Optimizing hepatocellular carcinoma disease staging systems by incorporating tumor micronecrosis: A multi-institutional retrospective study.

Tumor micronecrosis is a pathological feature that reflects malignant biological behavior in hepatocellular carcinoma (HCC). However, whether micronecrosis can optimize HCC staging systems remains unilluminated. A total of 1632 HCC patients who underwent curative hepatectomy in four institutions from January 2014 to December 2021 were enrolled in this study. Independent prognostic factors were identified, and optimized staging models were established using a training cohort (n = 934). The performance of optimized staging models was validated using an external cohort consisting of cases from three other institutions (n = 232). In addition, patients from our prospectively collected database (n = 379) tested the application effectiveness of the models. Harrel's c-statistics and the corrected Akaike information criterion (AICc) were used to assess the performance of staging models. In most of Barcelona Clinic Liver Cancer (BCLC) and tumor (T) stages, HCC patients with tumor micronecrosis showed poorer prognosis than those without. Tumor micronecrosis, microvascular invasion, multiple tumors and tumor size >2 cm were independent prognostic-related factors. The BCLC and T staging models incorporating tumor micronecrosis showed better performance than the original systems (c-statistic, 0.712 and 0.711 vs. 0.664 and 0.679; AICc, 2314.8 and 2322.3 vs. 2338.2 and 2338.1; respectively). Furthermore, the external validation cohort confirmed that the optimized staging models had improved efficiency compared with the original ones. Moreover, the prospective cohort demonstrated the applicability of the optimized staging systems. Tumor micronecrosis plays a stage-ascending role in HCC patients. The BCLC and T staging systems incorporating tumor micronecrosis can improve the prognosis stratification efficiency of patients.

Cuproptosis-related molecular patterns and gene (ATP7A) in hepatocellular carcinoma and their relationships with tumor immune microenvironment and clinical features.

BACKGROUND: Cuproptosis has been studied in various aspects as a new form of cell death. AIMS: We hope to explore the molecular patterns and genes related to cuproptosis in evaluating and predicting the prognosis of hepatocellular carcinoma (HCC), as well as the impact of tumor immune microenvironment. METHODS AND RESULTS: Sixteen cuproptosis related gene (CRGs) and cuproptosis related molecular and gene characteristics were comprehensively analyzed from 492 HCC samples. Cuproptosis related molecular patterns were generated by consensus clustering algorithm, including cuproptosis clusters, cuproptosis gene clusters (CGC) and cuproptosis score (CS). The characteristics of tumor microenvironment (TME) and tumor immune cells were described by the ssGSEA and ESTIMATE algorithms. Cuproptosis score was established to assess the clinical characteristics, prognostic and immunotherapy. The role and mechanism of CRG (ATP7A) in HCC, as well as its relationship with TME and immune checkpoints, have been further explored. The results of somatic mutation, copy number variations (CNV), and CRGs expression in HCC suggested the CRGs might participate in the HCC oncogenesis. The cuproptosis clusters were closely related to the clinical pathological characteristics, biological processes, and prognosis of HCC. The three CGC was revealed to be consistent with the three immune infiltration characterizations, including immune-high, immune-mid, and immune-low subtypes. Higher CS was characterized by decreased TMB, activated immunity, higher immune cell proportion score (IPS) and better overall survival (OS), which indicated higher CS was immune-high type and with better treatment effect and prognosis. The ATP7A had the highest hazard ratio (HR = 1.465, p < .001), was high expression in HCC tissues and with a shorter 5-year OS. Knocking down ATP7A could enhance intracellular copper concentration, cause a decrease in DLAT expression, and induce cuproptosis and inhibit cell proliferation and migration. ATP7A was also positively correlated with most cancer immune cells and immune checkpoints. CONCLUSION: Taken together, this research revealed the cuproptosis related molecular patterns and genes associated with the clinical pathological characteristics, TME phenotype and prognosis of HCC. The CS will further deepen our understanding of the TME characteristics of HCC, and the involvement of ATP7A in cuproptosis will provide new ideas for predicting HCC prognosis and immunotherapy.

