Synthetic miR-26a mimics delivered by tumor exosomes repress hepatocellular carcinoma through downregulating lymphoid enhancer factor 1.

BACKGROUND: The dysregulation of exosomal microRNAs plays an important role in the progression of hepatocarcinogenesis. In this study, we investigated the therapeutic potential of synthetic exosomal miR-26a against HCC cells and explored the feasibility of tumor-derived exosomes as drug delivery vehicles. METHODS: Proliferation and migration assays were performed to examine the effects of miR-26a on HCC in vitro. The direct target gene of miR-26a was identified through miRecords analysis and target validation. The transferring efficiency and anti-HCC effect of exosomes with different origin were studied and the optimal miR-26a delivery method was established and verified in vitro and in vivo. In addition, the relationships between prognosis of HCC patients and miR-26a expression in HCC serum and exosomes were retrospectively analyzed. RESULTS: Here, we found that tumor cell-derived exosomes were taken in preferentially by HCC cells and promoted HCC progression through Wnt pathway by low-density lipoprotein receptor-related protein 6 (LRP6). HCC cells with vacuolar protein sorting-associated protein 35 knocked down were adopted to generate engineered LRP6-exosomes. The engineered HCC-derived exosomes loading miR-26a inhibited HCC progression in vitro and in vivo effectively. Overexpression of miR-26a impaired the growth and migration of HCC by targeting lymphoid enhancer factor 1 (LEF1). Moreover, low expression of exosomal miR-26a was an independent prognostic factor for recurrence and survival in HCC patients. CONCLUSIONS: Our findings suggested the exosomal miR-26a could serve as a non-invasive prognostic marker for HCC patients. Genetically modified tumor-derived exosomes showed preferable transfection efficiency but reduced Wnt activity, which provides a novel therapeutic strategy for HCC.

Dissecting spatial heterogeneity and the immune-evasion mechanism of CTCs by single-cell RNA-seq in hepatocellular carcinoma.

Little is known about the transcriptomic plasticity and adaptive mechanisms of circulating tumor cells (CTCs) during hematogeneous dissemination. Here we interrogate the transcriptome of 113 single CTCs from 4 different vascular sites, including hepatic vein (HV), peripheral artery (PA), peripheral vein (PV) and portal vein (PoV) using single-cell full-length RNA sequencing in hepatocellular carcinoma (HCC) patients. We reveal that the transcriptional dynamics of CTCs were associated with stress response, cell cycle and immune-evasion signaling during hematogeneous transportation. Besides, we identify chemokine CCL5 as an important mediator for CTC immune evasion. Mechanistically, overexpression of CCL5 in CTCs is transcriptionally regulated by p38-MAX signaling, which recruites regulatory T cells (Tregs) to facilitate immune escape and metastatic seeding of CTCs. Collectively, our results reveal a previously unappreciated spatial heterogeneity and an immune-escape mechanism of CTC, which may aid in designing new anti-metastasis therapeutic strategies in HCC.

Circulating tumor cell detection and single-cell analysis using an integrated workflow based on ChimeraX® -i120 Platform: A prospective study.

Circulating tumor cell (CTC) analysis holds great potential to be a noninvasive solution for clinical cancer management. A complete workflow that combined CTC detection and single-cell molecular analysis is required. We developed the ChimeraX® -i120 platform to facilitate negative enrichment, immunofluorescent labeling, and machine learning-based identification of CTCs. Analytical performances were evaluated, and a total of 477 participants were enrolled to validate the clinical feasibility of ChimeraX® -i120 CTC detection. We analyzed copy number alteration profiles of isolated single cells. The ChimeraX® -i120 platform had high sensitivity, accuracy, and reproducibility for CTC detection. In clinical samples, an average value of > 60% CTC-positive rate was found for five cancer types (i.e., liver, biliary duct, breast, colorectal, and lung), while CTCs were rarely identified in blood from healthy donors. In hepatocellular carcinoma patients treated with curative resection, CTC status was significantly associated with tumor characteristics, prognosis, and treatment response (all P < 0.05). Single-cell sequencing analysis revealed that heterogeneous genomic alteration patterns resided in different cells, patients, and cancers. Our results suggest that the use of this ChimeraX® -i120 platform and the integrated workflow has validity as a tool for CTC detection and downstream genomic profiling in the clinical setting.

Detection of circulating tumour cells enables early recurrence prediction in hepatocellular carcinoma patients undergoing liver transplantation.

