Evaluation of the dose enhancement of combined ¹°B + ¹57Gd neutron capture therapy (NCT).

An innovative molecule, GdBLDL, for boron neutron capture therapy (BNCT) has been developed and its effectiveness as a BNCT carrier is currently under evaluation using in vivo experiments on small animal tumour models. The molecule contains both (10)B (the most commonly used NCT agent) and (157)Gd nuclei. (157)Gd is the second most studied element to perform NCT, mainly thanks to its high cross section for the capture of low-energy neutrons. The main drawback of (157)Gd neutron capture reaction is the very short range and low-energy secondary charged particles (Auger electrons), which requires (157)Gd to be very close to the cellular DNA to have an appreciable biological effect. Treatment doses were calculated by Monte Carlo simulations to ensure the optimised tumour irradiation and the sparing of the healthy organs of the irradiated animals. The enhancement of the absorbed dose due to the simultaneous presence of (10)B and (157)Gd in the experimental set-up was calculated and the advantage introduced by the presence of (157)Gd was discussed.

Testing and linearity calibration of films of phenol compounds exposed to thermal neutron field for EPR dosimetry.

This paper reports the preliminary results obtained by Electron Paramagnetic Resonance (EPR) measurements on films of IRGANOX® 1076 phenols with and without low content (5% by weight) of gadolinium oxide (Gd2O3) exposed in the thermal column of the Triga Mark II reactor of LENA (Laboratorio Energia Nucleare Applicata) of Pavia (Italy). Thanks to their size, the phenolic films here presented are good devices for the dosimetry of beams with high dose gradient and which require accurate knowledge of the precise dose delivered. The dependence of EPR signal as function of neutron dose was investigated in the fluence range between 10(11) cm(-2) and 10(14) cm(-2). Linearity of EPR response was found and the signal was compared with that of commercial alanine films. Our analysis showed that gadolinium oxide (5% by weight) can enhance the thermal neutron sensitivity more than 18 times. Irradiated dosimetric films of phenolic compound exhibited EPR signal fading of about 4% after 10 days from irradiation.

Comparative study of the radiobiological effects induced on adherent vs suspended cells by BNCT, neutrons and gamma rays treatments.

The present work is part of a preclinical in vitro study to assess the efficacy of BNCT applied to liver or lung coloncarcinoma metastases and to limb osteosarcoma. Adherent growing cell lines can be irradiated as adherent to the culture flasks or as cell suspensions, differences in radio-sensitivity of the two modalities of radiation exposure have been investigated. Dose related cell survival and cell cycle perturbation results evidenced that the radiosensitivity of adherent cells is higher than that of the suspended ones.

Toward a clinical application of ex situ boron neutron capture therapy for lung tumors at the RA-3 reactor in Argentina.

