Alexandre Radu

In vivo autofluorescence imaging of early cancers in the human tracheobronchial tree with a spectrally optimized system

The changes in the autofluorescence characteristics of the bronchial tissue is of crucial interest as a cancer diagnostic tool. Evidence exists that this native fluorescence or autofluorescence of bronchial tissues changes when they turn dysplastic and to carcinoma in situ. There is good agreement that the lesions display a decrease of autofluorescence in the green region of the spectrum under illumination with violet-light, and a relative increase in the red region of the spectrum is often reported. Imaging devices rely on this principle to detect early cancerous lesions in the bronchi. Based on a spectroscopic study, an industrial imaging prototype is developed to detect early cancerous lesions in collaboration with the firm Richard Wolf Endoskope GmbH, Germany. A preliminary clinical trial involving 20 patients with this spectrally optimized system shows that the autofluorescence can help to detect most lesions that would otherwise have remained invisible to an experienced endoscopist under white light illumination. A systematic off line analysis of the autofluorescence images pointed out that real-time decisional functions can be defined to reduce the number of false positive results. Using this method, a positive predictive value (PPV) of 75% is reached using autofluorescence only. Moreover, a PPV of 100% is obtained, when combining the white light (WL) mode and the autofluorescence (AF) mode, at the applied conditions. Furthermore, the sensitivity is estimated to be twice higher in the AF mode than in WL mode.

Photodynamic therapy of early squamous cell cancers of the esophagus

The conventional treatment for the cure of esophageal cancer is surgical resection. Esophageal cancer, when detected at an early stage, has a very good probability of being eradicated by less aggressive procedures, and photodynamic therapy has proven to be a safe and effective treatment modality in some carefully selected patients. The indications, outcomes, and future considerations regarding the use of photodynamic therapy for the treatment of superficial squamous cell carcinomas of the esophagus are discussed in this article.

Evaluation of the Photosensitizer Tookad® for Photodynamic Therapy on the Syrian Golden Hamster Cheek Pouch Model: Light Dose, Drug Dose and Drug–light Interval Effects¶

Abstract We have evaluated the efficacy of the new photosensitizer (PS) Tookad® in photodynamic therapy (PDT) in vivo. This PS is a palladium-bacteriopheophorbide presenting absorption peaks at 762 and 538 nm. The light dose, drug dose and drug injection–light irradiation interval (DLI), ranging between 100 and 300 J/cm2, 1 and 5 mg/kg and from 10 to 240 min, respectively, were varied, and the response to PDT was analyzed by staging the macroscopic response and by the histological examination of the sections of the irradiated cheek pouch. The level of PDT response, macroscopically and histologically, shows a strong dependence on the DLI, light dose and drug dose at the applied conditions in the normal hamster cheek pouch. A decay of the tissular response with increasing DLI is observed corresponding to a time of half-maximum response ranging from 10 to 120 min, depending on drug dose and light dose. The tissues affected at the lowest doses are predominantly the vascularized diffuse connective tissue situated between the inner and outer striated muscle (SM) layers as well as these muscle layers themselves. The highest response at the shortest DLI and the absence of a measurable response at DLI longer than 240 min at 300 J/cm2 and drug dose of 5 mg/kg are characteristics of a predominantly vascular effect of this PS. This observation suggests that Tookad® could be effective in PDT of vascularized lesions or pathologies associated with the proliferation of neovessels.

Photodynamic Therapy for 101 Early Cancers of the Upper Aerodigestive Tract, the Esophagus, and the Bronchi: A Single-Institution Experience

Cancer, when detected at an early stage, has a very good probability of being eradicated by surgery or radiotherapy. However, less aggressive treatments also tend to provide high rates of cure without the side effects of radical therapy. We report on the results of our clinical experience with photodynamic therapy (PDT) for the treatment of early carcinomas in the upper aerodigestive tract, the esophagus, and the tracheobronchial tree. Sixty-four patients with 101 squamous cell carcinomas were treated with three different photosensitizers: hematoporphyrin derivative (HPD), Photofrin II, and tetra (m-hydroxyphenyl)chlorin (mTHPC). Seventy-seven (76%) tumors showed a complete rsponse with no recurrence after a mean follow-up period of 27 months. There was no significant difference in terms of cure rates among the three dyes. However, mTHPC has a stronger phototoxicity and induces a shorter skin photosensitization than either of the other photosensitizers. There were eight major complications: three esophagotracheal fistulae after illumination with red light in the esophagus, two esophageal stenoses following 360° circumferential irradiation, and three bronchial stenoses. Illumination with the less penetrating green light and the use of a 180° or 240° windowed cylindrical light distributor render the risk of complications in the esophagus essentially impossible, without reducing the efficacy of the treatment. Therefore, PDT may be considered as a safe and effective treatment for early carcinomas of the upper aerodigestive tract, the esophagus, and the tracheobronchial tree.

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.

Selectivity of the photosensitiser Tookad for photodynamic therapy evaluated in the Syrian golden hamster cheek pouch tumour model.

