Significant response to pembrolizumab plus lenvatinib in Epstein-Barr-virus-associated intrahepatic cholangiocarcinoma: a case report.

BACKGROUND: The prognosis for advanced intrahepatic cholangiocarcinoma (iCCA) is poor, and there remains an urgent need to develop efficient systemic therapy. The efficacy of Pembrolizumab immunotherapy combined with lenvatinibin in iCCA is still unclear. The role of Epstein-Barr-virus (EBV) as a biomarker in iCCA for response to immunotherapy needs further exploration. CASE PRESENTATION: We report a case of a 60-year-old female with EBV-associated advanced iCCA (EBVaiCCA) who progressed after first-line therapy. She accomplished an available response to the combination therapy of pembrolizumab with lenvatinib, with overall survival of 20 months. CONCLUSIONS: As far as we know, this is the first case report about the application of Pembrolizumab with lenvatinib for EBVaiCCA patients. This case indicates that the combination of immunotherapy and antiangiogenic therapy provides a glimmer of hope for advanced EBVaiCCA patients.

Bioinformatics and system biology approaches to determine the connection of SARS-CoV-2 infection and intrahepatic cholangiocarcinoma.

INTRODUCTION: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of coronavirus disease 2019 (COVID-19), has infected millions of individuals worldwide, which poses a severe threat to human health. COVID-19 is a systemic ailment affecting various tissues and organs, including the lungs and liver. Intrahepatic cholangiocarcinoma (ICC) is one of the most common liver cancer, and cancer patients are particularly at high risk of SARS-CoV-2 infection. Nonetheless, few studies have investigated the impact of COVID-19 on ICC patients. METHODS: With the methods of systems biology and bioinformatics, this study explored the link between COVID-19 and ICC, and searched for potential therapeutic drugs. RESULTS: This study identified a total of 70 common differentially expressed genes (DEGs) shared by both diseases, shedding light on their shared functionalities. Enrichment analysis pinpointed metabolism and immunity as the primary areas influenced by these common genes. Subsequently, through protein-protein interaction (PPI) network analysis, we identified SCD, ACSL5, ACAT2, HSD17B4, ALDOA, ACSS1, ACADSB, CYP51A1, PSAT1, and HKDC1 as hub genes. Additionally, 44 transcription factors (TFs) and 112 microRNAs (miRNAs) were forecasted to regulate the hub genes. Most importantly, several drug candidates (Periodate-oxidized adenosine, Desipramine, Quercetin, Perfluoroheptanoic acid, Tetrandrine, Pentadecafluorooctanoic acid, Benzo[a]pyrene, SARIN, Dorzolamide, 8-Bromo-cAMP) may prove effective in treating ICC and COVID-19. CONCLUSION: This study is expected to provide valuable references and potential drugs for future research and treatment of COVID-19 and ICC.

Lymphoepithelioma-like neoplasm of the biliary tract with 'probable low malignant potential'.

AIMS: Lymphoepithelioma-like carcinomas (LELCs) are uncommon epithelial cancers characteristically showing two distinct components consisting of malignant epithelial cells and prominent dense lymphoid infiltrate. Hepatic LELCs consist of two types, the lymphoepithelioma-like hepatocellular carcinoma and lymphoepithelioma-like cholangiocarcinoma (LEL-CCA), with the latter being strongly associated with Epstein-Barr virus (EBV). METHODS AND RESULTS: We present a series of three cases of intrahepatic biliary EBV-associated LEL tumours in which the biliary epithelial component showed a distinctly benign appearance, instead of the usual malignant epithelial features of a typical CCA or EBV-associated LEL-CCA. In the lesions, the biliary epithelium showed interconnecting glands or cords of cells. All had a very low proliferation (Ki-67) index. Immunohistochemistry for IDH1 and TP53 performed on two cases was negative and molecular tests for EGFR and KRAS gene mutations performed on one were negative. Prognosis was very good in all three cases, with patients alive with no evidence of disease 24-62 months after surgery. Intriguingly, all three cases had co-infection of HBV and EBV. These cases are also discussed in the context of the 63 cases of LEL-CCA available in the literature, with a focus on epidemiology, clinicopathological features and potential research interests. CONCLUSIONS: Based on the distinct clinicopathological features and unique survival benefits, we believe these tumours represent the benign end of the spectrum of EBV-associated lymphoepithelial biliary carcinomas. Whether these tumours require a revision of the current nomenclature to 'lymphoepithelioma-like neoplasm of the biliary tract with probable low malignant potential' will require more detailed analysis with larger case-series.