PURPOSE: Many types of lung tumors have a very poor prognosis due to their spread in the whole organ volume. The fact that boron neutron capture therapy (BNCT) would allow for selective targeting of all the nodules regardless of their position, prompted a preclinical feasibility study of ex situ BNCT at the thermal neutron facility of RA-3 reactor in the province of Buenos Aires, Argentina. (l)-4p-dihydroxy-borylphenylalanine fructose complex (BPA-F) biodistribution studies in an adult sheep model and computational dosimetry for a human explanted lung were performed to evaluate the feasibility and the therapeutic potential of ex situ BNCT. METHODS: Two kinds of boron biodistribution studies were carried out in the healthy sheep: a set of pharmacokinetic studies without lung excision, and a set that consisted of evaluation of boron concentration in the explanted and perfused lung. In order to assess the feasibility of the clinical application of ex situ BNCT at RA-3, a case of multiple lung metastases was analyzed. A detailed computational representation of the geometry of the lung was built based on a real collapsed human lung. Dosimetric calculations and dose limiting considerations were based on the experimental results from the adult sheep, and on the most suitable information published in the literature. In addition, a workable treatment plan was considered to assess the clinical application in a realistic scenario. RESULTS: Concentration-time profiles for the normal sheep showed that the boron kinetics in blood, lung, and skin would adequately represent the boron behavior and absolute uptake expected in human tissues. Results strongly suggest that the distribution of the boron compound is spatially homogeneous in the lung. A constant lung-to-blood ratio of 1.3 ± 0.1 was observed from 80 min after the end of BPA-F infusion. The fact that this ratio remains constant during time would allow the blood boron concentration to be used as a surrogate and indirect quantification of the estimated value in the explanted healthy lung. The proposed preclinical animal model allowed for the study of the explanted lung. As expected, the boron concentration values fell as a result of the application of the preservation protocol required to preserve the lung function. The distribution of the boron concentration retention factor was obtained for healthy lung, with a mean value of 0.46 ± 0.14 consistent with that reported for metastatic colon carcinoma model in rat perfused lung. Considering the human lung model and suitable tumor control probability for lung cancer, a promising average fraction of controlled lesions higher than 85% was obtained even for a low tumor-to-normal boron concentration ratio of 2. CONCLUSIONS: This work reports for the first time data supporting the validity of the ovine model as an adequate human surrogate in terms of boron kinetics and uptake in clinically relevant tissues. Collectively, the results and analysis presented would strongly suggest that ex situ whole lung BNCT irradiation is a feasible and highly promising technique that could greatly contribute to the treatment of metastatic lung disease in those patients without extrapulmonary spread, increasing not only the expected overall survival but also the resulting quality of life.

Modeling radiation-induced cell death: role of different levels of DNA damage clustering.

Some open questions on the mechanisms underlying radiation-induced cell death were addressed by a biophysical model, focusing on DNA damage clustering and its consequences. DNA "cluster lesions" (CLs) were assumed to produce independent chromosome fragments that, if created within a micrometer-scale threshold distance (d), can lead to chromosome aberrations following mis-rejoining; in turn, certain aberrations (dicentrics, rings and large deletions) were assumed to lead to clonogenic cell death. The CL yield and d were the only adjustable parameters. The model, implemented as a Monte Carlo code called BIophysical ANalysis of Cell death and chromosome Aberrations (BIANCA), provided simulated survival curves that were directly compared with experimental data on human and hamster cells exposed to photons, protons, α-particles and heavier ions including carbon and iron. d = 5 µm, independent of radiation quality, and CL yields in the range ~2-20 CLs Gy(-1) cell(-1), depending on particle type and energy, led to good agreement between simulations and data. This supports the hypothesis of a pivotal role of DNA cluster damage at sub-micrometric scale, modulated by chromosome fragment mis-rejoining at micrometric scale. To investigate the features of such critical damage, the CL yields were compared with experimental or theoretical yields of DNA fragments of different sizes, focusing on the base-pair scale (related to the so-called local clustering), the kbp scale ("regional clustering") and the Mbp scale, corresponding to chromatin loops. Interestingly, the CL yields showed better agreement with kbp fragments rather than bp fragments or Mbp fragments; this suggests that also regional clustering, in addition to other clustering levels, may play an important role, possibly due to its relationship with nucleosome organization in the chromatin fiber.

The role of DNA cluster damage and chromosome aberrations in radiation-induced cell killing: a theoretical approach.

The role played by DNA cluster damage and chromosome aberrations in radiation-induced cell killing was investigated, assuming that certain chromosome aberrations (dicentrics, rings and large deletions, or 'lethal aberrations') lead to clonogenic inactivation and that chromosome aberrations are due to micrometre-scale rejoining of chromosome fragments derived from DNA cluster lesions (CLs). The CL yield and the threshold distance governing fragment rejoining were left as model parameters. The model, implemented as a Monte Carlo code called BIANCA (BIophysical ANalysis of Cell death and chromosome Aberrations), provided simulated survival curves that were compared with survival data on AG1522 and V79 cells exposed to different radiation types, including heavy ions. The agreement between simulation outcomes and experimental data suggests that lethal aberrations are likely to play an important role in cell killing not only for AG1522 cells exposed to X rays, as already reported by others, but also for other radiation types and other cells. Furthermore, the results are consistent with the hypothesis that the critical DNA lesions leading to cell death and chromosome aberrations are double-strand break clusters (possibly involving the ∼1000-10 000 bp scale) and that the effects of such clusters are modulated by micrometre-scale proximity effects during DNA damage processing.

