Gustavo Ferrín

Therapy with anti-tumor necrosis factor (TNF) has dramatically changed the natural history of Crohn’s disease (CD). However, these drugs are not without adverse events, and up to 40% of patients could lose efficacy in the long term. We aimed to identify reliable markers of response to anti-TNF drugs in patients with CD. A consecutive cohort of 113 anti-TNF naive patients with CD was stratified according to clinical response as short-term remission (STR) or non-STR (NSTR) at 12 weeks of treatment. We compared the protein expression profiles of plasma samples in a subset of patients from both groups prior to anti-TNF therapy by SWATH proteomics. We identified 18 differentially expressed proteins (p ≤ 0.01, fold change ≥ 2.4) involved in the organization of the cytoskeleton and cell junction, hemostasis/platelet function, carbohydrate metabolism, and immune response as candidate biomarkers of STR. Among them, vinculin was one of the most deregulated proteins (p < 0.001), whose differential expression was confirmed by ELISA (p = 0.054). In the multivariate analysis, plasma vinculin levels along with basal CD Activity Index, corticosteroids induction, and bowel resection were factors predicting NSTR.

Circulating tumor cells (CTCs), and particularly circulating cancer stem cells (cCSC), are prognostic biomarkers for different malignancies and may be detected using liquid biopsies. The ex vivo culture of cCSCs would provide valuable information regarding biological aggressiveness and would allow monitoring the adaptive changes acquired by the tumor in real time. In this prospective pilot study, we analyzed the presence of EpCAM⁺ CTCs using the IsoFlux system in the peripheral blood of 37 patients with hepatocellular carcinoma undergoing transarterial chemoembolization (TACE). The average patient age was 63.5 ± 7.9 years and 91.9% of the patients were men. All patients had detectable CTCs at baseline and 20 patients (54.1%) showed CTC aggregates or clusters in their peripheral blood. The increased total tumor diameter (OR: 2.5 (95% CI: 1.3–4.8), p = 0.006) and the absence of clusters of CTCs at baseline (OR: 0.2 (95% CI: 0.0–1.0), p = 0.049) were independent predictors of a diminished response to TACE. Culture of cCSC was successful in five out of thirty-three patients, mostly using negative enrichment of CD45⁻ cells, ultra-low adherence, high glucose, and a short period of hypoxia followed by normoxia. In conclusion, the identification of clusters of CTCs before TACE and the implementation of standardized approaches for cCSC culture could aid to predict outcomes and to define the optimal adjuvant therapeutic strategy for a true personalized medicine in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fifth most common neoplasm and a major cause of cancer-related death worldwide. There is no ideal biomarker allowing early diagnosis of HCC and tumor surveillance in patients receiving therapy. Liquid biopsy, and particularly circulating tumor cells (CTCs), have emerged as a useful tool for diagnosis and monitoring therapeutic responses in different tumors. In the present manuscript, we evaluate the current evidence supporting the quantitative and qualitative assessment of CTCs as potential biomarkers of HCC, as well as technical aspects related to isolation, identification, and classification of CTCs. Although the dynamic assessment of CTCs in patients with HCC may aid the decision-making process, there are still many uncertainties and technical caveats to be solved before this methodology has a true impact on clinical practice guidelines. More studies are needed to identify the optimal combination of surface markers, to increase the efficiency of ex-vivo expansion of CTCs, or even to target CTCs as a potential therapeutic strategy to prevent HCC recurrence after surgery or to hamper tumor progression and extrahepatic spreading.

Background: In patients with hepatocellular carcinoma (HCC), a complete clearance of circulating tumor cells (CTCs) early after liver transplantation (LT) or surgical resection (LR) could prevent tumor recurrence. Methods: prospective pilot study including patients with HCC who underwent LR or LT from September 2017 to May 2020. Enumeration of CTCs was performed in peripheral blood samples (7 mL) using the Isoflux^® system (Fluxion Biosciences) immediately before surgery, at post-operative day 5 and at day 30. A clinically relevant number of CTCs was defined as >30 CTCs/sample. Results: 41 HCC patients were included (mean age 58.7 ± 6.3; 82.9% male). LR was performed in 10 patients (24.4%) and 31 patients (75.6%) underwent LT. The main etiology of liver disease was chronic hepatitis C (31.7%). Patients undergoing LR and LT were similar in terms of preoperative CTC count (p = 0.99), but clearance of CTCs within the first month was more pronounced in the LT group. Clusters of CTCs at baseline were associated with incomplete clearance of CTCs at day 30 (54.2% vs. 11.8%, p = 0.005), which in turn impacted negatively on survival (p = 0.038). Conclusion: Incomplete clearance of CTCs after surgery could be a surrogate marker of HCC aggressiveness.

Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer and occurs mainly in patients with liver cirrhosis. The mammalian target of rapamycin (mTOR) signaling pathway is involved in many hallmarks of cancer including cell growth, metabolism re-programming, proliferation and inhibition of apoptosis. The mTOR pathway is upregulated in HCC tissue samples as compared with the surrounding liver cirrhotic tissue. In addition, the activation of mTOR is more intense in the tumor edge, thus reinforcing its role in HCC proliferation and spreading. The inhibition of the mTOR pathway by currently available pharmacological compounds (i.e., sirolimus or everolimus) is able to hamper tumor progression both in vitro and in animal models. The use of mTOR inhibitors alone or in combination with other therapies is a very attractive approach, which has been extensively investigated in humans. However, results are contradictory and there is no solid evidence suggesting a true benefit in clinical practice. As a result, neither sirolimus nor everolimus are currently approved to treat HCC or to prevent tumor recurrence after curative surgery. In the present comprehensive review, we analyzed the most recent scientific evidence while providing some insights to understand the gap between experimental and clinical studies.

(1) Background: The mammalian target of rapamycin (mTOR) pathway activation is critical for hepatocellular carcinoma (HCC) progression. We aimed to evaluate the mTOR tissue expression in liver transplant (LT) patients and to analyse its influence on post-LT outcomes. (2) Methods: Prospective study including a cohort of HCC patients who underwent LT (2012–2015). MTOR pathway expression was evaluated in the explanted liver by using the “PathScan Intracellular Signalling Array Kit” (Cell Signalling). Kaplan-Meier and Cox regression analyses were performed to evaluate post-LT HCC recurrence. (3) Results: Forty-nine patients were included (average age 56.4 ± 6, 14.3% females). Phospho-mTOR (Ser2448) was over-expressed in peritumoral tissue as compared with tumoral tissue (ΔSignal 22.2%; p < 0.001). The mTOR activators were also increased in peritumoral tissue (phospho-Akt (Thr308) ΔSignal 18.2%, p = 0.004; phospho-AMPKa (Thr172) ΔSignal 56.3%, p < 0.001), as they were the downstream effectors responsible for cell growth/survival (phospho-p70S6K (Thr389) ΔSignal 33.3%, p < 0.001 and phospho-S6RP (Ser235/236) ΔSignal 54.6%, p < 0.001). MTOR expression was increased in patients with multinodular HCC (tumoral p = 0.01; peritumoral p = 0.001). Increased phospho-mTOR in tumoral tissue was associated with higher HCC recurrence rates after LT (23.8% vs. 5.9% at 24 months, p = 0.04). (4) Conclusion: mTOR pathway is over-expressed in patients with multinodular HCC and is it associated with increased post-LT tumour recurrence rates.

: Hepatic steatosis is a common obesity-associated pathology characterized by the accumulation of fat within the liver, which can progress to liver fibrosis, cirrhosis and ultimately lead to hepatocellular carcinoma. Remarkably, obesity and cancer course with a profound dysregulation of the genetic expression patterns and, particularly, with the aberrant expression of splicing variants that could contribute to the aggressiveness and comorbidities of these pathologies. Since the appearance of alternative splicing variants could be related to a dysregulation of the cellular machinery responsible for this process ((spliceosome core elements and splicing factors (SFs)), the objective of the present study was to determine the association between the expression pattern of the components of this machinery and hepatic steatosis of obese patients. To this end, we collected clinical and demographic data and liver biopsy samples from obese women (IMC>30) with (n=32) and without (n=9) hepatic steatosis. RNA from liver samples was extracted and retrotranscribed to determine the expression levels of selected components of the major (n=13) and minor spliceosome (n=4), and associated SFs (n=28) using a qPCR-based array. Results revealed that the liver of steatotic patients exhibit a profound dysregulation of certain spliceosome components and SFs compared to non-steatotic patients (e.g. RNU6, SF3b1, etc.). Although these alterations were not associated with the hepatic steatosis level, the expression pattern of the components of this cellular machinery could define discrete groups of steatotic patients that presented similar alterations in certain spliceosome components and SFs. Interestingly, these groups of patients were also characterized by particular hepatic and clinical-metabolic alterations (e.g. ALT, hyperglicemia, hyperinsulinemia, etc.). Therefore, although further confirmatory studies are needed, these results could suggest that the development of hepatic steatosis and its associated comorbidities could be linked to the dysregulation of certain components of the splicing machinery, which may provide novel diagnostic/therapeutic tools for this pathology.

