Interstitial Photodynamic Therapy Using 5-ALA for Malignant Glioma Recurrences.

Interstitial photodynamic therapy (iPDT) using 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) as a cytotoxic photosensitizer could be a feasible treatment option for malignant gliomas. In a monocentric cohort of consecutive patients treated between 2006 and 2018, a risk profile analysis of salvage iPDT for local malignant glioma recurrences and associated outcome measures are presented here. It was considered indicated in patients with circumscribed biopsy-proven malignant glioma recurrences after standard therapy, if not deemed eligible for safe complete resection. A 3D treatment-planning software was used to determine the number and suitable positions of the cylindrical diffusing fibers placed stereotactically to ensure optimal interstitial irradiation of the target volume. Outcome measurements included the risk profile of the procedure, estimated time-to-treatment-failure (TTF), post-recurrence survival (PRS) and prognostic factors. Forty-seven patients were treated, of which 44 (median age, 49.4 years, range, 33.4-87.0 years, 27 males) could be retrospectively evaluated. Recurrent gliomas included 37 glioblastomas (WHO grade IV) and 7 anaplastic astrocytomas (WHO grade III). Thirty (68.2%) tumors were O-6-methylguanine-DNA methyltransferase (MGMT)-methylated, 29 (65.9%)-isocitrate dehydrogenase (IDH)-wildtype. Twenty-six (59.1%) patients were treated for their first, 9 (20.5%)-for their second, 9 (20.5%)-for the third or further recurrence. The median iPDT target volume was 3.34 cm3 (range, 0.50-22.8 cm3). Severe neurologic deterioration lasted for more than six weeks in one patient only. The median TTF was 7.1 (95% confidence interval (CI), 4.4-9.8) months and the median PRS was 13.0 (95% CI, 9.2-16.8) months. The 2- and 5-year PRS rates were 25.0% and 4.5%, respectively. The treatment response was heterogeneous and not significantly associated with patient characteristics, treatment-related factors or molecular markers. The promising outcome and acceptable risk profile deserve further prospective evaluation particularly to identify mechanisms and prognostic factors of favorable treatment response.

Spectrophotometric evaluation of hemolysis in plasma by quantification of free oxyhemoglobin, methemoglobin, and methemalbumin in presence of bilirubin.

Severe intravascular hemolysis leads to the simultaneous presence of free heme pigments (oxyhemoglobin, methemoglobin, and methemalbumin) and bilirubin in human plasma. Standard spectrophotometric methods used to assess in vivo hemolysis inadequately address this complex analytical situation. Thus, we propose a novel quantification algorithm to ensure the highest analytical specificity. A corresponding second-derivative fitting algorithm was validated according to the guideline of bioanalytical method validation from the European Medicines Agency using plasma specimens (n = 1759) spiked with different concentrations of oxyhemoglobin and methemoglobin. The results were compared to standard spectrophotometric quantification methods described by Harboe, Noe, and Fairbanks. Based on the second-derivative method, simultaneous quantification of oxyhemoglobin and methemoglobin/methemalbumin in samples with total bilirubin concentrations ≤4.9 mg/dL (83.8 µmol/L) provided robust results (inaccuracy ≤20%, imprecision ≤16%). Analyzing UV/VIS spectra of plasma from patients with confirmed severe intravascular hemolysis evidenced an underestimation of up to 33% for the combined free heme pigment content. The employed second-derivative algorithm allows for automated and highly specific quantification of the free heme pigment content in diluted human plasma, which cannot be realized with standard spectrophotometric evaluation methods. An Excel-based tool readily applicable to clinical datasets accompanies this manuscript.

Interrelation between Spectral Online Monitoring and Postoperative T1-Weighted MRI in Interstitial Photodynamic Therapy of Malignant Gliomas.

