Hepatocellular Carcinoma Recurrence Research Articles

Applying image features of proximal paracancerous tissues in predicting prognosis of patients with hepatocellular carcinoma

BackgroundMost of the methods using digital pathological image for predicting Hepatocellular carcinoma (HCC) prognosis have not considered paracancerous tissue microenvironment (PTME), which are potentially important for tumour initiation and metastasis. This study aimed to identify roles of image features of PTME in predicting prognosis and tumour recurrence of HCC patients. MethodsWe collected whole slide images (WSIs) of 146 HCC patients from Sun Yat-sen Memorial Hospital (SYSM dataset). For each WSI, five types of regions of interests (ROIs) in PTME and tumours were manually annotated. These ROIs were used to construct a Lasso Cox survival model for predicting the prognosis of HCC patients. To make the model broadly useful, we established a deep learning method to automatically segment WSIs, and further used it to construct a prognosis prediction model. This model was tested by the samples of 225 HCC patients from the Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC). ResultsIn predicting prognosis of the HCC patients, using the image features of manually annotated ROIs in PTME achieved C-index 0.668 in the SYSM testing dataset, which is higher than the C-index 0.648 reached by the model only using image features of tumours. Integrating ROIs of PTME and tumours achieved C-index 0.693 in the SYSM testing dataset. The model using automatically segmented ROIs of PTME and tumours achieved C-index of 0.665 (95% CI: 0.556–0.774) in the TCGA-LIHC samples, which is better than the widely used methods, WSISA (0.567), DeepGraphSurv (0.593), and SeTranSurv (0.642). Finally, we found the Texture SumAverage Skew HV on immune cell infiltration and Texture related features on desmoplastic reaction are the most important features of PTME in predicting HCC prognosis. We additionally used the model in prediction HCC recurrence for patients from SYSM-training, SYSM-testing, and TCGA-LIHC datasets, indicating the important roles of PTME in the prediction. ConclusionsOur results indicate image features of PTME is critical for improving the prognosis prediction of HCC. Moreover, the image features related with immune cell infiltration and desmoplastic reaction of PTME are the most important factors associated with prognosis of HCC.

Abstract 865: Single-cell sequencing identification of malignant cell subpopulations linked to recurrence in hepatocellular carcinoma

Abstract Introduction: Hepatocellular Carcinoma (HCC) is a highly malignant and challenging-to-treat tumor, with early recurrence affecting up to 35-50% of patients within two years after surgery, significantly impacting prognosis. Identifying high-risk patients for recurrence is critical for guiding postoperative adjuvant therapy. The rapidly advancing field of single-cell RNA sequencing technology offers an unprecedented opportunity to discover clinically relevant cell subpopulations. Here, we harnessed single-cell techniques to unveil a cell subpopulation closely linked with HCC recurrence, highlighting its clinical significance. Methods and Results: We conducted single-cell RNA sequencing on four HCC samples and harmonized data from three public datasets (GSE112271, GSE125449, CNP0000650) to elucidate the heterogeneity of malignant cells in HCC. Our investigation revealed a distinctive HCC subpopulation enriched in patients with early recurrence (11.4% vs. 3.8%, P=0.02). This subpopulation exhibited up-regulated STMN1 and EZH2, signifying high proliferative potential and a propensity for epithelial-mesenchymal transition (EMT). Applying the CIBERSORTx algorithm to the TCGA-LIHC dataset (N=323 patients), we inferred the proportion of this recurrence subpopulation among all malignant cells. Notably, patients with a higher proportion of this subpopulation (N=160) demonstrated significantly shorter recurrence-free survival (31.2 vs. 42.9 months, P=0.002), reduced median survival (45.9 vs 55.7 months, P=0.03) and a higher rate of microvascular invasion (35% vs. 23%, P=0.01). To validate this recurrence-related subpopulation in clinical samples, we conducted multiplex immunohistochemistry staining on paraffin-embedded (FFPE) HCC samples (including CD45, CD31, ARG1, STMN1, EZH2) and used machine learning for its identification. Our findings revealed that patients with a higher proportion of this recurrence subpopulation (N=40) experienced significantly shorter median survival (38.5 vs. 51.0 months, P<0.005) and progression-free survival (25.1 vs. 33.4 months, P=0.01) compared to a lower proportion (N=40). Multivariate Cox regression analysis confirmed that the proportion of this recurrence subpopulation is an independent predictor of worse prognosis (P=0.003). Conclusion: Through the power of single-cell RNA sequencing technology, we have unearthed and characterized a malignant cell subpopulation intricately connected with HCC recurrence. Importantly, this subpopulation can be identified in FFPE tissue samples and correlates robustly with patient prognosis. Our study underscores the transformative possibility of single-cell techniques in developing cell subpopulation-based biomarkers, emphasizing their clinical relevance and potential utility in guiding postoperative adjuvant therapy for HCC patients. Citation Format: Yucheng Dong, Xin Guo, Xuezhu Wang, Fan Yang, Yongchang Zheng, Yang Chen. Single-cell sequencing identification of malignant cell subpopulations linked to recurrence in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 865.

