The Use of Artificial Intelligence in the Liver Histopathology Field: A Systematic Review.

Digital pathology (DP) has begun to play a key role in the evaluation of liver specimens. Recent studies have shown that a workflow that combines DP and artificial intelligence (AI) applied to histopathology has potential value in supporting the diagnosis, treatment evaluation, and prognosis prediction of liver diseases. Here, we provide a systematic review of the use of this workflow in the field of hepatology. Based on the PRISMA 2020 criteria, a search of the PubMed, SCOPUS, and Embase electronic databases was conducted, applying inclusion/exclusion filters. The articles were evaluated by two independent reviewers, who extracted the specifications and objectives of each study, the AI tools used, and the results obtained. From the 266 initial records identified, 25 eligible studies were selected, mainly conducted on human liver tissues. Most of the studies were performed using whole-slide imaging systems for imaging acquisition and applying different machine learning and deep learning methods for image pre-processing, segmentation, feature extractions, and classification. Of note, most of the studies selected demonstrated good performance as classifiers of liver histological images compared to pathologist annotations. Promising results to date bode well for the not-too-distant inclusion of these techniques in clinical practice.

Noninvasive scores are poorly predictive of histological fibrosis in paediatric fatty liver disease.

OBJECTIVES: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in children. Roughly a quarter of paediatric patients with NAFLD develop nonalcoholic steatohepatitis and fibrosis. Here, we evaluated the diagnostic accuracy of previously published noninvasive fibrosis scores to predict liver fibrosis in a large European cohort of paediatric patients with NAFLD. METHODS: The 457 patients with biopsy-proven NAFLD from 10 specialized centers were included. We assessed diagnostic accuracy for the prediction of any (F ≥ 1), moderate (F ≥ 2) or advanced (F ≥ 3) fibrosis for the AST/platelet ratio (APRI), Fibrosis 4 score (FIB-4), paediatric NAFLD fibrosis score (PNFS) and paediatric NAFLD fibrosis index (PNFI). RESULTS: Patients covered the full spectrum of fibrosis (F0: n = 103; F1: n = 230; F2: n = 78; F3: n = 44; F4: n = 2). None of the scores were able to accurately distinguish the presence of any fibrosis from no fibrosis. For the detection of moderate fibrosis, area under the receiver operating characteristic curve (AUROC) were: APRI: 0.697, FIB-4: 0.663, PNFI: 0.515, PNFS: 0.665, while for detection of advanced fibrosis AUROCs were: APRI: 0.759, FIB-4: 0.611, PNFI: 0.521, PNFS: 0.712. Fibrosis scores showed no diagnostic benefit over using ALT ≤ 50/ > 50 IU/L as a cut-off. CONCLUSIONS: Established fibrosis scores lack diagnostic accuracy to replace liver biopsy for staging of fibrosis, giving similar results as compared to using ALT alone. New diagnostic tools are needed for Noninvasive risk-stratification in paediatric NAFLD.

Spermine oxidase induces DNA damage and sensitizes fusion negative rhabdomyosarcoma cells to irradiation.

Rhabdomyosarcoma (RMS) is a pediatric myogenic soft tissue sarcoma that includes fusion-positive (FP) and fusion-negative (FN) molecular subtypes. FP-RMS expresses PAX3-FOXO1 fusion protein and often shows dismal prognosis. FN-RMS shows cytogenetic abnormalities and frequently harbors RAS pathway mutations. Despite the multimodal heavy chemo and radiation therapeutic regimens, high risk metastatic/recurrent FN-RMS shows a 5-year survival less than 30% due to poor sensitivity to chemo-radiotherapy. Therefore, the identification of novel targets is needed. Polyamines (PAs) such as putrescine (PUT), spermidine (SPD) and spermine (SPM) are low-molecular-mass highly charged molecules whose intracellular levels are strictly modulated by specific enzymes. Among the latter, spermine oxidase (SMOX) regulates polyamine catabolism oxidizing SPM to SPD, which impacts cellular processes such as apoptosis and DNA damage response. Here we report that low SMOX levels are associated with a worse outcome in FN-RMS, but not in FP-RMS, patients. Consistently, SMOX expression is downregulated in FN-RMS cell lines as compared to normal myoblasts. Moreover, SMOX transcript levels are reduced FN-RMS cells differentiation, being indirectly downregulated by the muscle transcription factor MYOD. Noteworthy, forced expression of SMOX in two cell lines derived from high-risk FN-RMS: 1) reduces SPM and upregulates SPD levels; 2) induces G0/G1 cell cycle arrest followed by apoptosis; 3) impairs anchorage-independent and tumor spheroids growth; 4) inhibits cell migration; 5) increases γH2AX levels and foci formation indicative of DNA damage. In addition, forced expression of SMOX and irradiation synergize at activating ATM and DNA-PKCs, and at inducing γH2AX expression and foci formation, which suggests an enhancement in DNA damage response. Irradiated SMOX-overexpressing FN-RMS cells also show significant decrease in both colony formation capacity and spheroids growth with respect to single approaches. Thus, our results unveil a role for SMOX as inhibitor of tumorigenicity of FN-RMS cells in vitro. In conclusion, our in vitro results suggest that SMOX induction could be a potential combinatorial approach to sensitize FN-RMS to ionizing radiation and deserve further in-depth studies.

