Multidiameter Single-fiber Reflectance Research Articles

Detection of Early Esophageal Neoplastic Barrett Lesions with Quantified Fluorescence Molecular Endoscopy Using Cetuximab-800CW.

Esophageal adenocarcinoma causes 6% of cancer-related deaths worldwide. Near-infrared fluorescence molecular endoscopy (NIR-FME) uses a tracer that targets overexpressed proteins. In this study, we aimed to investigate the feasibility of an epidermal growth factor receptor (EGFR)-targeted tracer, cetuximab-800CW, to improve detection of early-stage esophageal adenocarcinoma. Methods: We validated EGFR expression in 73 esophageal tissue sections. Subsequently, we topically administered cetuximab-800CW and performed high-definition white-light endoscopy (HD-WLE), narrow-band imaging, and NIR-FME in 15 patients with Barrett esophagus (BE). Intrinsic fluorescence values were quantified using multidiameter single-fiber reflectance and single-fiber fluorescence spectroscopy. Back-table imaging, histopathologic examination, and EGFR immunohistochemistry on biopsy samples collected during NIR-FME procedures were performed and compared with invivo imaging results. Results: Immunohistochemical preanalysis showed high EGFR expression in 67% of dysplastic tissue sections. NIR-FME visualized all 12 HD-WLE-visible lesions and 5 HD-WLE-invisible dysplastic lesions, with increased fluorescence signal in visible dysplastic BE lesions compared with nondysplastic BE as shown by multidiameter single-fiber reflectance/single-fiber fluorescence, reflecting a target-to-background ratio of 1.5. Invisible dysplastic lesions also showed increased fluorescence, with a target-to-background ratio of 1.67. Immunohistochemistry analysis showed EGFR overexpression in 16 of 17 (94%) dysplastic BE lesions, which all showed fluorescence signal. Conclusion: This study has shown that NIR-FME using cetuximab-800CW can improve detection of dysplastic lesions missed by HD-WLE and narrow-band imaging.

Experimental validation of a recently developed model for single-fiber reflectance spectroscopy.

.Significance: We recently developed a model for the reflectance measured with (multi-diameter) single-fiber reflectance (SFR) spectroscopy as a function of the reduced scattering coefficient , the absorption coefficient , and the phase function parameter . We validated this model with simulations.Aim: We validate our model experimentally. To prevent overfitting, we investigate the wavelength-dependence of and propose a parametrization with only three parameters. We also investigate whether this parametrization enables measurements with a single fiber, as opposed to multiple fibers used in multi-diameter SFR (MDSFR).Approach: We validate our model on 16 phantoms with two concentrations of Intralipid-20% ( and at 500 nm) and eight concentrations of Evans Blue ( to at 605 nm). We parametrize as .Results: Average errors were 7% for , 11% for , and 16% with the parametrization of ; and 7%, 17%, and 16%, respectively, without. The parametrization of improved the fit speed 25 times (94 s to ). Average errors for only one fiber were 50%, 33%, and 186%, respectively.Conclusions: Our recently developed model provides accurate results for MDSFR measurements but not for a single fiber. The parametrization prevents overfitting and speeds up the fit.

Toward improved endoscopic surveillance with multidiameter single fiber reflectance spectroscopy in patients with Barrett's esophagus.

Patients with Barrett's esophagus are at an increased risk to develop esophageal cancer and, therefore, undergo regular endoscopic surveillance. Early detection of neoplasia enables endoscopic treatment, which improves outcomes. However, early Barrett's neoplasia is easily missed during endoscopic surveillance. This study investigates multidiameter single fiber reflectance spectroscopy (MDSFR) to improve Barrett's surveillance. Based on the concept of field cancerization, it may be possible to identify the presence of a neoplastic lesion from measurements elsewhere in the esophagus or even the oral cavity. In this study, MDSFR measurements are performed on non-dysplastic Barrett's mucosa, squamous mucosa, oral mucosa, and the neoplastic lesion (if present). Based on logistic regression analysis on the scattering parameters measured by MDSFR, a classifier is developed that can predict the presence of neoplasia elsewhere in the Barrett's segment from measurements on the non-dysplastic Barrett's mucosa (sensitivity 91%, specificity 71%, AUC = 0.77). Classifiers obtained from logistic regression analysis for the squamous and oral mucosa do not result in an AUC significantly different from 0.5.