Abstract 7665: Detection of γ-OHPdG as a hepatocellular carcinoma recurrence biomarker by CTCs and LC-MS/MS

Abstract Background: Hepatocellular carcinoma (HCC) is the third leading cause of cancer death globally. Recurrence risks following curative resection remain high, ~50% after three years and 75-100% over five years. A biomarker for HCC recurrence is urgently needed to help lead treatment strategies. We previously showed that hepatic γ-hydroxy-1, N2-propano-2′-deoxyguanosine (γ-OHPdG), a DNA adduct derived from lipid peroxidation, increased during hepatocarcinogenesis. Furthermore, in a retrospective study, higher γ-OHPdG levels in post-surgical liver tumor tissues were associated with poorer survival (p<0.0001) and shorter recurrence-free survival (p=0.007), suggesting that it may be a prognostic biomarker for HCC (Hepatology 2018). In this study, we aimed to test γ-OHPdG detected in livers after curative resection as a recurrence biomarker in a prospective study and, in addition, we developed a new non-invasive method for γ-OHPdG in circulating tumor cells (CTCs). This multicenter study seeks to validate γ-OHPdG as a biomarker for HCC recurrence. Methods: A total of 28 HCC patients undergoing hepatectomy at MedStar/Georgetown University Hospital, Johns Hopkins University Hospital, and the University of Maryland Medical Center were enrolled. γ-OHPdG was detected in DNA isolated from hepatic tumors and non-tumors by a previous liquid chromatograph-tandem mass spectrometry (LC-MS/MS) method and in HCC CTCs isolated from blood samples. CTCs were isolated using hepatocyte-specific rabbit anti-asialoglycoprotein-1 (ASGPR-1), followed by staining with mouse anti-γ-OHPdG antibodies. Clinical outcomes were correlated with γ-OHPdG in liver tissues and CTCs. Results: In our current cohort of 28 patients (mean age: 64.7 years; 82% male), five patients experienced HCC recurrence, with an estimated progression-free survival (PFS) rate of 85% over 8 months. γ-OHPdG levels (adducts/109 nucleotides) detected by LC-MS/MS varied widely in tumor tissues (5-56, mean: 11.8) and non-tumor tissue (7-71, mean: 20.6). Preliminary analysis showed shorter PFS associated with γ-OHPdG in non-tumors (cutoff: 16, p=0.0271). γ-OHPdG (arbitrary units x107) in CTCs also varied widely in the nucleus (0.95-18.84, mean: 2.85) and in the whole cell (0.16- 52.18, mean: 9.72). Total CTC γ-OHPdG intensity in both the nucleus (p=0.006) and in the whole cell (p=0.005) was associated with its levels in non-tumor tissue. Furthermore, a shorter PFS was associated with CTC count (cutoff: 270 cells, p=0.01), and total CTC whole cell γ-OHPdG intensity (cutoff: 21.17 × 107 arbitrary units, p=0.002). Conclusion: Preliminary results suggest that γ-OHPdG, measured in tissues by LC-MS/MS and in blood by CTC, predicts HCC recurrence. Once evaluated in all enrolled subjects, multiple variant analyses will be carried out to include stage, AFP, grade of differentiation, vascular invasion on surgical samples, and status of viral hepatitis infection. Citation Format: Mark Kuo, Merve Ozge Aktop, Elie Ghabi, Mahmoud A. Ouf, Margaret Carder, Vinona Muralidaran, Jhalen Ascue, Marketa Hlouskova, Pengfei Wu, Guang Cheng, Stephen S. Hecht, Hong-Bin Fang, Nader Hanna, Jin He, Alexander H. Kroemer, Monika Aggarwal, Aiwu R. He, Fung-Lung Chung. Detection of γ-OHPdG as a hepatocellular carcinoma recurrence biomarker by CTCs and LC-MS/MS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7665.

