The predictive value of the modified AFP model for liver transplantation outcomes in multinodular hepatocellular carcinoma patients.

BACKGROUND: There is a lack of studies focusing on the benefit of liver transplantation (LT) in hepatocellular carcinoma (HCC) patients with > 3 tumors. This study aims to establish a model to effectively predict overall survival in Chinese HCC patients with multiple tumors (> 3 tumors) who undergo LT. METHODS: This retrospective study included 434 HCC liver transplant recipients from the China Liver Transplant Registry. All HCC patients had more than 3 tumor nodules. Three selection criteria systems (i.e., AFP, Metroticket 2.0, and Up-to-7) were compared regarding the prediction of HCC recurrence. The modified AFP model was established by univariate and multivariate competing risk analyses. RESULTS: The AFP score 2 and the AFP score ≥ 3 groups had 5-year recurrence rates of 19.6% and 40.5% in our cohort. The prediction of HCC recurrence based on the AFP model was associated with a c-statistic of 0.606, which was superior to the Up-to-7 and Metroticket 2.0 models. AFP level > 1000 ng/mL, largest tumor size ≥ 8 cm, vascular invasion, and MELD score ≥ 15 were associated with overall survival. The 5-year survival rate in the modified AFP score 0 group was 71.7%. CONCLUSIONS: The AFP model is superior in predicting tumor recurrence in HCC patients with > 3 tumors prior to LT. With the modified AFP model, patients likely to derive sufficient benefit from LT can be identified.

E2F7 promotes mammalian target of rapamycin inhibitor resistance in hepatocellular carcinoma after liver transplantation.

The mammalian target of rapamycin (mTOR) pathway is frequently deregulated and has critical roles in cancer progression. mTOR inhibitor has been widely used in several kinds of cancers and is strongly recommended in patients with hepatocellular carcinoma (HCC) after liver transplantation (LT). However, the poor response to mTOR inhibitors due to resistance remains a challenge. Hypoxia-associated resistance limits the therapeutic efficacy of targeted drugs. The present study established models of HCC clinical samples and cell lines resistance to mTOR inhibitor sirolimus and screened out E2F7 as a candidate gene induced by hypoxia and promoting sirolimus resistance. E2F7 suppressed mTOR complex 1 via directly binding to the promoter of the TSC1 gene and stabilizes hypoxia-inducible factor-1α activating its downstream genes, which are responsible for E2F7-dependent mTOR inhibitor resistance. Clinically, low E2F7 expression could be an effective biomarker for recommending patients with HCC for anti-mTOR-based therapies after LT. Targeting E2F7 synergistically inhibited HCC growth with sirolimus in vivo. E2F7 is a promising target to reverse mTOR inhibition resistance. Collectively, our study points to a role for E2F7 in promoting mTOR inhibitor resistance in HCC and emphasizes its potential clinical significance in patients with HCC after LT.

USP22 promotes hypoxia-induced hepatocellular carcinoma stemness by a HIF1α/USP22 positive feedback loop upon TP53 inactivation.

OBJECTIVE: We aimed to elucidate the mutual regulation mechanism of ubiquitin-specific protease 22 (USP22) and hypoxia inducible factor-1α (HIF1α), and the mechanism they promote the stemness of hepatocellular carcinoma (HCC) cells under hypoxic conditions. DESIGN: Cell counting, migration, self-renewal ability, chemoresistance and expression of stemness genes were established to detect the stemness of HCC cells. Immunoprecipitation, ubiquitination assay and chromatin immunoprecipitation assay were used to elucidate the mutual regulation mechanism of USP22 and HIF1α. HCC patient samples and The Cancer Genome Atlas data were used to demonstrate the clinical significance. In vivo USP22-targeting experiment was performed in mice bearing HCC. RESULTS: USP22 promotes hypoxia-induced HCC stemness and glycolysis by deubiquitinating and stabilising HIF1α. As direct target genes of HIF1α, USP22 and TP53 can be transcriptionally upregulated by HIF1α under hypoxic conditions. In TP53 wild-type HCC cells, HIF1α induced TP53-mediated inhibition of HIF1α-induced USP22 upregulation. In TP53-mutant HCC cells, USP22 and HIF1α formed a positive feedback loop and promote the stemness of HCC. HCC patients with a loss-of-function mutation at TP53 and high USP22 and/or HIF1α expression tend to have a worse prognosis. The USP22-targeting lipopolyplexes caused high tumour inhibition and high sorafenib sensitivity in mice bearing HCC. CONCLUSION: USP22 promotes hypoxia-induced HCC stemness by a HIF1α/USP22 positive feedback loop on TP53 inactivation. USP22 is a promising target for the HCC therapy.

