The Role of Metabolic Reprogramming in the Tumor Immune Microenvironment: Mechanisms and Opportunities for Immunotherapy in Hepatocellular Carcinoma.

As one of the emerging hallmarks of tumorigenesis and tumor progression, metabolic remodeling is common in the tumor microenvironment. Hepatocellular carcinoma (HCC) is the third leading cause of global tumor-related mortality, causing a series of metabolic alterations in response to nutrient availability and consumption to fulfill the demands of biosynthesis and carcinogenesis. Despite the efficacy of immunotherapy in treating HCC, the response rate remains unsatisfactory. Recently, research has focused on metabolic reprogramming and its effects on the immune state of the tumor microenvironment, and immune response rate. In this review, we delineate the metabolic reprogramming observed in HCC and its influence on the tumor immune microenvironment. We discuss strategies aimed at enhancing response rates and overcoming immune resistance through metabolic interventions, focusing on targeting glucose, lipid, or amino acid metabolism, as well as systemic regulation.

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.

Identification of molecular characteristics of hepatocellular carcinoma with microvascular invasion based on deep targeted sequencing.

BACKGROUND: As an indicator of tumor invasiveness, microvascular invasion (MVI) is a crucial risk factor for postoperative relapse, metastasis, and unfavorable prognosis in hepatocellular carcinoma (HCC). Nevertheless, the genetic mechanisms underlying MVI, particularly for Chinese patients, remain mostly uncharted. METHODS: We applied deep targeted sequencing on 66 Chinese HCC samples. Focusing on the telomerase reverse transcriptase (TERT) promoter (TERTp) and TP53 co-mutation (TERTp+/TP53+) group, gene set enrichment analysis (GSEA) was used to explore the potential molecular mechanisms of the TERTp+/TP53+ group on tumor progression and metastasis. Additionally, we evaluated the tumor immune microenvironment of the TERTp+/TP53+ group in HCC using multiplex immunofluorescence (mIF) staining. RESULTS: Among the 66 HCC samples, the mutated genes that mostly appeared were TERT, TP53, and CTNNB1. Of note, we found 10 cases with TERTp+/TP53+, of which nine were MVI-positive and one was MVI-negative, and there was a co-occurrence of TERTp and TP53 (p < 0.05). Survival analysis demonstrated that patients with the TERTp+/TP53+ group had lower the disease-free survival (DFS) (p = 0.028). GSEA results indicated that telomere organization, telomere maintenance, DNA replication, positive regulation of cell cycle, and negative regulation of immune response were significantly enriched in the TERTp+/TP53+ group (all adjusted p-values (p.adj) < 0.05). mIF revealed that the TERTp+/TP53+ group decreased CD8+ T cells infiltration (p = 0.25) and enhanced PDL1 expression (p = 0.55). CONCLUSIONS: TERTp+/TP53+ was significantly enriched in MVI-positive patients, leading to poor prognosis for HCC patients by promoting proliferation of HCC cell and inhibiting infiltration of immune cell surrounding HCC. TERTp+/TP53+ can be utilized as a potential indicator for predicting MVI-positive patients and poor prognosis, laying a preliminary foundation for further exploration of co-mutation in HCC with MVI and clinical treatment.

Telomerase-related advances in hepatocellular carcinoma: A bibliometric and visual analysis.

