Multivariate analysis of breast tissue using optical parameters extracted from a combined time-resolved fluorescence and diffuse reflectance system for tumor margin detection.

Significance: Breast conservation therapy is the preferred technique for treating primary breast cancers. However, breast tumor margins are hard to determine as tumor borders are often ill-defined. As such, there exists a need for a clinically compatible tumor margin detection system. Aim: A combined time-resolved fluorescence and diffuse reflectance (TRF-DR) system has been developed to determine the optical properties of breast tissue. This study aims to improve tissue classification to aid in surgical decision making. Approach: Normal and tumor breast tissue were collected from 80 patients with invasive ductal carcinoma and measured in the optical system. Optical parameters were extracted, and the tissue underwent histopathological examination. In total, 761 adipose, 77 fibroglandular, and 347 tumor spectra were analyzed. Principal component analysis and decision tree modeling were performed using only TRF optical parameters, only DR optical parameters, and using the combined datasets. Results: The classification modeling using TRF data alone resulted in a tumor margin detection sensitivity of 72.3% and specificity of 88.3%. Prediction modeling using DR data alone resulted in greater sensitivity and specificity of 80.4% and 94.0%, respectively. Combining both datasets resulted in the improved sensitivity and specificity of 85.6% and 95.3%, respectively. While both sensitivity and specificity improved with the combined modeling, further study of fibroglandular tissue could result in improved classification. Conclusion: The combined TRF-DR system showed greater tissue classification capability than either technique alone. Further work studying more fibroglandular tissue and tissue of mixed composition would develop this system for intraoperative use for tumor margin detection.

Hyperspectral imaging assessment for radiotherapy induced skin-erythema: Pilot study.

Skin cancer (SC) is a widely spread disease in the USA, Canada, and Australia. Skin cancer patients may be treated by many different techniques including radiation therapy. However, radiation therapy has side effects, which may range from skin erythema to skin necrosis. As erythema is the early evidence of exposure to radiation, monitoring erythema is important to prevent more severe reactions. Visual assessment (VA) is the gold standard for evaluating erythema. Nevertheless, VA is not ideal, since it depends on the observer's experience and skills. Digital photography and hyperspectral imaging (HSI) are optical techniques that provide an opportunity for objective assessment of erythema. Erythema indices were computed from the spectral data using Dawson's technique. The Dawson relative erythema index proved to be highly correlated (97.1 %) with clinical visual assessment scores. In addition, on the 7th session of radiation therapy, the relative erythema index differentiates with 99 % significance between irradiated and non-radiated skin regions. In this study, HSI is compared to digital photography for skin erythema statistical classification.

Quantitative fluorescence imaging of point-like sources in small animals.

A planar imaging approach is described for the in vivo quantitative reconstruction of fluorescent point sources in small animals. The method uses the diffusion approximation as a forward model of light propagation from a point source in a homogeneous tissue to find source depth and strength. The tissue optical properties obtained from video reflectometry measurements were used to compensate for the effects of tissue heterogeneity. The method was evaluated on images of fluorescent sources implanted 2-8.5 mm deep in the thigh and abdomen of rats post mortem. In more than 70% of the total number of implants the source depth was retrieved with an error of less than 1 mm. The largest absolute error was 1.9 mm. In retrieving source strength, the errors ranged from 0.4% to 89% generally increasing with increased source depth.

Dual-modality optical biopsy of glioblastomas multiforme with diffuse reflectance and fluorescence: ex vivo retrieval of optical properties.