Effect of probe pressure on skin tissue optical properties measurement using multi-diameter single fiber reflectance spectroscopy

Multi-Diameter Single Fiber Reflectance spectroscopy (MDSFR) allows rapid and noninvasive extraction of tissue optical properties that requires the fiberoptic to be placed in contact with the tissue. To ensure a good optical contact, the application of probe pressure is inevitable and usually not well controlled. In this study, we investigated the effect of probe pressure by performing in vivo MDSFR measurements (400–1600 nm) on human inner forearm skin with controlled probe indentation and continuous pressure monitoring. For the probe pressures up to 28.4 s mm−2 (213 mmHg), the reduced scattering coefficient pivoted around 800 nm (increasing scattering slope) while the phase function related parameter decreased (up to 20%, depending on the wavelength). A significant decrease of hemoglobin concentration (∼81%), oxygen saturation (∼86%), apparent vessel diameter (∼100%) as well as the displacement of extracellular water (∼24%) was observed with increasing probe pressure. A correlation was observed between the changes in water volume fraction and phase function related parameter gamma . We theorize that the latter changes in phase function may be due to the induced deformation of the tissue structure and the displacement of extracellular water. The extensive analysis of the pressure induced changes in the measured MDSFR spectra and the related changes in optical properties demonstrate that the effects of probe pressure must be minimized to avoid bias in the results.

C-Met targeted fluorescence molecular endoscopy in Barrett's esophagus patients and identification of outcome parameters for phase-I studies.

Fluorescence molecular endoscopy (FME) is an emerging technique in the field of gastroenterology that holds potential to improve diagnosis and guide therapy, by serving as a 'red-flag' endoscopic imaging technique. Here, we investigated the safety, feasibility and optimal method of administration of EMI-137, targeting c-Met, during FME in Barrett's Esophagus (BE) and report several outcome parameters for early phase FME studies.Methods: FME was performed in 15 Barrett's neoplasia patients. EMI-137 was administered to three cohorts of five patients: 0.13 mg/kg intravenously (IV); 0.09 mg/kg IV or topically at a dose of 200 μg/cm BE (n=1) or 100 μg/cm BE (n=4). Fluorescence was visualized in vivo, quantified in vivo using multi-diameter single-fiber reflectance, single-fiber fluorescence (MDSFR/SFF) spectroscopy and correlated to histopathology and immunohistochemistry. EMI-137 localization was assessed using fluorescence microscopy.Results: FME using different IV and topical doses of EMI-137 appeared to be safe and correctly identified 16/18 lesions, although modest target-to-background ratios were observed (median range of 1.12-1.50). C-Met overexpression varied between lesions, while physiological expression in the stomach-type epithelium was observed. Microscopically, EMI-137 accumulated around the neoplastic cell membranes. We identified several outcome parameters important for the validation of EMI-137 for FME: 1) the optimal administration route; 2) optimal dose and safety; 3) in vivo FME contrast; 4) quantification of intrinsic fluorescence; 5) ex vivo correlation of fluorescence, histopathology and target expression; and 6) microscopic tracer distribution.Conclusions: C-Met targeted FME using EMI-137 may not be the ideal combination to improve BE surveillance endoscopies, however the identified outcome parameters may serve as a valuable guidance for designing and performing future early phase clinical FME studies, independent of which fluorescent tracer is investigated.

Multidiameter single-fiber reflectance spectroscopy of heavily pigmented skin: modeling the inhomogeneous distribution of melanin.

