Objective: When a stereotactic biopsy is taken to enable histopathological diagnosis of a suspected brain tumor, it is
essential to i) do this safely, that is not injure a major blood vessel and ii) to obtain relevant vital material from the
tumor. We are investigating the suitability of Indocyanine Green (ICG) fluorescence for blood vessel recognition and 5-
Aminolevulinic acid (5-ALA) induced Protoporphyrin IX (PpIX) fluorescence for identification of proliferative brain
Methods: A fiber-optic endoscopic approach was studied to generate and detect both fluorescence signals. PpIX
concentrations in brain tumors have been measured by chemical extraction. Preliminary equipment was studied in a
Results: PpIX-concentrations in glioblastoma tissue showed high inner- and inter-patient variability, but each patient
out of 15 with interpretable data showed at least one sample with a PpIX-concentration exceeding 2.4 μmol/l, which is
easily detectable by state-of-the-art fiberoptic fluorescence spectroscopy and imaging. The imaging fluoroscope with
30,000 pixels resolution could be introduced through a position controlled stereotactic needle. ICG-fluorescence from
vessels with diameters ≥ 0.1 mm can be detected with a contrast of 2-2.5 against surrounding tissue.
Conclusion: Fluorescence detection during stereotactic biopsy might increase safety and precision of the procedure
Objective: Provide preclinical data on the feasibility of 5-aminolevulinic acid (5-ALA) -based photodetection (PD) and
Photodynamic Therapy (PDT) of early childhood tumors.
Methods: Hepatoblastoma (HuH6), neuroblastoma (MHH-NB11) and N1-fibroblast cell lines were tested for their
relative capacities to synthesize Protoporphyrin IX (PpIX) from 5-ALA and for their susceptibility to PDT in vitro.
HuH6-cells were also inoculated in the peritoneum of rats. The pharmacokinetics of porphyrin accumulation was
measured in 9 rats by laparoscopic spectroscopy. 5-ALA was applied by i.p. injection of 500 mg/kg bw. In another 21
animals, tumors (n=20), liver (n=5) and peritoneum (n=4) were treated by PDT laparoscopically. 48 h after irradiation,
animals were again incubated with 5-ALA and then sacrificed and tissues were removed for further investigation.
Results: Both tumor cell lines showed higher levels of porphyrin fluorescence than the fibroblasts. Cell viability testing
proved the HuH6 cells to be most susceptible to PDT. Pharmacokinetic measurements of PpIX in xenografted tumors
showed a peak at 80-200 min after i.p. injection of 5-ALA. Irradiation resulted in pronounced photobleaching at all
irradiated sites and necrosis of tumor and liver tissue, whereas peritoneum appeared to remain unaffected. Necrosis
induced by PDT could be seen in fluorescence microscopy due to the lack of porphyrin synthesis in necrotic tissue after
the re-incubation with 5-ALA.
PpIX induced by administration of ALA is being successfully employed for tissue diagnosis and photodynamic therapy (PDT) of, for example, brain malignancies. To guide tissue biopsy by fluorescence during stereotaxy, correct quantification of the PpIX accumulation is required. However, the detected fluorescence intensity and spectral shape are influenced and distorted by the varying optical properties of tissue. Quantitative PpIX measurements thus need to disentangle these effects in order to provide the undistorted, intrinsic fluorescence. Numerous methods for obtaining the intrinsic fluorescence have been developed and optimized for certain fluorochromes. PpIX poses a particular case where excitation and fluorescence are spectrally well separated. Furthermore, the fluorescence appears within the red wavelength region where absorption in tissue is relatively weak.
Here, three experimental approaches towards assessing the intrinsic fluorescence for PpIX in homogeneous phantom materials for four subsets at tissue-like conditions, were tested and compared; 1) single-fiber with diameters 200-800 μm, 2) a two-fiber probe with evaluation based on an empirical ratio between fluorescence and reflectance signals or 3) a multi-fiber probe for combined fluorescence and reflectance measurements with evaluation based on a theoretical model of light propagation. All methods could be realized with an outer probe diameter of less than 1.5 mm, thus applicable during stereotaxy. Method 3 could quantify the PpIX concentration best, regarding all four subsets and thus covering a broad, physiologically relevant range of optical parameters. With accuracies between ± 3.2 % and ± 24.8 % for different subsets it was overall a great improvement to the accuracies resulting from calculations based on a plane wave geometry, which vary from ± 4.6 % to ± 84.3 %.
Protoporphyrin IX (PpIX) displays high tumour-selective uptake following oral administration of 5-aminolaevulinic acid
(ALA), a fact that is being exploited for the fluorescence-guided resection (FGR) and photodynamic therapy (PDT) of
human brain malignancies. A clinical procedure for interstitial PDT (iPDT) has been established including pre-treatment
planning, optical fiber insertion under stereotactic guidance and therapeutic irradiation at non-thermal fluence rates. We
have previously reported on median survival in the range of 15 months and the existence of some intriguing long-term
survivors (>5 years) following iPDT. Such successful treatments rely on for example sufficient light, PpIX and oxygen
levels. We have investigated the absolute PpIX concentration as well as the PDT-induced photobleaching kinetics in
brain tissues. Tissue samples acquired during FGR contained PpIX concentrations up to 28 μM. This observation implies
that ALA-induced PpIX levels are sufficient for inducing PDT effects in viable tumour tissue upon therapeutic
irradiation. However, regions of pre-existing necrosis were characterised by significantly lower photosensitiser levels.
Fluorescence spectroscopy was implemented in parallel to iPDT with the aim to employ PpIX photobleaching as a tool
for realtime treatment supervision and early treatment prognosis.
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/cm<sup>2</sup>. 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
Bladder cancer (BC) is among the most expensive oncological diseases. Any improvement in diagnosis or therapy
carries a high potential for reducing costs. Fluorescence cystoscopy relies on a selective formation of Protoporphyrin IX
(PpIX) or more general photoactive porphyrins (PAP) in malignant urothelium upon instillation of 5-aminolevulinic acid
(5-ALA) or its hexyl-derivative h-ALA. Fluorescence cystoscopy equipment has been developed with the aim to
compensate for the undesired distortion caused by the tissue optical properties by displaying the red fluorescence
simultaneously with the backscattered blue light. Many clinical studies proved a high sensitivity in detecting flat
carcinoma in situ and small papillary malignant tumours. As a result, recurrence rates were significantly decreased in
most studies. The limitation lies in a low specificity, caused by false positive findings at inflamed bladder wall. Optical
coherence tomography (OCT) is currently being investigated as a promising tool to overcome this limitation.
H-ALA-PDT (8 or 16 mM h-ALA in 50 ml instillation for 1-2 h, white light source, catheter applicator) has recently
been investigated in a phase I study. 17 patients were applied 100 J/cm<sup>2</sup> (3 patients received incrementing doses of 25 -
50 - 100 J/cm<sup>2</sup>) during approx. 1 hour irradiation time in 3 sessions, 6 weeks apart. PDT was performed without any
technical complications. Complete photobleaching of the PpIX-fluorescence, as intended, could be achieved in 43 of 45
PDT-sessions receiving 100 J/cm<sup>2</sup>. The most prominent side effects were postoperative urgency and bladder pain, all
symptoms being more severe after 16 mM h-ALA. Preliminary evaluation shows complete response assessed at 3
months after the third PDT-session (i.e. 6 months after first treatment) in 9 of 12 patients. 2 of these patients were free of
recurrence until final follow-up at 84 weeks.
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.