2638
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Materials/Methods: This is a multi-institutional case-control study comprehensively analyzing SNPs and comparing the alleles between the patients who developed grade 2 dermatitis (dermatitis group; n 77) and the control patients (control group; n 79). The patients of the dermatitis group were selected from the pool of more than 3,000 patients, reviewing the photographs of the skin and the radiation dose distribution charts, to exclude the confounding external factors. In patient selection for the control group, the patient-to-patient matching was employed, so that the two groups should be well balanced in terms of the major patient characteristics (e.g. patient’s age, menopausal status and pathological T/N classifications) and the therapeutic parameters (e.g. the institution, beam energy, radiation dose and fractionation). For the SNP analysis, DNA from each patient was subjected to genotyping of 3,144 SNPs covering 487 genes. Results: Two loci, the IL12RB2 gene locus and the ABCA1 gene locus, were shown to be associated with dermatits. In IL12RB2 gene, 5 SNPs of examined 13 SNPs showed significant correlation with dermatitis (p 0.0060 at lowest). The haplotype block analysis indicated that all of these 5 SNPs were located in a single haplotype block. A haplotype designated as H3 was associated with dermatisis (p0.004). As for ABCA1 gene, 50 SNPs were examined and 8 SNPs showed significant correlation with dermatitis (p 0.0038 at lowest). One of them was a cSNP causing amino acid substitution between Lys and Arg at the 219th amino acid. The haplotype block analysis revealed that the 8 SNPs were located in 2 haplotype blocks. Conclusions: The present study featured a more comprehensive SNPs analysis than any previous study. Since a considerable part of clinically observed hyperradiosensitivity may be regulated by integration of multiple minor alterations in genetic function, a comprehensive analysis, as was performed in the present study, may be suitable for identification of radiosensitive population of patients. We concluded that several SNPs in IL12RB2 and ABCA1 genes were associated with susceptibility of radiation induced dermatitis. This was suggested to be a non-random event, considering that these SNPs were located only in one or two haplotype blocks of the genes. Analyzing these SNPs may be a promising predictive assay to identify the radiosensitive patients, and to individualize radiotherapy. Author Disclosure: N. Oya, None; M. Isomura, None; S. Tachiiri, None; Y. Kaneyasu, None; M. Hareyama, None; N. Mitsuhashi, None; Y. Nishimura, None; T. Akimoto, None; Y. Miki, None; M. Hiraoka, None. 2638 Hedgehog Signaling Pathway as a Novel Molecular Target for Radiation Sensitization of Vascular Endothelium L. Geng, K. C. Cuneo, S. Konjeti, A. Fu, M. Cooper, D. E. Hallahan Vanderbilt University School of Medicine, Nashville, TN Purpose/Objective(s): Hedgehog (Hh) proteins are transmembrane receptors that play an essential role in pattern formation during embryogenesis. This signaling pathway has been shown to be associated with the proliferation and differentiation of normal tissue and cancer cells. Cyclopamine is an Hh signaling pathway antagonist and has been effective in inhibiting the growth of specific types of tumors. We studied the role of Hh signaling in endothelial cells and the efficacy of cyclopamine as a potential radiation sensitizer on both tumor cells and endothelium. Materials/Methods: Cultured endothelial cells (HUVEC) and two cancer cell lines, H460 (human lung adenocarcinoma) and B16F0 (murine melanoma), were used in this study. Northern blot assays for ptch expression were performed to determine the activity of Hh signaling in the cell lines. To test the effect of cyclopamine and radiation on endothelial function, tubule formation on Matrigel was studied using 3 Gy. Additionally, in vitro Brdu incorporation studies were performed using 5 nM recombinant Sonic hedgehog peptide (SHh) and/or 5 M cyclopamine. The treated cells were fixed and stained for flow cytometry or histological analysis. Brdu incorporation into nuclei was further used to study the effect of cyclopamine on proliferation in vivo using H460 and B16F0 hind limb tumor xenograph models. Results: Northern blot analysis of ptch expression revealed that the Hh signaling pathway was active in proliferating vascular endothelial cells. Tubule formation assays on Matrigel revealed a synergistic effect of cyclopamine and 3 Gy on endothelial capillary-like tubule formation. Brdu incorporation immunofluorescent staining assays showed a greater than 15% increase in HUVEC proliferation when the cells were stimulated with the recombinant SHh peptide (see figure). This increase was completely blocked by cyclopamine indicating that this pathway can be targeted in proliferating vascular endothelial cells. These results were confirmed with flow cytometry analysis of Brdu incorporation. Use of SHh peptide increased proliferation by 12% and this increase was attenuated by cyclopamine. In vivo studies further confirmed that cyclopamine affects the rate of endothelium and tumor cell (H460, B16F0) proliferation. Conclusions: This study identified an inducible hedgehog signaling pathway in endothelial cells. Furthermore, inhibiting hedgehog signaling with cyclopamine affected tumor cell and endothelial cell proliferation in vivo. The hedgehog signaling pathway antagonist cyclopamine had a synergistic effect with radiation on inhibiting endothelial cell tubule formation. S565 Proceedings of the 48th Annual ASTRO Meeting
Transcript of 2638
Materials/Methods: This is a multi-institutional case-control study comprehensively analyzing SNPs and comparing the allelesbetween the patients who developed �� grade 2 dermatitis (dermatitis group; n � 77) and the control patients (control group;n � 79). The patients of the dermatitis group were selected from the pool of more than 3,000 patients, reviewing thephotographs of the skin and the radiation dose distribution charts, to exclude the confounding external factors. In patientselection for the control group, the patient-to-patient matching was employed, so that the two groups should be well balancedin terms of the major patient characteristics (e.g. patient’s age, menopausal status and pathological T/N classifications) and thetherapeutic parameters (e.g. the institution, beam energy, radiation dose and fractionation). For the SNP analysis, DNA fromeach patient was subjected to genotyping of 3,144 SNPs covering 487 genes.
