Polymorphisms in Interleukin 13 Signaling and Interacting Genes Predict Advanced Fibrosis and Hepatocellular Carcinoma Development in Non-Alcoholic Steatohepatitis.

BACKGROUND
non-alcoholic steatohepatitis (NASH) recently headlined hepatocellular carcinoma (HCC) worldwide. This study aims to unveil the role of some unaddressed critical players that might aid in understanding, predicting, and targeting NASH and NASH-HCC.


METHODS
Serum interleukin 13 (IL-13) levels and single nucleotide polymorphisms (SNPs) within interleukin (IL)-13 rs20541, IL-13 receptors (IL-13R1) rs2248841, (IL-13R2) rs5946040, signal transducer activator of transcription 6 (STAT6) rs167769, yes-associated protein (YAP1) rs11225163, programmed death-ligand 1 (PD-L1) rs2282055, and programmed death-ligand 2 (PD-L2) rs7854413 genes were analyzed by qRT-PCR. Multiple stepwise regression analysis was performed on a cohort of 134 Egyptian male patients diagnosed with NASH and NASH-HCC.


RESULTS
higher serum alpha-fetoprotein (AFP) and higher serum IL-13 levels were directly associated with HCC development in NASH (odds ratio (OR) 19.6 and 1.9 p < 0.01). Reversibly, the presence of the C/C genotype in STAT6 rs167769 and the C allele carrier YAP1 rs11225163 were inversely associated with HCC in NASH patients (OR 0.015 and 0.047 p < 0.01). A predictive model was formulated with 97.5% specificity, 90.9% sensitivity, and 94.8% accuracy. Moreover, higher serum IL-13 levels and the presence of PD-L2 rs7854413 C allele carriers were associated with advanced fibrosis progression in NASH patients (OR 1.432 and 3.797 p < 0.01). Serum levels of IL-13 and C/C genotype in STAT6 rs167769 significantly increased the predictive capacity of serum AFP to predict HCC in F1-F2 and in F3-F4 fibrosis grades NASH patients.


CONCLUSION
association between serum IL-13 and PD-L2 rs7854413 polymorphism successfully predict advanced fibrosis in NASH. However, HCC development in NASH is associated with higher serum AFP, IL-13 levels, and STAT6 rs167769, YAP1 rs11225163 polymorphisms.


Introduction
Despite increasing awareness against obesity, it is still rising dramatically, precipitating multiple complications. Nowadays, non-alcoholic fatty liver disease (NAFLD), hepatic manifestation of hyperlipidemia, is considered the most common form of chronic liver disease [1]. Non-alcoholic fatty liver (NAFL) is a complex metabolic disease that starts with hepatic lipid accumulation. This aggravates liver inflammation (which can lead to hepatocyte injury and death), and one can end up with pericellular fibrosis. At this point, it turns into a more aggressive form termed non-alcoholic steatohepatitis (NASH) [2]. Thus, NASH is recognized as a multidisciplinary disease that has evolved from metabolic, inflammatory, and immune derangements. The progression of cirrhosis and hepatocellular carcinoma (HCC) from NASH is dramatically increasing, marking NAFLD as the second leading indication for liver transplantation [3]. outcomes [28]. Based on the aforementioned, investigating different SNPs in IL-13 gene, IL13Rα1 gene, IL13Rα2 gene, STAT6 gene, YAP gene, PD-L1, and PD-L2 genes with fibrosis progression and HCC development in NASH have not been studied yet. Accordingly, this study uniquely aims to underscore, for the first time, the influence of selected SNPs in fibrosis progression and HCC development in NASH Egyptian male patients.

