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Japanese Journal of Clinical Oncology Advance Access originally published online on May 18, 2007
Japanese Journal of Clinical Oncology 2007 37(4):245-249; doi:10.1093/jjco/hym022
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© 2007 Foundation for Promotion of Cancer Research

A Preliminary Experimental Study of Boron Neutron Capture Therapy for Malignant Tumors Spreading in Thoracic Cavity

Minoru Suzuki1,, Yoshinori Sakurai2, Shinichiro Masunaga1, Yuko Kinashi1, Kenji Nagata1, Akira Maruhashi2 and Koji Ono1

1 Particle Oncology Research Center
2 Division of Medical Physics, Research Reactor Institute, Kyoto University, Osaka, Japan

For reprints and all correspondence: Minoru Suzuki, Particle Oncology Research Center, Research Reactor Institute, Kyoto University, 2-1010, Asashiro-nishi, Kumatori-cho, Sennan-gun, Osaka 590-0494, Japan. E-mail: msuzuki{at}rri.kyoto-u.ac.jp

Received August 22, 2006; accepted November 24, 2006


   Abstract
TOP
 Abstract
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Conflict of interest statement
 References
 
Background: The purpose of the present study is to verify the treatment effects of boron neutron capture therapy (BNCT) in ectopic tumors implanted in the thoracic cavity mimicking malignant pleural mesothelioma (MPM).

Methods: The tumor model was created by implanting murine squamous cell carcinoma cells into the thoracic cavity. Mice were sorted into four groups: group I for non-treatment; group II for neutron irradiation; group III for {gamma}-ray irradiation; and group IV for BNCT irradiation. The antitumor effect was evaluated on the basis of the change in survival time. To assess the effects of BNCT on normal lung, non-tumor bearing mice were treated using the same method as done to the tumor-burdened mice.

Results: The BNCT group had a longer survival time of 31 days (range 5 – 60), which was significantly longer than that of the non-treated control group (P = 0.011), but not significantly different from that of the neutron and {gamma}-ray groups (P = 0.067 and 0.094, respectively). In the BNCT and neutron groups, incidence of minimal lung fibrosis was significantly higher compared with the non-treated control group (P = 0.003 and 0.04, respectively).

Conclusions: BNCT is a potentially promising treatment for malignant tumors spreading in the thoracic cavity such as MPM.

Key Words: BNCT • malignant pleural mesothelioma • experimental study.


   INTRODUCTION
TOP
 Abstract
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Conflict of interest statement
 References
 
Throughout the world, the incidence of malignant pleural mesothelioma (MPM) related to asbestos exposure is predicted to increase in the next 20–40 years (1), especially in the developing countries as a result of poor regulation and the proliferation of industrial use of asbestos (1). The prognosis of MPM has been dismal and the median survival is 9–12 months without intervention (2). Radiotherapy has been reported to be beneficial as a palliative therapy for MPM (3). However, radical radiotherapy with curative intent for MPM is impossible without destroying normal lung tissues, because MPM tends to spread throughout the entire pleural space and interlobar fissures.

Boron neutron capture therapy (BNCT) represents an interesting modality for selective irradiation of tumor cells and was first attempted some 40–50 years ago in the USA. Malignant gliomas and malignant melanomas have been treated with BNCT in Japan, Europe and the USA since the 1970s (4–6). BNCT facilities throughout the world includes seven research reactors; (i) Massachusetts Institute of Technology reactor (MTR); (ii) the FiR1 clinical reactor in Helsinki, Finland; (iii) R2-0 High Flux Reactor at Petten in the Netherlands; (iv) LVR-15 reactor at the Nuclear Research Institute in Rez, Czech Republic; (v) KUR in Kumatori, Japan; (vi) JRR4 at the Japan Atomic Energy Research Institute in Tokai-mura, Japan; and (vii) the RA-6 CNEA reactor in Bariloche, Argentina. In Japan, clinical trials on BNCT for recurrent head and neck tumor and multiple liver tumors started in 2001 (7).

