Radiation sterilisation of cultured human brain tumour cells for clinical immune tumour therapy.

The aim is to investigate the radiosensitivity of noninfected cultured human glioma cells to ascertain that intracutaneously administered cells are viable enough to produce interferon-gamma but not able to proliferate. Cell cultures were established from five patients undergoing brain tumour surgery. By karyotyping, we found four malignant (three glioblastoma multiforme (GBM), one giant cell glioma) and one normal. The cells were irradiated with (137)Cs-gamma rays at absorbed dose levels of 0, 20, 40, 60, 80, 100 and 120 Gy. The fraction of viable cells was examined by MTT incorporation assay. The average of the data obtained from three GBM cell cultures was fitted to an exponential model. The parameters were: extrapolation number n=0.85+/-0.10, mean lethal dose D(0)=12.4+/-3.2 Gy and an additional uncertainty parameter deltaS=0.14+/-0.03. By setting deltaS=0, the corresponding values of the parameters were n=0.86+/-0.16 and D(0)=30.0+/-8.1 Gy. The rate of proliferation was examined by (3)H-thymidine incorporation. The average of the proliferation data obtained from three GBM cell cultures was fitted to an exponential model yielding n=0.943+/-0.005 and D(0)=5.8+/-0.5 Gy for deltaS=0.057+/-0.005, and by setting deltaS=0, n=1.00+/-0.02 and D(0)=8.4+/-1.6 Gy. No outgrowth of plated cells was observed after 4 weeks at an absorbed dose of 100 Gy. This absorbed dose is recommended for irradiation of 2 x 10(6) glioma cells used for clinical immunisation.

Reference dosimetry at the neutron capture therapy facility at Studsvik.

The purpose of this publication was to present and evaluate the methods for reference dosimetry in the epithermal neutron beam at the neutron capture therapy facility at Studsvik. Measurements were performed in a PMMA phantom and in air using ionization chambers and activation probes in order to calibrate the epithermal neutron beam. Appropriate beam-dependant calibration factors were determined using Monte Carlo methods for the detectors used in the present publication. Using the presented methodology, the photon, neutron and total absorbed dose to PMMA was determined with an estimated uncertainty of +/- 5.0%, +/- 25%, and +/- 5.5% (2 SD), respectively. The uncertainty of the determination of the photon absorbed dose was comparable to the case in conventional radiotherapy, while the uncertainty of the neutron absorbed dose is much higher using the present methods. The thermal neutron group fluence, i.e., the neutron fluence in the energy interval 0-0.414 eV, was determined with an estimated uncertainty of +/- 2.8% (2 SD), which is acceptable for dosimetry in epithermal neutron beams.

Interaction between weak low frequency magnetic fields and cell membranes.

The question of whether very weak low frequency magnetic fields can affect biological systems, has attracted attention by many research groups for quite some time. Still, today, the theoretical possibility of such an interaction is often questioned and the site of interaction in the cell is unknown. In the present study, the influence of extremely low frequency (ELF) magnetic fields on the transport of Ca(2+) was studied in a biological system consisting of highly purified plasma membrane vesicles. We tested two quantum mechanical theoretical models that assume that biologically active ions can be bound to a channel protein and influence the opening state of the channel. Vesicles were exposed for 30 min at 32 degrees C and the calcium efflux was studied using radioactive (45)Ca as a tracer. Static magnetic fields ranging from 27 to 37 micro T and time varying magnetic fields with frequencies between 7 and 72 Hz and amplitudes between 13 and 114 micro T (peak) were used. We show that suitable combinations of static and time varying magnetic fields directly interact with the Ca(2+) channel protein in the cell membrane, and we could quantitatively confirm the model proposed by Blanchard.

Electrically mediated drug delivery for treatment of an adenocarcinoma transplanted into rat liver.

