Relapse Of Hepatocellular Carcinoma Research Articles

Long-term outcome of adjuvant radiotherapy upon postoperative relapse of centrally located hepatocellular carcinoma: a real-world study

Despite that surgical resection is widely regarded as the most effective approach to the treatment of liver cancer, its safety and efficacy upon centrally located hepatocellular carcinoma (HCC) remain unsatisfactory. In consequence, seeking an integrated treatment, like combined with adjuvant radiotherapy, to enhance the prognosis of patients is of critical importance. By recruiting patients undergoing surgical resection for centrally located HCC ranging from June 2015 to 2020, they were divided into liver resection combined with adjuvant radiotherapy (LR + RT) and mere liver resection (LR) groups. The calculation of propensity score and model of Cox proportional hazards regression were utilized. 193 patients were recruited in aggregation, containing 88 ones undergoing LR + RT, while 105 handled with LR. RT was verified to be an independent factor of prognosis for relapse (HR 0.60). In propensity-score analyses, significant association existed between adjuvant radiotherapy and better disease-free survival (DFS) (Matched, HR 0.60; Adjustment of propensity score, HR 0.60; Inverse probability weighting, HR 0.63). The difference of DFS was apparent within two groups (p value = 0.022), and RT significantly down-regulated early relapse (p value < 0.05) in subgroup analysis. The calculation of E-value revealed robustness of unmeasured confounding. The combination of liver surgical resection with RT is safe and effective towards patients with centrally located HCC, which would notably enhance the prognosis and decrease the early relapse of HCC.

Coordinated activation of DNMT3a and TET2 in cancer stem cell-like cells initiates and sustains drug resistance in hepatocellular carcinoma

BackgroundResistance to targeted therapies represents a significant hurdle to successfully treating hepatocellular carcinoma (HCC). While epigenetic abnormalities are critical determinants of HCC relapse and therapeutic resistance, the underlying mechanisms are poorly understood. We aimed to address whether and how dysregulated epigenetic regulators have regulatory and functional communications in establishing and maintaining drug resistance.MethodsHCC-resistant cells were characterized by CCK-8, IncuCyte Live-Cell analysis, flow cytometry and wound-healing assays. Target expression was assessed by qPCR and Western blotting. Global and promoter DNA methylation was measured by dotblotting, methylated-DNA immunoprecipitation and enzymatic digestion. Protein interaction and promoter binding of DNMT3a-TET2 were investigated by co-immunoprecipitation, ChIP-qPCR. The regulatory and functional roles of DNMT3a and TET2 were studied by lentivirus infection and puromycin selection. The association of DNMT and TET expression with drug response and survival of HCC patients was assessed by public datasets, spearman correlation coefficients and online tools.ResultsWe identified the coordination of DNMT3a and TET2 as an actionable mechanism of drug resistance in HCC. The faster growth and migration of resistant HCC cells were attributed to DNMT3a and TET2 upregulation followed by increased 5mC and 5hmC production. HCC patients with higher DNMT3a and TET2 had a shorter survival time with a less favorable response to sorafenib therapy than those with lower expression. Cancer stem cell-like cells (CSCs) displayed DNMT3a and TET2 overexpression, which were insensitive to sorafenib. Either genetic or pharmacological suppression of DNMT3a or/and TET2 impaired resistant cell growth and oncosphere formation, and restored sorafenib sensitivity. Mechanistically, DNMT3a did not establish a regulatory circuit with TET2, but formed a complex with TET2 and HDAC2. This complex bound the promoters of oncogenes (i.e., CDK1, CCNA2, RASEF), and upregulated them without involving promoter DNA methylation. In contrast, DNMT3a-TET2 crosstalk silences tumor suppressors (i.e., P15, SOCS2) through a corepressor complex with HDAC2 along with increased promoter DNA methylation.ConclusionsWe demonstrate that DNMT3a and TET2 act coordinately to regulate HCC cell fate in DNA methylation-dependent and -independent manners, representing strong predictors for drug resistance and poor prognosis, and thus are promising therapeutic targets for refractory HCC.

Targeting of CD25+ Activated T Cells in Gvhd-Associated Marrow Suppression and Cytopenias