BACKGROUND: As a critical early event in hepatocellular carcinogenesis, telomerase activation might be a promising and critical biomarker for hepatocellular carcinoma (HCC) patients, and its function in the genesis and treatment of HCC has gained much attention over the past two decades. AIM: To perform a bibliometric analysis to systematically assess the current state of research on HCC-related telomerase. METHODS: The Web of Science Core Collection and PubMed were systematically searched to retrieve publications pertaining to HCC/telomerase limited to "articles" and "reviews" published in English. A total of 873 relevant publications related to HCC and telomerase were identified. We employed the Bibliometrix package in R to extract and analyze the fundamental information of the publications, such as the trends in the publications, citation counts, most prolific or influential writers, and most popular journals; to screen for keywords occurring at high frequency; and to draw collaboration and cluster analysis charts on the basis of coauthorship and co-occurrences. VOSviewer was utilized to compile and visualize the bibliometric data. RESULTS: A surge of 51 publications on HCC/telomerase research occurred in 2016, the most productive year from 1996 to 2023, accompanied by the peak citation count recorded in 2016. Up to December 2023, 35226 citations were made to all publications, an average of 46.6 citations to each paper. The United States received the most citations (n = 13531), followed by China (n = 7427) and Japan (n = 5754). In terms of national cooperation, China presented the highest centrality, its strongest bonds being to the United States and Japan. Among the 20 academic institutions with the most publications, ten came from China and the rest of Asia, though the University of Paris Cité, Public Assistance-Hospitals of Paris, and the National Institute of Health and Medical Research (INSERM) were the most prolific. As for individual contributions, Hisatomi H, Kaneko S, and Ide T were the three most prolific authors. Kaneko S ranked first by H-index, G-index, and overall publication count, while Zucman-Rossi J ranked first in citation count. The five most popular journals were the World Journal of Gastroenterology, Hepatology, Journal of Hepatology, Oncotarget, and Oncogene, while Nature Genetics, Hepatology, and Nature Reviews Disease Primers had the most citations. We extracted 2293 keywords from the publications, 120 of which appeared more than ten times. The most frequent were HCC, telomerase and human telomerase reverse transcriptase (hTERT). Keywords such as mutational landscape, TERT promoter mutations, landscape, risk, and prognosis were among the most common issues in this field in the last three years and may be topics for research in the coming years. CONCLUSION: Our bibliometric analysis provides a comprehensive overview of HCC/telomerase research and insights into promising upcoming research.

Integrated plasma proteomics and N-glycoproteomics reveals alterations in the N-glycosylation in Chinese hepatocellular carcinoma patients.

Hepatocellular carcinoma (HCC) is a life-threatening disease that presents diagnostic challenges due to the absence of reliable biomarkers. Recently, plasma proteomics and glycoproteomics have emerged as powerful tools for identifying potential diagnostic biomarkers for various diseases. In this study, we conducted a comprehensive proteomic and glycoproteomic analysis of plasma samples from 11 HCC patients and 11 healthy control (HC) individuals. We identified 20 differentially expressed (DE) proteins and 32 DE intact glycosylated peptides (IGPs) that can effectively differentiate between HCC patients and HC samples. Our findings demonstrate that IGP profiles had better predictive power than protein profiles for screening HCC. Pathways associated with DE proteins and IGPs were identified. It was reported that the protein expression level of galectin 3 binding protein (LGALS3BP) and its N-linked glycosylation at the N398 and N551 sites might serve as valuable candidates for HCC diagnosis. These results highlight the importance of N-glycoproteomics in advancing our understanding of HCC and suggest possible candidates for the future diagnosis of this disease.

Long-term outcome of centrally located hepatocellular carcinoma treated by neoadjuvant radiotherapy and radical resection: a propensity score matched study.

Objective: Centrally located hepatocellular carcinoma (HCC) typically presents challenges in surgical intervention and is associated with a bleak prognosis. In order to address this pressing issue, it is imperative to identify a comprehensive treatment approach, such as neoadjuvant radiotherapy (neoRT), that can enhance the prognosis of patients diagnosed with centrally located HCC. Methods: Patients who had surgical resections for HCC between March 2015 and December 2020 were included in the study. Patients were assigned to either the neoRT combined with liver resection (neoRT+LR) group or the liver resection alone (LR) group. The study employed propensity-score analysis and Cox proportional-hazards regression models as research methodologies. Using the Kaplan-Meier method, overall survival (OS) and disease-free survival (DFS) were estimated in patients. Results: During the study, 162 patients were enrolled, with 41 receiving neoRT+LR and 121 receiving LR. The duration of the median follow-up period was 45 months. The 1-year, 3-year, and 5-year OS rates were 95, 70, and 70% for patients in the neoRT+LR group, and 82, 64, and 54% for patients in the LR group, respectively. The 1-year, 3-year, 5-year DFS rates were 71, 53, and 37% for patients in the neoRT+LR group, and 52, 38, and 34% for patients in the LR group, respectively. A successful matching of 37 patients was achieved through propensity-score analysis. OS and DFS after matching analysis was statistically different between the two groups ( P=0.0099, P=0.034, respectively). neoRT was an independent prognostic factor for OS and DFS [hazard ratio (HR)=0.47, 95% CI: 0.24-0.93; HR=0.56, 95% CI: 0.34-0.92, respectively]. According to matching analysis, there were no statistically significant differences observed in terms of baseline characteristics, surgical safety, and complications between the groups. Conclusion: Liver resection and neoRT can be advantageous for patients with centrally located HCC.

