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.

Metabolic dysfunction–associated fatty liver disease (MAFLD): an update of the recent advances in pharmacological treatment

Metabolic dysfunction–associated fatty liver disease (MAFLD) is nowadays considered the liver manifestation of metabolic syndrome. Its prevalence is increasing worldwide in parallel to the epidemic of diabetes and obesity. MAFLD includes a wide spectrum of liver injury including simple steatosis and non-alcoholic steatohepatitis (NASH) that may lead to serious complications such as liver cirrhosis and liver cancer. The complexity of its pathophysiology and the intricate mechanisms underlying disease progression explains the huge variety of molecules targeting diverse biological mechanisms that have been tested in preclinical and clinical settings in the last two decades. Thanks to the large number of clinical trials of the last few years, most of them still ongoing, the pharmacotherapy scenario of MAFLD is rapidly evolving. The three major components of MAFLD, steatosis, inflammation, and fibrosis seem to be safely targeted with different agents at least in a large proportion of patients. Likely, in the next few years more than one drug will be approved for the treatment of MAFLD at different disease stages. The aim of this review is to synthesize the characteristics and the results of the most advanced clinical trials for the treatment of NASH to evaluate the recent advances of pharmacotherapy in this disease. (AU)

Metabolic dysfunction-associated fatty liver disease (MAFLD): an update of the recent advances in pharmacological treatment.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is nowadays considered the liver manifestation of metabolic syndrome. Its prevalence is increasing worldwide in parallel to the epidemic of diabetes and obesity. MAFLD includes a wide spectrum of liver injury including simple steatosis and non-alcoholic steatohepatitis (NASH) that may lead to serious complications such as liver cirrhosis and liver cancer. The complexity of its pathophysiology and the intricate mechanisms underlying disease progression explains the huge variety of molecules targeting diverse biological mechanisms that have been tested in preclinical and clinical settings in the last two decades. Thanks to the large number of clinical trials of the last few years, most of them still ongoing, the pharmacotherapy scenario of MAFLD is rapidly evolving. The three major components of MAFLD, steatosis, inflammation, and fibrosis seem to be safely targeted with different agents at least in a large proportion of patients. Likely, in the next few years more than one drug will be approved for the treatment of MAFLD at different disease stages. The aim of this review is to synthesize the characteristics and the results of the most advanced clinical trials for the treatment of NASH to evaluate the recent advances of pharmacotherapy in this disease.

Inmunoterapia en el carcinoma hepatocelular / Immunotherapy in hepatocellular carcinoma

El carcinoma hepatocelular es el más frecuente de lostumores primarios del hígado y cuando alcanza un estadioavanzado, debe abordarse con tratamientos sistémicos.Hasta hace poco, las opciones disponibles se limitaban alos inhibidores de la tirosina-kinasa, cuyo efecto es modesto. El mejor conocimiento del microambiente tumoral y elpapel del sistema inmune en el control de la progresióntumoral han permitido desarrollar tratamientos dirigidoscontra los puntos de control inmunológico. Esta inmunoterapia logra tasas de respuesta tumoral más altas, que setraducen en una mayor supervivencia y que hoy constituyen la columna vertebral del tratamiento sistémico de estaenfermedad. Aunque se tolera bien en general, la inmunoterapia puede producir toxicidad importante derivadade la estimulación del sistema inmune. Y en el pacientecirrótico, el diagnóstico precoz y tratamiento de esta toxicidad supone un reto especial. Por ello, la investigacióncontinuada en el uso de estas nuevas terapias nos permitirá conocer mejor el perfil de los pacientes que más sebenefician de las mismas. (AU)

Interactive Role of Surrogate Liver Fibrosis Assessment and Insulin Resistance on the Incidence of Major Cardiovascular Events.

Introduction: The combination of easy-to-obtain validated biomarkers is interesting in the prognostic evaluation of patients at cardiovascular risk in a precision medicine scenario. The evaluation of the effect modification of insulin resistance and liver fibrosis with the Triglyceride-Glucose index (TyG) and Fibrosis-4 index (FIB4) might provide prognostic information in patients at cardiovascular risk. Patients and methods: A retrospective cohort study was performed with 2055 patients recruited in the Vascular Metabolic CUN cohort. The studied outcome was the incidence rate of major cardiovascular events (MACE). The Systematic Coronary Risk Evaluation (SCORE), FIB4 and TyG indexes were calculated according to validated formulas. Results: FIB4 and TyG showed a synergistic interaction using validated cut-offs for both indexes in the prediction of MACE (Hazard ratio (HR) 1.05 CI95% 1.01-1.08) which remained after adjustment by age, sex, SCORE subgroup, presence of diabetes, or previous MACE using standardized cut-off (HR 2.29 CI95% 1.33-3.94). Finally, a subgroup with significant TyG and FIB4 showed a higher cardiovascular risk in the study population (adjusted HR 3.34 CI 95% 1.94-5.77). Conclusion: The combined interpretation of TyG and FIB4 indexes might have a potential predictive value of major cardiovascular events.

