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We studied DSB induction and rejoining in the clamped and non-clamped transplanted tumors in mice leg after 80 keV/µm carbon ions and X-rays. The yields of DSB in the tumors were analyzed by a static-field gel electrophoresis. The OER of DSB after X-rays was 1.68 +/- 0.31, and this value was not changed by one hour rejoining time (1.40 +/- 0.26). These damages in oxygenated conditions were rejoined 60 to 70% within one hour in situ. On the other hand, no difference between X-rays and carbon ions was found for the induction and rejoining of DSB. Thus, the values of OER and rejoined fraction after carbon ions were similar to that after X-rays, and the calculated RBEs of carbon ion were around 1 under both oxygen conditions. The yields of DSB in vivo depend on exposure doses, oxygen conditions and rejoining time, but not the types of radiation quality.

Purpose: Respiration-gated irradiation for a moving target requires a longer time to deliver single fraction in proton radiotherapy (PRT). Ultrahigh dose rate (UDR) proton beam, which is 10-100 times higher than that is used in current clinical practice, has been investigated to deliver daily dose in single breath hold duration. The purpose of this study is to investigate the survival curve and relative biological effectiveness (RBE) of such an ultrahigh dose rate proton beam and their linear energy transfer (LET) dependence. Methods: HSG cells were irradiated by a spatially and temporally uniform proton beam at two different dose rates: 8 Gy/min (CDR, clinical dose rate) and 325 Gy/min (UDR, ultrahigh dose rate) at the Bragg peak and 1.75 (CDR) and 114 Gy/min (UDR) at the plateau. To study LET dependence, the cells were positioned at the Bragg peak, where the absorbed dose-averaged LET was 3.19 keV/um, and at the plateau, where it was 0.56 keV/um. After the cell exposure and colony assay, the measured data were fitted by the linear quadratic (LQ) model and the survival curves and RBE at 10% survival were compared. Results: No significant difference was observed in the survival curves between the two proton dose rates. The ratio of the RBE for CDR/UDR was 0.98+/-0.04 at the Bragg peak and 0.96+/-0.06 at the plateau. On the other hand, Bragg peak/plateau RBE ratio was 1.15+/-0.05 for UDR and 1.18+/-0.07 for CDR. Conclusions: Present RBE can be consistently used in treatment planning of PRT using ultrahigh dose rate radiation. Because a significant increase in RBE toward the Bragg peak was observed for both UDR and CDR, further evaluation of RBE enhancement toward the Bragg peak and beyond is required.

We aimed to determine the effect of carbon ion irradiation on the uptake of N-benzyl-N-11C-methyl-2-(7-methyl-8-oxo-2-phenyl-7,8-dihydro-9H-purin-9-yl)acetamide ([11C]DAC), a positron emission tomography (PET) ligand for the peripheral-type benzodiazepine receptor (PBR), in tumor cells and tumor-bearing mice. Spontaneous murine fibrosarcoma (NFSa) cells were implanted into the right hind legs of syngeneic C3H male mice. Conditioning irradiation with 290 MeV/u carbon ions was delivered to the 7- to 8-mm tumors In vitro uptake of [11C]DAC was measured in single NFSa cells isolated from NFSa-bearing mice after irradiation. In vivo biodistribution of [11C]DAC in NFSa-bearing mice was determined by small animal PET scanning and dissection. In vitro autoradiography was performed using tumor sections prepared from mice after PET scanning. In vitro and in vivo uptake of [11C]DAC in single NFSa cells and NFSa-bearing mice was significantly reduced by carbon ion irradiation. The decrease in [11C]DAC uptake in the tumor sections was mainly due to the change in PBR expression. In conclusion, [11C]DAC PET responded to the change in PBR expression in tumors caused by carbon ion irradiation in this study. Thus, [11C]DAC is a promising predictor for evaluating the effect of carbon ion radiotherapy.

To compare the biological effectiveness of 290 MeV/amu carbon-ion beams in Chiba, Japan and in Darmstadt, Germany, given that different methods for beam delivery are used for each.