The response to photodynamic therapy (PDT) with the photosensitiser (PS) Tookad® was measured in the Syrian hamster cheek pouch model on normal mucosae and chemically induced squamous cell carcinoma. This PS is a palladium-bacteriopheophorbide presenting absorption peaks at 538 and 762 nm. The light dose, drug dose and drug injection-light irradiation times (DLI), ranging between 100 and 300 J cm−2, 1–5 mg kg−1 and 10–240 min respectively, were varied and the response to PDT was analysed by staging the macroscopic response and by the histological examination of the sections of the irradiated cheek pouch. A fast time decay of the tissular response with drug dose of 1–5 mg kg−1 was observed for DLI ranging from 10 to 240 min and for light doses of 100–300 J cm−2 delivered at a light dose rate of 150 mW cm−2. A significantly higher level of tissular response was observed for squamous cell carcinoma compared to normal tissue. Nevertheless, the threshold level of the drug–light dose for a detectable response was not significantly different in the tumoral vs normal tissue. The highest response at the shortest DLIs and the absence of measurable response at DLI larger than 240 min at light dose of 300 J cm−2 and drug dose of 5 mg kg−1 reveals the predominantly vascular effect of Tookad®. This observation suggests that Tookad® could be effective in PDT of vascularised lesions.

Mucosal ablation with photodynamic therapy in the esophagus: optimization of light dosimetry in the sheep model

BACKGROUND Photodynamic therapy is an attractive technique for mucosal ablation in patients with superficial squamous cell carcinoma of the esophagus, or high-grade dysplasia or early stage adenocarcinoma arising in Barretts esophagus. Although illumination with green light is assumed to be safe, choice of the light has been empirical in clinical studies; light dose is often reduced to avoid potential complications. The present study assessed the safety of green and blue lights during photodynamic therapy in the esophagus by progressively administrating increasing doses in an attempt to standardize the dose and determine a safe upper limit. This would considerably simplify photodynamic therapy and improve therapeutic results. METHODS The sheep model was chosen because of similarities with humans regarding the thickness and histologic structure of the esophagus. Irradiation with a 180 degrees windowed cylindrical light distributor was performed in 19 sheep 4 days after injection of 0.15 mg/kg of tetra(m-hydroxyphenyl) chlorin. Light doses ranged from 10 to 500 J/cm(2) at 514 nm (argon ion laser) and from 5 to 250 J/cm(2) at 413 nm (krypton laser). RESULTS Follow-up endoscopies revealed a tissue response with a fibrinous area at almost all light doses, whereas application of extremely high light doses tended to induce circumferential necrosis with subsequent stenosis. Three months after irradiation with green light, histologic examination of the resected specimens revealed transmural scarring at doses higher than 100 J/cm(2). After illumination with blue light, partial or more extensive fibrosis of the muscular layer was observed only at light doses of 175 to 250 J/cm(2). CONCLUSIONS Application of high doses of green light after sensitization with tetra(m-hydroxyphenyl) chlorin led to severe complications in the esophagus of the sheep that are highly likely to occur in humans as well. Blue light causes significantly less damage than green light and may, therefore, be considered as an alternative for photodynamic therapy of early stage superficial esophageal cancer.

Fluorescence pharmacokinetics of Lutetium Texaphyrin (PCI-0123, Lu-Tex) in the skin and in healthy and tumoral hamster cheek-pouch mucosa

We have investigated the pharmacokinetics (PK) of Lutetium Texaphyrin (Lu-Tex), a second-generation photosensitizer, in the Syrian hamster cheek pouch early cancer model. Ten male hamsters, five with chemically induced early squamous cell cancer of the left cheek pouch, received an intracardiac injection of a 10 mg/ml Lu-Tex solution, resulting in a dose of 12 mg Lu-Tex per kg of bodyweight. The PK of the dye have been measured during the 24 h following the injection with an optical-fiber-based spectrofluorometer on the ventral skin, the healthy and the tumoral cheek-pouch mucosa. The Lu-Tex fluorescence is excited at 460 nm and detected around 740 nm. All the measurements yield very similar pharmacokinetic curves. The fluorescence intensity reaches a maximum between two and three hours after the injection and, at its maximum, it is consistently higher (up to 1.5 times) on the tumor than on the healthy mucosa. It remains smaller on the skin than on cheek-pouch mucosa. After 24 h, the Lu-Tex fluorescence is no longer detectable either on the skin, on the lesion or on the healthy mucosa. Moreover, Lu-Tex clearly displays a significant fluorescence selectivity between early carcinoma and healthy mucosa in this model. Furthermore, the inter-animal fluctuations of the fluorescence signal are small (+/-16% on the tumor-bearing mucosa). Eight-minute-long skin-irradiation tests have been performed 24 h after the injection of the Lu-Tex on the ventral skin of 16 additional animals with a solar simulator. No reaction is observed, either macroscopically or microscopically, which further demonstrates, as suggested by the fluorescence measurements, that this photosensitizer is significantly cleared from the skin after 24 h.

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.