Genomic and transcriptomic characteristics of 12 novel primary cell lines derived from three patients with cholangiocarcinoma.

Cholangiocarcinoma (CCA) is an aggressive bile duct malignancy with poor prognosis. To improve our understanding of the biological characteristics of CCA and develop effective therapies, appropriate preclinical models are required. Here, we established and characterized 12 novel patient-derived primary cancer cell (PDPC) models using multi-region sampling. At the genomic level of PDPCs, we observed not only commonly mutated genes, such as TP53, JAK3, and KMT2C, consistent with the reports in CCA, but also specific mutation patterns in each cell line. In addition, specific expression patterns with distinct biological functions and pathways involved were also observed in the PDPCs at the transcriptomic level. Furthermore, the drug-sensitivity results revealed that the PDPCs exhibited different responses to the six commonly used compounds. Our findings indicate that the established PDPCs can serve as novel in vitro reliable models to provide a crucial molecular basis for improving the understanding of tumorigenesis and its treatment.

DendroX: multi-level multi-cluster selection in dendrograms.

BACKGROUND: Cluster heatmaps are widely used in biology and other fields to uncover clustering patterns in data matrices. Most cluster heatmap packages provide utility functions to divide the dendrograms at a certain level to obtain clusters, but it is often difficult to locate the appropriate cut in the dendrogram to obtain the clusters seen in the heatmap or computed by a statistical method. Multiple cuts are required if the clusters locate at different levels in the dendrogram. RESULTS: We developed DendroX, a web app that provides interactive visualization of a dendrogram where users can divide the dendrogram at any level and in any number of clusters and pass the labels of the identified clusters for functional analysis. Helper functions are provided to extract linkage matrices from cluster heatmap objects in R or Python to serve as input to the app. A graphic user interface was also developed to help prepare input files for DendroX from data matrices stored in delimited text files. The app is scalable and has been tested on dendrograms with tens of thousands of leaf nodes. As a case study, we clustered the gene expression signatures of 297 bioactive chemical compounds in the LINCS L1000 dataset and visualized them in DendroX. Seventeen biologically meaningful clusters were identified based on the structure of the dendrogram and the expression patterns in the heatmap. We found that one of the clusters consisting of mostly naturally occurring compounds is not previously reported and has its members sharing broad anticancer, anti-inflammatory and antioxidant activities. CONCLUSIONS: DendroX solves the problem of matching visually and computationally determined clusters in a cluster heatmap and helps users navigate among different parts of a dendrogram. The identification of a cluster of naturally occurring compounds with shared bioactivities implicates a convergence of biological effects through divergent mechanisms.

Comut-viz: efficiently creating and browsing comutation plots online.

BACKGROUND: Comutation plot is a widely used visualization method to deliver a global view of the mutation landscape of large-scale genomic studies. Current tools for creating comutation plot are either offline packages that require coding or online web servers with varied features. When a package is used, it often requires repetitive runs of code to adjust a single feature that might only be a few clicks in a web app. But web apps mostly have limited capacity for customization and cannot handle very large genomic files. RESULTS: To improve on existing tools, we identified features that are most frequently adjusted in creating a plot and incorporate them in Comut-viz that interactively filters and visualizes mutation data as downloadable plots. It includes colored labels for numeric metadata, a preloaded palette for changing colors and two input boxes for adjusting width and height. It accepts standard mutation annotation format (MAF) files as input and can handle large MAF files with more than 200 k rows. As a front-end only app, Comut-viz guarantees privacy of user data and no latency in the analysis. CONCLUSIONS: Comut-viz is a highly responsive and extensible web app to make comutation plots. It provides customization for frequently adjusted features and accepts large genomic files as input. It is suitable for genomic studies with more than a thousand samples.

Comprehensive analysis of genomic alterations of Chinese hilar cholangiocarcinoma patients.

