Application of Graph Models to the Identification of Transcriptomic Oncometabolic Pathways in Human Hepatocellular Carcinoma.

Whole-tissue transcriptomic analyses have been helpful to characterize molecular subtypes of hepatocellular carcinoma (HCC). Metabolic subtypes of human HCC have been defined, yet whether these different metabolic classes are clinically relevant or derive in actionable cancer vulnerabilities is still an unanswered question. Publicly available gene sets or gene signatures have been used to infer functional changes through gene set enrichment methods. However, metabolism-related gene signatures are poorly co-expressed when applied to a biological context. Here, we apply a simple method to infer highly consistent signatures using graph-based statistics. Using the Cancer Genome Atlas Liver Hepatocellular cohort (LIHC), we describe the main metabolic clusters and their relationship with commonly used molecular classes, and with the presence of TP53 or CTNNB1 driver mutations. We find similar results in our validation cohort, the LIRI-JP cohort. We describe how previously described metabolic subtypes could not have therapeutic relevance due to their overall downregulation when compared to non-tumoral liver, and identify N-glycan, mevalonate and sphingolipid biosynthetic pathways as the hallmark of the oncogenic shift of the use of acetyl-coenzyme A in HCC metabolism. Finally, using DepMap data, we demonstrate metabolic vulnerabilities in HCC cell lines.

A Focus on the Pathophysiology of Adrenomedullin Expression: Endothelitis and Organ Damage in Severe Viral and Bacterial Infections.

Adrenomedullin (ADM) is a peptide hormone produced primarily in the adrenal glands, playing a crucial role in various physiological processes. As well as improving vascular integrity and decreasing vascular permeability, ADM acts as a vasodilator, positive inotrope, diuretic, natriuretic and bronchodilator, antagonizing angiotensin II by inhibiting aldosterone secretion. ADM also has antihypertrophic, anti-apoptotic, antifibrotic, antioxidant, angiogenic and immunoregulatory effects and antimicrobial properties. ADM expression is upregulated by hypoxia, inflammation-inducing cytokines, viral or bacterial substances, strength of shear stress, and leakage of blood vessels. These pathological conditions are established during systemic inflammation that can result from infections, surgery, trauma/accidents or burns. The ability to rapidly identify infections and the prognostic, predictive power makes it a valuable tool in severe viral and bacterial infections burdened by high incidence and mortality. This review sheds light on the pathophysiological processes that in severe viral or bacterial infections cause endothelitis up to the development of organ damage, the resulting increase in ADM levels dosed through its more stable peptide mid-regional proadrenomedullin (MR-proADM), the most significant studies that attest to its diagnostic and prognostic accuracy in highlighting the severity of viral or bacterial infections and appropriate therapeutic insights.

Prognostic value of ultra-low-pass whole-genome sequencing of circulating tumor DNA in hepatocellular carcinoma under systemic treatment.

BACKGROUND/AIMS: New prognostic markers are needed to identify patients with hepatocellular carcinoma (HCC) who carry a worse prognosis. Ultra-low-pass whole-genome sequencing (ULP-WGS) (≤0.5× coverage) of cell-free DNA (cfDNA) has emerged as a low-cost promising tool to assess both circulating tumor DNA (ctDNA) fraction and large structural genomic alterations. Here, we studied the performance of ULP-WGS of plasma cfDNA to infer prognosis in patients with HCC. METHODS: Plasma samples were obtained from patients with HCC prior to surgery, locoregional or systemic therapy, and were analyzed by ULP-WGS of cfDNA to an average genome-wide fold coverage of 0.3x. ctDNA and copy number alterations (CNA) were estimated using the software package ichorCNA. RESULTS: Samples were obtained from 73 HCC patients at different BCLC stages (BCLC 0/A: n=37, 50.7%; BCLC B/C: n=36, 49.3%). ctDNA was detected in 18 out of 31 patients who received systemic treatment. Patients with detectable ctDNA showed significantly worse overall survival (median, 13.96 months vs not reached). ctDNA remained an independent predictor of prognosis after adjustment by clinical-pathologic features and type of systemic treatment (hazard ratio 7.69; 95%, CI 2.09-28.27). Among ctDNA-positive patients under systemic treatments, the loss of large genomic regions in 5q and 16q arms was associated with worse prognosis after multivariate analysis. CONCLUSION: ULP-WGS of cfDNA provides clinically relevant information about the tumor biology. The presence of ctDNA and the loss of 5q and 16q arms in ctDNA-positive patients are independent predictors of worse prognosis in patients with advanced HCC receiving systemic therapy.