Hangzhou criteria as downstaging criteria in hepatocellular carcinoma before liver transplantation: A multicenter study from China.

BACKGROUND: The downstaging of hepatocellular carcinoma (HCC) has been confirmed to benefit liver transplantation (LT) patients whose tumors are beyond the transplantation criteria. Milan criteria (MC), a tumor size and number-based assessment, is currently used as the endpoint in these patients. However, many studies believe that tumor biological behavior should be added to the evaluation criteria for downstaging efficacy. Hence, this study aimed to explore the feasibility of Hangzhou criteria (HC), which introduced tumor grading and alpha-fetoprotein in addition to tumor size and number, as an endpoint of downstaging. METHODS: We performed a multicenter and retrospective study of 206 patients accepted locoregional therapy (LRT) as downstaging/bridge treatment prior to LT in three centers of China. RESULTS: Recipients were divided into four groups: failed downstaging to the HC (group A, n = 46), successful downstaging to the HC (group B, n = 30), remained within the HC all the time (group C, n = 113), and tumor progressed (group D, n = 17). The 3-year HCC recurrence probabilities of groups B and C were not significantly different (10.3% vs. 11.6%, P = 0.87). The HCC recurrent rate was significantly higher in group A (52.3%) compared with that in group B/C (P < 0.05). Seven patients (7/76, 9.2%) whose tumor exceeded the the HC were successfully downstaged to the MC, and 39.5% (30/76) to the the HC. In group B, 23 patients remained beyond the MC and their survivals were as well as those of patients within the MC. CONCLUSIONS: Compared to the MC, HC downstaging criteria can give more HCC patients access to LT and furthermore, the outcome of these patients is the same as those matching MC downstaging criteria. Hangzhou downstaging criteria therefore is applicable in clinical practice.

mTOR inhibitor reduces nontumour-related death in liver transplantation for hepatocellular carcinoma.

Sirolimus is a regularly applied immunosuppressant for patients undergoing liver transplantation (LT) for hepatocellular carcinoma (HCC). Sirolimus not only significantly inhibits HCC recurrence but also protects renal function. However, the improvement effect of sirolimus on nontumour-related death in patients is still unknown. The aim of our study was to investigate the therapeutic effect of sirolimus on nontumour-related deaths. In this study, we retrospectively enrolled 403 LT patients with HCC from January 1, 2015, to December 31, 2018. The median follow-up time was 47.1 months. The patients were divided into the sirolimus group (N = 184) and the sirolimus-free group (N = 219). There were no significant differences between the sirolimus group and the sirolimus-free group in survival (P = 0.054). In transplant patients who exceeded the Milan or Hangzhou criteria, the sirolimus group achieved higher survival than the sirolimus-free group (P = 0.005; P = 0.02). Moreover, multivariate analysis showed that sirolimus strongly reduced the hazard ratio (HR) for nontumour-related death in LT patients who exceeded the Milan (HR: 0.42; 95% CI: 0.18-1; P = 0.05) or Hangzhou criteria (HR: 0.26; 95% CI: 0.08-0.89; P = 0.032). HCC recurrence increased the risk of nontumour-related death. In conclusion, sirolimus-based immunosuppression can significantly reduce nontumour-related death in LT patients who exceed the criteria for transplantation. In addition, this finding will further promote the application of sirolimus after liver transplantation for hepatocellular carcinoma.

Ubiquitin-specific protease 22 promotes tumorigenesis and progression by an FKBP12/mTORC1/autophagy positive feedback loop in hepatocellular carcinoma.

Ubiquitin-specific protease 22 (USP22) has been identified as a potential marker for cancer stem cells in hepatocellular carcinoma (HCC). It can promote HCC stemness, which is considered a driver of tumorigenesis. Here, we sought to determine the role of USP22 in tumorigenesis, elucidate its underlying mechanism, and explore its therapeutic significance in HCC. As a result, we found that tissue-specific Usp22 overexpression accelerated tumorigenesis, whereas Usp22 ablation decelerated it in a c-Myc/NRasGV12-induced HCC mouse model and that the mammalian target of rapamycin complex 1 (mTORC1) pathway was activated downstream. USP22 overexpression resulted in increased tumorigenic properties that were reversed by rapamycin in vitro and in vivo. In addition, USP22 activated mTORC1 by deubiquitinating FK506-binding protein 12 (FKBP12) and activated mTORC1, in turn, further stabilizing USP22 by inhibiting autophagic degradation. Clinically, HCC patients with high USP22 expression tend to benefit from mTOR inhibitors after liver transplantation (LT). Our results revealed that USP22 promoted tumorigenesis and progression via an FKBP12/mTORC1/autophagy positive feedback loop in HCC. Clinically, USP22 may be an effective biomarker for selecting eligible recipients with HCC for anti-mTOR-based therapy after LT.

