Simon Wagner

Intraoperative detection of malignant gliomas by 5-aminolevulinic acid-induced porphyrin fluorescence

OBJECTIVE Survival after surgery and radiotherapy for the treatment of malignant gliomas is linked to the completeness of tumor removal. Therefore, methods that permit intraoperative identification of residual tumor tissue may be of benefit. In a preliminary investigation, we have studied the value of fluorescent porphyrins that accumulate in malignant tissue after administration of a precursor (5-aminolevulinic acid) for labeling of malignant gliomas in nine patients. METHODS Three hours before the induction of anesthesia, 10 mg 5-aminolevulinic acid/kg body weight was administered orally. Intraoperatively, red porphyrin fluorescence was observed with a 455-nm long-pass filter after excitation with violet-blue (375-440 nm) xenon light and was verified by analysis of fluorescence spectra. Fluorescing and nonfluorescing samples taken from the tumor perimeters were examined histologically or used to study the photobleaching of porphyrins by excitation light and white light from the operating microscope. Plasma and erythrocyte porphyrin levels were determined by fluorescence photometry. RESULTS Normal brain tissue revealed no porphyrin fluorescence, whereas tumor tissue was distinguished by bright red fluorescence. For a total of 89 tissue biopsies, sensitivity was 85% and specificity was 100% for the detection of malignant tissue. For seven of nine patients, visible porphyrin fluorescence led to further resection of the tumor. Under operating light conditions, fluorescence decayed to 36% in 25 minutes for violet-blue light and in 87 minutes for white light. Plasma and erythrocyte porphyrin contents increased slightly, without exceeding normal levels. CONCLUSION Our observations suggest that 5-aminolevulinic acid-induced porphyrin fluorescence may label malignant gliomas safely and accurately enough to enhance the completeness of tumor removal.

QUANTIFICATION OF 5-AMINOLEVULINIC ACID INDUCED FLUORESCENCE IMPROVES THE SPECIFICITY OF BLADDER CANCER DETECTION

PURPOSE 5-Aminolevulinic acid induced fluorescence endoscopy has outstanding sensitivity for detecting early stage bladder cancer. Nevertheless, a third of the lesions that show specific fluorescence are histologically benign. We decreased the false-positive rate of 5-aminolevulinic acid induced fluorescence endoscopy by incorporating protoporphyrin IX fluorescence quantification into the standard cystoscopy procedure. MATERIALS AND METHODS In 25 cases (53 biopsies) of a history of or suspicion for bladder cancer 5-aminolevulinic acid induced fluorescence endoscopy and fluorescence image quantification were performed. For fluorescence image quantification images obtained with a target integrating color charge-coupled device camera were digitized and stored in a personal computer. Red-to-blue ratios were calculated from fluorescence positive lesions and results were correlated with hematoxylin and eosin histology. RESULTS Malignant fluorescence positive lesions showed significantly stronger fluorescence intensity than fluorescing lesions with benign histology. A threshold was established that decreased the false-positive rate by 30% without affecting sensitivity. CONCLUSIONS Fluorescence image quantification is a new endoscopic method for objectively selecting multicolor fluorescence bladder lesion images for biopsy. It has the potential of eliminating human error by different surgeons with variable experience in fluorescence endoscopy.

5-Aminolaevulinic Acid (ALA) for the Fluorescence Detection of Bronchial Tumors.

At the moment only early detection of lung cancer offers a good prognosis for the patients. Conventional white light endoscopy is mostly insufficient for early diagnosis. Therefore we developed a system of fluorescence diagnosis using 5-aminolaevulinic acid (ALA) exogeneously applied. As precursor of the heme synthesis it is metabolized to protoporphyrin IX – a red fluorescent substance. Therefore protoporphyrin IX accumulates in tumorous and premalignant tissue, and can be directly visualized by fluorescence bronchoscopy. Excitation with blue light (380–435 nm) causes a red fluorescence, which can be detected after filtering most of the blue component with the naked eye or a camera system. After earlier work with laser systems and cold light sources we now use the system D-Light AF for the fluorescence diagnosis using ALA-induced protoporphyrin IX fluorescence.

Clinical Determination of Tissue Optical Properties in vivo by Spatially Resolved Reflectance Measurements

For many clinical light applications, such as photodynamic therapy (PDT), the therapeutic effect strongly depends on the light dose in a certain tissue depth. A measure for the attenuation and penetration of light in tissue is the optical penetration depth, which is derived from the tissue’s optical properties at a certain wavelength. Therefore, in vivo measurements to determine the optical properties were performed of the bladder wall (n = 12) and brain tissue (n = 11) on patients undergoing photodynamic therapy. The tip of a 400 μm bare fiber was placed in contact with the investigated tissue, either during open surgery (brain) or through the working channel of a cystoscope (bladder wall). Light of the wavelengths 420-450 nm, 532 nm, and 635 nm was coupled alternately into the fiber. The diffuse backscattered light was detected spatially resolved by means of a CCD camera. Additionally, the total diffuse reflectance of the tissue site was determined, by relating the white light spectra remitted from the tissue to that of a reflectance standard. These two independent measurements were fitted with Monte Carlo simulations. Thus, the reduced scattering and absorption coefficient could be obtained and the optical penetration depth was derived. The presented investigations showed that spatially resolved diffuse reflectance in combination with total diffuse remission provides a valuable method to determine tissue optical properties in vivo. Two human organs were analyzed with this technique and both, bladder wall tissue and brain tissue showed reproducible results.

