Real-time near infrared fluorescence (NIRF) intra-operative imaging in ovarian cancer using an αvβ3-integrin targeted agent

doi:10.1016/j.ygyno.2012.12.011

Gynecologic Oncology

Volume 128, Issue 3, March 2013, Pages 590-595

https://doi.org/10.1016/j.ygyno.2012.12.011 Get rights and content

Abstract

Background

In ovarian cancer, optimal cytoreductive surgery is of the utmost importance for long-term survival. The ability to visualize minuscule tumor deposits is important to ensure complete resection of the tumor. The purpose of our study was to estimate the in vivo sensitivity, specificity and diagnostic accuracy of an intra-operative fluorescence imaging system combined with an α_vβ₃-integrin targeted near-infrared fluorescent probe.

Method

Tumor bearing mice were injected intravenously with a fluorescent probe targeting α_vβ₃ integrins. Fluorescent spots and non-fluorescent tissue were identified and resected. Standard histopathology and fluorescence microscopy were used as gold-standard for tumor detection.

Results

Fifty-eight samples excised with support of intra-operative image-guided surgery were analyzed. The mean target to background ratio was 2.2 (SD 0.5). The calculated sensitivity of the imaging system was 95%, and the specificity was 88% with a diagnostic accuracy of 96.5%.

Conclusion

Near-infrared image-guided surgery in this model has a high diagnostic accuracy and a fair target to background ratio that supports the development towards clinical translation of α_vβ₃-integrin targeted imaging.

Highlights

► Near infrared fluorescence imaging with targeted probes enables tumor visualization. ► By using an avb3 integrin targeted fluorescent probe, tumor tissue can be detected with high accuracy. ► Near infrared fluorescence imaging combined with targeted probes can in may shift the paradigm of surgical oncology.

Introduction

The assessment of tumor invasion in surrounding tissues, the presence of metastases and lymph node involvement are major factors in the process of therapeutic decision making. While current established radiological and nuclear imaging modalities like CT, MRI, and PET are important in pre-operative staging, they are less suitable for real-time intra-operative assessment of tumor margins and metastases for practical reasons.

Ovarian cancer is the sixth most common cancer and the seventh cause of death from cancer in women worldwide [1]. The aim in the treatment of ovarian cancer is a radical tumor resection leaving no macroscopic residual tumor. Ovarian cancer patients without or with little macroscopic residual tumor have a better survival compared to patients with residual tumor [2]. Consequently, the current surgical goal for cytoreductive surgery is to achieve a complete resection of macroscopic tumor. Based on current detection and staging methods there is no evidence available that after technically well-performed cytoreduction followed by adjuvant chemotherapy, residual microscopic tumor deposits negatively affect survival. Nevertheless, one could easily imagine that microscopic tumor spots are left behind unnoticed and are indeed able to cause recurrent cancer.

It may be hypothesized that a substantial number of patients in whom microscopic tumor deposits are unnoticed (R1 resection), may benefit from an improvement in cytoreductive surgery to a microscopic (R0) resection.

Visual and tactile information is considered by many surgeons as an important feature in staging. However, this method is not ideal for detecting small volumes of disease and clusters of few cells; the human eye cannot visualize beyond the tissue surface and moreover it lacks sensitivity to molecular based features. There is no evidence in literature that does support the sensitivity, specificity and diagnostic accuracy of palpation in cancer surgery.

An intraoperative device able to detect tumor tissue with high specificity and sensitivity could be of great help to assist the surgeon in finding additional small tumor spots. These additional findings may lead to improved staging, facilitating a more patient-tailored treatment, in which over- but also under-treatment is prevented.

A promising new imaging technique is near infrared fluorescence imaging (NIRF). Variants of NIRF are already applied in the operating room using single fluorochrome for contrast enhancement in visualizing anatomic structures like lymph nodes, blood vessels, ureters and bile ducts [3], [4], [5]. Until recently no agents were available for tumor delineation. In 2011 our group published the first in human result of in vivo tumor specific imaging in ovarian cancer using a fluorescein labeled folate ligand targeting the folate receptor-α [6].

However, since the folate receptor-α is overexpressed by ovarian cancer but not uniformly in many other cancers there is a need for better more general applicable markers.

A promising tumor-specific target is the α_vβ₃ integrin. Integrins are expressed on the surface of several cancer cells and play an important role in cell adhesion, migration and events that regulate cell cycle progression and apoptosis [7], [8]. Moreover, the α_vβ₃ integrin protein has been associated with tumor progression and dissemination [9], [10], [11]. In a previous study it was possible to detect tumor positive resection margins and macroscopically invisible tumor spots, using a fluorescence epi-illumination imaging system in combination with a fluorescent α_vβ₃ integrin protein in a human breast cancer xenotransplant mouse model [12]. The primary aim of the study is to investigate the accuracy using a real time intraoperative NIRF technique in combination with a targeted α_vβ₃ integrin NIRF probe for real-time, intra-operative tumor detection in a human ovarian cancer xenotransplant mouse model. Additionally we present real-time fluorescent guided debulking surgery of peritoneal carcinomatosis in the same model.

Section snippets

Targeted fluorescence imaging agent

IntegriSense 680 (Perkin & Elmer Medical, Woburn, MA, USA) is a commercially developed fluorescent probe aimed at the α_vβ₃ integrin protein.