It is possible that BNCT may selectively destroy malignant MPM cells while sparing normal lung tissues. BNCT is based on a nuclear reaction in which atoms of the non-radioactive isotope 10B that have absorbed low-energy (<0.5 eV) neutrons disintegrate into alpha (4He) particles and recoiled lithium nuclei (7Li). These particles deposit high energies along their very short paths (<10 µm) (8). Malignant cells treated with 10B are thus destroyed following thermal neutron irradiation. If sufficient numbers of 10B atoms accumulate in the MPM cells, and the gradient of the amount of 10B atoms between the tumor cells and the surrounding normal tissues is large, selective BNCT will be successfully delivered to the tumor cells.

The key to the success of BNCT for MPM is that BNCT provides a large dose gradient between the tumor in the thoracic cavity and the adjacent normal lung tissues, though the sensitivity of MPM cells to BNCT is important. Therefore, we created a thoracic cavity tumor model mimicking MPM by inoculating mouse squamous cell carcinoma cells into the thoracic cavity. The purpose of the present study is to verify the treatment effects of BNCT in ectopic tumors implanted in the thoracic cavity.


   MATERIALS AND METHODS
TOP
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 Conflict of interest statement
 References
 
Animals and Tumor Cell Line
Eight- to nine-week-old female C3H/He mice were used for this study. SCCVII tumor cells (mouse squamous cell carcinoma) were maintained in Eagle's minimum essential medium supplemented with 292 mg/l glutamine and 12.5% fetal calf serum. Cells were collected from monolayer culture, and suspended in serum-free medium at a concentration of 1 x 107/ml. All procedures for animal experiments were carried out in accordance with the regulations of Kyoto University Research Reactor Institute regarding animal care and handling.

Inoculation
Mice were anesthetized by intraperitoneal injection of phenobarbital (50 mg/kg body weight) and tumor cells (1 x 106/0.1 ml) were injected into the left pleural cavity using a 27-gauge needle. In all of the transplanted mice, bloody pleural effusion and tumor nodules spreading in the thoracic cavity were observed 7 days after inoculation. The regional spread pattern mimicked the clinical presentation of human MPM.

Boron Compound and Measurement of Boron Concentrations
We used boronophenylalanine (BPA), which has been used in clinical trials. Because BPA is barely soluble in water, a BPA-fructose complex (BPA-F) was prepared at a concentration of 24.2 mg BPA/ml. On the seventh day after inoculation, BPA-F was administered intraperitoneally at a dose of 500 mg/kg. This dose is the same as that used for the patients in our clinical trials.

Mice were killed by cervical dislocation 30 min after BPA administration, and tumor and lung samples were collected for 10B measurement. The 10B concentrations in these tissues were measured by prompt {gamma}-ray spectrometry.

Irradiation and Dose Measurement
Treatments were carried out on the fifth day after inoculation. Mice were sorted into four groups: group I for non-treatment (control); group II for neutron irradiation; group III for {gamma}-ray irradiation; and group IV for BPA-BNCT irradiation. In group III, the {gamma}-ray radiation dose was 7.9 Gy, which was the same physical dose as that delivered by BPA-BNCT in group IV. In groups II and IV, irradiation was performed for 40 min. BPA was administered 30 min before irradiation. All procedures were performed under general anesthesia by intraperitoneal administration of phenobarbital (50 mg/kg body weight). 60Co-{gamma}-ray irradiation in group III was administered at a dose rate of 1.2 Gy/min. Thermal neutrons were delivered from the left side of the mouse body at 5 MW, at the heavy water facility of Kyoto University Research Reactor Institute. Each mouse was held within a specially designed acrylic cage during irradiation. Pb-block and LiF tiles (50-mm thick) were used to shield parts of the body other than the chest in {gamma}-ray and thermal neutron irradiation, respectively. Seven to ten mice were used in each treatment group.

Neutron fluences were measured by radioactivation of gold foils (3 mm diameter; 0.05 mm thick) on the body surface. Thermoluminesent dosimeters were used for {gamma}-ray dosimetry. To convert the physical doses (Gy) into photon-equivalent doses (Gy-Eq), the relative biological effectiveness (RBE) of each component of the beam, and the compound biological effectiveness (CBE) factors of the boron compounds, were used. CBE factors were used as an alternative to RBE in evaluating the biologically absorbed dose of BNCT, because different boron compounds, or the same compound, might have different effects on different tissues, because of variations in the microdistribution of the compounds and the morphological character of the target cells (8).