The Impact of Portal Hypertension Assessment Method on the Outcomes of Hepatocellular Carcinoma Resection: A Meta-Analysis of Matched Cohort and Prospective Studies.

OBJECTIVE: Examine portal hypertension (PHT) impact on postoperative and survival outcomes in hepatocellular carcinoma (HCC) patients after liver resection (LR), specifically exploring distinctions between indirect signs and invasive measurements of PHT. BACKGROUND: PHT has historically discouraged LR in individuals with HCC due to the elevated risk of morbidity, including liver decompensation (LD). METHODS: A systematic review was conducted using 3 databases to identify prospective-controlled and matched cohort studies until December 28, 2022. Focus on comparing postoperative outcomes (mortality, morbidity, and liver-related complications) and overall survival in HCC patients with and without PHT undergoing LR. Three meta-analysis models were utilized: for aggregated data (fixed-effects inverse variance model), for patient-level survival data (one-stage frequentist meta-analysis with gamma-shared frailty Cox proportional hazards model), and for pooled data (Freeman-Tukey exact and double arcsine method). RESULTS: Nine studies involving 1124 patients were analyzed. Indirect signs of PHT were not significantly associated with higher mortality, overall complications, PHLF or LD. However, LR in patients with hepatic venous pressure gradient (HVPG) ≥10 mm Hg significantly increased the risk of overall complications, PHLF, and LD. Despite elevated risks, the procedure resulted in a 5-year overall survival rate of 55.2%. Open LR significantly increased the risk of overall complications, PHLF, and LD. Conversely, PHT did not show a significant association with worse postoperative outcomes in minimally invasive LR. CONCLUSIONS: LR in the presence of indirect signs of PHT poses no increased risk of complications. Yet, in HVPG ≥10 mm Hg patients, LR increases overall morbidity and liver-related complications risk. Transjugular HVPG assessment is crucial for LR decisions. Minimally invasive approach seems to be vital for favorable outcomes, especially in HVPG ≥10 mm Hg patients.

Artificial intelligence-based pathology as a biomarker of sensitivity to atezolizumab-bevacizumab in patients with hepatocellular carcinoma: a multicentre retrospective study.

