Detection of viable human circulating tumor cells using telomerase-specific GFP-expressing bioengineered adenovirus in patients with gastric cancer: A feasibility study.

18 Background: The presence of circulating tumor cells (CTC) in the peripheral blood is associated with short survival and, therefore, the detection of CTC is clinically useful as prognostic factors of disease outcome and/or surrogate markers of treatment response. Recent technical advances in immunocytometric analysis and quantitative real-time PCR have made possible to detect a few CTC in the blood; however, there is no sensitive assay for detecting viable CTC. We developed a new approach to visually detect live CTC among millions of peripheral blood leukocytes using telomerase-specific replication-selective adenovirus expressing green fluorescent protein (GFP). Methods: We constructed a GFP-expressing attenuated adenovirus, in which the telomerase promoter regulates viral replication (OBP-401, TelomeScan). The detection method for viable human CTC in the peripheral blood involves a three-step procedure including the lysis of red blood cells, the subsequent addition of OBP-401 to the cell pellets, and the automated scan under the fluorescent microscope. We analyzed fresh blood samples collected from 37 patients with histologically confirmed gastric cancer. We further assessed the CTC dynamics in patients who were undergoing chemotherapy or surgery to demonstrate the clinical potential of our approach for monitoring treatment responses. Results: OBP-401 increased the signal-to-background ratio as a tumor-specific probe, because the fluorescent signal can be amplified only in viable human tumor cells by viral replication. Although the CTC level varied widely, ranging from 0 to 47 in 5-ml samples, 26 gastric cancer patients (70.3%) had more than one CTC; there was, however, no apparent relationship between CTC counts and TNM stages. Patients who had a recurrence of gastric cancer had decreased CTC counts after systemic chemotherapy. In the patients who underwent surgery, the CTC level dropped after complete resection. Conclusions: This GFP-expressing virus-based method is simple and allows precise enumeration of CTC, which might be useful for monitoring the efficacy of local and systemic treatments. [Table: see text]