[Confocal endomicroscopy of the lung: from the bronchus to the alveolus]. / Endomicroscopie confocale en pneumologie : de la bronche à l'alvéole.

INTRODUCTION: Fibred confocal fluorescence microscopy, also named probe based confocal laser endomicroscopy (pCLE), is a new endoscopic technique that can be applied for in-vivo microscopic imaging of both upper airways and distal lung structures during bronchoscopy. BACKGROUND: Two recent in-vivo human studies using pCLE at 488nm light excitation have described the normal fluorescence endomicroscopic features of the bronchial wall and the elastic framework of the alveolus. These studies have demonstrated that elastin, a major component of the bronchial basement membrane and of the acinar elastic framework, is the main endogenous fluorophore in the non-smoking population. In smoking subjects, the tobacco tar itself is highly fluorescent and, thereby, acts as an additional fluorophore, allowing study of the macrophage alveolitis associated with smoking. These studies have also confirmed the safety of this endoscopic procedure. VIEWPOINT: In the near future, confocal endomicroscopy of the airways should make it possible to investigate the semiology of focal and diffuse distal lung diseases, to characterize cancerous and precancerous lesions of both upper and distal airways and to study the lung microcirculation. These studies may also use exogenous molecular fluorescent probes, which will enable functional imaging of the lung structures in-vivo. CONCLUSION: Confocal endomicroscopy has the potential to explore accurately the peripheral lung in-vivo and may become a useful tool to improve endoscopic diagnosis of many lung diseases.

Human in vivo fluorescence microimaging of the alveolar ducts and sacs during bronchoscopy.

The aim of the present study was to assess fibred confocal fluorescence microscopy (FCFM) as a tool for imaging the alveolar respiratory system in vivo during bronchoscopy. A 488-nm excitation wavelength FCFM device was used in 41 healthy subjects including 17 active smokers. After topical anaesthesia, the 1.4-mm miniprobe was introduced into the bronchoscope working channel and advanced distally to the alveoli. Morphometric and cellular analyses were performed on selected frames harbouring a minimal compression effect. In vivo acinar microimaging was obtained from each lung segment except for the apical and posterior segments of both upper lobes. Reproducible patterns, corresponding to the elastic framework of the axial and peripheral interstitial systems, were recorded from 192 separate acini. The mean+/-sd thickness of the acinar elastic fibres was 10+/-2.7 microm. Alveolar mouth diameters (mean+/-sd 278+/-53 microm) were normally distributed but appeared smaller in the right upper lobe and right medial basal segment. Lobular microvessels (median diameter 90 microm) were equally distributed throughout the lungs. Alveolar macrophages were not detectable in nonsmokers, whereas a specific tobacco-tar-induced fluorescence was observed in smoking subjects, providing fine details of the alveolar walls and macrophages. A strong correlation was found between the number of cigarettes smoked per day and the amount of large and mobile macrophages observed in vivo, as well as with the intensity of the macrophage alveolitis. Fibred confocal fluorescence microscopy enables accurate exploration of the peripheral lung in vivo in both smokers and nonsmokers.

Endoscopic ultraviolet-induced autofluorescence spectroscopy of the esophagus: tissue characterization and potential for early cancer diagnosis.

BACKGROUND AND STUDY AIMS: Endoscopic identification of dysplasia and early carcinoma of the esophagus is difficult and is currently done through random pinch biopsies. This study assesses the potential of ultraviolet-induced autofluorescence spectroscopy for early diagnosis with special focus on Barrett's esophagus. PATIENTS AND METHODS: Measurements were performed on 24 patients using 330 nm light excitation. The determination of the spectral distribution typical of each histological tissue type was done using three fluorescence intensity ratios: RI = I390nm/I450nm; R2 = I550nm/I450nm; R3 - I390nm/I550nm. RESULTS: The spectral distribution of normal esophageal mucosa and specialized columnar Barrett's mucosa were similar. A strong modification of the spectral distribution was observed for high grade dysplasia and intramucosal carcinoma. Statistical analysis indicated that the spectral shape modification associated with neoplastic transformation was greater than intra- and interpatient spectral variations. These results allow the determination of discriminating criteria based on ratios R1 and R3. Using ratio R3, the spectroscopy-based diagnosis differentiated neoplastic tissue from normal esophageal mucosa and specialized columnar Barrett's mucosa with a sensitivity and specificity of 86% and 95 %, respectively. CONCLUSIONS: The use of ultraviolet autofluorescence spectroscopy should improve the diagnostic yield of standard endoscopy in patients with Barrett's esophagus.