Serum 25-hydroxyvitamin D and hyaluronic acid levels as markers of fibrosis in patients with chronic liver disease at the main tertiary referral hospital in Ghana: A case-control study design.

Background and Aims: Liver fibrosis leading to chronic liver disease (CLD) is a major cause of morbidity, mortality and health-care expenditure worldwide. The "gold standard" for diagnosis and staging of liver fibrosis is histological analysis of liver tissue obtained by liver biopsy, an invasive procedure. Therefore, there is the need to identify noninvasive and inexpensive markers for diagnosis and staging of liver fibrosis. This study aimed at evaluating the correlation of hyaluronic acid (HA) and 25-hydroxyvitamin D (25-OH vitamin D) serum levels as markers of fibrosis with histologically staged and graded liver biopsies obtained from CLD patients. Methods: This was a case-control study involving 40 CLD patients requiring liver biopsies and 40 controls. Liver biopsies were staged to determine the degree of fibrosis. Serum levels of 25-OH vitamin D and HA were determined using ELISA. Statistical analyses were performed to determine differences in HA and 25-OH vitamin D levels between controls and patients as well as to correlate the biomarkers with the stages of fibrosis. Results: CLD patients showed significant (p < 0.001) increase in the levels of AST, ALT, GGT, compared to the controls. Patients also had significantly (p < 0.001) lower serum 25-OH vitamin D and higher HA (p < 0.001) levels compared to the controls. Additionally, 25-OH vitamin D levels of the CLD patients were significantly different across the stages of liver fibrosis likewise serum HA levels. Furthermore, 25-OH vitamin D levels inversely correlated with the severity of liver fibrosis. A significant negative correlation (r = -0.33, p < 0.05) between CLD patients' HA and 25-OH vitamin D were found. Conclusion: CLD patients had significantly reduced serum 25-OH vitamin D and higher HA. Both markers correlated with the degree of liver fibrosis. These findings have major clinical translatable implication in the use of vitamin D supplementation in the management of CLD in Ghana.

The Role of the GH/IGF1 Axis on the Development of MAFLD in Pediatric Patients with Obesity.

The anomalies of the Growth Hormone (GH)/Insulin-like Growth Factor-1 (IGF1) axis are associated with a higher prevalence of Metabolic Associated Fatty Liver Disease (MAFLD) and with a more rapid progression towards fibrosis, cirrhosis, and end-stage liver disease. A total of 191 adolescents with obesity [12−18 years] were consecutively enrolled between January 2014 and December 2020 and underwent liver biopsy to diagnose MAFLD severity. In all patients GH, IGF1 and Insulin-like Growth Factor-Binding Protein 3 (IGFBP3) were measured. Patients with inflammation and ballooning have significantly lower values of GH and IGF1 than those without (GH: 5.4 vs. 7.5 ng/mL; IGF1 245 vs. 284 ng/mL, p < 0.05). GH and IGF1 were also negatively correlated with fibrosis' degree (r = −0.51, p = 0.001, and r = −0.45, p = 0.001, respectively). Only GH correlated with TNF-a (r = −0.29, p = 0.04) and lobular inflammation (r = −0.36, p = 0.02). At multivariate regression, both GH and IGF1 values, after adjustment for age, sex and BMI, were negatively associated with HOMA-IR but above all with fibrosis (GHâ†’ß = −2.3, p = 0.001, IGF1â†’ß = −2.8, p = 0.001). Even in the pediatric population, a reduction of GH input in the liver directly promotes development of de novo hepatic lipogenesis, steatosis, fibrosis and inflammation. The possible role of recombinant GH administration in adolescents with obesity and severe MAFLD deserves to be studied.

