t. kunisada 1 j. hasei 1 k. takeda 1 y. urata 3 t. fujiwara 2 t. ozaki 1
DESCRIPTION
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 - PowerPoint PPT PresentationTRANSCRIPT
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 Surgery2 Dept. of Gastroenterological SurgeryOkayama University, Okayama, Japan
3 Oncolys BioPharma, Inc., Tokyo, Japan
COI Disclosure Information
Toshiyuki Kunisada
I have no financial relationships to disclose.
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
Virus-based Capture System Detection of CTCs by Adenovirus-based Approach - TelomeScan System (Oncolys BioPharma)
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)
Telomerase Activity in Cancers
GGGTTAGGG CCCAATCCC 5’
TTAGGGTTA 3’
Telomere
chromosome
hTERT
AAUCCCAAUChTERC
hTEP1
ACA
TMG
3’
5’
Template
telomerase
OBP-401 (Telomescan®):GFP-expressing attenuated adenovirus vector
Adenovirus type 5
hTERT-promoter E1A IRES E1B
△E1▲
ITR ITR
(Kishimoto H et al. Nat Med, 12:1213-1219, 2006)△E3▲
GFP pACMV-
promoter
Telomerase activities in cancers
GGGTTAGGG CCCAATCCC 5’
TTAGGGTTA 3’
テロメア
chromosome
hTERT
AAUCCCAAUChTERC
hTEP1
ACA
TMG
3’
5’
Template
telomerase
Telomescan
Normal cellsTelomerase (-)
Tumor cellsTelomerase (+)
Infection
Telomescan
Infection Replication +GFP +
Replication -
GFP positivetumor cell
GFP negative
Tumor specific imaging system with Telomescan
GFP
regulates viral replication
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.
(Sasaki T et al. Gene Ther, 2013)
Telomescan could visualize bone and soft tissue sarcoma cell lines
Basic Study: Telomescan in Osteosarcoma Cells
Detection System of CTC Using Telomescan
Phase contrast
GFP
Step 1:Hemolytic reagent
Step 2:Telomescan
Step 3:Glass slide
Step 4:Fluorescence microscope
GFP-Positive CTCs !!
(7.5 mL)
Visualizing CTCs
(Kojima T et al. J Clin Invest, 2009)
The captured segmented tile images were joined together by an image acquisition and analysis software.
Scale bar :30 μm
We defined GFP-positive, CD45-negative, vimentin-positive and DAPI-positive cells as CTCs.
Methods: Staining for CTCs
Telomescan Hematopoietic marker
Mesenchymalmarker
Patients: 22 Cases
• Bone 6 Osteosarcoma 6
• Soft Tissue 16Myxofibrosarcoma 4DFSP 3Synovial sarcoma 2Well differentiated liposarcoma 2Myxoid liposarcoma 1Mixed liposarcoma 1Leiomyosarcoma 1MPNST 1 Extraskeletal osteosarcoma 1
Osteosarcoma
Extraskeletal OS
MyxofibrosarcomaDFSP
Synovial sarcoma
WD liposarcoma
Myxoid liposarcoma
Mixed liposarcoma
Leiomyosarcoma MPNST
0
1
2
3
4
5
6
7
CTC-NegativeCTC-Positive
Num
ber o
f pati
ent
Result: Positivity of CTCs
: 11 Pts: 11 Pts
0
1
2
3
4
5
6
16
17
Osteosarcoma DFSP MyxofibrosarcomaLeiomyosarcoma
MPNST
(cells)
Number of CTCs in CTC positive patients
11 Pts with CTC positive
Before After0
1
2
3
4
OsteosarcomaMyxofibrosarcomaMyxofibrosarcoma Myxofibrosarcoma Leiomyosarcoma
Before After0
1
2
3
4
Osteosarcoma
Osteosarcoma
Myxofibrosarcoma
Leiomyosarcoma
(cells) (cells)Surgery Chemotherapy
Change of CTC Number
MFS
MFS
OS
LMS
MFS
OS
OS
LMS
Pt who developed mets0
1
2
3
4
5
Num
ber o
f Pre
op. C
TCs
CTC at diagnosis and Metastasis
OS
OS
ES OS
LMS
MPNST5 patients developed lung metastasis.
4 of 11 patients with positive CTC developed lung metastases.
1 of 11 patients with negative CTC developed lung metastases.
0
1
2
3
4
5
6
Num
ber o
f CTC
s55 F Osteosarcoma of Pelvis
At diagnosis After surgery
MRI T2
Wide Resection
0
1
2
3
4
5
6
T1 enhance
Num
ber o
f CTC
s55 F Osteosarcoma of Pelvis
At diagnosis After surgery #1 Postop. Cx #3 Postop. Cx
Recurrence
CTC can be a biomarker for relapse after treatment.
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.