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Ataxia Telangiectasia Mutated (ATM) Gene Variants in American Indians
I. J. Radiation Oncology d Biology d Physics
S90
192
Volume 78, Number 3, Supplement, 2010
Ataxia Telangiectasia Mutated (ATM) Gene Variants in American Indians
D. G. Petereit1, A. Moser2, J. Hahn2, A. Boylan1, S. Kanekar1, M. Ritter2, S. Bentzen2, D. Koop1, J. Kaur3, M. Mehta2 1 3
Rapid City Regional Hospital, Rapid City, SD, 2University of Wisconsin School of Medicine and Public Health, Madison, WI, Mayo Clinic Comprehensive Cancer Center, Rochester, MN
Purpose/Objective(s): Approximately 5-10% of patients that undergo radiotherapy experience significant toxicities. A retrospective chart review of patients undergoing radiation therapy at our institution indicated that the American Indian (AI) population experienced increased toxicities, compared to the non-AI population. Homozygosity for mutations in ATM leads to extreme hypersensitivity to radiation; whereas, heterozygosity for an ATM mutation may be associated with an increased risk of breast cancer and an increased risk of radiation toxicities. To investigate a possible molecular mechanism for increased likelihood of ATM. Materials/Methods: DNA was isolated from blood samples collected from 100 AI and 100 non-AI undergoing radiation therapy for various cancers and a blinded assessment of ATM sequence alteration was conducted. Acute and intermediate term radiation toxicities were prospectively monitored and recorded for each patient. Direct sequencing of all of the coding exons of the ATM gene was performed on both forward and reverse strands. Sequence comparisons were made to a reference maintained at the ATM Project Website and in the published literature. The main outcome measures were prevalence of ATM SNPs for each patient. Results: No statistically significant differences for total prevalence of SNPs among AI (40%) and non-AI (48%) patients (p = 0.32) were identified. Five SNPs had a prevalence of .2%, of which 4 occurred at a rate of .5% in one or both groups. The prevalence of these could meaningfully be compared statistically in the two groups. The only statistically significant difference among the groups was the c.4138C.T SNP seen in 8% of AI versus 0% of non-AI patients (p = 0.007). However, this SNP is predicted using the Polyphen software tool not to affect protein function. The prevalence of those SNPs predicted to result in potentially deleterious missense mutations, was 28% among non-AI and 18% among AI (p = 0.13). of particular interest is SNP c.5557G.A, which had a prevalence of 25% in non-AIs versus 14% in AIs (p = 0.07). Three homozygous patients were identified for this SNP, all in the non-AI group. Conclusions: This study provides new information on the ethnic distribution of ATM SNPs in a regional US population and is the first to determine the prevalence of ATM SNPs in AI. Correlating the SNP data with available patient toxicity data is underway and will potentially reveal any important SNPs that can be predictive of radiosensitivity. There is no support in our study for the hypothesis that a difference in prevalence or type of ATM SNPs explains the putatively different spectrum of side effects after radiotherapy in AI versus non-AI patients. This study was funded by NIH grant 5 U56 CA099010. Author Disclosure: D.G. Petereit, None; A. Moser, None; J. Hahn, None; A. Boylan, None; S. Kanekar, None; M. Ritter, None; S. Bentzen, None; D. Koop, None; J. Kaur, None; M. Mehta, None.
193
High-dose Carbon Ion Beams and X-rays Induce Different Gene Expression Profiles in Human Radioresistant Glioma Cells with Mutant Type p53 In Vivo
M. Hasegawa1, I. Asakawa1, T. Tamamoto1, E. Katayama1, K. Inoue1, H. Okada2, S. Kato3, T. Murakami3 1 Nara Medical University, Kashihara, Japan, 2Higashiosaka City General Hospital, Higashiosaka, Japan, 3National Institute of Radiological Sciences, Chiba, Japan
Purpose/Objective(s): 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 beaminduced gene expression profiles in human radioresistant glioma cells with mutant-type p53 in vivo. Materials/Methods: 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. Results: 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. Conclusions: 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. Author Disclosure: M. Hasegawa, None; I. Asakawa, None; T. Tamamoto, None; E. Katayama, None; K. Inoue, None; H. Okada, None; S. Kato, None; T. Murakami, None.
