Piero Bruschi

Clinical lessons from the first applications of BNCT on unresectable liver metastases.

After a long series of studies on the effects of neutron irradiation of 10 B loaded neoplastic cells both in culture and in animal experiments, we started the clinical application of BNCT on humans affected by liver metastases of a radically resected colon adenocarcinoma. The procedure we adopted includes a first surgical phase, with hepatectomy; a radiotherapeutic phase, in which the isolated liver, washed and chilled, is extracorporeally irradiated with thermal neutrons; and then a second surgical phase for the reconnection of the liver to the patient. Until now two patients have been subjected to the BNCT treatment. The first one survived 44 months with a good quality of life, and died because of diffuse recurrences of his intestinal tumour. The second patient had the same early perioperative course, but after 33 days a worsening of a dilatative cardiomyopaty, from which he was suffering, determined a cardiac failure and eventually death. This clinical experience, although limited, has shown that extracorporeal neutron irradiation of the liver is a feasible procedure, able to ensure the complete destruction of liver metastases and a possible long lasting survival. In our patients neutron irradiation caused massive cellular necrosis highly specific to tumour cells, whereas normal cells were mostly spared. Nevertheless, the impact of such a traumatic operation on the patients organism must be taken into account. Finally, we have to be aware that the fight against tumour rarely leads to a complete victory. We now have an innovative weapon which is both powerful and partly unsettled: it must be refined and above all used.

Within- and among-tree variation in leaf morphology of Quercus petraea (Matt.) Liebl. natural populations

Abstract. Variation in leaf morphology of Quercus petraea in response to several ecological conditions has been studied extensively, although not explicitly in the context of within- and among-tree variation. This study examined leaf morphology and anatomy of Q. petraea, growing in five natural Italian populations adapted to different ecological environments, to understand the pattern of within- and among-tree variation in this species. We used an ANOVA model with both crossed and nested effects. All levels contributed significant components of variation. Within-tree variation due to branch position was large, particularly in thickness and productivity (40%). For 19 of 32 variables, the variation among trees was surprisingly lower than the within-tree variation explained by branch position. Trends in leaf morphology and anatomy with branch position exhibited the sun-shade dichotomy. Patterns of crown plasticity showed lower values in the two xeric populations. Results suggest the need for taxonomic studies to consider variation as a quantitative attribute of individual trees.

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.

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.

MRI-guided neutron capture therapy by use of a dual gadolinium/boron agent targeted at tumour cells through upregulated low-density lipoprotein transporters.

The upregulation of low-density lipoprotein (LDL) transporters in tumour cells has been exploited to deliver a sufficient amount of gadolinium/boron/ligand (Gd/B/L) probes for neutron capture therapy, a binary chemio-radiotherapy for cancer treatment. The Gd/B/L probe consists of a carborane unit (ten B atoms) bearing an aliphatic chain on one side (to bind LDL particles), and a Gd(III)/1,4,7,10-tetraazacyclododecane monoamide complex on the other (for detection by magnetic resonance imaging (MRI)). Up to 190 Gd/B/L probes were loaded per LDL particle. The uptake from tumour cells was initially assessed on cell cultures of human hepatoma (HepG2), murine melanoma (B16), and human glioblastoma (U87). The MRI assessment of the amount of Gd/B/L taken up by tumour cells was validated by inductively coupled plasma-mass-spectrometric measurements of the Gd and B content. Measurements were undertaken in vivo on mice bearing tumours in which B16 tumour cells were inoculated at the base of the neck. From the acquisition of magnetic resonance images, it was established that after 4-6 hours from the administration of the Gd/B/L-LDL particles (0.1 and 1 mmol kg(-1) of Gd and (10)B, respectively) the amount of boron taken up in the tumour region is above the threshold required for successful NCT treatment. After neutron irradiation, tumour growth was followed for 20 days by MRI. The group of treated mice showed markedly lower tumour growth with respect to the control group.

Design, development and characterization of multi-functionalized gold nanoparticles for biodetection and targeted boron delivery in BNCT applications

The aim of this study is to optimize targeted boron delivery to cancer cells and its tracking down to the cellular level. To this end, we describe the design and synthesis of novel nanovectors that double as targeted boron delivery agents and fluorescent imaging probes. Gold nanoparticles were coated with multilayers of polyelectrolytes functionalized with the fluorescent dye (FITC), boronophenylalanine and folic acid. In vitro confocal fluorescence microscopy demonstrated significant uptake of the nanoparticles in cancer cells that are known to overexpress folate receptors.

Geographical variation in morphology of Quercus petraea (Matt.) Liebl. as related to drought stress

Abstract Differences in phenotypic plasticity among six populations of Quercus petraea exposed to drought stress were examined. The study was performed as a common garden experiment with five‐year‐old seedlings in a greenhouse and a total of 13 morphological and micromorphological characters were scored for 24 seedlings from each population, in both control and droughted environments. The populations studied showed considerable differentiation for trait means while maintaining several of the morphological differences observed in the field. Since these differences in morphology were found in populations growing under the same conditions, it is probable that these are due to genetic differences. However, a high degree of phenotypic variation was found with significant population × water treatment interactions. Overall, this study shows that the origin of sessile oaks (xeric or mesic stands) determines the variation in phenotypic plasticity when plants are compared under different water regimes.

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.

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.

Leaf macro- and micro-morphological altitudinal variability of Carpinus betulus in the Hyrcanian forest (Iran)

We investigated the altitudinal variation of Carpinus betulus L. in the Hyrcanian forest using leaf macro-morphological and micro-morphological traits. We collected a total of 1600 leaves from two locations. In each location, we sampled six populations along an altitudinal gradient ranging from 100 m to 1,150 m. We found that trees in the higher elevations have smaller leaf lamina than those in the lower elevations. In contrast, leaf mass per area was high at low altitudes and increased newly at the higher ones. Stomatal dimension was negatively correlated with elevation, while stomatal density was positively correlated with elevation. We also found that two transects showed the same plasticity trend. Leaf area showed the highest plasticity, while the number of veins showed the lowest plasticity. This study shows that altitude, and related temperature and rainfall, represents an important driving force in Carpinus betulus leaf morphological variation. Moreover, our results suggest that leaf area, leaf mass per area and stomatal density could influence the species responses to different ecological conditions.