Application of a single-cell-RNA-based biological-inspired graph neural network in diagnosis of primary liver tumors.

Single-cell technology depicts integrated tumor profiles including both tumor cells and tumor microenvironments, which theoretically enables more robust diagnosis than traditional diagnostic standards based on only pathology. However, the inherent challenges of single-cell RNA sequencing (scRNA-seq) data, such as high dimensionality, low signal-to-noise ratio (SNR), sparse and non-Euclidean nature, pose significant obstacles for traditional diagnostic approaches. The diagnostic value of single-cell technology has been largely unexplored despite the potential advantages. Here, we present a graph neural network-based framework tailored for molecular diagnosis of primary liver tumors using scRNA-seq data. Our approach capitalizes on the biological plausibility inherent in the intercellular communication networks within tumor samples. By integrating pathway activation features within cell clusters and modeling unidirectional inter-cellular communication, we achieve robust discrimination between malignant tumors (including hepatocellular carcinoma, HCC, and intrahepatic cholangiocarcinoma, iCCA) and benign tumors (focal nodular hyperplasia, FNH) by scRNA data of all tissue cells and immunocytes only. The efficacy to distinguish iCCA from HCC was further validated on public datasets. Through extending the application of high-throughput scRNA-seq data into diagnosis approaches focusing on integrated tumor microenvironment profiles rather than a few tumor markers, this framework also sheds light on minimal-invasive diagnostic methods based on migrating/circulating immunocytes.

Proteomic and metabolomic features in patients with HCC responding to lenvatinib and anti-PD1 therapy.

Combination therapy (lenvatinib/programmed death-1 inhibitor) is effective for treating unresectable hepatocellular carcinoma (uHCC). We reveal that responders have better overall and progression-free survival, as well as high tumor mutation burden and special somatic variants. We analyze the proteome and metabolome of 82 plasma samples from patients with hepatocellular carcinoma (HCC; n = 51) and normal controls (n = 15), revealing that individual differences outweigh treatment differences. Responders exhibit enhanced activity in the alternative/lectin complement pathway and higher levels of lysophosphatidylcholines (LysoPCs), predicting a favorable prognosis. Non-responders are enriched for immunoglobulins, predicting worse outcomes. Compared to normal controls, HCC plasma proteins show acute inflammatory response and platelet activation, while LysoPCs decrease. Combination therapy increases LysoPCs/phosphocholines in responders. Logistic regression/random forest models using metabolomic features achieve good performance in the prediction of responders. Proteomic analysis of cancer tissues unveils molecular features that are associated with side effects in responders receiving combination therapy. In conclusion, our analysis identifies plasma features associated with uHCC responders to combination therapy.

Macro CD5L+ deteriorates CD8+T cells exhaustion and impairs combination of Gemcitabine-Oxaliplatin-Lenvatinib-anti-PD1 therapy in intrahepatic cholangiocarcinoma.

Intratumoral immune status influences tumor therapeutic response, but it remains largely unclear how the status determines therapies for patients with intrahepatic cholangiocarcinoma. Here, we examine the single-cell transcriptional and TCR profiles of 18 tumor tissues pre- and post- therapy of gemcitabine plus oxaliplatin, in combination with lenvatinib and anti-PD1 antibody for intrahepatic cholangiocarcinoma. We find that high CD8 GZMB+ and CD8 proliferating proportions and a low Macro CD5L+ proportion predict good response to the therapy. In patients with a poor response, the CD8 GZMB+ and CD8 proliferating proportions are increased, but the CD8 GZMK+ proportion is decreased after the therapy. Transition of CD8 proliferating and CD8 GZMB+ to CD8 GZMK+ facilitates good response to the therapy, while Macro CD5L+-CD8 GZMB+ crosstalk impairs the response by increasing CTLA4 in CD8 GZMB+. Anti-CTLA4 antibody reverses resistance of the therapy in intrahepatic cholangiocarcinoma. Our data provide a resource for predicting response of the combination therapy and highlight the importance of CD8+T-cell status conversion and exhaustion induced by Macro CD5L+ in influencing the response, suggesting future avenues for cancer treatment optimization.