Glioma itself accounts for 80% of all malignant primary brain tumors, and glioblastoma multiforme (GBM) accounts for 55% of such tumors. Diffuse reflectance and fluorescence spectroscopy have the potential to discriminate healthy tissues from abnormal tissues and therefore are promising noninvasive methods for improving the accuracy of brain tissue resection. Optical properties were retrieved using an experimentally evaluated inverse solution. On average, the scattering coefficient is 2.4 times higher in GBM than in low grade glioma (LGG), and the absorption coefficient is 48% higher. In addition, the ratio of fluorescence to diffuse reflectance at the emission peak of 460 nm is 2.6 times higher for LGG while reflectance at 650 nm is 2.7 times higher for GBM. The results reported also show that the combination of diffuse reflectance and fluorescence spectroscopy could achieve sensitivity of 100% and specificity of 90% in discriminating GBM from LGG during ex vivo measurements of 22 sites from seven glioma specimens. Therefore, the current technique might be a promising tool for aiding neurosurgeons in determining the extent of surgical resection of glioma and, thus, improving intraoperative tumor identification for guiding surgical intervention.

Matrix metalloproteinase-1 takes advantage of the induced fit mechanism to cleave the triple-helical type I collagen molecule.

The collagenases are members of the matrix metalloproteinase family (MMP) that degrade native triple-helical type I collagen. To understand the mechanism by which these enzymes recognize and cleave this substrate, we studied the substrate specificity of a modified form of MMP-1 (FC) in which its active site region (amino acids 212-254) had been replaced with that of MMP-9 (amino acids 395-437). Although this substitution increased the activity of the enzyme toward gelatin and the peptide substrate Mca-PLGL(Dpa)AR-NH2 by approximately 3- and approximately 11-fold, respectively, it decreased the type I collagenolytic activity of the enzyme to 0.13%. The replacement of Gly233, the only amino acid in this region of FC that is conserved in all collagenase family members, with the corresponding Glu residue in MMP-9 resulted in a substantial decrease in the type I collagenolytic activity of the enzyme without affecting its general proteolytic activities. The kinetic parameters of the FC/G233E mutant for the collagen substrate were similar to those of the chimeric enzyme. In addition, substituting Gly233 for Glu in the chimera increased the collagenolytic activity of the enzyme by 12-fold. Interestingly, replacing Glu415 in MMP-9 with Gly, its corresponding residue in FC, endowed the enzyme with type I collagenolytic activity. The catalytic activity of the MMP-9 mutant toward triple-helical type I collagen was 2-fold higher than that of the collagenase chimera. These data in conjunction with the X-ray crystal structure of FC indicate that Gly233 provides the flexibility necessary for the enzyme active site to change conformation upon substrate binding. The flexibility provided by the Gly residue is essential for type I collagenolytic activity.

Investigation of light propagation models to determine the optical properties of tissue from interstitial frequency domain fluence measurements.

Four models, standard diffusion approximation (SDA), single Monte Carlo (SMC), delta-P1, and isotropic similarity (ISM), are developed and evaluated as forward calculation tools in the estimation of tissue optical properties. The inverse calculation uses the ratio of the fluences and phase difference at two locations close to an intensity modulated isotropic source to recover the reduced scattering coefficient mus' and the absorption coefficient mua. Diffusion theory allows recovery of optical properties (OPs) within 5% for media with mus'mua>10. The performance of the delta-P1 model is similar to SDA, with limited enhanced accuracy. The collimation approximation may limit the use of the delta-P1 model for spherical geometry, and/or the fluence may not be accurately calculated by this model. The SMC model is the best, recovering OPs within 10% regardless of the albedo. However, the necessary restriction of the searched OPs space is inconvenient. The performance of ISM is similar to that of diffusion theory for media with mus'mua>10, and better for 15.

Comparison of the effect of mutant and wild-type p53 on global gene expression.