Purpose: To clarify the radiosensitivity of intratumor quiescent cells in vivo to accelerated carbon ion beams and reactor neutron beams. Methods and Materials: Squamous cell carcinoma VII tumor-bearing mice were continuously given 5-bromo-20-deoxyuridine to label all intratumor proliferating cells. Next, they received accelerated carbon ion or g-ray high-dose-rate (HDR) or reduced-dose-rate (RDR) irradiation. Other tumor-bearing mice received reactor thermal or epithermal neutrons with RDR irradiation. Immediately after HDR and RDR irradiation or 12 h after HDR irradiation, the response of quiescent cells was assessed in terms of the micronucleus frequency using immunofluorescence staining for 5-bromo-20-deoxyuridine. The response of the total (proliferating plus quiescent) tumor cells was determined from the 5-bromo-20-deoxyuridine nontreated tumors. Results: The difference in radiosensitivity between the total and quiescent cell populations after g-ray irradiation was markedly reduced with reactor neutron beams or accelerated carbon ion beams, especially with a greater linear energy transfer (LET) value. Clearer repair in quiescent cells than in total cells through delayed assay or a decrease in the dose rate with g-ray irradiation was efficiently inhibited with carbon ion beams, especially with a greater LET. With RDR irradiation, the radiosensitivity to accelerated carbon ion beams with a greater LET was almost similar to that to reactor thermal and epithermal neutron beams. Conclusion: In terms of tumor cell-killing effect as a whole, including quiescent cells, accelerated carbon ion beams, especially with greater LET values, are very useful for suppressing the dependency on the heterogeneity within solid tumors, as well as depositing the radiation dose precisely.

Background: The present retrospective cohort study was performed to determine the efficacy of contact-mode 1064 nm neodymium-yttrium-aluminum-garnet (Nd:YAG) laser laser for keloids and hypertrophic scars. The indication and limitations of this modality are discussed. Methods: The cohort consisted of 102 consecutive Japanese patients (23 males and 79 females) with keloids and hypertrophic scars for more than 1 year. They were treated every 3–4 weeks for 1 year with a long-pulsed 1064 nm Nd:YAG laser (Cutera, Brisbane, Calif.) in contact mode. Thirty-eight patients had hypertrophic scars and 64 had keloids. The scars were evaluated before the treatment commenced and 1 month after the last session by using the Japan Scar Workshop Scar Scale 2011. Recurrence was assessed at 6 months after the termination of treatment. Results: The average total Japan Scar Workshop score of the keloid and hypertrophic scar region groups dropped significantly after 1 year of treatment compared with before treatment (all P < 0.05). None of the hypertrophic scars or keloids deteriorated. However, 3 of the 34 anterior chest keloids (8.8%) did not respond. The following recurrence rates were observed 6 months after stopping laser treatment: 1 of the abdomen hypertrophic scars (4%), 18 of the anterior chest keloids (52.9%), 5 of the upper arm keloids (35.7%), and 4 of the scapula keloids (25%). Conclusions: Hypertrophic scars responded significantly better to 1064 nm Nd:YAG laser treatment than keloids. However, keloid recurrence occurred when there was remaining redness and induration, even if only a small part of the scar was affected.

A radiobiological method was developed to measure the initial growth of clonogenic tumor cells metastasizing to lung. Thorax of mice was externally irradiated by gamma rays after an intravenous transplantation of syngeneic fibrosarcoma cells. Lung colonies developed 11 days after irradiation were counted and provided surviving fractions. Survival curves moved downward when time interval between transplantation and radiation delayed from 1 to 21 hr, but shifted upward at 48 hr or later. Survival ratios at given doses and the extrapolation number of survival curves fitted to multi-target model were calculated, and plotted against time after the intravenous transplantation. Doubling times of 13.3 and 13.1 hr were obtained by use of the survival ratio and of the extrapolation number, respectively. This method is useful to measure growth dynamics of clonogenic tumor cells at the site of metastasized organ.

