{'fraction_non_alphanumeric': 0.0350971486925909, 'fraction_numerical': 0.019080379457687244, 'mean_word_length': 5.459295261239368, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 2, 'https://': 0, 'lorem ipsum': 0, 'www.': 3, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 2, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Non-alcoholic steatohepatitis and risk of hepatocellular carcinoma

Abstract The emergence of non-alcoholic fatty liver disease (NAFLD) as the leading chronic liver disease worldwide raises some concerns. In particular, NAFLD is closely tied to sedentary lifestyle habits and associated with other metabolic diseases, such as obesity and diabetes. At the end of the disease spectrum, non-alcoholic steatohepatitis (NASH) may progress to cirrhosis and hepatocellular carcinoma (HCC), representing a serious health problem to modern society. Recently, an increasing number of HCC cases originating from this progressive disease spectrum have been identified, with different levels of severity and complications. Updating the current guidelines by placing a bigger focus on this emerging cause and highlighting some of its unique features is necessary. Since, the drivers of the disease are complex and multifactorial, in order to improve future outcomes, having a better understanding of NASH progression into HCC may be helpful. The risks that can promote disease progression and currently available management strategies employed to monitor and treat NASH-related HCC make up the bulk of this review.


Introduction
Despite non-alcoholic fatty liver disease (NAFLD) affecting a great number of people (about one-quarter of the global population), only a small quantity of patients progresses beyond the initial stages of the disease into more severe conditions, with the onset of end-stage liver disease affecting less than 13% of NAFLD patients. [1] These stages are a reflection of the disease spectrum, characterized by specific histological signs, that range from non-alcoholic fatty liver (NAFL), to non-alcoholic steatohepatitis (NASH), to fibrosis and then cirrhosis. [2] Following this 'exacerbation path', after cirrhosis, patients develop hepatocellular carcinoma (HCC) and end-stage liver disease. Despite affecting a large portion of the population worldwide, we do not fully understand all factors contributing to disease progression, as well as the impact these factors have on clinical outcomes. NASH is defined by an accumulation of fat in hepatocytes (>5%) together with measurable signs of cell injury (hepatocyte ballooning and lobular inflammation) that are detected through histological examination. However, the fact that these histological changes to the normal liver cells can also be observed in alcoholic hepatitis, and that a liver biopsy is required for a definite diagnosis, indicates that the current diagnostic methodology has some limitations. [3][4][5][6][7][8][9] Recently, there has been a push towards updating the definition of NAFLD to a more accurate reflection of currently available knowledge. MAFLD, or metabolicassociated fatty liver disease, was proposed to an international panel of experts as an alternative nomenclature for NAFLD, where a consensus was reached regarding adopting this change, due to its many advantages such as positive diagnostic criteria, capturing the full spectrum of the disease (instead of just NASH and simple steatosis), as well as a concrete framework that includes other metabolic causes of fatty liver disease. [10,11] The diagnostic criteria of MAFLD are based on the presence of hepatic steatosis plus any of the next three criteria: elevated levels of body mass index (BMI), diabetes mellitus type 2, and evidence of metabolic dysregulation. [11,12] Obesity carries a negative impact in NASH patients, with elevated adiposity levels, on average, being associated with increased severity of fibrosis and inflammation. Likewise, diabetes (in particular type 2 diabetes), has been identified as a contributor to the development of NAFLD. Similarly, NAFLD being associated with an increased risk of diabetes has also been shown to be true. [13] As for HCC, it is more frequently associated with risk factors such as age (> 65 years old), diabetes, and metabolic syndrome. [14][15][16] This review aims to discuss the pathogenesis, disease progression, and current management of NASH-related HCC, which is a pertinent topic, now that NAFLD has become the most prevalent chronic liver disease in the world.