Introduction: Secondary cytopenias occurring after hematopoietic cell transplant (HCT) are an observed complication of graft-versus-host disease (GVHD). Causes include peripheral immune mediated destruction (hemolytic anemia or thrombocytopenia), usually associated with autoantibody production, and peripheral destruction associated with microangiopathy (TMA). However, cases have been observed where marrow production of the affected lineage is abnormal. Immune-mediated cases are treated with increased immunosuppression, e.g., steroids or calcineurin inhibitors (CNI), and TMA with complement inhibition. To better understand the mechanisms and treatment of hypoproductive cytopenias in GVHD, we combined analyses of hematopoietic stem and progenitor cells (HSPC) and peripheral blood and marrow activated T cells. We describe 3 cases of GVHD-associated cytopenias marked by HSPC depletion and presence of CD25 (IL-2R alpha subunit)-positive activated T cells in the marrow that responded to combinations of immunosuppression containing basiliximab (basil) anti-CD25 monoclonal antibody blockade. Methods/Results: We describe combined HSPC and T cell analyses in 3 patients (P) with severe GVHD-associated cytopenias. P1 was at 5 years post-HCT for acute lymphoblastic leukemia, P2 at 2 years post-HCT for severe aplastic anemia (SAA), and P3 at 1.5 months post-liver transplant for alcohol use disorder-associated cirrhosis and hepatocellular carcinoma ( Table 1). Because of refractory cytopenias, all were being considered for HCT. However, marrow analyses revealed that each had normal frequencies of phenotypic HSC, with variable decreases in cellularity and frequencies of committed progenitors ( Table 2). Chimerism analyses showed that 100% of CD34+ cells in P1 and P2 were of donor origin, and in P3 of host origin. All 3 marrows had activated CD3+ T cells per expression of CD69 and/or CD38, and frequency of CD25 on &amp;gt;10% of T cells. In addition, P1 had evidence of IgG bound to committed progenitor populations. The treatment strategy was to block IL-2 signaling with basil (induction of 20 mg on day 1, 4, 7, 14, 21, 28, followed by alternate week and then monthly dosing for 2-9 months), combined with suppression of IL-2 production with steroid and/or CNI. After induction, repeat analyses of the blood and marrow T-cells confirmed complete blockade of CD25. Therapy was also individualized for specific clinical conditions or as noted on marrow evaluation. P1 received prednisone, CNI, and rituximab for a presumed anti-HSPC autoantibody. At 9 months he was weaned off all therapy and remains on no immunosuppressive treatment 2 years later. P2 did not receive CNI due to chronic renal failure but received steroids and later sirolimus. At ~18 months, his cytopenias recurred, but subsequently responded to basil re-induction and steroids. P3 has received prednisone, CNI, and sirolimus indefinitely for prevention of liver rejection and/or hepatocellular carcinoma relapse. P1 had no viral reactivation; P2 had CMV pneumonia with his second course responsive to antiviral therapy, completion of basil, and tapering of steroids; and P3 had CMV reactivation responsive to antiviral therapy and EBV viremia. P3 has a recent diagnosis of EBV+ intrahepatic B cell lymphoma. In all cases, blood counts normalized except P2 who has anemia due to CKD. Discussion: HSPC analyses in these patients with GVHD and cytopenias demonstrate that peripheral changes are not due to loss of HSC, but instead to quantitative and/or qualitative defects in committed progenitor populations, likely mediated by activated CD25+ T cells. In addition, the presence of IgG on HSPCs in P1 demonstrates a novel role for antibody-mediated humoral suppression in some cases. The presence of HSC indicates that there may be no need for re-transplant. Previous studies of SAA have shown activated T cells to be drivers of aplasia and is the basis for immune suppressive therapy (anti-thymocyte globulin and CNI). Our analyses of patients with GVHD-associated cytopenias demonstrates that targeting of IL-2 signaling via combined CD25 receptor blockade and pharmacologic inhibition of IL-2 production can correct the marrow defects and cytopenias, albeit with need for attention to viral infections. Targeting CD25 in patients with evidence of CD25+ activated T cells is a rational approach to the difficult problem of GVHD-associated marrow suppression and cytopenias.

SETD1A drives stemness by reprograming epigenetic landscape in hepatocellular carcinoma stem cells.

Cancer stem cells (CSCs) are responsible for tumor progression and recurrence. However, the mechanisms regulating hepatocellular carcinoma (HCC) stemness remain unclear. Applying a genome-scale CRISPR knockout screen, we identify that the H3K4 methyltransferase SETD1A and other members of Trithorax group proteins drive cancer stemness in HCC. SETD1A is positively correlated with poor clinical outcome in HCC patients. Combination of SETD1A and serum AFP significantly improves the accuracy of predicting HCC relapse. Mechanistically, SETD1A mediates transcriptional activation of various histone-modifying enzymes, facilitates deposition of H3K4me3 and H3K27me3 and activates oncogenic enhancers and super-enhancers, leading to activation of oncogenes and inactivation of tumor suppressor genes simultaneously in HCC CSCs. In addition, SETD1A cooperates with PABPC1 to regulate H3K4me3 modification on oncogenes. Our data pinpoint SETD1A as a key epigenetic regulator driving HCC stemness and progression, highlighting the potential of SETD1A as a candidate target for HCC intervention and therapy.

RNA-Seq and Immune Repertoire Analysis of Normal and Hepatocellular Carcinoma Relapse After Liver Transplantation.