BACKGROUND: Cholangiocarcinoma (CCA) is a rare malignant tumor of the biliary system. The heterogeneity of CCA leads to the lack of effective targeted treatment for CCA subtypes. The molecular characteristic of hilar CCA (hCCA) is still unclear. METHODS: A total of 63 hCCA patients were enrolled from Shanghai Eastern Hepatobiliary Surgery Hospital. Formalin-fixed, paraffin-embedded tumor tissues, and matched blood were collected and deep sequencing targeting 450 cancer genes were performed. Tumor mutation burden (TMB) was measured by an algorithm developed in-house. Correlation analysis was performed by Fisher's exact test. RESULTS: The most commonly mutated genes were TP53 (51.7%), NF1 and KRAS (20%, for both), SMAD4 (16.7%), FAT3 and FRS2 (13.3%, for both), NF1 (11.7%), and KMT2C, MDM2, and ATM (10%, for each) in hCCA. ARID1A, GATA6, and PREX2 mutations commonly occurred in female and KMT2C mutations mainly occurred in patients under 60 years old. Statistical analysis showed the association between ARID1A mutation and tumor stage (P = 0.041) and between NF1 mutation and high TMB (P = 0.0095). Furthermore, ARID1B mutation was identified to associate with the poor prognosis of Chinese hCCA patients (P = 0.004). CONCLUSION: The mutational characterization of hCCA is different from both extrahepatic CCA and intrahepatic CCA. ARID1B is a potential biomarker for prognosis prediction of Chinese hCCA patients.

The Clinical Role of Preoperative Serum CA19-9 and Carcinoembryonic Antigen (CEA) Levels in Evaluating the Resectability of Advanced Gallbladder Cancer.

BACKGROUND The present study was designed to study the ability of preoperative serum concentrations of the tumor-associated biomarkers carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9) and adjusted CA19-9 to assess the resectability of advanced gallbladder cancer (GBC). MATERIAL AND METHODS This retrospective study included patients with potentially resectable stage II-IV (AJCC 8th) GBC examined at our institution between January 2012 and December 2016. Receiver operating characteristic (ROC) curve analysis was used to determine the predictive value and optimal cut-off point of tumor-associated biomarkers for curative resection. RESULTS Pathological examination of the 309 patients included in this study found that 169 (54.7%) underwent R0 (curative) resection, whereas 121 (39.2%) underwent R1/2 (non-curative) resection, and 19 (6.1%) were unresectable. The mean serum concentrations of CEA, CA19-9 and adjusted CA19-9 were significantly lower in patients who underwent R0 resection than in the other groups. ROC curve analysis showed that adjusted CA19-9 concentration was better able to predict resectability (area under the curve, 0.774; 95% confidence interval, 0.722-0.826; P<0.001) than total bilirubin, CEA, and CA19-9 concentrations. The optimal cut-off for adjusted CA19-9 concentration was 47.63 U/mL, which had a sensitivity of 69.82%, a specificity of 75%, a positive predictive value of 77.12% and a negative predictive value of 67.31%. CONCLUSIONS Adjusted CA19-9 concentration is an easily calculated parameter superior to CA19-9 and CEA concentrations in predicting the resectability of advanced gallbladder cancer.

Long non-coding RNA EPIC1 promotes cholangiocarcinoma cell growth.

Cholangiocarcinoma (CCA) is the as the most frequently observed biliary tract malignancy, which has low survival rate in addition to constrained treatment options. However, the fundamental molecular mechanism underlying malignant progression of CCA is quite ambiguous. Recent studies reported that long non-coding RNA (lncRNA) might play critical roles in regulating chemo-resistant of multiple types of cancer. In this study, our results indicate that the LncRNA-EPIC1 expression were significantly increased in cholangiocarcinoma tissues, compared to adjacent normal tissues. And also, its expression also increased in several CCA cancer cell lines than that in human normal immortalized cholangiocyte cell. Loss-and-gain of Lnc-EPIC1 contributes to the CCA cell growth, colony formation, cell apoptosis and also cell cycle. Myc has been reported to directly interact with Lnc-EPIC1 in several cancer cells. Myc targets, including Cyclin A/D and CDK9 were downregulated by Lnc-EPIC1 siRNA. Myc knockout also suppresses the CCA cell growth, colony formation and cell apoptosis. However, Lnc-EPIC1 knockdown failed to enhance the Myc-KO-induced suppression of CCA tumor progression. RNA immunoprecipitation (RIP) results showed the direct interaction between Lnc-EPIC1 and Myc. Taken together, our results show that Lnc-EPIC1 promotes CCA cancer progression by targeting Myc.

