Prognostic impact of the number of viable circulating cells with high telomerase activity in gastric cancer patients: A prospective study

  • Authors:
    • Hiroaki Ito
    • Haruhiro Inoue
    • Satoshi Kimura
    • Tohru Ohmori
    • Fumihiro Ishikawa
    • Keigo Gohda
    • Jun Sato
  • View Affiliations

  • Published online on: April 29, 2014     https://doi.org/10.3892/ijo.2014.2409
  • Pages: 227-234
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

The identification of circulating tumor cells (CTCs) in peripheral blood is a useful approach to estimate prognosis, monitor disease progression and measure treatment effects in several types of malignancies. We have previously used OBP-401, a telomerase-specific, replication-selective, oncolytic adenoviral agent carrying the green fluorescent protein (GFP) gene. GFP-positive cells (GFP+ cells) were counted under a fluorescence microscope. Our results showed that the number of at least 7.735 µm in diameter GFP+ cells (L-GFP+ cells) in the peripheral blood was a significant marker of prognosis in gastric cancer patients. However, tumor cells undergoing epithelial-mesenchymal transition (EMT) have been reported to be smaller in size than cells without EMT features; thus, CTCs undergoing EMT may escape detection with this technique. Therefore, in this study, we analyzed the relationship between patient outcome and the number of GFP+ cells of any size. We obtained peripheral blood samples from 65 patients with gastric cancer. After infection of OBP-401, GFP+ cells were counted and measured. The relationship between the number of GFP+ cells and surgical outcome was analyzed. The median follow-up period of the surviving patients was 36 months. A significant difference in overall survival was found between patients with 0-5 and patients with ≥6 L-GFP+ cells. No clear relationship was established between the number of small-sized GFP+ cells and patient prognosis. The number of L-GFP+ cells was significantly related to overall survival in patients with gastric cancer. The detection of L-GFP+ cells using OBP-401 may be a useful prognostic marker in gastric cancer.

Introduction

Distant metastasis is a strong prognostic factor in patients with solid tumors (13), and the presence of circulating tumor cells (CTCs) in peripheral blood indicates a systemic disease stage (4). The detection of CTCs in peripheral blood is useful for estimation of prognosis and monitoring of disease progression in breast, prostate, skin, colon and gastrointestinal malignancies. Although various methods have been developed to detect CTCs, the common techniques for the enrichment and detection of CTCs are density gradient separation (5,6), direct enrichment by filtration (7), immunomagnetic separation (8), flow cytometry (9), real-time reverse transcriptase polymerase chain reaction (RT-PCR) (10,11), and microchip technology (12). The CellSearch System (Veridex, LLC, Raritan, NJ, USA) (13) is based on immunomagnetic cell enrichment and is one of the most widely used automated techniques to enrich and detect CTCs (1416). The advantage of immunomagnetic cell separation is that CTCs can be visualized with a fluorescence microscope. Cells detected with antibodies against epithelial markers [epithelial cell adhesion molecules (EpCAMs)] are determined to be CTCs. During epithelial-mesenchymal transition (EMT), an important developmental process in CTCs (17), epithelial surface markers are suggested to decrease (18). Thus, CTCs undergoing EMT may escape detection by systems using epithelial markers.

Increased telomerase activity is a common characteristic of malignant tumors, and telomerase plays important roles in carcinogenesis and disease progression (19,20). Therefore, we have developed a novel detection system to enrich cells with high telomerase activity in peripheral blood samples from cancer patients. We used OBP-401 (TelomeScan, Oncolys BioPharma, Tokyo, Japan), which is a telomerase-specific, replication-selective modified viral agent 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 (21). We obtained 7.5-ml blood samples from 65 treatment-negative gastric cancer patients before surgery and 10 healthy volunteers (22). We detected viable CTCs in the blood samples after incubation with OBP-401. GFP-positive (GFP+) cells were detected in all blood samples. Since it has been reported that CTCs are larger than normal blood cells (23,24), we counted GFP+ cells having a diameter of at least 7.735 μm (L-GFP+ cells); this threshold was determined by receiver operating characteristic curve (ROC) analysis. As a result, there was a significant difference in overall survival between patients with 0–4, and those with ≥5 L-GFP+ cells in both the stage I–IV disease and stage II–IV advanced disease groups. On the other hand, the number of L-GFP+ cells showed no significant correlation to cancer stage. A pathological finding showed that the number of GFP+ cells was only significantly related to venous invasion, although there was a trend of higher number of L-GFP+ cells with disease progression (22).

