Coexistence of muscle atrophy and high subcutaneous adipose tissue radiodensity predicts poor prognosis in hepatocellular carcinoma

Introduction We aimed to assess the prognostic implications of muscle atrophy and high subcutaneous adipose tissue (SAT) radiodensity in patients with hepatocellular carcinoma (HCC). Methods In this retrospective study, muscle atrophy was assessed using the psoas muscle index (PMI) obtained from computed tomography. SAT radiodensity was evaluated based on radiodensity measurements. Survival and multivariate analyses were performed to identify factors associated with prognosis. The impact of muscle atrophy and high SAT radiodensity on prognosis was determined through survival analysis. Results A total of 201 patients (median age: 71 years; 76.6% male) with HCC were included. Liver cirrhosis was observed in 72.6% of patients, and the predominant Child–Pugh grade was A (77.1%). A total of 33.3% of patients exhibited muscle atrophy based on PMI values, whereas 12.9% had high SAT radiodensity. Kaplan–Meier survival analysis demonstrated that patients with muscle atrophy had significantly poorer prognosis than those without muscle atrophy. Patients with high SAT radiodensity had a significantly worse prognosis than those without it. Muscle atrophy, high SAT radiodensity, the Barcelona Clinic Liver Cancer class B, C, or D, and Child–Pugh score ≥ 6 were significantly associated with overall survival. Further classification of patients into four groups based on the presence or absence of muscle atrophy and high SAT radiodensity revealed that patients with both muscle atrophy and high SAT radiodensity had the poorest prognosis. Conclusion Muscle atrophy and high SAT radiodensity are significantly associated with poor prognosis in patients with HCC. Identifying this high-risk subgroup may facilitate the implementation of targeted interventions, including nutritional therapy and exercise, to potentially improve clinical outcomes.


Introduction
Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related deaths, and its incidence has been increasing globally (1).Multiple prognostic factors are critical in the prognosis of HCC.These include the tumor status, as per the Barcelona Clinic Liver Cancer (BCLC) classification, liver function assessment according to the Child-Pugh score and albumin-bilirubin grade, and the patient's general health status evaluated by the Eastern Cooperative Oncology Group performance status (2).While it is challenging to objectively assess a patient's performance status, body composition, particularly muscle mass, offers an objective estimate of a patient's physical, nutritional, and metabolic condition (3).Chronic liver diseases, including HCC, are known to cause secondary sarcopenia (4, 5); in addition, loss of muscle mass in patients with HCC is associated with a poorer prognosis and increased rates of recurrence (6)(7)(8).Moreover, in patients with chronic liver diseases, particularly those with liver cirrhosis (LC), the reported prevalence of sarcopenia is notably high and the loss of skeletal muscle mass progresses rapidly (7,(9)(10)(11).Therefore, it is crucial to assess muscle mass in patients with HCC to predict prognosis.
Recent studies suggest that aspects of body composition besides muscle mass also correlate with mortality in patients with decompensated cirrhosis and HCC (12).Factors such as subcutaneous adipose tissue (SAT) radiodensity (13, 14), intramuscular fat deposition, and visceral adiposity (15) have been reported as prognostic factors in patients with HCC.In particular, SAT radiodensity can be objectively measured by CT in Hounsfield units (HU), and average SAT radiodensity has been introduced as an indirect surrogate marker of adipose tissue quality (16).Consequently, the evaluation of body composition using CT, such as determining SAT radiodensity or detecting loss of skeletal muscle mass, is increasingly used as a non-invasive clinical tool with prognostic value.Importantly, early and appropriate identification of these abnormalities could enable interventions designed to rectify body composition abnormalities, potentially improving clinical outcomes (3).While a comprehensive analysis using SAT radiodensity and loss of skeletal muscle mass might predict prognosis in patients with HCC with greater accuracy, this approach is not yet fully clarified.In the present study, we aimed to evaluate the impact of muscle atrophy and SAT radiodensity on the prognosis of patients with HCC.