Postoperative radiotherapy following null-margin hepatectomy in patients with hepatocellular carcinoma adhering to the major vessels: A propensity score-matched survival analysis cohort study.

Background & Aims: This study aims to analyze the prognosis of null-margin (≤1.0 mm) hepatectomy (NH) in patients with hepatocellular carcinoma (HCC) adhering to the major vessels and explore the value of postoperative radiotherapy (RT) in these patients. Methods: HCC patients who underwent null-margin or wide-margin (≥1.0 cm) hepatectomy (WH) by our team from January 2008 to March 2016 were recruited and analyzed retrospectively. The patients were divided into the NH, NH + RT, and WH groups. Propensity score matching (PSM) was performed to balance baseline characteristics. Results: A total of 357 patients were recruited. Of these, 84, 49, and 224 patients were given NH alone, NH plus RT, and WH, respectively. After PSM, the 5-year overall survival (OS) and disease-free survival (DFS) rates of the NH group were significantly worse than those of the WH group (51.5 % vs. 71.4 %, P = 0.003; 32.2 % vs. 50.9 %, P = 0.005). The OS and DFS rates of the NH + RT group were significantly higher than those of the NH group (75.6 % vs. 56.1 %, P = 0.012; 46.6 % vs. 30.2 %, P = 0.015) and similar to those of the WH group (75.6 % vs. 75.1 %, P = 0.354; 46.6 % vs. 56.6 %, P = 0.717). In addition, patients in the NH + RT group experienced significantly lower early (P = 0.023) and intrahepatic (P = 0.015) recurrences than those in the NH group. Conclusions: Patients with HCC adhering to the major vessels who underwent NH alone had a poorer prognosis, and the addition of RT to NH provide a significant survival benefit for these patients, which may yield outcomes comparable to the efficacy of WH.

Clinical benefits of intraoperative radiotherapy for the recurrence of centrally located hepatocellular carcinoma with microvascular invasion.

BACKGROUND: Although the efficacy and safety of intraoperative radiotherapy (IORT) in the treatment of malignant tumours, such as breast cancer, have been documented, it remains unclear whether this treatment is effective for centrally located hepatocellular carcinoma (HCC) with microvascular invasion (MVI). AIMS: This study aimed to explore the efficacy and safety of IORT in the treatment of centrally located HCC with MVI. METHODS AND RESULTS: Patients with centrally located HCC, who underwent surgery between January 2016 and January 2020, were enrolled. The patient cohort was then allocated to two groups: those who underwent IORT combined with liver resection (IORT+LR); or LR alone (LR). Propensity score matching and Cox proportional hazards regression analyses were performed. The Kaplan-Meier method was used to estimate recurrence-free survival (RFS), and the log-rank test was used to determine whether RFS differed between the groups. Subgroup analysis was performed to evaluate differences in RFS and early recurrence rates in patients with different MVI grades. E-values were generated to measure the sensitivity to unmeasured confounding factors. In total, 97 patients were enrolled, 27 of whom underwent IORT+LR and 70 underwent LR alone. The 1-, 3-, and 5-year RFS rates in the IORT+LR group were 66%, 50%, and 32%, respectively, whereas those in the LR group were 54%, 37%, and 26%, respectively. After matching analysis, 23 patients were successfully matched, and RFS was found to be significantly different between the two groups (p = .04). IORT was an independent prognostic factor for RFS (hazard ratio 0.46 [95% confidence interval 0.21-0.99]). In subgroup analysis, RFS between the IORT+LR and LR groups was significantly different in patients with MVI (M1 grade) (p = .0067). The postoperative early recurrence rate was significantly reduced with IORT (p < .05). No serious complications were reported in either group following surgery. Based on E-values, the results appeared to be robust against unmeasured confounding factors. CONCLUSION: IORT+LR provided safe, feasible treatment for patients with centrally located HCC with MVI, along with an improvement in prognosis and lower early recurrence rates.