.When analyzing multidiameter single-fiber reflectance (MDSFR) spectra, the inhomogeneous distribution of melanin pigments in skin tissue is usually not accounted for. Especially in heavily pigmented skins, this can result in bad fits and biased estimation of tissue optical properties. A model is introduced to account for the inhomogeneous distribution of melanin pigments in skin tissue. In vivo visible MDSFR measurements were performed on heavily pigmented skin of type IV to VI. Skin tissue optical properties and related physiological properties were extracted from the measured spectra using the introduced model. The absorption of melanin pigments described by the introduced model demonstrates a good correlation with the co-localized measurement of the well-known melanin index.

Towards the Optical Detection of Field Cancerization in the Buccal Mucosa of Patients with Lung Cancer

INTRODUCTION: An increase in detection of early-stage asymptomatic lung tumors could increase the overall survival rate of lung cancer patients. A new approach to cancer (pre-)screening focusses on detecting field cancerization instead of the tumor itself. The objective of this study was to investigate the use of optical spectroscopy to detect field cancerization in the buccal mucosa of lung cancer patients. METHODS: Optical buccal mucosa measurements were performed in lung cancer patients and controls using multidiameter single-fiber reflectance spectroscopy. We analyzed whether the measured optical parameters could distinguish lung cancer patients from controls. RESULTS: Twenty-three lung cancer patients, 24 chronic obstructive pulmonary disease (COPD) control patients, and 36 non-COPD controls were included. The majority of tumors were non-small-cell lung carcinomas (96%) and classified as stage I (48%). The tissue scattering properties μs' and γ at 800 nm and the tissue bilirubin concentration were all near-significantly different (P=.072, 0.058, and 0.060, respectively) between the lung cancer and COPD group. μs' at 800 nm had a sensitivity of 74% and a specificity of 63%. The microvascular blood oxygen saturation of the lung cancer patients was also higher than the COPD patients (78% vs. 62%, P=.002), this is probably a consequence of the systemic effect of COPD. CONCLUSIONS: We have demonstrated that μs' at 800 nm is increased in the buccal mucosa of patients with lung cancer compared to controls with COPD. This might be an indication of field cancerization in the oral cavity of patients with lung cancer.

Optical pre-screening for laryngeal cancer using reflectance spectroscopy of the buccal mucosa.

A new approach in early cancer detection focuses on detecting field cancerization (FC) instead of the tumor itself. The aim of the current study is to investigate whether reflectance spectroscopy can detect FC in the buccal mucosa of patients with laryngeal cancer. The optical properties of the buccal mucosa of patients were measured with multidiameter single-fiber reflectance spectroscopy. The blood oxygen saturation and blood volume fraction were significantly lower in the buccal mucosa of laryngeal cancer patients than in non-oncologic controls. The data of these two parameters were combined to form a single 'biomarker α', which optimally discriminates these two groups. Alpha was lower in the laryngeal cancer group (0.28) than the control group (0.30, p = 0.007). Alpha could identify oncologic patients with a sensitivity of 78% and a specificity of 74%. These results might be the first step toward optical pre-screening for laryngeal cancer.

Optical detection of field cancerization in the buccal mucosa of patients with esophageal cancer.

IntroductionEsophageal cancer is an increasingly common type of neoplasm with a very poor prognosis. This prognosis could improve with more early tumor detection. We have previously shown that we can use an optical spectroscopy to detect field cancerization in the buccal mucosa of patients with laryngeal cancer. The aim of this prospective study was to investigate whether we could detect field cancerization of buccal mucosa of patients with esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC).MethodsOptical measurements were performed in vivo using a novel optical technique: multidiameter single-fiber reflectance (MDSFR) spectroscopy. MDSFR spectra were acquired by a handheld probe incorporating three fiber diameters. Multiple absorption and scattering parameters that are related to the physiological and ultrastructural properties of the buccal mucosa were derived from these spectra. A linear discriminant analysis of the parameters was performed to create a combined biomarker σ to discriminate oncologic from non-oncologic patients.ResultsTwelve ESCC, 12 EAC, and 24 control patients were included in the study. The median value of our biomarker σ was significantly higher in patients with ESCC (2.07 [1.93–2.10]) than control patients (1.86 [1.73–1.95], p = 0.022). After cross-validation σ was able to identify ESCC patients with a sensitivity of 66.7% and a specificity of 70.8%. There were no significant differences between the EAC group and the control group.ConclusionField cancerization in the buccal mucosa can be detected using optical spectroscopy in ESCC patients. This may be the first step towards non-invasive ESCC cancer screening.

