff values of LS and SS were assessed using the area under receiver operating characteristic curves (AUROC). The value with the highest Youden's Index (sensitivity + specificity − 1) was considered as the optimal cut-off. Competing risk Kaplan-Meier was applied to estimate RFS 51,52 ; and OS was estimated by Kaplan-Meier method and compared using Cox's proportional hazards model. 1:1 propensity score-matched analysis was also performed by greedy 8 to 1 digit match algorithm without replacement to reduce confounders, including tumor numbers, serum levels of AFP and total bilirubin, and viral hepatitis status. The factors associated with HCC recurrence and OS were identified by applying a multivariate forward stepwise logistic regression model using significant variables in the univariate analysis. We also performed two models in the multivariate analysis, dimensional ARFI velocity value in model I and dichotomous ARFI value in the model II, respectively. A two-tailed value of P < 0.05 was considered statistically significant. All statistical analyses were performed using IBM SPSS Statistics for Windows, version 21.0 (IBM Corp., Armonk, NY, USA).

Data availability
The datasets generated during and analyzed during the current study are available from the corresponding author on reasonable request.