BACKGROUND: In this study, electrochemotherapy (ECT), i.e. tumour treatment based on local augmentation of intracellular drug delivery from short, intense electric pulses, was evaluated in rats with an adenocarcinoma implanted into the liver. Tumour response and concentrations of macrophages and T-lymphocytes (CD4 and CD8) in and around the tumour were measured. MATERIALS AND METHODS: Rats were treated with permeabilizing electric pulses, bleomycin, or both, eight days after implantation of the tumour, while one group received sham treatment. RESULTS: Treatment with electric pulses and bleomycin resulted in a significantly reduced lesion volume and 92% cure rate (12 out of 13, p<0.0002 compared to the other treatment groups). The highest concentration of CD8 lymphocytes was found in tumours treated with electric pulses and bleomycin. Macrophages were found mainly in tumours treated with electric pulses, with or without bleomycin. CONCLUSION: Electrochemotherapy using millisecond exponential pulses and bleomycin is efficient in a rat liver tumour model and appears to stimulate the host's immune system.

A new antitumour treatment combining radiation and electric pulses.

AIM: To investigate the antitumour effect of radiation in combination with electropermeabilization on subcutaneous rat glioma tumours. MATERIALS AND METHODS: Sub-optimal radiation treatment was administered separately or in combination with electric pulses of high voltage to subcutaneous rat brain tumours. The treatment was repeated on four consecutive days and evaluated by TGD and microscopical examination. The tumours were stained for Factor VlII/von Willebrand Factor to investigate the effects on the tumour vasculature. RESULTS: Radiation and electric pulses applied concomitantly resulted in a cure rate of 67% (tumour free >80 days after treatment). Radiation-treated animals showed progressive disease. Histological and immunohistochemical examination of electric impulse-treated tumours showed instant and severe deteriorating effects on tumour vasculature. CONCLUSION: A distinct antitumour effect of the combined treatment of electric pulses and radiation treatment was observed. We believe that the tumouricidal effect arises from destruction of the tumour vasculature but also from DNA related damage from reactive oxygen formed by the electric pulses and the radiation treatment.

Toward clinical application of prompt gamma spectroscopy for in vivo monitoring of boron uptake in boron neutron capture therapy.

In boron neutron capture therapy (BNCT) the absorbed dose to the tumor cells and healthy tissues depends critically on the boron uptake. Pronounced individual variations in the uptake patterns have been observed for two boron compounds currently used in clinical trials. This implies a high uncertainty in the determination of the boron dose component. In the present work a technique known as prompt gamma spectroscopy (PGS) is studied that potentially can be used for in vivo and noninvasive boron concentration determination at the time of the treatment. The technique is based upon measurement of gamma rays promptly emitted in the 10B(n,alpha)7Li and 1H(n,gamma)2D reactions. The aim of this work is to prepare the present setup for clinical application as a monitor of boron uptake in BNCT patients. Therefore, a full calibration and a set of phantom experiments were performed in a clinical setting. Specifically, a nonuniform boron distribution was studied; a skin/ dura, a larger blood vessel, and tumor within a head phantom was simulated. The results show that it is possible to determine a homogeneous boron concentration of 5 microg/g within +/-3% (1 standard deviation). In the nonuniform case, this work shows that the boron concentration can be determined through a multistep measurement procedure, however, with a somewhat higher uncertainty (approximately 10%). The present work forms the basis for a subsequent clinical application of the PGS setup aimed at in vivo monitoring of boron uptake.

Distribution of bleomycin in a rat model.

Bleomycin has, in the years of developing electrochemotherapy (ECT), proven to be an extremely potent drug for this cancer treatment modality and is also the most frequently applied chemical agent. It is of importance to investigate the pharmacokinetics of bleomycin under normal conditions and particularly in combination with ECT.

Treatment of rat glioma with electrochemotherapy.

