Yukio Tsujino

Long-term prognostic impact of circulating tumour cells in gastric cancer patients

AIM To analyse the long-term prognostic impact of circulating tumour cells (CTCs) in gastric cancer patients who underwent surgery. METHODS A 7.5-mL peripheral vein blood sample was obtained from each patient with treatment-negative gastric adenocarcinoma before surgery. OBP-401, a telomerase-specific, replication-selective, oncolytic adenoviral agent carrying the green fluorescent protein gene, was used to label CTCs. Correlations between the number of CTCs and clinical end points were evaluated. RESULTS The median follow-up period of the surviving patients with gastric cancer was 60 mo. The CTC number tended to increase concomitantly with disease progression. The overall survival of patients with more than five CTCs in 7.5-mL of peripheral blood was lower than that of patients with five or less CTCs, although the difference was not significant (P = 0.183). A significant difference in relapse-free survival was found between patients with more than five and those with five or less CTCs (P = 0.034). CONCLUSION A lower number of CTCs was correlated with higher relapse-free survival rates in patients. Detection of CTCs using OBP-401 may be useful for predicting prognosis in gastric cancer.

Abstract 3160: Clinical utility of circulating tumor cell monitoring for therapeutic response in gastric cancer patients

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Circulating tumor cells (CTCs) in the peripheral blood are considered as a useful biomarker for disease outcome and treatment response because the presence of CTCs is associated with short survival. We previously established the biological imaging system to detect viable CTCs among millions of peripheral blood leukocytes using telomerase-specific replication-selective adenovirus expressing green fluorescent protein (GFP) (Kojima et al, J Clin Invest, 2010). In present study, we demonstrate the clinical potential of our method for monitoring the efficacy of treatment in patients with gastric cancer. Methods: We used a GFP-expressing adenovirus variant, in which the telomerase promoter regulates viral replication (OBP-401), to selectively label human tumor cell with fluorescence. Whole blood cells collected from 7.5ml of peripheral blood were infected OBP-401 after elimination of anti-adenovirus antibodies and incubated at 37 oC. Viruses were inactivated 24 hours after virus-infection and then red blood cells were lysed. The GFP-expressing cells were detected using fluorescence microscopy. Results: A total of 72 blood samples from 39 patients with histologically-confirmed gastric cancer (stage IA to IV) were analyzed. CTCs were detected in 32 of 39 patients (82%, range 1 to 40<) before treatment, however, there was no apparent relationship between the number of CTCs and clinical status. We further assessed the CTC dynamics in 24 patients who were undergoing surgery, chemotherapy or endoscopic submucosal dissection (ESD). In patients with early gastric cancer, 7 patients had decrease in CTC numbers after surgery or ESD, and 6 patients had decreased CTC numbers following temporary increase after treatment. As for advanced cancer, the number of CTCs dropped in 3 patients after complete resection and increased in 2 patients with poor prognosis despite chemotherapy. Conclusion: Our data suggest that patients with early gastric cancer also have CTCs in peripheral blood and the dynamic change of CTCs correlate with treatment response. Although numerous samples and long-term outcome should be analyzed, the enumeration of CTC using this GFP-expressing virus-based method might be useful for monitoring the efficacy of treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3160. doi:10.1158/1538-7445.AM2011-3160

Abstract LB-276: A novel approach using the telomerase-selective adenoviral marker (OBP-401) for detection of circulation tumor cells in breast cancer patients

