白井 克幸

The direct biological effects of radiation, particularly accelerated heavy particle ions, on neurons are not fully known. Hence, the direct effect of carbon-ion beams on immature neurons was investigated by comparing to the effect of X-rays in vitro using primary hippocampal neurons. Primary neurons were prepared from hippocampi of fetal rats at embryonic day 18 from timed pregnant Wistar rats and cultured with Banker's methods. At 7 Days In Vitro (DIV), the cells were irradiated with 140 kV X-ray and 18.3 MeV/amu carbon-ion beams (LET = 108 keV/mu m). The cells were fixed with 4% paraformaldehyde at 12 hours after irradiation. Then, the cells were treated with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and DAPI staining for measuring the percentage of apoptosis (apoptotic index: Al). Al in sham-irradiated hippocampal neurons was 18%. The value of Al (Als) of the cells irradiated with X-rays at 10 or 30 Gy were 15% or 23%, respectively. Al in cells irradiated with carbon-ion beams at 1 Gy, 3 Gy, 5 Gy and 10 Gy were 22%, 23%, 24% and 33%, respectively. Al was significantly increased by carbon-ion beams at 10 Gy (p < 0.001). The apoptosis of hippocampal neurons increased in a dose-dependent manner following both X-ray and carbon-ion beams irradiation. Carbon-ion beams were about 10-fold more effective than X-rays for apoptosis induction in immature hippocampal neurons.

World Health Organization (WHO) grade 3 glioma is one of the common brain tumors and has three main histological subtypes, including anaplastic astrocytoma (AA), anaplastic oligoastrocytoma (AOA) and anaplastic oligodendroglioma (AO). However, most previous studies have considered AOA and AO as one group because of the difficult differential diagnosis between AOA and AO. Therefore the prognostic difference among patients with these histological subtypes has been unclear. In this study, 68 patients with histologically proven WHO grade 3 glioma, consecutively received postoperative radiotherapy at the Gunma University Hospital, Japan, between 1983 and 2005, were investigated to assess the impact of histological subtype on the survival. The number of AA, AOA and AO patients was 41, 16 and 11, respectively. The mean and median follow-up periods were 72 and 48 months, respectively. The number of patients treated with gross total resection, partial resection and biopsy was 14, 38 and 16, respectively. The mean and median radiation doses were 58 +/- 5 Gy and 60 Gy, respectively. The 5-year overall survival rates of AA, AOA and AO were 21%, 38% and 80%, and median survival period were 16 months, 58 months and not reached, respectively. Univariate analysis showed that the histological subtype (P < 0.01) and extent of surgery (P < 0.01) were significant prognostic factors for survival. Selective comparison showed that overall survival of patients with AA was significantly worse than for those with AOA (P = 0.01) and AO (P < 0.01). The overall survival of patients with AO was better than for those with AOA; however, the difference was not statistically significant (P = 0.14). Multivariate analysis demonstrated that histological subtype, age and extent of surgery were the significant independent variable for survival (P < 0.01, P <0.01 and P = 0.04). In our study, histological subtype was one of the most important prognostic factors of WHO grade 3 glioma.

phosphorylated-Akt (pAkt) plays an important role in tumorigenesis through promotion of cell survival by inhibiting apoptosis and mediating cell proliferation Higher expression of pAkt has been reported to be associated with an unfavorable prognosis in several malignant tumors In this study, the prognostic value of pAkt expression was investigated in glioblastomas by using immunohistochemical methods Tissue sections obtained from 64 patients with glioblastoma were evaluated The mean and median follow-up period was 16 2 +/- 12 4 and 12 months. respectively (range. from 1 to 62 months) pAkt expression levels were determined by immunohistochemical staining and evaluated for cell positivity Positive staining was defined when more than 50% of the tumor cells were stained in each section The correlation between expression of pAkt and overall survival rate was assessed. Glioblastomas showed either or both cytoplasmic and nuclear positive findings for pAkt A total of 29 7% (19/64) of tissue specimens had greater than 50% positivity The median survival periods of the patients with pAkt positive and negative tumor were 10 and 14 months. respectively Two years overall survival rate of the pAkt positive and negative patients were 0% and 24 4%. respectively Survival rate of the patients with pAkt positive tumor was significantly lower than that of the patients with pAkt negative tumors (p = 0 004) Multivariate analysis showed that extent of surgery was the strongest factor for survival (p = 0 01) and the pAkt expression was the secondly strongest factor (p = 0.06) These results suggest that the higher expression of pAkt the poorer prognosis in patients with glioblastoma

