Branko Palcic

Detection and localization of early lung cancer by fluorescence bronchoscopy

Curative therapy is available for patients with Stage 0 lung carcinoma, with a >90% 5‐year survival rate. Promising chemopreventive agents also are under investigation currently to reduce the risk of lung carcinoma in high risk populations. However, preinvasive bronchial lesions (moderate to severe dysplasia and carcinoma in situ) are very small and thin. They are difficult to localize by conventional white‐light bronchoscopy. Fluorescence bronchoscopy is a new diagnostic tool for the detection of these preinvasive lesions.

Laser-induced fluorescence spectroscopy at endoscopy: tissue optics, Monte Carlo modeling, and in vivo measurements

The optical properties (absorption coefficient, scattering coefficient and the anisotropic factor of scattering) and fluorescence characteristics of normal and abnormal bronchial tissue were measured in vitro. After adding additional blood optical properties to in vitro optical properties of tissue, the in vivo bronchial fluorescence was simulated and analyzed by Monte Carlo modeling. The Monte Carlo simulation results showed that with an appropriate illumination and fluorescence collection geometry, the distortion of in vivo fluorescence spectra of tissue caused by variations of optical properties at different wavelengths could be much reduced. Based on these results, a spectrofluorometry system was developed for the collection of in vivo laser-induced fluorescence spectra of tissue during endoscopy. In comparing the in vivo fluorescence spectral shape of bronchial tissue collected by this system with the intrinsic one obtained in vitro, we found no obvious distortion in the in vivo spectra. This was completely consistent with the analysis of Monte Carlo modeling. The in vivo measurement results demonstrated that significant differences in fluorescence intensity between normal and diseased bronchial tissue (dysplasia, carcinoma in situ) can be used to differentiate them from each other. Also, changes in fluorescence intensity are more robust for detecting abnormal tissues than the differences in spectral characteristics.

SPECTROSCOPIC AND MICROSCOPIC CHARACTERISTICS OF HUMAN SKIN AUTOFLUORESCENCE EMISSION

To improve the understanding of human skin autofluorescence emission, the spectroscopic and microscopic characteristics of skin autofluorescence were studied using a combined fluorescence and reflectance spectroanalyzer and a fiber optic microspectrophotometer. The autofluorescence spectra of in vivo human skin were measured over a wide excitation wavelength range (350–470 nm). The excitation–emission matrices of in vivo skin were obtained. An excitation–emission maximum pair (380 nm, 470 nm) was identified. It was revealed that the most probable energy of skin autofluorescence emission photons increases monotonically and near linearly with increasing excitation photon energy. It was demonstrated that the diffuse reflectance, R, can be used as a first order approximation of the fluorescence distortion factor f to correct the measured in vivo autofluorescence spectra for the effect of tissue reabsorption and scattering. The microscopic in vitro autofluorescence properties of excised skin tissue sections were examined using 442 nm He–Cd laser light excitation as an example. It was demonstrated that the fluorophore distribution inside the skin tissue is not uniform and the shapes of the autofluorescence spectra of different anatomical skin layers vary. The result of this study confirms that the major skin fluorophores are located in the dermis and provides an excellent foundation for Monte Carlo modeling of in vivo autofluorescence measurements.

Optical properties of normal and carcinomatous bronchial tissue

To understand better the optical characteristics and autofluorescence properties of normal and carcinomatous bronchial tissue, we measured the absorption coefficient, scattering coefficient, and anisotropy factor from 400 to 700 nm. We made the measurements by using an integrating sphere with a collimated white-light beam to measure total reflectance and transmittance of samples. The unscattered transmittance of the samples was measured through polarized on-axis light detection. The inverse adding-doubling solution was utilized to solve the equation of radiative transfer and to determine the absorption coefficient and reduced scattering coefficient. The scattering coefficient and anisotropy factor were derived from the unscattered transmittance of the sample and the reduced scattering coefficient. The measured parameters allow us to simulate photon propagation in normal bronchial and tumoral tissue by using Monte Carlo modeling.

Diagnostic imaging of the larynx: autofluorescence of laryngeal tumours using the helium-cadmium laser.

The use of tissue autofluorescence for the detection and localization of cancer of the larynx is described. In this pilot study, eight patients with probable carcinoma of the vocal folds underwent laryngoscopy in which the tissue autofluorescence spectra of normal and pathologically confirmed tumour tissue were acquired in vivo. Fluorescence images of the suspect areas were also acquired using the LIFE system (Xillix Technologies Corp.). The results suggest that the autofluorescence properties of laryngeal tissue, under 442 nm illumination, are similar to those of bronchial tissue and that the LIFE system has the potential to increase the accuracy of staging of cancer of the larynx and also to allow earlier diagnosis of tumours and their recurrence.

Early localization of bronchogenic carcinoma.