Hepatocellular carcinoma (HCC) relapse is the main reason for the poor prognosis of HCC after Liver transplantation (LT). This study aimed to explore the molecular mechanisms and immune repertoire profiles of HCC relapse. RNA-seq of blood samples from patients with normal (n=12) and HCC relapse (n=6) after LT was performed to identify differentially expressed genes (DEGs) and key signalling pathways. The DEGs and immune genes were further analyzed by bioinformatics. TRUST4 was used to analyze the differences in the immune repertoire between the two groups. Another 11 blood samples from patients with HCC who had received LT were collected for RT-qPCR verification of key genes. A total of 131 upregulated and 157 downregulated genes were identified using RNA-seq, and GO enrichment analysis revealed that the top 15 pathways were immune-related. The PPI network identified 10 key genes. Immune infiltration analysis revealed a significant difference in the five immune cell types between the two groups. A total of 83 intersecting genes were obtained by intersecting DEGs and immune genes. 6 key genes, including MX1, ISG15, OAS1, PRF1, SPP1, and THBS1 were obtained according to the intersection of DEGs, PPI network top 10 genes and immune intersecting genes. Immune repertoire analysis showed that the usage frequency of variable (V) and joining (J) genes in the normal group was higher than that in the relapse group. RT-qPCR validation showed that the expression levels of key genes were consistent with the RNA-seq results. Our study identified key pathways and genes that could help determine whether transplant recipients are more prone to HCC relapse. Immune repertoire analysis revealed a difference in the usage frequency of VJ genes between the normal and relapse groups, providing a research direction for immunotherapy in patients with HCC relapse after liver transplantation.

Whole-exome sequencing reveals the metastatic potential of hepatocellular carcinoma from the perspective of tumor and circulating tumor DNA.

Relapse of hepatocellular carcinoma (HCC) due to vascular invasion is common, but the genomic mechanisms remain unclear, and molecular determinants of high-risk relapse cases are lacking. We aimed to reveal the evolutionary trajectory of microvascular invasion (MVI) and develop a predictive signature for relapse in HCC. Whole-exome sequencing was performed on tumor and peritumor tissues, portal vein tumor thrombus (PVTT), and circulating tumor DNA (ctDNA) to compare the genomic profiles between 5 HCC patients with MVI and 5 patients without MVI. We conducted an integrated analysis of exome and transcriptome to develop and validate a prognostic signature in two public cohorts and one cohort from Zhongshan Hospital, Fudan University. Shared genomic landscapes and identical clonal origins among tumor, PVTT, and ctDNA were observed in MVI ( +) HCC, suggesting that genomic changes favoring metastasis occur at the primary tumor stage and are inherited in metastatic lesions and ctDNA. There was no clonal relatedness between the primary tumor and ctDNA in MVI ( - ) HCC. HCC had dynamic mutation alterations during MVI and exhibited genetic heterogeneity between primary and metastatic tumors, which can be comprehensively reflected by ctDNA. A relapse-related gene signature named RGSHCC was developed based on the significantly mutated genes associated with MVI and shown to be a robust classifier of HCC relapse. We characterized the genomic alterations during HCC vascular invasion and revealed a previously undescribed evolution pattern of ctDNA in HCC. A novel multiomics-based signature was developed to identify high-risk relapse populations.

Efficacy and prognostic factors of repeated hepatectomy for postoperative intrahepatic recurrence of hepatocellular carcinoma undergoing initial hepatectomy.