Profiling of cell-free DNA methylation and histone signatures in pediatric NAFLD: A pilot study.

Nonalcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease in children and adolescents, increasing the risk of its progression toward nonalcoholic steatohepatitis (NASH), cirrhosis, and cancer. There is an urgent need for noninvasive early diagnostic and prognostic tools such as epigenetic marks (epimarks), which would replace liver biopsy in the future. We used plasma samples from 67 children with biopsy-proven NAFLD, and as controls we used samples from 20 children negative for steatosis by ultrasound. All patients were genotyped for patatin-like phospholipase domain containing 3 (PNPLA3), transmembrane 6 superfamily member 2 (TM6SF2), membrane bound O-acyltransferase domain containing 7 (MBOAT7), and klotho-ß (KLB) gene variants, and data on anthropometric and biochemical parameters were collected. Furthermore, plasma cell-free DNA (cfDNA) methylation was quantified using a commercially available kit, and ImageStream(X) was used for the detection of free circulating histone complexes and variants. We found a significant enrichment of the levels of histone macroH2A1.2 in the plasma of children with NAFLD compared to controls, and a strong correlation between cfDNA methylation levels and NASH. Receiver operating characteristic curve analysis demonstrated that combination of cfDNA methylation, PNPLA3 rs738409 variant, coupled with either high-density lipoprotein cholesterol or alanine aminotransferase levels can strongly predict the progression of pediatric NAFLD to NASH with area under the curve >0.87. Conclusion: Our pilot study combined epimarks and genetic and metabolic markers for a robust risk assessment of NAFLD development and progression in children, offering a promising noninvasive tool for the consistent diagnosis and prognosis of pediatric NAFLD. Further studies are necessary to identify their pathogenic origin and function.

Combination Treatment with Hydroxytyrosol and Vitamin E Improves NAFLD-Related Fibrosis.

Non-alcoholic fatty liver disease (NAFLD)-related liver fibrosis results in the encapsulation of injured liver parenchyma by a collagenous scar mainly imputable to hepatic stellate cells' activation. Approved pharmacological treatments against NAFLD-related fibrosis are still lacking, but natural compounds such as hydroxytyrosol (HXT) and vitamin E (VitE), are emerging as promising therapeutic opportunities. In this study, the potential anti-fibrotic effect of HXT + VitE combination therapy was investigated in vitro and in vivo. In particular, tumor growth factor (TGF)-ß-activated LX-2 cells as an in vitro model, and carbon tetrachloride plus a Western diet as a mice model were employed. The effect of HXT + VitE on fibrosis was also investigated in children with biopsy-proven NAFLD. Our results demonstrated that HXT + VitE caused a reduction of proliferation, migration, contractility, and expression of pro-fibrogenic genes in TGF-ß-activated LX-2 cells. HXT + VitE treatment also antagonized TGF-ß-dependent upregulation of pro-oxidant NOX2 by interfering with nuclear translocation/activation of SMAD2/3 transcription factors. The mouse model of NAFLD-related fibrosis treated with HXT + VitE showed a marked reduction of fibrosis pattern by histology and gene expression. Accordingly, in children with NAFLD, HXT + VitE treatment caused a decrease of circulating levels of PIIINP and NOX2 that was supported over time. Our study suggests that HXT + VitE supplementation may improve NAFLD-related fibrosis.

Higher Levels of Plasma Hyaluronic Acid and N-terminal Propeptide of Type III Procollagen Are Associated With Lower Kidney Function in Children With Non-alcoholic Fatty Liver Disease.

