The role of endobronchial ultrasound-guided transbronchial needle aspiration in the diagnosis of mediastinal lymphadenopathy

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a novel procedure for the diagnosis of mediastinal lymphadenopathy. Its utility in clinical practice for the diagnosis of patients presenting with mediastinal lymphadenopathy is unknown. This thesis describes the learning curve associated with EBUS-TBNA using cumulative sum analysis and then the diagnostic yield of EBUS-TBNA in different clinical scenarios. EBUS-TBNA was combined with standard bronchoscopy in patients with suspected sarcoidosis in a prospective trial. The role of EBUS-TBNA in patients with tuberculous lymphadenopathy and also patients with extra-thoracic malignancy was then clarified in multi-centre studies. A further prospective trial (REMEDY) aimed to ascertain whether mediastinoscopies could be prevented in patients presenting with isolated mediastinal lymphadenopathy. The utility of the specimens from EBUS-TBNA for sub-typing and genotyping of non-small cell lung cancer are also described in a multi-centre study. Finally, the results from a major multi-centre randomised controlled trial (Lung-BOOST) are presented, investigating whether EBUS-TBNA should be implemented as a first test in patients with suspected lung cancer. The data included in this thesis demonstrate that EBUS-TBNA has high diagnostic yield in patients with sarcoidosis, tuberculosis and extra-thoracic malignancy. For the first time, the REMEDY trial demonstrates that EBUS-TBNA can prevent 87% of mediastinoscopies in patients with isolated mediastinal lymphadenopathy. In patients with lung cancer, specimens from EBUS-TBNA are suitable for sub-typing and genotyping of NSCLC and results from the randomised Lung-BOOST trial demonstrate that when EBUS-TBNA is used as an initial investigation in patients with suspected lung cancer the time to treatment decision is significantly reduced

Multimodal Multispectral Optical Endoscopic Imaging for Biomedical Applications

Optical imaging is an emerging field of clinical diagnostics that can address the growing medical need for early cancer detection and diagnosis. Various human cancers are amenable to better prognosis and patient survival if found and treated during early disease onset. Besides providing wide-field, macroscopic diagnostic information similar to existing clinical imaging techniques, optical imaging modalities have the added advantage of microscopic, high resolution cellular-level imaging from in vivo tissues in real time. This comprehensive imaging approach to cancer detection and the possibility of performing an ‘optical biopsy’ without tissue removal has led to growing interest in the field with numerous techniques under investigation. Three optical techniques are discussed in this thesis, namely multispectral fluorescence imaging (MFI), hyperspectral reflectance imaging (HRI) and fluorescence confocal endomicroscopy (FCE). MFI and HRI are novel endoscopic imaging-based extensions of single point detection techniques, such as laser induced fluorescence spectroscopy and diffuse reflectance spectroscopy. This results in the acquisition of spectral data in an intuitive imaging format that allows for quantitative evaluation of tissue disease states. We demonstrate MFI and HRI on fluorophores, tissue phantoms and ex vivo tissues and present the results as an RGB colour image for more intuitive assessment. This follows dimensionality reduction of the acquired spectral data with a fixed-reference isomap diagnostic algorithm to extract only the most meaningful data parameters. FCE is a probe-based point imaging technique offering confocal detection in vivo with almost histology-grade images. We perform FCE imaging on chemotherapy-treated in vitro human ovarian cancer cells, ex vivo human cancer tissues and photosensitiser-treated in vivo murine tumours to show the enhanced detection capabilities of the technique. Finally, the three modalities are applied in combination to demonstrate an optical viewfinder approach as a possible minimally-invasive imaging method for early cancer detection and diagnosis

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