Predicting response to non-selective beta-blockers with liver-spleen stiffness and heart rate in patients with liver cirrhosis and high-risk varices.

INTRODUCTION: Non-selective beta-blockers (NSBB) are used for primary prophylaxis in patients with liver cirrhosis and high-risk varices (HRVs). Assessing therapeutic response is challenging due to the invasive nature of hepatic venous pressure gradient (HVPG) measurement. This study aims to define a noninvasive machine-learning based approach to determine response to NSBB in patients with liver cirrhosis and HRVs. METHODS: We conducted a prospective study on a cohort of cirrhotic patients with documented HRVs receiving NSBB treatment. Patients were followed-up with clinical and elastography appointments at 3, 6, and 12 months after NSBB treatment initiation. NSBB response was defined as stationary or downstaging variceal grading at the 12-month esophagogastroduodenoscopy (EGD). In contrast, non-response was defined as upstaging variceal grading at the 12-month EGD or at least one variceal hemorrhage episode during the 12-month follow-up. We chose cut-off values for univariate and multivariate model with 100% specificity. RESULTS: According to least absolute shrinkage and selection operator (LASSO) regression, spleen stiffness (SS) and liver stiffness (LS) percentual decrease, along with changes in heart rate (HR) at 3 months were the most significant predictors of NSBB response. A decrease > 11.5% in SS, > 16.8% in LS, and > 25.3% in HR was associated with better prediction of clinical response to NSBB. SS percentual decrease showed the highest accuracy (86.4%) with high sensitivity (78.8%) when compared to LS and HR. The multivariate model incorporating SS, LS, and HR showed the highest discrimination and calibration metrics (AUROC = 0.96), with the optimal cut-off of 0.90 (sensitivity 94.2%, specificity 100%, PPV 95.7%, NPV 100%, accuracy 97.5%).

MiR-3201 as a Prognostic Blood Biomarker for Curative Treatments in Hepatocellular Carcinoma.

Background: Hepatic resection, radiofrequency ablation (RF), and liver transplantation (LT) represent the only available curative treatments for early stage hepatocellular carcinoma (HCC). Various studies showed that the 5-year overall survival (OS) rate reaches ∼70% after resection and ∼60% after RF. Objective: To improve the success rate of curative therapies and consequently the OS, an improvement in patients' selection and management should be pursued. In this regard, microRNAs (miRNAs) can be helpful prognostic biomarkers. Materials and Methods: In this retrospective study, a miRNA array profiling was performed on 34 HCC blood samples which is collected before therapy (T0), 1 month (T1), and 6 months (T2) after curative treatments (resection and RF) to identify noninvasive biomarker candidates for therapy response and OS. MiRNAs were validated in 80 blood HCC samples using quantitative real-time PCR (qRT-PCR). Patients were divided into complete responder (CR) and partial responder and progressive disease (PRPD). Results: Among the selected miRNAs, miR-3201 is significantly associated with treatment response in the validation phase, showing a 23% reduction (P = .026) in CR compared to PRPD. MiR-3201 was able to distinguish CR from PRPD (area under the curve [AUC] = 0.69, 71% sensitivity, 70% specificity, P = .0036). Furthermore, lower levels of miR-3201 were associated with longer OS (hazard ratio [HR] = 2.61, P = .0006). Conclusions: Blood miR-3201 could be used as a prognostic biomarker for curative therapy response and OS in HCC.

Predictors of Hepatocellular Carcinoma Early Recurrence in Patients Treated with Surgical Resection or Ablation Treatment: A Single-Center Experience.

Introduction: Hepatocellular carcinoma (HCC) is the sixth most diagnosed malignancy and the fourth leading cause of cancer-related death worldwide, with poor overall survival despite available curative treatments. One of the most crucial factors influencing survival in HCC is recurrence. The current study aims to determine factors associated with early recurrence of HCC in patients with BCLC Stage 0 or Stage A treated with surgical resection or local ablation. Materials and Methods: We retrospectively enrolled 58 consecutive patients diagnosed with HCC within BCLC Stage 0 or Stage A and treated either by surgical resection or local ablation with maximum nodule diameter < 50 mm. In the first year of follow-up after treatment, imaging was performed regularly one month after treatment and then every three months. Each case was discussed collectively by the Liver Multidisciplinary Group to decide diagnosis, treatment, follow-up, and disease recurrence. Variables resulting in statistically significant difference were then studied by Cox regression analysis; univariately and then multivariately based on forward stepwise Cox regression. Results are represented in hazard ratio (H.R.) with 95% confidence interval (C.I.). Results: There was no statistically significant difference in recurrence rates (34.8 vs. 45.7%, log-rank test, p = 0.274) between patients undergoing surgical resection and local ablation, respectively. Early recurrence was associated with male gender (HR 2.5, 95% C.I. 1.9−3.1), nodule diameter > 20 mm (HR 4.5, 95% C.I. 3.9−5.1), platelet count < 125 × 103 cell/mm3 (HR 1.6, 95% C.I. 1.2−1.9), platelet-lymphocyte ratio < 95 (HR 2.1, 95% C.I. 1.7−2.6), lymphocyte-monocyte ratio < 2.5 (HR 1.9, 95% C.I. 1.4−2.5), and neutrophil-lymphocyte ratio > 2 (HR 2.7, 95% C.I. 2.2−3.3). Discussion and Conclusions: Our results are in line with the current literature. Male gender and tumor nodule dimension are the main risk factors associated with early HCC recurrence. Platelet count and other combined scores can be used as predictive tools for early HCC recurrence, although more studies are needed to define cut-offs.