BACKGROUND: Clinical benefits of atezolizumab plus bevacizumab (atezolizumab-bevacizumab) are observed only in a subset of patients with hepatocellular carcinoma and the development of biomarkers is needed to improve therapeutic strategies. The atezolizumab-bevacizumab response signature (ABRS), assessed by molecular biology profiling techniques, has been shown to be associated with progression-free survival after treatment initiation. The primary objective of our study was to develop an artificial intelligence (AI) model able to estimate ABRS expression directly from histological slides, and to evaluate if model predictions were associated with progression-free survival. METHODS: In this multicentre retrospective study, we developed a model (ABRS-prediction; ABRS-P), which was derived from the previously published clustering-constrained attention multiple instance learning (or CLAM) pipeline. We trained the model fit for regression analysis using a multicentre dataset from The Cancer Genome Atlas (patients treated by surgical resection, n=336). The ABRS-P model was externally validated on two independent series of samples from patients with hepatocellular carcinoma (a surgical resection series, n=225; and a biopsy series, n=157). The predictive value of the model was further tested in a series of biopsy samples from a multicentre cohort of patients with hepatocellular carcinoma treated with atezolizumab-bevacizumab (n=122). All samples in the study were from adults (aged ≥18 years). The validation sets were sampled between Jan 1, 2008, to Jan 1, 2023. For the multicentre validation set, the primary objective was to assess the association of high versus low ABRS-P values, defined relative to cross-validation median split thresholds in the first biopsy series, with progression-free survival after treatment initiation. Finally, we performed spatial transcriptomics and matched prediction heatmaps with in situ expression profiles. FINDINGS: Of the 840 patients sampled, 641 (76%) were male and 199 (24%) were female. Across the development and validation datasets, hepatocellular carcinoma risk factors included alcohol intake, hepatitis B and C virus infections, and non-alcoholic steatohepatitis. Using cross-validation in the development series, the mean Pearson's correlation between ABRS-P values and ABRS score (mean expression of ABRS genes) was r=0·62 (SD 0·09; mean p<0·0001, SD<0·0001). The ABRS-P generalised well on the external validation series (surgical resection series, r=0·60 [95% CI 0·51-0·68], p<0·0001; biopsy series, r=0·53 [0·40-0·63], p<0·0001). In the 122 patients treated with atezolizumab-bevacizumab, those with ABRS-P-high tumours (n=74) showed significantly longer median progression-free survival than those with ABRS-P-low tumours (n=48) after treatment initiation (12 months [95% CI 7-not reached] vs 7 months [4-9]; p=0·014). Spatial transcriptomics showed significantly higher ABRS score, along with upregulation of various other immune effectors, in tumour areas with high ABRS-P values versus areas with low ABRS-P values. INTERPRETATION: Our study indicates that AI applied on hepatocellular carcinoma digital slides is able to serve as a biomarker for progression-free survival in patients treated with atezolizumab-bevacizumab. This approach could be used in the development of inexpensive and fast biomarkers for targeted therapies. The combination of AI heatmaps with spatial transcriptomics provides insight on the molecular features associated with predictions. This methodology could be applied to other cancers or diseases and improve understanding of the biological mechanisms that drive responses to treatments. FUNDING: Institut National du Cancer, Fondation ARC, China Scholarship Council, Ligue Contre le Cancer du Val de Marne, Fondation de l'Avenir, Ipsen, and Fondation Bristol Myers Squibb Pour la Recherche en Immuno-Oncologie.

Mapping early serum proteome signatures of liver regeneration in living donor liver transplant cases.

The liver is the only solid organ capable of regenerating itself to regain 100% of its mass and function after liver injury and/or partial hepatectomy (PH). This exceptional property represents a therapeutic opportunity for severe liver disease patients. However, liver regeneration (LR) might fail due to poorly understood causes. Here, we have investigated the regulation of liver proteome and phosphoproteome at a short time after PH (9 h), to depict a detailed mechanistic background of the early LR phase. Furthermore, we analyzed the dynamic changes of the serum proteome and metabolome of healthy living donor liver transplant (LDLT) donors at different time points after surgery. The molecular profiles from both analyses were then correlated. Insulin and FXR-FGF15/19 signaling were stimulated in mouse liver after PH, leading to the activation of the main intermediary kinases (AKT and ERK). Besides, inhibition of the hippo pathway led to an increased expression of its target genes and of one of its intermediary proteins (14-3-3 protein), contributing to cell proliferation. In association with these processes, metabolic reprogramming coupled to enhanced mitochondrial activity cope for the energy and biosynthetic requirements of LR. In human serum of LDLT donors, we identified 56 proteins and 13 metabolites statistically differential which recapitulate some of the main cellular processes orchestrating LR in its early phase. These results provide mechanisms and protein mediators of LR that might prove useful for the follow-up of the regenerative process in the liver after PH as well as preventing the occurrence of complications associated with liver resection.

Synergistic effects of combined immunotherapy strategies in a model of multifocal hepatocellular carcinoma.