The biological effectiveness of carbon ions relative to γ rays (RBE) was compared between the tumor growth delay and an early skin reaction of syngeneic mice. The RBE was larger for a tumor than skin when irradiated with large doses of high-LET (linear energy transfer) carbon ions. The intra-track damage (a term of a linear quadratic model) of a tumor and skin increased equally with an increase of the LET, while the inter-track damage (β term) of skin alone increased with the LET. These data provide evidence that high-LET radiotherapy could achieve therapeutic gain by minimizing the difference in response to fractionated irradiation between the tumor and normal tissue.

We performed dynamic MRI to reveal the characteristic gadopentetate dimeglumine (Gd-DTPA) uptake in carbon-ion irradiated tumor and compare it with photon irradiation. Fibrosarcomas in C3H mice legs were irradiated with either 16 Gy of carbon ions (74 keV/mm) or an equivalent dose (30 Gy) of Cs-137 gamma-rays. Dynamic MRI was performed 1 or 6 days after irradiation when the tumors showed an initial growth delay or incipient regrowth, respectively. The enhancement pattern was visualized by mapping the maximum enhanced time (Tmax), relative signal intensity maximum (SImax), and time delay of starting enhancement (Td). Significantly larger Tmax and Td values were observed in the tumors 1 day after carbon-ion irradiation than in the nonradiated tumors (No-R) and tumors 1 day after gamma-ray irradiation. Among the selected pixels in the tumors 6 days after carbon irradiation, 77% had Tmax values of less than 120 sec, significantly more than in the No-R group. The Tmax maps for the tumors irradiated with gamma-rays showed a similar tendency to the carbon-irradiated ones, and only a significant difference was obtained between tumors 1 and 6 days after irradiation. Tmax and Td in the carbon-ion irradiated tumors were different from those in the gamma-ray-irradiated tumors. These treatment-specific kinetics may be useful in predicting the therapeutic efficacy of carbon-ion treatment.

It is well established that the RBE (relative biological effectiveness) for cell killing depends on LET (linear energy transfer), and that a maximum RBE is observed at ~150 keV/mm. However, the therapeutic gain depends on the ratio of the RBEs for the effects on the cancer cell population and the effects on normal tissues. The RBE of a given radiation quality depends on LET but also on dose, biological system and effect, and irradiation conditions. There is no data available to answer the question: which LET is suitable to improve the biological therapeutic gain of carbon ions? Here, we selected 3 different LET values of 290 MeV/u carbon ions, and compared the relative biological effectiveness between tumor-growth retardation and skin damage using a murine transplantable tumor. Larger RBE values for tumors after than the skin type were obtained when carbon-ions of intermediate LET were daily delivered for 2 through 5 fractions. The biological therapeutic gain would be high for the carbon-ion SOBP if the number of fractions were correctly selected in clinical trials.

In clinical use of heavy-ion beams, we cannot overlook skin response appearing in the acute period. We previously experimented effects of fractionated irradiation on the mouse skin with carbon beams of 290 MeV/u. Changing the number of fractions in therapy may alter the normal tissue damages; we have concluded that skin damage should be carefully studied when the number of fractions is changed in new clinical trials. It is still unclear whether any specific LETs cause such change in skin reaction. Here we irradiated mice feet with mono-energetic and SOBP carbon ions, to obtain dose response of early skin reaction at different LETs. With an increase of LET, repair of skin damage may be decreased. Required isoeffect total-dose increased linearly with the fraction numbers on a semi-logarithmic chart at low LET (20 and 40 keV/µm) SOBP beam. Similar behaviors were also found at high LET (60 and 80 keV/µm) beam when the fraction numbers were large. However, no increases of isoeffect dose were observed at few fractionations. The same tendency was observed for monoenergetic carbon beams. Douglas-Fowler s Fe-plot could not fit to the results for few fractionations at high-LET region. The a/b ratios obtained from the Fe-plot, the values were 71, 37, 11 and 2.9 for LETs at 20, 40, 60, and 80 keV/µm in the SOBP, respectively. We conclude that irradiation with few fractionation number at high-LET produced much stronger effect on skin. The LQ-model based Fe-plot could not fit skin reaction at few fractions at high-LET. This result may account for clinical carbon therapy of lung tumors that requires larger doses than doses predicted by extrapolating from fractionation date., Heavy Ion in Therapy and Space Radiation Symposium 2013 （HITSRS2013)

