Susanne Stocker

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

OBJECTIVEnSurvival 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.nnnMETHODSnThree 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.nnnRESULTSnNormal 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.nnnCONCLUSIONnOur observations suggest that 5-aminolevulinic acid-induced porphyrin fluorescence may label malignant gliomas safely and accurately enough to enhance the completeness of tumor removal.

In vitro and in vivo porphyrin accumulation by C6 glioma cells after exposure to 5-aminolevulinic acid

Several malignant tissues synthesize endogenous porphyrins after exposure to 5-aminolevulinic acid (5-ALA). The present experiments have been designed to elucidate whether the C6 glioma cell, a model cell for human malignant glioma, similarly synthesizes porphyrins when exposed to 5-ALA, and whether specific synthesis occurs when C6 cells are inoculated into rat brains to form a tumor. In this situation the blood-brain barrier may interfere with 5-ALA availability, and spreading of porphyrins with edema outside the tumor may occur. Flow cytometry is used to determine the course of cell volume and porphyrin fluorescence intensities in cultured C6 cells which are incubated in 1 mM 5-ALA. For the induction of experimental brain tumors, 10(4) untreated C6 cells are inoculated into the brains of rats. After 9 days animals receive 100 mg 5-ALA/kg body weight. Brains are removed after 3, 6, or 9 h and frozen coronal sections obtained for H/E staining or fluorescence spectography. Cultured C6 cells show a linear increase of protoporphyrin IX fluorescence after exposure to 5-ALA, which begins to plateau after 85 min. Marked fluorescence is also observed in solid and infiltrating experimental tumor. However, faint fluorescence also occurs in normal tissue, basal pia, choroid plexus, and, more obviously, in white-matter tracts bordering the tumor (maximal distance: 1.5 +/- 0.7 mm). The observations demonstrate that C6 cells synthesize protoporphyrin IX after exposure to 5-ALA in vitro and in vivo. However, when utilizing 5-ALA for fluorescence detection or photodynamic therapy of brain tumors, attention should be paid to the possibility of protoporphyrin IX occurring outside the tumor.

Pharmacokinetics of 5-aminolevulinic-acid-induced porphyrins in tumour-bearing mice

Photodynamic therapy and photodynamic diagnosis help to support efficient treatment of superficial and early-stage cancer. During the last few years, 5-aminolevulinic acid (5-ALA), a precursor of haemoglobin in the haem biosynthetic pathway, was used to stimulate endogenous porphyrin production. In the following the time dependence of 5-ALA-induced porphyrin concentration will be investigated on several tissues in an in-vivo tumour model. 5-ALA was administered intravenously at a concentration of 50 mg-1 body weight. According to a certain time schedule the animals were sacrificed and 12 different organs as well as the tumour were removed. During excitation with the violet light of a Kr+ laser, porphyrin fluorescence spectra in the range 550-750 nm could be detected on the tissue samples. The intensity of the emission spectra at lambda = 635 +/- 2 nm was taken as a measure of the porphyrin concentration. All tissues showed porphyrin fluorescence. Brightest fluorescence was found on the tumour. A maximum contrast of the fluorescence intensity between the tumour and the non-malignant organs of up to 30 was observed at 4-6 h post-injection. The kinetics of the porphyrin concentration depend on the organ. Simple mathematical models will be derived and discussed.

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

ObjectiveAminolevulinic 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.MethodsSpheroids were incubated for 4xa0h in 100xa0μg/ml ALA in 5% CO2 in room air or 95% O2 with subsequent irradiation using a diode laser (λxa0=xa0635xa0nm, 40xa0mW/cm2, total fluence 25xa0J/cm2). 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 8xa0days after PDT.ResultsPDT after incubation in 5% CO2 provided incomplete cell death and growth delay in spheroids of >350xa0μm diameter. However, complete cell death and growth arrest occurred in smaller spheroids (<350xa0μm). Incubation in 95% O2 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.ConclusionsPDT of experimental glioma results in rapid and significant cell death that could be verified as acute necrosis immediately after irradiation. This effect depended on O2 concentration and spheroid size.

In vivo fluorescence kinetics of porphyrins following intravesical instillation of 5-aminolaevulinic acid in normal and tumour-bearing rat bladders

The fluorescence kinetics of protoporphyrin IX (PPIX) following intravesical instillation of 5-aminolaevulinic acid (5-ALA) have been studied in vivo in a rat bladder tumour model. 5-ALA dissolved in NaHCO3 was intravesically instilled for 60 min in tumour-bearing and normal bladders of Wistar rats. The fluorescence was excited with the violet lines of a Kr(+)-laser and recorded in vivo by means of a fibre coupled optical multichannel analyser. The fluorescence emission bands of PPIX at lambda = 636 nm and lambda = 708 nm were detected in normal and tumorous urothelium after only 30 min. The maximum fluorescence intensity was obtained in tumorous and normal urothelium 3-4 h after instillation. The ratio of the fluorescence intensity in tumorous to normal urothelium decreased continuously from four to about two during the time range of 6 h. PPIX fluorescence following 5-ALA instillation could also be observed in kidney and liver. Fluorescence from further porphyrin species with emission bands at lambda = 617 nm and lambda = 682 nm was detected in the bladder, indicating an efflux of hydrophilic porphyrins from the hepatic pathway.

