Tanja Gabrecht

Photodynamic diagnosis of ovarian cancer using hexaminolaevulinate: a preclinical study

The unfailing detection of micrometastases during surgery of patients suffering from ovarian cancer is mandatory for the optimal management of this disease. Thus, the present study aimed at determining the feasibility of detecting micrometastases in an ovarian cancer model using the intraperitoneal administration of the photosensitiser precursor hexaminolaevulinate (HAL). For this purpose, HAL was applied intraperitoneally at different concentrations (4–12 mM) to immunocompetent Fischer 344 rats bearing a syngeneic epithelial ovarian carcinoma. The tumours were visualised laparoscopically using both white and blue light (D-light, Karl Storz, Tuttlingen, Germany), and the number of peritoneal micrometastases detected through HAL-induced photodiagnosis (PD) was compared to standard white light visualisation. Fluorescence spectra were recorded with an optical fibre-based spectrofluorometer and the fluorescence intensities were compared to the protoporphyrin IX (PpIX) fluorescence induced by 5-aminolevulinic acid under similar conditions. The number of metastases detected by the PD blue light mode was higher than when using standard white light abdominal inspection for all applied concentrations. Twice as many cancer lesions were detected by fluorescence than by white light inspection. The hexyl-ester derivative produced higher PpIX fluorescence than its parent substance aminolevulinic acid at the same concentration and application time. Fluorescence contrast between healthy and cancerous tissue was excellent for both compounds. To overcome poor diagnostic efficiency and to detect peritoneal ovarian carcinoma foci in the large surface area of the human peritoneal cavity, HAL fluorescence-based visualisation techniques may acquire importance in future and lead to a more correct staging of early ovarian cancer.

Comparison of ALA- and ALA hexyl-ester-induced PpIX depth distribution in human skin carcinoma.

Photodynamic therapy (PDT) based on the use of photoactivable porphyrins, such as protoporphyrin IX (PpIX), induced by the topical application of amino-levulinic acid (ALA) or its derivatives, ALA methyl-ester (m-ALA), is a treatment for superficial basal cell carcinoma (BCC), with complete response rates of over 80%. However, in the case of deep, nodular-ulcerative lesions, the complete response rates are lower, possibly related to a lower bioavailability of PpIX. Previous in vitro skin permeation studies demonstrated an increased penetration of amino-levulinic acid hexyl-ester (h-ALA) over ALA. In this study, we tested the validity of this approach in vivo on human BCCs. An emulsion containing 20% ALA (w/w) and preparations of h-ALA at different concentrations were applied topically to the normal skin of Caucasian volunteers to compare the PpIX fluorescence intensities with an optical fiber-based spectrofluorometer. In addition, the PpIX depth distribution and fluorescence intensity in 26 BCCs were investigated by fluorescence microscopy following topical application of 20% ALA and 1% h-ALA. We found that, for application times up to 24h, h-ALA is identical to ALA as a PpIX precursor with respect to PpIX fluorescence intensity, depth of penetration, and distribution in basal cell carcinoma, but has the added advantage that much smaller h-ALA concentrations can be used (up to a factor 13). We observed a non-homogenous distribution in BCCs with both precursors, independent of the histological type and depth of invasion in the dermis.

In vivo time-resolved spectroscopy of the human bronchial early cancer autofluorescence

Time-resolved measurements of tissue autofluorescence (AF) excited at 405 nm were carried out with an optical-fiber-based spectrometer in the bronchi of 11 patients. The objectives consisted of assessing the lifetime as a new tumor/normal (T/N) tissue contrast parameter and trying to explain the origin of the contrasts observed when using AF-based cancer detection imaging systems. No significant change in the AF lifetimes was found. AF bronchoscopy performed in parallel with an imaging device revealed both intensity and spectral contrasts. Our results suggest that the spectral contrast might be due to an enhanced blood concentration just below the epithelial layers of the lesion. The intensity contrast probably results from the thickening of the epithelium in the lesions. The absence of T/N lifetime contrast indicates that the quenching is not at the origin of the fluorescence intensity and spectral contrasts. These lifetimes (6.9 ns, 2.0 ns, and 0.2 ns) were consistent for all the examined sites. The fact that these lifetimes are the same for different emission domains ranging between 430 and 680 nm indicates that there is probably only one dominant fluorophore involved. The measured lifetimes suggest that this fluorophore is elastin.

