Yuko Kinashi

Radiobiological evidence suggesting heterogeneous microdistribution of boron compounds in tumors: Its relation to quiescent cell population and tumor cure in neutron capture therapy

PURPOSE The heterogeneous microdistribution of boron compounds in tumors and its significance on tumor cure were examined by a radiobiological procedure. The role of quiescent (Q) cells in tumor was especially investigated. METHODS AND MATERIALS 10B-enriched paraboronophenylalanine (BPA) and mercaptoundecahydrododecaborate (BSH) were administered to SCCVII tumor bearing C3H/He mice by intragastric and i.v. injections, respectively. The continued effects of these boron compounds with thermal irradiations were studied by using colony formation and tumor control assays. Their effects on Q cells were also analyzed by the combined method of micronucleus frequency assay and an identification of proliferating (P) cells by BUdR and anti-BUdR monoclonal antibody. RESULTS 10B-concentration after BPA (1,500 mg/kg) and BSH (75 mg/kg) administration were 11 ppm at 3 h and 10.5 ppm at 30 min, respectively. Cell survival decreased exponentially with an increment of neutron fluence (phi). The exponential parts of the curves were: -InSF = -0.052+ 13.0x10(13)phi, -InSF = -0.032+7.68X10(-13)phi, and -InSF = -0.0005+2.68x10(-13)phi for BPA-BNCT, BSH-BNCT, and NCT alone, respectively. Fifty percent tumor control was obtained at the influence of 10.2 x 10(12) n/cm2 in BPA-BNCT. On the other hand, 11.4 x 10(12) n/cm2 of neutrons had to be delivered in BSH-BNCT. The normal nuclear division fraction defined as the cell fraction that did not express micronuclei at first mitosis after treatment was investigated. The surviving cell fraction and the normal nuclear division fraction were regarded as equal in NCT alone. However, the normal nuclear division factor following BPA-BNCT was greater than the surviving cell fraction, and the difference increased with an increase in neutron fluence. In Q cells, BSH-BNCT yielded higher micronucleus frequency than BPA-BNCT and NCT alone. The frequencies in Q cells following BPA-BNCT and NCT alone were almost same as that in total cell population after NCT alone. CONCLUSIONS Our data suggested that BPA distributed in tumors hetergeneously. Q cells especially might not accumulate BPA. To decrease the possible disadvantage of BPA-BNCT, the combination of BPA and BSH or other neutron capture element that emit particles with longer ranges, for example, gadolinium, would have to be investigated.

The combined effect of boronophenylalanine and borocaptate in boron neutron capture therapy for SCCVII tumors in mice.

PURPOSE To increase the effect of boron neutron capture therapy (BNCT) on tumors in vivo, the combined effects of para-boronophenylalanine (BPA) and borocaptate sodium (BSH) were investigated. METHODS AND MATERIALS 10B-enriched BPA and BSH were administered to C3H/He mice bearing SCCVII tumors by intragastric and intravenous injections, respectively. The colony formation and tumor control assays were employed for investigating antitumor effects of BNCT. The extent of homogeneity of tumor cell killing effect was examined by the distribution of frequencies of binuclear cells (BNC) producing a certain number of micronuclei (0,1,2,--,> or =5) to total number of BNC and by the comparison between surviving cell fraction (SF) in colony formation assay and the normal nuclear division fraction (NNDF) at first mitosis following BNCT. RESULTS The relationships between SF and radiation dose in Gy (D) at around 10 ppm of 10B in tumors were as follow: -InSF = -0.101 + 0.648 Gy(-1) x D, 0.0606+0.435 Gy(-1) x D, and -0.0155 + 0.342 Gy(-1) x D for BPA, BPA + BSH, and BSH, respectively. In tumor control assay, BPA was also more effective than BSH, but the difference of effectiveness significantly decreased: 1.9 times more effective in colony assay vs. 1.2 times in tumor control assay. The most effective treatment to achieve tumor cure was BNCT using BPA + BSH, and it was 1.9 times more effective than BSH-BNCT. In BSH-BNCT, NNDF decreased exponentially with radiation dose and was equal to SF. However, NNDF following BPA-BNCT showed a biphasic decrease with radiation dose, and SF was much lower than NNDF. In the combination of BPA and BSH, the discrepancy between NNDF and SF decreased in comparison with BPA-BNCT. The distribution of frequency of BNC with a certain number of micronuclei to total BNC was very close to Poisson distribution in BSH-BNCT tumors; however, it deviated from the Poisson in BPA-BNCT tumors. In combination with BPA and BSH, the distribution showed an intermediate pattern. These findings indicate that BSH distributes homogeneously with a heterogeneous distribution of BPA in tumors, and the heterogeneous effect of BPA-BNCT was improved by the combination of two boron compounds. CONCLUSION The heterogeneous cell killing effect of BPA-BNCT was improved by the combination of BSH, and increased tumor control rates. Therefore, this combination may improve clinical outcome of BNCT although the effects on normal tissues have to be examined before clinical application.