Perioperative complication incidence and risk factors for retroperitoneal neuroblastoma in children: analysis of 571 patients.

BACKGROUND: Surgery plays an important role in the treatment of neuroblastoma. Perioperative complications may impact the course of neuroblastoma treatment. To date, comprehensive analyses of complications and risk factors have been lacking. METHODS: Patients with retroperitoneal neuroblastoma undergoing tumor resection were retrospectively analyzed between 2014 and 2021. The data collected included clinical characteristics, operative details, operative complications and postoperative outcomes. Risk factors for perioperative complications of retroperitoneal neuroblastoma were analyzed. RESULTS: A total of 571 patients were enrolled in this study. Perioperative complications were observed in 255 (44.7%) patients. Lymphatic leakage (28.4%), diarrhea (13.5%), and injury (vascular, nerve and organ; 7.5%) were the most frequent complications. There were three operation-related deaths (0.53%): massive hemorrhage (n = 1), biliary tract perforation (n = 1) and intestinal necrosis (n = 1). The presence of image-defined risk factors (IDRFs) [odds ratio (OR) = 2.09, P < 0.01], high stage of the International Neuroblastoma Risk Group staging system (INRGSS) (OR = 0.454, P = 0.04), retroperitoneal lymph node metastasis (OR = 2.433, P = 0.026), superior mesenteric artery encasement (OR = 3.346, P = 0.003), and inferior mesenteric artery encasement (OR = 2.218, P = 0.019) were identified as independent risk factors for perioperative complications. CONCLUSIONS: Despite the high incidence of perioperative complications, the associated mortality rate was quite low. Perioperative complications of retroperitoneal neuroblastoma were associated with IDRFs, INRGSS, retroperitoneal lymph node metastasis and vascular encasement. Patients with high-risk factors should receive more serious attention during surgery but should not discourage the determination to pursue total resection of neuroblastoma. Video Abstract (MP4 94289 KB).

WDR4/TRIM28 is a novel molecular target linked to lenvatinib resistance that helps retain the stem characteristics in hepatocellular carcinomas.

Hepatocellular carcinoma (HCC) is an aggressive malignancy with few effective treatment options. Lenvatinib is the first-line therapy for HCC but has only limited clinical benefit. Here, we explored the role and mechanism of the WD repeat domain 4 (WDR4) in lenvatinib resistance to improve clinical benefit. We found that lenvatinib-resistant HCC tissues/cells exhibited increased the N7-methylguanosine (m7G) modification and WDR4 expression. By a gain/loss of function experiment, we showed that WDR4 promoted HCC lenvatinib resistance and tumor progress both in vitro and in vivo. By proteomics analysis and RNA immunoprecipitation PCR, we found that tripartite motif protein 28 (trim28) was an important WDR4 target gene. WDR4 promoted TRIM28 expression, further affected target genes expression, and thus increased cell-acquired stemness and lenvatinib resistance. Clinical tissue data showed that TRIM28 expression was correlated with WDR4 levels, and the expression of both was positively correlated with poor prognosis. Our study provides new insight into the role of WDR4, suggesting a potential therapeutic target to enhance the lenvatinib sensitivity of HCC.

Oncogenic miR-93-5p/Gal-9 axis drives CD8 (+) T-cell inactivation and is a therapeutic target for hepatocellular carcinoma immunotherapy.

