Name: tissue-simulating phantoms for assessing potential near-infrared fluorescence imaging applications in breast cancer surgery
Uploaded: 2014-09-19T13:00:00
Description: Watch this Scientific Journal Video about Tissue-simulating Phantoms for Assessing Potential Near-infrared Fluorescence Imaging Applications in Breast Cancer Surgery at JoVE.com

This work was supported by a grant from the Jan Kornelis de Cock foundation.

1. Create Silicone Molds for Tumor-simulating Inclusions
<ol>
	<li>Collect solid items of the desired shape and size that can serve as models for tumor-simulating inclusions, e.g., beads or marbles.</li>
	<li>Thoroughly clean the tumor models. To ensure an easy removal from the silicone mold, the tumor models can be sprayed with anti-stick spray or covered with a thin layer of petroleum jelly or beeswax.</li>
	<li>Place each model in a separate thin walled square (plastic) box with a smooth surface. If necessa...

<ol>
	<li>Bellon, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ACR+Appropriateness+Criteria&#174;+Conservative+Surgery+and+Radiation+-+Stage+I+and+II+Breast+Carcinoma.">ACR Appropriateness Criteria&#174; Conservative Surgery and Radiation - Stage I and II Breast Carcinoma.</a> The Breast Journal. 17 (5), 448-455 (2011).</li><li>Kaufmann, M., Morrow, M., Von Minckwitz, G., Harris, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Biedenkopf+Expert+Panel+Members.+Locoregional+treatment+of+primary+breast+cancer.">The Biedenkopf Expert Panel Members. Locoregional treatment of primary breast cancer.</a> Cancer. 116, 1184-1191 (2010).</li><li>Pleijhuis, R. 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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+fluorescence+(NIRF)+imaging+in+breast-conserving+surgery:+assessing+intraoperative+techniques+in+tissue-simulating+breast+phantoms.">Near-infrared fluorescence (NIRF) imaging in breast-conserving surgery: assessing intraoperative techniques in tissue-simulating breast phantoms.</a> European Journal of Surgical Oncology. 37 (1), 32-39 (2011).</li><li>Baeten, J., Niedre, M., Dunham, J., Ntziachristos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+fluorescent+materials+for+Diffuse+Fluorescence+Tomography+standards+and+phantoms.">Development of fluorescent materials for Diffuse Fluorescence Tomography standards and phantoms.</a> Optics Express. 15 (14), 8681-8694 (2007).</li><li>Luker, G. D., Luker, K. 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We simulated potential clinical applications of NIRF-guided BCS through the use of breast-shaped phantoms with integrated tumor-simulating inclusions. Intraoperative tumor localization, NIRF-guided tumor resection, evaluation on the extent of surgery, and postoperative assessment of surgical margins were all found feasible using a custom-build NIRF camera system. Noninvasive detection of fluorescent tumor-simulating inclusions was only feasible for inclusions positioned in the phantom tissue at a depth of 2 cm or less. I...