Abstract P4-01-01: Phase II in-human dose escalation study of the optical molecular imaging tracer bevacizumab-800cw for molecular fluorescence guided surgery in primary breast cancer patients

Abstract Introduction Molecular Fluorescence Guided Surgery (MFGS) might be used for intraoperative detection of positive resection margins in breast conserving surgery (BCS) for the treatment of breast cancer. Currently, the presence of tumor positive resection margins can only be assessed ex vivo by histopathological analysis of the excised tissue, which takes up to 5 working days. The current study defined the optimal dose of the near-infrared (NIR) optical imaging tracer bevacizumab-800CW to enable intraoperative detection and image-guided pathology of tumor positive resection margins in breast cancer patients. Methods Molecular Fluorescence Guided Surgery during BCS was performed in subjects treated for primary breast cancer. The NIR optical imaging tracer bevacizumab-800CW targeting vascular endothelial growth factor A was administered intravenously three days prior to surgery in four escalating dose groups (4.5mg, 10mg, 25mg and 50mg). NIR fluorescent signals were detected real-time using an intraoperative fluorescence camera system (SurgVision BV). Standardized ex vivo analyses of tumor-to-normal ratios (TNR) were performed to define the optimal tracer dose using a BlackBox imaging system (SurgVision BV) for imaging fresh bread loaf slices, a NIR fluorescence flatbed scanner (Odyssey, Li-Cor) for imaging 10µm slices of formalin-fixed paraffin-embedded (FFPE) blocks, NIR Confocal Laser Endomicroscopy (CLE, Mauna Kea Technologies) and multi-diameter single fiber reflectance and single fiber fluorescence (MDSFR/SFF) spectroscopy quantification to enable correction for the influence of tissue optical properties on fluorescence in tumor and normal breast tissue. Results Currently, 12 subjects have been included and analyzed in four dosing groups. All tumors showed specific tracer uptake at macroscopic and microscopic level during ex vivo analyses, confirmed by histopathology. Quantification of NIR fluorescent signals showed higher TNRs by increasing doses up to 25mg. No further increase in TNR was seen in the 50mg dose group. CLE showed the feasibility of visualization of the tracer accumulation in tumor tissue compared to normal tissue at a microscopic level. MDSFR/SFF spectroscopy objectively confirmed the dose dependency up to 25mg. Conclusion and future perspective Intravenous administration of bevacizumab-800CW in doses up to 50mg is safe and highly tumor specific, showing a plateau of TNR at 25mg and 50mg. Further expansion of the dosing cohorts of 10mg and 25mg with additional seven patients per group will be performed to establish the optimal dose for MFGS during BCS for an upcoming phase III multicenter randomized controlled trial. By enabling MFGS during BCS the surgical treatment of primary breast cancer patients might be optimized by a reduced need for re-excision surgery and thereby reducing the risk of co-morbidity, psychological burden, poor cosmesis and healthcare costs. Citation Format: van Dam GM, Koller M, Qiu SQ, Linssen MD, de Vries J, Jansen L, Kelder W, de Jong JS, Jorritsma-Smit A, van der Vegt B, Robinson DJ, Nagengast WB. Phase II in-human dose escalation study of the optical molecular imaging tracer bevacizumab-800cw for molecular fluorescence guided surgery in primary breast cancer patients [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-01-01.

Sources of variability in the quantification of tissue optical properties by multidiameter single-fiber reflectance and fluorescence spectroscopy.