194
Defective DNA-PKcs Protein-Protein Interaction in LB0005 Cell Line from an Adult Cancer Patient with Late Radionecrosis
S. Loong1, S. Yap1, C. Boo1, P. Hande2, M. Paterson1, A. Price3, R. Baskar1, S. Yap1 1
National Cancer Centre, Singapore, Singapore, 2National University of Singapore, Singapore, Singapore, 3University of Edinburgh, Edinburgh, United Kingdom
S90
192
Volume 78, Number 3, Supplement, 2010
Ataxia Telangiectasia Mutated (ATM) Gene Variants in American Indians
D. G. Petereit1, A. Moser2, J. Hahn2, A. Boylan1, S. Kanekar1, M. Ritter2, S. Bentzen2, D. Koop1, J. Kaur3, M. Mehta2 1 3
Rapid City Regional Hospital, Rapid City, SD, 2University of Wisconsin School of Medicine and Public Health, Madison, WI, Mayo Clinic Comprehensive Cancer Center, Rochester, MN
Purpose/Objective(s): Approximately 5-10% of patients that undergo radiotherapy experience significant toxicities. A retrospective chart review of patients undergoing radiation therapy at our institution indicated that the American Indian (AI) population experienced increased toxicities, compared to the non-AI population. Homozygosity for mutations in ATM leads to extreme hypersensitivity to radiation; whereas, heterozygosity for an ATM mutation may be associated with an increased risk of breast cancer and an increased risk of radiation toxicities. To investigate a possible molecular mechanism for increased likelihood of ATM. Materials/Methods: DNA was isolated from blood samples collected from 100 AI and 100 non-AI undergoing radiation therapy for various cancers and a blinded assessment of ATM sequence alteration was conducted. Acute and intermediate term radiation toxicities were prospectively monitored and recorded for each patient. Direct sequencing of all of the coding exons of the ATM gene was performed on both forward and reverse strands. Sequence comparisons were made to a reference maintained at the ATM Project Website and in the published literature. The main outcome measures were prevalence of ATM SNPs for each patient. Results: No statistically significant differences for total prevalence of SNPs among AI (40%) and non-AI (48%) patients (p = 0.32) were identified. Five SNPs had a prevalence of .2%, of which 4 occurred at a rate of .5% in one or both groups. The prevalence of these could meaningfully be compared statistically in the two groups. The only statistically significant difference among the groups was the c.4138C.T SNP seen in 8% of AI versus 0% of non-AI patients (p = 0.007). However, this SNP is predicted using the Polyphen software tool not to affect protein function. The prevalence of those SNPs predicted to result in potentially deleterious missense mutations, was 28% among non-AI and 18% among AI (p = 0.13). of particular interest is SNP c.5557G.A, which had a prevalence of 25% in non-AIs versus 14% in AIs (p = 0.07). Three homozygous patients were identified for this SNP, all in the non-AI group. Conclusions: This study provides new information on the ethnic distribution of ATM SNPs in a regional US population and is the first to determine the prevalence of ATM SNPs in AI. Correlating the SNP data with available patient toxicity data is underway and will potentially reveal any important SNPs that can be predictive of radiosensitivity. There is no support in our study for the hypothesis that a difference in prevalence or type of ATM SNPs explains the putatively different spectrum of side effects after radiotherapy in AI versus non-AI patients. This study was funded by NIH grant 5 U56 CA099010. Author Disclosure: D.G. Petereit, None; A. Moser, None; J. Hahn, None; A. Boylan, None; S. Kanekar, None; M. Ritter, None; S. Bentzen, None; D. Koop, None; J. Kaur, None; M. Mehta, None.
193
High-dose Carbon Ion Beams and X-rays Induce Different Gene Expression Profiles in Human Radioresistant Glioma Cells with Mutant Type p53 In Vivo
M. Hasegawa1, I. Asakawa1, T. Tamamoto1, E. Katayama1, K. Inoue1, H. Okada2, S. Kato3, T. Murakami3 1 Nara Medical University, Kashihara, Japan, 2Higashiosaka City General Hospital, Higashiosaka, Japan, 3National Institute of Radiological Sciences, Chiba, Japan
Purpose/Objective(s): 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 beaminduced gene expression profiles in human radioresistant glioma cells with mutant-type p53 in vivo. Materials/Methods: 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. Results: 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. Conclusions: 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. Author Disclosure: M. Hasegawa, None; I. Asakawa, None; T. Tamamoto, None; E. Katayama, None; K. Inoue, None; H. Okada, None; S. Kato, None; T. Murakami, None.
194
Defective DNA-PKcs Protein-Protein Interaction in LB0005 Cell Line from an Adult Cancer Patient with Late Radionecrosis
S. Loong1, S. Yap1, C. Boo1, P. Hande2, M. Paterson1, A. Price3, R. Baskar1, S. Yap1 1
National Cancer Centre, Singapore, Singapore, 2National University of Singapore, Singapore, Singapore, 3University of Edinburgh, Edinburgh, United Kingdom