Liver stiffness as a predictor of hepatocellular carcinoma behavior in patients with hepatitis C related liver cirrhosis

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Liver stiffness as a predictor of hepatocellular carcinoma behaviour in patients with hepatitis C related liver cirrhosis Sameh A Lashen , Mohamed M. Elshafei , Fahmy H. Hablass , Eman A. Alsayed , Asmaa A. Hassan PII: DOI: Reference:

S1499-3872(19)30245-0 https://doi.org/10.1016/j.hbpd.2019.11.004 HBPD 235

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Hepatobiliary & Pancreatic Diseases International

Received date: Accepted date:

11 June 2019 25 November 2019

Please cite this article as: Sameh A Lashen , Mohamed M. Elshafei , Fahmy H. Hablass , Eman A. Alsayed , Asmaa A. Hassan , Liver stiffness as a predictor of hepatocellular carcinoma behaviour in patients with hepatitis C related liver cirrhosis, Hepatobiliary & Pancreatic Diseases International (2019), doi: https://doi.org/10.1016/j.hbpd.2019.11.004

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 First Affiliated Hospital, Zhejiang University School of Medicine in China. Published by Elsevier B.V. All rights reserved.

Manuscript type: Original Article/Liver Running title: Liver stiffness and HCC clinical behaviour Liver stiffness as a predictor of hepatocellular carcinoma behaviour in patients with hepatitis C related liver cirrhosis Authors: Sameh A Lashen1,*, Mohamed M Elshafei2, Fahmy H Hablass1, Eman A Alsayed1, Asmaa A Hassan3 (Lashen SA, Elshafei MM, Hablass FH, Alsayed EA, Hassan AA) City: Alexandria, Egypt Author Affiliations: 1 Department of Internal Medicine, Faculty of Medicine, University of Alexandria, Alexandria, Egypt 2 Department of Radiology, Faculty of Medicine, University of Alexandria, Alexandria, Egypt 3 Department of Biomedical Informatics and Medical Statistics, Medical Research Institute, University of Alexandria, Alexandria, Egypt * Corresponding Author: Sameh A Lashen (E-mail address: [email protected])

Received 11 June 2019 Accepted 25 November 2019

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CRediT authorship contribution statement: Sameh A Lashen: Conceptualization, Data curation, Formal analysis, Methodology, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing. Mohamed M Elshafei: Methodology, Validation, Visualization, Writing - original draft. Fahmy H Hablass: Data curation, Writing - original draft, Writing - review & editing. Eman A Alsayed: Data curation, Writing - original draft, Writing - review & editing. Asmaa A Hassan: Formal analysis, Validation. LSA is the guarantor. Funding: None. Ethical approval: The study was approved by the local ethics committee at Faculty of Medicine, University of Alexandria (http://www.med.alexu.edu.eg/ethics-committee/), Institutional Review Board number (IRB): 00007555; Federal Wide Assurance (FWA) number: 00015712; review serial number: 0302801. Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. Notes: The work has been published as an abstract in the United European Gastroenterology Journal after being presented as a poster presentation in the UEG conference, October 2018.

Abstract: Background: Risk stratification and prognostication of hepatocellular carcinoma (HCC) help to improve patient outcome. Herein we investigated the role of liver stiffness measurement (LSM) in the prediction of HCC behavior.

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Methods: Totally 121 naïve patients with HCC were included. HCC radiological evaluation and staging were done. LSM was measured using virtual touch quantification. Patients were divided into early to intermediate HCC (BCLC-0, A and B) and late HCC (BCLC-C and D). HCC was treated according to the BCLC stage. HCC recurrence-free interval was estimated. Results: The mean LSM inside the tumor was significantly lower than the peri-tumoral area and the cirrhotic non-cancerous liver parts (P<0.001). In late HCCs stage, the mean LSM inside the tumor and in the peri-tumoral tissue was lower than the corresponding values in the early to intermediate HCCs stage (P<0.001). LSM inside the tumor and in the peri-tumoral tissue negatively correlated with serum AFP, tumor vascular invasion, and stage (P<0.05). The recurrence-free interval was directly correlated to LSM inside the tumor and inversely to LSM in cirrhotic non tumorous liver part. Kaplan-Meier analysis showed that the recurrence-free interval was significantly longer in patients with LSM inside the tumor of ≥ 1.25 m/s compared to those with LSM inside the tumor of < 1.25 m/s. Conclusions: LSM can serve as a potential non-invasive predictor for HCC clinical behaviour and the recurrence-free interval following loco-regional treatments. Key words: Fibrosis; Hepatitis C; Hepatocellular carcinoma; Point shear wave; Liver cirrhosis

