Comparable Overall Survival in Patients with Hepatocellular Carcinoma Diagnosed within and outside a Surveillance Programme: The Potential Impact of Liver Cirrhosis.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death, and its incidence is rising. Mortality from HCC is predicted to increase by 140% by 2035. Surveillance of high-risk patients with cirrhosis or chronic liver disease may be one means of reducing HCC mortality, but the level of supporting evidence for international guidelines is low/moderate. This study explores the real-world experience of HCC surveillance at a tertiary referral centre. Electronic patient records for all new HCCs diagnosed between August 2012 and December 2021 were retrospectively reviewed. Patient and tumour characteristics were evaluated, including the co-existence of chronic liver disease, cancer treatment and survival, and categorised according to HCC diagnosis within or outside a surveillance programme. Patients with HCC who presented through surveillance had smaller tumours diagnosed at an earlier stage, but this did not translate into improved overall survival. All patients in surveillance had chronic liver disease, including 91% (n = 101) with cirrhosis, compared to 45% (n = 29) in the non-surveillance cohort. We propose that the immune dysfunction associated with cirrhosis predisposes patients to a more aggressive tumour biology than the largely non-cirrhotic population in the non-surveillance group.

Isolation of human intrahepatic leukocytes for phenotypic and functional characterization by flow cytometry.

With the growing appreciation of tissue-resident immunity, studying tissue-specific immune cells contributing to both homeostasis and disease is imperative. Here, we provide a protocol for the isolation of human intrahepatic leukocytes (IHL) maximizing viability, purity, and yield. Our protocol is scalable by tissue weight, allowing for reproducible and efficient IHL liberation suitable for functional characterization, cell isolation, and profiling by flow (or mass) cytometry. Furthermore, we provide a "guide" to determine an expected IHL yield per gram of tissue processed. For complete details on the use and execution of this protocol, please refer to Stegmann et al. (2016), Pallett et al. (2017), Easom et al. (2018), Swadling et al. (2020), Pallett et al. (2020), and Zakeri et al. (2022).

Characterisation and induction of tissue-resident gamma delta T-cells to target hepatocellular carcinoma.

Immunotherapy is now the standard of care for advanced hepatocellular carcinoma (HCC), yet many patients fail to respond. A major unmet goal is the boosting of T-cells with both strong HCC reactivity and the protective advantages of tissue-resident memory T-cells (TRM). Here, we show that higher intratumoural frequencies of γδ T-cells, which have potential for HLA-unrestricted tumour reactivity, associate with enhanced HCC patient survival. We demonstrate that γδ T-cells exhibit bona fide tissue-residency in human liver and HCC, with γδTRM showing no egress from hepatic vasculature, persistence for >10 years and superior anti-tumour cytokine production. The Vγ9Vδ2 T-cell subset is selectively depleted in HCC but can efficiently target HCC cell lines sensitised to accumulate isopentenyl-pyrophosphate by the aminobisphosphonate Zoledronic acid. Aminobisphosphonate-based expansion of peripheral Vγ9Vδ2 T-cells recapitulates a TRM phenotype and boosts cytotoxic potential. Thus, our data suggest more universally effective HCC immunotherapy may be achieved by combining aminobisphosphonates to induce Vγ9Vδ2TRM capable of replenishing the depleted pool, with additional intratumoural delivery to sensitise HCC to Vγ9Vδ2TRM-based targeting.

Monocyte dysfunction in decompensated cirrhosis is mediated by the prostaglandin E2-EP4 pathway.