Immune checkpoint-inhibitor combinations are the best therapeutic option for advanced hepatocellular carcinoma (HCC) patients, but improvements in efficacy are needed to improve response rates. We develop a multifocal HCC model to test immunotherapies by introducing c-myc using hydrodynamic gene transfer along with CRISPR-Cas9-mediated disruption of p53 in mouse hepatocytes. Additionally, induced co-expression of luciferase, EGFP, and the melanosomal antigen gp100 facilitates studies on the underlying immunological mechanisms. We show that treatment of the mice with a combination of anti-CTLA-4 + anti-PD1 mAbs results in partial clearance of the tumor with an improvement in survival. However, the addition of either recombinant IL-2 or an anti-CD137 mAb markedly improves both outcomes in these mice. Combining tumor-specific adoptive T cell therapy to the aCTLA-4/aPD1/rIL2 or aCTLA-4/aPD1/aCD137 regimens enhances efficacy in a synergistic manner. As shown by multiplex tissue immunofluorescence and intravital microscopy, combined immunotherapy treatments enhance T cell infiltration and the intratumoral performance of T lymphocytes.

Hepatocyte dedifferentiation profiling in alcohol-related liver disease identifies CXCR4 as a driver of cell reprogramming.

BACKGROUND & AIMS: Loss of hepatocyte identity is associated with impaired liver function in alcohol-related hepatitis (AH). In this context, hepatocyte dedifferentiation gives rise to cells with a hepatobiliary (HB) phenotype expressing biliary and hepatocyte markers and showing immature features. However, the mechanisms and impact of hepatocyte dedifferentiation in liver disease are poorly understood. METHODS: HB cells and ductular reaction (DR) cells were quantified and microdissected from liver biopsies from patients with alcohol-related liver disease (ArLD). Hepatocyte-specific overexpression or deletion of C-X-C motif chemokine receptor 4 (CXCR4), and CXCR4 pharmacological inhibition were assessed in mouse liver injury. Patient-derived and mouse organoids were generated to assess plasticity. RESULTS: Here, we show that HB and DR cells are increased in patients with decompensated cirrhosis and AH, but only HB cells correlate with poor liver function and patients' outcome. Transcriptomic profiling of HB cells revealed the expression of biliary-specific genes and a mild reduction of hepatocyte metabolism. Functional analysis identified pathways involved in hepatocyte reprogramming, inflammation, stemness, and cancer gene programs. The CXCR4 pathway was highly enriched in HB cells and correlated with disease severity and hepatocyte dedifferentiation. In vitro, CXCR4 was associated with a biliary phenotype and loss of hepatocyte features. Liver overexpression of CXCR4 in chronic liver injury decreased the hepatocyte-specific gene expression profile and promoted liver injury. CXCR4 deletion or its pharmacological inhibition ameliorated hepatocyte dedifferentiation and reduced DR and fibrosis progression. CONCLUSIONS: This study shows the association of hepatocyte dedifferentiation with disease progression and poor outcome in AH. Moreover, the transcriptomic profiling of HB cells revealed CXCR4 as a new driver of hepatocyte-to-biliary reprogramming and as a potential therapeutic target to halt hepatocyte dedifferentiation in AH. IMPACT AND IMPLICATIONS: Here, we show that hepatocyte dedifferentiation is associated with disease severity and a reduced synthetic capacity of the liver. Moreover, we identify the CXCR4 pathway as a driver of hepatocyte dedifferentiation and as a therapeutic target in alcohol-related hepatitis. Therefore, this study reveals the importance of preserving strict control over hepatocyte plasticity in order to preserve liver function and promote tissue repair.

Rescue of infant progressive familial intrahepatic cholestasis type 3 mice by repeated dosing of AAV gene therapy.