Using the thermal neutron fluence and the physical absorbed dose of {gamma}-rays, ETotal was calculated by the following equations:


Formula 022UM1

where D is physical absorbed dose (Gy), FEThermal is the thermal neutron fluence (cm –2), N is concentration (wt%), where N = 3.0% for tumor and 3.1% for normal lung, C is the 10B concentration (ppm), RBEThermal = 3.0, RBE{gamma} = 1.0 and CBE = 3.8 for tumor.

Evaluation of Antitumor Effect
The antitumor effect was evaluated on the basis of the change in survival time. The median survival time was calculated for each group. Kaplan–Meier curves were also constructed.

Assessment of Normal Lung Toxicity
To assess the effects of BNCT delivered to thoracic region on normal lung, non-tumor bearing mice were sorted into four groups: group I for non-treatment (control); group II for neutron irradiation; group III for {gamma}-ray irradiation; and group IV for BPA-BNCT irradiation. Mice in groups II, III and IV were treated using the same method as for the tumor-burdened mice mentioned above.

To evaluate normal lung toxicity, all mice were killed 6 months after the irradiations. The left lung was formalin-fixed and paraffin-embedded (n = 6–10 each group). Sections of 5-µm thickness were stained with hematoxylin and eosin (H&E) and Masson's trichrome to evaluate lung fibrosis. The Ashcroft score was used for semiquantitative assessment for lung fibrosis (9). A score of 0–1 was grouped as no fibrosis, 2–3 as minimal, 4–5 as moderate and 6–8 as severe fibrosis. Grading was performed by a pathologist without knowing the treatment.

Statistical Analysis
A log-rank test was performed to test for equality of the survival curves among the four groups. The differences in lung fibrosis score between the two groups were tested using Fisher's exact test.


   RESULTS
TOP
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 Conflict of interest statement
 References
 
Pharmacokinetics and Dosimetry
The results are reported as medians with interquartile ranges. 10B concentrations in tumor and normal lung were evaluated 30 min after BPA administration. Following administration of BPA at a dose of 500 mg/kg, 10B concentrations in the tumor and lung were 27.2 (20.9 – 30.4) ppm and 20.7 (14.5 – 22.0) ppm, respectively. The tumor/normal lung (T/N) 10B concentration ratio was 1.6 (1.4 – 1.6).

The average fluence of thermal neutrons and the {gamma}-ray dose rate on the body surface were 1.87 x 109 n/cm2/s and 2.24 x 10–4 Gy/s, respectively. Dose calculations were based on median boron concentrations of tumor (27.2 ppm) and normal lung (20.7 ppm). Physical and photon-equivalent doses for the tumor and the physical dose for the normal lung are summarized in Table 1. Because of uncertainties about the CBE for normal lung, the photon-equivalent dose for normal lung was not assigned.


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  		Table 1. Physical and estimated photon-equivalent doses delivered to the tumor and normal lung

 
Survival
Autopsy was performed on all mice. Bloody pleural effusion and pleural-based masses were observed in all mice (Fig. 1A). The regional spread pattern mimicked the clinical presentation of human MPM. Kaplan–Meier plots for survival data are shown in Fig. 1B. Median survival times from the day of treatment in the non-treated control, neutron irradiation and {gamma}-ray irradiation groups were 7 (range 6–22), 7 (range 6–12) and 12 (range 8–22) days, respectively. The BPA-BNCT group had a longer survival time of 31 days (range 5–60), which was significantly different to that of the non-treated control group (P = 0.011), but not significantly different to that of the neutron and {gamma}-ray groups (P = 0.067 and 0.094, respectively).


Figure 1
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  		Figure 1. (A) Pleural-based masses grown diffusely in the thoracic cavity (white arrows). Bloody pleural effusion was aspirated. (B) Kaplan–Meier survival curves. The survival times, in days after treatment, are shown for non-treated controls (solid circles), neutrons alone (open square), {gamma}-ray (open triangle) and BNCT (open cirlce).

 
Normal Lung Toxicity
As summarized in Table 2, neither treatment group had moderate or severe lung fibrosis.