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.

New developments in fluorescence detection of ALA-induced protoporphyrin IX for cancer localization

After the very promising clinical results for the detection of bladder cancer in urology, preclinical and clinical studies on aminolevulinic acid (5-ALA) induced protoporphyrin IX (PPIX) are preformed in various disciplines now. This paper provides a brief overview of the progress on 5-ALA assisted fluorescence diagnosis in urology, pulmonology, neurosurgery, gynecology and ENT performed in collaboration with the Laser Research Laboratory at the Department of Urology of the Ludwig-Maximilians-University in Munich. Five-ALA can be applied either topically or systemically to induce an intracellular accumulation of fluorescing PPIX. With appropriate dosage of 5-ALA, malignant tissue can be stained selectively, and irradiation with violet light excites a bright red fluorescence of the tumor. Optical properties of the tissue tend to hamper the precise identification and demarcation of suspect areas in fluorescence images. Multicolor remission and fluorescence imaging, therefore, seems to be indispensable for a reliable tumor localization.

Photodynamic investigation of mucosa-associated human papillomavirus efflorescences

Each year, < 30 million patients worldwide develop clinical human papillomavirus (HPV) infections in the form of condylomata acuminata. However, these productive infections only account for a small proportion of all HPV infections; far more infections either have a subclinical course or are latent, i.e. they cannot be diagnosed clinically [1]. Whereas latent HPV infections are, at most, theoretically responsible for direct sexual transmission to the partner or spreading of infection on the patients skin, the virulence of clinical and subclinical HPV infections has been con®rmed epidemiologically. Subclinical HPV infections, which are often not diagnosed and treated because they have been overlooked, are generally considered responsible for the high rates of recurrence and increasing prevalence of HPV infections. These subclinical HPV infections can be rendered relatively visible and thus treatable on the keratinized skin of the external genitals with 5% acetic acid and magni®cation techniques (a hand lens or colposcopy). In urology, such ef ̄orescences are found in the urethra; in otorhinolaryngology they are found in the oropharynx, and in gynaecology on the vagina and cervix. Herein we present a method for detecting mucosa-associated HPV ef ̄orescences, i.e. photodynamic diagnostics (PDD), using ̄uorescence after the topical application of 5-aminolaevulinic acid (ALA); examples of the clinical application of PDD in the urethra are presented.

Fluoreszenztumorlokalisierung nach Applikation von 5-Aminolävulinsäure — Technische Grundlagen

Nach Verabreichung von 5-Aminolavulinsaure (5-ALA) kommt es zu einer meist tumorselektiv erhohten intrazellularen Akkumulation von fluoreszierendem Protoporphyrin IX (PPIX). Durch eine endoskopische Fluoreszenzanregung von PPIX konnen somit Tumorgrenzen and Tumorfruhstadien besser aufgespurt werden [1]. Fur eine klinisch praktikable Fluoreszenzdarstellung erwies es sich als vorteilhaft, neben dem roten Fluoreszenzlicht von PPIX auch einen Teil des blauen Anregungslichts abzubilden. Damit ist nicht nur eine bessere Orientierung gewahrleistet, sondern auch eine vereinfachte Erkennung fluoreszenzpositiver Areale uber einen rot/blau Farbkontrast.

Fluoreszenztumorlokalisierung nach Applikation von 5-Aminolävulinsäure: Quantitative Bildanalyse

Die Fluoreszenzdiagnostik von Harnblasenkarzinom ist mit einer Rate von falsch fluoreszenzpositiven Befunden von ca. 35% behaftet. Mit Hilfe der Quantifizierung der Gewebefluoreszenz wird — an 19 Patienten and 41 Biopsien — gezeigt, das die Fluoreszenzintensitat falsch fluoreszenzpositiver Befunde signifikant medriger ist als der richtig positiver Befunde. Mit einem geeigneten Schwellwert konnen 33% der falsch positiven, das heist benignen Biopsien eindeutig von den malignen Biopsien differenziert werden. Eine Anhebung des Schwellenwertes, verbunden mit einer Reduktion der Rate der falsch positiven auf 66% erscheint moglich, wenn man morphologische Kriterien in der Begutachtung mit berucksichtigt.