Noninvasive urinary protein signatures combined clinical information associated with microvascular invasion risk in HCC patients.

BACKGROUND: Microvascular invasion (MVI) is the main factor affecting the prognosis of patients with hepatocellular carcinoma (HCC). The aim of this study was to identify accurate diagnostic biomarkers from urinary protein signatures for preoperative prediction. METHODS: We conducted label-free quantitative proteomic studies on urine samples of 91 HCC patients and 22 healthy controls. We identified candidate biomarkers capable of predicting MVI status and combined them with patient clinical information to perform a preoperative nomogram for predicting MVI status in the training cohort. Then, the nomogram was validated in the testing cohort (n = 23). Expression levels of biomarkers were further confirmed by enzyme-linked immunosorbent assay (ELISA) in an independent validation HCC cohort (n = 57). RESULTS: Urinary proteomic features of healthy controls are mainly characterized by active metabolic processes. Cell adhesion and cell proliferation-related pathways were highly defined in the HCC group, such as extracellular matrix organization, cell-cell adhesion, and cell-cell junction organization, which confirms the malignant phenotype of HCC patients. Based on the expression levels of four proteins: CETP, HGFL, L1CAM, and LAIR2, combined with tumor diameter, serum AFP, and GGT concentrations to establish a preoperative MVI status prediction model for HCC patients. The nomogram achieved good concordance indexes of 0.809 and 0.783 in predicting MVI in the training and testing cohorts. CONCLUSIONS: The four-protein-related nomogram in urine samples is a promising preoperative prediction model for the MVI status of HCC patients. Using the model, the risk for an individual patient to harbor MVI can be determined.

High spindle and kinetochore-associated complex subunit-3 expression predicts poor prognosis and correlates with adverse immune infiltration in hepatocellular carcinoma.

BACKGROUND: Spindle and kinetochore-associated complex subunit 3 (SKA3) is a malignancy-associated gene that plays a critical role in the regulation of chromosome separation and cell division. However, the molecular mechanism through which SKA3 regulates tumor cell proliferation in hepatocellular carcinoma (HCC) has not been fully elucidated. AIM: To investigate the molecular mechanisms underlying the role of SKA3 in HCC. METHODS: SKA3 expression, clinicopathological, and survival analyses were performed using multiple public database platforms, and the results were verified by Western blot and immunohistochemistry staining using collected clinical samples. Functional enrichment analyses were performed to evaluate the biological functions and molecular mechanisms of SKA3 in HCC. Furthermore, the Tumor Immune Estimation Resource and single-sample Gene Set Enrichment Analysis (ssGSEA) algorithms were utilized to investigate the abundance of tumor-infiltrating immune cells in HCC. The response to chemotherapeutic drugs was evaluated by the R package "pRRophetic". RESULTS: We found that upregulated SKA3 expression was significantly correlated with poor prognosis in patients with HCC. Multivariable Cox regression analysis indicated that SKA3 was an independent risk factor for survival. GSEA revealed that SKA3 expression may facilitate proliferation and migratory processes by regulating the cell cycle and DNA repair. Moreover, patients with high SKA3 expression had significantly decreased ratios of CD8+ T cells, natural killer cells, and dendritic cells. Drug sensitivity analysis showed that the high SKA3 group was more sensitive to sorafenib, sunitinib, paclitaxel, doxorubicin, gemcitabine, and vx-680. CONCLUSION: High SKA3 expression led to poor prognosis in patients with HCC by enhancing HCC proliferation and repressing immune cell infiltration surrounding HCC. SKA3 may be used as a biomarker for poor prognosis and as a therapeutic target in HCC.

