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.

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).

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.

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.

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.

Carborane derivatives loaded into liposomes as efficient delivery systems for boron neutron capture therapy.

Boron neutron capture therapy (BNCT) is an anticancer therapy based on the incorporation of (10)B in tumors, followed by neutron irradiation. Recently, the synthesis and delivery of new boronated compounds have been recognized as some of the main challenges in BNCT application. Here, we report on the use of liposomes as carriers for BNCT active compounds. Two carborane derivatives, i.e., o-closocarboranyl beta-lactoside (LCOB) and 1-methyl-o-closocarboranyl-2-hexylthioporphyrazine (H(2)PzCOB), were loaded into liposomes bearing different surface charges. The efficacy of these formulations was tested on model cell cultures, that is, DHD/K12/TRb rat colon carcinoma and B16-F10 murine melanoma. These induce liver and lung metastases, respectively, and are used to study the uptake of standard BNCT drugs, including borophenylalanine (BPA). Boron concentration in treated cells was measured by alpha spectrometry at the TRIGA mark II reactor (University of Pavia). Results showed high performance of the proposed formulations. In particular, the use of cationic liposomes increased the cellular concentration of (10)B by at least 30 times more than that achieved by BPA.

Natural hybridisation between Quercus petraea (Matt.) Liebl. and Quercus pubescens Willd. within an Italian stand as revealed by microsatellite fingerprinting.

Interspecific gene flow is frequently reported in the genus Quercus. However, interfertile oak species often seem to remain distinct, even within areas of sympatry. This study employed molecular markers to verify, at a fine scale, the presence of interspecific gene flow in a natural population of Quercus petraea and Quercus pubescens. Within a delimited area of 6 ha, all adult trees belonging to the studied oak complex and seeds from a subsample of such trees were collected and analysed using molecular microsatellite markers. A low interspecific genetic differentiation and a high level of interspecific genetic admixture suggested past hybridisation. Paternity inference of seeds allowed the estimation of pollination frequencies from the three groups of pollen donors (Q. petraea, Q. pubescens, intermediate). We also assayed pollen viability and germinability of each species group. We observed natural hybridisation between Q. petraea and Q. pubescens, with a predominant component in the direction Q. petraea --> Q. pubescens: Q. pubescens displayed a higher level of heterospecific pollination by Q. petraea (25.8%) and intermediate morphotypes (14.7%), compared to Q. petraea acting as pollen receptor (with less than 5% heterospecific pollinations). Intermediate 'mother trees' were pollinated in similar proportions by Q. petraea (23.1%), Q. pubescens (37.8%) and intermediate morphotypes (39.1%). The asymmetrical introgression observed for the studied generation may be caused, among other factors, by the relative abundance of trees from each species group in the studied area.

Calculations of dose distributions in the lungs of a rat model irradiated in the thermal column of the TRIGA reactor in Pavia.

To test the possibility to apply boron neutron capture therapy (BNCT) to lung tumors, some rats are planned to be irradiated in the thermal column of the TRIGA reactor of the University of Pavia. Before the irradiation, lung metastases will be induced in BDIX rats, which will be subsequently infused with boronophenylalanine (BPA). During the irradiation, the rats will be positioned in a box designed to shield the whole animal except the thorax area. In order to optimize the irradiation set-up and to design a suitable shielding box, a set of calculations were performed with the MCNP Monte Carlo transport code. A rat model was constructed using the MCNP geometry capabilities and was positioned in a box with walls filled with lithium carbonate. A window was opened in front of the lung region. Different shapes of the holder and of the window were tested and analyzed in terms of the dose distribution obtained in the lungs and of the dose absorbed by the radiosensitive organs in the rat. The best configuration of the holder ensures an almost uniform thermal neutron flux inside the lungs (Phi(max)/Phi(min)=1.5), an irradiation time about 10 min long, to deliver at least 40 Gy(w) to the tumor, a mean lung dose of 5.9+/-0.4 Gy(w), and doses absorbed by all the other healthy tissues below the tolerance limits.