Evading immune destruction is an emerging hallmark of cancer and a potential key step in tumorigenesis. Immune checkpoint blocker (ICB)-based combination therapies revolutionize the landscape of systemic therapy for HCC. However, the molecular underpinnings governing immune evasion and responses remain unclear. Our study aims to find new regulatory molecules that drive HCC immune escape and tumorigenesis and find new promising immunotherapeutic approaches for HCC. In our study, laser capture microdissection (LCM) and miRNA sequencing combined with in vitro and in vivo experiments identified miR-93-5p as a crucial initiating oncogene during liver progenitor cell (LPC) malignant transformation and immune escape. Mechanistically, miR-93-5p could directly target canonical tumour suppressors such as APC to promote LPC malignant transformation and hepatocarcinogenesis. More importantly, miR-93-5p could induce deviant GAL-9 augmentation to inactivate infiltrated CD8(+) T cells and induce immune evasion by targeting several epigenetic regulators, such as AEBP2, and then regulating H3K4me3/H3K27me3 bivalency. Experiments in C57BL/6 mice demonstrated that blockade of Gal-9 abrogated miR-93-5p-induced HCC progression and improved their prognosis. Clinically, we identified a unique subtype of HCC closely associated with high GAL-9 expression and anti-PD1 treatment resistance. Our study highlights the pivotal role of the miR-93-5p/Gal-9 axis in driving HCC immune escape and tumorigenesis. Blocking GAL-9 is an effective and promising immunotherapeutic approach for HCC.

Loss of 5-hydroxymethylcytosine induces chemotherapy resistance in hepatocellular carcinoma via the 5-hmC/PCAF/AKT axis.

Multidrug resistance is a major challenge in treating advanced hepatocellular carcinoma (HCC). Although recent studies have reported that the multidrug resistance phenotype is associated with abnormal DNA methylation in cancer cells, the epigenetic mechanism underlying multidrug resistance remains unknown. Here, we reported that the level of 5-hydroxymethylcytosine (5-hmC) in human HCC tissues was significantly lower than that in adjacent liver tissues, and reduced 5-hmC significantly correlated with malignant phenotypes, including poor differentiation and microvascular invasion; additionally, loss of 5-hmC was related to chemotherapy resistance in post-transplantation HCC patients. Further, the 5-hmC level was regulated by ten-eleven translocation 2 (TET2), and the reduction of TET2 in HCC contributes to chemotherapy resistance through histone acetyltransferase P300/CBP-associated factor (PCAF) inhibition and AKT signaling hyperactivation. In conclusion, loss of 5-hmC induces chemotherapy resistance through PCAF/AKT axis and is a promising chemosensitivity prediction biomarker and therapeutic target for HCC patients.

The clinical implications and molecular features of intrahepatic cholangiocarcinoma with perineural invasion.

BACKGROUND: Perineural invasion (PNI) is associated with metastasis in malignancies, including intrahepatic cholangiocarcinoma (ICC), and is correlated with poor prognosis. METHODS: The study included three large cohorts: ZS-ICC and TMA cohorts from our team, MSK cohort from a public database, and a small cohort named cohort 4. Prognostic implications of PNI were investigated in MSK cohort and TMA cohort. PNI-related genomic and transcriptomic profiles were analyzed in MSK and ZS-ICC cohorts. GO, KEGG, and ssGSEA analyses were performed. Immunohistochemistry was used to investigate the relationship between PNI and markers of neurons, hydrolases, and immune cells. The efficacy of adjuvant therapy in ICC patients with PNI was also assessed. RESULTS: A total of 30.6% and 20.7% ICC patients had PNI in MSK and TMA cohorts respectively. Patients with PNI presented with malignant phenotypes such as high CA19-9, the large bile duct type, lymph node invasion, and shortened overall survival (OS) and relapse-free survival (RFS). Nerves involved in PNI positively express tyrosine hydroxylase (TH), a marker of sympathetic nerves. Patients with PNI showed high mutation frequency of KRAS and an immune suppressive metastasis prone niche of decreased NK cell, increased neutrophil, and elevated PD-L1, CD80, and CD86 expression. Patients with PNI had an extended OS after adjuvant therapy with TEGIO, GEMOX, or capecitabine. CONCLUSION: Our study deciphered the genomic features and the immune suppressive metastasis-prone niche in ICC with PNI. Patients with PNI showed a poor prognosis after surgery but a good response to adjuvant chemotherapy.

Perioperative and oncologic outcomes of laparoscopic versus open liver resection for combined hepatocellular-cholangiocarcinoma: a propensity score matching analysis.