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Introduction The invasive nature of liver biopsy for diagnosing and grading of liver fibrosis and the possibility of sampling error necessitate the availability of non-invasive methods for fibrosis assessment [1]. In spite of the multiplicity of hepatocellular carcinoma (HCC) risk factors, liver fibrosis is the most important irrespective of its etiology [2-4]. For improvement of patients outcome, early detection and treatment of HCC are mandatory. Extensive investigations for screening, risk stratification and prognostication of HCC are essential [2]. In the last decade, image-based quantification of liver fibrosis by transient elastography (TE) has been introduced into practice. Several studies have addressed a high accuracy of TE in defining significant fibrosis (F > 2) and cirrhosis (F4) in chronic hepatitis C patients [5, 6]. In addition, it is more representative of the hepatic parenchyma, where it can evaluate a larger area compared to a single liver biopsy [5]. The risk for development of HCC has been assessed via liver stiffness measurement (LSM) in patients with hepatitis B (HBV) and hepatitis C virus (HCV) infections[7, 8]. Nevertheless, in most reports, the risk for HCC was only indirectly evaluated based on the value for liver cirrhosis as measured by TE. LSM related to HCC has not been directly evaluated [7, 9]. Shear wave elastography techniques have been applied in the real-time ultrasound systems, and their accuracy in the assessment of liver fibrosis has been addressed in several studies [10-14]. These techniques have the advantage of performing an examination in real-time. In the point shear wave elastography (pSWE), the shear wave elastometry is done at a certain tissue point by measurement of the speed of shear waves that are generated using acoustic

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radiation force impulse (ARFI) [15]. To our knowledge, no studies were directed to the evaluation of the impact of liver stiffness or tumor stiffness on HCC clinico-radiological characteristics or response to treatment.

Methods Patients Prospectively, our research included 128 naïve patients who were diagnosed as HCC on top of HCV related liver cirrhosis. Patients were recruited from the Hepatology Clinic at Alexandria Main University Hospital, Alexandria, Egypt. Patients with morbid obesity, marked ascites, seropositivity to HBV, other causes of chronic liver diseases, were excluded. We also excluded 7 patients with failed LSM. The study was approved by the local ethics committee at Faculty of Medicine, University of Alexandria (http://www.med.alexu.edu.eg/ethics-committee/), Institutional Review Board number (IRB): 00007555; Federal Wide Assurance (FWA) number: 00015712; review serial number: 0302801. Also, the study was conducted in accordance with the 1975 Helsinki Declaration and its later amendments as revised in 2008, and Good Clinical Practice guidelines. Informed consent was obtained from all patients included in the study. Patient evaluation All the patients were evaluated clinically. Liver tests profile and serum alfa-fetoprotein (AFP) were measured. The AST/platelet ratio index (APRI) was calculated (with a cut-off value of 1.5 to rule in cirrhosis) [16], and the severity of liver disease was assessed by Child-TurcottePugh classification (CTP). The diagnosis of HCC was based on the wash in/wash out pattern in

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triphasic CT. HCC evaluation included the number of lesions, maximum tumor diameter, vascular invasion, extra-hepatic nodal spread. The BCLC staging was used to evaluate the applicability of different treatment strategies [17]. Patients were classified as having early to intermediate stage HCC including BCLC stage 0, A and B, or as having late-stage HCC including BCLC stage C and D. Liver stiffness measurement Point shear wave elastography was performed by ACUSON TM S2000 ultrasound system (Siemens Mountain View, CA, USA), equipped with a 4C1 MHz frequency convex transducer, using the virtual touch quantification (VTQ) method. While patients were fasting for 6 hours, lying in decubitus (or 30o left lateral) position, and the right arm in maximal abduction, the transducer of VTQ® apparatus in the B-mode was placed between ribs, perpendicular to the liver capsule, and the liver was scanned to reach the most accessible positions. LSM was measured for the tumor mass, within 1 cm around the tumor, and at the non-malignant cirrhotic part of the liver (Fig. 1A and 1B). Measures were taken while the patient was holding the breath at the end of expiration. Verifying that the examined area was free of vascular and biliary structures and rib shadows were ascertained. LSM values were calculated as the average of 12 successful measurements and expressed as a meter per second (m/s). For every patient, the examination was annotated failed if the ratio of the number of valid measurements to the total number of measurements is < 60% [18]. After LSM, patients who were eligible for treatment were treated by appropriate modality according to the BCLC staging system, and after tumor ablation, the recurrence-free interval was calculated starting from last CT evidence of tumor ablation to 1st CT evidence of tumor recurrence or de novo lesions. Statistical analysis 6 / 27