Alanine aminotransferase and spleno-portal dynamics affect spleen stiffness measured by point shear-wave elastography in patients with chronic hepatitis C in the absence of significant liver fibrosis.

BACKGROUND: Spleen stiffness (SS) has gained a lot of interest in the context of liver cirrhosis and portal hypertension stratification. However, there is a paucity of data on confounding factors that may alter SS values. METHODS: Between January 2018 and October 2019, we enrolled 120 healthy subjects and 117 patients with hepatitis C virus (HCV) infection who did not have significant liver fibrosis (i.e., F0-1). Abdominal ultrasound evaluation was performed on each individual to measure portal vein diameter, portal flow velocity, spleen bipolar diameter, and splenic area. We also performed liver and spleen elastography. RESULTS: HCV patients had higher SS (p < 0.001), portal vein diameter (p = 0.031), portal flow velocity (p = 0.035), spleen bipolar diameter (p = 0.042) and area (p = 0.025), and ALT levels (p < 0.001). Linear regression models showed that SS increased by 3.220 kPa for each mm of portal vein diameter, by 0.7 kPa for each cm/s of portal flow velocity, by 2.239 kPa for each cm of spleen bipolar diameter, and by 0.233 kPa for each cm2 of spleen area. Patients with HCV infection were stratified according to median ALT levels (i.e. 32 IU/L). SS and spleno-portal axis parameters were significantly higher in patients with an ALT level > 32 IU/L. Besides, the relationship between SS and ALT was described by cubic polynomial regression according to the following equation: 11.735 + 0.404 (ALT)1 - 0.002 (ALT)2 + 4.26 × 10-6 (ALT)3. CONCLUSIONS: Our results bring new light to the role of inflammation as a confounding factor for SS measurement. Therefore, particular attention should be paid to serum transaminase for a correct evaluation of spleen elastography.

The role of elastography in alcoholic liver disease: fibrosis staging and confounding factors, a review of the current literature.

INTRODUCTION: Alcohol-related liver disease (ALD) was estimated to have a prevalence of 2% among the USA population. Since severe fibrosis in compensated patients is the main predictor of long-term survival, it is of utmost importance to early detect patients with severe fibrosis before decompensation occurs. Liver elastography has been used to stage liver fibrosis. However, there is a widespread lack in guidelines for the correct use of liver stiffness (LS) in ALD. EVIDENCE ACQUISITION: A structured search was carried out on MEDLINE/PubMed database. From the original 225 research articles identified, only 12 studies met the inclusion criteria, with 10 studies being eventually included. EVIDENCE SYNTHESIS: According to reported data, patients with aspartate aminotransferase (AST)>100 IU/L and 50 IU/L showed significantly higher values of LS if compared to patients with the same fibrosis stage. Also, excessive alcohol consumption greatly influences elastography, leading to false fibrosis staging. When LS values >5-6 kPa are detected, several aspects should be taken into account. First of all, the patient should be asked about the current alcohol consumption (i.e. active vs. abstinence, determination of abstinence period, and quantification of alcohol intake), and if the patient is an active drinker, liver elastography can be repeated after a complete abstinence period of at least two weeks. and if the patient is an active drinker, liver elastography can be repeated after a complete abstinence period of at least two weeks. Secondly, clinicians should check liver transaminases level, and if AST are above 100 IU/L, they should be aware of a possible overestimation of fibrosis. However, whether transaminases-adapted cut-off values should be used for ad-hoc decisions in patients with no time or option to withdraw from alcohol consumption is still a matter of debate. CONCLUSIONS: We hope that our review article may serve as a reference point in the prospect of futures guidelines.

Circulatory miRNA as a Biomarker for Therapy Response and Disease-Free Survival in Hepatocellular Carcinoma.

