Induction of immune mediators in glioma and prostate cancer cells by non-lethal photodynamic therapy.

BACKGROUND: Photodynamic therapy (PDT) uses the combination of photosensitizing drugs and harmless light to cause selective damage to tumor cells. PDT is therefore an option for focal therapy of localized disease or for otherwise unresectable tumors. In addition, there is increasing evidence that PDT can induce systemic anti-tumor immunity, supporting control of tumor cells, which were not eliminated by the primary treatment. However, the effect of non-lethal PDT on the behavior and malignant potential of tumor cells surviving PDT is molecularly not well defined. METHODOLOGY/PRINCIPAL FINDINGS: Here we have evaluated changes in the transcriptome of human glioblastoma (U87, U373) and human (PC-3, DU145) and murine prostate cancer cells (TRAMP-C1, TRAMP-C2) after non-lethal PDT in vitro and in vivo using oligonucleotide microarray analyses. We found that the overall response was similar between the different cell lines and photosensitizers both in vitro and in vivo. The most prominently upregulated genes encoded proteins that belong to pathways activated by cellular stress or are involved in cell cycle arrest. This response was similar to the rescue response of tumor cells following high-dose PDT. In contrast, tumor cells dealing with non-lethal PDT were found to significantly upregulate a number of immune genes, which included the chemokine genes CXCL2, CXCL3 and IL8/CXCL8 as well as the genes for IL6 and its receptor IL6R, which can stimulate proinflammatory reactions, while IL6 and IL6R can also enhance tumor growth. CONCLUSIONS: Our results indicate that PDT can support anti-tumor immune responses and is, therefore, a rational therapy even if tumor cells cannot be completely eliminated by primary phototoxic mechanisms alone. However, non-lethal PDT can also stimulate tumor growth-promoting autocrine loops, as seen by the upregulation of IL6 and its receptor. Thus the efficacy of PDT to treat tumors may be improved by controlling unwanted and potentially deleterious growth-stimulatory pathways.

Photodynamic diagnosis of prostate cancer using 5-aminolevulinic acid--first clinical experiences.

OBJECTIVES: To study the feasibility of 5-aminolevulinic-acid (5-ALA)-induced photodynamic diagnosis (PDD) for the evaluation of the surgical margins (SMs) during radical prostatectomy (RP) in patients with prostate cancer (PCa). METHODS: A total of 18 patients with histologically confirmed PCa (Gleason score 4 to 8, prostate-specific antigen 1 to 20 ng/mL) underwent RP. Of the 18 patients, 16 received 5-ALA (20 mg/kg) orally 2 hours before RP, and 2 served as controls without any application of 5-ALA. To study the protoporphyrin IX (PPIX) accumulation after application of 5-ALA, all harvested specimens were investigated by fluorescence microscopy and spectroscopy. In 10 of 16 patients, PDD of the SMs and the prostate was performed during RP using an incoherent light source filtered for efficient fluorescence excitation. RESULTS: In all 16 patients, who had received 5-ALA fluorescence microscopy showed a selective accumulation of PPIX in the PCa cells, and only weak PPIX fluorescence could be detected in benign epithelial cells and none in the adjacent stroma. The 2 patients, who had not received 5-ALA had no PPIX fluorescence in the prostate. Of 10 patients, 8 demonstrated fluorescence-negative and histologically confirmed negative margins during PDD, and 1 each demonstrated a fluorescence-positive SM that was also confirmed by histologic examination and a positive SM that was not confirmed by PPD. CONCLUSIONS: This is the first report of PDD for PCa using 5-ALA. These initial results have demonstrated that PPIX is selectively enhanced in malignant tissue, an essential prerequisite of PDD. Additional studies are warranted to validate these preliminary data and the efficacy of PDD for PCa during RP.

Long-sustaining response in a patient with non-resectable, distant recurrence of glioblastoma multiforme treated by interstitial photodynamic therapy using 5-ALA: case report.