Objective: Hyaluronic acid (HA) and N-terminal propeptide of type III procollagen (PIIINP) are two non-invasive biomarkers of liver fibrosis in non-alcoholic fatty liver disease (NAFLD). We examined the relationships of plasma levels of HA and PIIINP with kidney function in children with NAFLD. Methods: Plasma HA and PIIINP levels were measured using two commercially available enzyme-linked immunosorbent assay kits in a cohort of 106 Caucasian overweight or obese children with biopsy-proven NAFLD. Glomerular filtration rate (eGFR) was estimated using the Bedside Schwartz equation. Genotyping for the patatin-like phospholipase domain-containing protein-3 (PNPLA3) rs738409 variant was performed using an allelic discrimination assay. Results: Children with fibrosis F2 had significantly higher plasma PIIINP and HA levels than those with F0 or F1 fibrosis. Liver fibrosis was positively associated with plasma HA and PIIINP, as well as with the presence of the risk allele G of PNPLA3 rs738409 variant, and negatively with eGFR. Moreover, eGFR showed significant inverse associations with HA and PIIINP levels, as well as the presence of G of PNPLA3 rs738409, and liver fibrosis stage. Notably, our multivariable regression models showed that higher plasma PIIINP (standardized beta coefficient: -0.206, P = 0.011) and HA levels (standardized beta coefficient: -0.531, P < 0.0001) were associated with lower eGFR values, even after adjustment for age, sex, systolic blood pressure, PNPLA3 rs738409 genotype, and any stage of liver fibrosis. Conclusions: Higher levels of HA and PIIINP were associated with lower eGFR values in Caucasian children with biopsy-proven NAFLD, independently of PNPLA3 rs738409 genotype and other potential confounding factors.

Non-Invasive Diagnostic Test for Advanced Fibrosis in Adolescents With Non-Alcoholic Fatty Liver Disease.

Introduction: Non-alcoholic fatty liver disease (NAFLD) is a multifaceted disease that includes a wide spectrum of liver damage. The presence and the degree of fibrosis are considered important factors for the prognosis of NAFLD and in predicting the risk of developing cirrhosis. Our aim was to evaluate the usefulness of four fibrosis scores (aspartate aminotransferase/Platelet Index [APRI], FIB-4, NAFLD Fibrosis Score [NFS], and Hepamet) in predicting different degrees of fibrosis among children with biopsy-proven NAFLD. Methods: About 286 adolescents [mean age 14.3 years ± 2.5; 154 (53.6%) males], referred between January 2014 and December 2019, with biopsy-proven NAFLD were enrolled. Results: About 173 (60.4%) patients presented fibrosis at histological analysis. In particular: 140 (49.3%) patients had F = 1, 31 (10.8%), had F = 2 and 2 (0.66%) had F = 3. APRI (AUROC 0.619, 95% CI 0.556-0.679) and Hepamet (AUROC 0.778, 95% CI 0.722-0.828) scores had significant (p < 0.001) accuracy to distinguish subjects with fibrosis; while NFS and FIB-4 had not. APRI had a positive predictive value (PPV) of 62.77% (95% CI 57.96-67.35) and an negative predictive value (NPV) of 52.01% (95% CI 46.54-57.43); Hepamet a PPV of 63.24% (95% CI 59.95-66.41) and an NPV of 61.29% (52.9-69.01). Conclusions: Our study showed that Hepamet and APRI perform better than NFS and FIB-4 for identifying fibrosis in patients with NAFLD, but do not have PPVs so high to be considered diagnostic. Therefore, they cannot be employed, in children, for a certain diagnosis of fibrosis or its progression and cannot replace liver biopsy as the gold diagnostic standard. It is, therefore, necessary to continue to research and develop new markers of exclusive fibrosis.

LncOb rs10487505 variant is associated with leptin levels in pediatric non-alcoholic fatty liver disease.

