From nuclear track characterization to machine learning based image classification in neutron autoradiography for boron neutron capture therapy.

Knowledge of the 10B microdistribution is of great relevance in BNCT studies. Since 10B concentration assesment through neutron autoradiography depends on the correct quantification of tracks in a nuclear track detector, image acquisition and processing conditions should be controlled and verified, in order to obtain accurate results to be applied in the frame of BNCT. With this aim, an image verification process was proposed, based on parameters extracted from the quantified nuclear tracks. Track characterization was performed by selecting a set of morphological and pixel-intensity uniformity parameters from the quantified objects (area, diameter, roundness, aspect ratio, heterogeneity and clumpiness). Their distributions were studied, leading to the observation of varying behaviours in images generated by different samples and acquisition conditions. The distributions corresponding to samples coming from the BNC reaction showed similar attributes in each analyzed parameter, proving to be robust to the experimental process, but sensitive to light and focus conditions. Considering those observations, a manual feature extraction was performed as a pre-processing step. A Support Vector Machine (SVM) and a fully dense Neural Network (NN) were optimized, trained, and tested. The final performance metrics were similar for both models: 93%-93% for the SVM, vs 94%-95% for the NN in accuracy and precision respectively. Based on the distribution of the predicted class probabilities, the latter had a better capacity to reject inadequate images, so the NN was selected to perform the image verification step prior to quantification. The trained NN was able to correctly classify the images regardless of their track density. The exhaustive characterization of the nuclear tracks provided new knowledge related to the autoradiographic images generation. The inclusion of machine learning in the analysis workflow proves to optimize the boron determination process and paves the way for further applications in the field of boron imaging.

Enhanced Resolution of Neutron Autoradiography with UV-C Sensitization to Study Boron Microdistribution in Animal Models.

The assessment of boron microdistribution is essential to evaluate the suitability of boron neutron capture therapy (BNCT) in different biological models. In our laboratory, we have reported a methodology to produce cell imprints on polycarbonate through UV-C sensitization. The aim of this work is to extend the technique to tissue samples in order to enhance spatial resolution. As tissue structure largely differs from cultured cells, several aspects must be considered. We studied the influence of the parameters involved in the imprint and nuclear track formation, such as neutron fluence, different NTDs, etching and UV-C exposure times, tissue absorbance, thickness, and staining, among others. Samples from different biological models of interest for BNCT were used, exhibiting homogeneous and heterogeneous histology and boron microdistribution. The optimal conditions will depend on the animal model under study and the resolution requirements. Both the imprint sharpness and the fading effect depend on tissue thickness. While 6 h of UV-C was necessary to yield an imprint in CR-39, only 5 min was enough to observe clear imprints on Lexan. The information related to microdistribution of boron obtained with neutron autoradiography is of great relevance when assessing new boron compounds and administration protocols and also contributes to the study of the radiobiology of BNCT.

Measurement of an evaporation coefficient in tissue sections as a correction factor for 10B determination.

Boron neutron capture therapy (BNCT) is a cancer treatment option that combines preferential uptake of a boron compound in tumors and irradiation with thermal neutrons. For treatment planning, the boron concentration in different tissues must be considered. Neutron autoradiography using nuclear track detectors (NTD) can be applied to study both the concentration and microdistribution of boron in tissue samples. Histological sections are obtained from frozen tissue by cryosectioning. When the samples reach room temperature, they undergo an evaporation process, which leads to an increase in the boron concentration. To take this effect into account, certain correction factors (evaporation coefficients, CEv) must be applied. With this aim, a protocol was established to register and analyze mass variation of tissue sections, measured with a semimicro scale. Values of ambient temperature, pressure, and humidity were simultaneously recorded. Reproducible results of evaporation curves and CEv values were obtained for different tissue samples, which allowed the systematization of the procedure. This study could contribute to a more precise determination of boron concentration in tissue samples through the neutron autoradiography technique, which is of great relevance to make dosimetric calculations in BNCT.

Neutron autoradiography to study the microdistribution of boron in the lung.

In Argentina, a multi-institutional project has been established to assess the feasibility of applying BNCT ex-situ to the treatment of patients with multiple metastases in both lungs. Within this context, this work aims at applying the neutron autoradiography technique to study boron microdistribution in the lung. A comprehensive analysis of the different aspects for the generation of autoradiographic images of both normal and metastatic BDIX rat lungs was achieved. Histology, boron uniformity, optimal tissue thickness and water content in tissue were explored for the two types of samples. A qualitative and a quantitative analysis were performed. No heterogeneities in uptake were observed in normal lung. Conversely, samples with metastasis showed preferential boron uptake in the tumour areas with respect to surrounding tissue. Surrounding tissue would present a slightly higher uptake of boron than the normal lung. Quantitative results of boron concentration values and ratios determined by neutron autoradiography were obtained. In order to contribute to BNCT dosimetry, further analysis increasing the number of samples is warranted.

