[Tumors of the mouth cavity. Not all fluorescent tumors are malignant]. / Mundhöhlentumoren. Nicht alles was fluoresziert ist bösartig.

Early diagnosis and treatment of malignant tumors of the mouth and throat can greatly improve survival rates. Diagnostic fluorescence permits the reliable detection not only of oral cancer, but also of cancer precursor lesions. A problem here is the fact that although carcinomas can be detected with a high level of sensitivity, our own studies also demonstrate a considerable false-positive rate (35%). Possible reasons for this currently being considered are non-compliance by the patient with the required fasting period, poor mouth hygiene and the state after radiation therapy to the face. Under these circumstances, patient management is of particular importance.

Internalization of aggregated photosensitizers by tumor cells: subcellular time-resolved fluorescence spectroscopy on derivatives of pyropheophorbide-a ethers and chlorin e6 under femtosecond one- and two-photon excitations.

Amphiphilic sensitizers self-associate in aqueous environments and form aggregated species that exhibit no or only negligible photodynamic activity. However, amphiphilic photosensitizers number among the most potent agents of photodynamic therapy. The processes by which these sensitizers are internalized into tumor cells have yet to be fully elucidated and thus remain the subject of debate. In this study the uptake of photosensitizer aggregates into tumor cells was examined directly using subcellular time-resolved fluorescence spectroscopy with a high temporal resolution (20-30 ps) and high sensitivity (time-correlated single-photon counting). The investigations were performed on selected sensitizers that exhibit short fluorescence decay times (< 50 ps) in aggregated form. Derivatives of pyropheophorbide-a ether and chlorin e6 with varying lipophilicity were used for the study. The characteristic fluorescence decay times and spectroscopic features of the sensitizer aggregates measured in aqueous solution also could be observed in A431 human endothelial carcinoma cells administered with these photosensitizers. This shows that tumor cells can internalize sensitizers in aggregated form. Uptake of aggregates and their monomerization inside cells were demonstrated directly for the first time by means of fluorescence lifetime imaging with a high temporal resolution. Internalization of the aggregates seems to be endocytosis mediated. The degree of their monomerization in tumor cells is strongly influenced by the lipophilicity of the compounds.

[Visualizing carcinomas of the mouth cavity by stimulating synthesis of fluorescent protoporphyrin IX]. / Visualisierung von Karzinomen der Mundhöhle durch Stimulierung der Synthese von fluoreszierendem Protoporphyrin IX.

The fluorescence diagnosis based on the aminolavulinic acid-stimulated porphyrin synthesis allows the detection of superficial tumors in a very early stage even when they are very small tumors. A fluorescence diagnosis of tumors in the oral cavity can be simply performed by rinsing with a 0.4% ALA solution for 20 min. This topical application avoids systemic side effects such as skin sensitization. The red fluorescent areas are visible to the naked eye; only a blue light source for fluorescence excitation is necessary and a suitable red filter for observation. In our study on 56 patients suffering from carcinoma of the oral cavity, 96% of the histologically confirmed carcinoma could be visualized via fluorescence. In 3 patients additional tumors were detected via fluorescence that were not visible otherwise. However, many patients show fluorescent areas with no correlation to the histological finding. It was verified that bacteria from the oral cavity also produce PpIX after ALA incubation, which leads to false-positive findings. Reduction of the false-positive findings was achieved by rinsing the oral cavity with hydrogen peroxide and by mechanical plaque reduction. This improves the reliability of a fluorescence-guided biopsy. However, suppression of the bacteria fluorescence is necessary for clinical use of this diagnostic method.

Phototransformation of sensitisers: 3. Implications for clinical dosimetry.

Spectroscopic studies of aqueous solutions of haematoporphyrin-type sensitisers reveal that photobleaching during eposure to light is followed by the formation of stable red-absorbing photoproducts. Experiments in model systems (sensitisers bound to human serum albumin or in a suspension of resealed erythrocyte 'ghosts') and in tumour tissue show that similar photomodification takes place in all investigated environments. Loss of total absorption and emission intensities is accompanied by an increase of absorption in the red spectral region (630-650 nm) which is used for the treatment of tumours because of the deeper penetration of light into tissues. This should be taken into account when the duration of illumination is chosen to reach an appropriate photodynamic dose using Hp-type sensitisers in the photodynamic treatment of tumours.

5-aminolaevulinic-acid-induced formation of different porphyrins and their photomodifications.

The relative amounts of 5-aminolaevulinic acid (ALA)-induced hydrophobic and hydrophilic porphyrins produced in normal mouse tissue and solid Ehrlich ascites carcinoma in mice were investigated as a function of added glucose, light irradiation and restricted blood flow to the tumour. Protoporphyrin IX (PpIX) is the predominant porphyrin produced from exogenous ALA in tissues that have viable mitochondria. However, under special conditions which seem to be connected with a reduced viability of the mitochondria, the formation of water-soluble porphyrins can be observed in vivo. Fluorescence maxima of the water-soluble porphyrins are located in the region between 615 and 625 nm. Irradiation can further lead to the formation of PpIX photoproduct(s), showing a fluorescence emission band at 676 nm. The photobleaching of PpIX in normal tissue and tumour tissue is different under restricted blood flow. ALA-induced PpIX can be observed in the normal blood stream, and can be linked to a slower photobleaching than in tissue with restricted blood flow.

Formation of water-soluble porphyrins and protoporphyrin IX in 5-aminolevulinic-acid-incubated carcinoma cells.