Purpose: To assess the efficacy of radiation therapy (RT) by using intraluminal brachytherapy (IBT) combined with external beam RT (EBRT) for submucosal esophageal cancer. Methods and Materials: Between 1991 and 2005,59 consecutive patients received definitive RT without chemotherapy. IBT was performed after patients completed EBRT as a booster therapy for 17 patients, using low-dose-rate Cs-137 sources until 1997, and for 19 patients, using high-dose-rate Ir-192 sources thereafter. The long-term outcomes were investigated with a median follow-up time of 61 months. Results: Logoregional recurrences and distant metastases were observed in 14 patients and in 2 patients in the lung, respectively, and 5 patients were rescued by salvage treatments. The 5-year logoregional control and cause-specific survival rates were 75% and 76%, respectively. The 5-year cause-specific survival rate in the EBRT group was 62%, whereas the corresponding rate in the IBT group was 86% (p=0.04). Multivariate analysis revealed that IBT was the most powerful predictor of survival but did not reach a significant level (p=0.07). There were five esophageal ulcers in the IBT group, but no ulcers developed with small fractions of 3 Gy. Grade 2 or higher cardiorespiratory complications developed in 2 patients (5.6%) in the IBT group and in 3 patients (13.0%) in the EBRT group. Conclusions: Combining IBT with EBRT is suggested to be one of the preferable treatment modalities for medically inoperable submucosal esophageal cancer because of its preferable local control and survival probabilities, with appreciably less morbidity. (C) 2010 Elsevier Inc.

Okamoto, M., Suzuki, Y., Shirai, K., Mizui, T., Yoshida, Y., Noda, S., Al-Jahdari, W. S., Shirao, T. and Nakano, T. Effect of Radiation on the Development of Immature Hippocampal Neurons In Vitro. Radiat. Res. 172, 718-724 (2009). Little is known about the direct biological effects of radiation on immature neurons, despite its relevance to the mental retardation caused by irradiation of the brains of fetuses and children. In this study, we investigated the effects of radiation using primary cultured hippocampal neuronal cells with exclusion of glial cells, focusing on cell survival and structural development. Primary neurons were prepared from the hippocampi of fetal rats at embryonic day 18 and cultured according to Banker's methods. After incubation for 7 days, cells were irradiated with X rays and incubated continuously for 7 or 14 days. The number of neurons, their rate of apoptosis, and the patterns of expression of synaptic proteins on the neural dendrites were investigated by immunohistochemical methods. The total numbers of neurons were the same regardless of whether they were irradiated. The number of TUNEL-positive neurons, which can be considered as undergoing apoptosis, increased significantly in a dose-dependent fashion at both 7 and 14 days after irradiation. The mean numbers of clusters of synaptic proteins on neural dendrites, which are considered to represent their developmental level, decreased dose-dependently at both 7 and 14 days after irradiation. These results suggest that radiation not only induces apoptosis but also produces structural defects in the surviving neurons that may directly suppress neural development. (C) 2009 by Radiation Research Society