Design of a light delivery system for the photodynamic treatment of the Crohn's disease

Crohns disease is an inflammatory bowel disease originating from an overwhelming response of the mucosal immune system. Low dose photodynamic therapy (PDT) may modify the mucosal immune response and thus serve as a therapy for Crohns disease. Most patients with Crohns disease show inflammatory reactions in the terminal ileum or colon where PDT treatment is feasible by low-invasive endoscopic techniques. However, the tube like geometry of the colon, its folding, and the presences of multiple foci of Crohns lesions along the colon require the development of adequate light delivery techniques. We present a prototype light delivery system for endoscopic clinical PDT in patients with Crohns disease. The system is based on a cylindrical light diffuser inserted into a diffusing balloon catheter. Homogenous irradiation is performed with a 4 W diode laser at 635 nm. Light dosimetry is performed using a calibrated integrating sphere. The system can be used with conventional colonoscopes and colonovideoscopes having a 3.8 mm diameter working channel. The feasibility of PDT in colon with our prototype was demonstrated in first clinical trials.

Blue-violet excited autofluorescence spectroscopy and imaging of normal and cancerous human bronchial tissue after formalin fixation

Autofluorescence (AF) imaging is a powerful tool for the detection of (pre‐)neoplastic lesions in the bronchi. Several endoscopic imaging systems exploit the spectral and intensity contrast of AF between healthy and (pre‐)neoplastic bronchial tissues, yet, the mechanisms underlying these contrasts are poorly understood. In this report, the effect of formalin fixation on the human bronchi AF, hence on the contrast, was studied by spectrofluorometric point measurements and DAFE (Diagnostic AutoFluorescence Endoscopy) broad field imaging. Generally, formalin‐fixed samples have higher AF intensity than in vivo, whereas the emission spectral shape is similar. Additionally, the spectrofluorometric data showed a moderate decrease of the AF intensity on (pre‐)neoplastic lesions relative to the healthy bronchial samples. However, this decrease was lower than that reported from in vivo measurements. Neither spectral measurements nor imaging revealed spectral contrast between healthy bronchial tissue and (pre‐)neoplastic lesions in formalin. These results indicate that epithelial thickening and blood supply in the adjacent lamina propria are likely to play a key role in the generation of the AF contrast in bronchial tissues. Our results show that the AF contrast in bronchial tissues was significantly affected by standard, 10% buffered, formalin fixation. Therefore, these samples are not suited to AF contrast studies.

Optimized autofluorescence bronchoscopy using additional backscattered red light

Autofluorescence bronchoscopy (AFB) has been shown to be a highly sensitive tool for the detection of early endobronchial cancers. When excited with blue-violet light, early neoplasia in the bronchi tend to show a decrease of autofluorescence in the green region of the spectrum and a relatively smaller decrease in the red region of the spectrum. Superposing the green foreground image and the red background image creates the resultant autofluorescence image. Our aim was to investigate whether the addition of backscattered red light to the tissue autofluorescence signal could improve the contrast between healthy and diseased tissue. We have performed a clinical study involving 41 lung cancers using modified autofluorescence bronchoscopy systems. The lesions were examined sequentially with conventional violet autofluorescence excitation (430 nm+/-30 nm) and violet autofluorescence excitation plus backscattered red light (430 nm+/-40 nm plus 665 nm+/-15 nm). The contrast between (pre-)neoplastic and healthy tissue was quantified with off-line image analysis. We observed a 2.7 times higher contrast when backscattered red light was added to the violet excitation. In addition, the image quality was improved in terms of the signal-to-noise ratio (SNR) with this spectral design.

Improvement of the specificity of cancer detection by autofluorescence imaging in the tracheo-bronchial tree using backscattered violet light.

