Liver stiffness and insulin resistance in predicting recurrence for early stage hepatoma patients after curative resection

Curative resection is recommended for patient with early stage hepatocellular carcinoma (HCC), however, the prognosis is limited by high recurrence rate. This study was to investigate liver stiffness (LS) and metabolic factor in prediction of HCC recurrence for patients with early stage HCC who had undergone curative resection. Consecutive patients with suspicion of HCC who had undergone curative resection were prospectively enrolled. Transient elastography was performed to determine LS pre-operatively. The demographics, clinical characteristics and histological findings were recorded. All patients were followed up regularly until recurrence, death or last visit. Ninety-four patients with early stage HCC were enrolled. LS positively correlated with fibrosis stage (r = 0.666). In a median follow-up of 3.2 years, forty patients developed recurrences including 22 recurrences after 1-year post resection. The 5-year cumulative recurrence rate was 44.2%. LS was the independent factor associated with recurrence. Patients with LS > 8.5 kPa had higher 5-year cumulative recurrence rate (59.8% vs 25.1%, p = 0.007). For the prediction of recurrence after 1-year post resection, LS > 8.5 kPa (hazard ratio 2.72) and homeostatic model assessment for insulin resistance index (HOMA-IR) (hazard ratio 1.24) were independent factors in multivariate analysis. Those patients with both LS > 8.5 kPa and HOMA-IR > 2.3 had the highest recurrence rate after 1-year post resection.

. Therefore, the purpose of this study was to investigate whether LS and metabolic factor were useful in predicting tumor recurrence for patients with early stage HCC after curative resection.  (Table 1). Sixty patients underwent antiviral therapy including 34 HBV with oral nucleotide agents in complete virological response and 26 hepatitis C virus infection with interferon-based therapy in sustained virological response. LS was positively correlated with histological fibrosis stage (F) (Fig. 2). The median LS value was 5.9 kPa, 6.9 kPa, 8 Table 3). The performance of HOMA-IR was 0.553 in recurrence prediction and the optimal cutoff was 2.3 by ROC curve. With the cutoffs of 8.5 kPa and 2.3 for LS and HOMA-IR, patients with both LS and HOMA-IR more than the cutoffs had higher recurrence than those without (p = 0.002) (Fig. 3b).

Discussion
The risk of HCC recurrence after resection was bimodal with higher incidence within 1-year follow-up after resection 12,18 . In this prospective study, we demonstrated that LS by transient elastography was in correlation with fibrosis stage and useful in the prediction of all and recurrence after 1-year post resection in a cohort of 94 ALB (g/dL, median, range    www.nature.com/scientificreports/ patients in BCLC early stage HCC. However, the performance and prediction validities were not satisfactory. Patients with LS > 8.5 kPa had a higher 5-year cumulative recurrence rate than those without. In addition to LS, HOMA-IR was independently associated with recurrence after 1-year post resection. There were higher late recurrence rates for those patients with LS > 8.5 kPa and HOMA-IR > 2.3. The 5-year cumulative HCC recurrence rate was 50%-70% with intrahepatic locations in up to 66-70% after resection 8,10 . In this study cohort, the 5-year HCC recurrence rate was 44.2%, which was lower than that reported in the literature 10 . The proportions of patients with recurrence at intrahepatic location and in BCLC early stage pattern were 97.5% and 85%, which were higher than those reported in other studies 7,8,10 . The enrollment of an early stage and homogenous HCC patient cohort with regular follow-up might explain the lower recurrence rate and early-stage recurrence pattern in this study. In addition, 63.8% of patients underwent antiviral therapy pre-or post-operatively, which might also reduce HCC recurrence post-curative resection 19,20 . However, two patients with recurrences in BCLC stage C, including one intrahepatic recurrence with portal branch invasion and the other with extrahepatic metastasis without intrahepatic recurrence, seemed inevitable with current guidelines of optimal surveillance interval and timely recall policies, which might be explained by aggressive tumor behavior 10,21 . HCC recurrence is generally classified into early (within 1 or 2 years after curative resection) and late recurrence. While early recurrence is considered as resulting from intrahepatic metastasis by aggressive tumor behavior, late recurrence is de novo HCCs mainly owing to liver fibrosis background 10 . The predicting factors are tumor factors including tumor diameter, number, histology grade and microvascular invasion, and non-tumor factors including stage of liver disease. While liver stiffness was the only independent factor associated with recurrence after curative resection in this prospective cohort of patients, tumor factors were not. In this study, we enrolled patients with early stage HCC after curative resection and yielded a 1-year recurrence rate of 19.2%, lower than the 31% in a previous report 18 . The risk of intrahepatic metastasis might be low for this patient cohort with all early stage HCC and low histology grade, which explained low early recurrence rate after curative resection and no tumor factors associated with recurrence. However, liver advanced fibrosis or cirrhosis provided a background www.nature.com/scientificreports/ with higher hepatocarcinogenetic potential and resulted in higher development and recurrence post-curative treatment of HCC [4][5][6]18 . The incidence of de novo recurrence might continuously occur from early postoperative period until late period after resection 18 . In correlation with histological fibrosis stage, LS measured preoperatively was the independent factor in predicting all and late recurrences in this study. Our finding was compatible with that of the study from the same area 14 and different from the recent study result in which spleen stiffness was the only predictor of late recurrence, instead of LS 16 . In contrast to the studies enrolling not only patients in heterogenous stage but also with macrovascular invasions 14,16 , our study clarified this issue with a homogenous and early stage HCC cohort in which curative resections were indicated in the guidelines 4-6 . Therefore, our result is convincing and useful for HCC patients for whom curative resection was performed according to current recommendations. Although LS was useful in the prediction of HCC recurrence after resection, the performance and validities were not satisfactory in clinical practice. The prediction performance was 0.641 being similar to that in a previous study 14 . The optimal threshold in predicting recurrence varied for differences in the study populations. Instead of 13.4 kPa proposed in a previous study 14 , the optimal cutoff was 8.5 kPa, which might be owing to a lower proportion of patients in advanced fibrosis and cirrhosis stages (64.8% vs 81.2%) in this study cohort. However, both studies showed similar recurrence curves in demonstrating significant difference in recurrence after 1-year post resection stratified by 8.5 kPa and 13.4 kPa respectively. In addition to LS, insulin resistance by HOMA-IR was the independent factor associated with recurrence after 1-year post resection. Hyperinsulinemia has been considered to be involved in the progression and recurrence of HCC owing to the mitogenic and proliferative effects of insulin 22 . Insulin resistance has been identified as a risk factor of HCC development in chronic hepatitis C 22,23 . This study showed that the optimal cutoff of HOMA-IR index was 2.3, which was the same value proposed by the other study enrolling patients after curative treatments 24 .
Similar to other studies 23,24 , metabolic abnormalities including metabolic syndrome, diabetes, body mass index, waist and hyperlipidemia were not associated with recurrence in our study. We also demonstrated that combined HOMA > 2.3 and LS > 8.5 kPa identified those patients with high risk of HCC recurrence after 1-year post resection. Based on our study result, it might be beneficial for patients to improve insulin resistance and LS with life style changes and antiviral treatment in suppressing HCC recurrence. However, the risk prediction of HCC recurrence might be further improved with developments of risk scores or other models 25,26 . Table 3. Uni-and multi-variate analysis of factors associated with late recurrence (n = 75). B hepatitis B virus, C hepatitis C virus, NBNC non-hepatitis B virus and non-hepatitis C virus, LS liver stiffness, ICG indocyanine green, ALP alkaline phosphatase, HOMA-IR homeostatic model assessment for insulin resistance, *all characteristics in Table 1 were included in the univariable analysis and those with p values < 0.2 were entered into stepwise multivariable analysis. www.nature.com/scientificreports/ There were some limitations in this study. Transient elastography with M-probe was used for LS measurement, which might result in higher LS for patients with body mass index more than 30 kg/m 227 . Despite little effect on LS measurement due to small tumor diameter in our study, there might be over-or under-estimation of LS for patients with tumors located in the right liver. Spleen stiffness was not measured in this study. Most patients (92.6%) in this study were patients with chronic hepatitis B or C. Whether spleen stiffness and other etiologies were independent factors of HCC recurrence might need further study. For clinical practice, it might be necessary to validate the proposed cutoffs of LS and HOMA-IR in a large cohort study.
In summary, LS measured by transient elastography was the independent risk factor of HCC recurrence for patients with BCLC early stage HCC after curative resection. Insulin resistance by HOMA-IR was the independent factor for recurrence after 1-year post resection. With the cutoffs of 8.5 kPa and 2.3, LS and HOMA-IR stratified the risks of recurrence after 1-year post resection for early stage HCC after curative resection.