Pathogenesis
The path from NAFLD to HCC is underscored by multiple factors. From the simple accumulation of fat in hepatocytes to the more serious state of necroinflammation and fibrosis, it is currently understood that the foundation of all complications lies in the metabolic disturbance of NAFLD patients. [17] This section will explore the role of multiple factors in NAFLD development, such as insulin resistance, lipotoxicity, oxidative stress, and DNA damage response. [17] Insulin resistance Insulin resistance represents a growing problem that is exacerbated by the modern, western-influenced lifestyle and diet, and as the name states, implies a decrease in the effectiveness of the role that insulin molecules play in the regulation of glucose metabolism. Additionally, insulin resistance interferes in the metabolism of glucose, ketones, and lipids, generating an excess of free fatty acids (FFAs) that interfere with the normal function of the mitochondria in hepatocytes, playing a key role in the progression from NAFLD to HCC. [18,19] Initially, it was established that reduced insulin activation, together with lipid buildup, was associated with interference to the tricarboxylic acid (TCA) cycle, as well as, with increased production of reactive oxygen species (ROS). [18] In NASH patients, the production of ROS is almost doubled through a persistent increase in activity by the TCA cycle, despite suboptimal b-oxidation, insufficient lipid esterification, and impaired ketogenesis. [18,20,21] However, due to the adaptive capabilities of the hepatocytes to regulate their metabolism and to compensate for an initial disturbance (caused by increased FFAs and insulin resistance), changes in the TCA cycle may not be easily detected. [21] The problems caused by an increase in ROS, addressed in more detail in the oxidative stress segment, include inflammation, fibrogenesis, and DNA damage. Therefore, highlighting the importance of diabetes and insulin resistance in predicting more advanced stages of the disease, such as cirrhosis and HCC. [22][23][24] Lipotoxicity Following the changes introduced by the elevated levels of circulating FFAs, we can start to observe a change in the normal metabolic pathway, namely lipotoxicity. Lipotoxicity [25] is a phenomenon that occurs when elevated levels of free fatty acids interfere with the glucose metabolism pathway, which in turn, increases the production of toxic byproducts and the likelihood of tumor formation in the liver. [26] Due to the properties of liver tissue, namely its regenerative capabilities and its role in filtering the blood, the impact that NASH (a condition characterized by constant inflammation and, subsequently, fibrotic changes) has, is somewhat difficult to quantify. These properties also complicate the measurement of the impact on cellular DNA and, by consequence, its role in causing HCC. HCC cells show an upregulation of genes promoting de novo lipogenesis (a trait that is more predominant in cancer cells than in normal cells, which prefer to obtain fatty acids from circulation), [27,28] working together with the already accumulated lipids from steatosis, increasing ROS production, causing further oxidative stress in NASH patients, and, leading to HCC cells that adapt to the new lipotoxic environment (also known as lipid metabolic reprogramming [26] ). Due to the elevated energy requirements of tumor cells and the need to maintain high-energy metabolism, HCC cells can adopt a lipid-dependent metabolism, particularly when located in fatty liver tissue, which is abundant in lipids. [29] Understanding this process can provide an explanation to the limitation of current anti-angiogenesis medication (e.g., sorafenib and lenvatinib), as well as propose additional options for the treatment of NASH and prevent its progression to HCC. [20,26]

Oxidative stress
Oxidative stress is a syndrome that occurs when there is an increase in the production of free radicals (e.g. ROS) or cytotoxic oxidants (e.g. hydrogen peroxide), that damage tissues and cellular components due to unregulated oxidation. [30] This not only leads to hepatocyte apoptosis and consequently inflammation (due to immune response), but also fibrogenesis as well as DNA damage, as evidenced by studies reporting that NASH patients suffer more oxidative insults to hepatocytes than other diseases affecting the liver, particularly in patients having both NASH and HCC. [22,23] Normal physiological processes in the body produce a manageable sum of free radicals, the best example being ROS, which is a byproduct of aerobic respiration as part of adenosine