Background & Aims: Gene therapy using recombinant adeno-associated virus (rAAV) vector carrying multidrug resistance protein 3 (MDR3) coding sequence (AAV8-MDR3) represents a potential curative treatment for progressive familial intrahepatic cholestasis type 3 (PFIC3), which presents in early childhood. However, patients with the severest form of PFIC3 should receive treatment early after detection to prevent irreversible hepatic fibrosis leading ultimately to liver transplantation or death. This represents a challenge for rAAV-based gene therapy because therapeutic efficacy is expected to wane as rAAV genomes are lost owing to hepatocyte division, and the formation of AAV-specific neutralising antibodies precludes re-administration. Here, we tested a strategy of vector re-administration in infant PFIC3 mice with careful evaluation of its oncogenicity - a particular concern surrounding rAAV treatment. Methods: AAV8-MDR3 was re-administered to infant Abcb4 -/- mice 2 weeks after a first dose co-administered with tolerogenic nanoparticles carrying rapamycin (ImmTOR) given at 2 weeks of age. Eight months later, long-term therapeutic efficacy and safety were assessed with special attention paid to the potential oncogenicity of rAAV treatment. Results: Co-administration with ImmTOR mitigated the formation of rAAV-specific neutralising antibodies and enabled an efficacious second administration of AAV8-MDR3, resulting in stable correction of the disease phenotype, including a restoration of bile phospholipid content and healthy liver function, as well as the prevention of liver fibrosis, hepatosplenomegaly, and gallstones. Furthermore, efficacious repeat rAAV administration prevented the appearance of liver malignancies in an animal model highly prone to developing hepatocellular carcinoma. Conclusions: These outcomes provide strong evidence for rAAV redosing through co-administration with ImmTOR, as it resulted in a long-term therapeutic effect in a paediatric liver metabolic disorder, including the prevention of oncogenesis. Impact and implications: Redosing of gene therapy for inborn hepatobiliary disorders may be essential as effect wanes during hepatocyte division and renewal, particularly in paediatric patients, but the approach may carry long-term risks of liver cancer. Viral vectors carrying a therapeutic gene exerted a durable cure of progressive familial intrahepatic cholestasis type 3 in infant mice and reduced the risk of liver cancer only following a second administration.

Bariatric Surgery Is Associated with Alcohol-Related Liver Disease and Psychiatric Disorders Associated with AUD.

BACKGROUND/AIMS: Bariatric surgery can increase the risk of addictive disorders and nutritional deficiencies. The aim of this study was to evaluate the association between bariatric surgery and alcohol use disorder (AUD), alcohol-related liver disease (ALD), and psychiatric disorders associated with AUD. The impact of vitamin D deficiency in these associations was also investigated. METHODS: A cross-sectional study was performed using the National Inpatient Sample database and its ICD-9 codes information. Diagnostic and comorbidity data from hospital discharges were obtained from patients with bariatric surgery and other abdominal surgeries between 2005 and 2015. The two groups were then compared for alcohol-related outcomes after propensity-score matching. RESULTS: The final study cohort included 537,757 patients with bariatric surgery and 537,757 with other abdominal surgeries. The bariatric surgery group had an increased risk of AUD [odds ratio (OR): 1.90; 95% CI: 1.85-1.95], ALD [OR: 1.29; 95% CI: 1.22-1.37], cirrhosis [OR, 1.39; 95% CI: 1.37-1.42], and psychiatric disorders associated with AUD [OR, 3.59; 95% CI: 3.37-3.84]. Vitamin D deficiency did not impact in the association between bariatric surgery and AUD, ALD, or psychiatric disorders associated with AUD. CONCLUSIONS: Bariatric surgery is associated with an increased prevalence of AUD, ALD, and psychiatric disorders associated with AUD. These associations appear to be independent from vitamin D deficiency.

Identifying key multifunctional components shared by critical cancer and normal liver pathways via SparseGMM.

Despite the abundance of multimodal data, suitable statistical models that can improve our understanding of diseases with genetic underpinnings are challenging to develop. Here, we present SparseGMM, a statistical approach for gene regulatory network discovery. SparseGMM uses latent variable modeling with sparsity constraints to learn Gaussian mixtures from multiomic data. By combining coexpression patterns with a Bayesian framework, SparseGMM quantitatively measures confidence in regulators and uncertainty in target gene assignment by computing gene entropy. We apply SparseGMM to liver cancer and normal liver tissue data and evaluate discovered gene modules in an independent single-cell RNA sequencing (scRNA-seq) dataset. SparseGMM identifies PROCR as a regulator of angiogenesis and PDCD1LG2 and HNF4A as regulators of immune response and blood coagulation in cancer. Furthermore, we show that more genes have significantly higher entropy in cancer compared with normal liver. Among high-entropy genes are key multifunctional components shared by critical pathways, including p53 and estrogen signaling.

