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T. Kunisada 1 J. Hasei 1 K. Takeda 1 Y. Urata 3 T. Fujiwara 2 T. Ozaki 1

A detection system for circulating tumor cells using GFP expressing telomerase-specific replication-competent adenovirus in bone and soft tissue sarcoma. T. Kunisada 1 J. Hasei 1 K. Takeda 1 Y. Urata 3 T. Fujiwara 2 T. Ozaki 1 1 Dept. of Orthopaedic Surgery

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T. Kunisada 1 J. Hasei 1 K. Takeda 1 Y. Urata 3 T. Fujiwara 2 T. Ozaki 1

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  1. A detection system for circulating tumor cells using GFP expressing telomerase-specific replication-competent adenovirus in bone and soft tissue sarcoma T. Kunisada1J. Hasei1K. Takeda1 Y. Urata3T. Fujiwara2 T. Ozaki1 1 Dept. of Orthopaedic Surgery 2 Dept. of Gastroenterological Surgery Okayama University, Okayama, Japan 3OncolysBioPharma, Inc., Tokyo, Japan

  2. COI Disclosure InformationToshiyuki Kunisada I have no financial relationships to disclose.

  3. 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)

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

  5. 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 (OncolysBioPharma)

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

  7. 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) Telomerase activities in cancers Tumor specific imaging system with Telomescan chromosome Replication - GFPnegative Infection telomerase hTEP1 3’ Normal cells Telomerase (-) ACA 5’ TMG テロメア hTERT Telomescan Template AAUCCCAAUC hTERC Tumor cells Telomerase (+) GFP GGGTTAGGG CCCAATCCC 5’ TTAGGGTTA 3’ Infection Replication + GFP + GFP positive tumor cell

  8. 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.

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

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

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

  12. Patients: 22 Cases • Bone 6 Osteosarcoma 6 • Soft Tissue 16 Myxofibrosarcoma 4 DFSP 3 Synovial sarcoma 2 Well differentiated liposarcoma 2 Myxoidliposarcoma 1 Mixed liposarcoma 1 Leiomyosarcoma 1 MPNST 1 Extraskeletalosteosarcoma 1

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

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

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

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

  17. 55 F Osteosarcoma of Pelvis Wide Resection Number of CTCs MRI T2 At diagnosis After surgery

  18. 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

  19. 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.

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