BACKGROUND: Low and high leptin levels are associated with non-alcoholic fatty liver disease (NAFLD). The LncOb rs10487505 variant has been associated with body mass index (BMI), and the C allele was reported as leptin-lowering. We evaluated the association of rs10487505 with leptin levels, liver histology, and surgery-induced weight loss in youths with NAFLD. METHODS: One-hundred five obese youths with NAFLD, of whom 19 undergoing laparoscopic sleeve gastrectomy (LSG), were analyzed for rs10487505 and leptin circulating levels. RESULTS: The G allele frequency was lower in youths with NAFLD than in controls (p = 0.049). No difference was found in anthropometrics, biochemistry and histology between G allele carriers and CC homozygotes, except for leptin levels (p = 0.016). Leptin correlated with body weight, BMI, BMI-z score, waist circumference, insulin resistance/sensitivity, and triglycerides (p ≤ 0.01). A multivariable regression model including body weight and homeostasis model assessment of insulin resistance was a good predictor of plasma leptin (R2 = 0.45), and the addition of genotype to the model increased the R2 to 0.50. Following LSG, leptin levels and body weight were more reduced in G allele carriers (p < 0.05). CONCLUSIONS: LncOb rs10487505 variant was associated with pediatric NAFLD and high leptin levels, and with weight and leptin reduction after LSG in youths. IMPACT: The interplay of environment, genetics and epigenetics is crucial inflating the risk of non-alcoholic fatty liver disease (NAFLD). Several long non-coding RNA (LncRNAs) are found associated with NAFLD pathogenesis. Here, we evaluated the impact of the genetic variant rs10487505 in LncOb which is involved in the regulation of leptin gene expression. The LncOb rs10487505 is associated with increased levels of leptin, but not with liver histology, in youths with NAFLD. The LncOb rs10487505 was also associated with the significant decrease of leptin and body weight after bariatric surgery.

Epigenetic remodelling in human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer, being the sixth most commonly diagnosed cancer and the fourth leading cause of cancer-related death. As other heterogeneous solid tumours, HCC results from a unique synergistic combination of genetic alterations mixed with epigenetic modifications.In HCC the patterns and frequencies of somatic variations change depending on the nearby chromatin. On the other hand, epigenetic alterations often induce genomic instability prone to mutations. Epigenetics refers to heritable states of gene expression without alteration to the DNA sequence itself and, unlike genetic changes, the epigenetic modifications are reversible and affect gene expression more extensively than genetic changes. Thus, studies of epigenetic regulation and the involved molecular machinery are greatly contributing to the understanding of the mechanisms that underline HCC onset and heterogeneity. Moreover, this knowledge may help to identify biomarkers for HCC diagnosis and prognosis, as well as future new targets for more efficacious therapeutic approaches.In this comprehensive review we will discuss the state-of-the-art knowledge about the epigenetic landscape in hepatocarcinogenesis, including evidence on the diagnostic and prognostic role of non-coding RNAs, modifications occurring at the chromatin level, and their role in the era of precision medicine.Apart from other better-known risk factors that predispose to the development of HCC, characterization of the epigenetic remodelling that occurs during hepatocarcinogenesis could open the way to the identification of personalized biomarkers. It may also enable a more accurate diagnosis and stratification of patients, and the discovery of new targets for more efficient therapeutic approaches.

Comparison of the Lipidomic Signature of Fatty Liver in Children and Adults: A Cross-Sectional Study.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is an increasingly common condition in children characterised by insulin resistance and altered lipid metabolism. Affected patients are at increased risk of cardiovascular disease (CVD) and children with NAFLD are likely to be at risk of premature cardiac events. Evaluation of the plasma lipid profile of children with NAFLD offers the opportunity to investigate these perturbations and understand how closely they mimic the changes seen in adults with cardiometabolic disease. METHODS: We performed untargeted liquid chromatography-mass spectrometry (LC-MS) plasma lipidomics on 287 children: 19 lean controls, 146 from an obese cohort, and 122 NAFLD cases who had undergone liver biopsy. Associations between lipid species and liver histology were assessed using regression adjusted for age and sex. Results were then replicated using data from 9500 adults with metabolic phenotyping. RESULTS: More severe paediatric NAFLD was associated with lower levels of long chain, polyunsaturated phosphatidylcholines (pC) and triglycerides (TG). Similar trends in pC and TG chain length and saturation were seen in adults with hepatic steatosis; however, many of the specific lipids associated with NAFLD differed between children and adults. Five lipids replicated in adults (including PC(36:4)) have been directly linked to death and cardiometabolic disease, as well as indirectly via genetic variants. CONCLUSION: These findings suggest that, whilst similar pathways of lipid metabolism are perturbed in paediatric NAFLD as in cardiometabolic disease in adults, the specific lipid signature in children is different.

Circulating microRNAs as novel non-invasive biomarkers of paediatric celiac disease and adherence to gluten-free diet.

