The Linear-Quadratic (LQ) model is the most commonly used model for evaluating fractionation schedules in the clinical practice of radiation oncology; however, it has sometimes been reported that the biologically effective dose (BED) based on the LQ model is not adequate for hypofractionated radiation therapy at a high dose per fraction (Hypo-RT), like stereotactic irradiation (STI). On the other hand, there have been some reports that the LQ model is applicable to Hypo-RT. The aim of this study was to investigate the relationship between the effect of Hypo-RT and BED based on the LQ model in vivo. Glioblastoma with mutant-type p53 of human origin was subcutaneously transplanted into nude mice. Doses equivalent to 12 Gy/1 fraction (fx) were calculated according to the LQ model using an alpha/beta ratio of 10 Gy for the tumor (15.06 Gy/2 fx, 18.16 Gy/4 fx, 20.96 Gy/8 fx), and used to irradiate each tumor with X-rays (150 kV, 20 mA, 0.5 mmAl, and 0.1 mmCu filters). Twice daily irradiation schedules were utilized to reduce the effect of treatment time differences. Intervals of each irradiation were more than 10 hours. The irradiation started on the day the calculated tumor volume exceeded 200 mm3. Tumor sizes were measured every three days with calipers to evaluate the tumor growth delay (TGD). TGD for a 2- and 5-fold relative tumor size was defined as TGD2 and TGD5, respectively. Mean TGD2 of the unirradiated control, 12 Gy/1 fx, 15.06 Gy/2 fx, 18.16 Gy/4 fx, and 20.96 Gy/8 fx were 5.69, 22.64, 37.26, 36.81, and 28.96 days, respectively. Mean TGD5 of the control, 12 Gy/1 fx, 15.06 Gy/2 fx, 18.16 Gy/4 fx and 20.96 Gy/8 fx were 17.83, 48.03, 60.03, 60.04 and 64.96 days, respectively. Mean TGD2 and TGD5 of each irradiated group were longer than the control, and 15.06 Gy/2 fx, 18.16 Gy/4 fx and, 20.96 Gy/8 fx groups showed statistically significant differences, but no significant differences were shown among the irradiated groups (p > 0.05). This study suggests that the BED based on the LQ model may be applicable to Hypo-RT corresponding to 12 Gy/1 fx in vivo.

To investigate the changes of cancer stem cell marker expression following carbon ion (CI) beam or X-ray irradiation for human ependymoblastoma. Human ependymoblastomas with wild-type p53 were transplanted into nude mice and irradiated with CI beams (290 MeV/u, 6 cm spread-out Bragg peak) or 150-kV X-rays. Tumor diameters were measured by calipers for 6 weeks to evaluate tumor growth after 2, 4, 8, 16, or 24 Gy irradiation. Other tumors were excised for immunohistochemistry and cDNA microarray analysis 6, 24, or 48 hours after 2, 4, 8, 16, or 24 Gy irradiation. A part of each tumor was fixed in formalin and embedded in paraffin for histological examination. Apoptosis was estimated by the TUNEL assay, and CD133, CD44, Nestin, p53, PTEN, Ki67, and GFAP expressions were evaluated immunohistochemically. Total RNA was extracted from another part of each tumor, and cDNA microarray analysis, hierarchical clustering, gene ontology analysis, and pathway analysis were performed. Tumor growth delay was observed in every tumor group after irradiation compared with the unirradiated control group. Tumor regrowth was not observed in most of the tumors of 16- and 24-Gy groups following CI beam or X-ray irradiation. Apoptosis increased significantly 6 hours after CI beam or X-ray irradiation, but only a few apoptotic cells were found 48 hours and 6 weeks after irradiation. Significant changes in gene expression profiles were found following irradiation, and pathway analysis demonstrated that apoptosis, cell cycle, and p53 signaling pathways are involved; however, there were few differences between the profiles induced by CI beams and those by X-rays. Most of the cells were intensely Nestin-positive and weakly CD44-positive, and some of the tumor cells were weakly CD133-positive. Following 8 Gy or higher irradiation, Ki-67 labeling index decreased markedly (from 70.0% to 1.7%) and CD133-positive rate increased slightly, but no significant change was noted in CD44 or Nestin expressions. p53 signaling and apoptosis-related pathways were suggested to be involved following irradiation of ependymoblastomas with wild-type p53, and tumors showed a significant decrease in growth fraction and a slight increase of the CD133-positive rate after high-dose irradiation. These results suggest that CD133-positive and Ki-67-negative cells may be resistant to both X-rays and CI beams, and survive irradiation.

