Non-tumor Liver Research Articles

Increased expression of long non-coding RNA FIRRE promotes hepatocellular carcinoma by HuR-CyclinD1 axis signaling

There is a critical need to understand the disease processes and identify improved therapeutic strategies for hepatocellular carcinoma (HCC). The long noncoding RNAs (lncRNAs) display diverse effects on biological regulations. The aim of this study was to identify a lncRNA as a potential biomarker of HCC and investigate the mechanisms by which the lncRNA promotes HCC progression using human cell lines and in vivo. Using RNA-Seq analysis, we found that lncRNA FIRRE was significantly upregulated in hepatitis C virus (HCV) associated liver tissue and identified that lncRNA FIRRE is significantly upregulated in HCV associated HCC compared to adjacent non-tumor liver tissue. Further, we observed that FIRRE is significantly upregulated in HCC specimens with other etiologies, suggesting this lncRNA has a potential to serve as an additional biomarker for HCC. Overexpression of FIRRE in hepatocytes induced cell proliferation, colony formation, and xenograft tumor formation as compared to vector transfected control cells. Using RNA pull-down proteomics, we identified HuR as an interacting partner of FIRRE. We further showed that the FIRRE-HuR axis regulates cyclin D1 expression. Our mechanistic investigation uncovered that FIRRE is associated with an RNA binding protein HuR for enhancing hepatocyte growth. Together, these findings provide molecular insights into the role of FIRRE in HCC progression.

Oncogenic Roles of Laminin Subunit Gamma-2 in Intrahepatic Cholangiocarcinoma via Promoting EGFR Translation.

Intrahepatic cholangiocarcinoma (iCCA) is a highly lethal biliary epithelial cancer in the liver. Here, Laminin subunit gamma-2 (LAMC2) with important oncogenic roles in iCCA is discovered. In a total of 231 cholangiocarcinoma patients (82% of iCCA patients) across four independent cohorts, LAMC2 is significantly more abundant in iCCA tumor tissue compared to normal bile duct and non-tumor liver. Among 26.3% of iCCA patients, LAMC2 gene is amplified, contributing to its over-expression. Functionally, silencing LAMC2 significantly blocks tumor formation in orthotopic iCCA mouse models. Mechanistically, it promotes EGFR protein translation via interacting with nascent unglycosylated EGFR in the endoplasmic reticulum (ER), resulting in activated EGFR signaling. LAMC2-mediated EGFR translation also depends on its interaction with the ER chaperone BiP via their C-terminus. Together LAMC2 and BiP generate a binding "pocket" of nascent EGFR and facilitate EGFR translation. Consistently, LAMC2-high iCCA patients have poor prognosis in two iCCA cohorts. LAMC2-high iCCA cells are highly sensitive to EGFR tyrosine kinase inhibitors (TKIs) treatment both in vitro and in vivo. Together, these data demonstrate LAMC2 as an oncogenic player in iCCA by promoting EGFR translation and an indicator to identify iCCA patients who may benefit from available EGFR-targeted TKIs therapies.

Potential of Using qFibrosis Analysis to Predict Recurrent and Survival Outcome of Patients with Hepatocellular Carcinoma after Hepatic Resection

Background: There remains a lack of studies addressing the stromal background and fibrosis features and their prognostic value in liver cancer. qFibrosis can identify, quantify, and visualize the fibrosis features in biopsy samples. In this study, we aim to demonstrate the prognostic value of histological features by using qFibrosis analysis in liver cancer patients. Methods: Liver specimens from 201 patients with hepatocellular carcinoma (HCC) who underwent curative resection were imaged and assessed using qFibrosis system and generated a total of 33 and 156 collagen parameters from tumor part and non-tumor liver tissue, respectively. We used these collagen parameters on patients to build two combined indexes, RFS index and OS index, in order to differentiate patients with early recurrence and early death, respectively. The models were validated using the leave-one-out method. Results: Both combined indexes had significant prediction value for patients’ outcome. The RFS index of 0.52 well differentiates patients with early recurrence (p p = 0.02). Conclusions: Combined index calculated with qFibrosis from a digital readout of the fibrotic status of peri-tumor liver specimen in patients with HCC has prediction values for their disease and survival outcomes. These results demonstrated the potential to transform histopathological features into quantifiable data that could be used to correlate with clinical outcome.

