A detection system for circulating tumor cells using GFP expressing telomerase-specific replication-competent adenovirus in bone and soft tissue sarcoma.

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Presentation transcript:

A detection system for circulating tumor cells using GFP expressing telomerase-specific replication-competent adenovirus in bone and soft tissue sarcoma T. Kunisada1　J. Hasei1　K. Takeda1 Y. Urata3　T. Fujiwara2 T. Ozaki1 1 Dept. of Orthopaedic Surgery 2 Dept. of Gastroenterological Surgery Okayama University, Okayama, Japan 3 Oncolys BioPharma, Inc., Tokyo, Japan

COI Disclosure Information Toshiyuki Kunisada I have no financial relationships to disclose.

Pennacchioli, Clin Exp Metastasis (2012) Biomarker For Sarcoma Genetic specific aberrations: SYT-SSX1 etc. Vascular spread biomarkers: D-dimer etc. Notch pathway signaling miRNAs Circulating tumor cells (CTC) Pennacchioli, Clin Exp Metastasis (2012)

CTCs: Circulating Tumor Cells Tumor cells released from tumors into blood stream. Have a key role in future metastasis. (Chaffer CL and Weinberg RA, Science, 331:1559-1564, 2011)

The Detection of CTC Antibody-based Capture System Isolation of CTCs by Anti-EpCAM Antibody-based Approaches - CellSearch System (Veridex) - CTC-chip System - FACS System Cell Size-based Capture System Isolation of CTCs by Filter-based Approaches - Filtration Device (ScreenCell) - Flexible Micro Spring Array (FMSA) Device Electrical Charge-based Capture System Isolation of CTCs by Array-based Approaches - DEPArray (silicon biosystems) - ApoStream Technology (ApoCell) Virus-based Capture System Detection of CTCs by Adenovirus-based Approach - TelomeScan System (Oncolys BioPharma)

Telomerase Activity in Cancers GGGTTAGGG CCCAATCCC 5’ TTAGGGTTA 3’ Telomere chromosome hTERT AAUCCCAAUC hTERC hTEP1 ACA TMG 3’ 5’ Template telomerase

OBP-401 (Telomescan®): GFP-expressing attenuated adenovirus vector regulates viral replication hTERT- promoter CMV- promoter E1A IRES E1B GFP pA ITR ITR Adenovirus type 5 ▲ ▲ △E1 △E3 Telomescan (Kishimoto H et al. Nat Med, 12:1213-1219, 2006) Tumor specific imaging system with Telomescan Telomerase activities in cancers GGGTTAGGG CCCAATCCC 5’ TTAGGGTTA 3’ テロメア chromosome Infection Replication - GFP negative hTERT AAUCCCAAUC hTERC hTEP1 ACA TMG 3’ 5’ Template telomerase Normal cells Telomerase (-) Telomescan Tumor cells Telomerase (+) GFP Infection Replication + GFP + GFP positive tumor cell

Purpose To detect CTCs in peripheral blood collected from patients with bone and soft tissue sarcoma using Telomescan. To analyze the significance of the number of CTCs.

Basic Study: Telomescan in Osteosarcoma Cells Telomescan could visualize bone and soft tissue sarcoma cell lines (Sasaki T et al. Gene Ther, 2013)

Detection System of CTC Using Telomescan Step 1: Hemolytic reagent Step 2: Telomescan Visualizing CTCs Phase contrast (7.5 mL) Step 3: Glass slide Step 4: Fluorescence microscope The captured segmented tile images were joined together by an image acquisition and analysis software. GFP GFP-Positive CTCs !! (Kojima T et al. J Clin Invest, 2009)

Methods: Staining for CTCs Telomescan Hematopoietic marker Mesenchymal marker Scale bar :30 μm We defined GFP-positive, CD45-negative, vimentin-positive and DAPI-positive cells as CTCs.

Patients: 22 Cases Bone 6 Osteosarcoma 6 Soft Tissue 16 Myxofibrosarcoma 4 DFSP 3 Synovial sarcoma 2 Well differentiated liposarcoma 2 Myxoid liposarcoma 1 Mixed liposarcoma 1 Leiomyosarcoma 1 MPNST 1 Extraskeletal osteosarcoma 1

Result: Positivity of CTCs : 11 Pts : 11 Pts Number of patient

Number of CTCs in CTC positive patients (cells) 11 Pts with CTC positive Osteosarcoma DFSP Myxofibrosarcoma Leiomyosarcoma MPNST

Change of CTC Number Surgery Chemotherapy MFS MFS OS OS OS LMS LMS MFS (cells) (cells) MFS MFS OS OS OS LMS LMS MFS

CTC at diagnosis and Metastasis 5 patients developed lung metastasis. MPNST Number of Preop. CTCs OS OS LMS ES OS 4 of 11 patients with positive CTC developed lung metastases. 1 of 11 patients with negative CTC developed lung metastases.

55 F Osteosarcoma of Pelvis Wide Resection Number of CTCs MRI T2 At diagnosis After surgery

55 F Osteosarcoma of Pelvis T1 enhance Number of CTCs Recurrence CTC can be a biomarker for relapse after treatment. At diagnosis After surgery #1 Postop. Cx #3 Postop. Cx

Summary CTCs were detected in 11 of 22 patients with bone and soft tissue sarcoma using GFP-expressing Telomescan. 4 of 5 patients who developed lung metastases showed CTC-positive at diagnosis. Telomescan can be useful system to detect CTCs as a biomarker of bone and soft tissue sarcoma.