Tissue resident iNKT17 cells facilitate cancer cell extravasation in liver metastasis via interleukin-22.

During metastasis, cancer cells invade, intravasate, enter the circulation, extravasate, and colonize target organs. Here, we examined the role of interleukin (IL)-22 in metastasis. Immune cell-derived IL-22 acts on epithelial tissues, promoting regeneration and healing upon tissue damage, but it is also associated with malignancy. Il22-deficient mice and mice treated with an IL-22 antibody were protected from colon-cancer-derived liver and lung metastasis formation, while overexpression of IL-22 promoted metastasis. Mechanistically, IL-22 acted on endothelial cells, promoting endothelial permeability and cancer cell transmigration via induction of endothelial aminopeptidase N. Multi-parameter flow cytometry and single-cell sequencing of immune cells isolated during cancer cell extravasation into the liver revealed iNKT17 cells as source of IL-22. iNKT-cell-deficient mice exhibited reduced metastases, which was reversed by injection of wild type, but not Il22-deficient, invariant natural killer T (iNKT) cells. IL-22-producing iNKT cells promoting metastasis were tissue resident, as demonstrated by parabiosis. Thus, IL-22 may present a therapeutic target for prevention of metastasis.

Characteristics and outcomes of immunotherapy-related liver injury in patients with hepatocellular carcinoma versus other advanced solid tumours.

BACKGROUND & AIMS: Immune-related liver injury (irLI) is commonly observed in patients with cancer treated with immune checkpoint inhibitors (ICIs). We aimed to compare the incidence, clinical characteristics, and outcomes of irLI between patients receiving ICIs for hepatocellular carcinoma (HCC) vs. other solid tumours. METHODS: Two separate cohorts were included: 375 patients with advanced/unresectable HCC, Child-Pugh A class treated with first-line atezolizumab+bevacizumab from the AB-real study, and a non-HCC cohort including 459 patients treated with first-line ICI therapy from the INVIDIa-2 multicentre study. IrLI was defined as a treatment-related increase of aminotransferase levels after exclusion of alternative aetiologies of liver injury. The incidence of irLI was adjusted for the duration of treatment exposure. RESULTS: In patients with HCC, the incidence of any grade irLI was 11.4% over a median treatment exposure of 4.4 months (95% CI 3.7-5.2) vs. 2.6% in the INVIDIa-2 cohort over a median treatment exposure of 12.4 months (95% CI 11.1-14.0). Exposure-adjusted-incidence of any grade irLI was 22.1 per 100-patient-years in patients with HCC and 2.1 per 100-patient-years in patients with other solid tumours (p <0.001), with median time-to-irLI of 1.4 and 4.7 months, respectively. Among patients who developed irLI, systemic corticosteroids were administered in 16.3% of patients with HCC and 75.0% of those without HCC (p <0.001), and irLI resolution was observed in 72.1% and 58.3%, respectively (p = 0.362). In patients with HCC, rates of hepatic decompensation and treatment discontinuation due to irLI were 7%. Grade 1-2 irLI was associated with improved overall survival only in patients with HCC (hazard ratio 0.53, 95% CI 0.29-0.96). CONCLUSIONS: Despite higher incidence and earlier onset, irLI in patients with HCC is characterised by higher rates of remission and lower requirement for corticosteroid therapy (vs. irLI in other solid tumours), low risk of hepatic decompensation and treatment discontinuation, not negatively affecting oncological outcomes. IMPACT AND IMPLICATIONS: Immune-related liver injury (irLI) is common in patients with cancer receiving immune checkpoint inhibitors (ICIs), but whether irLI is more frequent or it is associated with a worse clinical course in patients with hepatocellular carcinoma (HCC), compared to other tumours, is not known. Herein, we compared characteristics and outcomes of irLI in two prospective cohorts including patients treated with ICIs for HCC or for other oncological indications. irLI is significantly more common and it occurs earlier in patients with HCC, also after adjustment for duration of treatment exposure. However, outcomes of patients with HCC who developed irLI are not negatively affected in terms of requirement for corticosteroid therapy, hepatic decompensation, treatment discontinuation and overall survival.

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.

Genetic variation in TERT modifies the risk of hepatocellular carcinoma in alcohol-related cirrhosis: results from a genome-wide case-control study.