Abstract 1481: Disparity landscapes of HBV integration in hepatocellular carcinoma: Mechanistic characterization and functional implications

Abstract Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and is a common malignancy worldwide. About half of all new liver cancers worldwide each year occur in China due to a high prevalence of hepatitis B virus (HBV) infection. HBV DNA integrates into the human genome, disrupting the endogenous tumor suppressors/regulatory genes or enhancing the activity of proto-oncogenes. It would be useful to examine the different NGS-based databases to provide a more unbiased and comprehensive survey of HBV integration. We aimed to take advantage of publicly available datasets of different regional cohorts to determine the disparity landscapes of integration events among sample cohorts, tissue types, chromosomal positions, individual host and viral genes, and genic locations. By comparing HCC tumor with non-tumorous liver, landscape of HBV integration was delineated at gene-independent and gene-dependent manners. Moreover, we performed mechanistic investigations on how HBV-TERT integration led to TERT activation and derived a score to predict patients’ prognostication according to their clonal disparity landscape of HBV integration. We revealed a global geographical disparity of HBV integration that the landscape of HBV integration between HCC and non-tumorous liver varied in regional cohorts, suggesting the different degrees of clonal enrichment. In the Mainland China, Hong Kong and Mongolia cohorts, HCC tumors had higher numbers of events than the non-tumorous livers, whereas in the French and US cohorts, the reverse was true. Moreover, most HBV integrations were positionally enriched at the telomeres and centromeres, and this highlighted the novel co-involvement of HBV integration at these regions, which likely introduces greater genomic instability. Furthermore, we constructed a large meta-cohort of multiple ethnicities to refine the landscape of HBV integration. This enabled the identification of events at key HCC-related genes (e.g., TERT, KMT2B, and CCNA2) and gene families (e.g., cyclin, cadherin, and contactin). As TERT is the most frequently integrated gene by HBV, we further investigated the mechanistic modulation of TERT transcription activation by combining and analyzing our in-house sample cohort with a previous report and luciferase reporter assays. This revealed the positive concurrent influence by both the orientation and relative distance of HBV integration (closer to transcription start site) from the TERT gene. Additionally, we observed clonal disparity of HBV integration among patients and the higher level of the clonal disparity score correlated with poor prognostication of HCC patients. Our study uncovered the different levels of clonal enrichment of HBV integration and identified mechanistic insights and prognostic biomarker. This strengthens our understanding of HBV-associated hepatocarcinogenesis. Citation Format: Xueying Lyu, Karen Man-Fong Sze, Joyce Man-Fong Lee, Abdullah Husain, Irene Oi-Lin Ng, Daniel Wai-Hung Ho. Disparity landscapes of HBV integration in hepatocellular carcinoma: Mechanistic characterization and functional implications [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 1481.

Abstract 2710: Combination CXCR4 and PD-1 blockade increases T cell infiltration and effector function in fibrolamellar carcinoma (FLC)

Abstract Introduction: Fibrolamellar carcinoma (FLC), a rare liver cancer affecting young patients without cirrhosis, has a poor prognosis and few systemic therapy options. Current evidence implicates an immunosuppressive tumor microenvironment (TME), including our preliminary data suggesting preferential localization of T cells in stromal bands. We hypothesized that dual blockade of C-X-C chemokine receptor type 4 (CXCR4) and programmed cell death protein (PD-1) would enhance T cell infiltration and activation, respectively, and thereby increase tumor killing. Methods: Flow cytometry and single-nuclear RNA sequencing (snRNA-seq) were used to analyze immune cells and evaluate gene expression, respectively, in patient-derived FLC and non-tumor liver (NTL). Immunohistochemistry (IHC) was used to assess CD3+ T cell localization in relation to CXCL12, the chemokine ligand of CXCR4. Tumor slice cultures (TSCs) from fresh FLC resection specimens (n=11) were treated with control (IgG1), a small molecule CXCR4 inhibitor (AMD3100), anti-PD-1 blocking antibody (αPD-1), or combination CXCR4 and PD-1 blockade (AMD3100/αPD-1). IHC, RNA expression (Nanostring), and live microscopy were used to evaluate T cell mobilization and function. Results: Flow cytometry analysis demonstrated fewer CD45+ cells (16% vs 54% of live cells, p=0.01) and heterogenous PD-1 expression on T cells in FLC compared with NTL. SnRNA-seq analysis showed that CXCR4 expression is upregulated in FLC lymphocyte and macrophage populations relative to NTL (15% vs 10% and 20% vs 5% expressed), and CXCL12 is expressed primarily in stellate cells. CD3+ T cells colocalized with CXCL12 in the FLC stroma on IHC. Multiplex IHC of treated TSCs revealed more T cells in the carcinoma (CK7+) than the stromal (SMA+) compartment after AMD3100 compared with IgG1 (63% vs 34% T cells in CK7+ compartment, p=0.05). Nanostring indicated upregulation of effector pathways (apoptosis, cytotoxicity, lymphocyte activation) after AMD3100/αPD-1 compared with IgG1. Time-lapse live microscopy revealed a greater percentage of apoptotic carcinoma cells (SR-FLICA+CK7+) colocalized with CD3+ T cells (37% vs 18%, p=0.005) and longer CD3+ T cell track length (27 vs 22 μm, p=0.03) after AMD3100/αPD-1 than before treatment. Cleaved caspase-3 (CC3) IHC demonstrated significantly increased tumor apoptosis after AMD3100/αPD-1 compared with IgG1 and monotherapy (53% vs 32% (IgG1), 39% (AMD3100), and 42% (αPD-1) CC3+ cells, p=0.002, p=0.03, p=0.04). Conclusion: The CXCR4/CXCL12 axis mediates T cell exclusion in the FLC TME. Combination CXCR4 and PD-1 blockade overcomes immunosuppression in FLC by mobilizing T cells into the carcinoma compartment and activating their effector function, with resultant tumor apoptosis. These findings support consideration of clinical trials testing drugs targeting CXCR4 and PD-1 in FLC. Citation Format: Lindsay K. Dickerson, Renske van den Bijgaart, Xiuyun Jiang, Sara K. Daniel, Jason A. Carter, Alaa Farghli, Kevin M. Sullivan, Yongjun Liu, Heidi L. Kenerson, Raymond S. Yeung, Teresa S. Kim, Ian N. Crispe, Praveen Sethupathy, Kevin C. Barry, Venu G. Pillarisetty. Combination CXCR4 and PD-1 blockade increases T cell infiltration and effector function in fibrolamellar carcinoma (FLC) [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 2710.