The analysis was done for 121 patients. The data were expressed as mean ± standard deviation (SD). Comparisons between groups were made by the Student’s t test or MannWhitney test as appropriate. Correlations between variables were done using the Pearson’s or Spearman Rho coefficient, when appropriate. The sensitivity and specificity of LSM by VQT in differentiating early-intermediate from late stages HCC was assessed by receiver-operating characteristic (ROC) curve. The area under the curve (AUC) and its 95% confidence interval (CI) were calculated. A binary logistic regression model using the “enter” method was applied for prediction of early-intermediate stage HCC, significant predictors by univariate analysis were entered in the model and predictors causing multi-collinearity problem were excluded. KaplanMeier survival analysis with log-rank test and Cox regression hazard models were applied for recurrence-free survival. Statistical significance was assessed at P < 0.05. All calculated P values were two-tailed.

Results Clinical laboratory parameters Our study included 121 patients with their baseline data shown in Table 1. Among our patients, 54 (44.6%) have early-intermediate stage HCC (BCLC 0, A or B), while 67 (55.4%) have late-stage HCC (BCLC C and D). In patients with early-intermediate stage HCC who were eligible for treatment, RFA was used in 6 (11.1%) patients, TACE in 36 (66.7%), and combined RFA+TACE in 12 (22.2%). We did not have cases of liver transplantation or hepatic resection. During the follow-up period after HCC complete ablation, the recurrence-free interval in this group ranged between 3-17 months. Patients in the late HCC group according to the BCLC

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algorithm were managed by the best supportive treatment or sorafenib according to the availability of resources (n=67). Patients with late HCC have a significantly lower platelet count and serum albumen compared to early-intermediate stage HCC (P<0.001), while they have significantly higher serum bilirubin, INR, Child-Pugh score, and AFP levels (P< 0.001, Table 2). LSM In all patients (n = 121), the LSM value inside the tumor, in the peri-tumoral area, and in the cirrhotic non-malignant parts were compared. The median value of the LSM inside the tumor was significantly lower than that in the peri-tumoral area [1.26 (0.51-3.71) m/s vs. 2.03 (1.104.39) m/s, P < 0.001, Z = -7.47] and in the cirrhotic non-malignant parts of the liver [1.26 (0.513.71) m/s vs. 2.73 (1.22-4.04) m/s, P < 0.001, Z = -10.1], while there was no statistically significant difference between the peri-tumoral area and the cirrhotic non-malignant parts [2.03 (1.10-4.39) m/s vs. 2.73 (1.22- 4.04) m/s, P = 0.14, Z = -1.46] (Fig. 2A). We compared the early-intermediate vs. late HCC subgroups as regards the LSM value. The LSM value inside the tumor in the late HCC group were significantly lower than their corresponding values in the early-intermediate HCC group [1.04 (0.51 – 2.38) m/s vs. 1.75 (0.63 – 3.71) m/s, P < 0.001, Z = 4.88]. Similarly, the median value of LSM in the peri-tumoral area in the late HCC group were significantly lower than their corresponding values in the early-intermediate HCC group [1.57 (1.10 – 3.92) m/s vs. 3.35 (1.27 – 4.39) m/s, P < 0.001, Z = -5.69], while there was no significant statistical difference between the two groups as regards LSM in the cirrhotic non-malignant part of the liver [2.70 (1.22 – 4.04) m/s vs. 2.78 (1.98 – 3.4) m/s, P = 0.36, Z = -0.91] (Fig. 2B). We subdivided all patients (n = 121) into two subgroups based on presence (n = 50) or 8 / 27