Our results (22) suggest that patients with L-GFP+ cells showed significant survival; however, other studies have shown that tumor cells undergoing EMT are smaller in size than cells without EMT features, because of changes in cell shape (25,26). Thus, CTCs undergoing EMT possibly escape detection using our technique. Therefore, we analyzed the relationship between the number of GFP+ cells of any size and patient outcome at a median-follow up of three years.

Materials and methods

Patients and healthy volunteers

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).

Virus

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.

Sample preparation and immunostaining

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.

Determination of GFP fluorescence intensity threshold

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.

Determination of cell size threshold

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).

Cell counting and analysis

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.

Statistical 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.

Results

Participant characteristics

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).

Table I.

Patient characteristics and pathological findings.

Table I.

Patient characteristics and pathological findings.

VariableNo. of patients
Gender
  Male46
  Female19
Age (years; mean, range)58.8 (33–76)
Gastrectomy
  Distal29
  Total32
  None4
Curability
  R057
  R10
  R28
TNM stage
  I40
  II6
  III10
  IV9
Depth of tumor invasion
  T136
  T28
  T39
  T412
Lymph node metastasis
  N039
  N15
  N26
  N315
Distant metastasis
  M056
  M19
Main histological typea
  Differentiated25
  Undifferentiated40
Lymphatic invasion
  L035
  L126
  LX4
Venous invasion
  V035
  V1-226
  VX4
Postoperative chemotherapy
  Yes (oral)19
  Yes (oral and infusion)9
  No37

a Well-differentiated or moderately differentiated adenocarcinoma and papillary adenocarcinoma were categorized as differentiated type. Signet-ring cell carcinoma, poorly differentiated adenocarcinoma, and mucinous adenocarcinoma were categorized as undifferentiated type.

Association of GFP-positive cells with pathological indices

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).

Relationship between the patient outcome and the number and size of GFP-positive cells

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).

Discussion

In this study, we analyzed the correlation between CTCs and prognosis in gastric cancer, which is the second leading cause of cancer-related death worldwide. The usefulness of the detection of CTCs in the diagnosis and estimation of prognosis has already been reported for breast (14,30), prostate (31), lung (32), and digestive tract (11,33) cancers. The results of the present study indicate that detection of CTCs may also be useful in the prognosis of gastric cancer.

This study showed two major findings. One was that the number of L-GFP+ cells is significantly associated with patient prognosis. In our previous study (22), the prognosis of the patients who had 5 or more GFP+ cells was significantly lower than that of the patients who had <5 L-GFP+ cells. In this study, we obtained a similar result showing that the prognosis of patients who had 6 or more L-GFP+ cells was significantly lower than that of patients who had <6 L-GFP+ cells.

Further, we determined whether the number of GFP+ cells of any diameter may be related to patient prognosis. Patients who had 17 or more GFP+ cells showed lower survival rate than those who had <17 GFP+ cells, although the difference was not significant. Since the combination of the number of total GFP+ cells and L-GFP+ cells showed a significant correlation with patient prognosis whereas the number of only L-GFP+ cells did not, we deemed the number of all GFP+ cells to be related to patient prognosis. On the other hand, the relationship between the number of S-GFP+ cells and prognosis was unclear. Although there was a significant difference in the prognosis between patients who had 29 or more S-GFP+ cells (n=2) and those who had <29 S-GFP+ cells (n=63), unequal numbers of patients were enrolled in the two groups. In our previous study (22), S-GFP+ cells were observed in the blood samples from healthy volunteers. Therefore, S-GFP+ cells may be detected as false-positive CTCs. There is possibility that OBP-401 infection caused increased telomerase activity in non-cancer cells.