In a former study, interstitial photodynamic therapy (iPDT) was performed on patients suffering from newly diagnosed glioblastoma (n = 11; 8/3 male/female; median age: 68, range: 40-76). The procedure includes the application of 5-ALA to selectively metabolize protoporphyrin IX (PpIX) in tumor cells and illumination utilizing interstitially positioned optical cylindrical diffuser fibers (CDF) (2-10 CDFs, 2-3 cm diffusor length, 200 mW/cm, 635 nm, 60 min irradiation). Intraoperative spectral online monitoring (SOM) was employed to monitor treatment light transmission and PpIX fluorescence during iPDT. MRI was used for treatment planning and outcome assessment. Case-dependent observations included intraoperative reduction of treatment light transmission and local intrinsic T1 hyperintensity in non-contrast-enhanced T1-weighted MRI acquired within one day after iPDT. Intrinsic T1 hyperintensity was observed and found to be associated with the treatment volume, which indicates the presence of methemoglobin, possibly induced by iPDT. Based on SOM data, the optical absorption coefficient and its change during iPDT were estimated for the target tissue volumes interjacent between evaluable CDF-pairs at the treatment wavelength of 635 nm. By spatial comparison and statistical analysis, it was found that observed increases of the absorption coefficient during iPDT were larger in or near regions of intrinsic T1 hyperintensity (p = 0.003). In cases where PpIX-fluorescence was undetectable before iPDT, the increase in optical absorption and intrinsic T1 hyperintensity tended to be less. The observations are consistent with in vitro experiments and indicate PDT-induced deoxygenation of hemoglobin and methemoglobin formation. Further investigations are needed to provide more data on the time course of the observed changes, thus paving the way for optimized iPDT irradiation protocols.

Fluorescence and Treatment Light Monitoring for Interstitial Photodynamic Therapy.

In interstitial photodynamic therapy, light is distributed to the tumor via light diffusers. The light dose and the related phototoxic effect achieved throughout the target volume critically depend on absorption, scattering and diffuser positioning. Using liquid tissue phantoms, we investigated the dependencies of treatment light transmission and protoporphyrin IX (PpIX) fluorescence on these parameters. This enabled monitoring hemoglobin oxygenation and methemoglobin formation during irradiation (635 nm, 200 mW cm-1 diffuser length). Starting with two parallel cylindrical diffusers at 10 mm radial separation, the light transmitted between the fibers was largely determined by the minimal distance between the diffusers, but rather insensitive to an additional axial displacement or tilting of one fiber with respect to the other. For fixed distance between the diffusor centers, however, tilting up to direct contact resulted in a 10-fold signal increase. For hemoglobin within erythrocytes, irradiation leads to photobleaching of PpIX without marked change in hemoglobin oxygenation until hemolysis occurs. Afterward, hemoglobin is rapidly deoxygenized and methemoglobin is formed, leading to a dramatic increase in absorption. For lysed blood, these effects start immediately. A comparison of intraoperative monitoring of the signals with the experimental results might help prevent insufficient treatment by reconsidering treatment planning or prolonging irradiation.

Rapid spectrophotometric quantification of urinary porphyrins and porphobilinogen as screening tool for attacks of acute porphyria.

Autosomal-dominant acute porphyria, a group of rare diseases, can lead to life-threatening neurovisceral attacks. No efficient screening test is available today. Elevated urinary porphobilinogen in addition to elevated porphyrins is highly specific for an attack of acute porphyria. This study proposes and evaluates a custom-made device, algorithm, and methods for a two-step quantification of urinary porphyrins and porphobilinogen. The first step is oxidation of the nonfluorescent porphyrinogens and subsequent fluorescence-spectroscopic determination of total urinary porphyrins (TUP) using second derivative spectral fitting. Photo-oxidation is compared with chemical oxidation methods. The second step is the quantification of porphobilinogen in case of elevated TUP. Heat-induced conversion products of porphobilinogen, namely uroporphyrin and porphobilin, are quantified by fluorescence and absorption spectroscopy. Results show that the preferred method combination is TUP quantification (lower limit of quantification: 0.2 µmol / L) after photo-oxidation with subsequent absorption-spectroscopic determination of porphobilin after heating for indirect quantification of porphobilinogen (quantification range: 0 to 20 mg / L). Urinary porphobilinogen and porphyrins of one acute porphyria patient were quantified with <10 % deviation from an external reference determination. The spectrophotometric approach requires only minimal sample processing and yields a result within 15 min, thus closing the screening gap for acute porphyria.