absence (n = 71) of tumoral portal vein invasion. The median (range) of LSM inside the tumor and in the peri-tumoral area were significantly lower in patients with portal vein invasion compared to patients without portal vein invasion [0.97 (0.51 – 2.38) m/s vs. 1.74 (0.63 – 3.71) m/s, P <0.001; and 1.42 (1.10 – 3.29) m/s vs. 3.29 (1.27 – 4.39) m/s, P < 0.001, respectively]. However, there was no significant difference between the two subgroups as regards the median value of LSM in the cirrhotic non-malignant liver parts [2.65 (1.22 – 3.43) m/s vs. 2.79 (1.98 – 4.04) m/s, P = 0.08]. Correlation of LSM to different study parameters The value of LSM inside the tumor showed negative correlation with the following parameters: serum AFP (r = - 0.57, P < 0.001), the number of HCC lesions (rs = - 0.36, P < 0.001), HCC maximum diameter (r = - 0.401, P < 0.001), CLIP (rs = - 0.45, P < 0.001), OKUDA ( rs = -0.27, P = 0.002), TNM ( rs = - 0.43, P < 0.001), and BCLC stages (rs = - 0.35, P < 0.001). The value of LSM in the peri-tumoral area showed negative correlation with serum AFP (r = - 0.49, P < 0.001), the number of HCC focal lesions (rs = - 0.55, P < 0.001), HCC maximum diameter (r = - 0.51, P < 0.001), CLIP (rs = - 0.57, P < 0.001), OKUDA (rs = - 0.46, P < 0.001), TNM (rs = - 0.55 , P < 0.001), and BCLC stages (rs = - 0.53, P < 0.001), while the value of LSM in the cirrhotic non-malignant part showed no significant correlation with all tumor-related parameters. ROC curve By plotting the ROC curve, at cut-off value of 1.25 m/s, LSM inside the tumor was found to have a sensitivity of 77.8% and a specificity of 71.6% in differentiating early-intermediate stages of HCC from late-stage HCC (AUC =0.76; 95% CI: 0.67-0.85). At cut-off values of 2.22

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m/s, LSM in the peri-tumoral area was found to have a sensitivity of 77.8% and a specificity of 73.1% in differentiating early-intermediate stages of HCC from late stage HCC (AUC = 0.80; 95% CI: 0.72-0.88), and at cut-off values of 2.72 m/s, LSM in the cirrhotic non-malignant part of the liver was found to have a sensitivity of 60% and a specificity of 53.1% in differentiating early-intermediate stages of HCC from late-stage HCC (AUC = 0.55; 95% CI: 0.45-0.65; Fig. 3). Predictors for tumor recurrence free interval Univariate analysis showed that the following parameters were significant predictors for the early recurrence of HCC following tumor ablation by loco-regional modalities: APRI score (P = 0.004), Child-Pugh class (P = 0.012), serum AFP (P < 0.001), LSM inside the tumor (P < 0.001), number and maximum diameter of the tumor (P < 0.05), and TNM (P < 0.001), CLIP (P = 0.004), and BCLC stages (P = 0.001). Cox-regression analysis models were run for predictors of recurrence free interval. We found that LSM inside the tumor (HR = 0.21; 95% CI: 0.10- 0.43; P < 0.0001), TNM staging (HR = 11.7; 95% CI: 1.81- 76.3; P = 0.01), BCLC staging (HR = 0.26; 95% CI: 0.06 - 0.98; P = 0.04), number of tumor foci (HR = 4.2; 95% CI: 1.66- 41.1; P = 0.01), and maximum size of the tumor (HR = 2.03; 95% CI: 1.21-3.41; P = 0.007) were predictors for recurrence free interval (Table 3). Kaplan-Meier with log-rank test was run for LSM inside the tumor at a cut-off value 1.25 m/s (as obtained from the ROC curve analysis). The recurrence-free interval was significantly longer in patients with LSM inside the tumor of ≥ 1.25 m/s [median (interquartile range, IQR) 14 months (7 – 15)] compared to patients with LSM inside the tumor of < 1.25 m/s [6 months (IQR: 5 – 6.9), P < 0.0001, Fig. 4A]. However, for the LSM at the peri-tumoral area, there was no

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statistically significant difference as regards the median duration of the recurrence-free interval at the cut-off value of 2.22 m/s (P = 0.78, Fig. 4B). For the LSM at the cirrhotic non-malignant part, the recurrence-free interval was significantly shorter in patients with LSM ≥ 2.72 m/s [7.5 months (IQR: 6-12)] compared to patients with LSM < 2.72 m/s [15 months (IQR: 6.38 – 16), P = 0.001, Fig. 4C]. Kaplan-Meier with log-rank test was run for the type of loco-regional treatment effect on the recurrence-free interval. There was no significant difference in the median duration of the recurrence-free interval among patients who were managed by RFA, TACE, or combined RFA+TACE (median, 15 months, 8 months, and 6 months, respectively) (P = 0.06, Fig. 4D).