Distribution of labelled anti-tenascin antibodies and fragments after injection into intact or partly resected C6-gliomas in rats

Introduction: For treatment of malignant glioma, radioimmunotherapy has become a valuable alternative for more than 2xa0decades. Surprisingly, very little is known about the distribution of intralesionally administered labelled antibodies or fragments. We investigated the migration of labelled antibodies and antibody fragments injected into intact and partly resected C6-glioma in rats at different times after injection. Materials and methods: Nine days after induction of a C6-glioma, 5xa0μl of 125I-labelled murine anti-tenascin antibodies (n=31) or 125I-labelled fragments of anti-tenascin antibodies (n=32) was injected slowly into the tumour (group I). In group II the tumour was subtotally resected 9xa0days after induction of the C6-glioma, and 24xa0h later the labelled antibodies (n=30) or fragments (n=12) were injected into the resection cavity. At 6, 24 or 48xa0h after the injection, animals were sacrificed, and brains removed. Distribution of labelled antibodies and fragments was determined by superimposing autoradiographs onto frozen sections and HE-stained neighbouring sections using a digital image analysing system. Results: After injection into intact C6-glioma, labelled antibodies covered a maximum distance of 3.2xa0±xa01.0, 4.1 ± 1.9 and 4.8xa0±xa00.9xa0mm after 6, 24 and 48xa0h, respectively; while labelled fragments were found at a distance of 6.7xa0mm (±1.1) after 24xa0h and 5.8xa0mm (±0.9) after 48xa0h (significant in univariate analysis). Following partial tumour resection, the respective distances at 24xa0h were 3 ± 0.4xa0mm for anti-tenascin antibodies and 3.4xa0±xa00.3xa0mm for Fab fragments. Conclusion: After injection into C6-glioma, labelled fragments are able to cover a greater distance than labelled antibodies. Injection of antibodies and fragments 1xa0day after tumour resection results in reduced velocity of both antibodies and fragments.

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.

Clinical experiences with fluorescence guided resection of malignant glioma

ABSTRACT Survival after surgery and radiotherapy of malignant glioma is closely linked to the completeness of tumor removal.Therefore methods are needed that permit intra-operative identification of tumor border lines. For tumor staining 5-ALAinduced PPIX is used. The influence of fluorescence guided resection on the survival is analyzed. 66 patients received 20mg/kg bodyweight 5-ALA per os 3 hours before anaesthesia. Fluorescence was visualized using a modified operatingmicroscope. Fluorescing tissue was removed as complete as possible. 72 hours after surgery MIII x97 images were taken andresidual enhancements quantified. Patients survival was analysed using the Kaplan-Meier method in correlation withKarnofsky-status, age, histology of tumor. These 66 patients were compared to 89 patients operated on between 1990 x97 1992. Complete resection ofmalignant and fluorescing tumor was obtained in 62 % of patients. Compared to the historicalgroup ofpatients survival using 5-ALA was significantly prolonged.Keywords: 5-aminolevulinic acid, fluorescence, malignant glioma

Intraoperativer Fluoreszenznachweis von 5-ALA-induziertem PPIX im malignen Gliom

Unter den Tumoren des zentralen Nervensystems weisen besonders die malignen Gliome (anaplastisches Astrozytom, Glioblastoma multiforme) eine sehr schlechte Prognose auf. Trotz Chirurgie mit kombinierter Radio- und Chemotherapie betragt die Uberlebensrate der betroffenen Patienten nach einem Jahr ca. 60% im Falle eines anaplastischen Astrozytoms, mit der Diagnose eines Glioblastoms etwa 27% (1).

Pharmacokinetic of 5-ALA-lnduced porphyrins in Tumourbearing Mice

Photodynamic diagnosis (POD) using fluorescence techniques is a sensitive method for localizing small tumours. Photodynamic therapy (PDT) add support to efficient treatment of superficial human malignancies [1). It was the aim of this study to investigate the time dependency of the porphyrin concentration in several types of tissues after i.v. application of S-ALA. Additionally the time dependency of the contrast between tumourous and non-malignant organs has been determined. A mathematical model was used to describe and to compare the observed kinetics.