OBJECTIVE: Hepatocellular carcinoma (HCC) often develops in patients with alcohol-related cirrhosis at an annual risk of up to 2.5%. Some host genetic risk factors have been identified but do not account for the majority of the variance in occurrence. This study aimed to identify novel susceptibility loci for the development of HCC in people with alcohol related cirrhosis. DESIGN: Patients with alcohol-related cirrhosis and HCC (cases: n=1214) and controls without HCC (n=1866), recruited from Germany, Austria, Switzerland, Italy and the UK, were included in a two-stage genome-wide association study using a case-control design. A validation cohort of 1520 people misusing alcohol but with no evidence of liver disease was included to control for possible association effects with alcohol misuse. Genotyping was performed using the InfiniumGlobal Screening Array (V.24v2, Illumina) and the OmniExpress Array (V.24v1-0a, Illumina). RESULTS: Associations with variants rs738409 in PNPLA3 and rs58542926 in TM6SF2 previously associated with an increased risk of HCC in patients with alcohol-related cirrhosis were confirmed at genome-wide significance. A novel locus rs2242652(A) in TERT (telomerase reverse transcriptase) was also associated with a decreased risk of HCC, in the combined meta-analysis, at genome-wide significance (p=6.41×10-9, OR=0.61 (95% CI 0.52 to 0.70). This protective association remained significant after correction for sex, age, body mass index and type 2 diabetes (p=7.94×10-5, OR=0.63 (95% CI 0.50 to 0.79). Carriage of rs2242652(A) in TERT was associated with an increased leucocyte telomere length (p=2.12×10-44). CONCLUSION: This study identifies rs2242652 in TERT as a novel protective factor for HCC in patients with alcohol-related cirrhosis.

Preliminary evidence of safety and tolerability of atezolizumab plus bevacizumab in patients with hepatocellular carcinoma and Child-Pugh A and B cirrhosis: A real-world study.

BACKGROUND AND AIMS: Atezolizumab plus bevacizumab (AtezoBev) is the standard of care for first-line treatment of unresectable HCC. No evidence exists as to its use in routine clinical practice in patients with impaired liver function. APPROACH AND RESULTS: In 216 patients with HCC who were consecutively treated with AtezoBev across 11 tertiary centers, we retrospectively evaluated treatment-related adverse events (trAEs) graded (G) according to Common Terminology Criteria for Adverse Events v5.0, including in the analysis all patients treated according to label (n = 202, 94%). We also assessed overall survival (OS), progression-free survival (PFS), overall response (ORR), and disease control rates (DCR) defined by Response Evaluation Criteria in Solid Tumors v1.1. Disease was mostly secondary to viral hepatitis, namely hepatitis C (n = 72; 36%) and hepatitis B infection (n = 35, 17%). Liver function was graded as Child-Pugh (CP)-A in 154 patients (76%) and CP-B in 48 (24%). Any grade trAEs were reported by 143 patients (71%), of which 53 (26%) were G3 and 3 (2%) G4. Compared with CP-A, patients with CP-B showed comparable rates of trAEs. Presence and grade of varices at pretreatment esophagogastroduodenoscopy did not correlate with bleeding events. After a median follow-up of 9.0 months (95% CI, 7.8-10.1), median OS was 14.9 months (95% CI, 13.6-16.3), whereas median PFS was 6.8 months (95% CI, 5.2-8.5). ORR and DCR were respectively 25% and 73%, with no difference across CP classes. CONCLUSIONS: This study confirms reproducible safety and efficacy of AtezoBev in routine practice. Patients with CP-B reported similar tolerability compared with CP-A, warranting prospective evaluation of AtezoBev in this treatment-deprived population.

Prognosis of patients with hepatocellular carcinoma treated with immunotherapy - development and validation of the CRAFITY score.