Study Population
One hundred and thirty-four well diagnosed male NASH and NASH-HCC Egyptian patients were enrolled in this study. They were recruited from a specialist, Dr. Yassin Abdel Ghaffar, from the Center for Liver Diseases and Researches in Cairo. The study was approved by the Research Ethics Committee, Faculty of Pharmacy, Ain Shams University, under memorandum no. ENREC-ASU-66. Informed consent was obtained from each patient for participation and publishing results. The study was carried out under the regulations and recommendations of the Declaration of Helsinki.
The population study included 55 male Egyptian Caucasian patients with primary HCC arising from a background of NASH. The diagnosis was determined through assessment according to the European Association for the Study of the Liver (EASL) guidelines and on histologic sampling [29]. The second group in this study included 79 male NASH patients. The diagnosis of NASH was established through radiological evidence of hepatic steatosis and on histological assessment when liver biopsy was available. NASH patients were sub-classified according to the degree of liver fibrosis: F1 and F2 as low fibrosis grade and F3 and F4 as high fibrosis grade. Histopathological data were collected when a liver biopsy was performed. Liver biopsy specimens were scored using the Meta-analysis of Histological Data in Viral Hepatitis (METAVIR) system [30] and the severity of steatosis was scored according to the validated Kleiner criteria [31]. All biopsies were evaluated and scored by the same pathologist.
Patients with alcohol intake, infected with viral hepatitis, diagnosed with an autoimmune disease, or subjected to drugs that induced liver injury were all excluded from the study. Baseline and clinical characteristics are summarized in Table 1. Basic anthropometric parameters as body mass index (BMI) was calculated.

Laboratory Analyses
Blood samples for hematological, biochemical analysis, and genomic DNA extraction were obtained after an overnight fast with the standard methods at the center's laboratory. For each patient, three blood samples were taken. Two samples were collected on EDTA vacutainer for whole blood preparation and the third sample was collected on plain vacutainer for serum separation. Whole blood was used for hematological analysis and genomic DNA extraction. Routine hematological analysis was done for the whole blood, and it included a complete blood picture (CBC). Serum was separated by centrifugation of the plain vacutainer at 4000 rpm for 10 min at 4 • C and then aliquoted and stored at −80 • C for subsequent use in biochemical tests. These tests included fasting blood glucose (FBG), serum transaminases (alanine aminotransferase (ALT), aspartate aminotransferase (AST)), total bilirubin, serum albumin, serum triglycerides, serum insulin, serum alpha-fetoprotein (AFP), and serum IL-13 (documented in Table 1).

Genomic DNA Extraction
Genomic DNA was extracted with QIAamp DNA Mini Kit protocol (QIAGEN, Santa Clarita, CA, USA) according to the manufacturer's instructions. DNA samples were subjected to DNA quantitation and purity assessment using the NanoDrop ® (ND)-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA).

Serum IL-13 and Serum Insulin Levels
Serum IL-13 was quantified using an enzyme-linked immunosorbent assay (ELISA) technique using a commercially available kit (Bioassay, Biotech, CO.; Ltd, Hangzhou, China). Serum insulin was also quantified using a commercially available ELISA kit (Nova Tec Immundiagnostica GmbH, Dietzenbach, Hessen, Germany). The homeostatic model assessment of insulin resistance (HOMA-IR) was calculated from fasting insulin and FBG by the following equation: HOMA-IR = fasting insulin (µU/mL) × FBG (mg/dL)/405 [35]. All ELISA procedures were done by Hyprep Automated ELISA system (Hyperion Inc., Miami, FL, USA) according to the manufacturer's instructions.

Statistical Analysis
IBM SPSS statistics (V. 22.0, IBM Corp., Armonk, NY., USA, 2013) was used for data analysis. The Shapiro-Wilk test was used to test the normal distribution of data. Continuous parametric variables were expressed as mean ± SD, while non-parametric data were expressed as median (range). Additionally, categorical variables were presented as frequencies (percentage). A comparison between two independent parametric variables were performed using Student's t-test, and Wilcoxon rank-sum test was carried out for comparison between two non-parametric variables. The Hardy-Weinberg equilibrium was assessed in the study population. A general linear model was used to control for potential confounders. Significant covariates at binary logistic regression analysis were included in a multivariate stepwise logistic regression model with a forward approach to identify independent predictors of fibrosis or cancer. Besides, any skewed data were logarithmically transformed before performing simple and multiple binary stepwise regression analyses. The area under the receiver operating characteristic curve (ROC) indicated the prediction capacity of this analysis. The results were reported as odds ratio (OR) and 95% confidence intervals (CIs). A difference of p < 0.05 was considered significant.