Results: Two loci, the IL12RB2 gene locus and the ABCA1 gene locus, were shown to be associated with dermatits. InIL12RB2 gene, 5 SNPs of examined 13 SNPs showed significant correlation with dermatitis (p � 0.0060 at lowest). Thehaplotype block analysis indicated that all of these 5 SNPs were located in a single haplotype block. A haplotype designatedas H3 was associated with dermatisis (p�0.004). As for ABCA1 gene, 50 SNPs were examined and 8 SNPs showed significantcorrelation with dermatitis (p � 0.0038 at lowest). One of them was a cSNP causing amino acid substitution between Lys andArg at the 219th amino acid. The haplotype block analysis revealed that the 8 SNPs were located in 2 haplotype blocks.
Conclusions: The present study featured a more comprehensive SNPs analysis than any previous study. Since a considerablepart of clinically observed hyperradiosensitivity may be regulated by integration of multiple minor alterations in geneticfunction, a comprehensive analysis, as was performed in the present study, may be suitable for identification of radiosensitivepopulation of patients. We concluded that several SNPs in IL12RB2 and ABCA1 genes were associated with susceptibility ofradiation induced dermatitis. This was suggested to be a non-random event, considering that these SNPs were located only inone or two haplotype blocks of the genes. Analyzing these SNPs may be a promising predictive assay to identify theradiosensitive patients, and to individualize radiotherapy.
Author Disclosure: N. Oya, None; M. Isomura, None; S. Tachiiri, None; Y. Kaneyasu, None; M. Hareyama, None; N.Mitsuhashi, None; Y. Nishimura, None; T. Akimoto, None; Y. Miki, None; M. Hiraoka, None.
2638 Hedgehog Signaling Pathway as a Novel Molecular Target for Radiation Sensitization of VascularEndothelium
L. Geng, K. C. Cuneo, S. Konjeti, A. Fu, M. Cooper, D. E. Hallahan
Vanderbilt University School of Medicine, Nashville, TN
Purpose/Objective(s): Hedgehog (Hh) proteins are transmembrane receptors that play an essential role in pattern formationduring embryogenesis. This signaling pathway has been shown to be associated with the proliferation and differentiation ofnormal tissue and cancer cells. Cyclopamine is an Hh signaling pathway antagonist and has been effective in inhibiting thegrowth of specific types of tumors. We studied the role of Hh signaling in endothelial cells and the efficacy of cyclopamine asa potential radiation sensitizer on both tumor cells and endothelium.
Materials/Methods: Cultured endothelial cells (HUVEC) and two cancer cell lines, H460 (human lung adenocarcinoma) andB16F0 (murine melanoma), were used in this study. Northern blot assays for ptch expression were performed to determine theactivity of Hh signaling in the cell lines. To test the effect of cyclopamine and radiation on endothelial function, tubuleformation on Matrigel was studied using 3 Gy. Additionally, in vitro Brdu incorporation studies were performed using 5 nMrecombinant Sonic hedgehog peptide (SHh) and/or 5 �M cyclopamine. The treated cells were fixed and stained for flowcytometry or histological analysis. Brdu incorporation into nuclei was further used to study the effect of cyclopamine onproliferation in vivo using H460 and B16F0 hind limb tumor xenograph models.
Results: Northern blot analysis of ptch expression revealed that the Hh signaling pathway was active in proliferating vascularendothelial cells. Tubule formation assays on Matrigel revealed a synergistic effect of cyclopamine and 3 Gy on endothelialcapillary-like tubule formation. Brdu incorporation immunofluorescent staining assays showed a greater than 15% increase inHUVEC proliferation when the cells were stimulated with the recombinant SHh peptide (see figure). This increase wascompletely blocked by cyclopamine indicating that this pathway can be targeted in proliferating vascular endothelial cells.These results were confirmed with flow cytometry analysis of Brdu incorporation. Use of SHh peptide increased proliferationby 12% and this increase was attenuated by cyclopamine. In vivo studies further confirmed that cyclopamine affects the rateof endothelium and tumor cell (H460, B16F0) proliferation.