Abstract 2695: A higher percentage of M2 macrophages in non-tumor part predict the tumor recurrence in patients with hepatocellular carcinoma after surgical resection

Abstract The primary hurdle in improving outcomes for hepatocellular carcinoma (HCC) patients after curative treatment is the high early recurrence rate. The existing literature indicates the presence of type 2 macrophages (M2), renowned for their immunosuppressive capabilities, has been implicated in creating a microenvironment conducive to tumor growth and recurrence. However, the intricate interplay involving M2 macrophages and the recurrence of HCC is underexplored. Therefore, we conducted an analysis involving HCC patients who underwent hepatectomy at our hospital between 2013 and 2017. We collected samples from both the tumor and non-tumor regions and subjected them to bulk RNA sequencing for comprehensive molecular profiling. The proportion of immune cells was calculated by CIBERSORTx.In our study, we enrolled fifty patients, with sixty percent presenting with cirrhosis and seventy-three percent having viral hepatitis. Twenty-three patients experienced tumor recurrence, with a median time to recurrence of 12.6 months. First, we found no correlation between five TIMR subtypes defined by TIMELASER (Xue R, Nature, 2022) and recurrence. However, as for the proportions of immune cell, a significantly lower NK/Treg ratio (p= 0.0163) and high within the resected tumor section in recurrent group was found. On the other hand, the ratio of M2 macrophages to total macrophages (M2/M) was significantly higher within the non-tumor region in patients experiencing tumor recurrence after hepatectomy (p=0.0357). Furthermore, the expression of the ACGT2 gene in non-tumor part, which strongly correlates with Treg function, was significantly higher (p=0.0056) in recurrent group. In addition, the innate T cells score was increased in non-tumor pat in recurrent group but with borderline significance (p=0.0628). However, in multivariable analysis, only a higher M2/M ratio within the non-tumor region (HR 3.756, p=0.0072) were identified as independent risk factors for tumor recurrence. We also found that patients with a higher M2/M ratio had a lower percentage of cirrhotic patients (p=0.0049) and less association with TIME-IA (immune activation) subtype of HCC by TIMELASER classification (p=0.0030). In conclusion, a higher M2/M ratio within the non-tumor region predict the tumor recurrence in patients with hepatocellular carcinoma. Citation Format: Po-Ting Lin, Wei-Ting Ku, Tsung-Han Wu, Cheng-Heng Wu, Chan-Keng Yang, Wei Teng, Yung-Chang Lin, Chun-Yen Lin. A higher percentage of M2 macrophages in non-tumor part predict the tumor recurrence in patients with hepatocellular carcinoma after surgical resection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2695.

Abstract 5010: Development of a universal cancer vaccine using common cancer antigens cocktail that targets various cancer patients and overcomes the heterogeneity of individual cancer

Abstract In clinical trials of the glypican-3 (GPC3)-derived peptide vaccine targeting hepatocellular carcinoma (HCC), hepatoblastoma, etc., peptide-specific CTL were detected in many patients. There have been some partial response advanced cancer cases, and it is expected to be effective in prolonging the prognosis after HCC surgery. Five children with refractory hepatoblastoma who had repeated relapses and remissions are all surviving for more than 9 years without relapse by only peptide vaccine therapy. On the other hand, several patients have experienced recurrence of hepatocellular carcinoma that does not express GPC3 despite the detection of a large number of peptide-specific CTLs in the blood after administration of peptide vaccines. GPC3 is highly expressed in many hepatocellular carcinomas, but there are GPC3-negative cancer cells, so it is difficult to prevent recurrence of hepatocellular carcinoma with a single type of GPC3 peptide vaccine. In other words, to cure cancer, it is necessary to combine multiple common cancer antigens. Through immunohistochemical analysis, we identified 10 common cancer antigens, including GPC3, that are frequently expressed in various solid cancers but rarely expressed in normal tissues. Five of these antigens are membrane protein antigens, and we have confirmed that at least one of these antigens, and often more than one, is expressed in many cancers investigated. We synthesized peptides predicted to be presented at HLA-A*24:02 or HLA-A*02:01 from the full-length amino acid sequences of 10 types of common cancer antigen proteins. These peptide vaccines were administered to HLA-A*24:02 or HLA-A*02:01 transgenic mice once a week for a total of three times, and a number of peptide-reactive CTLs were identified in the splenocytes. These peptides have better CTL-inducing ability than the peptides of the GPC3 peptide vaccine used in clinical trials. Among these 10 types of common cancer antigens, we will use 5 types of membrane protein common cancer antigens in particular to develop a cocktail mRNA vaccine with the goal of preventing cancer recurrence after surgery. Of course, it is also possible to develop cocktail peptide vaccines. At the same time, we are developing a cocktail CAR/TCR-T cell therapy targeting these common cancer antigens for the treatment of advanced cancer. Citation Format: Tetsuya Nakatsura, Kazumasa Takenouchi, Hiroki Kinoshita, Nobuo Tsukamoto, Kazunobu Ohnuki, Toshihiro Suzuki. Development of a universal cancer vaccine using common cancer antigens cocktail that targets various cancer patients and overcomes the heterogeneity of individual cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5010.