MicroRNA-125b predicts clinical outcome and suppressed tumor proliferation and migration in human gallbladder cancer.

We intended to investigate the functional role and clinical relevance of microRNA-125b in human gallbladder cancer. Quantitative real-time polymerase chain reaction was used to examine microRNA-125b expression in gallbladder cancer cell lines, and 79 pairs of gallbladder cancer and normal gallbladder clinical tissues. Clinical correlations between tumorous microRNA-125b expression and gallbladder cancer patients' clinicopathological variances or overall survivals were statistically analyzed. In gallbladder cancer cell lines, TYGBK-8 and G-415 cells, microRNA-125b was upregulated to examine its regulatory effect on gallbladder cancer proliferation and migration in vitro. MicroRNA-125b was significantly downregulated in gallbladder cancer cell lines and human gallbladder cancer tumors. MicroRNA-125b in gallbladder cancer was significantly correlated with patients' clinical stage, tumor differentiation, lymph metastasis, and tumor invasion. Low tumorous microRNA-125b expression was also found to be associated with poor overall survivals among gallbladder cancer patients. In vitro studies demonstrated that microRNA-125b upregulation significantly suppressed proliferation and migration in TYGBK-8 and G-415 cells. Tumorous microRNA-125b is an independent prognostic biomarker for patients with gallbladder cancer and possibly acts as a tumor suppressor in gallbladder cancer.

B. thetaiotaomicron-derived acetic acid modulate immune microenvironment and tumor growth in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide, and emerging evidence suggests that the gut microbiota may play a role in its development and progression. In this study, the association between B. thetaiotaomicron, a gut microbiota species, and HCC recurrence, as well as patient clinical outcomes, was investigated. It was observed that B. thetaiotaomicron-derived acetic acid has the potential to modulate the polarization of pro-pro-inflammatory macrophagess, which promotes the function of cytotoxic CD8+ T cells. The increased biosynthesis of fatty acids was implicated in the modulation of pro-inflammatory macrophages polarization by B. thetaiotaomicron-derived acetic acid. Furthermore, B. thetaiotaomicron-derived acetic acid was found to facilitate the transcription of ACC1, a key enzyme involved in fatty acid biosynthesis, through histone acetylation modification in the ACC1 promoter region. Curcumin, an acetylation modification inhibitor, significantly blocked the inhibitory effects of B. thetaiotaomicron and acetic acid on HCC tumor growth. These findings highlight the potential role of gut microbiota-derived acetic acid in HCC recurrence and patient clinical outcomes, and suggest a complex interplay between gut microbiota, immune modulation, fatty acid metabolism, and epigenetic regulation in the context of HCC development. Further research in this area may provide insights into novel strategies for HCC prevention and treatment by targeting the gut microbiota and its metabolites.

HBV infection effects prognosis and activates the immune response in intrahepatic cholangiocarcinoma.