Survivin is a member of the inhibitor of apoptosis family, and is expressed in various malignant tumors. Survivin overexpression has been reported to be a poorer prognostic factor in various malignancies. However, the prognostic value of survivin expression in patients with glioblastoma is still controversial. Therefore, in this study the role of survivin as a predictor for survival was investigated in patients with glioblastoma. Tissue specimens were obtained from 66 patients with glioblastoma treated with radiotherapy. Survivin expression was detected by an immunohistochemical method. Nuclear and cytoplasm survivin scores were defined by using the cell positivity and staining intensity. The scores were defined as follows, 0 (no staining), 1 (less than 50% of cell positivity and any staining), 2 (more than 50% of cell positivity and weak to moderate intensity) and 3 (more than 50% of cell positivity and strong intensity). The correlation between survivin scores and the overall survival rate was evaluated. Nuclear and cytoplasm survivin staining were noted in 47 and 58 patients, respectively. The number of patients with nuclear survivin score of 0, 1, 2 and 3, were 19 (28.8%), 26 (39.4%), 9 (13.6%) and 12 (18.2%), respectively. The 3-year overall survival rate of the nuclear survivin score 3 was 0%, significantly lower than the 11.6% of the nuclear survivin score a parts per thousand currency sign2 (P = 0.0003). Cytoplasm survivin score did not correlate with the prognosis. Nuclear survivin expression may be a useful biomarker for predicting prognosis in patients with glioblastoma.

Purpose: Recently carbon-ion beams have been reported to be remarkably effective for controlling various cancers with less toxicity and are thought to be a promising modality for cancer treatment. However, the biological effect of carbon-ion beams arising on normal neuron remains unknown. Therefore, this study was undertaken to investigate the effect of carbon-ion beams on neurons by using both morphological and functional assays. Materials and methods: Dorsal root ganglia (DRG) and sympathetic ganglion chains (SYMP) were isolated from day-8 and day-16 chick embryos and cultured for 20h. Cultured neurons were exposed to carbon-ion beams and X-rays. Morphological changes, apoptosis and cell viability were evaluated with the Growth Cone Collapse (GCC), Terminal deoxynucleotidyl Transferase (TdT)-mediated deoxyUridine TriPhosphate (dUTP) nick End Labeling [TUNEL] assay and 4-[3-(4-iodophenyl)- 2-(4-nitrophenyl)- 2H-5-tetrazolio]- 1,3-benzenedisulfonate [WST-1] assays, respectively. Results: Irradiation caused GCC and neurite destruction on a time- and irradiation dose-dependent manner. Changes in morphological characteristics were similar following either irradiation. Morphological and functional assays showed that day-8 neurons were more radiosensitive than day-16 neurons, whereas, radiosensitivity of DRG was comparable to that of SYMP. The dose-response fitting curve utilising both GCC and TUNEL labeling index showed higher relative biological effectiveness (RBE) values were associated with lower lethal dose (LD) values, while lower RBE was associated with higher LD values. Conclusion: Exposure to high-linear energy transfer (LET) irradiation is up to 3.2 more efficient to induce GCC and apoptosis, in early developed neuronal cells, than low-LET irradiation. GCC is a reliable method to assess the radiobiological response of neurons.

The growth cone is a structure at the terminal of a neurite that plays an important role in the growth of the neurite. The growth cone collapse assay is considered to be a useful method to quantify the effects of various factors on nerve tissue. Here, we investigated the effect of x-irradiation on growth cones and neurites and also the comparative radiosensitivity of different neurons. Dorsal root ganglia and sympathetic chain ganglion were isolated from day-8 and -16 chick embryos and cultured for 20 h. Neurons were then exposed to x-irradiation and morphological changes were quantitatively evaluated by growth cone collapse assay. Cell viability was examined using TUNEL and WST-1 assays. The results showed that radiation induced growth cone collapse and neurite retraction in a time- and exposure-responsive manner. Growth cone collapse, apoptosis and WST-1 assays showed that no significant difference between the neurons throughout the study period (p >= 0.5) after irradiation. Both types of day-8 neurons were more radiosensitive than day-16 neurons (p <= 0.05). The time course of the growth cone collapse was significantly correlated with the apoptotic and cell viability responses at different irradiation doses. Growth cone collapse may represent a useful marker for assaying the effect of x-irradiation on normal cell neurons.

