| Part Ⅰ The regularity of lymph node metastasis in biliary tract cancer and survival difference under different recurrence modesObjective Lymph node metastasis is a common metastasis of biliary tract tumors.The purpose of this study was to summarize the distribution of lymph nodes in biliary tract cancer at different stages and to evaluate the impact of different metastatic sites on survival.Methods The imaging records of patients with advanced biliary tract cancer who were first diagnosed and relapsed after surgery between 2015 and 2020 were retrospectively analyzed,and lymph nodes metastatic at different sites were identified and projected onto CT images of standard patients according to their anatomical locations.Assess the risk of lymph node involvement at different sites.Survival analysis was performed using the Kaplan-Meier method,and the impact of different recurrence sites on survival was analyzed using a log-rank test.The overall survival of patients with postoperative recurrence was calculated from the time of surgery until death or the most recent follow-up.Results A total of 61 advanced patients with lymph node metastasis were initially diagnosed,and 251 metastatic lymph nodes were identified.All lymph nodes were projected onto a standard patient CT image.These lymph nodes were mainly concentrated in regions 16a2(13%)and 16b1(17%),followed by regions 12(12%)and 8(9%).Subgroup analysis showed that the high involvement rate of lymph nodes in the 16b1 region was similar in biliary tract tumors at different sites.93 positive lymph nodes were identified in a cohort of 45 patients with recurrence after surgery.These lymph nodes are mainly distributed in region 16b1(33%)and 16a2(18%).The involvement rates of lymph node in regions 16al and 16b2 were relatively low,being 1%and 8%,respectively.In the postoperative gallbladder cancer subgroup,metastatic lymph nodes were found mainly in region 12(36%),followed by region 16b1(29%).In iCCA and eCCA,the involvement rates of lymph nodes in region 16b1 were both greater than 30%.Survival analysis showed that patients with PALN recurrence had a better prognosis than patients with other distant organ metastases(HR=6.50,95%CI 1.71-24.67,P=0.006);There was no significant difference in survival between patients with PALN recurrence and patients with intrahepatic metastasis alone(HR=0.84,95%CI 0.38-1.87,P=0.675).Conclusions In advanced and postoperative recurrent biliary tract cancer,the involvement rate of lymph nodes in the para aortic region is the highest,lymph nodes mainly concentrated in the 16b1 and 16a2 regions.In addition,this study demonstrated that the prognosis of patients with PALN recurrence is significantly better than that of patients with other distant organ recurrence,providing a survival basis to the reasonability of the para aortic region included in CTV.Part Ⅱ Recommendations for the delineation of paraaortic CTV in adjuvant radiotherapy based on the distribution pattern of PALNsObjective Although some recommendations have been reported on the delineation of CTV for BTC,the extent of paraaortic CTV remains unclear.This study will create a new mapping of paraaortic CTV based on the distribution pattern of paraaortic lymph nodes(PALN).Methods A cohort of 31 patients with advanced BTC with PALN metastasis was included.Referring to the anatomical location,these lymph nodes were depicted on a standard patient CT image.Measure the distance from the volumetric center of the lymph nodes to the edge of the aorta.Initially,CTV was expended by the average distance from the edge of the aorta to the center of the lymph node,and then the expansion was increased asymmetrically until the CTV range covered the clinically acceptable number of PALNs.Finally,the expansion margins calculated from the study cohort were used to evaluate the coverage of lymph node centers in the validation cohort.Results A total of 92 PALNs were found in the study cohort.All PALNs were projected onto axial CT image of the standard patient.PALNs were concentrated in the 16a2(36%)and 16b1(47%)regions.The upper boundary of 16a2 and the lower boundary of 16b1 were defined as the cranial and caudal border of para aortic CTV,respectively.The mean distance from the volume center of all lymph nodes in 16a2 and 16b1 to the proximal border of the aorta was 9 mm(range 4-24)in the front,7 mm(range 3-14)on the left,and 12 mm(range 5-29)on the right.In the validation cohort(n=14 patients,56 PALN),the mean distance from the center of the lymph node to the border of the aorta were 10 mm on the left(range 5-20)and right(range 6-23),and 9mm in front of the aorta(range 5-23).The CTV that formed by the expansion of 18 mm in the front,12 mm on the left and 24 mm on the right from the aorta got 96%(73/76)coverage of PALNs in the study cohort.When applied to the validation cohort,the coverage rate of the delineated CTV for recurrent PALN also reached 96%(47/49).Conclusions The involvement rates of PALN in 16a2 and 16b1 were highest.Based on the distribution of PALNs,a new and more accurate para aortic CTV was defined to provide a reference for adjuvant radiotherapy in BTC.Part Ⅲ Identifying prognostic biomarkers of biliary tract cancer associated with lymph node metastasis by bioinformaticsObjective Few studies have focused on molecular targets associated with lymph node