Objective: Revealing the single-cell immune ecosystems in true versus de novo hepatocellular carcinoma (HCC) recurrences could help the optimal development of immunotherapies. Design: We performed 5’and VDJ single-cell RNA-sequencing on 34 samples from 20 recurrent HCC patients. Bulk RNA-sequencing, flow cytometry, multiplexed immunofluorescence, and in vitro functional analyses were performed on samples from two validation cohorts. Results: Analyses of mutational profiles and evolutionary trajectories in paired primary and recurrent HCC samples using whole-exome sequencing identified de novo versus true recurrences, some of which occurred before clinical diagnosis. The tumour immune microenvironment (TIME) of truly recurrent HCCs was characterised by an increased abundance in KLRB1⁺CD8⁺ T cells with memory phenotype and low cytotoxicity. In contrast, we found an enrichment in cytotoxic and exhausted CD8⁺ T cells in the TIME of de novo recurrent HCCs. Transcriptomic and interaction analyses showed elevated GDF15 expression on HCC cells in proximity to dendritic cells, which may have dampened antigen presentation and inhibited antitumour immunity in truly recurrent lesions. In contrast, myeloid cells’ cross talk with T cells-mediated T cell exhaustion and immunosuppression in the TIME of de novo recurrent HCCs. Consistent with these findings, a phase 2 trial of neoadjuvant anti-PD-1 immunotherapy showed more responses in de novo recurrent HCC patients. Conclusion: True and de novo HCC recurrences occur early, have distinct TIME and may require different immunotherapy strategies. Our study provides a source for genomic diagnosis and immune profiling for guiding immunotherapy based on the type of HCC recurrence and the specific TIME.

Immune checkpoint inhibitors (ICIs) may lead to rejection and even graft loss of solid organ transplant recipients, making them not widely used in transplant patients. There is insufficient clinical experience in using ICIs as a bridging or downstaging therapy before transplantation. We performed a retrospective review of patients receiving programmed cell death 1 inhibitor (PD1) before liver transplantation for HCC in our center and analyzed the data of these patients with the purpose of investigating the safety and feasibility of preoperative PD1 inhibitor among liver transplant recipients and exploring the preoperative correlation ICIs and the postoperative risk of rejection and immune-related graft loss. A total of 16 patients enrolled in this study. Acute rejection occurred in 9 patients, with an incidence of 56.3%. The median time of rejection was 7 days after surgery. The median FK506 concentration at the time of rejection was 7.1 μg/L. All rejection reactions were reversed after adjusting the immunosuppression regimen. The interval between the last PD1 inhibitor and transplantation in the rejection group was shorter than that in the nonrejection group, and there was a statistical difference [21.0 (15.5–27.5) days vs. 60.0 (34.0–167.0) days, p=0.01]. In conclusion, PD1 inhibitor is a safe and feasible method for bridging or downstaging treatment before liver transplantation. Although preoperative PD1 inhibitor may increase the incidence of postoperative rejection, it is not associated with increased immune-related graft loss and patient death.

Chronic hepatitis B (CHB) is a major global health issue. The role of rare genetic variants in CHB has not been elucidated. We aimed to identify rare allelic variants predisposing to CHB. We performed exome sequencing in 50 CHB patients who had no identifiable risk factors for CHB and 40 controls who were healthy and hepatitis B surface antibody-positive, but had never received hepatitis B vaccination. We selected six rare variant alleles and followed up their association with disease status by Sanger sequencing in a case-control study comprising 1,728 CHB patients and 1,636 healthy controls. The latter had either not been immunized with hepatitis B vaccine or had uncertain vaccination status. Our results showed that transmembrane protein 2 p.Ser1254Asn, interferon alpha 2 p.Ala120Thr, its regulator NLR family member X1 p.Arg707Cys, and complement component 2 p.Glu318Asp were associated with CHB, with P values of <1.0 × 10−7, 2.76 × 10−5, 5.08 × 10−5, 2.78 × 10−4 and odds ratios (ORs) of 2.45, 4.08, 2.34, and 1.97, respectively. The combined P value was <2.0 × 10−16. As there has been no indication of immunological functions for the associated gene, transmembrane protein 2 , we further studied its expression by immunohistochemistry, real-time polymerase chain reaction, and western blotting. Our results showed that it was strongly expressed by healthy hepatocytes, but its expression was reduced in liver tissues with CHB, hepatitis B viral (HBV) genome-containing HepG2.2.15 cells, as compared with healthy liver tissues and non-HBV genome-containing HepG2 cells ( P = 0.022 and 0.0036, respectively). Conclusion : We identified four missense mutations associated with CHB, our results providing evidence for rare inborn genetic defects that contribute to increased host susceptibility to CHB.

