Clinical fluorescence spectroscopy and imaging for the detection of early carcinoma by autofluorescence bronchoscopy and the study of the protoporphyrin IX pharmacokinetics in the endometrium

The aim of this thesis is to optimise and gain fundamental information on two applications of photomedicine using fluorescence imaging and spectrofluorometry: (1) the detection of early bronchial cancer by autofluorescence imaging and (2) the endometrial ablation by photodynamic therapy (PDT) based on the use of Protoporphyrin IX (PpIX). Fluorescence imaging and spectroscopy require a fluorochrome localised within the tissue. The fluorochrome can either be endogenous (naturally synthesised in the body), endogenously induced (synthesised in the body from an administered drug), or exogenous (synthesised outside the body). This thesis concentrates on the clinical applications of the endogenous and an endogenously induced fluorochrome (PpIX). Therefore, this work has been divided into two parts according to the type of fluorochromes. The numerous endogenous fluorochromes occur naturally. They are collectively responsible for the fluorescence properties of biological tissues. This tissue's intrinsic fluorescence is also referred to as autofluorescence (AF). The AF of bronchial tissues, change when they become dysplastic or neoplastic. Early neoplastic or dysplastic lesions show an overall decrease in the AF intensity as well as a distorsion of the spectral shape. Endoscopic imaging devices rely on this principle to detect early neoplastic lesions in the tracheo-bronchial tree. The first part of this thesis describes our efforts to improve the performance of AFB and to gather insight into the mechanisms at the origin of the AF contrast in the bronchi. For this purpose, we conducted a number of clinical and ex vivo studies using imaging and spectrofluorometry. Our initial clinical imaging study revealed that the detection of a red background image instead of the red AF image increased the lesion-to-healthy tissue contrast by a factor of 2. This improvement has been implemented in an AFB device that is currently commercialised by the Richard Wolf Endoskope GmbH. In a seperate clinical imaging study we investigated the influence of the excitation wavelength on the AF contrast. Using a narrowband (6 nm FWHM) excitation around 410 nm resulted in a 1.5 times higher lesion-to-healthy tissue intensity contrast than observed with a comparable broadband (80 nm FWHM) excitation. A supplemental study showed that short wavelength blue backscattered light around 430 nm has the potential to discriminate true positive lesions (i.e. early neoplastic lesions detected positive with the AFB system) from false positive lesions (i.e. benign tissue changes detected positive with the AFB system). A spectrofluorometric ex vivo study was performed to gain insight on the mechanisms at the origin of these contrasts. Five principal mechanisms are discussed, namely changes of: (1) the fluorochrome's concentration, (2) the fluorochrome's metabolic status, (3) the fluorochrome's physico-chemical microenvironment, (4) the tissue architecture such as thickening of the epithelium, and (5) the concentration of light absorbing chromophores such as haemoglobin. We measured formalin fixed human bronchial tissue samples with an optical fibre based spectrofluorometer. The formalin fixed bronchial tissue samples showed a general decrease of the AF of early lesions compared to the healthy tissues. However, no distortion of the lesions' AF spectra with respect to that of the healthy tissues was observed. These results were confirmed by imaging of the tissue samples with our AFB system. The observations from these ex vivo studies together with results obtained in clinics with our imaging system lead us to conclude that the AF contrast can be attributed to a combined effect induced by: (1) changes in the architecture of superficial tissues and (2) the concentration and spatial distribution of haemoglobin in the submucosa. Furthermore, we investigated inter-patient variations of the bronchial AF to estimate their impact on the spectral/photonic design of AFB systems. An endoscopic reference with tissue-like optical and spectral properties was designed for this purpose. Surprisingly, the AF intensities in spectroscopy of the human bronchi showed only minor (< 30 %) variations from one individual to another. The exogenously induced fluorochrome Protoporphyrin IX (PpIX) is synthesised from 5-aminolaevulinic acid (5-ALA) in the haeme biosynthetic pathway. PpIX is widely used in PDT and fluoresence detection for both malignant and benign, lesions. The second part of this thesis deals with the pharmacokinetics of 5-ALA induced PpIX in the endometrium. The final goal of this study was the optimisation of the treatment protocol for photodynamic endometrial ablation to treat menorrhagia and hypermenorrhea. The PpIX build-up in the human endometrium was measured in vivo by spectrofluorometry following intra-uterine instillation of 5-ALA. An intra-uterine optical-fibre based probe was designed for this purpose. The PpIX pharmacokinetics showed important inter-patient and intra-patient variations regarding the time interval between the drug instillation and the maximal PpIX fluorescence. Indeed, we have found that this time interval ranges between 0.5 and 5 hours. The maximal measured PpIX fluorescence intensities varied by one order of magnitude from one patient to another. Finally, no correlation was found between the characteristics of the PpIX build-up and the patient's hormonal status

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 system

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

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

Influence of the menstrual cycle on aminolevulinic acid induced protoporphyrin IX fluorescence in the endometrium: in vivo study

BACKGROUND AND OBJECTIVES: In vitro studies indicated that compared to postmenopausal women, premenopausal women had increased aminolevulinic acid induced protoporphyrin IX (ALA-induced PpIX) fluorescence expression in the endometrium. The aim of this study was to evaluate menstrual cycle dependency of ALA-induced PpIX fluorescence in the endometrium in vivo. STUDY DESIGN/PATIENTS AND METHODS: Thirteen patients were included for in vivo spectrofluorometric measurements of ALA-induced PpIX in the endometrium and 51 patients for fluorescence hysteroscopy. Two milliliter of a 2% 5-ALA-solution at pH = 4.0 (ASAT AG/Zug, Switzerland) was topically administrated just before spectrofluorometry and 4 hours before hysteroscopy. Spectrofluorometry: Optical fiber based. Fluorescence hysteroscopy: STORZ-D-Light system (Storz, Tuttlingen, Germany). Histological classification of curettage and bioptic endometrial tissue stained with hematoxylin and eosin (H&E). RESULTS: Hysteroscopic and in vivo spectrofluorometric measurements showed an increase of ALA-induced PpIX fluorescence in the secretory and hyperplastic endometrium compared to proliferative and atrophic endometrium. CONCLUSIONS: The accuracy of fluorescence hysteroscopy and the success of the photodynamic endometrial ablation using ALA-induced PpIX may depend on the hormonal influence of the menstrual cycle. The mechanisms responsible for the increased ALA-induced PpIX fluorescence in the secretory versus proliferative phase of the menstrual cycle deserve further 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

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

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

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