1. 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

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

3. 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)

4. 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: , 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 (Oncolys BioPharma)

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

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: , 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

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 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)

11. 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.

12. Patients: 22 Cases Bone 6 Osteosarcoma 6 Soft Tissue 16
Myxofibrosarcoma 4
DFSP
Synovial sarcoma 2
Well differentiated liposarcoma 2
Myxoid liposarcoma 1
Mixed liposarcoma 1
Leiomyosarcoma 1
MPNST 1
Extraskeletal osteosarcoma 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 MFS MFS OS OS OS LMS LMS MFS
(cells)
(cells)
MFS
MFS OS OS OS
LMS
LMS
MFS

16. 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.

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.