Conclusions: This study identified an inducible hedgehog signaling pathway in endothelial cells. Furthermore, inhibitinghedgehog signaling with cyclopamine affected tumor cell and endothelial cell proliferation in vivo. The hedgehog signalingpathway antagonist cyclopamine had a synergistic effect with radiation on inhibiting endothelial cell tubule formation.
S565Proceedings of the 48th Annual ASTRO Meeting
Author Disclosure: L. Geng, None; K.C. Cuneo, None; S. Konjeti, None; A. Fu, None; M. Cooper, None; D.E. Hallahan, None.
2639 Risk of Second Malignant Neoplasm After Cancer in Childhood Treated by Radiotherapy: CorrelationWith the Integral Dose
T. V. F. Nguyen1, C. Rubino1, S. Guerin1, I. Diallo1, A. Samand1, M. Hawkins2, O. Oberlin1, D. Lefkopoulos1,F. De Vathaire1
1Institut Gustave Roussy, Villejuif, France, 2University of Birmingham, Birmingham, United Kingdom
Purpose/Objective(s): After successful treatment of malignant diseases in childhood, the problem of long-term complicationscame to the fore. Few studies have focused on the relationship between the radiation dose received and the risk of developinga second malignant neoplasm (SMN). Most of them estimated risk coefficients according to the radiation dose received at thesite of the second cancer: thus, the estimated relative risk (RR) is the ratio between the risk of developing a cancer at a givensite that received a given dose and the risk of developing a cancer at the same site in the absence of this dose of radiation.
Materials/Methods: This approach does not really correspond to needs in clinical practice, where a global evaluation of therisk is required. In order to investigate the influence of radiotherapy on the overall risk of SMN after a childhood cancer in acohort of 4401 patients treated in 8 centres in France and UK between 1947 and 1986, we used another dose distributionparameter, defined as the Integral Dose, which provides an estimate of the total energy (in joules) delivered to the patient’s bodyduring radiotherapy. We also calculated the integral dose per kilogram (kg) by dividing each integral dose by the patient’sweight in order to obtain an average dose (in grays) delivered throughout the body. The superiority of these new parametersin the risks estimate is that they take into account the deposit of a given dose to a given volume of the body whatever theirradiated site. In our cohort, we highlighted a significant linear relationship between the integral dose and the risk of SMN.
Results: According to the linear model, after adjustment on chemotherapy (mainly alkylating and intercalating agents), theexcess risk of SMN was of 0.008 per unity of the integral dose. This coefficient increased to 0.017 if a negative exponentialterm was taken into account. With the integral dose per kg, these coefficients became respectively 0.23 and 0.48. For 20% ofthe patients who received the higher dose, the risk of SMN, compared to the non-irradiated patients, was 5.43 and 4.97,respectively for the integral dose and the integral dose per kg. Nevertheless, if we focus on patients who were treated byradiotherapy, only those who had received the highest integral dose had a higher risk and indeed, among the other patients,including 80% of the variability of the integral dose, no increased risk was evidenced. One of the main reasons is probably dueto the wide heterogeneity in our cohort which included patients treated for all types of solid cancers and inadequate adjustmenton chemotherapy whose combinations varied considerably. Finally, the estimated risk with the integral dose, in our study,cannot be considered as a good predictor of a later risk of SMN.
Conclusions: Modern radiotherapy techniques such as intensity modulated radiation therapy (IMRT) or tomotherapy involvea greater number of treatment beams exposing a larger volume of normal tissue to lower doses. It is therefore of paramountimportance to take into account the irradiated volume when evaluating the long-term adverse effect of these radiotherapytechniques. Thus, other studies with the same study design are obviously needed to evaluate the usefulness of the integral doseas a new tool of decision-making aid for choosing between different radiation therapy plans.
Author Disclosure: T.V.F. Nguyen, None; C. Rubino, None; S. Guerin, None; I. Diallo, None; A. Samand, None; M. Hawkins,None; O. Oberlin, None; D. Lefkopoulos, None; F. De vathaire, None.
2640 p53-Regulated Transcriptional Response to Radiation in the Cerebral Cortex
J. F. Dorsey, J. P. Plastaras, W. S. El-Deiry
University of Pennsylvania, Philadelphia, PA
Purpose/Objective(s): Recent investigations have revealed that p53 activates downstream targets in a highly regulated,selective, and tissue-specific manner. We therefore sought to characterize the p53-mediated transcriptional response to radiationin the CNS with the goal of increasing our understanding of the therapeutic index of radiation and potential toxicities. Acomprehensive gene expression analysis was performed.
S566 I. J. Radiation Oncology ● Biology ● Physics Volume 66, Number 3, Supplement, 2006
