Abstract 2385: A highly sensitive detection system of genetic alterations in circulating tumor cells using a telomerase-specific replication-competent adenovirus

• Published in: Cancer research (Chicago, Ill.) Ill.), 2012-04, Vol.72 (8_Supplement), p.2385-2385
• Main Authors: Shigeyasu, Kunitoshi, Hashimoto, Yuuri, Morikawa, Tatsuya, Mori, Yoshiko, Sun, Dong-Sheng, Kagawa, Shunsuke, Uno, Futoshi, Tazawa, Hiroshi, Nagasaka, Takeshi, Kyo, Satoru, Mizuguchi, Hiroyuki, Urata, Yasuo, Fujiwara, Toshiyoshi
• Format: Article
• Language: English
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.AM2012-2385

Background: Malignant tumor cells often develop metastatic nodules at distant organs through circulation in blood flow and result in poor prognosis. Detection of circulating tumor cells (CTCs) is clinically an important issue to evaluate the metastatic potential of primary tumors in cancer patients. Furthermore, confirmation of genetic alterations in CTCs provides us the critical information to clarify the molecular mechanism in the acquisition of metastatic capability of tumor cells and to treat tumor metastasis by molecular targeted therapy. We recently developed a telomerase-specific replication-selective adenovirus (OBP-401; TelomeScan) expressing the green fluorescence protein (GFP). OBP-401-mediated GFP induction method was a simple and highly sensitive imaging system to detect a small population of viable CTCs in the peripheral blood. In this study, we assessed the potential of OBP-401-mediated CTC detection system to analyze the genetic alterations in CTCs. Methods: We used two types of human colon cancer cell lines SW480 and HCT116 that have the mutations in codon 12 and 13, respectively, of the K-ras gene. The detection method involved a 4-step procedure, including the lysis of red blood cells, the subsequent addition of OBP-401 to the cell pellets, the isolation of GFP-positive tumor cells using a fluorescence-activated cell sorting (FACS), and a confirmation of genetic mutations by direct sequencing. DNA was extracted from the captured cells using FACS and we investigated the mutation status in the K-ras gene by direct sequencing. Results: When only 10 cells of SW480 or HCT116 cells were added in a 5 ml of blood obtained from healthy volunteer, a total of 2-12 GFP-positive cells were captured from OBP-401-infected blood samples using FACS Aria. We confirmed the mutations in codon 12 and 13 of the K-ras gene in the FACS-captured cells from blood samples containing SW480 and HCT116 cells, respectively, by direct sequencing. Conclusion: These results suggest that OBP-401-mediated GFP induction and FACS-based capture system are useful methods to isolate small population of CTCs from peripheral blood samples and to evaluate the genetic alterations in CTCs. The combination method using OBP-401 and FACS would be a reliable strategy for the surveillance of genetic alterations in CTCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; C