Lymphoepithelioma-like Intrahepatic Cholangiocarcinoma Is a Distinct Entity With Frequent pTERT/TP53 Mutations and Comprises 2 Subgroups Based on Epstein-Barr Virus Infection.

The molecular characteristics of lymphoepithelioma-like intrahepatic cholangiocarcinoma (LELCC) remain elusive. We examined 27 LELCC cases through next-generation sequencing using a panel of genes commonly mutated in primary liver cancers. Alterations in BAP1, ARID1A, ARID2, and PBRM1 were detected through immunohistochemistry. Fluorescence in situ hybridization was performed to analyze FGFR2 fusions and CCND1 amplification. LELCC is histologically classified as predominantly undifferentiated or glandular. Epstein-Barr virus-encoded small RNA (EBER) expression was found in 16 LELCCs. Approximately 50% of LELCCs expressed programmed death-ligand 1 strongly. Notably, recurrent pTERT and TP53 mutations were detected in 9 (38%) and 8 (33%) tumors, respectively. Only 2 LELCCs exhibited loss of expression for PBRM1. Alterations in genes typically involved in intrahepatic cholangiocarcinoma, including IDH1, IDH2, ARID1A, ARID2, and BAP1, and FGFR2 fusions, were not identified. The 2-step clustering analysis showed 2 distinct subgroups in LELCC, which were separated by EBER expression. A meta-analysis of all reported cases (n=85) has shown that EBER+ LELCC is strongly associated with the female sex, younger age, and exhibited predominantly glandular differentiation (P=0.001, 0.012, and <0.001, respectively). Patients with EBER- LELCC were more likely to have viral hepatitis and cirrhosis (P=0.003 and 0.005, respectively). Genetic analysis demonstrated that EBER- LELCC was significantly associated with pTERT and TP53 mutations (P=0.033 and 0.008, respectively). In conclusion, LELCC is genetically distinct from intrahepatic cholangiocarcinoma. EBER- LELCC may exhibit a different pathogenesis from EBER+ LELCC. High programmed death-ligand 1 expression in LELCC has implications for potential immunotherapeutic strategies.

Synergistic impact of resection margin and microscopic vascular invasion for patients with HBV-related intrahepatic cholangiocarcinoma.

OBJECTIVES: The resection margin (RM) status and microscopic vascular invasion (MVI) are known prognostic factors for intrahepatic cholangiocarcinoma (ICC). An enhanced understanding of their impact on long-term prognosis is required to improve oncological outcomes. METHODS: A total of 711 consecutive patients who underwent curative liver resection for hepatitis B virus-related ICC were retrospectively analyzed. The different impact of the RM status (narrow, <1 cm, or wide, ≥1 cm) and MVI (positive, +, or negative, -) on overall survival (OS) and recurrence-free survival (RFS) were analyzed. RESULTS: The 1-, 3-, and 5-year OS rates were 67.6%, 42.5%, and 33.2% in wide RM & MVI (-), 58.0%, 36.1%, and 26.5% in narrow RM & MVI (-), 51.0%, 27.0%, and 24.3% in wide RM & MVI (+), and 39.0%, 20.4% and 14.3% in narrow RM & MVI (+) (p < 0.001). Multivariate analysis showed that RM & MVI were independent risk factors for the OS and RFS. CONCLUSION: Combined analysis of RM and MVI can better stratify the risks of postoperative death and recurrence in patients with HBV-related ICC, which may help subsequent adjuvant therapy and closer follow-up.

Comparing the clinicopathological characteristics of combined hepatocellular-cholangiocarcinoma with those of other primary liver cancers by use of the updated World Health Organization classification.