Postoperative recurrence of hepatocellular carcinoma (HCC) is associated with low survival rates. While HCC treatment options have expanded substantially, they are accompanied by several challenges. This study assessed the outcomes of repeated hepatectomy (RH) for postoperative intrahepatic recurrence of HCC among patients undergoing initial hepatectomy (IH) as well as independent risk factors for HCC recurrence among patients undergoing repeated hepatectomy (RH). Clinical data from 84 patients undergoing both IH and RH and 66 recurrent HCC patients who had received radiofrequency ablation (RFA) from July 2011 to September 2017 were retrospectively reviewed. The following groups were compared: (1) RH Group A (n = 84), (2) IH Group (n = 84, same individuals as RH Group A), (3) RH Group B (n = 45/84 from RH Group A), and (4) RFA Group (n = 66). The clinical pathology and operative characteristics of the patients in RH Group A were compared to those in the IH Group. Meanwhile, the clinical pathology and pre- and post-treatment features of the patients in RH Group B were compared to those in the RFA Group. The tumor-free survival time was compared between patients in RH Group A and the IH Group as well as between patients in RH Group B and the RFA Group. The independent risk factors for the 1-year postoperative tumor-free survival of RH Group A patients were investigated using univariate and multivariate analysis. Measures of clinical pathology, including AFP, Child-Pugh score, HBV-DNA, tumor number, liver cirrhosis, tumor differentiation, surgical approach, and TNM stage differed significantly between patients in RH Group A and the IH Group (all P < 0.05), with the exception of tumor number and tumor size (both P > 0.05). No significant differences were found in these measures between the patients in RH Group B and the RFA Group (all P > 0.05). While patients in the RH Group A had a longer operation time than those in the IH Group (4.35 ± 1.25 h vs. 3.55 ± 0.92 h, P < 0.001), the level of intraoperative bleeding was similar (400.00 ± 199.25 ml vs. 359.40 ± 213.37 ml, P = 0.204). RH Group B patients had a longer hospitalization time than those in the RFA Group (6.5 ± 0.8 d vs. 5.5 ± 1.1 d, P < 0.001), however, the difference in hospitalization costs was not statistically significant (29,009 ± 3,806 CNY vs. 29,944 ± 3,752 CNY, P = 0.202). Five-day post-operative serum biomarker levels, including direct bilirubin (DB) and albumin (ALB), were significantly higher in RH Group B than in the RFA Group (all P < 0.05), with the exception of ALT, AST, and total bilirubin (TB) (all P > 0.05). Patients in RH Group A had a lower tumor-free survival time than those in the IH Group (median: 12 vs. 22 months, P < 0.001), and patients in the RH Group B had a significantly higher tumor-free survival time than those in the RFA group (median: 15 months vs. 8 months, P < 0.001). Age ≥50 y, Child-Pugh class A, and negative HBV-DNA were independent risk factors that positively impacted the 1-year postoperative tumor-free survival rate of postoperative intrahepatic recurrent HCC patients undergoing RH (P < 0.001, respectively). Due to the potential of harm related to relapse of recurrent HCC for cancer patients, RH is a superior option. RH could offer better outcomes for recurrent HCC patients undergoing IH. Compared with lesion pathology, the better target organ of the liver will be key to ameliorating tumor-free survival for recurrent HCC patients undergoing RH.

Mitochondrial ROS driven by NOX4 upregulation promotes hepatocellular carcinoma cell survival after incomplete radiofrequency ablation by inducing of mitophagy via Nrf2/PINK1

BackgroundThe recurrence of hepatocellular carcinoma (HCC) after radiofrequency ablation (RFA) remains a major clinical problem. Cells that survive the sublethal heat stress that is induced by incomplete RFA are the main source of HCC relapse. Heat stress has long been reported to increase intracellular reactive oxygen species (ROS) generation. Although ROS can induce apoptosis, a pro-survival effect of ROS has also been demonstrated. However, the role of ROS in HCC cells exposed to sublethal heat stress remains unclear.MethodsHepG2 and HuH7 cells were used for this experiment. Insufficient RFA was performed in cells and in a xenograft model. ROS and antioxidant levels were measured. Apoptosis was analyed by Annexin-V/PI staining and flow cytometry. Protein expression was measured using western blotting. Colocalization of lysosomes and mitochondria was analyzed to assess mitophagy. Corresponding activators or inhibitors were applied to verify the function of specific objectives.ResultsHere,we showed that sublethal heat stress induced a ROS burst, which caused acute oxidative stress. This ROS burst was generated by mitochondria, and it was initiated by upregulated NOX4 expression in the mitochondria. n-acetylcysteine (NAC) decreased HCC cell survival under sublethal heat stress conditions in vivo and in vitro. NOX4 triggers the production of mitochondrial ROS (mtROS), and NOX4 inhibitors or siNOX4 also decreased HCC cell survival under sublethal heat stress conditions in vitro. Increased mtROS trigger PINK1-dependent mitophagy to eliminate the mitochondria that are damaged by sublethal heat stress and to protect cells from apoptosis. Nrf2 expression was elevated in response to this ROS burst and mediated the ROS burst-induced increase in PINK1 expression after sublethal heat stress.ConclusionThese data confirmed that the ROS burst that occurs after iRFA exerted a pro-survival effect. NOX4 increased the generation of ROS by mitochondria. This short-term ROS burst induced PINK1-dependent mitophagy to eliminate damaged mitochondria by increasing Nrf2 expression.

Multifunctional immunotherapeutic gel prevented postoperative recurrence of hepatocellular carcinoma

Postoperative recurrence of hepatocellular carcinoma (HCC) remains an important cause of treatment failure. To mitigate the relapse of HCC, we have constructed an immunotherapeutic in situ fibrin gel to stimulate antitumor immune responses following HCC surgical resection. The gel consists of hollow mesoporous Prussian blue nanoparticles (HMPB) co-loaded with anti-SIRPα antibodies and the colony-stimulating factor 1 receptor (CSF-1R) small molecule inhibitor BLZ945, and differentiation inducer all-trans retinoic acid (ATRA). After the gel is formed in the surgical cavity, HMPB are released locally and then BLZ945 and anti-SIRPα antibody block the MCSF-CSF-1R and CD47-SIRPα signal axes respectively, which can repolarize tumor-associated macrophages (TAMs) from a tumorigenic M2 phenotype to an antitumor M1 phenotype, thus restoring their ability to kill cancer cells. In addition, ATRA released from the gel leads to the differentiation of cancer stem cells (CSCs) and increases their sensitivity to immunotherapy. Our findings indicate that this in situ immunotherapeutic gel can reshape the postoperative tumor microenvironment (TME) and induce antitumor responses, thereby inhibiting postoperative tumor recurrence.