Extra-corporeal liver BNCT for the treatment of diffuse metastases: what was learned and what is still to be learned.

Almost eight years ago, in December 2001, we performed for the first time in the world thermal neutron irradiation on an isolated liver of a patient. The organ was affected by diffuse metastases of a colon carcinoma and had been previously loaded with a (10)B compound. In July 2003, the same procedure was applied again on a patient for the treatment of unresectable and incurable hepatic metastases of a carcinoma of the rectum. Both patients are dead at present. Now we can analyze in depth the clinical history of these patients and evaluate the effectiveness of this therapy. From this exciting experience we learned much, and we also found out about complications till then unknown, which need to be studied and addressed experimentally. Unfortunately we can base our conclusions just on the experience we had with these two patients. We could have been much more detailed and firm in our statements if the number of clinical cases was larger. The BNCT Pavia project has been suspended, but it is more than likely to resume in a short time. Good findings were many. The procedure is feasible; the original concept of complete immersion of the diseased liver in a homogeneous neutron field proved effective and winning. The tumor masses resulted completely necrotic and unknown metastases too appeared radically treated; healthy hepatic tissue was preserved from both morphological and functional points of view; no symptoms of cirrhosis appeared even four years after treatment. For the long term surviving patient, quality of life was excellent. Other findings require to be tackled in depth. The "post-irradiation syndrome" we observed in both patients, with identical symptoms and biochemical derangements, creates a dramatic--even though totally reversible--clinical condition, that is the probable cause of death for our second patient, suffering from cardiomyopathy, 33 days after treatment. For the first patient, recurrences were a late yet fatal complication, for which even a further surgical revision was ineffective. We offer some hypotheses about their origin and possible prevention.

Selective uptake of p-boronophenylalanine by osteosarcoma cells for boron neutron capture therapy.

Osteosarcoma is the most common non-hematologic primary cancer type that develops in bone. Current osteosarcoma treatments combine multiagent chemotherapy with extensive surgical resection, which in some cases makes necessary the amputation of the entire limb. Nevertheless its infiltrative growth leads to a high incidence of local and distant recurrences that reduce the percentage of cured patients to less than 60%. These poor data required to set up a new therapeutic approach aimed to restrict the surgical removal meanwhile performing a radical treatment. Boron neutron capture therapy (BNCT), a particular radiotherapy based on the nuclear capture and fission reactions by atoms of (10)B, when irradiated with thermal neutrons, could be a valid alternative or integrative option in case of osteosarcoma management, thanks to its peculiarity in selectively destroying neoplastic cells without damaging normal tissues. Aim of the present work is to investigate the feasibility of employing BNCT to treat the limb osteosarcoma. Boronophenylalanine (BPA) is used to carry (10)B inside the neoplastic cells. As a first step the endocellular BPA uptake is tested in vitro on the UMR-106 osteosarcoma cell line. The results show an adequate accumulation capability. For the in vivo experiments, an animal tumor model is developed in Sprague-Dawley rats by means of an intrafemoral injection of UMR-106 cells at the condyle site. The absolute amounts of boron loading and the tumor to normal tissue (10)B ratio are evaluated 2 h after the i.v. administration of BPA. The boron uptake by the neoplastic tissue is almost twice the normal one. However, higher values of boron concentration in tumor are requested before upholding BNCT as a valid therapeutic option in the treatment of osteosarcoma.

Feasibility study on the utilization of boron neutron capture therapy (BNCT) in a rat model of diffuse lung metastases.