Breast-conserving surgery (BCS) followed by radiotherapy is the standard treatment for breast cancer patients with T1-T2 breast carcinoma1,2. Inaccuracies in intraoperative assessment of the extent of surgery result in positive surgical margins in 20 to 40% of the patients who underwent BCS, necessitating additional surgical intervention or radiotherapy3,4,5. Although extensive resection of adjacent healthy breast tissue might reduce the frequency of positive surgical margins, this will also hamper cosmetic outcome and increase comorbidity6,7. Novel techniques are therefore needed that provide intraoperative feedback on the location of the primary tumor and the extent of surgery. Optical imaging, in particular near-infrared fluorescence (NIRF) imaging, might reduce the frequency of positive surgical margins following BCS by providing the surgeon with a tool for pre- and intraoperative tumor localization in real-time. Recently, our group reported on the first in-human trial of tumor-targeted fluorescence imaging in ovarian cancer patients, showing the feasibility of this technique to detect primary tumors and intraperitoneal metastases with high sensitivity8. Before proceeding to clinical studies in breast cancer patients, however, the feasibility of various tumor-targeted NIRF imaging applications in BCS can already be evaluated preclinically using phantoms.
The following research protocol describes the use of NIRF imaging in tissue-simulating breast phantoms containing fluorescent tumor-simulating inclusions9. The phantoms provide an inexpensive and versatile tool to simulate pre- and intraoperative tumor localization, real-time NIRF-guided tumor resection, assessment of the surgical margin status, and detection of residual disease. The gelatinous phantoms have elastic properties similar to human tissue and can be cut using conventional surgical instruments. During the simulated surgical procedure, the surgeon is guided by tactile information (in the case of palpable inclusions) and visual inspection of the operative field. In addition, NIRF imaging is applied to provide the surgeon with real-time intraoperative feedback on the extent of surgery.
It should be emphasized that NIRF imaging requires the use of fluorescent dyes. Ideally, fluorescent dyes should be used that emit photons in the near-infrared spectral range (650 - 900 nm) to minimize absorption and scattering of photons by molecules physiologically abundant in tissue (e.g., hemoglobin, lipids, elastin, collagen, and water)10,11. Moreover, autofluorescence (i.e., the intrinsic fluorescence activity in tissues due to biochemical reactions in living cells) is minimized in the near-infrared spectral range, resulting in optimal tumor-to-background ratios11. By conjugating NIRF dyes to tumor-targeted moieties (e.g., monoclonal antibodies), targeted delivery of fluorescent dyes can be obtained for intraoperative imaging applications.
As the human eye is insensitive to light in the near-infrared spectral range, a highly sensitive camera device is required for NIRF imaging. Several NIRF imaging systems for intraoperative use have been developed so far12. In the current study, we used a custom build NIRF imaging system that was developed for intraoperative application in collaboration with the Technical University of Munich. The system allows for simultaneous acquisition of color images and fluorescence images. To improve the accuracy of the fluorescence images, a correction scheme is implemented for variations in light intensity in tissue. A detailed description is provided by Themelis et al.13

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Bovine hemoglobin</td>
			<td>Sigma-Aldrich, Zwijndrecht, The Netherlands</td>
			<td>H2500</td>
			<td>Simulates absorption of photons in tissue&#160;</td>
		</tr>
		<tr>
			<td>Intralipid 20%</td>
			<td>Sigma-Aldrich, Zwijndrecht, The Netherlands</td>
			<td>I141</td>
			<td>Simulates scattering of photons in tissue</td>
		</tr>
		<tr>
			<td>Silicone A translucent 40 (2-components poly-addition silicone)</td>
			<td>NedForm, Geleen, The Netherlands</td>
			<td>N/A</td>
			<td>Package consists of components A and B, that should be mixed one on one (A:B=10:1).&#160; Link to manufacturers page: http://tinyurl.com/ncjq7jx</td>
		</tr>
		<tr>
			<td>Gelatine 250 Bloom</td>
			<td>Sigma-Aldrich, Zwijndrecht, The Netherlands</td>
			<td>48724</td>
			<td>Construction of breast-shaped phantoms</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Hispanagar, Burgos, Spain</td>
			<td>N/A</td>
			<td>Construction of tumor-simulating inclusions</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma-Aldrich, Zwijndrecht, The Netherlands</td>
			<td>T1503&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Hcl</td>
			<td>Sigma-Aldrich, Zwijndrecht, The Netherlands</td>
			<td>258148</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich, Zwijndrecht, The Netherlands</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH3</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>822335</td>
			<td>CAUTION: severe poison. The toxicity of this compound is comparable to that of soluble alkali cyanides and the lethal dose for an adult human is about 0.7 grams.</td>
		</tr>
		<tr>
			<td colspan="2">Examples of NIRF imaging devices for intraoperative application:</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T2 NIRF imaging platform&#160;</td>
			<td>SurgVision BV, Heerenveen, The Netherlands</td>
			<td>N/A</td>
			<td>Customized NIRF imaging system used in the current study. More details available at www.surgvision.com</td>
		</tr>
		<tr>
			<td>Photodynamic Eye</td>
			<td>Hamamatsu Photonics Deutschland GmbH, Herrsching am Ammersee, Germany</td>
			<td>PC6100</td>
			<td>www.iht-ltd.com</td>
		</tr>
		<tr>
			<td>FLARE imaging system kit</td>
			<td>The FLARE Foundation Inc, Wayland, MA, USA</td>
			<td>N/A</td>
			<td>www.theflarefoundation.org</td>
		</tr>
		<tr>
			<td>Fluobeam</td>
			<td>Fluoptics, Grenoble, France</td>
			<td>N/A</td>
			<td>www.fluoptics.com</td>
		</tr>
		<tr>
			<td>Artemis handheld camera</td>
			<td>Quest Medical Imaging BV, Middenmeer, the Netherlands</td>
			<td>N/A</td>
			<td>www.quest-mi.com</td>
		</tr>
		<tr>
			<td colspan="2">Examples of NIRF fluorescent dyes for intraoperative application:</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Indocyanine green</td>
			<td>ICG-PULSION,&#160; Feldkirchen, Germany</td>
			<td>PICG0025DE&#160;&#160;</td>
			<td>Clinical grade fluorescent dye for NIRF imaging used in the current study. More details available at www.pulsion.com</td>
		</tr>
		<tr>
			<td>IRDye 800CW NHS Ester</td>
			<td>LI-COR Biosciences, Lincoln, NE, USA</td>
			<td>929-70021</td>
			<td>www.licor.com</td>
		</tr>
	</tbody>
</table>