triphosphate synthesis in the mitochondria. However, NAFLD patients have excessive free fatty acids being metabolized by hepatic mitochondria, leading to incomplete b-oxidation, impaired ketogenesis, and overproduction of ROS. [18,20,21] This saturation of ROS, together with inflammation, seems to be more often associated with oncological outcomes, observed not only in NAFLD patients but also in hepatitis patients. [24,[31][32][33][34] Additionally, ROS production isn't limited to byproducts of aerobic respiration, since macrophages and neutrophils are capable of employing ROS for their cytotoxic properties. [35] Mitochondrial dysfunction can be interpreted as a response to the unhealthy environment faced by the hepatocytes in a fatty liver. The elevated amount of (FFAs) that the mitochondria have to process require an adjustment to the regular energy production mechanism, furthermore, the response that the hepatocytes have towards apoptosis, necrosis, and inflammation motivates mitochondria to undergo mutations that promote cell viability. So mutations that affect the Chinese Medical Journal 2021;134 (24) www.cmj.org development of insulin resistance, rate of hepatic fat buildup, and promote metabolic reprograming (e.g. impaired ketogenesis and increased glycolysis while in the presence of oxygen) will increase the risk of NASH-related HCC. [36] DNA damage response Regarding DNA damage response, several studies have shown that carcinogenesis might not only appear as a consequence of a mutation caused by direct damage to the DNA but also as a response to the damage that is misinterpreted by anti-cancer mechanisms (e.g., ROS triggering ATM [a protein kinase that activates tumor suppression], causing apoptosis of non-cancer cells). [37][38][39][40] Various mouse models investigating the liver also compared DNA repair enzymes with the impact of oxidation on DNA, proving an inverse relationship between the two, inferring that genetic inheritance or acquired genetic characteristics of DNA repair enzymes, that differ between individuals, may be the underlying cause of variations of susceptibility to HCC and worsening of NAFLD. [37,41] Genetic predisposition to hepatic fat accumulation in the form of single nucleotide polymorphisms (SNPs) also represents a significant risk regarding disease progression to HCC. [42,43] Lastly, overexpression of DNA repair mechanisms (e.g., DNAdependent protein kinase) that can occur when there is a greater accumulation of DNA damage (possibly triggered by an increased sensitivity of these repair mechanisms as a response), has been correlated with a worse survival rate. [17,44,45] Disease progression and risk factors

Fibrosis
Well-designed longitudinal cohorts featuring multiple liver biopsies reveal a more complex progression from NAFLD, to NASH, and fibrosis than the common linear sequence of events. Regarding fibrosis, for example, the time required to advance from one stage to the next requires almost double the amount of time for NAFLD patients when compared to NASH patients (14 and 7 years, respectively). This is of particular notice since fibrosis remains the most significant factor for predicting mortality in NAFLD patients, even when adjusting for confounding factors. [2,[46][47][48] Accumulation of fibrotic tissue is problematic, being the most important step toward cirrhosis and HCC. [49] These bands of collagen and fibrotic changes are formed as part of the normal healing process, but without being restricted by natural degradation rates, or with continuous injury, this tissue accumulates, increasing in density and entanglement as a result, between cell colonies, and interfering with liver architecture. [50] This leads to the isolation of hepatocytes into nodules, separated by these bands, reducing blood flow and disrupting normal liver function. The cells in charge of producing this fibrotic tissue are named hepatic stellate cells, which go from a standby state (quiescent) to actively secreting tumor growth factor-b, producing collagen and establishing an extracellular matrix. [51][52][53][54] Monitoring these changes can allow physicians to establish an accurate prediction of disease progression rate and assess the level of severity, particularly, when compared with liver steatosis and inflammation measurement, which are easily influenced by changes (i.e., medication or lifestyle intervention). [55,56] Genetic factors Many investigations during this past decade were dedicated to identifying (SNPs) or other genetic markers that demonstrate susceptibility to NAFLD, NASH, and HCC. Some examples of studied SNPs are phospholipase