Discussion Liver stiffness measurement - a non-invasive alternative for the assessment of the degree of liver fibrosis - has been explored in many cohort studies [10, 11, 19]. In addition, LSM was also evaluated as a predictor of HCC development in patients with liver cirrhosis, and liver stiffness based risk scores have been developed to predict HCC occurrence in patients with hepatitis B. Moreover; LSM >12.0 kPa using TE was an independent risk factor for new HCC development in patients with HCV [9, 20]. In spite of this, data about the role of LSM in prognostication of HCC itself is scarce, and to our knowledge, this is the first study to address the role of LSM using VTQ as a predictor for HCC clinico-biological behavior. In our study, the LSM inside the tumor and peri-tumoral area were significantly lower in patients with late stages of HCC than that in those with early to intermediate stage HCC, and the low LSM values were associated with worse characteristics of HCC including high serum AFP,

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larger tumor sizes and number, portal vein invasion, and late cancer stages with subsequent noneligibility for treatment. Point shear wave elastography generates a shear-wave inside the liver using a focused acoustic beam from an ultrasound transducer. These waves travel through the liver, and LSM is expressed as the velocity of transmission of the shear waves inside the liver (the stiffer the liver, the higher the velocity) [21]. The lower stiffness reading inside the tumor could be explained by the higher vascularity and the non-organized cellular structure due to the rapid growth of the tumor cells. These two characteristics would help the cancer cells to grow rapidly and to spread earlier compared to the less vascular, more fibrotic (and so, stiffer) tumor tissues. In addition, a rapidly growing tumor may have an internal breakdown due to an imbalance between the blood supply and cellular growth. Similarly, we proposed that the association between lower LSM at the peri-tumoral area and the worse HCC characteristics may be due to the loose peri-tumoral zone resulted from (a) tumor cell invasion of the nearby cirrhotic liver or microsatellite tumors, (b) the ramifying capillary network surrounding the highly vascular tumors [22], or (c) leaky peri-tumoral zone formed by substances released by aggressive HCCs e.g. metalloproteinases, which would help cancer cells to spread and get access to multi-centricity. We suggest that a lower peri-tumoral stiffness represents either a port for microsatellites extension (this may be missed and represent a source for early recurrence) or an indicator for more biologically aggressive cancer with a worse prognosis. It has been found that tumor blood supply may not be enough for rapidly growing HCCs; as a result, the enhanced neo-angiogenesis around the tumor leads to the formation of tumors with softer tissue structure, a finding linked to worse tumor characteristics [23, 24].

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However, opposing data about the influence of microenvironment stiffness on HCC growth and chemotherapy responsiveness were obtained from in vitro studies which showed that higher stiffness in the tumor supporting microenvironment was associated with a worse prognosis [25]. This influence may be attributed to the difference in the growth factors milieu and the cell cross-linkage pattern in the in vitro, rather than in vivo, studies. In our study, lower LSM at the cirrhotic non-malignant liver parts were associated with a higher incidence of vascular invasion by the tumor. A soft liver matrix/stiffness will facilitate cancer spread inside the liver including vascular channels. On the other hand, the higher LSM in the cirrhotic non-malignant part in our study was associated with a shorter recurrence-free interval. It has been shown previously that higher liver stiffness is associated with a higher incidence of HCC, but the ideal cut-off value of liver stiffness in the implementation of HCC surveillance program is still unknown [9]. Again, data available about liver cirrhosis stiffness and prognostication of HCC are scarce. An in-vitro study showed that increasing matrix stiffness promotes proliferation and chemotherapy non-responsiveness of cancer cells [25]. LSM using TE has been also investigated for prediction of HCC recurrence after liver resection where LSM was an independent predictor of recurrence, whereas histological fibrosis status was not [26]. These findings are in accordance with data obtained in the current study and reinforce the suggested possible role of LSM as a predictor for early occurrence/recurrence of HCC. After HCC ablation in our eligible patients, we found that the lower values of pre-ablation LSM inside the tumor were associated with shorter recurrence-free interval (irrespective to the type of the loco-regional treatment). Because a lower LSM was linked to poor tumor characteristics, this makes sense to be linked to a shorter recurrence-free interval. Highly vascular and aggressive tumors may be associated with the early micro-vascular spread and