BACKGROUND & AIMS: Immunotherapy with atezolizumab plus bevacizumab represents the new standard of care in systemic front-line treatment of hepatocellular carcinoma (HCC). However, biomarkers that predict treatment success and survival remain an unmet need. METHODS: Patients with HCC put on PD-(L)1-based immunotherapy were included in a training set (n = 190; 6 European centers) and a validation set (n = 102; 8 European centers). We investigated the prognostic value of baseline variables on overall survival using a Cox model in the training set and developed the easily applicable CRAFITY (CRP and AFP in ImmunoTherapY) score. The score was validated in the independent, external cohort, and evaluated in a cohort of patients treated with sorafenib (n = 204). RESULTS: Baseline serum alpha-fetoprotein ≥100 ng/ml (hazard ratio [HR] 1.7; p = 0.007) and C-reactive protein ≥1 mg/dl (HR, 1.7; p = 0.007) were identified as independent prognostic factors in multivariable analysis and were used to develop the CRAFITY score. Patients who fulfilled no criterion (0 points; CRAFITY-low) had the longest median overall survival (27.6 (95% CI 19.5-35.8) months), followed by those fulfilling 1 criterion (1 point; CRAFITY-intermediate; 11.3 (95% CI 8.0-14.6) months), and patients meeting both criteria (2 points; CRAFITY-high; 6.4 (95% CI 4.8-8.1) months; p <0.001). Additionally, best radiological response (complete response/partial response/stable disease/progressive disease) was significantly better in patients with lower CRAFITY score (CRAFITY-low: 9%/20%/52%/20% vs. CRAFITY-intermediate: 3%/25%/36%/36% vs. CRAFITY-high: 2%/15%/22%/61%; p = 0.003). These results were confirmed in the independent validation set and in different subgroups, including Child-Pugh A and B, performance status 0 and ≥1, and first-line and later lines. In the sorafenib cohort, CRAFITY was associated with survival, but not radiological response. CONCLUSIONS: The CRAFITY score is associated with survival and radiological response in patients receiving PD-(L)1 immunotherapy. The score may help with patient counseling but requires prospective validation. LAY SUMMARY: The immunotherapy-based regimen of atezolizumab plus bevacizumab represents the new standard of care in systemic first-line therapy of hepatocellular carcinoma (HCC). Biomarkers to predict treatment outcome are an unmet need in patients undergoing immunotherapy for HCC. We developed and externally validated a score that predicts outcome in patients with HCC undergoing immunotherapy with immune checkpoint blockers.

DNA Methylation Profiling of Human Hepatocarcinogenesis.

BACKGROUND AND AIMS: Mutations in TERT (telomerase reverse transcriptase) promoter are established gatekeepers in early hepatocarcinogenesis, but little is known about other molecular alterations driving this process. Epigenetic deregulation is a critical event in early malignancies. Thus, we aimed to (1) analyze DNA methylation changes during the transition from preneoplastic lesions to early HCC (eHCC) and identify candidate epigenetic gatekeepers, and to (2) assess the prognostic potential of methylation changes in cirrhotic tissue. APPROACH AND RESULTS: Methylome profiling was performed using Illumina HumanMethylation450 (485,000 cytosine-phosphateguanine, 96% of known cytosine-phosphateguanine islands), with data available for a total of 390 samples: 16 healthy liver, 139 cirrhotic tissue, 8 dysplastic nodules, and 227 HCC samples, including 40 eHCC below 2cm. A phylo-epigenetic tree derived from the Euclidean distances between differentially DNA-methylated sites (n = 421,997) revealed a gradient of methylation changes spanning healthy liver, cirrhotic tissue, dysplastic nodules, and HCC with closest proximity of dysplasia to HCC. Focusing on promoter regions, we identified epigenetic gatekeeper candidates with an increasing proportion of hypermethylated samples (beta value > 0.5) from cirrhotic tissue (<1%), to dysplastic nodules (≥25%), to eHCC (≥50%), and confirmed inverse correlation between DNA methylation and gene expression for TSPYL5 (testis-specific Y-encoded-like protein 5), KCNA3 (potassium voltage-gated channel, shaker-related subfamily, member 3), LDHB (lactate dehydrogenase B), and SPINT2 (serine peptidase inhibitor, Kunitz type 2) (all P < 0.001). Unsupervised clustering of genome-wide methylation profiles of cirrhotic tissue identified two clusters, M1 and M2, with 42% and 58% of patients, respectively, which correlates with survival (P < 0.05), independent of etiology. CONCLUSIONS: Genome-wide DNA-methylation profiles accurately discriminate the different histological stages of human hepatocarcinogenesis. We report on epigenetic gatekeepers in the transition between dysplastic nodules and eHCC. DNA-methylation changes in cirrhotic tissue correlate with clinical outcomes.

Impact of older age in patients receiving atezolizumab and bevacizumab for hepatocellular carcinoma.