Neutron Autoradiography Combined With UV-C Sensitization: Toward the Intracellular Localization of Boron.

Our group has reported the imprint formation of biological material on polycarbonate nuclear track detectors by UV-C exposure, which is used as an approach to simultaneously visualize cell imprints and nuclear tracks coming from the boron neutron capture reaction. Considering that the cell nucleus has a higher UV-C absorption than the cytoplasm and that hematoxylin preferentially stains the nucleus, we proposed to enhance the contrast between these two main cell structures by hematoxylin staining before UV-C sensitization. In this study, several experiments were performed in order to optimize UV-C exposure parameters and chemical etching conditions for cell imprint formation using the SK-BR-3 breast cancer cell line. The proposed method improves significantly the resolution of the cell imprints. It allows clear differentiation of the nucleus from the rest of the cell, together with nuclear tracks pits. Moreover, it reduces considerably the UV-C exposure time, an important experimental issue. The proposed methodology can be applied to study the boron distribution independently from the chosen cell line and/or boron compounds.

Electroporation optimizes the uptake of boron-10 by tumor for boron neutron capture therapy (BNCT) mediated by GB-10: a boron biodistribution study in the hamster cheek pouch oral cancer model.

Boron neutron capture therapy (BNCT) is a promising cancer binary therapy modality that utilizes the nuclear capture reaction of thermal neutrons by boron-10 resulting in a localized release of high- and low-linear energy transfer (LET) radiation. Electrochemotherapy (ECT) is based on electroporation (EP) that induces opening of pores in cell membranes, allowing the entry of compounds. Because EP is applied locally to a tumor, the compound is incorporated preferentially by tumor cells. Based on the knowledge that the therapeutic success of BNCT depends centrally on the boron content in tumor and normal tissues and that EP has proven to be an excellent facilitator of tumor biodistribution of an anti-tumor agent, the aim of this study was to evaluate if EP can optimize the delivery of boronated compounds. We performed biodistribution studies and qualitative microdistribution analyses of boron employing the boron compound sodium decahydrodecaborate (GB-10) + EP in the hamster cheek pouch oral cancer model. Syrian hamsters with chemically induced exophytic squamous cell carcinomas were used. A typical EP treatment was applied to each tumor, varying the moment of application with respect to the administration of GB-10 (early or late). The results of this study showed a significant increase in the absolute and relative tumor boron concentration and optimization of the qualitative microdistribution of boron by the use of early EP + GB-10 versus GB-10 without EP. This strategy could be a tool to improve the therapeutic efficacy of BNCT/GB-10 in vivo.

Extending neutron autoradiography technique for boron concentration measurements in hard tissues.

The neutron autoradiography technique using polycarbonate nuclear track detectors (NTD) has been extended to quantify the boron concentration in hard tissues, an application of special interest in Boron Neutron Capture Therapy (BNCT). Chemical and mechanical processing methods to prepare thin tissue sections as required by this technique have been explored. Four different decalcification methods governed by slow and fast kinetics were tested in boron-loaded bones. Due to the significant loss of the boron content, this technique was discarded. On the contrary, mechanical manipulation to obtain bone powder and tissue sections of tens of microns thick proved reproducible and suitable, ensuring a proper conservation of the boron content in the samples. A calibration curve that relates the 10B concentration of a bone sample and the track density in a Lexan NTD is presented. Bone powder embedded in boric acid solution with known boron concentrations between 0 and 100 ppm was used as a standard material. The samples, contained in slim Lexan cases, were exposed to a neutron fluence of 1012 cm-2 at the thermal column central facility of the RA-3 reactor (Argentina). The revealed tracks in the NTD were counted with an image processing software. The effect of track overlapping was studied and corresponding corrections were implemented in the presented calibration curve. Stochastic simulations of the track densities produced by the products of the 10B thermal neutron capture reaction for different boron concentrations in bone were performed and compared with the experimental results. The remarkable agreement between the two curves suggested the suitability of the obtained experimental calibration curve. This neutron autoradiography technique was finally applied to determine the boron concentration in pulverized and compact bone samples coming from a sheep experimental model. The obtained results for both type of samples agreed with boron measurements carried out by ICP-OES within experimental uncertainties. The fact that the histological structure of bone sections remains preserved allows for future boron microdistribution analysis.

Evaporation process in histological tissue sections for neutron autoradiography.