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synchronous/metachronous micro-satellites. This may draw attention to the possible use of LSM before planning to treat HCC. On the other hand, we found that higher LSM in the cirrhotic nonmalignant liver parts was linked to shorter recurrence-free survival. This is explained by the relation between degree of liver fibrosis (hence, the stiffness) and the risk of HCC occurrence/recurrence. Higher LSM means stiffer and more fibrotic liver, and the degree of fibrosis is a risk of earlier de novo tumor recurrence. [27] In HCC, it is clear that the large tumor size, multi-centricity, and vascular invasion already mean an advanced stage. Our study was to address the clinical significance of LSM in patients with early-intermediate HCC stages. LSM help us to predict the future behavior and recurrence of the tumor. LSM adds more certainty and predictability to the BCLC algorithm. LSM, not like biological behaviors, is a clinical approach which may be helpful for tumor treatment, especially with rising controversies about the BCLC treatment algorithm [28, 29]. Our data may have clinical implication in HCC treatment. Moreover; we highlighted the impact of liver stiffness on the clinical behavior of HCC. The limitation of our study is that we did not correlate LSM with either histological grading of the tumors or with the grade of hepatic fibrosis due to difficulty in obtaining liver biopsies. In conclusion, low LSM inside the HCC masses and at the peri-tumoral zone is a predictor of poor prognosis and shorter recurrence-free interval after HCC ablation. Also, the stiffness of cirrhotic liver tissues could predict the HCC recurrence-free interval. LSM inside HCC, at the peri-tumoral area, and at the cirrhotic non-malignant liver is a potential non-invasive marker for HCC prognostication and risk stratification especially in patients with early-

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intermediate HCC.

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Table 1. Clinico-laboratory characteristics of the patients included in the study (n=121) Parameters

Values

Age (yr)

54.5 ± 5.8

Sex Male

90 (74.4%)

Female

31 (25.6%

Hb (g/dL)

12.68 ±1.27 9

Platelets (×10 /L)

100.23 ±12.36

ALT (IU/L)

55.64 ±14.15

AST (IU/L)

85.17 ± 13.13

Albumin (g/dL)

3.20 ± 0.42

Bilirubin (mg/dL)

1.39 ± 0.53

INR

1.30 ± 0.17

Ascites Yes

18

Child-Pugh class A

96 (79.3%)

B

18 (14.9%)

C

7 (5.8%)

APRI score

2.16 ± 0.49

AFP (ng/mL)

855 ± 450

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Number of focal lesions

2.41 ± 1.57

Tumor maximum diameter (cm)

7.70 ± 3.28

PVT Yes

50

TNM stage I

74 (61.2%)

II

13(10.7%)

IIIA

12 (9.9%)

IIIB

49 (40.5%)

Okuda stage I

64 (52.9%)

II

38 (31.4%)

III

19 (15.7%)

BCLC stage (0/ A / B / C / D)

0/6 / 48 / 54 / 13

Treatment RFA

6 (5.0%)

TACE

36 (29.8%)

RFA+TACE

12 (9.9%)

BSC

67 (55.4%)

ALT:

alanine

aminotransferase;

APRI:

AST/platelet

ratio

index;

AST:

aspartate

aminotransferases; BCLC: the Barcelona-Clinic Liver Cancer; BSC: best supportive care; Hb: hemoglobin; INR: international normalization ratio; IQR: inter-quartile range; Max: maximum; Min: minimum; PVT: portal vein thrombosis; RFA: radiofrequency ablation; SD: standard deviation; TACE: trans-catheter arterial chemoembolization.