BACKGROUND: Celiac Disease (CD) is a multifactorial autoimmune enteropathy (with a prevalence of approximately 1% worldwide) that exhibits a wide spectrum of clinical, serological and histological manifestations. For the diagnosis of paediatric CD, the gold standard is the combination of serological tests (with high TGA-IgA values greater than 10 times the upper limit of normal) and duodenal biopsy (with a positive TGA-IgA but low titer). Therefore, a diagnostic test that totally excludes an invasive approach has not been discovered so far and the discovery of novel biological markers would represent an undoubted advantage for the diagnosis of CD and prognostic evaluation. MicroRNAs (miRNAs), small non-coding RNAs (18-22 nucleotides) that regulate gene expression at post-transcriptional level and play important roles in many biological processes, represent a novel class of potential disease biomarkers. Their presence in biological fluids (i.e., serum, plasma, saliva, urine) provides the opportunity to employ circulating miRNAs as novel non-invasive biomarkers. METHODS: In our prospective observational study, we examined the expression of circulating miRNAs in a cohort of CD patients (both at diagnosis and on gluten-free diet, respectively referred as CD and GFD) compared to healthy controls. By small RNA-Seq we discovered a set of circulating miRNAs that were further validated by qPCR with specific assays. FINDINGS: We found that out of the 13 miRNAs able to discriminate the three groups (i.e., CD, GFD and controls), three of them, namely miR-192-5p, miR-215-5p and miR-125b-5p (alone or in combination), were able to discriminate these three groups with high accuracy and specificity. INTERPRETATION: Our conclusions emphasize that these circulating miRNAs can be employed not only for the diagnosis of CD patients with a low TGA-IgA titer but also to monitor the adherence to a gluten-free diet by CD patients. In conclusion, we suggest the use of the circulating miRNAs identified in this work as a novel diagnostic and follow-up tool for paediatric CD. FUNDING: This work was supported by Fondazione Celiachia Onlus (FC) Grant n° 018/FC/2013 and by Italian Ministry of Health (Ricerca Corrente).

New Insights on the Nuclear Functions and Targeting of FAK in Cancer.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase over-expressed and activated in both adult and pediatric cancers, where it plays important roles in the regulation of pathogenesis and progression of the malignant phenotype. FAK exerts its functions in cancer by two different ways: a kinase activity in the cytoplasm, mainly dependent on the integrin signaling, and a scaffolding activity into the nucleus by networking with different gene expression regulators. For this reason, FAK has to be considered a target with high therapeutic values. Indeed, evidence suggests that FAK targeting could be effective, either alone or in combination, with other already available treatments. Here, we propose an overview of the novel insights about FAK's structure and nuclear functions, with a special focus on the recent findings concerning the roles of this protein in cancer. Additionally, we provide a recent update on FAK inhibitors that are currently in clinical trials for patients with cancer, and discuss the challenge and future directions of drug-based anti-FAK targeted therapies.

Changes in Total Homocysteine and Glutathione Levels After Laparoscopic Sleeve Gastrectomy in Children with Metabolic-Associated Fatty Liver Disease.

PURPOSE: Paediatric obesity is a well-known risk factor for metabolic-associated fatty liver disease (MAFLD). The aim of this study was to evaluate the effects of laparoscopic sleeve gastrectomy (LSG) on the levels of total homocysteine (tHcy) and total glutathione (tGSH) plasma levels in children with MAFLD. MATERIAL AND METHODS: Twenty-four children with severe obesity who underwent LSG were included in the study. The metabolic parameters, systemic inflammatory markers, one-carbon metabolism products, ultrasound and histological improvement were evaluated at baseline (T0M) and after 12 months from LSG (T12M). RESULTS: The patients exhibited a significant amelioration of several metabolic parameters at T12M. A significant reduction of steatosis was observed at ultrasound (from 72.7% of moderate-severe grade to 0% severe steatosis), accompanied by a statistically significant improvement of ballooning, portal and lobular inflammation and fibrosis. A statistically significant decrease of tumour necrosis factor circulating levels was also observed (T0M median = 290.3, IQR = 281.0-317.0 pg/mL; T12M median = 260.4, IQR = 240.0-279.0 pg/mL; p < 0.0001). After 12 months from LSG, a significant increase of mean plasma levels of tHcy(T0M mean = 15.7 ± 4.1 µmol/L; T12M mean = 21.1 ± 9.3 µmol/L; p = 0.0146) was also observed. The increase of tHcy showed no causal link with the improvement of MAFLD-related inflammatory, metabolic and histological pattern. CONCLUSION: LSG in children with obesity induces an improvement of MAFLD-related metabolic derangement and liver damage, but also a mild hyperhomocysteinemia that should be avoided to prevent cardiovascular risk.