Abstract 6982: The roles of NDRG1 in HCC progression and its clinical significance

Abstract Background: Hepatocellular carcinoma (HCC) is prevalent globally and the third leading cause of cancer-related mortality. N-myc downstream regulated 1 (NDRG1) was found to be tumor promoting in some cancers while suppressive in others. The expression of NDRG1 is induced by stress signals such as DNA damage and hypoxia. In HCC, it has been found to promote proliferation and metastasis. Contradictory results, however, were also suggested on the functions of NDRG1. Methods: Our own HCC cohort and multiple publicly available databases were examined for NDRG1 expression and clinicopathologic correlation. NDRG1 expression under hypoxic conditions was investigated. Various functional assays were performed for NDRG1-knockdown HCC cell lines Huh-7 and PLC/PRF/5. Results: With RT-qPCR analysis on our 102 pairs of patients’ HCC tumor and corresponding non-tumorous liver samples, we found NDRG1 to be upregulated in HCC tumors. Similarly, RNA-sequencing analysis comparing 41 pairs of samples confirmed NDRG1 enrichment in HCC tumors. The upregulation was significantly and positively correlated with the absence of tumor encapsulation and cellular differentiation. These were further supported by TCGA and ICGC databases, in which upregulation of NDRG1 was significantly correlated with poor HCC patient overall survival. Interestingly, in the TCGA database, only 17% of the HCC cases were inferred to have copy number amplification of NDRG1 gene while NDRG1 mutations were rarely found. As hypoxia is a major tumor microenvironmental factor in HCC, we investigated NDRG1’s expression level in HCC cells under hypoxic or normoxic conditions and found significant increase in NDRG1 expression under hypoxic environment. We further found that NDRG1 knockdown inhibited cell proliferation, self-renewal ability, and migration. RT-qPCR analysis found lower LGR5 and SOX2 expression upon NDRG1 knockdown. This is in line with the positive correlation between NDRG1 and various cancer stemness-related gene expression, including LGR5, CD24, MYC, SOX2, and NOTCH1, in TCGA database. Lastly, in the resected HCC tumors on which spatial transcriptomics using Visium 10X and single-cell RNA-sequencing using Chromium 10X were performed, we found NDRG1 enrichment in both tumors cells and cancer associated fibroblasts (CAFs). Conclusion: NDRG1 was found to be significantly upregulated in HCC cells in our in-house cohort and publicly available databases. NDRG1 consistently promotes various oncogenic features of HCC cells, including proliferation, self-renewal ability, and migration. In addition, NDRG1 was found to be enriched in both the HCC cells and CAFs. Citation Format: Jingyi Lu, Lu Tian, Yu-Man Tsui, Xia Wong, Qingyang Zhang, Daniel Wai-Hung Ho, Irene Oi-Lin Ng. The roles of NDRG1 in HCC progression and its clinical significance [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 6982.

