Oncologic surgery can greatly benefit from imaging techniques for the accurate identification of tumor-positive margins both intraoperatively and in resection specimens immediately following surgery. We have demonstrated clinically that fluorescence lifetime can significantly improve the accuracy for tumor vs. normal classification compared to fluorescence intensity in multiple cancer types using tumor targeted agents. Ongoing efforts by our group towards the translation of fluorescence lifetime imaging for intraoperative image guidance using exogenous agents will also be discussed.
SignificanceNear-infrared fluorescence imaging still lacks a standardized, objective method to evaluate fluorescent dye efficacy in oncological surgical applications. This results in difficulties in translation between preclinical to clinical studies with fluorescent dyes and in the reproduction of results between studies, which in turn hampers further clinical translation of novel fluorescent dyes.AimOur aim is to develop and evaluate a semi-automatic standardized method to objectively assess fluorescent signals in resected tissue.ApproachA standardized imaging procedure was designed and quantitative analysis methods were developed to evaluate non-targeted and tumor-targeted fluorescent dyes. The developed analysis methods included manual selection of region of interest (ROI) on white light images, automated fluorescence signal ROI selection, and automatic quantitative image analysis. The proposed analysis method was then compared with a conventional analysis method, where fluorescence signal ROIs were manually selected on fluorescence images. Dice similarity coefficients and intraclass correlation coefficients were calculated to determine the inter- and intraobserver variabilities of the ROI selections and the determined signal- and tumor-to-background ratios.ResultsThe proposed non-targeted fluorescent dyes analysis method showed statistically significantly improved variabilities after application on indocyanine green specimens. For specimens with the targeted dye SGM-101, the variability of the background ROI selection was statistically significantly improved by implementing the proposed method.ConclusionSemi-automatic methods for standardized quantitative analysis of fluorescence images were successfully developed and showed promising results to further improve the reproducibility and standardization of clinical studies evaluating fluorescent dyes.
A clinically-compatible imaging platform capable of performing widefield quantitative oxygenation and fluorescence imaging is presented with its potential for tissue status assessment in particular for blood perfusion and tumor margin assessment
Significance: Near-infrared (NIR) fluorescence imaging using indocyanine green (ICG) has proven to be a feasible application for real-time intraoperative assessment of tissue perfusion, although quantification of NIR fluorescence signals is pivotal for standardized assessment of tissue perfusion.
Aim: Four patients are described with possible compromised bowel perfusion after mesenteric resection. Based on these patients we want to emphasize the difficulties in the quantification of NIR fluorescence imaging for perfusion analysis.
Approach: During image-guided fluorescence assessment, 5 mg of ICG (2.5 mg / ml) was intravenously administered by the anesthesiologist. NIR fluorescence imaging was done with the open camera system of Quest Medical Imaging. Fluorescence data taken from the regions of interest (bowel at risk, transition zone of bowel at risk and adjacent normally perfused bowel, and normally perfused reference bowel) were quantitatively analyzed after surgery for fluorescence intensity-and perfusion time-related parameters.
Results: Bowel perfusion, as assessed clinically by independent surgeons based on NIR fluorescence imaging, resulted in different treatment strategies, three with excellent clinical outcome, but one with a perfusion related complication. Post-surgery quantitative analysis of fluorescence dynamics showed different patterns in the affected bowel segment compared to the unaffected reference segments for the four patients.
Conclusions: Similar intraoperative fluorescence results could lead to different surgical treatment strategies, which demonstrated the difficulties in interpretation of uncorrected fluorescence signals. Real-time quantification and standardization of NIR fluorescence perfusion imaging could probably aid surgeons in the nearby future.
Fluorescence-guided surgery give the surgeons extra input to improve the outcome of tumor resection procedures. However, the analysis of fluorescence images is qualitative and subjective inputs such as the surgeon’s perception and experience are considered when assessing the tumor margins. Objective indicators are needed to assess accurately the amount of fluorophore within the tissues. We developed a multimodal imaging platform capable of widefield quantitative fluorescence imaging for the use in a clinical environment. By mapping the fluorophore concentration, we offer an objective input for distinguishing healthy from diseased tissue and determining the resections margins in the optimal way: removing cancerous tissue while preserving healthy tissue and vital structures.