Boron neutron capture therapy outcomes for advanced or recurrent head and neck cancer

We retrospectively review outcomes of applying boron neutron capture therapy (BNCT) to unresectable advanced or recurrent head and neck cancers. Patients who were treated with BNCT for either local recurrent or newly diagnosed unresectable head or neck cancers between December 2001 and September 2007 were included. Clinicopathological characteristics and clinical outcomes were retrieved from hospital records. Either a combination of borocaptate sodium and boronophenylalanine (BPA) or BPA alone were used as boron compounds. In all the treatment cases, the dose constraint was set to deliver a dose <10–12 Gy-eq to the skin or oral mucosa. There was a patient cohort of 62, with a median follow-up of 18.7 months (range, 0.7–40.8). A total of 87 BNCT procedures were performed. The overall response rate was 58% within 6 months after BNCT. The median survival time was 10.1 months from the time of BNCT. The 1- and 2-year overall survival (OS) rates were 43.1% and 24.2%, respectively. The major acute Grade 3 or 4 toxicities were hyperamylasemia (38.6%), fatigue (6.5%), mucositis/stomatitis (9.7%) and pain (9.7%), all of which were manageable. Three patients died of treatment-related toxicity. Three patients experienced carotid artery hemorrhage, two of whom had coexistent infection of the carotid artery. This study confirmed the feasibility of our dose-estimation method and that controlled trials are warranted.

Experimental verification of beam characteristics for cyclotron-based epithermal neutron source (C-BENS).

A cyclotron-based epithermal neutron source has been developed for boron neutron capture therapy. This system consists of a cyclotron accelerator producing 1.1-mA proton beams with an energy of 30 MeV, a beam transport system coupled with a beryllium neutron production target, and a beam-shaping assembly (BSA) with a neutron collimator. In our previous work, the BSA was optimized to obtain sufficient epithermal neutron fluxes of ~10(9) cm(-2) s(-1) using a Monte Carlo simulation code. In order to validate the simulation results, irradiation tests using multi-foil activation at the surface of a gamma-ray shield located behind the collimator and water phantom experiments using a collimated epithermal neutron beam were performed. It was confirmed experimentally that the intensity of the epithermal neutrons was 1.2×10(9) cm(-2) s(-1).

The effects of boron neutron capture therapy on liver tumors and normal hepatocytes in mice.

To explore the feasibility of employing boron neutron capture therapy (BNCT) to treat liver tumors, the effects of BNCT were investigated by using liver tumor models and normal hepatocytes in mice. Liver tumor models in C3H mice were developed by intrasplenic injection of SCCVII tumor cells. After borocaptate sodium (BSH) and boronophenylalanine (BPA) administration, 10B concentrations were measured in tumors and liver and the liver was irradiated with thermal neutrons. The effects of BNCT on the tumor and normal hepatocytes were studied by using colony formation assay and micronucleus assay, respectively. To compare the effects of BSH‐BNCT and BPA‐BNCT, the compound biological effectiveness (CBE) factor was determined. The CBE factors for BSH on the tumor were 4.22 and 2.29 using D10 and D0 as endpoints, respectively. Those for BPA were 9.94 and 5.64. In the case of hepatocytes, the CBE factors for BSH and BPA were 0.94 and 4.25, respectively. Tumor‐to‐liver ratios of boron concentration following BSH and BPA administration were 0.3 and 2.8, respectively. Considering the accumulation ratios of 10B, the therapeutic gain factors for BSH and BPA were 0.7‐1.3 and 3.8‐6.6, respectively. Therefore, it may be feasible to treat liver tumors with BPA‐BNCT.