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.

Immunotherapy in hepatocellular carcinoma.

Hepatocellular carcinoma is a highly prevalent tumor worldwide and when it reaches an advanced stage, few systemic treatments are available to improve the survival of these patients. However, greater knowledge about the tumor microenvironment and the role of the immune system in the control of tumor progression has allowed the development of treatments targeting immune checkpoints, which result in encouraging tumor response rates and prolonged survival. Although most of these treatments are well tolerated, up to 20 % of patients may experience side effects derived from non-specific stimulation of the immune system. In the cirrhotic patient, the early diagnosis and treatment of such adverse events is particularly challenging. Therefore, the ongoing investigation on the use of these new therapies will allow us to better understand the profile of the patients who will benefit most.

Beneficial effect of ursodeoxycholic acid in patients with acyl-CoA oxidase 2 (ACOX2) deficiency-associated hypertransaminasemia.

BACKGROUND AND AIMS: A variant (p.Arg225Trp) of peroxisomal acyl-CoA oxidase 2 (ACOX2), involved in bile acid (BA) side-chain shortening, has been associated with unexplained persistent hypertransaminasemia and accumulation of C27-BAs, mainly 3α,7α,12α-trihydroxy-5ß-cholestanoic acid (THCA). We aimed to investigate the prevalence of ACOX2 deficiency-associated hypertransaminasemia (ADAH), its response to ursodeoxycholic acid (UDCA), elucidate its pathophysiological mechanism and identify other inborn errors that could cause this alteration. METHODS AND RESULTS: Among 33 patients with unexplained hypertransaminasemia from 11 hospitals and 13 of their relatives, seven individuals with abnormally high C27-BA levels (>50% of total BAs) were identified by high-performance liquid chromatography-mass spectrometry. The p.Arg225Trp variant was found in homozygosity (exon amplification/sequencing) in two patients and three family members. Two additional nonrelated patients were heterozygous carriers of different alleles: c.673C>T (p.Arg225Trp) and c.456_459del (p.Thr154fs). In patients with ADAH, impaired liver expression of ACOX2, but not ACOX3, was found (immunohistochemistry). Treatment with UDCA normalized aminotransferase levels. Incubation of HuH-7 hepatoma cells with THCA, which was efficiently taken up, but not through BA transporters, increased reactive oxygen species production (flow cytometry), endoplasmic reticulum stress biomarkers (GRP78, CHOP, and XBP1-S/XBP1-U ratio), and BAXα expression (reverse transcription followed by quantitative polymerase chain reaction and immunoblot), whereas cell viability was decreased (tetrazolium salt-based cell viability test). THCA-induced cell toxicity was higher than that of major C24-BAs and was not prevented by UDCA. Fourteen predicted ACOX2 variants were generated (site-directed mutagenesis) and expressed in HuH-7 cells. Functional tests to determine their ability to metabolize THCA identified six with the potential to cause ADAH. CONCLUSIONS: Dysfunctional ACOX2 has been found in several patients with unexplained hypertransaminasemia. This condition can be accurately identified by a noninvasive diagnostic strategy based on plasma BA profiling and ACOX2 sequencing. Moreover, UDCA treatment can efficiently attenuate liver damage in these patients.

The importance of liver functional reserve in the non-surgical treatment of hepatocellular carcinoma.

The aim of any oncological treatment is not just to eliminate the tumour, but to maximise patient survival and quality of life. Since the liver has a vital function, any radical treatment that severely compromises liver function will result in a shortening of life expectancy, rather than a prolongation. Furthermore, even non-severe liver damage may prevent the delivery of further effective therapies. This is particularly important in the case of hepatocellular carcinoma (HCC), as it is associated with underlying cirrhosis in most patients - cirrhosis itself is not only a potentially lethal disease and independent prognostic factor in HCC, but it also makes liver function fragile. Accordingly, some information about liver dysfunction is included in most staging systems for HCC and can be used to guide the selection of treatments that the functional liver reserve can tolerate. Unfortunately, the prediction of functional damage to the liver in the case of antitumor treatments is very challenging and still suboptimal in any given patient. Moreover, while the assessment of functional reserve can now be used to avoid postoperative liver failure in the surgical setting, its use has been less well clarified for non-surgical therapies, which is of particular relevance today, as several lines of effective non-surgical treatments, including systemic therapies, have become available. The present article will a) critically review the implications of the assessment of liver functional reserve in patients with HCC, b) illustrate the available tools to assess liver functional reserve and c) discuss the role of functional assessment for each type of non-surgical therapy for HCC.