Prediction of prognosis, immune infiltration, and personalized treatment of hepatocellular carcinoma by analysis of cuproptosis-related long noncoding RNAs and verification in vitro.

Background: The correlations between cuproptosis and long noncoding RNAs (lncRNAs) with the tumor microenvironment (TME), immunotherapy, and some other characteristics of hepatocellular carcinoma (HCC) remain unclear. Methods: Sixteen cuproptosis regulators and 356 cuproptosis-related lncRNAs (CRLnc) were identified from 374 HCC profiles in The Cancer Genome Atlas (TCGA) database. Six differentially expressed CRLnc were selected, and a prognostic risk model based on the CRLnc signature (CRLncSig) was constructed. The prognostic power of the model was verified. Moreover, a cuproptosis-related gene cluster (CRGC) was generated based on six lncRNAs and differentially expressed genes. The relationship between immune cell infiltration in the TME, immunotherapy, CRLncSig, and CRGC was demonstrated through various algorithms, Tumor Immune Dysfunction and Exclusion (TIDE), tumor mutational burden (TMB), etc. Potential drugs and sensitivity to those agents were evaluated for the risk model. LncRNA AL158166.1 was selected and verified in HCC tissues and cell lines, the impact of its knockdown and overexpression in HCC cells was examined, and the copper (Cu) concentration and the cuproptosis-related gene expression were detected. Results: A CRLncSig prognostic risk model with good predictive ability was constructed. The low-risk group had a longer overall survival (OS), lower tumor purity, more extensive immune cell infiltration, higher immune score, enrichment in immune-activated pathways, and more positive response to immunotherapy versus the high-risk group. CRGC-B exhibited the best OS and the lowest tumor stage; the immune cell infiltration analysis was similar to the low-risk group in CRLncSig. CRGC-B belonged to the "immune-high" group of the TME. The low-risk group had a higher TIDE score and susceptibility to antitumor drugs. The lncRNA AL158166.1 had the highest hazard ratio. The levels of AL158166.1 were higher in HCC tissues versus healthy tissues. Knockdown of AL158166.1 could lead to an increase in intracellular Cu concentration, induce DLAT low expression, and inhibit the proliferation and migration of HCC cells, whereas overexpression of AL158166.1 exerted the reverse effect. Conclusion: Overall, a new CRLncSig prognostic risk model and a cuproptosis-related molecular signature were constructed and evaluated. The model and signature were associated with the prognosis, immune infiltration, and immunotherapy of HCC. Inhibiting the lncRNA AL158166.1 may induce cuproptosis and showed potential for the inhibition of tumors. Evaluation of the CRLnc, CRLncSig, and CRGC may enhance our understanding of the TME, determine the effectiveness of immunotherapy, and act as a marker for the prognosis of HCC.

Low-Pass Genomic Sequencing Reveals Novel Subtypes of Pancreatic Cystic Neoplasms.

BACKGROUND: Over the years, the detection rate of pancreatic cystic neoplasms (PCNs) has significantly increased; however, the differential diagnosis and identification of high-risk PCNs remain challenging. We sought to investigate whether chromosomal instability (CIN) features in cell-free DNA in the cystic fluid of PCNs could help to identify high-risk PCNs. METHODS: Pancreatic cystic fluid samples from 102 patients with PCNs were intraoperatively collected for detection of CIN using an ultrasensitive chromosomal aneuploidy detector. Clinical and imaging data were retrospectively collected, and statistical analysis was performed to assess the potential role of CIN in clinical practice. RESULTS: CIN was investigated in a total of 100 patients. Sixteen of 26 serous cystic cystadenomas (SCAs) harbored deletions of chr3p and/or chr6p, whereas low rates of CIN were detected in mucinous cystic neoplasms. Most malignant PCNs presented with more than one type of CIN; amplification of chr1q and chr8q found in nine and seven of 11 malignant PCNs (81.8% and 63.6%), respectively, could aid in distinguishing high-risk IPMNs from low-risk ones, with a higher sensitivity than imaging. A combination of the mural nodule imaging feature and amplification of chr1q and chr8q achieved a sensitivity of 70.0% and a specificity of 82.4% in identifying high-risk IPMNs. CONCLUSIONS: Our work revealed the distinct CIN signature of different types of PCNs. Deletions of chr3p and chr6p defined a subtype of SCAs. Gains of chr1q and chr8q were associated with insidious malignant PCNs and helped identify high-risk IPMNs.

