The Current View of Nonalcoholic Fatty Liver Disease-Related Hepatocellular Carcinoma

Simple Summary The incidence of nonalcoholic fatty liver disease (NAFLD)-related hepatocellular carcinoma (HCC) is increasing. However, an effective screening or surveillance method is not established. Recently, the NAFLD/nonalcoholic steatohepatitis (NASH) guidelines of Japan were revised to incorporate new strategies and evidence for the management and surveillance of NAFLD/NASH. Advanced fibrosis and lifestyle-related and metabolic comorbidities, especially obesity and diabetes mellitus, are associated with HCC development. At the first screening, serum markers of hepatic fibrosis (hyaluronic acid, type IV collagen 7S, and mac-2 binding protein), or the fibrosis (FIB)-4 index or the nonalcoholic fatty liver disease fibrosis score (NFS), or a platelet count should be evaluated. When liver fibrosis is indicated, consultation with a gastroenterology specialist should be considered for the second screening. The risk of HCC should be stratified using the FIB-4 index or the NFS. Liver stiffness should be measured using vibration-controlled transient elastography in those at intermediate or high risk. Blood tests and imaging should be performed every 6–12 months in patients with advanced fibrosis for HCC surveillance. We review here what is known about NAFLD-HCC and provide perspectives for future research. Abstract Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome and can develop into hepatocellular carcinoma (HCC). The incidence of NAFLD-related HCC, which is accompanied by life-threatening complications, is increasing. Advanced fibrosis and lifestyle-related and metabolic comorbidities, especially obesity and diabetes mellitus, are associated with HCC development. However, HCC is also observed in the non-cirrhotic liver. Often, diagnosis is delayed until the tumor is relatively large and the disease is advanced; an effective screening or surveillance method is urgently required. Recently, the NAFLD/nonalcoholic steatohepatitis (NASH) guidelines of Japan were revised to incorporate new strategies and evidence for the management and surveillance of NAFLD/NASH. Fibrosis must be tested for noninvasively, and the risk of carcinogenesis must be stratified. The treatment of lifestyle-related diseases is expected to reduce the incidence of NAFLD and prevent liver carcinogenesis.


Introduction
The prevalence of nonalcoholic fatty liver disease (NAFLD) has been growing worldwide, and the incidence of this disease in Japan is estimated to be 29.7% [1]. NAFLD may give rise to hepatocellular carcinoma (HCC); such HCC is becoming more common [2]. However, the characteristics of, risk factors for, and prognosis of NAFLD-HCC have not been elucidated fully. The American Association for the Study of Liver Diseases [3], European Association for the Study of the Liver [4], and American Gastroenterological Association (AGA) [5] have published practice guidelines for NAFLD management. Recently, the Japanese NAFLD/nonalcoholic steatohepatitis (NASH) guidelines [6] were revised to

Characteristics and Pathogenesis of NAFLD-HCC
The "multiple parallel hits" hypothesis has been advanced to explain NASH onset [42]. Adipose tissue lipotoxicity, insulin resistance, lifestyle-related diseases, alcohol consumption, growth and sex hormone-related factors, menopause, aging, oxidative stress (disruption of iron and free fatty acid levels), and changes in the profiles of gut and oral bacteria and Helicobacter pylori are thought to contribute to NASH pathology and carcinogenesis. The hyperinsulinemia associated with insulin resistance may promote cell proliferation and trigger carcinogenesis. Cellular stress responses, including autophagy and endoplasmic reticulum-associated activity, may trigger NAFLD cytotoxicity [43]. In addition, innate immune reactions between the products of intestinal bacteria and the liver increase deoxycholic acid levels, triggering the senescence of hepatic stellate cells [44]. The senescence-associated secretory phenotype, which features the extracellular secretion of inflammatory cytokines (tumor necrosis factor-α and interleukin-6), chemokines, and extracellular matrix-degrading enzymes from senescent cells, may trigger liver carcinogenesis [45]. Furthermore, changes in the profiles of hepatokines, including angiopoietin-like factors and fibroblast growth factors [46], and acylcarnitine may be involved in carcinogenesis [47] Genetic influences on NASH and carcinogenesis have been reported. The gene encoding the patatin-like phospholipase domain-containing 3 (PNPLA3) protein is widely accepted to affect disease susceptibility. The frequency of "risky" alleles is elevated in patients with liver cancer; the gene is thus presumed to be associated with carcinogenesis [48]. Additionally, 17-beta hydroxysteroid dehydrogenase 13 (HSD17B13) was identified as having the protective role of NAFLD [49] and protecting against HCC development [50]. Lower HSD17B13 in non-cancerous tissues was associated with worse recurrence-free survival and overall survival in HCC patients. Although they mainly studied viral hepatitis-associated HCC, further study is required.
Like similar liver diseases, NAFLD-HCC is characterized by a trabecular phenotype [15]. In patients with a common subtype of NAFLD-HCC, Salomao et al. [51] reported cancers with the morphological characteristics of steatohepatitis and termed this phenotype steatohepatitic HCC (SH-HCC). The expression of markers of hepatocellular adenoma is not specific to NAFLD-HCC; but is evident in some patients with NAFLD-HCC [52]. Notably, the levels of serum amyloid A and c-reactive protein were increased significantly in patients with SH-HCC. In addition, HCC tumors in non-cirrhotic livers are large, and may have arisen from hepatocellular adenomas [31].