Radiomics-Based Classification of Tumor and Healthy Liver on Computed Tomography Images.

Liver malignancies, particularly hepatocellular carcinoma and metastasis, stand as prominent contributors to cancer mortality. Much of the data from abdominal computed tomography images remain underused by radiologists. This study explores the application of machine learning in differentiating tumor tissue from healthy liver tissue using radiomics features. Preoperative contrast-enhanced images of 94 patients were used. A total of 1686 features classified as first-order, second-order, higher-order, and shape statistics were extracted from the regions of interest of each patient's imaging data. Then, the variance threshold, the selection of statistically significant variables using the Student's t-test, and lasso regression were used for feature selection. Six classifiers were used to identify tumor and non-tumor liver tissue, including random forest, support vector machines, naive Bayes, adaptive boosting, extreme gradient boosting, and logistic regression. Grid search was used as a hyperparameter tuning technique, and a 10-fold cross-validation procedure was applied. The area under the receiver operating curve (AUROC) assessed the performance. The AUROC scores varied from 0.5929 to 0.9268, with naive Bayes achieving the best score. The radiomics features extracted were classified with a good score, and the radiomics signature enabled a prognostic biomarker for hepatic tumor screening.

The cytotoxic effects of prazosin, chlorpromazine, and haloperidol on hepatocellular carcinoma and immortalized non-tumor liver cells.

Liver cancer annually accounts for over 800,000 cases and 700,000 deaths worldwide. Hepatocellular carcinoma is responsible for over 80% of liver cancer cases. Due to ineffective treatment options and limited surgical interventions, hepatocellular carcinoma is notoriously difficult to treat. Nonetheless, drugs utilized for other medical conditions, such as the antihypertensive medication prazosin, the neuroleptic medication chlorpromazine, and the neuroleptic medication haloperidol, have gained attention for their potential anti-cancer effects. Therefore, this study used these medications for investigating toxicity to hepatocellular carcinoma while testing the adverse effects on a noncancerous liver cell line model THLE-2. After treatment, an XTT cell viability assay, cell apoptosis assay, reactive oxygen species (ROS) assay, apoptotic proteome profile, and western blot were performed. We calculated IC50 values for chlorpromazine and prazosin to have a molar range of 35-65µM. Our main findings suggest the capability of both of these treatments to reduce cell viability and generate oxidative stress in HepG2 and THLE-2 cells (p value < 0.05). Haloperidol, however, failed to demonstrate any reduction in cell viability revealing no antitumor effect up to 100µM. Based on our findings, a mechanism of cell death was not able to be established due to lack of cleaved caspase-3 expression. Capable of bypassing many aspects of the lengthy, costly, and difficult cancer drug approval process, chlorpromazine and prazosin deserve further investigation for use in conjunction with traditional chemotherapeutics.

Characterization of tumor microbiome and associations with prognosis in intrahepatic cholangiocarcinoma.

The tumor microbiome has been characterized in several malignancies; however, no previous studies have investigated its role in intrahepatic cholangiocarcinoma (ICC). Hence, we explored the tumor microbiome and its association with prognosis in ICC. One hundred and twenty-one ICC tumor samples and 89 adjacent normal tissues were profiled by 16S rRNA sequencing. Microbial differences between tumor and adjacent nontumoral liver tissues were assessed. Tumor microbial composition was then evaluated to detect its association with prognosis. Finally, a risk score calculated by the tumor microbiota was accessed by the least absolute shrinkage and selector operator method (Lasso) to predict prognosis of ICC. The tumor microbiome displayed a greater diversity than that in adjacent nontumoral liver tissues. Tumor samples were characterized by a higher abundance of Firmicutes, Actinobacteria, Bacteroidetes, and Acidobacteriota. Higher tumor microbial α diversity was associated with lymph node metastasis and predicted shortened overall survival (OS) and recurrence-free survival (RFS). A total of 11 bacteria were selected to generate the risk score by Lasso. This score showed potential in predicting OS, and was an independent risk factor for OS. In conclusion, our study characterized the tumor microbiome and revealed its role in predicting prognosis in ICC.