BACKGROUND & AIMS: Liver transplantation (LTx) is one of the most effective treatments for hepatocellular carcinoma (HCC); however, tumour recurrence after LTx often leads to poor outcomes. This study investigated the value of circulating tumour cells (CTCs) as a predictor of recurrence following LTx in patients with HCC. METHODS: This analysis included 193 patients with HCC who underwent LTx at our institute and accepted pre- and post-operative CTC detection; 38 were selected for serial CTC monitoring. The predictive value of CTCs for tumour recurrence in patients with HCC following LTx was evaluated. Single-cell whole genome sequencing was used to characterize CTCs. RESULTS: Overall, the CTC burden decreased after LTx (P < .05). Post-operative CTC count ≥ 1 per 5 mL peripheral blood was identified as a potential biomarker for predicting tumour recurrence after LTx, especially in patients with no detectable CTCs prior to LTx and negative tumour serological biomarkers. The predictive value of post-operative CTC count ≥ 1 per 5 mL blood was retained in patients who did not meet the Milan criteria, University of California San Francisco (UCSF) criteria, or Fudan criteria (all P < .05). Furthermore, post-operative serial CTC detection may be useful in post-surgical surveillance for HCC recurrence. CONCLUSIONS: CTCs may be a useful biomarker to evaluate recurrence risk following LTx in patients with HCC. Evaluation based on CTC detection may enhance the post-transplant management of HCC, and improve the therapeutic efficacy of LTx.

Effect of surgical margin on recurrence based on preoperative circulating tumor cell status in hepatocellular carcinoma.

BACKGROUND: High rates of recurrence after resection severely worsen hepatocellular carcinoma (HCC) prognosis. This study aims to explore whether circulating tumor cell (CTC) is helpful in determine the appropriate liver resection margins for HCC patients. METHODS: HCC patients who underwent liver resection were enrolled into training (n=117) or validation (n=192) cohorts, then classified as CTC-positive (CTC≥1) or CTC-negative (CTC=0). A standardized pathologic sampling method was used in the training cohort to quantify microvascular invasion (mVI) and the farthest mVI from the tumor (FMT). FINDINGS: CTC number positively correlated with mVI counts (r=0.655, P<0.001) and FMT (r=0.495, P<0.001). The CTC-positive group had higher mVI counts (P=0.032) and greater FMT P=0.008) than the CTC-negative group. In the CTC-positive group, surgical margins of >1 cm independently protected against early recurrence (training cohort, P=0.004; validation cohort, P=0.001) with lower early recurrence rates (training cohort, 20.0% vs. 65.1%, P=0.005; validation cohort, 36.4% vs. 65.1%, P=0.003) compared to surgical margins of ≤1 cm. No differences in postoperative liver function were observed between patients with margins >1 cm vs. ≤1 cm. Surgical margin size minimally impacted early postoperative HCC recurrence in CTC-negative patients when using 0.5 cm or 1 cm as the threshold. INTERPRETATIONS: Preoperative CTC status predicts mVI severity in HCC patients and is a potential factor for determining optimal surgical margin size to ensure disease eradication and conserve liver function. A surgical margin of >1 cm should be achieved for patients with positive CTC. FUNDING: A full list of funding bodies that contributed to this study can be found in the Acknowledgement section.

Integrated Bioinformatics analysis and clinical validation reveals that high expression of mucin 1 in intrahepatic cholangiocarcinoma predicts recurrence after curative resection.

Intrahepatic cholangiocarcinoma (ICC) is a cancer type with high malignancy and a current lack of biomarkers to predict recurrence. In the present study, to identify potential biomarkers, five ICC datasets from the Gene Expression Omnibus database were analyzed to construct initial datasets by using a robust rank aggregation approach. A total of 19 upregulated genes were identified in the initial datasets. The genes identified were then further analysed using data from The Cancer Genome Atlas. Only mucin 1 (MUC1) exhibited significance regarding differential expression and survival prediction. Finally, the expression levels of MUC1 were assessed using reverse transcription-quantitative PCR in 61 pairs of ICC tumor and matched non-cancerous samples. The expression of MUC1 was significantly elevated in ICC tissues compared with that in matched non-cancerous counterparts (P=0.001). Patients with high MUC1 expression levels had significantly shorter overall survival (OS, P=0.009) and recurrence-free survival (RFS, P=0.012). MUC1 was identified as an independent prognostic factor for OS [hazard ratio (HR)=2.364, 95%CI: 1.214-4.485; P=0.023] and RFS (HR=2.552 95%CI: 1.294-5.032; P=0.007) in the multivariate analysis. Using receiver operating characteristic analysis, a co-index including MUC1 had a high accuracy for predicting survival [MUC1 combined with serum levels of CEA and cancer antigen 19-9, and lymph node metastasis, area under curve (AUC)=0.746, 95%CI: 0.620-0.872] and recurrence (MUC1 combined with bile duct invasion and lymph node metastasis, AUC=0.729, 95%CI: 0.605-854). In conclusion, MUC1 is highly expressed in ICC tissue and is a potential prognostic biomarker and therapeutic target for ICC.