BACKGROUND: Laparoscopic liver resection (LLR) has now been established as a safe and minimally invasive technique that is deemed feasible for treating hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). However, the role of LLR in treating combined hepatocellular-cholangiocarcinoma (cHCC-CC) patients has been rarely reported. This study aimed to assess the efficacy of LLR when compared with open liver resection (OLR) procedure for patients with cHCC-CC. METHODS: A total of 229 cHCC-CC patients who underwent hepatic resection (34 LLR and 195 OLR patients) from January 2014 to December 2018 in Zhongshan Hospital, Fudan University were enrolled and underwent a 1:2 propensity score matching (PSM) analysis between the LLR and OLR groups to compare perioperative and oncologic outcomes. Overall survival (OS) and recurrence-free survival (RFS) parameters were assessed by the log-rank test and the sensitivity analysis. RESULTS: A total of 34 LLR and 68 OLR patients were included after PSM analysis. The LLR group displayed a shorter postoperative hospital stay (6.61 vs. 8.26 days; p value < 0.001) when compared with the OLR group. No significant differences were observed in the postoperative complications' incidence or a negative surgical margin rate between the two groups (p value = 0.409 and p value = 1.000, respectively). The aspartate aminotransferase (AST), alanine aminotransferase (ALT), and inflammatory indicators in the LLR group were significantly lower than those in the OLR group on the first and third postoperative days. Additionally, OS and RFS were comparable in both the LLR and OLR groups (p value = 0.700 and p value = 0.780, respectively), and similar results were obtained by conducting a sensitivity analysis. CONCLUSION: LLR can impart less liver function damage, better inflammatory response attenuation contributing to a faster recovery, and parallel oncologic outcomes when compared with OLR. Therefore, LLR can be recommended as a safe and effective therapeutic modality for treating selected cHCC-CC patients, especially for those with small tumors in favorable location.

The Intratumoral Bacterial Metataxonomic Signature of Hepatocellular Carcinoma.

Microbiota is implicated in hepatocellular carcinoma (HCC). The spectrum of intratumoral microbiota associated with HCC progression remains elusive. Fluorescence in situ hybridization revealed that microbial DNAs were distributed in the cytosol of liver hepatocytes and erythrocytes. Viable anaerobic or aerobic bacteria were recovered in HCC tissues by fresh tissue culture. We performed a comprehensive DNA sequencing of bacterial 16S rRNA genes in 156 samples from 28 normal liver, 64 peritumoral, and 64 HCC tissues, and the DNA sequencing yielded 4.2 million high-quality reads. Both alpha and beta diversity in peritumor and HCC microbiota were increased compared to normal controls. The most predominant phyla in HCC were Patescibacteria, Proteobacteria, Bacteroidota, Firmicutes, and Actinobacteriota. phyla of Proteobacteria, Firmicutes, and Actinobacteriota, and classes of Bacilli and Actinobacteria, were consistently enriched in peritumor and HCC tissues, while Gammaproteobacteria was especially abundant in HCC tissues compared to normal controls. Streptococcaceae and Lactococcus were the marker taxa of HCC cirrhosis. The Staphylococcus branch and Caulobacter branch were selectively enriched in HBV-negative HCCs. The abundance of Proteobacteria, Gammaproteobacteria, Firmicutes, Actinobacteriota, and Saccharimonadia were associated with the clinicopathological features of HCC patients. The inferred functions of different taxa were changed between the microbiota of normal liver and peritumor/HCC. Random forest machine learning achieved great discriminative performance in HCC prediction (area under the curve [AUC] = 1.00 in the training cohort, AUC = 0.950 for top five class signature, and AUC = 0.943 for the top 50 operational taxonomy units [OTUs] in the validation cohort). Our analysis highlights the complexity and diversity of the liver and HCC microbiota and established a specific intratumoral microbial signature for the potential prediction of HCC. IMPORTANCE Gut microbiome is an important regulator of hepatic inflammation, detoxification, and immunity, and contributes to the carcinogenesis of liver cancer. Intratumoral bacteria are supposed to be closer to the tumor cells, forming a microenvironment that may be relevant to the pathological process of hepatocellular carcinoma (HCC). However, the presence of viable intratumoral bacteria remains unclear. It is worth exploring whether the metataxonomic characteristics of intratumoral bacteria can be used as a potential marker for HCC prediction. Here, we present the first evidence of the existence of viable intratumoral bacteria in HCC using the tissue culture method. We revealed that microbial DNAs were distributed in the cytosol of liver hepatocytes and erythrocytes. We analyzed the diversity, structure, and abundance of normal liver and HCC microbiota. We built a machine learning model for HCC prediction using intratumoral bacterial features. We show that specific taxa represent potential targets for both therapeutic and diagnostic interventions.