BACKGROUND: The impact of HBV infection on the prognosis of patients with intrahepatic cholangiocarcinoma (ICC) remains uncertain, and the underlying mechanism has not been elucidated. This study aims to explore the potential mechanism via clinical perspectives and immune features. METHODS: We retrospectively reviewed 1308 patients with ICC treated surgically from January 2007 to January 2015. Then, we compared immune-related markers using immunohistochemistry staining to obtain the gene expression profile GSE107943 and related literature for preliminary bioinformatics analysis. Subsequently, we conducted a drug sensitivity assay to validate the role of TNFSF9 in the ICC organoid-autologous immune cell coculture system and in the patient-derived organoids-based xenograft platform. RESULTS: The analysis revealed that tumors in patients without HBV infection exhibited greater size and a higher likelihood of lymphatic metastasis, tumor invasion, and relapse. After resection, HBV-infected patients had longer survival time than uninfected patients (p<0.01). Interestingly, the expression of immune-related markers in HBV-positive patients with ICC was higher than that in uninfected patients (p<0.01). The percentage of CD8+ T cells in HBV-positive tissue was higher than that without HBV infection (p<0.05). We screened 21 differentially expressed genes and investigated the function of TNFSF9 through bioinformatics analyses. The expression of TNFSF9 in ICC organoids with HBV infection was lower than that in organoids without HBV infection. The growth of HBV-negative ICC organoids was significantly inhibited by inhibiting the expression of TNFSF9 with a neutralizing antibody. Additionally, the growth rate was faster in HbsAg (-) ICC patient-derived organoids-based xenograft model than in HbsAg (+) group. CONCLUSIONS: The activation of the immune response induced by HBV infection makes the prognosis of HBV-positive patients with ICC differ from that of uninfected patients.

MiR-20a triggers metastasis of gallbladder carcinoma.

BACKGROUND & AIMS: The dysfunction of miRNAs has been demonstrated to participate in the development of various tumors. However, whether miRNAs are involved in metastasis and progression of gallbladder carcinoma (GBC) remains unknown. METHODS: A new designed gain-of-function miRNA screening technology was applied to filter out pro-metastatic miRNAs in GBC. Their expression in GBC tissues was validated by real-time PCR. The biological functions of miRNAs were intensively studied by transwell, immunoblot, immunohistochemical, and in situ hybridization assays. Tumorigenicity and liver metastasis were further examined in nude mice. RESULTS: Of 880 miRNAs, 17 were filtered out as the prominent metastatic inducers of GBCs. Among them, the upregulation of pro-metastatic miR-20a was closely associated with local invasion, distant metastasis, and poor prognosis of 67 followed-up GBC patients, clinically. Patients with higher miR-20a expression exhibited worse overall survival (OS and median OS time was 5 and 20 months, respectively) than the lower expression group. A dramatically increased TGF-ß1 level was found in GBC patients, which was responsible for the elevation of miR-20a. The ectopic expression of miR-20a could induce epithelial-mesenchymal transition and enhance metastasis of GBC cells in vitro and in vivo, by directly targeting the 3' UTR of Smad7, and subsequently promoting nuclear translocation of ß-catenin. Conversely, the blockage of miR-20a by specific antagomir effectively restored the expression of Smad7 and attenuated TGF-ß-induced cell metastasis. CONCLUSIONS: TGF-ß1-mediated activation of the miR-20a/Smad7/ß-catenin axis plays a pivotal role in the pathogenesis and worse prognosis of GBCs and may serve as a potential therapeutic target in the future.

[Analysis of the efficacy of postoperative radiotherapy in gallbladder cancer].

OBJECTIVE: To summarize the experiences in gallbladder cancer treatment, evaluate the efficacy of postoperative radiotherapy, and investigate the method of improving the survival of gallbladder cancer patients. METHODS: One hundred and twenty-seven gallbladder cancer patients, treated in our center by radical resection (84 cases) and combined with postoperative radiotherapy (43 cases), between June 2003 to December 2009 were included in this study. Their clinical data and follow-up results were retrospectively analyzed. According to AJCC staging criteria, the survival time and 1-, 3- and 5-year survival rates of the surgery group and the postoperative radiotherapy group at the different pathological stages and resection margin status were compared. RESULTS: The median survival time of postoperative radiotherapy patients in stage III was 16.9 months, and the 1-year, 3-year, and 5-year survival rates were 55.7%, 23.5% and 18.2%, respectively, significantly higher than that of the simple operation group ( median survival time 14.3 months, and 1-year, 3-year, 5-year survival rates 42.7%, 22.6% and 16.7%, respectively) (P<0.05). The median survival time of postoperative radiotherapy patients in stage IV, the median survival time was 9.7 months in the postoperative radiotherapy group and 6.3 months in the simple surgery group, and the 1-year survival rates were 14.2% and 9.8%, the 3-year survival rates were 7.2% and 3.9%, the 5-year survival rates were 7.2% and 1.9%, respectively, all showing a statistically significant difference (P<0.05). Among the stage III and IVpatients, all the 1-, 3- and 5-year survival rates of the postoperative radiotherapy group were higher than that of the simple R0 and R1 surgical resection group (all P<0.05), but with a non-significant difference between the stageIandIIpatients (P>0.05). The main side effects in postoperative radiotherapy patients including nausea, vomiting and abdominal pain, all were successfully alleviated by symptomatic and supportive therapy, and the radiotherapy was successfully completed. CONCLUSIONS: With regard to the gallbladder cancer patients in stage III and IV, the survival rate can be obviously increased by postoperative radiotherapy. However, for patients in stageIand II, whether postoperative radiotherapy significantly improves the survival or not, needs to be further validated in larger scale studies.