BACKGROUND AND AIMS: Combination atezolizumab/bevacizumab is the gold standard for first-line treatment of unresectable hepatocellular carcinoma (HCC). Our study investigated the efficacy and safety of combination therapy in older patients with HCC. METHODS: 191 consecutive patients from eight centres receiving atezolizumab and bevacizumab were included. Overall survival (OS), progression-free survival (PFS), overall response rate (ORR) and disease control rate (DCR) defined by RECIST v1.1 were measured in older (age ≥ 65 years) and younger (age < 65 years) age patients. Treatment-related adverse events (trAEs) were evaluated. RESULTS: The elderly (n = 116) had higher rates of non-alcoholic fatty liver disease (19.8% vs. 2.7%; p < .001), presenting with smaller tumours (6.2 cm vs 7.9 cm, p = .02) with less portal vein thrombosis (31.9 vs. 54.7%, p = .002), with fewer patients presenting with BCLC-C stage disease (50.9 vs. 74.3%, p = .002). There was no significant difference in OS (median 14.9 vs. 15.1 months; HR 1.15, 95% CI 0.65-2.02 p = .63) and PFS (median 7.1 vs. 5.5 months; HR 1.11, 95% CI 0.54-1.92; p = .72) between older age and younger age. Older patients had similar ORR (27.6% vs. 20.0%; p = .27) and DCR (77.5% vs. 66.1%; p = .11) compared to younger patients. Atezolizumab-related (40.5% vs. 48.0%; p = .31) and bevacizumab-related (44.8% vs. 41.3%; p = .63) trAEs were comparable between groups. Rates of grade ≥3 trAEs and toxicity-related treatment discontinuation were similar between older and younger age patients. Patients 75 years and older had similar survival and safety outcomes compared to younger patients. CONCLUSIONS: Atezolizumab and bevacizumab therapy is associated with comparable efficacy and tolerability in older age patients with unresectable 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.

Genetic Variation in HSD17B13 Reduces the Risk of Developing Cirrhosis and Hepatocellular Carcinoma in Alcohol Misusers.

BACKGROUND AND AIMS: Carriage of rs738409:G in patatin-like phospholipase domain containing 3 (PNPLA3) is associated with an increased risk for developing alcohol-related cirrhosis and hepatocellular carcinoma (HCC). Recently, rs72613567:TA in hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) was shown to be associated with a reduced risk for developing alcohol-related liver disease and to attenuate the risk associated with carriage of PNPLA3 rs738409:G. This study explores the risk associations between these two genetic variants and the development of alcohol-related cirrhosis and HCC. APPROACH AND RESULTS: Variants in HSD17B13 and PNPLA3 were genotyped in 6,171 participants, including 1,031 with alcohol-related cirrhosis and HCC, 1,653 with alcohol-related cirrhosis without HCC, 2,588 alcohol misusers with no liver disease, and 899 healthy controls. Genetic associations with the risks for developing alcohol-related cirrhosis and HCC were determined using logistic regression analysis. Carriage of HSD17B13 rs72613567:TA was associated with a lower risk for developing both cirrhosis (odds ratio [OR], 0.79; 95% confidence interval [CI], 0.72-0.88; P = 8.13 × 10-6 ) and HCC (OR, 0.77; 95% CI, 0.68-0.89; P = 2.27 × 10-4 ), whereas carriage of PNPLA3 rs738409:G was associated with an increased risk for developing cirrhosis (OR, 1.70; 95% CI, 1.54-1.88; P = 1.52 × 10-26 ) and HCC (OR, 1.77; 95% CI, 1.58-1.98; P = 2.31 × 10-23 ). These associations remained significant after adjusting for age, sex, body mass index, type 2 diabetes, and country. Carriage of HSD17B13 rs72613567:TA attenuated the risk for developing cirrhosis associated with PNPLA3 rs738409:G in both men and women, but the protective effect against the subsequent development of HCC was only observed in men (ORallelic , 0.75; 95% CI, 0.64-0.87; P = 1.72 × 10-4 ). CONCLUSIONS: Carriage of variants in PNPLA3 and HSD17B13 differentially affect the risk for developing advanced alcohol-related liver disease. A genotypic/phenotypic risk score might facilitate earlier diagnosis of HCC in this population.

[Mild COVID-19 disease in healthcare workers at a German university clinic : The "first wave" at the University Medical Center Hamburg-Eppendorf]. / Milde COVID-19-Verläufe bei Mitarbeitenden einer Universitätsklinik : Die "erste Welle" am Universitätsklinikum Hamburg-Eppendorf.