Reprograming immune microenvironment modulates CD47 cancer stem cells in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common type of liver cancer. There are different therapeutic approaches to cancer eradication such as chemotherapy, radiotherapy, and surgery. The fuel for treatment resistance is heterogeneity, which also has significant effects on cancer therapies and biomarker research. One of the most common causes of cancer chemoresistance and relapse is the presence and percentage of cancer stem cells (CSCs), among them CD47+ CSCs. Besides, the change in the tumor microenvironment (TME) stands as one of the main factors for the failure of chemotherapeutic protocols. The current review aims to focus on how the change in immune mediators such as TGF-β, IL-10, IL-17, IDO, Gal-1, PD-L1, and CTLA-4 affect CD47+ CSCs in HCC and thus open a new era towards developing new approaches for prevention of HCC relapse and stemness through different immune modulation approaches. Then we investigate some drugs that have a dual effect on both TME and CD47 CSCs and thus the best choice in comorbidities.

Prognostic Value of Repeat Hepatectomy for Hepatocellular Carcinoma Patients

Background/Aim: The potential for repeat hepatectomy has been increasing in hepatocellular carcinoma (HCC) patients. Patients and Methods: To assess its significance, we examined the clinical features, surgical records, and survival of 279 HCC patients who underwent hepatectomy as primary treatment. Patients were divided into three groups: single hepatectomy without HCC relapse in 97 patients (Group 1), single hepatectomy with HCC relapse in 133 patients (Group 2) and repeat hepatectomy for HCC relapse in 49 patients (Group 3). Group 3 was divided into three subgroups. Multiple, larger size (&gt;5 cm), and confluent nodular HCC. Results: Child–Pugh B patients were significantly higher in Group 2 (p&lt;0.01). The prevalence of histological vascular involvement was significantly higher in Group 2 than in the other groups (p&lt;0.01). Although Group 2 showed significantly poorer survival (p&lt;0.01), the 10-year overall survival in Groups 1 and 3 was over 70% but not different between groups. The relapse-free survival period was significantly correlated with overall survival, and patients with a cancer-free period of over 36 months showed significantly better 10-year survival rates than those with a period of less than 36 months (p&lt;0.01). Patients undergoing 3 or more repeat hepatectomies also showed good survival periods. Conclusion: Careful follow-up and proper decision-making in terms of repeat hepatectomy with multimodal treatments including novel chemotherapies upon assessment of postoperative tumor relapse are important in HCC patients to prolong survival.

Preoperative Alpha-Fetoprotein and Radiological Total Tumor Diameter as Predictors of Hepatocellular Carcinoma Recurrence After Liver Transplantation

Liver transplantation is a unique treatment opportunity for patients with chronic liver disease and hepatocellular carcinoma (HCC). Selection of HCC patients for transplantation was revolutionized by Milan-based criteria, but tumor recurrence and shortage of organs are still a major concern. Nowadays, additional preoperative tumor parameters can help to refine the graft allocation process. The objective of this study was to evaluate the prognostic value and cut-off points of pretransplant serum alpha-fetoprotein (AFP) levels and radiological tumor parameters on liver transplantation outcomes. This is a single-team retrospective cohort of 162 consecutive deceased donor liver transplants (DDLT) with pathologically confirmed HCC. Pretransplant serum AFP levels and radiological tumor parameters were retrieved from a preoperative follow-up. Receiver-operating characteristics (ROC) curves were used to evaluate cut-off points for each outcome. Multivariate Cox regression model was used to assess the predictors of HCC relapse and recipient mortality. Twelve recipients (7.4%) had HCC recurrence after transplantation, with median survival time of 5.8 months. Pretransplant AFP ≥30 ng/mL (hazard ratio [HR]: 13.84, P=.003) and radiological total tumor diameter (TTD) ≥5 cm (HR: 12.89, P=.005) were independent predictors for HCC relapse. Moreover, pretransplant AFP ≥150 ng/mL was independently associated with recipient mortality (HR: 4.45, P=.003). Pretransplant AFP levels and radiological TTD were independently associated with HCC relapse and recipient mortality after DDLT, with different cut-off points predicting different outcomes. These findings may contribute to improving decision-making in the context of liver transplantation for HCC patients.