Liver transplantation is the most effective treatment of advanced liver disease, and the use of extended criteria donor organs has broadened the source of available livers. Although normothermic machine perfusion (NMP) has become a useful tool in liver transplantation, there are no consistent criteria that can be used to evaluate the viability of livers during NMP. This review summarizes the criteria, indicators, and methods used to evaluate liver viability during NMP. The shape, appearance, and hemodynamics of the liver can be analyzed at a macroscopic level, while markers of liver injury, indicators of liver and bile duct function, and other relevant indicators can be evaluated by biochemical analysis. The liver can also be assessed by tissue biopsy at the microscopic level. Novel methods for assessment of liver viability are introduced. The limitations of evaluating liver viability during NMP are discussed and suggestions for future clinical practice are provided.

Background: Mean platelet volume (MPV) to platelet count (PC) ratio (MPV/PC) is a useful indicator in several cancers. However, the role for MPV/PC ratio in esophageal squamous cell carcinoma (ESCC) is still controversial. Methods: A retrospective study was conducted including 277 resectable ESCC patients. The optimal cut-off values were calculated by the X-tile program. The receiver operator characteristic (ROC) curves were also created to show the candidate cut-off points. The comparisons between the X-tile plot and ROC curve were performed. The Kaplan-Meier method was utilized to analyze the cancer-specific survival (CSS). Prognostic factors for CSS were calculated with Cox regression univariate and multivariate analyses. Results: According to the X-tile program, the cut-off values for MPV, PC and MPV/PC ratio were 8.5 (fl), 200 (giga/l) and 0.04, respectively. However, the cut-off values for MPV, PC and MPV/PC ratio by the ROC curves were 8.25 (fl), 243.5 (giga/l) and 0.0410, respectively. The cut-off values were similar between the X-tile and ROC curve. A low MPV/PC ratio level (≤0.04) was associated with poor CSS (22.4% vs. 43.1%, P < 0.001). In multivariate analyses, we found that MPV/PC ratio was an independent predictor for CSS (P < 0.001). When we set the cut-off point using ROC curve, the MPV/PC ratio was still an independent predictor for CSS (P < 0.001). Conclusion: The MPV/PC ratio is a useful predictive indicator in patients with ESCC.

The shortage of transplant organs remains a severe global issue. Normothermic machine perfusion (NMP) has the potential to increase organ availability, yet its efficacy is hampered by the inflammatory response during machine perfusion. Mouse liver IRI models, discarded human liver models, and porcine marginal liver transplantation models were utilized to investigate whether FXR activation could mitigate inflammation-induced liver damage. FXR expression levels before and after reperfusion were measured. Gene editing and co-immunoprecipitation techniques were employed to explore the regulatory mechanism of FXR in inflammation inhibition. The expression of FXR correlates with the extent of liver damage after reperfusion. Activation of FXR significantly suppressed the inflammatory response triggered by IRI, diminished the release of pro-inflammatory cytokines, and improve liver function recovery during NMP, assisting discarded human livers to reach transplant standards. Mechanistically, FXR disrupts the interaction between p65 and p300, thus inhibiting modulating the nuclear factor kappa-B (NF-κB) signaling pathway, a key instigator of inflammation. Our research across multiple species confirms that activating FXR can optimize NMP by attenuating IRI-related liver damage, thereby improving the utilization of marginal livers for transplantation.