Microdosimetric measurements in the thermal neutron irradiation facility of LENA reactor.

A twin TEPC with electric-field guard tubes has been constructed to be used to characterize the BNCT field of the irradiation facility of LENA reactor. One of the two mini TEPC was doped with 50ppm of (10)B in order to simulate the BNC events occurring in BNCT. By properly processing the two microdosimetric spectra, the gamma, neutron and BNC spectral components can be derived with good precision (~6%). However, direct measurements of (10)B in some doped plastic samples, which were used for constructing the cathode walls, point out the scarce accuracy of the nominal (10)B concentration value. The influence of the Boral(®) door, which closes the irradiation channel, has been measured. The gamma dose increases significantly (+51%) when the Boral(®) door is closed. The crypt-cell-regeneration weighting function has been used to measure the quality, namely the RBEµ value, of the radiation field in different conditions. The measured RBEµ values are only partially consistent with the RBE values of other BNCT facilities.

Biodistribution of the boron carriers boronophenylalanine (BPA) and/or decahydrodecaborate (GB-10) for Boron Neutron Capture Therapy (BNCT) in an experimental model of lung metastases.

BNCT was proposed for the treatment of diffuse, non-resectable tumors in the lung. We performed boron biodistribution studies with 5 administration protocols employing the boron carriers BPA and/or GB-10 in an experimental model of disseminated lung metastases in rats. All 5 protocols were non-toxic and showed preferential tumor boron uptake versus lung. Absolute tumor boron concentration values were therapeutically useful (25-76ppm) for 3 protocols. Dosimetric calculations indicate that BNCT at RA-3 would be potentially therapeutic without exceeding radiotolerance in the lung.

Boron concentration measurement in biological tissues by charged particle spectrometry.

Measurement of boron concentration in biological tissues is a fundamental aspect of boron neutron capture therapy, because the outcome of the therapy depends on the distribution of boron at a cellular level, besides on its overall concentration. This work describes a measurement technique based on the spectroscopy of the charged particles emitted in the reaction (10)B(n,α)(7)Li induced by thermal neutrons, allowing for a quantitative determination of the boron concentration in the different components that may be simultaneously present in a tissue sample, such as healthy cells, tumor cells and necrotic cells. Thin sections of tissue containing (10)B are cut at low temperatures and irradiated under vacuum in a thermal neutron field. The charged particles arising from the sample during the irradiation are collected by a thin silicon detector, and their spectrum is used to determine boron concentration through relatively easy calculations. The advantages and disadvantages of this technique are here described, and validation of the method using tissue standards with known boron concentrations is presented.

Differential serotonin transport is linked to the rh5-HTTLPR in peripheral blood cells.

The human serotonin transporter (SERT) gene possesses a 43-base pair (bp) insertion-deletion promoter polymorphism, the h5-HTTLPR. Genotype at this locus correlates with variation in anxiety-related personality traits and risk for major depressive disorder in many studies. Yet, the complex effects of the h5-HTTLPR, in combination with closely associated single-nucleotide polymorphisms (SNPs), continue to be debated. Moreover, although SERT is of high clinical significance, transporter function in vivo remains difficult to assess. Rhesus express a promoter polymorphism related to the h5-HTTLPR. The rh5-HTTLPR has been linked to differences in stress-related behavior and cognitive flexibility, although allelic variations in serotonin uptake have not been investigated. We studied the serotonin system as it relates to the 5-HTTLPR in rhesus peripheral blood cells. Sequencing of the rh5-HTTLPR revealed a 23-bp insertion, which is somewhat longer than originally reported. Consistent with previous reports, no SNPs in the rh5-HTTLPR and surrounding genomic regions were detected in the individuals studied. Reductions in serotonin uptake rates, cell surface SERT binding, and 5-hydroxyindoleacetic acid/serotonin ratios, but not SERT mRNA levels, were associated with the rh5-HTTLPR short allele. Thus, serotonin uptake rates are differentiable with respect to the 5-HTTLPR in an easily accessible native peripheral tissue. In light of these findings, we foresee that primary blood cells, in combination with high sensitivity functional measurements enabled by chronoamperometry, will be important for investigating alterations in serotonin uptake associated with genetic variability and antidepressant responsiveness in humans.