AIMS: Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is an uncommon hepatic malignancy with a poor outcome. The 2019 World Health Organization (WHO) classification modified the definition and discarded the subtypes with stem cell features. However, the differences among cHCC-CCA, hepatocellular carcinoma (HCC), HCC with stem cell/progenitor features (HCCscf) and intrahepatic cholangiocarcinoma (iCCA) remain undetermined. The aim of this study was to investigate the characteristics of cHCC-CCA in comparison with those of other primary liver cancers by utilising the updated WHO classification. METHODS AND RESULTS: We retrospectively analysed 64 cHCC-CCA patients and 55 HCCscf patients from December 2007 to May 2018. Propensity score matching was conducted to compare these with HCC and iCCA patients. Clinicopathological characteristics, event-free survival and overall survival were evaluated with multivariate Cox proportional hazard regression. During a median follow-up of 55.9 months, cHCC-CCA patients had significantly poorer survival than HCCscf patients, and survival intermediate between that of HCC patients and that of iCCA patients. Hepatitis B virus (HBV) infection and high levels of tumour-infiltrating lymphocytes (TILs) were associated with favourable survival in cHCC-CCA patients. In the multivariate analysis, poor hepatic reserve, absence of HBV infection, stage IV disease and low levels of TILs were significant negative prognostic factors in cHCC-CCA patients. After being pooled with other primary liver cancers, cHCC-CCA and iCCA resulted in the worse survival. CONCLUSIONS: cHCC-CCA patients have survival intermediate between that of HCC patients and iCCA patients, and HBV infection and high levels of TILs predict favourable survival. Our study provides clinical correlations for the new 2019 WHO classification.

The CT and MRI Features of Primary Intrahepatic Lymphoepithelioma-Like Cholangiocarcinoma.

OBJECTIVE. The purpose of our study was to retrospectively characterize the CT and MRI features of primary intrahepatic lymphoepithelioma-like cholangiocarcinoma (LELCC). MATERIALS AND METHODS. Eleven patients (10 women and one man; age range, 30-63 years) with 11 pathologically proven LELCCs were enrolled retrospectively from April 2016 to December 2018. Triphasic enhanced images were obtained of all patients: MR images of five patients, CT images of five patients, and both CT and MR images of one patient. The clinical data and CT and MRI findings were reviewed. RESULTS. All LELCC cases were associated with Epstein-Barr virus (EBV) infection. Eight of the 11 patients had hepatitis B virus (HBV) infection. The tumor diameter ranged from 1.1 to 8.7 cm. All tumors were well defined with a smooth or lobulated margin. A cystic area was noted in two of the 11 tumors. After the administration of contrast material, the tumors showed homogeneous (n = 7) or heterogeneous (n = 4) hypervascular arterial enhancement and gradual washout, delayed central scar or irregular enhancement (n = 9), delayed circular thin or incomplete pseudocapsule enhancement (n = 7), and homogeneous hypointensity in the hepatobiliary phase (n = 2). No cirrhosis, focal dilatation of intrahepatic ducts, or satellite nodules were detected. Lymphadenopathy were detected in four patients, appearing as hypervascular enhancement and no necrosis (even in multiple nodes > 3 cm) or as moderate peripheral enhancement and necrosis. CONCLUSION. A liver mass in a middle-aged woman with EBV and HBV infection that appears on CT and MRI to have a well-defined boundary and a combination of hypervascularity, washout, delayed intratumoral enhancement, or pseudocapsule enhancement may suggest an imaging diagnosis of primary LELCC. More cases are needed to better understand this disease.

Hidden Carcinoma: Pitfalls in the Diagnosis of Lymphoepithelioma-Like Cholangiocarcinoma.

Lymphoepithelioma-like intrahepatic cholangiocarcinoma is a rare variant of cholangiocarcinoma that is associated with the Epstein-Barr virus. The intimate relationship between the malignant epithelial cells and the numerous lymphoid cells can make the diagnosis challenging on limited tissue samples. We present 2 cases in which the presence of a dense hematolymphoid infiltrate served to mask the diagnosis of carcinoma on initial frozen section and biopsy review, respectively. We bring awareness to this potential diagnostic pitfall and offer morphologic and immunohistochemical clues that may aid in recognition of this unusual and sometimes perplexing carcinoma.

Viral hepatitis B and C infections increase the risks of intrahepatic and extrahepatic cholangiocarcinoma: Evidence from a systematic review and meta-analysis.