Structural changes and anti-hepatocellular carcinoma activity of interferon-γ after interaction with sinensetin.

Exploring the interaction of small molecules with therapeutic proteins can provide useful information about development of ligand-protein complexes as synergistically therapeutic platforms. In this study, the interaction of sinensetin with human interferon gamma (IFNγ) was evaluated experimentally and theoretically. Also, the synergistic effects of IFNγ- sinensetin complex on the inhibition of hepatocellular carcinoma HepG2 cell proliferation were assessed by cell viability and quantitative real time PCR assays. It was realized that sinensetin interacts with IFNγ through a static quenching mechanism and hydrophobic forces mediated by presence of Lys55 and Lys58 amino acid residues in the binding site were the main contributing forces in the spontaneous formation of IFNγ-sinensetin complex. Also, the interaction of sinensetin with IFNγ did not induce a significant change in the secondary and tertiary structure of the protein. Cellular assays revealed a synergistic effect of sinensetin on IFNγ -triggered anticancer action in HepG2 cells through overexpression of caspase-3 mRNA and protein. In conclusion, this study may hold great promise for the development of potential ligand- protein complexes for therapeutic purposes.

Tertiary lymphoid structures predict the prognosis and immunotherapy response of cholangiocarcinoma.

Introduction: Cholangiocarcinoma (CCA) is a malignant tumor of the biliary epithelium with a poor prognosis. The lack of biomarkers to predict therapeutic response and prognosis is one of the major challenges for CCA treatment. Tertiary lymphoid structures (TLS) provide a local and pivotal microenvironment for tumor immune responses. The prognostic value and clinical relevance of TLS in CCA remain unclear. We aimed to explore the characteristics and clinical significance of TLS in CCA. Methods: We investigated the prognostic value and clinical relevance of TLS in CCA using a surgery cohort containing 471 CCA patients (cohort 1) and an immunotherapy cohort containing 100 CCA patients (cohort 2). Hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining were used to evaluate the maturity of TLS. Multiplex IHC (mIHC) was employed to characterize the composition of TLS. Results: Different maturity of TLS were observed in CCA tissue sections. Strong staining of the four-gene signature including PAX5, TCL1A, TNFRSF13C, and CD79A were found in TLS regions. A high density of intra-tumoral TLS (T-score high) were significantly correlated with longer overall survival (OS) both in CCA cohort 1 (p = 0.002) and cohort 2 (p = 0.01), whereas a high density of peri-tumoral TLS (P-score high) were associated with shorter OS in these two cohorts (p = 0.003 and p = 0.03, respectively). Conclusion: The established four-gene signature efficiently identified the TLS in CCA tissues. The abundance and spatial distribution of TLS were significantly correlated with the prognosis and immune checkpoint inhibitors (ICIs) immunotherapy response of CCA patients. The presence of intra-tumoral TLS are positive prognostic factors for CCA, which provide a theoretical basis for the future diagnosis and treatment of CCA.

A DNA-Methylation-Driven Genes Based Prognostic Signature Reveals Immune Microenvironment in Pancreatic Cancer.