Tumor micronecrosis predicts poor prognosis of patients with hepatocellular carcinoma after liver transplantation.

BACKGROUND: Tumor micronecrosis is a histopathological feature predicting poor prognosis in patients with hepatocellular carcinoma (HCC) who underwent liver resection. However, the role of tumor micronecrosis in liver transplantation remains unclear. METHODS: We retrospectively reviewed patients with HCC who underwent liver transplantation between January 2015 and December 2021 at our center. We then classified them into micronecrosis(-) and micronecrosis(+) groups and compared their recurrence-free survival (RFS) and overall survival (OS). We identified independent prognostic factors using Cox regression analysis and calculated the area under the receiver operating characteristic curve (AUC) to evaluate the predictive value of RFS for patients with HCC after liver transplantation. RESULTS: A total of 370 cases with evaluable histological sections were included. Patients of the micronecrosis(+) group had a significantly shorter RFS than those of the micronecrosis(-) group (P = 0.037). Shorter RFS and OS were observed in micronecrosis(+) patients without bridging treatments before liver transplantation (P = 0.002 and P = 0.007), while no differences were detected in those with preoperative antitumor therapies that could cause iatrogenic tumor necrosis. Tumor micronecrosis improved the AUC of Milan criteria (0.77-0.79), the model for end-stage liver disease score (0.70-0.76), and serum alpha-fetoprotein (0.63-0.71) for the prediction of prognosis after liver transplantation. CONCLUSION: Patients with HCC with tumor micronecrosis suffer from a worse prognosis than those without this feature. Tumor micronecrosis can help predict RFS after liver transplantation. Therefore, patients with HCC with tumor micronecrosis should be treated with adjuvant therapy and closely followed after liver transplantation. CLINICAL TRIALS REGISTRATION: Not Applicable.

Construction of a m5C-related long non-coding RNA signature for the prognosis of hepatocellular carcinoma.

RNA modification serves as a kind of posttranscriptional modification. Besides N6-methyladenosine (m6A), 5-methylcytosine(m5C) is also an important RNA modification. Long non-coding RNAs (lncRNAs) play an important role in tumor progression. Thus, we performed bioinformatic analysis to establish a m5C-related lncRNA signature(m5ClncSig) for hepatocellular carcinoma (HCC). The RNA sequencing data and clinical data were obtained from The Cancer Genome Atlas (TCGA) database. Pearson correlation coefficient analysis was applied to conduct m5C-related genes and m5C-related lncRNAs co-expressing network. Univariate Cox regression was used to screen the m5C-related lncRNAs with prognosis value. LASSO regression was applied to establish m5ClncSig. Functional analysis including KEGG and GO were performed. The relation between m5ClncSig and immunity was assessed by CIBERSORT and ESTIMATE. RP11-498C9.15 was selected for in vitro validation. A m5ClncSig was established containing 8 lncRNAs with significantly prognosis value. According to risk score calculated by m5ClncSig, high-risk group had worse clinical outcomes than low-risk group. The risk score was validated as an independent prognosis factor. Moreover, the abundances of 11 types of immune cells were significantly different between high-risk group and low-risk group while 8 immune-related genes expressed differently between these two groups. RP11-498C9.15 was validated as a risk factor in HCC progression.