The aim of this study was to investigate the efficacy of carbon ion beam (CI) for radioresistant human brain tumor (BT) cells expressing cancer stem cell (CSC) markers. Two human BTs, an ependymoblastoma (EB) with wild-type (wt) p53 and a glioblastoma (GB) with mutant-type (mt) p53, were transplanted into nude mice and were irradiated with CI beams (290 MeV/u, 6 cm spread-out Bragg peak) or 150 kV X-rays. Tumors were excised 6, 24, or 48 hours (h) after 2, 4, 8, or 16 Gy irradiation. Total RNA was extracted from a part of each tumor and cDNA microarray analysis, hierarchical clustering, gene ontology analysis, and pathway analysis were performed. The other part was fixed in formalin and embedded in paraffin. TUNEL assay was performed to evaluate apoptosis, and CD133, CD44, and Nestin expressions were evaluated immunohistochemically. Apoptosis increased significantly 6 h after CI beam or X-ray irradiation in EB, but only a few apoptotic cells were found 48 h after. This tumor showed significant changes in gene expression profiles following 2 Gy irradiation or more. There were few differences between the profiles induced by CI beams and those by X-rays. Pathway analysis demonstrated that apoptosis, cell cycle, and p53 signaling pathways are involved. Most of the EB cells were intensely Nestin-positive and weakly CD44-positive, and some of the tumor cells were weakly CD133-positive. Following irradiation, CD133-positive cells increased slightly, but no significant change was found in CD44 and Nestin expressions. In contrast, only a few apoptotic cells were found and few changes were shown in gene expression profiles following 2 Gy irradiation in GB; however, the tumor showed significant profile changes following 8 Gy or 16 Gy CI irradiation. Up- or down-regulation of p53, caspases, and Fas were not found, but p53R2, TRAIL-R, NF-kB, IkBa, and IAP were suggested to be involved. Profile changes induced by X-rays and carbon ion beams were different in part. Most of the GB cells were intensely CD44-positive and many cells were CD133-positive. The tumor cells were Nestin-positive, but weaker than EB. Following irradiation, CD133-positive cells increased slightly, but no significant change was found in CD44 and Nestin expressions. p53 signaling and apoptosis-related pathways were suggested to play important roles in radiation therapy for EB with wt p53. In contrast, anti-apoptotic or surviving pathways were suggested to be activated following irradiation in GB with mt p53. Both tumors showed a slight increase of CD133-positive cells following irradiation, although the expression patterns of CSC markers were different. These results suggested that CD133-positive cells are resistant to both X-rays and CI beams in these BTs.

Purpose/Objective(s)18F-fluorodeoxyglucose (FDG)-positron emission tomography/computed tomography (PET/CT) has been shown to be useful in the staging, treatment planning, and response evaluation of Hodgkin lymphoma and aggressive non-Hodgkin lymphoma, but its efficacy for indolent mucosa-associated lymphoid tissue (MALT) lymphoma has been suggested to be limited. The aim of this study was to evaluate the efficacy of FDG-PET in radiation therapy and post-therapeutic follow-up of indolent MALT lymphoma.Materials/MethodsFrom April 2005 to September 2010, 33 patients with MALT lymphoma (12 orbit, 10 stomach, 4 thyroid, and 7 others) received radiation therapy in our institute. Median age was 67 years old (34-80). There were 17 men and 16 women. FDG uptake in those cases was estimated visually and by using the standardized uptake value (SUV) before and after radiation therapy, respectively. Ki-67 labeling index (LI) was evaluated histologically if tissue sections were available to study the correlation between Ki-67 LI and SUV.ResultsMedian follow-up was 45 months (10-87). Twenty-three cases (72%) were FDG-positive. Positive cases in each organ were 8 (67%) orbit, 5 (56%) stomach, 4 (100%) thyroid, and 2 (100%) larynx. The other 5 cases in different organs showed positive FDG uptake (hypopharynx, lung, prostate, and thymus). The median of maximum SUV (SUVmax) was 6.4 (3.4-11.5). Small tumors showed a tendency to be false-negative. In the comparison of SUV and Ki-67 LI, a positive correlation between them was suggested. Tumors with SUV >5 often showed high Ki-67LI, indicating relatively aggressive growth. For those tumors, dose escalation was considered in radiation therapy planning. The median irradiated total dose was 36 Gy (32-40). In FDG-positive cases, complete response rate was 100% after the initial treatment. Positive accumulation of FDG disappeared visually in all cases and the mean SUV was decreased by 58% (32-78). Distant relapse occurred in 2 