Unannotated small RNA clusters associated with circulating extracellular vesicles detect early stage liver cancer.

OBJECTIVE: Surveillance tools for early cancer detection are suboptimal, including hepatocellular carcinoma (HCC), and biomarkers are urgently needed. Extracellular vesicles (EVs) have gained increasing scientific interest due to their involvement in tumour initiation and metastasis; however, most extracellular RNA (exRNA) blood-based biomarker studies are limited to annotated genomic regions. DESIGN: EVs were isolated with differential ultracentrifugation and integrated nanoscale deterministic lateral displacement arrays (nanoDLD) and quality assessed by electron microscopy, immunoblotting, nanoparticle tracking and deconvolution analysis. Genome-wide sequencing of the largely unexplored small exRNA landscape, including unannotated transcripts, identified and reproducibly quantified small RNA clusters (smRCs). Their key genomic features were delineated across biospecimens and EV isolation techniques in prostate cancer and HCC. Three independent exRNA cancer datasets with a total of 479 samples from 375 patients, including longitudinal samples, were used for this study. RESULTS: ExRNA smRCs were dominated by uncharacterised, unannotated small RNA with a consensus sequence of 20 nt. An unannotated 3-smRC signature was significantly overexpressed in plasma exRNA of patients with HCC (p<0.01, n=157). An independent validation in a phase 2 biomarker case-control study revealed 86% sensitivity and 91% specificity for the detection of early HCC from controls at risk (n=209) (area under the receiver operating curve (AUC): 0.87). The 3-smRC signature was independent of alpha-fetoprotein (p<0.0001) and a composite model yielded an increased AUC of 0.93. CONCLUSION: These findings directly lead to the prospect of a minimally invasive, blood-only, operator-independent clinical tool for HCC surveillance, thus highlighting the potential of unannotated smRCs for biomarker research in cancer.

Experimental Models of Liquid Biopsy in Hepatocellular Carcinoma Reveal Clone-Dependent Release of Circulating Tumor DNA.

Liquid biopsy, the molecular analysis of tumor components released into the bloodstream, has emerged as a noninvasive and resourceful means to access genomic information from cancers. Most data derived from translational studies showcase its numerous potential clinical applications. However, data from experimental models are scarce, and little is known about the underlying mechanisms and factors controlling the release of circulating tumor DNA (ctDNA) and cells (CTCs). This study aimed to model liquid biopsy in hepatocellular carcinoma xenografts and to study the dynamics of release of ctDNA and CTCs; this included models of intratumoral heterogeneity (ITH) and metastatic disease. We quantified ctDNA by quantitative polymerase chain reaction (PCR) targeting human long interspersed nuclear element group 1; targeted mutation analysis was performed with digital droplet PCR. CTCs were traced by flow cytometry. Results demonstrated the feasibility of detecting ctDNA, including clone-specific mutations, as well as CTCs in blood samples of mice. In addition, the concentration of ctDNA and presence of tumor-specific mutations reflected tumor progression, and detection of CTCs was associated with metastases. Our ITH model suggested differences in the release of DNA fragments impacted by the cell-clone origin and the treatment. Conclusion: These data present new models to study liquid biopsy and its underlying mechanisms and highlighted a clone-dependent release of ctDNA into the bloodstream.

Transcriptomic characterization of cancer-testis antigens identifies MAGEA3 as a driver of tumor progression in hepatocellular carcinoma.