Pancreatic cancer (PACA), which is characterized by an immunosuppressive nature, remains one of the deadliest malignancies worldwide. Aberrant DNA methylation (DNAm) reportedly influences tumor immune microenvironment. Here, we evaluated the role of DNA methylation driven genes (MDGs) in PACA through integrative analyses of epigenomic, transcriptomic, genomic and clinicopathological data obtained from TCGA, ICGC, ArrayExpress and GEO databases. Thereafter, we established a four-MDG signature, comprising GPRC5A, SOWAHC, S100A14, and ARNTL2. High signature risk-scores were associated with poor histologic grades and late TNM stages. Survival analyses showed the signature had a significant predictive effect on OS. WGCNA revealed that the signature may be associated with immune system, while high risk-scores might reflect immune dysregulation. Furthermore, GSEA and GSVA revealed significant enrichment of p53 pathway and mismatch repair pathways in high risk-score subgroups. Immune infiltration analysis showed that CD8+ T cells were more abundant in low score subgroups, while M0 macrophages exhibited an opposite trend. Moreover, negative regulatory genes of cancer-immunity cycle (CIC) illustrated that immunosuppressors TGFB1, VEGFA, and CD274 (PDL1) were all positively correlated with risk-scores. Furthermore, the four signature genes were negatively correlated with CD8+ lymphocytes, but positively associated with myeloid derived suppressor cells (MDSC). Conversely, specimens with high risk-scores exhibited heavier tumor mutation burdens (TMB) and might show better responses to some chemotherapy and targeted drugs, which would benefit stratification of PACA patients. On the other hand, we investigated the corresponding proteins of the four MDGs using paraffin-embedded PACA samples collected from patients who underwent radical surgery in our center and found that all these four proteins were elevated in cancerous tissues and might serve as prognostic markers for PACA patients, high expression levels indicated poor prognosis. In conclusion, we successfully established a four-MDG-based prognostic signature for PACA patients. We envisage that this signature will help in evaluation of intratumoral immune texture and enable identification of novel stratification biomarkers for precision therapies.

Novel protein kinase inhibitor TT-00420 inhibits gallbladder cancer by inhibiting JNK/JUN-mediated signaling pathway.

PURPOSE: This study aimed to investigate the efficiency of our chemically synthesized TT-00420, a novel spectrum-selective multiple protein kinase inhibitor, in cultured cells and animal models of gallbladder cancer (GBC) and explore its potential mechanism. METHODS: Multiple GBC models were established to assess the anti-tumor efficiency, toxicity, and pharmacokinetics of TT-00420. Integrated transcriptomic, proteomic and phosphoproteomic analysis was conducted to identify potential downstream effectors of TT-00420. Western blotting, qRT-PCR, nuclear-cytoplasm separation, and immunofluorescence were performed to confirm the multi-omic results and explore the molecular mechanism of TT-00420. Immunohistochemistry was used to detect FGFR1 and p-FGFR1 expression levels in GBC samples. Autodock software was utilized to investigate the potential binding mode between the TT-00420 and the human FGFR1. RESULTS: We found that TT-00420 exerted potent growth inhibition of GBC cell lines and multiple xenograft models. Treatment of mice with 15 mg/kg TT-00420 via gavage displayed a half-life of 1.8 h in the blood and rapid distribution to the liver, kidneys, lungs, spleen, and tumors at 0.25 h, but no toxicity to these organs over 2 weeks. Multi-omic analysis revealed c-Jun as a potential downstream effector after TT-00420 treatment. Mechanistically, TT-00420 showed rigorous ability to block FGFR1 and its downstream JNK-JUN (S63/S73) signaling pathway, and induce c-Jun S243-dependent MEK/ERK reactivation, leading to FASLG-dependent tumor cell death. Finally, we found that FGFR1 and p-FGFR1 expression was elevated in GBC patients and these levels correlated with decreased patient survival. CONCLUSIONS: TT-00420 shows potent antitumor efficacy and may serve as a novel agent to improve GBC prognosis.

Landscape of somatic alterations in large-scale solid tumors from an Asian population.