Materials and methods
In this retrospective study, we screened HCC patients who underwent proper CT imaging for evaluation of skeletal muscle mass and SAT radiodensity at Hokkaido University Hospital between July 2015 and May 2021.Patients were included if they were diagnosed with HCC according to Guidance by the American Association for the Study of Liver Diseases (17), had adequate clinical information and CT imaging for evaluation of skeletal muscle mass and SAT radiodensity, and were followed up for more than 3 months.We excluded patients with HCC who did not have adequate clinical and CT imaging information or were observed for a period of <3 months.
We collected the following clinical data: sex, age, etiology of chronic liver diseases, body mass index, blood test results, Child-Pugh class, TNM stage, BCLC class, presence of LC, and prognosis.In this study, LC was diagnosed based on liver biopsy, Fibroscan ® (Echosens, Paris, France) data, and/or radiologic findings, such as CT or magnetic resonance imaging, and laboratory data, as described in previous studies (18,19).We investigated the factors associated with prognosis in patients with HCC, including skeletal muscle mass and SAT radiodensity by multivariate analysis.Subsequently, we analyzed the impact of skeletal muscle mass and SAT radiodensity on prognosis of these patients.
The study protocol conformed to the ethical guidelines of the Declaration of Helsinki and was approved by the Ethics Committee of Hokkaido University Hospital.All patients provided written informed consent to participate.This study was registered at the University Hospital Medical Information Network Clinical Trials Registry as UMIN 000030755.

Skeletal muscle mass calculation by computed tomography imaging
In the present study, to assess muscle atrophy, skeletal muscle mass was evaluated using the psoas muscle index (PMI).PMI was calculated from CT imaging as follows: the sum of the L3 level crosssectional area of the right and left psoas muscle mass was identified by manual tracing and divided by height squared (cm 2 /m 2 ) (18).Muscle atrophy was also defined according to our previous study as follows: PMI values <3.74 cm 2 /m 2 for males, and < 2.29 cm 2 /m 2 for females (20).

SAT radiodensity calculation by CT imaging
SAT radiodensity was measured using abdominal plain CT scans taken at the third lumbar vertebra (L3) levels.CT values were measured for regions of interests of four circles on subcutaneous fat away from major vessels.The mean values of these four regions of interests were used as the regions of interest of SAT radiodensity (21).High SAT radiodensity was also defined according to a previous study as follows: High SAT radiodensity > −74 HU for males and > −83 HU for females (14).

Endpoint
The primary endpoint of this study was to identify the factors associated with overall survival.Overall survival was defined as the duration from the time of inclusion until death.Patients who were still alive or transferred other hospitals were censored on the date of the last follow-up registered in the medical record.

Statistical analysis
Continuous variables were analyzed using the Mann-Whitney U test.Categorical variables were analyzed using the Fisher's exact test.
Survival curves were constructed using the Kaplan-Meier method and compared using the log-rank test.Univariate Cox regression analysis was conducted for clinical factors and laboratory data; laboratory data that were included in the calculation of the Child-Pugh score and those that violated the proportional hazards assumption were excluded.Multivariate Cox regression analysis was conducted for factors showing significance (defined at p < 0.1) in the univariate analysis (22,23).All p-values were two-tailed, and the level of significance was set at p < 0.05.All statistical data were generated using Prism 7.03 (GraphPad Software, Inc., La Jolla, CA) and EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan).

Patient characteristics
Patients with HCC who visited Hokkaido University Hospital between July 2015 and May 2021 were screened for inclusion.Ultimately, 201 patients met the inclusion criteria and were included in this study (Figure 1).
The baseline characteristics of included patients with HCC are shown in Table 1.The median age of patients was 71 years (range: 20-90 years), and 76.6% of them were males.A total of 72.6% had LC.A total of 77.1% had Child-Pugh A. A total of 31.3 and 30.3% had BCLC stage 0 and A. Based on the PMI values, 33.3% patients had muscle atrophy.The definition of high SAT radiodensity in previous reports was adopted (SAT > −74 HU for males and > −83 HU for females) (14), and a total of 12.9% patients had high SAT radiodensity.

Comparison of prognosis between HCC patients with and without muscle atrophy
As shown in Figure 2, Kaplan-Meier survival analysis revealed that patients with muscle atrophy had a significantly poorer prognosis than those without muscle atrophy (log-rank p-value = 0.004; crude Cox regression hazard ratio [HR]: 1.878, 95% confidence interval [CI]: 1.18-2.99).The comparison between characteristics of patient with or without muscle atrophy are shown in Table 2. Patients with muscle atrophy exhibited significantly higher rates of high SAT radiodensity and lower body mass index compared to those without muscle atrophy.

Comparison of prognosis in patients with HCC with and without high SAT radiodensity
As shown in Figure 3, Kaplan-Meier survival analysis revealed that patients with high SAT radiodensity had a significantly poorer prognosis than those without it (log-rank p-value <0.001, crude Cox regression HR: 2.66, 95% CI: 1.18-5.96).Table 3 presents a comparison of characteristics between patients with and without high SAT radiodensity.Patients with high SAT radiodensity were significantly more likely to be male, have muscle atrophy and ascites, display lower body mass index, have reduced albumin levels, exhibit lower PMI in males, and have a worse Child-Pugh grade compared to those without it.