Cancer testis antigens (CTAs) are an extensive gene family with a unique expression pattern restricted to germ cells, but aberrantly reactivated in cancer tissues. Studies indicate that the expression (or re-expression) of CTAs within the MAGE-A family is common in hepatocellular carcinoma (HCC). However, no systematic characterization has yet been reported. The aim of this study is to perform a comprehensive profile of CTA de-regulation in HCC and experimentally evaluate the role of MAGEA3 as a driver of HCC progression. The transcriptomic analysis of 44 multi-regionally sampled HCCs from 12 patients identified high intra-tumor heterogeneity of CTAs. In addition, a subset of CTAs was significantly overexpressed in histologically poorly differentiated regions. Further analysis of CTAs in larger patient cohorts revealed high CTA expression related to worse overall survival and several other markers of poor prognosis. Functional analysis of MAGEA3 was performed in human HCC cell lines by gene silencing and in a genetic mouse model by overexpression of MAGEA3 in the liver. Knockdown of MAGEA3 decreased cell proliferation, colony formation and increased apoptosis. MAGEA3 overexpression was associated with more aggressive tumors in vivo. In conclusion MAGEA3 enhances tumor progression and should be considered as a novel therapeutic target in HCC.

Mutations in circulating tumor DNA predict primary resistance to systemic therapies in advanced hepatocellular carcinoma.

Little is known about the mutational landscape of advanced hepatocellular carcinoma (HCC), and predictive biomarkers of response to systemic therapies are lacking. We aimed to describe the mutational landscape of advanced HCC and to identify predictors of primary resistance to systemic therapies using circulating tumor DNA (ctDNA). We prospectively enrolled 121 patients between October 2015 and January 2019. We performed targeted ultra-deep sequencing of 25 genes and Digital Droplet PCR of TERT promoter, including sequential samples throughout treatment. Primary endpoint was progression-free survival (PFS) stratified by mutation profiles in ctDNA. Secondary endpoints were overall survival and objective response rate. The most frequent mutations in ctDNA of advanced HCC were TERT promoter (51%), TP53 (32%), CTNNB1 (17%), PTEN (8%), AXIN1, ARID2, KMT2D, and TSC2 (each 6%). TP53 and CTNNB1 mutations were mutually exclusive. Patients with mutations in the PI3K/MTOR pathway had significantly shorter PFS than those without these mutations after tyrosine kinase inhibitors (2.1 vs 3.7 months, p < 0.001), but not after immune checkpoint inhibition (CPI). WNT pathway mutations were not associated with PFS, overall survival, or objective response after CPI. Serial profiling of ctDNA in a subset correlated with treatment response. Mutation profiling of ctDNA in advanced HCC shows similar mutation frequencies for known HCC drivers compared to early stages and identifies predictive biomarkers of response to systemic therapies.

Brain ventricular enlargement in human and murine acute intermittent porphyria.

The morphological changes that occur in the central nervous system of patients with severe acute intermittent porphyria (AIP) have not yet been clearly established. The aim of this work was to analyze brain involvement in patients with severe AIP without epileptic seizures or clinical posterior reversible encephalopathy syndrome, as well as in a mouse model receiving or not liver-directed gene therapy aimed at correcting the metabolic disorder. We conducted neuroradiologic studies in 8 severely affected patients (6 women) and 16 gender- and age-matched controls. Seven patients showed significant enlargement of the cerebral ventricles and decreased brain perfusion was observed during the acute attack in two patients in whom perfusion imaging data were acquired. AIP mice exhibited reduced cerebral blood flow and developed chronic dilatation of the cerebral ventricles even in the presence of slightly increased porphyrin precursors. While repeated phenobarbital-induced attacks exacerbated ventricular dilation in AIP mice, correction of the metabolic defect using liver-directed gene therapy restored brain perfusion and afforded protection against ventricular enlargement. Histological studies revealed no signs of neuronal loss but a denser neurofilament pattern in the periventricular areas, suggesting compression probably caused by imbalance in cerebrospinal fluid dynamics. In conclusion, severely affected AIP patients exhibit cerebral ventricular enlargement. Liver-directed gene therapy protected against the morphological consequences of the disease seen in the brain of AIP mice. The observational study was registered at Clinicaltrial.gov as NCT02076763.

The Pattern of Progression Defines Post-progression Survival in Patients with Hepatocellular Carcinoma Treated with SIRT.