Reducing Postoperative Recurrence of Early‐Stage Hepatocellular Carcinoma by a Wound‐Targeted Nanodrug

New strategies to decrease risk of relapse after surgery are needed for improving 5‐year survival rate of hepatocellular carcinoma (HCC). To address this need, a wound‐targeted nanodrug is developed, that contains an immune checkpoint inhibitor (anti‐PD‐L1)and an angiogenesis inhibitor (sorafenib)). These nanoparticles consist of highly biocompatible mesoporous silica (MSNP) that is surface‐coated with platelet membrane (PM) to achieve surgical site targeting in a self‐amplified accumulation manner. Sorafenib is introduced into the MSNP pores while covalently attaching anti‐PD‐L1 antibody on the PM surface. The resulting nano‐formulation, abbreviated as a‐PM‐S‐MSNP, can effectively target the surgical margin when intraperitoneally (IP) administered into an immune competent murine orthotopic HCC model. Multiple administrations of a‐PM‐S‐MSNP generate potent anti‐HCC effect and significantly prolong overall mice survival. Immunophenotyping and immunochemistry staining reveal the signatures of favorable anti‐HCC immunity and anti‐angiogenesis effect at tumor sites. More importantly, microscopic inspection of a‐PM‐S‐MSNP treated mice shows that 2 out 6 are histologically tumor‐free, which is in sharp contrast to the control mice where tumor foci can be easily identified. The data suggest that a‐PM‐S‐MSNP can efficiently inhibit post‐surgical HCC relapse without obvious side effects and holds considerable promise for clinical translation as a novel nanodrug.

The monocyte to lymphocyte ratio not only at baseline but also at relapse predicts poor outcomes in patients with hepatocellular carcinoma receiving locoregional therapy

BackgroundMonocyte to lymphocyte ratio (MLR) represents a pro-inflammatory immune microenvironment. The aim of this study was to elucidate the effect of MLR and subsequent MLR when relapse occurred (R-MLR) on prognosis for hepatocellular carcinoma (HCC) after transcatheter arterial chemoembolization (TACE) combined with ablation.MethodsA prospective analysis was conducted on 606 patients with HCC who were treated with TACE combined with local ablation in Beijing You’an Hospital affiliated to Capital Medical University from January 1, 2012 to December 31, 2016. MLR or R-MLR were stratified according to the optimal cut-off values. The cumulative recurrence-free survival (RFS), overall survival (OS) , and recurrence-death survival (RDS) rates were calculated by Kaplan–Meier method. The Cox proportion hazard model and logistic regression analysis was conducted to screen for independent predictive factors for indicating early relapse and long-term prognosis.ResultsHigh MLR was significantly associated with relapse, early recurrence, and overall survival. After a median follow-up of 59.4 months, The cumulative 1-, 3-, 5-year RFS rates of low MLR were 74.6%, 43.8%, and 34.0%; while 66.1%, 32.2%, and 22.6% for high group (P < 0.001). There were also significant differences in corresponding OS rates of the two groups (P = 0.003). The cumulative 1-, 3-, 5-year OS rates of low R-MLR were 99.5%, 87.2%, 75.5%; while 98.3%, 78.3%, 61.7% for high group (P < 0.001). There were also significant differences in corresponding RDS rates in the two groups (P = 0.008). 436 patients were divided into four groups on the base of cut-off values of MLR and R-MLR (low-low, low–high, high-low, and high-high). The low-low group has shown better outcomes including the cumulative 1-, 3-, 5-year OS, and RDS rates(P < 0.001).ConclusionsHigh MLR was related to unfavorable outcome. Subsequent change of MLR between baseline and HCC relapse could indicate poor long-term survival after relapse.

Epigenetic regulation of ferroptosis via ETS1/miR-23a-3p/ACSL4 axis mediates sorafenib resistance in human hepatocellular carcinoma

BackgroundDrug resistance to sorafenib greatly limited the benefits of treatment in patients with hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) participate in the development of drug resistance. The key miRNA regulators related to the clinical outcome of sorafenib treatment and their molecular mechanisms remain to be identified.MethodsThe clinical significance of miRNA-related epigenetic changes in sorafenib-resistant HCC was evaluated by analyzing publicly available databases and in-house human HCC tissues. The biological functions of miR-23a-3p were investigated both in vitro and in vivo. Proteomics and bioinformatics analyses were conducted to identify the mechanisms that regulating miR-23a-3p. Luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay were used to validate the binding relationship of miR-23a-3p and its targets.ResultsWe found that miR-23a-3p was the most prominent miRNA in HCC, which was overexpressed in sorafenib non-responders and indicated poor survival and HCC relapse. Sorafenib-resistant cells exhibited increased miR-23a-3p transcription in an ETS Proto-Oncogene 1 (ETS1)-dependent manner. CRISPR-Cas9 knockout of miR-23a-3p improved sorafenib response in HCC cells as well as orthotopic HCC tumours. Proteomics analysis suggested that sorafenib-induced ferroptosis was the key pathway suppressed by miR-23a-3p with reduced cellular iron accumulation and lipid peroxidation. MiR-23a-3p directly targeted the 3′-untranslated regions (UTR) of ACSL4, the key positive regulator of ferroptosis. The miR-23a-3p inhibitor rescued ACSL4 expression and induced ferrotoptic cell death in sorafenib-treated HCC cells. The co-delivery of ACSL4 siRNA and miR-23a-3p inhibitor abolished sorafenib response.ConclusionOur study demonstrates that ETS1/miR-23a-3p/ACSL4 axis contributes to sorafenib resistance in HCC through regulating ferroptosis. Our findings suggest that miR-23a-3p could be a potential target to improve sorafenib responsiveness in HCC patients.