Comparison of overall survival among the four classified groups based on muscle atrophy and high SAT radiodensity
Given that high SAT radiodensity and muscle atrophy were found to be significant and independent prognostic factors in patients with HCC, we further classified the patients into four groups based on the presence or absence of muscle atrophy and high SAT radiodensity.As shown in Figure 4, Kaplan-Meier survival analysis revealed that the  overall survival among four groups was significantly different.In particular, the patients with muscle atrophy and high SAT radiodensity had a poor prognosis (median overall survival in the patients without muscle atrophy and with or without high SAT radiodensity was undefined, 4.29 years in the patients with muscle atrophy and without high SAT radiodensity, and 1.04 years in the patients with muscle atrophy and high SAT radiodensity; p = 0.002).

Stratified analysis
Based on the results presented above (Figure 4), we assessed the prognosis in the entire population, distinguishing between patients with both muscle atrophy and high SAT radiodensity, and those without both before conducting stratified analysis.Patients with both muscle atrophy and high SAT radiodensity had a significantly poorer prognosis than the other patients (log-rank p-value <0.001, Figure 5A; crude Cox regression HR: 4.53, 95% CI: 1.43-14.4).Subsequently, we conducted subgroup analyses based on clinical factors contributing to overall survival of patients with HCC.First, we performed the subgroup analysis stratified with Child-Pugh grade A, B, and C. A total of 77.1% patients had Child-Pugh grade A, and 22.9% had Child-Pugh grade B or C.Among the patients with Child-Pugh grade A, the patients with muscle atrophy tended to have a poor prognosis (log-rank p-value = 0.054, Supplementary Figure 2A; crude Cox regression HR: 1.66, 95% CI: 0.95-2.90),and the patients with high SAT radiodensity had a similar tendency toward a poorer prognosis (log-rank p-value = 0.116, Supplementary Figure 2B; crude Cox regression HR: 1.86, 95% CI: 0.67-5.14).Patients with both muscle atrophy and high SAT radiodensity had a significantly poorer prognosis than the other patients (log-rank p-value = 0.044, Figure 5B; crude Cox regression, HR: 3.48 95% CI: 1.03-11.70).Among the patients with Child-Pugh grade B or C, the patients with muscle atrophy tended to have a poorer prognosis (log-rank p-value = 0.054, Supplementary Figure 2C; crude Cox regression HR: 2.17, 95% CI: 0.95-4.91),and the patients with high SAT radiodensity had a significantly poorer prognosis (log-rank p-value = 0.001, Supplementary Figure 2D; crude Cox regression HR: 3.61, 95% CI: 1.06-12.29).Similar to patients with Child-Pugh grade A, patients with both muscle atrophy and high SAT radiodensity had a significantly poorer prognosis than other patients with Child-Pugh grade B or C (log-rank p-value <0.001, Figure 5C; crude Cox regression, HR: 10.43, 95% CI: 3.76-28.93).