TM6SF2/PNPLA3/MBOAT7 Loss-of-Function Genetic Variants Impact on NAFLD Development and Progression Both in Patients and in In Vitro Models.

BACKGROUND & AIMS: The I148M Patatin-like Phospholipase Domain-containing 3 (PNPLA3), the rs641738 in the Membrane bound O-acyltransferase domain containing 7-transmembrane channel-like 4 (MBOAT7-TMC4) locus, and the E167K Transmembrane 6 Superfamily Member 2 (TM6SF2) polymorphisms represent the main predisposing factors to nonalcoholic fatty liver disease (NAFLD) development and progression. We previously generated a full knockout of MBOAT7 in HepG2 cells (MBOAT7-/-), homozygous for I148M PNPLA3. Therefore, we aimed to investigate the synergic impact of the 3 at-risk variants on liver injury and hepatocellular carcinoma (HCC) in a large cohort of NAFLD patients, and create in vitro models of genetic NAFLD by silencing TM6SF2 in both HepG2 and MBOAT7-/- cells. METHODS: NAFLD patients (n = 1380), of whom 121 had HCC, were stratified with a semiquantitative score ranging from 0 to 3 according to the number of PNPLA3, TM6SF2, and MBOAT7 at-risk variants. TM6SF2 was silenced in HepG2 (TM6SF2-/-) and MBOAT7-/- (MBOAT7-/-TM6SF2-/-) through Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9). RESULTS: In NAFLD patients, the additive weight of these mutations was associated with liver disease severity and an increased risk of developing HCC. In HepG2 cells, TM6SF2 silencing altered lipid composition and induced the accumulation of microvesicular lipid droplets (LDs), whereas the MBOAT7-/-TM6SF2-/- cells showed a mixed microvesicular/macrovesicular pattern of LDs. TM6SF2 deletion strongly affected endoplasmic reticulum and mitochondria ultrastructures, thus increasing endoplasmic reticulum/oxidative stress. The mitochondrial number was increased in both TM6SF2-/- and MBOAT7-/-TM6SF2-/- models, suggesting an unbalancing in mitochondrial dynamics, and the silencing of both MBOAT7 and TM6SF2 impaired mitochondrial activity with a shift toward anaerobic glycolysis. MBOAT7-/-TM6SF2-/- cells also showed the highest proliferation rate. Finally, the re-overexpression of MBOAT7 and/or TM6SF2 reversed the metabolic and tumorigenic features observed in the compound knockout model. CONCLUSIONS: The co-presence of the 3 at-risk variants impacts the NAFLD course in both patients and experimental models, affecting LD accumulation, mitochondrial functionality, and metabolic reprogramming toward HCC.

Focal adhesion kinase inhibitor TAE226 combined with Sorafenib slows down hepatocellular carcinoma by multiple epigenetic effects.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common and lethal malignant tumours worldwide. Sorafenib (SOR) is one of the most effective single-drug systemic therapy against advanced HCC, but the identification of novel combination regimens for a continued improvement in overall survival is a big challenge. Recent studies highlighted the crucial role of focal adhesion kinase (FAK) in HCC growth. The aim of this study was to investigate the antitumor effects of three different FAK inhibitors (FAKi), alone or in combination with SOR, using in vitro and in vivo models of HCC. METHODS: The effect of PND1186, PF431396, TAE226 on cell viability was compared to SOR. Among them TAE226, emerging as the most effective FAKi, was tested alone or in combination with SOR using 2D/3D human HCC cell line cultures and HCC xenograft murine models. The mechanisms of action were assessed by gene/protein expression and imaging approaches, combined with high-throughput methods. RESULTS: TAE226 was the more effective FAKi to be combined with SOR against HCC. Combined TAE226 and SOR treatment reduced HCC growth both in vitro and in vivo by affecting tumour-promoting gene expression and inducing epigenetic changes via dysregulation of FAK nuclear interactome. We characterized a novel nuclear functional interaction between FAK and the NuRD complex. TAE226-mediated FAK depletion and SOR-promoted MAPK down-modulation caused a decrease in the nuclear amount of HDAC1/2 and a consequent increase of the histone H3 lysine 27 acetylation, thus counteracting histone H3 lysine 27 trimethylation. CONCLUSIONS: Altogether, our findings provide the first evidence that TAE226 combined with SOR efficiently reduces HCC growth in vitro and in vivo. Also, our data highlight that deep analysis of FAK nuclear interactome may lead to the identification of new promising targets for HCC therapy.