Transcriptomic analysis of graft liver provides insight into the immune response of rat liver transplantation.

Background: As an "immune-privileged organ", the liver has higher rates of both spontaneous tolerance and operational tolerance after being transplanted compared with other solid organs. Also, a large number of patients still need to take long-term immunosuppression regimens. Liver transplantation (LT) rejection involves varieties of pathophysiological processes and cell types, and a deeper understanding of LT immune response is urgently needed. Methods: Homogenic and allogeneic rat LT models were established, and recipient tissue was collected on postoperative day 7. The degree of LT rejection was evaluated by liver pathological changes and liver function. Differentially expressed genes (DEGs) were detected by transcriptome sequencing and confirmed by reverse transcription-polymerase chain reaction. The functional properties of DEGs were characterized by the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome pathway analyses. The cells infiltrating the graft and recipient spleen and peripheral blood were evaluated by immunofluorescence and flow cytometry. Result: A total of 1,465 DEGs were screened, including 1,177 up-regulated genes and 288 down-regulated genes. GO enrichment and KEGG pathway analysis indicated that DEGs were involved in several immunobiological processes, including T cell activation, Th1, Th2 and Th17 cell differentiation, cytokine-cytokine receptor interaction and other immune processes. Reactome results showed that PD-1 signaling was enriched. Further research confirmed that mRNA expression of multiple immune cell markers increased and markers of T cell exhaustion significantly changed. Flow cytometry showed that the proportion of Treg decreased, and that of PD-1+CD4+ T cells and PD-1+CD8+ T cells increased in the allogeneic group. Conclusion: Using an omic approach, we revealed that the development of LT rejection involved multiple immune cells, activation of various immune pathways, and specific alterations of immune checkpoints, which would benefit risk assessment in the clinic and understanding of pathogenesis regarding LT tolerance. Further clinical validations are warranted for our findings.

A Genomic Instability-Related Long Noncoding RNA Signature for Predicting Hepatocellular Carcinoma Prognosis.

Background: Long noncoding RNAs (lncRNAs) are found to be novel biomarkers for hepatocellular carcinoma (HCC) and play an important role in tumor progression. We established a genomic instability-related long noncoding RNA signature (GIlncSig) as an independent prognosis factor and also investigated its impact on prognosis significance. Method: Somatic mutation profiles, clinical characteristics, and RNA sequencing data were obtained from The Cancer Genome Atlas (TCGA) database. Lasso regression was used to construct GIlncSig. KEGG was used to identify the possible biological pathways. ESTIMATE and CIBERSORT algorithms were used to calculate the immune microenvironment scores and proportion of immune cells in HCC patients. The expression of LINC00501 was conducted by qRT-PCR. Cell proliferation was measured by EdU, CCK-8, and colony formation assay, and cell migration and invasion ability were measured by wound healing and transwell assay. Results: 135 genomic instability-related lncRNAs were identified, and GIlncSig was constructed using 13 independent lncRNAs with significant prognosis values. Based on the GIlncSig, high-risk group had worse clinical outcomes than low-risk group, while high-risk group also had higher UBQLN4, KRAS, ARID1A, and PIK3CA expression. Moreover, the efficiency of GIlncSig combining single-gene mutation was higher than single-gene mutation alone such as TP53. The results of CIBERSORT and ESTIMATE showed that GS group and GU group had significantly different immune infiltration. In addition, LINC00501 was identified as a potential biomarker in HCC with strong relationship with clinical characteristics. In vitro assays validated that LINC00501 promoted proliferation and migration of HCC cell lines. Conclusion: Our results showed that GIlncSig serves as a potential independent prognosis factor to predict HCC patients' prognosis for exploring potential mechanism and therapy strategy. Besides, LINC00501 plays an important role in the progression of HCC, which may be a potential therapy target.

Activation of the Aryl Hydrocarbon Receptor Ameliorates Acute Rejection of Rat Liver Transplantation by Regulating Treg Proliferation and PD-1 Expression.