Circulating tumor cells are an indicator for the administration of adjuvant transarterial chemoembolization in hepatocellular carcinoma: A single-center, retrospective, propensity-matched study.

BACKGROUND: High rates of postoperative tumor recurrence contribute to poor outcome in hepatocellular carcinoma (HCC). Here, we investigated whether circulating tumor cells (CTCs) status can predict the benefit of adjuvant transcatheter arterial chemoembolization (TACE) in patients with HCC. METHODS: The retrospective study enrolled 344 HCC patients with preoperative CTCs analysis. Clinical outcomes including recurrence and survival were compared between those who received and who did not receive adjuvant TACE. Similar comparisons were made for patients stratified according to CTC status (CTC-negative [CTC = 0], n = 123; CTC-positive [CTC ≥ 1], n = 221). Propensity score matching (PSM) strategy was adopted to offset differences between two groups. RESULTS: In the study cohort as a whole or in CTC-negative cohort, there were no observable differences in overall survival (OS) or time to recurrence (TTR) between TACE and control group (P > .05). In CTC-positive patients, PSM generated 64 patient pairs, and patients with adjuvant TACE had significantly better clinical outcomes (OS: not reached vs 36.4 months, P < .001; TTR: 45.8 vs 9.8 months, P < .001). Adjuvant TACE significantly reduced early recurrence (≤2 years) (64.1% vs 31.7%, P < .001) in CTC-positive patients. Notably, adjuvant TACE influenced TTR and OS even in subgroups of CTC-positive patients with low risk of recurrence according to traditional evaluation. CONCLUSIONS: Preoperative CTC status could serve as an indicator for the administration of adjuvant TACE in HCC patients. Adjuvant TACE benefits CTC-positive HCC patients mainly by reducing early recurrence.

Anlotinib suppresses tumor progression via blocking the VEGFR2/PI3K/AKT cascade in intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is a malignant tumor derived from bile duct epithelium. Its characteristics include an insidious onset and frequent recurrence or metastasis after surgery. Current chemotherapies and molecular target therapies provide only modest survival benefits to patients with ICC. Anlotinib is a novel multi-target tyrosine kinase inhibitor that has good antitumor effects in a variety of solid tumors. However, there are few studies of anlotinib-associated mechanisms and use as a treatment in ICC. In this study using in vitro experiments, we found that anlotinib had significant effects on proliferation inhibition, migration and invasion restraint, and cell-cycle arrestment. Anlotinib treatment affected induction of apoptosis and the mesenchymal-epithelial transition. Patient-derived xenograft models generated directly from patients with ICC revealed that anlotinib treatment dramatically hindered in vivo tumor growth. We also examined anlotinib's mechanism of action using transcriptional profiling. We found that anlotinib treatment might mainly inhibit tumor cell proliferation and invasion and promote apoptosis via cell-cycle arrestment by inactivating the VEGF/PI3K/AKT signaling pathway, as evidenced by significantly decreased phosphorylation levels of these kinases. The activation of vascular endothelial growth factor receptor 2 (VEGFR2) can subsequently activate PI3K/AKT signaling. We identified VEGRF2 as the main target of anlotinib. High VEGFR2 expression might serve as a promising indicator when used to predict a favorable therapeutic response. Taken together, these results indicated that anlotinib had excellent antitumor activity in ICC, mainly via inhibiting the phosphorylation level of VEGFR2 and subsequent inactivation of PIK3/AKT signaling. This work provides evidence and a rationale for using anlotinib to treat patients with ICC in the future.

Postoperative circulating tumor cells: An early predictor of extrahepatic metastases in patients with hepatocellular carcinoma undergoing curative surgical resection.