Restoring normal functioning and tissue healing after surgical intervention is, among others, critically dependent on tissue oxygenation and perfusion. Tissue necrosis, caused by inadequate tissue perfusion and/or oxygenation, is a common complication after surgical reconstruction of e.g. bowel anastomoses or skin defects. Currently, several optical techniques, which do not require administration of contrast agents, have been used to evaluate tissue perfusion and oxygenation. An emerging technique is hyperspectral imaging, which is capable to detect the scattering and absorption of light delivered to the tissue, caused by inhomogeneity of biological structures, such as haemoglobin, fat or water. Recently, a snapshot hyperspectral camera was developed to measure tissue oxygenation non-invasively using relevant wavelengths in the VIS-NIR region (450-950 nm). In this study, the effect of occlusion-reperfusion of the brachial artery on cutaneous blood oxygenation is explored in three human volunteers as assessed by a snapshot hyperspectral camera system. Furthermore, measurements of local changes in skin oxygenation and blood flow after applying a local vasodilator (capsaicin-based cream) and a local vasoconstrictor (brimonidine gel) are compared to measurements of an untreated area of the skin. Hyperspectral results are correlated to the haemoglobin oxygen saturation measured by an oximeter. Simultaneously with the hyperspectral measurements, real-time blood perfusion mapping is performed using Laser Speckle Contrast Imaging, which is able to measure cutaneous skin blood flow through analysis of the speckle pattern.
For solid tumors, such as breast cancer, surgery is usually the treatment of choice. For these operations, it is of utmost importance to remove the whole tumor, since tumor-positive resection margins may result in recurrent disease and impaired overall survival. Current optical imaging techniques using endogenous contrast - e.g. diffuse reflectance spectroscopy or optical coherence tomography – for detection of breast cancers are limited to point measurements or long acquisition times. Broadband hyperspectral cameras, which provide a complete spectral fingerprint of the object at pixel level, are needed for two-dimensional imaging of the operative region. An ex vivo study was conducted to evaluate the feasibility of hyperspectral imaging for breast cancer detection. Fresh (<3 hours after surgery) resected breast cancer slices were imaged with a snapshot hyperspectral camera, which has 41 spectral bands, equally distributed in the visible and near-infrared (VIS-NIR) range (450 – 950 nm). Supervised analysis was performed by using the pathology annotations and unsupervised analysis was performed by using the hierarchical stochastic neighbour embedding (h-SNE) algorithm. So far, nine resected specimens, of which six invasive carcinomas and two (partially) mucinous carcinomas, were imaged. Spectral differences were found between the non-tumor, malign and benign regions on the resected specimens. Furthermore, automatic feature classification using h-SNE was possible in selected cases. Hyperspectral imaging showed great potential for discrimination of benign and malign breast tissue by using specific wavelengths bands in an ex vivo setting. Further analysis will be performed to determine whether it is possible to select tumor-specific wavelengths.
The introduction of optical imaging by using near-infrared (NIR) light shines new light in the field of (oncologic) surgery. The use of non-specific fluorophores, such as Indocyanine Green (ICG) and Methylene Blue (MB) have already shown its value for different applications during image-guided surgery. Both ICG and MB are currently the only fluorophores approved by regulatory agencies for off-label use. ICG improves visibility of several solid tumors, sentinel lymph nodes, biliary ducts and can be used to evaluate tissue perfusion. MB could be used for ureter imaging and neuroendocrine or thyroid tumors detection. Recently, a shift to molecular imaging was made by the introduction of new NIR fluorophores (IRDye-800CW, ZW800-1), which could be conjugated to tumor or structure specific targets, such as proteins, antibodies, antibody fragments or nanoparticles. Several clinical trials showed detection of both tumor and metastases in patients with head-and neck, colorectal, pancreatic, ovarian, and renal tumors. Furthermore, nerve and ureter specific agents are (pre-)clinically evaluated, however more research is necessary to make these agents clinically available. Limitations of using NIR fluorescence imaging during surgery are the lack of quantification of fluorescence signals and limited penetration depth. Further optimization of NIR fluorescence imaging and evaluation of the clinical benefit for the patient are necessary steps to make NIR fluorescence guided surgery general applicable into surgical daily practice.
Endoscopic ultrasound-guided fine needle aspirations (EUS-FNA) of pancreatic masses suffer from sample errors and low-negative predictive values. Fiber-optic spectroscopy in the visible to near-infrared wavelength spectrum can noninvasively extract physiological parameters from tissue and has the potential to guide the sampling process and reduce sample errors. We assessed the feasibility of single fiber (SF) reflectance spectroscopy measurements during EUS-FNA of pancreatic masses and its ability to distinguish benign from malignant pancreatic tissue. A single optical fiber was placed inside a 19-gauge biopsy needle during EUS-FNA and at least three reflectance measurements were taken prior to FNA. Spectroscopy measurements did not cause any related adverse events and prolonged procedure time with ∼5 min. An accurate correlation between spectroscopy measurements and cytology could be made in nine patients (three benign and six malignant). The oxygen saturation and bilirubin concentration were significantly higher in benign tissue compared with malignant tissue (55% versus 21%, p=0.038; 166 μmol/L versus 17 μmol/L, p=0.039, respectively). To conclude, incorporation of SF spectroscopy during EUS-FNA was feasible, safe, and relatively quick to perform. The optical properties of benign and malignant pancreatic tissue are different, implying that SF spectroscopy can potentially guide the FNA sampling.
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