BACKGROUND: Healthcare workers are among the most exposed and potentially most threatened populations of the ongoing COVID-19 pandemic. Despite some reports on numbers of infections with SARS-CoV­2 in German healthcare workers, the courses of their clinical presentation when affected by COVID-19 are not well described. OBJECTIVE: In this contribution, characteristics and progressions of infected cases among healthcare workers at the University Medical Center Hamburg-Eppendorf during the first wave of the COVID-19 pandemic will be presented. METHODS: Between 1 July and 28 July 2020, 67 healthcare workers, who previously tested positive for SARS-CoV­2 via PCR, were invited via E­mail to participate in an anonymous online questionnaire; 39 persons participated. RESULTS: Participants (58%) were mostly ≤ 39 years old (64%) and female (70%). Most healthcare workers were involved in direct patient management (85%), including contact with SARS-CoV­2 positive patients (62%). All participants reported acute symptoms with a median duration of 19 days. The most frequent symptoms were fatigue (85%), anosmia (67%), cough (64%), headache (62%), and shortness of breath (51%). The disease course was mostly mild with low admission rates (5%). Ongoing symptoms lasting more than four weeks post-symptom-onset, particularly anosmia, fatigue, and shortness of breath, were reported by 38%. This group more frequently had pre-existing conditions (53% vs. 12%, p = 0.010), specifically hypertension (27% vs. 4%, p = 0.062). DISCUSSION: Healthcare workers reported mostly mild courses of COVID-19 despite increased contact with SARS-CoV-2 patients. However, some reported persistent symptoms months after infection.

Liquid biopsy in the clinical management of hepatocellular carcinoma.

With increasing knowledge on molecular tumour information, precision oncology has revolutionised the medical field over the past years. Liquid biopsy entails the analysis of circulating tumour components, such as circulating tumour DNA, tumour cells or tumour-derived extracellular vesicles, and has thus come as a handy tool for personalised medicine in many cancer entities. Clinical applications under investigation include early cancer detection, prediction of treatment response and molecular monitoring of the disease, for example, to comprehend resistance patterns and clonal tumour evolution. In fact, several tests for blood-based mutation profiling are already commercially available and have entered the clinical field.In the context of hepatocellular carcinoma, where access to tissue specimens remains mostly limited to patients with early stage tumours, liquid biopsy approaches might be particularly helpful. A variety of translational liquid biopsy studies have been carried out to address clinical needs, such as early hepatocellular carcinoma detection and prediction of treatment response. To this regard, methylation profiling of circulating tumour DNA has evolved as a promising surveillance tool for early hepatocellular carcinoma detection in populations at risk, which might soon transform the way surveillance programmes are implemented. This review summarises recent developments in the liquid biopsy oncological space and, in more detail, the potential implications in the clinical management of hepatocellular carcinoma. It further outlines technical peculiarities across liquid biopsy technologies, which might be helpful for interpretation by non-experts.

GALAD Score Detects Early Hepatocellular Carcinoma in an International Cohort of Patients With Nonalcoholic Steatohepatitis.

BACKGROUND & AIMS: The prevalence of nonalcoholic steatohepatitis (NASH) associated hepatocellular carcinoma (HCC) is increasing. However, strategies for detection of early-stage HCC in patients with NASH have limitations. We assessed the ability of the GALAD score, which determines risk of HCC based on patient sex; age; and serum levels of α-fetoprotein (AFP), AFP isoform L3 (AFP-L3), and des-gamma-carboxy prothrombin (DCP), to detect HCC in patients with NASH. METHODS: We performed a case-control study of 125 patients with HCC (20% within Milan Criteria) and 231 patients without HCC (NASH controls) from 8 centers in Germany. We compared the performance of serum AFP, AFP-L3, or DCP vs GALAD score to identify patients with HCC using receiver operating characteristic curves and corresponding area under the curve (AUC) analyses. We also analyzed data from 389 patients with NASH under surveillance for HCC in Japan, followed for a median of 167 months. During the 5-year screening period, 26 patients developed HCC. To compensate for irregular intervals of data points, we performed locally weighted scatterplot smoothing, linear regression, and a non-linear curve fit to assess development of GALAD before HCC development. RESULTS: The GALAD score identified patients with any stage HCC with an AUC of 0.96 - significantly greater than values for serum levels of AFP (AUC, 0.88), AFP-L3 (AUC, 0.86) or DCP (AUC, 0.87). AUC values for the GALAD score were consistent in patients with cirrhosis (AUC, 0.93) and without cirrhosis (AUC, 0.98). For detection of HCC within Milan Criteria, the GALAD score achieved an AUC of 0.91, with a sensitivity of 68% and specificity of 95% at a cutoff of -0.63. In a pilot Japanese cohort study, the mean GALAD score was higher in patients with NASH who developed HCC than in those who did not develop HCC as early as 1.5 years before HCC diagnosis. GALAD scores were above -0.63 approximately 200 days before the diagnosis of HCC. CONCLUSIONS: In a case-control study performed in Germany and a pilot cohort study in Japan, we found the GALAD score may detect HCC with high levels of accuracy in patients with NASH, with and without cirrhosis. The GALAD score can detect patients with early-stage HCC, and might facilitate surveillance of patients with NASH, who are often obese, which limits the sensitivity of detection of liver cancer by ultrasound.