Risk Factors for NAFLD-HCC
In a study of 622 patients with decompensated cirrhosis, Muto et al. [53] found that the risk factors for liver cancer were male sex, diabetes, high body mass index (BMI), AFP level ≥ 20 ng/mL, and low serum albumin level. A 16-year prospective follow-up study of 900,000 persons in the United States showed that among patients with BMIs ≥35 kg/m 2 , the relative risk of death from HCC was 4.52 (95% CI 1.13-2.05) in males and 1.62 (95% CI 1.40-1.87) in females [54]. Obesity is a risk factor for HCC and HCC-related death.

Treatment of NAFLD-HCC
NAFLD-HCC treatment is determined by liver function and the extent of HCC progression, in accordance with the Liver Cancer Practice Guidelines [60,61] and Barcelona Clinic Liver Cancer staging [62]. However, postoperative mortality is higher in patients with NAFLD-HCC than in those with HCV-HCC [62]. Vascular lesions (complications of lifestyle-related diseases) may be involved.
In patients with end-stage liver disease, NAFLD-HCC is an indicator for LT; NAFLD/NASH has been the fastest growing indicator for LT over the past 20 years [63,64]. Wong et al. [24] studied 61,868 patients who had undergone LT, including 10,061 HCC cases; the incidence of NASH-HCC increased from 8.3% in 2002 to 13.5% in 2012. About 50% of transplant recipients with NASH develop recurrent NAFLD, but the post-LT outcomes of patients with NAFLD are similar to those of patients without NAFLD [65].

Prevention of NAFLD-HCC
A meta-analysis of the effects of drugs used to treat lifestyle-related conditions on carcinogenesis showed that metformin taken by patients with type 2 diabetes was associated with a reduced risk of HCC [73]. In a large national cohort study, Kaplan et al. [74] found that metformin use was associated independently with a decline in overall mortality, but not liver-related mortality, HCC, or decompensation, in patients with cirrhosis. A large meta-analysis of 4298 HCC cases showed that statins reduced the HCC incidence by 37%, and that the treatment of lifestyle-related diseases may suppress HCC [75].
Recent analysis demonstrated that bariatric surgery was reduced NASH-HCC [76]. Bariatric group showed lower incidence of new-onset HCC (0.05% vs. 0.34%, p = 0.03) comparing to the propensity-matched control group. It might become an option for HCC prevention.