Autophagy and biotransformation affect sorafenib resistance in hepatocellular carcinoma.

As sorafenib is a first-line drug for treating advanced hepatocellular carcinoma, sorafenib resistance has historically attracted attention. However, most of this attention has been focused on a series of mechanisms related to drug resistance arising after sorafenib treatment. In this study, we used proteomic techniques to explore the potential mechanisms by which pretreatment factors affect sorafenib resistance. The degree of redundant pathway PI3K/AKT activation, biotransformation capacity, and autophagy level in hepatocellular carcinoma patients prior to sorafenib treatment might affect their sensitivity to sorafenib, in which ADH1A and STING1 are key molecules. These three factors could interact mechanistically to promote tumor cell survival, might be malignant features of tumor cells, and are associated with hepatocellular carcinoma prognosis. Our study suggests possible avenues of therapeutic intervention for patients with sorafenib-resistance and the potential application of immunotherapy with the aim of improving the survival of such patients.

Clinical Benefits of Neoadjuvant Radiotherapy on the Postoperative Recurrence of Centrally Located Hepatocellular Carcinoma: A Real-World Evidence Based on Phase II Clinical Trial.

Objective: Although surgical resection is one of the most effective way to treat liver cancer, its efficacy and safety in treatment of centrally located hepatocellular carcinoma (HCC) remains elusive. Therefore, it is very important to find a comprehensive treatment mode, such as radical resection combined with neoadjuvant radiotherapy (neoRT). Methods: The centrally located HCC patients who underwent radical resection from July 2015 to April 2021 were enrolled. According to whether the neoRT was implemented or not, these patients were allocated into neoadjuvant radiotherapy combined with liver resection (neoRT+LR) and liver resection alone (LR) group. The research method used propensity-score analysis and Cox proportional-hazards regression models. We generated an E-value to assess the sensitivity to unmeasured confounding. This study is a real-world, retrospective study based on phase II clinical trial. Results: A total of 168 patients were enrolled, including 38 patients treating with neoRT+LR and 130 patients with LR. The 1-, 3-, 5-year disease free survival (DFS) rates were 74%, 55% and 39% in the neoRT+LR group, and 44%, 28%, and 24% in the LR group, respectively. Neoadjuvant radiotherapy was an independent prognostic factor for postoperative recurrence ([HR]0.42, 95% CI [0.25, 0.69]). There was significant association between neoRT+LR and longer disease-free survival (Match, [HR] 0.43, 95% CI [0.24, 0.76]; GenMatch, [HR] 0.32, 95% CI [0.23, 0.43]; Adjusted for propensity score, [HR] 0.41, 95% CI [0.23, 0.73]; Inverse probability weighting, [HR] 0.38, 95% CI [0.22, 0.65], respectively). DFS before and after matching analysis was statistically different in two groups (p-value=0.005, p-value=0.0024, respectively). Neoadjuvant radiotherapy can significantly reduce the postoperative early recurrence (p-value <0.05). E-value analysis suggested robustness to unmeasured confounding. Conclusion: Liver resection combined with neoadjuvant radiotherapy was effective and safe for treatment of centrally located HCC patients, which improved the prognosis of patients and reduced the incidence of early recurrence.

Survival benefit of radiotherapy following narrow-margin hepatectomy in patients with hepatocellular carcinoma: A propensity score-matched analysis based on phase II study.