The mechanisms for "gain-of-function" phenotypes produced by mutant p53s such as enhanced proliferation, resistance to transforming growth factor-beta-mediated growth suppression, and increased tumorigenesis are not known. One theory is that these phenotypes are caused by novel transcriptional regulatory events acquired by mutant p53s. Another explanation is that these effects are a result of an imbalance of functions caused by the retention of some of the wild-type transcriptional regulatory events in the context of a loss of other counterbalancing activities. An analysis of the ability of DNA-binding domain mutants A138P and R175H, and wild-type p53 to regulate the expression levels of 6.9 x 10(3) genes revealed that the mutants retained only <5% of the regulatory activities of the wild-type protein. A138P p53 exhibited mostly retained wild-type regulatory activities and few acquired novel events. However, R175H p53 possessed an approximately equal number of wild-type regulatory events and novel activities. This is the first report that, after examination of the regulation of a large unfocused set of genes, provides data indicating that remaining wild-type transcriptional regulatory functions existing in the absence of counterbalancing activities as well as acquired novel events both contribute to the gain-of-function phenotypes produced by mutant p53s. However, mutant p53s are likely to be distinct in terms of the extent to which each mechanism contributes to their gain-of-function phenotypes.

Distortion correction and cross-talk compensation algorithm for use with an imaging spectrometer based spatially resolved diffuse reflectance system.

Optical spectroscopy of human tissue has been widely applied within the field of biomedical optics to allow rapid, in vivo characterization and analysis of the tissue. When designing an instrument of this type, an imaging spectrometer is often employed to allow for simultaneous analysis of distinct signals. This is especially important when performing spatially resolved diffuse reflectance spectroscopy. In this article, an algorithm is presented that allows for the automated processing of 2-dimensional images acquired from an imaging spectrometer. The algorithm automatically defines distinct spectrometer tracks and adaptively compensates for distortion introduced by optical components in the imaging chain. Crosstalk resulting from the overlap of adjacent spectrometer tracks in the image is detected and subtracted from each signal. The algorithm's performance is demonstrated in the processing of spatially resolved diffuse reflectance spectra recovered from an Intralipid and ink liquid phantom and is shown to increase the range of wavelengths over which usable data can be recovered.

Two-dimensional mapping of underdosed areas using radiochromic film for patients undergoing total skin electron beam radiotherapy.

PURPOSE: To demonstrate the viability of radiochromic film as an in vivo, two-dimensional dosimeter for the measurement of underdosed areas in patients undergoing total skin electron beam (TSEB) radiotherapy. The results were compared with thermoluminescent dosimeter measurements. METHODS AND MATERIALS: Dosimetry results are reported for an inframammary fold of 2 patients treated using a modified version of the Stanford six-position (i.e., six-field and dual-beam) TSEB technique. The results are presented as contour plots of film optical density and percentage of dose. A linear dose profile measured from film was compared with the thermoluminescent dosimeter measurements. RESULTS: The results showed that the percentage doses as measured by film are in good agreement with those measured by the thermoluminescent dosimeters. The isodose contour plots provided by film can be used as a two-dimensional dose map for a patient when determining the size of the supplemental patch fields. CONCLUSION: Radiochromic film is a viable dosimetry tool that the radiation oncologist can use to understand the surface dose heterogeneity better across complex concave regions of skin to help establish more appropriate margins to patch underdosed areas. Film could be used for patients undergoing TSEB for disorders such as mycosis fungoides or undergoing TSEB or regional skin electron beam for widespread skin metastases from breast cancer and other malignancies.

Modeling changes in the hemoglobin concentration of skin with total diffuse reflectance spectroscopy.

The ability to monitor changes in the concentration of hemoglobin in the blood of the skin in real time is a key component to personalized patient care. Since hemoglobin has a unique absorption spectrum in the visible light range, diffuse reflectance spectroscopy is the most common approach. Although the collection of the diffuse reflectance spectrum with an integrating sphere (IS) has several calibration challenges, this collection method is sufficiently user-friendly that it may be worth overcoming the initial difficulty. Once the spectrum is obtained, it is commonly interpreted with a log-inverse-reflectance (LIR) or "absorbance" analysis that can only accurately monitor changes in the hemoglobin concentration when there are no changes to the nonhemoglobin chromophore concentrations which is not always the case. We address the difficulties associated with collection of the diffuse reflectance spectrum with an IS and propose a model capable of retrieving relative changes in hemoglobin concentration from the visible light spectrum. The model is capable of accounting for concentration changes in the nonhemoglobin chromophores and is first characterized with theoretical spectra and liquid phantoms. The model is then used in comparison with a common LIR analysis on temporal measurements from blanched and reddened human skin.