Abstract 6871: Tumor-infiltrating mregDC restrains anti-tumor immunity to promote cancer relapse in HCC

Abstract Background and aims: Cancer relapse is one of the major causes of mortality in patients after curative resection of primary tumors. Furthermore, significant proportion of patients with recurrent tumor lack of effective treatments, thus novel therapeutic strategies are urgently needed. Although there have been several single-cell studies of primary tumor, functional roles and mechanism of tumor-infiltrating immune cells, especially those of myeloid cells in tumor relapse have remained to be elucidated. Methods: We analyzed the RNA profile of 16498 cells from 12 primary and 6 early-relapse HCC cases. Digital pathology, multiplex immunofluorescent staining and digital spatial profiling (DSP) of paired primary and relapsed tumors from 41 HCC patients were also performed as our validation set. The molecular mechanisms associated with DCs was further tested through ex vivo and in vivo experiments. Results: We performed single-cell clustering and visualized 3 DC clusters (cDC1, cDC2, and DC3). Compared with the other two subsets, the enrichment of DC3 in relapsed HCC was correlated with a higher second recurrence rate. Deeper inspections of the signature genes indicated this subset as ‘mregDC’, a DC subset enriched in maturation and regulatory molecules. Our results demonstrated that mregDC recruited CD161+CD8+ T cells that had an innate-like, low-cytotoxic, and low-clonal-expansion phenotype. CD161+CD8+ T cells secreted large amounts of TNF to activate non-canonical NF-κB pathway via tumor necrosis factor receptor 2 (TNFR2) and promoted the differentiation of immature DC into mregDC. This positive feedback loop between mregDC and CD161+CD8+ T cells shaped a dysfunctional anti-tumor immunity, facilitating cancer relapse in HCC. In vivo assay further indicated that therapy combining anti-TNFR2 and PD-L1 antibodies significantly inhibited HCC tumor growth via impeding the infiltration of mregDC compared with anti-PD-L1 monotherapy. We also found mregDC recruited FCN1+ monocyte, a MDSC-like cell population to create an immunosuppressive niche of relapsed HCC. Conclusions: We identified this population of mregDC with immunomodulatory function in recurrent HCC and demonstrated the role of these special DCs in shaping the immunosuppressive microenvironment of recurrent tumors. Our study suggests that mregDC may be a new target for the treatment of recurrent HCC in the future. Citation Format: Zefan Zhang, Jian Zhou, Jia Fan, Yu Zhong, Liang Wu, Yunfan Sun. Tumor-infiltrating mregDC restrains anti-tumor immunity to promote cancer relapse in HCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6871.

Everolimus Mitigates the Risk of Hepatocellular Carcinoma Recurrence after Liver Transplantation.

To obtain long-term data on the use of everolimus in patients who underwent liver transplantation for hepatocellular carcinoma, we conducted a retrospective, single-center analysis of adult recipients transplanted between 2013 and 2021. Patients on everolimus-incorporating immunosuppression were matched with those on tacrolimus using an inverse probability of treatment weighting methodology. Two propensity-matched groups of patients were thus compared: 233 (45.6%) receiving everolimus versus 278 (54.4%) on tacrolimus. At a median (interquartile range) follow-up of 4.4 (3.8) years after transplantation, everolimus patients showed a reduced risk of recurrence versus tacrolimus (7.7% versus 16.9%; RR = 0.45; p = 0.002). At multivariable analysis, microvascular infiltration (HR = 1.22; p < 0.04) and a higher tumor grading (HR = 1.27; p < 0.04) were associated with higher recurrence rate while being within Milan criteria at transplant (HR = 0.56; p < 0.001), a successful pre-transplant downstaging (HR = 0.63; p = 0.01) and use of everolimus (HR = 0.46; p < 0.001) had a positive impact on the risk of post-transplant recurrence. EVR patients with earlier drug introduction (≤30 days; p < 0.001), longer treatment duration (p < 0.001), and higher drug exposure (≥5.9 ng/mL; p < 0.001) showed lower recurrence rates versus TAC. Based on our experience, everolimus provides a reduction in the relative risk of hepatocellular carcinoma recurrence, especially for advanced-stage patients and those with earlier drug administration, higher drug exposure, and longer time on treatment. These data advocate for early everolimus introduction after liver transplantation to reduce the attrition rate consequent to chronic immunosuppression.

Predicting One-year Recurrence of HCC based on Gadoxetic Acid-enhanced MRI by Machine Learning Models.