BACKGROUND AND PURPOSE: We evaluated the postoperative radiotherapy (PORT) effects on hepatocellular carcinoma (HCC) prognosis and recurrence in patients who underwent narrow-margin (<1.0 cm) hepatectomy (NH). MATERIALS AND METHODS: This two-arm cohort study based on the phase II study compared NH with or without PORT in patients with HCC. All patients underwent NH; 76 patients who received PORT following NH in the phase II study were assigned to the NH + RT group, and 171 who underwent NH alone were assigned to the NH group. Propensity score matching (PSM) was used to balance clinicopathological characteristic differences between groups. RESULTS: Before PSM, the 5-year overall survival (OS) rates between groups differed significantly (72.2 % vs 60.7 %, P = 0.017). Moreover, the 5-year disease-free survival (DFS) rate was significantly higher in the NH + RT group (51.4 % vs 35.7 %, P = 0.002). After PSM, the between-group difference in OS rates remained high (P = 0.045); the 5-year OS rates were 74.7 % and 63.6 % in the NH + RT and NH groups, respectively. Similarly, the DFS rate remained significantly higher in the NH + RT group (P = 0.001); the 5-year DFS rates were 56.3 % and 31.6 %, respectively. Furthermore, both before and after PSM, patients in the NH + RT group showed significantly lower early, intrahepatic, and extrahepatic recurrence rates than those in the NH group. CONCLUSIONS: PORT may have significant OS and DFS benefits in patients with HCC undergoing NH.

Serum metabolic effects of corn oligopeptides with 7-day supplementation on early post-surgery primary liver cancer patients: a double-blind randomized controlled trial.

Background: Liver cancer as the main leading cancer has caused heavy burdens globally. The prognosis of liver cancer is closely related with postoperative nutrition support. Corn oligopeptides (COPs) are protein hydrolysates produced by enzymatic treatments, which have shown potential bioactivities, such as inhibiting angiotensin I-converting enzyme, resisting lipid peroxidation and anti-oxidant. However, the correlation between COPs and liver cancer patients is still unknown and the potential mechanism of COPs on liver cancer is unclear as well. The aim of this study was to assess effects of 7-day intervention of COPs after surgery on liver function and serum metabolic profiles of liver cancer patients. Methods: Patients were assigned into COPs intervention group (n=50) and control group (n=91) for 7 days. Investigations were scheduled at 1st day and 7th day after liver resection surgery respectively, mainly including anthropometric, biochemical indexes and liquid chromatography-mass spectrometry (LC/MS) analysis. Results: Seven-day supplementation of COPs on early post-surgery liver cancer patients down-regulated levels of alanine aminotransferase, aspartate aminotransferase, total bilirubin, direct bilirubin and up-regulated prothrombin time activity and prealbumin levels. LC/MS analysis revealed metabolic signatures including regulation of 16 metabolites, which was closely related with two metabolic pathways (nicotinate and nicotinamide metabolism, fatty acid metabolism). Conclusions: COPs supplementation has displayed the potentials on alleviating the injury of liver function and it may be due to regulation of fatty acid metabolism, nicotinate and nicotinamide metabolism, lipid peroxidation and anti-inflammatory action. More researches are warranted in future to confirm the exact mechanisms.

Phase 2 Evaluation of Neoadjuvant Intensity-Modulated Radiotherapy in Centrally Located Hepatocellular Carcinoma: A Nonrandomized Controlled Trial.