Glioblastoma multiforme continues to be a devastating disease despite modest improvements in survival achieved at present, and there is an urgent need for innovative treatment concepts. Five-aminolevulinic acid (ALA) is a drug which induces protoporphyrin IX accumulation in malignant gliomas and has been explored for fluorescence-guided resections of these tumors. ALA is also under investigation as a photosensitizer. We report a case of a patient with prior left frontal glioblastoma multiforme treated by surgery, radiation and chemotherapy, who developed a remote lesion in the left insula, which was refractory to secondary treatments. In a compassionate use setting she was treated by oral application of ALA (20 mg/kg bodyweight), and stereotactic phototherapy achieved by positioning four laser diffusors using 3-dimensional irradiation planning, and a 633 nm diode laser. The lesion disappeared 24 h after therapy. Circumferential contrast enhancement was observed at 72 h, which disappeared in the course of subsequent months. Edema resolved completely. The patient is still free of recurrence 56 months after treatment, demonstrating an impressive and long-lasting response to this novel mode of therapy.

Study of protoporphyrin IX (PpIX) pharmacokinetics after topical application of 5-aminolevulinic acid in urethral condylomata acuminata.

The pharmacokinetics of 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) in lesions of urethral condylomata acuminata were investigated. Sixty patients (20 to 60 years old, 48 male and 12 female) were divided randomly into five groups and received topic application of different concentrations of ALA solution (0.5%, 1%, 3%, 5% or 10%). Biopsy was performed between 1 and 7 h and specimens were subjected to histological, PpIX fluorescence and human papillomavirus (HPV) DNA typing analyses. Fluorescence examination confirmed that ALA-induced PpIX fluorescence was dominantly distributed in the HPV-infected epidermis. In contrast, only a minimal amount of PpIX fluorescence was detected in the dermis. The maximal fluorescence intensity was detected at 5 h incubation. Higher ALA concentration (e.g. 5% and 10%) produced a stronger intensity. These results suggest that the topical application of 5-10% ALA solution for 3-5 h is the optimal condition for the photodynamic therapy of urethral condylomata acuminata. The selective damage of the condylomata acuminata lesions in the epidermis without damaging the dermis ensures a better control of recurrence and side effects such as ulceration or scarring. DNA typing showed that all patients were positive for low risk-HPV DNA and among them 18.3% of patients harbored high risk-HPV DNA.

Interstitial photodynamic therapy of nonresectable malignant glioma recurrences using 5-aminolevulinic acid induced protoporphyrin IX.

BACKGROUND AND OBJECTIVE: Limited knowledge of the light and temperature distribution within the target volume in combination with non-selective accumulation of the applied photosensitizers (PS) has hampered the clinical relevance of interstitial photodynamic therapy (iPDT) for treatment of malignant glioma patients. The current pilot study focused on the development and the clinical implementation of an accurate and reproducible irradiation scheme for iPDT using 5-aminolevulinic acid (5-ALA) induced protoporphyrin IX (PPIX) as a selectively working PS. STUDY DESIGN/MATERIALS AND METHODS: Monte Carlo simulations of fluence rate and heat transport simulations were performed using the optical properties of normal brain tissue infiltrated by tumor cells (absorption coefficient micro(a) = 0.2 cm(-1), reduced scattering coefficient: micro'(s) = 20 cm(-1)). A modified 3-D treatment-planning software was used to calculate both, the treatment-volume and the exact position of the light diffusers within the lesion. The feasibility and the risk of iPDT were tested in 10 patients with small and circumscribed recurrent malignant gliomas. RESULTS: The optimum distance between the implanted light diffusers was determined to be 9 mm with regard to both fluence rate and temperature distribution. For this distance a temperature increase above 42 degrees C was not expected to occur. Up to six cylindrical light diffusers were stereotactically implanted to achieve a complete irradiation of the tumor volume, which was possible in every single patient (mean tumor volume: 5.9 cm3). The total applied light fluence was between 4,320 J and 11,520 J. Side effects of iPDT were not observed. Median survival was 15 months. CONCLUSION: 5-ALA iPDT in combination with a 3-D treatment-planning (which was based on optical and thermal simulations) is a safe and feasible treatment modality. The clinical impact of these findings deserves further prospective evaluation.

Two-photon photodynamic therapy of C6 cells by means of 5-aminolevulinic acid induced protoporphyrin IX.