Experimental recovery of intrinsic fluorescence and fluorophore concentration in the presence of hemoglobin: spectral effect of scattering and absorption on fluorescence.

The ability to recover the intrinsic fluorescence of biological fluorophores is crucial to accurately identify the fluorophores and quantify their concentrations in the media. Although some studies have successfully retrieved the fluorescence spectral shape of known fluorophores, the techniques usually came with heavy computation costs and did not apply for strongly absorptive media, and the intrinsic fluorescence intensity and fluorophore concentration were not recovered. In this communication, an experimental approach was presented to recover intrinsic fluorescence and concentration of fluorescein in the presence of hemoglobin (Hb). The results indicated that the method was efficient in recovering the intrinsic fluorescence peak and fluorophore concentration with an error of 3% and 10%, respectively. The results also suggested that chromophores with irregular absorption spectra (e.g., Hb) have more profound effects on fluorescence spectral shape than chromophores with monotonic absorption and scattering spectra (e.g., black India ink and polystyrene microspheres).

Assessing patient characteristics and radiation-induced non-targeted effects in vivo for high dose-rate (HDR) brachytherapy.

PURPOSE: To test whether blood, urine, and tissue based colony-forming assays are a useful clinical detection tool for assessing fractionated treatment responses and non-targeted radiation effects in bystander cells. MATERIALS AND METHODS: To assess patients' responses to radiation treatments, blood serum, urine, and an esophagus explant-based in vivo colony-forming assay were used from oesophageal carcinoma patients. These patients underwent three fractions of high dose rate (HDR) intraluminal brachytherapy (ILBT). RESULTS: Human keratinocyte reporters exposed to blood sera taken after the third fraction of brachytherapy had a significant increase in cloning efficiency compared to baseline samples (p < 0.001). Such results may suggest an induced radioresistance response in bystander cells. The data also revealed a clear inverse dose-rate effect during late treatment fractions for the blood sera data only. Patient characteristics such as gender had no statistically significant effect (p > 0.05). Large variability was observed among the patients' tissue samples, these colony-forming assays showed no significant changes throughout fractionated brachytherapy (p > 0.05). CONCLUSION: Large inter-patient variability was found in the urine and tissue based assays, so these techniques were discontinued. However, the simple blood-based assay had much less variability. This technique may have future applications as a biological dosimeter to predict treatment outcome and assess non-targeted radiation effects.

Relationship between mTHPC fluorescence photobleaching and cell viability during in vitro photodynamic treatment of DP16 cells.

An implicit dosimetric model has been proposed in which biological damage caused by photodynamic therapy (PDT) is monitored through the decrease in sensitizer fluorescence during treatment. To investigate this, in vitro experiments were performed in which DP16 cells were incubated in meta-tetra(hydroxyphenyl)chlorin (mTHPC) and then irradiated with 514 nm light. Photosensitizer concentration, fluence rate and oxygenation were independently controlled and monitored during the treatment. Fluorescence of mTHPC was continuously monitored via a charge-coupled device-coupled spectrometer during treatment and, at selected fluence levels, cell viability was determined using a trypan blue exclusion assay. The relationship of cell viability to normalized fluorescence was obtained for the different treatment conditions. The relationship was independent of cell medium oxygenation, treatment fluence rate and sensitizer incubation concentration except at a high mTHPC concentration (4 microg/mL). This relationship suggests that fluorescence bleaching may be used to predict mTHPC PDT damage in vitro.

The use of spatially resolved fluorescence and reflectance to determine interface depth in layered fluorophore distributions.

