Akinori Masago

Circulating tumor cells as a prognostic factor in patients with small cell lung cancer

The detection of circulating tumor cells (CTCs) in peripheral blood is currently an important field of study. Detection of CTCs by the OBP-401 assay (TelomeScan®) has previously been reported to be useful in the diagnosis, prognosis and evaluation of therapeutic efficacy in breast and gastric cancer. The aim of the present study was to evaluate the OBP-401 assay as a novel method of detecting CTCs of small cell lung cancer (SCLC) patients and to evaluate whether CTC count is associated with prognosis. Prospectively, 30 consecutively diagnosed SCLC patients who had commenced chemotherapy or chemoradiotherapy were enrolled as subjects of the current study. Peripheral blood specimens were collected from the SCLC patients prior to and following the initiation of treatment and the viable CTCs were detected in the specimens following incubation with a telomerase-specific, replication-selective, oncolytic adenoviral agent, which was carrying the green fluorescent protein gene. CTCs were detected in 29 patients (96%). The group of 21 patients with a CTC count of <2 cells/7.5 ml prior to treatment (baseline) had a significantly longer median survival time than the group of eight patients with a CTC count of ≥2 cells/7.5 ml prior to treatment (14.8 and 3.9 months, respectively; P=0.007). The results of a multivariate analysis showed that the baseline CTC count was an independent prognostic factor for survival time (hazard ratio, 3.91; P=0.026). Among the patients that achieved a partial response to treatment, patients who had a CTC count of <2 cells/7.5 ml following two cycles of chemotherapy tended to have a longer median progression-free survival compared with patients who had a CTC count of ≥2 cell/7.5 ml (8.3 and 3.8 months, respectively; P=0.07). Therefore, CTCs may be detected via OBP-401 assay in SCLC patients and the CTC count prior to treatment appears to be a strong prognostic factor.

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.