metastasis in biliary tract cancer.This section will explore prognostic biomarker associated with lymph node metastasis by bioinformatics methods to further enrich targeted treatment strategies for biliary tract cancer.Methods In this study,the core modules related to cancer were identified through a weighted gene co-expression network analysis(WGCNA).Conduct GO and KEGG enrichment analysis on the genes in the module.And then construct a PPI network and identify the potential key genes.Survival analysis of potential key genes through Kaplan Meier curve to determine hub genes.Then verify the differential expression of hub genes through public databases.Univariate and multivariate COX analysis was performed in a clinical cohort to further verify the relationship between hub gene and prognosis.The correlation between hub gene and lymph nodes was determined by chisquare test.The predictive ability of hub gene expression on lymph node metastasis was evaluated by ROC curve.Results The gene expression profiles of BTC obtained from TCGA were analyzed by WGCNA,and six coexpression modules were identified that were significantly correlated with phenotypes,among which the Turquoise module was highly positively correlated with cancer(0.97).This module contains a total of 650 genes.KEGG analysis showed that these genes were mainly concentrated in "cell cycle","ECM receptor interaction" and "oocyte meiosis" signaling pathways.Ten high degree genes were screened from the Turquoise module for survival analysis,and found that AURKB,CENPE and KIF23 were significantly correlated with the prognosis of BTC patients,therefore these three genes were selected as hub genes for further analysis.External databases showed that the mRNA and protein expressions of hub gene in biliary carcinoma tissues were significantly increased compared with non-tumor tissues.Moreover,52 BTC patients were divided into two groups based on expression of hub gene in postoperative tissue samples.Univariate analysis showed that AURKB>97.0456 and KIF23>38.444 were correlated with poor OS(HR=5.73,95%CI:2.0815.80,P<0.05;HR=3.28,95%CI:1.48-7.27,P<0.05).COX multivariate analysis showed that high AURKB and KIF23 expression were independent risk factors for prognosis in BTC patients(HR=3.88,95%CI:1.43-10.53,P=0.008;HR=2.88,95%CI:1.11-7.42,P=0.030).In addition,the KIF23 expression is closely related to Nstage(χ2=3.99,P=0.046),the area under the ROC curve was 0.692(P=0.029),which can effectively predict postoperative lymph node metastasis.Conclusions We identified KIF23,a prognostic factor associated with lymph nodes,as a candidate biomarker for biliary tract cancer,which provides a new molecular target and intervention strategy for the treatment of biliary carcinoma,and also provides a theoretical basis for further research.Part Ⅳ Exploration of molecular mechanism of hub gene KIF23 promoting disease progression in biliary tract cancerObjective The high expression of KIF23 is significantly associated with poor prognosis in biliary tract cancer.However,the mechanism of KIF23 in cholangiocarcinoma cells is still unknown.This section aims to explore the biological functions and possible regulatory mechanisms of KIF23 in cholangiocarcinoma cells.Methods The expression of KIF23 in HCCC9810 and RBE was studied using Western blotting and qRT-PCR.Small interfering RNA and overexpression plasmid were used to construct KIF23 silenced and overexpressed cell lines,respectively.CCK8,Transwell,cloning,and flow cytometry were used to detect the proliferation and migration of cholangiocarcinoma cells in vitro.Transcriptome sequencing and Western blotting were used to explore potential mechanism,Cistrome was used to predict the transcription factors of target genes,and ChIP experiment was used to verify the interaction between intracellular proteins and DNA.Results KIF23 was significantly expressed in HCCC9810 and RBE.After knockdown KIF23,the proliferation ability of HCCC9810 and RBE cells decreased.Transwell experiments showed that the migration ability of cells in si-KIF23 group was significantly reduced than that in si-NC group.The long-term effects of KIF23 on proliferation were evaluated by cloning assay.The number of colonies decreased significantly after KIF23 was knocked down.In addition,Cells transfected with small interfering RNA had a higher proportion of G2/M phase arrest in the cell cycle compared with siNC cells.The proliferation and migration ability of KIF23 overexpression HCCC9810 and RBE cells were higher than those in the Vector group,and the clonal formation test also confirmed that the number of colonies increased significantly when KIF23 overexpression.In RBE cells,knockdown of KIF23 increased the SGK3 expression,which inhibited the expression of FOXO3A,a key protein in the FOXO pathway.At the same time,the expression of KIF23 is directly regulated by the transcription factor FOXM1,which inhibits the expression of KIF23 by reducing the transcription of KIF23.Conclusions The expression of KIF23 influenced the proliferation and migration of bile duct cancer cells.Knockdown of KIF23 in cholangiocarcinoma affected the FOXO signaling pathway,and KIF23 expression was regulated by the transcription factor FOXM1.These results provided experimental basis for targeted KIF23 therapy. |
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