Photodynamic therapy (PDT) has received increased attention as a treatment modality for malignant tumors as well as non-oncologic diseases such as age-related macular degeneration (AMD). An alternative to excite the photosensitizer by the common one-photon absorption is the method of two-photon excitation (TPE). This two-photon photodynamic therapy has the potential of improving the therapeutic outcome due to a highly localized photodynamic effect. The present study investigated the two-photon excited PDT performing in vitro experiments where C6 rat glioma cells were irradiated with a pulsed and focused fs Ti:sapphire laser emitting light at 800 nm. The irradiance distribution of the laser beam was carefully analyzed before the experiment and the applied irradiance was known for each position within the irradiated cell layer. Cells were divided into four groups and one group was incubated with 5-ALA and irradiated 4-5h later. The survival of this group was tested after irradiation by means of ethidium bromide and acridine orange staining and compared to a control group, which was irradiated under the same conditions, but not incubated with 5-ALA before. Both groups showed necrotic areas depending on the applied irradiance, the value of which at the margin of the necrotic area could be deduced from its size. 5-ALA incubated cells became necrotic after irradiation with a mean irradiance above 6.1 x 10(10) W/cm(2), while non-incubated cells remained viable. Cells of both groups became necrotic when treated with an irradiance above 10.9 x 10(10) W/cm(2). The observed affected area of the cell layers was between 0.13 mm(2) and 1.10 mm(2). Since the irradiation of non-incubated cells below the mean power density of 10.9 x 10(10) W/cm(2) induced no necrosis, apparently no thermal damage was induced in the cells and necrosis of the 5-ALA incubated cells can be ascribed to the photodynamic effect induced by two-photon excitation. The successful photodynamic treatment of a large area of a monolayer cell culture induced by two-photon excitation offers new perspectives for photodynamic treatment modalities.

In-vivo kinetics of inhaled 5-aminolevulinic acid-induced protoporphyrin IX fluorescence in bronchial tissue.

BACKGROUND: In the diagnosis of early-stage lung cancer photosensitizer-enhanced fluorescence bronchoscopy with inhaled 5-aminolevolinic acid (5-ALA) increases sensitivity when compared to white-light bronchoscopy. This investigation was to evaluate the in vivo tissue pharmacokinetics of inhaled 5-ALA within the bronchial mucosa in order to define the time optimum for its application prior to bronchoscopy. METHODS: Patients with known or suspected bronchial carcinoma were randomized to receive 200 mg 5-ALA via inhalation 1, 2, 3, 4 or 6 hours before flexible fluorescence bronchoscopy was performed. Macroscopically suspicious areas as well as areas with visually detected porphyrin fluorescence and normal control sites were measured spectroscopically. Biopsies for histopathology were obtained from suspicious areas as well as from adjacent normal areas. RESULTS: Fluorescence bronchoscopy performed in 19 patients reveals a sensitivity for malignant and premalignant changes (moderate dysplasia) which is almost twice as high as that of white-light bronchoscopy, whereas specificity is reduced. This is due to false-positive inflammatory lesions which also frequently show increased porphyrin fluorescence. Malignant and premalignant alterations produced fluorescence values that are up to 5 times higher than those of normal tissue. According to the pharmacokinetics of porphyrin fluorescence measured by spectroscopy, the optimum time range for 5-ALA application is 80-270 min prior to fluorescence bronchoscopy, with an optimum at 160 min. CONCLUSION: According to our results we propose inhalation of 5-ALA 160 min prior to fluorescence bronchoscopy, suggesting that this time difference provides the best tumor/normal tissue fluorescence ratio.

Acute morphological sequelae of photodynamic therapy with 5-aminolevulinic acid in the C6 spheroid model.