BACKGROUND/AIMS: Previous study has shown a positive relationship between the hepatitis B virus (HBV) or hepatitis C virus (HCV) infection and cholangiocarcinoma (CCA); however, their correlation with different anatomical sites of CCA (i.e. ICC and ECC) has not been revealed. This study aims to evaluate the association of HBV or HCV infection with CCA, including the intrahepatic cholangiocarcinoma (ICC) and extrahepatic cholangiocarcinoma (ECC), and to determine the roles of α-1 fetoprotein (AFP), CA19-9, and lymph node involvement in CCA with HBV infection. MATERIALS AND METHODS: Relevant studies published between 2004 and 2016 were systematically searched and retrieved from PubMed, SpringerLink, and Science Direct using key terms such as "cholangiocarcinoma", "bile duct cancer", "extrahepatic cholangiocarcinoma", and "intrahepatic cholangiocarcinoma". The demographic, clinical, and laboratory data were extracted from the included studies, and the meta-analysis was performed using RevMan and STATA 11.0 software. RESULTS: A total of 13 studies with CCA matched the inclusion criteria in this meta-analysis, including 7,113 CCA patients and 24,763 controls. This meta-analysis showed that the HBV or HCV infections can significantly increase the risk of CCA, including ICC and ECC. In addition, the higher levels of AFP, lower levels of CA19-9, and lymph node involvement were detected in the CCA patients with HBV infection as compared to those without. CONCLUSION: The HBV and HCV infections significantly increased the risk of CCA, as well as ICC and ECC. The involvement of AFP, CA19-9, and lymph nodes may play an important role in the diagnosis of CCA.

[The research progress in clinicopathological characteristics and treatment of hepatitis B virus-associated intrahepatic cholangiocarcinoma].

Hepatitis B is a common and multiplex disease in China. Recently, epidemiological studies have shown that hepatitis B virus(HBV) infection is a high-risk factor for the development of intrahepatic cholangiocarcinoma(ICC). Based on a retrospective analysis of relevant literature of recent years, this article concludes that HBV-associated ICC is very different from other ICC regarding clinicopathological characteristics and treatment. Distinctive features of patients with HBV-associated ICC included younger age, preponderance of male patients, frequent elevation of alpha fetoprotein, decrease of CA19-9, frequent appearance of cirrhosis, infrequent lymph node metastasis and better prognosis. Because these clinicopathological features are similar to those of HBV-associated hepatocellular carcinoma(HCC), we speculate that the cell origin of HBV-associated ICC and HBV-associated HCC are consistent. In addition, this article also discusses that HBV-associated ICC should be treated with surgery-based comprehensive treatment in order to improve prognosis.

Role of Intravoxel Incoherent Motion in Discriminating Hepatitis B Virus-Related Intrahepatic Mass-Forming Cholangiocarcinoma from Hepatocellular Carcinoma Based on Liver Imaging Reporting and Data System v2018.

Backgroud: Intravoxel incoherent motion (IVIM) could be used to characterize benign and malignant hepatic lesions and predict the histological grade of hepatocellular carcinoma (HCC). To evaluate IVIM-derived parameters for differentiating between hepatitis B virus (HBV)-related intrahepatic mass-forming cholangiocarcinoma (IMCC) and HCC based on the Liver Imaging Reporting and Data System (LI-RADS) v2018. Materials and Methods: 20 IMCC patients and one-to-one matched control HCC patients were retrospectively assessed. IVIM scanning with 11 b-values (from 0 to 1500 s/mm2) was obtained using a 3.0-T magnetic resonance scanner. Apparent diffusion coefficient (ADC) and IVIM parameters, including diffusion coefficient (D), pseudodiffusion coefficient (D*), and perfusion fraction (f), were compared between IMCC and HCC. Receiver operating characteristic (ROC) curve analysis was performed to assess the diagnostic performances of ADC, D, f, and D*. The LI-RADS features and a final category were also compared using LI-RADS v2018. Results: ADC and D were significantly higher in IMCC than in HCC (p = 0.012 and p = 0.007, respectively); f was significantly higher in HCC than in IMCC (p = 0.004). The area under the ROC curve values for ADC, D, and f for differentiating HBV-related IMCC from HCC were 0.724, 0.753, and 0.741, respectively. Conclusion: The majority of HBV-related IMCCs can be categorized as LR-M by using LI-RADS. However, atypical IMCCs may be classified as non-LR-M. ADC, D, and f values may be helpful in differentiating HBV-related IMCC from HCC, and similar diagnostic performances were obtained for these values.