Boron determination in liver tissue by combining quantitative neutron capture radiography (QNCR) and histological analysis for BNCT treatment planning at the TRIGA Mainz.

The typical primary malignancies of the liver are hepatocellular carcinoma and cholangiocarcinoma, whereas colorectal liver metastases are the most frequently occurring secondary tumors. In many cases, only palliative treatment is possible. Boron neutron capture therapy (BNCT) represents a technique that potentially destroys tumor tissue selectively by use of externally induced, locally confined secondary particle irradiation. In 2001 and 2003, BNCT was applied to two patients with colorectal liver metastases in Pavia, Italy. To scrutinize the rationale of BNCT, a clinical pilot study on patients with colorectal liver metastases was carried out at the University of Mainz. The distribution of the (10)B carrier (p-borono-phenylalanine) in the liver and its uptake in cancerous and tumor-free tissue were determined, focusing on a potential correlation between the uptake of p-borono-phenylalanine and the biological characteristics of cancerous tissue. Samples were analyzed using quantitative neutron capture radiography of cryosections combined with histological analysis. Methodological aspects of the combination of these techniques and results from four patients enrolled in the study are presented that indicate that the uptake of p-borono-phenylalanine strongly depends on the metabolic activity of cells.

Cell death following BNCT: a theoretical approach based on Monte Carlo simulations.

In parallel to boron measurements and animal studies, investigations on radiation-induced cell death are also in progress in Pavia, with the aim of better characterisation of the effects of a BNCT treatment down to the cellular level. Such studies are being carried out not only experimentally but also theoretically, based on a mechanistic model and a Monte Carlo code. Such model assumes that: (1) only clustered DNA strand breaks can lead to chromosome aberrations; (2) only chromosome fragments within a certain threshold distance can undergo misrejoining; (3) the so-called "lethal aberrations" (dicentrics, rings and large deletions) lead to cell death. After applying the model to normal cells exposed to monochromatic fields of different radiation types, the irradiation section of the code was purposely extended to mimic the cell exposure to a mixed radiation field produced by the (10)B(n,α) (7)Li reaction, which gives rise to alpha particles and Li ions of short range and high biological effectiveness, and by the (14)N(n,p)(14)C reaction, which produces 0.58 MeV protons. Very good agreement between model predictions and literature data was found for human and animal cells exposed to X- or gamma-rays, protons and alpha particles, thus allowing to validate the model for cell death induced by monochromatic radiation fields. The model predictions showed good agreement also with experimental data obtained by our group exposing DHD cells to thermal neutrons in the TRIGA Mark II reactor of the University of Pavia; this allowed to validate the model also for a BNCT exposure scenario, providing a useful predictive tool to bridge the gap between irradiation and cell death.

Structure and stability of nickel/nickel oxide core-shell nanoparticles.