Role of Molecular Biomarkers in Liver Transplantation for Hepatocellular Carcinoma.

Patient selection and organ allocation for liver transplantation (LT) in patients with hepatocellular carcinoma (HCC) relies predominantly on clinical parameters, such as tumor burden (ie, radiological imaging). Patients transplanted within Milan criteria have outstanding outcomes with a 5- and 10-year survival of 70% and 55%, respectively. Tumor recurrence after transplantion is rare in these patients (10%); however, treatment options upon recurrence are generally limited, and outcomes are poor. There are also several studies showing how a subgroup of patients with tumors outside the Milan criteria might achieve comparable outcomes to patients within Milan criteria. In other words, the size and number of tumor nodules does not always reflect tumor biology, which could be better captured using molecular proxies for cancer aggressiveness. Over the last decade, we have significantly improved our understanding of the molecular landscape of early stage HCC. This includes the development of molecular classification, identification of prognostic and mutational signatures, and potential mechanisms of hepatocarcinogenesis. Some molecular markers have already proven useful to predict tumor-related outcomes in HCC patients after LT. Most of these analyses are limited to tissue-derived biomarkers, which limits their implementation in clinical practice because tissue biopsy is not required for HCC diagnosis. Minimally invasive alternative tools, such as liquid biopsy, are being increasingly explored and could help to individualize risk stratification for patients with HCC who will benefit from LT despite being outside the accepted clinical criteria.

Hepatocellular carcinoma: Intratumoral EpCAM-positive cancer stem cell heterogeneity identifies high-risk tumor subtype.

BACKGROUND: The translational interest in the intratumoral heterogeneity of hepatocellular carcinoma (HCC) has been increasing. The dismal prognosis of this pathology is linked to the features of the HCC harbouring cancer stem cells (CSC), represented by EpCAM-expression. However, the extent of the impact of intratumoral distribution of CSC-features, both on the recurrence after curative resection and on clinical outcome, remains unknown. To address this, we investigated the spatial heterogeneity of CSC-features with the aim of identifying the unique HCC patient subgroups amenable to adjuvant treatment. METHODS: We designed a tissue microarray (TMA) from patients who had received liver resection between 2011 and 2017. Tumor specimens were sampled at multiple locations (n = 3-8). EpCAM-positivity was assessed for intensity and proportion by applying a score dividing three groups: (i) negative (E-/-); (ii) heterogeneous (E-/+); and (iii) homogeneous (E+/+). The groups were further analysed with regard to time-to-recurrence (TTR) and recurrence-free-survival (RFS). RESULTS: We included 314 tumor spots from 69 patients (76.8% male, median age 66, liver cirrhosis/fibrosis 75.8%). The risk factors were alcohol abuse (26.2%), NASH (13.1%), HBV (15.5%), HCV (17.9%) and others (27.4%), representative of a typical Western cohort. E+/+ patients experienced significantly shorter TTR and RFS compared to E+/- and E-/- patients (TTR 5 vs. 19 months, p = 0.022; RFS 5 vs. 14 vs. 21 months, p = 0.016). Only homogeneous EpCAM-positivity correlated with higher AFP levels (> 400 ng/ml, p = 0.031). CONCLUSIONS: Spatial heterogeneity of EpCAM-expression was markedly present in the cohort. Of note, only homogeneous EpCAM-expression correlated significantly with early recurrence, whereas heterogeneous EpCAM-expression was associated with clinical endpoints comparable to EpCAM-negativity. We identified a unique HCC subtype associated with a high risk of tumor recurrence.

New systemic agents for hepatocellular carcinoma: an update 2020.