Extending the benefits of tumor molecular profiling for all cancer patients requires a comprehensive analysis of tumor genomes across distinct patient populations worldwide. In this study, we perform deep next-generation DNA sequencing (NGS) from tumor tissues and matched blood specimens from over 10,000 patients in China by using a 450-gene comprehensive assay, developed and implemented under international clinical regulations. We perform a comprehensive comparison of somatically altered genes, the distribution of tumor mutational burden (TMB), gene fusion patterns, and the spectrum of various somatic alterations between Chinese and American patient populations. Here, we show 64% of cancers from Chinese patients in this study have clinically actionable genomic alterations, which may affect clinical decisions related to targeted therapy or immunotherapy. These findings describe the similarities and differences between tumors from Chinese and American patients, providing valuable information for personalized medicine.

Cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy vs. cytoreductive surgery alone for intrahepatic cholangiocarcinoma with peritoneal metastases: A retrospective cohort study.

BACKGROUND: Cytoreductive surgery (CRS) combined with hyperthermic intraperitoneal chemotherapy (HIPEC) has survival benefits in patients with intraperitoneal malignant lesions, but there is no study specific to intrahepatic cholangiocarcinoma (ICC). PURPOSE: To compare the prognosis of patients with advanced ICC undergoing CRS + HIPEC compared with CRS alone. METHODS: This study was a retrospective cohort study of patients with advanced ICC treated at the Shanghai Eastern Hepatobiliary Surgery Hospital between 01/2014 and 12/2018. The patients were divided into either CRS + HIPEC or CRS group based on the treatment they received. Overall survival (OS), complications, hospital stay, biochemical indicators, tumor markers, and number of HIPEC were examined. RESULTS: There were 51 and 61 patients in the CRS + HIPEC and CRS groups, respectively. There were no differences between the groups regarding preoperative CA19-9 levels (421 ± 381 vs. 523 ± 543 U/mL, P = 0.208). The hospital stay was longer in the CRS + HIPEC group (22.2 ± 10.0 vs. 18.6 ± 7.6 days, P = 0.033). The occurrence of overall complications was similar in the two groups (37.2% vs. 34.4%, P = 0.756). The postoperative CA19-9 levels were lower in the CRS + HIPEC group compared with the CRS group (196 ± 320 vs. 337 ± 396 U/mL, P = 0.044). The median OS was longer in the CRS + HIPEC group than in the CRS group (25.53 vs. 11.17 months, P < 0.001). Compared with the CRS group, the CRS + HIPEC group showed a higher occurrence of leukopenia (7.8% vs. 0, P = 0.040) but a lower occurrence of total bilirubin elevation (15.7% vs. 37.7%, P = 0.032). CONCLUSION: CRS + HIPEC could be a treatment option for patients with advanced ICC, with improved OS and similar complications and adverse events compared with CRS alone.

Establishment and characterization of 38 novel patient-derived primary cancer cell lines using multi-region sampling revealing intra-tumor heterogeneity of gallbladder carcinoma.

Gallbladder carcinoma (GBC) is a lethal biliary tract malignant neoplasm. Patient-derived primary cancer cell lines (PDPCs) are appropriate models to explore biological characteristics and potential therapeutics; however, there is a lack of PDPCs in GBC. In this study, we aimed to establish and characterize the GBC PDPCs, and further investigated the intra-tumor heterogeneity (ITH). Multi-region sampling (3-9 regions) of the operable tumor tissue samples was used to establish PDPCs. Short tandem repeat genotyping for cell authentication and karyotyping was performed, followed by whole-exome sequencing and RNA sequencing to assess the ITH at the genetic and transcriptional levels, respectively. Thirty-eight PDPCs were successfully established from seven GBC patients and characterized. ITH was observed with a median of 38.3% mutations being heterogeneous (range, 26.6-59.4%) across all patients. Similar with other tumor types, TP53 mutations were always truncal. In addition, there were three genes, KMT2C, CDKN2A, and ARID1A, with truncal mutations in at least two patients. A median of 370 differentially expressed genes (DEGs) was identified per patient. Distinct expression patterns were observed between major histocompatibility complex (MHC) class I and II genes. We found the expression of MHC class II genes in the PDPC samples was closely regulated by CIITA, while that of MHC class I genes were not correlated with CIITA expression. The PDPCs established from GBC patients can serve as novel in vitro models to identify the ITH, which may pave a crucial molecular foundation for enhanced understanding of tumorigenesis and progression.