BACKGROUND Autofluorescence bronchoscopy (AFB) is a highly sensitive tool for the detection of early bronchial cancers. However, its specificity remains limited due to primarily false positive results induced by hyperplasia, metaplasia and inflammation. We have investigated the potential of blue-violet backscattered light to eliminate false positive results during AFB in a clinical pilot study. METHODS The diagnostic autofluorescence endoscopy (DAFE) system was equipped with a variable band pass filter in the imaging detection path. The backscattering properties of normal and abnormal bronchial mucosae were assessed by computing the contrast between the two tissue types for blue-violet wavelengths ranging between 410 and 490 nm in 12 patients undergoing routine DAFE examination. In a second study including 6 patients we used a variable long pass (LP) filter to determine the spectral design of the emission filter dedicated to the detection of this blue-violet light with the DAFE system. RESULTS (Pre-)neoplastic mucosa showed a clear wavelength dependence of the backscattering properties of blue-violet light while the reflectivity of normal, metaplastic and hyperplastic autofluorescence positive mucosa was wavelength independent. CONCLUSIONS Our results showed that the detection of blue-violet light has the potential to reduce the number of false positive results in AFB. In addition we determined the spectral design of the emission filter dedicated to the detection of this blue-violet light with the DAFE system.

Time-resolved autofluorescence spectroscopy of the bronchial mucosa for the detection of early cancer: Clinical results

Time-resolved measurements of endogenous tissue autofluorescence were carried out on the bronchial mucosa of 18 patients during endoscopy by the means of a optical fibre-based spectrometer. The objective was to assess the fluorescence lifetime as a new contrast parameter between normal and malignant tissue and to explain the origin of a previously observed contrast in fluorescence intensity. The intra- and interpatient variation of tissue autofluorescence intensity and decay on normal tissue was determined with the outcome that a strong fluctuation in autofluorescence intensity but not in lifetime was observed on the normal tissue. Preliminary results were obtained by comparing fluorescence decays on normal mucosa and dysplasia/carcinoma in situ. No significant change in fluorescence decay nor in spectrum between 510 and 650 nm was found. Measurements in parallel with an endoscopic autofluorescence imaging device, on the other hand, indicated a contrast in intensity and spectrum on the same lesions. This suggests that the spectral contrast might be due to an enhanced blood concentration in deeper lying layers of the lesion the optical fibre-based contact measurements are less sensitive to. The difference in intensity might be due to a lower concentration in fluorophores or to the thickening of the epithelium in the neoplastic mucous membrane. However, no indication for fluorescence quenching in the upper layers of the mucous membrane as the reason for the reduced fluorescence intensity was found. The fluorescence decays showed a quite stable behaviour with three decay times of 6.9 ns, 2.0 ns and 0.2 ns in the spectral range between 430 and 680 nm. This can be an indication that there is one dominant fluorophore involved, the calculated decay times suggest that it might be elastin. However, a slight spectral dependence of the fluorescence decays let presume that there is a contribution from other fluorophores, probably flavins and NADH.

Autofluorescence bronchoscopy: quantification of inter-patient variations of fluorescence intensity.

Autofluorescence (AF) from bronchial tissue is increasingly used for the endoscopic detection of early bronchial neoplasia. Several imaging systems are commercially available, all detecting the absolute or relative AF intensity and/or spectral contrasts between normal tissue and early neoplastic lesions. These devices have a high sensitivity for flat neoplasia, but the specificity remains limited. Variations in the AF intensity between individuals (inter-patient variations) is considered one of the most limiting factors. In the clinical study presented here, we quantified those variations using a non-invasive optical reference positioned in situ during AF bronchoscopy. The inter-patient variations in intensity on the main carina were in the order of 25– 30%. The results of this study are quite useful for improving and defining the design of the optical features (dynamic range, physical sensitivity) of AF detection systems.

Design of an endoscopic optical reference to be used for autofluorescence bronchoscopy with a commercially available diagnostic autofluorescence endoscopy (DAFE) system

We present the design of a sterilizable optical reference to characterize and quantify the inter-patient variations in tissue autofluorescence during autofluorescence bronchoscopy with Richard Wolfs diagnostic autofluorescence endoscopy (DAFE) system. The reference was designed to have optical and spectral properties similar to those of the human bronchial wall in spectral conditions corresponding to autofluorescence bronchoscopy conducted with the DAFE system (fluorescence excitation at 390-470 nm and red backscattering light at 590-680 nm). The references effective attenuation coefficient and reflectance were measured at 675 nm. In addition, its fluorescence emission spectrum was determined under 430 nm wavelength excitation. The reference is photostable, reproducible, biocompatible and small enough to be easily inserted through the working channel of a conventional bronchofibrescope. This cylindrical (length: 2 mm; diameter: 2 mm) optical reference was validated in a clinical environment.