BACKGROUND & AIMS: Infection is a major problem in advanced liver disease secondary to monocyte dysfunction. Elevated prostaglandin (PG)E2 is a mediator of monocyte dysfunction in cirrhosis; thus, we examined PGE2 signalling in outpatients with ascites and in patients hospitalised with acute decompensation to identify potential therapeutic targets aimed at improving monocyte dysfunction. METHODS: Using samples from 11 outpatients with ascites and 28 patients hospitalised with decompensated cirrhosis, we assayed plasma levels of PGE2 and lipopolysaccharide (LPS); performed quantitative real-time PCR on monocytes; and examined peripheral blood monocyte function. We performed western blotting and immunohistochemistry for PG biosynthetic machinery expression in liver tissue. Finally, we investigated the effect of PGE2 antagonists in whole blood using polychromatic flow cytometry and cytokine production. RESULTS: We show that hepatic production of PGE2 via the cyclo-oxygenase 1-microsomal PGE synthase 1 pathway, and circulating monocytes contributes to increased plasma PGE2 in decompensated cirrhosis. Transjugular intrahepatic sampling did not reveal whether hepatic or monocytic production was larger. Blood monocyte numbers increased, whereas individual monocyte function decreased as patients progressed from outpatients with ascites to patients hospitalised with acute decompensation, as assessed by Human Leukocyte Antigen (HLA)-DR isotype expression and tumour necrosis factor alpha and IL6 production. PGE2 mediated this dysfunction via its EP4 receptor. CONCLUSIONS: PGE2 mediates monocyte dysfunction in decompensated cirrhosis via its EP4 receptor and dysfunction was worse in hospitalised patients compared with outpatients with ascites. Our study identifies a potential drug target and therapeutic opportunity in these outpatients with ascites to reverse this process to prevent infection and hospital admission. LAY SUMMARY: Patients with decompensated cirrhosis (jaundice, fluid build-up, confusion, and vomiting blood) have high infection rates that lead to high mortality rates. A white blood cell subset, monocytes, function poorly in these patients, which is a key factor underlying their sensitivity to infection. We show that monocyte dysfunction in decompensated cirrhosis is mediated by a lipid hormone in the blood, prostaglandin E2, which is present at elevated levels, via its EP4 pathway. This dysfunction worsens when patients are hospitalised with complications of cirrhosis compared with those in the outpatients setting, which supports the EP4 pathway as a potential therapeutic target for patients to prevent infection and hospitalisation.

Biopsy for advanced hepatocellular carcinoma: results of a multicentre UK audit.

BACKGROUND: Advanced hepatocellular carcinoma (HCC) is commonly diagnosed using non-invasive radiological criteria (NIRC) defined by the European Association for the Study of the Liver or the American Association for the Study of Liver Diseases. In 2017, The National Institute for Clinical Excellence mandated histological confirmation of disease to authorise the use of sorafenib in the UK. METHODS: This was a prospective multicentre audit in which patients suitable for sorafenib were identified at multidisciplinary meetings. The primary analysis cohort (PAC) was defined by the presence of Child-Pugh class A liver disease and performance status 0-2. Clinical, radiological and histological data were reported locally and collected on a standardised case report form. RESULTS: Eleven centres reported 418 cases, of which 361 comprised the PAC. Overall, 76% had chronic liver disease and 66% were cirrhotic. The diagnostic imaging was computed tomography in 71%, magnetic resonance imaging in 27% and 2% had both. Pre-existing histology was available in 45 patients and 270 underwent a new biopsy, which confirmed HCC in 93.4%. Alternative histological diagnoses included cholangiocarcinoma (CC) and combined HCC-CC. In cirrhotic patients, NIRC criteria had a sensitivity of 65.4% and a positive predictive value of 91.4% to detect HCC. Two patients (0.7%) experienced mild post-biopsy bleeding. CONCLUSION: The diagnostic biopsy is safe and feasible for most patients eligible for systemic therapy.

Targeting human Acyl-CoA:cholesterol acyltransferase as a dual viral and T cell metabolic checkpoint.

Determining divergent metabolic requirements of T cells, and the viruses and tumours they fail to combat, could provide new therapeutic checkpoints. Inhibition of acyl-CoA:cholesterol acyltransferase (ACAT) has direct anti-carcinogenic activity. Here, we show that ACAT inhibition has antiviral activity against hepatitis B (HBV), as well as boosting protective anti-HBV and anti-hepatocellular carcinoma (HCC) T cells. ACAT inhibition reduces CD8+ T cell neutral lipid droplets and promotes lipid microdomains, enhancing TCR signalling and TCR-independent bioenergetics. Dysfunctional HBV- and HCC-specific T cells are rescued by ACAT inhibitors directly ex vivo from human liver and tumour tissue respectively, including tissue-resident responses. ACAT inhibition enhances in vitro responsiveness of HBV-specific CD8+ T cells to PD-1 blockade and increases the functional avidity of TCR-gene-modified T cells. Finally, ACAT regulates HBV particle genesis in vitro, with inhibitors reducing both virions and subviral particles. Thus, ACAT inhibition provides a paradigm of a metabolic checkpoint able to constrain tumours and viruses but rescue exhausted T cells, rendering it an attractive therapeutic target for the functional cure of HBV and HBV-related HCC.