Briefly, it is a targeted fluorescence agent comprising a potent, selective non-peptide small molecule integrin α_vβ₃ antagonist and an NIR fluorochrome with an excitation wavelength of 675 ± 5 nm and an emission wavelength of 693 nm.

Cell line

The luciferase transfected ovarian cancer cell line A2780.luc was used (kindly provided by Dr. S. de Jong, Medical Oncology,

Tumor growth

After 3–4 weeks the control mice with the subcutaneous tumor showed tumor growth (0.5 cm), based on previous experiments this was also the indication for the mice with i.p. inoculation to have significant intraabdominal tumor growth.

Imaging

In all mice (n = 6) suspected tumor tissue was detected by fluorescence imaging after laparotomy. All the suspicious fluorescent spots were removed by imaged-guided surgery using fluorescence epi-illumination imaging (FEI). If there were suspicious spots by visual

Discussion

The aim of this study was to estimate the diagnostic accuracy of a clinical multispectral intra-operative fluorescence camera system by using an α_vβ₃ targeted near-infrared fluorescent probe in a human ovarian cancer xenotransplant mouse model. The ultimate goal of the technique was to optimize surgical cytoreduction in this model as a step-up approach towards clinical development of the targeted probe.

The sensitivity and specificity of the system were evaluated by using histology as the gold

Conclusion

The high sensitivity, specificity and diagnostic accuracy of the described intra-operative optical imaging system in combination with a tumor-specific agent targeting α_vβ₃-integrin confirms the potential opportunities for the detection of residual tumor spots, loco-regional metastases and possible tumor margins in humans with disseminated disease in the abdominal cavity.

This approach could offer a practical feature for open and endoscopic imaging systems. When applied in a clinical setting,

Conflict of interest statement

None declared.

Acknowledgment

The authors would like to thank Prof. Dr. S. de Jong from the University Medical Center Groningen for providing the A2780.luc cell line. Claudia Mayerhofer is acknowledged for the cell culture.

References (23)

D.S. Chi et al.
What is the optimal goal of primary cytoreductive surgery for bulky stage iiic epithelial ovarian carcinoma (eoc)?
Gynecol Oncol
(2006)
A. Matsui et al.
Real-time, near-infrared, fluorescence-guided identification of the ureters using methylene blue
Surgery
(2010 Jul)
A. Matsui et al.
Real-time intra-operative near-infrared fluorescence identification of the extrahepatic bile ducts using clinically available contrast agents
Surgery
(2010 Jul)
R.O. Hynes
Integrins: a family of cell surface receptors
Cell
(1987)
M. Pignatelli et al.
Integrins and their accessory adhesion molecules in mammary carcinomas: loss of polarization in poorly differentiated tumors
Hum Pathol
(1992)
R.A. Sheth et al.
Improved detection of ovarian cancer metastases by intraoperative quantitative fluorescence protease imaging in a pre-clinical model
Gynecol Oncol
(2009)
E. Mery et al.
Intraoperative fluorescence imaging of peritoneal dissemination of ovarian carcinomas. A preclinical study
Gynecol Oncol
(2011 Jul)
D.M. Parkin et al.
Global cancer statistics, 2002
CA Cancer J Clin
(2005)
S.L. Troyan et al.
The flare() intraoperative near-infrared fluorescence imaging system: A first-in-human clinical trial in breast cancer sentinel lymph node mapping
Ann Surg Oncol
(2009 Oct)
G.M. van Dam et al.
Intraoperative tumor-specific fluorescence imaging in ovarian cancer by folate receptor-alpha targeting: First in-human results
Nat Med
(2011 Sep 18)

I. Pecheur et al.

Integrin alpha(v)beta3 expression confers on tumor cells a greater propensity to metastasize to bone

FASEB J

(2002)

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IntegriSense680 was applied for intraoperative NIRF imaging in a syngeneic rat model of colorectal metastases [54] (Table 6). IntegriSense680 was also applied as intraoperative imaging in the FGS of ovarian cancer with a diagnostic accuracy of 96.5% [55] (Table 6). Furthermore, IntegriSense750, which has a shorter half-life period in tissues than IntegriSense680, was invented to precisely identify in situ tumor nodes during FGS [56].
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Real-time near infrared fluorescence (NIRF) intra-operative imaging in ovarian cancer using an αvβ3-integrin targeted agent

Abstract

Background

Method

Results

Conclusion

Highlights

Introduction

Section snippets

Targeted fluorescence imaging agent

Cell line

Tumor growth

Imaging

Discussion

Conclusion

Conflict of interest statement

Acknowledgment

Gynecol Oncol

Surgery

Surgery

Cell

Hum Pathol

Gynecol Oncol

Gynecol Oncol

Global cancer statistics, 2002

CA Cancer J Clin

The flare() intraoperative near-infrared fluorescence imaging system: A first-in-human clinical trial in breast cancer sentinel lymph node mapping

Ann Surg Oncol

Intraoperative tumor-specific fluorescence imaging in ovarian cancer by folate receptor-alpha targeting: First in-human results

Nat Med

Integrin alpha(v)beta3 expression confers on tumor cells a greater propensity to metastasize to bone

FASEB J

Real-time near infrared fluorescence (NIRF) intra-operative imaging in ovarian cancer using an α_vβ_3-integrin targeted agent