The clinical outcome of hepatocellular carcinoma (HCC) treatment remains unsatisfactory, contributing to the high mortality of HCC worldwide. Circulating miRNAs have the potential to be a predictor of therapy response. Microarray profiling was performed in serum samples of 20 HCC patients before treatment. Circulating miRNAs associated with treatment response were validated in 86 serum HCC samples using the qRT-PCR system. Patients were treated either with curative treatments (resection or radiofrequency) or trans-arterial chemoembolization (TACE), and grouped according to therapy response in complete responders (CR) and partial responders or progressive disease (PRPD), following mRECIST criteria. Four miRNA candidates from the discovery phase (miR-4443, miR-4454, miR-4492, and miR-4530) were validated. Before therapy, miR-4454 and miR-4530 were up-regulated in CR to curative treatments (2.83 fold, p = 0.02 and 2.33 fold, p = 0.008, respectively) and were able to differentiate CR from PRPD (area under the curve (AUC) = 0.74, sens/spec 79/63% and AUC = 0.77, sens/spec 72/73%). On the contrary, miR-4443 was 1.95 times down-regulated in CR (p = 0.05) with an AUC of 0.72 (sens = 70%, spec = 60%) in distinguishing CR vs. PRPD). The combination of the three miRNAs was able to predict the response to curative treatment with an AUC of 0.84 (sens = 72%, spec = 75%). The higher levels of miR-4454 and miR-4530 in were associated to longer overall survival (HR = 2.79, p = 0.029 and HR = 2.97, p = 0.011, respectively). Before TACE, miR-4492 was significantly up-regulated in CR patients (FC = 2.67, p = 0.01) and able to differentiate CR from PRPD (AUC = 0.84, sens/spec 84.6/71%). We demonstrated that different miRNAs predictors can be used as potential prognostic circulating biomarkers according to the selected treatment for HCC.

Comparison between hepatocellular carcinoma prognostic scores: A 10-year single-center experience and brief review of the current literature.

BACKGROUND: Hepatocellular carcinoma (HCC) represents the most common primitive liver malignancy. A relevant concern involves the lack of agreement on staging systems, prognostic scores, and treatment allocation algorithms. AIM: To compare the survival rates among already developed prognostic scores. METHODS: We retrospectively evaluated 140 patients with HCC diagnosed between February 2006 and November 2017. Patients were categorized according to 15 prognostic scoring systems and estimated median survivals were compared with those available from the current medical literature. RESULTS: The median overall survival of the cohort of patients was 35 (17; 67) mo, and it was statistically different in relation to treatment choice, ultrasound surveillance, and serum alpha-fetoprotein. The Italian Liver Cancer (ITA.LI.CA) tumor staging system performed best in predicting survival according to stage allocation among all 15 evaluated prognostic scores. Using the ITA.LI.CA prognostic system, 28.6%, 40.7%, 22.1%, and 8.6% of patients fell within stages 0-1, 2-3, 4-5 and > 5 respectively. The median survival was 57.9 mo for stages 0-1, 43 mo for stages 2-3, 21.7 mo for stages 4-5, and 10.4 mo for stage > 5. The 1-, 3-, and 5-year survival rates were respectively 95%, 65%, and 20%, for stages 0-1; 94.7%, 43.9% and 26.3% for stages 2-3; 71%, 25.8% and 16.1% for stages 4-5; and 50%, 16.7% and 8.3% for stage > 5. At the same time, although statistically significant in prognostic stratification, the most commonly used Barcelona Clinic Liver Cancer system showed one of the most relevant differences in median survival, especially for stages A and C, when compared to the medical literature. In fact, 10.7%, 59.3%, 27.1%, 1.4%, and 0% of patients were stratified into stages 0, A, B, C, and D respectively. The median survival was > 81.1 mo for stage 0, 44.9 mo for stage A, 21.3 mo for stage B, and 3.1 mo for stage C. The 1-, 3-, and 5-year survival rates were respectively 86.7%, 60%, and 46.7% for stage 0; 91.6%, 50.6%, and 20.5% for stage A; 73.7%, 23.7% and 13.2% for stage B; and 2%, 0% and 0% for stage C. CONCLUSION: Survival analysis shows excellent prognostic ability of the ITA.LI.CA scoring system compared to other staging systems.

Evaluation of spleen stiffness in healthy volunteers using point shear wave elastography.