The possibility of using spatially resolved fluorescence and reflectance measurements to recover tissue optical properties, fluorophore concentration and the thickness of a superficial layer in a two-layer geometry was investigated. A diffusion theory model was used to fit reflectance and fluorescence data generated using Monte Carlo simulations or experimentally obtained using tissue-simulating phantoms. Initial analysis fitting diffusion theory generated data suggested that it should be possible to recover all parameters from a single set of spatially resolved fluorescence and reflectance measurements. However, when Monte Carlo or experimental data were fitted the results were less impressive. Overall, it was shown that there is a strong coupling between interface depth, fluorophore concentration and tissue absorption, especially at larger depths. The recovery of all input parameters from a single set of spatially resolved measurements was limited to interface depths less than 3 mm, which is a reasonable range for measuring fluorophore in skin. When the tissue optical properties and fluorophore concentrations were known, then the interface depth could be monitored with good accuracy in simulated serial measurements. These results may also point to deficiencies in the diffusion theory model that introduce significant errors in the fitted results.

Measurement of fluorophore concentrations and fluorescence quantum yield in tissue-simulating phantoms using three diffusion models of steady-state spatially resolved fluorescence.

Steady-state diffusion theory models of fluorescence in tissue have been investigated for recovering fluorophore concentrations and fluorescence quantum yield. Spatially resolved fluorescence, excitation and emission reflectance Carlo simulations, and measured using a multi-fibre probe on tissue-simulating phantoms containing either aluminium phthalocyanine tetrasulfonate (AlPcS4), Photofrin meso-tetra-(4-sulfonatophenyl)-porphine dihydrochloride The accuracy of the fluorophore concentration and fluorescence quantum yield recovered by three different models of spatially resolved fluorescence were compared. The models were based on: (a) weighted difference of the excitation and emission reflectance, (b) fluorescence due to a point excitation source or (c) fluorescence due to a pencil beam excitation source. When literature values for the fluorescence quantum yield were used for each of the fluorophores, the fluorophore absorption coefficient (and hence concentration) at the excitation wavelength (mu(a,x,f)) was recovered with a root-mean-square accuracy of 11.4% using the point source model of fluorescence and 8.0% using the more complicated pencil beam excitation model. The accuracy was calculated over a broad range of optical properties and fluorophore concentrations. The weighted difference of reflectance model performed poorly, with a root-mean-square error in concentration of about 50%. Monte Carlo simulations suggest that there are some situations where the weighted difference of reflectance is as accurate as the other two models, although this was not confirmed experimentally. Estimates of the fluorescence quantum yield in multiple scattering media were also made by determining mu(a,x,f) independently from the fitted absorption spectrum and applying the various diffusion theory models. The fluorescence quantum yields for AlPcS4 and TPPS4 were calculated to be 0.59 +/- 0.03 and 0.121 +/- 0.001 respectively using the point source model, and 0.63 +/- 0.03 and 0.129 +/- 0.002 using the pencil beam excitation model. These results are consistent with published values.

Haemoglobin oxygenation of a two-layer tissue-simulating phantom from time-resolved reflectance: effect of top layer thickness.

A dual wavelength time-resolved reflectance system was developed for monitoring haemoglobin saturation noninvasively. At each wavelength, the time-resolved reflectance data were fitted to a diffusion model of light propagation in a homogeneous, semi-infinite medium to yield the absolute scattering and absorption coefficients. The absorption coefficients were then used to calculate haemoglobin saturation. A two-layer phantom containing human erythrocytes in a scattering solution in the bottom layer was used to study system performance under more realistic conditions. The top layer was chosen to simulate either skin or fat and the oxygenation of the bottom layer, which corresponded to muscle, was controlled. The thickness of the fat layer was varied from 1.5 to 10 mm to investigate the effects of increasing the top layer thickness. These results, obtained with the simple diffusion model, were compared with simultaneous measurements of oxygenation made directly in the bottom layer. Errors in estimating haemoglobin saturation with this method ranged from 5-11% depending on the thickness of the top layer and its optical properties.