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  		Table 2. Lung fibrosis score

 
In the BNCT and neutron groups, incidence of minimal lung fibrosis was significantly higher compared with the non-treatment control group (P = 0.003 and 0.04, respectively). Comparing the BNCT group with the neutron and {gamma}-ray groups, there was no significant difference in incidence of minimal lung fibrosis (P = 0.41 and 0.21, respectively).


   DISCUSSION
TOP
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
Conflict of interest statement
 References
 
In this experimental study using mice that had tumors spreading in the thoracic cavity, we have shown that BNCT can significantly prolong survival time without causing severe lung fibrosis. MPM has a tendency to disseminate throughout the whole thoracic cavity and interlobar fissures. In conventional external radiotherapy, it is impossible to deliver a curative dose (>60 Gy) to MPM cells without causing fatal radiation pneumonitis at the same time. Theoretically, BNCT can achieve a large dose gradient between tumor cells and normal tissue if 10B atoms selectively accumulate in tumor cells. Results in this study support the possibility that BNCT may deliver a curative dose to MPM cells while maintaining the dose to normal lung to less than the tolerable dose.

Only one study, from a Massachusetts Institute of Technology group, is available on CBE factors for normal lung (10). According to this group, values of 1.50 and 2.20 were reported as the CBE factor and RBE factor for high linear energy transfer (LET) protons produced by the scattering of fast neutrons [1H(n, n)1H] and from the capture of thermal neutrons by nitrogen atoms [14N(n, p)14C], respectively. If these values are adopted for the dose conversion of physical dose to photon-equivalent dose for normal lung, 12.7 Gy-Eq were delivered to the normal lung in the BNCT-treated group. As shown in Table 1, the photon-equivalent dose delivered to the tumor in the BNCT-treated group was 43.3 Gy-Eq, which was 3.4 times larger than the dose to the normal lung (12.7 Gy-Eq), despite poor selective accumulation of BPA in this tumor model. This dose gradient between tumor and normal lung may have a significant therapeutic effect on survival time. If T/N 10B concentration ratios of 2.0 – 4.0 were achieved with BNCT for MPM, as experienced in clinical trials of BNCT for brain tumors and head and neck tumors, more promising results may be expected with BNCT for MPM.

SCCVII tumor is known to be radiation resistant due to its high expression of epidermal growth factor receptor (EGFR) (11). In an in vitro study, a mesothelioma cell line was shown to be more radiosensitive than non-small cell lung cancer cells, although not as sensitive as small cell carcinoma cells (12). In the present study, however, it was of utmost importance whether the spreading pattern of SCCVII cells in the thoracic cavity mimics the MPM. In the clinical situation, conventional radiotherapy is unable to deliver curative doses to MPM spreading throughout the entire pleural space and interlobar fissures without producing radiation pneumonitis in the adjacent lung. In BNCT, the radiation dose gradient between MPM and adjacent lung is provided by the gradient of the 10B concentration between the tumor and lung. Therefore, to investigate the therapeutic effect of BNCT for MPM, the model mice should have the tumors spreading in the thoracic cavity.

In this tumor model, contrary to our expectation, the median T/N 10B concentration ratio was 1.6. Pharmacokinetic studies were performed on the seventh day after inoculation, because this cell line was so aggressive that the median survival time was 12 days from inoculation in the non-treated control group (Fig. 1B). Small tumors developing in the thoracic cavity, with sizes ranging from 3 to 5 mm, were collected for the pharmacokinetic study. The immature vascularity of these tumors may lead to lower accumulation of BPA in the tumors.

One drawback of BNCT compared with conventional radiotherapy using photons is that thermal neutrons rapidly attenuate in the body because of their collisions with protons in tissue water (13). Poor penetration of thermal neutrons causes inadequate irradiation to the deep-seated tumors. MPM is located in the pleural space relatively near the body surface and is adjacent to the lung which has low proton density. Our previous dose-distribution study using the computed tomography images of MPM patients revealed that three-fractionated BNCT is possible to deliver curative doses to the tumors with keeping the dose delivered to the normal lung lower than the tolerance level (14). BNCT has the possibility of becoming a promising treatment for MPM from a viewpoint of dose-distribution analysis.