[Local curative treatment of superficial adenocarcinoma in Barrett's esophagus. First results of photodynamic therapy with a new photosensitizer]. / Traitement curatif local des adénocarcinomes superficiels sur endobrachyoesophage. Premiers résultats de thérapie photodynamique avec un nouveau photosensibilisateur.

Pre-cancerous lesions and mucosally confined superficial cancers can benefit from local therapy given with curative intent due to the absence of near metastatic lymph nodes. Photodynamic therapy (PDT) which acts by laser irradiation with an appropriate wave-length after administration of a photosensitiser retained preferentially by the cancerous tissue can destroy tumour cells selectively, but its efficiency depends upon the photosensitiser. The results presented concern 10 sites on Barrett's mucosa (BO). They consisted of either an association of intramucosal cancer (IMC) with high-grade dysplasia (HGD) or of high-grade dysplasia alone. The method consisted of intravenous injection of Temoporfin 0,15 mg/kg 4 days before irradiation of the lesion with a green laser light emitting 514 nm through a windowed diffuser. The light fluence was 75 J per cm2 and irradiation 100 mW per cm2. Irradiation time was 12,5 mn. Omeprazole was routinely prescribed after treatment at a dose of 40 mg daily. The follow-up protocol was 2 years with endoscopic surveillance at 2, 3, 6, 12, 18 and 24 months. Biopsies obligatory at 2 and 3 months were in fact carried out at all the other delays. Efficacy was judged on the absence of high-grade dysplasia or intra mucosal carcinoma on biopsies at treated sites. Undesirable side effects noted have been moderate for the most part. No stenosis appeared. Treatment has been 100% successful for the 10 lesion after 15 treatments with PDT. The follow up varies from 6-36 months and was more than 18 months for 6 lesions on 5 patients. Our series has demonstrated a great heterogeneity in lesions which were sometimes visible and highly localised, but more often invisible, multi-focal and diagnosable only by biopsy at different levels. In keeping with the literature and our experience, PDT has several advantages over the other locally curative therapies, mucosectomy and thermocoagulation. These are the possible treatments without general anaesthesia, selectively for cancer cells, an action on more extensive areas with eradication of non visible lesions. Temoporfin has contributed notably to the field of photodynamic therapy compared to previously used sensitisers. It is a pure, synthetic product which guarantees more reproducible results. Compared with Photofrin, Temoporfin has many advantages with smaller doses of drugs and less energy, better selectivity and rapid elimination which reduce the risk period for photosensitisation. The frequency of important undesirable side effects is diminished. Finally, it produces a consistent effect on the surface and in depth producing a complete reepithelialisation of the treated zones. Subject to validation of the method on a greater number of patients, the first results obtained on superficial cancer in Barrett's aesophagus allow us to propose green light Temoporfin PDT as an alternative first line therapy with curative intent.

Fluorescence and mass spectrometry studies of meta-tetra(hydroxyphenyl)chlorin photoproducts.

The meta-tetra(hydroxyphenyl)chlorin (m-THPC), a second-generation sensitizer used in photodynamic therapy (PDT), is currently under clinical trial. In vivo fluorometry provides direct evidence that photobleaching processes are induced at the tumor site during PDT. Photoproduct formation has thus to be taken into account to fully understand PDT treatment. A preliminary step is to determine the fluorescence characteristics of photoproducts formed in solution. Solutions of m-THPC irradiated at 514 nm have been separated by HPLC using absorption and fluorescence detection. Six main photoproducts have been isolated. According to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) results, five fluorescent photoproducts emitting at 652 nm have been attributed to three mono-, one di- and one tri-hydroxy derivatives (m/z 697, 713 and 729, respectively). Fluorescence characteristics of mono-hydroxy forms were found to be similar to those of m-THPC, whereas fluorescence yields in di- and tri-hydroxy derivatives were very low. Another product, corresponding to a MALDI-TOF MS main signal at m/z 542, showed an absorption spectrum maximum at 522 nm while a weak fluorescence was detected at 480 nm. The loss of the Soret band suggests that this photoproduct results from the opening of the reduced pyrrole ring. The part played by each of these products in the photobleaching phenomenon of m-THPC is discussed.