patients. In both cases, relapsed lesions were found by follow-up FDG-PET/CT regardless of any symptoms.ConclusionsThis study indicates that FDG-PET/CT may be useful for radiation therapy planning, response evaluation, and post-therapeutic follow-up for indolent MALT lymphoma. Purpose/Objective(s)18F-fluorodeoxyglucose (FDG)-positron emission tomography/computed tomography (PET/CT) has been shown to be useful in the staging, treatment planning, and response evaluation of Hodgkin lymphoma and aggressive non-Hodgkin lymphoma, but its efficacy for indolent mucosa-associated lymphoid tissue (MALT) lymphoma has been suggested to be limited. The aim of this study was to evaluate the efficacy of FDG-PET in radiation therapy and post-therapeutic follow-up of indolent MALT lymphoma. 18F-fluorodeoxyglucose (FDG)-positron emission tomography/computed tomography (PET/CT) has been shown to be useful in the staging, treatment planning, and response evaluation of Hodgkin lymphoma and aggressive non-Hodgkin lymphoma, but its efficacy for indolent mucosa-associated lymphoid tissue (MALT) lymphoma has been suggested to be limited. The aim of this study was to evaluate the efficacy of FDG-PET in radiation therapy and post-therapeutic follow-up of indolent MALT lymphoma. Materials/MethodsFrom April 2005 to September 2010, 33 patients with MALT lymphoma (12 orbit, 10 stomach, 4 thyroid, and 7 others) received radiation therapy in our institute. Median age was 67 years old (34-80). There were 17 men and 16 women. FDG uptake in those cases was estimated visually and by using the standardized uptake value (SUV) before and after radiation therapy, respectively. Ki-67 labeling index (LI) was evaluated histologically if tissue sections were available to study the correlation between Ki-67 LI and SUV. From April 2005 to September 2010, 33 patients with MALT lymphoma (12 orbit, 10 stomach, 4 thyroid, and 7 others) received radiation therapy in our institute. Median age was 67 years old (34-80). There were 17 men and 16 women. FDG uptake in those cases was estimated visually and by using the standardized uptake value (SUV) before and after radiation therapy, respectively. Ki-67 labeling index (LI) was evaluated histologically if tissue sections were available to study the correlation between Ki-67 LI and SUV. ResultsMedian follow-up was 45 months (10-87). Twenty-three cases (72%) were FDG-positive. Positive cases in each organ were 8 (67%) orbit, 5 (56%) stomach, 4 (100%) thyroid, and 2 (100%) larynx. The other 5 cases in different organs showed positive FDG uptake (hypopharynx, lung, prostate, and thymus). The median of maximum SUV (SUVmax) was 6.4 (3.4-11.5). Small tumors showed a tendency to be false-negative. In the comparison of SUV and Ki-67 LI, a positive correlation between them was suggested. Tumors with SUV >5 often showed high Ki-67LI, indicating relatively aggressive growth. For those tumors, dose escalation was considered in radiation therapy planning. The median irradiated total dose was 36 Gy (32-40). In FDG-positive cases, complete response rate was 100% after the initial treatment. Positive accumulation of FDG disappeared visually in all cases and the mean SUV was decreased by 58% (32-78). Distant relapse occurred in 2 patients. In both cases, relapsed lesions were found by follow-up FDG-PET/CT regardless of any symptoms. Median follow-up was 45 months (10-87). Twenty-three cases (72%) were FDG-positive. Positive cases in each organ were 8 (67%) orbit, 5 (56%) stomach, 4 (100%) thyroid, and 2 (100%) larynx. The other 5 cases in different organs showed positive FDG uptake (hypopharynx, lung, prostate, and thymus). The median of maximum SUV (SUVmax) was 6.4 (3.4-11.5). Small tumors showed a tendency to be false-negative. In the comparison of SUV and Ki-67 LI, a positive correlation between them was suggested. Tumors with SUV >5 often showed high Ki-67LI, indicating relatively aggressive growth. For those tumors, dose escalation was considered in radiation therapy planning. The median irradiated total dose was 36 Gy (32-40). In FDG-positive cases, complete response rate was 100% after the initial treatment. Positive accumulation of FDG disappeared visually in all cases and the mean SUV was decreased by 58% (32-78). Distant relapse occurred in 2 patients. In both cases, relapsed lesions were found by follow-up FDG-PET/CT regardless of any symptoms. ConclusionsThis study indicates that FDG-PET/CT may be useful for radiation therapy planning, response evaluation, and post-therapeutic follow-up for indolent MALT lymphoma. This study indicates that FDG-PET/CT may be useful for radiation therapy planning, response evaluation, and post-therapeutic follow-up for indolent MALT lymphoma.