Longevity and replenishment of human liver-resident memory T cells and mononuclear phagocytes.

The human liver contains specialized subsets of mononuclear phagocytes (MNPs) and T cells, but whether these have definitive features of tissue residence (long-term retention, lack of egress) and/or can be replenished from the circulation remains unclear. Here we addressed these questions using HLA-mismatched liver allografts to discriminate the liver-resident (donor) from the infiltrating (recipient) immune composition. Allografts were rapidly infiltrated by recipient leukocytes, which recapitulated the liver myeloid and lymphoid composition, and underwent partial reprogramming with acquisition of CD68/CD206 on MNPs and CD69/CD103 on T cells. The small residual pool of donor cells persisting in allografts for over a decade contained CX3CR1hi/CD163hi/CD206hi Kupffer cells (KCs) and CXCR3hi tissue-resident memory T cells (TRM). CD8+ TRM were found in the local lymph nodes but were not detected egressing into the hepatic vein. Our findings inform organ transplantation and hepatic immunotherapy, revealing remarkably long-lived populations of KCs and TRM in human liver, which can be additionally supplemented by their circulating counterparts.

Human Liver Memory CD8+ T Cells Use Autophagy for Tissue Residence.

Tissue-resident memory T cells have critical roles in long-term pathogen and tumor immune surveillance in the liver. We investigate the role of autophagy in equipping human memory T cells to acquire tissue residence and maintain functionality in the immunosuppressive liver environment. By performing ex vivo staining of freshly isolated cells from human liver tissue, we find that an increased rate of basal autophagy is a hallmark of intrahepatic lymphocytes, particularly liver-resident CD8+ T cells. CD8+ T cells with increased autophagy are those best able to proliferate and mediate cytotoxicity and cytokine production. Conversely, blocking autophagy induction results in the accumulation of depolarized mitochondria, a feature of exhausted T cells. Primary hepatic stellate cells or the prototypic hepatic cytokine interleukin (IL)-15 induce autophagy in parallel with tissue-homing/retention markers. Inhibition of T cell autophagy abrogates tissue-residence programming. Thus, upregulation of autophagy adapts CD8+ T cells to combat mitochondrial depolarization, optimize functionality, and acquire tissue residence.

Validation of CLIF-C ACLF score to define a threshold for futility of intensive care support for patients with acute-on-chronic liver failure.

BACKGROUND: Acute-on-chronic liver failure (ACLF) is a severe complication of cirrhosis and is defined by organ failure and high rates of short-term mortality. Patients with ACLF are managed with multiorgan support in the intensive care unit (ICU). Currently, it is unclear when this supportive care becomes futile, particularly in patients who are not candidates for liver transplant. The aim of this study was to determine whether the currently available prognostic scores can identify patients with ACLF in whom prolonged ICU care is likely to be futile despite maximal treatment efforts. METHODS: Data of 202 consecutive patients with ACLF admitted to the ICU at the Royal Free Hospital London between 2005 and 2012 were retrospectively analyzed. Prognostic scores for chronic liver diseases, such as Child-Pugh, Model for End-Stage Liver Disease (MELD), European Foundation for the study of chronic liver failure (CLIF-C) organ failure (OF), and CLIF-C ACLF, were calculated 48 hours after ICU admission and correlated with patient outcome after 28 days. RESULTS: The CLIF-C ACLF score, compared with all other scores, most accurately predicted 28-day mortality, with an area under the receiver operator characteristic of 0.8 (CLIF-C OF, 0.75; MELD, 0.68; Child-Pugh, 0.66). A CLIF-C ACLF score cutoff ≥ 70 identified patients with a 100% mortality within 28 days. These patients had elevated inflammatory parameters representing a systemic inflammatory response, most often renal failure, compared with patients below this cutoff. CONCLUSIONS: Patients with ACLF and high CLIF-C ACLF score (≥ 70) after 48 hours of intensive care may reach a threshold of futility for further ongoing intensive support. The best treatment options in this scenario remain to be determined but may include palliative care.