Hepatitis C Virus Infection and Cholangiocarcinoma: An Insight into Epidemiologic Evidences and Hypothetical Mechanisms of Oncogenesis.

Hepatitis C virus (HCV) infection is a global public health problem because it is a main cause of liver cirrhosis and hepatocellular carcinoma. This human oncogenic virus is also associated with the development of non-Hodgkin lymphoma and cholangiocarcinoma (CCA). The association between HCV infection and CCA has been examined in a number of epidemiologic studies. However, in vivo and in vitro results demonstrating the oncogenic mechanisms of HCV in CCA development and progression are insufficient. Here, we review the epidemiologic association of HCV and CCA and recent publications of studies of HCV infection of cholangiocytes and CCA cell lines as well as studies of viral infection performed with liver samples obtained from patients. In addition, we also discuss the preliminary results of in vitro assays of HCV protein expression in CCA cell lines. Finally, we discuss the hypothetical role of HCV infection in CCA development by induction of epithelial-mesenchymal transition and up-regulation of hedgehog signaling, and consequently biliary tree inflammation and liver fibrosis. Further studies are required to demonstrate these hypotheses and therefore to elucidate the mechanisms of HCV as a risk factor for CCA.

Hepatitis C virus infection and non-hepatocellular malignancies in the DAA era: A systematic review and meta-analysis.

BACKGROUND AND AIMS: Direct antiviral agents have greatly improved therapeutic options for chronic hepatitis C. Indeed, former "difficult-to-treat" patients can now be treated and can achieve sustained response. Hepatitis C virus (HCV) is associated with hepatocellular carcinoma and with B-cell non-Hodgkin lymphoma (B-NHL). Other malignancies have been reported to be associated with HCV infection albeit with various grades of evidence. Antineoplastic treatment is often reduced or suspended in HCV-positive cancer patients to avoid "HCV reactivation." In this setting, antiviral therapy combined with antineoplastic protocols may improve the outcome. For this reason, we conducted a systematic review and a meta-analysis to update the association between HCV infection and non-hepatocellular malignancies, and to shed light on the effects exerted by antiviral treatment on the natural history of oncological diseases. METHODS: Relevant studies were identified by searching PUBMED, EMBASE and MEDLINE up to 1 August 2018. Pooled risk estimates were calculated with random-effects models according to PRISMA guidelines. RESULTS: A total of 58 studies were included in the analysis: 27 studies of the association between HCV and B-NHL(OR 3.36; 95% CI 2.40-4.72;P < 0.00001);13 studies of the association between sustained virological response and progression-free survival (PFS) in B-NHL patients(OR 9.34; 95% CI 4.90-17.79; P < 0.00001); 13 studies of the association between HCV and intrahepatic-cholangio-carcinoma (OR 3.95;95% CI 2.25-6.94; P < 0.00001); and 5 studies of the association between HCV infection and pancreatic adeno-carcinoma(OR 1.60; 95% CI:1.25-2.04; P = 0.0002). CONCLUSIONS: This study updates the strong association between B-NHL and HCV infection, confirms the association between HCV and non-hepatocellular tumours, and demonstrates a very strong association between viral eradication and a better outcome of HCV-positive B-NHL.

Ultrasonographic findings of intrahepatic lymphoepithelioma-like cholangiocarcinoma associated with Epstein-Barr virus: Two cases report.