BACKGROUND: Postoperative extrahepatic metastases (EHM) contribute to a grim outcome in patients with hepatocellular carcinoma (HCC) who are undergoing curative surgical resection. The current study investigated the clinical value of circulating tumor cells (CTCs) in predicting EHM after curative surgery. METHODS: A total of 197 patients with HCC who were undergoing curative surgical resection were assigned to a retrospective training cohort (144 patients) or a prospective validation cohort (53 patients). The CELLSEARCH system was used for the detection of CTCs prior to surgical resection and 1 month thereafter. The cutoff value of CTCs was estimated using receiver operating characteristic analysis. Bonferroni correction was applied for multiple testing in a Cox proportional hazards regression model. RESULTS: In the training cohort, EHM was found to be associated with a higher postoperative CTC burden compared with no EHM (mean: 4.33 vs 0.52; P < .001). Receiver operating characteristic analysis demonstrated a postoperative CTC count ≥3 as the optimal cutoff value for the prediction of EHM. Patients with a postoperative CTC count ≥3 experienced a higher EHM risk (56.3% vs 5.5%) and a shorter median overall survival (31.25 months vs not reached) (all P < .001). The prognostic significance of a postoperative CTC count ≥3 also applied to patient subgroups with a low EHM risk, such as those with an α-fetoprotein level ≤400 ng/mL, absence of vascular invasion, well differentiation, and early tumor stage, and its predictive value was retained in patients with a continuous normal α-fetoprotein level during postoperative follow-up (all P < .05). The results were confirmed in the validation cohort. CONCLUSIONS: A postoperative CTC count ≥3 appears to be a surrogate marker for the prediction of EHM after curative surgical resection of HCC. More careful surveillance should be recommended to patients with a high CTC load to ensure the greater possibility of early interventions for postoperative EHM.

Deep learning-based automated detection of retinal diseases using optical coherence tomography images.

Retinal disease classification is a significant problem in computer-aided diagnosis (CAD) for medical applications. This paper is focused on a 4-class classification problem to automatically detect choroidal neovascularization (CNV), diabetic macular edema (DME), DRUSEN, and NORMAL in optical coherence tomography (OCT) images. The proposed classification algorithm adopted an ensemble of four classification model instances to identify retinal OCT images, each of which was based on an improved residual neural network (ResNet50). The experiment followed a patient-level 10-fold cross-validation process, on development retinal OCT image dataset. The proposed approach achieved 0.973 (95% confidence interval [CI], 0.971-0.975) classification accuracy, 0.963 (95% CI, 0.960-0.966) sensitivity, and 0.985 (95% CI, 0.983-0.987) specificity at the B-scan level, achieving a matching or exceeding performance to that of ophthalmologists with significant clinical experience. Other performance measures used in the study were the area under receiver operating characteristic curve (AUC) and kappa value. The observations of the study implied that multi-ResNet50 ensembling was a useful technique when the availability of medical images was limited. In addition, we performed qualitative evaluation of model predictions, and occlusion testing to understand the decision-making process of our model. The paper provided an analytical discussion on misclassification and pathology regions identified by the occlusion testing also. Finally, we explored the effect of the integration of retinal OCT images and medical history data from patients on model performance.

Chemotherapeutic perfusion of portal vein after tumor thrombectomy and hepatectomy benefits patients with advanced hepatocellular carcinoma: A propensity score-matched survival analysis.

BACKGROUND: Portal vein tumor thrombus (PVTT) is a common complication in hepatocellular carcinoma (HCC), signaling dismal outcomes. This study was conducted to evaluate the survival benefit of postoperative portal vein perfusion chemotherapy (PVC) in patients with HCC and PVTT. METHODS: A retrospective review was conducted in 401 consecutive patients with HCC and PVTT who underwent hepatic resection between January 2009 and December 2015 and 67 patients received adjuvant postoperative PVC. A propensity score matching (PSM) was used to match patients with and without PVC at a ratio of 1:1. RESULTS: After PSM, the median time to recurrence (TTR) and overall survival (OS) were significantly longer in PVC group compared with control group (12.3 vs 5.8 months, P = .001; 19.0 vs 13.4 months, P = .037; respectively). At 1, 2, 3, and 5 years, the cumulative recurrence rates in PVC group were 48.1%, 86.5%, 92.3% ,96.2%, respectively, with OS rates of 63.8%, 37.9%, 24.4%, 18.3%, respectively; whereas cumulative recurrence rates of 76.6%, 91.5%, 94.3%, and 97.2%, respectively and OS rates of 55.4%, 23.0%, 12.4%, and 12.4%, respectively were recorded for the control group. In multivariate analysis, postoperative PVC emerged as a significant predictor for TTR (hazard ratio [HR], 0.523; P = .001) and OS (HR, 0.591; P = .010). PVC could reduce early recurrence (≤1 year) rate after surgical resection (40.3% vs 64.2%, P = .006) and clinical outcomes were further enhanced by adding sorafenib to postoperative PVC. CONCLUSIONS: Compared with surgical resection alone, postoperative adjuvant PVC treatment boosts survival and reduces early tumor recurrences in patients surgically treated for HCC and PVTT.