BACKGROUND: Aryl hydrocarbon receptor (AhR) plays important roles in modulating immune responses. However, the role of AhR in rat liver transplantation (LT) has not been explored. METHODS: Safety and side effects of N -(3,4-dimethoxycinnamonyl) anthranilic acid (3,4-DAA) and 2-methyl-2H-pyrazole-3-carboxylic acid amide (CH223191) were evaluated. We used optimal doses of 2 drugs, 3,4-DAA, a drug used for mediating AhR activation, and CH223191, antagonist of AhR (3,4-DAA, CH223191, and 3,4-DAA + CH223191), intraperitoneally administered to recipients daily to investigate the role of AhR in the rat LT model. The recipient livers were used to observe the pathological changes, the cells infiltrating the graft, and changes of AhR and programmed death-1 (PD-1) by Western blot, real-time polymerase chain reaction, and immunofluorescence assays. The contents of Foxp3 + and PD-1 + T cells in the recipient spleen and peripheral blood mononuclear cells were evaluated by flow cytometry. In vitro, after isolating CD4 + T cells, they were treated with different AhR ligands to observe the differentiation direction and PD-1 expression level. RESULTS: The activation of AhR by 3,4-DAA prolonged survival time and ameliorated graft rejection, which were associated with increased expression of AhR and PD-1 in the livers and increased Foxp3 + T cells and PD-1 + T cells in recipient spleens, livers, and peripheral blood mononuclear cells. In vitro, primary T cells incubated with 3,4-DAA mediated increased proportion of Treg and PD-1 + T cells. However, the suppression of AhR with CH223191 reverses these effects, both in the LT model and in vitro. CONCLUSIONS: Our results indicated that AhR activation might reduce the occurrence of rat acute rejection by increasing the proportion of Treg and the expression of PD-1.

Lysophosphatidylcholine acyltransferase 1 promotes epithelial-mesenchymal transition of hepatocellular carcinoma via the Wnt/ß-catenin signaling pathway.

INTRODUCTION AND OBJECTIVES: Hepatocellular carcinoma (HCC) is one of the most malignant digestive tumors, and its insidious onset and rapid progression are the main reasons for the difficulty in effective treatment. Lysophosphatidylcholine acyltransferase 1 (LPCAT1) is a key enzyme that regulates phospholipid metabolism of the cell membrane. However, the mechanism by which LPCAT1 regulates HCC metastasis remains unknown. This study aimed to explore its biological function and potential mechanisms concerning migration and invasion in HCC. MATERIALS AND METHODS: LPCAT1 expression in HCC tissues and its association with clinical outcomes were investigated by western blotting and bioinformatic methods, respectively. The role of LPCAT1 in migration and invasion was assessed via Transwell assays. The expression pattern of epithelial-mesenchymal transition (EMT) markers was quantified by western blotting. The biological behaviors of LPCAT1 in vivo were evaluated using xenograft tumor models and caudal vein metastatic models. Signaling pathways related to LPCAT1 were predicted using gene set enrichment analysis (GSEA) and further confirmed by western blotting. RESULTS: LPCAT1 expression was significantly upregulated in HCC tissues and indicated a poor prognosis of HCC patients. Several EMT-related markers were found to be regulated by LPCAT1. HCC cells overexpressing LPCAT1 exhibited remarkably high migration and invasion capacities, upregulated expression of mesenchymal markers and reduced E-cadherin expression. In vivo, LPCAT1 promoted HCC pulmonary metastasis. Furthermore, the Wnt/ß-catenin signaling pathway was confirmed to be activated by LPCAT1. CONCLUSIONS: LPCAT1 could serve as a promising biomarker of HCC and as a novel therapeutic target for the treatment of metastatic HCC.

TOP2A promotes proliferation and metastasis of hepatocellular carcinoma regulated by miR-144-3p.