Gemcitabine-facilitated modulation of the tumor microenvironment and PD-1/PD-L1 blockade generate a synergistic antitumor effect in a murine hepatocellular carcinoma model

BackgroundGemcitabine can alter the immunogenic microenvironments, and the effect of gemcitabine plus programmed death-1 (PD-1)/ programmed death-ligand 1 (PD-L1) blockade in hepatocellular carcinoma (HCC) is investigated. MethodsSubcutaneous H22-green fluorescent protein (GFP) cells inoculation model was constructed and treated with gemcitabine, anti-PD-1 antibody (αPD-1), or the combination every four days when the tumor volumes reached about 50 mm3. Four days after the final treatment, primary tumor tissues were resected and dissociated, which were further subcutaneously injected on the contralateral side to construct the HCC relapse model. The infiltrated proportion of immune cells and PD-1 expression were quantified by flow cytometry. The relative content of transforming growth factor (TGF)-β, interleukin (IL)-12p70, and interferon (IFN)-γ were detected by the enzyme-linked immunosorbent assay (ELISA). Tumor volume and the number of tumor-free mice were evaluated. ResultsGemcitabine treatment can effectively increase the total proportion of infiltrating immune cells, reduce the proportion of myeloid-derived suppressor cells (MDSCs) and macrophages, and increase T cells proportion without significant growth inhibition. While after gemcitabine treatment, PD-L1 expression on tumor cells and PD-1 on T cells were significantly up-regulated. Subcutaneous tumors volume were reduced considerably after gemcitabine plus αPD-1 treatment compared with gemcitabine (P<0.01) or αPD-1 monotherapy (P<0.001) with the increased proportion of IL-2+CD8+T, CD8+T central memory cells (TCM), CD4 TCM, up-regulated IL12p70 and IFN-γ secretion, and down-regulated TGF-β. Gemcitabine plus αPD-1 blockade could inhibit the relapse tumor model as indicated with down-regulated tumor volume and increased number of tumor-free mice. ConclusionGemcitabine up-regulates the proportion of intratumor CD8+T and the relative expression of PD-1/PD-L1, and the combination of PD-1/PD-L1 blockade can further inhibit the growth and the relapse of HCC.

Finding the seed of recurrence: Hepatocellular carcinoma circulating tumor cells and their potential to drive the surgical treatment.

The treatment for hepatocellular carcinoma (HCC) relies on liver resection, which is, however, burdened by a high rate of recurrence after surgery, up to 60% at 5 years. No pre-operative tools are currently available to assess the recurrence risk tailored to every single patient. Recently liquid biopsy has shown interesting results in diagnosis, prognosis and treatment allocation strategies in other types of cancers, since its ability to identify circulating tumor cells (CTCs) derived from the primary tumor. Those cells were advocated to be responsible for the majority of cases of recurrence and cancer-related deaths for HCC. In fact, after being modified by the epithelial-mesenchymal transition, CTCs circulate as “seeds” in peripheral blood, then reach the target organ as dormant cells which could be subsequently “awakened” and activated, and then initiate metastasis. Their presence may justify the disagreement registered in terms of efficacy of anatomic vs non-anatomic resections, particularly in the case of microvascular invasion, which has been recently pointed as a histological sign of the spread of those cells. Thus, their presence, also in the early stages, may justify the recurrence event also in the contest of liver transplant. Understanding the mechanism behind the tumor progression may allow improving the treatment selection according to the biological patient-based characteristics. Moreover, it may drive the development of novel biological tailored tests which could address a specific patient to neoadjuvant or adjuvant strategies, and in perspective, it could also become a new method to allocate organs for transplantation, according to the risk of relapse after liver transplant. The present paper will describe the most recent evidence on the role of CTCs in determining the relapse of HCC, highlighting their potential clinical implication as novel tumor behavior biomarkers able to influence the surgical choice.