Importance: Centrally located hepatocellular carcinoma (HCC) is a special type of HCC whose outcome is unsatisfactory when treated with surgery alone. No standard adjuvant or neoadjuvant treatment for this disease has been established that improves clinical outcomes. Objective: To evaluate the effectiveness and safety of adding neoadjuvant intensity-modulated radiotherapy (IMRT) before surgery in patients with centrally located HCC. Design, Setting, and Participants: This phase 2, single-center, single-group prospective nonrandomized controlled trial was conducted between December 16, 2014, and January 29, 2019, at the Cancer Institute and Hospital of the Chinese Academy of Medical Sciences in Beijing, China. The last follow-up was on July 30, 2021. Patients with centrally located HCC who underwent neoadjuvant IMRT and surgery were included in the analysis. Interventions: Neoadjuvant IMRT followed by hepatectomy. Main Outcomes and Measures: The primary end point was 5-year overall survival (OS). The secondary end points were tumor response to IMRT, 5-year disease-free survival (DFS), and treatment-related adverse events. Results: Thirty-eight patients (mean [SD] age, 55.6 [9.3] years; 35 male [92.1%] individuals) completed the prescribed neoadjuvant IMRT without interruption. Radiographic tumor response to IMRT before surgery included partial response (16 [42.1%]) and stable disease (22 [57.9%]). Thirteen patients (34.2%) achieved major pathological response, of which 5 (13.2%) achieved pathologic complete response. With a median follow-up of 45.8 months, the median OS was not reached, and the OS rates were 94.6% at 1 year, 75.4% at 3 years, and 69.1% at 5 years. The median DFS was 45.8 months, and DFS rates were 70.3% at 1 year, 54.1% at 3 years, and 41.0% at 5 years. Radiotherapy-related grade 3 adverse events were observed in 3 patients (7.9%). Nineteen operative complications developed in 13 patients (34.2%), including grade I to II complications in 12 patients (31.6%) and grade IIIa complication in 1 patient (2.6%). No grade IIIb or higher operative complications were observed. Conclusions and Relevance: Results of this trial suggest that neoadjuvant IMRT plus surgery is effective and well-tolerated in patients with centrally located HCC. These data may inform a future randomized clinical trial of this new treatment strategy. Trial Registration: ClinicalTrials.gov Identifier: NCT02580929.

The role of intraoperative electron radiotherapy in centrally located hepatocellular carcinomas treated with narrow-margin (<1 cm) hepatectomy: a prospective, phase 2 study.

Background: Postoperative radiotherapy (RT) is known to play an important role in the treatment of hepatocellular carcinomas (HCCs), but the specific role of intraoperative electron radiotherapy (IOERT) in HCCs remains unclear. The aim of this study was to investigate the safety and efficacy of IOERT in centrally located HCCs treated with narrow-margin (<1 cm) hepatectomy. Methods: This was a single-center, phase 2, prospective non-randomized controlled study, including 268 patients with centrally located HCCs who underwent narrow-margin hepatectomy. The patients were subsequently allocated to the IOERT group (n=59) or to the control group (n=65). The primary outcome of the study was to compare recurrence-free survival (RFS) between the IOERT group and the control group, and the secondary outcome was to compare overall survival (OS) rate between the two groups. Results: Of 268 patients enrolled, a total of 124 were included in the study: 59 in IOERT group, 65 in control group. The 1-, 2-, 3-year RFS rates were 79.3%, 62.1% and 45.8% for patients in the IOERT group, and 47.6%, 28.6%, and 22.9% for patients in the control group, respectively (P=0.025). The 1-, 2-, and 3-year OS rates were 100.0%, 94.9%, and 83.7% for patients in the IOERT group, and 92.3%, 87.5%, and 79.4% for patients in the control group, respectively (P=0.314). Subgroup analysis of MVI (+) patients revealed that RFS and OS are significantly prolonged in the IOERT subgroup as compared to the control, whereas there was no significant difference of RFS and OS between the two groups in MVI (-) patients. Conclusions: IOERT for centrally located HCCs with concurrent narrow-margin hepatectomy was feasible and safe. Statistically better RFS rate was observed in the IOERT group compared to the control group. Subgroup analysis revealed that IOERT was more beneficial for postoperative survival of HCC patients with MVI. Trial Registration: ChiCTR-TRC-12002802; www.who.int/ictrp.

Comprehensive Analysis of DNA 5-Methylcytosine and N6-Adenine Methylation by Nanopore Sequencing in Hepatocellular Carcinoma.