Neoadjuvant transcatheter arterial chemoembolization and systemic chemotherapy for the treatment of undifferentiated embryonal sarcoma of the liver in children.

BACKGROUND: Undifferentiated embryonal sarcoma of the liver (UESL) is a rare and aggressive mesenchymal tumor in children. Herein, we describe our experience in neoadjuvant therapy (NAT) and subsequent surgery for the treatment of UESL in children. AIM: To evaluate the efficacy of NAT and explore a new choice for successful operation of UESL in children. METHODS: We retrospectively analyzed six patients newly diagnosed with unresectable UESL who received NAT and then surgery at our center between January 2004 and December 2019. The tumor was considered unresectable if it involved a large part of both lobes of the liver or had invaded the main hepatic vessels or inferior vena cava. The NAT included preoperative transcatheter arterial chemoembolization (TACE) and systemic chemotherapy. The patients were 4 boys and 2 girls with a mean age of 7 years. The longest tumor at presentation ranged from 8.6 to 14.8 cm (mean, 12 cm). Extrahepatic metastases were present in 2 cases. Preoperative systemic chemotherapy was administered 3 wk after TACE. Tumor resection was performed 3 wk after one or two cycles of NAT. The patients received systemic chemotherapy after surgery. RESULTS: All patients successfully underwent NAT and complete resection. The tumor volumes decreased by 18.2%-68.7%, with a mean decrease of 36% after 1 cycle of NAT (t = 3.524, P = 0.017). According to the Response Evaluation Criteria In Solid Tumors criteria, 4 patients had a partial response and underwent surgery, while 2 had stable disease and received another cycle of NAT before surgery. Massive tumor necrosis was seen on pathological examination of the surgical specimen: > 90% necrosis in two, > 50% necrosis in three, and 25% necrosis in 1, with an average of 71.8%. Post-NAT complications included fever, nausea and vomiting, and mild bone marrow suppression. Elevated alanine transaminase levels occurred in all patients, which returned to normal within 7-10 d after treatment. No cardiac or renal toxicity, severe hepatic dysfunction, bleeding and non-target embolization were observed in the patients. The median follow-up period was 8 years with an overall survival of 100%. CONCLUSION: NAT effectively reduced tumor volume, cleared the tumor margin, and caused massive tumor necrosis. This may be a promising choice for successful surgery of UESL in children.

KSR2-14-3-3ζ complex serves as a biomarker and potential therapeutic target in sorafenib-resistant hepatocellular carcinoma.