The aim of this study was to investigate carbon ion beam- and X-ray-induced gene expression profiles in human brain tumors in vivo. Tumors of human origin, an ependymoblastoma (EB) with wild-type (wt) p53, primitive neuroectodermal tumor (PNET) with wt p53, and a glioblastoma (GB) with mutant-type (mt) p53, were transplanted into nude mice subcutaneously, and the mice were irradiated with carbon ion beams (290MeV/u, 6 cm spread-out Bragg peak) or 200kV X-rays. Tumors were excised 4, 6, or 24 hours after 2, 4, 8, or 16 Gy irradiation. A part of each tumor was stored in RNA stabilization solution and total RNA was extracted, and cDNA microarray analysis, hierarchical clustering, pattern analysis, gene ontology analysis, and pathway analysis were performed. The other part of each tumor was fixed in formalin and embedded in paraffin for microscopic study, and the TUNEL assay was performed to evaluate apoptosis. In EB with wt p53 and PNET with wt p53, apoptosis increased significantly 4 or 6 hours after carbon ion beam or X-ray irradiation. These tumors showed significant changes in gene expression profiles following single-dose irradiation of 2Gy or more. There were few differences between the profiles induced by carbon ion beams and those by X-rays. Pathway analysis of up- and down-regulated genes demonstrated that apoptosis, cell cycle, and p53 signaling pathways are involved significantly. In GB with mt p53, only a few apoptotic cells were found and few changes were shown in gene expression profiles following 2Gy irradiation; however, the tumor showed significant changes of profiles 4, 6, or 24 hours after 8Gy or 16Gy following carbon ion irradiation. Up- or down-regulation of p53, caspases, Fas, and TNF were not found, but p53R2, p70S6K, TRAIL-R, NF-kB, IkBa, and IAP were suggested to be involved. Changes of profiles induced by X-rays were similar, but different from those by carbon ion in part. In the two tumors with wt p53, radiation-induced apoptosis was frequent and p53 signaling and apoptosis-related pathways were suggested to play important roles in radiation therapy. In contrast, in the glioblastoma with mt p53, radiation-induced apoptosis was rare and anti-apoptotic or surviving pathways were suggested to be activated following carbon ion irradiation.

