Detection And Genome Amplification Of Circulating Tumor Cells For Biliary Tumors

Circulating tumor cells (CTCs) are associated with the recurrence and metastasis of nalignant tumors, which has drawn wide attention in recent years, because of its mportant roles on the early-stage diagnosis, clinical tumor staging, and monitoring For the recurrence, as well as metastasis and drug resistance, prognosis judgment, and he development of new therapeutic targets. The detection of CTCs can realize a ninimized-trauma and dynamic monitor of tumors, so it is also called Liquid Biopsy. A variety of methods for the enrichment and separation of circulating tumor cells lave appeared. The capture, count and downstream analysis of circulating tumor cells lave already been widely used in the study of breast cancer, lung cancer, prostate ;ancer, colon cancer, pancreatic cancer and liver cancer.Biliary tumors, including the gall bladder cancer and cholangiocarcinoma, are a (>)ind of diseases with high invasiveness and malignancy, usually leading to a poor rognosis, which are threatening human’s life and health because of the dilemma of clinical diagnosis and treatment of them. However, there is still no systemic research (?) the circulating tumor cells in the patients suffering biliary tumors in the world.At present, except for the correlation of the number of CTCs and tumor staging, as well as prognosis, researchers also try to develop a stable system to prepare materials for downstream molecular analysis, through ex-vivo capture and amplification of the genome, such as digital PCR, whole genome sequencing, etc. The development and application of the platform in this study provides a stable procedure for ex vivo capture and amplification of the genome of CTCs, in order to facilitate the analysis of drug-resistance mutations, the dynamic monitoring of the disease process, the rediction of clinical outcome, the study of molecular changes in the course of invasion and metastasis, and the heterogeneity of tumor.Methods:1. Patients were enrolled according to the inclusion criteria, excluding the ones meeting the exclusion criteria. The frequency and time point were determined by the surgical situation (whether a curative resection was undertaken) and the pathological report, and the follow-up plan was made.2. All patients provided written informed consent. Circulating tumor cells were captured from the peripheral blood (elbow vein) using the wire developed by GILUPI.3. The cells attached to the wire were dyed with the fluorescent-label EpCAM, CK, CD45 monoclonal antibodies and Hoechst 33342, following the procedure of washing, fixation, permeabilization and blocking.4. Under fluorescence microscope, the cells on the wire were observed, photographed and identified through different channels, and then the count of circulating tumor cells was undertaken.5. The correlation between the number of CTCs and tumor staging, classification, disease progression and prognosis was analyzed. The clinical significance of the count of CTCs was explored.6. A variety of techniques of enrichment and separation of circulating tumor cells and the ex vivo capture and amplification of the genome were explored, including microdissection, single-cell micromanipulation with glass capillary, DEPArray system, the REPLI-g Sing Cell Kit, etc.Results:1. EpCAM was mostly highly or moderately expressed in the tissue of biliary malignant tumor.2. CTCs could be captured from the peripheral blood in part of patients suffering a biliary malignant tumor by placing the GILUPI wire into the elbow vein for 30 minutes, and identified and numbered by immunofluorescence technique. Besides, no complications and adverse reactions were observed in this study.3. The cut-off value of this detection was set at 2 CTCs:we defined it as a positive result when the number of CTCs was equal to or more than 2; a negative result when the number was less than 2. Finally, the positive rate in biliary malignant tumor was 52.5%, the number of CTCs was 2.95±3.78; and the positive rate in benign disease was 11%, the number of CTCs was 0.56±1.33.4. The CTCs amount in patients with a biliary malignant tumor on stage Ⅰ, Ⅱ, Ⅲ, Ⅳ was respectively 1.30±1.57,2.00±2.88,6.38±4.60,12; the positive rates was respectively 40%,43%,88%,1/1.5.91% patients with a biliary malignant tumor obtained a reduction of CTCs number after a curative resection.5. The positive predictive value of CTCs test for a hepatobiliary malignant tumor against a benign disease was 95.5%.7. With a flexible application of different techniques, including density gradient centrifugation, ISET, single-cell micromanipulation with glass capillary, microdissection, and DEPArray system, which owned different feature, we could realize a high throughput, precise, convenient, and undamaged manipulation for CTCs.8. The extraction and amplification of the genome of CTCs could be realized with a REPLI-g Single Cell Kit.Conclusion:1. Compared with the patients suffering biliary benign disease, the positive rate of CTCs in the ones with malignant tumors increased significantly, and so was the number of CTCs.2. The stage of biliary malignant tumors and the number of CTCs in these patients appeared a positive correlation.3. The detection of CTCs could play a vital role in the dynamic monitoring of tumor.4. The detection of CTCs before surgery could benefit the differential diagnosis between gall bladder cancer and xanthogranulomatous cholecystitis.5. To realize a high-throughput, precise, convenient, and undamaged manipulation for CTCs, a flexible application of different techniques should be performed.