The harmful effects of bile acid accumulation occurring during cholestatic liver diseases have been associated with oxidative stress increase and endothelial nitric oxide synthase (NOS-3) expression decrease in liver cells. We have previously reported that glycochenodeoxycholic acid (GCDCA) down-regulates gene expression by increasing SP1 binding to the NOS-3 promoter in an oxidative stress dependent manner. In the present study, we aimed to investigate the role of transcription factor (TF) AP-1 on the NOS-3 deregulation during GCDCA-induced cholestasis. The cytotoxic response to GCDCA was characterized by 1) the increased expression and activation of TFs cJun and c-Fos; 2) a higher binding capability of these at position -666 of the NOS-3 promoter; 3) a decrease of the transcriptional activity of the promoter and the expression and activity of NOS-3; and 4) the expression increase of cyclin D1. Specific inhibition of AP-1 by the retinoid SR 11302 counteracted the cytotoxic effects induced by GCDCA while promoting NOS-3 expression recovery and cyclin D1 reduction. NOS activity inhibition by L-NAME inhibited the protective effect of SR 11302. Inducible NOS isoform was no detected in this experimental model of cholestasis. Our data provide direct evidence for the involvement of AP-1 in the NOS-3 expression regulation during cholestasis and define a critical role for NOS-3 in regulating the expression of cyclin D1 during the cell damage induced by bile acids. AP-1 appears as a potential therapeutic target in cholestatic liver diseases given its role as a transcriptional repressor of NOS-3.

Obesity, a disease that is growing at epidemic proportions worldwide, is caused by a combination of genetic and lifestyle factors. One of the most common pathologies associated with obesity is hepatic steatosis, an accumulation of fat within the liver that can progress to liver fibrosis, cirrhosis, and ultimately lead to hepatocellular carcinoma. There is emerging evidence that alternative mRNA splicing, the key mechanism providing transcript and protein diversity, is dysregulated in many tissues under adverse metabolic conditions, such as obesity and cancer. Moreover, the splicing variants generated within this process could contribute to the aggressiveness and comorbidities of these diseases. We hypothesized that an alteration in the splicing machinery could occur in obese patients with hepatic steatosis, which might ultimately be associated with the progression to hepatic fibrosis/cirrhosis/carcinoma. To address this question, an array of selected components of the major (n=13) and minor spliceosome (n=4), and associated splicing factors (n=28) was developed, and their expression levels were evaluated using a Fluidigm methodology, in a series of liver samples from obese (BMI >30) women with (n=33) and without (n=9) liver steatosis. Results revealed that expression of a number of relevant splicing factors (SRSF2, SND1, SRRM1, PTBP1, KHDRBS1) and spliceosome components (SF3Btv2, U2AF1, U2AF2, RNU6) was altered in steatosis vs non-steatosis liver tissues. Interestingly, some of these changes were associated with relevant clinical parameters, including glucose levels (i.e. SRSF2, U2AF1/2, KHDRBS1), gamma-glutamyltransferase (SRSF2, U2AF1, KHDRBS1), HDL (U2AF2, SND1) as well as with insulin, cholesterol, creatinine and HOMA-IR (i.e. RNU6). Finally, ROC analysis revealed that the expression of specific splicing factors, especially SRSF2, U2AF2, PTBP1 and RNU6, can clearly predict patients with or without hepatic steatosis. In conclusion, the expression of specific splicing machinery components is significantly altered in the liver of obese patients with hepatic steatosis. Ongoing studies would clarify the potential pathological implications of these findings, which could help to predict a worsening in steatosis, and may provide novel diagnostic biomarkers and therapeutic tools for this disease.