Loco-regional recurrence is often observed after resection of non-small-cell lung cancer without evidence of distant metastases, but few studies have been performed to investigate the efficacy of radiation therapy for recurrence, especially in elderly patients. This retrospective study was conducted to assess the feasibility and efficacy of salvage radiation therapy for loco-regional recurrence of elderly non-small-cell lung cancer after resection. Between April 2007 and March 2011, 18 patients with loco-regional recurrence of elderly non-small-cell lung cancer after resection visited our hospital for definitive radiation therapy. We analyzed 17 patients (9 adenocarcinomas, 5 squamous cell carcinomas, and 3 large cell carcinomas), excluding one patient treated with radiation previously. Fifteen patients were treated by conventional radiation therapy with the total dose ranging from 60 to 70 Gy, and 2 patients by stereotactic radiation therapy with 60 Gy in 8 fractions and 60 Gy in 15 fractions, respectively. In patients treated with the conventional technique, the prophylactic mediastinal lymph nodes were included in the clinical target volume. Overall survival after the beginning of radiation therapy was estimated using the Kaplan-Meier method and adverse events were evaluated according to the CTCAE (version 4.0). Median age of patients was 78 (66-84) years old (y.o.), and twelve patients were late elderly (75 y.o. or more). Performance status (PS) of each patient was good (ECOG PS 0: 11, PS 1: 6), and pathological stages consisted of five 1A, two 1B, six 2A, and four 3A. Three patients received concurrent chemotherapy and six sequential. The other 8 patients were treated by radiation therapy alone. In 16 patients, radiation therapy was carried out completely according to the plan. In one patient, radiation therapy was interrupted at 62 Gy for Grade 3 radiation pneumonitis. No other severe non-hematologic toxicity (CTCAE Grade 3 or greater) was observed. The median follow-up period was 24.8 (7.5-51.9) months. The overall 1-year and 2-year Kaplan-Meier survival rates were 88% and 65%, respectively, and the median survival time (MST) was 27.6 months. In 12 late elderly patients, the overall 1-year and 2-year Kaplan-Meier survival rates were 83% and 57%, respectively, and the MST was 26.3 months. These results suggest that salvage radiation therapy for loco-regional recurrence of non-small-cell lung cancer after resection is feasible and will be promising for elderly patients with good PS.

We evaluated sequential dynamic contrast-enhanced CT (DCE-CT) scans to assess the possibility of early prediction of treatment responses by quantifying the tumor size reduction and the change in tumor enhancement during and after a course of radiotherapy (RT). Thirty-nine patients with non-small-cell lung cancer were treated with RT for initial treatment. DCE-CT scan was performed within one week before the beginning of treatment, after 17 or 18 fractions (34 or 36 Gy), and 1 week and 1 month after the end of RT. The correlation between the relative decrease in tumor diameter and that in the attenuation value was evaluated. Nineteen patients were evaluated in this study. The median tumor size was 39.5 mm at the start of treatment, 30.8 mm at 34-36 Gy, and 16.1 mm 1 month after the end of RT. The relative decrease in tumor diameter at 34-36 Gy well correlated with that 1 month after treatment (r = 0.85, r: Pearson's correlation coefficient, p < 0.001). Relative change in the attenuation value at the rim of the tumor at 34-36 Gy did not significantly correlate with the change in tumor diameter 1 month after the completion of RT, but in the center of the tumor, the change of the attenuation value in the delayed phase correlated with the change in tumor diameter. The decrease of tumor diameter during RT may be predictive of treatment response. The relative change of tumor enhancement in the center of the tumor in the delayed phase correlated with tumor shrinkage 1 month after the completion of RT.

Pancreatic cancer has a very poor prognosis and prolonged survival is achieved only by resection with macroscopic tumor clearance. Neoadjuvant treatment by chemoradiotherapy with gemcitabine for locally advanced pancreatic cancer is suggested to be a promising therapy for R0 resection (i.e. post-operative surgical margins showed no microscopic evidence of tumor cells), but the gemcitabine dose was reduced in many previous studies. This study was conducted to assess the feasibility and efficacy of neoadjuvant chemoradiotherapy with concurrent full-dose gemcitabine for such patients. Between October 2008 and July 2010, 41 new patients with pancreatic cancer visited our hospital for neoadjuvant chemoradiotherapy. We analyzed 36 patients (T3:27, T4:9) with locally advanced disease, excluding T2 patients (n = 3) and patients with previous chemotherapy (n = 2). In CT-based treatment planning, the clinical target volume (CTV) was defined as the gross tumor volume plus 5-mm margin added to the prophylactic lymph node area around celiac artery origin and superior mesenteric artery origin. The planning target volume (PTV) was defined as the CTV plus set-up margin. We irradiated patients with a four-field beam arrangement (50Gy in 25 fractions) or intensity modulated radiation technique (54Gy in 27 fractions) with concurrent weekly full-dose (1000mg/m2) gemcitabine. Thirty-three patients (92%) completed concurrent chemoradiation. Median PTV was 135 (range 94-306) cc, and no severe non-hematologic toxicity (Grade 4 or greater) was observed. The median cumulative dose of gemcitabine was 5000 (range 3000-8000) mg/m2 with a withdrawal period of median 1 (range 0-4) week because of hematologic toxicity. Thirty-one patients were underwent surgery, and 26 (72%) of all patients had R0 resections. According to the Evans' grading system for the chemoradiation treatment effect, Grade 1, 2A, 2B, 3, 4 and 5 were 1 (3%),14 (47%),11 (37%),2 (7%) and 2 (7%),respectively, except for one exploratory laparotomy. The completion rate of chemoradiation was 92% with no severe non-hematologic toxicity, and the R0 resection rate was 72%. This result suggests that neoadjuvant chemoradiotherapy with concurrent full-dose gemcitabine for locally advanced pancreatic cancer may be feasible and promising.