TP53/mTORC1-mediated bidirectional regulation of PD-L1 modulates immune evasion in hepatocellular carcinoma.

BACKGROUND: Immunotherapy has facilitated great breakthroughs in the treatment of hepatocellular carcinoma (HCC). However, the efficacy and response rate of immunotherapy are limited and vary among different patients with HCC. TP53 mutation substantially affects the expression of immune checkpoint molecules in multiple cancers. However, the regulatory relationship between programmed death ligand 1 (PD-L1) and TP53 is poorly studied in HCC. We aimed to elucidate the regulatory mechanism of PD-L1 in HCC with different TP53 statuses and to assess its role in modulating immune evasion in HCC. METHODS: HCC mouse models and cell lines with different TP53 statuses were constructed. PD-L1 levels were detected by PCR, western blotting and flow cytometry. RNA-seqencing, immunoprecipitation, chromatin immunoprecipitation and transmission electron microscopy were used to elucidate the regulatory mechanism in HCC with different TP53 status. HCC mouse models and patient with HCC samples were analyzed to demonstrate the preclinical and clinical significance of the findings. RESULTS: We report that loss of p53 promoted PD-L1 expression and reduced CD8+ T-cell infiltration in patient with HCC samples and mouse models. Mammalian target of rapamycin (mTOR) pathway was activated in p53-loss-of-function HCC or after knocking down TP53. The transcription factor E2F1 was found to bind to the p53 protein in TP53 wild-type HCC cells, and inhibiting mammalian target of rapamycin complex 1 (mTORC1) disrupted this binding and enhanced E2F1 translocation to the nucleus, where it bound to the PD-L1 promoter and transcriptionally upregulated PD-L1. In p53-loss-of-function HCC cells, autophagosomes were activated after mTORC1 suppression, promoting the degradation of PD-L1 protein. The combination of mTOR inhibitor and anti-PD-L1 antibody enhanced CD8+ T-cell infiltration and tumor suppression in TP53 wild-type HCC mouse models, but no benefit was observed in p53-loss-of-function HCC mouse models. In patients with TP53 wild-type HCC, PD-L1 levels were significantly higher in the high E2F1 group than in the low E2F1 group, and the low E2F1 level group had significantly superior survival. CONCLUSION: We revealed the bidirectional regulatory mechanism of PD-L1 mediated by TP53/mTORC1 in HCC. The combination of mTOR inhibitor and anti-PD-L1 antibody could be a novel precise immunotherapy scheme for TP53 wild-type HCC.

Downstaging treatment for patients with hepatocelluar carcinoma before transplantation.

Liver transplantation (LT), one of the radical methods of treating liver cancer, has brought new hope for the treatment of unresectable liver cancer. Currently, patients who meet transplant criteria can achieve a favorable prognosis, but those who exceed transplant criteria tend not to have very satisfactory outcomes. For patients whose tumor burden exceeds the transplant criteria, downstaging treatment is a promising method to reduce tumor burden to within the transplant criteria that may lead to good posttransplant survival. Multiple treatments, such as transcatheter arterial chemoembolization (TACE), transarterial radioembolization (TARE), percutaneous ethanol injection (PEI), and radiofrequency ablation (RFA), have been used as downstaging treatments. However, there are still some issues that limit the effectiveness of downstaging treatments, such as the inclusion criteria for downstaging, which the choice of downstaging treatment method, and the endpoint of downstaging, all of which are worthy of further discussion. Based on the published literature, this review discusses these issues.

Sirolimus-based immunosuppression improves the prognosis of liver Transplantation Recipients with low TSC1/2 expression in hepatocellular carcinoma beyond the Milan Criteria.