BACKGROUND: Kinase suppressor of Ras 2 (KSR2) is a regulator of MAPK signaling that is overactivated in most hepatocellular carcinoma (HCC). We sought to determine the role of KSR2 in HCC pathogenesis. METHODS: We tested the level of KSR2 in HCC tissues and cell lines by tissue microarray, qPCR, and western blotting. Functionally, we determined the effects of KSR2 on the proliferation, migration, and invasion of HCC cells through colony formation assays, scratch assays, transwell migration assays, and xenograft tumor models. Co-immunoprecipitation (co-IP) experiments were used to assess the interaction of phospho-serine binding protein 14-3-3ζ and KSR2, and the effects of this interaction on growth and proliferation of human HCC cells were tested by co-overexpression and knockdown experiments. Additionally, we used flow cytometry to examine whether the KSR2 and 14-3-3ζ interaction conveys HCC resistance to sorafenib. RESULTS: KSR2 was significantly upregulated in HCC tissues and cell lines, and high KSR2 expression associated with poor prognosis in HCC patients. KSR2 knockdown significantly suppressed HCC cell proliferation, migration, and invasion in vitro and in vivo. Mechanistically, co-IP experiments identified that 14-3-3ζ complexed with KSR2, and elevated 14-3-3ζ increased KSR2 protein levels in HCC cells. Importantly, Kaplan-Meier survival analysis showed that patients with both high KSR2 and high 14-3-3ζ expression levels had the shortest survival times and poorest prognoses. Interestingly, HCC cells overexpressing both KSR2 and 14-3-3ζ, rather than either protein alone, showed hyperactivated MAPK signaling and resistance to sorafenib. CONCLUSIONS: Our results provide new insights into the pro-tumorigenic role of KSR2 and its regulation of the MAPK pathway in HCC. The KSR2-14-3-3ζ interaction may be a therapeutic target to enhance the sorafenib sensitivity of HCC.

Current applications and future perspective of CRISPR/Cas9 gene editing in cancer.

Clustered regularly interspaced short palindromic repeats (CRISPR) system provides adaptive immunity against plasmids and phages in prokaryotes. This system inspires the development of a powerful genome engineering tool, the CRISPR/CRISPR-associated nuclease 9 (CRISPR/Cas9) genome editing system. Due to its high efficiency and precision, the CRISPR/Cas9 technique has been employed to explore the functions of cancer-related genes, establish tumor-bearing animal models and probe drug targets, vastly increasing our understanding of cancer genomics. Here, we review current status of CRISPR/Cas9 gene editing technology in oncological research. We first explain the basic principles of CRISPR/Cas9 gene editing and introduce several new CRISPR-based gene editing modes. We next detail the rapid progress of CRISPR screening in revealing tumorigenesis, metastasis, and drug resistance mechanisms. In addition, we introduce CRISPR/Cas9 system delivery vectors and finally demonstrate the potential of CRISPR/Cas9 engineering to enhance the effect of adoptive T cell therapy (ACT) and reduce adverse reactions.

Amplification of spatially isolated adenosine pathway by tumor-macrophage interaction induces anti-PD1 resistance in hepatocellular carcinoma.

BACKGROUND: Immune checkpoint blockade resistance narrows the efficacy of cancer immunotherapies, but the underlying mechanism remains elusive. Delineating the inherent mechanisms of anti-PD1 resistance is important to improve outcome of patients with advanced HCC. METHOD: The level of cricTMEM181 was measured in HCC patients with anti-PD1 therapy by RNA sequencing and then confirmed by qPCR and Sanger sequencing. Immune status in tumor microenvironment of HCC patients or mice models was evaluated by flow cytometry and IHC. Exosomes from HCC cell lines were isolated by ultracentrifugation, and their internalization by macrophage was confirmed by immunofluorescence. The underlying mechanism of HCC-derived exosomal circTMEM181 to macrophage was confirmed by SILAC, RNA FISH and RNA immunoprecipitation. The ATP-ADO pathway amplified by HCC-macrophage interaction was evaluated through ATP, AMP and ADO measurement and macrophage-specific CD39 knockout mice. The role of circTMEM181 in anti-PD1 therapy and its clinical significance were also determined in our retrospective HCC cohorts. RESULTS: Here, we found that circTMEM181 was elevated in hepatocellular carcinoma (HCC) patients responding poorly to anti-PD1 therapy and in HCC patients with a poor prognosis after operation. Moreover, we also found that high exosomal circTMEM181 favored the immunosuppressive microenvironment and endowed anti-PD1 resistance in HCC. Mechanistically, exosomal circTMEM181 sponged miR-488-3p and upregulated CD39 expression in macrophages. Using macrophage-specific CD39 knockout mice and pharmacologic approaches, we revealed a novel mode of anti-PD1 resistance in HCC. We discovered that cell-specific CD39 expression in macrophages and CD73 expression in HCC cells synergistically activated the eATP-adenosine pathway and produced more adenosine, thereby impairing CD8+ T cell function and driving anti-PD1 resistance. CONCLUSION: In summary, HCC-derived exosomal circTMEM181 contributes to immunosuppression and anti-PD1 resistance by elevating CD39 expression, and inhibiting the ATP-adenosine pathway by targeting CD39 on macrophages can rescue anti-PD1 therapy resistance in HCC.