Accurate prediction of recurrence risk after resction in patients with Hepatocellular Carcinoma (HCC) may help to individualize therapy strategies. This study aimed to develop machine learning models based on preoperative clinical factors and multiparameter Magnetic Resonance Imaging (MRI) characteristics to predict the 1-year recurrence after HCC resection. Eighty-two patients with single HCC who underwent surgery were retrospectively analyzed. All patients underwent preoperative gadoxetic acidenhanced MRI examination. Preoperative clinical factors and MRI characteristics were collected for feature selection. Least Absolute Shrinkage and Selection Operator (LASSO) was applied to select the optimal features for predicting postoperative 1-year recurrence of HCC. Four machine learning algorithms, Multilayer Perception (MLP), random forest, support vector machine, and k-nearest neighbor, were used to construct the predictive models based on the selected features. A Receiver Operating Characteristic (ROC) curve was used to assess the performance of each model. Among the enrolled patients, 32 patients experienced recurrences within one year, while 50 did not. Tumor size, peritumoral hypointensity, decreasing ratio of liver parenchyma T1 value (ΔT1), and α-fetoprotein (AFP) levels were selected by using LASSO to develop the machine learning models. The area under the curve (AUC) of each model exceeded 0.72. Among the models, the MLP model showed the best performance with an AUC, accuracy, sensitivity, and specificity of 0.813, 0.742, 0.570, and 0.853, respectively. Machine learning models can accurately predict postoperative 1-year recurrence in patients with HCC, which may help to provide individualized treatment.

Evaluation of a delayed liver transplantation strategy for patients with HCC receiving bridging therapy: the DELTA-HCC study

To maximize utility and prevent premature liver transplantation (LT), a delayed LT strategy (DS) was adopted in France in 2015 in patients listed for any single HCC treated with resection or thermal-ablation during waiting phase, postponing LT until recurrence. The purpose of this study was to evaluate DS to make sure that it did not hamper pre and post-LT outcomes in DS patients. Patients listed for HCC in France between 2015 and 2018 were studied. After data extraction from the national LT database, 2,025 patients were identified and classified according to 6 groups: single tumor entering DS, single tumor not entering DS, multiple tumors, no curative treatment, untreatable HCC or T1 tumors. 18-months Kaplan-Meier estimates of drop-out for death, too sick to be transplanted or tumor progression before LT, 5-year post-LT HCC recurrence and post LT-survival rates were compared. Median waiting-time in DS group was 910 days. Pre-LT drop-out probability was significantly lower in DS compare to other groups (13% vs 19%, p=0.0043) and significantly higher in the T1 group (25.4%, p=0.05). Post-LT HCC-recurrence rate in multiples nodules group was significantly higher (19.6%, p= 0.019) and post-LT 5-year survival did not differ among groups with 74% in DS group (p=0.22). The DELTA HCC study shows that DS does not negatively impact neither pre- nor post-LT patients 'outcomes, and has the potential to redistribute organs to patients in more urgent need of LT. It can reasonably be proposed and pursued. The unexpected high risk of drop out in T1 patients seems related to the MELD-based driving rules underserving this subgroup, calling for revision of allocation rules. To maximize utility and prevent premature liver transplantation (LT), a delayed LT strategy (DS) was adopted in France in 2015. It consists in postponing LT until recurrence in patients listed for any single HCC curatively treated by surgical resection or thermal ablation. The DELTA HCC study was conducted to evaluate this nationwide strategy. It shows in a non-US, European LT program that DS:- does not negatively impact pre- nor post-LT patients 'outcome,- concerns up to 20% of LT candidates-has therefore the potential to redistribute organs to patients in more urgent need of LT. Such a delayed strategy can reasonably be pursued and extended to other LT programs. Of note, an unexpected high risk of drop out in T1 patients, seemingly related to MELD-based offering rules which underserve these patients, calls for further scrutinization and revision of allocation rules in this subgroup.

Safety and efficacy of early corticosteroid withdrawal in liver transplant recipients: A randomized controlled trial.

Prolonged use of steroids after liver transplantation (LT) significantly increases the risk of diabetes or cardiovascular disease, which can adversely affect patient outcomes. Our study evaluated the effectiveness and safety of early steroid withdrawal within the first year following LT. This study was conducted as an open-label, multicenter, randomized controlled trial. Liver transplant recipients were randomly assigned to one of the following two groups: Group 1, in which steroids were withdrawn two weeks posttransplantation, and Group 2, in which steroids were withdrawn three months posttransplantation. This study included participants aged 20 to 70 years who were scheduled to undergo a single-organ liver transplant from a living or deceased donor at one of the four participating centers. Between November 2012 and August 2020, 115 patients were selected and randomized into two groups, with 60 in Group 1 and 55 in Group 2. The incidence of new-onset diabetes after transplantation (NODAT) was notably higher in Group 1 (32.4%) than in Group 2 (10.0%) in the per-protocol set. Although biopsy-proven acute rejection, graft failure, and mortality did not occur, the median tacrolimus trough level/dose/weight in Group 1 exceeded that in Group 2. No significant differences in safety parameters, such as infection and recurrence of hepatocellular carcinoma, were observed between the two groups. The present study did not find a significant reduction in the incidence of NODAT in the early steroid withdrawal group. Our study suggests that steroid withdrawal three months posttransplantation is a standard and safe immunosuppressive strategy for LT patients.

Preoperative prediction for early recurrence of hepatocellular carcinoma using machine learning-based radiomics.