OBJECTIVE: Aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) may represent a treatment option for malignant brain tumors. We used a three-dimensional cell culture system, the C6 glioma spheroid model, to study acute effects of PDT and how they might be influenced by treatment conditions. METHODS: Spheroids were incubated for 4 h in 100 microg/ml ALA in 5% CO(2) in room air or 95% O(2) with subsequent irradiation using a diode laser (lambda = 635 nm, 40 mW/cm(2), total fluence 25 J/cm(2)). Control groups were "laser only", "ALA only", and "no drug no light". Annexin V-FITC, a marker used for detection of apoptosis, propidium iodide (PI), a marker for necrotic cells and H 33342, a chromatin stain, were used for morphological characterization of PDT effects by confocal laser scanning and fluorescence microscopy. Hematoxylin-eosin staining and TdT-FragEL (TUNEL) assay were used on cryosections. Growth kinetics were followed for 8 days after PDT. RESULTS: PDT after incubation in 5% CO(2) provided incomplete cell death and growth delay in spheroids of >350 microm diameter. However, complete cell death and growth arrest occurred in smaller spheroids (<350 microm). Incubation in 95% O(2) with subsequent PDT resulted in complete cell death and growth arrest regardless of spheroid size. In incompletely damaged spheroids viable cells were restricted to spheroid centers. The rate of cell death in all control groups was negligible. Cell death was accompanied by annexin/PI costaining, but there was also evidence for annexin V-FITC staining without PI uptake. CONCLUSIONS: PDT of experimental glioma results in rapid and significant cell death that could be verified as acute necrosis immediately after irradiation. This effect depended on O(2) concentration and spheroid size.

High-level virtual reality simulator for endourologic procedures of lower urinary tract.

OBJECTIVES: To analyze the limitations of existing simulators for urologic techniques, and then test and evaluate a novel virtual reality (VR) simulator for endourologic procedures of the lower urinary tract. Surgical simulation using VR has the potential to have a tremendous impact on surgical training, testing, and certification. Endourologic procedures seem to be an ideal target for VR systems. METHODS: The URO-Trainer features genuine VR, obtained from digital video footage of more than 400 endourologic diagnostic and therapeutic procedures, as well as data from cross-sectional imaging. The software offers infinite random variations of the anatomy and pathologic features for diagnosis and surgical intervention. An advanced haptic force feedback is incorporated. Virtual cystoscopy and resection of bladder tumors were evaluated by 24 medical students and 12 residents at our department. RESULTS: The system was assessed by more than 150 international urologists with varying experience at different conventions and workshops from March 2003 to September 2004. Because of these evaluations and constant evolutions, the final version provides a genuine representation of endourologic procedures. Objective data are generated by a tutoring system that has documented evident teaching benefits for medical students and residents in cystoscopy and treatment of bladder tumors. CONCLUSIONS: The URO-Trainer represents the latest generation of endoscopy simulators. Authentic visual and haptic sensations, unlimited virtual cases, and an intelligent tutoring system make this modular system an important improvement in computer-based training and quality control in urology.

Photodynamic therapy of prostate cancer by means of 5-aminolevulinic acid-induced protoporphyrin IX - in vivo experiments on the dunning rat tumor model.

OBJECTIVE: In order to expand the use of photodynamic therapy (PDT) in the treatment of prostate carcinoma (PCA), the aim of this study was to evaluate PDT by means of 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PPIX) in an in vivo tumor model. METHODS: The model used was the Dunning R3327 tumor. First of all, the pharmacokinetics and the localization of PPIX were obtained using fluorescence measurement techniques. Thereafter, PDT using 150 mg 5-ALA/kg b.w. i.v. was performed by homogenous irradiation of the photosensitized tumor (diode laser lambda = 633 nm). The tumors were resected 2 days post-PDT and the extent of the necrosis was determined histopathologically. RESULTS: The kinetics of PPIX fluorescence revealed a maximum intensity in the tumor tissue within 3 and 4.5 h post-application of 5-ALA. At this time, specific PPIX fluorescence could be localized selectively in the tumor cells. The PDT-induced necrosis (n = 18) was determined to be 94 +/- 12% (range 60-100%), while the necrosis of the controls (n = 12) differs significantly (p < 0.01), being less than 10%. CONCLUSION: These first in vivo results demonstrate the effective potential of 5-ALA-mediated PDT on PCA in an animal model.

Comparison of thermal tissue effects induced by contact application of fiber guided laser systems.