Study Characteristics of NASH and NASH-HCC Patients
One hundred and thirty-four patients were recruited in this study. The clinical and demographic characteristics of NASH-HCC and NASH patients are provided in Table 1. Patients were classified as follows: 58.9% (n = 79) were classified as NASH and 41% (n = 55) were classified as NASH-HCC. The univariate regression analysis showed that patients with NASH-HCC were significantly older than NASH patients (OR = 1.073, 95% CI = 1.012-1.138, p = 0.017). Additionally, higher serum AFP levels and higher serum IL-13 levels were significantly associated with NASH-HCC at (OR = 7.641, 95% CI = 3.6-16.18, p < 0.001) and (OR = 1.616 95%, CI = 1.3-2.008, p < 0.001), respectively. This study showed significant difference between lower fibrosis grades F1-F2, advanced fibrosis grades F3-F4, and cirrhosis among NASH and NASH-HCC patients at p < 0.001. Whereas, 51.8% showed lower fibrosis grades F1-F2 in NASH and 48.1% showed advanced fibrosis grades F3-F4. However, cirrhosis was absent among NASH patients. On the other hand, 90.9% were confirmed to be cirrhotic among NASH-HCC patients and only 9% were of advanced fibrosis grade F3-F4, with complete absence of lower fibrosis grades, among NASH-HCC patients.

Association of Adjusted Univariate Significant Parameters with HCC Development in NASH Patients
To determine whether the significant univariate parameters influence the development of HCC in NASH patients after adjustment of significant covariate as age. A general linear model was performed to control the confounding variable. The results in Table 3 showed that all adjusted values for serum AFP, serum IL-13, the presence of G/G genotype in IL-13 rs20541, the presence of T/T genotype in IL-13R2 rs5946040, the presence of T/T genotype in PD-L1 rs2282055, the absence of C/C genotype STAT6 rs167769, and the absence of C allele carrier in YAP1 rs11225163 were all still significantly associated to HCC development in NASH with p < 0.05.

IL-13/STAT6 Signaling Axis and YAP1 Are Critical Players for HCC Development in NASH Patients
This study aimed to formulate a predictive model using all adjusted significant univariate variables. Multiple stepwise logistic regression analysis was performed after controlling for significant confounder (age), as shown in Table 3, and showed that only higher serum AFP levels and higher serum IL-13 levels were found to be directly related to HCC development (OR = 19.6, p < 0.001) and (OR = 1.9, p = 0.005), respectively. On the other hand, the presence of the C/C genotype in STAT6 rs167769 and the carrier state of the C allele in YAP1 rs11225163 were inversely associated with HCC in NASH patients (OR = 0.015, p < 0.001) and (OR = 0.047, p = 0.004), respectively. A predictive equation predicting the probability for HCC development in NASH patients (P) was estimated with formula: where the substitution values were as follow: a: serum IL-13 levels (ng/L), b: log serum AFP levels (ng/mL), c: age of the patients in years, d: STAT6 rs167769 C/C genotype carrier=1, non-carrier=0, e: YAP1 rs11225163 C allele carrier = 1, non-carrier = 0.
The ROC was plotted in accordance with the same model as expressed in Figure 1. The predictive capacity of the final model was excellent as indicated by the area under the ROC curve 0.964 (95% CI = 0.92-0.999). Where the best cut-off value for this model was 0.511. This means that patients found above this cut-off value might experience HCC development on NASH background. Multiple stepwise logistic regression analysis showed that this model presented showed specificity of 97.5% and significantly increase the sensitivity of serum AFP levels from 60% to 90.9% as well as increase the accuracy of serum AFP levels from 83.6% to 94.8%.

IL-13/PD-L2 Are Crucial for Fibrosis Progression in NASH Patients
Multivariate binary logistic regression analysis was made, and found that older age is not associated with advanced fibrosis progression at p = 0.335. On the other hand, higher serum IL-13 levels and the presence of PD-L2 rs7854413, C allele carriers states are directly associated with advanced fibrosis progression in NASH patients (OR = 1.432 95%, CI = 1.022-2.008, p = 0.037) and (OR = 3.797, 95% CI = 1.216-11.875, p = 0.022), respectively, as shown in Table 5. The ROC was plotted in accordance with the same model showed that the area under the ROC curve was 0.783 (95% CI = 0.703-0.862) with 77.5% specificity, 72.2 % accuracy, and 66.7% sensitivity.

