Abstract 2232: Circulating tumor cells in peripheral venous blood of primary lung cancer patients: detection using adenovirus GFP-labeling
Authors: Satoh Yukitoshi , Matsui Yoshio , Ogawa Fumihiro , Amano Hideki , Hara Hidenori , Nezu Kenji , Iyoda Akira , Yoshimura Hirokuni , Fukui Tomoya , Igawa Satoshi , Katagiri Makoto , Masuda Noriyuki , Masago Akinori , Tsujino Yukio 
Published: February 2011

Abstract
Background: Circulating tumor cells (CTCs) in peripheral venous blood (PVB) are considered as a potential surrogates for distant metastases, a critical factor influencing decision making and therapy of primary lung cancer (PLC) patients. We here evaluated viable CTCs in PLC patients using a new assay with telomerase-specific replication-selective adenovirus expressing green fluorescent protein (GFP).
Methods: From May to October 2009, a total of 58 consecutive PLC patients (22 women and 36 men) who underwent any treatment were included. CTCs in PVB were quantitatively examined before treatment. For this, 7.5 ml aliquots of PVB were collected using CPD-anticoagulant agent and incubated at 37oC with OBP-401® (Oncolys BioPharma) viruses for 24 hours. Adenovirus OBP-401® features a human telomerase reverse transcriptase gene promoter inserted upstream of the E1 genes, along with a GFP gene driven by a CMV promoter. Whole blood cells were then stained using a LIVE/DEAD Fixable Red Dead Cell Stain Kit. Immunohistochemistry was performed on slides using a tyramide signal amplification kits with Alexa 350-labeled tyramide as the substrate for horse radish peroxidase. Anti-CEA and anti- HRP-goat anti-mouse were applied as primary and secondary antibodies, respectively. Two slides for each patient were also processed for fluorescence microscopy. Software Image-Pro Plus Ver. 6.0 (Media Cybernetics) was used to count the number of GFP expressing cells and measure GFP intensity. GFP positive cells were assumed to have at least 200,000 MEFL, which is the cut-off level of GFP intensity previously obtained using lung, breast and gastric cell lines, or more. Dead cells were assumed to be 20 micrometers diameter or more.
Results: Fifty patients were non-small cell lung cancers (NSCLCs) and 6 were small cell cancers (SCLCs). Fifty one (88%) patients had positive GFP results, rates being more frequent with NSCLCs (48/52) than SCLCs (3/6). None of 72 healthy controls had GFP positive cells in blood samples. The mean GFP positive cell count was 5.62 (range, 0 to 24). As for clinical stages, cells were detected in 85% for Stage IA, 100% each for Stage IB, IIB and IIIA, 77% for IIIB, and 78% for IV. There was no significant correlation with the stage, and between GFP-positive cell count and any other patient characteristic. As for dead cell analysis, 36 (62%) had positive results, with a mean count of 4.5 (range, 0 to 24).
Conclusions: Our results indicated that detection of viable CTCs with our GFR-expression virus-based method provides useful and precise information for PLC patients. Thus, we suggest that such examination should be performed even for patients with early stage disease before beginning any treatment. Furthermore, long term outcome of enrolled patients should be analyzed to assess the prognostic impact.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2232.