Recently, a multidiameter single-fiber reflectance and fluorescence spectroscopy device has been developed that enabled us to extract the autofluorescence of tissue that is corrected for the optical properties. Such a system has been incorporated in the population-based Rotterdam Study to investigate the autofluorescence of the skin. Since the device will be used by different operators over many years, it is essential that the results are comparable between users. It is, however, unclear how different methods of handling the probe might influence the outcome. Variability of blood oxygen saturation, blood volume fraction and vessel diameter, average gamma, reduced scattering coefficient at 800 nm, and integrated intrinsic fluorescence measured in three volunteers were assessed within and between eight untrained users. A variability of less than one standard deviation from the group mean was defined as an acceptable limit. Three mature volunteers were also included to assess the intrauser variability of mature skin. The variation in the measured parameters suggests that variation is dominated by tissue heterogeneity. Most users measured within one standard deviation of the group mean. Notably, corrected intrinsic fluorescence showed low intra- and interuser variability. These results strongly suggest that variability is mostly caused by tissue heterogeneity and is not user induced.

Method for rapid multidiameter single-fiber reflectance and fluorescence spectroscopy through a fiber bundle

We have recently demonstrated a means for quantifying the absorption and scattering properties of biological tissue through multidiameter single-fiber reflectance (MDSFR) spectroscopy. These measurements can be used to correct single-fiber fluorescence (SFF) spectra for the influence of optical properties, enabling quantification of intrinsic fluorescence. In our previous work, we have used a series of pinholes to show that selective illumination and light collection using a coherent fiber bundle can simulate a single solid-core optical fiber with variable diameter for the purposes of MDSFR spectroscopy. Here, we describe the construction and validation of a clinical MDSFR/SFF spectroscopy system that avoids the limitations encountered with pinholes and free-space optics. During one measurement, the new system acquires reflectance spectra at the effective diameters of 200, 600, and 1000 μm, and a fluorescence spectrum at an effective diameter of 1000 μm. From these spectra, we measure the absolute absorption coefficient, μ(a), reduced scattering coefficient, μ'(s'), phase function parameter, γ, and intrinsic fluorescence, Qμ(a,x)(f), across the measured spectrum. We validate the system using Intralipid- and polystyrene sphere-based scattering phantoms, with and without the addition of the absorber Evans Blue. Finally, we demonstrate the combined MDSFR/SFF of phantoms with varying concentrations of Intralipid and fluorescein, wherein the scattering properties are measured by MDSFR and used to correct the SFF spectrum for accurate quantification of Qμ(a,x)(f).

In vivo quantification of the scattering properties of tissue using multi-diameter single fiber reflectance spectroscopy

Multi diameter single fiber reflectance (MDSFR) spectroscopy is a non-invasive optical technique based on using multiple fibers of different diameters to determine both the reduced scattering coefficient (μs′) and a parameter γ that is related to the angular distribution of scattering, where γ = (1-g2)/(1-g1) and g1 and g2 the first and second moment of the phase function, respectively. Here we present the first in vivo MDSFR measurements of μs′(λ) and γ(λ) and their wavelength dependence. MDSFR is performed on nineteen mice in four tissue types including skin, liver, normal tongue and in an orthotopic oral squamous cell carcinoma. The wavelength-dependent slope of μs′(λ) (scattering power) is significantly higher for tongue and skin than for oral cancer and liver. The reduced scattering coefficient at 800 nm of oral cancer is significantly higher than of normal tongue and liver. Gamma generally increases with increasing wavelength; for tumor it increases monotonically with wavelength, while for skin, liver and tongue γ(λ) reaches a plateau or even decreases for longer wavelengths. The mean γ(λ) in the wavelength range 400-850 nm is highest for liver (1.87 ± 0.07) and lowest for skin (1.37 ± 0.14). Gamma of tumor and normal tongue falls in between these values where tumor exhibits a higher average γ(λ) (1.72 ± 0.09) than normal tongue (1.58 ± 0.07). This study shows the potential of using light scattering spectroscopy to optically characterize tissue in vivo.