Comparing Different Fibrosis Grades in NASH with HCC Development
Multiple multinomial logistic regression analysis was done after adjusting significant cofounder (age) to compare advanced fibrosis grades F3-F4 with HCC development in NASH, as well as to compare lower fibrosis grades F1-F2 with HCC development in NASH. It was found that by comparing advanced fibrosis grades F3-F4 with HCC-NASH, serum AFP level has a moderate predictive power, where the area under the ROC curve was 0.751 (95% CI = 0.651-0.851) with 76.9% specificity, 69.1% accuracy, and 63.6% sensitivity. Interestingly, this study found that higher serum IL-13 levels and higher serum AFP levels were associated with cancer (OR = 1.625, 95% CI = 1.077-2.451, p = 0.021) and (OR = 8.231, 95% CI = 2.668-25.394, p < 0.001) respectively. However, the presence of the C/C genotype in STAT6 rs167769 was inversely associated with HCC development, compared to advanced fibrosis grades F3-F4 (OR = 0.029, 95% CI = 0.004-0.197, p < 0.001). This model showed strong predictive capacity with the area under the ROC curve 0.936 (95% CI = 0.885-0.986) and 87.2% specificity, 88.3% accuracy, and 89.1% sensitivity.
Similarly, the association of HCC development with lower fibrosis grades F1-F2 in NASH patients showed, also, that serum AFP levels alone has moderate predictive power, where the area under the ROC curve was 0.769 (95% CI = 0.67-0.868) with 92.5% specificity, 75.8% accuracy, and 63.6% sensitivity. However, IL-13 and STAT6 significantly improved the prediction power for HCC development in F1 and F2 NASH patients. This study showed that higher serum IL-13 levels and higher serum AFP levels were directly associated with cancer (OR = 2.036, 95% CI = 1.384-3.076, p = 0.001) and (OR = 9.151, 95% CI = 2.94-28.425, p < 0.001), respectively. While, the presence of C/C genotype in STAT6 rs167769 was inversely associated with HCC development (OR = 0.063, 95% CI = 0.013-0.308, p = 0.001). This model showed strong predictive capacity with area under the ROC curve 0.948 (95% CI = 0.903-0.993) and 87.5% specificity, 89.5% accuracy, and 90.9% sensitivity. This highlights the important role played by IL-13/STAT6 axis in cancer development in NASH, in both lower and advanced fibrosis grades patients.

