This study is an interim analysis of our prospective preliminary study on CTCs from 65 patients with treatment-negative gastric adenocarcinoma, who underwent surgery at the Digestive Disease Center of the Showa University Northern Yokohama Hospital between April 2010 and May 2011, and from whom we extracted peripheral blood samples before treatment. The inclusion criteria were: i) histologically proven adenocarcinoma of the stomach by endoscopic biopsy; ii) clinical solitary tumor; iii) no prior endoscopic resection, chemotherapy, or radiotherapy; iv) ages, 20–80 years; v) Eastern Cooperative Oncology Group performance status (27) of 0 or 1; vi) sufficient organ function; and vii) written informed consent. The exclusion criteria were: i) synchronous or metachronous malignancy; ii) pregnant or breast-feeding women; iii) active or chronic viral hepatitis; iv) active bacterial or fungal infection; v) diabetes mellitus; vi) systemic administration of corticosteroids; and vii) unstable hypertension. The pathologic stage of the disease was determined according to the seventh edition American Joint Committee on Cancer (AJCC)/International Union Against Cancer (UICC) TNM classification system (28). The depth of the tumor invasion in four patients without gastrectomy and the regional lymph node status of seven patients without sufficient lymphadenectomy were surgically diagnosed.

All the patients were checked regularly every three months in our hospital after surgery. The patients also underwent endoscopy and computed tomography at least once a year, according to their disease stage and course. Healthy volunteers were also recruited to act as controls. All healthy volunteers were employees of Sysmex Corporation, which included seven men (mean age, 31.4 years; range, 24–39 years) and three women (mean age, 33.7 years; range, 26–48 years). All volunteers underwent medical check-ups upon employment and annually; check-ups included medical interviews, auscultation, chest radiography, and blood and urine analyses. In addition, individual interviews were done before sample collection; any volunteer who was currently receiving medical treatment, pregnant, or breast-feeding or who had donated blood within the past month was excluded.

The study was approved by the Institutional Review Board of the Showa University, Northern Yokohama Hospital (no. 0903-03). The study protocol was explained to the patients and volunteers before written informed consent was obtained. This study was registered with the University Hospital Medical Information Network in Japan (no. 000004026).

OBP-401, a telomerase-specific, replication-selective adenoviral agent in which the TERT promoter element drives the expression of the EIA and EIB genes and into which the GFP gene is integrated, was used. The sensitivity and specificity of the assay using OBP-401 have been reported previously by Kim et al (29). The test was repeated five times. In the sample containing one MDA-MB-468 (breast carcinoma) cell and 7.5-ml blood, the numbers of GFP⁺ cells were one, one, one, two, and three; in the sample containing 20 MDA-MB-468 (breast carcinoma) cells, the numbers of GFP⁺ cells were 15, 17, 19, 22, and 24. Viral samples were stored at −80°C.

Details of sample preparation and assay have been described in our previous study (22). A 7.5-ml peripheral vein blood sample was obtained from each patient before surgery and from each volunteer. The samples were drawn into tubes containing citric acid, phosphoric acid, and dextrose and stored at 4°C. The assay was started within 48 h of sample collection. The samples were centrifuged for 5 min at 540 x g, and the plasma phase was removed. The cells were then washed four times with phosphate-buffered saline (PBS) and twice with Roswell Park Memorial Institute medium. The samples were infected with 4×10⁸ plaque-forming units (PFU) of OBP-401 virus by incubation in the medium for 24 h at 37°C. Dead cells were stained with the red-fluorescent reactive dye L23102 (Life Technologies, Carlsbad, CA, USA), OBP-401 was inactivated, and cells were fixed with 2% paraformaldehyde for 20 min at room temperature. The samples were treated with a surface-active agent (Emalgen 2025G; Kao Chemicals, Tokyo, Japan) for 10 min at 40°C to degrade red blood cells. Phycoerythrin-labeled anti-human CD45 antibody (BioLegend, San Diego, CA, USA) was diluted 1:5, and Pacific Blue-labeled anti-human CD326 (EpCAM) antibody (BioLegend) was diluted 1:10 in PBS containing 2% fetal bovine serum. Cells were incubated with the diluted antibodies for 30 min at 25°C. After being washed with PBS containing 2% fetal bovine serum, the cells were mounted on two glass slides for microscopic analysis.