The first attempt to apply electrochemotherapy (ECT) to the brain was reported in 1993 by Salford et al. 1993 (1). They managed to significantly prolong the survival of RG2 glioma bearing Fischer-344 rats by 200% by iv administration of bleomycin followed by intracranial electrochemotherapy with exponential decaying pulses. In collaboration with the department of tumor immunology, Lund University, an ethyl-nitroso-urea induced rat glioma cell line (N32) is developed that produces glioma of malignant astrocytoma type with only half the growth rate of the RG2 cells (2). The N32 tumor implanted in rats was treated with intracranial electrochemotherapy and enhanced uptake of [111In]bleomycin from intracranial ECT was also demonstrated with a scintillation camera (3). (111)In-labeled bleomycin has been used to investigate the uptake and retention after ECT treatment of subcutaneous N32 tumors (4).

Studies of in vivo electropermeabilization by gamma camera measurements of (99m)Tc-DTPA.

A protocol was developed to study the drug uptake from in vivo electropermeabilization at different settings of parameters influencing the uptake efficiency. Radiolabelled diethylenetriaminepentaacetic acid (DTPA) was used to trace the distribution and internalization of a hydrophilic drug after in vivo electropermeabilization. Skeletal muscle tissue in rat was treated with permeabilizing electric pulses before or after intravenous administration of (99m)Tc-DTPA. The drug accumulation in the treated volume was subsequently evaluated with a scintillation camera. The dependence of uptake on field strength and duration of the applied electric pulses was investigated for exponentially decaying pulses and square wave pulses. Further, the uptake dependence on time interval between injection and pulsation was studied as well as the uptake dependence on the number of pulses applied in a single electropermeabilization treatment. Dynamic gamma camera studies were performed to quantify the time scale of the drug uptake in electropermeabilized tissue.

Simplified treatment planning for interstitial laser thermotherapy by disregarding light transport: a numerical study.

BACKGROUND AND OBJECTIVE: The objective was to investigate the effect of light transport on the temperature distribution and the coagulated volume under conditions relevant to interstitial laser thermotherapy (ILT) of tumors in the human liver. STUDY DESIGN/MATERIALS AND METHODS: Temperature distributions and coagulated volumes produced with a diffusing laser fiber or a conductive heat source, at equal output power, were numerically calculated for tissue with different optical penetration depths. Four irradiation times (5, 10, 20, and 30 min) were studied. A three-dimensional finite-element model was used to calculate the temperature distribution during heating with four conductive heat sources (no light emission). Results were compared with measured temperature distributions during laser irradiation in a gel phantom with known optical properties. RESULTS: Numerical calculations showed that the influence of light transport on the coagulated volume was negligible in tissue with optical penetration depths below 3-4 mm at all studied irradiation times. The phantom experiment indicated good agreement with the calculated temperature distribution, both with a single diffusing laser fiber and with four fibers. CONCLUSION: Light transport influences coagulated volumes only slightly under conditions presented in this work, which is relevant to ILT of tumors in the human liver.

MRI thermometry in phantoms by use of the proton resonance frequency shift method: application to interstitial laser thermotherapy.

In this work the temperature dependence of the proton resonance frequency was assessed in agarose gel with a high melting temperature (95 degrees C) and in porcine liver in vitro at temperatures relevant to thermotherapy (25-80 degrees C). Furthermore, an optically tissue-like agarose gel phantom was developed and evaluated for use in MRI. The phantom was used to visualize temperature distributions from a diffusing laser fibre by means of the proton resonance frequency shift method. An approximately linear relationship (0.0085 ppm degrees C(-1)) between proton resonance frequency shift and temperature change was found for agarose gel, whereas deviations from a linear relationship were observed for porcine liver. The optically tissue-like agarose gel allowed reliable MRI temperature monitoring, and the MR relaxation times (T1 and T2) and the optical properties were found to be independently alterable. Temperature distributions around a diffusing laser fibre, during irradiation and subsequent cooling, were assessed with high spatial resolution (voxel size = 4.3 mm3) and with random uncertainties ranging from 0.3 degrees C to 1.4 degrees C (1 SD) with a 40 s scan time.