A Co-Expression Network Reveals the Potential Regulatory Mechanism of lncRNAs in Relapsed Hepatocellular Carcinoma

BackgroundThe mechanistic basis for relapsed hepatocellular carcinoma (HCC) remains poorly understood. Recent research has highlighted the important roles of long non-coding RNAs (lncRNAs) in HCC. However, there are only a few studies on the association between lncRNAs and HCC relapse.MethodsDifferentially expressed lncRNAs and mRNAs between a primary HCC group and relapsed HCC group were identified using the edge R package to analyze the GSE101432 dataset. The differentially expressed lncRNAs and mRNAs were used to construct a lncRNA–mRNA co-expression network. Weighted gene co-expression network analysis followed by Gene Ontology (GO) enrichment analyses were conducted on the database. Furthermore, correlation and survival analyses were performed using The Cancer Genome Atlas database, and expression in the clinical samples was verified by qRT-PCR. Thereafter, we inputted the genes from the two groups into the HCC TNM stage and tumor grade database from TCGA. Finally, we performed Kaplan–Meier survival analysis on the lncRNAs related to relapsed HCC.ResultsIn this study, lncRNAs and mRNAs associated with HCC relapse were identified. Two gene modules were found to be closely linked to this. The GO terms in the yellow and black modules were related to cell proliferation, differentiation, and survival, as well as some transcription-related biological processes. Through qRT-PCR, we found that the expression levels of LINC00941 and LINC00668 in relapsed HCC were higher than those in primary HCC. Further, mRNA levels of LOX, OTX1, MICB, NDUFA4L2, BAIAP2L2, and KCTD17 were changed in relapsed HCC compared to levels in primary HCC. In addition, we verified that these genes could predict the overall survival and recurrence-free survival of HCC. Moreover, we found that LINC00668 and LINC00941 could affect tumor grade and TNM stages. In total, we identified and validated two lncRNAs (LINC00941 and LINC00668) and six mRNAs (LOX, MICB, OTX1, BAIAP2L2, KCTD17, NDUFA4L2) associated with HCC relapse.ConclusionIn summary, we identified the key gene modules and central genes associated with relapsed HCC and constructed lncRNA–mRNA networks related to this. These genes are likely to have potential prognostic value for relapsed HCC and might shed new light on novel biomarkers or diagnostic targets for relapsed HCC.

Recurrence of Hepatocellular Carcinoma After Liver Transplantation is Associated with Episodes of Acute Rejections.

PurposeThe impact of acute rejection (AR) after liver transplantation (LT) for hepatocellular carcinoma (HCC) on patient outcome is uncertain. This aim of this study is to investigate whether AR is associated with HCC relapse and overall survival.Patients and MethodsPatients undergoing LT for HCC between 2001 and 2015 were retrospectively analyzed with regard to histopathological proven AR within the median time until recurrence. Cox’s regression analysis was conducted revealing risk factors for HCC recurrence.ResultsHCC recurred in 47 of 252 analyzed patients with a median time to recurrence of 20 months. Patients with AR (28.6%) had a significantly higher frequency of recurrence compared to patients without AR (13.0%, p=0.002). Multiple Cox regression analyses identified AR within 20 months to be an independent risk factor for HCC recurrence both as dichotomized (HR=2.91, 95%CI: 1.30–6.53; p=0.009) and as a continuous variable (HR=1.81, 95%CI: 1.28–2.54; p=0.001). HCC recurrence and AR were associated with higher grades of liver fibrosis one year after LT, when compared to patients without AR (p=0.019).ConclusionOur results demonstrate an association of AR with HCC recurrence after LT with implications for intervals of monitoring in tumor surveillance. Graft fibrosis and immune mechanisms are potentially related and causal interactions are worth further investigation.

Development and Validation of a Machine Learning Prognostic Model for Hepatocellular Carcinoma Recurrence After Surgical Resection.

Surgical resection remains primary curative treatment for patients with hepatocellular carcinoma (HCC) while over 50% of patients experience recurrence, which calls for individualized recurrence prediction and early surveillance. This study aimed to develop a machine learning prognostic model to identify high-risk patients after surgical resection and to review importance of variables in different time intervals. The patients in this study were from two centers including Eastern Hepatobiliary Surgery Hospital (EHSH) and Mengchao Hepatobiliary Hospital (MHH). The best-performed model was determined, validated, and applied to each time interval (0–1 year, 1–2 years, 2–3 years, and 3–5 years). Importance scores were used to illustrate feature importance in different time intervals. In addition, a risk heat map was constructed which visually depicted the risk of recurrence in different years. A total of 7,919 patients from two centers were included, of which 3,359 and 230 patients experienced recurrence, metastasis or died during the follow-up time in the EHSH and MHH datasets, respectively. The XGBoost model achieved the best discrimination with a c-index of 0.713 in internal validation cohort. Kaplan-Meier curves succeed to stratify external validation cohort into different risk groups (p < 0.05 in all comparisons). Tumor characteristics contribute more to HCC relapse in 0 to 1 year while HBV infection and smoking affect patients’ outcome largely in 3 to 5 years. Based on machine learning prediction model, the peak of recurrence can be predicted for individual HCC patients. Therefore, clinicians can apply it to personalize the management of postoperative survival.