KPNA3 Confers Sorafenib Resistance to Advanced Hepatocellular Carcinoma via TWIST Regulated Epithelial-Mesenchymal Transition.

Sorafenib, a multikinase inhibitor, is a new standard treatment for patients with advanced hepatocellular carcinoma (HCC). However, resistance to this regimen is frequently observed in clinical practice, and the molecular basis of this resistance remains largely unknown. Herein, the antitumor activity of sorafenib was assessed in 16 patient-derived xenograft (PDX) models of HCC. Gene expression analysis was conducted to identify factors that promote sorafenib resistance. Quantitative RT-PCR and immunoblotting were used to determine gene expression and activation of signaling pathways. Cell proliferation, clone formation, and transwell assays were conducted to evaluate drug-sensitivity, proliferation, and invasiveness, respectively. Kaplan-Meier analysis was used to evaluate the predictive power of biomarkers for sorafenib response. Differential gene expression analysis suggested that sorafenib resistance correlated with high karyopherin subunit alpha 3 (KPNA3) expression. Overexpression of KPNA3 in HCC cells enhanced tumor cell growth and invasiveness. Interestingly, KPNA3 was found to trigger epithelial-mesenchymal transition (EMT), a key process mediating drug resistance. On a mechanistic level, KPNA3 increased phosphorylation of AKT, which then phosphorylated ERK, and ultimately promoted TWIST expression to induce EMT and sorafenib resistance. Moreover, retrospective analysis revealed that HCC patients with low KPNA3 expression had remarkably longer survival after sorafenib treatment. Finally, we have identified a novel KPNA3-AKT-ERK-TWIST signaling cascade that promotes EMT and mediates sorafenib resistance in HCC. These findings suggest that KPNA3 is a promising biomarker for predicting patient responsiveness to sorafenib. Targeting KPNA3 may also contribute to resolving sorafenib resistance in HCC.

Clinical Characteristics and Prognostic Factors of Patients with Intrahepatic Cholangiocarcinoma with Fever: A Propensity Score Matching Analysis.

BACKGROUND: Patients with intrahepatic cholangiocarcinoma (ICC) rarely present fever as the initial symptom. We aimed to identify clinical characteristics and prognostic factors for these feverish patients. SUBJECTS, MATERIALS, AND METHODS: This study retrospectively reviewed 31 patients with ICC with fever (≥38.0°C) treated at our hospital between January 2002 and December 2014. A propensity score was used to match patients with and without fever at a ratio of 1:2. RESULTS: Patients with ICC with fever had higher serum γ-glutamyl transferase and carcinoembryonic antigen levels, larger tumors, poorer tumor differentiation, and worse prognosis (all p < .05) than those without fever. This was supported by propensity score matching (PSM) analysis. Univariate and multivariate analyses indicated that microvascular invasion, hilar lymph node metastasis, and temperature ≥ 38.6°C were related to prognosis. Patients with ICC with fever had higher levels of leucocytes, neutrophils, neutrophil-to-lymphocyte ratio (NLR), and platelet-to-lymphocyte ratio (PLR) in peripheral blood before and after PSM analysis. Body temperature positively correlated with leucocytes (r = 0.599, p < .001), neutrophils (r = 0.644, p < .001), NLR (r = 0.681, p < .001), and PLR (r = 0.457, p = .010). CONCLUSION: Patients with ICC with fever ≥38.0°C and ≥38.6°C had poor and extremely poor prognosis, respectively. Radical surgical treatment may improve the prognosis of patients with ICC with fever <38.6°C. However, systemic therapy (e.g., anti-inflammatory and immune therapy) may be preferable to surgery for these patients with fever ≥38.6°C. IMPLICATIONS FOR PRACTICE: Patients with intrahepatic cholangiocarcinoma (ICC) with fever (≥38.0°C) as the initial symptom are extremely rare. Because their symptoms are similar to those of liver abscess, diagnosis is challenging, and most of these patients are already at an advanced stage at the time of diagnosis. Patients with ICC with fever had different clinical characteristics and worse prognosis than those without fever. The prognosis of those with temperature <38.6°C would be improved by timely surgical intervention. Those with fever ≥38.6°C had an extremely dismal outcome, although they all received radical surgical treatment. New therapeutic strategies are needed to improve survival for patients with ICC with temperature ≥38.6°C.