Purpose/Objective(s)High LET carbon ion beams are often suggested to be more effective for radioresistant tumors than low LET X-rays; however, high-grade gliomas remain resistant to any radiotherapy. The aim of this study was to investigate carbon ion beam-induced gene expression profiles related to radiosensitivity or radioresistance in human glioblastoma cells in vivo.Materials/MethodsAn ependymoblastoma with wild-type (wt) p53, a primitive neuroectodermal tumor with wt p53, and a glioblastoma with mutant-type (mt) p53 were transplanted into nude mice subcutaneously and irradiated with carbon ion beams (290MeV/u, 6 cm spread-out Bragg peak) or 200kV X-rays. These tumors were excised 4, 6, and 24 hours after 2Gy of single dose irradiation. Additionally, the glioblastoma was examined 4 and 6 hours after 2, 8, or 16Gy irradiation to evaluate the dose-response relationship, and furthermore 4, 6 or 24 hours after 16Gy irradiation to evaluate the time-course following high-dose irradiation. Part of each tumor was fixed in formalin and embedded in paraffin for microscopic study. The TUNEL assay was performed to evaluate the induction of apoptosis. The other part of each tumor was stored in RNA stabilization solution and total RNA was extracted for GeneChip expression microarray analysis. Hierarchical clustering, gene ontology analysis, and pathway analysis were also performed.ResultsApoptosis increased significantly 4 or 6 hours after 2Gy irradiation in tumors with wt p53. Significant changes in gene expression were shown, and pathway analysis of up- or down-regulated genes demonstrated that apoptosis, the cell cycle, and the p53 signaling pathways were involved. There was little difference between the gene expression profiles induced by carbon ion beams and those by X-rays. In contrast, apoptosis increased only slightly in the glioblastoma with mt p53, and very few changes in gene expression profiles was found after 2Gy irradiation. However, significant profile changes were shown 4, 6, or 24 hours after 8Gy or 16Gy irradiation, and those induced by carbon ion beams were different from those by X-rays. In addition, p53, caspases, Fas, and TRAIL were not involved in the pathways, but up-regulation of the inhibitor of apoptosis proteins (IAP) and nuclear factor-kappaB (NF-kB) were shown 4 or 6 hours after irradiation.ConclusionsActivation of IAP and NF-kB was suggested to be correlated with radioresistance to high LET carbon ion beams in the glioblastoma with mt p53. Purpose/Objective(s)High LET carbon ion beams are often suggested to be more effective for radioresistant tumors than low LET X-rays; however, high-grade gliomas remain resistant to any radiotherapy. The aim of this study was to investigate carbon ion beam-induced gene expression profiles related to radiosensitivity or radioresistance in human glioblastoma cells in vivo. High LET carbon ion beams are often suggested to be more effective for radioresistant tumors than low LET X-rays; however, high-grade gliomas remain resistant to any radiotherapy. The aim of this study was to investigate carbon ion beam-induced gene expression profiles related to radiosensitivity or radioresistance in human glioblastoma cells in vivo. Materials/MethodsAn ependymoblastoma with wild-type (wt) p53, a primitive neuroectodermal tumor with wt p53, and a glioblastoma with mutant-type (mt) p53 were transplanted into nude mice subcutaneously and irradiated with carbon ion beams (290MeV/u, 6 cm spread-out Bragg peak) or 200kV X-rays. These tumors were excised 4, 6, and 24 hours after 2Gy of single dose irradiation. Additionally, the glioblastoma was examined 4 and 6 hours after 2, 8, or 16Gy irradiation to evaluate the dose-response