Tobias Beck

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.

Fluorescence-guided resections and photodynamic therapy for malignant gliomas using 5-aminolevulinic acid

Oral application of 20 mg/kg bw of 5-aminolevulinic acid results in a highly specific accumulation of fluorescent and phototoxic Protoporphyrin IX in malignant glioma tissue. Surgical removal with fluorescence guidance is studied in a phase III clinical trial, adjuvant Photodynamic Therapy (PDT) to the surgical cavity is in phase II and for interstitial PDT of recurrent gliomas, a phase I/II study has started. Fluorescence guided resections have been shown to be safe and effective in augmenting neurosurgical removal of malignant gliomas in 52 consecutive patients. Intra-operative fluorescence spectroscopy showed statistically significant higher sensitizer accumulation in vital brain tumor versus the infiltration zone and in the infiltration zone versus adjacent normal brain, which contained very little PPIX. This is promisingly exploited for PDT - both to the surgical cavity by surface irradiation and for stereotactically guided interstitial irradiation.

ALA-mediated fluorescence-guided resection (FGR) and PDT of glioma

A summary of clinical trials employing photodynamic diagnosis (PDD) and photodynamic therapy (PDT) for the diagnosis and treatment of brain malignancies is presented. Intra-cavity PDT has been performed within the surgical cavity following FGR, employing oral administration of 5-aminolevulinic acid (5-ALA), either targeting fluorescing tissue regions that were not removed during FGR due to safety reasons (referred to as focal PDT, n=20) or illuminating the entire resection cavity (referred to as integral PDT, n=9). Both approaches proved technically feasible and safe. Spectroscopic measurements performed pre-, during and post-PDT revealed Protoporphyrin IX (PpIX)-photobleaching of more than 95% after the delivery of 200 J/cm2. This light dose did not induce any side effects. Furthermore, interstitial PDT (iPDT) has been employed within one feasibility trial (n=10) and one Phase I/II trial (n=15). Here, three to six cylindrical light diffusors (20-30 mm length, 200 mW/cm, 720 J/cm) were positioned within the target tissue under stereotactic guidance. Pre-treatment planning was performed with the intent to target the entire tumour volume with a sufficient light dose while also minimising the risk of any light-induced temperature increase. For the feasibility trial patients with small, recurrent gliomas were included, resulting in a median survival of 15 months as well as some unexpected longterm survivals (up to 5 years). The Phase I/II trial employed the same clinical procedures. Here, the 12-month survival was 35% and the median progression-free survival was 6 months. In summary, stereotactic iPDT in combination with treatment-planning could be shown to be a safe and feasible treatment modality. These trials are presently being extended to also include on-line monitoring of PpIX fluorescence and photobleaching kinetics. Preliminary data has revealed dramatically different PpIX levels and photobleaching kinetics. Such data could possibly be employed for realtime treatment monitoring and as an early prognostic marker for the PDT response.

Ex-vivo investigations on endoluminal vein treatment procedures

An ex-vivo model was developed for experimental evaluation of endoluminal thermal procedures for the occlusion of saphenous veins. The model consists of the subcutaneous foot vein from freshly slaughtered cows. Using this model primary and acute effects and initial mechanisms on vein vessel could be studied. In this study different energy sources (laser and radiofrequency generator), different energy application parameters (velocity, fluence, fluence rate, temperature) were compared. The dependency of using bare fibre and cylindrical diffusors could be investigated with respect to the induced effects on the vessels wall. Contraction of the vessels were measured and investigated macroscopically and microscopically as well as by means of optical coherence tomography. As a result an optimized treatment protocol could be developed and discussed with respect to the induced effects.

Fluorescence guided resection for malignant glioma using 5-aminolevulinic acid

Background: Oral application of 20 mg/kg body weight of 5-aminolevulinic acid (ALA) results in a highly specific accumulation of fluorescent Protoporphyrin IX (PpIX) in malignant glioma tissue. In a clinical phase II study, Photodynamic Therapy (PDT) to postsurgical tumor-remnants was monitored by fluorescence spectroscopy and photobleaching of PpIX was detected. Methods: Fluorescence spectra were measured from the tumor remnant pre and at intervals during PDT with 100, 150 and 200 J/cm2 and from adjacent normal cortex. The spectra obtained were fitted with spectra from pure PpIX, autofluorescence and photoproducts, the fit-parameters quantifying the contributions of these components to the measured spectrum. Results: The PpIX-fluorescence bleached to 8%, 16% and 1% of the initial intensity for the 100, 150 and 200 J/cm2-groups (median values). Photoproduct formation was minimal. PpIX-fluorescence of normal cortex was 6% of tumor remnant fluorescence (median, range: 0% to 49%).

A fluorescence diagnostic system detecting cancer-specific enzymatic activities: preliminary results

Colorectal carcinoma is one of the most frequent and deadliest tumors in the western world. The visualization of cancer-specific enzymatic activities could possibly improve sensitivity and specificity as compared to classical white-light endoscopy. DNase X, which is typically found in early lesions, and TKTL1, which identifies aggressive carcinomas with a high metastatic potential, could potentially constitute such cancer-specific enzymes. Here, fluorescent dyes have been developed in order to specifically detect these enzymatic activities. A fiber-based system was developed for the detection of small concentrations of fluorescent dyes in scattering and absorbing media. With the use of the reflectance spectrum and a theoretical model for the light distribution, the intrinsic fluorescence is assessed from the raw fluorescence. The resulting intrinsic spectrum shows only a weak dependence on the optical properties of the sample and its intensity correlates with the fluorophore concentration. Thus, small concentrations and small variations in the concentrations of the fluorescent dye can be measured. In conclusion, the presented fluorescence diagnostic system in combination with new fluorescent probes has the potential to distinguish between cancerous tissue samples with high enzymatic activity and non-cancerous tissue samples with lower enzymatic activity.