To carry out the radio-microsurgery study using silkworm, Bombyx mori, we have already developed the specific irradiation systems for eggs and third to fifth instar larvae. In this study, a modified application consisting of the first instar silkworm larvae was further developed using heavy-ion microbeams. This system includes aluminum plates with holes specially designed to fix the first instar silkworm larvae during irradiation, and Mylar films were used to adjust energy deposited for planning radiation doses at certain depth. Using this system, the suppression of abnormal proliferation of epidermal cells in the knob mutant was examined. Following target irradiation of the knob-forming region at the first instar stage with 180-μm-diameter microbeam of 220 MeV carbon (^<12>C) ions, larvae were reared to evaluate the effects of irradiation. The results indicated that the knob formation at the irradiated segment was specially suppressed in 5.9, 56.4, 66.7 and 73.6% of larvae irradiated with 120, 250, 400 and 600 Gy, respectively, but the other knob formations at the non-irradiated segments were not suppressed in either irradiation. Although some larva did not survive undesired non-targeted exposure, our present results indicate that this method would be useful to investigate the irradiation effect on a long developmental period of time. Moreover, our system could also be applied to other species by targeting tissues, or organs during development and metamorphosis in insect and animals.

Aim: The aim of this study was to examine the clinical usefulness of cytokeratin 19 fragments (CYFRA 21-1) compared with squamous cell carcinoma (SCC) antigen in patients with esophageal cancer treated with radiation therapy. Methods: Fifty-one patients with stage I-IV esophageal cancer were evaluated. CYFRA 21-1 and SCC antigen serum levels were measured at the start and the end of radiation therapy. Results: CYFRA 21-1 (&gt; 3.5 ng/mL) and SCC antigen (&gt; 1.5 ng/mL) before radiation therapy were elevated in 63% and 53% of the patients, respectively. The CYFRA 21-1 levels were significantly correlated with TNM stages, tumor depth and lymph node metastasis (P = 0.0003, P = 0.019 and P = 0.019, respectively), whereas no correlation was observed between SCC antigen and these factors. The values of CYFRA 21-1 in all patients who survived without recurrence were under the cutoff level at the end of treatment, but the values in all patients with locoregional recurrence were above the level. However, there was no significant correlation between SCC antigen level at the end of treatment and any clinical outcome. Conclusions: The results suggest that the evaluation of CYFRA 21-1 would be useful not only for assessment before radiation therapy but also for monitoring after radiation therapy in the treatment for esophageal cancer.

Objective. Survivin is a member of the inhibitors of apoptosis and has been implicated in both the regulation of cell division and the suppression of apoptosis. Over-expression of cytoplasmic survivin correlates with an unfavorable prognosis in many malignant tumors. However, the prognostic value of nuclear survivin expression is still equivocal. Here, we investigated the prognostic value of survivin expression in cervical cancer treated with radiation therapy. Methods. Tissue sections were obtained from 72 patients with cervical squamous cell carcinoma treated with radiation therapy alone. Survivin expression levels were determined by imintmohistochemical staining and evaluated for cell positivity. The correlation between survivin expression and clinical outcome endpoints including cause-specific survival and local control were evaluated. Results. A total of 14% (10/72) of tissue specimens had greater than 5% nucleus positivity, while 47% (34/72) had greater than 50% cytoplasmic positivity. Local control rate of the cytoplasmic survivin-negative tumors was 94%, significantly higher than the 76% of the positive tumors (p=0.046). Local control rate of the nuclear survivin-positive and cytoplasmic survivin-negative patients was 95%, significantly higher than the 74% of the other patients (p=0.02). In contrast, no significant correlation was noted between survivin expression and disease-free survival. Conclusions. The cytoplasmic survivin expression alone and the combination of nuclear and cytoplasmic expression were suggested to be predictors for local control in patients with cervical squamous cell carcinoma treated with radiation therapy alone. (c) 2006 Elsevier Inc. All rights reserved.