The bioconversion of 5-aminolevulinic acid (ALA) into hydrophobic protoporphyrin IX and other water-soluble porphyrins was investigated in Ehrlich ascite carcinoma (EAC) cells and in a myeloma cell line. The effects of irradiation (514 nm), temperature, incubation time and added glucose on the relative porphyrin concentrations (protoporphyrin vs. water-soluble porphyrins) were examined. Variations in these parameters induced a change in the amount of water-soluble porphyrins relative to protoporphyrin IX. The main component of the hydrophilic porphyrins was found to be uroporphyrin (Up), with minor components of coproporphyrin (Cp) and other carboxyporphyrins. The enhanced production of water-soluble porphyrins appears to be associated with alterations in the activities of the various enzymes in the heme biosynthetic pathway, resulting, for example, in the reduction in the activity of mitochondrial enzymes.

Variation in the fluorescence decay properties of haematoporphyrin derivative during its conversion to photoproducts.

Haematoporphyrin derivative (HpD) photoproducts are formed in aqueous solutions during light exposure in the presence of oxygen. The evaluation of the fluorescence decay of the photoproduct-enriched HpD solution shows an increase in the short-lived components, especially about 2 ns, in comparison with HpD without photoproducts. The bleaching of the HpD fluorescence and the photoproduct formation by the fluorescence-exciting radiation has to be taken into account in the evaluation of stationary as well as time-resolved fluorescence measurements.

[Laser-induced fluorescence diagnosis of tumors exemplified by solid Ehrlich carcinoma]. / Laserinduzierte Tumor-Fluoreszenzdiagnostik am Beispiel des soliden Ehrlichkarzinoms.

The laser-induced fluorescence of hematoporphyrin derivative (HpD) sensitized experimental Ehrlich carcinoma on mice is investigated. The fluorescence is excited with the 364-nm argon laser line. The tumor fluorescence spectra are recorded in the red spectral region. The HpD induced fluorescence of the tumors is superimposed on the autofluorescence of tissue. The tumor fluorescence shows a maximum 4 h after i.p. application of HpD. The fluorescence intensity is reduced by strong excitation causing bleaching of the HpD absorption. The fluorescence spectrum is also changing and the generation of a fluorescent photoproduct is observed.

[Photochemotherapy of experimental tumors in animals using various photosensitizers]. / Photochemotherapie an tierexperimentellen Tumoren mit verschiedenen Photosensibilisatoren.

Different colouring matters with high molar extinction coefficient were tested at the Friedrich Schiller University with regard to their photodynamic efficacy. Test system was the rapid-growing solid Ehrlich carcinoma. Methylen-blue, hematoporphyrin derivative Halle and chlorophyllin produce a markedly reduction of the tumor weight. In the presence of hypoxic areas an additional application of nitroimidazols is possible. A pilot laser unit for photochemotherapy of urinary bladder tumors is presented.

[Laser-assisted transurethral resection of ureteral occlusions]. / Lasergestützte transurethrale Resektion von Ureterverschlüssen.

After transurethral resection across the ureteral ostium a scared stenosis or complete occlusion is possible and its transurethral recanalization is dangerous. Therefore, a new concept with utilisation of laser light is presented. After percutaneous renal punction the laser light fiber is placed antegrade into the ureter near the stenotic ostium and the stenosis is resected transurethrally under fluoroscopy onto the laser light spot. Thereafter a splint is placed transrenal into the bladder for 3 weeks. The presented technique could minimize the operative expense.

In vivo autofluorescence investigations on animal tumors.

The in vivo fluorescence behavior of unmarked tissue (autofluorescence) of tumor-bearing mice was investigated. An argon-ion laser was used as the excitation source emitting radiation at 351 nm and 364 nn. Fluorescence bands in the red spectral range were recorded. The spectral distribution of the fluorescence of the solid Ehrlich carcinoma was different from that of the neighboring tissue.

Photodynamic therapy with haematoporphyrin derivative on mice with solid Ehrlich carcinomas.

The tumor-toxic effect of the photosensitizer HpD/Halle activated by laser radiation was demonstrated. Solid Ehrlich carcinoma was used as model. Investigations into the tumor growth, the HpD-distribution after different kinds of drug administration by means of fluorescence measurements, the phototoxicity and the reduction of tumor masses after HpD application and laser irradiation were carried out.

[Hematoporphyrin derivative-induced tumor fluorescence]. / Hämatoporphyrinderivat-induzierte Tumorfluoreszenz.

Detection of HpD-sensitized tumour was successful by use of HBO 200 mercury lamp, Argon ion laser, as well as for N2-pumped dye laser. After 20 hours period of HpD application the localization of the Ehrlich ascites tumour in mice was identified macroscopically and by spectroscopy. To exclude red emissions of detection sources the use of dye lasers is recommend.

Photochemotherapy of animal tumors with the photosensitizer methylene blue using a krypton laser.

The cytotoxic effect of the photosensitizer Methylene Blue activated with long wavelength laser radiation has been investigated using mice with solid Ehrlich carcinomas. A krypton laser was used as a light source emitting laser radiation at 647 nm and 676 nm. The red spectral range is important for photochemotherapy because it is easily transmitted through tissue. A significant tumor reduction, including complete tumor destruction, has been achieved.

Intracavity pulse compression with glass: a new method of generating pulses shorter than 60 fsec.

The introduction of a glass prism in a ring dye laser is shown to provide simultaneous wavelength selection and pulse compression.