PURPOSE: In patients with advanced hepatocellular carcinoma (HCC) treated with sorafenib, post-progression survival (PPS) is marked by the pattern of progression. Our aim was to assess the influence of the pattern of progression to selective internal radiotherapy (SIRT) in PPS among patients with HCC. METHODS: A retrospective analysis of patients treated with SIRT between 2003 and 2015 was conducted, excluding those with a single nodule < 5 cm or with metastases. Four patterns of progression to SIRT were defined: target tumour growth, non-target tumour growth, new intrahepatic disease, and new extrahepatic disease. PPS was calculated from the time of progression based on RECIST 1.1 criteria. RESULTS: Out of the 102 patients who met the selection criteria, 76 progressed after a median follow-up of 15 months. Median PPS was 6.5 months (95% CI 3.8-9.3 months). Patients who progressed at pre-existing lesions had a better PPS (median 12.5 months) than those who progressed with new lesions inside or outside the liver (median 4.2 months) (p = 0.02). In a Cox model adjusted by liver function and systemic inflammation, the pattern of progression had a hazard ratio of 1.64 (95% CI 0.92-2.93; p = 0.093). CONCLUSION: In a cohort of HCC patients treated with SIRT, the pattern of progression associated with worst survival was the development of new intrahepatic lesions or extrahepatic metastases.

A phenotypical map of disseminated hepatocellular carcinoma suggests clonal constraints in metastatic sites.

AIMS: Access to tissue in patients with hepatocellular carcinoma (HCC) is limited compared to other malignancies, particularly at advanced stages. This has precluded a thorough characterisation of molecular drivers of HCC dissemination, particularly in relation to distant metastases. Biomarker assessment is restricted to early stages, and paired primary-metastatic comparisons between samples from the same patient are difficult. METHODS AND RESULTS: We report the evaluation of 88 patients with HCC who underwent autopsy, including multiregional sampling of primary and metastatic sites totalling 230 nodules analysed. The study included morphological assessment, immunohistochemistry and mutation status of the TERT promoter, the most frequently mutated gene in HCC. We confirm a strong predilection of HCC for lung dissemination, including subclinical micrometastases (unrecognised during imaging and macroscopic examinations) in 30% of patients with disseminated disease. Size of dominant tumour nodule; multinodularity; macrovascular invasion; high histological, nuclear and architectural grades; and cellular crowding were associated with the presence of extrahepatic metastasis. Among the immunohistochemistry markers tested, metastatic nodules had significantly higher K19 and EpCAM expression than primary liver tumours. Morphological and immunohistochemical features showed that metastatic HCC could be traced back to the primary tumour, sometimes to a specific hepatic nodule. CONCLUSIONS: This study suggests limited heterogeneity in metastatic sites compared to primary tumour sites.

High-density single cell mRNA sequencing to characterize circulating tumor cells in hepatocellular carcinoma.

Patients with hepatocellular carcinoma (HCC) release tumor cells to the bloodstream, which can be detected using cell surface markers. Despite numerous reports suggest a direct correlation between the number of circulating tumor cells (CTCs) and poor clinical outcomes, few studies have provided a thorough molecular characterization of CTCs. Due to the limited access to tissue samples in patients at advanced stages of HCC, it is crucial to develop new technologies to identify HCC cancer drivers in routine clinical conditions. Here, we describe a method that sequentially combines image flow cytometry and high density single-cell mRNA sequencing to identify CTCs in HCC patients. Genome wide expression profiling of CTCs using this approach demonstrates CTC heterogeneity and helps detect known oncogenic drivers in HCC such as IGF2. This integrated approach provides a novel tool for biomarker development in HCC using liquid biopsy.

A pilot study of ultra-deep targeted sequencing of plasma DNA identifies driver mutations in hepatocellular carcinoma.

Cellular components of solid tumors including DNA are released into the bloodstream, but data on circulating-free DNA (cfDNA) in hepatocellular carcinoma (HCC) are still scarce. This study aimed at analyzing mutations in cfDNA and their correlation with tissue mutations in patients with HCC. We included 8 HCC patients treated with surgical resection for whom we collected paired tissue and plasma/serum samples. We analyzed 45 specimens, including multiregional tumor tissue sampling (n = 24), peripheral blood mononuclear cells (PMBC, n = 8), plasma (n = 8) and serum (n = 5). Ultra-deep sequencing (5500× coverage) of all exons was performed in a targeted panel of 58 genes, including frequent HCC driver genes and druggable mutations. Mutations detected in plasma included known HCC oncogenes and tumor suppressors (e.g., TERT promoter, TP53, and NTRK3) as well as a candidate druggable mutation (JAK1). This approach increased the detection rates previously reported for mutations in plasma of HCC patients. A thorough characterization of cis mutations found in plasma confirmed their tumoral origin, which provides definitive evidence of the release of HCC-derived DNA fragments into the bloodstream. This study demonstrates that ultra-deep sequencing of cfDNA is feasible and can confidently detect somatic mutations found in tissue; these data reinforce the role of plasma DNA as a promising minimally invasive tool to interrogate HCC genetics.