Composite depth dose measurement for total skin electron (TSE) treatments using radiochromic film.

Total skin electron (TSE) radiotherapy is routinely used to treat cutaneous T-cell lymphomas and can be implemented using a modified Stanford technique. In our centre, the composite depth dose for this technique is achieved by a combination of two patient positions per day over a three-day cycle, and two gantry angles per patient position. Due to patient morphology, underdosed regions typically occur and have historically been measured using multiple thermoluminescent dosimeters (TLDs). We show that radiochromic film can be used as a two-dimensional relative dosimeter to measure the percent depth dose in TSE radiotherapy. Composite depth dose curves were measured in a cylindrical, polystyrene phantom and compared with TLD data. Both multiple films (1 film per day) and a single film were used in order to reproduce a realistic clinical scenario. First, three individual films were used to measure the depth dose, one per treatment day, and then compared with TLD data; this comparison showed a reasonable agreement. Secondly, a single film was used to measure the dose delivered over three daily treatments and then compared with TLD data; this comparison showed good agreement throughout the depth dose, which includes doses well below 1 Gy. It will be shown that one piece of radiochromic film is sufficient to measure the composite percent depth dose for a TSE beam, hence making radiochromic film a suitable candidate for monitoring underdosed patient regions.

Comparison of monitor unit calculations performed with a 3D computerized planning system and independent "hand" calculations: results of three years clinical experience.

A comparison of the monitor unit calculations of a commercial 3D computerized treatment planning system (TPS) with "hand" calculations from lookup tables was made for a large number of clinical cases (greater than 13 500 treatment fields). Differences were analyzed by treatment site for prostate, rectum, cranium, and breast. The 3D TPS monitor unit calculation was systematically higher than the "hand" calculation by an amount that depended on the complexity of the treatment geometry. For simple geometries the mean difference was 1% and was as high as 3% for more complicated geometries. The higher value was attributed to an accumulation of differences introduced by multiple factors in the monitor unit calculation. Careful attention to factors such as patient contour could reduce the mean difference. "Hand" calculations were shown to be an accurate and useful tool for verification of TPS monitor unit calculations.

Evaluation of a ferrous benzoic xylenol orange transparent PVA cryogel radiochromic dosimeter.

A stable cryogel dosimeter was prepared using ferrous benzoic xylenol orange (FBX) in a transparent poly-(vinyl alcohol) (PVA) cryogel matrix. Dose response was evaluated for different numbers of freeze-thaw cycles (FTCs), different concentrations of PVA, and ratios of water/dimethyl sulfoxide. Linear relationships between dose and absorbance were obtained in the range of 0-1000 cGy for all formulations. Increasing the concentration of PVA and number of FTCs resulted in increased absorbance and sensitivity. The effects of energy and dose rate were also evaluated. No significant dose rate dependence was observed over the range 1.05 to 6.33 Gy min(-1). No energy response was observed over photon energies of 6, 10, and 18 MV.

Inexpensive diffuse reflectance spectroscopy system for measuring changes in tissue optical properties.

The measurement of changes in blood volume in tissue is important for monitoring the effects of a wide range of therapeutic interventions, from radiation therapy to skin-flap transplants. Many systems available for purchase are either expensive or difficult to use, limiting their utility in the clinical setting. A low-cost system, capable of measuring changes in tissue blood volume via diffuse reflectance spectroscopy is presented. The system consists of an integrating sphere coupled via optical fibers to a broadband light source and a spectrometer. Validation data are presented to illustrate the accuracy and reproducibility of the system. The validity and utility of this in vivo system were demonstrated in a skin blanching/reddening experiment using epinephrine and lidocaine, and in a study measuring the severity of radiation-induced erythema during radiation therapy.