The analysis of the distribution and density of nuclear tracks forming an autoradiography in a nuclear track detector (NTD) allows the determination of 10B atoms concentration and location in tissue samples from Boron Neutron Capture Therapy (BNCT) protocols. This knowledge is of great importance for BNCT dosimetry and treatment planning. Tissue sections studied with this technique are obtained by cryosectioning frozen tissue specimens. After the slicing procedure, the tissue section is put on the NTD and the sample starts drying. The thickness varies from its original value allowing more particles to reach the detector and, as the mass of the sample decreases, the boron concentration in the sample increases. So in order to determine the concentration present in the hydrated tissue, the application of corrective coefficients is required. Evaporation mechanisms as well as various factors that could affect the process of mass variation are outlined in this work. Mass evolution for tissue samples coming from BDIX rats was registered with a semimicro analytical scale and measurements were analyzed with software developed to that end. Ambient conditions were simultaneously recorded, obtaining reproducible evaporation curves. Mathematical models found in the literature were applied for the first time to this type of samples and the best fit of the experimental data was determined. The correlation coefficients and the variability of the parameters were evaluated, pointing to Page's model as the one that best represented the evaporation curves. These studies will contribute to a more precise assessment of boron concentration in tissue samples by the Neutron Autoradiography technique.

Reprint of Inter-comparison of boron concentration measurements at INFN-University of Pavia (Italy) and CNEA (Argentina).

An inter-comparison of three boron determination techniques was carried out between laboratories from INFN-University of Pavia (Italy) and CNEA (Argentina): alpha spectrometry (alpha-spect), neutron capture radiography (NCR) and quantitative autoradiography (QTA). Samples of different nature were analysed: liquid standards, liver homogenates and tissue samples from different treatment protocols. The techniques showed a good agreement in a concentration range of interest in BNCT (1-100 ppm), thus demonstrating their applicability as precise methods to quantify boron and determine its distribution in tissues.

Inter-comparison of boron concentration measurements at INFN-University of Pavia (Italy) and CNEA (Argentina).

An inter-comparison of three boron determination techniques was carried out between laboratories from INFN-University of Pavia (Italy) and CNEA (Argentina): alpha spectrometry (alpha-spect), neutron capture radiography (NCR) and quantitative autoradiography (QTA). Samples of different nature were analysed: liquid standards, liver homogenates and tissue samples from different treatment protocols. The techniques showed a good agreement in a concentration range of interest in BNCT (1-100ppm), thus demonstrating their applicability as precise methods to quantify boron and determine its distribution in tissues.

Assessing advantages of sequential boron neutron capture therapy (BNCT) in an oral cancer model with normalized blood vessels.

BACKGROUND: We previously demonstrated the therapeutic success of sequential boron neutron capture therapy (Seq-BNCT) in the hamster cheek pouch oral cancer model. It consists of BPA-BNCT followed by GB-10-BNCT 24 or 48 hours later. Additionally, we proved that tumor blood vessel normalization with thalidomide prior to BPA-BNCT improves tumor control. The aim of the present study was to evaluate the therapeutic efficacy and explore potential boron microdistribution changes in Seq-BNCT preceded by tumor blood vessel normalization. MATERIAL AND METHODS: Tumor bearing animals were treated with thalidomide for tumor blood vessel normalization, followed by Seq-BNCT (Th+ Seq-BNCT) or Seq-Beam Only (Th+ Seq-BO) in the window of normalization. Boron microdistribution was assessed by neutron autoradiography. RESULTS: Th+ Seq-BNCT induced overall tumor response of 100%, with 87 (4)% complete tumor response. No cases of severe mucositis in dose-limiting precancerous tissue were observed. Differences in boron homogeneity between tumors pre-treated and not pre-treated with thalidomide were observed. CONCLUSION: Th+ Seq-BNCT achieved, for the first time, response in all treated tumors. Increased homogeneity in tumor boron microdistribution is associated to an improvement in tumor control.

Neutron autoradiography to study boron compound microdistribution in an oral cancer model.

PURPOSE: We previously reported the therapeutic efficacy of Sequential Boron Neutron Capture Therapy (Seq-BNCT), i.e., BPA (boronophenylalanine) - BNCT followed by GB-10 (decahydrodecaborate) - BNCT 1 or 2 days later, in the hamster cheek pouch oral cancer model. We have utilized the neutron autoradiography methodology to study boron microdistribution in tissue. The aim was to use this method to evaluate if the distribution of GB-10 is altered by prior application of BPA-BNCT in Sequential BNCT protocols. MATERIALS AND METHODS: Extensive qualitative and quantitative autoradiography analyses were performed in the following groups: G1 (animals without boron); G2 (animals injected with BPA); G3 (animals injected with GB-10); G4 (same as G3, 24 h after BPA-BNCT); and G5 (same protocol as G4, 48 h interval). RESULTS: A detailed study of boron localization in the different tissue structures of tumor, premalignant and normal tissue in the hamster cheek pouch was performed. GB-10 accumulated preferentially in non-neoplastic connective tissue, whereas for BPA neoplastic cells showed the highest boron concentration. Boron distribution was less heterogeneous for GB-10 than for BPA. In premalignant and normal tissue, GB-10 and BPA accumulated mostly in connective tissue and epithelium, respectively. CONCLUSIONS: BPA-BNCT could alter boron microlocalization of GB-10 administered subsequently. Boron targeting homogeneity is essential for therapeutic success.