CTLA-4 Synergizes With PD1/PD-L1 in the Inhibitory Tumor Microenvironment of Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is highly invasive and carries high mortality due to limited therapeutic strategies. In other solid tumors, immune checkpoint inhibitors (ICIs) target cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD1), and the PD1 ligand PD-L1 has revolutionized treatment and improved outcomes. However, the relationship and clinical significance of CTLA-4 and PD-L1 expression in ICC remains to be addressed. Deciphering CTLA-4 and PD-L1 interactions in ICC enable targeted therapy for this disease. In this study, immunohistochemistry (IHC) was used to detect and quantify CTLA-4, forkhead box protein P3 (FOXP3), and PD-L1 in samples from 290 patients with ICC. The prognostic capabilities of CTLA-4, FOXP3, and PD-L1 expression in ICC were investigated with the Kaplan-Meier method. Independent risk factors related to ICC survival and recurrence were assessed by the Cox proportional hazards models. Here, we identified that CTLA-4+ lymphocyte density was elevated in ICC tumors compared with peritumoral hepatic tissues (P <.001), and patients with a high density of CTLA-4+ tumor-infiltrating lymphocytes (TILsCTLA-4 High) showed a reduced overall survival (OS) rate and increased cumulative recurrence rate compared with patients with TILsCTLA-4 Low (P <.001 and P = .024, respectively). Similarly, patients with high FOXP3+ TILs (TILsFOXP3 High) had poorer prognoses than patients with low FOXP3+ TILs (P = .021, P = .034, respectively), and the density of CTLA-4+ TILs was positively correlated with FOXP3+ TILs (Pearson r = .31, P <.001). Furthermore, patients with high PD-L1 expression in tumors (TumorPD-L1 High) and/or TILsCTLA-4 High presented worse OS and a higher recurrence rate than patients with TILsCTLA-4 LowTumorPD-L1 Low. Moreover, multiple tumors, lymph node metastasis, and high TumorPD-L1/TILsCTLA-4 were independent risk factors of cumulative recurrence and OS for patients after ICC tumor resection. Furthermore, among ICC patients, those with hepatolithiasis had a higher expression of CTLA-4 and worse OS compared with patients with HBV infection or undefined risk factors (P = .018). In conclusion, CTLA-4 is increased in TILs in ICC and has an expression profile distinct from PD1/PD-L1. TumorPD-L1/TILsCTLA-4 is a predictive factor of OS and ICC recurrence, suggesting that combined therapy targeting PD1/PD-L1 and CTLA-4 may be useful in treating patients with ICC.

CircRNA UBAP2 serves as a sponge of miR-1294 to increase tumorigenesis in hepatocellular carcinoma through regulating c-Myc expression.

Circular RNAs (circRNAs) are a class of regulatory RNAs with complex roles in healthy and diseased tissues. However, the oncogenic role of circRNAs in hepatocellular carcinoma (HCC) remains poorly understood, including the mechanisms by which the circular ubiquitin-binding associated protein 2 (circUBAP2) contributes to tumorigenesis. We analyzed the expression of circUBAP2 in 20 paired samples of HCC and healthy tissue as well as in seven HCC cell lines via quantitative real-time polymerase chain reaction. Functional experiments, such as CCK8 viability assays, colony formation assays, wound healing, transwell assays and flow cytometry, were conducted to assess the effects of circUBAP2 in vitro. To further elucidate the mechanisms by which circUBAP2 acts, we conducted dual-luciferase assays, western blots, RNA pull-down assays and rescue experiments. CircUBAP2 was highly upregulated in most HCC tissues and was associated with poor prognosis. HCC patients with high circUBAP2 expression had greater vascular invasion and worse differentiation. Functionally, circUBAP2 overexpression enhanced HCC cell proliferation, migration and invasion and inhibited apoptosis. Furthermore, we found that circUBAP2 upregulated c-Myc expression by sponging miR-1294, thus contributing to hepatocarcinogenesis. Inhibiting circUBAP2 expression in HCC attenuated the oncogenic effects of c-Myc. These findings suggest that circUBAP2 promotes HCC growth and metastasis. CircUBAP2 may have value as an independent prognostic biomarker or as a new target for the treatment of HCC.