Thermal conductivity of uterine tissue in vitro.

Thermotherapy of the uterus has emerged as an alternative to hysterectomy in the treatment of menorrhagia, from whence it follows that the thermal properties of uterine tissue have become of importance. This study presents measurements of the thermal conductivity and the water content of uterine tissue in vitro. A steady-state thermal conductivity apparatus, based on the comparison of test samples with a material with known thermal conductivity, is described. Measurements were conducted on tissue samples from eleven patients, directly after hysterectomy. Samples with and without endometrium, as well as coagulated samples, were examined. The thermal conductivity of myometrial tissue was found to be 0.536 +/- 0.012 W m(-1) K(-1) (mean +/- 1 SD) and the corresponding water content was 81.2 +/- 1.5% (mean +/- 1 SD). Measurements on samples with both endometrium and myometrium showed similar thermal conductivity (0.542 +/- 0.008 W m(-1) K(-1), mean +/- 1 SD) and water content (81.6 +/- 0.7%, mean +/- 1 SD). It was also indicated that coagulation causes dehydration, resulting in a lower thermal conductivity.

Feedback interstitial diode laser (805 nm) thermotherapy system: ex vivo evaluation and mathematical modeling with one and four-fibers.

BACKGROUND AND OBJECTIVE: In this study a newly developed microprocessor controlled power regulation and thermometry system integrated with a diode laser (805 nm wavelength) was evaluated with respect to temperature distribution, effectiveness of regulation, and ability to predict temperature distributions by computer simulation. STUDY DESIGN/MATERIALS AND METHODS: Experiments were performed in ground bovine muscle using either a single laser fiber or four-fibers. The target temperature at one (feedback) thermistor, placed 5 mm from one of the laser fibers, was set to 50 degrees C and was maintained by means of stepwise power regulation. The temperature distribution was monitored using multiple thermistor probes. A numerical model based on the bioheat equation was used to calculate the temperature distributions. RESULTS: Temperature regulation was excellent with a tendency towards better regulation in the four-fiber than in the single-fiber experiments. Agreement between calculated and measured temperatures was good. The coagulated (> 55 degrees C) and hyperthermic (> 45 degrees C) volumes were 6 and 10-11 times larger, respectively, with four-fibers than with a single fiber. CONCLUSION: It is concluded that the stepwise power regulation system was efficient in maintaining a stable target temperature. The results indicate that the system can produce lesion volumes adequate for treating a relatively large tumor in a single session and that computer simulation may be useful for predicting temperature distribution.

Quantification of low-velocity motion using a navigator-echo supported MR velocity-mapping technique: application to intracranial dynamics in volunteers and patients with brain tumours.

Gradient-echo pulse sequences with velocity-encoding gradients of 22.5-25 mT/m, were used for brain-motion and CSF-flow studies. To reduce motion artifacts, a phase-correction technique based on navigator echoes was evaluated. Three patients with right-sided parietal tumours were investigated; one astrocytoma grade III-IV, one astrocytoma grade I-II and one benign meningioma. In healthy volunteers, a maximal brain-tissue velocity of (0.94 +/- 0.26) mm/s (mean +/- 1SD) was observed, which is consistent with previously presented results. The phase correction was proven useful for reduction of artifacts due to external head movements in modulus and phase images, without loss of phase information related to internal motion. The tissue velocity within the astrocytomas was low during the entire cardiac cycle. An abnormally high rostral velocity component was, however, observed in the brain tissue frontal to the astrocytomas. In all patients, an abnormal CSF flow pattern was observed. The study of brain motion may provide further understanding of the effects of tumours and other pathological conditions in the brain. When considering intracranial motion as a source of error in diffusion/perfusion MRI, the present study suggests that a pathology can alter the properties of brain motion and CSF flow considerably, leading to a more complex impact on diffusion/perfusion images.