Discussion
In this study, we demonstrated that muscle atrophy and high SAT radiodensity could significantly and independently predict prognosis in patients with HCC.Moreover, we highlighted the importance of evaluating both muscle atrophy and high SAT radiodensity as prognostic factors in patients with HCC because coexistence of muscle atrophy and high SAT radiodensity could predict quite poor prognosis in patients with HCC.Identifying this high-risk subgroup may facilitate the implementation of targeted interventions, including nutritional therapy and exercise, to potentially improve clinical outcomes.Prognosis in patients with HCC is reported to be associated with functional liver reserve, TNM staging, BCLC class, and performance status.In addition to these factors, loss of skeletal muscle mass is reported to be an important factor associated with prognosis in patients with HCC (8,(24)(25)(26).Furthermore, the importance of assessing adipose tissue-related parameters such as intramuscular fat, visceral fat, and SAT radiodensity for predicting prognosis has been recently reported (12-15, 27-29).Among these studies, Ebadi et al. conducted a study on cirrhotic patients, half of which had HCC, and reported that SAT density could stratify prognosis.Moreover, Ebadi et al. suggested that morphological rearrangements of SAT, diagnosed by SAT density, might occur prior to the loss of SAT mass ( 14).In the present study, we found that HCC patients with high SAT radiodensity had significantly worse prognosis, which was consistent with the results of previous studies.
SAT plays a pivotal role in lipid storage and energy homeostasis and is now recognized for its multifaceted endocrine function, with adipokines playing a role in the regulation of diverse metabolic and inflammatory processes (30).High SAT radiodensity not only indicates a reduced SAT volume but also implies remodeling of adipose tissue, characterized by morphological changes such as atrophy, evidenced by smaller and shrunken adipocytes, expanded interstitial space, and infiltration of mononuclear cells.Therefore, it is suggested that high SAT radiodensity reflects the severe energy depletion associated with cirrhosis, leading to unfavorable clinical outcomes (14).Survival rates in patients with normal and high subcutaneous adipose tissue (SAT) radiodensity Kaplan-Meier estimates of overall survival stratified according to SAT radiodensity in patients with HCC.The median overall survival is 6.02 years in patients with normal SAT radiodensity and 1.80 years in patients with high SAT radiodensity.Patients with high SAT radiodensity have a poorer prognosis than those with normal SAT radiodensity (log-rank p-value <0.001; crude Cox regression hazard ratio: 2.66, 95% confidence interval: 1.18-5.96).HCC, hepatocellular carcinoma; SAT, subcutaneous adipose tissue.This study highlighted that multivariate analysis revealed both muscle atrophy and high SAT radiodensity significant and independent poor prognostic factors.Furthermore, the coexistence of muscle atrophy and high SAT radiodensity was found to be a strong and significant predictor of poor prognosis in patients with HCC.To date, there have been limited studies performing simultaneous analysis of skeletal muscle mass and adipose tissue-related parameters as prognostic factors in HCC.Lim et al. reported that muscle depletion combined with visceral adiposity was a crucial prognostic factor for predicting survival in older patients with HCC who underwent transarterial chemoembolization (27).Additionally, Saeki et al. demonstrated that the absence of muscle depletion combined with a high visceral fat area could serve as a novel biomarker for patients with advanced HCC treated with sorafenib (28).Thus, combining assessment tools for muscle mass-and adipose tissue-related parameters may provide highly accurate stratification of HCC mortality risk.In patients with gastric cancer, subcutaneous but not visceral adipose tissue is a predictive marker of prognosis (31).This study is the first to demonstrate stratification of HCC prognosis based on a combination of muscle atrophy and SAT radiodensity.Furthermore, among Child-Pugh A patients, prognosis could not be stratified by the presence of muscle atrophy or high SAT radiodensity alone; however, the combination of muscle atrophy and SAT radiodensity proved effective in stratifying patients with poor prognosis.Therefore, simultaneous evaluation of muscle atrophy and high SAT radiodensity may hold clinical significance.
In patients with BCLC class B or more, muscle atrophy could not predict prognosis, whereas high SAT radiodensity could stratify prognosis.The reason why muscle atrophy could not predict the prognosis in those patients remains unclear; however, is a hypothesis.In patients with BCLC class B or more, muscle mass had already decreased due to advanced HCC, and thus, prognosis could be stratified using only SAT radiodensity.In addition, the combination of muscle atrophy and SAT radiodensity more clearly stratified the prognosis.It has been reported that adipose tissue remodeling is observed at the early stages of cachexia ( 14).Thus, this study's observation might reflect the presence of cancer-related cachexia, which results in poor prognosis.
Skeletal muscle mass is measured using bioelectrical impedance analysis (BIA), CT imaging with specialized software, CT imaging using a simple CT method and PMI calculation, and dual-energy X-ray absorptiometry.Among these, CT is commonly used for diagnostic purposes, recurrence screening, and evaluation of treatment response in patients with HCC (2,17,32).Our recent study highlighted that, for an accurate evaluation of relative changes in skeletal muscle mass in patients with chronic liver diseases, including HCC, CT imaging is a suitable method compared to BIA (33).In addition, CT imaging allows for the simultaneous evaluation of SAT radiodensity and muscle area.Therefore, CT imaging is a valuable tool not only for assessing HCC but also for evaluating the nutritional Our study had several limitations that need to be acknowledged.First, it was a retrospective study conducted at a single center.In addition, due to the nature of the study, certain clinical were either lacking or incomplete.Therefore, further validation of our results via a prospective multicenter study is required.
In conclusion, muscle atrophy and high SAT radiodensity are significantly associated with poor prognosis in patients with HCC.Our findings highlight the importance of evaluating muscle atrophy and high SAT radiodensity as prognostic factors in patients with HCC.This study is the first to report the significant negative impact of both muscle atrophy and high SAT radiodensity on survival in patients with HCC.In particular, for patients with muscle atrophy and high SAT radiodensity, interventions such as nutritional therapy and exercise may have the potential to improve their clinical outcomes.