INTRODUCTION AND OBJECTIVES: This study aims to measure the values of spleen stiffness (SS) in healthy subjects, the inter-operator agreement in SS measurement, and to detect statistically significant correlations between SS and age, sex, weight, BMI, portal vein dynamics and splenic dimensions. MATERIALS AND METHODS: The study included 100 healthy volunteers who had no substantial alcohol intake (<30g/daily for man, <20g/daily women), were negative on hepatitis B, hepatitis C, HIV blood serology, and had any history of lymphoproliferative disorders. Abdominal ultrasound, liver and spleen elastography were performed on each patient to search for focal splenic lesions, bile tract or portal vein dilatation, moderate/severe liver steatosis, and to measure liver and spleen stiffness. RESULTS: The mean value was 18.14 (±3.08) kPa. In the group of men (n=49), the mean was 17.73 (±2.91) kPa, whereas in the group of women (n=51) it was 16.72 (±3.32) kPa. Statistical analyses showed no correlation between spleen stiffness and sex, age, weight, and BMI. Regarding their splenoportal axis, statistically significant differences in SS were found in the means of the two subgroups of subjects stratified by their portal flow velocity (p=0.003) and spleen area (p<0.001). Spearman's rank showed a weak association between SS and portal flow velocty (r=0.271) and splenic area (r=-0.237). ICC showed excellent (0.96) inter-operator agreement and Bland-Altman plot demonstrated no systematic over/under-estimation of spleen stiffness values. CONCLUSIONS: Our results may serve as a reference point in the evaluation of SS especially in patients affected by advanced liver disease.

α-ketoglutarate dehydrogenase inhibition counteracts breast cancer-associated lung metastasis.

Metastasis formation requires active energy production and is regulated at multiple levels by mitochondrial metabolism. The hyperactive metabolism of cancer cells supports their extreme adaptability and plasticity and facilitates resistance to common anticancer therapies. In spite the potential relevance of a metastasis metabolic control therapy, so far, limited experience is available in this direction. Here, we evaluated the effect of the recently described α-ketoglutarate dehydrogenase (KGDH) inhibitor, (S)-2-[(2,6-dichlorobenzoyl) amino] succinic acid (AA6), in an orthotopic mouse model of breast cancer 4T1 and in other human breast cancer cell lines. In all conditions, AA6 altered Krebs cycle causing intracellular α-ketoglutarate (α-KG) accumulation. Consequently, the activity of the α-KG-dependent epigenetic enzymes, including the DNA demethylation ten-eleven translocation translocation hydroxylases (TETs), was increased. In mice, AA6 injection reduced metastasis formation and increased 5hmC levels in primary tumours. Moreover, in vitro and in vivo treatment with AA6 determined an α-KG accumulation paralleled by an enhanced production of nitric oxide (NO). This epigenetically remodelled metabolic environment efficiently counteracted the initiating steps of tumour invasion inhibiting the epithelial-to-mesenchymal transition (EMT). Mechanistically, AA6 treatment could be linked to upregulation of the NO-sensitive anti-metastatic miRNA 200 family and down-modulation of EMT-associated transcription factor Zeb1 and its CtBP1 cofactor. This scenario led to a decrease of the matrix metalloproteinase 3 (MMP3) and to an impairment of 4T1 aggressiveness. Overall, our data suggest that AA6 determines an α-KG-dependent epigenetic regulation of the TET-miR200-Zeb1/CtBP1-MMP3 axis providing an anti-metastatic effect in a mouse model of breast cancer-associated metastasis.

Sema3F (Semaphorin 3F) Selectively Drives an Extraembryonic Proangiogenic Program.

OBJECTIVE: Molecular pathways governing blood vessel patterning are vital to vertebrate development. Because of their ability to counteract proangiogenic factors, antiangiogenic secreted Sema3 (class 3 semaphorins) control embryonic vascular morphogenesis. However, if and how Sema3 may play a role in the control of extraembryonic vascular development is presently unknown. APPROACH AND RESULTS: By characterizing genetically modified mice, here, we show that surprisingly Sema3F acts instead as a selective extraembryonic, but not intraembryonic proangiogenic cue. Both in vivo and in vitro, in visceral yolk sac epithelial cells, Sema3F signals to inhibit the phosphorylation-dependent degradation of Myc, a transcription factor that drives the expression of proangiogenic genes, such as the microRNA cluster 17/92. In Sema3f-null yolk sacs, the transcription of Myc-regulated microRNA 17/92 cluster members is impaired, and the synthesis of Myc and microRNA 17/92 foremost antiangiogenic target Thbs1 (thrombospondin 1) is increased, whereas Vegf (vascular endothelial growth factor) signaling is inhibited in yolk sac endothelial cells. Consistently, exogenous recombinant Sema3F inhibits the phosphorylation-dependent degradation of Myc and the synthesis of Thbs1 in mouse F9 teratocarcinoma stem cells that were in vitro differentiated in visceral yolk sac epithelial cells. Sema3f-/- mice placentas are also highly anemic and abnormally vascularized. CONCLUSIONS: Sema3F functions as an unconventional Sema3 that promotes extraembryonic angiogenesis by inhibiting the Myc-regulated synthesis of Thbs1 in visceral yolk sac epithelial cells.