Sorafenib extends the survival time of patients with multiple recurrences of hepatocellular carcinoma after liver transplantation.

AIM: to determine the efficacy and toxicities of sorafenib in the treatment of patients with multiple recurrences of hepatocellular carcinoma (HCC) after liver transplantation in a Chinese population. METHODS: twenty patients with multiple recurrences of HCC after liver transplantation were retrospectively studied. They received either transarterial chemoembolization (TACE) or TACE combined with sorafenib. RESULTS: the median survival times (MST) after multiple recurrences was 14 months (TACE+sorafenib group) and 6 months (TACE only group). The difference was significant in MST between the two groups (P=0.005). The TACE + sorafenib group had more stable disease (SD) patients than the TACE group. The most frequent adverse events of sorafenib were hand-foot skin reaction and diarrhea. In the univariate analysis, preoperative bilirubin and CHILD grade are found to be significantly associated with tumor-free survival time, the survival time after multiple recurrences and overall survival time. TACE+sorafenib group showed a better outcome than single TACE treatment group. In the multivariate COX regression modeling, the preoperative high CHILD grade was found to be a risk factor of tumor-free survival time. In addition, the preoperative high bilirubin grade was also found to be a risk factor of survival time after recurrence and overall survival time. Furthermore, survival time after recurrence and overall survival time were also associated with therapeutic schedule, which was indicated by the GROUP. CONCLUSION: Treatment with TACE and sorafenib is worthy of further study and may have more extensive application prospects.

Effect of miR­449a­mediated Notch signaling pathway on the proliferation, apoptosis and invasion of papillary thyroid carcinoma cells.

The present study aimed to investigate the effect of miR­449a­mediated Notch signaling pathway on the proliferation, apoptosis and invasion of papillary thyroid carcinoma cells. Human papillary thyroid carcinoma cell line TPC­1 was selected, and cells were grouped and transfected: Control group (without any treatment), negative control (NC) group (transfection with NC plasmid), miR­449a mimic group (transfection with miR­449a mimic), miR­449a inhibitor group (transfection with miR­449a inhibitor), DAPT group (addition of γ­secretase inhibitor DAPT to inhibit the Notch signaling pathway), and miR­449a inhibitor + DAPT group (transfection with miR­449a inhibitor and addition of DAPT). The target relationship between miR­449a and Notch1 was detected by dual­luciferase reporter assay. qRT­PCR and western blotting were used to assess the expression of miR­449a, Notch1 and Jagged1 in cells. Cell proliferation was detected using EdU; the cell cycle and apoptosis were detected by flow cytometry; cell invasion ability was detected by Transwell assay. PCNA, MMP­2, MMP­9, Bcl­2 and Bax mRNA and protein expression were assessed by qRT­PCR and western blotting. The results revealed that miR­449a negatively regulated Notch1. Compared with the control group, there was significantly increased miR­449a expression in the miR­449a mimic group, and there was significantly decreased expression of Notch1, Jagged1, PCNA, MMP­2, MMP­9 and Bcl­2, increased Bax, reduced cell proliferation, increased G1­phase cell fraction, decreased S­phase cell fraction, an increased apoptosis rate, and decreased invasion ability in the miR­449a mimic group and DAPT group (all P<0.05). However, the results in the miR­449a inhibitor group were the opposite of those in miR­449a mimic group (all P<0.05). There was no significant difference in cell proliferation, apoptosis and invasion in the NC group and miR­449a inhibitor + DAPT group compared to the control group (all P>0.05). miR­449a overexpression can inhibit Notch signaling pathway, thereby inhibiting the proliferation and invasion of papillary thyroid carcinoma cells and promoting cell apoptosis.