Ph. Monnier

Photodetection and photodynamic therapy of "early" squamous cell carcinomas of the pharynx, oesophagus and tracheo-bronchial tree

The efficacy of photodynamic therapy (PDT) alone was evaluated on 41 ‘early’ squamous cell carcinomas of the pharynx (10), oesophagus (15) and tracheo-bronchial tree (16). All lesions but two were synchronous second primaries in ENT-patients suffering from a more extensive cancer, governing the overall oncological prognosis.Photofrin I (3 mg/kg) or Photofrin II (2 mg/kg) were injected 72 h prior to the red light irradiation, supplied by an argon pumped dye laser. A diffusing cylinder was used to obtain a homogeneous light distribution at the tumour site (60 J to 150 J/cm2). In the oesophagus and bronchi, the results are good for cancers staged in situ or microinvasive at endoscopy (two recurrencies for 23 lesions treated). For more advanced cancers (submucosal in the oesophagus or invading the bronchial cartilage), the results are less satisfactory (three recurrencies for eight lesions treated). In the pharynx where light dosimetry is more difficult, the rate of recurrencies is higher (3/10 lesions treated). In the bronchi (one case) and oesophagus (one case), the longest disease-free survival is now 5 years.The irradiation of a non-cancerous zone of normal buccal mucosa on 25 patients having received HPD showed necrosis in all cases with light doses as low as 50mW/cm2 for 20 min (60 J cm−2), even with Photofrin II.We encountered six complications (three cicatricial stenosis, two fistulae, one severe sunburn), most of them resulting from the lack of selectivity of HPD. According to these experiments, PDT is efficient at destroying early squamous cell carcinomas in the pharynx, oesophagus and bronchi, but the tumour selectivity of HPD is poor in the digestive tract lined with squamous cell epithelium. The only hope for the future lies in the synthesis of a more selective and more stable photosensitizer. This discussion reviews possible directions of research for the development of new dyes (cationic dyes, dyes attached to monoclonal antibodies, etc), for PDT and hyperthermia, for photodetection of early cancers using a fluoro-endoscope, and finally, for tumour depth profiling in hollow organs using lasers of different wavelengths.

In vivo autofluorescence spectroscopy of human bronchial tissue to optimize the detection and imaging of early cancers

We are developing an imaging system to detect pre-/early cancers in the tracheo-bronchial tree. Autofluorescence might be useful but many features remain suboptimal. We have studied the autofluorescence of human healthy, metaplastic and dysplastic/CIS bronchial tissue, covering excitation wavelengths from 350 to 480 nm. These measurements are performed with a spectrofluorometer whose distal end is designed to simulate the spectroscopic response of an imaging system using routine bronchoscopes. Our data provide information about the excitation and emission spectral ranges to be used in a dual range detection imaging system to maximize the tumor vs healthy and the tumor vs. inflammatory/metaplastic contrast in detecting pre-/early malignant lesions. We find that the excitation wavelengths yielding the highest contrasts are between 400 and 480 nm with a peak at 405 nm. We also find that the shape of the spectra of healthy tissue is similar to that of its inflammatory/metaplastic counterpart. Finally we find that, when the spectra are normalized, the region of divergence between the tumor and the nontumor spectra is consistently between 600 and 800 nm and that the transition wavelength between the two spectral regions is around 590 nm for all the spectra regardless of the excitation wavelength, thus suggesting that there might be one absorber or one fluorophore. The use of backscattered red light enhances the autofluorescence contrast.

Optimizing light dosimetry in photodynamic therapy of early stage carcinomas of the esophagus using fluorescence spectroscopy

Under standardized conditions (drug and light dose, timing), the result of the photodynamic therapy (PDT) of carcinomas of the esophagus with tetra(meta‐hydroxy‐phenyl)chlorin (mTHPC) shows large variations between patients.

Clinical photodynamic therapy for superficial cancer in the oesophagus and the bronchi: 514 nm compared with 630 nm light irradiation after sensitization with Photofrin II.