Endometrial destruction by hyperthermia--a possible treatment of menorrhagia. An experimental study.

BACKGROUND: Treatment of menorrhagia by heat-destruction of the endometrium, intended to be an alternative to hysterectomy, was investigated in an experimental study. METHOD: A specially designed catheter (CavatermTM) with a silicone balloon containing a self-regulating containing heating element is inserted into the uterus, filled with glycine to a pressure of around 180 mmHg and heated to about 75 degrees C. We investigated the treatment effect in vitro in five extirpated uteri (series A) and in vivo in three patients treated peroperatively just before hysterectomy, temperatures being monitored in the surrounding tissues (series B). In both series we monitored the following variables: heating-power, balloon-pressure, temperature of the heating element (around 85 degrees C) and the temperatures at five locations from top to bottom of the balloon surface. RESULTS: After 30 min in vitro treatment at 75 degrees C, the endometrium was partly destructed, and condensed. Histological examination showed smooth muscle cells to be destroyed to a depth of 2-5 mm close to the endometrium. With in vivo treatment for 30 min at a heating power of about 20 W (resulting in balloon surface temperatures of 58-65 degrees C), the increases in temperature of the surrounding tissue were too small to measure accurately (+/-1 degree C). Histological examination showed destruction of cells in the corpus uteri to a maximum depth of 8 mm. CONCLUSION: Findings in in vitro and in vivo experiments suggest that 30 min heating of the endometrium to 58-65 degrees C with an intrauterine silicone balloon filled with a liquid to a pressure of 180 mmHg exerts therapeutic effects on both endometrium and uterine cavity smooth muscle cells without damage to surrounding tissues.

Temperature control and light penetration in a feedback interstitial laser thermotherapy system.

The aim of this study was to describe the performance of a closed loop interstitial laser thermotherapy system in processed liver and to demonstrate its suitability for treating a vascularized tumour in vivo. The thermotherapy system consisted of an Nd: YAG laser and a temperature feedback circuit including an automatic thermometry system and thermistor probes. Experiments in processed liver were performed with a sapphire probe and temperature control at a distance of 10 mm. In most experiments at 1-2 W, and in half of the experiments at 3 W, there was no carbonization, a moderate change in the light penetration and excellent control of the temperature. In experiments with output powers of 4-5 W there was carbonization with rapid deterioration of light penetration and impaired control of the temperature. Carbonization affected the distribution of temperatures, which were lower below, and higher above, the laser tip in experiments with carbonization as compared to experiments without carbonization. Treatment of an adenocarcinoma implanted into rat liver was performed at 2 W with a bare fibre and without blood inflow occlusion. The feedback thermistor probe was placed 3 mm outside the margin of the tumour (largest diameter 9.5 +/- 0.3 mm (mean +/- SEM)). Temperature control and light penetration characteristics were similar to those found in vitro. No tumour could be demonstrated at sacrifice 6 days later. It is concluded that a closed loop feedback system can produce stable and reproducible local hyperthermia, that it performs better when carbonization is avoided and light penetration is preserved and that it has a great potential for interstitial thermotherapy of malignant tumours.

A stochastic model for subcellular dosimetry in boron neutron capture therapy.

The therapeutic effectiveness of boron neutron capture therapy is highly dependent on the microscopic distribution of the administered boron compound. Two boron compounds with different uptake mechanisms in the tumour cells may thus cause effects of different degrees even if the macroscopic boron concentrations in the tumour tissue are the same. This difference is normally expressed quantitatively by the so-called relative local efficiency (RLE). In this work, a stochastic model for the subcellular dosimetry has been developed. This model can be used to calculate the probability for an energy deposition above a certain threshold level in the cell nucleus due to a single neutron capture reaction. If a threshold cell-kill function is assumed, and if the dose is low enough that multiple energy depositions are rare, the model can also be applied to calculations of the survival probability for a cell population. Subcellular boron distributions in rats carrying RG 2 rat gliomas were measured by subcellular fractionation after administration of two different boron compounds: a sulphydryl boron hydride (BSH) and a boronated porphyrin (BOPP). Based on these data, the RLE factors were then calculated for these compounds using the stochastic model.