The results of a combined x-ray photoelectron spectroscopy (XPS) and high resolution transmission electron microscopy (HR-TEM) study of Ni nanoparticles (NP), before and after oxidation, are presented. An experimental set-up was realized for the preparation and study of pre-formed NP films, concentrating the attention on Ni NP in the diameter range between 4 and 8 nm. The XPS data were taken in situ from NPs after different stages of oxidation, including controlled dosing of O(2) gas in the experimental system and exposure to the atmosphere. The Ni 2p structure is a combination of spectra from metallic Ni in the NP core and from the oxide shell. The signal from the NP core was observed even for samples after exposure to air. From the comparison of HR-TEM experimental images with theoretical simulations, it was found that the Ni NP core has a regular multitwinned icosahedral structure, composed of single-crystal tetrahedra with (111) faces. The NiO phase is clearly observed forming islands on the NP surface.

Simulation of the neutron flux in the irradiation facility at RA-3 reactor.

A facility for the irradiation of a section of patients' explanted liver and lung was constructed at RA-3 reactor, Comisión Nacional de Energía Atómica, Argentina. The facility, located in the thermal column, is characterized by the possibility to insert and extract samples without the need to shutdown the reactor. In order to reach the best levels of security and efficacy of the treatment, it is necessary to perform an accurate dosimetry. The possibility to simulate neutron flux and absorbed dose in the explanted organs, together with the experimental dosimetry, allows setting more precise and effective treatment plans. To this end, a computational model of the entire reactor was set-up, and the simulations were validated with the experimental measurements performed in the facility.

Dose estimation in B16 tumour bearing mice for future irradiation in the thermal column of the TRIGA reactor after B/Gd/LDL adduct infusion.

To test the efficacy of a new (10)B-vector compound, the B/Gd/LDL adduct synthesised at Torino University, in vivo irradiations of murine tumours are in progress at the TRIGA Mark II reactor of the Pavia University. A localised B16 melanoma tumour is generated in C57BL/6 mice and subsequently infused with the adduct. During the irradiation, the mouse will be put in a shield to protect the whole body except the tumour in the back-neck area. To optimise the treatment set-up, MCNP simulations were performed. A very simplified mouse model was built using MCNP geometry capabilities, as well as the geometry of the shield made of 99% (10)B enriched boric acid. A hole in the shield is foreseen in correspondence of the back-neck region. Many configurations of the shield were tested in terms of neutron flux, dose distribution and mean induced activity in the tumour region and in the radiosensitive organs of the mouse. In the final set-up, up to five mice can be treated simultaneously in the reactor thermal column and the neutron fluence in the tumour region for 10 min of irradiation is of about 5×10(12) cm(-2).

A micro-PET/CT approach using O-(2-[18F]fluoroethyl)-L-tyrosine in an experimental animal model of F98 glioma for BNCT.

The present study focuses on a micro-PET/CT application to be used for experimental Boron Neutron Capture Therapy (BNCT), which integrates, in the same frame, micro-CT derived anatomy and PET radiotracer distribution. Preliminary results have demonstrated that (18)F-fluoroethyl-tyrosine (FET)/PET allows the identification of the extent of cerebral lesions in F98 tumor bearing rat. Neutron autoradiography and α-spectrometry on axial tissues slices confirmed the tumor localization and extraction, after the administration of fructose-boronophenylalanine (BPA). Therefore, FET-PET approach can be used to assess the transport, the net influx, and the accumulation of FET, as an aromatic amino acid analog of BPA, in experimental animal model. Coregistered micro-CT images allowed the accurate morphological localization of the radiotracer distribution and its potential use for experimental BNCT.

1H and 10B NMR and MRI investigation of boron- and gadolinium-boron compounds in boron neutron capture therapy.

(10)B molecular compounds suitable for Boron Neutron Capture Therapy (BNCT) are tagged with a Gd(III) paramagnetic ion. The newly synthesized molecule, Gd-BPA, is investigated as contrast agent in Magnetic Resonance Imaging (MRI) with the final aim of mapping the boron distribution in tissues. Preliminary Nuclear Magnetic Resonance (NMR) measurements, which include (1)H and (10)B relaxometry in animal tissues, proton relaxivity of the paramagnetic Gd-BPA molecule in water and its absorption in tumoral living cells, are reported.