BACKGROUND AND OBJECTIVES: Laser light of various wavelengths is being used for surgical procedures in otolaryngology. Apart from well-known fiber guided laser systems such as Nd:YAG- and Ho:YAG-lasers, newly developed diode-laser systems of different wavelengths have recently become popular in surgery. In order to compare the effects of fiber guided laser light with respect to their induced tissue effects, these laser systems have been studied and compared under reproducible test conditions. STUDY DESIGN/MATERIALS AND METHODS: The laser fibers of four common medical laser systems (Ho:YAG- (lambda = 2,080 nm), Nd:YAG- (lambda = 1,064 nm), and diode-laser (lambda = 830 and 940 nm)) were fixed to a computer controlled stepper motor. The laser light was applied in contact mode onto ex vivo muscle tissue, using identical power settings and a reproducible application procedure (application velocity, application angle) under constant conditions (temperature of tissue and volume). The size of the thermal effects on the tissue (e.g., coagulation, ablation, and carbonization zones) were measured and photographed via optical microscopy. RESULTS: Depending on the laser wavelength used, the experimental results proved different degrees of tissue responses. Nd:YAG- and diode-lasers provided for only low coagulation effects in the depth of the tissue, but produced severe carbonization at the surface. Ho:YAG-laser light revealed the highest ablation capabilities of the lasers investigated in addition to large coagulation zones which were of larger extent than those produced by Nd:YAG- and diode-laser light. CONCLUSIONS: Contact treatment by Ho:YAG-laser light might provide for a precise and effective tissue reduction in a bloodless manner because of its high ablation and coagulation capabilities, especially if large volumes are treated and structures beneath are non-critical. In comparison, Nd:YAG- and diode-laser treatment in contact application showed low thermal tissue effects (i.e., coagulation) in the depth, resulting from a high power loss caused by the development of large carbonization zones at the surface of the tissue. Therefore, the degree of blood-perfusion and the capability of vessel-closure induced by these lasers should be taken into account. The presented investigation also revealed that in contact mode, the tested laser systems produced tissue effects, which were highly different from those already described for applications in non-contact mode. Physicians who are performing laser treatments in close boundaries must be aware that changing from non-contact to contact mode in laser application greatly influences the resulting tissue effects.

Ultraviolet-excited (308 nm) autofluorescence for bladder cancer detection.

OBJECTIVES: To assess the ability of ultraviolet laser-induced autofluorescence for the diagnosis of transitional cell carcinoma of the bladder. METHODS: We studied 43 patients undergoing transurethral resection with recurrent transitional cell carcinoma of the bladder. Guided through 5-aminolevulinic acid-induced fluorescence endoscopy, the autofluorescence of the red fluorescing areas and the adjacent tissue and inconspicuous-appearing mucosa were measured spectroscopically. The autofluorescence excitation was carried out with a xenon chloride excimer laser operating at 308 nm (AF308). For the evaluation of the autofluorescence spectra, an intensity ratio (335/430 nm) was calculated and correlated with the histologic results of the biopsies taken. RESULTS: We analyzed the AF308 spectra of 114 biopsies (21 malignant, 93 benign). The autofluorescence intensity ratios for the benign lesions were a factor of 2 to 7 higher than carcinoma in situ and neoplastic tissue. Therefore, 20 of 21 neoplastic lesions were detected as true positive by AF308. A sensitivity and specificity for AF308 of 95% and 77%, respectively, could be calculated. The sensitivity and specificity for 5-aminolevulinic acid-induced fluorescence endoscopy was 90% and 61%, respectively. By combining the two methods, we calculated a sensitivity of 90% and a specificity of 84%. CONCLUSIONS: The combination of a sensitive imaging technique such as 5-aminolevulinic acid-induced fluorescence endoscopy and a more specific spectral fluorescence probe technique with autofluorescence at 308 nm is a very efficient procedure in the detection of transitional cell carcinoma of the bladder.

Kinetics of 5-aminolevulinic acid-induced fluorescence in organ cultures of bronchial epithelium and tumor.