domaincontaining 3 (PNPLA3), [57][58][59] transmembrane 6 superfamily member 2 (TM6SF2), [58,60] 17b-Hydroxysteroid dehydrogenase 13 (HSD17B13) [61,62] and membrane-bound Oacyltransferase domain containing 7 (MBOAT7). [63,64] Among these, PNPLA3 has the most concrete correlation with fibrosis and fatty hepatocytes, based on histology grading. [65,66] PNPLA3, TM6SF2, and MBOAT7 represent genetic variations related to fat build-up in liver cells while an inhibited HSD17B13 variant was associated with a reduced risk of NAFL to NASH. [42] Alteration to the standard lipid metabolism can promote metaplastic changes in hepatocytes. [67] Due to the quick nature of tumor growth in cancer, the aggregated mass often outgrows vascularization, resulting in a low oxygen environment. [68] To overcome this cause of cell death, the tumor will modify its genetic expression (e.g. upregulating HIF-2a [EPAS1] gene, which is a part of a family of genes of key importance to hypoxic environment response) which in turn, can increase its lethality, resistance to treatment and expanded metastasis regions. [69][70][71][72][73][74] HIF-2a, due to its role in promoting disease progression in a subset of patients with both steatosis and HCC, could be a valuable target for the treatment of NAFLD-related HCC. [75] Desterke et al released a study that attempted to identify all genes involved in NASH progression, as well as genes/proteins involved in the transition from NASH to HCC, using data-mining and text-mining, finding 25 genes for NASH development and 44 genes/proteins in the advancement from NASH to HCC. [76] Initially, to find out which genes were relevant for the study, the authors searched for genes that were expressed distinctly between NASH patients and obese patients with healthy livers. Of the 25 genes found for NASH, 22 were upregulated and three were downregulated (when compared with healthy obese patients). Following this step, patients were then organized into three subsets: healthy obese, NAFL + obese, and NASH groups. Furthermore, the gene YWHAZ [77] was responsible for leading a precancerous pathway according to the analysis results. [76] This study, not only narrowed down the list of lipid-related genes that are associated with disease progression from NAFL to HCC but also, for the first time, organized these genes together with their roles and interactions mapped out in a "canonical pathway". [76] The pathway clarifies the role that these genes have in increasing the synthesis of triglycerides, cholesterol, and de novo fatty acids; in promoting inflammatory processes and chemo-attraction; higher FFA accumulation due to the disruption of the mitochondria and its beta-oxidation; and, faster development of insulin resistance and cancer progression. [76] To prove these findings, an additional stratified test was performed, using new patients subdivided into healthy Chinese Medical Journal 2021;134 (24) www.cmj.org controls, NAFL and NASH. Three genes were selected to test for inflammation and cancer progression (YWHAZ, SMPD2, CCL2), and the other three genes (FASN, CIDEC, VLDLR) were selected for testing the metabolism and formation of lipid droplets. The results displayed significantly higher activity in the NASH group than in the other two groups. [76] Besides the mentioned 25 genes involved in the progression from NAFL to NASH, 5 additional genes had a role in cancer development. So to comprehend the roles these 30 genes play in the progression from NASH to HCC, datasets from Gene Expression Omnibus (GEO), [78] in the National Center for Biotechnology Information, and from The Cancer Genome Atlas project consortium [79] were selected to perform an analysis. [76] 13 genes had roles related to lipid metabolism and accumulation, as well as inflammation and cancer processes. The later genes were observed in increased quantities in both cirrhotic and HCC patients, with a marked decrease in lipid accumulation genes, which might be related to the metabolic alterations that liver neoplastic cells undergo, using the stored fat for energy production. [69] 5 genes in particular (DGAT1, FASN, YWHAZ, LPL, and IRS2) were identified as upregulated in over 4% of HCC patients, indicating a less optimistic prognosis, with increased possibilities of a relapse. From data-mining studies like this, we can conclude that patients identified with the genes specified in this study possess an increased risk of disease progression since the pathway established ascertains a connection to most processes associated with steatosis, inflammation, insulin resistance, and HCC. [76] Wu et al found