In order for boron neutron capture therapy (BNCT) to be eligible for application in lung tumour disease, three fundamental criteria must be fulfilled: there must be selective uptake of boron in the tumour cells with respect to surrounding healthy tissue, biological effectiveness of the radiation therapy and minimal damage or collateral effects of the irradiation on the surrounding tissues. In this study, we evaluated the biological effectiveness of BNCT by in vitro irradiation of rat colon-carcinoma cells previously incubated in boron-enriched medium. One part of these cells was re-cultured in vitro while the other was inoculated via the inferior vena cava to induce pulmonary metastases in a rat model. We observed a post-irradiation in vitro cell viability of 0.05% after 8 days of cell culture. At 4 months follow-up, all animal subjects in the treatment group that received irradiated boron-containing cells were alive. No animal survived beyond 1 month in the control group that received non-treated cells (p<0.001 Kaplan-Meier). These preliminary findings strongly suggest that BNCT has a significant lethal effect on tumour cells and post irradiation surviving cells lose their malignant capabilities in vivo. This radio-therapeutic potential warrants the investigation of in vivo BNCT for lung tumour metastases.

Neutron autoradiography imaging of selective boron uptake in human metastatic tumours.

The ability to selectively hit the tumour cells is an essential characteristic of an anti-tumour therapy. In boron neutron capture therapy (BNCT) this characteristic is based on the selective uptake of (10)B in the tumour cells with respect to normal tissues. An important step in the BNCT planning is the measurement of the boron concentration in the tissue samples, both tumour and healthy. When the tumour is spread through the healthy tissue, as in the case of metastases, the knowledge of the different kinds of tissues in the sample being analysed is crucial. If the percentage of tumour and normal tissues cannot be evaluated, the obtained concentration is a mean value depending on the composition of the different samples being measured. In this case an imaging method that could give information both on the morphology and on the spatial distribution of boron concentration in the sample would be a fundamental support. In this paper, the results of the boron uptake analysis in the tumour and in the healthy samples taken from human livers after boron phenylalanine (BPA) infusion are shown; boron imaging was performed using neutron autoradiography.

How to study boron biodistribution in liver metastases from colorectal cancer.

The purpose of this study was to evaluate boron distribution for a safe and effective BNCT (Boron Neutron Capture Therapy) of liver metastases. Samples both from healthy and tumour liver parenchyma were analysed, after i.v. boron administration, by: alpha particles counting under neutron irradiation; morphological analysis by standard haematoxylin-eosin staining; neutron autoradiography. Our method was unaffected by the cytological heterogeneity inside tumour nodules; it demonstrated selective boron distribution in tumour tissue and predicted estimated mean therapeutic doses in tumour and safety doses in healthy tissue. The time interval for efficient BNCT was 2 to 4 hours after i.v. boron administration.

Response of Quercus pubescens leaves exposed to geothermal pollutant input in southern Tuscany (Italy).

The paper reports a case of evident and widespread leaf damage on trees in southern Tuscany (Central Italy) attributed to the input of pollutants produced in a geothermal area. The main potentially phytotoxic substances are boron and hydrogen sulphide. Trees affected are conifers as well as both evergreen and deciduous broadleaves. In the present study the possible impact of geothermal pollutants on Quercus pubescens leaves has been considered. Leaf samples coming from three sampling locations (S1 inside the geothermal area; S2 on the margins; S3 outside) and three consecutive dates (June, July and August) were analyzed for the following parameters: sulphur and boron concentration; leaf area; leaf mass per area; chlorophyll fluorescence (Fv/Fm); chlorophyll a, chlorophyll b and carotenoid concentrations. Anatomical and ultrastructural observations were also performed. In all sampling location sulphur and boron concentrations are greater than the background values recorded in southern Tuscany in a previous survey. The sulphur concentration in leaves was higher in S1 than S2 and S3, but did not increase throughout the survey period. Boron reached the greatest concentrations in S2 and showed a continuous increase over the study period. Leaves subjected to a higher load of pollutants were smaller in size (in terms of leaf area), but were more sclerophyllous. Damaged chloroplasts and reduced Fv/Fm values were observed at S1 and S2, but chlorophyll concentration values were higher at S1. Such an apparent anomaly can possibly be explained by the onset of compensation and recovery mechanisms. Foliar injuries appeared to be related to boron concentration.