Discussion
Obesogenic cancers are currently at the top of public health concern. Accounting for 40% of cancer diagnoses in the United States, obesity and being overweight are now recorded as significant contributing factors to cancer incidences and death [36]. Being historically related to viral hepatitis, liver cancer becomes markedly alarmed by NASH with the introduction of novel accessible antiviral treatments [37].
Despite a consistent relationship between NASH and HCC development, no approved pharmacological therapy has yet been introduced. This challenges researchers to tackle the exact mechanisms for understanding disease pathogenesis and progression. Recently, attention has been raised to type 2 immunity and its crucial contributions to pathological fibrosis and cancer development in multiple organs [7]. IL-13, a type 2 immunity effector, is directly involved in tissue repair and regeneration following injury; however, when persistently activated, it transforms these tissue-regenerative responses into progressive fibrotic disorder with unknown clear mechanism or signaling [38]. Accordingly, this research aimed to address and underscore the role of IL-13, as well as its receptors and some of the signaling genes and integrated crosstalk genes, in the prediction of advanced fibrosis progression and HCC development in NASH.
This study comparatively investigated well-characterized male NASH patients with different fibrosis grades and NASH-HCC patients. This study found a significant direct association between higher serum IL-13 levels and cancer progression in NASH. Interestingly, recent reports proved that IL-13 may play a crucial role in other tumors. Whereas, IL-13 was proved to be associated with obesity-related colorectal tumorigenesis [39]. Inflammation provoked by obesity notably increased expression of IL-13, which in turn leads to increased expression of IL-13R1, ending up with activation of downstream phosphorylation of the transcription factor STAT6, suggesting a possible mechanism of carcinogenesis in colon cancer [40]. Our study agreed with this report, as higher serum IL-13 and polymorphism at STAT6 rs167769 were reported to be the strongest variables associated with cancer progression in NASH patients, regardless of their fibrosis stages. In accordance, evidence has suggested that STAT6 is involved in the HCC process and may predict a worse prognosis in patients with HCC [41]. However, this study specifically introduced the C/C genotype carrier in the STAT6 rs167769 gene as a predictive marker with a strong inverse relation to HCC development in NASH patients, irrespective of their fibrosis stage. More interestingly, higher IL-13 levels and the absence of the STAT6 rs167769 C/C genotype also significantly predict HCC development in NASH when compared to low F1 and F2 patients, and advanced fibrosis grades, F3 and F4 patients. This confirms that IL-13/STAT6 plays a major role in HCC development in NASH. The underlined mechanism for STAT6 rs167769 polymorphism is still unclear. Whereas, some studies showed that the T alleles of STAT6 rs167769 could increase STAT6 promoter activity, which further increases the STAT6 signaling axis in dermatitis [42]. Hence, it is suggested that C allele has a protective role via decreasing STAT6 signaling activation. Other studies confirmed that SNP at STAT6 rs167769 is strongly associated with STAT6 expression in the blood and lungs [43]. More interestingly, previous reports suggest that STAT6 rs167769 SNP is significantly associated with immune function via decreasing interferon gamma (IFNγ) production [44]. Notably, it was previously found that diets rich in fat significantly suppress natural killer T (NKT) cell-derived IFNγ production, but significantly enhance the production of IL-13, which activates carcinogenesis in NASH [45]. Another possible explanation for our finding of STAT6 association with HCC development in NASH was supported by a recent study, which showed that variant of STAT6 rs167769 is strongly associated with eosinophilic esophagitis [46]. Interestingly, it was confirmed recently that progression of NASH was significantly associated with increasing eosinophilic type 2 liver inflammation in experimental, as well as in human patient biopsies [6]. All these evidences provided a possible explanation of our finding of IL-13/STAT6 rs167769 association with HCC in NASH. However, functional analysis studies are required to understand the exact mechanism of action.
Interestingly, this study found a significant association of the hippo signaling pathway represented through YAP1, a downstream effector of hippo signaling, with HCC development in NASH patients. This study showed that C allele carriers in YAP1 rs11225163 are protected from HCC development in NASH. The hippo signaling seems to be the ideal candidate pathway that governs hepatocyte proliferation during regeneration [47]. Our results are in alignment with a recent report, which proved that progression HCC in NASH was attributed to YAP1 [48]. Surprisingly, this study sheds light on the possible integration of IL-13/STAT6 axis and Hippo signaling through YAP1 in cancer progression in NASH. In accordance, it has been recently proven that YAP1 expression is differentially regulated by IL-13, which, upon STAT6 activation, enhances the expression of the M2-macrophage associated genes, which in turn promotes tissue repair and control inflammation in inflammatory bowel syndrome [49]. However, future studies are required to confirm our findings for HCC development in NASH patients.
It is important to mention that by classifying NASH patients into low F1 and F2 fibrosis grades and high F3 and F4 fibrosis grades, C allele carriers in YAP1 rs11225163 lost its significant association with HCC development in NASH. This finding might be explained by the absence of cirrhosis among NASH patients. On the other side, 90.9% of NASH-HCC were cirrhotic. Interestingly, 66.6% of the T/T genotype in YAP1 rs11225163 were cirrhotic NASH-HCC patients. While, only 33.3% of the T/T genotype in the YAP1 rs11225163 gene were in NASH patients. Upon classifying NASH into lower (F1 and F2) and higher grades (F3 and F4), all T/T genotype in the YAP1 rs11225163 gene were of higher fibrosis grades (F3 and F4). This highlights that YAP1 rs11225163 might be associated with cirrhosis in NASH-HCC. However, the sample size decreased significantly upon subclassification of NASH patients into low and high fibrosis grades. That is why YAP1 rs11225163 polymorphism lost its association with cancer upon subclassification. However, this finding might indicate that YAP1 might lie at the intercept between severe advanced fibrosis/cirrhosis and HCC development in NASH. In agreement, previous studies showed that YAP activation in HSCs and hepatocytes could possibly increase carcinogenesis in cirrhotic livers [47,50]. However, a larger sample size is required to confirm this suggestion.