that tumor suppressor Zinc fingers and homeoboxes 2 has a key role in regulating hepatic fat homeostasis, both in healthy and neoplastic cells, observed in the charges resulting from alterations in the expression of this gene. [80] Fujiwara et al [81] tested that downregulating carnitine palmitoyltransferase 2 not only allowed HCC to resist lipotoxic effects but also increase the generation of hepatic cancer cells. Other mutations types of mutation, such as telomerase reverse transcriptase (TERT) promoter mutations are additional risk factors for HCC since overexpression of telomerase is observed in 90% of human tumors. [82,83] These occur frequently in HCC patients (from 60% to 90%), with a few studies using TERT mutation as a therapeutic target with little success. [84] However, Akuta et al [85] have proven the feasibility of TERT promoter mutation (TERT C228T) in diagnosing primary HCC, which performed better than alpha-fetoprotein (AFP). Liver cancer diagnosis in family members can be correlated with an increased probability of developing HCC, with both inherited and acquired factors promoting carcinogenesis. [86,87] In addition to determining how different ethnicities have different inherent susceptibilities, NAFLD inheritability has been discussed in multiple twin studies. [88][89][90][91] Furthermore, a study performed on an ethnically diverse cohort, comparing Hispanic, Caucasian, and African Americans found that a higher prevalence of NAFLD manifests in Hispanics and is the least prevalent in African Americans. [92] A possible explanation for this distribution could lie in the metabolism of fat, wherein Western cohorts, it was verified that NAFLD patients, who were ethnically African American, have lower levels of TGs and higher high-density lipoprotein cholesterol when compared with that of Hispanics and Caucasians. [92] Hormonal factors Besides the often discussed metabolic causes of NAFLD, endocrine diseases (commonly responsible for hormonal imbalance) and non-pathological conditions, like aging and menopause, seem to have a clear impact on the disease prevalence and outcome severity.
Sex has long been identified as an independent risk factor for the development of NAFLD and is addressed as such in multiple studies, acknowledging that male patients have a higher risk of developing NAFLD and have a more rapid progression to more advanced stages of the disease when compared to female patients. Furthermore, HCC also has a higher onset in males than females. [56,93] However, this comparison is often performed without distinguishing menopause status, which represents a shift in the hormonal balance of women. When comparing pre-menopausal with post-menopausal women, the latter have a higher prevalence of NAFLD. Additionally, women that undergo HRT (hormone replacement therapy) seem to have lower NAFLD prevalence when compared with women that didn't, suggesting the possibility that these hormones might have a protective effect in NAFLD. [94][95][96][97][98] Regarding HCC, a study that analyzed the protective effect of sex in overall survival in 39,345 patients, observed that this effect was more predominant in women between the ages of 18 to 44. [99] This implies that improving the current guidelines concerning sex as a risk factor might provide a more accurate assessment of disease prevalence and risk outcome.
Thyroid hormone levels can also have a protective effect in NAFLD, as observed in studies relating hyperthyroidism with lower NAFLD prevalence. Hypothyroidism has been identified in multiple studies as having an increased risk of prevalent NAFLD, however, several studies seem to have conflicting results regarding which thyroid hormones are relevant (subclinical type vs. overt type hypothyroidism), which hormone levels are significant, and some publications conclude that there is no correlation to NAFLD prevalence with either type. [100][101][102][103][104] The current lack of consensus reflects that there is room for further investigation, and the role that thyroid function has in risk prediction should not be ignored. Regarding HCC, a study performed by Pinter et al found that elevated free tetraiodothyronine (T4) was associated with a poor survival outcome while increased levels of thyroidstimulating hormone (TSH) were correlated with bigger tumors (despite being unrelated to survival). [105] Sahin et al observed that elevated free triiodothyronine (T3) was associated with HCC progression. [106] As such, thyroid function can be considered a potential risk factor for NAFLD, as well as a possible marker to measure disease progression in HCC.