Patients and methods
Patients. This study was approved by the Institutional Review Board of Chang Gung Memorial Hospital (IRB number: 101-1075A3) and conducted in accordance with the 1975 Declaration of Helsinki. Consecutive patients fulfilling all inclusion and exclusion criteria were enrolled prospectively. Patients with suspicion of HCC who had undergone curative resection and Child-Pugh classification A liver function reserve signed informed consents. The inclusion criteria were patients with HCC in BCLC very early or early stage. The exclusion criteria were patients with history of hepatic malignancy or underwent two-stage resection. While the demographics and baseline clinical characteristics including metabolic syndrome, lipid profiles and homeostatic model assessment for insulin resistance index (HOMA-IR) were recorded before resection, the histological characteristics of tumors and surrounding normal hepatic parenchyma were recorded after resection. All patients were followed up regularly with imaging studies including hepatic ultrasonography, computed tomography and magnetic resonance imaging. The patients were followed up until tumor recurrence, death or end of 2018. All patients signed informed consents before enrollment.
Liver stiffness measurement. LS was measured by using transient elastography (FibroScan, Echosens, Paris, France) with an M-probe before resection. It was performed in an overnight fasting state and by an experienced technician. The right lobe of the liver was assessed through the intercostal space while the patients were lying in a supine position with their right arms at maximal abduction. LS results were expressed as a median value with an interquartile range (IQR) in kilopascal (kPa) 28 . The results were considered reliable only when 10 successful shots, a successful rate more than 60%, and the IQR-to-liver stiffness ratio < 0.30 had been obtained.
Statistical analysis. Quantitative variables were expressed with mean ± standard deviation or median with a range. Qualitative variables were expressed as absolute and relative frequencies. While Mann-Whitney U test and Student t test were used for comparisons of quantitative variables, Chi-square and Fisher's exact tests were used in categorical variables. The correlations between liver stiffness values and histological fibrosis were analyzed with Spearman's rank correlation method. Cox regression model was performed to determine independent factors associated with tumor recurrence. Hazard ratios (HR) and corresponding 95% confidence intervals (CI) were indicated 29 . The diagnostic performance of independent factor in predicting tumor recurrence was evaluated by receiver operating characteristic (ROC) curve. The area under the ROC curve (AUROC) and the 95% confidence interval were used as indexes of accuracy. The optimal cutoff values were determined with Youden index from ROC curve 30 . All data were recorded and analyzed using the SPSS v18 software package (SPSS Inc, Chicago, IL, USA); all p values were derived from 2-tailed tests, and a level of < 0.05 was accepted as statistically significant.