Establishment Of The Methodology Of Circulating Tumor Cells Detection And Its Clinical Application For Pancreatic Cancer

Background Pancreatic cancer(PC)was regarded as a lethal malignancy with a poorer outcome than most other solid tumors.Because of the hidden symptoms,the early diagnosis is still a challenge in clinical treatment of pancreatic cancer,and only 15%-20%patients with pancreatic cancer are considered resectable when first diagnosed.So how to early detect and monitor it is an urgent problem in present.Circulating tumor cells(CTCs),which are tumor cells fall off from solid tumor masses and travel into peripheral blood circulation,have been used as a promising biomarker to monitor chemotherapeutic efficacy in prostate cancer,breast cancer and colorectal cancer.Circulating tumor cells(CTCs)is an emerging non-invasive biomarker for tumor detection.However,the false negative is a main problem in traditional Cell Search System to detect CTCs,which use Ep CAM and CK as marker.Method 1.Patients and samples collection.Forty pancreatic cancer patients,8 pancreatic benign disease and 35 healthy donors were accessed to our research at Tianjin Medical University Cancer Institute and Hospital from April 2014 to July 2016.Among them.All the patients were diagnosed with pancreatic adenocarcinoma by pathological analysis but received no chemotherapy.2.Detection of CTCs by NE-FISH.The primary method of CTCs Negative enrichment was removing hematopoietic cells as much as possible by density gradient centrifugation and blood cellular markers.Anti-CD45 and anti-CK18 would be used to label WBCs and CTCs,respectively.Dry and fix the enriched cells on the slide at 31 centigrade overnight and then dehydrate the cells by anhydrous ethanol.10 ?l chromosome enumeration probes 8 were used to cover to the cells on the slide.Then denature at 76? for 5 minutes,hybridize at 37? for 90 minutes.The final step was nuclear staining by 4',6-diamidino-2-phenylindole(DAPI),then observe the cells under the fluorescence microscope.Result 1.Cell types were divided into five categories by immunofluorescence and in situ hybridization results which are type-A: CK18-,CD45-,DAPI+,CEP-8?2;type-B: CK18+,CD45-,DAPI+;type-C: CK18-,CD45+,DAPI+;type-D: CK18-,CD45-,DAPI+,CEP-8=2;type-E: circulating tumor microembolus(CTMs).type-A,type-B and type-E were confirmed as CTCs.Type-C was confirmed as hematopoietic cells.Type-D was confirmed as indeterminate cells.2.PC patients exhibited more CTCs than control group whether in the median or the positive rate.Receiver Operator Characteristic(ROC)curve was used to reveal the 77.5% sensitivity and 79.1% specificity of CTCs as a biomaker to diagnose pancreatic cancer when the cuttoff value was decided as 1.5 CTCs/7.5 m L(AUC=0.83,95% CI 0.747–0.913).3.The 1-year survival in the group of CTC<3 was significantly higher than that of CTC?3(p=0.043).In addition,we analyzed the role of chromosomal instability in CTCs detection.Interestingly,the group of triploid CTC?3 showed a shorter 1-year survival(p=0.0279)and overall survival(p=0.0188)than that with triploid CTC<3.The proportion of triploid detected by the NE-i FISH was significantly decreased after chemotherapy(?2=30.381,p<0.001).4.CTM has been detected in 6 patients out of 40 patients and they were all metastatic pancreatic cancer(stage ?).CTM=0 group exhibited significantly longer survival than CTM>0 group,with the median of 8.27 months versus 6.6 months(p=0.0277).Conclusions NE-FISH system significantly improved the positive detection rate of diverse CTCs.The diagnostic rate of PC reached to 97.5% when combined CTCs?2 and CA19-9>37?mol/L.Triploid CTCs could be a marker for diagnose and outcome prediction of PC patients.CTM is a potential indicator of chemotherapeutic effect in advanced cancer patient.