The threshold for GFP fluorescence intensity was determined as previously reported (22). Briefly, ∼30,000 cultured cells were added into 7.5-ml blood samples from healthy volunteers, which were mixed with various cancer cell lines: A549 (lung carcinoma), HepG2 (hepatocellular carcinoma), HEC-1 (endometrial carcinoma), KATO-III (gastric carcinoma), SBC-3 (small cell lung carcinoma), LNCaP (prostate adenocarcinoma), MDA-MB-MB468 (breast carcinoma), and OVCAR-3 (ovarian carcinoma); the cell lines were cultured according to the vendor’s specifications. The blood samples were assayed using CTC detection assay, and the detectable cells were counted by fluorescence microscopy. More than 100 cells were analyzed in each sample. The GFP signal intensity threshold was determined to be 2.85×10⁷ mean equivalent fluorochrome on the basis of the minimal GFP intensity level observed in the blood samples mixed with the cell lines. In addition, there was no significant difference of cell size between the cell before and after OBP-401 infection.

In our previous study (22), various sizes of GFP⁺ cells were observed in each sample, making it difficult to identify representative GFP⁺ cells for comparison between patients and healthy volunteers. Therefore, to establish a constant value, we used the optimum threshold derived from the ROC analysis based on cell size, that is, 7.735 μm, as the threshold to define GFP-positive CTCs. In this study, we categorized GFP⁺ cells into two groups: smaller (S-GFP⁺ cells) or larger (L-GFP⁺ cells) than 7.735 μm in diameter (Fig. 1).

All GFP⁺ cells on the two slides were analyzed using a computer-controlled fluorescence microscope (IX71, Olympus, Tokyo, Japan); the observer was blinded to the sample detail. S-GFP⁺ cells with fluorescent emissions ≥2.85×10⁷ mean equivalent fluorochrome were counted as GFP⁺ cells. GFP⁺ cells included epithelial marker-positive and epithelial marker-negative cells because tumor cells undergoing EMT have been reported to be epithelial marker, such as EpCAM and cytokeratin, negative (18). CD45⁺ cells were excluded from the analysis.

All statistical analysis was performed using JMP Pro 10.0.0.2 (SAS Institute, Cary, NC, USA). Parametric comparisons were done using analysis of variance, and nonparametric comparisons were done using the Wilcoxon and Kruskal-Wallis tests. ROC curve analysis was performed to examine the relationship between patient outcome and the number of GFP⁺ cells. The log-rank test was also used to calculate overall and relapse-free survival rates. Cox proportional hazards analysis was used to investigate risk factor for survival; P≤0.05 was considered statistically significant.

The clinicopathological characteristics of 65 patients (46 men and 19 women; mean age 60.7 years; range 33–76 years) are summarized in Table I. The median follow-up period of surviving patients was 36 months. Fifty-seven of the 65 patients underwent pathological curative surgery, and of these patients, nine experienced disease recurrence. Fourteen patients died. Twenty-nine patients had distal gastrectomy, 32 had total gastrectomy, and four had exploratory laparotomy. Twenty-eight of the 65 patients received chemotherapy after surgery, 19 patients received oral chemotherapy (S-1), and 9 received oral chemotherapy combined with infusion (S-1/cisplatin and S-1/docetaxel).

Comparison of GFP⁺ cells between healthy volunteers and patients are shown in Fig. 2. The numbers of GFP⁺ cells (any size) and S-GFP⁺ cells in the samples from the health volunteers were significantly higher than the ones of the patients (P=0.038 and 0.006). There was no significant difference in L-GFP⁺ cells between the samples from healthy volunteers and the ones from the patients (P=0.760).