One limitation of our study was that the metastatic potential of the detected CTCs was not determined. Our results suggested L-GFP+ cells to be a predictive and prognostic marker; however, further study is needed to determine the metastatic potential of L-GFP+ cells. On the other hand, S-GFP+ cells may contain a small population of CTCs with metastatic potential including tumor cells with EMT. It was suggested that the CTCs with EMT were included in both of S-GFP+ cells and L-GFP+ cells in this study. Clearly, more studies in a larger population of patients, and with different cancer types, are needed to clarify the clinical applicability of CTC detection. Thus, further studies should analyze the functions of viable CTCs after cell sorting, and identify CTCs with metastatic potential using additional tools such as DNA ploidy analysis (34,35). Furthermore, gene expression profiling of viable CTCs, dead cells, primary tumors, and metastatic tumors will also provide important insight into the mechanisms of cancer metastasis. Finally, the results of the present study indicate that CTCs are useful as predictors of disease progression in gastric cancer patients, but they do not constitute an independent prognostic factor.

The number of detected L-GFP+ cells showed a significant relationship with prognosis in gastric cancer. However, the study used a short follow-up period and only a small number of participants. In addition, whether all GFP+ cells have true metastatic potential was unclear. Further studies are warranted to confirm the findings of this study.

Acknowledgements

This study was supported in part by a Grant-in-Aid for Challenging Exploratory Research (23659308) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT). We are grateful to all the patients and volunteers who donated blood for this study. We would like to thank Professor Toshiyoshi Fujiwara (Okayama University Graduate School of Medicine, Okayama, Japan) for helpful comments and suggestions; Mr. Yasuo Urata (Oncolys BioPharma, Tokyo, Japan) for supplying the OBP-401; Dr Yukio Tsujino, Dr Toshiyuki Ozawa, and Dr Akinori Masago (Sysmex Corporation, Kobe, Japan) for their valuable support; and the clinical staff.

References

1. 

Kowalski LP: Lymph node metastasis as a prognostic factor in laryngeal cancer. Rev Paul Med. 111:42–45. 1993.PubMed/NCBI

2. 

Nakane Y, Okamura S, Masuya Y, Okumura S, Akehira K and Hioki K: Incidence and prognosis of para-aortic lymph node metastasis in gastric cancer. Hepatogastroenterology. 45:1901–1906. 1998.PubMed/NCBI

3. 

Arai Y, Kanamaru H, Yoshimura K, Okubo K, Kamoto T and Yoshida O: Incidence of lymph node metastasis and its impact on long-term prognosis in clinically localized prostate cancer. Int J Urol. 5:459–465. 1998. View Article : Google Scholar : PubMed/NCBI

4. 

Liotta LA, Kleinerman J and Saidel GM: Quantitative relationships of intravascular tumor cells, tumor vessels, and pulmonary metastases following tumor implantation. Cancer Res. 34:997–1004. 1974.PubMed/NCBI

5. 

Hanahan D and Weinberg RA: The hallmarks of cancer. Cell. 100:57–70. 2000. View Article : Google Scholar

6. 

Gertler R, Rosenberg R, Fuehrer K, Dahm M, Nekarda H and Siewert JR: Detection of circulating tumor cells in blood using an optimized density gradient centrifugation. Recent Results Cancer Res. 162:149–155. 2003. View Article : Google Scholar : PubMed/NCBI

7. 

Vona G, Sabile A, Louha M, et al: Isolation by size of epithelial tumor cells: a new method for the immunomorphological and molecular characterization of circulating tumor cells. Am J Pathol. 156:57–63. 2000. View Article : Google Scholar

8. 

Talasaz AH, Powell AA, Huber DE, et al: Isolating highly enriched populations of circulating epithelial cells and other rare cells from blood using a magnetic sweeper device. Proc Natl Acad Sci USA. 106:3970–3975. 2009. View Article : Google Scholar : PubMed/NCBI

9. 

He W, Wang H, Hartmann LC, Cheng JX and Low PS: In vivo quantitation of rare circulating tumor cells by multiphoton intravital flow cytometry. Proc Natl Acad Sci USA. 104:11760–11765. 2007. View Article : Google Scholar : PubMed/NCBI

10. 