BACKGROUND: 5-Aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PPIX) fluorescence improves the differentiation of tumor and normal tissue in the bladder, skin and brain. OBJECTIVE: The kinetics of 5-ALA-induced protoporphyrin IX (PPIX) fluorescence in organ cultures of normal human bronchial epithelium and cocultures of bronchial epithelium and tumor have been studied. METHODS: Cultured biopsies of bronchial epithelium were exposed for 5 or 15 min, or continuously to 5-ALA. PPIX fluorescence was quantified for up to 300 min by spectroscopy. Cocultures of normal bronchial epithelium and a non-small-cell lung cancer cell line (EPLC-32M1) were incubated with 5-ALA. Space-resolved fluorescence microscopy was used to quantify PPIX fluorescence kinetics in the tumor and normal epithelium. RESULTS: In cultures of normal epithelium, PPIX fluorescence kinetics were shown to depend on the duration of exposure to 5-ALA. There was a trend to higher fluorescence intensities with longer exposure times. In cocultures of bronchial epithelium and tumor, increases of fluorescence intensity were significantly greater in the tumor. Best tumor/normal tissue fluorescence ratios were found between 110 and 160 min after exposure to 5-ALA. CONCLUSION: Data obtained in this coculture system of bronchial epithelium and tumor is valuable to optimize modalities of fluorescence bronchoscopy for the diagnosis of early bronchial carcinoma.

Autofluorescence spectroscopy for the diagnosis of cervical intraepithelial neoplasia.

OBJECTIVE: To assess the feasibility of autofluorescence spectroscopy in the diagnosis of cervical intraepithelial neoplasia (CIN) using broadband light excitation. DESIGN: Feasibility study. SETTING: Colposcopy clinic of an university hospital. POPULATION: Sixty-eight patients at risk for CIN. METHODS: After excitation with a broadband light between 375 and 440 nm, spectral distribution of native tissue fluorescence (autofluorescence) was acquired from 685 cervical sites for the localisation and differentiation of CIN, and compared with colposcopically directed biopsy and human papillomavirus (HPV) DNA testing. MAIN OUTCOME MEASURE: Detection of CIN. RESULTS: The evaluation of spectral measurements revealed significantly lower autofluorescence values for CIN 3 lesions compared with normal tissue (P < 0.001), and compared with CIN 1 or CIN 2 (P < 0.002). High grade CIN lesions (CIN 2/3) presented with a significant reduced autofluorescence compared with CIN 1 (P < 0.002). Patients with a positive HPV DNA testing showed a significantly lower autofluorescence than patients tested negative for HPV DNA (P < 0.05). Severe inflammation such as chronic cervicitis may lead to false positive results. CONCLUSIONS: Autofluorescence spectroscopy represents an interesting approach for the detection of cervical neoplasia. Using an excitation wavelength band between 375 and 440 nm, significant differences between normal and precancerous lesions of the cervix can be seen.

Comparison of laser induced effects on hyperplastic inferior nasal turbinates by means of scanning electron microscopy.

BACKGROUND AND OBJECTIVE: Various laser systems have been used for volume reduction of hyperplastic inferior nasal turbinates. Many studies have thus reported about clinical experiences concerning short and long term results. Although there are also some clinical investigations on histological changes after laser treatment, there is a lack of examinations by means of scanning electron microscopy (SEM) in order to especially investigate laser induced superficial tissue effects (ablation, carbonization, and coagulation). MATERIALS AND METHODS: Directly following operative excision, three hyperplastic nasal turbinates were treated by laser light of five common medical laser systems and their corresponding wavelengths, using identical laser parameters (irradiance, laser spot diameter) and a reproducible application procedure. After passing through an ethanol dehydration process, the thermal effects on the tissue (carbonization, coagulation, and ablation) were investigated and photographed via SEM. RESULTS: Depending on the laser wavelength used, tissue responses which could be observed by SEM greatly differ with respect to ablation, coagulation, and carbonization zones. While the CO(2) laser showed a precise cutting of tissue via ablation with only slight thermal effects on the adjoining tissue, Argon-ion-, Nd:YAG-, and diode-lasers provided for an intense destruction of both the surrounding, superficial respiratory epithelium, and the tissue in the depth of the turbinates, resulting from the development of rather large carbonization and coagulation zones. Ho:YAG-laser light induced precise vaporization in addition to remarkable superficial coagulation of the tissue, which was of smaller extent than after treatment with Nd:YAG- and diode-laser, but comparable to the Argon-ion-laser. CONCLUSIONS: In the course of this study SEM proved to be especially suitable for the examination of tissue changes on the surface of laser treated turbinates (i.g., destruction of respiratory epithelium). According to our results, Argon-ion-, Nd:YAG-, and diode-lasers seem to be useful for an effective and bloodless reduction of hyperplastic turbinates. However, broad coagulation zones may lead to consecutive complications and side effects. Due to its good ablation and superficial coagulation capabilities, Ho:YAG-laser light also seems to be usable for an effective turbinate reduction, but its low thermal effects in the depth of the tissue may lead to bleedings during treatment. Laser light of the CO(2) laser showed the lowest thermal tissue effects of the lasers investigated and therefore holds the risk of bleedings during treatment and the need of more than one therapeutic session to get a sufficient result. This investigation revealed that physicians, who are performing endonasal laser treatment, must be aware of the varying tissue effects of different medical laser systems to get a more aimed effect and to avoid intra- and post-operative complications (e.g., Rhinitis sicca, bone sequestration of the turbinate, bleedings, ...).