PURPOSE OF REVIEW: Despite major therapeutic improvements in most cancer entities, hepatocellular carcinoma (HCC) has remained a dismal disease. In fact, incidence and mortality are increasing in many parts of the world, including the United States. Given that a number of systemic agents has recently been tested positive in phase 3 clinical trials, the objective of this review is to summarize the current treatment landscape for advanced HCC. RECENT FINDINGS: Following the positive SHARP trial in 2008, sorafenib has been the only systemic agent for advanced HCC for almost a decade. However, in first line, lenvatinib was tested noninferior to sorafenib, and most recently, the combination of atezolizumab with bevacizumab was tested superior to sorafenib. In second line, regorafenib, cabozantinib, and ramucirumab (the latter for patients with AFP ≥400 ng/ml) have shown prolonged overall survival compared with placebo. SUMMARY: Systemic treatment options for advanced HCC have substantially increased over the past years. The combination of atezolizumab and bevacizumab will likely become the new standard of care as it is the first treatment to report improved overall survival compared with sorafenib and the first, and so far only, positive phase 3 clinical trial for an immune-checkpoint inhibitor-containing regimen in advanced HCC.

High rates of cirrhosis and severe clinical events in patients with HBV/HDV co-infection: longitudinal analysis of a German cohort.

BACKGROUND: Chronic hepatitis delta virus (HDV) infection causes severe liver disease which often leads to cirrhosis and hepatocellular carcinoma (HCC). Aim of this study was to establish the disease severity and prognostic factors for disease outcome by analysing frequencies of clinical events and their correlation with baseline virological and biochemical parameters as well as interferon and nucleos(t)ide analogue treatment choice. METHODS: We studied a single-centre cohort of 49 anti-HDAg-positive patients with HBsAg persistence for at least 6 months. Virological and biochemical parameters, interferon and nucleos(t)ide analogue treatment choice as well as clinical events during follow-up were analysed by retrospective chart review (mean follow-up time 3 years, range 0.25-7.67 years). RESULTS: Severe clinical events occurred in 11/49 hepatitis D patients, including HCC (8/49), death (8/49) or liver transplantation (2/49). HCCs only occurred secondary to liver cirrhosis and their event rates in this cohort of hepatitis D patients did not differ from a matched HBV mono-infected cohort with comparable frequency of liver cirrhosis. A stepwise multivariate logistic regression revealed low platelet count (p = 0. 0290) and older age (p = 0.0337) correlating most strongly with overall clinical events, while serum HDV RNA positivity at baseline did not correlate with any clinical outcome. Interferon-free but not nucleos(t)ide analogue-free patient care correlated with the occurrence of HCC at logistic regression, although only 3/18 interferon-treated patients demonstrated repeatedly negative HDV PCR results post therapy. CONCLUSIONS: Our data indicate that progressive liver disease at baseline plays a major role as predictive factor for overall clinical outcome of hepatitis D patients. In particular, HCC risk may not be underestimated in hepatitis D virus RNA negative hepatitis D patients with advanced liver fibrosis.

Detection of a Broad Range of Low-Level Major Histocompatibility Complex Class II-Restricted, Hepatitis Delta Virus (HDV)-Specific T-Cell Responses Regardless of Clinical Status.

Background: This study aimed to comprehensively define the breadth and specificity of the hepatitis delta virus (HDV)-specific T-cell response in patients at different stages of chronic coinfection with hepatitis B virus (HBV). Methods: Following in vitro stimulation with an overlapping set of 21 HDV-specific 20mer peptides and exogenous interleukin 2, HDV-specific CD4+ and CD8+ T-cell responses of 32 HDV-infected patients were analyzed by enzyme-linked immunospot analysis and intracellular cytokine staining for interferon γ production at the single-peptide level. Additionally, HLA-binding studies were performed both in silico and in vitro. Results: We were able to detect ≥1 T-cell response in >50% our patients. Interestingly, there was no significant difference between the breadth of the response in patients positive and those negative for HDV by PCR. HDV-specific T-cell responses focused on 3 distinct HDV-specific epitopes that were each detected in 12%-21% of patients-2 HLA class II-restricted epitopes (amino acids 11-30 and 41-60) and 1 major histocompatibility complex class I-restricted epitope (amino acids 191-210). In in vitro HLA-binding assays, the 2 CD4+ T-cell specificities (amino acids 11-30 and 41-60) showed promiscuous binding to multiple HLA-DR molecules. Conclusions: This comprehensive characterization of HDV T-cell epitopes provides important information that will facilitate further studies of HDV immunopathogenesis.

Heterozygous carriage of the alpha1-antitrypsin Pi*Z variant increases the risk to develop liver cirrhosis.