relationship, and furthermore 4, 6 or 24 hours after 16Gy irradiation to evaluate the time-course following high-dose irradiation. Part of each tumor was fixed in formalin and embedded in paraffin for microscopic study. The TUNEL assay was performed to evaluate the induction of apoptosis. The other part of each tumor was stored in RNA stabilization solution and total RNA was extracted for GeneChip expression microarray analysis. Hierarchical clustering, gene ontology analysis, and pathway analysis were also performed. An ependymoblastoma with wild-type (wt) p53, a primitive neuroectodermal tumor with wt p53, and a glioblastoma with mutant-type (mt) p53 were transplanted into nude mice subcutaneously and irradiated with carbon ion beams (290MeV/u, 6 cm spread-out Bragg peak) or 200kV X-rays. These tumors were excised 4, 6, and 24 hours after 2Gy of single dose irradiation. Additionally, the glioblastoma was examined 4 and 6 hours after 2, 8, or 16Gy irradiation to evaluate the dose-response relationship, and furthermore 4, 6 or 24 hours after 16Gy irradiation to evaluate the time-course following high-dose irradiation. Part of each tumor was fixed in formalin and embedded in paraffin for microscopic study. The TUNEL assay was performed to evaluate the induction of apoptosis. The other part of each tumor was stored in RNA stabilization solution and total RNA was extracted for GeneChip expression microarray analysis. Hierarchical clustering, gene ontology analysis, and pathway analysis were also performed. ResultsApoptosis increased significantly 4 or 6 hours after 2Gy irradiation in tumors with wt p53. Significant changes in gene expression were shown, and pathway analysis of up- or down-regulated genes demonstrated that apoptosis, the cell cycle, and the p53 signaling pathways were involved. There was little difference between the gene expression profiles induced by carbon ion beams and those by X-rays. In contrast, apoptosis increased only slightly in the glioblastoma with mt p53, and very few changes in gene expression profiles was found after 2Gy irradiation. However, significant profile changes were shown 4, 6, or 24 hours after 8Gy or 16Gy irradiation, and those induced by carbon ion beams were different from those by X-rays. In addition, p53, caspases, Fas, and TRAIL were not involved in the pathways, but up-regulation of the inhibitor of apoptosis proteins (IAP) and nuclear factor-kappaB (NF-kB) were shown 4 or 6 hours after irradiation. Apoptosis increased significantly 4 or 6 hours after 2Gy irradiation in tumors with wt p53. Significant changes in gene expression were shown, and pathway analysis of up- or down-regulated genes demonstrated that apoptosis, the cell cycle, and the p53 signaling pathways were involved. There was little difference between the gene expression profiles induced by carbon ion beams and those by X-rays. In contrast, apoptosis increased only slightly in the glioblastoma with mt p53, and very few changes in gene expression profiles was found after 2Gy irradiation. However, significant profile changes were shown 4, 6, or 24 hours after 8Gy or 16Gy irradiation, and those induced by carbon ion beams were different from those by X-rays. In addition, p53, caspases, Fas, and TRAIL were not involved in the pathways, but up-regulation of the inhibitor of apoptosis proteins (IAP) and nuclear factor-kappaB (NF-kB) were shown 4 or 6 hours after irradiation. ConclusionsActivation of IAP and NF-kB was suggested to be correlated with radioresistance to high LET carbon ion beams in the glioblastoma with mt p53. Activation of IAP and NF-kB was suggested to be correlated with radioresistance to high LET carbon ion beams in the glioblastoma with mt p53.