Reversing immunosuppression in the tumor microenvironment of fibrolamellar carcinoma via PD-1 and IL-10 blockade

Fibrolamellar carcinoma (FLC) is a rare liver tumor driven by the DNAJ-PKAc fusion protein that affects healthy young patients. Little is known about the immune response to FLC, limiting rational design of immunotherapy. Multiplex immunohistochemistry and gene expression profiling were performed to characterize the FLC tumor immune microenvironment and adjacent non-tumor liver (NTL). Flow cytometry and T cell receptor (TCR) sequencing were performed to determine the phenotype of tumor-infiltrating immune cells and the extent of T cell clonal expansion. Fresh human FLC tumor slice cultures (TSCs) were treated with antibodies blocking programmed cell death protein-1 (PD-1) and interleukin-10 (IL-10), with results measured by cleaved caspase-3 immunohistochemistry. Immune cells were concentrated in fibrous stromal bands, rather than in the carcinoma cell compartment. In FLC, T cells demonstrated decreased activation and regulatory T cells in FLC had more frequent expression of PD-1 and CTLA-4 than in NTL. Furthermore, T cells had relatively low levels of clonal expansion despite high TCR conservation across individuals. Combination PD-1 and IL-10 blockade signficantly increased cell death in human FLC TSCs. Immunosuppresion in the FLC tumor microenvironment is characterized by T cell exclusion and exhaustion, which may be reversible with combination immunotherapy.

Integrative single-cell transcriptomic analyses reveal the cellular ontological and functional heterogeneities of primary and metastatic liver tumors

BackgroundThe global cellular landscape of the tumor microenvironment (TME) combining primary and metastatic liver tumors has not been comprehensively characterized.MethodsBased on the scRNA-seq and spatial transcriptomic data of non-tumor liver tissues (NTs), primary liver tumors (PTs) and metastatic liver tumors (MTs), we performed the tissue preference, trajectory reconstruction, transcription factor activity inference, cell–cell interaction and cellular deconvolution analyses to construct a comprehensive cellular landscape of liver tumors.ResultsOur analyses depicted the heterogeneous cellular ecosystems in NTs, PTs and MTs. The activated memory B cells and effector T cells were shown to gradually shift to inhibitory B cells, regulatory or exhausted T cells in liver tumors, especially in MTs. Among them, we characterized a unique group of TCF7+ CD8+ memory T cells specifically enriched in MTs that could differentiate into exhausted T cells likely driven by the p38 MAPK signaling. With regard to myeloid cells, the liver-resident macrophages and inflammatory monocyte/macrophages were markedly replaced by tumor-associated macrophages (TAMs), with TREM2+ and UBE2C+ TAMs enriched in PTs, while SPP1+ and WDR45B+ TAMs in MTs. We further showed that the newly identified WDR45B+ TAMs exhibit an M2-like polarization and are associated with adverse prognosis in patients with liver metastases. Additionally, we addressed that endothelial cells display higher immune tolerance and angiogenesis capacity, and provided evidence for the source of the mesenchymal transformation of fibroblasts in tumors. Finally, the malignant hepatocytes and fibroblasts were prioritized as the pivotal cell populations in shaping the microenvironments of PTs and MTs, respectively. Notably, validation analyses by using spatial or bulk transcriptomic data in clinical cohorts concordantly emphasized the clinical significance of these findings.ConclusionsThis study defines the ontological and functional heterogeneities in cellular ecosystems of primary and metastatic liver tumors, providing a foundation for future investigation of the underlying cellular mechanisms.

MicroRNAs as Plasma Biomarkers of Hepatocellular Carcinoma in Patients with Liver Cirrhosis-A Cross-Sectional Study.

Ultrasound screening for hepatocellular carcinoma (HCC) in patients with liver cirrhosis has a poor sensitivity for small tumors. Circulating microRNAs (miRNAs) have been explored as HCC biomarkers, but results are diverging. Here, we evaluate if miRNAs up-regulated in HCC tissue can be detected in plasma and used as screening biomarkers for HCC. In this cross-sectional study, plasma, HCC tissue and surrounding non-tumorous liver tissue were collected from liver resections. Tissue miRNAs were identified and quantitated by RNA-sequencing analysis, and the fold-changes between HCC and surrounding liver tissue were calculated. The miRNAs up-regulated in HCCs were then re-analyzed in plasma from the same patients, and the miRNAs with the highest plasma levels were subsequently measured in plasma from an independent cohort of patients with cirrhosis or HCC. In tissues from 84 resected patients, RNA-sequencing detected 197 differentially expressed miRNAs, 40 of which had a raw count above 200 and were analyzed in plasma from the same cohort. Thirty-one miRNAs were selected for further analysis in 200 patients with HCC or cirrhosis. Of these, eleven miRNAs were significantly increased in HCC as compared to cirrhosis patients. Only miR-93-5p and miR-151a-3p were significantly associated with HCC, with an AUC of 0.662. In comparison, alpha-fetoprotein and des-gamma-carboxy prothrombin yielded an AUC of 0.816, which increased to 0.832 if miR-93-5p and miR-151a-3p were added. When including sex and age, the addition of miR-93-5p and miR-151a-3p did not further improve the AUC (from 0.910 to 0.911). In conclusion, micro-RNAs up-regulated in HCCs are detectable in plasma but have a poor performance as screening biomarkers of HCC.

