Distant metastasis of a solid tumour is a strong prognostic factor [1–3]. The existence of circulating tumour cells (CTCs) in peripheral blood suggests that a patient is in a systemic disease phase . The identification of CTCs in peripheral blood is a useful approach to estimate prognosis, monitor disease progression, and measure treatment effects in breast, prostate, skin, colon and gastrointestinal malignancies. Therefore, various methods have been developed to detect CTCs, and are occasionally used in combination. Common techniques for the enrichment and detection of CTCs are density gradient separation , direct enrichment by filtration [6, 7], immunomagnetic separation , flow cytometry , real-time reverse transcriptase polymerase chain reaction (RT-PCR) [10, 11], and microchip technology .
Cell enrichment by density gradient separation is performed using commercial kits such as OncoQuick® (Greiner, Frickenhausen, Germany)  and Lymphoprep® (Nycomed, Oslo, Norway) . Density gradient separation is based on the theory that different types of cells can be separated according to their density. Therefore, it is difficult to extract all CTCs because of cell migration. RT-PCR is one of the most common methods of tumour cell detection because of its high sensitivity and specificity, assuming adequate primer and probe design. However, false-positive results may occur because of its technical delicacy and high sensitivity [15, 16].
Immunomagnetic cell enrichment, such as that performed by the CellSearch System® (Veridex, LLC, Raritan, NJ, USA) , is currently the most commonly used technique to enrich and detect CTCs [18–21]. The advantage of immunomagnetic cell separation is that CTCs can be visualised with a fluorescent microscope. Cells detected with antibodies against epithelial markers (epithelial cell adhesion molecules; EpCAMs) are determined to be CTCs. Therefore, this technique can provide false-positive results based on normal epithelial marker expression by non-tumour cells, and false-negative results can arise based on the lack of selective marker expression on tumour cells. As a result of the limitations associated with the above approaches, a new technique is needed to detect viable CTCs precisely.
Telomerase plays important roles in carcinogenesis, cancer invasion, and metastasis [22–24]. We have developed a technique to exploit high telomerase activity in cells. This technique uses a telomerase-specific, replication-selective modified viral agent (OBP-401; TelomeScan®, Oncolys BioPharma, Tokyo, Japan) in which the human telomerase reverse transcriptase (TERT) gene promoter is inserted into the E1 region, and the green fluorescent protein (GFP) gene is placed under the control of the cytomegalovirus promoter in the E3 region as a marker of viral replication . It has been reported that OBP-401 can be used to detect viable CTCs among normal blood cells [26, 27].
Here, we applied the assay to detect viable CTCs with the potential for metastasis in gastric cancer patients. We detected GFP-positive CTCs. In contrast, it is possible that non-cancer cells emit GFP fluorescence after OBP-401 infection . Therefore, we selected cells larger than a threshold determined by comparison between healthy volunteers and patients, because CTCs are larger than normal blood cells [7, 29, 30]. We studied the association of GFP-positive CTC number with survival and pathological indices of disease progression.