Purpose: To evaluate late rectal bleeding and genitourinary (GU) morbidity in patients consecutively treated with combined high-dose-rate (HDR) brachytherapy and external beam radiation therapy (EBRT). Materials and Methods: Data from 80 patients treated consecutively from October 2000 to May 2004 were analyzed. The median age was 69 years old, median follow-up 31 months, ranging from 17-59 months. All patients received endocrine therapy before radiation therapy. The patients were divided into low-, intermediate- and high-risk groups (4/24/52 patients) according to the risk factors defined by T-classification, PSA and Gleason score. Fractionation schedules for HDR brachytherapy were prospectively changed, and EBRT was fixed with 3 Gy fractions to 51 Gy. The distribution of fractionation was scheduled as follows; 5 Gy&times;5 times in 14 patients, 7 Gy&times;3 times in 19 patients, and 9 Gy&times;2 times in 47 patients. The rectal bleeding was graded using the toxicity criteria of the Radiation Therapy Oncology Group and European Organization for Research and Treatment of Cancer while the genitourinary morbidities were graded using the toxicity criteria of the Common Terminology Criteria for Adverse Events v.3.0.<br>Results: Grade 2 or worse rectal bleeding developed in 9 patients (11.3%) with the 2-year actuarial probability at 11.2%. Grade 2 and 3 rectal bleeding was recognized in 8 and 1 patients, respectively. Grade 3 morbidity developed in the biopsied sites that were performed in the other hospital. No significant difference was observed in any HDR brachytherapy fractionation schedule. Grade 2 or worse GU morbidities were recognized in 30 patients (37.5%), consisting of 29 Grade 2 patients and 1 Grade 3 patient. Twenty-one patients in Grade 2 morbidity had an increase in the frequency of urination or nocturia, and urethral strictures developed in 3 patients. The 3-year actuarial probability of urethral stricture was 6.0%. One patient experienced Grade 3 incontinent. No Grade 4 GU complications was observed.<br>Conclusion: HDR brachytherapy combined with hypofractionated EBRT for localized prostate cancer is feasible considering severity of late rectal and genitourinary morbidity.

Japanese randomized trials showed that there was a significant impact on survival from stage I adenocarcinoma (AD) of the lung by adjuvant chemotherapy with uracil-tegaful after complete resection but there was no effect for patients with squamous cell carcinoma (SQ). The purpose of this study was to examine the correlation of tumor histology and clinical outcome of radiation therapy (RT) for stage I non-small-cell lung cancer (NSCLC) and to consider the necessity of adjuvant chemotherapy after RT for these patients. The subjects were 83 patients, 54 with SQ and 29 with AD; they had received definitive RT with the total dose ranging from 60 to 80 Gy with conventional fractionation at a daily dose of 2 Gy. The differences between SQ and AD with respect to survival and recurrence pattern were investigated. The 5-year overall survival and cause-specific survival rates were 26.5% and 49.1%, respectively. No difference in survival was observed between SQ and AD patients, and the recurrence rates were almost identical (44% for SQ and 45% for AD). However, the 5-year primary control rate of SQ was significantly poorer than that of AD (SQ: 61.5%; AD: 87.6%; p = 0.03). Conversely, the 5-year metastasis-free survival rate of SQ was significantly better than that of AD (SQ: 88.2%; AD: 53.0%; p = 0.005). The different failure pattern, according to tumor histology, indicates that taking into consideration the difference in their clinical behaviors would also be important for planning RT and surgery for early lung cancer.