Liver volumetry in cirrhotic patients with or without hepatocellular carcinoma: Its correlation with Child-Pugh, model for end-stage liver diseases and indocyanine green dye test.

To evaluate the correlation between non-tumoral liver volume (NTLV) by computed tomography (CT) volumetry and indocyanine green retention at 15minutes (ICG-r15%), Child-Pugh score (CTP) and model for end-stage liver diseases (MELD) score in cirrhotic patients having hepatocellular carcinoma (HCC) (group A) and in cirrhotics without HCC (group B). As many as 111 consecutive patients with liver cirrhosis, who underwent triple-phase CT abdomen, were retrospectively included in our study. They were classified into group A (cirrhosis with HCC, n = 69) and group B (cirrhosis only, n = 42). Segmental liver volume, tumor and NTLV were calculated using Myrian XP-Liver segmentation software. In group B, NTLV was the same as the total liver volume (TLV). The correlation of NTLV with ICG-r15%, CTP and MELD scores was analyzed using appropriate correlation tests for each group. NTLV had a good and significant negative correlation with ICG-r15% (ρ = - 512; p < 0.001) in group A, but not in group B. It also had a significant negative correlation with CTP (ρ = - 251; p = 0.038) and MELD (ρ = - 323; p = 0.007) scores only in group A. Furthermore, ICG-r15% had a good and significant positive correlation with CTP and MELD scores in both groups (p < 0.05). NTLV showed a significant negative correlation with ICG-r15% in cirrhotic patients with HCC, but not in cirrhotic patients without HCC. Therefore, CT volumetry can be a valuable tool to predict the functional hepatic volume in patients of cirrhosis with HCC subjected for hepatectomy, where a facility of ICG-r15% is not available. However, further studies are needed to validate our findings in cirrhotic only patients.

Intra-arterial Peptide Receptor Radionuclide Therapy for the Treatment of Hepatic Neuroendocrine Tumor Metastases: Hope or Hype?

Peptide receptor radionuclide therapy (PRRT) confers significant progression-free survival advantage for patients with small bowel grade 1 and 2 well-differentiated neuroendocrine tumors (WD NET). PRRT may also be clinically beneficial for patients with NET of pancreatic, bronchial, and other sites of origin; patients with paragangliomas; as well as for patients with well-differentiated grade 3 NET. Direct intra-arterial (IA) administration of PRRT into the hepatic artery for patients with NET liver metastases may result in higher radiopharmaceutical dose and longer dwell time in the liver tumors while relatively sparing non-tumor liver tissue and other organs such as the kidneys and bone marrow when compared with intravenous (IV) administration. This review summarizes currently available data on IA and IV PRRT dose distribution, reports safety and efficacy of IA PRRT, and proposes future research questions.

Serologic, radiographic, and tissue-based markers associated with major pathologic response after treatment with neoadjuvant immunotherapy in patients with resectable hepatocellular carcinoma.

561 Background: Neoadjuvant immunotherapy can induce a major pathologic response (MPR) in patients with resectable hepatocellular carcinoma (HCC), which may be associated with prolonged recurrence-free survival. This study aimed to understand which variables correlate with achieving an MPR. Methods: Patients with resectable HCC who received either neoadjuvant nivolumab plus ipilimumab or nivolumab alone and underwent surgery were included. 18 patients had baseline and post-treatment computed tomography, alpha-fetoprotein (AFP), and elastography. 17 of these patients had baseline and post-treatment tissue available for immunohistochemistry. Patients were classified into two groups: MPR, defined as necrosis of &gt;70%, and no MPR. Data was summarized using descriptive statistics and compared using Wilcoxon rank sum test. P value &lt;0.05 was considered statistically significant. Results: Statistically significant differences in objective response (OR) and changes in CD8, Granzyme B, and PD-1 expression were identified upon comparison of patients with an MPR to those without an MPR (Table 1). Additionally, numerical differences in baseline tumor size, change in AFP, and both non-tumor and tumor liver fibrosis (at baseline, after treatment, and interim change) were identified upon comparison of patients with an MPR to those without an MPR. Conclusions: Our data show that large baseline tumor size, greater OR, reduction in AFP, increase in liver and tumor fibrosis, and increase in CD8, Granzyme B, and PD-1 expression after receipt of neoadjuvant immunotherapy are associated with achieving an MPR in patients with resectable HCC. [Table: see text]

Rapid predictive dosimetry for radioembolization.