NAFLD-HCC Screening and Surveillance
NAFLD/NASH-HCC is often diagnosed when the tumor is relatively large and the disease is thus advanced [15]. Bertot et al. [77] reported that patients with incidentally diagnosed NAFLD cirrhosis exhibited decreased platelet counts and international normalized ratios (both p < 0.05) and were more likely to have HCC (12%). In contrast, a surveillance group (who underwent liver ultrasound every six months) had no HCC complication. Screening and surveillance are necessary. As the cancer risk associated with NAFLD/NASH is minimal, regular surveillance of low-risk groups is inefficient and uneconomical. The risk factor for liver carcinogenesis in patients with NAFLD/NASH is advanced liver fibrosis; such patients require strict follow-up by a general physician applying the algorithm of the revised Japanese guidelines (Figure 1) [6]. At the first screening, serum markers of hepatic fibrosis (hyaluronic acid, type IV collagen 7S, and mac-2 binding protein), or the FIB-4 index or the nonalcoholic fatty liver disease fibrosis score (NFS), or a platelet count should be evaluated. When liver fibrosis is indicated, consultation with a gastroenterology specialist should be considered for the second screening.
Castera et al. [78] found that such simple, inexpensive, and widely available assays had high negative predictive values, effectively ruling out advanced fibrosis. Patients at low risk of advanced fibrosis (FIB-4 score < 1.3 or the NFS score < −1.455) do not require further assessment. Liver stiffness should be measured using vibration-controlled transient elastography in those at intermediate (FIB-4 score 1.3 to 2.66 or NFS score −1.455 to 0.674) or high (FIB-4 score ≥ 2.67 or NFS score ≥ 0.675) risk. Although no NAFLD/NASH-HCC surveillance method has been established, blood tests and imaging should be performed every 6-12 months in patients with advanced fibrosis.
Recently, the AGA proposed an HCC surveillance method for patients with NAFLD and liver cirrhosis, or severe fibrosis revealed by noninvasive testing [5]. When the quality of ultrasonography is suboptimal for screening of HCC, future screening by either computed tomography (CT) or magnetic resonance imaging (MRI) every six months are recommended. The GALAD score (based on age, sex, and AFP, AFP-L3, and DCP levels) is also used to estimate fibrosis [79]. In terms of imaging, abdominal ultrasonography is cost effective and noninvasive, but its diagnostic sensitivity is poor in obese patients [80]. Although CT is useful, patients with NAFLD often have renal disorders contraindicating the use of contrast media, and antidiabetic drugs (especially biguanide) must be stopped a few days before contrast enhancement. Although MRI detects HCC with high sensitivity, it is costly and its contraindications include pacemaker presence and renal dysfunction. The establishment of an efficient, cost-effective method for the screening of those at high risk of HCC is desirable. Figure 1. The algorithm for hepatocellular carcinoma screening and surveillance contained in the revised Japanese guidelines [6]. AST, aspartate aminotransferase; ALT, alanine aminotransferase; BMI, body mass index; FIB-4, Fibrosis-4; NFS, nonalcoholic fatty liver disease fibrosis score; IFG, impaired fasting glucose; NAFLD, nonalcoholic fatty liver disease; HCC, hepatocellular carcinoma; US, ultrasonography.

Perspectives for Future Research
The development of tyrosine kinase inhibitors and immune checkpoint inhibitors affected a paradigm shift in HCC treatment [81,82]. However, the molecular and biological mechanisms underlying NAFLD-HCC development are not fully understood. Shimada et al. [83] classified HCCs by reference to genetic and immune profiles. Proteins that play key roles in HCC development would be valuable biomarkers for NAFLD-HCC screening. Molecular profiling might reveal target molecules for NAFLD-HCC therapy. It might be able to apply for personalized therapy and also when loco-regional treatment is not feasible. In addition, extracellular vesicles (EVs) [84][85][86] and CRISPR-Cas9-mediated genomic editing [87] are new tools for cancer therapy. EVs have shown great potential as drug delivery systems; exosomal micro-RNAs from HCC cells enhance transformed celllike growth of recipient cells. Genomic editing of genes such as PNPLA3 may potentially treat NAFLD-HCC.
In terms of NAFLD-HCC prevention, metformin and pioglitazone reduced NAFLD-HCC development in patients with type 2 diabetes; statins were effective in those with dyslipidemia. Several novel NAFLD treatment agents are under evaluation. Future studies will explore new monotherapies and combination therapies.

Conclusions
The incidence of NAFLD-HCC will increase as obesity increases. Efficient surveillance is essential. Fibrosis should be evaluated noninvasively and the risk of cancer should be stratified. The treatment of lifestyle-related diseases prevents liver carcinogenesis. Further studies should seek to identify more noninvasive markers of fibrosis and NAFLD-HCC.
Author Contributions: Conception, and design: T.K. Drafting of the manuscript: T.K. and K.T. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.

Institutional Review Board Statement:
The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of TWMU.
Informed Consent Statement: Informed consent was obtained from the patient for the publication of our study.

Data Availability Statement:
The datasets used and/or analyzed in this study are available from the corresponding author upon reasonable request.