Induction of DNA Double-Strand Breaks and Cellular Migration Through Bystander Effects in Cells Irradiated With the Slit-Type Microplanar Beam of the Spring-8 Synchrotron

PURPOSE To determine whether glioma cells irradiated with a microplanar X-ray beam exert bystander effects. METHODS AND MATERIALS Microplanar beam irradiation of glioma cells in vitro was done using the SPring-8 synchrotron radiation facility. The amount of DNA double-strand breaks (dsbs) was measured by the fluorescence intensity of phosphorylated H2AX or the number of 53BP1 foci. The dose distribution in a cell population exposed to a single microplanar beam was determined by the amount of phosphorylated H2AX-positive cells. Bystander effects were determined by counting the number of 53BP1 foci in nonirradiated cells treated with conditioned medium from cultures of irradiated cells. RESULTS More DNA dsbs were detected in cells adjacent to an area irradiated by the single beam than in cells in distant, nonirradiated areas as a result of bystander effects caused by scattered X-rays and DNA dsbs. In support of this, more 53BP1 foci were observed in nonirradiated, conditioned medium-treated cells than in control cells (i.e., cells not treated with irradiation or conditioned medium). These results suggest that DNA dsbs were induced in nonirradiated cells by soluble factors in the culture medium. In addition, we observed cellular migration into areas irradiated with peak doses, suggesting that irradiated cells send signals that cause nonirradiated cells to migrate toward damaged cells. CONCLUSIONS Bystander effects are produced by factors secreted as a result of slit-type microplanar X-ray beam irradiation.

A novel concept of treatment of diffuse or multiple pleural tumors by boron neutron capture therapy (BNCT)

Two patients, one with malignant pleural mesothelioma and one with a malignant short spindle cell tumor, received boron neutron capture therapy (BNCT). In each case, the tumors regressed or remained stable in size for 3-6 months following BNCT. No acute or late adverse events higher than grade 2 were observed.

Treatment results of boron neutron capture therapy using intra-arterial administration of boron compounds for recurrent head and neck cancer

The effect of boron neutron capture therapy (BNCT) is correlated with the density of boron in the tumour. BNCT using intra-arterial administration of boron compounds was performed for recurrent head and neck cancer. Of the five patients treated, one achieved a complete response and four achieved a partial response. There was one case of transient headache but no severe adverse effects were observed. The advantages of using an intra-arterial administration route for BNCT, which causes the selective killing of tumour cells, might offer a new option in the treatment of recurrent head and neck malignancies. These promising results require further verification and optimization of the BNCT schedule; however, dose escalation would appear to be justified because the toxicity appears to be very low.

Mutagenic effects at HPRT locus induced in Chinese hamster ovary cells by thermal neutrons with or without boron compound

CHO cells were exposed to thermal neutrons and their mutation frequency was determined. The Kyoto University Research Reactor (KUR), which has a very low level of contamination by gamma-rays and fast neutrons was used as a thermal neutron source. Cells were irradiated in the presence or absence of boric acid to determine mutation frequency and cell survival. Thermal neutron irradiation was 2.5 times as mutagenic as gamma-irradiation without boron. In the presence of boron, however, thermal neutron irradiation was from 4.2 to 4.5 times as mutagenic as gamma-irradiation. When the mutation frequency was plotted against the survival fraction, a higher degree of mutagenicity was observed in the presence than in the absence of boron. These results suggest that the enhancement of thermal neutron-induced mutation with boron is strongly associated with alpha-particles released by 10B(n, alpha)7 Li reaction.

The Micronucleus Assay of Lymphocytes Is a Useful Predictive Assay of the Radiosensitivity of Normal Tissue: A Study of Three Inbred Strains of Mice

Radiation sensitivity was measured by the micronucleus assay in T lymphocytes of the spleen and in fibroblasts derived from three inbred strains of mice, BALB/c, A/J and C3H/He. A linear correlation between the surviving fraction and the micronucleus frequency was obtained. There were clear differences in the radiosensitivity of T lymphocytes and of fibroblasts from the different strains of mice. T lymphocytes and fibroblasts from BALB/c mice were more radiosensitive than those from A/J and C3H/He mice. To explore the correlation between the radiosensitivity of T lymphocytes and of fibroblasts in vitro and the reaction of normal tissue to radiation, the survival ofjejunal crypt stem cells from the three mouse strains was determined using a microcolony assay. The jejunal crypt cells of BALB/c mice were more radiosensitive than those of A/J and C3H/He mice. Thus the order of radiosensitivity of T lymphocytes and fibroblasts in vitro corresponds to the sensitivity ofjejunal crypt cells in vivo. Our results suggest that determining the radiosensitivity of T lymphocytes and fibroblasts in vitro may be useful in predicting the magnitude of radiation-induced damage in the small intestine.