The performance of a fluorescence imaging device was compared with conventional white-light bronchoscopy in 100 patients with lung cancer, 46 patients with resected stage I non-small cell lung cancer, 10 patients with head and neck cancer, and 67 volunteers who had smoked at least 1 pack of cigarettes per day for 25 years or more. Using differences in tissue autofluorescence between premalignant, malignant, and normal tissues, fluorescence bronchoscopy was found to detect significantly more areas with moderate/severe dysplasia or carcinoma in situ than conventional white-light bronchoscopy with a similar specificity. Multiple foci of dysplasia or cancer were found in 13–24% of these individuals. Fluorescence bronchoscopy may be an important adjunct to conventional bronchoscopic examination to improve our ability to detect and localize premalignant and early lung cancer lesions.

Reconstruction of in vivo skin autofluorescence spectrum from microscopic properties by Monte Carlo simulation

The in vivo skin autofluorescence spectrum was reconstructed by Monte Carlo simulation using microscopic fluorophore distributions and intrinsic fluorescence spectra measured from excised skin tissue sections as well as employing published skin tissue optical parameters. The theoretical modeling took into account the light-tissue interactions of scattering, absorption, and regeneration of fluorescence photons. The modification of the intrinsic spectra by tissue optical properties to generate the in vivo spectrum observed at the tissue surface can be represented by a fluorescence detection efficiency function (eta) which equals the integral of the product of the excitation light distribution inside the tissue and the fluorescence escape efficiency. Comparison of the reconstructed in vivo spectrum with the measured spectra showed good agreement, outside of the blood absorption bands, suggesting that (i) the theoretical modeling, (ii) the skin optical parameters used, and (iii) the measured microscopic morphology and spectral data are consistent. The divergence which exists over the strong blood absorption wavelength band (530-600 nm) suggests that the effect of blood contents on in vivo tissue optical properties deserves further investigations.

Image Feature Extraction in the Last Screening Mammograms Prior to Detection of Breast Cancer

Image feature extraction was utilized to retrospectively analyze screening mammograms taken prior to the detection of a malignant mass for early detection of breast cancer. The mammograms of 58 biopsy proven breast cancer patients were collected. In each case, the mammograms taken 10 to 18 months prior to cancer detection were evaluated. For each of the two mammographic projections of the abnormal breast, two regions were marked: 1) region one, which corresponded to the site where the malignant mass subsequently developed and 2) a region which appeared similar to region one on the same mammogram. On each projection of the normal breast a third region which corresponds to region one but on the opposite breast was also marked (mirror-image site). Sixty-two texture and photometric image features were then calculated for all of the marked areas. A stepwise discriminant analysis showed that six of these features could be used to best distinguish between the normal and abnormal regions. The best linear classification function resulted in a 72% average classification. At its current stage, the system can be used by a radiologist to examine any pattern in a mammogram. The regions which are flagged by the system have a 72% chance of developing a malignant mass by the time of the next screening. Therefore, further evaluation of these patients (e.g., a screening examination sooner than the normal one year interval) could result in earlier detection of breast cancer. The ultimate goal is to run the system automatically over the whole mammogram and flag any suspicious area.

Detection of squamous cell cancer and pre-cancerous lesions by imaging of tissue autofluorescence in the hamster cheek pouch model

Early detection of invasive and pre-invasive neoplasms of the aerodigestive tract will ultimately improve the management of patients with these lesions. This paper describes the use of quantitative fluorescence imaging of early squamous cell carcinomas in an animal model. Dysplasia, carcinoma in situ and invasive cancers were imaged exploiting tumour autofluorescence. Mapped biopsies were obtained from areas imaged determining a sensitivity of 100% and specificity of 80%. Autofluorescence imaging is an excellent method of detecting neoplasms of the aerodigestive tract.

Malignancy associated changes in bronchial epithelial cells and clinical application as a biomarker

A total of 74 bronchial brushing specimens, 24 from patients with advanced stage cancer, eight from patients with CIS, 31 from patients with atypical metaplasia and 11 from normal subjects were examined for the existence of malignancy associated changes (MAC). Conventional fiberoptic bronchoscopy and fluorescence endoscopy was carried out on every case. Each case was classified according to the highest grade of abnormality diagnosed by bronchial biopsy of the suspect areas. During the endoscopy examination, a bronchial brushing specimen was obtained from a visually normal area very remote from the abnormal area as possible such as the opposite lung or another lobe. The bronchial brushing specimens were fixed, mounted and stained by a DNA specific method and approximately 1500 images of individual nuclei per case were captured by an automated high resolution image cytometry. For each of these images, more than 100 nuclear features such as size, shape and chromatin spatial organization were calculated. Discriminant function analysis revealed nuclear features which differentiated between normal bronchial cell nuclei from the normal subjects and ostensively normal nuclei (MAC cell nuclei) from the lung cancer patients. The best discrimination was achieved when the frequency of individual cells expressing MAC was 50% or greater. With this threshold, 75% of the patients with invasive cancer and CIS were correctly classified. Fifty percent of those with severe or moderate atypia and 35% with mild atypia were also MAC positive. The frequency of cells expressing MAC also increased as the degree of abnormality of the groups increased. MAC may be a useful criterium to determine biological behavior of the intra-epithelial (pre-invasive) neoplasia.