The aim of this study was to clarify the RBE (relative biological effectiveness) and OER (oxygen enhancement ratio) for cell killing and micronucleus formation in clamped and non-clamped transplanted solid tumors in mice leg after 290 MeV/n carbon-ion beams and X-rays. Furthermore, single-cell suspensions were prepared by tumors and were used directly to determine the radiation response of oxic cells. Squamous cell carcinoma (SCC VII) cells were transplanted into the right hind legs of syngeneic C3H male mice. Irradiation with either carbon-ion beams with a 6-cm spread-out Bragg peak (46 and 80 keV/µm) or X-rays was delivered to 5-mm diameter tumors. Single-cell suspensions were prepared from excised and trypsinized tumors, and were used for in vivo-in vitro colony formation assay to obtain cell-survival curves. Moreover, a large portion of single-cell suspensions were incubated with cytochalasin-B to examine the dose-response curves for micronucleus (MN) formation. The RBE and OER for cell survival were evaluated by D10 values (the dose required to reduce surviving fraction down to 10%), and the RBE and OER for MN formation were evaluated by D0 values (the dose corresponding to 37% retained binucleated cell without micronuclei. The RBEs for survival and MN formation increased with increasing LET. These maximum values were 1.7-2.4 at an LET of 80 keV/µm. The OERs for in vivo (clamped tumor / non-clamped tumor) were smaller than that for in vitro (clamped tumor / single-cell suspension) in both endpoints, and the dependence of OERs for in vivo toward LET was small in comparison to the OERs for in vitro., The 10th International Symposium on Chromosomal Aberrations

[Background and Purpose] The fractionated irradiation is standard protocol for radiotherapy including carbon-ion beam (C-ions) therapy. There are big differences between single and fractionation, for example on DNA damage repair, reoxygenation of hypoxic region, etc. It is necessary to get the results using fractionated irradiation to know the effects in clinical. The purpose of this study is to examine the effects of fractionated irradiation for metastasis using C-ions and X-rays. [Materials and Methods] A mouse osteosarcoma cells, LM8 having highly metastatic potential were used. C-ions irradiation (290 MeV/u) was performed at the center position of 6cm-SOBP, and X-rays were used as reference beam. The interval among each fraction was 24 hours, and 1 to 5 fractions were used. [in vitro] The cytotoxic effects were examined using colony formation assay, and the anti-metastatic effects were examined by the inhibition of migration and invasion activities using Boyden chamber assay and Matrigel invasion assay, respectively. [in vivo] LM8 tumors inoculated into right hind leg of mice were irradiated at 12days after inoculation. Radiosensitivity for individual cell in a tumor was examined by in vivo-in vitro assay. The metastatic effects were analyzed with the spontaneous lung metastasis model. [Results] [in vitro] After X-rays fractionation, cell survival and metastatic potentials (migration and invasion activities) were increased depend on the fraction number, and the degree was significant at low dose region. On the other hand, the enhancement was not significant after C-ions fractionation. [in vivo] The survival of cells in local tumor and the number of lung metastatic nodules were increased with the increment of the fraction number after fractionated irradiation. The degree of increase was more remarkable by X-rays than C-ions. The RBE values of C-ions were increased with fraction number on both cell killing and suppression of metastasis, and the values were larger on metastasis suppression than on cell killing. [Conclusion] It is confirmed that the anti-metastatic effects showed the dependency of the fraction number as well as cell killing effects. The degree of the dependency was more significant for X-rays compared with C-ions with high LETs than X-rays with low LETs. It was suggested that C-ions therapy was superior to photon therapy not only on tumor cell killing but also on metastasis suppression even for clinical fractionated irradiation., 39th Annual Meeting of the European Radiation Research Society

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