Purpose: To evaluate the incidence of Grade 2 or worse rectal bleeding after high-dose-rate (HDR) brachytherapy combined with hypofractionated external-beam radiotherapy (EBRT), with special emphasis on the relationship between the incidence of rectal bleeding and the rectal dose from HDR brachytherapy. Methods and Materials: The records of 100 patients who were treated by HDR brachytherapy combined with EBRT for &gt;= 12 months were analyzed. The fractionation schema for HDR brachytherapy was prospectively changed, and the total radiation dose for EBRT was fixed at 51 Gy. The distribution of the fractionation schema used in the patients was as follows: 5 Gy X 5 in 13 patients; 7 Gy X 3 in 19 patients; and 9 Gy X 2 in 68 patients. Results: Ten patients (10%) developed Grade 2 or worse rectal bleeding. Regarding the correlation with dosimetric factors, no significant differences were found in the average percentage of the entire rectal volume receiving 30%, 50%, 80%, and 90% of the prescribed radiation dose from EBRT between those with bleeding and those without. The average percentage of the entire rectal volume receiving 10%, 30%, 50%, 80%, and 90% of the prescribed radiation dose from HDR brachytherapy in those who developed rectal bleeding was 77.9%, 28.6%, 9.0%, 1.5%, and 0.3%, respectively, and was 69.2%, 22.2%, 6.6%, 0.9%, and 0.4%, respectively, in those without bleeding. The differences in the percentages of the entire rectal volume receiving 10%, 30%, and 50% between those with and without bleeding were statistically significant. Conclusions: The rectal dose from HDR brachytherapy for patients with prostate cancer may have a significant impact on the incidence of Grade 2 or worse rectal bleeding. (c) 2006 Elsevier Inc.

X-irradiation to neuronal progenitor cells causes brain dysfunctions, such as a mental retardation, in adulthood. However, little has been known about the degree of radiosensitivity of neurons in the developmental stages at which they are most vulnerable. In this study we compared the effect of irradiation on mature neurons with that on immature neurons. Primary dissociated neuronal cultures were prepared from fetal rat hippocampi of embryonic day 18. X-irradiations were performed on the cultured cells at 7 or 21 days in vitro (DIV), and the cells were fixed at 12 or 24 h after irradiation. Then the cells were stained with 4',6-diamidino-2-phenylindole (DAPI) or terminal deoxynucleotidyl transferase- mediated dUTP nick end labeling (TUNEL). The apoptotic changes were measured quantitatively by nuclear pyknosis and DNA fragmentation-both characteristic morphological changes of apoptosis. Light microscopy with differential interference contrast showed that 30 Gy of irradiation increased cellular shrinkage in 7-DIV neurons but not in 21 -DIV neurons. Quantitative analysis using DAPI imaging showed that 30 Gy of irradiation significantly enhanced pyknotic changes in 7-DIV neurons after 24 h. In contrast, this irradiation did not enhance any pyknotic changes in 21 -DIV neurons after 24 h. Further TUNEL staining also showed that the irradiation did not enhance any DNA fragmentation in nuclei of 21-DIV neurons after 24 h. Hence, we showed that the radiosensitivity of 21 -DIV postmitotic neurons was significantly lower than that of 7-DIV neurons, indicating that the susceptibility of such neurons depends on their developmental stage. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

Purpose: To evaluate the advantages of anatomy-based inverse optimization (IO) in planning high-close-rate (HDR) brachytherapy. Methods and Materials: A total of 114 patients who received HDR brachytherapy (9 Gy in two fractions) combined with hypofractionated external beam radiotherapy (EBRT) were analyzed. The dose distributions of HDR brachytherapy were optimized using geometric optimization (GO) in 70 patients and by anatomy-based 10 in the remaining 44 patients. The correlation between the dose-volume histogram parameters, including the urethral dose and the incidence of acute genitourinary (GU) toxicity, was evaluated. Results: The averaged values of the percentage of volume receiving 80-150% of the prescribed minimal peripheral dose (V-80-V-150) of the urethra generated by anatomy-based 10 were significantly lower than the corresponding values generated by GO. Similarly, the averaged values of the minimal dose received by 5-50% of the target volume (D-5-D-50) obtained using anatomy-based IO were significantly lower than those obtained using GO. Regarding acute toxicity, Grade 2 or worse acute GU toxicity developed in 23% of all patients, but was significantly lower in patients for whom anatomy-based IO (16%) was used than in those for whom GO was used (37%), consistent with the reduced urethral dose (p &lt; 0.01). Conclusion: The results of this study suggest that anatomy-based 10 is superior to GO for dose optimization in HDR brachytherapy for prostate cancer. (c) 2006 Elsevier Inc.