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: The detection of circulating tumor cells (CTCs) is a potential method to predict survival of metastatic breast cancer patients as well as outcomes of early breast cancer patients. However, no method for CTCs has yet proven to be the golden standard. We developed a new approach for detecting CTCs using the telomerase-selective adenoviral marker (OBP-401, Oncolys Biopharma). This marker contains the replication cassette, in which the human telomerase reverse transcriptase promoter drives expression of E1 genes, and the green fluorescent protein (GFP) gene for monitoring viral replication. Thus, OBP-401 infects viable cancer cells and replicates in them under the telomerase activity, and we can detect viable CTCs as GFP-positive cells. Methods: This system is consisted of the following steps; (1) virus-infection for 7.5 ml whole blood (incubated with 4 × 10sup8 Plaque Forming Unit OBP-401 virus for 24 hours at 37 Celsius), (2) dead cell staining using [L23102][1] (invitrogen), (3) virus-inactivation and RBC elimination, and (4) detection of GFP expressing cells using fluorescence microscopy. In a preclinical study, the sensitivity of this system was assessed using cell lines. Next, we conducted feasibility studies for CTCs detection in 80 healthy individuals, 70 metastatic, and 27 early breast cancer patients. In metastatic and early breast cancer patients, we compared the sensitivity of this system with that of CellSearch® (Veridex). GFP-positive cells (viable CTCs) and [L23102][1] expressing cells measuring ≥ 20 m in diameter (dead CTCs) were considered as CTCs in this system. Preliminary data: The sensitivity of this system, which was determined by a serial dilution of MDA-MB-468 cells against healthy volunteers blood, was 1 cell per 7.5 ml. Neither viable nor dead CTCs were detected in any of healthy controls. Of 50 metastatic patients, 12% were primary breast cancers with stage IV disease, 24% were in the 1st line chemotherapy setting, and 42 % were heavily pretreated with chemotherapy. The sensitivities of tumor markers (CEA and CA15-3), CellSearch, and OBP-401 were 78%, 54%, and 66%, respectively. 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 LB-276. [1]: /lookup/external-ref?link_type=GEN&access_num=L23102&atom=%2Fcanres%2F70%2F8_Supplement%2FLB-276.atom

Abstract 2232: Circulating tumor cells in peripheral venous blood of primary lung cancer patients: detection using adenovirus GFP-labeling

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC 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.

Detection of Circulation Tumor Cells Using Telomerase-Selective Adenoviral Marker (OBP-401®) in Breast Cancer Patients.

Background: The detection of circulating tumor cells (CTCs) is a potential method to predict survival of metastatic breast cancer patients as well as outcomes of early breast cancer patients. However, no method for CTCs has yet proven to be the golden standard. We developed a new approach for detecting CTCs using telomerase-specific replication-competent adenovirus expressing green fluorescent protein (GFP) (OBP-401®, Oncolys Biopharma™).Methods: OBP-401® contains the replication cassette, in which the human telomerase reverse transcriptase promoter drives expression of E1 genes, and the GFP gene for monitoring viral replication. This system is consisted of the following steps; (1) virus-infection for 7.5 ml whole blood (incubated with 4 X 108 Plaque Forming Unit OBP-401 virus for 24 hours at 37 Celsius), (2) dead cell staining using L23102 (invitrogen™), (3) virus-inactivation and RBC elimination, and (4) detection of GFP expressing cells using fluorescence microscopy. In a preclinical study, the sensitivity of this system was assessed using cell lines. Next, we conducted feasibility studies for CTCs detection in 80 healthy individuals, 50 metastatic, and 27 early breast cancer patients. In metastatic and early breast cancer patients, we compared the sensitivity of this system with that of CellSearch® (Veridex™) and tumor makers (CEA and CA15-3). GFP-positive cells (viable CTCs) and L23102 expressing cells measuring ≥ 20µm in diameter (dead CTCs) were considered as CTCs in this system.Results: The sensitivity of this system, which was determined by a serial dilution of MDA-MB-468 cells against healthy volunteer9s blood, was 1 cell per 7.5 ml. No CTCs were detected in any of healthy controls. Of 50 metastatic patients, 12% were primary breast cancers with stage IV disease, 24% were in the 1st line chemotherapy setting, and 42% were heavily pretreated with chemotherapy. The sensitivities of tumor markers, CellSearch®, and OBP-401® were 78%, 54%, and 66%, respectively. Neither CTCs detected with CellSearch® nor OBP-401® were significantly associated with clinicopathologic parameters. However, CTCs-positivity detected with CellSearch® were strongly associated with CA15-3 positivity (p = 0.003). Of 14 patients with normal CA15-3 levels, CellSearch® detected CTCs in three patients (21%) but OBP-401® in nine patients (64%). The sensitivity of the combination of tumor markers and OBP-401® was 92%. In 27 early breast cancers (Stage1 7, StageII 17, StageIII 3), three patients were treated with neoadjuvant chemotherapy (NAC). All blood samples were drawn before surgery or NAC. The sensitivities of tumor markers, CellSearch®, and OBP-401® were 7%, 0%, 67%, respectively. There were no significant correlations between CTCs detected with OBP-401® and clinicopathologic features.Conclusion: OBP-401® showed no false positive in healthy controls, and a high sensitivity for CTCs detection, particular in metastatic breast cancer patients with normal 15-3 levels and early breast cancer patients, compared with CellSearch®. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3012.