RATIONALE: Epstein-Barr virus (EBV)-related lymphoepithelioma-like cholangiocarcinoma (LELCC) is an extremely rare primary liver tumor with nonspecific clinical manifestations. The clinicopathological features of EBV-associated LELCC have been reported in a few cases. But reports of the tumor's imaging characteristics, particularly ultrasonographic findings, are very rare. PATIENT CONCERNS: The first patient was a 64-year-old man with left upper quadrant pain and no nausea and dizziness for about 3 months. The second patient was a 40-year-old man, had an incidental finding of a hepatic tumor in a routine health checkup at a local hospital. DIAGNOSES: In the first patient, the abdominal ultrasound demonstrated a slightly heterogeneous hypoechoic nodule in segment 3 of the liver. The nodule was about 2.0 cm × 1.7 cm in size, with a clear margin and regular shape. Color Doppler flow imaging (CDFI) revealed no blood flow signals in this nodule. According to the clinical information and imaging features, it was difficult to determine the diagnosis of the nodule. In the second patient, gray-scale ultrasound revealed a slightly heterogeneous hypoechoic mass measuring 3.5 cm × 2.5 cm with well-defined margin and regular shape at the superior segment of the left hepatic lateral lobe. There was a blurrily hypoechoic halo around the mass. In contrast-enhanced ultrasound (CEUS), the mass was homogeneous hyperenhancement in the arterial phase. In the portal phase and late phase, the center enhancement of the mass washed out gradually, presenting hypoenhancement, Therefore, the tumor was diagnosed as malignancy. INTERVENTION: Finally, a laparoscopic left hepatic lateral lobectomy was performed in the first patient. The second patient underwent a left hepatectomy with cholecystectomy. OUTCOMES: The first patient has been alive without recurrence or distant metastases for 11 months since the surgery. The second patient received routine follow-up after surgery. Until now, he has been tumor-free for 32 months. LESSONS: We mainly focus on the ultrasound characteristics of EBV-associated LELCC, especially its enhancement patterns on CEUS, which may provide valuable information for diagnosis of the LELCC. When a liver tumor with typical CEUS patterns of malignancy is found in middle-aged adults with EBV positive, the possibility of EBV-related LELCC should be considered.

Specific prognostic factors in hepatitis B virus-related and non-hepatitis B virus-related intrahepatic cholangiocarcinoma after macroscopic curative resection.

BACKGROUND: The hepatitis B virus (HBV)-related intrahepatic cholangiocarcinoma (iCCA) was recognized as a unique subtype of iCCA, within particular features in demography, clinicopathology, and genealogy. However, how they predict prognosis, in particular, for HBV- and non-HBV-related iCCA is still unclear. METHODS: Demographic, clinicopathologic, and genetic features were retrospectively collected and reviewed to determine the specific prognostic factors, precisely predicting the overall survival (OS) in HBV-related (n = 119) and non-HBV-related ( n = 149) iCCA patients, respectively. RESULTS: In HBV-related iCCA, TP53 mutation, vascular invasion, extrahepatic metastasis, and serum levels of alpha-fetoprotein (AFP) were independent prognostic factors for OS. In non-HBV-related iCCA, RAS/ RAF mutation and lymphatic metastasis independently predicted the OS of patients. Tumor differentiation and serum levels of CA19-9 were significantly associated with OS in both HBV- and non-HBV-related iCCA patients. In a subset analysis, TP53 and RAS/RAF mutations were consistently related to poorer outcome in HBV- and non-HBV-related iCCA, respectively. CONCLUSIONS: The HBV- and non-HBV-related iCCA have different prognostic factors for the OS.

EBV as a potential risk factor for hepatobiliary system cancer: A meta-analysis with 918 cases.

OBJECTIVES: Hepatobiliary system cancer, which includes hepatocellular carcinoma (HCC), cholangiocarcinoma, and gallbladder carcinoma, has an increase of incidence and mortality due to various risk factors. Epstein-Barr virus (EBV) is associated with various types of lymphomas and carcinomas, which is also acknowledged as the first-discovered human tumor virus. Despite this, there is no systematic analysis about the relationship between the infection of EBV and hepatobiliary system cancer. The aim of this meta-analysis is to explore the significance of EBV infection in the development of hepatobiliary system cancer by evaluating the EBV infection ratio. METHODS: A systematic search of PubMed, Embase, Cochrane Library, as well as China National Knowledge Infrastructure (CNKI), Chongqing VIP, Wan Fang, and China Biology Medicine databases was conducted. The EBV infection ratio and 95% confidence intervals (CIs) in hepatobiliary system cancer was evaluated. The I2 statistic was used to represent heterogeneity. Through meta-regression, stratified analyses were applied to find out heterogeneity's sources. Odds ratios (ORs), 95% CIs of EBV infection in case-control studies were calculated. RESULTS: Altogether, 15 studies were included containing a total of 918 cases and 157 controls. The whole infection ratio of EBV was 23% (95% CI: 13%, 33%, I2 = 95.7%, P < 0.001) among all the patients. Comparable EVB infection ratios were observed in hepatobiliary system cancer as divided into different subtypes. The five case-control studies were epitomized to a pooled OR of 9.35 (95%CI: 2.95, 29.61, I2 = 20.1%, P < 0.286). CONCLUSION: EBV may be a potentially risk factor in the process of hepatobiliary system cancer. The prospective molecular mechanism remains to be explored.