Economics of today's busy clinical practice demand both time and cost-efficient methods of predictive dosimetry for liver radioembolisation. A rapid predictive schema adapted from the Medical Internal Radiation Dose (MIRD) method i.e., Partition Model, has been devised that can be completed within minutes. This rapid schema may guide institutions that do not have access to software capable of comprehensive auto-segmentation of lung, tumour and non-tumorous liver, or where rigorous artery-specific tomographic predictive dosimetry is unfeasible for the routine clinical workflow. This rapid schema is applicable to any beta-emitting radiomicrosphere, although absorbed dose-response thresholds will differ according to device. Sampling errors in lung, tumour and non-tumorous liver will compound and propagate throughout this schema. This rapid schema achieves efficiency in lieu of accuracy. The user must be mindful of potentially large sampling errors and assumes all responsibility. Any suspicion of significant error requires the user to revert back to standard-of-care methods.

PD1 and TIM3 Expression is Associated with Very Early Hepatocellular Carcinoma Recurrence After Percutaneous Thermal Ablation.

Percutaneous thermal ablation (PTA) is a cornerstone in the management of early-stage hepatocellular carcinoma (HCC). However, intrahepatic distant recurrence (IDR) occurs in the majority of patients after PTA. The aim of this study was to evaluate the immune signature associated with very early IDR. Thirty-one patients (26 men, 5 women; mean age:72.4 ± 8.6; age range:57-86 years) who underwent PTA for HCC were included in this study. After PTA for HCC, patients were followed and later divided into two groups: a "very early recurrence" group in case of IDR within 12 months after PTA, and a "prolonged recurrence-free" group in case of no recurrence before 12 months of follow-up. Freshly harvested intratumoral and nontumoral liver tissues and peripheral blood were obtained before PTA and explored by multiparametric flow cytometry. The frequency of PD1+CD4+ T cells was higher in the early recurrence group than in the prolonged recurrence-free group in the peripheral blood (24.3%, IQR: 22.3-36.5 vs 14.0%, IQR: 11.5-16.4, p<0.0001), in the nontumoral liver (37.9%, IQR: 36.0-50.0 vs 22.5%, IQR: 18.0-29.9, p=0.0004), and in the tumor (37.6%, IQR: 32.3-39.3 vs 24.0%, IQR: 20.0-30.3, p=0.0137). Similarly, the frequency of TIM+CD8+ T cells was higher in the very early recurrence group in the peripheral blood (p=0.0021), non-tumoral liver (p=0.0012), and tumor (p=0.0239). The expression of immune checkpoint molecules, such as PD1 and TIM3 on T cells identified HCC patients at risk of very early IDR after PTA who would likely benefit from adjuvant immunotherapy. Thus, our study contributes to a better understanding of the potential association of PTA with adjuvant immunotherapies.

Comprehensive Analysis of Angiogenesis and Ferroptosis Genes for Predicting the Survival Outcome and Immunotherapy Response of Hepatocellular Carcinoma.

Angiogenesis and ferroptosis are both linked to hepatocellular carcinoma (HCC) development, recurrence, and medication resistance. As a result, a thorough examination of the link between genes associated with angiogenesis and ferroptosis and immunotherapy efficacy is required to improve the dismal prognosis of HCC patients. The molecular subtypes were found using a non-negative matrix factorization technique (NMF) based on the genes associated with angiogenesis and ferroptosis. Based on the differentially expressed genes (DEGs) screed between different molecular subtypes, an angiogenesis and ferroptosis-related prognostic stratification model was built using LASSO-COX regression, random forest technique, and extreme gradient boosting (XGBoost), which was further validated in the ICGC and GSE14520 databases. The impact of this model on tumor microenvironment (TME) and immunotherapy sensitivity was also investigated. The expression levels of candidate genes were detected and validated by Real-Time PCR and immunohistochemistry between liver cancer tissues and adjacent non-tumor liver tissues. Both angiogenesis and ferroptosis-related genes can significantly divide HCC patients into two subgroups with different survival outcomes, mutation profiles, and immune microenvironments. We screened six core genes (SLC10A1, PAEP, DPYSL4, MSC, NQO1, and CD24) for the construction of prognostic models by three machine learning methods after intersecting DEGs between angiogenesis and ferroptosis-related subgroups. In both the TCGA, ICGC, and GSE14520 datasets, the model exhibits high prediction efficiency based on the analysis of KM survival curves and ROC curves. Immunomodulatory genes analysis suggested that the model could be used to predict which patients are most likely to benefit from immunotherapy. Furthermore, the transcriptional expression levels of SLC10A1 in the validation experiment matched the outcomes derived from public datasets. We identified a new angiogenesis and ferroptosis-related signature that might offer the molecular characteristic information needed for an efficient prognostic assessment and perhaps tailored treatment for HCC patients.