A computer simulation program for MR imaging: application to RF and static magnetic field imperfections.

A computer simulation program capable of demonstrating various artifacts, such as image distortion caused by metallic implants in MR imaging, is presented. The structure of the program allows for the implementation of various imaging situations as long as spins only experience weak interaction, i.e., the Bloch equations are obeyed. The raw data are obtained by repeatedly applying the Bloch equations to the magnetization vector of each point of the simulated object, throughout the pulse sequence. With only a limited number of spins in each voxel, the effects of intravoxel dephasing and rephasing require special attention, and algorithms for this have been implemented.

Performance of sulfhydryl boron hydride in patients with grade III and IV astrocytoma: a basis for boron neutron capture therapy.

This study investigated the rationale of boron neutron capture therapy (BNCT) for the treatment of Grade III and IV astrocytoma. The European Community joint research program on BNCT plans to use sulfhydryl boron hydride (BSH) in clinical trials. The work presented here, examines the performance of BSH in eight patients with Grade III and IV astrocytoma using a measurement technique which precisely correlates the boron uptake with the histology of the tumor and the peritumoral brain. Astrocytomas are exceptionally heterogeneous and spread migrating tumor cells into the surrounding brain. The patients were infused with 50 mg BSH per kilogram of body weight at 12, 18, 24 or 48 hours before surgery. At the time of operation, specimens were obtained of the tumor, skin, muscle, dura, blood, urine, and, when surgically possible, the brain adjacent to tumor. In three patients the intracellular boron distribution was investigated by subcellular fractionation. The blood clearance was biphasic with half-lives of 0.6 and 8.2 hours. After 3 days, approximately 70% of the dose injected was excreted in the urine. The maximum boron concentration in the tumor was 20 ppm, 12 hours after the infusion. The tumor-to-blood ratios ranged between 0.2 and 1.4, with the highest values after 18 to 24 hours. In the brain specimens the boron concentration never exceeded 1 ppm. This work confirms a selective uptake of boron in the tumor compared to the surrounding brain and that boron, to some extent, is incorporated in the tumor cells.

A comparative study on the pharmacokinetics and biodistribution of boronated porphyrin (BOPP) and sulfhydryl boron hydride (BSH) in the RG2 rat glioma model.

Boron neutron capture therapy is a treatment modality for cancer that depends on the specific uptake of boron by the tumor cells. The infiltrative growth of malignant gliomas requires that boron reach and accumulate in migrating cells outside the margin of the tumor; thus, it is important that the biodistribution of new boron compounds is also studied in the surrounding healthy brain tissue. This study is undertaken in the present work, in which the biodistribution and pharmacokinetics of sulfhydryl boron hydride (BSH) and boronated porphyrin (BOPP) in the RG2 rat glioma model are investigated. This model mimics the characteristics of human glioma with cells migrating into the surrounding brain. The animals were infused intravenously with either BSH (25 micrograms or 175 micrograms of boron per gram of body weight) or BOPP (12 micrograms of boron per gram body weight). For the low dose of BSH, the maximum tumor-boron content was 8 ppm at approximately 9 hours after the infusion with a tumor-to-blood ratio of 0.6. At the higher dose, the corresponding figures were 15 ppm after 12 hours with a tumor-to-blood ratio of 0.5. For BOPP, a tumor-boron concentration of 81 ppm was achieved 24 hours after the infusion and sustained in that range for at least 72 hours. The tumor-to-blood ratio at 24 hours was slightly above 6, but continued to increase as the blood was cleared. These results indicate that both compounds are spread into the normal brain tissue following the same pathways as the migrating tumor cells and in this way can be taken up even in distant tumor cell foci.