[Establishment of Cas9 stably expressed human hepatocellular carcinoma and cholangiocarcinoma cell lines].

Objective: To facilitate using the CRISPR/Cas9 gene editing system in human liver and gallbladder cancer cells, we established Cas9 stably expressed human liver and gallbladder cancer cell lines, and validated the gene editing activity of Cas9. Methods: Human liver cancer cell lines (Huh7, PLC/PRF/5, HepG2, Hep3b, SK-HEP-1 and Li-7), human cholangiocarcinoma cells (RBE) and human gallbladder cancer cells (GBC-SD) were infected with 3 Cas9-expressing lentivirus vectors (pLv-EF1α-Cas9-Flag-Neo, pLv-EF1α-Cas9-Flag-Puro, Cas9m1.1), respectively, and Cas9 stably expressed colonies were screened and selected. We extracted the genomic DNA and protein, validated the stable expression of Cas9 by using genomic polymerase chain reaction (PCR) and western blot. Three of cell lines were further infected with Lv-EF1α-mCherry. Then mCherry positive cells were sorted by flow cytometry and infected with designed guide RNA (gRNA) vectors which targeted mCherry gene. Subsequently the gene editing activity of Cas9 was detected by genomic PCR, fluorescence microscopic observation and flow cytometry analysis. Results: One hundred Cas9-expressing human liver and gallbladder cancer cell lines were selected. Among them, 35 cell lines expressed Cas9-Neo, 25 expressed Cas9-puro, and 40 expressed mutant Cas9 (mCas9). We also established 3 cell lines with stable expression of mCherry (Huh7-mCas9-M, PLC/PRF/5-Cas9-M and SK-HEP-1-Cas9-M). The results of genomic PCR and sequencing showed that by lentiviral infection with 2 types of designed gRNA, the long fragment deletion of mCherry gene was found in these 3 cell lines. Moreover, mCherry(-)EGFP(+) cells infected with 2 types of gRNA were observed by fluorescence microscope. The results of flow cytometry showed that mCherry(-)EGFP(+) cells accounted from 0.3% to 93.6%. Conclusion: We successfully establish 100 human liver and gallbladder cancer cell lines with stable expression of Cas9 protein and validate their activities of gene editing.

Low-level clonal FGFR2 amplification defines a unique molecular subtype of intrahepatic cholangiocarcinoma in a Chinese population.

Intrahepatic cholangiocarcinoma (ICC) is a subtype of primary liver cancer rarely curable by surgery that is increasing rapidly in incidence. Chromosomal translocations and amplifications of the fibroblast growth factor receptor 2 (FGFR2) locus are present in several kinds of tumors including ICC, but their incidence has not been assessed in Chinese patients. Using break-apart probes and by determining the ratios of FGFR2/chromosome enumeration probe (CEP) 10 double-color probes, we evaluated 122 ICCs for the presence of FGFR2 translocations and amplifications, respectively, by fluorescence in situ hybridization. We further determined FGFR2 protein expression by immunohistochemistry and analyzed the clinicopathologic records of the patients. Eight tumors (6.6%) had FGFR2 translocations, whereas 15 (12.3%) had low-level FGFR2 amplification. Interestingly, the tumors that showed both translocation and low-level amplification frequently were of the mass-forming type. Compared with the ICCs with normal FGFR2s, tumors with amplifications secreted less mucus (P = .017) and typically were accompanied by hepatitis B virus infection (P = .004). Tumors with low-level amplification generally were of lower stage (P = .013) and associated with better overall survival (P = .017). As tumors with FGFR2 amplification exhibit different biology from lesions with a normal gene, low-level amplification of FGFR2 may play an important role in tumor progression and may be a marker for targeted therapy.