tissue-simulating phantoms for assessing potential near-infrared fluorescence imaging applications in breast cancer surgery

Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery (BCS). Optical imaging, in particular near-infrared fluorescence (NIRF) imaging, might reduce the frequency of positive surgical margins following BCS by providing the surgeon with a tool for pre- and intraoperative tumor localization in real-time. In the current study, the potential of NIRF-guided BCS is evaluated using tissue-simulating breast phantoms for reasons of standardization and training purposes.
Breast phantoms with optical characteristics comparable to those of normal breast tissue were used to simulate breast conserving surgery. Tumor-simulating inclusions containing the fluorescent dye indocyanine green (ICG) were incorporated in the phantoms at predefined locations and imaged for pre- and intraoperative tumor localization, real-time NIRF-guided tumor resection, NIRF-guided evaluation on the extent of surgery, and postoperative assessment of surgical margins. A customized NIRF camera was used as a clinical prototype for imaging purposes.
Breast phantoms containing tumor-simulating inclusions offer a simple, inexpensive, and versatile tool to simulate and evaluate intraoperative tumor imaging. The gelatinous phantoms have elastic properties similar to human tissue and can be cut using conventional surgical instruments. Moreover, the phantoms contain hemoglobin and intralipid for mimicking absorption and scattering of photons, respectively, creating uniform optical properties similar to human breast tissue. The main drawback of NIRF imaging is the limited penetration depth of photons when propagating through tissue, which hinders (noninvasive) imaging of deep-seated tumors with epi-illumination strategies.

Results from this study have been previously reported elsewhere9.
Our data show that NIRF imaging can be applied to detect fluorescent tumor-simulating inclusions in tissue-simulating breast phantoms, simulating NIRF-guided breast-conserving surgery in breast cancer patients. Using our phantom model, we found intraoperative tumor localization, NIRF-guided tumor resection, intraoperative assessment of surgical cavity margins, and detection of residual disease to be feasible (...

Watch this Scientific Journal Video about Tissue-simulating Phantoms for Assessing Potential Near-infrared Fluorescence Imaging Applications in Breast Cancer Surgery at JoVE.com

Tissue-simulating Phantoms for Assessing Potential Near-infrared Fluorescence Imaging Applications in Breast Cancer Surgery

Near-infrared fluorescence (NIRF) imaging may improve therapeutic outcome of breast cancer surgery by enabling intraoperative tumor localization and evaluation of surgical margin status. Using tissue-simulating breast phantoms containing fluorescent tumor-simulating inclusions, potential clinical applications of NIRF imaging in breast cancer patients can be assessed for standardization and training purposes.

Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery ...

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