To develop a contrast-enhanced computed tomography (CECT) based radiomics model using machine learning method and assess its ability of preoperative prediction for the early recurrence of hepatocellular carcinoma (HCC). A total of 297 patients confirmed with HCC were assigned to the training dataset and test dataset based on the 8:2 ratio, and the follow-up period of the patients was from May 2012 to July 2017. The lesion sites were manually segmented using ITK-SNAP, and the pyradiomics platform was applied to extract radiomic features. We established the machine learning model to predict the early recurrence of HCC. The accuracy, AUC, standard deviation, specificity, and sensitivity were applied to evaluate the model performance. 1,688 features were extracted from the arterial phase and venous phase images, respectively. When arterial phase and venous phase images were employed correlated with clinical factors to train a prediction model, it achieved the best performance (AUC with 95% CI 0.8300(0.7560-0.9040), sensitivity 89.45%, specificity 79.07%, accuracy 82.67%, p value 0.0064). The CECT-based radiomics may be helpful to non-invasively reveal the potential connection between CECT images and early recurrence of HCC. The combination of radiomics and clinical factors could boost model performance.

Transcriptomic sequencing analysis of key long noncoding RNAs and mRNAs expression profiles in postoperative recurrence of hepatocellular carcinoma.

Recurrence is the main cause of death in hepatocellular carcinoma (HCC) patients after liver resection. The long non-coding RNAs (lncRNAs) have been reported participated in progression and prognosis of HCC, however, the vital role of lncRNA in postoperative recurrence of HCC has rarely been systematically identified. RNA-sequencing (RNA-seq) was performed between orthotopic model of HCC and hepatoma postoperative recurrent model to comprehensively analyze the integrated transcriptome expression profiles of lncRNA and mRNA. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) was then conducted to quantify the expression levels of DElncRNAs and their target mRNAs. In our study, 211 lncRNAs (P-value < 0.05) and 1125 mRNAs (P-adjust < 0.05) were significantly differentially expressed (DE) between two groups. Moreover, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that DElncRNAs and DEmRNAs were mainly enriched in lipid metabolism, including Arachidonic acid metabolism, PPAR signaling pathway, Steroid hormone biosynthesis, Linoleic acid metabolism, Inflammatory mediator regulation of TRP channels, and Fatty acid degradation. Furthermore, we constructed lncRNA-mRNA interaction networks and protein-protein interaction (PPI) network, and verified by qRT-PCR, suggesting that increased DEIncRNAs (XLOC_063499 and XLOC_042016) may prevent HCC recurrence after surgery by upregulating on targeted cytochrome P450 (CYP) family genes in the lipid metabolism pathway, such as cyp3a16, cyp3a44, cyp2c39, cyp2c40 and cyp2c68. Overall, Our findings provided new insights for further investigation of biological function in lncRNA related HCC recurrence.

Infiltrative Vessel Co-optive Growth Pattern Induced by IQGAP3 Overexpression Promotes Microvascular Invasion in Hepatocellular Carcinoma.

Microvascular invasion (MVI) is a major unfavorable prognostic factor for intrahepatic metastasis and postoperative recurrence of hepatocellular carcinoma (HCC). However, the intervention and preoperative prediction for MVI remain clinical challenges due to the absent precise mechanism and molecular marker(s). Herein, we aimed to investigate the mechanisms underlying vascular invasion that can be applied to clinical intervention for MVI in HCC. The histopathologic characteristics of clinical MVI+/HCC specimens were analyzed using multiplex immunofluorescence staining. The liver orthotopic xenograft mouse model and mechanistic experiments on human patient-derived HCC cell lines, including coculture modeling, RNA-sequencing, and proteomic analysis, were used to investigate MVI-related genes and mechanisms. IQGAP3 overexpression was correlated significantly with MVI status and reduced survival in HCC. Upregulation of IQGAP3 promoted MVI+-HCC cells to adopt an infiltrative vessel co-optive growth pattern and accessed blood capillaries by inducing detachment of activated hepatic stellate cells (HSC) from the endothelium. Mechanically, IQGAP3 overexpression contributed to HCC vascular invasion via a dual mechanism, in which IQGAP3 induced HSC activation and disruption of the HSC-endothelial interaction via upregulation of multiple cytokines and enhanced the trans-endothelial migration of MVI+-HCC cells by remodeling the cytoskeleton by sustaining GTPase Rac1 activity. Importantly, systemic delivery of IQGAP3-targeting small-interfering RNA nanoparticles disrupted the infiltrative vessel co-optive growth pattern and reduced the MVI of HCC. Our results revealed a plausible mechanism underlying IQGAP3-mediated microvascular invasion in HCC, and provided a potential target to develop therapeutic strategies to treat HCC with MVI.