TM2D1 contributes the epithelial-mesenchymal transition of hepatocellular carcinoma via modulating AKT/ß-catenin axis.

Various epidemiology studies showed the correlation between Alzheimer's disease (AD) and low incidence of cancer. However, the etiology underlying etiology of AD-related carcinogenesis remains largely elusive. Our study focused on characterizing the role of TM2D1 (TM2 domain containing 1) in hepatocellular carcinoma. TM2D1 is also known as ß-amyloid peptide binding protein and is critical to the pathogenesis of AD. We found that TM2D1 is increasingly expressed in HCC tumors relative to the peritumoral tissues of the matched tumors and high TM2D1 expression predicts unfavorable clinical outcomes. TM2D1 overexpression induced HCC cell proliferation, migration and invasion, which was related to the epithelial-mesenchymal transition (EMT) observed in these cells. Conversely, TM2D1 depletion led to opposite phenotype in HCC. Mechanistically, we found that TM2D1 promoted Akt and ß-catenin hyper-activation, which corresponded with molecular marker change in EMT signaling pathway. Taken together, our results indicated that TM2D1 played an important role in the EMT process in HCC cells by activating AKT and ß-catenin signaling and may become a promising therapeutic target in HCC.

Do the existing staging systems for primary liver cancer apply to combined hepatocellular carcinoma-intrahepatic cholangiocarcinoma?

BACKGROUND: The incidence of combined hepatocellular carcinoma-intrahepatic cholangiocarcinoma (cHCC-ICC) is relatively low, and the knowledge about the prognosis of cHCC-ICC remains obscure. In the study, we aimed to screen existing primary liver cancer staging systems and shed light on the prognosis and risk factors for cHCC-ICC. METHODS: We retrospectively reviewed 206 cHCC-ICC patients who received curative surgical resection from April 1999 to March 2017. The correlation of survival measures with the histological types or with tumor staging systems was determined and predictive values of tumor staging systems with cHCC-ICC prognosis were compared. RESULTS: The histological type was not associated with overall survival (OS) (P = 0.338) or disease-free survival (DFS) (P = 0.843) of patients after curative surgical resection. BCLC, TNM for HCC, and TNM for ICC stages correlated with both OS and DFS in cHCC-ICC (all P < 0.05). The predictive values of TNM for HCC and TNM for ICC stages were similar in terms of predicting postoperative OS (P = 0.798) and DFS (P = 0.191) in cHCC-ICC. TNM for HCC was superior to BCLC for predicting postoperative OS (P = 0.022) in cHCC-ICC. CONCLUSION: The TNM for HCC staging system should be prioritized for clinical applications in predicting cHCC-ICC prognosis.

Paraplegia after transcatheter artery chemoembolization in a child with clear cell sarcoma of the kidney: A case report.

BACKGROUND: Transcatheter arterial chemoembolization (TACE) is a common treatment for inoperable malignant renal tumors. However, a series of complications may follow the TACE treatment. Spinal cord injury caused by the embolization of intercostal or lumbar arteries is extremely rare. CASE SUMMARY: We describe a case with quite uncommon spinal cord injury after TACE in a 3-year-old child with clear cell sarcoma of the kidney. Sensory impairment beneath the T10 dermatomes and paraplegia on the day after TACE were found in this patient. Unfortunately, sustained paraplegia still existed for more than 2 mo after TACE despite the large dose of steroids and supportive therapy. CONCLUSION: We should draw attention to an uncommon complication of paraplegia after TACE treatment in malignant renal tumors. Although it is rare, the result is disastrous.