Photodynamic therapy (PDT) for cancer in the oesophagus and bronchi with red (630 nm) light may occasionally lead to wall perforation and fistula. Therefore, we investigated the clinical use of a less penetrating wavelength (514 nm) for the curative treatment of nine superficial carcinomas in the oesophagus and bronchi after photosensitization with Photofrin II. Tumours without infiltration beyond the submucosa in the oesophagus and beyond the lamina propria in the bronchi were considered as superficial cancers. The outcome and complications were compared with those of 13 superficial cancers treated with PDT and 630 nm light. In addition, we evaluated histologically the extent of the long-term tissue damage and scarring following treatment of six oesophageal cancers with either green or red light. At first endoscopic control, 7-10 days after PDT, tissue necrosis simply matched the illuminated area, without evidence of selective tumour damage. Six of nine tumours treated with 514 nm light had a complete response compared with nine of 13 after 630 nm irradiation. No perforation or fistula occurred in either treatment group. However, severe chest pain and fever with or without pleural effusion, consistent with occult perforation, were observed in three patients after 630 nm illumination in the oesophagus. Histologically, fibrous scarring in the three distinct sites treated with green light was limited to the superficial layers of the oesophagus. After red light treatment, transmural fibrosis with marked thinning of the oesophageal wall was evident in two of the three specimens available for inspection. These results indicate that PDT with 514 nm light has the potential to cure superficial cancer in the oesophagus and bronchi with essentially the same probability of success as red light. In the oesophagus, green light prevents deep tissue damage, thus reducing the risk of perforation.

Production of the free radicals O-2(.-) and OH by irradiation of the photosensitizer zinc(II) phthalocyanine

Zinc(II) phthalocyanine (ZnPC) is a new photosensitizer currently undergoing phase I and II clinical trials at Lausannes CHUV hospital for the photodynamic therapy (PDT) of early cancer in the upper aerodigestive tract. Activated oxygen species other than singlet oxygen produced during the photosensitization of ZnPC in liposomes have been examined by electron paramagnetic resonance (EPR) spin trapping and by the cytochrome c reduction method. Visible light irradiation of ZnPC associated with liposomes in the presence of the spin trap 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) gives an EPR spectrum characteristic of the DMPO-hydroperoxyl radical spin adduct (DMPO-.OOH). Superoxide anion attains a level of 1 microM min-1 20 min after the start of irradiation as determined by the superoxide dismutase (SOD)-inhibitable reduction of cytochrome c. The yield of O2.- is strongly enhanced by physiological electron donors. An EPR spectrum characteristic of the DMPO-hydroxyl radical spin adduct (DMPO-.OH) is also observed. The addition of dimethyl sulphoxide or ethanol produces additional hyperfine splittings due to the respective hydroxyalkyl radical products, indicating the presence of free .OH. DMPO-.OH is significantly inhibited by desferrioxamine or catalase. Conversely, this adduct is enhanced by hydrogen peroxide. These data demonstrate the ability of ZnPC in liposomes to photoreact effectively by an electron transfer mechanism. Such type I processes may add to the effects of singlet oxygen in ZnPC-mediated PDT.

Photodynamic therapy of early squamous cell carcinoma with tetra(m-hydroxyphenyl)chlorin: optimal drug-light interval.