Lifestyle choices
Of all risk factors known to affect the disease progression of NASH, routine lifestyle behavior is expected to have one of the most important impacts on outcome and severity. A correlation was observed between NAFLD and living locations in proximity to a large variety of food options, Chinese Medical Journal 2021;134 (24) www.cmj.org consumption of processed foods, unbalanced diets leaning heavily towards high meat/fat to fresh fruit ratio, and little regular exercise. [107][108][109][110][111][112] Although the strength of such observations might vary in different countries, there is enough evidence to establish a strong association between an unhealthy lifestyle and the presence of NAFLD. [91] Epidemiological studies Studies documenting prevalence data of NAFLD-related HCC are less rigorous. A relative level of selection bias seems to be applied in available data, particularly when access to large databases is not possible. [113][114][115][116] Cryptogenic cirrhosis shows many similarities with NASH-related cirrhosis (leading to an underestimation of NAFLD), and with an increased awareness of the disease, it could inflate the growing curve representing the number of patients diagnosed every year for the past decades. [115,117] A United States study collected information on 170,540 adult patients from a transplant waitlist database. [118] This data was collected over a span of 15 years (2002-2017), and 17% of the patients were listed with HCC, and of those patients, roughly 16% had no discernible etiology. However, 24,431 patients had identifiable causes, of which, 2520 patients (10.3%) were identified as having NASH as the cause of HCC. This study features a very large sample size of NASH-HCC patients, and it allows us to find an approximate proportion of NASH within the varied etiologies of HCC, in an American population. [118] Information regarding the distribution of NASH-related HCC patients is not easily available, particularly in such a large sample size.
Other publications that feature extensive data regarding the epidemiology of NASH in the context of hepatocellular carcinoma focus more extensively on NASH-related cirrhosis rather than HCC, [119] identifying NASH as the third most common risk factor for liver cirrhosis. [120] Since cirrhosis is a valuable factor in increased mortality of liver patients, both findings implicate the importance that NASH has not only as an earlier predictor of mortality risk, but its role in the cause of mortality as well.
NASH-related HCC also fuels debate and discord regarding its monitoring and management. Since the progressive nature of this metabolic disease has already been established, the need to closely monitor patients can have a very important impact on management and early intervention. On the other hand, since the number of NAFLD patients that end up developing HCC is quite low in proportion, it can be argued that repetitive monitoring might be costly and devoid of practicality, that unless the liver reaches the stage of cirrhosis, monitoring for HCC is possibly premature. [121] However, documented reports of significant correlation between non-cirrhotic steatosis/ steatohepatitis patients and HCC have been established as well, showing that NASH alone could progress to HCC, contradicting the idea that pre-cirrhosis monitoring is premature. [122][123][124] Sahil et al [125] reports that, in the absence of cirrhosis, NAFLD and metabolic syndrome are the most common causes of HCC, when compared with viral hepatitis and other causes. In NAFLD-related HCC patients, this study showed that around one-third (34.6%), had no evidence of cirrhosis. However, in this study, there was no assessment of the level of threat implied by noncirrhotic NAFLD-related HCC, and the benefits that screening non-cirrhotic NAFLD patients will have in treatment and disease outcome when weighed against the added cost (caused by the large increase in the number of patients to screen). A different study observed that, frequently, tumor size found in non-cirrhotic HCC patients appeared to be larger. [124] This could be a result of late detection of the tumor, due to its asymptomatic nature, which could be an argument for more aggressive screening. This hypothesis is also presented by Dasari et al [126] when investigating the overall survival post-surgery of noncirrhotic and cirrhotic HCC patients. Regarding liver transplantation, its viability as a treatment option is limited by very high recurrence rates (50%), organ donor shortage, rejection or immunosuppressant complications, cost, and possible surgery complications. [126] Further studies are required in order to establish a more reasonable guideline, improve the current diagnostic/ monitoring methods, and develop a more thorough understanding of the mechanisms that dictate neoplastic transformation of hepatocytes. When comparing the severity of HCC between NAFLD patients and viral hepatitis patients, the