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.