A comparative study of normal inspection, autofluorescence and 5-ALA-induced PPIX fluorescence for oral cancer diagnosis.

Fluorescence diagnosis aims to improve the management of oral cancer via early detection of the malignant lesions and better delimitation of the tumor margins. This paper presents a comparative study of normal inspection, combined fluorescence diagnosis (CFD) and its 2 main components, autofluorescence and 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PPIX) fluorescence. Biopsy-controlled fluorescence imaging and spectral analysis were performed on a total of 85 patients with suspected or histologically proven oral carcinoma both before and after topical administration of 5-ALA (200 mg 5-ALA dissolved in 50 ml of H(2)0). Fluorescence excitation was accomplished using filtered light of a xenon short arc lamp (lambda = 375-440 nm). As for CFD, a "streetlight" contrast (red to green) was readily found between malignant and healthy tissue on the acquired images. In terms of tumor localization and delimitation properties, CFD was clearly favorable over either normal inspection or its 2 components in fluorescence imaging. The performance of CFD was found to be impeded by tumor keratinization but to be independent of either tumor staging, grading or localization. In spectral analysis, cancerous tissue showed significantly higher PPIX fluorescence intensities and lower autofluorescence intensities than normal mucosa. There is a great potential for CFD in early detection of oral neoplasms and exact delimitation of the tumors' superficial margins and an advantage over white light inspection and each of its 2 main components. The method is noninvasive, safe and easily reproducible.

In vitro photodynamic therapy of nasopharyngeal carcinoma using 5-aminolevulinic acid.

The purpose of this study was to investigate the potential use of 5-aminolevulinic acid (5-ALA, 5-amino-4-oxovaleric acid) induced protoporphyrin IX (PPIX) for photodynamic therapy (PDT) of nasopharyngeal carcinoma (NPC) and its related mechanisms of inducing cell death. PPIX biosynthesis at I to 8 h after incubation of a cultured NPC cell line (HNE1) with 5-ALA (10-5,000 microg ml(-1)) was determined via fluorescence analysis HNEI cells were irradiated at 4 h after incubation with 5-ALA (10-200 microg ml(-1)) by diode laser (lambda = 630 nm) at various energy levels (1-50 J cm(-2)). The survival rates at 6, 12, 24 and 48 h after PDT were determined by MTT assay. Mechanisms of PDT-induced cell death were investigated via Anncxin-V/propidium iodide staining and DNA electrophoresis After incubation with 5-ALA, a time- and dose-dependent increase of cellular PPIX-fluorescence was recorded up to a threshold concentration of 1,000 microg ml(-1) 5-ALA, above which a decline of fluorescence intensities occurred. Similar values of PPIX-fluorescence were found at 100-1,000 microg ml(-1) of 5-ALA. Unlike sole incubation with 5-ALA or sole laser irradiation, the combination of both factors lead to a significant, concentration-, energy- and time-dependent increase of cell death (p < 0.01). At 100 microg ml(-1) ALA and 10 J cm 2 laser irradiation, cellular survival was <5% after 48 h. More than 80% of induced cell deaths thereby occurred via apoptosis within the first 12 h following irradiation; necrosis was accountable for less than 20%. High level induction of apoptosis by 5-ALA-PDT was confirmed by DNA electrophoresis. Our investigations show promising results of 5-ALA based PDT of nasopharyngeal carcinoma in vitro and set the basis for future studies in tumor models or humans, respectively.