The optimal drug-light interval for effective photodynamic therapy (PDT) of early squamous cell carcinomas was evaluated with tetra(m-hydroxyphenyl)chlorin (mTHPC) by means of two complementary modalities: irradiation tests and ex vivo fluorescence microscopy. A Syrian hamster cheek pouch tumour model was used in these experiments. Photodynamic therapy on both tumour-bearing and contralateral healthy cheek pouch mucosae was performed at 650 nm and 514 nm. Light doses of 12 J cm(-2) were delivered at a light dose rate of 150 mW cm(-2) and light doses of 80 J cm(-2) were delivered at a light dose rate of 100 mW cm(-2) respectively, at these two wavelengths, between 6 h and 12 days after the injection of 0.5 mg kg(-1) body weight mTHPC. Two histologically different types of tissue damage were observed: first, a non-selective and non-specific ischaemic vascular necrosis for the cases in which PDT took place during the first 48 h after the injection of the dye and, second, tissue-specific PDT damage, as a coagulation necrosis, when PDT took place more than 72 h after injection of the dye. The time-dependent biodistribution of mTHPC investigated by fluorescence microscopy shows a weak and non-significant difference in relative fluorescence intensities between early SCC and healthy mucosae. Up to 2 days after the injection, the drug is mainly localized in the endothelial cells of the blood vessels. After this period, the dye accumulates in the squamous epithelia with a concentration peaking at 4 days. At all time points, a weak fluorescence intensity is observed in the underlying lamina propria and striated muscle. The information obtained from these studies could well be relevant to clinical trials as it suggests that time delays between 4 and 8 days after i.v. injection should be optimal for PDT of early malignancies in hollow organs.

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.

Optimizing light dosimetry in photodynamic therapy of the bronchi by fluorescence spectroscopy

Under identical conditions (drug and light dose, timing), the results of photodynamic therapy (PDT) of carcinomas of the bronchi with tetra(meta-hydroxyphenyl)chlorin (mTHPC) show large variations between patients. Before patients underwent PDT treatment, the mTHPC level was measured in the lesion, the normal surrounding tissue and the oral cavity, with an apparatus based on fluorescence spectroscopy. The fluctuations in degree of tissue reaction and tumour destruction between patients could be explained by individual variations in the mTHPC level in the mucosa of the bronchi. The patients who showed the highest mTHPC fluorescence signal also had the strongest response to PDT. In addition, a correlation between the mTHPC level in the oral cavity and bronchial mucosa was found. It is concluded that PDT can be improved by measuring the mTHPC level in the bronchi or the oral cavity before treatment by fluorescence spectroscopy, and then by adjusting the light dose to be applied to the observed mTHPC level.

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.

Time‐dependent Biodistribution of Tetra(m‐hydroxyphenyl)chlorin and Benzoporphyrin Derivative Monoacid Ring A in the Hamster Model: Comparative Fluorescence Microscopy Study

The pharmacokinetics of the photosensitizer used play a key role in the understanding of the mechanism of photodynamic therapy‐induced damage. Fluorescence microscopy was used to compare time‐dependent biodistribution of tetra(m‐hydroxyphenyl)chlorin (mTHPC) and benzoporphyrin derivative monoacid ring A (BPD‐MA) in different hamster tissues, including an early, chemically induced, squamous cell carcinoma. Following injection of 0.5 mg/kg body weight of mTHPC and 2.0 mg/kg BPD‐MA, groups of three animals were sacrificed at different time points and a series of fluorescence micrographs from different excised organs were analyzed. The highest fluorescence intensities of mTHPC were observed at 96 h for squamous epithelia and skin and at 48 h for smooth muscle. There is no real peak of BPD‐MA fluorescence between 30 min and 3 h in the basal epithelial layers, fibroconnective tissue, muscles or blood vessels. At 4 h after injection, the fluorescence level of BPD‐MA decreased and at 24 h it had returned to background level in all observed tissues. The significantly faster clearance of BPD‐MA is the principal advantage as compared to mTHPC. However, similar localization patterns in different tissues with essentially vascular affinity represent a possible disadvantage for treating early malignancies with BPD‐MA as compared to mTHPC, which is mainly localized in various epithelia. For both photosensitizers no significant selectivity between early squamous cell carcinoma and healthy mucosae is seen. Pharmacokinetic studies of different photosensitizers in an appropriate animal model are essential for selecting new‐generation photosensitizers with the most favorable localization for photodynamic therapy of early malignancies in hollow organs.