In conclusion, BNCT has the possibility to be a promising treatment for malignant tumors spreading in thoracic cavity such as MPM. We are currently preparing an orthotopic MPM tumor model using a human MPM cell line. Further investigation of the therapeutic effect of BNCT using the orthotopic MPM model is warranted.


   Conflict of interest statement
TOP
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Conflict of interest statement
References
 
None declared.


   Acknowledgment
 
We thank Dr Manabu Fukumoto for histopathological scoring of lung tissue microslides.

This study was partially supported by a Grant-in-Aid for Scientific Research (18591379) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.


   References
TOP
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Conflict of interest statement
 References
 
1 Peto J, Decarli A, La Vecchia C, Levi F, Negri E. The European mesothelioma epidemic. Br J Cancer (1999) 79:666–72.[CrossRef][Web of Science][Medline]

2 Robinson BW, Musk AW, Lake R. Malignant mesothelioma. Lancet (2005) 366:397–408.[CrossRef][Web of Science][Medline]

3 Gordon W Jr, Antman KH, Greenberger JS, Weichselbaum RR, Chaffey JT. Radiation therapy in the management of patients with mesothelioma. Int J Radiat Oncol Biol Phys (1982) 8:19–25.[Web of Science][Medline]

4 Haritz D, Gabel D, Huiskamp R. Clinical phase-I study of Na2B12H11SH (BSH) in patients with malignant glioma as a precondition for boron neutron capture therapy (BNCT). Int J Radiat Oncol Biol Phys (1994) 28:1175–81.[Web of Science][Medline]

5 Diaz AZ. Assessment of the results from the phase I/II boron neutron capture therapy trials at the Brookhaven National Laboratory from a clinician's point of view. J Neurooncol (2003) 62:101–9.[CrossRef][Medline]

6 Fukuda H, Hiratsuka J, Kobayashi T, Sakurai Y, Yoshino K, Karashima H, et al. Boron neutron capture therapy (BNCT) for malignant melanoma with special reference to absorbed doses to the normal skin and tumor. Australas Phys Eng Sci Med (2003) 26:97–103.[Medline]

7 Kato I, Ono K, Sakurai Y, Ohmae M, Maruhashi A, Imahori Y, et al. Effectiveness of BNCT for recurrent head and neck malignancies. Appl Radiat Isot (2004) 61:1069–73.[CrossRef][Web of Science][Medline]

8 Coderre JA, Morris GM. The radiation biology of boron neutron capture therapy. Radiat Res (1999) 151:1–18.[Web of Science][Medline]

9 Ashcroft T, Simpson JM, Timbrell V. Simple method of estimating severity of pulmonary fibrosis on a numerical scale. J Clin Pathol (1988) 41:467–70.[Abstract/Free Full Text]

10 Kiger JL, Kiger WS, Patel H, Binns PJ, Riley KJ, Hopewell JW, et al. Effects of boron neutron capture irradiation on the normal lung of rats. Appl Radiat Isot (2004) 61:969–73.[CrossRef][Web of Science][Medline]

11 Akimoto T, Hunter NR, Buchmiller L, Mason K, Ang KK, Milas L. Inverse relationship between epidermal growth factor receptor expression and radiocurability of murine carcinomas. Clin Cancer Res (1999) 5:2884–90.[Abstract/Free Full Text]

12 Sterman DH, Albelda SM. Advances in the diagnosis, evaluation, and management of malignant pleural mesothelioma. Respirology (2005) 10:266–283.[CrossRef][Web of Science][Medline]

13 Sakurai Y, Kobayashi T. Controllability of depth dose distribution for neutron capture therapy at the Heavy Water Neutron Irradiation Facility of Kyoto University Research Reactor. Med Phys (2002) 29:2338–50.[CrossRef][Web of Science][Medline]

14 Suzuki M, Sakurai Y, Masunaga S, Kinashi Y, Nagata K, Maruhashi A, et al. Feasibility of boron neutron capture therapy (BNCT) for malignant pleural mesothelioma from a viewpoint of dose distribution analysis. Int J Radiat Oncol Biol Phys (2006) 66:1584–9.[Web of Science][Medline]


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This Article
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