Poor Outcomes of Patients With NAFLD and Moderate Renal Dysfunction or Short-Term Dialysis Receiving a Liver Transplant Alone.

The outcomes of patients with moderate renal impairment and the impact of liver disease etiology on renal function recovery after liver transplant alone (LTA) are largely unknown. We explored whether NAFLD patients with pre-LTA moderate renal dysfunction (GFR 25-45 ml/min/1.73 m2) may be more susceptible to develop post-LTA severe renal dysfunction (GFR<15 ml/min/1.73 m2) than ALD patients, as well as other overall outcomes. Using the UNOS/OPTN database, we selected patients undergoing liver transplant for NAFLD or ALD (2006-2016), 15,103 of whom received LTA. NAFLD patients with moderate renal dysfunction were more likely to develop subsequent GFR<15 ml/min/1.73 m2 than ALD patients (11.1% vs. 7.38%, p < 0.001). Patients on short-term dialysis pre-LTA (≤12 weeks) were more likely to develop severe renal dysfunction (31.7% vs. 18.1%), especially in NAFLD patients, and were more likely to receive a further kidney transplant (15.3% vs. 3.7%) and had lower survival (48.6% vs. 50.4%) after LTA (p < 0.001 for all). NAFLD was an independent risk factor for post-LTA severe renal dysfunction (HR = 1.2, p = 0.02). NAFLD patients with moderate renal dysfunction and those receiving short-term dialysis prior to LTA are at a higher risk of developing subsequent severe renal dysfunction. Underlying etiology of liver disease may play a role in predicting development and progression of renal failure in patients receiving LTA.

Nivolumab after selective internal radiation therapy for the treatment of hepatocellular carcinoma: a phase 2, single-arm study.

PURPOSE: To evaluate the safety and efficacy of selective internal radiation therapy (SIRT) in combination with a PD-1 inhibitor in patients with unresectable hepatocellular carcinoma (uHCC) and liver-only disease ineligible for chemoembolization. PATIENTS AND METHODS: NASIR-HCC is a single-arm, multicenter, open-label, phase 2 trial that recruited from 2017 to 2019 patients who were naïve to immunotherapy and had tumors in the BCLC B2 substage (single or multiple tumors beyond the up-to-7 rule), or unilobar tumors with segmental or lobar portal vein invasion (PVI); no extrahepatic spread; and preserved liver function. Patients received SIRT followed 3 weeks later by nivolumab (240 mg every 2 weeks) for up to 24 doses or until disease progression or unacceptable toxicity. Safety was the primary endpoint. Secondary objectives included objective response rate (ORR), time to progression (TTP), and overall survival (OS). RESULTS: 42 patients received SIRT (31 BCLC-B2, 11 with PVI) and were followed for a median of 22.2 months. 27 patients discontinued and 1 never received Nivolumab. 41 patients had any-grade adverse events (AE) and 21 had serious AEs (SAE). Treatment-related AEs and SAEs grade 3-4 occurred in 8 and 5 patients, respectively. Using RECIST 1.1 criteria, ORR reported by investigators was 41.5% (95% CI 26.3% to 57.9%). Four patients were downstaged to partial hepatectomy. Median TTP was 8.8 months (95% CI 7.0 to 10.5) and median OS was 20.9 months (95% CI 17.7 to 24.1). CONCLUSIONS: The combination of SIRT and nivolumab has shown an acceptable safety profile and signs of antitumor activity in the treatment of patients with uHCC that were fit for SIRT. TRIAL REGISTRATION NUMBER: NCT03380130.

