Circulating Tumor Endothelial Cells With Stemness Indicate Prognosis Of Pancreatic Ductal Adenocarcinoma And Are Characterized By Single-cell Whole Exome Sequencing

BackgroundPancreatic ductal carcinoma(PDAC)is a malignant digestive system tumor with extremely poor prognosis,and it's also prone to vascular infiltration or distant metastasis.There is a lack of effective peripheral markers to assess prognosis.Circulating tumor endothelial cells(CTECs)are mature endothelial cells shed from damaged tumor vessels into circulation.CTECs have been used in a variety of cancers as indicators for assessing prognosis,evaluating tumor angiogenic capacity and indicating drug efficacy.However,CTECs were rarely reported in PD AC.In addition,the downstream analysis of CTECs remains difficult due to its low total amount in peripheral blood and low capture efficiency.Single-cell whole exome sequencing technology based on next-generation sequencing(NGS)can accurately complete whole exome amplification,obtain ideal sequencing depth,analyze tumors from the perspective of individual cells,and perform analysis in circulating rare cells such as circulating tumor cells(CTCs).This technique has been applied in studies of CTCs,but has not yet been applied to the study of CTECs.It is important to explore the molecular characteristics and the key mutations of CTECs at single cell level.Since angiogenesis plays an important role in the growth and invasion of cancers,and CTECs are important indicator to assess the endothelial function of tumors,it is important to investigate the relationship between angiogenesis and prognosis in pancreatic cancer for the treatment and evaluation of tumors.Objective1.To detect CTECs in peripheral blood of patients with PD AC using subtraction enrichment and immunostaining-fluorescence in situ hybridization(SE-iFISH)technique,and explore the correlation between CTECs and prognosis.2.To investigate the significant mutations and exome characteristics of CTECs in pancreatic cancer using single-cell whole exome sequencing.3.To investigate the relationship between tumor cell sternness marker CD44,angiogenesis and prognosis.Methods1.The SE-iFISH platform was applied to detect and enumerate CTECs.Immunofluorescence was used to identify phenotypes of CTECs.Univariate and multivariate Cox proportional hazards models were employed to investigate prognostic factors for disease-free survival(DFS)or overall survival(OS).2.CTECs and leukocytes were isolated at single-cell level.Whole-exome sequencing(WES)was applied to detect single-cell samples(CTECs and leukocytes),formalin-fixed and paraffin-embedded(FFPE)of primary tumor.Somatic single nucleotide variant(SNV),insertion and deletion(InDel),copy number variation(CNV)and mutational signatures were identified in the samples.Significantly mutated genes and driver mutations were revealed by bioinformatics analysis.3.CD44,a cancer stem cell(CSC)marker and CD31 were measured in primary tumor of patients with PD AC by immunohistochemical(IHC)staining.We evaluated the staining score of CD44 and microvascular density(MVD)assessed by CD31.We also analyzed the relationship among CD44,MVD and patients' prognosis.Results1.We detected CTECs levels in 73 patients before surgery,at postoperative day 7(POD7)and postoperative month 1(POM1)using SE-iFISH system.The median follow-up was 10.8 months.We found that history of diabetes(P=0.017),number of positive lymph nodes(P<0.017),preoperative CD44+CTEC levels(P=0.004),and POM1 CA19-9 levels(P<0.001)were independent prognostic factors for DFS after radical surgery.History of diabetes(P=0.004)and POM1 CA19-9 levels(P=0.026)were independent prognostic factors for OS.2.A total of 131,783 SNVs and 24,809 InDels were detected in 4 FFPE samples and 13 matched CTEC samples.While the CNV pattern was consistent among primary tumors,the CNV pattern among CTECs was different from it of primary tumors and showed more heterogeneity.We found that C>T/G>A mutations increased and T>G/A>C mutations decreased in all samples,which were significantly similar to mutational signature 4 and 5 from COSMIC database.1,505 significantly mutated genes(SMGs)such as TTN,CMYA5,AHNAK,and 770 driver mutations such as PRRC2A,SRCAP,RNF213,AHNAK,HERC1 were identified.We also found CD44+CTECs-specific SMGs such as UBR4?TTN?TEP1 and CD44+CTECs-specific diver mutations such as CUX1?MYH11?DCTN1.3.IHC analysis of CD44 and CD31 were performed in 39 PD AC primary tumor samples.We found that tumor diameter(P=0.020),differentiation of tumor(P=0.010)and CD44 staining index(P<0.001)were independent prognostic factors for DFS.CD44 staining index(P<0.001)was also an independent prognostic factor for OS.CD44 expression was significantly associated with low-grade differentiated tumor(correlation coefficient=0.339,P=0.035)and MVD(correlation coefficient=0.485,P=0.002).ConclusionPreoperative CD44+CTEC levels were independent prognostic factors for DFS after radical surgery.The CNV pattern among CTECs showed more heterogeneity and difference comparing to it of primary tumors.Mutational signature 4 and 5,CD44+CTECs-specific significantly mutated genes such as UBR4?TTN?TEP1 may play an important role.CD44 expression in primary tumor of PD AC was an independent prognostic factor for both DFS and OS.CD44 expression was significantly associated with low-grade differentiated tumor and MVD.