lifespan of NAFLD patients was significantly reduced, while those with NAFLD-related HCC carried a worse outcome in terms of mortality rate. [121] Disease management Disease progression in NAFLD patients is relatively slow, with most patients spending years without any symptoms. This added to the fact that the main preventative measure of progression is introducing changes to lifestyle habits, and on a consistent basis to make a significant difference, which can be highly dependent on the patient motivation to maintain such a healthy lifestyle. The management of NAFLD/NASH-related HCC is not yet addressed by any of the major guidelines, this is mainly due to the fact that when the consensus was established regarding HCC management, its main cause was viral hepatitis [17,127,128] and that no approved pharmacologic agents can be directed for NASH patients. Comparatively, viral hepatitis is much simpler to manage, unlike NASH, where it usually requires targeting other associated complications and comorbidities. As mentioned above, current evidence suggests a difference in the outcome between NAFLDrelated HCC and HCC of other etiologies. [121] Studies regarding the establishment of a stage-based approach to diagnosis and treatment of NAFLD, as well as the introduction of novel, noninvasive biomarkers to correctly identify the histological progress of the disease, have been performed. [129][130][131][132] From lipid signatures specific to NASH to biomarkers detecting advanced fibrosis, and recommending, for example, the introduction of pharmacological treatment during the intermediate stage, screening for HCC and esophageal varices at the late stage of NASH, and lifestyle interventions and diet regimen control across all stages. [129,[132][133][134] Beyond laboratory tests, imaging technology has also been a focus when tackling the noninvasive diagnosis and staging of NAFLD. [49,131] Abdominal ultrasound (US) examination is a common test used to screen and monitor HCC, particularly if the patient liver has already shown signs of fibrotic tissue, with followup examinations being repeated in periods between 4 and 6 months. According to the American Association for the Study of Liver Diseases guideline, [128] computer tomography (CT) or magnetic resonance imaging should be used for further verification for HCC if abnormal US and AFP levels were found in severe fibrosis patients (stages [3][4]. Confirming the diagnosis of NASH, however, still requires performing a liver biopsy, which remains the diagnostic 'gold-standard', despite its invasiveness and cost. [117,135,136] Abdominal US often lacks sensitivity, as ultrasound waves are influenced by large amounts of abdominal fat, which may compromise their accuracy, particularly in patients with very elevated (BMI).
Furthermore, since the results obtained can vary depending on the experience of the operator, US accuracy in detecting NASH becomes less rigorous. [127,137] Cost-effectiveness is very important when discussing the feasibility of monitoring techniques, particularly in diseases like NASH, that are complex in management as well as requiring long-term follow-ups. Therefore, studies aiming to document factors that can increase accuracy, improve risk assessment and reduce cost, and therapeutic targets are needed. [17] Certain cancer-targeted biomarkers such as circulating tumor DNA, or extracellular vesicles, [138,139] showed potential screening capabilities for HCC, and research related to telomere shortening has found indications of the dysfunction of hepatocytes, generation of fibrosis, and interference in the metabolic pathway of lipids, all factors related to disease progression risks. [17,140] Despite liver biopsy being the 'gold standard' of NAFLD and NASH diagnosis, when possible, HCC should be screened by imaging techniques. While liver biopsy still has the highest diagnostic accuracy, HCC is easy to notice under contrastenhanced imaging when compared to NAFLD, but more importantly, the invasiveness of the biopsy introduces a risk of spreading the cancer cells and bleeding, while risking incorrect assessment due to sampling location. [141,142] Future progress will likely phase out biopsies and expensive imaging tests, in favor of targeted cellular components, such as proteomic and genomic measurements, and cost-effective serum exams.

Treatment
Treatment strategy guidelines for HCC traditionally follow the format of matching treatment options to disease stages, like the Barcelona Clinic Liver Cancer guidelines, however, studies that have more flexible approaches to treatments, where administering therapy from the next stage of the disease concurrently with the current stages' treatment have provided different advantages and disadvantages to the established stage hierarchy system. [143] Furthermore, changes to the current therapy methods will be reliant on either the development of new strategies or an increase in the feasibility of existing ones.