There was no significant relationship between the number of GFP⁺ cells (any size, P=0.329), S-GFP⁺ cells (P=0.424) and L-GFP⁺ cells (P=0.213), and cancer stage (Fig. 3A). Although no statistical significance was observed, the number of GFP⁺ cells (any size) and S-GFP⁺ cells tended to increase with the progression of the primary tumor (Fig. 3B). However, the number of GFP⁺ cells in the samples from the node-positive patients was greater than that in the node-negative patients, there was no significant difference (Fig. 3C). Compared with the patients without distant metastases, those with distant metastases had relatively higher numbers of GFP⁺ cells (Fig. 3D). The numbers of GFP⁺ cells were similar in the samples from patients with and without lymphatic invasion (Fig. 3E). For venous invasion, the number of L-GFP⁺ cells in the samples from the patients with invasion was significantly higher than that in patients without invasion (P=0.031) (Fig. 3F).

The numbers of the detected GFP⁺ cells in the peripheral blood samples are shown in Fig. 4. The mean value of GFP⁺ cells with any size, <7.735 μm and >7.735 μm were 23.8, 19.0 and 4.8 in the samples from healthy volunteers, and 24, 19 and 5 were prescribed cutoff values of GFP⁺ cells with any size, <7.735 μm and >7.735 μm. The overall survival rate of patients who had 24 or more GFP⁺ cells was lower than that of patients who had <24 GFP⁺ cells (P= 0.281) (Fig. 4A); however, the difference was not significant. The overall survival rate of patients who had 20 or more GFP-positive S-GFP⁺ cells also tended to be lower than that of patients who had <20 GFP-positive S-GFP⁺ cells (P=0.327) (Fig. 4B). Although there was no significant difference, the overall survival rate of patients who had 5 or more L-GFP⁺ cells was lower than that of patients who had <5 L-GFP⁺ cells (P=0.148) (Fig. 4C).

We performed ROC analysis to determine another cutoff values. The ROC analysis showed that the numbers of GFP⁺ cells (P= 0.241, AUC 0.546, cutoff 17, sensitivity 55.6%, and specificity 68.8%) and L-GFP⁺ cells (P=0.770, AUC 0.548, cutoff 6, sensitivity 44.4%, and specificity 81.3%) in the samples from the deceased patients were higher than those in the samples from the surviving patients (Fig. 5A and B), although the difference was not significant. No particular tendency was observed in GFP-positive S-GFP⁺ cells (P=0.159, AUC 0.557, cutoff 29, sensitivity 22.2%, and specificity 100%) (Fig. 5C). Based on these results, 17 and 6 were prescribed second cutoff values of GFP⁺ cells with any size and >7.735 μm. The overall survival rate of patients who had 17 or more GFP⁺ cells was lower than that of patients who had <17 GFP⁺ cells (P=0.067) (Fig. 6A); however, the difference was not significant. The overall survival rate of patients who had 6 or more L-GFP⁺ cells was significantly lower than that of patients who had <6 L-GFP⁺ cells (P=0.037) (Fig. 6B). Moreover, the overall survival rate of patients who had both 17 or more GFP⁺ cells and 6 or more L-GFP⁺ cells was significantly lower than that of patients who had <17 GFP⁺ cells or <6 L-GFP⁺ cells (P=0.004) (Fig. 6C). Seven of 16 (43.8%) patients who had both 17 or more GFP⁺ cells and 6 or more L-GFP⁺ cells, and 7 of 49 (14.3%) patients who had <17 GFP⁺ cells or <6 L-GFP⁺ cells deceased. In the 57 patients who underwent curative surgery, the relapse-free survival rate of the patients who had 17 or more GFP⁺ cells was lower than that of patients who had <17 GFP⁺ cells (P=0.130) (Fig. 7A); however, the difference was not significant. Although there was no significant difference, the relapse-free survival rate of patients who had 6 or more L-GFP⁺ cells was also lower than that of patients who had <6 L-GFP⁺ cells (P= 0.124) (Fig. 7B). The relapse-free survival rate of the patients who had both 17 or more GFP⁺ cells and 6 or more L-GFP⁺ cells was significantly lower than that of the patients who had <17 GFP⁺ cells or <6 L-GFP⁺ cells (P=0.015) (Fig. 7C).