Ito H, Kanda T, Nishimaki T, Sato H, Nakagawa S and Hatakeyama K: Detection and quantification of circulating tumor cells in patients with esophageal cancer by real-time polymerase chain reaction. J Exp Clin Cancer Res. 23:455–464. 2004.PubMed/NCBI

11. 

Honma H, Kanda T, Ito H, et al: Squamous cell carcinoma-antigen messenger RNA level in peripheral blood predicts recurrence after resection in patients with esophageal squamous cell carcinoma. Surgery. 139:678–685. 2006. View Article : Google Scholar

12. 

Nagrath S, Sequist LV, Maheswaran S, et al: Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature. 450:1235–1239. 2007. View Article : Google Scholar : PubMed/NCBI

13. 

Cohen SJ, Punt CJ, Iannotti N, et al: Relationship of circulating tumor cells to tumor response, progression-free survival, and overall survival in patients with metastatic colorectal cancer. J Clin Oncol. 26:3213–3221. 2008. View Article : Google Scholar : PubMed/NCBI

14. 

Riethdorf S, Fritsche H, Muller V, et al: Detection of circulating tumor cells in peripheral blood of patients with metastatic breast cancer: a validation study of the CellSearch system. Clin Cancer Res. 13:920–928. 2007. View Article : Google Scholar : PubMed/NCBI

15. 

Davis JW, Nakanishi H, Kumar VS, et al: Circulating tumor cells in peripheral blood samples from patients with increased serum prostate specific antigen: initial results in early prostate cancer. J Urol. 179:2187–2191. 2008. View Article : Google Scholar

16. 

Hou JM, Greystoke A, Lancashire L, et al: Evaluation of circulating tumor cells and serological cell death biomarkers in small cell lung cancer patients undergoing chemotherapy. Am J Pathol. 175:808–816. 2009. View Article : Google Scholar : PubMed/NCBI

17. 

Ksiazkiewicz M, Markiewicz A and Zaczek AJ: Epithelial-mesenchymal transition: a hallmark in metastasis formation linking circulating tumor cells and cancer stem cells. Pathobiology. 79:195–208. 2012. View Article : Google Scholar : PubMed/NCBI

18. 

Gorges TM, Tinhofer I, Drosch M, Roese L, Zollner TM, Krahn T and von Ahsen O: Circulating tumour cells escape from EpCAM-based detection due to epithelial-to-mesenchymal transition. BMC Cancer. 12:1782012. View Article : Google Scholar : PubMed/NCBI

19. 

Kim NW, Piatyszek MA, Prowse KR, et al: Specific association of human telomerase activity with immortal cells and cancer. Science. 266:2011–2015. 1994. View Article : Google Scholar : PubMed/NCBI

20. 

Blackburn EH: Telomere states and cell fates. Nature. 408:53–56. 2000. View Article : Google Scholar : PubMed/NCBI

21. 

Fujiwara T, Kagawa S, Kishimoto H, et al: Enhanced antitumor efficacy of telomerase-selective oncolytic adenoviral agent OBP-401 with docetaxel: preclinical evaluation of chemovirotherapy. Int J Cancer. 119:432–440. 2006. View Article : Google Scholar

22. 

Ito H, Inoue H, Sando N, et al: Prognostic impact of detecting viable circulating tumour cells in gastric cancer patients using a telomerase-specific viral agent: a prospective study. BMC Cancer. 12:3462012. View Article : Google Scholar

23. 

Lin HK, Zheng S, Williams AJ, et al: Portable filter-based microdevice for detection and characterization of circulating tumor cells. Clin Cancer Res. 16:5011–5018. 2010. View Article : Google Scholar : PubMed/NCBI

24. 

Zheng S, Lin HK, Lu B, Williams A, Datar R, Cote RJ and Tai YC: 3D microfilter device for viable circulating tumor cell (CTC) enrichment from blood. Biomed Microdevices. 13:203–213. 2011. View Article : Google Scholar : PubMed/NCBI

25. 

Thiery JP: Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer. 2:442–454. 2002. View Article : Google Scholar : PubMed/NCBI

26. 