Comparison of absorbed doses to the tumoral and non-tumoral liver in HCC patients undergoing 99mTc-MAA and 90Y-microspheres radioembolization.

This study aimed to determine the absorbed doses in the tumoral-liver and non-tumoral liver of hepatocellular carcinoma (HCC) patients undergoing radioembolization with Yttrium-90 (90Y) resin microspheres, and compared with those derived from 99mTc-MAA using the partition model. A total of 42 HCC patients (28 males and 14 females, mean age 65 ± 11.51years) who received 45 treatment sessions with 90Y-microspheres between 2016 and 2021 were included. Pre-treatment 99mTc-MAA and post-treatment 90Y-bremsstrahlung SPECT/CT were acquired for each patient. Semi-automated segmentation of regions of interest (ROIs) was performed using MIM Encore software to determine the tumor-liver ratio (TLR) encompassing the liver volume, tumoral-liver, and lungs, and verified by both nuclear medicine physician and interventional radiologist. A partition dosimetry model was used to estimate the administered activity of 90Y-microspheres and the absorbed doses to the tumoral-liver and non-tumoral liver. The student's paired t test and Bland-Altman plot were used for the statistical analysis. The mean TLR values obtained from 99mTc-MAA SPECT/CT and 90Y-bremsstrahlung SPECT/CT were 4.78 ± 3.51 and 2.73 ± 1.18, respectively. The mean planning administered activity of 90Y-microspheres based on 99mTc-MAA SPECT/CT was 1.56 ± 0.80GBq, while the implanted administered activity was 2.53 ± 1.23GBq (p value < 0.001). The mean absorbed doses in the tumoral-liver estimated from 99mTc-MAA and 90Y-bremsstrahlung SPECT/CT were 127.44 ± 4.36Gy and 135.98 ± 6.30Gy, respectively. The corresponding mean absorbed doses in the non-tumoral liver were 34.61 ± 13.93Gy and 55.04 ± 16.36Gy. This study provides evidence that the administered activity of 90Y-microspheres, as estimated from 90Y-bremsstrahlung SPECT/CT, was significantly higher than that estimated from 99mTc-MAA SPECT/CT resulted in increased absorbed doses in both the tumoral-liver and non-tumoral liver. However, 99mTc-MAA SPECT/CT remains a valuable planning tool for predicting the distribution of 90Y-microspheres in liver cancer treatment.

Prophylactic Medication during Radioembolization in Metastatic Liver Disease: Is It Really Necessary? A Retrospective Cohort Study and Systematic Review of the Literature.

Trans-arterial radioembolization is a well-studied tumoricidal treatment for liver malignancies; however, consensus and evidence regarding periprocedural prophylactic medication (PPM) are lacking. A single-center retrospective analysis from 2014 to 2020 was performed in patients treated with 90Y-glass microspheres for neuroendocrine or colorectal liver metastases. Inclusion criteria were the availability of at least 3 months of clinical, biochemical, and imaging follow-up and post-treatment 90Y-PET/CT imaging for the determination of the whole non-tumorous liver absorbed dose (Dh). Logistic regression models were used to investigate if variables (among which are P/UDCA and Dh) were associated with either clinical toxicity, biochemical toxicity, or hepatotoxicity. Additionally, a structured literature search was performed in November 2022 to identify all publications related to PPM use in radioembolization treatments. Fifty-one patients received P/UDCA as post-treatment medication, while 19 did not. No correlation was found between toxicity and P/UDCA use. Dh was associated with biochemical toxicity (p = 0.05). A literature review resulted in eight relevant articles, including a total of 534 patients, in which no consistent advice regarding PPM was provided. In this single-center, retrospective review, P/UDCA use did not reduce liver toxicity in patients with metastatic liver disease. The whole non-tumorous liver-absorbed dose was the only significant factor for hepatotoxicity. No standardized international guidelines or supporting evidence exist for PPM in radioembolization.