Immunotherapy has untapped potential but its reliability has still not been proven and it is still being tested in clinical trials. [144] Meanwhile, the greatest progress made in HCC management has been the improvement in the diagnostic methods and screening, that allow for the established treatment methods to have a greater impact on survivability by treating the tumor in earlier stages, since HCC is a type of cancer that was conventionally identified in later stages of the disease. [145] Established treatment methods for NAFLD-related HCC can be divided into three categories: lifestyle changes, surgery, and pharmacology. Lifestyle changes (mainly diet control and frequent exercise) have a positive effect on slowing or reversing NASH progression, but once HCC is confirmed, more significant measures need to be adopted. Further study on the impact that behavioral changes have in NAFLD-related HCC patients is necessary.
Surgical options for HCC patients include liver resection, ablation, chemoembolization, radioembolization, and liver transplantation. The liver resection is limited to smaller-scaled tumors as a general rule, but the thresholds for operable size and the number of tumors can vary in different countries, which in accordance with EASL-EORTC guidelines, can reach 5 year survival rates between 60% and 80%. [127,146,147] Thermal ablation is mainly used in early HCC and embolization can be used from early to advanced HCC. [148] Radiofrequency ablation (RFA) has been thoroughly researched, and its performance grants 5-year survival rates of about 40% to 70% by itself, however, combination with other methods (e.g. chemoembolization) has been investigated as well. [127,149] Transcatheter arterial chemoembolization (TACE) plus CT-guided (RFA) has produced very good results, with an overall survival rate of 96.1%, 76.7%, and 41.3%, for 1, 3 and 5 years, respectively. However, it has yet to be validated in Western centers. [150,151] Liver transplant carries fewer risks for the patient when compared with resection, and may sometimes help treat the underlying cause for liver disease. [152] The Milan criteria, featuring over 17 thousand patients, established a 5-year survival rate ranging between 65% and 78%. [153] However, the main disadvantages of transplantation are donor shortage and possible immune response/rejection. [154] Drug treatment for end-stage HCC patients can be subcategorized into first-line and second-line drugs. Firstline includes two tyrosine kinase inhibitors (TKIs), sorafenib [155] (established) and lenvatinib [156] (novel), with a life expectancy increase of about 10 weeks. Additionally, atezolizumab and bevacizumab have been administered as a combination therapy in a Phase III study, obtained FDA approval, with 'overall survival' numbers superior to sorafenib. [157,158] Second-line drugs include regorafenib (TKI), [159] cabozantinib (TKI), [160] and ramucirumab (anti-angiogenic), [148] with an average increase of life expectancy of 3.6, 10.2, and 8.5 months, respectively. [161] Ramucirumab is a phase III trial drug that presents an alternative to sorafenib, being effective in patients with AFP levels over 400, but no difference in efficacy. Nivolumab [162] on the other hand, belongs to the novel immunotherapy category, with an overall response rate of 20%, currently in phase III clinical trial, performing significantly better than sofenib. [163,164]

Conclusion
The continuous rise in prevalence of NAFLD patients worldwide warrants the need to coordinate appropriate healthcare measures. However, the lack of disruptive symptoms renders its early development relatively unnoticed by those affected, and thus conducive to the progression of simple steatosis to NASH and/or fibrosis, and ultimately evolving into HCC. The role played by lipotoxicity, insulin resistance and mitochondrial activity reveal a possible route of pharmacology research for NASH management. However, the mechanism underlying the neoplastic change among NAFLD patients is poorly understood; therefore, future studies are suggested to focus on NASH-related HCC investigations.

Funding
This work was supported by grants from the National Natural Science Foundation of China (No. 82070588), High Level Creative Talents from the Department of Public Health in Zhejiang Province (No. S2032102600032), Project of New Century 551 Talent Nurturing in Wenzhou. This work is a part of the PERSONS study.

Conflicts of interest
None.