FIGURE 5
FIGURE 5 Survival rates in all patients and in those stratified by Child-Pugh grade.(A) Kaplan-Meier analysis is performed to stratify patients with and without muscle atrophy and high SAT radiodensity.The median overall survival is 1.04 years in patients with both muscle atrophy and high SAT radiodensity and 6.02 years in the other patients.Patients with muscle atrophy and high SAT radiodensity have a poorer prognosis than the other patients (log-rank p < 0.001; crude Cox regression HR: 4.53, 95% CI: 1.43-14.4).(B) Among patients with Child-Pugh grade A, Kaplan-Meier analysis is performed to stratify patients with and without muscle atrophy and high SAT radiodensity.The median overall survival is 1.79 years in patients with both muscle atrophy and high SAT radiodensity and not reached in the other patients.Patients with both muscle atrophy and high SAT radiodensity have a poorer prognosis than the other patients (log-rank p-value = 0.044; crude Cox regression HR: 3.48, 95% CI: 1.03-11.70).(C) Among the patients with Child-Pugh grade B or C, Kaplan-Meier analysis is performed to stratify patients with and without muscle atrophy and high SAT radiodensity.The median overall survival is 0.40 years in patients with both muscle atrophy and high SAT radiodensity and 4.25 years in the other patients.Patients with both muscle atrophy and high SAT radiodensity have a significantly poorer prognosis than the other patients (log-rank p-value <0.001; crude Cox regression HR: 10.43, 95% CI: 3.76-28.93).CI, confidence interval; HCC, hepatocellular carcinoma; HR, hazard ratio; SAT, subcutaneous adipose tissue.

FIGURE 6
FIGURE 6Survival rates stratified according to BCLC class.(A) Among patients with BCLC class 0 or A, Kaplan-Meier analysis is performed to stratify patients with and without muscle atrophy and high SAT radiodensity.The median overall survival is 1.80 years in patients with both muscle atrophy and high SAT radiodensity and not reached in the other patients.Patients with muscle atrophy and with high SAT radiodensity have a poorer prognosis than the other patients (log-rank p-value <0.001; crude Cox regression HR: 4.51, 95% CI: 1.72-11.85).(B) Among patients with BCLC class B, C, or D, Kaplan-Meier analysis was performed to stratify patients with and without muscle atrophy and high SAT radiodensity.The median overall survival is 0.40 years in patients with both muscle atrophy and high SAT radiodensity and 2.18 years in the other patients.Patients with both muscle atrophy and high SAT radiodensity have a poorer prognosis than the other patients (log-rank p-value <0.001; crude Cox regression HR: 5.36, 95% CI: 2.34-12.28).CI, confidence interval; HCC, hepatocellular carcinoma; HR, hazard ratio; SAT, subcutaneous adipose tissue.

TABLE 2
Baseline clinical and biochemical characteristics of patients with or without muscle atrophy.
Data are presented as number, percentages of patients or median (range) values.BCLC, the Barcelona Clinic Liver Cancer; F, female; HBV, hepatitis B virus; HCV, hepatitis C virus; NBNC, non-HBV non-HCV; PMI, psoas muscle mass index; M, male; SAT, subcutaneous adipose tissue.

TABLE 3
Baseline clinical and biochemical characteristics of patients with normal SAT or high SAT.
Data are presented as number, percentages of patients or median (range) values.BCLC, the Barcelona Clinic Liver Cancer; F, female; HBV, hepatitis B virus; HCV, hepatitis C virus; NBNC, non-HBV non-HCV; PMI, psoas muscle mass index; M, male; SAT, subcutaneous adipose tissue.

TABLE 4
Prognostic factors in patients with HCC.
ALT, alanine aminotransferase; BCLC, the Barcelona Clinic Liver Cancer; Plt, platelet counts SAT, subcutaneous adipose tissue.FIGURE 4Comparison of overall survival among the four classified groups based on muscle atrophy and SAT radiodensity.The median overall survival is not reached in patients without muscle atrophy and normal SAT radiodensity and patients without muscle atrophy and high SAT radiodensity; it is 4.30 years in patients with muscle atrophy and normal SAT radiodensity and 1.04 years in patients with muscle atrophy and high SAT radiodensity.Overall survival is significantly different among the four classified groups based on muscle atrophy and SAT radiodensity (p = 0.002).HCC, hepatocellular carcinoma; SAT, subcutaneous adipose tissue.10.3389/fnut.2023.1272728Frontiers in Nutrition frontiersin.orgstatus of patients with HCC.Additionally, even muscle mass was assessed using the simple method (n = 145) (34), which is a simpler CT-based method than PMI, patients with both muscle atrophy and high SAT had a poor prognosis (log-rank p-value <0.001, Supplementary Figure 1; crude Cox regression HR: 4.79, 95% CI: 1.48-15.5).