Use of a coherent fiber bundle for multi-diameter single fiber reflectance spectroscopy

Multi-diameter single fiber reflectance (MDSFR) spectroscopy enables quantitative measurement of tissue optical properties, including the reduced scattering coefficient and the phase function parameter γ. However, the accuracy and speed of the procedure are currently limited by the need for co-localized measurements using multiple fiber optic probes with different fiber diameters. This study demonstrates the use of a coherent fiber bundle acting as a single fiber with a variable diameter for the purposes of MDSFR spectroscopy. Using Intralipid optical phantoms with reduced scattering coefficients between 0.24 and 3 mm−1, we find that the spectral reflectance and effective path lengths measured by the fiber bundle (NA = 0.40) are equivalent to those measured by single solid-core fibers (NA = 0.22) for fiber diameters between 0.4 and 1.0 mm (r ≥ 0.997). This one-to-one correlation may hold for a 0.2 mm fiber diameter as well (r = 0.816); however, the experimental system used in this study suffers from a low signal-to-noise for small dimensionless reduced scattering coefficients due to spurious back reflections within the experimental system. Based on these results, the coherent fiber bundle is suitable for use as a variable-diameter fiber in clinical MDSFR quantification of tissue optical properties.

Quantification of the reduced scattering coefficient and phase-function-dependent parameter γ of turbid media using multidiameter single fiber reflectance spectroscopy: experimental validation

Multidiameter single fiber reflectance (MDSFR) spectroscopy is a method that allows the quantification of μs' and the phase-function-dependent parameter γ of a turbid medium by utilizing multiple fibers with different diameters. We have previously introduced the theory behind MDSFR and its limitations, and here we present an experimental validation of this method based on phantoms containing a fractal distribution of polystyrene spheres both in the absence and presence of the absorber Evans Blue.

Measurement of tissue scattering properties using multi-diameter single fiber reflectance spectroscopy: in silico sensitivity analysis

Multiple diameter single fiber reflectance (MDSFR) measurements of turbid media can be used to determine the reduced scattering coefficient (μ′s) and a parameter that characterizes the phase function (γ). The MDSFR method utilizes a semi-empirical model that expresses the collected single fiber reflectance intensity as a function of fiber diameter (dfiber), μ′s, and γ. This study investigated the sensitivity of the MDSFR estimates of μ′s and γ to the choice of fiber diameters and spectral information incorporated into the fitting procedure. The fit algorithm was tested using Monte Carlo simulations of single fiber reflectance intensities that investigated biologically relevant ranges of scattering properties (μ′s ∈ [0.4 – 4]mm−1) and phase functions (γ ∈ [1.4 – 1.9]) and for multiple fiber diameters (dfiber ∈ [0.2 – 1.5] mm). MDSFR analysis yielded accurate estimates of μ′s and γ over the wide range of scattering combinations; parameter accuracy was shown to be sensitive to the range of fiber diameters included in the analysis, but not to the number of intermediate fibers. Moreover, accurate parameter estimates were obtained without a priori knowledge about the spectral shape of γ. Observations were used to develop heuristic guidelines for the design of clinically applicable MDSFR probes.

Method to quantitatively estimate wavelength-dependent scattering properties from multidiameter single fiber reflectance spectra measured in a turbid medium

This study utilizes experimentally validated Monte Carlo simulations to identify a mathematical formulation of the reflectance intensity collected by a single fiber probe expressed in terms of the reduced scattering coefficient (μs'), fiber diameter d(fiber), and a property of the first two moments of the scattering phase function (γ). This model is then utilized to accurately obtain wavelength-dependent estimates of μs'(λ) and γ(λ) from multiple single fiber spectral measurements of a turbid medium obtained with different diameters. This method returns accurate descriptions (mean residual <3%) of both μs' and γ across the biologically relevant range.