This study found a significant contribution of IL-13 rs20541, IL-13R2 rs5946040, and PD-L1 rs2282055 at univariate analysis with cancer progression in NASH. However, the contribution of IL-13/STAT6 and YAP1, together with serum AFP, are proven to have the upper hand in predicting cancer development in NASH. In fact, serum AFP is a well-known classical marker for HCC [51]. Its level was strongly proved to be associated with cancer progression in NASH, as highlighted by this study. However, combining IL-13/STAT6 and YAP1 increases the sensitivity and accuracy of serum AFP, which again highlights the critical role played by variables in predicting HCC development in NASH. Additionally, combining IL-13/STAT6 with serum AFP also increased predictive power of serum AFP and increased its specificity, sensitivity, and accuracy for predicting HCC development in high fibrosis grades F3 and F4 NASH patients. In addition, combining IL-13/STAT6 with serum AFP also increased predictive capacity of serum AFP and increased its sensitivity and accuracy in predicting HCC development in low fibrosis grades, F1 and F2 NASH patients.
Interestingly, this study highlighted the importance of formulating a predictive model for HCC development in the absence of higher fibrosis grades in NASH. Whereas, a significant association was found between higher serum AFP and higher serum IL-13 levels with HCC development in F1 and F2 NASH patients. While, the presence of C/C genotype in STAT6 rs167769 was inversely associated with HCC development in F1 and F2 NASH patients. This predictive model showed excellent predictive capacity improving the predictive capacity of serum AFP alone. It is important to note that all NASH-HCC patients in this study were cirrhotic or with advanced fibrosis grades. It agreed with a previous study predicting HCC development in NASH [52]; however, previous studies reported that HCC can develop de novo in patients with NASH without the presence of cirrhosis [53,54]. Therefore, this study might aid in understanding the mechanism of progression of HCC in absence of fibrosis grades in the future.
The second aim of this study was to investigate the possible role played by our studied variables for fibrosis progression in NASH. Interestingly, this study showed that higher serum IL-13 is significantly associated with advanced fibrosis grades in NASH. This comes in alignment with previous reports, which showed the disturbed healing process in NASH contributed to fibrosis progression via chronic activation of type 2 inflammation manifested by increased serum IL-13 levels [9,10]. It was explained by overproduction of Th2 cytokines, which typically promote B-cell hyperactivity and humoral immune responses, whereas T cell hyperactivity and inflammation frequently associated with an excess of Th1 and Th17 cytokines at earlier stages of the diseases [55]. However, IL-4/IL-13-secreting invariant nature killer T cells (iNKT) were prevalent later in the disease [56].
Additionally, this study also highlighted the contribution of PD-L2 C allele carriers together with serum levels of IL-13 for advanced fibrosis progression in NASH. There are a multitude of mechanisms that dampen hepatic immunity upon hepatocyte inflammation due to abnormal lipid accumulation and peroxidation in NASH. Of these lies the involvement of NKT cells at the center of interest, by limiting T cell responses and inducing the upregulation of the inhibitory molecules PD-L1 and PD-L2 on dendritic cells (DC) [57]. This interplay among innate lymphocyte populations switches the convert of a quiescent immune environment into a cellular battlefield during chronic inflammatory diseases, such as NASH [58]. Thus, this study adds evidence for the possible involvement of PD-L2 and IL-13 in advanced fibrosis progression in NASH.
In summary, this study reports a striking association between serum levels of IL-13 and SNP at PD-L2 rs7854413 with advanced fibrosis in NASH patients, independent with other variables. This study uniquely formulated a predictive model with moderate predictive capacity for advanced fibrosis progression in NASH. Additionally, this study showed a significant association between serum levels of IL-13, serum AFP levels, and SNPs at STAT6 rs167769 and YAP1 rs11225163 with HCC development in NASH. Serum levels of IL-13, serum AFP levels, and SNPs at STAT6 rs167769 are associated with HCC development when compared to lower and higher fibrosis grades in NASH patients. These predictive models significantly increased the predictive capacity of serum AFP for HCC prediction in NASH. These data highlight the importance of understanding the contribution of IL-13 and its integrating and signaling genes in NASH-HCC pathogenesis, and if validated, may contribute to a tailored, personalized approach to cost effective surveillance and detection, possibly targeting NASH-HCC patients. Therefore, this study strongly recommends further investigation using a multi-racial validation cohort, required to explore clinical significance. Additionally, functional studies are required to investigate the mechanisms of PD-L2/IL-13/STAT6 and YAP1 in fibrosis progression and HCC development in NASH.

Conclusions
Stratifying patients based on their genetic background is a reliable cornerstone approach for better understanding and targeting of diseases, especially multidisciplinary diseases with complex pathophysiology and integrated signaling crosstalk, such as NASH. This study sheds light on the possible integration of different pathways in fibrosis progression and HCC development in NASH. Finally, this study adds clinical proof for the important role that is played by type 2 immunity and the aberrant healing process for advanced fibrosis progression and HCC development in NASH.
Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.