DNA methylation is a widespread epigenetic signal in human genome. With Nanopore technology, differential methylation modifications including 5-methylcytosine (5mC) and 6-methyladenine (6mA) can be identified. 5mC is the most important modification in mammals, although 6mA may also function in growth and development as well as in pathogenesis. While the role of 5mC at CpG islands in promoter regions associated with transcriptional regulation has been well studied, but the relationship between 6mA and transcription is still unclear. Thus, we collected two pairs of tumor tissues and adjacent normal tissues from hepatocellular carcinoma (HCC) surgical samples for Nanopore sequencing and transcriptome sequencing. It was found that 2,373 genes had both 5mC and 6mA, along with up- and down-regulated methylation sites. These genes were regarded as unstable methylation genes. Compared with 6mA, 5mC had more inclined distribution of unstable methylation sites. Chi-square test showed that the levels of 5mC were consistent with both up- and down-regulated genes, but 6mA was not significant. Moreover, the top three unstable methylation genes, TBC1D3H, CSMD1, and ROBO2, were all related to cancer. Transcriptome and survival analyses revealed four potential tumor suppressor genes including KCNIP4, CACNA1C, PACRG, and ST6GALNAC3. In this study, we firstly proposed to combine 5mC and 6mA methylation sites to explore functional genes, and further research found top of these unstable methylation genes might be functional and some of them could serve as potential tumor suppressor genes. Our study provided a new solution for epigenetic regulation research and therapy of HCC.

DNA N6-Adenine methylation in HBV-related hepatocellular carcinoma.

DNA methylation on N6-adenine (6mA) has recently been found to be a potential epigenetic marker in prokaryotes and eukaryotes. However, its distribution patterns and potential functions in human tumorigenesis remain largely unknown. Here, we reported global profiling of 6mA sites in the genome of hepatocellular carcinoma at single-nucleotide resolution using Nanopore sequencing. 6mA was widely distributed throughout the human genome. The 6mA sites were related to the porphyrin and chlorophyll metabolism in autosomes and were related to oxidative phosphorylation and ATP metabolism in mitochondria. AGG was the most significant motif associated with 6mA modification and the prevalent motifs in tumors were mainly distributed in mitochondria. The density of 6mA was related to the activation of gene transcription and 6mA density in repetitive sequences decreased in hepatocellular carcinoma. DNA 6mA methylation modification may also be a potential biomarker for cancer diagnosis and treatment.

Research progress regarding programmed cell death 1/programmed cell death ligand 1 inhibitors combined with targeted therapy for treating hepatocellular carcinoma.

In recent years, a number of targeted therapeutic agents have achieved success in phase III trials in patients with advanced hepatocellular carcinoma (HCC), including sorafenib, lenvatinib, and regorafenib. Immunotherapy is considered to be an effective treatment for advanced HCC. Immune checkpoint inhibitors targeting programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) are important antitumor immunotherapy agents that represent breakthroughs in the treatment of advanced HCC. However, treating advanced HCC is still a great challenge, and the need for new treatments remains urgent. This review briefly summarizes the research progress in the use of PD-1/PD-L1 inhibitors combined with targeted therapy for treating HCC.

Long-read sequencing reveals the structural complexity of genomic integration of HBV DNA in hepatocellular carcinoma.

The integration of HBV DNA into the human genome can disrupt its structure in hepatocellular carcinoma (HCC), but the complexity of HBV genomic integration remains elusive. Here we applied long-read sequencing to precisely elucidate the HBV integration pattern in the human hepatocellular genome. The DNA library was sequenced using the long-read sequencing on GridION and PacBio Sequel II, respectively. The DNA and mRNA were sequenced using next-generation sequencing on Illumina NextSeq. BLAST (Basic Local Alignment Search Tool) and local scripts were used to analyze HBV integration patterns. We established an analytical strategy based on the long-read sequences, and analyzed the complexity of HBV DNA integration into the hepatocellular genome. A total of 88 integrated breakpoints were identified. HBV DNA integration into human genomic DNA was mainly fragmented with different orientations, rarely with a complete genome. The same HBV integration breakpoints were identified among the three platforms. Most breakpoints were observed at P, X, and S genes in the HBV genome, and observed at introns, intergenic sequences, and exons in the human genome. Tumor tissue harbored a much higher integrated number than the adjacent tissue, and the distribution of HBV integrated into human chromosomes was more concentrated. HBV integration shows different patterns between cancer cells and adjacent normal cells. We for the first time obtained the entire HBV integration pattern through long-read sequencing and demonstrated the value of long-read sequencing in detecting the genomic integration structures of viruses in host cells.