Long-term survival analysis of ultrasound-guided percutaneous microwave ablation for hepatocellular carcinoma conforming to the Milan criteria: primary versus recurrent HCC

Background This study compared long-term outcomes between patients with initial hepatocellular carcinoma (IHCC) and those with recurrent HCC (RHCC) treated with microwave ablation (MWA). Methods This retrospective study included 425 patients with HCCs (294 IHCCs and 131 RHCCs) within the Milan criteria who were treated with ultrasound-guided percutaneous MWA between January 2008 and November 2021. All patients with RHCC had previously undergone MWA for initial HCC. Overall survival (OS) and recurrence-free survival (RFS) rates were compared between the IHCC and RHCC groups before and after propensity score matching (PSM). Results Before matching, the 1-, 3-, 5-, and 10-year OS rates in the IHCC group were 95.9%, 78.5%, 60.2%, and 42.5%, respectively, which were significantly higher than those in the RHCC group (93.8%, 70.0%, 42.0%, and 6.6%, respectively). This difference remained significant after PSM. However, subgroup analyses suggested that there were no significant differences in OS rates between IHCC and RHCC in patients with solitary HCC ≤3.0 cm, AFP ≤200 ng/mL, ablative margins ≥0.5 cm, or Albumin-Bilirubin (ALBI) grade 1. RFS was significantly higher in IHCC than in RHCC before and after PSM, as well as in subgroup analyses. ALBI grade (hazard ratio (HR), 2.38; 95% CI: 1.46–3.86; p < 0.001), serum AFP level (HR, 2.07; 95% CI: 1.19–3.62; p = 0.010) and ablative margins (HR, 0.18; 95% CI: 0.06-0.59; p = 0.005) were independent prognostic factors for OS of RHCC. Serum AFP(HR, 1.29; 95% CI: 1.02–1.63, p = 0.036) level was the only factor associated with RFS in RHCC. Conclusions MWA yielded comparable OS in IHCC and RHCC patients with solitary HCC ≤3.0 cm, AFP ≤200 ng/mL, ablative margins ≥0.5 cm, or ALBI grade 1.

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.

Optimal treatment strategy and prognostic analysis of salvage liver transplantation for patients with earlyhepatocellular carcinoma recurrence after hepatectomy.

We aimed to investigate the prognostic factors for salvage liver transplant in patients with early hepatocellular carcinoma recurrence after hepatectomy. This retrospective analysis included 53 patients who underwent salvage living-donor liver transplantation between January 2007 and January 2018. There were 24 and 29 patients in the early (recurrence ≤24months after primary liver resection) and the late recurrence groups, respectively. In the multivariate Cox regression model, pre-liver transplant downstaging therapy, early recurrence (ER) after primary liver resection , and recurrence-to-liver-transplant ≥12months were independent risks to predict recurrent hepatocellular carcinoma recurrence after salvage living-donor liver transplantation. Compared with the late recurrence group, the ER group showed lower disease-free survival rates (p<0.001); however, the overall survival rates did not differ between the two groups (p=0.355). The 1-, 3-, and 5-year disease-free survival rates were 83.3%, 70.6%, and 66.2%, and 96.0%, 91.6%, and 91.6% in the early and late recurrence groups, respectively. When stratified by recurrence-to-liver transplant time and pre-liver transplant downstaging therapy in the ER group, disease-free survival and overall survival rates were significantly different. ER after primary liver resection with advanced tumor status and a longer period of recurrence-to-liver-transplant (≥12months) have a negative impact on salvage liver transplant. Our findings provide novel recommendations for treatment strategies and eligibility for salvage liver transplant candidates.

Three-dimensional morphology scoring of hepatocellular carcinoma stratifies prognosis and immune infiltration

BackgroundThe morphological attributes could serve as pivotal indicators precipitating early recurrence and dismal overall survival in hepatocellular carcinoma (HCC), and quantifying morphological features may better stratify the prognosis of HCC. ObjectiveTo develop a radiomics approach based on 3D tumor morphology features for predicting the prognosis of HCC and identifying differentially expressed genes related to morphology to guide HCC treatment. Materials and methodsRetrospective study of 357 HCC patients. Radiomic features were extracted from MRI tumor regions; 14 morphology-related features predicted early HCC recurrence and patient stratification via LASSO-Cox modeling. Overall survival (OS) and recurrence-free survival (RFS) were analyzed. RNA sequencing from the Cancer Imaging Archive (TCIA) examined drug sensitivity and stratified HCC using morphological immunity genes, validating recurrence and prognosis. ResultsPatients were split into training (n = 225), test (n = 132), and 50 TCIA dataset cohorts. Two features (Maximum2DdiameterColumn, Sphericity) in Cox regression stratified patients into high/low-risk Morphological Radiological Score (Morph-RS) groups. Significant OS and RFS were seen across all sets. Differentially expressed genes focused on T cell receptor signaling; low-risk group had higher T cells (P = 0.039), B cells (P = 0.041), NK cells (P = 0.018). SN-38, GSK2126458 might treat high-risk morphology. Morphology-immune genes stratified HCC, showing significant RFS/OS differences. ConclusionTumor Morph-RS effectively stratifies HCC patients' recurrence and prognosis. Limited immune infiltration seen in Morph-RS high-risk groups signifies the potential of employing tumor morphology as a potent visual biomarker for diagnosing and managing HCC.