Phosphodiesterase 4D Depletion/Inhibition Exerts Anti-Oncogenic Properties in Hepatocellular Carcinoma.

Isoform D of type 4 phosphodiesterase (PDE4D) has recently been associated with several human cancer types with the exception of human hepatocellular carcinoma (HCC). Here we explored the role of PDE4D in HCC. We found that PDE4D gene/protein were over-expressed in different samples of human HCCs compared to normal livers. Accordingly, HCC cells showed higher PDE4D activity than non-tumorigenic cells, accompanied by over-expression of the PDE4D isoform. Silencing of PDE4D gene and pharmacological inhibition of protein activity by the specific inhibitor Gebr-7b reduced cell proliferation and increased apoptosis in HCC cells, with a decreased fraction of cells in S phase and a differential modulation of key regulators of cell cycle and apoptosis. PDE4D silencing/inhibition also affected the gene expression of several cancer-related genes, such as the pro-oncogenic insulin growth factor (IGF2), which is down-regulated. Finally, gene expression data, available in the CancerLivER data base, confirm that PDE4D over-expression in human HCCs correlated with an increased expression of IGF2, suggesting a new possible molecular network that requires further investigations. In conclusion, intracellular depletion/inhibition of PDE4D prevents the growth of HCC cells, displaying anti-oncogenic effects. PDE4D may thus represent a new biomarker for diagnosis and a potential adjuvant target for HCC therapy.

The rs599839 A>G Variant Disentangles Cardiovascular Risk and Hepatocellular Carcinoma in NAFLD Patients.

BACKGROUND AND AIMS: Dyslipidemia and cardiovascular diseases (CVD) are comorbidities of nonalcoholic fatty liver disease (NAFLD), which ranges from steatosis to hepatocellular carcinoma (HCC). The rs599839 A>G variant, in the CELSR2-PSRC1-SORT1 gene cluster, has been associated CVD, but its impact on metabolic traits and on the severity liver damage in NAFLD has not been investigated yet. METHODS: We evaluated the effect of the rs599839 variant in 1426 NAFLD patients (Overall cohort) of whom 131 had HCC (NAFLD-HCC), in 500,000 individuals from the UK Biobank Cohort (UKBBC), and in 366 HCC samples from The Cancer Genome Atlas (TCGA). Hepatic PSRC1, SORT1 and CELSR2 expressions were evaluated by RNAseq (n = 125). RESULTS: The rs599839 variant was associated with reduced circulating LDL, carotid intima-media thickness, carotid plaques and hypertension (p < 0.05) in NAFLD patients and with protection against dyslipidemia in UKBBC. The minor G allele was associated with higher risk of HCC, independently of fibrosis severity (odds ratio (OR): 5.62; 95% c.i. 1.77-17.84, p = 0.003), poor prognosis and advanced tumor stage (p < 0.05) in the overall cohort. Hepatic PSRC1, SORT1 and CELSR2 expressions were increased in NAFLD patients carrying the rs599839 variant (p < 0.0001). SORT1 mRNA levels negatively correlated with circulating lipids and with those of genes involved in lipoprotein turnover (p < 0.0001). Conversely, PSRC1 expression was positively related to that of genes implicated in cell proliferation (p < 0.0001). In TCGA, PSRC1 over-expression promoted more aggressive HCC development (p < 0.05). CONCLUSIONS: In sum, the rs599839 A>G variant is associated with protection against dyslipidemia and CVD in NAFLD patients, but as one it might promote HCC development by modulating SORT1 and PSRC1 expressions which impact on lipid metabolism and cell proliferation, respectively.