OBJECTIVE: Homozygous alpha1-antitrypsin (AAT) deficiency increases the risk for developing cirrhosis, whereas the relevance of heterozygous carriage remains unclear. Hence, we evaluated the impact of the two most relevant AAT variants ('Pi*Z' and 'Pi*S'), present in up to 10% of Caucasians, on subjects with non-alcoholic fatty liver disease (NAFLD) or alcohol misuse. DESIGN: We analysed multicentric case-control cohorts consisting of 1184 people with biopsy-proven NAFLD and of 2462 people with chronic alcohol misuse, both cohorts comprising cases with cirrhosis and controls without cirrhosis. Genotyping for the Pi*Z and Pi*S variants was performed. RESULTS: The Pi*Z variant presented in 13.8% of patients with cirrhotic NAFLD but only in 2.4% of counterparts without liver fibrosis (p<0.0001). Accordingly, the Pi*Z variant increased the risk of NAFLD subjects to develop cirrhosis (adjusted OR=7.3 (95% CI 2.2 to 24.8)). Likewise, the Pi*Z variant presented in 6.2% of alcohol misusers with cirrhosis but only in 2.2% of alcohol misusers without significant liver injury (p<0.0001). Correspondingly, alcohol misusers carrying the Pi*Z variant were prone to develop cirrhosis (adjusted OR=5.8 (95% CI 2.9 to 11.7)). In contrast, the Pi*S variant was not associated with NAFLD-related cirrhosis and only borderline with alcohol-related cirrhosis (adjusted OR=1.47 (95% CI 0.99 to 2.19)). CONCLUSION: The Pi*Z variant is the hitherto strongest single nucleotide polymorphism-based risk factor for cirrhosis in NAFLD and alcohol misuse, whereas the Pi*S variant confers only a weak risk in alcohol misusers. As 2%-4% of Caucasians are Pi*Z carriers, this finding should be considered in genetic counselling of affected individuals.

The burden of hepatitis E among patients with haematological malignancies: A retrospective European cohort study.

BACKGROUND & AIMS: The burden of hepatitis E virus (HEV) infection among patients with haematological malignancy has only been scarcely reported. Therefore, we aimed to describe this burden in patients with haematological malignancies, including those receiving allogeneic haematopoietic stem cell transplantation. METHODS: We conducted a retrospective, multicentre cohort study across 11 European centres and collected clinical characteristics of 50 patients with haematological malignancy and RNA-positive, clinically overt hepatitis E between April 2014 and March 2017. The primary endpoint was HEV-associated mortality; the secondary endpoint was HEV-associated liver-related morbidity. RESULTS: The most frequent underlying haematological malignancies were aggressive non-Hodgkin lymphoma (NHL) (34%), indolent NHL (iNHL) (24%), and acute leukaemia (36%). Twenty-one (42%) patients had received allogeneic haematopoietic stem cell transplantation (alloHSCT). Death with ongoing hepatitis E occurred in 8 (16%) patients, including 1 patient with iNHL and 1 patient >100 days after alloHSCT in complete remission, and was associated with male sex (p = 0.040), cirrhosis (p = 0.006) and alloHSCT (p = 0.056). Blood-borne transmission of hepatitis E was demonstrated in 5 (10%) patients, and associated with liver-related mortality in 2 patients. Hepatitis E progressed to chronic hepatitis in 17 (34%) patients overall, and in 10 (47.6%) and 6 (50%) alloHSCT and iNHL patients, respectively. Hepatitis E was associated with acute or acute-on-chronic liver failure in 4 (8%) patients with 75% mortality. Ribavirin was administered to 24 (48%) patients, with an HEV clearance rate of 79.2%. Ribavirin treatment was associated with lower mortality (p = 0.037) and by trend with lower rates of chronicity (p = 0.407) when initiated <24 and <12 weeks after diagnosis of hepatitis E, respectively. Immunosuppressive treatment reductions were associated with mortality in 2 patients (28.6%). CONCLUSION: Hepatitis E is associated with mortality and liver-related morbidity in patients with haematological malignancy. Blood-borne transmission contributes to the burden. Ribavirin should be initiated early, whereas reduction of immunosuppressive treatment requires caution. LAY SUMMARY: Little is known about the burden of hepatitis E among patients with haematological malignancy. We conducted a retrospective European cohort study among 50 patients with haematological malignancy, including haematopoietic stem cell transplant recipients, with clinically significant HEV infection and found that hepatitis E is associated with hepatic and extrahepatic mortality, including among patients with indolent disease or among stem cell transplant recipients in complete remission. Hepatitis E virus infection evolved to chronic hepatitis in 5 (45.5%) patients exposed to a rituximab-containing regimen and 10 (47.6%) stem cell transplant recipients. Reducing immunosuppressive therapy because of hepatitis E was associated with mortality, while early ribavirin treatment was safe and effective.