Integrated Transcriptomic and Proteomic Analysis Identifies Plasma Biomarkers of Hepatocellular Failure in Alcohol-Associated Hepatitis.

Alcohol-associated hepatitis (AH) is a form of liver failure with high short-term mortality. Recent studies have shown that defective function of hepatocyte nuclear factor 4 alpha (HNF4a) and systemic inflammation are major disease drivers of AH. Plasma biomarkers of hepatocyte function could be useful for diagnostic and prognostic purposes. Herein, an integrative analysis of hepatic RNA sequencing and liquid chromatography-tandem mass spectrometry was performed to identify plasma protein signatures for patients with mild and severe AH. Alcohol-related liver disease cirrhosis, nonalcoholic fatty liver disease, and healthy subjects were used as comparator groups. Levels of identified proteins primarily involved in hepatocellular function were decreased in patients with AH, which included hepatokines, clotting factors, complement cascade components, and hepatocyte growth activators. A protein signature of AH disease severity was identified, including thrombin, hepatocyte growth factor α, clusterin, human serum factor H-related protein, and kallistatin, which exhibited large abundance shifts between severe and nonsevere AH. The combination of thrombin and hepatocyte growth factor α discriminated between severe and nonsevere AH with high sensitivity and specificity. These findings were correlated with the liver expression of genes encoding secreted proteins in a similar cohort, finding a highly consistent plasma protein signature reflecting HNF4A and HNF1A functions. This unbiased proteomic-transcriptome analysis identified plasma protein signatures and pathways associated with disease severity, reflecting HNF4A/1A activity useful for diagnostic assessment in AH.

Design and validation of a tunable inertial microfluidic system for the efficient enrichment of circulating tumor cells in blood.

The analysis of circulating tumor cells (CTCs) in blood is a powerful noninvasive alternative to conventional tumor biopsy. Inertial-based separation is a promising high-throughput, marker-free sorting strategy for the enrichment and isolation of CTCs. Here, we present and validate a double spiral microfluidic device that efficiently isolates CTCs with a fine-tunable cut-off value of 9 µm and a separation range of 2 µm. We designed the device based on computer simulations that introduce a novel, customized inertial force term, and provide practical fabrication guidelines. We validated the device using calibration beads, which allowed us to refine the simulations and redesign the device. Then we validated the redesigned device using blood samples and a murine model of metastatic breast cancer. Finally, as a proof of principle, we tested the device using peripheral blood from a patient with hepatocellular carcinoma, isolating more than 17 CTCs/ml, with purity/removal values of 96.03% and 99.99% of white blood cell and red blood cells, respectively. These results confirm highly efficient CTC isolation with a stringent cut-off value and better separation results than the state of the art.

Immunotherapies for hepatocellular carcinoma and intrahepatic cholangiocarcinoma: Current and developing strategies.

Liver cancer including hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA) is the third leading cause of cancer-related deaths worldwide. HCC arises from hepatocyte or hepatic stem cells, while iCCA originates from biliary epithelial cells, and the respective biological context are very different. Despite screening programs, the diagnosis of liver cancer is in most cases made when curative treatments such as surgery or ablation are not possible. In 2020, after a decade of using only tyrosine kinase inhibitors (TKI), a combination of an immune-check point inhibitor (ICI) and a VEGF antagonist proved superior to a TKI as first line therapy of advanced HCC. In 2022, the addition of an ICI to standard chemotherapy demonstrated an improvement of patient survival in iCCA. Moreover, ICI offer an unprecedented rate of durable responses to HCC and iCCA patients. Nevertheless, still two thirds of patients do not respond to ICI-based combinations, and research efforts are focused on deciphering the mechanisms of immune evasion of these lethal cancers. Reliable predictive and prognostic biomarkers are still lacking, but the molecular phenotyping of the tumor microenvironment is currently providing potential candidates for patient stratification. In this review, we will summarize the current knowledge on the immune biology of the liver, the discovery of cell-intrinsic and immune cell-mediated mechanisms of immune evasion by means of high-resolution single cell data, the main targets of current immunotherapy approaches, and the recent milestones in immunotherapy of HCC and iCCA.