BACKGROUND: The use of the immunosuppressive agent sirolimus (SRL) following liver transplantation (LT) in patients with hepatocellular carcinoma (HCC) is controversial. Sirolimus is a typical mammalian target of rapamycin (mTOR) inhibitor, and tuberous sclerosis 1-tuberous sclerosis 2 complex (TSC1/TSC2) is an important negative effector in the mTOR pathway. In this study, we investigated the effect of SRL-based immunosuppression on the prognosis of LT recipients with HCC beyond the Milan criteria based on TSC1/2 expression and explored the effect of TSC1 on HCC in vitro and in vivo. METHODS: We retrospectively analyzed 120 HCC patients who underwent LT in our hospital between January 1, 2015 and December 30, 2018. All patients had HCC beyond the Milan criteria and were divided into the SRL group (n = 50) and non-SRL group (n = 70). TSC1/2 expression levels in paraffin-embedded tissues were determined by immunohistochemistry (IHC) and then analyzed as subgroups. Overall survival (OS) and disease-free survival (DFS) were analyzed using the Kaplan-Meier method. TSC1 expression was silenced in Huh-7 and Bel-7402 cell lines for further cell function experiments. RESULTS: 88.3% of patients were HBV LT recipients. The SRL group exhibited better DFS and OS compared to the non-SRL group (P = 0.02, P = 0.003). Subgroup (TSC1-based or TSC2-based) analyses revealed that patients with low TSC1 or TSC2 expression benefited from sirolimus (DFS: P = 0.046, OS: P = 0.006 for TSC1; DFS: P = 0.05, OS: P = 0.003 for TSC2) compared with patients with high expression. TSC1 knockdown in Huh-7 and Bel-7402 HCC cell lines activated the mTORC1 pathway and enhanced cell proliferation, migration and sensitivity to SRL in vitro and in vivo. CONCLUSION: TSC1/2 expression could be used to predict the prognosis of patients with HCC beyond the Milan criteria who underwent SRL-based immunosuppression following LT. TSC1 knockdown promoted HCC malignancy and enhanced sensitivity to SRL.

Self-Activated Cascade-Responsive Sorafenib and USP22 shRNA Co-Delivery System for Synergetic Hepatocellular Carcinoma Therapy.

Resistance to sorafenib severely hinders its effectiveness against hepatocellular carcinoma (HCC). Cancer stemness is closely connected with resistance to sorafenib. Methods for reversing the cancer stemness remains one of the largest concerns in research and the lack of such methods obstructs current HCC therapeutics. Ubiquitin-specific protease 22 (USP22) is reported to play a pivotal role in HCC stemness and multidrug resistance (MDR). Herein, a galactose-decorated lipopolyplex (Gal-SLP) is developed as an HCC-targeting self-activated cascade-responsive nanoplatform to co-delivery sorafenib and USP22 shRNA (shUSP22) for synergetic HCC therapy. Sorafenib, entrapped in the Gal-SLPs, induced a reactive oxygen species (ROS) cascade and triggered rapid shUSP22 release. Thus, Gal-SLPs dramatically suppressed the expression of USP22. The downregulation of USP22 suppresses multidrug resistance-associated protein 1 (MRP1) to induce intracellular sorafenib accumulation and hampers glycolysis of HCC cells. As a result, Gal-SLPs efficiently inhibit the viability, proliferation, and colony formation of HCC cells. A sorafenib-insensitive patient-derived xenograft (PDX) model is established and adopted to evaluate in vivo antitumor effect of Gal-SLPs. Gal-SLPs exhibit potent antitumor efficiency and biosafety. Therefore, Gal-SLPs are expected to have great potential in the clinical treatment of HCC.

Sirolimus-based immunosuppression improves outcomes in liver transplantation recipients with hepatocellular carcinoma beyond the Hangzhou criteria.

BACKGROUND: The administration of calcineurin inhibitors (CNIs) posttransplant has been implicated as an independent risk factor for the recurrence of hepatocellular carcinoma (HCC) after liver transplantation (LT). The new immunosuppressive agent sirolimus (SRL) acts as a primary immunosuppressant or antitumor agent. In this study we investigated the effect of sirolimus-based immunosuppression compared to CNIs (non-SRL) on the outcomes of LT candidates with HCC. METHODS: We retrospectively analyzed 204 HCC patients who underwent LT in our hospital between January 2, 2014 and December 10, 2017. The median of the follow-up duration of patients was 24.5 months. The patients were divided into a sirolimus (SRL) group (76 patients) and a non-sirolimus (non-SRL) group (128 patients). Patients exceeding the LT criteria were analyzed as subgroups. Disease-free survival (DFS) and overall survival (OS) after tumor recurrence were compared using the Kaplan-Meier method. Univariate and multivariate Cox analyses were used to compare OS between the SRL and non-SRL groups. RESULTS: The SRL group achieved better OS compared to the non-SRL group, while there was no significant difference in DFS. Subgroup (Milan criteria-based or Hangzhou criteria-based) analyses revealed that patients exceeding, rather than meeting, the Milan or Hangzhou criteria benefited from SRL (exceeding the Milan criteria: P=0.002; exceeding the Hangzhou criteria: P<0.001). There was no significant difference in OS between the SRL group and the non-SRL group that met the Milan or Hangzhou criteria. CONCLUSIONS: SRL can improve survival outcomes in LT patients with HCC exceeding the Hangzhou criteria.