Liver Stiffness Values to Predict Occurrence and Recurrence of Hepatocellular Carcinoma.

Globally, liver cancer is the third most frequent etiology of cancer death, with the rates of occurrence of both new cases and mortality estimated to increase. Given the availability of multiple treatments, interdisciplinary management of the patient is crucial. Moreover, the diagnostic assessment of patients with severe liver fibrosis is essential for the staging of HCC and liver cirrhosis and early diagnosis of HCC. In this context, non-invasive evaluation plays a critical role in identifying prognostic factors of clinical application for the surveillance of the occurrence or recurrence of HCC. The new frontiers of transient elastography have become a useful tool to assess the risk of HCC occurrence and recurrence. There has been a major increase in studies investigating the cutoff liver stiffness value that best predicts the need for monitoring for the onset of HCC. Therefore, this review discusses the new advances that have occurred in the last four years on HCC, highlighting the new frontiers of non-invasive evaluation of HCC subjects, with particular attention regarding the clinical application of liver stiffness assessment for de novo HCC and predicting recurrence in patients with chronic HCV achieving sustained virological response after treatment with direct antiviral agents.

TGF-β1/SMAD3-driven GLI2 isoform expression contributes to aggressive phenotypes of hepatocellular carcinoma

Hedgehog signaling is activated in response to liver injury, and modulates organogenesis. However, the role of non-canonical hedgehog activation via TGF-β1/SMAD3 in hepatic carcinogenesis is poorly understood. TGF-β1/SMAD3-mediated non-canonical activation was found in approximately half of GLI2-positive hepatocellular carcinoma (HCC), and two new GLI2 isoforms with transactivating activity were identified. Phospho-SMAD3 interacted with active GLI2 isoforms to transactivate downstream genes in modulation of stemness, epithelial-mesenchymal transition, chemo-resistance and metastasis in poorly-differentiated hepatoma cells. Non-canonical activation of hedgehog signaling was confirmed in a transgenic HBV-associated HCC mouse model. Inhibition of TGF-β/SMAD3 signaling reduced lung metastasis in a mouse in situ hepatic xenograft model. In another cohort of 55 HCC patients, subjects with high GLI2 expression had a shorter disease-free survival than those with low expression. Moreover, co-positivity of GLI2 with SMAD3 was observed in 87.5% of relapsed HCC patients with high GLI2 expression, indicating an increased risk of post-resection recurrence of HCC. The findings underscore that suppressing the non-canonical hedgehog signaling pathway may confer a potential strategy in the treatment of HCC.

Hepatitis C Virus-Related One-Year Hepatocellular Carcinoma Recurrence After Directly Acting Antivirals: A Randomized Controlled Trial.

Available data on hepatocellular carcinoma (HCC) recurrence after direct-acting antivirals (DAAs) treatment for hepatitis C virus (HCV) are conflicting. No randomized trials were done. This study aims to compare the 1-year HCC recurrence rates in patients who received DAAs after tumor ablation versus those who postponed HCV treatment for 1year. Included patients were randomized after complete HCC ablation into two groups: a postponed DAAs group for whom DAAs initiation was postponed for 12months and a DAAs group who were given sofosbuvir/velpatasvir. Patients were followed for 1year. Eighty-four HCV patients with a mean age of 56.35 ± 8.12years were included; 78.57% of them were males. The number of lesions per patient ranged from 1 to 3 lesions, and the size of the largest lesion ranged from 1.5 to 5cm. There were no statistically significant differences between both groups regarding baseline characteristics. In the DAAs group (43 patients), 11 patients had HCC recurrence, while 25 patients in the postponed DAAs group (41 patients) had HCC recurrence. Using Kaplan-Meier analysis, the 1-year recurrence-free survival (RFS) was significantly higher in the DAAs group (72.2% vs. 38%, P = 0.001). On multivariate analysis, both higher albumin levels (HR 0.147, 95% CI 0.066-0.329) and receiving DAAs (HR 0.358, 95% CI 0.176-0.730) 1year after ablation were associated with significantly lower recurrence. Direct-acting antiviral usage after complete hepatocellular carcinoma ablation significantly decreases the 1-year HCC recurrence rates, but the risk of recurrence is still not eliminated.The study registration number on clinicaltrials.gov : NCT04653818 (initial release on 28/11/2020).