A new animal model of atrophy-hypertrophy complex and liver damage following Yttrium-90 lobar selective internal radiation therapy in rabbits.

Lobar selective internal radiation therapy (SIRT) is widely used to treat liver tumors inducing atrophy of the treated lobe and contralateral hypertrophy. The lack of animal model has precluded further investigations to improve this treatment. We developed an animal model of liver damage and atrophy-hypertrophy complex after SIRT. Three groups of 5-8 rabbits received transportal SIRT with Yttrium 90 resin microspheres of the cranial lobes with different activities (0.3, 0.6 and 1.2 GBq), corresponding to predicted absorbed radiation dose of 200, 400 and 800 Gy, respectively. Another group received non-loaded microspheres (sham group). Cranial and caudal lobes volumes were assessed using CT volumetry before, 15 and 30 days after SIRT. Liver biochemistry, histopathology and gene expression were evaluated. Four untreated rabbits were used as controls for gene expression studies. All animals receiving 1.2 GBq were euthanized due to clinical deterioration. Cranial SIRT with 0.6 GBq induced caudal lobe hypertrophy after 15 days (median increase 34% -ns-) but produced significant toxicity. Cranial SIRT with 0.3 GBq induced caudal lobe hypertrophy after 30 days (median increase 82%, p = 0.04). No volumetric changes were detected in sham group. Transient increase in serum transaminases was detected in all treated groups returning to normal values at 15 days. There was dose-dependent liver dysfunction with bilirubin elevation and albumin decrease. Histologically, 1.2 GBq group developed permanent severe liver damage with massive necrosis, 0.6 and 0.3 GBq groups developed moderate damage with inflammation and portal fibrosis at 15 days, partially recovering at 30 days. There was no difference in the expression of hepatocyte function and differentiation genes between 0.3 GBq and control groups. Cranial SIRT with 0.3 GBq of 90Y resin microspheres in rabbits is a reliable animal model to analyse the atrophy-hypertrophy complex and liver damage without toxicity.

Hospital preparedness for mass gathering events and mass casualty incidents in Matera, Italy, European Capital of Culture 2019.

INTRODUCTION: Mass Casualty Incidents (MCI) may occur during Mass Gathering Events (MGE). A failure to prepare and train the health care system for potential MCI, can cause chaos and delays in the response, leading to an increased morbidity and mortality. Education and training of staff are crucial for preparedness. In Italy, hospital Emergency Plans for Massive Influx of Injured (in Italian designated with the acronym PEMAF) are mandatory since the '90's. However, when available, they are usually poorly known by the staff, rarely reviewed and validated. In 2014, Matera, a city in Southern Italy, was designated as the European Capital of Culture for 2019. As a result, we took this opportunity to revise the "Madonna delle Grazie" PEMAF and to start a program for increasing the awareness of the plan among the medical staff and provide specific training for MCI management. MATERIAL & METHODS: The PEMAF was reviewed through simulations that involved the entire staff. A partnership with the International Association for Medical Response to Major Incidents & Disasters (MRMI) led to the support of experts and to the organization of residential courses based on the MAss Casualty SIMulation tool (MACSIM®). In total, six residential educational events of MACSIM-PEMAF were organized. Individual capacity was tested before and after the education through self-administered semi-quantitative questionnaires. RESULTS: All the available resources were mapped and the functional areas identified. Alert, coordination and command sequences were defined. The communication network was improved. Documentation and registration systems were developed. Standard operational procedures (action cards) were created for the key positions. The knowledge and capacity to function in active roles during a MCI was improved among the participants in the educational program. CONCLUSIONS: MGE are great opportunities for the development of the hosting community but also represent an increased risk of MCI. Preparedness is mandatory for health care systems. The educational format MACSIM-PEMAF seems to be adequate to review and improve the existing plans and transfer specific skills to attendants.