Circulating osteopontin per tumor volume as a prognostic biomarker for resectable intrahepatic cholangiocarcinoma.

BACKGROUND: The role of osteopontin (OPN) in intrahepatic cholangiocarcinoma (ICC) remains controversial. This study aimed to explore the prognostic value of OPN in patients with ICC undergoing curative resection. METHODS: Patients undergoing curative resection from 2005 to 2016 were identified for inclusion in this retrospective study. The expression level of OPN in tumors was measured in each of the 228 patients by immunohistochemistry. Circulating OPN in serum was tested in 124 patients by ELISA. Tumor volume was calculated according to preoperative imaging or operation record. Proliferation assay, wound healing assay, and invasion assay were performed to investigate the biological function. RESULTS: Low expression of OPN in tissue was associated with lymph node metastasis (P=0.009) and shorter overall survival (OS) (P=0.001). A low level of circulating OPN/volume was associated with multiple tumors (P<0.001), vascular invasion (P=0.027), visceral peritoneal perforation (P=0.001), and lymph node metastasis (P=0.002). It was also able to predict the invasive behavior, lymph node metastasis, and early recurrence with the area under the receiver operating curve (AUC) of being 0.719, 0.708 and 0.622 respectively. Patients with a low level of circulating OPN/volume had shorter OS (P=0.028) and disease-free survival (DFS) (P=0.004) and could benefit from adjuvant chemotherapy (P=0.011). Compared with negative controlled cells, ICC cell lines, which expressed more OPN, showed a decelerated proliferation rate, the weaker ability of migration and invasion, while the opposite was true for the cells expressed less OPN. MMP1, MMP10, and CXCR4 were negatively regulated by OPN. CONCLUSIONS: A low level of circulating OPN/volume could indicate aggressive characteristics, along with poor prognosis and efficacy of adjuvant chemotherapy in ICC patients. Over expression of OPN may inhibit phenotypes facilitating ICC metastasis by negatively regulating MMP1, MMP10, and CXCR4.

Intrahepatic cholangiocarcinoma patients without indications of lymph node metastasis not benefit from lymph node dissection.

BACKGROUND: To investigate the necessity of routine lymph node dissection (LND) in intrahepatic cholangiocarcinoma (ICC) patients without indications of lymph node metastasis (LNM) preoperatively. METHODS: 422 consecutive ICC patients who undergone curative resection from January 2009 to December 2014 were enrolled and categorized as two groups (hepatectomy only or hepatectomy plus LND). Clinicopathologic data was compared between the groups by χ2 or Fisher's exact test. Overall survival (OS) and recurrence-free survival (RFS) were calculated by the Kaplan-Meier method and differences were analyzed using the log-rank test. Cox regression model was adopted for multivariable analysis. RESULTS: The median OS time of all 422 patients was 41.4 months. One-, 3-, and 5-year OS was 67%, 47%, and 35%, respectively. A total of 73 patients had undergone curative resection combined with LND, of whom 20.5% (15/73) were confirmed lymph node positive pathologically. The clinicopathologic characteristics between LND and control groups showed no significant differences. Of the 422 patients, 271 patients had recurrence. The recurrence rates were 65.8% for the LND group and 63.9% for the non-LND group. Survival analysis revealed that, neither the OS (LND vs. non-LND: 32.2 months vs. 46.2 months; p = 0.16) nor the RFS (LND vs. non-LND: 23.1 months vs. 17.0 months; p = 0.09) had significant difference. Multivariate analysis revealed that tumor size, tumor number, carbohydrate antigen19-9, carcinoembryonic antigen, and gamma-glutamyl transpeptidase were independent predictive factors for OS and RFS. CONCLUSION: Routine LND may not improve survival in resectable ICC patients with negative LNM diagnosis before operation.