Brabletz T, Hlubek F, Spaderna S, Schmalhofer O, Hiendlmeyer E, Jung A and Kirchner T: Invasion and metastasis in colorectal cancer: epithelial-mesenchymal transition, mesenchymal-epithelial transition, stem cells and beta-catenin. Cells Tissues Organs. 179:56–65. 2005. View Article : Google Scholar : PubMed/NCBI

27. 

Oken MM, Creech RH, Tormey DC, Horton J, Davis TE, McFadden ET and Carbone PP: Toxicity and response criteria of the Eastern Cooperative Oncology Group. Am J Clin Oncol. 5:649–655. 1982. View Article : Google Scholar : PubMed/NCBI

28. 

Sobin LH, Gospodarowicz MK and Wittekind C: International Union Against Cancer: TNM Classification of Malignant Tumours. 7th edition. Chichester, West Sussex, UK; Hoboken, NJ: Wiley-Blackwell; 2010

29. 

Kim SJ, Masago A, Tamaki Y, et al: A novel approach using telomerase-specific replication-selective adenovirus for detection of circulating tumor cells in breast cancer patients. Breast Cancer Res Treat. 128:765–773. 2011. View Article : Google Scholar

30. 

Cristofanilli M, Budd GT, Ellis MJ, et al: Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med. 351:781–791. 2004. View Article : Google Scholar : PubMed/NCBI

31. 

Moreno JG, Miller MC, Gross S, Allard WJ, Gomella LG and Terstappen LW: Circulating tumor cells predict survival in patients with metastatic prostate cancer. Urology. 65:713–718. 2005. View Article : Google Scholar : PubMed/NCBI

32. 

Krebs MG, Sloane R, Priest L, et al: Evaluation and prognostic significance of circulating tumor cells in patients with non-small-cell lung cancer. J Clin Oncol. 29:1556–1563. 2011. View Article : Google Scholar : PubMed/NCBI

33. 

Katsumata K, Sumi T, Mori Y, Hisada M, Tsuchida A and Aoki T: Detection and evaluation of epithelial cells in the blood of colon cancer patients using RT-PCR. Int J Clin Oncol. 11:385–389. 2006. View Article : Google Scholar : PubMed/NCBI

34. 

Bonsing BA, Beerman H, Kuipers-Dijkshoorn N, Fleuren GJ and Cornelisse CJ: High levels of DNA index heterogeneity in advanced breast carcinomas. Evidence for DNA ploidy differences between lymphatic and hematogenous metastases Cancer. 71:382–391. 1993.PubMed/NCBI

35. 

Klijanienko J, el-Naggar AK, de Braud F, et al: Tumor vascularization, mitotic index, histopathologic grade, and DNA ploidy in the assessment of 114 head and neck squamous cell carcinomas. Cancer. 75:1649–1656. 1995. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

July-2014
Volume 45 Issue 1

Print ISSN: 1019-6439
Online ISSN:1791-2423

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Ito H, Inoue H, Kimura S, Ohmori T, Ishikawa F, Gohda K and Sato J: Prognostic impact of the number of viable circulating cells with high telomerase activity in gastric cancer patients: A prospective study. Int J Oncol 45: 227-234, 2014.
APA
Ito, H., Inoue, H., Kimura, S., Ohmori, T., Ishikawa, F., Gohda, K., & Sato, J. (2014). Prognostic impact of the number of viable circulating cells with high telomerase activity in gastric cancer patients: A prospective study. International Journal of Oncology, 45, 227-234. https://doi.org/10.3892/ijo.2014.2409
MLA
Ito, H., Inoue, H., Kimura, S., Ohmori, T., Ishikawa, F., Gohda, K., Sato, J."Prognostic impact of the number of viable circulating cells with high telomerase activity in gastric cancer patients: A prospective study". International Journal of Oncology 45.1 (2014): 227-234.
Chicago
Ito, H., Inoue, H., Kimura, S., Ohmori, T., Ishikawa, F., Gohda, K., Sato, J."Prognostic impact of the number of viable circulating cells with high telomerase activity in gastric cancer patients: A prospective study". International Journal of Oncology 45, no. 1 (2014): 227-234. https://doi.org/10.3892/ijo.2014.2409