High LET beams have different radiobiological effects on tumors compared with X-rays and their efficacy for radioresistant tumors with mutant-type (mt) p53 is often suggested. The aim of this study was to investigate carbon ion beam-induced gene expression profiles in human radioresistant glioma cells with mutant-type p53 in vivo. Three tumors of human origin, an ependymoblastoma (EB) with wild-type (wt) p53, a primitive neuroectodermal tumor (PNET) with wt p53, and a glioblastoma (GB) with mt p53, were transplanted into nude mice subcutaneously, and irradiated with carbon ion beams (290MeV/u, 6 cm spread-out Bragg peak) or 200kV X-rays. These tumors were excised 4, 6, or 24 hours after 2 Gy of single dose irradiation. Additionally, GB tumors were excised 6 hours after 2, 8, or 16 Gy irradiation. A part of each tumor was fixed in formalin and embedded in paraffin for microscopic study. Hematoxylin and eosin staining, and the TUNEL assay were performed to evaluate microscopic morphological changes and the induction of apoptosis. The other part of each tumor was stored in RNA stabilization solution, and total RNA was extracted for cDNA microarray analysis. GeneChip Human Genome U133 Plus 2.0 array (Affymetrix) was utilized to compare gene expression profiles following carbon ion beam or X-ray irradiation. Hierarchical clustering of the gene expression, gene ontology analysis, and pathway analysis were also performed. In EB and PNET with wt p53, apoptosis increased 4 and 6 hours after 2 Gy irradiation, and significant changes in gene expression profiles were shown simultaneously. There was little difference between the gene expression profiles induced by carbon ion beams and those by X-rays. Pathway analysis of up-regulated and down-regulated genes demonstrated that apoptosis, p53 signaling pathway, and cell cycle are involved significantly (p = 0.000). In contrast, GB with mutant-type p53 showed much less change in gene expression profiles following 2 Gy irradiation; however, significant changes in gene expression profiles were induced by 8 Gy or 16 Gy irradiation, and the profiles by carbon ion beams were significantly different from those by X-rays. These profiles were different from those of the tumors with wt p53, and many genes, not included in the p53 signaling pathway, are suggested to be involved. The present study demonstrated that high-dose carbon ion beams induce different gene expression profiles in human radioresistant glioma cells with mt p53 compared with X-rays, though most profiles induced by carbon ion beams and X-rays are similar in radiosensitive tumors with wt p53.

We evaluated sequential dynamic enhanced CT (DCE-CT) scans to assess the possibility of early prediction of treatment outcomes by quantifying the tumor size reduction and the change in tumor enhancement during and after a course of radiotherapy (RT). From January to December 2009, 32 patients with non-small-cell lung cancer (NSCLC) were treated with RT for initial treatment under the following protocol. DCE-CT scan was performed within one week before the beginning of the treatment, after 17 or 18 fractions (34 or 36 Gy), and 1 week after and 1 month after the end of the RT. The maximum diameter and the attenuation value of the tumor were measured. Two regions of interest in both the center and edge of each tumor were examined. The correlation between the relative decrease in tumor diameter and that in the attenuation value was evaluated. Of the 32 patients treated in this period, 2 died during the course of RT, and 2 aborted treatment because of adverse effects. In 3 patients, contrast-enhanced CT scans were not obtained because of renal dysfunction. In 1 patient with a 10 mm tumor, the attenuation value could not be measured in two regions. In 2 patients, CT scans were not obtained because of technical issues. In 5 patients, CT scans were not performed under this protocol. One patient was lost to follow-up. The other 16 patients were evaluated in this study. The median age was 72.5 years (range: 41-79) and 1 patient was female. The median radiation dose was 66 Gy (range: 60-70). Chemotherapy was given to 13 patients. The clinical stage was IIB in 3 patients, IIIA in 6 patients, IIIB in 6 patients and IV in 1 patient. The individual tumor size ranged from 11.0 to 72.9 mm at the start of treatment. The median tumor size was 36.4 mm before treatment, 32.6 mm (mean regression rate [MRR]: 0.90) at 34-36 Gy, 24.9 mm (MRR: 0.68) 1 week after the end of RT, and 20.7 mm (MRR: 0.57) 1 month after the end of RT, respectively. All but 3 tumors regressed at 34-36 Gy (more than 8%) and 1 month after the end of RT by more than 30%. Three tumors regressed less than 8% at 34-36 Gy. The relative decrease in tumor diameter at 34-36 Gy was well correlated with that 1 month after treatment (r = 0.84, r: Pearson's correlation coefficient, p < 0.001). Relative changes in the attenuation value in the center and at the edge of the tumors at 34-36 Gy did not correlate with the change in tumor diameter 1 month after the completion of RT in the early phase (r = -0.17, 0.26) or in the delayed phase (r = -0.48, 0.05). The relative decrease of tumor diameter at 34-36 Gy may be predictive of treatment outcome; however, no correlation between the relative early change of tumor enhancement and tumor shrinkage was suggested.