From radiation-induced chromosome damage to cell death: modelling basic mechanisms and applications to boron neutron capture therapy.

Cell death is a crucial endpoint in radiation-induced biological damage: on one side, cell death is a reference endpoint to characterise the action of radiation in biological targets; on the other side, any cancer therapy aims to kill tumour cells. Starting from Lea's target theory, many models have been proposed to interpret radiation-induced cell killing; after briefly discussing some of these models, in this paper, a mechanistic approach based on an experimentally observed link between chromosome aberrations and cell death was presented. More specifically, a model and a Monte Carlo code originally developed for chromosome aberrations were extended to simulate radiation-induced cell death applying an experimentally observed one-to-one relationship between the average number of 'lethal aberrations' (dicentrics, rings and deletions) per cell and -ln S, S being the fraction of surviving cells. Although such observation was related to X rays, in the present work, the approach was also applied to protons and alpha particles. A good agreement between simulation outcomes and literature data provided a model validation for different radiation types. The same approach was then successfully applied to simulate the survival of cells enriched with boron and irradiated with thermal neutrons at the Triga Mark II reactor in Pavia, to mimic a typical treatment for boron neutron capture therapy.

Boron uptake measurements in a rat model for Boron Neutron Capture Therapy of lung tumours.

Lung carcinoma is the leading cause of cancer mortality in the Western countries. Despite the introduction over the last few years of new therapeutic agents, survival from lung cancer has shown no discernible improvement in the last 20 years. For these reasons any efforts to find and validate new effective therapeutic procedures for lung cancer are very timely. The selective boron uptake in the tumour with respect to healthy tissues makes Boron Neutron Capture Therapy a potentially advantageous option in the treatment of tumours that affect whole vital organs, and that are surgically inoperable. To study the possibility of applying BNCT to the treatment of diffuse pulmonary tumours, an animal model for boron uptake measurements in lung metastases was developed. Both healthy and tumour-bearing rats were infused with Boronophenylalanine (BPA) and sacrificed at different time intervals after drug administration. The lungs were extracted, and prepared for boron analysis by neutron autoradiography and α-spectroscopy. The boron concentrations in tumour and normal lung were plotted as a function of the time elapsed after BPA administration. The concentration in tumour is almost constant within the error bars for all the time intervals of the experiment (1-8 h), while the curve in normal lung decreases after 4 h from BPA infusion. At 4 h, the ratio of boron concentration in tumour to boron concentration in healthy lung is higher than 3, and it stays above this level up to 8 h. Also the images of boron distribution in the samples, obtained by neutron autoradiography, show a selective absorption in the metastases.

In vitro and in vivo studies of boron neutron capture therapy: boron uptake/washout and cell death.

Boron neutron capture therapy (BNCT) is a binary radiotherapy based on thermal-neutron irradiation of cells enriched with (10)B, which produces α particles and (7)Li ions of short range and high biological effectiveness. The selective uptake of boron by tumor cells is a crucial issue for BNCT, and studies of boron uptake and washout associated with cell survival studies can be of great help in developing clinical applications. In this work, boron uptake and washout were characterized both in vitro for the DHDK12TRb (DHD) rat colon carcinoma cell line and in vivo using rats bearing liver metastases from DHD cells. Despite a remarkable uptake, a large boron release was observed after removal of the boron-enriched medium from in vitro cell cultures. However, analysis of boron washout after rat liver perfusion in vivo did not show a significant boron release, suggesting that organ perfusion does not limit the therapeutic effectiveness of the treatment. The survival of boron-loaded cells exposed to thermal neutrons was also assessed; the results indicated that the removal of extracellular boron does not limit treatment effectiveness if adequate amounts of boron are delivered and if the cells are kept at low temperature. Cell survival was also investigated theoretically using a mechanistic model/Monte Carlo code originally developed for radiation-induced chromosome aberrations and extended here to cell death; good agreement between simulation outcomes and experimental data was obtained.