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Table 2. Comparison between early and late hepatocellular carcinoma patients as regards clinicolaboratory parameters Parameters

Early-intermediate HCC (n=54)

Late HCC (n=67)

P value*

Age (yr)

53.44 ± 5.64

55.27 ±5.84

0.08

Platelet (×109 /L)

104.81 ±8.28

96.54 ±13.85

< 0.001

ALT (IU/L)

58.05 ±13.53

53.70 ±14.43

0.09

AST (IU/L)

88.35 ±8.45

82.61 ±15.53

0.016

Albumen (g/dL)

3.38 ±0.34

3.06 ±0.43

< 0.001

Bilirubin (mg/dL)

1.17 ±0.17

1.58 ±0.64

< 0.001

INR

1.25 ±0.16

1.35 ±0.16

0.001

CTP class

-

-

< 0.001¶

CTP score

5.67 ±0.48

6.66 ±1.49

< 0.001

APRI score

2.11 ±0.21

2.20 ±0.64

0.34

AFP (ng/mL)

435.41 ±254.56

1193.43 ±234.64

< 0.001

Tumor number

1.22 ±0.42

3.37 ±1.50

< 0.001

Tumor size (cm)

4.44 ±1.07

10.27 ±1.81

< 0.001

*: Independent sample T-test; ¶: Fisher exact test; values are expressed as mean ± SD. AFP: alfafetoprotein; ALT: alanine aminotransferase; APRI: AST/platelet ratio index; AST: aspartate aminotransferases; CTP: Child-Turcotte-Pugh; INR: international normalization ratio.

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Table 3: Univariate and multivariate analysis for predictors of recurrence-free interval: Univariate analysis

Cox-regression analysis

Parameter P value

P value

HR (95% CI)

APRI score

0.004

-

-

Child-Pugh class

0.012

-

-

Serum AFP

< 0.001

-

-

LSM inside the tumor

< 0.001

<0.0001

0.21 (0.10-0.43)

Number of tumor foci

< 0.05

0.01

4.2 (1.66 – 41.1)

Tumor maximum diameter

< 0.05

0.007

2.03 (1.21 – 3.41)

TNM stage

< 0.001

0.01

11.7 (1.81 – 76.3)

CLIP stage

0.004

-

-

BCLC stage

0.001

0.04

0.26 (0.06 – 0.98)

AFP: alfa fetoprotein; APRI: aspartate aminotransferase/platelet ratio index; BCLC: BarcelonaClinic Liver Cancer; CLIP: Cancer of the Liver Italian Program; LSM: liver stiffness measurement; TNM: tumor node metastasis staging.

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Figures legend

Fig. 1. Image showing how to measure the liver stiffness using acoustic radiation force impulse ultrasound probe. A: Inside the hepatocellular carcinoma mass; B: At the zone within 1 cm around the tumor mass. The mean of twelve readings is recorded.

Fig. 2. Liver stiffness measurements. A: Inside the hepatocellular carcinoma (HCC) mass, in the peri-tumoral area, and in the non-malignant cirrhotic liver part; B: Early-intermediate versus late HCC.

Fig. 3. Receiver operating characteristic curve (ROC): the sensitivity and specificity of liver stiffness measurement (LSM) at a cut-off values of 1.25 m/s, 2.22 m/s, and 2.72 m/s inside the tumor, in the peri-tumoral tissue, and in cirrhotic non-malignant liver parts respectively have sensitivities and specificities of 77.8% and 71.6% (AUC = 0.76), 77.8% and 73.1% (AUC = 0.80), and 60.0% and 53.1% (AUC = 0.55) respectively in differentiating early from late HCC.

Fig. 4. Kaplan-Meier analysis of recurrence-free interval based on liver stiffness measurement (LSM). A: Inside the tumor showing that the recurrence-free interval was longer in patients with LSM inside the tumor of ≥ 1.25 m/s vs. patients < 1.25 m/s (P < 0.0001); B: At the peri-tumoral area showing that there was no statistically significant difference in the recurrence-free interval at the cut-off value of 2.22 m/s (P = 0.78); C: At the cirrhotic non-malignant liver part showing that the recurrence-free interval was shorter in patients with LSM ≥ 2.72 m/s vs. patients < 2.72 m/s (P = 0.001); D: Based on treatment modality for hepatocellular carcinoma showing that there was no significant difference in the recurrence-free interval between patients who were treated by radiofrequency ablation (RFA), or trans-arterial chemoembolization (TACE), or combination of both (P = 0.06).

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