Topoisomerase II alpha (TOP2A) is an important nuclear protein which is found in various types of cancers. Whether TOP2A plays an important role in hepatocellular carcinoma (HCC) remains unclear. Through bioinformatic analysis and clinical specimen verification, we found that TOP2A is highly expressed in HCC and is associated with poor prognosis. Knockdown and overexpression of TOP2A can respectively inhibit or promote proliferation, metastasis and invasion of HCC cells in vitro and in vivo. Mechanismly, TOP2A activates cell cycle progression from G2 to M phase by inhibiting the phosphorylation of CHK1 and promotes Epithelial-to-mesenchymal transition (EMT) process. We further confirmed that TOP2A is a direct target of miR-144-3p whose overexpressing can partially reverse the effect of TOP2A in HCC cells. Our data suggested that TOP2A functions by promoting the proliferation, migration, invasion and EMT process of HCC and can be considered as a potential target for the treatment of HCC.

Correlation analysis of RDM1 gene with immune infiltration and clinical prognosis of hepatocellular carcinoma.

PURPOSE: Liver hepatocellular carcinoma (LIHC) is one of the most common primary malignant liver tumors worldwide. The RAD52 motif-containing protein 1 (RDM1) has been shown to play a role in mediating DNA damage repair and homologous recombination. The present study was designed to determine the expression of RDM1 and its prognostic value as well as its relationship with immune infiltration in LIHC patients. METHODS: Oncomine and Tumor Immunoassay Resource were used to assess the expression of RDM1. PrognoScan and Kaplan-Meier bioinformatics database were used to analyze the impact of clinical influencing factors on prognosis. Finally, the Tumor Immune Assessment Resource (TIMER) and Gene Expression Analysis Interactive Analysis (GEPIA) databases were used to detect the correlation between the expression of RDM1 and expression of marker genes related to immune infiltration. Immunohistochemistry (IHC) method was used to detect the expression level of RDM1 in 90 cases of hepatocellular carcinoma and adjacent normal liver tissues. RESULTS: RDM1 expression was up-regulated in most cancers. The expression of RDM1 was remarkably higher than that of the corresponding normal control genes in LIHC tissues. The increase in RDM1 messenger RNA (mRNA) expression was closely related to the decreases in overall survival (OS) and progression-free survival (PFS). Additionally, the increase in RDM1 mRNA expression was closely related to the infiltration levels of macrophages, CD8+ T cells and B cells and was positively correlated with a variety of immune markers in LIHC. CONCLUSION: The findings of the present study demonstrate that RDM1 is a potentially valuable prognostic biomarker that can help determine the progression of cancer and is associated with immune cell infiltration in LIHC.

TDO2 Promotes the EMT of Hepatocellular Carcinoma Through Kyn-AhR Pathway.

Tryptophan 2,3-dioxygenase (TDO2), an enzyme involved in tryptophan (Trp) metabolism has been linked with some malignant traits of various cancers. Kyn, the main product of Trp metabolism pathway catalyzed by TDO2 and indoleamine 2,3-dioxygenase (IDO) in tumor cells, was also demonstrated to activate aryl hydrocarbon receptor (AhR), which may regulate cancer growth and invasion in some malignancies. However, whether TDO2 participates in the metastasis and invasion of HCC has not been explored before. The underlying mechanism played by TDO2 in this process still requires further investigation. Here, we demonstrated that overexpression of TDO2 correlates with advanced stage or malignant traits in HCC patients. Knockdown or inhibition of TDO2 suppressed the migration and invasion of HCC cells in vitro and in vivo. Epithelial to mesenchymal transition (EMT) is an essential program happened in the initial phase of cancer metastasis. We found that in HCC cells, TDO2 promoted the EMT process evidenced by altered levels of biomarkers for EMT. Mechanically, TDO2 regulated the Kyn production in HCC cell via activated aryl hydrocarbon receptor (AhR). Together, these results indicate that TDO2 promotes the EMT of hepatocellular carcinoma through activating Kyn-AhR pathway, thereby participating in the metastasis and invasion of HCC.