Bleeding Risk with Invasive Procedures in Patients with Cirrhosis and Coagulopathy.

PURPOSE OF REVIEW: Previous perceptions of cirrhosis as a hypocoagulable state have resulted in empirical blood product transfusions prior to invasive procedures. We evaluate procedure-related bleeding risks in patients with cirrhosis, assess the utility of conventional and newer global coagulation tests, and explore evidence surrounding prophylactic transfusion strategies. RECENT FINDINGS: Recent literature supports the concept of a rebalanced, albeit fragile, haemostasis equilibrium in cirrhosis, with a potential hypercoagulable tendency in stable patients. Standard coagulation tests provide a poor reflection of bleeding risks and yet are relied upon for transfusion thresholds. Consequently, a sizeable proportion of patients receive unnecessary blood products. The role of viscoelastic tests to guide transfusions requires further evaluation. In stable cirrhotic patients, procedure-related bleeding rates appear low. Prophylactic transfusion strategies based on arbitrary thresholds lack evidence of clinical benefit. There is a pressing need for point-of-care coagulation tests that represent the complex coagulopathy of cirrhosis and well-powered randomised controlled trials to develop evidence-based pre-procedure transfusion guidelines.

Diagnostic imaging and radiation exposure in inflammatory bowel disease.

Diagnostic imaging plays a key role in the diagnosis and management of inflammatory bowel disease (IBD). However due to the relapsing nature of IBD, there is growing concern that IBD patients may be exposed to potentially harmful cumulative levels of ionising radiation in their lifetime, increasing malignant potential in a population already at risk. In this review we explore the proportion of IBD patients exposed to high cumulative radiation doses, the risk factors associated with higher radiation exposures, and we compare conventional diagnostic imaging with newer radiation-free imaging techniques used in the evaluation of patients with IBD. While computed tomography (CT) performs well as an imaging modality for IBD, the effective radiation dose is considerably higher than other abdominal imaging modalities. It is increasingly recognised that CT imaging remains responsible for the majority of diagnostic medical radiation to which IBD patients are exposed. Magnetic resonance imaging (MRI) and small intestine contrast enhanced ultrasonography (SICUS) have now emerged as suitable radiation-free alternatives to CT imaging, with comparable diagnostic accuracy. The routine use of MRI and SICUS for the clinical evaluation of patients with known or suspected small bowel Crohn's disease is to be encouraged wherever possible. More provision is needed for out-of-hours radiation-free imaging modalities to reduce the need for CT.

Risk factors for endoscopic sedation reversal events: a five-year retrospective study.

OBJECTIVE: Conscious sedation is widely used in endoscopic practice but is not without risk. We aimed to determine the frequency of sedation complications requiring reversal, and to identify potential patient and procedural risk factors. DESIGN: A retrospective study of all gastrointestinal endoscopic procedures performed under conscious sedation, in a large three-campus tertiary referral endoscopic centre, between 12 October 2007 and 31 December 2012 (n=52 553). Flumazenil or naloxone administration was used as a marker of sedation complications requiring reversal. Reversal cases were analysed for associations with sedation dose, patient American Society of Anesthesiologists (ASA) grade, age and type of procedure undertaken. RESULTS: In total, 149 sedation reversals occurred, representing 0.28% of all sedated endoscopic procedures carried out. Endoscopic Retrograde Cholangiopancreatography (ERCP) and increasing patient ASA grade were positively associated with sedation reversal (p<0.05). Mean midazolam dose was highest for ERCP (4.9±2.9 mg) and lowest for flexible sigmoidoscopy (1.7±0.6 mg; p<0.01). Mean opioid dose (calculated as pethidine equivalent) was highest for ERCP (62.9±38.7 mg) and lowest for gastroscopy (6.9±13.5 mg; p<0.01). Maximum doses of midazolam or opioid recommended by the British Society of Gastroenterology were exceeded in 7.4% and 14.1% of reversals, respectively. CONCLUSIONS: ERCP procedures and higher patient ASA grade were associated with an increased risk of conscious sedation-related complications requiring reversal. In these high-risk groups, alternative sedation strategies should be considered and tested. Prospective studies are needed to further explore risk factors that may help predict adverse sedation outcomes.